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PMC10000755 | The purpose of the present study was to evaluate the prognostic role of C-reactive protein (CRP) in children with Ewing's sarcoma. We conducted a retrospective study on 151 children undergoing multimodal treatment for Ewing's sarcoma in the appendicular skeleton from December 1997 to June 2020. Univariate Kaplan-Meier analyses of laboratory biomarkers and clinical parameters showed that CRP and metastatic disease at presentation were poor prognostic factors associated with overall survival and disease recurrence at 5 years (p < 0.05). A multivariate Cox regression model showed that pathological CRP (>=1.0 mg/dL) [HR of 3.67; 95% CI, 1.46 to 10.42] and metastatic disease [HR of 4.27; 95% CI, 1.58 to 11.47] were associated with a higher risk of death at 5 years (p < 0.05). In addition, pathological CRP (>=1.0 mg/dL) [HR of 2.66; 95% CI, 1.23 to 6.01] and metastatic disease [HR of 2.56; 95% CI, 1.13 to 5.55] were associated with a higher risk of disease recurrence at 5 years (p < 0.05). Our findings demonstrated that CRP was associated with the prognosis of children with Ewing's sarcoma. We recommend pre-treatment measurement of the CRP in order to recognize children with Ewing's sarcoma who are at greater risk of death or local recurrence. inflammatory biomarkers C-reactive protein Ewing's sarcoma long bones appendicular skeleton survival prognosis local recurrence children This research received no external funding. pmc1. Introduction Ewing's sarcoma is a highly aggressive malignant small round-cell tumor that mainly affects children, adolescents and young adults. It predominantly occurs in the bones and occasionally in the soft tissues . In the pre-chemotherapeutic era, the overall survival at 10 years was approximately 10% . With the introduction of multimodal treatment, combining surgery and/or radiotherapy with systemic chemotherapy, the prognosis improved to 65-75% at 10 years for patients with localized disease, and 30% for those with metastatic disease . Although there are no doubts that the presence of metastatic disease at presentation is the main negative prognostic factor, for patients with localized disease, the prognostic significance of several prognostic factors is still uncertain . A raised level of C-reactive protein (CRP) has been found to be an indicator of poor prognosis in several cancers, including esophageal carcinoma, carcinoma of the stomach, colorectal cancer, hepatocellular carcinoma, renal cancer, and pancreatic tumor . The prognostic value of CRP has also been evaluated in patients affected by bone and soft tissue sarcomas, without differentiating the various histotypes that have distinct treatments and prognoses . The only studies that analyzed CRP in patients with Ewing's sarcoma had important limitations . Nakamura et al. analyzed patients without rule out the presence of inflammatory conditions . Li et al. analyzed patients with Ewing's sarcoma who underwent a heterogeneous treatment with many patients not receiving chemotherapy and the variables analyzed did not include chemotherapy-induced tumor necrosis and tumor volume . Xu et al. only analyzed patients with vertebral or sacral Ewing's sarcoma, which are usually associated with worse prognosis . Recently, two other studies analyzed the possible association between pathological CRP and prognosis in patients with Ewing's sarcoma, but the authors did not analyze tumor size, other inflammatory biomarkers, and did not consider the subgroup analysis of localized patients and metastatic patients . The purpose of the present study was to evaluate the prognostic role of CRP in children with Ewing's sarcoma in relation to previously cited variables, including age, gender, tumor volume, tumor site, and chemotherapy-induced necrosis. The identification of reliable prognostic factors could help to identify high-risk patients, which may require a different treatment and follow up. 2. Materials and Methods An independent ethics committee approved this study, which was registered with ClinicalTrials.gov (NCT05100368). We conducted a retrospective study of 362 patients undergoing multimodal treatment for Ewing's sarcoma in the appendicular skeleton from December 1997 to June 2020. We investigated potential clinical and laboratory prognostic factors. The prognostic clinical and laboratory factors considered for the analysis were chosen based on previous studies on bone and soft tissue sarcoma survival factors . The inclusion criteria were patients younger than 18 years old with a pathologically confirmed diagnosis of Ewing's sarcoma of the long bones that were treated at our institution. Exclusion criteria were patients who underwent previous treatments including chemotherapy, radiotherapy and surgery (118); patients who had clinical evidence of infection or inflammatory disease before the biopsy (2); patients who underwent any major surgical treatment in the previous 30 days from the diagnosis; patients with incomplete clinical or laboratory data (83); and patients who were alive with a follow-up shorter than 24 months (8). One hundred and fifty-one patients were eligible for the study . There included 40 females (26.5%) and 111 males (73.5%), with a mean age of 13 years (ranging from three to 18 years). The mean follow-up was 75 months (ranging from 24 to 224 months). The most common tumor site was the pelvis (25.8%), followed by the femur (23.8%) and the tibia (21.2%). A chest Computed Tomography and PET or bone scan were performed for disease staging. One hundred and eight patients (71.5%) had localized disease, and 43 patients (28.5%) had metastatic disease at presentation. The most common metastatic site was the lung (55.8%), followed by the bones (18.6%). A multidisciplinary team (according to European guidelines) made decisions about treatment. Radiotherapy was considered if patients were not candidates for surgery, if there was a positive surgical margin after excision, or if the tumor had a poor response to chemotherapy (<90% tumor necrosis). Each patient underwent chemotherapy and surgical treatment in our institute but received radiotherapy at their local oncology hospitals. One hundred and fourteen patients (75.5%) underwent surgical treatment as local treatment, 34 patients (22.5%) received definitive radiotherapy as local treatment, and three patients (2%) only received chemotherapy. Neoadjuvant chemotherapy according to protocols used at the time of treatment was provided to all patients (Table 1). The histopathological diagnosis was made in our institute. The surgical margins were wide in 106 patients (93%), marginal in four patients (3.5%), and intralesional in four patients (3.5%). The response to chemotherapy was good (tumor necrosis >= 90%) in 62 patients (54.4%) and poor (tumor necrosis < 90%) in 52 patients (45.6%). We analyzed the following prognostic factors: patient age, gender, tumor site, tumor size (>=200 mL or <200 mL), metastasis at presentation, treatment methods (neoadjuvant or adjuvant chemotherapy, radiotherapy, surgery), and response to chemotherapy. We also analyzed several inflammatory biomarkers suggested in previous studies: CRP, neutrophil count, lymphocyte count, monocyte count, platelet count, serum hemoglobin, alkaline phosphatase, lactate dehydrogenase (LDH), neutrophil-lymphocyte ratio (NLR), and platelets-lymphocyte ratio (PLR) . Tumor volume was calculated as an ellipsoid mass according to previous studies . Inflammatory biomarkers were measured before biopsy (Table 2). NLR was calculated by dividing the neutrophil count by the lymphocyte count ; PLR was calculated by dividing the platelet count by the lymphocyte count . The laboratory biomarkers were measured as part of a routine biochemical examination before any treatment, including chemotherapy, radiotherapy and surgery. We defined the cut off value for each biomarker (normal versus high or low) according to previous studies on the prognosis of bone and soft tissue sarcomas . Pathological CRP was defined as values > 1 mg/100 mL . A high level of NLR was defined as >2.38 . The AU Beckman Coulter 680 analyzer was used for the CRP analysis and a Dasit system XN-1000 was used for the remaining laboratory biomarkers . Surveillance for local and systemic recurrence consisted of clinical and radiologic assessments (radiograph of the surgical site and chest Computed Tomography) every 3 months for the first 2 years, then every 6 months up to 5 years, and annually thereafter. Statistical Analysis Statistical analyses were performed with the use of JMP(r), Version 12.0.1. SAS Institute Inc., Cary, NC, USA, 1989-2007 and R version 3.4.2. (Comprehensive R Archive Network). Overall survival time was set as the interval between the date of diagnosis and the date of the latest follow-up or death. Recurrence-free survival time was set as the interval between the date of diagnosis and the date of the latest follow-up or local versus distant recurrence. CRP has been used to investigate a possible association with 5-year overall survival and 5-year recurrence-free survival, separately in the group of patients with metastatic disease at presentation and in the group with localized disease. Survival curves were estimated with the Kaplan-Meier method. The log-rank test was used to compare survival between groups. A further analysis was conducted testing all the recorded laboratory biomarkers and clinical factors in the group of patients treated with surgery as the local treatment, including both patients with metastatic and localized disease, in order to investigate a possible association with 5-year overall survival and 5-year recurrence free survival. Survival curves were estimated with the Kaplan-Meier method. The log-rank test was used to compare survivorship between groups. A multivariate Cox regression model was estimated with the parameters that were statistically significant at the univariate analysis. Hazard ratios and their corresponding 95% CI were estimated, and p values < 0.05 were considered significant. 3. Results The 5-year overall survival was 68.5% (95% CI 60.2-75.8): 102 patients (67.5%) were alive, while 49 patients (32.5%) died. Eighty-five patients (56.3%) were alive with no evidence of disease, 13 patients (8.6%) were alive with no evidence of disease after a recurrence, and four patients (2,6%) were alive with disease. The 5-year recurrence-free survival was 60.1% (95 CI 51.6-67.9): 60 patients (39.7%) developed a recurrence of disease (nine patients had a local recurrence, 42 patients had distant metastasis and nine patients had both local recurrence and distant metastasis). A univariate Kaplan-Meier analysis showed that CRP was a negative prognostic factor associated with overall survival at 5 years both in patients with metastatic and localized disease, p = 0.010 and p = 0.004, respectively (Table 3). In the group of patients with localized disease, patients with pathological CRP had a poorer 5-year overall survival than patients with a normal CRP (p = 0.004). The overall survival at five years was 65.6% (95% CI 47.9 to 79.8) in patients with pathological CRP and 92.7% (95% CI 79.6 to 97.6) in those with normal CRP . In the group of patients with metastatic disease at presentation, patients with pathological CRP had a poorer 5-year overall survival than patients with normal CRP (p = 0.010). The overall survival at five years was 12.5% (95% CI 4.1 to 32.4) in patients with pathological CRP, and 50% (95% CI 24.4 to 75.6) in those with normal CRP . CRP was a negative prognostic factor associated with recurrence-free survival at 5 years in the group of patients with localized disease (p = 0.024). The estimated 5-year recurrence-free survival was 54.8% (95% CI 37.4 to 71.1) in patients with pathological CRP, and 79.5% (95% CI 65.1 to 89.0) in those with normal CRP . A further evaluation was performed in the group of patients treated with surgery as a major local treatment, including both patients with metastatic and localized disease (Table 4). Univariate Kaplan-Meier analyses of laboratory biomarkers and clinical parameters showed that CRP, metastatic disease at presentation, and radiotherapy were poor prognostic factors associated with overall survival at 5 years (p < 0.05). A multivariate Cox regression model of these three negative prognostic factors at univariate analysis was performed (Table 5): pathological CRP (>=1.0 mg/dL) [HR of 3.67; 95% CI, 1.46 to 10.42] and metastatic disease [HR of 4.27; 95% CI, 1.58 to 11.47] were associated with a higher risk of death at 5 years (p < 0.05). In addition, pathological CRP, metastatic disease, radiotherapy and NLR were associated with recurrence-free survival at 5 years (Table 6). Pathological CRP and metastatic disease remained significant in the multivariate Cox analysis for both overall survival and recurrence-free survival. A multivariate Cox regression model of negative prognostic factors was performed (Table 7): pathological CRP (>=1.0 mg/dL) [HR of 2.66; 95% CI, 1.23 to 6.01] and metastatic disease [HR of 2.56; 95% CI, 1.13 to 5.55] were associated with a higher risk of disease recurrence at 5 years (p < 0.05). 4. Discussion Elevated levels of inflammatory biomarkers and poor prognosis have been reported not only in patients with cancers, but also in patients with bone and soft tissue sarcomas . Although metastatic disease at presentation is a well-known negative prognostic factor in patients with Ewing's sarcoma, the prognostic significance of inflammatory biomarkers is still uncertain . Our findings showed that CRP was a negative prognostic factor in children with Ewing's sarcoma. We reported that pathological CRP and metastatic disease were both associated with overall survival and recurrence-free survival. These results may suggest that pathological CRP seems to be related to aggressive tumor behavior, with a higher risk of local recurrence and metastasis. Our study has a few limitations. First, its retrospective nature is a major limitation. Second, although this study was performed in a single institution, patients were probably treated differently over the years. Third, we had a relatively small series of patients because of the rarity of Ewing's sarcoma; the outcome of the study could change with a larger patient population. Nakamura et al. retrospectively analyzed 318 patients with bone sarcomas between 2003 and 2010: pathological CRP was a negative prognostic factor of survival and local recurrence. They also analyzed the association between CRP, survival and local control for patients with Ewing's sarcoma, showing that pathological CRP was a negative prognostic factor for survival . In contrast, pathological CRP was not a negative prognostic factor for local control . One of the limitations of this study is that the authors did not analyze the presence of systemic disease that may be associated with higher levels of inflammatory biomarkers . Patients with sarcoma may have concurrent morbidity that causes a rise in their CRP; therefore, in our study we excluded patients who had clinical evidence of infection or inflammatory disease. Our findings showed that pathological CRP was a negative prognostic factor for both survival and disease recurrence in children with Ewing's sarcoma. Li et al. retrospectively analyzed 122 patients with Ewing's sarcoma between 2009 and 2015, showing that several inflammatory prognostic biomarkers, including CRP, were correlated with the survival of patients with Ewing's sarcoma . The major limitation of this study was that the authors did not analyze the tumor size and the response to chemotherapy that are well-known prognostic factors in patients with Ewing's sarcoma; in addition, the authors analyzed both patients with metastatic and those with localized disease as a single group . In our study, we separately analyzed patients with metastatic disease and localized disease. To the best of our knowledge, this is the first study that confirms that pathological CRP is associated with poor prognosis both in patients with metastatic disease and localized disease. In addition, the tumor size and the response to chemotherapy were analyzed in patients treated surgically, confirming that pathological CRP was associated with poor survival in children with Ewing's sarcoma. Xu et al. retrospectively analyzed 83 patients with Ewing's sarcoma of the spine between 2007 and 2016, reporting that the presence of metastasis and a CRP/albumin ratio < 1.5 were negative prognostic factors for overall survival. The major limitation of the study is that the authors only analyzed patients with Ewing's sarcoma of the spine, and most patients did not receive chemotherapy prior to surgery . However, we confirm these data by showing that pathologic CRP and metastatic disease are associated with overall survival in patients with Ewing's sarcoma of not only the spine but also of the long bones. Consalvo et al. retrospectively analyzed 40 patients with Ewing's sarcoma, showing that CRP was different in patients with a poorer prognosis and in patients with distant metastases, whereas CRP was not different in patients with local recurrence. The major limitation of the study is that the authors did not analyze the tumor size or other inflammatory biomarkers, and the study did not consider the subgroup analysis of localized patients and metastatic patients. In addition, 13 patients did not undergo surgical treatment, and thus the relatively small number of the remaining patients may have influenced the statistical analysis . Del Baldo et al. al retrospectively analyzed 89 pediatric patients with Ewing's sarcoma, showing that LDH and CRP were associated with a poorer prognosis at univariate analysis, while only LDH remained associated with a poorer prognosis at multivariate analysis . As in the previous study, the authors did not analyze tumor size, other inflammatory biomarkers, and did not consider the subgroup analysis of localized patients and metastatic patients. In addition, Consalvo et al. and Del Baldo et al. used 0.5 mg/dL instead of 1 mg/dL as a cutoff for physiological CRP, unlike recent literature regarding prognostic inflammatory biomarkers for musculoskeletal tumors . Laboratory biomarkers are known to be prognostic factors for some malignancies ; however, very few studies have investigated the prognostic value of serum biomarkers in patients with Ewing's sarcoma . Most of these tested biomarkers are related to a systemic inflammatory process, and this is not surprising, since it is known that a systemic inflammation can be associated with cancer development and progression . CRP is the only single biomarker that, according to the literature, is prognostic of both bone and soft tissue sarcomas as well as for cancer . Avnet et al. reported that tumor acidosis could be directly associated with tumor invasion . Cancer seems to induce permanent inflammation, and cancer-associated inflammation could eventually lead to tumor progression and metastasis . 5. Conclusions Our findings showed that CRP was associated with the prognosis of children with Ewing's sarcoma of the long bones. We recommend pre-treatment measurement of the CRP in order to recognize children with Ewing's sarcoma who are at greater risk of death or local recurrence. Multicenter prospective studies are necessary to confirm these preliminary results in a larger patient population. Acknowledgments The authors thank the patients and their families. Author Contributions Conceptualization, C.E., M.T., S.T. and A.F.M.; methodology, C.E., M.T., B.B. and M.C.; validation, C.E., M.T., B.B. and M.C.; formal analysis, C.E., M.T., B.B. and M.C.; investigation, C.E. and M.T.; data curation, M.T., S.B. and M.C.; writing--original draft preparation, C.E. and M.T.; writing--review and editing, C.E., M.T., S.T. and A.F.M.; visualization, C.E. and S.B.; supervision, M.M. and D.M.D. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Ethical approval for this study was obtained from the Institutional Review Board of the Istituto Ortopedico Rizzoli on 2 October 2021 (ClinicalTrials.gov NCT05100368). Informed Consent Statement Informed consent was not applicable, as the study is retrospective. Data Availability Statement The data analyzed can be found in the electronic medical records of the patients who participated in the study. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Flow diagram of children with Ewing's sarcoma treated at our institution during the time of study. Figure 2 Kaplan-Meier curve showing the overall survival for patients affected by localized Ewing's sarcoma with normal and raised C-reactive protein (CRP). Figure 3 Kaplan-Meier curve showing the overall survival for patients affected by metastatic Ewing's sarcoma with normal and raised C-reactive protein (CRP). Figure 4 Kaplan-Meier curve showing the recurrence-free survival for patients affected by localized Ewing's sarcoma with normal and raised C-reactive protein (CRP). cancers-15-01573-t001_Table 1 Table 1 Patient data. Variable Localized Metastatic Overall Patients (n) 108 43 151 Gender (n) Male 79 (73.1%) 32 (74.4%) 111 (73.5%) Female 29 (26.9%) 11 (25.6%) 40 (26.5%) Age (years) 12.7 (3-18) 13.9 (6-18) 13.1 (3-18) Tumor location (n) Extremities 81 (75.0%) 26 (60.5%) 107 (70.9%) Trunk 27 (25.0%) 17 (39.5%) 44 (29.1%) Tumor size (n) >200 mL 31 (28.7%) 29 (67.4%) 60 (39.7%) <200 mL 70 (64.8%) 9 (20.9%) 79 (52.3) missing 7 (6.5%) 5 (11.7%) 12 (7.9%) Surgery (n) Yes 95 (88.0%) 19 (44.2%) 114 (75.5%) No 13 (22.0%) 24 (55.8%) 37 (24.5%) Radiotherapy (n) None 83 (76.9%) 15 (34.9%) 98 (64.9%) Exclusive 13 (12.0%) 21 (48.8%) 34 (22.5%) Before surgery 6 (5.5%) 2 (4.6%) 8 (5.3%) Adjuvant 6 (5.5%) 5 (11.6%) 11 (7.3%) Neoajuvant CHT (n) Yes 108 (100%) 42 (97.7%) 150 (99.3%) No 0 1 (2.3%) 1 (0.7%) cancers-15-01573-t002_Table 2 Table 2 Prognostic factors associated with overall survival in children with localized or metastatic disease. Localized Metastatic Overall Variables Raised CRP Normal CRP p-Value Raised CRP Normal CRP p-Value Raised CRP Normal CRP p-Value n. Patients (%) 37 60 p = 0.025 (Proportion Z-Test) 28 12 p = 0.017 (Proportion Z-Test) 65 (43.0) 72 (47.7) p = 0.608 (Proportion Z-Test) Gender n. (%) p = 0.238 (Fisher's Exact Test) p = 1.000 (Fisher's Exact Test) p = 0.249 (Fisher's Exact Test) Male 30 (81.1) 41 (68.3) 21 (75.0) 9 (75.0) 51 (78.5) 50 (69.4) Female 7 (18.9) 19 (31.7) 7 (25.0) 3 (25.0) 14 (21.5) 22 (30.6) Mean age (min-max) 13.4 (3.0-18.0) 12.4 (3.0-18.0) p = 0.224 (t-Test) 14.5 (6.0-18.0) 12.3 (6.0-18.0) p = 0.055 (t-Test) 13.8 (3.0-18.0) 12.3 (3.0-18.0) p = 0.021 (t-Test) n. Tumor location (%) p = 0.020 (Fisher's Exact Test) p = 0.152 (Fisher's Exact Test) p < 0.001 (Fisher's Exact Test) Extremities 24 (64.9) 52 (86.7) 15 (53.6) 10 (83.3) 39 (60.0) 62 (86.1) Trunk 13 (35.1) 8 (13.3) 13 (46.4) 2 (16.7) 26 (40.0) 10 (13.9) Tumor size p = 0.004 (Fisher's Exact Test) p = 0.027 (Fisher's Exact Test) p < 0.001 (Fisher's Exact Test) >200 mL 16 (43.2) 10 (16.7) 22 (78.6) 5 (41.7) 38 (58.5) 15 (20.8) <200 mL 18 (48.6) 46 (76.7) 3 (10.7) 5 (41.7) 21 (32.3) 51 (70.8) cancers-15-01573-t003_Table 3 Table 3 Univariate Kaplan-Meier analysis of C-reactive protein (CRP) as a prognostic factor for overall survival and recurrence-free survival. Localized Metastatic Pathological CRP Normal CRP Log Rank Test Raised CRP Pathological CRP Log Rank Test 5 years overall survival (95% CI) 65.6% (47.9-79.8) 92.7% (79.6-97.6) 0.004 12.5% (4.1-32.4) 50.0% (24.4-75.6) 0.010 5 years recurrence-free survival (95% CI) 54.8% (37.4-71.1) 79.5% (65.1-89.0) 0.024 13.0% (4.3-33.5) 45.5% (20.3-73.2) 0.076 cancers-15-01573-t004_Table 4 Table 4 Univariate Kaplan-Meier analyses of laboratory biomarkers and clinical parameters for overall survival at 5 years. Variables n Death in 5 Years p-Value (Log-Rank Test) Sex 0.771 Male 70 16 Female 19 5 Tumor location (n) 0.595 Extremities 75 17 Trunk 14 4 Tumor size 0.309 >200 mL 30 8 <200 mL 49 9 CRP 0.003 Pathological 34 14 Normal 47 6 Monocyte count 0.482 Raised 50 13 Normal 39 8 Neutrophile count 0.445 Raised 8 3 Normal 80 18 Lymphocyte count 0.199 Raised 16 3 Normal 73 18 Haemoglobin 0.570 Low 22 6 Normal 67 15 LDH 0.264 Raised 22 7 Normal 54 14 ALP 0.993 Raised 9 2 Normal 80 19 NLR 0.054 Raised 34 12 Normal 55 9 PLR 0.634 Raised 55 14 Normal 34 7 Radiotherapy 0.010 Yes 15 7 No 74 14 Metastasis at diagnosis <0.0001 Yes 19 11 No 70 10 cancers-15-01573-t005_Table 5 Table 5 Multivariate Cox regression model of metastasis at diagnosis, radiotherapy and C-reactive protein (CRP) for overall survival at 5 years. Variables HR (95% CI) p-Value (Log-Rank Test) Metastasis at diagnosis 4.27 (1.58-11.47) 0.004 Radiotherapy 1.7 (0.57-4.86) 0.310 Pathological CRP 3.67 (1.46-10.42) 0.005 cancers-15-01573-t006_Table 6 Table 6 Univariate Kaplan-Meier analyses of laboratory biomarkers and clinical parameters associated with disease recurrence. Variables n Recurrence in 5 Years p-Value (Log-Rank Test) Sex 0.999 Male 74 27 Female 20 7 Tumor location (n) 0.184 Extremities 79 27 Trunk 15 7 Tumor size 0.736 >200 mL 31 11 <200 mL 41 17 CRP 0.002 Pathological 35 19 Normal 49 11 Monocyte count 0.531 Raised 52 20 Normal 42 14 Neutrophile count 0.305 Raised 10 5 Normal 84 29 Lymphocyte count 0.467 Raised 6 3 Normal 88 31 Haemoglobin 0.955 Low 23 8 Normal 71 26 LDH 0.576 Raised 23 9 Normal 66 23 ALP 0.995 Raised 9 3 Normal 85 31 NLR 0.026 Raised 36 18 Normal 58 16 PLR 0.803 Raised 57 21 Normal 37 13 Radiotherapy 0.050 Yes 17 9 No 77 25 Metastasis at diagnosis 0.001 Yes 19 12 No 75 22 cancers-15-01573-t007_Table 7 Table 7 A multivariate Cox regression model of metastasis at diagnosis, radiotherapy and C-reactive protein (CRP) associated with disease recurrence. Variables HR (95% CI) p-Value (Log-Rank Test) Metastasis ad diagnosis 2.56 (1.13-5.55) 0.024 Radiotherapy 1.86 (0.69-4.49) 0.201 Pathological CRP 2.66 (1.23-6.01) 0.012 NRL 1.72 (0.79-3.74) 0.165 Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. |
PMC10000756 | Healthcare (Basel) Healthcare (Basel) healthcare Healthcare 2227-9032 MDPI 10.3390/healthcare11050769 healthcare-11-00769 Article Common Traditions, Practices, and Beliefs Related to Safe Motherhood and Newborn Health in Morocco Moujahid Chaimae Conceptualization Methodology Software Validation Formal analysis Investigation Data curation Writing - original draft Writing - review & editing 1* Turman Jack E. Jr. 23 Amahdar Loubna 1 Mohamed Abdel-Latif Academic Editor Ma Ping Academic Editor Chen Lei-Shih Academic Editor 1 Laboratory of Health Sciences and Technologies, Higher Institute of Health Sciences, Hassan First University of Settat, Settat 26000, Morocco 2 Department of Social and Behavioral Sciences, Richard M. Fairbanks School of Public Health, Indiana University, Indianapolis, IN 46202, USA 3 Department of Pediatrics, School of Medicine, Indiana University, Indianapolis, IN 46202, USA * Correspondence: [email protected]; Tel.: +212-063-797-3599 06 3 2023 3 2023 11 5 76930 1 2023 23 2 2023 02 3 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). The cultural context influences women's antenatal care and postpartum knowledge. This study aims to determine the traditional practices related to maternal health in Morocco. We conducted in-depth qualitative interviews with 37 women from three different Moroccan regions on the first postpartum day. We used thematic content to analyze data, and an a priori coding framework was created utilizing the pertinent literature. Beliefs regarding pregnancy and postpartum positively affect maternal health, such as family support, prolonged rest for health recovery, and specific dietary precautions according to the mode of delivery of the new mother. However, some practices may negatively affect maternal health, such as cold postpartum treatment through traditional medicine and not seeking prenatal care after the first pregnancy experience. Such practices include painting newborns with henna, using kohl and oil to hasten the umbilical cord's descent, and producing solutions based on chicken throat to cure respiratory ailments in newborns that might harm their health. maternal health postpartum practices healer faith witchcraft Morocco This research received no external funding. pmc1. Introduction Morocco has been able to progress in reducing maternal mortality. According to the World Health Organization, this progress reached 61.8% in Morocco between 1990 and 2015 . Achieving the SDG target of a global MMR below 70 requires reducing the global MMR by an average of 7.5% annually between 2016 and 2030 . This progress requires more than three times the 2.3% annual rate of reduction observed globally between 1990 and 2015. Wide disparities in maternal health between urban and rural areas must be addressed to meet these goals . Most maternal and newborns deaths are caused by sociocultural variables, such as cultural and religious influences, and other social factors that impact personal choices. Every culture has certain enduring customs that represent societal values and have been passed down from generation to generation . Women are influenced by cultural and traditional norms of their ethnic origin regardless of whether they reside in an urban or rural location. This influences how they adjust to the postpartum time, and how they see providing postpartum maternal and infant care . We grounded our work on a theoretical framework based on an ontological starting point and understanding of the lifeworld of the women as created from their behaviors, attitudes, practices, and perceptions and community and gender norms . To improve Moroccan maternal and child health outcomes, it is imperative to investigate how these women view their prenatal and postpartum situations . The interaction between study participants and the researcher serves as the epistemological starting point for knowledge creation about their lived prenatal and postpartum experiences. It is possible that lifeworld knowledge is so ingrained in the culture that community members are unaware of its consequences for women's and children's health. Our study helps in revealing this for populations in Morocco to improve outcomes for women, children, and families, and their healthcare team. To identify harmful traditional practices and provide guidance on counseling women during antenatal and postnatal education to improve maternal and infant health, nurses and midwives would benefit from understanding traditional practices and beliefs related to maternal and infant care. There is, however, little research on Moroccan customs associated with the postpartum period . In this study, we sought to understand the traditional practices of women regarding maternal health during pregnancy and after delivery. 2. Materials and Methods 2.1. Setting This study was performed in hospitals from three provinces between March and September 2021. The ethics committee of the Faculty of Medicine and Pharmacy of Rabat, Morocco approved the study (reference CERB: 45/21). 2.2. Participants Participants were 37 postpartum married women from the Essaouira (n = 13), Settat (n = 13) and Sale (n = 11) provinces. Each selected woman had at least one child and was accepted to participate in the study. The participants were 17-37 years old, and they had had at least one experience with childbirth. Two were employees, ten were illiterate, and four had had a miscarriage experience. All women lived at home with their husbands, and most of them with the husband's family. 2.3. Materials The materials consisted of in-depth interviews with participants during their postpartum stays, written documents (personal notes and comments), and observations along the study were adopted. The interviewer used thematic guides covering the following aspects: the new mother's diet, rest, and activity, the newborn's diet, beliefs, and practices related to the health of the mother-newborn couple, and the perception of their benefits and drawbacks. We conducted the interviews in an Arabic dialect, and each interview lasted between 40 and 90 min. All interviews were recorded with the permission of the interviewees to be transcribed and analyzed afterward. 2.4. Data Analysis A digital voice recorder and a smartphone were used to record the qualitative data. Data analyses were performed concurrently with data collection with the goal of concluding the study when new obtained data no longer provided additional insights. The method involved reading and rereading the interviews and focusing on the identification of themes linked to intrapartum and postpartum continuum of care beliefs and practices at each research setting. Data collected in the local language were first translated into French and then to English by two researchers; the interviewer and another person compared the translations for consistency. Using computer-assisted qualitative data analysis software (CAQDAS) MAXQDA 2020, we coded the verbatim transcripts of interviews and field notes to identify emerging themes. We classified and organized data using a conceptual framework on the basis of developing themes, concepts, and categories . The writers and investigators reviewed and reread the transcripts individually to discover emergent patterns, and a framework was constructed on the basis of these themes. To build explanations and uncover relationships, data segments were used for each item to characterize comparable and different beliefs and find associations. From preliminary reading of the transcripts, a coding scheme for the key themes and subthemes was created. The coding scheme specified the numerous codes used during coding, their definitions, when to use and when not to use such a code, and examples of statements to consider while coding into certain codes. These codes were converted into software nodes. Following that, all transcripts were read line by line within the MAXQDA 2020 program, and important elements of the respondents' utterances were coded into existing and new nodes. The numerous codes were classified into themes on the basis of codes to reflect the content of the material obtained and memos written on certain pertinent topics. 3. Results The following section illustrates the significance of themes from the interviews that presented the living experiences and understanding of 37 Moroccan women's cultural and traditional health beliefs related to pregnancy, childbirth, and newborn health. Three major themes emerged from the analysis: cultural practices related to pregnancy, cultural practices related to safe motherhood, and cultural practices described to newborn health. 3.1. Cultural Practices Related to Pregnancy If the first pregnancy is safe, the later pregnancies do not need to be followed up. In contrast, a sex change between two successive pregnancies negatively affects the woman's health, and she would suffer in the subsequent pregnancy: "... This time I had pain in my belly because I got pregnant with a boy after a girl. It says that is because of a change of sex of the baby between two successive pregnancies..."--Khadija. Protecting pregnant women from nasty smells, intense emotions, and other unpleasant things is essential. They should eat nourishing food; the husband should bring back the food for which the pregnant woman asks. Otherwise, stains would appear on the newborn's body. The neighbors and the family should help the pregnant woman with the housework, especially in the first and last trimesters. Muscular uterine contractions are a sign of childbirth, weak contractions are a sign of difficulty in birth, with the mother-in-law giving advice. "Before the delivery, I smoked myself with the peganum "lharma", my mother-in-law insisted on thwarting contraction to facilitate and accelerate the delivery."--Zahra. 3.2. Cultural Practices Described to Safe Motherhood 3.2.1. Witchcraft and Sorcery--"Chaawada" Women believe in the practice of witchcraft that negatively affects the newborn and new mother's health; participants confirmed that a course related to witchcraft exists. Chaawada witchcraft is a practice from which the mother-newborn couple has not been spared, so the woman hides the newborn from the hospital to the house from visitors, except for the closest family who can see the baby. "We must be careful from the puerperal blood "dem nfass", the woman must do her preventive measures so that only people whom trust take care of its sanitary napkins... We risk people hurting us and doing sorcery by using the napkin of the new mother delivered. What can they do with the blood of a new mother? To separate the woman from her husband and not to have more children..." --Latifa 3.2.2. Bad Eyesight Some women find that the Quran is an effective way to protect the new mother "nafssa" and the newborn. Without the need to practice customs to fight against the evil eye, others mentioned that the mother or the mother-in-law had the right to practice her traditions to protect the newborn and the new mother. Fumigations based on alum and peganum "chebba" and "harmal" are necessary during the seven days of postpartum each day before sunset. "... We put 101 cynoge in the newborn's right hand, and they fumigate it with alum and peganum CHEBBA and HARMAL, which protects the newborn against the evil eye."--Ghizlan. 3.2.3. The 40 Days of Postpartum--"Trabaina" In the Moroccan context, the new mother should not leave her home before the 40th day, "trabaina". The newborn's vaccine comes initially in the first 40 days of postpartum. Someone in the family oversees bringing the newborn back to the medical facility for the vaccine. "... the newly delivered woman should not go out before the 40 days of postpartum it is for its health so that it does not have the bad eye, she should not speak too much... she should not walk nor make the household... she should just rest and behave sick." --Khdija The postpartum consultation is no longer necessary when she feels well. The first 40 days of postpartum, "trabaina", is a vulnerable period where the new mother is exposed to any risk of complications "one foot in the ground and the other in the grave". Once she exceeds the 40 days, there is no more risk, and the woman returns to everyday life. She can go out and travel with her baby and resume household chores: "... and I return to the housework after I finish 40 days of postpartum."--Samira. 3.2.4. Postpartum Hemorrhage--"Lfayda" Postpartum hemorrhage, "lfayda", is a sign of danger. It is a complication that requires care in a health facility; women say that the bleeding kills the nafssa if she is not hospitalized, and the risk of death is significant. Hospital delivery is preferable to home delivery; the traditional midwife can no longer manage the situation in case of postpartum hemorrhage. 3.2.5. The "Nfass", "Bard Nfass", or "Tajoughit" Cold The cold of "nfass" or "bard nfass" negatively affects the health of the new mother, the "nafssa". If she is exposed to the cold during the postpartum period, does not warm her body, and does not tighten her head scarf, whatever the season, she loses consciousness and becomes insane. It is irreversible, and "bard nfass" can kill the woman. Otherwise, it causes side effects in the long term, such as chronic migraine in its mildest forms. There are two types of "bard nfass", the one attacks the head and the second affects the uterus--"iwalda"; warming via preparations based on medicinal plants is an effective way to treat it. "The woman must pay attention and take her measures of protection against the cold... if she had a cold after having sweated, she will have disabling complications going until death, 'kaytnfakh liha rasha o tkdar tmout'." --Saadia "The cold of postpartum 'tajoughit' or 'tajdayt' or 'tajjayt' is hazardous for the health of the new mother. It is not treated in the hospital by medicines, it requires a tradition such as Timija and Tasrghint. The woman must smoke these plants to treat the cold that attacks her after the childbirth until the tears flow from her eyes. Women must tighten her head very well with a napkin, she doesn't expose to the light." --Habiba 3.2.6. Nutrition of "Nafssa" Women say that "rfissa" is the principal meal of the new mother in the Moroccan setting. The soup of chickens in the open air, spices "lmsakhen", and raw and cooked eggs, milk, and peppergrass seeds serve to warm the new mother's body and the production of breastmilk. Only women who have given birth via vaginal delivery without episiotomy have the right to eat these dishes to warm the body to avoid the risk of suture disunion and bleeding. "Labrik" is an herbal broth whose essential plant is Alpinia galanga "khounjlan" and Madder "lfoua," which helps in eliminating blood debris from the uterine cavity after birth. Meals composed of grilled liver and minced meat help restore blood lost during delivery. "I must eat "rfissa" to regain her physical well-being, but now that I have had a cesarean section, I should not eat the hot foods that include "lmsakhen" since it promotes bleeding... When I gave birth to my first child, they gave me the same as other women; I ate hot meals." --Ghizlan "... so, they prepared me a mist of medicinal plants that warms the womb "lwalda" it is a "lbrik," it is a broth-based on khounjlan and el foua "this broth increases the blood fluidity ... and elfoua treats anemia and jaundice... The khounjlan sterilizes the uterine body of the woman." --Malika 3.2.7. New Mother's Bath: The Day of Tightening--"Nhar L Hzam" Therapeutic herbs are used to treat postbirth pain. Hot baths are not recommended for women delivered with a Cesarean section or episiotomy. Nafssa believes that, after birth, the bones of the woman's body move and hot bath with body massage help to returns the bones to their original position. "... The Tayaba goes up on the back of Nafssa, makes the massage, and tightens the body from feet to head so that the bones can return to their place, because the woman's bones shift after the delivery."--Ilham 3.3. Cultural Practices Related to Newborn Health 3.3.1. New Clothes Are Bad Luck The first clothes that the newborn wears should be old and have been used by a living baby, as there is the belief that new clothes are a stroke of bad luck, and the mother should not buy new clothes until she delivers her newborn alive; they do not advance joy anymore. 3.3.2. Sorra The participants say that, if someone enters the couple's mother-baby room and wears anything tight, the newborn will have "sorra", a deformation and swelling of the child's head with an increased cranial perimeter... Women bring their babies to the fqih (religious healer) or rebagha (healer woman) three times and treat sorra with inflamed sticks of white marrube and mariout or henna. "... When my children have the sorra, we take them to the fkih who has the baraka to do the necessary and burn them with something. It is my mother-in-law who takes care of this task."--Halima. 3.3.3. LAGHROUR An oily preparation based on argan oil, butter, olive oil, seven types of medicinal plants, and hull throat is essential for every birth; a spoonful of this preparation helps in clearing lung secretions and lubricating the throat of the newborn. ""Laghrour" is prepared either with argan oil, butter "zebda l beldia", or olive oil it is a mixture of plants such as lavender, garlic, thyme, Marrubium... with the throat of the chicken, it is a beneficial treatment for respiratory crises, influenza, nasal obstruction, for respiratory attacks, flu... it is given to the baby in the 3rd day after birth" --Latifa 3.3.4. Umbilical Care Umbilical care with oil and henna powder or with kohl accelerates the dryness and then fall of the umbilical cord when medical treatment is not effective. 3.3.5. Ritual Practices On the seventh postpartum day, the mother-in-law submerges the newborn's body in henna. To eliminate the skin's desquamation and prevent neonatal icterus, the baby takes a bath only after one day of this ritual practice. Participants indicated that honey is equivalent to all necessary vaccination doses. "... at the 7th day, the whole body of the newborn is taken out in the henna, and then we dry it with a towel without washing it. We do this practice to eliminate his skin's dermal cortex and prevent jaundice. My mother and mother-in-law prepare a collyrium based on saffron at home to cure the baby's eye infections..." --Halima 4. Discussion 4.1. Cultural Practices Related to Pregnancy Pregnancy is a privileged event in Moroccan society; the pregnant woman receives help during pregnancy from those around her and eats nourishing food. Eating protein-rich foods, family care, and support for Moroccan women throughout pregnancy and after birth are all likely beneficial from health and social standpoints. Similar findings were seen in other cultures in Zambia and China . Morocco has seen a decline in the utilization of health services by women during pregnancy, according to an ENPSF study . Connecting pregnancy pain with infant sex change is one of the beliefs that might have a detrimental impact on maternal health. Health professionals must emphasize these sociocultural behaviors during health education and devise focused interventions. In the Moroccan context, heat-up contraction and fumigations stimulate contractions and strengthen them; this was also reported by to hasten the active phase before going to hospital. This also contributes to the first delay in maternal mortality, as mentioned by several studies . 4.2. Cultural Practices Related to Safe Motherhood Traditional methods were extensively used to protect new mothers from health problems such as postpartum hemorrhage and colds. Some foods were forbidden for women who had given birth through Cesarean section or episiotomy because hot dish msakhen caused bleeding. Women in Morocco rely on traditional medicine to heal colds or bard nfass. Our findings are consistent with what Obermeyer has discovered in Morocco and other cultures over the last two decades . Warmth is vital for the new mother. Only women who have given birth without an episiotomy benefit from a warm bath with massage and manipulation. Participants globally reported the same practice (kayjmaao laadam) . The care and support of the family towards the new mother during the postpartum period are a ritual practice. A mother's sanitary napkin is used in witchcraft to separate women from their husbands. In postpartum, new mother "nafssa" has a 40-day confinement and must not leave her bed for seven days. This is to prevent the "nfass bard" cold, bad eyesight, and sorcery. Fumigating the mother of the couple's newborn and passing a clove of garlic around the room of the mother and her newborn, and putting it under her bed are also practices that help in fighting against bad odors that occur during this period. Our findings are supported by those of Raven and Obermeyer . In general, mothers and mothers-in-law are the most influential people in recommending traditional practices such as eating "msakhen", much hot food to warm the body, and recovering energy after giving birth and producing milk. These results are similar to those of Vietnamese women . 4.3. Cultural Practices Related to Newborn Health Fumigation-based chebba and lharmal are practiced every day before sunset. Successive crying without cause is a sign of the evil eye and sorra. Some ritual practices could negatively affect the newborn's health, such as applying olive oil and khol to dry and make the navel fall quickly. Comparable findings have been reported in Morocco since the 20th century . Applying henna on the newborn's body, which can distort the early detection of neonatal jaundice, is one of the most practiced rituals in the studied regions. Infant jaundice is a significant global cause of neonatal illness and death. It is a primary reason for hospitalization in the first week of birth worldwide, and one of the most prevalent causes of neurodevelopmental problems in developing nations . 5. Study Limitation This study was carried out as a descriptive research design using a sample of 37 women drawn from three separate hospitals in three distinct regions of Morocco. As a result, the current study's findings solely apply to women who took part in Morocco. Because convenience samples were employed, the findings may not be generalizable. How the women learned about these behaviors or who proposed them was not addressed. However, because the participants' age, educational level, employment, and number of children varied, a rather comprehensive picture of cultural postpartum beliefs and behaviors among Moroccan women in the provinces of Sale, Settat, and Essouira was acquired. More studies should be conducted on the education of healthcare personnel to enable them to give culturally appropriate care to mothers and newborns. 6. Conclusions This research aids in raising the knowledge and understanding of maternal and newborn care in the Moroccan cultural setting. This new understanding has applications in instruction and research. The research also gives information to Moroccan groups that work to improve the health of mothers and children. This report promotes more research on this subject. It is critical to thoroughly understand the traditions and customs that regulate Moroccan families and women about motherhood, and how these traditions impact the lives of these women and their children. Postpartum cultural beliefs and practices are common for Moroccan women and their babies. They are transmitted from mother to daughter from generation to generation. Many practices are beneficial to the health and wellbeing of mothers and babies, but some can be harmful. To reduce the power of these harmful traditional beliefs and practices regarding maternal health, the sensitization of women and the community at large to basic knowledge on the dangers associated with unskilled service uptake is critical, allowing for women to be aware of the consequences of harmful practices toward newborns and mothers, and adhere postnatal prenatal care. These requirements make it imperative that knowledge of cultural values be included in the training of midwives, nurses, and other professionals to increase their cultural awareness and skill opportunities for culturally appropriate care. Midwives in particular and medical staff should know the cultural practices adopted by society, so they can deliver culturally appropriate messages to women, and convince them on how certain practices and beliefs can affect their health and the health of newborns. Acknowledgments We would like to thank everyone who took part in this study. We would also like to acknowledge Morocco's National Center for Scientific and Technical Research in Rabat. Author Contributions Conceptualization, C.M. and J.E.T.J.; methodology, C.M., J.E.T.J. and L.A.; formal analysis, C.M. and J.E.T.J.; investigation, C.M.; data curation, C.M. and L.A.; writing--original draft preparation, C.M. and J.E.T.J.; writing--review and editing, C.M., J.E.T.J. and L.A.; visualization, J.E.T.J. and L.A.; supervision, L.A. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The study was conducted in accordance with the Declaration of Helsinki and approved by Ethics Committee of the Faculty of Medicine and Pharmacy of Rabat, Morocco (referred as CERB: 45/21). Informed Consent Statement Informed consent was obtained from all subjects involved in the study. Data Availability Statement Data are accessible upon reasonable request from the respective author. Conflicts of Interest The authors declare no conflict of interest. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. References 1. 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PMC10000757 | Cells Cells cells Cells 2073-4409 MDPI 10.3390/cells12050736 cells-12-00736 Review Current State and Future Directions in the Diagnosis of Amyotrophic Lateral Sclerosis Vidovic Maximilian 1* Muschen Lars Hendrik 2 Brakemeier Svenja 3 Machetanz Gerrit 4 Naumann Marcel 5 Castro-Gomez Sergio 678* Romano Maurizio Academic Editor 1 Department of Neurology, University Hospital Carl Gustav Carus, Technische Universitat Dresden, 01307 Dresden, Germany 2 Department of Neurology, Hannover Medical School, 30625 Hannover, Germany 3 Department of Neurology and Center for Translational Neuro and Behavioral Sciences (C-TNBS), University Hospital Essen, 45147 Essen, Germany 4 Department of Neurology, Klinikum Rechts der Isar, Technical University of Munich, 81675 Munich, Germany 5 Translational Neurodegeneration Section "Albrecht Kossel", Department of Neurology, University Medical Center, University of Rostock, 18147 Rostock, Germany 6 Department of Neurodegenerative Disease and Geriatric Psychiatry/Neurology, University Hospital Bonn, 53127 Bonn, Germany 7 Institute of Physiology II, University Hospital Bonn, 53115 Bonn, Germany 8 Department of Neuroimmunology, Institute of Innate Immunity, University Hospital Bonn, 53127 Bonn, Germany * Correspondence: [email protected] (M.V.); [email protected] (S.C.-G.) 24 2 2023 3 2023 12 5 73604 2 2023 22 2 2023 23 2 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by loss of upper and lower motor neurons, resulting in progressive weakness of all voluntary muscles and eventual respiratory failure. Non-motor symptoms, such as cognitive and behavioral changes, frequently occur over the course of the disease. Considering its poor prognosis with a median survival time of 2 to 4 years and limited causal treatment options, an early diagnosis of ALS plays an essential role. In the past, diagnosis has primarily been determined by clinical findings supported by electrophysiological and laboratory measurements. To increase diagnostic accuracy, reduce diagnostic delay, optimize stratification in clinical trials and provide quantitative monitoring of disease progression and treatment responsivity, research on disease-specific and feasible fluid biomarkers, such as neurofilaments, has been intensely pursued. Advances in imaging techniques have additionally yielded diagnostic benefits. Growing perception and greater availability of genetic testing facilitate early identification of pathogenic ALS-related gene mutations, predictive testing and access to novel therapeutic agents in clinical trials addressing disease-modified therapies before the advent of the first clinical symptoms. Lately, personalized survival prediction models have been proposed to offer a more detailed disclosure of the prognosis for the patient. In this review, the established procedures and future directions in the diagnostics of ALS are summarized to serve as a practical guideline and to improve the diagnostic pathway of this burdensome disease. amyotrophic lateral sclerosis ALS motor neuron disease MND diagnosis diagnostics initiatives Alle-Lieben-Schmidt e.V., Alzheimer Forschung e.V21060 the intramural BONFOR program of the University Hospital Bonn2021-1A-12 Clinician Scientist Program of the University Medical Center RostockS.C.-G. is supported by the initiatives Alle-Lieben-Schmidt e.V., Alzheimer Forschung e.V (Grant 21060) and the intramural BONFOR program of the University Hospital Bonn (Grant 2021-1A-12). M.N. is supported by the Clinician Scientist Program of the University Medical Center Rostock. pmc1. Introduction The diagnosis of amyotrophic lateral sclerosis (ALS) remains an enormous challenge not only to general physicians, but also to specialized neurologists. ALS, a fatal neurodegenerative and the most frequent motor neuron disease (MND), is primarily characterized by progressive weakness of voluntary muscles due to degenerating motor neurons in the brain, brainstem and spinal cord. Considered to be a multisystem disorder, it can also be accompanied by non-motor symptoms, such as behavioral and cognitive impairment, and even manifest as an overlap syndrome with signs of frontotemporal dementia (FTD), known as ALS-FTD . The clinical, genetic and neuropathological heterogeneity, and the resemblance to other neuromuscular diseases, especially early in the disease's course, commonly described as ALS mimics, often requires the application of additional diagnostic methods. Given the short median survival of 2 to 4 years and a diagnostic delay of 10 to 16 months , there is an urgent need for optimizing diagnostic accuracy to yield a faster and more reliable recognition of the disease. This, in turn, may provide access to novel therapeutic agents and participation in clinical trials at an early disease stage. In recent decades, the diagnosis of ALS was mainly based on clinical findings with support of electrophysiological, imaging and laboratory techniques to exclude other diseases. Recent efforts have focused on establishing simplified, more practice-oriented diagnostic guidelines, as well as investigating the potential of specific fluid biomarkers, functional brain imaging and additional measures, such as predictive models, to hasten diagnosis, improve diagnostic accuracy and help outline the disease trajectory for the individual patient. This review aims to present the current state of ALS diagnosis and outline how recent findings and evolving concepts may show where it is headed in the future. 2. Clinical Presentation It is essential to record the clinical findings and their course as accurately as possible. Therefore, a thorough exploration of the patient's history of symptoms and a complete physical and neurological examination should be considered as the first step in the diagnosis of ALS. The clinical hallmarks of ALS are related to the impairment of voluntary muscles, resulting in progressive weakness of the limbs, speech and swallowing dysfunction and respiratory failure with concomitant signs, such as muscle atrophy, fasciculations and increased muscle tone. These motor dysfunctions are derived from the combined impairment of the upper motor neurons (UMN) in the motor cortex of the brain and the lower motor neurons (LMN) in the brainstem and the spinal cord . Though generally unaffected, there is also evidence of oculomotor, sphincter and autonomic dysfunction . Initially, the disease typically presents itself either with asymmetrical focal muscle weakness in the upper or lower limbs in spinal onset ALS or with speech and swallowing difficulties due to facial, tongue and pharyngeal muscle weakness in bulbar-onset ALS . Disease propagation preferably follows an organized contiguous pattern with first symptoms in one limb, subsequently spreading to the contralateral limb and, later, to adjacent regions . However, disease progression is highly variable and frequently shows a non-linear decline . The anatomically separated body regions (or segments) are defined as the bulbar, cervical, thoracic and lumbar regions, with the latter three comprising the spinal regions . The clinical signs of UMN and LMN involvement according to each of the body regions are presented in Figure 1. The notable clinical heterogeneity of motor manifestations frequently leads to controversy in the description of phenotypes as opposed to the determination of the diagnosis of ALS. Distinguishing these phenotypes is of relevance, as they are associated with various disease progression rates and survival times . The different clinical phenotypes of ALS are generally described with regard to the extent of UMN and LMN impairment, its distribution and progression to the different body segments . 2.1. Spinal-Onset ALS ALS with spinal onset is defined by focal weakness in distal muscle groups of the limbs and simultaneous UMN and LMN involvement. With up to 82% of all ALS patients , it is the most common phenotype, termed typical or classical ALS. Distal segments of the upper or lower limbs are affected in a focal manner at the onset of the disease. Characteristically, thenar eminence with the abductor pollicis brevis (APB) and the first dorsal interosseus (FDI) muscles are more affected compared to the hypothenar muscle abductor digiti minimi (ADM), referred to as split-hand syndrome . Onset in the dominant hand is thought to be predominant . Lower-limb weakness typically becomes apparent as it causes an unsteady gait due to weak foot dorsiflexion . Notably, UMN dysfunction is not always easily identified in wasted or atrophic muscles of the limbs, particularly in the early stages of the disease . 2.2. Bulbar-Onset ALS ALS with bulbar onset, or bulbar ALS, is the second leading phenotype with initial motor dysfunction in the bulbar region. Speech difficulties and frequent choking with concomitant hypersalivation are the cardinal presenting symptoms. Both LMN and UMN impairment are present, causing tongue wasting with fasciculations, facial spasticity and pseudobulbar affect in the early stages of the disease. Propagation to other spinal regions is evident later in the disease's course . Bulbar UMN involvement, also known as pseudobulbar palsy, can become clinically indicated by emotional lability, accompanied by excessive crying or laughing response to minor stimuli. This is termed the "pseudobulbar affect". Other bulbar UMN signs include facial spasticity and slowed spastic movement of the tongue, whereas slurred speech, wasting of the tongue and fasciculations indicate bulbar LMN impairment . Frontal release signs, such as the palmomental reflex, may also indicate bulbar involvement . In contrast to the often used phrase "progressive bulbar palsy", "ALS with bulbar onset" appears to be the more convenient term, as it represents a phenotypic description rather than a diagnostic label, suggesting no progression to other body regions . 2.3. Progressive Muscular Atrophy Progressive muscular atrophy (PMA) presents with clinically isolated LMN impairment of the anterior horn cells and brainstem motor nuclei. However, subclinical UMN impairment can be detected in the early stages of the disease . Although PMA tends to have a slower disease progression than classical ALS , 20 to 30% of the patients may develop ALS with clinically evident UMN impairment within 5 to 10 years from the disease's onset. It is still controversially debated whether PMA should be considered a variant of ALS . 2.4. Primary Lateral Sclerosis Primary lateral sclerosis (PLS) is characterized by progressive isolated UMN dysfunction detectable in at least two regions (thoracic region will not be considered) for at least two years. LMN dysfunction is absent, whereas minimal signs of denervation (positive sharp waves or fibrillation potentials) on electromyography (EMG) are permitted. Whether PLS constitutes a separate disease entity or rather represents a clinically benign variant of ALS remains controversial . 2.5. Flail-Arm-Syndrome In flail-arm-syndrome (FAS), also known as Vulpian Bernhardt's type, progressive, proximal and symmetrical weakness of both upper limbs caused by LMN impairment is predominantly apparent. Motor symptoms in bulbar muscles or lower limbs are unaffected from 12 to 20 months after the onset of upper limb symptoms . LMN involvement is predominant, whereas UMN involvement can be occasionally present in lower limbs . FAS represents a rather benign phenotype with a median survival time of 4 years and a 10 year survival rate of 17% . 2.6. Flail-Leg-Syndrome Analogous to FAS, flail-leg-syndrome (FLS) presents itself with progressive and symmetrical weakness of both lower limbs, whereas distal muscle groups are typically affected and LMN involvement outweighs UMN involvement . Other segments are clinically spared for a mean of 16 months after the disease's onset. Unlike FAS, FLS has a similar prognosis as spinal-onset ALS, with a median survival time of 3 years and a 10-year survival rate of 13% . 2.7. Axial or Respiratory-Onset ALS Axial-onset ALS initially presents itself with weakness of trunk muscles. Typically, paravertebral muscles are affected, resulting in bent posture, axial instability and dropped head syndrome . However, weakness of the thoracic muscles can be rather difficult to recognize . In respiratory-onset ALS patients suffer from dyspnoea and orthopnoea at the beginning of the disease, caused by weakness of the respiratory muscles and the diaphragm, which is also anatomically related to the thoracic region . The prognosis is poor due to early respiratory failure and complications such as pneumonia . There is no reliable method to detect thoracic UMN involvement . However, brisk and deep abdominal reflexes, particularly with diminished or absent superficial abdominal reflexes, might be suggestive signs . 2.8. Hemiplegic ALS (Mill's Syndrome) This very rare phenotype is defined by slowly progressive, unilateral muscle weakness in the limbs alongside clinically predominant UMN signs, such as pathological deep tendon reflexes (DTR) and pyramidal tract signs. The onset may either occur in the upper limbs with subsequent descending propagation to the lower limbs or vice versa . As is the case with PLS, there is an ongoing debate regarding whether Mill's syndrome should be considered a distinct clinical entity in the spectrum of motor neuron diseases or an ALS variant . 3. Diagnostic Criteria 3.1. The El Escorial Criteria (1994) and Revised El Escorial Criteria (2000) The El Escorial criteria were proposed as the first consensus diagnostic criteria for ALS in 1994 and were primarily designed for clinical trials and scientific research purposes . However, they were increasingly applied in clinical practice due to the lack of any more reliable diagnostic procedures . According to the criteria, clinical evidence of UMN and LMN impairment in four anatomically segmented body regions (bulbar, cervical, thoracic, lumbar) is surveyed for the diagnosis. Additionally, the progressive spreading of symptoms and the absence of electrophysiological and neuroimaging evidence of other causing diseases must be fulfilled. Patients are stratified into four categories of diagnostic certainty based on the number of affected regions and the extent of motor neuron involvement: suspected, possible, probable and definite ALS . The criteria were revised (termed as revised El Escorial/Airlie House criteria) in 2000 to improve diagnostic sensitivity (Table 1). Diagnostic categories were refined with the following terms: clinically definite, clinically probable, clinically probable-laboratory supported and clinically possible ALS, while the category of suspected ALS was excluded. The electrophysiological examination was implemented to support clinical findings and identify LMN involvement, with signs of active and chronic denervation by EMG and the exclusion of motor neuropathy by nerve conduction studies . 3.2. The Awaji Criteria (2008) The Awaji criteria, published in 2008, elaborated on the electrophysiological findings in the diagnosis of ALS (Table 1). Adapting the basic principles of the revised El Escorial criteria, evidence for chronic neurogenic damage in needle EMG is of equal significance as clinical signs for LMN involvement. Also, fasciculation potentials are equivalent to fibrillation potentials and positive sharp waves, implying that acute denervation if chronic neurogenic change on needle EMG is present. Consequently, the diagnostic categories were redetermined as follows: clinical definite ALS, clinically probable ALS and clinically possible ALS. The category of probable laboratory-supported ALS was discarded . A meta-analysis proved a better diagnostic performance of the Awaji criteria with a sensitivity of 81.1% compared to the 62.2% of the revised El Escorial criteria, facilitating earlier diagnosis of ALS . 3.3. The Gold Coast Criteria (2020) Although achieving improvement in diagnostic accuracy, both the revised El Escorial and the Awaji criteria are associated with various difficult aspects. Their complexity can be rather misleading and vulnerable to erroneous in their application, confirmed by a low test-retest as well as inter-rater reliability . Also, the division into diagnostic categories may falsely suggest a predictive value about the actual occurrence of the disease. Furthermore, both criteria are limited in providing a prognostic value, as their diagnostic categories do not provide any relation to disease progression . Ultimately, the category of possible ALS with isolated UMN signs in two regions exacerbates the debate about PLS being a separate entity or a prolonged form of ALS. Considering these issues, the Gold Coast criteria were introduced to further simplify the diagnostic approach by dichotomizing diagnostic categories into ALS and non-ALS (Table 2) . Studies evaluating the feasibility of the Gold Coast criteria to date have shown an increase in diagnostic sensitivity with largely preserved high specificity . 4. Clinical Assessments of Disease Severity and Progression To evaluate the functional status of patients, the ALS functional rating scale (ALSFRS) and its revised form (ALSFRS-R) were established. This self-assessment questionnaire contains items on bulbar functions, fine motor tasks, gross motor tasks and respiratory functions rated on a five-point scale from 0 to 4, with a maximum of 48 points . The ALSFRS-R has become the gold standard tool to assess physical decline and rate of disease progression not only in clinical routine, but also in therapeutic trials. New staging systems, such as King's staging system , Milano Torino Staging System (MiToS) or Rasch overall ALS disability scale (ROADS) , have been recently developed to overcome methodological issues of the ALSFRS-R and improve reliability and measurement of therapeutic monitoring . 5. Pre-Symptomatic ALS Since most recent pharmacological studies have yet failed to demonstrate an improvement of motor function or extend survival, much effort has been made to identify ALS patients in the early stages of the disease. The concept of pre-symptomatic ALS with mild motor impairment (MMI) has been discussed. Benatar and colleagues proposed a concept of ALS as a biological entity with a continuum of symptoms rather than a clinical syndrome alone. Therefore, there may be a pre-symptomatic phase with mild and/or unspecific symptoms that do not allow for a distinct differential diagnosis of MND until definite clinical signs of ALS occur and the ALS diagnosis according to recent diagnostic criteria is established . In a small cohort of 20 pre-symptomatic patients with a known ALS gene mutation, there was found different mild motor symptoms, i.e., very mild focal weakness not resulting in disability, deep tendon hyperreflexia or EMG abnormalities, such as ongoing denervation. According to the underlying gene mutation, MMI onset takes place 1 month up to 10 years before ALS diagnosis has been established. While for some mutations the duration of the prodromal phase and disease was consistent (e.g., SOD1 A4V and I113T), a rather long prodrome over 4 years followed by a rapid disease course within about 15 months of the disease's onset to permanent ventilation was reported. Conversely, FUS mutation carriers exhibit a relatively short prodromal stage, shorter than 1 year, followed by a disease course over 2.4 years . Outside familial ALS, it remains a major concern to identify patients according to potential risk factors for developing sporadic ALS. Prospective population-based studies may be a useful tool to explore MMI in potential future ALS patients. 6. Cognitive and Behavioral Assessment Historically defined as a pure motor neuron disease, ALS is now considered a complex multisystemic disorder not only occurring with motor impairment, but also with non-motor features, particularly cognitive and behavioral changes . Cognitive and behavioral impairment have been extensively reported in ALS, occurring in up to 50% of patients, with approximately 10% exhibiting the full symptoms of FTD, predominantly the behavioral variant . This is attributable to underlying aggregation of ubiquitinated transactive response DNA binding protein 43 (TDP-43), which has been identified as the common pathological hallmark and possible mechanism . Foremost, deficits are observed in the domains of executive and language functions . Since these cognitive and behavioral changes have an impact on prognosis, disease progression and caregiver burden, neuropsychological assessment has been emphasized in the diagnostic routine of ALS. As a result, the Strong criteria as diagnostic consensus criteria for the diagnosis of frontotemporal dysfunction in ALS were published. Patients meeting the criteria for cognitive or behavioral dysfunction are labelled as ALS with cognitive (ALS-ci) and behavioral impairment (ALS-bi) or both (ALS-bci) . Criteria for FTD are implemented to stratify patients as ALS-FTD . Many neuropsychological batteries, such as the Frontal Assessment Battery (FAB), the Montreal Cognitive Assessment Test (MoCa) or the Mini-mental State Assessment (MMSE) have been in clinical use. Disease-specific assessments have later been developed to counteract motor disabilities in ALS . With the Edinburgh Cognitive and Behavioral ALS Screen (ECAS), a disease-specific assessment tool for determining the presence, severity and type of cognitive and behavioral changes in ALS, and its differentiation from other disorders is available. It consists of tasks testing for ALS-specific (executive function, language, fluency, social cognition) and non-ALS-specific (memory, visuospatial functions) cognitive changes and can be administered by neuropsychological and non-neuropsychological professionals within approximately 15 to 20 min . A systematic review comparing different cognitive assessments for ALS entitled the ECAS promises to be the most suitable screen to detect cognitive or behavioral changes in ALS . 7. Technical Diagnostic Tools Electrodiagnostic (ED) studies are established and fundamental for the correct diagnosis of MND, particularly for the exclusion of disease mimics such as multifocal motor neuropathy (MMN). Nerve conduction studies are required to demonstrate an affection of motor nerves, indicated by reduced compound muscle action potential, prolonged distal motor latency and decreased conduction velocity without evidence for conduction blocks. This is complemented by needle EMG examination evaluating signs of acute and chronic denervation. In recent years, much effort has been invested in the development of more sophisticated approaches to diagnose MND with higher accuracy at an early stage in the course of the disease. As to the point of differentiation from disease mimics, the nerve ultrasound has become a powerful tool to aid in the diagnostic work-up when ED studies remain inconclusive . While MMN, for instance, usually presents itself with enlarged cross-sectional areas (CSA) of the nerves, this is not found in MND . Indeed, the CSA appears reduced in MND, which is associated with clinical affection of the respective innervated muscle, yielding a higher diagnostic accuracy . Analysis of the vagus nerve (VN) has become another interesting application of nerve ultrasound in ALS, showing a reduced CSA of the VN on both sides in ALS, independent of clinical symptoms . This could add another pathophysiological hint as to the involvement of the autonomous system, which was also reported to be affected in ALS, although only to a mild extent and without a correlation with disease severity . To further determine structural deficits of the peripheral nervous system suggestive of axonal degeneration in ALS, different magnetic resonance imaging (MRI) techniques were reported to add diagnostic value and work as a surrogate for disease progression, as measured by the ALSFRS . Beyond the pure structural point of view, modern MRI sequences allow a functional-connectivity assessment, such as DTI (diffusion tensor imaging), which has been thoroughly investigated as a potential biomarker for ALS in the past years . It is a measure for the unguided diffusion of water according to the Brownian movement, allowing assessment of the direction of the flux. It is usually numerically expressed by the fractional anisotropy, which can vary between 0 (pure spherical diffusion) and 1 (diffusion is strictly directed in one direction). More importantly, this technique has been extensively investigated in the field of MND and applied to determine the deterioration of both the peripheral and central motor nervous systems in ALS . While routine MRI of the central nervous system is essential for regular diagnostic work-up, it often appears unremarkable, shows T2 hyperintensities along the corticospinal tract (CST) or shows atrophy of the precentral gyrus, which are, however, of unclear specificity . In contrast, defined multiparametric MRI measurements, including DTI, allow for a much more accurate and reliable diagnostic workflow. However, it needs to be highly standardized . On the other hand, these different imaging modalities offer a vast range of different data types that can be integrated into a multiparametric model acquired by machine learning algorithms, which could be a very powerful approach to making an unsupervised diagnosis based on neuroimaging data . However, such elaborated MRI approaches require a high level of expertise and technical equipment, which is a limiting factor. Nonetheless, multiparametric MRI assessment including DTI should be accounted as a facultative biomarker for the diagnosis of MND in the future. Furthermore, in addition to the growing importance of fluid biomarkers for neuroinflammation in ALS, positron emission tomography (PET) imaging with highly specialized tracers indicating glial activation has caught attention. Standard [18F]Fluorodeoxyglucose-PET (FDG-PET) demonstrates distinct patterns of hypometabolism in the frontotemporal cortex and diverse areas of hypermetabolism (e.g., spinal cord, cerebellum ). However, tracers indicating inflammatory processes might reflect the disease course in a much more reliable way. The [11C]-PBR28 tracer binds to the 18pkD translocator protein (TSPO), which is located in glial cells and can indicate inflammatory activation. TSPO is enriched in the precentral gyrus of patients with ALS, correlating with the disease burden of upper motor neuron symptoms . Although having shown no correlation to disease progression in a longitudinal observation of patients with ALS, it still shows potential as a very specific biomarker. Broader studies including patients with fast disease progression and correlations to neuroinflammatory biomarkers are required to foster the concept of neuroinflammation as a diagnostic tool in ALS. 8. Genetic Testing Most ALS cases are sporadic and cannot be accounted for by a single genetic mutation, but a multitude of monogenic causes for ALS have been identified . They are thought to constitute 5 to 10% of all ALS cases. More than 30 potentially causative genes have been identified, and the large majority are inherited in an autosomal-dominant manner. Mutations in most of these genes are very infrequent and most Mendelian ALS cases are due to mutations in chromosome 9 open reading frame 72 (C9orf72), superoxide dismutase 1 (SOD1), fused-in sarcoma (FUS) and TAR DNA binding protein (TARDBP). It is important to note that up to 20% of patients with a negative family history have been reported to harbor a causative genetic variant . In a survey published in 2017, 90% of caregivers reported offering genetic testing to patients with familial ALS and 50% to patients with sporadic ALS. Most commonly, SOD1, C9orf72, TARDBP, and FUS were tested . With more widespread availability of genetic testing in general and next-generation sequencing (NGS) in particular, it seems likely that the number of caregivers offering genetic testing has increased and that NGS approaches are used more frequently. The diagnostic yield is certainly higher using these approaches and progress in the analysis of sequencing data will lead to the identification of even more variants with effect on ALS risk, especially structural variations and intronic variants . In contrast to other rare diseases, in the vast majority of ALS cases genetic testing will not be used to establish a diagnosis. However, genetic testing in patients with ALS does have a number of important implications:Exclusion of genetic disorders mimicking ALS, such as spinal and bulbar muscular atrophy (SBMA) which is caused by a polyglutamine expansion in the androgen receptor (AR) gene and can be mistaken for LMN-predominant ALS . Other examples include, but are not limited to, adult-onset spinal muscular atrophy, a number of hereditary spastic paraparesis (HSP) subtypes and adult polyglucosan body disease (APBD) ; Identification of patients who are eligible for trials targeting specific mutated genes, such as SOD1, C9orf72, Ataxin 2 (ATXN2) and FUS . For patients with SOD1 mutations, tofersen, an antisense oligonucleotide reducing SOD1 protein synthesis, was shown to reduce neurofilament light chain levels in plasma and is already available in many countries in an early access program ; Counselling of family members concerning predictive testing. Predictive testing always needs thorough counselling, especially when there are no measures to prevent the disease, as is currently the case for ALS . However, family members may benefit from the knowledge gained from genetic testing, either because they are relieved when they do not harbor the mutation or because they are able to plan ahead for specific life decisions. This does also include preimplantation genetic diagnosis if a desire to have children is present . Also, the exemplary ATLAS trial is already trying to closely follow up asymptomatic carriers of disease-causing mutations to initiate therapy on the basis of biomarker-defined phenoconversion and before the advent of clinical symptoms for an optimal disease-modifying effect . Approaches like these will likely be applied more often as more gene-specific therapies become available; Identification of genetic mutations may help in the prediction of the clinical course of the disease, which is of importance for patient and caregiver counselling (see prediction models section). It should be mentioned that, with the advent of NGS techniques, the identification of variants of uncertain significance (VUS) can be challenging when counselling patients and relatives . In one study, 47% of variants in ALS patients with potential functional significance were classified as VUS . Segregation analysis, consultation of population databases and even functional analysis will often leave uncertainty concerning the pathogenicity of VUS and thorough pre-test counselling is therefore of utmost importance so patients can anticipate possible results . Apart from disease-causing mutations, genome-wide association studies (GWAS) have identified a number of variants that modify the risk to develop ALS. They offer valuable insights into ALS pathogenesis and may help in the identification of treatment targets, but are not currently of additional diagnostic value . In the future, polygenic risk scores (PRS) may also be of help in predicting disease progression, as one study already hinted at an association of a PRS and cognitive decline in ALS, or help stratify ALS patients according to underlying disease processes using pathway-specific PRS, as has been suggested for Parkinson's disease . 9. Diagnostic and Prognostic Value of Fluid Biomarkers 9.1. Neurofilaments Neurofilaments (Nf) emerged as one of the most promising biomarkers of ALS in the recent past. Considered an indicator of ongoing neuronal or axonal injury, the role of Nf in the pathophysiology of ALS remains unclear . Elevated Nf can be found in several neurological diseases, including neurodegenerative disorders such as atypical Parkinson syndromes or FTD, as well as infectious or inflammatory disorders, such as multiple sclerosis (MS) or Creutzfeld-Jakob disease (CJD) . Therefore, interpretation of Nf elevation should always be performed in the context of the primary suspected diagnosis and clinical finding. As of now, examination of the neurofilament light chain (NfL) and the phosphorylated heavy chain of the neurofilament (pNfH), both in the cerebrospinal fluid (CSF) and serum, are established methods and comparable with regard to diagnostic utility. Both NfL and pNfH values in the CSF and serum strongly correlate . Numerous studies found a significant elevation of Nf in ALS patients compared to both healthy and disease controls . Additionally, the site of symptom onset or presence of ALS-related gene mutations, such as C9Orf72, SOD1 or FUS, are associated with higher Nf values. ALS patients with known bulbar onset or with a mutation in the C9Orf72 tend to have higher Nf levels . The same applies to ALS patients with more regions involved or present upper motor neuron signs at the time of diagnosis . One major issue in the diagnosis of ALS is the date of diagnosis, since a proper diagnosis of ALS is established up to 10 to 12 months after the first symptoms emerge . In pre-symptomatic patients with a known ALS-related mutation, elevated Nf values can be found up to 12 months before patients develop the first ALS-related symptoms . Before that, Nf values are comparable to healthy controls . After symptom onset, the longitudinal analysis revealed almost stable values for neurofilaments over the course of the disease . The increase of Nf from baseline prior to symptom onset depends on the respective mutation found, i.e., in ALS patients with known SOD1 mutation, approximately 6 to 12 months in advance of the first emergence of ALS symptoms. This is compared to 2 to 3.5 years in ALS patients with mutations in the FUS gene or C9Orf72 with hexanucleotide (G4C2)n repeat expansion (HRE) . Although these measurements are currently only available for a few patients with inherited ALS, they can most likely apply to sporadic ALS patients to an extent, as neuronal and axonal injury precedes the onset of motor symptoms in sporadic patients as well. Nf may shorten the diagnostic delay by up to 3 months in patients with suspected ALS . The diagnostic sensitivity and specificity of Nf measurement depend, among other things, on the measurement of either NfL or pNfH and the assay used, yet efforts have been made to standardize procedures for comparable results among different laboratories . Steinacker and colleagues first demonstrated a sensitivity of 77% and a specificity of 88% for CSF NfL values using a cut-off of 2200 pg/mL to distinguish ALS patients from ALS mimics. For CSF pNfH values above 560 pg/mL, a robust sensitivity of 83% and a specificity of 80% were found. Subsequent studies confirmed the usability of NfL and pNfH by applying comparable cut-off values for CSF NfL and CSF pNfH in the differential diagnosis of ALS (Table 3). The examination of serum NfL demonstrated comparable results for sensitivity and specificity . However, pNfH examination in the serum seems to be inferior regarding differential diagnosis . Besides the differential diagnostic value of the Nf measurement, several studies addressed the prognostic and therapeutic aspects of Nf in ALS patients. Elevated Nf positively correlates with disease progression rate . Conversely, ALS patients with longer disease duration display lower Nf levels . Additionally, high Nf levels are associated with shorter survival . However, findings considering the therapeutic implications of Nf are ambiguous. In both phase 1/2 and phase 3 of tofersen trials, an antisense oligonucleotide (ASO) targeting SOD1 messenger RNA (mRNA) transcripts in ALS associated with mutations in SOD1, there was a significant reduction of NfL levels in the CSF and plasma, but no improvement in clinical endpoints could be demonstrated . On the other hand, Riluzole, currently the only approved treatment for ALS, does not alter Nf levels after treatment initiation . Nevertheless, findings regarding Nf in the therapy of other neurologic disorders are encouraging. In relapse-remitting MS, Nf may serve as a marker for treatment response for different disease-modifying therapies . In other motor neuron diseases, such as spinal muscular atrophy (SMA), a significant reduction of plasma pNfH was found after treatment initiation with nusinersen, an ASO targeting SMN1 splicing, in children with SMA . Therefore, the role of Nf as a marker for therapeutic response remains to be determined. 9.2. Inflammatory Biomarkers The pathophysiology of ALS is characterized not only by neurodegenerative but also by inflammatory processes involving glial cells of the central nervous system and peripheral circulating immune cells . Genetic alterations linked to ALS, e.g., in SOD1 and C9orf72, are also associated with the dysregulation of immune processes . However, the dysfunction of autophagy and glial cells was also present in ALS patients without these genetic alterations . It is assumed that anti-inflammatory processes predominate at the onset of the disease, while proinflammatory processes become relevant in later stages, accelerating motor neuron injury . This marks the relevance of inflammatory biomarkers, which promise to provide information on disease stage, progression rate as well as pathophysiological and potential protective mechanisms. Several studies have investigated patterns of blood immune cells, such as granulocytes and T cells, as possible diagnostic biomarkers in ALS patients, finding altered leukocyte phenotypes . Others found differentially regulated soluble factors such as interleukin (IL)-6, IL-8, tumor necrosis factor (TNF) and interferons . They generally suggest differential immune regulation in ALS compared to healthy individuals. However, findings are heterogeneous and quite variable between studies and different methodological approaches. It is especially difficult to distinguish ALS from mimics, as they are often inflammatory diseases. More detailed data were provided by studies on a transcriptional level, including the finding that proinflammatory gene profiles had higher expression levels of IL-8, FBJ murine osteosarcoma viral oncogene homolog B (FOSB), cluster of differentiation 83 (CD83), suppressor of cytokine signaling 3 (SOCS3), chemokine (C-X-C motif) ligand 1 (CXCL1) and CXCL2 in monocytes of ALS patients compared to healthy controls . Nevertheless, these inflammatory diagnostic biomarkers are far from making their way into clinical use. Similarly, they have not been proven to provide a prognostic value. Most studies failed to find a significant correlation between common clinical progression parameters such as the ALSFRS-R and blood concentrations of anti-inflammatory cytokines in ALS patients . Other groups found correlations between the ALSFRS-R and survival on the one hand and seemingly protective monocyte and T-cell immune profiles on the other . In addition to this, a higher number of pro-inflammatory differentially-expressed genes in monocytes of ALS patients was associated with faster disease progression . Another potential inflammatory biomarker for ALS is neopterin, which is secreted by macrophages under interferon-gamma influence from stimulated T lymphocytes, is an indicator of general immune system activation and is renally excreted. In urine, it can thus be examined non-invasively without great effort. Higher concentrations were found in ALS patients than in patients with other neurological diseases, such as multiple sclerosis, and in healthy controls . A higher neopterin level in ALS patients was associated with more severe symptoms evaluated by the ALSFRS-R . In addition to peripherally circulating inflammatory biomarkers, there are CNS-specific ones that represent microglial and astrocyte-derived inflammation, which, however, are more difficult to access and measure. Up-regulation of activated microglia and astrocytes producing pro-inflammatory cytokines was found in the spinal cord tissue of ALS patients . SOD1-mutated mouse microglia were found to express predominantly anti-inflammatory markers like chitinase-like 3 (Ym1), cluster of differentiation 163 (CD163) and brain-derived neurotrophic factor (BDNF) mRNA and fewer proinflammatory markers like NADPH oxidase 2 (Nox2) mRNA at disease onset than later in disease progression, consistent with observations of other peripheral inflammatory markers during disease course . Astrocytes and microglia have been shown to interact and alter each other's phenotype through the release of inflammatory mediators affecting disease progression in a mouse model . As a representation of neuroinflammatory involvement not only in the spinal cord, but also in the motor cortex in early stages of ALS with TDP-43 pathology, activated astrocytes and microglia were detected in this brain area in patients and a TDP-43 mouse model. It was also demonstrated that cells of the primary motor cortex express the monocyte chemoattractant protein-1 (MCP1), a ligand for C-C chemokine receptor 2+ (CCR2+) monocytes infiltrating the CNS, driving the immune response in this area . Recently, some approaches detected the above-explained inflammatory processes non-invasively using functional imaging, e.g., using positron emission tomography . 9.3. Chitinase, Tau Protein, TDP-43, Creatine Kinase and Other Fluid Biomarkers Elevations of chitinases (CHIs) and chitinase-like proteins (CLPs) have also been found in the CSF of ALS patients. CHIs are hydrolytic enzymes, widely distributed in nature, that metabolize chitin, the most abundant polysaccharide in nature and essential structural component of several organisms, including arthropods, protozoan parasites, nematodes, bacteria and fungi. Despite the absence of endogenous chitin, mammals express true CHIs with enzymatic activity and homologous structurally related CLPs lacking enzymatic activity but bind chitin with high affinity . Despite its implication in several neurological diseases, the function of CHIs and CLPs in the CNS is still not completely understood. Chitotriosidase-1 (CHIT1) has been found only in microglia and CNS infiltrating peripheral macrophages . Chitinase-3-like protein 1 (CHI3L1) has been mostly found in reactive astrocytes . Little is known about the role of Chitinase-3-like protein 2 (CHI3L2) in physiologic and pathologic conditions. Increased CHIT1, CHI3L2 and CHI3L2 expression or CSF levels have been reported in various neuroinflammatory conditions . CHIs and CLPs have been recently investigated. Thompson and colleagues showed that CHIT1, CHI3L1 and CHI3L2 were elevated in the CSF of patients with ALS compared with healthy controls and ALS-mimics. CHIT1 and CHI3L2 were elevated in ALS compared with PLS . Additionally, the CHIT1 response appears to be an attribute of the late pre-symptomatic to early symptomatic phases in patients carrying mutations in C9orf72 or SOD1 . Other markers commonly used for the diagnosis of other neurodegenerative diseases have also been studied in the context of ALS. For example, the microtubule-associated protein Tau, commonly found elevated in Alzheimer's disease, increases significantly in the CSF of ALS patients. Significantly higher levels of total Tau (tTau) and lower phosphorylated Tau (pTau)/tTau ratio have been found in ALS patients in comparison with healthy controls in observational studies . b-Amyloid, another Alzheimer's disease-related biomarker, is elevated in the CSF of ALS patients and seems to predict shorter survivals correlating with the ALSFRS-R at baseline . On the other hand, the soluble amyloid precursor protein (sAPPb) appears reduced in the CSF of ALS and FTD patients, correlating with cognitive performance . TDP-43 has also demonstrated some prognostic value in ALS patients. Several studies have reported elevated CSF TDP-43 levels in patients with ALS . Similarly, significantly increased levels of TDP-43 and pTDP-43 have been found in plasma of the ALS patients. Especially, the pTDP-43/TDP-43 ratio appears to distinguish individuals with ALS from healthy controls . High levels of miR-181, a highly conserved non-coding RNA molecule enriched in neurons, predict a greater than a two-fold risk of death in ALS patients. The molecule miR-181 performed similarly to NfL, and when combined, miR-181 + NfL show a superior prognostic value . Concerning non-neuronal related biomarkers, higher levels of creatine kinase (CK) are often found in ALS patients, especially in those with slow progression, correlating to lower ALSFRS-R scores. Higher CK blood concentration is likely linked to longer survival . In contrast to the cardiac troponin I (cTnI), serum concentration of cardiac troponin T (cTnT) is elevated in the serum of ALS patients. Both are common biomarkers in the initial approach of myocardial infarction. This is especially true in patients with a spinal onset (AUC 0.87; 0.78-0.94), and it can thus differentiate ALS from other neurodegenerative diseases and ALS mimics . CSF levels of the basic fibroblast growth factor (bFGF) are increased in ALS and correlate with disease duration and survival . Similarly, the perivascular fibroblast marker Secreted Phosphoprotein 1 (SSP1, Osteopontin) increased in plasma of ALS patients in four independent cohorts. Increased levels of SPP1 at disease diagnosis predicted shorter survival as well . 9.4. Frontiers in Fluid Biomarkers Despite the value as a diagnostic tool showed by increasing research on Nf, many other neurological disorders present elevated Nf in serum and CSF, decreasing its specificity in the ALS diagnosis. Currently, great effort is exerted in clinical research to find ALS-specific biomarkers that indicate the onset of pathological events in pre-symptomatic or prodromal phases of the disease. Among the most promising is the translation products of the C9orf72 intronic expansion, poly-GP dipeptide repeats, which are increased in the CSF of pre-symptomatic patients of C9orf72-associated ALS. Similarly, higher levels of the poly-GP proteins were also found in peripheral mononuclear cells of pre-symptomatic C9orf72 mutation carriers . Recently, it was found that the nuclear TDP-43 suppresses cryptic exon-splicing events of some ALS-associated genes, such as UNC13A. This repression is lost in the ALS/FTD pathology, as extranuclear TDP-43 is a specific hallmark of these disorders. The early identification of such cryptic exon-splicing variants or their translational products represents one of the most promising and specific biomarkers for identifying disease onset . 10. Predictive Models "Prognosis can no longer be relegated behind diagnosis and therapy in high-quality neurologic care" . With a diagnosis of ALS, the question of prognosis almost automatically arises. However, the disease course is highly variable. This poses a problem when discussing prognosis with the individual patient, but also when dealing with high variability in the design and evaluation of disease-modifying trials . Biomarkers such as neurofilaments and rating scales such as the ALSFRS-R at baseline, the ALSFRS-R decline from disease onset to the test date, the initial clinical presentation or specific genetic mutations are all individually associated with disease progression and survival time . The multitude of parameters that can be evaluated in patients with ALS calls for approaches that incorporate many factors influencing the disease course to provide a more accurate estimate of how the disease will likely progress in the individual patient. The ENCALS survival prediction model uses eight predictors to define five groups with distinct survival outcome, which was defined as the time between symptom onset and non-invasive ventilation > 23 h/day, tracheostomy or death . Clinical parameters, such as bulbar vs. non-bulbar onset, forced vital capacity, the age of onset and the diagnostic delay and the presence of C9orf72 repeat expansion, were used in the model. Patients with a predicted brief disease course had a median of 17.7 months from symptom onset to the composite survival outcome, while the median was 91.0 months for the group with a very long course. A qualitative study looking at the impact of personalized prognosis using the ENCALS survival prediction model found that it can be discussed with "minimal adverse emotional impact" and may help facilitate planning of the future . However, the quotes of patients, relatives and caregivers in reaction to the prediction, probably not unexpectedly, show varying reactions. More research is needed to assess the impact of personal prediction models in ALS. Additionally, while the ENCALS model at least incorporated one non-clinical parameter by using the C9orf72 repeat expansion as a predictor, many studies trying to predict ALS or trying to identify ALS subgroups are still only or mostly using clinical parameters . We are convinced that, in the future, the combination of clinical signs, genetic testing and biomarkers, such as neurofilaments, will offer the patient an informed prognostic estimation after establishing a diagnosis of ALS. Establishing and refining experimental disease models will be crucial for obtaining information about the underlying heterogenous disease-causing mechanisms and identifying new potential diagnostic and therapeutic targets in ALS, particularly in its sporadic forms. While transgenic animal models for familiar ALS with the disease-causing mutation have provided insights into pathogenesis and potential therapeutic targets, identifying the underlying disease and causes of sporadic forms remains challenging. Currently, various in vitro cellular models with induced pluripotent stem cells (iPSCs) and organoids from donors with sporadic ALS show phenotypic differences in the pattern of neuronal organization and degeneration, protein aggregation, cell death, as well as in onset and progression . The further development of these methods in combination with advanced omics technologies, such as single-cell sequencing and Deep Learning algorithms, will allow precise, accurate and reliable decoding of patient-specific cellular and genetic dysfunction, leading not only to an individual molecular diagnosis but a solid predictive model for applying personalized therapies . 11. Conclusions The diagnosis of ALS is currently primarily based on clinical aspects as options for early detection, such as biomarkers with high diagnostic accuracy, are lacking. The ongoing development of new and innovative diagnostic tools, however, promises major advances that will fundamentally impact future clinical practice and research in this field . Expert clinical assessment remains essential in diagnosing ALS. Its heterogeneous clinical presentation and multisystemic complexity can be challenging and requires interdisciplinary assessment of cognitive and behavioral aspects in addition to pure motor impairments. Electrophysiological assessments as well as imaging techniques, such as functional MRI and PET, are becoming increasingly sensitive and can aid in early diagnosis and, in particular, differentiation from related diseases. In addition to established Nf in the diagnostic workup, other biomarkers of several systems, ranging from inflammatory to degenerative types, are emerging. Ideally, biomarkers need to be easy to obtain and minimally invasive, improve diagnostic accuracy, facilitate early detection of ALS and enable the monitoring of disease progression. This would aid in enrolling patients in new clinical trials even in pre-symptomatic phases, evaluating new treatments and optimizing treatment plans and disease management. Early identification of affected and pre-symptomatic individuals may prevent prolonged diagnostic procedures in the future and greatly improve the potential efficacy of therapeutics by extending and advancing the treatment window. Genetic testing is becoming more feasible and frequently used. It promises a better endophenotypic classification of patients based on their neurobiological disease correlates and thus may enable targeted treatment and improved prediction of individual disease courses. Ultimately, the integration of various diagnostic methods and all collected patient data will be key to deriving patterns towards more personalized diagnostics with the future opportunity of evaluating individual prognosis as well as directing patients towards treatment studies or approved therapeutic strategies. Acknowledgments This article was developed as a result of the 2nd ALS Young Investigators Academy, which took place at the Klinikum rechts der Isar of the Technical University of Munich on 21-22 October 2022, with generous support of ITF Pharma. Author Contributions Conceptualization, M.V., S.C.-G., G.M., S.B., M.N. and L.H.M.; writing--original draft preparation, M.V.; writing--review and editing, M.V., S.C.-G., G.M., S.B., M.N. and L.H.M.; visualization, S.C.-G. and L.H.M. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement No new experimental data were created or analyzed in this study. Data sharing is not applicable to this article. Conflicts of Interest L.H.M. reports personal fees from Biohaven Pharmaceuticals and a speaker honorarium from Forum fur Medizinische Fortbildung, both outside the submitted work. Abbreviations ALSFRS-R Revised Amyotrophic Lateral Sclerosis Functional Rating Scale ADM abductor digiti minimi ALS amyotrophic lateral sclerosis ALS-bci ALS with cognitive and behavioral impairment ALS-bi ALS with behavioral impairment ALS-ci ALS with cognitive impariment APB abductor pollicis brevis APBD adult polyglucosan body disease AR androgen receptor ASO antisense oligonucleotide ATXN2 Ataxin 2 Ab Amyloid b bFGF basic fibroblast growth factor C9orf72 chromosome 9 open reading frame 72 CD83 cluster of differentiation 83 CHI3L1 chitinase-3-like protein 1 CHI3L2 chitinase-3-like protein 2 CHIT1 chitotriosidase 1 CJD Creutzfeld-Jakob disease CK creatine kinase CLP chitinase-like proteins CSA cross-sectional areas CSF cerebrospinal fluid CST corticospinal tract cTnT cardiac troponin T CXCL1 chemokine (C-X-C motif) ligand 1 CXCL2 chemokine (C-X-C motif) ligand 2 DTI diffusion tensor imaging DTR deep tendon reflex ECAS Edinburgh Cognitive and Behavioral ALS Screen ED electrodiagnostic EMG electromyography FAB Frontal Assessment Battery FAS flail-arm-syndrome FDG-PET [18F]Fluorodeoxyglucose-PET FDI first dorsal interosseus FLS flail-leg-syndrome FOSB FBJ murine osteosarcoma viral oncogene homolog B FTD Frontotemporal dementia FUS fused-in sarcoma GWAS Genome-wide association studies HRE hexanucleotide (G4C2)n repeat expansion HSP hereditary spastic paraparesis IL-18 interleukin-18 IL-8 interleukin-8 iPSCs induced pluripotent stem cells LMN lower motor neurons MCP-1 monocyte chemoattractant protein-1 MiToS Milano Torino Staging System MMI mild motor impairment MMN multifocal motor neuropathy MMSE Mini Mental State Assessment MND motor neuron disease MoCa Montreal Cognitive Assessment Test MRI magnetic resonance imaging MS multiple sclerosis Nf neurofilaments NfL neurofilament light chain NGS next-generation sequencing PET positron emission tomography PLS primary lateral sclerosis PMA progressive muscular atrophy pNfH phosphorylated neurofilament heavy chain Poly-GP arginine containing dipeptide repeat polymers PRS polygenic risk scores pTau phosphorylated Tau ROADS Rasch overall ALS disability scale sAPPb soluble amyloid precursor protein SBMA spinal and bulbar muscular atrophy SMA spinal muscular atrophy SOCS3 suppressor of cytokine signaling 3 SOD1 superoxide dismutase 1 SPP1 secreted Phosphoprotein 1 TARDBP TAR DNA binding protein TDP-43 transactive response DNA binding protein 43 TNF-a tumor necrosis factor TSPO 18pkD translocator protein t-Tau total Tau UMN upper motor neurons Vn vagus nerve Figure 1 Clinical signs of UMN and LMN involvement according to body regions. DTR: Deep tendon reflex; LMN: Lower motor neurons; UMN: Upper motor neurons. Created by Castro-Gomez with "BioRender.com"; accessed on 4 February 2023. Figure 2 Current state of and future directions for the diagnosis of amyotrophic lateral sclerosis. A delayed diagnosis of ALS is still currently performed using clinical criteria such as Revised El Escorial, but the determination of fluid biomarkers such as Nf is accelerating an earlier identification of the disease. In the future, the availability of more specific biomarkers related to disease pathology onset will allow early risk assessment and definitive diagnosis in patients in prodromal phases. Additionally, an ideal biomarker should develop preventive therapies and monitor disease activity in patients under novel causative therapies. ALSFRS-R: Revised Amyotrophic Lateral Sclerosis Functional Rating Scale; Ab: Amyloid b; bFGF: basic fibroblast growth factor; CHI3L1: chitinase-3-like protein 1; CHI3L2: chitinase-3-like protein 2; CHIT1: chitotriosidase 1; CK: creatinine kinase; cTnT: cardiac troponin T; IL-6: interleukin-6; IL-8: interleukin-18; MCP-1: monocyte chemoattractant protein-1; MMI: mild motor impairment; NfL: neurofilament light chain; pNfH: phosphorylated neurofilament heavy chain; Poly-GP: arginine containing dipeptide repeat polymers; pTau: phosphorylated tau; SPP1: secreted phosphoprotein 1; TNF-a: tumor necrosis factor; t-Tau: total tau. Created by Castro-Gomez with "BioRender.com"; accessed on 04 February 2023. cells-12-00736-t001_Table 1 Table 1 The revised El Escorial criteria (2000) and the Awaji criteria (2008). Clinically definite ALS Clinical or electrophysiological * evidence of UMN and LMN involvement in bulbar region and >=2 spinal regions or Clinical or electrophysiological * evidence of UMN and LMN involvement in 3 spinal regions Clinically probable ALS Clinical or electrophysiological * evidence of UMN and LMN involvement in >=2 regions with UMN signs rostral to LMN signs Clinically probable Laboratory-supported ALS Clinical evidence of UMN and LMN involvement in 1 region or Clinical evidence of isolated UMN involvement in 1 region with electrophysiological evidence of LMN involvement in >=2 regions Clinically possible ALS Clinical or electrophysiological * evidence of UMN and LMN involvement in 1 region or Evidence of isolated UMN involvement >=2 regions or Evidence of LMN involvement rostral to UMN involvement ALS: Amyotrophic lateral sclerosis; UMN: upper motor neuron; LMN: lower motor neuron; * additional feature for Awaji criteria; only included in revised El Escorial criteria. cells-12-00736-t002_Table 2 Table 2 The Gold Coast criteria (2020). Progressive motor impairment and Clinical or electrophysiological UMN and LMN involvement in >=1 region or only LMN involvement in >=2 regions and Exclusion of other diseases UMN: upper motor neuron; LMN: lower motor neuron. cells-12-00736-t003_Table 3 Table 3 Sensitivity and specificity of Neurofilament in CSF. Reference values for neurofilaments in the cerebrospinal fluid. Demonstrated are sensitivity, specificity and positive and negative predictive values for different neurofilament cut-off values for distinguishing ALS patients from either ALS mimics, disease controls or healthy controls. Neurofilament Cut-Off [pg/mL] Sensitivity (95% CI) [%] Specificity (95% CI) [%] NPV (95% CI) [%] PPV (95% CI) [%] ALS vs. Study NfL >2200 77 (71-82) 88 (79-94) 56 (48-65) 95 (91-98) ALS mimics >2200 - 85 (79-90) 75 (69-80) 87 (81-91) Other controls pNfH >560 83 (78-88) 80 (70-88) 62 (52-71) 93 (88-95) ALS mimics >560 - 77 (71-83) 79 (72-84) 82 (77-86) Other controls NfL >3819 88.4 (78.8-94) 84.7 (76.8-90.2) - - Disease controls >2453 85.4 78 - - ALS mimics pNfH >618 94.2 (86-97.7) 74.8 (66-81.9) - - Disease controls >768 90.7 88 76 - ALS mimics NfL 1431 79 (66.1-87.6) 86.4 (75.7-93.6 - - Controls pNFH 568.5 78.7 (67.7-87.3) 93.3 (85.1-97.8) - - Controls Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). 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PMC10000758 | Diagnostics (Basel) Diagnostics (Basel) diagnostics Diagnostics 2075-4418 MDPI 10.3390/diagnostics13050859 diagnostics-13-00859 Article Attention Deep Feature Extraction from Brain MRIs in Explainable Mode: DGXAINet Tasci Burak Massimi Luca Academic Editor Vocational School of Technical Sciences, Firat University, Elazig 23119, Turkey; [email protected] 23 2 2023 3 2023 13 5 85924 1 2023 09 2 2023 17 2 2023 (c) 2023 by the author. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). Artificial intelligence models do not provide information about exactly how the predictions are reached. This lack of transparency is a major drawback. Particularly in medical applications, interest in explainable artificial intelligence (XAI), which helps to develop methods of visualizing, explaining, and analyzing deep learning models, has increased recently. With explainable artificial intelligence, it is possible to understand whether the solutions offered by deep learning techniques are safe. This paper aims to diagnose a fatal disease such as a brain tumor faster and more accurately using XAI methods. In this study, we preferred datasets that are widely used in the literature, such as the four-class kaggle brain tumor dataset (Dataset I) and the three-class figshare brain tumor dataset (Dataset II). To extract features, a pre-trained deep learning model is chosen. DenseNet201 is used as the feature extractor in this case. The proposed automated brain tumor detection model includes five stages. First, training of brain MR images with DenseNet201, the tumor area was segmented with GradCAM. The features were extracted from DenseNet201 trained using the exemplar method. Extracted features were selected with iterative neighborhood component (INCA) feature selector. Finally, the selected features were classified using support vector machine (SVM) with 10-fold cross-validation. An accuracy of 98.65% and 99.97%, were obtained for Datasets I and II, respectively. The proposed model obtained higher performance than the state-of-the-art methods and can be used to aid radiologists in their diagnosis. XAI Densenet201 GradCam INCA SVM brain tumor This research received no external funding. pmc1. Introduction The incidence of cancer, which is one of these diseases, is increasing day by day. Cancer prevents cells in the body from growing normally, causing damage to tissues. According to the GLOBOCAN 2020 data created by the International Agency for Research on Cancer (IARC), cancer incidence and mortality rates show approximately 19.3 million new cancer cases in 185 countries worldwide. It is estimated that these cancer cases will be 30.2 million in 2040. Brain tumors from cancer types were 308 thousand in 2020. While 168 thousand of these cases are men, 140 thousand are women. It has been reported that brain tumor cases are estimated to be around 435 thousand in 2040. In addition, according to the data collected by IARC in 2020, the number of people who died from brain tumors in the 0-85 age range is 251 thousand. It has been reported that the number of deaths in 2040 is estimated to be around 368,000 . A brain tumor is a mass development within the brain that is created by the tissues surrounding the brain or skull and directly impacts human existence. These masses may be benign or cancerous. Brain tumors cause uneven growth inside the brain and exert pressure on the meninges. As a result of pressure, the brain experiences various problems that impair its ability to manage the body. At the onset of such symptoms as dizziness, headaches, fainting, paralysis, etc., in people, scenarios arise. Malignant tumors, as opposed to benign tumors, develop unevenly and destroy the surrounding tissues. Surgical methods often treat brain tumors. Surgery is located in a crucial region if the tumor is removed. However, medicines, radiation, etc., are preferable . There are more than 120 different types of brain tumors in the world. Therefore, the tumor classification and grading system developed by the World Health Organization (WHO) is used today to predict the outcome of brain tumors, standardize communication, and plan treatment . Generally, the cells obtained by biopsy are examined, and tumors are classified according to cell type and grade. The cell type refers to the cells that are the origin of the tumor, and nearly half of the primary brain tumors grow from glial cells. Grade refers to how the tumor cells look under the microscope and indicates aggressiveness . In the WHO grading system, there is a scaling by malignancy between benign grade I and rapidly growing and difficult-to-diagnose grade IV based on identifying different histopathological groups . In addition, depending on their origin, brain tumors are divided into primary brain tumors or metastatic brain tumors. In primary cells, the cells originate from brain tissue cells. In metastatic brain tumors, the cells become a cancerous structure in another body organ and spread to the brain . Early diagnosis plays a crucial role in the treatment of diseases and the prevention of severe symptoms. Medical imaging techniques such as magnetic resonance imaging (MRI) , computed tomography (CT), single-photon emission computed tomography, Single-Photon Emission Computed Tomography (SPECT), and positron emission tomography (PET). Brain anatomy can be studied with . The advantage of MRI over other medical imaging techniques is that it does not expose the patient to radiation . Another significant advantage is that it offers better opportunities for imaging the anatomical structure of tissues, thanks to its ability to be unaffected by the human body. MRI is a medical method that can safely distinguish an anatomical structure from other anatomical structures by using radio waves in the strong magnetic field generated by magnets . As a reflection of the rapid increase in the human population in the health sector, the amount of clinical data that needs to be handled by healthcare professionals has increased tremendously. Thus, most healthcare professionals analyze laboratory data and medical images in their daily clinical routines to investigate the presence of various diseases. Due to the increasing complexity, the increase in the specialists' workload paves the way for the formation of critical errors in clinical decisions. Factors such as fatigue and inexperience may cause diseases to be misdiagnosed, areas without lesions to be evaluated as lesions, and especially malignant lesions to be interpreted as benign . Le et al. stated in a retrospective study that the error rate of radiological examinations was around 30%, while the daily error rate of radiologists was between 3-5%.Obermeyer et al. , stated that the number of diagnostic errors increased significantly, and there was not enough intervention that could reduce the errors. For this reason, computerized diagnostic systems need to be developed to assist healthcare professionals in their decisions. Explainable artificial intelligence techniques can be described as a set of techniques and methods that enable humans to interpret and understand the results of artificial intelligence. The need for explicable artificial intelligence also differs according to the target of artificial intelligence and the field in which it is applied. The need for explanation is low in a model that predicts whether an image is a cat or a dog, while the need for explanation is high in a model that predicts whether a patient has cancer. Computerized diagnostic systems are widely used to detect and diagnose breast cancer, lung cancer, prostate cancer, skin lesions, stroke, Alzheimer's disease, and many more. Thanks to such developed systems, patterns related to diseases that experts miss in some cases can be captured with high sensitivity by computer vision techniques. Thus, it is possible to reduce healthcare professionals' workload, use time more effectively, and increase diagnostic accuracy . The development of computer diagnostic systems is significant for underdeveloped and developing countries with insufficient experts. Studies on deep learning-based brain tumor detection in the literature are given below. Raza et al. , removed the last five layers of GoogLeNet to create DeepTumorNet. Instead of these five layers, 15 new layers were added. The created hybrid CNN network was compared with nine different pre-trained models. accuracy, precision, recall, and F1 score were 99.67%, 99.6%, 100%, and 99.66%, respectively. Khazaee et al. , used the brats 2019 dataset to classify high-grade gliomas (HGG) and low-grade gliomas (LGG). 13,233 HGG and 13,671 LGG MR images were used in the EfficientNetB0 pre-trained model. In total, 80% of 26,904 MR images were used for training and 20% for validation. Accuracy, precision, sensitivity, and specificity were obtained at 98.87%, 98.98%, 98.86%, and 98.79%, respectively. Hamdaoui et al. , classified HGG and LGG using pre-trained models VGG16, VGG19, MobileNet, InceptionV3, Xception, InceptionResNetV2, DenseNet121. The Brats 2019 dataset was used as the dataset. Accuracy, precision, sensitivity, and F1-score 98.06%, 98.67%, 98.33%, and 98.62% were obtained, respectively. Tandel et al. attempted to detect tumors from brain MRI images. The authors used four different datasets in their work. ResNet50, AlexNet, ResNet18, VGG16, and GoogleNet pre-trained models were used. Majority voting was applied to their predictions from these five pre-trained models. For the 1st dataset of normal and tumor classes, accuracy, sensitivity, and specificity 96.51%, 96.76%, and 96.43% were obtained with 5-fold CV, respectively. For the second dataset consisting of AST-II and AST-III classes, accuracy, sensitivity, and specificity 97.70%, 94.63%, and 99.44% 5-fold CV were obtained, respectively. For the 3rd dataset consisting of OLI-II and OLI-III classes, accuracy, sensitivity, specificity 100%, 100%, and 100% 5-fold CV results were obtained, respectively. Accuracy, sensitivity, and specificity 98.43%, 98.33%, and 98.57%, respectively, with 5-fold CV, were obtained for the 4th dataset, which consisted of HGG and LGG classes. Rizwan et al. performed brain tumor classification using the Gaussian Convolutional Neural Network (GCNN). Datasets consisting of two different class T1 painful MR images were used. The classes of the first dataset are pituitary, glioma, and meningioma tumors. The classes of the second Dataset are Grade-two, Grade-three, and Grade-four. 99.80% and 97.14% accuracy were obtained for the first and second datasets, respectively. Tariciotti et al. classified primary central nervous system lymphoma, glioblastoma, and solitary brain metastasis. 70% of the images in the dataset were used for training and 30% for testing. ResNet101 used the pre-trained network model. Their studies obtained an accuracy of 94.72%. Majib et al. , used a Kaggle dataset consisting of 253 brain MRI images. In their study, they analyzed sixteen pre-trained models. Among these pre-trained models, VGG-SCNet was used. With Stacked Classifier, F1 scores, precision, and recall were obtained at 99.20%, 99.20%, and 99.10%, respectively. Mehrotra et al. , classified benign and malignant tumors. Flipped image, mirrored image, noisy image, and 45deg rotated preprocesses were applied to the dataset. AlexNet, GoogleNet, SqueezeNet, Resnet101 and Restnet 50 CNN networks were used. 99.04% accuracy was achieved with PT-CNN(AlexNet). Kaur et al. performed brain tumor classification using Inceptionv3, InceptionResNetV2, VGG-16, Resnet101, VGG-19, Alexnet, Resnet50, GoogLeNet, and Resnet101. Three different datasets were used. 60% of the dataset was used for training and 40% for testing. Accuracy of 100%, 94% and 95.92% was achieved for the three datasets, respectively. Begum and Lakshmi , proposed a deep learning model for anomaly detection in brain MRI images. The method consists of 4 stages as feature extraction, feature selection, classification and segmentation stages. After the texture feature extraction process, feature reduction is performed with the oppositional gravity search algorithm, oppositional gravitational search algorithm. The reduced feature set was classified with the recursive neural network Recurrent Neural Network (RNN), and tumors in MRI images thought to contain anomalies were extracted with modified region growing algorithm. In their study, they achieved 96.26% accuracy. Saucedo et al. , diagnosed a brain tumor using the Grad-CAM-CNN explainable artificial intelligence model. With their proposed method, they achieved 97.11% accuracy, 95.58% sensitivity and 96.81% specificity. Etminani et al. , used 3D 18F-FDG-PET images to feed the input of the 3D-CNN network. Occlusion and Grad-CAM XAI methods were used. For CN, the F1 Score was 84.00% on the 3D model, 59.00% on Resnet50 and 59.00% on InceptionV3. Kaur et al. , provided segmentation of medical images by combining the segmentation power of U-Net and the explainability of the Xception network with Grad-CAM features. In the 3D-IRCADb-01 dataset, 97.73% dice results were obtained. 1.1. Novelties and Contributions Novel sides of this research:- We have used deep learning as preprocessing model to create an XAI model. - A patch-based deep feature extraction model has been proposed. - A new XAI strategy has been used for brain tumor classification. 1.2. Contributions - Brain tumor classification is a hot-topic research area for biomedical image classification, and XAI models have been proposed in this area to assist medical professionals. However, there are limited XAI models. In this research, we have proposed an XAI model to fill this gap and increase classification performance. - To show the superiority of our proposal, we have used two public brain tumor image datasets. These datasets contain four and three categories. We obtained a pre-trained network by using a dataset with four classes and DenseNet201. Using this pretrained DenseNet201, preprocessing and feature extraction layers of our model have been created. In this respect, a deep learning-based cognitive model has been created. Moreover, our model attained superior classification performances than other state-of-art models. 2. Materials We have used two datasets in this research to show the general classification ability of the proposed model. The used both datasets are publicly available, and these are brain tumor datasets. These datasets were downloaded from Figshare and Kaggle platforms. There are three classes in the Figshare dataset, and the Kaggle dataset has four classes. Therefore, we used Kaggle Dataset for training. Sample images of these datasets are demonstrated in Figure 1. The details of these datasets are given below. 2.1. Dataset I The dataset consists of four classes of axial, sagittal, and coronal section images. The data is divided into training and test data. The glioma tumor class contains 100 images in the test folder and 826 images in the training folder. The meningioma tumor class contains 115 images in the test folder and 822 images in the training folder. There are 105 images in the test folder and 395 images in the training folder for the no-tumor class. The pituitary tumor class contains 74 images in the test folder and 827 images in the training folder. In this study, the images in the training and test folders were combined. In total, there are 926 images in the glioma tumor class, 937 in the meningioma tumor class, 500 in the no-tumor class, and 901 in the pituitary tumor class. The sum of the number of images in all classes is 3264 . 2.2. Dataset II The dataset consists of three classes of axial, sagittal, and coronal section images. In the meningioma class, 82 patients had 708 MR images. Of the meningioma tumor class images, 209 are axial, 268 are coronal, and 231 are sagittal. There are 1426 MR images of 89 patients in the glioma tumor class. Of the glioma tumor class images, 494 are axial, 437 are coronal, and 495 are sagittal. There are 930 MR images of 62 patients in the pituitary tumor class. Of the pituitary tumor class images, 291 are axial, 319 are coronal, and 320 are sagittal. The dataset consists of 3064 T1-weighted contrast-enhanced MR images of 233 patients . 3. The Proposed DenseNet201 and Grad-Cam-Based Brain Tumor Detection Model In this research, we have proposed a new explainable artificial intelligence (XAI) model for brain tumor classification. We have used two public datasets with three and four classes, respectively. Our model is a deep model, and we have used DenseNet201 for training. This model consists of 5 phases, and these phases are (i) training using DenseNet201, (ii) segmentation of tumor areas using Grad-Cam model and trained network, (iii) exemplar deep feature extraction using region of interest (ROI) and average pooling layer of the trained DenseNet201, (iv) feature selection using INCA and (v) classification with support vector machine (SVM) with 10-fold cross-validation. A graphical demonstration of the proposed XAI model is demonstrated in Figure 2. The general steps of the proposed model are given below. Step 1: Train dataset I using DenseNet201 since Dataset I has four classes. Step 2: Read each image from the datasets. Step 3: Apply Grad-Cam and obtain a score map. Step 4: Segment the ROI using a score map. Step 5: Resize ROI to 224 x 224. Step 6: Apply patch division to the obtained ROI-segmented image-in Step 5. Herein, the size of the patch is selected as 28 x 28. Step 7: Extract features from each patch using global average pooling of the trained DenseNet201 (in Step 1). Step 8: Merge the generated features. Step 9: Choose the most informative features by deploying the INCA selector. Step 10: Classify the selected/chosen features using an SVM classifier with a 10-fold CV. The hyperparameters of the used SVM have been optimized using Bayesian optimization. These ten steps have been defined in the proposed model. The steps per the phases have been given as follows. Step 1: Training, steps 2-8: patch-based deep feature extraction, Step 9: Feature selection, and Step 10: Classification. A detailed explanation of these phases is given below. 3.1. Training Dataset DenseNet201 is among the popular convolutional neural networks (CNN) in the literature. DenseNet CNN models generally consist of Dense blocks and transition layers between Dense blocks, apart from the input layer and prediction layers. The combining feature of dense blocks is formulated as given in equation 1, where x is the output feature maps, H is the layer, and i is the current number of layers. Because each layer is composed of dense blocks, the properties of all the layers that came before it is reused. Consequently, the problem of vanishing gradients is alleviated, and feature propagation is strengthened. In addition, the utilization of a limited number of filters brought about a reduction in the total number of parameters utilized by the model. Dense blocks consist of consecutive batch normalization, ReLU, and convolution operations. While the size of the feature maps remains constant within dense blocks, the number of filters varies between blocks (3 x 3-1 x 1). Between dense blocks, there are layers called transition layer, which includes 1 x 1 convolution and 2 x 2 average pooling operations. The growth rate is a hyperparameter that regulates how much information is added to the network at each layer. Concatenate is performed for this operation. DenseNet201 size is 80 MB. The number of parameters is approximately 20 million. The layer depth is 708. The image size used in the network login is 224 x 224 . (1) xi=Hi([x0,x1,...xi-1]) This work requires a pre-trained network for creating a deep feature engineering model. Therefore, DenseNet201 has created a pre-trained model using brain tumor images. The pre-trained networks have generally trained on ImageNet1k, but ImageNet1k is not related to MR images. Therefore, we used Dataset 1 to obtain a pre-trained network. The parameters used for training are given below (Table 1). The elapsed time for the training to finish is 476 min and 57 s. Accuracy and loss curves for training and validation are demonstrated in Figure 3. 3.2. Preprocessing The explainable artificial intelligence (XAI) statement supports not only the function of the algorithm in producing output but also in communicating to the user how the system obtains a certain result. Recently, Gradient Weighted Class Activation Mapping (Grad-CAM) has been used to provide visual explanation and interpretability of artificial intelligence predictions . The score map preprocess obtained with Grad-Cam is shown in Figure 4. Grad-CAM determines the difference between a differentiable output, such as a class score, and the convolutional features in the chosen layer . The neuron weights are found by adding the gradients over space and time. After that, these weights are put to use to combine the activation maps linearly and determine which features are most significant when it comes to producing a forecast. Assume you have a 2-D image classification network with output oc, indicating the class c score, and you wish to compute the Grad-CAM map for a convolutional layer with k feature mappings (channels), Bi,jk where i,j indices the pixels . (2) bkc=1Nijoc Bi,jk The neuron weight is where N represents the total number of pixels in the feature map. The Grad-CAM map is, after that, a weighted mixture of the feature maps with a ReLU:(3) M=RELU(kbkcBk) The ReLU activation ensures that you only receive features that provide value to the class of interest. As a result, the output is a heat map with the same dimensions as the feature map for the selected class. The Grad-CAM map is then upsampled to the size of the input data. In this phase, our main objective is to detect tumor areas from MR images. We have used Grad-Cam to segment ROI. In the first step of this phase, we read each image from Dataset 1 and Dataset 2. The pre-trained DenseNet201 with Dataset 1 and Grad-Cam has generated a score map (hot map). By using hot areas, the ROI has been obtained. Examples of ROI and hot map images obtained with Grad-Cam are shown in Figure 5. 3.3. Feature Extraction A deep feature extraction model has been presented in this work. Patch-based deep feature extraction has been used to provide a high classification ability of the patch-based model in our model. To create a feature engineering model, we need a feature extraction function. We have used a deep feature extraction function, and this deep feature extraction function is the created pretrained DenseNet201. We have used the global average pooling layer to extract features; by using this layer, 1920 features have been extracted from a patch. The steps of our proposed feature extraction model have been listed below. 1: Resize ROIs to 224 x 224 sized images. 2: Apply the fixed-size patch division and obtain 64 patches from an ROI. Herein, 28 x 28 sized patches have been used. We tested patches with variable sizes, and the best accurate patch size is obtained as 28 x 28. 3: Extract deep features from ROI. This feature vector is the first feature vector with a length of 1920. 4: Extract deep features from each patch. By using patched, 64 more feature vectors with lengths of 1920 have been generated. 5: Merge 65 feature vectors generated to create the final feature vector with a length of 124,800 (=1920 x 65). 3.4. Feature Selection The k-NN technique was the foundation for developing a non-parametric and embedded method known as the Neighboring Component Analysis (NCA). The NCA algorithm's primary objective is to learn the feature weighing vector by optimizing the classification accuracy with an optimum editing parameter. This is accomplished through the learning process. NCA provides information on essential qualities in addition to ranking those features is one of the advantages of using NCA . NCA is a particularly useful feature selection model among the many different feature selection methods. However, it cannot calculate the optimum number of features to include. Because of this, a variant of neighborhood component analysis (NCA) called iterative neighborhood component analysis (INCA) was utilized. This is the form of NCA that can count the number of features. INCA can perform an iterative feature selection procedure in conjunction with an error calculator, which enables the automatic selection of the optimum number of features. To attain the best possible degree of accuracy in classification, these feature generation and selection algorithms aim first to generate functional characteristics and then pick the most distinguishable ones. After analyzing the accumulated loss data, the smallest error values and most advantageous characteristics are chosen. An interval for iteration has been set to cut down on the amount of time required by INCA, an iterative selector with high temporal complexity. INCA was utilized as the error calculator in this investigation, with the lower and higher bounds of the classifier and iteration set to 100 and 900, respectively. kNN had the following properties: k-value: 1, distance metric: Euclidean, voting: None, and k-fold cross-validation: 10. 3.5. Classification Bayesian optimization is used in this study to adjust the parameters of the SVM classifier. Bayesian optimization (BO) is a sequential experiment design method for the global optimization of functions with unknown input-to-output relationships. BO uses a sequential optimization process that iteratively decides which new data points to evaluate based on the given inputs and updates the model of the optimized objective function. BO can find the optimum value with fewer experiments compared to conventional experimental design strategies. For this reason, it is often used to optimize functions that are expensive to evaluate . Considering the cost and time of experiments with these parameters, it is important to reach the optimum formulation with fewer experiments. The goal function for Bayesian optimization was developed by employing the training dataset as well as the validation dataset as inputs. The validation dataset's classification error is the value that is returned by the objective function after a convolutional neural network has been trained. Because Bayesian optimization chooses the optimum model based on the error rate in the validation dataset, over-learning likely occurred in the final mesh when applied to the validation dataset. To circumvent this problem, the final model under consideration is put through its paces on an independent test dataset to determine the generalization error. The objective function performs the following steps:- As input for optimization, variable class values are used. The objective function is defined in the objective function with the valid values of the optimization variables in a table where each column name matches the variable name. - Training options for network architecture and optimization are defined. - The network is trained and validated. - The trained network is saved with training options on validation error and optimization. Within the scope of this research, we implemented the INCA, SVM, and bayesian optimization methodologies to develop a novel feature engineering model. We achieved good classification performance by utilizing a shallow SVM classifier in conjunction with our feature engineering model. The hyperparameters of the fine-tuned SVM obtained with 100 iterations for a dataset I are shown in Figure 6. The hyperparameters of the fine-tuned SVM obtained with 100 iterations for Dataset II are shown in Figure 7. The hyperparameters used for datasets I and II are tabulated in Table 2. Best point hyperparameters for datasets I and II were calculated using bayesian optimization. Best point hyperparameters had 98.6% accuracy with SVM for Dataset I and 100% accuracy with SVM for Dataset II. 4. Experimental Results The proposed XAI-based brain tumor classification model is programmed using MATLAB2021 on a personal computer with 64 GB of memory, an Intel i9-11900 processor, and a Windows 10 operating system without running parallel operations or needing graphics or tensor processing units. Standard performance metrics used include F1-score, specificity, precision, accuracy, and recall. Different options were tested to decide which split: ratio or k-fold CV to use. First, classification is completed using SVM for datasets I and II from 2-fold CV to 10-fold CV. Obtained results are shown in Figure 8. In addition, datasets I and II were classified using SVM using six different split: ratio ratios In the literature, studies were carried out using different split ratio ratios . The accuracy values obtained with the split ratio ratios of 10:90, 20:80, 25:75, 30:70, 40:60, and 50:50 are shown in Figure 9. As a result, CV for Dataset I and dataset II has been chosen in this study because it gives the highest accuracy value. 124,800 features were obtained from DenseNet201 with the help of 28 x 28 patches. These features are selected with the help of INCA. With INCA, 231 features were selected for Dataset I, and 220 features were selected for Dataset II. The selected features were classified by running 100 times with SVM 10 fold CV. Confusion matrices of the highest accuracy achieved are shown in Figure 10. Table 3 shows the accuracy values of different patch sizes and classifiers with 10-fold CV. The SVM classifier was chosen because it was more successful than the KNN, tree, and ANN classifiers. In addition, the highest accuracy values were obtained with the patch size 28 x 28 SVM classifier and 10-fold CV. In the proposed method, the features obtained with INCA were classified by running 100 times with SVM 10-fold CV. TP, TN, FN, and FP values were obtained from the confusion matrix obtained. Accuracy, precision, recall, and F1-Score values were calculated with the results obtained 100 times. Calculated values (mean +- sd) are tabulated in Table 4. In addition, some misclassified images for dataset I are shown in Figure 11. 5. Discussion Detection or classification of brain tumors is crucial in saving patients' lives. Therefore, in this study, we presented the INCA feature selective-based SVM classification model using brain MR images. This model is a CNN-based decision support system with explainable artificial intelligence (XAI). Two datasets with four classes and three classes were used. Dataset I DenseNet201 with four classes was trained with a CNN network. This trained network was used to segment the MR images using Grand-Cam datasets and obtain features. The 124,800 features obtained were selected with INCA. Finally, an accuracy of 98.65% for Dataset I and 99.97% for Dataset II were obtained using SVM classifier with 10-fold cross-validation. The summary of comparison with the state-of-the-art techniques is shown in Table 5. As can be seen in Table 5, brain tumor detection datasets with three and four classes were used. The proposed method was applied for the Kaggle brain tumor dataset I, which consists of four classes with 3264 images, and provided 98.65% accuracy. In addition, the proposed method was applied to the figshare brain tumor dataset I, which consists of three classes with 3064 images, and provided 99.97% accuracy. Datasets I and II, widely used in the literature, were used to compare the success of the proposed method. Basaran et al. used a traditional machine learning which is a time-consuming and tedious task. Authors in used CNN architectures and shallower mesh. In , a 5-fold CV was used, and the results obtained with their method were lower than the proposed model. Different split: ratio ratios were used in . The results obtained are lower than the results of our proposed model. Although 10-fold CV was used as in the method in , the accuracy obtained with Inception-v3 was lower. 6. Conclusions In this study, we aimed to classify brain tumors using XAI. DenseNet201 architecture. End-to-end training was performed using the DenseNet201 architecture. This trained architecture was used to segment the MR images using Grand-Cam datasets and obtain features. The segmented images were resized to 224 x 224. Then the images were divided into 28 x 28 patches. The attributes of the split patches were extracted using DenseNet201 trained with Dataset I. Extracted features were selected with the INCA feature selector and classified using SVM with a 10-fold CV. The proposed method obtained 98.65% accuracy for Dataset I and 99.97% for Dataset II. To support the physicians in diagnosing, XAI has been employed to discover unknown information from medical images. The solution we developed based on datasets labeled by publicly available experts provided positive results regarding the brain tumor classification targeted in this study and outperformed existing methods in delivering superior results. In the future, we plan to conduct a multidisciplinary study with specialist doctors and make comparative studies using SHAP, LIME, and GradCam tools on new data sets. In addition, XAI can be integrated into different diagnostic applications (Eye, Skin lesions, other cancer types, etc.). Institutional Review Board Statement Not applicable. Informed Consent Statement Informed consent was obtained from all subjects involved in the study. Data Availability Statement In this paper, the dataset is publicly available. Conflicts of Interest The author declares no conflict of interest. Figure 1 Sample images used in the two datasets. Dataset I: Kaggle dataset and this dataset have been used for training, Dataset II: Figshare dataset. Figure 2 Graphical representation of the presented XAI model. Figure 3 Training and validation curves of the DenseNet201 for Dataset I. Figure 4 Illustration of preprocessing. Figure 5 Examples of ROI and hot map images. Figure 6 Graph of classification errors versus iterations obtained using SVM for dataset 1. Figure 7 Graph of classification errors versus iterations obtained using SVM for dataset 2. Figure 8 Accuracy graph obtained using SVM classifier for various k-fold CVs. Figure 9 Accuracy graph obtained using SVM for various split ratios. Figure 10 Confusion matrices obtained using SVM with 10-fold CV. Legend: (a) Dataset I 1 = Glioma Tumor, 2 = Meningioma Tumor, 3 = No Tumor, 4 = Pituitary Tumor, CV (b) Dataset II Legend 1 = Meningioma Tumor, 2 = Glioma Tumor, 3 = Pituitary Tumor. Figure 11 Few misclassified images using Densenet201. diagnostics-13-00859-t001_Table 1 Table 1 Training Parameters. Solver Basic Sequence Advanced Solver sgdm Validation Frequency 1 Sequence Length Longest L2 Regularization 0.0001 Initial Learn Rate 0.01 Max Epochs 20 Sequence Padding Value 0 Gradient Threshold Method I2norm Iteration 170 Mini Batch Size 128 Sequence PaddingDirection right Shuffle every Epoch diagnostics-13-00859-t002_Table 2 Table 2 Summary of hyperparameters used for datasets I and II. Hyperparameters Dataset I Dataset II Multiclass method One-vs-All One-vs-One Box Constraint level 616.6747 987.8273 Kernel Scale 2.2347 - Kernel Function Gaussian Linear Standardize data False False diagnostics-13-00859-t003_Table 3 Table 3 Classification performance obtained for the proposed method using various classifiers. Datasets Patch Size SVM (%) Cubic KNN (%) Fine Tree (%) ANN (%) Dataset I 28 x 28 98.65 96.30 91.30 97.60 56 x 56 97.60 96.00 86.30 96.00 112 x 112 96.70 95.40 85.90 95.60 Dataset II 28 x 28 99.97 96.10 99.93 98.50 56 x 56 99.93 95.00 95.60 96.50 112 x 112 96.50 95.70 85.30 95.60 diagnostics-13-00859-t004_Table 4 Table 4 Classification metric calculations based on proposed feature extraction and INCA feature selection (%). Datasets Accuracy (% +- sd) Precision (% +- sd) Recall (% +- sd) F1-Score (% +- sd) Dataset I Glioma Tumor 98.42 +- 0.11 97.99 +- 0.17 97.59 +- 0.19 98.40 +- 0.26 Dataset I Meningioma Tumor 97.63 +- 0.18 97.99 +- 0.23 97.28 +- 0.23 Dataset I No Tumor 99.08 +- 0.12 99.06 +- 0.22 99.10 +- 0.12 Dataset I Pituitary Tumor 99.30 +- 0.09 99.36 +- 0.16 99.24 +- 0.11 Dataset II Meningioma Tumor 99.96 +- 0.01 100 +- 0.00 100 +- 0.00 100 +- 0.00 Dataset II Glioma Tumor 99.92 +- 0.02 100 +- 0.00 99.96 +- 0.01 Dataset II Pituitary Tumor 100 +- 0.00 99.98 +- 0.03 99.94 +- 0.02 diagnostics-13-00859-t005_Table 5 Table 5 Comparison of results with the state-of-the-art techniques. Ref. (Year) Dataset Model Split: Ratio Results (%) Alanazi et al. (2022) Dataset I Dataset II 22 Layer CNN 80:20 Dataset I Accuracy = 95.75 Precision = 95.15 Sensitivity = 96.32 Dataset II Accuracy = 96.90 Basaran et al. (2022) Dataset I Gray level co-occurrence matrix (GLCM), Local Binary Pattern (LBP), AlexNet, VG16, EfficientNet, ResNet, PSO, GA, ABC, SVM 5 Fold CV Accuracy = 98.22 Precision = 97.84 Sensitivity = 98.27 Specificity = 99.43 F1 Score = 98.04 Belciug et al. (2022) Dataset I DE/CNN Train:2870 image Test:394 image Accuracy = 90.04 Amou et al. (2022) Dataset II Modified VGG16 CNN 90:10 Accuracy = 98.70 Precision = 98.30 Sensitivity = 98.60 F1 Score = 98.60 Asthana et al. (2022) Dataset II Hanman-Renyi transform, HSTC Classifier 5 Fold CV Accuracy = 98.91 Sensitivity = 98.92 Specificity = 99.47 Rasool et al. (2022) Dataset II Google-Net + SVM 80:20 Accuracy = 98.10 Precision = 98.20 Recall = 98.10 Kumar et al. (2021) Dataset II ResNet-50 CNN with global average pooling added 5 Fold CV Accuracy = 97.08 Precision = 98.3 Sensitivity = 98.6 F1 Score = 98.6 Diaz-Pernaz et al. (2021) Dataset II Multi-pathway CNN 5 Fold CV Accuracy = 97.30 Noreen et al. (2021) Dataset II Transfer learning with Inception-v3 Ensemble2 model 10 Fold CV Accuracy = 93.79 Kakarla et al. (2021) Dataset II Average pooling convolutional neural network (AP-CNN) model 5 Fold CV Accuracy = 97.42 Precision = 97.41 Recall = 97.42 Alhassan et al. (2021) Dataset II Hard swish-based RELU activation function-CNN 80:20 Accuracy = 98.60 Precision = 99.60 Recall = 98.60 F1 Score = 99.00 Gab et al. (2021) Dataset II PGGAN-augmentation VGG19 + Bi-GRU Training:70 Test:15 Validation:15 Accuracy = 98.54 Precision = 97.69 Sensitivity = 97.69 Specificity = 98.93 F1 Score = 97.69 Gu et al.(2021) Dataset II Custom CNN model (Convolutional dictionary learning with local constraint (CDLLC) 5 Fold CV Accuracy = 96.39 Precision = 94.61 Sensitivity = 94.64 F1 Score = 94.70 Noreen et al.(2020) Dataset II Transfer learning with Inception-v3, Densenet201 80:20 Inception-v3 Accuracy = 99.34 Densenet201 Accuracy = 99.51 Ghassemi et al.(2020) Dataset II DCGAN CNN-GAN 5 Fold CV Accuracy = 95.60 Precision = 95.29 Sensitivity = 94.91 Specificity = 97.69 F1 Score = 95.10 Ismael et al.(2020) Dataset II ResNet50 CNN model 80:20 Accuracy = 99.00 Precision = 99.00 Recall = 99.00 F1 Score = 99.00 Proposed Model Dataset I Dataset II Exemplar, DenseNet201, GradCam,SVM 10 Fold CV Dataset I Accuracy = 98.65 Pituitary Precision = 99.34 Recall = 99.56 F1 Score = 99.45 Dataset II Accuracy = 99.97 Meningioma Precision = 100 Recall = 100 F1 Score = 100 Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). 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PMC10000759 | Healthcare (Basel) Healthcare (Basel) healthcare Healthcare 2227-9032 MDPI 10.3390/healthcare11050631 healthcare-11-00631 Case Report Management of Bilateral Quadriceps Tendon Ruptures Post Total Knee Arthroplasty by Kesler Technique Using Fiber Tape AlShaafi Waleed Ahmad 1 Alqahtani Mohammed Hassan 2 Assiri Abdullah Hassan 2 Alqhtani Abdulrhman Abdullah 2 Tedla Jaya Shanker 3* Motlag Dhuha Saeed 2 van Zundert Andre Academic Editor 1 Consultant Orthopedics and Arthroplasty, Saudi German Hospital-Aseer, Abha 61411, Saudi Arabia 2 Department of Orthopedics, Aseer Central Hospital, Abha 61411, Saudi Arabia 3 Department of Medical Rehabilitation Science, College of Applied Medical Sciences, King Khalid University, Abha 61421, Saudi Arabia * Correspondence: [email protected]; Tel.: +966-538901083 21 2 2023 3 2023 11 5 63121 12 2022 16 2 2023 19 2 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). Total knee arthroplasty is an effective way to manage osteoarthritis patients surgically. However, patients may encounter post-surgical complications, such as quadriceps rupture in rare instances, in addition to surgical complications. In our clinical practice, we encountered a 67-year-old Saudi male patient with a rare bilateral quadriceps rupture two weeks post-total knee arthroplasty. The cause of the bilateral rupture was a history of falls in both knees. The patient was reported to our clinic with clinical features like pain in the knee joint, immobility, and bilateral swelling in the knees. The X-ray did not show any periprosthetic fracture, but an ultrasound of the anterior thigh revealed a complete cut of the quadriceps tendon on both sides. The repair of the bilateral quadriceps tendon was done by direct repair using the Kessler technique and was reinforced with fiber tape. Following knee immobilization for six weeks, the patient began intensive physical therapy management to decrease pain, enhance muscle strength, and increase range of motion. After rehabilitation, the patient regained a complete range of motion in the knee and improved functionality, and he could walk independently without crutches. total knee arthroplasty quadriceps tendon ruptures surgery graft Kesler technique fiber tape King Khalid UniversityR.G.P. 1/130/43 This research was funded by the Deanship of Scientific Research, King Khalid University, Abha, Kingdom of Saudi Arabia, with grant number R.G.P. 1/130/43. pmc1. Introduction Osteoarthritis is the most common degenerative disease-causing articular dysfunction; the accompanying pain and limited range of motion can eventually lead to decreased functional capacity and reduced quality of life . Osteoarthritis is one of the most challenging disorders for the orthopedic rehabilitation team. Due to the lack of any disease-modifying medications, severe pain, limitation in the range of motion, difficulty in walking, and inability to perform day-to-day activities, most patients will ultimately undergo surgery . One of the most effective surgical options for patients with severe knee osteoarthritis is total knee arthroplasty . Postoperatively, after total knee arthroplasty, there are some common complications, such as infection, venous thromboembolism, the persistence of pain, and mortality . However, rupture of the bilateral quadriceps post total knee arthroplasty is a rare complication, but it leads to serious functional limitations . The knee's extensor mechanism involving the quadriceps muscle is crucial for locomotion. The four muscles of the quadriceps (namely, the vastus medialis, vastus lateralis, vastus intermedius, and rectus femoris), tendon of the quadriceps, patella, patellar tendon attachment to tibia, and retinaculum are the critical components of knee extension . A concurrent, unexpected quadriceps tendon rupture on both sides after total knee arthroplasty is an unlikely orthopedic injury . A disturbance in the extensor mechanism is a severe consequence of total knee arthroplasty, with a reported incidence of 0.17 to 2.5 percent . A disruption of the quadriceps tendon is commonly related to the failure of surgical technique or patient-related factors . A dysfunction in the extensor mechanism post-total knee arthroplasty may be caused by numerous factors ranging from medically induced reasons to post-traumatic disturbance. Such dysfunction in the knee extensor mechanism ranges across acute, subacute, and chronic stages . Several surgical options have been proposed for quadriceps repair. The gap between the incised quadriceps muscle, the patient's general demographic characteristics, the duration of injury, and tissue pathophysiology all help to inform the type of surgery required. In some cases, direct repair by getting the two ends of the muscle together with sutures will be sufficient, whereas in other cases there may be a need to use an external material other than the same muscle; among alternative materials, the most common are allografts where the muscle graft is obtained from another human, an autograft where the graft is obtained from other muscles in the same human, and synthetic mesh, which is an artificial material used to reinforce the surgical repair . In this case study, we describe a case presented in our clinical setting two weeks after total knee arthroplasty; we saw a 67-year-old Saudi man with a rare bilateral quadriceps rupture. The bilateral rupture was provoked by a history of falling on both knees. The bilateral quadriceps tendons were repaired directly using the Kessler approach and strengthened with fiber tape. In the Kessler repair, a knot is used to secure the suture to the tendon at each of its four corners. This knot stops the suture from slipping inside the tendon material . The Kessler suture procedure is simple and time efficient, which is an essential feature during surgery . The Kessler approach using fiber wire suture significantly enhances the mechanical qualities of intra-synovial tendon repair and lowers the possibility of postoperative gapping and rupture . The objective of our case study is to provide more details regarding the case in terms of the patient's mechanism of injury, area of the damage in the muscle, diagnosis procedures used, surgical procedure, and the patient's recovery journey after rehabilitation. 2. Case Description 2.1. Pre-Intervention Phase A 67-year-old Saudi male patient visited our outpatient department with knee joint pain on both sides and a diagnosis of bilateral knee osteoarthritis. His preoperative examinations were standard except for a deficiency in vitamin D, which was corrected by oral supplement. The patient was a known diabetic and hypertensive but had no history of steroid use or diagnosis of rheumatoid arthritis. His bilateral knee joint X-rays revealed the severity of the disease and osteoarthritic changes, as shown in Figure 1. Subsequently, the patient underwent total bilateral knee arthroplasty by the standard midline approach on the para patellar region by Zimmer, USA, without lateral retinacular release. The patient's surgery was successful; his postoperative knee joint X-rays can be seen in Figure 2. The patient revisited our clinic two weeks after the surgery with pain in both knees and the inability to walk following a fall on his operated knees. A physical assessment showed swelling on both knees, no gross joint deformity, a bilateral supra-lateral gap, and tenderness. Although a clinical examination of the knee joint failed to show the extensor mechanism, it was distrusted by a positive leg-raising test that was conducted bilaterally, as shown in Figure 3. Radiological screening with a bilateral knee X-ray showed no periprosthetic fracture, as seen in Figure 4. A lateral view of the knee joint X-rays also revealed no acute fracture but showed a lower positioning of the patella, known as patella baja. An ultrasound report of the quadriceps disclosed a complete bilateral rupture of the quadriceps tendon . On the ultrasound, the gap between two ruptured segments of the quadriceps was less; however, intraoperatively, when we observed the gap between the segments, it was larger, with a 1.4 cm defect in the right quadriceps and a 2.4 cm defect on the left side. 2.2. Intervention Phase The subject underwent surgery to repair the disrupted quadriceps. Anesthesia was administered using the spinal route, and we started surgery with prophylactic antibiotic medications and applied a tourniquet bilaterally. Using the sterile technique of prepping and draping at the surgical site, we identified the previous midline incision site and extended the incision proximal to the previous one in order to identify the quadriceps rupture. During the procedure, the surgeons observed a mid-substance complete tear of the right quadriceps and a distance complete tear of the left quadriceps, as shown in Figure 6. However, the left quadriceps tear was distal compared with the right side. Commonly in these situations, we use Ethibond for suturing and repairing purposes; however, upon considering the patient's characteristics, such as age, weight, and muscle weakness, we intended to secure it better. Hence, the surgeons decided to use an innovative method where the primary direct repair of the bilateral quadriceps tendon was done utilizing the Kessler technique using fiber tape (Braided Polyblend Suture) provided by Arthrex(r). The intraoperative post-suture quadriceps muscle can be seen in Figure 7. 2.3. Post-Intervention Phase and the Results The patient was on a knee immobilizer for six weeks, and during immobilization, isometric exercises for quadriceps muscle were encouraged. Post immobilization, the patient came to the outpatient department for follow-up and was referred for regular physical therapy sessions. After six weeks, he began pain management using transcutaneous electrical stimulation. In addition, muscle strengthening was done using a quadriceps table in the therapy sessions and using weight cuffs at home in the available ranges. The muscle strength was measured using a Baseline Hydraulic Push-Pull Dynamometer of 250 pounds with a dial (analog) gauge (Model: FEI-12-0394, Fabrication Enterprises Inc., USA). The maximum resistance that the patient could exert was assessed; this resistance measured in pounds is called a one-repetition maximum. The forty percent load of this one repetition maximum was used as the training weight initially. In our case, in the sixth week, the patient could exert around eight pounds as the one repetition maximum, and his training weight was approximately three pounds. This training intensity was progressively increased in the later stages by increasing the weight and repetitions. More details of the load percentage progression, number of repetitions per set, number of sets per session, and number of sessions per week are described in Table 1. Gradual range of motion exercises were performed with knee flexion 0-30, 0-60, 0-90, and 0-120 degrees, followed by full flexion. Along with these open kinematic strengthening exercises, we performed close kinematic eccentric muscle strengthening by quadriceps lunge exercises. Finally, the patient obtained a good range of motion three months post-repair, with excellent results in function. His straight leg-raising test can be seen in Figure 8 for both legs (Video S1). Before sending this research study for publication, the authors obtained written informed consent from the patient, and his identification and medical details were maintained confidentially. 3. Discussion Quadriceps rupture following a complete knee replacement is an uncommon but catastrophic impediment that can develop in any patient . The quadriceps muscle is an essential muscle for maintaining a standing posture. When a complete rupture happens bilaterally, it is devastating for patients because it is so painful that patients cannot sit, stand, walk, reach, or bend. Unless the damage is repaired bilaterally, the subjects cannot do their activities of daily living and participate in any of their life-related skills. After our study's surgical repair of the quadriceps tendon, we obtained an excellent outcome with an improved range of motion and function after six months. Many individuals with complete extension mechanism disruption have required surgical intervention, with varied results . Upon examining the literature, we discovered that quadriceps tendon repair generally has a good outcome . Primary repair of acute quadriceps rupture at the tendon level is understood as repair with sufficient tissue within two weeks of injury. This type of repair has the best results and has only a two percent reoperation rate . The treatment for complete disruptions is determined by the damage's place, degree, and duration, the subjects' overall health and expectations, and the performing surgeon's proficiency and capability . Principle repair using cerclage wire or suture augmentation, followed by restriction of the knee's range of movement in full extension, is the most frequently described procedure . Ormaza et al. conducted a case series on managing chronic quadriceps rupture post-total knee arthroplasty using synthetic mesh. They had three subjects who had chronic partial unilateral quadriceps tears and underwent surgical repair using a suture reinforced with synthetic mesh after the failure of conservative management. The patients improved significantly after this procedure . In our case, the quadriceps injury was unique because it was an acute injury that happened within two weeks of total knee arthroplasty; it happened bilaterally. In addition, the primary direct repair was done by the unique Kessler technique of fiber tape suturing. Even though there are several surgical options available, like allograft, autograft, and synthetic mesh, for repairing quadriceps rupture, the surgeons in the current study had considered Kessler's technique because of the following reasons. First, the extent of the injury was less than 5 cm; if there is a more significant difference between two ends of the ruptured quadriceps muscle, then it is challenging to approximate them and perform the direct primary repair without the use of any external materials. Second, the injury was acute within two weeks after total knee replacement, it was bilateral, and the patient was old and already had a bilateral total knee replacement, hence the outlook for performing autograft or allograft and having a subsequent favorable level of recovery was very poor . Since there is no bone fragment (such as, for example, in patellar tendon ruptures), the use of Illizarov surgery is also not possible because the ruptured segments are soft tissues . Post-repair outcomes are linked to the period between injury and repair; patients with immediate primary repair are more likely to experience a favorable functional outcome, full range of motion, and strong quadriceps. We believe that in our case, the immediate direct repair using the Kessler technique with reinforced fiber tape, along with good rehabilitation and patient factors, made it possible for excellent improvements in the patient's recovery of function. Like all other case studies, we also have a similar limitation: the lack of generalizability of results. We advise that the effect of the same methodology and technique should be further investigated in the form of case series and experimental studies. We could not use structured assessments, such as scales or questionnaires, to identify this patient's functional and quality of life composite scores. Hence, future studies should consider incorporating more objective evaluations for assessing the function and participation of the patients. 4. Conclusions After three months of follow-up, we could see that the patient is walking independently and can raise their leg in the air. Currently, after two and half years of injury, the patient is walking independently and has had no re-injuries. There were no complications reported after this surgery. Hence, we assume that this rare condition, like bilateral quadriceps rupture post-total knee replacement, can be surgically repaired directly with the help of the Kessler technique using simple fiber tape without any grafts or mesh materials. Acknowledgments We sincerely thank the patient and his relatives for cooperating with us while conducting this research. Supplementary Materials The following supporting information can be downloaded at Video S1: Patient lifting right and left lower extremity after the surgery. Click here for additional data file. Author Contributions Conceptualization: W.A.A. and M.H.A.; methodology: A.H.A., A.A.A., J.S.T. and D.S.M.; data curation: W.A.A. and M.H.A.; writing--original draft preparation: J.S.T. and D.S.M.; writing--review and editing: A.H.A. and A.A.A.; funding acquisition: J.S.T. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Informed consent was obtained from the subject involved in the study. Data Availability Statement Data is available with the corresponding author mentioned in this research paper. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Patient's X-rays showing osteoarthritic knee changes. Figure 2 Showing postoperative knee joint in anterior-posterior and lateral views. Figure 3 The patient showing disruptive extensor mechanism by right positive leg-raising test. The patient is trying to lift the right leg; the thigh portion is lifting from the bed surface, but the heel is not lifting due to a complete rupture in the quadriceps muscle. Figure 4 The patient's standing bilateral knee X-ray did not reveal any periprosthetic fracture. Figure 5 Ultrasound report of the quadriceps tendon with complete bilateral rupture. R: Indicates right side; L: Indicates left side. Figure 6 Demonstrating quadriceps rupture intraoperatively. Figure 7 Representing the intra-operative repair of the quadriceps muscle. Figure 8 Showing the active hip knee extension in the left and right lower extremity. healthcare-11-00631-t001_Table 1 Table 1 Training details of the high sitting quadriceps and hamstrings resistance exercise. 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PMC10000760 | Front Plant Sci Front Plant Sci Front. Plant Sci. Frontiers in Plant Science 1664-462X Frontiers Media S.A. 10.3389/fpls.2023.1118383 Plant Science Review Tomato responses to salinity stress: From morphological traits to genetic changes Rosca Mihaela + Mihalache Gabriela + Stoleru Vasile * Department of Horticultural Technologies, Faculty of Horticulture, "Ion Ionescu de la Brad" Iasi University of Life Sciences, Iasi, Romania Edited by: Maria Celeste Pereira Dias, University of Coimbra, Portugal Reviewed by: Suriyan Cha-um, National Science and Technology Development Agency (NSTDA), Thailand; Jose Ramon Acosta Motos, Catholic University San Antonio of Murcia, Spain *Correspondence: Vasile Stoleru, [email protected] +These authors have contributed equally to this work and share first authorship This article was submitted to Plant Abiotic Stress, a section of the journal Frontiers in Plant Science 10 2 2023 2023 14 111838307 12 2022 26 1 2023 Copyright (c) 2023 Rosca, Mihalache and Stoleru 2023 Rosca, Mihalache and Stoleru This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Tomato is an essential annual crop providing human food worldwide. It is estimated that by the year 2050 more than 50% of the arable land will become saline and, in this respect, in recent years, researchers have focused their attention on studying how tomato plants behave under various saline conditions. Plenty of research papers are available regarding the effects of salinity on tomato plant growth and development, that provide information on the behavior of different cultivars under various salt concentrations, or experimental protocols analyzing various parameters. This review gives a synthetic insight of the recent scientific advances relevant into the effects of salinity on the morphological, physiological, biochemical, yield, fruit quality parameters, and on gene expression of tomato plants. Notably, the works that assessed the salinity effects on tomatoes were firstly identified in Scopus, PubMed, and Web of Science databases, followed by their sifter according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guideline and with an emphasis on their results. The assessment of the selected studies pointed out that salinity is one of the factors significantly affecting tomato growth in all stages of plant development. Therefore, more research to find solutions to increase the tolerance of tomato plants to salinity stress is needed. Furthermore, the findings reported in this review are helpful to select, and apply appropriate cropping practices to sustain tomato market demand in a scenario of increasing salinity in arable lands due to soil water deficit, use of low-quality water in farming and intensive agronomic practices. abiotic stress PRISMA salt stress screening of salinity effects tomato alleviation of salinity effects pmcIntroduction Tomatoes (Solanum lycopersicum L.) are widely consumed worldwide as fresh or processed food products (e.g. canned tomatoes, sauce, juice, ketchup, soup, etc.) (Campestrini et al., 2019; Li et al., 2021) ranking second in the top of the most consumed vegetables in the United States of America, after potatoes (Reimers and Keast, 2016). These fruits have a high content of nutrients and bioactive substances (De Sio et al., 2021; Ali et al., 2021a) that are beneficial for a healthy body, a healthy skin, and weight loss, and which may ameliorate or prevent various human chronic degenerative diseases (Ali et al., 2021a). Tomato fruits are rich in carotenoids (e.g. b-carotenoids and lycopene), ascorbic acid (vitamin C), tocopherol (vitamin E), and bioactive phenolic compounds such as quercetin, kaempferol, naringenin and lutein, caffeic, ferulic and chlorogenic acids (Dasgupta and Klein, 2014; Mihalache et al., 2020; Stoleru et al., 2020; Murariu et al., 2021). The carotenoids from tomatoes are known to display anticancer properties and to be excellent deactivators of reactive oxygen species (ROS) (e.g. for singlet oxygen (1O2) and peroxyl radical (ROO*)) (Campestrini et al., 2019; Stoleru et al., 2020). Lycopene, which is an antioxidant, might protect the cells against oxidative damage and prevent cardiovascular disease and various types of cancer (e.g. prostate, breast, lung, bladder, ovaries, colon, as well as pancreas cancer) (Dasgupta and Klein, 2014). Li et al. (2021) ascertained in their study that the consumption of tomatoes provides about 85% of the daily dose of lycopene required by the population of North America and 56-97% in five European countries. According to FAOSTAT database, in 2020 about 251,687,023 tonnes of tomatoes were harvested from 6,163,463 hectares worldwide, with a yield average of 40.84 tonnes/ha (FAOSTAT, 2022). In 2020, the European Community reported a production of 16,657,000 tonnes, of which 9,801,000 tonnes were processed and 6,856,000 tonnes were consumed fresh. Compared to the previous year, EU production increased by almost 1%. In the last 10 years, the average annual tomato production in the EU was 16,474,000 tonnes, with the lowest value recorded in 2012 and 2013 (15,082,000 tonnes) and the highest in 2016 (17,862,000 tonnes) (European Commission, 2021). Annually, a wide variety of factors can affect tomato yield and fruit nutritional quality (Inculet et al., 2019). Among these factors, the salt content in soil and water used in irrigation stands out. According to Shrivastava and Kumar (2015) "worldwide 20% of total cultivated and 33% of irrigated agricultural lands are afflicted by high salinity". Furthermore, by the year 2050 more than 50% of the arable land will probably become saline soils as a consequence of weathering of native rocks, irrigation with saline water, climate change projections predicting increasing drought events forcing farmers to make use of salty water, and intensive agronomic practices. The Food and Agriculture Organization of the United Nations ascertained that every year soil salinization takes 1.5 million ha of farmland out of production and annually decreases the production potential by up to 46 million ha per year. In sum, soil salinization has been causing annual losses in agricultural productivity estimated to be US $ 31 million (FAO, 2022). Tanji (2002) defined the salinity as "concentration of dissolved mineral salts present in soils (soil solution) and waters". In small amounts, the dissolved salts are vital for the normal plant growth and development, but at high levels, they become harmful and often cause the death of plants (Nebauer et al., 2013). Sodium chloride is the most common salt detected in salty soils and waters, along with the chloride, sulfate, and carbonate salts of calcium, magnesium, and sodium (Nebauer et al., 2013; Riaz et al., 2019). Soil and water salinization generally occurs naturally, but the human factor via land clearing and inappropriate irrigation practices emphasizes this phenomenon. The soil is generally considered salt-affected when its electrical conductivity (EC) is above 4 dS*m-1. The soil salinity can be also increased by rainwater, which according to Riaz et al. (2019) can contain even 650 mg*kg-1 NaCl. Salinity induces various deleterious effects on plants which are forced to react. Depending on the post-exposure phase, plant responses induced by salinity can be grouped into (Negrao et al., 2017; Isayenkov and Maathuis, 2019): (I) the ion-independent response which occurs in the first hours-days after exposure and is characterized by stomatal closure and inhibition of cell expansion mainly in the shoot, and general plant growth; (II) the ion-dependent response which takes place over days or even weeks and is characterized by the slowdown of the metabolic processes, premature senescence, and ultimately cell death. Plant adaptation to saline stress depends significantly on a multitude of physiological and molecular mechanisms which are classified into three main categories: osmotic tolerance, ion exclusion, and tissue tolerance (Munns and Tester, 2008; Roy et al., 2014; Isayenkov and Maathuis, 2019). Under salinity stress, the plants maintain their growth and development, by tolerating the water loss, preserving the leaf expansion and stomatal conductance (osmotic tolerance), avoiding the accumulation of Na+ ions in the shoots and leaves at toxic concentrations (by ion exclusion) and protecting the plant cells against the toxic action of Na+ through its removal from the cytosol and subsequent sequestration in vacuoles (tissue tolerance) (Munns and Tester, 2008; Hasegawa, 2013; Roy et al., 2014). A range of transporters and their controllers at both plasma membrane and tonoplast levels are involved in ion exclusion and tissue tolerance. The ways of plants react to salinity stress at molecular, cellular, metabolic, and physiological levels, as well as the mechanisms involved in salinity tolerance are far from being completely understood (Gupta and Huang, 2014; Maathuis, 2014). Under osmotic stress, the cell expansion in root tips and young leaves is immediately reduced and stomatal closure is induced. Plant tolerance to salt is mediated by various biochemical pathways that support water retention and/or acquisition, protection of chloroplast functions and the maintenance of ion homeostasis (Ludwiczak et al., 2021). Proline, glycine-betaine and soluble sugars are the main osmoprotectants synthesized by plants to balance the osmotic difference between the cell's surroundings and the cytosol and to protect the cell structure (Gupta and Huang, 2014; Sharma et al., 2019). According to Roy et al. (2014), the action of the tolerance mechanisms is highly dependent on the salinity level. For example, the Na+ exclusion is more effective in conditions of high salinity, while osmotic tolerance may be the most important tolerance mechanism at moderate salinity. In Figure 1 the possible adaptive responses of plants to salt stress is schematically shown (Horie et al., 2012; de Oliveira et al., 2013). Figure 1 Plant adaptive responses to salt stress. Plant exposure to salinity causes negative effects on their growth and development, even leading to their death. The first visible sign of salinity stress in plants is usually stunted growth, with plant leaves often colored in bluish-green (Zahra et al., 2020). Toxicity of Na+ occurs with time and after a great concentration increase of these ions in the older leaves which causes their premature death (Hasegawa, 2013). Salinity induces osmotic stress, excessive uptake of sodium and chloride ions (cytotoxicity), and nutritional imbalance, impairing the plant growth and development (Zahra et al., 2020; Ludwiczak et al., 2021). Plant exposure to saline stress also causes oxidative stress due to the generation of reactive oxygen species (ROS) (Isayenkov and Maathuis, 2019). High levels of salt cause physiological dysfunctions, affect photosynthesis, respiration, starch metabolism, and nitrogen fixation, and lead to reduced crop yield (Zahra et al., 2020). Salt accumulation inside the plant tissues above the tolerance limits leads to several negative changes in plant morphology, physiology, biochemistry and crop productivity. Salinity reduces water availability for plant use and due to unfavorable osmotic pressure, the roots are unable to absorb the water (Shrivastava and Kumar, 2015). According to Hasegawa (2013), Na+ causes the destabilization of membranes and proteins and negatively affects the fundamental cellular and physiological processes, mainly the division and expansion, primary and secondary metabolism, and mineral nutrient homeostasis. In addition, Na+ competes with K+ uptake causing K+ deficiency. The adverse effects of soil salinity on plants have been proven to be caused not only by Na+ cations but also by (Acosta-Motos et al., 2017). It has been reported in various studies that from having a toxic effect on plants, it also is a beneficial element for higher plants. As a micronutrient, the leaf osmotic potential and turgor, stimulates growth in plants by increasing the leaf area and biomass, and improves the photosynthetic performance of plants (Colmenero-Flores et al., 2019; Franco-Navarro et al., 2019; Wu and Li, 2019). Geilfus (2018) stated that 0.2-2 mg g-1 fresh weight of act in stabilizing the oxygen-evolving complex of photosystem II, maintaining the electrical potential in cell membranes, regulating tonoplast H+-ATPase and enzyme activities. Na+ cations are usually more toxic than chlorine anions in plants, but Wu and Li (2019) asserted that the salinity effects observed in soybeans and avocado were mainly due to . High concentrations of nitrogen or phosphorus deficiency, interfered with photosystem II (PSII) quantum yield and photosynthetic electron transport rate, and induced necrotic lesions, resulting in the symptom of leaf-tip burning and impairment of photosynthesis and growth (Teakle and Tyerman, 2010; Wu and Li, 2019). Due to both Na+ and , high levels of salt can induce a large number of negative effects on tomato plants: alteration of phenological development, replacement of nutrients with sodium and chloride ions, osmotic inhibition, photosynthetic reduction, nutrient deficiencies or imbalances, changes in gene expression and protein synthesis, and negative effects on crop productivity . Salinity affects almost all aspects of plant growth including germination, vegetative growth and reproductive development. Plants are generally more sensitive to salinity during germination and early growth, and excessive accumulation of sodium in cell can rapidly lead to osmotic stress and cell death (Shrivastava and Kumar, 2015). Figure 2 Salinity effects on tomato plants. According to Ibrahim (2018) and Zaki and Yokoi (2016), tomato is a moderately tolerant species to salinity, and seed germination, plant growth and fruit development are just affected by high salinity levels. The response to salinity depends mainly on the tomato genotype (Zaki and Yokoi, 2016) and it has been demonstrated that salt tolerance is controlled by several gene families (Ali et al., 2021a). Studies conducted so far have highlighted that the different levels of salts in soil or in the irrigation water can induce changes in plant morphology, physiology, and biochemistry, with particular consequences on yield and fruit quality. The knowledge of the salinity effects on tomato plants and fruits is an asset in the selection and application of the appropriate crop practices to fulfill tomato market demand. The assessment of the tomato responses to salinity stress is the main focus of this review, which was achieved through: (i) identification in Scopus, PubMed, and Web of Science databases of research works that assessed the effects induced by salinity on tomatoes, followed by (ii) their sifter according to PRISMA guideline and (iii) emphasis of the salinity effects on morphology, physiology, biochemistry, yield, fruit quality and gene expression of tomato plants induced by different levels of salts in water and soil. Bibliographic research and data collection The problem of plant salinity stress has attracted the attention of many researchers who have been focusing on this topic. The main research approaches refer both to the effects of salinity on plant growth and development and to the possible strategies to increase plant tolerance to salinity. In this study, only original scientific papers which were published in the last 10 years, in peer-reviewed journals, and underlying the individual salinity effects on morphology, physiology, biochemistry, yield, fruit quality, and gene expression of tomato plants induced by different levels of Na, K and Mg salts in water and soil were included. PRISMA guideline (Page et al., 2021) was used in this review to extract from Scopus, Web of Knowledge and PubMed databases the scientific papers focused on the assessment of the effects induced by salinity on tomato plants. The key expression "tomato salinity effects" was used to identify the scientific papers and the search returned 529, 751, and 178 articles in Scopus, Web of Science, and PubMed databases respectively, published in the last 10 years. According to the PRISMA flow diagram after repetitive publications removal, 964 scientific papers were considered in the screening step. Following a careful reading of titles and abstracts, 435 articles were identified as incompatible with the search topic. Subsequently, the full texts of the left papers were downloaded and assessed to identify the works eligible with the established criteria. After an extensive screening, 11 papers in another language than English, 23 articles without full text, 250 articles focused on the methods and practices that could increase the tomato tolerance to saline stress, and 99 items for other reasons (e.g., reviews, inadequate experimental criteria data, book chapters, conference papers, are not highlighted the salinity effects, etc.) were removed. Figure 3 Preferred Reporting Items for Systematic Reviews and Meta-Analyses PRISMA flow diagram for the targeted systematic review. Finally, only 146 original articles were eligible based on the inclusion criteria. The detailed analysis of these articles led to the following results : 14 articles focused on salinity's impact on seed germination; in 92 articles the plant/parts of the plant height, fresh/dry weight, leaf area, and/or flower/ branch number depending on salinity level in the soil or water were measured; in 87 articles the physiological parameters related to photosynthesis, osmosis, nutrients uptake, and water content in plant parts were evaluated; in 81 articles the biochemical activity of tomato plants under saline stress was assessed. The main parameters analyzed were enzymatic activity, proteins, sugars and other compound synthesis, hormonal levels, and/or molecular biology analyses. and only in 51 scientific papers, the impact of saline stress on yield and/or fruit quality was studied. Figure 4 The number of relevant articles (A) which underline the salinity impact on tomato morphology, physiology, biochemistry or/and yield and fruits and (B) published annually starting from 2012. Out of the 146 full articles assessed for eligibility, only 98 studies were included in the reference list, following the evaluation of the information reported by the proposed objectives. In the last 10 years, at least 12 articles focusing on the impact of salinity on tomato morphology, physiology, biochemistry, and yield have been published annually in Scopus, Web of Science, and PubMed databases, respectively . Morphological changes of tomato plants under salinity stress Salinity strongly influences all the aspects of a tomato plant's life, producing changes even in the morphological characteristics. In general, the morphology of a plant is a reflection of its environmental conditions, proving information about its metabolic function. Increases in salt content and in particular of sodium chloride in the growing environment can significantly affect the plant's physical appearance, but also the germination traits of tomato seeds. In the study conducted by Sholi (2012), it was reported that the increase of NaCl concentration in the 1/2 MS solidified medium delayed the seed germination of all four tomato cultivars: Jenin 1, Hebron, Ramallah and Maramand. The experiments were done in Petri dishes and incubated in the light at 23degC. The medium with the corresponding salt concentration was solidified with 8 g L-1 agar. At 0 mM NaCl the time required for germination of 50 % of 'Jenin 1' seeds was 2.45 days, but at 100 mM NaCl the same germination rate was reached in 8.51 days. At 150 mM NaCl the germination of 'Jenin 1', 'Hebron' and 'Maramand' cultivar seeds were completely inhibited. Similar results were obtained by Abdel-Farid et al. (2020), who observed that a salinity level of 50, 100 and 200 mM, NaCl reduced significantly the germination rate of tomato seeds, while at 100 and 200 mM NaCl the germination of tomato seeds was completely inhibited. The authors explained that the delay in seed germination may be due to the impairment of enzyme activity by the partially osmotic or ion toxicity. Gonzalez-Grande et al. (2020) found that 85 mM NaCl reduced the seed germination rate of tomato cultivar Rio Grande by 6.4% compared with the control (0 mM). At 171 and 257 mM NaCl the germination was severely affected, the rate being lower than 2.8%. The experiments were done in sterile Petri dishes on filter papers. Paradoxically, at 100 mM NaCl, Tanveer et al. (2020) reported a germination rate of 80% for tomato seeds. In the study of Adilu and Gebre (2021), a delay in seed germination with salinity increase was observed, the mean germination times (days) for the four selected tomato varieties (Sirinka, Weyno, ARP D2, and Roma VF) were 10.70, 8.72, 7.31, and 6.85 days respectively at 4 dS m-1 and 5.79, 5.69, 4.68, 5.09 days respectively at 0 dS m-1. According to Adilu and Gebre (2021) a low level of NaCl induces seed dormancy while a high level inhibits seed germination. Gonzalez-Grande et al. (2020); Abdel-Farid et al. (2020) and Adilu and Gebre (2021) explained that the reduction in germination rate and percentage under salt stress can be linked to a decrease in water potential gradient among seeds and their surrounding medium. Furthermore, the osmotic and toxic effects of NaCl affect the enzyme activation during seed germination and the gibberellin acid content. Regarding the salinity effects on plant morphology, changes can appear in all stages of plant development, affecting the plant height, root/shoot ratio, leaf area, number of branches, or the number of leaves/flowers per plant. The studies focusing on the salinity effects on tomato plants showed that the intensity of plant morphology changes depends on the salt level in the growing environment. In addition, each cultivar/hybrid responds differently to saline stress. Assimakopoulou et al. (2015) assessed the responses of three cultivars (Santorini Authentic, Santorini Kaisia and Chios) and four hybrids of cherry tomato (Cherelino F1, Scintilla F1, Delicassi F1, and Zucchero F1) at 0, 75 and 150 mM NaCl in a mix of loamy soil and perlite (3:1 v/v). The results of this study showed that cultivar Chios was the most affected at 150 mM and its total plant dry weight decreased by 65.37% and the root/upper plant part ratio in terms of fresh weight from 0.09 to 0.03. The total plant dry weight of the other cherry tomato cultivars was reduced by 52.52-56.52% at the highest salinity level compared to the lowest level. Assimakopoulou et al. (2015) stated that the growth inhibition was due to the toxicity of Na+ ions and to the nutritional imbalance induced by salinity. Samarah et al. (2021) assessed the tomato seedling growth in response to four saline water solutions of NaCl (0, 5, 10, and 15 dS m-1). The seedlings at 15 dS m-1 had a mean length of 3.8 cm and a dry weight of 9 mg, showing a longer length and weight at 0 dS m-1 (16.2 cm and 45 mg/seedling, respectively). The harmful effects of salinity on leaf area, leaf number, and leaf length also increase with the salt concentration rise, according to the studies performed by Babu et al. (2012); De Pascale et al. (2012); Hossain et al. (2012); Lovelli et al. (2012); Sanchez et al. (2012); Martinez et al. (2014); Al Hassan et al. (2015); Abouelsaad et al. (2016); Parvin et al. (2016); Chaichi et al. (2017); Rahman et al. (2018); Abdelaziz and Abdeldaym, (2019); Maeda et al. (2020). The cultivar Raf exposed at a salinity level of 5.5 dS m-1 had 2708 cm2 for the leaf area, but at 11 dS m-1 the leaves were smaller, and their leaf area decreased to 1815 cm2 (Sanchez et al., 2012). According to De Pascale et al. (2012), the saline water with an electrical conductivity of 4.4 dS m-1 used in tomato irrigation reduced the leaf number per plant from 82.6 at 48.9 and their leaf area with 47.55%, compared to the control. In their study, Babu et al. (2012) assessed the morphological changes induced by salinity on tomato cultivar PKM 1 based on leaf area, dry matter weight percentage, plant height and number of fruits per plant. Irrigation during 90 days with water containing NaCl at the concentrations of 0, 25, 50, 100,150, and 200 mM immediately after sowing caused negative changes in tomato plants. For example, it was found that the treatment with 200 mM NaCl reduced the plant leaf area by 43.91% and the fruit number per plant to 4 compared to 15 in the control. In addition, at this concentration, the plant height was 76.17 cm shorter compared to the control. In another study, irrigation with water having EC between 1.75 and 10.02 dS m-1 produced significant effects on specific leaf area, number of nodes per stem, fresh weight of roots/shoots/leaves, and length of primary roots/stem of the tomato cultivars Roma and Rio Grande (Prazeres et al., 2013). Increasing the NaCl concentration, in the irrigation water up to 3.22 dS m-1 led to an increase in the fresh weight of cultivar Roma leaves (by 84.7 g per plant), but at a higher NaCl concentration the leaf weight was reduced by 2.98-31.33 g. At 5.02 dS m-1 the leaf weight per plant was 157.80 g, with a non-significant reduction induced by salinity compared to the control whose leaf weight was 160.78 g per plant. In contrast, the fresh weight of the stems and roots decreased with the NaCl content increase in irrigation water. For cultivar Rio Grande the water EC higher than 1.75 dS m-1 had a positive effect on the fresh weight of roots, shoots and leaves, on specific leaf areas, number of nodes per stem and length of primary roots and stem (Prazeres et al., 2013). Several other studies have shown that the salt variation in the growing medium caused negative or positive changes in fresh biomass, plant height, root/shoot ratio, leaf areas, number of branches, and number of leaves/flowers per plant. In this respect, the results of some studies which assessed the morphological changes in tomato plants under salinity stress have been reported in Table 1 . Reducing plant height, leaf area, leaf number, and leaf length under salt stress conditions may be an adaptive morphological strategy to limit the water loss through transpiration. However, it could also be the result of the toxicity of Na+ and accumulated in cells, which slow the cell growth of young leaves (Negrao et al., 2017). Table 1 Morphological changes in tomato plants under salinity stress. Tomato cultivar Salinity level Plant height (cm) Dry weight (g) Fresh weight (g) Root/Shoot Leaf area/Leaf area index Reference unit value PKM 1 0-200 mM NaCl 161.88 \ 85.71 9.94 \ 2.79 cm2 18.24 \ 10.23 Babu et al. (2012) Tainan ASVEG No. 19 0, 50, 150 mM NaCl - 58.9 \ 23.0 278.4 \ 137 0.4 / 0.5 - - Liu et al. (2014) Hualien ASVEG No. 21 - 66.1 \ 40.4 345.7 \ 234.2 0.2 / 0.6 - - Taiwan Seed ASVEG No. 22 - 59.0 \ 38.0 290.3 \ 232.6 0.4 / 0.5 - - Bush Beef Steak 0, 6 dS m-1 NaCl 61 \ 60 38.5 \ 31.1 - 0.15 \ 0.14 - - Chaichi et al. (2017) CF Momotaro York 1.2, 6.0 dS m-1 NaCl 78.4 / 79 stems 99.6 \ 97.1 0.64 / 0.85 m2 m-2 1.8 \ 1.7 Maeda et al. (2020) Endeavour 128.9 / 131.2 stems 109.8 \ 96.1 0.53 / 0.83 2.2 \ 1.8 Tomato 0, 100 mM NaCl 47 \ 35 3.53 \ 1.31 13.3 \ 3.72 0.15 / 0.26 cm2 10.5 \ 5.2 Tanveer et al. (2020) Roma 1.75 - 3.22 dS m-1 - - 386.88 / 448.03 - - Prazeres et al. (2013) Rio Grande - - 333.80 / 351.88 - - Tomato 0, 2, 4, 6, 8 dS m-1 NaCl 88.50 / 90 (0-4 dS m-1) 90 \ 75.25 (4-8 dS m-1) 16.48 \ 6.375 (stem) - - cm2 187.2 \ 166.2 (Parvin et al., 2015) |, Increase in parameter value; |, decrease in parameter value. The same authors interestingly focused on tissue and cellular levels to assess the morphological alterations caused by salinity in tomato plants. In this respect, Bogoutdinova et al. (2016) investigated the cell organization of the epidermis and parenchyma cortical tissues of tomato hypocotyl under different levels of NaCl in vitro. The size of the intercellular spaces in the cortical parenchyma as well as the average cross-sectional areas and shape of epidermal and cortical parenchyma hypocotyl cells of tomato line YaLF and cultivar Rekordsmen were significantly affected by the addition of NaCl to the culture medium. At 250 mM NaCl, the highest increase in the cell areas of tomato line YaLF was observed and the epidermal cell became angular in contours. Physiological changes under salinity stress Plant physiological processes are very sensitive to all environmental changes. Variations in NaCl and other salt levels in soil or hydroponic cultivation have a strong impact on the physiology of plants. Depending on the stress duration and severity, changes that can occur in the physiological processes affect plant growth, development, and productivity. The studies done on tomatoes in the last 10 years highlighted a negative influence of salinity stress on the physiological parameters such as photosynthetic rate, transpiration, stomatal conductance, chlorophyll content and mineral uptake (Hossain et al., 2012; Lovelli et al., 2012; Giannakoula and Ilias, 2013; Maeda et al., 2020; Yang et al., 2021). For instance, Maeda et al. (2020) reported that the increase of Enshi nutrient solution EC from 1.2 to 6 dS m-1 caused the reduction of: photosynthetic rate by 10.2 % and 12.4 %, respectively, in tomato leaves of cultivars CF Momotaro York and Endeavour; transpiration rate and stomatal conductance by 26.9% and 23.4%, respectively, in the cultivar CF Momotaro York, and by 24.6% and 24.1%, respectively, in the cultivar Endeavour. At 6 dS m-1, the stomatal conductance of tomato leaves grown in silt loam soil was 0.03 mol m-2 s-1, i.e., 0.05 mol m-2 s-1 lower than in control (EC= 0 dS m-1 Na) (Parvin et al., 2016). Marsic et al. (2018) reported that the photosynthetic and transpiration rates as well as stomatal conductance were lower in the leaves of tomato cultivars Belle and Gardel raised in hydroponics with electrical conductivity of 6 dS m-1, compared to 2 dS m-1. The photosynthetic and transpiration rates and stomatal conductance of cultivar Belle leaves were lower by 44.1%, 52.9% and 90%, respectively, than the control, and by 40.3%, 48.6% and 91.3%, respectively compared to cultivar Gardel. According to Marsic et al. (2018), the decreased values of these parameters could be due to the stomatal closure induced by water deficit. Like the photosynthesis rate, the chlorophyll synthesis in tomato plant leaves can be negatively affected by the exposure to high salt levels (Giannakoula and Ilias, 2013; Taheri et al., 2020). This may happen due to metabolic disorders which result in decreased chloroplast activity and photosynthesis, increased chlorophyllase enzyme activity, and respiration, followed by reduced chlorophyll contents (Taheri et al., 2020). Singh et al. (2016) found in their study that the chlorophyll content in 'Lakshmi' tomato leaves was reduced from 0.996 mg g-1 to 0.751 mg g-1 when the NaCl level increased from 0 to 0.5 g kg-1 in soilless cultivation. The same trend was observed in chlorophyll b synthesis, whose content decreased by 27.73% compared to the control. In another study carried out on the tomato cultivar Super Chef grown in hydroponics, the total chlorophyll content decreased by 40.93% at 120 mM NaCl compared to the control (0 mM NaCl) (Taheri et al., 2020). The effects of salinity on photosynthesis processes in tomatowere evaluated in various studies by chlorophyll fluorescence. This type of analysis offers information on energy transfer in the photosynthetic apparatus and the related photosynthetic processes, mainly about the activity of photosystem II (PSII). PSII is a membrane protein complex whose active centers exist as dimers in the thylakoid membranes of grana stacks. It is known that PSII has the function to catalyze light-induced water oxidation in oxygenic photosynthesis and in this way light energy is converted into biologically useful chemical energy (Khorobrykh, 2019; Rantala et al., 2021). Shin et al. (2020) used chlorophyll fluorescence to assess the PSII activity in the leaves of cultivars 'Dafnis', 'Maxifort', 'BKO' and 'B-blocking' irrigated with saline water. At 400 mM (the maximum concentration of NaCl in saline water) the chlorophyll fluorescence decrease ratio (Rfd) was the parameter whose levels were most negatively affected, followed by the maximum quantum yield of PSII photochemistry (Fv/Fm). The chlorophyll fluorescence parameters, such as the coefficient of photochemical quenching of variable fluorescence based on the puddle model of PSII (qP) and coefficient of nonphotochemical quenching of variable fluorescence (qN) showed moderate negativechanges due to the salt level increase in irrigation water, whereas the quantum yield of nonregulated energy dissipation in PSII Y(NPQ) showed a significant increment at the higher salt concentration compared to control. Gong et al. (2013) reported that the values of Fv/Fm parameter and the actual quantum efficiency of photosynthetic system II (FPSII) in cv. 'Jinpeng No. 1' decreased with increasing levels of salt in the hydroponic media. For the non-photochemical quenching (NPQ) parameter was noticed that an increase in salt level led to an increase in its value, the highest being recorded at 100 mM. According to Zhao et al. (2019) the qP parameter measures the openness of PSII centers and reflects the conversion efficiency of the captured light quantum into chemical energy, while qN assesses the rate constant for heat loss from PSII. Fv/Fm parameters give information about the maximum light energy conversion efficiency of PSII after adaptation to darkness and NPQ reflects the level of excess energy dissipation as heat. Using the FPSII parameter of chlorophyll fluorescence is assessed the actual photochemical efficiency when the PSII reaction center is partly shut down under light. Thereby, as Tsai et al. (2019) and Zhao et al. (2019) stated, the changes observed in the chlorophyll fluorescence parameters under salt stress are the results of the membrane system stability disturbance (especially the damage of thylakoid membrane), the aggravation of the PSII reaction center and disturbances in PSII performance, which diminished the photosynthesis. More results on the changes induced by saline stress on photosynthetic rate, transpiration, stomatal conductance and chlorophyll content in tomato leaves have been included in Table 2 . Table 2 Photosynthetic rate, transpiration, stomatal conductance and chlorophyll content in tomato leaves under salinity stress. Tomato cultivar Growth system Salt Salinity level (mM) Photosynthetic rate (mmol CO2 m-2s-1) Transpiration (mmol H2O m-2s-1) Stomatal conductance (mol m-2s-1) Chlorophyll (mg/g) Reference BINATomato-5 Hydroponic cultivation with vermiculite and half-strength Hoagland's nutrient solution NaCl 0, 60, 120 mM 12.2 \ 4.9 0.27 \ 0.151 - Hossain et al. (2012) Tomato Hydroponic system with aerated Hoagland nutrient solution NaCl 0, 100, 150 mM 31.3 \ 24.1 10.6 \ 5.8 0.6 \ 0.2 - Lovelli et al. (2012) Tomato Sandy loam soil NaCl 0, 100, 150 mg/L 25.2 \ 5.90 3.7 \ 1.39 0.5 \ 0.04 12.8 \ 7.0 Giannakoula and Ilias (2013) Belladonna Hydroponic system with perlite and standard nutrient solution NaCl 0, 50 mM 25.70 / 27.28 - 0.55 \ 0.50 - Costan et al. (2020) Super Chef Hydroponic system with aerated half-strength Hoagland's nutrient solution NaCl 0-120 mM - 1.373 \ 0.811 1.373 \ 0.811 Taheri et al. (2020) Tomato Silt loam (sand 20.84 %, silt 57.46 % and clay 21.7 %) NaCl 0, 2, 4, 6, 8 dS m-1 - - 0.08 \ 0.02 - Parvin et al., (2015) Moneymaker Coconut coir fiber irrigated with Hoagland no. 2 solution NaCl 0, 75 mM 8.5 \ 6.1 - 0.41 \ 0.20 - Renau-Morata et al. (2017) Tomato Sandy loam soil NaCl 0.6, 2, 4, 6 dS m-1 11.10 \ 8.86 2.65 \ 2.26 0.114 \ 0.092 39.81 \ 37.74 Yang et al. (2020) CF Momotaro York Coconut fiber and rice husks in hydroponic system with Enshi nutrient solution NaCl 1.2, 6.0 dS m-1 14.7 \ 13.2 5.2 \ 3.8 0.47 \ 0.36 - Maeda et al. (2020) Endeavour 17.7 \ 15.5 5.7 \ 4.3 0.54 \ 0.41 - Belle Vermiculite and rockwool flocks in hydroponic system with standard nutrient solution NaCl 2, 4, 6 dS m-1 30.6 \ 17.1 25.7 \ 12.1 4.0 \ 0.4 - Marsic et al. (2018) Gardel 32.5 \ 19.4 17.6 \ 15.7 4.6 \ 0.4 - Ailsa Craig Hydroponic cultivation with half-strength Hoagland's nutrient solution NaCl 0, 125 mM 5.97 \ 2.74 - 0.51 \ 0.25 - Gharbi et al. (2017a) Lakshmi Sand NaCl 0, 0.3, 0.5 g/kg 27 \ 21 - - 0.996 \ 0.751 (Chl a) 0.256 \ 0.185 (Chl b) Singh et al. (2016) Tomato Perlite-vermiculite (1:3 v/v) with half-strength modified Hoagland nutrient solution NaCl 0, 40, 80, 160 mM - - - 0.645 \ 0.618 Martinez et al. (2014) |, Increase in parameter value; |, decrease in parameter value. Frequently, salinity increase can lead to a reduction in the essential minerals content such as calcium, potassium or magnesium and, consequently, to a nutritional imbalance. Calcium is one of the structural components of cell walls and membranes and serves as a second messenger in a variety of processes (Thor, 2019; Bang et al., 2021). By transduction, integration and incoming signals multiplication, the calcium links the environmental stimuli with the physiological responses of plants (Bang et al., 2021). Potassium ensures optimal plant growth, acts as an activator of dozens of important enzymes and enhances plant yield. For example, potassium plays an important role in protein synthesis, sugar transport, N (nitrogen) and C (carbon) metabolism, photosynthesis, cell osmotic pressure regulation and maintaining the balance between cations and anions in the cytoplasm (Xu et al., 2020). Magnesium in plant tissue is the central element of the tetrapyrrole ring of the chlorophyll molecule and, therefore, its deficiency leads to a chlorophyll synthesis decrease and to the impairment of normal plant growth and development. Magnesium also acts as an activator or cofactor of enzymes involved in carbohydrate metabolism (Guo et al., 2015; Bang et al., 2021). Therefore, a deficiency of these minerals in the plant tissues can cause negative effects on growth and development (Bang et al., 2021). In tomato plants, the essential mineral uptake in soil or hydroponic cultivation can be significantly affected by saline stress (Sanchez et al., 2012; Nebauer et al., 2013; Assimakopoulou et al., 2015; Javeed et al., 2021). The results of studies presented in Table 3 show that high salt levels in the growing culture can cause a lower uptake of calcium, potassium and sometimes of magnesium ions (Sanchez et al., 2012; Nebauer et al., 2013; Assimakopoulou et al., 2015; Parvin et al., 2016). Nebauer et al. (2013) reported in their study that regardless of the salt applied (NaCl, Na2SO4, MgCl2 or MgSO4), a level of 100 mM in soil reduced the Ca uptake by 48.75 to 71.26% in tomato cultivar Marmande RAF and by 12.28 to 38.60% in cultivar Daniela. Moreover, the amount of K in plants was lower by up to 68.05% at 100 mM MgSO4 in cv. Marmande RAF leaves and by up to 42.67% at 100 mM MgCl2 or 100 mM MgSO4 in cv. Daniela leaves. Decreases in the content of aforementioned minerals were also reported by Manan et al. (2016); Gharbi et al. (2017a); Rodriguez-Ortega et al. (2019) or Borbely et al. (2020). Therefore, it can be stated that salinity limits the assimilation of essential minerals in the tomato plant tissue and the physiological processes are adversely affected by these deficiencies. However, there are studies that showed that potassium, calcium and magnesium content in tomato leaves increased under salt stress (Costan et al., 2020; Javeed et al., 2021). For example, the content of calcium increased from 6.66 mg g-1 to 11.03 mg g-1 and of potassium from 36.68 mg g-1 to 71.51 mg g-1 in the fresh leaves of cultivar Rio Grande, grown in hydroponics with nutrients solution and seawater (5%, 10 % and 20%), and an EC of the growing media between 0.41 and 8.14 dS m-1 (Javeed et al., 2021). The high content of calcium and magnesium ions in tomato leaves under saline stress could be due to the higher uptake affinity for these ions rather than for Na+ or Cl- (Al-Ghumaiz et al., 2017). According to Al-Ghumaiz et al. (2017), the tolerant plants under salinity stress can exclude the Na+ ions from their shoots or blades while maintaining high levels of K+. Table 3 Mineral accumulation in tomato leaves under salinity stress. Tomato cultivar Growth system Salinity level Unit Mineral content Reference Na+ Mg2+ Ca2+ K+ Hydroponic system with perlite and nutrient solution 5.5 and 11 dSm-1 NaCl g/kg d.w. Leaves Sanchez et al. (2012) 2.71 / 8.17 3.23 / 3.58 24.80 \ 23.35 9.90 \ 7.98 13.49 / 28.82 2.98-2.98 10.95 \ 8.13 Stem 2.58 / 6.55 1.93 \ 1.81 6.96 \ 6.26 14.79 \ 12.41 15.27 / 21.58 3.32 / 3.42 1.83 \ 1.60 Marmande RAF Soil 25-100 mM NaCl mmol/g d.w. 0.20 / 0.158 0.33 \ 0.21 0.87 \ 0.45 0.72 \ 0.36 0.49 / 2.22 0.19 \ 0.16 0.37 \ 0.25 Nebauer et al. (2013) 25-100 mM Na2SO4 0.20 / 2.04 0.33 \ 0.25 0.87 \ 0.42 0.72 \ 0.43 0.49 / 0.44 0.19 \ 0.10 0.37 / 3.48 25-100 mM MgCl2 0.20 \ 0.12 0.33 / 1.56 0.87 \ 0.25 0.72 \ 0.31 0.49 / 3.85 0.19 \ 0.06 0.37 \ 0.16 25-100 mM MgSO4 0.20 \ 0.10 0.33 / 1.36 0.87 \ 0.37 0.72 \ 0.23 0.49 \ 0.44 0.19 \ 0.10 0.37 / 2.96 Daniela 25-100 mM NaCl 0.07 / 0.65 0.17-0.17 0.57 \ 0.50 0.60 \ 0.50 0.26 / 1.29 0.15 / 0.20 0.29 \ 0.16 25-100 mM Na2SO4 0.07 / 0.60 0.17 \ 0.15 0.57 \ 0.45 0.60 \ 0.42 0.26 / 0.68 0.15 / 0.17 0.29 / 0.86 25-100 mM MgCl2 0.07 \ 0.05 0.17 / 1.17 0.57 \ 0.47 0.60 \ 0.35 0.26 / 3.44 0.15 \ 0.10 0.29 \ 0.15 25-100 mM MgSO4 0.07-0.07 0.17 / 0.42 0.57 \ 0.35 0.60 \ 0.35 0.26 \ 0.24 0.15 / 0.20 0.29 / 1.01 *Cherelino loamy soil and perlite (3:1 v/v) and Hoagland No 2 nutrient solution 0, 75, 150 mM NaCl g/kg d.w. 5.40 / 38.10 11.00 \ 10.50 66.00 \ 53.70 25.10 \ 14.30 3.20 / 4.50 3.30 \ 2.30 - Assimakopoulou et al. (2015) *Scintilla 5.60 / 39.50 12.00 \ 9.80 68.10 \ 52.80 24.80 \ 18.20 2.70 / 5.80 1.70 / 1.90 *Delicassi 3.70 / 35.80 11.20 \ 10.10 59.10 \ 55.40 26.20 \ 22.10 2.00 / 5.10 2.20 / 2.80 *Zucchero 5.10 / 46.60 11.10 \ 8.90 59.20 \ 44.70 26.90 \ 14.50 2.30 / 4.30 2.40 / 2.70 *Chios 13.40 / 50.20 12.40 \ 9.40 64.30 \ 46.00 18.60 \ 7.90 2.30 / 5.70 2.80 \ 2.30 *Santorini Authentic 6.60 / 52.10 10.40 \ 8.40 53.60 \ 43.90 17.20 \ 11.40 2.40 / 6.70 2.60 \ 1.60 *Santorini Kaisia 6.70 / 48.50 12.30 \ 8.40 65.30 \ 43.20 15.80 \ 13.60 2.10 / 6.60 1.50 / 1.80 Tomato silt loam 0, 2, 4, 6, 8 dSm-1 % d.w. 0.24 / 0.61 - 0.65 \ 0.48 1.04 \ 0.75 - - - Parvin et al. (2016) Rio grande Hydroponic system with aerated Hoagland solution 0.41, 2.91, 5.96, 8.14 dS m-1 seawater mg/g f.w. 11.9 / 56.15 9.85 \ 1.35 6.66 / 11.03 36.68 / 71.51 8.59 / 123.47 - Javeed et al. (2021) Belladonna Hydroponic system with perlite and standard nutrient solution 0, 50 mmol/L g/kg d.w. 1.51 / 8.69 3.53 / 4.07 16.20 / 19.86 33.64 \ 24.78 - 3.90 / 5.06 - Costan et al. (2020) d.w., dry weight; f.w., fresh weight. |, Increase in parameter value; |, decrease in parameter value; -, no change in parameter value. Salinity effects on the biochemical parameters of tomato plants and fruits Besides affecting the morphological and physiological status, saline stress can also influence the biochemical reactions of plants. Many studies have shown that high salt concentrations cause biochemical imbalances resulting in low plant productivity (Kusvuran et al., 2016). Tomato plants, though considered moderately sensitive to saline stress, show many changes at the biochemical level such as increases or decreases in the accumulation of hormones, reactive oxygen species (ROS) or antioxidants. These changes have been mainly recorded when NaCl has been used as a salt stressor, in concentrations varying between 25 and 600 mM ( Table 4 ). Table 4 Salinity impact on the biochemical parameters in tomato plants and fruits. Tomato cultivar/variety Salinity treatment Salt application Plant part Enzymatic/non-enzymatic activity References Perfectpeel 100 mM, 150 mM NaCl 10 days after transplantation leaves, roots - ABA accumulation in leaves and roots. Lovelli et al. (2012) PKM 1 25, 50, 100, 150 and 200 mM NaCl immediately after sowing leaves, fruits - ABA, IAA accumulation in leaves; proline in fruits. Babu et al. (2012) Solanum chilense, Ailsa Craig 125 mM NaCl 23 days old leaves - decrease of total auxins (both cultivars) - increase of ABA (both cultivars), total jasmonates (Ailsa Craig), benzoic acid, total gibberellins, total jasmonates, cytokinins (Solanum chilense) Gharbi et al. (2017a) Solanum chilense, 125 mM NaCl 23 days old leaves, roots - increases for salicylic acid, ethylene, Spm (leaves) and Spd (roots) - decreases for Put (leaves and roots) - no impact on Spd (leaves), salicilyc acid and Spm (roots) Gharbi et al. (2017b) Ailsa Craig - no significant effect on `Ailsa Craig` Gran Brix, Marmande Raf 100 mM NaCl 38 days after germination and was maintained for 15 days. - increase of phytohormones: cytokinins (trans-zeatine and isopentenyl adenine), gibberellins (GA4), salicylic acid (Grand Brix); cytokinins (trans-zeatine and isopentenyl adenine), ACC, jasmonic acid, salicylic acid (Marmande), ABA - increase of the H2O2 (Marmande), LOX (Grand Brix), antioxidant enzymes, MDA -decrease of ACC, jasmonic acid, H2O2 (Grand Brix), LOX, (Marmande), catalase, O2- - no effect on gibberellins (Marmande) de la Torre-Gonzalez et al. (2017b) Micro-Tom 120 mM NaCl 20 days after cultivation roots - increase of MDA, carbonyl groups, glutathione reductase, glutathione peroxidase, nitric oxide. - decrease of ascorbate, glutathione, NADP-isocitrate dehydrogenase (NADP-ICDH), glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), GSNO reductase, catalase. Manai et al. (2014) Cerasiforme 40, 80 and 160 mM NaCl 4 days after transplantation leaves - increase of SOD up to 80 mM; decrease at 160 mM - decrease of APX Martinez et al. (2012) Cerasiforme S. chilense Dun. 40, 80 and 160 mM NaCl 18 days after sowing leaves - no effect on total soluble proteins, MDA, caroteinoids and GR. - decrease in activity for CAT, APX (S. lycopersicum L. var. cerasiforme) - increase in activity for dehydroascorbate reductase (S. lycopersicum L. var. cerasiforme), APX and SOD (160 mM NaCl) for S. chilense Dun. Martinez et al. (2014) Puangphaka 5, 10, 25, 50 and 100 mM NaCl starting from seed inoculation roots - 7 days: SOD - highest at 25 mM, lowest at 100 mM, at 10 mM, lowest at 50 mM and GPx - highest at 5 mM, lowest at 50 mM - 14 days: SOD - highest at 5 mM, lowest at 10 mM, CAT - highest at 5 mM, lowest at 25 mM and GPx - highest at 100 mM, lowest at 50 mM - 21 days: SOD - highest at 50 mM, lowest at 25 mM, CAT - highest at 10 mM, lowest 100 mM and GPx - highest at 10 mM, lowest at 50 mM. - the highest CAT activity at 14 days, medium at 7 days, lowest at 21 days. Srineing et al. (2015) stems - 7 days: SOD - highest at 25 mM, lowest at 100 mM, CAT - highest at 5 mM, lowest 25 mM and GPx - highest at 50 mM, lowest at 25 mM, - 14 days: SOD - highest at 5 mM, lowest at 10 mM, CAT - highest at 100 mM, lowest 5-50 mM and GPx - highest at 5 mM, lowest at 25 mM, - 21 days: SOD - highest at 50 mM, lowest at 25 mM, CAT - highest at 10 mM, lowest 100 mM and GPx - highest at 25 mM, lowest at 5 mM, - the highest CAT activity at 7 days, medium at 14 days, lowest values at 21 days. Cerasiforme 150, 300 and 450 mM NaCl 62 days after sowing leaves - no effect at 150 and 300 mM at 25 days after starting the experiment for MDA - increase for MDA at 450 mM NaCl (25 days after starting the experiment), for all the concentrations at 33 days. - decrease for total carotenoids, except 150 and 300 mM, 25 days after treatment - increase for total phenolics and flavonoids, except 150 mM, 25 days after treatment, in the case of phenolics. - decrease in TSS at 300 and 450 mM NaCl (25 and 33 days after treatment) Al Hassan et al. (2015) Microtom 50, 150 mM NaCl The stage of six leaves leaves - increase of phenols at 150 mM Bacha et al. (2017) Rio grande, Savera 25, 50, 75, 100 and 125 mM NaCl 10 days after transplantation leaves - increase of catalase activity, peroxidase activity, total free amino acids, proline Manan et al. (2016) Tomato NaCl:Na2SO4 9:1 molar ratio 4 weeks after sowing roots, stem, leaves - increases of proline in the roots, stem and leaves - no change in the total sugar concentration Wang et al. (2015) Ciettaicale, San Marzano 300, 450 and 600 mM NaCl leaves, roots - increase of total antioxidant capacity at 600 mM in leaves and gradual decrease in roots for both cultivars - increase of carotenoids until 450 mM for San Marzano, gradual increase for Ciettaicale - increase of total soluble sugars in leaves of both cultivars, gradual decrease for Ciettaicale and increase until 450 mM for San Marzano in roots Moles et al. (2016) Tomato 25, 50, 100 and 200 mM NaCl 27 days after plantation leaves - increase of flavonoids, phenolics, saponin - decrease of proline, carotenoids - no effect on total antioxidant capacity Abdel-Farid et al. (2020) Roma, SuperMarmande 100, 200 mM NaCl 10 days after germination leaves - decrease of protein content at 100 mM (Roma), 100 and 200 mM (SuperMarmande). - upregulation of proteins involved in energy and carbon metabolism, photosynthesis, ROS scavenging and detoxification, stress defence and heat shock proteins, amino acid metabolism and electron transport Manaa et al. (2013a) Castle rock, Edkawi 50, 100, 150, 200 and 300 mM NaCl 5 days after germination seedling - accumulation of proteins at 50 mM, decrease at higher concentrations for Castle rock, constant accumulation for Edkawi at 100 - 200 mM, decrease at 300 mM NaCl Khalifa (2012) BINATomato-5 60, 120 mM NaCl 30 days after germination leaves, roots - accumulation of glutamate, proline, glycin, serine, alanine, protease, glutamate synthase, Fd-dependent glutamate synthase, NADP-dependent isocitrate dehydrogenase, glutamate dehydrogenase. - decrease of nitrate and nitrite reductase, soluble protein. - no change for NADH-dependent glutamate synthase Hossain et al. (2012) Gran brix, Marmande Raf 100 mM NaCl 38 days after germination and was maintained for 15 days. leaves and roots - increase in citrate synthase, malate dehydrogenase, phosphoenolpyruvate carboxylase, isocitrate dehydrogenase, citrate, malate and oxalate for Gran brix, in citrate synthase for Marmande Raf - decrease of malate dehydrogenase, phosphoenolpyruvate carboxylase, isocitrate dehydrogenase for Marmande Raf - increase of citrate (Marmande Raf) and malate for Grand brix - decrease of malate and oxalate for Marmande Raf de la Torre-Gonzalez et al. (2017a) Ciettaicale, San Marzano 25 mM NaCl Starting from sowing seeds - increase of endo-b-mannanase, b-mannosidase, catalase, total antioxidant capacity (Ciettaicale), TSS, H2O2 - decrease of endo-b-mannanase, b-mannosidase, a-galactosidase, catalase, total antioxidant capacity (San Marzano), starch Moles et al. (2019) ABA, abscisic acid; IAA, indole acetic acid; Spm, spermine; Spd, spermidine; Put, putrescine; ACC, aminocyclopropane-1-carboxylic acid; H2O2 - Hydrogen Peroxide; LOX, lipoxygenase; MDA, malondialdehyde; NADP, nicotinamide adenine dinucleotide phosphate; GSNO, S-Nitrosoglutathione; SOD, superoxide dismutase; APX, ascorbate peroxidase; GR, glutathione reductase; CAT, catalase; GPx, glutathione peroxidase; TSS, total soluble solids; Fd- - ferredoxin dependent; NADH, nicotinamide adenine dinucleotide. In general, the plants respond to the salinity stress in two phases: in the first, which lasts for days or weeks, the effect of osmotic stress is predominant; in the second, of weeks to months duration, the ionic toxicity effect of leaf salt accumulation affects plant growth. In the first phase, the phytohormones play an important role in regulating plant growth. For instance, abscisic acid (ABA) under saline conditions can accumulate in tomato leaves and/or roots, as a response to the low soil water potential, causing stomatal closure, thus affecting the photosynthesis or enhancing the root growth (Babu et al., 2012; Lovelli et al., 2012; Gharbi et al., 2017a; de la Torre-Gonzalez et al., 2017b). Indole acetic acid (IAA) is another hormone that is usually highly synthesized under saline stress, alleviating the negative effects of osmotic and oxidative stress, being involved in all aspects of the plant, from germination to vegetative growth and flowering. The accumulation of IAA was recorded in tomato leaves exposed to salt concentrations varying from 25 mM NaCl to 100 mM NaCl (Babu et al., 2012; de la Torre-Gonzalez et al., 2017b). However, decreases or no change in the total auxins were found by Gharbi et al. (2017a), in S. chilense and cultivar Ailsa Craig at 125 mM NaCl or by de la Torre-Gonzalez et al. (2017b) in cultivar Marmande at 100 mM NaCl. Other phytohormones studied in relation to saline stress in tomato are salicylic acid, polyamines (Put, Spd and Spm), ethylene, benzoic acid, total jasmonates, total gibberellins, cytokinins or aminocyclopropane-1-carboxylic acid (ACC, the ethylene precursor), whose content has shown very changeable responses to salinity. The content of phytohormones has been found highly dependent on the cultivar, salt concentration or plant part. For instance, the bioactive gibberellin GA4 accumulated in the cultivar Grand Brix, but not in Marmande; the total jasmonates increased in the leaves of cultivar Ailsa Craig, but remained unchanged in the roots ( Table 4 ) (de la Torre-Gonzalez et al., 2017b; Gharbi et al., 2017b, 2017a). Under salinity stress, but not only, plants increased the content of ROS, causing oxidative damages. Regarding tomato, the studies have mainly focused on the activity of malondialdehyde (MDA, a lipid peroxidation marker), carbonyl groups, H2O2, lipoxygenase (LOX). Their accumulation can lead to the inhibition of plant growth and development, and plant death. Increases in ROS content in tomato plants were reported at low levels of salinity (25 mM NaCl), in cultivar Ciettaicale, for hydrogen peroxide, but also at high levels of salinity (450 mM NaCl) in the variety cerasiforme for MDA (Al Hassan et al., 2015; Moles et al., 2019). The duration of exposure to salinity is an important factor in ROS accumulation, as suggested by Al Hassan et al. (2015), who recorded a significant increase in MDA content 33 days after starting the treatment but not after 25 days. Cultivar also plays a key role: the exposure of tomato cultivar Micro-Tom to NaCl (120 mM) or of Marmande and Grand Brix (100 mM NaCl) led to an increase in MDA and carbonyl groups or H2O2 and LOX contents, while at 40, 80 and 160 mM NaCl the MDA content in S. chilense Dun. and variety cerasiforme was not affected (Manai et al., 2014; Martinez et al., 2014; de la Torre-Gonzalez et al., 2017b). In order to prevent the negative effects of ROS, plants produce enzymatic and non-enzymatic compounds such as: ascorbic acid, phenols, ascorbate peroxidase (APX), superoxide dismutase (SOD), glutathione reductase (GR), catalase (CAT), peroxidase (POD), glutathione peroxidase (GPx), plasma glutathione peroxidase (GSHPx) etc., which play a key role in cell protection against the oxidative stress (Kusvuran et al., 2016). In tomato subjected to saline stress, the antioxidant production can vary depending on cultivar, salt concentration, plant age or part. For instance, in a study done on cerasiforme variety subjected to 40, 80 and 160 mM NaCl, the enzymatic activity of SOD increased at 40 and 80 mM NaCl, then decreased at 160 mM, while the APX activity decreased regardless of the salt concentration (Martinez et al., 2012). In another study, where tomato cultivar Micro-Tom was subjected to 120 mM NaCl, the activity/content of ascorbate, glutathione (GSH), NADP-isocitrate dehydrogenase (NADP-ICDH), glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), S-nitrosoglutathione (GSNO) reductase and CAT decreased, while the activity of GR and GPx increased, suggesting a negative impact of the salinity stress on the redox status and NO metabolism (Manai et al., 2014). Interesting findings were made by Srineing et al. (2015), in a study in vitro on the cultivar Puangphaka treated with NaCl at concentrations ranging between 5 - 100 mM. The authors analyzed the activity of SOD, CAT and GPx (roots and stem) at different time intervals: 7, 14, 21 days after incubation. The results showed differences in enzyme activity depending on plant age and part (roots or stems) ( Table 4 ). The influence of the salt and the exposure time on total carotenoids, total phenolics, total flavonoids and TSS was also analyzed by Al Hassan et al. (2015) in cerasiforme variety exposed to 150, 300 and 450 mM NaCl. The results showed that regardless of the time of treatment (25 or 33 days) the content of total carotenoids significantly decreased at all the concentrations, except for 150 and 300 mM, 25 days after treatment, while the content of the total phenolics and flavonoids significantly increased at all the salt concentrations, except for 150 mM, 25 days after treatment, in the case of phenolics. In another study, where the tomato plants of cultivar Microtom were exposed shorter to NaCl stress (14 days) the phenols increased to 150 mM NaCl (Bacha et al., 2017). Changes in the antioxidant activity were also reported by Martinez et al. (2014); Manan et al. (2016) and de la Torre-Gonzalez et al. (2017b), included in Table 4 . Salinity stress is known to produce a C shortage in plants, stimulating the synthesis of C-rich compounds such as trehalose, mannitol, sorbitol or proline, involved in the osmotic adjustment mechanism to stressful conditions. Moreover, the N status is affected because of the influence on NO4+ uptake. Hossain et al. (2012) and Manai et al. (2014) reported that the activity of enzymes involved in the N absorption was affected by saline stress: a decrease was recorded for nitrate and nitrite reductase or nitric oxide (NO), suggesting a negative impact on the NO metabolism under salinity stress, while an increase was recorded for protease, glutamate synthase and Fd-dependent glutamate synthase, NADP-dependent isocitrate dehydrogenase, and glutamate dehydrogenase. No change was observed for NADH-dependent glutamate synthase. Most of the studies carried out on different tomato cultivars, varieties or genotypes (e.g. BINATomato-5, PKM1, Cerasiforme, Rio grande, Savera, Ciettaicale or San Marzano) reported increases in the proline, glycine betaine, serine, alanine, or total soluble sugars contents under different NaCl concentrations, as a result of osmotic adjustments (Babu et al., 2012; Hossain et al., 2012; Al Hassan et al., 2015; Manan et al., 2016; Moles et al., 2016). Increases in the proline content in the roots, stems and leaves of tomato plants, but not of the total soluble sugars, were also recorded in the case of combined salt stress, consisting of NaCl:Na2SO4 in a molar ratio of 9:1 (Wang et al., 2015). By contrast, a decrease in the proline content was reported by Abdel-Farid et al. (2020), in a pot experiment, where tomato plants were treated with 25, 50, 100, 200 mM NaCl. The decrease was explained by taking into consideration the replacement of the proline by another osmoprotectant under saline conditions. The salinity stress can also affect the protein content of plants. A study performed on two tomato cultivars (Castle rock and Edkawi) with different tolerance to salinity showed an accumulation of proteins (the large chloroplast subunit (RbcL), structural maintenance of chromosomes (SMC) protein, a protein from the plasma membrane, and transcription factors) at 50 mM NaCl in both cultivars, a gradual decrease at higher salt concentration for Castle rock and an approximately constant accumulation for Edkawi at 100, 150, 200 mM NaCl, followed by a decrease to 300 mM NaCl. According to the authors, the accumulation of RbcL at 50 mM NaCl in the cultivar Castle rock might be the result of Rubisco degradation under saline stress, as this cultivar is more sensitive to salinity. The better tolerance to salt stress of cultivar Edkawi is demonstrated by better retention of Rubisco content, chromosome segregation and up-regulation of ion pump proteins (Khalifa, 2012). In another study carried out on the cultivar BINATomato-5 the soluble protein content decreased by 25.64% at 60 mM NaCl and by 42.75% at 120 mM NaCl (Hossain et al., 2012). A decrease in protein content was also observed by Manaa et al. (2013a) in the leaves of two tomato cultivars (Roma - salt tolerant, SuperMarmande - salt sensitive), at 100 and 200 mM NaCl. The same author conducted leaf proteomic analysis, identifying 26 proteins involved in energy and carbon metabolism, photosynthesis, ROS scavenging and detoxification, stress defense and heat shock proteins, amino acid metabolism and electron transport. The majority of the proteins identified were upregulated as a consequence of saline stress. Variations in protein abundance were also reported in the fruits of two tomato cultivars (Cervil and Levovil), which were correlated to the salt treatments and the fruit ripening stage. Most of the proteins identified were associated with carbon and energy metabolism, salt stress, oxidative stress, and the ripening process (Manaa et al., 2013b). In general, the content of soluble proteins represents an indicator of plant physiological status under stress, having an important role in osmotic adjustments, and providing storage for different forms of nitrogen. Depending on the cultivar, the soluble proteins can decrease as a result of protein synthesis inhibition and/or protein hydrolysis or can increase through the production of new stress-related proteins (Ahmad et al., 2016). Salinity stress can also have no impact on the protein content, as recorded by Martinez et al. (2014), in a study done on S. chilense Dun. and variety cerasiforme at 40, 80, or 160 mM NaCl. Salinity can also affect the carboxylate metabolism and organic acid production, depending on the cultivar as demonstrated by (de la Torre-Gonzalez et al., 2017a) ( Table 4 ). High activity of the enzymes involved in the carboxylate metabolism enhances tomato resistance to salinity due to the activation of osmotic adjustments mechanism of response which helps the plant to adapt to stressful conditions. Also, high organic acid concentrations are necessary for enhancing the plant's tolerance to salinity, taking into account their important role in different biochemical pathways, such as energy production or amino-acid biosynthesis. In addition, Moles et al. (2019) showed that NaCl can influence the activity of the cell wall enzymes (endo-b-mannanase, b-mannosidase, a-galactosidase) involved in seed germination. Under 25 mM NaCl, the concentration of endo-b-mannanase and b-mannosidase increased in cultivar Ciettaicale, and decreased in cultivar San Marzano affecting the seed germination. Reyes-Perez et al. (2019) stated that acid and alkali phosphatase, trypsin, lipase, b-galactosidase, and esterase can be used as biomarkers for NaCl-stress tolerance in tomato. Salinity effects on tomato gene expression In general, salinity stress, like other abiotic stresses, determines changes in the gene expression of plants. The knowledge of the gene expression as a result of salt stress is still limited, but mostly refers to changes in transcription factors (Devkar et al., 2020). Tomato research regarding the effect of salinity on gene expression has been carried out on different cultivars and focused mostly on the effect of NaCl applied at the concentration range between 50 and 500 mM ( Table 5 ). The results suggested changes in the expressions of genes involved in cell wall construction, biosynthesis of volatiles and secondary metabolites, protein synthesis, transport activity, etc. for the plants subjected to salinity stress. Table 5 Salinity stress-related genes in tomato plants. Tomato cultivar/accessions Salinity level Stress-related genes References Micro-Tom 100-400 mM NaCl 4CL3, PAL6, CHI1, CHI2, HQT, XTH4, XTH20, XTH16, EXPA4, EXPA5, EXPA18, FLA 2, FLA10, FLA11, TPS, FPS, LEA, LOX, HSF30 Hoffmann et al. (2021) Yanfen 210 10%, 20%, 30%Seawater SlGA20OX1, SlMYB13, SlCI-2, SlHYD, SlPCC27-04, SlMYB48, SlAPRR5, SlMFS Mu et al. (2021) Ailsa Craig 150 mM NaCl SlSOS2, P5CS, SlDREB2 Coyne et al. (2019) Tomato 0, 50, 75 mM CS, AH, PDH, PAP, ALDH, DGD, LAT, DGK, FAD, LCS, ACOX, PHS, AOS, FPS, MK, GPS Zhang et al. (2018) New Yorker 0.2 M NaCl/0.02 M CaCl2 NCED1, TAS14 Pye et al. (2018) Manitoba 100 mM NaCl NRT1.1, NRT1.2, AMT1.1, AMT1.2, Gs1 Abouelsaad et al. (2016) Solanum chilense LA 1938, LA 1959; S. chmielewskii LA 1325, LA 2695; S. corneliomuelleri GI 568, PI 126443; S. galapagense LA 0532, LA 0317; S. habrochaites G156, LA 2167; S. habrochaites glabratum LA 2860, PI 126449; S. lycopersicum Abigail F1, LA 3320, LA 2711 and Arbasson F1; S. neorickii LA 2194; S. pennellii LA 1340, LA 1522; S. pennellii puberulum LA 1302; S. peruvianum LA 2548; S. pimpinellifolium OT 2209, LA 1245 100mM NaCl P5CS, NHX1, NHX3, HKT1;1, HKT1;2, SOS1 Almeida et al. (2014b) Arbasson 0, 5, 75 mM NaCl HKT1;2 Almeida et al. (2014a) Ailsa Craig 100, 200, 300, 400, 500 mM NaCl SlERF5 Pan et al. (2012) Rio Fuego 100 mM NaCl SlGSTU23, SlGSTU26, SlGSTL3, SlGSTT2, SlDHAR5, SlGSTZ2 Csiszar et al. (2014) San Miguel, Perfect peel HF1, Mouna HF1 150 mM NaCl WRKY (8, 31, 39), ERF (9, 16, 80), LeNHX (1, 3, 4), HKT (Class 1) Gharsallah et al. (2016) Mircrotom 0, 10, 20, 25, 30 mM Na+ LeHAK5 Bacha et al. (2015) Microtom 250 mM NaCl SlARF1, SlARF4, SlARF8A, SlARF19, SlARF24 Bouzroud et al. (2018) 4CL3, 4-coumarate-CoA ligase; PAL6, phenylalanine ammonia lyase; CHI1, CHI2, chalcone isomerase; HQT - hydroxycinnamoyl-CoA quinate transferase; XTH4, XTH20, XTH16, xyloglucan endo-transglucosylase or hydrolase; EXPA, expansins; FLA, fasciclin-like arabinogalactan proteins; TPS, terpene synthase; FPS, farnesyl diphosphate synthase; LEA, late embryogenesis abundant proteins; LOX, lipoxygenase genes; HSF30, heat shock transcription factor; SlGA20OX1, gibberellin 20-oxidase1 gene; SlMYB, transcription factors of the MYB family; SlCI-2, proteinase inhibitor; SlHYD, SlMFS, genes related to membrane; SlPCC27-04, desiccation-related protein; SlAPRR5, response regulator; SlSOS2, Solanum lycopersicum Salt-Overly-Sensitive 2; P5CS, pyrroline 5-carboxylate synthase, SlDREB2, Solanum lycopersicum Dehydration Responsive Element Binding 2; CS, citrate synthase; AH, aconitate hydratase; PDH, pyruvate dehydrogenase; PAP, phosphatidate phosphatase, ALDH, phosphatidate phosphatase, DGD, digalactosyldiacylglycerol synthase, LAT, lysophospholipid acyltransferase, DGK, diacylglycerol kinase, FAD, fatty acid desaturase, LCS, long chain acyl-CoA synthetase, ACOX, peroxisomal acyl-CoA oxidase, PHS, beta-phellandrene synthase, AOS, allene oxide synthase, MK, mevalonate kinase, GPS, geranyl pyrophosphate synthase; NCED1, 9-cis-epoxycarotenoid dioxygenase; TAS14, tomato dehydrin gene; NRT, nitrate transporters; AMT, ammonium transporters; Gs1, glutamine synthetase; NHX, Na+/H + Antiporters; HKT1;1; HKT1;2, sodium transporter; SOS1, Salt-Overly-Sensitive 1; SlERF5, ethylene response factors; SlGSTU23, SlGSTU26, SlGSTL3, SlGSTT2, SlDHAR5, SlGSTZ2, Solanum lycopersicum glutathione-S-transferase genes; ERF , ethylene responsive factor HKT, Histidine Kinase Transporter; LeHAK5, potassium transporter; SlARF, Solanum lycopersicum auxin response factor. In a study with the cultivar Micro-Tom subjected to NaCl at 100, 200 and 400 mM, the genes responsible for the phenylpropanoid pathway (4CL3 = 4-coumarate-CoA ligase, PAL6 = phenylalanine ammonia lyase, CHI1 and CHI2 = chalcone isomerase, HQT = hydroxycinnamoyl-CoA quinate transferase), xyloglucan endo-transglucosylase or hydrolase (XTH4, XTH20, XTH16) activities, or enzymatic response to reactive oxygen species (ROS, SOD genes), were up-regulated in the top younger leaflets as compared to the older ones situated at the bottom of tomato plants, indicating an increase in the lignification process and flavonoid synthesis, a strengthening in the mechanical cell wall properties and an intensification in SOD production, an enzyme involved in the response to ROS as a result of the salinity stress. Furthermore, in the top leaflets of stressed plants, the expression of expansins (EXPA4, EXPA5, EXPA18), genes involved in cell wall reshaping, fasciclin-like arabinogalactan proteins (FLA 2, FLA10, FLA11) involved in keeping the plasma membrane and cell wall in close contact, and volatile organic compounds' synthesis (TPS, FPS) were down-regulated, suggesting an increase in the salt sensitivity, as plant growth was stopped, as well as the production of terpene synthase (TPS) or farnesyl pyrophosphate synthase (FPS). Changes in the gene expression were also recorded in the bottom leaflets, with the LEA and LOX genes up-regulated, indicating an accumulation in late embryogenesis abundant (LEA) proteins responsible for membrane maintenance and ion-sequestering properties, as well as in lipoxygenases, markers for cell membrane damage. Other up-regulated genes in the salt-stressed tomato plants were those coding for heat shock transcription factor HSF30 (Hoffmann et al., 2021). In another experiment, in which tomato cultivar Yanfen 210 was treated with seawater at different concentrations (10%, 20% and 30%), a significant differential change was recorded in the expression of 509 genes, 40.67% of which were up-regulated, while 59.33% down-regulated. The highlighted genes were responsible for biological processes (i.e. metabolic process, cellular process or single organism process), cellular components (i.e. cell, cell part, membrane, organelle, etc.) or molecular functions (i.e. catalytic activity, binding, transporter activity, etc.). Notably, the SlGA20OX1gene expression was down-regulated, thus affecting the production of gibberellin and plant growth. Down-regulations were also observed for SlMYB13, part of MYB family transcription factors involved in biological and developmental processes, cell morphology, biological stress response, primary and secondary metabolism adjustment, SlCI-2 gene involved in the inhibition of proteinase activity or SlHYD gene responsible for the activity of cell membrane. On the other hand, over-expressions were observed for SlPCC27-04 gene coding for plant desiccation-related proteins, SlMYB48 gene responsible for ABA signaling, SlAPRR5 gene known to control the time of the flowering process, the circadian rhythms or the photomorphogenesis, or SlMFS gene involved in the membrane activity (Mu et al., 2021). Zhang et al. (2018), investigating the effect of NaCl on the volatile compound emission of tomato plants, found the expression of 18 genes down-regulated, thus affecting the biosynthesis of isopentenyl diphosphate isomerase, geranyl pyrophosphate synthase, sesquiterpene synthase, b-phellandrene synthase, terpene synthase 1, 28, 38 or farnesyl pyrophosphate synthase 1. Out of a total 7210 differentially expressed after NaCl exposure, of which 1208 were over-expressed and 6200 were down-expressed, other 3454 genes were related to plant-pathogen interaction, RNA-transport or hormone signal transduction. Changes in the expression of hormone-related genes were also recorded by Pye et al. (2018) in the roots of the cultivar New Yorker. The treatment with NaCl and CaCl2 led to an increased expression of two ABA-related genes: NCED and TAS14. An interesting finding was made by Coyne et al. (2019), who observed a correlation between the expression of some genes and the circadian rhythms. The gene coding for sodium or hydrogen antiporter and an enzyme for proline synthesis, SlSOS2 and P5CS, were expressed only in the morning, while SlDREB2 encoding a transcription factor responsible for the response of tomatoes to salinity was expressed only in the evening. Due to this behavior, tomato, but also other species, might be able to keep the balance of the endogenous systems to circadian rhythms. Almeida et al. (2014b) also reported an overexpression of P5CS gene which led to an accumulation of proline and Na+ in the leaves of five weeks old tomato plants, but not in the roots. The same authors observed a higher expression of NHX1 and NHX3 genes correlated with a lower Na+ accumulation in leaves, and a higher Na+ accumulation in roots; the expression of HKT1;2 gene in the roots was positively correlated with the amount of Na+ in leaves and stems, but not in the roots, where other genes were responsible for the accumulation of Na+ (HKT1;1). Changes in the expression of HKT1;2 gene due to salinity stress was also recorded in the cultivar Arbasson where an increase in the gene expression in stems and roots was recorded along with increased salinity stress. In leaves, the accumulation of Na+ was correlated with a low expression of HKT1;2 genes (Almeida et al., 2014a). The role of HKT1;1 and HKT1;2 in the ion homeostasis in tomato leaves and stems was also confirmed by Asins et al. (2013). Jaime-Perez et al. (2017) demonstrated in transgenic tomato plants the importance of HKT1;2 gene in Na+ homeostasis and salinity tolerance. The same genes (HKT1;1 and HKT1;2) along with LeNHX1, LeNHX3, LeNHX4, SIWRKY8, SIWRKY31, SIWRKY39 (WRKY gene family) and ERF transcription factors were reported to be highly expressed in a study carried out by Gharsallah et al. (2016) on three tomato genotypes. The salinity stress can also affect the expression of genes related to nitrogen uptake and transport. In this respect, Abouelsaad et al. (2016) demonstrated a decrease in the expression of mRNA of nitrate transporters NRT1.1 and NRT1.2 in both cultivars Manitoba and S. pennellii. The same authors observed a higher expression of remarkable affinity ammonium transporters (AMT1.1 and AMT1.2) in Manitoba and a down-regulation of the Gs1 gene (cytosolic glutamine synthetase) in S. pennellii. Other genes whose expression was changed by salt stress are: SlERF5 gene, part of ERF family gene, which has an important role in the ethylene and abscisic acid signaling pathway (Pan et al., 2012); SlGSTU23, SlGSTU26, SlGSTL3, SlGSTT2, SlDHAR5, SlGSTZ2 involved in primary metabolism, regulation of plant growth and development, anthocyanin's absorption, detoxification of toxic compounds (xenobiotic, lipid peroxides), etc. (Csiszar et al., 2014); LeHAK5 gene whose expression was significantly decreased when the Na+ concentration was increased (Bacha et al., 2015); SlARF1, SlARF4, SlARF8A, SlARF19 and SlARF24 which were upregulated in response to salinity stress (Bouzroud et al., 2018). The gene RBCL (large subunit RUBISCO) whose level of expression was not different as a result of salinity stress, in the presence or absence of ABA synthesis, but whose protein it encodes, showed a significant decrease (Poor et al., 2019). Salinity impact on yield and fruit quality High levels of sodium chloride in soil or in nutritional medium highly affect plant physiological and biochemical processes as well as gene expression, with effects on plant morphology, but also on yield and fruit quality. Most of the research carried out with tomato suggested a positive or no impact of salinity on fruit quality ( Table 6 ). Therefore, increases are reported in the lycopene content (De Pascale et al., 2012; Islam et al., 2018; Sellitto et al., 2019), sugar (De Pascale et al., 2012; Islam et al., 2018; Marsic et al., 2018; Botella et al., 2021), total soluble solids (TSS), titratable acidity (TA), organic acids (OA), fruit firmness (Cantore et al., 2012; De Pascale et al., 2012; Martinez et al., 2012; Liu et al., 2014; Zhai et al., 2015; Pengfei et al., 2017; Islam et al., 2018; Rodriguez-Ortega et al., 2019; Maeda et al., 2020; Botella et al., 2021) or cuticle thickness (Agius et al., 2022). According to Agius et al. (2022) a salinity level of up to 5 dS m-1 in nutrient solutions may enhance the fruit quality. In a study conducted by Cantore et al. (2012) on two tomato cultivars, salinity increased the content of TSS and had no significant effect on the ascorbic acid content or the TA. Martinez et al. (2012) showed no change in the TSS and TA content at 40 or 80 mM NaCl. At a salinity level of 6.8 dS m-1 in soil, the TSS and TA contents in fruits of Buran F1 grafted on Maxifort are higher compared to the values determined in fruits grown in soil with the EC of 1.7 dS m-1 (Pasalic et al., 2016). Zhang et al. (2016) reported that the salt enrichment in nutrient solution also leads to an increase in the acidity of the tomato fruit. Islam et al. (2018); Costan et al. (2020) and De Pascale et al. (2012) found in their studies that the total soluble solids (Brix index) and citric acid content increased in tomato fruits with salinity increase. In the fruits of tomato cultivar Unicorn the total soluble solids (Brix index) and citric acid content increased by 22% and 20% per dS m-1 (Islam et al., 2018). Improvement of fruit quality as a result of salinity was also reported by: Ahmed et al. (2017); Pengfei et al. (2017) in cultivar Pepe; Rodriguez-Ortega et al. (2019) in tomato cultivar Optima; Maeda et al. (2020) in the two tomato cultivars CF Momotaro York and Endeavour. The main factors influencing the fruit quality under salinity stress are harvest day, salinity distribution in the soil or the growth stage (Iglesias et al., 2015; Chen et al., 2016; Zhang et al., 2017). In a study conducted with 4 tomato varieties (Raf, Delizia, Conquista, Tigre) subjected to salinity stress, the content of TSS was significantly decreased when the fruits were harvested 136 days after transplant for cultivar Raf and 90 and 104 days for Delizia; a significant increase of TSS was recorded for Conquista 150 days after transplant and Tigre 136 days (Iglesias et al., 2015). By testing the effect of the uneven vertical distribution of soil salinity on the tomato quality of cultivar Yazhoufenwang, Chen et al. (2016) showed that the content of TSS, OA and vitamin C increased with the soil salt concentration in the upper layer. Zhang et al. (2017), demonstrated that the salinity stress applied from flowering until the fruiting stage improves the TSS content. However, negative effects of high salt levels can be found in the mineral content of tomato fruits. Studies conducted by De Pascale et al. (2012); Hernandez-Hernandez et al. (2018); Islam et al. (2018); Costan et al. (2020) showed that under salinity stress, the mineral content in tomato fruits ( Table 7 ), especially of calcium and potassium, can decrease. Table 6 Salinity impact on yield and citric acid, lycopene, soluble solids contents in tomato fruits. Tomato cultivar/ hybrid/variety Salinity level Citric acid Lycopene mg/kg f.w. Soluble solids (degBrix) Fruit weight (g/fruit) No. of fruits/plant Total yield References Unit Value Unit Value Tampico F1 0.5 - 4.4 dS-m-1 g/kg f.w. 3.2 / 3.4 12.7 / 14.3 4.93 / 5.82 69.1 \ 55.5 21.7 \ 17.2 tonnes/ha 65.0 \ 50.0 De Pascale et al. (2012) Unicorn 2.5 - 7.5 mS*cm-1 % 0.65 / 0.76 115.1 / 137.9 7.66 / 8.01 13.17 \ 11.22 - - - Islam et al. (2018) Belladonna 0, 50 mM % 2.25 / 3.68 159 \ 155 3.85 / 6.60 204.1 \ 109.1 22.4 / 26.6 kg/plant 4.54 \ 2.9 Costan et al. (2020) Cerasiforme (Alef) mM mEq/L 3.90 / 7.75 - 5.63 / 7.78 21.9 \ 14.3 13.3 \ 12.0 - - Martinez et al. (2012) Tainan ASVEG No. 19 0-150 mM % 0.62 / 0.93 - 9.2 / 10.7 7.3 \ 5.4 - g/plant 243.9 \ 48.8 Liu et al. (2014) Hualien ASVEG No. 21 0.33 / 1.20 - 7.4 / 13.2 8.7 \ 4.6 - 78.7 \ 6.9 Taiwan Seed ASVEG No. 22 0.47 / 0.86 - 8.6 / 12.9 8.1 \ 3.6 - 155.5 \ 19.3 Rio Grande 0 - 90 mM - - - 7.74 / 8.87 - 18.89 \ 13.00 kg/pot 0.91 \ 0.51 Naeem et al. (2020) Rio Grande 0 - 60 mM - - - 6.13 / 8.24 - 15.44 \ 12.33 kg/plant 1.28 \ 1.13 Alam et al. (2020) d.w., dry weight; f.w., fresh weigh. |, Increase in parameter value; |, decrease in parameter value. Table 7 Salinity impact on mineral content in tomato fruit. Tomato cultivar/hybrid/variety Salinity level Unit Minerals References N P K Ca Na Mg Tampico F1 0.5, 2.3, 4.4 dS-m-1 % d.w. 2.29 \ 1.98 0.275 \ 0.233 4.042 \ 3.392 0.319 \ 0.288 - - De Pascale et al. (2012) Unicorn 2.5, 5, 7.5 mS cm-1 % d.w. - 0.637 \ 0.287 2.44 \ 2.10 0.127 \ 0.087 - 0.13 / 0.17 Islam et al. (2018) Belladonna 0, 50 mmol/L g/kg d.w. 17.61 / 17.78 1.38 / 1.58 24.05 \ 21.72 0.82 \ 0.55 0.62 / 1.31 0.59 / 0.62 Costan et al. (2020) Durinta F1 7, 21, 37, 49, 64 mM % d.w. 2.22 \ 2.02 - 3.91 \ 3.55 0.13 / 0.15 0.08 / 0.26 0.15 \ 0.12 Giuffrida et al. (2009) Huno F1 0, 100 mM g/kg d.w. 23.14 \ 21.78 - 9.16 \ 8.31 6.34 \ 2.92 0.98 / 4.30 2.31 / 2.34 Hernandez-Hernandez et al. (2018) d.w.,- dry weight. |, Increase in parameter value; |, decrease in parameter value. Regarding tomato yield under saline stress, the Division of Agriculture and Natural Resources of University of California specifies that a soil salinity of 7.6 dS m-1 may reduce both tomato plant emergence and crop yield by 50% (Division of Agriculture and Natural Resources, 2022), but these effects are closely related to the tomato cultivar. The study performed by De Pascale et al. (2012) showed that at 4.4 dS m-1 the mean fruit weight, the number of fruits per plant and the total yield of tomato decreased compared to the control (0.5 dS m-1) by 19.68%, 20.74%, and 23.07%, respectively. According to Islam et al. (2018) an increase in soil salinity from 2.5 at 7.5 dS m-1 causes a 14.81% reduction in the mean fruit weight of the cultivar Unicorn. In addition, Liu et al. (2014) reported that the yield of three cherry tomato cultivars grown inpeat moss, perlite and sand mix (2:1:1) was affected differently by the same levels of salinity. At 150 mM NaCl the mean fruit weight of Tainan ASVEG No. 19, Hualien ASVEG No. 21 and Taiwan Seed ASVEG No. 22 was reduced by 26.03%, 47.13%, and 55.56% respectively, compared to the control, and the total yield decreased from 243.9, 78.7 and 155.5 g/plant to 48.8, 6.9, and 19.3 g/plant, respectively. Costan et al. (2020) reported that, although the number of fruits per plant increased with the salinity rises in the hydroponic system (from 0 at 50 mM), the yield of the tomato cultivar Belladonna was reduced by more than 36%. Noshadi et al. (2013) found the highest yield (47.15 t*ha-1) was recorded when the irrigation water EC was of 2 dS m-1. At 0.6 dS m-1, 38.02 t*ha-1 were harvested and at 4 dS m-1 about 31.57 t*ha-1, whereas the lowest yield was at 8 dS m-1 EC (21.20 t*ha-1). Therefore, according to the results of the latter study, a slightly saline soil or hydroponic cultivation can enhance tomato yield. Recommendations for alleviating the effects of salinity on tomato The negative effects of salinity on tomato plants can be alleviated by using different strategies like plant priming or genetic modification. Plant priming represents a promising method to reduce the time required for a plant exposed to abiotic stress to respond efficiently to the stressor and, thereby, to increase the tolerance to stress conditions (Aranega-Bou et al., 2014). Effective priming agents against salt stress in tomato, which have been studied over years are elements (Fe, Si, K, N), plant growth regulators (ACC, IAA, SA, melatonin), reactive species (S-nitrosoglutathione, sodium hydrosulfide, sodium nitroprusside), vitamins (ascorbic acid - AsA), aminoacids, natural extracts (seaweed), polymers (chitosan), osmoprotectants (glycine betaine, proline), polyamines (spermidine) or plant growth promoting microorganisms (bacteria, fungi or arbuscular mycorrhizal fungi) (Choudhary et al., 2022; Gedeon et al., 2022; Zulfiqar et al., 2022). The results showed in most of the cases an enhancement of the tolerance of plants to various concentrations of salt, by decreasing the osmotic stress, enhancing the activity of the antioxidant system, increasing the growth and yield or by improving the fruit quality. For instance, the application of Fe increased the ascorbic acid content in the fruits of tomato along with the increment in salinity level; the Si addition stimulated an early accumulation of TSS in the fruits of tomatoes, but did not influence the quality of the taste; in another study, the presence of Si decreased the SOD activity, suggesting a reduction in ROS production; also, the treatment with Si increased the b-carotene and vitamin C content; the addition of 5 mM K+ regulated the ascorbate-glutathione cycle, the activity of antioxidant enzymes, the carbohydrate metabolism and increased the proline content; nitrogen applied at different concentrations (25, 75, 150 kg N ha-1) had a positive impact on the proline content and on the activity of P5CS enzyme, also affected the activity of various enzymes: proline dehydrogenase, nitrate reductase, nitrite reductase, glutamine synthetase and glutamate synthase, glutamate dehydrogenase under NaCl stress (Tantawy et al., 2013; Iglesias et al., 2015; Muneer and Jeong, 2015; Singh et al., 2016; Costan et al., 2020; Khan et al., 2021). The application of plant growth regulators such as ACC decreased the osmotic stress in 'Ailsa Craig' tomato cultivar; spraying the tomato plants with IAA (100 and 200 ppm) increased the TSS content of fruit juice and the chlorophyll content of the leaves; the exogenous application of salicylic acid decreased the ethylene synthesis and increased the polyamine endogenous concentration; in another study, salicylic acid applied foliar increased the TSS and the vitamin C content; the treatment of the seeds with salicylic acid (1 mM) and H2O2 (50 mM) increased the TSS, proteins, POD, CAT, SOD and MDA content; the treatment with 20 and 50 mM melatonin improved the activity of the antioxidant system, the proline and carbohydrate metabolism, also the ascorbate/reduced glutathione cycle in 'Five Start' tomato cultivar; in another studies, melatonin improved the root architecture, reduced the production of reactive oxygen species, enhanced the activity of enzymatic antioxidants and the photosynthesis (Gharbi et al., 2016; Gaba et al., 2018; Siddiqui et al., 2019; Alam et al., 2020; Altaf et al., 2020, 2021; 2022b; Borbely et al., 2020; Naeem et al., 2020; Hu et al., 2021; Ali et al., 2021b). The application of S-nitrosoglutathione and NaHS promoted the accumulation of NO and H2S, alleviating the deleterious effects of oxidative stress; the use of sodium nitroprusside increased the content of non-enzymatic and enzymatic antioxidants, up-regulated the NO level in leaves, enhanced the activity of Calvin cycle, overcame the stomatal limitations and protected the photosystem II from damages (da-Silva et al., 2018; Taheri et al., 2020; Li et al., 2022). Alves et al. (2021) by soaking the tomato 'Micro-Tom' seeds for one hour in 100 mM AsA, observed that the tolerance of plants to salt stress was enhanced by modulating the antioxidant mechanisms. The content of CAT, APX, POX, GPX, GR, GSH, SOD, chlorophyll, and carotenoids in the leaves of primed plants was higher than in the control. Chen et al. (2021) by spraying 0.5 mmol/L AsA solution on the leaves of cv. 'Ligeer87-5' exposed at 100 mmol/L NaCl reported an attenuation of the photoinhibition and oxidative stress damage in chloroplasts, dissipation of excitation energy in PSII antennae, stimulation of chlorophyll synthesis and reduction of damaging effects on photosynthesis in tomato leaves. The foliar application of an aminoacid (Botamisol as free L-amino acids) at different concentrations (0, 2, 4 g*L-1) increased the proline level in the leaves of tomato plants exposed to salinity (8 and 10 dS*m-1) (Jannesari et al., 2016). The application of a seaweed extract (100 mL of P. gymnospora 0.2% w/v) improved the growth, yield and quality of 'Rio Fuego' tomato cultivar (Hernandez-Herrera et al., 2022). The use of chitosan solution at different concentrations (0.03% and 0.05% or 50, 100 and 150 mg/L) for spraying the tomato leaves, enhanced the salt tolerance of tomato at 100 mM NaCl applied as a root drench, promoted the growth and development of plants and increased the chlorophyll contents (Ullah et al., 2020; Ozkurt and Bektas, 2022). The exogenous application of spermidine (Spd) on tomato cv. 'Ailsa Craig' seedlings grown under salt stress resulted in higher photosynthesis and biomass, better ionic and osmotic homeostasis, and enhanced ROS scavenging capacity (Raziq et al., 2022). Siddiqui et al. (2017) found that the chlorophyll a and b contents, proline, activity of CAT, SOD, POD, GR and APX were increased and H2O2 and MDA production in tomato var. Five Star was reduced as a result of exogenous spermidine application on seedlings. The foliar application of 10 and 20 mg/L proline during the flowering stage of cultivars 'Rio Grande' and 'Heinz-227' led to an increase in the dry mass of leaves, stems and roots, improved various chlorophyll fluorescence parameters, increased the potassium and phosphorous content and reduced the accumulation of Na+ in different organs, compared with control (Kahlaoui et al., 2014). The effects of the exogenous application of glycine betaine (GB) on different tomato cultivars have been assessed in a few studies and both positive and negative correlations were found between GB exogenous application and salt tolerance in tomato. Chen et al. (2009) found that the exogenous use of 5 mM GB in half-strength Hoagland could alleviate the salt stress effects in tomato cv. 'F144' and cv. 'Patio' through changing the expression abundance of some proteins. Sajyan et al. (2019) irrigated the tomato 'Sila' plants with saline water (with EC between 2 and 10 dS m-1) and exogenous GB in various doses (4.5, 6 and 7.5 g/L) and observed a positive effects on leaf number, stem diameter, number of flowers, number of fruits, no evident effects on the number of clusters, fruit set, the weight of individual fruit, yield and fruit diameter were observed and a reduction in the fruit ripening process at 7.5 g/L GB. Plant growth-promoting rhizosphere bacteria (PGPB) can alleviate the effects induced by salt stress by production of phytohormone (e.g. auxin, cytokinin, and abscisic acid), ACC-deaminase, ammonia, IAA, extracellular polymeric substance (EPS), induction of synthesis of plant osmolytes and antioxidant activity, increasing the essential nutrient uptake or/and by reducing ethylene production (Kumar et al., 2020). Sphingobacterium BHU-AV3 (Vaishnav et al., 2020), Bacillus megaterium strain A12 (Akram et al., 2019), Enterobacter 64S1 and Pseudomonas 42P4 (Perez-Rodriguez et al., 2022), Bacillus aryabhattai H19-1 and Bacillus mesonae H20-5 (Yoo et al., 2019) are some of the PGPB that have been proved to increase tomato tolerance to salt stress. For example, inoculation of tomato cv. 'Kashi amrit' plants with Sphingobacterium BHU-AV3 exhibited a less senescence in plants exposed to 200 mM NaCl, being determined that the proline content was increased, ion balance was maintained and the ROS was lower compared to the non-inoculated plants. In BHU-AV3-inoculated plant leaves superoxide content, cell death and lipid peroxidation were significantly reduced (Vaishnav et al., 2020). Enterobacter 64S1 and Pseudomonas 42P4 under salt stress reduced electrolyte leakage and lipid peroxidation and increased chlorophyll quantum efficiency (Fv/Fm), proline and antioxidant nonenzymatic compounds (carotenes and total phenolic compounds) contents in tomato leaves (Perez-Rodriguez et al., 2022). A combination of arbuscular mycorrhizal fungi (Claroideoglomus etunicatum, Funneliformis mosseae, Glomus aggregatum, Rhizophagus intraradices), bacteria and fungi (Trichoderma, Streptomyces, Bacillus, Pseudomonas) improved the tomato fruit quality and the antioxidant content of 'Pixel F1' tomato cultivar exposed to soils electrical conductivity of 1.5, 3.0, 4.5, and 6.0 (Sellitto et al., 2019). Some researchers have focused not only on assessing the individual effects of a potential priming agent against salt stress in tomato plants, but also their combined effect. For example, Attia et al. (2021) studied the effects of foliar application of chitosan dissolved in acetic acid (Ch ACE), ascorbic acid (Ch ASC), citric acid (Ch CIT) and malic acid chitosan (Ch MAL) on tomato cultivar 023 irrigated with saline water (100 mM NaCl). These treatments alleviated the negative effects of salinity on tomato plants by increasing the photosynthetic pigments, osmoprotective compounds, and potassium content and lowering MDA, H2O2 and Na+ levels in leaves. Chanratana et al. (2019) used as a bioinoculant chitosan-immobilized aggregated Methylobacterium oryzae CBMB20 to improve the salt tolerance of cv. 'Yeoreum Mujeok Heukchima' and the results showed that plant dry weight, nutrient uptake, photosynthetic efficiency, and the accumulation of proline have been enhanced. Furthermore, the oxidative stress exerted by salt stress was alleviated and the electrolyte leakage and the excess Na+ influx into the plant cell were reduced. Tomato genetic modification techniques have already proven their efficiency and accuracy in protecting plants against salinity stress by improving their genome. Gene transformation, gene editing, quantitative trait loci (QTLs) analysis, gene-pyramiding, and genetic engineering (overexpression) are some examples of molecular genetic tools that have helped in the development of salt-tolerant tomato plants. Gene transformation has mainly focused on transferring genes of various origins, which can be good candidates to increase the tolerance to salinity stress, into tomato plants. Salt tolerant tomato plants were successfully obtained by Gilbert et al. by transferring the gene HAL1 from Saccharomyces cerevisiae, involved in Na+ transport and K+ regulation, which improved the in vivo and in vitro salt tolerance of transgenic tomato plants, by promoting the retention of K+ and the growth of the plants (Gisbert et al., 2000); by Goel et al., who demonstrated that by transforming the tomato cultivar 'Pusa Ruby' with the bacterial codA gene from Arthrobacter globiformis encoding for choline oxidase, the production of glycine betaine was induced, the content of relative water, chlorophyll and proline increased, also the overall tolerance of the plants under saline stress was improved (Goel et al., 2011); by Jia et al., who transferred the BADH gene from Atriplex hortensis in 'Bailichun' tomato cultivar, obtaining a normal growth and development of the plants treated with 120 mM NaCl (Jia et al., 2002); by Li et al., who isolated the SpPKE1 a lysine-, proline-rich type gene from the abiotic resistant Solanum pennellii LA0716 and transferred it to S. lycopersicum cv. M82 or by transferring the Osmotin gene from tobacco into tomato plants, an increased tolerance to salt stress was obtained, highlighted by better cell signaling, ROS scavenging, the content of carbohydrates, amino acids, polyols and performance of the antioxidant and photosynthetic systems (Goel et al., 2011; Li et al., 2019a; Rao et al., 2020). The only genetic editing technique that has been reported to be used in improving the tomato tolerance to salinity is clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 (CRISPR-associated nuclease 9) a modern, easy and very effective genome editing tool (Salava et al., 2021; Altaf et al., 2022a). However, the researches on increasing tomato tolerance to salt stress by using CRISPR/Cas9 are still limited. So far, this tool was used to precisely edit the hybrid proline-rich proteins domain (HyPRP1) involved in different biotic and abiotic responses. The deletion of the SlHyPRP1 negative-response domain led to salt tolerance as high as 150 mM NaCl, improving the germination and the growth of the plants (Tran et al., 2021). The same results were obtained earlier by Li et al., who also observed that by silencing the negative regulator HyPRP1 the expression of the genes responsible for the production of SOD and CAT was enhanced (Li et al., 2016). In addition, CRISPR/Cas9 technology was used to knock out the SlABIG1 gene in tomato exposed to salinity, resulting plants with improved chlorophyll and proline content, photosynthetic system, root dry weight and decreased concentrations of ROS, MDA and Na+ (Ding et al., 2022). By using the same tool, Wang et al., demonstrated the importance of the plasma membrane Na+ /H+ antiporter SlSOS1 in the salt tolerance of tomato, by creating two mutant alleles (Slsos1-1 and Slsos1-2) which showed a significant increase in the Na+/K+ ratio and the salt sensitivity, as compared with the wild type (Wang et al., 2021). Bouzroud et al., by generating tomato SlARF4-crispr (arf4-cr) plants showed the importance of Auxin Response Factor 4 (ARF4) in the tolerance of tomato plants to salinity (Bouzroud et al., 2020). Regarding the other two known genetic editing techniques (zinc finger nucleases - ZFNs and Transcription Activator-Like Effector Nucleases - TALENs) no reports are available on tomato tolerance (Salava et al., 2021; Altaf et al., 2022a). Due to the QTLs mapping, different loci related to the oxidative defence system, Na+/K+ homeostasis, or developmental stages were identified in playing an important role in increasing the tomato tolerance to salinity. Therefore, Frary et al., identified 125 QTLs for antioxidant compounds under saline and non-saline conditions in S. pennellii tomato introgression lines, and their parental lines, salt-resistant wild tomato (S. pennellii LA716) and the salt sensitive cultivated S. lycopersicum Mill. cv. M82 that could be beneficial in developing salt-tolerant cultivars. Under the salt stress (150 mM NaCl), the wild tomato and different introgression lines accumulated more antioxidant compounds (phenolics, flavonoids, SOD, CAT, APX) than the cultivated tomato (Frary et al., 2010). The same wild tomato ascension, the wild S. lycopersicoides LA2951 and two introgression lines derived from them were used to identify QTLs for tolerance to salinity in the seedling stage by Li et al. Four major QTLs were detected on chromosomes 6, 7 and 11 in S. pennellii IL library, while in S. lycopersicoides IL library, six major QTLs were found on chromosomes 4, 6, 9 and 12. The authors concluded the possibility to create hybrids with QTLs coming from these two ascensions (Li et al., 2011). Foolad et al., detected and validated a number of five QTLs for tomato salt tolerance during vegetative growth in a population (BC1) resulted from the crosses between the breeding line NC84173 (Lycopersicon esculentum Mill.) and L. pimpinellifolium (Jusl.) Mill. accession LA722. One minor QTLs was identified on chromosome 3 in the interval CT82-TG515, two major QTLs on chromosomes 1 and 5, and the other two on chromosomes 6 and 11 (Foolad et al., 2001). Villalta el al., found QTLs for salt tolerance during reproductive stage in two populations of F7 tomato lines (P and C) resulted from 'cerasiforme' variety (salt sensitive genotype), as female parent, and two lines tolerant to salt tolerant, as male parents: S. pimpinellifolium, the P population (142 lines), and S. cheesmaniae, the C population (116 lines). The authors suggested that the QTLs detected by them can be used to increase the fruit yield of tomato plants under salt stress, being good candidates for increasing the tomato tolerance to salinity. The QTLs for fruit yield were detected in chromosome 5, the specific loci being fn5.2 and tw8.1 found in C population and fn10.1 which overlaps tw10.1 and fw8.1 loci in P population. Under saline conditions the fruits set percentage per truss, fruit number per plant and the total fruit weight per plant increased (Villalta et al., 2007). Other candidates for QTL can be those associated with Na+/K+ homeostasis are the genes encoding HKT1-like transporters (SlHKT1;1 and SlHKT1;2), with tonoplast NHX Na+/H+-antiporters (SlNHX3 and SlNHX4), with the content of a-tocopherol in tomato fruits (chromosomes 6 and 9), or with tocopherol biosynthesis (chromosomes 7, 8, and 9) (Egea et al., 2022). Gene pyramiding, which consists in combining multiple traits in a single genotype, represents another method that can help to obtain tomato plants tolerant to salinity stress, but the researches are still limited. Some strategies that have been proposed refer to pyramiding the ascorbic acid (AsA) biosynthetic pathway, the ascorbate-glutathione pathway, or different QTLs. For improving the AsA content in tomato, Li et al. pyramided the biosynthetic genes involved in the D-Man/L-Gal pathway of ascorbate, resulting the pyramiding lines GDP-Mannose 3',5'-epimerase (GME) x GDP-d-mannose pyrophosphorylase (GMP), GDP-l-Gal phosphorylase (GGP) x l-Gal-1-P phosphatase (GPP) and GME x GMP x GGP x GPP. The results showed increased concentrations of total ascorbate in leaves and fruits and improved AsA transport capacity, light response and salinity stress tolerance. In addition, the fruit weight (significantly decreased in GGP x GPP lines), fruit size (significantly decreased in GMP x GME and GGP x GPP lines), and soluble solid (significantly increased in GMP x GME and GMP x GME x GGP x GPP lines) were affected by pyramiding maybe because of the influence of different primary metabolism pathways (sugar, acid, and cell wall metabolism) as stated by the authors (Li et al., 2019b). By pyramiding the genes of ascorbate-glutathione pathway, isolated from Pennisetum glaucoma (Pg) (PgSOD, PgAPX, PgGR, PgDHAR and PgMDHAR) Raja et al., obtained tomato lines with better germination rate, survival rate, photosynthetic and antioxidant activity, reduced ROS production, and membrane disruption, under 200 mM NaCl (Raja et al., 2022). Pyramiding QTLs can be an effective method to improve the tomato salt tolerance. The pyramiding of QTLs takes place by using a marker assisted selection (MAS). Some authors proposed the use of different QTLs associated with salt tolerance during seed germination or vegetative growth in tomato (Foolad, 2004). Another way to enhance the tomato salt tolerance is to overexpress specific genes that can increase the tomato tolerance to salt stress. Some authors highlighted the importance of various genes in the salt stress in transgenic plants and, in this respect, Hu et al. (2014) demonstrated that the overexpression of LeERF1 and LeERF2 genes have a positive impact on tomato plants exposed to salinity stress. Good results regarding different physiological and biochemical parameters (i.e. root length, chlorophyll, proline and antioxidant enzymes contents) were obtained in the transgenic tomato, where the expression of other genes related to salinity stress was up-regulated (RBOHC, TAS14, HVA22, PR5 and LHA1). The overexpression of SlERF5 gene (ethylene response factor) in transgenic tomato led to an increased tolerance to salinity by improving the relative water content (Rao et al., 2020). Albacete et al. (2014) recorded improved fruit yield, hormone concentrations, and sugar content in transgenic tomato due to the overexpression of a gene coding for isopentenyl transferase, an enzyme involved in cytokines biosynthesis - IPT gene and a cell wall invertase gene - CIN1. Cai et al., 2016 showed the importance of SlDof22 gene, coding for Dof proteins responsible for abiotic stress response, gibberellins regulation, and evolution of cell cycle, in improving the tomato tolerance to salinity stress. Other genes whose expression increased the tomato plant biomass production and yield under salinity stress were CDF3, which regulated important genes for redox homeostasis, photosynthesis process or primary metabolism (Renau-Morata et al., 2017). NAC transcription factor SlTAF1 is another gene described as a good candidate for increasing the salinity tolerance of tomato and other species. It's silencing in transgenic plants increased the damages related to salinity (Devkar et al., 2020). Conclusions and future perspectives Soil salinity represents one of the main causes of agricultural yield losses worldwide. Natural factors such as topography, and type of geological material, but especially anthropogenic activities like inappropriate agricultural practices (i.e. excessive fertilization, irrigation without proper drainage, and leaching) intensify the soil salinization process. Plants are directly impacted by the increases in soil salt concentration through reduced water and nutrient uptake by roots. In tomato plants, salinity stress affects positively ornegatively the germination process, the morphological traits, the physiological features, the biochemical and molecular parameters, and also the yield. Usually, the germination, morphology and physiology of tomato plants are negatively influenced by the saline stress. When the soil salinity increases, its water potential drops to a point close to the root water potential, slowing down the process of water uptake by roots, thus causing drought stress-related symptoms. Also, in saline soils, nutrients in the form of cations (Mg+, Ca+, K+, NH4+ ) and anions ( NO3- , PO43- ) compete with Na+ and be transported inside the plant. Na+ competes with NH4+ and K+ cations decreasing their absorption, while with decreasing its uptake. Therefore, along with a deficiency in the nutrient uptake, ion toxicity takes place due to excessive concentrations of Na+ and Cl-, consequently affecting plant growth and development. Regarding the effects on gene expression, the salinity stress can down-regulate or up-regulate the expression of genes in tomato plants. A similar situation also occurs with regard to the biochemical parameters which can either be enhanced by the saline stress or can be decreased. Generally, most of the increases and the decreases recorded for the biochemical parameters and the down-regulation of genes represent adaptive responses to stress by plants that try to improve their homeostasis and resistance. However, the decreases can also be the result of biochemical pathways dysregulations. The quality of tomato fruit benefits from saline conditions in most cases, maybe due to lower water content and accumulation of biomolecules such as sugars, amino acids, and inorganic solutes that contribute to osmotic adjustments. The results of the studies carried out over the last 10 years have shed more light on the impact that saline stress can have on tomato plants. However, for a clearer image of the effects of salinity on tomato plants, more studies should be carried out in the field, in salt-affected soils, taking into account the individual and cumulative interactions of the factors involved. The deleterious effects of salinity on tomato plants can be alleviated by using different strategies like plant priming or genetic modification techniques. The results are very promising, but at this moment, they are relatively limited and at their beginnings. In addition, most of the research has focused on developing salt-resistant tomato plants and testing them for the needed characters, but to develop commercial lines, research carried out in saline fields are needed. Considering the FAO predictions that by 2050 more than 50% of arable land will become saline, urgent measures should also be taken to reduce the salinization process such as better water drainage and leaching of salts; a decrease in the quantity and number of fertilizers applied and water used in irrigation; proper crop selection or reduction of the degree of tillage systems. Therefore, researchers should focus more their attention on methods to desalinate the soils, on studies regarding the development of fertigation schemes that promote a better management of water and fertilizers applied according to the plant requirements, on the production of new varieties resistant to salinity, or in improving the existing species. Author contributions VS, GM and MR: Conceptualization. MR and GM: Formal analysis. MR and GM: Investigation. MR and GM: Writing--original draft preparation. VS: Supervision and validation. VS, GM and MR: Writing - review & editing. MR and GM contributed equally to this work and share first authorship. All authors have read and agreed to this version of the manuscript. All authors contributed to the article and approved the submitted version. Acknowledgments The authors are grateful to prof. Gianluca Caruso for his time and efforts in assisting with the proofreading of the manuscript. Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Publisher's note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. References Abdelaziz M. E. Abdeldaym E. A. (2019). 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PMC10000761 | Diagnostics (Basel) Diagnostics (Basel) diagnostics Diagnostics 2075-4418 MDPI 10.3390/diagnostics13050953 diagnostics-13-00953 Article Novel Host Response-Based Diagnostics to Differentiate the Etiology of Fever in Patients Presenting to the Emergency Department + Atallah Johnny Conceptualization Methodology Investigation Data curation Writing - original draft Writing - review & editing Visualization 12 Ghebremichael Musie Methodology Software Formal analysis Writing - review & editing 13 Timmer Kyle D. Methodology Writing - review & editing 12 Warren Hailey M. Formal analysis Writing - review & editing 12 Mallinger Ella Methodology Writing - review & editing 12 Wallace Ellen Methodology Resources Writing - review & editing 4 Strouts Fiona R. Conceptualization Methodology Investigation Resources Writing - review & editing Supervision Project administration 4 Persing David H. Methodology Resources Writing - review & editing 4 Mansour Michael K. Conceptualization Investigation Resources Data curation Writing - original draft Writing - review & editing Supervision Project administration Funding acquisition 12* Sessa Francesco Academic Editor 1 Department of Medicine, Harvard Medical School, Boston, MA 02115, USA 2 Infectious Diseases Division, Massachusetts General Hospital, Boston, MA 02114, USA 3 Ragon Institute of MGH, MIT and Harvard, Cambridge, MA 02138, USA 4 DH Diagnostics LLC, Brea, CA 92821, USA * Correspondence: [email protected]; Tel.: +1-617-726-6726 + Novel rapid host-response diagnostic modality for febrile syndromes. 02 3 2023 3 2023 13 5 95327 1 2023 28 2 2023 01 3 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). Fever is a common presentation to urgent-care services and is linked to multiple disease processes. To rapidly determine the etiology of fever, improved diagnostic modalities are necessary. This prospective study of 100 hospitalized febrile patients included both positive (FP) and negative (FN) subjects in terms of infection status and 22 healthy controls (HC). We evaluated the performance of a novel PCR-based assay measuring five host mRNA transcripts directly from whole blood to differentiate infectious versus non-infectious febrile syndromes as compared to traditional pathogen-based microbiology results. The FP and FN groups observed a robust network structure with a significant correlation between the five genes. There were statistically significant associations between positive infection status and four of the five genes: IRF-9 (OR = 1.750, 95% CI = 1.16-2.638), ITGAM (OR = 1.533, 95% CI = 1.047-2.244), PSTPIP2 (OR = 2.191, 95% CI = 1.293-3.711), and RUNX1 (OR = 1.974, 95% CI = 1.069-3.646). We developed a classifier model to classify study participants based on these five genes and other variables of interest to assess the discriminatory power of the genes. The classifier model correctly classified more than 80% of the participants into their respective groups, i.e., FP or FN. The GeneXpert prototype holds promise for guiding rapid clinical decision-making, reducing healthcare costs, and improving outcomes in undifferentiated febrile patients presenting for urgent evaluation. host-response diagnostics gene signatures fever febrile syndromes National Institutes of HealthRO1 AI132638 Danaher CorporationThis work was supported, in part, by the National Institutes of Health RO1 AI132638 and an unrestricted grant from Danaher Corporation, both to M.K.M. pmc1. Introduction Fever is among the most common presentations to healthcare facilities and urgent care services . Fever has been linked to multiple disease processes that include malignancy, autoimmune diseases, sterile inflammatory processes, and, most commonly, infectious etiologies . Confirming the definitive etiology of fever in hospital-admitted adult patients remains a significant challenge for patient management and represents a large investment in hospital resource utilization . When suspecting an infectious process, it is particularly challenging that clinical manifestations of microbial infection can range across a broad spectrum of nonspecific symptoms. Clinically, the detrimental impact of missed or delayed diagnoses can lead to antibiotic overuse, increased antimicrobial resistance, increased length of stay, and ultimately unfavorable health effects . Economically, increased healthcare costs, prolonged hospitalization periods, and the excessive utilization of hospital resources directly result from poorly performing diagnostics . The need to determine the etiology of fever in febrile patients at an accurate and rapid rate necessitates improved diagnostic modalities for better medical decision-making and management . Pathogen-based diagnostic testing remains the current standard of care when dealing with suspected infections. However, as with any other diagnostic modality, pathogen-specific diagnostics have shortcomings. Depending on the pathogen-based diagnostic test chosen, these challenges include both low sensitivity or low specificity, especially for the commonly used blood cultures, often leaving the healthcare provider with a conundrum regarding the nature of the microbe detected, whether it is commensal or contaminant versus a true invasive pathogen . Thus, as an adjunct to pathogen-specific diagnostics, host immunodiagnostics shows promise. These techniques involve assays such as reverse transcription-polymerase chain reaction (RT-PCR) to measure specific host gene expression signatures and transcripts, which can inform the host's susceptibility and response to infection , allowing for the integration of multiple determinations into single predictive models for accurate diagnosis and disease prognosis. Numerous gene signatures have been linked to the direct response of the immune system to different etiologies, including viral and bacterial infections. For example, the Interferon Regulatory Factor 9 (IRF-9) and Lymphocyte Antigen 6 Family Member E (LY6E) play an essential role in anti-viral immunity, including virus-mediated activation of interferons . On the other hand, other genes, such as Integrin Subunit Alpha M (ITGAM), have been shown to regulate neutrophil migration and mediate the adhesion of neutrophils to pathogens leading to pathogen clearance in bacterial infections . Other assays aimed at understanding the host response to different etiologies include flow cytometry. Flow cytometry can serve as an assay for stratification and differentiating the etiology of fever. Surface markers such as CD64 on neutrophils and CD169 on monocytes have been shown to serve as sensitive markers to differentiate bacterial infections from viral infections This prospective study aims to understand the transcriptional gene changes of circulating white blood cells in admitted patients with undifferentiated febrile syndromes. Using a PCR-based prototype assay (GeneXpert) capable of accurately measuring five mRNA transcripts directly from whole blood, the primary objective of this study is to determine the ability to differentiate infectious versus non-infectious febrile syndromes in patients as compared to the results of traditional pathogen-based microbiology tests in an adjudicated cohort. Additionally, we explored surface maker determination using flow cytometry to detect potential surface markers that predict the etiology of fever, we aim to evaluate the sensitivity and accuracy of such markers to understand febrile syndromes better. Finally, patient pathways from the Emergency Department (ED) arrival to the inpatient unit admission were collected to map out and determine critical time points where such host-based diagnostic may have the highest potential for improving clinical decision-making for febrile patients. 2. Materials and Methods 2.1. Study Design and Study Participants The study included 100 febrile patients recruited from a single site at Massachusetts General Hospital (MGH), and 22 healthy controls enrolled through an on-campus primary care clinic. The study was conducted according to the guidelines of the Declaration of Helsinki, and approval was obtained from the MGH institutional review boards (IRB) protocol, approval number: 2021P0003374. Informed consent was obtained from all subjects participating in the study. The research investigators designed the study, collected the data, and performed the analysis. DH Diagnostics LLC provided unrestricted funding and the GeneXpert(r) system required to complete the study but was not involved in data interpretation, analysis, or assembling the manuscript. 2.2. Patients Potential subjects were detected daily through generated screening EPIC reports that detect patients admitted to the MGH ED with a documented fever. Patients were enrolled within 24 h after ED presentation, and blood samples were collected within 48 h after ED presentation. Patients were considered eligible for recruitment if they were greater than 18 years of age, had a documented fever >38 degC, and had an ongoing work-up to determine the etiology of the fever initiated. Patients were excluded from the study in the case of pregnancy. Subjects were enrolled from March 2022 till October 2022. 2.3. The Prototype Host Response Assay Protocol A prototype host response assay that integrates sample preparation and RT-PCR to measure 6-mRNA genes on the GeneXpert system was developed. The prototype assay measures five mRNA targets (RUNX1, LY6E, IRF9, ITGAM, PSTPIP2) and a control (ABL1) chosen from the literature based on their combined ability to distinguish infectious from non-infectious illness in patients with fever . Assay testing was performed in the Division of Infectious Diseases at Massachusetts General Hospital in Boston, MA. A total of 20 cc of venous blood was collected in ethylenediaminetetraacetic acid (EDTA) tubes from the enrolled subject. A total of 200 mL of blood was added to the Xpert Lysis Reagent. Next, 1 mL of blood/lysis mixture was added to the GeneXpert cartridge and loaded into the GeneXpert system for processing. The turnaround time for assay results is 35 min. . 2.4. Preparation of Human Neutrophils Healthy blood donors consented under the MGH IRB protocol, approval number: 2018P001283. Whole blood was collected in EDTA-coated vacutainers (Beckton Dickinson, Franklin Lakes, NJ, USA) and subsequently centrifuged at 1500x g for 15 min. Buffy coat was collected, and neutrophil isolation was performed using the negative selection EasySep Direct Human Neutrophil Isolation Kit, according to the manufacturer's instructions (STEMCELL Technologies, Seattle, WA, USA). Wright-Giemsa staining was performed after the isolation process to confirm neutrophil purity from the isolation kit. Flow cytometry was also used to verify a high neutrophil purity from the isolation procedure (>=94% neutrophil purity). Cell concentration and viability were measured by staining the cells with a 1:10 dilution of acridine orange/propidium iodide followed by automatic cell counting using the LUNA fl Dual Fluorescence Cell Counter (Logos Biosystems, Annandale, VA, USA) (>=99% live). 2.5. Flow Cytometry Isolated neutrophils (50,000 cells) were stained in fluorescent activated cell sorting (FACS) buffer containing 2% heat-inactivated fetal bovine serum (FBS) (Life Technologies, Dun Laoghaire, Ireland) and 1 mM EDTA (Life Technologies) in phosphate buffer saline (PBS) without calcium and magnesium (Corning, Corning, NY, USA). Cells were stained with antibodies for 30 min at 4 degC in FACS buffer containing (BV421) anti-CD10 (1:200 dilution; clone HI10a; BioLegend, San Diego, CA, USA) and (BV786) anti-CXCR4 (1:100 dilution; clone12G5; BioLegend) and (AF647) anti-CD64 (1:100 dilution; clone 10.1; BioLegend) or (BV786) anti-CD63 (1:100 dilution; clone H5C6; BioLegend) and (APC) anti-CD66b (1:800 dilution; clone G10F5; BioLegend), or (BV421) anti-CD62L (1:200 dilution; clone DREG-56; BioLegend), and (PE/Dazzle) anti-CD32 (1:200 dilution; clone FUN-2; BioLegend). Data were acquired on a BD FACSCelesta (BD Biosciences, San Jose, CA, USA) with a BVR laser configuration (488 nm, 405 nm, 640 nm). Before recording data, gates were prepared so that 10,000 neutrophil events could be collected. FCS files were exported from BD FACSDiva Software (BD Biosciences) in a 3.0 format. FCS files were analyzed using FlowJo v.10 software (BD Biosciences). 2.6. Outcomes The primary outcome of the study is to determine the efficiency of the GeneXpert prototype assay monitoring host-response gene signatures in correctly predicting the etiology of fever when compared to microbiology results. The expression of the genes stratified by the different groups measured by delta Ct (ABL1 Ct value--Target Ct value), the correlation of the five genes with each other, and their predictive performance of the etiology of fever will be evaluated. Microbiology results were defined as the results of blood and urine cultures and viral panels. Febrile patients were categorized among two groups based on their infection status, and healthy controls were categorized as a third group. Febrile Positive (FP) group (n = 74): Febrile patients with confirmed positive microbiology results and clinically adjudicated febrile patients with suspected infections by clinical assessment despite the absence of positive microbiology results were categorized into one group of positive composite outcomes labeled FP. Clinical adjudication was performed by manual physician chart review. Febrile Negative (FN) group (n = 26): Febrile patients with negative microbiology results and the absence of suspected infection by clinical assessment were categorized into a second group labeled FN. Healthy Control (HC) group (n = 22): Healthy subjects presenting to the primary clinic for routine annual laboratory testing were categorized into a third group labeled HC . 2.7. Statistical Analyses Descriptive measures such as median, interquartile range, frequencies, and percentages were used to summarize the data. Exact binomial confidence intervals were used to estimate confidence intervals for sensitivities and specificities. Wilcoxon rank-sum and Fisher exact tests were used to compare continuous and categorical study variables, respectively, between febrile positive and negative patients. For comparing continuous outcomes among the three groups (healthy, febrile negative, and febrile positive patients), the Kruskal-Wallis test with Dunn's post-hoc analysis was used. Spearman's rank correlation was used to assess the strength and direction of association between the study variables. Univariate logistic regression models were utilized to assess the predictors of being a febrile positive. The least absolute shrinkage and selection operator (Lasso) logistic regression algorithm was performed to select the most predictive variables of febrile positive. We estimated the predictive accuracy of the selected variables in distinguishing class membership (febrile negative or positive) using several machine-learning algorithms, including linear discriminant analysis (LDA), quadratic discriminant analysis (QDA), k-nearest neighbor (KNN), support vector machine (SVM), classification tree (CART), AdaBoost (ADA), neural networks (NNET), random forest (RF), Gaussian process and logistic regression. The leave-one-out cross-validation procedure was used to estimate the performance of the classifier algorithms. We used the algorithm with the highest cross-validated area under the receiver operating curves to evaluate the performance of the selected variables as biomarkers of febrile-positive patients. Statistical analyses were performed using the R package version 4.2.1 and SAS software version 9.4 (SAS Institute, Cary, NC, USA). All p values were 2-sided and considered statistically significant if <0.05. 3. Results 3.1. Patients The median time for running the assay for all study subjects after blood collection was 3 h, and the average time was 3.4 h. The baseline characteristics of the patients are shown in Table 1. A total of n = 122 subjects were enrolled in the study and divided into three groups. The first group was the FP group, which comprised 74 patients (60.7%). Among the FP group, 45 patients (60.8%) were males. The confirmed infections included 57 bacterial and 17 viral infections. The bacterial infections included 17 urinary tract infections, 24 bloodstream infections, three cellulitis infections, two cholangitis infections, two pelvic inflammatory disease infections, one hepatic abscess infection, seven community-acquired pneumonia infections, and one endocarditis infection. The urinary tract and bloodstream infections were caused by Gram-positive (43%) and Gram-negative pathogens (57%). On the other hand, the viral infections included eight SARS-CoV-2 (COVID-19) infections, three influenza A infections, three rhinovirus infections, one human metapneumovirus infection, one Epstein-Barr virus (EBV) infection, and one parainfluenza virus infection. The median age was 63 (interquartile range, 44-73). Thirty percent (29.7%) of the patients had a body-mass index (the weight in kilograms divided by the square of the height in meters) >30. In total, 13.5% of the patients had known diabetes mellitus, and 10.9% had a history of previous lung disease (e.g., asthma, COPD). A total of 15 patients (20.3%) had a history of malignancy, 22 patients (29.7%) had an active malignancy, 17 patients (23%) had congestive heart failure, 20 patients (27%) had a history of recurrent infections, and two patients (2.7%) had liver disease/cirrhosis. A total of 48 patients (64.9%) did not require supplemental oxygenation at day 1, 23 patients (31.1%) were receiving supplemental oxygen at <=6 L per minute, delivered by nasal cannula, to maintain an oxygen saturation >92%, and three patients (4%) were receiving high-flow oxygen. The median white blood cell (WBC) count for the FP group was 12.71 x 109/L (interquartile range: 6.2-16.2 x 109), and the median C-reactive protein (CRP) was 149.25 mg/L (interquartile range: 54.3-261.3 mg/L). The second group was the FN group, which comprised 26 patients (21.3%). Among the FN group, 17 patients (65.4%) were males. The median age was 57.5 (interquartile range, 27-66). The number of patients with a body-mass index (the weight in kilograms divided by the square of the height in meters) >30 was 7 (26.9%). In total, 15.4% of the patients had known diabetes mellitus, and 3.8% had a history of previous lung disease (e.g., asthma, COPD). One patient (3.8%) had a history of malignancy, ten patients (38.5%) had an active malignancy, two patients (7.7%) had congestive heart failure, four patients (15.4%) had a history of recurrent infections, and two patients (7.7%) had liver disease/cirrhosis. A total of 23 patients (88.4%) did not require supplemental oxygenation on day 1, 2 patients (7.7%) were receiving supplemental oxygen at <=6 L per minute, delivered by nasal cannula, to maintain an oxygen saturation >92%, and 1 patient (3.8%) required mechanical ventilation. The median white blood cell (WBC) count for the FN group was 9.37 x 109/L (interquartile range: 4.72-12.56 x 109), and the median C-reactive protein (CRP) was 96.15 mg/L (interquartile range: 47.55-148.1 mg/L). The third group was the HC group, consisting of 22 patients (18%). Among the HC group, six patients (27.3%) were males. The median age was 57 (interquartile range, 43-68). The number of patients with a body-mass index (the weight in kilograms divided by the square of the height in meters) >30 was 8 (36.4%). In total, 18.2% of the patients had known diabetes mellitus, three patients (13.64%) had a history of malignancy, and one patient (4.5%) had an active malignancy. (See Table 1). 3.2. Network Structure Correlation of the Five Genes in the FP, FN, and HC Groups To understand the network structure of the five genes, correlation plots between the gene signatures delta Ct in the three different groups were performed. The results showed that the network structure representing the correlation of the five genes for febrile patients with positive microbiology (FP) or with negative microbiology (FN) using the GeneXpert prototype assay was significantly more robust than the network structure of the five genes in the HC group. In the febrile patients' (FP and FN) group, a strong correlation with a positive correlation coefficient is demonstrated between the five genes. The FP group's five genes (ITGAM, IRF-9, LY6E, PSTPIP2, and RUNX1) were significantly correlated with each other (p-value < 0.001). Similarly, the FN group's five genes (ITGAM, IRF-9, LY6E, PSTPIP2, and RUNX1) were also significantly correlated with each other (p-value < 0.001). No significant difference in the association among the five genes was noted between the two groups of febrile patients. On the other hand, in the HC group, a weak network structure with low correlation coefficients was noted between the five genes. Except for LY6E and IRF-9, which showed a significant correlation with a p-value < 0.05, the other gene signatures do not demonstrate any significant correlation. 3.3. Expression Profiles of the Genes among Febrile and Non-Febrile Cohorts To further investigate how the etiology of fever changes the host immune response, we next compared the gene expression profiles of matched subjects from the three different patient groups. We compared the expression of the five genes across the three different groups. Our analysis revealed that patients with confirmed infections (FP group) form a distinct cluster with higher expression of the five genes, demonstrating the effect of bacterial and viral infections on modifying the host response in febrile subjects. The analysis identified higher expression of the five genes in the FP group as compared to the FN and HC groups. Two clusters of genes were noted in the FP group. ITGAM, RUNX1, and PSTPIP2 formed one cluster with high expression in subjects with confirmed bacterial infections, whereas LY6E and IRF-9 formed another cluster with high expression, mainly in patients with confirmed viral infections. Moreover, for patients in the HC group, the expression levels of the five genes are lower than in other groups and are detected at low levels, implicating the role of fever on the immune response. . 3.4. Predictive Performance of the GeneXpert Assay Figure 5 displays the odds of being febrile with positive microbiology. In addition to the five genes, several other covariates, including age, gender, disease severity, cancer status, durations of fever, presence of fever on blood collection day, and antibiotics administration duration, were considered. The higher expression of the five genes was associated with higher odds of being febrile-positive patients: IRF-9 (OR = 1.750, 95% CI = 1.16 to 2.638), ITGAM (OR = 1.533, 95% CI = 1.047 to 2.244), PSTPIP2 (OR = 2.191, 95% CI = 1.293 to 3.711), and RUNX1 (OR = 1.974, 95% CI = 1.069 to 3.646) had a significant expression with positive infection status. The odds of being febrile-positive were associated with higher values of the LY6E gene, although it did not reach statistical significance (OR = 1.11, 95% CI: 0.91-1.35). Considering the differences in antibiotic exposure duration from the ED admission to the time of blood collection, and the potential effects of longer antibiotic usage on altering the gene expression profiles, stratification by antibiotic duration was also performed. Two groups of subjects were evaluated: those receiving antibiotics for two or fewer days before collecting blood and running the assay, and those receiving antibiotics for more than two days before collecting blood and running the assay. The results demonstrate that subjects receiving antibiotics for two or fewer days before running the assay (OR = 6.682, 95% CI = 2.452 to 18.206) were more significantly associated with positivity in infection status. The difference in the distribution of the fives genes varied by the duration of antibiotics usage, as shown in the density plots displayed in Figure 6. As expected, the differences between gene expression of febrile-positive and febrile-negative subjects are more noticeable in the shorter antibiotic usage category. The other parameters did not demonstrate any statistically significant association with infection status. We used machine-learning algorithms to develop a classifier model to classify study participants into groups based on the significant variables presented in Figure 5. The classifier model assessing the performance of the five genes in subjects receiving antibiotics for less than two days correctly classified more than 80% of the participants into their respective groups, i.e., FP or FN groups. The classifier correctly classified 65% [95% CI: 0.54-0.75] of the FP subjects and 89% [95% CI: 0.71-0.96] of the FN subjects. 3.5. Predictive Performance of Surface Markers to Determine Infection Status Using flow cytometry to detect specific markers that can predict infection in 20 febrile patients of our study subjects (13 FP and 7 FN) and 9 HC subjects, three different surface markers, CD10 (percent positive), CD64 mean fluorescence intensity (MFI), and CXCR4 MFI demonstrated a significant distinction between the FP and FN groups. The expression of CD10 and CXCR4 were significantly lower in the FP group compared to the FN group. In contrast, the expression of CD64 was significantly higher in the FP group compared to the FN group . 3.6. Patient Care Pathways Different pathways mapping the clinical course of patients (n = 70) from the point of the initial evaluation in the ED arrival until the inpatient unit admission were collected to determine critical time points in clinical decision-making. Such pathways provided a framework where the implementation of the GeneXpert prototype assay could be best utilized. Average times to first labs, imaging, cultures, and antibiotics were collected for FP patients with bacterial infections (n = 44), FP patients with viral infections (n = 12), and FN patients (n = 14). The following timepoints were collected for the enrolled subjects prior to the establishment of a definite diagnosis. All patients were being managed for possible infections based on their febrile illness. The results show that the average times to the first labs are 1.09 h, 1.3 h, and 2 h for patients with bacterial, viral, and no infections, respectively. The average times for the first cultures performed were 2 h, 2.1 h, and 2.8 h for patients with bacterial infections, viral infections, and no infections, respectively. The average times to first imaging performed were 2.4 h, 2.6 h, and 3.5 h for patients with bacterial infections, viral infections, and no infections, respectively. Finally, the average times to first antibiotics administration were 4.9 h, 4.2 h, and 3.9 h for patients with bacterial infections, viral infections, and no infections, respectively. There were no significant differences between any of the average times among the three different groups indicating the need for earlier diagnostic to guide better management of febrile etiologies. It is noteworthy to mention that out of twelve patients with viral infections, eight patients (67%) received antibiotics in the ED prior to viral diagnostics indicating a viral pathogen. Four out of these eight (50%) were continued on antibiotics after the viral diagnostics turned positive due to concern for bacterial superinfection. Out of 14 patients with no infections (FN), 7 patients (50%) received antibiotics in the ED before ruling out infectious etiologies, and out of 44 patients with bacterial infections, 43 patients (97.7%) received antibiotics in the ED. . Moreover, the average time from ED arrival to inpatient admission was collected for these patients. The average times to admission were 21.1 h, 18.6 h, and 23.4 h for patients with bacterial infections, viral infections, and no infections, respectively. . 4. Discussion In this prospective study, a robust predictive performance in differentiating the etiology of febrile syndromes has been demonstrated by the GeneXpert prototype assay. Such results reveal promise for using this assay for accurate diagnostic outcomes in the future. The rapid turnaround time of the assay (35 min) and the simple sample preparation protocol could provide additional potential for future implementation of such a modality from both clinical and possibly economic perspectives. Notably, the role of each of the five genes (ITGAM, IRF-9, LY6E, PSTPIP2, and RUNX1) has been linked to response to infection in the literature. To start with, ITGAM is known to promote the adherence of monocytes and macrophages and to mediate the uptake of opsonized particles and pathogens . In fact, in one recent study, using a four-gene signature that includes ITGAM and three other genes has demonstrated a promising model to diagnose patients with sepsis . Additionally, IRF-9 has also been shown to be an integral transcription factor in mediating the type I interferon antiviral response, and the expression of IRF-9 plays an essential role in antiviral immunity . In a recent case report, a five-year-old child with IRF-9 deficiency experienced severe influenza pneumonitis, further highlighting IRF-9's role in antiviral immunity . Similarly, Ly6E genes have also been shown to possess an antiviral regulation response. Ly6E confers critical antiviral functions by restricting the entry of human coronaviruses, including SARS-CoV, MERS-CoV, and SARS-CoV-2, by interfering with spike protein-mediated membrane fusion . Interestingly, our results revealed two gene expression clusters in patients with confirmed infections. ITGAM, RUNX1, and PSTPIP2 formed one cluster with high expression in subjects with confirmed bacterial infections, whereas LY6E and IRF-9 formed another cluster with high expression in patients with confirmed viral infections. These findings further support the different roles of the host-response gene signatures regarding the etiology of infections. In this study, flow cytometry was performed to evaluate the role of host surface markers in response to infections. The results showed that three different surface markers, CD10, CD64, and CXCR4, significantly differentiate between the presence and absence of infection in patients with febrile illnesses. In a previous study, the role of CD10 expression in sepsis patients was described, where CD10 and CD66b were shown to be effective biomarkers and good predictors for early bacterial infections in patients with suspected sepsis. When compared to the performance of procalcitonin and CRP, the accuracy of CD10 and CD66b expression for predicting bacterial infections was significantly higher, with a sensitivity of 86.5% and a specificity of 90.3%. . Other studies have described the role of CD64 in differentiating bacterial and viral infections . The immunologic assays performed in this study were exploratory. Consequently, future considerations directed at understanding the role of cell surface markers in the setting of infections can provide insight into developing a combined model that detects gene signatures and surface markers for more accurate diagnostic performance. Larger sample sizes will be ultimately needed to run such a classifier model. Thus, from a clinical perspective, the development of this promising five-gene signature assay has the potential to serve as a guide for better patient outcomes. The rapid and accurate differentiation between infectious and non-infectious etiologies in patients presenting with fever can lead to more optimal administration of antibiotics and a reduction in antimicrobial resistance . Additionally, the results from this study suggest a potential effect of longer antibiotic administration duration on the expression profiles of the five genes. The prototype assay demonstrated more accurate performance for febrile subjects receiving shorter antibiotic duration before running the assay , attributed to possible gene expression changes in response to exposure to antimicrobials. Moreover, patient pathways collected in this study show that 50% of patients with no infections and 67% of patients with viral infections receive unnecessary antibiotics in the ED due to the absence of a rapid modality that can differentiate the etiology of fever . The implications of our findings suggest that the optimal implementation of such a diagnostic would be in the ED setting before the administration of antibiotic therapy. Such an implementation has the potential to improve patient outcomes and provide optimal antibiotic use. The reduction in antibiotic usage, in addition to reducing antimicrobial resistance, ultimately leads to a reduction in antibiotics-associated gastrointestinal, dermatologic, musculoskeletal, hematologic, renal, cardiac, and neurologic adverse events . Finally, from an economic perspective, such a modality could provide valuable decision-making guidance regarding unnecessary hospital admissions for the management of infections for patients with low suspicion of infectious etiologies. In a recent study of inpatient hospital costs for COVID-19 patients in the United States, the overall median cost of a hospital stay per day was shown to be more than $1700 USD, while the overall median cost of an ICU admission per day was shown to be approximately $3000 USD . Thus, the reduction in patients' hospital length of stay duration could potentially lead to significant healthcare savings . Additional reductions in healthcare costs associated with adopting such an assay include a decline in the number of blood cultures drawn and fewer antibiotics being prescribed, decrease in unnecessary laboratory tests, imaging, and procedures . Future interventional trials will be required for validation. In short, attempts have been focused on using the host response as a reference for a more personalized approach to precision medicine, taking into account its impact. By understanding the mechanisms underlying the host-immune response using specific signatures and robust diagnostics, we can achieve better medical decision-making guidance that ultimately leads to better patient outcomes. Limitations Our study should be interpreted in the setting of several limitations. One significant limitation is regarding the classification of subjects into different groups. Patients with suspected, and often apparent, infections such as cellulitis or community-acquired pneumonia had no cultures taken and no confirmed positive results. Thus, for the accurate classification of these patients, the clinical assessment of the medical team was followed. Moreover, the variability in the duration of antibiotic therapy received by the patients before the blood sample collection may present a confounding variable that alters the host's immune response. Additionally, the GeneXpert prototype demonstrated lower predictive performance for patients on longer antibiotic duration suggesting a limited optimal timing for implementation of this diagnostic. Another limitation of the study is that the GeneXpert is designed to detect the expression of genes that point toward bacterial and viral infections. Its performance in detecting fungal and parasitic infections is yet to be evaluated. The lack of an adequate sample size to perform subgroup analysis between bacterial and viral infections poses an additional limitation. Larger cohorts will be required to perform any subgroup analysis for differentiating the microbiological etiology. Finally, the results presented in this manuscript are based on a single study center. A multi-center study might be essential to attain a larger and more diverse sample size. 5. Conclusions The implementation of novel rapid host-based diagnostics, such as the GeneXpert prototype assay and flow cytometry for patients with febrile syndromes has the potential to reduce adverse events, decrease the misuse of antibiotics, and lower the rate of emerging antimicrobial resistance. Furthermore, such modalities have the potential to reduce healthcare costs and inform clinicians about the optimal utilization of resources from an economic perspective. Future directions could be directed toward launching an interventional trial to assess the efficacy of the assay in reducing healthcare costs and patient adverse outcomes. Additionally, enrolling a larger sample size for subgroup analysis to assess the performance of the GeneXpert in differentiating bacterial vs. viral infections will be necessary. Finally, the future development of an exploratory model using the multi-modality host-based assays including flow markers and best-performing gene signatures may provide improved diagnostic accuracy in the management of the undifferentiated febrile patient. Author Contributions Conceptualization, M.K.M., J.A. and F.R.S.; methodology, J.A., K.D.T., E.M., E.W., F.R.S., D.H.P. and M.G. data curation, J.A. and M.K.M.; formal analysis, M.G. and H.M.W.; investigation, M.K.M., J.A. and F.R.S.; resources, M.K.M., E.W., F.R.S. and D.H.P.; software, M.G. and H.M.W.; writing--original draft preparation, J.A. and M.K.M.; writing--review & editing, M.K.M., J.A., E.W., F.R.S., D.H.P. and M.G.; supervision, M.K.M.; project administration, M.K.M. and F.R.S.; funding acquisition, M.K.M. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The study was conducted according to the guidelines of the Declaration of Helsinki, and approval was obtained from the MGH institutional review boards (IRB) protocol, approval number: 2021P0003374, date of approval: 22 December 2021. Informed Consent Statement Informed consent was obtained from all subjects involved in the study. Data Availability Statement The data underlying this article are available in the article. Conflicts of Interest M.K.M. reports consultation fees from Vericel, Pulsethera, NED biosystems, GenMark Diagnostics, Clear Creek Bio, and Day Zero Diagnostics; unrestricted grant support from Thermo Fisher Scientific, Genentech and Danaher Corporation; and medical editing/writing fees from UpToDate, outside the submitted work. E.W., D.H.P. and F.R.S. are employees at Cepheid and Danaher Corporation. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors declare no restrictions on the availability of materials or information. Grant number and sources of support: this work was supported, in part, by the National Institutes of Health RO1 AI132638 and by an unrestricted grant from Danaher Corporation, both to M.K.M. Abbreviations RT-PCR Reverse transcription-polymerase chain reaction IRF-9 Interferon Regulatory Factor 9 Ly6E Lymphocyte Antigen 6 Family Member E ITGAM Integrin Subunit Alpha M ED Emergency Department IRB Institutional review boards MGH Massachusetts General Hospital HC Healthy controls EDTA Ethylenediaminetetraacetic acid SNI Symptomatic but non-infectious PSTPIP2 Proline-Serine-Threonine Phosphatase Interacting Protein 2 FACS Fluorescent activated cell sorting FBS Fetal bovine serum PBS Phosphate buffer saline ABL1 ABL proto-oncogene 1, non-receptor tyrosine kinase FP Febrile positive FN Febrile negative EBV Epstein-Barr virus LDA Linear discriminant analysis QDA Quadratic discriminant analysis KMN K-nearest neighbor SVM Support vector machine CART Classification tree ADA AdaBoost NNET Neural networks RF Random Forest CRP C-reactive protein WBC White blood cell OR Odds ratio CI Confidence Interval MFI Mean fluorescence intensity Figure 1 Steps for running the prototype GeneXpert assay. Figure 2 Study Flowchart. Figure 3 Correlation plots between the five genes in (A) FP group, (B) FN group, and (C) HC group. The size of the circle indicates the strength of the correlation, with the larger circle representing a stronger correlation. The value inside the circle represents the significance level (*** < 0.001; ** < 0.01, * < 0.05). Figure 4 Heat map of the five expressed genes from the FP group, FN group, and HC group. Rows represent the subjects, and columns represent genes. Gene expression levels are color-coded; red and blue represent high (+4) and low (-4) expression levels, respectively. Figure 5 Forest plot presenting odds of FP. Figure 6 Density plots displaying distributions of the five genes in the FP, FN, and HC groups classified by antibiotic duration status. Figure 7 Distribution of surface markers using flow cytometry for FP, FN, and HC group. Figure 8 (a) Primary care pathways for the FP, and FN groups from ED arrival to admission represent the time to first labs, first cultures, first imaging, and first antibiotics. Large circles with black borders represent average hours. Small circles represent the timepoints for each subject for the FP and FN groups. (b) Average time from ED arrival to admission for the FP and FN groups. Red circles represent average hours for time to admission. Blue circles represent the time from ED arrival to admission for each subject for the FP and FN groups. diagnostics-13-00953-t001_Table 1 Table 1 Patient demographics and characteristics. Characteristic Febrile Positive Febrile Negative Healthy Controls Total No. (%) No. (%) No. (%) No. (%) (n = 74) (n = 26) (n = 22) (n = 122) Demographics Age, Median (IQR) 63 (44-73) 57.5 (27-66) 57 (43-68) 60.5 (41-70) Men 45 (60.8) 17 (65.4) 6 (27.3) 68 (55.7) BMI > 30 22 (29.7) 7 (26.9) 8 (36.4) 37 (30.3) Race Non-Hispanic 72 (97.3) 26 (100) 22 (100) 120 (98.36) Hispanic 2 (2.7) 0 (0) 0 (0) 2 (1.64) Oxygen Requirement at day 1 No requirement 48 (64.9) 23 (88.4) 22 (100) 93 (76.2) Nasal Cannula 23 (31.1) 2 (7.7) 0 (0) 25 (20.5) High Flow Nasal Cannula 3 (4) 0 (0) 0 (0) 3 (2.5) Intubation 0 (0) 1 (3.8) 0 (0) 1 (0.8) Medical History Diabetes 10 (13.5) 4 (15.4) 4 (18.2) 18 (14.8) COPD 8 (10.9) 1 (3.8) 0 (0) 9 (7.4) History of Malignancy 15 (20.3) 1 (3.8) 3 (13.64) 19 (15.6) Hypertension 25 (33.8) 7 (26.9) 5 (22.7) 37 (30.3) Organ Transplant 7 (9.5) 0 (0) 0 (0) 7 (5.7) Coronary artery disease 7 (9.5) 0 (0) 0 (0) 7 (5.7) Heart Failure 17 (23) 2 (7.7) 0 (0) 19 (15.6) Active Malignancy 22 (29.7) 10 (38.5) 1 (4.5) 33 (27) CKD 9 (12.2) 0 (0) 1 (4.5) 10 (8.2) Liver Disease/Cirrhosis 2 (2.7) 2 (7.7) 0 (0) 4 (3.3) Recurrent Infections 20 (27) 4 (15.4) 0 (0) 24 (19.7) Substance use disorder 2 (2.7) 2 (7.7) 0 (0) 4 (3.3) Neurological Disease 9 (12.2) 2 (7.7) 0 (0) 11 (9) Median Laboratory Values (IQR) WBC Count - x 109/L 12.71 (6.2-16.2) 9.37 (4.72-12.56) N/A 11.45 (5.76-15.78) C-reactive protein 149.25 (54.3-261.4) 96.15 (47.55-148.1) N/A 136.05 (54.3-242.7) Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). 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PMC10000762 | Healthcare (Basel) Healthcare (Basel) healthcare Healthcare 2227-9032 MDPI 10.3390/healthcare11050682 healthcare-11-00682 Article The Mobile Patient Information Assistant (PIA) App during the Inpatient Surgical Hospital Stay: Evaluation of Usability and Patient Approval Gortz Magdalena Conceptualization Methodology Formal analysis Writing - original draft Visualization Project administration 12* Wendeborn Antonia Investigation Data curation Writing - review & editing 3 Muller Michael Software Resources Writing - review & editing Visualization 4 Hohenfellner Markus Conceptualization Resources Writing - review & editing Supervision 1 Maglaveras Nicos K. Academic Editor 1 Department of Urology, University Hospital Heidelberg, 69120 Heidelberg, Germany 2 Junior Clinical Cooperation Unit 'Multiparametric Methods for Early Detection of Prostate Cancer', German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany 3 Medical Faculty, Ruprecht-Karls University of Heidelberg, 69117 Heidelberg, Germany 4 mbits Imaging GmbH, 69115 Heidelberg, Germany * Correspondence: [email protected] 25 2 2023 3 2023 11 5 68226 12 2022 14 2 2023 21 2 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). Mobile eHealth apps are becoming increasingly important tools in healthcare management, capable of providing education and support at any time. There is little knowledge about surgical patients' appreciation and use of these apps. The objective of this study was to develop and evaluate a user-friendly medical app (PIA; Patient Information Assistant) for providing individual patient information before and after inpatient urological surgery. Twenty-two patients aged 35 to 75 years were provided with timely information, push notifications, and personalized agendas (e.g., date of presentation, time of surgery, time of doctor's consultation, imaging appointment) via the PIA app. Of the 22 patients, 19 evaluated the PIA app in terms of usage and usability, benefits, and potential for improvement. Of the study participants, 95% did not need any assistance to use the app, 74% confirmed that the PIA app made them feel better informed and more satisfied with their hospital stay, and 89% stated that they would like to re-use the PIA app and support the general use of medical apps in healthcare. Thus, we created an innovative digital health information tool, allowing targeted support for doctor-nurse-patient communication and offering great potential for patient support before and after surgery. Our study revealed that use of an app during the surgical hospital stay is readily accepted and benefits patients by acting as an additional informative tool. electronic health hospital medical app mobile health surgery telemedicine mbits imaging GmbHThis research received no external funding. The app development was completely financed by the company mbits imaging GmbH to explore and evaluate the business case of an app that supports patients' surgical journey. pmc1. Introduction The healthcare industry is on the precipice of widespread digitization. The last decade brought great increases in electronic health record adoption and attestation to the use of defined documentation practices that are key to realizing the value of technology-enabled healthcare . Health information technology describes the hardware, software, users, implementation, inputs, data, and outputs of computerized systems in the healthcare delivery environment. It takes many forms in healthcare organizations, such as patient health records, patient portals, computerized physician order entry systems, electronic prescribing, lab and radiology information systems, clinical data repositories, business intelligence applications, and health information exchanges . The increasing adoption of various health information technologies has created new channels for doctor-patient communication, especially when the communication of information is critically important, such as during cancer therapy. By improving access to care, health information technology solutions were proven to enhance care delivery efficacy and make patients feel more confident and empowered, which supported their active involvement in the process, creating better relationships with the healthcare team . Other promising areas of health information technology applications are the use of telehealth for pediatric patients with medical complexity and the use of wearable healthcare devices for the self-monitoring of geriatric patients on a real-time basis . Health information technology does offer the potential for transforming the healthcare delivery system and improving the health of patients, including those with urologic diseases . In 2012, the European Commission announced the eHealth Action Plan 2012-2020 as an agenda to empower patients and healthcare workers, link up devices and technologies, and invest in research towards the personalized medicine of the future . Because of its ease of use and broad acceptance, mHealth is considered to be a valuable tool in the implementation of patient-centered care, which has become a central goal of healthcare systems and international standards . Precise definitions of eHealth and mHealth vary because they are applied in differing contexts and because of the ongoing advancements of information technologies that lead to new applications for eHealth and mHealth . However, eHealth can be defined broadly as "the use of information and communications technology, especially the Internet, to improve or enable health and healthcare" and mHealth, which is a subdivision of eHealth, as "medical and public health practice supported by mobile devices, such as mobile phones, patient monitoring devices, personal digital assistants, and other wireless devices" . mHealth includes all telecommunication devices for the transfer of healthcare information between participants at different locations, especially when referring to services that are mobile and wireless . eHealth and mHealth tools have the potential to improve the quality of healthcare and reduce healthcare costs . Educating patients with timely medical information delivered via their smartphones improves their levels of knowledge, treatment adherence, satisfaction, and clinical outcomes, as well as having positive effects on healthcare economics . mHealth tools have further value because only a limited amount of new medical information can be correctly processed by the patient after a consultation, and mHealth apps can be used at any time and in any place . This is particularly relevant in surgical treatment: healthcare professionals provide patients with information about treatment options, outcomes, how to prepare for surgeries, and advice for the recovery phase. Apps allow patients to revisit comprehensive medical information as often as they need, and push notifications allow healthcare providers to actively educate patients with timely information when details become relevant . Consequently, ongoing advances in smartphone technology and healthcare apps could play important roles in enhancing patient-centered care in the future. There are currently over 300,000 mHealth apps available in the Apple App Store and Google Play Store . An important motivator for health app users is the accessibility of specific information that can increase their knowledge about their conditions. However, it is important to ensure that this information is up-to-date, trustworthy, and valid . Despite the increasing mHealth market, there is a lack of involvement of healthcare professionals in the design of medical apps, including in urological surgery . Previous research showed that the number of trustworthy and high-quality apps on offer is extremely low. In former studies, most of the available medical apps were considered inappropriate for use in daily clinical practice, or their purpose had not been evaluated in studies . In Germany, a previous multicenter study reported that, so far, only a minority of the population used medical apps. In detail, even as much as approximately 90% of the patients owned a smartphone, while only 11% of the patients reported previous medical app usage, and only 3.5% of the patients received an app recommendation from a physician . With the PIA (Patient Information Assistant) app, we developed a user-friendly mobile app for patients in need of inpatient urological surgery to provide high-quality, timely information in the perioperative period. The purpose of the app was to be a 24-hour companion to help prepare patients for hospitalization, enhance patient knowledge about the ward routine, and encourage patients to follow postoperative behavioral recommendations at home. The main goals of this study were to assess the user-friendliness and the patient's acceptance of the PIA app, as well as positive and negative user experiences. 2. Materials and Methods 2.1. App Development The PIA app was developed in cooperation between the Urology Clinic of Heidelberg University Hospital and the software developer mbits imaging GmbH. The design and implementation process of the PIA app was based on well-established structured software design principles. First, the basic needs and requirements from the perspective of the clinicians were assessed by contextual inquiries with the different stakeholders. Contextual inquiries are a tool within the software design process in which a potential user of the system is interviewed on the distinctive steps of the corresponding real-world process . The notable idea of the contextual inquiries is to decouple the interviewee from the technical aspects of the software that will be developed. By employing this tactic, requirements are recorded solely on the causal and temporal conditions of a real-world process rather than on technical implementations that are often confused with user needs. Second, a specification was created from the process definition which described the different parts of the app. The specification, therefore, contained a formal description of the tech stack that would be used alongside with implementation details such as the program flow and the user interface of the app. To address the importance of an adequate user interface for patients, several sketches were created and reworked prior to any implementation. All stakeholders and the development team refined the specification draft during several main meetings until there was a common agreement on a final version. Doctors from the Urology Clinic at Heidelberg University Hospital were the initiators and drivers of the app development, and they played a major role in determining the content of the app. All content of the app was designed with the experience of the urological doctors and nurses at the respective hospital who knew what patient information was important, often forgotten, or frequently requested. The content of the PIA app was created specifically for the various urological surgeries conducted in the hospital by the medical team. The pure implementation took approximately two weeks of full-time work by a team of two experienced developers. The whole process was accompanied by several meetings with clinicians to review different implementation versions. Briefly, there were three main alpha versions of the app or "release candidates" until a final version was reached. The versions were tested on a small, selected cohort of patients before the study was conducted. The feedback and improvement recommendations from these test runs were incorporated into the development of the final version of the PIA app. The final version was then validated by the clinicians, i.e., whether it could fulfil the real-world process requirements. Afterwards, the deployment of the PIA app in the app stores was initiated. The development was completely financed by the company mbits imaging GmbH to explore and evaluate the business case of an app that supports patients' surgical journey. The time of the whole development process--which started with a kick-off meeting and ended with the release of the first app version in the respective app stores--was approximately six months. 2.2. Software Technology The PIA app was built using two major software technologies: "Flutter" and "Firebase" . Flutter provided software tools for creating apps on all operating systems, especially mobile platforms, while ensuring compliance with modern user interface guidelines. Firebase was a Google technology that represented a "backend as a service" platform. The platform, therefore, provided functionality for authentication, data validation, push notifications, and scalable storage of data of any kind in one system. The location of the physical storage of data could be chosen within the platform. For the PIA app, all data were stored on German servers. Furthermore, the communication between the PIA app and the Firebase database occurred in real time and was encrypted at all times, which ensured live updates of messages while protecting them from unauthorized access. To comply with General Data Protection Regulation requirements, the PIA app provided all functions required for the patient to make use of their proper rights: a privacy statement describing the information collected and how it was processed, and the processed data could be viewed, transferred, and deleted at any time. 2.3. Recruitment of Participants and Questionnaire Fifty consecutive patients with planned admissions for inpatient urological surgery at the Urology Clinic of Heidelberg University Hospital were screened in April 2021. The inclusion criteria of the study were that the patient was admitted for surgery on a scheduled basis, used a smartphone in their daily life, was an adult capable of giving consent, and had sufficient German language skills. The app was provided to the study participants from their first presentation at Heidelberg University Hospital on the day of surgical preparation until several weeks after the inpatient surgery. The study participants were asked to download the PIA app for free onto their smartphones from Google Play Store for Android and from Apple App Store for iOS. As there is no established, standardized questionnaire for the evaluation of medical apps, despite the continuous development of new apps , a questionnaire was developed for the evaluation of the PIA app. In designing the questionnaire, the MAUQ questionnaire for patient apps was used as a guideline and adapted for the PIA app. This questionnaire included the categories of general patient information, information about the hospital stay, satisfaction with the app, evaluation of the user interface as well as applicability. Patients were asked to fill out a first questionnaire containing demographic characteristics upon study inclusion. A second questionnaire was provided to be filled out at the end of the app's use after discharge, including closed-ended questions about usability, usage data, and perceived benefits, and open-ended questions about the strengths and weaknesses of the PIA app. Important values were satisfaction, supportiveness, reusability, and recommendability of the app. The PIA app was provided as an alternative source of information in the study. The standard education and information provided by physicians and nurses of Heidelberg University Hospital was maintained for the participants in the study. Push messages about the latest updates or developments in the ward routine were sent to the patients by an undergraduate (AF) under direct doctors' supervision. Timely information was obtained by involving key hospital staff in the conduct of the study (including ward physicians, surgeons, surgical coordinators, nursing management). The entire study implementation and the sending of personalized push notifications to the patients were continuously supervised by MG. 2.4. Ethical and Legal Framework Data were collected prospectively, and the study was approved by the Ethical Committee of the University of Heidelberg (Approval No. S-778/2019). All study participants gave their informed consent. Patient participation was voluntary; the study did not change the standard therapy, but merely supplemented it. Consent could be withdrawn by the patient at any time, without giving reasons and without disadvantages for further medical care. In the event of withdrawal from the study, data obtained would be deleted at the request of the patient. The names of patients and all other confidential information were subject to medical confidentiality and the provisions of the General Data Protection Regulations. After registering and accepting the privacy policy in the PIA app, patients could log into the app with their email address and user password. The PIA app used a multilevel security concept in which, ultimately, only the user could access the data on their smartphone using a password of their choice. In addition, end-to-end asymmetric data encryption was used, data were pseudonymized after collection, and (encrypted) data were automatically deleted after completion of the study. 2.5. PIA App Content Hospitals and operating rooms are logistically complex to run. The more insight patients are given into the hospital's procedures and regulations, the better they can adapt to the processes. When background information regarding medical interventions is given to patients, they better understand the reasonableness of the recommendations, and treatment adherence is increased. The medical team, on the other hand, can be relieved from the need to repetitively explain the same standard information to every patient (e.g., the procedures on the ward) through an app, which would give them more time for the individual engagement with the patient. In addition, for patients, a hospital stay is often an exceptional situation, which leads to them forgetting information that was communicated verbally. As a result, operations may have to be cancelled (e.g., if patients have accidentally continued to take their anticoagulants) or the maximum success of the operation cannot be achieved (e.g., because patients forget post-operative behavioral instructions). The PIA app was designed to provide the necessary information during the hospital stay as well as preoperative information to help prepare for hospitalization, and discharge information for the rehabilitation process at home. The app was intended to be a 24-hour companion to provide optimal patient information and to improve patient knowledge and self-management. . The PIA app features had the aim of an optimized patient education and information flow, with a smaller knowledge deficit for the patients before and after medical treatment. Information tailored to the patient was to be made accessible at any time and in any place. The PIA app contained three tabs: one for general information, one for a personal agenda, and one for push notifications. The content of the app was offered in German. General information in the app included, among others, an overview of the required pre-operative examinations (e.g., anesthesia, electrocardiography), items to carry to the hospital, a location plan in the hospital, a daily schedule for the ward (doctor's visits, doctor's time for discussions with relatives, visiting hours), latest COVID-19 rules, an introduction to the medical team, the use of multimedia in the patient's room, the preparation for the surgery including storage of valuables, post-operative medication including pain killers, contact details of support services (inside and outside the hospital), and information about the social services. Regarding the personal agenda, patients received perioperative appointments in a calendar in the PIA app, such as date and place of presentation in the hospital, time of surgery, date and place of imaging appointments, time of doctor's consultation for discharge examinations, date and place of re-presentation in the hospital. A few examples of push notifications that were sent as a reminder or update to the patients included: which and when long-term medication was necessary to discontinue, at what time point it was needed to fast before surgery, timely information about delays in the surgical schedule, what the goals were for the first post-operative days, why specific examinations were carried out, what to remember at home after the respective operation, when to present again in the hospital for follow-up, what the long-term behavioral recommendations were to avoid a recurrence (e.g., stone metaphylaxis, pelvic floor training). Sample screenshots of the mobile health application PIA demonstrate the user experience and highlight the app's functions. After the patient logged in, the PIA app offered three tabs: one for general information, one for individual push notifications, and one for a personal agenda. The app pages were translated into English; the original app tabs can be found in the Supplementary Material. 3. Results 3.1. Study Population Common reasons for exclusion of patients from the proof-of-concept study were not carrying a smartphone to the hospital, lack of German language, or lack of interest in the study . Nineteen of the twenty-two patients who were provided with the newly developed PIA app evaluated the app at the end of the usage period. They evaluated the PIA app via a questionnaire assessing functionality, suitability, and utility from a patient perspective. The median age of the study cohort was 50 years old; the youngest study participant was 35 and the oldest was 75 years old. In total, five out of nineteen patients were female and fourteen out of nineteen patients were male. The median length of hospital stay was 5 days (Table 1). The most common surgeries were tumor removal from the kidney or prostate, and kidney stone removal. 3.2. Patient Evaluation of the PIA App Regarding usability, the study participants confirmed the easy operability of the PIA app, with 18 out of 19 patients (95%) confirming that the app was easy to use (fourteen patients completely agreed, four patients agreed, one patient partially agreed, and none disagreed). Similarly, 18 out of 19 patients (95%) did not need any assistance to begin using the PIA app . The structuring of the various functions of the app was found to be comprehensible and clear . The aim of this question was to ask the patients about the interface of the app and the organization of the information in the app, so that they could easily find the information they needed. In terms of innovation, none of the patients had previously used a medical app in another hospital. None of the patients had any safety concerns about using the PIA app, and all the study participants felt secure during the study period, in the way that they believed that the data they shared in the app was securely stored . In terms of benefits for the patients, fourteen out of nineteen patients (74%) agreed or completely agreed that the provision of the PIA app made them feel better informed during their hospital stay, with five out of nineteen patients (26 %) partially agreeing and none disagreeing. Fourteen out of nineteen patients (74%) stated that they were more satisfied with their overall hospital stay because of the provision of the PIA app. The study participants expressed that they would like to use the PIA app in a further hospital stay (thirteen patients completely agreed, four patients agreed, two patients partly agreed, and none disagreed). In general, the study patients said that they would like to use the PIA app regularly in healthcare (eleven patients completely agreed, six patients agreed, two patients partly agreed, and none disagreed). The evaluation of the app was obtained after use via structured questionnaires. A total of 19 out of 22 recruited patients gave feedback about the PIA app, which is shown above. The participants' answers are given as the number of patients who chose each multiple-choice answer. A total of 13 of the 19 study patients who filled out the questionnaire completed the free-text evaluation. In the free-text evaluation, suggestions for improvements from the study's participants included delivery of push messages to the mobile phone in standby mode, directly receiving the results of examinations via the app (e.g., blood tests), and receiving more detailed descriptions on planned interventions. Further feedback about the PIA app is illustrated in Figure 4. 4. Discussion Patients reported a general lack of readily available information, e.g., on medical procedures, side effects, and the logistics of care . mHealth holds great potential in healthcare as a low-threshold offer to inform patients in the hospital, and as a companion at home before and after treatment. In this proof-of-concept project, we developed and evaluated, to the best of our knowledge for the first time, a user-friendly app to provide patients with timely information before and after inpatient urological surgery. Our study revealed several major results. First, the use of the PIA app was both highly innovative and desirable for patients. Of the fifty patients screened in this study, only four were not included due to a lack of interest. Another 24 patients could not be included in the study because they did not speak sufficient German or did not carry a functioning mobile device with them. None of the study participants used a medical app in another hospital before; however, after using the PIA app, they clearly expressed their recommendation for the increased use of the PIA app in healthcare. Second, our study results confirmed that the use of the PIA app was unproblematic and did not require a need for assistance. Third, the evaluation results revealed that patients had no privacy or security concerns about the digital technology of the medical app PIA. There are not just potential benefits by mHealth applications, but patients can also have severe direct and indirect privacy concerns and the fear of discrimination; for example, through the employer or health insurance company. Potential difficulties for the acceptance of mHealth applications are the patients' concerns about data security, the consequences of potential data theft, and data sales to third parties. Users of medical apps rely on the ethical operation of app services and need to trust the apps they use . Fourth, the vast majority of patients confirmed that the provision of the PIA app made them feel better informed and that they were more satisfied with their overall hospital stay because of the provision of the PIA app. More and more patients, as well as hospital staff, possess a smartphone. By adding the concept of timing to patient education, it is possible to increase the likelihood that patients will receive the right information at the right time . The digitalization of the healthcare sector can help to create healthcare that is more predictive, preventive, personalized, and participatory , while simultaneously relieving medical staff of the need to repetitively provide standard information to the patients, ensuring that they can spend their time on more individual engagements with patients. The more efficient use of information and personnel can contribute to long-term cost reductions as well as the optimization of work processes at hospitals. Smartphones and their apps are appealing to healthcare providers, as they provide intuitive user interfaces and allow for easier interaction with patients , particularly in the postoperative outpatient setting. mHealth can facilitate virtual means of contact, which is a viable solution when surgical patients live in remote areas, and in situations such as pandemics (e.g., COVID-19) . Thus, innovative apps, such as the PIA app for urological surgery patients, have the potential to create benefits for patients, physicians, hospitals, and public health. Within the scope of mHealth, smartphones are increasingly being utilized for patient education, disease prevention, medical diagnosis, and disease monitoring. The COVID-19 outbreak has further led to the acceptance of new strategies such as telemedicine as pragmatic approaches and alternatives to doctor consultations . Successful examples of mHealth interventions include medication adherence (e.g., improving the cardiometabolic profile of patients with hypertension, or monitoring the opioid use of patients with chronic noncancer pain) and promoting smoking cessation . Further examples of mHealth applications include follow-up assessments after hospital discharge and inpatient stroke rehabilitation and timely postoperative care education after total knee replacement . In urological settings, the feasibility of an app for postoperative behavioral recommendations after radical prostatectomy was demonstrated previously . The PIA app distinguished itself from previous works by its timely support of surgical patients not only in the outpatient setting but also during the hospital stay. In other medical disciplines, there are already promising research results on patient support provided by medical apps during the inpatient hospital stay. For example, to address the challenges of communication and remote rounding during COVID-19, a mobile app was developed to communicate changes in hospital policies and enable direct telephonic communication between clinical team members and hospitalized patients in terms of inpatient telehealth . In another study, to facilitate the daily life of the physician and patients with diabetes, medical apps to improve the inpatient glycemic management were described. The applications identified for in-hospital glycemic management promoted better glycemic mean and lower risk of hypoglycemia than usual management . In a psychiatric inpatient unit, a recent study suggested that there is significant untapped potential for utilizing smartphone applications for psychiatric monitoring and treatment, with a majority of patients expressing interest in using mental health apps in the future . These research results demonstrate that the use of health applications to support inpatient therapy is on the rise to be incorporated into medical practice with user activity metrics suggesting that mHealth solutions will remain important. In line with our study results, medical apps can be tools to improve the quality of patient care and can have sustained value among clinicians in communicating with inpatients . However, the number of studies is still scarce. Going further, envisioning several dimensions for expanding the PIA app in subsequent projects is straightforward. Now that we have confirmed the usability and patient approval of the PIA app in our initial proof-of-concept cohort, the next step is to investigate the benefit of the app in a prospective randomized study. The potential added value and increased productivity should be evaluated by patients, doctors, and nursing staff alike. Connecting the PIA app to existing hospital information systems through the Internet of Things has the potential to further optimize timely patient information and can be a step towards a smart hospital . In the near future, we expect eHealth and mHealth to become revolutionary in healthcare. Major industry directions are health data curation and enrichment, AI-enabled diagnostic interpretation, and an application marketplace for healthcare providers . Real-time monitoring devices can gather live patient data from sensors/wearables and send inputs to a mobile medical app on a smartphone, server, or network to support clinical decision making . This new wave of big-data-powered personalized risk assessment tools can help healthcare professionals to make better, more individualized decisions. The standardized capture of patient-reported outcome data with minimal resource utilization over an mHealth application can be used for quality assessment, optimization, and research. mHealth will likely assist in the transition from isolated healthcare sources with inaccessible proprietary information to an integrated paradigm of continuous care built around the individual, with an improvement in health outcomes and a reduction in costs . The limitations of our study include that this study was conducted solely in the field of urology surgery in the German healthcare system. It was limited to 22 German-speaking patients in a single inpatient surgical hospital in Germany. Our analysis of the PIA app was an initial exploratory study focusing on the evaluation of the app's usability and quality. As there has only been little research on mHealth applications for inpatient support, the exact requirements and level of acceptance of a medical app for inpatient surgical patients were unknown. The present proof-of-concept study allowed us to assess the integrability of the PIA app into daily clinical routine, to evaluate its usability and approval, to become aware of problems that could occur during implementation, as well as to contribute to the evidence base. A successful initial proof-of-concept clears the way for setting up larger clinical studies and to establish the PIA app in clinical routine . So far, the widespread use of mobile apps on smartphones and tablets has not become reality in inpatient hospital care in German hospitals. Initial evidence of potential benefits from mHealth apps is required to overcome challenges that hinder the wider adoption of apps (e.g., regulatory, financial, and organizational issues). First promising descriptive results enable a process to implement, supervise, and evaluate clinical mHealth apps in larger cohorts . Because of the limited scope of application in our study, the gained evidence was only representative for the respective target group. For instance, population groups such as non-German speakers or patients with visual issues did not meet study requirements and were not included. Thus, of the 50 screened patients, only 22 took part in the study, as the other patients did not carry a mobile device to the hospital, did not speak sufficient German, or were not interested in the study participation, resulting in selection bias. With the small size of this proof-of-concept study, the generalizability of our findings to the overall population must be handled with particular caution. This necessitates the need to explore any additional requirements that may be needed for medical apps in emerging nations or more rural nations and healthcare systems, particularly as it relates to technology literacy and internet access. A future mixed-methods project could be conducted to gain a broader perspective on user experience and usability of the PIA app by involving participants from various cities, states, and countries. A mixed-methods research design would systematically combine quantitative and qualitative methods for a comprehensive understanding of the research questions. It would enable to move beyond 'what works', documented by measurable parameters in quantitative research, and understand 'what works for whom, why, how, and when', through qualitative research in different populations . Moreover, it is crucial to evaluate whether the requirements of patients for mHealth apps vary in disciplines other than surgery. A further limitation of our study includes the utilized survey tool and the challenge of applying validated criteria for the evaluation of medical apps. The assessment of the PIA app focused on obtaining patient feedback through a survey; however, studies that rely on self-reporting of patients are particularly prone to recall bias. In addition, the questionnaire, which was applied in the study, and which was developed based on the MAUQ questionnaire for patient apps , has not yet been validated. Future directions may include the design of validated instruments to evaluate the vast diversity of mHealth formats. Finally, it must be emphasized that some patients may not own a smartphone and cannot be reached via mHealth apps. This is changing with smartphone ownership on the rise; however, no patient should be left out. It is important to be aware that mHealth tools are designed to support and enhance, but they will probably never fully replace direct consultation with a healthcare professional. 5. Conclusions Via the introduction of the medical app PIA, we created an innovative digital health information tool that offered great potential for patient assistance before and after surgery and for targeted support of doctor-nurse-patient communication in the hospital. The German patients of our exploratory proof-of-concept study approved the use of the PIA app during their surgical hospital stay, as the provision of the app made them feel better educated about hospital procedures and medical background. The broad majority of the study participants confirmed the easy usability of the app and supported the general future use of the PIA app in clinical routine. Supplementary Materials The following supporting information can be downloaded at: Structure and design of the PIA app in German. Click here for additional data file. Author Contributions Conceptualization, M.G. and M.H.; methodology, M.G.; software, M.M.; formal analysis, M.G.; investigation, A.W.; resources, M.M. and M.H.; data curation, A.W.; writing--original draft preparation, M.G.; writing--review and editing, A.W., M.M. and M.H.; visualization, M.G. and M.M.; supervision, M.H.; project administration, M.G. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethical Committee of the University of Heidelberg (Approval No. S-778/2019). Informed Consent Statement Informed consent was obtained from all participants involved in the study. Data Availability Statement Not applicable. Conflicts of Interest M.M. is chief executive officer at mbits imaging GmbH (Heidelberg, Germany). The other authors declare no conflict of interest. Figure 1 Structure, design, and functionality of the PIA app. Figure 2 Flow chart for inclusion of study participants. Figure 3 Evaluation of patient satisfaction with the PIA app. Figure 4 Individual patient feedback on the PIA app. healthcare-11-00682-t001_Table 1 Table 1 Study population. Study Cohort Patients evaluating the app, n 19 Female participants, n 5 Male participants, n 14 Age, yr 1, median (IQR 2) 50 (44-57) Length of hospital stay, days, median (IQR) 5 (4-7) Endourological intervention 6 Robot-assisted, minimally invasive surgery 6 Open surgical intervention 7 1 yr = year; 2 IQR = interquartile range. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. 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PMC10000763 | Diagnostics (Basel) Diagnostics (Basel) diagnostics Diagnostics 2075-4418 MDPI 10.3390/diagnostics13050973 diagnostics-13-00973 Article The Use of Visual Analysis for Gait and Foot Posture in Children with Developmental Dysplasia of the Hip Vasilcova Veronika Conceptualization Methodology Validation Formal analysis Investigation Resources Data curation Writing - original draft Writing - review & editing Project administration 12* AlHarthi Moqfa Methodology Formal analysis Writing - original draft Writing - review & editing 2 Jawadi Ayman H. Investigation Writing - original draft Writing - review & editing Supervision 3 Zvonar Martin Resources Writing - original draft Writing - review & editing Supervision 4 Terpos Evangelos Academic Editor 1 Department of Kinanthropology, Faculty of Sport Science, Masaryk University in Brno, Kamenice 753/5, 62500 Brno, Czech Republic 2 Pediatric Rehabilitation Department, King Abdullah Specialized Children's Hospital, P.O. Box 22490, Riyadh 11426, Saudi Arabia 3 College of Medicine, King Saud bin Abdul-Aziz University for Health Sciences, P.O. Box 22490, Riyadh 11426, Saudi Arabia 4 Division of Sport Motorics and Methodology in Kinanthropology, Department of Kinesiology, Faculty of Sport Science, Masaryk University in Brno, Kamenice 753/55, 62500 Brno, Czech Republic * Correspondence: [email protected] 03 3 2023 3 2023 13 5 97301 1 2023 25 2 2023 27 2 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). Background: Developmental dysplasia of the hip (DDH) is recognized as a leading cause of significant long-term complications, including inaccurate gait patterns, persistent pain, and early regressive joint disorder, and it can influence families functionally, socially, and psychologically. Methods: This study aimed to determine foot posture and gait analysis across patients with developmental hip dysplasia. We retrospectively reviewed participants referred to the pediatric rehabilitation department of KASCH from the orthopedic clinic between 2016 and 2022 (patients born 2016-2022) with DDH for conservative brace treatment. Results: The foot postural index for the right foot showed a mean of 5.89 (n = 203, SD 4.15) and the left food showed a mean of 5.94 (n = 203, SD 4.19). The gait analysis mean was 6.44 (n = 406, SD 3.84). The right lower limb mean was 6.41 (n = 203, SD 3.78), and the left lower limb mean was 6.47 (n = 203, SD 3.91). The correlation for general gait analysis was r = 0.93, presenting the very high impact of DDH on gait. Significant correlation results were found between the right (r = 0.97) and left (r = 0.25) lower limbs. Variation between the right and left lower limb p-values was 0.88 (p < 0.05). DDH affects the left lower limb more than the right during gait. Conclusion: We conclude that there is a higher risk of developing foot pronation on the left side, which is altered by DDH. Gait analysis has shown that DDH affects the right lower limb more than the left. The results of the gait analysis showed gait deviation in the sagittal late stance phases. DDH gait analysis foot posture FPI-6 conservative treatment hip surgery The authors received no financial support for the research. pmc1. Introduction Developmental dysplasia of the hip (DDH) is a common disorder associated with significant impairment of the hip joint . Early recognition of DDH can prevent malformation and enhance the appropriate anatomy of the hip, thereby leading to recovery without the need for surgery . The prevalence rate for DDH varies within countries and continents from 1-34/1000 (cases per birth) . In 2020, Sadat-Ali published 10.46/1000 cases per birth as the latest prevalence of DDH in the Kingdom of Saudi Arabia (KSA) . However, the incidence may vary in specific areas such as the Middle East, the gulf area, and Italy . The three leading pediatric hip conditions (PHCs) are Perthes, Developmental Dysplasia of the Hip (DDH), and Slipped Upper Femoral Epiphyses (SUFE), which are all perceived as causes of early degenerative hip osteoarthritis in adults . If DDH is not diagnosed and treated early, the long-term outcomes of late treatment lead to unfavorable consequences . Late-diagnosed or persistent DDH is increasingly recognized as a leading cause of significant long-term morbidity, including: pathological gait pattern, chronic pain, and early regressive joint disease, and can affect families functionally, socially, and psychologically . A hip shift is a common complication in children with a neurology diagnosis, which can lead to hip dislocation . This shift can have a negative effect on a range of motion, motor abilities, and personal care skills and can be linked to pain . Children typically are born with normal anatomic hip alignment . While growing, hip development may be afflicted for various reasons. In combination with muscle inequality and spasticity, decreased weight bearing has often been raised as the primary contributor to hip displacement . It can decrease the femoral head's contact with the acetabulum, leading to a reduced range of motion and bone deformities such as coxa valga, femoral anteversion, and acetabular dysplasia . If DDH is diagnosed early and treated accordingly and constantly, the treatment outcomes are positive. Any incorrect use of rigid orthosis can cause complications such as avascular necrosis of the femoral head (AVN) or lead to paralysis of the femoral and obturator nerves. The need to immobilize the child's legs to restrain active lower limb movement raises inquiry among parents and physicians, as they fear it may cause a delay in the child's gross motor development, especially sitting, standing, and walking . Pediatric foot alignment has been described with footprint assessments in many studies. All through early development, children's feet develop a medial longitudinal arch, which is different from the adult community. Children's foot posture must be assessed in every developmental stage with the presence/absence of fundamental elements--hypotonia or hypermobility . In the available literature, we found a limited number of analyses concerning the influence of applying an abduction brace on gross motor development in children with DDH. The latest was published by Zgoda et al., 2010 . This study aimed to determine the foot posture and gait analysis patterns across patients with developmental hip dysplasia receiving brace treatment. The main hypothesis for this study, congenital dislocation of the hip affects the foot and lower limb position unilaterally. 2. Materials and Methods 2.1. Study Area This study was conducted at KASCH of the Ministry of National Guard Health Affairs (MNGHA), Riyadh, Kingdom of Saudi Arabia (KSA). KASCH is the nation's most advanced children's hospital and the first medical referral institute that served pediatric patients in the KSA. 2.2. Study Design Patients referred to the pediatric rehabilitation department of KASCH from the orthopedic clinic between 2016 and 2022 (patients born 2016-2022) with DDH for conservative treatment were screened as Acute cases (Category A). The principal investigator gathered demographic information from the Best Care medical system and a family member, the parent. The results had been passed on for statistical interpretation. Cohort study: We retrospectively collected all patients from the Pediatric Physiotherapy Department in KASCH receiving conservative treatment for DDH. All individuals were sampled, and information about their past assessments and investigations were collected. 2.3. Sampling Technique Demographic information were collected and entered into the working sheet. Inclusion and exclusion criteria were applied to clear data according to our project description. The principal investigator prepared patient lists with phone numbers, and the co-investigators contacted all parents to check if they were willing to participate in our project and bring their child to the Pediatric Physiotherapy Outpatient Clinic at KASCH for Zebris gait and foot assessment. The physiotherapy and orthopedic consultant who participated in our project as co-investigators renewed physiotherapy referrals for patients if they were discharged from our services. Participants This study was carried out in the gait analysis laboratory at King Abdullah Specialized Children's Hospital (KASCH), Riyadh, KSA. We called patients retrospective from 2016-2022. Parents signed a written consent form approved by the ethics commission. Inclusion criteria: could independently walk for ten steps, were diagnosed with DDH by an orthopedic physician, and received a physiotherapy referral for abduction braces treatment. Exclusion criteria: could not walk for ten steps, were diagnosed with DDH. 2.4. Protocol Tools The foot posture was assessed barefoot, in a relaxed standing position using the standard protocol for the Foot Postural Index 6 (FPI-6). Gait analysis was carried out in the gait laboratory under the supervision of the principal investigator, barefoot, on a hard floor, for at least ten steps. The FPI-6 evaluates the multi-segmental nature of foot posture in all three planes and does not require specialized equipment. Each item of the FPI-6 scores between minus two and plus two, with a total of six items referring to positions of the forefoot, mid-foot and hind-foot, and the three planes of motion: talar head palpation; the symmetry of the infra-lateral malleolus curvature; inversion/eversion of the calcaneus; prominence in the region of the talonavicular joint; height of the medial longitudinal arch; abduction/adduction of the forefoot. The FPI-6 score spectrum goes from minus twelve--highly supinated to plus twelve--highly pronated . The Wee Glasgow Gait Index (WeeGGI) is a gait analysis and screening tool for the many clinicians who do not have access to gait laboratories and special equipment to benefit their clinical decision-making . The WeeGGI focuses on eleven gait domains, designed by the neuro-biomechanics team at Westmark, Glasgow, which are the most compatible with a normal gait. The clinician presents a diagram for each parameter with a choice of three stick figures, each of which has explicit descriptors and or values: 0 for the standard or neutral position--indicates the ideal position for the joint or segment at the specific point in the gait sequence; 1 for mild deviation--indicates a mild deviation from the expected position; 2 for gross deviation--indicates a gross deviation. A score applies to each outcome, and a single grade must be selected for each parameter. The score for all eleven parameters should be estimated. The higher the score, the higher the standard gait alteration, which leads to a higher indication for physiotherapy intervention . 2.5. Ethical Approval The study was conducted according to the Declaration of Helsinki and was approved by the Institutional Review Board from KAIMRC, Riyadh, KSA, with memo Ref. No. IRBC/1747/21, study No. SP21R/364/06 on 23 August 2021 and by The Masaryk University Research Ethics Committee, Brno, Czechia, Ref. No. EKV-2021-018, proposal No. 0107/2021 on 31 May 2021. 2.6. Statistical Methods The statistical analysis was conducted using TIBCO StatisticaTM 14.0.1 (StatSoft, EN version, San Diego, CA, USA). For assessing the normality of distribution, we applied the Shapiro-Wilk test. To estimate the intergroup differences in the non-parametric distribution, we applied the Mann-Whitney test. The differences at p < 0.05 were defined as statistically significant. 3. Results In our population study of 203 children, the mean age was 20.09 months, ranging from 11 (2.5%) to 132 (0.5%) months. All patients were referred to the physiotherapy department and diagnosed by orthopedic physicians with DDH for treatment with abduction braces. This included 143 patients treated conservatively only with abduction braces (70.4%), 32 presented after surgical hip reduction and with casting (15.7%), were referred for abduction braces after cast removal, and 28 children who were seen for brace treatment with another diagnosis (13.7%), such as neurology, rheumatology, ophthalmology, cardiac, and other orthopedic diagnoses. The study population gender distribution was 165 females and 38 males. We evaluated 406 feet and lower limbs in this study. All patients were assessed for foot posture using FPI-6 and gait analysis using the WeeGGI index (Table 1). The FPI-6 mean score for n = 406 feet was 5.9 (SD 4.16). Pronation is considered when FPI >= +6 and standard foot position is FPI < 6. Our group presented with pronation of both feet at 58.12% (236) and standard foot position had 41.87% (170) . Generally, our study group was at high risk of developing foot pronation. These results also show the difference between the right and left foot. The FPI-6 for the right foot showed a mean of 5.89 (n = 203, SD 4.15) and the left foot showed a mean of 5.94 (n = 203, SD 4.19) (Table 1). We concluded that there is a higher risk of developing foot pronation on the left side, which is altered by DDH. Foot supination (FPI >= -1) was not detected. Right foot pronation with a score of FPI >= +6 was observed in 58.6% (119, n = 203) of children, normal foot position of FPI < 6 was observed in 41.3% (84, n = 203), and supination of FPI >= -1 was not detected in any participants. For the left foot, a normal position of FPI < 6 was observed in 42.3% (86, n = 203) of children, pronation was observed in 57.6% (117, n = 203), and supination of FPI >= -1 was not detected . WeeGGI gait analysis was measured during the same session as the foot postural index; patients independently walked barefoot on the hard floor for at least ten steps (Table 1). The WeeGGI mean was 6.44 (n = 406, SD 3.84), which can be considered a deviation from the regular gait pattern. The right foot mean score was 6.41 (n = 203, SD 3.78), and the left foot mean score was 6.47 (n = 203, SD 3.91). The correlation for general gait analysis was r = 0.93, presenting very high impact of DDH on gait. Significant correlation was found between the right r = 0.97 and left r = 0.25 lower limbs. Gait analysis showed that DDH affects the right lower limb more than the left. The sign test revealed difference between the right and left lower limbs with a p-value of 0.88 (p < 0.05). DDH affects the left lower limb more than the right during gait . Confounding Results Additional statistical analyses were completed for conservative treatment with abduction braces and surgical treatment of DDH. Testing for foot posture and gait, the H0 (H0: There will be a difference in the foot posture and gait analysis between the conservative and surgical treatment of DDH) foot posture/gait was the same for conservative and surgical treatment of DDH. This study contained 143 patients who were treated conservatively, aged from 11 to 48 months, with a mean age of 16.22 months. The gender distribution was 31 (17.7%) males and 144 (82.2%) females. Post-hip surgery, 32 patients agreed to enter this study. The mean age was 19.6, the highest age was 48 and younger patient was 11 months old. We compared WeeGGI and FPI-6 results for the right and left limbs between the conservative treatment group and the post-hip-surgery participants (Table 2). FPI-6 results showed no difference between the brace and operative DDH treatments (p < 0.05). The mean for the right foot in the conservative treatment group was 5.21 (SD 4.07, n = 143), while the mean in the surgery group was 7.25 (SD 4.11, n = 32). The mean for the left foot in the conservative treatment group was 5.25 (SD 4.14), while the mean in the surgery group was 7.28 (SD 4.04). A result of FPI-6, indicate foot pronation, in participants after surgical treatment for DDH (Table 2). We tested each part of the eleven domains of WeeGGI, gait analysis. There was a significant difference in two WeeGGI gait domains of the right leg and four parts of the analysis on the left leg. Marked tests are significant at p < 0.05. The right lower limb showed diversity in the:sagittal mid-stance on the right ankle, with a mean of 0.39 (SD 0.73) in the conservative treatment group and a mean of 0.50 (SD 0.67) in the surgery treatment group, and a p-value of 0.02; sagittal late stance thigh attitude, with a mean of mean 0.51 (SD 0.79) in the conservative treatment group and a mean of 0.65 (SD 0.90) in the surgery treatment group, and a p-value of 0.01. The left lower limb showed diversity in the:sagittal initial contact knee analysis, with a mean of 0.55 (SD 0.49) in the conservative treatment group and a mean of 0.78 (SD 0.49) in the surgery treatment group, and a p-value of 0.03; sagittal mid-stance on the left ankle, with a mean of 0.39 (SD 0.73) in the conservative treatment group and a mean of 0.56 (SD 0.80) in the surgery treatment group, and a p-value of 0.01; sagittal mid-late stance shank attitude, with a mean of 0.48 (SD 0.76) in the conservative treatment group and a mean of 0.68 (SD 0.89) in the surgery treatment group, and a p-value of 0.03; sagittal late stance thigh attitude, with a mean of 0.43 (SD 0.49) in the conservative treatment group and a mean of 0.59 (SD 0.49) in the surgery treatment group, and a p-value of 0.01. Results of the gait analysis showed gait deviation in the sagittal late stance phases. 4. Discussion This project has shown the effect of DDH on gait and foot posture in pediatric patients after conservative treatment. The foot posture showed difference between conservative and surgical treatment of DDH. It is crucial to correct pathology in the hip, in this case, DDH, which affects gait and foot posture on the opposite leg due to the possibility of shifting body weight to create a higher burden on a healthy lower hip and foot. It is commonly claimed that children with DDH who have not been treated start walking slightly later than healthy children . This delay is about 2-3 months and usually does not exceed the average age of children starting to walk . Dunn estimated that 20% of children with undiagnosed and untreated DDH do not start walking until 18 months . Kamath and Bennet have demonstrated that the mean walking age in children not treated for this disorder was 13 months (with range of 9.5-18 months), which was, on average, one month later than in healthy children . Zgoda et al.'s outcomes demonstrate that the abduction brace treatment does not appear to affect the child's gross development. Though the three-week delay in walking is statistically significant, it does not influence their further locomotor development . This study did not analyze the age when the child started to walk. We had patients starting DDH treatment at different age groups; the mean of the gait analysis was 20.09 months in the leading group and 16.22 months in the confounding group, which is lower than Dunn's 1990 finding of 18 months but higher than Zgoda's finding of 13 months. The youngest patient was 11 months old . Gait analysis should be the golden standard for DDH patients. The visual gait analysis is now added as an outcome measure for all patients with DDH. Reduced hip range of motion while sitting, standing, and walking is ordinary. In 2014, Larnert et al. defined an association between hip dislocation and pelvic obliquity, windswept deformity, and scoliosis . Our study showed no difference or pathology in the coronal mid-stance pelvic obliquity. In 2019, Gijon-Nogueron et al. published an article describing a significant difference (p < 0.01) between countries . The mean for Spanish children was FPI = 4.00 (2.9), the mean for UK children was FPI = 4.9 (3.3), and the mean for Australian children was FPI = 4.7 (3.1). The general trend showed FPI scores declining in some countries . The Saudi population in our study had a mean of FPI = 5.92 (4.16), which is higher than the average stated in Gijon-Nogueron et al.'s publication. The opportunity to access average FPI scores empowers clinicians to inform parents of what is 'average' and what is 'normal' at any age, it has been statistically proven . After this project, the FPI-6 was uploaded to the pediatric physiotherapy outcome measures at KASCH. Wenger et al. found that the percentage of high dislocations was 5.5% for children diagnosed in their first year, 31% when diagnosed in the second year, and 67% when diagnosed after the second year, showing that hip dislocations worsened with time . As soon as the hip reduction is associated, there is a lower risk of avascular necrosis of the femoral head after treatment . Our study had one female (0.49%) with AVN complications resolving in Perthes. Patients screened and diagnosed early and treated conservatively have a success rate of 90%. Within Saudi Arabia, there is a wide-ranging variability of presentations from newborn to six years of age, making treatment decisions difficult and outcomes unpredictable . In our practice, the physiotherapist receives patients for abduction braces treatment as the first or second choice after the Pavlik harness or surgical intervention. According to Mulpuri et al., some studies revealed that the initial treatment method was splinting, with discrepancies between the younger and older groups regarding surgical management . Some studies of limited strength have examined the use of rigid and soft splints; however, the results are inconsistent with small effect sizes . With our project regarding the prevalence and outcomes of DDH in physiotherapy practice, we want to highlight the importance of rehabilitation, as Jennings et al. wrote in 2016: "DDH is a poorly presumed disorder as evidenced by the profusion of literature, both recent and historical." . The main limitations of this study were lack of probability, different physiotherapists with variable years of experience assessing participants' gaits and foot posture, child presentation during analysis. 5. Conclusions We concluded that there is a higher risk of developing foot pronation on the left side, which is altered by DDH. Gait analysis has shown that DDH affects the right lower limb more than the left. The results of the gait analysis showed gait deviation from the regular gait pattern in the sagittal late stance phase. Author Contributions V.V.: principal investigator, conceptualization, data curation, formal analysis, investigation, methodology, project administration, resources, software, validation, visualization, writing--original draft, writing--review, and editing. M.A.: co-investigator, data curation, formal analysis, investigation, project administration, writing--original draft, writing--review, and editing. A.H.J.: project coordinator at KASCH, pediatric orthopedic consultant, supervision, writing--review, and editing. M.Z.: Masaryk University--project supervisor and coordinator, co-investigator, formal analysis, project administration, supervision, writing--review, and editing. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The study was conducted according to the Declaration of Helsinki and approved by the Institutional Review Board from KAIMRC, Riyadh, KSA, with memo Ref. No. IRBC/1747/21, study No. SP21R/364/06 on 23 August 2021 and by The Masaryk University Research Ethics Committee, Brno, Czechia, Ref. No. EKV-2021-018, proposal No. 0107/2021 on 31 May 2021. Informed Consent Statement Informed consent was distributed and signed by every parent whose child participated in the study before all diagnostic and therapeutic procedures. All data related to this publication are in an anonymous form. Data Availability Statement V.V. had full access to the data for this study and took responsibility for the integrity of the data and the accuracy of data analysis. Conflicts of Interest The authors declared no potential conflict of interest concerning this article's research, authorship, and publication. Figure 1 Histogram of all FPI-6 analyses. Figure 2 FPI-6 results from the right and left foot. Figure 3 Correlation dialog between the right and left lower limbs. diagnostics-13-00973-t001_Table 1 Table 1 Descriptive statistics. Variable N Mean Minimum Maximum Std.Dev. Age 203 20.09 11.00 132.00 17.37 FPI-6 all 406 5.91 0.00 12.00 4.16 FPI-6 Right 203 5.89 0.00 12.00 4.15 FPI-6 Left 203 5.94 0.00 12.00 4.19 WeeGGI all 406 6.44 0.00 20.00 3.84 WeeGGI Right 203 6.41 0.00 20.00 3.78 WeeGGI Left 203 6.47 0.00 20.00 3.91 diagnostics-13-00973-t002_Table 2 Table 2 Foot and gait analysis results for the conservative treatment and surgery groups. Variables Valid N Mean Minimum Maximum Std. Dev. FPI-6 general score conservative Rt 143 5.21 0.00 12.00 4.07 FPI-6 general score surgery Rt 32 7.25 0.00 12.00 4.11 FPI-6 general score conservative Lt 143 5.25 0.00 12.00 4.14 FPI-6 general score surgery Lt 32 7.28 0.00 12.00 4.04 WeeGGI general score conservative Rt 143 5.76 0.00 12.00 3.55 WeeGGI general score surgery Rt 32 7.21 0.00 13.00 2.80 WeeGGI general score conservative Lt 143 5.65 0.00 12.00 3.65 WeeGGI general score surgery Lt 32 7.78 0.00 13.00 3.01 Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. References 1. Zgoda M. Wasilewski P. Wasilewska I. Golicki D. Influence of the treatment of developmental dysplasia of the hip by the abduction brace on locomotor development in children J. Child. Orthop. 2010 4 9 12 10.1007/s11832-009-0219-0 19937280 2. Jennings H.J. Gooney M. O'Beirne J. Sheahan L. Exploring the experiences of parents caring for infants with developmental dysplasia of the hip attending a dedicated clinic Int. J. Orthop. Trauma Nurs. 2016 5 48 53 10.1016/j.ijotn.2016.10.004 27979662 3. Topak D. Seyithanoglu M. Dogar F. Karadeniz A.A. Tanriverdi B. Ozan F. Bilal O. Are vitamin D and vitamin D receptor levels different in children with developmental dysplasia of the hip? J. Orthop. Surg. Res. 2021 16 24 10.1186/s13018-020-02162-y 33413534 4. Al-Essa R.S. Aljahdali F.H. Alkhilaiwi R.M. Philip W. Jawadi A.H. Khoshhal K.I. Diagnosis and treatment of developmental dysplasia of the hip: A current practice of pediatric orthopedic surgeons J. Orthop. Surg. 2017 25 2309499017717197 10.1177/2309499017717197 5. Pollet V. Percy V. Prior H.J. Relative Risk and Incidence for Developmental Dysplasia of the Hip J. Pediatr. 2017 181 202 207 10.1016/j.jpeds.2016.10.017 27866823 6. Zamborsky R. Kokavec M. Harsanyi S. Attia D. Danisovic L. Developmental Dysplasia of Hip: Perspectives in Genetic Screening Med. Sci. 2019 7 59 10.3390/medsci7040059 7. Sadat-Ali M. Developmental Dysplasia of the Hip (DDH) in Saudi Arabia: Time to Wake up. A Systematic Review (1980-2018) Open J. Epidemiol. 2020 10 125 131 10.4236/ojepi.2020.102011 8. Gambling T. Long A.F. Development and validation of a patient-centered outcome measure for young adults with pediatric hip conditions: The "Quality of Life, Concerns and Impact Measure" Patient Relat. Outcome Meas. 2019 10 187 204 10.2147/PROM.S192672 31388318 9. Buonsenso D. Curatola A. Lazzareschi I. Panza G. Morello R. Marrocco R. Valentini P. Cota F. Rendeli C. Developmental dysplasia of the hip: Real world data from a retrospective analysis to evaluate the effectiveness of universal screening J. Ultrasound 2021 24 403 410 10.1007/s40477-020-00463-w 32356221 10. Gmelig Meyling C. Ketelaar M. Kuijper M.A. Voorman J. Buizer A.I. Effects of Postural Management on Hip Migration in Children with Cerebral Palsy: A Systematic Review Pediatr. Phys. Ther. 2018 30 82 91 10.1097/PEP.0000000000000488 29578990 11. Gijon-Nogueron G. Martinez-Nova A. Alfageme-Garcia P. Montes-Alguacil J. Evans A.M. International normative data for pediatric foot posture assessment: A cross-sectional investigation BMJ Open 2019 9 e023341 10.1136/bmjopen-2018-023341 30987983 12. Keenan A.M. Redmond A.C. Horton M. Conaghan P.G. Tennant A. The Foot Posture Index: Rasch analysis of a novel, foot-specific outcome measure Arch. Phys. Med. Rehabil. 2007 88 88 93 10.1016/j.apmr.2006.10.005 17207681 13. Tennant N. Wiggins L. Read H. Meadows B. The Wee Glasgow Gait Index--A Gait Screening Tool APCP J. 2012 3 39 48 Available online: (accessed on 31 December 2022) 14. Sharaf I. Tachdjian's Pediatric Orthopaedics: From the Texas Scottish Rite Hospital for Children Malays. Orthop. J. 2015 9 53 10.5704/MOJ.1503.012 15. Dunn P.M. Is late walking a marker of congenital displacement of the hip? Arch. Dis. Child. 1990 65 1183 1184 10.1136/adc.65.10.1183-c 16. Kamath S.U. Bennet G.C. Does developmental dysplasia of the hip cause a delay in walking? J. Pediatr. Orthop. 2004 24 265 10.1097/01241398-200405000-00005 15105720 17. Larnert P. Risto O. Hagglund G. Wagner P. Hip displacement in relation to age and gross motor function in children with cerebral palsy J. Child. Orthop. 2014 8 129 134 10.1007/s11832-014-0570-7 24595560 18. Wenger D. Duppe H. Nilsson J. Tiderius C.J. Incidence of Late-Diagnosed Hip Dislocation After Universal Clinical Screening in Sweden JAMA Netw. Open 2019 2 e1914779 10.1001/jamanetworkopen.2019.14779 31702798 19. Stein-Zamir C. Volovik I. Rishpon S. Sabi R. Developmental dysplasia of the hip: Risk markers, clinical screening and outcome Pediatr. Int. 2008 50 341 345 10.1111/j.1442-200X.2008.02575.x 18533949 20. Mulpuri K. Schaeffer E.K. Kelley S.P. Castaneda P. Clarke N.M. Herrera-Soto J.A. Upasani V. Narayanan U.G. Price C.T. IHDI Study Group What Is the Impact of Center Variability in a Multicenter International Prospective Observational Study on Developmental Dysplasia of the Hip? Clin. Orthop. Relat. Res. 2016 474 1138 1145 10.1007/s11999-016-4746-y 26891895 21. Wilkinson A.G. Sherlock D.A. Murray G.D. The efficacy of the Pavlik harness, the Craig splint and the von Rosen splint in the management of neonatal dysplasia of the hip. A comparative study J. Bone Jt. Surg. 2002 84 716 719 10.1302/0301-620X.84B5.0840716 |
PMC10000764 | Foods Foods foods Foods 2304-8158 MDPI 10.3390/foods12050909 foods-12-00909 Article Impact of Cavitation Jet on the Structural, Emulsifying Features and Interfacial Features of Soluble Soybean Protein Oxidized Aggregates Guo Yanan Conceptualization Software Writing - original draft Liu Caihua Data curation Wang Yichang Investigation Writing - review & editing Ren Shuanghe Visualization Zheng Xueting Methodology Zhang Jiayu Investigation Cheng Tianfu Investigation Guo Zengwang Supervision * Wang Zhongjiang Project administration Funding acquisition Xie Jianhua Academic Editor Zhang Yanjun Academic Editor Yu Hansong Academic Editor Shen Mingyue Academic Editor College of Food Science, Northeast Agricultural University, Harbin 150030, China * Correspondence: [email protected] 21 2 2023 3 2023 12 5 90905 2 2023 18 2 2023 20 2 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). A cavitation jet can enhance food proteins' functionalities by regulating solvable oxidized soybean protein accumulates (SOSPI). We investigated the impacts of cavitation jet treatment on the emulsifying, structural and interfacial features of soluble soybean protein oxidation accumulate. Findings have shown that radicals in an oxidative environment not only induce proteins to form insoluble oxidative aggregates with a large particle size and high molecular weight, but also attack the protein side chains to form soluble small molecular weight protein aggregates. Emulsion prepared by SOSPI shows worse interface properties than OSPI. A cavitation jet at a short treating time (<6 min) has been shown to break the core aggregation skeleton of soybean protein insoluble aggregates, and insoluble aggregates into soluble aggregates resulting in an increase of emulsion activity (EAI) and constancy (ESI), and a decrease of interfacial tension from 25.15 to 20.19 mN/m. However, a cavitation jet at a long treating time (>6 min) would cause soluble oxidized aggregates to reaggregate through an anti-parallel intermolecular b-sheet, which resulted in lower EAI and ESI, and a higher interfacial tension (22.44 mN/m). The results showed that suitable cavitation jet treatment could adjust the structural and functional features of SOSPI by targeted regulated transformation between the soluble and insoluble components. soybean protein soluble oxidized aggregates emulsifying properties rheological properties cavitating jet the National Natural Science Foundation32202228 National key R&D plan2021YFD2100401 National key R&D plan2022YFF1100603 Heilongjiang Province key R&D planGA21B001 Heilongjiang Province Major Achievements Transformation ProjectCG19A002 China Fund on the surface of a postdoctoral project2022M721995 Heilongjiang Province million project2021ZX12B02 This research was funded by The National Natural Science Foundation, grant number "32202228"; National key R&D plan, grant number "2021YFD2100401"; National key R&D plan, grant number "2022YFF1100603"; Heilongjiang Province key R&D plan, grant number "GA21B001"; Heilongjiang Province Major Achievements Transformation Project, grant number "CG19A002"; China Fund on the surface of a postdoctoral project, grant number "2022M721995" and Heilongjiang Province million project, grant number "2021ZX12B02". pmc1. Introduction Soybean is an important crop with seeds that contain abundant protein of approximately 40% . The soybean protein has different physiological impacts including dropping blood lipids, blood pressure, and inhibiting cardiovascular and cerebrovascular disease indirectly . Therefore, soybean proteins have been extensively exploited in food and feed plants due to their superior nutritious rate, high functional features, and low price . Studies have revealed that in 2019, global soy production reached 366.67 million tons , which caused huge storage and transportation pressures. In addition, soy protein is vulnerable to oxidative attack during storage and transportation. The parties within the molecule re-syndicate to create oligomers following disclosure, owing to the oxidative denaturation of soy protein, which further forms macromolecular aggregates due to hydrophobicity and electrostatic attraction . It is challenging to use oxidized soy protein in food manufacturing, because the formation of insoluble aggregates in protein aggregates is a significant factor in the loss of some biological and functional features of proteins, for instance, protein solubility, emulsifying effects, and emulsifying stability . The physical control of oxidized protein aggregates is currently the subject of extensive research. The degree of whey protein isolate (WPI) aggregation's cross-linking could be controlled, and its gel and emulsifying characteristics could be improved, expanding the use of WPI in food processing, according to . A decrease in the aggregate concentration of b-conglycinin and a rise in the size of the solvable accumulates for glycinin and soy protein insulate were found by Keerati-U-Rai et al. (2009) , who also established that dynamic extra-pressure homogenization triggered a transformation from insoluble aggregate to soluble aggregate. Additionally, Cao et al. (2021) discovered that using ultrasound might modify the intermolecular interactions, alter the shape and accumulation of oxidized quinoa proteins, and increase the quantity of soluble aggregates, refining the functional attributes of the quinoa proteins. In a study by Zhang (2020) , it was demonstrated that ultrasonic treatment could prevent casein molecules from self-aggregating in a solution, as well as deteriorate the accumulation brought on by interfacial adsorption through foam fractional process, resulting in an improved protein aggregate function. Physical fields can therefore cause subunit dissociation and aggregation to directly regulate the protein structure, which eventually results in an improvement in the functional characteristics of protein aggregates. However, because of their high power requirements and limited effort capabilities in food processing procedures, high-pressure homogenization and ultrasonic processes were unable to be extensively utilized. A cavitation jet is a water jet that can produce the cavitation effect; it can induce the rapid vaporization of the liquid to form many cavitation bubbles. After the liquid flows into the high-pressure zone, these cavitation bubbles will collapse and extinguish, resulting in the generation of an extra-velocity turbulent shear and a substantial pressure differential and molecular impact, which could cause big particles to break up into smaller ones and the structure of food to be refined, which will affect its functional characteristics . The cavitation jet technique provides benefits over alternative mechanical treatments, including ease of use, speedy processing, low processing temperatures, and cheap processing costs . Thus, in the realm of food processing, the cavitation jet treatment may be employed as an effective and energy-saving processing method. Cavitating jets could alter the structure and characteristics of proteins, as well as eliminate the hydrophobic and electrostatic connections between molecules. Based on this, researchers have shown that cavitation treatments may change the structures of the protein isolate and promote emulsifying characterizations . The previous research results revealed that the appropriate time of cavitation jet treating could damage the structure of protein-oxidized accumulates and improve the emulsification and interface properties. In addition, this outcome might be associated with the regulation of the cavitation jet on the oxidized masses and the induction of conversion between the soluble and insoluble oxidized aggregates. Nevertheless, in the current work, the research on the transformation law between the soluble and insoluble components of protein oxidative aggregates was less. It is limited by the tender of the cavitation jet physical field in the governing of the protein accumulates and the analysis of its mechanism. Thus, in this study, soybean protein was utilized as the investigation entity, and 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) was employed to create an oxidation aggregation system of soy protein. By mimicking the definite creation of oxidized protein accumulates in the means of factory storing, the oxidized protein accumulates were treated with several cavitation jet times (0-15 min), and the soluble aggregates with the cavitation jet treatment were obtained during the centrifugation. We studied the change of the structure and the emulsifying and interfacial descriptions of the soluble component in protein accumulates after cavitation jet treatment. The mechanisms of the cavitation jet governing the oxidative accumulates of soybean protein and transformation law between soluble and insoluble components were explained at the molecular level. This might cause enhancements in the function of the cavitation jet in the soy protein plant, and deliver a theoretic base for the claim of the development, alterations, storing, and shipping of the soybean protein stuffs. 2. Materials and Methods 2.1. Materials Shandong Yuwang ecological food industry Plant Limited provided the soy protein isolate (92.4% protein) (Shandong, China). From Beijing Dingguo Changsheng Biotechnology Co. Ltd., chemicals such as 2,2'-dithiobis(5-nitropyridine) (DTNP) and 8-Anilino-1-Naphthalene Sulfonate (ANS) were acquired (Beijing, China). 2.2. Formulation of Soluble Soybean Protein Oxidized Accumulates According to a prior work, the soluble oxidized accumulates of soybean protein (SOSPI) were produced . To generate a 10 mg mL-1 soybean protein mix, the soybean protein was liquified in a phosphate buffer solution (PBS) with a phosphate dose of 0.01 mol L-1, a pH of 7.2, and 0.5 mg mL-1 of NaN3. The final concentration of AAPH was increased to 0.5 mmol L-1 by adding AAPH. The soybean protein oxidized accumulate mix with a 12 h oxidation period was prepared after oxidating treatment for 12 h at 37 degC and became murky. Dialysis was performed at 4 degC for 72 h using a 14,000 kDa dialysis bag, and the deionized water (dH2O) was replaced every 6 h. The samples were gathered and given the designation OSPI after spray drying. The soybean protein oxidized accumulate mix underwent a 12 h oxidation process before standing centrifugated at 4 degC for 20 min at a rapidity of 9000 rpm to discrete the soluble components from the insoluble components. The samples were gathered and given the name SOSPI after spray drying. 2.3. Formulation of Samples for Cavitation Jet Treatment The 2 L OSPI (25 degC, 10 mg mL-1) was poured into the SL-2 cavitation jet machine (Zhongsen Huijia Technology Development Co., Ltd., Beijing, China) to treat at 80 MPa for six diverse times: 2, 4, 6, 8, 10, and 15 min. The SOSPI was liquified in 0.01 mol/L PBS (pH 7.2, comprising 0.5 mg mL-1 NaN3). After treatment, the protein was immediately chilled in an ice bath for 15 min, and trailed by 20 h of centrifugal treatment at 4 degC and 9000 rpm to remove the insoluble parts. Spray drying was used to create all sample solutions, which were given the names SCOSPI-2 min, SCOSPI-4 min, SCOSPI-6 min, SCOSPI-8 min, SCOSPI-10 min, and SCOSPI-15 min. Three groups of parallel samples were taken. 2.4. Measuring the Particle Size Dispersal Based on the technique labeled by Ma et al. (2019), the particle size dispersal (PSD) was estimated via a laser scattering Mastersizer S (Malvern, UK) and a 300 inverse Fourier lens with the relief of a He-Ne laser l = 633 . The protein's refractive index was 1.33 when the amount was made at room temperature (RT, 25 +- 2 degC). Before measurement, the samples were diluted with dH2O to 50 mg/mL, and the particle sizes ranged between 0-10,000 mm. 2.5. Measurement of the Molecular Weight Circulation Following Ma et al. (2019) , examples of soybean protein were examined using an HPLC unit (Milford, MA, USA). Briefly, the molecular weight of the proteins at 280 nm was determined via a Waters 2175 UV finder (Milford, MA, USA). 2.6. Measurement of the Fourier Transform Infrared Spectroscopy (FTIR) Spectroscopy A Bruker Vertex 70 was used to analyze the materials using Fourier transform infrared (FTIR) (Bruker Optics GmbH, Ettlingen, Germany). At 0.5 cm-1 tenacity and RT (25 +- 2 degC), a total of 64 scans were found between 4000 and 400 cm-1. The secondary structure was determined using the FTIR spectra's secondary-derivation and deconvolution processes, and it was based on the amide I band (1600-1700 cm-1). According to Tang et al. (2009), the method involved the secondary structure of the proteins being examined using Peakfit Ver., 4.12 software, and the algorithm utilized was Gaussian peak fitting . 2.7. Measuring of the Fluorescence Emission Spectra According to the technique used by Jiang et al. (2014), the fluorescence emission spectra of the materials were found via a Hitachi F-7000 fluorescence spectrophotometer (Hitachi Inc., Tokyo, Japan) . The soybean protein trials were thinned in 0.01 mol L-1 PBS to a protein dose of 0.2 mg mL-1 to produce emission spectra at an excitation wavelength of 295 nm and from 300 to 400 nm. By employing a fixed 5 nm for both the emission and excitation in triplicate, the bandwidths were attained. 2.8. Measurement of the Sulfhydryl Content According to the Wu et al. (2019) approach, the amounts of disulfide bonds and free sulfhydryl (SH) assemblies were measured . DTNP was used in a variation of Ellman's approach to ascertain the SH cluster insides in the trial. The molar extinction constant (13,600 M-1 cm-1) was utilized to represent the SH contents as a nmol mg-1 protein. 2.9. Measuring of the Transmission Electron Microscopy (TEM) TEM was dedicated by utilizing a previously described technique . After being diluted 350 times in dH2O, the sample was dispensed in 30 mL droplets and applied on a carbon net (200 mesh). The surplus was wiped away using permeable paper after 120 s. The net was air-dehydrated on sieve paper after the samples were dyed for 3 min with a 2% uranyl acetate solution. Benefitting a TEM-JEM-1230 (JEOL, Tokyo, Japan) with a hastening voltage of 80 kV, the morphology of the sample was examined. 2.10. Measuring of the Emulsifying Activity Index (EAI) and Emulsion Solidity Index (ESI) The Kevin et al. (1978) approach was used to evaluate the EAI and ESI . A high-rapidity homogenizer (T-25 homogenizer, IKA, Staufen, Germany) was used to combine a 15 mL sample of a 0.1% (w/v) protein mix with 5 mL of maize oil at 7200x g for 10 min to create an emulsion. The emulsion was then detached from the lowest of the centrifuge tubes and normalized for 0 and 30 min before being diluted 100 times with 5 mL 0.1% sodium dodecyl sulfate. A spectrophotometer was used to test the absorbance at 500 nanometers (Beckman DU 500, Fullerton, CA, USA). The EAI and ESI were stated as:EAI(m2/g)=2x2.303xDFxA0(1-th)xCxphx10000ESI(%)=A0A0-A30x10 where A0 is the absorbance at 0 min of the thinned emulsion, DF is the dilution aspect (x100), c is the model dose (g mL-1), ph is the pictorial path, th is the portion of the oil (0.25), and A30 is the absorbance after 30 min. 2.11. Measurement of the Confocal Laser Scanning Microscope (CLSM) The Leica TCS SP2 CLSM was used to study the microstructure of emulsions. To create an emulsion, 15 mL of a 0.1% (w/v) protein mix was normalized with 5 mL of maize oil at 7200x g for 30 min. A 1 mL of emulsion was added to the dye (40 mL), which included 0.02% Nile red dye and 0.1% Nile blue dye. After that, a coverslip was put on top of the colored emulsion in the middle of the slide. To prevent the water from evaporating, silicone oil was sprayed to the superiority of the coverslip. The emphasis plane was originally changed following an inspection with a 100x impartial lens, while the slide was mounted on a laser confocal microscope phase. Pre-examining was performed with Ar ion at 488 nm and a He/Ne ion laser at 633 nm. A fluorescence figure was composed with a visualizing intensity of 1024 x 1024. 2.12. Measuring of the Quantity of Adsorbed Proteins at Interface (AP%) According to Liang and Tang, the amount of adsorbed proteins at the interface (AP%) of these emulsion samples was calculated . A 10,000 g centrifuge was used to spin each new emulsion (1 mL) for 45 min at RT. A cream coat (or concerted oil droplets) at the upper of the tube and the aqueous stage of the emulsion at the bottom were visible after centrifugation. A 0.22 mm filter was utilized to sieve the supernatant after the cream layer was delicately detached using a syringe (Millipore Corp.). The Lowry technique was utilized to estimate the filtrate's protein content, with a BSA serving as the reference. To estimate the protein intensity (Cs) in the upper phase, the initial protein mix was likewise centrifuged under identical circumstances. The AP (%) was expressed as:AP (%)=CS-CfC0x100 where Cs is the content of preliminary protein solution in the supernatant (mg), Cf is the content of protein in filtrate after centrifugation (mg), and C0 is the preliminary protein intensity of the protein mixes concerned for the emulsion formulation (mg). 2.13. Measurement of the Interfacial Tension Various materials' surface tension was estimated via an automated surface tensiometer (DCAT21, Data Physics Instruments GmbH, Filderstadt, Germany). A total of 20 mL of the sample mix was then put into a 25 mL cylinder after the protein model had first been dissolved in dH2O (1%, m/v). The apparatus's measuring variety was always between 1 and 100 mN m-1, with a SD that never went beyond 0.03 mN m-1. 2.14. Measurement of the Viscoelastic Properties The Sun et al. (2012) approach is used to assess the viscoelastic characteristics of emulsions . An RST-CPS rheometer was used to measure the sample emulsions' rheological characteristics (Brookfield, Middleboro, MA, USA). At a temperature of 40 degC, the samples were sandwiched between two parallel plates with 1 mm space among them. A strain examining the analysis performed at an incidence of 1 Hz was used to identify the linear viscoelastic area of each sample. Each protein sample's elastic and storage moduli were determined in the linear viscoelastic area. 2.15. Measurement of the Apparent Viscosity Rendering to the technique delineated by Swa et al. (2020), rheological tests were carried out via an AR 1500 regulated stress rheometer (TA, West Sussex, UK) outfitted with cone and bowl geometries (40 mm, angle 1deg, and gap 0.100 mm) . The same technique was used to create the sample emulsions. The sample emulsions were divided into 2.0 mL aliquots and placed on the stage for measurement at 25 +- 0.1 degC. After 5 min, the viscosity ranged from 0 to 200 s-1. Using the program, the measuring was performed in triplicate. We matched the investigational flow curves to Sisko's pattern that provided the finest fit and was signified by:e=e0+Kgn-1 where e is the ostensible viscosity (Pa*s), e0 is the vintage ostensible viscosity (Pa*s), K is the consistency index (Pa*sn), g is the shear ratio (s-1), and n is the performance index (dimensionless). 2.16. Statistical Analysis Statistical assessment was accomplished via SPSS ver. 20.0. The outcomes were imperiled to Duncan's multiple series and ANOVA tests. All the rates gained are stated as the mean +- SD in triplicate. A p-value <= 0.05 was measured significantly. 3. Results 3.1. Particle Size Distribution and Molecular Weight Circulation The SEC-HPLC and particle size dispersal can characterize the molecular weight, size, and aggregation degree of the soluble components in soybean protein oxidized aggregates treated by cavitation jet. It can be seen from Figure 1 and Figure 2 and Table 1 that, equated with SPI, the particle size of OSPI showed a unimodal particle size and lifted to the right, meaning the average particle size increased significantly. Furthermore, the elution time of the first molecular weight peak of OSPI diminished and the peak quantity increased. However, as a soluble component in OSPI, the particle size of SOSPI displayed a bimodal particle size, the initial particle size peak transferred to the left, and the average particle size decreased. The elution time of the first molecular weight peak of SOSPI increased and the peak area decreased. The results revealed that after the oxidation treatment, the oxidized accumulates with a huge particle size and a high molecular weight were insoluble aggregates, and the soluble components were proteins with a small particle size and a low molecular weight. Radicals in the oxidative environment could induce proteins to form insoluble oxidative aggregates through covalent crosslinking, but they will also attack protein side chains to form small molecular weight soluble proteins . With the increase of the cavitation jet treatment time, the retention time of the initial elution peak and the peak area of the protein components with a small molecular weight of SCOSPI decreased, and the particle size peak of SCOSPI lifted to the right. When the cavitation jet treatment time was 8 min, the first particle size peak of SCOSPI moved to the maximum right, and the average particle size achieved the highest. The outcomes displayed that the molecular weight and particle size of SCOSPI with the cavitation jet treatment increased, and the low molecular weight and small particle size protein components declined. The cavitation jet treatment could promote the depolymerization of insoluble aggregates in OSPI and transform them into soluble oxidized aggregates through high shear and cavitation effects, developing an increase of the particle size and molecular weight of the soluble oxidized accumulates . Moreover, the cavitation jet would intensify the collision between the small molecule soluble aggregates, and then polymerize into a bigger particle size and molecular weight soluble protein molecule, resulting in the reduction of small molecular weight protein components . When the cavitation jet treatment time exceeded 8 min, the first particle size peak of SCOSPI moved to the left and the retaining time of the initial elution peak and the peak area of protein substances with small molecular weight of SCOSPI amplified, indicating that when the treatment time was too long, the protein molecular weight of SCOSPI decreased and the small molecular weight protein component increased. The thermal effect and free radical effect of the cavitation jet, on the one hand, could promote the further aggregation among proteins to form insoluble aggregates, which were removed by centrifugation. On the other hand, it would split some peptide chains, resulting in soluble protein components dominated by small molecular weight and particle size protein molecules . Combined with the research of the team in the early stage , cavitation jets can break losing the disulfide bonds and protein skeleton structures that declined the amassed sizes and molecular weights of oxidized aggregates. However, how these components of protein aggregates mutually transform is unclear. Through the particle size and molecular weight of this research, we can obtain that a cavitation jet can also induce the insoluble aggregates to break down under high shear stress and transform into soluble aggregates, ensuing in the rise of the particle size and molecular weight of the solvable accumulates. Consequently, a suitable cavitation jet treatment could adjust the structural and functional attributes of OSPI by inducing the cleavage of insoluble oxidized aggregates and transforming them into soluble aggregate components. 3.2. FTIR Spectroscopy Fourier transform infrared spectroscopy can be utilized to elucidate the secondary structure change of proteins during aggregation and disaggregation . Figure 3 is the FTIR spectra, and Table 2 is the secondary structure of oxidized accumulates and soluble oxidized aggregates after the cavitation jet treatment. Oxidized treatment raised the compounds of b1, b-turn, and g-random coil in the OSPI and declined the compounds of a-helix. Compared with OSPI, the components of b1 and g-random coil in SOSPI declined, and the compounds of a-helix increased. a-helix has structured secondary structures featured by high inflexibility and recurrence structure, while g-random coil has unordered secondary structures featured by plasticity and the deficiency of a recurrence structure . The marker structure of aggregation (b1) is created by molecular interactions during protein oxidation . Changes in the constancy of the H-bond between the amino parties and the polypeptide chain's carbonyl parties are primarily responsible for the changes in the amount of a-helices . Since the hydrogen connection among the amino and carbonyl groups in the polypeptide chain is unstable, oxidation may attack the amino acid residues in the primary peptide chain, reducing the amount of a-helix present. The spatial structure of a protein heavily influences its functional activities, and proteins with a suitably organized and compact structure exhibit beneficial functional behaviors . Compared with other samples, the lowest a-helix content of OSPI referred that excessive oxidation would seriously demolish the ordered structure of protein. This might be one of the important reasons for the decline of OSPI functional activity . Comparing the results of OSPI and SOSPI, we can find that oxidized protein with several b1 existed in OSPI, while SOSPI has more rigid and ordered structures. With the increase of the cavitation jet treatment time, b1 of SCOSPI increased first, then decreased and then increased, and other structures showed no obvious regular change trend. Combined with the outcomes of particle size and molecular weight, the superior pressure and superior shear strengths of the cavitation jet at a fleeting treatment time could lead to the cleavage of protein accumulates by weakening the protein-protein interactions and induce insoluble aggregates with high contents of b1 to transform into soluble aggregates resulting in the increase of the b1 contents . Nevertheless, after the treatment time of the cavitation jet exceeded 6 min, the components of b1 of SCOSPI decreased first and then increased. The cavitation jet with long treatment time could induce the soluble aggregate in OSPI to aggregate further, due to the thermal impact and extra-speed instability and formed the insoluble aggregates with high b1 components which were centrifuged and removed, resulting in the decrease of the b1 content. When the cavitation jet treatment time was 8-15 min, the b1 content of SCOSPI increased. Combined with the previous research results of the team during this timeframe, the b1 of the protein oxidized accumulates and soluble aggregates both increased, which showed that continuously extreme cavitation jet treatment can cause the formation of more b1 structures with the aggregation characteristics of soluble and insoluble components. Integrated with the particle size and molecular weight findings, we could find that the particle size and molecular weight of SCOSPI decreased with a long cavitation jet treatment time. This showed that the cavitation jet could depolymerize the soluble components and at the same time could induce the aggregation reaction between protein molecules, resulting in more b1 structures. The above results showed that the control of cavitating jet is an extremely complex process. The cavitation jet might dynamically govern the depolymerization and reaggregation of soluble soybean protein oxidized accumulates through the transformation of the protein spatial structure. 3.3. Fluorescence Emission Spectra Fluorescence spectra can characterize the polarity changes of aromatic amino acids in the microenvironment, so as to predict alterations in the tertiary structure of soluble protein aggregates . Figure 4 is the intrinsic fluorescence spectra of the soluble soybean protein oxidized aggregates. Compared with SPI, the fluorescence intensity of OSPI declined significantly and lmax was blue shifted. Compared with OSPI, the fluorescence intensity of SOSPI raised and lmax was red shifted. Free radicals in the oxidizing situation could induce the crosslinking, condensation, and nucleation of SPI, and then form the protein aggregation with a tighter structure . Comparing the fluorescence spectrum of OSPI and SOSPI, we could find that the components with tighter structures in the OSPI components exist in insoluble oxidized aggregates. On the other hand, this showed that the change of the structural tightness degree could reflect the conformational change law of the transformation from a soluble protein to an insoluble protein. With the prolongation of the cavitation jet treatment time, SCOSPI lmax initially raised and then declined, and achieved the supreme when the treatment time was 6 min. This showed that the cavitation jet could alter the spatial structure of SCOSPI to regulate the functional activity. The cavitation jet could cleave and break some insoluble soybean protein oxidation aggregates and induce the creation of soluble oxidation accumulates with a loose structure and a larger particle size. This could cause the increase of the number and exposed degree of aromatic amino acid elements of SCOSPI; to show polar solute circumstances, a red move of lmax of SCOSPI was noted. However, with the further extension of the treatment time, some soluble oxidized aggregates of the soybean protein regrouped and transformed into insoluble aggregates, which were centrifuged, resulting in the reduction of the number of aromatic amino acid elements of SCOSPI . In addition, the other soluble aggregates could form b1 structures and aggregate through covalent cross-linking and hydrophobic interaction in the collision, so that the aromatic amino acids of SCOSPI were buried in the structure . Consequently, excessive cavitation jet treatment will, through this dual effect of aggregation and depolymerization together, cause the lmax of SCOSPI blue shift. 3.4. Sulfhydryl Content SH/SS replacement reactions play a key role in protein accumulation, which can reproduce the impact of physical fields on the depolymerization mechanisms of protein. As shown in Table 3, the free sulfhydryl and total sulfhydryl quantity of OSPI decreased, and the disulfide bond quantity increased compared with SPI. However, compared with OSPI, the free sulfhydryl content of SOSPI rose, and the disulfide bond content decreased. The oxidation treatment could promote the creation of disulfide bonds via the disulfide/sulfhydryl switch reaction. More disulfide bonds reflect the tighter spatial structure of soybean protein, so that the oxidation increased the tightness of the soybean protein molecular space structure . This showed that oxidation could adjust the compactness of the protein structure by changing the disulfide bond, thus affecting its functional activity . In addition, the total sulfhydryl content was also decreasing, and the decay of free sulfhydryl quantity was higher than the raise of disulfide bond content, signifying that oxidation also had a non-reversible oxidation reaction on the soybean protein, inducing the transformation of free sulfhydryl into sulfur compounds without a disulfide bond . For soluble components in OSPI, namely SOSPI, the disulfide bond quantity decreased. Combined with the results of the particle size and fluorescence emission spectra, the oxidized treatment diminished the particle size and amplified the fluorescence intensity of SOSPI, indicating that the oxidized masses of soybean protein after the oxidation treatment were mainly insoluble aggregates, and most of the soluble components show a small particle size and a loose and unfolded structure . With the addition of the cavitation jet treatment time, the contents of free sulfhydryl, total sulfhydryl, and disulfide bonds of SCOSPI amplified first and then declined, but the processing time, corresponding to a maximum value of the three, is inconsistent. This is because there was more than one conversion reaction between free sulfhydryl and disulfide bonds in the system. The cavitation jet treatment could break the core aggregation skeleton of SCOSPI, destroy the intermolecular force and spatial structure, and induce the conversion of disulfide bonds into free sulfhydryl groups, resulting in the increase of the free sulfhydryl content of the soluble oxidized aggregates. At the identical time, combining with the increase particle size findings of SOSPI after the cavitation jet, cavitation could also promote the transformation from insoluble aggregates with high disulfide bonds content into soluble aggregates, so the number of soluble aggregates increased , which instigated the expansion of disulfide bond content and free sulfhydryl content of soluble oxidized aggregates. Nevertheless, when the cavitation jet treatment time was too long, high intensity, long-time cavitation, turbulence, and thermal effects would cause the re-aggregation of soluble aggregates and also cause the cracking of all accumulates, which was an irreversible denaturation for protein . To be more specific, when the treatment time was maximized, the cavitation jet would destroy the spatial structure and intermolecular force of the oxidized aggregates, resulting in the reduction of the disulfide bond content, which is well matched with the finding of a decreased particle size. However, the free sulfhydryl groups, formed by the disulfide bond breaking in OSPI, would aggregate with each other to form SCOSPI with a tighter spatial structure, ending in a reduction in the quantity of free sulfhydryl groups. This is matched with the findings of FTIR spectroscopy and fluorescence emission spectra. At the same time, cavitation jet also induced the irreversible reaction of protein sulfhydryl groupings to generate the sulfur-comprising components with non-disulfide bonds. Moreover, the conversion of soluble oxidized aggregates to insoluble oxidized aggregates will also lead to the fluctuation of the disulfide bond and free sulfhydryl quantity. These factors together caused the reduction of free sulfhydryl and disulfide bond quantity. The inconsistent processing time, corresponding to the maximum value of the free sulfhydryl, total sulfhydryl, and disulfide bond, showed that the process of aggregation and depolymerization and the conversion between soluble and non-soluble was a very complex process and needs further research. 3.5. Transmission Electron Microscopy (TEM) To better understand SPI and compare the differences among SPI, OSPI, SOSPI, and SCOSPI, the apparent morphology was visualized by TEM, as shown in Figure 5. Compared with SPI, the aggregation degree of OSPI was increased, and OSPI formed a dense network structure with intense central part. The skeleton structure of SOSPI mainly presented short and small wormlike structures. Oxidation led to the conformational changes of SPI and exposed the side chain groups of hydrophobic aliphatic and aromatic amino acids entrenched within, inducing cross-linking aggregation through hydrophobic interaction. Furthermore, they can also attack the sulfhydryl groups of proteins and convert them into disulfide bonds, showing insoluble protein accumulates with a large particle size and highly cross-linked clusters in OSPI . However, SOSPI showed short rod protein molecules with a small particle size; this is because the proteins with a high degree of cross-linking were transformed into insoluble aggregates and removed by centrifugation , as the soluble components of the protein are mainly in the shape of short and small rods. With the increase of the cavitation jet treatment time from 2 min to 8 min, the aggregation degree of SCOSPI increased. Most of the protein aggregates heavily bonded, which consisted of agglomerated smaller worm-like particles, and the skeleton structure became larger and more branches appeared. This is well matched with the outcomes of the particle size and molecular weight. On the one hand, it might be because the cavitation jet broke the disulfide bond of the aggregates, and the insoluble aggregation with large, clustered morphology, resembling those of compact reticulation, was cracked. Then, the insoluble aggregation transformed into soluble aggregates, which led to the amplification of the number of soluble aggregates in the supernatant and presented a cluster structure. On the other hand, it might be that under the cavitation treatment, the fragmentation of the skeleton structure increased. This result promoted the mutual collision between soluble protein molecules and the binding probability of free sulfhydryl clusters, resulting in the enlargement and additional branches of the originally short rod-shaped skeleton structure . However, with the further conservation of the cavitating jet treatment time, the mesh skeleton structure was seriously broken and gradually transformed into a short bar structure. When the treatment time reached 15 min, the mesh structure disappeared, and the skeleton structure presented a slender bar. Combined with the above results, the cavitation jet has the dual effects of breaking and reassembling the protein skeleton structure. In addition, it also induces a mutual transformation between the soluble and insoluble aggregates. Therefore, the long-time cavitation jet can induce the soluble aggregates to transform into insoluble aggregates and be removed by centrifugation, and decrease the content of the soluble aggregates. In addition, under high temperatures, great pressure, and the shear force conditions of the long-time cavitation jet, the skeleton structure of the protein was broken. These two works together resulted in the decrease of the cementation and intercross network structure of the soluble aggregates, and the formation of a small and slender skeleton structure. 3.6. Emulsion Capacity and Stability Due to its good emulsifying activity, proteins are usually used in food emulsions and artificial fats. However, emulsifying features differ both on the capability of the protein adsorbed on the oil droplet superficially and the protein intermolecular binding, and it is related to the shape, size, and superficial hydrophobicity of the protein molecules . The EAI and ESI of the emulsions stabilized by the soluble soybean protein oxidized accumulates are shown in Table 4. The oxidization treatment decreased the EAI and ESI of emulsions steadied by OSPI and SOSPI. In addition, the EAI of SOSPI was superior to that of SPI, but the ESI was relatively inferior. The oxidation treatment could form a highly ordered intermolecular b-sheet between proteins, which were poor supports to the flexible body. The molecular flexibility of OSPI decreased and formed insoluble oxidized aggregates whose structure is difficult to relax, resulting in the reduction of protein interface activity and the decline of the binding ability between the protein and oil, which induced the EAI and ESI of OSPI, inferior than that of SPI . However, compared with the OSPI, the SOSPI contained abundant free sulfhydryl groups and short worm-like skeleton structures. In the process of forming the emulsions, the SOSPI moved to the interface in smaller particle size aggregates, which increased the exchange area between the protein and the oil-water interface and made SOSPI easier to absorb and relax at the interface, so that the EAI of SOSPI were higher than that of OSPI. Nevertheless, it is difficult for proteins with a miniature particle size to adsorb stably on the interface for a long time, resulting in a decrease of the ESI of SOSPI. With the extension of the cavitation jet treatment time, the EAI and ESI of SOSPI initially amplified and then declined, and achieved the highest when the cavitation jet treatment time was 6 min. The high pressure, shear, and cavitation effects shaped by the cavitating jet could cleave oxidized aggregates and induce some insoluble aggregates to change into soluble aggregates, so that the content of the soluble protein accumulates increased. The cavitation jet could also abolish the intermolecular binding of SCOSPI, and the structure of SCOSPI is altered. These two results result in the amplification of the number of exterior hydrophobic parties and polar groups, particle size, and molecular flexibility of SCOSPI to improve the emulsifying activity and emulsifying constancy . Additionally, the intercross networks structure, cross-connected by the protein, were useful in the creation of the emulsion . Combined with the TEM results, it can be found that after the cavitation jet treatment, the mesh skeleton structure of SCOSPI became larger than was beneficial to the formation and stability of the emulsion. However, when the cavitation jet treating time exceeded 6 min, the long-time high temperature, pressure, and shear force produced by cavitation jet impacted the hydrophilic and hydrophobic clusters and interior binding of the protein molecules, which unfavored the aptitude of SCOSPI to adsorb at the oil-water interface. In addition, during the long time cavitation jet, the SCOSPI gradually formed a small molecular protein with a more highly ordered b-sheet structure, difficult relaxation, and low molecular flexibility, resulting in the reduction of the emulsifying activity and emulsifying stability of SCOSPI . 3.7. Confocal Laser Scanning Microscope (CLSM) The CLSM was utilized to explore the fluctuations in the microstructure of the soybean protein emulsion after pretreating it with the cavitation jet, as displayed in Figure 6. The green fluorescence in CLSM micrographs signified the protein piece, the red fluorescence represented the soybean oil, and the vivid yellow fluorescence signified the proteins adsorbed on the oil droplets. The small spherical droplets were evenly distributed throughout the emulsion system, which was prepared from SPI. After the oxidation treating, the flocculation of the emulsion dewdrops prepared from OSPI and SOSPI were serious, and the red areas in the unceasing phase increased significantly and gathered on the surface of emulsion droplets, showing the phenomenon of oil-water separation. The oil-water separation of OSPI was more serious than SOSPI. The oxidative treatment caused the accumulation and solubility of the protein molecules to decrease, causing difficulty for the formation of steady interfacial film in the emulsification procedure, thus showing a red oil droplet accretion area in a large aggregation area . Nevertheless, compared with OSPI, the particle size and skeleton structure of SOSPI were smaller, and it had better adsorption ability at the oil-water interface. Most of the oil droplets are encapsulated in the emulsion droplets prepared by SCOSPI, and there was only a small-scale accumulation of red oil droplets. This shows that macromolecules and insoluble protein oxidation aggregates formed during the oxidation were the key to causing significant decline in emulsifying activity. Therefore, regulating the macromolecules and insoluble proteins aggregates could improve the function activity of OSPI. During the treatment of the cavitation jet, the green area of the emulsion prepared with SCOSPI in the CLSM image increased gradually, and the red area gradually decreased and showed the wrapped state in the green area, and a steady protein interfacial film was shaped at the oil-water interface. The cavitation jet treatment could induce the insoluble oxidation aggregates, which are difficult to relax at the interface, to transform into soluble aggregates with smaller steric hindrance and a more flexible structure, as well as to expand the adsorption, relaxation, and reordering effects of SCOSPI at the interface, so as to improve the steadiness of the oil-water interface . In addition, the number of surface hydrophobic groups and polar groups in SCOSPI were increased, which promoted additional protein molecules to be adsorbed at the oil-water interface to produce a steadier interfacial film, and could improve the interfacial activity and emulsifying features . Therefore, the proteins discolored green were consistently and firmly spread across the oil droplets that efficiently avoided coalescence. However, with the further delay of the cavitation jet treatment time, in the CLSM images of the emulsion prepared by SCOSPI, the red area of oil increased gradually, and the green area of the protein was mainly concentrated. Excessive cavitation jet treatment would increase the content of the anti-parallel intermolecular b-sheet, surface hydrophobicity, and z-potential reduction of SCOSPI, and the specific surface area declined, which was not encouraging for distribution to crossing and extension on the oil-water interface . Finally, the binding capacities of SCOSPI to oil were weakened, resulting in the increase of free oil droplets and serious oil-water separation. A cavitation jet can affect the flocculation and stability of the emulsion by regulating the transformation between insoluble and soluble aggregates. 3.8. Quantity of Adsorbed Proteins at Interface (AP%) The quantity of the adsorbed proteins at the interface has a key impact on the constancy of the emulsion. The higher interfacial protein amount, the stronger capability of protein adsorption to the oil-water interface . As shown in Figure 7, the AP% of the emulsion made by OSPI and SOSPI decreased compared with SPI, and the AP% of the emulsion made by SOSPI was lower than OSPI. It is generally considered that proteins undergo a certain amount of structural extension and relaxation when dissolved in an aqueous solution . The more flexible the structure of the proteins, the better it is for structural expansion and the more prone they are to bulk diffusion, adsorption at the interface, unfolding, and rearrangement, inducing the increase of AP% . During oxidation, the level of protein accumulation creased, and the structural flexibility of highly clustered OSPI was reduced and its rigidity was enhanced, which was not favorable to the expansion and adsorption at the interface, resulting in the decrease of AP% . In addition, the decrease of AP% might be largely ascribed to the fabrication of the bridged emulsions, e.g., two particular oil droplets allocated an identical protein particle layer, facilitated by the oxidization-induced aggregation of SPI . At the time of the formulation of the emulsion, large molecules can be transported to the oil-water interface in preference to small molecules because of the convective mass transport effect of high-pressure homogenization, i.e., large molecules can adsorb more quickly than small molecules . Compared with OSPI, SOSPI had a small particle size and low molecular weight, which affected the protein adsorption ratio to the oil-water interface, subsequent in lower AP% . However, the protein with a small particle size, low molecular weight, and a high solubility contributes to improving the connection region with the oil-water interface and the affinity of protein to the interface; foremost there was only a small-scale accumulation of red oil droplets in the emulsion prepared by SOSPI. Therefore, the AP% is not the only consideration in deciding the characteristics of emulsion. With the expansion of the cavitation jet treatment time, the AP% of SCOSPI boosted first and then diminished, and touched the highest when the treatment time was 6 min. The high-velocity turbulent flow, high-speed shearing, and large pressure produced by the cavitation jet acted on cross-linked aggregates to realize the transformation from insoluble aggregates to soluble aggregates and directional regulation for amorphous soluble aggregation, which improved the soluble protein content and molecular flexibility of the soluble proteins. It promoted more proteins with structural relaxation and overall flexibility to be adsorbed, unfolded, and rearranged at the interface. Then, it enhanced the AP% of the emulsion prepared by SCOSPI, and increased to interfacially prepare and bulk stabilize the oil-water systems . Nevertheless, when the cavitation jet treating time was too long, the majority of the soluble protein components were dominated by a small molecular weight and particle size protein molecules, and the content of b1 in SCOSPI was increased, designating that the content of the methodical structure augmented and that the structure was relatively tight and complex, which was not favorable for the adsorption and unfolding of protein at the oil-water interface and the formation of a dense interface interfacial film. These results together led to the decrease of AP% of emulsion prepared by SCOSPI . 3.9. Interfacial Tension A key element in the investigation and analysis of emulsion stability is the interfacial tension at the liquid-liquid interface, which can also describe the exterior activity of proteins at the oil-water interface . As shown in Figure 8, the interfacial tension of the emulsion prepared with natural soy protein was 21.29 mN/m. After the oxidation treatment, the interfacial tension of the emulsions made by OSPI and SOSPI were raised, and SOSPI was higher than OSPI. The oxidation treatment would promote a protein aggregate to form insoluble oxidation aggregates with a larger particle size, low molecular flexibility, and poor solubility . In addition, the exact surface region of the protein molecules was reduced, and the steric hindrance was increased, which were not beneficial to the adsorption and reordering of protein at the oil-water interface, resulting in an increase in the protein interfacial tension . The soluble components in SOSPI were more easily dissolved in the water phase, the particle size was lesser, and the ordered structure was greater. The ability to form stable interfacial film was significantly reduced, resulting in a higher interfacial tension and lower emulsion stability. After the cavitation jet treatment, the interfacial tension of SCOSPI decreased first and was then amplified, achieving the bottom when the treatment time was 6 min. Combined with the results of the particle distribution and TEM, it could be seen that the particle size of SCOSPI increased and the protein skeleton widened, indicating that the cavitation jet could break the insoluble soybean protein oxidation aggregate and transform it into soluble oxidation aggregate, resulting in the increase of hydrophobic clusters of soluble protein components and a more complex conformational space . Protein molecules were not easily soluble in the aqueous phase, which improved the expansion and reordering of the protein molecules at the interface and declined the interfacial tension . However, when the cavitation jet treatment time was too long, the quantity of b1 increased and the g-random coil (Table 2) decreased. Additionally, particle size (Table 1) was reduced and soluble oxidized aggregates with a more orderly structure and lower molecular flexibility were formed, with the result that adsorption energy barricaded at the boundary was higher, and the adsorption efficacy was decreased. This affected the adsorption and evolving of the protein at the oil-water interface and caused an increase of interfacial tension . Combined with the results of EAI, ESI, CLSM, and AP%, we can find that when the cavitation jet treatment was 6 min, the soluble soybean protein oxidized aggregates showed the best emulsification interface characteristics. This provided a simple and effective technology for the application of soybean protein in the food industry. 3.10. Viscoelastic Properties Rheological quantities deliver evidence on the physical performance and steadiness of lotion . Elastic modulus G' is a measurement of elasticity and signifies the storing modulus of the energy of stress that could be reinstated when the stress is liberated, while the viscous modulus G'' signifies the viscous substances, which assumes the flow defiance of the sample . G' and G'' of emulsion are shown in Figure 9. Both the elastic modulus (G') and viscous modulus (G'') gradually amplified within the oscillation frequency range. In all samples, the G' was superior to the G'' and exhibited an elastic character. It showed that the protein on the interface formed a viscoelastic adsorbed film and suggested an elastic network structure of emulsion. The G' and G'' of the emulsion formulated by OSPI were both higher than SPI, while the SOSPI showed the opposite results. The rheological features of the interface layer were mainly impacted by the hydrophobic interaction and disulfide bonds among proteins adsorbed at the oil-water interface . After the oxidation treatment, the quantity of the disulfide bonds in the protein aggregates increased significantly. In addition, under the hydrophobic interaction, they were bound to the proteins that have been adsorbed on the interface layer. Therefore, the formation of the oxidative aggregates enhanced the binding between protein molecules at the interface, thus amending the interfacial elastic modulus of the emulsion prepared by OSPI. However, the soluble soybean protein aggregates were transformed into insoluble oxidized aggregates after the oxidation treatment. The soluble components with smaller molecular proteins were more likely to dissolve in the water phase and were unable to shape a protein-bound film, resulting in a decline in G' and G'' of emulsion equipped by SOSPI . With the cavitation jet treatment, the G' of the emulsion fabricated by SCOSPI increased first and then declined, and the G'' presented no obvious change. With the delay of cavitation jet treatment time, the skeleton of soluble oxidized aggregates became wider, the particle size was larger, the exposure of hydrophilic and lipophilic groups in components raised, the electrostatic revulsion among emulsion droplets was also amplified, the protein content adsorbed on the oil-water interface layer was more and more, and the thickness of the interfacial film slowly augmented, subsequent to the increase of interfacial elastic modulus. When the cavitation jet treating time was maximized, the SCOSPI through b1 would form protein aggregates with a high aggregation degree, small particle size, and weak reticular structure, which had negative impacts on the interface activity and resulted in the decrease of G' . 3.11. Apparent Viscosity Figure 10 and Table 5 depict the emulsions' rheological behavior. All the emulsions' flow curves could be matched with Sisko's model. With flow performance indices fluctuating from 0.059 to 0.271, all the emulsions displayed shear-thinning behavior. Intermolecular bindings among aggregated molecules, which result in the creation of weak transient networks, may be the motive of shear-thinning actions for the stabilized emulsions . Emulsions steadied by OSPI exhibited a higher ostensible viscosity and K than those stabilized by SPI, but those steadied by SOSPI had an inverse relationship between their apparent viscosity and K. The volume proportion of the dispersed phase and the size of the combinations created from the proteins determined the rheological parameters of the emulsion. The higher the number and size of these masses, the higher the viscosity . This led to a higher initial apparent viscosity in the emulsion made from oxidized soy protein aggregates. After the oxidation process, the initially soluble oxidized aggregates eventually changed into insoluble oxidized aggregates. A reduction in the K and apparent viscosity resulted from the remaining soluble components, which were primarily made up of tiny protein molecules that tended to dissolve in the aqueous phase and enclose oil droplets . The ostensible viscosity and K of the emulsion produced by SCOSPI grew initially and subsequently dropped with the length of the cavitation jet treatment time. Particle size and TEM results show that an effective cavitation jet treatment could encourage the conversion of insoluble aggregates into soluble aggregates, increase the particle size and skeleton structure of soluble soybean protein oxidation masses, and ultimately, increase the ostensible viscosity and K of the emulsion created by SCOSPI. However, the prolonged cavitation jet treating period might lead to an increase in insoluble aggregates that were unfavorable to the steadiness of the emulsion, lowering K and its apparent viscosity. By modifying the structure and content of the soy protein soluble oxidized accumulates and controlling the reciprocal transformation of protein constituents, the cavitation jet treatment can alter the rheological characteristics of the emulsion. 4. Conclusions Oxidized treatment influenced the structure of the SOSPI, causing a decline in their emulsifying properties and interfacial features. A cavitation jet at a short treating time can break the insoluble soybean protein oxidation aggregate and transform it into a soluble oxidation aggregate, causing the expansion of particle size, protein skeleton, and disulfide bond content. This also improved the emulsion activity and state of SOSPI and raised the quantity of adsorbed proteins at the interface while decreasing the interfacial tension of the emulsion. A long cavitation jet treatment time could induce the soluble oxidized aggregate to gradually form a small molecular weight protein with difficult relaxation and low molecular flexibility, which were not favorable to the solidity of emulsion, resulting in the decrease of EAI, ESI, apparent viscosity, K, and an increase of interfacial tension. Acknowledgments The authors would like to thank the National Natural Science Foundation , the National key R&D plan [2021YFD2100401], the National key R&D plan [2022YFF1100603], the Heilongjiang Province key R&D plan [GA21B001] the Heilongjiang Province Major Achievements Transformation Project [CG19A002], the China Fund on the surface of a postdoctoral project [2022M721995], and the Heilongjiang Province million project [2021ZX12B02] for the support. Author Contributions Y.G.: Conceptualization, Software, Writing-original draft. C.L.: Data curation. Y.W.: Investigation, Writing. S.R.: Visualization. X.Z.: Methodology. J.Z.: Investigation. T.C.: Investigation. Z.G.: Supervision. Z.W.: Funding acquisition, Project administration. All authors have read and agreed to the published version of the manuscript. Data Availability Statement The data presented in this study are available on request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Particle size distribution (PSD) of natural, oxidized soybean protein, and the cavitation jet treated on soluble soybean protein oxidized accumulates at several times (2, 4, 6, 8, 10, and 15 min). Figure 2 SEC profiles of natural, oxidized soybean protein, and cavitation jet treated on soluble soybean protein oxidized accumulates at several times (2, 4, 6, 8, 10, and 15 min). Figure 3 FTIR spectra of natural, oxidized soybean protein, and cavitation jet treated on soluble soybean protein oxidized accumulates at several times (2, 4, 6, 8, 10, and 15 min). Figure 4 Fluorescence emission spectra of natural, oxidized soybean protein, and cavitation jet treated on soluble soybean protein oxidized accumulates at several times (2, 4, 6, 8, 10, and 15 min). Figure 5 Backbone structure of natural, oxidized soybean protein, and cavitation jet treated on soluble soybean protein oxidized accumulates at several times (2, 4, 6, 8, 10, and 15 min). Figure 6 CLSM micrographs of natural, oxidized soybean protein, and cavitation jet treated on soluble soybean protein oxidized accumulates at several times (2, 4, 6, 8, 10, and 15 min). Note: (a) represents the proteins, (b) represents the oil droplets, and (c) represents the proteins adsorbed on the oil droplet. Figure 7 AP% of emulsions of natural, oxidized soybean protein, and cavitation jet treated on soluble soybean protein oxidized accumulates at several times (2, 4, 6, 8, 10, and 15 min). Note: Values with a different letter(s) indicate a significant difference at p <= 0.05. Figure 8 Interfacial tension of emulsions of natural, oxidized soybean protein, and cavitation jet treated on soluble soybean protein oxidized accumulates at several times (2, 4, 6, 8, 10, and 15 min). Note: Values with a different letter(s) indicate a significant difference at p <= 0.05. Figure 9 Viscoelastic properties of natural, oxidized soybean protein, and cavitation jet treated on soluble soybean protein oxidized accumulates at several times (2, 4, 6, 8, 10, and 15 min). Figure 10 Apparent viscosity and shear rate relationship of emulsions of natural, oxidized soybean protein, and cavitation jet treated on soluble soybean protein oxidized accumulates at several times (2, 4, 6, 8, 10, and 15 min). foods-12-00909-t001_Table 1 Table 1 Particle size and protein dispersibility index (PDI) of natural, oxidized soybean protein, and cavitation jet treated on soluble soybean protein oxidized accumulates at several times (2, 4, 6, 8, 10, and 15 min). Samples Particle Size (nm) PDI SPI 181.59 +- 2.77 h 0.76 +- 0.11 a OSPI 3836.18 +- 66.82 a 0.19 +- 0.03 d SOSPI 97.38 +- 1.63 i 0.17 +- 0.01 d SCOSPI-2 min 551.03 +- 5.07 e 0.14 +- 0.02 ef SCOSPI-4 min 639.27 +- 18.31 d 0.11 +- 0.04 f SCOSPI-6 min 705.81 +- 15.33 c 0.29 +- 0.04 c SCOSPI-8 min 776.14 +- 11.91 b 0.15 +- 0.01 e SCOSPI-10 min 538.15 +- 9.52 f 0.28 +- 0.03 c SCOSPI-15 min 443.96 +- 22.28 g 0.33 +- 0.01 b Note: Comparisons were carried out between values of the same column; values with a different letter(s) indicate a significant difference at p <= 0.05. foods-12-00909-t002_Table 2 Table 2 Secondary structure content of natural, oxidized soybean protein, and cavitation jet treated on soluble soybean protein oxidized accumulates at several times (2, 4, 6, 8, 10, and 15 min). Content (%) Anti-Parallel Intermolecular b-Sheet (b1) Parallel Intermolecular b-Sheet (b2) Intramolecular b-Sheet a-Helix b-Turn g-Random Coil Wavenumber (cm-1) 1608-1622 1682-1700 1622-1637 1646-1662 1662-1681 1637-1645 SPI 9.27 +- 0.28 d 15.95 +- 0.23 a 18.41 +- 0.18 a 26.35 +- 0.18 c 21.33 +- 0.17 f 8.69 +- 0.15 e OSPI 13.58 +- 0.19 a 7.16 +- 0.18 f 17.18 +- 0.13 b 17.82 +- 0.19 d 26.66 +- 0.20 e 17.41 +- 0.20 a SOSPI 9.05 +- 0.26 d 9.74 +- 0.22 b 13.58 +- 0.25 f 27.84 +- 0.19 a 28.28 +- 0.18 d 10.51 +- 0.22 b SCOSPI-2 min 11.28 +- 0.08 c 8.54 +- 0.26 d 13.38 +- 0.07 g 26.27 +- 0.08 c 30.61 +- 0.13 b 9.92 +- 0.17 c SCOSPI-4 min 12.70 +- 0.10 b 7.84 +- 0.19 e 13.80 +- 0.26 e 24.52 +- 0.19 e 31.82 +- 0.02 a 9.32 +- 0.18 d SCOSPI-6 min 12.77 +- 0.01 b 9.02 +- 0.25 c 13.58 +- 0.05 f 23.36 +- 0.04 f 31.92 +- 0.22 a 9.35 +- 0.06 d SCOSPI-8 min 11.42 +- 0.19 c 9.83 +- 0.01 b 15.80 +- 0.19 c 22.72 +- 0.14 g 29.66 +- 0.22 c 10.57 +- 0.16 b SCOSPI-10 min 12.78 +- 0.06 b 6.78 +- 0.22 g 13.70 +- 0.03 ef 26.68 +- 0.20 b 30.73 +- 0.24 b 9.33 +- 0.30 d SCOSPI-15 min 13.51 +- 0.13 a 6.76 +- 0.05 g 15.36 +- 0.15 d 25.09 +- 0.06 d 29.79 +- 0.15 c 9.49 +- 0.18 d Note: Comparisons were carried out between values of the same column; values with a different letter(s) indicate a significant difference at p <= 0.05. foods-12-00909-t003_Table 3 Table 3 Sulfhydryl content of natural, oxidized soybean protein, and cavitation jet treated on soluble soybean protein oxidized accumulates at several times (2, 4, 6, 8, 10, and 15 min). Samples Free Sulfhydryl (nmol/mg) Total Sulfhydryl (nmol/mg) Disulfide Bond (nmol mg-1) SPI 11.72 +- 0.15 a 15.64 +- 0.32 a 1.96 +- 0.11 c OSPI 4.05 +- 0.23 h 10.78 +- 0.14 g 3.37 +- 0.13 a SOSPI 8.06 +- 0.22 e 10.99 +- 0.12 f 1.47 +- 0.16 d SCOSPI-2 min 8.65 +- 0.17 c 11.95 +- 0.16 e 1.65 +- 0.17 cd SCOSPI-4 min 8.92 +- 0.21 b 12.56 +- 0.18 c 1.82 +- 0.13 c SCOSPI-6 min 8.87 +- 0.19 bc 13.29 +- 0.22 b 2.21 +- 0.15 b SCOSPI-8 min 8.21 +- 0.14 d 12.15 +- 0.19 d 1.97 +- 0.16 c SCOSPI-10 min 7.65 +- 0.16 f 10.69 +- 0.15 g 1.52 +- 0.16 d SCOSPI-15 min 7.15 +- 0.18 g 10.07 +- 0.16 h 1.46 +- 0.19 d Note: Comparisons were carried out between values of the same column; values with a different letter(s) indicate a significant difference at p <= 0.05. foods-12-00909-t004_Table 4 Table 4 Emulsion capacity and solidity of natural, oxidized soybean protein, and cavitation jet treated on soluble soybean protein oxidized accumulates at several times (2, 4, 6, 8, 10, and 15 min). Samples EAI/(m2*g-1) ESI/min SPI 91.61 +- 1.17 a 186.19 +- 3.89 a OSPI 46.97 +- 2.24 g 137.20 +- 3.51 e SOSPI 89.92 +- 2.37 a 126.75 +- 3.18 f SCOSPI-f-2 min 57.17 +- 1.89 f 149.08 +- 3.15 d SCOSPI-f-4 min 71.92 +- 1.91 e 156.24 +- 2.89 c SCOSPI-f-6 min 86.24 +- 2.39 b 163.17 +- 2.68 b SCOSPI-f-8 min 81.64 +- 2.64 c 152.56 +- 3.37 cd SCOSPI-f-10 min 76.95 +- 2.19 d 147.89 +- 2.90 d SCOSPI-f-15 min 72.68 +- 2.64 e 140.27 +- 3.64 e Note: Comparisons were carried out between values of the same column; values with a different letter(s) indicate a significant difference at p <= 0.05. foods-12-00909-t005_Table 5 Table 5 Fitting result of Sisko's model of emulsion of natural, oxidized soybean protein, and cavitation jet treated on soluble soybean protein oxidized accumulates at several times (2, 4, 6, 8, 10, and 15 min). Samples K (Pa*sn) n R2 SPI 0.066 0.271 0.99 OSPI 0.626 0.059 0.99 SOSPI 0.021 0.169 0.99 SCOSPI-2 min 0.539 0.183 0.99 SCOSPI-4 min 1.380 0.216 0.99 SCOSPI-6 min 2.673 0.219 0.99 SCOSPI-8 min 3.343 0.173 0.99 SCOSPI-10 min 3.123 0.154 0.99 SCOSPI-15 min 2.676 0.152 0.99 Note: The K signifies the consistency index. The n signifies the behavior index. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. References 1. Boerma H.R. Managing Inputs for Peak Production American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America Madison, WI, USA 2004 15 20 2. Caponio G.R. Wang H. Ciaula A.D. Angelis M. Portincasa P. 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PMC10000765 | Parathyroid tumors are mostly sporadic but can also occur in familial forms, including different kinds of genetic syndromes with varying phenotypes and penetrance. Recently, somatic mutations of the tumor suppressor gene PRUNE2 were found to be frequent in parathyroid cancer (PC). The germline mutation status of PRUNE2 was investigated in a large cohort of patients with parathyroid tumors from the genetically homogenous Finnish population, 15 of which had PC, 16 atypical parathyroid tumors (APT), and 6 benign parathyroid adenomas (PA). Mutations in previously established hyperparathyroidism-related genes were screened with a targeted gene panel analysis. Nine PRUNE2 germline mutations with a minor allele frequency (MAF) of <0.05 were found in our cohort. Five of these were predicted to be potentially damaging and were identified in two patients with PC, two with APT, and three with PA. The mutational status was not associated with the tumor group nor related to the clinical picture or severity of the disease. Still, the frequent finding of rare germline mutations of PRUNE2 may point to the gene playing a role in the pathogenesis of parathyroid neoplasms. primary hyperparathyroidism parathyroid carcinoma whole-exome sequencing PRUNE2 germline mutation Helsinki University HospitalTYH2019254 Finska LakaresallskapetThis work was financially supported by the Helsinki University Hospital Research Funds (TYH2019254, to C.S.-J.) and Finska Lakaresallskapet (to C.S.-J.). pmc1. Introduction Parathyroid carcinoma (PC) is a rare cause of primary hyperparathyroidism (PHPT). In Finland, around 2-3 new cases are diagnosed every year . In contrast, PHPT due to benign adenomas is very common with an incidence of 1-2/1000 people . The clinical picture of PC is similar to that of PHPT caused by benign parathyroid adenomas (PA). However, PC tumors are often larger than benign adenomas, and the clinical picture is usually more severe. PHPT due to benign adenomas is most common in postmenopausal women (gender ratio 3:1), while for PC, there is no gender difference, and incidence peaks at 40-50 years . The diagnosis of PC is set based on unequivocal invasive characteristics on histopathological examination. Surgery is the only possible curative treatment. Of note, so called en bloc surgery, with the removal of the ipsilateral thyroid lobe and parathyroid gland gives the patient a better prognosis . Atypical parathyroid tumors (APT), also called atypical parathyroid adenomas, are tumors which lack invasive histological characteristics but feature properties such as intratumoral fibrosis and nuclear atypia not regularly found in conventional parathyroid adenomas . Around 5% of parathyroid adenomas (up to 15%, according to some studies) can be classified as APT . The vast majority of PC cases are sporadic (80-90%) and associated with somatic mutations, although PC can also be hereditary . Familial PHPT can occur as isolated PHPT or syndromes, presenting with both benign parathyroid adenomas as well as PC. Germline inactivation of the CDC73 gene, coding for the protein parafibromin, is associated with hyperparathyroidism jaw-tumor syndrome (HPT-JT). The parafibromin protein functions as a tumor suppressor, and HPT-JT presents with early-onset PHPT, ossifying jaw fibromas, as well as tumors of the kidney and uterus. However, CDC73 mutation carriers display incomplete penetrance, and germline CDC73 mutations can also cause isolated familial PHPT without the HPT-JT phenotype . Up to 30% of PC patients carry inactivating germline CDC73 mutations . Due to incomplete penetrance, these PC patients do not always present with a family history of PHPT. Somatic inactivation of CDC73 in tumor DNA is common in sporadic PC, occurring in up to 75% of cases . Parathyroid neoplasms can also be associated with multiple endocrine neoplasia (MEN) syndromes MEN1, MEN2A, and MEN4, caused by mutations in the genes MEN-1, RET, and CDKN1B, respectively . While loss-of-function mutations in calcium-signaling-related genes, such as CASR, GNA11, and AP2S1, are associated with familial hypocalciuric hypercalcemia (FHH) and neonatal severe hyperparathyroidism (NSHPT), these mutations are not associated with parathyroid neoplasms . Non-syndromic hereditary hyperparathyroidism, called familial isolated hyperparathyroidism (FIHP), can be caused by the incomplete manifestation of mutations of syndrome-related genes such as MEN-1, CDC73, GCM2, CDKN1A, CDKN1B, or CDKN2C . However, up to 70% of FIHP cases lack mutations in known susceptibility genes, indicating that there are many factors not yet discovered in the pathogenesis of parathyroid tumors . Additional germline mutations reported in PC patients include genes PRUNE2, CCD1, ADCK1, and genes of the PI3K/AKT/mTOR pathway . Prune homolog 2 with BCH domain (PRUNE2, also known as BMCC1) (9q21.2, ENSG00000106772) belongs to the B-cell CLL/lymphoma 2 and adenovirus E1B 19kDa interacting family. Members of this gene family are involved in several cellular processes, such as apoptosis, cell transformation, and synaptic function . PRUNE2 is a tumor suppressor gene of particular significance shown in prostate cancer, where its expression is regulated by prostate cancer antigen 3 (PCA3), a non-protein coding gene located on the opposite DNA strand in an intron of PRUNE2 . PCA3 has been investigated as a biomarker in prostate cancer . Increased PCA3 levels have also been found in other cancers such as choriocarcinoma as well as ovarian and thyroid cancer, indicating involvement of PRUNE2 in the pathogenesis of these cancers . Increased PRUNE2 protein levels are associated with a favorable prognosis in neuroblastoma and leiomyosarcoma and have also been associated with lower tumorigenic activity in colorectal cancer . Somatic PRUNE2 mutations have been reported in up to 18% of sporadic PCs. These mutations, in combination with the loss of heterozygosity (LOH) events in the tumors, suggest PRUNE2 involvement in the pathogenesis of PC . Moreover, Yu et al. identified a rare germline missense mutation in one PC patient without other known PC-driving mutations. The remaining wild-type allele was silenced in the patient's tumor by LOH, a feature characteristic of tumor suppressor genes. However, the role of PRUNE2 in the genetic predisposition of PC is yet to be confirmed . As somatic PRUNE2 mutations are considered significant in the pathogenesis of PC, we wanted to study whether germline PRUNE2 mutations might be identified in patients diagnosed with aggressive parathyroid tumors in the genetically homogenous Finnish population, an ideal population for the discovery of rare genetic defects of monogenic diseases . 2. Materials and Methods Our study cohort consists of 37 patients with PHPT, 15 of which have been diagnosed with PC, 16 with APT, and 6 with PA. Three patients are known to have an ethnicity other than Finnish. All patients have been diagnosed and treated for PHPT during the years 2004-2021 at the Endocrine Department of Helsinki University Hospital. Clinical history, surgical and pathological reports as well as laboratory results at diagnosis were collected from the Helsinki University Central Hospital databases. Patients with previously known CDC73 mutations were reported earlier and are not included in this study . PC patient 152164 is the mother of APT patient 152177; otherwise, the patients are not related to each other and do not have known family history of PHPT. All patients were summoned for laboratory tests. Serum ionized calcium (S-Ca-ion) as well as fasting plasma parathyroid hormone (fP-PTH), serum vitamin D (S-D-25-OH), and serum creatinine were also analyzed to assess possible recurrence of primary hyperparathyroidism, as some patients had not been participating in regular follow-ups. For some patients, due to problems with sample delivery, DNA was not extracted from whole blood but from saliva samples sent to the patients' home addresses, using the Ora-Collect OCR-100 sample collection kit (Ottawa, Ont., Canada)). All patients gave their written informed consent to study participation. The study protocol was approved by the Ethics Committee of the Helsinki University Hospital (Dnro 1803/2018). EDTA whole-blood samples were sent to Blueprint Genetics (www.blueprintgenetics.com, accessed on 3 January 2023, Helsinki, Finland)) for DNA extraction and whole exome sequencing (WES). WES was performed using BpG according to in-house methods and sequence alignment to human reference genome GRCh37/hg19 with Illumina's bcl2fastq2 Software v2.20 (Illumina, Inc. San Diego, CA, USA). Mutations in previously known PHPT-related genes were analyzed and reported according to a customized extended BpG hyperparathyroidism gene panel (www.blueprintgenetics.com, accessed on 3 January 2023, Helsinki, Finland, including genes AIP, AIRE, AKAP9, AP2S1, APC, BRCA2, CASR, CDC73, CDKN1A, CDKN1B, CDKN2B, CDKN2C, CEP152, CTNNB1, DICER1, EZH2, FLNA, GCM2, GNA11, KDM5C, MEN1, MTOR, NF1, NTRK1, PIK3CA, PTEN, PTH, RB1, RET, SDHA, SETD1B, SMARCA4, STK11, TERT, TNRC6A, TP53, TRPV6, TSC1, TSC2, WT1, and ZEB1. The gnomAD38 v.2.1.1 database (accessed on 3 January 2023)) was used as a resource for population frequencies of the germline mutations. Previously known mutation consequences were assessed through the ClinVar database (accessed on 3 January 2023)) . For assessing the pathogenicity of the variants PolyPhen-239 v.2.2.3 (accessed on 4 January 2023)) and SIFT v5.1.1 (accessed on 4 January 2023)) mutation consequence predictors were used. PhyloP conservation scores were gathered from the UCSC database (PhyloP version 3.19, accessed on 4 January 2023)) . Protein sequence and structural information about the PRUNE2 gene was gathered from the Ensembl database (v.108) . Visualization of PRUNE2 single nucleotide variants and small insertions/deletions were performed with the in-house developed analysis and visualization program BasePlayer . Minimum coverage for variant calling was set at four reads, and the mutated allele was required to be present in at least 20% of the reads. Output processing and analysis was performed using RStudio v.1.1.463 (RStudio, Inc., Boston, MA, USA) and IBM SPSS Statistics v.27 (SPSS, Inc., Chicago, IL, USA). p-values of <0.05 (two-tailed) were considered statistically significant. The kh2-test with Fisher's exact test, as appropriate, was used to investigate differences in categorical variables between groups, while the Kruskal-Wallis test was used for continuous variables. For PRUNE2 mutations, those with a frequency of less than 5% in the global and/or Finnish population are reported. Genome position is reported according to PRUNE2 transcript ENST00000376718. 3. Results 3.1. Overview of the Main Study Results and Patient Characteristics A flowchart depicting the study design and the main findings is shown in Figure 1. The patient characteristics are shown in Table 1. 3.2. Hyperparathyroidism Gene Panel Findings Gene panel findings in the cohort are listed in Table 2. The BpG custom hyperparathyroidism gene panel (www.blueprintgenetics.com, accessed on 3 January 2023, Helsinki, Finland did not reveal any additional germline CDC73 mutations in our cohort. One PC patient (152164) is the mother of one of the APT patients (152177). Otherwise, the patients are not related to each other and do not have any known cases of hyperparathyroidism in their respective families. Three PC patients are known to have an ethnicity other than Finnish. All patients were alive at follow-up. More than one surgery was performed on nine PC patients; five patients had local or distant PC recurrence. Additional en bloc surgery was performed as a preventative measure on four PC patients, as en bloc was not performed as the primary surgery. Three APT patients had more than one surgery; in all these cases, additional surgery was performed due to persistent hypercalcemia after the initial surgery, or the patient presented with additional parathyroid pathology (adenoma or hyperplasia). None of these patients were carriers of any potentially damaging germline mutations found in this study. One PC patient (patient ID 152172) was found to have a likely pathogenic heterozygous germline MEN-1 mutation (c.1280G > T, p.Ser427Ile, ENST00000312049.6), although displayed no other clinical features of the MEN1 syndrome . One APT patient had a family history of MEN1, but no pathogenic MEN-1 variants were detected in this patient. The custom WES panel revealed monosomy X (Turner syndrome) in one APT patient (patient ID 152142) as an incidental or secondary finding (ISF) with clinical significance . Moreover, a heterozygous germline APC missense mutation (c.2222A > G, p.Asn741Ser, rs150209825) was found in PA patient 152129. The variant is considered of unknown significance (VUS) . This patient does not have any history of other neoplasia, and the possible family history of cancer is unknown. A 5'UTR variant of the APC gene (c.-128G > A, rs543098847) was found in PC patient 152174. This multiallelic single nucleotide variant is considered likely benign VUS (accessed on 3 January 2023)). A RET mutation (c.604G > A, p.Val202Met, rs751572082) of unknown significance was identified in PC patient 152165. An amino acid deletion of BRCA2 with unknown significance (c.3900_3902del, p.Met1300_Thr1301delinsIle, rs397507697) was found in APT patient 152175, and APT patient 152146 had a VUS missense AIP mutation (c.940C > T, p.Arg314Trp, rs375740557). In PA patient 152125, a multiallelic 5'UTR SDHA variant (c.-11C > T, rs1396057630) was identified. 3.3. PRUNE2 Mutations Altogether, 25 non-synonymous PRUNE2 germline variants were found in the patient cohort. Out of those, nine variants had a minor allele frequency (MAF) <0.05 in a control population (accessed on 21 December 2022)) . The details on these nine PRUNE2 variants are listed in Table 3. The nine variants were distributed among twelve patients, of which three had PC, six APT and three PA. None of the patients with rare PRUNE2 variants had mutations of previously established PHPT-related genes (Table 2). All discovered mutations were heterozygous missense changes. Altogether, seven patients harbored a PRUNE2 variant in silico predicted to be likely deleterious . Interestingly, the germline variant p.Ser595Tyr found in APT patient 152142 was previously also identified in prostate cancer patients . Otherwise, the rare PRUNE2 germline variants found in our cohort have not been associated with any kind of pathology (accessed on 3 January 2023)). APT patient 152142 with Turner syndrome, who was 59 years old at the time of her diagnosis, had three different germline PRUNE2 variants; however, only (c.1784G > T, p.Ser595Tyr) was predicted to be damaging (Table 3). More than one PRUNE2 mutation was also found in patients 152127 (PA, 38 years old at diagnosis) and 152171 (PC, 66 years at diagnosis). Similarly, in these cases, only one mutation per patient was predicted to be pathogenic. In addition, patients 152155 (PC, 49 years at diagnosis), 152121 (PA, 44 years at diagnosis), 152123 (PA, 54 years at diagnosis), and 152131 (APT, 63 years at diagnosis) each harbored one PRUNE2 germline mutation that was predicted to be damaging (Table 3). The location of the PRUNE2 mutations found in our cohort in relation to the PRUNE2 gene and previously found PRUNE2 mutations in PC (somatic and germline) are visualized in Figure 2. Most mutations were located in exon 8, the longest exon of the gene, but mutations were also observed in exons 3, 9, and 12. The cohort was screened for gene variants of ADCK1, CCD1, FAT3, and THRAP3 that have previously been associated with PC as described in the literature but that were not included in the BpG hyperparathyroidism gene panel assessment . No previously described variants of these genes were found in our cohort. PRUNE2 mutation status did not correlate with clinical parameters such as severity of disease (Ca-ion or PTH levels at diagnosis), tumor size, or parafibromin staining on immunohistochemistry, neither by looking at all rare prune mutations in our cohort nor by separately analyzing the variants considered damaging. Neither were the PRUNE2 mutations associated with a lower age at diagnosis. 4. Discussion The majority of PC cases are associated with somatic alterations, although hereditary predisposition also plays a role in the genesis of this rare malignancy . Somatic recurrent PRUNE2 mutations have been reported in up to 18% of PC cases. A reported germline PRUNE2 mutation and inactivation of the wild-type allele by LOH in the patient's tumor indicates that PRUNE2 alterations might predispose to PC . To clarify the contribution of inherited PRUNE2 mutations in the development of parathyroid tumors, the germline mutation status of PRUNE2 was analyzed in a cohort of 37 mostly Finnish PHPT patients. We identified nine rare PRUNE2 germline variants (MAF < 0.05) in twelve patients, of which five mutations were predicted to be deleterious by disrupting the function of the PRUNE2 protein. These five mutations were distributed among seven patients, including individuals from all tumor groups (PC, APT, and PA). Interestingly, one of the mutations (c.270C > T, p.Asp90ASn) was shared between three unrelated patients: two with PC and one with PA. The MAF of the variant was 0.0148 among the Finnish controls and 0.0092 in the global control population (Table 3). This recurrent mutation might be a Finnish founder mutation, and enrichment of the mutant allele in our patient cohort may imply a causal relationship between the c.270C > T (p.Asp90ASn) mutation and the disease phenotype. The homogenous Finnish population has a unique genetic background, and founder mutations exist at high frequencies . As such, Finnish population-based cohorts are valuable for the discovery of the causative genetic mutations of monogenic diseases. Most of the rare PRUNE2 variants found in our cohort are localized to PRUNE2 exon 8, which is, by far, the largest exon of the gene, harboring approximately half of all the amino acids in its sequence. The mutations found in our study do not seem to be targeting any specific regions or previously established domains of PRUNE2 . According to the literature, germline mutations of the PRUNE2 gene have been very scarcely investigated in other forms of cancers, despite somatic mutations playing a role in the pathogenesis of prostate cancer, leiomyosarcoma, and colorectal cancer among others . Of note, a recent study found PRUNE2 germline mutations in 2.8% of patients with familial prostate cancer, proposing PRUNE2 as a new prostate cancer predisposition gene . The PRUNE2 germline variant c. 1784G > T (p.Ser595Tyr) found in our APT patient was also identified in the familial prostate cancer study. This mutant allele was classified as a variant of unclear association with the prostate cancer risk . The patients with rare PRUNE2 germline mutations did not carry any germline mutations in previously established PHPT-driving genes. One patient was found to have monosomy X/Turner syndrome. This is relevant in the setting of parathyroid tumors due to the X chromosome harboring loci for the FLNA and KDM5C genes. The FLNA protein participates in the regulation of the calcium-sensing receptor and has been associated with increased aggressiveness in a wide range of cancers . FLNA expression has also been investigated in parathyroid tumors, with unclear conclusions . Inactivation of the KDM5C gene encoding for the histone demethylase protein JARID1C is frequent in renal cell carcinoma, and somatic KDM5C mutations have also been found in PC . The monosomy X patient is thus susceptible to somatic mutations in these genes. One patient (152172) was discovered to have a likely pathogenic mutation of the MEN-1 gene. Other mutations of the same codon (c.1281T > A, p.Ser427Arg, rs1114167528) were previously reported in several MEN1 patients . The family history of the MEN-1 mutation-positive patient identified in this study is not known. The patient has no other medical history of cancer except for PC, and the age at diagnosis (67 years) was not conspicuous. Still, due to the likely pathogenicity of this gene mutation, further clinical follow-up might be needed. Similarly, despite the germline APC mutations discovered in our cohort not being previously reported to be associated with pathogenicity, these patients and their relatives might also require further investigations, as APC mutations are so pronouncedly associated with hereditary colorectal malignancy . Patients with previously known CDC73 mutations were excluded from this study, but as germline CDC73 mutations are also quite common in patients with sporadic PC it is perhaps rather surprising that none of the patients in the present cohort were found to carry CDC73 mutations. As CDC73 mutations may underlie both benign and malignant parathyroid tumors with varying penetrance and phenotypes, one can, therefore, speculate that mutations of the PRUNE2 gene could also give rise to similarly varying parathyroid tumor phenotypes with similarly varying penetrance . The incomplete penetrance manifested by PRUNE2 germline defects might be the reason for the observed lack of family history of the disease. The shortcoming of the study was the lack of tumor material; hence, the investigation of the biallelic inactivation of PRUNE2 in tumors was not achievable. Our patient cohort is of a reasonable size considering the rarity of PC. Another strength of our material is the detailed clinical, surgical, and histopathological characterization of the patients. However, the patient number is too small for the relevant assessment of the possible relationships between gene variants and clinical or biochemical parameters. All our patients were alive at the time of the study, with a median follow-up time of 7 years. Globally, the 5-year survival of PC is considered around 85%, indicating that our patients have had a rather favorable course of disease . En bloc surgery was performed in 13 of our 15 PC patients (86%), either as primary or secondary surgery. The excellent prognosis of the patients likely reflects the high awareness of PC as a cause of PHPT in our tertiary centre and the close collaboration with our endocrine surgeons, preventing diagnostic delay, as well as en bloc surgery, ensuring margin-free resection. Recently, margin-free resection was reported to predict excellent long-term outcomes in PC . In this study, we report that Finnish PHPT patients with CDC73 mutation-negative parathyroid tumors frequently display rare germline mutations in the PRUNE2 tumor suppressor gene. However, further work is needed to examine whether PRUNE2 plays a causative role in the genetic predisposition of parathyroid neoplasia. Clarification of this question would require additional sample sets and more extensive molecular workup. The identification of new parathyroid tumor-predisposing genes is important to improve the risk assessment of patients, and it would enable targeted testing of family members at risk. 5. Conclusions Rare germline PRUNE2 variants are frequent in Finnish patients with parathyroid neoplasms, regardless of tumor type (PC, APT, or PA). Further studies are needed to clarify the role of PRUNE2 in patients with parathyroid tumors. Acknowledgments We wish to thank Blueprint Genetics (BpG) for DNA extractions, WeS sequencing, and gene panel analysis. We also want to thank the study nurse Hanna Talosela for her excellent assistance in patient-related matters, including paperwork and practicalities. Author Contributions Conceptualization, S.S., A.K. and C.S.-J.; Methodology, S.S., E.R., A.K. and C.S.-J.; Formal analysis, S.S., A.K. and C.S-J.; Investigation, S.S., E.R. and C.S.-J.; Resources, C.S.-J.; Writing--original draft, S.S.; Writing--review & editing, S.S., E.R., A.K. and C.S.-J.; Supervision, A.K. and C.S.-J.; Project administration, C.S.-J.; Funding acquisition, C.S.-J. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of the Helsinki University Hospital (Dnro 1803/2018). Informed Consent Statement Written informed consent was obtained from all study subjects. Data Availability Statement The data is available on request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Flowchart of PRUNE2 germline mutation screening in patients with parathyroid tumors. The whole exome sequencing (WES) was performed from normal DNA. After WES analysis, the variant calls were filtered to have MAF < 0.05. Mutation consequences were assessed using pathogenicity predictor software and the ClinVar database. Five potentially deleterious PRUNE2 mutations were identified, distributed among seven PHPT patients. PC = parathyroid cancer; APT = atypical parathyroid tumor; PA = parathyroid adenoma; MAF = minor allele frequency. Figure 2 Schematic depiction of the PRUNE2 gene and its exons. PRUNE2 variants found in our cohort are annotated above the gene, and previously identified PRUNE2 mutations (somatic and germline) in PC patients are annotated below the gene. The respective sizes of the exons are not to scale. Somatic mutations are marked with an asterisk (*). Mutations predicted to be damaging are marked in bold. cancers-15-01405-t001_Table 1 Table 1 Characteristics of the patient cohort. PC APT PA Total p-Value n. 15 16 6 37 Sex (m:f) 8:7 5:11 0:6 13:25 0.054 Age at diagnosis (years, median, range) 61 (17-76) 60 (33-80) 58 (38-73) 60 (17-80) S-Ca-ion at diagnosis (mmol/L, median, range) 2.05 (1.62-2.58) 1.73 (1.32-2.39) 1.50 (1.41-1.64) 1.8 (1.32-2.58) <0.001 * S-PTH at diagnosis ng/L (median, range) 1743 (358-4000) 330 (73-3500) 138 (63-222) 502 (63-4000) <0.001 * More than one surgery for PHPT (n. of patients) 9 3 0 8 0.271 Hypercalcemic crisis or hypercalcemia requiring in-hospital treatment 10 6 0 16 0.015 * Palpable neck mass 2 2 0 4 0.689 Renal failure (transient or permanent serum creatinine elevation) 5 4 0 9 0.320 Skeletal manifestation (osteoporosis or osteitis fibrosa cystica) 4 6 1 11 0.713 Number of patients with rare PRUNE2 mutations 3 7 3 13 0.343 p-values marked with an asterisk (*) are considered statistically significant. PC; parathyroid carcinoma; PA: Parathyroid adenoma; APT: atypical parathyroid tumor; PHPT: primary hyperparathyroidism. cancers-15-01405-t002_Table 2 Table 2 Results of the customized Blueprint Genetics (BpG) hyperparathyroidism gene panel analysis. Patient ID Diagnosis Age at Diagnosis Ethnicity (If Other Than Finnish) Mutated Gene Mutation Predicted Consequence 152155 PC 49 152161 PC 61 Russian 152163 PC 45 152164 PC 53 152165 PC 35 RET c.604G > A p.Val202Met VUS 152168 PC 71 152170 PC 71 152171 PC 66 152172 PC 67 Estonian MEN1 c.1280G > T, p.Ser427Ile Likely pathogenic 152174 PC 65 APC 5'UTR variant c.-128G > A VUS 156889 PC 76 167873 PC 40 167874 PC 17 171375 PC 46 Russian 177360 PC 72 152131 APT 63 152133 APT 80 152134 APT 52 152135 APT 56 152136 APT 68 152137 APT 60 152140 APT 70 152142 APT 59 Monosomy X (Turner syndrome) 152143 APT 50 152146 APT 52 AIP c.940C > T, p.Arg314Trp VUS 152149 APT 67 152169 APT 56 152175 APT 70 BRCA2 Inframe deletion c.3900_3902del p.Met1300_Thr1301delinsIle VUS 152176 APT 64 152177 APT 33 177359 APT 49 152121 PA 44 152123 PA 54 152125 PA 73 SDHA 5'UTR variant c.-11C > T VUS 152127 PA 38 152128 PA 68 152129 PA 61 APC c.2222A > G p.Asn741Ser VUS cancers-15-01405-t003_Table 3 Table 3 Identified rare PRUNE2 germline mutations (MAF < 0.05) according to underlying tumor in 37 patients with primary hyperparathyroidism. Patient ID Dg (Age at Onset/Sex) PRUNE2 Mutation (Exon) * MAF (Finnish) * MAF (Global) Predicted Effect SIFT /Polyphen Phylop Conservation Score rs Number 152155 PC (49/F) c.270C > T, Asp90Asn (3) 0.01477 0.009245 Deleterious/probably damaging 5.56559 rs41304230 152164 PC (53/F) c.7719C > T Ala2573Thr (9) 0.01656 0.02567 Tolerated/benign 0.280654 rs56261747 152171 PC (66/M) c.270C > T, Asp90Asn (3) 0.01477 0.009245 Deleterious/probably damaging 5.56559 rs41304230 c.3842G > A, Ser1281Phe (8) 0.01263 0.01024 Tolerated/possibly damaging 2.67864 rs41310047 152131 APT (63/M) c.2547TC > AA +, p.Glu2516Leu (9) 0.001639, 0.001641 0.001639, 0.001641 Deleterious/probably damaging 0.104701, 0.36863 rs187947807, rs190606277 152134 APT (52/F) c.2784T > C, Lys928Glu (8) 0.03 0.03 Tolerated/benign 0.747535 rs41289953 152140 APT (70/F) c.2784T > C, Lys928Glu (8) 0.03 0.03 Tolerated/benign 0.747535 rs41289953 152142 APT (59/F) c.1784G > T, Ser595Tyr (8) 0.01447 0.001681 Deleterious/probably damaging 2.36166 rs201792781 c.7707T > C, Arg2569Gly (9) 0.01659 0.02568 Tolerated/benign 1.07244 rs41288767 c.7719C > T, Ala2573Thr (9) 0.01656 0.02567 Tolerated/benign 0.280654 rs56261747 152149 APT (67/F) c.2784T > C, Lys928Glu (8) 0.03 0.03 Tolerated/benign 0.747535 rs41289953 152176 APT (64/F) c.2784T > C, Lys928Glu (8) 0.03 0.03 Tolerated/benign 0.747535 rs41289953 152121 PA (44/F) c.3339C > T, Asp1113Asn (8) 0.004178 0.0032776 Deleterious/possibly damaging 4.12181 rs200875180 152123 PA (54/F) c.270C > T, Asp90Asn (3) 0.01477 0.009245 Deleterious/probably damaging 5.56559 rs41304230 152127 PA (38/F) c.8711C > A, Arg2904Ile (12) 0.0162 0.003269 Deleterious/probably damaging 1.7718 rs80290481 c.7719C > T, Ala2573Thr (9) 0.01656 0.02567 Tolerated/benign 0.280654 rs56261747 c.7707T > C, Arg2569Gly (9) 0.01659 0.02568 Tolerated/benign 1.07244 rs41288767 * (accessed on 4 January 2023); + combined missense GAa > TTa variant. PhyloP: (version 3.19, accessed on 4 January 2023) (GRCh37/Hg19). MAF: mutant allele frequency; PC; parathyroid carcinoma; PA: parathyroid adenoma; APT: atypical parathyroid tumor. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. |
PMC10000766 | Diagnostics (Basel) Diagnostics (Basel) diagnostics Diagnostics 2075-4418 MDPI 10.3390/diagnostics13050984 diagnostics-13-00984 Article CD24 Gene Expression as a Risk Factor for Non-Alcoholic Fatty Liver Disease Amin Mona A. 1* Ragab Halla M. 2 Abd El Maksoud Nabila Investigation 2 Elaziz Wafaa Abd 2 Fierbinteanu-Braticevici Carmen Academic Editor 1 Department of Internal Medicine-Hepato-Gastroenterology, Kasr Al-Ainy, Cairo University, Cairo 42403, Egypt 2 Department of Biochemistry, Biotechnology Research Institute, National Research Centre, Dokki, Giza 12622, Egypt * Correspondence: [email protected] or [email protected] 04 3 2023 3 2023 13 5 98417 1 2023 25 2 2023 01 3 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). In light of increasing NAFLD prevalence, early detection and diagnosis are needed for decision-making in clinical practice and could be helpful in the management of patients with NAFLD. The goal of this study was to evaluate the diagnostic accuracy of CD24 gene expression as a non-invasive tool to detect hepatic steatosis for diagnosis of NAFLD at early stage. These findings will aid in the creation of a viable diagnostic approach. Methods: This study enrolled eighty individuals divided into two groups; a study group included forty cases with bright liver and a group of healthy subjects with normal liver. Steatosis was quantified by CAP. Fibrosis assessment was performed by FIB-4, NFS, Fast-score, and Fibroscan. Liver enzymes, lipid profile, and CBC were evaluated. Utilizing RNA extracted from whole blood, the CD24 gene expression was detected using real-time PCR technique. Results: It was detected that expression of CD24 was significantly higher in patients with NAFLD than healthy controls. The median fold change was 6.56 higher in NAFLD cases compared to control subjects. Additionally, CD24 expression was higher in cases with fibrosis stage F1 compared to those with fibrosis stage F0, as the mean expression level of CD24 was 7.19 in F0 cases as compared to 8.65 in F1 patients but without significant difference (p = 0.588). ROC curve analysis showed that CD24 CT had significant diagnostic accuracy in the diagnosis of NAFLD (p = 0.034). The optimum cutoff for CD24 was 1.83 for distinguishing patients with NAFLD from healthy control with sensitivity 55% and specificity 74.4%; and an area under the ROC curve (AUROC) of 0.638 (95% CI: 0.514-0.763) was determined. Conclusion: In the present study, CD24 gene expression was up-regulated in fatty liver. Further studies are required to confer its diagnostic and prognostic value in the detection of NAFLD, clarify its role in the progression of hepatocyte steatosis, and to elucidate the mechanism of this biomarker in the progression of disease. NAFLD gene expression CD24 Egypt's National Research Centre for financiallyWe express our gratitude to Egypt's National Research Centre for financially funding the current study. pmc1. Introduction NAFLD is a clinico-pathologic syndrome that encompasses various medical entities, including simple fatty liver or simple steatosis, nonalcoholic steatohepatitis (NASH), cirrhosis, and its complications . NAFLD now affects up to 25% of people around the world. The highest prevalence rate is in the Middle East (32%), followed by South America (30%), while the lowest is in Africa (13%). It also accounts for 2% of total deaths . The increase in NAFLD prevalence parallels the rise in obesity and is tightly associated with metabolic comorbidities (diabetes, hypertension, insulin resistance, and dyslipidemia). It also places patients at higher risk for progressive liver disease . It became clear that, as with different complex multisystem disorders, NAFLD is triggered by a variety of underlying mechanisms; the most important one of them is the alterations in hepatic and extra-hepatic lipid metabolism . The study of genetic factors in NAFLD is a rapidly growing field, as they determine not only the response of different individuals to excess caloric consumption, but also the resulting metabolic derangements . Cluster of differentiation 24 (CD24) is a glycophosphatidylinositol (GPI)-anchored mucin-like cell surface glycoprotein, encoded by a gene located on chromosome 6. It is expressed on mature granulocytes and B cells and regulates growth and differentiation signals to these cells. Accumulating evidence showed that abnormal over-expression of this protein is a prognostic factor in many types of cancers, resulting in cancer cell growth, proliferation, and metastasis . The expression of the cell surface molecule CD24 has previously been shown to identify a subset of adipocyte progenitor cells that is crucial for the reconstitution of white adipose tissue (WAT) function in vivo, as well as a particular regulator of adipogenesis in vitro . Recently, CD24 has been identified as a possible biomarker for distinguishing NAFLD/NASH. It was concluded that the mRNA expression of CD24 is upregulated in the fatty liver . Additionally, Feng et al., (2021) detected that CD24 was positively associated with NAFLD severity, and it could also differentiate mild NAFLD patients from severe NAFLD patients . Therefore, the present study aimed to identify the association between gene expression of CD24 and early stage of NAFLD. 2. Subjects and Methods The present study is a prospective study that was carried out on 80 subjects who attended outpatient clinics of the Internal Medicine Department of Kasr Al Ainy Hospital Cairo, Egypt during the period from May 2019 to December 2020 either for general health checks or to identify and treat the complications of other metabolic disorders such as diabetes or obesity. The selected subjects were divided into two groups according to the sonographic findings of steatosis: 40 NAFLD patients with bright liver echogenicity and 40 healthy subjects with normal liver echogenicity. All cases have age ranging between 19 to 56 years old. Those with clinical, biochemical, or histological evidence of cirrhosis, those with known causes of liver disease [viral hepatitis B and C, autoimmune hepatitis, primary biliary cirrhosis, haemochromatosis or Wilson disease], those with history of current or past excessive alcohol drinking as defined by an average daily consumption of more than 20 g alcohol, drug-induced liver disease, pregnant women and patients on hormonal contraceptive drugs (oral, parenteral), hormone replacement therapy were excluded from the study. The study was approved by Medical Research Ethical Committee of the National Research Center, Cairo, Egypt (Approval No.19-001), and informed consent was obtained from all patients. All patients were evaluated by history and clinical examination and measurement of anthropometric parameters, such as weight (kg), height (m), body mass index (BMI; kg/m2), waist circumference (cm), and mid-arm circumference (cm). Body mass index (BMI) was determined by dividing weight by square height (kg/m2). BMI is calculated as weight in kilograms divided by the height in metres squared. According to WHO, People with BMI = 18.5-24.9 have normal weight, people with BMI = 25.0-29.9 were classified overweight, while people with BMI >= 30 kg/m2 defines obese. BMI is calculated as weight in kilograms divided by the height in metres squared. According to WHO, in adults, overweight is defined as a BMI of 25-29.9, while a BMI >= 30 kg/m2 defines obesity. Waist circumference (WC) was obtained from each subject by measuring at the midpoint between the lower rib margin and the iliac crest using a conventional tape graduated in centimeters (cm). Mid-arm circumference was measured as the right upper arm measured at the midpoint between the tip of the shoulder and the tip of the elbow (olecranon process and the acromium). Cases were divided according to their previous diagnosis or levels of fasting blood sugar: a fasting blood sugar level less than 115 mg/dL is considered normal or prediabetes. While, if the fasting blood sugar level is 126 mg/dL or higher, the patient was diagnosed diabetic. Complete blood count was determined using the automated hematology analyzer SF-300 (Sysmex Corporation, Japan). Additionally, liver enzymes (ALT, AST, ALP, GGT), serum albumin, prothrombin time, INR, serum creatinine, lipid profile, and fasting blood sugar were measured to all individuals according to the manufacture instructions. The reagents were purchased from Spectrum Company, Cairo, Egypt. NAFLD fibrosis score (NFS), FIB-4, and Fast score were calculated as mentioned previously by Angulo et al. (2007) and Cales et al. (2009) to assess fibrosis of the NAFLD patients' group. NFS score = -1.675 + 0.037 x age [y] + 0.094x BMI [kg/m2] + 1.13 x IFG/diabetes [yes = 1, no = 0] + 0.99 x AST/ALT ratio - 0.013 x platelet count [x109/L] - 0.66 x albumin [g/dL] FIB-4 score = Age [y] x AST [U/L]/platelet [x109/L] x ALT [U/L] FAST score was calculated according to Newsome et al., (2020) as: FAST = {exp (-1.65 + 1.07 x ln (LSM) + 2.66 x 10-8 x CAP3 - 63.3 x AST-1)}/{1 + exp (-1.65 + 1.07 x ln (LSM) + 2.66 x 10-8 x CAP3 - 63.3 x AST-1)}(1) Abdominal ultrasonography was performed to all individuals using the 3.5 MHz probe of Logic 6 of a General Electric machine. 2.1. Liver Stiffness Measurement (LSM) and Controlled Attenuation Parameter (CAP) Fibroscan (M probe, Echosens, Paris) was carried out by an experienced examiner in all patients (with at least 6 h of fasting) in left lateral position and the median liver stiffness of the 10 successful measurements fulfilling the criteria (success rate of greater than 60% and interquartile range/median ratio of <30%) were noted (in kPa). The final CAP value, which ranges from 100 to 400 (dB/m), is the median of individual measurements. As an indicator of variability, the ratio of the IQR of CAP values to the median (IQR/MCAP) was calculated. The operator was blinded to the patients' clinical data. According to the manufacturer's instructions, in addition to previous studies, the stages of fibrosis (F0: 1-6, F1: 6.1-7, F2: 7-9, F3: 9.1-10.3, and F4: >=10.4) were defined in kPa . Moreover, steatosis stages (S0: <215, S1: , S2: 253-296, S3: >296) were defined in dB/m . 2.2. Sample Collection 10 mL venous blood were drawn from all study participants in the morning after a 12 h fast; a portion of the blood was collected on EDTA tube for the extraction of RNA and for the determination of routine blood pictures (CBC) by Sysmex, the automated hematology analyzer SF-300, which was produced by Sysmex Corporation, Japan. The other portion was left to clot at room temperature. Serum was separated by centrifuging for 10 min at 3000 rpm. Sera were used immediately for other biochemical investigations including aspartate aminotransferase (AST), alanine aminotransferase (ALT), bilirubin, serum albumin, fasting blood glucose, cholesterol, triglycerides, HDL-C, and LDL-C according to the manufacturer's instructions. The reagents were purchased from Spectrum Company, Cairo, Egypt. 2.3. CD24 Gene Expression by Quantitative Real Time-PCR (qRT-PCR): Total RNA was isolated from whole blood using GeneJET Whole Blood RNA Purification Mini Kit (Thermo Scientific, Lithuania) following the manufacturer's suggestions. 2.4. Reverse Transcription for cDNA Synthesis and Quantitative Real-Time PCR (RTqPCR) Reverse transcription (RT) was performed to obtain cDNA from 400 ng of purified RNA using the High-Capacity cDNA Reverse Transcription Kits (Applied Biosystem, Lithuania) with random hexamers according to the manufacturer's suggestions. A value of 10 mL of the 2X-RT master mix was pipetted into each tube and then 10 mL of RNA sample was added to it and mixed well. The tubes were centrifuged to spin down the content and to eliminate any air bubbles. After that, the tubes were placed on the PCR machine (Cleaver Scientific, UK) programmed as follows: 25 degC, 10 min, 37 degC, 120 min, and 85 degC, 5 min. After detection of cDNA concentration and purity, they were stored in -20 degC until carryover quantitative real-time PCR (QRT-PCR). CD-24 gene expression for enrolled samples was quantified using PowerUp SYBR Green master mix (2x) (ThermoFisher Scientific, Lithuania). The sequences for used primers were as follows: Primer Primer Sequence CD24 Forward primer 5'-ACC CAC GCA GAT TTA TTC CA-3' CD24 Reverse primer 5'-ACC ACG AAG AGA CTG GCT GT-3' b-actin Forward primer 5'-TGA GCG CGG CTA CAG CTT-3' b-actin Reverse primer 5'-TCC TTA ATG TCA CGC ACG ATT T-3' PCR amplification was carried out in 20 mL reaction volume containing 1 mL cDNA, 10 mL PowerUp SYBR Green master mix, 7 mL nuclease-free water, and 1 mL of gene-specific forward and reverse primers as mentioned in table. The reaction was run in the Rotor-Gene Q instrument, (QIAGEN). Fluorescence measurements were made in every cycle, and the thermal profile was used as the follows: The amplification program included a UDG activation at 50 degC with a 2-min hold, and a dual-lock DNA polymerase at 94 degC with a 3-min hold, followed by 45 cycles with denaturation at 94 degC for 30-s, annealing at 55 degC for 30-s, and extension at 72 degC for 30-s. The expression levels of CD-24 in tested samples were expressed in the form of CT (cycle threshold) value, which was calculated based on threshold cycle (Ct) values, corrected by b-actin expression, with the following equation. The relative amount of CD-24 = 2-DDCt; DDCt = [DCt of cases - DCt of control]; [DCt = Ct (CD-24) - Ct (b-actin)]. The following primers were used in the quantitative real-time PCR analyses. 2.5. Statistical Analysis SPSS version 16.0 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis with a two-side significant criterion at p < 0.05. The clinical data were expressed as mean +- SD (continuous, normally distributed variables). Categorical data were summarized as percentages. The significance for the difference between groups was determined by using a two-tailed Student's t-test. Additionally, qualitative variables were assessed by chi-squared kh2-test. Correlations between different parameters were performed using Pearson's and spearman's correlation coefficients. A receiver operating characteristic (ROC) curve was plotted to assess the diagnostic power of CD24 in NAFLD and controls, and the area under the curve (AUC) greater than 0.5 considered to be statistically significant. The probability (p) values of <=0.05 were considered statistically significant and indicated, while p > 0.05 was considered statistically not significant and indicated NS. 3. Results The present study is a case-control study recruited 80 adult subjects, (28 males and 52 females). Their age ranged from 19 to 56 years. The demographic, anthropometric, clinical, and biochemical characteristics of both groups (NAFLD and controls) are summarized in Table 1. Patients with NAFLD were significantly older than controls (mean age 42.18 +- 11.1 4 y vs. 29.65 +- 6.63 y, p < 0.0001). There were more males in the control group (45%), but the majority was females in the NAFLD group (75%). NAFLD patients exhibited a higher mean BMI (31.8 +- 2.9 kg/m2) than the control group (23.76 +- 1.4 kg/m2) (p < 0.001). Patients with NAFLD had a higher prevalence of hypertension and diabetes mellitus in comparison to healthy controls (p < 0.001) (Table 1). Among studied NAFLD patients, 22.5% had a family history of diabetes, and 30% had family history of liver disease, and 62.5% of NAFLD cases (n = 25) have enlarged liver size on ultrasound. The mean serum fasting blood glucose was significantly higher in NAFLD patients than that in controls (122.6 +- 40.97 vs. 96.03 +- 7.77); (p < 0.001). In addition, hemoglobin levels were lower in NAFLD cases (11.56 +- 1.4 (g/dL) than in healthy controls (12.81 +- 1.06 (g/dL), (p < 0.001). No significant difference was observed in total leucocytic count (TLC) and platelet count between the NAFLD and control groups (p > 0.05). NAFLD patients had significantly higher serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and gamma-glutamyl transferase (GGT) compared to healthy controls (p < 0.001). On the other hand, the mean albumin level was almost normal (3.8 +- 0.38 g/dL) in the NAFLD group. There was a significant elevation in total cholesterol, triglycerides, and LDL-cholesterol among NAFLD patients compared to controls, while there was significant decrement in HDL in the NAFLD group as opposed to controls (p < 0.05). Table 2 shows clinical and biochemical characteristics of participants stratified by sex and presence/absence of NAFLD. In both sexes, participants with NAFLD were older, had a higher BMI, as well as a higher prevalence of diabetes. Levels of hemoglobin was significantly lower in female cases compared to male cases in NAFLD group (p = 0.001). However, ALT and AST levels were significantly higher in male NAFLD cases compared to female NAFLD casess (p = 0.009 and p = 0.038; respectively) (Table 2). The mean Fibroscan value in all NAFLD patients was 5.1 +- 0.99 (kPa), indicating that all patients had mild fibrosis with a stage less than 2. Thirty patients had fibrosis belonging to stage 0, while the rest had fibrosis stage 1. Mean Fibroscan values for cases with fibrosis stages 0 and 1 were 4.7 +- 0.67 and 6.5 +- 0.3 (kPa), respectively. There was a statistically significant difference in liver stiffness measurements in patients with stage 0 fibrosis as compared to stage 1 fibrosis (p < 0.001). In addition, there was a stepwise increase in Cap score parallel to the increase in severity of liver fibrosis (p < 0.001) (Table 3). This study showed that both NFS and FIB-4 score were similar in patients with fibrosis stages 0 and those with fibrosis stages 1 (p > 0.05). This may be due to that all cases included in our study have mild fibrosis. Additionally, performances of FIB-4 and NFS to rule in advanced fibrosis are rather inadequate, meaning that further assessment with another test is needed in case of positive results. According to the RT-PCR results, it was detected that expression of CD24 was significantly higher in patients with NAFLD than healthy controls. The median fold change in the expression of CD24 was 6.56 higher in NAFLD cases compared to control subjects . The present study showed higher expression of CD24 in female cases with NAFLD compared to male cases (fold change was 6.9 in females vs 4.4 in males, but without significant difference; p = 0.262) . Additionally, CD24 expression was higher in cases with fibrosis stage F1 compared to those with fibrosis stage F0, as the mean expression level of CD24 was 7.19 in F0 cases as compared to 8.65 in F1 patients, but without significant difference (p = 0.588). Furthermore, there was no difference in CD24 fold change between overweight patients (median fold change = 9) and obese cases (median fold change = 5.89) (p = 0.447) . Additionally, the median fold change in CD24 in diabetic cases was seven compared to 5.13 in non-diabetic cases (p = 0.609) . 3.1. Evaluation of the Diagnostic Accuracy of CD24 Gene Expression for Distinguishing Patients with NAFLD from Healthy Controls Figure 5 illustrates the ROC plots to assess the diagnostic accuracy of CD24 CT to distinguish patients with NAFLD from healthy controls. ROC curve analysis showed that CD24 CT had significant diagnostic accuracy in the diagnosis of NAFLD (p = 0.034). ROC curve showed the optimum cutoff for CD24 was 1.83 for distinguishing patients with NAFLD from healthy control with sensitivity 55% and specificity 74.4%; and an area under the ROC curve (AUROC) 0.638 (95% CI: 0.514-0.763). 3.2. Correlation between Different Non-Invasive Fibrosis Markers and CD24 Gene Expression Table 4 shows the correlation of Kpa, CAP, FAST, NFS, and FIB-4 with CD 24 gene expression. Pearson's correlation test showed positive significant correlation between CD24 and NFS (r = 0.356, p = 0.001). By binary logistic regression analysis, none of the examined parameters found to be significant determinant of NAFLD after adjusting the effects of potential cofounders of age, gender, suffering of diabetes, and BMI, respectively (Table 5). 4. Discussion NAFLD is known nowadays as the most common liver disorder in the 21st century. It is diagnosed by the presence of more than 5% fat accumulation in liver cells without excess alcohol consumption or secondary causes of fat accumulation in the background. Approximately 25% of the world's adult's population has NAFLD, and the prevalence is still increasing . NAFLD may eventually deteriorate to HCC as a result of excessive lipid accumulation, liver cell damage, immune system dysfunction, which leads to scarring, and permanent liver damage . In light of increasing NAFLD prevalence, early detection and diagnosis are needed for decision-making in clinical practice and could be helpful in the management of patients with NAFLD. The present study showed a significant trend of elder age with the progression of non-alcoholic fatty liver disease. This finding substantiates previous findings in the literature, which suggested that the prevalence of NAFLD increases with increasing age . The present study showed that, regarding gender distribution, there were more males in the control group (45%) compared to the NAFLD group (25%), but the majority was females in the NAFLD group (75%). These results revealed that there was no statistically significant difference between both studied groups according to gender as p = 0.061. The explanation for the gender difference is different distributions of fat mass by gender, e.g., more abdominal visceral adipose tissue in male and more subcutaneous adipose tissue mass in female. Additionally, previous results showed that Hispanic women having a higher risk for NAFLD compared to men, whereas, for the non-Hispanic population, the prevalence of NAFLD is more frequent in males . Additionally, Lonardo et al. mentioned that gender is one of the main cause of variation in NAFLD risk factors. They also detected that NAFLD is more common and more severe in men than women. However, it is more common in women after menopause, indicating that estrogen may be beneficial . In the current study, the incidence of NAFLD has been increasing in concert with the presence of multiple metabolic disorders, such as dyslipidemia, diabetes, hypertension, and visceral obesity. As expected, the incidence of diabetes and hypertension was significantly higher in patients suffering from NAFLD. This is in good agreement with previous studies that mentioned impaired glucose tolerance as an independent risk factor for the progression of NAFLD . According to the International Diabetes Federation (IDF), the prevalence of DM among Egyptian adults is 15.2%, which may be an underestimation . Lonardo et al. reported that patients with T2DM had 80% higher liver fat contents compared to non-diabetic patients . Additionally, Lee, et al., (2019), mentioned that compared to the general population (around 25%), 50% to 70% of people with diabetes have NAFLD, and NAFLD severity (including fibrosis) tends to be worsened by the presence of diabetes . Additionally, another study carried out on the Egyptian college students showed that around 1 in 3 had steatosis, and 1 in 20 had fibrosis. The prevalence of NAFLD in young adults was estimated to be 31.6%, which is perfectly in line with the 31.8% prevalence rate found in a meta-analysis of numerous epidemiological studies across general Middle Eastern populations. It is known that the Middle East and North Africa region has one of the highest prevalence rates of NAFLD globally, and that Egypt ranked among the highest 10 nations with obesity prevalence. Combing both may explain our unexpected observation. In our cohort, 59 (49.2%) of participants were overweight or obese . NAFLD is caused by a variety of different molecular pathways and cellular alterations. The molecular pathways of NAFLD pathogenesis in the liver have been identified in several studies. The major genes linked to illness development and the underlying functional pathways are yet unknown, and whether the differentially expressed CD24 is involved in hepatic lipid metabolism is still unclear. Microarray technologies have revealed a large number of new molecular markers (DNA, RNA, and protein) in recent years. Further research is needed to confirm the clinical utility of these impending novel indicators in relation to hepatic steatosis. CD24 is one of these markers, which was recently reported by Huang et al. as a possible biomarker in the course of hepatocyte steatosis . Various studies have recently discovered that CD24 expression is relatively high in many human malignancies, including HCC . Additionally, CD24 overexpression has been correlated with increased invasiveness, proliferation, and metastasis . It was previously identified that a subpopulation of adipocyte progenitor cells with the expression of the cell surface molecule CD24 being necessary for reconstitution of white adipose tissue function in vivo as well as being a key regulator of adipogenesis in vitro . In our study, we investigated the association between CD24 gene expression and the prevalence of NAFLD. The current study found that CD24 gene expression was considerably greater in NAFLD cases compared to controls, and the normalized CD24 gene expression in NAFLD was up-regulated 6.56-fold. These findings suggest that the CD24 gene is important in the development of NAFLD. This could be related to CD24 gene expression's impact on the immune/inflammatory response via T-cell activation . Several immune cell-mediated inflammatory processes are involved in NAFLD and its progression to NASH. They also influence the generation of cytokines by necrotic liver cells . This confirms the previous results detected by Feng et al., who observed the up-regulation of CD24 gene expression in the livers of HFD-induced NAFLD mice and in cultured HepG2 cells exposed to glucolipotoxicity (palmitic acid or advanced glycation end products) . Up until now, the precise role and the underlying mechanisms of CD24 in NAFLD progression remain unclear. However, Huang and his colleague identified the prominent correlation between CD24 and NAFLD/NASH. They mentioned that CD24 could play a key role in one of the pathways that may cause IR and may induce NAFLD/NASH in humans including ["glycolysis/gluconeogenesis", "p53 signaling pathway" and "glycine", serine and threonine metabolism . Additionally, CD24 expression was higher in cases with fibrosis stage F1 compared to those with fibrosis stage F0, as the mean expression level of CD24 was 7.19 in F0 cases as compared to 8.65 in F1 patients, but without significant difference (p = 0.588). This may be because that all cases included in the present study have mild fibrosis. This results most be confirmed by other studies based on large number of samples and on patients with severe stage of fibrosis. The changes in liver tissue-transcriptome in a subset of 25 mild-NAFLD and 20 NASH biopsies were examined in a cross-sectional study. CD24 was revealed to be one of five differentially expressed genes (DEGs) positively linked with disease severity and to be main classifiers of mild and severe NAFLD . Additionally, CD24-positive cells isolated from hepatocellular carcinoma cell lines exhibited stemness properties, such as self-renewal, chemotherapy resistance, metastasis, and tumorigenicity . These results indicate that CD24 may play a role in hepatocyte injury and promote regeneration during the development and progression of NAFLD. Another Egyptian study detected that CD24 polymorphism 170 CT/TT may affect the incidence of infection with CHC, as well as HCC . They revealed that the P170T allele, which is expressed at a higher level than P170C, encodes a certain protein, which is responsible for the progression of chronic HCV infection by affecting the efficiency of cleavage of posttranslational GPI. Additionally, Robert and Pelletier (2018) showed that the P170T allele affects the progression of chronic HCV infection through posttranslational mechanisms . Another study by Kristiansen et al. (2010) also suggested that CD24 SNPs are prognostic markers for hepatic carcinoma . Interestingly, CD24 was also up-regulated in the NAFLD patients with type 2 diabetes than its expression in non-diabetic cases, but without significant difference. Another study carried out by Shapira et al. (2021) reported that CD24 may negatively regulate peroxisome proliferator-activated receptor gamma (PPAR-g) expression in male mice. This gene is a regulator of adipogenesis that plays a role in insulin sensitivity, lipid metabolism, and adipokine expression in adipocytes. Furthermore, they concluded the association between the CD24 and insulin sensitivity, suggesting its possible mechanism for diabetes . 5. Conclusions The current study found CD24 gene expression was considerably greater in NAFLD cases compared to controls. This could indicate that CD24 may contribute to hepatic steatosis, but a current study showed that it cannot be used as an independent predictor of NAFLD. Further studies are required to confer its diagnostic and prognostic value in the detection of NAFLD, as well as to clarify its role in the progression of hepatocyte steatosis in patients with advanced stage of fibrosis and to elucidate the mechanism of this biomarker in the progression of disease. However, our study is limited because of the small sample size, because all participants in this study have early stage of NAFLD, and because accurate diagnosis of liver fibrosis or hepatocellular injury are invasive and very expensive. Although abdominal ultrasonography has low sensitivity for detecting mild-NAFLD as reported in the previous literature, it is the best low-cost available non-invasive technique to detect NAFLD. Because of ethical considerations, we did not rely on the liver biopsy for diagnosis, as none of the patients had clinical manifestations. Moreover, the studied patients considered themselves healthy and refused to undergo further invasive investigations, including pathological examinations via liver biopsy to detect fibrosis. Acknowledgments We acknowledge the National Research Centre for technical and financially supporting the current research. Author Contributions H.M.R., W.A.E. and N.A.E.M.: conceptualized the study; H.M.R., M.A.A. and N.A.E.M.: supervision, contributing to experiments design, reviewing, and editing and final approval of the version to be submitted for publication; M.A.A.: samples collections, visualization and investigation, supervision and revised the manuscript; W.A.E.: performed the experiments, analyzed the data, drafted and revised the manuscript. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The study protocol was approved by the Medical Research Ethical Committee--National research center, Cairo, Egypt (Approval No.19-001). Informed Consent Statement Written informed consent was obtained from all participants. Data Availability Statement The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Comparison between CD24 gene expression in both NAFLD and control. The median fold change was 6.56 higher in NAFLD cases compared to control subjects. Figure 2 Gender difference in CD24 gene expression in relation to NAFLD. Expression of CD24 was higher in female than male but without significant difference. Figure 3 Comparison between CD24 gene expression in both overweight and obese NAFLD patients. There was no difference in CD24 fold change between overweight patients (median fold change = 9) and obese cases (median fold change = 5.89). Figure 4 Comparison between median CD24 gene expression in both non-diabetic and diabetic NAFLD patients. The median fold change in CD24 in diabetic cases was seven compared to 5.13 in non-diabetic cases. Figure 5 ROC curve of CD24 gene expression to differentiate between NAFLD cases from health controls. diagnostics-13-00984-t001_Table 1 Table 1 Characteristics of Study Participants According to NAFLD Status. Variable NAFLD Group (N = 40) Control Group (N = 40) p-Value Groups Age (Yrs.) 42.18 +- 11.14 29.65 +- 6.63 <0.001 ** Gender a 0.061 Male/Female 10/30 18/22 Percentage of Male (25%) (45%) Hypertension a 0.006 ** Absent 33 (82.5%) 40 (100%) Present 7 (17.5%) 0 (0%) Diabetes a <0.001 ** Absent 20 (50%) 40 (100%) Oral hypoglycemic 9 (22.5%) 0 (0%) on insulin 11 (27.5%) 0 (0%) Family history of diabetes a 1.000 No 31 (77.5%) 31 (77.5%) Yes 9 (22.5%) 9 (22.5%) Family history of liver diseases a 0.805 No 28 (70%) 29 (72.5%) Yes 12 (30%) 11 (27.5%) BMI (kg/m2) 32.42 +- 3.59 23.66 +- 1.3 <0.001 ** Waist circumference (cm) 117.9 +- 9.4 74.3 +- 6.8 <0.001 ** Mid-arm circumference (cm) 31.8 +- 4.3 25.5 +- 1.5 <0.001 ** Ultrasound finding a Enlarged liver size 25 (62.5%) 0 (0%) <0.001 ** Laboratory variables Fasting blood glucose (mg/dL) 122.6 +- 40.97 96.03 +- 7.77 <0.001 ** HB (g/dL) 11.56 +- 1.4 12.81 +- 1.06 <0.001 ** Platelets count (103/mL) 236 +- 72.78 260.63 +- 56.79 0.096 Total leucocytic count (103/mL) 6.79 +- 1.91 7.24 +- 1.7 0.273 ALT (U/L) 47.9 +- 18.3 30.6+- 4.9 <0.001 ** AST (U/L) 33.55 +- 14.9 23.6 +- 4.6 <0.001 ** Total bilirubin (mg/dL) 0.76 +- 0.24 0.68 +- 0.2 0.132 ALP (U/L) 155.7+- 37.5 115.02+- 15.2 <0.001 ** GGT (U/L) 91.1+- 64.2 34.5+- 9.2 <0.001 ** Total Protein (g/dL) 8.1+- 0.2 7.9+- 0.24 0.007 ** INR 1.05 +- 0.1 1.0 +- 0.0 0.003 ** Serum Albumin (g/dL) 3.8 +- 0.38 3.8 +- 0.3 0.939 Cholesterol (mg/dL) 150.88 +- 32.4 101.65 +- 19.76 <0.001 ** Triglycerides (mg/dL) 168.5 +- 44.15 143.9 +- 29.7 0.005 ** LDL (mg/dL) 133.7 +- 34.6 104.02 +- 16.1 <0.001 ** HDL (mg/dL) 48.4 +- 15.5 55 +- 12.7 0.04 * BMI, body mass index; Hb, hemoglobin; ALT, alanine aminotransferase; AST, aspartate aminotransferase; GGT, gamma-glutamyl transferase; ALP, alkaline phosphatase; HDL, high-density lipoproteins; LDL, low-density lipoproteins. All data are displayed as mean and standard deviation. (SD) unless otherwise indicated. a Categorical data were summarized as percentages. Differences were analyzed with kh2 (chi square) tests and Fisher's exact test. * = significant p value (<=0.05), ** = highly significant p value (<=0.01). diagnostics-13-00984-t002_Table 2 Table 2 Clinical and biochemical characteristics of participants stratified by sex and presence/absence of NAFLD. Variable NAFLD Group (N = 40) p-Value Control Group (N = 40) p-Value Groups Male N = 10 Female N = 30 Male N = 18 Female N = 22 Age (Yrs.) 44.8 +- 9.1 41.3 +- 11.7 0.396 28.5 +- 4.7 30.59 +- 7.8 0.305 Hypertension 0.338 - Absent 7 (70%) 26 (86.7%) 18 (100%) 22 (100%) Present 3 (30%) 4 (13.3%) 0 (0%) 0 (0%) Diabetes 0.322 - Absent 7 (70%) 13 (43.3%) 18 (100%) 22 (100%) Oral hypoglycemic 1 (10%) 8 (26.7%) 0 (0%) 0 (0%) on insulin 2 (20%) 9 (30%) 0 (0%) 0 (0%) Family history of diabetes 0.827 0.970 No 8 (80%) 23 (76.7%) 14 (77.8%) 17 (77.3%) Yes 2 (20%) 7 (23.3%) 4 (22.2%) 5 (22.7%) Family history of liver diseases 0.231 0.173 No 9 (90%) 19 (63.3%) 11 (61.1%) 18 (81.8%) Yes 1 (10%) 11 (36.7%) 7 (38.9%) 4 (18.2%) BMI (kg/m2) 32.51 +- 3.1 32.39 +- 3.8 0.929 23.95 +- 1.1 23.4 +- 1.33 0.183 Laboratory variables Fasting blood glucose (mg/dL) 108.7 +- 38.9 127.23 +- 41.2 0.220 93.78 +- 6.3 97.86 +- 8.5 0.098 HB (g/dL) 12.72 +- 1.03 11.17 +- 1.3 0.001 ** 12.9 +- 0.99 12.7 +- 1.1 0.572 Platelets count (103/mL) 252.3 +- 73.3 230.57+- 73.03 0.421 270.9 +- 64.8 252.2 +- 49.3 0.305 Total leucocytic count (103/mL) 7.18 +- 1.69 7.07 +- 1.9 0.875 6.7 +- 1.2 7.3 +- 1.83 0.253 ALT (U/L) 60.7 +- 18.6 43.7 +- 16.4 0.009 * 29.9+- 5.1 31 +- 4.8 0.489 AST (U/L) 44.7 +- 18.9 29.8 +- 11.4 0.038 * 22.8 +- 3.9 24.3 +- 5 0.312 Total bilirubin (mg/dL) 0.82 +- 0.25 0.74 +- 0.24 0.331 0.69 +- 0.2 0.66 +- 0.2 0.605 ALP (U/L) 156.4+- 40.4 155.5 +- 37.2 0.949 117.3+- 15 113.1 +- 15.4 0.390 GGT (U/L) 111.1+- 61.9 84.4 +- 64.5 0.259 35.6+- 10.3 33.5 +- 8.3 0.488 Total Protein (g/dL) 8.1+- 0.34 8.04+- 0.23 0.574 7.8+- 0.24 7.9 +- 0.24 0.277 Serum Albumin (g/dL) 3.8 +- 0.37 3.8 +- 0.39 0.962 3.8 +- 0.34 3.8 +- 0.26 0.489 Cholesterol (mg/dL) 158 +- 22.4 148.5 +- 35.15 0.429 105.1 +- 22.1 98.8 +- 17.6 0.323 Triglycerides (mg/dL) 146.3 +- 52.4 175.83 +- 39.3 0.066 152.2 +- 25.8 137.14 +- 31.5 0.112 LDL (mg/dL) 128.4+- 34.2 135.5 +- 35.2 0.582 105.11+- 17 103.9 +- 15.7 0.811 HDL (mg/dL) 46.5 +- 20.17 49+- 14.02 0.665 52.4 +- 6.5 57.09 +- 15.9 0.253 BMI, body mass index; Hb, hemoglobin; ALT, alanine aminotransferase; AST, aspartate aminotransferase; GGT, gamma-glutamyl transferase; ALP, alkaline phosphatase; HDL, high-density lipoproteins; LDL, low-density lipoproteins. * = significant p value (<=0.05), ** = highly significant p value (<=0.01). diagnostics-13-00984-t003_Table 3 Table 3 Results of Fibroscan, CAP, NAFLD Fibrosis Score, FIB 4, and FAST Score in NAFLD Group. Group I Total NAFLD Patients N = 40 Fibrosis Stage (F0) N = 30 Fibrosis Stage (F1) N = 10 p-Value Fibroscan (kPa) Mean +- SD 5.1 +- 0.99 4.7 +- 0.67 6.5 +- 0.3 <0.001 ** Range 3.8-6.9 3.8-5.9 6.2-6.9 CAP (dB/m) Mean +- SD 263.9 +- 11.61 260.4 +- 10.9 274.2 +- 6.44 <0.001 ** Range 242-286 242-286 265-286 FAST score Mean +- SD 0.203 +- 0.139 0.2377 +- 0.14 0.099 +- 0.09 0.001 ** Range 0.04-0.47 0.05-0.47 0.04-0.25 NAFLD fibrosis score 0.147 Mean +- SD -1.37 +- 1.38 -1.19 +- 1.4 -1.92 +- 1.01 Range -4.1-1.22 -4.1-1.22 -3.2-0.33 FIB 4 Mean +- SD 0.919 +- 0.46 0.96 +- 0.5 0.81 +- 0.28 0.376 Range 0.33-2.39 0.33-2.39 0.36-1.14 ** = highly significant p value (<=0.01). diagnostics-13-00984-t004_Table 4 Table 4 Correlation between different non-invasive fibrosis markers and CD24 gene expression. Parameters CD24 Gene Expression r p-Value Kpa (kPa) -0.070 0.677 CAP(dB/m) -0.050 0.764 FAST -0.006 0.970 NFS 0.356 0.001 ** FIB-4 0.090 0.432 ** = highly significant p value (<=0.01). diagnostics-13-00984-t005_Table 5 Table 5 Binary logistic regression analysis using NAFLD as the dependent variable after adjusting the major confounders. Variables Coefficient Sig. Odds Ratio age 0.695 0.999 2.003 sex -8.443 0.999 0.000 DM 27.061 1.000 565,692,536,831.086300 BMI -5.426 0.997 227.262 Fold change of CD24 1.372 1.000 3.943 Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. References 1. Sherif Z.A. 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PMC10000767 | Diagnostics (Basel) Diagnostics (Basel) diagnostics Diagnostics 2075-4418 MDPI 10.3390/diagnostics13051003 diagnostics-13-01003 Article Comparison of Ultra-Magnifying Endocytoscopic and Hematoxylin-Eosin-Stained Images of Lung Specimens Kobayashi Misato Conceptualization Methodology Validation Investigation Writing - original draft Visualization 1 Kurimoto Noriaki Conceptualization Methodology Validation Investigation Writing - original draft Visualization Project administration 1* Tanino Ryosuke Conceptualization Methodology Software Validation Formal analysis Writing - original draft Visualization 1 Shiratsuki Yohei Investigation 1 Okuno Takae Investigation 1 Nakao Mika Resources 1 Hotta Takamasa Resources 1 Tsubata Yukari Writing - review & editing Supervision Project administration 1 Nagasaki Makoto Resources 2 Nishisaka Takashi Resources 3 Isobe Takeshi Writing - review & editing Supervision Project administration 1 Lung Maria Li Academic Editor Ko Josephine Academic Editor 1 Division of Medical Oncology & Respiratory Medicine, Department of Internal Medicine, Faculty of Medicine, Shimane University, Izumo 693-8501, Japan 2 Division of Pathology, National Hospital Organization Hamada Medical Center, Hamada 697-8511, Japan 3 Department of Pathology and Laboratory Medicine, Hiroshima Prefectural Hospital, Hiroshima 734-8530, Japan * Correspondence: [email protected]; Tel.: +81-853-20-2580 06 3 2023 3 2023 13 5 100326 1 2023 01 3 2023 02 3 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). Endocytoscopy enables real-time observation of lesions at ultra-magnification. In the gastrointestinal and respiratory fields, endocytoscopic images are similar to hematoxylin-eosin-stained images. This study aimed to compare the nuclear features of pulmonary lesions in endocytoscopic and hematoxylin-eosin-stained images. We performed an endocytoscopy to observe resected specimens of normal lung tissue and lesions. Nuclear features were extracted using ImageJ. We analyzed five nuclear features: nuclear number per area, mean nucleus area, median circularity, coefficient of variation of roundness, and median Voronoi area. We conducted dimensionality reduction analyses for these features, followed by assessments of the inter-observer agreement among two pathologists and two pulmonologists to evaluate endocytoscopic videos. We analyzed the nuclear features of hematoxylin-eosin-stained and endocytoscopic images from 40 and 33 cases, respectively. Endocytoscopic and hematoxylin-eosin-stained images displayed a similar tendency for each feature, despite there being no correlation. Conversely, the dimensionality reduction analyses demonstrated similar distributions of normal lung and malignant clusters in both images, thus differentiating between the clusters. The diagnostic accuracy of the pathologists was 58.3% and 52.8% (k-value 0.38, fair), and that of the pulmonologists was 50% and 47.2% (k-value 0.33, fair). The five nuclear features of pulmonary lesions were similar in the endocytoscopic and hematoxylin-eosin-stained images. endocytoscopy nuclear feature peripheral pulmonary lesion lung cancer This research received no external funding. pmc1. Introduction Lung cancer is a leading cause of death in Japan. Identifying driver variants is essential for selecting appropriate targeted therapy for lung cancer . Bronchoscopy facilitates pathological diagnosis and detection of oncogenes in biopsy specimens . Endobronchial ultrasonography is performed in the clinical setting to determine the appropriate biopsy site in peripheral lesions. However, this site often cannot be directly observed during a biopsy, resulting in uncertainty concerning the presence of sufficient tissue for a pathological diagnosis for detecting oncogenes from the target lesion. Endocytoscopy (ECS) enables the observation of a lesion at high magnification (approximately 450x) on a monitor by placing the objective lens in direct contact with the target lesion, with a depth of field of approximately 30 mm from the surface. ECS has been used in the gastrointestinal field in clinical practice, particularly for esophageal, stomach, and colon lesions, with methylene blue staining or crystal violet and methylene blue double staining . ECS demonstrates a diagnosis rate comparable to that of biopsy for colorectal lesions, with a high level of diagnostic sensitivity and specificity . Endocytoscopic images of colorectal lesions are similar to their hematoxylin-eosin (H&E)-stained images , and colorectal endocytoscopic classification is used for the diagnosis . Nevertheless, some respiratory field studies have reported endocytoscopic observations , and no quantitative studies have compared the endocytoscopic findings with the pathologic findings. This study aimed to obtain the endocytoscopic findings of pulmonary lesions for quantitative comparison of the nuclear features with those of H&E-stained images. We also intended to examine the inter-observer agreement of endocytoscopic videos for investigating the diagnostic ability of ECS. 2. Materials and Methods 2.1. Study Design This prospective, single-center, observational study was performed at the Shimane University Hospital. We conducted this study from September 2019 to September 2021 in 40 patients who underwent surgical resection of pulmonary lesions at the Department of Cardiothoracic Surgery, Division of Thoracic Surgery, Shimane University. Patients aged 20 years or older who underwent surgical resection of an intrathoracic nodule or mass and gave written informed consent were included. The exclusion criterion was any patient considered inappropriate at the discretion of the doctor in charge. The target sample size was 50 participants. The enrolment period was from October 2019 to March 2020. We analyzed the data until September 2021. The study was approved by the Medical Research Ethics Committee of the Shimane University Faculty of Medicine (approval number: 4020) and registered in the UMIN Clinical Trials Registry (UMIN 000038136). Informed consent was obtained from all patients before lung resection. The study was conducted in accordance with the tenets of the Declaration of Helsinki. 2.2. Endocytoscopy We used a catheter-type endocytoscope (prototype XEC-300-2, Olympus Medical Systems, Tokyo, Japan) with a length of 380 cm and an outer diameter of 3.2 mm at the tip. An endocytoscopic image can provide an observation field, an observation depth, and a horizontal resolution of 300 x 300 mm, 0-30 mm, and 4.2 mm, respectively, with 450-fold magnification on a 14-inch monitor . 2.3. Acquisition of the Endocytoscopic Images Computed tomography (CT) images showed the location of a target lesion . Thoracic surgeons performed surgical resection of the lesion. A pathologist cut into the center of the lesion in the resected specimens. In cases that required rapid intraoperative diagnosis, the part that remained following the removal of a portion of the resected tissues for frozen sections by the pathologist was used for endocytoscopic observation . Without staining, no relevant findings regarding the lesion were revealed. Methylene blue staining revealed dark-blue cell nuclei at the same lesion site . Therefore, we stained the cut surface with a drop of 0.25-0.5% methylene blue using a 1-mL syringe with a 23-G needle. The tip of the endocytoscope was placed directly on the cut surface, and we immediately observed the stained lesion on the monitor screen. Additional drops of methylene blue were added for large lesions as necessary. We attempted to observe the normal lung area in a similar manner. These observations were recorded using a video recorder (IMH-20; Olympus Medical Systems). The procedure was completed within 3 h of the resection. The specimens were fixed with formalin and stained with H&E. 2.4. Analyzing Nuclear Features 2.4.1. Nuclear Extraction ImageJ 1.53o (National Institutes of Health, Bethesda, MD, USA) was used for image quantification. We obtained H&E-stained images of the pathologic tissues, and every image was processed using the ImageJ "subtract background" feature (parameters: rolling ball radius = 20, light background; the other functions were unused). Endocytoscopic images of the normal lung tissue and lesions were obtained from the endocytoscopic observation videos using Edius 8 Pro version 8.53 (Grass Valley, Montreal, Canada). Every image was cropped at the observation area (x = 780, y = 188, w = 660, and h = 680) using ImageJ. Dark endocytoscopic images (mean gray value < 50) were excluded owing to the difficulty in nuclear extraction. We extracted the stained nuclear component using Colour Deconvolution 1.7 plugin. The H&E (Colour_1) and Masson Trichrome (Colour_3) vectors were used for the H&E-stained and endocytoscopic images, respectively. We performed subsequent processing in the following order: median (parameter: radius = 3), subtract background (parameters: rolling ball radius = 20, light background; the other functions were unused), auto local threshold (parameters: method = Phansalkar, radius = 20 [H&E images] or 10 [ECS images], parameter_1 = 0, parameter_2 = 0, white objects on black background), despeckle, and watershed (parameters: iterations = 1, count = 1, EDM output = overwrite; the other functions were unused). Moreover, we obtained Voronoi diagram images using Voronoi processing (with the same parameters as those used for the watershed process) and a nuclear-extracted image. 2.4.2. Quantification of the Nuclear Features To obtain the nuclear features, we performed an Analyze Particles (parameters: size = 0 to infinity, circularity = 0.00-1.00) command using nuclear-extracted images (nucleus area, nuclear number, circularity [(4p x area)/(perimeter2)], roundness [(4 x area)/(p x major axis2)]) and Voronoi diagram images (Voronoi area). Moreover, we calculated the nuclear number per area, mean nucleus area (the sum of the nucleus area/nuclear number), median circularity, coefficient of variation (CV) of roundness, and median Voronoi area of each image. 2.4.3. Two-Dimensional Projection of Five Nuclear Features Min-max normalized values of the nuclear features were calculated using the following formula: normalized values = (value - minimum value)/(maximum value - minimum value). The distinct nuclear trajectory was two-dimensionally mapped using principal component analysis (PCA) and nonlinear uniform manifold approximation and projection (UMAP) to reduce the dimension from the normalized values of the nuclear number per area, mean nucleus area, median circularity, CV of roundness, and median Voronoi area. 2.5. Endocytoscopic Observation and Inter-Observer Agreement Two pathologists and two pulmonologists reviewed the recorded videos of the endocytoscopic observation and evaluated the lesions. The observers were requested to classify malignancy upon identifying unequal nuclei size and uneven distribution of the nuclei in the video. When none or one of the two findings was present, the lesion was classified benign by endocytoscopic ("ECS-benign"). On identifying a lesion as malignant, the observers were requested to differentiate it as lung adenocarcinoma by ECS ("ECS-adeno") or another malignancy by ECS ("ECS-non-adeno"), according to the following criteria. ECS-adeno showed enlarged and irregularly sized nuclei piled up on the alveolar wall. Conversely, ECS-non-adeno was classified as malignancy, except adenocarcinoma, for unevenly distributed enlarged and irregularly sized nuclei, without piling up on the alveolar wall. Accuracy (acc) was calculated using the following formula: acc = (number of classified cases matched with pathology)/(number of cases). Inter-observer agreement was determined using Kappa statistics. All of the observers were blinded to the information regarding the patients who participated in the endocytoscopic observation. 2.6. Statistical Analyses We performed the Kruskal-Wallis test with Dunn's multiple comparison test and a simple linear regression using GraphPad Prism 9.3.1 (GraphPad, San Diego, CA, USA). The results were considered statistically significant at p < 0.05. Correlations between the nuclear features of the endocytoscopic and H&E-stained images were calculated using the Pearson correlation coefficient. Kappa statistics were calculated using JMP Pro 14.2.0 (SAS Institute, Cary, NC, USA). Agreements between observers were defined based on k values as follows: poor, k < 0; slight, k = 0-0.20; fair, k = 0.21-0.40; moderate, k = 0.41-0.60; substantial, k = 0.61-0.80; and almost perfect, k = 0.81-1.00. 3. Results 3.1. Cases and Their Diagnosis In total, 40 lesions from 40 cases were diagnosed histopathologically . Of the 40 lesions, 36 were malignant (20 lung adenocarcinomas, five squamous cell carcinomas, 4 cases of other types of lung cancer [two large-cell neuroendocrine carcinomas, one carcinoid, and one pleomorphic carcinoma], six metastatic lung tumors [five colorectal cancers and one pancreatic cancer], and one diffuse large B-cell lymphoma). The four benign cases consisted of two granulomas, one lung cyst, and one intrapulmonary lymph node. Thirty-eight lesions and 38 normal lung tissue specimens were observed by ECS . No patient had a history of anticancer therapy or chest radiation therapy. 3.2. ECS and H&E Findings of Representatives Next, we compared endocytoscopic and H&E-stained images for each specimen. ECS displayed alveolar cell nuclei and slightly stained alveolar septa in an endocytoscopic image of normal lung tissue, similar to that of the H&E-stained image . Figure 3B depicts an adenocarcinoma. The nodule was 18 x 10 mm in size. The ECS displayed enlarged and irregularly shaped nuclei piled up on the alveolar walls. For the same case, H&E staining displayed cells with a high nuclear-cytoplasmic ratio. Moreover, large nuclei increased along the alveolar walls, thus corresponding to lung adenocarcinoma. Figure 3C depicts a squamous cell carcinoma. The nodule was 20 x 15 mm in size. ECS revealed enlarged, irregularly shaped nuclei forming nests and partially overlapping. H&E staining revealed tumor cells forming nests, thereby corresponding to squamous cell carcinoma. Figure 3D depicts a case of rectal cancer metastasis. The nodule comprised a whitish solid part and a yellowish part, with a total size of 8 x 6 mm. ECS revealed an increase in the number of palisading nuclei. H&E-stained tumor cells were lined with irregularly shaped nuclei and formed atypical gland ducts, which correspond to the metastasis of rectal cancer. Figure 3E depicts a round and black intrapulmonary lymph node measuring 7 x 7 mm in size. ECS revealed clusters of small round nuclei. H&E staining displayed small lymphocytes, which correspond to the intrapulmonary lymph nodes. Figure 3F depicts a case of necrotizing granuloma. The nodule was whitish, soft, and 15 x 9 mm in size. ECS revealed few nuclei with fibrotic fibers, whereas H&E staining revealed proliferated macrophages, fibroblasts, and collagen fibers, thus corresponding to necrotizing granuloma. The aforementioned endocytoscopic findings were similar to those of H&E-stained imaging. 3.3. Comparison of the Nuclear Features of the Normal Lung, Benign, and Malignant Lesions To identify the features obtained from H&E-stained and endocytoscopic images, we determined the nuclear features of the normal lung, benign lesions, and malignant lesions via image analyses. All 40 lesions were included in the analysis of the H&E-stained images . Figure 4A depicts representative images of nuclear extraction and the quantification of nuclear features from an H&E-stained image of lung adenocarcinoma. Nuclear feature analysis revealed that the nuclear number per area and mean nucleus area were significantly higher in the malignant lesions than in the normal lung tissues . Moreover, the median circularity, CV of roundness, and median Voronoi area were significantly higher in normal tissues than in malignant lesions. The nuclear number per area was also higher, and the median Voronoi area was lower in benign lesions than in normal lung tissues . Similarly, we performed nuclear extraction and the quantification of the nuclear features from the endocytoscopic images . Thirty-three (29 malignant and four benign lesions) of 40 lesions were included in the analysis of the endocytoscopic images, and 19 of 40 normal lung tissues were included in the analysis of the endocytoscopic images . Consistent with the H&E-stained image analysis, the mean nuclear number per area and mean nucleus area was significantly higher in the malignant lesions than in the normal lung tissues . Moreover, the median circularity, CV of roundness, and median Voronoi area were significantly higher in the normal tissues than in the malignant lesions . All five features were concordant between H&E-stained and endocytoscopic images. Value ranges of the normal lung tissues and malignant lesions were not thoroughly separated in each comparison. 3.4. Comparisons of Nuclear Features between H&E-Stained and Endocytoscopic Images We compared the values of H&E-stained and endocytoscopic nuclear features extracted from similar specimen areas of the same cases where an H&E-stained and endocytoscopic image were both obtained (malignant: 29 cases; benign: 4 cases; normal lung tissue: 19 cases) . However, no feature values were correlated between the H&E-stained and endocytoscopic images. To obtain the data trend for all five nuclear features, we presented heat maps of the normalized nuclear feature values in the H&E-stained and endocytoscopic images . Subsequently, we performed the PCA and UMAP , respectively, using normalized values. PCA and UMAP clustering and embedding revealed analogous cluster locations between the H&E-stained images (malignant: 36 cases; benign: 4 cases; normal lung tissue: 40 cases) and endocytoscopic images (malignant: 29 cases; benign: 4 cases; normal lung tissue: 19 cases). We distinguished the normal lung-prone and malignant lesion-prone areas in the UMAP clusters . UMAP clustering separated the normal and malignant clusters more than PCA did. Thus, the five nuclear features displayed similar trends in both H&E staining and ECS. 3.5. Inter-Observer Agreement in the Endocytoscopic Image Evaluation To investigate agreements between observers of ECS, the pathologists and pulmonologists evaluated 36 cases from the recorded endocytoscopic videos . The typical lesions of "adenocarcinoma" and "squamous cell carcinoma" of the lungs obtained in this study (one of the 20 adenocarcinoma cases and one of the five squamous cases) were presented to each observer as labeled training videos before evaluation. Two of the labeled training videos were excluded from the evaluation. We assessed the diagnostic accuracy and inter-observer agreement between the three categories (ECS-adeno, ECS-non-adeno, and ECS-benign) . The total diagnostic accuracy of pathologists 1 and 2 was 58.3% (21/36) and 52.8% (19/36), respectively, and their inter-observer agreement was 0.38, i.e., fair. The total diagnostic accuracy of pulmonologists 1 and 2 was 50% (18/36) and 47.2% (17/36), respectively, and their inter-observer agreement was 0.33, i.e., fair. The diagnostic accuracy of each category (adeno, non-adeno, and benign) is also shown in Figure 7B. ECS-adeno was the best-matched category. Inter-observer agreement between each two of the four observers was between 0.31 and 0.43, i.e., fair or moderate. 4. Discussion This study revealed that the five nuclear features (nuclear number per area, mean nucleus area, median circularity, CV of roundness, and median Voronoi area) significantly differed between the malignant lesions and normal lungs in both the H&E-stained and endocytoscopic images. Shibuya et al. reported that ECS during bronchoscopy could distinguish between normal bronchial mucosa, bronchial squamous dysplasia, and squamous cell carcinoma. They inserted the tip of the catheter-type endocytoscope through the biopsy channel of the bronchoscope and placed it on the bronchial surface, close to the site of interest. Endocytoscopic imaging of squamous cell carcinoma showed that cellular densities were increased and that the nuclear-cytoplasmic ratio was high and variable . Shah et al. reported that ECS could distinguish normal epithelium from dysplasia and cancer . Despite similarities between H&E-stained and endocytoscopic findings , they have not been quantitatively evaluated. To our knowledge, this is the first report of ECS in various types of pulmonary lesions and a quantitative investigation of endocytoscopic images. We found that five nuclear features are beneficial for distinguishing between normal and malignant tissue in the resected specimens in this study. All five features were concordant between the H&E-stained and endocytoscopic images. The lower median Voronoi area may reflect the uneven distribution of malignant cells. In contrast, larger nucleus areas and lower median circularity were greatly related to the morphological changes of the malignant cell nuclei. Malignant lesions showed lower CV of roundness than normal lung tissue. The cells in normal lung tissue comprise various types, such as fibroblast, macrophages, and alveolar epithelial cells, and these nuclear shapes differ from one another. However, malignant lesions lose cell type diversity and have high cellular clonality. The nuclear size in the endocytoscopic images tended to be smaller than that in the H&E-stained images. The low-contrast endocytoscopic images with high background might provide less nuclear detection, thereby resulting in lower mean nuclear area and nuclear number per area than those of the existing nuclei. Despite significant differences in the five nuclear features between the malignant lesions and normal lungs in the H&E-stained and endocytoscopic images, all five nuclear features were not correlated with the H&E-stained and endocytoscopic images in each feature of the same lesion. Wang et al. reported that the combinations of some extracted nuclear features from the H&E-stained images could predict recurrence in patients with lung cancer who underwent surgery . Wolberg et al. presented 10 nuclear features obtained from breast cancer lesions useful in predicting whether the lesion was malignant or benign . Similarly, in this study, the combination of all five nuclear features provided information on the malignant and normal lung cells since the UMAP-mapped data locations of the malignant lesions and normal lungs of the endocytoscopic images were considerably similar to those of the H&E-stained images. In clinical practice, pathologists determine the malignancy of a lesion based on the combination of multiple features of the cells and extracellular matrix. Our findings might also provide evidence for the importance of the five nuclear features in distinguishing lung malignancy. In this study, we used H&E-stained images as the gold standard for comparison with the endocytoscopic images. We presented five representative cases with similar nuclear distribution and structures in both images, and ECS seems to be available for easily recognizing irregular nuclear distribution and piled-up nuclei on the alveolar wall. However, the in vivo usage of ECS in lungs poses some challenges owing to a thick-diameter bronchoscope, breathing movement, and DNA damage attributed to methylene blue staining . Considering ex vivo application, ECS will likely be useful in determining whether the specimen of the pulmonary lesions is qualified for pathologic diagnosis during bronchoscopy. Evaluating the nuclear features of biopsy specimens using ex vivo ECS may reduce the number of biopsies by assessing the specimen's amount and quality. In addition, ex vivo ECS may assist in the rapid pathologic diagnosis of tissues obtained by surgery, bronchoscopic biopsy, or CT-guided biopsy. This study is essential for developing endocytoscopic applications in the respiratory field. In confocal laser endomicroscopy, Takemura et al. reported moderate inter-observer agreement among two pulmonologists who evaluated the cell density and nuclear size disparity of transbronchial biopsy specimens . We examined the inter-observer agreement to investigate the diagnostic ability of ECS for surgically resected specimens. Although the pathologists are experts in pathological images, agreements were only fair between the pulmonologists and between the pathologists that carried out an ECS evaluation. As they have little experience observing endocytoscopic videos with only two training videos, training could improve the diagnostic ability of ECS. In addition, as a new approach in the gastroenterology field, crystal violet and methylene blue double staining and artificial intelligence (AI) are used to improve the diagnostic ability of ECS . Mori et al. reported on a computer-aided diagnosis using ECS and demonstrated a sensitivity and accuracy of 92.0% and 89.2%, respectively, for the classification of colorectal polyps . AI plays an important role in pathology, such as the diagnosis of prostate cancer , breast cytology , and immunohistochemistry applications . Moreover, AI may facilitate the on-site identification of histological types of biopsy specimens based on the nuclear features of endocytoscopic images. This study has some limitations. First, several endocytoscopic images of normal lung areas were not used in the analyses because they were too dark. Second, we obtained only four cases of benign lesions owing to the low number of surgeries for benign diseases that included granuloma, lung cysts, and intrapulmonary lymph nodes. The features of benign lesions could not be shown. In conclusion, the nuclear features of pulmonary lesions and normal lung tissue were similar in both the endocytoscopic and H&E-stained images. Endocytoscopic imaging could provide nuclear information similar to that of H&E-stained images. Acknowledgments We wish to thank T Kubonoya (Olympus Medical Systems, Tokyo, Japan) for his technical assistance. The endocytoscopy system was borrowed from Olympus Medical Systems. Author Contributions Conceptualization, M.K., N.K. and R.T.; methodology, M.K., N.K. and R.T.; software, R.T.; validation, M.K., N.K. and R.T.; formal analysis, R.T.; investigation, M.K., N.K., Y.S. and T.O.; resources, M.N. (Mika Nakao), T.H., M.N. (Makoto Nagasaki) and T.N.; writing--original draft preparation, M.K., N.K. and R.T.; writing--review and editing, Y.T. and T.I.; visualization, M.K., N.K. and R.T.; supervision, Y.T. and T.I.; project administration, N.K., Y.T. and T.I. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The study was conducted in accordance with the Declaration of Helsinki and approved by the Medical Research Ethics Committee of the Shimane University Faculty of Medicine (approval number: 4020), registered in the UMIN Clinical Trials Registry (UMIN 000038136). Informed Consent Statement Informed consent was obtained from all subjects involved in the study. Data Availability Statement The data that support the findings of this study are available from the corresponding author, Noriaki Kurimoto, upon reasonable request. Conflicts of Interest Misato Kobayashi has no conflict of interest. Noriaki Kurimoto received personal fees from Olympus Corporation. Noriaki Kurimoto received personal fees from Eli Lilly Japan K.K. and Chugai Pharmaceutical Co., Ltd., outside of the submitted work. Ryosuke Tanino has no conflict of interest. Yohei Shiratsuki has no conflict of interest. Takae Okuno has no conflict of interest. Mika Nakao has no conflict of interest. Takamasa Hotta has no conflict of interest. Yukari Tsubata received grants from ONO PHARMACEUTICAL CO., LTD., and Pfizer Health Research Foundation., outside the submitted work. Yukari Tsubata received personal fees from DAIICHI SANKYO COMPANY, LIMITED, AstraZeneca K.K., Kyowa Kirin Co., Ltd., TAIHO PHARMACEUTICAL CO., LTD., Bristol Myers Squibb., and Chugai Pharmaceutical Co., Ltd., outside the submitted work. Makoto Nagasaki has no conflict of interest. Takashi Nishisaka has no conflict of interest. Takeshi Isobe received grants from KONICA MINOLTA, INC., IQVIA Services JAPAN K.K., DAIICHI SANKYO COMPANY, LIMITED, and Insmed Incorporated., outside the submitted work. Takeshi Isobe received personal fees from AstraZeneca K.K. and Nippon Boehringer Ingelheim Co., Ltd., outside of the submitted work. Figure 1 Endocytoscopy (ECS) overview for resected lung specimens. (A) Appearance of an endocytoscope (Source: courtesy of Olympus Medical Systems). (B) High-resolution computed tomography of a patient with adenocarcinoma. (C) Macroscopic appearance of a lesion (white arrows: adenocarcinoma) resected from the patient. Scale bar: 10 mm. (D) Endocytoscopic images of the lesion with and without methylene blue staining. Figure 2 Study overview. All 40 cases were included in the analysis of the H&E-stained images. Thirty-eight cases provided endocytoscopic images. Thirty-three lesions (malignant = 29, benign = 4) and 19 normal lung tissues were included in the nuclear feature analysis of the endocytoscopic images after excluding endocytoscopic images that were too dark to extract the nucleus using ImageJ. In endocytoscopic observation, 36 cases were used to assess inter-observer agreement, excluding two cases as the labeled training videos. Figure 3 Macroscopic appearance, endocytoscopic images, and hematoxylin-eosin (H&E)-stained images of the resected lesions. (A) Normal lung tissue (yellow arrows: alveolar septa). (B) Adenocarcinoma. (C) Squamous cell carcinoma. (D) Lung metastasis from rectal cancer. (E) Intrapulmonary lymph node. (F) Necrotizing granuloma. Figure 4 Nuclear feature extraction from the hematoxylin-eosin (H&E)-stained images of the formalin-fixed, paraffin-embedded resected lung sections. (A) Nuclear extraction and analyses of the H&E-stained image. Scale bar, 50 mm. (B) Box plot of the nuclear features extracted from the H&E-stained images of the normal lung (n = 40), benign (n = 4), and malignant (n = 36) areas from the resected specimens (Kruskal-Wallis test with Dunn's multiple comparison test). ns, not significant. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Figure 5 Nuclear feature extraction from the endocytoscopic images of methylene blue-stained resected lung specimens. (A) Nuclear extraction and analyses of the endocytoscopic image. Scale bar, 50 mm. (B) Box plot of nuclear features extracted from the endocytoscopic images of the normal lung (n = 19), benign (n = 4), and malignant (n = 29) areas from the resected specimens (Kruskal-Wallis test with Dunn's multiple comparison test). ns, not significant. * p < 0.05, ** p < 0.01, *** p < 0.001. Figure 6 Comparison of the extracted nuclear features between the hematoxylin-eosin (H&E)-stained and endocytoscopic images in the resected lung specimens. (A) Linear regression analysis of each nuclear feature between the H&E-stained and endocytoscopic images (normal lung tissues, benign, and malignant lesions) using specimens of the same patients. CV, coefficient of variation. r, Pearson correlation coefficient (n = 52, simple linear regression). (B) Heatmaps of the min-max normalized nuclear feature values. Letters represent nuclear features: a, nuclear number per area; b, mean nucleus area; c, median circularity; d, CV of roundness; e, median Voronoi area. (C) Dimension reduction plots in nuclear features of the H&E-stained and endocytoscopic images using clustering embedding of principal component (PC) analysis (top-left) and uniform manifold approximation and projection (UMAP) clustering embedding with cosine distance as the metric. Figure 7 Evaluation of the endocytoscopic images of the methylene blue-stained resected lung specimens by two pathologists and two pulmonologists. (A) Schema of the evaluation of endocytoscopic images. (B) Diagnostic accuracy of the four observers compared with pathology. (C) The Kappa coefficient determining inter-observer agreement between each two of the four observers in the three categories (ECS-adeno, ECS-non-adeno, and ECS-benign). Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). 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PMC10000768 | Diagnostics (Basel) Diagnostics (Basel) diagnostics Diagnostics 2075-4418 MDPI 10.3390/diagnostics13050991 diagnostics-13-00991 Case Report Secondary Breast Malignancy from Renal Cell Carcinoma: Challenges in Diagnosis and Treatment--Case Report Spasic Marko 12 Zaric Dusan 3 Mitrovic Minja 4 Milojevic Sanja 5 Nedovic Nikola 4 Sekulic Marija 6 Stojanovic Bojan 12 Vulovic Dejan 17 Milosevic Bojan 12* Milutinovic Filip 8 Milosavljevic Neda 9 Speeckaert Marijn Academic Editor 1 Department of Surgery, Faculty of Medical Sciences, University of Kragujevac, 34000 Kragujevac, Serbia 2 Clinic for General Surgery, University Clinical Centre, 34000 Kragujevac, Serbia 3 Clinic for Urology, Clinical Hospital Centre "Dragisa Misovic", 11000 Belgrade, Serbia 4 Faculty of Medical Sciences, University of Kragujevac, 34000 Kragujevac, Serbia 5 Centre for Radiology, University Clinical Centre, 34000 Kragujevac, Serbia 6 Department of Hygiene and Ecology, Faculty of Medical Sciences, University of Kragujevac, 34000 Kragujevac, Serbia 7 Centre for Plastic Surgery, University Clinical Centre, 34000 Kragujevac, Serbia 8 Clinic for Urology, University Clinical Centre, 34000 Kragujevac, Serbia 9 Centre for Radiation Oncology, University Clinical Centre, 34000 Kragujevac, Serbia * Correspondence: [email protected] 05 3 2023 3 2023 13 5 99130 1 2023 11 2 2023 28 2 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). Renal cell carcinoma represents about 2% of all malignant tumours in adults. Metastases of the primary tumour in the breast make up to about 0.5-2% of the cases. Renal cell carcinoma metastases in the breast are extremely rare and have been sporadically recorded in the literature. In this paper, we present the case of a patient with breast metastasis of renal cell carcinoma 11 years after primary treatment. Case presentation: An 82-year-old female who had right nephrectomy due to renal cancer in 2010 felt a lump in her right breast in August 2021, whereby a clinical examination revealed a tumour at the junction of the upper quadrants of her right breast, about 2 cm, movable toward the base, vaguely limited, and with a rough surface. The axillae were without palpable lymph nodes. Mammography showed a circular and relatively clearly contoured lesion in the right breast. Ultrasound showed an oval lobulated lesion of 19 x 18 mm at the upper quadrants, with strong vascularisation and without posterior acoustic phenomena. A core needle biopsy was performed, and the histopathological findings and obtained immunophenotype indicated a metastatic clear cell carcinoma of renal origin. A metastasectomy was performed. Histopathologically, the tumour was without desmoplastic stroma, comprising predominantly solid-type alveolar arrangements of large moderately polymorphic cells, bright and abundant cytoplasm, and round vesicular cores with focally prominent nuclei. Immunohistochemically, tumour cells were diffusely positive for CD10, EMA, and vimentin, and negative for CK7, TTF-1, renal cell antigen, and E-cadherin. With a normal postoperative course, the patient was discharged on the third postoperative day. After 17 months, there were no new signs of the underlying disease spreading at regular follow-ups. Conclusion: Metastatic involvement of the breast is relatively rare and should be suspected in patients with a prior history of other cancers. Core needle biopsy and pathohistological analysis are required for the diagnosis of breast tumours. secondary malignancy breast renal cell carcinoma metastasis metastasectomy This research received no external funding. pmc1. Introduction Kidney cancer accounts for approximately 5% of cancer incidence worldwide, with higher incidence and mortality rates in men. This difference can be explained by genetics, sex hormones, and modifiable risk factors such as uncontrolled hypertension, alcohol, and obesity . The most common type of kidney cancer in the cortex is renal cell carcinoma (RCC), accounting for approximately 2% of adult malignancies and encompassing 80-85% of all renal neoplasms, predominantly the clear cell RCC subtype (75-85%). Other RCC subtypes include papillary, chromophobe, oncocytoma, and other . Renal pelvis cancer characteristics resemble those of urothelial cancer . In most cases, RCC is diagnosed incidentally, and about 10% of patients experience the classic triad: palpable mass, flank pain, and haematuria. Other common symptoms include weight loss, fatigue, fever, and others, with 20% of patients diagnosed with a variety of paraneoplastic syndromes . Renal cell cancer differs in biology and behaviour, reflecting multiple genetic foundations and well-defined hereditary renal cancer syndromes. The practical significance of this in terms of treatment is limited, while certain renal cell neoplasms are subject to specific therapy recommendations . The most common metastasis in renal cell carcinoma occurs in the lungs, followed by the involvement of the bones, liver, lymph nodes, adrenal glands, and brain . About one-third of patients with renal cell carcinoma have metastases at the time of diagnosis or within months or years after renal cell cancer treatment . For patients with localised disease, the recommended treatment is surgery, with no benefit from adjuvant therapy. In advanced settings, a combination of tyrosine kinase inhibitor and PD-1/PD-L1 inhibitor shows benefits in terms of increasing response rates and prolonging progression-free survival . When considering treatment options, the recommendations by the International Metastatic Renal Cell Carcinoma Database Consortium (IMDC), which stratifies patients with metastatic disease into three subgroups--good-, intermediate-, and poor-risk groups, should be reviewed . Breast cancer is the most common cancer in the world, with a 47.8% incidence rate and 2,261,419 cases in 2020, according to the Global Cancer Observatory (GLOBOCAN) . Breast metastases from extramammary tumours are rare, with an incidence ranging from 0.5% to 2% in clinical reports on all breast neoplasms; in most cases, they are accompanied by a poor prognosis. Diagnosed breast masses in up to 50% of cases may be the first sign of another primary tumour . It is recommended that treatment and its sequencing follow the guidelines for the primary tumour type . Metastases in the breast from renal cell carcinoma are extremely rare, with only a few sporadic cases reported in the literature . In this study, we report a rare case involving a female patient who underwent a nephrectomy and, 11 years later, was diagnosed with a breast metastasis from the renal cell carcinoma. Based on the literature research we performed, this is the first case from Serbia to be published. 2. Case Presentation Data from the patient's complete medical record, which included clinical and radiological findings from the treatment of RCC, as well as clinical breast examinations, breast ultrasound, mammography, CT findings of the chest, abdomen, and pelvis, and histopathological findings, were used for writing this case report. The patient gave her written consent to publish the data. An 82-year-old female patient underwent complete diagnostics due to the appearance of haematuria, and it was determined that she had a primary tumour of the right kidney. No distant metastases on other organs were observed. The oncology MDT suggested a radical right nephrectomy. According to the American Joint Committee on Cancer (AJCC), 7th edition, the disease was Stage I, T1bN0M0. She had regular urological follow ups until August 2021, when she felt the lump in her right breast and consulted a doctor. Clinical breast examinations detected a 2 cm movable toward the base, vaguely limited, rough-surfaced lump, without clear-cut borders at the junction of the upper quadrants of the right breast. No other palpable tumour lesions were observed in the rest of the right breast or in the left breast. The axillary lymph nodes were not detected. Mammography was performed and showed a mass with well-defined borders in the right breast at the 12-1 o'clock position, 4 cm from the nipple . Suspicious breast calcifications were not detected, and the axillae were without significant lymph nodes. Skin and subcutaneous tissue were without signs of infiltration. Ultrasound of the right breast was performed, showing an oval lobulated lesion of 19 x 18 mm at the upper quadrants with strong vascularisation, with no posterior acoustic phenomena . A 14-gauge core needle biopsy was performed under local anaesthesia, and three samples of the tumour mass were obtained. The examined material contained a section of breast tissue, with fibroadipose tissue permeated with tubular pseudoglandular arrangements, round and polygonal cells, distinctly bright cytoplasm, and uniform centrally placed nuclei with a pronounced capillary network between them. Immunohistochemically, tumour cells express EMA, AE1/3, CD10, and vimentin, and a moderate amount of CK7, but do not express ER, PR, or HER2. Proliferation index Ki-67 expression was 10%. Histopathologically and immunohistochemically, the tumour was metastatic RCC. A multislice CT scan of the chest, abdomen, and pelvis w performed when staging the cancer diagnosis. This showed a hypervascular tumour 2.2 x 2.4 cm, whereas the axillae were without significant lymph nodes . There were no signs of right kidney cancer recurrence, and the other findings of the chest, abdomen, and pelvis showed no signs of disease. The patient was presented to a urology and breast multidisciplinary team, and a metastasectomy was suggested. A partial right breast resection and a histological and immunohistochemical analysis of the sample were performed . Histopathologically, the tumour was without desmoplastic stroma and mainly comprised solid-type alveolar arrangements of large moderately polymorphic cells, bright and abundant cytoplasm, and round vesicular cores with focally prominent nuclei . Immunohistochemically, the tumour cells were diffusely positive for CD10, EMA (epithelial membrane antigen), and vimentin, and negative for CK7, TTF-1, renal cell antigen, and E-cadherin with positive and negative, and internal and external controls for the given antibodies . The aforementioned was a histological confirmation of metastatic renal cell carcinoma. Due to a normal postoperative course, the patient was discharged on the third postoperative day. The postoperative multidisciplinary team suggested regular follow-up appointments. After 17 months, no new signs of underlying disease spread were observed. The patient felt well, with a good (ECOG = 1) performance status. 3. Discussion Breast metastases from extramammary primary malignancies are rare where both breasts are equally involved, with uncommon bilateral involvement. In about 85% of cases, solitary lesions are found in the breast . Breast metastases, although rare, mainly originate from melanoma, lymphoma, and leukaemia, as well as the ovaries, lungs, and stomach . Disease manifestation can vary, showing signs and symptoms of primary neoplasms and/or metastatic breast lesions, and is influenced by dissemination . Clinical practice and the literature show that renal cell carcinoma can metastasize to any part of the body, but unusual metastatic sites, as described in published case reports, are the ovaries, pancreas, spleen, and head and neck organs, such as the thyroid . Routine surveillance imaging does not include the aforementioned organs, which delays the identification of metastatic disease, resulting in patients being diagnosed after exhibiting symptoms . The recurrence risk for RCC is highest after treatment, with median relapse time within the first two years, but many case reports, especially those with unusual metastatic sites, have reported disease recurrence after more than a decade after initial surgical treatment, where surveillance imaging does not provide for recommendations , as in our case, with recurrence 11 years after nephrectomy. Similar data are shown in other published cases that report RCC metastasis in the breast. The route for metastatic tumour cells includes the right ventricle of heart, then through the inferior vena cava, passing into the pulmonary circulation, and eventually the breast. The paravertebral venous plexus can be involved in cancer cell transport as well . Regardless of the primary malignancy source, the presence of breast metastasis is associated with a poor outcome, with a mean survival of 10.9 months . The available published case reports (PubMed database) show that all patients reported in the cases were women. Clinically, breast metastatic lesions, unlike primary tumours, do not involve the skin, nor do they cause nipple retraction or discharge , whereas the involvement of an axillary lymph node is variable . Primary breast malignancies in most of the cases have spiculated lesions, microcalcifications, architectural distortion, and asymmetrical density , while radiographic findings of secondary tumours showed features that are not metastases-specific--nonspiculated lesions, oval-shaped, without calcifications, and well-circumscribed, similar to most benign tumours , except when they have rich blood flow, as is in this case, where lesions show increased vascularity. In most of the cases, clear cell RCC is hypervascular , due to a defective von Hippel Lindau (VHL) tumour suppressor gene causing the upregulation the vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF), which promote survival, tumour cell growth, and angiogenesis . Pathology analysis of our case showed metastatic RCC, initially treated with radical nephrectomy 11 years prior to the current presentation, whereas immunohistochemistry revealed positive staining for CD10, EMA, and vimentin, which was consistent with the diagnosis of RCC , and negative for CK7, TTF-1, renal cell antigen, and E-cadherin. Metastatic disease in RCC, regardless of the site, can occur in synchronous form--at the time of the primary cancer diagnosis, or metachronous form (as in the presented case), when metastases are diagnosed in a later follow up . In the Republic of Serbia, according to real-world data, there were 1155 newly diagnosed patients per year (in 2020), with a 5-year prevalence of 35.56 per 100,000 people with RCC . However, this is first recorded case of isolated, metachronous RCC in breast tissue in Serbia. Patients with synchronous metastasis have a worse cancer-specific survival rate compared with that for the metachronous pattern of the disease . Early suspicion and disease recurrence confirmation are fundamental for future therapeutic management. The standard diagnostic battery of tests includes, as described in our case, breast imaging and radiographic body evaluation to determine whether the disease is oligoprogressive. Over the past years, due to an increase in patients with breast masses requiring precise and individual diagnostics testing, the necessity for advanced diagnostics has arisen. Breast tomosynthesis overcomes the limitations of mammography due to the ability to provide three-dimensional information (using a lower dose), enhancing the early detection of breast neoplasms. Dedicated breast computed tomography (DBCT) is a technique comparable to magnetic resonance imaging (MRI); however, it is possible to perform it without breast compression, and it is not limited by breast density or implants. Breast MRI is recommended in diagnosis, staging, and at follow up, using diffusion-weighted imaging (DWI). Utilising apparent diffusion coefficient (ADC) values, this shows a promising ability to characterise breast lesions . The use of artificial intelligence (AI), such as algorithms and computational models, may represent a future answer to challenges in precise breast lesion diagnosis and facilitate treatment decisions. Due to the rare occurrence of this case presentation, no accepted consensus or specific management treatment guidelines exist. Metastatic breast RCC is treated following principles that are used to treat other metastatic lesions , whereby surgical resection may be a recommended management option for patients with isolated metastases, especially in those with a prolonged metachronous disease-free interval . In less than 15% of cases, a complete response to nonsurgical treatment was achieved, while surgical treatment is associated with better overall survival . Patients with metastatic RCC have a poor prognosis, and their overall survival differs in the literature, but without considering treatment modalities, patients had a median survival of 5.9 months (for the high-risk group) to 50.6 months (for the low-risk group) , while our patient continued to undergo regular follow-up examinations and showed no evidence of disease 17 months after the diagnosis of the metastatic disease. There has been a rapid expansion of therapeutic options for these patients in the last few years, in particular targeted immunotherapy, with CR rates suggesting that it may be possible to achieve a survival benefit . Furthermore, stereotactic ablative radiotherapy is safe and efficacious for RCC oligometastases, with a high one-year local control (at 90%) and can be recommended as an option in selected cases , either alone or in combination with systemic treatment . 4. Conclusions Metastases of primary tumours in the breast are extremely rare. Due to the rare occurrence of this case presentation, there are no studies with larger patient numbers and randomised therapeutic options or any accepted consensus or specific treatment guidelines, despite rapidly growing therapeutic options for these patients over the last decade. It is necessary to consider this rare localisation of metastases, especially in patients with a history of treated malignancies of other primary localisations. To establish the correct diagnosis of a metastatic tumour in the breast, core needle biopsy, pathohistological analysis, and immunohistochemical analysis are mandatory because they indicate the type and origin of the metastatic tumour in the breast so that adequate modalities of systemic or surgical treatment can be applied. Author Contributions M.S. (Marko Spasic) was the patient's surgeon, manuscript drafting. N.N., D.Z. and M.S. (Marija Sekulic) design and critical review. B.M., B.S. and D.V. contributed to manuscript drafting. M.M. and S.M. reviewed the literature. F.M. and N.M. were responsible for the revision of the manuscript for important intellectual content. All authors contributed to the article and approved the submitted version. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The publication of Case Reports does not require our Institutional Review Board approval. Informed Consent Statement Written informed consent was obtained from the patient to publish this paper. Data Availability Statement Not applicable. Conflicts of Interest The authors declare no conflict of interest. Figure 1 The cranial caudal (CC) view and the mediolateral oblique (MLO) view showing a mass with well-defined borders in the right breast, without suspicious breast calcifications or axillar lymph nodes. Figure 2 Ultrasound of the right breast showing an oval lobulated lesion of 19 x 18 mm with strong vascularisation, without posterior acoustic phenomena (A); multislice CT scan of the chest showing the right breast's hypervascular tumour of 2.2 x 2.4 cm, whereas the axillae were without significant lymph nodes (B). Figure 3 Macroscopic findings showing the tumour in the right breast with clear boundaries, yellow brown in colour, and 18 mm in diameter. Figure 4 Histological examination, haematoxylin and eosin staining (HE), and immunohistochemical staining with magnification x200. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. References 1. Peired A.J. Campi R. Angelotti M.L. Antonelli G. Conte C. Lazzeri E. Becherucci F. Calistri L. Serni S. Romagnani P. Sex and Gender Differences in Kidney Cancer: Clinical and Experimental Evidence Cancers 2021 13 4588 10.3390/cancers13184588 34572815 2. Padala S.A. Barsouk A. Thandra K.C. Kalyan S. Mohammed A. Vakiti A. Rawla P. Barsouk A. Epidemiology of renal cell carcinoma World J. Oncol. 2020 11 79 87 10.14740/wjon1279 32494314 3. Christensen M. Hannan R. 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PMC10000769 | Diagnostics (Basel) Diagnostics (Basel) diagnostics Diagnostics 2075-4418 MDPI 10.3390/diagnostics13050882 diagnostics-13-00882 Article The Adaptive Force as a Potential Biomechanical Parameter in the Recovery Process of Patients with Long COVID Schaefer Laura V. 123* Bittmann Frank N. 23 Rodriguez Bill Academic Editor Dipalma Gianna Academic Editor 1 Health Education in Sports, Department of Sports and Health Sciences, University of Potsdam, 14476 Potsdam, Germany 2 Regulative Physiology and Prevention, Department of Sports and Health Sciences, University of Potsdam, 14476 Potsdam, Germany 3 Practice for Integrative Medicine Bittmann, 14467 Potsdam, Germany * Correspondence: [email protected] 25 2 2023 3 2023 13 5 88219 11 2022 20 2 2023 23 2 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). Long COVID patients show symptoms, such as fatigue, muscle weakness and pain. Adequate diagnostics are still lacking. Investigating muscle function might be a beneficial approach. The holding capacity (maximal isometric Adaptive Force; AFisomax) was previously suggested to be especially sensitive for impairments. This longitudinal, non-clinical study aimed to investigate the AF in long COVID patients and their recovery process. AF parameters of elbow and hip flexors were assessed in 17 patients at three time points (pre: long COVID state, post: immediately after first treatment, end: recovery) by an objectified manual muscle test. The tester applied an increasing force on the limb of the patient, who had to resist isometrically for as long as possible. The intensity of 13 common symptoms were queried. At pre, patients started to lengthen their muscles at ~50% of the maximal AF (AFmax), which was then reached during eccentric motion, indicating unstable adaptation. At post and end, AFisomax increased significantly to ~99% and 100% of AFmax, respectively, reflecting stable adaptation. AFmax was statistically similar for all three time points. Symptom intensity decreased significantly from pre to end. The findings revealed a substantially impaired maximal holding capacity in long COVID patients, which returned to normal function with substantial health improvement. AFisomax might be a suitable sensitive functional parameter to assess long COVID patients and to support therapy process. Adaptive Force maximal isometric Adaptive Force holding capacity muscle function long COVID post COVID syndrome muscle weakness fatigue neuromuscular control biomechanical parameter Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)491466077 The publication was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)-Project number 491466077. pmc1. Introduction Long term sequelae of SARS-CoV-2 infections increasingly challenge medical, social and economic systems worldwide. Different terms are used to define persisting post-infectious symptoms, such as 'long COVID', 'post-COVID syndrome', 'post-acute COVID' or 'persistent post-COVID', mostly depending on the duration of symptoms after acute infection. For simplification, the term 'long COVID' will be used in the following for patients suffering from symptoms at least 4 weeks after acute infection. Reports on the amount of patients with at least one persistent symptom after SARS-CoV-2 infection range from 10% to 57%, or even up to 87% in hospitalized patients, depending on the time span after acute infection or hospitalization vs. non-hospitalization . Long COVID occurs in 10% to 35% of non-hospitalized patients , which is most important, since only 5% to 7% of patients are hospitalized . Current data show a lower rate of long COVID after infection with omicron variants than with delta (4.5% vs. 10.8%) . Infection severity is considered to not be a major factor for the development of long COVID . According to 'COVID-19 data Explorer' from Johns Hopkins University, more than 570 million SARS-CoV-2 cases were confirmed worldwide (Europe, Asia, North America, South America, Africa, Australia) from 22 January 2020 to 28 July 2022. Assuming that 10% of them develop long term sequelae, more than 57 million people suffer or suffered from long COVID. The socioeconomic relevance becomes clear. The medical community is mainly describing the characteristics of long COVID, but the pathomechanisms or causality are not sufficiently known . Furthermore, there is a lack of diagnostic and therapeutic approaches, which are urgently needed to intercept the large amount of sick leave . Post-infectious syndromes have been studied for decades and they are known to emerge after different viral infections . They partly result in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) , which is closely connected to long COVID. Symptoms of long COVID range from fatigue, tiredness, muscle weakness, joint/muscle pain, cognitive impairments ('brain fog'), depression, anxiety, dyspnoea, chest pain/tightness, cough, loss of taste/smell, headache, cardiac symptoms, insomnia, diarrhoea and more . As can be seen, different systems are involved, including the respiratory, cardiovascular, musculoskeletal, integumentary, gastrointestinal, endocrine and neurological systems . A dysfunction of the autonomous nervous system (ANS) has been discussed as a cause for the symptoms . However, the diagnosis of post-infectious syndromes is difficult and is usually based on a diagnosis of exclusion . Patients frequently report that they are not taken seriously by their doctors , which even increases the helplessness and anxiety. A possible supportive diagnostic approach could be to investigate the neuromuscular system since muscle weakness and musculoskeletal pain occur frequently in long COVID patients. Some researchers examined the maximal voluntary isometric contraction (MVIC, e.g., hand grip force) in patients with post-infectious syndromes . Two studies reported non-significant differences between patients and controls regarding the MVIC of the quadriceps femoris muscle (90deg knee flexion) or of elbow flexors (90deg elbow flexion, maximal supination forearm), respectively . Two further studies revealed a significantly reduced hand grip force in ME/CFS . However, in Meeus et al., gender effects were not considered . Females were overrepresented in ME/CFS patients vs. controls (96% vs. 62%) , which might explain the lower strength. The findings are inconclusive and highlight that common maximal strength assessments might not be that appropriate to investigate muscle dysfunction in post-infectious states. The Adaptive Force (AF) was inaugurated as a special neuromuscular function, which was found to be sensitive to stimuli . The AF characterizes the capacity of the neuromuscular system to adapt to external varying forces in an isometric holding manner . It can be assessed by a technical device using pneumatics or by an objectified manual muscle test (MMT) using a handheld device which measures dynamics and kinematics during the MMT . For the latter, it was shown that the maximal isometric AF (AFisomax; maximal holding capacity) was significantly reduced in reaction to negative stimuli, such as unpleasant emotional imagery or odors in healthy participants . AFisomax immediately decreased by perceiving the negative stimulus and switched back instantaneously to baseline values by applying the positive stimulus. The peak value (maximal AF; AFmax) was reached during the subsequent eccentric action and was similarly high as for the baseline and positive stimuli. For baseline or under positive stimuli, the muscles remained stable during the whole force increase up to AFmax (AFisomax >= 99% of AFmax). Thereby, AFisomax was similar to AFmax of unstable muscles. Hence, the maximal force was not influenced by the stimuli but the isometric holding function. In other words, under disturbing influences, the isometric holding capacity broke down to a significantly low level but the maximal strength was not affected. This was interpreted as a high sensitivity of AFisomax with respect to possibly impairing stimuli . Neurophysiological explanations were given previously. This longitudinal study aimed to investigate the AF in patients with long COVID in a non-clinical setting. For that, AF parameters were assessed at three time points in the course of long COVID: (1) in the long COVID state (pre), (2) after the first treatment (post) and (3) with substantial health improvement (recovery; end). The individual treatments received were not part of the study. It was not a clinical trial; therefore, it was not aimed to measure treatment efficacy. However, the treatments were queried and described to gain an impression of potentially helpful approaches without any claim of evidence. Based on the current scientific knowledge of AF and of therapeutical experience, the main hypotheses were (1) the holding capacity would be significantly reduced in patients with long COVID and then it would stabilize, thus increase, during the recovery process. (2) AFmax would show no significant differences between the time points. (3) AF at onset of oscillations (AFosc) would be significantly higher in long COVID state compared to post and end. The study provides early data on AF in long COVID patients. If the hypotheses are positively verified, AF might be used as a supportive biomechanical parameter to examine patients with long COVID. Furthermore, AF could help to find the appropriate treatment approach, which will be explained and discussed. 2. Materials and Methods This longitudinal non-clinical study investigated patients in a long COVID state and in the course of their recovery process. Patients were not approached directly. They consulted the practice for Integrative Medicine (Potsdam, Germany; complementary medicine) out of their own personal initiative. If they were diagnosed with post-COVID syndrome or long COVID, they were invited to participate in the study. Regardless of their response, AF data were measured anyway for diagnostic purposes in daily practice. The treatments were neither subject nor part of the investigation. We only aimed to investigate the AF in those patients and its behavior during the recovery process. Therefore, a control group was not included. The measurements took place at the practice of Integrative Medicine and were conducted by researchers from the University of Potsdam (Potsdam, Germany). 2.1. Patients Until July 2022, 37 patients diagnosed with long COVID attended the above-mentioned practice for consultation and the AF was measured initially using a handheld device. The only inclusion criterion was the medical diagnosis 'post-COVID-syndrome' or 'long COVID', which was received from medical doctors before the patients visited the practice. Exclusion criteria were pre-existing complaints of arm, shoulder, hip or knee of the measured side. Seventeen patients were included in this study since they reported a substantially improved or regained health state by July 2022. The remaining 20 patients were still in therapy or cancelled further therapy because of various reasons (long distance between home and the practice, difficulties in finding appointments, other ongoing treatments/rehabilitation or unknown reasons). Of the 17 included patients, 14 were female (age: 44.43 +- 14.78 yrs., body height: 168.75 +- 5.23 cm, body mass: 69.93 +- 13.18 kg) and three were male (49.00 +- 7.94 yrs., 187.5 +- 3.54 cm, 94.75 +- 0.35 kg). Further information is given in the Results section (intensity of acute infection, duration from acute SARS-CoV-2 infection to input measurement, symptoms and others). The study was conducted according to the Declaration of Helsinki and permission from the local ethics committee of the University of Potsdam (Germany) was given (no. 70/2021, date: 16 February 2022). Each participant gave written informed consent. 2.2. Questionnaires The patients filled out two questionnaires. The first one assessed information with respect to acute SARS-CoV-2 infection: duration, medical diagnosis and examination, symptoms and degree of severity (0 = symptom free, 1 = mild, 2 = moderate, 3 = severe but without hospitalization, 4 = hospitalization without intensive care, 5 = hospitalization with intensive care without invasive ventilation, 6 = intensive care with invasive ventilation); as well as concerning long COVID state: period between acute infection and onset of long COVID, periods of improvement, symptoms, diagnosis, medical examinations, experiences with health care, treatments and their effects. The second questionnaire queried the intensity of common symptoms during long COVID using a scale from 0 (no) to 10 (very strong). The assessed symptoms were fatigue, breathing difficulties, cough, chest pain, chest tightness, memory/concentration problems, headache, muscle pain, fast/strong heartbeat, loss of smell/taste, depression/anxiety, fever, dizziness and post-exertion malaise. Professional and personal stress levels were also queried. The questionnaire was filled out for the following time points: (1) retrospectively for the pre-COVID baseline (before acute SARS-CoV-2 infection), (2) in long COVID state (time of input measurement; pre), (3) 1 day after first treatment (post) and (4) after recovery/with substantial health improvement (output measurement; end). 2.3. Handheld Device to Measure the Adaptive Force The AF of the hip and elbow flexors of one side was assessed by the objectified MMT using the handheld device which was used in previous studies . . It records force and position simultaneously and has proven to be reliable and valid . Strain gauges (co. sourcing map, model: a14071900ux0076, precision: 1.0 +- 0.1%, sensitivity: 0.3 mV/V) and kinematic sensor technology (Bosch BNO055, 9-axis absolute orientation sensor, sensitivity: +-1%) are implemented inside the device. The reaction force between tester and the patient's limb, as well as the linear accelerations and angular velocity were captured during the MMT. The sampling rate was 180 Hz. The data were buffered, A/D converted and sent via Bluetooth 5.0 to a tablet. An app (Sticky Notes, comp.: StatConsult) saved the transmitted data . 2.4. Manual Muscle Test to Assess the Adaptive Force: Procedure and Setting For testing the AF, the MMT in the form of a 'break test' was performed , since it enables a flexible and time-saving approach. This is especially necessary in fatigued long COVID patients. The MMT aims to assess the patient's neuromuscular capacity to adapt to an external force increase. It does not test the maximal strength of the patient in the sense of MVIC. MMT characteristics were described previously . The starting positions of MMT of elbow and hip flexors, including the application of the handheld device, are illustrated in Figure 1c,d (according to ). The patient laid supine. The starting position for the elbow flexor test was 90deg elbow flexion and maximal supination of the forearm . For the hip flexor test, hip and knee angles were ~90deg . The contact with the handheld device was at the distal forearm or thigh, respectively. The contact points were marked and the lever was measured from the lateral epicondyle of the humerus and trochanter major, respectively, to the respective contact point for the standardization of retests. The tester applied a smoothly increasing force on the participant's limb in the direction of muscle lengthening until a considerably high force level was reached. The patient had the task to maintain the starting position in an isometric holding manner for as long as possible. The patient is supposed to react and adapt to the applied force, but the patient was not allowed to push against the tester (for explanation see ). The whole MMT lasted ~4 s. The MMTs were rated subjectively by the tester: 'unstable': the muscle started to lengthen during the force increase, hence, the patient was not able to maintain the isometric position. In that case, the maximal holding capacity (AFisomax) was lower than AFmax, which was then reached during eccentric muscle action. 'Stable': the patient was able to maintain the isometric position until an oscillating force equilibrium occurred at a considerably high force level; in that case, the maximal AF (AFmax) was reached under isometric conditions (AFmax = AFisomax). Healthy persons usually show such stable adaptation (AFisomaxAFmax >= 99%) . 'Unclear': the muscle was neither completely stable nor unstable; slight suspensions were present. A reproducible force application is a necessary precondition for valid data. Experienced testers are able to perform reliable force profiles over time . Both testers (female, 36 years, 168 cm, 55 kg, 9 yrs. of MMT experience; male, 65 years, 185 cm, 87 kg, 26 yrs. of MMT experience) who assessed the AF of the patients in the present study, had previously proven their ability to test reproducibly . Moreover, the force profiles over time matched precisely between both testers . 2.5. Procedure At the first appointment, the patient was examined by means of the MMT by one of the two testers. This tester also conducted all subsequent MMTs of the same patient. Four muscle groups of the lower and upper extremities on both sides were assessed manually (without handheld device), respectively, to obtain an overall impression of the neuromuscular functionality. Then, the input measurements (pre) were performed: AF of hip and elbow flexors of one side was recorded utilizing the handheld device for objectification during the MMT. Patients chose the side to measure, in case of complaints, the complaint-free side was used. Both muscle groups were measured consecutively in alternating order three times, each starting with hip flexors (~1 min resting period between trials). The subjective assessment of the performed MMT by the tester was noted (0 = unstable; 1 = stable, 2 = unclear). Subsequently, the patients received their individual treatment which was not part of the study. Following this treatment (~1 h after input measurements), the AF of hip and elbow flexors was measured again (post) according to the procedure of the pre-measurements. A treatment period of varying duration and number of treatments were prescribed for each patient. During this phase the patients received their individual treatments, which they would have received anyway regardless of the study. The patients were prompted to contact the tester as soon as they felt substantially better or recovered. Then, a final appointment was scheduled for the end measurements (end), that followed the same measuring procedure as for the pre/post measurements. It should be emphasized that no treatment was given at the final appointment prior to the end measurements. 2.6. Data Processing and Statistical Analyses Data processing and evaluation were performed according to Schaefer et al. in NI DIAdem 2020 (National Instruments, Austin, TX, USA). The recorded data (force and gyrometer signals) were transferred from the measuring app to NI DIAdem. They were interpolated (1 kHz) and filtered (Butterworth, filter degree 5, cut-off frequency 20 Hz). For visualization proposed, the angular velocity was additionally filtered (degree: 3, cut-off: 10 Hz) to smoothen the oscillations. The following AF parameters were captured for further evaluation:Maximal Adaptive Force (AFmax): AFmax (N) refers to the peak value of a trial. This could have been reached either during isometric or eccentric muscle action. 2. Maximal isometric Adaptive Force (AFisomax): AFisomax stands for the highest force value under isometric conditions. It was defined as the force at the moment in which the gyrometer signal increased above zero (breaking point). This indicated a yielding of the limb and, accordingly, muscle lengthening. If the gyrometer signal oscillated around zero during the entire trial, AFmax = AFisomax. Thus, the muscle length stayed stable during the whole MMT until the peak force value was reached (stable MMT). If the muscle started to lengthen in the course of MMT, AFisomax was reached during the force increase prior to the peak value. This points out that the position of the limb has to be considered to assess AFisomax. In 1 of 256 trials, AFisomax could not be determined because of peculiarities in the curve shape (excluded from evaluation). The ratio of AFisomax to AFmax (%) was additionally calculated per trial. 3. Adaptive Force at the moment of onset of oscillations (AFosc): AFosc (N) characterizes the force at the moment in which oscillations start to appear regularly (onset of oscillations). Previous studies showed that both interacting partners develop an oscillating force equilibrium, especially during stable MMTs. This was indicated by oscillations which arose in the force signal mostly in phase 3 of MMT (linear increase) showing a clearly distinguishable regular oscillatory behavior. During unstable MMTs, this oscillatory up swing was missing or occurred attenuated on a considerably higher force level. To evaluate AFosc, the force signal was checked for oscillations (force maxima) appearing sequentially during the force increase. If four maxima with a time distance dx < 0.15 s appeared consecutively, the force value of the first maximum was defined as AFosc. Time delta dx < 0.15 s was chosen due to the knowledge that mechanical muscle oscillations occur ~10 Hz . In case no such oscillatory onset occurred, AFosc = AFmax. In 2 of 256 trials, AFosc could not be clearly determined, hence, they were excluded from evaluation. Ratios of AFosc to AFmax (%), as well as AFosc to AFisomax (%) were calculated per trial. The latter is based on previous findings that for stable MMTs, AFosc arose on a lower level than AFisomax, and for unstable MMTs, oscillations occurred--if at all--after that breaking point. 4. Slope of force rise: The slope of force increase prior to the breaking point (AFisomax) of all trials was evaluated to control the force application by the tester. This has to be similar between the trials for a valid comparison. The difference quotient m = y2-y1x2-x1 was used to calculate the slope, whereby x refers to time and y to the respective force values. The reference points (time, force) were 70% and 100% of the averaged AFisomax of all of the assessed unstable MMTs of one patient. The decadic logarithm was taken from the slope values (lg(N/s)) since the force rise was exponential. In 11 of 256 trials, the slope could not be determined since oscillations occurred too intensively which would have distorted the slope value. Arithmetic means (M), standard deviation (SD), coefficient of variation (CV) and 95% confidence intervals (CIs) were calculated per parameter, muscle and time point (pre, post, end) in Microsoft Excel (Microsoft 365, Redmond, WA, USA, Microsoft Corp). Statistical evaluation was performed with IBM SPSS Statistics 28 (Windows, Armonk, NY, USA, IBM Corp). All parameters (AFmax, AFisomax, AFosc, their ratios and slope) were checked for normal distribution by a Shapiro-Wilk test. In case of normal distribution, a repeated measures ANOVA (RM ANOVA) was used to compare the three time points (pre, post, end). In case the Mauchly test of sphericity was significant, the Greenhouse-Geisser correction was chosen (FG). For post-hoc tests, a Bonferroni correction was applied (adjusted p values are given by padj). Effect size eta squared (e2) was given for the RM ANOVA. For pairwise comparisons, the effect size Cohen's dz was used, which was interpreted as small (0.2), moderate (0.5), large (0.80) or very large (1.3) . Since the RM ANOVA is known to be robust against violation of the normal distribution , a Friedman test was only executed to compare the three time points if more than one dataset (pre, post or end) was not normally distributed (applied for the ratio of AFisomax to AFmax). The Bonferroni post-hoc test was used for pairwise comparisons (padj) and the effect size Pearson's r was calculated by r = zn in Microsoft Excel. Significance level was a = 0.05. In addition to the AF parameters, the intensities of the different queried symptoms were evaluated by calculating M and SD. Those values were also compared between the three time points using a Friedman test. Furthermore, the percentage of patients who stated their respective symptoms with an intensity of at least 2 was calculated for descriptive purposes. 3. Results 3.1. Number of Trials and Subjective MMT Ratings by the Testers The hip flexors were measured in all 17 patients at the three time points (pre, post, end). The measurements of the elbow flexors were only completed in 14 patients due to reasons such as lack of time, shoulder complaints, too exhausted or similar. In total, 144 MMTs were performed using the handheld device for hip flexors and 118 for elbow flexors. One patient was only tested twice for both muscles because of a lack of time. In two other patients, hip and elbow flexors were only measured twice at pre and post because of tiredness. The hip flexors of another patient were assessed only once at end because of hip pain. In total, 141 valid trials were gathered for the evaluation of hip flexors and 115 for elbow flexors, since technical issues occurred in six trials (3x hip, 3x elbow). In one patient, four trials of hip flexors were performed at the end because the device indicated an error, but the data were nevertheless transferred and, therefore, used for evaluation. The female tester assessed ten patients and the male tester assessed seven. The number of MMTs rated as 'unstable', 'stable' and 'unclear' by the testers is given in Table 1. As can be seen, all MMTs (100%) were rated as 'unstable' at pre. This reflects that elbow and hip flexors started to lengthen during the force increase, thus, the patients were not able to adapt their muscle length and force adequately in an isometric position during the external force increase. At post and end, the majority of MMTs were rated as 'stable' for both muscles (elbow: 92.1% and 97.6%, respectively; hip: 89.4% and 95.9%, respectively). This indicates that the patients were able to adapt to the external force increase in an isometric holding position in the vast majority of MMTs, and the muscles did not yield during the force rise. In total, six MMTs were rated as 'unclear' (elbow: 2.6% at post; hip: 6.4% at post, 4.1% at end). This highlights that the MMTs could not be rated as completely stable. The testers mostly described that the muscle showed stronger suspensions than usual for stable MMTs or that the muscle started to yield at a very high force level (especially in comparison to the pre trials). Those subjective ratings should be verified using the data from the handheld device. 3.2. Parameters of Adaptive Force in the Course of Long COVID Figure 2 exemplifies the force and gyrometer signals of three MMTs of elbow and hip flexors, respectively, of one female patient (24 years, 168 cm, 65 kg) tested by the male tester at the pre, post and end time points. As can be seen in the uppermost graphs of Figure 2, force rises at pre, post and end can be regarded as similar except for one curve at pre. The single values of each parameter and patient are provided in the Supplementary Materials. Table 2 displays the group averages and statistical results. 3.2.1. Slope of Force Increase Slope was similar for elbow and hip flexors and did not differ significantly between the three time points, neither for elbow, nor for hip flexors. For the latter, the RM ANOVA was close to significant. The lowest slope was present for pre. Thus, at post and end, the challenge for patients to adapt to the external load can be assumed as even higher. The slope can be interpreted as statistically similar between the time points, which is the prerequisite for comparison of the AF parameters. 3.2.2. Maximal Adaptive Force and Maximal Isometric Adaptive Force The AFmax did not differ significantly between the three time points for both muscles . As can be seen in Table 2, AFmax was considerably high in the pre measurements. One female patient (outlier) showed an extremely low AFmax in the pre trials, with only 61.43 +- 4.86 N for elbow and 67.38 +- 8.66 N for hip flexors. At post, she could increase her AFmax immediately to 146.44 +- 22.05 N for elbow and to 162.58 +- 26.01 N for hip flexors. In her case, the AFmax at pre amounted to only 42% of AFmax at post for elbow and 41% for hip flexors, respectively. This is usually not expected and will be discussed later. The other patients showed AFmax values between 145.83 and 295.05 N for elbow and 124.10 and 257.27 N for hip flexors. For timepoints post and end the AFmax was considerably high for all patients . The AFmax at pre related to post amounted averagely 100 +- 14% for elbow and 106 +- 24% for hip flexors, respectively (excl. outlier). Similar for post vs. end with 100 +- 10% for elbow and 102 +- 15% for hip, respectively. Hence, AFmax seems to not be appropriate to reflect the testers' MMT ratings adequately, which showed clear differences between pre and post and pre and end, as well as similar ratings between post and end (Table 1). It has to be mentioned that for all pre trials, AFmax was reached during muscle lengthening, whereby for the majority of the post and end trials, AFmax arose during isometric muscle action. Therefore, the suggested main parameter to quantify the manually found difference is the maximal force during the isometric muscle action (holding capacity; AFisomax). The AFisomax showed clearly lower values at pre vs. post/end, with a significant main effect in the RM ANOVA . The pairwise comparisons revealed significantly lower values for pre vs. post (elbow: t(13) = -11.144, padj < 0.0001, d = 2.978; hip: t(16) = -10.228, padj < 0.0001, d = 2.481; one-tailed) and for pre vs. end (elbow: t(13) = -12.140, padj < 0.0001, d = 3.245; hip: t(16) = -10.007, padj < 0.0001, d = 2.427, one-tailed). Post vs. end did not differ significantly (elbow: padj = 1.000; hip: padj = 1.000). For elbow flexors, four patients showed an AFisomax below 60 N at pre (range 20.56 to 58.66 N), which has to be considered as extremely low. Hereby, the outlier mentioned above, showed the lowest value. Another patient showed a very high AFisomax = 229.94 N. The others ranged from 62.21 to 156.30 N. All patients showed considerably high AFisomax values at post and end . For hip flexors, three patients showed AFisomax < 60 N at pre (range: 27.36 to 52.00 N), whereby the mentioned outlier again showed the lowest value. The highest AFisomax for pre was 166.73 N, which was reached by the same patient who showed the highest value for elbow flexors. At post and end, AFisomax was considerably high for all patients. This is mirrored by the ratio of AFisomax to AFmax . For elbow flexors, it ranged from 23% to 78% at pre, 89% to 100% at post and 97% to 100% at end; for hip flexors, it ranged from 28% to 69% at pre, 87% to 100% at post and 98% to 100% at end. The patients started to lengthen their muscles already at 47 +- 16% of their maximal force (AFmax) for elbow flexors and at 49 +- 12% for hip flexors in the pre trials. Some patients showed an extremely low ratio in single MMTs. The lowest ratios were 14% for elbow and 15% for hip flexors. In 15 of 36 MMTs (elbow) and 13 of 46 MMTs (hip), the ratio amounted less than 40%. In 11 and 13 MMTs, respectively, it was >60%. The others were in-between 40% and 60%. At post, already 12 of 14 patients were able to generate at least 98% of AFmax for elbow flexors, two patients reached lower values (89% and 96%). It was similar for hip flexors, whereby 14 of 17 patients were able to demand at least 98% of AFmax, three showed lower values (87%, 93% and 97%). In all end trials, the patients were able to reach 100% of AFmax in an isometric holding position, except for two patients who showed values of ~97% or ~98% for elbow flexors and one who reached ~98% for hip flexors. 3.2.3. Onset of Oscillations during Force Increase The AFosc revealed a significant main effect in the RM ANOVA for both muscles (Table 2). For elbow flexors, oscillations started at an 18% and 17% higher force level comparing pre vs. post and pre vs. end, respectively. The pairwise comparisons missed significance after the Bonferroni correction . For hip flexors, oscillations occurred at a 43% and 52% higher force level comparing pre vs. post and pre vs. end, respectively. Pairwise comparisons were highly significant (pre vs. post: t(16) = 5.997, padj < 0.0001, d = 1.454; pre vs. end: t(16) = 5.892, padj < 0.0001, d = 1.429). Post vs. end did not differ significantly (padj = 1.000) . The above-mentioned outlier regarding AFmax showed an extremely low AFosc for both muscles at pre with 60.84 (elbow) and 66.48 N (hip). For elbow flexors, AFosc was 100.94 N at post and 100.90 N at end. For hip flexors AFosc was similarly low comparing pre (66.48 N), post (62.88 N) and end (69.89 N). The other patients showed AFosc of elbow flexors in the range of 145.69 to 272.70 N at pre, 62.98 to 217.69 N at post and 52.00 to 236.84 N at end. For hip flexors, it ranged from 116.90 to 242.17 N at pre, from 62.88 to 188.86 N at post and from 29.86 to 191.76 N at end. The between-patients CV for pre, post and end was large with ~31 +- 2% for elbow and ~32 +- 6% for hip flexors. The intraindividual CV was considerably lower with 5.8 +- 2.8% (pre), 12.1 +- 8.3% (post) and 12.8 +- 5.3% (end) for elbow flexors and 6.1 +- 4.1%, 12.3 +- 7.03% and 16.5 +- 15.1%, respectively, for hip flexors. The ratio of AFosc to AFmax was clearly and significantly higher at pre vs. post and pre vs. end for both muscles (elbow: pre vs. post: t(13) = 5.455, padj < 0.0001, d = 1.458; pre vs. end: t(13) = 5.863, padj < 0.0001, d = 1.567; hip: pre vs. post: t(16) = 8.306, padj < 0.0001, d = 2.014; pre vs. end: t(16) = 9.876, padj < 0.0001, d = 2.395). No significant differences were present comparing post vs. end. Displayed by the ratio of AFosc to AFisomax, the oscillations arose consistently after the breaking point (AFisomax) at pre. At post and end, they occurred before AFisomax . Only in one MMT of elbow flexors at post, the oscillations arose just with AFisomax (AFosc/AFisomax = 100%). The RM ANOVA revealed a significant main effect for both muscles (Table 2). Pairwise comparisons were highly significant for pre vs. post (elbow: t(13) = 5.918, padj < 0.0001, d = 1.582; hip: t(16) = 8.905, padj < 0.0001, d = 2.160) and pre vs. end (elbow: t(13) = 5.892, padj < 0.0001, d = 1.575; hip: t(17) = 8.979, padj < 0.0001, d = 2.178). Post vs. end showed no significant differences (padj = 1.000 for both muscles). 3.3. Patients Characteristics Regarding Long COVID The patients' professions were teachers/educators (6) students/trainees (2), IT specialist (1), editor (1), lawyer (1), filmmaker (1), social insurance clerk (1), physiotherapist (1), business economist (1), manager of a coronavirus test center (1) and pensioner (1). From the 16 employed patients, 14 were unable to work because of long COVID at the first appointment (pre), one had just started to work again and one had no sick leave at all. At timepoint end, eight of the 14 incapacitated patients were working again and six wanted to return to work again soon. One was still on sick leave. The acute SARS-CoV-infection lasted on average 15.29 +- 9.40 days (range: 7 to 40, n = 17). The median of acute infection severity was 2.25 (n = 16). One patient was admitted to hospital due to vertigo, another because of a suspected heart attack (nevertheless, she rated the intensity with 1). Overall, infections could be interpreted as mild. The duration from acute infection to input measurement (pre) was on average 274.88 +- 210.70 days (range: 32 to 688). From pre to end, the duration amounted to 71.06 +- 44.43 days (range: 26 to 166 days). The patients had on average 3.29 +- 1.79 (range: 1 to 7, n = 17) treatment appointments at the practice from pre to end. At end, four patients were completely recovered and required no further appointments. Thirteen patients reported to feel sustainably better but wanted to receive further treatments. For 10 of those 13 patients the therapy phase was completed after an average of 2.80 +- 1.99 further treatments. Three patients were still in therapy (July 2022), since they had not regained their full quality of life back or they wanted to stabilize their health further, especially with regard to mental stability. The patients reported a large variety of symptoms in the long COVID state, which were not all regarded in the questionnaire. Beyond the queried symptoms, the patients reported recurrent 'crashes', (muscle) weakness, joint stiffness, limb heaviness, feeling of whole body paralysis, brain fog to black outs, aphasia, forgetfulness, slowed reaction, sensitivity to stimuli, such as light/noise, hypersomnia or sleeping problems, vertigo, nausea, diarrhea, sore throat, ague, strong sweating, impaired vision, olfactory hallucination, body aches (back/shoulder/neck/heart/lung/tooth/eyes), tingles in the nerves/limbs/head/tongue, cold hands/feet, increased convulsion tendency, internal vibrating, inner restlessness, being phlegmatic, high level of irritability, fast overload, mental imbalance, depression, tachycardia, extrasystoles, hair loss, eczema, herpes, reactivated Epstein-Barr virus infection and tinnitus. Figure 6a illustrates the percentage of patients who stated the respective symptoms with an intensity of at least 2. As can be seen, no patient reported to have chest pain/tightness, cough, dizziness, loss of smell/taste or fever with such an intensity before COVID. However, the majority of symptoms was already present in some patients--at least slightly--before COVID infection (7.14% to 35.71%, n = 14). Depression/anxiety showed the highest percentage before COVID. This is in line with the statements regarding job-related and personal stress before COVID (Table 3), which were rated with an intensity of >=2 in 90.91% and 100%, respectively (n = 11). Those values declined in long COVID state to 85.71% (n = 14, three patients did not check the boxes because of sick leave) and 88.24% (n = 17), respectively; at timepoint end, they amounted to 30.33% and 50.00% (n = 12), respectively. Absolute symptom intensities are displayed in Table 3 and Figure 6b. The Friedman test was significant for each symptom. From 'before COVID' to 'long COVID state', the intensities increased significantly for each symptom (p < 0.001 to 0.024), except for fever (p = 0.202). In long COVID state, all patients reported to suffer from fatigue, post-exertion malaise and breathing difficulties with an intensity of at least 1. Most prominent were post-exertion-malaise and fatigue with an intensity of 8.1 and 7.8, respectively. The other symptoms did not occur in each patient, whereby fever was the rarest (four patients). Comparing 'long COVID state' vs. end, the intensity of all symptoms declined, most of them significantly (p < 0.001 to 0.031), except for fever (p = 0.281) and loss of smell/taste (p = 0.062) . The symptom intensities did not differ significantly between 'before COVID' and end (p = 0.202 to 0.922), whereby for concentration/memory problems, significance was just missed with p = 0.050. The latter was still present in 11 of 13 patients (three of them stated an intensity of 1, one patient of 9). Another patient stated that an elevated temperature was partly still present at timepoint end when he was physically active (rated fever with intensity 3); after one further treatment this was resolved, too. Although the individual treatments were not part of the evaluation, they should be reported briefly. Fifteen of 17 patients filled out the questionnaire with respect to their diagnosis and therapies prior to the first appointment. At least seven patients had large diagnostic assessments in centers or rehabilitation facilities for long COVID, the others stated to have received assessments from medical specialists (pulmonologists, internists and similar). The initiated treatments ranged from rehabilitation measures, such as physiotherapy including lymph drainage, manual therapy/massage, reflective breathing therapy, hot role and exercise therapy to (hyperbaric) oxygen therapy, ergotherapy, psychotherapy or psychological guidance, behavioral therapy, speech therapy, concentration training, pharmacological approaches (antibiotics, cortisone, asthma inhaler) to dietary changes. The most common advice from medical specialists was 'pacing'. According to the patients' statements, this was very frustrating. Four patients stated that they received no advice or arranged therapy from medical specialists. They were told to rest or were not taken seriously. The majority of patients reported having the impression that conventional medicine had no treatment approach and some reported that medical specialists were 'clueless'. Others stated that as soon as no somatic reason for their condition could be found, the patients were diagnosed with a psychosomatic disorder. Nevertheless, some patients reported positive effects regarding reflective breathing therapy and for psychological guidance to cope with the condition. Regarding physiotherapeutic measures, the effects varied widely. Some patients reported at least a supporting effect regarding manual therapy which helped to reduce some pain in the short-term. Others stated that lymph drainage worsened the condition. Some also stated that exercise therapy helped for their musculature and cardiovascular system, however, others reported a deterioration after low-intensity exercise therapy. The mentioned helplessness and--if at all--mostly short-term supportive therapies led the patients to try interventions on their own initiative. Those included supplements (mostly vitamins, trace elements), walks, relaxation techniques or meditation, planning of daily routine, concentration training and rest. At least two patients went to naturopaths or specialists in traditional Chinese medicine. However, none of those measures led to the desirable improvement of their health condition. That is why the patients were seeking other approaches and made an appointment at the practice, where the AF measurements took place. Two of the 17 patients were transferred from a pulmonologist, the others came via other ways. The patients were still partly undergoing therapies elsewhere. The additive treatment at the practice for integrative medicine involved an individual approach based on the muscular holding capacity. Some regularities regarding the applied treatments were found. For each patient, an individualized pulsed electromagnetic field therapy (PEMF) was applied. Based on several studies , an influence on the ANS is assumed. For a single case, the PEMF therapy in long COVID was recently described . Moreover, 11 of 17 patients were treated for mental stress (persisting or post traumatic situations) using an individualized treatment approach. The lymphatic system was treated in seven of 17 patients using manual methods, as well as individualized PEMF. In some cases, osteopathic and chiropractic techniques for the cranial and/or the musculoskeletal system were applied. 4. Discussion The present pilot study investigated the AF of elbow and hip flexors via an objectified MMT in patients with long COVID at three time points: during long COVID state (pre), after the first treatment (post) and after recovery/substantial health improvement (end). The additionally received individual treatments of the patients were not part of the study and were only included descriptively. The evaluation of the slope of applied force rise by the tester revealed a non-significant difference between the three time points. Therefore, the results are based on reproducible force increases and can be regarded as valid. The results supported the hypotheses and will be discussed with regard to different physiological and practical aspects. 4.1. Comparison of the Subjective Ratings of the Manual Muscle Test and Measured AF The MMT was comprehensibly criticized to be subjective. The applied force increase as well as the ratings of MMTs are based on the manual ability and 'feeling' of the tester. By measuring the dynamics and kinematics during the MTT, the force increase and breaking point can be objectified. The question remains whether the measured AF parameters support the subjective MMT ratings of the tester. Since the results of elbow and hip flexors showed similar characteristics, they will be considered together. All 84 MMTs at pre were assessed as 'unstable' by the testers. The MMTs at post and end were rated as 'stable' in the majority of trials (164 of 173), as 'unstable' in four of 173 cases and as 'unclear' in six of 173 cases. Regarding MMTs rated as either 'unstable', 'stable' or 'unclear', the ratio of AFisomax to AFmax amounted to 50.27 +- 13.15%, 99.69 +- 0.64% or 97.95 +- 2.61%, respectively. It can be concluded that the testers' MMT ratings were in accordance with the measured AF. Under unstable conditions, AFisomax was only half as high as for the stable tests. The AF values rated as unclear in the MMTs were rather in accordance with the stable ones. Obviously, they showed a high AFisomax. However, during the MMT, the testers felt higher suspensions and the muscle resistance felt 'softer'. The values of stable MMTs support the previous findings, in which the ratio of AFisomax to AFmax was >=99% . Unstable MMTs previously revealed values of ~56%, which is slightly higher than the ~50% found here. This might be attributable to the fact that the previous studies were performed on healthy participants who were affected temporarily by unpleasant odors or imagery. Unhealthy individuals with long COVID seem to show--at least in part--even stronger impairment of muscular adaptation. Some patients showed extremely low AFisomax values; the lowest was 15% for AFmax for hip flexors and 14% for elbow flexors. This is interpreted as a--partly extremely--impaired muscular adaptation, probably due to the long COVID state. However, it cannot be stated whether their muscular adaptation was already impaired before SARS-CoV-2 infection. Based on the findings, it can be concluded that the MMT ratings of both experienced testers were strongly in accordance with the measured AF values. Therefore, AF measured by the objectified MMT seems to be a suitable biomechanical parameter to evaluate the muscular function in adaptation to an external increasing force. 4.2. Adaptive Force in the Recovery Process of Long COVID Fatigue is considered as the main symptom of long COVID . The link between fatigue and muscle weakness has already been raised previously . That is why maximal strength is partly investigated in post-infectious syndromes or ME/CFS. As was mentioned in the introduction, the findings have been inconclusive until now . In the presented study, AFmax did not differ significantly between the three time points, as was assumed. Since AFmax was previously found to be similar to MVIC , the assumption that maximal forces (eccentric/MVIC) might not be suitable parameters to investigate patients in post-infectious states is supported. However, one outlier existed here, who showed extremely low AFmax values at pre. This could be a hint that some individuals suffering from post-infectious syndromes or ME/CFS may also have significantly reduced common maximal strengths, as was found in . Nevertheless, the results of AFmax can also explain, why other investigations did not find such differences . The findings for AFisomax suggest that the holding capacity seems to be a more sensitive biomechanical parameter to assess muscle function. AFisomax was significantly lower with very large effect sizes for pre vs. post and pre vs. end, whereby post vs. end did not differ significantly, according to the hypothesis. In the long COVID state (pre), the patients were not able to maintain an isometric position while trying to adapt to the increasing applied force. Muscles gave way at less than half of the maximal AF. Hence, patients could not exert their maximal strengths at this stage. This was further supported by the ratio of AFisomax to AFmax, which was significantly reduced at pre. As the main result of the study, AFisomax turned out to be a sensitive parameter for a long COVID state, because 100% of the patients showed initially clear instability (this was also the case for the other 20 patients measured in long COVID state, but who were not included in the study). To the authors' knowledge, only one study assessed muscle strength in SARS-CoV-2 patients . MVIC was measured directly at the discharge of elderly hospitalized patients. Thereby, 73% and 86% of patients showed a 'weakness' for biceps brachii and quadriceps femoris muscles, respectively. Muscle weakness was defined as strength which "was inferior to 80% of the predicted normal value" based on Andrews et al. . However, those patients are not comparable with those included in the study, since measurements were executed at the end of acute infection following a period of hospitalization (averagely 20.7 days). More than 90% received oxygen supply and all of them were pharmacologically treated. Jakel et al. reported a sensitivity of ~70% and ~86% for maximal hand grip strength in CFS/ME patients aged 20-39 years and 50-59 years, respectively (prior to the COVID-19 pandemic) . AFisomax of all long COVID patients responded immediately following the treatment at the first appointment with a clear and significant increase. This instant change leads to the assumption that AFisomax does not reflect the maximal strength capacity but a functional aspect of motor control that can be influenced by stimuli. It can switch immediately from instability to stability or vice versa. This was shown in previous studies involving healthy participants . The health condition of the long COVID patients in this study was not improved directly after the first treatment (except for one patient), but the motor control already clearly responded. It is hypothesized that the motor reaction could have been a first hint at a helpful intervention, at least in a share of subjects. The actual causality remains unclear. There could have been helpful treatment methods, but also possible mental factors, such as an empathetic atmosphere or the like. The significant differences between pre and end revealed that the holding capacity was not only substantially improved, but even fully normalized until recovery. This result has to be discussed independently of the possible causations of the improvement. Because the study was non-clinical, no control group was included. Therefore, the reasons for improvement of health conditions and AF parameters remain unclear. Considering the queried symptoms, it was visible that they behave inversely proportional to the holding capacity: at pre, the symptom intensity was significantly higher in most items compared to timepoint end (p < 0.001 to 0.031; except for fever (p = 0.281) and loss of smell/taste (p = 0.062)), whereby AFisomax and the ratio of AFisomax to AFmax was significantly lower at pre vs. end with large effect sizes of > 2.42. This indicates an inverse correlation of the health condition and holding capacity. The directionality and causation of this connection can only be assumed. Since the holding capacity was improved already directly after the first treatment (post), the holding capacity cannot be a direct indicator for the improvement in health. Moreover, it remains unclear whether that observed instant improvement was sustainable. It seems to be likely that the motor response was a more transitional phenomenon at the beginning. MMTs at following treatment appointments showed a fallback to muscular instability for the most patients; however, this was not verified by objective measures. Because the output-measurements (end) were not carried out after an immediately preceding treatment, the observed stability could be interpreted as a part of the improved health state. We assume that the holding capacity is regained prior to the decrease of symptom intensity. Hence, after suitable treatments, the functionality is first restored. A probable improvement in the health condition is time-delayed and might possibly depend on the sustainability of this regained functionality, mirrored by the stable muscle function. 4.3. Neurophysiological Considerations with Respect to the Reaction of AF in Long COVID The discussion on the etiology of long COVID should not be opened here in detail. Brain stem dysfunction , a reduced cerebral blood flow and the involvement of the ANS were discussed. Recently, preinfection psychological distress was reported as a risk factor for long COVID . This is in line with the self-reported stress prior to acute infection regarding the patients in the present study. Central structures, such as the brain stem, thalamus, basal ganglia, cerebellum, inferior olivary nucleus, cingulate cortex and more are involved in processing and controlling nociception, emotions and motor control . Hence, the influence of possibly interfering inputs in the complex control circuitries of motor function are conceivable. The adaptive holding capacity in reaction to an external increasing force was suggested to be especially vulnerable regarding such stimuli. The length-tension control with respect to an increasing external load challenges the regulation and control processes of motor control in a specific way (for detailed discussion see ). Therefore, it is conceivable that a health state, such as long COVID, can influence the holding capacity. Based on the findings of previous studies on the influence of emotions on AF in healthy participants and on long-term practical experience that mental stress can reduce the holding capacity, we assume that the motor output in the sense of AF could have been impaired already prior to SARS-CoV-2 infection because of mental stress. This might have affected the functionality of the human system on different levels. Especially an impairment of the immune system is known to be associated with mental distress . Hence, the individually perceived mental stress could have diminished the resilience of the individual with regard to the virus and, probably, could have impeded the recovery of the acute infection, resulting in long COVID. Wang et al. highlighted that the findings that psychological distress is a risk factor for long COVID "should not be misinterpreted as supporting a hypothesis that post-COVID-19 conditions are psychosomatic". We concur with this statement. From our point of view, mental stress might lead to disbalances of different bodily systems, e.g., the immune system or the ANS . This, in turn, could lower the resilience and might favor long COVID. We interpret the long COVID state rather as a sign of dysfunction. The found instability of the holding muscle function might be a part of the complex physiological functional disbalance in long COVID patients. The onset of oscillation (AFosc) might also reflect an impaired functionality. The neuromuscular system is known to be characterized by oscillations. AFosc was significantly higher for pre vs. post and pre vs. end, as was hypothesized. Moreover, in all of the 84 MMTs of elbow and hip flexors at pre (rated as unstable), oscillations arose--if at all--after the breaking point, thus during muscle lengthening. For the remaining 173 MMTs at post and end (mostly rated as stable), the up-swing of oscillations arose regularly during isometric actions. Those findings support the previous ones that, in case of stability, oscillations occur during isometric muscle action; in case of instability, they do not arise. This indicates that oscillations might be a prerequisite for the stable adaptation in the sense of AF, as was suggested previously . The evidence consolidates that oscillations are playing a major role in the neuromuscular adaptation with respect to external forces. Based on the connection of physiological disbalances and motor control, the AF might be a suitable biomechanical parameter to check for such functional impairments. Due to the immediate response of the holding capacity to supporting or disrupting inputs, the recovery process of long COVID could also be controlled, and a potentially supportive therapy approach might be ascertained by assessing the holding capacity. 4.4. Limitations One limitation was the non-standardized duration from post to end measurements. Due to the individual recovery process, this limitation is difficult to resolve. The duration depended on the self-reported health state of the patients. This self-report is another limitation. Further studies could include a more quantitative assessment of the health state. However, the individual feeling of health is the most important one, also for return to work. Furthermore, the study was not blinded. The testers were aware of the patients' health state. However, the evaluation of the slope and AFmax revealed statistically similar values between the three time points. Only AFisomax, as well as AFosc, showed significant differences between pre vs. post and pre vs. end. This strongly indicates that the AF assessment was not influenced by lack of blinding. 5. Conclusions The investigation of the AF in patients with long COVID and in the course of their recovery process revealed that the holding capability was significantly reduced in long COVID state and was stabilized after the first treatment and with substantial health improvement. AFmax did not reflect this difference. The holding capacity seems to be sensitive but is assumed to be not specific for long COVID. Nonetheless, its assessment might support the diagnostics of long COVID and especially the choice of the individual helpful therapy approach, since the holding function can switch immediately from instability to stability. This should be used to identify a treatment tailored to the patient's individual conditions and requirements. It is concluded that the assessment of AFisomax could be a supportive biomechanical parameter to assess the functional health state, follow up and recovery process in patients with long COVID. The next step should be to investigate the mentioned treatment approaches in a clinical design. Based on the present study, it cannot be judged whether the treatments were the reason for the recovery. Possibly, other received treatments or a spontaneous recovery over time could have led to the improved health state. In case the treatment approaches are verified positively, this would be a big step towards diagnostics and therapy with regard to long COVID. This would have major socioeconomic implications. Acknowledgments We acknowledge the student assistant who participated in evaluating the AF measurements. Furthermore, we like to thank all patients for their interest and participation in the study. Supplementary Materials The following supporting information can be downloaded at: Table S1: Patient characteristics, COVID specific durations and number of treatments; Table S2: Intensity of symptoms; Table S3: Ratings of the manual muscle tests by the tester; Table S4: Maximal adaptive force of elbow flexors; Table S5: Maximal isometric adaptive force of elbow flexors; Table S6: Adaptive force at onset of oscillations of elbow flexors; Table S7: Slope of force increase for elbow flexors; Table S8: Maximal adaptive force of hip flexors; Table S9: Maximal isometric adaptive force of hip flexors; Table S10: Adaptive force at onset of oscillations of hip flexors; Table S11: Slope of force increase for hip flexors. Click here for additional data file. Author Contributions Conceptualization, F.N.B. and L.V.S.; methodology, F.N.B. and L.V.S.; software, L.V.S.; validation, L.V.S. and F.N.B.; formal analysis, L.V.S.; investigation, L.V.S. and F.N.B.; data curation, L.V.S.; writing--original draft preparation, L.V.S.; writing--review and editing, L.V.S. and F.N.B.; visualization, L.V.S.; supervision, F.N.B.; project administration, F.N.B. and L.V.S. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The study was conducted in accordance with the Declaration of Helsinki and approved by the ethics committee of University of Potsdam, Germany (protocol code 70/2021, 16 February 2022). Informed Consent Statement Informed consent was obtained from all subjects involved in the study. Written informed consent has been obtained from the patients to publish this paper. Data Availability Statement The data presented in this study are available in the article and Supplementary Materials. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Setting. (a) Handheld device; (b) force increase during manual muscle test (MMT), including the decisive phases, as was suggested to be optimal by Bittmann et al. and Schaefer et al. ; starting positions of MMTs of (c) elbow flexors and (d) hip flexors. Figure 2 Exemplary signals of force and angular velocity during AF measurements of one female patient (24 yrs., 168 cm, 65 kg) recorded during three MMTs of elbow (left) and hip flexors (right) at time points pre, post and end (according to Schaefer and Bittmann ). Figure 3 Slope of force increase during the AF assessment. Displayed are arithmetic means, standard deviations (error bars) and 95% CIs of the logarithmic slope (lg(N/s)) of force increase during manual muscle tests for elbow (a) and hip flexors (b) at each time point pre (red), post (grey) and end (blue). RM ANOVA was non-significant, p-values of RM ANOVA are given. Figure 4 Maximal Adaptive Force and maximal isometric Adaptive Force. Displayed are the arithmetic means, standard deviations (error bars) and 95% CIs of the AF parameters of both muscles at each time point: pre (red), post (grey) and end (blue). Elbow flexors: (a) maximal Adaptive Force (AFmax); (b) maximal isometric AF (AFisomax) and (c) ratio of AFisomax to AFmax; hip flexors: (d) AFmax; (e) AFisomax and (f) ratio of AFisomax to AFmax. The adjusted p-values (Bonferroni correction) for the pairwise comparison of the RM ANOVA, as well as of the Friedman test and the respective effect sizes Cohen's d or Pearson's r are given in case of significance. Figure 5 Adaptive Force at onset of oscillations. Displayed are the arithmetic means, standard deviations (error bars) and 95% CIs of AF parameters with regard to the onset of oscillations during MMT for both muscles at each time point: pre (red), post (grey) and end (blue). Elbow flexors: (a) AF at onset of oscillations (AFosc); (b) ratio of AFosc to AFmax and (c) ratio of AFosc to AFisomax; hip flexors: (d) AFosc; (e) ratio of AFosc to AFmax and (f) ratio of AFosc to AFisomax. Adjusted p-values (Bonferroni correction) and effect sizes Cohen's d are given in case of significance. Figure 6 Symptom intensity. (a) Percentage of patients who rated the symptom intensity with >=2. (b) Arithmetic means of the symptom intensity on the scale from 0 (no) to 10 (very strong). Both are given for each symptom and time point: before COVID (grey, n = 14), during long COVID state (red, n = 14, corresponds to pre measurements), and end (blue, n = 13, corresponds to end measurements). diagnostics-13-00882-t001_Table 1 Table 1 Subjective ratings of the manual muscle tests by the testers. The number of MMTs assessed as unstable, stable or as unclear for hip and elbow flexors for each time point is given. Hip Flexors (n = 144) Elbow Flexors (n = 118) MMT Rating pre Post End pre Post End unstable 48 2 0 39 2 1 stable 0 42 47 0 35 40 unclear 0 3 2 0 1 0 diagnostics-13-00882-t002_Table 2 Table 2 Parameters of the Adaptive Force (AF) of elbow and hip flexors. Arithmetic means +- standard deviations (M +- SD), lower and upper borders of 95% confidence intervals (CIs) and values of the RM ANOVA (F-statistics, degrees of freedom (df), significance p and effect size e2) or of the Friedman test (z, df, p, effect size Kendall's W) are given for all AF parameters at each time point (pre: in long COVID state-before treatment, post: directly after treatment, end: recovery) of elbow (n = 14) and hip flexors (n = 17). Parameters: maximal AF (AFmax) (N), maximal isometric AF (AFisomax) (N), ratio AFisomax to AFmax (%), AF at onset of oscillations (AFosc) (N) and the ratios of AFosc to AFmax (%) and AFosc to AFisomax (%), as well as the slope of force rise (lg(N/s)). Parameter Time Point M +- SD 95%-CI F (df1,df2) or z (df) Significance p e2/Kendall's W elbow flexors (n = 14) AFmax (N) pre 177.02 +- 53.47 149.01; 205.03 1.054 (1.43,18.63) a 0.345 - post 184.74 +- 39.02 164.27; 205.15 end 187.87 +- 52.00 160.63; 215.11 AFisomax (N) pre 87.92 +- 54.41 59.42; 116.42 114.772 (2,26) <0.0001 0.898 post 182.26 +- 38.58 162.05; 202.47 end 187.22 +- 52.15 159.90; 214.53 Ratio AFisomax to AFmax (%) pre 46.58 +- 15.91 38.25; 54.91 25.064 (2) b <0.0001 0.895 b post 98.73 +- 3.01 97.15; 100.31 end 99.62 +- 0.96 99.12; 100.13 AFosc (N) pre 170.95 +- 49.17 145.20; 196.71 5.274 (2,26) 0.012 0.289 post 144.54 +- 44.83 121.06; 168.03 end 146.51 +- 48.64 121.04; 171.99 Ratio AFosc to AFmax (%) pre 96.87 +- 2.85 95.38; 98.36 23.403 (2,26) <0.0001 0.643 post 76.95 +- 11.89 70.73; 83.18 end 76.98 +- 11.09 71.17; 82.79 Ratio AFosc to AFisomax (%) pre 258.83 +- 110.74 200.82; 316.84 34.701 (1.02,13.19) a <0.0001 0.727 post 78.06 +- 12.30 71.62; 84.50 end 77.28 +- 10.92 71.55; 83.00 Slope lg(N/s) pre 1.85 +- 0.23 1.73; 1.98 1.282 (2,26) 0.294 - post 1.87 +- 0.18 1.78; 1.97 end 1.90 +- 0.21 1.79; 2.01 hip flexors (n = 17) AFmax (N) pre 174.98 +- 50.03 148.77; 148.77 0.015 (2,32) a 0.952 - post 175.67 +- 40.95 154.22; 197.12 end 174.21 +- 46.78 149.71; 198.72 AFisomax (N) pre 88.30 +- 40.67 66.99; 109.61 88.739 (1.47,23.45) a <0.0001 0.847 post 173.30 +- 41.75 151.43; 195.18 end 174.06 +- 46.80 149.54; 198.58 Ratio AFisomax to AFmax (%) pre 49.25 +- 12.01 42.96; 55.54 32.109 (2) b <0.0001 0.944 b post 98.54 +- 3.44 96.74; 100.35 end 99.91 +- 0.39 99.70; 100.11 AFosc (N) pre 167.10 +- 43.80 144.16; 190.04 27.952 (2,32) <0.0001 0.636 post 116.47 +- 39.63 95.71; 137.23 end 110.06 +- 40.81 88.68; 131.44 Ratio AFosc to AFmax (%) pre 95.19 +- 5.59 92.26; 98.12 53.417 (2,32) <0.0001 0.77 post 65.62 +- 11.56 59.57; 71.68 end 62.01 +- 13.74 54.81; 69.21 Ratio AFosc to AFisomax (%) pre 223.06 +- 69.65 186.57; 259.54 78.199 (1.07,17.11) a <0.0001 0.83 post 66.88 +- 13.18 59.97; 73.78 end 62.07 +- 13.78 54.85; 69.29 Slope lg(N/s) pre 1.85 +- 0.18 1.75; 1.94 3.260 (1.45,21.73) a 0.071 - post 1.93 +- 0.15 1.85; 2.00 end 1.89 +- 0.14 1.81; 1.97 a Greenhouse-Geisser correction was applied for the RM ANOVA. b Friedman test was performed with effect size Kendall's W. Significant results are displayed in bold. diagnostics-13-00882-t003_Table 3 Table 3 Stress level and symptom intensity. Given are the arithmetic means, standard deviations (M +- SD) and range including the number of patients (n) for job-related and personal stress level, as well as for the intensity of the queried common long COVID symptoms (from 0 = no, 10 = very strong) for the different time points before COVID infection (retrospectively), during long COVID state (corresponds to pre) and at recovery/substantial heath improvement (end). The values of the Friedman test comparing the three time points, significance p and effect size Kendall's W are given. The results of the pairwise comparisons are indicated in superscript. Stress M +- SD (Range, n) Before COVID Long COVID State (pre) End Friedman Test Significance p Effect Size Kendall's W Stress level job-related 4.23 +- 2.56 (0-9, n = 11) 5.64 +- 2.95 (0-10, n = 14 *) 2.29 +- 3.17 (0-8, n = 12) 0.667 0.717 - Stress level personal life 3.77 +- 2.70 (2-10, n = 12) 4.76 +- 2.75 (0-10, n = 17) 3.29 +- 3.53 (0-9, n = 12) 4.056 0.132 - Symptoms M +- SD (range) n = 14 n = 14 n = 13 Depression/anxiety 1.43 +- 2.21 (0-8) 3.96 +- 3.78 (0-10) 1.58 +- 2.33 (0-7) 9.389 0.009 1,2 0.361 Fatigue 0.43 +- 0.76 (0-2) 7.75 +- 2.50 (1-10) 2.23 +- 2.67 (0-7.5) 22.217 <0.001 1,2 0.855 Post-exertion malaise 0.57 +- 1.40 (0-5) 8.14 +- 1.96 (3-10) 2.23 +- 3.06 (0-9) 20.311 <0.001 1,2 0.781 Muscle pain 0.29 +- 0.61 (0-2) 5.64 +- 4.27 (0-10) 1.65 +- 2.81 (0-8) 14.800 0.001 1,2 0.569 Chest pain/tightness 0.00 +- 0.00 3.43 +- 3.01 (0-9.5) 0.88 +- 1.23 (0-4) 18.667 <0.001 1,2 0.718 Breathing difficulties 0.29 +- 0.61 (0-2) 4.29 +- 2.37 (1-8) 1.23 +- 1.36 (0-3) 23.106 <0.001 1,2 0.889 Cough 0.14 +- 0.36 (0-1) 2.23 +- 3.00 (0-10) 0.46 +- 0.97 (0-3) 16.267 <0.001 1,2 0.678 Strong/fast heartbeat 0.36 +- 1.08 (0-4) 4.93 +- 4.03 (0-10) 0.69 +- 1.18 (0-3) 17.882 <0.001 1,2 0.688 Concentration/memory problems 0.43 +- 0.76 (0-2) 5.96 +- 3.20 (0-10) 2.88 +- 2.60 (0-9) 23.130 <0.001 1,2 0.890 Dizziness 0.14 +- 0.36 (0-1) 4.71 +- 3.81 (0-10) 0.92 +- 1.98 (0-7) 16.800 <0.001 1,2 0.646 Headache 0.64 +- 1.28 (0-4) 5.29 +- 4.07 (0-10) 0.81 +- 1.28 (0-3) 18.242 <0.001 1,2 0.702 Loss of smell or taste 0.00 +- 0.00 4.39 +- 4.85 (0-10) 1.35 +- 3.16 (0-10) 13.923 0.001 1 0.536 Fever 0.00 +- 0.00 2.14 +- 3.66 (0-10) 0.23 +- 0.83 (0-3) 7.538 0.023 0.290 * Three patients made no statement because of sick leave. 1 The pairwise comparison of 'before COVID' vs. 'long COVID state' was significant: p < 0.05. 2 The pairwise comparison of 'long COVID state' vs. end was significant: p < 0.05. 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PMC10000770 | Cells Cells cells Cells 2073-4409 MDPI 10.3390/cells12050743 cells-12-00743 Article Multiparametric Profiling of Neutrophil Function via a High-Throughput Flow Cytometry-Based Assay Timmer Kyle D. Conceptualization Methodology Formal analysis Data curation Writing - original draft Writing - review & editing 1 Floyd Daniel J. Methodology Formal analysis Data curation Writing - original draft Writing - review & editing 1 Scherer Allison K. Methodology Formal analysis Data curation Writing - original draft Writing - review & editing 12 Crossen Arianne J. Methodology Formal analysis Data curation Writing - original draft Writing - review & editing 1 Atallah Johnny Methodology Formal analysis Writing - original draft Writing - review & editing 12 Viens Adam L. Conceptualization Methodology Formal analysis Writing - original draft Writing - review & editing 1 Sykes David B. Conceptualization Methodology Formal analysis Writing - original draft Writing - review & editing 2345 Mansour Michael K. Conceptualization Formal analysis Resources Writing - original draft Writing - review & editing Visualization Supervision Project administration Funding acquisition 123* Prince Lynne Academic Editor Rossi Adriano Academic Editor Hazeldine Jon Academic Editor 1 Department of Medicine, Division of Infectious Diseases, Massachusetts General Hospital, Boston, MA 02114, USA 2 Harvard Medical School, Boston, MA 20114, USA 3 Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA 02114, USA 4 Harvard Stem Cell Institute, Cambridge, MA 02114, USA 5 Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02114, USA * Correspondence: [email protected]; Tel.: +1-617-726-6726 25 2 2023 3 2023 12 5 74319 11 2022 03 2 2023 14 2 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). Neutrophils are a vital component of the innate immune system and play an essential function in the recognition and clearance of bacterial and fungal pathogens. There is great interest in understanding mechanisms of neutrophil dysfunction in the setting of disease and deciphering potential side effects of immunomodulatory drugs on neutrophil function. We developed a high throughput flow cytometry-based assay for detecting changes to four canonical neutrophil functions following biological or chemical triggers. Our assay detects neutrophil phagocytosis, reactive oxygen species (ROS) generation, ectodomain shedding, and secondary granule release in a single reaction mixture. By selecting fluorescent markers with minimal spectral overlap, we merge four detection assays into one microtiter plate-based assay. We demonstrate the response to the fungal pathogen, Candida albicans and validate the assay's dynamic range using the inflammatory cytokines G-CSF, GM-CSF, TNFa, and IFNg. All four cytokines increased ectodomain shedding and phagocytosis to a similar degree while GM-CSF and TNFa were more active in degranulation when compared to IFNg and G-CSF. We further demonstrated the impact of small molecule inhibitors such as kinase inhibition downstream of Dectin-1, a critical lectin receptor responsible for fungal cell wall recognition. Bruton's tyrosine kinase (Btk), Spleen tyrosine kinase (Syk), and Src kinase inhibition suppressed all four measured neutrophil functions but all functions were restored with lipopolysaccharide co-stimulation. This new assay allows for multiple comparisons of effector functions and permits identification of distinct subpopulations of neutrophils with a spectrum of activity. Our assay also offers the potential for studying the intended and off-target effects of immunomodulatory drugs on neutrophil responses. neutrophil function flow cytometry high throughput assay neutrophil augmentation National Institutes of Health/National Institute of Allergy and Infectious DiseasesR01 AI132638 This research was funded by the National Institutes of Health/National Institute of Allergy and Infectious Diseases Grant R01 AI132638 (to M.K.M.). pmc1. Introduction Neutrophils possess a repertoire of functions as the first line of defense in controlling invading pathogens. While the absolute neutrophil count is of obvious importance, the functional capacity of neutrophils to properly execute these functions is also critical for the prevention of disease . Despite having normal or greater than normal neutrophil counts, studies suggest select patient cohorts face an increased risk of infection due to inherited or acquired defects in neutrophil function, including those with diabetes , cirrhosis , chronic granulomatous disease , and recipients of organ transplants . Interrogating neutrophil functions can help to explain why neutrophils may be ineffective in controlling pathogens in these patients. For example, neutrophils from patients with diabetes have decreased reactive oxygen species (ROS) production, chemotaxis, phagocytosis, and neutrophil recruitment. Shedding light on these impairments may explain patients' increased vulnerability to infection . In addition to genetic and metabolic causes, many approved therapeutics can have unintended side effects leading to neutrophil dysfunction. Bruton's tyrosine kinase (Btk) inhibitors, such as ibrutinib, a backbone in treating chronic lymphocytic leukemia , impairs neutrophil function . Patients on treatment with ibrutinib have neutrophils with diminished phagocytosis, ROS generation, and cytokine production, which may explain the increased incidence of invasive aspergillosis in this patient population . Developing an assay that rapidly and efficiently analyzes a diverse array of neutrophil functions could improve our understanding of neutrophil dysfunction in various disease settings and highlight potential off-target effects of medications on neutrophil function. Flow cytometry is commonly used for the concurrent measurement of multiple parameters on large populations of cells. Reactive dyes, fluorescent pathogens, analyte capture beads, and antibody labeling of cell surface proteins allow one to quantify various neutrophil functions rapidly and can be accomplished within hours of blood collection . Flow cytometry-based multifunctional assays have identified aberrant neutrophil activity in specific subpopulations of patients such as those with community-acquired pneumonia , HIV , or severe-injury-related trauma . By quickly identifying patients with neutrophil dysfunction, interventions can be promptly employed to manage at-risk patients. To date, the breadth of measurements in such assays has been limited to two functions: phagocytosis and oxidative burst . Specific protocols increase the study dimensions with parallel assays or downstream secondary analyses of supernatant to measure the release of soluble factors . A high throughput assay that measures multiple functions remains an unmet need for granulocyte phenotyping. We present a microtiter plate assay for simultaneously measuring four neutrophil functions, including phagocytosis, ROS generation, ectodomain shedding, and degranulation. The selected functions are cardinal features of neutrophil response. Ectodomain shedding, an often-overlooked neutrophil function, refers to the cleavage of specific motif-bearing surface proteins by selective proteases, most often of the a disintegrin and metalloproteases (ADAMs) family . These changes to the neutrophil surface (ectodomain) influence various neutrophil activities such as cell-cell adhesion and rolling through the cleavage of L-selectin (CD62L) , opsonin recognition through the cleavage of the Fc receptor CD16b , paracrine signaling through the cleavage of tumor necrosis factor alpha (TNFa) and the TNFa receptors , and triggering neutrophil activation through the cleavage of the receptor TREM-1 . CD62L is rapidly shed within minutes of stimulation and is a classic and early indicator of neutrophil activation . Additionally, CD62L, CD16b, TNFa, and the TNFa receptor are cleaved by the same metalloprotease, ADAM17 . Thus, CD62L is an ideal marker for measuring ectodomain shedding because it acts as a metric for other surface changes and is an early sign of neutrophil activation. Degranulation is another critical function in neutrophil pathogen response. Neutrophils possess granules that release preformed enzymes into the extracellular environment during degranulation . These granules contain antimicrobials such as myeloperoxidase (MPO), lysozyme, lactoferrin, mixed metalloprotease 9, and neutrophil elastase . Granule subsets are categorized based on their contents and are released in a regulated order: secretory vesicles, tertiary, secondary, and finally primary granules . We chose to analyze secondary granule release, through expression of CD66b, since it requires greater activation than secretory vesicles and tertiary granules and because these granules contain multiple antimicrobial peptides such as lactoferrin, neutrophil gelatinase-associated lipocalin (NGAL), and prodefensin . Here, we describe a new high-throughput assay that quantifies four neutrophil functions simultaneously from a single sample. By selecting compatible fluorescence read-outs, we minimize spectral overlap and demonstrate the potential to detect increases and decreases in neutrophil activity following treatment with cytokines, kinase inhibitors, and common immunomodulatory drugs. Our assay provides a convenient workflow for multiple rounds of priming or inhibition, pathogen co-culture, antibody labeling, and data acquisition within the same reaction well. 2. Materials and Methods 2.1. Reagents Flow cytometry staining buffer (FACS buffer) was prepared with 2% heat-inactivated fetal bovine serum (FBS) (Life Technologies, Carlsbad, CA, USA) and 1 mM EDTA (Life Technologies) in phosphate-buffered saline (PBS) without calcium and magnesium (Corning, Corning, New York, NY, USA). Common immunosuppressive drugs were used to demonstrate the ability to test off-target therapeutic effects on neutrophil functions including cyclosporine A (10 mg/mL, Selleckchem, Houston, TX, USA) and mycophenolic acid (30 mM, Selleckchem). Cytokines used included G-CSF (100 ng/mL, Peprotech, Cranbury, NJ, USA), IFNg (100 ng/mL, Peprotech), TNFa (10 ng/mL, Peprotech), and GM-CSF (10 ng/mL, Peprotech). Chemical inhibitors included diphenyleneiodonium for ROS inhibition (DPI, 10 mM, Selleckchem), TMI-005 for inhibition of CD62L shedding (2.5 mM, Cayman Chemical, Ann Arbor, MI, USA), PP1 for Src inhibition (10 mM, Cayman Chemical), R406 for Syk inhibition (20 mM, Selleckchem), and IBT for Btk inhibition (1 mM, Cayman Chemical). Lipopolysaccharide (LPS) from E. coli strain K12 was purchased from InvivoGen (San Diego, CA, USA). Complete RPMI (cRPMI) was prepared from RPMI (Corning), 10% FBS, 2 mM L-glutamine (Life Technologies), and 1% penicillin-streptomycin (Thermo Fisher Scientific, Waltham, MA, USA). 2.2. Preparation of Human Neutrophils Healthy blood donors were consented under the Massachusetts General Hospital Institutional Review Board-approved protocol (2019P002840). Whole blood was collected in EDTA-coated vacutainers (Beckton Dickinson, Franklin Lakes, NJ, USA) and subsequently centrifuged at 1500xg for 15 min. Buffy coat was collected, and neutrophil isolation was performed using the negative selection EasySep Direct Human Neutrophil Isolation Kit, according to the manufacturer's instructions (STEMCELL Technologies, Seattle, WA, USA). Wright-Giemsa staining was performed after the isolation process to confirm neutrophil purity from the isolation kit. Flow cytometry was also used to verify a high neutrophil purity from the isolation procedure (>=94% neutrophil purity). Cell concentration and viability were measured by staining the cells with a 1:10 dilution of acridine orange/propidium iodide followed by automatic cell counting using the LUNA fl Dual Fluorescence Cell Counter (Logos Biosystems, Annandale, VA, USA) (>=99% live). Neutrophils were resuspended in cRPMI at a concentration of 2 x 106 cells/mL. 2.3. Preparation of C. albicans Wildtype strain SC5314 Candida albicans was purchased from the American Type Culture Collection (American Type Culture Collection, Manassas, VA, USA). SC5314 constitutively expressing far-red fluorescent protein (C. albicans iRFP) was kindly donated by Robert Wheeler (University of Maine, Orono, ME) . C. albicans was grown in YPD liquid media (yeast extract, peptone, dextrose) containing 1% yeast extract (Acros Organics, Fair Lawn, NJ, USA), 2% peptone (BD Biosciences, San Jose, CA, USA), and 2% dextrose (Sigma-Aldrich). C. albicans was cultured overnight at 30 degC on a rotating culture wheel (Thermo Fisher Scientific). The following day, C. albicans was removed from the wheel, washed twice with PBS and resuspended in PBS. C. albicans was counted using the LUNA automatic cell counter and kept on ice until the time of the assay. 2.4. Neutrophil-Candida Co-Incubation Drugs, cytokines, or appropriate vehicles were prepared at a 2X concentration in cRPMI, with a maximum DMSO concentration of 0.1% v/v. In a 96-well V bottom polypropylene plate (Corning) the perimeter and outer wells were moated with 200 mL sterile PBS. Next, 50 mL of cRPMI or 50 mL of 2X drug or cytokine was mixed with 50 mL of neutrophil stock in a reaction well. The plate was sealed with breathable film (VWR, Radnor, PA) and placed in an incubator at 37 degC, 5% CO2 for 30 min to one hour. A 30-min cytokine incubation was chosen based on previous time courses. Neutrophils incubated with immunomodulatory drugs for 1 h, as this was the midpoint seen in similar published studies . After the neutrophils were incubated, 20 mL of C. albicans at the desired multiplicity of infection (MOI) was added to the appropriate wells and mixed by pipetting. Immediately following, 30 mL of dihydrorhodamine 123 (DHR123) (5 mM, Thermo Fisher Scientific) was added to the wells and mixed well by pipetting. The plate was sealed and returned to the incubator for 30 min for coincubation with C. albicans. After the 30 min, the plate was placed on ice for 10 min in the dark and prepared for flow cytometry. During rescue studies, neutrophils were first treated with kinase inhibitors for 30 min in an incubator. LPS (400 ng/mL) was then added to the well and the plate was returned to the incubator for an additional 45 min prior to co-culturing neutrophils with C. albicans for 30 min. 2.5. Flow Cytometry Cells were pelleted at 4 degC and stained in 50 mL of cold FACS buffer containing (BV605) anti-human CD62L antibody (1:200 dilution; clone DREG-56; BioLegend, San Diego, CA, USA) and (BV421) anti-CD66b (1:200 dilution; clone 6/40c; BioLegend). The cells were incubated for 30 min at 4 degC in the dark. Cells were rinsed with 150 mL cold FACS buffer, centrifuged at 4 degC and resuspended in 150 mL cold FACS buffer. The 96 well plate was left on ice until just prior to data acquisition on a BD FACSCelesta (BD Biosciences, San Jose, CA, USA) with a blue, violet, red (BVR) laser configuration with specific wavelengths at 488 nm, 405 nm, 640 nm respectively. Bandpass filters for the Celesta include 450/40, 525/50, 610/20, 660/20, 780/60 for the 405 nm laser; 530/30, 575/26, 610/20, 695/40 for the 488 nm laser; and 670/30, 730/45, 780/60 for the 640 nm laser. Before recording data, gates were prepared so that 7000 neutrophil events could be collected. Compensation was performed with single color controls and was calculated using BD FACSDiva Software (BD Biosciences). A compensation matrix demonstrating the spectral overlap values can be seen in Supplementary Table S1. FCS files were exported from BD FACSDiva Software in a 3.0 format. Analysis of FCS files was performed using FlowJo v.10 software (BD Biosciences). T-distributed stochastic neighbor embedding (tSNE) was performed in FlowJo on gated neutrophils and calculated with the four neutrophil function flow cytometer parameters. Heat map statistics for iRFP (phagocytosis), DHR123 (ROS), CD62L expression (shedding), and CD66b expression (degranulation) were then overlayed on tSNE plots. Fluorescence minus one (FMO) controls were performed to ensure minimal spectral overlap. For each FMO control, the multiparametric assay was run equivalently but the flow cytometry preparation excluded one fluorescent marker while retaining the others. Supplementary Table S2 provides further information on the fluorescence reagents and detection. 2.6. Statistical Analysis All statistical analyses for normality and significance were performed on GraphPad Prism 9 (San Diego, CA) using ordinary one-way ANOVA. A p-value greater than 0.05 was considered nonsignificant (ns). 3. Results Neutrophils employ a variety of effector functions to combat pathogens including phagocytosis, oxidative burst, degranulation, cytokine release, ectodomain shedding, and NETosis as illustrated in Figure 1. Here, we demonstrate a simple multiparametric assay to examine four neutrophil functions to Candida albicans challenge. Our assay is performed in a 96 well plate format and requires 1 x 105 cells per well. Neutrophils isolated from healthy donors or patients can be pre-incubated with test biologics or small molecules to assess their influence on neutrophil functions. Following pre-incubation, fluorescent C. albicans are introduced along with DHR123 as an indicator for ROS production. Following co-incubation with C. albicans, neutrophils are immunostained for CD62L and CD66b. CD62L allows for the detection of ectodomain changes and elevation of CD66b serves as a metric for secondary granule release . A stepwise flow cytometric gating strategy identified neutrophils. Total events were gated by forward and side scatter properties to target live cells with the correct size and granularity. This gate removed most free-floating C. albicans and non-granulocytes . It is important to note that this initial gate was generous because activated neutrophils engaging in the various functions tend to increase in size (FSC) and granularity (SSC). Single cells were identified, and neutrophils were selected by their expression of the neutrophil-specific surface protein CD66b . Gates were set by comparing fluorescence minus one control to unstained as well as unstimulated stained samples. Phagocytosing neutrophils were gated as iRFP(+) events, ROS producing neutrophils were gated as DHR123(hi) events, shedding neutrophils were gated as CD62L(-) events, and degranulating neutrophils were gated as CD66b(hi) events. Neutrophil functions were analyzed simultaneously or individually . Increasing the ratio of C. albicans to neutrophils in the assay format increased the frequency of cells responding in terms of ectodomain shedding, phagocytosis, degranulation, and ROS production . The multiplicity of infection (MOI) of 1, 2, 4, and 8 were statistically different in the frequencies of neutrophils engaging in phagocytosis, ROS generation, and secondary granule release , while CD62L shedding plateaued between MOI or 4 and MOI of 8 . Vastly different levels of neutrophil activity were achieved simply by increasing the ratio of C. albicans to neutrophils. Using an MOI of 8 triggered robust neutrophil responses resulting in 77% phagocytosis and 79% secondary granule release compared to 33% and 29%, respectively, at an MOI of 2 . Many neutrophil functional studies have examined phagocytosis and ROS production , while our assay simultaneously measures four canonical neutrophil functions. The multiparametric nature of the assay reveals that not all neutrophils perform the exact coordination of functional responses . For example, shedding and degranulation may occur regardless of whether a neutrophil is phagocytosing and generating ROS. Further, some neutrophils may be performing all four simultaneous functions (population b) while others have not begun any of the measured responses (population a). We additionally, determined the overlap of neutrophils that could participate in more than one function. Figure 3B summarizes the proportion of individual neutrophils engaging in multiple simultaneous functions after co-culture with C. albicans. This presentation of the data reveals relationships between neutrophil functions. For example, there was great overlap between phagocytosing and ROS-generating neutrophils. Most phagocytosing neutrophils also generated ROS; however, only about 60% of degranulating neutrophils were simultaneously phagocytosing C. albicans. Furthermore, our assay allows one to measure the frequency of neutrophils participating in four concurrent functions. At an MOI of 1, on average, 40% of phagocytosing neutrophils participate in all four functions simultaneously compared to 82% at MOI of 4. Each flow cytometer is equipped with multiple lasers and filters that allow different fluorescent panel possibilities. The cytometer configuration is carefully paired with fluorescent markers to minimize spectral overlap and the possibility of false positives. To ensure our multiparametric assay accurately detected individual functions, gates were set using fluorescence minus one (FMO) controls. In the degranulation (BV421), ROS (DHR123), and phagocytosis (iRFP) FMO controls, there were fewer than 0.4% positive events for each function . In the ectodomain shedding (BV605) FMO control, 99.8% of neutrophil events were negative for CD62L expression . Our assay reliably measures individual functions, and results are not confounded by spectral overlap. An essential application of the multiparametric assay is to screen potential bioactive small molecule drugs for neutrophil-modulating effects. We selected a panel of compounds known to influence immune function. These chemicals are inhibitors of ROS production (DPI) , and ectodomain shedding (TMI-005) , as well as two clinically relevant compounds used for immune suppression in transplant patients, mycophenolate (MPA) and cyclosporine A (CSA) . Treatment with compounds for one hour before C. albicans co-culture did not significantly affect neutrophil phagocytic ability. DPI was the only chemical to profoundly affect ROS generation, decreasing the number of ROS-positive cells by 87% . Ectodomain shedding was influenced by many of the small molecule compounds tested. The ADAM17 inhibitor TMI-005, nearly abolished ectodomain shedding. Treatment with MPA did not cause significant impairments to neutrophil functions. CSA however did cause a striking 55% reduction in ectodomain shedding and 45% reduction in secondary granule release. The small molecules tested resulted in unique changes to specific neutrophil behaviors and highlight the value of determining the precise functional area of neutrophil impairment. To demonstrate the ability to measure neutrophil augmentation, we pre-stimulated neutrophils with cytokines, including granulocyte colony-stimulating factor (G-CSF), interferon-gamma (IFNg), granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor-alpha (TNFa). These four inflammatory cytokines significantly increased the frequency of neutrophils engaging in phagocytosis, ROS generation, ectodomain shedding, and secondary granule release relative to the vehicle . The response was nonuniform and cytokine-specific; IFNg elicited the least improvement in neutrophil function. While IFNg increased secondary granule release, the response was less than that of G-CSF, GM-CSF, or TNFa stimulation. Additionally, some of these cytokines began to trigger neutrophil responses in the absence of C. albicans co-culture. TNFa and GM-CSF produced significantly more ectodomain shedding at rest . Interestingly, GM-CSF and TNFa encouraged degranulation in approximately 80% of neutrophils cultured with C. albicans , nearly a sevenfold increase over the vehicle control. TNFa stimulation was unique in that it induced significant secondary granule release in the absence of a pathogen, a result seen in other studies . We also sought to determine how the assay might be used to study the recovery of functional responses in dysfunctional or attenuated neutrophils. Inhibition of downstream kinases (e.g., Btk, Syk, Src) within the Dectin-1 signal transduction pathway can render neutrophils unresponsive to fungal pathogens such as C. albicans or Aspergillus fumigatus . Syk and Btk inhibition can completely block neutrophil functions, including swarming, phagocytosis, oxidative burst, and cytokine production, even when challenged with C. albicans or A. fumigatus . In the context of our assay, the inhibition of Btk, Syk, and Src, prevented phagocytosis, ROS production, ectodomain shedding, and secondary granule release . Neutrophil inhibition by ibrutinib (IBT) or R406 can be overcome by alternate pathway stimulation (e.g., TLR stimulation using LPS) before challenging with C. albicans . LPS stimulation alone did not trigger ROS production in pre-treatment conditions when C. albicans was not present . However, when inhibited neutrophils were stimulated with LPS and challenged with C. albicans, phagocytosis, and ROS production were significantly restored . All LPS-treated neutrophil conditions had near unanimous shedding of CD62L , and secondary granule release was improved with LPS stimulation . These results suggest that the multiparametric assay can measure inhibition, augmentation, and subsequent pathogen challenge in the presence of crucial kinase inhibitors. 4. Discussion While the absolute number of circulating neutrophils is critical for pathogen control, the neutrophil function is another crucial factor that is less easily quantified. Microbial killing assays provide information on the overall ability of neutrophils to recognize and eliminate pathogens, though they do not explain which specific functions contribute to pathogen detection and containment. In studying how specific disease states and small molecules may influence pathogen killing, we wished to quantify multiple neutrophil functions simultaneously. Here, we have demonstrated a simple high-throughput multiparametric flow cytometer-based assay that permits studying four canonical neutrophil functions: phagocytosis, ROS production, ectodomain shedding, and secondary granule release. Furthermore, we demonstrated our assay's clinical relevance and sensitivity in detecting improvements and diminution of neutrophil activities by pre-stimulating neutrophils with biological and small molecule agents. Our study does have limitations. There is growing evidence of neutrophil heterogeneity in peripheral blood , suggesting there may be populations with distinct functional potentials . Our multiparametric assay did not include markers, such as CD10, to identify left shift neutrophils, an immature subset known to behave differently than mature circulating neutrophils . Our study focused on healthy individuals who tend to contain scarce populations of immature neutrophils in circulation . While it is unlikely that a left-shift neutrophil population significantly influenced the results demonstrated here, future work with the multiparametric assay should incorporate heterogeneity markers to fractionate results into additional unique subsets. Our assay is not exhaustive of all neutrophil functions, and future iterations could be expanded to define additional neutrophil responses . The most versatile flow cytometers can be equipped with up to nine lasers and corresponding detectors, allowing for the theoretical recognition of ~40 different parameters. Our assay did not include a measurement of NETosis, a well-defined mechanism for pathogen control. During NETosis, neutrophils ensnare the microbe in a conglomerate of nuclear material laced with high concentrations of antimicrobial proteins . NETosis can be quantified by flow cytometry using cell impermeable nucleic acid dyes such as SYTOX and fluorochrome-conjugated antibodies targeting MPO and/or citrullinated histone 3 . In addition to pathogen elimination, measuring relative neutrophil extracellular trap formation is also essential for understanding potential tissue damage in the host. Excessive NET formation is implicated in ongoing endothelial tissue damage and can contribute to microthromboses . Further adaptations to the panel could also include readouts of other granule types. For example, primary (also known as azurophilic) granule release results in the extracellular expulsion of toxic proteins such as proteinase 3, neutrophil elastase, and MPO . Primary granule release can be measured using fluorochrome conjugated antibodies against CD63 which is not traditionally expressed on the neutrophil surface but is deposited on the cellular membrane upon primary granule fusion . In developing future multiparametric assays to measure neutrophil function, primary granules are of key interest in bacterial pathogenesis. Some pathogens inhibit primary granule release as an immune evasion strategy . Furthermore, the recognition of excessive primary degranulation of neutrophil elastase has been implicated in host tissue damage . Alternatively, secretory granule release can be measured by similarly detecting increases in the surface level expression of CD11b or CD35, though these responses can be non-specifically provoked making them markers of general neutrophil activation . The discarded reaction media from the neutrophil-pathogen co-culture contains informative metabolites and biomolecules that could shed light on specific neutrophil responses including the measurement of cytokines involved in cell-cell communication. Advancements in multiplex cytokine panels could allow for multiple soluble analyte measurements from microtiter volumes, making it compatible with the miniaturized design of our assay. Shed compounds such as CD62L, TREM-1 or the release of factors like G-CSF, or other pro and anti-inflammatory cytokines could be measured by bead capture and quantified by flow cytometry . In our assay, we used live C. albicans marked by the constitutive expression of a fluorescent protein. Enforced expression of fluorescent proteins by pathogens are useful tools however, genetic manipulation may introduce additional changes from the parental strain . In our FMO experiment, lower rates of all functions were observed, when the nonfluorescent parental strain of C. albicans was incubated with neutrophils. This result was obtained on multiple separate occasions and highlights a limitation in utilizing fluorescent protein expressing pathogen. Live fluorescent pathogen culture is not feasible in all lab settings and instead, uncolored pathogens may be conveniently FITC stained or surface labeled using succinimidyl ester-based reactions . Live pathogens may also not be feasible, or approved, for use in cytometer or within every flow cytometry core facility. If so, the quantification of phagocytosis can be adapted to measure the uptake of alternative targets such as fixed or heat-killed samples. Additionally, inert fluorescent bioparticles or fluorescent beads coated with microbial antigens could also substitute the use of live pathogens . The multiparametric assay accurately measures augmented neutrophil functions. Cytokine therapies have been explored for multiple patient diagnoses, such as cirrhosis and cases of neutrophil dysfunction . Cytokine screens in our multiparametric assay could identify the most effective treatments for enhancing or attenuating neutrophil activity for specific diagnoses and therefore increase patient outcomes. Our assay design may also be applied to studies investigating the role of endogenous cytokine levels in tissue-specific microenvironments in the setting of diseases such as cystic fibrosis or cancer . Newly evolving biologic and small molecules are constantly being developed for the treatment of a variety of autoimmune disorders and malignancies. Small molecule kinase inhibitors have revolutionized such treatments; however, they can have off-target effects and can leave the patient vulnerable to invasive fungal disease . We showed that three kinase inhibitors (IBT, R406, and PP1) eliminate neutrophil responses to C. albicans but that function can be restored with cytokines and growth factors. In the development of novel drugs, our assay could be used to detect negative consequences of kinase inhibiting therapeutics on neutrophil function and potential ways to overcome function inhibition. In addition to kinase inhibitors, we tested two FDA-approved drugs, CSA and MPA, that are frequently used to suppress T and B cells in transplant patients. The calcineurin inhibitor, CSA, suppressed innate immune cell function including neutrophil ectodomain shedding and secondary granule release. Other studies have also observed inhibitory effects of CSA on neutrophil function in vitro and in vivo, including reductions in primary granule release, neutrophil migration, and ROS production . Our assay did not detect a decrease in ROS production though it appears that CSA diminishes ROS production in response to some but not all neutrophil-stimulating molecules . This distinction may also reflect the type of ROS detected, specifically those from external release versus internal signaling . As our assay detects functions simultaneously, one can observe changes to coupled mechanisms. Figure 3 shows a substantial overlap between individual cells capable of phagocytosis and ROS production. The strong association between phagocytosis and ROS-producing cells can be explained by the fact that NOX2 is activated upon phagosome formation . When neutrophils were treated with the ROS inhibitor DPI, phagocytosis was relatively unchanged, but ROS was nearly abolished, highlighting the ability to investigate uncoupled functional processes. Our assay yields reproducible results and possesses numerous applications for studying functional deficits. Larger donor enrollment could establish a healthy neutrophil function range from which aberrant neutrophil activity could be identified for clinical applications. We showed that our multiparametric assay could detect function improvement upon pre-incubation with inflammatory cytokines. Our assay may therefore be compatible with studies looking to augment neutrophil responses in patients experiencing neutrophil dysfunction or recurrent opportunistic infections . In conclusion, our multiparametric assay demonstrates the utility of simultaneous measurement of several neutrophil functions that can be used for patient and population cohort immune profiling, assessment of biological or small molecule impact on granulocyte function as well as determination of novel approaches to augmentation of patients with dysfunctional neutrophils for improved health outcomes. The potential addition of metrics such as NETosis and cytokine release can render these assays more informative. Supplementary Materials The following supporting information can be downloaded at: Supplementary Table S1. Compensation matrix with spectral overlap values calculated using BD FACSDiva software; Supplementary Table S2. Fluorescence reagent descriptions. Click here for additional data file. Author Contributions Conceptualization, M.K.M., D.B.S., K.D.T.;Methodology, K.D.T., A.K.S., A.L.V., D.B.S., M.K.M.; Validation, K.D.T., D.J.F., A.K.S., A.J.C.; Formal analysis, K.D.T., D.J.F., A.K.S., A.J.C., J.A. Investigation, K.D.T., D.J.F., A.J.C., A.L.V. Resources, M.K.M. Data curation, K.D.T., D.J.F., A.L.V. Writing-original draft preparation, K.D.T., M.K.M. Writing-review and editing, K.D.T., D.J.F., A.K.S., A.J.C., J.A., A.L.V., D.B.S., M.K.M. Visualization, K.D.T. Supervision, M.K.M. Project administration, M.K.M. Funding acquisition, M.K.M. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement This study was conducted in accordance with the Dec-laration of Helsinki, and approved by the Institutional Review Board of Massachusetts General Hospital (protocol code 2019P002840 approved 16 December 2019). Informed Consent Statement Informed consent was obtained from all subjects involved in the study. Data Availability Statement Access to raw data files is available upon request. Conflicts of Interest M.K.M. reports consultation fees from Vericel, NED Biosystems, GenMark Diagnostics, Safi Biosolutions and Day Zero Diagnostics; grant support from Thermo Fisher Scientific and Genentech; and medical editing/writing fees from UpToDate, outside the submitted work. M.K.M. also reports patents 14/110,443 and 15/999,463 pending. The other authors have no financial conflicts of interest. Figure 1 Measurable neutrophil functions by flow cytometry. Human neutrophils perform a variety of functions in response to pathogen sensing. Beneath each category or subcategory are surface proteins, soluble factors, dyes, or pathogenic insults that can be used to detect an array of neutrophil functions in a multiparametric flow cytometer-based assay. Illustration by Nicole Wolf, MS, (c)2022. ([email protected]) Printed with permission. Figure 2 Multiple neutrophil functions can be measured using a multiparametric flow assay. (A) Schematic representation of assay workflow. Neutrophils are incubated with cytokines or drugs prior to the addition of ROS detection dye DHR123 and co-culture with iRFP expressing fungal pathogen C. albicans. After co-incubation, neutrophils are immunostained for the detection of secondary granule release and ectodomain shedding. Neutrophils were analyzed by flow cytometry for the frequency of functional behaviors. (B) Flow cytometry serial gating strategy for detection of neutrophil functions. Total events were first gated by scatter to target events with the proper size and granularity of neutrophils. Next, doublets were excluded, and finally, neutrophils were selected by gating for the expression of the neutrophil specific surface protein, CD66b (arrows designate sequential analysis). All function gates were set using fluorescence minus one controls, stained, unstimulated samples. (C-F) Percentage of total neutrophils exhibiting phagocytosis (C), ROS generation (D), ectodomain shedding (E), or secondary granule release (F) in response to increasing ratios of C. albicans. Four different multiplicities of infection (MOI), 1, 2, 4, 8 were tested. Data are represented as mean +- SD; n = 3 per group in each experiment per donor; experiments were repeated six times with six different donors. Data were analyzed by one-way ANOVA with a Tukey posttest. * p < 0.03, **** p < 0.0001. Illustration by Nicole Wolf, MS, (c)2022. ([email protected]) Printed with permission. Figure 3 The multiparametric assay allows for the detection of neutrophils performing combinations of simultaneous functions. (A) tSNE projection of neutrophils incubated for 30 min with MOI of 4 C. albicans. Heatmap statistics of each flow cytometry parameter were individually overlayed to visualize low functioning (a) and multifunctioning (b) neutrophil subpopulations. (B) Neutrophils were co-cultured with C. albicans (MOI of 2) for 30 min. Neutrophil populations can be compared for activity in multiple functions. Above, neutrophils are gated on a single-parent function. Subsequently, we show the frequency of neutrophils from the parent function concurrently exhibiting a second function. Each row represents the positive population of neutrophils for a given parent function. Each column represents the mean percentage of neutrophils from the parent gate undergoing an additional function. n = 3 per experiment per donor; experiments were repeated four times with four different donors. Figure 4 Fluorescence minus one (FMO) controls prove no confounding spectral overlap. (A,B) Resting neutrophils (no C.a) and C. albicans challenged neutrophils (w/C.a.) were stained (A) and compared to FMO controls (B), for which one fluorescent marker was removed at a time while the others were held constant. Red box denotes the FMO and the corresponding channel. Figure 5 The multiparametric assay screens for modulation of neutrophil function by therapeutic and inhibitory drugs. Neutrophils were pre-treated with functional inhibitors TMI-005 or diphenyleneiodonium (DPI) or the immunosuppressive drugs cyclosporine A (CSA) or mycophenolate (MPA) for one hour. Neutrophils were then co-cultured with C. albicans (MOI of 3) for 30 min. The frequency of total neutrophils performing phagocytosis, ROS generation, ectodomain shedding, or secondary degranulation was measured. Data are represented as mean +- SD; n = 3 per group in each experiment per donor; experiments were repeated three times with three different donors. Data were analyzed by one-way ANOVA with a Tukey posttest. *** p < 0.0002, **** p < 0.0001, ns = p > 0.05. Figure 6 The multiparametric assay can detect functional augmentation. Neutrophils were pre-treated with pro-inflammatory cytokines G-CSF, IFNg, GM-CSF, TNFa or vehicle control for 30 min to prime the neutrophils for enhanced functional output before coincubation with C. albicans (MOI of 2). The frequency of total neutrophils performing phagocytosis (A), ROS generation (B), ectodomain shedding (C), or secondary degranulation (D) was measured at rest (Rest) and after co-culture (C.a.). Data are represented as mean +- SD; n = 3 per group in each experiment per donor; experiments were repeated three times with three different donors. Data were analyzed by one-way ANOVA with a Tukey posttest. ** p < 0.0021, **** p < 0.0001. Figure 7 Multiparametric assay provides a simple format for measuring recovery of function in chemically inhibited neutrophils. Neutrophils were pre-treated with kinase inhibitors ibrutinib (IBT), R406, PP1 or DMSO vehicle control (Vehicle) for 30 min in the reaction well. Lipopolysaccharide (LPS, LPS + C.a.) or the LPS vehicle (Rest, C.a.) was then spiked into the reaction well and neutrophils incubated for an additional 45 min. Finally, neutrophils were co-cultured with C. albicans (MOI of 4) for 30 min (C.a., LPS + C.a.). The frequency of total neutrophils performing phagocytosis (A), ROS generation (B), ectodomain shedding (C), or secondary degranulation (D) was measured at rest (Rest) and after co-culture (C.a.). Data are represented as mean +- SD; n = 3 per group in each experiment per donor; experiments were repeated two times with two different donors. Data were analyzed by one-way ANOVA with a Tukey posttest. **** p < 0.0001. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). 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PMC10000771 | Background: In recent years, the completeness of ethnicity data in the English cancer registration data has greatly improved. Using these data, this study aims to estimate the influence of ethnicity on survival from primary malignant brain tumours. Methods: Demographic and clinical data on adult patients diagnosed with malignant primary brain tumour from 2012 to 2017 were obtained (n = 24,319). Univariate and multivariate Cox proportional hazards regression analyses were used to estimate hazard ratios (HR) for the survival of the ethnic groups up to one year following diagnosis. Logistic regressions were then used to estimate odds ratios (OR) for different ethnic groups of (1) being diagnosed with pathologically confirmed glioblastoma, (2) being diagnosed through a hospital stay that included an emergency admission, and (3) receiving optimal treatment. Results: After an adjustment for known prognostic factors and factors potentially affecting access to healthcare, patients with an Indian background (HR 0.84, 95% CI 0.72-0.98), Any Other White (HR 0.83, 95% CI 0.76-0.91), Other Ethnic Group (HR 0.70, 95% CI 0.62-0.79), and Unknown/Not Stated Ethnicity (HR 0.81, 95% CI 0.75-0.88) had better one-year survivals than the White British Group. Individuals with Unknown ethnicity are less likely be diagnosed with glioblastoma (OR 0.70, 95% CI 0.58-0.84) and less likely to be diagnosed through a hospital stay that included an emergency admission (OR 0.61, 95% CI 0.53-0.69). Conclusion: The demonstrated ethnic variations associated with better brain tumour survival suggests the need to identify risk or protective factors that may underlie these differences in patient outcomes. brain tumours overall survival ethnicity health inequalities cancer registry The author(s) received no specific funding for this work. pmc1. Background Each year, over 5000 new cases of primary brain tumours are diagnosed in the United Kingdom (UK) . In particular England has become both more multicultural in recent decades and seen a steady increase in the incidence of malignant primary brain tumours . One study considering broad ethnic groups found higher incidence rates of 4.8 per 100,000 population from 2001 to 2007 for people from White ethnic group compared to those from South Asian, Black, and Chinese ethnic groups (respective rates of 3.1, 2.8, and 2.7 per 100,000 population) . English population-based studies have reported ethnic differences in the incidence of most cancers with individuals from non-White groups generally having a lower cancer risk than the White Group . Survival for the four common cancers has been widely reported , but not examined in detail for brain tumours using the well-defined ethnicity information now available. A small study of high-grade gliomas in South-East England 2000-2009 , reported that patients of White and Not Known ethnicities had the worse survival for all tumour groups after adjusting for sex, age, morphology, socio-economic deprivation, and co-morbidity. The improved and detailed National Health Service (NHS) data on ethnicity captured by the National Disease Registration Service, which is part of NHS England, provided an opportunity to explore the impact of ethnicity on brain tumour survival. This resulted from major efforts across the NHS to increase self-reporting of this variable. With data collected on over 300,000 cancer cases in England each year, this is also the first English study to consider the more detailed classifications for malignant primary brain tumours including all gliomas, primary central nervous system lymphoma (PCNSL), as well as unclassified malignant brain neoplasms. It aims to examine the possible effect of ethnicity on the route or pathway taken to diagnosis and of receiving optimal treatment. A better understanding of any ethnic inequalities in brain cancer could potentially lead to improved treatment or services for these patients. 2. Data and Methods 2.1. Study Population Data on all adult patients diagnosed with a malignant primary brain tumour during 2012-2017, who are resident in England and registered with a general practitioner (GP), were extracted from the English cancer registration data. 2.2. Selection of Cases Cases for this study were identified using the International Classification of Diseases [version 10] (ICD-10) tumour site C71. For those with PCNSL, ICD-10 code site was used along with the morphology codes for lymphoma. Other inclusion criteria were cases having a complete tumour registration and known sex. The brain tumour morphological subtypes considered in this study were based on the 2016 WHO Classification of Tumours of the Central Nervous System . WHO updated this classification in 2021; however, the changes have minimal effect on the analysis of this data. Due to sample sizes, histological tumour subtypes were grouped as follows: glioblastoma, anaplastic astrocytoma, astrocytoma NOS, oligodendroglioma, PCNSL, malignant glioma, and unclassified malignant. Data on all tumours were extracted from the English cancer registration irrespective of their pathological confirmation--gliomas without a specified classification or as unclassified malignant neoplasms were included. In addition, glioblastomas with a pathological confirmation were included but tumours of benign, uncertain, and metastatic nature were not included. Molecular data are not available for this study cohort. Inpatient hospital episodes statistics (HES) data were linked to the cancer registration data from 2012. These records include ethnicity data that are almost always self-reported upon admission to NHS hospitals. The categories of ethnicities were as follows: White British, Bangladeshi, Indian, Pakistani, Chinese, Black African, Black Caribbean, and Unknown/Not Stated, and due to small numbers in these groups--White Irish and Any Other White were grouped together as Any Other White, and Mixed Ethnic Groups and Any Other Ethnic Group were grouped as Other Ethnic Group. Socio-economic deprivation was measured using the income domain of the index of multiple deprivation (IMD) 2015, divided into quintiles across England and Wales, and assigned to cases using postcode of residence at diagnosis. Charlson co-morbidity score was based on conditions occurring within one year of the cancer diagnosis date. The conditions were weighted according to their severity and scores were grouped as 0 (where none were recorded), 1, and 2 or more. Route to Diagnosis is defined as the sequence of interactions between the patient and the NHS, leading to a cancer diagnosis . This is identified using an algorithm linking various sources based on the setting of diagnosis, and the pathway and referral route into secondary care. Information on surgical resections, chemotherapy, and radiotherapy treatments received within the first 18 months following diagnosis were also extracted. Treatment options were categorised to reflect clinical practice as: radiotherapy only, chemotherapy only, surgical resection only, all three treatments given as surgical resection followed by radiotherapy and chemotherapy (optimal treatment), radiotherapy plus chemotherapy, surgical resection plus radiotherapy, surgical resection plus chemotherapy, and no treatment. Surgical resections did not include cases with biopsies only. 2.3. Data Analysis We first extracted 27,934 records, cleaning the dataset to exclude duplicated cases, those without the required brain tumour morphology, with unknown vital status, or who were registered by death certificate only (DCO) . Survival time was calculated from the date of diagnosis until date of death with a survival period of up to one year. To retain 145 patients who died on their date of diagnosis, we added half a day to their survival time. The final study population included 24,319 cases. Initially, we examined the distribution of patients by demographic factors (age, sex, area of residence, and socio-economic status), co-morbidity, tumour morphology, route to diagnosis, and treatment factors. Univariate and multivariate Cox proportional hazards regressions were then used to estimate hazard ratios (HR) and their 95% confidence intervals (95% CI) for the survival of each ethnic group up to one year following diagnosis. The follow-up period ended on 31 December 2018. kh2 Tests estimated the p-values for trend and heterogeneity, excluding unknown categories. We then carried out a sensitivity analysis in which each variable was adjusted to identify how much variation it contributed to the model, and as a result we finally focused the analysis on age, sex, co-morbidity, socio-economic deprivation, tumour morphology, route to diagnosis, and treatment received. Due to the high fatality of malignant primary brain tumours, cancer-specific survival was not studied, as this is similar to overall survival. Logistic regression was used to generate odds ratios (OR) (and their 95% CI) for each ethnic group of (1) being diagnosed with pathologically confirmed glioblastoma, (2) being diagnosed during a hospital stay that included an emergency admission, and (3) receiving optimal treatment (surgical resection followed by radiotherapy and chemotherapy). ORs were adjusted for age, sex, socio-economic deprivation, co-morbidity, morphology, route to diagnosis (patient's pathway to diagnosis), and treatment. All analyses were performed using Stata Software, version 16 (StataCorp, TX, USA). 2.4. Ethical Approval Data for this study were collected and analysed under the National Disease Registries Directions 2021, made in accordance with sections 254(1) and 254(6) of the 2012 Health and Social Care Act. Further ethical approval for this study was not required per the definition of research according to the UK Policy Framework for Health and Social Care Research. 3. Results Data from 24,319 patients with a malignant primary brain tumour diagnosed between 2012 and 2017 in England were included. Table 1 displays the distribution of patient, tumour and clinical characteristics, and univariate and mutually adjusted HRs. Brain tumour diagnosis increased with age, peaking at 65-74 years with most patients being men (58.0% n = 14,094). In absolute numbers, it was more frequent in people living in Southeast England, an area that is highly populated and more ethnically diverse. Overall, the most aggressive morphology, glioblastoma, was the most common type (60.7% n = 14,768). The Kaplan-Meier analysis for brain tumour morphology demonstrates glioblastoma as having a very high mortality, followed by malignant glioma (7.0% of all cases, n = 1709) and unclassified malignant tumours (11.1% of all cases, n = 2707) (log-rank test, p < 0.001). Over one half of cases (53.2% n = 12,926) were diagnosed during a hospital stay that included an emergency admission, with most patients receiving either the optimal treatment (23.0% n = 5585), or no treatment (34.9% n = 8483). In the univariate analysis, each of the covariates was correlated with survival. The effects of age, sex, and co-morbidity were attenuated in the mutually adjusted analyses. Almost all patients (95.6%) were recorded as having a known ethnicity. The most common ethnic group representing 85.5% (n = 20,795) of the patients was the White British Group, followed by 4.2% (n = 1018) from the Any Other White Group and 2.8% (n = 674) from Other Ethnic Group. The more specific ethnic groups were less common with 1.3% (n = 321) of patients defining themselves as Indian, 0.8% (n = 186) as Pakistani, and less than 0.4% as Bangladeshi (n = 30), Chinese (n = 37), Black African (n = 84), and Black Caribbean (n = 94) (Table 2). The univariate model for ethnicity showed a survival difference and the mutually adjusted model demonstrated that patients with Other Ethnic Group and Unknown/Not Stated Ethnicity had a 18% and 23% decreased risk of death from any cause, respectively, compared to the White British Group. In a sensitivity analysis, the association of survival with age seemed to disappear in most non-white ethnic groups. This could be explained by the younger age of these groups leading to a lower median age at diagnosis than for the White British population (Table 2). The effect on survival in the Unknown/Not Stated Group was less sensitive to statistical adjustment by age, as the median age was older than for the White British Group. After fully adjusting for age, sex, co-morbidity, socio-economic deprivation, tumour morphology, route to diagnosis and treatment received, patients from the Indian Group (HR 0.84, 95% CI 0.72-0.98), Any Other White (HR 0.83, 95% CI 0.76-0.91), Other Ethnic Group (HR 0.70, 95% CI 0.62-0.79) and Unknown/Not Stated Ethnicity (HR 0.81, 95% CI 0.75-0.88), had better one-year survivals than the White British Group (Table 3). There was no difference between the White British Group and the remaining Bangladeshi, Pakistani, Chinese, Black Caribbean, and Black African Ethnic minority groups. The ethnic difference in survival was further explored by investigating whether there was any interaction between ethnicity and glioblastoma diagnosis, route or pathway to diagnosis, and optimal treatment received (Table 4). The Any Other White Group were more likely to be diagnosed through a hospital stay that included an emergency admission (OR 1.16, 95% CI 1.02-1.33). The Other Ethnic Group were nearly a third more likely to receive the diagnosis of glioblastoma (OR 1.28, 95% CI 1.04-1.56) than the White British Group. However, individuals with Unknown/Not Stated Ethnicity had the most favourable prognosis and were less likely to be diagnosed with a glioblastoma (OR 0.70, 95% CI 0.58-0.84), less likely to be diagnosed through a hospital stay that included an emergency admission (OR 0.61, 95% CI 0.53-0.69), and more likely to receive the optimal treatment option for their other-than-glioblastoma diagnosis (OR 0.39, 95% CI 0.31-0.49). 4. Discussion 4.1. Main Findings This study of 24,319 people residing in England and diagnosed with a brain tumour between 2012 and 2017 shows better one-year survival for patients from Indian, Any Other White, Other Ethnic Groups, and Unknown/Not Stated Ethnic Groups than for the White British Group (HR 0.84 (95% CI 0.72-0.98), HR 0.83 (95% CI 0.76-0.91), HR 0.70 (95% CI 0.62-0.79), and HR 0.81 (95% CI 0.75-0.88), respectively). The survival analysis was adjusted for age, sex, co-morbidity, socio-economic deprivation, tumour morphology, route to diagnosis, and treatment received. Individuals with Unknown/Not Stated Ethnicity had the best prognoses and as a group were less likely be diagnosed with glioblastoma or to be diagnosed through a hospital stay, including an emergency admission. 4.2. Comparisons to Other Findings In comparison to the smaller study by Ratneswaren et al. (2014), which was limited to high-grade glioma patients living in South East England , our current study was able to incorporate additional factors that may influence the impact of ethnicity on survival. In this larger national dataset, the heterogenous ethnicities were categorised into better defined groups for a precise analysis. We also found the Indian and Other Ethnic Group had a better survival than the White British Group. However, we identified the Unknown/Not Stated Ethnic Group having a better one-year survival than the White British Group, in contrast to the reverse finding in the earlier study. This could be due to a higher proportion of unknown ethnicity data, which was 21.7% compared to only 4.4% in the current study. US population-based studies have also reported racial and ethnic variations in brain tumour incidence and survival. Most have shown that Caucasian people have poorer survival outcomes compared to Black/African Americans and Asian/Pacific Islander Americans . Other work, however, has reported that African Americans have an increased risk of death from malignant brain tumour compared to Caucasians and other race and ethnicities , which was explained further by an interaction between race and surgery type. 4.3. Interpretations and Implications In this study, we have demonstrated that the White British Ethnic Group has a poorer survival compared to other ethnic groups. An English paper by Maile et al. (2016) has reported incidence data broadly similar to the US finding that patients from White Ethnic Groups were significantly more likely to develop glioblastoma than other racial/ethnic groups . They did not evaluate survival; however, their results could help explain the association between White British ethnicity and a higher risk of mortality from high-grade glioma. Increasing age is known to be a poor prognostic factor for patients with malignant brain tumour . The demography of ethnic minorities in England reflects the fact that people from these groups are younger and congregate in major cities, such as London, compared to other areas of England. A cohort study from the US also identified that patients of Hispanic background were diagnosed at a younger age compared to non-Hispanic Whites and had an improved overall survival . A report by The King's Fund suggested that, overall, people from ethnic minorities have poorer access to UK healthcare compared to the White British Groups , and this could correlate with fewer individuals from these being registered with the NHS. As a result, the probability of White people being diagnosed with a glioma could be increased due to their greater use of diagnostic tools, even from a young age, and therefore, they have a greater risk of ionising radiation exposure . Since our finding of better survival for patients from Any Other White and Unknown/Not Stated Ethnic Group is new, it needs further exploration. One explanation could be that these individuals travel to their countries of origin for better healthcare and social support following their diagnosis, which could mean that their deaths abroad were not formally registered in the English system . Brain tumours are considered difficult to diagnose , as these cancers tend to (1) involve 3 or more GP visits before diagnosis and (2) are likely to present as an emergency ; both could lead to poorer outcomes . The potential impact of family support on outcome might also differ between ethnic groups. People from minority ethnic groups, particularly those from Asian backgrounds, are more likely to be surrounded by extended families compared to the nuclear family structure, typical of the White British Group; it is possible that extended families may be more likely to recognise subtle signs of a brain tumour, including neuro-cognitive changes, as well as possible recurrences and encourage earlier diagnosis. The current standard therapy for gliomas consists of surgical resection followed by adjuvant chemotherapy and radiation, prolonging median overall survival to 15 months for glioblastomas , and is represented in this study by the optimal treatment option. From our results, we demonstrated that the Unknown/Not Stated Ethnic Group are less likely to receive optimal treatment, but that could be due to the lower chance of being diagnosed with a glioblastoma. Studies investigating possible factors explaining brain tumour occurrence have identified genes that could be associated with glioma development and tumours carrying the worst prognosis . The presence of such genes and their specific alterations could perhaps explain the differences in prognosis by ethnicity. Epigenetic age acceleration, which is the difference between age predicted by DNA methylation and chronological age, has been linked with many cancers . A recent study by Crimmins et al. (2021), which examined epigenetic clocks to evaluate a linkage with race/ethnicity, found that the majority of clocks indicated slower epigenetic ageing among Hispanic and African American individuals compared to White individuals . The reports of differing incidence and survival by racial/ethnic groups, make it important to explore possible genetic alterations and variation in signalling pathways to identify and compare polymorphisms between ethnic minority and White individuals . For example, one study found a 42% reduction of risk of glioma in patients with a history of diabetes , and a recent meta-analysis confirmed this inverse relationship where elevated blood sugar, or a previous history of diabetes, are inversely associated with risk of glioma . In England, people of Black (African and Caribbean) and South Asian (Bangladeshi, Indian, and Pakistani) backgrounds are at higher risk of developing type 2 diabetes from a younger age compared to those of a White background , and this could possibly be associated with the decreased glioma mortality observed here. The better survival for Indian individuals after adjusting for other factors was of particular interest and may suggest that there are other more specific influencing factors. As there were no significant differences between Indian and White British Groups in terms of patient characteristics, tumour morphology, and route or pathway to diagnosis, further explanation is needed to justify the difference when treatment received is added to the Cox proportional regression model. Due to curcumin's anti-tumoural effects on glioma cells in preclinical in vitro and in vivo , we speculate that an Indian diet that usually includes curcumin might play a role in their prolonged brain tumour survival, perhaps linked to a better response to treatment. A recent study in India evaluated the molecular biomarkers of brain tumours in Indian patients and reported a high prevalence of isocitrate dehydrogenase 1 (IDH1) mutation in astrocytoma and glioblastoma in these patients . The presence of IDH1 mutation correlates with a survival benefit and is more common among glioblastomas progressing from a lower grade glioma compared with 5-10% of de novo glioblastomas . Consequently, the association between individuals of an Indian background and improved survival could be related in some way to this mutation, in addition to, or perhaps independent of, the therapeutic potential of curcumin. Obtaining detailed information on molecular, genetics, and lifestyle or environmental factors could enable us to further compare outcomes with other populations. 4.4. Strengths and Limitations The extended period of time covered by this study, along with the greatly improved cancer registration data in recent years for England, have meant that more detailed ethnic groups can be analysed. The previously reported NCIN incidence data for the years 2002 to 2006, and other related studies, found a quarter of cancer patients had unknown (i.e., missing) ethnicity information . While cancer registration acknowledges that ethnicity data may not be self-reported and may possibly have been derived from already held information, the very large increase in completeness means we can be confident in these analyses. In these new data, only 4% of patients had unknown/not stated ethnicity information and were analysed separately to get a better understanding of this individual group. Our study also considered the importance of analysing more defined ethnic groups, as significant heterogeneity of risk for many cancers can be seen particularly among those from Black and South Asian Groups . Another strength of this study is the adjustment for many prognostic variables that could vary by ethnicity and prognosis. However, other and unknown factors could still be relevant including patient's performance status, tumour location within the brain, extent of tumour excision, and more interestingly, molecular biomarkers and genetic information. Our study had some limitations. Reporting patterns of incidence for the histological subtypes by ethnicity would have strengthened the study; however, this was restricted by the small number of patients in each subgroup. In addition, limited numbers of patients in the mixed ethnic groups and those from a White Irish background meant they had to be combined within Any Other Ethnic and Any Other White Groups respectively. Additionally, cancer registration did not collate information that could possibly influence survival, such as recurrences, glioblastoma progressing from a low-grade glioma, or biopsy-only--and hence, we observed a high proportion of patients (34.9%) who had no surgical resection but could possibly just had a diagnostic biopsy. 5. Conclusions To obtain a better understanding of potential ethnic differences in malignant primary brain tumour survival, we carried out a detailed evaluation of factors, including age at diagnosis, sex, co-morbidity, socio-economic deprivation, histologic tumour subtype (which is correlated with tumour grade), route or pathway to diagnosis, and treatment options that might affect prognosis. After controlling for these variables, we found that patients from Indian, Any Other White, Other Ethnic Groups, and Unknown/Not Stated Ethnic Groups, had better one-year survival compared to the White British Group. To determine whether biological, behavioural, or clinical factors are driving these survival differences, more data on patients' clinicopathological characteristics are therefore needed. This will help us better understand any ethnic inequalities in brain cancer and identify improvements to the health service for specific groups. Acknowledgments The data are collated, maintained, and quality assured by the National Disease Registration Service, which is part of NHS England. We thank Sally Vernon, Vivian Mak and Victoria Coupland for help extracting the data. Author Contributions H.A.W., H.M. and E.A.D. conceived and designed the study, H.A.W. analysed the data, and all authors were involved in the interpretation of the results and in drafting and reviewing the manuscript. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement All investigations were carried out according to the Declaration of Helsinki. Informed Consent Statement Not applicable. Data Availability Statement Data may be obtained from a third party and are not publicly available. The data that support the findings of this study are available from NHS England but restrictions apply to the availability of these data, which were used under license for the current study, and so are not publicly available. The authors do not own these data, and therefore are not permitted to share or provide these data other than in scientific communication format. Conflicts of Interest The authors declare that they have no conflict of interest. Figure 1 Selection of cases for analysing the influence of ethnicity on malignant primary brain tumour survival in England, 2012-2017 with follow-up to 2018 (DCO = Death Certificate Only). Figure 2 Kaplan-Meier survival curves by brain tumour morphology, England 2012-2017. cancers-15-01464-t001_Table 1 Table 1 Distribution of covariates in survival analysis of malignant primary brain tumours patients diagnosed in England, 2012-2017 with follow-up to 2018. Univariate and mutually adjusted Cox proportional hazards regression analyses to estimate hazard ratios. Cases % Deaths Univariate Mutually Adjusted HR (95% CI) HR (95% CI) Age at diagnosis <=34 1549 6.4 538 1.00 1.00 35-44 1662 6.8 802 1.44 (1.22, 1.70) 1.54 (1.30, 1.82) 45-54 3158 13.0 2278 2.84 (2.46, 3.26) 2.64 (2.29, 3.04) 55-64 4993 20.5 4285 4.89 (4.28, 5.59) 3.95 (3.45, 4.53) 65-74 6667 27.4 6184 8.11 (7.11, 9.26) 4.95 (4.32, 5.67) 75-84 4738 19.5 4635 15.69 (13.75, 17.91) 5.31 (4.63, 6.09) >=85 1552 6.4 1545 26.21 (22.81, 30.12) 6.37 (5.52, 7.36) Trend kh2 (1) = 7273.09; p < 0.001 kh2 (1) = 1279.79; p < 0.001 Sex Male 14,094 58.0 11,828 1.00 1.00 Female 10,225 42.1 8439 1.07 (1.03, 1.10) 0.96 (0.92, 0.99) Heterogeneity kh2 (1) = 15.63; p < 0.001 kh2 (1) = 7.60; p < 0.001 Area of residence East Midlands 2268 9.3 1913 1.24 (1.16, 1.34) 1.24 (1.15, 1.34) East of England 2837 11.7 2404 1.41 (1.31, 1.51) 1.18 (1.10, 1.27) London 2573 10.6 1970 1.00 1.00 North East 1414 5.8 1212 1.35 (1.24, 1.47) 1.22 (1.12, 1.33) North West 3318 13.6 2747 1.21 (1.13, 1.30) 1.36 (1.27, 1.46) South East 4094 16.8 3415 1.27 (1.19, 1.36) 1.10 (1.03, 1.18) South West 2895 11.9 2483 1.38 (1.29, 1.48) 1.15 (1.07, 1.24) West Midlands 2409 9.9 2041 1.31 (1.22, 1.41) 1.34 (1.24, 1.44) Yorkshire and The Humber 2511 10.3 2082 1.31 (1.22, 1.41) 1.17 (1.09, 1.26) Heterogeneity kh2 (8) = 126.13; p < 0.001 kh2 (8) = 110.88; p < 0.001 Socio-economic deprivation 1 least deprived 5615 23.1 4705 1.00 1.00 2 5607 23.1 4724 1.07 (1.03, 1.13) 1.04 (0.99, 1.09) 3 4985 20.5 4169 1.09 (1.04, 1.14) 1.06 (1.01, 1.11) 4 4308 17.7 3548 1.09 (1.03, 1.14) 1.08 (1.03, 1.14) 5 most deprived 3804 15.6 3121 1.09 (1.04, 1.15) 1.17 (1.11, 1.24) Trend kh2 (1) = 11.53; p < 0.001 kh2 (1) = 31.88; p < 0.001 Charlson co-morbidity score 0 20,400 83.9 16,660 1.00 1.00 1 2108 8.7 1901 1.60 (1.52, 1.69) 1.07 (1.01, 1.12) 2+ 1811 7.5 1706 2.19 (2.07, 2.31) 1.11 (1.05, 1.17) Trend kh2 (1) = 1031.64; p < 0.001 kh2 (1) = 16.55; p < 0.001 Morphology Glioblastoma 14,768 60.7 13,619 1.00 1.00 Astrocytoma, Anaplastic 1058 4.4 675 0.40 (0.36, 0.44) 0.58 (0.53, 0.65) Astrocytoma, NOS 1306 5.4 557 0.20 (0.18, 0.23) 0.20 (0.18, 0.23) Oligodendroglioma 1279 5.3 321 0.11 (0.09, 0.13) 0.12 (0.10, 0.15) PCNSL 1492 6.1 1124 0.97 (0.91, 1.04) 0.67 (0.62, 0.72) Malignant Glioma 2707 11.1 2386 1.68 (1.60, 1.76) 0.67 (0.63, 0.70) Unclassified Malignant 1709 7.0 1585 2.48 (2.34, 2.61) 0.79 (0.75, 0.84) Heterogeneity kh2 (6) = 3293.59; p < 0.001 kh2 (6) = 1345.58; p < 0.001 Treatment Radiotherapy only 2176 9.0 1964 1.00 1.00 Chemotherapy only 916 3.8 642 0.65 (0.59, 0.72) 0.80 (0.72, 0.90) Surgical resection only 2317 9.5 1563 0.86 (0.80, 0.93) 1.56 (1.45, 1.68) Surgical resection + Radiotherapy + Chemotherapy 5585 23.0 4200 0.24 (0.22, 0.26) 0.23 (0.21, 0.25) Radiotherapy + Chemotherapy 1994 8.2 1588 0.40 (0.37, 0.44) 0.44 (0.41, 0.48) Surgical resection + Radiotherapy 2525 10.4 2127 0.63 (0.58, 0.67) 0.57 (0.53, 0.61) Surgical resection + Chemotherapy 323 1.3 227 0.46 (0.39, 0.54) 0.72 (0.60, 0.85) No treatment 8483 34.9 7956 2.51 (2.38, 2.65) 2.47 (2.33, 2.62) Heterogeneity kh2 (7) = 9705.48; p < 0.001 kh2 (7) = 6350.45; p < 0.001 Route to Diagnosis Emergency presentation 12,926 53.2 11,622 1.00 1.00 GP referral 4833 19.9 3668 0.55 (0.52, 0.57) 0.73 (0.70, 0.76) Inpatient elective 743 3.1 595 0.49 (0.44, 0.54) 0.87 (0.78, 0.96) Other outpatient 4791 19.7 3569 0.46 (0.44, 0.48) 0.80 (0.77, 0.84) Two Week Wait (TWW) Urgent referral 428 1.8 355 0.62 (0.55, 0.70) 0.82 (0.72, 0.93) Unknown 598 2.5 458 0.69 (0.62, 0.77) 0.60 (0.54, 0.67) Heterogeneity (excluding unknown) kh2 (4) = 1646.89; p < 0.001 kh2 (4) = 230.11; p < 0.001 cancers-15-01464-t002_Table 2 Table 2 Survival analysis of 24,319 patients diagnosed with malignant primary brain tumour in England, 2012-2017, by ethnicity. Univariate Mutually Adjusted Ethnic Group Number of Patients % Number of Deaths Median Age Median Survival/Months (95% CI) HR (95% CI) HR (95% CI) White British 20,795 85.5 17,653 66.0 17.0 (16.6, 17.5) 1.00 (Ref) 1.00 (Ref) Bangladeshi 30 0.1 22 61.5 18.0 (8.5, 85.5) 0.98 (0.62, 1.53) 1.19 (0.76, 1.88) Indian 321 1.3 231 59.0 27.7 (23.4, 36.0) 0.72 (0.62, 0.84) 0.89 (0.76, 1.03) Pakistani 186 0.8 130 56.0 26.0 (19.1, 34.5) 0.75 (0.61, 0.91) 0.95 (0.77, 1.16) Chinese 37 0.2 26 57.0 25.3 (11.7, 50.7) 0.85 (0.56, 1.30) 1.16 (0.76, 1.76) Black African 84 0.4 59 53.5 29.8 (20.8, 47.7) 0.67 (0.50, 0.91) 0.98 (0.72, 1.33) Black Caribbean 94 0.4 78 60.5 19.6 (13.2, 33.8) 0.85 (0.65, 1.11) 0.86 (0.66, 1.12) Any Other White 1018 4.2 747 61.0 26.7 (24.0, 31.5) 0.73 (0.67, 0.80) 0.88 (0.81, 0.96) Other Ethnic Groups 674 2.8 445 53.0 40.5 (36.5, 47.4) 0.53 (0.47, 0.60) 0.77 (0.68, 0.87) Unknown/Not Stated 1080 4.4 876 68.0 10.2 (9.4, 11.8) 1.25 (1.16, 1.35) 0.82 (0.76, 0.88) Heterogeneity (excluding unknown) kh2 (8) = 182.58; p < 0.001 kh2 (8) = 29.78; p < 0.001 cancers-15-01464-t003_Table 3 Table 3 Survival analysis of different ethnic groups of patients diagnosed with malignant primary brain tumour in England, 2012-2017. Adjusted for Age, Sex and Socioeconomic Deprivation, Co-morbidity and Morphology and Route to Diagnosis and Treatment Received Ethnic Group HR (95% CI) p-Value HR (95% CI) p-Value HR (95% CI) p-Value HR (95% CI) p-Value HR (95% CI) p-Value White British 1.00 (Ref) 1.00 (Ref) 1.00 (Ref) 1.00 (Ref) 1.00 (Ref) Bangladeshi 1.47 (0.94, 2.30) 0.094 1.24 (0.79, 1.95) 0.341 1.15 (0.74, 1.81) 0.531 1.15 (0.73, 1.80) 0.546 1.05 (0.67, 1.65) 0.824 Indian 0.92 (0.79, 1.07) 0.290 0.89 (0.76, 1.04) 0.142 0.91 (0.78, 1.06) 0.213 0.90 (0.77, 1.05) 0.178 0.84 (0.72, 0.98) 0.025 Pakistani 1.16 (0.95, 1.42) 0.153 1.00 (0.82, 1.23) 0.989 1.04 (0.85, 1.27) 0.707 1.00 (0.82, 1.23) 0.984 0.96 (0.78, 1.17) 0.658 Chinese 1.29 (0.85, 1.96) 0.231 1.24 (0.82, 1.88) 0.315 1.22 (0.80, 1.85) 0.356 1.23 (0.81, 1.86) 0.342 1.09 (0.72, 1.66) 0.690 Black African 1.08 (0.80, 1.46) 0.611 0.98 (0.72, 1.33) 0.888 0.91 (0.67, 1.24) 0.551 0.90 (0.66, 1.22) 0.496 0.87 (0.64, 1.17) 0.355 Black Caribbean 1.02 (0.78, 1.33) 0.868 0.90 (0.69, 1.17) 0.416 0.84 (0.65, 1.10) 0.207 0.82 (0.63, 1.07) 0.153 0.81 (0.62, 1.06) 0.126 Any Other White 0.91 (0.83, 0.99) 0.034 0.90 (0.82, 0.98) 0.014 0.90 (0.83, 0.98) 0.019 0.89 (0.81, 0.97) 0.007 0.83 (0.76, 0.91) <0.001 Other Ethnic Groups 0.84 (0.74, 0.94) 0.003 0.80 (0.71, 0.90) <0.001 0.80 (0.71, 0.90) <0.001 0.79 (0.70, 0.89) <0.001 0.70 (0.62, 0.79) <0.001 Unknown/Not Stated 1.14 (1.06, 1.23) <0.001 1.17 (1.09, 1.26) <0.001 1.11 (1.03, 1.19) 0.008 1.10 (1.02, 1.18) 0.016 0.81 (0.75, 0.88) <0.001 Heterogeneity (excluding unknown) kh2 (8) = 20.85; p < 0.001 kh2 (8) = 23.81; p < 0.001 kh2 (8) = 23.76; p < 0.001 kh2 (8) = 25.32; p < 0.001 kh2 (8) = 56.43; p < 0.001 The HR were adjusted for age and sex, and then, they were sequentially adjusted for socio-economic and co-morbidity, morphology, route to diagnosis, and treatment received. cancers-15-01464-t004_Table 4 Table 4 Odds ratios of malignant primary brain tumour patients in England 2012-2017, by ethnicity, diagnosed with glioblastoma, diagnosed as emergency through hospital stay and receiving optimal treatment. Percentage (%) of patients within each ethnic group. Pathologically Confirmed Glioblastoma Diagnosis 1 Diagnosed through a Hospital Stay That Included an Emergency Admission 2 Optimal Treatment 3 Ethnic Group % Patients OR (95% CI) p Value % Patients OR (95% CI) p Value % Patients OR (95% CI) p Value White British 56.0 1.00 53.2 1.00 24.8 1.00 Bangladeshi 56.7 1.77 (0.79, 3.99) 0.168 50.0 0.80 (0.38, 1.69) 0.559 13.3 0.42 (0.13, 1.31) 0.136 Indian 54.1 1.14 (0.86, 1.52) 0.360 53.9 1.19 (0.94, 1.50) 0.148 24.9 0.81 (0.61, 1.08) 0.150 Pakistani 40.6 0.74 (0.51, 1.08) 0.121 59.1 1.34 (0.98, 1.83) 0.068 18.3 0.66 (0.44, 1.00) 0.049 Chinese 48.6 1.22 (0.54, 2.77) 0.638 48.6 0.90 (0.45, 1.79) 0.765 18.9 0.53 (0.22, 1.28) 0.156 Black African 48.7 1.10 (0.63, 1.92) 0.741 54.8 1.18 (0.75, 1.84) 0.474 20.2 0.68 (0.38, 1.23) 0.203 Black Caribbean 51.8 0.96 (0.56, 1.65) 0.893 56.4 1.06 (0.69, 1.63) 0.799 27.7 1.27 (0.75, 2.14) 0.377 Any Other White 54.1 1.14 (0.96, 1.34) 0.124 54.6 1.16 (1.02, 1.33) 0.029 26.6 0.95 (0.81, 1.12) 0.545 Other Ethnic Groups 54.2 1.28 (1.04, 1.56) 0.017 51.0 1.10 (0.94, 1.30) 0.233 31.0 0.99 (0.82, 1.19) 0.911 Unknown/Not Stated 33.6 0.70 (0.58, 0.84) <0.001 50.1 0.61 (0.53, 0.69) <0.001 9.4 0.39 (0.31, 0.49) <0.001 1 Adjusted for age, sex, socio-economic deprivation, co-morbidity, route to diagnosis, treatment. 2 Adjusted for age, sex, socio-economic deprivation, co-morbidity, morphology, treatment. 3 Adjusted for age, sex, socio-economic deprivation, co-morbidity, morphology, route to diagnosis. 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PMC10000772 | Int J Environ Res Public Health Int J Environ Res Public Health ijerph International Journal of Environmental Research and Public Health 1661-7827 1660-4601 MDPI 10.3390/ijerph20053789 ijerph-20-03789 Article Healthcare Resource Consumption and Related Costs in Patients on Antiretroviral Therapies: Findings from Real-World Data in Italy Perrone Valentina Conceptualization Methodology Writing - review & editing 1 Dovizio Melania Conceptualization Writing - original draft 1 Sangiorgi Diego Methodology Software Data curation 1 Andretta Margherita Data curation 2 Bartolini Fausto Data curation 3 Cavaliere Arturo Data curation 4 Ciaccia Andrea Data curation 5 Chinellato Alessandro Data curation 6 Costantini Alberto Data curation 7 Dell'Orco Stefania 8 Ferrante Fulvio Data curation 9 Gentile Simona Data curation 10 Lavalle Antonella Data curation 10 Moscogiuri Rossella Data curation 11 Mosele Elena Data curation 12 Procacci Cataldo Data curation 13 Re Davide Data curation 14 Santoleri Fiorenzo Data curation 7 Roccia Alessandro Validation Resources Supervision 15 Maggiolo Franco Methodology Validation Visualization 16 Degli Esposti Luca Conceptualization Validation Supervision Project administration Funding acquisition 1* Cao Wangnan Academic Editor 1 CliCon S.r.l. Societa Benefit--Health, Economics & Outcomes Research, 40137 Bologna, Italy 2 Azienda ULSS 8 Berica, 36100 Vicenza, Italy 3 USL Umbria 2 Terni, 05100 Terni, Italy 4 ASL Viterbo, 01100 Viterbo, Italy 5 Servizio Farmaceutico Territoriale ASL Foggia, 71121 Foggia, Italy 6 Azienda ULSS 3 Serenissima, 30174 Mestre, Italy 7 ASL Pescara, 65100 Pescara, Italy 8 ASL Roma 6, 00041 Albano Laziale, Italy 9 ASL Frosinone, 03100 Frosinone, Italy 10 Direzione Generale per la Salute Regione Molise, 86100 Campobasso, Italy 11 ASL Taranto, 74121 Taranto, Italy 12 UOC Assistenza Farmaceutica Territoriale, Azienda ULSS 7 Pedemontana, 36061 Bassano del Grappa, Italy 13 Dipartimento Farmaceutico ASL BAT, 76125 Trani, Italy 14 ASL Teramo, 64100 Teramo, Italy 15 Gilead Sciences S.r.l., 20124 Milano, Italy 16 ASST Papa Giovanni XXIII, 24127 Bergamo, Italy * Correspondence: [email protected] 21 2 2023 3 2023 20 5 378931 1 2023 16 2 2023 17 2 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). This real-world analysis conducted on administrative databases of a sample of Italian healthcare entities was aimed at describing the role of therapeutic pathways and drug utilization in terms of adherence, persistence, and therapy discontinuation in HIV-infected patients under antiretroviral therapies (ART) and Tenofovir Alafenamide (TAF)-based regimens on healthcare resource consumption and related direct healthcare costs. Between 2015 and 2019, adults (>=18 years) prescribed with TAF-based therapies were identified and characterized in the year prior to the first prescription (index-date) for TAF-based therapies and followed-up until the end of data availability. Overall, 2658 ART-treated patients were included, 1198 of which were under a TAF-based regimen. TAF-based therapies were associated with elevated percentages of adherence (83.3% patients with proportion of days covered, PDC > 95% and 90.6% with PDC > 85%) and persistence (78.5%). The discontinuation rate was low in TAF-treated patients, ranging from 3.3% in TAF-switchers to 5% in naive. Persistent patients had lower overall mean annual healthcare expenditures (EUR 11,106 in persistent vs. EUR 12,380 in non-persistent, p = 0.005), and this trend was statistically significant also for costs related to HIV hospitalizations. These findings suggest that a better therapeutic management of HIV infection might result in positive clinical and economic outcomes. antiretroviral therapies (ART) human immunodeficiency virus (HIV) tenofovir alafenamide (TAF)-based regimens drug utilization adherence persistence healthcare resource consumption direct healthcare costs real-word evidence Gilead Sciences S.r.l.Gilead Sciences S.r.l. purchased the study report that is the basis for this manuscript. This manuscript was developed with Gilead Sciences S.r.l. and CliCon S.r.l. Societa Benefit. The views expressed here are those of the authors and not necessarily those of the supporters. The agreement signed by CliCon S.r.l. and Gilead Sciences S.r.l. does not create any entityship, joint venture, or any similar relationship between parties. CliCon S.r.l. is an independent company. Neither CliCon S.r.l. nor any of their representatives are employees of Gilead Sciences S.r.l. for any purpose. The journal fee was funded by Gilead Sciences S.r.l. pmc1. Introduction The development and introduction of antiretroviral therapies (ART) and then the availability of combination ART in the clinical practice has dramatically changed the course of human immunodeficiency virus (HIV) infection from an acute to a chronic disease . Nowadays, life expectancy for people living with HIV (PLWH) is up to that of the general population in those patients who receive a prompt diagnosis and early treatment and achieve adequate viral-load suppression . International guidelines strongly recommend initiating an ART combination soon after diagnosis in accordance with patient needs, attitudes, and readiness . The main goal of ART is to inhibit viral replication, to control the progression of HIV-infection into AIDS, to improve the overall clinical outcomes, to prevent the development of antiretroviral resistance, and to reduce HIV transmission . According to the World Health Organization (WHO), adherence is defined as "the extent to which a person's behaviour--taking medication, following a diet, and/or executing lifestyle changes, corresponds with agreed recommendations from a health care provider" . Adherence to ART plays a key role in maximizing the viral suppression. Historically, the threshold for adherence was set at >=95% based on studies conducted on the early era of ART . With the second generation of ART, the cut-off level of adherence needed to achieve HIV viral suppression is shifting to lower values such as 80-85% that still allow to achieve a successful immunosuppression . Nevertheless, long-lasting worldwide experience has shown that maintaining an optimal level of adherence to ART is still an unsolved clinical problem . Thus, improving adherence to ART represents an important goal for both patients' outcomes and healthcare savings in terms of resource utilization and related costs . Among the most widely used ART combinations for PLWH, the Tenofovir Alafenamide (TAF)-based regimen has been shown to achieve a successful viral load suppression, but the results can be influenced by drug administration schedule. Complex regimens often remain among the few therapeutic options for PLWH with previous ART failure or resistance, but they are associated with an increased rate of adverse events and drug-drug interactions, ultimately leading to a higher risk of discontinuation. Conversely, ART simplification could lead to improved adherence levels, lower discontinuation rates, and higher patient satisfaction, feasibly due to the advantage of taking a single pill . Therapeutic persistence, defined as the time that a patient remains on ART before interrupting or switching to another regimen, is also a crucial factor to ensure successful management of HIV, and the discontinuation of therapy has been related not only to lack of sustained viral suppression but also to toxicity or simplification independently from the level of adherence . The progress made with HIV therapy is reflected on the shifting paradigm of the economic burden of HIV care, which was previously due mostly to hospitalization and management of opportunistic illness. The introduction of potent combinations of ART that elongated the life expectancy of PLWH made a substantial contribution in decreasing HIV-related hospitalizations and made HIV one of the most expensive chronic diseases, driven mostly by drug costs . This real-world analysis was aimed at describing the demographic and clinical characteristics of patients treated with ART and evaluating therapeutic pathways and drug utilization. A special focus was placed on adherence, persistence, and therapy discontinuation of Tenofovir Alafenamide (TAF)-based regimens either as multi-tablet regimens (MTR) or single-tablet regimens (STR) among naive patients and those with previous ART who switched for the first time to TAF-based regimens and the resulting impact on healthcare resource consumptions and related direct costs for the Italian National Health Service (INHS). 2. Materials and Methods 2.1. Data Source The present retrospective observational analysis was based on data collected from the administrative databases of a sample of Italian Local Health Units (LHUs), covering around 5.52 million health-assisted subjects belonging to Apulia, Abruzzo, Molise, Campania, Lazio, Umbria, Liguria, Piedmont, and Veneto regions. The following databases were used for the analysis: (1) demographic database to obtain data on age, gender, and mortality; (2) pharmaceutical database for information related to drug prescriptions, such as anatomical-therapeutic chemical (ATC) code, number of packages, number of units per package, costs, and prescription date; (3) hospitalization database, including all hospitalization data with discharge diagnosis codes classified according to the International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM), diagnosis-related group (DRG), and DRG-related charge; and (4) outpatient specialist services database, reporting data on prescriptions, type, description of diagnostic tests, and specialist visits. To guarantee patients' privacy, an anonymous univocal numeric code (patient ID) was assigned to each health-assisted subject by the LHUs. The patient ID code allowed the electronic linkage between the databases. The anonymous code of the patient ensures the anonymity of the extracted data in full compliance with UE Data Privacy Regulation 2016/679 ("GDPR") and Italian D.lgs. n. 196/2003, as amended by D.lgs. n. 101/2018. All the results of the analyses were produced as aggregated summaries, which could not be connected, either directly or indirectly, to individual patients. 2.2. Design of the Analysis Population Included All adult (>=18 years old) patients prescribed with ART between January 2015 and December 2019 were screened. Among them, patients with TAF-based therapies were identified during all inclusion period by presence of the following drugs: TAF (ATC code J05AF13); emtricitabine and tenofovir alafenamide (ATC code J05AR17); emtricitabine, tenofovir alafenamide, elvitegravir, and cobicistat (ATC code J05AR18); emtricitabine, tenofovir alafenamide, and rilpivirine (ATC code J05AR19); emtricitabine, tenofovir alafenamide, and bictegravir (ATC code J05AR20); and emtricitabine, tenofovir alafenamide, darunavir, and cobicistat (ATC code J05AR22). Patients with TAF-based therapies were defined as naive if they did not present any ART prescription in the 12 months before starting TAF-based therapies and as TAF-switchers if they had switched from a non-TAF-based to a TAF-based regimen. Patients who moved to another LHU during the period considered in the analysis or with just one ART prescription or with prescription gaps greater than 12 months between subsequent prescriptions were excluded from the analysis. The date of the first prescription for TAF detected within the inclusion period was defined as the index date. Patients' clinical characteristics were evaluated during at least 12 months prior to the index date (characterization period), while patients were followed-up until the end of database availability, which was at least 12 months for each patient. 2.3. Variables Used in the Analysis At baseline, the following comorbidities were evaluated: depression, respiratory disease, renal failure, alcohol/drugs abuse, cardiovascular disease, neoplastic diseases, diabetes, dyslipidemia, HBV/HCV, and hypertension (codes for identification are reported in Supplementary Table S1). Comorbidity profile was scored using a modified version of the Charlson Comorbidity Index (CCI) not accounting for HIV . Drug utilization of TAF-based regimens was evaluated during follow-up. Adherence was measured as proportion of days covered (PDC), namely the ratio between the number of days of medication supplied and days of therapy, multiplied by 100 . To define adherent patients, two thresholds were selected: the most conservative one, i.e., PDC > 95%, and a PDC > 85%, which represents a newly hypothesized threshold for adherence in view of novel ART entering the market . Persistence to TAF-based regimens was defined as presence of TAF-based regimens prescriptions or any ART in the last quarter of 1-year follow-up. Discontinuation was defined as absence of any ART in the last quarter of follow-up. Switch was identified as a change from TAF-based to a not-TAF-based regimen during follow-up. Changes to a different TAF-based scheme or from TAF-MTR to TAF-STR were not considered a switch of therapy. The analysis of healthcare resource consumptions was reported as mean annual number of the following items for each alive patient: the mean annual number of prescription for HIV-related drugs, mean annual number of other medications, mean annual number of HIV-related hospitalization (DRG 488, 489, 490), mean annual number of hospitalizations for other causes, and mean annual number of outpatient specialist services (visits/diagnostic tests). The related direct healthcare costs in euros (EUR) sustained by INHS were retrieved from the administrative flows of the LHUs involved, which collect data for reimbursement purposes. The mean annual healthcare costs per patient were calculated as overall mean annual costs and divided by cost item, namely drug treatment (HIV-related drugs and other drugs), hospital admissions (HIV-related hospitalization and other hospitalizations), and outpatient specialist services. Healthcare resource consumptions and costs were stratified by persistence and level of adherence. Outliers (patients for whom total cost exceeds the mean cost plus 3-fold standard deviation) were excluded from cost analysis. Drug costs were calculated based on the purchase price by INHS. Hospitalization costs were determined using DRG tariffs. The costs of outpatient services (visits/tests) were defined according to tariffs applied by each region. 2.4. Statistical Analysis Continuous variables are reported as mean +- standard deviation and categorical variables as numbers and percentages. A generalized linear model (GLM) with gamma distribution and identity link function was developed to assess the relationship between adherence level and non-ART costs. Statistical significance was accepted at p < 0.05. All the analyses were performed using Stata SE version 12.0 (StataCorp, College Station, TX, USA). 3. Results Overall ART-Treated Population A total of 2658 adult patients treated with ART were included in the analysis, and their demographic and clinical characteristics in the overall population and by calendar year are shown in Table 1. In the total inclusion period, the mean age was 48.6 years, with the majority of patients in the age range of 36-55 years (61.6%). Male gender was more represented (71.6%). Analyzing the demographic characteristics by each year of inclusion, the male gender remained more frequent (71-73%), and a slight increase in age was noticed over the years. In the overall inclusion period, the most frequent comorbidities were hypertension (19.5%), followed by respiratory disease (15.9%) and dyslipidemia (11.2%) and mild average CCI (0.2) with 87.2% of patients with CCI score 0. The pattern of comorbidity distribution analyzed by each calendar year mirrored that of the total inclusion period. Patients Treated with TAF-Based Regimens The analysis focused on 1198 patients prescribed a TAF-based regimen during inclusion period. Table 2 provides the demographic and clinical characteristics of overall TAF-treated patients and in subgroups divided into MTR (N = 514, 43%), STR (N = 684, 57%), naive (N = 478, 40%), and TAF-switchers (N = 720, 60%). Adherence and persistence in the subgroups and in the total sample population were then investigated. In detail, the proportion of patients adherent to TAF-based regimens was 83.3% with a PDC threshold above >95%; specifically, it was higher among TAF-switchers (85.6%), followed by TAF-STR (84.4%), TAF-MTR (81.9%), and naive (79.9%). The proportion of adherent patients with PDC > 85% was 90.6% (92.8% among TAF-switchers, 90.9% in TAF-STR, 90.1% in TAF-MTR, and 87.2% in naive). Data on persistence revealed that the proportion of patients persistent to TAF-based regimens was 78.5% and reached 96% when persistence to any ART was analyzed. Switch to another ART not TAF-based occurred in 18% of patients, and 4% discontinued the therapy. The stratification in subgroups confirmed in general this overall trend, in particular for the rate of discontinuation that ranged between 3.3% (TAF-switchers) and 5% (naive). Of note, patients under MRT regimen compared to those with STR showed a greater tendency to switch (30.4% vs. 8.8%, respectively) and a lower persistence on TAF-based therapy (66.5% vs. 87.4%, respectively). Overall persistence for TAF naive patients was 74.3%. The healthcare resource consumption during first year of follow-up for TAF-based regimes patients evaluated in the last 3 years of inclusion (2017-2018-2019) showed the highest numbers for ART prescriptions and specialistic visits/diagnostic tests, followed by other drug prescriptions, with a decreasing trend over time (Supplementary Table S2A). The related mean annual total costs per patient are consistent with the data of healthcare resource consumption . A decreasing trend over the years was observed, with mean annual total costs per patients of EUR 15,493 for patients included in 2017, EUR 12,060 for those included in 2018, and EUR 10,216 for those included in 2019, mostly burdened by ART expenses. The evaluation of the role of persistence on healthcare resources consumptions during the first year of follow-up in patients with TAF-based regimen revealed that the most relevant consumptions were attributable to ART prescriptions and specialistic visits/diagnostic tests and, lastly, other drug prescriptions to a higher extent in non-persistent patients (Supplementary Table S2B). Regarding the cost analysis shown in Figure 2, patients persistent to TAF-based therapies had significantly lower mean annual costs than the non-persistent ones (EUR 11,106 vs. EUR 12,380, p = 0.005), and this difference was mostly appreciable in the cost item related to HIV hospitalization. Moreover, when the analysis was focused only on patients with HIV-related hospitalization, the mean costs were found to be higher for non-persistent patients (N = 25) compared to persistent ones (N = 79) (EUR 5996 and EUR 2448, respectively; data not shown). The healthcare resources consumption in TAF-treated patients stratified by adherence <80%, 81-95%, and >95% indicated ART prescriptions and specialistic visits/diagnostic tests as the most impactive items (Supplementary Table S2C). Consistently, during the first year of follow-up, from PDC < 80% to PDC > 95%, the total costs tended to rise, but this effect was mostly due to the higher burden of drug expenses that reflect the growing consumption with increasing adherence . Healthcare resources consumptions in non-adherent (PDC < 80%) patients treated with ART other than TAF and TAF-based regimens highlighted that even in non-adherent patients, the majority of consumptions were imputable to ART prescriptions and specialistic visits/diagnostic tests although less markedly in the patients receiving a TAF combination (Supplementary Table S2D). Cost analysis extended to the overall observation period in TAF-based regimens is shown in Figure 4. The mean annual healthcare direct costs by adherence (PDC > 95%) confirmed that the overall costs tended to be higher in ART-adherent compared to TAF-adherent treatment (EUR 8981 vs. EUR 8523, p = 0.070, nearly significant), mostly driven by the expenses for other drugs (EUR 1059 vs. EUR 632, p < 0.001) and specialistic visits and outpatient services (EUR 814 vs. EUR 638, p = 0.025). The GLM model showed that the mean yearly non-ART costs increased with older age, specifically by EUR 1364 in patients aged between 51 and 65 years (p < 0.001) and EUR 2265 in those over 65 (nearly significant, p = 0.079). A worse comorbidity profile was also significantly predictive of higher non-ART costs (EUR 1736 for CCI unit increment, p = 0.027) (Supplementary Table S3). 4. Discussion The current awareness of the management of HIV patients chronically treated with ART and the subsequent rebounds on healthcare resource consumptions and expenditures is highly variable across the different countries . The present analysis was conducted in a setting of real-life clinical practice in Italy to describe the state-of-art of HIV-infected patients treated with ART and more specifically with TAF-based regimens, evaluate therapeutic pathways and drug utilization, and give special attention to the impact of adherence, persistence, and therapy discontinuation of TAF-based regimens on HIV management and direct healthcare costs. The demographic characteristics of the overall ART population reported a male predominance and a mean age of around 49 years, which is in line with other Italian real-world studies on HIV . Hypertension and dyslipidemia were among the most frequent comorbidities detected; a French study also observed these two conditions to be highly prevalent in PLWH . We then focused on the 1198 patients prescribed with TAF-based regimen during the inclusion period in order to investigate in this specific group the management of treatments in terms of adherence, persistence, and therapy discontinuation and ultimately how their drug utilization could affect healthcare consumptions and costs. There is large body of evidence from national and international studies that, despite the undeniable progression and growing availability of pharmacological options for HIV patients, the goal of maintaining an adequate therapy adherence in the long term is still far from being reached . This point deserves great efforts from the scientific community since adherence to ART therapy has essential repercussions on viral load control and thus patients' clinical outcomes but also on the consequent savings in terms of healthcare resource consumptions and expenditures for the national health systems . In our study, a higher proportion of adherent patients was found among patients with STR rather than MTR regimens, which is in line with the evidence reported in the literature on higher adherence to STR than MTR . Interestingly, the comparison between naive and TAF-switchers showed greater adherence levels in switchers regardless of the cutoff considered (PDC > 95% or PDC > 85%). This is in line with another real-world study in which patients naive to ART were found less likely to be adherent . Furthermore, a meta-analysis showed that treatment-experienced patients had the highest pooled odds ratio for optimal adherence vs. suboptimal even though this tendency was not statistically significant . Older studies based on first-generation ART suggested that adherence could be unchanged or decreased after change of therapy and that ART switch should trigger intervention to reassess adherence , while most recent studies have observed an increase in the number of patients with improved adherence after switching to a second line . Persistence to TAF-based regimens mirrored the trend observed for adherence, with a high proportion of persistent patients among those with STR or switchers. Sutton et al found in a cohort of U.S veterans a proportion of patients persistent to TAF-based regimens ranging from 64-73% for STR and 46-58% for MTR. These values, however, referred to naive patients, and this could explain the difference with our findings. Indeed, naive patients had a trend of less persistence than those who were experienced (i.e., switchers to TAF). To the best of our knowledge, there are limited data on the actual economic burden for patients with TAF-based regimens in Italy, while the literature reports an estimation of cost-effectiveness or budget-impact analysis on such therapies . The latter estimated a direct healthcare cost for year 2018 spanning from EUR 8928 to EUR 11,200, which, although lower than our results, is, however, based in real-life settings . In all the scenarios depicted, cost analysis revealed that during the first year of follow-up, the weightiest item on the annual healthcare direct cost per patient was that for ART, which is consistent with previously published data for European countries . The downward trend observed over the last 3 years of inclusion was mainly due to a halving in the costs of other drugs, hospitalizations, and outpatients' services through the years, suggesting that the introduction of innovative ART on the market could influence the costs for the management of patients. Persistence to TAF-based therapies was associated with significantly lower mean annual costs, in particular for the cost item related to HIV hospitalization. Moreover, the comparison of mean annual healthcare direct costs by adherence (PDC > 85% vs. PDC < 85%) in TAF-based regimen revealed that increased costs not related to ART were observed in non-adherent patients; once again, this trend was evident for HIV hospitalizations. In this regard, Toh et al. explored the economic burden of AIDS-defining illnesses, which are still a major threat for PLWH despite the improvement of life expectancy thanks to ART: the authors found that improved level of adherence is related to an increase in the medical costs but could lead to savings from lower incidence rates of AIDS-defining illnesses and the related costs they carry . Considering the mean annual health care resource consumptions and costs during all available follow-up in TAF-treated patients with respect to adherence, we found tendentially increased consumptions and costs with growing adherence levels but this might be explained by the obviously higher burden of drug expenses in adherent patients, which is in line with the literature . Multivariate analysis revealed that older age and more complex comorbidity profile were significant predictors of healthcare costs other than ART, adding to the growing body of evidence that aging is one of the upcoming challenges for HIV management and one of the factors that will contribute to increased HIV care costs . Indeed, PLWH age while taking the life-long ART and therefore will have to address issues related to a multi-comorbidity profile such as polypharmacy, polydoctoring, and iatrogenic diseases . These findings should be interpreted in the light of some limitations related to the observational retrospective design of the analysis, which was based on data extracted from administrative databases. Thus, there might be lacking or insufficient clinical information on a number of other potential confounders (i.e., disease severity, comorbidities, previous virological failures/resistance mutations) that may have influenced the results. Secondly, data on the reasons for changing regimens (switch or discontinuation) are not reported within the database as well as the causes of non-adherence since not-measurable variables as patient attitudes towards medication or social status are not present; similarly, adherence and persistence were evaluated based on the drug dispensed; therefore, the actual use by the patient is unknown. Ultimately, indirect costs or out-of-pocket costs could not be evaluated, as administrative databases contain data on healthcare resources reimbursed by INHS. 5. Conclusions This real-world analysis provided an in-depth characterization of patients prescribed TAF-based regimens in terms of drug utilization and how this could have affected healthcare resource consumptions and costs. Higher levels of adherence and persistence to TAF-based regimens were detected among users of STR rather than MTR and among experienced patients that switched from an a TAF-based treatment compared to naive patients. A declining trend over the years in healthcare resource consumption and the related direct costs could be suggestive of the changing of costs related to the management of HIV patients due to the advent of novel therapeutic options. The total healthcare costs were mainly driven by ART-related costs, which represent almost 70% of the total expenditure. Patients that showed a higher level of TAF persistence and adherence were reported to have a reduced healthcare cost related to HIV hospitalizations compared to those who were non-persistent or not adherent to TAF-based therapies, suggesting that the optimization of the drug utilization may have a positive impact not only from a clinical point of view but from the perspective of the sustainability of the INHS as well. Supplementary Materials The following supporting information can be downloaded at: Table S1. Codes used for comorbidities identification. Table S2: Healthcare resources consumption in patients with TAF-based regimen during first year of follow-up by (A) year of inclusion (2017-2018-2019), (B) persistence, (C) adherence (PDC > 95%), and (D) non-adherence (PDC < 80% in patients treated with . TAF-based regimens). Table S3. Generalized linear model (GLM) for predictors of mean annual non-ART healthcare costs (EUR) during first year of follow-up for patients with TAF-based regimens. Click here for additional data file. Author Contributions Conceptualization, V.P., A.R., M.D. and L.D.E.; methodology, D.S.; validation, L.D.E. and F.M.; formal analysis, D.S.; investigation, V.P. and M.D.; resources, M.A., F.B., A.C. (Arturo Cavaliere), A.C. (Andrea Ciaccia), A.C. (Alessandro Chinellato), A.C. (Alberto Costantini), S.D., F.F., S.G., A.L., R.M., E.M., C.P., D.R. and F.S.; data curation, M.A., F.B., A.C. (Arturo Cavaliere), A.C. (Andrea Ciaccia), A.C. (Alessandro Chinellato), A.C. (Alberto Costantini), S.D., F.F., S.G., A.L., R.M., E.M., C.P., D.R. and F.S.; writing--original draft preparation, V.P. and M.D.; writing--review and editing, all authors; visualization, A.R., L.D.E. and F.M.; supervision, L.D.E. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee) of Comitato Etico Lazio I (protocol 1080, approval date: 23 September 2020), Comitato Etico Interprovinciale Area I (protocol 140/CE/2020 and 141/CE/2020, approval date: 30 November 2020), Nucleo Ricerca Clinica ULSS Berica (protocol 106370, approval date: 5 November 2020), Comitato Etico Sezione Area Centro Regione Calabria (protocol 212, approval date: 23 June 2020), Comitato Etico Lazio II (protocol 0179046/2020, approval date: 28 October 2020, protocolo 0216084/2020 approval date 16 December 2020, protocol 0031200/2021, approval date 10 February 2021), Comitato Etico Azienda Sanitaria Regionale Molise (approval date 27 October 2020), Comitato Etico Per le Sperimentazioni Cliniche della Provincia di Vicenza (protocol 0036999, approval date 13 April 2021), Comitato Etico per la Sperimentazione Clinica della provincia di Venezia e IRCCS S. Camillo (28 July 2020), Comitato Etico Delle Province di Chieti e Pescara (protocol 7, approval date 18 March 2021), Comitato Etico per le province di L'Aquila e Teramo (protocol number 11, approval date 24 March 2021), Comitato Etico Regionale Umbria (protocol 19414/20/ON, approval date 16 September 2020), Comitato Indipendente di Etica Medica (protocol 48144, approval date 28 May 2021). Informed Consent Statement Informed consent was not required since obtaining it is impossible for organizational reasons (pronouncement of the Data Privacy Guarantor Authority, General Authorization for personal data treatment for scientific research purposes--n.9/2014). Data Availability Statement All data used for the current study are available upon reasonable request to CliCon S.r.l., which is the body entitled to data treatment and analysis by Local Health Units. Conflicts of Interest A.R. is an employee of Gilead Sciences S.r.l.; V.P., M.D., D.S., L.D.E. are employees of CliCon s.r.l. Societa Benefit at the time of the analysis. CliCon s.r.l. is an independent company. The agreement signed by Clicon S.r.l. and Gilead Sciences Srl does not create any entity, joint venture or any similar relationship between parties. Neither CliCon S.r.l. nor any of their representatives are employees of Gilead Sciences Srl for any purpose.The remaining authors report no other conflicts of interest in this work. Figure 1 Mean annual health care costs (EUR) in patients with TAF-based regimen during first year of follow-up for the inclusion years 2017-2018-2019. Figure 2 Mean annual health care costs (EUR) during first year of follow-up for patients with TAF-based regimen based on persistence. Figure 3 Mean annual health care costs (EUR) during first year of follow-up for patients with TAF-based regimen based on adherence. Figure 4 Mean annual health care costs (EUR) during all available follow-up for patients with TAF-based regimens and based on adherence (PDC > 95%). ijerph-20-03789-t001_Table 1 Table 1 Demographic and clinical characteristics of the overall ART-treated patients and divided by calendar year. Continuous variables are reported as mean +- standard deviation and categorical variables as numbers and percentages in brackets. 2015 2016 2017 2018 2019 Overall Patients, n 966 814 1566 2198 1583 2658 Age, years 48.7 +- 10.2 48.9 +- 10.5 49.5 +- 10.7 50.4 +- 10.7 51.0 +- 10.9 48.6 +- 11.0 Age < 36 years 103 (10.7%) 89 (10.9%) 177 (11.3%) 228 (10.4%) 161 (10.2%) 349 (13.1%) Age 36-55 years 649 (67.2%) 533 (65.5%) 963 (61.5%) 1303 (59.3%) 860 (54.3%) 1637 (61.6%) Age > 55 years 214 (22.2%) 192 (23.6%) 426 (27.2%) 667 (30.3%) 562 (35.5%) 672 (25.3%) Male gender 691 (71.5%) 598 (73.5%) 1129 (72.1%) 1563 (71.1%) 1145 (72.3%) 1903 (71.6%) Depression 68 (7.0%) 73 (9.0%) 122 (7.8%) 172 (7.8%) 134 (8.5%) 196 (7.4%) Respiratory disease 152 (15.7%) 127 (15.6%) 351 (22.4%) 397 (18.1%) 212 (13.4%) 422 (15.9%) Renal failure 8 (0.8%) 14 (1.7%) 27 (1.7%) 35 (1.6%) 18 (1.1%) 35 (1.3%) Alcohol/drug abuse 30 (3.1%) 30 (3.7%) 55 (3.5%) 99 (4.5%) 73 (4.6%) 104 (3.9%) Cardiovascular disease 19 (2.0%) 20 (2.5%) 63 (4.0%) 83 (3.8%) 62 (3.9%) 65 (2.4%) Diabetes 34 (3.5%) 35 (4.3%) 71 (4.5%) 92 (4.2%) 77 (4.9%) 93 (3.5%) Dyslipidemia 131 (13.6%) 101 (12.4%) 225 (14.4%) 290 (13.2%) 205 (13.0%) 298 (11.2%) HBV/HCV 71 (7.3%) 73 (9.0%) 154 (9.8%) 196 (8.9%) 136 (8.6%) 207 (7.8%) Hypertension 178 (18.4%) 178 (21.9%) 355 (22.7%) 524 (23.8%) 399 (25.2%) 519 (19.5%) Charlson Comorbidity Index 0.2 +- 0.6 0.1 +- 0.5 0.2 +- 0.5 0.2 +- 0.5 0.1 +- 0.5 0.2 +- 0.5 0 840 (87.0%) 737 (90.5%) 1354 (86.5%) 1924 (87.5%) 1418 (89.6%) 2318 (87.2%) 1 96 (9.9%) 60 (7.4%) 172 (11.0%) 220 (10.0%) 137 (8.7%) 275 (10.3%) >=2 30 (3.1%) 17 (2.1%) 40 (2.6%) 54 (2.5%) 28 (1.8%) 65 (2.4%) Note: HBV, hepatitis B virus; HCV, hepatitis C virus; NR, not reported (for data privacy when <4 patients are involved). ijerph-20-03789-t002_Table 2 Table 2 Demographic and clinical characteristics of patients with TAF-based regimen (overall and stratified by MTF/STR and naive/switchers). Continuous variables are reported as mean +- standard deviation and categorical variables as numbers and percentages in brackets. Characteristics TAF-MTR TAF-STR Naive TAF-Switchers Overall Patients, n 514 684 478 720 1198 Age 49.7 +- 9.8 48.0 +- 11.6 47.3 +- 11.5 49.7 +- 10.) 48.7 +- 10.9 Age < 36 years 48 (9.3%) 117 (17.1%) 87 (18.2%) 78 (10.8%) 165 (13.8%) Age 36-55 years 332 (64.6%) 381 (55.7%) 281 (58.8%) 432 (60.0%) 713 (59.5%) Age > 55 years 134 (26.1%) 186 (27.2%) 110 (23.0%) 210 (29.2%) 320 (26.7%) Male 378 (73.5%) 501 (73.2%) 354 (74.1%) 525 (72.9%) 879 (73.4%) Depression 39 (7.6%) 49 (7.2%) 35 (7.3%) 53 (7.4%) 88 (7.3%) Respiratory disease 96 (18.7%) 105 (15.4%) 76 (15.9%) 125 (17.4%) 201 (16.8%) Renal failure 7 (1.4%) NR 4 (0.8%) 6 (0.8%) 10 (0.8%) Alcohol/drug abuse 28 (5.4%) 22 (3.2%) 11 (2.3%) 39 (5.4%) 50 (4.2%) Cardiovascular disease 20 (3.9%) 16 (2.3%) 10 (2.1%) 26 (3.6%) 36 (3.0%) Diabetes 20 (3.9%) 25 (3.7%) 15 (3.1%) 30 (4.2%) 45 (3.8%) Dyslipidemia 52 (10.1%) 61 (8.9%) 34 (7.1%) 79 (11.0%) 113 (9.4%) HBV/HCV 49 (9.5%) 59 (8.6%) 28 (5.9%) 80 (11.1%) 108 (9.0%) Hypertension 117 (22.8%) 117 (17.1%) 80 (16.7%) 154 (21.4%) 234 (19.5%) Cancer 28 (5.4%) 16 (2.3%) 11 (2.3%) 33 (4.6%) 44 (3.7%) Charlson Comorbidity Index 0.2 +- 0.7 0.1 +- 0.4 0.2 +- 0.7 0.1 +- 0.5 0.2 +- 0.6 0 450 (87.5%) 615 (89.9%) 422 (88.3%) 643 (89.3%) 1065 (88.9%) 1 52 (10.1%) 56 (8.2%) 45 (9.4%) 63 (8.8%) 108 (9.0%) >=2 12 (2.3%) 13 (1.9%) 11 (2.3%) 14 (1.9%) 25 (2.1%) Note: HBV, hepatitis B virus; HCV, hepatitis C virus; MTR, multi-tablet regimen; NR, not reported (for data privacy when <4 patients are involved); STR, single-tablet regimen; TAF, Tenofovir Alafenamide. 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PMC10000773 | Purposes: To establish the appropriate staging system and assess the role of curative thyroidectomy alone (Surgery) vs. involved-site radiation therapy after open biopsy (OB-ISRT) in stage IE mucosa-associated lymphoid tissue (MALT) lymphoma. Methods: We examined the Tokyo Classification as a modified classification. This retrospective cohort study included 256 patients with thyroid MALT lymphoma; 137 underwent standard therapy (i.e., OB-ISRT) and were enrolled for the Tokyo classification. Sixty stage IE patients with the same diagnosis were examined to compare Surgery with OB-ISRT. Results: Overall survival (p = 0.0092) and relapse-free survival (0.00113) were significantly better in stage IE vs. stage IIE under the Tokyo classification. No OB-ISRT and Surgery patients died, but three OB-ISRT patients relapsed. The incidence of permanent complications was 28% in OB-ISRT (mainly dry mouth) and 0% in Surgery (p = 0.027). The number of painkiller prescription days was significantly greater in OB-ISRT (p < 0.001). During follow-up, the rate of the new appearance/change of the low-density area in the thyroid gland was significantly higher in OB-ISRT (p = 0.031). Conclusions: The Tokyo classification allows an appropriate discrimination between stages IE and IIE MALT lymphoma. Surgery can provide a good prognosis in stage IE cases; it also avoids complications, shortens painful periods during treatment, and simplifies ultrasound follow-up. mucosa-associated lymphoid tissue lymphoma primary thyroid lymphoma involved-site radiation therapy thyroidectomy This research received no external funding. pmc1. Introduction Primary thyroid lymphoma (PTL) accounts for only 1-2% of extranodal lymphoma and <5% of thyroid malignancies , and it characteristically occurs in the setting of Hashimoto's disease . PTL is almost exclusively non-Hodgkin's, B-cell lymphoma. The most common subtype of PTL is diffuse large B-cell lymphoma (DLBCL) (50-70% of PTLs), followed by mucosa-associated lymphoid tissue (MALT) lymphoma (10-50%) . The therapeutic strategy of primary thyroid MALT lymphoma is not well-established, in part because of primary thyroid MALT lymphoma's rarity. Treating PTL thus usually follows non-organ-specific guidelines, such as the U.S. National Comprehensive Cancer Network (NCCN) clinical practice guidelines for B-cell lymphomas , in which the standard and preferred initial therapy for limited-stage non-gastric MALT lymphoma is involved-site radiation therapy (ISRT) after an open biopsy (OB-ISRT). The guidelines note that surgery may be considered for certain sites including the thyroid. No staging system for primary thyroid MALT lymphoma exists. The Ann Arbor classification system was initially developed for staging Hodgkin's disease but has some use in non-Hodgkin lymphoma . After the advent of computed tomography (CT), CT was included in lymphoma staging at the 1989 Cotswolds meeting . Fluorodeoxyglucose (FDG) positron emission tomography (PET)/CT technology is now available in many countries, and in the PET/CT era, the staging for non-Hodgkin lymphoma is the Lugano classification , which is based on the Ann Arbor classification. These staging systems have been used clinically for primary thyroid MALT lymphoma, but they cannot distinguish between stage IE and stage IIE appropriately in terms of prognosis, as we have de-scribed . Ito Hospital (Tokyo) specializes in the treatment of thyroid diseases and has long collected clinical data on PTL cases . The objectives of the present study were to (i) establish the appropriate staging system for primary thyroid MALT lymphoma, and (ii) assess the role of curative thyroidectomy in patients with stage IE primary thyroid MALT lymphoma compared to OB-ISRT. 2. Materials and Methods 2.1. Assessment of a Modified Staging System for Limited-Stage Thyroid MALT Lymphoma Study Design and Patients We conducted a retrospective cohort study of patients with limited-stage primary thyroid MALT lymphoma at Ito Hospital (Tokyo) between January 1990 and October 2021 after obtaining Institutional Ethics Board approval (no. 169). Patients with limited-stage MALT lymphoma were included for the modified staging system's assessment. The preferred initial therapy for limited-stage non-gastric MALT lymphoma is OB-ISRT . Our exclusion criteria were thus patients who had undergone (1) a thyroidectomy instead of an open biopsy for diagnosis or (2) initial treatment(s) other than OB-ISRT. The pathological diagnoses were based on the World Health Organization classification of lymphoid neoplasms. 2.2. The Conventional and Novel Staging Systems and the Data Collection Stage IE primary thyroid MALT lymphoma is defined as involvement of the thyroid alone; stage IIE is defined as the major site of involvement being in the thyroid gland with associated regional lymph node involvement (cervical and superior mediastinal areas). To divide limited-stage MALT lymphoma into clinical stage IE and stage IIE, the conventional staging systems, i.e., the Lugano classification and the Ann Arbor classification system with Cotswold modifications , recommend using CT and/or PET/CT. The Lugano classification states that unexplained node enlargement detected by a CT scan and/or increased FDG uptake shown by PET/CT in the regional lymph nodes indicates clinical stage IIE; however, the classification does not mention what size of lymph node is considered an unexplained enlarged lymph node. The conventional staging systems thus involve qualitative and subjective judgments. Moreover, the coexistence of Hashimoto's disease makes it difficult to distinguish 'involved' from 'inflamed' lymph nodes. A minimal axial diameter of 10 mm is generally thought to be the most accurate size criterion for predicting lymph node metastasis . After we held a discussion in Tokyo among leading endocrinologists, a hematologist, endocrine surgeons, a radiologist, and a pathologist regarding the appropriate objective staging system, we defined 'clinical involvement of the regional lymph nodes of the thyroid' as follows as the "Tokyo classification": (1) a minimal axial dia. >=10 mm on CT, and/or (2) increased uptake on 18F-FDG PET and/or 67Ga-citrate scintigraphy (Table 1). For a comparison with this new definition, we collected data on the patients' stages from their medical records in our hospital's PTL database; those stages were based on one of the two above-described conventional staging systems. 3. Comparison of OB-ISRT and Surgery 3.1. Study Design and Patients We next compared surgery (i.e., curative thyroidectomy) alone with OB-ISRT in the stage IE patients defined using the Tokyo classification. Because a thyroidectomy without regional lymph node dissection is not curative for stage IIE, we did not include stage IIE patients in this analysis. Initially, at Ito Hospital, all patients who underwent a thyroidectomy for limited-stage primary thyroid MALT lymphoma also underwent post-operative radiotherapy. After 2012, we provided a therapeutic option: no ISRT after a curative thyroidectomy for stage IE MALT lymphoma. Given this background, the inclusion criteria were patients with stage IE MALT lymphoma who had undergone OB-ISRT or a thyroidectomy alone during the period between January 2012 and October 2021. The exclusion criteria were: (1) patients whose data concerning treatment complications could not be collected (e.g., lack of radiation therapy complication information because the patient underwent an open biopsy at Ito Hospital but ISRT at another institution), and (2) patients who had another disease associated with lymphadenopathy (other than autoimmune thyroid disease [Graves' disease and Hashimoto's disease]). 3.2. Study Variables The patient demographics included age, sex, the coexistence of Hashimoto's disease, hypothyroidism before treatment, and lesion location. We also analyzed variables pertaining to treatment-related complications, the new appearance or change of a low-density area (LDA) on the ultrasound post-treatment, the performance of a biopsy (Bx)/fine-needle aspiration (FNA) or scintigraphy due to the suspicion of recurrence, and cancers after treatment. These data were collected retrospectively from the medical records. Radiation-induced complications were graded using CTCAE ver. 5.0 . Most of the patients with ISRT underwent radiotherapy with 30 Gy at Ito Hospital, but some patients underwent ISRT at other institutions nearby. Figure 1 shows the typical radiation treatment planning for stage IE primary thyroid MALT lymphoma at Ito Hospital. 3.3. Statistical Analyses We used the kh2-test or Fisher's exact test to compare nominal data, and we used the Kaplan-Meier method and stratified log-rank tests for the overall survival (OS) and relapse-free survival (RFS) rates. For all procedures, p-values < 0.05 were considered significant. The statistical analyses were conducted using STATA software ver. 15.0 (Stata, College Station, TX, USA). 4. Results 4.1. The Modified Staging System for Limited-Stage Thyroid MALT Lymphoma The selection process is summarized in Supplementary Figure S1. Between January 1990 and October 2021, 256 patients were diagnosed with limited-stage MALT lymphoma at Ito Hospital, and 119 were excluded from the study based on the exclusion criteria: thyroidectomy performed for diagnosis (n = 59), and 60 patients underwent initial treatments other than ISRT after open biopsy (combined modality treatments, n = 50; observations, n = 4; chemoimmunotherapies, n = 4; and rituximab, n = 2). Thus, 137 patients were included in the analyses. Supplementary Table S1 summarizes these patients' baseline characteristics: median age, 71 years; 85.4% were female; 87.6% had Hashimoto's disease; and 2.9% had Grave's disease. Figure 2 depicts the patients' OS and RFS rates, comparing the rates of the patients with stage IE and stage IIE disease classified with one of the conventional staging systems or the Tokyo classification. When the staging was done with the conventional staging systems, there was no significant difference in the OS of the stages IE and IIE patients (hazard ratio [HR] 3.05, 95% confidence interval [CI]: 0.87-10.69; p = 0.0673), although the OS in stage IIE tended to be worse compared to that in stage IE. The RFS was also not significantly different between stages IE and IIE under the conventional staging systems (HR 1.76, 95%CI: 0.69-4.53; p = 0.232). After the restaging using the Tokyo classification, the 10-year OS rates were 96.8% and 95.0% for stage IE, and 78.1% and 78.1% for stage IIE, respectively (HR 4.29, 95%CI: 1.30-14.14; p = 0.0092). The 10-year RFS rates were 94.7% and 89.7% for stage IE and 74.1% and 69.8% for stage IIE, respectively (HR 3.18, 95%CI: 1.24-8.17; p = 0.00113) under the Tokyo classification. After the restaging using the Tokyo classification, no upstaging was detected, but downstaging from stage IIE to stage IE was observed in 17 patients (33.3%). No relapse was detected in these 17 cases with a median follow-up of 8.1 years. 4.2. OB-ISRT vs. Surgery To assess the advantages/disadvantages of curative thyroidectomy for primary thyroid MALT lymphoma, we next compared surgery alone to OB-ISRT in the stage IE patients. Supplementary Figure S2 summarizes the selection process. Under the Tokyo classification, 73 stage IE patients underwent either OB-ISRT or surgery alone between January 2012 and October 2021. We excluded 13 of these patients based on the exclusion criteria and compared the final totals in the Surgery (n = 14) and OB-ISRT (n = 46) groups. Supplementary Table S2 provides the patients' baseline characteristics. There were no significant between-group differences in age, sex, or the coexistence of Hashimoto's disease. Both radiation therapy and thyroidectomy have treatment-specific complications. For radiation therapy to the cervical area, well-known radiation-induced complications are dermatitis, oral mucositis, dry mouth, dysphagia, esophagitis/pharyngitis, and hoarseness. Possible surgical complications after thyroidectomy are hypoparathyroidism and recurrent laryngeal nerve paralysis (RLNP). Both treatments also pose a risk of thyroid dysfunction. Table 2 and Supplementary Table S3 summarizes the treatment-related complications that occurred in the Surgery and OB-ISRT groups. Regarding the complications other than thyroid function, all 46 patients (100%) in the OB-ISRT group suffered from a treatment-related complication, whereas surgical complications occurred in 36% of the Surgery group (p < 0.001). Radiation-induced esophagitis/pharyngitis (98% of the patients), dysphagia (85%), dermatitis (54%), dry mouth (50%), hoarseness (33%), and oral mucositis (11%) were observed. Almost all of the cases of dermatitis and/or hoarseness were CTCAE grade 1 and did not require any specific treatment, but many of the cases of esophagitis/pharyngitis, dysphagia, dry mouth, and/or oral mucositis were CTCAE grade 2 and needed additional treatment. Most of the radiation-induced complications improved with time, but 28% of patients with OB-ISRT had permanent complications, most of which were dry mouth. One patient in the OB-ISRT group had post-operative bleeding and required re-operation after the open biopsy. In the Surgery group, all cases of surgical complications were transient hypoparathyroidism (incidence rate: 36%), and no patients faced permanent hypoparathyroidism or post-operative RLNP. The incidence of permanent complications other than thyroid function was thus significantly higher in the OB-ISRT group compared to the Surgery group (p = 0.027). The number of painkiller prescription days was significantly greater in the OB-ISRT group vs. the Surgery group (p < 0.001). Concerning complications associated with thyroid function, only 15% and 21% of the patients in the OB-ISRT and Surgery groups, respectively, were euthyroid after treatment (p = 0.685). There were also no significant differences between the groups in terms of the incidence of new hypothyroidism or in the rate of patients whose levothyroxine dose was increased after treatment. The patients' post-treatment courses are described in Table 3. During the follow-up, the rate of a new appearance or change of an LDA in the thyroid gland after OB-ISRT was significantly higher than that after surgery alone (48% vs. 14%; p = 0.031). However, there were no significant differences between the OB-ISRT and Surgery groups in the post-treatment performance of Bx/FNA (15% vs. 7%) or scintigraphy (11% vs. 0%, respectively). After the initial treatment for lymphoma, three patients in the OB-ISRT group had cancers (lung, gastric, and colorectal cancers, respectively). During the follow-up (93.3% follow-up rate with an average follow-up of 5.12 years), none of the patients died, and three OB-ISRT patients had a relapse of MALT lymphoma (two in the thyroid gland and one in a lymph node) . 5. Discussion The results of our analyses demonstrate that the modified staging system (i.e., the Tokyo classification) is suitable for primary thyroid MALT lymphoma compared to the conventional staging systems (Ann Arbor and Lugano), which are non-organ-specific systems. Under the Tokyo classification, both curative thyroidectomy alone and OB-ISRT provided a satisfactory prognosis. Because the morphological and immunohistological findings are sometimes insufficient for a confident diagnosis in terms of regional lymph nodes, the pathological diagnosis of lymph node involvement in MALT lymphoma is more difficult than that of lymph node involvement in DLBCL. Moreover, FDG PET/CT has limited sensitivity in the assessment of MALT lymphoma because of the low metabolic rate of this malignancy and the high rate of concurrent Hashimoto's disease . For these reasons, in patients with primary thyroid MALT lymphoma, it is difficult to determine the benign vs. malignant status of lymph nodes precisely by imaging examinations or by pathological surveys. There is thus a possibility that some of the present patients classified as having stage IE disease under our modified staging system were not truly stage IE (i.e., had lymph node involvement). However, this issue is not a concern in clinical settings, because patients who undergo a curative thyroidectomy without additional therapy for small (<10 mm) lymph nodes have good prognoses. This may be because MALT lymphoma is an indolent and low-grade malignancy. Using the Tokyo classification, 33.3% of the stage IIE cases in this study were restaged as stage IE, and it would thus be possible to reduce the irradiation field size for these patients. There is currently no consensus as to which treatment is the best for stage IE primary thyroid MALT lymphoma. Our present analyses of stage IE patients with primary thyroid MALT lymphoma revealed that both curative thyroidectomy alone and OB-ISRT provided satisfactory overall and relapse-free survival. It is thus reasonable for physicians to present both treatments as therapeutic options to these patients. Because ISRT and thyroidectomy have different types of treatment-related complications, it is difficult to state which treatment is better in terms of complications. Nonetheless, our present findings demonstrate that thyroidectomy alone was superior in terms of permanent complications and pain duration compared to OB-ISRT. In this series, radiation-induced permanent complications (mainly dry mouth) occurred in approx. one in three to four patients. Most patients who have primary thyroid MALT lymphoma are in the seventh and eighth decades of life , and dry mouth is known to cause an impaired quality of life among older people . Our finding that the patients who underwent OB-ISRT used painkillers for a longer period seems to have been caused by the longer-term treatment of fractionated radiation compared to surgery. Regarding thyroid function after treatment, the present results revealed that neither surgery nor OB-ISRT is particularly better than the other treatment. Although an appropriate radiation dose for primary thyroid MALT lymphoma has not been established, the NCCN guidelines recommend 24-30 Gy for MALT lymphoma . In a second analysis of the present patient series, the median radiation dose was approx. 30 Gy (Supplementary Table S2). A reduction of the radiation dose (e.g., to 24 Gy) could thus be considered toward the goal of reducing radiation-associated complications. However, an increase in the relapse rate within the thyroid gland due to a radiation dose reduction may be possible, as a few patients in the present OB-ISRT group had a relapse. As noted in a textbook, the tolerance doses for 5% and 50% xerostomia in 5 years are 32 and 46 Gy, respectively . In the present patient series, though the median radiation dose was ~30 Gy and the irradiation to parotids was minimized in most cases , the incidence of dry mouth was higher than expected. If a patient's clinical features and/or the results of an FNA suggest a lesser possibility of DLBCL, an open biopsy and thyroidectomy tend to be preferred compared to a core-needle biopsy, to ensure that aggressive histologies are not missed since the more limited core-biopsy specimen may not be representative of the entire tumor . These patients thus need to undergo surgery (i.e., open biopsy or thyroidectomy) at least one time. Compared to a thyroidectomy, an open biopsy cannot avoid blind manipulation. For some surgeons, this causes anxiety about injuring organs behind the thyroid such as the trachea. In our present series, a serious surgical complication (Clavien-Dindo classification >=grade III) occurred not in the Surgery group but in the OB-ISRT group. An open biopsy is thus not always a less-invasive and safer surgical procedure compared to thyroidectomy. The potential advantages of a curative thyroidectomy are (i) easy ultrasound follow-up due to the removal of LDA(s) in the thyroid gland, (ii) a more definite removal of lymphoma within the thyroid gland, and (iii) the removal of the risk factor of lymphoma when a total thyroidectomy is performed. After ISRT, an LDA inside the thyroid gland does not disappear soon and remains in some cases. In such cases, careful observation of the lesions is required. For this reason, the removal of LDA(s) by a thyroidectomy provides the possibility of relieving both patients' and physicians' psychological stress caused by LDAs remaining after treatment. Moreover, a curative thyroidectomy may help rule out histological transformation. Rajamaki et al. reported that a rebiopsy based on a high SUVmax in FDG-PET/CT was valuable in detecting the transformation of follicular lymphoma , but it is not yet clear whether FDG-PET/CT can detect the transformation of a primary thyroid MALT lymphoma. Helicobacter pylori plays a major role in the pathogenesis of gastric MALT lymphoma, and antibiotic therapy for H. pylori is recommended for H. pylori-positive gastric MALT lymphoma . By the same principle, the removal of thyroiditis, i.e., a total thyroidectomy, might have potential merit for preventing a relapse inside the thyroid gland because primary thyroid MALT lymphoma characteristically occurs in the setting of Hashimoto's disease. Compared to total thyroidectomy, the advantages of hemithyroidectomy are (i) the avoidance of post-operative hypoparathyroidism and bilateral vocal cord paralysis, and (ii) the possibility of maintaining euthyroid status after surgery. Study Limitations Despite our encouraging results, some study limitations should be noted: (i) We did not assess whether curative thyroidectomy following an open biopsy is an acceptable treatment. (ii) The data on radiation-induced complications were collected from the patients' medical records and not questionnaires completed by the patients. (iii) We did not evaluate the appropriate radiation dose for each stage of primary thyroid MALT lymphoma. All of the patients in the Surgery group underwent one-stage surgery, i.e., a thyroidectomy, for diagnosis and treatment, and none of the patients underwent a thyroidectomy alone after a diagnosis by open biopsy. However, we feel that performing a thyroidectomy after a diagnosis by open biopsy is also an acceptable therapeutic option. In addition, an open biopsy exposes only the front side of the thyroid and does not expose the recurrent laryngeal nerve (RLN); a subsequent thyroidectomy can thus be performed without adhesion around the RLN . We thus do not think that a thyroidectomy after an open biopsy increases the incidence of complications compared to the one-stage surgery. This was a retrospective study and did not use any questionnaires completed by the patients to collect the data on radiation-induced complications. Hospital records usually underestimate the true number of complications. As noted above, we could not assess the appropriate radiation dose with a good balance of reducing radiation-associated complications and avoiding relapse within the thyroid gland because few patients in this series received 24 Gy radiotherapy. Because ISRT is the preferred initial therapy in the NCCN guidelines, the present Surgery group was quite smaller than the OB-ISRT group (14 vs. 46 patients, respectively). 6. Conclusions Our study results demonstrated that the modified staging system (the Tokyo classification) allowed us to distinguish between stage IE and stage IIE. Although our findings regarding ISRT side effects may have limited informative value due to this study's retrospective nature, approx. one in three to four patients had radiation-induced permanent complications (mainly dry mouth) in this series. The use of the Tokyo classification revealed that (i) curative thyroidectomy provides a good prognosis that is equivalent to that provided by OB-ISRT, and it also avoids permanent dry mouth, shortens painful periods during treatment, and simplifies the ultrasound follow-up in stage IE patients; and (ii) approx. three in 10 stage IIE patients were reclassified as stage IE and would thus be able to choose thyroidectomy alone as a therapeutic option, although, in the NCCN guidelines, ISRT is the preferred initial therapy for limited-stage non-gastric MALT lymphoma. Further research is necessary to determine the precise potential implications of both curative thyroidectomy and the clinical usage of the Tokyo classification. Supplementary Materials The following supporting information can be downloaded at: Table S1. Baseline characteristics of the patients with OB-ISRT (n = 137). Table S2. Comparison of the baseline characteristics and initial treatment characteristics between the OB-ISRT and Surgery groups. Table S3. Details of permanent complications in the patients with OB-ISRT (n = 13). Figure S1. Flow chart of patient enrollment in the first study for the assessment of a modified staging system. CMT: combined modality therapy, ISRT: involved site radiation therapy, MALT: mucosa-associated lymphoid tissue. Figure S2. Flow chart of patient enrollment in the second study for the comparison of OB-ISRT and surgery. RT: radiation therapy. Click here for additional data file. Author Contributions Conceptualization, Y.S., N.W., N.S. (Naoko Saito), H.N., and K.K.; methodology, Y.S., N.W., N.S. (Naoko Saito), H.N., and K.K.; validation, Y.S., N.W., N.S. (Nami Suzuki), N.S. (Naoko Saito), H.T., K.K., K.Y., C.M., J.A., K.Y.H., M.M., M.F., A.Y., R.O., C.T., A.S., K.M., W.K., M.N., J.Y.N., K.S., and K.I.; formal analysis, Y.S.; data curation, N.W. and N.S. (Nami Suzuki); writing--original draft preparation, Y.S.; writing--review and editing, N.W., N.S. (Nami Suzuki), N.S. (Naoko Saito), H.T., K.K., K.Y., C.M., J.A., K.Y.H., M.M., M.F., A.Y., R.O., C.T., A.S., K.M., W.K., M.N., J.Y.N., K.S., and K.I.; visualization, Y.S.; supervision, K.I.; project administration, N.W. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional Ethics Board of Ito Hospital (no. 169. Approval date: 27 August 2016). Informed Consent Statement Informed consent was obtained from all subjects involved in the study. Data Availability Statement The study's data are available upon request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Typical radiation treatment planning for stage IE primary thyroid mucosa-associated lymphoid tissue (MALT) lymphoma at Ito Hospital. Tables of color correspondence (A) Radiation dose distribution on the treatment planning CT image. The lower right is the corresponding table between color lines in (A-C) and structure names. Orange line: the clinical target volume (CTV). Red line: the planning target volume (PTV). (B) Beam's eye view demonstrates that the radiation protocol for stage IE minimizes salivary gland irradiation outside of the PTV. (C) The cumulative dose-volume histogram (DHV) also shows that the radiation doses toward the right and left parotids (yellowish-green curves) are minimized. Figure 2 The overall survival (OS) and relapse-free survival (RFS) curves for the patients with stage IE and stage IIE primary thyroid mucosa-associated lymphoid tissue (MALT) lymphoma classified with the conventional staging systems (A,B) and the modified staging system (the Tokyo classification) (C,D). * p < 0.05. + p < 0.001. Figure 3 Relapse-free survival curves for the stage IE primary thyroid MALT lymphoma patients who underwent OB-ISRT and those who underwent Surgery after an open biopsy or thyroidectomy. cancers-15-01451-t001_Table 1 Table 1 Modified staging system for primary thyroid MALT lymphoma: The Tokyo Classification. Stage Definition CT-Based Staging PET/CT-Based Staging a IE Involvement of the thyroid alone Minimal axial dias. of all regional lymph nodes are <10 mm No regional lymph node with increased FDG uptake IIE Major site of involvement in the thyroid with associated regional lymph node A minimal axial dia. of a regional lymph node is >=10 mm Increased FDG uptake in a regional lymph node a In the cases in which PET/CT is not available (such as old cases), using 67 Ga-citrate scintigraphy instead is acceptable. CT: computed tomography, FDG: fluorodeoxyglucose, MALT: mucosa-associated lymphoid tissue, PET: positron emission tomography. cancers-15-01451-t002_Table 2 Table 2 Comparison of treatment-related complications between the OB-ISRT and Surgery groups. OB-ISRT n = 46 Surgery n = 14 p Complications without thyroid function Any complications 46 (100) 5 (36) <0.001 + Radiation-induced complications, n (%): G1/G2/G3 a Dermatitis 25 (54): 24/1/0 - Oral mucositis 5 (11): 0/5/0 - Dry mouth 23 (50): 5/18/0 - Dysphagia 39 (85): 9/30/0 - Esophagitis/Pharyngitis 45 (98): 4/41/0 - Hoarseness 15 (33): 12/2/1 - Complications associated with open biopsy Post-operative bleeding 1 (2) Surgical complications Transient hypoparathyroidism - 5 (36) RLNP - 0 (0) Permanent complications 13 (28) 0 (0) 0.027 * Prescribed painkillers, days 12 (3, 18) 2 (2, 5) <0.001 + Complications associated with thyroid function Euthyroid after treatment 7 (15) 3 (21) 0.685 New hypothyroidism 16 (35) 7 (50) 0.305 Increased LT4 dose after treatment 31 (67) 10 (71) 1.000 Data are number (%) or median (IQR). * p < 0.05. + p < 0.001. a CTCAE ver. 5.0 grade 1, 2, and 3. CTCAE: Common Terminology Criteria for Adverse Events, IQR: interquartile range, LT4: levothyroxine, OB-ISRT: involved-site radiation therapy after open biopsy, RLNP: recurrent laryngeal nerve paralysis. cancers-15-01451-t003_Table 3 Table 3 Comparison of the post-treatment course between the OB-ISRT and Surgery groups. OB-ISRT n = 46 Surgery n = 14 p New appearance or change of low-density area on US 22 (48) 2 (14) 0.031 * Suspected recurrence 8 (17) 1 (7) 0.671 Performance of Bx or FNA due to suspicion of recurrence 7 (15) 1 (7) 0.667 Performance of scintigraphy due to suspicion of recurrence 5 (11) 0 (0) 0.329 Cancers other than lymphoma after treatment 3 (7) 0 (0) 1.000 Data are number (%). * p < 0.05. Bx: biopsy, FNA: fine needle aspiration, OB-ISRT: involved-site radiation therapy after open biopsy, US: ultrasound. 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PMC10000774 | Foods Foods foods Foods 2304-8158 MDPI 10.3390/foods12051010 foods-12-01010 Article Analytical Purity Determinations of Universal Food-Spice Curcuma longa through a QbD Validated HPLC Approach with Critical Parametric Predictors and Operable-Design's Monte Carlo Simulations: Analysis of Extracts, Forced-Degradants, and Capsules and Tablets-Based Pharmaceutical Dosage Forms Mohammed Hamdoon A. Methodology 12 Alsahabi Dhafer S. Methodology Data curation 3 Hegazy Amira M. Software 4 Khan Riaz A. Methodology 1* Ahmed Adel M. Validation 5* Beccaria Marco Academic Editor De Luca Chiara Academic Editor 1 Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, Qassim University, Qassim 51452, Saudi Arabia 2 Department of Pharmacognosy and Medicinal Plants, Faculty of Pharmacy, Al-Azhar University, Cairo 11371, Egypt 3 PharmD Graduate, College of Pharmacy, Qassim University, Qassim 51452, Saudi Arabia 4 Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, Beni-Suef University, Beni-Suef 62574, Egypt 5 Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, South Valley University, Qena 83523, Egypt * Correspondence: [email protected] (R.A.K.); [email protected] (A.M.A.) 27 2 2023 3 2023 12 5 101014 12 2022 24 1 2023 30 1 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). Applications of analytical quality by design (QbD) approach for developing HPLC (High Performance Liquid Chromatography) methods for food components assays, and separations of complex natural product mixtures, are still limited. The current study developed and validated, for the first time, a stability-indicating HPLC method for simultaneous determinations of curcuminoids in Curcuma longa extracts, tablets, capsules, and curcuminoids' forced degradants under different experimental conditions. Towards separation strategy, critical method parameters (CMPs) were defined as the mobile phase solvents' percent-ratio, the pH of the mobile phase, and the stationary-phase column temperature, while the peaks resolution, retention time, and the number of theoretical plates were recognized as the critical method attributes (CMAs). Factorial experimental designs were used for method development, validation, and robustness evaluation of the procedure. The Monte Carlo simulation evaluated the developing method's operability, and that ensured the concurrent detections of curcuminoids in natural extracts, commercial-grade pharmaceutical dosage-forms, and the forced degradants of the curcuminoids in a single mixture. The optimum separations were accomplished using the mobile phase, consisting of an acetonitrile-phosphate buffer (54:46 v/v, 0.1 mM) with 1.0 mL/min flow rate, 33 degC column temperature, and 385 nm wavelength for UV (Ultra Violet) spectral detections. The method is specific, linear (R2 >= 0.999), precise (% RSD < 1.67%), and accurate (% recovery 98.76-99.89%), with LOD (Limit of Detection) and LOQ (Limit of Quantitation) at 0.024 and 0.075 mg/mL for the curcumin, 0.0105 mg/mL and 0.319 mg/mL for demethoxycurcumin, and 0.335 mg/mL and 1.015 mg/mL for the bisdemethoxycurcumin, respectively. The method is compatible, robust, precise, reproducible, and accurately quantifies the composition of the analyte mixture. It exemplifies the use of the QbD approach in acquiring design details for developing an improved analytical detection and quantification method. drug screening chemical stability quality by design curcuminoids full factorial design Monte Carlo simulations critical method parameters critical method attributes method operable design region Plackett-Burman design This research received no external or internal funding. pmc1. Introduction The universal spice turmeric, botanically named Curcuma longa, family Zingiberaceae, is well known as the curry-coloring spice. It is frequently used as the commonest food-additive throughout the globe. Its use is much prevalent in the oriental worlds of India, China, Indo-China, and the Mediterranean regions. The dried root powder of the plant is commercially available at groceries and spice shops. The herb powder is consumed for several culinary purposes to induce color, spiciness, taste, fragrance, and flavor . The edible herbal powder possesses several biological activities, i.e., anticancer, antimicrobial, antiviral, anti-inflammatory, and wound-healing . Curcuminoids are the main constituents in root powder and are considered responsible for the pharmacological efficacy of the powder. The primary constituents are curcumin, demethoxycurcumin, and bisdemethoxycurcumin , while different structural degradants have also been identified . The structural similarities among the phytoconstituents, and the sensitivity of the Curcuma powder exposure to light, moisture, and other unnatural and forced conditions, including the powder's proneness to degradation over time, lead to compositional changes . This situation has raised issues about the purity, efficiency, and overall quality of the powder for human consumption. Several studies related to analytical purity, composition profile of the powder, and curcumin-based commercial products containing curcumin and other curcuminoids, including their degradants, have led to various investigations for developing isolation-purification, impurity profiling, structural analyses, componential assays, and contents-tracing through spectro-analytical and chromatographic techniques development. A number of reports on HPLC (High Performance Liquid Chromatography) analyses of the Curcuma powder under different experimental conditions, including bio-analytical procedures, instrumental backgrounds, and mobile and stationary chromatographic phases are known . The current approach aimed to develop a QbD (Quality by Design) analytical purity analysis method with different factors playing a part in the separation of compounds of the Curcuma powder mixture. These critical factors in conjunction with simulations of the resolution phenomenon and methods design approach indicators were employed to arrive at an optimally resolved chromatographic analysis of the Curcuma powder components, and their degradants. The current method also aimed to analyze the purity conditions of the commercially available Curcuma powder formulations of tablets and capsules. Nonetheless, a plethora of work has recommended the HPLC technique with UV (Ultra Violet) spectrum-based detections, as one of the best-suited analytical methods for analyzing the high contents of curcuminoids in the curcumin powder, and its different formulation forms. However, certain limitations related to a comparatively longer time of analysis as a consequence of longer retention time, the need for a complicated gradient-elution-based mobile phase solvent system, higher margins in LOD (limit of detection), and high mobile-phase flow rates , have necessitated improvements in the HPLC-based analytical methodology. Another analytical target needing attention is the simultaneous detection of curcuminoid products, namely, curcumin, demethoxycurcumin, and bisdemethoxycurcumin. The detection of curcuminoids degradants, i.e., ferulic acid, ferulic aldehyde, ferulic methane, and vanilla, are also some of the most common degradation products needing attention. An approach was developed to overcome the separations, and detection pitfalls underplaying in the curcumin's components resolution, retention time, and possibilities in the gradient-based mobile phase's componential variability for its effective and optimum flow rate, as well as temperature, and pH fluctuations. The critical method parameters (CMPs), i.e., the resolutions among the components, their retention times, the number of involved theoretical plates, and the method's operable designs, are some of the parameters that were considered as the factors in the method development. For experimental set-up, for example, pH, temperature effectiveness in the stationary phases during separations, and the mobile phase components' variability were selected to find an optimized region of maximum operability conditions for the analytical method. The method development was formulated to achieve the analytical target profiles of the curcuminoid products and their degradants in the separation. Monte Carlo simulations validated the critical parameters for the developing method and its design factors. It paved the way for the QbD approach to a validated method development, which was needed for making the procedure capable of resolving the closely eluted components, and for better quantitation in the given curcumin powder of fresh and degraded samples. The analytical target profiling of these separations of the curcuminoid products and their degradants was achieved through the designated HPLC-based separations. It also incorporated the critical method attributes (CMAs), leading to the identification of the associated CMPs responsible for better separations, through maneuverability in the resolution, retention times, and the conditions of pH, as well as the flow-rate and polarity controls through the composition of the mobile phase. The adeptness to the variability of the parameters on separation, the method's working feasibility, separation out-reach of the method without losing the analytical targets, precision, and robustness were addressed and confirmed through the quality risk assessment, a key component of the QbD approach. The approach was traversed through the CMPs. The Method Operable Design Region (MODR) factors for the risk assessment of the quality components were also investigated. The MODR approach was used for the development of an RP-HPLC (Reverse Phase High-Performance Liquid Chromatography) method for the simultaneous determinations of the curcuminoids, and their degradants. The starting step was to examine, and extrapolate the prior knowledge about the analytical behavior of these products, and define the analytical target profile (ATP) of the products under investigation. The quality risk-assessment tools were used to define the CMPs through the application of the design of experiment (DoE) way, which resulted in defining the MODR in the ensuing analysis for developing the analytical separation, and detection, with clear limits of detections. The robustness of the method was achieved with a well-defined design space, together with a viable analytical procedure to analyze the curcuminoids, their degradants in the commercial powders, different dosage forms, and other pharmaceutical formulations, including the nutraceutical/food-additive samples. 2. Materials and Methods 2.1. Chemicals and Reagents All reagents used in this study were of analytical grade. Acetonitrile (ACN) and methanol (MeOH) were purchased from Fisher Scientific; Milli-Q water used to prepare buffer solutions was obtained by a Millipore(r) purification system (Bedford, MA, USA). K2HPO4, phosphoric acid, sodium acetate, and acetic acid were purchased from Merck (Darmstadt, Germany); turmeric rhizome was purchased from the herbal drug market. Standards of curcumin, demethoxycurcumin, and bisdemethoxycurcumin (>98% in purity) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 2.2. Extraction of Curcuma longa Constituents High-grade Curcuma longa was purchased from the local market as rhizomes and reduced to a fine powder using a mechanical device. The extraction procedure was conducted to measure the effect of the solvent on the chromatographic performance of the analysis method. Acetone and ethanol (100 mL) were selected separately for extractions of two sets of 1.0 g Curcuma longa powder for 20 min, using an ultrasound bath (40 kHz and 135 W, model Max Clean 1400, Unique, Sao Paulo, Brazil). After completing the volume up to the mark with the mobile phase, 2.0 mL was diluted with the mobile phase into a 10 mL volumetric flask. The samples were then filtrated through a 0.45 um nylon syringe (Sinergia Cientifica, Campinas, Brazil), and packed into HPLC vials before injection into the HPLC system. 2.3. Preparation of Stock Solutions The methanolic stock solutions of curcumin, demethoxycurcumin, and bisdemethoxycurcumin (10.0 mgmL-1) were prepared separately in methanol, and the standard working solutions were prepared through dilution with the mobile phase. Stock solutions were stored in an amber flask at -40 degC until chromatographic analysis. 2.4. Preparation of Sample Solution 2.4.1. Curcumin Capsules Five capsules (Solgar(r) (Leonia, NJ, USA) containing full spectrum curcumin as soft gel capsules containing 48 mg of curcuminoids) were accurately weighed, and mixed with 20 mL of methanol in a 100 mL volumetric flask. The mixture was sonicated for 20 min, and after cooling, the solution was completed to the volumetric mark with the mobile phase. A total of 2.0 mL from the solution was diluted with the mobile phase into a 100 mL volumetric flask. The sample was filtered through a 0.45-mm before its injection into the HPLC system. 2.4.2. Curcumin Tablets Five tablets (Longvida(r), Noblesville, IN, USA) with optimized curcumin containing 1000 mg of turmeric) were accurately weighed, and finely powdered. 1.0 gm of powder was weighed and transferred to a 100 mL volumetric flask, to which 20 mL of methanol was added. The mixture was sonicated for 20 min, and after cooling, the solution was completed up to the flask's mark with the mobile phase. A total of 2.0 mL of the solution was placed in a 10 mL volumetric flask and diluted with the mobile phase. The solution was finally filtered using a 0.45 mm membrane filter before its injection into the HPLC system. 2.5. Chromatographic Conditions Chromatographic analyses were performed on an Alliance 2685/2489 separation system (Waters, Milford, CT, USA). Chromatographic separations were achieved using a Waters Symmetry(r) C18 column {150 mm x 4.6 mm, 5 mM (meter) particle size}. The appropriate wavelength for HPLC analysis of the curcuminoids was selected using Varian Cary 50 Spectrophotometer which provided the full UV-VIS (Ultra Violet-Visible) spectra (200-800 nm). Based on that, 385 nm was selected as the detection wavelength, and the injection volume was kept at 10 mL. The final selected working condition, where the mobile phase consisted of a mixture of ACN-K2HPO4 with pH adjusted 2.7, with a 54:46, v/v ratio. The flow rate was set at 1.0 mL min-1, and the column temperature was kept at 33 degC. 2.6. Forced Degradation Studies 2.6.1. Acid and Base Degradations The acid and base degradations were performed by transferring 1.0 mL of curcuminoids stock solution (1.0 mg mL-1) to a 10.0 mL amber volumetric flask, and diluting with 1 mL of 1.0 M HCl, and 1 mL of 1.0 N NaOH (separately). The flasks were placed at 80 degC for 2 h in the dark. After that, the solution was cooled, neutralized with 1.0 mL of 1.0 N NaOH, or 1.0 mL of 1.0 N HCl, diluted with the mobile phase, filtered through a 0.45 mm syringe filter, and injected into the HPLC system. 2.6.2. Sunlight-Exposed Degradation A 1.0 mL of the stock solution (1.0 mg mL-1) was placed with 1.0 mL distilled water in a 10 mL transparent volumetric flask. The flask was placed under sunlight for 6 h. After that, the solution was cooled and filtered through a 0.45 mm syringe filter, before its injection into the HPLC system. 2.6.3. Oxidative Degradation Oxidative degradation was performed by mixing 1.0 mL of the stock solution (1.0 mg mL-1) with 5.0 mL of hydrogen peroxide 5% solution. The solution was kept at RT (Room Temperature) in the dark for 4 h. After that, the solution was filtered through a 0.45 mm syringe filter, before its injection into the HPLC system. 2.6.4. UV Irradiation Degradation A total of 1.0 mL of curcuminoids stock solution (1.0 mg mL-1) in a 10 mL transparent volumetric flask was exposed to UV light (365 nm) in a photo-stability chamber for 4 h. After that, the solution was filtered through a 0.45 mm syringe filter before its injection into the HPLC system. 2.6.5. Thermal Degradation Curcuminoids powder was stored in an oven at 80 degC for 2.0 h to study dry-heat degradation. After that, the solution (1.0 mg mL-1) was prepared and diluted before its injection into the HPLC system. 2.6.6. Validation of the Method Validation studies were conducted using the optimized assay conditions according to ICH guidelines Q2 (R1) concerning linearity, detection limit, quantitation limit, accuracy, precision, and robustness. 2.7. Software Empower-2 software was used for monitoring the HPLC signals. Design-Expert (r) 8.0.7.1 (Stat-Ease Inc., Minneapolis, MN, USA) was used for experimental design, data analysis, and calculations of desirability functions. Companion software (M/s Minitab Inc., State College, PA, USA) was used to perform Monte Carlo simulations. 3. Results and Discussion 3.1. Curcumin, and Curcuminoids' Physico-Chemical Properties, and Elution Profile Curcuminoids are polyphenolic compounds composed of two differently placed ortho-methoxyphenyl moieties connected by an a,b-unsaturated-b-diketone structural part . At the neutral pH and RT, the curcuminoids are water-insoluble, but they exhibit solubility under alkaline conditions. The products are relatively unstable and degrade rapidly upon exposure to sunlight as well as at neutral and alkaline conditions. However, the products are comparatively more stable under acidic conditions . The environmental, naturally initiated, and forced-degradation products were identified as vanillin, ferulic acid, ferulic aldehyde, and ferulic (feruloyl) methane, together with auto-oxidized dicyclopentadiene as the degradants in the curcuminoids powder. The relative concentrations of these degradants differed according to the conditions, and durations of the conditions of pH and temperature, as also observed earlier . The lipophilic nature, the levels of lipophilicity, solubility behavior, partition coefficients of the products, and degradants played an important part in the separation of these components in the powdery mixture analysis. A better understanding of the retention times of the products was assessed by the MarvinSketch(r) software v 20.11.0. (Marvin (chemaxon.com), which predicted the lipophilicity, log P, and distribution coefficients, log D, of the curcuminoid products. The curcumin showed log P at 4.12, log D at 3.76, the demethoxycurcumin's log P at 4.28, and log D at 3.92, while the bisdemethoxycurcumin's log P was predicted to be 4.44, and log D was predicted as 4.44, which suggested the elution order of these products from the RP-HPLC, wherein the bisdemethoxycurcumin suggested to be eluted first, followed by demethoxycurcumin, and lastly the curcumin. The predicted values of log P and log D for the expected degradations ranged between 1.18 and 2.14, and 1.22 and 2.14, respectively, and it was indicated that the low polarity degradation products would elute before the curcuminoid products . As mentioned, the acidic pH is better at solubilizing, and is suitable for best separation on the RP-HPLC column owing to the higher pKa values; e.g., for curcuminoids, it ranged from 8.38 to 10.41. Furthermore, a literature survey revealed that pH 3.0 was frequently used for the separation of curcuminoids . Henceforth, a range of pH between 2.5 and 3.5 was chosen for further investigations. Preliminary experimentations on ACN, and methanol, as an organic modifier of the mobile phase, showed better results with ACN. Potassium phosphate was tested as a buffer, and a 10 mM concentration was selected for the separation purpose. The influence of the maximum wavelength absorbance and mobile phase flow rate was examined, and the best results were obtained at a 1.0 mL min-1 flow rate with 385 nm wavelength for detection. 3.2. Analytical Target Profiling The analytical target profile (ATP) was developed and validated to be a robust, efficient, and stability-indicating method, with accuracy > 99%, and a precision limit <2% for the simultaneous separations of the curcuminoids (RS >= 1.2), with the shortest possible analysis time in the run-of of the RP-HPLC during the study. The CMAs were analyzed for material resolution between the curcumin and demethoxycurcumin, together with separations between the demethoxycurcumin and bisdemethoxycurcumin, respectively, as denoted by resolution 1 (RS1) and RS2. The retention time (RT) of curcumin (RTC), the retention time of the demethoxycurcumin (RT-DMCMN), and the retention time of bisdemethoxycurcumin (RT-BDMCMN), as well as the number of theoretical plates for curcumin separation (TPCMN), and the number of theoretical plates required for demethoxycurcumin (TPDMCMN) and bisdemethoxycurcumin (TPBDMCMN) separations, were all considered . For the beforehand test, the fallibility of these CMAs and CMPs was chosen for the RP-HPLC run, and a quality risk assessment (QRA) was performed on the parameters. The QRA work helped to ensure the achievement of the intended analytical target profile of well-resolved conditions for the HPLC run, with proper separations of the peaks, and the feasibly separated retention times, wherein the supportive roles of the mobile phases were also envisioned. These requisites were validated through the QbD approach by predicting the probable fallacy of the method in any of the domains of these parameters to achieve the superior resolution, as the method was intended to achieve . To identify the risk associated with the approach involving the CMPs in achieving the target analytical profile, an Ishikawa diagram was developed . It was determined that the pH of the mobile phase, column temperature, and the gradient mobile phase's composition as the ratio of the organic solvent were among the critical parameters supporting the requisite. The DoE (Design of Experiment) methodologies were employed to study the suggested parameters (Table 1). These parameters, with their settings of the level of operation, were selected for the QRA. A 23 full fractional design (FFD) with a total of eight experiments and three center points was planned and conducted, wherein each run was repeated in triplicate. All the runs were randomized to avoid systematic error . The design expert software was used to analyze the obtained data (Table S1). The ANOVA was performed, and the results (Table 2) showed that all F-values were large enough, which confirmed that the models were significant. The obtained p-values (< 0.0001) indicated that the models explained a significant portion of the variability. The adj. R2 (adjusted R-squares) were higher than 0.987, and that indicated that the experimental data had a good fit with the second-order polynomial, and the grouping variables were approximately 98.7% of the variation observed. The adequate (adeq.) precision values were greater than 4, and therefore the models were relevant for the separation process. The coefficient of variation (CV) for all the models was less than 10%, thereby indicating feasible and good model reproducibility. The regression's lack of fit was determined by performing an F-test, and a good fit was assumed for all models, as the p-value was >0.0779 (p-value for lack of fit testing need be >0.05, non-significant). 3.3. Method's Risk Assessments The pH suggested a positive effect on the retention times of all the curcuminoids, while the column temperature and percentage of ACN had negative effects. The column temperature exerted a positive effect on the resolutions (RS1, RS2), while the buffer pH had negative effects. The number of theoretical plates was suggested to negatively affected the resolutions and retention times of all the curcuminoids (Table S2). An optimization step to gauge the predictability of the experimental region of the CMPs was run using Derringer's desirability functions. The best value for each parameter was optimized to find the desirable chromatographic conditions . For better visualization of the results (Table 3), a 3D plot of the overall desirability function is presented in Figure 3. 3.4. Method's Critical Parameters and Design Space An optimized value for the CMPs was established. A design space was generated that overlaid all the contours of the predicted as well as the acceptable limits within the design range and the allowed limits. The yellow region refers to the area in which all the CMPs were bound within the designed and predicted values of the parameters. The optimized design space overlaid (yellow) area was selected, corresponding to intersects, to choose the method's operable design's region (MODR), also defined by pH (2.5-2.9), temperature (32-35 degC), and the acetonitrile composition (54-57%) as represented in the blue box for the choice of the parameters validated these inputs, wherein the predictive errors were computed through the probability to reach the desired objectives of the experimental levels of the CMPs, as well as the accepted limits of the method's attributes by evaluating the capacity index (Cpk) value of the process . 3.5. Monte Carlo Simulations, Plackett-Burman Design, Robustness, LOD, and LOQ The MODR is considered an area of robustness , wherein each point can be chosen as a working point; however, the practical considerations are of utmost priority as they fall within the common area between MODR and the designated experimental condition decided based on these designs encompassing the MODR. Some verification points (test points) were chosen before selecting a working point to verify the prediction. The practicability of the parameters was established from the obtained specifications within the design space limitations. The choices were guided through the temperature gradient feasible to be practically adopted later. In this overall context, the optimum conditions were selected whereby the mobile phase was composed of 54% acetonitrile (46% water) containing 10 mM phosphate buffer, pH at 2.7, and the column temperature at 33 degC. Figure 6 showed the experimental result of the RP-HPLC run (full-time run, 20 minutes chromatogram, view available in Supplementary File), and the resultant chromatogram under these designed, simulated, and validated conditions. The robustness of the experimental method was again tested by employing the Plackett-Burman design (Table 5), wherein the effectiveness of the four critical method parameters was crisscrossed on eight critical method attributes. The selected parameters, i.e., the pH of the mobile phase (2.5-2.9), the ACN percentage (54-57%), column temperature (32-35 degC), and the buffer concentrations (8-12 mM), showed that in all the experiments, the effects of small changes in all of these parameters were producing insignificant effects (p-value > 0.05), and therefore, the method was considered robust. The accuracy and precision of the designed and tested method were experimentally evaluated in the HPLC runs by injecting different concentrations of the curcumin (1, 10, 20 mg/mL), desmethoxycurcumin (1, 5, 9 mg/mL), and bisdemethoxycurcumin (1, 2, 4 mg/mL) in triplicate. The averaged area was determined, and the % recoveries and % RSD were calculated (Table S3). The obtained % of RSD was less than two, thus indicating a good precision of the method. The good recoveries (98.76-99.89%) from the method also suggested an excellent accuracy of the developed method. The linearity of the developed method was established by analyzing the standard solutions, also in triplicate, of curcumin, demethoxycurcumin, and bisdemethoxycurcumin. Regression analysis showed good linearity, as indicated by the correlation coefficient values (>0.999). The LOD and LOQ were calculated as 3.3 s/s for LOD and 10 s/s for LOQ, where s is the standard deviation of the response and s is the slope, determined from the corresponding calibration curve. From Table 6, the LOD was determined as 0.024, 0.0105, and 0.335 mg mL-1 for the curcumin, demethoxycurcumin, and bisdemethoxycurcumin, respectively, whereas LOQ was found to be 0.075, 0.319, and 1.015 mg mL-1 for curcumin, demethoxycurcumin, and bisdemethoxycurcumin, respectively. These values indicated the acceptable sensitivity of the method to analyze the curcumin, demethoxycurcumin, and bisdemethoxycurcumin. 3.6. Previous Works, Present Method, and Analysis of Samples Moreover, the present work was also compared with previous reports. The comparisons concerning the stationary phase, retention time, elution mode, detection wavelength, and limit of detection were carried out. The analysis is presented in Table 7, which showed that most of the reported methods used 1.5-2.0 mL min-1 as flow rates and consumed moderate quantities of expensive HPLC-grade solvents. The retention time of these methods ranged between 11 and 28 min . In retrospect, these methods are expensive and time-consuming. Additionally, in most cases the LOD is not provided , probably, owing to the poor separation. In some cases, the peaks were also not well resolved and the detection limits were unacceptably high. The separation time was 2.5 min when the UPLC system was used, but this system is expensive and not available in most of the labs. Additionally, in one method , the separation time is 3.0 min on the accucore(c) column at high column temperature (40 degC) with the use of a high percentage of organic solvent. foods-12-01010-t007_Table 7 Table 7 A comparison between the reported methods of curcuminoids separation by HPLC with the present work. No. Column RT Elution Mode Detection Wavelength Drawbacks Ref. 1. Partisphere5 WCX 21.0 Isocratic 280 nm High separation time; catalytic effect of stationary phase; forced degradation; photo-oxidative; no detection limit 2. C18 14.0 Gradient 250, 425 nm Carried at 48 degC; curcuminoids as one peak; no detection limit 3. C18 7.0 Isocratic 425 nm low resolution; no detection limit 4. C18 10.0 Isocratic 420 nm High separation time 5. TSK-GEL, ODS 80Ts 10.0 Isocratic 405 nm Detection limits not provided 6. C18 3.0 Isocratic 420 nm Poor base-line; overlapping peaks 7. C18 15.0 Isocratic 425 nm High separation time; high cost 8. C18 28.0 Isocratic Fluorescence High separation time; high cost 9. C18 2.5 Gradient 420 nm Expensive instrument; gradient elution 10. C18 11.0 Gradient 420 nm Gradient elute; no detection limit 11. C18 17.0 Gradient 426 nm High separation time; no detection limit 12. C18 12.0 Isocratic 425 nm Curcuminoids as one peak 13. C18 14.0 Gradient 244, 360 nm Retention times too close; gradient elution; detection limits not given 14. Chromolith 21.0 Isocratic 425 nm High retention time; high cost; retention times too close 15. C18 20.0 Isocratic 425 nm High retention time; Forced degradation studies: Acid/base/oxidation/thermal/photo 16. C18 and Accucore(r) 3.0 Isocratic 428 nm The high percent of organic modifier, column's high temp 17. RP-phenyl 10.5 Isocratic 360 nm High retention time 18. C18 18.0 Gradient 420 nm High retention time; complex gradient 19. C18 8.0 Isocratic 370 nm Only for CMN 20. C18 16.0 Isocratic 420 nm High retention time; high cost; no stability studies 21. Fused C18 3.0 Gradient 240-600 Complex gradient; no stability studies 22. C18 24.5 Isocratic 425 nm High retention time; high cost 23. C18 11.0 Gradient 425 nm High retention time; high cost; complex gradient 24. C18 12.0 Isocratic 425 nm High retention time; high column temp 25. C18 8.0 Isocratic 425 nm Long time for column conditioning and baseline stability; curcuminoids as one peak; Degradation studies: Acid/base/oxidation/thermal/ photo 26. C18 3.0 Gradient 425 nm Complex gradient; high column temp 27. C18 12.0 Gradient 420 nm High retention time; complex gradient On the other hand, the current method is stability indicating, which showed sharp peaks with good resolution, although, for certain analyses, the resolution was less clear, probably owing to the column's stationary phase materials' physico-chemical conditions, which are not a contributor from the QbD approach, and different factors considered, including the mobile phase characteristics. Additionally, the current approach needed a short experimental time and provided a new detection wavelength. The implementation through concepts of the QbD approach made the current method novel, as well as owing to its tested workability based on design concepts through Method Operable Design Region (MODR) analysis. The pharmaceutical dosage forms of the curcumin formulations were also tested for their chromatographic behavior. Solgar(r) (full spectrum soft gel curcumin capsules x 5) containing 48 mg of curcuminoids, and Longvida(r) (optimized curcumin tablets x 5) containing 1 g of turmeric powder were analyzed and compared with the ethanol and acetone extracts of the commercially available Solgar(r) product. A comparative analysis of the Solgar(r) product, and acetone and ethanol extracts of the commercial powder showed higher RT respectively at 5.90 and 5.80 minutes for the curcumin, and RT for demethoxycurcumin and bisdemethoxycurcumin was observed at 5.37 and 4.86 min., respectively, for the Solgar(r), and acetone and ethanol extracts of the commercial powder. The componential weight ratio of the Solgar(r) product was lesser than the commercial powder compounds, demethoxycurcumin, and bisdemethoxycurcumin . The acetone and ethanol extractions showed no variation in the contents ratio. A comparison of the currently developed and reported methods of analytical and viability studies is presented in Table 8 for further comparison. An absence of new peaks in the pharmaceutical dosage forms chromatograms indicated that no interference was present from the additives and excipients of the formulation. As observed, the quantities of the three curcuminoids were variable, but in the same trend of occurrence as the curcumin, desmethoxycurcumin, and bisdemethoxycurcumin, respectively, in all the samples wherein the contents were found present in the ratio of curcumin > demethoxycurcumin > bisdemethoxycurcumin, as also present in the commercial curcumin powder extracts by the acetone and ethanol and the Solgar(r) sample. The comparison further validated the method's reproducibility and non-interference of the capsules and tablets' additives and excipients present in the formulations. The forced-degradation studies were also carried out on the curcumin powder's extract to produce the possible degradants and test their chromatographic behavior using the developed HPLC method in a simultaneous run of the method, and check its workability under the presence of other degradant materials obtained through various degrading conditions. The acid-induced degradants showed small-scale degradation (4.92%) with degradation peaks at 2.06 and 2.85 min, while the base-catalyzed degradation products were represented in Figure 7b, wherein a high level of degradation (79%) was observed, which also produced a new peak at 2.86 min. The thermal degradation of curcumin showed much less degradation (4.21%) and produced new peaks showing up at 1.74-3.60 min retention time in the chromatogram . Under sunlight, a high degradation of curcuminoids was observed (47.2%), while oxidative conditions upon reacting with H2O2 produced 11.16% of degradation that were detected between 2.34 and 3.59 min of retention times. The UV-irradiation-based degradation was the lowest . The degradation of curcuminoids under different stress conditions was evaluated to develop a stability-indicating method for the simultaneous determination of the curcuminoids in the Curcuma longa extract and pharmaceutical dosage forms. The acidic hydrolysis of the curcuminoids showed a small degradation (4.92%), and the degradation peaks were observed at 2.06 and 2.85 min. However, the alkaline hydrolysis of the curcuminoids showed higher degradation (21.45%), thereby producing a new peak showing at 2.86 min in the chromatogram of the degraded products with an area almost identical to the peak that appeared at its specific retention time . The oxidative degradation studies with H2O2 showed mild degradation of curcuminoids (11.16%), whereby the four degradation product peaks were observed between 2.34 and 3.59 min. Under irradiation at 254 nm, the HPLC chromatogram displayed a small-scale degradation of curcuminoids (2.6%) with the appearance of three degradation peaks ranging between 2.36 and 3.86 min . For thermal degradation, small degradation (4.21%) was observed and the peaks were seen between 1.74 and 3.60 min. The highest degradation (47.2%) was observed under the sunlight. 4. Conclusions The QbD approach was used to develop an HPLC-based detection and quantitation of curcuminoids and their forced-degradation products in a commercially available curcumin powder. The method was advanced on inputs from the requisites of the separation parameters of the products that relied on the critical attributes of resolutions of peaks in the chromatogram, retention times of the products and degradants. It also estimated the requirements of the theoretical plates for each constituent's separation, suitable pH, and composition of the mobile phase, the composition of the mobile phase solvents, together with the temperature of the column during separation. The method was found to be robust in analysis, and the Monte Carlo simulations predicted the feasibility of the operational domains of the method's factors-based values for experimental purposes, which itself was validated in several of the laboratory runs of the curcuminoid products and its degraded materials. The generated data, parametric perturbations, operational region's predicted values and the products analyses unequivocally verified that the developed method was specific for the determination of curcuminoids, and is free from the interference of degradation products or overlapping of the peaks in the chromatogram. However, the overlapping is caused by column void and physical conditions of the stationary phase. The resolution was for the most part was clear, and the method was found to be stable in commercial products, capsules, tablets, and curcumin powder extracts analyses. The total separation time was less than 7.0 min, which contributed to the increased productivity of the method in comparison to the previously reported methods for the products. The QbD approach helped to identify the sources of the method's variability. It also helped in controlling the risk parameters to design a better, stable, and robust method, thereby extending the lifecycle of the currently developed analytical method, which also provided simultaneous separations of the curcuminoid products and their degradants under various conditions. The QbD approach also enhanced the method development capability and reduced the time and operational cost of the chromatographic process. It also increased the safety of the analysis. This approach is also known as Analytical Quality by Design (AQbD) and can be developed for other mixtures and pharmaceutical products of nutraceutical and extract-based origins. Moreover, the current method development provides an approach for the analysis of food components, other natural as well as synthetic drugs and their impurity profiling, and degradants analysis, including the analysis in clinical settings. The approach can also help to purify, identify, compare, and quantify, as well as bulk isolate, the principal and individual components of a mixture in a robust and feasible manner. The method also laid down the approach details towards a designated time-defined optimum resolution for complex and labile mixtures, a comparison-based validation of the componential analysis, and the resolution of light, heat and pH sensitive materials. The current method developmental approach, and the method itself may find broader applicability for the analytical chemist, clinical and forensic analyst, and analytical toxicologist, as the case and requirements may be. Apart from clinical studies involving HPLC, medico-legal comparatives for components and techniques involving competitive protein binding assays and HPLC comparisons for sensitivity, and LOQ and LOD-based analysis, immunoassays confirmation and comparisons of material analysis through HPLC are of importance and various comparisons with other techniques for purification and componential analysis techniques and parables have been undertaken in the past. This method may provide a better HPLC based analysis based on QbD approach. Not to mention, the accuracy of the HPLC chromatographic technique is still of unparalleled importance for pharmaceutical, and other genres of analytical chemists, and the approach can serve to show the prime importance of the singular method of analysis, the HPLC/RP-HPLC, for purity and contents qualitative and quantitative analysis. Acknowledgments The researchers would like to thank the Deanship of Scientific Research, Qassim University for funding the publication of this project. Supplementary Materials The following supporting information can be downloaded at: Table S1: Design matrix with factorials and responses; Table S2: Model equations for all other responses; Model equations for all other responses; Table S3: Intraday and interday accuracy and precision of curcuminoids; Figure S1: Full time (20 min.) HPLC chromatogram obtained under the optimum conditions of Solgar(r) Curcumin Full Spectrum (A); Curcuma longa after extraction with acetone (B); Curcuma longa after extraction with ethanol (C). Click here for additional data file. Author Contributions Conceptualization, H.A.M., R.A.K. and A.M.A.; methodology, H.A.M., D.S.A., R.A.K. and A.M.A.; software, A.M.A., H.A.M., A.M.H. and R.A.K.; validation, H.A.M., A.M.A. and R.A.K.; formal analysis, A.M.A., H.A.M., D.S.A., A.M.H. and R.A.K.; investigation, A.M.A., D.S.A., H.A.M., A.M.H. and R.A.K.; resources, H.A.M.; A.M.A., D.S.A. and R.A.K.; data curation, H.A.M., A.M.H., D.S.A., R.A.K. and A.M.A.; writing--original draft preparation, D.S.A., A.M.A., H.A.M. and R.A.K.; writing--review and editing, H.A.M., A.M.A., A.M.H. and R.A.K.; funding acquisition, H.A.M., D.S.A., A.M.A. and R.A.K. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement Data are contained in the manuscript and are provided in the Supplementary File. Conflicts of Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Figure 1 Chemical structure of curcuminoids, and their common degradants. Figure 2 Ishikawa diagram showing the factors and parameters (boldface) affecting the HPLC separation. Figure 3 A 3D plot of Derringer's desirability function in correlation with variable acetonitrile and pH (A), pH and temperature of the separation column (B), and temperature and acetonitrile composition % (C). Figure 4 (a) Design space overlay incorporating the CMPs. (b) Design space generated for robust chromatography for curcuminoids. Figure 5 Monte Carlo simulation histograms of CMAs, and Cpk values with upper (right side row), and lower (left side row) specifications limits. The resolutions (Rs), retention time (RT), and theoretical plates (TP) were simulated for the curcumin (C), bisdemethoxycurcumin (BD), and desmethoxycurcumin (DC). Figure 6 HPLC chromatogram obtained under the optimum conditions. Figure 7 HPLC chromatograms of curcuminoids, (a) after exposure to acid degradation; (b) after exposure to base degradation; (c) after exposure to thermal degradation. Figure 8 HPLC chromatograms of curcuminoids after exposure to (a) UV-irradiation; (b) degradation after exposure to sunlight; (c) degradation after exposure to oxidative conditions. foods-12-01010-t001_Table 1 Table 1 The selected factors and their levels. Chromatographic Factors Levels Used in the Experiments Low Medium High pH 2.5 3 3.5 % ACN 50 60 70 Temperature (deg C) 25 30 35 foods-12-01010-t002_Table 2 Table 2 The statistical summary of all the responses. Response F Model p-Value % CV R2 (R-Squared) Adj R2 Adeq. Precision RS1 344.27 <0.0001 3.76 0.992 0.989 39.93 RS2 651.13 <0.0001 2.51 0.994 0.991 59.83 RTCMN 316.27 <0.0001 4.79 0.991 0.988 36.76 RTDMCMN 410.47 <0.0001 4.05 0.993 0.991 42.37 RTBisdemethoxycurcumin 397.74 <0.0001 3.89 0.993 0.991 42.25 TPCMN 315.96 <0.0001 2.6 0.991 0.988 47.00 TPDMCMN 196.14 <0.0001 8.22 0.987 0.982 34.37 TPBisdemethoxycurcmin 662.65 <0.0001 4.87 0.996 0.994 56.61 RS stands for Resolution, RT stands for Retention Time, and TP stands for Theoretical Plates. foods-12-01010-t003_Table 3 Table 3 The optimization standards for each CMA. CMAs Upper Limit Lower Limit Goal Results Predicted Experimental Resolution between curcumin and demethoxycurcumin (RS1) 2.12 0.85 >1.2 1.87 2.05 Resolution between demethoxycurcumin and bisdemethoxycurcumin (RS2) 2.04 0.83 >1.2 1.77 1.88 Curcumin RT (minutes) 6.51 2.38 Maximize 4.65 4.86 Demethoxycurcumin RT (min.) 7.18 2.51 In range 5.47 5.36 Bisdemethoxycurcumin RT (min.) 7.9 2.66 Minimize 6.02 5.93 Number of theoretical plates of curcumin 5800 655 >2000 4818 5122 Number of theoretical plates of demethoxycurcumin 4644 679 >2000 3937 4144 Number of theoretical plates of bisdemethoxycurcumin 3699 2637 >2000 4905 5249 foods-12-01010-t004_Table 4 Table 4 CMPs values obtained by the Monte Carlo simulation method and the predicted separation model's summary. No. CMPs Distribution Type CMPs Range CMAs Specification Limits Low High Low High 1. Acetonitrile composition (%) 54 57 RS1 1.2 12 2. pH 2.5 2.9 RS2 1.2 12 3. Temperature 32 35 RT Curcumin 2 (min) 7 (min) RT Demethoxycurcumin 2 (min) 7 (min) RT Bisdemethoxy curcumin 2 (min) 7 (min) TP Curcumin 2000 >=2000 TP Demethoxy curcumin 2000 >=2000 TP Bisdemethoxy curcumin 2000 >=2000 Predicted model's summary foods-12-01010-t005_Table 5 Table 5 The experimental plan and responses of the Plackett-Burman design for robustness test. Run CMPs Variations CMAs Responses 1 2 3 4 1 2 3 4 5 6 7 8 pH Acetonitrile % Temperature degC Buffer mM Resolution 1 Resolution 2 RT(BDMCR) RT (DMCR) RT(CR) TP (BDMCR) TP (DMCR) TP (CR) 1 2.5 54 35 8 1.84 1.729 4.878 5.373 5.919 4806 3970 4915 2 2.9 54 35 12 1.84 1.728 4.88 5.374 5.923 4808 3966 4919 3 2.9 54 35 12 1.84 1.728 4.88 5.374 5.924 4809 3965 4920 4 2.9 57 32 8 1.834 1.719 4.877 5.368 5.918 4805 3968 4918 5 2.9 57 35 8 1.836 1.722 4.875 5.369 5.917 4801 3965 4911 6 2.5 54 32 8 1.838 1.726 4.88 5.374 5.92 4810 3973 4920 7 2.5 57 32 12 1.836 1.721 4.877 5.369 5.916 4805 3967 4909 8 2.5 54 32 12 1.839 1.726 4.881 5.374 5.921 4811 3974 4917 9 2.5 57 35 8 1.837 1.724 4.874 5.368 5.914 4800 3964 4908 10 2.9 57 32 12 1.835 1.72 4.878 5.37 5.919 4806 3969 4913 11 2.9 54 32 8 1.836 1.724 4.881 5.375 5.923 4811 3968 4919 12 2.5 57 35 12 1.838 1.725 4.875 5.368 5.915 4801 3965 4909 13 2.7 55.5 33.5 10 1.836 1.721 4.874 5.374 5.915 4813 3977 4916 14 2.7 55.5 33.5 10 1.833 1.726 4.879 5.369 5.921 4807 3971 4923 foods-12-01010-t006_Table 6 Table 6 Statistical parameters for individual calibration curves. Parameters Curcumin Demethoxycurcumin Bisdemethoxycurcumin Linearity (mg/mL) 0.76-24.5 0.51-10.0 0.13-4.3 Slope 2,222,250 345,7396 1,077,486.9 Intercept 499,471.1 -414,324.79 -133,404.51 R2 0.9997 0.9997 0.9986 LOD 0.024 0.0105 0.335 LOQ 0.075 0.319 1.015 foods-12-01010-t008_Table 8 Table 8 Application of the proposed HPLC method for the analysis of the contents of curcuminoids (mg/mL) in Curcuma longa powder extract and pharmaceutical dosage forms. Source/Dosage Form API Content (mg/gm) +- S.D. t-Value * F-Value * Present Method (n = 3) Reported Method , (n = 3) Solgar(r) Full Spectrum capsules Curcumin 27.15 +- 0.35 27.62 +- 0.26 1.87 1.76 Demethoxycurcumin 15.6 +- 0.24 15.86 +- 0.15 1.02 2.67 Bisdemethoxycurcumin 3.78 +- 0.17 3.80 +- 0.18 0.11 1.05 Longvida(r) Optimized Curcumin tablets Curcumin 21.84 +- 0.03 21.86 +- 0.08 0.309 5.37 Demethoxycurcumin 13.6 +- 0.12 13.62 +- 0.11 0.241 1.11 Bisdemethoxycurcumin 3.36 +- 0.07 3.36 +- 0.06 0.341 1.31 Ethanol extracts Curcumin 31.71 +- 0.60 31.54 +- 0.09 2.62 1.83 Demethoxycurcumin 9.81 +- 0.13 9.84 +- 0.14 0.322 1.21 Bisdemethoxycurcumin 3.84 +- 0.15 3.77 +- 0.25 0.372 2.59 Acetone extracts Curcumin 20.28 +- 0.17 20.40 +- 0.44 0.454 6.37 Demethoxycurcumin 8.37 +- 0.15 8.52 +- 0.36 0.659 5.09 Bisdemethoxycurcumin 3.97 +- 0.12 3.96 +- 0.10 0.175 1.31 * Tabulated values at 95% confidence limit, t = 2.78 and F = 6.39. 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PMC10000775 | Objectives. To externally validate and assess the accuracy of a previously trained fully automatic nnU-Net CNN algorithm to identify and segment primary neuroblastoma tumors in MR images in a large children cohort. Methods. An international multicenter, multivendor imaging repository of patients with neuroblastic tumors was used to validate the performance of a trained Machine Learning (ML) tool to identify and delineate primary neuroblastoma tumors. The dataset was heterogeneous and completely independent from the one used to train and tune the model, consisting of 300 children with neuroblastic tumors having 535 MR T2-weighted sequences (486 sequences at diagnosis and 49 after finalization of the first phase of chemotherapy). The automatic segmentation algorithm was based on a nnU-Net architecture developed within the PRIMAGE project. For comparison, the segmentation masks were manually edited by an expert radiologist, and the time for the manual editing was recorded. Different overlaps and spatial metrics were calculated to compare both masks. Results. The median Dice Similarity Coefficient (DSC) was high 0.997; 0.944-1.000 (median; Q1-Q3). In 18 MR sequences (6%), the net was not able neither to identify nor segment the tumor. No differences were found regarding the MR magnetic field, type of T2 sequence, or tumor location. No significant differences in the performance of the net were found in patients with an MR performed after chemotherapy. The time for visual inspection of the generated masks was 7.9 +- 7.5 (mean +- Standard Deviation (SD)) seconds. Those cases where manual editing was needed (136 masks) required 124 +- 120 s. Conclusions. The automatic CNN was able to locate and segment the primary tumor on the T2-weighted images in 94% of cases. There was an extremely high agreement between the automatic tool and the manually edited masks. This is the first study to validate an automatic segmentation model for neuroblastic tumor identification and segmentation with body MR images. The semi-automatic approach with minor manual editing of the deep learning segmentation increases the radiologist's confidence in the solution with a minor workload for the radiologist. tumor segmentation independent validation external validation neuroblastic tumors deep learning automatic segmentation PRIMAGE826494 This study was funded by PRIMAGE (PRedictive In-silico Multiscale Analytics to support cancer personalized diagnosis and prognosis, empowered by imaging biomarkers), a Horizon 2020|RIA project (Topic SC1-DTH-07-2018), grant agreement no: 826494. pmc1. Introduction Image segmentation consists of the identification, delineation, and labeling of the voxels that belong to a region of interest . In medical cancer images, this method is a cornerstone in the pipeline that enables the seamless extraction of quantitative radiomic features that can potentially be considered as imaging biomarkers if linked to clinical endpoints, as well as the assistance of image-guided measurement of treatment response. Segmentation is usually performed manually, which is a long-lasting observer-dependent process with inter and intra-observer variability . In recent years, several studies have developed automatic tools for facilitating and standardizing this process . Most of these solutions are based on deep-learning segmentation algorithms, usually built on convolutional neural networks (CNNs) . A new CNN-based algorithm called nnU-Net was recently developed. In contrast to existing methods, it consists of an automatic deep learning-based segmentation framework that automatically configures itself, including preprocessing, network architecture, training, and postprocessing. It adjusts to any new dataset, outperforming most previous approaches . External validation is a fundamental step before any artificial intelligence (AI) solution is applicable to clinical practice, checking for the reproducibility and generalizability of dealing with different patients, institutions, and scanners. This independent validation consists of testing the novel algorithm in a set of new patients to determine whether the tool works to an acceptable degree, as the solution might have been overfitted , usually performing worse in the external validation than in the internal evaluation process . Despite the development of several segmentation tools in recent years, only a few were externally validated , while the majority are designed as proof-of-concept methodological feasibility studies . A recent analytical study showed that only 6% of the publications that evaluate the performance of AI algorithms for diagnostic analysis of medical images were designed with robust external validation performance . There is a need to demonstrate adequate generalizability of AI models through external validation in independent institutions to bridge the gap between research and clinical application, requiring software as a Medical Device regulatory specifications. Within an international research project on pediatric cancer and imaging , a model to detect and segment neuroblastic tumors on MR images based on the state-of-the-art deep learning architecture nnU-Net has been developed . The CNN had a median DSC of 0.965 +- 0.018 (median +- Interquartile Range (IQR)) compared to manual segmentations for a training set of 106 MR sequences (cross-validation) and a Dice Similarity Coefficient (DSC) of 0.918 +- 0.067 (median +- IQR) for an internal independent validation set of 26 MR sequences . The aim of this study was to perform an independent validation of the automatic deep learning architecture nnU-Net previously developed and tested, proposing a novel semiautomatic segmentation methodology. We hypothesize that the state-of-the-art deep learning framework nnU-Net has excellent performance for automatically detecting and segmenting neuroblastic tumors on MR images in a different population and MR equipment than the ones used to train the tool. 2. Materials and Methods 2.1. Participants The data set was collected within the scope of the PRIMAGE (PRedictive In-silico Multiscale Analytics to support cancer personalized diagnosis and prognosis, Empowered by imaging biomarkers) project . A pediatric radiologist with 6 years of expertise and previous experience on segmentation tasks visually reviewed all the cases from the PRIMAGE Platform to exclude those that were used for the training and internal validation of the segmentation tool , those images that did not have an objectifiable tumor (such as the brain in an abdominal tumor or exams after treatment with no tumor rest), exams with important artifacts, and those not orientated in the transverse plane . Finally, 535 MR exams from 300 children were used for the independent external validation of the segmentation tool. This retrospective multicenter international series compilation was made with 300 pediatric patients with neuroblastic tumor diagnosis and pathological confirmation between 2002 and 2022. All the included patients had undergone an MR exam of the anatomical region of the tumor at diagnosis (n = 290) or after initial treatment with chemotherapy (n = 36, as 26 of the included patients had studied at diagnosis and after treatment). None of them was used for the development of the published segmentation neural network . Most of the patients were included in two clinical trials: Society of Paediatric Oncology European Neuroblastoma Network (SIOPEN). High-Risk Neuroblastoma Study (HR-NBL1/SIOPEN) (n = 119) with patients from 12 countries, led by St. Anna Children's Cancer Research Institute (Vienna, Austria), and SIOPEN European Low and Intermediate Risk Neuroblastoma Protocol clinical trial (LINES/SIOPEN), led by La Fe University and Polytechnic Hospital (Valencia, Spain) (n = 107). In addition, patients who are not included in any of the above-mentioned clinical studies were also recruited from different European hospitals or clinical research institutions that collaborate in the PRIMAGE project: 62 from the German Neuroblastoma Registry NB2016 of the GPOH, led by the department of Pediatric Oncology at the University Children's Hospital Cologne and, nine from Spain (La Fe University and Polytechnic Hospital, Hospital La Paz, Hospital Nino Jesus, Hospital Son Espases, Hospital Reina Sofia) and three from Italy (Pisa University Hospital, Istituto Gaslini). The study had the institutional Ethics Committee approvals from all involved institutions. MR images and related patients data from all partners were pseudonymized with the European Unified Patient Identity Management (EUPID) system enabling a privacy-preserving record-linkage documentation connection and a secure data transition to the PRIMAGE framework. Age at diagnosis was 18 +- 32 months (mean +- SD), range of 0 to 212 months, with a balanced gender distribution (155 girls and 145 boys). Tumor histology was neuroblastoma (263 cases), ganglioneuroblastoma (27 cases), and ganglioneuroma (10 cases). 2.2. MR Imaging Automatic labeling of MR series using ML methods on DICOM metadata was applied to the dataset available on the PRIMAGE platform. From the 558 patients available at the PRIMAGE platform (October 2022), 300 with an MR exam at diagnosis were selected. Cases with an MR performed after initial treatment with chemotherapy and tumoral rests were also included. MR images accounted for a high data acquisition variability, including different institutions with large heterogeneity in scanner vendors, protocols, and tumor location. MR exams were acquired with a 1.5 Tesla (n = 435) or 3 Tesla (n = 100) scanners, manufactured by either General Electric Healthcare (Signa Excite HDxt, Signa Explorer, Discovery, Genesis; n = 105); Siemens Medical (Aera, Skyra, Symphony, Avanto, Magneto Espree, TrioTim, Sonata, Spectra, Verio, Harmony; n = 318); Philips Healthcare (Intera, Achieva, Ingenia, Panorama; n = 109) or Canon (Titan; n = 3). All exams included a spin echo T2 weighted (T2wSE) or T2* weighted gradient echo (T2*wGE) sequence, with or without fat suppression (FS or STIR). Among the 535 used sequences, 307 were T2wSE, 11 T2*wGE FS, 176 T2wSE-FS, and 41 T2wSE-STIR. Chest images were obtained with respiratory synchronization. The Mean acquired FOV was 410 mm (median 440, range 225-500 mm). 2.3. Study Design In order to achieve a reproducibility validation of the performance of the previously trained deep learning nnU-Net architecture, the heterogeneity of the dataset was increased by including all the 535 available transversal T2/T2*w MR sequences from 300 patients. The trained 3D segmentation tool was applied to all these cases within the PRIMAGE platform. The radiologist reviewed each sequence, blinded to the segmentation result, and visually localized the tumor and its boundaries. Then, she reviewed the segmentation masks in all the slices of the MR exams, visually validating the performance of the net and ensuring that the tumor was well segmented. Manual mask adjustment was performed when necessary. The time used by the radiologist for visually validating and manually editing the mask was recorded. Both tumor location and treatment initiation were recorded. At the MR sequence level, tumor location was abdominopelvic (430 sequences: 269 in the adrenal gland, 143 abdominal, and 18 with pelvic location) or cervicothoracic (105 sequences: 95 of them thoracic, and 10 cervical). The influence of the initial chemotherapy treatment in the final segmentation results was analyzed by comparing the results at two different moments in the course of the illness, defined as time points: sequences of patients at diagnosis (n = 486) and sequences of patients after treatment (n = 49). The dataset used for validation was completely independent of the one included in the training and tuning of the published segmentation neural network . The age at diagnosis was lower in the validation set (mean of 18 +- 32 months vs. 37 +- 42 months for training-tuning). Regarding the technique, in training set, images performed after treatment were not included, while 9% of the sequences used for validation were obtained after chemotherapy treatment. Some differences were found in the manufacturer distribution, with an increase in the amount of Siemens studies included for validation and the introduction of Canon. Finally, T2wSE STIR and T2*wGE FS were included in the analysis of validation sequences. (Supplementary Materials Table S1). 2.4. Convolutional Neural Network Architecture The automatic segmentation model employed a 3D self-configuring framework for medical segmentation, nnU-Net. The net was previously trained with a cross-validation strategy developed in a cohort of 106 MR sequences of 106 patients and internally validated in a second cohort of 26 cases. The model training was performed along 1000 epochs with 250 iterations each and a batch size of 2. The loss function to optimize each iteration was based on the DSC. A z-score normalization was applied to the images . This automatic segmentation algorithm was encapsulated in a docker container and integrated into the PRIMAGE platform. Then, all the cases were executed on a Kubernetes-based architecture (Quibim Precision v2.9) as batch processes. The output of these processes was the primary tumor mask, accessible from the DICOM viewer integrated into the platform for its final review. 2.5. Metrics Different metrics have been described for addressing segmentation problems in order to compare the automatic segmentation with the human-defined manual ground truth. Since metrics have different properties, the following ones were selected: The DSC, a spatial overlap index, is the most used metric in validating medical volume segmentations, allowing direct comparison between ground truth and automatic segmentations, and is also widely used to measure reproducibility . It ranges from 0 (no spatial overlap between two sets) to 1 (complete perfect overlap), being expressed as:DSC=2TP2TP+FP+FN The Jaccard index (JAC) is a spatial overlap metric defined as the intersection between the masks of the two datasets divided by their union :JAC=TPTP+FP+FN The Hausdorff distance (HD) is a spatial distance metric based on calculating the distances between all pairs of voxels from each mask (A or B), measuring the maximum distance of a segmentation mask (A) to the nearest point in the other mask (B). The HD measures the maximum distances between each point and is defined by : HD(A, B) = max(h(A, B), h(B, A)), where h(A,B)= max min ||a-b||, and a A, b B. The ROC AUC metric (sensitivity against 1-specificity) when a test segmentation is compared to ground truth is defined as :AUC=1-12(FPFP+TN+FNFN+TP) The true negatives have a large impact on the result since the background (normally the largest part of the image) contributes to the agreement . Therefore, two additional spatial overlap-based metrics that do not include the true negatives were considered for the comprehension of the direction of the encountered errors: the modified false positive rate (FPRm) and false negative rate (FNR) of the automatic masks compared to the manually curated ground truth . The FPRm considered those voxels that were identified by the automatic net as a tumor but corresponded to other structures (FP), divided by the voxels that actually corresponded to the manually curated ground truth mask (TP + FN voxels). FPRm=FPTP+FN The FNR of the automatic segmentation to the ground truth considered voxels belonging to the tumor that the net did not include as such, divided by the ground truth voxels. FNR=FNTP+FN=1-Sensitivity For consistency reasons, these last two metrics are expressed as 1-self, resulting in a maximum of 1 for a complete voxel-wise agreement and a minimum of 0 for a null similitude. A simultaneous analysis (ANOVA) of the influence on the DSC of location, timepoint, magnetic field, and sequence weighting), was done. 2.6. Time Sparing Time measurements (in seconds) were annotated by the radiologist when reviewing and adjusting the automatic masks (visual evaluation and manual evaluation). 3. Results 3.1. External Validation Results From the 535 T2/T2*w MR sequences, the DSC was 0.997; 0.944-1.000 (median; Q1-Q3) and the JAC 0.996; 0.894-1.000 (median; Q1-Q3), both results being considered as excellent (Table 1). The mean +- SD HD was 7.081 +- 19.999 mm, with a small maximum distance of the automatic segmentation mask to the nearest point in the corrected mask. The mean AUC ROC was 0.930, showing an excellent agreement. The mean +- SD 1-FPRm was 0.847 +- 1.123, resulting in a high concordance between the net and the manual correction according to the non-tumor-included voxels. The mean +- SD 1-FNR was 0.917 +- 0.215, meaning that the net did not miss an important amount of tumor during the segmentation. Some examples of the segmentation performance in different cases are shown in Figure 2. A descriptive sub-analysis attending outliers were performed. On a subpopulation analysis, 14% (74 from 535) of MR sequences had a DSC value < 0.8. These cases with high variability were visually analyzed by the radiologist to identify reasons for the low level of agreement. Complete failure was defined in 32 cases having a DSC < 0.19 (6%), reflecting an unsuccessful performance of the net: in 18 cases, the DSC was 0 as the network did not segment anything, while in 14 cases, the network segmented lymph nodes (10 cases) or structures such as non-suppressed fat (4 cases). In 42 cases (8%), the DSC between 0.2 and 0.8 showed that the network was able to identify the tumor, but the segmentation was quite incomplete. In these cases, the mean +- SD tumor volume was 121.140 mm3 +- 281.210, smaller compared to the mean volume of the well-segmented tumors (194.660 mm3 +- 222.750). The proportion of cases after chemotherapy was larger (16 cases in this group vs. 33 cases in the well-segmented group). Six cases had cervicothoracic location, while 68 were abdominopelvic (with a slightly higher proportion of abdominopelvic cases compared to the well-segmented group). Regarding the magnetic field, 63 were acquired on a 1.5T equipment, while 11 were performed on a 3T; 40 sequences were weighted on T2 SE, four on STIR, 29 were T2 SE FS, and 1 T2 GE, showing similar proportions compared to the well-segmented group. A descriptive analysis stratifying the results of the DSC regarding the location (abdominopelvic or cervicothoracic), timepoint (diagnosis or treatment), magnetic field strength (1.5T or 3T) and sequence weighting (T2 SE, T2 SE FS, T2 GE, STIR) was performed (Table 2). The results for the mean DSC are shown in Table S2. A simultaneous analysis (ANOVA) of the influence on the DSC of these four factors was performed (location, timepoint, magnetic field, sequence weighting), showing that none of the factors or their interactions had a significant influence on the DSC (Table S3). Despite not showing significant differences, the median DSC at diagnosis was 0.999, and after treatment was 0.902. The mean +- SD volume of tumors at diagnosis was larger 210.389 mm3 +- 227.830) than after treatment (43.467mm3 +- 49.745). Examples of the performance of the automatic segmentation tool and manual edition in two cases at different time points are shown in Figure 3. 3.2. Time Sparing The mean time necessary for visual revision of the generated masks when no editing was necessary was 7.8 +- 7.5 s (mean +- SD). Only 136 masks required further correction, and the time required for this manual edition was 124 +- 120 s (mean +- SD). The median DSC of the 136 edited masks was 0.887 +- 0.499 (median; Q1-Q3). 4. Discussion This multicentric international study, including an independent and heterogeneous data set of MR images from children with neuroblastic tumors, confirms and validates the performance of a trained ML tool to identify and delineate the lesions. The nnU-Net segmentation masks were visually validated by an experienced pediatric radiologist, with a median DSC of 0.997. Among the 535 T2/T2*w MR sequences, only 14% (74 cases with a DSC < 0.8) needed manual editing, and in only 6% (18 sequences), the tool failed to segment. The 136 manually adjusted masks had a quite good median DSC of 0.887; 0.562-0.990 (median; Q1-Q3), facilitating the radiologist tasks . The automatization of the segmentation process has the added value over the manual method that it is deterministic, always providing the same outcomes given the same input images, improving repeatability . The visual validation and manual editing of the automatic masks improve the usability of the method, so this semiautomatic approach is a reliable method that could be integrated into the radiology workflow even in large datasets . Besides, this methodology implies an important reduction in the time required for segmentation tasks. In previous work, the mean time required to segment 20 neuroblastic tumor cases manually from scratch was 56 min . With this approach, the mean time required for the manual edition was 124 +- 120 s (mean +- SD), resulting in a 96.5% of time reduction. To our knowledge, this is the first study to perform an international, multicenter, and multivendor external validation of an automatic segmentation model for neuroblastic tumor identification and segmentation with body MR images. Previous studies investigated the development of semiautomatic segmentation algorithms for neuroblastic tumors on CT or MR images , making use of different imaging processing tools. Nevertheless, apart from our work, there is no preceding the literature addressing either the performance of a CNN-based solution in neuroblastic tumors nor the validation of a segmentation methodology. Despite the lack of the literature regarding neuroblastic tumor automatic segmentation architectures, the deep learning tool nnU-Net has set a new state-of-the-art in numerous segmentation problems surpassing most existing approaches , and as it has been proven, displays strong generalization characteristics. Multiple AI processing tools have been developed, described, and published, but only a few have undergone external validation. Validation in an independent real-world dataset is a fundamental process in order to determine the accuracy of a model and to estimate its reproducibility and generalizability, which are essential in order to base clinical decisions on correct prediction models. Thus, this study represents an important step in the process of implementation of a new tool. Among the few preceding works that have addressed the issue of external validation of automatic segmentation algorithms in different areas, some studies have explored lesions such as glioblastoma , focal cortical dysplasia , or liver segments . The most frequent limitation regarding external validation is the inclusion of a small number of subjects. As an example, the work addressing segmentation of craniopharyngioma on MR using a U-Net-based deep convolutional neural network performed an independent validation with 38 patients , while a study on the impact of manual and automatic segmentations on the radiomics models to predict the response of locally advanced rectal cancers the number of patients in the independent validation dataset was 28 cases . External validation for demonstrating the robustness of newly developed radiomic tools used a series of 88 patients to predict biochemical recurrence in prostate cancer . Our series is one of the largest using real-world cases for independent validation. This work is a translational multicenter and multivendor study and has demonstrated the reproducibility of a segmentation CNN tool, following international recommendations for biomarker validation . Other studies designed to validate deep learning segmentation algorithms are single-center, as one work aimed to quantify the skeletal muscle index and sarcopenia in metastatic renal carcinoma . As expected, our study shows that the automatic tool performs worse after chemotherapy treatment (median DSC 0.902 after treatment vs. 0.999 at diagnosis), although the difference is not statistically significant. The reduction in the tumor volume and border delimitation after neuroblastoma treatment may influence the performance of the net. Besides, previous works have demonstrated changes in radiomic features in other tumors, such as pancreatic cancer . There are some limitations to this study, as only one experienced radiologist reviewed the automatic segmentations, so interobserver variability was not tested. Nevertheless, this is not a clinically relevant limitation as a high concordance between observers (with a median DSC overlap index of 0.969) performing manual segmentation in neuroblastoma has been described . Another bias is that the CNN was trained with MR images in the transversal plane, and the validation has only been performed on transversal images. In case of low generalizability of the model, future work could include fine-tuning the nnU-Net tool to perform segmentations in sagittal and coronal planes. This robust, repeatable, and automatized segmentation algorithm based on the state-of-the-art nn-Unet with final visual validation improves the consistency of the data extraction and strengthens the workflow of imaging tumor segmentation. In conclusion, this international, multicentric, and multivendor independent validation study shows that a previously trained automatic nnU-Net tool is able to locate and segment neuroblastic tumors on T2/T2* weighted MR images in 97% of the cases, despite the body location and MR equipment characteristics. To our knowledge, this is the first study to independently validate an automatic segmentation model for neuroblastic tumor identification and segmentation with body MR images. The semi-automatic approach with minor manual editing of the DL segmentation increases the radiologist's confidence in the solution and significantly reduces their required involvement. Supplementary Materials The following supporting information can be downloaded at: Table S1. Table showing the difference in population and technique of the original training dataset and validation. The population had a similar distribution except for the age at diagnosis, which was lower in the validation set. The variables referred to the technique shown in the validation sequences after treatment. The distribution of the manufacturer and new protocols were included (T2wSE STIR and T2*wGE FS); in Table S2. Stratified descriptive analysis of the DSC results regarding timepoint (diagnosis or treatment), location (abdominopel-vic or cervicothoracic), magnetic field 1.5T or 3T, and sequence (T2 SE, T2 SE FS, T2 GE, STIR); Table S3. Results obtained from the ANOVA test were performed to show the influence of the different variables and their interactions (timepoint, location, magnetic field strength, and sequence) on the dependent variable DSC. None of the factors or their interactions had a significant influence on the DSC. Click here for additional data file. Author Contributions Conceptualization, L.C.-A. and L.M.-B.; Data curation, D.V.-C. and A.G.-M.; Formal analysis, D.V.-C., L.C.-A., A.J.-P. and J.M.C.S.; Investigation, L.C.-A.; Methodology, D.V.-C., L.C.-A., A.J.-P., J.M.C.S. and L.M.-B.; Project administration, L.M.-B.; Resources, C.S.-N., B.M.d.l.H., S.T.-M., V.D., U.P., T.S., E.N., A.A.-B., A.C., B.H. and R.L.; Software, A.G.-M. and M.F.-P.; Supervision, L.C.-A., J.M.C.S., A.A.-B. and L.M.-B.; Validation, D.V.-C.; Writing--original draft, D.V.-C.; Writing--review & editing, L.C.-A., A.J.-P., J.M.C.S., M.F.-P., B.M.d.l.H., S.T.-M., V.D., A.A.-B., A.C., B.H. and L.M.-B. All authors have read and agreed to the published version of the manuscript. Conflicts of Interest The authors of this manuscript declare relationships with the following companies: QUIBIM SL. Ethics Statements This study has been approved by the Hospital's Ethics Committee (The Ethics Committee for Investigation with medicinal products of the University and Polytechnic La Fe Hospital, ethic code: 2018/0228). Figure 1 Figure depicting the study design. Transversal MR sequences were used for validation of the automatic segmentation tool for patients with neuroblastic tumors. After applying some exclusion criteria, a total amount of 300 patients, including 535 MR T2 sequences, were used for external validation, 466 sequences at diagnosis and 49 after treatment. Figure 2 Examples of the automatic segmentation masks before and after manual edition in four different cases with heterogeneous location and imaging acquisition to show the performance of the automatic segmentation architecture and the comparison of the masks after manual correction. Figure 3 Examples of the automatic segmentation performance and manual edition in two cases (Case 1: abdominal tumor and Case 2: thoracic tumor) at different time points and performed with diverse equipment. cancers-15-01622-t001_Table 1 Table 1 Performance metrics for the multicentric MR studies (n = 535) considering DSC, JAC, HD, AUC ROC, 1-FPRm, and 1-FNR. From the 535 T2/T2*w MR sequences, the DSC was 0.997; 0.944-1.000 (median; Q1-Q3) and the JAC 0.996; 0.894-1.000 (median; Q1-Q3), obtaining. DSC JAC HD AUC ROC 1-FPRm 1-FNR Median 0.997 0.996 0.000 0.999 1.000 1.000 IQR 0.944-1.000 0.894-1.000 0.000-3.000 0.973-1.000 0.996-1.000 0.969-1.000 Mean 0.887 0.862 7.081 0.930 0.847 0.917 SD 0.262 0.279 19.999 0.191 1.123 0.215 cancers-15-01622-t002_Table 2 Table 2 Descriptive analysis of the different metrics used: DSC, JAC, AUC ROC, 1-FPRm, and 1-FNR. Results are depicted for timepoint (diagnosis or treatment), location (abdominopelvic or cervicothoracic), magnetic field 1.5T or 3T, and sequence (T2 SE, T2 SE FS, T2 GE, STIR). DSC at diagnosis was 0.999, and after treatment was 0.902. DSC JAC HD AUC ROC 1-FPRm 1-FNR Tumor at diagnosis (n = 486) Median 0.999 0.997 0.000 0.999 1.000 1.000 Q1-Q3 0.964-1.000 0.930-1.000 0.000-2.207 0.978-1.000 0.997-1.000 0.976-1.000 Mean 0.901 0.879 7.115 0.931 0.853 0.923 SD 0.250 0.266 20.720 0.196 1.112 0.208 Tumor after chemotherapy (n = 49) Median 0.902 0.821 2.803 0.999 1.000 1.000 Q1-Q3 0.755-0.220 0.607-0.360 0.000-6.000 0.910-0.148 0.968-0.102 0.821-0-295 Mean 0.752 0.691 6.737 0.926 0.785 0.854 SD 0.334 0.344 10.553 0.137 1.245 0.275 Cervicothoracic (n = 105) Median 0.999 0.999 0.000 0.999 1.000 1.000 Q1-Q3 0.975-1.000 0.951-1.000 0.000-2.000 0.998-1.000 0.979-1.000 0.999-1.000 Mean 0.960 0.938 3.933 0.994 0.928 0.988 SD 0.109 0.145 11.712 0.023 0.212 0.046 Abdominopelvic (n = 430) Median 0.997 0.995 0.000 0.999 1.000 1.000 Q1-Q3 0.929-1.000 0.868-1.000 0.000-3.527 0.973-1.000 0.996-1.000 0.975-1.000 Mean 0.869 0.843 7.849 0.923 0.814 0.916 SD 0.284 0.300 21.484 0.208 1.251 0.222 1.5T (n = 434) Median 0.998 0.996 0.000 1.000 1.000 1.000 Q1-Q3 0.945-1.000 0.896-1.000 0.000-2.979 0.968-1.000 0.996-1.000 0.961-1.000 Mean 0.879 0.855 7.372 0.925 0.841 0.911 SD 0.275 0.291 20.622 0.197 1.174 0.223 3T (n = 101) Median 0.995 0.990 0.000 0.999 1.000 1.000 Q1-Q3 0.943-1.000 0.892-1.000 0.000-3.000 0.989-1.000 0.997-1.000 0.982-1.000 Mean 0.918 0.888 5.944 0.951 0.871 0.942 SD 0.196 0.220 17.251 0.163 0.884 0.177 T2 SE (n = 307) Median 0.997 0.994 0.000 0.999 1.000 1.000 Q1-Q3 0.951-1.000 0.906-1.000 0.000-2.855 0.971-1.000 0.995-1.000 0.951-1.000 Mean 0.904 0.877 6.078 0.938 0.873 0.907 SD 0.232 0.253 17.649 0.170 0.828 0.249 T2 SE FS (n = 176) Median 0.998 0.997 0.000 0.999 1.000 1.000 Q1-Q3 0.928-1.000 0.865-1.000 0.000-3.577 0.973-1.000 0.997-1.000 0.983-1.000 Mean 0.849 0.827 9.737 0.907 0.769 0.917 SD 0.317 0.327 24.984 0.239 1.618 0.226 STIR (n = 41) Median 0.999 0.999 0.000 0.999 1.000 1.000 Q1-Q3 0.933-1.000 0.875-1.000 0.000-2.207 0.991-1.000 0.999-1.000 0.983-1.000 Mean 0.911 0.881 3.393 0.976 0.991 0.952 SD 0.210 0.241 8.908 0.081 0.026 0.162 T2 GE FS (n = 11) Median 0.996 0.992 0.000 0.998 1.000 1.000 Q1-Q3 0.993-1.000 0.986-1.000 0.000-0.433 0.957-1.000 0.872-1.000 0.921-1.000 Mean 0.929 0.901 7.499 0.943 0.792 0.888 SD 0.176 0.229 18.535 0.109 0.480 0.217 Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). 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PMC10000776 | Tumour necrosis factor alpha (TNFa) is a multifunctional cytokine that plays a pivotal role in apoptosis, cell survival, as well as in inflammation and immunity. Although named for its antitumor properties, TNFa also has tumour-promoting properties. TNFa is often present in large quantities in tumours, and cancer cells frequently acquire resistance to this cytokine. Consequently, TNFa may increase the proliferation and metastatic potential of cancer cells. Furthermore, the TNFa-driven increase in metastasis is a result of the ability of this cytokine to induce the epithelial-to-mesenchymal transition (EMT). Overcoming the resistance of cancer cells to TNFa may have a potential therapeutic benefit. NF-kB is a crucial transcription factor mediating inflammatory signals and has a wide-ranging role in tumour progression. NF-kB is strongly activated in response to TNFa and contributes to cell survival and proliferation. The pro-inflammatory and pro-survival function of NF-kB can be disrupted by blocking macromolecule synthesis (transcription, translation). Consistently, inhibition of transcription or translation strongly sensitises cells to TNFa-induced cell death. RNA polymerase III (Pol III) synthesises several essential components of the protein biosynthetic machinery, such as tRNA, 5S rRNA, and 7SL RNA. No studies, however, directly explored the possibility that specific inhibition of Pol III activity sensitises cancer cells to TNFa. Here we show that in colorectal cancer cells, Pol III inhibition augments the cytotoxic and cytostatic effects of TNFa. Pol III inhibition enhances TNFa-induced apoptosis and also blocks TNFa-induced EMT. Concomitantly, we observe alterations in the levels of proteins related to proliferation, migration, and EMT. Finally, our data show that Pol III inhibition is associated with lower NF-kB activation upon TNFa treatment, thus potentially suggesting the mechanism of Pol III inhibition-driven sensitisation of cancer cells to this cytokine. RNA polymerase III cancer TNFa NF-kB First TEAM programme of the Foundation for Polish SciencePOIR.04.04.00-00-3FD3/17 European Union under the European Regional DevelopmentThis study was supported by POIR.04.04.00-00-3FD3/17 grant (for D.G.) within the First TEAM programme of the Foundation for Polish Science, co-financed by the European Union under the European Regional Development. pmc1. Introduction Colorectal cancer (CRC) is the third-most commonly diagnosed cancer and the second leading cause of cancer deaths worldwide . CRC is very heterogeneous molecularly, and a wide range of causative genetic aberrations have been identified, including mutations, loss of heterozygosity and epigenetic changes. Although CRC has a substantial heritable component, most CRC cases are sporadic . Moreover, CRC is one of the best examples of the involvement of chronic inflammation in the development of sporadic and heritable forms of this disease . Chronic inflammation triggered by microbial infection, autoimmune diseases, or other pathologies raises the risk of tumorigenesis. Inefficient clearance of infection during chronic inflammation is a major cause of tissue damage and reconstitution. During this process, reactive oxygen species accumulate, leading to DNA damage and mutation. Moreover, cells are continuously proliferating to maintain tissue homeostasis under inflammatory conditions, which can be a major driving force for transforming initial tumour cells . Finally, it is now clear that the tumour microenvironment, which is primarily orchestrated by inflammatory cells, is an indispensable participant in the neoplastic process. Tumour-infiltrating immune cells produce cytokines that activate various transcription factors, which regulate cancer cell survival, growth, proliferation, epithelial-mesenchymal transition (EMT), and metastasis . Interleukin-6 and tumour necrosis factor alpha (TNFa) are cytokines considered to be important players in colorectal cancer development and progression . TNFa is a multifunctional cytokine primarily produced by macrophages and other immune system cells, as well as some non-immune cells, although to a lesser extent . TNF-a was initially discovered and named according to its ability to induce the necrosis of transplanted sarcomas in mice . TNFa appeared to work when injected directly into tumours in high doses, however, its severe toxicity, when administrated systemically, almost entirely hampered its usage in cancer therapy. The only successful therapeutic intervention is the local administration of TNFa (usually in combination with chemotherapy) via isolated limb perfusion to treat soft tissue sarcomas . Apart from its toxicity, TNFa is now believed to have also pro-tumorigenic properties . Consistently this cytokine is frequently present in the tumour microenvironment, and tumour cells usually acquire resistance to TNFa-induced cell death . TNFa signals through two cell surface receptors, TNFR1 and TNFR2, which differ in their signalling activity and expression pattern . TNFR1 is expressed in almost all cell types, whereas the expression of TNFR2 is limited to immune cells and a few other cell types. TNFR1 and TNFR2 have similar extracellular TNF-binding domains, which equally efficiently bind both transmembrane and soluble forms of TNFa. Interestingly, transmembrane TNFa strongly activates signalling through both TNFR1 and TNFR2, and soluble TNFa triggers signalling only through TNFR1 . Signalling through TNFR1 is usually pro-apoptotic, whereas signalling through TNFR2 is usually anti-apoptotic , which is a result of structural differences in the cytoplasmic domains of these receptors. TNFR1 contains a cytoplasmic death domain, which is not present in TNFR2 . Upon TNF-a binding to TNFR1, the adaptor protein TNFR1-associated death domain protein (TRADD) is recruited to the cytoplasmic death domain of the receptor along with Receptor Interacting Protein Kinase 1 (RIPK1) and TNF receptor-associated factor 2 (TRAF2). Then, the ubiquitin ligase cellular inhibitors of apoptosis 1/2 (cIAP1/2) are recruited to form the so-called complex I. Within this complex, cIAPs attach ubiquitin chains to themselves and other subunits, leading to the recruitment of the linear ubiquitin chain assembly complex (LUBAC). Linear ubiquitin chains deposited by LUBAC on the complex I components constitute a docking platform for Transforming growth factor-b-activated kinase 1 (TAK1), TAK1-binding proteins 2/3 (TAB2/3), and the inhibitor of kB kinase (IKK) subunit, NEMO. Subsequently, TAK1 phosphorylates IKK, which leads to the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) transcription factor (for review, see ). NF-kB is a crucial transcription factor mediating inflammatory signals. It has also been suggested to have a wide-ranging role in tumour progression, including acceleration of cell proliferation, inhibition of apoptosis, promotion of cell migration and invasion, and stimulation of angiogenesis and metastasis . There are five mammalian members of the NF-kB family of transcription factors, RelA (p65), RelB, c-Rel, NF-kB1 (p50/p105), and NF-kB2 (p52/p100) . NF-kB DNA binding activity consists of many possible heterodimers, although p50/RelA heterodimers are most commonly observed . Under normal cellular conditions, NF-kB binds to and is negatively regulated by the inhibitor of kappa B (IkB) in the cytoplasm. Following an inflammatory stimulus, IkB is phosphorylated by IkB kinase (IKK) and undergoes proteasomal degradation. This allows NF-kB to translocate to the nucleus, where it regulates the transcription of a wide variety of target genes that induce inflammation, proliferation, and cell survival . Notably, inhibition of this NF-kB-mediated pro-survival response may destabilise the TNFR1-bound complex I and lead to the formation of the pro-apoptotic complex II, consisting of TRADD, RIPK1, FAS-associated death domain protein (FADD), and caspase-8 . When formed, complex II triggers apoptosis. The pro-inflammatory and pro-survival function of NF-kB can be disrupted by blocking macromolecule synthesis. Consistently, general RNA or protein synthesis inhibitors Actinomycin D (ActD) or cycloheximide (CHX), respectively, strongly sensitise cells to TNFa-induced cell death . In mammalian cells, there are three DNA-dependent RNA polymerases (Pols). Pol I synthesises ribosomal RNA (rRNA), whereas Pol II synthesises mostly mRNAs and long non-coding RNA and micro RNAs. Pol III synthesises several essential components of the protein biosynthetic machinery, including tRNA, 5S rRNA, 7SL RNA, and a subset of small non-coding RNAs required for the maturation of other RNA molecules (U6 RNA). These untranslated RNAs are essential for cell growth, proliferation, and immune responses . Moreover, an elevated Pol III activity is a recurring feature of murine and human tumours, and inhibition of Pol III has anti-tumorigenic effects . ActD inhibits the activity of all three RNA polymerases, with Pol I being the most sensitive . It is believed that this drug sensitises the cells to TNFa mainly through the prevention of Pol II-dependent gene expression (regulated by NF-kB) . While this is most likely the case, no studies have directly explored the possibility that specific inhibition of Pol III activity sensitises cancer cells to TNFa. In the current study, we show that in colorectal cancer cells, Pol III inhibition augments the cytotoxic and cytostatic effects of TNFa. We also show that Pol III inhibition blocks TNFa-induced migration and EMT. Importantly, Pol III inhibition alone has very little effect on the cells. Finally, we show that Pol III inhibition impinges on NF-kB activity, which may potentially explain the sensitisation of cancer cells to TNFa. 2. Materials and Methods 2.1. Cell Culture Cells were cultured in a humidified incubator with 5% CO2 at 37 degC. HCT116 (ATCC(r) CCL-247TM) and LoVo (ATCC(r) CCL-229TM) colorectal cancer cells were grown in DMEM supplemented with 2 mM L-glutamine, penicillin (100 U/mL), streptomycin (100 U/mL), and 10% foetal bovine serum (FBS), unless otherwise stated. Generated cell lines were cultured in the same medium. Cell lines were routinely tested for mycoplasma presence using the MycoSpy detection kit (Biontex, Munich, Germany, Cat. No. M020-025). When indicated, cells were treated with DMSO (PanReac, Darmstadt, Germany, Cat. No. A3672,0100), TNF-a (Peprotech, London, UK, Cat. No. 300-01A), and ML-60218 (Merck, Darmstadt, Germany, Cat. No. 557403). The cells were plated 24 h before the experiment, followed by treatment with DMSO, TNFa, ML-60218 alone, or TNFa and ML-60218 simultaneously for the indicated time. 2.2. MTT Assay Cell metabolic activity was evaluated using the MTT assay. This assay is based on the conversion of MTT (3-(4,5-dimethyl thiazolyl)-2,5-diphenyltetrazolium bromide) to a blue/purple formazan crystal by NADPH or NADH produced by dehydrogenase enzymes in metabolically active cells. HCT116 and LoVo cells were seeded at a density of 5 x 103 cells per well in 96-well plates and incubated at 37 degC overnight. After 24 h, cells were treated with DMSO, TNFa, ML-60218 alone, or TNFa and ML-60218 simultaneously. The MTT assay was performed according to the manufacturer's instructions (Promega, Madison, WI, USA, Cat. No. PRG8080). Briefly, MTT reagents were added (final concentration of 0.5 mg/mL) to each well. The microplate was incubated at 37 degC in 5% CO2 for 4 h (until the formazan crystals appeared). After incubation, 100 mL of solubilisation buffer was added to each well. Following complete solubilisation, the plate was read at 590 nm using a microplate reader (Beckman Coulter, Brea, CA, USA, DTX880). 2.3. Clonogenic Cell Survival Assay The cells were seeded at a density of 5 x 104 cells for each condition on a 6 cm dish. The following day, the cells were treated with DMSO, TNFa, ML-60218 alone, or TNFa and ML-60218 simultaneously. Following the removal of the medium, the cells were gently rinsed with PBS, and a new medium devoid of the medication was introduced. The plates were then kept in the incubator in the regular medium for 7 days. Then, the medium was removed, and the cells were washed with PBS, fixed, and stained with a crystal violet solution (0.05% crystal violet by volume, 1% formaldehyde, 1x PBS, and 1% methanol). Following a PBS washing, the cells were allowed to dry at room temperature before being imaged. 2.4. Stable Cell Lines HCT116 cell lines stably overexpressing tRNAiMet or tRNAeMet were generated using lentiviral transduction. HEK293T cells were transfected with pLHCX, pLHCX-tRNAiMet, or pLHCX-tRNAeMet plasmids along with the lentiviral packaging vectors psPAX2 and pDM2.G. psPAX2 and pDM2.G were a gift from Didier Trono (Addgene plasmid #12260; (Accessed on 1 December 2022); RRID: Addgene_12260, Addgene plasmid #12259; (Accessed on 1 December 2022); RRID: Addgene_12259). After 48 h, the medium containing lentiviral particles was collected and filtrated using sterile 0.45-mm filters (Merck, Darmstadt, Germany, Cat. No SLHP033RS or Sarstedt, Numbrecht, Germany, Cat. No. 83.1826). The medium filtrated was used to infect HCT116 cells that were cultured in 6-well plates. The cell lines stably expressing pLHCX, pLHCX-tRNAiMet, and pLHCX-tRNAiMet were selected with puromycin (1 mg/mL) until there were no live cells on the control plate. Pools of cells were used for the experiments. 2.5. Protein Extracts and Western Blotting Cells were washed with ice-cold phosphate-buffered saline (PBS) and harvested by scraping directly into lysis buffer (100 mM NaCl, 50 mM HEPES (pH 7.9), 1 mM EDTA, protease and phosphatase inhibitor, 0.05% NP-40, 0.1% SDS). Extracts were sonicated using a Bioruptor (Diagenode) and spun for 20 min at 13,000 rpm at 4 degC. Supernatants were collected, and protein concentration was assessed using the Pierce BCA protein assay (Thermo Scientific, Waltham, MA, USA, Cat. No. 23225). A total of 25 mg of proteins was separated on SDS polyacrylamide gels, transferred to a PVDF (G.E. Healthcare, Chicago, IL, USA, Cat. No. 10600021) or nitrocellulose membrane (G.E. Healthcare, Cat. No. GE10600001), and incubated with antibodies in 5% w/v skimmed milk in Tris-buffered saline-Tween (TBST) and then probed with the appropriate antibodies. The antibodies used are listed in Supplementary Table S1. Original Western blots can be found in Supplementary Figure S1. 2.6. Fractionation Cells were plated at a density of 4 x 106 cells/dish in a 10 cm dish, treated as described in the figure legend, and harvested. Fractionation and nuclei isolation was performed as described in . 2.7. RNA Isolation and cDNA Synthesis Total RNA was isolated from cells using RNA Extracol (EURx, Gdansk, Poland, Cat. No. E3700) according to the manufacturer's instructions. Then, 100 ng of RNA was used for cDNA synthesis using a QuantiTect reverse transcriptase kit (Qiagen, Hilden, Germany, Cat. No. 205314). To increase the efficiency of cDNA synthesis from tRNAs, oligonucleotides specific to the 3' end of tRNA were added to the reaction mixture (Supplementary Table S2), each at the final concentration of 1 mM as described before . The oligonucleotide sequences used for qPCR are listed in Supplementary Table S3. 2.8. Quantitative PCR Quantitative PCR was performed using a Roche Light Cycler 480 System. The thermal cycling conditions were composed of 20 s at 95 degC, 30 s at 61 degC, and 20 s at 72 degC. After PCR amplification, each sample was subjected to a melting curve analysis to confirm that a single product with the predicted melting curve characteristics was achieved. Each sample was run in technical duplicate or triplicate. A non-reverse transcriptase control, a no-template control, and cDNA dilutions for the standard curve were all present on each plate. The effectiveness of PCR ranged from 90% to 100%. The Light Cycler 480 Software was used to process the data, and Microsoft Excel was used for further analysis. 2.9. Scratch Wound Assay HCT116 and LoVo cells were seeded at a density of 5 x 104 cells/well (100 mL/well) in each well of the Image-lock 96-well plate (Sartorius, Gottingen, Germany). The cells were allowed to settle at ambient temperature for 10 min, and then the plates were kept in a 37 degC incubator with 5% CO2 overnight. The next day the Image-lock plate was carefully removed from the incubator, and a 96-well Wound-maker (Sartorius) was used to create wounds in all wells simultaneously. Immediately after making the wound, cells were washed twice with PBS and replenished with a medium containing DMSO, TNFa, ML-60218 alone, or TNFa and ML-60218. The cells were then kept in the IncuCyte Live cell imaging system and monitored for 72 h at 3 h intervals. Cell migration was analysed using IncuCyte 2019B Rev2 software (Sartorius). 2.10. Cell Proliferation Assay The cells were seeded at a density of 5 x 103 cells per well in a 96-well plate and allowed to attach overnight at 37 degC in the incubator. Then, the cells were treated with DMSO, TNFa, ML-60218 alone, or TNFa and ML-60218 simultaneously, and the plates were then kept in the IncuCyte S3 Live-Cell Analysis System (Sartorius) to monitor the confluency for 48 h at 3 h intervals. Cell confluency was analysed using IncuCyte 2019B Rev2 software. 2.11. Propidium Iodide Exclusion Assay For cell death assessment, HCT116 cells were treated with DMSO, TNFa, ML-60218 alone, or TNFa and ML-60218 simultaneously for 24 h. After treatment, both the adherent and floating cells were harvested into 15 mL falcon tubes. The cells were then washed with ice-cold PBS. Then, the cells were resuspended in 1 mL of PBS solution containing propidium iodide and transferred into a FACS tube. The flow cytometry analysis was performed using an Attune NxT flow cytometer (Thermo Scientific). Where required, the cells were pre-treated for 3 h with broad-spectrum caspase inhibitor Q-VD-OPh and processed for cell death analysis. 2.12. Cell Death Assay Using IncuCyte The cells were seeded at a density of 5 x 103 cells per well in a 96-well plate and allowed to attach overnight at 37 degC in the incubator. The cells were then treated with DMSO, TNFa, ML-60218 alone, or TNFa and ML-60218 in the presence of Sytox Green (Thermo Scientific, Cat. No. S34860) in the medium. The plates were then kept in the IncuCyte S3 Live-Cell Analysis System (Sartorius). The number of Sytox green positive (dead) cells was acquired at 48 h of treatment. The cell death was calculated by normalising the number of Sytox green positive cells to the cell confluency. The analysis was performed using IncuCyte 2019B Rev2 software. 2.13. NF-kB Induction Reporter Assay HCT116-DualTM cells (Invivogen, San Diego, CA, USA) were used to assess NF-kB activity. HCT116-DualTM cells express a secreted embryonic alkaline phosphatase (SEAP) reporter gene under the control of NF-kB binding sites. HCT116-DualTM cells were cultured in a medium containing heat-inactivated (56 degC for 30 min) serum, seeded on a 96-well plate, and allowed to attach overnight in the incubator. Following incubation, the cells were treated with DMSO, TNFa, ML-60218 alone, TNFa, and ML-60218 simultaneously for 24 h. Then, the samples were processed according to manufacturer's instructions. The absorbance at 655 nm was measured using a microplate reader (Beckman Coulter, DTX880). 2.14. Confocal and High-Content Microscopy Immunofluorescence staining was used to detect E-cadherin expression in HCT116 and LoVo cells. Cells were grown on coverslips for 24 h. Following appropriate treatment, immunostaining was performed according to a previously described procedure . The E-cadherin antibodies were purchased from Cell Signaling Technology (Mouse mAb #14472), and the Alexa FluorTM 488-conjugated secondary antibodies were purchased from Thermo Scientific (Goat anti-mouse IgG1# A-21121). Nuclei were stained with Hoechst dye (1:5000, Hoechst 33342). High-content cell imaging was performed using a ScanR automated microscope (Olympus) with a UPlanSApo 20.0x objective. Image analysis was performed using ScanR 2.7.2 software (Olympus, Tokyo, Japan). Representative images were taken with a Nikon C1 confocal laser scanning microscope with Plan Apo 60.0x/1.40 NA with an oil objective. The images were processed using Nikon EZ-C1 software. 3. Results 3.1. Inhibition of RNA Polymerase III Augments TNFa-Induced Cytotoxic and Cytostatic Effects in CRC Cells To test whether Pol III inhibition sensitises colorectal cancer cells to TNFa treatment, we first investigated the viability of cells using an MTT colorimetric assay. HCT116 and LoVo human colorectal cancer cells were treated with DMSO, TNFa, RNA polymerase III inhibitor (ML-60218) alone, or a combination of TNFa and ML-60218. Treatment of HCT116 cells with TNFa alone slightly, and not statistically significantly, increased their viability . ML-60218 treatment alone did not affect the cells, although it downregulated Pol III activity . The combination of TNFa and ML-60218, however, markedly reduced the viability of HCT116 cells. In LoVo cells, similar results were obtained, with the exception that TNFa significantly increased cell viability . We also monitored cell proliferation in real time. In HCT116 cells, the treatment with TNFa or ML-60218 alone led to a slight decrease in proliferation . However, the combination of TNFa and ML-60218 strongly inhibited cell proliferation. In LoVo cells, TNFa substantially increased, whereas ML-60218 modestly decreased cell proliferation . Notably, the combination of TNFa and ML-60218 strongly suppressed the proliferation of LoVo cells. In the case of HCT116 cells, the proliferation results do not fully overlap with MTT assay data, which showed a modest and statistically non-significant increase in cell viability upon TNFa treatment. In proliferation experiments, we observed the reverse effect. We speculate that in these cells a combination of effects occurs: a slight increase in proliferation and concomitant induction of cell death in some cells (see below). As a consequence, during the early stages of cell death, some cells detach from the bottom of the plate under TNFa treatment (we see an increase in the number of floating cells). Floating cells, not necessarily dead yet, are out of the focus in the IncuCyte device while still contributing to overall metabolic activity in the MTT assay. Nevertheless, MTT and proliferation assay results are consistent in both cell lines upon concomitant treatment with TNFa and ML-60218 and show the detrimental effect of the treatment. To further support our observations, we also performed clonogenic assays. Treatment with TNFa or ML-60218 alone slightly, and consistently with proliferation results, decreased colony formation by HCT116 cells . Furthermore, the combination of TNFa and ML-60218 led to even more potent inhibition of the colony-forming ability of these cells. In LoVo cells, treatment with TNFa alone significantly increased the number of colonies, whereas the addition of ML-60218 had a slightly opposite effect . ML-60218 alone had no apparent effect on colony formation in LoVo cells. Altogether, these data suggest that RNA Polymerase III inhibition augments the cytostatic/cytotoxic effect of TNFa in HCT116 cells and completely reverses the proliferation-stimulating effect of this cytokine in LoVo cells. 3.2. Combination of RNA Polymerase III Inhibitor with TNFa Induces Apoptosis in HCT116 Cells The observed decrease in the overall viability of the cells (the MTT assay) or the cell number (proliferation monitoring) may stem from a reduction of cell proliferation, increased cell death, or a combination of both. Indeed, upon microscopic inspection, we noticed some cell death in HCT116 cells treated with TNFa, which was substantially increased upon concomitant treatment with ML-60218. Therefore, we sought to look in more detail whether ML-60218 augments the cytotoxic effects of TNFa. To this end, HCT116 and LoVo cells were treated as described above. The propidium iodide (PI)-exclusion method combined with FACS showed that in HCT116 cells, treatment with ML-60218 does not affect cell viability, whereas TNFa treatment slightly induces cell death . However, the combination of TNFa and ML-60218 leads to a significant induction of cell death. When treated with TNFa, LoVo cells are difficult to detach from tissue culture dishes. The attempts to prolong the incubation with trypsin lead to a substantial decrease in cell viability, which hinders the usage of PI-exclusion combined with FACS. We, therefore, monitored the cell death in LoVo cells using the IncuCyte live-cell imaging system. In this case, as a reference, we included cells treated with a combination of TNFa and cycloheximide, which triggers rapid cell death . Of note, we did not use IncuCyte for monitoring cell death in HCT116 cells, because when they die, they detach from the bottom of the plate, become out of focus, and cannot be counted. The results showed that in LoVo cells, TNFa treatment alone slightly induces cell death and that the combination of TNFa and ML-60218 does not further potentiate this effect . ML-60218 alone had minimal impact on the survival of LoVo cells. Thus, in LoVo cells, ML-60218 treatment does not potentiate TNFa-induced cell death as in HCT116 cells but instead has a cytostatic effect. We then sought to investigate further the type of death the HCT116 cells undergo. TNFa induces apoptosis mediated by caspases , and we explored this possibility. Western blotting analysis showed that concomitant treatment of cells with ML-60218 and TNFa leads to the cleavage of caspase 8, the most upstream protease participating in the activation cascade responsible for death receptor-induced cell death . We also observed executioner caspase 7 and PARP cleavage, a hallmark of apoptosis . Furthermore, while TNFa alone slightly induced PARP and caspase 7 and 8 cleavage, ML-60218 had no effect. These results are in agreement with PI-exclusion experiments. Finally, to further validate that the observed cell death type is apoptosis, the HCT116 cells were pre-treated with a broad-spectrum caspase inhibitor, Q-VD-Oph (Quinoline-Val-Asp-Difluorophenoxymethylketone) . Then, the cells were treated as above. PI-exclusion experiments showed that inhibition of caspases blocked the cell death induced by TNFa and, most importantly, by concomitant treatment of cells with TNFa and ML-60218 . Altogether, these data suggest that inhibition of RNA Polymerase III enhances TNFa-induced apoptosis in HCT116 cells. The data presented so far show that in the HCT116 cells, which are slightly sensitive to TNFa, Pol III inhibition strongly augments the cytotoxicity of this cytokine. On the other hand, in the LoVo cells where TNFa does not induce cell death but rather stimulates their proliferation, Pol III inhibition has a cytostatic effect, only marginally causing cell death. 3.3. RNA Polymerase III Inhibition Affects the Levels of TNFa-Induced Cell Cycle Progression Markers in CRC Cells TNFa is known to promote cancer cell proliferation , which we could observe in our experiments with LoVo cells. More importantly, we could also see a decrease in cell proliferation when additional treatment with Pol III inhibitor was introduced . Cyclin D1 is directly implicated in stimulating the proliferation of cells, and it was shown to be upregulated by TNFa . We, therefore, asked whether ML-60218 treatment can block TNFa-induced upregulation of cyclin D1 protein levels. To address this, we treated LoVo and HCT116 cells with DMSO, TNFa, ML-60218, or TNFa simultaneously with ML-60218. Please note that since HCT116 cells are slightly sensitive to TNFa, from this point forward, the experiments with these cells were performed using a lower concentration of this cytokine to avoid the confounding effect of cell death. The results showed that while TNFa treatment induced cyclin D1 protein levels both in LoVo and HCT116 cells, the addition of ML-60218 completely blocked this effect . ML-60218 treatment alone has a very modest, if any, impact on cyclin D1 protein levels. These data show that inhibition of RNA Polymerase III blocks TNFa-driven induction of cell proliferation marker, cyclin D1, in CRC. The results also suggest that the inhibition of cell proliferation upon combined treatment of cells with TNFa and ML-60218 may result from decreased cyclin D1 protein levels. 3.4. Inhibition of Pol III Decreases TNFa-Induced Migration in CRC Cells The ability of cancer cells to migrate is crucial for metastasis . It has been reported that TNFa enhances the migration of cancer cells, including colorectal cancer cells . We employed a scratch wound-healing assay to investigate whether Pol III inhibition affects TNFa-induced alterations in cell migration. HCT116 and LoVo cells were treated with DMSO, TNFa, ML-60218 alone, or TNFa simultaneously with ML-60218. Please note that for the HCT116 cell line, similarly as for proliferation assays, the lower concentration of TNFa was used to decrease potential cell death. Consistently with literature data, we observed that TNFa enhanced the speed of wound closure for both cell lines tested, although it was less apparent for the LoVo cell line . ML-60218 treatment alone had little effect on HCT116 cell migration (statistically non-significant downregulation was observed at 48 h and 72 h of treatment). In LoVo cells, ML-60218 treatment led to the modest inhibition of cell migration. Importantly, the combination of TNFa with ML-60218 significantly decreased the migratory potential of HCT116 cells . In this case, the migration was even slower than in control, DMSO-treated samples. In LoVo cells, although a similar effect was observed, ML-60218 only partially blocked the TNFa-induced increase of migration . Overall, these data suggest that Pol III inhibition decreases TNFa-induced migration of CRC cells. The additive effect of TNFa and Pol III inhibitor in HCT116 cells may result from the higher sensitivity of these cells to the combination of treatments. 3.5. RNA Polymerase III Inhibition Blocks TNFa-Induced Suppression of E-Cadherin Expression in HCT116 Cells During the epithelial-to-mesenchymal transition (EMT), the expression of epithelial markers decreases while the expression of mesenchymal markers increases . E-cadherin is an adherens junction protein that is one of the critical elements involved in forming intercellular contacts in epithelial cells. Downregulation of E-cadherin is frequently observed in epithelial tumours and is a hallmark of EMT . TNFa potentiates EMT in several cancers by downregulating epithelial markers (e.g., E-cadherin) and stimulating the expression of mesenchymal markers . Given our results showing an effect of Pol III inhibition on TNFa-induced cell migration, we sought to determine whether it is associated with changes in EMT. To address this, we monitored the E-cadherin levels. HCT116 and LoVo cells were treated with DMSO, TNFa, ML-60218 alone, or TNFa simultaneously with ML-60218. In agreement with the literature data, Western blotting and immunofluorescence experiments showed that TNFa markedly downregulates the expression of E-cadherin in HCT116 cells . Notably, this effect was entirely blocked by ML-60218. In LoVo cells, we could not detect E-cadherin by Western blotting, and only a weak signal using immunofluorescence was observed. This is consistent with previous data showing much lower levels of E-cadherin in LoVo cells as compared to HCT116 cells . Nevertheless, a slight downregulation of E-cadherin was observed in TNFa-treated cells . Similarly, as in HCT116 cells, ML-60218 blocked the effect of TNFa in LoVo cells . There is a clear difference in regards to E-cadherin levels between HCT116 and LoVo cells. It is, therefore, plausible that LoVo cells have partially gone through EMT and have more mesenchymal-like characteristics as compared to HCT116 cells, which display a more epithelial-like phenotype . We, therefore, tested the mRNA levels of Fibronectin 1, a mesenchymal marker, which is an essential component of the extracellular matrix that links collagen fibres to integrins on the surface of the cells . RT-qPCR experiments showed that in HCT116 cells, Fibronectin 1 mRNA levels were below the detection threshold. In LoVo cells, however, we could observe an increase in Fibronectin 1 mRNA levels in TNFa-treated cells . Notably, concomitant treatment of cells with ML-60218 blocked the effect of TNFa. ML-60218 alone also slightly reduced the levels of Fibronectin 1 mRNA. Overall, these data and data regarding cell migration show that inhibition of Pol III activity blocks TNFa-induced EMT in colorectal cancer cells. Of note, the presence of easily detectable Fibronectin 1 mRNA in LoVo cells and its lack in HCT116 cells further suggests that LoVo cells have more mesenchymal-like characteristics as compared to HCT116 cells. 3.6. Elevated Expression of Initiator Methionine tRNA Does Not Contribute to the Increased Proliferation and Migration of HCT116 Cells We previously showed that TNFa induces Pol III activity in macrophages . It has also been reported that increased initiator methionine tRNA (tRNAiMet) expression may drive cancer cell proliferation and migration . Therefore, we speculated that the increased proliferation and migration of CRC cells treated with TNFa may also result from increased tRNAiMet levels and that ML-60218 would act by preventing tRNAiMet upregulation. We first assessed whether the levels of tRNAiMet in HCT116 and LoVo cells treated with TNFa were upregulated. We also tested whether ML-60218 blocks this effect, if any. The data show that in HCT116 cells, TNFa treatment significantly upregulated tRNAiMet levels, which was blocked by Pol III inhibitor . In LoVo cells, this effect was also visible. However, it was less pronounced and did not reach statistical significance . We then sought to determine whether overexpression of tRNAiMet in HCT116 can stimulate their proliferation and migration. We prepared cell lines stably overexpressing tRNAiMet or, as controls, cells overexpressing elongator methionine tRNA (tRNAeMet) or cells harbouring an empty vector. Using RT-qPCR, we confirmed the efficiency of tRNAiMet overexpression . However, our results show that tRNAiMet overexpression did not affect either the proliferation or migration of HCT116 cells . Thus, these data suggest that it is unlikely that the observed effects of TNFa on colorectal cancer cell proliferation and migration are solely a result of higher tRNAiMet expression. Consequently, we also conclude that the impact of ML-60218 on the TNFa-treated cells is most likely not exclusively dependent on the prevention of tRNAiMet upregulation. 3.7. Inhibition of RNA Polymerase III Blocks TNFa-Induced NF-kB Activation NF-kB is an inducible nuclear transcription factor involved in immune responses, cell proliferation, and apoptosis. NF-kB is strongly activated in response to TNFa and contributes to cell survival and proliferation . NF-kB controls the expression of several genes encoding anti-apoptotic proteins, such as c-FLIP, Bcl-2, Bcl-xL, and cIAP2 , as well as proteins involved in proliferation, such as cyclin D1 . Disruption of NF-kB activity either by genetic manipulations or by chemical inhibition of IKK kinase, which activates this transcription factor, renders the cells highly susceptible to TNFa-induced cell death . We thus speculated that Pol III inhibition might affect the activity of NF-kB and abolish its protective and pro-proliferative functions. To validate this possibility, nuclear extracts were prepared from HCT116 cells treated with DMSO, TNFa, ML-60218, or TNFa simultaneously with ML-60218 and analysed by Western blotting. The results show that a small amount of p65, an NF-kB subunit, is present in the nucleus, even in the unstimulated control cells , which is consistent with the notion that some cancer cells may display constitutive activity of the NF-kB pathway . Nevertheless, we could observe even higher nuclear levels of p65 in TNFa-treated cells as compared to the control . Notably, ML-60218 treatment blocked the TNFa-induced localisation of p65 to the nucleus in HCT116 cells. ML-60218 alone had a minimal and not statistically significant effect on p65 localisation to the nucleus. Neither ML-60218 alone nor the combination of ML-60218 and TNFa downregulated total cellular levels of p65. We further validated these observations using HCT116-DualTM cells (Invivogen) designed to monitor the NF-kB signal transduction pathway. The cells were treated as above, and the activity of the secreted embryonic alkaline phosphatase was assessed colorimetrically. In response to TNFa, the NF-kB activity was strongly induced in these cells , whereas the addition of ML-60218 partially blocked this effect. Please note that the effect in reporter cells is less robust as compared to the effect on p65 localisation. This is most likely because secreted embryonic alkaline phosphatase accumulates in the medium over time, whereas the fractionation shows a snapshot of p65 localisation. Nevertheless, these data suggest that Pol III inhibition partially blocks the activation of NF-kB in response to TNFa. We also assessed the nuclear localisation of p65 in LoVo cells. In contrast to HCT116, the nuclear levels of p65 were high in the control cells, and we did not see an increase after TNFa treatment . The constitutive activation of NF-kB in these cells was observed previously ; thus, it is possible that TNFa treatment is not able to stimulate p65 nuclear localisation further. Nevertheless, treatment of cells with the combination of TNFa and ML-60218 significantly downregulated nuclear p65 levels. We also observed slight downregulation of nuclear p65 levels in ML-60218-treated LoVo cells. These data further suggest that the inhibition of Pol III affects NF-kB activity. Of note, the high activity of NF-kB in LoVo cells may explain their resistance to TNFa-induced cell death and low levels of E-cadherin. cFLIP and cIAP1/2 encode anti-apoptotic proteins, and their expression is regulated by NF-kB . Given the altered localisation of the NF-kB subunit, p65, we asked whether the expression of cFLIP and cIAP1/2 would also be altered. We could not detect cFLIP or cIAP2 proteins using Western blotting. We also tested the mRNA levels of cFLIP and cIAP2 using RT-qPCR and found the Cp values were very high. We thus concluded they are not expressed or are expressed at very low levels in HCT116 cells. We could, however, detect cIAP1 protein. The Western blotting showed that cIAP1 is slightly but not statistically significantly downregulated upon TNFa treatment . ML-60218 alone did not affect cIAP1 protein levels. However, the combination of ML-60218 and TNFa substantially downregulated the levels of cIAP1 protein. Thus, the strong downregulation of cIAP1 may potentially explain increased cell death upon concomitant treatment with ML-60218 and TNFa. 4. Discussion TNFa treatment alone very rarely induces cancer cell death, as the cells acquire resistance to this cytokine . The cytotoxicity of TNFa can be enhanced by treating cells with, for example, translation or transcription inhibitors. TNFa is frequently present in tumours in large quantities, which can be exploited by delivering drugs that sensitise cells to this cytokine. Our results show that inhibition of Pol III may serve as a potential therapeutic intervention. Importantly, our data suggest that Pol III inhibitor alone has minimal impact on the CRC cells and is mainly limited to anti-proliferative and anti-migratory properties, resembling the effects of some commonly used anti-cancer drugs . In our hands, Pol III inhibition does not induce cell death in colorectal cancer cells. These observations may be somewhat counterintuitive, given the housekeeping role of Pol III products. However, it is essential to note that Pol III products are abundant and relatively stable. Thus, the effect of Pol III inhibition may be modest, not immediate, and observed under specific environmental conditions, e.g., inflammatory response. Indeed, we previously showed that inhibition of Pol III in macrophages hampers their pro-inflammatory response upon treatment with lipopolysaccharides, a cell wall component of gram-negative bacteria . Thus, Pol III inhibition may affect a specific subset of proteins, for example, those with a high turnover rate (short half-life). This would be significantly exacerbated in conditions of higher protein synthesis demand, like immune response triggered by TNFa. The Pol III inhibition would then rather affect cellular signalling pathways. The plausibility of this scenario is substantiated by the fact that in the case of E-cadherin, we observe a lack of its TNFa-induced downregulation when cells are additionally treated with a Pol III inhibitor . The lower NF-kB nuclear localisation in cells treated with a combination of TNFa and ML-60218 may also partially explain the phenotypes observed in our current work. Firstly, NF-kB is known to positively regulate cyclin D1 through direct binding within the CCND1 gene promoter . Thus, NF-kB inactivation may prevent cyclin D1 upregulation and cell cycle acceleration, a phenomenon observed by us and previously by others . Secondly, our results show that in cells treated with TNFa alone, there is a substantial downregulation of E-cadherin, which is blocked by additional treatment of cells with Pol III inhibitor. This observation is consistent with previous evidence showing that NF-kB negatively regulates E-cadherin levels by inducing the expression of transcriptional repressors ZEB1 and TWIST1 . Moreover, TWIST1 promotes EMT and enhances the motility of several cancer cells . Thus, it is possible that ML-60218 indirectly affects TWIST1 by blocking NF-kB activity and altering the migratory potential of colorectal cancer cells. Whether this is the case remains to be elucidated. It has been known for over two decades now that inhibition of NF-kB strongly sensitises cells to TNFa-induced cell death . Generally, the fate of the cell treated with TNFa depends on the balance between pro-apoptotic and anti-apoptotic signalling. High activity of NF-kB confers the resistance of cells to this cytokine, and insufficient NF-kB activation tips the balance towards cell death. This is because NF-kB controls the expression of several anti-apoptotic genes, including c-FLIP, B-cell lymphoma-2 (BCL-2), BCL-xL, and cIAP1/2 . Thus, the enhanced apoptosis we observe in cells upon concomitant treatment with TNFa and ML-60218 may result from lower levels of anti-apoptotic proteins, whose gene expression is regulated by NF-kB. Alternatively, decreased Pol III activity may directly impinge on the synthesis of these anti-apoptotic proteins. Pol III inhibition could especially affect MCL-1 and BCL-xL, which have short half-lives . A subtle change in tRNA repertoire could slow down the synthesis of these proteins, tip the balance, and push the cells towards cell death. Further studies are needed to unequivocally determine the mechanism whereby ML-60218 sensitizes the cells to TNFa and whether the downregulation of NF-kB contributes to the observed phenotypes. 5. Conclusions The current study shows that treating colorectal cancer cells with ML-60218 augments prototypical functions of TNFa. In particular, we showed that ML-60218 treatment increases the cytotoxic and cytostatic effects of TNFa. Furthermore, ML-60218 blocks TNFa-induced cell migration and EMT. The observed effects correlate with the lower activity of NF-kB. Notably, ML-60218 seems not to have a cytotoxic effect on colorectal cancer cells. Given that TNFa is present in the tumour microenvironment, the administration of ML-60218 systemically could have an effect locally in the tumours, thus having a lower impact on other tissues. Indeed, this is a beneficial setting from the standpoint of potential therapeutical application. Interestingly, in recent years, RNA polymerase I (Pol I), which is responsible for synthesising ribosomal RNA, and Pol III, considered a housekeeping enzyme, have emerged as a promising anti-cancer target . Given the stability of Pol I and Pol III products, inhibiting these key enzymes is not necessarily linked with extensive cytotoxicity. The high dependence of cancer cells on increased translation rates may render them particularly vulnerable to Pol I/III inhibition while sparing the normal cells. Moreover, the tumour microenvironment, where inflammatory and proliferative signalling dominates and increased translation occurs, may also be a susceptibility spot where the inhibition of Pol I/III would have a beneficial outcome. We hope our results will encourage other scientists to investigate further Pol III inhibitors as potential anti-cancer drugs. Of particular need now is research involving animal models. Supplementary Materials The following supporting information can be downloaded at: Figure S1: Original Western blots; Figure S2: ML-60218 inhibitor downregulates Pol III activity in colorectal cancer cells; Figure S3: Overexpression of initiator methionine tRNA does not contribute to the increased proliferation and migration of CRC cells; Table S1: The antibodies used in this study; Table S2: The tRNA-specific oligonucleotides used for cDNA synthesis. Table S3: The PCR primers used in this study. Click here for additional data file. Author Contributions Conceptualization, D.G.; investigation, H.G.N., A.J.; writing--original draft preparation, D.G., H.G.N.; writing--review and editing, D.G.; supervision, D.G.; funding acquisition, D.G. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement The data presented in this study are available in this article and Supplementary Material. Conflicts of Interest The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. Figure 1 Concomitant treatment with TNFa and RNA Polymerase III inhibitor decreases the viability of colorectal cancer cells. (a) HCT116 cells were treated with 0.1% DMSO (control), 40 ng/mL of TNFa, 30 mM of RNA polymerase III inhibitor, ML-60218 (Pol III INH), or both TNFa and ML-60218 for 48 h; (b) LoVo cells were treated with 0.1% DMSO (control), 20 ng/mL of TNFa, 30 mM ML-60218, or both TNFa and ML-60218 for 72 h. (a,b) Cell viability was determined using the MTT colorimetric assay. N = 3. (c,d) The proliferation of cells was monitored using the IncuCyte S3 live-cell analysis system. The scans were performed every 3 h for a total of 48 h. N = 3. The shaded area represents 95% confidence intervals. (e,g) Clonogenic assay. The cells were treated as in a and b, but only for 48 h. After treatment, the cells were washed and allowed to grow in a regular medium for one week, and then the cells were stained using crystal violet. (f,h) The percentage of colony area from e and g was assessed by ImageJ software. N = 3. The error bars represent the standard deviation. Statistical analysis was performed using one-way ANOVA followed by a post hoc Tukey test. * p-value < 0.05; ** p-value < 0.01; *** p-value < 0.001; n.s., non-significant. Figure 2 RNA Polymerase III inhibition sensitises HCT116 cells to TNFa-induced apoptosis. HCT116 cells were treated with 0.1% DMSO (control), 40 ng/mL of TNFa, 30 mM of RNA polymerase III inhibitor, ML-60218, or both TNFa and ML-60218 for 24 h. The cells were collected and stained with Propidium Iodide and then subjected to flow cytometry analysis. N = 5. (b) LoVo cells were seeded on a 96-well plate and treated with 0.1% DMSO (control), 20 ng/mL of TNFa, 30 mM of RNA polymerase III inhibitor, ML-60218, or both TNFa and ML-60218 in the presence of Sytox Green and monitored in the IncuCyte Live-Cell imaging system. As a control, concomitant treatment with cycloheximide (5 mg/mL) and TNFa (20 ng/mL) was used. The data shown were acquired at 48 h of treatment. The number of Sytox green-positive (dead) cells was normalised to the confluence (see Materials and Methods for further details). (c,d) HCT116 cells were treated with 0.1% DMSO (control), 40 ng/mL of TNFa, 30 mM of RNA polymerase III inhibitor, ML-60218, or both TNFa and ML-60218 for 16 h. Total protein was isolated from the cells, resolved on SDS-PAGE gels, and analysed using antibodies against indicated proteins by Western blotting. Actin was used as a loading control. Representative Western blots show indicated protein levels. N = 3. (e) HCT116 cells were left untreated (No treatment) or pre-treated with 20 mM Q-VD-Oph for 3 h and further treated as in (a). The cells were trypsinised after 24 h, stained with propidium iodide, and then subjected to flow cytometry analysis. N = 3. The error bars represent the standard deviation. Statistical analysis was performed using one-way ANOVA followed by a post hoc Tukey test (a,b) or a Bonferroni post hoc test (e).* p-value < 0.05; ** p-value < 0.01; *** p-value < 0.001; ns, non-significant. Figure 3 RNA Polymerase III inhibition affects the levels of TNFa-induced cell cycle markers in CRC cells. (a) HCT116 and (c) LoVo cells were treated with 0.1% DMSO (control), 20 ng/mL of TNFa, 30 mM of RNA polymerase III inhibitor, ML-60218 (Pol III INH), or both TNFa and ML-60218 for 24 h or 48 h, respectively. Total protein was isolated from the cells, resolved on SDS-PAGE gels, and analysed using antibodies against indicated proteins by Western blotting. (b,d) The densitometry quantification of Western blots from panels a and c. The protein levels were normalised to actin. N = 3. Error bars represent the standard deviation. Statistical analysis was performed using one-way ANOVA followed by the post hoc Tukey test. * p-value < 0.05; n.s., non-significant. Figure 4 RNA Polymerase III inhibition decreases TNFa-induced migration in CRC cells. Scratch wound-healing assay. (a) HCT116 (c) and LoVo colorectal cancer cells were seeded in 96-well plates and grown overnight in the medium containing 2% serum. Then, the wounds were created, and the cells were treated with DMSO (control), 20 ng/mL of TNFa, 30 mM of RNA polymerase III inhibitor, ML-60218 (Pol III INH), or both TNFa and ML-60218 for 72 h. The wound closing was monitored using the IncuCyte S3 Live-Cell imaging system. Images of the cells were captured every 3 h. N = 3 (HCT116), N = 5 (LoVo). (b,d) Quantification of the relative wound density at indicated time points. Error bars represent the standard deviation. Statistical analysis was performed using two-way repeated measures ANOVA followed by a Bonferroni post-hoc test. * p-value < 0.05; ** p-value < 0.01; *** p-value < 0.001; n.s., non-significant between treatments. Figure 5 RNA Polymerase III inhibition blocks TNFa-induced downregulation of E-cadherin levels. (a,b) HCT116 cells were treated with 0.1% DMSO (control), 20 ng/mL of TNFa, 30 mM of RNA polymerase III inhibitor, ML-60218, or both TNFa and ML-60218 for 24 h. (a) Total protein was isolated from the cells, resolved on SDS-PAGE gels, and analysed using antibodies against indicated proteins by Western blotting. N = 3. (b) The Western blots from panel a were quantified by densitometry using ImageJ Software. The protein expression was normalised to actin. (c,d) HCT116 or (e-g) LoVo cells were treated with 0.1% DMSO (control), 10 ng/mL of TNFa, 30 mM of RNA polymerase III inhibitor, ML-60218, or both TNFa and ML-60218 for 72 h. (c,e) Confocal microscopy analysis of E-cadherin (green). Nuclei were stained with Hoechst (blue). (d,f) Quantitative analysis of E-cadherin levels was performed using high-content screening microscopy (ScanR). (g) RT-qPCR analysis of Fibronectin 1 mRNA. RNA levels were normalised to GAPDH and RPLP0 mRNAs. N = 3. Error bars represent the standard deviation. Statistical analysis was performed using: (b,g) one-way ANOVA followed by the post hoc Tukey test; (d,f) Kruskal-Wallis test followed by the post hoc Dunn test. * p-value < 0.05; ** p-value < 0.01; *** p-value < 0.001; ns, non-significant. Figure 6 RNA Polymerase III inhibition decreases TNFa-induced activation of the NF-kB transcription factor. (a,b) HCT116 cells were treated with 0.1% DMSO (control), 20 ng/mL of TNFa, 30 mM of RNA polymerase III inhibitor, ML-60218, or both TNFa and ML-60218 for 24 h. (c) cells were treated as in panel (a). The colorimetric assay was used to assess the activity of a secreted embryonic alkaline phosphatase, which reflects the NF-kB activity (see Materials and Methods for further details). N = 3. (d,e) LoVo cells were treated with 0.1% DMSO (control), 20 ng/mL of TNFa, 30 mM ML-60218, or both TNFa and ML-60218 for 48 h. (a,d) Total cellular and nuclear extracts were prepared from cells, resolved on SDS-PAGE gels, and analysed using antibodies against indicated proteins by Western blotting. Representative Western blots showing the levels of indicated proteins. (b,e) The densitometry quantification of Western blots from nuclear fraction from panels (a,d). The protein levels were normalised to histone H3. N = 3 (HCT116), N = 2 (LoVo). (f,g) HCT116 cells were treated with 0.1% DMSO (control), 40 ng/mL of TNFa, 30 mM of RNA polymerase III inhibitor, ML-60218, or both TNFa and ML-60218 for 16 h. Total cellular extracts were prepared from cells, resolved on SDS-PAGE gels, and analysed using antibodies against indicated proteins by Western blotting. Representative Western blots showing the levels of indicated proteins. (g) The densitometry quantification of Western blots from panel (f). The protein levels were normalised to actin. N = 4. The error bars represent the standard deviation. Statistical analysis was performed using one-way ANOVA followed by a post hoc Tukey test * p-value < 0.05; *** p-value < 0.001; ns, non-significant. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). 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PMC10000777 | Healthcare (Basel) Healthcare (Basel) healthcare Healthcare 2227-9032 MDPI 10.3390/healthcare11050747 healthcare-11-00747 Article Sleep-Disordered Breathing among Saudi Primary School Children: Incidence and Risk Factors Alwadei Saleh H. 1* Alsaeed Suliman Methodology 234 Masoud Ahmed Ibrahim Methodology Visualization 5 Alwadei Farhan Methodology Resources Visualization 1 Gufran Khalid Writing - original draft Writing - review & editing 1 Alwadei Abdurahman Conceptualization Methodology Software 6 Lupi Saturnino Marco Academic Editor Wieckiewicz Mieszko Academic Editor 1 Department of Preventive Dental Sciences, College of Dentistry, Prince Sattam Bin Abdulaziz University, Alkharj 11942, Saudi Arabia 2 Preventive Dental Sciences Department, College of Dentistry, King Saud Bin Abdulaziz University for Health Sciences, Riyadh 11481, Saudi Arabia 3 King Abdullah International Medical Research Center, Riyadh 11481, Saudi Arabia 4 Ministry of the National Guard Health Affairs, Riyadh 11426, Saudi Arabia 5 Department of Orthodontics, Faculty of Dentistry, King Abdulaziz University, Jeddah 21589, Saudi Arabia 6 Department of Pediatric Dentistry and Orthodontics, College of Dentistry, King Saud University, Riyadh 11545, Saudi Arabia * Correspondence: [email protected] 03 3 2023 3 2023 11 5 74711 1 2023 22 2 2023 28 2 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). This study aimed to identify the incidence and risk factors of sleep-disordered breathing (SDB) using an Arabic version of the pediatric sleep questionnaire (PSQ). A total of 2000 PSQs were circulated to children aged 6-12 years who were randomly selected from 20 schools in Al-Kharj city, Saudi Arabia. The questionnaires were filled out by the parents of participating children. The participants were further divided into two groups (younger group: 6-9 years and older group: 10-12 years). Out of 2000 questionnaires, 1866 were completed and analyzed (93.3% response rate), of which 44.2% were from the younger group and 55.8% were from the older group. Among all the participants, a total of 1027 participants were female (55%) and 839 were male (45%) with a mean age of 9.67 +- 1.78 years. It showed that 13% of children were suffering from a high risk of SDB. Chi-square test and logistic regression analyses within this study cohort showed a significant association between SDB symptoms (habitual snoring; witnessed apnea; mouth breathing; being overweight; and bedwetting) and risk of developing SDB. In conclusion: habitual snoring; witnessed apnea; mouth breathing; being overweight; and bedwetting strongly contribute the to development of SDB. SDB pediatric sleep questionnaire sleep apnea prevalence This research received no external funding. pmc1. Introduction Many airway dysfunctions, including obstructive sleep apnea (OSA) and primary snoring, are caused by sleep-disordered breathing (SDB) . OSA is a severe form of SDB characterized by upper airway obstruction, either partial or intermittent, that interrupts the normal sleeping pattern . Among children, SDB is linked with growth retardation, behavioral problems, disturbances in cognitive development, failure to thrive, and attention deficit/hyperactivity disorder . The myriad of SDB symptoms in children also include abnormal breathing, snoring, sweating, aggressiveness, irritability, hyperactivity, sleepiness, excessive fatigue, memory impairments, and poor school performance, among many others . Furthermore, the craniofacial morphology associated with SDB in children includes retrognathic mandible, increased lower facial height, narrow maxillary arch with a high vault, posterior crossbite, anterior open bite, and restriction in the upper airway space . The prevalence of pediatric OSA ranges from 1-6%, while habitual snoring varies by definition and ranges from 4-17% . A previous study in southern Italy found that 4.9% school children were possessed habitual snoring and among them only 1% had OSA . Another Turkish study stated that the prevalence of 6-13 years children's snoring habit is 7% . Moreover, 11.4%, 12% and 27.6% prevalence rates of snoring habit were observed in the Indian, Chinese and Brazilian populations, respectively . In Saudi Arabia, the prevalence among adults and children leans toward the higher end of the range reported in the literature . However, the majority of children with OSA remain undiagnosed . In order to diagnose OSA in children, nocturnal sleep-based polysomnography (PSG) is widely used and is considered the gold standard to diagnose OSA . Regardless of its diagnostic advantages, PSG presents challenges related to its cost, time, complexity, and availability/access which necessitates the use of an efficient screening test that may prompt early detection and treatment . As dentists and selected dental specialists obtain radiographic x-rays regularly and examine children daily, they could effectively identify the children who are at risk of SDB . This is especially true given the previously mentioned association between SDB and craniofacial growth and development, as well as the proven positive responses to various treatment modalities ranging from interceptive orthodontics to orthognathic surgery . Although thorough clinical history and physical examination are an integral part of best practice in any healthcare profession including assessment of SDB, they may not be sufficient to identify children suspected (or at high risk) of OSA . It is important to consider efficient, reliable, and accurate screening tests for timely diagnosis and management (including referral) of children with SDB since it might prevent associated comorbidities . Pediatric sleep questionnaires have been developed for the screening of SDB in children and adolescents, and epidemiological studies have shown them to be clinically significant and relevant . As subjective parent report tools, sleep questionnaires are apprehensive about the risk factors and sign symptoms of SDB and OSA . Among these questionnaires, the pediatric sleep questionnaire-22 (PSQ-22) has been validated in groups of referred snoring children and controls, showing excellent specificity and sensitivity for identifying children with OSA . Therefore, this study aimed to determine the incidence of SDB and identify related risk factors among primary school children in Al-Kharj, Saudi Arabia. 2. Materials and Methods This was a cross-sectional study that was conducted from September 2018 to December 2018 in Al-Kharj city, Saudi Arabia. Al-Kharj city is situated in the Riyadh province of Saudi Arabia with about 376,325 residents (accessed on 21 November 2017)). Ethical approval was obtained from the College of Dentistry Research Centre at Prince Sattam Bin Abdulaziz College of Dentistry (Registration No: 1439-03-003). In addition, permission to conduct this research was also obtained from the authority of the Ministry of Education. A stratified randomization technique was used to list the schools to be included in this study to ensure the proper sample representation in the Al-Kharj. A free online randomization software (accessed on 21 November 2017)) was used to select 20 schools (10 boys' schools and 10 girls' schools). A formal letter was sent to the principal of each school to obtain permission by explaining the purpose of the study. Once the school authorities agreed to participate in the current research, they were requested to provide a list of primary school students with an assigned number corresponding to each student. Then, a randomization table was used to select the students. A required sample size of 454 subjects was estimated based on a statistical power calculation described by Pourhoseingholi and colleagues (2013) , considering 0.20 non-responses, a confidence level of 95%, a precision of 0.04, and 20% prevalence SBD as reported in Saudi-based literature using PSQ . Each child was given a folder in order to obtain permission from the parents which included: (1) a cover page explaining the aim of the study, the significance of the study, and the confidentiality measures taken to protect collected information, (2) a consent form, and (3) the PSQ. Parents/guardians were asked to observe their child's sleep pattern for one week before filling out the PSQ to improve response accuracy. Children aged 6-12 years who presented a consent form signed by their parents were included in the current study. The participants were divided into two groups by age: the younger group (age 6-9 years) and the older group (age 10-12 years). A total of 2000 folders containing a cover letter, a consent form, and the PSQ were distributed to randomly selected children, and the folders were collected a week later. The PSQ was previously validated by Chervin et al. (2000); therefore, no validation was required for this study. Moreover, the Arabic version of PSQ was validated by Baidas et al. (2019); therefore, the Arabic form of PSQ was used in this study in order to explain the PSQ properly by the parents . The PSQ contains 22 items, and each item consists of three options to respond to with the following options: 'yes' = 1; 'no' = 0; and 'don't know' = missing. If participants scored >=8 items to 'yes', they would be considered at high risk of SDB, whilst if they scored <8 items to 'yes', they would be considered at a low risk of SDB. Statistical Analysis Statistical analysis was performed using SPSS software version 28 (IBM Corp. Armonk, NY, USA). The demographic of the children and the prevalence rate of SDB was assessed with descriptive statistics. A Chi-square test was performed to identify the differences in demographic variables and SDB symptoms related to the risk factors. For the Chi-square test (2 x 2), demographic variables were dichotomized as (male/female) for gender and (older/younger) for age, when the test was performed to identify differences in being at high risk of developing SBD (yes/no). Moreover, possible risk factors for the SDB were assessed by binary logistic regression. The p-value was set to <0.05 as statistically significant. 3. Results A total of 1866 parents out of 2000 agreed to participate and complete the questionnaire (93.3%). The mean age of the participants was 9.67 +- 1.78 years. Among all the participants, a total of 1027 children were female (55%) and 839 were male (45%). The younger group and older group consisted of a total of 44.2% and 55.8% of participants, respectively. The outcome of the PSQ scoring among all the participants was presented in Table 1. Based on the PSQ scores, a total of 243 children (13%) were categorized as high risk of SBD. Table 2 showed that the most prevalent symptom of SBD is mouth breathing (14.4%) and the least prevalent symptom is witnessed apnea (6.6%). The Chi-square test showed that there were significant differences between gender (females/males) and age (younger/males) in relation to being at high risk of developing SBD (p < 0.05). Moreover, there was a significant difference between high and low-risk children concerning habitual snoring, mouth breathing, witnessed apnea, being overweight, and bedwetting (Table 2). Table 3 presented that binary logistic regression exhibited no significant association with gender in terms of developing SDB. However, the younger age group is significantly associated with the risk of developing SDB. The risks of developing SDB were 1.43 times higher in younger children compared to older children. In addition, children with habitual snoring, witnessed apnea, mouth breathing, being overweight, and bedwetting were at 8.9 times, 2.15 times, 6.6 times, 4.57 times, and 4.81 times higher risk of developing SDB, respectively (Table 3). 4. Discussion The current study determined the incidence of SDB and identified related risk factors among primary school children in Al-Kharj, Saudi Arabia with the Arabic version of PSQ, which is the most used pediatric sleep questionnaire . The original PSQ was translated into the Arabic language by Baidas et al. (2018), which assessed 1350 Saudi children, with 91% of the questionnaire reporting good concordance . The risk of undiagnosed and untreated pediatric OSA could result in significant medical comorbidities including, but not limited to, cardiovascular, cognitive, metabolic, and growth hormone dysfunction . The lack of awareness of pediatric SDB is one of the main barriers for families to seeking proper care, which starts with a formal diagnosis by a sleep physician using PSG followed by proper treatment . This is the first population-based study in Al-Kharj which determined the incidence of SDB among school-going children. The response rate of the current study is 93.3% from a total of 1866 participants which is relatively large compared to previous studies , and somewhat comparable to a recent Saudi study . Such a high response rate (93.3%) might be attributed to the way the questionnaire was distributed through school principals who are figures of authority at their schools. The prevalence of SDB varies by definition and ranges from 4-17% . The main finding of the study showed that 13% of participants with an age limit of 6-12 years were at a high risk of SDB. Additionally, 14.4% were considered mouth breathing, and 6.6% had witnessed OSA. In comparison to previous Saudi studies that used PSQ and included children with similar age ranges and comparable gender distribution exhibited 21% and 23% of high-risk SBD which is higher compared to the current study . Moreover, samples from the previous studies reported participants with habitual snoring are more reported (10.7% and 15.9%) and witnessed apneas reported to a lesser extent (3.4% and 4%) compared to the present study . The variation between our findings and Baidas et al. (2018) is due to differences in the operational definition of what constitutes habitual snoring. Moreover, Al Ehaideb et al. (2021) conducted a PSQ-based survey study of 285 Saudi children seeking orthodontic treatment and found that 47.7% were at high risk of developing SDB . They also reported 11.3% and 11.6% of their sample to have habitual snoring and witnessed apnea, respectively . These larger numbers could be because their sample was collected from an orthodontic clinic at a tertiary public hospital that receives referrals of cases with moderate or severe forms of malocclusions. Globally, studies that used the PSQ reported the prevalence of children at high risk of developing SDB to range from 7.9% to 12.8% . The current study shows that there is a significant difference (p = 0.009) in SBD risk factors between younger and older groups. Additionally, it also showed that younger children were 1.43 times more likely to be at high risk of developing SDB compared to older children. In children, enlarged adenoids are the main reason for developing SDB and adenoidectomy/tonsillectomy is considering the primary treatment of SDB in children . Adenoids reach their maximum size between the ages of 5 and 7 and begin to shrink afterward . Therefore, enlarged adenoids were more likely to be found in the younger age group in our sample aged 6 to 9 years, and this might be the reason why the younger age group exhibited increased risks of developing SDB. In addition to enlarged adenoids, obesity has also been considered a possible cause of OSA. This study showed that there was a significant association between the high-risk group and children being overweight as perceived by their parents, which agrees with other studies conducted in Saudi Arabia . In terms of gender distribution, this study showed a significant association between females and OSA, which is opposite to the reported male predilection of pediatric OSA in other studies . The reason why there are usually more males affected by OSA is suggested to be due to the differences in the puberty age between males and females, as females enter puberty first. This variation in OSA prevalence between males and females usually increases as they age . This finding reinforced the information from the previous studies where a significant difference between gender and OSA was observed, including children older than 12-13 years , while studies that did not show a significant association between gender and OSA were mostly limited to the younger age group . This might explain why the current study, which was limited to children younger than 12 years old, was not in line with other studies in terms of the association between males and OSA. Nonetheless, it is important to note that the unique contribution of gender in the regression model was not significant in relation to being at high risks of developing SBD, controlling for other variables (including age, habitual snoring, witnessed apnea, parent perception of the child being overweight, and bedwetting). Therefore, cautious interpretation is warranted regarding this study findings in terms of gender association with, and contribution to, being at high risk of developing SBD. It is not surprising that this study showed a significant association between high risks of developing SBD and snoring as snoring is one of the main symptoms of SDB and OSA . Similar findings were observed in other previous studies . Hence, parents need to appreciate the importance of seeking medical care when their child snores during sleeping. In the future, it would be worthwhile to explore whether the parents who participated in the study have considered medical care for their children, especially those with a high risk of pediatric SDB. One of the common misapplications of PSQ questionnaires in the literature is the interpretation of a "yes" response to question no. 6 in the first domain (Table 1), as many authors have interpreted this as the presence of OSA . It is recommended that authors use the term "witnessed apnea" for "yes" responses to this question instead of the inaccurate assumption that the child has OSA or is considered at high risk to develop SDB. The current study also showed that the high-risk group (19.3%) is more associated with OSA compared to the low-risk group (4.7%). Additionally, children with witnessed apneas were 2.84 times more likely to be at high risk for SDB. However, not every child with witnessed apnea was at high risk for SDB. There were 77 children who had witnessed apneas yet were considered low risk for SDB based on the answers to the remaining questions. This study consists of some risk of bias due to the nature of its methodology. Moreover, being overweight was assessed by the parent's perception. Using the body mass index would have been more objective yet was much harder to do especially given the large sample used in the current study. Additionally, having a PSG would have given a definitive OSA diagnosis. A future study could include a subgroup of the total sample to assess the prevalence more accurately. Although it is not the aim of the study, public awareness programs regarding snoring for children and the risk of pediatric SDB and its symptoms need to be implemented in schools as many local studies have reported similar findings of high rates of snoring and witnessed apnea. 5. Conclusions In conclusion, 13% of the school-going children in Al-Kharj Saudi Arabia are at high risk of developing SDB at a younger age. Moreover, habitual snoring, mouth breathing, being overweight, bedwetting, and witnessed apnea were more prevalent in children with a high risk of SDB. Thus, the importance of further exploration of SDB among Saudi school-going children needs to be recognized, strategized, and materialized. Acknowledgments The authors want to thank Abdulrahman Hattan, Khalid Faqihi, and Ali Alhawiatan for their help with data collection. This study is supported via funding from Prince Sattam bin Abdulaziz University project number (PSAU/2023/R/1444). Author Contributions Conceptualization, S.H.A. and A.A.; methodology, S.A., A.I.M., F.A. and A.A.; software, S.H.A. and A.A.; validation, S.H.A. and A.A.; formal analysis, S.H.A., K.G. and A.A.; investigation, S.H.A., K.G. and A.A.; resources, S.H.A. and K.G.; data curation, S.H.A.; writing--original draft preparation, S.H.A., K.G. and A.A.; writing--review and editing, S.H.A., K.G. and A.A.; visualization, S.H.A., K.G., A.A., F.A. and A.I.M. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement This statement is to signify that the authors obtained permission from the Research Ethical Committee at Prince Sattam Bin Abdulaziz College of Dentistry to conduct the study (IRB #1439-03-003). We, the authors, declare that the consents were obtained from parents/guardians, and this research did not pose any risk to the participants, in accordance to the Helsinki Declaration. Also, we would like to mention that the data set used in this study is available upon request with the Corresponding Author. Informed Consent Statement Informed consent was obtained from all subjects involved in the study. Data Availability Statement Not applicable. Conflicts of Interest The authors declare no conflict of interest. healthcare-11-00747-t001_Table 1 Table 1 Affirmative responses to PSQ questions including frequency and percentages. Domain Question n (%) Snoring/breathing problems Snores more than half the time during sleep 202 (10.8) Always snores during sleep 167 (8.9) Snores loudly 199 (10.7) Heavy or loud breathing 230 (12.3) Difficulty in breathing 159 (8.5) Has stopped breathing during sleep 124 (6.6) Mouth breathing during the day 268 (14.4) Dry mouth upon waking 304 (16.3) Wets bed, walks during sleep or wakes up scared during the night 209 (11.2) Daytime sleepiness and development Wakes up unrefreshed 447 (24.0) Wakes up with a headache 180 (9.6) Difficult to wake the child up 435 (23.3) Sleepiness during the day 340 (18.2) Sleepiness during the day noticed by the teacher 179 (9.6) Has stopped growing at a normal rate 122 (6.5) Parent perception of the child being overweight 219 (11.7) Inattention/hyperactivity Does not respond quickly when spoken to 297 (15.9) Difficulty in organizing and managing tasks 328 (17.6) Easily distracted by external stimuli 435 (23.3) Seems restless and moves when seated 234 (12.5) Looks in a hurry all the time 761 (40.8) Interrupts others during speech 369 (19.8) Number of children at high risk of sleep-disordered breathing (eight or more yes responses) 243 (13) n; Total number, %; percentage. healthcare-11-00747-t002_Table 2 Table 2 Chi-square test results of differences in demographic variables and SDB symptoms related to SDB risk. Variable All Children Low Risk High Risk p-Value n (%) n (%) n (%) Personal Characteristics Sex Male 839 (45.0) 745 (45.9) 94 (38.7) 0.035 Female 1027 (55.0) 878 (54.1) 149 (61.3) Age group, years 6-9 825 (44.2) 700 (43.1) 125 (51.4) 0.015 10-12 1041 (55.8) 923 (56.9) 118 (48.6) SDB symptoms Habitual snoring Yes 167 (8.9) 101 (6.2) 66 (27.2) <0.001 * Witnessed apnea Yes 124 (6.6) 77 (4.7) 47 (19.3) <0.001 * Mouth breathing Yes 268 (14.4) 145 (8.9) 123 (50.6) <0.001 * Parent perception of the child being overweight Yes 219 (11.7) 139 (8.6) 80 (32.9) <0.001 * Bedwetting Yes 209 (11.2) 127 (7.8) 82 (33.7) <0.001 * SDB: sleep-disordered breathing, n; total number, %; percentage, *; significant difference (<0.05). healthcare-11-00747-t003_Table 3 Table 3 Binary logistic regression analysis of sleep-disordered breathing risk. 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PMC10000778 | Diagnostics (Basel) Diagnostics (Basel) diagnostics Diagnostics 2075-4418 MDPI 10.3390/diagnostics13050870 diagnostics-13-00870 Article Comprehensive Analysis of Prognosis and Immune Landscapes Based on Lipid- Ferroptosis-Associated Signature in Uterine Corpus Endometrial Carcinoma Yang Pusheng Conceptualization Methodology Validation Data curation Writing - original draft 1+ Lu Jiawei Conceptualization Methodology Software 23+ Zhang Panpan Validation Data curation 1+ Zhang Shu Writing - review & editing Funding acquisition 1* Boichuk Sergei Academic Editor Kiyamova Ramziya Academic Editor 1 Shanghai Key Laboratory of Gynecology Oncology, Department of Gynecology and Obstetrics, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, China 2 Department of Gastroenterology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 201620, China 3 Shanghai Key Laboratory of Pancreatic Diseases, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 201620, China * Correspondence: [email protected] + These authors contributed equally to this work. 24 2 2023 3 2023 13 5 87012 1 2023 13 2 2023 21 2 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). (1) Background: The effect of tumor immunotherapy is influenced by the immune microenvironment, and it is unclear how lipid metabolism and ferroptosis regulate the immune microenvironment of uterine corpus endometrial carcinoma (UCEC). (2) Methods: Genes associated with lipid metabolism and ferroptosis (LMRGs-FARs) were extracted from the MSigDB and FerrDb databases, respectively. Five hundred and forty-four UCEC samples were obtained from the TCGA database. The risk prognostic signature was constructed by consensus clustering, univariate cox, and LASSO analyses. The accuracy of the risk modes was assessed through receiver operating characteristic (ROC) curve, nomogram, calibration,, and C-index analyses. The relationship between the risk signature and immune microenvironment was detected by the ESTIMATE, EPIC, TIMER, xCELL, quan-TIseq, and TCIA databases. The function of a potential gene, PSAT1, was measured by in vitro experiments. (3) Results: A six-gene (CDKN1A, ESR1, PGR, CDKN2A, PSAT1, and RSAD2) risk signature based on MRGs-FARs was constructed and evaluated with high accuracy in UCEC. The signature was identified as an independent prognostic parameter and it divided the samples into low-risk groups. The low-risk group was positively associated with good prognosis, high mutational status, upregulated immune infiltration status, high expression of CTLA4, GZMA and PDCD1, anti-PD-1 treatment sensitivity, and chemoresistance. (4) Conclusions: We constructed a risk prognostic model based on both lipid metabolism and ferroptosis and evaluated the relationship between the risk score and tumor immune microenvironment in UCEC. Our study has provided new ideas and potential targets for UCEC individualized diagnosis and immunotherapy. lipid metabolism ferroptosis immunotherapy prognostic marker uterine corpus endometrial carcinoma National Natural Science Foundation of China82273007 This research was funded by a grant from the National Natural Science Foundation of China (Grant No. 82273007 to S.Z.). pmc1. Introduction Uterine corpus endometrial carcinoma (UCEC) is one of the most common gynecologic malignancies, with an increasing incidence of about 1% per year . Approximately 15% of UCEC patients are diagnosed at an advanced stage, and approximately 15-20% of patients will experience relapse after primary surgical treatment . Although surgery, carboplatin/paclitaxel systemic chemotherapy, and hormone therapy are effective treatments, patients with advanced disease, recurrence, or drug resistance still have poor prognoses . In recent years, it has been reported that patients with advanced endometrial cancer may benefit from immunotherapy. The main immunotherapy approaches include immune checkpoint inhibitors (ICIs), adoptive cell transfer (ACT), cancer vaccines, and lymphocyte-promoting cytokines. For example, dostarlimab, a drug that inhibits the programmed cell death 1 and programmed cell death ligand 1 pathway, can improve the prognosis of patients receiving platinum chemotherapy or progressive mismatch repair deficiency endometrial cancer . However, the effect of immunotherapy is not ideal due to the complexity of the immune microenvironment and differences in the response to immunotherapy . Therefore, it is vital to identify potential diagnostic and prognostic targets or risk signatures and to tailor individualized immunotherapy strategies for improving the outcomes of UCEC patients. Obesity is an independent risk factor for UCEC . Almost all UCEC patients with obesity have altered lipid metabolism . Tan et al. built an 11 lipid metabolism gene (LMG) signature to reflect the prognosis of UCEC patients . Lipids are susceptible to oxidation by oxygen free radicals. Overproduction and elimination failure of lipid peroxidation are the main reasons for the novel iron-dependent cell death ferroptosis . Liu et al., Wang et al., and Wei et al. constructed a ferroptosis-related gene signature to predict the prognosis of UCEC patients . Lipid synthesis, storage, and degradation processes can be regulated by ferroptosis . Iron depletion leads to a large amount of lipid accumulation in breast cancer cells . Iron accumulation is due to altered lipid metabolism associated with increased oxidative stress in myelodysplastic syndromes . Ferroptosis is closely associated with lipid metabolism pathways . Inhibiting b-oxidation can restore tumor cell sensitivity to ferroptosis . Upregulating stearoyl CoA desaturase 1 (SCD1), the rate-limiting enzyme in fatty acid synthesis, increases the resistance of tumor cells to ferroptosis. Increasing evidence suggests that lipid metabolism and ferroptosis closely affect each other . However, the interaction and shared role of ferroptosis and lipid metabolism in UCEC remains unclear. In the present study, we aimed to construct a prognostic risk signature based on both lipid metabolism and ferroptosis to comprehensively analyze their combined effects on UCEC. We screened six risk genes (CDKN1A, ESR1, PGR, CDKN2A, PSAT1, and RSAD2) as reliable diagnostic and prognostic biomarkers and divided UCEC patients into low-risk groups based on their risk score. Then, we estimated differences in immune score, immune infiltration, immune checkpoint, immunotherapy, and chemotherapy response between the low-risk groups. The findings provide a new idea for individualized therapy strategies to improve the prognosis of UCEC patients. 2. Materials and Methods 2.1. Dataset Information Sequencing RNA data (HTSeq-FPKM) and clinical information were obtained from The Cancer Genome Atlas (TCGA) database, and 579 cases were selected for study, including 544 UCEC samples and 35 normal samples. The detailed clinical information of the UCEC patients is shown in Table S1. 2.2. Extraction of Lipid-Metabolism-Related and Ferroptosis-Associated Genes Lipid-metabolism-related genes (LMRGs) were collected from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and the Molecular Signatures Database (MSigDB), including the GSEA, HALLMARK, and REACTOME databases . The detailed gene sets are shown in Table S2. A total of 1457 genes were selected for analyses after removing duplicate genes (Table S3). In addition, we downloaded 288 ferroptosis-associated genes (FAGs) from the FerrDb database accessed on 1 June 2022). After removing the replicates, 259 individual FAGs were used for further investigation. 2.3. Construction of the LMRG and FAR Prognostic Signature The evaluation of the differentially expressed LMRGs (DE-LMRGs) was performed using the default settings for the "lmFit", "eBayes", and "topTable" functions in the "limma" R package. The screening criteria were p < 0.05, |Log2 Fold Change (FC)| > 1, and a false discovery rate (FDR) < 0.05. Then, univariate Cox regression analysis was applied to determine LMRGs with overall survival (OS) in UCEC by using the coxph function in the "survival" R package at p < 0.05. The molecular classification of DE-LMRGs in UCEC was analyzed by the "ConsensusClusterPlus" R package. Principal component analysis (PCA) was performed to identify the grouping ability of our model with the R package "stats". Then, the FAGs interacted with the results of the consensus clustering approach, and the genes of interaction were selected for further study. We performed univariate cox and least absolute shrinkage and selection operator (LASSO) analyses to identify significant prognostic genes based on both LMRGs and FARs with a threshold of p < 0.05. Then, a risk score signature was created by considering the estimated cox regression correlation coefficients and the expression values of the optimized LMRGs and FARs. The formula is risk score = Si1expGenei*coeffi. According to the median value of the calculated risk scores from the TCGA-UCEC, the patients were divided into high-risk groups. The prognostic ability and stability of the signature was measured by the Kaplan-Meier (K-M) analysis, multivariate Cox regression analysis, and receiver operating characteristic (ROC) curve with the "Survival" and "sevivalROC" R package (p < 0.05). 2.4. Functional Enrichment Analysis To examine the distinction between the low-risk group of our model, we further carried out gene set variation analysis (GSVA) using the "GSVA" function with method parameters (min.sz = 10, max.sz = 500, verbose = TRUE) of the "GSVA" R package, and conducted KEGG pathway analysis and Gene Ontology (GO) analysis via the "clusterProfiler (version 3.14.3)" R package (p < 0.05). 2.5. Tumor Mutational Burden (TMB) Analysis We downloaded the somatic mutation data from TCGA. Using Perl, we calculated the TMB value of each sample and divided all samples into low-TMB groups based on the median TMB . Then, K-M analysis was used to assess survival differences between the groups. We also calculated the expression differences in TMB between the low-risk groups and analyzed the relationship between TMB and the risk score (p < 0.05) 2.6. Immune Infiltration of the Prognostic Model The CIBERSORT algorithm was utilized to evaluate the 22 types of immune fractions between the low-risk groups, and the results were visualized with the "vioplot" R package. Then, we used the Tumor Immune Estimation Resource (TIMER) to evaluate correlations between expression of six model genes and the immune infiltration level of tumor-infiltrating immune cells. We also analyzed the relationship between innovative targeted therapy and risk prognostic models. The Wilcoxon test was used to detect expression of potential immune checkpoints between the high-risk and low-risk groups (p < 0.05). Furthermore, we downloaded clinical data from The Cancer Immunome Atlas (TCIA) to predict the response to immune checkpoint blockade (CTLA-4 and PD-1) in patients in the low-risk groups by the immunophenoscore. In addition, according to the Genomics of Drug Sensitivity in Cancer (GDSC) database, the R package "pRRophetic" was used to measure the half-maximal inhibitory concentration (IC50) of chemotherapeutic drugs. 2.7. Cell Culture The UCEC cell lines Ishikawa, HEC-1A, HEC-1B, and ECC-1 were obtained from the American Type Culture Collection (ATCC). The HEC-1A cell lines were cultured in McCoy's 5A (Gibco, New York, NY, USA) supplemented with 10% fetal bovine serum (FBS, Biological Industries, Kibbutz Beit-Haemek, Israel) and 1% penicillin/streptomycin (P/S); the others were cultured in RPMI 1640 culture medium with 10% FBS and 1% P/S. All of the cells were cultured at 37 degC in a humidified incubator under 5% CO2. 2.8. Small Interfering RNA (siRNA) Transfection The siRNA PSAT1 and scrambled control sequences were obtained from Gene Pharma (Shanghai, China). The details of the sequences are as follows: si-PSAT1-1: forward 5'-CAGUGUUGUUAGAGAUACAdTdT-3', reverse 5'-UGUAUCUCUAACAACACUGdTdT-3'; si-PSAT1-2: forward 5'-GCUGUUCCAGACAACUAUAdTdT-3', reverse 5'-UAUAGUUGUCUGGAACAGCdTdT-3'. siRNA transfection was carried out using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). 2.9. Quantitative Real-Time PCR (qRT-PCR) Total RNA was extracted using TRIzol reagent (Sangon Biotech, Shanghai, China) after transfecting siRNA for 48 h, and reverse transcription was performed using PrimeScriptTM RT Reagent Kit (TAKARA, RR047A). QRT-PCR was conducted with the SYBR Green qPCR Supermix kit (Invitrogen). The primers used were purchased from Tsingke Biotechnology Co (Beijing, China), as follows: PSAT1 Forward 5'-ACTTCCTGTCCAAGCCAGTGGA-3'; PSAT1 Reverse 5'-CTGCACCTTGTATTCCAGGACC-3'; GAPDH Forward 5'-GGAGCGAGATCCCTCCAAAAT-3'; GAPDH Reverse 5'-GGCTGTTGTCATACTTCTCATGG-3'. 2.10. Western Blot Analysis Total proteins were obtained from cells using PIPA buffer (New Cell & Molecular Biotech, Suzhou, China) at 72 h after siRNA transfection, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, New York, NY, USA). The membranes were blocked using 5% BSA for at least 1 h at room temperature and incubated with PSAT1 (10501-1-AP, Proteintech, Wuhan, China) or GAPDH (10494-1-AP, Proteintech) at 4 degC overnight. The next day, the membranes were incubated with secondary antibody (GB23303, Servicebio, Shanghai, China) for 1 h at room temperature, and bands were detected by chemiluminescence. 2.11. Cell Proliferation Assay Cell proliferation was detected by the Cell Counting Kit-8 assay (CCK-8) and colony formation assay. For CCK-8, the cells were seeded into 96-well plates at a density of 2000 cells/well after 72 h of transfection. At the indicated time, CCK-8 solution (10 mL) was added to each well of the culture medium. Cell viability was measured using an automatic enzyme-linked immune detector after incubation for 1 h. For the colony formation assay, 1000 transfected cells were seeded into six-well plates for 10-14 days, and the culture medium was changed every three days. After staining with 0.1% crystal violet and photographing, cell colonies were statistically analyzed by the t-test. 2.12. Cell Migration and Invasion Assay Cell migration and invasion were assessed using 24-well transwell chambers (8 mm; Millipore). In brief, a sample of 4 x 104 cells suspended in 200 mL serum-free medium was seeded in the upper chamber, and the lower chamber contained 600 mL medium with 10% FBS. After 48 h, the chambers were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet dye for 30 min. The upper chamber cells were wiped off and then photographed and counted under a microscope. For the invasion assays, Matrigel (BD, biocoat, #358248) was used to coat the upper chamber, after which the cells were seeded; the next step was the same as above. 2.13. Statistical Analysis Bioinformatic statistical analyses were performed using R (v.3.6.1) software. Pearson correlation analysis was employed for correlation analysis between TMB and the risk model. All of the in vitro experiments were independently performed in triplicate and analyzed by the t-test. Data were analyzed using the IBM SPSS Statistics 22 and visualized in GraphPad Prism 9. The values were presented as the mean +- standard deviation (SD). p < 0.05 was considered statistically significant. 3. Results 3.1. Identification and Clustering of LMRGs A brief workflow of this research is presented in Figure S1. We screened 1457 LMRGs for differential expression analysis and identified 88 differentially expressed LMRGs (DE-LMRGs) with the "limma" R package based on 544 UCEC samples and 35 normal samples from TCGA. The boxplot of the expression patterns of the 88 DE-LMRGs is shown in Figure 1A. KEGG analysis and GO analysis showed that these significant genes mainly participate in lipid metabolic processes . Then, univariate cox hazards regression and Kaplan-Meier (K-M) analyses were utilized to screen out prognostic LMRGs based on TCGA, and we obtained six risk genes and three protective genes for survival . The consensus clustering approach was used to divide the UCEC samples with the non-negative matrix factorization (NMF) algorithm. Based on LMRGs expression, the optimal clustering stability was confirmed when K = 3 . We also performed principal component analysis (PCA), which showed the good grouping ability of our clustering . Therefore, all of the UCEC samples were divided into three clusters, and the heatmap showed lower expression for the DE-LMRG genes in Cluster A . Moreover, K-M analysis indicated a significant difference in OS among the three subgroups, with the patients in Cluster A having the best prognosis . By further analyzing the clinical characteristics among the three clusters, we found that patients in Cluster C had an older age and a higher grade and stage . 3.2. Signature Construction Based on LMRGs and FAGs Differentially expressed genes among the three clusters were obtained from consensus clustering analysis and intersected with FAGs. Then, we obtained both lipid metabolism-related and ferroptosis-associated genes (LMG-FAGs) . We performed overall-survival-based univariate regression analysis on the lipid-metabolism-related and ferroptosis-associated genes (LMG-FAGs) obtained through consensus clustering analysis. This approach revealed 211 LMG-FAGs associated with the prognosis of endometrial cancer, and we classified them into 87 risk genes and 124 protective genes according to the hazard ratio (HR) and p value (Table S4, p < 0.05). To avoid overfitting and bias, the results of univariate regression analysis were subjected to LASSO regression analysis using the "glmnet" R package, and the accuracy of the model was tested by cross-validation . Hence, a six-gene prognostic risk model was established by the following formula: risk score = [CDKN1A expression x (-0.02353)] + [CDKN2A expression x (0.11554)] + [ESR1 expression x (-0.05874)] + [PGR expression x (-0.11493)] + [PSAT1 expression x (0.05505)] + [RSAD2 expression x (0.01431)]. We analyzed the relationship between different risk scores and patient follow-up times, events, and expression changes of individual genes, and it was observed that with an increase in the risk score, the survival rate of patients decreased significantly. CDKN1A, ESR1, and PGR were found to be protective factors that showed downregulated expression with increased risk scores; CDKN2A, PSAT1, and RSAD2 showed the opposite result . Furthermore, we detected expression levels and performed multivariate Cox regression and K-M survival analyses on the six independent prognostic genes. The results indicated that high expression of CDKN1A, ESR1, and PGR was related to better prognosis, whereas high expression of CDKN2A, PSAT1, and RSAD2 was not . According to the median cut-off value of the risk score, the low-risk groups were established to differentiate the UCEC patients in TCGA, and the high-risk patients had a worse prognosis than the low-risk patients . Then, time-dependent ROC analysis was applied to evaluate the prediction capacity of the signature, with an area under the receiver operating characteristic curve (AUC) of 0.67, 0.70, and 0.70 at 365, 1905, and 1825 days, respectively . 3.3. Prognosis and Validation of the FAG-Based Signature To assess the accuracy of the model, we evaluated the performance of this signature with regard to pathological features (age, grade, and stage). The results indicated that high risk was significantly associated with older age and higher grade and stage . Then, the pathological features were added for univariate and multivariate cox regression, and the forest plot showed that age, grade, and stage were still independent prognostic factors, which means that the signature had high accuracy . In addition, we built a nomogram to predict the 1-year, 3-year, and 5-year survival probability of UCEC patients based on all of the above prognostic elements , and the calibration plot showed a C-index of 0.767 (0.741-0.793), indicating that the nomogram had good predictive ability . 3.4. DEG and Functional Enrichment Analyses of the Signature To investigate the relationship between the six genes in the risk model, we constructed a protein-protein interaction (PPI) network and analyzed the correlations . The results showed that PSAT1 and RSAD2 were more independent and less associated with other genes. Next, a volcano plot and heatmap showed the DEGs between the two risk groups; 81 genes were upregulated and 195 genes were downregulated . The PPI network of the DEGs is depicted in Figure S4C. To reveal the underlying biological characteristics associated with the risk scores, KEGG and GO analyses were performed based on DEGs between the low-risk groups. The results indicated that pathways such as kinase and peptidase regulation, apparatus morphogenesis, cell cycle regulation, viral infection, and antiviral innate immune response were highly enriched . In addition, we performed GSVA to probe differences in pathways between the two risk groups. As illustrated in the heatmap in Figure 4E, pathways related to lipid metabolism and ferroptosis, such as "tyrosine metabolism", "fatty acid metabolism", "alpha linolenic acid metabolism", and "DNA replication", were significantly enriched (p < 0.05). 3.5. Relationship between the Tumor Mutational Burden (TMB) and the Risk Model TMB, the somatic coding errors, is generally considered high when >10 or >16 mutations/megabase DNA are present . Recently, TMB is thought to be closely related to the survival prognosis of tumor patient . To examine in more depth how well the risk-prognosis model predicts tumor development, we investigated its relationship with TMB. First, correlation analysis showed that the TMB level had a negative association with the LMRG-FAG risk score , and the high-risk group showed lower TMB levels . We also investigated the survival of patients with different TMB statuses by K-M analysis, and the results demonstrated that the patients in the low-TMB group had poor prognostic outcomes . In addition, mutation information of the genes in the high-TMB groups was explored using a waterfall chart, and PTEN (58.2%), PIK3CA (48.7%), TTN (44.5%), ARID1A (43.5%), and TP53 (36.4%) were the top five mutated genes . We further studied and classified the mutation information, variant type, and SNV class, and the results demonstrated that missense mutations, single nucleotide polymorphism (SNP), and C > T accounted for the largest proportion . The number of altered bases in each sample and the mutation types in different colors are shown in Figure S5D,E; mutation information for the six risk genes [PGR (37%), ESR1 (33%), RSAD2 (27%), PSAT1 (18%), CDKN1A (14%), and CDKN2A (5%)] is provided in Figure S5F. Recently, multiple pieces of research have illustrated that TMB is closely associated with tumor immune cell infiltration and affects the efficacy of immunotherapy . Therefore, we evaluated the value of TBM in the complexity of the tumor immune microenvironment. We discovered that most immune cells had a positive correlation with the TMB level, especially T cells CD8+, T cells CD4+, and B cells . In addition, T cells CD8+, T cells CD4+ memory activated, T cells CD4+ memory resting, and T cells regulatory had higher expression in the high-TMB group compared to the low-TMB group , suggesting that TMB may have an effect on the immune response. 3.6. Immune Infiltration Associated with the LMRG-FAG-Based Signature Recent studies have shown that lipid metabolism and ferroptosis are important components of the tumor microenvironment and are strongly associated with tumor immune activities . We first used ESTIMATE to determine the relationship of tumor immune infiltration between the two risk groups. The stromal, immune score, and ESTIMATE score were significantly downregulated in the high-risk group . Then, the CIBERSORT algorithm was applied to detect the composition of the 22 immune cells in UCEC patients . A boxplot demonstrated that the difference in the distribution of the 10 immune-infiltrating cells between the two risk groups was significant. The naive B cells, memory B cells, resting CD4 memory T cells, regulatory T cells (Tregs), and resting dendritic cells had low expression in the high-risk group compared to the low-risk group. Meanwhile, the follicular helper T cells, monocytes, M1 macrophages, activated dendritic cells, and M2 macrophages were significantly upregulated in the high-risk group compared to the low-risk group . We also analyzed immune infiltration using the EPIC, TIMER, xCELL, and quanTIseq databases, which fully confirmed the six-gene prognostic risk signature to be closely related to immune activity . In addition, the TIMER database was utilized to assess the relationship between the six risk genes and tumor-infiltrating immune cells. The results showed that only RSAD2 correlated positively with B cells (cor = 0.1858, p = 0.0015); except for RSAD2, the other genes were significantly associated with CD8+ T cells . 3.7. Immunotherapy and Chemotherapy in Different Risk Groups Recently, immune checkpoints have been identified as key targets of immunotherapy, and immune checkpoint inhibitors (ICIs) are regarded as an effective therapeutic strategy for patients with advanced disease . Therefore, we identified potential relationships between the expression of immune checkpoint molecules and our risk model. The results showed that IDO1 and LAG3 expression was significantly increased in the high-risk group compared with the low-risk group, while the expression of CTLA4, GZMA and PDCD1 was obviously decreased in the high-risk group compared with the low-risk group . Then, we conducted immunophenoscore (IPS) analysis to predict immunotherapy response. As shown in Figure 6F, low-risk patients were more sensitive to anti-PD-1 therapy (p < 0.05), suggesting that immunotherapy of blocking CTLA-4 and PDCD1 may be more beneficial for patients in the low-risk group. Since chemotherapy is the main treatment for advanced and recurrent UCEC, we evaluated the response of chemotherapeutics to UCEC patients using the pRRophetic algorithm based on our signature and found that the estimated IC50 of typical chemotherapy drugs (cisplatin, paclitaxel, doxorubicin, and etoposide, etc.) were significantly higher in the low-risk group . For the other 40 chemotherapy and small molecule drugs, such as lenalidomide, gefitinib, AMG.706, and JNK inhibitor VIII, patients in the high-risk group were identified as being more sensitive . Thus, we indicated that patients with low risk scores were more resistant to chemotherapy than those with high risk scores, but they were more sensitive to anti-PD-1 therapy. In addition, patients in the high-risk group were better suited for chemotherapy. These results may have important implications for individualized immunotherapy in patients with advanced UCEC. 3.8. In Vitro Function of the Risk Gene PSAT1 in UCEC Cells To further validate the ability of risk signatures to predict prognosis, we investigated protein expression of the six risk genes between normal and UCEC tissues with the CPTAC and HPA (Human Protein Atlas) databases , and the results corresponded with previous analysis. Combined with prognostic analysis and literature searches, we selected PSAT1 for further in vitro functional assays. We identified the mRNA and protein expression of PSAT1 in four UCEC cell lines (Ishikawa, HEC-1A, HEC-1B, and ECC1), and Ishikawa and HEC-1B cells were selected for subsequent studies . Next, we knocked down PSAT1 with siRNA, and the efficiency was verified by qPCR and Western blot analysis . CCK-8 and colony formation assays showed that knockdown of PSAT1 significantly suppressed the proliferation of Ishikawa and HEC-1B cells . In addition, the migration and invasion of the two cell lines were also apparently inhibited after PSAT1 knockdown, as determined by transwell assays . These results demonstrate that the risk gene PSAT1 significantly promotes progression of UCEC and may affect the prognosis of UCEC patients. 4. Discussion UCEC is one of the most lethal gynecological malignancies. Although many studies over the past decades have sought to improve treatment efficacy, patients with advanced and recurrent disease still have poor prognosis . With the rise and application of immunotherapy, it is insufficient to estimate the prognosis of UCEC patients based on traditional clinicopathological stage . Therefore, our study included the tumor immune microenvironment and immunotherapy in UCEC based on both lipid metabolism and ferroptosis to select more effective prognostic targets and guide individualized treatment of patients. Previous studies have established prognostic models of lipid metabolism or ferroptosis in UCEC . However, they only took a single influencing factor into account, and the complex tumor microenvironment was not considered. In our study, we comprehensively considered the interrelationship between lipid metabolism and ferroptosis, based on which a prognostic model of six genes was constructed. We deeply explored the relationship between the model risk score and the tumor immune microenvironment. We found that infiltration of B cells, T cells, and NK cells and expression of the immune checkpoints (CTLA4, GZMA, and PDCD1), as well as sensitivity and chemotherapy resistance to anti-PD-1 treatment in UCEC patients were closely related to the risk scores of the prognostic model. Moreover, in vitro experiments demonstrated that one of the potential targets, PSAT1, promoted the proliferation, migration, and invasion of UCEC cells. Our experiments provide new ideas and a basis for individualized immunotherapy for UCEC patients and provide a potential target for UCEC therapy. In the present study, we obtained genes associated with both lipid metabolism and ferroptosis by consensus clustering analysis. After LASSO Cox regression, we constructed a prognostic signature containing six risk genes (CDKN1A, ESR1, PGR, CDKN2A, PSAT1, and RSAD2) based on LMG-FAGs. K-M survival analysis, ROC curves, a nomogram, and calibration identified that the signature had high predictive ability. Estrogen receptor 1 (ESR1) and a progesterone receptor (PGR) were reported to participate in lipid metabolism by encoding estrogen or steroid receptors to promote tumor progression . Cyclin-dependent kinase inhibitors 1A and 2A (CDKN1A and CDKN2A) have been identified as ferroptosis-related genes in recent studies and can be regarded as biomarkers that influence the tumor microenvironment . Radical s-adenosyl methionine domain containing 2 (RSAD2) is an interferon-stimulated gene that exerts antiviral effects by dysregulating cellular lipid metabolism . Phosphoserine aminotransferase 1 (PSAT1) has been reported to affect the progression of various cancers by participating in lipid metabolism processes . In conclusion, the six-gene prognostic model showed a significant correlation with lipid metabolism or ferroptosis. In our study, these six genes were used for risk scoring, and each UCEC patient was categorized into two risk groups according to the risk score. We then explored the pathological features of the risk signature, with the high-risk group being related to older age and higher grade and stage. We also found that knockdown of PSAT1 inhibited the proliferation, migration, and invasion of UCEC cells, enhancing the reliability of our model. Subsequently, we comprehensively analyzed the impact of the risk signature on UCEC. A total of 276 genes were identified to be closely related to the risk score. GO, KEGG, and GSVA analyses based on the signature demonstrated that pathways associated with lipid metabolism and ferroptosis were significantly enriched, which also confirmed the accuracy of our signature. TMB is reported to correlate highly with tumor progression; for example, gastrointestinal tumor patients with low TMB have lower objective response rates and shorter progression-free survival , and high TMB is a poor prognostic factor for non-small cell lung cancer . We found that TMB levels had a negative relationship with the LMRG-FAG risk model, which means that patients with low risk and high a mutational burden have a better prognosis in UCEC. Because surgery and chemoradiotherapy have limited effects in patients with advanced and recurrent UCEC and traditional pathological staging has an insufficient ability to estimate prognosis, we focused on the relationship of the LMRG-FAG-based risk model with immunotherapy. Stromal, immune, and ESTIMATE scores were significantly downregulated in the high-risk group, indicating that lipid metabolism and ferroptosis are significantly associated with the immune status of UCEC. CIBERSORT algorithm analysis showed that the distribution of 10 immune cells varied between the low-risk groups, with antitumor cells (B cells, T cell CD8, and monocytes, etc.) present at higher abundance in the low-risk group. According to the results, we suggest that the risk score is associated with immune infiltration and immune status in UCEC. Adverse T cell regulatory pathways tend to be overactive when cancer occurs. CTLA-4 inhibits the immune response at the early stage of T cell induction, and PDCD1 prevents T cell function in peripheral tissues in the later stages . Recently, immune checkpoint blockade, one of the major immunotherapy methods, has proven to be an effective strategy for enhancing the effector activity and clinical impact of anti-tumor T cells . Among the ICIs, blocking CTLA-4 and PDCD1 are the two most eminent approaches. CTLA-4 and PDCD1 blockade could induce tumor immunity by improving effector T cell activity or consuming Treg . In 2011, Ipilimumab, a CTLA-4 inhibitor, was approved for melanoma . In 2017, the PDCD1 inhibitor pembrolizumab was approved for UCEC patients with microsatellite instability, and half of the patients benefited from it . Since the predictive value of immune checkpoints has been demonstrated in a variety of human malignancies, we then explored immune checkpoint expression between the two risk groups to guide individualized immunotherapy for UCEC patients. The expression of CTLA4, GZMA and PDCD1 was significantly upregulated in patients with low risk scores, and IDO1 and LAG3 were increased in the high-risk group. Therefore, we indicated that specially blocking CTLA-4 and PDCD1 immunotherapy would be more effective for patients in the low-risk group. Meanwhile, we detected the difference in sensitivity to PD-1 and CTLA-4 inhibitors, and the results indicated that low-risk patients were more sensitive to anti-PD-1 therapy, meaning that immunotarget therapy was more effective in low-risk patients. Accordingly, our risk signature has a certain guiding role in the anti-PD-1 immunotherapy of UCEC patients. Interestingly, high-risk patients were more sensitive to traditional chemotherapeutic agents and small molecule inhibitors such as cisplatin, paclitaxel, AMG.706, and ABT.888. Hence, patients in the high-risk group were more likely to benefit from chemotherapy and our signature can be used to guide personalized treatment of UCEC patients. However, it is undeniable that our study also has some limitations. First, the study data were obtained from only TCGA, and we did not verify the accuracy of our model with more cohorts. Second, immunotherapy and chemosensitivity analyses were only derived from the transcriptome, and we still need to obtain more prospective experimental data to support the findings. Finally, as a potential therapeutic target, the molecular mechanism underlying the risk-related gene PSTA1 needs to be further clarified. 5. Conclusions Consequently, we constructed a risk prognostic model based on both lipid metabolism and ferroptosis to deeply analyze the relationship between lipid metabolism, ferroptosis and gene mutation, immune infiltration, immunotherapy, and chemotherapy in UCEC patients and provided potential biomolecules and a preliminary basis for individualized treatment of patients. Supplementary Materials The following supporting information can be downloaded at: Click here for additional data file. Author Contributions Conceptualization, methodology, and software: P.Y. and J.L.; validation and data curation: P.Y. and P.Z.; writing--original draft preparation: P.Y.; writing--review and editing and funding acquisition: S.Z. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Our study is based on open source data, so there are no ethical issues and other conflicts of interest. Thus, our study was exempted from the approval of local ethics committees. Data Availability Statement Publicly available datasets were analyzed in this study. These data can be found here: accessed on 1 June 2022 (TCGA); accessed on 1 June 2022 (GEO); accessed on 1 June 2022 (FerrDb); accessed on 1 June 2022 (MSigDB). Conflicts of Interest The authors declare no conflict of interest. Figure 1 Identification and clustering of LMRGs: (A) expression of 88 DE-LMRGs between the UCEC samples and normal samples. * p < 0.05, ** p < 0.01, and *** p < 0.001. (B) Consensus clustering map of NMF clustering, and the optimal cluster number was three (k value = 3). (C) Principal component analysis (PCA) showed that the three clusters in the consensus clustering approach were robustly segregated. (D) Kaplan-Meier curve survival (K-M) analysis of patients in the three clusters, and patients in Cluster A had a better prognosis. p < 0.001. Figure 2 Constructing the LMR-FAG-related risk score. (A) Venn diagram showing lipid-me tabolism-related and ferroptosis-associated genes (LMR-FAGs). (B,C) LASSO coefficient profiles and cross-validation were used to identify LMR-FAG-related genes. (D) Distributions of risk scores, survival status, and expression levels of six prognostic genes in UCEC. (E) The K-M survival analysis demonstrated that patients in the low-risk group had a better prognosis. p < 0.001. (F) Time-dependent ROC curve and AUC of the prognostic signature in UCEC patients from TCGA. Figure 3 Establishment of a predictive nomogram. (A-C). The pathological characteristics age, grade, and stage in the low-risk groups. Patients in the high-risk group had an older age and higher grade and stage. (D) Univariate and multivariate Cox regression analyses of the signature and clinical characteristics. (E) Nomogram to predict 1-year, 3-year, and 5-year overall survival times. (F) Calibration curve to assess the accuracy of the nomogram. The C-index was 0.767 (0.741-0.793). Figure 4 DEGs and functional enrichment of the signature. (A,B) The volcano plot and heatmap show the details of different genes between the low-risk groups. (C,D) KEGG and GO functional enrichment analysis in terms of differentially expressed genes. (E) Gene set variation analysis between low-risk patients based on TCGA-UCEC. Figure 5 Relationship between the tumor mutational burden (TMB) and the risk model. (A) Relationship between TMB and risk score. TMB was negatively associated with risk score. (R = -0.13, p < 0.05). (B) The result showed that patients in high-risk group had lower TMB levels. p < 0.05 (C) The K-M analysis presents the difference in overall survival between the high-TMB groups. In addition, patients with high TMB had a better prognosis, p < 0.05. (D) The waterfall plot shows the mutation information of the top 20 genes in each UCEC sample. Figure 6 Immune landscape associated with the FAG-based signature. (A-C) Differences in stromal score, immune score, and ESTIMATE score between the low-risk groups. (Wilcoxon, p < 0.05). (D) The difference in 22 immune infiltrating cells in the TCGA-UCEC samples between the low-risk groups was analyzed by the Wilcoxon test. (E) The connection between immune checkpoint molecules (CTLA4, GZMA, IDO1, LAG3, and PDCD1) and risk scores. (F) Prediction of immunotherapy response to anti-PD-1 and anti-CTLA4 in patients in different risk groups. (G) Estimated IC50 values of four typical immunotherapy drugs (cisplatin, paclitaxel, doxorubicin, and etoposide) between the high-risk groups. * p < 0.05, ** p < 0.01, and *** p < 0.001. Figure 7 The risk gene PSAT1 promotes UCEC cells' proliferation, migration, and invasion. (A,B) mRNA and protein levels of PSAT1 were measured by qPCR and Western blotting in four UCEC cell lines. (C,D) The efficiency of the knockdown of PSAT1 in Ishikawa and HEC-1B cells were measured by qPCR and Western blotting. (E,F) CCK-8 and colony formation assays showed that the proliferation ability of UCEC cells was decreased after PSAT1 knockdown. 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PMC10000779 | Foods Foods foods Foods 2304-8158 MDPI 10.3390/foods12051108 foods-12-01108 Article Study on the Structure, Function, and Interface Characteristics of Soybean Protein Isolate by Industrial Phosphorylation Guo Yanan Conceptualization Software Writing - original draft 1 Liu Caihua Data curation 1 Ma Yitong Investigation Writing - review & editing 1 Shen Lulu Visualization 1 Gong Qi Methodology 1 Hu Zhaodong Investigation 1 Wang Zhongjiang Investigation 1 Liu Xin Investigation 1 Guo Zengwang Supervision 1* Zhou Linyi Project administration Funding acquisition 2* Strzelczak-Cybulska Agnieszka Academic Editor 1 College of Food Science, Northeast Agricultural University, Harbin 150030, China 2 College of Food and Health, Beijing Technology and Business University, Beijing 100048, China * Correspondence: [email protected] (Z.G.); [email protected] (L.Z.) 05 3 2023 3 2023 12 5 110807 2 2023 24 2 2023 02 3 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). The impacts of industrial phosphorylation on the structural changes, microstructure, functional, and rheological features of soybean protein isolate (SPI) were spotlighted. The findings implied that the spatial structure and functional features of the SPI changed significantly after treatment with the two phosphates. Sodium hexametaphosphate (SHMP) promoted aggregation of SPI with a larger particle size; sodium tripolyphosphate (STP) modified SPI with smaller particle size. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) results showed insignificant alterations in the structure of SPI subunits. Fourier transform infrared (FTIR) and endogenous fluorescence noted a decline in a-helix quantity, an amplification in b-fold quantity, and an increase in protein stretching and disorder, indicating that phosphorylation treatment fluctuated the spatial structure of the SPI. Functional characterization studies showed that the solubility and emulsion properties of the SPI increased to varying degrees after phosphorylation, with a maximum solubility of 94.64% for SHMP-SPI and 97.09% for STP-SPI. Emulsifying activity index (EAI) and emulsifying steadiness index (ESI) results for STP-SPI were better than those for SHMP-SPI. Rheological results showed that the modulus of G' and G'' increased and the emulsion exhibited significant elastic behavior. This affords a theoretical core for expanding the industrial production applications of soybean isolates in the food and various industries. phosphorylation soybean protein isolate structure functional characteristics interfacial activity Heilongjiang Province million project2021ZX12B02 Key and R&D projects in Shandong Province2022CXGC010603 National key R&D plan2021YFD2100401 National Natural Science Foundation32202228 Heilongjiang Province key R&D planGA21B001 Heilongjiang Province Major Achievements Transformation ProjectCG19A002 China Fund on the surface of a postdoctoral project2022M721995 This research was funded by the Heilongjiang Province million project [2021ZX12B02], Key and R&D projects in Shandong Province [2022CXGC010603], the National key R&D plan [2021YFD2100401], the National Natural Science Foundation , the Heilongjiang Province key R&D plan [GA21B001], the Heilongjiang Province Major Achievements Transformation Project [CG19A002], and the China Fund on the surface of a postdoctoral project [2022M721995]. pmc1. Introduction Food nutrition and safety are becoming a major concern as a result of the world population's fast expansion . A key dietary element in people's everyday lives is soy protein, one of the sources of high-quality plant protein. It is employed in many different food items with rich nutritional content and potent functional qualities . Soybeans have a higher protein level than both cereals and potatoes, and they are filled with all the important amino acids that the human body needs. It can be utilized as a replacement for animal proteins in the food sector since it has nutritional and biological benefits similar to those of animal proteins . However, natural soy protein isolate contains a large number of hydrophobic groups, which seriously affects the solubility and emulsification of soy protein, resulting in its functional properties not being able to be well developed, limiting the application of soy protein in industry . Phosphorylation has been demonstrated to be an effective assay for enhancing the functional features of food proteins . Besides the structural and functional features, the biological roles of food proteins can also be conferred or enhanced by phosphorylation methods . Inorganic phosphates have a long history of being used safely in food, which provides more evidence for their safety. Phosphoryl chloride, phosphorus pentoxide, cyclic phosphoric acid trona, phosphoric acid and trichloro acetonitrile (as a coupling agent), monophenyl phosphonic acid di-chloro compound, sodium tripolyphosphate, and sodium hexametaphosphate are among the chemicals frequently used in food protein phosphate. At present levels, which result in intakes substantially below the acceptable daily intake (ADI) of 70 mg-1 kg-1 d-1, adding inorganic phosphates directly to food is unlikely to have a negative impact on people . Studies on the phosphorylation modification of dietary proteins have been conducted recently. According to Walz et al. , adding the right proportions of sodium hexametaphosphate (SHMP) to the sausage batter can prevent efflorescence from growing on dry fermented sausages. According to research by Matheis , phosphorylation with POCI3 (in the presence and absence of important amino acids) may be a useful technique for enhancing the nutritional value and functional qualities of dietary proteins. According to research by Cen et al. , phosphorylation modification of fish gelatin protein (GLP) considerably improved gel strength, textural characteristics, emulsification, and emulsification stability. GLP's phosphorylation with ultrasound assistance hastened the transition from helix to sheet and random coil. Hu et al. investigated that the use of ultrasound-assisted phosphorylation increased the surface hydrophobicity, protein structure, and lowered protein aggregation to enhance the emulsifying characteristics of goose liver protein. Hu et al. made it possible for us to comprehend the relationship between myofibrillar protein and sodium tripolyphosphate (STPP), tetrasodium pyrophosphate (TSPP), SHMP, and morphology in greater detail. They also showed that phosphates significantly increased gel complexity and decreased average roughness, resulting in a more well-organized and well-built protein gel. However, the studies of the above scientists are based on a laboratory basis and the laboratory results and industrial production cannot be reproduced with great variability , which is not a good guide to the application of phosphate modification in industrial production. This is because phosphorylation modification of SPI in the laboratory is performed by adding phosphate to the rehydrated SPI. Industrial phosphorylation modification of SPI, on the other hand, is performed by adding phosphate directly in the neutralization modification tank during the production of SPI . Therefore, in this study, based on the traditional industrial extraction of SPI, we add phosphate to the neutralization modification tank to phosphorylate SPI to simulate the industrial production of phosphorylated modified soybean protein. Additionally, we study the effect of industrial phosphorylation modification on the structural, functional, and interfacial properties of SPI, and this study provides guidance for the industrial production process of phosphorylated modified soybean isolate. 2. Materials and Methods 2.1. Materials The Yuwang Group (Dezhou, China) supplied soy flakes without fat (52.27% crude protein concentration). From Beijing Dingguo Changsheng Biotechnology Co. Ltd., chemicals such as 2,2'-dithiobis(5-nitropyridine) (DTNP) and 8-Anilino-1-Naphthalene Sulfonate (ANS) were acquired (Beijing, China). 2.2. Phosphorylation Treatment of SPI 2.2.1. Treatment of Proteins with Sodium Hexametaphosphate An amount of 10% (w/v) defatted soy flake solution was adjusted to pH 8.0 and stirred for 2 h to extract protein and then centrifuged (13500x g, 30 min) at 4 degC. The supernatant was adjusted to pH 4.5 with 2 mol/L HCl and centrifuged (3300x g, 20 min). The pellet was washed twice with distilled water and put into neutralization and modification tank. Sodium hexametaphosphate (SHMP) was added to the neutralization and modification tank so that the percentage of SHMP mass in the solids mass of soy protein solution was 0.4, 0.8, 1.2, 1.6, and 2.0%, respectively. The samples were spray-dried after mixing well to obtain SHMP phosphorylated modified SPI samples, noted as SHMP-0.4%, SHMP -0.8%, SHMP-1.2%, SHMP-1.6%, and SHMP-2.0%, respectively, while the sample not including the adding of sodium hexametaphosphate (SHMP) was utilized as the control one and recorded as SPI. 2.2.2. Treatment of Proteins with Sodium Tripolyphosphate An amount of 10% (w/v) defatted soy flake solution was adjusted to pH 8.0 and stirred for 2 h to extract protein and then centrifuged (13500x g, 30 min) at 4 degC. The supernatant was adjusted to pH 4.5 with 2 mol/L HCl and centrifuged (3300x g, 20 min). The pellet was washed twice with distilled water and put into neutralization and modification tank. Sodium tripolyphosphate (STP) was added to the neutralization and modification tank so that the mass of sodium tripolyphosphate (STP) as a percentage of the mass of solids in soy protein sol was 0.5, 1.0, 1.5, 2.0, and 2.5%, mixed well and spray-dried to obtain STP phosphorylated modified SPI samples, recorded as STP-0.5%, STP-1.0%, STP-1.5%, STP-2.0%, and STP-2.5%, respectively, while the sample without the addition of sodium tripolyphosphate (STP) was used as the control one and recorded as SPI. 2.3. Determination of the Level of Phosphorylation The phosphorylation degree was measured using the protocol of Ai and Jiang with minor adjustments. The phosphorylation level was conveyed as g of phosphorus per g of protein, using the molybdenum blue colorimetric assay to estimate the phosphorus quantity of the samples and employing unmodified SPI as a blank. 2.4. Particle Size and Turbidity Circulation The protein samples were suspended in H2O to prepare a 0.01 g mL-1 mix and then added to the measurement cell. The refractive index of the particles was decided at 1.460, where it was adjusted for the dispersant at 1.330, and the particle size distribution characteristics and protein dispersion index (PDI) were estimated via a nanoparticle size and possibility analyzer. 2.5. Zeta Potential and Surface Hydrophobicity (H0) The protein samples were suspended in H2O to prepare a 0.01 g mL-1 mix and the z-potential of each sample was determined via a nanoparticle size and capacity analyzer at a cuvette length of 1 cm and a spacing of 0.4 cm. The hydrophobicity of the protein surface was estimated by reference to the assay of Ma et al. with minor adjustments. Different samples were dispersed in dH2O and then these sample mixes were thinned with dH2O to a range of concentrations of 0.02, 0.04, 0.06, 0.08, and 0.10 mg mL-1. A 5 mL amount of different concentrations was taken and 20 mL of 8-anilinonaphthalene-1-ate (ANS) mix of 8 mmol L-1 was added. The samples were mixed thoroughly and placed under light-proof conditions for 15 min. The fluorescence strength of the samples was estimated via a fluorescence spectrophotometer. The concentration of protein is the horizontal axis, the relative fluorescence intensity is the horizontal axis, and the slope of the fitted line is the hydrophobicity mark (H0). 2.6. Free Sulfhydryl (SH) and Disulfide (SS) Bonds The determination of protein free sulfhydryl groups was based on the approach of Yan et al. with minimal adjustments. The free sulfhydryl quantity of the protein samples was determined using the DTNB (5,5'-dithiobis-(2-nitrobenzoic acid)) method. The protein sample was first prepared in dH2O to 0.01 g mL-1, then 40 mL of the protein mix was blended with 160 mL of Tris-Gly Urea buffer, mixed well, and then 8 mL of DTNB reagent was inserted and the reaction was run for 20 min at room temperature (RT). The free sulfhydryl concentration was determined as:SHF=73.53xDxA412C where SHF--molar concentration of free sulfhydryl mass, mmol g-1; 73.53--molar extinction coefficient of DTNB reagent; D--dilution multiple; A412--absorbance value of the sample; C--protein dose, mg mL-1. A 1.0 mL sample of 0.01 g mL-1 protein mix was inserted to 10.0 mL of Tris-Gly Urea buffer followed by 0.2 mL of b-mercaptoethanol and then responded at 25 degC for 1 h. At the edge of the reaction, 100 mL of 12% trichloroacetic acid (TCA) was added for 1 h, followed by centrifugation at 4000 rpm for 10 min. The supernatant was then showered again with 100 mL of 12% TCA and centrifuged three times. After dissolving the precipitate with 10 mL Tris-Gly Urea buffer and adding 0.1 mL of 4 mg mL-1 DTNB, it was mixed well and reacted for 20 min at 25 degC. After the reaction, the absorbance value at 412 nm was quickly estimated and the total sulfhydryl quantity and disulfide bond content were calculated as:SHT= 73.53xA412C SS=SHT-SHF2 where SHT--mass molar dose of total sulfhydryl clusters, mmol g-1; SS--mass molar dose of disulfide bonds, mmol g-1; 73.53--molar extinction coefficient of DTNB reagent; A412--absorbance value of the sample; C--protein dose, mg mL-1. 2.7. Solubility The solubility of proteins was estimated by reference to the approach of Kornet et al. . The protein was configured with dH2O to a protein solution of 0.01 g mL-1. The protein mix was centrifugated at 1000x g for 30 min at RT. The amount of protein contained in the upper phase was measured for M1 and a standard curve was made via bovine serum protein as the ideal protein for M2. Protein solubility was then expressed as: Solubility = M1/M2 x 100% 2.8. Size Exclusion Chromatography (SEC) The experimental approach of Liu et al. was modified for the purpose of determining the molecular weight of the proteins. The protein samples were processed to 0.01 g mL-1 and dissolved in dH2O. High-performance liquid chromatography (HPLC) was utilized to analyze the molecular weight distribution. An Advanced Bio SEC column was used at a temperature of 25 degC, with dH2O as the mobile phase, a flow rate of 0.8 mL min-1, a sample injection volume of 20 mL, and a detection wavelength of 280 nm. At 280 nm, the detecting wavelength was set. 2.9. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) The protein was dispersed in dH2O to form a 0.01 g mL-1 protein solution by the method of Ma et al. . The protein blend (200 mL) was blended with 50 mL of "5 x protein loading buffer solution" and the sample was simmered for 5 min. The sample volume was 5 mL for marker and 10 mL for sample. The gel was first run at 50 V for 30 min, then at 80 V for a further 30 min, and finally at 120 V to stop electrophoresis when the sample turned to the bottom side of the gel. After electrophoresis stopped, the gel was removed and the gel was stained with G-250 stain for 30 min, after which the stain was rinsed off with dH2O until the background became apparent. The gels were snapped with a gel imager. 2.10. Fourier Transform Infrared (FTIR) With a few minor adjustments to Peng's experimental procedure , Fourier infrared spectroscopy was used to identify the secondary structure of the proteins. A 100 mg sample of KBr and a 1 mg protein sample were properly combined before being pressed. The absorption spectra were obtained with a shift of 64 scans and a resolution of 4 cm-1 over the wavelength range of 4000-400 cm-1. Peakfit 4.2.0 software was used to calculate the relative content of each secondary structure by using the integral area. Of these, the band from 1646 to 1662 cm-1 could be assigned to the a-helix. The bands from 1608 to 1622 cm-1 could be assigned to the anti-parallel intermolecular b-sheet (b1), and the bands from 1622 to 1637 cm-1, as well as bands from 1682 to 1700 cm-1, could be assigned to the intramolecular b-sheet and parallel intermolecular b-sheet (b2) of the protein, respectively. The band from 1637 to 1645 cm-1 could be assigned to the random coil structure, while the bands from 1662 to 1681 cm-1 could be assigned to the b-turn. 2.11. Intrinsic Fluorescence The approach of Zhao et al. was used to determine the intrinsic fluorescence of proteins, with a few minor adjustments. A 0.1% protein solution was created by dissolving the protein sample in dH2O. The excitation and emission slits were both set at 5 nm, the scanning speed was 12,000 nm min-1, and the excitation wavelength was 290 nm. The scanning divergence wavelength ranged from 300 to 400 nm. 2.12. Transmission Electron Microscope (TEM) The microstructure of the protein was examined via transmission electron microscopy. The proteins were dispersed in distillation to form a 0.01 g mL-1 protein mix, then an appropriate amount of protein mix was added dropwise to the copper netting, and an appropriate amount of 1% phosphotungstic acid solution (ready-to-use) was added dropwise to stain the copper mesh for 60 s before the protein solution dried completely. After drying, the microstructure of the protein was later examined by TEM with a hastening voltage of 80 kV. 2.13. Characteristics of Emulsion 2.13.1. Preparation of Emulsion To create a protein mix with a concentration of 0.01 g mL-1, the protein sample was dispersed in H2O. To create an emulsion, a 3:1 mixture of the protein solution and soybean oil was dispersed at a high rapidity of 12,000 r min-1 for 1 min. 2.13.2. Emulsifying Activity Index (EAI) and Emulsifying Steadiness Index (ESI) of Emulsion The emulsion emulsification activity and emulsion stability were estimated with slim adjustments following the protocol of Yan et al. . A 7 mL amount of soybean oil and 21 mL of 0.01 g mL-1 protein solution were mixed and dispersed at high speed for 1 min at 12,000 r min-1, so 50 mL of the emulsion was combined to 5 mL of 0.1% sodium dodecyl sulfate (SDS) mix from the bottom of the blend at 0 and 10 min, respectively. The absorbance was recorded at 500 nm to obtain the emulsification action index (EAI) and emulsion solidity indicator (ESI). The EAI and ESI are calculated as:EAI= 2x2.303xDFxA0(1-th)xcxLx10000 ESI= 10xA0 A0-A10 where DF--100, dilution multiple; th--0.25, volume fraction of oil phase; c--protein concentration, g mL-1; L--cuvette light range with 0.001 m. 2.13.3. Morphology Examination of Emulsion Emulsion globules were examined via confocal laser scanning microscopy (CLSM). The product was homogenized 3 times at high pressure using a high-pressure homogenizer at 80 Mpa. Nile blue and Nile red were prepared into 1% and 0.1% staining solution, respectively, using isopropyl alcohol, and sifted via a 0.22 mm purify tissue to remove the residue. The emulsion was diluted 5 times and 1 mL was taken, then 45 and 40 mL of Nile blue and red were added, respectively, and agitated for 30 s and stained for 30 min, protected from light. At the end of staining, 3 mL of the emulsion was positioned on a slip. 2.13.4. Interfacial Protein Intensity (G) of Emulsion A 5 mL amount of the emulsion was centrifuged at 10,000x g for 1 h in a 10 mL centrifuge tube. After centrifugation, the subnatant was eliminated by needle and sifted via a 0.45 mL layer and collected. The protein content of the original emulsion and the remainder was estimated using a BCA kit. The interfacial protein content was calculated as:G=WT-WSWT where WT--protein quantity of the emulsion, mg; WS--mass quantity of the protein in the filtrate, mg. 2.13.5. Interfacial Tension of Emulsion Various materials' surface tension was estimated via an automated surface tensiometer (DCAT21, Data Physics Instruments GmbH, Stuttgart, Germany) . An amount of 20 mL of the sample mix was then put into a 25 mL cylinder after the protein sample had first been dissolved in dH2O (1%, m/v). The apparatus's measuring variety was always between 1 and 100 mN m-1, with an SD that never went beyond 0.03 mN m-1. 2.13.6. Rheological Measurement The determination of the rheology of the emulsions was based on the experimental method of Sun and Arntfield with slight adjustments. High-pressure homogenized emulsions were positioned between two analogous plates of 40 and 1 mm spacing, and frequency scanning tests were accomplished in the assortment of shear frequency 0.1-10 Hz to analyze the variation of elastic modulus (G') and viscous modulus (G'') of the emulsion samples as well as shear frequency. 2.14. Statistical Analysis All investigations in this study were repeated 3 times and all investigational statistics were portrayed as mean +- SD. The data were analyzed for significance of differences using SPSS ver., 22.0 software, Origin 2021 software for graphing, and p < 0.05 was the significance test. 3. Results 3.1. Degree of Phosphorylation One of the control markers for detecting phosphate on modified soy protein during industrialization is the degree of phosphorylation. As seen in Figure 1, the degree of phosphorylation of SPI followed a trend of initially rising and subsequently decreasing with the addition of phosphate. When they were 1.2% and 2.0%, respectively, the addition of SHMP and STP had achieved its limit. The STP group had a greater level of phosphorylation than the SHMP group. The possibility is that more phosphate groups bind to protein because of the increased likelihood of collision between phosphate groups and the amino acids serine, threonine, and tyrosine in soybean protein at higher phosphate concentrations. However, if the concentration of phosphate is too high, the phosphate groups attached to the altered protein molecules will increase the electrostatic repulsion between protein molecules, leading to a spatial effect that decreases the likelihood of binding phosphate groups to soy protein molecules, which is unfavorable to the phosphorylation process . The SHMP-1.2% group has a lower level of phosphorylation than the STP-2.0% group, which suggests that cyclic metaphosphate and protein react with less efficiency than linear STP . Cyclic metaphosphates, on the other hand, are said to be more effective in chemical reactions (compared to straight chain polyphosphates). Our experimental findings were consistent with earlier ones. According to research by Hu et al. , the structural characteristics and composition of proteins may also affect how protein molecules and phosphate groups interact. The environment during protein modification affects the free radical activity in the SPI molecule, and the microenvironments provided by SHMP and STP vary during the phosphorylation of soy proteins. Therefore, the phosphorylation in the STP group is greater than that in the SHMP group because the affinity between SPI and STP is larger than that of SHMP. 3.2. Particle Size Distribution and Turbidity Analysis The impacts of various doses of SHMP (0, 0.4, 0.8, 1.2, and 1.6%) and STP (0.5, 1.0, 1.5, and 2.0%) treatments on the mean particle size and turbidity of the SPI samples are portrayed in Figure 2 and Table 1. The mean particle size and turbidity of SHMP-SPI were larger than those of the control, while the opposite was true for STP-SPI. Triplet distribution curves of approximately 90, 800, and 2100 nm were observed in the SHMP-SPI. The STP samples showed a bimodal distribution curve. The research conducted by Sung et al. showed that the phosphate group of phosphate can selectively induce the reaction to change the protein structure by binding to -OH and -NH2 on the edge chain of the protein. Thus, in the industrial spray-drying of modified soy protein, the selective induction of the phosphate group of SHMP with hydroxyl groups and -NH2 clusters on the adjacent chain of SPI leads to a partial unfolding of the SPI molecular structure, which promotes the cross-linking of SPI and SPI in the subsequent process because of its ring structure. This leads to an expansion in the size of all SHMP-SPI compared to the unmodified treated SPI particles. As the SHMP dose raises from 0.4% to 1.2%, the average particle size of SHMP-SPI starts to show a decreasing trend. The increase in SHMP concentration increases the number of charged groups on the surface of SPI molecules by cross-linking more phosphate groups, which leads to the formation of more electrostatic repulsions around the SPI molecules. This inhibits the intermolecular interaction forces of SHMP-SPI and increases the spatial potential resistance effect between protein particles , causing a decrease in particle size and turbidity. However, as the SHMP dose expanded from 1.2 to 2.0%, the average particle size and turbidity of SHMP-SPI started to show an increasing trend. Xiong et al. demonstrated that an extra phosphate dose can cause the creation of new and large protein accumulates by the enhancement of the binding effect of protein molecules during phosphorylation. Therefore, when the addition amount of SHMP is too high, the electrostatic repulsion between SPI molecules initiated by the addition of SHMP cannot eventually overwhelm the attraction (Van der Waals force and hydrophobicity) and an aggregation phenomenon will take place, resulting in the increase in SHMP-SPI particle size, turbidity, and uneven distribution. While STP is a chain-like phosphate, SHMP has stronger affinity and higher phosphorylation compared with SPI. STP can also augment the electrostatic revulsion and steric hindrance impact of SPI molecule surface by changing the non-covalent interaction within the molecule, resulting in the lessening of particle size and turbidity of STP-SPI. Moreover, too high an STP concentration will also lead to the enhancement of SPI molecular interaction, developing in the increase in particle size and turbidity. However, it is worth noting that the particle size of STP-SPI is always lower than that of SPI, while that of SHMP-SPI is much higher. This indicates that SHMP phosphorylation promotes SPI aggregation to form large-size SPI, while STP phosphorylation results in SPI disaggregation to form small particles with more uniform distribution. The reason may be that the modification of SPI by phosphate is obviously related to the kinds, spatial structure, and molecular properties of phosphate, and SHMP has a ring structure, and the chain length is longer than STP with a chain structure. Consequently, it might be assumed that the impact of SHMP was lower to that of STP due to the longer chain length. Jin et al. reached the same conclusion by studying the impact of phosphate on the gelling features of heat-induced egg white gel. 3.3. SDS-Polyacrylamide Gel Electrophoresis The reduction agent (b-mercaptoethanol) disrupts the hydrophobic interactions between the disulfide bond and the SPI subunits, leading to subunit dissociation, so that SDS-PAGE can essentially signify the differences in the creation of the SPI subunits by phosphate . As can be seen in Figure 3, the overall protein's structure was not altered by the phosphorylation modification, and the disappearance of the A subunit (SHMP-1.2% group) was more likely due to the cross-linking of non-disulfide covalent bonds ; phosphorylation treatment changed one mutual state between subunits and substrates, but had no significant effect on subunits , and the remainder of the groups showed insignificant variations between bands of the same molecular weight, implying that the peptide groups in the SPI were not damaged by the introduction of phosphate and that they still maintained their primary structural integrity . Blended with the findings of particle size and turbidity tests, the phosphorylation treatment promoted the aggregation or depolymerization of SPI molecules from SPI molecules, indicating that phosphate can significantly affect the stability of proteins in aqueous solution. 3.4. Fourier Transform Infrared Both STP and SHMP exhibit comparable properties in their infrared spectra. As seen in Figure 4, SPI exhibits three key distinguishing peaks: the amide I band (mostly C=O stretching) at 1480-1575 cm-1; the amide II band (C-N stretching combined with N-H bending) at 1600-1700 cm-1; and the amide A band (related with N-H stretching coupled with H-bonding) at 3273 cm-1 . Protein structural changes are indicated by variations in spectral peak location and intensity . The stretching vibration of P-O in PM-SPI samples, in contrast, was primarily responsible for the STP-SPI's unique absorption peak at 895 cm-1 and rising intensity with increasing STP concentration. Electrostatic interactions between proteins and other components were linked to changes in amide I and II . Table 2 displays the secondary structures of the STP and SHMP alterations. After phosphorylation, the a-helical content of SPI decreased while the b-fold and irregular curl content increased. As phosphate addition increased, the a-helical content of SPI displayed a trend of first increasing and then decreasing while the irregular curl content displayed a trend of first increasing and then decreasing. Its irregular curl content peaked when SHMP and STP were added at 1.2% and 2.0%, respectively, and the STP-2.0% group was higher than the STP. Protein aggregation and the side chains of amino acids may have changed, which is why the secondary structure of SPI changed before and after alteration . Amide-I is characteristic of the coiled-coil composition of proteins, and the overview of phosphate may affect the a-helical coiled-coil structure of proteins primarily by increasing the repulsive forces between charged residues in the protein chain . Phosphorylation of STP also stemmed in a decline in the amides I and II band peak amplitude. These alterations suggest that these reactive groups are reduced by binding to the phosphate group. Changes in the side chain rearrangement or secondary structure of peptides in proteins result in changes in peak intensity and peak position. The phosphorylation reaction acts on only a few particular amino acid remains, not most of them. Both the e-NH2 of Lys residues and the hydroxyl of Ser or Thr remains show the greatest effects . It can be speculated that a C-N-P or C-O-P bond may have been established between SPI and STP or SHMP. This is the same result as that reported by Zhao et al. . 3.5. Intrinsic Fluorescence Emission Spectra When the milieu changes, inherent fluorescence might react, and the phosphate-based tertiary structure of SPI Try persists. According to the findings in Figure 5, there was a tendency for the fluorescence intensity of SHMP-SPI and STP-SPI to increase and subsequently decrease when the phosphate concentration increased. Although there was a minor blue shift, the fluorescence intensity of SHMP-SPI did not significantly alter with SHMP concentration. However, the total fluorescence intensity of STP-SPI was higher than that of SHMP-SPI, and the fluorescence lightness of STP-SPI showed a considerable change with increasing STP concentration as well as a substantial blue shift. A strong negative association between the amount of a-helices and the surface hydrophobicity was found by Kato et al. . A decrease in structural ordering, as shown by a decrease in the a-helical structure of the secondary structure, is caused by phosphorylation treatment, which also causes an increase in the hydrophobic region within the SPI molecule. Exposure of the hydrophobic sites within the molecule may also cause the aromatic amino acid residues in the soy protein aggregates to move from a hydrophilic to a hydrophobic environment , leading to the blue-shift phenomenon. The fluorescence strength and supreme excitation wavelength (lmax = 340.7) of the SHMP-treated protein did not alter more than that of the STP-treated protein (lmax = 335.6); this is due to the combination of different modes of aggregation occurring between SPI molecules and the unfolding of the SPI molecular structure after phosphate treatment reduces the internal burst, and SPI molecules are more likely to form large aggregates and partial expansion of the molecules during SHMP treatment, reducing the acquaintance of the protein interior color amino acid remains, so the fluorescence intensity changes are not as pronounced as for STP-SPI. When the phosphate concentration continues to increase, the aggregation behavior leads to more tryptophan residues buried, and a too-high concentration of phosphate leads to SPI re-aggregation and increased spatial site resistance, resulting in fluorescence burst and reduced fluorescence strength . The results of endogenous fluorescence suggest that SHMP and STP modification can cause changes in the conformation of SPI tertiary structure. 3.6. Surface Hydrophobicity and z-Potential The surface hydrophobicity (H0) and zeta potential of proteins play a crucial part in determining the structural conformation of phosphorylated modified SPI by judging the changes in intermolecular interaction forces and can influence the functional properties of modified SPI . The surface hydrophobicity and z-potential of the two phosphate-modified soybean proteins is shown in Table 1. Both SHMP and STP treatments stemmed in an expansion and then a decline in the surface hydrophobicity and z-potential of SPI. Compared to SPI, the H0 of SPI reached the maximum at SHMP-1.2% and STP-2.0% and the z-potential of SPI was highest at SHMP-1.6% and STP-2.0%. It is hypothesized here that the disclosure of hydrophobic groupings and the expansion in surface charge number are caused by alterations in the secondary and tertiary structure of SPI. The addition of phosphate leads to the denaturation/unfolding of the SPI molecule subjecting the hydrophobic sites and hydrophobic and charged groups. Moreover, phosphate colonization also causes a growth in the negative charge groups on the protein exterior , which leads to a corresponding rise in hydrophobicity and z-potential on the phosphate-modified SPI surface. However, overly high concentrations of phosphate may alter the interaction between protein side chain groups and phosphate through inter-molecular aggregation and swelling of SPI and intra-molecular hydrophobic association aggregation of SPI, inhibiting additional disclosure of hydrophobic and charged parties, and as a substitute reburying them within the protein molecule through aggregation , reducing the surface hydrophobicity and z-potential values. However, the extent of H0 and z-potential changes were more pronounced in STP-SPI compared to SHMP-SPI. This may be due to the increase in the disordered structure of SPI during STP phosphorylation treatment, which improves the flexibility of SPI molecules by forming a more extended and sparser structure . The above results together contribute to make some hydrophilic groups of STP-SPI such as - -OH and hydrophobic regions will be exposed to a higher degree, leading to a more pronounced degree of H0 and changes. The STP-SPI particles with higher zeta potential have increased electrostatic repulsion between ions and the system is relatively stable because their charges inhibit the aggregation of SPI and enhance their stability . This causes the protein molecules in STP-SPI to show small particle dispersion in the liquid that is reliable with the particle size findings. 3.7. Free Sulfhydryl Groups and Disulfide Bonds The sulfhydryl disulfide exchange (SH/SS) reaction is an important factor in altering the spatial structure of industrially phosphorylated modified SPI and plays an essential function in intermolecular accumulation and depolymerization of SPI . The results for free sulfhydryl groupings and di-sulfide bonds for SHMP-SPI and STP-SPI are shown in Table 1. After the industrial phosphorylation modification, both the free sulfhydryl groups of SPI revealed a tendency of rising and then declining, and the trend in disulfide bonds revealed a tendency of declining and then expanding. As the concentration of SHMP increased from 0.4 to 1.2%, the free sulfhydryl content increased from 2.91 to 14.79 umol g-1, whereas the free sulfhydryl quantity of STP-SPI did not increase drastically. In contrast, the disulfide bond content of STP-SPI decreased more than that of SHMP-SPI. Phosphorylation appears to lead to exposure of the -SH group in the SPI and disruption of the disulfide bond due to the negative charge introduced by phosphorylation and the subsequent shift in the unfolding structure of the SPI . The accumulation of phosphate leads to a relaxing impact on the SPI structure by increasing the negative charge on the SPI exterior, exposing the buried sulfhydryl groups in the protein in the process of reducing the size of the SPI particle, leading to an expansion in cross-linking of free sulfhydryl groups and the breaking of disulfide bonds, the breaking of which stabilizes the SPI accumulates, leading to an expansion in the rearrangement and hydrophobicity of the SPI particles. However, the change in -SH content was more pronounced in the SHMP-SPI group, which may be due to the structural unfolding of soy protein, causing the conversion of other sulfur-containing compounds into free sulfhydryl groups in addition to disulfide bonds . The transformation of free sulfhydryl groupings into disulfide bonds resulted in the burial of some free -SH groups by hydrophobic interactions and the phosphorylation of some free -SH groups to form intermolecular -SS. This agrees with the particle size and hydrophobicity results. The results suggest that phosphorylation can regulate the polymerization and depolymerization between SPI molecules that in turn influences the functional features of the protein , and similar conclusions were reached by the study. 3.8. Transmission Electron Microscope Figure 6 shows the TEM snaps of SHMP-SPI and STP-SPI. The backbone morphology of the protein was examined by TEM. As can be noted from the state portrayed in Figure 6-SPI, the native SPI is in small, aggregated bundles and after industrial phosphorylation treatment, the SHMP-0.4% group shows strong black centers of aggregation, and the stacked SHMP-SPI shows a high skeletal rigidity forming a multi-branched structure. With increasing concentration of SHMP, the SPI aggregates showed depolymerization and repolymerization, while STP-SPI also showed aggregation-depolymerization-repolymerization, but STP-SPI showed smaller aggregates and did not show a large aggregation structure like that of SHMP-SPI, the white particles being the SPI particles suspended in solution. During industrial phosphorylation, phosphate and protein interact primarily through hydrophobic and electrostatic interactions, with phosphate acting as a bridge to polymerize protein molecules . 3.9. Solubility The solubility of SHMP-SPI and STP-SPI is shown in Table 3. The phosphorylated SPI is more soluble than the unphosphorylated SPI. The STP-SPI group showed a greater improvement in solubility than the SHMP-SPI group, with the soybean isolate having the maximum solubility (97.09%) in the STP-2.0% treatment group and the SPI having the lowest solubility (93.64%) in the SHMP-1.2% treatment group. The SPI side chain groups undergo extensive anionic phosphate group introduction during industrial phosphorylation, and the phosphate groups connected to the SPI molecules are able to make many hydrogen bonds with water molecules . The electrostatic repulsion between the SPIs in aqueous solution is increased by increasing the negative charge introduced by the phosphate group along the surface of the SPIs . High phosphate concentrations limit the interaction between the inhibited protein and H2O molecules, causing phosphate and SPI molecules to compete with H2O molecules and reducing SPI solubility . The STP-2.0% group had the highest solubility, which could be attributed to the linear structure of STP, the formation of more H-ion ligands in the STP phosphate group during industrial phosphorylation, the presence of more positive and negative charges on the surface of the protein molecules, an increase in the number of H-bonding interactions between SPI molecules, the thickness of the hydration layer, and the ability of hydrophilic and hydration repulsion to repel one another . 3.10. Emulsifying Action Index and Emulsion Solidity Index The EAI and ESI of STP and SHMP at different concentrations are shown in Table 3. With increasing phosphate addition, the emulsifying action (EAI) and emulsion solidity (ESI) of SPI revealed a tendency of first rising and then declining; the highest rates were reached when SHMP and STP were added at 1.2% and 2.0%, respectively, and the STP-2.0% group was better than the SHMP-1.2% group. Moderate phosphorylation led to unfolding of the protein secondary structure, displaying more phosphorylation sites and hydrophobic groupings, and increasing the intermolecular flexibility of the SPI . During emulsion preparation, the high-pressure homogeneous sheer force of the SPI facilitated diffusion to the interface and increased the interfacial protein content, and the phosphorylated SPI facilitated protein adsorption to the oil-water interface and oil droplet dispersion . Alternatively, the improved water solubility of SPI also has an impact on improved emulsification, as the SPI molecular aggregates tend to adhere to the oil-water interface and form a thicker adsorption layer, changing the amphiphilic nature of the SPI attached to the oil-water interface, resulting in better emulsifying activity . The adding of phosphate leads to increased irregular curl content and increased structural flexibility of the SPI structure, which reduces the surface tension of the emulsion and makes it simpler to create emulsion droplets, while increasing the electrostatic repulsion between droplets, making it easier for droplets to disperse, thus significantly improving emulsion stability . This is a good illustration of how phosphorylation can quicken the rise in electrostatic repulsion, thus promoting the dispersion of emulsion droplets and inhibiting the incorporation of oil droplets. Smaller particle sizes and higher z-potential values have been reported to increase electrostatic repulsion and spatial site resistance among droplets, and phosphorylation-promoted adsorption layers lead to increased electrostatic repulsion at interfacial proteins, preventing aggregation and flocculation of emulsion particles . Emulsion stability is one of the most improved properties, as confirmed by the data in Table 3. 3.11. Confocal Laser Scanning Microscopy The microstructure of emulsion particles has been frequently displayed using a confocal laser scanning microscope, which can immediately display the particle size and dispersion state . The red fluorescent core and green, fluorescent periphery in the CLSM micrographs indicate the oil and protein fractions, respectively. As shown in Figure 7, the size of the phosphorylated SPI droplets decreased, the emulsion was more uniformly dispersed, and the droplet shape tended to be regular spherical compared to the emulsion formed by the non-phosphate modified SPI. With increasing phosphate addition, the droplet dispersion revealed a trend of expanding and then declining. The highest dispersion of the emulsions was observed in the SHMP-1.6% and STP-2.0% groups, and the emulsions were more dispersed in the STP-2.0% group than in the SHMP-1.6% group. The phosphorylation treatment leads to the aggregation of protein molecules, resulting in the rearrangement and relaxation of the interface during emulsification, which fails to form a stable interfacial film, resulting in large areas of red oil droplet aggregation. With the addition of phosphate to increase the surface charge of SPI, the resulting interfacial protein film is able to induce strong enough electrostatic repulsion in adjacent emulsion droplets to offer better spatial solidity and inhibit accumulation and binding between emulsions . On the other hand, the phosphate modification led to the stretching and unfolding of the SPI structure, the exposure of internal lipophilic groups, and the improvement of the flexible structure, which promoted the molecular interaction between the phosphorylated SPI and SPI at the oil-water interface, contributing to the adsorption of SPI at the oil-water interface and the foundation of a stiffer interfacial film, while inhibiting the flocculation of the emulsion droplets . This also explains the minor particle size and improved stability of SPI emulsions after phosphorylation. 3.12. Emulsion Rheology By applying different degrees of oscillation frequency strain to the emulsion, elastic modulus G' is a measurement of elasticity and represents the storage modulus of the energy of stress, which can be restored when the stress is released, while the viscous modulus G'' represents the viscous components which assumes the flow resistance of the sample. G' and G'' are used to reflect the viscoelasticity of the emulsion, which reflects the structural characteristics and intermolecular interactions of the SPI molecules at the water-oil interface after phosphorylation, which is one of the key factors determining emulsion stability . Figure 8 shows the change in dynamic modulus as the phosphorylated SPI emulsion frequency scanned. The energy storage modulus (G') and loss modulus (G'') of all emulsion samples expanded with increasing frequency sweep and tended to increase and then decrease with increasing phosphate concentration. The energy storage modulus (G') and loss modulus (G'') reach their maximum values when SHMP and STP are added at 1.6% and 1.5%, respectively, with G'' values reliably higher than G'' values, implying that the elastic properties dominate and that the emulsion forms a better elastic network structure . The binding among protein molecules via covalent and noncovalent bonds has an important influence on the maintenance of the gel network. Phosphate-modified SPI particles have a higher z-potential and higher hydrophobicity (Table 1), which enhances covalent and non-covalent interactions among proteins . The SPI molecular chain is unfolded by phosphorylation ; more protein is adsorbed at the oil-water interface and wraps around the oil droplet, increasing the stabilizing effect on the oil droplet and the resulting increase in emulsion viscosity, which increases the energy storage modulus and loss modulus of the emulsion, resulting in improved emulsion stability . At the identical frequency, the G' values of both SHMP-SPI and STP-SPI were greater than those of the control group, and the G'' values were substantially greater than those of the control group, indicating that the phosphorylated SPI had better viscosity performance. At the same time, the viscosity of the SHMP-modified SPI performed better than that of the STP-modified SPI. The range of G' dynamics in the SHMP-SPI group was superior to that in the STP-SPI group, while the range of G'' dynamics in the STP-SPI group was superior to that in the SHMP-SPI group, indicating that the larger molecular weight SPI promoted more entanglement in the emulsion . At the same concentration, the higher-molecular-weight SHMP-SPI occupied more space and was more likely to meet other molecules, reducing molecular mobility and increasing viscoelasticity. This explains very well the effect of the two phosphate modifications on SPI. 3.13. Interfacial Tension The interfacial tension of emulsions can characterize the ease of emulsion formation and infer the adsorption and exchange behavior of SPI at the emulsion interface, which is related to the diffusion rate of SPI molecules and their own adsorption capacity. As can be seen from Table 3, the addition of phosphate reduced the interfacial tension and indicated an inclination to decrease and then boost with increasing phosphate addition. The interfacial tension of the STP-2.0% group was shorter than that of the SHMP-1.2% group. On the one hand, phosphates are highly hydrophilic and after industrial phosphorylation, the surface hydrophobicity of SPI molecules increases and the hydrophilic/hydrophobic ratio reaches equilibrium, increasing the rearrangement efficiency of SPI molecules in the interfacial layer after adsorption to the interfacial layer, promoting rapid protein adsorption at the oil-water interface, and reducing the interfacial tension . Alternatively, the integration of the phosphate group increases the net charge on the superficial layer of the SPI molecule and the increased electrostatic repulsion leads to a variation in protein compliance, deconvolution of the a-helix, and a disheveled structure that enables speedy conformational change of the protein at the oil-water interface, exposing the hydrophobic amino acids from the interior structure and, thus, reducing the interfacial tension . It is commonly assumed that the increased b-fold content encourages interactions among proteins at the oil-water interface, adding to the creation of denser and stiffer interfacial films , which also leads to a reduction in interfacial tension. Excess phosphate inhibits the dissociation of SPI molecules, reducing the net surface charge, and electrostatic repulsion is insufficient to maintain the stability of the system, leading to aggregation of SPI, encapsulation of hydrophobic groups, and a decrease in surface hydrophobicity. This reduces the rate of SPI adsorption to the interfacial layer and the ability to cross-link with the oil phase and increases the mutual repulsion of the water and oil phases at the contact surface, with a further increase in interfacial tension values. The lower interfacial tension in the STP 2.0% group compared to the SHMP 1.2% group may be due to the smaller molecular size of the proteins in the STP 2.0% group. Proteins with a smaller molecular size and more relaxed conformation have a greater ability to reduce interfacial tension, which is more conducive to rapid protein adsorption at the water/oil interface . The results suggest that the reduction in interfacial tension of the phosphate modified SPI emulsion is one of the key factors contributing to the increase in EAI. 4. Conclusions The structural and functional features of SPI treated with STP and SHMP during industrial production were investigated. It was shown that the absolute values of a-helix content, endogenous fluorescence intensity, surface hydrophobicity, and z-potential of SPI in both SHMP and STP groups tended to increase and then decrease with increasing phosphate addition, reaching maximum values at 1.2% and 2.0% for SHMP and STP, respectively. Microscopic results showed that the SHMP-modified SPI exhibited a large and aggregated rigid skeleton structure, while STP exhibited small and black aggregates. The emulsifying activity and emulsion solidity of both SHMP-SPI and STP-SPI demonstrated a tendency of rising and then declining with increasing phosphate adding. The emulsions of SHMP-SPI and STP-SPI have a predominantly elastic behavior and show an increasing trend in viscosity. The phosphorylation of SHMP cross-linked the protein, facilitating the formation of many micelles in solution. These micelles interacted to form a mesh structure and increase the viscosity of the system, making it suitable for use as a thickener in food processing. Phosphorylation of STP reduces the size of protein molecules and increases intermolecular interactions, allowing it to be utilized as a dispersing additive in food processing. This study provides a reference to produce functional properties of industrially modified soy isolate proteins for the food industry. Author Contributions Y.G.: Conceptualization, Software, Writing-original draft. C.L.: Data curation. Y.M.: Investigation, Writing. L.S.: Visualization. Q.G.: Methodology. Z.H.: Investigation. Z.W.: Investigation. X.L.: Investigation. Z.G.: Supervision. L.Z.: Funding acquisition, Project administration. All authors have read and agreed to the published version of the manuscript. Data Availability Statement The data presented in this study are available in [Study on the structure, function, and interface characteristics of soybean protein isolate by industrial phosphorylation]. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Effect of phosphate addition on phosphorylation level of soybean protein isolate. Note: Values with a different letter(s) indicate a significant difference at p <= 0.05. Figure 2 Effect of phosphate addition on particle size distribution of soybean protein isolate. Figure 3 Effect of phosphate addition on gel electrophoresis of soybean protein isolate. Figure 4 Effect of phosphate addition on FTIR spectra of soybean protein isolate. Figure 5 Effect of phosphate addition on intrinsic fluorescence emission spectra of soybean protein isolate. Figure 6 Effect of phosphate addition on backbone structure of soybean protein isolate. Figure 7 Effect of phosphate addition on microstructure of emulsion prepared by soybean protein isolate. Figure 8 Effect of phosphate addition on rheology properties of emulsion prepared by soybean protein isolate. foods-12-01108-t001_Table 1 Table 1 Effects of phosphate on average particle size, PDI, turbidity, surface hydrophobicity, z-potential, free sulfhydryl groups, and disulfide bonds of soybean protein isolate. Sample Concentration Average Particle Size (nm) PDI Turbidity Surface Hydrophobicity z-Potential (mV) Free Sulfhydryl Groups (mmol/g) Disulfide Bonds (mmol/g) SPI 0 408.82 +- 2.18 fA 0.45 +- 0.02 aAB 0.195 +- 0.007 dA 310.80 +- 6.56 eF -20.80 +- 0.23 eF 1.2133 +- 0.0125 eD 13.5053 +- 0.1990 aA SHMP 0.4% 1341.01 +- 7.35 a 0.38 +- 0.05 b 0.252 +- 0.002 a 484.98 +- 7.37 d -23.34 +- 0.21 d 2.1268 +- 0.1760 c 12.0783 +- 0.4114 b 0.8% 1140.12 +- 5.56 b 0.31 +- 0.04 c 0.241 +- 0.004 b 945.99 +- 8.00 c -25.86 +- 0.10 c 2.9100 +- 0.2110 d 10.3210 +- 0.4780 c 1.2% 567.91 +- 3.56 e 0.18 +- 0.01 e 0.229 +- 0.005 c 1183.00 +- 8.82 a -29.63 +- 0.18 a 14.7899 +- 0.1210 a 8.6773 +- 0.2212 e 1.6% 664.47 +- 4.19 d 0.26 +- 0.02 d 0.236 +- 0.001 b 984.00 +- 7.46 b -29.45 +- 0.12 a 13.2934 +- 0.2930 b 8.6791 +- 0.2103 e 2.0% 759.64 +- 10.21 c 0.24 +- 0.03 d 0.237 +- 0.002 b 950.00 +- 7.90 c -28.78 +- 0.13 b 6.0904 +- 0.3010 c 9.1565 +- 0.3980 d STP 0.5% 299.91 +- 2.12 B 0.36 +- 0.02 C 0.089 +- 0.005 B 1109.10 +- 18.45 E -25.31 +- 0.23 E 1.3533 +- 0.0124 C 12.1268 +- 0.2212 B 1.0% 297.73 +- 4.21 B 0.41 +- 0.02 B 0.082 +- 0.001 C 1274.30 +- 16.99 C -29.82 +- 0.10 D 1.4068 +- 0.0134 B 10.2836 +- 0.1836 C 1.5% 290.37 +- 3.33 C 0.26 +- 0.02 D 0.080 +- 0.002 C 1479.60 +- 18.31 B -31.45 +- 0.13 C 1.5059 +- 0.0132 A 9.9836 +- 0.1735 D 2.0% 266.73 +- 5.63 E 0.11 +- 0.02 E 0.060 +- 0.003 D 1864.50 +- 27.17 A -35.01 +- 0.12 A 1.4259 +- 0.0101 B 7.5765 +- 0.1134 F 2.5% 281.53 +- 4.87 D 0.46 +- 0.02 A 0.068 +- 0.006 D 1210.50 +- 22.80 D -33.68 +- 0.25 B 1.4118 +- 0.0099 B 8.3846 +- 0.1239 E Note: Comparisons were carried out between values of the same column; values with a different lowercase letter (s) indicate a significant difference at p <= 0.05 between the sample with SHMP; values with a different capital letter (s) indicate a significant difference at p <= 0.05 between the sample with STP. foods-12-01108-t002_Table 2 Table 2 Effect of phosphate addition on the secondary structures of soybean protein isolate. Secondary Structure Content (%) b-Sheet/% a-Helix/% b-Turn/% Random Coil/% Sample Concentration SPI 0 26.20 +- 0.35 eC 36.18 +- 0.05 aA 15.12 +- 0.03 eA 22.50 +- 0.23 eF SHMP 0.4% 30.39 +- 0.17 d 25.11 +- 0.01 b 22.50 +- 0.13 a 22.00 +- 0.27 e 0.8% 31.43 +- 0.12 b 23.74 +- 0.31 d 21.33 +- 0.21 c 23.50 +- 0.12 c 1.2% 37.34 +- 0.27 a 20.05 +- 0.14 e 3.81 +- 0.32 f 38.80 +- 0.22 a 1.6% 31.17 +- 0.98 c 23.64 +- 0.03 d 22.19 +- 0.09 b 23.00 +- 0.23 d 2.0% 31.05 +- 0.54 c 24.55 +- 0.06 c 20.31 +- 0.16 d 24.09 +- 0.03 b STP 0.5% 26.52 +- 0.84 C 27.24 +- 0.12 C 12.78 +- 0.24 C 33.46 +- 0.01 D 1.0% 27.05 +- 0.07 B 27.14 +- 0.04 CD 11.66 +- 0.51 E 33.75 +- 0.07 C 1.5% 27.24 +- 0.15 B 27.04 +- 0.08 D 12.15 +- 0.20 D 34.27 +- 0.12 B 2.0% 28.39 +- 0.37 A 25.83 +- 0.08 E 10.98 +- 0.35 F 34.80 +- 0.11 A 2.5% 25.55 +- 0.28 D 27.67 +- 0.02 B 13.22 +- 0.02 B 33.12 +- 0.04 E Note: Comparisons were carried out between values of the same column; values with a different lowercase letter (s) indicate a significant difference at p <= 0.05 between the sample with SHMP; values with a different capital letter (s) indicate a significant difference at p <= 0.05 between the sample with STP. foods-12-01108-t003_Table 3 Table 3 Effect of phosphate addition on solubility, emulsifying action, emulsion solidity, and interfacial tension of soybean protein isolate. Sample Concentration Solubility (%) (EAI) (m2/g) (ESI) (min) Interfacial Tension SPI 0 85.86 +- 0.49 dE 25.53 +- 0.123 eE 18.51 +- 0.82 eF 18.470 +- 0.124 aA SHMP 0.4% 88.44 +- 0.28 c 30.00 +- 0.201 d 35.91 +- 1.90 d 16.406 +- 0.163 b 0.8% 90.26 +- 0.26 b 33.00 +- 0.167 c 60.89 +- 1.66 c 14.726 +- 0.142 cd 1.2% 94.64 +- 0.25 a 35.82 +- 0.158 a 87.56 +- 1.00 a 13.340 +- 0.212 e 1.6% 90.35 +- 0.27 b 35.15 +- 0.103 b 72.80 +- 0.90 b 14.570 +- 0.225 d 2.0% 86.72 +- 0.19 d 34.65 +- 0.191 b 68.31 +- 0.99 b 15.013 +- 0.109 c STP 0.5% 89.19 +- 0.26 D 27.00 +- 0.15 D 46.63 +- 1.11 E 15.175 +- 0.205 B 1.0% 90.68 +- 0.21 C 30.00 +- 0.35 C 70.00 +- 1.38 D 14.246 +- 0.109 C 1.5% 93.55 +- 0.20 B 35.00 +- 0.29 B 78.35 +- 1.00 C 12.365 +- 0.102 D 2.0% 97.09 +- 0.21 A 39.50 +- 0.38 A 95.00 +- 1.21 A 11.555 +- 0.163 E 2.5% 94.53 +- 0.24 B 38.47 +- 0.44 A 90.00 +- 1.09 B 12.887 +- 0.109 D Note: Comparisons were carried out between values of the same column; values with a different lowercase letter (s) indicate a significant difference at p <= 0.05 between the sample with SHMP; values with a different capital letter (s) indicate a significant difference at p <= 0.05 between the sample with STP. 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PMC10000780 | Cells Cells cells Cells 2073-4409 MDPI 10.3390/cells12050705 cells-12-00705 Article Stem Cell Factor and Granulocyte Colony-Stimulating Factor Promote Remyelination in the Chronic Phase of Severe Traumatic Brain Injury Qiu Xuecheng Conceptualization Methodology Formal analysis Investigation Data curation Writing - original draft Writing - review & editing Visualization 1 Ping Suning Methodology Formal analysis 1 Kyle Michele Methodology 1 Chin Lawrence Conceptualization Funding acquisition 1 Zhao Li-Ru Conceptualization Resources Writing - review & editing Supervision Project administration Funding acquisition 1* Wang Zhouguang Academic Editor 1 Department of Neurosurgery, State University of New York Upstate Medical University, Syracuse, NY 13210, USA * Correspondence: [email protected]; Tel.: +1-315-464-8470; Fax: +1-315-464-5504 23 2 2023 3 2023 12 5 70530 12 2022 14 2 2023 17 2 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). Severe traumatic brain injury (TBI) causes long-term disability and death in young adults. White matter is vulnerable to TBI damage. Demyelination is a major pathological change of white matter injury after TBI. Demyelination, which is characterized by myelin sheath disruption and oligodendrocyte cell death, leads to long-term neurological function deficits. Stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) treatments have shown neuroprotective and neurorestorative effects in the subacute and chronic phases of experimental TBI. Our previous study has revealed that combined SCF and G-CSF treatment (SCF + G-CSF) enhances myelin repair in the chronic phase of TBI. However, the long-term effect and mechanism of SCF + G-CSF-enhanced myelin repair remain unclear. In this study, we uncovered persistent and progressive myelin loss in the chronic phase of severe TBI. SCF + G-CSF treatment in the chronic phase of severe TBI enhanced remyelination in the ipsilateral external capsule and striatum. The SCF + G-CSF-enhanced myelin repair is positively correlated with the proliferation of oligodendrocyte progenitor cells in the subventricular zone. These findings reveal the therapeutic potential of SCF + G-CSF in myelin repair in the chronic phase of severe TBI and shed light on the mechanism underlying SCF + G-CSF-enhanced remyelination in chronic TBI. traumatic brain injury chronic phase SCF G-CSF demyelination remyelination National Institutes of Health/National Institute of Neurological Disorders and Stroke in the United StatesR01NS118166 This study was supported by the National Institutes of Health/National Institute of Neurological Disorders and Stroke in the United States (R01NS118166). pmc1. Introduction Traumatic brain injury (TBI) causes a primary traumatic axon injury and secondary injury in white matter tracts . TBI has become a major cause of death and long-term disability among children and young adults . Epidemiological data on TBI from the Center for Disease Control and Prevention of the United States reveal about 223,135 TBI-related hospitalizations in 2019 and 64,362 TBI-related deaths in 2020 . Approximately 10-15% of patients with TBI have more severe injuries and require long-term care , which causes a high economic burden. Growing clinical evidence has shown long-term white matter impairments in TBI patients , even in mild cases . Myelin is a major component of white matter. The myelin sheath enwrapping axons is comprised of oligodendrocyte processes. Post-TBI white matter injury involves both traumatic axon injury (TAI) and myelin pathology. Myelin is vulnerable to damage from TAI after TBI . Persistent accumulation of myelin debris after TAI has been found in a rat model of TBI . In TBI patients, chronic inflammation in the white matter with white matter degeneration persists years after TBI . Disrupted myelin-released myelin-associated proteins such as Nogo, oligodendrocyte myelin glycoprotein, and myelin-associated glycoprotein negatively mediate axonal regeneration and neural plasticity after injury . Numerous studies have shown that TBI induces widespread oligodendrocyte death and demyelination, followed by concomitant oligodendrogenesis and remyelination . Manipulating oligodendrogenesis and remyelination may provide a novel therapeutic target to promote white matter integrity and improve neurological function recovery after brain injury . Stem cell factor (SCF) and granulocyte-colony stimulating factor (G-CSF) were originally discovered as hematopoietic growth factors to regulate and maintain hematopoiesis . Both SCF and G-CSF can pass through the blood-brain barrier in intact animals , revealing the possibility of SCF and G-CSF in regulating physiological function in the brain. Increasing evidence has shown that SCF and G-CSF can enhance angiogenesis, neurogenesis, neurite outgrowth, neuronal network remodeling, and neuronal function in neurological disorders . It has been reported that SCF and G-CSF treatments attenuate white matter loss and increase the proliferation of intrinsic oligodendrocyte precursor cells in animal models of spinal cord injury . Our previous study has also revealed that the combined treatment of SCF and G-CSF (SCF + G-CSF) in the chronic phase of severe TBI enhances remyelination and improves neurological functional recovery . However, the time-dependent effects of SCF + G-CSF treatment in promoting myelin repair in the chronic phase of severe TBI remains unclear. In the present study, young adult mice with a severe TBI received 7-day treatments of SCF + G-CSF initiated at 3 months post-TBI. The myelin changes in the ipsilateral white matter were assessed 1 day, 21 weeks, and 36 weeks after SCF + G-CSF treatment. The possible mechanism of the SCF + G-CSF-enhanced myelin repair in the chronic phase of severe TBI was also explored. 2. Materials and Methods All procedures of animal experiments were approved by the Institutional Animal Care and Use Committee of SUNY Upstate Medical University and performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. 2.1. Controlled Cortical Impact Model of TBI The controlled cortical impact (CCI) TBI model was used in this study for making a severe TBI model in young adult mice as described in our previous studies . Briefly, C57BL/6J male mice (The Jackson Laboratory, Bar Harbor, ME, USA) at the age of 12 weeks were anesthetized with Avertin (Sigma-Aldrich, St. Louis, MO, USA. 0.4 g/kg body weight, i.p.). The heads of mice were fixed on a stereotaxic frame, and a 4-mm-diameter cranial window centered at 2 mm lateral to the bregma on the right side of the skull was created. The brain was impacted using a 3-mm diameter flat tip with a 4deg medial angle, 2-mm depth strike from the surface of the dura, at a 1.5 m/s strike velocity and 8.5 s contact time. As sham controls, mice were handled in the same manner but without craniotomy and impact. After surgery, mice were placed on a homeothermic blanket (37 degC) to prevent post-anesthesia hypothermia. An antibiotic (ampicillin, 20-100 mg/kg, s.c.) and analgesic (sustained-release buprenorphine, 0.6 mg/kg, s.c.) were injected before and after surgery, respectively. 2.2. Experimental Design To carry out a time course experiment for assessing the therapeutic effects of SCF + G-CSF on myelin repair in the chronic phase of TBI, a total of 43 male mice were divided into three time points: 1 day, 21 weeks, and 36 weeks after SCF + G-CSF treatment. Each time point has three groups: a sham group, a TBI-vehicle control group, and a TBI-SCF + G-CSF treatment group. The SCF + G-CSF treatment was initiated 3 months after TBI, corresponding to the chronic phase of TBI. Mice were subcutaneously injected daily with recombinant mouse SCF (200 mg/kg/day, diluted with saline, PeproTech, Cranbury, NJ, USA) and recombinant human G-CSF (50 mg/kg/day, diluted with 5% dextrose, Amgen, Thousand Oaks, CA, USA), or an equal volume of vehicle solution for 7 days according to our previous TBI study . The experimental flowchart is shown in Figure 1. Mice were euthanized at the selected time points: 1 day (sham, n = 5; TBI-vehicle, n = 4; TBI-SCF + G-CSF, n = 5), 21 weeks (sham, n = 6; TBI-vehicle, n = 6; TBI-SCF + G-CSF, n = 6) and 36 weeks (sham, n = 3; TBI-vehicle, n = 4; TBI-SCF + G-CSF, n = 5) post treatment. Mice with health problems during the experiment were excluded from this study. 2.3. Immunofluorescence Staining Mice were anesthetized with Ketamine (90 mg/kg) and Xylazine (9 mg/kg) (i.p.) and transcardially perfused with phosphate-buffered saline (PBS) containing heparin (10 U/mL, Sagent Pharmaceuticals, Schaumburg, IL, USA), followed by 10% neutral buffered formalin (Sigma-Aldrich, St. Louis, MO, USA) perfusion. Brains were removed, post-fixed in the same fixative solution overnight at 4 degC and placed in 30% sucrose solution in PBS. After being fully dehydrated, the brains were embedded in O.C.T. compound (Thermo Fisher Scientific, Waltham, MA, USA) on dry ice and sectioned at a thickness of 30 mm using a Leica cryostat. For free-floating immunofluorescence staining, brain sections were washed with PBS (pH 7.4) and incubated in PBS with 10% normal donkey serum, 0.3% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA), and 1% IgG-free bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, MO, USA) for 1 h at room temperature to block non-specific binding. Brain sections were incubated with the primary antibodies including rabbit anti-MBP (1:500, Abcam, Cambridge, England), rabbit (1:500, ThermoFisher, Waltham, MA, USA), goat anti-Olig2 (1:500, Novus Biologicals, Centennial, CO, USA), and mouse anti-Sox2 (1:500, T Thermo Fisher Scientific, Waltham, MA, USA) overnight at 4 degC. After rinsing with PBS, brain sections were incubated with the corresponding secondary antibodies including donkey anti-rabbit IgG conjugated with Alexa fluor 488 or 594 (1:300, Invitrogen, Waltham, MA, USA), donkey anti-goat IgG conjugated with Alexa fluor 488 or 594 (1:300, Invitrogen), or donkey anti-mouse IgG conjugated with Alexa fluor 488 or 594 (1:300, Invitrogen) for 2 h at room temperature in the dark. After rinsing with PBS, brain sections were mounted on Superfrost Plus Slides (Thermo Fisher Scientific, Waltham, MA, USA) with VECTASHIELD HardSet Antifade Mounting Medium (Vector Laboratories, Newark, CA, USA). 2.4. Quantification of Immunofluorescence Staining Two or three brain sections (from Bregma 0.14 mm to 0.86 mm) from each mouse were selected for immunofluorescence staining and quantification. To quantify the percentage of MBP+ area, MBP immunofluorescence staining in brain sections was captured with a confocal microscope (Zeiss LSM780) through a 40x objective. The images were taken in the areas adjacent to the cavity of TBI (i.e., within 500 mm to the cavity). The percentage of MBP positive area was analyzed using ImageJ software. When quantifying the percentage of MBP positive areas in the striatum, the regions without axon bundles were chosen and analyzed using ImageJ. To quantify the Olig2+ cells in the external capsule (EC) region, the EC region was selected using the tool "polygon selections" in ImageJ and the Olig2+ cells in the selected area were manually counted using the tool "Multi-point" in ImageJ. The Olig2+ cells were normalized by the area of the selected region. To quantify the number of Olig2+Sox2+ or Olig2+Ki67+ double positive cells in the subventricular zone (SVZ), the entire SVZ was captured with a confocal microscope (Zeiss LSM780) under a 20x objective lens using "Tile Scan" mode. The immunofluorescence double positive cells in the SVZ were manually counted using the tool "Multi-point" in ImageJ and normalized by the length of the counted SVZ. 2.5. Culture of Oligodendrocyte Progenitor Cells Isolated from Adult Mouse Brain Adult C57BL/6J mice (male, 3-4 months old) were decapitated after being anesthetized with Ketamine (90 mg/kg) and Xylazine (9 mg/kg) (i.p.). The brains were removed quickly and placed into ice-cold Hibernate E (Thermo Fisher Scientific, Waltham, MA, USA). The forebrain was dissected, and the meninges were mechanically removed. The forebrain was then mechanically minced into 1 mm3 pieces. The tissue pieces were spun down in a centrifuge at 100x g for 1 min. The pellet was mixed with 10 mL of dissociation solution (0.2% papain containing 1.1 mM EDTA, 0.067 mM mercaptoethanol, and 5.5 mM cysteine-HCl in DMEM high glucose medium) and incubated for 60 min at 37 degC on a shaker (50 rpm). The digestion was stopped by adding ice-cold Hanks' Balanced Salt Solution (HBSS). The mixture was centrifuged at 200x g for 3 min at room temperature. The pellet was resuspended in 10 mL ice-cold Hibernate E medium. To obtain a single cell suspension, the tissue suspension was triturated 10 times using a 5-mL serological pipette and subsequently three 1-mL pipette tips another 10 times. The tissue suspension was then allowed to sediment for 1 min, and the supernatant was filtered through a 70 mm cell strainer to obtain the single-cell suspension. To get more cells, 5 mL of fresh ice-cold Hibernate E medium was added to the sediment, and the above procedures were repeated. The collected cell suspension was mixed with isotonic Percoll (GE Healthcare, Chicago, IL, USA. 90% Percoll 10% 10x PBS, pH 7.2) at a final concentration of 22.5% and centrifuged at 800x g for 20 min without break at room temperature. The myelin debris and supernatant were discarded. The brain cell-containing phase (about the last 2 mL) and cell pellet were resuspended in PBS, transferred to a fresh 15 mL tube, and centrifuged at 300x g for 5 min at room temperature. The supernatant was discarded. The cell pellet was resuspended in red blood cell lysis buffer (BD Bioscience, San Jose, CA, USA) and incubated for 1 min at room temperature to remove red blood cells. Thereafter, 10 mL of modified washing buffer (MWB) containing 2 mM EDTA, 2 mM Na-Pyruvate, and 0.5% BSA in D-PBS (pH 7.2) was added to the cell suspension and spun down using a centrifuge at 300x g for 5 min at room temperature. The cell pellets were resuspended in 0.1 mL of MWB. To acquire purified OPCs, anti-A2B5 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) were used for magnetic-activated cell sorting according to the manufacturer's instructions. The sorted A2B5 positive OPCs were flushed out of the column with pre-warmed, CO2 and O2 pre-equilibrated OPC growth medium containing DMEM high glucose medium (Thermo Fisher Scientific, Waltham, MA, USA), 60 mg/mL N-Acetyl cysteine (Sigma-Aldrich, St. Louis, MO, USA), 10 mg/mL human recombinant insulin (Thermo Fisher Scientific, Waltham, MA, USA), 1 mM sodium pyruvate (Thermo Fisher Scientific, Waltham, MA, USA), 50 mg/mL apo-transferrin (Sigma-Aldrich, St. Louis, MO, USA), 16.1 mg/mL putrescine (Sigma-Aldrich, St. Louis, MO, USA), 40 ng/mL sodium selenite (Sigma-Aldrich, St. Louis, MO, USA), 60 ng/mL progesterone (Sigma-Aldrich, St. Louis, MO, USA), and 330 mg/mL bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) supplemented with epidermal growth factor (EGF) (10 ng/mL, Thermo Fisher Scientific, Waltham, MA, USA), basic-fibroblast growth factor (b-FGF) (20 ng/mL, Thermo Fisher Scientific, Waltham, MA, USA), and platelet-derived growth factor AA (PDGF-AA) (20 ng/mL, Sigma-Aldrich, St. Louis, MO, USA) . The OPCs were seeded onto PDL-coated (100 mg/mL, Sigma-Aldrich, St. Louis, MO, USA) wells of a 12-well plate at the concentration of 0.5 x106 OPCs per well. The cell medium was half exchanged every 3 days and passaged after confluent. To identify the purity of the cultured OPCs, the passaged cells were seeded on PDL-coated coverslips for 24 h, and immunostained with rabbit anti-NG2 (1:300, Millipore, St. Louis, MO, USA), goat anti-Olig2 (1:500, Novus Biologicals, Centennial, CO, USA), and rabbit anti-Sox2 (1:300, Millipore, St. Louis, MO, USA) using the above-mentioned immunofluorescence staining protocol. The cells were then imaged under a confocal microscope (LSM 780) using a 20x objective lens. 2.6. Proliferation and Differentiation Assay of Oligodendrocyte Progenitor Cells For proliferation assays, OPCs (passage 1) were cultured in the OPC growth medium with/without SCF + G-CSF (20 ng/mL) for 24 h. BrdU (Roche, Indianapolis, IN, USA. 20 ng/mL) was then added in the medium 6 h before fixation. For immunofluorescence staining, the cells were fixed with 10% neutral buffered formalin for 10 min at room temperature. After washing with PBS, the cells were treated with 2N HCl for 30 min at room temperature to hydrolyze DNA for BrdU staining. After rinsing with PBS, the non-specific staining was blocked with 10% normal donkey serum in PBS with 1% BSA and 0.3% triton for 1 h and then incubated with mouse anti-BrdU (1:1000, Roche, Indianapolis, IN, USA), rabbit anti-Ki67 (1:600, ThermoFisher Scientific, Waltham, MA, USA), and goat anti-Olig2 (1:500, Novus Biologicals, Centennial, CO, USA) overnight at 4 degC. The next day, the cells were rinsed with PBS 3 times and incubated with donkey anti-mouse IgG conjugated with Alexa fluor 488, donkey anti-rabbit IgG conjugated with Alexa fluor 594, or donkey anti-goat IgG conjugated with Alexa fluor 647 at room temperature for 2 h. After rinsing with PBS, coverslips were mounted with DAPI mounting medium and imaged with a confocal microscope (LSM 780) using a 20x objective lens. For differentiation assays, OPCs (passage 1) were cultured in the OPC growth medium without all growth factors. OPCs were treated with medium alone as a vehicle control, triiodothyronine (T3, 40 ng/mL, Sigma-Aldrich, St. Louis, MO, USA) as a positive control, or SCF + G-CSF (20 ng/mL) for 48 h. Cells were fixed with 10% neutral formalin solution for 10 min and rinsed with PBS three times. The fixed cells were immunostained with rabbit anti-MBP (1:500, Abcam, Cambridge, UK). The cells were imaged with an Olympus fluorescence microscope using a 20x objective lens. The cell that was immunopositive for MBP and counterstained with an intact cell nucleus (DAPI positive staining) was identified as a MBP positive cell. The percentage of MBP positive cells was calculated by the ratio of the number of the MBP positive cells to the total number of the intact cell nuclei. 2.7. Statistical Analysis Data are presented as the mean +- standard error of the mean (SEM). All statistical analyses were performed in GraphPad Prism (GraphPad Software, Inc., La Jolla, CA, USA). Before the analysis of parametric data, Shapiro-Wilk tests were performed to ensure the normality of the data. For multiple groups, data were analyzed using either one-way or two-way ANOVA followed by Tukey's post hoc test or Fisher LSD tests and presented as significant differences when the p values reached statistical significance. Unpaired Student's t-tests were used for two group comparisons. Two-tailed t-tests were used throughout, and statistical significance was defined as p < 0.05. For correlation analyses, we used Pearson's correlation coefficient test (linear regression). 3. Results 3.1. SCF + G-CSF Treatment Increases Remyelination in the Chronic Phase of Severe TBI Demyelination, which has been observed in both human TBI patients and rodent TBI models, has become a therapeutic target for TBI . White matter tracts are highly vulnerable to brain injury . It has been reported that oligodendrocyte apoptosis remains up to at least 3 months in the ipsilateral external capsule which is the subcortical region directly next to the cortical injury site in a mouse model of TBI . Our previous study has revealed that post-TBI demyelination occurs in the ipsilateral striatum and hippocampus, and that SCF + G-CSF treatment shows superior efficacy in enhancing remyelination than that of SCF and G-CSF treatment alone in the chronic phase of severe TBI . In the present study, we determined the time-course changes of demyelination in the ipsilateral external capsule and striatum and explored the timing effects of SCF + G-CSF treatment on myelin repair in the chronic phase of severe TBI. Myelin basic protein (MBP), as a myelin marker, was used for detecting TBI-caused demyelination and SCF + G-CSF-induced remyelination in the chronic phase of severe TBI. Compared with the sham group, the percentage of MBP+ area was significantly decreased in the ipsilateral external capsule and striatum in all TBI mice, including the mice in both TBI-vehicle and TBI-SCF + G-CSF groups at 1 day, 21 weeks, and 36 weeks post treatment (i.e., 13, 34, and 49 weeks after TBI, respectively) . In addition, in all TBI mice, the percentage of MBP+ area in both the ipsilateral external capsule and striatum were also significantly decreased at 49 weeks post-TBI compared with 34 weeks after TBI . These findings indicate that severe TBI causes persistent and progressive demyelination in the white matter. In the ipsilateral external capsule, the MBP+ area in the TBI-SCF + G-CSF group showed an increasing trend at 21 weeks post treatment and significant increases at 36 weeks post treatment compared to the TBI-vehicle group . In the ipsilateral striatum, the SCF + G-CSF treatment significantly increased the MBP+ area at 1 day and 36 weeks post treatment and showed an increasing trend at 21 weeks post treatment compared with the TBI-vehicle group . These findings indicate that SCF + G-CSF treatment enhances remyelination in the white matter during the chronic phase of severe TBI. The time-course data showed that the MBP expression in both the ipsilateral external capsule and striatum had no significant differences between 1 day and 21 weeks after treatment (i.e., between 13 and 34 weeks after TBI) in both the TBI-vehicle and TBI-SCF + G-CSF groups , suggesting that progressive demyelination may not occur yet during this particular period after severe TBI. However, the MBP expression was significantly decreased at 36 weeks post treatment (i.e., 49 weeks after TBI) in both the TBI-vehicle group and TBI-SCF + G-CSF group , indicating that progressive demyelination happens in the late chronic phase of severe TBI. Altogether, severe TBI leads to long-term and progressive demyelination in the white matter. SCF + G-CSF treatment in the chronic phase of severe TBI shows a long-term effect in enhancing remyelination in the ipsilateral external capsule and striatum. 3.2. SCF + G-CSF Treatment Increases Olig2 Positive Cells in the Ipsilateral External Capsule in the Chronic Phase of Severe TBI Oligodendrocyte transcription factor 2 (Olig2) expressed in both the oligodendrocyte progenitor cells (OPCs) and mature oligodendrocytes plays an important role in the generation of oligodendrocytes . Myelin sheath is comprised of oligodendrocyte processes. Myelination is largely dependent on oligodendrocytes . The increases in OPCs and oligodendrocytes are indicators of a cellular response for remyelination after TBI . To determine whether oligodendrocyte generation is involved in the SCF + G-CSF-enhanced remyelination in the white matter in the chronic phase of severe TBI, we quantified the number of Olig2+ cells in the ipsilateral external capsule over the time course. We observed that the number of Olig2+ cells in the TBI-SCF + G-CSF group was significantly increased at 1 day, 21 weeks, and 36 weeks post treatment compared with the TBI-vehicle group , suggesting that the SCF + G-CSF-induced persistent increases of Olig2+ cells in the ipsilateral external capsule may play a reparative role in enhancing remyelination in the chronic phase of severe TBI. We also observed interesting findings showing that at 21 weeks post treatment, the number of Olig2+ cells in both the TBI-vehicle control and TBI-SCF + G-CSF groups was significantly greater than in the sham control group, while the TBI-SCF + G-CSF group showed the greatest increases of Olig2+ cells in the ipsilateral external capsule compared with both the sham and TBI-vehicle control groups , suggesting that the TBI-caused increases of Olig2+ cells at this particular time are further enhanced by SCF + G-CSF treatment. We also found that in both the TBI-vehicle and TBI-SCF + G-CSF groups, the number of Olig2+ cells at 21 weeks post treatment was significantly greater than those at 1 day and 36 weeks post treatment . These findings suggest that the high number of Olig2+ cells appearing at 21 weeks post treatment (i.e., 34 weeks after TBI) may play a role in maintaining myelin at this time point, and that the decreased number of Olig2+ cells showing at 36 weeks post treatment (i.e., 49 weeks after TBI) could be responsible for the big fall of myelin (severe demyelination) in this late chronic phase of severe TBI. Our observation indicates that the Olig2+ cells in the ipsilateral external capsule are dynamically changing in the chronic phase of severe TBI, which is aligned with the processes of progressive demyelination in the chronic phase of severe TBI. 3.3. SCF + G-CSF Treatment Increases Oligodendrocyte Progenitor Cells in the Subventricular Zone in the Chronic Phase of Severe TBI Neural stem cells (NSCs) have been reported to contribute to brain remodeling following TBI . The proliferation of progenitor cells in the subventricular zone (SVZ) is vigorously increased days after TBI, and the progenitor cells migrate from the SVZ to the injury sites in response to TBI . As mentioned earlier, the increase in OPCs is a cellular response to remyelination after TBI. We then sought to determine the efficacy of SCF + G-CSF treatment in increasing OPCs in the SVZ of the TBI brain in the chronic phase. To do so, Olig2 and Sox2 antibodies were used for detecting the OPCs in the SVZ. Sox2 is a marker for both NSCs and OPCs . We found that the number of Olig2/Sox2 double-positive OPCs in the contralateral SVZ of SCF + G-CSF-treated TBI mice was significantly greater than in the sham and TBI-vehicle control groups at 1 day post treatment . At 21 weeks post treatment, the number of Olig2+/Sox2+ OPCs in the ipsilateral SVZ of the SCF + G-CSF-treated TBI mice was significantly increased compared with the mice in the TBI-vehicle control group . However, there were no significant differences among all groups at 36 weeks post treatment . Altogether, our findings suggest that SCF + G-CSF treatment increases the number of OPCs in the SVZ in the chronic phase of severe TBI, and that this effect could last for more than 5 months. To validate these findings, we performed double immunofluorescence staining using Ki67 (a proliferation marker) and Olig2 antibodies to detect the proliferation of OPCs in the SVZ at 1 day post treatment. In both the contralateral and ipsilateral SVZ, the number of Olig2/Ki67 double immunopositive cells was significantly increased in the TBI-SCF + G-CSF group compared with the TBI-vehicle and sham control groups , suggesting robust effects of SCF + G-CSF treatment in enhancing the proliferation of OPCs in the bilateral SVZ in the chronic phase of severe TBI. Correlation analyses demonstrated that the number of Olig2+/Ki67+ proliferating OPCs in the ipsilateral SVZ showed a positive correlation with the percentage of MBP+ area in the ipsilateral external capsule but it did not reach a statistically significant level (p = 0.0928). However, there was a significantly positive correlation between the number of Olig2+/Ki67+ proliferating OPCs in the ipsilateral SVZ and the percentage of MBP+ area in the ipsilateral striatum , indicating that the proliferation of OPCs in the SVZ is linked to the remyelination in the white matter during the chronic phase of severe TBI. SCF + G-CSF-enhanced proliferation of OPCs in the SVZ could contribute to increased remyelination in the white matter of the TBI brain in the chronic phase. 3.4. SCF + G-CSF Treatment Promotes Oligodendrocyte Progenitor Cell Proliferation and Differentiation In Vitro To validate the in vivo data, we carried out an in vitro study through which we sought to examine the efficacy of SCF + G-CSF treatment in promoting the proliferation and differentiation of cultured primary OPCs. Before starting the experiment, we evaluated the purity of the cultured OPCs using OPC markers. As shown in Figure 7A,B, the percentages of NG2+ cells, Olig2+ cells, and Sox2+ cells were 97.2%, 96.6%, and 94.5%, respectively, suggesting cultured primary OPCs with high purity that can be used for further study. The proliferation of OPCs was assessed 24 h after SCF + G-CSF treatment. The OPCs treated with SCF + G-CSF showed significant increases in the percentage of BrdU+/Olig2+ cells and Ki67+/Olig2+ cells , indicating that SCF + G-CSF treatment can enhance OPC proliferation. We then determined the efficacy of SCF + G-CSF treatment on OPC differentiation. OPCs were cultured in the OPC differentiation medium (i.e., OPC medium without OPC growth factors). As a positive control, some OPCs were treated with T3. After SCF + G-CSF or T3 treatments for 48 h, the differentiated oligodendrocytes were detected by immunofluorescence staining MBP. The percentage of MBP+ cells in T3-treated OPCs was significantly increased compared with medium controls and SCF + G-CSF treatment . Furthermore, SCF + G-CSF treatment significantly increased the percentage of MBP+ cells compared to the medium controls , suggesting that SCF + G-CSF treatment can also promote the differentiation of OPCs into oligodendrocytes. 4. Discussion Our study has demonstrated that persistent and progressive myelin loss with a temporary increase in oligodendrocytes exists in the chronic phase of severe TBI. The combination of SCF and G-CSF treatment in the chronic phase of severe TBI attenuates the TBI-induced myelin loss and enhances remyelination in the ipsilateral external capsule and striatum, which is associated with SCF + G-CSF-enhanced OPC proliferation and oligodendrocyte generation. Myelin impairments disrupt the axonal integrity and neuronal signaling conduction, which may lead to cognitive decline, motor dysfunction, and emotional symptoms commonly observed after TBI . It is plausible that SCF + G-CSF-improved neurological function recovery in chronic TBI could be linked to the enhancement of remyelination in the chronic phase of severe TBI. Our findings reveal the potential role of OPCs and oligodendrocytes in SCF + G-CSF-enhanced myelin repair in the chronic phase of severe TBI. White matter contains myelinated axons which are highly vulnerable to damage after TBI. White matter injury is associated with long-term impairments of neurological function after TBI . Myelin pathology is the key feature of white matter injury post TBI. Post-TBI demyelination that has been observed in both humans and rodent TBI models is linked to long-term deficits in sensorimotor and cognitive function . Since most experimental TBI studies target demyelination/remyelination in the white matter days and weeks after TBI , it remains largely unknown whether white matter demyelination occurs months after severe TBI in the chronic phase. Our findings from examining the MBP expression in the ipsilateral external capsule and striatum indicate that progressive demyelination in the white matter persists up to more than one year after severe TBI, which exhibits an exacerbating myelin loss at 12.25 months post severe TBI. It has been shown that the apoptosis of oligodendrocytes starts in the acute phase of TBI and remains at least three months post TBI , which may contribute to the persistent and progressive demyelination in the chronic phase of TBI. It has been thought that chronic demyelination is an irreversible pathology leading to progressive functional decline . In the present study, our data have revealed that treatment with SCF + G-CSF in the chronic phase of severe TBI could reverse the chronic demyelination by enhancing remyelination, which provides the feasibility of repairing the damaged myelin in the chronic phase of TBI and reveals a long-term therapeutic window for myelin repair after TBI. The myelin sheath is comprised of oligodendrocyte processes. Myelination is largely dependent on oligodendrocytes . Increased OPCs and oligodendrocytes are the indicators of a cellular response for remyelination after TBI . Olig2 is the oligodendrocyte lineage transcription factor and is expressed in both OPCs and oligodendrocytes . In the present study, we found that the number of Olig2+ cells in the ipsilateral external capsule was increased 1 day, 21, and 36 weeks after SCF + G-CSF treatment in the chronic phase of severe TBI. This observation indicates a long-term effect of SCF + G-CSF treatment in enhancing oligodendrocyte generation in the white matter tract, which lays a vital foundation to support remyelination in the chronic phase of severe TBI. Our findings also showed that the number of Olig2+ cells in the ipsilateral external capsule was increased at 34 weeks post TBI (i.e., 21 weeks post treatment) and then dropped down at 12.25 months post TBI (i.e., 36 weeks post treatment) in all TBI mice. This dropping down could be attributed to the exhaustion of oligodendrogenesis which has remained active during the long-term stimulation of demyelination or the interaction with age-related decline in oligodendrogenesis . Interestingly, MBP+ myelin also dramatically decreased in the ipsilateral external capsule at 12.25 months post TBI for all TBI mice compared to the level shown at 34 weeks post TBI, which is consistent with the dropping down pattern of Olig2+ cells in the same region. These findings confirm the tight link between the ability of oligodendrogenesis and the severity of post-TBI demyelination. The SVZ is a persistent source of OPCs in the adult brain. The OPCs in the SVZ of the adult brain can proliferate, migrate, and differentiate into mature oligodendrocytes which restore impaired myelin sheaths in injuries and diseases of the central nervous system (CNS) . Studies in both human severe TBI and animal models of TBI have shown increases in OPCs days, weeks, and even up to 3 months after TBI, and the TBI-increased OPCs have been proposed to play a role in myelin repair . In the present study, we have demonstrated that SCF + G-CSF treatment initiated at 3 months after severe TBI promotes OPC proliferation and increases the number of OPCs in the SVZ at 1day post-treatment. It has been reported that injections of SCF + G-CSF 11-20 days after spinal cord injury in mice promote the proliferation of OPCs . OPCs express the receptor of SCF named C-KIT which loses its expression in OPCs when OPCs differentiate into mature oligodendrocytes , while the G-CSF receptor is expressed in the oligodendrocytes , indicating that SCF and G-CSF may play different roles in OPC proliferation and differentiation for myelin repair. The data from our in vitro study further confirmed the direct role of SCF + G-CSF in promoting OPC proliferation and differentiation. In fact, fewer than 4% of OPCs differentiated following SCF + G-CSF treatment in vitro, suggesting that either OPCs did not differentiate sufficiently at 48 h or that SCF + G-CSF is more essential in boosting OPC proliferation than differentiation. However, it will be further confirmed in a long-term differentiation study in the future. SCF and G-CSF may also be indirectly involved in myelin repair by improving the microenvironment, such as anti-inflammation, neuroprotection, and brain-derived neurotrophic factor (BDNF) production . It has been shown that G-CSF protects oligodendrocytes from death via the suppression of inflammatory cytokines and up-regulation of anti-apoptotic proteins in spinal cord injury . Taken together, SCF and G-CSF may enhance myelin repair after CNS injury through direct and indirect mechanisms. Our previous studies have demonstrated that SCF + G-CSF treatment in both the subacute phase and the chronic phase of TBI improves neurological function recovery and ameliorates pathological progression post injury . The present study provides additional evidence supporting the long-term effects of the SCF + G-CSF treatment on myelin repair after severe TBI. However, the molecular mechanism underlying the SCF + G-CSF-enhanced myelin repair in the chronic phase of severe TBI remains unclear. Our previous studies have shown that SCF + G-CSF treatment activates MEK/ERK/p53 and PI3K/AKT/NF-kB/BDNF signal pathways to enhance neurite growth . Further studies need to be performed to elucidate whether the same molecular mechanism is involved in the SCF + G-CSF-enhanced myelin repair, and whether SCF + G-CSF treatment could be applied in other demyelination-related diseases. 5. Conclusions In the present study, we demonstrate that persistent myelin impairment exists in the chronic phase of severe TBI and that SCF + G-CSF treatment in the chronic phase of severe TBI enhances remyelination in the ipsilateral white matter. Furthermore, SCF + G-CSF treatment in the chronic phase of severe TBI promotes the proliferation of OPCs in the SVZ and increases OPCs and mature oligodendrocytes in the ipsilateral white matter, suggesting that the OPCs in the SVZ may migrate to the demyelinated sites and differentiate into mature oligodendrocytes to repair the TBI-induced demyelination. Acknowledgments We are grateful to Karen Hughes for her assistance in proofreading the manuscript and to Lisa Grace for her administrative assistance. Author Contributions Conceptualization, L.-R.Z., L.C. and X.Q.; methodology, X.Q., S.P. and M.K.; formal analysis, X.Q. and S.P.; investigation, X.Q.; resources, L.-R.Z.; data curation, X.Q.; writing--first draft preparation, X.Q.; writing--review and editing, L.-R.Z.; visualization, X.Q.; supervision, L.-R.Z.; project administration and funding acquisition, L.-R.Z. and L.C. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement This study was conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of SUNY Upstate Medical University (IACUC#495, 22 January 2021) (IACUC#361, 21 May 2019). Informed Consent Statement Not applicable. Data Availability Statement Not applicable. Conflicts of Interest The authors declare no conflict of interest. Figure 1 A schematic flowchart of the experiment. The mice were treated with SCF + G-CSF (SG) for 7 days at 3 months after TBI. The immunofluorescence (IF) staining on brain sections was performed at 1 day, 21 weeks, and 36 weeks post-treatment, respectively. Figure 2 Persistent and progressive white matter demyelination in the ipsilateral external capsule and striatum 13, 34, and 49 weeks after TBI (i.e., at 1 day, 21 weeks, and 36 weeks post-treatment, or 3.25, 8.5, and 12.25 months after TBI). MBP+ area: immunopositive area by immunostaining of myelin basic protein (MBP). Two-way ANOVA followed by Tukey post hoc test. Time point difference: * p < 0.05, ** p < 0.01; Sham vs. TBI: ++++ p < 0.0001. Figure 3 SCF + G-CSF treatment increases remyelination in the chronic phase of severe TBI. (A,B) Representative images of MBP immunopositive myelin in the ipsilateral external capsule (A) and striatum (B) at 1 day, 21 weeks, and 36 weeks post-treatment. (C-F) Quantitative data of analysis of the MBP immunopositive area in the ipsilateral external capsule (C,D) and striatum (E,F). Two-way ANOVA followed by Tukey post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Scale bars: 10 mm. SG: SCF + G-CSF. EC: external capsule. Figure 4 SCF + G-CSF treatment increases Olig2-immunopositive cells in the ipsilateral external capsule in the chronic phase of severe TBI. (A) Representative images of Olig2-immunopositive oligodendrocyte progenitor cells and oligodendrocytes in the ipsilateral external capsule (EC, between two dashed lines) at 1 day, 21 weeks, and 36 weeks post-treatment. (B) Quantitative data of analysis of the Olig2-immunopositive cells in the ipsilateral external capsule at 1 day, 21 weeks, and 36 weeks post-treatment in the sham, TBI-vehicle, and TBI-SCF + G-CSF groups. (C) Quantitative data of analysis of the Olig2-immunopositive cells in the ipsilateral external capsule in the sham, TBI-vehicle, and TBI-SCF + G-CSF groups at different time points. Two-way ANOVA followed by Tukey post hoc test. * p < 0.05, ** p < 0.01, **** p < 0.0001. Scale bar: 20 mm. SG: SCF + G-CSF. Figure 5 SCF + G-CSF treatment increases the number of oligodendrocyte progenitor cells (OPCs) in the subventricular zone (SVZ) in the chronic phase of severe TBI. (A) Representative images of Olig2+Sox2+ double-positive OPCs (the arrow-indicated yellow cells) in the contralateral and ipsilateral SVZ at 1 day, 21 weeks, and 36 weeks post-treatment. (B-D) Quantitative data of analysis of the number of OPCs in the SVZ at 1 day (B), 21 weeks (C), and 36 weeks (D) post-treatment in three experimental groups. Two-way ANOVA followed by Fisher's LSD test. * p < 0.05. Scale bars: 20 mm. SG: SCF + G-CSF. Figure 6 SCF + G-CSF treatment promotes the proliferation of oligodendrocyte progenitor cells (OPCs) in the subventricular zone (SVZ) in the chronic phase of severe TBI. (A) Representative images of proliferating OPCs (the arrow-indicated yellow cells co-expressing Olig2 and Ki67) in the contralateral and ipsilateral SVZ at 1day post-treatment. (B,C) Quantitative data of analysis of the number of Olig2+Ki67+ double positive OPCs in the contralateral SVZ (B) and Ipsilateral SVZ (C) at 1 day post-treatment. One-way ANOVA followed by Fisher's LSD test. * p < 0.05, ** p < 0.01, *** p < 0.001. Scale bars: 100 mm. SG: SCF + G-CSF. (D) Correlation between MBP+ area in the ipsilateral external capsule (EC) and the number of Olig2+Ki67+ double positive OPCs in the ipsilateral SVZ. (E) Correlation between MBP+ area in the ipsilateral striatum and the number of Olig2+Ki67+ double positive OPCs in the ipsilateral SVZ. Pearson's correlation coefficient test (linear regression) was used for correlation analysis. Figure 7 SCF + G-CSF treatment promotes the proliferation and differentiation of oligodendrocyte progenitor cells (OPCs) in vitro. (A) Representative images show that the primary cultured OPCs express NG2, Olig2, and Sox2. DAPI: nuclear counterstain. (B) The bar graph shows the percentage of NG2+, Olig2+, and Sox2+ cells in the primary cultured OPCs, indicating the high purity of the cultured OPCs. Sample size: n = 4. (C) Representative images show the proliferating OPCs (BrdU+ Olig2+ or Ki67+ Olig2+ double-positive cells) at 24 h post-treatment. Scale bars in panels A and C: 50 mm. (D,E) Quantitative data of analysis of the number of BrdU+/Olig2+ (D) and Ki67+/Olig2+ (E) proliferating OPCs in the medium control and SCF + G-CSF treatment groups. Unpaired Student's t-test, * p < 0.05, n = 4. (F) Representative images show the differentiated oligodendrocytes (MBP+) 48 h after treatment. 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PMC10000781 | Healthcare (Basel) Healthcare (Basel) healthcare Healthcare 2227-9032 MDPI 10.3390/healthcare11050720 healthcare-11-00720 Review Metabolic Syndrome and Chronic Disease Risk in South Asian Immigrants: A Review of Prevalence, Factors, and Interventions Mahadevan Meena 1* Bose Mousumi 1 Gawron Kelly M. 2 Blumberg Renata 1 Alikari Victoria Academic Editor Mantoudi Alexandra Academic Editor 1 Department of Nutrition and Food Studies, Montclair State University, Montclair, NJ 07043, USA 2 San Diego Post-Acute Center, El Cajon, CA 92020, USA * Correspondence: [email protected]; Tel.: +1-973-655-7574 01 3 2023 3 2023 11 5 72022 12 2022 21 2 2023 26 2 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). South Asians (SAs) are among the fastest-growing ethnic groups in the U.S. Metabolic syndrome (MetS) is a condition that is characterized by multiple health factors that increase the risk for chronic diseases, such as cardiovascular disease (CVD) and diabetes. MetS prevalence among SA immigrants ranges from 27-47% in multiple cross-sectional studies using different diagnostic criteria, which is generally higher compared to other populations in the receiving country. Both genetic and environmental factors are attributed to this increased prevalence. Limited intervention studies have shown effective management of MetS conditions within the SA population. This review reports MetS prevalence in SAs residing in non-native countries, identifies contributing factors, and discusses ways to develop effective community-based strategies for health promotion targeting MetS among SA immigrants. There is a need for more consistently evaluated longitudinal studies to facilitate the development of directed public health policy and education to address chronic diseases in the SA immigrant community. metabolic syndrome chronic disease South Asian immigrants acculturation This research received no external funding. pmc1. Introduction Metabolic syndrome (MetS) is a cluster of chronic disease risk factors, including abdominal obesity, hypertension, dyslipidemia, and impaired glucose tolerance . Because MetS is directly related to risk for both cardiovascular disease (CVD) and type 2 diabetes mellitus , it can incur a high cost to both individuals and society. Reports from the International Diabetes Federation (IDF) have estimated that, in 2021 alone, the global healthcare costs associated with diabetes were USD 966 billion and are expected to grow to a projected rate of USD 1054 billion by 2045 . As such, MetS and its related chronic conditions are public health issues of global significance. While the prevalence of MetS across different ethnic groups varies widely , the IDF estimates that, from 2011-2016, the overall prevalence of MetS in the United States was 36.9% . Currently, there are distinct criteria for MetS that have been established by several institutions (Table 1). The most widely used definitions have been developed by the World Health Organization (WHO); the National Cholesterol Education Program Adult Treatment Panel (NCEP ATP III); the American Health Association (AHA), in conjunction with the National Heart, Lung, and Blood Institute (NHLBI); and the IDF . Recently, the IDF and the AHA/NHLBI released a joint statement unifying several definitions into one consensus set of criteria . All of these definitions incorporate cutoff points for body mass index (BMI) and waist circumference (WC), blood glucose and lipid levels, and blood pressure. Additionally, the consensus definition uses ethnic-specific criteria for WC, including distinct cutoff points for the Asian population. The term "South Asian" (SA) is typically used to refer to individuals with ethnic origins in India, Pakistan, Bangladesh, Nepal, Bhutan, Maldives, or Sri Lanka. Over the last two centuries, individuals of South Asian (SA) origin, particularly those originating from India and Pakistan, have immigrated to different countries, including the U.S. According to census data, there were over 4.5 million SA immigrants living in the U.S. in 2022, making this the fastest growing ethnic group in the country . Studies have reported disparities in chronic disease between SA immigrants and the general U.S. population. Despite a lower average BMI , SA immigrants have higher prevalence rates of both diabetes and CVD compared to the majority population. Individuals of SA descent show higher amounts of abdominal fat and stronger insulin resistance when compared to individuals of European descent , suggesting an increased risk for metabolic syndrome. However, there are few large-scale studies examining MetS in the SA immigrant population. The purpose of this review is to compile and summarize studies focusing on MetS in SAs residing in non-native countries and to explore the factors that may influence the high burden of chronic disease observed in this population. Additionally, this review seeks to identify existing interventions and make suggestions for further research and practice to more effectively address the chronic disease burden in this burgeoning, yet underserved community. 2. Materials and Methods For the determination of MetS prevalence, factors, and interventions among SA immigrants, a literature search was performed with the PubMed (National Library of Medicine, Bethesda, MD, USA) search engine. Multiple PubMed literature searches were performed using the following search terms and combinations contained in all fields of publications: ("Metabolic Syndrome"), ("Metabolic Syndrome" AND "South Asians"), ("Metabolic Syndrome" AND "South Asian Immigrants"), ("Metabolic Syndrome" AND "India"), ("Metabolic Syndrome" AND "Pakistan"), ("Metabolic Syndrome" AND "Bangladesh"), ("Metabolic Syndrome" AND "Sri Lanka"), ("Metabolic Syndrome" AND "Nepal"), ("Metabolic Syndrome" AND "Bhutan"), and ("Metabolic Syndrome" AND "Maldives"). Additional searches were performed to obtain articles pertaining to factors associated with MetS, such as acculturation, diet, physical activity, genetic/biochemical, and other psychosocial and environmental factors (e.g., food access and food availability). Finally, searches were also performed to identify interventions and lifestyle approaches to treat and manage MetS in these groups. This review method allowed for an in-depth examination of articles . As the focus of this review was SA immigrants, exclusion criteria were studies that did not relate to any of the aforementioned countries of origin, or the prevalence, contributing factors, or interventions targeting MetS in this population. 3. Results The Prevalence of MetS in SAs residing in non-native countries: Studies examining the prevalence of MetS in SAs residing in non-native countries have reported rates ranging from 20-51% (Table 2). A large 2005 study (n = 1603) conducted in the U.K. reported a prevalence of 31-46% among SA immigrants, which was significantly higher than that among European subjects (9-18% prevalence, p < 0.001) . A subset of that population, consisting of only males (n = 1420), showed a MetS prevalence of 44.6%; a higher prevalence compared to Europeans was still observed . Another U.K. study found that MetS prevalence in SAs (n = 245) was nearly double what was observed among white subjects (39% vs. 20%, p < 0.001) . Smaller studies in the U.S. and the Netherlands reported similar findings. In contrast, one study in Canada found that there were no significant differences in MetS prevalence between SAs (25.9%, n = 342) and whites (22%, n = 326) . The specific countries of origin were not described in this study. Among the large-scale studies conducted in the U.S. (n = 1403), one study showed an overall prevalence of 47% in a population of Sas residing in the country . An earlier study observing Sas in the U.S. (n = 1445) found the prevalence of MetS to be 27% , while another report found that, among a population of 997 SAs living in the U.S., the prevalence of MetS was 37.6% . These, and other large-scale studies in the U.S., have been comprised of SAs mostly of Indian descent . One study involving Bangladeshi men living in Texas, USA (n = 91) reported MetS prevalence at 38% using NHLBI/AHA guidelines , while another showed that the highest prevalence of MetS was among Bangladeshi participants . This suggests that the degree of variability in prevalence reported in these studies may be related to the ethnicity or country of origin of the participants. Prevalence of MetS in SAs residing in native countries: A few studies have examined the prevalence of MetS among SAs residing in their native countries (Table 3). Researchers have suggested that the rising prevalence can be attributed to the tremendous economic growth brought on by rapid transitions in nutrition, culture, and the environment in their native countries, especially over the past three decades. For example, some studies show that increased access to processed and refined foods , a decline in the availability and accessibility of healthier food options , along with a concurrent increase in sedentary behavior , are the results of globalization and industrialization . Ultimately, these factors have led to an imbalance in energy intake and expenditure among its citizens . Other environmental factors, such as an increase in air pollution, which is described as having a negative impact on the pathways regulating macronutrient metabolism , and psychosocial factors, such as depression, anxiety, and a lack of social support , have also been implicated in the development of chronic disease risks in these groups. More research examining these factors is necessary to get a clearer picture of whether or not disparities in the prevalence of MetS are due to differences in ethnicity. Differences in MetS in SAs between native and non-native countries: Few studies have compared differences in risks for chronic disease among SA immigrants and their contemporaries residing in their native countries. These studies have consistently reported a higher BMI and waist circumference among SA immigrants compared to their native counterparts . A 2011 meta-analysis of 10 studies reported that indices of obesity were greater in migrant Indian populations compared to native Indians . Other studies have noted a higher risk for insulin resistance, diabetes, CVD, dyslipidemia, and hypertension among SA immigrants compared to the non-native country's majority population . In South Asians, polymorphisms for the gene encoding Apolipoprotein A-I, a protein component of high-density lipoprotein (HDL) particles, were significantly associated with MetS as well as low HDL levels, suggesting that the racial disparity of MetS between SAs and other races may be due to genetic differences . While genetic predisposition is noteworthy, it does not provide a complete picture of the factors that contribute to greater risk for MetS in SA immigrants and their descendants. The sections below describe these factors. Acculturation: The migration process has been linked to the increased prevalence of chronic diseases among SAs in non-native countries, suggesting the strong role of acculturation . Acculturation is defined as the process by which a particular culture adopts the tenets and behaviors of a new culture, typically due to immigration . Studies of immigrant cultures in the U.S. show that acculturation and its multiple dimensions might play a significant role in the development of chronic disease . Acculturation and diet: Dietary acculturation refers to the process by which immigrants adopt the food habits of their host culture over a period of time . Dietary behavior and the duration of residence in the U.S. have been identified as strong predictors of MetS in some SA immigrants . For instance, several studies show that the adoption of a predominantly western diet, which is typically positively associated with increased duration of residence, contributes to the increased chronic disease burden in this group . In one study, a high protein intake was related to an increased risk for diabetes, increased BMI, and higher waist circumference among SA immigrants living in America . Of the total 146 participants in this study, 85 reported eating meat, while the rest adhered to a lacto-vegetarian diet (n = 29), or a lacto-ovo-vegetarian diet (n = 32). Among the meat eaters, the source of protein intake (vegetable, fish, or animal) was not associated with diabetes status. Compared to the meat eaters, however, both of the vegetarian diets were associated with lower insulin resistance. Also, those maintaining one of the two vegetarian diets had resided in the U.S. for a shorter duration than those who ate meat. An earlier study among Sri Lankan and Pakistani immigrants living in Norway found similar results, although a better understanding of the Norwegian language was associated with lower fat consumption . It is worth noting here that using BMI as a proxy for obesity has several limitations. Unlike bioelectrical impedance, which measures both body fat and muscle mass, BMI is based solely on height and weight. Therefore, it is considered an indirect measure of body fat . Additionally, it fails to reflect the differences and changes in these two components that may occur with gender, age, or physical activity level . Among athletes, for instance, a higher weight may be due to higher muscle mass, thus resulting in overestimation . Finally, two people may have considerably different BMIs, despite having identical or nearly identical percentages of body fat . For these reasons, the use of BMI as a measure of obesity can misclassify, resulting in bias when estimating effects related to obesity. Nevertheless, these findings warrant further research, as they suggest that a transition away from a plant-based diet may contribute to increased chronic disease risk. Acculturation and physical activity: Within a small population of Indian immigrants in California, U.S., even a moderate amount of physical activity was correlated with a lower prevalence of MetS in men . Another study reported that duration of exercise was a statistically accurate predictor of MetS in SA immigrant women, but not in men . The decrease in physical activity was associated with increases in both anthropometric (BMI and waist circumference) and biochemical (glucose and triglycerides) measures of MetS risk in this study. Increased acculturation was significantly associated with vigorous and moderate leisure-time physical activity, but not with light physical activity among Indian immigrants living in Canada . A study of Indian immigrants in New Zealand that used pedometer measurements also reported decreased steps with increased acculturation . These studies emphasize the role of physical activity in possibly mitigating the risk of MetS. However, the conflicting results with respect to gender and exercise intensity warrant further investigation. Interventions for MetS: Several researchers have demonstrated the effectiveness of /or physical activity-based interventions in reducing the prevalence of MetS and chronic disease risks among various SA immigrant groups . A randomized, controlled trial involving Pakistani immigrant men living in Norway (n = 150) found that after a 5-month intervention promoting physical activity (both cardiorespiratory and strength training), there was a slight decrease in the prevalence of MetS, and a significantly greater reduction in waist circumference and serum insulin concentration in the intervention group compared to the control group . Another randomized, controlled trial involving Pakistani immigrant women living in Norway (n = 198) found that a 7-month intervention promoting both diet and physical activity resulted in a significant reduction in risk for Type 2 diabetes and MetS . A smaller study of Pakistani immigrant women living in Australia (n = 40) also found that a 24-week intervention promoting healthy dietary behaviors and regular physical activity significantly decreased the participants' BMI, blood pressure, cholesterol, and glucose levels compared to baseline . Studies involving Indian immigrants have utilized a wide range of intervention strategies to address MetS, including nutrition education sessions on incorporating more plant-based protein sources, such as nuts ; exercise ; or a combination of exercise-based approaches . Some of these interventions were delivered in a variety of formats, including one-on-one counseling sessions; group education workshops involving friends, peers, or family members; offered in the privacy of a participant's home; or offered in a faith-based setting, such as a Hindu temple. Some educational components were also tailored to be more /or gender-specific, while others were tailored for audiences with low levels of literacy. Regardless of the approach, there were improvements noted across a range of parameters, including BMI, blood pressure, blood glucose, and waist circumference . A review of randomized control trials from India showed that, in addition to diet, incorporating regular physical activity resulted in marked changes in risk factors for MetS, including a decreased BMI, waist circumference, and serum triglycerides and increased HDL-c levels . Literature examining the impact of such interventions on MetS among other SA groups in their native countries is limited. Nevertheless, these data show that lifestyle interventions have the potential to positively impact MetS among SAs regardless of their country of residence. 4. Discussion Gaps in prevalence: The harmonized consensus definition for MetS has recommended that waist circumference cutoff in Asian Americans be set at >=90 cm for men, and >=80 cm for women as a risk factor for MetS, which is in contrast to the values established by the WHO, NCEP ATP III, and the AHA/NHLBI, which are set to be higher at >=102 cm for men and >=88 cm for women, regardless of ethnicity . These differences in criteria make it difficult to compare the prevalence of MetS across studies using different MetS definitions; this may explain the variability in these reports among SAs. In studies that describe MetS in SAs across multiple MetS definitions, disparate prevalence proportions have been reported within the same population . Therefore, the discrepancies between MetS definitions need to be considered when comparing the prevalence of MetS in SAs across different studies. Additionally, most studies covered in this review are cross-sectional, suggesting a need for more longitudinal, prospective studies of MetS in SAs to develop more ethnic-specific criteria and apply these criteria in the formulation of effective treatment and prevention strategies for chronic disease in SAs. Future studies on MetS in the SA population warrant the application of a consistent MetS definition that takes into consideration ethnic-specific criteria, such as that of waist circumference in the IDF and harmonized consensus definitions. Gaps in interventions: The studies reviewed thus far demonstrate that lifestyle interventions focusing on healthy eating and/or physical activity may be effective in preventing and treating MetS, in general. A scoping review comparing physical activity levels and their impact on MetS among Indian, Pakistani, and Bangladeshi immigrants settled in the UK describes these differences . While levels of physical activity were generally lower compared to the majority population among all three groups, Bangladeshis had the lowest levels of physical activity, while Indians had the highest. In all three groups, women were less physically active than men, and older adults were the least physically active. Another review on cardio-metabolic risk factors among SA labor migrants who were hired for semi-skilled or unskilled jobs in the Middle East reported a high prevalence of being overweight/obese and related chronic diseases, such as diabetes and hypertension. Despite the high burden, there was a lack of focus on screening and inadequate provisions for health care . These data show that educational components within interventions that are targeted toward reducing the rising burden of MetS in SAs need to vary depending on the group's unique contextual needs; universal approaches that fail to consider ethnic and other societal inequities may fail in their compliance and overall effectiveness, likely widening the racial disparities in MetS prevalence. Most studies have grouped all SA immigrants together, even though there may be considerable differences in each ethnic group's nutrition and lifestyle behaviors. Furthermore, the majority of the participants in these studies have been Indians. This group tends to be over-represented in South Asian chronic disease research. Some studies have alluded to an inverse relationship between socioeconomic status and chronic disease in SAs living in non-native countries . Studies on the relationship between mental health and chronic disease in this population have been inconclusive . The influence of factors, such as health care access and differences in chronic disease risks due to occupational category or ethnicity, have not been studied sufficiently in this group either. Published literature that recognizes these differences and has reported the results separately is either limited or outdated . Implications: Adopting a lower BMI cut-off for SA immigrants would serve to increase opportunities for improved diagnosis and intervention. However, a cross-sectional survey of primary care physicians practicing in a major southern city in the U.S. found only 9% of physicians reported measuring waist circumference, and only 21% of physicians were aware of ethnicity-specific guidelines. Most lacked the knowledge and training to appropriately assess overweight/obesity and related chronic disease risks in SA immigrants . These data highlight the need for more culturally sensitive clinical strategies to reduce the burden of MetS in SA immigrant communities across the U.S. . The socio-ecological model has been recognized as a systematic and coordinated approach to understanding and reducing disease risks, particularly among underserved and vulnerable population groups . The model assumes that individuals are more likely to sustain disease treatment and management requirements within a comprehensive network that considers their individual (demographic characteristics, knowledge, attitudes, and beliefs), interpersonal (social support and size of social networks), environmental (availability of healthy food options and other resources), and institutional (public health policies) needs . There appears to be a scientific gap, however, in developing and testing relevant policies and programs for SA immigrants that are designed around the constructs of the socio-ecological model. While most healthcare organizations provide basic services, they are largely based on models that focus on a single disease risk and do not address the personal, familial, or social needs of the various ethnic groups within this community. Few programs have addressed cultural relevancy in screening, diagnostic, and treatment tools. As summarized in Figure 2, translational and prospective research that provides a clear evaluation of the importance of ethnic-specific guidelines for diet and physical activity may ultimately help reduce the chronic disease burden in SA immigrants. An optimal case management model for SA immigrants might be one that includes components that are not only culturally competent but ones that also strengthen and facilitate an individual's environmental, psychological, and social networks . For example, economic and zoning policies that ensure the availability of healthy foods and affordable preventive services in the neighborhoods in which these immigrant communities live and congregate may decrease risks for chronic diseases in the long term. Components that focus on personal control and self-esteem can help an individual regain better control over his or her health and attain weight management goals more effectively. Service facilities consisting of support staff that are more vigilant, especially to immigrant clients' needs, may be critical to removing institutional barriers and building on assets to reduce the prevalence of MetS in this vulnerable population. 5. Conclusions The toll of MetS and related chronic disease risks among individuals of South Asian origin is a rising public health problem. There is a need for an innovative treatment and management approach that balances ethnic-specific guidelines and recommendations with cultural and social norms and preferences, disease severity, disparities in access to neighborhood resources, and social support networks. Such an approach might affect SA immigrants to better engage in healthy behaviors. Acknowledgments The authors would like to profusely thank Valerie Kwong for her assistance. Without her commitment and dedication, this manuscript would not be in its current form. The authors would also like to thank Matthew Lock, Rory K. Koleman, Heidi Harris, and Eugenie Verdel for their assistance. Author Contributions Conceptualization, M.M. and M.B.; methodology, M.B. and K.M.G.; formal analysis, M.B. and K.M.G.; investigation, M.B. and K.M.G.; resources, M.M., M.B. and R.B.; data curation, M.B. and K.M.G.; writing--M.B. and M.M.; writing--review and editing, M.M.; visualization, M.B. and M.M.; supervision, M.M. and M.B.; project administration, M.M. and M.B. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement Not applicable. Conflicts of Interest The authors declare no potential conflict of interest with respect to the research, authorship, and/or publication of this article. Figure 1 Prisma-flow diagram for literature review. Figure 2 Conceptual model for data gathering for MetS and chronic disease risks in South Asian immigrants. healthcare-11-00720-t001_Table 1 Table 1 Major definitions/criteria for metabolic syndrome. Institution, Year Consensus definition, developed by IDF and AHA/NHLBI, 2009 IDF, 2005 AHA/NHLBI, 2004 NCEP ATP III, 2001 EGIR, 1999 WHO, 1998 BMI N/A >=30 kg/m2 N/A >=25 kg/m2 N/A >=30 kg/m2 Central Obesity WC cutoffs specific to the ethnicity being developed, currently using IDF criteria WC for European: >=94 cm (men) >=80 cm (women) WC for Asian: >=90 cm (men) >=80 cm (women) WC for all: >=102 cm (men) >=88 cm (women) WC for all: >=102 cm (men) >=88 cm (women) WC for all: >=94 cm (men) >=80 cm (women) WHR >=0.90 (men) >=0.85 (women) Fasting Glucose >=100 mg/dL >=100 mg/dL >=100 mg/dL >=110 mg/dL >=110 mg/dL >=100 mg/dL (as criteria for IR) TGL >=150 mg/dL >=150 mg/dL >=150 mg/dL >=150 mg/dL >=150 mg/dL >=150 mg/dL HDL-C <40 mg/dL (men) <50 mg/dL (women) <40 mg/dL (men) <50 mg/dL (women) <40 mg/dL (men) <50 mg/dL (women) <40 mg/dL (men) <50 mg/dL (women) <39 mg/dL <40 mg/dL (men) <50 mg/dL (women) Blood Pressure >=130/85 mm Hg >=130/85 mm Hg >=130/85 mm Hg >=130/85 mm Hg >=140/90 mm Hg, or hypertensive drug usage >=140/90 mm Hg Abbreviations: IDF, International Diabetes Foundation; AHA, American Heart Association; NHLBI, National Heart, Lung, and Blood Institute; NCEP, National Cholesterol Education Program; ATP, Adult Treatment Panel; EGIR, European Group for the Study of Insulin Resistance; WHO, World Health Organization; BMI, body mass index; WC, waist circumference; WHR, waist-hip ratio; IR, insulin resistance; TGL, triglycerides; HDL-C, high-density lipoprotein cholesterol. healthcare-11-00720-t002_Table 2 Table 2 Prevalence of MetS in SA in non-native countries. Author, Year (SA Group Studied) Non-Native Country Criteria Used Prevalence Khan et al., 2016 (Pakistan, India, Bangladesh, Nepal, Iran, Sri Lanka, Afghanistan, Bhutan) US Modified harmonized definition by IDF and NHLBI 47%; Highest prevalence among Bangladeshi men Garduno-Diaz et al., 2013 (India, Pakistan) UK IDF 20% Andersen et al., 2012 (Pakistan) Norway IDF 47-51% Dodani et al., 2011 (India) US IDF, WHO, NCEP ATP III, 29.7% (IDF), 13.3% (WHO), 40% (NCEP ATP III) Flowers et al., 2010 (India, Pakistan, Sri Lanka) US IDF 27%; Prevalence significantly higher in men (31%) than in women (17%) Misra et al., 2010 (India) US IDF, NCEP ATP III 37.6% (IDF), 32.4% (NCEP ATP III); Prevalence significantly increased with age in women, but not with men Telle-Hjellset et al., 2010 (Pakistan) Norway IDF 41% Rianon et al., 2009 (Bangladesh) US Modified AHA/NHLBI 38% Balusubramanyam et al., 2008 (India) US NCEP ATP III 32%; Prevalence was higher in the older population Ajjan et al., 2007 (India, Pakistan, Bangladesh) US IDF 39%; Prevalence was significantly higher compared to those of Caucasian descent Williams et al., 2007 (India, Pakistan, Bangladesh, Sri Lanka) UK NCEP ATP III 22.2% Forouhi et al., 2006 (India, Pakistan, Bangladesh) UK IDF 44.6%; Prevalence was significantly higher in South Asians compared to Europeans Tillin et al., 2005 (India, Pakistan, Bangladesh) UK WHO, NCEP ATP III 46% Male, 31% Female (WHO), 29% Male, 32% Female (NCEP ATP III); Prevalence, using both criteria, was significantly higher compared to Europeans Misra et al., 2005 (India) US NCEP ATP III 33.9% Anand et al., 2003 (Not specified) Canada NCEP ATP III 25.8%; Prevalence was significantly higher compared to Chinese immigrants, but not those of European descent healthcare-11-00720-t003_Table 3 Table 3 Prevalence of MetS in SAs in native countries. Author, Year Native Country Criteria Used Prevalence/Contributing Factors Adil et al., 2023 Pakistan NCE ATP III 28.8% Sundarakumar et al., 2022 India NCEP ATP III 46.2% (rural) 54.8% (urban) Ali et al., 2020 Bangladesh NCE ATP III 22% Subramani et al., 2019 India NCE ATP III, IDF 72.7% (NCEP ATP III) 50.2% (IDF) DeSilva et al., 2019 Sri Lanka IDF 47.2% Mehata et al., 2018 Nepal NCE ATP III, IDF 15% (NCEP ATP III) 16% (IDF) Sinha et al., 2013 India NCEP ATP III, IDF 29.6% (NCEP ATP III) 20.4% (IDF) Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. References 1. Grundy S.M. 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PMC10000782 | Colorectal cancer (CRC) is a leading cause of cancer-related mortality and chemoresistance is a major medical issue. The epithelial-to-mesenchymal transition (EMT) is the primary step in the emergence of the invasive phenotype and the Hedgehog-GLI (HH-GLI) and NOTCH signaling pathways are associated with poor prognosis and EMT in CRC. CRC cell lines harboring KRAS or BRAF mutations, grown as monolayers and organoids, were treated with the chemotherapeutic agent 5-Fluorouracil (5-FU) alone or combined with HH-GLI and NOTCH pathway inhibitors GANT61 and DAPT, or arsenic trioxide (ATO) to inhibit both pathways. Treatment with 5-FU led to the activation of HH-GLI and NOTCH pathways in both models. In KRAS mutant CRC, HH-GLI and NOTCH signaling activation co-operate to enhance chemoresistance and cell motility, while in BRAF mutant CRC, the HH-GLI pathway drives the chemoresistant and motile phenotype. We then showed that 5-FU promotes the mesenchymal and thus invasive phenotype in KRAS and BRAF mutant organoids and that chemosensitivity could be restored by targeting the HH-GLI pathway in BRAF mutant CRC or both HH-GLI and NOTCH pathways in KRAS mutant CRC. We suggest that in KRAS-driven CRC, the FDA-approved ATO acts as a chemotherapeutic sensitizer, whereas GANT61 is a promising chemotherapeutic sensitizer in BRAF-driven CRC. colorectal cancer signaling pathways chemoresistance epithelial-to-mesenchymal transition organoids Sapienza University of Rome2021 Sapienza Ateneo 2021RM12117A7F986146 Istituto Pasteur Italia--Fondazione Cenci Bolognetti20203AMKTW Project LOCALSCENTA0375-2020-36549 Call POR-FESR "Gruppi di Ricerca 2020"This work was supported by Sapienza University of Rome (Ateneo Project 2021) (A.V.); Sapienza Ateneo 2021 RM12117A7F986146 (L.M.); Istituto Pasteur Italia--Fondazione Cenci Bolognetti (E.F.); PRIN PRIN2020 20203AMKTW (F.B.); Project LOCALSCENT, Grant PROT. A0375-2020-36549, Call POR-FESR "Gruppi di Ricerca 2020" (G.G., M.L.). pmc1. Introduction Colorectal cancer (CRC) is the third most frequent cancer and the second cause of cancer-related death worldwide . Mutations in KRAS and BRAF oncogenes represent the most common genetic drivers in CRC. Indeed, KRAS and BRAF mutations occur in 40% and 10% of CRC, respectively , and they are both associated with a poor outcome . Even though they both belong to the MAPK pathway, KRAS and BRAF mutations are mutually exclusive in CRC and these two types of cancer are characterized by distinct clinical and molecular features. BRAF-mutant CRC often displays genome-wide hypermethylation, high microsatellite instability and mutation rates, while KRAS mutant CRC is associated with lower levels of microsatellite instability and gene methylation . First-line and palliative treatments for metastatic CRC, bearing KRAS or BRAF mutations, include the cytotoxic chemotherapeutic agent 5-fluorouracil (5-FU) ; however, patients often present disease recurrence after 5-FU therapy . Chemoresistance is conferred by a plethora of mechanisms, including the modulation of signaling pathways involved in the emergence of the cancer stem features and epithelial-to-mesenchymal transition (EMT) . Other mechanisms for resistance to therapy include the inhibition of apoptosis driven by upregulation of autophagy , metabolic reprogramming , upregulation of molecules involved in drug efflux and drug metabolism and activation of alternative pathways . Hedgehog-GLI (HH-GLI) and NOTCH signaling are pivotal developmental pathways involved in the regulation of multiple biological and pathological processes. The canonical HH-GLI pathway is activated upon the interaction between the extracellular ligands Shh, Ihh and Dhh and the receptor Patched (PTCH), which in turn derepresses Smoothened (Smo), thus activating the transcription factors GLI1, GLI2 and GLI3. Activated GLI translocate into the nucleus where they bind to DNA and activate the transcription of target genes . In cancers, GLI1 can also be activated in a non-canonical way by the "oncogenic load" of the cancer cell . NOTCH cascade is activated upon binding of ligands Jag1, Jag2, Dll1, Dll3 and Dll4 to NOTCH receptors (from 1 to 4). The binding leads to proteolytic cleavages of the NOTCH receptor, releasing the NOTCH intracellular domain (ID) into the cytoplasm. Then, NOTCH ID migrates into the nucleus where, in complex with CBF1 (also known as RPBJ), it activates its transcriptional program . Downstream target genes include HES1, which is involved in EMT and transcriptionally regulates ATP-binding cassettes transporters (ABC transporters), involved in multidrug resistance . Interestingly, deregulation of the NOTCH pathway was described in numerous cancerous and non-cancerous diseases, with its role being highly context-dependent . Deregulated HH-GLI is involved in the development and maintenance of numerous cancers and, together with NOTCH signaling, plays a crucial role in the maintenance of stem cells of the intestinal epithelia . The crosstalk of HH-GLI and NOTCH signaling is fundamental for spinal cord patterning , and several previous reports highlighted how several molecules belonging to the NOTCH pathway regulate the key components of the HH-GLI pathway and vice versa, as reviewed Kumar et al. . Both HH-GLI and NOTCH pathways were described as deregulated and associated with poor prognosis in CRC . In this context, our previous work has described a chemoresistance mechanism operated by the HH-GLI signaling in CRC, where chemotherapy treatment resulted in aberrant activation of the HH-GLI pathway which in turn led to the transcription of ATP-binding cassette transporters (ABC transporters), involved in multidrug resistance . Therefore, our current work aimed to evaluate the role of HH-GLI and NOTCH signaling pathways as regulatory molecular mechanisms responsible for chemotherapy resistance in models of BRAF-driven CRC. 2. Materials and Methods 2.1. Cell Cultures and Treatments HCT116 (KRAS G13D mutant) and HT29 (BRAF V600E mutant) were obtained from American Type Culture Collection (ATCC) and grown in DMEM high glucose (supplemented with 10% (v/v) fetal bovine serum, 1% (v/v) penicillin (50 U mL-1)--streptomycin (50 U mL-1)--and 2 mM L-glutamine. Cells were routinely checked for mycoplasma contamination by testing with PCR Mycoplasma Detection Kit (Cat. G238, ABM, Richmond, BC, Canada). Cells were treated with 10 mM GANT61 (ENZO Lifesciences, New York, NY, USA), 10 mM DAPT (Merk Life Science S.r.l., Milan, Italy), 10 mM Arsenic Trioxide (ATO) (Merck, Merk Life Science S.r.l., Milan, Italy) and 10 mM 5-Fluorouracil (5-FU). For combined treatments, GANT61 and DAPT or Arsenic Trioxide (ATO) were administered to the cells 24 h before 5-FU. 2.2. Cell Viability by Trypan Blue Exclusion Assay Cell proliferation was assessed by trypan blue dye exclusion test using 0.4% (w/v) Trypan Blue solution (Merk Life Science S.r.l., Milan, Italy). Blue-stained cells were scored as non-viable and unstained cells were scored as viable cells. The percentage of viable cells was obtained as the ratio between the percentage of viable cells in treated cells versus control. 2.3. Transwell Invasion Assay Transwell invasion assay was performed using Corning(r) Transwell(r) chambers (8 mm pore size, Corning(r)). HCT116 and HT29 cells (2.5 x 104 in each well) were seeded in the upper chambers of the 48-well plates (Corning, Somerville, MA, USA) while lower chambers were filled with 1 mL of medium with indicated treatments. Cells in the lower chambers were fixed with 95% ethanol for 10 min, stained with crystal violet and counted. 2.4. Western Blot Cells were lysed as previously described . Lysates were separated on 8% acrylamide gel and immunoblotted using standard procedures . Primary antibodies were Anti-GLI1 (L42B10, Cell Signalling Technology Inc., Boston, MA, USA), anti-PARP p85 Fragment (G7341, Promega, Madison, WI, USA) and anti-Cleaved NOTCH1 (D3B8, Cell Signalling Technology Inc., Boston, MA, USA). HRP-conjugated secondary antisera (Santa Cruz Biotechnology, Shanghai, China) were used, followed by enhanced chemiluminescence (ECL Amersham, Merk Life Science S.r.l., Milan, Italy). 2.5. RNA Isolation and Real Time qPCR cDNA was obtained as described earlier . RNA expression was analyzed on cDNAs using the ViiATM 7 Real-Time PCR System, SensiFASTTM Probe Lo-ROX (Bioline, Memphis, TN, USA), TaqMan gene expression assay according to the manufacturer's instructions (Life Technologies, Waltham, MA, USA). mRNA quantification was expressed in arbitrary units, as the ratio of the sample quantity to the calibrator or to the mean values of control samples. All values were normalized to three endogenous controls: HPRT, GAPDH and b-ACTIN. Primers for gene expression are listed in Supplementary Table S1. Gene expression of GLI1, HES1, c-MET, ABCG2, CD133, KRAS, BRAF, HPRT, GAPDH and b-ACTIN was assessed using Life technologies "best coverage" assays (Life Technologies, Waltham, MA, USA). 2.6. Organoids Organoids were produced by seeding 1500 cells per well. Cells were mixed with 33% growth-factor-reduced phenol red-free Matrigel (Corning, Somerville, MA, USA). Cultures were grown using a flat-bottom 24-well microplate in advanced DMEM-F12 (Cat. 12634010, Gibco, Waltham, MA, USA) supplemented with Epidermal growth factor and Fibroblast growth factor both at final concentrations of 20 ng/mL. For in vivo live imaging experiments, GFP-labelled organoids were obtained by transducing HCT116 with PLKO lentiviral particles carrying pTWEEN-GFP vector. Transduced green fluorescent cells were selected by cell sorting and used for organoids production. 2.7. Whole Mount Immunofluorescence Organoids were fixed with 4% paraformaldehyde and permeabilized with Triton X-100 in PBS (Sigma-Aldrich, St. Louis, MO, USA). Organoids were stained with anti-vimentin (ab11256, ABCAM, Cambridge, UK) antibody. Nuclei were DAPI-counterstained. Phalloidin was used for f-actin staining. Images were acquired using an LSM 900 (Zeiss, Milan, Italy) laser scanning confocal microscope with 40x/0.75 NA objective. Images were analyzed by using the program Zeiss ZEN 2.3 blue edition (accessed on 10 September 2022)). 2.8. Datasets and In Silico Analyses Datasets available on R2 platform (accessed on 15 December 2021)) were interrogated to evaluate GLI1 and NOTCH1 correlation in patients carrying BRAF or KRAS mutations. In detail, Tumor Colon Mutation status (Core Exon)--Sieber--211--rma_sketch--huex10p investigated gene correlation between GENE/REPORTER1: GLI1 and GENE/REPORTER2: NOTCH1, in 29 samples of CRC-carrying braf_v600e mutation; Tumor Colon (after surgery)--Beissbarth--363--custom--4hm44k investigated gene correlation between GENE/REPORTER1: GLI1 and GENE/REPORTER2: NOTCH1, in 32 samples of CRC carrying kras_g13d mutation. 2.9. Single-Particle Tracking Analysis Single-particle tracking (SPT) diffusibility analysis was performed in five steps. In the first step, single cells were detected from the time-lapse movies of oHCT116-treated and control group organoids using Imaris spot model. Spots were taken in each frame and were linked to the spots corresponding to the same cell in the successive frame. Frame-to-frame tracking was implemented using the linear assignment problem (LAP) method . In the third step, MSD, the mean square distance travelled by a cell given a certain time interval , was computed from single trajectories, as described by Michalet X . In the fourth step, the diffusion parameter D was calculated for each tracked cell. To this end, the MSD was plotted for different time intervals (Dt) for each cell trajectory and the slope was computed using the Least Squares Method. In the fifth step, the Kolmogorov-Smirnov test was applied on the diffusion parameters D, obtained from 5-FU-treated oHCT116 and control group organoids. 2.10. Statistical Analysis Results are representative of at least three independent experiments and are expressed as means +/- SD. Differences were analyzed using One-way ANOVA and Two-way ANOVA tests where appropriate, using the GraphPad Prism software Version 8.0. Adjusted p-values of less than 0.05 were considered as statistically significant. 3. Results 3.1. HH-GLI and NOTCH Signaling Pathways Sustain Resistance to 5-FU in KRAS Mutant CRC Cells 5-fluorouracil (5-FU) is a chemotherapeutic agent used for adjuvant and palliative treatment of CRC; however, patients often present disease recurrence . Therefore, we evaluated the role of HH-GLI and NOTCH signaling pathways as molecular mechanisms responsible for chemotherapy resistance. KRAS mutant HCT116 CRC cells were treated with 5-FU, alone or in combination with the HH-GLI inhibitor GANT61 and/or the NOTCH inhibitor DAPT. Our results showed that GLI1 and NOTCH1 ID were significantly upregulated after 5-FU treatment . HH-GLI inhibition by GANT61 resulted in the downregulation of GLI1 and, interestingly, in the upregulation of NOTCH1 ID; vice versa, NOTCH inhibition by DAPT resulted in the downregulation of NOTCH1 ID and the upregulation of GLI1 . We then determined the effects of treatments with the combination of HH-GLI inhibitor, NOTCH inhibitor and 5-FU. NOTCH1 ID expression was impaired in all combined treatments that included DAPT (DAPT+GANT61, DAPT+5-FU and DAPT+GANT61+5FU), while it was unaffected by the combination of 5-FU+GANT61. On the other hand, GLI1 was downregulated by GANT61 alone, as well as when combined with 5-FU and 5-FU+DAPT, while the combination of NOTCH inhibition and 5-FU failed to inhibit GLI1. Overall, our results show that GLI1 and NOTCH1 ID levels were concomitantly significantly downregulated only after the combined treatment of the chemotherapeutic agent 5-FU together with the inhibition of HH-GLI and NOTCH. In addition, we observed that the combination of HH-GLI and NOTCH pathway inhibition prevents the GLI1 upregulation and NOTCH1 activation induced by 5-FU. To determine the effects of treatments on apoptosis, levels of cleaved PARP (c-PARP) were evaluated; our results show that c-PARP was significantly induced by the combination of 5-FU and GANT61 or DAPT, and the three drugs combined . We further analyzed the effects of treatments on cell viability, and we found it significantly impaired in cells treated with the combination of GANT61 and DAPT and with the combination of 5-FU with either GANT61 or DAPT or the combination of the three drugs . To discern potential interdependence between the HH-GLI and NOTCH signaling pathways in mutant KRAS CRC cells, we analyzed GLI1 and NOTCH1 levels in an available cohort of CRC patients carrying this mutation (Tumor Colon (after surgery)--Beissbarth--363--custom--4hm44k; accessed on 15 December 2021) and no correlation was found We therefore envisioned a model where the oncogenic force of the driver gene KRASG13D sustains both HH-GLI and NOTCH pathways and both pathways need to be targeted to achieve a successful impairment of cells after chemotherapy. Hence, to clarify if the KRASG13D driver mutation sustained expression of GLI1 and NOTCH, we performed silencing of KRAS in HCT116 , which resulted in the significant downregulation of GLI1 and NOTCH1 ID protein levels . KRAS silencing was also accompanied by a significant downregulation of ABCG2 and HES1, target genes of HH-GLI and NOTCH1 ID, respectively . Arsenic Trioxide (ATO) is an organic compound approved for the therapy of adult patients with acute promyelocytic leukemia and was shown to successfully inhibit both HH-GLI and NOTCH pathways . ATO'sability to inhibit both GLI1 and NOTCH ID levels was confirmed in the KRASG13D-driven CRC model . As previously shown, 5-FU alone was able to upregulate both GLI1 and NOTCH1 ID, while the combination with ATO impaired both signaling pathways . Cleaved-PARP levels showed that apoptosis was significantly increased by the combination of ATO and 5-FU, while we observed a non-significant trend in ATO-treated cells . A pivotal feature of CRC aggressiveness relies on the epithelial-to-mesenchymal transition (EMT), a process that includes the acquisition by cancer cells of properties including motility and migration, early steps in cancer invasion and metastasis. Therefore, we investigated whether the targeting of HH-GLI and NOTCH could impair KRAS mutant CRC's migratory ability. We investigated the effects of the combined treatment of 5-FU and ATO on the migration ability of HCT116 cells. We observed that the migration was unaffected by 5-FU treatment, while it was impaired with ATO treatment and was completely abrogated after ATO plus 5-FU combined treatment . Then, we evaluated epithelial differentiation through E-cadherin levels, which increased after the combined treatment of ATO and 5-FU . 3.2. HH-GLI Signaling Pathway Sustains Resistance to 5-FU in BRAF Mutant CRC Cells BRAF V600E is the activating driving mutation in 10% of CRC and correlates with poor prognosis, however targeted therapy against the mutation was proven ineffective and first-line treatment includes cytotoxic chemotherapy ; thus, we investigated the role of the HH-GLI and NOTCH signaling pathways in 5-FU chemotherapy resistance in BRAF mutant HT29 cells. HT29 cells were treated with 5-FU alone or in combination with the HH-GLI inhibitor GANT61 and the NOTCH1 inhibitor DAPT . We observed that 5-FU induced upregulation of GLI1 and NOTCH1. GANT61 treatment resulted in the downregulation of both GLI1 and NOTCH1 ID, while DAPT treatment caused the downregulation only of NOTCH1 ID, without exerting any effect on GLI1 levels compared to control cells. The combination of GANT61 and DAPT successfully targeted both GLI1 and NOTCH1 ID. The combined treatment of GANT61 plus 5-FU was able to revert the 5-FU-induced upregulation of GLI1 and NOTCH1 ID, and the combined treatment of DAPT and 5-FU was able to revert the 5-FU-induced upregulation of NOTCH1 ID and partially of GLI1. Only when both HH-GLI and NOTCH pathways were inhibited together with 5-FU treatment were both GLI1 and NOTCH1 ID significantly downregulated . Apoptosis was evaluated through c-PARP levels; treatment with 5-FU and single inhibition of HH-GLI and NOTCH1 failed to induce apoptosis; c-PARP levels indeed increased only when cells were treated with GANT61 in combination with 5-FU, or with the combination of the three drugs . We then investigated cell viability and our results showed a significant impairment after GANT61 treatment, alone or in combination with 5-FU . Based on these results, chemotherapy resistance to apoptosis in BRAF V600E mutated cells seems to be driven by the HH-GLI signaling, which in turn sustains the activation of the NOTCH pathway. To gain more insight into the interdependence between the HH-GLI and NOTCH pathways, we interrogated GLI1 and NOTCH1 levels in a cohort of CRC patients carrying BRAFV600E mutation (Mutation status (Core Exon)--Sieber--211--rma_sketch--huex10p; accessed on 15 December 2021) and found a positive and significant correlation between GLI1 and NOTCH1 . The above presented data suggest an upstream role of HH-GLI in the regulation of NOTCH signaling in the BRAF-driven CRC model. To investigate whether BRAFV600E acted as a driver on the regulation of HH-GLI and NOTCH, we performed BRAF silencing . BRAF silencing resulted in decreased GLI1 and NOTCH1 ID protein levels . We also evaluated mRNA levels of HH-GLI and NOTCH1 ID readout, ABCG2 and HES1, respectively, and both were significantly decreased after BRAF silencing . The above-reported data demonstrate that chemotherapy stress induced increased levels of both HH-GLI and NOTCH1 pathways in the BRAF-driven CRC model. Interestingly, we observed that the GLI1 inhibitor GANT61 was also able to decrease NOTCH1 ID levels; conversely, the NOTCH1 inhibitor DAPT did not affect GLI1 levels. Since we observed that the targeting of HH-GLI was able to indirectly also target the NOTCH pathway, we wondered if the combination of 5-FU and GANT61 could affect cell motility, a key feature of EMT and therefore of CRC aggressiveness. Our experiments showed that 5-FU did not affect cell motility, while GANT61 resulted in decreased cell motility, which was further impaired by the combination of GANT61 with 5-FU . Then, we investigated the expression of two HT29 cell-specific epithelial differentiation markers, Axin and Muc2. We observed upregulation of Axin only after combined treatment, while Muc2 was affected by both 5-FU and GANT61 alone and by their combination , suggesting that treatments enhance the differentiated phenotype. 3.3. 5-FU Increases Motility of CRC Organoids The previous set of experiments allowed us to point out the role of HH-GLI and NOTCH pathways as regulators of EMT in KRAS mutant and BRAF mutant CRC, a key feature of chemoresistance . Organoid models in pre-clinical studies have become widespread due to their high reproducibility and high similarity to in vivo models . Indeed, cell features and behavior depend on the architecture of the cell population, e.g., the cell-cell contact, the stiffness of the extracellular matrix and the interaction with the microenvironment. All these conditions concur with specific characteristics related to cell polarity, stemness and differentiation status. Thus, to obtain CRC organoids, we seeded HCT116 and HT29 cells in Matrigel and after 7 days we observed organoid growth, as shown in Figure 3A,B. We compared basal levels of GLI1 and NOTCH1 in organoids and in 2D monolayer and our results reported higher GLI1 and HES1 expression levels in organoids, indicating that both pathways were more active in organoids compared to monolayer cellular models . We then evaluated levels of the EMT marker c-MET in both organoids and monolayers and observed that c-MET was expressed at higher levels in organoids . Since our results showed that 5-FU was not able to impair the migratory ability of CRC , and that CRC patients often present disease progression despite chemotherapy, we wondered if 5-FU itself favored aggressiveness in organoids, unleashing the migratory potential. Increased motility and migration capacity are features of EMT, thus we performed in vivo live cell imaging in the KRASG13D-driven CRC organoid model, the HCT116-derived organoids (oHCT116) at basal state and after 5-FU treatment (Supplementary Material Supplementary Video S1). To investigate the behavior of CRC cells within organoids, we investigated the diffusion parameters that allow the motility of individual cells to be quantified. The diffusion parameters from the oHCT116 control or 5-FU-treated organoids are reported along with the single cell trajectories that were used for the calculation of the diffusion parameters . Interestingly, 5-FU-treated oHCT116 cells mostly present lower diffusion parameters compared with CTRL , with a long tail corresponding to a sub-group of cells presenting very high diffusion . Based on these results, we believe that cells with augmented motility after chemotherapy represent a subset of aggressive cells able to initiate the metastatic process. 3.4. HH-GLI and NOTCH Inhibition Impairs 5-FU-Driven Mesenchymal Phenotype in KRASG13D-Driven CRC Organoids We then proceeded to investigate the inhibition of HH-GLI and NOTCH by using ATO in combination with 5-FU in KRAS-driven CRC organoids, oHCT116. Treatment with 5-FU alone did not affect organoid growth, while organoids treated with ATO were significantly smaller; the association of 5-FU and ATO further impaired organoid growth . Expression levels of the EMT marker c-MET, cancer stemness markers ABCG2 and CD133a, which is both HH-GLI target and cancer stemness marker, were significantly decreased in the combined treatment of 5-FU and ATO . Interestingly, ATO was able to counteract the 5-FU-driven upregulation of ABCG2. Mesenchymal features were also investigated by the immunofluorescence of the EMT marker vimentin, whose levels increased after 5-FU treatment and were reduced when organoids were treated with ATO alone or in combination with 5-FU. Of note F-actin, revealed by phalloidin staining, underwent a marked rearrangement in 5-FU-treated oHCT116, where cells lost their pseudopodia, probably due to a modification in the cell polarity . 3.5. HH-GLI Inhibition Impairs 5-FU-Driven Mesenchymal Phenotype in BRAFV600E-Driven CRC Organoids We then investigated the effects of 5-FU alone or in combination with the HH-GLI blockade in the BRAFV600E-driven CRC organoids (oHT29). Our results showed that the size of oHT29 treated with 5-FU did not differ from the control group, while organoids treated with GANT61 were smaller in size and the combination of 5-FU and GANT61 strongly impaired organoid growth . Gene expression analysis showed that the levels of cancer stem cell and EMT markers ABCG2, CD133 and c-MET significantly increased after chemotherapy treatment and were impaired by HH-GLI inhibition and the combination of 5-FU and GANT61 . To better investigate EMT, we performed whole-mount immunofluorescence staining for the mesenchymal marker vimentin and observed that vimentin levels were upregulated in 5-FU-treated organoids, they decreased with GANT61 and were strongly impaired in the combined treatment . Altogether, our experiments show that in KRAS-driven and BRAF-driven CRC, the HH-GLI and NOTCH pathways sustain the resistance to 5-FU through the activation of the EMT. Of note, ATO, the drug targeting both HH-GLI and NOTCH pathways, reverted the mesenchymal phenotype, therefore supporting the action of the chemotherapeutic drug. 4. Discussion Despite recent advances in cancer therapy, CRC is still among the prevalent causes of cancer-related death . Even though medical research has focused on identifying genetic mutations linked to CRC progression and tumor prognosis to improve patient treatment, drug resistance often occurs. One of the mechanisms conferring drug resistance is the misactivation of evolutionarily conserved pathways, such as Wingless (WNT) , phosphoinositide-3-kinase , extracellular signal-regulated kinase (ERK) , nuclear factor-kB (NF-kB) and the Hedgehog-GLI (HH-GLI) signaling pathway . The HH-GLI pathway has a crucial role in correct embryonic development and plays a role in the physiological maintenance of many tissues, including the colonic mucosa . While canonical activation of the HH-GLI pathway transduces the signal through the Hedgehog/PTCH/SMO/GLI axis, non-canonical regulation of GLI is external to Hedgehog signaling. Of note, it was demonstrated that transforming growth factor-beta (TGF-b) , epidermal growth factor receptor (EGFR) , mitogen-activated protein kinases (MAPK) , b-arrestin and WNT/b-catenin were able to induce the expression of GLI, regardless of SMO activation. Since both canonical and non-canonical routes culminate with the activation of the GLI1 transcriptional program, GLI1 inhibition could be useful to prevent chemoresistance in cancer cells. Our group has previously demonstrated that HH-GLI signaling regulates the expression of ATP-binding cassette transporters (ABC transporters), which are correlated to multidrug resistance in cancer cells, providing a rationale for the consideration of the HH-GLI pathway as a therapeutic target in CRC . NOTCH signaling has been reported to play a crucial role in the development of the normal mucosa and its aberrant activation is related to carcinogenesis in CRC. HH-GLI and NOTCH signaling pathways together with the WNT and BMP pathways are responsible for the development of intestinal mucosa, which is the innermost layer of the colon. Stem cells, transit amplifying cells and terminally differentiated secretory cells or enterocytes, concur in the formation of the structural unit of the colon, known as the crypt of Lieberkuhn . A recent paper showed that the HH-GLI blockade with GANT61 was able to inhibit NOTCH and WNT/b-catenin in cellular models of CRC . Since the HH-GLI and NOTCH pathways play a fundamental role in the correct patterning of the colonic mucosa and HH-GLI is upregulated by chemotherapeutic stress, we wondered whether HH-GLI and NOTCH crosstalk could be involved in the resistance mechanism of CRC cells related to 5-FU chemotherapeutic stress. The results of this study show how the HH-GLI and NOTCH pathways sustain CRC chemoresistance in different ways depending on the driver oncogene mutation. In detail, in KRASG13D-driven HCT116 cells we observed an upregulation of HH-GLI and NOTCH pathways after 5-FU and the inhibition of HH-GLI resulted in increased levels of NOTCH1 ID and vice versa . These results, coupled with the interrogation of public datasets suggested that the HH-GLI and NOTCH signaling pathways are connected in a positive feedback loop aiming to escape apoptosis induced by 5-FU . Importantly, the combined inhibition of HH-GLI and NOTCH was able to impair EMT, shown both as an impairment of transwell migration ability and with EMT markers in organoids . ATO, which was used to target both HH-GLI and NOTCH pathways, has been approved by the FDA for the therapy of adult patients with acute promyelocytic leukemia (APL). A phase I trial investigating the co-administration of ATO and 5-FU/Leucovorin in patients with advanced/relapsed CRC showed that ATO was well tolerated and that in some patients it was associated with therapeutic response and increased survival; a later study investigated GLI1 levels in biopsies from the above-mentioned clinical trial and found that it resulted to be down-modulated after ATO administration. Of note, data on the mutational status of enrolled patients are not available . In BRAFV600E-driven CRC, both pathways were upregulated after 5-FU treatment, but importantly GANT61 downregulated not only its specific target GLI1 but also NOTCH , suggesting an upstream role of HH-GLI over the NOTCH pathway , thus explaining the positive correlation between these two signaling pathways . Importantly, HH-GLI inhibition was able to impair EMT features, both in monolayer and organoids . 5. Conclusions In conclusion, our study describes for the first time two distinct models for BRAF-driven CRC where the HH-GLI and NOTCH signaling pathways play different roles in the chemoresistance and mesenchymal phenotype of CRC . Indeed, we described that in KRASG13D-driven CRC, chemotherapy resistance is directed by the concurrent activation of the HH-GLI and NOTCH pathways and the inhibition of both is crucial to revert the resistant phenotype. Conversely, in BRAFV600E-mutated CRC, the resistance to apoptosis induced by chemotherapy is mainly sustained by the HH-GLI signaling pathway. The implications of this novel information can be far-reaching if taken into consideration for the management of CRC patients, providing clinicians with further tools for the development of more effective treatment plans. Acknowledgments T.M.A. is fellow of Network Oncology and Precision Medicine, Dpt. Experimental Medicine, Sapienza University of Rome. Graphical abstract and Figure 6 were created with BioRender.com. Supplementary Materials The following supporting information can be downloaded at Supplementary Table S1. List of gene expression primers for quantitative real-time PCR. Gene expression of GLI1, HES1, c-MET, ABCG2, CD133, KRAS, BRAF, HPRT, GAPDH and b-ACTIN was assessed using Life Technologies "best coverage" assays (Life Technologies). Supplementary Figure S1. (A) Quantitative real-time PCR of KRAS in HCT116 cells after KRAS silencing (siKRAS) and control group (siCTRL). (B) Western blot analysis of KRAS, GLI1 and NOTCH1 ID in HCT116 cells after KRAS silencing (siKRAS) and control group (siCTRL). (C) Quantitative real-time PCR of ABCG2 and HES1 in HCT116 cells after KRAS silencing (siKRAS) and control group (siCTRL). (D) mRNA levels of E-cadherin (ECAD) expressed in arbitrary units in HCT116 treated with 5-FU, ATO, combined treatment and control group (CTRL). Data are representative of three independent experiments * p < 0.05 versus control; ** p < 0.01 versus 5-FU (Two-way ANOVA test). Supplementary Figure S2. (A) Quantitative real-time PCR of BRAF in HCT116 cells after BRAF silencing (siBRAF) and control group (siCTRL). (B) Western blot analysis of GLI1 and NOTCH1 ID in HT29 cells after BRAF silencing (siBRAF) and control group (siCTRL). (C) Quantitative real-time PCR of ABCG2 and HES1 in HT29 cells after BRAF silencing (siBRAF) and control group (siCTRL). (D) mRNA levels of AXIN and MUC2 expressed in arbitrary units, in HT29 treated with 5-FU, GANT61, the combined treatment and control group (CTRL). Data are representative of three independent experiments * p < 0.05; ** p < 0.01 (Two-way ANOVA test). Supplementary Figure S3. The plots show how the mean square displacement (MSD) changes for different time intervals (Dt) for each tracked single cell trajectory from either the HCT116 control group 3D organoids (CTRL) or the HCT116 5-FU-treated group (5-FU). Supplementary Figure S4. Uncropped full scan for Figure 1 (panels refer to main figure panel). Supplementary Figure S5. Uncropped full scan for Figure 2 (panels refer to main figure panel). Supplementary Video S1. time lapse of gfp transduced oHCT116 at basal state (oHCT116-gfp CTRL); Supplementary Video S2. treated with 5-FU (oHCT116-gfp 5-FU); length: 6 h. Click here for additional data file. Author Contributions Conceptualization, A.C. and A.P.; methodology, G.G.; validation, G.C., S.T. and T.M.A.; formal analysis, Z.M.B., G.G., M.L. and L.M.; investigation, A.C., G.C., S.T., F.B., A.D.F., L.C., T.M.A., Z.S., M.A.V.; resources, M.L., E.D.S., M.A.V.; writing--original draft preparation, A.C., Z.M.B., A.P.; writing--review and editing, G.C., A.V., E.D.S., E.F. and L.M.; visualization, A.C., A.P. and L.M.; supervision, E.F., A.P.; project administration, A.P.; funding acquisition, M.L., F.B., A.V., E.F. and L.M. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement All relevant data are included in the manuscript. Conflicts of Interest The authors declare no conflict of interest. Figure 1 HH-GLI and NOTCH signaling pathways sustain resistance to 5-FU in KRAS mutant CRC cells. (A) Western blot analysis of NOTCH1 Intracellular domain (NOTCH1 ID), GLI1 and cleaved-PARP (c-PARP) in HCT116 cells after 5-FU treatment in combination with GANT61 and DAPT. Numbers indicate intensity ratio of bands. Bar graphs show densitometric quantification of the band intensity values normalized to the loading control. * p < 0.05; ** p < 0.01; *** p < 0.001 versus control; SS p < 0.05 versus 5-FU; SSSS p < 0.01; SSSSSS p < 0.001 (Two-way ANOVA test). Uncropped full scan in Supplementary Figure S4. (B) Evaluation of cell viability by Trypan Blue exclusion assay in HCT116 after 5-FU treatment with or without GANT61 and/or DAPT; * p < 0.05, ** p < 0.01 versus CTRL (One-way ANOVA test). (C) Correlation analysis between GLI1 and NOTCH1 expression from dataset interrogated on R2 platform, as indicated in main text. (D) Western blot analysis of GLI1, NOTCH1 ID and c-PARP in HCT116 cells treated with 5-FU and ATO. Numbers indicate intensity ratio of bands. Bar graphs show densitometric quantification of the band intensity values normalized to the loading control; * p < 0.05; ** p < 0.01; (Two-way ANOVA test). (E) Transwell invasion assay in HCT116 cells treated with 5-FU, ATO and the combined treatment and control group (CTRL). Scale bar 150 mm. ** p < 0.01; *** p < 0.001; versus control; SSSSSS p < 0.001 (One-way ANOVA test). Figure 2 HH-GLI signaling pathway sustains resistance to 5-FU in BRAF mutant CRC cells. (A) Western blot analysis of NOTCH1 Intracellular domain (NOTCH1 ID), GLI1 and cleaved-PARP (c-PARP) in HT29 cells after 5-FU treatment in combination with GANT61 and DAPT. Numbers indicate intensity ratio of bands. Bar graphs show densitometrically quantified band intensity values normalized to the loading control; * p < 0.05 ** p < 0.01; *** p < 0.001; SS p < 0.05 versus 5-FU; SSSS p < 0.01 (Two-way ANOVA test). Uncropped full scan in Supplementary Figure S5. (B) Evaluation of cell viability by Tripan Blue exclusion assay in HT29 after 5-FU treatment in combination with GANT61 and DAPT; * p < 0.05, ** p < 0.01 versus CTRL (One-way ANOVA test). (C) Correlation analysis between GLI1 and NOTCH1 expression from dataset interrogated on R2 platform as indicated in main text. (D) Transwell invasion assay in HT29 cells treated with 5-FU, GANT61, the combined treatment and control group (CTRL); Scale bar 150 mm; * p < 0.05, ** p < 0.01 versus CTRL (One-way ANOVA test). Figure 3 5-FU increases motility in CRC organoids. (A) Workflow for CRC organoid growth. (B) Brightfield image of HCT116 and HT29 organoids (respectively oHCT116 and oHT29) after 7 days of culture. (C) Quantitative real-time PCR of HES1, GLI1, c-MET expressed in HCT116 and HT29 cultured in monolayer and as organoids; * p < 0.05 versus CTRL; ** p < 0.01 (Two-way ANOVA test). (D) Fluorescent images of GFP-labeled HCT116 on sequential hours, scale bar 200 mm. Supplementary Videos S1 and S2 of time lapse experiments are available in Supplementary Figure S3A,B. (E) Violin plot of the diffusion parameters obtained from the single cell trajectories for CTRL and 5-FU-treated organoids. Kolmogorov-Smirnov test p-value 0.0032. Figure 4 HH-GLI and NOTCH inhibition impairs 5-FU-driven mesenchymal phenotype in KRASG13D-driven CRC organoids. (A) Brightfield image of oHCT116 treated with 5-FU, ATO, their combination and the control group (CTRL); Scale bar 150 mm; * p < 0.05 versus CTRL; SSSS p < 0.01 versus 5-FU (One-way ANOVA test). (B) Quantitative real-time PCR of stem markers expressed in oHCT116 treated with 5-FU, ATO, their combination and the control group (CTRL). mRNA levels of ABCG2, CD133a and c-MET expressed in oHCT116 were expressed in arbitrary units; * p < 0.05 versus CTRL; SSSS p < 0.01 versus 5-FU (Two-way ANOVA test). (C) Whole-mount immunofluorescence staining of oHCT116 stained with phalloidin-594 (F-actin, red), vimentin (mesenchymal marker, green) and DNA (DAPI). Images were analyzed by using the program Zeiss ZEN 2.3 blue edition. Scale bar 200 mm. Figure 5 HH-GLI inhibition impairs 5-FU-driven mesenchymal phenotype in BRAFV600E-driven CRC organoids. (A) Brightfield image of oHT29 treated with 5-FU, GANT61, their combination and the control group (CTRL); Scale bar 150mm; * p < 0.05 versus Ctrl; SSSS p < 0.01 versus 5-FU (One-way ANOVA test). (B) Quantitative real-time PCR of ABCG2, CD133a and c-MET expressed in oHT29 were expressed in arbitrary units. Data are representative of three independent experiments, * p < 0.05 versus Ctrl; SSSS p < 0.01 versus 5-FU (Two-way ANOVA test). (C) Whole-mount immunofluorescence of HT29 organoids stained using phalloidin-594 (f-actin, red), vimentin (mesenchymal marker, green) and DNA (DAPI). Images were analyzed by using the program Zeiss ZEN 2.3 blue edition. Scale bar 200 mm. Figure 6 Hedgehog-Gli and NOTCH pathways sustain chemoresistance and the mesenchymal phenotype in CRC. Model of the activity of the Hedgehog-GLI and NOTCH pathways after chemotherapy stress in BRAFV600E and KRASG13D models. In KRAS-driven CRC, the chemotherapy stress activates both HH-GLI and NOTCH, which independently sustain the EMT program; in BRAF-driven CRC, chemotherapy stress induces the activation of the HH-GLI pathway, which in turn sustains the activation of NOTCH1 signaling, determining the acquisition of the EMT phenotype. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. |
PMC10000783 | Cells Cells cells Cells 2073-4409 MDPI 10.3390/cells12050785 cells-12-00785 Review Various AAV Serotypes and Their Applications in Gene Therapy: An Overview Issa Shaza S. 1 Shaimardanova Alisa A. 2 Solovyeva Valeriya V. 2 Rizvanov Albert A. 2* Pistello Mauro Academic Editor Chen Shih-Heng (David) Academic Editor 1 Department of Genetics and Biotechnology, St. Petersburg State University, 199034 St. Petersburg, Russia 2 Institute of Fundamental Medicine and Biology, Kazan Federal University, 420008 Kazan, Russia * Correspondence: [email protected]; Tel.: +7-(905)-3167599 01 3 2023 3 2023 12 5 78529 12 2022 22 2 2023 26 2 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). Despite scientific discoveries in the field of gene and cell therapy, some diseases still have no effective treatment. Advances in genetic engineering methods have enabled the development of effective gene therapy methods for various diseases based on adeno-associated viruses (AAVs). Today, many AAV-based gene therapy medications are being investigated in preclinical and clinical trials, and new ones are appearing on the market. In this article, we present a review of AAV discovery, properties, different serotypes, and tropism, and a following detailed explanation of their uses in gene therapy for disease of different organs and systems. AAV gene therapy approach serotype tropism gene therapy medication Kazan Federal University Strategic Academic Leadership ProgramPRIORITY-2030 This article has been supported by the Kazan Federal University Strategic Academic Leadership Program (PRIORITY-2030). pmc1. Introduction Thanks to the rapid progress in biomedical technologies, large developments of novel high-tech treatment approaches have become possible today for several human diseases. However, despite scientific discoveries in the field of gene and cell therapy, some diseases still have no effective treatment. Advances in genetic engineering methods have enabled the development of effective gene therapy methods for various diseases based on adeno-associated viruses (AAVs). AAVs are effectively used to treat a number of genetic and acquired human diseases, more specifically, monogenic hereditary ones, i.e., diseases caused by mutations in one gene. Today, many AAV-based gene therapy medications are being investigated in preclinical and clinical trials, and new ones are appearing on the market for the treatment of numerous human diseases, including those that were previously considered incurable. To date, there are only a few approved AAV-based gene therapy medications. For example, the US Food and Drug Administration (FDA) recently approved "Hemgenix", a drug for the treatment of hemophilia type B, which is caused by congenital deficiency of factor IX. The drug is an AAV serotype 5 (AAV5), carrying the gene of deficient factor IX. As for hemophilia type A, Roctavian has also recently been approved by the FDA as an AAV5-based gene therapy vector, carrying the deficient factor VIII gene, driven by a liver-selective promoter . Another breakthrough gene therapy medication is "Zolgensma", which was intended for spinal muscular atrophy (SMA) treatment, a disease characterized by degeneration of the anterior horns motor neurons in the spinal cord resulting in a loss of their functions. Zolgensma is an AAV-serotype-9 (AAV9)-based vector encoding complementary DNA (cDNA) of the survival motor neuron 1 gene (survival motor neuron, SMN1). The drug was approved by the FDA in May 2019 for the treatment of type 1 SMA. Following intravenous administration, AAV9 crosses the blood-brain barrier (BBB) and provides targeted delivery of the SMN gene to neurons. It has been shown that intravenous administration of Zolgensma leads to an improvement in patients' motor skills, along with a decrease in the clinical manifestations of SMA . Another FDA-approved AAV-based gene therapy medication is Luxturna, which is intended for the treatment of retinal dystrophy caused by biallelic mutations in the RPE65 gene (an enzyme of retinal cells involved in light-sensitive pigment regeneration). Drug delivery to the retinal cells leads to synthesis restoration of normal RPE65 protein, which is necessary for the regeneration of the photosensitive pigment . The very first AAV-based gene therapy medication to be registered is Glybera, which was approved by the European Commission for the treatment of a rare hereditary disease, lipoprotein lipase deficiency . Nevertheless, despite the successful development of AAV-based gene therapy medications, many preclinical and clinical studies still get suspended or terminated, without resulting in a drug registration, and therefore, many diseases, especially the rare ones, remain untreated. One explanation for that could be the lack of knowledge in gene therapeutics, in particular, features of AAV vectors. To tackle this issue, further research is needed to achieve better optimization and higher effectiveness of AAV-based gene therapy approaches. This review discusses various AAV serotypes, some features of infection, tissue and organ tropism, efficacy in many human diseases, etc. 2. Brief History of AAVs' Discovery AAVs are small non-enveloped DNA viruses belonging to the Parvoviridae family, that were first isolated in 1965, as a contaminant in preparations of a simian adenovirus (Ad) . The viruses were found incompetent to productively infect cells without a co-infection by a helper virus, usually an Ad or any type of herpesviruses, and thereby they were named as "adeno-associated", and classified into the Dependovirus genus . After being regarded as defective viruses for a long time due to their codependency, later studies on AAVs disproved this theory and showed that they rather launch a latent infection in the host cell, that could convert to productive infection under stress . Although AAVs have a high seroprevalence in humans (it has been estimated that 50% to 96% of the human population is seropositive for the second serotype of AAV (AAV2) depending on age and ethnic group) , they, however, were not linked to any disease neither in humans, nor in any other species . Different AAVs have not only been detected in primates isolates , but also from avian , caprine , bovine , and equine stocks . 3. Properties, Structure, and Genome Organization of AAVs Aside from AAV5 being the most divergent, all AAVs share a similar structure and properties . AAVs are easy to manipulate, as their particles can maintain biological stability in extreme conditions of pH and temperature . They share a genome of approximately 4.7 kb single-stranded DNA packed into an icosahedral, non-enveloped capsid with a diameter of 20-25 nm . The AAV genome consists mainly of two viral genes: rep (replication) and cap (capsid), flanked by inverted terminal repeats (ITRs) . As the ITRs have a palindromic nucleotide sequence, they create characteristic T-shaped hairpin structures, providing essential structural elements for viral genome replication and packaging . ITRs also play a regulatory role in viral gene expression and host genome integration . The open reading frame (ORF) of rep encodes several nonstructural proteins that are required for gene regulation, replication, transcription, and encapsidation , while the ORF of cap encodes three structural proteins: virion protein 1 (VP1), VP2, and VP3, with a molar ratio of 1:1:10 in AAV particles . Distinct tissue tropism of different AAV serotypes results from variations in the processing of this cap ORF, leading to variant immune and transduction profiles . 4. AAV Serotypes and Tropism Depending on their serotype, AAVs can have specific tropism for specific organs and tissues of the body. There are different AAV serotypes that vary in many aspects. Next, each serotype will be separately discussed. Table 1 summarizes the characteristics and properties of AAV serotypes, and Figure 1 demonstrates their variant tropisms. 4.1. AAV1 The exact origin of the first serotype of AAV (AAV1) is still unknown, as it was not initially isolated from tissues, but as a contaminant of Ad stocks , and its antibodies were found both in humans and non-human primates (NHPs) . This serotype uses sialic acid as its primary cellular surface receptor , and AAV receptor (AAVR) as a coreceptor . According to Rabinowitz J.E. et al., AAV1 does not bind heparin as it lacks R585 and R588, the amino acid residues required for such binding, and thereby it cannot be purified using heparin . Zolotukhin S. et al., developed a protocol for AAV1 chromatographic purification by iodixanol gradient centrifugation and anion-exchange chromatography . It can also be purified using mucin columns, as it can bind the sialic acid residues in mucin . Moreover, recombinant AAV1 (rAAV1) was not found to contain any detectable post-translational modifications (PTMs), according to a systematic analysis conducted on ten AAV serotypes by Mary B. et al. , and it was the first viral vector to be approved for use in gene therapy . In 1999, Xiao W. et al., conducted a study to investigate viral vectors for gene therapy, as a result of which, AAV1 was found to be the most efficient serotype for skeletal muscles' transduction . Many studies have since confirmed AAV1 high tropism to skeletal muscles of murine, canine, and NHP origins, compared to other serotypes . AAV1 was also found to achieve an efficient transduction of neurons and glial and ependymal cells in the murine brain . Moreover, it was found to be able to effectively transduce the heart , endothelial and vascular smooth muscles , and retina . 4.2. AAV2 AAV2 is considered the most studied serotype among all AAVs . It was first discovered in 1965 as a contaminant of simian Ad preparations . Later, in 1998, its primary cellular receptor, heparan sulfate proteoglycan (HSPG), was identified by Summerford C. and Samulski R.J. , and the amino acid residues providing its affinity to HSPG were suggested, afterwards, as R585 and R588 . Accordingly, rAAV2 can be purified using heparin column affinity chromatography . Nevertheless, the binding of AAV2 to its primary receptor was found to be insufficient for cell entry, so several coreceptors were later identified for it , including the human fibroblast growth factor receptor 1 (FGFR1) , aVb5 and a5b1 integrins , hepatocyte growth factor receptor (HGFR) , laminin receptor (LR) , and CD9 . The capsid of rAAV2 is reported to acquire multiple PTMs, including ubiquitination, phosphorylation, SUMOylation, and multiple-site-glycosylation . As the most studied serotype, AAV2, in fact, has shown various tropisms for several tissues in NHP, murine, canine, avian, and human cell types, including renal tissue , hepatocytes , retina , non-mitotic cells of the central nervous system (CNS) , and skeletal muscles . Nonetheless, broader tissue tropisms of AAV2 were enabled by the further innovation of mosaicism and cross-packaging (or cross-typing) of AAVs (explained in detail later in this article), where the viral genome of one serotype could be packaged into a capsid of another type, providing a wider transduction spectrum 4.3. AAV3 The third serotype of AAV (AAV3) was originally isolated from humans . Similar to AAV2, this serotype uses HSPG, FGFR1, and LR receptors , along with the human HGFR (hHGFR) receptor . Iodixanol gradient ultracentrifugation along with ion exchange chromatography have been used for AAV3 purification . PTMs of rAAV3 capsid include acetylation, phosphorylation, and glycosylation . Due to its inadequate transduction efficiency in vitro and in murine cell lines, AAV3 was mostly overlooked as a choice for gene therapy . However, as it has been later found to use hHGFR as a coreceptor, it showed extremely efficient transduction of human liver cancer cells as well as human and NHP hepatocytes . Since this selective tropism of AAV3 has been discovered, various studies have aimed at optimizing strategies to generate rAAV3 vectors with a higher transduction efficiency . The developed strategies suggested different approaches, mainly, capsid modification of AAV3 vectors and modification of hHGFR expression levels, along with the tyrosine kinase activity associated with it . AAV3 was also found to have specific tropism to cochlear inner hair cells, showing high in vivo transduction efficiency in a murine model . 4.4. AAV4 The fourth serotype of AAV (AAV4) is considered one of the most antigenically distinct serotypes . It has reportedly originated in NHPs , mainly in green African monkeys , as antibodies to its viral particles have been detected in their sera . A study of the AAV4 structure showed that its capsid surface topology shares a significant similarity with that of human parvovirus B19 and Aleutian mink disease virus . AAV4 uses a-2,3-O-linked sialic acid for cell binding and infection ; accordingly, mucin columns can be used for AAV4 purification, based on its ability to bind sialic acid residues in mucin . In addition, as this serotype lacks heparin-binding activity, it cannot be purified using heparin column affinity chromatography like AAV2, however, ion-exchange chromatography procedures have been developed and proven a high purification efficiency . The only reported PTM of rAAV4 is ubiquitination of its capsid proteins . AAV4 is suggested to be able to transduce human/NHP cells, as well as cells of murine and canine origins . The specific tropism of AAV4 results in a transduction efficacy of specific cell types in the mammalian central nervous system (CNS), mainly the ependymal cells . Moreover, following subretinal delivery, AAV4 has showed stable transduction of retinal pigmented epithelium (RPE) cells of rodent, canine, and nonhuman primate models, a distinctive feature enabled by the specificity of its capsid . In a murine model, AAV4 has also shown a significant transduction rate of kidney, lung, and heart cells . 4.5. AAV5 As it was first isolated in 1983 from male genital lesions, AAV5 became the only AAV serotype to be isolated directly from a human tissue . This serotype is considered the most genetically divergent of all AAVs , with a variety of unique characteristics, such as the distinct size and function of its ITR regions , utilizing herpes simplex virus (HSV) as its helper virus for human infections , and using an atypical endocytic route as a pathway for viral entry . Another distinctive feature of AAV5 is its ability to transduce cells that cannot be transduced with AAV2, an exclusive advantage for gene therapy uses . AAV5 was also found to use sialic acid as its primary receptor , along with platelet-derived growth factor receptors (PDGFR) a and b as coreceptors . Similar to AAV1 and AAV4, mucin columns can be used for AAV5 purification , and ion-exchange chromatography procedures have also been developed for that aim . Capsid proteins of rAAV5 are reported to undergo multiple PTMs, including ubiquitination, phosphorylation, SUMOylation, and glycosylation . AAV5 proved to have a significant transduction efficiency for murine retinal cells , mainly for photoreceptor cells . In addition, AAV5 tropism has been investigated in the murine brain, and proved, as a result, a transduction competence for multiple neural cell types, including Purkinje cells, stellate, basket, and Golgi neurons, and it was able to reach to the inferior colliculus and ventricular epithelium . AAV5 is also known for its efficient transduction of murine airway epithelia by apical infection , vascular endothelial cells, and smooth muscles . It is also reported to have tropism for liver cells in mice . 4.6. AAV6 The classification of the sixth serotype of AAV (AAV6) is still a matter of controversy, as it presents high genomic similarity with both AAV1 and AAV2 serotypes, however, it has still been assigned its own serotype numbering . AAV6 has a serological profile almost identical to that of AAV1, and shares its sequence of coding region with a homology percentage of 99%, along with multiple regions identical to those of AAV2 . Accordingly, it was suggested to be a naturally occurring hybrid resulting from homologous RECOMation between AAV1 and AAV2 . AAV6 was first isolated from a human Ad preparation , and similar to AAV1, was found to bind sialylated proteoglycans, mainly a2,3-/a2,6-linked sialic acid, as its primary receptor, as well as binding heparan sulfate . As for its coreceptor, it binds epidermal growth factor receptor (EGFR) . The only reported PTM of rAAV6 is acetylation of its capsid proteins . Similar to the previously described serotypes, AAV6 can be purified by either heparin or mucin column affinity chromatography, as it can bind both . AAV6 is reported to have tropism for a variety of tissues, including airway epithelia of murine and canine models , murine liver cells , and skeletal muscles of murine and canine models, with a transduction efficiency even higher than that of AAV2 , cardiomyocytes in murine , porcine , canine , and in sheep models. 4.7. AAV7 The seventh serotype of AAV (AAV7) was first isolated in 2002 from NHP tissues, specifically, from Rhesus macaque monkeys . Its mechanisms of cell binding and cell entry are still unknown , but it is established that this serotype does not bind heparin, or any other glycan in general . Capsid proteins of rAAV7 undergo multiple PTMs, including glycosylation, primarily, along with phosphorylation, SUMOylation, and acetylation . A study by Calcedo R et al., investigated the epidemiology of AAV-neutralizing antibodies in the worldwide population, and found that the seroprevalence of AAV7 antibodies is relatively low in humans, an advantage of this serotype to be used in clinical applications . Viral vectors based on AAV7 proved a high efficiency of transduction for skeletal muscle cells in murine models, similar to that achieved by AAV1, and higher than AAV2 . This serotype also proved a strong tropism to hepatocytes in murine and human tissues. In CNS of NHPs, AAV7 viral vectors were found to achieve a robust transduction mainly in cortical and spinal tissues . Moreover, AAV7-based viral vectors can, reportedly, achieve a significantly high transduction rate of murine neurons and photoreceptor cells in the retina both in vivo and ex vivo . AAV7 vectors also appear to have a limited tropism to vascular endothelial cells, that could be relatively enhanced through proteasome inhibition , and an in vivo transduction preference for epicardium cells in murine cardiac tissue . 4.8. AAV8 Similar to AAV7, the eighth serotype of AAV (AAV8) was first isolated in 2002 from Rhesus macaque monkeys . As a primary receptor, AAV8 binds LR, the same receptor used by AAV2 and AAV3 . Various procedures for rapid and scalable purification of AAV8 have been developed since its discovery, including, for example, dual-ion-exchange chromatography , or iodixanol gradient centrifugation . Phosphorylation, glycosylation, and acetylation are the three PTMs reported for rAAV8 capsid proteins . AAV8 is best known for its strong tropism to liver cells, and accordingly, its transduction efficiency of hepatocytes, which is far stronger and faster than those of all other AAV serotypes in different models, including murine, canine, and NHP . Following systemic delivery in murine models, AAV8 was proven to be the most efficient serotype for transduction of both skeletal and cardiac muscles, owing to its ability to cross the blood vessel barrier, a feature that both AAV1 and AAV6 lack, limiting their efficiency of muscle transduction to local delivery only . AAV8 could also achieve a successful in vivo transduction of murine pancreatic cells, following localized delivery . Moreover, a high-rate transduction of murine renal cells could be reached by local direct delivery of AAV8 viral vectors into the kidney tissue . AAV8 was also found to achieve an efficient transduction of different cells in the murine retina, including amacrine, Muller, and putative bipolar cells, along with some horizontal cells and cells in the ganglion cell layer (GCL) . AAV8 transduction efficiency appears to be susceptible to proteasome levels in some tissues, and accordingly, could be increased using proteasome inhibitors . 4.9. AAV9 The ninth serotype of AAV (AAV9) was first identified in a human isolate in 2004, and was named a new serotype as it had a serological profile distinct from the previously known AAVs, however, it was suggested to be closely related to clades containing AAV7 and AAV8 . As a primary receptor, AAV9 uses terminal N-linked galactose , and it is also suggested to bind a putative integrin, along with LR as coreceptors . Scalable simple purification protocols have been developed for AAV9 purification, including ion-exchange chromatography and sucrose gradient centrifugation . Capsid of rAAV9 has one of the highest totals of PTMs, including multiple ubiquitination, phosphorylation, SUMOylation, and glycosylation modifications, along with acetylation . In most tissues, AAV9 seems to achieve cell transduction with efficiency superior to other AAVs . For example, in a study aimed to investigate AAV1-9 distribution following systemic delivery in a murine model, AAV9 has shown rapid-onset, the best genome distribution, and the highest protein levels, in comparison with all other AAVs . Moreover, in the CNS of murine, NHP, and feline models, it has a unique feature compared to other serotypes, as it can cross the BBB and transduce not only neuronal but also non-neuronal cells, including astrocytes, that cannot usually be transduced by other AAVs , also showing tropism to photoreceptor cells in the retina . Viral vectors based on AAV9 have also proven to be more efficient than those of AAV1 and AAV8 (in some cases 5-10-fold higher than AAV8), for murine, NHP, and porcine cardiac muscle transduction , enabled with another distinctive feature of AAV9--its ability to traverse the physical barrier of vascular system endothelia . Another example of AAV9 superiority over other AAVs was presented in a study by Inagaki K et al., where the serotype achieved robust transduction of murine hepatocytes, skeletal muscles, and pancreatic cells . AAV9-based viral vectors also appear to be tropic to murine photoreceptor cells , renal tubular epithelium cells , Leydig cells in the testicular interstitial tissue , and alveolar and nasal epithelia . 4.10. AAV10 and AAV11 The ninth and tenth serotypes of AAV (AAV10 and AAV11) were first found and described in 2004 in NHP isolates, namely from cynomolgus monkeys, with capsid proteins of great resemblance to AAV8 and AAV4, respectively , resulting in serological cross-reactivity with those two serotypes . However, antisera against AAV10 and AAV11 were not found to have any cross-reactivity against those of AAV2, which recommended them as good viral vector candidates for gene therapy in individuals having antibodies against the latter . It remains unknown what cellular receptors and coreceptors AAV10 and AAV11 use for cell binding and entry ; therefore, procedures describing their purification protocols are generally based on iodixanol gradient centrifugation . Similar to AAV9, capsid of rAAV10 has one of the highest totals of PTMs, including mainly multiple glycosylation and phosphorylation modifications, along with ubiquitination, SUMOylation, and acetylation . A study investigating biological distribution of both AAV10 and AAV11 in monkeys suggested them to be tropic to NHP intestinal cells, hepatocytes, lymph nodes, and less frequently, to renal cells and adrenal glands . AAV10 was also suggested to have tropism to murine small intestine and colon cells . Compared to AAV8 and AAV9, AAV10 appears to have the largest tropism range to murine retinal cells, as it can reportedly transduce a variety of cell types, including RPE, cells in the ganglion cell layer, several cell types in the inner nuclear layer, photoreceptors, and a highly efficient transduction of horizontal cells . Following intravenous delivery, AAV10 was found to target murine liver and lung cells , however, upon localized delivery, it transduced murine renal and pancreatic cells . As for AAV11, it was found to have mild tropism to NHP CNS, mainly to the cerebrum and spinal cord . A recent study of neural gene therapy using rAAV11 found that murine projection neurons and astrocytes could also be targeted using this serotype . 4.11. AAV12 The twelfth serotype of AAV (AAV12) was first isolated from a simian Ad stock, and then characterized as a novel serotype, as it exhibited distinctive biological and serological properties . Although it was proven that AAV12 does not use heparan sulfate proteoglycans or sialic acids for attachment and cell entry, it remains unknown how exactly it binds target cells . However, according to a study investigating components of a potential receptor complex for AAV12, mannose and mannosamine were suggested as components of such complex, as they inhibited AAV12 cell transduction . Moreover, being resistant to neutralization by human antibodies, AAV12 represents a good candidate for human gene therapy applications . AVB Sepharose affinity chromatography has been used for purification of AAV12 . In murine models, it has shown tropism to salivary glands and muscles . It has also shown strong, localized in vivo tropism to murine nasal epithelia following intranasal administration . 4.12. AAV13 The thirteenth serotype of AAV (AAV13) is another simian Ad that appears to bind HSPG, although its primary cell receptor remains unknown . It was also found to share structural similarity with AAV2 and AAV3, making it the closest related AAV to those two serotypes, with a capsid conserving all AAV capsids' structural features , but there are only limited data on this serotype tropism and transduction efficiency . 4.13. Novel Hybrid AAV Vectors In addition to the natural AAV serotypes described above, novel AAV vectors have been developed during the last two decades, and are still being developed . Using different engineering strategies, novel hybrid vectors have been generated in order to enhance their transduction, modulate their immunogenicity, or limit their tropism to specific cells . There has been different types of such engineered novel vectors, including mosaic, chimeric, and combinatorial vector libraries . Mosaic vectors have multiple subunits of various serotypes in their capsid, chosen according to their properties of receptor binding and intracellular trafficking . In chimeric virions, the capsid usually has modified protein generated by domain swapping and DNA shuffling strategies to alter specific amino acids . Combinatorial vector libraries also use DNA shuffling and error-prone PCR methods to generate AAV libraries of novel serotypes with mixed genomes . 5. AAV as Viral Vectors for Gene Therapy Applications A variety of AAV features have made it an appealing viral vector candidate to be used in gene therapy applications. To begin with, AAVs are non-pathogenic viruses, since no diseases have been linked to them . AAV vectors have also proven their ability to provide stable integration into the target genome , and such feature is highly needed for gene therapeutic uses considering the high level of expression that could be maintained as a result , without affecting the target cell functions in the long term . Moreover, having only ITRs, AAV vectors cannot interfere with the inserted gene regulation either . More specifically, a unique feature of AAV vectors, contrary to all currently available gene-editing platforms, is utilizing the homologous recombination pathway, which does not involve exogenous nucleases, providing, therefore, a highly accurate editing process that preserves genome integrity without adding to the mutational burden of the target site . Thanks to such unique gene-editing properties, AAV vectors are currently the leading platform for in vivo gene therapy delivery . Another attractive feature is the very broad range of cells, tissues, and hosts that AAV can efficiently transduce in vivo and in vitro , including dividing and non-dividing cells in humans , and NHP , murine , canine , feline , and a variety of other models. In addition to the genetic properties, AAVs' distinct physical properties represent more reasons why they should be used for gene therapy, such as easy manipulation, resistance to pH changes, heat changes, and detergents . Next, we present various studies that investigated AAV vectors for a wide range of gene therapy applications (reviewed in Table 2). 5.1. AAV Viral Vectors for Gene Therapy of the CNS AAV-mediated CNS gene therapy was first believed to predominantly target neurons, with lesser chance to affect other cells and tissues in the CNS , a fact that was later proven to be inaccurate in many studies . For a variety of neurodevelopmental and neurodegenerative diseases, AAV-mediated gene therapy has been tested using different administration routes, including intraparenchymal, intrathecal, intracerebroventricular, and intracisternal injection, many of which have shown promising results . Parkinson's disease (PD) has been one of the most studied neurological disorders as a target for AAV-mediated gene therapy . In a phase I clinical trial, Kaplitt et al., investigated an AAV-mediated gene therapy approach for advanced PD patients, where serotype 2 was used as a vector for unilateral subthalamic delivery of the glutamic acid decarboxylase (GAD) gene . Providing a significant improvement in motor function scores up to 12 months after surgery, the approach was found to be safe and well-tolerated in patients. A similar double-blind, controlled, randomized clinical trial conducted later by LeWitt et al., investigated the same AAV2-GAD vector for bilateral subthalamic delivery in advanced PD patients, and showed similar results of safety and improvement of motor function . Bartus et al., also tested the bilateral stereotactic delivery of AAV2-neurturin in PD patients of an open-label clinical trial, the initial obtained data of which supported the feasibility, safety, and good tolerance of the approach as a potential treatment for PD . However, bilateral intra-striatal infusion of an AAV2 vector containing the aromatic L-amino acid decarboxylase (AADC) gene in moderately advanced PD patients led to an improvement in PD rating scales that was associated with a risk of intracranial hemorrhage in patients, along with headaches . As for sustainable transgene expression, a clinical trial conducted by Mittermeyer G. et al., investigated the potentials of rAAV2 carrying the aromatic L-amino acid decarboxylase gene (rAAV2-AADC) . In the trial, 10 patients with moderately advanced PD received bilateral infusions of recombinant vector into the putamin, showing good tolerance and a stable expression of transgene that lasted for the following 4 years, although higher vector doses were suggested for further studies. In children with AADC deficiency, the same delivery route for the same vector (rAAV2-AADC) was also assessed in an open-label phase I/II clinical trial . The therapy was well-tolerated in general, providing evidence for potential improvement of motor function. Similarly, spinal muscular atrophy (SMA) has always represented an attractive candidate for gene therapy, being caused by a single gene defect that affects the survival motor neuron (SMN) protein . Therefore, SMA has been suggested as a target for AAV-mediated therapeutic approaches, mainly using AAV9 . An open-label, phase I clinical trial was designed by AveXis, Inc. in 2014 to assess the safety and efficacy of AA9-SMN as a treatment for SMA . Following single-dose intravenous administration of the vector, a significant improvement of the motor function in all 15 patients was observed, reflected by their ability to perform different activities, such as unassisted sitting and walking, oral feeding, and speaking, with no reported motor function regression at the two-year follow-up . A long-term safety assessment, however, was recommended. Lysosomal storage diseases (LSDs), another group of neurodegenerative diseases, have also been extensively targeted by AAV-mediated gene therapy . One example of which is mucopolysaccharidosis type VII (MPS VII or Sly disease), a disease that results from genetic deficiency of beta-glucuronidase (GUSb) . In the murine model, AAV-mediated gene therapy has been shown to provide stable expression of the deficient enzyme upon single administration, that was adequate for phenotype correction in the liver and most of the neuraxis . As for neonatal murine model, intravenous delivery of rAAV-GUSb proved to yield therapeutic levels of the deficient enzyme in multiple organs, including ones of the CNS, with the gene expression not being affected by rapid growth and differentiation of tissues . The canine model of MPS VII has also shown promising therapy results following rAAV-GUSb intrathecal injection of serotypes 9 and the NHP-derivate AAVrh10 , as high expression levels of GUSb were detected in the CNS tissues, with the enzyme in brain tissue homogenates showing over 100% normal activity. Another type of MPS, MPS IIIA, has also been assessed for gene therapy in the canine model using AAV9 carrying the deficient enzyme's gene . Following intra-cerebrospinal fluid (CSF) delivery, the administered vector provided sustained and widely distributed transgene expression with no toxicity for a duration of seven years after therapy. Metachromatic leukodystrophy (MLD) is also one of the LSDs that results from arylsulfatase A (ARSA) enzyme deficiency and has been targeted by AAV-mediated therapeutic approaches . In the murine model of MLD, as well as in NHPs, serotypes 1, 5, 9, and rh10 carrying the ARSA gene have provided supporting results of their therapeutic potentials as gene therapy viral vectors . GM2 gangliosidoses is another example of lysosomal storage diseases that have been assessed for AAV-mediated gene therapy, including Tay-Sachs disease (TSD) and Sandhoff disease (SD) . Preclinical data from many in vivo studies have provided evidence of AAVs' efficacy for GM2 gangliosidoses in murine , ovine , and feline models. Clinically, there has been trials applying AAV-mediated gene therapy for different LSDs throughout the last decades with variant outcomes . In 2018, four children with asymptomatic or early-stage MLD underwent a phase I/II trial for gene therapy using AAVrh.10-hARSA . Although there was a significant elevation in ARSA levels recorded in the CSF following intracerebral vector delivery, no clinical improvement was noticed compared to the control group. As for gangliosidosis, the first clinical trial for gene therapy of Tay-Sachs disease using an AAV vector was conducted by Taghian et al., in 2020 . The treatment proved to be safe for the two treated children and resulted in a broad distribution of the transgene in CNS. Canavan disease (CD) is another rare inherited leukodystrophy belonging to LSDs, that has been targeted by AAV-mediated gene therapy . CD results from aspartoacylase (ASPA) enzyme deficiency, that leads to N-acetylaspartic acid (NAA) accumulation in the brain, causing white matter degeneration . In a murine model, rAAV2-ASPA delivery into the striatum and thalamus of the brain resulted in an increased ASPA activity and, thereby, decreased NAA accumulation and white matter degeneration. However, areas remote from the injection site, such as the cerebellum, were not affected . In 2002, Janson et al., suggested a clinical protocol using rAAV2-ASPA for gene therapy in CD patients , and currently there are several ongoing clinical trials testing different serotypes of rAAV (including 2, 9, and olig001), carrying the ASPA gene for CD gene therapy . Krabbe disease, also known as globoid cell leukodystrophy (GLD), causes demyelination in a similar way. The diseases results from deficiency of the lysosomal enzyme galactocerebrosidase (GALC), leading to accumulation of its substrate, psychosine, which affects both the central and peripheral nervous systems . During the last two decades, a large number of in vivo studies have been conducted to investigate different AAV serotypes as viral vectors for gene therapy of GLD in different animal models, including murine and canine. Intracerebral delivery of rAAV1-GALC has been tested in the murine model of GLD in an in vivo study conducted by Rafi et al. . The approach resulted in sustained expression of the deficient enzyme, and thereby positively affected myelination status, and animals' lifespan. However, both treated and untreated mice died with similar symptoms, suggesting that the used approach should be initiated prior to symptoms' onset. rAAV2/5-GALC has also shown therapeutic efficacy in the murine model of GLD following intracranial delivery . In the study conducted by Lin et al., in 2011, mice treated with rAAV2/5-GALC showed wide dispersion of the GALC transgene across the CNS, reaching areas remote from the injection site . Moreover, rAAV2/5-GALC delivery resulted in a reduced loss of oligodendrocytes and Purkinje cells, along with a significant improvement of neuromotor function and a prolonged lifespan of treated mice. Karumuthil-Melethil et al., conducted another preclinical study using recombinant vectors AAV9, AAVrh10, and AAV-Olig001, carrying the GALC gene, for GLD gene therapy in the murine model, following lumbar intrathecal delivery . All three serotypes provided wide distribution of the transgene across the CNS and liver, resulting in a significant improvement of myelination, and a prolonged lifespan, with AAV9 being the most effective when combined with bone marrow transplantation. AAV9 and AAVrh10 have also been tested for GLD therapy in the canine model . Combined intravenous and intracerebroventricular delivery of AAVrh10 encoding canine GALC (AAVrh10-cGALC) resulted in delayed symptoms onset, prolonged lifespan, correction of biochemical defects, and also positively affected neuropathology in treated animals . Similarly, AAV9-cGALC has shown promising therapeutic potency in the GLD canine model . Reportedly, intrathecal delivery of AAV9-cGALC resulted in increased activity of the deficient enzyme and, therefore, normal levels of its substrate. It also improved myelination, and decreased inflammation both in the CNS and PNS, which, along with prevention of clinical neurological dysfunction, resulted in a significantly prolonged lifespan of treated dogs, compared to the control group. However, sufficient dosing was found to be critical, as high doses significantly extended the lifespan even for post-symptomatic subjects, and a 5-fold lower dose of the vector resulted in an attenuated form of disease . For ocular diseases, AAV viral vectors have also provided great therapeutic opportunities both in vivo and in human clinical trials . In a study by Petrs-Silva et al., the efficacy of intraocular transduction of rAAV serotypes 2, 8, and 9 was investigated in mice for targeting ocular neurovascular and retinal diseases . Besides transduction of retinal cells, both serotypes 8 and 9 efficiently transduced the ganglion cell layer, providing evidence for their potentials in therapeutic approaches of ocular diseases. Moreover, AAV2/7 and AAV2/8 hybrid serotypes have achieved high transduction rates of the murine photoreceptors, showing promising potentials for treatment of inherited photoreceptor diseases . AAV2/6 has also shown an efficient transduction of murine cone photoreceptors following subretinal injection . In humans, AAV2/4 has been used in a clinical trial for treatment of childhood blindness caused by Leber Congenital Amaurosis (LCA) disease, mainly by a mutation in the retinal pigment epithelium-specific protein (RPE65) . Over a follow-up period between 1 and 3 years, subretinal injection of AAV2/4-RPE65 showed good tolerance both systemically and locally, with no adverse effects, and resulted in an improvement of visual function presented by different parameters in different patients, including improvement of visual acuity and color vision, as well as a reduction of visual fatigue or photophobia . Another example of AAVs' applications in ocular disease therapy is a phase I/II clinical trial by MacLaren R et al., that has aimed to treat blindness caused by choroideremia, an X-linked recessive disease that results from a mutation affecting retinal escort protein 1 (REP1) . In the trial, AAV2-REP1 resulted in improvement of retinal sensitivity in all six patients, following subretinal injection, with two of them having significant increases in visual acuity, supporting further consideration of the tested therapeutic approach . Various other clinical trials have applied AAV-mediated gene therapy for ocular diseases, and many of which have presented promising results, using mainly AAV2 and AAV8 (detailed in Table 2). Similar to ocular diseases, hearing disorders have also been suggested for AAV-mediated gene therapy . In a porcine model, Lalwani A. et al., tested AAV9 intracochlear delivery to assess its efficacy as a vector for gene therapy of hearing disorders, and demonstrated, as a result, transgene expression in the inner ear of animals following administration . 5.2. AAV Viral Vectors for Gene Therapy of Respiratory Diseases For over 20 years, AAV vectors have been vastly investigated for gene therapeutics of respiratory diseases, both in preclinical experiments and human clinical trials . However, a key limitation was that many AAV serotypes cannot efficiently transduce airway epithelial cells through the apical surface, suggesting molecular modifications of such serotypes to enhance their transduction efficiency . Cystic fibrosis (CF) represents one of the most studied diseases as a target for AAV-mediated gene therapy . In rabbits, AAV has proven to promote an efficient and stable gene transfer of the cystic fibrosis transmembrane conductance regulator (CFTR) gene into the airway epithelium, indicating, as a result, the vector potential to be used for gene therapy . Subsequently, since 1998, clinical trials have started using recombinant AAV viral vectors to target different sites of the airway epithelium of CF patients, using variant administration routes . The very first clinical trial to do so used the maxillary sinuses for delivery of recombinant AAV2 containing the CFTR gene (AAV2-CFTR) in ten CF patients, demonstrating a safe, successful transduction of targeted cells and a detected function restoration of the sinuses . Soon after, a following phase II double-blind clinical trial tested unilateral administration of the same vector into the sinus for 23 CF patients, with an in-patient control, achieved by administering a placebo drug into the other sinus . The approach again showed safety and good tolerance, although it did not confirm clinical effectiveness of the treatment. Aitken et al., also tested AAV2-CFTR in a phase I clinical trial for twelve mild CF patients using aerolization by nebulation for delivery, confirming the approaches safety, but failing to yield an effective clinical treatment . Further trials on AAV2-CFTR with single or repeated dosing have yielded similar results of safety and good tolerance, providing little evidence of clinical treatment after intranasal and endobronchial delivery . In order to optimize such therapeutic approaches and produce a functional CFTR in CF patients, further modifications of recombinant AAV vectors have since been developed . Alpha-1 antitrypsin (a1AT or AAT) deficiency is another lung and liver disease that has been extensively studied as a target for AAV-mediated gene therapy both in vivo and in humans . Reportedly, intravenous delivery of recombinant AAV vector carrying the human alpha-1 antitrypsin gene (AAV-hAAT) in the murine model resulted in potentially therapeutic serum levels of AAT . Further research found that intrapleural delivery of rAAV2-hAAT or rAAV5-hAAT could achieve higher AAT levels both in lungs and serum compared to intramuscular delivery in C57BL/6 mice, with the rAAV5 showing 10-fold higher effectiveness than rAAV2 . However, rAAV6/2-hAAT has been shown to transduce murine lung cells even more efficiently than rAAV5 both in vivo (in murine lung cells) and in vitro (in human airway epithelial cell culture) . Similarly, rAAV8-hAAT has also been shown to provide murine lung cells' transduction superior to that of rAAV5 following intratracheal delivery, as it resulted in 6-fold and 2.5-fold AAT levels in serum and broncho-alveolar fluid, respectively , and a high transduction rate of murine hepatocytes . Chiuchiolo M et al., also proved the safety of rhAAV10-hAAT as a viral vector for treatment of AAT in wild-type murine and NHP models . Besides, the delivered vector resulted in persistent expression of the transgene in chest cavity cells of both models, suggesting the efficacy of the tested therapeutic approach . Examples of other rAAV vectors' therapeutic potentials for AAT treatment have been shown in a variety of other preclinical studies, including rAAV1-AAT, rAAV2/9-AAT, and rAAV6 . AAVs have also been tested to develop therapeutic approaches for other lung diseases, such as asthma and surfactant B deficiency . A preclinical study by Zavorotinskaya et al., investigated the efficacy of rAAV carrying the interleukin 4 gene (IL-4) as a vector for gene therapy of allergic asthma, upon intratracheal delivery into the murine model . Showing a significant inhibition of airway eosinophilia and mucus production along with a reduction in airway hyper-responsiveness and asthma-associated cytokines levels, obtained data suggested rAAV efficacy for gene therapy of the studied disease. Kang et al., have recently tested an engineered rAAV6/2 vector carrying human or murine surfactant protein B gene (SFTPB) in a murine model . Mutations of this gene cause surfactant protein B deficiency (SPB) in humans, a rare genetic condition with a very poor prognosis . In their study, Kang et al., demonstrated efficient transduction of the airway and alveolar epithelium by engineered rAAV6/2-SFTPB following intratracheal administration into a murine model of SPB. As a result, the administered vector was well-tolerated with no adverse effects. Moreover, rapid, long-term restoration of the deficient SPB protein was reported, along with an improvement of lung function, leading, subsequently, to an extended survival . 5.3. AAV Viral Vectors for Gene Therapy of Muscle Diseases Having a large, body-distributed mass, and myofibers with a long half-life time makes muscles an attractive target for gene therapy, also considering the minimal invasiveness of the intramuscular delivery route . Muscular dystrophies (MD) constitute a large group of muscle diseases that have been studied for a long time and proved to be a good target for AAV-mediated gene therapy . For example, AAV1 carrying the follistatin gene, an antagonist of muscle growth negative regulator, has been shown to promote sustained improvement in muscle size and strength in NHPs following intramuscular administration . There has also been many reports of both histopathological and functional sustained muscle restoration as a result of using AAV-mediated therapeutic approaches in animal models of Duchenne muscular dystrophy (DMD) , and Limb-Girdle muscular dystrophies (LGMD) . As for clinical research, there have been several registered clinical trials of AAV-mediated gene therapy for muscular dystrophies . The first clinical study was a randomized, double-blind, placebo-controlled phase I clinical trial, conducted in 2012 by Bowles D et al., which used AAV2.5 carrying the mini-dystrophin gene for intramuscular administration in DMD patients . As a result, the administered vector was found to be safe and well-tolerated, and transgene DNA was detected in all patients. Similarly, other clinical trials were started in 2017 and 2018 to test AAV-micro-dystrophin efficacy for treatment of DMD (details in Table 2). 5.4. AAV Viral Vectors for Gene Therapy of Cardiovascular and Blood Diseases Cardiovascular diseases make another attractive target for gene therapy, being a leading cause of death globally with their high incidence and mortality rates . As mentioned before, AAV serotypes 6, 8, and 9 have been shown to efficiently transduce cardiac cells in different animal models, and accordingly, they have been used for therapeutic purposes in different cardiovascular diseases . In the search for an effective gene therapy platform for heat failure (HF), White J et al., published a study in 2011, in which they suggested a novel technique for AAV-mediated myocardial gene therapy using molecular cardiac surgery . Using AAV6 in an ovine model, the suggested approach resulted in a global transgene expression, that was cardiac-tropic and substantially more robust and targeted, compared to that of intramuscular or intracoronary injection. Furthermore, a randomized phase I/II AAV1-based clinical trial for heart failure treatment was conducted in 2013, where the researchers used a sarcoplasmic reticulum calcium ATPase gene (SERCA2a), the product of which was suggested to play a key role in HF pathology . As a result, adverse effects, including death, were found to be highest in the placebo group, and lowest in the high-dose group, with evidence of long-term transgene expression. However, in the low-dose and mid-dose groups, adverse effects were also found to be high but delayed. Hemophilia, an inherited disease that is caused either by deficiency of blood clotting factor VIII (type A) or IX (type B), is another example of diseases that have been targeted by AAV-mediated gene therapy . rAAV6 and 8 expressing the canine gene of FVIII have been shown to restore physiologic levels of the deficient factor in a canine model for three years following intravenous administration, without any toxicity or immune reactions . The same vectors could result in a similar effect in the murine model; however, neutralizing antibodies against cFVIII were detected in the mice sera . As for hemophilia B, intravenous administration of rAAV8 and 9 carrying the IX factor gene in a murine model resulted in a significant increase in transgene expression and therefore in IX factor levels, with a decreased proinflammatory risk . 5.5. AAV Viral Vectors for Gene Therapy of Liver Diseases AAV-based gene therapy plays a significant role in liver diseases, as it is, in some cases, an alternative to the only effective therapy, which is liver transplantation . In addition to the previously mentioned diseases affecting the liver, that have been targeted by AAV-base gene therapy, such as CF, AAT deficiency, and hemophilia, there are other liver diseases that have been targeted and continue to be, using AAV8, mainly. Wilson's disease (WD) is a rare autosomal recessive disease caused by mutations in the copper transporter gene, ATP7B, resulting in copper accumulation mainly in the liver, along with some other organs . A recent in vivo study, conducted by Murillo et al., proved the efficacy of an AAV-based gene therapy for WD in a murine model of the disease . The vector used in the study was AAV8 carrying an optimized short version of the ATP7B gene (AAV8-mini ATP7B) adjusted to the right size, so it could be produced and delivered more efficiently. As a result, intravenous administration of the recombinant vector to 12-week-old WD mice could restore copper homeostasis, with 20% hepatocyte transduction being sufficient for correction. Crigler-Najjar syndrome (CNs) type 1 is another autosomal recessive liver disease caused by deficiency of uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1), which leads to severe inherited unconjugated hyperbilirubinemia . There is currently an ongoing clinical trial investigating AAV8-UGT1A1 as a vector for gene therapy in children with CNs ; however, as the preclinical data of the trial suggest a negative effect of liver cells' proliferation on the efficacy of gene therapy in children, Shi et al., suggested to optimize the approach by starting therapy at a specific age and combining it with an immune suppression regimen . In their in vivo study, Shi et al., found that a stable correction was achieved when AAV8-UGT1A1 was administered to CNs rats at the 28th postnatal day, coupled with a rapamycin-based immunosuppression regimen delivered intraperitoneally. 5.6. AAV Viral Vectors for Gene Therapy of Endocrine Disorders Being a major health issue for humans all around the world, with relatively high prevalence estimates, endocrine disorders have always been in the focus of research for therapeutic approaches and techniques . Accordingly, rapid developments of diagnostic techniques, along with the better understanding of endocrine disorders' pathophysiology and molecular bases, helped in developing different therapies, such as hormonal replacement therapies, for example . However, as these approaches have mostly focused on improving the patient's quality of life, reducing or reversing symptoms, rather than targeting the underlying defect, the resulting effect has not always been sufficient, which highlighted the need for gene therapy approaches, including AAV-based ones . Type 1 diabetes mellitus (T1DM) is one of the endocrine autoimmune disorders that has been extensively studied as a target of gene therapy . T1DM is characterized by self-destruction of insulin-secreting islet b cells, resulting from a wide variety of causative factors . In non-obese diabetic (NOD) mice, administration of recombinant adeno-associated virus serotype 8 carrying DNA of mouse insulin promoter (dsAAV8-mIP) has been shown to prevent hyperglycemia in a dose-dependent manner . High levels of mouse interleukin 10 (mIL-10) achieved following rAAV2-IL-10 intramuscular administration also proved to have a positive effect in NOD mice by decreasing autoimmunity, and thereby hyperglycemia . Similarly, negative regulation of the immune response by programmed death ligand 1 (PDL1) was achieved in NOD mice following intraperitoneal delivery of AAV8-PDL1, which protected b cells . Currently, there is an ongoing clinical trial investigating AAV8 containing a transgene for the fragment antigen-binding region of anti-vascular endothelial growth factor (anti-VEGF fab), as a vector for gene therapy of diabetic retinopathy, delivered in the suprachoroidal space . Autoimmune polyglandular syndrome type-1 (APS1) is another disorder characterized by multiple endocrine abnormalities, resulting from a monogenic defect in the autoimmune regulator (AIRE) gene . Recently, Almaghrabi tested AAV9-AIRE as a potential gene therapy for APS1 . As a result, AAV9-AIRE, following intra-thymic administration in a murine model, showed high transduction efficiency, along with restoration of AIRE expression in the thymus. Subsequently, a significant reduction of serum auto-antibodies was detected in treated mice, with a relatively normal tissue morphology showing no lymphocytic infiltrations. 5.7. AAV Viral Vectors for Gene Therapy of Cancer Despite the variety of therapeutic approaches developed so far for cancer, including chemotherapy, radiotherapy, surgery, and different medications, cancer is still a huge health issue and a leading cause of death worldwide . Several AAV-mediated cancer gene therapy approaches have been reported so far, including suicide gene, RNA-interference, and anti-angiogenesis gene therapies . In AAV-mediated suicide gene therapy, an AAV-based system with herpes simplex virus type-1 thymidine kinase and ganciclovir (AAVtk/GCV) has been used. This system was found to exhibit a significant in vitro and in vivo tumor-suppressor efficacy in human head and neck cancer xenografts in a murine model , and murine models of bile duct cancer and bladder carcinoma . As for AAV-mediated RNA-interference cancer gene therapy, an AAV vector is used to provide a stable expression of short hairpin-structured RNA (shRNA) for gene knockdown applications . This approach could induce a strong androgen receptor (AR) gene silencing in a murine model of prostate cancer following intravenous administration . It has also been tested in a murine model of hepatocellular carcinoma and was found to induce tumor-specific apoptosis, resulting in a significant inhibition of cancer cell proliferation without toxicity . AAV-mediated anti-angiogenesis cancer gene therapy targets factors that can regulate a cancer cell angiogenesis process. VEGF, being an important factor for angiogenesis, has been targeted in a murine model of breast carcinoma using AAV2 carrying transgene of VEGF Trap protein (a pseudo high-affinity receptor) . The approach resulted in significant suppression of tumor growth and prevention of spontaneous pulmonary metastases. Another factor that has been targeted by this approach is pigment epithelium-derived factor (PEDF), a highly potent angiogenesis inhibitor. In a murine colorectal peritoneal carcinomatosis model, AAV2 carrying the human gene of PEDF was found to induce significant tumor suppression, inhibit metastases, and prolong survival time . Similarly, kallistatin is another anti-angiogenesis factor that has been targeted using an AAV-mediated murine model of hepatocellular carcinoma, and it provided a similar efficacy as AAV-kallistatin-induced potent cancer cell apoptosis . 6. Challenges and Limitations of AAVs for Gene Therapy AAV-Mediated Applications Despite the many advantages AAVs provide as viral vectors for gene therapy of various diseases, and the many improvements and molecular modifications they have undergone since their first discovery, AAVs still have some limitations that can result in impeding impacts on their applications in some cases. One example of such major limitations is the limited cloning capacity of AAVs, that is ~4.5 Kb . Moreover, AAVs need to converse into double-stranded DNA, and may cause a delay in transgene expression following delivery, compared to double-stranded vectors . Although it can be dose-dependent in some cases, preexisting immunity against some serotypes can also be a limiting factor . Preexisting immunity, or an immunity response occurring after the first introduction of a viral vector, can be a major obstacle in the development of gene therapy approaches based on AAV or other viral vectors, and their application in clinical practice. Neutralizing antibodies are often found in the human population with a high prevalence that varies depending on the virus serotype and the geographic location of the population. Moreover, in a number of diseases, multiple administration of an AAV-based drug may be required, which can result in developing an immune response against the viral vector. Neutralizing antibodies can have a significant impact on the efficiency of gene transfer in gene therapy. Consequently, many patients with genetic diseases will not be able to benefit from such gene therapy medications. Furthermore, although AAVs have not been linked to human pathologies or diseases, wild-type AAVs are widely distributed in the human population, and despite having a low immunogenicity profile, an immune response was observed in humans as a result of gene therapy using AAVs . To date, the impact of such anti-AAV immune response on gene therapy is still not entirely understood, however, its mechanisms are being actively explored. For example, systemic delivery of AAV vectors, while having neutralizing antibodies, has been shown to result in accumulation of vector genomes in lymphoid organs. Whereas the efficiency of liver transduction while having binding antibodies (indicative of a previous infection, but not neutralizing the virus), on the contrary, increases . The role of neutralizing antibodies in activating the complement system has also been studied . Individuals seropositive for AAV have high titers of IgG1, IgG2, and IgG3. It is known that IgG1, IgG2, and IgM highly correlate with the level of neutralizing antibodies, and IgG1 levels increase after gene therapy with AAV . Additionally, IgG3 correlates with the level of T-cell reactivity against AAV vectors . From a clinical point of view, neutralizing antibody titers are of a great significance, as having neutralizing antibodies against a specific AAV serotype highly affects gene therapy effectiveness upon using this serotype. Although methods for detecting neutralizing antibodies are poorly standardized, existing research data indicate that the prevalence of neutralizing antibodies against AAV2 is about 40% . It has been shown that neutralizing antibodies can trigger elimination of the vector by the immune system. At the same time, viral vectors can accumulate in the spleen, not reaching the liver . Therefore, researching mechanisms to establish and develop effective methods for preexisting immunity assessment can help overcome many problems arising in gene therapy applications. Tackling such an urgent problem can expand the possibilities of therapy for many incurable human hereditary diseases . Another challenge is the need to deeply understand the exact mechanisms and pathways of AAV cellular uptake, trafficking, and transduction, which are still unknown for most AAV serotypes . Such understanding can help in addressing and explaining the changes of transduction potency and efficacy that can be found between different species in different in vivo studies, or between treatment efficacy in vivo and in clinical trials . In other words, differences between humans and various animal models in terms of body size, genomic and anatomic specificity, immunogenicity, and disease process should be taken into account, as they can affect the effective translation of preclinical investigations . cells-12-00785-t001_Table 1 Table 1 Characteristics of different AAV serotypes. AAV Serotype Primary Receptor Other/Coreceptors Post Translational Modifications Recommended Purification Method Tropism AAV1 Sialic acid AAV receptor (AAVR) - iodixanol gradient centrifugation, anion-exchange chromatography, and mucin column affinity chromatography Skeletal muscles , heart , glial and ependymal cells in the murine brain , endothelial and vascular smooth muscles , retina AAV2 HSPG FGFR1, aVb5 and a5b1 integrins, HGFR, LR, and CD9 ubiquitination, phosphorylation, SUMOylation, and multiple-site-glycosylation heparin column affinity chromatography renal tissue , hepatocytes , retina , non-mitotic cells of central nervous system (CNS) , and skeletal muscles AAV3 HSPG FGFR1, LR, and HGFR acetylation, phosphorylation, and glycosylation iodixanol gradient centrifugation human liver cancer cells as well as human and NHP hepatocytes , murine cochlear inner hair cells . AAV4 Sialic acid - ubiquitination ion-exchange chromatography, mucin column affinity chromatography ependymal cells of mammalian CNS , RPE cells of the retina (canine, rodent, and NHP origins) , murine kidney, lung, and heart cells . AAV5 Sialic acid PDGFR (a and b) ubiquitination, phosphorylation, SUMOylation, and glycosylation ion-exchange chromatography, mucin column affinity chromatography murine: retinal cells , mainly photoreceptors , airway epithelia , liver cells , vascular endothelial cells and smooth muscles , and neurons (murine and NHP) AAV6 Sialic acid and HSPG EGFR acetylation heparin or mucin column affinity chromatography airway epithelia of murine and canine models , murine liver cells , skeletal muscles of murine and canine models , cardiomyocytes in murine , porcine , canine , and in sheep models. AAV7 - - glycosylation, phosphorylation, SUMOylation, and acetylation - murine skeletal muscle cells , murine and human hepatocytes , murine and NHPs CNS , murine photoreceptor cells , murine vascular endothelial cells (limited tropism) , murine epicardium cells . AAV8 LR - phosphorylation, glycosylation, and acetylation Dual-ion-exchange chromatography, iodixanol gradient centrifugation murine, canine, and 115 hepatocytes , murine skeletal and cardiac muscles , murine pancreatic cells , murine renal cells , and different cells in the murine retina AAV9 terminal N-linked galactose putative integrin, LR glycosylation, ubiquitination, phosphorylation, SUMOylation, and acetylation Sucrose gradient centrifugation, and ion-exchange chromatography murine, NHP, and feline neuronal and non-neuronal cells, including astrocytes , murine and NHP retinal photoreceptors cells , murine, NHP, and porcine cardiac muscle tissue , murine hepatocytes, skeletal muscles, and pancreatic cells , photoreceptor cells , renal tubular epithelium cells , Leydig cells in the testicular interstitial tissue , and alveolar and nasal epithelia . AAV10 - - glycosylation, ubiquitination, phosphorylation, SUMOylation, and acetylation iodixanol gradient centrifugation NHP intestinal cells, hepatocytes, lymph nodes, and less frequently, renal cells and adrenal glands , murine small intestine and colon cells , retinal cells (including RPE, cells in the ganglion cell layer, several cell types in the inner nuclear layer, photoreceptors, and a highly efficient transduction of horizontal cells) , murine liver cells, lung cells , renal, and pancreatic cells . AAV11 - - - iodixanol gradient centrifugation NHP intestinal cells, hepatocytes, lymph nodes, and less frequently, renal cells and adrenal glands , murine projection neurons and astrocytes , and mild tropism to NHP CNS (cerebrum and spinal cord, mainly) . AAV12 mannose and mannosamine have been suggested as components of a potential receptor complex - AVB Sepharose affinity chromatography murine salivary glands and muscles , murine nasal epithelia (mainly after intranasal administration) . AAV13 - HSPG - iodixanol gradient centrifugation, heparin column affinity chromatography - cells-12-00785-t002_Table 2 Table 2 Preclinical and clinical studies using AAVs as viral vectors for gene therapy of different diseases. Gene Therapy Target Disease ** AAV Viral Vector Study Type Outcome Ref. CNS PD AAV2-GAD phase I clinical trial for advanced PD patients safe and well-tolerated approach, providing significant improvement in motor function scores up to 12 months after unilateral subthalamic delivery double-blind, controlled, randomized clinical trial for advanced PD patients safe and well-tolerated approach, along with improved motor function scores following bilateral subthalamic delivery AAV2-neurturin open-label clinical trial for PD patients suggested feasibility, safety, and good tolerance of the approach rAAV2-AADC clinical trial for moderately advanced PD patients good tolerance, improvement on motor rating scales, however, accompanied with an increased risk of intracranial hemorrhages and headache good tolerance and a stable expression of the transgene that lasted for the following 4 years, although higher vector doses were suggested for further studies AADC deficiency rAAV2-AADC open-label, phase I/II trial in children good tolerance in general, with evidence for potential improvement of motor function SMA AA9-SMN open-label, phase I clinical trial significant improvement of the motor function in all 15 patients following single-dose intravenous administration, reflected by their ability to perform different activities, such as unassisted sitting and walking, oral feeding, and speaking, with no reported motor function regression at two-year follow-up. A long-term safety assessment, however, was recommended. LSD MPS VII (rAAV-GUSb) in vivo study, murine model stable expression of the deficient enzyme upon single administration, that was adequate for phenotype correction in the liver (rAAV-GUSb) in vivo study, neonatal murine model therapeutic levels of the deficient enzyme in multiple organs, including ones of the CNS, with the gene expression not being affected by rapid growth and differentiation of tissues rAAV9-GUSb and rAAVrh10-GUSb in vivo study, canine model significantly high expression levels of GUSb in the CNS tissues, with the enzyme in brain tissue homogenates showing over 100% normal activity MPS IIIA AAV9-Sgsh (canine sulfamidase gene) in vivo study, canine model sustained and widely distributed transgene expression with no toxicity for a duration of 7 years after therapy MLD AAV1-ARSA in vivo study, murine model significant elevation of ARSA levels and activity, resulting in reduction of accumulated sulfatides AAV5-ARSA in vivo study, murine model rapid, abundant, and sustained restoration of ARSA levels in the brain and brainstem up to 15 months after administration, reduction of accumulated sulfatides, and preservation of neurologic function. AAV5-ARSA in vivo study, NHP model good tolerance, distribution of the transgene in the brain with elevated activity of the deficient enzyme AAV9-ARSA in vivo study, neonatal murine model global expression of the transgene in the brain and spinal cord, along with muscles and heart, inhibition of sulfatide accumulation, and improvement of neurologic/motor function AAVrh10-ARSA AAVrh10-ARSA AAVrh10-ARSA in vivo study, murine model correction of sulfatide accumulation following single administration, with a transduction efficacy higher than that of AAV5 as it transduced both neurons and oligodendrocytes in vivo study, NHP model good tolerance, neuroinflammation 3 months following the fifth dose but none after the first, detection of transgene expression after the first dose along with increased ARSA activity, and detection of the enzyme in other organs but not in gonads clinical trial for children with asymptomatic or early-stage MLD significant elevation in ARSA levels in the cerebrospinal fluid (CSF) following intracerebral vector delivery, but no clinical improvement has been noticed compared to the control group. GM2-gangliosidose rAAV2-HEXA + rAAV2-HEXB in vivo study, murine model delay of disease clinical onset, maintained motor function, good tolerance, stable and abundant levels of the deficient enzyme, resulting in a significant reduction of gangliosides' storage in the CNS AAVrh8-HEX in vivo study, feline model prevention or reduction of tremors that is characteristic of improvement in the neurologic function, and a signification elevation of the deficient enzyme levels (HEX), resulting in a significant reduction of gangliosides' storage in different tissues of the CNS. AAVrh8-HEXA + AAVrh8-HEXB in vivo study, ovine model delay of disease clinical onset and progression, improved neurologic function and clinical biomarkers. However, lifespan was not significantly higher. AAVrh8-HEXA + AAVrh8-HEXB first clinical trial for children with TSD good tolerance and broad distribution of the transgene in the CNS CD rAAV2-ASPA in vivo study, murine model increased ASPA activity and, thereby, decreased NAA accumulation and white matter degeneration. However, areas remote from injection site, such as the cerebellum, were not affected GLD rAAV1-GALC in vivo study, murine model sustained expression of the deficient enzyme, improved myelination status, and prolonged lifespan. However, both treated and untreated mice died with similar symptoms, suggesting that the used approach should be initiated prior to symptoms' onset. rAAV2/5-GALC in vivo study, murine model wide dispersion of GALC transgene across the CNS reaching areas remote from the injection site, reduced loss of oligodendrocytes and Purkinje cells, along with a significant improvement of neuromotor function and a prolonged lifespan of treated mice. AAV9-GALC AAVrh10-GALC AAVOlig001-GALC in vivo study, murine model All three serotypes provided wide distribution of the transgene across the CNS and liver, resulting in a significant improvement of myelination, and a prolonged lifespan, with AAV9 being the most effective when combined with bone marrow transplantation. AAVrh10-cGALC in vivo study, canine model delayed symptoms onset, prolonged lifespan, correction of biochemical defects, and a positive effect on neuropathology in treated animals . AAV9-cGALC in vivo study, canine model increased activity of the deficient enzyme and, therefore, normal levels of its substrate, improved myelination, and decreased inflammation both in the CNS and PNS, which, along with prevention of clinical neurological dysfunction, resulted in a significantly prolonged lifespan of treated dogs, compared to the control group. However, sufficient dosing was found to be critical, as high doses significantly extended the lifespan even for post-symptomatic subjects, and a 5-fold lower dose of the vector resulted in an attenuated form of disease . Retinal degeneration and ocular neurovascular diseases AAV2, 8, and 9 in vivo study, C57BL/6 mice transduction of retinal cells, as well as efficient transduction of ganglion cell layer by AAV8 and AAV9 Inherited photoreceptor diseases hybrid serotypes AAV2/7 and AAV2/8 in vivo study, C57BL/6 mice high transduction rates of the murine photoreceptors Retinal blindness caused by LCA AAV2/6 in vivo study, wild-type 129Sv/Ev (Taconic) mice efficient transduction of murine cone photoreceptors following subretinal injection AAV2/ clinical trial for patients with LCA systemic and local good tolerance with no adverse effects, along with improvement of visual function presented by different parameters in different patients, including improvement of visual acuity and color vision, as well as reduction of visual fatigue or photophobia, over a follow-up period between 1 and 3 years Retinal blindness caused by choroideremia phase I/II clinical trial improvement of retinal sensitivity in all six patients, following subretinal vector delivery, with two of them having significant increases in visual acuity, supporting further consideration of the tested therapeutic approach. RPE65-mediated inherited retinal dystrophy AAV2-hRPE65v2 open-label, randomized, controlled phase III trial good tolerance with no adverse effects, restoration of RPE65 enzymatic activity reflected by significant and sustained improvement in light perception and navigational abilities Leber Hereditary Optic Neuropathy (LHON) rAAV2-ND4 (gene encoding nicotinamide adenine dinucleotide dehydrogenase subunit IV) open-label, phase I/II randomized clinical trial good tolerance, although a mild, intraocular inflammation was detected after vector administration, but it was responsive to treatment and suggested to be overcome in further studies by systemic vector delivery instead of local. open-label, phase I clinical trial minor adverse events, improvement in visual activity in some but not all patients, no detection of vector DNA in patients' blood samples RPGR-related X-linked retinitis pigmentosa AAV8-RPGR phase I/II clinical trial no adverse effects other than steroid-responsive subretinal inflammation following administration of higher doses, sustained improvements in visual function in 6/18 patients. X-Linked Retinoschisis AAV8-RS1 (Retinoschisin gene) phase I/IIa single-center, open-label, clinical trial good tolerance in general, although steroid-responsive, dose-related inflammation was observed, and a dose-related increase of systemic antibodies against AAV8, but none against RS1 CNGA3-linked achromatopsia AAV8-CNGA3 nonrandomized controlled clinical trial no substantial safety issues, successful targeting of cone photoreceptors reflected by reported improvement of color vision ability, along with improvements in visual acuity and contrast sensitivity (although a cause-effect relationship was not established) Hearing disorders AAV9 in vivo study, porcine model persistent expression of the transgene within the mammalian inner ear following intracochlear delivery Respiratory organs (airway epithelia) CF AAV2-CFTR in vivo study-rabbits efficient and stable gene transfer of CFTR into airway epithelium, indicating, as a result, the vector potential to be used for gene therapy phase I clinical trial for CF patients safety, successful transduction of targeted cells, and a detected function restoration of sinuses. phase II, double-blind, randomized, placebo-controlled clinical trial for CF patients safety and good tolerance, but no effective clinical treatment of disease was achieved phase I clinical trial for mild CF patients safety, but no effective clinical treatment of disease was achieved a1AT deficiency rAAV-hAAT in vivo study, intravenous delivery into murine model Serum levels of AAT that are potentially therapeutic rAAV2-hAAT and rAAV5-hAAT in vivo study, intrapleural vs. intramuscular delivery into murine model AAT lung and serum levels higher than the ones achieved by intravenous delivery, with rAAV5 showing 10-fold higher effectiveness than rAAV2 rhAAV10-hAAT in vivo study, intrapleural delivery into murine and NHP models safety, and persistent expression of the transgene in the chest cavity cells of both models rAAV6/2-hAAT in vivo study, intratracheal delivery into C57/Bl6 mice, and in vitro study, cultures of human airway epithelial cells lung cells' transduction, even more efficient than rAAV5 both in vivo and in vitro rAAV8-hAAT in vivo study, intratracheal delivery into C57/Bl6 mice lung cells' transduction superior to that of rAAV5, as it resulted in 6-fold and 2.5-fold higher AAT levels in serum and broncho-alveolar fluid, respectively. rAAV2/8-hAAT in vivo study, intravenous delivery into C57/Bl6 mice high transduction rate of hepatocytes rAAV1-AAT in vivo study, intramuscular delivery into C57/Bl6 mice and rabbits dose-dependent inflammatory infiltrates at injection sites not affecting expression of transgene, along with dose-dependent detection of vector DNA in most animals, both at injection sites and in distal organs rAAV2/9-AAT in vivo study, intratracheal delivery into C57/Bl6 mice efficient, relatively stable transduction of alveolar and nasal epithelia, that was not affected by high levels of neutralizing antibodies upon following re-administration rAAV6-hAAT in vivo study, nasal delivery into C57/Bl6 mice, and intratracheal delivery into canine model therapeutic hAAT concentrations in both studied animal models, with levels being higher in lungs than serum, accompanied with an immune response against the vector capsid in some animals despite being immunosuppressed Allergic asthma rAAV-IL-4 in vivo study, intratracheal delivery into Balb/cByJ mice significant inhibition of airway eosinophilia and mucus production along with a reduction in airway hyper-responsiveness and asthma-associated cytokine levels SPB rAAV6/2-SFTPB in vivo study, intratracheal delivery into murine model efficient transduction of airway and alveolar epithelium, good tolerance of administered vector with no adverse effects, rapid and long-term restoration of the deficient SPB protein, along with an improvement of lung function, leading, subsequently, to an extended survival Muscles Degenerative muscle disorders AAV1-FS344 (follistatin gene) in vivo study, NHP model safety and good tolerance of administered vector, promotion of sustained improvement in muscle size and strength DMD rAAV6-micro-dystrophin in vivo study, dystrophin/utrophin double-knockout murine model no serious adverse events, improvement of muscle function along with prolongation of lifespan, explained by sustained restoration of deficient protein (dystrophin) in the respiratory, cardiac, and limb muscles rAAV1-mini-dystrophin in vivo study, dystrophin/utrophin double knockout murine model prolongation of lifespan, highly efficient expression of transgene, improvement in muscle histopathology and function, reflected by improved growth and motility, along with prevention of spine and extremities' deformation AAV2.5-mini-dystrophin Randomized, double-blind, placebo-controlled phase I clinical trial safety and good tolerance of the vector, and detection of transgene DNA in all patients following intramuscular administration AAV9-micro-dystrophin phase I/II open-label, randomized, controlled clinical trial safety and successful expression of transgene AAV-rh74-micro-dystrophin phase I/II, open-label, non-randomized clinical trial safety and successful expression of transgene, along with improvement of motor function LGMD rAAV8-hd-SG (human d-sarcoglycan gene) in vivo study, murine model prolongation of lifespan, efficient and sustained transduction of cardiac and skeletal muscles along with improvement in their histopathology and function rAAV1-ha-SG in vivo study, murine model efficient and sustained expression of the transgene, histological and functional improvement of musculature reflected by correction of contractile force deficits and stretch sensibility, along with increase of animal activity rAAV-hg-SG in vivo study, murine model significantly efficient transduction of muscle fibers, and general histopathological improvement, that were achieved only upon early intervention Cardiovascular system HF AAV6-EGFP in vivo study, ovine model global expression of the transgene, that was cardiac-tropic and substantially more robust and targeted, compared to that of intramuscular or intracoronary injection. AAV1-SERCA2a randomized phase I/II clinical trial highest adverse effects, including death, in the placebo group, and lowest in the high-dose group, with evidence of long-term transgene expression. However, in the low-dose and mid-dose groups, adverse effects were found to be high but delayed. Hemophilia A rAAV6-cFVIII and rAAV8-cFVIII in vivo study, murine and canine models restoration of physiologic levels of the deficient factor in the canine model for three years following intravenous administration, without any toxicity or immune reactions. A similar effect in the murine model was found, however, with detection of neutralizing antibodies against cFVIII in the mice sera Hemophilia B rAAV8-FIX and rAAV9-FIX in vivo study, murine model significant increase in transgene expression and therefore in IX factor levels, with decreased proinflammatory risk following intravenous administration Liver WD AAV8-mini ATP7B in vivo study, murine model restoration of copper homeostasis, with 20% hepatocyte transduction being sufficient for correction CNs ongoing clinical trial - in vivo study, murine model stable correction was achieved when the vector was administered at the 28th postnatal day, coupled with a rapamycin-based immunosuppression regimen delivered intraperitoneally Endocrine system T1DM dsAAV8-mIP in vivo study, murine model prevention of hyperglycemia in a dose-dependent manner rAAV2-IL-10 in vivo study, murine model a positive effect, decreasing autoimmunity, and thereby hyperglycemia AAV8-PDL1 in vivo study, murine model pancreatic b cells' protection AAV8-anti-VEGF fab ongoing clinical trial for diabetic retinopathy patients - APS1 AAV9-AIRE in vivo study, murine model high transduction efficiency, along with restoration of AIRE expression in the thymus, following intra-thymic administration, and a subsequent significant reduction of serum auto-antibodies, with relatively normal tissue morphology showing no lymphocytic infiltrations. Cancer laryngeal cancer cell line (HEp-2) AAVtk/GCV system in vitro and in vivo study, murine model significant tumor-suppressor efficacy in human head and neck cancer xenografts, and markedly prolonged survival bile duct cancer AAVtk/GCV system in vivo study, murine model increased anti-tumor effect upon simultaneous administration with 5-fluorouracil, and better survival bladder carcinoma AAVtk/GCV system in vivo study, murine model control of tumor cell growth, a strong anti-tumor efficacy prostate cancer AAV2-ARHP8 in vivo study, murine model strong androgen receptor (AR) gene silencing following intravenous administration hepatocellular carcinoma AAV8-miR-26a in vivo study, murine model Induction of tumor-specific apoptosis, resulting in a significant inhibition of cancer cell proliferation without toxicity breast carcinoma AAV2-VEGF Trap in vivo study, murine model significant suppression of tumor growth and prevention of spontaneous pulmonary metastases colorectal peritoneal carcinomatosis AAV2-hPEDF in vivo study, murine model significant tumor suppression, inhibition of metastases, and prolonged survival time hepatocellular carcinoma AAV-kallistatin in vivo study, murine model induced potent cancer cell apoptosis, and prolonged survival time ** Disease abbreviations: PD: Parkinson's disease, AADC deficiency: amino acid decarboxylase deficiency, SMA: spinal muscular atrophy, MPS: mucopolysaccharidosis, MLD: metachromatic leukodystrophy, CD: Canavan's disease, GLD: globoid cell leukodystrophy, LCA: Leber congenital amaurosis, CF: cystic fibrosis, a1AT deficiency: Alpha-1 antitrypsin deficiency, SPB deficiency: surfactant protein B deficiency, DMD: Duchenne muscular dystrophy, LGMD: Limb-Girdle muscular dystrophies, HF: heart failure, WD: Wilson's disease, CNs: Crigler-Najjar syndrome, T1DM: Type 1 diabetes mellitus, APS1: autoimmune polyglandular syndrome type-1. 7. Conclusions AAVs were discovered over five decades ago and have since represented a potent tool for gene therapy, that still needs to be better understood and developed for broader and larger therapeutic applications. Further optimizations should cover vector design, tropism modifications, and delivery routes. In this article, we have covered different serotypes of adeno-associated viruses and their applications in gene therapy for different diseases in preclinical and clinical studies, with a brief overview of AAVs' limitations and challenges. Acknowledgments We thank the Center for Precision Genome Editing and Genetic Technologies for Biomedicine, IGB RAS, for scientific advice on development of gene therapy drugs. Author Contributions Conceptualization, S.S.I., A.A.S. and V.V.S.; writing--original draft preparation, S.S.I. and A.A.S.; writing--review and editing, V.V.S.; supervision, A.A.R. All authors have read and agreed to the published version of the manuscript. 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PMC10000784 | Foods Foods foods Foods 2304-8158 MDPI 10.3390/foods12050971 foods-12-00971 Review Insights into Ultrasonication Treatment on the Characteristics of Cereal Proteins: Functionality, Conformational and Physicochemical Characteristics Wang Yang 1 Liu Jiarui 1 Zhang Zhaoli 1* Meng Xiangren 1 Yang Tingxuan 1 Shi Wangbin 1 He Ronghai 2* Ma Haile 2 Silva Filipa V. M. Academic Editor 1 College of Tourism and Cooking & College of Food Science and Engineering, Yangzhou University, Yangzhou 225127, China 2 School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, China * Correspondence: [email protected] (Z.Z.); [email protected] (R.H.); Tel.: +86-(511)-8878-0174 (R.H.) 24 2 2023 3 2023 12 5 97108 2 2023 22 2 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). Background: It would be impossible to imagine a country where cereals and their byproducts were not at the peak of foodstuff systems as a source of food, fertilizer, or for fiber and fuel production. Moreover, the production of cereal proteins (CPs) has recently attracted the scientific community's interest due to the increasing demands for physical wellbeing and animal health. However, the nutritional and technological enhancements of CPs are needed to ameliorate their functional and structural properties. Ultrasonic technology is an emerging nonthermal method to change the functionality and conformational characteristics of CPs. Scope and approach: This article briefly discusses the effects of ultrasonication on the characteristics of CPs. The effects of ultrasonication on the solubility, emulsibility, foamability, surface-hydrophobicity, particle-size, conformational-structure, microstructural, enzymatic-hydrolysis, and digestive properties are summarized. Conclusions: The results demonstrate that ultrasonication could be used to enhance the characteristics of CPs. Proper ultrasonic treatment could improve functionalities such as solubility, emulsibility, and foamability, and is a good method for altering protein structures (including surface hydrophobicity, sulfhydryl and disulfide bonds, particle size, secondary and tertiary structures, and microstructure). In addition, ultrasonic treatment could effectively promote the enzymolytic efficiency of CPs. Furthermore, the in vitro digestibility was enhanced after suitable sonication treatment. Therefore, ultrasonication technology is a useful method to modify cereal protein functionality and structure for the food industry. ultrasonication cereal proteins functionality conformational physicochemical characteristics National Natural Science Foundation of China32102134 Key R & D plan of Shandong Province2022CXGC010603 Jiangsu Agriculture Science and Technology Innovation FundCX(22)3073 Yangzhou University High-level Talent Research Fund Project137012132 Jiangsu University Student Training Program202211117088Y This research was funded by National Natural Science Foundation of China (32102134), Key R & D plan of Shandong Province (2022CXGC010603), Jiangsu Agriculture Science and Technology Innovation Fund (CX(22)3073), and Yangzhou University High-level Talent Research Fund Project (137012132), Jiangsu University Student Training Program (202211117088Y), and sponsored by a project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). pmc1. Introduction The global population is increasing and is forecasted to reach 10 billion by 2050. To meet the growing population, cereals, one of the most basic foodstuffs around the world, provide energy and nutrients to the global population . Wheat, rice, barley, rye, oat, maize (corn), millet, and sorghum are the most common staple cereals . The fact that cereals is a larger share of sustainable agriculture adds to the importance of cereal and cereal-based products. Cereal provides considerable carbohydrates, protein, dietary fiber, and bioactive nutrients that our bodies require for growth and metabolism . Cereal proteins (CPs), a prior micronutrient of cereal, are gaining more attention from scholars working in the food industry because of their nutritional benefits and functional components. CPs are primarily applied in food processing owing to their nutritious values and functional characteristics . They are available for solubility, foaming, emulsifying, and gelling applications . CPs, particularly gluten, are utilized in many products and byproducts, such as leavened and unleavened bread, noodles, and cookies . However, the characteristics of proteins must be stable during mixing, heating, desiccating, storage, and physical, chemical, or enzymatic modifications, maintaining their structure and not damaging their physicochemical properties . With the increasing demands of consumers for nutritious foodstuffs, emerging pretreatment technologies have been applied to food science such as ultrasonication, high-pressure processes, pulsed electric fields, microwaves, irradiation, low-temperature plasma, and radio frequencies. Studies such as these have drawn increasing attention . These technologies play a critical role in maintaining food quality and safety with high nutritional merits and minimal damage over conventional food-processing techniques . Ultrasonic technology has attracted much attention in the food industry, mainly because it is considered an innovative technology to achieve higher-quality and -functionality processed foods with ecofriendly chemistry and cost-effective industrialization . Ultrasound is a mechanical wave at a higher frequency than that of the level of human hearing (>16 kHz) that can be categorized into two types, low-intensity high-frequency, and high-intensity low-frequency ultrasound. Low-intensity high-frequency ultrasound (100 kHz-1 MHz, power < 1 W/cm2) is most often used for nondestructive analytical food testing, quality evaluation, and process control to ensure the quality and safety of food. High-intensity low-frequency ultrasound (18 kHz-100 MHz, power > 1 W/cm2), on the other hand, is mainly used to physically or chemically alter the properties of food in food processing such as extraction, protein enzymolysis, peptides, and modifications . Therefore, the ultrasonic technique is commonly utilized in food processing as it is a proven effective approach to modify protein properties via cavitation . Studies were conducted on the influence of sonication on the physicochemical characteristics and structural features of CPs . However, there has been no a systematic review concerning this subject. Therefore, the objective of this review is to winnow all the eligible articles from different databases via appropriate keywords and a comprehensive search. On the basis of the analytical and synthesis results, we summarize how sonication treatment affected the functional and structural characteristics of CPs. The review provides indepth information on sonication's influence in CPs research and guide the food industry in making strategic decisions. 2. Search Program and Selection Standards In this review, all related articles were retrieved through a comprehension search. There are several main hotspots related to ultrasound, ultrasonic practices, or ultrasonication in the databases with the subject classification of "food science technology", such as "protein", "high-intensity ultrasound", "functional properties", "structural characteristics", and "physical properties". On the basis of these articles, the co-occurrence network of numerous keywords in bibliographic data is exhibited from 2014 to 2022 via VOSviewer software . As shown in Figure 1, ultrasound has been widely used in the modification of CPs in recent years. All related studies were selected after examining their title, abstract, and full content, and ultimately on the basis of inclusion and exclusion criteria. Inclusion criteria were as follows: (1) they reported on the functional, structural, and physicochemical characteristics, including solubility, foaming and emulsification, surface-hydrophobicity, sulfhydryl group, particle-size, conformational-structure, microstructural, enzymolytic, and in vitro digestive properties; (2) ultrasonic treatment was involved; (3) published date from 2000 to 2022; (4) no language limitations; (5) they included CPs. Moreover, the following were the exclusion criteria: (1) ultrasonic treatment not mentioned; (2) CPs were omitted; (3) publishing date before 2000. There was evidence that different ultrasonic conditions, such as power density, frequency, temperature, amplitude, and sonication time, could lead to different results. Consequently, the final results were systematically analyzed and summarized. 3. Results and Discussions 3.1. Changes in the Functionality of CPs via Ultrasound 3.1.1. Solubility The solubility of CPs, as one kind of functionality, plays a major role in the quality of food products. The solubility of proteins refers to the extent to which proteins were denaturized and aggregated. This may impact other functional characteristics, such as foaming and emulsifying properties . Therefore, the other technofunctional characteristics of proteins and their application may be influenced by their solubility within the food industry. Reasonable ultrasonic conditions are used to enhance solubility up to a certain point. The influences of ultrasound on CP solubility can be ascribed to conformational variations in the (partial unfolding of the) protein structure caused by the implosion of cavitation bubbles. Physical perturbations help in breaking up hydrogen and hydrophobic bonds, exposing the buried hydrophilic groups to the surrounding water. Thus, the interaction between protein molecules and water is promoted, enhancing protein solubility . A study by Wang et al. demonstrated that, in comparison with the rice bran protein without ultrasonic treatment, the solubility of rice bran protein with ultrasound (20 kHz; 6 mm diameter titanium probe with a depth of 1 cm; URBP) had obviously been increased via the enhancement of ultrasonic power. The solubility of URBP reached the maximum when the ultrasonic power was 200 W and the ultrasonic time was 10 min. Other findings regarding ultrasonic treatment of CPs exhibited a homotrend of an increase in protein solubility, such as protein isolates (BPIs) , rice dreg protein isolates (RDPIs) , sorghum kafirin , and oat protein isolates (OPIs) , via various types of ultrasonic devices. The decrease in the particle size and partial unfolding of proteins during ultrasonication increased the charged groups on the surface of the protein. This phenomenon also has significant influence on protein-water and protein-protein interactions via hydrogen bonds, hydrophobicity, and electrostatic forces, and as a result, causes protein dispersion and increases protein solubility. Figure 2 presents an illustration of the cavitation effect. It is also essential for maintaining a consistent temperature with ultrasonication when investigating protein dissolution . Similar results utilizing sonication are reported in Table 1. Nevertheless, studies have demonstrated that the solubility of CPs significantly decreases when they are exposed to excessive high-intensity ultrasound. For example, Chen et al. observed the impact of the solubility of rice protein (RP)-dextran conjugates treated with ultrasound-assisted glycation (URPDCs) (25 kHz; power: 400, 500, and 600 W). When the ultrasonic power increased (under 700 W), the solubility increased significantly. The solubility reached up to 88.5% at 600 W and then reduced at ultrasonic power of 700 W. Cavitation generated by ultrasonic treatment likely enhanced the proteins' solubility, whereas excessively high ultrasonic power could cause protein aggregation, restraining the reaction. Proteins were more susceptible to aqueous solutions because of the broken covalent bonds and more hydrophobic groups caused by acoustic cavitation. Therefore, larger aggregates are formed through hydrophobic bonds, destabilizing dissociated proteins and thus reducing solubility . The majority of the available literature on ultrasonication regards the frequency range of 20-50 kHz and moderate power under 700 W; ultrasonic treatment may effectively promote the solubility of CPs. 3.1.2. Emulsifying Properties Emulsifying properties play a crucial role in the application of proteins as surfactant substances. Because of the amphiphilic nature of proteins, the solid homogeneous emulsion of a protein can be established in an oil-water system via surface active agents , thus improving the emulsification attributes, including the emulsifying activity index (EAI) and emulsion stability index (ESI). In addition, the modification of CPs' emulsification properties dramatically influences the protein size, conformation, surface hydrophobicity, and molecule flexibility under an ultrasonic field . Modifications in the emulsifying characteristics of an isolated cereal protein caused by sonication were evaluated. Cavitation efficiency may affect emulsification during sonication. Zhang et al. significantly increased the EAI and ESI of wheat gliadin (WG) and green wheat gliadin (GG) treated with sonication with various power inputs. The increase in the emulsification characteristics of WG and GG via sonication was related to the smaller particles with the help of sonication acoustic cavitation. With the extension of acoustic time or/and ultrasonic power, the dispersed phase volume and bubble population increased, increasing the shear forces transferred through the rapid collapse of bubbles. Further, the disruption of oil droplets became more favorable, thereby strengthening emulsion stability . In another study, Hu et al. identified an association between protein secondary and tertiary structural variation, and improved the emulsifying properties. When a-helix and b-sheets are influenced by ultrasonication, the ultrasound-treated protein has a better effective potential for adsorption capacity on the interface of oil/water. The particle size was reduced with ultrasonication, which increased the ratio of the surface area to the volume, thus increasing the emulsification characteristics . Further, the proteins' surface hydrophobicity that is increased with ultrasonic treatment can also act as a driving force for a reduction in the tension on the oil-water interface. Hence, the protein absorption rate was increased, which helped in rendering the films rigid through hydrophobic/hydrophilic groups. As a result of these alterations, proteins treated with sonication may be more easily emulsified. Furthermore, high-intensity ultrasound can enhance emulsion homogenization by pretreating the proteins; it is widely applied in food processing. We show previous results from other authors in Table 1. The results show that ultrasonic treatment such as at a 20 or 25 kHz frequency and with power of 100 or 300 W could lead to a significant increase in emulsification efficiency. In contrast, excessive ultrasonic power input might cause a loss in the emulsification properties of the protein. Wang et al. found that the emulsification characteristics of rice bran protein decreased slightly as ultrasonic power increased between 450 and 600 W. The opposite might be attributed to the intensified sonochemical effects induced by excessive power disrupting the protein's secondary structure, resulting in the flocculation of interfacial proteins and the aggregation of emulsion droplets. Therefore, it is important to select the appropriate ultrasonic power and frequency levels for different CPs to maintain an equilibrium between the exposure of hydrophobic/hydrophilic groups and protein aggregation to achieve excellent emulsification performance. 3.1.3. Foaming Properties Foaming characteristics are largely determined via molecular movement, penetration, and rearrangement at the air/water interface, mainly applied in food processing. Foaming capacity (FC) depends on the protein dispersion, unfolding, and repositioning at the gas/solvent interface to decrease tension at the interface, while foaming stability (FS) is mostly dependent on the formation of a cohesive, robust layer around air bubbles. There are verified relationships among the foaming properties, structural flexibility, and surface hydrophobicity of proteins . After sonication, the rapid diffusion of molecules in the air/liquid interface and molecular rearrangement allow for cohesive viscoelastic films to entrap air, which can modify the foaming properties. On this basis, there are also close relationships between foaming properties and other properties, such as particle size, surface hydrophobicity, molecular weight, and structural flexibility . Appropriate ultrasonic modes can improve the FC of CPs. Akharume et al. reported that FC improved significantly under a long treatment for both the prolamin and glutelin fractions via sonication (20 kHz; 12.70 mm probe; 100%, 75%, 50% amplitude; 5, 10 min). FC was enhanced from 3.83 to 10.33% for the Dawn prolamin and from 22.50 to 34.33% for the plateau glutelin. In addition, FS increased from 57.50 to 100% at 52.72 W for 5 min. Comparable observations were reported in earlier studies: rice bran protein , millet protein concentrate (MPC) , and foxtail millet concentrates . Study outcomes are listed in Table 1. Ultrasonication treatment enhanced the foaming properties with a frequency of 20 kHz and power range of 100-600 W. The observed improvement in the foaming characteristics was attributed to the cavitation effect of sonochemical action. The surface hydrophobicity and molecular flexibility of protein molecules were improved. The particles of proteins were also distributed more evenly, and particle size was reduced. These changes might have led to a rapid enhancement of the adsorption ability on the gas/liquid interface and thus resulted in greater foaming capacity. Furthermore, ultrasonic treatment could induce changes in the conformation of protein molecules, namely, the partial exposure of the protein structure and hydrophobic amino acid residuals to polymerize viscoelastic films at the air/water interface . However, excessive ultrasonic treatment should be noted considering that the reaggregation of proteins induces the desorption of protein molecules at the air-water interface . Meanwhile, the foaming properties of protein might be influenced by other sonication parameters, such as intensity (W/cm2) and power density (W/mL). 3.2. Surface Hydrophobicity (H0) Hydrophobic interactions play an essential role because they are highly associated with the content of hydrophobic amino acids in the food system . Hydrophobicity (H0) is partly responsible for the conformational structures of proteins and protein correlations, such as polar-nonpolar group interactions and complex formation . The H0 of the protein significantly increased as hydrophobic groups were exposed to ultrasonic cavitation, which impacted the functionality of the proteins. With the prolongation of ultrasonic treatment, the spatial structure of the protein changed, exposing more hydrophobic amino acid residues and increasing the surface hydrophobicity because ultrasonic waves can break the hydrogen bonds, electrostatic interactions, and hydration between protein molecules, exposing the hydrophobic groups buried inside the protein molecules. For example, Zhou et al. evaluated the functional impact of ultrasonication (25 +- 1 degC initial temperature; 30 min; power 600 W) on the surface hydrophobicity of defatted wheat germ protein (DWGP) and indicated that the fluorescence peak intensity (420-540 nm) of DWPG increased gradually from 63.7 to 573.25 W/cm2 via sonication. Wang et al. also examined a similar finding and reported an increase in H0 with ultrasound-treated rice bran protein. Later, Yang et al. found that the increase in H0 could have been attributed to the cavitation action of ultrasound that had generated the turbulent shear force, microflow, and other effects. The initially buried hydrophobic regions in the interior of the molecule were effectively revealed to the hydrophilic surrounding medium through strong cavitation effects. Further, cavitation actions can destroy the protein molecules, shrinking the particles from large aggregates into smaller fragments, hence improving the hydrophobic surface of the protein . Thus, as demonstrated in the studies shown in Table 1, ultrasonic treatment with a frequency range of 20-40 kHz and power range of 80-400 W could enhance the H0 of CPs to varying degrees. 3.3. Sulfhydryl (SH) and Disulfide Bond (SS) Content The sulfhydryl (SH) and disulfide (SS) groups are widely acknowledged as critical functional groups in protein molecules. Both play essential roles in maintaining the stability of protein structures, and their ratio can also influence the functionality of proteins . The modification of the free sulfhydryl content of protein molecules could be directly related to the denaturation degree of proteins. For example, Yang et al. found that, at ultrasonic power intensity from 40 to 100 W/L, the SH of defatted wheat germ protein increased significantly (p < 0.05). The highest increment in SH at 60 W/L was 53.20 mmol/g, an increase of 43.21%, which remained steady with increasing ultrasonic power density. The increase in SH can be attributed to the stretching of the protein and its internal sulfhydryl group being exposed to its external surface. The possible reason was that the buried sulfhydryl groups could be unfolded, accompanied by a reduction in protein size, disrupted by the high pressure and shear force caused by sonication . Later, another study by Qin et al. reported that the number of free SH groups in soy protein isolate/wheat gluten mixture increased during the dual modification of protein under high-intensity ultrasound and microbial transglutaminase (MTGase) cross-linking. Similarly, this trend is consistent with the work by Zhang et al. for wheat gliadin. Their study indicated that SH content reached the maximum (10.99 mmol/g) with ultrasonic power of 400 W. Another study, by Zhang et al. , examined the effect of sonication (20, 28, 35, 40, and 50 kHz; power density 400 W/L) on the SH and SS of rice dreg protein isolates. The results showed that sonication treatment caused an increase in the total sulfhydryl group and free sulfhydryl content, and a decrease in SS bonds, especially at a frequency of 20/40 kHz. Later, Liu et al. studied the effect of sonication time (5, 10, 15 min) and power (130, 160, 200 W) on the SH and SS of yellow dent corn-separated protein. The works indicated that the SH content increased by 37.21%, and the SS content decreased by 43.66% via sonication (15 min, 200 W). This was attributed to ultrasonic cavitation, which induced the sulfhydryl group to be exposed to the protein surface. In other words, CPs treated with sonication may exhibit an increase in SH due to the oxidation of hydroxyl radicals generated by cavitation. Meanwhile, the disintegration of SS destroyed the protein conformation and hydrophobic groups initially inside, and the protein molecule was exposed to the external surface more . 3.4. Particle Size The particle size of proteins is an essential factor influencing protein functionality. A (controlled or moderate) sonication treatment could reduce the particle size of CPs through protein aggregations. The smaller particles of proteins could be attributed to the disruptive effects of sonication acoustic cavitation. Cavitation damages the electrostatic interactions and hydrogen bonds between CPs molecules, resulting in protein molecules aggregating into smaller fragments . Similar study outcomes of CPs via sonication displayed a decrease in the particle size under various ultrasonic conditions. For example, as reported by Sharma et al. , ultrasonic treatment (amplitude: 5 and 10%; duration: 5, 10, and 20 min) significantly decreased (p < 0.05) the particle size of foxtail millet protein, and the decrease continued with the increase in ultrasonic time. Moreover, Zhang et al. indicated that, as the ultrasonic power was from 0 to 400 W, the average particle size of wheat gliadin and green wheat gliadin by sonication was reduced by 42.1% and 32.2%, respectively. Further, O'Sullivan et al. (2016) also found that ultrasonic treatment (20 kHz, 34 W/cm-2 for 2 min) could significantly reduce the particle size of wheat protein. The tendency was in concordance with the reports by Qin et al. , Sun et al. , Wang et al. , and Jin et al. . In addition, when ultrasonic treatment is combined with other treatments, the particle size of proteins might be altered. For instance, Zhang et al. concluded that the particle size of rice dreg protein isolates decreased from 330.8 to 219.6 nm after ultrasound-assisted alkali treatment. However, various trends in the particle size of CPs were reported in several studies. Wang et al. found that, as ultrasonic power increased to 500 W, there was an increase in the particle size of rice bran protein. The phenomenon may have been due to the reaggregation of small particles through the thermal effect generated by the ultrasound. This result agreed with Jiang et al. , who confirmed an increase in the particle size of black bean protein isolates via ultrasonication (20 kHz, 450 W). The enhancement in particle size might be related to the repolymerization of aggregates through noncovalent and covalent interactions. In general, when ultrasound is applied, appropriate frequency and power parameters could reduce the particle size of CPs. The particle size might have been enhanced, but ultrasonic power was excessive . 3.5. Conformational Structures In addition to modifying the functional characteristics properties of the protein, ultrasonication can alter its structural characteristics. In accordance with the progressive state of the spatial arrangement of polypeptide chains, the protein structure is categorized into primary, secondary, and tertiary structures. The protein structure is differently affected by ultrasonic treatment on the basis of the types and conditions of ultrasonic treatment. As shown in Figure 3, when the treated protein was exposed to ultrasonic cavitation, alterations only happened in the secondary and higher-order structure of the protein except for the primary . Once the noncovalent interactions between proteins and polysaccharides are modified by cavitation and shearing forces, and hydrophobic and hydrogen bonds are broken, thereby contributing to a variety of corresponding conformational changes (such as unfolding, denaturation, and reaggregation) and sequential alterations in technofunctional and nutritional properties . 3.5.1. Primary Structure Proteins are composed of amino acid sequences as their primary structure. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis is the main method used to examine the changes in subunits of CPs following sonication. According to Wang et al. , there was no noticeable alteration in the subunit of rice dreg protein isolates via sonication (20, 28, 35, 40, 52 kHz). This phenomenon was attributed to alterations in the molecular weight of the proteins, which was not reflected in the electrophoretic spectrum of the protein. Li et al. found that sonication did not alter oat protein composition when the oat protein was subjected to high-intensity ultrasound (20 kHz, 80 W) for 5 min at 70% amplitude. However, some studies are in disagreement with these viewpoints. For example, Jhan et al. showed that the main polypeptide band was around 20 kDa for all native proteins. and that the molecular weight (27-10 kDa) of sorghum protein nanoparticles was reduced following sonication-assisted nanoreduction. This might be attributed to the breakdown of intermolecular hydrogen and hydrophobic bonds, decreasing the molecular weight of the proteins. Furthermore, Nazari et al. indicated that there was a reduction in the molecular weight (40-50 kDa) of millet protein concentrate after ultrasonic treatment (20 kHz, 73.95 W/cm2 for 12.5 min). So, sonication has various effects on the primary structure of proteins due to various ultrasonic conditions. 3.5.2. Secondary Structure The secondary structure of the protein is formed by primary polypeptides on a nascent protein in distinctively coiled aqueous environment. The secondary structure of proteins is characterized through the ratios of a-helix, b-sheet, b-turn, random coil, and unordered groups. Those movements could be transformed by each other to some extent after ultrasonic treatment. Therefore, sonication could alter the protein secondary structure. For instance, Sullivan et al. reported that there was an increase in the amount of unordered or random coils of purified kafirins, while the a-helix content decreased after ultrasonic treatment (20 kHz +- 50 kHz at 40% amplitude for 10 min). In addition, the work of Zhang et al. concluded that the a-helical content of gluten protein was decreased, while the b-sheet and b-turn contents were increased after multifrequency sonication treatment (28, 40, and 80 kHz). A similar phenomenon was studied by Wang et al. regarding corn gluten meal protein, and Liu et al. regarding yellow dent corn separated protein. In the former study, low-power density ultrasound (20/40 kHz, 100 W/L, 20 min, 5:2 s/s) altered the secondary conformational structure of corn gluten meal protein, leading to a reduction in a-helix and b-turn, and an increase in b-sheet and random coil. In the latter study, there was a decrease in a-helix and b-turn, and an enhancement in b-sheet and random coil via sonication (200 W, 15 min), resulting in transforming the yellow dent corn separated protein structure from order into disorder. Furthermore, these observations are in line with earlier studies on wheat gluten by Qin et al. , and Zhang et al. . On the basis of the aforementioned studies, even though the secondary structure might be influenced by the sonication device, intensity, time, and frequency, a protein could undergo some changes in the secondary structures, and exhibit a looser and more flexible structure following ultrasonication induced by cavitation. 3.5.3. Tertiary Structure The protein's tertiary structure is preferred three-dimensional arrangement of the folded polypeptide chains. Compared with a protein secondary structure, a tertiary structure directly affects the functional characteristics of the proteins. Tryptophan residues are essential indicators for characterizing the tertiary structure . Alterations in the tertiary structures of wheat gliadin (GG) and green wheat gliadin (WG) with different ultrasonic conditions were investigated by Zhang et al. . By means of fluorescence spectroscopy, they found that the fluorescence intensity of GG and WG decreased significantly after ultrasound. This might be ascribed to the bubbles transferring through the sound wave, and the location was instantaneously heated. Cavitation generated by the bubble burst could have unfolded and exposed the protein structure during ultrasonic treatment. Moreover, a slight red shift was observed in the maximal fluorescence emission wavelengths of GG and WG: 3 and 2 nm, respectively. In this experiment, an increase in the polarity of the tryptophan residue microenvironment was demonstrated, thereby indicating that ultrasonic treatment enhanced the tertiary structure formation of the GG and WG molecules. A similar conclusion was reached by Su et al. , who found that mass transfer effects enhanced by ultrasound might cause transient bubbles. When the bubbles collapsed, vast reactive free radicals could have led to the modification of amino acid side chains, which would change three-dimensional folded structures. Therefore, ultrasound has the ability to irreversibly alter and adapt the three-dimensional structure of CPs to emulsify oils, yielding a stable structure for up to years. Qu et al. also mentioned that ultrasound-treated (20/28 kHz, 150 W/L) CPs had shown increased absorption intensity at 275 nm. and a new structure had been shaped. Thus, ultrasonic working parameters such as ultrasonic power, time, and temperature might influence the tertiary structure of proteins. 3.6. Microstructure Having undergone ultrasonic treatment, the structure of CPs was reduced from large and interconnected aggregates to small and dense fragments with loose and granular distribution . The microstructure of CPs was influenced according to the cereal protein type and source, besides ultrasonic parameters and processing conditions. For instance, Jin et al. reporte that the turbulent and cavitation forces could fracture the macroparticles and further change the surface state of the proteins. According to this study, ultrasonication caused cavitation bubbles and microstreaming effects that disrupted the formation of protein aggregates. The aggregation of fragments might be attributed to the destruction of cross-links between amino acid residues in protein containing S-S bonds, hydrophobic bonds, and Van der Waals interactions generated by ultrasonic radiation . A similar phenomenon was observed by Zhang et al. . The researchers studied the effects of alternate dual-frequency ultrasound on the structure of wheat gluten, and found that sonication caused alterations in the structure, which displayed irregular agglomerates. The findings from their work indicated that the values of roughness (Ra, Rq) greatly increased with sonication (20/35 kHz, 150 W/L, 10 min). Likewise, Wang et al. demonstrated that sonicated rice dreg protein isolated with multifrequency countercurrent S-type sonication displayed more uniform protein fragments and a looser structure under ultrasonic conditions (20/40 kHz, 60 W/L, 20 min). Their findings also agreed with the results of rice proteins obtained by Yang et al. , and Wang et al. . Apart from the modification of CPs via ultrasonic treatment alone, ultrasound combined with alkali treatment, one of the synergistic methods of ultrasonic modification aided by other chemical and biological means, has gained considerable attention. For example, Zhang et al. observed that, after sonication-assisted alkali treatment, the structure of rice dreg protein isolates loosened and disordered, exhibiting more irregular fragments and microparticles. Li et al. performed a similar study . The alterations in the microstructure may be ascribed to the generation of cavitation bubbles that disrupt protein aggregation, thereby destroying the cross-linking reaction between protein molecules. Meanwhile, variation in the morphological structures might be attributed to the unfolding and partial denaturation of the tertiary protein structure, liberating more hydrophobic bonds, thereby leading to different changes in the CPs' functionality. 3.7. Enzymatic Hydrolysis Enzymatic hydrolysis is broadly applied to enhance the functional quality of proteins, which is beneficial to biological substances in the human body. Compared to traditional enzymatic hydrolytic technology, ultrasound-assisted enzymolysis has better advantages, such as easier control, shorter processing time, and more convenient operation . Ultrasonic pretreatment could generate special acoustic cavitation that produces high-intensity shearing force, free radicals, and shock waves, leading to protein denaturation to release hydrophilic groups, thereby affecting the solubility, bioactivity, and enzymatic efficacy of proteins . A study by Qu et al. examined the influence of sweep frequency and pulsed ultrasound (24 +- 2 kHz, 24 W) on the enzymatic hydrolytic efficiency and activity of ACE inhibitory peptides from wheat germ protein. In that study, ultrasound-assisted enzymatic hydrolysis could change a conformational molecule and improve the efficiency of enzymatic hydrolysis. This observation indicated that ultrasound could increase enzymatic activity on the basis of reasonable conformational alterations in the protein. These results agree with those of Li et al. , who indicated that sonication (28 kHz; 2 cm deep inserted with ultrasonic probe) could shorten the enzymolytic action time of rice protein (RP) and improve the efficiency of enzymatic hydrolysis. Compared with untreated RP, the structural surface of enzymolytic residues became uniform with many small fragments. Alterations in the structures of hydrolytic residues via sonication resulted in the improvement of enzymatic efficacy. The results agreed with those by Li et al. for rice protein. Similarly, a recent study by Wang et al. found that the enzymolytic efficiency of corn gluten meal was enhanced via sonication (sequential double frequency of 20/40 kHz). Enzymolytic efficiency reached a maximum of 15.99% with a protein dissolution rate of 61.69%. The mechanism could be ascribed to the collapse of cavitation bubbles formed by sonication at a frequency of 20 kHz, providing new cavitation nuclei for the ultrasound at 40 kHz, ultimately resulting in an increase in cavitation bubbles. Figure 4 is a diagram of the whole process of ultrasonically treated corn gluten meal. Jin et al. used ultrasonic sweeping frequency (28 +- 2/68 +- 2 kHz, 80 W/L, 40 min) to study the enzymolysis of corn gluten meal, and their results indicated that ultrasound could accelerate enzymolysis, leading to an increase in the affinity between enzyme and substrate. Further, a similar phenomenon was shown after ultrasonic treatment in other CPs, such as defatted corn germ protein , defatted wheat germ protein , and oat-isolated protein . Therefore, whether ultrasonication conditions are a single, dual, or triple ultrasonic frequency, ultrasound can be considered a promising treatment technique for enhancing the enzymatic efficacy of CPs. 3.8. In Vitro Digestion The digestibility of protein mainly reflects the extent to which protein in food has been absorbed and utilized by the human body in the digestive tract. Food protein digestibility is related to the susceptibility of a protein to proteolysis. Increased digestibility meant that it could be better hydrolyzed by pepsin and trypsin, and have higher nutritional value. According to previous studies, ultrasound-assisted treatment is commonly used to optimize CPs and CP-based products so as to increase their digestive value within the human gut. For instance, Hassan et al. investigated the influence of ultrasonically treated sorghum grain protein on in vitro digestibility. In that study, ultrasound (40% amplitude for 5 min) showed significantly higher digestibility (64.70 +- 0.50%) than that of other ultrasonic conditions (60% amplitude for 10 min). Compared with the control, germination was significantly promoted by sonication, thereby improving protein digestibility. Moreover, Li et al. investigated the influences of various ultrasonication working modes on the in vitro digestibility of rice protein. According to that study, mono-, dual-, and triple-frequency ultrasonication significantly improved the simulated gastrointestinal digestion product. An increase in hydrolyzed protein content promoted the contact activity of an enzyme involved in proteolysis, subsequently boosting the simulated gastrointestinal digestion process. In another study, wheat gliadin and green wheat gliadin treated with ultrasound (400 W, 300 W) improved in vitro digestibility by 12.75% and 11.03%, respectively . Similarly, Jin et al. showed that sonication (20 kHz, pulsed on-time 10 s/off-time 5 s, amplitude 60% for 10 min) enhanced the in vitro digestibility of buckwheat protein, which increased by 41% compared to the untreated samples. This trend could be attributed to the alternations in protein structure by acoustic cavitation effects. Nevertheless, high-intensity ultrasonic treatment could decrease the in vitro digestibility of proso millet bran protein when the ultrasonic conditions were a frequency of 24 kHz, power of 400 W, and amplitude of 100% for 20 min. The decrease in the in vitro digestibility was caused by the temperature increase during high-intensity ultrasonic treatment. Generally, moderate sonication could influence the digestibility and nutritional quality of CPs. 4. Conclusions and Future Trends Sonication treatment has many advantages in terms of enhancing the functionality, conformation, enzymolysis, and digestive properties of CPs. As discussed in this review, the modification of CPs with ultrasonication technology mainly improved the functional, conformational, and physicochemical characteristics of CPs. In this regard, sonication could alter protein structural characteristics, including surface hydrophobicity, particle size, conformational (primary, secondary, and tertiary) structures, and microstructures, to improve the functionality of CPs. Moreover, alterations in the protein molecules with ultrasonic acoustic cavitation can influence the physicochemical properties, such as bu improving enzymatic efficacy and in vitro digestibility. In addition, the impact of sonication on the CP structure are related to the type of ultrasonic device/equipment, ultrasonic treatment conditions, and processing intensity. Therefore, the specific mechanism of ultrasonic processing technology or various types of ultrasonic equipment on CPs should be further studied. At the same time, it is necessary to recognize the dynamic change process of CP structures via sonication through new technologies, and precisely control the spatial structure, which is conducive to improving the functionality of CPs. Lastly, research on large-scale industrialization should also be strengthened. Author Contributions Conceptualization, Y.W., J.L., Z.Z., X.M., T.Y. and R.H.; Investigation, Y.W.; Writing--original draft, Y.W.; Validation, J.L., X.M., T.Y. and H.M.; writing--review and editing, J.L. and W.S.; Project administration, Z.Z. and R.H.; Supervision, Z.Z. and R.H.; Formal analysis, H.M. All authors have read and agreed to the published version of the manuscript. Data Availability Statement No new data were created or analyzed in this study. Data sharing is not applicable to this article. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Visualization of trends in the modification of cereal proteins with ultrasound for enhancing functional properties. Figure 2 Schematic illustration of the ultrasonic generation of bubble cavitation and collapse . Figure 3 Sequential alterations in the primary, secondary, tertiary and quaternary structures of a protein exposed to ultrasonic treatment . Figure 4 Diagram of the whole process of ultrasonically treated corn gluten meal (CGM) . Note: The proposed mechanism schematic diagram of ultrasonicated CGM: (I)-Potein by ultrsound; (II)-Formation of tiny bubbles; (III)-Formation of cavitation bubbles; (IV)-Implosion of cavitation bubbles; (V)-Generation of jet beam; (VI)-Hole corrosion. Dual-frequency slit ultrasound (DFSU) equipment: 1. Sample inlet; 2. Sample outlet; 3. Ultrasonic transducer; 4. Waste outlet; 5. Slit cavity; 6. Treat liquid; 7. Magnetic stirrer; 8. Ultrasonic generator; 9. Programmable Logic Controller (PLC) control panel. foods-12-00971-t001_Table 1 Table 1 Summary of representative research on the functionality changes in modified CPs after ultrasonic treatment. Proteins Ultrasonic Conditions Solubility Emulsibility Foamability Surface Hydrophobicity References Foxtail millet protein concentrate A 1 cm diameter probe; amplitude: 5, 10%; duration: 5, 10, 20 min; room temperature. Increased Increased Increased Increased Sharma et al. (2022) Barely protein isolate (BPI) A 13 mm diameter stainless steel probe; frequency: 20 kHz; amplitude: 100%; Increased -- -- -- Silventoinen et al. (2020) Corn gluten meal protein (CGM) frequency: 20/40 kHz; power density: 60, 80, 100, 120, 160, 200 W/L; time: 10-40 min; temperature: 20-45 degC; pulse period: 5:1-5:5 s/s Increased -- -- Increased Wang et al. (2020) Rice protein (RP) Frequency: 25 kHz; power: 400, 500, 600 W; time: 15, 20, 25 min; temperature: 75, 80, 85 degC. Increased -- -- -- Chen et al. (2022) Rice bran protein (BRP) A 6 mm diameter titanium probe; frequency: 20 kHz; time: 10, 20 min; power:100, 200 W. Increased Increased -- -- Wang et al. (2021) Millet protein concentrate (MPC) A 3 mm diameter titanium sonotrode probe; frequency: 20 kHz; power: 100 W; amplitude: 20%, 60%, 100%; time: 5, 12.5, 20 min; temperature: 20-30 degC. Increased Increased Increased -- Nazari et al. (2017) Oat protein isolate (OPI) Frequency: 20 kHz; power: 80 W; amplitude: 70%; time: 5 min; temperature: 20 degC Increased -- -- Increased Li et al. (2021) Sorghum kafirin Frequency: 20 kHz +- 50 kHz; time: 5, 10 min; temperature: 4 degC. Increased -- -- -- Sullivan et al. (2018) Rice bran protein hydrolysate (RBPH) Frequency: 20 kHz; pulse period: 6 s (4 s on, 2 s off); power: 0, 150, 300, 450, 600 W. -- Increased -- -- Wang et al. (2022) Rice protein (RP) Frequency: 25 kHz; power: 400, 500, 600 W; time: 15, 20, 25 min; temperature: 75, 80, 85 degC. -- Increased -- -- Chen et al. (2022) Wheat protein isolate (WHPI) Frequency: 20 kHz; amplitude: 95%. -- Increased -- -- O'Sullivan et al. (2016) Quinoa protein isolate (QPI) Power: 100 W; temperature: 25 degC; time: 20 min -- Increased -- -- Xin-Sheng Qin et al. (2018) Rice bran protein Power: 100, 200, 300 W; time: 10, 20 min -- -- Increased -- Sun et al. (2021) Rice protein (RP) Frequency: 20 kHz; power: 600 W; temperature: 50 degC; time: 60 min -- -- Increased -- Zhang et al.(2017) Proso millet protein Amplitude: 50%, 75%, 100%; frequency: 20 kHz; time: 5, 10 min. Increased -- -- -- Akharume et al. (2022) Yellow dent corn separated protein Power: 130, 160, 200 W; time: 15 min -- -- -- Increased Liu et al. (2021) Rice bran protein (RBP) Frequency: 20 kHz; pulse period 6 s (4 s on, 2 s off) -- -- Increased -- Wang et al. (2021) Rice dreg protein isolates (RDPI) Frequency: 20, 28, 35, 40, 50 kHz; power density: 400 W/L -- -- -- Increased Zhang et al. (2020) Wheat gluten (WG) Frequency: 40 kHz; power: 400 W; time: 0, 10, 20, 30, 40 min -- -- -- Increased Qin et al. 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PMC10000785 | Healthcare (Basel) Healthcare (Basel) healthcare Healthcare 2227-9032 MDPI 10.3390/healthcare11050644 healthcare-11-00644 Article Psychological Experience of Smoking Addiction in Family and Friends of Schizophrenic Adults Who Smoke Daily: A Qualitative Study Caponnetto Pasquale Conceptualization Writing - original draft 12 Maglia Marilena Writing - review & editing 12 Gulizia Annalisa Data curation 1 Prezzavento Graziella Chiara Writing - review & editing 1* Polosa Riccardo Supervision 23 Quattropani Maria Catena Supervision 1 Signorelli Maria Salvina Supervision 3 Giansanti Daniele Academic Editor 1 Department of Educational Sciences, University of Catania, 95121 Catania, Italy 2 Center of Excellence for the Acceleration of Harm Reduction (COEHAR), University of Catania, 95121 Catania, Italy 3 Department of Clinical and Experimental Medicine, University of Catania, 95121 Catania, Italy * Correspondence: [email protected] 22 2 2023 3 2023 11 5 64415 1 2023 13 2 2023 20 2 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). The smoking addiction of patients with severe mental disorders has consequences not only for the patients but also for the people around them. This is qualitative research on family and friends of patients with Schizophrenia spectrum disorders to investigate their perception and vision of smoking, its impact on the patients' physical and mental health, and the possible attempts to combat addiction. The research also investigates the participants' views on electronic cigarettes as a means of replacing traditional cigarettes and helping the patient to quit smoking. The survey method used was a semi-structured interview. The answers were recorded, transcribed and analyzed with the technique of thematic analysis. The results of this study show that the view of most participants on smoking is negative (83.3%), although not all of them consider smoking cessation treatments for these patients of primary importance (33.3%). Nevertheless, a good number of them have tried to intervene spontaneously with their own resources and strategies (66.6%). Finally, low-risk products, and in particular electronic cigarettes, are considered by many participants as a useful alternative to traditional cigarettes in patients with schizophrenia spectrum disorders. About the meaning that cigarettes can assume for the patient, recurring themes emerge: they are considered as a way to manage nervousness and tension or as a means to contrast daily monotony and boredom or repeat usual gestures and habits. schizophrenia smoking addiction e-cigarette thematic analysis qualitative research This research received no external funding. pmc1. Introduction Schizophrenia is a chronic mental disorder, considered one of the first causes of mental disability worldwide . Onset usually occurs between late adolescence and early adulthood. Symptoms persist for the whole life cycle; for this reason, it is one of the most serious and disabling psychological pathologies. The reality of the patient suffering from schizophrenia is altered in many aspects: the perceptive area is affected by hallucinations and the thought by delusions. This condition pours on the emotional and social front, where the patient manifests flattening and reduction of emotional expression and abnormal social behavior. Regarding the etiopathogenesis of schizophrenia, today's science tends to support a polygenic hypothesis, so mutations of different genes, often rare, appear as determinants. In a study published by Purcell, genetic sequences of 2500 subjects in the schizophrenic population were compared with subjects in the normal population. Thanks to this comparison between several alterations of dozens of genes, it has been seen that the genes coding for the proteins involved in the formation of ion channels and those responsible for synaptic transmission are those that have a more important role . Today, researchers have found that the most important genes playing a significant part in the onset of schizophrenia are COMT, NRG1, DISC1, and SETD1A, while the most relevant environmental risk factors affecting genetic predisposition are pre and peri-natal complications, substance abuse and stressful events . The abuse of drugs, alcohol, and nicotine is particularly widespread among patients suffering from schizophrenia spectrum disorders, more than among the general population. As a result of substance use, there may be a worsening of psychotic symptoms, non-adherence to treatment, interaction with prescribed agents as well as an increase in violent behavior and suicide with premature mortality. These critical issues have been reported by several studies, highlighting the importance of finding solutions that can help patients improve their quality of life and that of the people around them, such as family and friends . Focusing on smoking addiction, some studies have demonstrated that this problem affects between 60-90% of people with schizophrenia, compared to 15-24% of the non-psychiatric adult population . From the literature, it emerges that genetic factors contribute more than all other factors, such as environmental or stress-related ones, to the comorbidity between smoking and schizophrenia, although it is necessary to underline the importance of further research to better understand the complex relationship between these two disorders . The association between smoking and mental health conditions becomes stronger depending on the severity of the disease and seems to vary depending on the actual health conditions, so a person who has no physical or psychological health problems will be less likely to take substances . People with schizophrenia spectrum disorder have an increased mortality risk if compared to the non-psychiatric adult population, and the risk is even higher in people with a double diagnosis of Schizophrenia and Substance use disorder . In a large-scale follow-up study, Callaghan and colleagues also found that tobacco-related conditions included about 53 of total deaths in the schizophrenic cohort, 48% in the bipolar cohort, and 50% in the depressive cohort . This is further confirmed by Carney's studies which showed that people with schizophrenia spectrum disorders have a significantly higher prevalence of cancer, respiratory disease, and cardiovascular disease than the general population . Regarding the effects that smoking can have on pathology among smokers with schizophrenia spectrum disorders, the association with depressive symptoms, increased hospitalization, stress, poor treatment results, low quality of life and improved psychotic symptoms has been noted . The treatment of smoking addiction in people suffering from a psychiatric disorder is more complex than it is in non-psychiatric patients, and it is often neglected for other seemingly more important problems. Scientific literature has shown that the most effective treatments for these patients include nicotine replacement therapy (NTR), Bupropion, and behavioral interventions (CBT) . In addition to these treatments, electronic cigarettes can be considered a low-risk substitute for traditional cigarettes, as has been demonstrated recently. Researchers have shown that the use of electronic cigarettes in patients with schizophrenia caused a 50% reduction in daily tobacco cigarette consumption in 50% of participants (from 30-15 cigs/day), whereas 14.3% of participants quit completely by week 52 . Treatments are very important not only to improve the quality of life of these patients but also to help their family and friends who experience a condition of psychological discomfort due to cigarette abuse by a family member. A study by Budiono indicates that family psychoeducation on the nature of schizophrenia is effective in positively changing family reactions and indirectly increasing patients' adherence to prescribed treatment . Family members help and support the patient and assist in ensuring drug adherence. In our previous research, we analyzed the point of view of healthcare personnel dealing with schizophrenic patients who smoke and live in assisted therapeutic communities. Our results showed that all specialists agree on e-cigarettes as good alternatives to cigarettes. The positive view on electronic cigarettes is reinforced by other factors, such as that they don't release bad smells and don't stain or burn clothes and linens . The current research has focused on family and friends of patients diagnosed with schizophrenia to better understand the impact that cigarette smoking has on the thoughts and emotions that relatives experience every day. We have studied the views of family members on traditional cigarettes, but also their consideration of e-cigarettes and the impact those could have in helping patients to decrease or stop smoking. We also investigated if participants had ever considered it appropriate to intervene in an attempt to reduce the number of cigarettes smoked by their family members and examined the risk perception of smoking on health. 2. Materials and Methods This qualitative research was carried out between November 2021 and June 2022. It involved family and friends of patients with schizophrenia spectrum disorders living in two assisted therapeutic communities. The sample consisted of 30 participants, including 17 women and 13 men, who were selected by the non-probability sampling technique widely used in qualitative research, so participants are selected based on the basis of the research questions: be friends or relatives of adults who smoke with schizophrenia . The methodological orientation was the analysis of the contents. No subjects refused to participate. Some participants were recruited by telephone, while another part was recruited from within the same communities. This is because some of them had returned to their homes or had been hospitalized for health problems. The inclusion criterion consisted in having a kinship/friendship/sentimental bond, such as to guarantee a thorough knowledge of the patients, their pathology and their level of dependence. The participants were informed about the objectives of this qualitative analysis:The perception of family and friends of patients suffering from schizophrenia on smoking and its impact on physical and mental health; Their contribution to promoting or containing smoking addiction of the family member; Their perception of electronic cigarettes as an alternative to traditional cigarettes to reduce or eliminate dependency. Based on the research questions, we developed more detailed questions that were asked through a semi-structured interview (Table 1). The interviews lasted about 10 min, depending on the subject's answers. Brief information was collected about the participants (Table 2): sex, degree of kinship or friendship. Then, they were informed about the purpose of our research, and they were asked for permission to record the interview, ensuring that the audio recording was used for the sole purpose of transcription and analysis. The semi-structured interviews were conducted individually. All interviews were recorded and subsequently transcribed. The length of the interviews ranged from 5 to 10 min, depending on the responses of participants. Audio recording has been very important for data collection purposes; in fact, during the same interview, it was preferred not to take notes of the answers in order to keep the interaction as natural and smooth as possible and be able to adapt the subsequent question to the previous answer of the participant. We used the method of Thematic Analysis to identify, analyze, and report the issues within the data, as well as to provide a rich and detailed but complex data report . The Thematic Analysis model consists of six phases:Familiarization: requires the researcher to develop a thorough knowledge of the data, familiarize himself with the material collected, reading the transcripts several times, with the aim of going beyond the most obvious meanings; Generation of codes: means identifying elements of interest, summarizing them and identifying them by codes, understood as words or short sentences. Braun and Clarke argue at this stage the importance of looking beyond the overt meaning of the participants' answers to questions to also address the implicit and latent meaning; First generation of themes: Topics are being identified based on how the codes are combined with each other, so we proceed by identifying broader themes that group together different codes. This creates a list of themes with the corresponding codes; Revision of themes: This phase is very important to verify that the themes have remained consistent and data-related, plus it serves to rework the initial themes and decide whether to expand them into more units, delete them, group multiple themes into a larger unit or keep them as they are; Definition and naming of themes: each theme, which is identified in its essential characteristics, is assigned a label to name it; Production of the Report: A final report is produced at this stage, focusing more on the themes that answer the research questions, developing them and including extracts from the interviews to make the meaning of the themes and the report explicit, which must have been maintained between these and the data initially collected (Table 3). 3. Results Most participants (25 out of 30) have a negative view of smoking, whether they are people who smoke daily or not. The same believe that the impact of cigarette smoking traditions is strongly negative on physical health, being the cause of several respiratory and cardiovascular disorders in patients. Some of them (18 out of 25) identify a correlation between the state of mental health of the patient and cigarette smoking, reporting that the patient has a greater need to smoke in moments of discomfort or nervousness. Participants who do not believe that smoking has a negative impact are all people who smoke daily (5 out of 30). They tend to underestimate the problem of the addiction, not attributing to smoking the responsibility of any type of symptom of the patients. Instead, as emerges from the interviews, they are more concerned about other types of problems, such as the unpredictable behavior of the patient, so they do not think the smoking addiction is a priority to be addressed. No participant reports a positive view of cigarette addiction. According to family and friends, recurrent themes emerge of cigarettes as a way to manage nervousness and tension or as a means to contrast daily monotony and boredom or repeating gestures and habits. Twenty participants out of the total believe they have provided help to the patient, trying to make them stop smoking, intervening more or less directly (Theme 2). Some say they have recommended quitting, and others have intervened by interfering with the purchase of cigarettes or by establishing with the patient a maximum number of cigarettes a day, or even proposing alternatives to traditional cigarettes. Ten patients did not intervene for different reasons: being people who smoke themselves, they did not think they could help the patient; they did not consider smoking a major problem; they did not believe that the patient could ever stop smoking. Only three participants believe that the patient can quit smoking, 20 argue that the patient can at least reduce, and seven report the impossibility that the patient would stop or reduce the habit of smoking. All participants reported knowing about e-cigarettes (Theme 3). Twenty of them think that e-cigarettes are less harmful than traditional cigarettes, seven felt unable to make a judgment because they said they did not have sufficiently thorough knowledge, while 3 participants considered e-cigarettes more dangerous than traditional cigarettes. Regarding the possibility of using e-cigarettes in an attempt to help the patient to quit smoking, 13 participants declared that they could be useful, while the remaining part believes they are not. The analysis of the three themes was carried out after transcribing the interviews. Most participants, including adult who smoke daily, believe they have made a positive contribution to reducing patient dependency with either directional or authoritarian interventions. A small portion of the sample has never intervened for several reasons; they believe that any attempt is useless because they do not consider smoking such a problem as to require intervention or because, being people who smoke themselves, they don't feel able to help another person with the same problem. Most participants believe that with the help of specialists, patients can quit smoking or at least reduce it, while a relatively small part of the sample thinks that this cannot happen. 4. Discussion The influence of family or other significant people seems to be very important in two main aspects of dependence: first of all, in the development of the addiction and secondarily in the treatments and the interventions to contrast it. About the first point, many theories and studies have shown how parenting has a strong impact in predicting exposure to deviant companions during adolescence and so in increasing the risk of developing deviant behaviors, such as smoking addiction and drugs dependencies . Besides, several pieces of evidence testify to the role of the attachment relationship between members of the family and children. Both in the development of psychopathology and in the growth of dependence, systemic theory, in fact, has made several contributions to the understanding of addiction phenomena by shifting the focus of analysis from the subject to the family context, and in particular to the styles of attachment in intra-family relation . Many studies have shown that safe attachment is an important protective factor against the development of mental disorders, while unsafe styles are associated with their development and consumption behaviors associated with addiction to tobacco and other substances . A large body of research has provided evidence about the link between parental smoking and smoking in adolescents. Cognitive theories have tried to explain the reason behind this phenomenon, elaborating a research line that describes an explicit transmission path involving conscious elaboration and explicit decision-making processes. Theories that focus on health behavior, such as Social Cognitive Theory, assume that the decision to engage in substance use is based on a rational assessment of the positive and negative consequences. According to these theories, explicit cognitions are important predictors of the decision to engage in certain behaviors . The observation of a certain behavior by a model figure forms cognition and, therefore, leads to the adoption and imitation of the same behavior . Recent literature describes a second possible pathway that also considers the role of implicit smoking-related cognitions: implicit transmission. It involves the formation of cognitive processes that are more automatic and less readily accessible by introspection or self-report . The achievement of the patient's desired outcome can be considered a phenomenon widely studied in social psychology on self-fulfilling prophecy . In this perspective, the beliefs that everyone has about themselves are strongly influenced by the expectations and beliefs of others. So applying this hypothesis to our research context, the beliefs of others, especially people considered as meaningful to the patient, like friends or family, can affect the real possibility of quitting smoking for the patient. Regarding the second point, concerning the role of the family in the treatment of dependence, it is known that the behavior of one member of the family influences the behavior of other members. So, according to the theory of the role model, individuals compare themselves with people that are significant to them, and these people are more likely to influence their behavior. For this reason, it is appropriate to think that the influence of family and other significant people may be important in the treatment of tobacco addiction because they are seen as models to imitate and to listen to. So, in an intervention program to limit cigarette consumption in schizophrenic patients, it can be very fruitful to include families and work with them to help the patients to achieve the goal of reducing or quitting smoking. The main objectives should be to make families conscious of their active role and their influence in this process and work on their perception of the risks associated with tobacco addiction. It has also been demonstrated that a positive and supportive environment in the planning of the intervention can be useful to help the patient not to feel alone and helpless. Therefore, also thanks to the support of their family and friends, people with schizophrenia can proceed toward recovery and improve the quality of their leaves, which is the main goal of treating both schizophrenia and addiction . Depending on the results of the current research, it might be interesting to conduct a large study focused on the perception of low-risk products also among doctors and health professionals. The lack of accurate communication from scientists may contribute to the confusion about the health risks deriving from e-cigarettes and their contribution to helping to quit the smoking habit. Electronic cigarettes could be, instead, a valuable ally in the fight against smoking addiction, especially in patients suffering from schizophrenia. Promoting their use might avoid authoritarian methods that would end up exacerbating the paranoid characteristics of the disorder. The use of e-cigarettes could give the patients the possibility to maintain a habit, which in many cases is an important part of their lives, while helping them to reach the first goal, which is risk reduction. Eliminating common misconceptions about e-cigarettes is an important step that health professionals should consider in order to effectively educate the general population by providing the correct information about the risks of vaping compared to the risk of traditional tobacco. Some principles and solutions that should guide policymakers towards a more reasonable regulation of e-cigarettes include the concept of harm reduction, the possibility of de-normalization of smoking, the availability of low-risk products, the importance of proportionate taxation and of consumer perspectives, and the value of a reassessment of the role of non-tobacco flavors . It is necessary to spread correct information about devices without combustion because, as evidenced by our research, behaviors, as well as the beliefs of patients, are often influenced by those of people significant to them. Several studies have shown that the harm perception of electronic cigarettes can deter people who smoke currently from starting or continuing the use of electronic cigarettes . This perception can also discourage a complete switch from cigarettes to electronic cigarettes among adults who smoke. E-cigarettes share many similarities with smoking in the behavioral aspect of their use . As confirmed by the data from clinical trials , users report the necessity of using an e-cigarette for a long period to reduce cigarette consumption or quit smoking or relieve tobacco withdrawal symptoms , with much-reduced health risks . Moreover, the fact that they can be used in many areas where smoking is prohibited, that they have a very competitive price, and are perceived as a much less harmful alternative to tobacco smoking , increases their popularity. As Farsalinos demonstrated in his study, over 48 million people in the EU have ever used e-cigarettes, and 7.5 million currently use them . Between 2012 and 2017, the proportion of those currently smoking tobacco in the EU who had ever tried to quit tobacco smoking using e-cigarettes increased from 7.1% to 15.6%, respectively . The percentages of subjects who vape more than others are young, as shown by the many studies conducted in different parts of Western countries. Clinicians and health policy experts who fight against the risks related to smoking cannot consider this phenomenon as an opportunity to improve people's health. Increasing evidence confirms that harm reduction through the transition from traditional cigarettes to electronic products could represent an important stage in the path of smoking cessation , so it is equally good to promote a total cessation of smoking even with the abandonment of hard reductions tools, which although it is well known to represent an improvement from the point of view of danger and harmfulness about traditional tobacco, they must only represent a stage in the path of cessation and not a final point of arrival. However, the literature suggests with increasing evidence that the health risks are significantly lower than those of traditional smoking among adults who cannot or do not want to quit . Innovation in the electronic vapor category is likely to further reduce health risks in favor of maximizing the health benefits of consumers switching from traditional to electronic cigarettes . Instead, for people who wish to stop vaping, we must provide cessation programs that are effective, accessible, and well-publicized . In the context of medical care, it is necessary to be clear about the benefits and options available for tobacco cessation, and test-based methods for this purpose should be easily accessible at the national level and free of charge. 5. Conclusions This qualitative study compared the vision of 30 participants who had a relationship of kinship, friendship or sentimental nature on traditional cigarette smoking and low-risk products, including electronic cigarettes, in patients suffering from Schizophrenia spectrum disorders. We have also investigated their beliefs about tobacco abuse and new treatments, including the use of electronic cigarettes. The results of this study show that the view of most participants on smoking is negative (83.3%, 25 out of 30), but despite this, not everyone considers the treatment of tobacco in patients with the above disorders to be of primary importance (33.3%, 10 out of 30; 66.6%, 20 out of 30). Finally, low-risk products and, in particular, electronic cigarettes are viewed by a good number of participants as a useful alternative to traditional cigarettes in the fight against smoking in patients suffering from Schizophrenia Spectrum disorders. Our research is certainly not without flaws or limitations. The small number of the sample does not allow us to generalize the data, and therefore, we do not have the certainty that what has been found is valid for the entire population that the sample represents. Another potential limitation could be that we are personally involved in data collection, as this increases the possibility of unconsciously influencing responses. The risk is to administer the questions, suggesting answers rather than letting them reflect the real perceptions of the participants. To avoid this, we tried to ask the questions in a similar way, keeping the same form, that is, the one reported in the interview. Future research will help us to fill these gaps and explore the phenomenon under study by using other types of research tools. Acknowledgments Thanks to all the authors for their participation and careful supervision in the drafting of this work. Author Contributions P.C. is responsible for the conceptualization of the research and for drafting the original draft; A.G. collected the data; M.M. and G.C.P. are responsible for the modification of the final manuscript; R.P., M.S.S. and M.C.Q. are responsible for the supervision. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The study was conducted in accordance with the Declaration of Helsinki. Informed Consent Statement Informed consent was obtained from all subjects involved in the study according to the principles of the general data protection regulations (GDPR). Data Availability Statement Not applicable. Conflicts of Interest P.C. has been affiliated with the CoHEAR since December 2019 in a pro bono role. He is the co-author of a protocol paper supported by an investigator-Initiated study award program established by Philip Morris International in 2017. The other authors have no conflicts of interest to declare. R.P. is a full-time employee of the University of Catania, Italy. In relation to his work in the area of tobacco control and respiratory diseases, R.P. has received lecture fees and research funding from Pfizer, GlaxoSmithKline, CV Therapeutics, NeuroSearch A/S, Sandoz, MSD, Boehringer Ingelheim, Novartis, Duska Therapeutics, and Forest Laboratories. He has also served as a consultant for Pfizer, Global Health Alliance for treatment of tobacco dependence, CV Therapeutics, NeuroSearch A/S, Boehringer Ingelheim, Duska Therapeutics, Forest Laboratories, ECITA (Electronic Cigarette Industry Trade Association, in the UK) and Health Diplomat (a consulting company that delivers solutions to global health problems with special emphasis on harm minimization). Lecture fees from a number of European electronic cigarette industry and trade associations (including FIVAPE in France and FIESEL in Italy) were directly donated to vaper advocacy non-profit organizations. He is currently a scientific advisor for LIAF, Lega Italiana Anti Fumo (Italian acronym for Italian Anti-Smoking League) and Head of the European Technical Committee for standardization on "Requirements and test methods for emissions of electronic cigarettes" (CEN/TC 437; WG4). All other authors have no conflict of interest to declare. healthcare-11-00644-t001_Table 1 Table 1 Questions for research objectives. First Objective 1. What do you think about smoking? 2. How dangerous do you consider this addiction to health? 3. Are you a person who smokes, or have you ever smoked? 4. How do you assess the physical and mental health of your family member/friend? 5. Do you think his/her health is related to your addiction to smoking? 6. Did you ever notice if the times when the addiction was more or less accentuated coincided with periods when the patient's health was different? 7. Do you think there are times when the patient needs to smoke more? 8. Do you think that cigarette use can relieve or accentuate some symptoms of the patient's mental illness? 9. What do you think cigarette represents for the patient? Second Objective 1. If you are a person who smokes daily, do you think this will affect your familiar's addiction? If so, how? 2. Have you ever tried to help him/her reduce or stop smoking? How? 3. Do you think he/she can actually do that? Third Objective 1. Do you know about electronic cigarettes? 2. Do you think it is less, equally or more dangerous than traditional cigarettes? 3. Do you think it is a good alternative to help patients quit smoking? healthcare-11-00644-t002_Table 2 Table 2 Participants' characteristics. Gender Degree of Kinship Degree of Friendship Male n(%) 13 (43.34%) Fathers 2 (6.65%) Friends 6 (20%) Brothers 2 (6.65%) Companions 3 (10%) Husbands 0 (0%) Female n (%) 17 (56.66%) Mothers 5 (16.65%) Friends 0 (0%) Wives 2 (6.65%) Companions 5 (16.65%) Sisters 5 (16.65%) Sample Size (n) = 30; Standard deviation (s.d.) = 1.669; Signify (x) = 3.75; Variance (s2) = 2.79; Coefficient variance = 0.4451; Average standard error (SE) = 0.59. healthcare-11-00644-t003_Table 3 Table 3 Themes and codes. Research Questions Themes Codes The perception of family and friends about smoking and its impact on the patient's health Positive, negative, or neutral perceptions of traditional cigarettes - Participants who smoke daily/Participants who do not smoke - Assessment of the risk of smoking to physical and mental health - Relationship between difficult times and increased prevalence of cigarette addiction - Meaning which patient attributes to the cigarette The contribution of family and friends in promoting or containing patient's tobacco dependence Positive, negative, or neutral impact on dependence - Presence/absence of support for smoking cessation - Belief that patients can reduce or quit smoking Family and friends' perception of e-cigarettes and their contribution to reducing addiction Perception of the electronic cigarette - Knowledge about electronic cigarette - Impact of e-cigarettes on health - Assessment of electronic cigarettes as a valid means of containing addiction or not Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). 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PMC10000786 | Delta like canonical notch ligand 4 (Dll4) expression levels in tumors are known to affect the efficacy of cancer therapies. This study aimed to develop a model to predict Dll4 expression levels in tumors using dynamic enhanced near-infrared (NIR) imaging with indocyanine green (ICG). Two rat-based consomic xenograft (CXM) strains of breast cancer with different Dll4 expression levels and eight congenic xenograft strains were studied. Principal component analysis (PCA) was used to visualize and segment tumors, and modified PCA techniques identified and analyzed tumor and normal regions of interest (ROIs). The average NIR intensity for each ROI was calculated from pixel brightness at each time interval, yielding easily interpretable features including the slope of initial ICG uptake, time to peak perfusion, and rate of ICG intensity change after reaching half-maximum intensity. Machine learning algorithms were applied to select discriminative features for classification, and model performance was evaluated with a confusion matrix, receiver operating characteristic curve, and area under the curve. The selected machine learning methods accurately identified host Dll4 expression alterations with sensitivity and specificity above 90%. This may enable stratification of patients for Dll4 targeted therapies. NIR imaging with ICG can noninvasively assess Dll4 expression levels in tumors and aid in effective decision making for cancer therapy. cancer tumor microenvironment modifier notch-DLL4 consomic xenograft model machine learning binary classification dynamic enhanced NIR imaging indocyanine green time series tumor detection NIH/NCI2R01CA193343 This work was supported by funding from NIH/NCI 2R01CA193343 to the MCW Cancer Center. pmc1. Introduction Breast cancer heterogeneity has been extensively studied and it has enabled the classification and categorization of tumors into molecular subtypes depending on the overexpression of antigens or hormone receptors on tumor cells . Identification of tumor subtypes improves cancer patients' care and prognosis by tailoring therapies to the subtypes . Breast and many other cancers are highly heritable, yet most causative variants are unknown, and most of the known risk variants are considered tumor-cell-autonomous, with far less emphasis placed on identifying the role of germline variants impacting the tumor microenvironment (TME). The TME is a complex and dynamic system that includes cancer cells, stromal cells, blood vessels, and extracellular matrix and plays a significant role both in tumor cell proliferation and in radiotherapy delivery and response . There is growing evidence that heritable modifiers of the tumor microenvironment can profoundly impact tumor behavior and response to diagnostic and therapeutic interventions . Tumor blood vessels have abnormal structure and function, which leads to heterogeneity in blood perfusion both temporally and spatially . This heterogeneity has multiple adverse consequences, including limiting the access of blood-borne drugs and effector immune cells to poorly perfused regions of tumors . As a result, these areas become hypoxic and have low extracellular pH . Hypoxia has been shown to play a significant role in tumor progression and metastasis by inducing genetic instability, angiogenesis, immunosuppression, inflammation, and resistance to cell death by apoptosis and autophagy . Anti-angiogenic drugs are designed to target the vasculature in order to starve tumors and prevent them from growing. However, recent studies have shown that the efficacy of these drugs may be limited by specific biomarkers and pathways associated with resistance . For example, it has been shown that some patients may not benefit from anti-VEGF therapies if they have elevated levels of plasma sVEGFR1 . Similar outcomes have been observed with increased levels of SDF1a and anti-VEGF therapies . Further the vascular TME and therapy response differs in primary tumor and metastasis sites and the anatomic location . Thus, characterizing angiogenesis in tumors holistically may have therapeutic implications. The process of tumor angiogenesis is closely regulated by a balance between promoting and suppressing angiogenic factors . Delta like canonical notch ligand 4 (Dll4) is a protein-coding gene that provides instructions for making a protein part of a signaling pathway known as the notch pathway, which is essential for the normal development of many tissues throughout the body, affecting cell functions , modulating tumor angiogenesis , promoting vessel maturation, and inhibiting vessel sprouting by inducing apoptosis of tip endothelial cells (TECs) . Dll4 is overexpressed in various types of cancer, including breast, ovarian, and colorectal cancer, and has been shown to promote tumor angiogenesis, growth, and metastasis by interacting with receptors on endothelial cells (ECs) . Blockade of Dll4 activity results in enhanced vessel sprouting and increased vascular permeability , but anti-Dll4 therapy has not been universally successful, as Dll4 has been shown to have both pro-tumorigenic and anti-tumorigenic effects depending on the context of its expression . We recently reported that Dll4 expression on the host TME rather than on tumor cells determines the EPR or enhanced permeation and retention effect in breast tumor xenografts and thus governs nanomedicine delivery and therapy response . Despite the increasing evidence about the function of germline genetic modifiers, such as Dll4, in TME heterogeneity and enhanced permeability and retention (EPR) effects, the underlying influencers have mainly remained unexplored because of the lack of a systematic approach to studying them. Therefore, we developed the Consomic Xenograft Model (CXM) as a strategy for mapping heritable modifiers of TME heterogeneity. In the CXM, human breast cancer cells are orthotopically implanted into consomic xenograft host strains. These strains are derived from two parental strains with different susceptibilities to breast cancer. Salt-sensitive (SS) rats were employed as a tumor promoting strain, while Brown Norway (BN) rats were used as a tumor suppressing strain. A sequence of consomic strains were generated with chromosomes of SS rats replaced by those of BN rats one at a time and used for breast tumor xenograft studies . Because the host backgrounds genetically differ by one chromosome, whereas the tumor cells are unvaried, any observed phenotypic differences are due to TME modifier(s) and can be linked to a single chromosome. These modifiers can be further localized by congenic mapping (inbred strains containing a given sub-chromosomal region in their genome). By combining CXM with dynamic epifluorescence near-infrared (DE-NIR) imaging, systemic injections of indocyanine green (ICG) through a tail vein in tumor-bearing rats, and multiparametric analysis of pharmacokinetic modeling, we localized and identified the function of the vascular-specific Dll4 allele on rat chromosome 3 (RNO3) as a heritable host TME modifier of EPR . The SS. strain with low-level expression of Dll4 (referred to as Dll4-) had significant tumor growth inhibition compared with the parental with higher expression of Dll4 (Dll4+), despite a paradoxical increase in tumor blood vessel density in Dll4+. Further analysis revealed that the changes in the Dll4+ tumors were accompanied by altered expression of Dll4, which was previously linked with nonproductive angiogenesis. Additionally, Dll4 was found to be co-localized within a host TME modifier locus (Chr3: 95-131 Mb) identified by congenic mapping and correlated with the phenotypic differences observed at the consomic level . The inheritance of functionally different Dll4 alleles can influence the efficacy of nanoparticle (NP) therapy, and previous results indicate that inherited microvascular distribution patterns, rather than overall NP uptake, ultimately determine the effectiveness of NP-mediated photothermal therapy (PTT). Consequently, patients with high endothelial Dll4 expression can be selected for treatment with anti-Dll4 targeted nanoparticles as opposed to patients with low Dll4 expression, where PEGylated nanoparticles will provide sufficient therapy response . Recent advances in dynamic vascular imaging techniques, such as DCE-MRI and perfusion computed tomography, have facilitated the investigation of the time kinetics of a contrast agent to extract multiple vascular parameters and have been successfully applied in clinical trials of anti-angiogenic drugs . However, these techniques have certain drawbacks, including a lack of high temporal resolution and the need for a heavy hardware system with sophisticated analysis software. Dynamic NIR fluorescence imaging, on the other hand, offers a sufficient and effective alternative to other dynamic vascular imaging techniques for characterizing germline-dependent vascular phenotypes . This has led to the combination of these modalities, such as in the paired agent MRI-coupled fluorescence tomography approach for noninvasive quantification of paired-agent uptake in response to anti-angiogenesis therapy in vivo . As the field of artificial intelligence continues to advance, researchers are increasingly utilizing AI techniques, particularly machine learning, to develop predictive models that can support effective decision making in various domains including cancer therapy selection . Previous research has investigated the use of machine learning algorithms to analyze near-infrared (NIR) signal intensity and perfusion patterns to differentiate between healthy, benign, and malignant tissue . This work demonstrated that the signal intensity time course of an FDA-cleared near-infrared dye ICG inflow during the wash-in phase and ICG outflow during the wash-out phase could serve as significant markers for tissue distinction. This finding offers a new method for noninvasive tissue distinction and has prognostic potential . However, there remains a need for further exploration of the use of machine learning for classifying host genetic tumor microenvironment (TME) modifiers and predicting therapy responses based on dynamic contrast-enhanced imaging of tumors, particularly DE-NIR fluorescence imaging data . 2. Hypothesis and Objective We hypothesize that the observation of subtle differences in vasculature structure and perfusion patterns characterized by ICG inflow and outflow using DE-NIR imaging could be used to differentiate between inherited tumor vascular microenvironment differences, such as Dll4 expression levels. We propose an experimental framework to noninvasively assess Dll4 expression levels in tumors based on the NIR signal intensity time course of perfusion patterns characterized by ICG time kinetics to develop a predictive model to support effective decision making in cancer therapy. Herein, we used two rat-based CXM strains of breast cancer, SSIL2Rg-(Dll4+) and SS.BN3IL2Rg- (Dll4-) , as well as eight congenic xenograft strains, CG1-CG8 , to assess the impact of germline TME vascular heterogeneity on the signal intensity of DE-NIR imaging with systemically delivered ICG. Principal component analysis (PCA)-based decomposition of time-dependent epifluorescence videos (image stacks) was used for visualization and anatomical segmentation of tumors, liver, lungs, and fat pads . In addition, we utilized modified principal component analysis (PCA)-based anatomical segmentation techniques to identify and analyze regions of interest (ROIs) representing potential tumors within the current dataset. To gather further information, we calculated the average NIR intensity for each ROI by analyzing the brightness of individual pixels at each time interval, resulting in a series of intensity measurements for each ROI. From this analysis, several easily interpretable features were extracted, including the slope of the initial uptake of ICG, the time it takes to reach peak perfusion, and the rate of ICG intensity changes once the half-maximum intensity is reached (which, to the best of our knowledge, has not been previously reported in the literature). We then applied a subset of machine learning algorithms, including Support Vector Machines (SVMs), Naive Bayesian Classifiers (NBCs), Generalized Additive Models (GAMs), Decision Trees (DTs), Nearest Neighbors (NN), and Logistic Regression (LR) to select the most discriminative features for classification. The performance of the model was evaluated using confusion matrix, receiver operating characteristic curve (ROC), and the area under the curve. To further evaluate our hypothesis of detecting Dll4 expression levels from DE-NIR imaging and test the generalizability of our framework, we conducted a secondary performance evaluation method using congenic groups with high and low Dll4 expression levels. The classification models were trained based on the selected features, and the performance of the model was tested on the remaining congenic groups. We demonstrate that robust ML methods can identify the alterations in host Dll4 expression from the tumor dynamic imaging datasets, and thus these methods can potentially stratify patients for Dll4 targeted therapies. 3. Materials and Methods All methods have been carried out in accordance with relevant guidelines and regulations. Approved protocols by the Medical College of Wisconsin Institutional Biosafety Committee (IBC) and Institutional Animal Care and Use Committee (IACUC) were followed. All live animal experiments are reported per the ARRIVE guidelines' recommendations . All results were rigorously adjusted for multiple comparisons. 3.1. Animals All animal protocols employed in this study were approved by the Institutional Animal Care and Use Committee (IACUC), Medical College of Wisconsin (MCW). The MCW has an Animal Welfare Assurance (Assurance number D16-00064 (A3102-01)) on file with the Office of Laboratory Animal Welfare, National Institutes of Health (NIH). Animal experiments were performed according to the relevant guidelines and regulations and in compliance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no.85-23, revised 1996). SS.BN3 rats were developed as part of the Consomic Xenograft Model at the Medical College of Wisconsin (MCW) . SS and SS.BN3 rats were purchased from the Rat Research Models Service Center at the Medical College of Wisconsin . All rats were provided reverse osmosis hyper-chlorinated water ad libitum. All animal experiments were performed on anesthetized animals. The animal was placed in a transparent induction chamber to induce anesthesia. Isoflurane was delivered through a precision vaporizer and compressed O2 to the chamber. For induction, the percentage of isoflurane was up to 5%. Once the animal was unconscious, it was removed from the chamber. The unconscious animal was then placed on a warm surface and fitted with a nose cone attached to the vaporizer in the presence of a scavenging system and oxygen source. At this point, the concentration of isoflurane was reduced to this level that maintained the correct anesthesia plane, usually between 0.5 and 3%. After the end of the experiment, or when other criteria for animal protocols were justified, rats were euthanized. Rats were placed in an approved euthanasia chamber and exposed to CO2 from a compressed gas cylinder until the animal was no longer breathing. To ensure death in rats, a pneumothorax was created via thoracotomy for rats weighing more than 200 g. For rats weighing less than 200 g, a pneumothorax was created, or a cervical dislocation was performed. As previously described , consomic strains (SS and SS.BN3 rats) were generated by sequentially replacing SS chromosomes with the outbred wild-type and tumor-resistant strain of Brown Norway (BN) rats referred to as SS.BN#, which are reported for their tumorigenic potential, where # refers to the chromosome number. These parental SS and consomic SS.BN# strains were genetically ablated by knocking down the IL2Rg gene to allow the grafting and growth of human cancer cell lines. Such immunocompromised strains are labeled as SSIL2Rg-(Dll4+) and SS.BN#IL2Rg-(Dll4-). Previous research has localized inherited modifier(s) of TME vascular heterogeneity to RNO3 by CXM mapping and further narrowed by congenic mapping to a 36 Mb locus containing Dll4 alleles with distinct vascular expression patterns in the SS. (Dll4-) and SSIL2Rg- (Dll4+) rat strains, and verified via species-specific RNA sequencing and immunohistochemistry that strains inheriting the SS Dll4 allele on chromosome 3 have higher Dll4 expression on tumor-associated endothelium and that the blood vessel tortuosity and dysfunction increased in . Since there are many other candidate alleles on chromosome 3 that could also potentially account for the observed differences in therapeutic efficacy between the SS. , to further investigate the potential contribution of Dll4 to inherited tumor vascular heterogeneity, eight novel SS. xenograft host strains (CG1 to CG8) were constructed by introgression of the F1 progeny and F2 generation to capture different regions of RNO3 by marker-assisted selection, as described previously . The exclusion congenic mapping localized a 7.9 Mb candidate region (marked by the SSLP marker D3Mgh11) that was associated with inherited tumor vascular heterogeneity and contained the Dll4 locus. As a result, eight new congenic strains CGN(s-e) were generated, where N (1 to 8) refers to the congenic group, while s and e refer to the starting and ending of Simple Sequence Length Polymorphism (SSLP) marker regions, respectively. This resulted in generating CG1(D3Rat26-D3Mgh30), CG2(D3Rat222-D3Got42), CG3(D3Rat222-D3Mco33), CG4(D3Rat164-D3Rat218), CG5(D3Rat26-D3Mco218), CG6(D3Rat86-D3Rat218), CG7(D3Mgh13-D3Rat218), and CG8(D3Rat160-D3Rat218) congenic groups . 3.2. Cell Culture and Triple-Negative Breast Cancer Xenografts As previously described , triple-negative breast cancer MDA-MB-231 cells were maintained in DMEM media (Sigma, Burlington, MA, USA) supplemented with 10% FBS (Gibco, New York, NY, USA) and 1% penicillin and streptomycin (Lonza, Cohasset, MN, USA) and incubated in 5% CO2 at 37 degC. These cells (6 x 106) in 50% Matrigel were orthotopically implanted into the mammary fat pads (MFP) of 6-week-old female Dll4+ (n = 8) and Dll4- (n = 17) rats and eight congenic strains CG1 (n = 19), CG2 (n = 2), CG3 (n = 26), CG4 (n = 12), CG5 (n = 28), CG6 (n = 12), CG7 (n = 5), and CG8 (n = 4) . Tumors were treated after 10 days of implantation at an approximate size of 600 mm3, consistent across all rat strains. 3.3. In Vivo NIR Fluorescence Imaging A customized NIR imaging system was assembled for imaging the rats. A bifurcated optical fiber bundle was used to deliver 785 nm excitation light (~5 mW/cm2 power at the surface, diode laser, ThorLabs Inc., Newton, NJ, USA) from two positions for uniform illumination of the entire rat body surface. A 16-bit deep-cooled intensified charge-coupled device camera (PIMAX4 ICCD, Princeton Instruments, Trenton, NJ, USA) equipped with 830 nm long-pass filter positioned following a holographic notch rejection filter in the optical path (ThorLabs Inc.) was used to image the rats through computer-controlled LightField(r) software (Teledyne Princeton Instruments, Trenton, NJ, USA) . Dynamic contrast-enhanced NIR fluorescence imaging was performed on anesthetized rats, as reported previously for 800 nm NIR imaging . In this study, the setup was used for imaging the whole body. A total of 133 rats (Dll4+ (n = 8), Dll4- (n = 17)) and eight congenic strains CG1 (n = 19), CG2 (n = 2), CG3 (n = 26), CG4 (n = 12), CG5 (n = 28), CG6 (n = 12), CG7 (n = 5), and CG8 (n = 4) were imaged. NIR imaging was performed for approximately 6 min following ICG injection with the CCD array hardware binned to 256 x 256 with a frame rate of 10.6 fps. A total of 3000 frames were captured for each imaging case, including about 50 frames for background correction. ICG (MP Biomedicals) was delivered in an intravenous bolus of 1200 mM ICG/200 g body weight into the tail vein via a catheter with a 32-gauge needle tip connected to a syringe pump (Harvard Apparatus PHD 2000 syringe pump, Holliston, MA, USA) operated at a speed of 0.2 mL/s. The injected volume was calibrated to provide a body-weight-equilibrated dose to each rat. 3.4. Denoising and Motion Correction Image processing and data analysis were performed in MATLAB (R2021b MATHWORKS Inc.) software. The time-dependent image frames were assembled as 3D arrays (two spatial and one temporal dimensions) for all animals. A custom-designed breathing correction method with a low-pass temporal filter combined with a 1D wavelet-based denoising was used to filter the high-frequency jitter generated by the animal's respiratory motion from the fluorescence kinetic sequences of each pixel, as described previously . An average of pre-ICG injection frames (acquired in the ~5 s before ICG injection) was used as background, incorporating contributions from CCD noise and excitation light leakage from emission filters and subtracted from all the frames. (Refer to Video S1 for respiratory motion corrected time course images of ICG biodistribution). 3.5. Principal Component Analysis for Extraction of Spatial Patterns of Internal Organs First, motion correction and background subtraction were performed on the imaging data described in the previous steps. This was carried out to remove any potential artifacts or noise that could affect the accuracy of the principal component analysis. Next, the data were decomposed by PCA along the time dimension using MATLAB software following the previously published methods . This resulted in converting the imaging data to a k-component vector for each pixel, where k is the number of time-frames in the original dataset. The PCA on the dynamic fluorescence image was used to extract spatial patterns of internal organs linked to statistically similar kinetic behaviors. This was carried out by comparing the k-component vectors for each pixel and identifying those that displayed similar patterns over time. The contribution of the first six principal components on a time basis is illustrated in Figure S1. 3.6. PCA Ranking Tumor Detection The ROI detection module employed three steps in order to identify the tumor area in images : (1) spatial alignment, (2) PCA ranking and selection, and (3) ROI selection and masking. First, images were registered to a reference image using a rigid body transformation in order to ensure consistent spatial alignment for the detection of the tumor area in subsequent steps. The variability in the visual appearance of internal organs or tumors within certain principal components necessitated the adaptation of existing methods for ranking normalized components based on their two-dimensional cross-correlation (2DCC) with a reference image containing the tumor. This was achieved by generating a stack of the first ten normalized principal components and applying a two-dimensional cross-correlation function (xcorr2 in MATLAB) to each component. The principal components were ranked according to the correlation scores obtained and a semi-automatic algorithm was then utilized to carry out the subsequent two steps for tumor ROI generation. To select the appropriate principal component for tumor identification, the algorithm prioritized the PC with the highest likelihood of containing the tumor tissue based on the ranking from the previous step. Once the proper PC was selected, we used 2DCC to estimate the tumor's location in the frame. This was performed by creating a stack of four reference images using PCs that clearly visualized the tumor and generated a bounding box around the tumor. Next, we applied basic morphological dilation and image arithmetic operations to emphasize the tumor's boundaries. A threshold was then applied to separate the tumor from the background. Finally, we used Blob Analysis (Computer Vision ToolboxTM, MATLAB, MathWorks, MA, USA) to extract the centroid and exact location of the tumor in the frame, generating a region of interest (ROI). The use of a graphic user-interface-enabled semi-automatic platform allowed for real-time evaluation and adjustment of the algorithm's performance if necessary. 3.7. Video Processing The underlying assumption for the NIR fluorescence intensity video analysis is that intensity was proportional to the ICG concentration in the tissue, which has been shown in the literature . From each available video, p, between 4 and 5 fluorescence time series are extracted, one based on the tumor ROI generated from earlier steps, and between three and four from mammary fat pads (MFPs) . For each ROI, r, and each included time step, t, the mean brightness of the pixels inside that ROI in the NIR video is stored as Ip,r(t). The result is a collection of time series Ip,r(t), where p ranges from 1 to N (the number of animals in each group in our dataset), r ranges from 4 to the number of ROIs generated in the specific animal (up to 5, 1 tumor and 3 or 4 fat pads), and t from 0 to 3000 . 3.8. Peak and Latency Estimation First, we started with smoothing the data to exclude any potential noise from motion artifacts. The smoothing was conducted using a Savitzky-Golay filter of order 3 with window length 31 . The parameters for this filter were determined through manual annotation of peak and latency in a subset of the data, with the goal of minimizing average estimation error. Subsequently, we used a MATLAB function to identify the time point of maximum intensity, which corresponded to the peak of the fluorescence signal. We employed a custom latency detector script that analyzed the smoothed derivative of the curves and identified the first "robust" zero crossing as the latency point (Lp,r(t)) . 3.9. Feature Design The features were chosen in two steps as previously described : first, the following characteristic numbers were chosen for each normalized time series individually. The time to peak (TTPp,r) is simply the time difference between the peak intensity time point and the latency Lp,r. The upslope (Up,r) is computed as (1) Up,r=1-Ip,r(Lp,r)TTPp,r, which is the average slope between initial ICG arrival and the peak. The downslopes (DS) are the downslopes between the peak and S seconds further:(2) Dp,r=1-Ip,r(Lp,r+TTPp,r+S)S,S{2,4,5,6,8,10,12,14,16,18,20,23,25,30,35,40,50,60,70,80}. The time ratio (TR) is the ratio between TTPp and when Ip(t) reaches half the peak values. The half intensity forward (HIF) is the average slopes between when Ip,r(t) reaches half the peak values (T1/2p,r) and S seconds further, so (3) HIFp,r=1-Ip,r(Lp,r+T1/2p,r+S)I,S{2,4,5,6,8,10,12,14,16,18,20,23,25,30,35,40,50,60,70,80}. To increase the robustness of estimated downslope-based and HIF-based features, we introduced a window around Lp,r+TTPp,r+S and Lp,r+T1/2p,r+S time steps taking the median of those downslopes:(4) DSp,r,avg=median sWindow D(S+s)p,r, (5) HIFp,r,avg=median sWindow D(S+s)p,r,Window={-1.5,-1.3,...,1.3,1.5}seconds. These features provide insight into the tumor's ICG uptake and decay. U and TTP relate to the initial uptake of ICG, while DS relates to the decay of ICG fluorescence . TR is a measure of the temporal inhomogeneity of the initial uptake, and HIF is a measure of the temporal inhomogeneity of both the initial uptake and decay of ICG fluorescence. To address inter-animal variation, we propose a feature design that relates the features of tumor intensity (Fr=tumor) to the features of healthy tissue intensity (Fr=fatpad) in the same animal. The median value of a feature across the healthy tissue (fat pad) is chosen as a reference value, and each feature is defined as its percentage difference to that reference value. This results in a single normalized feature for each animal. By using the median as an average that is robust to outliers, our feature design allows for a more accurate comparison of tumor intensity across different animals. (6) Fn =Ftumor-median(Ffat pad)median(Ffat pad), 3.10. Classification Algorithms and Feature Selection The feature extraction and design required specialized knowledge of the tumor microenvironment (TME) and its impact on near-infrared intensity. However, the classification based on the inheritances of Dll4 can be considered a standard binary classification problem with feature selection as a sub-problem: given the features of an animal for which the Dll4 inheritance is unknown, we want to assign the label "Dll4high" or "Dll4low" to it. We also want to investigate which small subset of features performs best . We restricted ourselves to a subset of available ML algorithms which were reported to perform best with intensity time series classification. , We excluded neural networks from consideration and evaluated Support Vector Machines (SVMs), Naive Bayesian Classifiers (NBs), Generalized Additive Models (GAMs), Decision Trees (DTs), Nearest Neighbors (NN), and Logistic Regression (LR). 3.11. Primary Classification DTs and SVMs using the full set of 86 features were trained. Each ML model was tuned to the training set in an internal cross-validation procedure of 10-fold. This process was repeated 20 times, and the performance metrics are reported as classifier performance. One of the key metrics used to evaluate the performance of a classification algorithm is accuracy, which measures the proportion of correctly classified instances in the dataset. Other important metrics include sensitivity, which measures the proportion of positive instances that were correctly classified, and specificity, which measures the proportion of negative instances that were correctly classified. In our case, we are interested in classifying animals as either Dll4high or Dll4low, and we use the average metric (Ascore) for each pair of groups to evaluate the performance of the different classification algorithms. The Ascore for a given pair of groups is calculated as the average of accuracy, sensitivity, and specificity, as follows:(7) Ascore (Dll4high| Dll4low)=Accuracy+Sensitivity+Specificity3, 3.12. Congenic Pair Selection In this study, we describe a two-step method for selecting congenic pairs with high and low Dll4 expression levels for feature selection and hypothesis testing. This method was used to identify well-behaved pairs for binary classification and to identify the pair with the highest classification performance: The primary selection of congenic pairs is based on their primary classification scores (Ascore) and their performance against parental strains using all available features. The secondary selection is based on the classification performance using the 2 best-performing features. The congenic pair selection process involved the selection of all possible combinations of congenic pairs with high and low Dll4 expression levels with n > 5, resulting in a total of 12 pairs: Dll4+|Dll4-, Dll4+|CG3-, Dll4+|CG4-, Dll4-|CG1+, Dll4-|CG5+, Dll4-|CG6-, CG1+|CG3-, CG1+|CG4-, CG3-|CG5+, CG3-|CG6+, CG4-|CG5+, CG4-|CG6+. It should be noted that we only included subgroups with n > 5 in our analysis, to avoid introducing noise into the feature selection process. This is because smaller sample sizes can be more prone to variability and may not represent the larger population . Therefore, we focused on more significant subgroups to ensure our feature selection process was robust and reliable. These 12 pairs have gone through our primary classification algorithm with a split of 75%-25% for testing and training with seeded randomization and proportional distribution of each group in training and testing datasets. Each ML model was tuned to the training set in an internal cross-validation procedure of 10-fold and evaluated by its performance on the test set. This process was repeated 20 times, and the best Ascore was reported as the metric of classifier performance. The result of this step was used to identify well-behaved congenic pairs for binary classification by calculating the separation score (Sscore) for each pair of congenic groups with CG#+ and CG#- as below, which is bound between 0 and 1 and reported in Table 1:(8) Sscore (CG#+| CG#-)=Ascore (Dll4+| CG#-)+Ascore (CG#+| Dll4-)+2* Ascore (CG#+| CG#-)4, The congenic pairs with Sscore above 80% were selected for secondary congenic pair selection. For the secondary selection, we evaluated the classification performance (Ascore) of each pair determined this time by using the two most effective features as described in Section 3.13. We used a 75%-25% split for testing and training, with randomization to ensure a proportionate representation of each group in both datasets. The pair with the highest classification performance, Ascore, based on the two most effective features was selected for the final classification model training. To ensure a high-quality classification model, we set a minimum threshold for the Ascore of 0.70 for inclusion in the feature selection process. Congenic groups with an Ascore below 0.70 were considered to have an insignificant contribution to the classification model and were excluded from further consideration in the feature selection process. This approach allowed us to focus on the most informative feature pairs, improving the overall classification performance of our machine learning model. Each machine learning model was tuned to the training set using a 10-fold cross-validation procedure and evaluated based on its performance on the test set. We repeated this process 20 times and reported the best Ascore as the classifier's performance. 3.13. Feature Selection and Secondary Classification We selected the best pair of features in terms of achieved sensitivity, specificity, and accuracy by a two-step procedure as previously reported: DTs and SVMs using the full set of 86 features were trained, and recursive feature elimination (RFE) was performed to refine a much smaller set of best-performing features . RFE is a widely used machine learning classification algorithm that helps in reducing the dimensionality of feature space and selecting a small subset of features that yield the best classification performance. This was achieved through an iterative procedure that uses a ranking criterion to eliminate features one or more at a time. The RFE algorithm started by selecting a subset of features and training a model on this subset. The features were then ranked based on their contribution to the model's performance, and the least important feature was eliminated. The process was then repeated with the remaining features, and the best subset of features was selected based on a model selection criterion . One of the main advantages of RFE is that it helps to reduce the risk of overfitting when the number of features is large, and the number of training patterns is comparatively small . This is because the algorithm selects only a subset of features that are relevant to the classification task, and this helps to avoid the inclusion of irrelevant and redundant features. RFE can be used in conjunction with other techniques such as regularization and support vector machines (SVMs) to further improve the performance of the classification model. In addition, projection methods such as principal component analysis can reduce the feature space's dimensionality before applying RFE . We used a k-fold cross-validation strategy to assess the performance of our model. We also reserved a portion of the training data for primary testing of the model after hyperparameter optimization. Our experiments were conducted with 20 random splits of the training and test datasets, and the mean performance metrics were reported for sensitivity, specificity, and accuracy as Ascore. To facilitate the interpretation of our results, we limited the number of final features to two. Furthermore, given the small size of our dataset, there was no justification for using high-dimensional feature spaces. 3.14. Data Augmentation The use of data augmentation has become a popular technique in machine learning and deep learning, especially in the field of computer vision. Data augmentation involves applying random transformations to the training dataset to increase its diversity and improve the performance of a model. In this study, we used data augmentation on raw near-infrared (NIR) image stacks to evaluate the robustness of a classification model. We used a dataset of 3000 frames of the original raw 256 x 256 NIR images for this part of our study. These images were augmented using TensorFlow and the Keras API, which allowed us to apply random transformations to the dataset. The transformations included random rotation followed by a horizontal flipping, and up to 2% rescaling. 3.15. Training and Testing Dataset The final training and testing dataset for the machine learning models was determined by the outcome of the congenic pair selection and feature selection steps. This dataset included all congenic groups except for Dll4+ and Dll4-, as well as the selected CG#+ and CG#- groups in the previous step. This step was conducted separately for the original dataset and augmented dataset. The models were trained using 10-fold cross-validation and a portion of the training data was reserved for testing after hyperparameter optimization, with 25% for the original dataset and 20% for the augmented dataset. The performance of the models was assessed using a confusion matrix, receiver operating characteristic curve (ROC), and the area under the curve. 3.16. Statistical Analysis Repeated measures models are a powerful tool in statistical analysis that allow researchers to study the effects of different factors on a given outcome while accounting for the inherent dependence of multiple measurements taken on the same subject. In this study, a mixed effects model with appropriate time varying covariates was used to analyze the average fluorescence intensity of indocyanine green (ICG) in the tumor with multiple measurements per subject, with the subject number serving as the repeated measure indicator and the rat strain serving as a covariate. This allows for flexible time-based modeling when using multiple measures, likely dependent from the same animal . Customized scripts in MATLAB were used to generate the fitted coefficients, covariance parameters, design matrix, error degrees of freedom, and within-subjects factor names for the repeated measures model. The output was then analyzed with a multiple comparison of the estimated marginal means based on the variable strain, using the Tukey-Kramer test statistic . This allowed estimation of multiplicity-adjusted p-values for the post hoc comparisons, which indicate whether the groups significantly differed with respect to strain. The data were then visualized as a p-value matrix, providing a clear illustration of the significant differences between groups. 3.17. Data Availability This study employed the established consomic rat models SS and SS.BN3 as well as our congenic strains CG1 to CG8. The publicly accessible and NIH-supported Rat Genome Database (rgd.mcw.edu) catalogs has tools to explore the genotype and phenotype information for the SS (Dll4+) and SS.BN3 (Dll4-) and congenic strains under strain records: Dll4+ (RGDID:61499), Dll4- (RGDID:1358154), CG1 (RGDID:155782881), CG2 (RGDID:155782883), CG3 (RGDID:155782884), CG4 (RGDID:155791428), CG5 (RGDID:155791426), CG6 (RGDID:155791430), CG7 (RGDID:155791429), and CG8 (RGDID:155791427). 4. Results and Discussion 4.1. Dynamic Contrast-Enhanced NIR Fluorescence Imaging and Tumor Detection Dynamic contrast-enhanced NIR fluorescence imaging has been widely used for tumor detection in various studies . The use of NIR imaging allows for the visualization of internal organs and tissues without the need for invasive procedures, which can be particularly useful in detecting tumors due to their vascular heterogeneity compared to surrounding healthy tissues. In previous studies, the use of principal component analysis (PCA) on the time domain of dynamic fluorescence images was utilized to extract spatial patterns of internal organs linked to statistically similar kinetic behaviors, such as liver, kidneys, lungs, and various tumors . However, this technique required manual inspection and selection of proper principal components, which was time consuming and prone to human error and bias. In order to overcome the limitations present in the current dataset, we implemented a modified method that utilizes near-infrared imaging and principal component analysis to detect tumors with high accuracy and without the need for manual correction . The use of principal component analysis in this context not only allows for dimensionality reduction and noise removal but also enhances the robustness and efficiency of the method. Our study also implemented a novel method of ranking PCA components based on the 2D cross-correlation of a reference image containing the tumor. This added to the simplicity and computational efficiency of the framework. However, it should be noted that this method may not be effective for detecting tumors with random locations. On the other hand, it could be useful for detecting tumors or tissues of interest with high localization, such as the lungs, liver, and kidney, and lesions in breast tissue. Overall, our method shows potential for improving the accuracy and efficiency of tumor detection using NIR imaging and PCA . However, further experimentation is needed to expand the framework to a general tumor detection algorithm. 4.2. Dll4 and Its Effect on the NIR Time Series The analysis of the average fluorescence intensity of indocyanine green (ICG) in the tumor tissue of Dll4+ and bearing triple-negative breast cancer (TNBC) tumors revealed that ICG uptake occurred more rapidly in and was retained for longer periods of time compared to Dll4+ hosts . This indicates systemic differences in vascular function between the two rat strains. Our previous histological data showed that Dll4+ tumors have a higher vascular density and tortuosity, indicating a genetic microenvironment that promotes nonproductive angiogenesis . This is further supported by the slower ICG wash-out observed in the Dll4+ tumors. These findings provide insight into the effects of host genetics on tumor angiogenesis and suggest potential therapeutic targets for TNBC. In order to further investigate the role of Dll4 in vascular function in tumors, we divided chromosome 3 into regions with and without the Dll4 gene in congenic rat strains and then examined the ICG fluorescence intensity of tumors in Dll4-high and Dll4-low rats (Video S2) . Our findings reveal significant systemic differences in vascular function between tumors in Dll4+ and (parental strains), indicating the critical role of the Dll4 gene in tumor angiogenic response . However, analysis of the ICG fluorescence intensity of tumors for individual strains reveals more complex behavior than the obvious differences in wash-in and wash-out patterns observed between Dll4+ and Dll4-. This supports the need for further investigation into the impact of Dll4 on NIR time series signatures and the potential use of Dll4-directed therapies for cancer treatment. It is worth noting that although there are significant differences in Dll4-low vs. Dll4-high rat strains (when all the strains of Dll4 expression levels are combined), they are inconsistent with the observations made in Dll4+ and . These results have significant implications for developing novel therapies that target Dll4 and other host TME modifiers involved in angiogenesis, as they demonstrate the critical role of these genes in tumor vascular function and angiogenic response. Additionally, our research further highlights the capricious nature of the NIR signal, which is influenced by various heritable tumor microenvironments across different groups, as shown in Figure 4b,c. We aim to illustrate and categorize the impact of the Dll4 expression level on the NIR signal through this erratic behavior. We used a repeated measures model to analyze the average fluorescence intensity of ICG in the tumor over time, with the rat strain serving as a covariate. Figure 5a,b show the estimated response covariances matrix, which is the covariance of the repeated measures. The higher values in this matrix indicate the time points at which groups experience the greatest differences. By projecting the diagonal of the covariance matrix onto the time axis , we were able to visualize the amount of difference between groups over time. This projection, when compared to the average fluorescence intensity of ICG in the tumor , showed the strongest differences between groups at the points where the NIR signal regions from half of its peak value to the peak value and at the tail of the curve, which are measures of the temporal inhomogeneity of the initial uptake and the decay of ICG fluorescence, were found to be particularly useful in discriminating between groups with different levels of Dll4 expression. This projection of the diagonal of the estimated response covariances matrix on the time curve can be used in feature design to focus on regions with the maximum amount of useful information for discriminating between groups and, subsequently, between classes with different levels of Dll4 expression. This could potentially improve the accuracy of tumor classification and ultimately improve therapy outcomes. Our repeated measures model, which included responses as measurements and strains as predictor variables, allowed us to conduct multiple comparisons of estimated marginal means between groups. The resulting p-value matrix revealed significant differences in estimated marginal means between the Dll4+ and , with a p-value of 4.71 x 10-7. In addition, we observed significant differences between Dll4+ and CG3, CG4, and CG8, with p-values of 1.67 x 10-5, 7.03 x 10-7, and 2.18 x 10-3, respectively. For each group pair with high and low Dll4 expression levels, the separation score was calculated. First each of Dll4+|CG#-, CG#+|Dll4-, and CG#+|CG#- went through our classification algorithm with 10-fold cross-validation using Nearest Neighborhood, Linear SVM, RBF SVM, Decision Tree, Naive Bayes, and Logistic Regression models. The highest average classification metrics (Accuracy + Specificity + Sensitivity)/3 for Dll4+|CG#-, CG#+| CG#+|CG#-) was used to calculate the separation score (Score Dll4+|CG#- + Score CG#+|Dll4- + 2 x Score CG#+|CG#-)/4. Furthermore, our analysis showed significant differences between CG5 and CG6, with p-values of 8.70 x 10-3 and 2.58 x 10-4, respectively. This supports the hypothesis that Dll4 expression levels can act as a heritable TME modifier on NIR time series intensity. However, the smallest p-value between Dll4+ and that there are other factors on chromosome 3, in addition to Dll4, that contribute to the observed differences in the NIR time series signature between these groups. In contrast, no significant differences were found between the congenic strains with low levels of Dll4 expression (CG2, CG3, CG4, CG7, and CG8). This further supports the notion that Dll4 plays a crucial role in determining tumor vascular function and NIR time series intensity. Among the congenic groups, the most significant differences were observed between CG5, CG6, and CG3, CG4 from the Dll4-high and Dll4-low groups, respectively. Notably, the differences were most significant between CG4 and CG6, with a p-value of 0.0003. This suggests that very narrow regions of differences on chromosome 3 between these two groups, one containing Dll4 and the other lacking it, have a significant effect on the NIR time series signature. 4.3. Primary Classification and Congenic Dissimilarity The relationship between Dll4 expression and classification performance was analyzed using a total of 12 congenic pairs with n > 5 based on their levels of Dll4 expression (Dll4+|Dll4-, Dll4+|CG3, Dll4+|CG4, Dll4-|CG1, Dll4-|CG5, Dll4-|CG6, CG1|CG3, CG1|CG4, CG3|CG5, CG3|CG6, CG4|CG5, and CG4|CG6). The pairs were then subjected to a primary classification algorithm and their mean performance metrics, the Ascore, were calculated and reported in Table 1. The congenic pairs with low levels of Dll4 expression showed a mean Ascore of 0.91 +/- 0.01, indicating a high level of classification performance when compared to the Dll4+ parental strain. In contrast, the congenic pairs with high levels of Dll4 expression showed a mean Ascore of 0.8 +/- 0.05 when classified against the strain. Among the congenic pairs, the CG5|CG4 pair demonstrated the highest Ascore of 0.8, followed by the CG6|CG4 and CG6|CG3 pairs with Ascore values of 0.78 and 0.77, respectively. The results of the Ascore calculation are visualized in Figure 5e through a Sankey diagram. To account for potential differences between the congenic pairs and the parental pairs, the Sscore was calculated. The CG5|CG4, CG6|CG3, and CG6|CG4 pairs showed the highest Sscore values of 0.84, 0.84, and 0.85, respectively, and were selected for the feature selection step. These results align with the multiple comparison of estimated marginal means between groups, indicating that CG5|CG4, CG6|CG3, and CG6|CG4 show the strongest differences in classification performance. 4.4. Feature Selection RFE is a wrapper method that evaluates the entire classification algorithm and has shown improved classification accuracy and reduced overfitting compared to other feature selection methods . However, RFE can be sensitive to noise and irrelevant features, leading to suboptimal feature subsets and reduced classification performance. Additionally, RFE is computationally intensive, which can pose a challenge for large datasets with a high number of features. Despite these limitations, RFE remains a valuable tool for selecting an optimal subset of features that maximizes classification performance . To address these limitations, we performed feature selection in two steps to optimize the selection process and improve the performance of the classifier. First, we used RFE to select only two features out of the 86 available features for congenic pairs Dll4+|Dll4-, Dll4+|CG4, CG5|Dll4-, CG5|CG4, CG6|Dll4-, and CG6|CG4. The CG3 and its combinations (CG6|CG3, CG5|CG3, and Dll4+|CG3) were dropped from the feature selection process as the performances of the classifiers, the Ascore, using only two features were below 0.70, and lower than the other strains. The congenic pairs CG5|CG4 and CG6|CG4 as well as the parental and consomic group Dll4+| through our feature selection algorithm, and for each pair the two best-performing classification algorithms based on Ascore and associated feature pair were reported (Table 2). The highest Ascore for the two best-performing models for CG5|CG4 was 0.78 +- 0.04 compared to CG6|CG4 with an Ascore of 0.72 +- 0.19 and 0.72 +- 0.22, resulting in the selection of CG5|CG4 for final congenic pair selection. Finally, from each pair of Dll4+|Dll4-, Dll4+|CG4, CG5|Dll4, and CG4|CG5, four of the best performing features regardless of the ML model were chosen and were used as a collection of features for the final feature selection (Table 2). A combination of parental and consomic groups and the final selected congenic pair (Dll4+, Dll4-, CG5, and CG4) was used to select the final feature pair out of the 16 selected features, resulting in the selection of HIF5_avg and HIF50_avg as the best-performing features. 4.5. Performance of the Classification Models Based on the Selected Features To evaluate the performance of the selected features, we trained datasets consisting of all the remaining congenic groups excluding the Dll4+, Dll4-, CG4, and CG5 (CG1 to CG3 and CG5 to CG8) using 10-fold cross-validation and keeping 25% of the dataset for testing the trained models. This allowed us to assess the generalizability of our model and test it on previously unseen datasets. The results of this step are reported as a confusion matrix, ROC curve, and AUC , as well as general classification metrics (Table 3). The best-performing models based on the selected features were SVM and KNN, with sensitivity and specificity of 1.00 and 0.81 and 1.00 and 0.75, respectively. In order to further assess the effectiveness of our model, the selected features, and the generated congenic pair, we generated an augmented dataset consisting of all remaining congenic pairs excluding Dll4+, Dll4-, CG4 and CG5 (CG1 to CG3 and CG5 to CG8, with random variations in rotation, horizontal flip, and limited scaling (up to +-2%) to increase the diversity of the dataset. This resulted in a total of 606 data points. The performance of the models was evaluated using 10-fold cross-validation and a 20% hold out. The results of this step were reported as a confusion matrix, ROC curve, AUC , and overall classification metrics (Table 4). The best-performing models based on the selected features were SVM and KNN, with sensitivity and specificity of 0.97 and 0.91, and 0.97 and 0.92, respectively. These results align closely with the performance of the models over the original dataset, indicating the generalizability of our framework. It is noteworthy that of the 16 most contributing features used to select the final feature pair, 12 were the newly proposed HIF features, and the other 4 were DS features, which we previously reported . Additionally, the HIF features were amongst the best features for identifying genetic TME modifiers. The relationship between covariance of the repeated measures and optimal feature design in machine learning classification algorithms is an essential factor in developing effective classification algorithms. Combined with our recent report , our analysis found that the DS and HIF features, which are generated in regions where the NIR signal varies from half of its peak value to the peak value and at the tail of the curve, were particularly effective in discriminating between benign/malignant tumors (DS features) and groups with different levels of Dll4 expression (HIF features). Furthermore, the projection of the covariance matrix onto the time axis revealed similar regions, indicating a relationship between this projection and optimal feature design. These findings have significant implications for feature design in machine learning classification algorithms. By focusing on the regions with the greatest amount of useful information for discrimination, we can design features specifically to capture these differences and improve the accuracy of tumor classification. This can ultimately lead to better therapy outcomes for patients. It is worth noting that this relationship between the covariance of the repeated measures and optimal feature design is not limited to HIF features and the specific context of our analysis. In general, considering the covariance of repeated measures can provide valuable information for identifying key regions and designing effective features for machine learning classification algorithms. 5. Conclusions Dynamic vascular imaging techniques such as DCE-MRI and perfusion CT are used to extract multiple vascular parameters and have been used in clinical trials of anti-angiogenic drugs. However, these techniques have limitations, such as low temporal resolution and the need for specialized hardware and software . To overcome these limitations, dynamic near-infrared (NIR) fluorescence imaging can serve as an effective alternative for characterizing germline-dependent vascular phenotypes. It can be combined with other modalities, such as in a paired-agent or multimodal MRI and fluorescence tomography approaches for noninvasive quantification of response to anti-angiogenesis therapy and classifying in vivo vascular phenotypes . Furthermore, DE-NIR imaging, as a potential alternative for characterizing germline-dependent vascular phenotypes in preclinical models, can be extended to clinical modalities upon validation with cross-sectional dynamic contrast enhanced imaging. The present study proposes that by combining DE-NIR imaging and machine learning algorithms with consomic xenograft models with human tumors, the role of inherited notch protein Dll4 (rat variant of delta like canonical ligand 4) expression specifically in the host vascular microenvironment can be studied. Specifically, in the context of breast cancer, where different genetic subtypes can impact treatment outcomes, identifying patients with high or low DLL4 (human variant of delta like canonical ligand 4) expression levels through noninvasive imaging could assist in selecting personalized treatment options. Nonetheless, the study authors acknowledge notable differences between the rat model utilized in the study and the human system, which could affect the generalization of the findings to human cases, as in human tumors DLL4 expression maybe be both on tumor cells and host vasculature, whereas in our CXM model, we focused specifically on the inherited variation in rat-derived host vasculature Dll4 expression in human xenograft tumors. Such differences may result in amplification or suppression of vascular phenotype responses if both tumor cells and the host microenvironment express high levels or contrasting levels of DLL4. However, even in that case, dynamic imaging will be useful in identifying patients likely to respond better to DLL4 targeted therapies. Future studies will be necessary to validate this study's findings, to assess the reliability and validity of the developed imaging and machine learning algorithms in a large and diverse patient population to determine if contrast agent kinetic profiles observed in human DCE-MRI or dyna-CT imaging datasets for primary and/or metastatic disease differ in human patients with high vs. low DLL4 expression. In the metastatic setting, where surgery is no longer an option, a machine-learning-enabled analysis of dynamic contrast-enhanced imaging will be valuable to assess the expression levels of DLL4 and guide therapy selection, especially in cases where a biopsy is not taken or if biopsy results are inconclusive . Human anti-DLL4 antibodies have been reported for cancer treatment . In one study, immunotoxin DLL4Nb-PE was developed, potentially as a cell cytotoxic agent and angiogenesis maturation inhibitor . Another study successfully developed a bispecific monoclonal antibody that targets both human DLL4 and VEGF and showed efficacy in inhibiting proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVEC) . In a phase 1a trial, navicixizumab, a bispecific antibody that inhibits DLL4 and VEGF, was tested in refractory solid tumor patients and showed the potential to inhibit tumor growth . While DLL4 blockade is an attractive therapy, long-term extended use of DLL4 mAbs has demonstrated concerning off-target effects . Pharmacokinetic modulation of DLL4 mAbs may reduce off-target effects , such as via short-term administration or by focusing on patients where dynamic contrast imaging indicates a high DLL4 vascular phenotype. As we have shown in prior work, high vascular DLL4 expressing tumors may also be susceptible to DLL4 targeted nanomedicine or as combination therapy with anti-DLL4 monoclonal antibodies with nanomedicine drugs such as nab-paclitaxel (Abraxane) or Liposomal Doxorubicin (DoxilTM). The use of noninvasive DE-NIR imaging to detect heritable TME modifiers is significant for several reasons. First, this method allows for the identification of potential modifiers without the need for invasive procedures, reducing the potential for discomfort and complications for patients. Second, the use of machine learning and DE-NIR imaging to develop a predictive model for cancer nanomedicine therapy can support effective decision making in the treatment process. While data processing and preparation and algorithm training can be complex, the resulting algorithms are simple and allow for the prediction of heterogeneity in a single step using ROI brightness measurements. Interestingly, traditional features such as time-to-peak and upslope do not appear in our selection of the most discriminative features. However, two novel features derived from HIF (HIF5_avg and HIF50_avg), which is a measure of the temporal inhomogeneity of both the initial uptake and decay of ICG fluorescence, were identified. It is important to note that the training and testing sets used in this study are minimal, and therefore the high accuracy rates obtained should be interpreted with caution. Further research with larger datasets will be necessary to assess the reliability and validity of these findings with confidence. We have reported novel dynamic enhanced near-infrared (NIR) fluorescence imaging and machine learning algorithms to noninvasively assess Dll4 expression levels in tumors. Our results showed that observation of subtle differences in vasculature structure and perfusion patterns characterized by ICG time kinetics could be used to differentiate between inherited tumor vascular microenvironment differences, such as Dll4 expression levels. Additionally, our analysis demonstrated the importance of considering the covariance of the repeated measures in the design of features for machine learning classification algorithms. By utilizing this information, we can improve the accuracy of tumor classification and ultimately improve therapy outcomes for patients. To summarize, based on our recent study, we investigated the impact of genetically heterogeneous notch-Dll4 inheritance on the contrast agent uptake and clearance in triple-negative breast cancer xenografts. The differences in Dll4 inheritance have been shown to impact nanomedicine biodistribution, pharmacokinetics, and therapy response in our prior work. Thus, our results indicated that imaging can be potentially employed for selecting patients for Dll4-directed therapies by identify host microenvironments with low-expressing Dll4 inheritance. This further suggests that the success of nanomedicine might depend on hereditary tumor microenvironment genes, regardless of tumor type. Additionally, host genes such as Dll4 can affect individual differences in NP uptake and response to NP-mediated therapies, providing the potential for more effective personalized Dll4 targeted nanomedicine for therapy-resistant hosts. Further studies are needed to validate these findings and explore the potential clinical applications of this approach. Supplementary Materials The following supporting information can be downloaded at: Figure S1: Principal component analysis; Video S1: RespMotionCorretion; Video S2: ICG_NIR_All_Strains. Click here for additional data file. Author Contributions Conceptualization, S.S. and A.J.; Data curation, S.S. and J.J.; Formal analysis, S.S. and A.J.; Funding acquisition, A.J.; Investigation, S.S.; Methodology, S.S., A.B., A.G., M.F. and A.J.; Project administration, A.J.; Resources, A.J.; Software, S.S., M.Z. and J.E.; Supervision, S.Z. and A.J.; Validation, S.S.; Visualization, S.S.; Writing--original draft, S.S.; Writing--review and editing, J.E., A.B. and A.J. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement All methods have been carried out in accordance with relevant guidelines and regulations. Approved protocols by the Medical College of Wisconsin Institutional Biosafety Committee (IBC) and Institutional Animal Care and Use Committee (IACUC) were followed. All live animal experiments are reported per the ARRIVE guidelines' recommendations. Informed Consent Statement Not applicable. Data Availability Statement This study employed the established consomic rat models SS and SS.BN3 as well as our congenic strains CG1 to CG8. The publicly accessible and NIH-supported Rat Genome Database (rgd.mcw.edu) catalogs has tools to explore the genotype and phenotype information for the SS (Dll4+) and SS.BN3 (Dll4-) and congenic strains under strain records Dll4+ (RGDID:61499), Dll4- (RGDID:1358154), CG1 (RGDID:155782881), CG2 (RGDID:155782883), CG3 (RGDID:155782884), CG4 (RGDID:155791428), CG5 (RGDID:155791426), CG6 (RGDID:155791430), CG7 (RGDID:155791429), and CG8 (RGDID:155791427). Conflicts of Interest The authors declare no conflict of interest. Figure 1 (a) Schematic representation of the SS(Dll4+) and SS.BN3(Dll4-) genomes modified by TALEN-mediated editing of the IL2Rg gene, showing chromosomes derived from SS (red) or BN (blue). (b) Schematic representing congenic strains and vascular-specific Dll4 protein expression, generated by introgressing BN(Dll4-) chromosome 3 (blue) into the (red) via marker-assisted breeding. Thin black bars represent confidence intervals of chromosomal regions that could be BN or SS. The horizontal black line indicates the location of vascular-specific Dll4 protein on chromosome. The dotted lines represent the location of SSLP markers. (c) Transgenically labeled human MDA-MB-231 triple-negative breast cancer cells were orthotopically implanted in the mammary fat pad of parental, consomic, and congenic rats. (d) Schematic diagram of the dynamic epifluorescence NIR imaging setup. A bifurcated optical fiber bundle delivers 785 nm excitation light for uniform illumination of the rat body surface. A 16-bit deep-cooled intensified charge-coupling device camera was used to image the rats through computer-controlled software. Figure 2 (a) Diagram of the process for detecting tumors and generating regions of interest (ROIs). The ROI detection module consists of three steps: spatial alignment, PCA ranking and selection, and ROI selection and masking. First, NIR time series data are registered to a reference image using a rigid body transformation. Next, PC reconstructions are ranked based on their 2D cross-correlation with a reference image featuring a clearly visible tumor. The tumor's location and a bounding box (shown in yellow) are estimated based on the 2D cross-correlation score of the selected PC and reference images featuring different tumors. The tumor's boundaries are enhanced and distinguished from the background through morphological dilation and thresholding. The resulting ROI is used to mask the NIR time series data. (b) Plot of the mean intensity of ICG biodistribution fluorescence kinetic data over 300 s for the tumor (red) and four fat pads (blue), normalized to a range of 0 to 1. Figure 3 Feature extraction. The normalized mean intensity of ICG taken over ROIs is plotted for the first 75 s (blue). The time series was smoothed using a Savitzky-Golay filter of order 3 with window length 31 (red). The insets show enlarged curve regions to visualize the characteristics and relationships of the features. S = {2, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 23, 25, 30, 35, 40, 50, 60, 70, 80}. Figure 4 (a) Color-coded and merged principal component images were used to anatomically segment tumors from a single view/projection for rat strains with high (red) or low (black) levels of Dll4 expression. The top 3 ranked PCs for detecting tumors are merged as red, green, and blue channels of a true color (RGB) image. Tumors are outlined in yellow. (b-d) Mean intensity of ICG biodistribution fluorescence kinetic data over 140 s for (b) parental (Dll4+) and consomic (Dll4-) rats, (c) all Dll4-high and Dll4-low rats, and (d) all strains, plotted and normalized to a range of 0 to 1. Shaded regions show s.e.m. Figure 5 (a,b) The covariance parameter of a mixed-effects model for 1400 timesteps with appropriate time-varying covariates, used to analyze the average fluorescence intensity of ICG in the tumor with multiple measurements per subject, with the subject number serving as the repeated measure indicator and the rat strain serving as a covariate. (c) Projection of the diagonal of the covariance parameter on the time axis. (d) Multiplicity-adjusted p-value matrix showing significant differences between groups based on multiple comparison of estimated marginal means using the Tukey-Kramer test statistic. (e) Sankey diagram showing differences in strains with high and low levels of Dll4 expression based on the Ascore (mean of accuracy, sensitivity, and specificity in the primary classification of each pair); 0.65 was subtracted from all the Ascore values for better visualizations of differences. Figure 6 Performance of selected features on original dataset. The confusion matrix and ROC plots for training and testing of best-performing models based on HIF5_avg and HIF50_avg features with 10-fold cross-validation. The dataset includes all rat strains except Dll4+, Dll4-, CG4-, and CG5+. An amount of 25% of the dataset was used for holdout testing. Top row: SVM model. Bottom row: Nearest Neighborhood. Figure 7 Performance of selected features on augmented dataset. The confusion matrix and ROC plots for training and testing of best-performing models based on HIF5_avg and HIF50_avg features with 10-fold cross-validation. The dataset includes all rat strains except Dll4+, Dll4-, CG4-, and CG5+. 25% of the dataset was used for holdout testing. Top row: SVM model. Bottom row: Nearest Neighborhood. cancers-15-01460-t001_Table 1 Table 1 Separation score for group pairs. Groups Average Classification Metrics (Ascore) Separation Score (Sscore) Dll4+|+|+||CG3 0.9 0.74 0.72 0.775 CG1|CG4 0.92 0.74 0.75 0.7875 CG5|CG3 0.9 0.83 0.61 0.7925 CG6|CG3 0.9 0.86 0.778 0.8495 CG5|CG4 0.92 0.83 0.8 0.845 CG6|CG4 0.92 0.86 0.78 0.855 cancers-15-01460-t002_Table 2 Table 2 Primary validation and feature selection. Groups Feature Best Average Best Accuracy Best Sensitivity Best Specificity Alg. Value(std) Alg. Value(std) Alg. Value(std) Alg. Value(std) Dll4+|, HIF12_avg DT 0.8666 (0.2309) DT 1 (0) DT 0.6 (0.5026) DT 1 (0) HIF6_avg, HIF50_avg LR 0.7597 (0.1412) LR 0.8682 (0.1468) LR 0.6 (0.5026) DT 0.9391 (0.116) Dll4+|CG3 HIF300_avg, HIF200_avg RBF SVM 0.6415 (0.3196) RBF SVM 0.5914 (0.0983) L SVM 1 (0) KNN 0.9166 (0.1147) HIF300_avg, TR_rel LR 0.6453 (0.1036) LR 0.6176 (0.1624) LR 0.85 (0.2665) KNN 0.9083 (0.1147) Dll4+|CG4 HIF30_avg, D16_avg DT 0.9175 (0.0303) DT 0.9125 (0.0915) L SVM 0.95 (0.0888) NB 1 (0) HIF8_avg, D6_avg RBF SVM 0.88125 (0.1254) RBF SVM 0.89375 (0.1174) L SVM 1 (0) NB 0.85 (0.2016) CG5|, HIF16_avg DT 0.8251 (0.0698) DT 0.8292 (0.1093) L SVM 1 (0) NB 0.845 (0.1952) HIF8_avg, HIF25_avg KNN 0.8044 (0.1505) KNN 0.8201 (0.0919) L SVM 1 (0) NB 0.7925 (0.2014) CG5|CG3 HIF300_avg, HIF400_avg L SVM 0.5683 (0.4037) LR 0.5292 (0.1507) L SVM 1 (0) KNN 0.85 (0.1613) HIF300_avg, HIF50_avg LR 0.5919 (0.4037) LR 0.5626 (0.1652) L SVM 1 (0) KNN 0.8333 (0.1324) CG5|CG4 D18_avg, HIF4_rel KNN 0.7811 (0.0485) KNN 0.7948 (0) L SVM 0.8928 (0) DT 0.7272 (0) D18_avg, HIF4_avg KNN 0.7811 (0.0485) KNN 0.7948 (0) L SVM 0.8928 (0) DT 0.7272 (0) CG6|, D2_avg KNN 0.8685 (0.0404) KNN 0.8755 (0.1180) L SVM 0.95 (0.1574 KNN 0.905 (0.1422) D40_avg, HIF5_rel KNN 0.8482 (0.01263) KNN 0.8440 (0.1386) RBF SVM 0.9375 (0.1293) KNN 0.8383 (0.1643) CG6|CG3 HIF300_avg, HIF350_avg L SVM 0.5729 (0.3995) KNN 0.5318 (0.1215) L SVM 1 (0) NN 0.8583 (0.1733) HIF300_avg, HIF180_avg RBF SVM 0.5890 (0.2399) RBF SVM 0.5570 (0.1259) L SVM 1 (0) KNN 0.85 (0.2222) CG6|CG4 HIF8_avg, HIF23_avg KNN 0.7278 (0.1917) KNN 0.7437 (0.0904) RBF SVM 1 (0) NB 0.7285 (0.2853) HIF6_avg, HIF50_avg NB 0.7280 (0.2263) NB 0.7468 (0.0982) L SVM 1 (0) DT 0.6071 (0.1846) For each group pair with high and low Dll4 expression levels, 2 sets of feature pairs and best-performing algorithms listed. For each algorithm, accuracy, sensitivity, specificity metrics, and average metric (Accuracy + Specificity + Sensitivity)/3 shown. Alg.: classifier algorithm, KNN: Nearest Neighborhood, L SVM: Linear SVM, RBF SVM: Radial Basis Function kernel SVM, DT: Decision Tree, NB: Naive Bayes, LR: Logistic Regression models. cancers-15-01460-t003_Table 3 Table 3 Performance of selected features. SVM KNN Measure Training Testing Training Testing Sensitivity 0.9310 1.0000 0.8108 1.0000 Specificity 0.8182 0.8182 0.9286 0.7500 Precision 0.8710 0.7500 0.9677 0.6250 Negative Predictive Value 0.9000 1.0000 0.6500 1.0000 False-Positive Rate 0.1818 0.1818 0.0714 0.2500 False Discovery Rate 0.1290 0.2500 0.0323 0.3750 False-Negative Rate 0.0690 0.0000 0.1892 0.0000 Accuracy 0.8824 0.8824 0.8431 0.8235 F1 Score 0.9000 0.8571 0.8824 0.7692 Matthews Correlation Coefficient 0.7600 0.7833 0.6758 0.6847 The training and testing metrics of the best-performing models based on the selected features HIF5_avg and HIF50_avg with 10-fold cross-validation are shown. The training dataset includes features extracted from the original NIR time series data of all congenic groups except CG5+ and CG4-, and 25% of the dataset was used for holdout testing of the models. SVM: Support Vector Machine, KNN: Nearest Neighborhood. cancers-15-01460-t004_Table 4 Table 4 Performance of selected features on augmented dataset. SVM KKN Measure Training Testing Training Testing Sensitivity 0.9253 0.9756 0.9702 0.9762 Specificity 0.9132 0.9125 0.9211 0.9241 Precision 0.8564 0.8511 0.867 0.8723 Negative Predictive Value 0.9562 0.9865 0.9832 0.9865 False Positive Rate 0.0868 0.0875 0.0789 0.0759 False Discovery Rate 0.1436 0.1489 0.133 0.1277 False Negative Rate 0.0747 0.0244 0.0298 0.0238 Accuracy 0.9175 0.9339 0.9381 0.9421 F1 Score 0.8895 0.9091 0.9157 0.9213 Matthews Correlation Coefficient 0.8254 0.8625 0.8705 0.8793 The training and testing metrics of the best-performing models based on the selected features HIF5_avg and HIF50_avg with 10-fold cross-validation are shown. The training dataset includes features extracted from the original NIR time series data of all congenic groups except CG5+ and CG4-, and 25% of the dataset was used for holdout testing of the models. SVM: Support Vector Machine, KNN: Nearest Neighborhood. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. |
PMC10000787 | Foods Foods foods Foods 2304-8158 MDPI 10.3390/foods12051022 foods-12-01022 Article Influence of Pig Genetic Line and Salt Reduction on Peptide Production and Bioactivity of Dry-Cured Hams Munoz-Rosique Beatriz Conceptualization Methodology Writing - original draft 1+ Hernandez-Correas Noelia Conceptualization Software Formal analysis Writing - original draft 2*+ Abellan Adela Validation Investigation 2 Bueno Estefania Validation Resources 2 Gomez Rafael Investigation Data curation Visualization 3 Tejada Luis Validation Writing - review & editing Supervision Project administration Funding acquisition 2 1 Departamento de Calidad, AromaIberica Serrana, S.L. Ctra. Fuente Alamo, Km 17.4, 30332 Murcia, Spain 2 Departamento de Tecnologia de la Alimentacion y Nutricion, Universidad Catolica de Murcia, Campus de los Jeronimos, 30107 Murcia, Spain 3 Departamento de Bromatologia y Tecnologia de los Alimentos, Universidad de Cordoba, Campus de Rabanales, Edificio Darwin, 14014 Cordoba, Spain * Correspondence: [email protected] + These authors contributed equally to this work. 28 2 2023 3 2023 12 5 102226 1 2023 17 2 2023 25 2 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). Ham (Jamon) is a product of great value in Spanish gastronomy, although experts have recommended reducing its consumption due to its high salt content and its relationship with cardio-vascular diseases due to the increase in blood pressure it may cause. Therefore, the objective of this study was to evaluate how the reduction of salt content and the pig genetic line influence bioactivity in boneless hams. For this purpose, 54 hams were studied, 18 boneless Iberian hams (RIB), 18 boneless white hams from commercial cross-bred pigs (RWC), and 18 salted and traditionally processed Iberian hams (TIB) to check if the pig genetic line (RIB vs. RWC) or the processing (RIB vs. TIB) affect the peptide production and bioactivity of the hams. The pig genetic line significantly affected the activity of ACE-I and DPPH, with RWC having the highest ACE-I activity and RIB having the highest antioxidative activity. This coincides with the results obtained in the identification of the peptides and the bioactivity analysis performed. Salt reduction positively affected the different hams, influencing their proteolysis and increasing their bioactivity in traditionally cured hams. bioactive peptide salt reduction proteolysis deboned ham Iberian ham antioxidant angiotensin-I converting enzyme (ACE) Ministerio de Ciencia, Innovacion y Universidades (Spain)Desarrollo de un nuevo jamon iberico deshuesado bajo en sodio y rico en peptidos bioactivosRTC-2017-6319 RETOS-COLABORACION 2017 This research was funded by the project "Desarrollo de un nuevo jamon iberico deshuesado bajo en sodio y rico en peptidos bioactivos" (RTC-2017-6319, RETOS-COLABORACION 2017). Ministerio de Ciencia, Innovacion y Universidades (Spain). The authors have not stated any conflicts of interest. pmc1. Introduction In recent years, due to the increasing concern of the population for health and nutrition, there is a tendency to investigate and demonstrate the added value and nutritional properties of certain foods. Therefore, the term "Functional Foods" is becoming increasingly prevalent and these foods could prevent the appearance or improve symptoms of certain chronic diseases . Meat products have also been studied for this bifunctionality , and numerous proposals have been reformulated to reduce or even eliminate certain components such as salt or fat, seeking to improve the nutritional profile of the food. One of the most important challenges in the food industry is the development of functional foods that maintain the organoleptic characteristics necessary to meet consumer demands, especially regarding flavor and texture Bioactive peptides are a group of biological molecules normally buried in the structure of parent proteins that become active after the cleavage of the proteins. During the ham curing process, the action of endopeptidases (capthesins and calpains) and exoproteases is crucial for the formation of peptides. Capthepsins and calpeins begin their action during the cold phase of the dryer (post-salting stage), but the increase in salt concentration, together with drying, causes their activity to decrease. However, exoprotease activity is highly favored when the temperature is increased in the dryer above 25 degC (temperature-dependent activity) and is maintained until the most advanced stages of the process. Intense proteolysis is thus triggered, leading to the hydrolysis of proteins and the release of non-protein nitrogenous compounds (NPNs). Due to this process, free amino acids and small peptides accumulate. These would comprise 2-20 amino acids (AA) with a molecular mass less than 6000 Da . The natural generation of these bioactive peptides is a consequence of the intense proteolysis of muscle peptidases produced during the processing of cured ham . However, there is still little information on the amount of these peptides in the final product. Due to the large number of bioactive peptides, the low abundance of each one and their presence within a complex matrix such as dry-cured ham makes their extraction and analysis difficult . Peptides modify the texture of the cured meat and influence the aroma at the end of the processing . Numerous studies have described the beneficial properties that these peptides may have on health. The influence they may have on certain diseases of chronic evolution has been evaluated, exerting antioxidant activity , antihypertensive activity , immunomodulatory , appetite regulator activity , or antidiabetogenic activity ; with good results and evidence for the ability to inhibit the angiotensin-I converting enzyme (ACE-I) . The direct or indirectly prescription of these peptides, which are naturally derived from food, could treat certain diseases and thus circumvent adverse effects secondary to the use of artificial drugs. Other authors described strategies to increase the consumption of meat products in meals, to add precursor proteins of these functional peptides to certain meals or to directly add the peptides, after studying their encapsulation . Salt plays a fundamental role during the salting period of cured ham. Water retention capacity, texture, flavor, pH, and proteolysis are parameters directly influenced by salt content . Despite being one parameter that defines the quality of cured ham, and being physiologically necessary for the proper function of an organism , the current excessive consumption of high salt products, such us ultra-processed foods has caused current human populations to exceed the necessary daily intake of this mineral, causing hypertension, directly related to cardiovascular diseases and other diseases of risk . Therefore, numerous studies are being conducted on various foods to reduce their salt content. In the case of ham, salt reduction directly influences proteolysis, which is greaterwhen the salt content is lower, and which, together with water activity, will result in a softer texture of the ham, which, as a consequence, will alter the quality of the final product . The increase in proteolysis leads to protein degradation, resulting in the generation of free AA and peptides through the proteolytic action of endopeptidases that can have biological activity, which can counteract and/or prevent diseases . Scant research has been performed on meat-derived peptides, and the degree to which salt reduction has influenced the generation of peptides or the generation of bioactive peptides has not been investigated. Numerous authors have identified peptides with antihypertensive and antioxidant activity in different types of cured ham that show in vitro ACE inhibitory activity, most important in the peptides Ala-Ala-Pro-Leu-Ala-Pro and Ile-Ala-Gly-Arg-Pro . Some of the identified peptides showed multifunctional activity, i.e., some showed antioxidant or anti-inflammatory activity besides antihypertensive activity . Some trials have been conducted with peptides found in ham to test whether they had bioactivity. A study conducted in hypertensive rats showed a significant reduction in systolic pressure after administering a peptide with antihypertensive activity (ACE inhibitor) eight hours earlier . Furthermore, this multifunctional activity had been recognized from bioactive peptides in other foods , most commonly generated from hydrophobic waste . Despite advances made in this field, the effect of salt reduction in ham on peptide generation and bioactivity has not been studied. Furthermore, no studies have been found comparing the peptides present in Iberian ham with those present in white ham, and no studies have attempted to identify peptides in boneless hams. Therefore, the objective of this study was to evaluate the influence of the reduction of salt content and pig genetic line on peptide production and bioactivity in boneless cured hams. 2. Materials and Methods For this study, 54 hams were selected from different genetic lines of Iberian pigs (thirty-six hams with a racial percentage of 50% Iberian and 50% Duroc) and white pigs (eighteen hams from crossbreeding Landrace x Large White or Hampshire). The hams from the Iberian pork genetic line were distributed in six batches of six hams. Half were processed on bone traditionally (18 TIB hams) and were the control batches. The other half of the Iberian hams (18 RIB hams) and the three batches of white hams (18 RWC hams) were freshly deboned and subjected to the new process developed to achieve salt reduction (RIB and RWC, respectively). To evaluate the effect of processing (deboning and salt reduction), TIB and RIB hams were compared, so only salt reduction in the Iberian hams was compared. To study the effect of the genetic line, RIB and RWC hams were compared. To compare hams from different genetic lines, the percentage of loss was taken as a reference. During the processing phases, each sample was weighed in triplicate to determine the percentage weight loss of each ham respect to the initial fresh weight of each piece. We considered the optimal curing moment, or the final product, when the ham reached 38% of weight loss. The experimental design is shown in Figure 1. Fresh hams were deboned and salted using sea salt and nitrifying salts. These hams were kept in a cold room at 3 degC for an established period (0.8 days per kilogram of ham weight). The hams were then removed and washed with water, following the normal curing process . The next stage (rest or post-salting) was conducted at 3 degC, gradually increasing this temperature to 6 degC until the percentage of weight loss of the hams rose to 18%. When this phase ended, the temperature was increased to 28 degC, reaching its completion when the hams reached a loss of 38%. Once the hams were cured, 18 hams were selected (six TIB hams, six RIB hams and six RWC hams). The samples were taken during the drying stage (33% weight loss) and final product stage (38% weight loss). All samples were taken when the ham reached the required percentage of weight loss. To avoid damage to the piece during the sampling process, for all the analysis all samples were taken from the femoral muscle (biceps) using a stainless steel cylinder with a diameter of 2 cm. After, samples were kept refrigerated until their analysis. 2.1. Non-Protein Nitrogenous Compounds To prepare the extracts, 2 g of sample were weighed in an Erlenmeyer flask, after which 30 mL of distilled water were added, and agitated for 15-20 min in a magnetic stirrer. Then, 15 mL of 20% trichloroacetic acid was added and shaken for 10 min. The content of the Erlenmeyer was filtered using a funnel and filter paper in a 50 mL volumetric flask. After filtration, the flask was filled with distilled water . Finally, 10 mL of the extract were used for the Kjeldahl method . 2.2. Antioxidant Activity The determination of antioxidant activity was conducted following the method of Bersuder et al. (1998) with minor variations. First, a standard was created with the TROLOX reagent (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), which is an analog of vitamin E with antioxidant capacity, used to react with the DPPH radical (2,2-diphenyl-1-picrylhydrazyl). The stock solution of TROLOX (2 Mm) was prepared by weighing 12.5 mg of TROLOX and diluting it in 25 mL of ethanol. A second 0.1 mM stock solution of 10 mL was then prepared from the first stock solution of 2 Mm. This second stock solution was prepared with 0.5 mL of the first stock solution and 9.5 mL of ethanol. The TROLOX concentrations of the straight standard were 5, 10, 15, 20, 40, and 50 mM. Two milliliters of each of the concentrations were prepared from the second 0.1 mM stock solution to prepare the standard line, obtaining the following linear equation: y = 1.01x + 10.224 (R2 = 0.9988). The antioxidant activity of the samples was then determined using a 0.02% (w/v) solution of the DPPH radical in ethanol. In Eppendorf tubes, 500 mL of ethanol, 500 mL of sample, and 125 mL of 0.02% (w/v) DPPH solution were added. For the blank, 500 mL of ethanol, 500 mL of water, and 125 mL of DPPH were used. Samples were incubated for 1 h in the dark at room temperature. The samples were then centrifuged at 10,000x g for 2 min and their absorbance was measured at 517 nm. % DPPH RSA = (Abs Blank - Abs Sample)/Abs Blank x 100 where, % DPPH RSA is % DPPH radical scavenging activity. Abs blank is the absorbance of DPPH with water instead of hydrolysate and Abs sample is the absorbance of DPPH radical in the presence of hydrolysate. The analyses for the different hydrolysates were performed three times. To calculate the IC50 (concentration of our sample that inhibits 50% of the DPPH radical) of our sample, we use the equation of the TROLOX standard we performed previously. 2.3. Angiotensin-I-Converting Enzyme Inhibitory Activity The ACE-I activity of the hydrolysates was conducted according to the spectro-photometric method of Cushman and Cheung (1971) , modified by Miguel et al. (2004) . For this purpose, 40 mL of each hydrolysate was incubated at 37 degC with 100 mL of 5 mM HHL dissolved in 0.1 M borate buffer and 0.3 M NaCl (pH 8.3). Next, 2 mU of ECA was added to the substrate. Thirty minutes later, 150 mL of 1 M HCl was added. The formed hippuric acid was recovered with 1000 mL ethyl acetate and centrifuged 10 min at 4000x g and the organic phase (800 mL) was collected. The ethyl acetate was removed by bringing the temperature to 95 degC. The resulting hippuric acid was resuspended in 1000 mL of distilled water and the absorbance was measured at 228 nm. ACE inhibitory activity was determined by the following equation (1) ACE inhibitory activity (%)=(Acontrol-Ablank)-(Asample-Ablank)(Acontrol-Ablank)x100 where, Acontrol is the measure of hippuric acid produced by the action of uninhibited ACE, Asample is the measure of hippuric acid produced by the action of ACE with the sample, and Ablank is the measure of unreacted HHL. To calculate the IC50, (the peptide concentration required for inhibit 50% of ACE activity), dilutions were prepared at different concentrations of the hydrolysate and the percentage inhibition was calculated for each concentration tested. Subsequently, the percentage of ACE-I activity versus the concentration of hydrolysate used (mg peptides/mL) was plotted. The equation of the line (y = ax + b) was obtained and the concentration of the hydrolysate giving an inhibition activity of 50%, i.e., IC50 = (50 - b)/a, was calculated. 2.4. Peptide Identification The identification was determined using tandem mass spectroscopy (MS) analysis using non-liquid chromatography from the NPN fraction obtained in Section 2.1. The methodology used was described by Bueno-Gavila et al. (2019). The identification of the peptide sequences of the hydrolysates of the different hams was conducted at the Proteomics and Bioinformatics Unit of the University of Cordoba, Spain. MS2 spectra were found with SEQUEST HT against the UnitProtKB database. The peptides of each hydrolysate were identified and quantified using the peptide spectral matches (PSM). Quantification values were normalized, focusing on the total PSM for all peptides in the sample. Thus, the quantification of a single peptide was comparable between those of the different samples. Additionally, we performed a search for each of the identified peptides in the BIOPEP-UWM database . Two types of searches were performed: identification of activated biopeptides in the sample and identification of potential biopeptides containing fragments of bioactive sequences in their primary structure. Data analysis was performed with R (version 3.4.1; accessed on 17 December 2022). 2.5. Statistical Analysis All analyses of our samples were performed in triplicate. Statistical analysis of our samples was performed using SPSS software (version 21.0, IBM Corporation, Armonk, NY, USA). To evaluate whether salt reduction and deboning affected the different analyses, a one-way ANOVA between RIB and TIB hams was performed. The effect of breed was also evaluated using a one-way ANOVA between RIB and RWC hams. When the effect of transformation or breed was significant (p < 0.05), the results were compared using a Fisher's LSD test. 3. Results and Discussion 3.1. Evaluation of Antihypertensive Activity (ACE-I) in Hams with Different Curing Losses (33% and 38%) Angiotensin-I-converting enzyme (ACE-I) is one of the key enzymes in the regulation of blood pressure, given its participation in the renin angiotensin-aldosterone system (RAAS) . Figure 2 indicates the evolution of ACE inhibitory activity throughout the assay as a function of peptide concentration in hams with different curing loss. Table 1 and Table 2 indicate the effect of pig genetic line and processing on ACE inhibitory activity, represented as the concentration of peptides necessary (mg/mL) to inhibit 50% of this activity (IC50). All samples showed ACE inhibitory activity, which increased with increasing concentration of the peptides. This could be due to the presence of small peptides smaller than 3 kDa . The ham that showed the highest ACE-I activity (IC50) was RIB33, having a greater potential to control diseases associated with the cardiovascular system . The IC50 in the final product was lower (higher activity) in Iberian hams than in white hams (Table 1), probably due to the longer curing time used in Iberian hams, consistent with what has been observed in other studies . However, when the weight loss is 33%, no significant differences were observed between genetic lines (Table 2). The processing method did not significantly influence ACE-I activity in Iberian hams (p >= 0.05), although it was slightly higher in salt-reduced hams (RIB38) (Table 1). A study conducted at the Catholic University of Murcia (UCAM) showed that the consumption of cured ham rich in bioactive peptides has a positive influence on the regulation of glycaemia and cholesterolemia in healthy patients, so that far from being a restricted food, its regular consumption has a positive effect on modifiable risk factors associated with premature cardiovascular disease . Table 3 presents the results of the effect of processing time on the production of ACE inhibitory activity. In RWC, hams with a weight loss of 33% have greater antihypertensive activity than those with a 38% weight loss (p <= 0.05). However, in Iberian hams, processing time does not imply greater ACE-I activity. In Serrano and Panxian hams, some have observed that this activity increases significantly in the last curing phase . Furthermore, other authors have also observed this behavior for dipeptide AA, which increases its activity by 40% from 6 months to 12 months of ham curing . Because ACE-I has been detected in the hams studied, it could counteract the harmful effects of sodium in the body . 3.2. Antioxidant Activity The DPPH radical study to evaluate the antioxidant activity of samples has been described as a suitable procedure for this purpose . Cured ham has been identified as a source of peptides with antioxidant activity . Despite this, no studies have evaluated antioxidant activity in salt-reduced hams. DPPH scavenging activity is also a commonly used technique to evaluate antioxidant capacity. This activity is directly associated with hydrophobic AA in peptides, so these AA will exist in antioxidant peptides . Figure 3 indicates the evolution of in vitro antioxidant activity as a function of the peptide concentration of the hams with different processing; all samples show higher antioxidant activity as the concentration of peptides increases. RWC38 has higher antioxidant activity, reaching 75% inhibition. In RIB38, we also observed an increase in antioxidant activity as the curing process progressed, higher than the healing process, higher than in RIB33. TIB38 shows lower antioxidant activity than RWC38 and is like RIB38. The ham with the lowest antioxidant activity was RIB33 in all the peptide concentrations we studied. Table 4 and Table 5 indicate the in vitro DPPH radical (antioxidant) scavenging activity of the ham in the drying and final phases, respectively. The concentration (mg/mL) of each NPN needed to inhibit 50% of the antioxidant activity (IC50) was evaluated. All the samples studied showed antioxidant activity both in the drying phase and in the final product. Table 4 indicates the IC50 values obtained for each sample and the effect of pig genetic line and processing on the antioxidant activity of the TIB, RIB, and RWC hams. Genetic line significantly influenced the uptake of the DPPH radical in these samples (p <= 0.05), as did RIB33 and RWC33 (Table 5). However, salt reduction and deboning did not influence the antioxidant activity of the samples, although it was higher in TIB38. The RWC38 hams have the highest antioxidant activity because they reached the IC50 with a lower peptide concentration (0.155 +- 0.013 mg/mL). These data coincide with the higher proteolysis index obtained in white hams in a previous study due to the higher activity of cathepsins and calpains of this breed . In Serrano hams, peptides have been identified with an IC50 at a concentration of 1.5 mg/mL . Furthermore, Jinhua hams in eastern China, managed an IC50 at a lower concentration of 1 mg/mL . However, in subsequent studies, Jinhua hams achieved an IC50 at 2.5 mg/mL, whereas Xuanwei hams required a concentration of 4.5 mg/mL . In contrast to this study, others have shown that meat from purebred and Duroc-crossed Iberian pigs would be less predisposed to oxidation than those from white pig breeds . Others claim that meat products such as Iberian ham have a greater antioxidant capacity than fresh ham products before being cured, or other foods such as red wine . Table 6 shows that, for both salt-reduced Iberian and white hams, the increase in curing time significantly affects the antioxidant capacity of the samples (p <= 0.05), being higher in RIB38 and RWC38. This could be due to the increase observed in proteolytic activity in the later stages of curing , often related to the increase in temperature . The results show that the antioxidant capacity of the hams increases as the curing process progresses and is not affected by the reduction in the Iberian ham. Therefore, cured hams would be a good source of antioxidant activity despite containing pro-oxidant agents such as salt and heme and even reactive oxygen species (ROS), which can cause cell damage . 3.3. Bioactive Peptide Sequencing The peptides present in the samples of the hams from the five batches studied (RIB38, RIB33, RWC38, RWC33, and TIB38) were sequenced by LC-MS/MS analysis. Table 7 indicates the number of sequenced peptides per sample. The ham with the highest number of sequenced peptides was RWC38, RIB33 had the lowest number of peptides sequenced, and RIB38 presented a greater number of peptides than TIB38. This coincides with the values of non-protein nitrogen and the proteolysis index (PI) obtained in a previous study in hams with a loss of 38%, where the highest and lowest NPN and PI were found in salt-reduced white hams (RWC38) and traditionally cured hams (TIB38), respectively . In this study, no peptides already obtained from the database were found among the peptides obtained in the proteomic study. Therefore, their bioactivity has not been demonstrated in previous studies. In other studies, identical sequences were found in cured ham, for example, KAAAAP, AAPLAP, and KPVAAP, with origin in different types of myosin protein, were identified as the peptides with the highest ACE-I activity in Teruel PDO ham , and are also present in Serrano ham . Their stability and their retention of bioactivity during processing and after in vitro digestion were examined. In vivo studies showed that the AAATP peptide had the highest antihypertensive activity, lowering systolic blood pressure with a short-term effect . Furthermore, other sequences with antihypertensive activity were identified, such as ASGPINFT and DVITGA (both also derived from myosin protein). In another study, AAATP with the KA dipeptide had DPP4 inhibitory activity that would contribute to improving the concentration of glucose in the blood . The antioxidant power is another bioactivity studied in traditional Serrano ham . The SAGNPN peptide has been identified to have the greatest capacity to donate electrons, neutralizing the oxidative capacity, even more than the peptides synthesized ; furthermore, the peptide GLAGA had the highest reducing power . Moreover, SNAAC and AEEEYPDL, identified in the cured ham, had high antioxidant activity . Numerous bioactive peptides with a high antihypertensive power have been identified in Iberian ham, which are higher than those in Serrano ham. The sequences that are repeated most frequently, which coincide with the BIOPEP database, are PPK, PAP, and AAP . However, the following dipeptides, such as EA, with ACE-I activity, or PP and VE, which showed DPP4-inhibitory activity have also been sequenced . Dipeptides with anti-inflammatory and cardiovascular protective activity (PA, GA, VG, EE, ES, DA, and DG) have been identified in hams with reduced salt content, besides contributing to the product aroma and flavor . However, no studies have been found in fresh deboned and salt-reduced Iberian or white ham. Study of Putative Activity Peptide Sequences A search has been conducted for peptide precursors that may contain biopeptides in their sequence and could theoretically be activated after digestion. This technique is useful for very small sequences (less than seven Amino Acids) and by using the proteomics procedure, it is impossible to detect them. To contextualize the type of bioactivity of the samples, a Z-scoring was performed to plot the variation between samples regarding the mean of the different activities (heatmap). The results are shown in Figure 4; a higher intensity red color means that this activity will be over-represented regarding the mean of the five samples. Bioactivities are grouped according to the intensity of occurrence in each sample. In addition, the succession of rows and columns is rearranged to avoid intersection of the dendrogram lines. Blue lines represent the value of the coefficient. Individually, we have represented in which sample each group of activities stands out for each group or clusters , each corresponding to a group of bioactivities. In RWC33, the main activity is immunostimulatory. No studies have been found on the presentation of this bioactivity in cured ham. RWC38 showed the highest antioxidant activity. These results coincide with the activity observed in vitro using DPPH (Table 4). Other bioactivities that stand out in this sample are those of neuropeptide activation, hypolipemic, anti-inflammatory, anti-cancer, and hypotensive activities. Antioxidant and hypotensive activity have also been well studied in white pig hams. Several studies confirm the occurrence of these bioactivities in ham . Recently, peptides with anti-inflammatory activity have been identified in Xuanwei hams, showing reduced symptoms of inflammatory bowel disease in mice, and it has been pro-posed that these peptides could be a functional drug in patients suffering from this disease . In RIB33 hams, the predominant activities are stimulatory, immunomodulatory, a CaMPDE inhibitor, a DPP4 inhibitor, antithrombotic, and ACE-I, consistent with our results for antihypertensive activity (Table 2), where RIB33 had the highest ACE-I. Likewise, the Iberian ham showed greater ACE-I activity compared to the traditional Serrano hams . Antihypertensive activity is well studied in ham . There are studies that would claim that Serrano ham would be a good source of DPP4 and that these peptides could be an adjunct in the treatment of type 2 diabetes . RIB38 hams have HMG-CoA reductase inhibitory, regulatory, and immunological activity. HMG-CoA reductase inhibitors play an important role in the control of hyper-cholesterolemia and, indirectly, in the control of the onset of cardiovascular disease. Other studies have found dipeptides such as DA, DD, EE, ES, and LL in cured ham, which have been identified as the main inhibitors of this coenzyme . Furthermore, TIB38 hams stand out for their binding, ubiquitin mediator protein activator, renin inhibitor, dipeptidyl peptidase III inhibitor, and embryotoxic activity, bioactivities that have not yet been studied. Each group of bioactivities was represented by a color . In Figure 5, we can observe six clusters, one for each group of bioactivities, where the values of that group of bioactivities are quantified for each sample. Because bioactive sequence fragments are found in the samples, a spider web plot with normalized quantification of the peptide precursors of the five hams is shown in Figure 6. This distribution allows differentiation between hams according to activity. The potential bioactivity of the peptides identified in each sample is reflected by using the same scale and amplitude and the same scale and width of the axis, allowing comparison between them. Cured ham is considered a good source of different bioactive peptides that have important functional activities, such as the inhibition of the angiotensin converting enzyme, hypoglycemic, and anti-inflammatory activities . 3.4. Bioactivity Analysis Based on Amino Acid Composition The composition of Amino Acid (AA) used to analyze the bioactivity of the samples was conducted on 38% cured hams, as these had the best organoleptic characteristics and, therefore, would be destined for the end consumer. In bioactivity studies, it is important to consider the structural properties of sequences . Certain characteristics, such as size, hydrophobicity, and composition, may influence the stability or bioavailability of the peptides. Approximately 20 sequences were selected from those identified in each ham with less than 1.5 kDa and with a maximum of 12 AA in their chain. Processing time causes the size of the peptides to decrease and increases the antioxidant activity of the peptides , as short AA sequences are more likely to be bioactive . In addition, over 50% of the AAs in the chain should be hydrophobic, as this contributes to antioxidant activity . The presence of AAs A, D, E, G, L, P, and V confers antioxidant and antihypertensive activity on the peptide sequence , and this activity is directly related to the molecular weight of the peptide sequence . However, the presence of H, Y, W, F, M, and C could inhibit free radicals by direct electron transfer . The amino acid sequences of the peptides identified from salt-reduced Iberian hams (RIB) are shown in Table 7. 3.5. Identification of Peptides Present in RIB Hams The AA sequences of the peptides identified from the hydrolysates of salt-reduced Iberian hams (RIB) are shown in Table 8. Antioxidant activity is highly present among the selected sequences. Some have over 50% of the peptides that provide antioxidant activity. The LDLALEKD, AAFPPDVGGN, AGNPDLVLPV, and AFGPGLEGGL peptides stand out for having over 80% of AAs that would favor antioxidant activity, with AFGPGLEGGL having the highest antioxidant activity (90% of its AAs). The AAFPPDVGGN and AFPPDVGGN have been identified as present in pork and six sequences containing them have been found (AFPPDVGGN, AAFPPDVGGN, AFPPDVGGNV, AAFPPDVGGGGNV, AFPPDVGGGGNVD, and AAFPPDVGGGGNVD). The peptides FPPDVGGN and FPPDVGGNVD originating from the protein could also be derived from these sequences, identified as myosin . From the action of the enzyme, dipeptidyl peptidase could be released from some sequences as the VD dipeptide, which would have DPP4 inhibitory activity and, therefore, anti-diabetic activity . The most prominent sequence is CLFVCR, as it has 83% of hydrophobic AAs, 67% of AAs conferring ACE-I activity, and 50% of AAs scavenging free radicals. ACE-I activity would be more present in sequences containing hydrophobic AA residues in the three C-terminal positions . For this sample, the sequence AGNPDLVLPV has three hydrophobic AAs at the C-terminus. The dipeptide WK could be extracted from longer peptides originating from b-enolase, such as DGADFAKW (Table 8). This dipeptide has been identified as an inhibitor of DPP4 . Likewise, the sequences LIGIEVPH, IDLIEKPM, FDKIEDMA, WNDEIAPQ, and DLDISAPQ originate from the IE and SI dipeptides of the a-enolase protein; they have been described as DPP4-inhibitory peptides, respectively . These dipeptides could be responsible for the high antihypertensive activity observed in this study for sample RIB38 (Table 1). Recently, some dipeptides related to anti-inflammatory activity, which could confer cardiovascular protection, have been identified in salt-reduced cured hams . These dipeptides are PA, GA, DA, and DG and could be derived from sequences found in RIB38 (ALQPALKF, WNDEIAPQ, MADTFLEH, DLDISAPQ, DGADFAKW, MADTFLEH, and AGNPDLVLPV), with GA being mainly identified in the study. Table 9 indicates the prominent peptide sequences detected in the ham samples of traditionally cured Iberian ham (TIB). In these samples, six of the selected sequences presented over 80% of the AAs that could provide antioxidant activity to the product (AFPPDVGGNV, AAFPPDVGGN, DVVLPGGNL, VAVGDKVPAD, DIAVDGEPLG AGNPDLVLPV, and AFGPGLEGGL). RIB38 has the highest antioxidant activity. However, the sequence that stands out for having the highest amount of hydrophobic peptides is ILPGPAPW. This peptide comprises the Pro-Ala-Pro sequence, one of the most repeated sequences among the bioactive peptides described in the literature , which would confer good antioxidant activity to the sample . Furthermore, these sequences could contribute to the bioactivity described for TIB38 ham . Four sequences (ILPGPAPW, VMGAPGAPM, GDLGIEIPA, and AGNPDLVLPV) have three hydrophobic AAs at the C-terminus, and are therefore more likely to develop ACE-I activity . The AGNPDLVLPV sequence matches that found in RIB38. In the GDLGIEIPA and IELIEKPM sequences, we can find the dipeptide IE dipeptide related to ACE-I bioactivity . The same six sequences identified in RIB38 have also been found in TIB38 (AAFPPDVGGNV, AAFPPDVGGN, AFPPDVGGNVD, AAFPPDVGG-NVD, AFPPDVGGN, and AFPPDVGGNV), have been identified in pork, and could have inhibited DPP4 derived from the dipeptide DV . From a comparison study between traditional and salt-reduced cured hams, di-peptides such as DA, PA, and VG would be present in a higher proportion in traditional cured hams . The last two sequences of Table 9 could derive from the peptides found in sample TIB38 and could contribute to its anti-inflammatory and antihypertensive activity. Other sequences, which were identified in this study, are GA (ACE and DPP4 inhibitory activities) and DG (ACE-I activity). The selected AA sequences of salt-reduced white hams (RWC) are shown in Table 10. The sequences that stand out for having over 80% of AAs and confer antioxidant activity are DLAEDAPW and AEVIALPVE. The latter sequence is also present in RIB38, with the highest antioxidant activity. The sequence that stands out for having the highest amount of hydrophobic AAs is ILPGPAPW, the same as TIB38, and has one of the most repeated sequences among bio-active peptides (PAP) . Furthermore, this sequence has 75% of AAs that confer antioxidant activity, three hydrophobic AAs at the C-terminus that confer ACE-I activity, and 13% of AAs that could inhibit free radicals. However, six peptide sequences with three hydrophobic AAs at the C-terminus (ILPGPAPW, AVIGPSLPL, VMGAPGAPM, ISAPSADAPM, DLAEDAPW, and GDLGIEIPA) were found in the RWC38 samples, which conferred ACE-I activity. However, in RIB38, of the sequences selected, none had over two hydrophobic AAs at the C-terminus. The sequences ILPGPAPW, VMGAP-GAPM, and GDLGIEIPA match TIB38. Furthermore, the LKGADPEDVITGA and GADPEDVITGA would contain the bioactive peptide DVITGA in their chain, related to high ACE-I activity due to the presence of AA alanine at the C-terminus . Despite this, the RWC38 ham showed the least antihypertensive activity ; it would be necessary to study if these peptides confer ACE-I activity and in what quantity they are present. There are more sequences from which the dipeptide IE could be derived, already described as a precursor of this bioactivity . The sequence identified in RWC38, FKAEEEYPDLS, once digested, could cause the peptide AEEEYPDL, derived from protein creatine kinase and identified as a potent antioxidant . Using multiple reaction monitoring (MRM), it was quantified at a concentration of 0.148 fg/g in cured ham . This could explain why the RWC38 hams showed the highest antioxidant activity and the highest rate of proteolysis obtained in white hams . In RWC38 hams, the same six sequences described in RIB38 and TIB38 (AAFPPDVGGNV, AAFPPDVGGNV, AFPPDVGGNVD, AAFPPDVGGNVD, AFPPDVGGNV, and AFPPDVGGNV) have been identified. However, this would explain the difference in proteolytic activity between different pig genetic lines and influence of processing, because salt-reduced Iberian hams (RIB38 and RIB33) had the highest DPP4 inhibitory activity, lower than white hams (RWC38 and RWC33) and traditional Iberian ham (TIB38). In a recent study of salt-reduced white ham, hydrophobic PA dipeptides (related to bitter taste and with ACE-I and anti-inflammatory activity) and VG (related to bitter and umami taste and with ACE-I activity) were identified that could be derived from the sequences identified in our sample . Different unique and common sequences that could act as peptide precursors and that have been identified in the samples would be responsible for the bioactivities found in the different types of ham. The results show that in RIB38 ham, the precursors found could be responsible for its high antihypertensive capacity, noting that the change in processing varies the sequences identified in both samples (RIB38 and TIB38). Furthermore, peptides already referenced in the literature have been found in the RWC38 ham, including a sequence that gives rise to a potent antioxidant peptide (AEEEYPDL) that would explain its increased bioactivity. However, none of these co-inciding peptides are found in Iberian hams. 4. Conclusions Salt-reduced boneless hams presented a higher concentration of peptides and higher bioactivity compared to traditionally cured hams, due to higher proteolysis. The salt-reduced white ham had the highest antioxidant activity and the salt-reduced Iberian ham the highest antihypertensive activity. Antioxidant activity was significantly influenced by pig genetic line and antihypertensive activity was modified by pig genetic line in the final product. In addition, salt-reduced white ham had the greatest hypolipidemic, anti-inflammatory, and anticarcinogenic activity. However, salt-reduced Iberian ham stood out for its significant HMG-CoA reductase inhibitory, regulatory, and immunological activity. Salt reduction has had a positive influence on the bioactivity of the hams, with salt-reduced hams, both Iberian hams and hams from white pigs, having the highest bioactivity compared to traditionally cured Iberian hams. In all types of hams, peptide precursors sequenced could give rise to sequences identified as bioactive in literature, with white pig hams showing the highest quantity. The dipeptide DV is present among the precursors of all hams and the bioactive peptides DVITGA and AEEEYPDL in the precursors of the reduced white hams. Acknowledgments The authors thank all the colleagues who support our work on a daily basis. We also thank Phillip John Bentley Bruce for the English revision of the manuscript. Author Contributions B.M.-R.: Methodology, Validation, Formal analysis, Investigation, Writing--review & editing, Visualization. N.H.-C.: Methodology, Validation, Formal analysis, Investigation, Writing--review & editing, Visualization. A.A.: Formal analysis, Investigation, Resources, Writing--original draft. E.B.: Investigation. R.G.: Investigation. L.T.: Supervision, Project administration, Conceptualization, Resources, Writing--original draft. All authors have read and agreed to the published version of the manuscript. Data Availability Statement Data is contained within the article. Conflicts of Interest The authors declare that they have no known competing financial interest or personal relationship that could have appeared to influence the work reported in this paper. Figure 1 Outline of the experimental design. Figure 2 Evolution of ACE-I activity as a function of peptide concentration in hams with 33% and 38% curing loss. Figure 3 Evolution of in vitro antioxidant activity as a function of peptide concentration in hams with 33% and 38% curing loss. Figure 4 Heatmap and dendrogram of bioactivities of the different ham samples studied. Quantification of bioactivity is regarding the mean. The grouping relationship between the groups of activities is defined. Figure 5 Representation of the bioactivity in each sample for each cluster of activities. Figure 6 Standardized quantification of peptide precursors (x103) in different cured ham samples. foods-12-01022-t001_Table 1 Table 1 Effect of pig genetic line and processing on ACE-I activity in hams with 38% cure loss. Dry-Cured Ham Type p-Value Formulation TIB RIB RWC Genetic Line Processed IECA (IC50) 0.249 +- 0.018 b 0.220 +- 0.012 b 0.335 +- 0.017 a 0.025 0.387 One-way ANOVA. a,b Values within a row with different superscripts differ significantly at p <= 0.05 (Fisher LSD Test). TIB: traditional Iberian dry-cured ham; RIB: reduced Iberian dry-cured ham; RWC: reduced white pig commercial crosses. ACE inhibitor (IC50): peptide concentration required (mg/mL) to inhibit 50% of ACE activity. p-Value production line: one-way ANOVA RIB vs. RWC; p-value processed: one-way ANOVA TIB vs. RIB (p-value significant at p <= 0.05). SEM: standard error of the mean. foods-12-01022-t002_Table 2 Table 2 Effect of pig genetic line on ACE-I activity in hams with a 33% curing loss. Dry-Cured Ham Type p-Value Formulation RIB RWC Genetic Line IECA (IC50) 0.215 +- 0.043 a 0.230 +- 0.014 a 0.7550 One-way ANOVA. a Values within a row with different superscripts differ significantly at p <= 0.05 (Fisher LSD Test). RIB: reduced Iberian dry-cured ham; RWC: reduced white pig commercial crosses dry-cured ham. ACE inhibitor (IC50): peptide concentration required (mg/mL) to inhibit 50% of ACE activity. p-value production line: One-way ANOVA between RIB and RWC (p-value significant at p <= 0.05). SEM: standard error of the mean. foods-12-01022-t003_Table 3 Table 3 Effect of the decrease in ACE-I activity of salt-reduced Iberian and white hams (RIB and RWC). Dry-Cured Ham Type RIB RWC IECA (IC50) 33% reduction 0.215 +- 0.043 a 0.230 +- 0.014 a 38% reduction 0.220 +- 0.012 b 0.335 +- 0.017 a p-value reduction 0.486 0.001 One-way ANOVA. a,b Values within a row with different superscripts differ significantly at p <= 0.05 (Fisher LSD Test). RIB: reduced Iberian dry-cured ham; RWC: reduced white pig commercial crosses dry-cured ham. ACE inhibitor (IC50): peptide concentration required (mg/mL) to inhibit 50% of ACE activity. p-Value reduction: one-way ANOVA 38% reduction vs. 33% reduction. The results are expressed in mg/mL as mean +- SEM. SEM: standard error of the mean. foods-12-01022-t004_Table 4 Table 4 Effect of pig genetic line and processing on antioxidant activity in hams with a curing loss of 38%. Dry-Cured Ham Type p-Value Formulation TIB RIB RWC Processed Genetic Line DPPH (IC50) 0.173 +- 0.054 a 0.199 +- 0.048 a 0.155 +- 0.013 a 0.455 0.048 One-way ANOVA. a Values within a row with different superscripts differ significantly at p <= 0.05 (Fisher LSD Test). TIB: traditional Iberian dry-cured ham; RIB: reduced Iberian dry-cured ham; RWC: reduced white pig commercial crosses. DPPH (IC50): peptide concentration required (mg/mL) to inhibit 50% of the antioxidant activity. p-value production line: One-way ANOVA between TIB, RIB, and RWC (p-value significant at p <= 0.05). SEM: standard error of the mean. foods-12-01022-t005_Table 5 Table 5 Effect of pig genetic line on the antioxidant activity of hams with a 33% curing loss. Dry-Cured Ham Type p-Value Formulation RIB RWC Genetic Line DPPH (IC50) 1.888 +- 0.041 b 0.197 +- 0.013 a 0.001 One-way ANOVA. a,b Values within a row with different superscripts differ significantly at p <= 0.05 (Fisher LSD Test). RIB: reduced Iberian dry-cured ham; RWC: reduced white pig commercial crosses dry-cured ham. DPPH (IC50): peptide concentration required (mg/mL) to inhibit 50% of the antioxidant activity. p-value production line: One-way ANOVA between RIB and RWC (p-value significant at p <= 0.05). SEM: standard error of the mean. foods-12-01022-t006_Table 6 Table 6 Effect of reduction in the DPPH activity of salt-reduced Iberian and white hams (RIB and RWC). Dry-Cured Ham Type RIB RWC DPPH (IC50) 33% reduction 1.888 +- 0.041 b 0.197 +- 0.013 b 38% reduction 0.199 +- 0.048 a 0.155 +- 0.013 a p-value reduction 0.000 0.043 One-way ANOVA. a,b Values within a row with different superscripts differ significantly at p <= 0.05 (Fisher LSD Test). RIB: reduced Iberian dry-cured ham; RWC: reduced white pig commercial crosses dry-cured ham. DPPH (IC50): peptide concentration required (mg/mL) to inhibit 50% of the antioxidant activity. p-value reduction: one-way ANOVA 38% reduction vs. 33% reduction. The results are expressed in mg/mL as mean +- SEM. SEM: standard error of the mean. foods-12-01022-t007_Table 7 Table 7 Number of peptides sequenced per sample. Sample Number of Peptides Sequenced RIB38 979 RIB33 602 RWC38 1053 RWC33 780 TIB38 904 foods-12-01022-t008_Table 8 Table 8 Identification of amino acid sequences of peptides present in salt-reduced Iberian ham (RIB) using LC-MS/MS. N Peptide Seq Exp. Mass Protein Source Acc 1 CLFVCR 750.37 Ubiquitin-associated and SH3 domain containing B F1S9R5 2 DLDISAPQ 858.43 Calsequestrin F1RJW7 3 AFPPDVGGN 873.41 Myosin regulatory light chain 2 Q5XLD2 4 ALQPALKF 887.53 Superoxide dismutase A0A287A4Z2 5 LYKVAVGF 896.52 Alpha-amylase I3LSA5/F1S574/F1S573 6 FDKPVSPL 902.5 Creatine Kinase M-type/Mitochondrial Creatine kinase 2 Q5XLD3/Q2HYU1 7 DGADFAKW 909.41 Fructose-biphosphate aldolase A0A287A1V5 8 AAVKELATL 915.55 Carboxypeptidase B F1SKC7 9 LDLALEKD 916.5 Alpha-amylase I3LSA5/F1S574/F1S573 10 AFGPGLEGGL 917.47 Filamin C F1SMN5 11 AAFPPDVGGN 944.45 Myosin regulatory light chain 2 Q5XLD2 12 DNDIMLIK 961.5 Uncharacterized Protein A0A287B5W2 13 MADTFLEH 963.42 Pyruvate Kinase A0A287B8G0 14 MIADYLNK 967.49 Alpha-amylase I3LSA5/F1S574 15 FDKIEDMA 968.44 Myosin 2-4-7 F1SS64/Q9TV62/A0A2867PQ9 16 LGIDVWEH 968.48 Superoxide dismutase A0A287A4Z2 17 WNDEIAPQ 972.44 Phosphoglycerate mutase B5KJG2 18 AFPPDVGGNV 972.48 Myosin regulatory light chain 2 Q5XLD2 19 AGNPDLVLPV 994.56 Beta-enolase A0A287AZR0 20 AAFPPDVGGNV 1043.52 Myosin regulatory light chain 2 Q5XLD2 21 AFPPDVGGNVD 1087.51 Myosin regulatory light chain 2 Q5XLD2 22 AAFPPDVGGNVD 1158.54 Myosin regulatory light chain 2 Q5XLD2 foods-12-01022-t009_Table 9 Table 9 Identification of the amino acid sequences of peptides present in traditional Iberian ham (TIB) using LC-MS/MS. N Peptide Seq Exp. Mass Protein Source Acc 1 VMGAPGAPM 830.39 Uncharacterized Protein F1RVL5 2 ILPGPAPW 850.48 PDZ and LIM domain protein 3 Q6QGC0 3 DIDLSAPQ 858.42 Calsequestrin F1RJW7 4 AFPPDVGGN 873.41 Myosin regulatory light chain 2 Q5XLD2 5 GDLGIEIPA 884.47 Pyruvate kinase A0A287B8G0 6 IGIGPGGVIGA 910.54 Uncharacterized protein A0A287AXV0 7 AFGPGLEGGL 917.47 Filamin C F1SMN5 8 AAFPPDVGGN 944.45 Myosin regulatory light chain 2 Q5XLD2 9 TVPPAVPGIT 951.55 Fructose-biphosphate aldolase A0A287A1V5 10 VAVGDKVPAD 970.52 Calcium-transporting ATPase A0A287APK5 11 AFPPDVGGNV 972.48 Myosin regulatory light chain 2 Q5XLD2 12 IELIEKPM 972.54 Myosin 2-4 F1SS65/Q9TV62 13 DVVVLPGGNL 982.56 DJ-1 Protein Q0R678 14 HMWPGDIK 983.48 Alpha-amylase I3LSA5/F1S574 15 FNVIQPGPI 984.55 2,4-dienoyl-CoA reductase 1 D6QST6 16 DIAVDGEPLG 985.48 Peptidyl-prolyl cis-trans isomerase A P62936 17 AGNPDLVLPV 994.56 Beta-enolase A0A287AZR0 18 RIPADVDPL 995.55 Alpha-crystallin B chain A0A287ATJ4 19 NGAHIPGSPF 996.49 Filamin C F1SMN5 20 AAFPPDVGGNV 1043.52 Myosin regulatory light chain 2 Q5XLD2 21 AFPPDVGGNVD 1087.51 Myosin regulatory light chain 2 Q5XLD2 22 AAFPPDVGGNVD 1158.54 Myosin regulatory light chain 2 Q5XLD2 foods-12-01022-t010_Table 10 Table 10 Identification of the amino acid sequences of peptides present in salt-reduced White ham (RWC) using LC-MS/MS. N Peptide Seq Exp. Mass Protein Source Acc 1 VMGAPGAPM 829.39 Uncharacterized protein F1RVL5 2 AKLPADTE 843.44 Triosephosphate isomerase A0A286ZRV2 3 SGMNVARL 846.43 Pyruvate Kinase A0A287B8G0 4 ILPGPAPW 849.48 PDZ and LIM domain protein C Q6QGC0 5 AFPPDVGGN 873.41 Myosin regulatory light chain 2 Q5XLD2 6 LIGIEVPH 876.51 Uncharacterized Protein F1RK48 7 IKIIAPPE 879.55 Actin Alpha skeletal muscle P68137 8 GDLGIEIPA 883.47 Pyruvate kinase A0A287B860 9 ALQPALKF 886.53 Superoxide dismutase A0A287A4Z2 10 LYKVAVGF 895.52 Alpha-Amylase I3LSA5 11 DLAEDAPW 915.40 Filamin C F1SMN5 12 AAFPPDVGGN 944.45 Myosin regulatory light chain 2 Q5XLD2 13 LSVEAPLPK 952.57 Heat shock protein beta-1 A0A2C9F366 14 ISAPSADAPM 958.45 Glyceraldehyde-3-phosphate dehydrogenase A0A287BG23 15 IELIEKPM 971.54 Myosin-2 F1SS65 16 AFPPDVGGNV 972.48 Myosin regulatory light chain 2 Q5XLD2 17 AEVIALPVE 981.55 Cut a divalent cation tolerance homolog F1RZR6 18 LVIIEGDLE 999.56 Tropomyosin alpha 3-chain A0A287AID2 19 AAFPPDVGGNV 1043.52 Myosin regulatory light chain 2 Q5XLD2 20 GADPEDVITGA 1044.48 Myosin regulatory light chain 2 Q5XLD2 21 AFPPDVGGNVD 1087.51 Myosin regulatory light chain 2 Q5XLD2 22 AAFPPDVGGNVD 1158.54 Myosin regulatory light chain 2 Q5XLD2 23 LKGADPEDVITGA 1285.66 Myosin regulatory light chain 2 Q5XLD2 24 FKAEEEYPDLS 1327.61 Creatine Kinase M-type Q5XLD3 Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). 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PMC10000788 | Diagnostics (Basel) Diagnostics (Basel) diagnostics Diagnostics 2075-4418 MDPI 10.3390/diagnostics13050827 diagnostics-13-00827 Article Mobile Cone-Beam CT-Assisted Bronchoscopy for Peripheral Lung Lesions Salahuddin Moiz Methodology Formal analysis Data curation Writing - review & editing 1 Bashour Sami I. 1 Khan Asad Conceptualization Methodology Writing - review & editing 1 Chintalapani Gouthami Conceptualization Methodology Writing - review & editing 2 Kleinszig Gerhard Conceptualization Methodology Writing - review & editing 3 Casal Roberto F. 1* Carretta Angelo Academic Editor 1 Department of Pulmonary Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA 2 Siemens Medical Solutions USA Inc., Malvern, PA 19355, USA 3 Siemens Healthcare GmbH, 91301 Forchheim, Germany * Correspondence: [email protected] 21 2 2023 3 2023 13 5 82730 1 2023 08 2 2023 17 2 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). Peripheral bronchoscopy with the use of thin/ultrathin bronchoscopes and radial-probe endobronchial ultrasound (RP-EBUS) has been associated with a fair diagnostic yield. Mobile cone-beam CT (m-CBCT) could potentially improve the performance of these readily available technologies. We retrospectively reviewed the records of patients undergoing bronchoscopy for peripheral lung lesions with thin/ultrathin scope, RP-EBUS, and m-CBCT guidance. We studied the performance (diagnostic yield and sensitivity for malignancy) and safety (complications, radiation exposure) of this combined approach. A total of 51 patients were studied. The mean target size was 2.6 cm (SD, 1.3 cm) and the mean distance to the pleura was 1.5 cm (SD, 1.4 cm). The diagnostic yield was 78.4% (95 CI, 67.1-89.7%), and the sensitivity for malignancy was 77.4% (95 CI, 62.7-92.1%). The only complication was one pneumothorax. The median fluoroscopy time was 11.2 min (range, 2.9-42.1) and the median number of CT spins was 1 (range, 1-5). The mean Dose Area Product from the total exposure was 41.92 Gy*cm2 (SD, 11.35 Gy*cm2). Mobile CBCT guidance may increase the performance of thin/ultrathin bronchoscopy for peripheral lung lesions in a safe manner. Further prospective studies are needed to corroborate these findings. cone beam CT bronchoscopy peripheral lung tumors This research received no external funding. pmc1. Introduction Lung cancer is the number one cause of cancer-related mortality for both men and women throughout the world . Five-year survival in lung cancer is largely dependent on the stage of the disease at the time of diagnosis, ranging from 55% in early stages to 4% in metastatic disease . Thus there is an effort to detect lung cancer at an early stage with annual screening by low-dose chest CT . The combination of lung cancer screening with low-dose chest CT and the wide-spread use of chest CT scans has led to an increase in the number of detected lung nodules. Diagnosis of peripherally located lung nodules can be achieved with a variety of techniques: bronchoscopy, CT-guided transthoracic needle biopsy, and video-assisted thoracoscopic surgery (VATS) . With a favorable safety profile and the ability to provide nodal staging of lung cancer when indicated, the use of bronchoscopy to diagnose peripheral lung tumors has substantially grown in the past two decades. Navigational technologies (electromagnetic and non-electromagnetic) and, more recently, robotic bronchoscopy, have been added to the bronchoscopists' armamentarium. These technologies have been demonstrated to provide a greater diagnostic yield than the use of thin/ultrathin (T/UT) bronchoscopy with radial-probe endobronchial ultrasound (RP-EBUS) . However, these technologies are costly (requiring capital equipment purchase and disposables for every case) and not widely available outside the United States of America. As we have previously described, there are three main phases during peripheral bronchoscopy: navigation, confirmation, and acquisition . For long, RP-EBUS has been the sole method to confirm that a target has been reached during bronchoscopy. Unfortunately, RP-EBUS is not an accurate method of target confirmation, as has been highlighted by recent work from our group demonstrating that atelectasis occurs very commonly during bronchoscopy under general anesthesia, and can easily create false-positive RP-EBUS images . Thus the interest in the use of cone-beam CT (CBCT) guidance, in particular for the phase of confirmation, to compensate for the suboptimal performance of RP-EBUS . Multiple studies have demonstrated that CBCT can increase the yield of different navigational techniques . However, most of these studies were performed with "fixed" CBCTs. This type of CBCTs is typically located outside of the bronchoscopy suites (interventional radiology suites, hybrid operating rooms), and most pulmonologists do not have access to it. Hence, the interest in "mobile" CBCT (m-CBCT)--a more affordable alternative which has the advantage of being able to be shared amongst multiple hospital service lines, can be easily "rolled" in and out of most bronchoscopy suites and can provide both two dimensional (2D) and three dimensional (3D) images . The combination of m-CBCT along with the readily available T/UT bronchoscopes and RP-EBUS has been briefly described in case reports and a small case series . Data on diagnostic performance and radiation exposure are needed before adopting this new imaging technology to guide our bronchoscopies. Here, we report our initial experience with the use of m-CBCT in combination with T/UT bronchoscopy and RP-EBUS for patients with peripheral lung lesions, and its efficacy and safety. 2. Materials and Methods 2.1. Study Center and Population After obtaining IRB approval (2021-0268), consecutive patients were captured retrospectively through electronic medical records at The University of Texas MD Anderson Cancer Center between June 2020 and March 2021. Bronchoscopies performed for the diagnosis of peripheral lung lesions (located in the outer two-thirds of the lungs) with the combination of thin (BF-P190F; Olympus, Tokyo, Japan) or ultra-thin scopes (BF-MP190F; Olympus, Tokyo, Japan), RP-EBUS (UM-S20-17S, Olympus, Tokyo, Japan), and m-CBCT (Cios Spin, Siemens Healthineers, Forchheim, Germany) were included in this analysis. 2.2. Procedures Bronchoscopies were performed under general anesthesia, without a ventilatory strategy to prevent atelectasis (data not available at that time), through a laryngeal-mask airway (LMA) in most cases, with 100% FiO2 and 0-5 cmH2O of positive-end of expiration pressure (PEEP). Five different physicians performed bronchoscopies. These physicians operated the m-CBCT C-arm themselves. A combination of sampling tools including needle (21 G PeriView FLEX needle; Olympus, Tokyo, Japan), cytology brush (BC-202D, Olympus, Tokyo, Japan), and biopsy forceps (FB-231D, Olympus, Tokyo, Japan) was utilized at the discretion of the operators. Broncho-alveolar lavage was performed when infection was suspected. Rapid on-site cytology examination (ROSE) was present for all cases. The m-CBCT C-arm was utilized for both fluoroscopy imaging (2D) and CT scans (3D) at the discretion of the operator. Using its laser projection, the m-CBCT C-arm was isocentered on the target lesion. The m-CBCT scan consisted of a 30 s spin, with the target nodule positioned in the center and C-arm rotating around the patient acquiring 400 projection images in fixed angular intervals that are reconstructed into multiplanar cross-sectional CT-like images. Each m-CBCT spin was performed with a ventilatory breath hold of 30 s (time required for spin). During spins, the bronchoscope was fixed in position with an articulating arm that attaches to the bronchoscopy boom (The Arm, Neuwave Medical Inc., Madison, WI, USA), so that all personnel could exit the room (except for one anesthesia staff that remained behind a protective shield) . Patients were recovered per standard of care and all patients underwent post-procedural chest X-ray to rule out pneumothorax at the end of the recovery period. 2.3. Study Definitions Our primary endpoint was "diagnostic yield", defined as the number of patients in whom diagnostic samples are obtained divided by the total number of patients undergoing bronchoscopy. Samples were considered diagnostic if they demonstrated malignancy or a benign (but abnormal) process. Samples showing blood, bronchial cells, macrophages, and non-specific acute or chronic inflammation were considered "non-diagnostic". Benign tumors, specific infections or granulomatous inflammation were considered diagnostic (provided that further biopsies, surgery or clinical-radiographical follow-up agreed with this). All patients with samples that did not show malignancy were further assessed by either CT-guided fine needle aspiration (FNA), surgery, or radiographic follow-up at the discretion of their managing physicians. Chart records and radiographic follow-up were reviewed 18 months post-bronchoscopy. The main secondary endpoints included sensitivity for malignancy, complications, and radiation exposure. The gold standard to calculate sensitivity for malignancy was either surgical pathology (from lung resection when available), CT-guided FNA, or 18-month clinical and radiographic follow-up. We defined sensitivity as true positives (TP)/true positives (TP) + false negatives (FN) with the disease being malignancy of any type. All bronchoscopic samples showing malignancy were considered TP. Cases where bronchoscopic samples were not diagnostic of malignancy and malignancy was later confirmed by either CT-guided FNA, surgery, or radiographical progression of disease were considered FN. Intra-bronchoscopy and post-bronchoscopy complications were extracted from medical records. Fluoroscopy time, number of m-CBCT spins, and radiation exposure associated with each were recorded. Radiation exposure measured as the dose area product (DAP) was defined as product of dose and beam area (Gy cm2) and it was measured using an ionization chamber placed between the X-ray tube/collimator setup and the patient. Other relevant data collected included demographics, patient characteristics, target characteristics (anatomic location, distance to the pleura, size, radiographic characteristics, presence of bronchus sign), and procedure characteristics (e.g., duration which was defined as first scope "in" to last scope "out" and concomitant mediastinal staging). Descriptive statistics (frequencies, proportions, means, standard deviations, medians, and range) were provided for patient, target, procedure characteristics, and radiation exposure. Diagnostic yield and sensitivity for malignancy with 95% confidence intervals were reported. SAS 9.4 (SAS Institute INC, Cary, NC, USA) was used for data analysis. 3. Results A total of 51 patients were included in the analysis. The mean target size was 2.6 cm (SD, 1.3 cm) and the mean distance to the pleura was 1.5 cm (SD, 1.4 cm) (see patient and target characteristics in Table 1). Procedure characteristics are depicted in Table 2. The most utilized sampling tool was the needle (94% of the procedures). The median procedural time was 85 min (range of 24-144 min), which included the time required for mediastinal staging which was performed in 33 patients (65%). The diagnostic yield was 78.4% (95 CI, 67.1-89.7%), and the sensitivity for malignancy was 77.4% (95 CI, 62.7-92.1%). Specific diagnoses are described in Table 3. Malignancy was detected in 24 patients and missed in 11 patients. Sixteen patients had benign diagnoses. The only recorded complication was one case of pneumothorax requiring a chest tube. The median fluoroscopy time was 11.2 min (range, 2.9-42.1) and the median number of CT spins was 1 (range, 1-5). The mean fluoroscopy time was 13.59 min (range, 2.9 to 42.1). Fluoroscopy and CBCT (3D spins)-associated radiation exposure is described in Table 4. 4. Discussion This is the largest report on the specific combination of T/UT bronchoscopy, RP-EBUS, and m-CBCT, and the largest one to report radiation exposure associated with m-CBCT. When comparing with prior literature of UT/T bronchoscopy and RP-EBUS, the addition of m-CBCT seems to positively impact its results in terms of diagnostic yield and sensitivity for malignancy . Procedures were safe (only one pneumothorax) with acceptable levels of radiation exposure (approximately two-thirds of DAP was secondary to fluoroscopy and one-third due to the m-CBCT spins). Navigational bronchoscopy platforms, robotic bronchoscopy platforms, use of tomosynthesis, augmented fluoroscopy, and fixed CBCT are rapidly evolving and increasing our reach for peripheral lung lesions. Unfortunately, these pieces of equipment and the required disposables are too costly and not within reach to the majority of bronchoscopists world-wide. Because of this, it is imperative to improve the performance of the simpler and readily available tools such as the use of T/UT bronchoscopes. A large multicenter randomized controlled trial of standard fluoroscopy guided bronchoscopy versus thin bronchoscope with RP-EBUS by Tanner and coworkers reported a fair diagnostic yield of 49% with thin bronchoscopy/RP-EBUS . This same study showed one of the greatest gaps reported in the literature between navigation and diagnostic yield. Of 179 patients who underwent RP-EBUS either because of randomization or subsequent crossover, 174 (97%) had ultrasound "confirmation" of lesion localization, with a concentric image seen in 113 (65%). Yet, diagnostic yield was 50% for concentric and 31% for eccentric lesions. A potential partial explanation for this enormous gap between navigational yield and diagnostic yield is the "pseudo-confirmation" of target reach given by falsely positive RP-EBUS images (attributed to atelectasis, clotting, etc.). Of course, factors associated with the sampling tools they utilized may have had a role as well in this enormous gap. As mentioned before, electromagnetic navigational bronchoscopy has also demonstrated a diagnostic yield around 70%, close to what we have shown in this study . Nevertheless, from the cost-effective aspect, the need of specific disposable navigational tools, and sometimes the need for repeat chest CT with specific slice thickness, makes these navigational techniques less feasible for many bronchoscopists throughout the world. Lastly, transthoracic needle aspiration--with a higher yield, lower cost, and higher rate of pneumothorax--is unable to provide nodal staging, making it a less attractive option for patients with suspected or known lung cancer. The need for a more precise and certain method for confirmation of navigation success has led our group and many others to investigate the additional value of CBCT guidance during peripheral bronchoscopy. One of the initial studies to combine T/UT bronchoscopes with fixed CBCT came from our own group . We performed a prospective pilot study (n = 20 patients) in which we assessed the effect on navigational and diagnostic yield provided by the addition of fixed-CBCT imaging to the standard 2D fluoroscopy and T/UT bronchoscopy combination. CBCT imaging resulted in a 25% absolute increase in navigational yield (from 50 to 75%, p = 0.02), and 20% absolute increase in diagnostic yield (from 50 to 70%, p = 0.04). The median fluoroscopy time was 8.6 min (range, 5-15.4), and the median number of CT scans was 1.5 (range, 1-2). The mean DAP from the total exposure was 64.57 Gy*cm2 (range, 6.14-114.89 Gy*cm2), and originating in the CBCTs 50.45 Gy*cm2 (range, 5.43-66.75 Gy*cm2). Both diagnostic and navigational yield and overall radiation exposure figures were not that distant from our current study. Of note, opposite to our current study, most radiation exposure originated from the CBCTs, instead of originating from 2D fluoroscopy. A more recent report of T/UT bronchoscopy along with fixed CBCT and "augmented fluoroscopy" (Artis zeego, Siemens Healthineers, Forchheim, Germany) is that of DiBardino and coworkers . This study retrospectively analyzed three cohorts of patients: UT bronchoscope + fixed CBCT + RP-EBUS (n = 30); thin or therapeutic bronchoscope + fixed CBCT + RP-EBUS (n = 27); thin or therapeutic bronchoscope + RP-EBUS (n = 59). Diagnostic yields were 85.0% (95% CI, 68.6% to 100%), 68.3% (95% CI, 50.1% to 86.6%), and 44.5% (95% CI, 31.0% to 58.0%), respectively. The median pulmonary lesion diameter was 1.95 cm (interquartile range, 1.5 to 2.75 cm). Virtual navigational bronchoscopy (VNB) (Archimedes, Broncus Medical Inc., San Jose, CA, USA) was most utilized in the thin or therapeutic bronchoscope + RP-EBUS group (45.8% vs. 18.5%, p = 0.02; 45.8% vs. 13.3%, p = 0.002) compared with the thin or therapeutic bronchoscope + fixed CBCT + RP-EBUS and UT bronchoscope + fixed CBCT + UTB + RP-EBUS groups, respectively. They reported a median radiation dose in the fixed-CBCT groups of 70.42 Gy*cm2 (IQR: 42.49 to 99.70). Their study differed from our current one in multiple ways: they utilized augmented fluoroscopy with their fixed CBCT in all cases and they allowed for virtual navigational bronchoscopy. While their reported diagnostic yield is only slightly higher than ours, their mean radiation exposure is substantially higher. This could be explained, in part, by the fact that the operators obtained an extra scan to corroborate tool in lesion. Data on m-CBCT guiding T/UT bronchoscopy are truly scant. The first report is in the form of a "research letter" by Avasarala and coworkers . They reported on the use of an m-CBCT (Cios Spin, Siemens Medical Solutions, Malvern, PA, USA) as a guide for peripheral bronchoscopy in eight patients with an average lesion size of 2.6 cm (electromagnetic navigational bronchoscopy, superDimension Navigation System version 7.0; Medtronic, Minneapolis, MN, USA was used in two cases). Though results are restricted by the sample size, they reported a mean radiation exposure per procedure of 40.92 Gy*cm2, quite similar to ours. The only additional report on the m-CBCT guiding T/UT bronchoscopy combination (same m-CBCT system) that we could find in the literature is that of Sadoughi and coworkers, which consists of a case series of four patients . The use of a different m-CBCT system, the O-arm O2 Imaging System (Medtronic, Minneapolis, MN, USA), was reported in combination with electromagnetic navigational bronchoscopy (EMN) (superDimension Navigation System version 7.0; Medtronic, Minneapolis, MN, USA), and RP-EBUS by Cho and coworkers . In this very small report, the average nodule size was 2.1 cm, and only two of the six cases were diagnostic. Unfortunately, DAP was not reported in this case series for comparison, they only reported an estimated average effective dose per 3D spin of 3.73 mSv. Mobile-CBCT guidance use has been reported with more sophisticated bronchoscopy technologies such as electromagnetic navigation and robotic bronchoscopy . In a very interesting brief research report, Chan and coworkers reported the use of EMN with both mobile-CBCT (Cios Spin, Siemens Healthineers, Forchheim, Germany) and floor-mounted CBCT (syngo DynaCT(r) of Artis Zeego by Siemens Healthineers) in a hybrid operating room . They utilized this combination for a total of 11 procedures: 5 diagnostic biopsies, 4 transbronchial microwave ablations, and 2 cases of dye marking followed by surgery. They intentionally utilized both CBCT systems in order to compare image quality and ease of use. Navigation success was achieved in four out of five cases. The authors reported comparable ease-of-use, and they stated that the m-CBCT unit was able to identify most lung lesions, especially the larger and denser nodules. In two cases of ground glass opacities (8 and 13 mm in diameter), the authors describe that these lesions were faintly seen with m-CBCT and clearly seen with their floor-mounted counterpart. An elegant study by Reisenauer and coworkers reported the combination of m-CBCT (Cios Spin, Siemens Healthineers, Forchheim, Germany) and Shape-Sensing Robotic-Assisted Bronchoscopy (SSRAB) (Ion Endoluminal Robotic Bronchoscopy System, Intuitive Surgical, Sunnyvale, CA, USA) . The objectives of this small prospective pilot study were to assess the ability of m-CBCT to demonstrate tool in lesion, to calculate CT-to-body divergence, and report diagnostic yield and radiation exposure. A total of 30 nodules were sampled, with a median size of 17.5 mm (SD, 6.8) in the largest dimension. The mean airway generation was 7, with a mean distance to pleura of 14.9 mm (range, 1-45.8 mm). Bronchus sign was present in 40% of patients. In 100% of procedures, the proceduralist was able to navigate to the lesion, with a mean number of m-CBCT spins of 2.5 (SD of 1.6). Nineteen (63.3%) cases had an eccentric RP-EBUS signal, 13.3% had a concentric signal, and 23.3% had no signal with RP-EBUS. The total mean fluoroscopy time was 8.7 min (range, 2-27 min) and the total mean DAP was 50.3 Gycm2 with an average of 2.5 spins overall for all cases. The authors reported a diagnostic yield of 93.3%, with a true positive rate 73.3% (22/30 cases) with a 6.7% false negative rate (2/30 cases), and an overall sensitivity for malignancy of 91.7%. These promising results once again show how image guidance with m-CBCT can improve the navigational and diagnostic yield of the most sophisticated bronchoscopy technologies, such as this new robotic platform. The fact that a mean of 2.5 spins were necessary to reach lesions and obtain diagnosis shows that in many of these cases success would have not occurred without this image guidance helping correct the CT to body divergence. With our aging population and the wide-spread use of chest CT, we are bound to diagnose more and more often early-stage lung cancer in medically inoperable patients. In the past few years, bronchoscopists have ventured into the field of therapeutic bronchoscopy with the development of several different modalities for bronchoscopic ablation of lung cancer or lung metastases . While these different ablative techniques are still at a very early investigational level, they do have something in common, which is the need for a real-time imaging modality that can show the location of the ablative probe with respect to the target, vital structures, and the pleura . Whereas fixed-CBCT has been utilized in most reported experiments, Chan and coworkers described the use of m-CBCT in four cases of transbronchial microwave ablations, and Chen and coworkers described another case of transbronchial microwave ablation of a 15 mm left upper lobe ground glass opacity . With comparable image quality, m-CBCT will likely allow more centers throughout the world to slowly adopt bronchoscopic ablative techniques once they are demonstrated to be safe and effective. Our study has a few limitations. At the time these bronchoscopies were done, not all m-CBCT images were being recorded and some proceduralists were not obtaining an extra spin to document tool in lesion (TIL). Thus, we do not have information with regards to TIL, which we believe is key when utilizing CBCT guidance. As described in the methods, ROSE was utilized in all cases. ROSE may not be available in some institutions, and it may have increased our diagnostic yield. Our clinical and radiographic follow-up for suspected benign lesions was 18 months. Though we considered this appropriate and in line with other publications, slow or non-growing adenocarcinomas of the lung could be missed with this observation period. 5. Conclusions Bronchoscopic navigational platforms have been shown to improve the yield of peripheral bronchoscopy but they are also costly and not available world-wide. In lieu of the new evidence suggesting that RP-EBUS may be an inaccurate confirmatory tool for peripheral bronchoscopy, image guidance with m-CBCT may be key to improving the performance of the widely available thin and ultrathin bronchoscopes. Our report, the largest of its kind, suggests that m-CBCT guidance may increase the performance of thin and ultrathin bronchoscopes for peripheral lung lesions in a safe manner. Further prospective studies are needed to corroborate these findings. Author Contributions Conceptualization, M.S., G.C., G.K. and R.F.C.; methodology, M.S., S.I.B., A.K., G.C., G.K. and R.F.C.; formal analysis, M.S., G.C. and R.F.C.; investigation, M.S., S.I.B., A.K., G.C., G.K. and R.F.C.; data curation, M.S., G.C. and R.F.C.; writing--original draft preparation, M.S., G.C. and R.F.C.; writing--review and editing, all authors.; supervision, R.F.C. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of The University of Texas MD Anderson Cancer Center (protocol number 2021-0268). Informed Consent Statement Patient consent was waived by the Institutional Review Board due to retrospective nature of the study. Data Availability Statement Not applicable. Conflicts of Interest M.S., S.I.B. and A.K. have no conflicts of interest. G.C. and G.K. are employees of Siemens. R.F.C. has received research grants from Siemens, Nanobiotix, and Olympus. He is a paid consultant for Olympus, Intuitive Surgical, and Siemens. Figure 1 Bronchoscopy suite set up with m-CBCT. The red arrow corresponds to the metallic arm holding the bronchoscope during cone beam CT scan. diagnostics-13-00827-t001_Table 1 Table 1 Patient and target characteristics. Characteristics N = 51 Gender - Female - Male 26 (51) 25 (49) Smoking History - Never - Ex-smoker - Current 18 (35) 24 (47) 9 (18) Prior Malignancy - Lung cancer - Others 10 (20) 20 (40) ECOG Median (range) 1 (0-2) ASA Score Median (range) 3 (2-4) Target Size (cm) Mean (SD) 2.6 (1.3) Target Characteristics - Solid - Semi-solid 37 (73) 14 (27) Target Location - Right Upper Lobe - Right Middle Lobe - Right Lower Lobe - Left Upper Lobe - Left Lower Lobe 21 (41) 3 (6) 5 (10) 17 (33) 5 (10) Bronchus Sign 39 (75) Distance to Pleura (cm) Mean (SD) 1.5 (1.4) (ECOG = Eastern Cooperative Oncology Group; ASA = American Society of Anesthesiology; SD = standard deviation). diagnostics-13-00827-t002_Table 2 Table 2 Procedure Characteristics. Procedure Characteristics N = 51 Artificial Airway - Laryngeal Mask Airway - Endotracheal Tube 44 (86) 7 (14) Sampling Tools * - Needle - Forceps - Cytology Brush 48 (94) 29 (57) 20 (21) Radial-Probe EBUS - Eccentric view - Concentric view - Image not recorded 29 (57) 13 (25) 9 (18) Presence of Atelectasis by CT 24 (47) Procedure Time (min) Median (range) 85 (24-144) Mediastinal Staging 33 (65) Fluoroscopy Time (min) Median (range) 11.2 (2.9-42.1) Number of CBCT Spins Median (range) 1 (1-5) * More than one tool was utilized in most cases. RP-EBUS = radial-probe endobronchial ultrasound; CBCT = Cone-Beam Computed Tomography. diagnostics-13-00827-t003_Table 3 Table 3 Diagnosis Obtained with Bronchoscopy. Malignancy (n = 24) Benign (n = 16) Non-Diagnostic Sample (n = 11) Lung Cancer Adenocarcinoma Squamous Cell Carcinoma Non-Small Cell (not specified) Small Cell Lung Cancer Carcinoid Others Lymphoma Melanoma Renal Cell Carcinoma 14 3 1 1 1 2 1 1 Infectious/Presumed Infectious Granulomas Aspergilloma Coccidioidomycosis Necrotizing pneumonia Organizing pneumonia Histoplasmosis Staphylococcus Aureus pneumonia Others Hamartoma Post-Radiation Scar 4 2 2 2 2 1 1 1 1 Follow-Up Diagnosis Lung cancer Prostate cancer Lymphoma Granulomatous inflammation Subsequently resolved opacity 5 1 1 1 3 Confirmed with CT-FNA, resolved or improved during 18-month follow-up. diagnostics-13-00827-t004_Table 4 Table 4 Fluoroscopy and Cone-Beam CT-Associated Radiation Exposure. 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PMC10000789 | Healthcare (Basel) Healthcare (Basel) healthcare Healthcare 2227-9032 MDPI 10.3390/healthcare11050697 healthcare-11-00697 Article A New Artificial Intelligence Approach Using Extreme Learning Machine as the Potentially Effective Model to Predict and Analyze the Diagnosis of Anemia Saputra Dimas Chaerul Ekty Conceptualization Methodology Software Validation Formal analysis Data curation Writing - original draft Visualization 1 Sunat Khamron Conceptualization Methodology Software Validation Formal analysis Investigation Resources Data curation Writing - review & editing Visualization Supervision Project administration 1 Ratnaningsih Tri Conceptualization Validation Formal analysis Investigation Resources Data curation Writing - review & editing Visualization Supervision Project administration Funding acquisition 2* Rodriguez-Gonzalez Alejandro Academic Editor Santosh KC Academic Editor Shen Linlin Academic Editor Sicilia Rosa Academic Editor Almeida Joao Rafael Academic Editor 1 Department of Computer Science and Information Technology, College of Computing, Khon Kaen University, Khon Kaen 40000, Thailand 2 Department of Clinical Pathology and Laboratory Medicine, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta 55281, Indonesia * Correspondence: [email protected] 26 2 2023 3 2023 11 5 69718 1 2023 09 2 2023 16 2 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). The procedure to diagnose anemia is time-consuming and resource-intensive due to the existence of a multitude of symptoms that can be felt physically or seen visually. Anemia also has several forms, which can be distinguished based on several characteristics. It is possible to diagnose anemia through a quick, affordable, and easily accessible laboratory test known as the complete blood count (CBC), but the method cannot directly identify different kinds of anemia. Therefore, further tests are required to establish a gold standard for the type of anemia in a patient. These tests are uncommon in settings that offer healthcare on a smaller scale because they require expensive equipment. Moreover, it is also difficult to discern between beta thalassemia trait (BTT), iron deficiency anemia (IDA), hemoglobin E (HbE), and combination anemias despite the presence of multiple red blood cell (RBC) formulas and indices with differing optimal cutoff values. This is due to the existence of several varieties of anemia in individuals, making it difficult to distinguish between BTT, IDA, HbE, and combinations. Therefore, a more precise and automated prediction model is proposed to distinguish these four types to accelerate the identification process for doctors. Historical data were retrieved from the Laboratory of the Department of Clinical Pathology and Laboratory Medicine, Faculty of Medicine, Public Health, and Nursing, Universitas Gadjah Mada, Yogyakarta, Indonesia for this purpose. Furthermore, the model was developed using the algorithm for the extreme learning machine (ELM). This was followed by the measurement of the performance using the confusion matrix and 190 data representing the four classes, and the results showed 99.21% accuracy, 98.44% sensitivity, 99.30% precision, and an F1 score of 98.84%. anemia extreme learning machine beta thalassemia trait iron deficiency anemia hemoglobin E complete blood count This research received no external funding. pmc1. Introduction The main task of the circulatory system is to allow the flow of blood, oxygen, and nutrients to all cells and tissues in the body . However, there are disorders of the circulatory system, better known as blood disorders, in which blood circulation is obstructed Blood disorders commonly experienced by humans include anemia, hemophilia , and blood clots , as well as blood cancers such as leukemia , lymphoma , and myeloma . A blood disorder is a condition that affects the ability of blood to function properly in humans , with most of these disorders having the capacity to reduce the number of cells, proteins, platelets, or nutrients in the blood, thereby impairing its function . It is important to note that most of these problems are caused by abnormalities in certain genes and can be passed down via families . Some medical issues such as drug use and lifestyle also lead to blood abnormalities . It has been reported that anemia is the most common blood disorder seen in humans . Anemia has been defined as a decrease in red blood cells, hemoglobin, and the blood's ability to carry oxygen throughout the body . It is a serious and persistent issue affecting individuals worldwide . The prevalence of anemia among Indonesian women of reproductive age is shown in Figure 1 to exceed the global incidence . A previous study also noted that iron deficiency is the major cause of anemia in every part of the globe . As previously stated, this disease is the substantial reduction in the number of red blood cells circulating inside the body , thereby leading to a great decrease in the ability of the blood to transport oxygen . The diagnosis of anemia is usually confirmed by the concentration of hemoglobin in the blood or the hematocrit, which is the ratio of the number of red blood cells to the total volume of a blood sample . A patient with hemoglobin or hematocrit values that are more than two standard deviations below the normal range is believed to have anemia . Meanwhile, the blood's hemoglobin and hematocrit levels may not adequately reflect the severity of the anemia in a patient with a low RBC mass who is also suffering from hypovolemia-caused dehydration-induced plasma volume loss because the values are likely to fall within the normal range . There are many people suffering from anemia in Indonesia . Iron is believed to be an essential component of several enzymes; it has a role in the formation of hemoglobin in the human body . This means its deficiency can cause anemia . A survey conducted showed that the frequency of anemia in the Indonesian population was anticipated to grow by 0.8% in 2019 reaching 31.20% of the population . The prevalence of anemia sufferers worldwide increased by 0.3% in 2019 to 29.90% . It has also been noted that abnormal production of alpha (a)- or beta (b)-globin chains is the root cause of thalassemia, which is a hematological disease that runs in families, usually passed down from one generation to another . Serum ferritin levels , serum iron , total iron binding capacity, and transferrin saturation percentage are the tests most often used to confirm the presence of IDA, as presented in Table 1 . The identification of BTT and HbE is normally performed through Hb tests using high-performance liquid chromatography, capillary/hemoglobin electrophoresis, or DNA analysis . The application of DNA analysis is not accessible in normal labs due to the need for specialized equipment; in addition, it is time-consuming and expensive . When a patient is assumed to be anemic, doctors often prescribe a hematology test cassette that includes the diagnosis . The high cost of this laboratory test can exhaust patients' resources and government funds, thereby precipitating a financial crisis in the national healthcare systems of middle-income nations. Therefore, a web-based application is proposed in this study to assist doctors in prescribing cost-effective and sensible laboratory tests for the diagnosis of anemia to aid in the rational use of laboratories by clinicians. Anemia is a significant threat to public health on a global scale, and its incidence is disproportionately higher among young children and pregnant women. According to the World Health Organization (WHO), 42% of children under the age of five and 40% of pregnant women worldwide are affected by anemia . Indonesia Family Life Surveys (IFLS) reported that the prevalence among Indonesian children, adolescents, women, and men continued to fall from 1997 to 2008 as indicated in Figure 2 . The anticipated decline among non-pregnant women in Southeast Asia between 1995 and 2011 was predicted to be 8% less than the 9.4% decline among non-pregnant women between 1997 and 2008 as reported by IFLS . It was also discovered that the reduction among pregnant women during the period was comparable to IFLS (7.8%) and Southeast Asian (9%) estimates . However, the decline among children under the age of five was much greater in the IFLS (14.6%) than in Southeast Asia (4%) . The Fick equation is normally used to determine the flow of oxygen to a certain bodily region using three independent variables, including the blood flow , the concentration of red blood cells , and the portion of hemoglobin that has released oxygen on its journey from the arteries to the veins . The oxygen-carrying capacity of anemic individuals' blood was discovered to be diminishing while the remaining two variables underwent compensatory modifications as illustrated in Equation (1) and in the further discussion in . (1) where DPG is diphosphoglycerate, Asat is arterial blood, and Vsat is venous blood. In anemic individuals, the blood flow to crucial organs such as the heart, brain, liver, and kidneys is boosted, while the blood supply to less vital organs is diminished . The anemic patient appears pale because blood is taken from the skin to ensure the vital organs continue to get sufficient oxygen . Moreover, cardiac output is projected to be lower at rest and greater during exercise in individuals with mild or severe anemia compared to healthy individuals . It has also been reported that severe anemia has the ability to increase resting cardiac output in individuals with coronary artery disease or other preexisting cardiovascular diseases, thereby increasing their risk of developing high-output heart failure . It was discovered, as shown in Table 1, that indices and discriminant formulas had promising prediction outcomes in several investigations, but the forecast findings for different populations remain unsatisfactory, particularly when assessing the efficacy of the methodologies used. Some research was observed to be contentious due to gender, age, or ethnic variances . Meanwhile, machine learning-based strategies are believed to have a short-term translational effect. This is indicated by their most significant applications in the field of biomedicine such as medical diagnostics, radiological diagnostics, and medication synthesis. Therefore, it is feasible to create discriminant models using machine learning software approaches to undertake large-scale assessments of laboratory data. It is pertinent to state that machine learning is one of the subfields under the umbrella of artificial intelligence . Artificial intelligence (AI) is defined as the effort of computers to simulate human cognitive processes . This is observed in signal processing, voice recognition, expert systems, and natural language processing. The continuous development of interest in AI has enhanced competition among businesses to showcase the AI features of their goods and services . It is important to note that the creation and training of machine learning and deep learning algorithms require the application of specialized hardware and software . This is mainly due to the fact that the operation of AI systems entails the intake of massive quantities of labeled training data as well as the analysis of the data for correlations and patterns to predict future states . AI programming focuses on three cognitive skills, which include learning , reasoning , and self-correction . This aspect of AI programming involves the collection of data and the development of rules to translate the data into actionable knowledge. Algorithms provide computer systems with detailed instructions to perform a certain task . It is important to state that AI technology is experiencing a time of rapid expansion, but few are aware it has several subfields, one of which is deep learning . The subfields and branches were developed to reduce the very large scope of AI for the purpose of development or research . It is anticipated that AI has the ability to expedite the process of discovering human issues. For example, Laengsri et al. classified 6935 data obtained from the Medical Laboratory Service Centre, Faculty of Medical Technology, Mahidol University between July 2014 and September 2016 as either thalassemia trait or iron deficiency anemia using k-nearest neighbor, decision tree, random forest, artificial neural network, and support vector machine methods. It was discovered that the decision tree algorithm attained a maximum degree of accuracy of 98.03%. The study used seven hematology analyzer-generated features to determine the existence of anemia in individuals. This is necessary because it is difficult to determine the kind of anemia present in a patient using only a blood sample. The findings are expected to allow medical personnel to conduct further diagnostic tests without difficulty and also to ensure a more precise and specific diagnosis . Another study also applied extreme learning machines and regularized extreme learning machines to anemia cases . A total of 342 patients, including 152 with beta-thalassemia-type anemia obtained from the Elazig Public Health Laboratory between 1 December 2016 and 23 May 2019 and 190 with iron deficiency anemia obtained from the Elazig City Hospital Biochemistry Laboratory between 1 March 2018 and 31 July 2018, were studied. The investigation considered a large number of other variables such as gender, in addition to the findings of the clinical pathology test. The regularized extreme learning machine approach produced a 95.59%accuracy rate by combining the k-nearest neighbor, support vector machine, extreme learning machine, and regularized extreme learning machine methods . This present research focuses on establishing an AI model to rapidly, precisely, and reliably diagnose anemia. The process involves classifying anemia into four types, which include beta thalassemia trait (BTT), hemoglobin E (HbE), iron deficiency anemia (IDA), and combination (BTT and IDA or HbE and IDA) using the extreme learning machine approach. Previous studies have shown the relevance of data mining and increasing computing capacity in several biological applications. Therefore, this research seeks to develop a trustworthy and interpretable computational model through the following: (a) collection of clear and dependable laboratory datasets for training and validation, (b) demonstration of dataset characteristics or descriptors to predict the intrinsic properties, and (c) development of a simple and interpretable model. 2. Related Work Support vector machines (SVM), naive Bayes (NB), decision trees (DT), k-nearest neighbor (KNN), multilayer perceptron (MLP), hybrid classifier machine learning, average ensemble (AE), genetic algorithm convolutional neural network (GA-CNN), genetic algorithm stacked-encoder (GA-SAE), support vector machines (SVM), and random forest (RF) are different types of AI. Several studies have been published on the application of machine learning to categorize different kinds of anemia, as indicated by , which forecasted data in the form of a complete blood count (CBC) and constructed a model to identify anemia. Hemoglobin level estimation is an important step in any task related to blood analysis , and it also determines whether a person is anemic. A study used blood test characteristics and applied a machine learning model to calculate hemoglobin levels and identify anemia. The dataset used consists of 9004 data with 75%, or 6753, for training and 25%, or 2251, for testing. A total of three machine learning algorithms--including DT, NB, and NN, as well as a combination of all three approaches known as a hybrid classifier--were applied. Moreover, the MAE and RMSE methodologies were used to assess the performance of the approach, and the MAE results showed that the hybrid classifier had 0.083, the best RMSE value of 0.015, and an accuracy of 0.996% . Tremendous advances in the healthcare industry have resulted in the production of significant data in everyday life . There is a need to extract information for analysis, prediction, recommendation, and decision-making purposes. It was discovered in the realm of medical research that the prediction of disease is essential to design effective prevention and treatment methods. The presentation of wrong information occasionally leads to death. Therefore, a recent study applied 200 CBC data fields obtained from the Pathology Centre and Laboratory Test Centre, as well as RF, C4.5, and NB, which are considered three distinct types of machine learning. K-fold cross-validation and mean absolute error were both used at different stages of the model evaluation process. It was discovered that the C4.5 approach produced the most precise answers, with a precision percentage of 96.0909 and an absolute mean error of 0.0333 . Anemia was also found to be a severe public health problem, particularly for children, in Bangladesh . Thus, the prediction of illness is essential to formulate community and healthcare policy as well as to forecast resource planning. The study used the common risk variables to determine the appropriate machine learning method to predict anemia status in children (under five years) . The 2013 data containing all relevant characteristics for the children, obtained through a nationally representative cross-sectional study conducted by the Bangladesh Demographic and Health Survey (BDHS) in 2011, were used. The investigation employed six techniques, which included the LDA, CART, KNN, SVM, RF, and LR, and they were assessed using the confusion matrix, accuracy, sensitivity, and specificity. The findings showed that the CART approach yielded the greatest evaluation scores of 62.35%, 71.54%, and 53.52% . It is important to note that "deep learning" and "machine learning" are interchangeable when discussing artificial intelligence (AI) . Deep learning is established based on the concept of creating learning algorithms or models that can simulate the human brain . Humans use neurons in their brains to process information, while deep learning algorithms utilize artificial neural networks to perform the same function . Some recent studies used deep learning to enhance the process of identifying anemia in patients. The single red blood cell count imaging data of 130 individuals with sickle cell anemia (SCA) were surveyed and discovered to exceed 9000 single red blood count image data of patients . SCA is a severe hematological illness that often leads to lifelong hospitalization and, in some circumstances, death . It is important to note that the manual location and classification of aberrant cells in the blood films of SCA patients is time-consuming, difficult, and error-prone, and it requires the skill of a hematologist. The study used the AlexNet deep learning model, and the accuracy was recorded to be 95.92%, sensitivity was 77%, specificity was 98.82%, and precision was 90% based on the assessment conducted using the confusion matrix . Deep learning algorithms are gaining importance in the prognosis and prediction of diseases using patients' data . It is pertinent to state that the lack of prompt diagnosis and treatment of anemia can lead to a life-threatening illness . Therefore, several artificial intelligence algorithms have been employed to forecast nutritional anemia cases, especially those related to iron deficiency . Each algorithm was observed to be optimized for a certain subset of data, and this means there is a need to develop new processing techniques. The trend was identified in a previous study where the properties of each dataset are unique, and the size was governed by the number of records and variables specific to the dataset . The strategy blends machine and deep learning to improve the identification process. These were observed to be in the form of genetic algorithm (GA), stacked autoencoder (SAE), and convolutional neural network (CNN) methods, which were used to predict the HGB, nutritional or iron deficiency, B12 deficiency, and folate deficiency anemia as well as to examine individuals without the illness . Moreover, a confusion matrix was used to assess the model, and the greatest level of accuracy for the GA-CNN algorithm was recorded to be 98.5%; the F1 score was 98.8%, sensitivity was 98.7%, and precision was 99.00% . Hemoglobin, a protein contained in red blood cells, is important for the transport and storage of oxygen throughout the body . It has been reported to have the ability to preserve its elasticity, spherical form, and stability in healthy individuals . This is the reason it can float above the red blood cells, but its structure does not ameliorate the symptoms of sickle cell disease . The phenomenon is associated with red blood cells that are twisted and blocked with fluid. It is also important to note that blood circulation is hindered by dysfunctional cells. This is dangerous and has the ability to lead to a range of symptoms, including excruciating pain, organ damage, and even heart attacks . It also has the potential to reduce the average human lifespan. Sickle cell disease identified at an early stage can be treated with antibiotics, vitamins, blood transfusions, painkillers, and other medications. However, the manual grading, diagnosis, and cell counts are time-intensive, and this poses a risk of inaccurate data and misclassification because a single sample usually comprises millions of red blood cells. This is the reason the application of data mining techniques is considered effective and efficient in determining the status of sickle cells inside the human body . An example of this is the adoption of a robust and rapid MLP (multilayer perceptron) classification algorithm to separate sickle cell anemia (SCA) patients into three groups, and the method was observed to surpass the constraints of the manual methods. It was discovered that there are three different types of red blood cells, which include normal, sickle, and thalassemic cells ; this discovery was followed by the application of the confusion matrix to analyze the performance of the MLP approach. The results obtained using the 1387 datasets gathered between August 2017 and August 2019 showed a correctness score of 96.04% while the 100 most recent datasets obtained from the Thalassemia and Sickle Cell Society (TSCS) in Rajendra Nagar, Hyderabad, Telangana, India from September 2019 to August 2020 showed 99% . 3. Materials and Methods 3.1. Data Collection This research was conducted using 165 females and 25 males between the ages of 15 and 41 diagnosed with different kinds of anemia. The data used were compiled by the Clinical Pathology Laboratory at Dr. Sardjito General Hospital in Yogyakarta, Indonesia, and the Department of Clinical Pathology and Laboratory Medicine of the Faculty of Medicine, Public Health, and Nursing at Universitas Gadjah Mada. Moreover, a hematological measure was generated from patients with BTT, IDA, HbE, and a combination of BTT and IDA or HbE and IDA. It is important to note that the Medical and Health Research Ethics Committee (MHREC) of the Faculty of Medicine, Public Health, and Nursing at Dr. Sardjito General Hospital, Universitas Gadjah Mada, issued an ethical letter for the conduct of this research, with the identifier KE/FK/1255/EC/2021. The parameters used include the RBC, Hb, HCT, MCV, MCH, and MCHC in addition to RDW. A total of 24 patients were diagnosed with BTT, 41 with HbE, 104 with IDA, and 21 with the combination method. The definitions of several acronyms used during the investigation are presented in Table 2. 3.2. Research Flow The data derived from the results of a full blood count performed in the laboratory using Advia and Sysmex hematology analyzers produced seven primary characteristics. Moreover, serum ferritin was applied to acquire the gold standard from IDA while hemoglobin electrophoresis was used for BTT and HbE, and the data obtained were examined further and placed in the database based on the flow presented in Figure 3. The seven characteristics previously identified were processed in the database, and the data were labeled by clinical pathology physicians. The data put into the database were preprocessed through cleansing, deletion, the MinMax scaler, and the LabelEncoder. The remaining data were divided into 67%training and 33%testing. Furthermore, the ELM algorithm was used to train the data, which were subsequently applied as the standard to grade the test data. The doctor was involved in the process to provide training courses based on the findings from the laboratory tests. This was followed by the application of the ELM algorithm to classify the data, and its performance was also evaluated. The performance results were further used by the clinical pathology doctor to analyze the data once more to ensure transparency and accountability of the categorization. 3.3. Extreme Learning Machine The research on the predictive capacities of feedforward neural networks has been mathematically centered on two aspects. The first is the simultaneous estimate of the number of inputs while the second is the estimation within a certain period. Thus, the focus of several studies has been on the feedforward neural networks, as indicated by those conducted on the extensive approximation capabilities of typical multilayer feedforward neural networks . Due to their benefits, these networks have been extensively adopted across a variety of commercial sectors over the last few decades. These benefits include the capability to predict complex nonlinear mappings using the available input samples, as well as to provide models for an extraordinarily high number of natural and artificial occurrences, which are considered problematic for standard parametric techniques designed for such events . The single hidden layer feedforward networks, also known as SLFNs, are among the most well-known feedforward neural networks, and their learning and fault tolerance properties have been the topic of discussion in both theoretical and practical studies . The recent development of the extreme learning machine (ELM) neural algorithm for SLFNs was used to improve their performance. It is a novel training method that is exceedingly efficient and effective as indicated in Figure 4. The SLFNs were used in this research to analyze anemia data. It is pertinent to note that the behavior of a linear function as a sum of all linear functions in the network is identical to that of a perceptron regardless of the number of layers comprising the neural network . Thus, a linear function can be described, but there is a possibility of obtaining a nonlinear outcome when an attempt is made to imitate reality. Therefore, a nonlinear activation function was included in the model. It is also pertinent to note that when a network with several layers fails to provide the desired output, the weights and biases need to be modified. The absence of an activation function can cause a change, such as a switch in the neuron signal from 0 to 1, a huge shift, with each neuron feeding a few neurons in the next layer causing a few more neurons to flip. This means minute modifications to the weights and biases used can have a dramatic effect on the end conclusions. Therefore, an activation function was applied to the neuron's output, and small changes in the function's weights can lead to moderate changes in the output. Moreover, the sigmoid function receives any number between -infinity and +infinity, but its output is always between 0 and 1. The Adam optimization method is used. This method is the optimal method for these research data in order to obtain optimal performance results. Table 3 explains the mathematical notation used in the ELM formula. In Equation (2), xi represents the input vector, oj is the output vector, bj=[bj1,bj2,...,bjm]T indicates the output layer's density, wj=[wj1,wj2,...,wjn]T represents the difference in weight between the input and hidden layers, bj is the function's threshold, and g(.) is the function of activation. Moreover, the output matrix of hidden layers H and output-hidden layer weights b for the given input-output sample pairs allows the ELM to obtain an output calculated as Hb=O as indicated in Equation (3), (2) oj=j=1Nbjg(j=1Nwjxi+bj), where wj and bj are randomly generated learning parameters of hidden jth nodes, bj are the links connecting hidden jth nodes and output nodes, and g is the sigmoid activation function for ELM. The wj . xi part becomes the product of the parts of wj and xi. Equation (3) is, therefore, presented as follows:(3) Hb=O where (4) H=[g(w1x1+b1)g(wNx1+bN)g(w1xn+b1)g(wNxn+bN)]nxN (5) b=[b1TbNT]Nxm, and (6) O=[O1TONT]Nxm, and H is referred to here as the output matrix of the hidden layer, (7) b^=HTt where HT is the generalized Moore-Penrose inverse of H and t is the target class/data label. Therefore, the output weights were calculated using a mathematical transformation that eliminates the need for a lengthy training phase requiring repeated updates of the network's parameters through suitable learning parameters such as learning rate and iteration. It is possible to implement the ELM method in two simple steps, which include the training and testing steps. Training Data Step 1 : Prepare a training data matrix X of N number with features of d. Step 2 : Prepare training data target label t. Step 3 : Determine the number of neurons H in the hidden layer. Step 4 : Create a matrix of initial weight values w of size Hxd. Step 5 : Fill w with a random value. Step 6 : Calculate the output hidden layer initialization matrix, Hinit=X.wT (8) Step 7 : Calculate the hidden layer output matrix using a sigmoid function. Step 8 : Count H+, H+=(HT.H)-1.HT (9) Step 9 : Calculate output weight, b=H+.t (10) Step 10 : Calculate output value, O=H.b (11) Testing Data Step 1 : Prepare a testing data matrix X of N number with features of d. Step 2 : Calculate the output initialization matrix for the hidden layer using step 6. Step 3 : Calculate the output matrix for the hidden layer using step 7. Step 4 : Calculate the output value using step 10. 3.4. Blood All blood cells in the body, as shown in Table 4, are derived from pluripotent stem cells located in bone marrow . It is important to note that one of the basic activities of red blood cells is to transfer oxygen from the lungs to the tissues and also to move carbon dioxide in the opposite direction . Moreover, the platelets, which are vital to hemostasis, circulate for just ten days, but red blood cells have a lifetime of four months . It has also been stated that different kinds of phagocytes--including neutrophils, eosinophils, basophils, monocytes, and lymphocytes--comprise white blood cells . The B cells are responsible for the creation of antibodies while the T cells are in charge of immunological responses and defending against viruses and other foreign cells . The white blood cells are present in the blood's white component to combat illnesses caused by bacteria and fungus. Furthermore, the lymphocytes are responsible for the generation of antibodies. Previous studies have also shown that white blood cells have a relatively lengthy lifetime . Red blood cells are the most numerous blood cells , and they appear as biconcave discs densely packed with cytoplasm rich in the oxygen-carrying protein hemoglobin on smears of human peripheral blood . They have a clever structure that allows them to perform their primary functions of transporting oxygen from the lungs to the tissues in the body's periphery and transporting carbon dioxide from the tissues in the body's periphery to the lungs, where it can be expelled via respiration. This means red blood cells facilitate the exchange of oxygen and carbon dioxide between the lungs and peripheral tissues of the body . They also have an average lifespan of 120 days . Meanwhile, platelets which are also known as thrombocytes are microscopic, fully nucleated, and granular-colored cell fragments. They are usually released by the megakaryocytes in bone marrow and play a key part in the control of hemostasis together with the clotting factors of plasma . Platelets have a ten-day lifespan . It has also been discovered that there are several varieties of white blood cells . These include the granulocytes, which are bone marrow-derived, short-lived cells that look identical on a peripheral smear . Neutrophils, sometimes referred to as polymorphonuclear leukocytes, are the most prevalent kind of white blood cell, which possess between three and five lobes on their nucleus and an abundance of light purple granules in their cytoplasm . They are phagocytes that provide defense against a variety of acute pathogens . Monocytes are the biggest white blood cells, ranging from 12 to 20 mm in diameter . They have a folded or kidney-shaped nucleus and an abundance of light blue cytoplasm with a modest number of extremely tiny granules . Monocytes, like neutrophils, are extremely phagocytic, although they vary from neutrophils in a crucial aspect . They primarily develop into relatively long-lived macrophages capable of recognizing "danger" signals created by infection or tissue damage upon emigration into tissues . Meanwhile, eosinophils with a diameter of 12 to 15 have two nuclear lobes and an abundance of red cytoplasmic granules (as befits the cell named after Eos, goddess of the dawn) . They have a crucial role in some chronic immunological responses, including those linked with worm infections, asthma, and certain forms of allergic reactions . The rarest of the granulocytes is basophil, and its nucleus is enveloped by numerous dark blue cytoplasmic granules . They have a diameter of 12 to 15 mm , and many of the circumstances linked with an increase in eosinophil counts are also related to a small rise in basophil numbers . It was also discovered that the mononuclear cells, another kind of white blood cell, lack the segmented nucleus typical of granulocytes . Furthermore, lymphocytes are an essential part of the adaptive immune system and are found to be approximately the same size, 7 to 9 mm in diameter, as a typical red blood cell while at rest and feature a spherical, compact nucleus with minimal cytoplasm . However, the active cells have the potential to grow to a maximum size of 20 mm and also have a small number of granules in addition to the expanded nucleus and copious cytoplasm . Unless the cells are evaluated for the presence of certain lineage-specific markers, it is impossible to tell with absolute certainty whether circulating lymphocytes are B cells, T cells, or natural killer cells. This is due to the fact that the lymphocytes circulating in the blood can be any of these three types . The immune system also has the ability to "remember" the pathogen exposures from many years ago since it has the necessary foundation due to its longevity . 3.5. Anemia Anemia is usually defined through the blood hemoglobin level which is below what is considered normal for a person's age and gender, as indicated in Table 5. The results can vary across labs, but the average values for adult men and women are fewer than 135 g/L and 115 g/L, respectively . The existence of less than 110 g/L for children between the ages of 2 and puberty implies anemia , and because newborns have high hemoglobin levels, the minimum acceptable threshold at birth is 140 g/L . The World Health Organization classifies individuals as having anemia when their hemoglobin levels fall below 130 g/L for males and 120 g/L for women . This scenario shows that approximately 40% of the world's population was expected to suffer from anemia in 2019. There is a higher prevalence in females than males of any age, and in children less than five years old. Moreover, the greatest occurrence throughout the globe has been reported in Sub-Saharan South Asia, and Central, West, and East Africa . The primary causes were found to be iron deficiency (hookworms, schistosomiasis), sickle cell disease, thalassemia, malaria, and chronic diseases . Physicians usually inquire about the patient's medical and family history, conduct a physical examination, and perform some tests including a full blood count to diagnose anemia . More concern is usually placed on the hematocrit and hemoglobin levels, as well as the total number of red blood cells present in the patient's blood, as indicated in Figure 5a. The natural differences between the quantity of blood components present in males and females are presented in Table 4 . It is important to note that the blood counts can possibly be lower in those engaging in or those who have engaged in significant physical activity, particularly in pregnant women or the elderly . Smoking and being at higher altitudes can also increase the number . The testing process usually requires analyzing the size and content of red blood cells as well as the shape and color deviations. A doctor can also prescribe further tests to establish the underlying reason and occasionally examine a sample of bone marrow to determine the existence of anemia, as indicated in Figure 5b . Some patients can exhibit symptoms such as shortness of breath (particularly during physical exercise), weakness, tiredness, palpitations, and headaches . Other symptoms--such as heart failure, angina pectoris, intermittent claudication, and disorientation--are more prevalent among the elderly . Moreover, vision impairment due to retinal hemorrhages can be a serious consequence of anemia, particularly when it develops rapidly . These signals can be classified as either generic or particular. It is also important to note that pallid mucous membranes and nail beds, as shown in Figure 6, are prominent indicators of a hemoglobin concentration below 90 g/L. It is pertinent to state that the color of a person's skin is not a reliable indicator, but tachycardia, pulse rate, cardiomegaly, and a systolic flow murmur indicate hyperdynamic circulation, particularly at the apex. The symptoms of congestive heart failure can also manifest at any age, but they are more prevalent in older people. Furthermore, certain symptoms are linked to each subtype of anemia, such as koilonychia, sometimes referred to as "spoon nails", with iron deficiency, jaundice with hemolytic or megaloblastic anemia, foot ulcers with sicklecell and other hemolytic anemias, and skeletal abnormalities with thalassemia major . Koilonychia is usually caused by the deficiency of iron in the body and is classified as a disorder characterized by inwardly curled nails resembling spoons . It is important to note that megaloblastic anemia is the most prevalent kind. The conjunction of anemic symptoms with severe infection or spontaneous bruising shows the presence of neutropenia or thrombocytopenia, potentially due to bone marrow failure . People with blood hemoglobin levels below the values considered normal for their age and gender are believed to have anemia. Moreover, individual cell size can be used to assess when red blood cells are macrocytic, normocytic, or microcytic. It is also pertinent to state that the cause of anemia can be diagnosed in part by examining the reticulocyte count, the red blood cell shape, and any changes to the white blood cell and/or platelet count . The common clinical manifestations include exertional dyspnea, pale mucous membranes, and tachycardia , while the other symptoms associated with some forms of anemia include jaundice and leg ulcers . The aspiration or trephine biopsy of bone marrow can also be used to investigate anemia and a variety of other hematological disorders . It is also possible to conduct specialized examinations such as immunology and cytogenetics on the cells recovered . 4. Experimental Results The experiment involved using an ELM model to identify and categorize illness in a dataset of individuals with beta thalassemia trait, iron deficiency anemia, hemoglobin E, and the combinations previously defined. The model parameters utilized are listed in Table 2. The real anemic dataset was used and split into training and testing sets during the experiment, and the classification method applied was evaluated using a Python-written confusion matrix. The process was conducted on an Apple M1 machine with 512GB internal memory and 8GB RAM. 4.1. Evaluation Model The data used were classified into test and training data, and they were both evaluated using the confusion matrix model. This was necessary due to the feasibility of determining the accuracy of classification algorithms using an industry-standard technique. It was discovered that the dataset had five separate classifications, which included the BTT, IDA, HbE, and their combinations. The training data accounted for 67% of the entire data set for the inquiry while test data made up the remaining 33%. The assessment conducted was based on the accuracy, precision, sensitivity, and F1 score of the classification algorithm as listed in Equations (12)-(15). The method of value distribution is highlighted in Table 6. Table 6 shows that positive data correctly classified by the system are referred to as the "true positive" (TP), negative data correctly identified as negative are referred to as the "true negative" (TN), negative data incorrectly perceived as positive are known as "false negatives" (FN), and "positive" data incorrectly recognized as "positive" are "false positives" (FP). These values were further used to determine the accuracy, precision, recall, and F1 scores through the following formulas. (12) Accuracy: TP+TNTP+TN+FP+FN (13) Precision: TPTP+FP (14) Sensitivity: TPTP+FN (15) F1-Score: 2xRecall x PrecisionRecall + Precision 4.2. Experimental Results of Extreme Learning Machine The ELM classification model applied to categorize the anemia dataset used a single feedforward network with a hidden layer implementation (SLFNs). This strategy reduced the processing time required for the concealed layer. The usefulness of the model was assessed based on accuracy, precision, sensitivity, and the F1 score in classifying anemic datasets. Table 7 shows the model used in the ELM. The findings of the ELM performance model are presented in Table 8, and it was discovered that it performed best on the four-class anemia dataset, with 99.21% accuracy, 99.30% precision, 98.44% sensitivity, and 98.84% F1 score. The confusion matrix for the model is presented in Table 9, with each row representing an instance of the prediction class while each column indicates an instance of the actual class. The RF approach use n estimators = 400, max features = auto, and entropy. Although entropy is more sophisticated than the Gini index, entropy provides ideal results. In contrast, the KNN technique employs several experiments, including Euclidean distance to determine the distance between classes, and K = 15, which is derived from the K error rate calculation. Several tests were conducted by employing polynomial kernels, RBF kernels, and linear kernels in the SVM approach. In linear kernels, optimal outcomes were obtained. The ELM model, which is the approach described in this work, employed the sigmoid activation function, with the number of hidden layers (9) modified based on the number of inputs and outputs, followed by gradient descent to optimize the weights. ELM was used to optimize the classification process for anemic datasets in order to improve the success rate of the approach as indicated in Table 9. The performance index of each class and the recommended strategy with the highest rate of success are, therefore, presented in Table 10. It was discovered that the random forest, k-nearest neighbor, support vector machine, and extreme learning machine techniques provided the most accurate predictions for the beta thalassemia trait and iron deficiency anemia classes. Moreover, the forecasts for the hemoglobin E and the combination classes were rather correct. 5. Discussion It is very dangerous in the field of medicine to erroneously identify healthy individuals with sickness and vice versa due to the possibility of severe repercussions. This has led to an increase in the usage of data mining technologies for a reliable diagnosis. Therefore, this research used a model of an extreme learning machine to reliably detect and diagnose anemia as well as construct a decision support system to aid clinicians in making decisions. A total of 127 training and 63 test data were employed, and it was discovered that the ELM approach performed much better than RF, KNN, and SVM as indicated by its 99.21% accuracy, 98.44% sensitivity, 99.30% precision, and 98.84% F1 score compared to RF's 77.01% accuracy, 90.83% precision, 78.40% recall, and 80.99% F1 score as well as KNN's 65.42%, 59.40%, 62.81%, and 51.74%, respectively. A previous study by used 6935 data with 986 variables and applied two of the five techniques, KNN (92.36%) and RF (94.16%), to classify BTT and IDA into two groups. Another study by predicted the risk of childhood anemia using several machine learning techniques including KNN and RF. The results showed that KNN had a classification accuracy of 61.95%, a sensitivity of 65.85%, and a specificity of 58.20%while RF had 68.5%, 70.7%, and 66.4%, respectively. This means the overall performance of RF was better than KNN in all three aspects. Another research conducted in 2020 showed that RELM had an accuracy of 95.59% when applied to separate 342 patient records into two types of anemia, IDA and BTT. It is important to note that the ELM method was also applied in the research. Thus, studies have been conducted on the ELM approach, and the concept has progressed to the point where it has shifted from a single hidden layer to a 100-node multilayer hidden layer. This is known as the enhanced improved multilayer extreme learning machine (IML-ELM) with the neural activity occurring both during and after training in the proposed network architecture. Moreover, each layer of the first IML-ELM (IML-ELM1) network was assigned an orthonormal random connection weight while only the very first layer of the second iteration of the IML-ELM contained the random orthonormal connection weights (IML-ELM2). The output weight matrix of the layer was used to calculate the connection weights. The application of the IML-ELM2 assignment method considerably reduced the amount of time required for calculations, and the root mean square error test was observed to have produced 0.627977, 0.104272 (83%), and 0.092685 (85%) . The three studies conducted by used several machine and deep learning techniques, and RELM was reported to have the highest level of performance with 95.59% in distinguishing two forms of anemia. The outcomes of this experiment conducted using the ELM method have been encouraging, with the anemia classified into four separate subtypes, thereby increasing the diagnostic accuracy to 99.21%, precision to 99.30%, sensitivity to 98.44%, and F1 score to 98.84%. Table 11 compares the findings of this research with those from previous studies based on the accuracy metric. It is important to note that this research divided the patients into four distinct groups, including BTT, IDA, HbE, and combinations, and the differences between these groups were categorized with a greater degree of precision than previous approaches, as indicated by the 99.21% recorded for each class. Future studies are expected to focus on analyzing the characteristics considered to be the most important components of anemia to ensure an easier diagnosis process for physicians using a more ideal system. There is also the need for a technique to identify and recommend appropriate anemia datasets using deep learning. 6. Conclusions It is difficult to distinguish between BTT, IDA, and HbE, as well as combinations of these three variables, due to the variability of the anemia-afflicted population. The introduction of computer models was observed to have ensured the rapid screening of anemia at a lower cost. This research provided a summary of the findings of the health system analysis as well as the challenges and barriers encountered throughout the globe in treating anemia patients by using a thorough analysis. Therefore, an ELM approach was applied to expedite the identification of different kinds of anemia. The method using 190 data and seven parameters was found to have an accuracy, sensitivity, and precision of 99.21%, 98.44%, and 99.30%, respectively, as well as an F1 score of 98.84% using a confusion matrix. This means it has a high performance and can be applied quickly and at a cheaper cost. The ELM approach is believed to have the capacity to supplement current indices and formulas developed to aid in the operations of healthcare professionals. Acknowledgments This work was supported in part by Khon Kaen University Grant for ASEAN Countries and China under the KKU Active Recruitment Project 2022, and in part by the Advance Smart Computing Laboratory, College of Computing, Khon Kaen University. We acknowledge PT Abbott and PT Saba Indomedika for assistance with hematology examination reagents and CV Dlastika for assistance with hemoglobin analysis reagent examination. Author Contributions Conceptualization, D.C.E.S., K.S. and T.R.; methodology, D.C.E.S. and K.S.; software, D.C.E.S. and K.S.; validation, D.C.E.S., K.S. and T.R.; formal analysis, D.C.E.S., K.S. and T.R.; investigation, K.S. and T.R.; resources, K.S. and T.R.; data curation, D.C.E.S., K.S. and T.R.; writing--original draft preparation, D.C.E.S.; writing--review and editing, K.S. and T.R.; visualization, D.C.E.S., K.S. and T.R.; supervision, K.S. and T.R.; project administration, K.S. and T.R.; funding acquisition, T.R. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Medical and Health Research Ethics Committee (MHREC) Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada-Dr. Sardjito General Hospital (protocol code KE/FK/1255/EC/2021 and 22 November 2021). Informed Consent Statement Informed consent was obtained from all subjects involved in the study. Written informed consent has been obtained from the patient(s) to publish this paper. Data Availability Statement Not applicable. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Adapted from Ref. about prevalence of anemia in Indonesia and worldwide: A case problem in women of reproductive age, 2000 to 2019. Copyright (2019) World Health Organization (via World Bank). Figure 2 Adapted from Ref. about during the second, third, and fourth waves of the Indonesia Family Life Surveys (IFLS), the prevalence of anemia among children, women, and men were evaluated. These waves occurred from 2005 to 2008. Copyright (2015), Jonathan S Barkley, Katherine L Kendrick, Karen Codling, Siti Muslimatun, and Helena Pachon. Figure 3 Research flow. Figure 4 ELM architecture. Figure 5 Adapted from Refs. and about complete blood count (CBC) test (a) and bone marrow aspirate (b). Both tests are usually used to further identify anemia. Figure 6 Reproduced with permission from Paul A. H. Moss Victor Hoffbrand, Hoffbrand's Essential Haematology 7th Edition; published by John Wiley and Sons, 2015 about conjunctival mucosa (a) and nail beds (b) of two patients with severe anemia (hemoglobin of 60 g/L) . healthcare-11-00697-t001_Table 1 Table 1 Adapted from Ref. about tools to analyze different types of anemia. Copyright (2015), D. Provan, T. Baglin, I. Dokal, and J. de Vos. Type Analyte Anemia Blood Hemoglobin Hemoglobinopathy: Thalassemia Trait; Hemoglobin E; Hemoglobin S Hemoglobin/Capillary Electrophoresis Iron Deficiency Serum Ferritin and Serum Transferrin Receptor (sTfR) Folate Deficiency Erythrocyte Folate Vitamin B12 Deficiency Serum Cyanocobalamin Inflammation Serum C-Reactive Protein (CRP) healthcare-11-00697-t002_Table 2 Table 2 List of laboratory examination abbreviations used in this study. Full Name Abbreviation Red Blood Cell RBC Hemoglobin Hb Hematocrit HCT Mean Corpuscular Volume MCV Mean Corpuscular Hemoglobin MCH Mean Corpuscular Hemoglobin Concentration MCHC Red-Cell Distribution Width RDW b-Thalassemia Trait BTT Iron Deficiency Anemia IDA Hemoglobin E HbE healthcare-11-00697-t003_Table 3 Table 3 Explanation of mathematical notation. Variable Definition O Output layers b Weight of output layer g Activation functions w Weight vector between the input and hidden layers x Input vector b Threshold functions H Number of neurons in hidden layers t Target class Hinit Output hidden layer initialization matrix HT H healthcare-11-00697-t004_Table 4 Table 4 Adapted from Ref. about blood cells. Copyright (2016), A. V. Hoffbrand and P. A. H. Moss. Cell Type Diameter Lifespan in Blood Number of Cells Function Red cells 6-8 120 days Male: 4.5-6.5x106 Female: 3.9-5.6x106 Conveyance of oxygen and carbon dioxide Platelets 0.5-3.0 10 days 140-400x103 Hemostasis Phagocytes Neutrophils 12-15 6-10 h 1.9-7.6x103 (48-76%) Protection against organisms such as bacteria and fungi Monocytes 12-20 20-40 h 0.2-0.8x103 (2.5-8.5%) Protection against organisms such as bacteria and fungi Eosinophils 12-15 Days 0.04-0.44x103 (<5%) Protection against parasites Basophils 12-15 Days 0.01-0.1x103 (<1.5%) Release histamine for inflammatory responses Lymphocyte B T 7-9 (resting) 12-20 (active) Weeks or years 1.5-3.5x103 (18-41%) B-cells: Releases antibodies and assists activation of T-cells. T-cells: Protection against viruses; immune function. healthcare-11-00697-t005_Table 5 Table 5 Normal values for blood cells . Type Males Females Hemoglobin (g/L) 135.0-175.0 115.0-155.0 Erythrocytes (x102L) 4.5-6.5 3.9-5.6 Hematocrit (%) 40-52 36-48 Mean Corpuscular Volume (fL) 80-95 Mean Corpuscular Hemoglobin (pg) 27-34 Leucocytes 50-150 Total (x109L) 4.0-11.0 Neutrophils (x109L) 1.8-7.5 Monocytes (x109L) 0.2-0.8 Eosinophils (x109L) 0.04-0.44 Basophils (x109L) 0.01-0.1 Lymphocyte (x109L) 1.5-3.5 Platelets (x109L) 150-400 Serum Ferritin (mg/L) 40-340 14-150 Serum Vitamin B12 (ng/L) 160-925 (20-680 pmol/L) Serum Folate (mg/L) 3.0-15.0 (4-30 nmol/L) Red Cell Folate (mg/L) 160-640 (360-1460 nmol/L) healthcare-11-00697-t006_Table 6 Table 6 Adapted from Refs. about confusion matrix. Real True False Class Prediction True TP FN False FP TN healthcare-11-00697-t007_Table 7 Table 7 Extreme learning machine models. Parameters Extreme Learning Machine Target (RMSE) 0.001 Inputs 7 Outputs 4 Hidden layers 1 Training data 128 Testing data 62 Hidden layer neurons 9 Output layer neurons 4 Activation function Sigmoid healthcare-11-00697-t008_Table 8 Table 8 Performance results of the ELM model. Split Data Model Accuracy (%) Precision (%) Sensitivity (%) F1 Score (%) 67% (128 Data) Train-33% (62 Data) Test Extreme Learning Machine 99.21 99.30 98.44 98.84 healthcare-11-00697-t009_Table 9 Table 9 Confusion matrix results from the proposed model compared to other methods. Model Classes BTT IDA HbE Combination Random Forest BTT 6 0 0 0 IDA 0 13 4 0 HbE 1 0 34 0 Combination 0 1 2 2 K-Nearest Neighbor BTT 5 1 0 0 IDA 0 13 4 0 HbE 1 2 32 0 Combination 0 2 3 0 Support Vector Machine BTT 6 1 0 4 IDA 0 13 2 1 HbE 1 1 27 5 Combination 0 0 2 0 Extreme Learning Machine BTT 5 0 0 0 IDA 0 15 1 0 HbE 0 0 35 0 Combination 0 0 0 7 healthcare-11-00697-t010_Table 10 Table 10 Index of performance results for each class in each method. Model Classes Accuracy (%) Precision (%) Sensitivity (%) F1 Score (%) Random Forest BTT 100 85.71 100 92.30 IDA 88.57 92.86 76.47 83.87 HbE 96.55 85 97.14 90.66 Combination 90.91 100 40 57.14 K-Nearest Neighbor BTT 89.70 83.34 83.34 83.34 IDA 87.88 72.23 76.47 37.14 HbE 89.28 82.05 91.43 86.49 Combination 85.29 0 0 0 Support Vector Machine BTT 96.61 85.71 54.54 66.61 IDA 93.44 86.67 81.25 83.87 HbE 82.54 87.10 79.41 83.08 Combination 80.95 0 0 0 Extreme Learning Machine BTT 100 100 100 100 IDA 98.44 100 93.75 96.77 HbE 98.41 97.22 100 98.59 Combination 100 100 100 100 healthcare-11-00697-t011_Table 11 Table 11 Competitive results from other methods. Authors Year Data Size Number of Classes Method Accuracy (%) Meena et al. 2019 259,627 4 Decision Tree 97.35 Sow et al. 2019 6935 4 ANN, SVM, RF, and NB 94.74 Laengsri et al. 2019 186 2 DT, KNN, RF, ANN, and SVM 98.03 Ayyildiz and Tuncer 2019 342 2 SVM and KNN 96.20 Kilicarslan et al. 2020 15,300 5 GA-CNN and GA-SAE 98.50 Cil et al. 2020 342 2 ELM, RELM, SVM, and KNN 95.59 Tyas et al. 2020 7108 9 Multilayer Perceptron 93.77 De and Chakraborty 2021 200 2 LR, RF, NB, MLP, DT, and KNN 92.00 Fu Yi-Kai et al. 2021 350 3 Support Vector Machine 76.00 Dejene et al. 2022 11,174 4 RF, Extreme Gradient Boosting, and Cat Boost 97.56 Memmolo et al. 2022 1000 2 DT, DA, NB, SVM, KNN, and Ensemble Learning 84.30 Memmolo et al. 2022 1000 5 DT, DA, NB, SVM, KNN, and Ensemble Learning 69.50 Islam et al. 2022 3020 2 LR, LDA, KNN, SVM, QDA, NN, CART, and RF 81.29 Proposed Model 2023 190 4 ELM 99.21 Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). 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PMC10000790 | Diagnostics (Basel) Diagnostics (Basel) diagnostics Diagnostics 2075-4418 MDPI 10.3390/diagnostics13050850 diagnostics-13-00850 Article Retinitis Pigmentosa Associated with EYS Gene Mutations: Disease Severity Staging and Central Retina Atrophy Placidi Giorgio Conceptualization Software Investigation Data curation Writing - original draft Writing - review & editing Project administration 12+ Maltese Paolo Enrico Methodology Software Writing - review & editing 3+ Savastano Maria Cristina Conceptualization Formal analysis Writing - original draft Writing - review & editing 12* D'Agostino Elena Investigation Data curation 1 Cestrone Valentina Investigation Data curation 1 Bertelli Matteo Methodology 345 Chiurazzi Pietro Methodology Supervision 67 Maceroni Martina Investigation Writing - review & editing 12 Minnella Angelo Maria Investigation Writing - review & editing 12 Ziccardi Lucia Writing - review & editing 8 Parisi Vincenzo Writing - review & editing 8 Rizzo Stanislao Conceptualization Supervision Project administration 129 Falsini Benedetto Conceptualization Methodology Software Formal analysis Investigation Writing - original draft Writing - review & editing Project administration 12 Pinilla Isabel Academic Editor 1 Ophthalmology Unit, Fondazione Policlinico Universitario Agostino Gemelli IRCCS, Largo Gemelli 8, 00168 Rome, Italy 2 Ophthalmology Unit, Catholic University of the Sacred Heart, Largo Francesco Vito 1, 00168 Rome, Italy 3 MAGI'S LAB, 38068 Rovereto, Italy 4 MAGI EUREGIO, 39100 Bolzano, Italy 5 MAGISNAT, Atlanta Tech Park, 107 Technology Parkway, Peachtree Corners, GA 30092, USA 6 Medical Genetics, Fondazione Policlinico Universitario Agostino Gemelli IRCCS, Largo Gemelli 8, 00168 Rome, Italy 7 Genomic Medicine, Catholic University of the Sacred Heart, Largo Francesco Vito 1, 00168 Rome, Italy 8 IRCCS-Fondazione Bietti, 00198 Rome, Italy 9 Consiglio Nazionale delle Ricerche, Istituto di Neuroscienze, 56127 Pisa, Italy * Correspondence: [email protected]; Tel.: +39-0630-154-928 + These authors contributed equally to this work. 23 2 2023 3 2023 13 5 85031 12 2022 07 2 2023 13 2 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). Background. Eyes shut homolog (EYS) gene mutations are estimated to affect at least 5% of patients with autosomal recessive retinitis pigmentosa. Since there is no mammalian model of human EYS disease, it is important to investigate its age-related changes and the degree of central retinal impairment. Methods. A cohort of EYS patients was studied. They underwent full ophthalmic examination as well as assessment of retinal function and structure, by full-field and focal electroretinograms (ERGs) and spectral domain optical coherence tomography (OCT), respectively. The disease severity stage was determined by the RP stage scoring system (RP-SSS). Central retina atrophy (CRA) was estimated from the automatically calculated area of the sub-retinal pigment epithelium (RPE) illumination (SRI). Results. The RP-SSS was positively correlated with age, showing an advanced severity score (>=8) at an age of 45 and a disease duration of 15 years. The RP-SSS was positively correlated with the CRA area. LogMAR visual acuity and ellipsoid zone width, but not ERG, were correlated with CRA. Conclusions. In EYS-related disease, the RP-SSS showed advanced severity at a relative early age and was correlated with the central area of the RPE/photoreceptor atrophy. These correlations may be relevant in view of therapeutic interventions aimed at rescuing rods and cones in EYS-retinopathy. retinal degeneration EYS gene disease staging multimodal imaging OCT subretinal illumination electroretinography Retina Italia ODVUnrestricted grant for Retinitis Pigmentosa studies from Retina Italia ODV. pmc1. Introduction Pathogenic variants in the eyes shut homolog (EYS) gene are estimated to affect at least 5% of patients with autosomal recessive retinitis pigmentosa (arRP) . The gene is defective in several arRP populations worldwide . EYS encodes a large extracellular protein that in Drosophila promotes the formation of epithelial lumina, a selective space inside the rhabdomeres which isolates individual photoreceptor cells and is useful for their development . In humans, the function of EYS is not yet fully understood. However, the protein product is thought to play a role in stabilizing ciliary axonemes in rods and cones and it is involved in the maintenance of photoreceptor cells . Since there is no mammalian model of human EYS disease, it is important to investigate its age-related retinal changes in both structure and function, as well as the degree of central retina involvement. Recently, Iftikhar et al. developed a simple and easily applicable classification of disease severity in RP patients, with a score based on best corrected visual acuity (BCVA), Goldmann visual field diameter and ellipsoid zone (EZ) width. This RP stage score system (RP-SSS) appeared to be readily applicable to different subtypes of RP. In particular, our group recently applied the RP-SSS to the clinical and morphological features of USH2A related retinal degeneration. The USH2A severity score was reliably correlated with patient age as well as with several morphologic and functional parameters of retinal disease . In a previous study by Mcguigan et al. , EYS patients showed typically RP features. Although a residual foveal cone function could still be detected into the fourth, fifth or sixth decade of life, the outer nuclear layer (ONL) thickness and EZ width progressively decreased. Determining the degree of retinal atrophy involving retinal pigment epithelium (RPE)/photoreceptors in the macular region may be particularly relevant either for visual prognosis or for the effects of potential treatments rescuing photoreceptors. An estimate of the area of central macular atrophy (CRA) can be obtained by the automatic determination of the area of sub-RPE illumination (SRI). The SRI identifies bright areas of increased light transmission beneath the RPE, indicating RPE/outer retina atrophy, averaged over a circular area of 5 mm around the fovea by the automated spectral domain optical coherence tomography (SD-OCT) software. In age-related macular degeneration, the SRI was used to measure the area of RPE and outer retinal atrophy (RORA), established from an international consensus by Guymer et al. . The aim of the present study was to determine the disease severity stage in a cohort of ArRP patients carrying pathogenic variants in the EYS gene and to estimate, in these patients, the extent of CRA by using the automated SRI measurements. 2. Materials and Methods This clinical study was performed in compliance with the ICH Guidelines for Good Clinical Practice, adhered to the tenets of the Declaration of Helsinki (1991) and was approved by the Ethics Committee/Institutional Review Board of the Catholic University of Rome, Italy (protocol #8383/15). After a detailed explanation regarding the study procedures, written informed consents for clinical and molecular analyses were obtained from all the adult subjects or relatives when the patient was a minor. All the reported clinical data were retrospectively re-evaluated. 2.1. Subjects We enrolled 17 patients (8 male, 9 female; mean age: 50.5 years.; SD: +-14.7) affected by RP due to variants in the EYS gene and followed at the Center for Inherited Retinal Degenerations of Fondazione Policlinico A. Gemelli, IRCCS. All patients were evaluated between November 2013 and December 2021 and met the following inclusion criteria: (1) clinical and genetic diagnosis of EYS-related RP; (2) good cooperation in psychophysical testing; (3) dioptric media clean enough to perform Spectral Domain-Optical Coherence Tomography (SD-OCT). Exclusion criteria were the presence of: (1) concomitant ocular (e.g., amblyopia, glaucoma) and systemic diseases; (2) poor cooperation in psychophysical testing; (3) severe ocular media opacities. EYS-related RP diagnosis was confirmed through a genetic test and, whenever possible, a segregation analysis on available family members was performed. Seven patients were homozygous and the remaining eight were compound heterozygous for EYS variants. Molecular genetic data are reported in Table 1. The pathogenic variants in the EYS gene were detected using the Next Generation Sequencing (NGS) technology. NGS was performed on a MiSeq personal sequencer (Illumina, San Diego, CA), following the molecular and bioinformatic strategy that we previously published . Multiplex ligation-dependent probe amplification (MLPA) (www.mrc-holland.com, (accessed on 1 December 2017) was also performed in one patient using the Beckman Coulter CEQ 8000 sequencer, and revealed the presence of a deletion in heterozygosity (c. (2135 _2204) _ (2351_2469) del) resulting in the loss of the exons 14 and 15. All variants identified were evaluated according to American College of Medical Genetics and Genomics (ACMG) guidelines with the help of the VarSome online tool (accessed on 24 May 2022) . Molecular genetic data for each patient are detailed in Table 1. 2.2. Clinical Assessment and Functional Evaluation All patients underwent a full ophthalmologic examination including detailed family history, anterior segment biomicroscopy, intraocular pressure measurement, BCVA measured with ETDRS charts, Goldmann visual field using the V/4e target, SD-OCT with measurement of the EZ extension, scotopic and photopic full-field electroretinogram (ERG) recordings and direct and indirect ophthalmoscopy. All patients had a typical RP phenotype. The data collected allowed the determination of the disease severity stage of all enrolled patients according to the cumulative score (CS) and grade indicated by Iftikhar et al . Sub-groups of patients underwent a more detailed comprehensive electro-functional study. A total of 12 out of 17 patients performed 30 Hz submicrovolt flicker ERG, (Retimax Advanced Plus, CSO, Scandicci, Italy)) with the assessment of response variability and signal-to-noise ratio (S/N), as already described in a previous article by Falsini et al. . Macular cone-mediated focal ERG (FERG) was recorded in 7 out of 17 patients using a published technique . Briefly, FERGs were recorded monocularly in response to a flickering uniform red field stimulus superimposed on an equiluminant steady adapting background. Off-line discrete Fourier analysis quantified the peak-to-peak amplitude of the response first harmonic at 41 Hz. 2.3. Morphological Analysis Using Retinal Advanced Multimodal Imaging All patients underwent advanced retinal analysis by SD-OCT using Zeiss Cirrus 5000-HD-OCT Angioplex, sw version 10.0, (CarlZeiss, Meditec, Inc., Dublin, CA, USA). In two patients, the advanced imaging was not possible due to unstable fixation. A High-Definition 5-Line Raster and a macular map (6 x 6 mm Macular Cube 512 x 128) were acquired. In order to obtain the most reliable measurements, two independent operators (M.C.S. and M.M.) measured the residual EZ extension on OCT horizontal scans using a caliper, as previously described . The agreement rate between the two independent experts was equal to 89% (95% confidence interval = 79-98%). The EZ extension was determined by the retinal points where the temporal and nasal EZ borders met the RPE becoming indistinguishable. The Advanced RPE post-processing analysis was used to automatically determine areas of sub-RPE illumination (SRI, measured in mm2) for increased light penetration through atrophic OR, RPE and choriocapillaris, by means of the sub-RPE algorithm. An automated SD-OCT software allowed detection of RPE atrophy in a 5 mm circular area around the fovea. The SRI was used to measure the RPE and outer retinal atrophy (RORA), as established in an international consensus by Guymer et al. . RORA corresponds to a region of signal hyper-transmission into the choroid resulting from the interruption of the RPE and OR and can be classified as complete and incomplete. If the signal hyper-transmission into the choroid does not exceed an area of 250 microns, RORA is defined as incomplete (iRORA). RORA is complete (cRORA) when this value is higher and corresponds to geographic atrophy (GA). 2.4. Statistical Analysis We analyzed both right and left eyes. In this study, we considered only the results from the right eyes for statistical analysis in order not to overestimate the statistical significance and p-values. Analysis from right and left eyes showed substantially similar results. The data were analyzed by parametric or non-parametric analyses, depending on their distribution. Both Pearson's correlation and Spearman rank order correlation were used. ERG data were log transformed to better approximate normal distribution. A p value of less than 0.05 was considered statistically significant. diagnostics-13-00850-t001_Table 1 Table 1 Molecular Genetic Data of EYS patients. ID Sex Nucleotide Change Amino Acid Change Allele State Varsome ACMG Criteria dbSNP rs References 1 M c.8411_8412insTT p.(Thr2805*) HOM LP PVS1 PM2 NA 2 F c.8598del p.(Gly2867Valfs*5) HOM P PVS1 PM2 PP5 rs1050742628 NA 3 M c.8161_8165del p.(Gln2721Alafs*24) HET LP PVS1 PM2 NA NA c.9405T>A p.(Tyr3135*) HET P PVS1 PM2 PP5 rs137853190 4 M c.5928-2A>G HOM P PVS1 PM2 PP5 rs181169439 5 M c.5621dup p.(Pro1875Thrfs*8) HET LP PVS1 PM2 NA c.8411_8412insTT p.(Thr2805*) HET LP PVS1 PM2 NA 6 M c.8565_8568del p.(Asn2855Lysfs*5) HET P PVS1 PM2 PP5 rs1216993077 NA c.4073del p.(Pro1358Glnfs*23) HET LP PVS1 PM2 NA NA 7 F c.5644+5G>T HOM LP PM2 PP3 NA NA 8 F c.4045C>T p.(Arg1349*) HET P PVS1 PM2 PP5 rs930421180 c.4350_4356del p.(Ile1451Profs*3) HET PVS1 PM2 PP5 rs761238771 9 F c.7919G>A p.(Trp2640*) HOM P PVS1 PM2 PP5 rs527236066 10 F c.403_423delinsCTTTT p.(Thr135Leufs*26) HET P PVS1 PM2 PP5 rs1582376398 c.(2135_2204)_(2351_2469)del HET LP PVS1 PM2 NA NA 11 F c.(2137+1_2138-1)_(2259+1_2260-1)del HOM LP PVS1 PM2 NA 12 M c.4045C>T p.(Arg1349*) HET P PVS1 PM2 PP5 rs930421180 c.9299_9302del p.(Thr3100Lysfs*26) HET P PVS1 PM2 PP5 rs769824975 13 F c.9328G>A p.(Gly3110Ser) HOM VUS PM2 PM5 PP3 NA NA 14 c.5621dup p.(Pro1875Thrfs*8) HET LP PVS1 PM2 NA c.8411_8412insTT p.(Thr2805*) HET LP PVS1 PM2 NA 15 M c.5928-2A>G HOM P PVS1 PM2 PP5 rs181169439 16 F c.4219C>T p.(Gln1407*) HET LP PVS1 PM2 rs1421392730 NA del ex32-35 HET LP PVS1 PM2 NA NA 17 M c.1852G>A p.(Gly618Ser) HET VUS PM2 PP5 BP4 rs142450703 c.1561_1563del p.(Asn521del) HET VUS PM2 PM4 PP5 rs747069281 NA c.2309A>C p.(Gln770Pro) HET VUS PM2 BP4 rs398123574 Legend: F, female; M, male; HET, heterozygous; HOM, homozygous; in italics, in cis variants; NA, not available; VUS, variant of unknown significance; LP, likely pathogenic; P, pathogenic. 3. Results Clinical results of individual patients are reported in Table 2. The results of RP-SSS, ERGs and iRORA area are reported in Supplementary Material (Table S1). Figure 1 shows typical macular appearance in an EYS patient with a mild (Pt #3) and an advanced RP-SSS (Pt #1) according to the staging classification by Iftkhar . In the advanced patient EZ is not detectable and ONL thickness is markedly reduced in comparison to the patient with an early stage of the disease. Figure 1 shows the SRI analysis in the macula obtained from the moderate (A) and the advanced (B) EYS patient. SRI area increases with disease severity. Patient A has a RP-SSS of 1 and 12 corresponding to grade 1 and 4, respectively. This picture is associated with changes in the sub-RPE slab (areas of increased SRI) within the 5 mm circle outlined in white. The red line shows the atrophic area closest to the fovea. The staging score was significantly correlated with age and disease duration (r = 0.54, p < 0.01) as shown in Figure 2A,B. An age of 45 years and a disease duration of 15 years corresponded to the appearance of an advanced RP score (>=8). The data showed a trend to saturate after the age of 50, indicating a ceiling effect. Figure 3 shows a scattergram of SRI area as a function of staging score. It can be noted that SRI area, and consequently the CRA severity, was positively correlated with severity score (r = 0.5, p < 0.05). SRI did not show any significant correlation with 30 Hz Flicker or focal ERG amplitude. Figure 4A,B shows scattergrams of SRI area as a function of LogMAR acuity and EZ extension. SRI area was significantly correlated with both measurements (r = 0.5, p < 0.01). 4. Discussion The present study was designed to evaluate the severity stage of EYS disease patients, as determined by the RP-SSS , and to estimate the area of CRA (from a standardized method measuring the area of SRI in the macula) in relation to disease stage in the same patients. The results of our study showed a correlation between the stage of RP and the age and disease duration of patients. Such correlation revealed a severe stage at a relatively early age of 45, supporting the severity of the molecular pathology underlying this sub-type of RP. Previous scientific investigations concerning structure and function correlations in patients with biallelic mutations in the EYS gene support a rapid disease progression as a function of time. McGuigan et al. evaluated a cohort of 15 patients by chromatic static perimetry, SD-OCT, and enface autofluorescence imaging reporting anomalies in the outer nuclear retinal layer of the central retina with some exceptions in the foveal region. Furthermore, an analysis of the perifoveal region as a function of time showed that photoreceptor structural loss was followed by dysmorphology of the inner retina and loss of retinal pigment epithelial integrity. Based on their results, arRP caused by EYS mutation was considered a more rapidly progressive disease compared to other gene mutations causing arRP, such as the USH2A and MAK variants. Some authors suggested that a different phenotype and progression in EYS-related retinal degeneration could be related to the specific EYS genotype . However, due to the limited number of patients examined, such correlations could not be verified and confirmed in the present study. An important finding of this study is represented by the correlation of CRA area with RP-SSS. The increase in CRA area was positively correlated with a greater RP-SSS value, indicating an increase in the disease severity. In addition, it was correlated inversely with the EZ width and positively with LogMAR acuity. Although other morphological predictor factors of atrophy progression in OCT analysis have been reported , RORA is the result of multiple information on the status of EZ, outer segments of the photoreceptors, interdigitation zone and RPE-Bruch's membrane complex . Recently, RORA assessment has been considered not only for early diagnosis but also as a prognostic factor of AMD progression . Indeed, the possibility of evaluating the progression of central atrophy growth could become a key element in the evolutionary study of RP. Currently, the detection of RORA finds an important application in eyes with age-related macular degeneration (AMD) . Automated identification of RORA using machine learning has also been described . Extending the present approach to inherited retinal diseases would be important for predicting their natural history. Indeed, we already demonstrated in other studies how the SRI area, as a result of RORA, was correlated with the disease severity stage in patients with USH2A-related Retinitis Pigmentosa . Although EYS-related RP represents a rare disease, our sample did not include a high number of patients. This constitutes a limitation to our study. Further comparative studies using a similar methodology would be desirable in order to identify useful diagnostic and prognostic criteria to study the natural history of the EYS-related disease, which is currently still difficult to predict. In our study population, no correlations were found between the stage of the disease and the ERG, both focal and 30 Hz flicker ERG. Although in most cases the degenerative evolution of the disease proceeds from rod to cone photoreceptors and the cone loss is considered a secondary occurrence, the obtained ERG data show that a severe cone dysfunction may be present early in EYS patients. Similar to what is observed in patients with USH2A mutations , our study patients showed severely abnormal 30 Hz flicker ERG and fERG, well in advance of other cone-related visual tests, such as visual acuity. This finding supports an early involvement of cone driven responses in EYS-related RP. This hypothesis is in agreement with McGuigan et al. who reported the constant presence of anomalies in the outer nuclear retinal layer of the central retina in a cohort of patients with EYS mutations, except in some where only the foveal region appeared still preserved. It is important to specify that, although the number of patients who underwent a FERG in this study was 7 out of 15, all patients had abnormal ERG responses. Additionally, 30 Hz Flicker ERG showed markedly reduced responses in all patients and was unrelated to other parameters. This latter finding indicates that in our patients a severe extrafoveal cone dysfunction was present, too. Although no treatments are available for EYS-related RP, many novel therapeutic approaches could represent potential therapeutic options in the future . In view of the above, the correct identification of useful outcome measures to evaluate the effectiveness of potential therapies in upcoming clinical trials is a goal to be achieved. Studies in this regard are still few and more will have to be conducted to strengthen our hypothesis. The data reported in this study qualifies the SRI in the detection of iRORA as an important parameter concerning the rate of progression of retinal degeneration caused by biallelic mutations in the EYS gene. 5. Conclusions This research has provided new information concerning patients affected by Retinitis Pigmentosa associated with EYS gene mutations. We used a known RP severity score and validated techniques for the detection of morpho-functional correlations. In particular, in EYS-related RP the severity score was positively correlated with the central retinal atrophy, the central visual acuity and the EZ extension and increases with age and disease duration. In contrast, due to the intrinsic characteristics of the disease, functional measures did not appear sensitive enough to reveal staging-based changes. Given the small size of our sample, further studies concerning the natural history of this kind of retinal dystrophy would be desirable. Acknowledgments Supported by an unrestricted grant from Retina Italia ODV to BF Supported by ERN-eye (BF and SR). The contribution of Fondazione Bietti in this paper was supported by the Ministry of Health and Fondazione Roma. Supplementary Materials The following supporting information can be downloaded at: Table S1: Results of RP-SSS, ERGs and iRORA area of individual patients. Click here for additional data file. Author Contributions Conceptualization, B.F., G.P., S.R. and M.C.S.; methodology, B.F., P.C., M.B. and P.E.M.; software, P.E.M., B.F. and G.P.; formal analysis, B.F. and M.C.S.; investigation, B.F., G.P., E.D., V.C., A.M.M. and M.M.; data curation, G.P., E.D. and V.C.; writing--original draft preparation, G.P., M.C.S. and B.F.; writing--review and editing, B.F., G.P., M.C.S., P.E.M., A.M.M., M.M., L.Z. and V.P.; supervision, S.R. and P.C.; project administration, B.F., S.R. and G.P. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement This study was approved by the Ethics Committee/Institutional Review Board of the Catholic University of Rome, Italy (protocol #8383/15). This research adhered to the tenets of the Declaration of Helsinki. Informed Consent Statement Informed consent was obtained from all patients, after full and detailed explanation of the goals and procedures of the study. Data Availability Statement Data available from authors. Conflicts of Interest The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. Abbreviations ACMG American College of Medical Genetics and Genomics AMD Age-related macular degeneration arRP Autosomal recessive retinitis pigmentosa BCVA Best corrected visual acuity CRA Central retina atrophy cRORA Complete RORA CS Cumulative score ERG Electroretinogram EYS Eyes shut homolog EZ Ellipsoid zone F Female FERG Focal ERG GA Geographic atrophy HET Heterozygous HOM Homozygous iRORA Incomplete RORA LE Left eye LogMAR Logarithm of minimum angle of resolution LP Likely pathogenic M Male MLPA Multiplex ligation-dependent probe amplification NA Not available NGS Next Generation Sequencing OCT Optical coherence tomography ONL Outer nuclear layer P Pathogenic RE Right eye RORA RPE and outer retinal atrophy RPE Retinal pigment epithelium RP-SSS RP stage scoring system SD-OCT Spectral domain optical coherence tomography SRI Sub-retinal pigment epithelium illumination VF Visual Field VUS Variant of unknown significance Figure 1 Images OCT B scan, sub-RPE slab and RPE profile of two patients with early stage RP (A) and advanced stage RP (B). In early stage RP (A), the ellipsoid zone is preserved in the foveal and parafoveal regions. In advanced RP (B) the almost complete loss of the ellipsoid zone and marked reduction in the outer nuclear layer are observable. Figure 2 Cumulative score (CS) from the right eye of each EYS patient plotted as a function of age at testing (A) and disease duration (B). It can be noted that the score increases linearly with both parameters. The r value is 0.54 (p < 0.01). Figure 3 Results of correlation analysis between SRI and CS. SRI showed a positive correlation with CS. The r value is 0.5 (p < 0.05). Figure 4 Correlation analysis between SRI and BCVA (a) and EZ extension (b). SRI area gradually increases as visual acuity and/or ellipsoid zone extension decreases. diagnostics-13-00850-t002_Table 2 Table 2 Demographic and clinical data of studied patients. Nr Sex Onset Age of Assessment Disease Duration RE LE BCVA (LogMAR) VF EZ BCVA (LogMAR) VF EZ 1 M 25 46 21 -0.50 21 0 -1.0 25 0 2 F 42 75 33 -2.70 0 0 -0.50 10 0 3 M 39 43 4 0.00 124 5251 -0.10 131 5053 4 M 18 42 24 -3.70 0 0 -3.70 0 0 5 M 17 30 13 -0.50 50 3321 -0.18 43 641 6 M 22 26 4 0.00 129 3576 0.00 133 3525 7 F 20 62 42 -0.92 17 2084 -0.80 18 1337 8 F 14 26 12 0.00 79 2197 0.00 80 2577 9 F 25 49 24 -0.70 120 2085 -0.50 107 1556 10 F 25 46 21 -0.30 62 1549 -0.30 60 1581 11 F 43 68 25 -2.70 17 0 -2.70 0 0 12 M 39 49 10 -0.50 30 4075 -0.40 16 4313 13 F 30 65 35 -0.70 18 0 -0.50 16 0 14 F 30 51 21 -0.10 22 2018 0.00 25 2448 15 M 14 69 55 -3.70 0 0 -3.70 0 N/A 16 F 26 51 25 -0.30 19 0 -0.18 20 3395 17 M 45 61 16 -2.70 0 0 -2.70 0 0 Legend: F, female; M, male; RE, right eye; LE, left eye; BCVA, best corrected visual acuity; VF, visual field; EZ, ellipsoid zone. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. References 1. Littink K.W. van den Born L.I. Koenekoop R.K. Collin R.W.J. Zonneveld M.N. Blokland E.A.W. Khan H. Theelen T. Hoyng C.B. Cremers F.P.M. Mutations in the EYS Gene Account for Approximately 5% of Autosomal Recessive Retinitis Pigmentosa and Cause a Fairly Homogeneous Phenotype Ophthalmology 2010 117 2026 2033.e7 10.1016/j.ophtha.2010.01.040 20537394 2. Audo I. Sahel J.-A. Mohand-Said S. Lancelot M.-E. Antonio A. Moskova-Doumanova V. Nandrot E.F. Doumanov J. Barragan I. Antinolo G. EYS Is a Major Gene for Rod-Cone Dystrophies in France Hum. Mutat. 2010 31 E1406 E1435 10.1002/humu.21249 20333770 3. Numa S. 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PMC10000791 | J Synchrotron Radiat J Synchrotron Radiat J. Synchrotron Rad. Journal of Synchrotron Radiation 0909-0495 1600-5775 International Union of Crystallography 36891842 ok5086 10.1107/S1600577523000619 JSYRES S1600577523000619 Research Papers Photon-shot-noise-limited transient absorption soft X-ray spectroscopy at the European XFEL Transient XAS at EuXFEL Le Guyader Loic a* Eschenlohr Andrea b Beye Martin c Schlotter William d Doring Florian e Carinan Cammille a Hickin David a Agarwal Naman a Boeglin Christine f Bovensiepen Uwe b Buck Jens c Carley Robert a Castoldi Andrea g D'Elia Alessandro h Delitz Jan-Torben a Ehsan Wajid a Engel Robin c Erdinger Florian i Fangohr Hans ajk Fischer Peter i Fiorini Carlo g Fohlisch Alexander l Gelisio Luca a Gensch Michael mn Gerasimova Natalia a Gort Rafael a Hansen Karsten c Hauf Steffen a Izquierdo Manuel a Jal Emmanuelle o Kamil Ebad a Karabekyan Suren a Kluyver Thomas a Laarmann Tim cp Lojewski Tobias b Lomidze David a Maffessanti Stefano c Mamyrbayev Talgat e Marcelli Augusto qrs Mercadier Laurent a Mercurio Giuseppe a Miedema Piter S. ac Ollefs Katharina b Rossnagel Kai tu Rosner Benedikt e Rothenbach Nico b Samartsev Andrey a Schlappa Justine a Setoodehnia Kiana a Sorin Chiuzbaian Gheorghe o Spieker Lea b Stamm Christian v Stellato Francesco w Techert Simone c Teichmann Martin a Turcato Monica a Van Kuiken Benjamin a Wende Heiko b Yaroslavtsev Alexander a Zhu Jun a Molodtsov Serguei a David Christian e Porro Matteo ax Scherz Andreas a a European XFEL, Holzkoppel 4, 22869 Schenefeld, Germany b Faculty of Physics and Center for Nanointegration Duisburg-Essen (CENIDE), University Duisburg-Essen, Lotharstrasse 1, 47057 Duisburg, Germany c Deutsches Elektronen-Synchrotron DESY, Notkestrasse 85, 22607 Hamburg, Germany d Linear Coherent Light Source, SLAC National Accelerator Lab, 2575 Sand Hill Rd, Menlo Park, CA 94025, USA e Paul Scherrer Institute, 5232 Villigen PSI, Switzerland f Universite de Strasbourg, CNRS, Institut de Physique et Chimie des Materiaux de Strasbourg, UMR 7504, F-67000 Strasbourg, France g Politecnico di Milano, Dip. Elettronica, Informazione e Bioingegneria and INFN, Sezione di Milano, Milano, Italy h IOM-CNR, Laboratorio Nazionale TASC, Basovizza SS-14, km 163.5, 34012 Trieste, Italy i Institute for Computer Engineering, University of Heidelberg, Mannheim, Germany j Max-Planck Institute for the Structure and Dynamics of Matter, Luruper Chaussee 149, 22761 Hamburg, Germany k University of Southampton, Southampton SO17 1BJ, United Kingdom l Institute for Methods and Instrumentation for Synchrotron Radiation Research (PS-ISRR), Helmholtz-Zentrum Berlin fur Materialien und Energie GmbH (HZB), Albert-Einstein Strasse 15, 12489 Berlin, Germany m Institute of Optical Sensor Systems, DLR (German Aerospace Center), Rutherfordstrasse 2, 12489 Berlin, Germany n Institute of Optics and Atomic Physics, Technische Universitat Berlin, Strasse des 17 Juni 135, 10623 Berlin, Germany o Sorbonne Universite, CNRS, Laboratoire de Chimie Physique-Matiere et Rayonnement, LCPMR, 75005 Paris, France p The Hamburg Centre for Ultrafast Imaging CUI, Luruper Chaussee 149, 22761 Hamburg, Germany q INFN - Laboratori Nazionali di Frascati, via Enrico Fermi 54, 00044 Frascati, Italy r RICMASS - Rome International Center for Materials Science Superstripes, 00185 Rome, Italy s Istituto Struttura della Materia, CNR, Via del Fosso del Cavaliere 100, 00133 Rome, Italy t Institute of Experimental and Applied Physics, Kiel University, 24098 Kiel, Germany u Ruprecht Haensel Laboratory, Deutsches Elektronen-Synchrotron DESY, 22607 Hamburg, Germany v Department of Materials, ETH Zurich, 8093 Zurich, Switzerland w Physics Department, University of Rome Tor Vergata and INFN-Sezione di Roma Tor Vergata, Via della Ricerca Scientifica 1, 00133 Roma, Italy x Department of Molecular Sciences and Nanosystems, Ca' Foscari University of Venice, 30172 Venice, Italy Kvashnina K. Editor ESRF - The European Synchrotron, France Correspondence e-mail: [email protected] 01 3 2023 20 2 2023 20 2 2023 30 Pt 2 s230200 284300 08 11 2022 24 1 2023 (c) Loic Le Guyader et al. 2023 2023 This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-BY) Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited. A full version of this article is available from Crystallography Journals Online. A beam-splitting off-axis zone plate setup to measure transient X-ray absorption spectroscopy is presented, as implemented at the Spectroscopy and Coherent Scattering instrument at the European X-ray Free-Electron Laser. Femtosecond transient soft X-ray absorption spectroscopy (XAS) is a very promising technique that can be employed at X-ray free-electron lasers (FELs) to investigate out-of-equilibrium dynamics for material and energy research. Here, a dedicated setup for soft X-rays available at the Spectroscopy and Coherent Scattering (SCS) instrument at the European X-ray Free-Electron Laser (European XFEL) is presented. It consists of a beam-splitting off-axis zone plate (BOZ) used in transmission to create three copies of the incoming beam, which are used to measure the transmitted intensity through the excited and unexcited sample, as well as to monitor the incoming intensity. Since these three intensity signals are detected shot by shot and simultaneously, this setup allows normalized shot-by-shot analysis of the transmission. For photon detection, an imaging detector capable of recording up to 800 images at 4.5 MHz frame rate during the FEL burst is employed, and allows a photon-shot-noise-limited sensitivity to be approached. The setup and its capabilities are reviewed as well as the online and offline analysis tools provided to users. transient absorption soft X-ray spectroscopy European XFEL Volkswagen FoundationDeutsche ForschungsgemeinschaftSFB 1242 This research was partly supported by the Maxwell computational resources operated at Deutsches Elektronen-Synchrotron DESY, Hamburg, Germany. LLG acknowledges the Volkswagen-Stiftung for the financial support through the Peter-Paul-Ewald Fellowship. This research was funded in part by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) - Project-ID 278162697 - SFB 1242. pmc1. Introduction X-ray absorption spectroscopy is one of the most widely used techniques at synchrotron radiation facilities around the world for investigating the local structure and electronic properties of atoms in solids and molecules on surfaces or in solutions (Bianconi & Marcelli, 1992 >; Stohr, 1992 >; Bokhoven & Lamberti, 2016 >). Its implementation at free-electron lasers (FELs) opens the possibility of performing high-resolution spectroscopy like at synchrotrons with the added advantage of accessing ultrafast dynamics on the femtosecond timescale. Transient X-ray absorption spectroscopy (XAS) allows, for example, monitoring electron-hole dynamics (Boeglin et al., 2010 >; Zurch et al., 2017 >; Britz et al., 2021 >), electron localization (Stamm et al., 2007 >; Lojewski et al., 2022 >), on-site Coulomb repulsion (Baykusheva et al., 2022 >), lattice excitation (Rothenbach et al., 2019 >, 2021 >), magnetic order (Agarwal, 2022 >) and ultrafast phase transitions (Cavalleri et al., 2005 >). Monochromatic soft X-ray pulses of a few nJ of energy are easily delivered by FELs and contain a few 107 photons, which is several orders of magnitude more than the intensity delivered by synchrotron-based femto-slicing facilities (Holldack et al., 2014 >). At the photon shot-noise limit, a single-shot signal-to-noise ratio (SNR) at the level of a few thousands is thus achievable. However, the X-ray pulses generated by self-amplified spontaneous emission (SASE) at a FEL feature very high pulse-to-pulse intensity fluctuations after a monochromator (Saldin et al., 1998 >). It is therefore essential to normalize the transmitted signal by measuring the incoming radiation intensity before the sample (I 0). The main challenge in measuring femtosecond XAS in the soft X-ray regime, where small changes in the spectra have to be detected, is precisely this normalization scheme. XAS at FELs was pioneered by Bernstein et al. (2009 >), where the I 0 normalization was achieved by using a half of a sample such that one half of the X-ray beam was propagating through the sample while the other half was propagating freely. A Ce-doped yttrium aluminium garnet (Ce:YAG) scintillator screen placed in front of an intensified charge-coupled-device camera (ICCD) was used for detection. This approach relies on spatial beam coherence and pointing stability of the FEL beam. An alternative approach consists of using a transmission grating to create copies of the incoming beam with the different diffraction orders and using the +1st grating order to measure the sample transmission and the -1st grating order to measure I 0 (Katayama et al., 2013 >, 2016 >; Brenner et al., 2019 >; Engel et al., 2020 >, 2021 >). The advantage here is that the beam intensities are linked by the grating element. Later, the sensitivity of this method was improved by combining the transmission-grating approach with a focusing zone plate component. As the beams propagate towards the detector, they are focused in front of the sample and then expand and illuminate many more pixels, thereby increasing the maximum number of photons that can be detected without saturating the detector (Schlotter et al., 2020 >). In our improved scheme, we use an off-axis zone plate (Buzzi et al., 2017 >; Jal et al., 2019 >; Rosner et al., 2020 >) which gives the possibility of separating the different zone plate orders on the detector, as we discuss below. In this article, we review the scheme as implemented at the Spectroscopy and Coherent Scattering (SCS) instrument at the European XFEL. In Section 2, the setup is described. First, an overview of the setup and its capabilities is given in Section 2.1, followed by the design choices, specifications and fabrication details of the employed diffractive optics in Section 2.2. In Section 2.3, we review the different control aspects necessary to collect data efficiently during an experiment. Finally, in Section 2.4, we detail the beam propagation calculator that we provide to users in order to design their samples to be compatible with this setup. In Section 3, we detail the data analysis steps required to make the best use of the collected data. In Section 3.1, we introduce basic statistical concepts. In Section 3.2, we discuss the imaged beam on the detector, which leads to the flat-field correction in Section 3.3. In Section 3.4, we describe the non-linear correction and how it is calculated and applied to the data. In Section 3.5, we discuss how close these different corrections bring us to the photon shot-noise limit. In Section 3.6, we describe the offline analysis procedure available to the users and, in Section 3.7, we describe the tools we provide for the analysis during the experiment as the data are being collected. In Section 4, we showcase some examples of experiment results, starting, in Section 4.1, with the transient XAS in NiO and the impacts of the different corrections on the data. Finally, in Section 4.2, we discuss the sensitivity limits of the current setup in terms of X-ray fluence (Section 4.2.1), repetition rate (Section 4.2.2) and typical sample systems that can and cannot be measured currently with this setup (Section 4.2.3). 2. Setup 2.1. Overview The setup implemented at the SCS instrument at the European XFEL is schematically shown in Fig. 1 >. The X-ray pulses are generated in the SASE3 undulator system (UND). The X-ray photon energy is determined by the fixed electron bunch acceleration energy and the variable undulator gap. The X-rays are then monochromated with the help of a variable-line-spacing grating (MONO) combined with an exit slit (ES) (Gerasimova et al., 2022 >). The monochromatic X-ray pulses propagate through the beam-splitting off-axis zone plate (BOZ) optics. It consists of a transmission grating and a focusing zone plate in a single element. The grating splits the initial beam into three beams of approximately equal intensity. The zone plate focuses these beams shortly before the sample (SAM) and the resulting X-ray spot size on the membrane is typically 50 mm x 50 mm. The transmitted beams further expand downstream and are detected on a single monolithic sensor of the DSSC1 detector placed at 5.4 m. The sensor is 3 cm high and 6.2 cm wide and populated by 128 by 256 pixels. The high sensitivity of this measurement scheme is assured by using a low-noise detector and illuminating many pixels to achieve a high signal-to-noise ratio. The method's sensitivity is then mainly limited by the number of photons detected, as we show below. The DSSC can record up to 800 frames at 4.5 MHz during the FEL train, meaning an effective repetition rate of 8 kHz. The sample consists of an array of X-ray transparent membranes, with each of the three X-ray beams passing through a separate membrane window. The middle membrane window consists of the bare substrate, while the right and left membranes each consist of the thin film under investigation. To record a spectrum, the X-ray photon energy can be scanned by varying together the undulator gap, the monochromator energy and the BOZ position along the X-ray beam, with the help of the Distributed Object Oriented Control System (DOOCS) (Grygiel et al., 1996 >) and the Karabo control system (Hauf et al., 2019 >), as shown in Fig. 1 > by dash-dotted lines connecting blue double-headed arrows. Finally, for stroboscopic or single-shot pump-probe experiments, an optical pump laser (OL) can be focused onto one of the membranes to excite it (Pergament et al., 2016 >). The time delay between the optical pump and X-ray probe pulses can be controlled by an optical delay line (not shown in Fig. 1 > for simplicity). By defining regions of interest, the transmitted intensity of each of the three separated beams can be computed from the DSSC detector images. The XAS of the unexcited sample can be determined from the intensity of the beam going through the bare membrane (grating 0th order) and the beam passing through the unexcited sample (grating +1st order), as shown by the blue curve in the bottom-left plot in Fig. 1 >. Similarly, the XAS of the excited sample can be determined simultaneously, as shown by the orange curve in the bottom-right plot in Fig. 1 >. Finally, the pump-induced XAS change can be determined as well simultaneously from the intensity of the beam going through the unexcited sample and the beam going through the excited sample, as shown by the green curve in the bottom-center plot in Fig. 1 >. In the next sections, we present the design and fabrication of the BOZ optics, the different control aspects necessary to collect a spectrum, and, finally, the tools available to users to aid in the design of samples for this setup. 2.2. Diffractive optics Horizontally, the grating structure of the BOZ optics splits the beam into different orders. The period of the grating structure is chosen to provide an angular separation of the diffraction orders of 3.1 mrad, which is just sufficient to prevent the beams from overlapping as they propagate 5.4 m up to the DSSC detector placed at the end of the SCS experiment hutch. This gives, for example, a grating structure period of 465 nm to operate around the Ni L 3,2 edges at 860 eV. To detect as many photons as possible without saturating the detector, the beams have to be as large as feasible. This means that the focal length of the Fresnel zone plate component of the BOZ optics should be chosen as small as possible. For this setup, we have chosen a focal length of 250 mm to ensure that the sample can be placed just upstream of the zone-plate focus in its most upstream position. Then, by using the 190 mm scanning range of the sample along the beam propagation, we can control the spot size of the X-rays on the sample, from tight focus at the zone plate focus to much larger beams in the most downstream position. Similarly to the grating structure, the Fresnel zone plate structure creates diffraction orders. Considering only the lowest diffraction orders, we have the +1st zone plate order, which is focused downstream at the zone plate focus, the 0th zone plate order, which is unfocused and simply propagates through, and the -1st zone plate order, which is diverging. With an on-axis zone plate, all these different orders would spatially overlap on the detector (Schlotter et al., 2020 >). Here, we use an off-axis part of the zone plate, as schematically shown in Fig. 1 > with the beam hitting the bottom instead of the center of the zone plate, to vertically separate the different orders on the detector. In our setup, this off-axis component, measured as the distance between the BOZ optics center and the optical axis for the zone plate, is chosen to be 0.55 mm. For the Ni L 3,2 edges at 860 eV, this gives an off-axis Fresnel zone plate structure with an outermost zone width of 179 nm for a zone plate aperture of 0.8 mm x 0.8 mm. The intensity ratio of the three focused beams can be controlled by two different design parameters in the BOZ pattern as described in the pattern-inversion method and the pattern-shift method (Doring et al., 2020 >). We applied the latter method with a shift parameter of s = 0.32, as it gives higher overall efficiency. The BOZ elements were made from single-crystal silicon membranes (Doring et al., 2020 >). The 1 mm-thick silicon membranes (Norcada Inc., Edmonton, Canada) were sputter-coated with a 10 nm chromium layer and then spin-coated with a 70 nm polymethylmethacrylate (PMMA) resist. After electron-beam lithography (Vistec EBPG5000+, operated at 100 keV electron energy) of the BOZ patterns and subsequent development, the resist patterns were transferred into the Cr mask by reactive ion etching in a Cl2/O2 plasma. After removal of the PMMA resist in acetone, the pattern was etched down to a depth of about 700 nm into the silicon membranes by reactive ion etching in an SF6/C4F8/O2 plasma. Finally, the Cr mask layer was removed to yield pure silicon structures. 2.3. Controls To record an XAS spectrum, the monochromator (Gerasimova et al., 2022 >) is scanned continuously back and forth between two energy endpoints. The 120 m-long undulator system is controlled through the DOOCS control system (Karabekyan et al., 2012 >, 2013 >). A DOOCS middlelayer (ML) server provides an interface to specify the undulator photon energy and, in turn, controls the gap size of each undulator. During a scan, the Karabo control system ensures that the undulator photon energy follows the monochromator photon energy through a feedback loop that interfaces the DOOCS ML server through Karabo-DOOCS bridging software. This combination allows spectra covering tens of eV to be recorded. However, to maintain full lasing of the undulators, the relative variation in the undulator deflection parameter DK/K and, respectively, the relative change in the magnetic field strength DB/B for the undulator system should not exceed the Pierce or FEL parameter r (Pierce, 1950 >; Bonifacio et al., 1984 >; McNeil & Thompson, 2010 >). In the case of the SASE3 undulator system, this relative change of the deflecting parameter DK/K should not exceed the value of 1 x 10-3. The magnitude of the magnetic field depends on the undulator gap g and the undulator period lu and is described by an exponential decay according to the expression where, in the case of the U68 undulator with period lu = 68 mm, the parameters have the values a = 3.214, b = -4.623 and c = 0.925. By applying the partial differential method with respect to changes of the gap Dg, the boundary condition for full lasing can be determined, giving Assuming that the working range of the U68 undulator gap is 10-25 mm, the maximum deviation of the gap between undulators of one system should not exceed Dg = 15-17 mm to maintain the full lasing condition. If the undulator system is set to follow the monochromator with a scanning speed of 1 eV s-1, we need to determine the undulator gap scanning speed. From the undulator resonance equation for the first harmonic at small observation angles, where g is the relativistic Lorentz factor for the electron, we can use the the partial differential method with respect to the undulator gap change. The resulting gap velocity values for 10 mm and 25 mm undulator gap are 10 mm s-1 and 2 mm s-1, respectively. Measurements made on a system of four undulators showed that, even without forced synchronization of the axes of the undulators, the maximum deviation for a gap velocity of 0.856 mm s-1 corresponds to 40 mm (Karabekyan et al., 2013 >). It was also shown that this dependence is close to linear. By linear approximation for a gap velocity of 10 mm s-1, it could be concluded that the maximum deviation of the gap of undulators in one system would not exceed 0.5 mm. This value confirms that, by coupling the monochromator axis and the gap axes of the undulator system, the full on-the-fly lasing condition for soft X-ray beamlines can be achieved, even with a few seconds delay in communication between the undulator system and the monochromator. One drawback of employing diffractive optics is that their properties are wavelength-dependent. For the BOZ, this means that both the focal distance and the grating diffraction angle are proportional to the photon energy. During an extended energy scan, both the X-ray spot size and the beam pointing on the sample can vary significantly. To compensate for these effects, we use a three-axis linear piezo-motor stage to displace the BOZ along the X-ray beam to a position calculated from the monochromator readback energy. This ensures that the X-ray spot size and position on the sample remain constant during the energy scan. The change in BOZ position along the X-ray beam Dz due to a change in photon energy DE can be calculated from the zone plate focal length f at the design energy E 0 using As an example, considering an energy scan spanning both Ni L 3,2 edges from 845 eV to 875 eV for a zone plate with a design energy of E 0 = 860 eV with a focal length of f = 230 mm, the change in BOZ position along the X-ray beam Dz is 8 mm. 2.4. Beam propagation In contrast to the setup of Schlotter et al. (2020 >), where independent manipulators were used to align individual sample and reference membranes in the beam, the setup at the SCS instrument has only one sample manipulator. Therefore, the sample has to be precisely designed to fit with the three-beam geometry from the start. To facilitate this, we publicly provide the BOZcalc Python package (SCS, 2022a >) to calculate the beam propagation and display projections at the sample and detector plane. For example, in Fig. 2 >, the horizontal and vertical beam profiles as a function of the distance from the interaction point, starting from the horizontal and vertical intermediate source points up to the DSSC detector, are displayed. From the intermediate source point, the X-ray beam propagates down to the SCS instrument and is slightly focused by the Kirkpatrick-Baez mirrors (Mercurio et al., 2022 >) up to the BOZ. After that, the beam is strongly focused and then expands until it reaches the DSSC detector. The advantage of using a zone plate with an off-axis component becomes evident in Fig. 2(b) >, where the undulators' fundamental (red) and the second harmonic (blue) are spatially separated. This allows the sample to be used as a harmonic sorting aperture, blocking the unwanted undulator second-harmonic contribution that is not always suppressed by the beamline offset mirrors. In Figs. 3 >(a) and 3(b), the results of a calculation, using the BOZcalc Python package, of the three beams' positions and shapes at the sample plane, respectively, at the detector plane are shown. All the calculations are performed within a Jupyter notebook. In the sample plane in Fig. 3 >(a), a membrane array is displayed as a series of black rectangles. The etching facets on the back of the substrate are represented as gray dashed lines. The red squares indicate the expected beam size for the focused 1st order beam of the zone plate at the given sample position. The overlapping gray squares represent the unfocused 0th order of the zone plate. The blue squares are the expected beam position and size of the second-harmonic beam from the undulator. In this arrangement, the unwanted second-harmonic radiation would be blocked by the sample frame, while the fundamental beam would propagate through the sample membranes. In the detector plane shown in Fig. 3 >(b), the black rectangle represents the DSSC module used to record the beams' intensity, while the green shapes represent the DSSC filter mount that is opaque to X-rays. The red squares show how the three beams expand before reaching the DSSC to fill the sensor area after being focused by the zone plate in front of the sample. It is worth noting that the undulators' second harmonics would overlap with the other beams of interest if they were not blocked by the sample. It is also worth mentioning that the grating 2nd order, zone plate 2nd order of the second undulator harmonic, will exactly overlap with the grating 1st order, zone plate 1st order of the undulator fundamental, and therefore cannot be separated with an aperture. However, the intensity of the 2nd grating order is determined by the duty cycle of the grating structure and is exactly zero for an even duty cycle. As a consequence, the pattern shift method shows the important advantage of suppressing a potential contamination of the signal by the second-harmonic beams appearing in the 2nd grating order (Doring et al., 2020 >). The unfocused beams from the 0th zone plate order are the barely visible small spots seen on the filter mount near the bottom of the DSSC module. It is also important to block these beams as they would otherwise saturate the illuminated pixels. The input controls of the BOZcalc calculator can be interactively adjusted. This allows, for example, to quickly check the expected beam size at different sample distance. Having presented the setup and the tools available to users to design samples compatible with this setup, we now detail the data processing and analysis required. 3. Data processing 3.1. Statistics The absorption of light propagating through a sample with thickness d is given by the Beer-Lambert law, where I 0 is the incoming photon intensity, I t is the transmitted photon intensity after the sample, m is the inverse of the absorption length, and d is the sample thickness. The sample transmission is T = I t/I 0, and the X-ray absorption A is The other important quantity to determine is the SNR of the measurement, which is given by propagating uncertainties (Meija & Mester, 2007 >; Schlotter et al., 2020 >), where |...| is the absolute value, s is the standard deviation, and is the covariance. This last term is essential as it reduces drastically the variance of the measured ratio, as both I t and I 0 measurements fluctuate mainly due to a common cause which is the large intensity fluctuation of the monochromitized SASE pulses. To determine the photon shot-noise limit, we consider the ideal case where the incoming intensity is constant, and the only source of noise comes from measuring the I t and I 0 intensities in the detector. This leads to = , where N 0,t is the number of photons. In addition, there is no longer any correlation between the two intensity measurements such that = 0. Simplifying equation (8) in the photon shot-noise limit gives where N is the number of photons in the I 0 beam and T is the average transmission. For a weakly absorbing sample with a transmission close to 1, the SNR limit is thus , while for a strongly absorbing sample, i.e. with a transmission of 0.1, the SNR limit will be dominated by the noise in the transmitted beam with fewer photons, giving a reduced SNR limit of in this example. When data are recorded, several shots are taken at the same photon energies (or time delay) and have to be averaged together. The first approach is to compute the transmission T i for each of the M shots and average them together, such that T = . The uncertainty is simply given by the standard deviation s T of T i and the single-shot SNR is obtained from the left-hand side of equation (8), However, shots with weak intensity, due to the high fluctuations introduced by monochromatizing the SASE pulses, tend to be noisier and dominate the uncertainty in the measurement. A better approach is to first compute the summed incoming and transmitted intensities before calculating the averaged transmission T w, Here, we are simply summing up the photons detected over many shots together. This is identical to computing a weighted average of T i with I 0i as weight, i.e. intense shots should contribute more and weak shots should contribute less to the mean. The uncertainty in the measurement is now given by the weighted standard deviation, with = and = . The single-shot weighted SNR is then 3.2. Imaging To characterize the sensitivity of the setup, we collect a set of data without samples in the beam in which case the transmission is known to be exactly T = 1. In Fig. 4 >(a), a single SASE pulse is imaged on the DSSC sensor, showing the three characteristic beams from the diffractive optics. To simplify the discussion in the rest of the manuscript, we identify these three beams by their -1st, 0th and +1st grating order alone, omitting to mention their +1st zone-plate order part. The shape of each beam is roughly a square and is determined by the aperture size of the zone-plate optics. A four-slit system directly upstream of the diffractive optics is used to control the incoming beam size and to ensure separation of the diffracted beams on the DSSC detector. The average dark-corrected image over a 5 min-long dataset consisting of 3000 FEL trains with 15 X-ray pulses per train is shown in Fig. 4 >(b). The first point to note is that both the -1st and the +1st grating orders are somewhat weaker than the 0th grating order in the middle. This is due to the design of the diffractive optics and the resulting diffraction efficiency in each order, which can be tuned (Doring et al., 2020 >). The second point to note is that the beam intensity of the right beam decreases on its right side and, similarly, the left beam intensity decreases on its left side. We suspect this effect to be an intrinsic property of the diffractive optics and not a fabrication issue, as it occurs for all the optics we tested. The variation of intensity across different beams makes the measured transmission spatially dependent and deviating from 1.0. In the next section, we detail how this effect can be corrected. 3.3. Flat-field correction The ratios between each beam and the 0th grating order beam are shown in Fig. 4 >(c). One notices the presence of gradients in the -1st/0th and the +1st/0th ratios, with values ranging from 0.55 up to 0.85. The orientation of the gradients is along the diagonal direction with respect to the square shape of the beams, and they appear to be mirror images of each other. To correct for the gradients, we fit a plane defined by ax + by + cz + d = 0, where x and y are the horizontal and vertical positions, respectively, of a pixel in the sensor and z is the value of the ratio computed for the corresponding pixel. We can impose a horizontal mirror symmetry such that both gradients are fitted with only four fitting parameters, a, b, c and d, in total. The result of the fitting is shown in Fig. 4 >(d), where a value of 1.0 is set everywhere, except where the -1st and +1st beams are located, in which case the fitted plane and its mirror are evaluated. Dividing the data shown in Fig. 4 >(b) by the normalization shown in Fig. 4 >(d) results in the data shown in Fig. 4 >(e). The imaged beams now appear as three identical copies of the same initial beam image, as we would expect from the property of a grating. We call this normalization step the flat-field correction. To better understand the statistical distribution of the data, we extract the intensity of the three beams by defining three regions of interest (ROIs) around each beam and summing up the measured pixel values within them after dark correction. We then compute the three possible ratios -1st/0th, +1st/0th and -1st/+1st grating orders as a function of the intensity in the 0th grating order for every pulse in every train in the dataset. The resulting two-dimensional histograms are shown in Fig. 5 >. The column on the left in Fig. 5 > labeled 'raw' shows data that are only dark-corrected. We can see that they are quite dispersed. In these plots, the data in red show the pulses where at least one pixel is saturated, making them not reliable, while the data in blue show no saturation of the DSSC detector. On each plot, the SNR given by equation (10) and weighted SNR (SNRw) given by equation (13) are shown for the non-saturated data. After applying the flat-field correction, which compensates for some diffraction-efficiency variation in the diffractive optics, we obtain the data shown in the middle column in Fig. 5 >, labeled 'flat-field'. We can see that the improvement is significant and that these first corrected data are much less dispersed. We also see a large increase in SNRw by a factor of six for the -1st/+1st ratio, from 46 to 299. For the two other ratios, the improvement is more moderate and only a factor of two. In addition, the measured transmission now clearly appears non-linear as a function of the intensity in the 0th order, a feature that was in part hidden in the noise before. In practice, the zone plate can be slightly rotated with respect to the DSSC sensor. This can be taken into account in the fitting procedure of the flat-field correction by lifting the horizontal symmetry requirement at the cost of doubling the number of fitting parameters from four to eight. In addition, a ratio is only properly defined where we have enough intensity; otherwise, it creates outliers. The process of finding ROIs encompassing bright enough pixels and excluding outliers can be time consuming. To solve this problem, a more reliable approach was derived. The idea is based on the change displayed by flat-field-corrected data as compared with the raw data and is shown in Fig. 5 >. If we bin the data in k = 40 small intervals of intensity in the 0th order, then, within each of these intervals, the spread in the data for each of the three ratios is reduced by the flat-field correction. This spread is naturally measured with the standard deviation over each k interval, s k . To fit the flat-field correction, we introduce the following criterion J ff, where the term in aff is the sum over all k intervals of the standard deviation for all three ratios. The term in (1 - aff) is a regularization term to keep the mean <...> of each of the three ratios around unity. Minimizing J ff as a function of the eight fitting parameters (four for each plane without mirror symmetry) proved to be more reliable and gave better estimates of the flat-field correction compared with fitting a plane to the ratio of the mean images of each beam. In practice, the fitting procedure converges within a few iterations and a regularization parameter aff of about 0.1 works well and is not critical. By making the k intervals small enough, we can be sure that the non-linear trend, visible in Fig. 5 > for the flat-field-corrected data, is not contributing to J ff. While the improvement brought in by the flat-field correction in the data is very significant, it is clear that the non-linearity observed in Fig. 5 > needs to be addressed if we want to make the best use of the available data. 3.4. Non-linear correction The mini-silicon drift detector (MiniSDD) camera is a linear system with an analog chain linearity error better than 0.25% (Grande et al., 2019 >) and an analog-to-digital converter (ADC) with integral non-linearity (INL) and differential non-linearity (DNL) better than 0.5 least significant bits (LSB) and 0.32 LSB on the full range, respectively (Hansen et al., 2013 >). Nevertheless, the remaining non-linearity that we observe needs to be addressed to further improve the data quality. Here, we assume as a first approximation that the DSSC single-pixel response is non-linear as a function of the incoming photon intensity. Moreover, we assume that this non-linearity can be corrected by a pixel-independent non-linear correction function F nl that only deviates slightly from the ideal linear detector response. We model F nl(x) over the integer range from 0 to 511, representing the 9 bits of the DSSC pixel output values, as a piecewise constant function composed of S segments. It starts from a user-defined low level L and goes up to a high level H. In practice, we have S = 80, L = 40, which is below the dark pedestal, and H = 511. To apply this correction function to the collected raw data output of the DSSC, we proceed with the following algorithm: (1) Replace integer value x with float value F nl(x) in both dark run data and run data. (2) Subtract from the run data the pulse-resolved mean dark value. (3) Divide run data by flat-field normalization. (4) Sum run data pixel values over each ROI. We then compute the weighted variance according to equation (12) for each of the three intensity ratios measured in each ROI. The goal is then to fit the pixel-independent non-linear correction function F nl in order to maximize the SNRw of the -1st/0th and the +1st/0th ratios. For this, we calculate the following criterion J nl, with anl being a user-defined parameter between 0 and 1 controlling the strength of the regularization term. This term prevents the fitting from diverging to an unrealistic non-linear correction function by keeping the correction cost, i.e. the deviation from the ideal detector response, as small as possible. In practice, we use anl = 0.5 as a default value. We then minimize J nl as a function of the S piecewise constant values modeling F nl. This computation typically takes 2 to 8 h on a single node on the Maxwell computational resources operated at DESY and accessible to the users of the European XFEL. In Fig. 6 >, the fitted non-linear correction deviation F nl(x) - x is shown and is indeed small. Here, a maximum deviation of less than 1 for an input value of about 80 is seen. As the DSSC was operated at a frame rate of 4.5 MHz, the actual input value recorded in this dataset does not extend beyond 280 (Porro et al., 2021 >). This explains why the deviation is zero in the range 280 to 511. The inset in Fig. 6 > shows the evolution of each component of the minimization criterion J nl as a function of the fitting iteration number, with SNRw being 1/[10-8 J nl(anl = 0)]1/2 and the correction cost being J nl(anl = 1). One can see that, within a few iterations, SNRw increases significantly for a very moderate increase in the correction cost. With further iterations, SNRw increases slightly to a plateau, at the cost of a much larger increase in the correction cost. At this stage, the only gain in minimizing J nl is by reducing the correction cost, as shown by the small reduction around iteration 15 while SNRw remains constant. Overall, within 25 iterations, the fitting has converged. In Fig. 5 >, the data corrected for dark, flat-field and non-linear response are shown in the right column labeled 'non-linear'. It is evident from these plots that the data are now much more linear, with the exception of the saturated data in red, which are discarded from further analysis in any case. We see an increase by a factor of 2.5 in SNRw with the addition of the non-linear correction for the -1st/0th and the +1st/0th ratios. For the -1st/+1st ratio, the gain is much smaller. This is easy to understand, considering that, since the +1st and -1st are very similar in intensity, their ratio is largely independent of detector non-linearity. This, in turn, motivates the omission of the -1st/+1st ratio in equation (15). Interestingly, we note that discarding the saturated pulses when computing the -1st/+1st ratio might not always be the best strategy, as these data do not apparently deviate significantly from the non-saturated data. This could be because the increase in beam intensity is well determined by the many non-saturated pixels and not dominated by a few saturated pixels in each beam, in contrast to beams of dissimilar intensity, where saturation occurs only in one of the beams. In summary, combining a flat-field and a non-linear correction that can be efficiently calculated, we significantly improved the collected data. In the next section, we discuss how close the corrected data are to the photon shot-noise limit. 3.5. Photon shot-noise limit To address the question of how close the corrected data are to the photon shot-noise limit, we plot in Fig. 7 > the inverse of the standard deviation of the data binned as a function of the intensity in the 0th order. The data, which are only dark-corrected, are shown as blue dotted lines. Data that are additionally flat-field corrected are shown as orange dash-dotted lines. Data that are also non-linear corrected are shown as continuous green lines. The photon shot-noise limit given by equation (9) is shown as red dashed lines in Fig. 7 >. With the flat-field correction, data are already approaching the photon shot-noise limit closely. The effect of correcting for non-linearity is not visible in this plot as we only show the inverse of the standard deviation of the binned data and not the systematic deviation from 1. From Fig. 7 >, we conclude that we are making efficient use of every photon detected by the DSSC detector, using the detailed data correction steps. Before looking at actual time-resolved transient XAS measurements on the sample and confirming that the data treatment gives sensible results, we discuss the different corrections we apply to the data and their origin. At the moment, we lack a predictive model for the position-dependent diffraction efficiency of the zone plate, which we correct with the flat-field correction. Nevertheless, it seems that the plane approximation to that unknown dependence is sufficient. For the non-linear correction, the MiniSDD DSSC pixel response is linear within the expected margins (Grande et al., 2019 >; Hansen et al., 2013 >; Porro et al., 2021 >), but, as we have shown, the data quality can be further improved by correcting the remaining non-linear behavior with a pixel-independent non-linear correction function. Finally, we point out that taking the necessary calibration data, consisting of a dark run of 1 min and a run without sample for a few minutes, is very quick to acquire. Moreover, the computed correction should remain valid unless the X-rays or the BOZ are realigned due to a beam pointing drift, in which case a new set of calibration data needs to be taken. Fortunately, the data processing that we detailed allows us to completely mitigate these effects and to reach the desired regime, where the sensitivity of the setup is only limited by the number of detected photons. 3.6. Offline analysis The analysis procedure, detailed in the previous section, is made publicly available to all users as routines in the SCS toolbox Python package (SCS, 2022c >), with example Jupyter notebooks readily available in the online documentation (SCS, 2022b >). The workflow is quite simple and detailed here. First, we use a dedicated notebook to calculate the flat-field and non-linear correction on a set of data recorded without a sample. This computation takes several hours for the non-linearity correction but only a few tens of minutes for the flat-field correction. The result is saved in a small JavaScript Object Notation (JSON) file that can be used later on to process data, both for the offline analysis and for the online analysis. To speed up the data analysis during the beam time, an intermediate JSON correction file is saved as soon as the flat-field correction is finished. Second, the processing of data recorded with a sample is split into two parts, each having its dedicated notebook. The first part consists of processing the DSSC data, applying all the detailed corrections and computing the intensity in each beam, and finally saving the result in an intermediate small data file. The second part is to load one or several of these small data files and to compute the XAS spectra or time delay traces with a binning procedure. This part can be easily modified and adapted by the users to their needs during and after the beam time. Offline analysis programs and notebooks make use of European XFEL's Extra-data package (Fangohr et al., 2018 >), which provides convenient access to the data files written at the European XFEL. The Python-based Extra-data framework (Extra-data, 2022 >) makes data available through common data science tools and objects such as NumPy's arrays (Harris et al., 2020 >), xarray (Hoyer & Hamman, 2017 >) and dask array (Dask, 2016 >). In particular, it is thanks to the multiprocessing capability offered by dask array that the computation time of the non-linearity correction could be reduced from days to just a few hours. Jupyter notebooks are used by European XFEL users and staff to explore and analyze experiment data (Fangohr et al., 2020 >). The JupyterHub installation of the Maxwell cluster provides remote execution of Jupyter notebooks using the Maxwell resources and thus provides an alternative to remote X, FastX or other remote-access technologies. This is of particular value, as the datasets recorded at the European XFEL can be so large -- up to a petabyte for a five-day-long beam time recording the full DSSC detector with 800 frames per train -- that they typically stay at the facility and need to be analyzed remotely after the beam time. Here, for experiments employing a single DSSC module and recording a few tens of pulses per train, the amount of data generated is more moderate, of the order of a few tens of terabytes per beam time. The use of Jupyter notebooks can also help to make data analysis and publications more reproducible (Beg et al., 2021 >). 3.7. Online analysis FEL beam times are both expensive and limited. It is therefore crucial for users to be able to analyze the data in real time in order to steer the experiment and maximize the scientific output. Karabo is designed to support concurrent initial analysis during data acquisition (Hauf et al., 2019 >; Fangohr et al., 2018 >). It is a distributed software that consists of small pluggable components, so-called devices, that represent various components: a detector, a piece of equipment such as a sensor, or a control and analysis procedure such as a scanning routine. Karabo also includes a graphical user interface that allows feedback on the control system. There are different possibilities to achieve real-time data analysis at the European XFEL, which is developing a related Karabo device that describes the analysis (Flucke et al., 2020 >) or connecting an external application via a bridge (Fangohr et al., 2018 >). The DSSC detector produces up to 800 images per train with a data rate of 1 GB s-1 for a single module. To ensure low-latency data processing, we have used EXtra-metro (Schmidt, 2022 >), a framework developed in-house with intrinsic parallelization, which enables fast and reliable online previews of various analysis routines. These routines are generated by interpreting a Python script, where the analysis procedures are described. During the experiment, the detector images are recorded while scanning either the X-ray photon energy or the pump laser time delay parameters. These data are simultaneously collected by EXtra-metro directly from the control system using the data pipelines for large detector data and from the central messaging broker for the control data. The analysis routines defined in the SCS toolbox package (Section 3.6) are then applied to the received data, using the pre-calculated non-linear and flat-field corrections, in a train-by-train manner. The processed data are then displayed in the Karabo graphical user interface. Custom widgets are developed for further data visualization and interaction using the GUI extensions (Flucke et al., 2020 >). Fig. 8 >(a) shows a dark-corrected detector module with three overlayed ROIs that define the intensities of the pumped, unpumped and reference signals. These ROIs, along with other analysis parameters such as bin spacing and pulse selection, can also be modified during the run time. In addition to showing the DSSC image and ROIs, the vertical and horizontal projections of the intensity are displayed and are used during the initial zone plate alignment to find the center of the X-ray beam. Finally, the resulting X-ray absorption spectra of the signals are shown in Fig. 8 >(b) and are further discussed in the next section. 4. Results 4.1. Transient XAS It is essential to verify the effect of the different levels of data correction on actual time-resolved data. To demonstrate this, we selected an extended XAS spectrum measured on a NiO thin film sample at the Ni L 3,2 edge. The sample was excited above its band gap by an optical laser pulse with 266 nm wavelength, 50 fs pulse duration and 5 mJ cm-2 peak fluence. The time delay between optical pump and X-ray probe was fixed to 1.0 ps. During the FEL train, 18 pairs of optical pump/X-ray probes were used, with 17.8 ms separation between them, corresponding to an effective repetition rate of 56 kHz during the train. In Fig. 9 >(a) the XAS and in Fig. 9 >(b) the pump-induced change in XAS at the Ni L 3,2 edges are shown as continuous blue lines and labeled as 'raw' for data that are only dark-corrected. The energy range of these spectra spans 30 eV and is divided in 300 bins of 0.1 eV width. The data were collected for 30 min, which corresponds to 6 s per bin. With 10 trains per second at the European XFEL, and 18 X-ray pulses per train for this measurement, we collected a thousand X-ray pulses per bin. This provides a noise reduction of 30 which, together with a single-shot SNR of 300, gives a final noise of 10-4 which is necessary to record the smaller features in the transient XAS signal shown in Fig. 9 >(b). Discussions of the physics behind these NiO XAS and transient XAS are beyond the scope of this article and will be published separately. However, we can briefly discuss the XAS and transient XAS measured. The XAS is characteristic of NiO and its multiplet structure (Regan et al., 2001 >; de Groot et al., 2021 >) and comparable with the spectrum measured at Synchrotron SOLEIL (Rothenbach, 2020 >). In the simplest picture, the transient XAS probes the holes left by excited electrons after interaction with the pump laser and is observed as an increase in absorption. At the same time, the states filled by excited electrons give a reduction of the XAS (Stamm et al., 2007 >; Boeglin et al., 2010 >; Willems et al., 2020 >; Hennes et al., 2020 >; Le Guyader et al., 2022 >). The amplitude of the transient XAS is of the order of 5% of the static XAS at most, as seen in Fig. 9 >(b). Although small, it is above the noise in the measurement even for the 'raw' data that are only dark-corrected. Here, we define noise as the apparent random fluctuations in data points that are close to each other. The situation improves drastically using the flat-field corrected data, which are shown as a dashed orange line in Fig. 9 >. Over the whole spectrum, the noise level in the transient XAS is significantly reduced compared with the uncorrected data. In the XAS spectra, this reduction is also visible in the inset in Fig. 9 >(a), which shows a zoomed region on the flat continuum transitions part of the XAS between the L 3 and L 2 edges. We note that, for this dataset, the characterization run without sample in the beam was not recorded at the time. To compute the flat-field and non-linear corrections, we instead selected in the data the shots falling in the flat pre-edge region below 848 eV. We do not expect the analysis to be significantly affected by this, as confirmed in experiments conducted later. The data, which are in addition corrected for non-linearity, are shown as a dot-dashed green line in Fig. 9 >. For the XAS, a further improvement in the data is visible in the zoomed inset, where the curve is now very smooth with negligible noise remaining. For the transient change, there is almost no visible difference between flat-field correction and non-linear correction. This is what one would expect from the results shown in Fig. 5 >, where the improvement with the non-linear correction is limited for the -1st/+1st order, which directly probes the transient XAS change, while the improvement is much larger for the other two ratios probing the unpumped and pumped XAS spectra. While it is evident that each additional correction improves the data quality with a significant noise reduction, there are also some systematic deviations. This is shown by the fact that the three different levels of corrections do not result in curves overlapping each other in Fig. 9 >. Therefore, we have to discuss the implications of each correction on the data. The flat-field correction ensures that the measured intensity is independent of the FEL intensity profile impinging on the zone plate. Clearly, the transmission of the sample should not depend on the pulse-to-pulse fluctuating FEL intensity profile; therefore, data that are not corrected by the flat field cannot be trusted. Similarly, the non-linear correction ensures that the measured quantity does not depend on the X-ray intensity, so data that are not corrected for the remaining DSSC non-linearity cannot be trusted either. Are there systematic variations in flat-field and non-linearity corrected data that require additional correction? There is one, which is visible in Fig. 9 >(b), where the baseline of the transient XAS seems to shift away from zero. This is probably related to the flat-field correction, which is calculated at a fixed photon energy. However, the zone-plate properties depend on the photon energy. This can be corrected with an additional step, where we record an XAS spectrum without sample, from which the linear background can be extracted and subtracted from flat-field and non-linearity corrected data. Such data are not available for this particular dataset, so this correction cannot be applied here. It is, however, now part of the standard measuring protocol. Overall, we have shown that the different data corrections allow the extraction of the most information from every photon detected and the recording of XAS and transient XAS with excellent SNR, approaching the photon shot-noise limit. 4.2. Setup limits As we have shown, the BOZ setup at SCS allows recording XAS with sensitivity reaching the photon shot-noise limit. Detecting more photons or increasing the repetition rate of the experiment are two simple means by which we can increase the statistics. In this section, we thus discuss the limits in terms of X-ray photons that we can use and the limits in terms of repetition rate. Finally, given these limits, we review which sample systems can be measured with this setup. 4.2.1. X-ray fluence If our setup is photon shot-noise limited, then counting more photons by increasing the beam intensity would directly translate into a better signal with less noise, as confirmed by Fig. 7 >. This is true until we reach saturation of pixels in the DSSC detector too frequently, as such data have to be discarded from the analysis, thereby reducing the final statistics. In practice, the X-ray intensity is adjusted such that few percents of the shots are saturated. If the intensity of the monochromatic X-ray could be made more stable, for example by employing a self-seeding scheme (Serkez et al., 2013 >), frequent pixel saturation could be avoided while collecting intense shots more regularly, resulting in higher final photon counts. Given this pixel saturation and the lack of available soft X-ray seeding scheme at SCS, the only way to increase the intensity would be to enlarge the beam even more on the DSSC sensor. Here, we reach two limits with the current setup. First, expanding the beam even more means that we need to either move the DSSC further downstream or use a BOZ with a shorter focal length. However, the DSSC detector is already placed as far downstream as possible given the current size of the SCS experiment hutch. Using a BOZ with a shorter focal length would reduce the space available between the BOZ and the sample to couple in the optical pump laser. Moreover, given that, with the current setup, we are nearly fully illuminating the sensor, as seen in Fig. 4 >, expanding the beam further would require a larger monolithic sensor. Clearly, given that the DSSC detector is currently the only detector capable of recording up to 800 pulses per train at 4.5 MHz repetition rate that can be delivered by the European XFEL at SCS (Porro et al., 2021 >), we are at the limit. We could only make use of higher beam intensity with a new detector having higher saturation limits, a larger continuous sensor, or smaller pixels with similar electron well depth. However, detecting more photons is not the only aspect we should discuss here. With increasing the X-ray intensity, eventually non-linear X-ray absorption effects will set in, where the X-ray pulse modifies the sample that it probes (Wu et al., 2016 >; Higley et al., 2019 >). In Fig. 10 >, we show the X-ray fluence as a function of the X-ray spot size on the sample and the number of photons in the beam for photons with 1 keV energy. Non-linear X-ray phenomena set in at fluences of a few mJ cm-2 (Wu et al., 2016 >; Higley et al., 2019 >), so we need to stay below 0.1 mJ cm-2 to be on the safe side. With a typical beam size of 30 mm x 30 mm, the beam intensity should stay below a few 106 photons. This is already the range we reached, as shown in Fig. 5 >, so we are close to the limit here as well. It is possible, in principle, to increase the X-ray beam size on the sample by simply moving it further downstream of the zone-plate focus. However, a larger X-ray beam size means an even larger optical pump spot size, which results in slower heat dissipation. As discussed in the next section, slow heat dissipation limits, in turn, the number of X-ray pulses that can be used per train such that the beneficial effect of increased statistics per shot with increased spot size might be compensated by a reduced number of shots due to sample heating. 4.2.2. Repetition rate In stroboscopic pump-probe experiments, heat dissipation is a known issue limiting the effective repetition rate at which data can be collected. This is particularly the case here, as the samples are X-ray transparent thin membranes, which limit the heat dissipation to the two in-plane dimensions. Moreover, the X-ray pulse pattern at the European XFEL, with its trains of X-ray pulses at up to 4.5 MHz, leaves very limited time between the X-ray pulses in the train for heat dissipation to take place. In Fig. 11 >(a), the transient change in XAS is shown as a function of the photon energy around the Ni L 3 absorption edge in a 20 nm Ni sample for different ranges of optical pump/X-ray probe pulse pairs within each train. A clear trend is visible, where the change in XAS increases with the pulse pair number. In a simple picture, we can interpret this change in XAS as a change in electron population around the Fermi level. The integral of the absolute change in XAS over the spectral range measured is then proportional to the deposited energy in the system. We plot this deposited energy as a function of the pulse pair number in the train, for different sample stacks, as shown in Fig. 11 >(b). The same Ni film is deposited on membranes made of silicon, silicon nitride and diamond, with and without a Cu heat sink for each case. The diamond membranes are prepared by chemical vapor deposition (CVD). All data in Fig. 11 > appear to be linear, i.e. each optical pump pulse in the train adds energy to the system that does not dissipate completely before the next pulse arrives, leading to a temperature increase of the sample. We fitted these data with a straight line and extracted the slope and intercept, as listed in Table 1 >. In the ideal case, the sample would be efficiently excited with the first pump pulse, meaning that we would measure a large intercept. At the same time, the sample would cool down efficiently until the next pump pulse arrives, meaning that we would measure the same excited sample for subsequent pulses without any heat accumulation, resulting in the slope being zero. Therefore, an optimal sample stack combines a large intercept and a small slope. To characterize this property of the sample stack, we introduce a figure of merit as the ratio of the intercept and the slope. The larger this number, the better suited the sample stack is for repetitive pump-probe experiments within the train. Looking at Table 1 >, we can see that the worst sample would be the Si substrate. Indeed, for this sample stack, we had to reduce the fluence by a factor of two, otherwise the sample would break, suggesting that heat accumulation in this sample is strong. For the Si3N4 sample, the presence of the Cu heat sink does not improve the performance, but both are some of the best sample stacks. For the CVD diamond substrate, the Cu heat sink improves the performance by a factor of two but does not perform better than Si3N4-based stacks. In all the cases presented here, the heat accumulation is a measurable effect on top of the transient change in XAS. If no further analysis is possible, we are forced to limit the number of pulses used in the experiment and separate them as far as possible, given the European XFEL pulse pattern, which in practice is often 10 to 20 pulses per train. However, if one can assume the response of the system to be linear both on the transient change and on the heat accumulation change, then the two contributions can be disentangled and possibly more pulses per train can be used with improved statistics. Having discussed the limits in terms of X-ray fluence and repetition rate in the previous section, we now discuss from which classes of sample systems can we expect a measurable signal level. 4.2.3. Sensitivity limits To make the best use of the setup and the detected photons, the intensity of the three beams on the DSSC should be similar. This way, the full dynamic range of the DSSC pixels can be used, from the dark pedestal level up to the saturation level or to the 4.5 MHz digitization cutoff. If the three beam intensities are not well balanced, the efficiency of the setup will be reduced, as already discussed with equation (9). In the case of a strongly absorbing sample at a resonant edge, the measurement will be limited both by saturation of the DSSC detector on the pre-edge region and by low transmitted intensity at the resonance. In practice, these considerations limit the sample thickness to one or two absorption lengths at most. In other cases, one might be interested in systems that are much more diluted. These are the cases considered in Table 2 >. We begin with the case of a 20 nm-thick Fe film, which corresponds to one absorption length at the L 3 resonance. The static XAS signal is thus 1, as shown in Table 2 >. For the noise level, we can estimate it to be similar to a no-sample case, as the change in the number of detected photons will be moderate. This gives us a single-shot SNR of 250. For the other cases that we consider, the absorption is smaller, so the number of detected photons, and therefore the noise, will be constant. The only thing that changes is the level of signal. For example, for this 20 nm Fe film, we can roughly estimate the transient XAS to be around 5% of the static XAS. In other words, the signal reduces by a factor of 20, giving a single-shot SNR of 12, as shown in Table 2 >. Ideally, we would like to measure the transient XAS with an overall SNR greater than 3, which can be achieved in this case with a single shot, as indicated in the last column of Table 2 >. If we now consider the case of an Fe monolayer, the static XAS signal scales down by a factor of 50. This results in a single-shot SNR of 5 for the XAS and 0.2 for the transient XAS. To achieve an SNR of 3 on the transient XAS, we have to average 225 pulses. This is between 22.5 s of data acquisition if the sample can only be pumped at 10 Hz and 1 s and if the sample can be pumped with 20 shots during one FEL train. If we now consider the case of a single layer of molecules containing a single Fe central atom, we would have tens to hundred times less absorbing atoms than in the Fe monolayer case, which means hundreds to tens of thousands more shots and longer acquisition required to reach the target SNR, making this kind of experiment challenging even with many pulses per train. Finally, we have described in this article the case of a homogeneous sample, where both the excited and unexcited sample membranes can be prepared identical. In practice, samples are often inhomogeneous, such that the transient XAS computed from the excited and unexcited membrane might not be meaningful. In such cases, the transient XAS can be measured with this setup using the excited and reference membranes, by alternating pumped and unpumped shots during the FEL train. The benefits of photon-shot-noise-limited detection and shot-by-shot normalization remain. 5. Conclusion The beam-splitting off-axis zone plate setup, which is available to users at the SCS instrument at the European XFEL, was presented in detail. We showed that two essential data correction steps are necessary to make the best use of the collected photons: a flat-field normalization that compensates for the inhomogeneous diffraction efficiency of the diffractive optics employed, and a correction of the remaining DSSC non-linearity. Remarkably, with these two corrections, the resulting data are shown to be close to the photon shot-noise limit. In addition, we reviewed several tools that we provide to the users, namely a beam propagation calculator to help users design their samples to be compatible with the fixed beam position of this setup, the complete analysis procedure in the form of a Python package and associated Jupyter notebooks, and finally the online analysis framework to process live the measured data with all the levels of correction available. We showed an example of transient XAS in NiO at the Ni L 3,2 edges with unprecedented data quality, and discussed the effect of the different corrections. Finally, we reviewed the current limits of the existing setup in terms of the number of photons on the sample, the repetition rate that thin X-ray transparent samples can accommodate, and the signal level for increasingly dilute systems. We finally conclude that the current setup is as good as it can be with the current detector, and that we can measure transient XAS down to a few tens of layers of molecules. Further improvement in the sensitivity of the setup may come from the implementation of a self-seeding scheme, or by using an improved detector, such as the DEPFET DSSC, featuring a lower readout noise and higher dynamic range than the current MiniSDD DSSC we used here. With the installation of the Apple-X helical afterburners in the near future, linear-polarization-dependent transient XAS experiments, such as X-ray magnetic circular dichroism and X-ray magnetic linear dichroism, will become possible. Plans to adapt this setup to flat liquid jet experiments to investigate, for example, optically driven transitions in molecules in solution are ongoing. Finally, we note that the data processing that we presented provides a clear path for a data reduction strategy that can be applied on the fly, as the needed data corrections are computationally fast. This means that this method is compatible with quasi-continuous operation at several tens to hundreds of kHz. Such a regime will become available with forthcoming FEL accelerators that are being developed and would be ideal for experiments on X-ray transparent thin solid samples. 6. Data availability All data are available in the main text or the supporting information. The raw data generated at the European XFEL for the experiment UP2161 used in Fig. 11 > are available at For the experiment UP2712 used in Figs. 4 >, 5 >, 6 > and 7 >, raw data are available at For the experiment UP2589 used in Figs. 1 > and 9 >, raw data are available at Supplementary Material European XFEL for the experiment UP2161: European XFEL for the experiment UP2712: European XFEL for the experiment UP2589: We acknowledge European XFEL in Schenefeld, Germany, for provision of X-ray free-electron laser beam time at Scientific Instrument SCS (Spectroscopy and Coherent Scattering) and would like to thank the staff for their assistance. We thank Carsten Deiter, Carsten Broers, and Alexander Reich for technical support. We thank Kurt Ament for proofreading the article. Open access funding enabled and organized by Projekt DEAL. Figure 1 Scheme of the beam-splitting off-axis zone plate (BOZ) setup which is described in detail in the main text. Figure 2 (a) Horizontal and (b) vertical beam propagation from the intermediate source point to the detector. The position of the sample is shown as a dashed vertical line. The inset in each figure shows a zoomed-in region around the sample position. The red-filled areas represent the 707 eV photon beams from the fundamental harmonic of the undulators. The blue-filled areas represent the beams at 1414 eV photon energy originating from the second harmonic of the undulators. The vertical separation between fundamental and second-harmonic beams near the zone-plate focus arising from the off-axis component of the diffractive optics can be used to block the unwanted radiation. Figure 3 BOZ calculator showing the beams at (a) the sample and (b) the detector planes. The focused 1st zone-plate order beam footprint at the respective positions is shown as red for the fundamental and blue for the second harmonic. The 0th zone plate order is shown as gray. Individual membrane windows and the etching lines are shown in (a) as continuous and dashed lines, respectively. In (b) the DSSC sensor is represented by the black rectangle and the DSSC filter mount is represented by the green shapes. Figure 4 (a) Dark-corrected single-pulse image of the DSSC sensor showing the three imaged beams (-1st, 0th and +1st order). (b) Dark-corrected average image of a single DSSC sensor showing the three imaged beams based on a 5 min-long data acquisition recording 3000 FEL trains with 15 X-ray pulses per train. (c) Image showing each beam normalized by the 0th order beam. (d) Fitted flat-field correction. (e) Dark-corrected and flat-field-corrected average image in a single DSSC sensor. The three imaged beams now appear as identical copies of the initial beam. Figure 5 Two-dimensional histograms of the three ratios (-1st/0th grating order in the top row, 1st/0th in the middle row, and -1st/1st in the bottom row) as a function of the number of photons in the 0th order for dark-corrected data in the left 'raw' column, for the dark-corrected and flat-field-corrected data in the middle 'flat-field' column, and finally for the dark-corrected, flat-field-corrected and non-linear-corrected data in the right 'non-linear' column. Data that contain saturated pixels are shown as red, as opposed to blue where no saturation occurs. In each plot, the SNR and weighted SNR (SNRw) of the non-saturated events are indicated. Figure 6 Fitted non-linear deviation from the ideal detector response F nl(x) - x as a function of the DSSC pixel values. Inset: the evolution of the weighted SNR (blue squares) and the correction cost (orange circles) as a function of the fitting iteration number. Figure 7 SNR (inverse of the standard deviation) of the data binned as a function of the intensity in the 0th order for (a) the -1st/0th order, (b) the +1st/0th order and (c) the -1st/+1st order. Data that are only dark-corrected are shown as dotted blue lines. Data that are also flat-field corrected are shown as dot-dashed orange lines. Data that are also corrected for non-linearity are shown as continuous green lines. The photon shot-noise limit is shown as dashed red lines. Figure 8 (a) Dark-corrected single-train image of the DSSC sensor in the Karabo GUI. The overlayed regions of interest define the intensity of the imaged beams and can be modified using the EXtra-metro parameter fields or by user interaction in the GUI. The projections along x and y axes are also plotted as guides for optimal zone plate alignment. (b) XAS spectra of the three ratios after an energy scan displayed in the Karabo GUI. The panel also contains the analysis parameter fields that can be changed during run time. Figure 9 (a) XAS and (b) transient change in XAS at the Ni L 3,2 edges of a NiO thin film. The data were recorded at a fixed time delay of 1.0 ps, with a peak fluence of 5 mJ cm-2 and a pump wavelength of 266 nm. Data that are only dark-corrected are shown as continuous blue lines and labeled 'raw'. Data that are additionally flat-field corrected are shown as dashed orange lines and labeled 'flat-field'. Data that are also corrected for non-linearity are shown as dot-dashed green lines and labeled 'non-linear'. Figure 10 X-ray fluence on the sample, in mJ cm-2, as a function of the beam size and the number of 1 keV photons in the beam. A limit of 0.1 mJ cm-2, ensuring negligible non-linear X-ray absorption effects, is shown as white color. Values below and above this limit are shown in blue and red, respectively. Figure 11 (a) Transient change in XAS in the Ni/Si3N4 sample as a function of photon energy for selected pulses in the train as given by the pulse range in the corresponding legend. The time delay was 0.5 ps, the laser fluence was 7 mJ cm-2, and the pump wavelength was 800 nm. The repetition rate of the 32 pulses during the FEL train was 280 kHz. (b) Integral of the absolute change in XAS as a function of the pulse number in the train, ranging from 1 to 32. The points are the measured data, while the lines are linear fits. Table 1 Slope and intercept and their ratio (intercept/slope) fitted from the integral of the absolute change in XAS as a function of the pulse number in the train shown in Fig. 11 > The samples are 20 nm Ni film capped with 2 nm MgO and grown on different sample stacks listed below with thickness in nm. Membrane Heat sink Slope (10-3) Intercept (10-2) Ratio Si3N4(200) -- 2.8 4.7 16.9 Si3N4(200) Cu(100) 1.9 3.2 16.9 Si(200) -- 6.3 1.9 3.0 Si(200) Cu(100) 1.7 1.0 5.7 CVD diamond(100) -- 2.6 2.3 9.3 CVD diamond(100) Cu(100) 2.9 4.8 16.6 Table 2 Single-shot static and transient XAS signal as well as SNR for different cases To achieve an SNR greater than 3 for the transient XAS signal, the minimal number of shots is given in the last column. Static Transient Sample Signal SNR Signal SNR Minimal shots Fe (20 nm) 1 250 0.05 12 1 Fe monolayer (0.287 nm) 2 x 10-2 5 10-3 0.2 225 Molecule on surface 10-3-10-4 0.25-0.025 5 x 10-5-5 x 10-6 10-2-10-3 105-106 1 DSSC stands for DEPFET sensor with signal compression, with DEPFET standing for depleted p-channel field-effect transistor. However, the DSSC detector that was employed in this article is using a mini-silicon drift detector (MiniSDD) sensor and is described in detail by Porro et al. (2021 >). 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PMC10000792 | J Synchrotron Radiat J Synchrotron Radiat J. Synchrotron Rad. Journal of Synchrotron Radiation 0909-0495 1600-5775 International Union of Crystallography 36891858 yi5132 10.1107/S1600577523000814 JSYRES S1600577523000814 Short Communications Mapping the 3D position of battery cathode particles in Bragg coherent diffractive imaging 3D positioning of battery cathode particles in Bragg CDI Shabalin A. G. ab* Zhang M. c Yao W. c Rysov R. b Ren Z. b Lapkin D. b Kim Y.-Y. b Assalauova D. b Mukharamova N. b Sprung M. b Vartanyants I. A. b Meng Y. S. c Shpyrko O. G. a a Department of Physics, University of California San Diego, La Jolla, CA 92093-0319, USA b Deutsches Elektronen-Synchrotron DESY, Notkestrasse 85, 22607 Hamburg, Germany c Department of NanoEngineering, University of California San Diego, La Jolla, CA 92093-0448, USA Yabashi M. Editor RIKEN SPring-8 Center, Japan Correspondence e-mail: [email protected] 01 3 2023 16 2 2023 16 2 2023 30 Pt 2 s230200 445448 12 7 2022 30 1 2023 (c) A. G. Shabalin et al. 2023 2023 This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-BY) Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited. A full version of this article is available from Crystallography Journals Online. A method to determine the 3D position of particles in Bragg coherent diffractive imaging experiments is proposed. Test measurements demonstrate depth-resolution with a precision of 20 mm along the beam. In Bragg coherent diffractive imaging, the precise location of the measured crystals in the interior of the sample is usually missing. Obtaining this information would help the study of the spatially dependent behavior of particles in the bulk of inhomogeneous samples, such as extra-thick battery cathodes. This work presents an approach to determine the 3D position of particles by precisely aligning them at the instrument axis of rotation. In the test experiment reported here, with a 60 mm-thick LiNi0.5Mn1.5O4 battery cathode, the particles were located with a precision of 20 mm in the out-of-plane direction, and the in-plane coordinates were determined with a precision of 1 mm. extra-thick battery cathodes Bragg coherent X-ray diffractive imaging battery cathodes Bragg diffraction sphere of confusion 3D mapping US Department of Energy, Office of ScienceDE-SC0001805 DE-EE0008442 Funding for this research was provided by the US Department of Energy, Office of Science (contract no. DE-SC0001805). The electrode fabrication effort is supported by the US Department of Energy's Office of Energy Efficiency and Renewable Energy (EERE) and US Army Tank and Automotive Research Development and Engineering Command (TARDEC) under the award number DE-EE0008442. pmc1. Introduction Imaging of local strain in the bulk of polycrystalline samples requires a probe with high penetration depth and sensitivity to the crystal structure deformations at the nanoscopic level. This became possible with major advances in synchrotron instrumentation, in particular coherent scattering methods that have been developed within the last two decades. Bragg coherent diffractive imaging (CDI) (Robinson et al., 2001 >; Miao et al., 2002 >; Pfeifer et al., 2006 >; Robinson & Harder, 2009 >) is now established as a powerful tool for imaging the structure deformation and structure defects in individual nanocrystals (Ulvestad et al., 2015 >; Kim et al., 2021 >). As the crystals are usually diverse, several particles at different locations are measured to collect sufficient statistical information across the sample (Singer et al., 2018 >). The precise location of the measured particles is usually unknown in such experiments, and thus homogeneity of the sample is often assumed. For systems in which the material response is not uniform, it is important to obtain the location information. For example, in Li-ion batteries with extra-thick cathodes, the charging behavior is expected to depend on the depth under the cathode surface (Zheng et al., 2012 >; Lee et al., 2018 >). Strengthening the capabilities of operando Bragg CDI with the possibility to map the measured particles would provide a missing link between the performance of individual nanoparticles and the 3D structure of extra-thick electrodes. As a general solution to this problem, here we suggest a method to determine the 3D position of the measured particles in Bragg CDI experiments. Our method has some similarities with the procedure involving the detection of rotational centers from cross-correlations in micro-tomography (Pan et al., 2012 >), which uses optical contrast as a measure of alignment, whereas our work uses the integrated intensity of a Bragg diffraction spot. The fundamental difference is that in tomography the full 180deg angular range is available for sample rotations. In Bragg CDI, each particle is observed only in the angular range of about 1deg in which the Bragg condition is satisfied. Despite this limitation on the angular range, in which the particle is observed in the beam, our method allows us to determine where exactly along the beam the particle is. This adds new information to the Bragg CDI method. 2. Method In a typical Bragg CDI experiment, a finite crystalline sample is illuminated with a focused X-ray beam and oriented to fulfill the Bragg diffraction condition (Vartanyants & Yefanov, 2015 >). The resulting far-field interference pattern around the Bragg peak is recorded with a 2D detector (see the schematic in Fig. 1 >). Different cross-sections through reciprocal space are accessed by rocking the sample in the beam so that a typical dataset consists of a few tens of diffraction patterns (Williams et al., 2003 >). In such a geometry, the crystal is on the instrument axis of rotation aligned to be perpendicular to the X-ray beam. Since the particle must stay in the beam during the scan, bringing the particle precisely to the instrument axis of rotation is an important step. To describe the alignment procedure, let us assume that the nanoparticle is initially located off the axis of rotation. First, we adjust the rotation of the sample (o) and the translations perpendicular to the beam (x and y) to maximize the diffracted intensity of the peak. Then, o is scanned near the position of the highest intensity (o p ), as shown in Fig. 2 >(a). For two angular positions on the opposite sides of the resulting intensity profile (o1 and o2), we maximize the intensity by scanning the x spatial position. By that we determine how much the sample moved [see Fig. 2 >(b)]. Finally, we move the sample along the beam by These steps are repeated until Dz = 0, which means the particle is located exactly at the axis of rotation. 3. Experiment We performed our experiment at the P10 Coherence Applications beamline of PETRA III facility (Hamburg, Germany), using an X-ray beam with a photon energy of 11.89 keV that was focused to a 2 mm x 2 mm spot. The sample was an Li-ion battery cathode consisting of a roughly 60 mm-thick layer of LiNi0.5Mn1.5O4 (LNMO) material deposited on a roughly 20 mm-thick Al foil. The cathode was mounted in a way that the X-ray beam was incident normal to the front side (LNMO surface) leaving Al foil downstream as shown in Fig. 1 >. First, we selected an Al (111) reflection and measured 12 particles for positional reference to use our mapping method. After that, we switched to the LNMO (111) reflection and measured 30 LNMO particles. Then, to confirm that choice of reflection does not affect the consistency of the results, we switched to the LNMO (222) reflection and measured four more particles. The resulting 3D positions are presented in Figs. 3 >(a) and 3 >(b). We can see that the z positions of LNMO particles fit well into the 60 +- 20 mm range. The Al particles appear within the expected size of the sample and fit into the 20 +- 30 mm range. The positions of Al particles are less precise due to the much lower quality of the Bragg peaks that were less intense and would often overlap. This can be explained by the significantly smaller grain size of Al. Overall, the positions are consistent with the cathode geometry and dimensions. We did not apply any bias when choosing the particles and selected them using only two criteria. The first was that there should be no peak overlap with other particles in the beam, and this should not depend on the position in the sample because the sample material was pristine and therefore uniform. The second was high peak intensity, which should also not depend on the position, as the total optical path would be roughly the same for any position along the beam, at least for scattering angles in the range 10-15deg, used in our work. Using equation (1), the precision of the positioning in our method can be estimated from the angular difference Do (0.8deg in our case) and the precision of the measurement of Dx (about 0.3 mm in our case). This provides 20 mm precision along the beam, which is confirmed by the experimental data presented in Figs. 3 >(a) and 3 >(b). Instrumental inaccuracies of the rotational and translational positioning can potentially bring systematic errors to our method. At the P10 beamline, we used a six-circle Huber diffractometer with high-precision translational piezo mechanisms. Beforehand, for the measurements, we aligned the beam and the axis of rotation using a metal pin with sharp edges. Considering that all sample rotations in the method were performed within a narrow range of about 1deg, we can confidently state that, throughout the measurements, the axis of rotation remained in the beam center with a precision better than 1 mm. Therefore, instrumental errors did not contribute significantly in our case. 4. Discussion and conclusions In our experiment, it was difficult to find particles with isolated and intense Bragg peaks because the diffraction rings from such a thick sample were densely populated with peaks. In future work, this can be mitigated by employing a smaller X-ray beam and choosing higher-order reflections. We note that the low transmission of X-rays in thick samples makes alignment difficult, thus limiting the applications of our method. Future advances in coherent scattering methods toward using higher photon energies would help overcome this problem. We suggest that our method can be used to explore the difference in charging behaviors of underlying nanoparticles at different depths in extra-thick Li-ion battery cathodes. On one hand, such cathodes are significantly thicker than the depth-resolution of the method (20 mm). On the other hand, they are still thin enough for a hard X-ray beam to penetrate. Such an experiment could help explain the origin of thick electrode electrochemical performance deterioration. We proposed a simple method for mapping the 3D position of particles in Bragg CDI experiments, which is based on the precise alignment of the selected particles with the instrument axis of rotation. We carried out an experiment in which positions of LiNi0.5Mn1.5O4 battery cathode particles were measured with a precision of 1 mm in the plane perpendicular to the beam and 20 mm in the direction along the beam. In future experiments, our method can help to study inhomogeneous systems in which the structural behavior of composing nanocrystals depends on their location in the sample interior. We acknowledge DESY (Hamburg, Germany), a member of the Helmholtz Association HGF, for the provision of experimental facilities. Parts of this research were carried out at the PETRA III synchrotron facility and we would like to thank all the beamline staff for assistance in using the Coherence Application beamline P10. Open access funding enabled and organized by Projekt DEAL. Figure 1 Schematic of the depth-resolved Bragg CDI approach. Before the measurements, the axis of rotation of the instrument (o) is aligned to intersect with the focused X-ray beam. The sample (in our case, a battery cathode in a coin cell) is searched for particles that have bright and isolated Bragg peaks on the 2D detector. We use the right-hand coordinate system with the z axis along the direction of the X-ray beam and the y axis aligned with the sample rotation axis. Figure 2 Alignment for the longitudinal position in the depth-resolved Bragg CDI. (a) After the x and y coordinates have been aligned, the Bragg peak intensity is scanned with the o-scan rotation providing the rocking curve data. Then, x profiles are measured on the opposite sides of that curve: o1 and o2. (b) If the particle was seated off the axis of rotation, the x position of the peak would shift between these two scans. The misalignment along the beam direction is calculated as the amount of that shift divided by the angular difference expressed in radians. Figure 3 Measured 3D positions of particles in a 60 mm-thick battery cathode. The cathode consisted of the LNMO material (yellow area), deposited on 20 mm-thick Al foil (gray area). Slopes of the red and black dashed contours illustrate the thickness uncertainties for the LNMO and Al layers, respectively, which are estimated to be about 10 mm on each side. LNMO (111) (red markers) and LNMO (222) (blue markers) reflections were used. For Al, we used the (111) reflection (shown by black markers). (a) yz projection. (b) xz projection. References Kim, Y. Y., Keller, T. F., Goncalves, T. J., Abuin, M., Runge, H., Gelisio, L., Carnis, J., Vonk, V., Plessow, P. N., Vartaniants, I. A. & Stierle, A. (2021). Sci. Adv. 7, 757. Lee, B.-S., Wu, Z., Petrova, V., Xing, X., Lim, H.-D., Liu, H. & Liu, P. (2018). J. Electrochem. Soc. 165, A525-A533. Miao, J., Ishikawa, T., Johnson, B., Anderson, E. H., Lai, B. & Hodgson, K. O. (2002). Phys. Rev. Lett. 89, 088303. Pan, Y., De Carlo, F. & Xiao, X. (2012). Proc. SPIE, 8313, 645-653. Pfeifer, M. A., Williams, G. J., Vartanyants, I. A., Harder, R. & Robinson, I. K. (2006). Nature, 442, 63-66. Robinson, I. & Harder, R. (2009). Nat. Mater. 8, 291-298. Robinson, I. K., Vartanyants, I. A., Williams, G., Pfeifer, M. & Pitney, J. (2001). Phys. Rev. Lett. 87, 195505. Singer, A., Zhang, M., Hy, S., Cela, D., Fang, C., Wynn, T., Qiu, B., Xia, Y., Liu, Z., Ulvestad, A., Hua, N., Wingert, J., Liu, H., Sprung, M., Zozulya, A. V., Maxey, E., Harder, R., Meng, Y. S. & Shpyrko, O. G. (2018). Nat. Energy, 3, 641-647. Ulvestad, A., Singer, A., Clark, J. N., Cho, H. M., Kim, J. W., Harder, R., Maser, J., Meng, Y. S. & Shpyrko, O. G. (2015). Science, 348, 1344-1347. Vartanyants, I. A. & Yefanov, O. M. (2015). X-ray Diffraction. Modern Experimental Techniques, ch. 12, pp. 341-384. Singapore: Pan Stanford Publishing. Williams, G. J., Pfeifer, M. A., Vartanyants, I. A. & Robinson, I. K. (2003). Phys. Rev. Lett. 90, 175501. Zheng, H., Li, J., Song, X., Liu, G. & Battaglia, V. S. (2012). Electrochim. Acta, 71, 258-265. |
PMC10000793 | J Synchrotron Radiat J Synchrotron Radiat J. Synchrotron Rad. Journal of Synchrotron Radiation 0909-0495 1600-5775 International Union of Crystallography 36891860 yi5133 10.1107/S1600577522012085 JSYRES S1600577522012085 Beamlines The beam transport system for the Small Quantum Systems instrument at the European XFEL: optical layout and first commissioning results Beam transport system for the SQS instrument Mazza Tommaso a* Baumann Thomas M. a Boll Rebecca a De Fanis Alberto a Grychtol Patrik a Ilchen Markus ab Montano Jacobo a Music Valerija ac Ovcharenko Yevheniy a Rennhack Nils a Rivas Daniel E. a Rorig Aljoscha a Schmidt Philipp a Usenko Sergey a Ziolkowski Pawel a La Civita Daniele a Vannoni Maurizio a Sinn Harald a Keitel Barbara b Plonjes Elke b Jastrow Ulf Fini b Sorokin Andrey b Tiedtke Kai b Mann Klaus d Schafer Bernd d Breckwoldt Niels efg Son Sang-Kil e Meyer Michael a* a European XFEL, Holzkoppel 4, 22869 Schenefeld, Germany b Deutsches Elektronen-Synchrotron DESY, Notkestr. 85, 22607 Hamburg, Germany c Department of Physics, University of Kassel, Heinrich-Plett-Strasse 40, 34132 Kassel, Germany d IFNANO Institut fur Nanophotonik Gottingen e.V., Hans-Adolf-Krebs-Weg 1, 37077 Gottingen, Germany e Center for Free-Electron Laser Science CFEL, Deutsches Elektronen-Synchrotron DESY, Notkestr. 85, 22607 Hamburg, Germany f Department of Physics, Universitat Hamburg, Notkestr. 9-11, 22607 Hamburg, Germany g The Hamburg Centre for Ultrafast Imaging, Luruper Chaussee 149, 22761 Hamburg, Germany Yabashi M. Editor RIKEN SPring-8 Center, Japan Correspondence e-mail: [email protected], [email protected] 01 3 2023 03 2 2023 03 2 2023 30 Pt 2 s230200 457467 08 9 2022 22 12 2022 (c) Tommaso Mazza et al. 2023 2023 This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-BY) Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited. A full version of this article is available from Crystallography Journals Online. The soft X-ray beam from the SASE3 source at the European XFEL is delivered to the SQS instrument by a beam transport system. The system layout and performances are reported. The Small Quantum Systems instrument is one of the six operating instruments of the European XFEL, dedicated to the atomic, molecular and cluster physics communities. The instrument started its user operation at the end of 2018 after a commissioning phase. The design and characterization of the beam transport system are described here. The X-ray optical components of the beamline are detailed, and the beamline performances, transmission and focusing capabilities are reported. It is shown that the X-ray beam can be effectively focused as predicted by ray-tracing simulations. The impact of non-ideal X-ray source conditions on the focusing performances is discussed. free-electron lasers X-ray beam transport microfocusing atomic, molecular and optical science Deutsche ForschungsgemeinschaftSFB-925 - project 170620586 Volkswagen FoundationPeter-Paul-Ewald Fellowship Horizon 2020 Framework Programme, European Metrology Programme for Innovation and Research730872 Deutsche ForschungsgemeinschaftCUI: Advanced Imaging of Matter - EXC 2056 - project ID 390715994 MM and TM acknowledge support by the Deutsche Forschungsgemeinschaft (DFG) (SFB-925, project No. 170620586). MI, PS and VM acknowledge funding by the Volkswagen Foundation for a Peter-Paul-Ewald Fellowship. BK and EP acknowledge funding from the European Union's Horizon 2020 Research and Innovation program under grant agreement No. 730872. NB acknowledges support from the Cluster of Excellence 'CUI: Advanced Imaging of Matter' of the DFG (EXC 2056, project ID 390715994). pmc1. Introduction The Small Quantum Systems (SQS) instrument at the European XFEL (Decking et al., 2020 >) is dedicated to the investigation of atoms, molecules, clusters and biomolecules in the gas phase. It uses the beam from the SASE3 soft X-ray undulator between the carbon and argon K edges, in the range 260-3200 eV. The instrument, described in detail by Mazza et al. (2012 >) and Meyer et al. (2023 >), was commissioned in 2018 (Meyer et al., 2023 >) and the results of the first successful user experiments have been published (Kastirke et al., 2020a >,b >; Eichmann et al., 2020 >; Mazza et al., 2020 >; LaForge et al., 2021 >; Jahnke et al., 2021 >; Li et al., 2021 >, 2022a >,b >; Boll et al., 2022 >; Feinberg et al., 2022 >). Experiments at the SQS instrument exploit the intense, short and coherent X-ray pulses for non-linear phenomena studies (Mazza et al., 2020 >; Eichmann et al., 2020 >; LaForge et al., 2021 >; Boll et al., 2022 >), for time-resolved experiments following dynamical processes on the femtosecond timescale (Kastirke et al., 2020b >; Jahnke et al., 2021 >; Grychtol et al., 2021 >; Rivas et al., 2022 >) and for investigations using coherent scattering techniques (Ekeberg et al., 2022 >; Feinberg et al., 2022 >). This paper is dedicated to the description of the X-ray beam transport system in use during the commissioning phase and the first three user runs until the beginning of 2020, and reports on its performance, evaluated from the results obtained during its commissioning. It comprises three main sections. In Section 2, the optical layout of the beamline is shown and the different beam transport configurations are described. In Section 3, we report on the calculated and measured beamline transmission for the different beam transport configurations, which sorts them according to the different use cases. In Section 4, we report on the micro-focusing performances of the system, which have been both simulated by ray tracing and measured by direct as well as indirect methods. 2. Optical layout of the beamline The optical system transporting and focusing the free-electron laser (FEL) radiation extends over more than 400 m from the last SASE3 undulator section to the SQS experiment interaction region. The main FEL beam parameters are listed in Table 1 >. The optical system, schematically represented in Fig. 1 >(a), consists of six high-quality grazing-incidence mirrors, which are listed along with their characteristics in Table 2 >. The surface error of the mirrors, in the nm range, is at the state of the art, especially for such long substrates (Vannoni & Freijo-Martin, 2017 >, 2019 >). The first four of these mirrors, named M1, M2, M3 and M4, are located in an underground tunnel. The last two, named horizontal focusing mirror (HFM) and vertical focusing mirror (VFM), build up a Kirkpatrick-Baez (KB) mirror system and are located in the SQS instrument hutch. All mirrors are coated with a 50 nm-thick layer of boron carbide (B4C). The mirrors M1 and M2 constitute a horizontal chicane, which filters out the high-photon-energy background (spontaneous radiation, bremsstrahlung radiation) as well as contributions from higher harmonics of the FEL fundamental wavelength. The grazing-incidence angle of both mirrors can be set to any value between 9 and 20 mrad by changing their pitch and their relative transverse position. Working points have been defined at 9, 13 and 20 mrad. The beam divergence increases monotonically with the wavelength (Table 1 >), so that at low photon energies the geometric transmission is effectively increased by increasing the incidence angles, which give a larger projected clear aperture. At the same time, the range of the transmitted photon energy by the reflectivity of the mirrors increases with decreasing incidence angle, with a cutoff changing from about 1.4 keV to >3 keV for an incidence angle varying from 20 mrad to 9 mrad. M1 is a flat mirror; M2 is equipped with a mechanical bender inducing a cylindrical curvature (Vannoni et al., 2016 >) which is varied with the incidence angle to focus the beam horizontally at a distance of 90 m from the mirror, or 374 m from the source. This feature makes the size of the beam footprint at the position of HFM comparable with the projected clear aperture of the mirror itself (see Fig. 1 > and Table 2 >). The mirrors M3 and M4 constitute a vertical chicane. Two different substrates can be placed in the M3 position depending on the beam wavelength, with an incidence angle of 20 mrad for low photon energies (low-energy pre-mirror, LEPM) and 9 mrad for high photon energies (high-energy pre-mirror, HEPM). Both pre-mirrors have a spherical shape with a fixed radius, to focus the beam at a distance of 100 m from the mirror. M4 is a plane mirror which can be replaced by a variable line spacing (VLS) grating if a monochromatic beam is required by the beam users; the spectral band is selected in this case by a vertical slit located at the focal position of M3. The grating and the slit constitute the SASE3 soft X-ray monochromator (Gerasimova et al., 2022 >). In addition, the focusing provided by M3 narrows the beam also vertically, such that, similarly to the horizontal direction, the beam size at the position of VFM is made compatible with the projected clear aperture of the mirror itself (see Fig. 1 > and Table 2 >). Depending on the experimental requirements in terms of photon energy range, various beam transport configurations are then defined, combining the different set points of the horizontal and vertical chicane. The beam transport performances regarding transmission are presented for selected configurations in Section 3. All the transport configurations deliver the beam to the same position in the instrument hutch, where it is focused into the experimental region by the last two mirrors. The HFM and VFM mirrors are a pair of elliptical substrates with fixed radii, installed in a KB configuration. They are dedicated to microfocusing the beam into the experimental region of the SQS instrument. Their design parameters are a HFM = 28 m, b HFM = 9.41 m, a VFM = 15 m, b VFM = 5.34 m, where a and b are, respectively, the semi-major and semi-minor axes of the elliptical profiles in the meridional direction. These correspond to a source distance to the mirror center p 0 = 52.7 m (28 m) and an image distance q 0 = 3.3 m (2.0 m) for the HFM (VFM) with an incidence angle at the mirror center of th0 = 9 mrad for both HFM and VFM (Peatman, 1997 >). The mirrors are used to image the virtual sources given by the horizontal and vertical foci generated along the tunnel by M2 and M3, called intermediate foci (IMFH, IMFV), into the experimental region of the SQS instrument. The mismatch between the respective p parameters, stated above, and the distances IMFH - HFM = p H = 72 m and IMFV - VFM = p V = 46 m is compensated, approximating the elliptical profile to a cylindrical profile, by detuning the incidence angle from the set value th0 by Dth, with (Peatman, 1997 >) resulting in the incidence angles listed in Table 2 >. Because of this adjustment, the beam profile is affected by minor spherical aberrations. The impact of these on the focal spot size is discussed in Section 4. The KB mirrors with fixed radii have been recently replaced in the SQS instrument by a pair of mechanically bendable mirrors, aiming for better focusing performances and higher operational flexibility. The new KB system will be described elsewhere (Mazza et al., 2023 >). 3. Beamline transmission The beam transport layout described in Section 2 is designed to provide high transmission for the X-ray beam for a photon energy range spanning more than one order of magnitude (Table 1 >). The transmission is limited on the low-photon-energy side by the large beam size, due to the increased divergence, exceeding the finite size of the mirrors, and on the high-energy side by the cutoff determined by the grazing-incidence angle. The transmission was calculated for different beam transport configurations as the product of the combined reflectivity of the six mirrors and the geometric acceptance of the beamline. The mirror reflectivity was calculated using the X-ray Database of the Center for X-ray Optics (The Center for X-ray Optics, 2022 >). Photon beams with horizontal polarization and energies between 0.25 and 4 keV were reflected by a single layer of B4C with a thickness of 50 nm on a Si substrate under incidence angles of 9, 13 and 20 mrad. The density of the substrate is assumed to be equal to the bulk value. For the coating density, a value of 2.37 g cm-3 was used. The coating density of the mirrors was measured by X-ray reflectometry using a laboratory source with a sub-nm accuracy on smaller superpolished silicon substrates coated together with each mirror (Stormer et al., 2018 >). The geometric acceptance of the beamline was obtained by analytically calculating the propagation of a beam with source size and divergence as given in Table 1 > through the optical layout schematically represented in Fig. 1 >. The transmission was also experimentally determined by simultaneously measuring the energy per pulse upstream and downstream of the six mirrors by means of gas monitor detectors (XGMs) (Baumann et al., 2023 >; Sorokin et al., 2019 >), one located before any optical element and the other in the experimental hutch after the last reflecting mirror. The measurements were performed for selected beam transport configurations over a wide range of photon energies. Measured and calculated transmission values are shown in Fig. 2 >(a). The transmission is high over a large range, and the comparison shows a good agreement between predicted and observed beamline transmission over the whole range. The curves represent the calculated beamline transmission for the four standard beam transport configurations, in which either the HEPM or the LEPM is used in the vertical chicane; in combination with the HEPM, the horizontal chicane is set to an incidence angle of either 9 or 13 mrad, whereas in combination with the LEPM the horizontal chicane is set to an incidence angle of either 13 or 20 mrad. The full lines represent the transmission given by the product of the reflectivity and geometric acceptance contributions. The dashed lines give the reflectivity contribution to the overall transmission, showing that for photon energies above 2 keV, where the beam size is small enough, the geometric acceptance is essentially equal to one. The geometric acceptance at low photon energies drops because of the increasing beam size given by its larger divergence (Table 1 >). At high energies, the transmission is cut by a drop in reflectivity above a photon energy value which depends on the incidence angle. The points represent measurements performed under different beam transport configurations, to be compared with the corresponding calculated curves. The measurements have been performed changing also the opening conditions of a double slit system located right after the source, before the upstream XGM, called the synchrotron radiation aperture (SRA). With a small enough SRA, only the central part of the beam is propagated, so that geometric losses play no role in determining the measured transmission. The measurement performed under such conditions, with an SRA of 1 mm x 1 mm, is labeled 'data2' and can then be compared with the calculated reflectivity contribution. The ratio between the measurements and the calculated transmission is shown in Fig. 2 >(b). The measured transmission is on average between 10 and 20% lower than calculated, likely due to degradation of the mirror coatings induced by X-ray exposure. The stronger reduction of the transmission observed when using the LEPM would be in this case consistent with the more intensive use that this substrate had undergone at the time of the measurements. Also, the reflectivity of the peripheral parts of the mirrors, which are not probed by the measurement made using a narrow SRA, is possibly less degraded due to the milder X-ray exposure compared with the central part, which could explain the higher ratio between experiment and calculations observed for the measurement with larger SRA ('data1') compared with the measurement with narrower SRA ('data2'). Fig. 2 >(c) shows the calculated transmission curves in a logarithmic scale. This representation gives a semi-quantitative picture of how the different beam transport configurations are effective in suppressing contributions from higher harmonics of the FEL fundamental. An experimental investigation quantifying these contributions from the SASE3 undulator will be published elsewhere (Baumann et al., 2023 >). 4. Focusing performance The expected spatial distribution of the X-rays in the focal spot produced by the KB mirrors has been calculated by ray-tracing simulations using the SHADOW code (Sanchez del Rio et al., 2011 >). The source was modeled as a Gaussian beam originating at the end of the last undulator cell. A source with a finite longitudinal extension, accounting for the situation in which the FEL exponential power growth saturates before the last undulator cell is reached, was modeled as the superposition of point-like sources located at the end of each of the last n undulator cells, with the number n as well as the relative intensity of the sources set as variable parameters in the simulations. The source size and divergence were set based on the expressions given in Table 1 >. The beam was propagated along the SASE3 beamline, described in Section 2 (see Fig. 1 > and Table 2 >), adjusting the incidence angle of the HFM and VFM mirrors to compensate the mismatch between their design profile and the beamline geometry. The contribution from the measured polishing error of the mirrors (see Table 2 >) was explicitly included in the simulations as an additional surface error. As ray-tracing simulations do not include diffraction effects, to account for the diffraction contribution given by the finite size of the mirrors to the beam profile at the focal position a random distribution was added to the rays' position in both directions of the focal plane. The probability for this distribution follows the single slit diffraction intensity and is given by with = CAth/(l q)x where q is the mirror-focus distance, l is the X-ray wavelength, CA is the mirror clear aperture, th is the grazing-incidence angle and x is the displacement from the beam axis in either the horizontal or the vertical direction. The beam intensity profile in the focal position, simulated under the assumption of a source with no longitudinal extension located at the end of the last undulator cell for a photon energy of 1.05 keV, is shown in Fig. 3 >(a). The predicted beam size is approximately 1.5 mm full width at half-maximum (FWHM), matching the requirement for a micrometric focus which is necessary for performing non-linear studies. The polishing error and the finite mirror size diffraction contributions, estimated from the squared difference between the simulations with and without inclusion of the respective effects, amount to 0.55 and 0.73 mm FWHM, respectively (see Fig. 4 >). According to expression (2), diffraction effects dominate the beam size in the low end of the photon energy range and become smaller as the wavelength decreases. The asymmetric tail observed on the negative side in both horizontal and vertical directions is due to aberrations given by the deviation of the mirror surface profile from the ideal one, given by the mismatch between the elliptical shape of the HFM and VFM mirrors and the beamline geometry. This mismatch is compensated only in the first order by detuning the incidence angle of the focusing mirrors (see Section 2). The FEL radiation generated by the undulators is commonly assumed to originate from the last cell of the undulator array, which in the case of SASE3 consists of 23 cells spaced 6.1 m from each other (Fig. 1 >). This point, identified as the radiation source, can be effectively focused in the experiment by an optical system based on reflective elements like the one described here, by adapting the radius in the case of bendable mirrors or by adapting the grazing-incidence angle as for the present case. If the upstream cells yield non-negligible radiation, these contributions will be focused at different positions along the propagation direction, being delivered out of focus to the desired image position. Such a situation is realized when the FEL gain saturation level is reached at an undulator cell n sat located upstream of the last undulator cell N, n sat < N: the undulator gain curve I(n) grows less than exponentially for the downstream radiators, I(n)/I(n - 1) < I(n - 1)/I(n - 2) for n > n sat, and the radiation yield from each of the downstream cells becomes comparable, I(n) - I(n - 1) I(n - 1) - I(n - 2) for n > n sat (Milton et al., 2001 >). Fig. 3 >(b) shows the beam intensity profile in the focal position simulating an extended source consisting of ten points located at the end of each of the last ten undulator cells, each radiating with the same intensity. This provides a rough modeling for the situation in which the FEL gain saturation is reached by the 14th of the 23 cells of the SASE3 undulator. Coherence effects are neglected in the ray-tracing simulations. The beam FWHM is approximately the same as in Fig. 3 >(a), but the asymmetric tail is much more pronounced, resulting in a broad, low-intensity underlying contribution with a size of several micrometres. The photon density distribution can be modeled as the sum of two contributions, a narrower one G1, with total integral E G1, width w G1 and maximum f G1, coming mostly from the imaging of the in-focus radiator, and a broader background G2 with total integral E G2, width w G2 and maximum f G2 yielded by the radiating cells located further away. Fitting the distribution with the superposition of two Gaussians (Fig. 4 >) shows that for the present simulation conditions the contribution from G2 is higher than that from G1 (E G2/E G1 = 1.89) but the contribution from G2 to the peak fluence is minor (f G2/f G1 = 0.21), because of the larger width of the second Gaussian with respect to the first (w G2 = 4.1 mm, w G2/w G1 = 3.0). The experimental focusing conditions were optimized during the commissioning phase of the instrument. After aligning the beamline elements in the tunnel, focusing was optimized by fine tuning the incidence angle (pitch) of the fixed-radii KB mirrors and in addition by adjusting the roll of the HFM to get the two mirrors to be orthogonal to each other. For the horizontal (vertical) direction, around the nominal focal distance a variation of 1 mrad in the incidence angle would move the focus longitudinally by 0.38 mm (0.29 mm), and accordingly in the transverse direction by 6.6 mm (4 mm), so when adjusting the focal distance by several centimetres a translation in the order of 1 mm of the focal position in both directions in the beam plane would need to be accounted for. Ray-tracing simulations show that the aberrations given by the mismatch between the mirror profiles and the beamline geometry are minimized, albeit not completely removed, when the image distance set by the incidence angle detuning is equal to the nominal (design) image distance of the mirrors, which was set as the design distance for the position of the interaction region. The optimization and then the characterization of the beam focusing was aided by different diagnostics tools. 4.1. Wavefront sensor The spatial distribution of the beam intensity in the focal position was reconstructed from wavefront measurements performed using a Hartmann-type wavefront sensor (WFS) (Keitel et al., 2016 >). The WFS is based on an array of 75 mm-diameter holes, electroformed on a quadratic pattern with a 250 mm pitch in a 20 mm-thick Ni foil, the Hartmann plate. The Hartmann plate is exposed to the beam 2.3 m downstream of the focal position and is placed upstream of the CCD chip coated with an EUV-to-VIS quantum converter P43 (1392 x 1040 pixels with 6.45 mm x 6.45 mm pixel size; field of view of 8.9 mm x 6.7 mm) at a distance of ~200 mm. As described in detail by Keitel et al. (2016 >), the plate splits the incoming beam into an array of sub-beams which are recorded by the CCD. The displacement of a spot centroid position with respect to a previously measured reference plane wave, divided by the camera-plate distance, yields the local radiation angle and thereby the local wavefront gradient inside one sub-aperture. The wavefront is reconstructed from these local gradients using mathematical algorithms. The extracted information allows one to compute relevant beam parameters as well as to back-propagate the beam spatial profile to the focal position. Measurements were performed at a photon energy of hn = 1.05 keV by attenuating the X-ray beam, optimized for high pulse energy, by several orders of magnitude, to preserve the Hartmann plate and the coated CCD camera from damage. A representative reconstructed single-shot intensity distribution of the X-ray beam in the focal plane is shown in Fig. 3 >(c). The photon beam distribution at the focus position was determined by numerically back-propagating the beam obtained from the WFS measurement of the wavefront phase and intensity after optimization of the focusing conditions. The optimization was performed by tuning the pitch and roll of the KB mirrors, aiming for minimizing the Zernike coefficients provided by the WFS algorithm which quantifies the optical aberrations of the wavefront. The intensity distribution is characterized by a sharp 'hot-spot' contribution G1, with a FWHM of 1.4 mm, superposed on a broad background G2 (FWHM = 9 mm), as given by the double 2D Gaussian fit (see Fig. 4 >). We then describe the X-ray density distribution measured by the WFS using the same double Gaussian model described above; from a double Gaussian fit analysis performed on a data set of 20 single FEL shots we get the statistically more robust estimates for the parameters w G1 = 1.9 mm, w G2 = 8 mm, w r = w G2/w G1 = 4.2, E G2/E G1 = 2.6, f r = f G2/f G1 = 0.15. 4.2. Charge-state distribution based on light atoms The size of the beam profile was also indirectly characterized by determining the incident photon fluence distribution from a quantitative comparison between theoretical and experimental charge-state distributions (CSDs) that are generated by X-ray multiphoton multiple ionization of Ar atoms. The CSDs, measured by ion time-of-flight mass spectrometry (Meyer et al., 2023 >), are modeled using the XATOM package (Son et al., 2018 >), which calculates the atomic electronic structure and simulates their ionization dynamics under intense FEL pulses, with calibrated beam parameters. We employed an extended version of XCALIB (Toyota et al., 2019 >) to calibrate the fluence distribution at the focal spot, utilizing a range of pulse energies (Breckwoldt et al., 2023 >). The numerical method is described in detail by Toyota et al. (2019 >). In brief, for a given photon energy the XATOM toolkit is used to calculate the ion yield for different charge states as a function of the photon fluence over a broad range. Instead of mapping the spatial fluence distribution as a function of position, XCALIB combines the theoretical CSDs to model the experimental CSDs with a few fitting parameters assuming a specific form of the spatial fluence profile in the focus position, giving access to the beam spatial distribution quantities, including the beam size. We measured the CSDs of Ar atoms undergoing X-ray multiphoton multiple ionization at photon energies between 1200 and 1700 eV. For each photon energy, the pulse energy on target was tuned over a range of more than one order of magnitude between 0.1 and 2 mJ with the aid of a gas attenuator device (Sinn et al., 2012 >). The theoretical CSDs are fitted to experimental CSDs with two different fluence distribution models, assuming that the beam spatial profile did not change when attenuating the X-ray beam, so retrieving a unique set of parameters for each photon energy and a relation between the experimental pulse energies delivered to the sample {E pulse} and the peak fluence in the focal plane {F peak}. The simplest model would be the beam spatial distribution described by a single Gaussian, so that the E pulse values would be scaled to the F peak values according to F peak = . The parameter w, representing the beam FWHM at the focal position, would be the only fitting parameter in this case. Based on the results from ray-tracing simulations and on the observation from the WFS measurement, following the method depicted by Toyota et al. (2019 >) we instead describe the fluence spatial distribution in the focal plane by a superposition of two Gaussian distributions with partial yields E G1 and E G2, with which, expressed by the terms introduced in the previous subsection, f r = f G2/f G1, w r = w G2/w G1 and E G2/E G1 = , reads which is equivalent to equation (17d) of Toyota et al. (2019 >), as E pulse is the pulse energy on target, so the quantity T entering the expression of Toyota et al. (2019 >) is set equal to 1. The scaling between the E pulse values and the F peak values in the focal plane is then given by three independent parameters: the width of the G1 Gaussian contribution w (= w G1), the ratio between the peak fluence of the two Gaussian contributions f r and the ratio between the width of the two contributions w r . The photon budget is distributed between the two contributions according to E G2/E G1 = . The fluence distribution in the interaction region, which is limited along the beam direction z by the acceptance of the ion spectrometer, actually depends on the position z according to w = w 0 [1 + (2z/d)2]1/2, where d is the depth of the beam focus. A comparison (not shown here) between the calculations based on a 3D modeling including the variation of the intensity distribution along the beam path and a simplified 2D calculation demonstrates that this variation can be safely neglected for the present case, and a model assuming constant conditions along the propagation axis can be used. Fig. 5 > shows an example time-of-flight mass spectrum from Ar at a given pulse energy and the measured CSDs of Ar for selected charge states over a range of pulse energies, at a photon energy of 1200 eV. The CSDs have been fitted by theory to yield the photon energy dependent parameters w, f r and w r reported in Table 3 >. The fitting procedure is described in detail by Breckwoldt et al. (2023 >). 4.3. Focusing performance results overview The charge-state distribution characterization results based on multiphoton ionization are in very good quantitative agreement with both the outcome of the ray-tracing simulations and the results from the wavefront sensor characterization. Both experiments and simulations clearly show that the beam transport system can deliver the X-rays from the SASE3 undulator into the experiment focused down to below 1.5 mm FWHM, enabling one to perform non-linear studies on small quantum systems. With a pulse energy of 1 mJ and the parameters reported in Sections 4.1 and 4.2, an energy fluence of 0.12 to 0.16 mJ mm-2 is obtained in the focal position. Using a double Gaussian fluence distribution for modeling the CSD of Ar ions and the WFS measurement, we get the indication that the beam profile is characterized by the presence of a narrow contribution with a linear size between 1 and 1.5 mm over a broader term with a size from two to three times larger. From the ray-tracing simulation results, it is natural to consider the narrow contribution, modeled by the first or smaller Gaussian, as being made by the fraction of the photon beam that is properly focused in the intended position by the KB mirrors. Fig. 6 > summarizes the beam size results obtained from the WFS and CSD characterization, where 'beam size' is the FWHM of the narrow contribution, or first Gaussian, compared with the beam size evaluated from the ray-tracing simulations performed assuming a source with no longitudinal extension [see Figs. 3 >(a) and 4 >(a)]. The results from both characterizations reproduce very well on a quantitative level the results from the simulations. The CSD characterization was moreover performed over a relatively broad photon energy range, reproducing experimentally the decreasing trend of the simulated beam size as a function of photon energy, which is mainly due to the decreasing relevance of diffraction effects. Our experimental results show that even with state-of-the-art high-quality focusing mirrors, the beam intensity profile in the focal position can be different from an ideal micrometre-sized Gaussian. Based on ray-tracing simulations, we can assess that the contribution from aberrations due to the mirror surface quality is marginal, and the shape is likely due to the non-ideal properties of the beam source, which becomes significantly extended in the longitudinal direction when the FEL saturation conditions are reached before the last undulator cell. This feature, which should be considered to be common to most of the XFEL experiments performed, optimizing the photon yield from the undulator, has a relevant impact on non-linear studies, which require high photon density conditions. The presence of a broad low-intensity pedestal in the beam spatial distribution, modeled as a double Gaussian spatial profile (Toyota et al., 2019 >), has already been observed in gas-phase atomic and molecular experiments, with good agreement between experiment and theory (Toyota et al., 2019 >; Murphy et al., 2014 >; Rudenko et al., 2017 >; Rudek et al., 2018 >). 5. Conclusions and outlook The SQS instrument at the European XFEL started user operation at the end of 2018, after a few weeks of commissioning dedicated to the basic equipment, with subsequent effort aimed at expanding the offer in equipment and methodologies. The beam transport system has been designed to cope with the requirement for tightly focused X-ray beams to be delivered to the instrument over a very broad range of available photon energies. The characterization for both the beamline transmission and the focusing performances, reported here, has clearly shown that these requirements have been fully met. The beam transport system has been recently upgraded with a pair of mechanically bendable KB mirrors replacing the fixed-radii substrates described in this work. The new system will allow much more flexible operation conditions, enabling, in addition, movement of the focal position of the beam to different positions along the instrument, so allowing the simultaneous installation of different end-stations. The experimental data shown here are available at We acknowledge the European XFEL in Schenefeld, Germany, for the provision of XFEL beam time at the SQS instrument and thank the staff for their assistance. We thank S. Serkez for the insightful discussions. The authors acknowledge the contribution of the entire European XFEL team, in particular the machine crew, the diagnostics group and the X-ray optics group. Open access funding enabled and organized by Projekt DEAL. Figure 1 (a) Schematic representation of the beam transport layout of the SQS branch of the SASE3 beamline (Tschentscher et al., 2017 >), which is described in detail in the text. The distance of each optical element to the source point is given at the bottom of the scheme. (b) The beam propagation conditions are schematically represented for both the vertical and the horizontal directions. The beam diverges from the source point until it reaches the first focusing element, which is M2 in the horizontal and M3 in the vertical direction. The beam converges horizontally and vertically on two different positions along the beamline, labeled HIMF and VIMF (horizontal and vertical intermediate focus, respectively), located at 90 m and 100 m distance from the respective mirrors. These positions are then the virtual sources imaged by the HFM (horizontal focusing mirror) and VFM (vertical focusing mirror) into the focal position where the experiment takes place. The dashed vertical lines indicate the position of the focusing mirrors and of the (virtual) sources and images in the vertical and horizontal directions. Figure 2 (a) Calculated and measured beamline transmission, including all six optical elements, as a function of photon energy for different beam transport configurations. The dashed curves represent the reflectivity contribution to the transmission, whereas the full lines represent the total transmission given by the product of the reflectivity and the geometric acceptance contributions. Different colors of the curves correspond to different incidence angle settings for the horizontal (H) and vertical (V) chicanes. Yellow: H = 20 mrad, V = 20 mrad; red: H = 13 mrad, V = 20 mrad; blue: H = 13 mrad, V = 9 mrad; green: H = 9 mrad, V = 9 mrad. The points represent transmission measurements. Green squares ('data1'): H = 9 mrad, V = 9 mrad; gray circles ('data2'): H = 9 mrad, V = 9 mrad, probing reflectivity only (see text); red diamonds ('data3'): H = 13 mrad, V = 20 mrad. (b) Ratio between the measured and the respective calculated transmission values shown in (a). (c) Calculated beamline transmission, in logarithmic scale, showing the transmission above the cut-off energy, which determines the fraction of the original higher harmonics contribution from the undulator, which is delivered to the experiment (Baumann et al., 2023 >). Figure 3 Ray-tracing simulations results compared with a wavefront measurement of the beam profile in the focal position at hn = 1050 eV. For all three beam profile images the color scale goes from 0 (blue) to 1 (red), as each image is normalized to the maximum value. (a) Beam profile from ray-tracing simulations performed assuming a Gaussian-shaped source with size and divergence as per Table 1 > with no extension in the longitudinal direction. (b) Beam profile from ray-tracing simulations performed assuming a source extending over the last ten undulator cells (see text). (c) Beam profile as obtained from the wavefront sensor measurement. Figure 4 Curves (a), (b) and (c) in black are the projection in the vertical direction of the focal beam profiles shown in Figs. 3 >(a), 3(b) and 3(c), respectively. The projection of the profile obtained from a simulation assuming a source without extension in the longitudinal direction [(a), black] is compared with the projection of the same profile, assuming no diffraction effects from the finite length of the mirrors [(a), green], and assuming no surface error and no diffraction effects [(a), blue]. The projection of the profiles obtained from a simulation assuming an extended source over the last ten undulator cells [(b), black] and the projection of the profile measured by the wavefront sensor [(c), black] are compared with the projection of a fitting distribution made of the sum of two 2D Gaussian profiles [(b) and (c), red]. The projections of the individual Gaussian contributions are also plotted as dashed red lines. Figure 5 Left: a representative time-of-flight mass spectrum of multiphoton ionized Ar atoms. Photon energy was hn = 1200 eV, pulse energy was E p = 1.0 mJ, corresponding to 5 x 1012 photons per pulse. Right: measured CSDs as a function of pulse energy at hn = 1200 eV for selected charge states compared with the best-fitting curves from the XATOM calculations, yielding the parameters reported in the first row of Table 3 >. Figure 6 Black line: beam size (FWHM) as a function of photon energy estimated from the ray-tracing simulated intensity distribution under the assumption that the FEL source is located at the end of the last undulator, with no longitudinal extension. The simulations are performed accounting for the surface error of the mirrors and taking into account the diffraction effects, as described in the text. Red star: beam size from the WFS characterization, taken from the FWHM of the smaller Gaussian contribution to the double Gaussian fit of the 2D intensity distribution [see Figs. 3 >(c) and 4 >(c)]. Blue dots: beam size from the CSD characterization, taken from the FWHM of the smaller Gaussian contribution to the double Gaussian model for the fluence distribution used to fit the CSDs collected as a function of photon energy, see Table 3 >. Table 1 FEL beam parameters The photon energy ranges are limited for each electron energy set point on the low end by the minimum size of the undulator gap, on the high end by the lack of yield due to the open gap. As such, the lower limit is a hard one, whereas the higher one depends on the self-amplified spontaneous emission level. The source size and divergence FWHM are calculated according to the expressions size = 6log(6000l [nm]) mm and divergence = 14.1/hn [keV]0.75 mrad, respectively (Sinn et al., 2011 >). Electron beam energy (GeV) 16.5 14 11.5 8 Photon energy (keV) 0.92-4.0 0.66-2.9 0.5-1.96 0.26-0.95 Source size, FWHM (mm) 57 mm @ 0.5 keV 51 mm @ 1.5 keV 48 mm @ 2.5 keV Source divergence, FWHM (mrad) 24 mrad @ 0.5 keV 10 mrad @ 1.5 keV 7 mrad @ 2.5 keV Pulses s-1 10000 (achieved), 27000 (design) Pulse energy (mJ) 12 (achieved), 10 (design) Table 2 Parameters describing the beamline mirrors Beam footprints, given as FWHM, on the mirrors are not necessarily round; the given values are to be intended for the respective orientation (e.g. for HFM the beam given footprint is horizontal). Slope error values [root mean square (RMS)] for the tunnel mirrors are from Gerasimova et al. (2022 >). Distance from source (m) Clear aperture (w x l) (mm) Incidence angle (mrad) Shape Orientation Curvature radius (km) Tangential slope error RMS (nrad) Beam footprint at hn = 0.5, 1.6, 2.7 keV (mm) M1 281 20 x 800 9, 13, 20 Planar H - 56 6.5, 2.7, 1.9 M2 283.9 20 x 800 9, 13, 20 Spherical (bendable) H 15.03, 10.40, 6.76 58 6.5, 2.7, 1.9 M3-LEPM 300 20 x 500 20 Spherical (fixed) V 7.62 63 6.8, -, - M3-HEPM 300 20 x 500 9 Spherical (fixed) V 16.49 37 -, 2.9, 2.0 M4 301 20 x 500 9, 20 Planar V - 140 6.8, 2.9, 2.0 HFM 444.8 20 x 600 9.15 Elliptical (fixed) H 0.69 81 5.1, 2.2, 1.5 VFM 446.1 20 x 400 9.24 Elliptical (fixed) V 0.415 71 3.1, 1.3, 0.9 Table 3 Best-fitting parameters modeling the experimental CSDs measured at photon energies between 1200 and 1700 eV with the double Gaussian superposition described in the text hn (eV) w (mm) f r w r 1200 1.55 0.65 2.2 1250 1.70 0.60 2.0 1300 1.70 0.55 2.0 1350 1.45 0.70 2.2 1400 1.48 0.60 2.2 1450 1.43 0.65 2.2 1500 1.41 0.65 2.2 1550 1.40 0.65 2.2 1600 1.26 0.70 2.4 1650 1.26 0.70 2.4 1700 1.25 0.75 2.4 References Baumann, T. 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PMC10000794 | J Synchrotron Radiat J Synchrotron Radiat J. Synchrotron Rad. Journal of Synchrotron Radiation 0909-0495 1600-5775 International Union of Crystallography 36891849 vl5003 10.1107/S1600577522012024 JSYRES S1600577522012024 Research Papers Effects of synchrotron-based X-rays and gold nanoparticles on normal and cancer cell morphology and migration AuNPs and cancer cell morphology and migration Shahhoseini Elham a Nakayama Masao b Panettieri Vanessa c Hall Chris d Feltis Bryce e Geso Moshi a* a Medical Radiation, RMIT University, 289 McKimmies Road, Bundoora, Victoria 3083, Australia b Division of Radiation Oncology, Kobe University, 7-5-2 Kusunokicho Chuou-ku, Kobe 650-0017, Japan c Alfred Health Radiation Oncology, 55 Commercial Road, Melbourne, Victoria 3004, Australia d ANSTO, Australian Synchrotron, 800 Blackburn Road, Clayton, Victoria 3168, Australia e Human Bioscience, RMIT University, 289 McKimmies Road, Bundoora, Victoria 3083, Australia Aranda M. A. G. Editor University of Malaga, Spain Correspondence e-mail: [email protected] 01 3 2023 13 1 2023 13 1 2023 30 Pt 2 s230200 359367 31 7 2022 21 12 2022 (c) Elham Shahhoseini et al. 2023 2023 This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-BY) Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited. A full version of this article is available from Crystallography Journals Online. The effects of synchrotron-based kilovoltage X-rays on a cell's morphology and motility are investigated. It has been shown lately that gold nanoparticles (AuNPs) and ionizing radiation (IR) have inhibitory effects on cancer cell migration while having promoting effects on normal cells' motility. Also, IR increases cancer cell adhesion with no significant effects on normal cells. In this study, synchrotron-based microbeam radiation therapy, as a novel pre-clinical radiotherapy protocol, is employed to investigate the effects of AuNPs on cell migration. Experiments were conducted utilizing synchrotron X-rays to investigate cancer and normal cell morphology and migration behaviour when they are exposed to synchrotron broad beams (SBB) and synchrotron microbeams (SMB). This in vitro study was conducted in two phases. In phase I two cancer cell lines - human prostate (DU145) and human lung (A549) - were exposed to various doses of SBB and SMB. Based on the phase I results, in phase II two normal cell lines were studied: human epidermal melanocytes (HEM) and human primary colon epithelial (CCD841), along with their respective cancerous counterparts, human primary melanoma (MM418-C1) and human colorectal adenocarcinoma (SW48). The results show that radiation-induced damage in cells' morphology becomes visible with SBB at doses greater than 50 Gy, and incorporating AuNPs increases this effect. Interestly, under the same conditions, no visible morphological changes were observed in the normal cell lines post-irradiation (HEM and CCD841). This can be attributed to the differences in cell metabolic and reactive oxygen species levels between normal and cancer cells. The outcome of this study highlights future applications of synchrotron-based radiotherapy, where it is possible to deliver extremely high doses to cancer tissues whilst preserving surrounding normal tissues from radiation-induced damage. synchrotron-based X-rays cell migration microbeam radiation therapy pmc1. Introduction Synchrotron-generated X-ray beams represent a valuable tool for radiation biology studies. They can be used in a broad range of bio-medical research fields from soft tissue imaging to radiation therapy (Ventura, 2019 >; Pelka, 2008 >). Synchrotron radiation (SR) generates high-intensity and coherent X-ray beams with dose rates up to 20000 Gy s-1 (Pelka, 2008 >). As a comparison an X-ray tube can generate a maximum dose rate of 100 Gy s-1 close to the output window (Pelka, 2008 >). The brightness of synchrotron X-ray beams makes them suitable for the production of monochromatic beams. After monochromation of the white beam, the intensity is lowered by two to six orders; however, the intensity is still high enough to be suitable for a number of radiobiology studies and it is still higher than that of the polychromatic beam generated by conventional medical X-ray tubes. The natural properties of SR, such as low divergence and associated lateral coherence, make it suitable for generating fine and spatially divided radiation beams such as those used in microbeam radiation therapy (MRT). The combination of high intensity and fine radiation fields enables the delivery of a high dose of radiation in a very small radiation field. This can be as small as 25 mm to the target with a minimum amount of scatter to the surrounding areas (Hall & Lewis, 2019 >). Pre-clinical in vivo and in vitro studies with such irradiation have shown the reduction of normal tissue damage while destroying the cancerous tissue (Bouchet et al., 2013 >; Laissue et al., 2001 >, 2007 >; Smyth et al., 2018 >; Yang et al., 2014 >). A range of theories have been reported as the cause for the different behaviours of normal and cancer cells. The phenomenon occurs when the cells are irradiated with a combination of high dose rate (HDR) X-rays and a micro-sized beam. For instance, it has been reported that the high tolerance of normal cells exposed to MRT may be attributed to differences in normal and cancer cell migration or motility (Crosbie et al., 2010 >). Despite several extensive studies on the effects of MRT on normal and cancer cells and tissues, the direct relationship between in vitro and in vivo studies remains unclear and the underlying mechanisms which cause these outcomes are not well understood (Engels et al., 2020 >). In this in vitro study, we have used the unique properties of SR, i.e. high dose rates and low divergency, to enable us to irradiate a small area of monolayer cultured normal and cancer cells. Post-irradiation we studied the cells' behaviour within the radiation field and compared with that of the cells outside the field. In the micro-beam irradiation experiments our dosimetry results for peak-to-valley dose ratio (PVDR) confirmed that the valley dose is negligible compared with that in the peak. We conducted an innovative micrography method (utilizing SBB with 500 mm width) to visualize the effects of a high-intensity 90 kV X-ray beam on the morphology and motility of cultured cells with and without inclusion of AuNPs. Our results were compared with those from cells irradiated with synchrotron microbeam (SMB). These experiments showed that the changes in cells' morphology become visible with synchrotron broad beam (SBB) at doses greater than 50 Gy. The inclusion of gold nanoparticles (AuNPs) increases this effect. Interestly, no visible morphological changes were observed in normal cell lines up to 96 h post-irradiation. This may be explained by the differences in the cell metabolic and reactive oxygen species level between normal and cancer cells (Mapuskar et al., 2019 >). 2. Experimental details 2.1. Cell lines Four different cancer cell lines were used - human prostate epithelial cancer DU145 (ATCC(r)HTB-81TM; Manassas, VA, USA), human lung epithelial cancer A549 (ATCC(r)CCL-185TM; Manassas, VA, USA), human primary melanoma MM418-C1 (RRID:CVCL_C843, provided by A/Pro. Moshi Geso), and human colorectal adenocarcinoma SW48 (ATCC(r)CCL-231TM; Manassas, VA, USA) - along with two different normal cell lines - human epidermal melanocytes HEM (ATCC(r)PCS-200-013TM; Manassas, VA, USA) and human primary colon epithelial CCD841 CoN (ATCC(r)CRL-1790TM; Manassas, VA, USA). To examine the effects of AuNPs and/or ionizing radiation (IR) on viability, a cell proliferation and viability assay (MTS) was performed. More details on the cell culture and viability assays can be found in our previous publication (Shahhoseini et al., 2019 >). 2.2. AuNPs preparation and cellular uptake To prepare a range of concentrations of gold nanoparticles, the original AuNP (Yaphank, NY, USA) solution was diluted using cell culture media. The final concentration was 0.197 mg ml-1. To determine the cellular uptake of AuNPs, inductively coupled plasma mass spectrometry (ICP-MS) was performed. More details on the AuNPs' preparation protocols and the ICP-MS measurement method are found in our previous publication (Shahhoseini et al., 2019 >). 2.3. Cell exposure with SR Cell irradiations were performed at hutch 2B on the Imaging and Medical Beamline (IMBL) at the ANSTO Australian Synchrotron. Two different radiation treatments were applied for all cell types - control groups with no AuNPs and treated groups which were incubated with 1 mM AuNPs, having 15 nm diameter, for 24 h prior to irradiation. Cells were placed in 25 cm2 (T25) flasks until a monolayer of 80-90% confluency was achieved. The samples were then exposed to radiation doses ranging from 50 to 1000 Gy of synchrotron-based 90 kV X-rays. The width of the radiation field size for SBB was 500 mm. This aimed to resemble the size of a typical scratch made in a migration assay. For the microbeam the collimation was an SMB grid, 25 mm peak area and 175 mm pitch. The irradiated samples were incubated for 24 h after irradiation and gently washed with phosphate buffered saline (PBS) to remove the dead cells from the exposed areas. The samples were then observed using a live image microscope for 96 h. 2.4. Radiation setup As the SR beam orientation is naturally horizontal, the cells samples had to be placed vertically, perpendicular to the radiation beam. To avoid the cells being out of the culture media during the irradiation time, the flasks were completely filled with media and placed against the radiation beam as shown in Fig. 1 >. The flasks were ~34 m from the source of radiation in hutch 2B at IMBL. In this study, an SBB was arranged by passing the SR beam through a tungsten carbide slit of width 500 mm and height 2 mm. The SMB was produced by passing the SR beam through a multi-slit collimator which formed microbeams with 25 mm width and 500 mm pitch (Stevenson et al., 2017 >). Fig. 2 > shows a schematic diagram of the radiation setup for SBB and SMB. The samples were placed on a motion stage which sets the flasks in the beam. The flasks were moved from top to bottom through the beam to expose the confluent area of the flask to the beam. The delivered dose was calculated based on the measured dose rate of 261.54 Gy s-1 and the sample vertical speed. Fig. 3 > shows the relationship between the delivered dose and the speed of the sample, where 2.5. Dose distribution and validation The dose distribution for both SBB and SMB was measured using a PTW microDiamond detector (Damodar et al., 2018 >) and was verified using GAFchromicTM HD-V2 films. The films were placed on the surface of the 25 cm2 flasks prior to irradiation. 2.5.1. Dose distribution in SBB measured with microDiamond detectors and GAFchromicTM HD-V2 films The original dose measurement was performed by the IMBL team using a PTW microDiamond detector and calibrated electrometer. This detector measures the dose in an area of 0.5 mm x 2 mm at 20 mm depth of the sample. Based on this measurement, dose profiles were plotted. As seen in Fig. 4 >, the dose out of the radiation field drops dramatically. About 50 mm away from the radiation field the dose is negligible. To verify the delivered dose during irradiation, GAFchromicTM HD-V2 films were used. The distribution of the radiation dose was checked using ImageJ (c) software (Rasband, 1997-2018 >) to quantify the optical density of the film which is directly correlated with the dose. The pixel values were plotted against position. Shown in Fig. 5 > are the dose/darkness profiles plotted using ImageJ (c) for a typical GAFchromicTM film exposed to 100 Gy SBB radiation dose. 2.5.2. Dose distribution in SMB measured with microDiamond detectors and GAFchromicTM HD-V2 films The same procedure was followed for the SMB irradiations. Fig. 6 > shows the dose distribution measured by the PTW microDiamond detector. The plot includes three typical peaks (25 mm) and valley (175 mm) areas. The distribution of the radiation dose was verified using ImageJ (c) software and the darkness of the film (directly correlated to the dose) was plotted against the distance. Fig. 7 > shows the dose/darkness profile plotted by ImageJ (c) for typical GAFchromicTM files exposed to 100 Gy SMB radiation dose. 3. Results Cell irradiation with SBB and SMB at the Australian Synchrotron were conducted in two phases. In phase I, two different cancer cell lines, i.e. human lung (A549) and human prostate (DU145), with and without AuNPs treatment, were irradiated with 90 kV beam with doses ranging from 50 to 1000 Gy. Based on these results, in phase II we conducted similar experimental protocols to irradiate two different normal cell lines: human epidermal melanocyte (HEM) and human primary colon epithelial (CCD841) The results were compared with their cancerous counterparts: human primary melanoma (MM481) and human colorectal adenocarcinoma (SW48), respectively. In this section, results for all cell types irradiated with SBB are presented, followed by SMB irradiation of the same cells. 3.1. Human lung cancer cells (A549) irradiated with SBB Monolayers of A549 cells grown in T25 flasks were exposed to SBB (width: 500 mm) and SR beam of various doses, i.e. 50, 100, 500 and 1000 Gy. To observe radiation-induced morphological changes in cells, the samples were incubated for 24 h after the irradiation, and then washed with PBS to remove dead cells from the culture medium. As seen in Fig. 8 >, no visible changes were observed in the cells irradiated with 50 and 100 Gy of SBB beams. Cells located in the radiation field exposed to 500 Gy, Fig. 8(C), show morphological changes. Based on these results, a dose of 1000 Gy was chosen to conduct the rest of the experiments. The boundaries of the irradiated area are marked with orange lines in the following figures. The flask surface is almost covered with damaged cells. Despite PBS wash, most of them are still attached to the polystyrene surface of the T25 flasks. Cells irradiated with 1000 Gy were damaged and killed, and after PBS wash some of the dead cells were removed from the exposed area in a way that still no clear gap is seen in the image, Fig. 8(D). As seen in Fig. 9 >, A549 cells in two groups, i.e. control and treated with 1 mM AuNPs, were irradiated with 1000 Gy SBB and observed for morphological changes over time post-irradiation. 3.2. Human prostate cancer cells (DU145) irradiated with SBB A similar procedure was followed for human prostate (DU145) cancer cells. DU145 cells were irradiated with various doses, i.e. 50, 100, 500 and 1000 Gy. Based on the morphological changes of the cells after the exposure (Fig. 10 >), 1000 Gy was chosen to create a cell gap on the cells. As seen in Fig. 11 >, exposure of 1000 Gy SBB to DU145 in the control group (with no AuNPs) resulted in an almost clear cell gap 24 h post-irradiation. However, in the treated group (with 1 mM AuNPs) there are still some damaged cells attached to the flask surface. 3.3. Human epidermal melanocytes (HEM) and human primary melanoma (MM418-C1) irradiated with SBB A similar procedure as given in Section 3.1 was followed for human epidermal melanocyte (HEM) and its cancerous counterpart human primary melanoma (MM418). Both cell types were partitioned into the control (with no AuNPs) and those treated (with 1 mM AuNPs). Due to longer proliferation times for HEM, this observation was continued for 96 h post-irradiation. As seen in Figs. 12 > and 13 >, MM418 cells located in radiation fields show visible morphological deformation in both control and treated groups such that the radiation field is clearly recognisable; its size is consistent with the actual radiation field which was 500 mm. Interestingly, HEM cells after exposure to 1000 Gy dose did not show any visible morphological damage in either the control (with no AuNPs) or those treated with 1 mM AuNPs groups. 3.4. Human primary colon epithelial (CCD841) and human colorectal adenocarcinoma (SW48) irradiated with SBB A similar procedure as given in Section 3.1 was followed for human primary colon epithelial (CCD841) and its cancerous counterpart, human colorectal adenocarcinoma (SW48). Both cell types were divided into the control with no AuNPs and those treated with 1 mM AuNPs. Due to longer proliferation times for CCD841 this observation was continued for 96 h post-irradiation. As seen in Figs. 14 > and 15 >, SW48 cells located in radiation fields show visible morphological deformation in both the control and the treated groups such that the radiation field is clearly recognisable and its size is consistent with the actual radiation field which was 500 mm. Of interest was that CCD841 cells after exposure to 1000 Gy dose did not show any visible morphological damage in either the control (with no AuNPs) or those treated with 1 mM AuNPs groups. A tabulated summary of the results for all experimental cell lines irradiated with SBB is given in Table 1 >. 3.5. Cell irradiation with SMB A similar procedure to that described previously was used for irradiation with SMB (Section 3.1) to irradiate all six cell types, i.e. human prostate cancer (DU145), human lung cancer (A549), human primary melanoma (MM418), human colorectal adenocarcinoma (SW48), human epidermal melanocyte (HEM) and colon human primary colon epithelial (CCD8). The cells were irradiated with doses of 50, 500 and 1000 Gy (the maximum possible dose that can be delivered using the MRT collimator) with grid size of 175 mm (valley)/25 mm (peak). Follow-up microscopy observations up to 96 h post-irradiation did not show any visible deformation or morphology changes in any of the cell lines (either in the control or in the AuNPs treated groups). 4. Discussion The main aim of this study was to investigate the effects of both SBB and SMB radiation on cancer and normal cell morphology and motility via in vitro based investigations. All cancer cell lines used in this study (DU145, A549, MM418 and SW48) showed a similar dose-dependent response to SBB. None of them showed any visible morphological deformation or changes after being exposed to up to 50 Gy doses. However, by increasing the dose to greater than 50 Gy, the cells located within the radiation field (500 mm) showed visible radiation-induced damage consistent with an apoptosis pattern, i.e. shrinking size and rounding shape with condensed cytoplasm (Larson & Banks, 2020 >). As seen in a phase contrast micrograph of MM418 (Fig. 16 >), after exposure to 1000 Gy dose, the cells within the radiation field 24 h post-exposure are demonstrating radiation-induced apoptosis. Conversely, both normal cell lines used in our study, which were exposed to the same SSB and SMB dose range, demonstrate no visible radiation-induced morphological changes up to 1000 Gy. These cells were observed for longer than for the cancer cells (96 h) to ensure any late cell apoptosis was not missed. It should be noted here that doses in such very narrow fields are much less effective than in broad beams - for instance, 50 Gy of a broad beam is sufficient to kill almost all cancer cells. Radiation-induced morphological changes in cancer and normal cells might be affected by their size compared with the radiation field size. It is known that cancer cells are normally presented in various sizes - they can be larger or smaller than normal cells (Eldridge, 2017 >). As seen in Fig. 17 >, there is a significant difference between cell sizes in normal cell lines, i.e. HEM and CCD841, and in the cancer cell lines, i.e. MM418 and SW48. Normal cell lines are about five times larger than their cancerous counterparts which may cause their different responses to the same radiation dose with the same radiation field size. The difference between the sizes of the cancer and normal cells and the radiation field size can be quantified by the following equation, Therefore, the number of cells within a unit length of the radiation field can be estimated as follows. For HEM: (~500)/500 1; for MM418: (~500)/100 5; for CCD841: (~500)/500 1; for SW48: (~500)/50 10. Based on the results, for normal cell lines (HEM and CCD841), on average, one cell can barely be covered or exposed in a 500 mm SBB radiation field and in the case of SMB this decreases to an even smaller fraction (~10%) of a cell body. This is significantly smaller than the population of cancer cells that can be exposed in the same field area. Therefore, the hit probability in the case of the normal cell's nucleus/DNA is lower due to the geometrical exposed cross section of the cells. In addition, different responses to the radiation in the normal cells compared with the cancer cells can be attributed to the difference in cell-division cycle checkpoints amongst normal and cancer cells. The G1 phase is involved in cell growth and synthesis of the required proteins and S phase involved in DNA replication. A cell in the G2 phase enters further growth and then starts the M phase in which it undertakes cell division (Pawlik & Keyomarsi, 2004 >). A cell's DNA is more radio-sensitive during the M (mitosis) phase and less at the end of the S and during the G2 phase. The normal cell lines (HEM and CCD841) that were used in this study exhibit longer doubling times (almost threefold longer) compared with their cancerous counterparts which means longer G1, S and G2 phases. Therefore, for these cells, there is a higher chance of being exposed during the most radio-resistant phases than for cancer cells. This in vitro study showed that, under the same AuNPs treatment regimen and SBB radiation dose, lethal damage to cancer cells is significantly more pronounced compared with to normal cells, which highlights the promising role of synchrotron-based X-rays in future radiotherapy. Figure 1 Setup for irradiating the cell samples with kilovoltage SR beams. (A) 25 cm2 flask filled completely with culture media, (B) SR (broad beam), (C) the cells attached to the flask's internal surface. Figure 2 Schematic diagram of the irradiation setup for SBB and SMB. (A) Cell sample, (B) broad beam slit, (C) microbeam collimator, (D) synchrotron pink-beam, (E) beryllium window. Figure 3 Relationship between the vertical speed of the sample (mm s-1) and delivered dose (Gy). Figure 4 Dose distribution in SBB measured by IMBL PTW microDiamond. The delivered dose was 100 Gy, and the radiation field was 500 mm. Figure 5 Radiation field exposed to 100 Gy of 90 kV SBB. (A) Dose profile plotted by ImageJ (c) and (B) GAFchromicTM HD-V2 film. Figure 6 Dose distribution in SMB measured by IMBL PTW microDiamond. The delivered dose was 100 Gy with peak (25 mm) and valley (175 mm). Figure 7 Radiation field exposed to 100 Gy of 90 kV SMB. (A) Dose profile plotted by ImageJ (c) and (B) GAFchromicTM HD-V2 film. Figure 8 Human lung cancer A549 monolayer cells 24 h after being exposed to SBB with field size of 500 mm. The radiation fields are marked between orange lines: (A) 50 Gy, (B) 100 Gy, (C) 500 Gy and (D) 1000 Gy. Figure 9 Human lung A549 cancer cells irradiated with 1000 Gy SBB. The radiation field is marked by orange dotted lines. (A1) Control (no AuNPs) 24 h, (A2) control (no AuNPs) 48 h, (B1) treated group (with 1 mM AuNPs) 24 h and (B2) treated group (with 1 mM AuNPs) 48 h post-irradiation with PBS wash. Figure 10 Human prostate cancer DU145 monolayer cells 24 h after being exposed to 500 mm SBB. The radiation fields are marked between orange lines. (A) 50 Gy, (B) 100 Gy, (C) 500 Gy and (D) 1000 Gy. Figure 11 Human prostate DU145 cancer cells irradiated with 1000 Gy SBB. The radiation field is marked by orange dotted lines. (A1) Control (no AuNPs) 24 h, (A2) control (no AuNPs) 48 h, (B1) treated group (with 1 mM AuNPs) 24 h and (B2) treated group (with 1 mM AuNPs) 48 h post-irradiation with PBS wash. Figure 12 Human epidermal melanocytes HEM and human primary melanoma MM418-C1 monolayer 24 h after being exposed to 1000 Gy 90 kVp SBB (width 500 mm). Both cell types are control with no AuNPs treatment. The boundaries of the radiation fields are marked with orange lines. (A1) HEM immediately after exposure, (A2) 24 h, (A3) 48 h and (A4) 96 h after exposure. (B1) MM418-C1 immediately after exposure, (B1) 24 h, (B2) 48 h and (B4) 96 h after exposure. Figure 13 Human epidermal melanocytes HEM and human primary melanoma MM418-C1 monolayer 24 h after being exposed to 1000 Gy 90 kVp SBB (width 500 mm). Both cell types are treated with 1 mM AuNPs. The boundaries of the radiation fields are marked with orange lines. (A1) HEM immediately after exposure, (A2) 24 h, (A3) 48 h and (A4) 96 h after exposure. (B1) MM418-C1 immediately after exposure, (B1) 24 h, (B2) 48 h and (B4) 96 h after exposure. Figure 14 Human primary colon epithelial CCD841 and human colorectal adenocarcinoma SW48 monolayer 24 h after being exposed to 1000 Gy 90 kVp SBB (width 500 mm). Both cell types are control with no AuNPs treatment. The radiation fields boundaries are marked with orange lines. (A1) CCD841 immediately after exposure, (A2) 24 h, (A3) 48 h and (A4) 96 h after exposure. (B1) SW48 immediately after exposure, (B1) 24 h, (B2) 48 h and (B4) 96 h after exposure. Figure 15 Human primary colon epithelial CCD841 and human colorectal adenocarcinoma SW48 monolayer 24 h after being exposed to 1000 Gy 90 kVp SBB (width 500 mm). Both cell types are treated with 1 mM AuNPs. The radiation fields boundaries are marked with orange lines. (A1) CCD841 immediately after exposure, (A2) 24 h, (A3) 48 h and (A4) 96 h after exposure. (B1) SW48 immediately after exposure, (B1) 24 h, (B2) 48 h and (B4) 96 h after exposure. Figure 16 Morphological changes in MM418 cells 24 h after they were irradiated with 1000 Gy. The area enclosed between orange lines is exposed to 90 kVp SBB. (A) Apoptotic cells in irradiated area. (B) Viable cells in unirradiated area. Figure 17 Phase-contrast 2D micrographs of HEM, MM418-C1, CCD841 and SW48. (A) HEM individual cell with average size/length of ~500 mm, (B) MM418-C1 individual cells with average size/length of ~100 mm, (C) CCD841 individual cell with average size/length of ~500 mm and (D) SW48 individual cells with average size of ~50 mm. Table 1 A summary of the results of microscopic observation 24 h post-irradiation of DU145, A549, HEM, MM418, CCD841 and SW48 in control and treated groups irradiated with various doses of SBB Cell type SBB dose (Gy) Experimental groups Morphological changes within the exposed area Cell gap created DU145 (prostate cancer) 50 Control No No 1 mM AuNPs No No 100 Control ~10% No 1 mM AuNPs ~10% No 500 Control ~50% No 1 mM AuNPs ~50% No 1000 Control Almost all cells Yes 1 mM AuNPs Almost all cells Yes A549 (lung cancer) 50 Control No No 1 mM AuNPs No No 100 Control ~10% No 1 mM AuNPs ~10% No 500 Control ~50% No 1 mM AuNPs ~50% No 1000 Control Almost all cells No 1 mM AuNPs Almost all cells No EM (skin normal) 50 Control No No 1 mM AuNPs No No 100 Control No No 1 mM AuNPs No No 500 Control No No 1 mM AuNPs No No 1000 Control No No 1 mM AuNPs No No MM418 (skin cancer) 50 Control No No 1 mM AuNPs No No 100 Control ~10% No 1 mM AuNPs ~10% No 500 Control ~50% No 1 mM AuNPs ~50% No 1000 Control Almost all cells No 1 mM AuNPs Almost all cells No CCD841 (colon normal) 50 Control No No 1 mM AuNPs No No 100 Control No No 1 mM AuNPs No No 500 Control No No 1 mM AuNPs No No 1000 Control No No 1 mM AuNPs No No SW48 (colon cancer) 50 Control No No 1 mM AuNPs No No 100 Control ~10% No 1 mM AuNPs ~10% No 500 Control ~50% No 1 mM AuNPs ~50% No 1000 Control Almost all cells No 1 mM AuNPs Almost all cells No References Bouchet, A., Lemasson, B., Christen, T., Potez, M., Rome, C., Coquery, N., Le Clec'h, C., Moisan, A., Brauer-Krisch, E., Leduc, G., Remy, C., Laissue, J. 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PMC10000795 | J Synchrotron Radiat J Synchrotron Radiat J. Synchrotron Rad. Journal of Synchrotron Radiation 0909-0495 1600-5775 International Union of Crystallography 36891863 ay5608 10.1107/S1600577522012127 JSYRES S1600577522012127 Beamlines Sample-minimizing co-flow cell for time-resolved pump-probe X-ray solution scattering Sample-minimizing co-flow cell Kosheleva Irina a* Henning Robert a Kim Insik a Kim Seong Ok b Kusel Michael c Srajer Vukica a a BioCARS, Center for Advanced Radiation Sources, The University of Chicago, 9700 South Cass Ave, Bld 434B, Lemont, IL 60439, USA b Department of Chemistry, Korea Advanced Institute of Science and Technology, E6-6 #513, 291 Daehak-ro, Daejeon, Yuseong-gu 34141, Republic of Korea c Kusel Design, 12 Coghlan Street, Niddrie, Wurundjeri Country 3042, Australia Amemiya Y. Editor University of Tokyo, Japan Correspondence e-mail: [email protected] 01 3 2023 01 2 2023 01 2 2023 30 Pt 2 s230200 490499 14 10 2022 22 12 2022 (c) Irina Kosheleva et al. 2023 2023 This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-BY) Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited. A full version of this article is available from Crystallography Journals Online. For irreversible or slow reactions studies by time-resolved pump-probe X-ray solution scattering, the standard sample cell and protocol for data collection at the BioCARS 14-ID beamline potentially require up to hundreds of milligrams of sample. A sheath co-flow cell, described here, allows such solution scattering measurements to be conducted with the sample consumption reduced by more than ten times compared with standard protocols at this beamline. A fundamental problem in biological sciences is understanding how macromolecular machines work and how the structural changes of a molecule are connected to its function. Time-resolved techniques are vital in this regard and essential for understanding the structural dynamics of biomolecules. Time-resolved wide-angle X-ray solution scattering has the capability to provide a multitude of information about the kinetics and global structural changes of molecules under their physiological conditions. However, standard protocols for such time-resolved measurements often require significant amounts of sample, which frequently render time-resolved measurements impossible. A cytometry-type sheath co-flow cell, developed at the BioCARS 14-ID beamline at the Advanced Photon Source, USA, allows time-resolved pump-probe X-ray solution scattering measurements to be conducted with sample consumption reduced by more than ten times compared with standard sample cells and protocols. The comparative capabilities of the standard and co-flow experimental setups were demonstrated by studying time-resolved signals in photoactive yellow protein. sheath co-flow cell time-resolved pump-probe X-ray solution scattering sample minimization National Institute of General Medical SciencesGM118217 Rama RanganathanUS Department of Energy, Office of ScienceDE-AC02-06CH11357 Advanced Photon SourceThe following funding is acknowledged: National Institute of General Medical Sciences (grant No. GM118217 to Rama Ranganathan); US Department of Energy, Office of Science (contract No. DE-AC02-06CH11357 to Advanced Photon Source). pmc1. Introduction Biomolecules undergo structural changes in response to modulations in their environment, for example temperature, concentration of protons (pH) or ions of metals, ligand binding, light absorption, electric field and other stimuli. Recording the molecular structural response as a function of time after such modulations allows the molecular structural intermediates during the reaction pathway, their time courses and free-energy landscape to be determined (Thompson et al., 2019 >; Ihee et al., 2005 >; Schmidt et al., 2013 >; Akiyama et al., 2002 >; Andersson et al., 2009 >; Kim et al., 2012a >). It is favourable to know the molecular structural response in most detail. Time-resolved crystallography provides atomic resolution structures of such molecular intermediates (Branden & Neutze, 2021 >; Srajer & Schmidt, 2017 >) but large functional structural changes may not be accessible due to crystal packing forces. In addition, the structural dynamics in the crystal may differ from the dynamics in the solution environment (Cho et al., 2016 >). X-ray solution scattering has the advantage of probing a wider range of structural dynamics in molecules. However, due to the random orientation of molecules in solution, their interference pattern is averaged azimuthally, so only radial structural information is preserved (Putnam et al., 2007 >; Koch et al., 2003 >). Macromolecules operate over large structural and temporal scales, from electron transfer processes on the sub-picosecond time scale to large conformational changes that may continue for hundreds of seconds and longer. Examples include protein folding and conformational dynamics (Wee et al., 2012 >; Srajer & Royer, 2008 >; Olmos et al., 2018 >; Hsu et al., 2021 >), ligand and RNA/DNA binding (Wee et al., 2012 >; Tokuda et al., 2018 >), signal transduction (Cho et al., 2016 >; Takala et al., 2014 >), electron transfer mechanisms (Rimmerman et al., 2018 >; Cammarata et al., 2008 >) etc. The most common way to study molecular structural dynamics in solution is by mixing components or changing the pH or ionic strength of the solvent etc. and following the reaction by using optical or X-ray scattering methods. If molecules of a solute or a solvent are photosensitive by nature or by design, the reaction can be initiated by a laser pulse, and then probed optically or by X-rays. This time-resolved technique is referred to as pump-probe X-ray solution scattering. Advances in improving the brilliance of X-ray sources, combined with fast detectors and technical advances in X-ray focusing, made possible the use of microfluidic mixing devices for studying molecular dynamics by time-resolved small-angle X-ray scattering (SAXS). Significant efforts have been made to improve laminar and turbulent mixers (Koster et al., 2008 >; Inguva et al., 2018 >; Kathuria et al., 2013 >; Mizukami & Roder, 2022 >; Ghazal et al., 2016 >, Park et al., 2006 >; Plumridge et al., 2018 >). The time resolution in both turbulent and laminar devices is limited by the diffusion time across the channel, and time series are collected by scanning the mixing cell across the X-ray beam. Advances in the design of turbulent mixers allowed the time resolution to be increased in mixing experiments to ~30-50 ms (Graceffa et al., 2013 >; Akiyama et al., 2002 >; Matsumoto et al., 2007 >). Turbulent mixing demands high flow rates to achieve breaking the liquid into small eddies (Graceffa et al., 2013 >) for fast mixing. As a result, turbulent mixing requires very high sample consumption. Therefore, most mixing experiments performed using this technique use commercially available samples. In laminar mixers, sample consumption is small compared with turbulent mixing. Typically, in laminar mixers, the central sample stream is focused hydrodynamically by a series of buffer streams. Laminar mixers require very narrow sample streams, typically only several micrometres in diameter, to decrease the diffusion time. Several types of laminar mixers have been developed by different groups (Brody et al., 1996 >; Pollack et al., 2001 >; Koster et al., 2008 >; Park et al., 2006 >; Plumridge et al., 2018 >). So far, sub-millisecond time resolution in laminar mixers has been achieved (Park et al., 2006 >), but the millisecond range is typical. Microfluidic mixers, turbulent or laminar, remain custom-built state-of-the-art devices, micro-machined, etched or printed (Ghazal et al., 2016 >; Lu et al., 2016 >, Plumridge et al., 2018 >). The reaction photo-initiation method employed in pump-probe time-resolved X-ray solution scattering experiments is distinctly different from the reaction initiation by mechanical mixing in mixing devices, either turbulent or laminar. Subsequently, the design requirements for those two types of apparatuses are also incisively different (Huyke et al., 2020 >). In this contribution, we discuss solely the pump-probe time-resolved X-ray solution scattering (TRXSS) technique. In TRXSS, a reaction in a molecule is triggered by an external short stimulus pump, often a laser pulse, and is probed by a delayed short X-ray pulse probe. Then the sample volume is refreshed and the process is repeated. We refer to the frequency at which the pump-probe is repeated as the repetition rate of the experiment. A complete time-resolved pump-probe experiment typically involves collecting scattering data at a number of time delays between pump and probe pulses. The length of the X-ray probe or the length of the laser pump, whichever is longer (typically the X-ray probe pulse is longer), defines the time resolution. At dedicated time-resolved pump-probe beamlines, a difference technique is used to measure scattering before and after reaction initiation with great accuracy and with a time resolution of 100 ps (at fourth generation synchrotron sources) or tens of femtoseconds (at X-ray free-electron lasers) (Kim et al., 2011 >, 2012a >; Cammarata et al., 2008 >; Lee et al., 2021 >; Levantino et al., 2015a >). To achieve the best time resolution at the BioCARS 14-ID beamline at the Advanced Photon Source (APS), USA, we use a picosecond or nanosecond laser pulse as a pump and a single polychromatic X-ray pulse of 100 ps duration as a probe. This permits a reaction to be probed in the time window of 100 ps to a few hundreds of nanoseconds. However, even with the high polychromatic X-ray flux at 14-ID, a single 100 ps X-ray pulse is not sufficient to achieve the necessary signal-to-noise ratio and a number of pump-probe cycles have to be repeated and data accumulated. For a typical experiment, one needs to accumulate data from thousands of single X-ray pulses per image and hundreds of images to achieve the signal-to-noise required to see differences in SAXS scattering curves. The standard capillary cell diameter used in such experiments at 14-ID at 12 keV is ~0.5-1 mm. For irreversible reactions or reactions with slow initial-state recovery such as enzymatic reactions, the sample has to be refreshed between pump-probe cycles. When experiments are conducted with a standard capillary flow cell, the sample consumption for the complete experiment can be as high as several grams of protein. This is an unsustainable amount for protein produced in a research lab. So far, most experiments for pump-probe TRXSS have been performed with X-ray radiation stable samples that undergo reversible reaction, such as photoactive yellow protein, cytochrome c, myoglobin etc. However, many biological reactions are irreversible or have very long relaxation times. To render those irreversible reaction studies possible, we have developed a sample-minimizing cytometry-style sheath co-flow device suitable for pump-probe TRXSS experiments. 2. Sheath co-flow cell description In the co-flow cell, a sample solute is injected through a tube or a needle into a capillary filled with a solvent. The co-flow-type cell has been widely used in Taylor dispersion analysis, cytometry, cell sorting or alignment, fluorescence flow-induced dispersion analysis, time-resolved spectroscopy mixing experiments and other applications (Ghazal et al., 2016 >; Buell & Jensen, 2021 >; Gang et al., 2018 >; Wang et al., 2014 >; Calvey et al., 2019 >; Hamadani & Weiss, 2008 >; Huyke et al., 2020 >). Co-flow mixing cell designs were also employed in time-resolved solution scattering mixing experiments (Pollack et al., 2001 >; Park et al., 2006 >, 2008 >; Plumridge et al., 2018 >). In static X-ray solution scattering experiments, a co-flow device was used for decreasing radiation damage of the samples during static SAXS measurements (Kirby et al., 2016 >). At BioCARS, the goal of introducing the co-flow cell design was to minimize sample consumption in time-resolved pump-probe X-ray solution scattering measurements. The cell has to satisfy several requirements. The sample and the buffer flow in the cell must be laminar in the range of velocities required in a typical pump-probe experiment with repetition rates up to 1 kHz. In addition, it has to be compatible with anaerobic sample studies and deliver stable sample streams of a variety of sizes, which match the sizes of the X-ray and laser beams at the beamline. The sample cell has to be robust in handling, made from parts available commercially and be compatible with BioCARS laser configurations (collinear and orthogonal to the X-ray beam), X-ray path and visualization optics. In partnership with Kusel Design (Australia), we designed and produced such a robust flow cell, which was integrated into the BioCARS 14-ID beamline. A drawing of the co-flow cell is shown in Fig. 1 >. The device has a modular design. The main body of the sample consists of a quartz capillary with diameter of 0.6 mm and wall thickness of 10 mm. The capillary inlet is connected to the upper chamber (A). The chamber houses a stainless-steel needle with ~200 mm orifice for sample delivery (B); the needle position has manual adjustment screws (C) which allow the centre of the needle to be aligned in the capillary. The upper chamber also connects to a buffer line (D) and a vent line (E). All connections accept standard 1/16-inch outer-diameter tubing fittings. The capillary outlet attaches to the bottom enclosure (F) and to a discard line (G). The connection of the sample cell capillary to the inlet and outlet enclosures are sealed with Viton O-rings. The whole assembly is mounted in a rigid stainless-steel enclosure (I) which has openings for access to pump laser beam, probe X-ray beam and viewing optics. The sample and the buffer are pumped in a controlled manner through the flow cell. An important feature of the design is that it constitutes a closed volume, essential in the experiments, which require an anaerobic sample environment (Rimmerman et al., 2018 >; Kim et al., 2011 >; Levantino et al., 2015b >). The flow dynamics of incompressible liquids in a co-flow capillary system are well understood (Lu et al., 2016 >). When fluid is incompressible, has constant viscosity and is in a laminar regime, the liquid flows in parallel layers without interaction between them, and each layer flows with different velocity along the same direction. Neglecting the effects of gravity, one can obtain equations for the buffer and sample flow velocities (Landau & Lifshitz, 1987 >), where R is the radius of the capillary, R s is the radius of the sample core, ms is the sample viscosity, mb is the buffer viscosity, and p/z is the pressure drop in the capillary. Then the ratio of buffer to sample flow rates (Q b and Q s) follows the simple equation In time-resolved pump-probe experiments, the X-ray pulse has to probe the sample volume in which the reaction was initiated by the laser pulse. To ensure the most homogeneous laser pulse exposure of the sample volume probed by the X-ray pulse, the laser beam size is normally slightly larger than the X-ray beam size. Therefore, the average linear speed of the sample refreshment is defined by the laser spot size and the time between the pump-probe cycles (repetition rate of an experiment). As seen from equations (1) and (2), the linear sample speed and sample core diameter uniquely define the buffer linear speed. Fig. 2 >, top panel, shows calculated speeds for sample and buffer solutions that are needed for sample refreshment for typical repetition rates. Speeds are shown as a function of the distance from the centre of the capillary. The calculations were performed for the 0.6 mm capillary device and a target 0.3 mm sample core diameter, 0.25 mm laser beam size, and repetition rates ranging from 1 Hz to 1 kHz. In the calculations, viscosities of the sample and the buffer are assumed to be equal and gravity is neglected. If the gravitational force is parallel to the direction of the flow, differences in density of the sample solution and buffer solute cause small corrections to calculated values (Giorello et al., 2020 >). In the case when the gravitational force is perpendicular to the direction of the flow, the result may be complicated. For small molecules, this may cause mixing in the vertical direction, as a function of distance from the sample injection. For the time-resolved pump-probe measurements, it is important that the flow remains laminar and there is no intermixing between the layers. In this regard, an important characteristic of the device is the Reynolds number, Re. When Re is low, viscous forces dominate and liquid moves in a laminar manner. If Re is greater than ~2 x 103 (Squires & Quake, 2005 >) the flow becomes turbulent. For the velocities shown in Fig. 2 >, and assuming a kinematic viscosity of water at 20degC of ~1 mm2 s-1, the co-flow device has an Re value below 200; therefore, the laminar condition is well satisfied. With low sample speeds, diffusion will play a larger contribution in the accuracy of time-resolved pump-probe measurements. The translational diffusion coefficient is typically 10-5 cm2 s-1 for small molecules and 10-7 cm2 s-1 for large macromolecules. For lower repetition rates, and therefore small sample linear speeds, the diffusion length for species with high diffusivity may become comparable with the sample core size, effectively causing sample dilution. This dilution does not constitute a problem if the sheath buffer is matching the protein buffer and if dilution does not change the solute-solvent and solute-solute interactions. In a co-flow cell capillary, only the buffer is in contact with the capillary walls and only the buffer velocity approaches zero. Under such conditions, there is no capillary wall fouling and there is no sample radiation damage in the proximity of the capillary walls (Kirby et al., 2016 >). Fig. 2 >, lower panel, shows the time required to replenish the liquid sample inside the capillary as a function of the distance from the capillary centre. The time was calculated for the 0.6 mm capillary and a laser beam size of 0.25 mm at repetition rates of 1 Hz and 1 kHz. In laminar flow devices, the sample refreshment time at the capillary wall approaches infinity so the sample is not refreshed between the pump-probe cycles. This portion of the sample experiences multiple pump-probe cycles during the data collection. Fig. 2 > shows that as much as ~15% of the total sample in the proximity of the capillary wall may not be properly refreshed. This portion of the sample experiences radiation damage and contributes to the low scattering vector region of the difference signal. In addition, if the relaxation time of the molecular reaction is longer than the repetition rate of the experiment, the sample will not be excited by the laser pump and therefore will be excluded from the reaction course. Both contributions will change the magnitude and the time course of the measured time-resolved structural changes. Although these have not caused issues for most experiments at BioCARS, the effects were observed in some cases. In the co-flow device, because the sample occupies only the central portion of the capillary, these effects are significantly minimized, or eliminated. 3. Data collection To characterize the co-flow cell performance, we compared time-resolved scattering data for the photoactive yellow protein (PYP) collected with the co-flow cell and with a standard flow cell. PYP is a small photoreceptor protein. The photocycle reaction in PYP starts with absorption of blue light by a pCA chromophore molecule and isomerization of the chromophore from trans to cis. The structural signal then propagates through multiple intermediate states, which are well characterized spectroscopically and structurally (Ihee et al., 2005 >; Kim et al., 2012a >; Schotte et al., 2012 >; Schmidt et al., 2013 >; Meyer et al., 1987 >; Ujj et al., 1998 >). The sample in this study was 1.5 mM PYP solution in a buffer of 20 mM Tris, pH 7.0, 150 mM NaCl. PYP was prepared as previously described (Yang et al., 2017 >). For the co-flow cell data collection, the matching 20 mM Tris, 150 mM NaCl buffer at pH 7.0 was used. Time-resolved pump-probe X-ray solution scattering data were collected at BioCARS 14-ID-B beamline at the APS, in standard 24-bunch operating mode of the storage ring. The beamline has two pairs of Kirkpatrick-Baez mirror systems capable of focusing X-ray beam down to ~15 mm x 20 mm (V x H) at the sample position, and timing shutters which can deliver on demand a single 100 ps X-ray pulse or a longer X-ray pulse sequence, with frequency of up to 1 kHz (Graber et al., 2011 >). In standard time-resolved solution scattering configuration at 14-ID-B, polychromatic X-ray beam is used at peak energy 12 keV and bandpass of ~2.5%. A single 100 ps polychromatic X-ray pulse delivers 5 x 109 photons. Standard data collection requires 50-100 images per one time delay, with approximately 2000 single 100 ps X-ray pulses per image. For such data collection, sample consumption can be as high as 20-40 ml per one time delay when a standard diameter flow cell is used. Because the velocity of the sample replacement at the walls of the capillary approaches zero, it is not uncommon to expend even more sample to ensure proper sample replacement between pump-probe cycles. The sample environments of the experiment in the standard flow cell and co-flow cell data collection modes are shown in Fig. 3 >. An Opotek nanosecond laser with 7 ns pulse duration tuned to 450 nm was used to initiate the reaction in the sample. The X-rays were focused to a spot size of 35 mm x 35 mm (FWHM). The scattering pattern was recorded using a Rayonix HS-340 detector in 2 x 2 binning mode (pixel size 88.6 mm). A cone purged with He gas was mounted on the face of the detector to minimize air scattering. Solution scattering was measured in the range of the scattering vector q = 4p sin(th)/l (where 2th is the scattering angle and l is the X-ray wavelength) from 0.02 A-1 to 2.8 A-1. To enable direct comparison between the measurements, data collection in standard mode and co-flow mode were carried out at the same repetition rates of 20 Hz and 5 Hz. At 5 Hz we collected a time delay series consisting of longer pump-probe delays: -10 ms, -5 ms, 5 ms (standard mode only), 50 ms (co-flow mode only), 500 ms, 5 ms, 50 ms and 150 ms. The 5 Hz repetition rate (200 ms between pump-probe sequences) for these time delay series was dictated by the longest time delay in the series (150 ms). Given that we probed longer pump-probe time delays, we also used longer X-ray exposures as probes. Rather than a single 100 ps X-ray pulse, we used an X-ray probe consisting of 24 consecutive 100 ps X-ray pulses, with total duration of 3.6 ms. We refer to this probe as a 24-pulse X-ray exposure. At a faster 20 Hz repetition rate we collected shorter pump-probe time delays: -5 ms, -10 ms, 5 ns (co-flow mode only), 50 ns (standard mode only), 500 ns and 500 ms. The short time delays in this series required using a single 100 ps X-ray pulse as a probe (using several consecutive X-ray pulses as a probe was not possible in this case as pulses are spaced at 150 ns in the APS storage ring mode we used). We refer to this probe as a single-pulse X-ray exposure. A repetition rate of 20 Hz was determined by the maximum repetition rate of the Opotek nanosecond laser we used. We would like to emphasize here that, for both 5 Hz and 20 Hz data collections, one pump-probe cycle and therefore one X-ray exposure (as defined above for two repetition rates) is not sufficient for recording an image with acceptable signal-to-noise level. Pump-probe cycles have to be repeated and accumulated prior to an image readout. Details of how this is accomplished for standard and co-flow cells are described in Sections 3.1 and 3.2, respectively. Laser-OFF images were obtained at -5 ms and -10 ms time delays, meaning that first the X-ray image was collected and then the laser pulse arrived after a specified time delay. X-ray solution scattering is known to be very sensitive to temperature, with a significant temperature structural signal contribution at high scattering angles, reflecting changes in interatomic distances between the solvent molecules. We use laser-OFF negative time delays to keep capillary temperature conditions as close as possible to the laser-ON pump-probe measurements and to minimize possible experimental errors due to capillary local temperature instability. During initial data reduction, geometrical and polarization corrections were applied; then images were integrated radially to obtain 1D scattering curves and normalized on the isosbestic point of the solvent in the scattering vector q range 1.4-1.65 A-1. For each laser-ON pump-probe scattering curve, the appropriate laser-OFF scattering curve was subtracted. This difference technique mitigates experimental errors caused by the slow changes in experimental conditions such as small temperature drifts, beam positions instabilities etc. The common time delays were used to scale data collected at different frequencies. 3.1. Standard data collection mode In standard data collection mode, the nanosecond laser was focused to an elliptical spot size of 120 mm x 500 mm (FWHM), with power density of 1 mJ mm-2 and the shorter axis of the elliptical spot along the length of the capillary. Polychromatic X-ray beam was used in the direction perpendicular to the laser beam and the direction of the capillary cell. The laser and X-rays were aligned to overlap spatially at the sample position. In this setup we used a standard flow cell of 0.5 mm-diameter quartz capillary and a wall thickness 10 mm. The observed structural signal was maximized by collecting data at 0.15 mm from the top of the capillary. To minimize radiation damage of the sample and to ensure full sample recovery between the pump-probe cycles, the cell was translated through the X-ray beam with step size 0.25 mm at 5 Hz or 20 Hz repetition rates (see details below). After the capillary scan, 20 ml of sample in the capillary cell was refreshed, and the process was repeated until the desired quantity of the total X-ray pulses per image was achieved. Then the image was read by the detector. In total, the number of single 100 ps X-ray pulses per image equals the product of the number of single X-ray pulses in an X-ray exposure used for a pump-probe step, the number of pump-probe steps per capillary scan, and the number of capillary scans per image. For shorter time delays, we used a 20 Hz repetition rate, a single-pulse exposure per pump-probe step and 107 pump-probe steps per capillary scan. After the capillary scan, the sample was replenished. This procedure was repeated ten times before the image was finally read. In this case, the total number of single 100 ps X-ray pulses per image was therefore 1 single pulse per exposure x 107 pump-probe steps per capillary x 10 capillary scans. Therefore 1070 single 100 ps X-ray pulses were used per image, with a total of 10 x 20 ml = 200 ml of sample solution per image. For 5 Hz data collection, we used a 24-pulse exposure per pump-probe step (see description in Section 3) and also used 107 pump-probe steps per capillary scan. In this case the image was read after each scan. The total number of single 100 ps X-ray pulses per image was 24 single pulses per exposure x 107 pump-probe steps per capillary, and therefore 2568 single 100 ps X-ray pulses per image. In this case 20 ml of sample per image was used (see Table 1 > for details). For each time delay, we collected and averaged 50 images at both repetition rates. 3.2. Sheath co-flow cell data collection mode For co-flow data collection, given the vertical mount of the co-flow cell, the laser light was delivered in near-collinear geometry, at ~15deg with respect to the X-ray beam direction. The laser was focused into a 150 mm x 150 mm spot with laser power density of 1 mJ mm-2. The sample solution and the buffer were pumped through the capillary cell using a CP Dual Syringe (Cole-Parmer) which allows two liquids with different flow rates to be delivered simultaneously. The sample solution was injected through a needle in the centre of the co-flow cell while buffer was delivered through the buffer port as described above. To avoid sample flow instabilities, the solutions were degassed by bubbling nitrogen through the liquids for 20 min prior to data collection. The co-flow cell was used in the vertical geometry so the gravity force was collinear with the directions of sample and buffer flows. The flow rate of the sample delivery pump was chosen so that fresh sample was delivered at each pump-probe cycle, and the targeted sample core was 300 mm in diameter. The buffer flow rate was calculated using equation (3). The sample core size was measured by scanning the capillary horizontally across the beam and measuring the SAXS signal, and then the capillary was centred. As for the standard cell, for shorter time delays we used a 20 Hz repetition rate, but in this case with 2000 pump-probe cycles of single X-ray pulse exposures (see Section 3). For longer time delays, we used 5 Hz repetition rate with a 24-pulse exposure (see Section 3) and 107 exposures per image, so the total number of single 100 ps X-ray pulses per image was 2568. As for the standard data collection, for each time delay we collected and averaged 50 images at both repetition rates. For 5 Hz data collection, the sample flow rate was set to 5.45 ml min-1 and the buffer flow rate to 10 ml min-1. For 20 Hz data collection, the sample flow rate was 20.57 ml min-1 and the buffer flow rate was 28 ml min-1. The sample core was measuring ~270 mm for the 5 Hz and ~300 mm for the 20 Hz repetition rates. The measurements were performed at approximately 12 mm from the sample injection point. To ensure that the X-ray beam probes the sample volume excited by the laser beam, the laser beam position was calculated and offset for each time delay by data collection software. During switching of the frequency for the data collection in the co-flow cell, the sample and the buffer flow rates must change accordingly. During such switching, small variations in the sample core diameter are possible and the probed sample volume can change slightly. To account for the possible change of the X-ray path length in the sample at different repetition rates, a common data point at 500 ms time delay was collected to ensure proper scaling of the data. A summary of the data collection parameters for the standard and co-flow cells is shown in Table 1 >. The table presents the repetition rate of the experiment, time delays used in the experiment, details of the sample refreshment, number of 100 ps X-ray pulses per image and sample volume consumption per image. As described in Section 3, for each recorded image we used repeated pump-probe cycles with single-pulse X-ray exposures for 20 Hz data collection (short pump-probe time delays) and 24-pulse X-ray exposures for 5 Hz data collection (longer pump-probe time delays). At both repetition rates for each time delay we collected and averaged 50 images for both standard cell and co-flow cell. 4. Results and discussion Fig. 4 > shows difference signals qDS(q,t) as a function of scattering vector q. The difference signal was obtained by subtracting the laser-OFF reference scattering curve measured at -5 ms time delay from the scattering curves with the laser-ON positive time delays, containing the structural signal. The top panel shows difference data collected with the standard flow cell, and the middle panel shows difference data collected with the co-flow cell at different time delays. The data are presented on the same scale for direct comparison and are offset for clarity. The error band in the data represents root mean square deviation (RMSD) errors, obtained from averaging the data at each time delay. No median filters, sorting or outlier rejections were applied in the process of data averaging. The signal-to-noise level for the data collected at 20 Hz (blue lines) at high scattering vectors (q > 0.4 A-1) is smaller in the standard flow cell data than in other difference scattering curves. At 20 Hz, we performed measurements which demand single 100 ps X-ray pulse exposures. With the standard cell, these measurements take a long time because the sample has to be replenished multiple times in the capillary cell between the cell scans. Due to beam time limitations, the total number of 100 ps X-ray pulses per image that we used for the standard cell at 20 Hz was by a factor of >=2 smaller compared with other data collection modes (see Table 1 >). In particular, with the standard flow cell at 20 Hz we used 1070 single 100 ps X-ray pulses per image (see Section 3.1), compared with 2000 single 100 ps X-ray pulses per image for the co-flow cell (see Section 3.2; for other details see Table 1 >). At low scattering vectors (q < 0.3 A-1) for the co-flow cell the RMSD errors of the averaged difference scattering curves are larger than in the standard cell measurements (Fig. 4 >). The signal-to-noise at lower scattering vectors is on average about four times larger for the standard cell than for the co-flow cell. We calculated the mean of the RMSD errors for averaged difference scattering curves for the 0.03 A-1< q < 0.3 A-1 region and determined that in this q region RMSD errors for all co-flow measurements exceed the RMSD errors in standard measurements by about two times. This is higher than expected, based on the differences in the sample volume in the two setups. We attribute this additional noise in the co-flow cell measurements to instabilities in the delivery of solutions caused by the syringe pump, which we used in the measurements. Despite this fact, the averaged scattering difference curves collected with both sample cells agree very well. The bottom panel of Fig. 4 > compares difference scattering curves for 500 ns, 500 ms and 5 ms time delays. The curves for different time delays are offset for clarity. To emphasize the agreement between difference scattering curves from standard cell and co-flow cell measurements, the curves are presented using different absolute scales, shown on the left and right y axes. The scale on the left y axis corresponds to difference scattering curves measured in the standard cell axis. The scale on the right y axis corresponds to difference scattering curves measured in the co-flow cell. We observe excellent agreement in the pairs of difference scattering curves collected in the standard and co-flow configurations at the same time delays. In Fig. 4 >, normalized time-resolved difference scattering signals for the co-flow cell are two times smaller than those for the standard cell. Such a difference is the result of the data normalization procedure. A standard procedure for the data normalization in TRXSS is normalization using the isosbestic region of the solvent q range of 1.4-1.6 A-1. In the co-flow cell, all molecules of the buffer solvent, from the sample solution and from the sheath buffer, contribute to such signal. Therefore, normalized in this way, the structural signal appears proportionally smaller. In general, this is not a problem for the further data analysis. The scattering fingerprints of the intermediates are obtained by kinetic modelling and global analysis of the time-resolved scattering data (Cho et al., 2016 >). A static scattering pattern of the protein ground state should be measured during the time-resolved (standard or co-flow) experiment. Then absolute scattering curves of the intermediates are acquired in the regular manner by adding the protein ground state scattering pattern and difference scattering pattern of the intermediate scaled for 100% photoexcitation (Cho et al., 2016 >). Subsequently, the protein shape is modelled by using regular methods of static SAXS, or molecular dynamics simulations methods. Time-resolved scattering data were collected at only seven time delays, since detailed structural analysis of the PYP intermediate states was not the goal. However, we still performed global kinetic modelling of the available data for comparison purposes. For our analysis, we limited the scattering data to the q range 0.02-1.0 A-1, the portion of the scattering curve where scattering is determined by structural changes of protein and protein-solvent interactions. For data analysis we used the ReactLab Kinetics program which is capable of modelling kinetic reactions of proteins of first and second order and is available commercially ). The result of the SVD analysis for both data collections indicated the presence of three intermediates. We modelled the data by following (Cho et al., 2016 >) and applying a serial kinetic model A - B - C - D where A, B and C are intermediate species in the course of the reaction and D is the final ground PYP state. The population courses and difference scattering patterns of the reaction intermediates obtained by the kinetic modelling are shown in Fig. 5 > (in the bottom panels for the standard cell and in the top panels for the co-flow cell). Both data protocols result in very similar scattering patterns of the intermediates, similar time constants and population courses. In particular, from global kinetic analysis, the time constants between states in the reaction A - B - C - D are comparable for the standard flow cell and the co-flow cell: 10 ms, 1.96 ms, 150 ms and 70 ms, 2.2 ms, 250 ms, respectively. The differences in the time constants are likely attributed to sparse time-delay points and a minor difference is the time-delay sequence in the collected time domain (Table 1 >). Overall, the difference scattering patterns of the intermediates and their population's time courses are in agreement with those observed in detailed time-resolved SAXS studies of the PYP photocycle (Cho et al., 2016 >; Kim et al., 2012b >). The final signalling state is observed with a relaxation time of 250 ms in the co-flow cell and 150 ms in the standard cell experiment. Those values are comparable with the relaxation time of the signalling state of 280 ms in the work of Cho et al. (2016 >), where a sequential model was also used to describe the PYP dynamics. The most important comparison of the two devices is sample consumption. For the data collection described above, 57 ml of sample was used with the standard flow cell (7 ml for 5 Hz and 50 ml for 20 Hz data collection) and 9.5 ml was used for the co-flow cell (660 ml for 5 Hz and 8.8 ml for 20 Hz data collection). A comparison of the calculated sample consumption in a typical time-resolved pump-probe solution scattering experiment for capillaries of different sizes and for the co-flow device is presented in Table 2 >. We show the calculated sample consumption for the standard cell capillary experiment in orthogonal laser beam/X-ray beam geometry for measurements conducted at 0.15 mm from the top of the capillary cell and compare it with the sample consumption of the co-flow cell. Both are for 20 Hz data collection used for short time delays and more demanding in terms of sample consumption (see Table 1 >). Owing to concerns about replenishing a liquid in the proximity of the capillary wall for the standard cell, we adopted the methodology of scanning the capillary through the series of pump-probe cycles, and then replenishing the sample (see also Section 3.1). To ensure maximum efficiency of the sample replenishing and to mitigate scattering structural difference errors due to the sample velocity profile at the proximity of the capillary wall, the pumped sample volume is typically two times larger than the calculated volume of the capillary cell device. In Table 2 >, column 5, we show the sample volumes per image in the standard cell and co-flow cell configurations. In column 6, we show the ratio of the sample volume per image in the standard cell and co-flow cell configurations. For the calculations in column 6, the co-flow sample volume was adjusted to account for X-ray sample path differences in the capillaries and in the co-flow cell, and to achieve identical total X-ray sample path per image in both devices. The experimental ratio values are substantially larger than the theoretical gains due to the adopted data collection protocol as described above. 5. Conclusions We present a novel design of a sheath co-flow cell for minimizing sample consumption in time-resolved pump-probe X-ray solution scattering measurements. This design makes it feasible to study protein structural dynamics for irreversible reactions. By using photoactive yellow protein, we demonstrated that standard data collection at BioCARS 14-ID beamline and data collected with the sheath co-flow cell give similar difference scattering patterns for reaction intermediates and similar time constants from the kinetic analysis. We show that the co-flow cell uses significantly less sample than the standard capillary cell employed in BioCARS time-resolved pump-probe X-ray solution scattering experiments. Identical sample X-ray paths and exposures in both configurations can result in data with similar signal-to-noise levels providing improved stability of the pumps that deliver sample solution and sheath solvent buffer. Further advances can be achieved by improving the focusing X-ray optics and decreasing the X-ray and the laser beam sizes, and by utilizing novel integrating detectors with improved X-ray sensitivity. 6. Data availability Authors will make available images for time-resolved measurements with photoactive yellow protein, averaged difference scattering patterns and results of the kinetic modelling upon reasonable request. We would like to thank Nigel Kirby, Timothy Ryan and Nathan Cowieson for the helpful discussions. Time-resolved set-up at Sector 14 was developed in part through a collaboration with Philip Anfinrud (NIH/NIDDK). Figure 1 Drawing of the co-flow cell for time-resolved pump-probe X-ray solution scattering Figure 2 Velocity (top panel) and time of sample refreshment (bottom panel) as functions of distance from the centre of the capillary are shown for different repetition rates of the experiment. For the co-flow cell a buffer, velocities of which are shown in blue, occupies the outer portion of the capillary cell. Figure 3 Sample environments for the standard flow cell (left panel) and co-flow cell (right panel). Figure 4 Difference signal qDS(q,t) as a function of scattering vector q, measured in the standard cell (top panel) and co-flow cell (middle panel). Black lines represent data collected at 5 Hz, blue lines those at 20 Hz. The bottom panel compares the shapes of difference scattering curves for the co-flow (black, right y axis) and standard (red, left y axis) data collections at selected time delays. Figure 5 Right panels show difference scattering patterns as a function of scattering vector for PYP reaction intermediates A (blue), B (red), C (black): co-flow cell (top, right), standard cell (bottom, right). Time courses for the intermediate populations are shown in the left panels: co-flow cell (top, left), standard cell (bottom, left). The population time course of the ground state D is shown by the dashed lines. The difference scattering curve of the ground state D is equal to zero and is not shown. Table 1 Summary of data collection parameters in standard and co-flow data collection modes Standard data collection Repetition rate (Hz) Time delays Sample refreshment method Capillary scans per image 100 ps X-ray pulses per image Sample volume per image (ml) 5 -10 ms, -5 ms, 5 ms, 500 ms, 5 ms, 50 ms, 150 ms Capillary scan, then refresh 20 ml 1 2568 20 20 -5 ms, -10 ms, 50 ns, 500 ns, 500 ms Capillary scan, then refresh 20 ml 10 1070 200 Co-flow data collection Repetition rate (Hz) Time delays Sample refreshment method 100 ps X-ray pulses per image Sample volume per image (ml) 5 -10 ms, -5 ms, 50 ms, 500 ms, 5 ms, 50 ms, 150 ms Flow 2568 1.89 20 -5 ms, -10 ms, 5 ns, 500 ns, 500 ms Flow 2000 35.3 Table 2 Calculated sample consumption in standard cell and co-flow cell data collection Capillary diameter (mm) Laser beam geometry relative to X-ray beam X-ray path (mm) Volume per image at 20 Hz (ml) Experimental V st/V co per image, corrected Standard scan 1 Orthogonal 0.15 mm depth 0.714 1800 21.5 Standard scan 0.5 Orthogonal 0.15 mm depth 0.45 400 7.6 Standard scan 0.3 Orthogonal 0.15 mm depth 0.3 140 4 Co-flow 0.3 Pseudo-collinear 0.3 35 1 References Akiyama, S., Takahashi, S., Kimura, T., Ishimori, K., Morishima, I., Nishikawa, Y. & Fujisawa, T. 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PMC10000796 | J Synchrotron Radiat J Synchrotron Radiat J. Synchrotron Rad. Journal of Synchrotron Radiation 0909-0495 1600-5775 International Union of Crystallography 36891861 ok5084 10.1107/S160057752300019X JSYRES S160057752300019X Beamlines MAXPEEM: a spectromicroscopy beamline at MAX IV laboratory MAXPEEM: a spectromicroscopy beamline at MAX IV laboratory Niu Yuran a Vinogradov Nikolay a Preobrajenski Alexei a Struzzi Claudia a Sarpi Brice a Zhu Lin a Golias Evangelos a Zakharov Alexei a* a MAX IV Laboratory, Lund University, Box 118, 22100 Lund, Sweden Kvashnina K. Editor ESRF - The European Synchrotron, France Correspondence e-mail: [email protected] 01 3 2023 03 2 2023 03 2 2023 30 Pt 2 s230200 468478 27 10 2022 09 1 2023 (c) Yuran Niu et al. 2023 2023 This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-BY) Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited. A full version of this article is available from Crystallography Journals Online. The design and performance of a new dedicated spectromicroscopy beamline at the Swedish national synchrotron (MAX IV) is presented. MAXPEEM, a dedicated photoemission electron microscopy beamline at MAX IV Laboratory, houses a state-of-the-art aberration-corrected spectroscopic photoemission and low-energy electron microscope (AC-SPELEEM). This powerful instrument offers a wide range of complementary techniques providing structural, chemical and magnetic sensitivities with a single-digit nanometre spatial resolution. The beamline can deliver a high photon flux of >=1015 photons s-1 (0.1% bandwidth)-1 in the range 30-1200 eV with full control of the polarization from an elliptically polarized undulator. The microscope has several features which make it unique from similar instruments. The X-rays from the synchrotron pass through the first beam separator and impinge the surface at normal incidence. The microscope is equipped with an energy analyzer and an aberration corrector which improves both the resolution and the transmission compared with standard microscopes. A new fiber-coupled CMOS camera features an improved modulation transfer function, dynamic range and signal-to-noise ratio compared with the traditional MCP-CCD detection system. X-ray photoelectron microscopy ACLEEM micro-ARPES XMCD Vetenskapsradet2013-44950-102300-94 This work has been financially supported by the Swedish Research Council (the transfer package project; grant no. 2013-44950-102300-94). pmc1. Introduction In the last decades, photoemission combined with high spatial resolution has seen remarkable growth (Bauer, 2014 >). Photoemission electron microscopy (PEEM) is an important surface characterization tool. Similar to conventional optical microscopy, it employs full-field microscopy, which utilizes photo-exited electrons to form the image. Owing to major instrument development in the last few decades, especially with the incorporation of low-energy electron microscopy (LEEM), energy filters and synchrotron light sources, PEEM has become a powerful instrument that can provide various contrast mechanisms, multiple operation modes, and complementary measurement capabilities with spatial, momentum, energy and temporal resolutions. Synchrotron-based PEEM (XPEEM) has been extensively used in a wide range of disciplines, e.g. materials science, nano-science, heterogeneous catalysis, corrosion science, biology and biomineral science, to image structural, chemical, electronic and magnetic properties of the surface and nanostructures (Shi et al., 2018 >; Laverock et al., 2018 >; Ali-Loytty et al., 2018 >; Bommanaboyena et al., 2021 >; Rullik et al., 2016 >; Hjort et al., 2014 >; Fitzer et al., 2016 >; Metzler et al., 2007 >). To perform such measurements with single-digit nanometre resolution, one has to design a beamline with high photon density flux and install a state-of-the-art low-energy electron microscope with a high-resolution detection system. In this paper, we outline the design and characteristics of the MAXPEEM beamline which houses an aberration-corrected low-energy electron microscope and demonstrate its performances. The aberration corrector not only allows the user to improve the spatial resolution but also increases the microscope transmission. In turn, the higher transmission enables the user to decrease the total flux on the sample, thus diminishing the space-charge effect and beam damage at the surface. The MAXPEEM beamline is located at the 1.5 GeV synchrotron ring of the MAX IV laboratory, Sweden. Many synchrotron facilities in the world have PEEM beamlines but only a few are equipped with an aberration corrector (AC). At Diamond Light Source (beamline I06; Dhesi et al., 2010 >), an aberration-corrected low-energy electron microscope (AC-LEEM) has recently been installed but no performance data are available yet. At NSLS II (Reininger et al., 2012 >), which hosts a similar instrument, the main difference is the spot size (50 mm) and grazing incidence illumination. At Shanghai Synchrotron Radiation Facility (Xue et al., 2014 >), an aberration-corrected photoemission electron microscope (AC-PEEM) is used for time-resolved photoelectron microscopy (Li et al., 2019 >). There are also two non-commercial aberration-corrected PEEM instruments: one is SMART type at BESSY II [a complete version with LEEM function and a dedicated Omega-type energy filter (Schmidt et al., 1998 >)] and the other one is PEEM3 at the Advanced Light Source (beamline 11.0.1.1). Among all these microscopes MAXPEEM is unique due to the small photon beam spot size, which enables higher spatial resolution measurements. Another feature of the MAXPEEM beamline is the normal incidence geometry of the incoming photon beam. 2. Beamline overview The MAXPEEM beamline consists of an elliptically polarizing undulator as a light source, a collimated plane-grating monochromator with corresponding focusing and refocusing optics, and aberration-corrected spectroscopic photoemission and low-energy electron microscope (AC-SPELEEM) as the endstation. 2.1. Insertion device The source for the MAXPEEM beamline is an elliptically polarizing undulator (EPU) of Apple-II type (Sasaki et al., 1993 >). The insertion device is called EPU58 and has 42 periods and a period length of 58 mm. The maximum radiated power is 1.46 kW (planar phase, 500 mA ring current). The estimated maximum flux into the beamline is >=1015 photons s-1 (0.1% bandwidth)-1. The undulator was first tuned and characterized on a magnetic bench prior to installation on the ring. Under normal operation, the undulator demonstrates that its performance is in very good agreement with simulations [Fig. 1 >(a)] except for a small energy shift attributed to the imperfection in the mutual alignment of the insertion device and the beamline (Tanaka & Kitamura, 2001 >). The measured undulator spectrum of the first harmonics effectively reproduces interference fringes [inset, Fig. 1 >(a)]. The fundamental harmonics (linearly polarized light) cover the photon energy range 30-350 eV. When the EPU58 is tuned to produce circularly polarized light only the first harmonic has intensity on the optical axis and covers the photon energy range 40-300 eV. For higher harmonics, as shown in Fig. 1 >(b), the undulator must be tuned to produce elliptically polarized light and the degree of circular polarization will be less than 100%. The undulator also works in the inclined mode, in which it is possible to produce linearly polarized light at an arbitrary azimuth angle, i.e. from -90deg to +90deg. The degree of polarization in the helical mode as well as the linear polarization direction in the inclined mode have been characterized by a multilayer-based soft X-ray polarimeter (Grizolli et al., 2016 >). For example, Fig. 1 >(c) shows that, in the inclined mode, as the multilayer analyzer rotates around the optical axis, the X-ray intensity follows a sine function nicely. The measured angles of the polarization are consistent with the simulated angles. 2.2. Optical layout The optical layout of the MAXPEEM beamline is shown schematically in Fig. 2 >. The first optical element in the beamline is a cylindrical mirror (M1) at 15000 mm after the EPU source. The mirror collimates the beam vertically before the monochromator. The size of M1 determines the acceptance of the beamline, which is selected to be 0.80 mrad (H) x 1.36 mrad (V). Horizontal acceptance is chosen to be relatively large, 8s (s = 0.1 mrad is the standard deviation of the angular opening of the first undulator harmonic at the lowest photon energy of 30 eV). For accepting 99.7% of the monochromatic radiation a fan of 6s would be sufficient, but even larger horizontal acceptance makes the thermally induced deformation smoother in the central area, which is irradiated by the light of interest. The need for full illumination of M1 determines its length to be 350 mm. The high vertical acceptance follows from the fact that it is difficult to make a mirror narrower than 20 mm, and at a distance of 15000 mm this corresponds to 1.36 mrad. The water-cooled apertures in front of the monochromator can reduce both horizontal and vertical acceptance further. The monochromator is SX-700 PGM from the beamline I311 at the old MAX laboratory; it consists of a plane mirror (M2) and three interchangeable plane gratings (PGs). The dispersed radiation from the grating is focused both vertically and horizontally at the exit slit by a toroidal focusing mirror (M3). This mirror has to accept the fan of 6s (because heat deformations are not as important as they are for M1), setting the length of M3 to 320 mm. The final refocusing is accomplished by a single ellipsoidal mirror (M4) deflecting the beam horizontally. The optical aperture of M4 is 260 mm x 20 mm, accepting 85% intensity at 30 eV and more at higher energies (a longer M4 would provide slightly more flux at low energies but would also increase the level of slope errors, the price, and require a larger and more expensive mirror vessel and mechanics). The image at the exit slit plane is demagnified by a factor of ten at the sample position. The grazing incidence angles for M1, M3 and M4 are all 2deg. 2.3. Mirror units The mirror system is designed based on an idea to improve its vibrational behavior by making the movable parts as small and as lightweight as possible, avoiding stacked motion stages, and keeping the distance between a massive support block and the movable elements as short as possible. This approach has been reached by having a small cylindrical mirror chamber held and moved by five driven and one non-driven legs in a rectangular parallel kinematic arrangement. This system is able to determine all degrees of freedom. By driving the legs separately or in a combined way all three rotations, the vertical linear motion and the horizontal transversal motion can be achieved. The horizontal longitudinal direction is fixed and not motorized. The mirror motion is fully automated and incorporated into the beamline control system. 3. Beamline performance The spot size at the sample position is around 15 mm (H) x 15 mm (V) (with a 0.15 mm exit slit opening vertically and horizontally) and can be reduced down to approximately 10 mm (H) x 4 mm (V) by closing the exit slit further. The relatively large horizontal spot size results from the expected tangential slope errors on M4 (0.7 arcsec r.m.s.). The spot size can also be increased up to approximately 40 mm x 40 mm by changing the yaw rotation angle of the M4 mirror, thus defocusing the photon beam. The measured and calculated optimal spot size is presented in Fig. 3 > (Klementiev & Chernikov, 2014 >). The beamline photon flux within the main energy range 30-545 eV for the low-density (LD) grating (300 lines mm-1) and 70-1100 eV for the high-density (HD) grating (1221 lines mm-1) is shown in Fig. 4 >. The photon energy resolution of the beamline was measured by studying the absorption spectrum of molecular nitrogen recorded with a gas cell. The ion yield spectrum at the N1s - 1pg* excitation region is shown in the inset of Fig. 4 >. The spectrum has been measured with the HD grating at the smallest vertical exit slit (10 mm). The instrumental broadening was estimated by calculating the intensities of the first valley in the spectrum and the third peak (Chen & Sette, 1989 >). A Gaussian broadening of 60 meV gives a resolving power of the monochromator of about 7000 for the HD grating. This result is consistent with its previous calibration of energy resolution carried out at the old I311 beamline from which the SX-700 monochromator was moved. The measured and calculated energy resolution in the whole photon energy range (50-1000 eV) was presented in an earlier publication (Nyholm et al., 2001 >). The energy resolution DE changes as E 3/2 when the monochromator is operated in the fixed focus mode. The resolving power for the LD grating is 3x lower but is still sufficient for most of the measurements since it matches the energy resolution of the microscope. 4. Beamline and microscope control Initially, we had three different control systems at the MAXPEEM beamline. The beamline control system (the source and the optics) is built using Tango ) and uses a Python-based Sardana framework (Coutinho et al., 2011 >) to communicate with the IcePAP controllers (Janvier et al., 2013 >). The microscope has its own software (U-view2002+LEEM2000) developed by the microscope company Elmitec GmbH ). The camera and detector (TVIPS GmbH, ) have standard software for electron microscopes (TVIPS GmbH). First, we incorporated the detector software into the U-view program and then arranged a communication of the U-view program with the beamline control system when steering the undulator and monochromator is needed as, for example, in the acquisition of X-ray absorption spectra. 5. Endstation: aberration-corrected SPELEEM microscope The microscope combines the ability to perform XPEEM (X-ray photoemission electron microscopy), XAS (X-ray absorption microspectroscopy), small-spot XPS (X-ray photoelectron micro-spectroscopy), XPD (X-ray photoelectron diffraction, including micro-ARPES), LEEM and LEED (low-energy electron microscopy and diffraction, respectively). A schematic of the microscope is shown in Fig. 5 >. Component 6 in the figure is the R100 energy analyzer which was recently upgraded to R200. All images presented in the manuscript were collected with the old R100 analyzer and better performance is expected with the new energy filter. A large multi-purpose preparation and experimental ancillary chamber is attached to the microscope. It has in situ sample preparation facilities including material deposition, LEED measurements, gas dosing, sputtering and annealing up to 2000 K. The system is also compatible with the Universal Sample Holder (USH) system (Omicron flag-type adapter), allowing sample transfer via a Ferrovac ultrahigh vacuum (UHV) 'suitcase' between the microscope and other external UHV systems, e.g. a scanning tunneling microscope (STM) facility. The sample is hosted in the analysis chamber (or so-called main chamber) of the AC-SPELEEM microscope with a unique light incidence geometry - the photons from the synchrotron impinge the sample surface at normal incidence. This helps to avoid undesirable shadowing effects at 3D structures which is a problem for oblique-angle illumination. Another advantage of the normal incidence geometry is much better focusing in the horizontal direction, therefore, increasing the useful flux density at the sample and enabling total flux density to be lowered which then diminishes the space-charge effect. One consequence of this geometry is a potential decrease in photoionization cross-section due to the 90deg angle between the polarization vector and the direction of photoelectrons (Yeh & Lindau, 1985 >). In the main chamber, live imaging is possible not only at varying temperatures, from 89 K to 1600 K, but also under varied external conditions, i.e. magnetic field, electric potential or current and material/gas deposition. In this section, we describe the microscope performance. The microscope has an aberration corrector to compensate for the spherical and chromatic aberrations of the objective lens. If the corrector is on, the second beam-splitter directs the electron beam from the intermediate imaging column into the mirror column which is a tetrode electrostatic mirror. In this way, a consistent improvement in lateral resolution and transmission is achieved. The beam separator design also allows us to switch off the mirror when the electron beam is deflected directly into the imaging column, energy analyzer and detector. The mirror-on configuration is more time-consuming and is used when higher spatial resolution and/or higher transmission is needed. Below we present the performance of the AC microscope with some application examples. 5.1. New fiber-coupled CMOS detector: TVIPS F216 Nowadays, most PEEM/LEEM systems use the MCP-CCD system to convert the final image from electrons into a grayscale image that can be shown and stored on a PC. However, the resolution of such a detector system is limited to about 130 mm, which corresponds to about 300 effective pixels across the 40 mm diameter of the MCP (Shimizu et al., 2006 >; Moldovan et al., 2008 >). The dominant mechanism of image degradation is the lateral spread of the secondary electrons in the phosphor screen. With the new generation of AC-SPELEEM microscopes, the old MCP-CCD detector will limit the overall system performance. To overcome this problem, several types of solid-state detectors that convert the electron events directly into electronic signal were once tested in PEEM/LEEM systems after many years of application in X-ray diffractometers and transmission electron microscopes (Tinti et al., 2017 >; Tromp, 2015 >; van Gastel et al., 2009 >). Recently, a more economic camera system that combines the traditional scintillator and the latest CMOS chip was adopted by several LEEM/PEEM systems (Janoschka et al., 2021 >; see also ). In such systems, the 20 keV electrons first generate fluorescent photons on the scintillator layer. These photons are then transferred through the optical-fiber coupling unit to the CMOS chip. The detector is not bakeable but is very compatible with UHV as only the scintillator layer and the optical fiber array are inside the UHV chamber. After installing the detector into a pre-baked chamber that has a base pressure of 7 x 10-11 Torr, the chamber pressure decreases to a low -10 scale within one or two days. The detector used in our microscope (TVIPS F216 model) with 16 mm physical pixel size has demonstrated 4x higher resolution as well as two magnitudes higher dynamic range than the traditional MCP-CCD system. These improvements are shown in Fig. 6 >. 5.2. Aberrations in the Elmitec microscope Correction of main aberrations (spherical and chromatic) in the electron microscope is one of the most significant breakthroughs in improving the spatial resolution of the instrument. The spherical (C 3) and chromatic aberrations (C c) of the cathode lens on the image side are well understood and are given by Tromp (2011 >): C c = - L(E/E 0)1/2 + C cm and C 3 = L(E/E 0)1/2 + C 3m, where L is the sample-objective distance (2-3 mm) and C cm and C 3 m are the chromatic and spherical aberrations of the magnetic part of the objective lens, respectively. E and E 0 are the electron landing energy and the final energy after acceleration, respectively. For the Elmitec instrument, the corresponding simulations are presented as solid black dots and curves in Figs. 7 >(a) (C 3) and 7 >(b) (C c). There is an elegant way to measure spherical aberrations using a micro-spot LEED with an illumination area on the order of 250-500 nm, where the image location of the diffracted beam is observed in the Gaussian image plane [so-called real space micro-LEED (Tromp, 2013 >)]. Fig. 7 >(c) shows a schematic of the experiment (the corrector was off), and the experimental data are presented in Figs. 7 >(d) and 7 >(a) (red dots). Using a small illumination aperture and slight sample defocusing allows us to highlight the effect of aberrations. The pattern in Fig. 7 >(d) looks like a diffraction pattern but in fact it is a real space image formed by electron beams with radial displacements that depend on the diffracted angles. The experimental spherical aberration coefficients of the microscope are in satisfactory agreement with the theoretical simulations [Fig. 7 >(a)]. With all the aberration coefficients the user can estimate the gain in the spatial resolution and the microscope transmission. From Fig. 7 >(e), based on a simple analytical calculation, the user can observe if the third-order spherical and chromatic aberrations are completely removed, we could gain 4-5x not only in spatial resolution but also in electron transmission owing to the larger size of the contrast aperture. 5.3. Experimental tests 5.3.1. Test of the aberration corrector Before we discuss the performance of the aberration corrector, it is worth mentioning the spatial resolution that can be obtained without the corrector and which other factors (aside from aberrations) are crucial for the ultimate performance of the microscope. At high magnifications, a stable environment, first of all, mechanical noises, temperature and electronic stability, are the key factors for a good microscope performance. This means that a short acquisition time and subsequent drift correction of the stack of images can markedly improve the image quality. This method is particularly useful for XPEEM imaging because of low signals. For the XPEEM image, a large (several hundred) stack of images was collected at the shortest possible exposure time of the detector [e.g. 1 s in Fig. 8 >(a)]. Then, an image drift-correction procedure was applied to the whole stack and after that the images were integrated (Schindelin et al., 2012 >; Tseng et al., 2012 >). Without a corrector, the spatial resolution in the XPEEM mode is about 10 nm [Fig. 8 >(a)] whereas in LEEM mode it is about 5 nm [Fig. 8 >(b)] (Niu et al., 2017 >). In the case of XPEEM, it is very important to keep the photon flux as low as possible to diminish the space-charge effect. Space-charge effects always occur when there is a very high electron density in the (photo)electron beam. Very often it is observed in the microscopes installed at a synchrotron (Schmidt et al., 2013 >; Locatelli et al., 2011 >) due to the pulse nature of the photon source. The space-charge effect first occurs at the sample surface before the acceleration and then in the electron crossovers in the imaging column. Low photon flux at the sample, i.e. by detuning the undulator gap, is the main factor in eliminating the space-charge effect [e.g. compare Figs. 9 >(a) and 9 >(b)]. In fact, in addition to the aforementioned method of image acquisition, the good resolution of Fig. 8 >(a) benefits greatly from the very careful control of space-charge effects. When the aberration-corrector is on, an extra space-charge effect occurs in the mirror column, where electrons decelerate and the space-charge effect there is even more severe than at the surface [e.g. compare Figs. 9 >(b) and 9 >(c)]. To diminish the space charge in the microscope, especially in the mirror column, the user has to decrease the electron beam flux inside the column. One way to do this is to introduce a field-limiting aperture (selected area aperture, SAA) during the course of image acquisition as observed in the comparison of Figs. 9 >(c) and 9 >(d) [as well as Figs. 9 >(e) and 9 >(f) with core-level photoelectrons]. The drawback of putting SAA in the XPEEM mode is image vignetting if the sampling area is less than the field of view (FoV). For example, in Fig. 9 >(d) [as well as Fig. 9 >(f)], a 100 mm SAA was inserted to reduce the space charge, which in fact reduces the visible size of the image from the original FoV of 10 mm to 5 mm. In our microscope, we introduced another remedy to mitigate the space-charge effect in the mirror column: a knife-limiting edge. This edge is installed in the middle of the intermediate imaging column, which is close to a dispersive plane of the electron beam. It cuts secondary photoelectrons thus significantly improving the quality of core-level XPEEM images [e.g. compare Figs. 9 >(e) and 9 >(g)]. Fig. 9 >(h) demonstrates the enhanced action of both the SAA and the knife in the aberration-corrected XPEEM imaging mode. Using the corrector and limiting the electron beam in the microscope, we can easily obtain a moderate resolution of 50 nm with much higher transmission. As shown in Fig. 10 >, the gain in transmission is about 3-4x and roughly scales with the opening of the contrast aperture (CA = 70 mm for the corrector on compared with 30 mm for the corrector off case). Experimentally, we observe that the photon flux has to be reduced to obtain a decent spatial resolution in both (corrector on and off) modes. The ultimate resolution with the corrector on is not necessarily better than the case when the corrector is off due to space-charge effects and other factors. Nevertheless, the gain in transmission with the corrector on can offset the signal loss quite well due to the reduction of photon flux and make the acquisition time shorter, especially when only a moderate resolution is needed. 5.3.2. Micro-ARPES mode of the microscope An X-ray photoemission electron microscope (X-PEEM) equipped with a hemispherical energy analyzer is capable of fast acquisition of momentum-resolved photoelectron angular distribution patterns in a complete cone (Zakharov et al., 2011 >). We have applied this technique to observe the 3D (E, kx , ky ) electronic band structure of p-n junctions in germanium intercalated graphene; the sampling area can be on a sub-micrometre scale. Fig. 11 >(a) displays a LEEM image of the surface where the contrast can be attributed to different doping (one and two monolayers of intercalated germanium underneath a free-standing graphene layer). At fixed photon energy it is possible to sweep kinetic energies and obtain an energy slice of the 2D image (kx , ky ) in reciprocal space [Figs. 11 >(b) and 11 >(c)]. Using the stack of images acquired at different kinetic energies it is possible to plot 'standard' E(k) dependencies at different areas on the surface [marked by the red and yellow circles in the LEEM image; Figs. 11 >(d) and 11 >(e)]. 5.3.3. XMCD and XMLD measurements at MAXPEEM The normal incidence geometry of the photon beam at the MAXPEEM beamline is unique among similar beamlines around the world. It favors studies with out-of-plane magnetic moments in X-ray magnetic circular dichroism (XMCD) experiments. This is also the beamline of choice when in-plane magnetic moments in X-ray magnetic linear dichroism (XMLD) experiments are performed. Owing to the normal incidence geometry, the gain in signal is about a factor of four for XMCD, whereas, for in-plane sensitive XMLD, measurement is simplified as no sample rotation is needed. In Fig. 12 > the magnetic domain patterns of a multilayer Pt/Co sample are presented. The magnetic domains with down-magnetic moments were imaged by using the XMCD signal. The magnetic domain patterns can be altered by an external out-of-plane magnetic field produced with our magnetic sample holder. The photon flux of pure circular polarization mode only exists in the first harmonics which are within the energy interval 40-300 eV. In addition, the merit flux of elliptically polarized light occurs at a higher odd harmonic spectrum. The user has to optimize the flux and polarization rate on the different phasing positions of the undulator. The least photon flux of higher harmonic spectra occurs around the phasing position of 21 mm. The data in Fig. 12 > were collected at a phasing position of 16 mm (i.e. degree of polarization 0.86) which gives an optimal tradeoff between the beamline flux and degree of circular polarization. Each XMCD image in Fig. 12 > only took 80 s due to the 100% sensitivity from the normal incidence of the photon beam. With XMLD, as shown in Fig. 13 >, not only the antiferromagnetic domains of a Mn2Au film but also the ferromagnetic domains of the Permalloy layer on its top can be imaged. The resemblance of the two sets of domains demonstrates that the exchange coupling between the layers effectively copies the antiferromagnetic domain from Mn2Au to the ferromagnetic Permalloy layer (Bommanaboyena et al., 2021 >). It is worth emphasizing that the normal incidence of the photon beam means neither in-plane ferromagnetic components nor out-of-plane antiferromagnetic components would be sensed in the current design of MAXPEEM. Nevertheless, the two examples above have proved the particular usefulness of MAXPEEM in XMCD and XMLD. 6. Conclusions The MAXPEEM beamline is a dedicated beamline for high spatial resolution spectro-microscopy measurements. The aberration-corrected SPELEEM microscope offers a wide range of complementary techniques providing structural, chemical and magnetic sensitivity with a single-digit nanometre spatial resolution. The aberration corrector improves both the resolution and the transmission provided that the space-charge effect in the mirror column is diminished by limiting the size of the electron beam in the intermediate imaging column of the microscope. The very unique geometry of the incoming photon beam (normal incidence) implies much more effective sample illumination and the absence of shadow effects characteristic for grazing incidence geometry. In addition, it favors studies with out-of-plane magnetic moments in XMCD and in-plane magnetic moments in XMLD experiments. The beamline reaches low photon energies suitable for valence band and micro-ARPES studies. The initial user operation shows that the beamline meets the design parameters very well and performs as expected. We thank the past and present staff of the MAX IV laboratory for all the help and participation in the beamline construction, commissioning operation, and transfer of the microscope and monochromator from the old facility. The authors are grateful to Professor Ernst Bauer for the idea of limiting space-charge effects in the intermediate imaging column. We would also like to thank Torsten Franz and Florian Schutz from Elmitec Elektronenmikroskopie GmbH for their technical support as well as Viacheslav Sachenko for his calculation support. Finally, we would like to thank Martin Pavelka, Hermann Durr and Stefano Bonetti for providing the Co/Pt multilayer sample and for fruitful discussions. Figure 1 (a) Simulated and measured undulator spectra in planar mode for an undulator gap of 30 mm. The beamline opening is 0.1 mrad. The monochromator grating features 1221 lines mm-1. The inset shows the agreement between the simulated and measured spectra for the first harmonics at 115 eV (logarithmic scale on the intensity axis); the small energy shift has been corrected. The experimental undulator spectrum was measured right after the exit slit of the beamline. (b) Calculated map of the undulator spectra showing the degree of circular polarization of the photon beam at the fixed phase of 14 mm. The locations of the third and fifth harmonics are indicated by the blue curves. (c) Reflected X-ray intensities versus azimuth angles of the multilayer analyzer in the polarimeter when the undulator, i.e. the gap and phase, were configured to several simulated polarization angles in the inclined mode. The photon energy is 708 eV, and 0deg (90deg) means the polarization of the X-rays is linearly horizontal (vertical). Figure 2 Optical layout of the MAXPEEM beamline with a single refocusing ellipsoidal mirror (M4). Distances are given in metres (not to scale). Figure 3 Beam profile at the sample position of the MAXPEEM beamline. (a) Simulation using X-ray tracing software (Klementiev & Chernikov, 2014 >), photon energy 40 eV; the energy slit is 150 mm (H) x 150 mm (V). The color code is the energy dispersion that scales with the photon energy. It is +-7 meV at 40 eV photon energy. (b) Experimental beam profile in the photoelectron microscope at 43 eV photon energy. The beamline energy slit is 150 mm x 150 mm and the signal is from secondary photoelectrons discriminated by the energy analyzer of the microscope, with an energy window of 0.2 eV. Figure 4 Measured photon flux after the exit slit for two different gratings, 300 lines mm-1 and 1221 lines mm-1. The beamline opening is 0.15 mrad, the energy slit is 150 mm (H) x 300 mm (V). Inset: ion yield spectrum at the N1s -absorption edge of N2. Figure 5 Schematic of the AC-SPELEEM endstation. (1) Preparation chamber, (2) main chamber, (3) first beam separator, (4) second beam separator, (5) AC mirror, (6) energy analyzer, (7) TVIPS-F216 detector (CMOS-camera), (8) UV lamp, and (9) electron gun (LaB6) and the illumination column. Figure 6 (a) LEEM and (b) micro-spot LEED images from an epitaxial mono-/bi-layer graphene sample grown on SiC to show the resolution and dynamic range of TVIPS-F216. The image size of (a) is 20 mm x 20 mm. The upper inset in (a) is the magnified image of the area selected in red (1484 nm x 1484 nm) and the profile across the mono-/bi-layer boundary (yellow line) is shown in the lower inset of (a). The resolution of the detector is determined to be about 2.1 pixels, which corresponds to 32.8 nm. The inset in (b) shows the profile along the red line across the LEED spots [note that panel (b) is given on a logarithmic scale]. Figure 7 Aberration coefficients and spatial resolution in Elmitec LEEM. Panels (a) and (b) show calculated spherical (C3) and chromatic (Cc) aberration coefficients at three different energies 1 eV, 10 eV and 30 eV along with the interpolation. The red squares in (a) are the experimentally measured C3 for different energies with real space micro-LEED. (c) Schematic of the real space micro-LEED for measuring spherical aberrations in the microscope. (d) Experimental real space image of the Si(111) surface, with electron energy 5.4 eV, illumination aperture 10 mm, defocus 25 mA. (e) Comparison of the resolution and transmission for the Elmitec microscope with and without the aberration corrector. Electron energy E = 10 eV, and the electron energy spread is 0.25 eV. The correction was carried out by removing only the third-order spherical and chromatic aberrations. Figure 8 High-resolution secondary (a) XPEEM and (b) LEEM images of Sn/SnO x intercalated graphene. For the XPEEM image, every frame was acquired with 1 s exposure time in the stack of 185 images. Before integration, the images in the stack were drift-corrected. For the LEEM image, the image was integrated from eight frames with 1 s exposure time without drift correction (note that the single step is clearly visible in both images). Figure 9 Space-charge effects manifested in the XPEEM images obtained under different conditions. Top row: secondary XPEEM images: (a) high photon flux of 4.2 x 1013 photons s-1, (b) low photon flux of 1.4 x 1013 photons s-1 in the mirror-off mode, (c) same as (b) but in the mirror-on mode, (d) same as (c) with insertion of the SAA (100 mm). Lower row: Si2p core-level XPEEM images, (e) same as (c), (f) with the SAA inserted, (g) with the knife aperture inserted (no SAA), and (h) with both the SAA and the knife inserted. All XPEEM images presented are 2.5 mm x 2.5 mm, cropped from raw images with FoV = 10 mm. The sample is monolayer graphene with a few bilayer islands grown on SiC (0001). In the mirror-on (mirror-off) mode, the 70 mm (30 mm) CA was always used. The photon energy used for all images was 150 eV. The integration time for (a)-(d) was 3.2 s and (e)-(h) was 320 s. Figure 10 Si2p core-level XPEEM images taken in the (a) mirror-off and (b) mirror-on modes with both the SAA and the knife inserted. For better illustration, the original gray images were converted with a fake-color map. The average intensities of the two images are 34.7 and 120.8, respectively, as different CAs were used: 30 mm in (a) and 70 mm in (b). [Note that image (b) is a copy of the image in Fig. 9 >(h).] Figure 11 (a) LEEM image of Ge-intercalated graphene. The contrast in the image can be attibuted to different doping, black areas are p-type doped and white areas are n-type doped. On the right, selected area micro-ARPES data are shown: panels (b) and (c) are constant energy scans close to the Fermi level; panels (d) and (e) show the E(k) band structure (Dirac cone) from the marked areas (red and yellow) in (a). The sampling area is about 1.5 mm and the photon energy is 42.3 eV. Figure 12 Magnetic domain patterns from XMCD measurements in a Pt /Co multilayer sample with an out-of-plane magnetic moment. Images were taken at the remanent state after applying out-of-plane magnetic fields of 0 mT (left), 25 mT (middle) and 72 mT (right). The images were taken with a photon energy of 780 eV (L 3 peak in the Co XAS spectrum) and a kinetic energy of 1.6 eV. 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PMC10000797 | J Synchrotron Radiat J Synchrotron Radiat J. Synchrotron Rad. Journal of Synchrotron Radiation 0909-0495 1600-5775 International Union of Crystallography 36891853 tv5043 10.1107/S1600577522012176 JSYRES S1600577522012176 Research Papers High-energy X-ray micro-laminography to visualize microstructures in dense planar objects High-energy X-ray micro-laminography Hoshino Masato a* Uesugi Kentaro a Imai Takuya b a Japan Synchrotron Radiation Research Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5198, Japan b Fukui Prefectural University, 4-1-1 Matsuoka-kenjojima, Eiheiji, Fukui 910-1195, Japan Stevenson A. Editor Australian Synchrotron, Australia Correspondence e-mail: [email protected] 01 3 2023 03 2 2023 03 2 2023 30 Pt 2 s230200 400406 12 5 2022 26 12 2022 (c) Masato Hoshino et al. 2023 2023 This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-BY) Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited. A full version of this article is available from Crystallography Journals Online. The development of synchrotron-radiation-based high-energy X-ray micro-laminography to visualize microstructures in dense planar objects such as planar fossils is described. High-energy X-ray micro-laminography has been developed to observe near-surface structures in dense planar objects that are not suitable for observation by X-ray micro-tomography. A multilayer-monochromator-based high-intensity X-ray beam with energy of 110 keV was used for high-energy and high-resolution laminographic observations. As a demonstration of high-energy X-ray micro-laminography for observing dense planar objects, a compressed fossil cockroach on a planar matrix surface was analyzed with effective pixel sizes of 12.4 mm and 4.22 mm for wide field of view and high-resolution observations, respectively. In this analysis, the near-surface structure was clearly observed without undesired X-ray refraction-based artifacts from outside of the region of interest, a problem typical in tomographic observations. Another demonstration visualized fossil inclusions in a planar matrix. Micro-scale features of a gastropod shell and micro-fossil inclusions in the surrounding matrix were clearly visualized. When observing local structures in the dense planar object with X-ray micro-laminography, the penetrating path length in the surrounding matrix can be shortened. This is a significant advantage of X-ray micro-laminography where desired signals generated at the region of interest including optimal X-ray refraction effectively contribute to image formation without being disturbed by undesired interactions in the thick and dense surrounding matrix. Therefore, X-ray micro-laminography allows recognition of the local fine structures and slight difference in the image contrast of planar objects undetectable in a tomographic observation. high-energy X-ray micro-laminography fossils X-ray refraction contrast pmc1. Introduction X-ray micro-laminography (micro-LG) utilizing a synchrotron radiation X-ray source as well as a laboratory source has been employed as a non-destructive three-dimensional (3D) imaging technique for planar objects as a substitute for conventional X-ray micro-tomography (micro-CT) (Helfen et al., 2005 >, 2012 >, 2013 >; Fisher et al., 2019 >; Wood et al., 2019 >). In the tomographic observation of a planar object, its planar shape essentially hinders visualization of the inner structure due to poor X-ray transmission along the longitudinal direction and a geometrical restriction caused by the relation between the effective field of view (FOV) in the projection image and the sample size in the longitudinal direction. In comparison, X-ray micro-LG enables us to observe the region of interest even for a large-area sample by simply tilting the rotational axis of the sample, which is generally set perpendicular to the planar surface. In previous studies, X-ray micro-LG has been applied to observations of crack initiation and propagation (Moffat et al., 2010 >; Xu et al., 2010 >; Tsuritani et al., 2015 >). In addition, some studies involving cultural heritage and thin biological samples have been successfully conducted with X-ray micro-LG (Krug et al., 2008 >; Verboven et al., 2015 >). Phase-contrast X-ray micro-LG based on a grating interferometer has also been developed to apply to polymer-based samples (Helfen et al., 2009 >; Harasse et al., 2011 >; Altapova et al., 2012 >). In the case of dense and thick planar objects such as flat fossils, X-ray micro-LG can also be a promising tool for assessing non-destructive 3D analysis. Since the biological structures in a planar fossil specimen are often preserved parallel to the planar surface of the rock that contains them, such a specimen can be a good candidate for observation with X-ray micro-LG. Although a few studies have reported the laminographic observation of fossils, the applicability of X-ray micro-LG has not been fully explored with a thorough description of the technical background investigation (Houssaye et al., 2011 >; Zuber et al., 2017 >). The challenge has been that a dense, flattened macroscopic fossil generally requires high-energy X-rays with extensive spatial resolution for practical observation of its inner structures. So far, micro-fossils and high-density materials that can be trimmed into small pieces have been successfully measured with synchrotron-radiation-based high-resolution X-ray micro-CT using lower-energy X-rays (Donoghue et al., 2006 >; Tafforeau et al., 2006 >; Yin et al., 2015 >). However, micro-structures in a macro-scale fossil might have a significant impact on understanding the paleobiology of extinct organisms. From this perspective, the development of a high-resolution X-ray imaging technique that potentially serves non-destructive 3D analyses for such planar macroscopic fossils has been desired. In the case of high-resolution measurements in the high-energy region, the performance greatly depends on the X-ray photon flux density. High-intensity and high-energy X-rays obtained from large-sized synchrotron radiation facilities are basically suitable for such analyses. In addition, X-ray micro-imaging systems using synchrotron radiation are superior compared with laboratory-based systems in terms of spatial coherence even in the high-energy region. When fossil samples are observed with high-energy X-rays, the effective enhancement of the image contrast is crucial to differentiate fossil inner structures from other, non-biological, micro-scale materials. In this regard, a high degree of the spatial coherence in synchrotron radiation is useful to support high-contrast observations using proper edge-enhancement caused by X-ray refraction (Wilkins et al., 1996 >; Yagi et al., 1999 >). In this study, synchrotron-radiation-based X-ray micro-LG employing X-rays higher than 100 keV has been developed for the purpose of observing dense planar objects. To demonstrate its applicability to planar objects such as planar fossils, the image quality obtained from X-ray micro-LG was compared with that obtained from a conventional tomographic geometry to address the validity of the laminographic observation for planar fossil specimens in terms of the image contrast and the spatial resolution. 2. Development of high-energy X-ray micro-LG 2.1. Measurement setup High-energy X-ray micro-LG was developed at the medium-length bending-magnet beamline BL20B2 at SPring-8 (Goto et al., 2001 >). A double-multilayer monochromator (DMM) optimized for an output energy of 110 keV was used to utilize high-energy X-rays with higher photon flux density. The energy resolution (DE/E) was 0.9%. Lower-energy components from total reflection were reduced by a 0.3 mm-thick copper filter. Details of the DMM are given elsewhere (Koyama et al., 2023 >). Downstream experimental hutches at BL20B2 located more than 200 m from the source were used in this study. A long propagation distance from the source to the experimental hutches makes it possible to use an X-ray beam with higher spatial coherence which is more advantageous for observing fossil specimens with X-ray refraction contrast, namely X-ray phase contrast. A goniometer was installed on the sample stage in order to tilt the rotational axis of the sample for X-ray micro-LG. The goniometer could be operated with a tilt angle range from 0deg, which is normal to the optical axis of the X-ray beam, to 30deg for X-ray micro-LG. Accordingly, a laminographic setup could be quickly switched to a tomographic one for comparison of resulting images of the same sample. X-ray transmission images were recorded with a lens-coupled visible-light conversion-type X-ray imaging detector (Uesugi et al., 2011 >). For a wide FOV measurement up to 50 mm in width, a dedicated X-ray detector for high-energy X-ray micro-imaging developed in our previous study was used (Hoshino et al., 2020 >). As a scintillating material, a Lu3Al5O12 (LuAG) transparent ceramics plate of thickness 500 mm was employed (Kameshima et al., 2019 >). A visible-light image demagnified by the lens system was detected with a high-definition CMOS camera [C13949-50U; 4096 (H) x 3008 (V) pixels, 3.45 mm pixel-1, 12-bit ADC; Hamamatsu Photonics]. The propagation distance from the sample to the detector was set to 8 m to utilize the X-ray refraction contrast effectively. The effective pixel size and the width of the projection image were 12.4 mm and 50.8 mm, respectively. On the other hand, a high-resolution X-ray imaging detector was also prepared to demonstrate the high-resolution observation for micro-scale structures within the fossil specimens. In this high-resolution detector, an AA60 beam-monitor (Hamamatsu Photonics) was used. Additionally, as a scintillating material, a LuAG single-crystal plate of thickness 500 mm was employed. As in the wide FOV measurement, a high-definition CMOS camera was used for high-resolution observations. In these observations, the propagation distance from the sample to the detector was set to 3 m. The effective pixel size was 4.22 mm. A filtered back-projection (FBP) method considering the tilt angle of the rotational axis was used for reconstructing a sectional image in X-ray micro-LG (Hoshino et al., 2011a >). A conventional FBP method was used for X-ray micro-CT. In both reconstructions under the FBP method, a Chesler filter was applied to the projection images. In this study, phase retrieval was not employed to directly utilize the edge-enhancement effect by X-ray refraction in the reconstructed images. 2.2. Specimens Specimens employed in this study include FPDM-I-145 and FPDM-I-2337 deposited in Fukui Prefectural Dinosaur Museum (FPDM), Katsuyama, Fukui, central Japan. FPDM-I-145 contains a fossilized cockroach (Blattaria indet.) from the late Aptian Crato Formation, Araipe Basin, Brazil. The Crato Formation is known for its high-fidelity preservation of flattened fossil insects. They are preserved in forms of pyrite and goethite, replacing the original cuticle, and of calcium phosphate, replacing the original internal soft tissues (Barling et al., 2020 >), as FPDM-I-145 most probably is. These fossilized insects are preserved within finely laminated limestones that tend to split into a form of plates, revealing insect fossils on its surface. FPDM-I-145 is 78.1 mm long, 68.8 mm wide and 7.9 mm thick, including the fossilized cockroach and matrix preserving it [Fig. 1 >(a)]. FPDM-I-2337 is platy and contains abundant invertebrate remains from the Middle Permian Akasaka Limestone, Gifu, central Japan. The fossil remains are preserved as dark calcium carbonate and are largely composed of gastropods including Vebericochilis sp. FPDM-I-2337 is 81.7 mm long, 57.7 mm wide and 6.9 mm thick at the thickest part (at which the largest individual of Vebericochilis sp. is preserved). Numerous micro-fossils are observed on the matrix surface [Fig. 1 >(b)]. In X-ray micro-LG, FPDM-I-145 was employed to demonstrate its effectiveness for the observation of a flattened fossil preserved on a planar surface, and FPDM-I-2337 for the observation of characteristic inner structures inside the dense planar object. These samples were held with a commercially available plastic container with elastic membranes (Boxel - Precision Instrument Transport Storage Container, No.32, AS ONE, Japan) which supported the specimens even under the tilted condition in the laminographic observation. In the following measurements, the rotational axis was set to be perpendicular to the planar surface of each specimen. 3. Results Fig. 2 >(a) shows the experimental arrangement of FPDM-I-145 on the sample stage. The measurement conditions in the wide FOV observation for FPDM-I-145 with X-ray micro-LG were as follows: 7200 projections from 360deg rotation, 150 ms exposure time and 30deg rotational axis tilt angle. In a laminographic measurement, an effective vertical field of view (v_FOV) of more than h_FOVsinPh is basically required in the projection images to reconstruct a full-field sectional image, where h_FOV is the horizontal field of view that is equal to the width of the sectional image, and Ph is the tilt angle of the rotational axis. Since h_FOV was 50.8 mm in the wide FOV observation, v_FOV should be larger than 25.4 mm for a 30deg tilt angle. However, the effective vertical width of the high-energy X-ray beam at the sample position was limited to approximately 10 mm. To overcome this limitation, the sample was scanned along the vertical direction step by step at every 360deg rotation as shown in Fig. 2 >(b). FPDM-I-145 was scanned five times with a stepping amount of 8 mm per step. Then, five-part sub-projection images were combined before reconstruction to make a single projection image with v_FOV of 40 mm. A sectional image of FPDM-I-145 parallel to the planar surface is shown in Fig. 3 >(a). For comparison, the same specimen was analyzed under tomographic conditions (Ph = 0) with the only difference being the tilt of the rotational axis. The following measurement conditions for X-ray micro-CT were applied: number of projections 3600 from 180deg rotation and exposure time 150 ms. A sectional image obtained from X-ray micro-CT at the same sectional plane is shown in Fig. 3 >(b). In the tomographic image, part of the elastic membrane that would have been located near the sectional plane could be seen as indicated by arrows. In addition, some artifacts from undesired interactions with the structures near the surface significantly affect the image quality (see the video in the supporting information). The penetration path length of the X-ray beam in the sample tends to be longer under tomographic conditions so the rotational axis is set to be normal to the planar surface. Therefore, the required signals from the region of interest can be disturbed by any material in the beam path outside of it, causing a noisy image due to a decrease in transmission and overlapping of streak-like artifacts. Consequently, the image quality defined by the spatial resolution and the image contrast is degraded. In comparison, micro-scale morphologies of FPDM-I-145 are clearly observed in X-ray micro-LG. The aid of the high-resolution detector reveals the veins in the wing membranes and ventral side of the abdomen covered by the matrix. Additionally, we conducted high-resolution X-ray micro-LG with the following experimental conditions: number of projections 7200 from 360deg rotation, exposure time 200 ms and tilt angle 30deg. A high-resolution sectional image around the abdomen is shown in Fig. 3 >(c). Micro-structures of the abdomen in the ventral side, which X-ray micro-CT failed to capture, is clearly visualized. From this point of view, localized high-resolution observations and multi-scale observations even in a large-scale sample are outstanding features of X-ray micro-LG. To demonstrate the observation of fossils within a dense planar matrix, FPDM-I-2337 was analyzed with the high-resolution detector. The measurement conditions in high-resolution X-ray micro-LG were as follows: number of projections 7200 from 360deg rotation, exposure time 200 ms and tilt angle 30deg. A sectional image of a Vebericochilis shell is shown in Fig. 4 >(a). Micro-scale morphology in the shell and small fossil remains in the surrounding matrix indicated by arrows in Fig. 4 >(a) are clearly visualized. The small remains in the matrix likely represent fragmentary invertebrate shells and micro-fossils including foraminifera typical of Akasaka Limestone. For comparison, the same region of interest was observed with X-ray micro-CT; the measurement conditions were as follows: number of projections 3600 from 180deg rotation and exposure time 200 ms. A sectional image obtained from X-ray micro-CT is shown in Fig. 4 >(b). The image quality is degraded by lower X-ray transmission and undesired interactions similar to the images obtained from FPDM-I-145. The magnified images shown in Figs. 4 >(c) and 4 >(d) indicate the difference in the image quality in more detail. For example, filamentous structures as well as the matrix infilling the shell indicated by arrows are distinguishable only in X-ray micro-LG. Notably, the angular increments in both tomographic and laminographic measurements were equal at 0.05deg per projection. Nevertheless, significant improvement of the image quality resulted by simply tilting the rotational axis. 4. Discussions In this study, the validity and superiority of high-energy X-ray micro-LG for observing planar fossils are successfully demonstrated by comparing the image quality in sectional images obtained from X-ray micro-LG and micro-CT. As shown in Figs. 3 >(b) and 4 >(b), a long penetration path length in the matrix surrounding the region of interest causes significant degradation of the image quality due to undesired interactions. A major factor degrading the image quality appears to be a decrease in the effective signals due to insufficient transmission along the surrounding matrix. In this regard, X-ray micro-LG was intrinsically valid for such a measurement because the penetration path length in the surrounding matrix is greatly shortened. In addition, the tilted layout in X-ray micro-LG is capable of effectively using X-ray refraction generated around the region of interest to enhance the image contrast without disturbance from the surrounding matrix. On the other hand, streak-like artifacts that might be caused by undesired X-ray refraction from the planar surface in the tomographic condition degrade the quality of the sectional images near the surface as shown in Fig. 3 >(b) (see also the video in the supporting information). As for the streak-like artifacts, it is well known that they occur along the back-projection angle in the tomographic reconstruction when the projection image contains areas with significantly different absorption coefficients. In this case, only a sectional plane that includes such areas is affected. In comparison, refracted X-rays from the planar surface near the sectional plane under reconstruction may also affect the resultant image even though they do not originate from the structure in the plane. This is a result of the strong edge-enhancement caused by X-ray refraction spreading over several pixels in the projection image when the X-ray beam path is parallel to the surface. When a planar object is measured under X-ray micro-CT using X-rays with high degrees of spatial coherence, especially in the case where a region of interest is found near the surface, the influence of such artifacts must always be taken into account even if the observation is made with high-energy X-rays. In comparison, no projection image generally includes the angular region where the incident X-ray beam becomes parallel to the planar surface in X-ray micro-LG. This feature saves the image quality in the sectional image from undesired interactions caused outside the region of interest and enables high-resolution observations focused on the micro-structures of planar objects. In this study, a commercially available plastic container with elastic membranes was used to hold indefinite-shaped planar specimens. The study demonstrates that the holding performance of the container is adequately applicable to high-resolution observation with an effective pixel size of 4.22 mm, albeit a limitation is the size of the container. While the elastic membrane was vsible in the sectional image of FPDM-I-145 under X-ray micro-CT, as in Fig. 3 >(b), the X-ray micro-LG observation under the same experimental settings except for a tilt of the rotational axis did not cause such an issue, likely benefitting from a specific effect in the laminographic measurement. In X-ray micro-LG, the elastic membrane covered the whole area of the region of interest in the projection images. Therefore, the membrane was regarded as a transparent and uniform material on the projection images like a thin filter and caused no interaction with the X-ray beam. In addition, all interactions of the X-ray beam and the membrane are generally small in the 110 keV high-energy region compared with in the lower-energy region. Consequently, the membrane located near the sectional plane did not provide any effect on the sectional image as shown in Fig. 3 >(a). In comparison, the membrane presented in the X-ray micro-CT sectional image was likely caused by strong X-ray refraction generated at the boundary between the membrane and the air in the angular region where the membrane became parallel to the X-ray beam. With respect to more generalized measurements for planar specimens and more irregularly shaped specimens, such as those containing protrusions, a high-performance 3D printer combined with a precision 3D outline scanner might be useful for creating a dedicated sample holder. The female holder precisely adapted to the outline of the indefinite-shaped specimen should enable a higher spatial resolution to be achieved, even under the tilted laminography layout In the X-ray micro-LG observation, the artifacts specific to laminography have been discussed in previous studies (Helfen et al., 2005 >, 2006 >; Hoshino et al., 2011b >; Xu et al., 2012 >). The boundary structure along the rotational axis, which is normal to the planar surface, becomes unclear due to missing information in the Fourier domain in the laminography reconstruction. In addition, an inherent artifact also tends to appear at the cross section away from the central sectional plane. In this case, a distinct structure in the central sectional plane tends to appear as isotropic blurred images in other sectional planes along the rotational axis. However, no significant impact was detected to recognize the micro-structures in the current study as shown in Figs. 3 > and 4 >. Since typical fossil specimens and their matrices are composed of minerals with similar material features, little change in density or absorption coefficients is present, causing little influence of the blurred images derived from the distinct structure. Thus, the material features in fossil specimens positively affected the quality of the observation with minimal effects from the artifacts specific to X-ray micro-LG, while measurements using synchrotron radiation with high degrees of spatial coherence are necessary for visualization of such similar material features. Moreover, micro-scale morphologies in compressed planar specimens can be observed in the sectional plane normal to the rotational axis in which the influence of artifacts specific to laminography can be reduced. Therefore, it can be concluded that high-energy X-ray micro-LG is a compatible method for analyzing microscopic morphologies and inner structures of planar fossil specimens and fossils within planar matrices. On the other hand, it would be more effective to use an iterative reconstruction technique to reduce artifacts specific to laminography (Fisher et al., 2019 >). This approach would allow for a wider range of application of high-energy X-ray micro-LG to various materials. X-ray micro-LG has numerous applications for visualizing the micro-scale morphology of compressed fossil specimens within a planar matrix. As in FPDM-I-145 from the Crato Formation, multiple well-known fossil sites produce animal remains that are preserved flattened, sometimes with internal organs and epidermal structures, on finely laminated rocks that typically break into a planar matrix with such remains exposed on top (e.g. Burgess Shale, Bear Gulch Limestone, Holzmaden Shale, Solnhofen Limestone, Yixian Formation, Santana Group and Messel Oil Shale). Under such preservation, physical observation of the unexposed side of the specimen is virtually impossible without removing the matrix completely, which involves serious risk of damaging it. Previously, tomographic analyses on flattened fossils on a planar matrix failed to capture their morphological details due to X-ray artifacts and inadequate X-ray transmission along the direction parallel to the matrix plane. X-ray micro-LG can negate such problems and can be applied to numerous specimens that are flattened onto planar matrices. 5. Conclusion High-energy X-ray micro-LG at an X-ray energy of 110 keV has been developed, and two specimens with planar fossil materials were observed to demonstrate the applicability of the technique to dense planar objects for which X-ray micro-CT fails to capture adequate structural information. While the present study focuses on planar fossil specimens, the high-penetration power of the high-energy X-rays has potential for various applications. As further applications using high-energy X-ray micro-LG, non-destructive testing of electronic devices and in situ observation of a fuel cell battery would be potential candidates. Since localized high-resolution observations can be regarded as outstanding features of X-ray micro-LG, upgrading the system so that it is capable of conducting measurements with an effective pixel size of 1 mm or smaller will be an interesting approach to reveal micrometre-scale features for geological, biological and engineering investigations. Supplementary Material Supplementary Figures S1, S2 and S3. DOI: 10.1107/S1600577522012176/tv5043sup1.pdf Click here for additional data file. Video comparing sequential slices of -145 along the depth direction. DOI: 10.1107/S1600577522012176/tv5043sup2.avi The authors would like to thank Mr Tomoki Fukui for his technical support. This work was carried out with the approval of the SPring-8 Program Committee (2019B1303, 2019B1304 2021A1262, 2021B1398). Drs Kaito Asato (Fukui Prefectural Dinosaur Museum) and Noboru Oyama (Kyushu University) helped the authors to identify and describe the specimens. Figure 1 Photographs of the specimens. (a) FPDM-I-145 preserved on the planar surface. (b) FPDM-I-2337 preserving various invertebrate remains including gastropods. Figure 2 (a) Measurement setup for X-ray micro-laminography of planar specimens. In this photograph, the tilt angle of the rotational axis was set to 30deg from the normal direction to the X-ray beam. (b) Schematic drawing of the wide FOV observation with a vertical sample scan. Figure 3 Sectional images of Blattaria indet. (FPDM-I-145) analyzed by (a) X-ray micro-LG and (b) X-ray micro-CT, in which the same sectional planes are presented. The arrows indicate part of the elastic membrane that hold the specimen. (c) High-resolution sectional image of Blattaria indet. (FPDM-I-145) observed with an effective pixel size of 4.22 mm. Figure 4 Sectional images of a Vebericochilis shell in FPDM-I-2337 analyzed with (a) X-ray micro-LG and (b) X-ray micro-CT. The arrows in (a) indicate small fossil remains in the surrounding matrix. Magnified images at the upper middle region in sectional images obtained from (c) X-ray micro-LG and (d) X-ray micro-CT. The arrows in (c) and (d) indicate filamentous structures and the matrix infilling the shell. References Altapova, V., Helfen, L., Myagotin, A., Hanschke, D., Moosmann, J., Gunneweg, J. & Baumbach, T. (2012). Opt. Express, 20, 6496-6508. Barling, N., Martill, D. M. & Heads, S. W. (2020). Cretac. Res. 116, 104608. Donoghue, P. C. J., Bengtson, S., Dong, X., Gostling, N. J., Huldtgren, T., Cunningham, J. A., Yin, C., Yue, Z., Peng, F. & Stampanoni, M. (2006). Nature, 442, 680-683. Fisher, S. L., Holmes, D. J., Jorgensen, J. S., Gajjar, P., Behnsen, J., Lionheart, W. R. B. & Withers, P. J. (2019). Meas. Sci. Technol. 30, 035401. 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PMC10000798 | J Synchrotron Radiat J Synchrotron Radiat J. Synchrotron Rad. Journal of Synchrotron Radiation 0909-0495 1600-5775 International Union of Crystallography 36891847 yn5092 10.1107/S1600577523000796 JSYRES S1600577523000796 Research Papers Monitoring surface dynamics of electrodes during electrocatalysis using in situ synchrotron FTIR spectroscopy Monitoring surface dynamics of electrodes Cheng Weiren ab++ Xu Yanzhi a++ Yang Chenyu a Su Hui a Liu Qinghua a* a National Synchrotron Radiation Laboratory, University of Science and Technology of China, Hefei, Anhui 230029, People's Republic of China b Institute for Catalysis, Hokkaido University, Sapporo 001-0021, Japan Yamamoto M. Editor RIKEN SPring-8 Center, Japan Correspondence e-mail: [email protected] ++ These authors contributed equally to this work. 01 3 2023 16 2 2023 16 2 2023 30 Pt 2 s230200 340346 29 4 2022 30 1 2023 (c) Weiren Cheng et al. 2023 2023 This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-BY) Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited. A full version of this article is available from Crystallography Journals Online. A novel bifunctional in situ Fourier transform infrared (FTIR) cell has been elaborately designed and prepared. By coupling with single-reflection infrared mode, a facile and general in situ synchrotron FTIR spectroscopic method has been developed based on the FTIR cell for surface dynamic studies of electrodes during electrolysis. Monitoring the surface dynamics of catalysts under working conditions is important for a deep understanding of the underlying electrochemical mechanisms towards efficient energy conversion and storage. Fourier transform infrared (FTIR) spectroscopy with high surface sensitivity has been considered as a powerful tool for detecting surface adsorbates, but it faces a great challenge when being adopted in surface dynamics investigations during electrocatalysis due to the complication and influence of aqueous environments. This work reports a well designed FTIR cell with tunable micrometre-scale water film over the surface of working electrodes and dual electrolyte/gas channels for in situ synchrotron FTIR tests. By coupling with a facile single-reflection infrared mode, a general in situ synchrotron radiation FTIR (SR-FTIR) spectroscopic method is developed for tracking the surface dynamics of catalysts during the electrocatalytic process. As an example, in situ formed key *OOH is clearly observed on the surface of commercial benchmark IrO2 catalysts during the electrochemical oxygen evolution process based on the developed in situ SR-FTIR spectroscopic method, which demonstrates its universality and feasibility in surface dynamics studies of electrocatalysts under working conditions. in situ cell FTIR spectroscopy synchrotron radiation surface dynamics electrocatalysis National Natural Science Foundation of ChinaU1932212 The following funding is acknowledged: National Natural Science Foundation of China (grant No. U1932212); Natural Science Foundation of Anhui Province (2208085J01). pmc1. Introduction High-performance electrochemical devices, such as electrolyzers, fuel cells, Zn-air batteries etc., are very important for the blossoming of a modern sustainable energy industry (Zhou et al., 2022 >; Cheng et al., 2019 >; Pomerantseva et al., 2019 >). To develop efficient electrochemical devices, one of the key challenges is to design advanced catalysts with high activity and durability to overcome the substantial thermodynamic reaction barriers on their anodes/cathodes (Pomerantseva et al., 2019 >; Cheng et al., 2021 >). Monitoring the dynamic evolution of electrodes, especially surface species, under working conditions in these electrochemical devices will be very helpful for understanding their intrinsic energy conversion mechanisms for design, synthesis and screening of advanced electrocatalysts (Miyanaga, 2021 >; Su et al., 2020 >; Cheng et al., 2019 >). However, it is still very challenging to promptly probe the progress of reactive intermediates over the electrode surface during the electrocatalytic process due to their low concentration and the substantial influence of aqueous electrolytes (Li et al., 2020 >; Catlow et al., 2020 >). Hence, a cutting-edge measuring technique with an ultra-high surface sensitivity is urgently needed for surface dynamics studies during electrocatalysis. Fourier transform infrared (FTIR) spectroscopy, which is highly sensitive to surface adsorbed species, has been widely adopted to detect reactants or products on the surface of nanomaterials in various catalytic reactions (Catlow et al., 2020 >; Li et al., 2020 >; Marcelli et al., 2012 >). Moreover, with molecular fingerprint identification of surface active intermediates and relatively facile operation, in situ FTIR spectroscopy has been considered a reliable and powerful tool for tracking the surface species evolution of electrodes during electrocatalysis in relation to Raman spectroscopy and X-ray photoelectron spectroscopy (Catlow et al., 2020 >; Han et al., 2021 >; Huang et al., 2020 >). For in situ FTIR measurements during electrolysis, it is a rather challenging task to acquire reliable and high-quality FTIR data due to the dramatic infrared (IR) adsorption of water molecules in aqueous electrolytes (Li et al., 2020 >; Nayak et al., 2018 >). A well designed in situ FTIR cell coupled with a reasonable IR reflection mode will be an effective approach to avert the overwhelmingly weak IR signal of interest in the background IR adsorption of aqueous solutions during in situ FTIR tests (Zhang et al., 2014 >; Nakamura & Nakato, 2004 >; Zhang et al., 2018 >). Indeed, a very delicate IR window of ZnSe or diamond single-crystal prisms, which possesses a unique trapezoid section, has been carefully developed for attenuated total reflection infrared (ATR-IR) spectroscopy tests, where the incident IR beam would undergo multiple total reflections within the ZnSe/diamond prisms before being collected by deuterated triglycine sulfate (DTGS) or mercury cadmium telluride (MCT) detectors (Nakamura & Nakato, 2004 >; Zhang et al., 2018 >; Nayak et al., 2013 >). Based on a traditional thermal IR source (Globar), weak IR signals of the targeted surface species can be effectively intensified by approximately one order of magnitude through a multiple reflection mode, which is beneficial for acquiring high-quality FTIR data during in situ tests. It is notable that a special optical system is highly imperative for realizing multiple total IR reflections within the ZnSe or diamond single-crystal prisms, which undoubtedly complicates the operation process of the ATR-IR measurements (Zhang et al., 2018 >; Li et al., 2020 >). Moreover, direct deposition of catalysts onto the IR cell window of ZnSe or diamond prisms as film-like electrodes is required for an effective ATR-IR test, which is difficult and inconvenient to realize for common powdered catalysts. Relative to conventional Globar sources, a synchrotron radiation (SR) IR source shows substantial merits of two to three orders of magnitude greater brightness for SR-based FTIR (SR-FTIR) spectroscopy when the IR aperture size is cut down to a scale of several micrometres (Hu et al., 2020 >; Li et al., 2020 >; Lee et al., 2020 >; Kamel et al., 2021 >). Therefore, based on the brighter synchrotron IR source, it is potentially possible to acquire high-quality FTIR spectra under aqueous conditions via a single-reflection mode. For the implementation of effective in situ SR-FTIR tests, the design of a smart in situ FTIR cell that is compatible with SR IR beamline configurations and the single-reflection mode is absolutely imperative (Diaz-Lopez et al., 2020 >; Li et al., 2020 >), but has been rarely reported. Herein, a novel bifunctional in situ FTIR cell has been elaborately designed and developed for practical in situ SR-FTIR measurements. It is compatible with a single-reflection synchrotron FTIR mode concurrently developed by our group and successfully commissioned at the IR beamline BL01B of National Synchrotron Radiation Laboratory (NSRL), China. Thanks to a unique screw assembly of the IR window, the water film over the surface of working electrodes can be facilely and rationally tuned on a micrometre scale in the well designed in situ FTIR cell. Moreover, dual electrolyte/gas channels within the in situ FTIR cell enable it to act as a bifunctional in situ cell for tracking the surface dynamics of catalysts in various electrochemical reactions of interest that may or may not involve gaseous reactants. Taking commercial benchmark IrO2 catalysts as an example, we promptly track the reactive intermediate evolution over the surface of IrO2 catalysts during the intriguing electrochemical oxygen evolution reaction (OER) using the well designed in situ FTIR cell coupled with the developed single-reflection SR-FTIR method. Based on the in situ FTIR observations, it is revealed that the formation of a key *OOH intermediate during the OER may be promoted by the newly formed surface Ir--O bonds during the OER, resulting in fast OER kinetics for commercial IrO2 catalysts in alkaline solution. This result demonstrates the feasibility of the developed in situ synchrotron FTIR spectroscopic method in dynamic studies of advanced catalysts during electrocatalysis. Compared with traditional FTIR spectroscopy, SR-FTIR with single-reflection IR mode is a more promising approach to acquire reliable and effective IR signals of key intermediates over an electrode surface during electrochemical reactions in a relatively easy operation manner. 2. In situ FTIR cell 2.1. Schematic design As shown in Figs. 1 >(a) and 1(b), the well designed in situ FTIR cell consists of four main parts: base, main body, top cap and IR window. All these components are processed using polyether ether ketone (PEEK) as the original material, which possesses robust acid-base resistance (pH = 1-14) and can work normally under a wide temperature range of -100degC to 260degC (Ma et al., 2020 >; Ling et al., 2020 >). For the IR window, a round ZnSe film, of diameter ~14 mm and thickness ~1 mm, is fixed at the end of a hollow cylindrical PEEK tube; IR radiation can pass through the tube and ZnSe film from the inside to easily arrive at the working electrode. The working electrode is prepared by homogeneously coating catalysts onto a square hydrophobic conductive carbon cloth, which will be placed as close as possible to the ZnSe film of the IR window [Fig. 1 >(b)]. Note that the IR window is attached to the main body via a thread assembly, where a special thread spacing of approximately 0.2 mm is adopted; a thread feed of about 5 mm is expected to move the IR window forward/backward to/from the working electrode with every 10deg rotation of the cylindrical IR window. This is very helpful to precisely adjust the thickness of the electrolyte layer among the ZnSe film of the IR window and the working electrode in the in situ FTIR cell. As presented in Fig. 1 >(a), there is an electrolyte chamber within the main body of the cell with a volume of approximately 3.0 ml around the assembled IR window. Coupled with the electrolyte inlet and outlet, a flow field of electrolytes surrounding the catalyst layer of the working electrode is created in the main body [Fig. 1 >(b)], which is beneficial for avoiding the mass transport limitation during electrolysis (Li et al., 2020 >; Chen et al., 2020 >). Moreover, a gas transport channel is situated just below the working electrode [Fig. 1 >(b)], from which gaseous products during the electrocatalytic process can readily escape. In the meantime, if necessary, gaseous reactants can be pumped into the cells directly through the gas channel and are then easily accessible to the working electrodes during electrocatalytic reactions that involve gaseous reactants; for example, electrochemical O2 reduction, CO2 reduction and N2 fixation reactions. The well designed electrolyte and gas channels enable the in situ FTIR cell to act as a bifunctional cell for dynamic studies of various important electrocatalytic processes with or without gaseous reactants. In addition, a reference electrode and a counter electrode are placed in the electrolyte chamber through the designed holes in the main body, which will be coupled with the working electrode to form the desirable three-electrode system for the in situ electrochemical measurements. 2.2. Entity The components of the base, main body and top cap can be easily assembled to generate the preliminary in situ FTIR cell using four screw fasteners, as shown in Fig. 1 >(c). It is worth noting that the square working electrode with length size of 40 mm will be positioned between the electrolyte chamber and the gas channel, where the hydrophobic carbon-cloth substrate can serve as a useful membrane to prevent the aqueous solution escaping from the electrolyte chamber (Nagy et al., 2020 >; Xing et al., 2021 >). Meanwhile, a translucent rectangular silicone rubber washer is coupled with the carbon-cloth substrate to further seal the electrolyte chamber and avoid leakage of aqueous electrolyte. Copper tape is placed on top of the carbon-cloth substrate and is tightly connected to it through the four fastening screws; this will serve as the current collector bridging the working electrode and the electrochemical workstation. The IR window is directly screwed into the main body via a thread assembly, where the distance between the IR window and working electrode is tunable by adjusting the number of turns and rotation angle. To clearly depict each component, the assembled in situ FTIR cell is placed upright, rather than lying down, on a table in the right-hand image of Fig. 1 >(c). The two holes on the left-hand side of the assembly are for connecting to a peristaltic pump or gas cylinder via small plastic pipes to form the electrolyte circulating or gas pumping systems, respectively, while the holes on the top of the assembly are for Ag/AgCl reference and carbon counter electrodes. Accordingly, a light and handy cubic-like in situ FTIR cell of size ~60.0 mm x 60.0 mm x 29.0 mm (length x width x height) is developed, which is very easy to assemble and transport, for in situ FTIR measurements. 3. Implementing in situ FTIR measurements 3.1. Materials The commercial IrO2 catalysts, Nafion 117 (~5 wt%) and KOH reagent (99.9%) were purchased from Sigma-Aldrich. The carbon and Ag/AgCl electrodes were obtained from Gaossunion Photoelectric Technology Co. Ltd, Tianjin, China. The hydrophobic carbon cloth (HCP330P) and conductive copper tape were bought from Shanghai Hesen Electrical Co. Ltd, China. The peristaltic pump and small plastic pipes were obtained from Shanghai Kamoer Co. Ltd, China. Nitrogen (99.9%) was supplied by Nanjing Special Gas Co. Ltd, China. Laboratory-made deionized water (DI) was used in electrode preparation and electrochemical measurements. All chemicals and commercial IrO2 catalysts were used directly without further purification or treatments. 3.2. Electrochemical measurement Electrochemical oxygen evolution measurements were carried out in a 1.0 M KOH aqueous solution in the in situ FTIR cell using a three-electrode electrochemical workstation (CHI760D, CH Instruments), where commercial IrO2 catalysts, carbon rod and Ag/AgCl were adopted as the working, counter and reference electrodes, respectively. All the potentials were converted using a reversible hydrogen electrode (RHE) based on the following equation: E RHE = E Ag/AgCl + (0.059 x pH) + 0.197 V (Garcia & Koper, 2018 >; Brewer et al., 2012 >). The overpotential during the OER was determined using the following equation: e = E RHE - 1.23 V (Cheng et al., 2020 >; Garcia & Koper, 2018 >). Linear sweep voltammetry (LSV) curves were recorded in the potential range 1.0-1.7 V versus RHE at a scan rate of 10 mV s-1. Regarding the preparation of the working electrode, 5 mg IrO2 catalysts were dispersed into a mixed solution of 250 mL DI water, 700 mL ethanol and 50 mL Nafion solution via ultrasonication for 30 min. Afterwards, 900 mL of the resultant catalytic ink was loaded onto a piece of carbon cloth (4 cm x 4 cm) with an effective loading area of 3 cm x 3 cm and a loading mass of 0.5 mg cm-2. This as-obtained IrO2 catalyst supported on carbon cloth served as the working electrode after drying naturally at room temperature for 6 h. 3.3. FTIR spectra collection 3.3.1. Measurement setup An in situ single-reflection synchrotron FTIR method has been developed by our group for dynamic studies over the solid-liquid interface of electrodes during electrolysis, based at the infrared beamline BL01B of Hefei Light Source (HLS) at NSRL, China (Su et al., 2020 >; Cheng et al., 2019 >). After the upgrade of HLS in 2016, this infrared beamline was reconstructed with upgraded station performance, where IR radiation is extracted from a bending magnet with a superior acceptance angle of 65 mrad x 55 mrad (H x V) and excellent flux of approximately 1013 photons s-1 (0.1% bandwidth)-1 (Hu et al., 2020 >). Moreover, an advanced Bruker 70v FTIR spectrometer (Bruker Corporation, Ettlingen, Germany) with a KBr beam-splitter and various detectors (a liquid-nitrogen-cooled MCT detector was used here) are available at this IR endstation. By coupling with an infrared Bruker Hyperion 3000 microscope, it can provide a broad range of ~650-4000 cm-1 and a high spectral resolution of 0.25 cm-1 for IR spectroscopy measurements at the micrometre scale. As shown in Fig. 2 >(a), the in situ FTIR cell lies on the working platform of the microscope, with the IR window facing up and close to the objective of the microscope. For the electrolyte circulation system, a peristaltic pump is connected to the in situ cell using small plastic pipes; 150 mL 1.0 M KOH electrolyte contained in a glass bottle forms the external solution reservation. The working electrode is connected to the electrochemical workstation using copper tape, as presented in Figs. 2 >(a) and 2(c), where the counter and reference electrodes are linked to the electrochemical workstation simultaneously. It is noted that the incident IR radiation is almost perpendicular to the surface of the working electrode with a unique incident angle of about 83deg, and the reflected IR signals will be collected directly after a single reflection on the surface of the catalysts [Fig. 2 >(b)]. The IR signals are then recorded and analyzed by the IR signal collection system, and the desirable FTIR spectra are displayed on a computer screen. As illustrated in Fig. 2 >(c), the IR window of the in situ cell is tight to the objective of the microscope, which is useful for shortening the IR light path in air to mitigate the influence of external noise, and is also beneficial for precisely focusing the incident IR beam on the surface of the targeted catalysts. 3.3.2. Measurement procedure Before in situ FTIR measurements were made, highly reflective Au substrate was placed at the catalyst position in the in situ cell (Fig. 1 >) without electrolyte and the corresponding FTIR spectrum was collected as a reference spectrum in single-reflection mode. Subsequently, FTIR spectra of targeted samples are measured under similar conditions to clarify the intrinsic IR characteristic peaks of the targeted catalysts. During the in situ FTIR tests, a FTIR spectrum of the targeted catalyst is firstly collected for the catalyst in electrolyte without applied potentials; this serves as the background spectrum for those acquired under various applied potentials during the electrochemical process. For each selected potential, a constant potential method is adopted, where the potential is applied to the working electrodes in the circulating KOH electrolyte for about 20 min before collection of the FTIR spectrum. As an example, the surface dynamics of commercial IrO2 catalysts during the OER process are promptly monitored using the in situ FTIR cell coupled with the developed single-reflection synchrotron FTIR method. The KOH electrolyte in the glass bottle is first bubbled with nitrogen gas for 30 min to fully purge the dissolved O2 in the solution before commencing electrolyte circulation. To acquire high-quality and reliable FTIR data, the working electrode is placed tight to the ZnSe film of the IR window in the in situ cell, where there is a water film thickness of about 10 mm over the working electrode surface due to capillary permeation of the porous carbon-cloth substrate. Meanwhile, the incident IR beam is meticulously focused on the targeted IrO2 catalysts that are supported on the carbon-cloth, with a micrometre-scale rectangular light spot of 10 mm x 10 mm. Subsequently, LSV curves were recorded for the IrO2 catalysts during the OER to carefully determine suitable applied voltage parameters for the in situ FTIR measurements. In general, FTIR spectra are collected at: the open circuit voltage (OCV); the pre-catalytic stage; the onset potential (corresponding to 1 mA cm-2); the highly efficient catalytic stage. During the in situ FTIR test, a constant potential method is adopted and the electrolyte in the in situ cell is kept in close circulation with a flow rate of 50 mL s-1. Typically, for the collection of each FTIR spectrum, the selected constant potential is applied to the working electrodes in the circulating KOH electrolyte for 20 min as a pre-treatment step. After that, the FTIR spectra are collected for the IrO2 catalysts in the range 650-4000 cm-1 with a step interval of 2.0 cm-1 by averaging 256 scans. Moreover, the acquired FTIR data are further processed using OPUS software (Bruker Optik GmbH, Ettlingen, Germany) to obtain a series of desirable transmission FTIR spectra (Ji et al., 2020 >; Kong & Liu, 2021 >). 4. Results The LSV curve in Fig. 3 > shows that the commercial rod-like IrO2 catalyst has high OER activity in alkaline solution with an onset potential of 1.55 V versus RHE (at 1 mA cm-2) and a good overpotential (e) of about 0.41 V at 5 mA cm-2 (corresponding to a potential of 1.64 V versus RHE), which is consistent with previous reports (Plevova et al., 2021 >; Zheng et al., 2022 >; Zagalskaya & Alexandrov, 2020 >). This result confirms the practicability of the in situ FTIR cell for electrochemical measurements when electrolyte circulation is rationally used. Based on the LSV results in Fig. 3 >, the following suitable voltage parameters for the subsequent in situ FTIR test are carefully determined: the OCV (point A; 1.29 versus RHE); the potential at the pre-catalytic stage (point B; 1.40 versus RHE); the onset potential (point C; 1.55 V versus RHE); and the potential at 5 mA cm-2 (point D; 1.60 V versus RHE). For the FTIR spectra illustrated in Fig. 4 >, there are three dominant IR vibrational bands located at ~2030, 2160 and 2320-2360 cm-1 for the IrO2 catalysts at OCV, which are well assigned to the stretching modes of bridge-bonded and linear-bonded *CO on the surface of the carbon-based substrate and the stretching model of gaseous CO2 in air, respectively (Pate et al., 2022 >; Zhang et al., 2021 >; Zeng et al., 2022 >). When the potential is increased to 1.40 V, no additional vibrational band is obtained for IrO2 catalysts relative to that at OCV, indicating that the surface adsorbed species of electrodes at the pre-catalytic stage are similar to those under OCV. Notably, a relatively weak IR vibrational band appears at about 801 cm-1 at the enhanced potential of 1.55 V, which may be derived from post-formed Ir--O bonds on the surface of the IrO2 catalyst (Su et al., 2021 >; Cao et al., 2019 >; Jiang et al., 2020 >). Interestingly, a strong newly formed IR vibrational band is concurrently observed at about 1079 cm-1 at 1.55 V, which is attributed to the formation of a key *OOH intermediate on the surface of the IrO2 catalysts (Su et al., 2021 >; Zhang et al., 2014 >; Czioska et al., 2021 >). Moreover, these IR vibrational bands at 801 and 1079 cm-1 are retained when the applied potential is further increased to 1.60 V. These results suggest that the in situ formed Ir--O bonds on the surface of the IrO2 catalyst can effectively trigger the formation of the key *OOH during OER for efficient electrochemical oxygen evolution, where the generation of the key *OOH intermediate may be the rate-determining step for the IrO2 catalyst during OER in alkaline solution (Czioska et al., 2021 >; Naito et al., 2021 >; Jiang et al., 2020 >). To exclude the influence of irreversible structural changes of the catalyst itself, the applied potential is restored to OCV from 1.60 V for the IrO2 catalyst (Su et al., 2021 >; Ji et al., 2020 >). It can be seen in Fig. 5 > that the IR vibrational bands at ~801 and 1079 cm-1, associated with surface Ir--O bonds and the key *OOH formation, have completely disappeared. Moreover, FTIR spectroscopy under the restored OCV shows a similar trend in shape and frequency to that at OCV. This indicates the reversible surface species evolution over the solid-liquid interface of electrodes during OER, and further confirms the formation of the key *OOH intermediate during OER for the IrO2 catalyst. Accordingly, it can be revealed that the potential-dependent surface Ir--O bonds may be very helpful for promoting the generation of the key *OOH intermediation during OER, resulting in fast 4 kinetics for the IrO2 catalyst in alkaline solution. 5. Conclusion A novel bifunctional in situ FTIR cell has been meticulously designed and prepared, and is compatible with synchrotron-radiation-based IR sources. With well designed IR window and dual electrolyte/gas channels, this in situ FTIR cell can be potentially adopted in surface dynamics studies during various electrochemical processes with or without gaseous reactants. By coupling with a single-reflection synchrotron FTIR method, the surface species evolution of commercial benchmark IrO2 catalyst is promptly monitored during OER using the in situ FTIR cell. The feasibility and practicability of the in situ FTIR cell in dynamics studies are rationally demonstrated by the successful observation of the key *OOH intermediate over the IrO2 electrode surface during OER. The in situ synchrotron FTIR spectroscopic method developed in this work would be very helpful for surface dynamic investigations during important electrocatalytic processes, such as electrocatalytic hydrogen evolution, CO2 reduction, N2 fixation etc. Figure 1 Schematics of the in situ FTIR cell observed from the side (a) and from the front (b). (c) Photographs of the in situ FTIR cell parts (left) and fully assembled (right). Figure 2 (a) Photograph of the equipment used for the in situ SR-FTIR measurements. (b) Enlarged photograph of the testing microscope instrumentation. (c) Enlarged photograph of the in situ cell, where WE, CE and RE are the working, counter and reference electrodes, respectively. Figure 3 LSV curve recorded using the in situ cell without IR correction. The insert shows a transmission electron microscope image of the commercial IrO2 catalysts. Figure 4 FTIR spectra of the commercial IrO2 catalysts during the OER. 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PMC10000799 | J Synchrotron Radiat J Synchrotron Radiat J. Synchrotron Rad. Journal of Synchrotron Radiation 0909-0495 1600-5775 International Union of Crystallography 36891850 rv5166 10.1107/S1600577522011833 JSYRES S1600577522011833 Research Papers Development of serial X-ray fluorescence holography for radiation-sensitive protein crystals Developing sXFH for radiation-sensitive protein crystals Ang Artoni Kevin R. a*++ Umena Yasufumi b Sato-Tomita Ayana c Shibayama Naoya c Happo Naohisa d Marumi Riho a Yamamoto Yuta a Kimura Koji a Kawamura Naomi e Takano Yu f Matsushita Tomohiro g Sasaki Yuji C. h Shen Jian-Ren i Hayashi Kouichi ae a Department of Physical Science and Engineering, Nagoya Institute of Technology, Gokiso, Showa, Nagoya 466-8555, Japan b Synchrotron Radiation Research Center, Nagoya University, Furo, Chikusa, Nagoya 466-8603, Japan c Division of Biophysics, Department of Physiology, Jichi Medical University, Yakushiji, Shimotsuke, Tochigi 329-0498, Japan d Department of Computer and Network Engineering, Graduate School of Information Sciences, Hiroshima City University, Asa-Minami-ku, Hiroshima 731-3194, Japan e Japan Synchrotron Radiation Research Institute (JASRI), Sayo, Hyogo 679-5198, Japan f Graduate School of Information Sciences, Hiroshima City University, Asa-Minami-ku, Hiroshima 731-3194, Japan g Graduate School of Science and Technology, Nara Institute of Science and Technology, Ikoma 630-0192, Japan h Graduate School of Frontier Sciences, University of Tokyo, Kashiwa, Chiba 277-8561, Japan i Research Institute for Interdisciplinary Science and Graduate School of Natural Science and Technology, Okayama University, Tsushima Naka, Okayama 700-8530, Japan Strange R. W. Editor University of Essex, United Kingdom Correspondence e-mail: [email protected] ++ Current address: Toyota Technological Institute, Nagoya, Japan. 01 3 2023 20 1 2023 20 1 2023 30 Pt 2 s230200 368378 17 7 2022 12 12 2022 (c) Artoni Kevin R. Ang et al. 2023 2023 This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-BY) Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited. A full version of this article is available from Crystallography Journals Online. In this work, serial X-ray fluorescence holography is developed and its capabilities are demonstrated by obtaining hologram patterns from the protein crystal Photosystem II before any signs of radiation-induced damage. This new technique paves the way for future experiments on protein crystals that aim to clarify the local atomic structure of their functional metal clusters. X-ray fluorescence holography (XFH) is a powerful atomic resolution technique capable of directly imaging the local atomic structure around atoms of a target element within a material. Although it is theoretically possible to use XFH to study the local structures of metal clusters in large protein crystals, the experiment has proven difficult to perform, especially on radiation-sensitive proteins. Here, the development of serial X-ray fluorescence holography to allow the direct recording of hologram patterns before the onset of radiation damage is reported. By combining a 2D hybrid detector and the serial data collection used in serial protein crystallography, the X-ray fluorescence hologram can be directly recorded in a fraction of the measurement time needed for conventional XFH measurements. This approach was demonstrated by obtaining the Mn Ka hologram pattern from the protein crystal Photosystem II without any X-ray-induced reduction of the Mn clusters. Furthermore, a method to interpret the fluorescence patterns as real-space projections of the atoms surrounding the Mn emitters has been developed, where the surrounding atoms produce large dark dips along the emitter-scatterer bond directions. This new technique paves the way for future experiments on protein crystals that aim to clarify the local atomic structures of their functional metal clusters, and for other related XFH experiments such as valence-selective XFH or time-resolved XFH. X-ray fluorescence holography atomic structures protein structures Japan Society for the Promotion of ScienceJP16KT0058 JP17H06372 17H05219 26105005 26105006 20H05878 20H05881 20H05883 20H05884 This work was supported by the Japan Society for the Promotion of Science (JSPS) Grants-in-Aid for Scientific Research (grant nos. JP16KT0058; JP17H06372) and Grant-in-Aid for Scientific Research on Innovative Areas '3D Active-Site Science' (grant nos. 17H05219; 26105005; 26105006) and Grant-in-Aid for Transformative Research Areas (A) 'Hyper-Ordered Structures Science' (grant nos. 20H05878; 20H05881; 20H05883; 20H05884). The XFH experiments were performed in the SPring-8 Synchrotron Radiation Facility in Japan with the approval of JASRI (proposal nos. 2015A1071; 2016A1391; 2016A2514; 2016B1328; 2017A2529; 2017A1137; 2017B1355; 2018A1097; 2018A2519; 2018B1153; 2019A1230; 2019B1149; 2019B1151). pmc1. Introduction Atomic resolution holography techniques, such as photoelectron holography, X-ray fluorescence holography and neutron holography, are a family of powerful experimental techniques that allow the direct probing of the local structures around atoms of a target element (Daimon, 2018 >, 2020 >). These techniques are capable of simultaneously recording the intensity and phase of the scattered beams, allowing the direct, model-free extraction of the 3D positions of the scatterer atoms around the emitter atoms. X-ray fluorescence holography (XFH) (Tegze & Faigel, 1996 >; Faigel & Tegze, 1999 >; Hayashi et al., 2012 >; Hayashi & Korecki, 2018 >), in particular, is a robust and powerful technique that has been used to reveal the local structures around active sites in various functional materials. In XFH, atoms of a target element are excited by an incident X-ray, which then emit fluorescent X-rays. These fluorescent X-rays act as a reference wave, which is then scattered by the surrounding atoms (object wave). The interference between the reference and object waves stores information about the 3D arrangement of the scatterers. This interference pattern can be treated as a hologram pattern, where the 3D arrangement of atoms can be directly reconstructed using Barton's method (Barton, 1991 >), which is a Fourier transform based algorithm, or advanced reconstruction algorithms such as the L1-regularized linear regression method (Matsushita, 2018 >). While almost all reported XFH experiments have been on inorganic functional materials, XFH is also expected to be capable of resolving the local structures around metal atoms in organic materials. This was first proposed back in 1996 (Fadley & Len, 1996 >); however, experimental difficulties in dealing with organic samples have significantly stalled progress in this field. These difficulties included low metal concentrations, large complicated unit cells, samples consisting mostly of light elements and the susceptibility of these samples to radiation damage. However, recent advances in synchrotron radiation facilities, X-ray detectors and cryogenic cooling have provided possible solutions to these challenges. Work on adapting XFH for organic samples started recently, with the first bioXFH setup for protein crystal samples developed and tested on human hemoglobin (Hb) crystals in 2016 (Sato-Tomita et al., 2016 >). The experimental difficulties were addressed by using large P41212 Hb crystals, a toroidal graphite energy analyzer crystal to collect and focus the fluorescent X-rays, an N2 cold-gas flow system and the introduction of a new kh-circle stage and sample holder. While the experiment successfully prevented radiation damage to the protein crystal, the atomic reconstruction has not yet been fully interpreted. The resulting reconstructed atomic image is a complicated superposition of the 16 different Fe local environments, requiring more sophisticated methods of analysis. A more recent attempt on sperm whale myoglobin, a simpler crystal with the space group P21, successfully obtained the reconstructed image (Sato-Tomita et al., 2022 >). In this work, the atomic image around the Fe heme was reconstructed from the measured holograms, and some of the atomic images reflected the actual atomic positions. Recently, clear atomic reconstruction was obtained and interpreted from XFH experiments on the organic superconductor k-(BEDT-TTF)2Cu[N(CN)2]Br or k-Br (Ang et al., 2021 >). k-Br is an organic charge-transfer salt that is widely studied in strongly correlated electron physics, where an Anderson-type metal-insulator transition is induced by the introduction of random defects by X-ray irradiation (Sano et al., 2010 >). Atomic reconstructions from XFH, molecular dynamics simulations and hologram calculations have shown experimental evidence for the previously proposed 'bond-shifted' model (Kang et al., 2017 >). Diffraction images and resistivity measurements before and after XFH experiments showed no additional radiation damage. Contrary to the complicated local structures around the Fe heme in hemoglobin or myoglobin, there are only four inequivalent Cu sites in k-Br and they all lie in the anion layer, significantly simplifying the interpretation of the atomic reconstructions. To date, all bioXFH experiments reported have been performed using the bioXFH apparatus, in which the angular dependence of the fluorescent X-rays is recorded as the sample is rotated point-by-point along the polar and azimuthal angles, taking several hours to record a full hologram pattern (Sato-Tomita et al., 2016 >, 2022 >; Ang et al., 2021 >). For more robust crystals like hemoglobin or myoglobin, cooling the sample to 100 K and controlling the photon flux enable collection of the hologram pattern before the onset of radiation-induced global damage. No changes in the diffraction patterns were observed after recording the holograms. However, at these dosage levels, specific damage around water molecules and active redox sites can still occur (Garman, 2010 >), and this bioXFH apparatus is no longer appropriate for these kinds of sensitive protein crystals. An example of sensitive protein crystals is the protein membrane complex Photosystem II [PSII (Dau & Haumann, 2008 >; Shen, 2015 >)]. The oxygen-evolving complex (OEC) of PSII is the catalytic center of the photosynthetic oxidation of water by green plants. In this process, the OEC, which consists of an Mn4CaO5 cluster, cycles through a series of redox states as described by the Kok cycle, where these states are designated as S i (where i = 0-4). The structure of PSII, and its OEC, have been extensively studied by synchrotron X-ray diffraction (XRD) with gradually increasing resolution (Zouni et al., 2001 >; Kamiya & Shen, 2003 >; Ferreira et al., 2004 >; Guskov et al., 2009 >; Umena et al., 2011 >). The Mn4CaO5 cluster consists of a cubane-like structure, with one Ca and three Mn atoms (Mn1D, Mn2C, Mn3B) occupying four corners, and O occupying the other four, while the last Mn atom (Mn4A) is connected to the cubane by two di-m-oxo-bridges [Fig. 1 >]. Although the PSII structure obtained at 1.9 A resolution provided remarkable details (Umena et al., 2011 >), the average Mn-ligand and Mn-Mn distances were slightly longer than those extracted from extended X-ray absorption fine structure [EXAFS (Yano, Pushkar et al., 2005 >; Dau et al., 2008 >; Glockner et al., 2013 >)]. Recently, the 'low-dose' structure of PSII was revealed using extremely low-dose synchrotron XRD (Tanaka et al., 2017 >), and the 'damage-free' structure was obtained from a combination of large crystals and the femtosecond X-ray pulses of an X-ray free-electron laser (XFEL) (Suga et al., 2015 >), which provided shorter Mn-Mn distances, consistent with those obtained from EXAFS studies. The valence of each Mn ion in the Mn4CaO5 cluster of PSII S1 [2 x Mn(III), 2 x Mn(IV)] have been asserted based on the results of experiments such as X-ray absorption spectroscopy [XAS (Dau et al., 2008 >; Glockner et al., 2013 >; Roelofs et al., 1996 >; Yachandra et al., 1996 >; Robblee et al., 2001 >], Fourier transform infrared spectroscopy (Chu et al., 2001 >, 2004 >; Debus et al., 2005 >), various electron paramagnetic resonance (EPR) spectroscopy techniques (Kulik et al., 2007 >; Cox et al., 2011 >; Stich et al., 2011 >; Asada et al., 2013 >), through the analysis of the Jahn-Teller distortion effects from XRD studies (Suga et al., 2015 >) and from theoretical calculations. These results suggest that the typical doses in protein crystallography experiments reduce, or partially reduce, the Mn ions and change the local structure around Mn, making the determination of the structural arrangement and the valence states of Mn difficult. In this work, we develop a novel approach to determine the local structure of the metal clusters in protein crystals. By adapting XFH for low doses, the local structure of these metal clusters can be directly obtained. To minimize X-ray exposure of the crystals during the XFH measurements, the holograms can be directly imaged using a 2D hybrid pixel detector allowing much faster data acquisition. We have previously demonstrated the direct imaging of the Fe Ka holograms of the mixed-valence compound magnetite [Fe3O4 (Ang et al., 2018 >)]. Building from this work, the principles of serial crystallography were incorporated into the experiment to further reduce the X-ray exposure of the protein crystal samples. This new approach is demonstrated on large PSII S1 crystals, where the crystal structure and Mn valence states are already well established in the literature. By scanning the irradiation point across the surface of several PSII crystals, the holograms were directly imaged using a 2D hybrid pixel detector prior to the onset of X-ray-induced reduction of the metal clusters. By interpreting the holograms in terms of dips in the fluorescent X-ray intensity in the forward-scattering directions, real-space projections of the arrangement of the atoms in the Mn clusters were obtained. Furthermore, we attempted valence-sensitive XFH by tuning the incident X-ray energy based on the small shifts in the Mn K-edge of the different Mn ions. The real space projections in the Mn(III) and Mn(IV) holograms showed modest indications of the different local structure around these ions. These results demonstrate a new and straightforward approach for XFH experiments on highly sensitive protein crystals and the future possibility of simultaneous valence-selective XFH experiments on the metal clusters in protein crystals. 2. Material and methods 2.1. X-ray fluorescence holography measurements Serial X-ray fluorescence holography (sXFH) experiments were performed on BL39XU of SPring-8, Japan, using X-rays focused by Kirkpatrick-Baez (KB) mirrors (Suzuki et al., 2013 >). Using the KB mirrors and a variable Al film attenuator, the beam spot size and the X-ray photon flux were set to 7 mm x 10 mm and ~2 x 109 photons s-1, respectively. To perform sXFH experiments, the bioXFH apparatus previously described by Sato-Tomita et al. (2016 >) was modified to allow the scanning of the irradiation point, and the use of a 2D X-ray detector. A schematic of the sXFH apparatus is shown in Fig. 2 >. The sXFH apparatus consists of a precision motorized four-axis (YZ-swivel-tilt) sample stage that is mounted on the kh-circle stage of the bioXFH apparatus (Sato-Tomita et al., 2016 >). This entire assembly is mounted on a 2th goniometer, where the sample stage assembly and the detector assembly can be rotated independently. The samples are cooled to 100 K using a liquid-nitrogen gas-flow system (Cryostream 800, Oxford Cryosystems, Inc.) with the cryostream nozzle set according to the protocols for cryogenic X-ray crystallography. The scanning of the irradiation point necessary for these experiments is incompatible with inverse-mode XFH, where the sample is rotated point-by-point along the th axis (0-75deg) and ph axis (0-360deg) as the fluorescent X-ray is recorded. In normal-mode XFH, the hologram can be directly imaged with a hybrid pixel detector (Ang et al., 2018 >; Bortel et al., 2019 >), allowing faster data acquisition. To record the holograms, a Medipix3-based quad chip version of the Merlin 2D detector system (Quantum Detectors) was used (Plackett et al., 2013 >; Ballabriga et al., 2018 >). This is the same detector used in a previous valence-sensitive normal-mode XFH experiment (Ang et al., 2018 >), and in a related experiment on Kossel lines (Bortel et al., 2016 >; Faigel et al., 2016 >). The detector features high spatial resolution and dynamic range (256 x 256 pixels x 24 bit at 110 mm pixel size), which makes it suitable for holography experiments. The much larger EIGER X 1M 2D detector has also been used in a normal-mode XFH apparatus optimized for speed, where statistically relevant Ni holograms from an NiO crystal were recorded in a single image taken in 1 s (Bortel et al., 2019 >). However, the Merlin quad chip detector allows two simultaneous energy thresholds to measure photons in a narrow-energy-window mode, which allows the user to record element-selective holograms from more complicated samples. In this setup, the Merlin detector was set 30 mm from the sample. A Cr filter (4 mm film on a 8 mm Kapton sheet) was placed in front of the detector array to block the incident X-rays. Additional shielding was also set in front of the detector to prevent unwanted scattering reaching the detector. 2.2. Data collection Each PSII crystal is scanned along the y and z directions as indicated in Fig. 2 >(a), with step sizes of 30 mm and 40 mm, respectively, to distribute the dosage and minimize radiation damage (Yano, Kern et al., 2005 >). Images of the fluorescence intensity patterns were recorded at each point at an integration time of 10 s, resulting in an average dose of 0.15 MGy per point, as calculated using the program RADDOSE (Zeldin et al., 2013 >). Table 1 > summarizes the experimental conditions of the sXFH measurements on PSII. The fluorescence intensity patterns were recorded using the 24 bit differential mode of the Merlin detector, with two simultaneous energy thresholds set at 5.15 keV and 6.15 keV. The fluorescence spectra at the detector position were confirmed with an SDD detector (XR-100SDD, Amptek Co. Ltd). A typical fluorescence spectrum from a PSII crystal, taken at an incident X-ray energy of 6.565 keV, is shown in Fig. 3 >(a), where the Mn Ka and Ca Ka peaks and a peak from the incident X-ray can be clearly observed. The Ca Ka peak contains signals from both the Ca atoms in the OEC of PSII and the Ca atoms in the cryoprotectant. An additional fluorescence peak at ~4.5 keV was also detected and was attributed to the Litholoop. Aside from the energy-windowing from the simultaneous energy thresholds, a Cr thin-film filter was also placed in front of the detector to block the scattered incident X-rays. 2.3. Hologram data processing Before data processing, the raw images (energy-windowed 5.15-6.15 keV) obtained from each PSII crystal are first integrated, and any hot or dead pixels are removed using a 3 x 3 median filter. Figs. 3 >(b)-3(e) show a typical hologram pattern obtained from a PSII crystal at different stages of data processing. To extract the hologram pattern from the fluorescence images, several data processing steps are needed. These are described in more detail by Ang et al. (2018 >); Bortel et al. (2019 >); and Matsushita, Muro, Matsui et al. (2020 >). First, all the images taken while scanning the irradiation point from each sample are integrated [Fig. 3 >(b)]. Then, each image (I n ) is normalized using a normalizing pattern (P), obtained by integrating several images taken as the sample is slowly rotated in-plane. By rotating the sample, the holographic signal is averaged out in P, and only the low-frequency fluorescence background attributed to the sample-detector geometry remain. By normalizing each image pixel-by-pixel, H n = I n /P [Fig. 3 >(c)]; this low-frequency background and any inhomogeneity in the sensitivity of the detector pixels can be removed. An additional histogram filter was applied to reduce noise. The images H n , which are gnomonic projections, 2D projections of the spherical hologram pattern on the detector surface, are converted to a spherical projection [Fig. 3 >(d)]. To further remove low-frequency fluorescence background signals, the images are flattened by applying a Gaussian convolution on the images (G n ), and dividing the original image with the background kh n = H n /G n . The images are then rotated based on their orientations, as determined from the indexing of the diffraction images obtained from each sample. At this point, the fluorescence intensity oscillates around 1. The final hologram fragments from the different samples are composed into one hologram kh. Finally, a constant 1 is subtracted, and a low pass filter (s = 10deg) is applied. To expand the recorded hologram in k-space, symmetry operations were applied based on the symmetry of the PSII S1 crystal. 2.4. Hologram pattern simulations The intensity of the X-ray fluorescence holograms, kh(k), is expressed as where r e is the free electron radius, f j is the atomic structure factor of the jth atom, and th rj is the angle between k and r j . For organic materials, however, the dynamic fluctuations caused by thermal vibrations are stronger than in inorganic materials, making it necessary to consider the effects of thermal vibrations. Thermal vibrations can be considered as atoms oscillating around their ideal positions with a Gaussian distribution with a standard deviation, s, given as <u 2> /6 = s2/2. This introduces an additional term in the holographic oscillation (Matsushita, Muro, Yokoya et al., 2020 >), To simplify the calculations and reduce the computational times, the holograms were calculated using clusters constructed from only the Mn, O and Ca atoms within the OEC of PSII, based on atomic positions in the PDB entry 3wu2. For the total Mn Ka holograms, 32 clusters, each centered on an Mn emitter atom, were created and used for the calculations. The angular positions (th, ph) of the forward scattering (FS) dips for all Mn and Ca scatterer atoms from each Mn emitter atom were also calculated using the atomic positions in 3wu2 and are indicated by the purple and green circles overlaid on the hologram patterns. 2.5. Sample purification and crystallization For the XFH experiments, the holograms were measured from many large isomorphous PSII crystals. The XFH signal from the sample was weak due to the low-dose irradiation that was necessary to avoid the X-ray reduction of the Mn atoms in the Mn4CaO5 cluster as much as possible. The samples were extracted from the thermophilic cyanobacteria, Thermosynechococcus vulcanus, and the PSII crystals were prepared based on the methods reported by Kawakami & Shen (2018 >). To maintain the isomorphism between PSII crystals, the crystals were replaced with different concentrations of the cryo-protectant solutions step-by-step using a gentle dialysis method. The post-crystallization process using dialysis membrane (MWCO6000-8000 purchased from Spectra/Por dialysis) with a molecular weight cutoff of 6-8 kDa pore size was divided into six steps, in which each step was treated every hour, from the first crystal solution containing 10% PEG3000 to the final cryo-protectant solution containing 25% PEG3000 (polyethlyne glycol) and 20% dimethyl sulfoxide (DMSO). When a PSII crystal is frozen by flash-cooling, excess cryo-protectant solution covering the surface of a PSII crystal must be excluded, as this causes the attenuation of X-ray fluorescence. Therefore, the PSII crystal was placed on a mesh loop of diameter 400 mm or 1000 mm with 40 mm x 40 mm spacing, and the excess solution was absorbed from the back side by filter paper before flash-cooling the PSII crystal. To obtain different sections of the hologram pattern, PSII crystals oriented in different directions were prepared. 2.6. Evaluation of radiation damage To evaluate the radiation damage introduced into the samples during the XFH experiments, the XANES spectra taken before and after the XFH scans are compared. The 'before' spectrum was obtained at a point on the surface of the sample outside the XFH scan region, and another spectrum was obtained from the center of the sample after the XFH scans with 1 eV steps and 2 s integration time. The Mn K-edge inflection point is determined from the zero point in the second derivative. 2.7. Determination of crystal orientation The orientation of each PSII crystal relative to the Merlin detector was obtained from the indexing of the diffraction images taken by the Pilatus 100 K. Diffraction images were taken at a wavelength of 1.89 A and indexed using XDS (Kabsch, 2010 >). 3. Results 3.1. Serial X-ray fluorescence holography Fig. 4 >(a) shows typical Mn K-edge absorption spectra of a PSII S1 crystal taken before and after the XFH experiment, which shows no observable shift in the Mn K-edge. The inflection points (zero point of the second derivative) at 6.551 keV agree with the previously reported inflection points for the S1 state (Roelofs et al., 1996 >; Robblee et al., 2001 >). These results demonstrate that the measures taken to minimize radiation damage were sufficient to prevent radiation-induced reduction of the Mn ions in the OEC. Fig. 4 >(b) shows the Mn Ka hologram integrated from nine PSII crystals. The hologram pattern appears to be dominated by the FS dips of the holographic oscillations (black in the color scale). The application of the low pass filter, the larger thermal vibration in protein crystals and the mostly light elements surrounding the Mn4CaO5 cluster (water molecules and amino-acid residues) suggest that the hologram consists mostly of holographic signal from scatterers near the Mn emitters. To investigate how the larger thermal vibrations in proteins affect the holograms, the holograms were calculated with and without root-mean-square displacements of the scatterers due to static positional fluctuations within the crystal and dynamic fluctuations from thermal vibrations. The root-mean-square displacements were introduced into the hologram calculation by assuming isotropic vibrations of the scatterer atoms relative to the fixed emitter (<u 2> Rel), which is represented by a Gaussian distribution with a standard deviation s. First, Fig. 5 >(a) shows the holographic oscillations calculated from a simple Mn-Mn dimer system using different relative root-mean-square displacement values, <u 2> Rel, whereas Fig. 5 >(b) shows the corresponding Debye-Waller factor or damping term. The holograms are composed of minima in the FS direction, surrounded by concentric higher-order interference rings, as shown in the inset of Fig. 5 >(b). As the <u 2> Rel values increase, the backscattering is suppressed and the FS dips become the prominent features of the hologram patterns. Next, the Mn Ka holograms of PSII were calculated. Since PSII has a large unit cell (122.2 A x 228.5 A x 286.4 A) and consists mostly of light elements, only the atoms within the Mn4CaO5 cluster are used for the calculations. The atomic positions were extracted from the PDB entry 3wu2 (Umena et al., 2011 >). The total calculated hologram is the superposition of the hologram patterns calculated for each of the 32 Mn atoms in a PSII unit cell. Each hologram was calculated using the Mn, Ca and O atoms located within the same Mn4CaO5 cluster as the emitter Mn (Fig. 1 >). Fig. 5 >(c) shows the Mn Ka hologram of PSII calculated without any thermal vibrations. With increasing <u 2> Rel values, the similarities with the experiment become more apparent. In Figs. 5 >(d)-5(f), a series of holograms were calculated with different <u 2> Rel values. As the <u 2> Rel value increases, the positive signals (bright yellow) in the hologram patterns are suppressed, and in Fig. 5 >(f), with an extreme <u 2> Rel = 2.00 A2, the pattern is reduced to a large dark feature that bears strong resemblance to feature A in Fig. 4 >(b). To determine the best <u 2> Rel value, an R-factor analysis is performed (Kuznetsov et al., 2014 >). The R-factor describes the agreement between the calculated and experimental hologram patterns, where a smaller R-factor means a better agreement. Fig. 6 >(a) shows the dependence of the R-factor on <u 2> Rel. The minimization of the R-factor shows that the hologram calculated with <u 2> Rel = 0.35 A2 shows the best agreement with the experiment. This calculated hologram pattern is shown in Fig. 6 >(b) and the experimental hologram is shown again in Fig. 6 >(c) for comparison and discussion. There are several key features in the calculated hologram that are also clearly observed in the experimental hologram [highlighted by the dashed white lines in Figs. 6 >(b) and 6 >(c)]: region A, the large, dark and dog-bone-shaped region; region B, the bright feature observed at an azimuthal angle of 45deg; and region C, a combination of dark and bright features. For further interpretation of these features, the FS directions of all Mn and Ca scatterers were calculated from each Mn emitter and are superimposed on the lower images of Figs. 6 >(b) and 6 >(c). The angular positions of the circles on the images indicate the angular directions of the FS dips, and the relative sizes of the circles indicate the relative distances of the scatterer from the Mn emitters. These FS dips can be thought of as real-space projections of the positions of the scatterers around the emitter. The origin of region A becomes apparent from the superimposed FS directions, where a dense collection of FS directions appear over the dark regions of the experimental hologram pattern in Fig. 6 >(c). Among these is the FS direction marked as (1) in Fig. 6 >(c), where a dark dip in the hologram pattern is observed in both the experiment and the calculation. This FS direction corresponds to an Mn4A-Mn3B emitter-scatterer pair where the Mn-Mn distance is 2.89 A. The short emitter-scatter distance will result in a lower-frequency holographic signal and a larger FS dip (amplitude and width). On the other hand, some FS directions appear as bright signals in both the experiment and the calculation, an example of which is the FS dip marked as (2). This occurs when the FS dip interferes with the first interference ring of an adjacent FS dip. 4. Discussion The good agreement of the experimental results with the calculated hologram and calculated FS directions in Figs. 6 >(b) and 6 >(c) is a strong indication that the projection pattern obtained using sXFH contains structural information about the Mn4CaO5 cluster of PSII. Furthermore, the fact that the data were collected before any shifts in the Mn absorption spectra shows the tremendous potential of this method in studying the local structures of metal clusters in proteins before any radiation-induced damage. In the current study, we interpreted the hologram patterns as real-space projections of the local structure around the emitter by analyzing the FS dips. This is similar to the tomographic interpretation of the directional fine structure in the absorption of white X-rays (Korecki & Materlik, 2001 >; Korecki et al., 2006 >, 2009 >), also later termed white XFH (Dul & Korecki, 2012 >; Dabrowski et al., 2013 >). By employing polychromatic X-rays, a decrease in the coherence length results in the suppression of the higher-order interference fringes in the higher scattering angles, while the FS dip, which is largely energy independent, remains relatively unchanged. In this work, the combination of the large positional fluctuations in the PSII crystal and the application of the low pass filter on the hologram pattern also results in the suppression of the higher-order interference fringes while leaving the FS dip unchanged. Furthermore, the FS dip approach used in this study can be thought of as the X-ray fluorescence analog of X-ray photoelectron diffraction (XPD) (Kuznetsov et al., 2014 >). In XPD, the local structure information is extracted by analyzing the forward-focusing peaks (FFP) in the angular distribution of the photoelectron intensity. Using photoelectrons with high kinetic energies allows the FFP to dominate the XPD patterns and a real-space projection of the scatterers around the emitter can be obtained. XPD has been extensively used in studying local structures in 2D layers, thin films and interfaces. Aside from the FS dips, other features such as Kossel lines (Bortel et al., 2016 >; Faigel et al., 2016 >) or X-ray standing wave lines that may also appear in the hologram can also be used to extract structural information about the sample. To improve the accuracy of hologram calculations used in this study, the effects of the positional fluctuations of the scatterers were directly introduced into the calculations by a Debye-Waller factor or damping term [Fig. 5 >(b) and equation (2)]. Until now, the effects of atomic fluctuations have been introduced into the hologram calculations by introducing a distribution of the atomic positions in the atomic model used in the calculations. This can include random Gaussian distributions in the atomic positions (Hayashi et al., 2014 >; Hosokawa et al., 2013 >; Kimura et al., 2020 >) or, alternatively, using atomic positions extracted from molecular dynamics simulations (Ang et al., 2021 >). Although these are good approximations that can result in the adequate reproduction of the atomic reconstruction, using a DWF or damping term directly in the hologram calculation is a simpler and more direct way to calculate the hologram pattern. In the hologram calculations in Fig. 6 >, a root-mean-square displacement value of khH = 0.35 A2 was used. This value obtained from the R-factor analysis in Fig. 6 >(a) is relative and the average <u 2> of the atoms in the Mn4CaO5 cluster can be obtained using (Kimura et al., 2020 >) resulting in an average <u 2> = 0.29 A2, or an average B-factor of 23.25 A2 for all the atoms in Mn4CaO5. This is comparable with the average R-factors of the Mn atoms of the synchrotron radiation [B-factor ~26 A2 (Umena et al., 2011 >)] or XFEL [B-factor ~24 A2 (Suga et al., 2015 >)] structures, suggesting that, aside from dynamic fluctuations caused by thermal vibrations at 100 K, the atoms in the Mn4CaO5 cluster also have large positional fluctuations within the crystal. Though we were only able to extract average isotropic fluctuations in this work, we expect that in future work, the radial and angular positional fluctuations of Mn atoms can be extracted from more accurate hologram patterns, as this has already been demonstrated in XFH experiments on inorganic samples and organic crystals (Hosokawa et al., 2013 >; Hayashi et al., 2014 >; Ang et al., 2021 >; Kizaki et al., 2022 >). Once this is realized, valuable information to help understand the mechanisms of the oxidation process in PSII will be obtainable. Aside from the Mn Ka hologram obtained in Fig. 4 >(b), the experimental configuration will also allow valence-selective XFH. By carefully tuning the incident X-ray energy based on the small shifts in the Mn K-edge of the different Mn ions, valence-sensitive hologram patterns can be recorded. We performed preliminary valence-selective XFH experiments on the same PSII samples by recording an additional hologram pattern at 6.551 keV [below the Mn(IV) K-edge, but above the Mn(III) edge]. Fig. 7 > shows the high (khH) and low (khL) energy Mn Ka holograms taken at 6.565 keV and 6.551 keV, respectively. Both hologram patterns were taken from the same set of samples, and the only difference is the incident X-ray energy. The difference in the incident X-ray energies will result in a small change in the Mn(III):Mn(IV) contribution ratio, which means that, aside from small differences, the khH and khL holograms should be almost the same. At 6.551 keV, the absorption and the subsequent fluorescence from Mn(IV) is suppressed, resulting in a hologram pattern that is mostly from the Mn(III) ions. The total hologram in Fig. 7 >(a), taken at an incident X-ray energy of 6.565 keV [above the Mn K-edge of both Mn(III) and Mn(IV)], kh H, is a superposition of hologram contributions from all Mn ions, khMn(III) and khMn(IV). Using the known Mn valence distribution of PSII S1 [2 Mn(III) and 2 Mn(IV) (Suga et al., 2015 >)], the total hologram can be expressed as At the lower incident X-ray energy of 6.551 keV, both khMn(III) and khMn(IV) signals are suppressed due to the lower X-ray absorption, and the hologram kh L can be expressed as where the a and b parameters represent the decrease in the absorption spectra of khMn(III) and khMn(IV) relative to the absorption at 6.565 keV. From equations (3) and (4), khMn(III) can be numerically extracted using and subsequently khMnIV can be obtained using khH, khMn(III) and equation (4). From the normalized intensity of the absorption spectra in Fig. 4 >(a), the a and b parameters are estimated to be 0.75 and 0.55, respectively. Using these parameters, the khMn(III) and khMn(IV) hologram patterns were extracted and are shown in Figs. 8 >(a) and 8 >(b). To calculate the hologram patterns for Mn(III) or Mn(IV), only 16 clusters were used, with Mn emitters at the known sites of either the Mn(III) or Mn(IV) ions. Using the same khH parameter used for the total hologram pattern in Fig. 6 >(b), the hologram patterns were calculated for Mn(III) and Mn(IV) emitters in PSII and are shown in Figs. 8 >(c) and 8 >(d). The FS directions are once again superimposed on the holograms. From the calculations of the hologram patterns and the FS directions, a distinct difference in the khMn(III) and khMn(IV) holograms can be observed: the large dark feature (region A) in khMn(IV) that results from the dense arrangement of the FS directions. For the khMn(III) hologram, there is no clear distinct feature in the hologram, the FS directions are evenly distributed. This difference between khMn(III) and khMn(IV) can also be observed in the experimentally extracted holograms. The dark region in khMn(IV), marked as region D in Fig. 8 >(b), is clearly observed in both the experimentally extracted and the calculated khMn(IV) hologram patterns. On the other hand, the experimentally extracted khMn(III), like the calculations, shows no distinct features. These results show that, while the agreement between the experiment and the calculations are not clear as shown in the the total hologram in Fig. 6 >, the distinct features in the hologram patterns can allow differentiation of the khMn(III) and khMn(IV) hologram patterns. In the case of the S2 state, where only one Mn(III) remains in the OEC, the distinction between khMn(III) and khMn(IV) will be clearer, making XFH analysis more straightforward. Thus, analysis of the khMn(III) and khMn(IV) hologram patterns can be an alternative approach to experimentally determining which Mn site is the remaining Mn(III). In our experiment, a careful compromise had to be made between the total X-ray dose on the sample, the total photons collected by the 2D detector and the limited experiment times at synchrotron radiation facilities. Even under such limiting experimental conditions, we could already reach a certain agreement between the experimental and calculated holograms, as shown and discussed in the preceding sections. However, further optimization of the experiment would be necessary to obtain more accurate valence-selective holograms, because they must extract the small difference in the holograms taken at two different energies. By increasing the total photons collected during the experiment, either through longer exposure times, larger single-crystal samples or by measuring samples, the quality of the experimental hologram might be significantly improved. Alternatively, novel approaches to the extraction of the khMnIII and khMnIV holograms from the low-energy holograms recorded in the experiment can also be developed. The subtraction in equation (6) can result in the propagation of uncertainty, and this can be seen in the higher contrast scale used in Fig. 8 >. Several valence-selective XFH experiments have already been reported for inorganic crystals: a direct-imaging experiment using the same 2D hybrid pixel detector on magnetite (Fe3O4) (Ang et al., 2018 >) and conventional XFH experiments using 0D detectors (avalanche photodiode or SDD) on yttrium oxide thin films (YO/Y2O3) and YbInCu4 (Stellhorn et al., 2017 >; Hosokawa et al., 2019 >, 2020 >). In the experiments on Fe3O4, the difference in the Kossel line features between the low-energy hologram patterns clearly show the valence selectivity (Ang et al., 2018 >). Atomic reconstructions of these valence-selective hologram patterns have resulted in mixed results. Ang et al. (2018 >) numerically extracted the Fe(II) hologram using an equation similar to equation (6), and, although the reconstruction can be distinguished from the reconstruction obtained from the total [Fe(II) and Fe(III)], the quality of the results shows that it will be difficult to use the same approach on samples with larger and more complicated structures. Hosokawa et al. (2020 >) used a different approach to extract the Yb(II) hologram, where the Yb(II) hologram was recorded at an energy where only the Yb 2p 3/2 electrons in Yb(II) were excited and emitted fluorescent X-rays. The reconstructions obtained showed the expected f.c.c. structure around Yb(III), whereas the reconstructed atomic image around Yb(II) showed large positional fluctuations. In principle, this would be a reasonable approach to obtain valence-sensitive holograms. However, for samples with low concentration of metal emitters, or for radiation-sensitive samples such as metalloproteins, it will be experimentally difficult to obtain valence-selective holograms using this approach. 5. Conclusions In this work, we develop a novel approach for obtaining the local atomic structure around metallic clusters in protein crystals. By employing a 2D hybrid pixel detector to directly image the hologram patterns, a statistically significant hologram pattern was obtained in serial data acquisition mode, similar to those used in serial protein crystallography. The Mn Ka hologram was recorded from PSII crystals prior to the onset of radiation-damage-induced reduction of the Mn4CaO5 clusters. The good agreement between the calculated and experimental hologram patterns and the analysis of the FS directions show that the recorded hologram pattern can be treated as a real-space projection of the atoms around the Mn emitters in the Mn4CaO5 clusters. Furthermore, our new approach also allows valence-sensitive XFH experiments on protein crystals. Our preliminary results show that, by tuning the incident X-ray energy, the holograms from Mn(III) and Mn(IV) were extracted, and some distinct features observed in both the experimental and the calculated holograms allow some differentiation between them. Though additional data processing and more advanced reconstruction algorithms will be necessary to selectively reconstruct the local atomic structure around either Mn(III) or Mn(IV) from the holograms, the analysis of the FS patterns in the hologram, in combination with R-factor analysis, may allow the determination of which Mn sites each Mn ion occupies. This paves the way for future valence-sensitive XFH studies of other metal clusters in proteins and, considering that the valence states of these metal clusters play key roles in the functions of these proteins, there can be significant developments unlocked by further refinement of this approach. Figure 1 Crystal structure of PSII determined from protein X-ray crystallography (PDB entry 3wu2), with the relative positions of the Mn4CaO5 clusters highlighted. Inset: the Mn4CaO5 cluster. Figure 2 sXFH experiment to visualize the local structure around metal atoms in protein crystals at 100 K. (a) Schematic of the sXFH measurements. (b) Photograph of the experimental apparatus taken at BL39-XU, SPring-8. Reproduced with permission from The Japan Society of Applied Physics (Copyright 2020) (Ang et al., 2020 >). Figure 3 (a) Typical fluorescence spectra from a PSII crystal showing the Mn Ka and Ca Ka peaks, and from an empty Litholoop mesh taken at an incident X-ray energy of 6.565 keV. A typical Mn Ka hologram image from a PSII crystal at different stages of data processing: (b) the total image from a single crystal, (c) normalized by dividing with background image, (d) after converting to a spherical projection, and (e) after flattening and rotating the pattern to the correct orientation. Figure 4 Mn K-edge absorption spectra and Mn Ka holograms of PSII. (a) Mn absorption spectra of PSII taken before and after the sXFH experiment and various reference Mn oxide powders. (b) Mn Ka hologram obtained at an incident X-ray energy of 6.565 keV (khH). Figure 5 Evolution of XFH patterns with increasing root-mean-square displacements. (a) Holographic oscillations as a function of scattering angle simulated from a simple Mn-Mn dimer at different relative root-mean-square displacement values, khH, and (b) their corresponding damping factors. Inset: spherical projection of the hologram calculated from the Mn-Mn dimer. (c)-(f) Evolution of the Mn Ka holograms calculated from the OEC of PSII using different khH values: (c) Mn Ka holograms simulated without thermal vibrations, and those with khH = 0.35 A2 (d), khH = 0.60 A2 (e) and khH = 2.00 A2 (f). Figure 6 R-factor analysis between the calculated and experimental hologram patterns. (a) R-factor as a function of the relative mean-square displacements, <u 2> Rel, of the scatterer atoms used in the Mn Ka hologram calculations. (b) Calculated hologram with <u 2> Rel = 0.35 A2 and (c) the experimental hologram. The dashed white lines are visual guides for features discussed in the text. Circular marks indicate the FS directions of Mn (purple) and Ca (green) scatterers calculated from each Mn emitter from PDB entry 3wu2. Figure 7 Mn Ka hologram obtained at (a) a high incident X-ray energy of 6.565 keV (kh H) and (b) a low incident X-ray energy of 6.551 keV (kh L). Figure 8 Valence-sensitive Mn Ka hologram patterns khMn(III) and khMn(IV). (a) khMn(III) and (b) khMn(IV) hologram patterns extracted numerically from the experimental holograms using (a, b) = (0.75, 0.55) as determined from Mn absorption spectra. Calculated (c) khMn(III) and (d) khMn(IV) holograms with <u 2> Rel = 0.35 A2. The FS directions of neighboring Mn and Ca atoms were calculated from either khMn(III) (Mn1D and Mn4A sites) or khMn(IV) (Mn2C and Mn3B sites) emitters and are indicated by the purple (Mn) or green (Ca) circles. Table 1 Summary of the experimental conditions of the sXFH measurements on PSII Unit-cell parameters Total photons collected (x106) Sample (A) (deg) No. of scan positions At khH, 6.565 keV At khL, 6.551 keV 001 a = 123.807, b = 228.591, c = 286.008 a = b = g = 90 231 4.30 3.58 002 a = 125.444, b = 228.707, c = 286.563 a = b = g = 90 144 3.17 2.59 003 a = 124.75, b = 229.485, c = 285.433 a = b = g = 90 72 2.19 1.70 004 a = 124.234, b = 228.938, c = 285.951 a = b = g = 90 80 2.22 1.65 005 a = 134.579, b = 227.073, c = 286.66 a = b = g = 90 179 2.12 1.77 006 a = 121.072, b = 227.512, c = 286.063 a = b = g = 90 138 1.76 1.43 007 a = 123.024, b = 228.847, c = 286.000 a = b = g = 90 77 2.01 1.57 008 a = 124.049, b = 229.029, c = 290.245 a = b = g = 90 143 4.64 3.74 009 a = 121.633, b = 228.184, c = 286.12 a = b = g = 90 164 5.07 3.76 References Ang, A. K. R., Marumi, R., Sato-Tomita, A., Kimura, K., Happo, N., Akagi, K., Sasaki, T. & Hayashi, K. (2021). Phys. Rev. B, 103, 214106. Ang, A. K. 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PMC10000800 | J Synchrotron Radiat J Synchrotron Radiat J. Synchrotron Rad. Journal of Synchrotron Radiation 0909-0495 1600-5775 International Union of Crystallography 36891845 ve5163 10.1107/S1600577522012115 JSYRES S1600577522012115 Research Papers High-efficiency focusing and imaging by dielectric kinoform zone plate lenses with soft X-rays Dielectric kinoform zone plate lenses Tong Xujie a Chen Yifang a* Xu Zijian b Li Yijie a Xing Zhenjiang b Mu Chengyang a Zhao Jun ab Zhen Xiangjun b Mao Chengwen b Tai Renzhong b a Nanolithography and Application Research Group, School of Information Science and Technology, Fudan University, Shanghai 200433, People's Republic of China b Shanghai Synchrotron Radiation Facility, Shanghai Advanced Research Institute, Chinese Academy of Sciences, Shanghai 201210, People's Republic of China Svensson S. Editor Uppsala University, Sweden Correspondence e-mail: [email protected] 01 3 2023 17 2 2023 17 2 2023 30 Pt 2 s230200 319326 18 6 2022 22 12 2022 (c) Xujie Tong et al. 2023 2023 This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-BY) Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited. A full version of this article is available from Crystallography Journals Online. The effects of zone materials and zone shapes on the focusing/imaging quality of dielectric kinoform zone plates have been theoretically investigated by a modified thin-grating-approximation method. Greyscale electron beam lithography was applied to generate 3D kinoform zones in HSQ and PMMA for soft X-rays. The dielectric kinoform zone plate lens demonstrates high diffraction efficiency well beyond conventional X-ray optics. With fast advances in enhancing the focusing/imaging resolution of Fresnel zone plate lenses toward sub-10 nm, low diffraction efficiency in connection with their rectangular zone shape still remains a big issue in both soft and hard X-ray microscopy. In hard X-ray optics, encouraging progress has recently been reported in our earlier attempts of high focusing efficiency by 3D kinoform shaped metallic zone plates, formed by greyscale electron beam lithography. This paper addresses our efforts towards high focusing/imaging efficiency by developing a novel dielectric kinoform zone plate lens for soft X-rays. The effects of the zone materials and zone shapes on the focusing/imaging quality were first theoretically investigated by a modified thin-grating-approximation method, revealing superior efficiencies of dielectric kinoform zone plates over rectangular ones in metals. Optical characterizations of replicated dielectric kinoform zone plates by greyscale electron beam lithography demonstrate a focusing efficiency of 15.5% with a resolution of 110 nm in the water window of X-rays. Apart from high efficiency, the novel kinoform zone plate lenses developed in this work exhibit significant advantages over conventional zone plates, i.e. simplified process, low cost and no need for a beamstop. dielectric kinoform zone plate lens greyscale electron beam lithography high-efficiency focusing by soft X-rays X-ray microscopy Science and Technology Commission of Shanghai Municipality19142202700 Yifang ChenNational Natural Science Foundation of ChinaU1732104 61927820 11875316 U1832146 Zijian XuZijian XuZijian XuZijian XuThe following funding is acknowledged: Science and Technology Commission of Shanghai Municipality (grant No. 19142202700 to Yifang Chen); National Natural Science Foundation of China (grant No. U1732104 to Zijian Xu; grant No. 61927820 to Zijian Xu; grant No. 11875316 to Zijian Xu; grant No. U1832146 to Zijian Xu). pmc1. Introduction Soft X-ray microscopy is one of the most important nanoprobe techniques (Chu et al., 2008 >; Kirz, 1974 >; Spector, 1997 >; Selin et al., 2015 >), being widely applied in the development of, but not limited, biological science (Kapishnikov et al., 2017 >; Dehlinger et al., 2020 >), material science (Le et al., 2020 >) and nanotechnology (de Smit et al., 2008 >). Especially in the 2.3-4.4 nm wavelength range (the 'water window'), it is particularly adequate for high-resolution imaging of biological specimens (Kepsutlu et al., 2020 >; Chiappi et al., 2016 >) because of the high optical contrast of the X-rays in this energy range between carbon and water present in testing materials. Good quality imaging of bio-specimens requires high photon flux for reducing the exposure time to avoid damage to cells by soft X-ray irradiation, demanding both high resolution and high focusing efficiency of the lenses. Conventional lenses are metal-based binary Fresnel zone plates (FZPs) (Keskinbora et al., 2018 >; Sanli et al., 2018a >; Belkhou et al., 2015 >; Chao et al., 2009 >; Moldovan et al., 2018 >; Mohacsi et al., 2017 >), being widely applied in X-ray microscopes. Unfortunately, their rectangular zone shape and metal material with high absorption coefficient limits their diffractive focusing efficiency within 10% in the water window band (Chao et al., 2005 >; Marschall et al., 2017 >; Rosner et al., 2020 >; Lindblom et al., 2009 >; Reinspach et al., 2011 >). It is understood that a zone plate with parabolic zone shape, known as a kinoform lens (Di Fabrizio, 1994 >), should, in principle, possess superior focusing/imaging efficiencies over rectangular lenses for both soft and hard X-rays. However, the construction of such lenses with 3D zone shape has met big challenges in both design and fabrication technique. To optimize the zone shape for maximizing efficiency, the standard beam propagation method (BPM) needs to be modified so that one can govern the focusing performance by utilizing the best possible material as well as the best designed zone structure. In the technical development, 3D lithography should be innovated for transferring the designed profile into 3D kinoform zone plate (KZP) lenses on a nanometre scale. So far, despite substantial reported attempts, significant progress at soft X-ray wavelengths has still not been seen. The development of one-dimensional (1D) kinoform lenses is inhibited by the limited aspect ratio in the deep silicon etch or LIGA technique (Fu et al., 2017 >; Zhang et al., 2016 >; Simons et al., 2016 >). 3D nanoprinting has been attempted using kinoform lenses (Yu et al., 2020 >; Sun et al., 2014 >; Xie et al., 2020 >) in organic materials for visible and near-infrared wavelengths, but not in the X-ray regime. Femtosecond two-photon 3D nanoprinting was applied for plastic kinoform lenses (Sanli et al., 2018b >) with an efficiency of 20% in the soft X-ray region (1.5 keV) but the resolution achieved was merely 558 nm. Metal kinoform lenses based on focused ion beam (FIB) lithography (Keskinbora et al., 2015 >) have also been attempted, resulting in 15% efficiency and 400 nm resolution at 1100 eV. The efficiency was constrained by their rough surface, and further enhancement of both the numerical aperture and the resolution would be almost impossible because of the limited writing field with FIB. Therefore, to the best of our knowledge, a kinoform zone plate lens capable of high-efficiency focusing with advanced resolution in the soft X-ray region, especially in the water window band, has still not been achieved. In the hard X-ray region, however, a trapezoid-kinoform zone plate was recently reported in our earlier work using greyscale electron beam lithography (GS-EBL) combined with gold (Au) electroplating, based on the theoretical design of the zone shape using our modified BPM method (Tong et al., 2022 >). In this work, inspired by our success in the hard X-ray region, a novel dielectric kinoform zone plate lens made of low-loss material, such as HSQ (hydrogen silsesquioxane) and PMMA (polymethyl methacrylate), for soft X-ray focusing/imaging with high efficiency has been developed. State-of-the-art 3D GS-EBL was applied to generate the theoretically designed kinoform shape of the zones. Using our modified BPM, the efficiencies were quantitatively compared with various lens materials including both metals and dielectrics, and an optimized kinoform zone profile suitable for GS-EBL was calculated. Optical characterizations, using a scanning X-ray microscope (STXM), of the fabricated dielectric kinoform zone plate lenses demonstrate a peak focusing efficiency of 15% in the water window band. Structural analysis of the X-ray tested kinoform zone plate lenses with HSQ using a high-resolution scanning electron microscope (SEM) shows no sign of deformation in the zone structure. The progress achieved in this work lays a solid foundation for constructing a new generation of soft X-ray lenses with both high-focusing/imaging efficiencies and high resolution. 2. Effects of lens structure and material on focusing efficiency The lens structure and the material properties characterized by the refractive index, n = 1 - d - ib, are the two most important factors determining the focusing efficiency for a phase zone plate. In the soft X-ray region, Au zone plates work mostly in binary mode because of the high absorption of light, giving rise to the theoretical limit of efficiency of 10% (Kirz, 1974 >). On the other hand, zone plates made of dielectric materials such as HSQ and PMMA have larger phase shifts (1 - d) but lower absorption (b) than those made of metals, making phase modulation possible for soft X-rays. Therefore, dielectric zone plates enjoy a theoretical limit of efficiency up to 40% (Kirz, 1974 >), provided that the zones are both tall enough to meet the coherent diffraction on the focusing spot and robust enough to withstand X-ray radiation. Furthermore, if the dielectric zones are shaped as kinoform ones, even higher efficiency limits beyond 40% are expected (Yan, 2010 >; Lassaline et al., 2020 >). Theoretical calculations of focusing efficiencies by lenses of different materials (Au, HSQ and PMMA) as well as different zone shapes (rectangular and kinoform) were carried out using our modified BPM (Tong et al., 2022 >). X-ray propagation inside the lens was calculated using our earlier developed BPM-QDHT method (Tong et al., 2022 >) (where QDHT stands for quasi-discrete Hankel transform). Considering that the transmitted relative light intensities and efficiencies are not influenced by the incident light intensity, the incident light field for all lenses is simplified to a plane wave with unit amplitude. The calculated wavefields propagating in 3D space through the HSQ-KZP and Au-FZP are shown in Fig. 1 >. In the binary FZP, as schematically illustrated in Fig. 1 >(a), the lens thickness was chosen for peak efficiency and the outermost zone width is 100 nm for both zone plates. It can be seen in Fig. 1 >(a) that the light through the centre of the binary FZP is not diffracted illumination, and must be blocked by a beamstop to ensure essential contrast. All these energy losses are responsible for the efficiency reduction in FZPs. However, by contrast, in the HSQ-KZP configuration [Fig. 1 >(b)], all the zones with the kinoform shape, including the central ones, diffract the incoming light onto the focus point, giving rise to an intensity much higher than that in the binary FZP [Fig. 1(c)]. Fig. 1 >(d) presents the efficiencies calculated using our BPM-QDHT method for the Au-FZP and the KZP for two different materials, HSQ and PMMA, at 500 eV. When the lens thickness reaches 250 nm, the Au-FZP collects much more light than the dielectric KZPs because its Au zone thickness satisfies the integers of the p phase shift at 500 eV for the efficiency maxima, which makes Au-FZP a phase-shifting zone plate; whereas, the same thickness for dielectric KZPs is not large enough to satisfy the integers of the p phase shift so the lenses fail to diffract most of the light onto the focus spot. When the thickness of the lens is further increased, high absorption of light disables the Au-FZPs' ability to modulate the phase, so the lenses work in binary mode. As a result, the efficiency saturates at a low level of 6.7%, as presented by the brown curve in Fig. 1(d). However, on the other hand, the dielectric KZPs with low loss meet the integer multiple phase shift for coherent diffraction when the thickness increases to 1-1.5 mm and the efficiency reaches a high of 18.7%, as shown in Fig. 1(d), well beyond that of Au binary zone plates by a factor of two to three. It is commonly understood that the morphology of fabricated KZPs inevitably differs from the theoretically designed kinoform profile due to its complex three-dimensional topography and process limitations (Sanli et al., 2018b >; Keskinbora et al., 2015 >; Takeuchi et al., 2012 >) such as the proximity effect (Chen, 2015 >) in the EBL process. To calculate the focusing/imaging efficiencies of a zone plate lens with kinoform-like morphology, a modified thin grating approximation (Kirz, 1974 >; Di Fabrizio & Gentili, 1999 >; Tatchyn et al., 1984 >) is proposed in this work. Based on the variational formulation of zone plate theory (Tatchyn et al., 1984 >), a more general formula for a wide energy band with varying height in zones has been developed in this work. The incident light field has wavelength l and amplitude C; the number of zones is N. The refractive index of the material used in the optical element is expressed as n = 1 - d - ib, where 1 - d and b represent the real and imaginary parts of the refractive index of the material, respectively. According to the height function t i (th) of the ith zone [Fig. 2 >(a)], the corresponding phase shift function of the ith zone can be expressed as Then the amplitude A i of the ith zone according to the thin grating approximation formula can be expressed as where th is the optical path length difference to the focal spot over one ring period. Since the initial luminous flux received by each zone is the same, the total efficiency of the final optical element is the average of the efficiencies of all the wavebands. According to different height functions of the 1-N zones, the amplitude of each zone can be obtained. Then the overall efficiency, FE, can be expressed as Fig. 2 >(a) presents the replicated morphology of a typical HSQ-KZP with kinoform-like profiles by GS-EBL. In order to calculate its theoretical efficiency, each zone is divided into three areas. The shallower area usually produces gaps in region I, arising from over development or insufficient exposure dose. Due to proximity effects, the sidewalls are no longer vertical in region III. The corresponding amplitudes in the three regions were calculated independently and the phase shift function can be expressed as Here, structural parameters m and n describe the relative position of the gap and the top of the KZP, respectively, in one zone (0 < m < n < 1). Ph0 is the maximum phase shift of the lens, Ph0 = . t max is the maximum height of the ith zone. Generally speaking, in the central area of the lens, m = 0-0.1, n = 0.8-0.9. In the peripheral area, m = 0.3-0.5, n = 1 (details can be found in Fig. S4 of the supporting information). We set different structural parameters m and n and repeat the efficiency calculation using equations (1) to (3). Fig. 2 >(b) presents the calculated efficiency of HSQ-KZPs of thickness 700 nm as a function of m and n at 500 eV. The lens can reach the highest efficiency when n = 1, m = 0.2, implying that the peripheral sidewall of each zone is completely vertical with a 20% gap left. The 15-25% gap in each ridge is beneficial to the efficiency improvement (Tatchyn et al., 1984 >) and leaves a comfortable margin to tolerate the GS-EBL fabrication errors in generating 3D kinoform shapes. In addition, it is worth noting that when the verticality of the sidewall on the outer edge of each zone becomes poor (e.g. n = 0.8), the efficiency of the KZP drops by 10%, as shown in Figs. 2 >(d) and 2(e). Therefore, to achieve efficient focusing, each zone of the KZP needs to have a vertical edge, which will be discussed in the fabrication part of this paper (Section 3). According to the structural parameters that can be practically realized by GS-EBL, Figs. 2 >(c), 2(d) and 2(e) present the calculated focusing efficiencies of Au-FZP, PMMA-KZP and HSQ-KZP, respectively, in the soft X-ray region under ideal n and m parameters. KZP lenses in both HSQ and PMMA demonstrate much higher focusing efficiencies than when Au is used in the water window band, although higher thickness is necessary, which is consistent with the results calculated by the BPM-QDHT method. Therefore, for soft X-rays, kinoform or kinoform-like zones with dielectric materials should be the premier choice for diffractive phase lenses with high efficiencies over metallic ones which are absorptive to X-rays. 3. 3D kinoform zone plate lenses by e-beam direct write To demonstrate high-efficiency focusing and imaging with soft X-rays, dielectric KZP lenses were fabricated by direct e-beam write using 3D GS-EBL in SiO x -based HSQ and PMMA, respectively. To achieve the parabolic shape for the zones in HSQ-KZP as schematically illustrated in Fig. 3 >(a), exposure dose distributions were calculated using TRACER and LAB software, delivered by GenLsys Ltd, according to the desired profiles of the lens zones. Fig. 3 >(b) presents the calculated dose distribution by TRACER, seen as circles with regularly varying diameter on a 2D plane. Each zone consists of sub-zones with single pixel width of 20 nm, as shown in the inset of Fig. 3 >(b). Each sub-zone has a dosage factor ranging from 0 to 1. In order to pursue the kinoform-like topography, a large dose was assigned to the outermost circle of each zone in the hope of obtaining vertical sidewalls; meanwhile, the two innermost circles remained unexposed to obtain a gap. The overall spatial distribution of charge to be applied for generating KZP lenses is shown in Fig. 3 >(c). In order to maintain the sloping shape of the periphery and to reduce structural distortions, the greyscale proximity effect correction (GS-PEC) was also applied. By adjusting the charge distribution in the resists, the exposure dose on the outermost circle of each zone was almost doubled to obtain sharp standing triangles. Detailed exposure dose information for PMMA-KZP can be found in the supporting information. In greyscale e-beam lithography, 750 nm-thick positive tone HSQ (Fox-15) and 1000 nm-thick PMMA (350 K) were spin-coated on 100 nm-thick SiN x membranes, followed by a soft bake in an oven at 180degC for 1 h. E-beam exposure was carried out by using a state-of-the-art beam-writer, JBX6300 FS (see the supporting information for details). After development in TMAH:H2O (1:3) for HSQ and MIBK:IPA (1:3) for PMMA, respectively, PMMA-based 3D kinoform lenses were formed on 100 nm-thick free-standing SiN x membranes. Figs. 3 >(d)-3(g) present the replicated KZP lenses with diameter of 100 mm and outermost zone-width of 100 nm in HSQ and PMMA. Accompanying KZPs with a trench across the lens centre were also prepared for inspecting the profiles of the lens zones. Clear kinoform-like profiles can be observed, especially for the HSQ-KZP lenses. Additional ribs were facilitated for the PMMA-KZP to prevent the zones from collapsing. 4. High efficiency focusing/imaging by kinoform zone plate lenses High-efficiency focusing/imaging by the fabricated kinoform zone plate lenses was demonstrated at Shanghai Synchrotron Radiation Facility using in-house-developed STXMs with soft X-rays provided by beamline BL08U1A. Both the HSQ-KZP lens and the PMMA-KZP lens with 100 nm resolution were tested (see the supporting information for details of the optical-characterization setup). The standard testing samples, Siemens stars, were prepared in-house with a resolution of 30 nm and height of 1 mm. Fig. 4 > presents TXM images of the testing samples (Siemens star) by the fabricated HSQ-KZP lenses at 500 eV. Clear images can be seen in Figs. 4 >(a) and 4 >(b). To further demonstrate the 100 nm resolution of the image, in-house-made gratings with variable pitches were scanned by the focused X-ray beam, as shown in Figs. 4 >(c) and 4(d). Resolved peaks with 100 nm pitch indicate the resolution capability of the HSQ-KZP lens. Furthermore, the focusing spot dimensions of the first order were measured, using the knife-edge method, by scanning the focused spot on the line edge of the Siemen star. Figs. 5 >(a) and 5 >(b) present scanned curves using the spots focused by the HSQ-KZP and the PMMA-KZP lens, respectively. The first-order derivatives of the scanned curves give rise to a full width at half-maximum (FWHM) of 110 nm for the HSQ-KZP lens and 125 nm for the PMMA-KZP lens. Efficiencies in the energy range 0.2-1.0 keV were also measured, as shown in Figs. 5 >(c) and 5 >(d) for the HSQ-KZP lens and the PMMA-KZP lens, respectively. A peak efficiency of 15.5% at 0.45 keV was measured for the HSQ-KZP, which is about 95% of the theoretical value [Fig. 5 >(c)]. Such an efficiency enhancement by the kinoform-like profile is also repeated by the PMMA-based lens, as shown in Fig. 5 >(d). The efficiency peak of the PMMA-KZP lens is around 13.5%, which is about 80% of the theoretical value, showing a larger deviation than that of the HSQ-KZP. This is mainly caused by the structural deformation under X-ray radiation in efficiency tests, as shown in Figs. 5 >(e) and 5 >(f). After 12 h exposure to X-rays during the measurement, it was found that the ridges and the ribs collapsed in the outer area of the PMMA-based lens, indicating that PMMA-KZP was unable to survive the X-ray radiation. But no sign of X-ray radiation induced damage was observed on HSQ-KZP lenses after over 48 h exposure to X-rays for the efficiency measurement. 5. Conclusions Based on our earlier success in pursuing high-efficiency focusing in hard X-ray optics by metallic kinoform zone plate lenses, this work further tackles the same issue in soft X-ray microscopy by developing dielectric kinoform zone plate lenses. Theoretical calculations using the earlier developed QDHT-BPM method prove that dielectric KZPs are able to diffract incident light in the central zones to the focal plane, which is usually blocked by a beamstop in conventional Fresnel zone plates. By extending the thin grating approximation for rectangle zones to that for 3D kinoform profiles, systematic comparisons between the dielectric KZPs and metallic FZPs show significantly enhanced focusing/imaging efficiencies of dielectric kinoform zone plates over metallic ones with rectangular shape. Greyscale electron beam lithography was successfully applied to generate the 3D kinoform zone plates in both HSQ and PMMA for the first time. As high as 15.5% efficiency with a resolution of 100 nm was achieved by the fabricated dielectric KZP lenses in the water window of X-rays, in which the central zones are used for diffracting light instead of being blocked by a beamstop. Structural inspection by a scanning electron microscope shows that the HSQ kinoform zone plate lenses are robust enough to withstand X-ray radiation. The progress made in this work opens a new avenue toward high-efficiency focusing/imaging in soft X-ray microscopy and the next step is to enhance the resolution with such a novel zone plate lens. 6. Related literature The following reference, not cited in the main body of the paper, have been cited in the supporting information: Henke et al. (1993 >). Supplementary Material Sections S1 to S3, Figures S1 to S4, Table S1. DOI: 10.1107/S1600577522012115/ve5163sup1.pdf The authors thank the BL08U1A beamline and BL15U1 beamline of the Shanghai Synchrotron Radiation Facility for providing the beam time. Data underlying the results presented in this paper are not publicly available at this time but may be obtained from the authors upon reasonable request. Figure 1 Comparisons of wavefield propagations between the HSQ kinoform zone plate (HSQ-KZP) and the metal zone plate (Au-FZP). The wavefield propagation is calculated using the beam propagation method combined with the quasi-discrete Hankel transform [BPM-QDHT (Tong et al., 2022 >)]. The resolution of the Au-FZP (a) and the HSQ-KZP (b) is 100 nm with a diameter of 100 mm. The thickness of the Au-FZP and HSQ-KZP is 250 nm and 750 nm, respectively. The diameter of the beamstop for the FZP is 50 mm. (c) Comparison of point-spread functions of the focused light by both the Au-FZP and the HSQ-KZP, respectively, at the focal plane. (d) The calculated focusing efficiencies of the Au-FZP, HSQ-KZP and PMMA-KZP for soft X-rays (500 eV) using the BPM-QDHT approach. Figure 2 Theoretically calculated focusing efficiencies of kinoform zone plates suitable for GS-EBL for soft X-rays. (a) Illustrations of zone profile for KZPs. The thickness is shown in terms of the phase shift. An ideal kinoform lens has a vertical sidewall on one side; a fabricated kinoform zone plate, e.g. HSQ-KZP, has slopes on both sidewalls. Each zone of a real KZP is then divided into three regions with different transition function F(th). m and n describe the relative position of the gap and the top of the KZP in the zone (0 < m < n < 1). (b) The calculated efficiency of the HSQ-KZP with thickness of 700 nm as a function of m and n at 500 eV, based on the thin grating approximation. (c-e) Theoretical calculations of the focusing efficiency of Au-FZP, PMMA-KZP (m = 0.2, n = 1) and HSQ-KZP (m = 0.2, n = 1) based on the thin grating approximation as a function of both the photon energy and the lens thickness in the soft X-ray region (0-1 keV). Figure 3 Overview of the fabrication procedures. (a) Schematic illustration of greyscale electron beam lithography (GS-EBL) for the HSQ-KZP lens as an example. (b) The calculated dose gradient pattern for the HSQ-KZP lens by GS-EBL. (c) Dose distribution curves for the HSQ-KZP in which the greyscale proximity effect correction (GS-PEC) was applied. (d) Micrographs by SEM of the cross-sectional view at a tilt of 45deg for the fabricated KZP lenses in HSQ. (e) The whole fabricated HSQ-KZP lens. (f) Close-up view at a tilt of 75deg of the replicated HSQ-KZP zones. (g) Cross-sectional view at a tilt of 45deg of the PMMA-KZP lens. Figure 4 Results of soft X-ray imaging tests for the fabricated HSQ kinoform zone plate. (a) SEM image (left) of the in-house-made Siemens stars with resolution of 30 nm and soft X-ray image (right) of the same target at 500 eV with photon flux of 5 x 109 photons s-1. The dwelling time is 1 ms and step size is 50 nm, using the HSQ-KZP lens as focusing component. (b) SEM image and soft X-ray STXM image of the magnified central part of the Siemens star, using the HSQ-KZP lens at 500 eV with dwelling time of 1 ms and 25 nm step size. The colour bars in (a) and (b) are normalized photon flux. (c) Image of an in-house-made grating with variable pitches, on which the red arrow line shows the scanning with a dwelling time of 2 ms and step size of 30 nm, using the HSQ-KZP lens as focusing component. (d) Intensity profile of the test grating over 50 pitches as outlined in (c) by the red arrow line. Figure 5 (a, b) Focusing spot profiles for soft X-rays by the HSQ-KZP and the PMMA-KZP lens measured by knife-edge scan method with dwell time of 1 ms and step size of 25 nm, where the dots are the raw data, the solid lines are the fitting curves and the dash lines are the first-order differentials of the raw data. The first-order derivatives of the scanned curves give rise to a full width at half-maximum (FWHM) of 110 nm for the HSQ-KZP lens and 125 nm for the PMMA-KZP lens. (c) Comparison between the measured focusing efficiencies of HSQ-KZP and the theoretical ones using the BPM-QDHT approach. 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PMC10000801 | J Synchrotron Radiat J Synchrotron Radiat J. Synchrotron Rad. Journal of Synchrotron Radiation 0909-0495 1600-5775 International Union of Crystallography 36891848 wz5028 10.1107/S1600577523000462 JSYRES S1600577523000462 Research Papers Microfluidic rotating-target device capable of three-degrees-of-freedom motion for efficient in situ serial synchrotron crystallography Microfluidic rotating-target device for serial crystallography Zhao Feng-Zhu ab++ Wang Zhi-Jun c++ Xiao Qing-Jie c++ Yu Li d Sun Bo c Hou Qian e Chen Liang-Liang a Liang Huan a Wu Hai d Guo Wei-Hong a He Jian-Hua f Wang Qi-Sheng c* Yin Da-Chuan a* a School of Life Sciences, Northwestern Polytechnical University, Xi'an 710072, People's Republic of China b School of NCO, Army Medical University, Shijiazhuang 050081, People's Republic of China c Shanghai Advanced Research Institute, Chinese Academy of Sciences, Shanghai 201800, People's Republic of China d Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, People's Republic of China e School of Materials Science and Engineering, Northwestern Polytechnical University, Xi'an 710072, People's Republic of China f The Institute for Advanced Studies, Wuhan University, Wuhan 430072, People's Republic of China Wang M. Editor Paul Scherrer Institute, Switzerland Correspondence e-mail: [email protected], [email protected] ++ These authors contributed equally to this work. 01 3 2023 15 2 2023 15 2 2023 30 Pt 2 s230200 347358 08 11 2022 17 1 2023 (c) Feng-Zhu Zhao et al. 2023 2023 This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-BY) Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited. A full version of this article is available from Crystallography Journals Online. A novel microfluidic rotating-target device capable of three-degrees-of-freedom motion has been proposed, and its feasibility for sample delivery was successfully verified at a synchrotron radiation facility. This device guarantees the full utilization of crystals and only 0.1 mg of protein is consumed in collecting a complete dataset. There is an increasing demand for simple and efficient sample delivery technology to match the rapid development of serial crystallography and its wide application in analyzing the structural dynamics of biological macromolecules. Here, a microfluidic rotating-target device is presented, capable of three-degrees-of-freedom motion, including two rotational degrees of freedom and one translational degree of freedom, for sample delivery. Lysozyme crystals were used as a test model with this device to collect serial synchrotron crystallography data and the device was found to be convenient and useful. This device enables in situ diffraction from crystals in a microfluidic channel without the need for crystal harvesting. The circular motion ensures that the delivery speed can be adjusted over a wide range, showing its good compatibility with different light sources. Moreover, the three-degrees-of-freedom motion guarantees the full utilization of crystals. Hence, sample consumption is greatly reduced, and only 0.1 mg of protein is consumed in collecting a complete dataset. sample delivery system serial synchrotron crystallography microfluidic rotating-target circular motion in situ diffraction National Key Research and Development Plan of China2017YFA0504901 National Natural Science Foundation of China82172063 Innovation Capability Support Program of Shaanxi2020TD-042 Science and Technology Program of Ali Region, TibetQYXTZX-AL2022-07 The following funding is acknowledged: National Key Research and Development Plan of China (grant No. 2017YFA0504901); National Natural Science Foundation of China (grant No. 82172063); Innovation Capability Support Program of Shaanxi (grant No. 2020TD-042); Science and Technology Program of Ali Region, Tibet (grant No. QYXTZX-AL2022-07). pmc1. Introduction The development of serial crystallography (SX) offers new opportunities to the fields of crystallography, biology and medicine. The advantages of SX include structural determination at room temperature (Ayan et al., 2022 >) at crystal sizes down to the micrometre or even submicrometre level. Furthermore, SX is an ideal tool for carrying out high-resolution time-resolved studies (Pearson & Mehrabi, 2020 >; Orville, 2020 >; Dods et al., 2021 >; Pandey et al., 2020 >), such as enzymatic reactions and light activation of proteins (Kwon et al., 2021 >; Li et al., 2021 >; Poddar et al., 2022 >; Yun et al., 2021 >; Oda et al., 2021 >; Suga et al., 2019 >; Wiedorn et al., 2018 >; Olmos et al., 2018 >). SX experiments can also be used for de novo phase studies (Lawrence et al., 2020 >; Nass et al., 2020 >; Huang et al., 2018 >) and the structural analysis of viruses (Roedig et al., 2017 >; Meents & Wiedorn, 2019 >; Lawrence et al., 2015 >). SX experiments can be performed at room temperature thanks to the mechanism of 'diffraction before destruction' in serial femtosecond crystallography (SFX) (Neutze et al., 2000 >) and the ability to spread the dose over multiple crystals in serial synchrotron crystallography (SSX) (Ebrahim et al., 2019 >). The rise and development of SX has also promoted a renaissance of room-temperature crystallography because conformation heterogeneity related to function can be observed at room temperature but might be changed under cryogenic conditions (Fraser et al., 2011 >; Russi et al., 2017 >; Echelmeier et al., 2020 >). In addition, studies have shown that global radiation damage and specific radiation damage at room temperature exhibit less decoupling than those under cryogenic conditions (Gotthard et al., 2019 >). Many related technologies have been developed for better SX practices, including crystal detection technology (Cheng et al., 2020 >), in situ diffraction devices and sample delivery systems. The sample delivery system is one of the key links in the implementation of SX research, and it is also an important tool for diversified experiments using SX technology. Practically, the sample delivery system is also the main limiting factor affecting the application of SX and has been increasingly studied by researchers. A number of sample delivery systems which can be categorized as liquid jet technology and fixed-target technology as well as hybrid methods have been developed (Echelmeier et al., 2019 >; Zhao et al., 2019 >; Cheng, 2020 >; Grunbein & Kovacs, 2019 >; Echelmeier et al., 2020 >; Ren et al., 2020 >; Monteiro et al., 2020 >; Li et al., 2018 >). In addition, various novel sample delivery systems, including upgrades to existing sample delivery systems, are constantly emerging for better SX practice (Gilbile et al., 2021 >; Nam, 2022 >; Martiel et al., 2021 >; Doppler et al., 2022 >; Lee et al., 2022 >; Patel et al., 2022 >; Vakili et al., 2022 >; Hammarstrom et al., 2022 >). These devices can be applied to different light sources, including X-ray free-electron laser (XFELs) and synchrotron radiation (SR) sources, or both, depending on their delivery speed. In general, XFEL sources require a higher sample delivery speed, and SR sources require a slower sample delivery speed to obtain effective diffraction data that can be used for structural analysis. In addition, experiments using XFELs with a low repetition rate also require late sample delivery. Since there are far more SR sources than XFEL sources worldwide, the development of SSX has provided opportunities for more researchers to conduct SX experiments, thereby providing new opportunities for the widespread application of SX (Lan et al., 2018 >). With the increase in SSX experiments, there is an increasing demand for developing sample delivery systems that demonstrate low consumption and high efficiency for SR sources; moreover, SFX experiments also have a variety of sample delivery system requirements. Therefore, the trend continues for developing sample delivery devices suitable for SR sources, XFEL sources, or both. In our previous research, we developed a circular-motion-based sample delivery device; that is, a microfluidic rotating-target device capable of two-degrees-of-freedom motion (2-DOF-MRT), and its sample delivery performance was verified at the Shanghai Synchrotron Radiation Facility (SSRF). The sample delivery study of the 2-DOF-MRT device pioneered a new sample delivery mode based on circular motion for the first time, which was different from the existing technologies that adopted linear or multisegment linear motion for sample delivery. The circular motion feature allows the sample delivery speed to be easily adjusted over a wide range, giving the 2-DOF-MRT device the potential to be applied in many different SR facilities (Zhao et al., 2020 >). Inspired by oscillating serial crystallography (Guo et al., 2018 >; Gati et al., 2014 >; Wierman et al., 2019 >), we believe that the crystal still has the ability to provide effective diffraction frames after a single short-term exposure of the SR source (depending on the radiation dose rate). However, in our study of the 2-DOF-MRT device, each crystal contributed at most one effective diffraction frame. If these crystals are rotated, more than one diffraction frame from the same crystal can be collected. Therefore, we propose to add a rotational degree of freedom based on the 2-DOF-MRT device, that is, a microfluidic rotating-target device capable of three-degrees-of-freedom motion (3-DOF-MRT), including two rotational degrees of freedom and one translational degree of freedom. Then, multiple effective diffraction frames can be collected from each crystal to further reduce sample consumption and achieve the goal of collecting a complete dataset from a single microfluidic sample plate. Herein, a 3-DOF-MRT device is developed and successfully tested on beamline BL18U1 of SSRF by using lysozyme as a test model. This device combines oscillating serial crystallography and circular-motion-based serial crystallography and exhibits attractive features; for example, the circular motion ensures that the delivery speed can be adjusted over a wide range [0-20 revolutions min-1 (hereafter RPM) - the corresponding sample speed range is approximately 0-1.36 x 10-2 m s-1], showing its good compatibility with different light sources. The in situ diffraction avoids the need for crystal harvesting. The 3-DOF-MRT guarantees the full utilization of crystals. Hence, sample consumption is greatly reduced, and only 0.1 mg protein is consumed for collecting a complete dataset within 6.5 h. Finally, we discuss the selection of motion control parameters for SSX research with this device to provide a reference for its application. 2. Materials and methods 2.1. Design of the 3-DOF-MRT device The design of the 3-DOF-MRT device involves two parts: a microfluidic sample plate and a motion control system. As shown in Fig. 1 >(a), the purpose of the 3-DOF-MRT device is to control the microfluidic sample plate containing crystals to rotate (r 1 and r 2) and translate (t) through a 3-DOF motion control system to realize efficient sample delivery. The overall exterior of the 3-DOF-MRT device is shown in Fig. 1 >(b). 2.1.1. Microfluidic sample plate The microfluidic sample plate is the carrier that drives the movement of crystals, and its structural design was embodied in the 2-DOF-MRT device (Zhao et al., 2020 >). The important feature of the microfluidic sample plate is its disk-like geometry that is suitable for circular motion. Moreover, the microfluidic sample plate has a channel with a depth of 140 mm for carrying crystals on the micrometre scale. In fact, it is possible to manufacture smaller microfluidic channels to accommodate smaller crystals by choosing thinner double-sided tape. For example, 3M can offer products as thin as 10 mm, which means that the available crystal size can be reduced to approximately 10 mm. The microfluidic sample plate was divided into an upper unit and lower unit for assembly according to Fig. 1 >(c). The upper unit was obtained by placing double-side tape, a polymethyl methacrylate (PMMA) plate, double-sided tape and Mylar film layer-by-layer, and the lower unit was obtained by placing double-side tape, Mylar film and a PMMA plate layer-by-layer. The crystallization solution was loaded into the microfluidic channel of the lower unit by pipette, and then a complete microfluidic sample plate was assembled by affixing the upper unit to the lower unit. To adapt to the new motion control strategy, the size and appearance of the microfluidic sample plate were adjusted based on our previous study. Details of the size of the microfluidic sample plate are presented in Fig. 1 >(d). 2.1.2. 3-DOF motion control system The 3-DOF motion control of the microfluidic sample plate relies on the combination of a motor and goniometer in function and structure. The microfluidic sample plate was installed on the motor shaft through a hole machined in its middle that matched the motor shaft. We designed and machined a 'connector' to join the motor to the magnetic base (CrystalCap Copper Magnetic). Thus, the entire 3-DOF-MRT device could be installed in the diffraction facility through the magnetic base. Due to space limitations, it is not possible to directly apply the rotational (r 2) degree of freedom to the 2-DOF-MRT device. Moreover, the microfluidic sample plate of 2-DOF-MRT was not on the same axis as the goniometer; thus, the rotation angle resolution was not accurate when rotated by the goniometer. Therefore, in the design of the 3-DOF-MRT device, a bent 'connector' was designed, as shown in Fig. S1 of the supporting information, so that the microfluidic sample plate and goniometer were on the same axis to achieve precise control of the rotational (r 2) degree of freedom. As shown in Fig. 1 >(a), the rotational (r 1) degree of freedom (red arrow) was controlled by a motor. By selecting motors with different models, the rotational speed could be controlled in different ranges. The translational degree of freedom (green arrow) was controlled by the goniometer, and the goniometer was programmed to translate along the z-axis to realize the control of translational motion. The second rotational (r 2) degree of freedom (blue arrow) was also controlled by the programmed goniometer, which controlled the rotational (r 2) degree of freedom by programming the goniometer to rotate. Fig. 2 > shows physical images of the 3-DOF-MRT device and its status used for the SSX experiment on BL18U1 of the SSRF. It can be seen that the designed device was completely compatible with the diffraction environment. In this study, the rotational (r 1) motion controlled by the motor was carried out at a speed of 1/6 RPM. A translation movement was performed after one circle of rotation; that is, translation occurred once every 6 min, with a translation step of 100 mm; a total of seven translations occurred. Then, the goniometer was used to reverse translate to the initial position and perform one rotational (r 2) motion at an angle resolution of 1deg. The above rotation and translation motion was repeated until enough diffraction data were obtained for analysis. In our experiment, a total of seven (r 2) rotations were performed, and diffraction data from -4deg to 3deg (as determined by the goniometer) were collected. An animation demonstration of the motion control scheme is displayed in Movie S1 of the supporting information. 2.2. Manufacturing of the 3-DOF-MRT device The manufacture of the 3-DOF-MRT device involved the manufacture of a microfluidic sample plate and a 'connector', as well as the assembly of a microfluidic sample plate and the entire device. The Mylar film, double-sided tape and a PMMA plate were chosen as the materials for the microfluidic sample plate. The Mylar film was purchased from Shanghai East Electronic Co. Ltd., and its thickness was 3.6 mm. The double-sided tape (3M9500PC) was purchased from the 3M Company, and its thickness was 140 mm, which could provide a spacer for microlevel crystal growth. The PMMA plate was purchased from Xi'an Shuguang Polymethyl Methacrylate Products Co. Ltd, and its thickness was 1 mm, providing sufficient stiffness. In addition, the PMMA plate exhibited good processability and could flexibly cope with complex processing requirements. Polylactic acid (PLA) was selected as the material for manufacturing the 'connector' by 3D printing. The motor (AZH1G5) was purchased from Zhaowei Electromechanical Co. Ltd, and controlled the rotational degrees of freedom; its speed adjustment range was 0-20 RPM with a rotation angle resolution of 0.05deg. The double-sided tape and PMMA plate were manufactured by a computer numerical control (CNC) laser processing machine (R60 series) according to the model as shown in Fig. 1 >(d). After that, the Mylar film and processed double-sided tape as well as the processed PMMA plate were assembled according to the steps shown in Fig. 1 >(c). The 'connector' was 3D printed by a Makerbot Replicator 2 device according to the 3D model as shown in Fig. S1 of the supporting information, and then the motor and magnetic base were connected through the 'connector'. 2.3. Crystallization Lysozyme purified by our laboratory was used for in situ crystallization following the microbatch method. The lysozyme was dissolved in ultrapure water to obtain a 40 mg ml-1 protein solution, and NaCl was dissolved in ultrapure water at a concentration of 50 mg ml-1 to obtain a precipitant solution. Then, the protein solution and the precipitant solution were mixed in equal volumes to obtain the crystallization solution. After that, the crystallization solution was centrifuged at 10000 RPM for 1 min. Then, 5 mL of the supernatant of the crystallization solution was pipetted into the microfluidic channel, and the crystallization solution was guided to the entire microfluidic channel through the pipette tip so that the crystallization solution uniformly nucleated in the microfluidic channel. Finally, the upper unit covered the lower unit containing the crystallization solution to seal the microfluidic channel, and the microfluidic sample plate was placed in an incubator at 20degC for 36 h. 2.4. Data collection of the SX experiment The SX experiment was carried out on BL18U1 at the SSRF equipped with a PILATUS3 X 6M detector. Regarding data collection, the X-ray photon energy was 12.6 keV with a full width at half-maximum (FWHM) of 30 mm x 30 mm (H x V) and a beam flux of 5 x 1011 photons s-1. The exposure time for each detector frame was 0.3 s. The data acquisition rate was 2 Hz (0.5 s per frame) because the data acquisition included the time to open and close the shutter. The dose rate calculated by RADDOSE-3D was 20 kGy s-1 (Bury et al., 2018 >; Dickerson & Garman, 2021 >). Regarding the 3-DOF-MRT device, the rotating speed (r 1) was 1/6 RPM (the corresponding linear velocity range was 96.0-113.4 mm s-1 because the inner and outer radii of the microfluidic channel were 5.5 mm and 6.5 mm, respectively), the step length was 100 mm (with seven steps overall), and the rotational angle (r 2) was 1deg per time from -4deg to 3deg (as determined by a goniometer). More details are listed in Table S1. Before data collection, the microfluidic channel was focused through the control interface of the beamline station. 2.5. SX experiment data processing and structural solution First, the SX data sets were processed through the CrystFEL (version 0.8.0) program, including indexing the data using indexamajig, scaling using partialator, calculating the figures of merit by the compare_hkl (R split, CC1/2 and CC*) and check_hkl [signal-to-noise ration (SNR), multiplicity and completeness] programs, and exporting the data to XSCALE (Kabsch, 2010 >) through the create-xscale script for further structural refinement (White, 2019 >; White et al., 2012 >, 2016 >). Then, Phenix (version 1.14-3260) was used for molecular replacement (Adams et al., 2010 >; Liebschner et al., 2019 >) and refinement with 193L as a model (Vaney et al., 1996 >). Coot (version 0.8.2) was used to manually correct the protein structure (Emsley et al., 2010 >). CCP4 (version 7.0) was used to calculate the 2F o-F c and F o-F c electron density maps (Winn et al., 2011 >). Finally, the structure and electron density maps were imaged by PyMOL (version 2.2.0) ). 3. Results 3.1. Effect of rotation on crystal distribution Lysozyme crystals with an average size of approximately 100 mm x 100 mm x 100 mm and an average distribution density of approximately 15 crystals mm-2 in the microfluidic channel were obtained through the in situ microbatch method. During sample delivery, the stable position and uniform distribution of crystals in the microfluidic channel were critical. The movement of crystals in the device caused by sedimentation and centrifugal force may interfere with the diffraction pattern, spot size and dimensions, and even data quality. To test the influence of the adopted motion control strategy on the distribution of crystals in the microfluidic channel, we carried out different rotation speed tests (1/6 RPM, 1/5 RPM, 1/4 RPM, 1/3 RPM, 1/2 RPM, 1 RPM, 2 RPM, 4 RPM, 6 RPM, 12 RPM and 20 RPM) and performed statistical analysis on the distribution of crystals. The corresponding centrifugal acceleration range of the crystals was approximately 1.67 mm s-2 (1/6 RPM) to 28 mm s-2 (20 RPM). In this test, each speed was used to make five full rotations, and three sets of repeated tests were performed. After the rotations were completed, the same position was selected to take a picture and count the percentage of crystals remaining at the initial position. The distribution of crystals in the microfluidic channel before and after rotation at a speed of 1/6 RPM is shown in Fig. 3 >(a). We located the position of the crystal, as shown in Fig. 3 >(b). The blue dots indicate that the crystals remained in their initial position, and the red dots mean that the crystals deviated from their initial position. The percentage of crystals remaining at the initial position is shown in Fig. 3 >(c). The crystal position was stable at low speeds. As the rotation speed increased, the percentage of crystals that remained at the initial position gradually decreased. When the rotation speed reached 4 RPM, the percentage of crystals remaining at the initial position no longer changed with the increase in rotation speed. The crystal could not be maintained at the initial position because the centrifugal force and gravity of crystals, as well as the friction between the crystals and inner wall of the device, could not stay balanced during the movement, as analyzed in Fig. S2 and Section S1 of the supporting information. In this study, 1/6 RPM was selected as the experimental speed to adapt multiangle SSX research. Under these conditions, approximately 79% of the crystals stably remained at their initial position, and no obvious crystal aggregation occurred. If a faster rotation speed or delivery speed is required, the force of the crystal needs to be considered comprehensively to avoid the influence of crystal aggregation on data analysis. An ideal solution may be to constrain the crystal distribution by referring to the strategy of the sample holder (Baxter et al., 2016 >; Illava et al., 2021 >; Nam et al., 2021 >). 3.2. Analysis of background scattering As one of the commonly used X-ray transparent films, Mylar film exhibits excellent X-ray transmittance, ultralow X-ray absorption and very weak background scattering. More importantly, it is a chemically inert material that will not affect the physical and chemical environment for crystal growth (Broecker et al., 2016 >, 2018 >). Considering these advantages, we chose Mylar film with a thickness of 3.6 mm as the diffraction window material for this research, and its X-ray transmittance exceeded 99.9% at an energy level of 12.6 keV (Zhao et al., 2020 >). Background scattering analysis was carried out using ADXV (version 1.9.14). Fig. 4 >(a) shows the representative diffraction frames and their corresponding magnifications of the crystal, solution, device and air samples, respectively. Fig. 4 >(b) shows the radial profiles of the background scattering intensities of the solution (gray line), device (orange line) and air (blue line) from 0 to 1200 pixels along the y-axis of the frame, which is indicated by the red arrow in the diffraction frame. The crystal diffraction frame was derived from the in situ grown crystal in the microfluidic channel, which meant that the diffraction intensity came from the crystal, two layers of Mylar film and some crystallization solution around the crystal. The diffraction peak from the crystal was strong enough, although there was some background scattering from the Mylar film and crystallization solution. Then, we analyzed the background scattering of the Mylar film (device) and solution (solution). As a control, we also analyzed the background scattering of air (air). The diffraction frame and radial profile of the device showed weak and thin scattering signals at 5.4 A and 2.1 A. The diffraction frame and radial profile of the solution showed another scattering signal between 3.0 and 3.5 A. The scattering signal generated from the solution is stronger, while, compared with the strong peak of crystal, the scattering signal generated by the solution did not affect the data analysis. 3.3. Data collection analysis and structural refinement 3.3.1. Data collection analysis The SX experiment was carried out on BL18U1 at the SSRF, and 44459 diffraction images from one microfluidic sample plate were collected in approximately 6.5 h, resulting in a sample consumption of 5 mL of crystallization solution or 0.1 mg of protein. Details of the data collection analysis are listed in Table 1 >. The indexamajig program of CrystFEL was used to index all the obtained diffraction data, resulting in an effective indexed diffraction frame of 4470 with a corresponding indexable rate of 10.73%. The average distribution density of crystals in the microfluidic channel is approximately 15 crystals mm-2, resulting in only approximately 15% of the microfluidic channel being covered by crystals. The index rate of 10.73% indicates that most crystals can be hit and obtain efficiently indexable diffraction frames. After that, the partialator program was used for scaling. The figures of merit were calculated through the compare_hkl and check_hkl programs, yielding R split, CC1/2, CC* and SNR values of 27.52% (53.41%), 88.47% (57.70%), 96.89% (85.54%) and 3.98 (2.45), respectively. To monitor the changes in the diffraction data during the experiment, we performed data collection analysis on different subsets that were grouped as shown in Fig. 5 >(a). The data collection analysis of different subsets was performed following the above steps. Fig. 5 >(b) and Table S2 show the indexable rates of the different subsets. The indexable rate was the highest when the rotation (r 2) angle was 0deg, that is, when the X-ray beam was orthogonal to the microfluidic sample plate. The statistics of the indexable rate during the rotation process are shown in Fig. 5 >(c), and the trend derives from the accumulation of diffraction data. These results regarding the indexable rate indicate that the implementation of the rotation degree of freedom (r 2) may cause a slight decrease in the indexable rate, which may result from the misalignment of some crystals after rotation. Figs. 5 >(d)-5(f) present the change in the figures of merit (completeness, multiplicity and R split) with the accumulation of diffraction data. More details of the data collection analysis are reported in Table S3. The completeness and multiplicity exhibited obvious increases with the accumulation of diffraction data. R split exhibits a better trend with the accumulation of diffraction data. We drew a radar graph to visually exhibit the changes in the different figures of merit. It can be understood that the larger the graph area composed of different figures of merit, the better the data quality. Notably, the subset of D1-5 had a resolution of 2.05 A, while the resolution of D1-6, D1-7 and D1-8 was 2.15 A, which may indicate that the quality of the crystals deteriorated during the data collection process. In addition, the SNRs of subsets D1 and D1-2 are higher. This result comes from the fact that their cutoff resolution is low because the data completeness of the high-resolution shell is insufficient. Of course, the result also comes from the fact that the signals are from relatively fresh crystals. 3.3.2. Structural refinement Structural refinement was performed using Phenix. The PDB entry 193L was selected as a model for molecular replacement and refinement. After that, the protein structure was manually corrected using Coot. Finally, the resolution of lysozyme was processed to 2.15 A with a final R work/R free of 18.74%/20.00%. The refined structure was deposited in the PDB database with the code 7dln. The average B value of our data is 77.3 A2. Typically, SX obtains a higher B value than traditional single-crystal room-temperature diffraction due to the intensity merging method in SX (Stellato et al., 2014 >). More details of the structural refinement are listed in Table 1 >. Fig. 6 >(a) shows the lysozyme structure and the enlargement of typical residues as well as their electron density maps. We also analyzed the structure from the D1-3, D1-4, D1-5, D1-6 and D1-7 subsets. The detailed structural information is reported in Table S4. The corresponding structures and electron density maps are presented in Fig. S3. 3.4. Radiation damage analysis Radiation damage, as one of the largest obstacles to obtaining the precise structure of biological macromolecules at room temperature, has been greatly alleviated since the advent of SX. We performed Fourier difference maps calculations of disulfide bonds to evaluate the radiation damage. There are four disulfide bonds in lysozyme (Cys6--Cys127, Cys30--Cys115, Cys64--Cys80 and Cys76--Cys94) that are susceptible to radiation damage. The FFT program of CCP4 (version 7.0) was used to calculate the 2F o-F c and F o-F c electron density maps. Then, the 2F o-F c and F o-F c electron density maps of disulfide bonds were imaged by PyMOL (version 2.2.0), as shown in Fig. 6 >(b). Regarding the analyzed structures in this study, no obvious F o-F c electron density was found at a contour level of -3.0s. This result indicated that the lysozyme structure obtained by our proposed device and method did not suffer serious radiation damage. However, an increase in the rotation (r 2) range will inevitably increase the radiation damage. Thus, it is necessary to consider the cumulative effect of the radiation dose to avoid or reduce radiation damage. In addition, radicals will be generated by radiation damage by exposing crystals continuously. Therefore, the proposed method should be applied to samples sensitive to radiation damage with caution. 4. Discussion 4.1. Comparison of 3-DOF-MRT and 2-DOF-MRT Compared with our previous SX study on the 2-DOF-MRT device (Zhao et al., 2020 >), this 3-DOF-MRT device exhibits a significantly reduced sample consumption and shortened time requirement; moreover, the indexable rate clearly increases, as reported in Table S5. These results benefit from the 3-DOF motion control strategy, which enables the full application of crystals. In addition, adequate adaptation of the 3-DOF-MRT device to the diffraction environment contributes to obtaining diffraction frames that are easier to process by software. For example, there is no strong diffraction ring that comes from the platinum aperture in the study of the 3-DOF-MRT device, while that is observed in the study of the 2-DOF-MRT device. In the study of the 2-DOF-MRT, the capillary in the diffraction facility is lowered to make enough space, which leads to strong diffraction rings coming from the platinum aperture. 4.2. Analysis of 3-DOF-MRT device performance The rotation speed range of the motor used in the 3-DOF-MRT device is 0-20 RPM, which means that the corresponding sample delivery speed range was approximately 0-1.36 x 10-2 m s-1 because the outer radius of the microfluidic channel is 6.5 mm. Theoretically, the range of the rotational speed can be from 0 to very large values, depending on the capability of the hardware. However, we need to consider the centrifugal acceleration that different samples can withstand, depending on the features of different protein crystals. Although the 3-DOF-MRT device has made great progress compared with the 2-DOF-MRT device, collecting a full dataset in 6.5 h is extremely long, both for single cryo-frozen crystallography and for SX. However, the effective time to acquire a complete dataset can be drastically improved at a microfocus beamline with a faster detector. For example, if a data collection rate of 100 Hz (0.01 s frame-1) is applied, the data collection time can be reduced to less than 10 min. 4.3. Strategy of motion control parameters selection Sample delivery technology based on circular motion is worthy of promotion. The selection of motion control parameters [rotation (r 1) speed, translation step length and rotation (r 2) mode] is discussed to provide a reference. The rotation speed corresponds to the sample delivery speed. If the X-ray beam contacts the crystal completely during a single exposure, this contact is regarded as effective diffraction. The model presented below would be an ideal choice for the sample delivery speed, where L crystal is the size of the crystal in the delivery direction, L exposure is the distance of the crystal delivery during exposure, L gap is the distance of the crystal delivery during the delay time for the detector collecting data, T exposure is the exposure time, V is the sample delivery speed, and L beam is the size of the X-ray beam. Under these conditions, each crystal can collect at least one and up to two diffraction frames, as shown in Fig. 7 >(a). The model presented below is an ideal choice for the translation step length, where is the size of the crystal in the translation direction and L step is the translation step length. Under the recommended translation step length, each crystal can collect at least one and up to two diffraction frames, as shown in Fig. 7 >(b). Since the shape of different crystals varies, the crystal size in the delivery direction and translation direction is not intuitive. It will be more instructive to use the size of the inscribed square as the size of the crystal in the delivery and translation directions. To more conveniently use this method, the length of the short side of the crystal can be directly used as the crystal size in the delivery direction and translation direction. In this study, the rotation (r 2) of the crystal is continuously performed in one direction. Relevant experimental data show that the highest indexable rate will be obtained when the X-ray beam is orthogonal to the microfluidic sample plate; thus, the rotation range should not be too large. Therefore, we demonstrate another reference rotation mode, that is, alternate rotation, as shown in Fig. 7 >(c). Starting from the middle, follow the number signs to alternately swing in two directions, which may help to obtain better diffraction results. In addition, for smaller crystals, there is a typical problem of crystal settling, which can be weakened by alternate rotation. 5. Conclusions Sample delivery based on circular motion has become a valuable mode because its delivery speed can be adjusted flexibly and made applicable to many different platforms; therefore, it is worthy of further development to fully utilize the advantages. In this paper, oscillating serial crystallography and circular-motion-based serial crystallography have been combined, and a novel microfluidic rotating-target device capable of three-degrees-of-freedom motion has been developed. By collecting multiple diffraction frames from each crystal in the microfluidic channel, the sample consumption can be greatly reduced, and the efficiency of sample delivery can be highly improved. In addition, the new device retains the technical advantages of in situ diffraction and room-temperature structural determination. Furthermore, the device is easy to manufacture and can be conveniently implemented on synchrotron radiation facilities. Due to these advantages, we strongly recommend the practical utilization of this technology in SSX. Supplementary Material Click here for additional data file. mmcif file. DOI: 10.1107/S1600577523000462/wz5028sup1.mcf Structure factors: contains datablock(s) r7dlnsf. DOI: 10.1107/S1600577523000462/wz5028sup2.hkl Click here for additional data file. Movie S1. An animation demonstration of the motion control scheme. DOI: 10.1107/S1600577523000462/wz5028sup3.avi Supporting Figures S1 to S3; Tables S1 to S5. DOI: 10.1107/S1600577523000462/wz5028sup4.pdf We thank the staff from beamline BL18U1 of the National Facility for Protein Science in Shanghai (NFPS) at Shanghai Synchrotron Radiation Facility for assistance during data collection. Figure 1 Design of the 3-DOF-MRT device. (a) Overall design of the 3-DOF-MRT device. r 1: rotational degree of freedom; t: translational degree of freedom; r 2: rotational degree of freedom. (b) Schematic diagram of the overall exterior of the 3-DOF-MRT device. (c) Structure and assembly process of the microfluidic sample plate. (d) Size of the microfluidic sample plate. Figure 2 Physical images of the 3-DOF-MRT device and its status when used for the SSX experiment. (a) Front view of the 3-DOF-MRT device. The red arrow indicates the rotational (r 1) degree of freedom, the green arrow indicates the translational degree of freedom, and the blue arrow indicates the rotational (r 2) degree of freedom. (b) Side view of the 3-DOF-MRT device. (c) Top view of the 3-DOF-MRT device. Figure 3 Analysis of crystal distribution. (a) Optical imaging of the crystal morphology and its distribution in the microfluidic channel before and after rotation at a speed of 1/6 RPM. (b) Schematic diagram of crystal positioning. (c) Percentage of crystals remaining at the initial position. Figure 4 Background scattering analysis. (a) Representative diffraction frames and their corresponding magnifications of the crystal, solution, device and air samples. (b) Radial profiles of the background scattering intensities of the solution (gray line), device (orange line) and air (blue line) (0 to 1200 pixels along the y-axis of the frame as indicated by the red arrow). Figure 5 Data collection analysis. (a) Strategy of subset grouping. (b) Indexable rate of different subsets. (c) Indexable rate changes as the diffraction data accumulated. Changes in the figures of merit: (d) completeness, (e) multiplicity and (f) R split of the different subsets as the diffraction data accumulated. (g) Radar graph of the different subsets plotted by the different figures of merit. The scale of the axis is from the inside to the outside. Figure 6 Structural analysis. (a) Lysozyme structure and the enlargement of typical residues as well as their electron density maps obtained through the data set of D1-8. (b) 2F o-F c (blue mesh, 1.5s) and F o-F c (red mesh, -3.0s) electron density maps of the disulfide bond in lysozyme. Figure 7 Recommended sample delivery speed, step length and rotational (r 2) mode. (a) Recommended sample delivery speed. L crystal is the size of the crystal in the delivery direction, L exposure is the distance of the crystal delivery during exposure, and L gap is the distance of the crystal delivery during the delay time for the detector collecting data. (b) Recommended step length. is the size of the crystal in the translation direction, and L step is the translation step length. (c) Recommended rotational (r 2) modes, including continuous rotation in one direction and alternating rotation in two directions. Table 1 Data collection analysis and structural refinement for lysozyme (D1-8) Crystal size (mm) 100 x 100 x 100 Exposure time (s) 0.3 Wavelength (A) 0.98 Photon energy (keV) 12.6 FWHM (H x V) (mm) 30 x 30 Beam flux (photons s-1) 5 x1011 No. of indexed / collected images / indexable rate 4770 / 44459 / 10.73% Detector distance (mm) 300 Resolution (A) 34.24-2.15 (2.23-2.15) Space group P43212 a, b, c (A) a, b, g (deg) 79.8, 79.8, 38.7 90, 90, 90 R split (%) 27.52 (53.41) CC1/2 (%) 88.47 (57.70) CC* (%) 96.89 (85.54) I/s(I) (SNR) 3.98 (2.45) Completeness (%) 99.26 (97.20) R work / R free (%) 18.74 / 20.00 Average B value (A2) 77.3 Total number of reflections 6887 Number of reflections in refinement 6200 Number of free reflections in refinement 687 Number of atoms 1050 Protein 1000 Water and others 50 R.m.s deviations from ideal values Bonds (A) 0.0092 Angles (deg) 1.15 Ramachandran plot statistics (%) Favored 96.06 Allowed 3.94 Disallowed 0 PDB entry 7dln References Adams, P. 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PMC10000802 | J Synchrotron Radiat J Synchrotron Radiat J. Synchrotron Rad. Journal of Synchrotron Radiation 0909-0495 1600-5775 International Union of Crystallography 36891852 gy5038 10.1107/S1600577522012103 JSYRES S1600577522012103 Research Papers Hard X-ray full-field nanoimaging using a direct photon-counting detector Hard X-ray full-field nanoimaging Flenner Silja a* Hagemann Johannes b Wittwer Felix b++ Longo Elena aSS Kubec Adam cP Rothkirch Andre b David Christian c Muller Martin a Greving Imke a a Helmholtz-Zentrum Hereon, Max-Planck-Strasse 1, 21502 Geesthacht, Germany b Center for X-ray and Nano Science - CXNS, Deutsches Elektronen-Synchrotron - DESY, Notkestrasse 85, 22607 Hamburg, Germany c Paul Scherrer Institut, Forschungsstrasse 111, 5232 Villigen, Switzerland Bergamaschi A. Editor Paul Scherrer Institut, Switzerland Correspondence e-mail: [email protected] ++ Current address: NERSC, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA. SS Current address: Elettra - Sincrotrone Trieste SCpA, 34149 Basovizza, Trieste, Italy. P Current address: XRnanotech GmbH, Forschungsstrasse 111, 5232 Villigen, Switzerland. 01 3 2023 01 2 2023 01 2 2023 30 Pt 2 s230200 390399 05 5 2022 22 12 2022 (c) Silja Flenner et al. 2023 2023 This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-BY) Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited. A full version of this article is available from Crystallography Journals Online. A direct photon-counting detector was used for different nanoimaging phase contrast techniques, increasing the temporal resolution. Full-field X-ray nanoimaging is a widely used tool in a broad range of scientific areas. In particular, for low-absorbing biological or medical samples, phase contrast methods have to be considered. Three well established phase contrast methods at the nanoscale are transmission X-ray microscopy with Zernike phase contrast, near-field holography and near-field ptychography. The high spatial resolution, however, often comes with the drawback of a lower signal-to-noise ratio and significantly longer scan times, compared with microimaging. In order to tackle these challenges a single-photon-counting detector has been implemented at the nanoimaging endstation of the beamline P05 at PETRA III (DESY, Hamburg) operated by Helmholtz-Zentrum Hereon. Thanks to the long sample-to-detector distance available, spatial resolutions of below 100 nm were reached in all three presented nanoimaging techniques. This work shows that a single-photon-counting detector in combination with a long sample-to-detector distance allows one to increase the time resolution for in situ nanoimaging, while keeping a high signal-to-noise level. nanotomography full-field X-ray microscopy near-field holography near-field ptychography Zernike phase contrast single-photon-counting detector phase contrast Deutsche Forschungsgemeinschaft192346071 Silja FlennerThe authors gratefully acknowledge financial support from the Deutsche Forschungsgemeinschaft (DFG) (project No. 192346071, SFB 986, project Z2). pmc1. Introduction Hard X-ray nanoimaging and nanotomography are powerful and frequently used tools in many research areas such as materials science, biology, geology and medical science. A classical method for full-field X-ray tomography at the nanoscale is based on a transmission X-ray microscope. It typically consists of condenser optics with a central stop focusing the incoming X-ray beam onto the sample. The image formation is achieved by an X-ray objective lens placed behind the sample, projecting the image onto the detector. However, especially for low-absorbing biological samples, contrast and dose are major issues when it comes to absorption-based X-ray imaging. High X-ray energies allow for a lower total dose, but require phase contrast methods to visualize low-absorbing specimens at high spatial resolution. For a transmission X-ray microscope, the method of choice is Zernike phase contrast (ZPC) (Zernike, 1942 >; Schmahl et al., 1994 >) which can easily be implemented into an existing transmission X-ray microscope by inserting a phase ring in the back-focal plane of the objective lens. The advantage of ZPC is that the contrast of the image is directly enhanced so no phase retrieval step is necessary. In a transmission X-ray microscope, the resolution is limited by the numerical aperture (NA) of both the condenser optics NAcon and objective lens NAobj. In the case of a similar NA, the resolution is improved by a factor of two: d = 1.22l/(NAcon + NAobj) = 0.61l/NA. For a transmission X-ray microscope based on Fresnel zone plates (FZPs), the resolution is simply limited by the outermost zone width d r of the FZP and beamshaping condenser: d = 0.61d r (Attwood, 2007 >; Born et al., 1999 >). Near-field holography (NFH) (Cloetens et al., 1999 >) is another phase contrast method. In combination with nano-focusing optics it allows imaging at the nanoscale, offering high flexibility with a scalable field of view (FOV) and magnification, quantitative phase retrieval and a large working distance. The latter allows one to integrate sample environments, e.g. for in situ experiments, more easily. In NFH, the magnification is achieved by placing the sample in the divergent beam of the focusing optics. In this case, the contrast in the measured hologram is formed by propagation of the wavefield behind the object in free space. In the NFH setup, no optics are placed behind the sample. Such optics usually have a limited efficiency and can introduce further artifacts (e.g. aberrations). Therefore, NFH is also referred to as a lens-less imaging scheme which makes NFH also very dose efficient. The resolution of NFH is limited by the focal spot size. Near-field ptychography (NFP) (Stockmar et al., 2013 >; Xu et al., 2020 >) offers the opportunity to increase the FOV compared with plain NFH and to obtain a reconstruction without prior sample knowledge, also for non-uniform illuminations. For NFP, holographic projections are recorded at different lateral sample positions. With a large and sensitive detector the scattering information outside the primary beam area can be exploited. Thus, the resolution is no longer limited by the focal spot size, but by the achievable numerical aperture of the detector. Especially at synchrotron radiation sources these full-field methods offer fast nanotomography for both transmission X-ray microscopy (TXM) (Ge et al., 2018 >; Flenner et al., 2020c >) and NFH (Villanova et al., 2017 >). From the point of view of data analysis, the noise level of a detector, i.e. the contrast-to-noise ratio (CNR), is very important, especially if a high time resolution is required. Two types of detector chips are commonly in use: charge-coupled device (CCD) systems usually have a lower noise level, but suffer from long readout and dead-times, making them less suitable for fast tomography. Complementary metal-oxide-semiconductor (CMOS)-based cameras on the other hand offer a much faster readout and small pixel sizes and are therefore well suited for fast nanoimaging approaches (Alle et al., 2016 >; Flenner et al., 2020c >). Often a detector system is used where the scintillator is coupled to a light optical system, which magnifies the image onto the camera chip. Hence, many photons are lost in the complex system for light optical magnification which increases the noise level significantly. Also systems without a light optical magnification are in use, e.g. the Hamamatsu CMOS (C12849), where the scintillator is directly coupled to the chip via a fiber optical plate. Here, a long sample-to-detector distance is needed to achieve a sufficient magnification in the X-ray regime due to the small divergence of the focused X-ray beam. This makes the system more efficient, but the noise level, mainly a combination of detector readout noise and photon noise, is still not negligible (Flenner et al., 2022 >). One approach to improve the image quality in full-field imaging techniques is the use of single-photon-counting pixel detectors (Manolopoulos et al., 1999 >; Ponchut et al., 2021 >). Such detectors provide a noiseless readout and can threshold photons within a specific energy range, making them highly attractive for fast tomography approaches. In addition, they offer an almost single-pixel point spread function (PSF) (Donath et al., 2013 >). In comparison, CMOS cameras have a PSF of 2 to 3 pixels, limiting the spatial resolution. Among the commercially available single-photon-counting detectors, the X-Spectrum LAMBDA detector (Pennicard et al., 2013 >, 2014a >,b >) based on the MEDIPIX3 sensor (Ballabriga et al., 2013 >; Manolopoulos et al., 1999 >) currently offers the smallest pixel size (55 mm) and has been used for imaging techniques such as far-field ptychography (Wilke et al., 2014 >). Other single-photon-counting detectors with larger pixel size are also used for imaging, e.g. the Dectris Eiger with a pixel size of 75 mm (Dinapoli et al., 2011 >; Schropp et al., 2020 >). In addition, photon-counting detectors are frequently used in benchtop devices and for medical purposes to reduce dose and scan time (Ballabriga et al., 2021 >). However, the physical pixel sizes of commercially available detectors are currently limited to >50 mm (Ballabriga et al., 2021 >). Therefore, full-field nanoimaging applications in the hard X-ray regime using photon-counting detectors require a much stronger demagnification of the pixel size compared with CMOS cameras with much smaller pixels in the range of 5 to 10 mm. This demagnification can for example be achieved via a large sample-to-detector distance. At the imaging beamline P05 at PETRA III (DESY, Hamburg), a sample-to-detector distance of up to 22 m is accessible. In this article, TXM measurements using a direct photon-counting detector are shown and compared with the performance of a conventional CMOS system. In addition, NFH and NFP are presented and evaluated using a LAMBDA detector. 2. Materials and methods The experiments were performed at the P05 nanotomography endstation which is operated by the Helmholtz-Zentrum Hereon at the storage ring PETRA III at DESY, Hamburg, Germany (Ogurreck et al., 2013 >; Greving et al., 2017 >, 2018 >). The nanotomography endstation is located at the first experimental hutch (EH1) of the imaging beamline P05 [Fig. 1 >(a)]. The beamline also hosts a microtomography experiment located in a second experimental hutch (EH2). Since only one experiment can run at the same time, the detector can be placed in EH2, enabling a sample-to-detector distance of up to 22 m [Fig. 1 >(a)]. This allows for a high magnification in the X-ray regime, enabling nanoimaging without additional light optical magnification. The different imaging techniques were performed at a photon energy of 11 keV, monochromatized by a channel-cut Si-111 monochromator. A Si-based LAMBDA 750k single-photon-counting detector with a pixel size of 55 mm x 55 mm was used (Pennicard et al., 2013 >, 2014a >,b >). The detector area consists of 12 individual chips of 256 x 256 pixels, arranged on a 2 x 6 grid. At the edges between adjacent chips there are larger pixels of size 55 mm x 165 mm (edges) and 165 mm x 165 mm (corners). These are split into three pixels inside the detector software, resulting in a final image size of 1556 x 516 pixels. The LAMBDA was used in 24 bit mode (16.7 million photons per pixel) which enables a maximum frame rate of 1000 frames per second (full frames). For comparison, an X-ray sCMOS camera (Hamamatsu C12849-101U, 6.5 mm pixel size, 2048 x 2048 pixels, 16 bit image depth) with a 10 mm Gadox scintillator was used. The Hamamatsu's dynamic range is reduced due to readout noise to a range of 18000:1 at a frame rate of 30 frames per second (full frames). 2.1. Zernike phase contrast setup The TXM setup for ZPC is shown in Fig. 1 >(b). The beamshaping condenser (1.8 mm diameter) (Vartiainen et al., 2014 >) and the beamstop (800 mm diameter) create a hollow cone illumination and focus the beam on a 50 mm x 50 mm spot in the sample plane. Order sorting apertures (OSA) block the higher diffraction orders of the beamshaper. An FZP is placed behind the sample and serves as an objective lens. Depending on the desired FOV and magnification, FZPs with different diameters and outermost zone width d r can be chosen. A rotating piece of paper is mounted in front of the microscope in order to decrease the impact of disturbing phase effects originating from the high coherence of the beam. For ZPC, phase rings matching the aperture of the FZP and the beamshaper are placed in the back-focal plane of the objective lens. The detector is mounted in the adjacent experimental hutch at a distance of 20.7 m, utilizing the high magnification in the X-ray regime. At this distance, the image created by the FZP has a size of approximately 14 mm x 14 mm and therefore covers the whole detector chip of the Hamamatsu detector or a single chip of the LAMBDA detector. A FOV of approximately 256 x 256 pixels on the LAMBDA is projected on a single chip. When placed in the FOV, the large pixels between the chips could induce ring-like artifacts in the tomographic reconstruction. Details of the different configurations for TXM are given in Table 1 >. 2.2. Near-field holography setup and ptychographic measurements The NFH setup at the P05 nanoimaging endstation is based on a single FZP [Fig. 1 >(c)] (Flenner et al., 2020a >). The FZP with a diameter of 300 mm and outermost zone width of d r = 50 nm focuses the beam to a small spot of 1.22d r = 61 nm which limits the achievable resolution. A beamstop is placed behind the FZP, covering half of the FZP and blocking the direct beam. In the focal distance of 133 mm, OSAs block the higher diffraction orders of the FZP. For the holotomographic measurements, the sample was placed at a defocus distance y 1 depending on the desired FOV and magnification. In the case of a cone beam setup the magnification M is given by M = y 2/y 1, where y 2 is the detector distance [Fig. 1 >(c)]. Thus with y 1 the magnification or FOV can be adjusted to the needs of the experiment. More details of the measurements can be found in Table 2 >. For the NFP measurements, we kept the same setup as for NFH. The samples were placed at a defocus distance of 36.8 mm and scanned across the beam in a 6 x 6 raster. The step size was 1 mm and each point was randomly offset horizontally and vertically by up to 1 mm to avoid scan grid artifacts. Each image was exposed for 1 s. The images from the LAMBDA were padded to a size of 1024 x 1024 pixels. Because the detector panel of the LAMBDA is only 516 pixels high, the parts of the image outside the detector area were filled with zeros. Further details of the measurements are given in Table 3 >. In ptychographic imaging, a structured illumination can improve the reconstruction (Stockmar et al., 2013 >). To this end, for the scan of the spider hair, a piece of paper was inserted in front of the FZP to add diversity into the illuminating wavefront. 2.3. Data processing The three different phase contrast methods require slightly different processing protocols. This includes a phase retrieval step in the case of NFH and NFP, while the ZPC data, after the flat-field correction, can be directly processed to obtain the tomographic reconstruction. For NFH, the phase reconstructions were obtained from a single distance measurement by an iterative alternating projections algorithm based on the work of Wittwer et al. (2022 >), integrated in the HoloTomoToolbox (Lohse et al., 2020 >). Prior to the reconstruction, the projections have been corrected with a dynamic flat-field correction based on a principal components analysis (PCA) (Van Nieuwenhove et al., 2015 >; Hagemann et al., 2021 >). For NFP, the phase retrieval was carried out via the refractive ptychographic iterative engine (refPIE) (Wittwer et al., 2022 >), using the Fresnel propagator instead of the usual Fraunhofer propagator. Due to the required precision of the sample positioning system used for scanning, a position refinement was needed in the reconstruction process (Schropp et al., 2013 >). We accelerated the reconstruction by using Nesterov-accelerated gradients (Maiden et al., 2017 >). The tomographic reconstructions of the TXM and holography scans were obtained using the gridrec algorithm (Dowd et al., 1999 >) with a Shepp-Logan filter implemented in tomopy (Gursoy et al., 2014 >). The CNR of the TXM tomograms was calculated by the following equation (Muhogora et al., 2008 >), where I mat and I bg are the mean gray values in the material and the background, and smat and sbg are the standard deviations for these regions. The ratio is calculated on the unfiltered TXM slices, using the mask of the segmented volume. The resolution was estimated via the Fourier ring correlation (FRC, 2D) and Fourier shell correlation (FSC, 3D). For the tomograms, the projections were divided in two stacks and the corresponding reconstructions were then used as an input for the FSC. For the 2D phase reconstructions, several holograms were taken with the same exposure time. For NFP, the scans were split into two halves and reconstructed separately. The half-period resolution was calculated from the FRC/FSC curves using the half-bit threshold criterion (Van Heel & Schatz, 2005 >). 2.4. Samples Two different samples were used. The first one, a low-absorbing biological sample, was a spider attachment hair (Roscoe & Walker, 1991 >; Niederegger & Gorb, 2006 >; Schaber et al., 2019 >; Flenner et al., 2020b >). These approximately 10 mm-wide and up to 1 mm-long hairs enable spiders to walk upside down on many surfaces. The hairs are built up hierarchically: a single attachment hair located at the feet of the spider is covered by several hundreds of smaller hairs (microtrichia) with diameters of 150 nm to 300 nm, ending in spatula-shaped adhesive tips. These fine elements make the spider attachment hair an ideal test sample for phase contrast nanoimaging. The second, larger and more complex sample was a tardigrade. These species are able to survive under extreme conditions and are approximately 50 mm to 150 mm in diameter and up to 800 mm long (Gross et al., 2019 >). The tardigrade is therefore well suited as a test sample for tomography with a large FOV and region of interest (ROI). 3. Results In the following, a comprehensive comparison of the three methods, ZPC, NFH and NFP, is given. 3.1. Zernike phase contrast For the first time, a photon-counting detector was used for a transmission X-ray microscope at a synchrotron radiation source. Fig. 2 > shows reconstructed slices of a tardigrade derived from data taken with two different detector types with exactly the same experimental setup, i.e. distances and optics. Here, optics with 50 nm outermost zone width and an FZP with a diameter of 150 mm were used. More details of the measurements can be found in Table 1 >. Figs. 2 >(a) and 2 >(b) were recorded with the LAMBDA detector, while Fig. 2 >(c) was acquired with the Hamamatsu camera. A magnified image of the ROI, indicated by the red boxes, is shown in Figs. 2 >(d)-2 >(f). Both scans were recorded with the same number of projections (1600) but different exposure times of 0.5 s (Hamamatsu) and 0.2 s (LAMBDA) due to the lower intrinsic noise of the LAMBDA detector. The effective pixel size reached with this setup was 21.4 nm for the Hamamatsu detector and 181 nm for the LAMBDA photon-counting detector; this corresponds to the ratio of the physical pixel sizes (6.5 mm for the Hamamatsu and 55.0 mm for the LAMBDA). Covering the full detector chip of the Hamamatsu (2048 x 2048 pixels) and a single chip on the LAMBDA (256 x 256 pixels) yields a comparable FOV - 44 mm and 46 mm, respectively. Due to a PSF of 2 to 3 pixels of the Hamamatsu detector, the images are usually binned in order to increase the CNR and decrease the storage space, without losing spatial resolution. At the same time, the LAMBDA data can be upsampled by a factor of four [Figs. 2 >(b), 2 >(e)], yielding a comparable effective pixel size of 45.3 nm. Upsampling of the projections prior to the reconstruction yields additional details, as the tomography scan was heavily oversampled, and features can be recognized more easily (Dudak et al., 2017 >). The image contrast is higher in the LAMBDA scan, whereas the resolution is still higher in the Hamamatsu scan: the effective pixel size for the LAMBDA (181 nm) is almost a factor of two larger compared with the half-period resolution calculated from FRC for the sCMOS camera (98 nm). The limiting factor for achieving a higher spatial resolution is currently the large physical pixel size of single-photon-counting detectors. Three different approaches to overcome this limitation will be discussed in the following: (i) decreasing the radius r of the FZP, (ii) decreasing the outermost zone width d r of the FZP, and (iii) increasing the FZP-to-detector distance y. All three approaches result in a higher magnification in the X-ray regime, which can be calculated by where l is the wavelength of the X-rays. For typical values, refer to Table 1 >. The first option (i) is the utilization of an objective FZP with a smaller diameter while keeping the same d r , i.e. NA. In order to achieve a resolution comparable with that of the sCMOS detector, a diameter of 83 mm is required. One disadvantage of this approach, however, is that the focal distance is reduced, limiting the space between the objective lens and the sample, hindering the implementation of larger in situ environments. The second option (ii) to increase the X-ray magnification is to reduce the outermost zone width of the optics, thereby increasing the NA. Here, also the theoretical resolution can be pushed further from 30.5 nm to 18.3 nm, as the resolution limit d of TXM is determined by the outermost zone width d r of the optics (d = 0.61d r ). However, also here the focal distance is drastically reduced. In addition, the focal depth is also reduced, e.g. from 44 mm (d r = 50 nm) to only 16 mm (d r = 30 nm). For tomography, a focal depth smaller than the FOV is problematic since the entire sample needs to be in focus for the image to be considered as a simple projection of the specimen. From the fabrication side, the achievable aspect ratio of the FZP structures is limited. This means that the structure height for FZPs with smaller outermost zone width is lower, resulting in a less efficient focusing and thus less flux on the detector. To achieve an effective pixel size of 98 nm with this approach, an outermost zone width of 13 nm would be required, which is currently not feasible from the fabrication side. The third approach (iii) to gain a higher magnification with ZPC is to simply increase the distance from the sample to the detector. For example, to achieve an effective pixel size of 98 nm with the LAMBDA, a distance of 36 m is needed. Such large sample-to-detector distances are currently not feasible at P05 or, to our knowledge, at any other nanoimaging beamline. In order to achieve a high spatial resolution with the LAMBDA, we combined the approaches of (i) and (ii) and used an FZP with smaller diameter and smaller outermost zone width. Since not all arbitrary sizes and outermost zone widths of FZPs were available, an FZP with d r = 30 nm and a diameter of 120 mm was chosen, yielding an effective pixel size of 85 nm. Although the effective pixel size is still much larger than for the Hamamatsu detector and only a single chip is used (256 x 256 pixels), the resolution turned out to be sufficient to resolve the fine microtrichia in the spider attachment hair [Fig. 3 >(a)]. High-contrast tomograms with low noise can be acquired much faster, even with the lower efficiency of the 30 nm optics compared with the 50 nm optics. Fig. 3 >(a) shows the reconstructed slices of a spider attachment hair for different scan times, from 6 s up to 6 min. Details of the parameters of the scan series can be found in Table 4 >. With an extent of 100 pixels, the spider hair requires approximately 314 projections for a fully sampled tomogram. Apart from the 6 s scan, all scans fulfill the Crowther criterion. In the very short scans (6 and 18 s) the reconstructions suffer from a high noise level. However, already in the 36 s scan, single microtrichia are clearly resolved. The noise in this scan could be easily removed using, for example, an iterative non-local means filter (Bruns et al., 2017 >) or a machine learning based approach (Pelt & Sethian, 2018 >; Hendriksen et al., 2020 >; Flenner et al., 2022 >). The slices obtained from scans with longer acquisition time show a decreasing noise level and a high CNR [Fig. 3 >(b)]. The CNR increases only slightly after 60 s total scan time. At this scan time, 423 photons per pixel were measured in a single projection on average, corresponding to 38.9 x 104 photons per pixel in the full tomogram (Table 1 >). After 36 s scan time (11.5 x 104 photons per pixel for a tomogram), the FSC curves show correlations up to the highest sampled frequency, indicating that the resolution is now limited by the effective pixel size rather than by noise [Fig. 3 >(c)]. However, due to limited sampling, meaning the effective pixel size, the interpretation of FSC curves at these high frequencies is ambiguous (Van Heel & Schatz, 2005 >). A scan time longer than 360 s is therefore not recommended, as it increases the risk of additional sample movements and therefore artifacts in the reconstruction. The CNR maximum for the Hamamatsu detector is reached only after 15 to 30 min using significantly more efficient optics (Flenner et al., 2020c >). For comparison: the tardigrade in Fig. 2 > was measured in 360 s with 3540 photons per pixel (5.66 x 106 photons per pixel for a tomogram). This high number of photons was achieved due to much more efficient optics. Thus, the scan time could have been reduced by a factor of roughly 15. In conclusion, the scan time can be significantly reduced compared with the sCMOS detector, thanks to the photon-counting detector. This also enables the stitching of sub-tomograms to larger volumes of specimens such as biological tissue (Longo et al., 2020 >; Topperwien et al., 2019 >). Fig. 4 > shows a slice of a large volume of a rat lung. For details of sample preparation, refer to Longo et al. (2020 >). The tomogram shows a volume of 105 mm x 105 mm x 105 mm obtained from 3 x 3 x 3 = 27 individual tomography scans with an effective pixel size of 181 nm and a step size of 35 mm. The scans were individually reconstructed using the above-described procedure and stitched with the 3D stitching plugin (Preibisch et al., 2009 >) available in FIJI (Schindelin et al., 2012 >). This enables a high-resolution reconstruction of relevant tissue volumes at short acquisition times while at the same time being non-destructive without any requirements of staining or slicing. The obtained 3D volumes can be used to complement classical 2D histology, opening the door for correlative imaging techniques (Topperwien et al., 2020 >; Albers et al., 2021 >). 3.2. Near-field holography Near-field holotomography offers quantitative imaging and at the same time the dose is significantly reduced compared with TXM as no optics are placed behind the sample. A single hologram is sensitive to noise, since the finest fringes, which determine the resolution, need to be sampled with a sufficiently high CNR for the phase reconstruction. Therefore, the resolution has a strong relation to the number of photons, i.e. dose on the sample (Rudolph et al., 1990 >; Kirz et al., 1995 >; Howells et al., 2009 >; Hagemann & Salditt, 2017 >; Du et al., 2020 >). Using a single-photon-counting detector allows for testing the theoretical fluence-resolution predictions, i.e. the number of photons per pixel needed to achieve a certain resolution in an experiment. We recorded multiple flat fields and holograms of an object at different exposure times in the range from 3 ms to 8000 ms. The holograms were flat-field corrected using the PCA method (Van Nieuwenhove et al., 2015 >; Hagemann et al., 2021 >), where the PCA was computed separately for each exposure time. Note that the division of the two noisy images is a situation which in general is not treated in today's existing theoretical studies. The reconstructions were obtained from the flat-field corrected holograms with an iterative projection algorithm (Wittwer et al., 2022 >; Hagemann et al., 2021 >) using a self-refining support and constraints for the physically allowed values for absorption and phase shifts. The low-absorbing spider hair can be handled as a pure phase object so the allowed values for the absorption were set to zero. The measured 2D resolution as a function of number of photons per pixel is displayed in Fig. 5 >(a). The number of photons per pixel was calculated by averaging over a series of flat fields for each exposure time. The half-period resolution was calculated on 30 reconstructed projections via the FRC for each exposure time [Fig. 5 >(b)]. Again, we observe correlations up to the highest sampled frequencies. From the fit of the data we observe a scaling of the resolution d as a function of the number of photons per pixel proportional to d -3.2+-0.3. This trend continues even at the highest sampled frequencies close to the pixel size limit. This value is well between calculations of the scaling from coherent (d -4) (Starodub et al., 2008 >) and incoherent (d -3) (Shen et al., 2004 >) cases. Note that the current magnitude projection used in the algorithm is implemented in a straightforward manner and does not take the noise present in the measurements into account. Because of too sparse sampling of the exposure times, the number of photons necessary to achieve the best (detector limited) resolution is determined from the fit to be 460 photons per pixel, corresponding to an exposure time of 150 ms. Exemplary reconstructions for selected exposure times are shown in Fig. 5 >(c). Even for very short exposure times of only 3 ms, the outlines of the spider hair can be identified by eye [Fig. 5 >(c)]. Single microtrichia can already be resolved at lower spatial resolution with an exposure time of 30 ms. The higher sensitivity of the LAMBDA detector allows one to significantly reduce the exposure time needed for a single hologram, compared with a sCMOS camera (Flenner et al., 2020a >). Two test samples were measured at different magnifications, and details of the measurements can be found in Table 2 >. The phase reconstructions of the test samples are shown in Figs. 6 >(a) and 6 >(b). An effective pixel size of 168 nm was obtained for the spider hair at a defocus distance of 62.8 mm. Due to the small PSF of the LAMBDA, the microtrichia with diameters between 150 and 300 nm can clearly be resolved [Fig. 6 >(a)]. Since the tardigrade is not a pure phase object, the phase reconstruction is more challenging. Here, an effective pixel size of 98 nm was achieved. The phase could be reconstructed from only one distance, i.e. with a single exposure, revealing the inner structures of the tardigrade's head [Fig. 6 >(b)]. In conclusion, NFH in combination with photon-counting detectors offers a fast and low-dose imaging method, especially well suited for low-absorbing biological specimens. 3.3. Near-field ptychography NFP (Stockmar et al., 2013 >, 2015 >; Xu et al., 2020 >) offers two advantages over NFH: (i) the FOV is no longer limited by the beam size, because the sample is scanned transversally to the X-ray beam; (ii) ptychography is able to separate the illuminating wavefront from the transmission of the sample and therefore does not require flat fields and can be used even for non-planar wavefronts (Maiden & Rodenburg, 2009 >). As the spider hair is smaller than the FOV and only weakly phase shifting, the NFP reconstruction is similar to the NFH result, see Fig. 6 >(c). Considering also the longer scan time due to the stepping of the sample and therefore also higher dose, NFH is more suited for such small samples. In contrast, the reconstruction of the tardigrade sample highlights the strength of NFP [Fig. 6 >(d)]. The FOV is larger and every part of the head is reconstructed with more fidelity than in NFH. While the NFH reconstruction requires manual tuning of phase and absorption constraints, NFP can reconstruct the tardigrade without prior knowledge of the sample. The calculated resolution is similar for both methods. The strength of the ptychographic measurements is that both object and illumination function are reconstructed. This reduces the influence of a non-uniform illumination on the sample reconstruction caused by a non-ideal flat-field correction. In ptychography, it is not only the diffraction inside the direct beam that contributes to the reconstruction, but also the scattering signal around it. Typically, the direct beam is three orders of magnitude more intense than the scattering around it. Here, a photon-counting detector shows its full potential due to its high dynamic range and single-photon sensitivity. Currently, the instrument at P05 is not optimized for ptychographic imaging in terms of motor movement and accessible scattering angle. The detector aperture is slightly larger than the direct beam and most of the scattered photons are blocked due to the limited diameter of the beam pipes, which connect the two experimental hutches. A larger pipe and detector aperture would allow one to extend the FOV and improve the resolution due to the accessibility of larger scattering vectors. 4. Conclusion and outlook We presented a transmission X-ray microscope setup using a single-photon-counting detector. The combination of a large sample-to-detector distance with the LAMBDA detector allows one to realize small effective pixel sizes, significantly reduced scanning times for tomography and at the same time a high contrast-to-noise ratio. The reduced scan time leads to less dose on the sample and less drift during the measurement. In NFH, extremely short exposure times are possible. While the outlines of the samples are already visible at 3 ms, a phase reconstruction of a projection with reasonable quality can be obtained at only 30 ms exposure time. At exposure times of 150 ms, the resolution is no longer limited by photon noise, but by the sampling of the detector (effective pixel size). NFP shows its potential especially for large samples with higher absorption and a strong phase shift. Here, the reconstruction quality can be improved and the FOV can be enlarged compared with NFH, while the spatial resolution is similar, as also stated by Monaco et al. (2022 >). A new detector aiming to break the pixel size limit of 50 mm is currently under development (Dinapoli et al., 2014 >; Ramilli et al., 2017 >). In addition to the small pixel size of only 25 mm, it offers sub-pixel interpolation. In the future, such a detector has great potential to improve the achievable spatial resolution, especially in TXM mode, if readout speed is drastically improved. The presented setups have been developed at the third-generation storage ring PETRA III. Meanwhile, the first fourth-generation synchrotron radiation sources have started operation, e.g. MAX IV and ESRF-EBS. An upgrade is also planned for PETRA III aiming towards a diffraction-limited storage ring. The new technical developments allow for a significant reduction of the emittance in the horizontal direction, increasing the spectral brightness by one to two orders of magnitude (Schroer et al., 2018 >). The higher coherent flux at fourth-generation sources further increases the time resolution for the imaging modalities presented in this work. Although direct converting detectors without the use of light optical systems offer the great advantage of efficient X-ray detection, there is an issue which cannot be solved easily: the FOV that can be captured with the detector is limited by the achievable numerical aperture in the hard X-ray regime and hence by the X-ray optics and the microscope geometry. Therefore, a large sample-to-detector distance is a key factor in pushing the spatial resolution even further. In particular, the presented holotomography setup will benefit from the higher coherence at a fourth-generation source: the focal spot size of an FZP is influenced by the coherence properties of the beam. Currently, also the size of the FZP is limited by the coherence length in the horizontal direction at the experiment (105 mm) (Flenner et al., 2020a >). Due to the higher coherence available at PETRA IV, a larger FZP can be used than at PETRA III, resulting in a significantly higher flux at the detector. In conclusion, the upgrade to a fourth-generation synchrotron radiation source in combination with single-photon-counting detectors will open the door to even faster, dose-optimized and low-noise nanotomography, well suited for in situ applications. This research was supported in part through the Maxwell computational resources operated at DESY. The authors thank Clemens Schaber, Vladimir Gross and Lucie Sancey for providing the samples. AK is an employee of XRnanotech GmbH. Open access funding enabled and organized by Projekt DEAL. Figure 1 (a) Overview of the beamline P05 with relevant hardware components and their distances to the source. The nanotomography setup is located in the first experimental hutch (EH1) while the camera is placed in the second experimental hutch (EH2). (b) Zernike phase contrast implemented in a standard transmission X-ray microscope and (c) near-field holography setup using a Fresnel zone plate (FZP) as focusing optics. This setup was also used for the near-field pytchography measurements. Figure 2 Reconstructed slices of the same tardigrade specimen imaged with Zernike phase contrast and the same optics but recorded with two different detectors. (a) Recorded with the LAMBDA. (b) Projections recorded with the LAMBDA and upsampled by a factor of four prior to the tomographic reconstruction. (c) Tomographic slice acquired with the Hamamatsu camera. Panels (d)-(f) show the magnified ROI indicated by the red boxes in (a)-(c). Note that the sample has been removed from the rotation axis between the two scans and may not show the exact same region. No filtering or further post-processing applied. The scale bar indicates 10 mm. Figure 3 Fast Zernike phase contrast tomography with a photon-counting detector. (a) Reconstructed slices of a spider attachment hair with different scan times. (b) CNR for the different scan times. For details of the fit, refer to Flenner et al. (2020c >). A high CNR is already reached after 60 s. (c) Half-period resolution as calculated from the FRC. The effective pixel size (85 nm, solid horizontal line) limits the resolution after 36 s scan time. The scale bar indicates 2 mm. Figure 4 Slice of a stitched volume of a rat lung. The volume was obtained from 27 individual scans with a step size of 35 mm. The scale bar indicates 10 mm. Figure 5 (a) Half-period resolution of holographic reconstructions (2D projections) as a function of the number of photons per pixel. (b) Estimation of the half-period resolution via the FRC for different exposure times. (c) Phase reconstructions of a spider attachment hair for different exposure times ranging from 3 to 3000 ms. At 300 ms, the detector sampling limits the achievable resolution. The scale bar indicates 5 mm. Figure 6 Phase reconstructed projections of two test samples, a spider attachment hair [(a), (c)] and a tardigrade head [(b), (d)], obtained with different phase contrast methods using a LAMBDA detector. (a), (b) Near-field holography and (c), (d) near-field ptychography. Note: (b) and (d) might not show exactly the same angle and region, since the sample was remounted between the two experiments. The scale bar indicates 5 mm. Table 1 Parameters of the TXM (Zernike phase contrast) measurements for the low-resolution and high-resolution setups Parameter TXM low resolution TXM high resolution Sample Tardigrade Spider hair Energy 11 keV 11 keV Sample-detector distance 20.4 m 20.4 m FZP diameter 150 mm 120 mm FZP d r 50 nm 30 nm Theoretical resolution limit 30.5 nm 18.3 nm Effective pixel size CMOS 21.4 nm 10 nm+ Effective pixel size LAMBDA 181 nm 85 nm Magnification 303 647 Half-period resolution CMOS (3D) 98 nm - Scan time at resolution limit (3D) - 60 s + This value was calculated from the parameters and not experimentally verified. Table 2 Parameters of the near-field holography measurements Parameter Near-field holography Sample Spider hair/tardigrade Energy 11 keV Sample-detector distance 20.6 m FZP diameter 300 mm FZP d r 50 nm Resolution limit (focus size limited) 61 nm Defocus distance of sample y 1 62.8 mm/36.8 mm Effective pixel size 168 nm/98 nm Exposure time at resolution limit (2D) 300 ms Table 3 Parameters of the near-field ptychography measurements Parameter Near-field ptychography Sample Spider hair/tardigrade Energy 11 keV Sample-detector distance 20.45 m FZP diameter 300 mm FZP d r 50 nm Defocus distance of sample y 1 36.8 mm Resolution (2D) 177 nm/115 nm Scan points 6 x 6 Step size (random) 1 +- 0.5 mm Exposure time per point 1000 ms Table 4 Parameters of the tomographic scans displayed in Fig. 3 > Scan time (s) No. of projections Exposure time (ms) Angular speed (deg s-1) Photons per pixel (tomogram) 6 182 10 30 1.0 x 104 18 338 30 10 5.7 x 104 36 682 30 5 11.5 x 104 60 919 75 3 38.9 x 104 180 1041 150 1 88.1 x 104 360 1109 300 0.5 190.6 x 104 References Albers, J., Svetlove, A., Alves, J., Kraupner, A., di Lillo, F., Markus, M. 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PMC10000803 | J Synchrotron Radiat J Synchrotron Radiat J. Synchrotron Rad. Journal of Synchrotron Radiation 0909-0495 1600-5775 International Union of Crystallography 36891851 vl5004 10.1107/S1600577522011705 JSYRES S1600577522011705 Research Papers In situ synchrotron X-ray multimodal experiment to study polycrystal plasticity In situ synchrotron multimodal study of polycrystal plasticity Ribart Clement a* King Andrew b Ludwig Wolfgang cd Bertoldo Joao P. C. a Proudhon Henry a a Mines Paris, PSL University, Centre des Materiaux (MAT), UMR7633 CNRS, 91003 Evry, France b Synchrotron SOLEIL, L'Orme des Merisiers, BP 48, 91192 Gif-sur-Yvette, France c Universite de Lyon, INSA-Lyon MATEIS CNRS UMR 5510, 69621 Villeurbanne, France d European Synchrotron Radiation Facility, BP 220, 38043 Grenoble, France Aranda M. A. G. Editor University of Malaga, Spain Correspondence e-mail: [email protected] 01 3 2023 20 1 2023 20 1 2023 30 Pt 2 s230200 379389 10 8 2022 06 12 2022 (c) Clement Ribart et al. 2023 2023 This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-BY) Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited. A full version of this article is available from Crystallography Journals Online. In situ diffraction contrast tomography has been deployed at the Psiche beamline of Soleil. The evolution of lattice rotation in a 2000 grain region of a titanium sample under tension is studied. The microstructure of polycrystals is known to govern the performance of structural materials. This drives the need for mechanical characterization methods capable of probing large representative volumes at the grain and sub-grain scales. In this paper, the use of in situ diffraction contrast tomography (DCT) along with far-field 3D X-ray diffraction (ff-3DXRD) at the Psiche beamline of Soleil is presented and applied to study crystal plasticity in commercially pure titanium. A tensile stress rig was modified to comply with the DCT acquisition geometry and used for in situ testing. DCT and ff-3DXRD measurements were carried out during a tensile test of a tomographic Ti specimen up to 1.1% strain. The evolution of the microstructure was analyzed in a central region of interest comprising about 2000 grains. Using the 6DTV algorithm, DCT reconstructions were successfully obtained and allowed the characterization of the evolution of lattice rotation in the entire microstructure. The results are backed up by comparisons with EBSD and DCT maps acquired at ESRF-ID11 that allowed the validation of the orientation field measurements in the bulk. Difficulties at the grain boundaries are highlighted and discussed in line with increasing plastic strain during the tensile test. Finally, a new outlook is provided on the potential of ff-3DXRD to enrich the present dataset with access to average lattice elastic strain data per grain, on the possibility of performing crystal plasticity simulations from DCT reconstructions, and ultimately on comparisons between experiments and simulations at the scale of the grain. polycrystalline materials in situ mechanical testing multi-modal synchrotron experiments diffraction contrast tomography 3DXRD SafranThe authors acknowledge the funding of the BIGMECA research initiative funded by Safran. Synchrotron Soleil and ESRF are aknowledged for providing beam time under proposal numbers 20191646 and ma4925, respectively. pmc1. Introduction Establishing microstructure-property relationships remains a critical engineering challenge for advanced structural materials. Metals display a heterogeneous polycrystalline organization which governs their performance and thus drives the need for mechanical characterization methods capable of probing large representative volumes at the grain and sub-grain scales. For many years, research teams have studied mechanisms closely related to these scales such as crystal plasticity, damage, fatigue or crack propagation (Pearson, 1975 >; Jones & Hutchinson, 1981 >; Roters et al., 2010 >; Pineau et al., 2016 >). A large range of characterization techniques is available to probe deformation mechanisms linked to the material microstructure. Historically, investigations were either limited to surfaces (Gourgues, 2002 >; Wang et al., 2010 >; Guo et al., 2014 >; Chen & Daly, 2018 >; Chen et al., 2018 >) or required destructive operations (Echlin et al., 2012 >; Rowenhorst et al., 2020 >). Recent progress in synchrotron (Maire & Withers, 2014 >; Nygren et al., 2020a >) and laboratory X-ray techniques (Bachmann et al., 2019 >) has paved the way to a paradigm shift sometimes called 'diffraction microstructure imaging' (DMI), leading to increasingly complex multimodal in situ experiments that allow observations to be made non-destructively and concurrently on several scales, resulting in significantly richer datasets. In particular, diffraction contrast tomography (DCT), a near-field variant of 3DXRD (Poulsen et al., 2001 >), allows reconstruction of mesoscopic digital grain maps (on the order of 1 mm3) on which simulations can be computed directly (Proudhon et al., 2016 >; Shade et al., 2019 >). The convergence of experimental and numerical data leads to unified but massive datasets (Sangid, 2020 >); yet this wealth of information can render manual post-processing untractable. Moreover, modalities are often acquired independently which further hinders analysis. Note that efforts in the scanning electron microscope (SEM) community, also driven by equipment manufacturers, have led to successful correlative experiments using microstructural information in 3D using serial sectioning methods (Burnett et al., 2014 >; Charpagne et al., 2021 >). Several recent studies took advantage of DMI for the study of polycrystal plasticity, either with single techniques (Miller & Dawson, 2014 >; Pagan et al., 2017 >; Hektor et al., 2019 >) or relying on a multimodal approach while focusing on a few grains with manual (Proudhon et al., 2018 >; Sangid et al., 2020 >; Nygren et al., 2019 >, 2020b >) or statistical analysis (Nervo et al., 2014 >; Wang et al., 2021 >). These remain difficult studies, limited in several aspects by access to one of the few synchrotron beamlines where DMI is possible, the limited number of samples one can test during beam time, and most notably by the time needed to analyze the data produced. Regarding the latter, the absence of a stable multimodal framework in the community clearly limits the diffusion and use of these promising techniques. On the other hand, continuous technological progress is expected to overcome these obstacles. Specifically, the recent ESRF EBS upgrade represents a significant leap with two orders of magnitude improvement in brightness, signal focusing, and spatial and time resolution (Cho, 2020 >). This leads the way for unmatched in situ testing opportunities. For instance, the duration of a single DCT scan has dramatically reduced from 1 h to 3 min. Other synchrotrons such as SOLEIL have already scheduled similar upgrades for the coming years. As part of an effort to promote these developments, the present paper aims to introduce the deployment of an in situ (or 4D) X-ray multimodal technique involving DCT for users on the Psiche beamline at Synchrotron SOLEIL. In addition, the ability to acquire and reconstruct 3D polycrystalline grain maps of initial and deformed microstructures with up to 103 grains is demonstrated in hexagonal titanium, allowing access to lattice curvature evolution over large datasets in the bulk of the material. 2. Materials A commercially pure a-phase grade 2 titanium (CP-Ti) was used for this study (Barkia et al., 2015 >; Marchenko et al., 2016 >). It has a hexagonal close-packed structure with a c/a ratio of 1.586. The material was obtained from TIMET in the form of a 1.6 mm-thick rolled sheet with an initial average grain size of 15 mm and typical texture of cold-rolled Ti (Keeler & Geisler, 1956 >). The chemical composition of the batch was reported as follows (in wt%): 0.14 Fe, 0.005 C, 0.08 O, 0.008 N. Prior to sample preparation, a heat treatment was applied in order to increase the grain size to 50 mm over 24 h (below the b-transus at 855degC) with 10 l min-1 argon flux and followed by air quenching. Samples were machined by electron discharge machining (EDM) along the rolling direction (RD) with a dog-bone shape (20 mm long with a 5 mm gauge length) and an initial 600 mm square cross section. In this case a volume of 1 mm height contains around 10 000 grains. Circular pin holes of 1 mm diameter were drilled symmetrically in each of the specimen heads to apply the mechanical load. All four sample faces were then pre-polished by mechanical grinding with a 1200 grit sandpaper. One face was further polished with EBSD (electron back-scattered diffraction) quality by an additional 2400 and 4000 grit pre-polishing followed by a 25 h vibratory OPS cycle using a QATM Qpol Vibro polisher and 50% Eposil M/50% distilled water solution. Eventually, fiducial micro-indents were added close to the center of this face [shown in Fig. 4(a)] to define a region of interest (ROI) for the present study. Reference SEM imaging covering the central zone delimited by the indents was then obtained by a mosaic of secondary electron (SE) images and EBSD. Prior to the synchrotron in situ experiment, reference tensile curves up to 3% total strain were obtained via laboratory tensile tests. After the in situ test, new reference acquisitions were performed for validation of the Psiche DCT data. The ROI of the sample was scanned again with DCT at the ID11 beamline of the ESRF using a limited aperture to reduce diffraction spot overlap. Post mortem EBSD was also carried out (after re-polishing the front face of the sample over a depth of 40 mm). 3. In situ experimental testing Experiments were carried out on the Psiche beamline to implement the in situ multimodal acquisition with DCT, far-field 3DXRD (ff-3DXRD) and phase contrast tomography (PCT), each technique providing complementary information on the deformation event during the mechanical test. The beamline was configured with a 40 keV monochromatic beam. An overview of the multimodal experimental setup is shown in Fig. 1 >. The near-field detection system used for PCT and DCT acquisitions is composed of a 0.8 mm x 1 mm tungsten beamstop, a 50 mm-thick LuAG scintillator, a 45deg angle deflecting mirror and a 4608 x 2592 pixel Hamamatsu ORCA-Lightning Digital CMOS camera mounted with a 5x optical magnification lens giving an effective pixel size set to 1.087 mm. The far-field acquisition system is made of an attenuation block composed of two 10 mm-thick glass slabs to avoid saturating the detector, a 2048 x 2048 pixel Perkin-Elmer XRD 1621 CN3 X-ray detector with a 200 mm pixel size and a lead beam stop directly mounted on the detector screen. Note that in the present work the DCT and ff-3DXRD scans were carried out sequentially but in principle could be performed simultaneously using a semi-transparent mirror. The in situ test rig Bulky, designed at the Centre des Materiaux, was used for the experiment [see Fig. 2 >(a) and the work by Pelerin et al. (2019 >) for more details on the stress rig]. The anchoring system has been modified by integrating a quartz tube (8 mm in diameter and 1 mm thick) in order to comply with the DCT geometry, by allowing the detector to be brought as close as 5 mm to the rotation axis [Fig. 2 >(b)]. The tensile setup was calibrated with a 500 lb load cell purchased from Futek. Bulky was installed on the tomograph of the experimental hutch of the Psiche beamline and the sample was carefully mounted to avoid causing deformation prior to loading. Scans were performed in the undeformed configuration and after two different loading steps (0.7% and 1.1% total strain) in the selected ROI. At each step, a large-field-of-view PCT scan was performed first (2 mm in height), with the detector positioned 30 mm behind the sample while removing the beamstop and switching the camera to high dynamic range mode. The sample was rotated over 360deg around the tomograph vertical axis to acquire 1000 images. The initial PCT acquisition allowed the generation of a high-definition absorption contrast tomographic volume which can be used as a mask for the DCT reconstruction. In addition, the fiducial markers were visible in the reconstructed volume and were used to position the sample accurately to ensure that the same zone was illuminated at each step. Moreover, the distance between markers in the intermediate PCT reconstructions provided a direct measurement of macroscopic strain. At each loading step, following the PCT, the detector was moved 8.5 mm behind the sample and centered in order to align the beam stop with the X-ray beam. The camera was switched to low dynamic range, the rotation speed was set to 0.05deg s-1 and 1 s exposure time was used for each radiograph. Two 1060 mm x 278 mm box beam aperture DCT scans were performed with 50 mm overlap, resulting in a total acquired height of 470 mm. A total of 7200 diffraction images per scan over 360deg were collected with an integration step of 0.1deg, taking 2 h and producing 150 Gb of raw data. The far-field detector assembly was placed 1 m downstream of the sample, the near-field detector was moved aside and ff-3DXRD scans could be performed with the same illumination as DCT; 3600 diffraction images were taken over 360deg with an integration step of 0.05deg. Each scan took 15 min and represents 60 Gb of data. The complete experimental procedure is summarized in Fig. 3 >. 4. Volume reconstruction 4.1. PCT reconstruction The PCT acquisition scans were reconstructed with a filtered back-projection algorithm using PyHST2 (Mirone et al., 2014 >). A Paganin filter (Paganin et al., 2002 >) was also used to enhance contrast in the reconstruction. Fiducial indents are easily detected in the PCT reconstruction which allows the strain to be measured directly at each step. 4.2. DCT reconstruction at Psiche Currently, DCT reconstructions are tightly linked to the ESRF computing infrastructure, using the Matlab code developed by the team working at the Materials Science beamline ID11 [ (Ludwig et al., 2009 >; Reischig et al., 2013 >; Vigano et al., 2014 >)]. The key reconstruction steps consist of background correction and normalization of the collected images, diffraction spot segmentation, spot-pair matching, grain indexing, and grain-by-grain tomographic reconstruction. Microstructures exhibiting negligible intragranular orientation spread can be reconstructed using the 3D-DCT (single orientation) approach which is based on the algebraic reconstruction algorithm [simultaneous iterative reconstruction technique (SIRT)], implemented in the ASTRA open source tomography library (van Aarle et al., 2016 >; Palenstijn et al., 2011 >). For materials with non-negligible orientation spread within the grains, the 6D-DCT approach can be used in order to capture the intra-granular orientation field. This approach is based on an in-house implementation of the Chambolle-Pock optimization algorithm (Chambolle & Pock, 2016 >) and total variation (TV) regularization of the solution (Vigano et al., 2014 >). Under full-field illumination, grain maps can be generated up to about 2% total strain. Reconstructions were managed with the DCT code hosted at the ESRF. This required a change in file format and transfer of the data during the experiment. In addition the code was updated to take into account the specifics of the acquisition chain of the Psiche beamline. In parallel, efforts are ongoing to convert the current code to Python while accelerating reconstruction speed and improving user experience in a Jupyter Lab environment. Currently the pre-processing and segmentation steps have been implemented at Psiche. Resulting data can be input into the existing Matlab code to complete the DCT reconstruction. With this new pipeline, an acquisition consisting of 3600 images can be processed in less than 1 h (about 15 min for pre-processing and 30 min for segmentation) which can be up to one order of magnitude faster with respect to the current DCT code. In order to ensure that DCT reconstructions are ready for mechanical simulations using finite element or FFT methods, additional numerical cleaning operations are performed with the Python pymicro package ). This includes morphological cleaning to eliminate small-artifact grains and final-grain dilation. 5. Results In this section, the reconstructed data of DCT scans in the initial undeformed and subsequent deformed states for the same ROI are presented. Eventually both configurations are qualitatively mutually compared with EBSD measurements. 5.1. DCT reconstructions Raw DCT volume outputs from both SIRT and 6DTV reconstruction algorithms for the undeformed state are presented in Fig. 4 > to assess the performance of the DCT reconstructions with the present setup at Psiche. Fig. 4 > shows the results with the two algorithms side by side. Both algorithms reconstruct grains individually after the indexing phase leading to the same number of grains (1853). But not only does the 6D-DCT algorithm provide the full orientation field, it also improves the grain shapes significantly compared with EBSD. This is due to the fact that 6D-DCT accounts for local variations of the diffraction geometry. As a result it correlates more diffraction information which results in a more reliable grain shape. The expense is a more computationally intensive reconstruction: 10 h for SIRT and about 100 h for 6DTV [using 8 Intel Xeon cores with 256 Gb of RAM; note that for the 6DTV reconstruction the back-projection operations are handed over to the Astra toolbox (Palenstijn et al., 2011 >) which runs on a Nvidia Titan X GPU card while the rest of the algorithm runs on a CPU]. For the remainder of this paper, the 6DTV reconstruction algorithm will be used. Fig. 5 > shows the DCT reconstructions at each step of the tensile test. As deformation proceeds, some grains are no longer indexed due to excessive overlap between diffraction spots. At step 1, 98% of the grains compared with the undeformed configuration can be reconstructed. At step 2, this reduces to 77%. In addition, the shape of reconstructed grains degrades as deformation proceeds, so that we lose more data at grain boundaries. For a more precise comparison of the performance of the reconstructions, we display slices corresponding to the re-polished EBSD surface, which have been extracted from the DCT volume data [see Fig. 5 >(b)]. The 6DTV microstructure was numerically dilated to generate the final volume [Fig. 5 >(c), step 0]. Note that only the undeformed state needs to be numerically dilated since it will be used later for crystal plasticity simulations (not discussed in this paper). Fig. 6 > provides a further demonstration of the improved performance of the 6DTV algorithm in the case of a deformed microstructure. A forward simulation of the reconstructed data has been performed with a post-processing module available in the ESRF program in order to generate virtual spots which are compared with the experimental acquisition (first row in Fig. 6 >). The comparison with forward-simulated diffraction spots calculated for a constant (grain average) orientation in the grain shows that we lose much of the correspondence with experimental data (middle row). Indeed, the intensity distributions in the diffraction spots encode both grain shape and the local orientation field. As deformation proceeds, the information of the grain shape becomes progressively convoluted with the orientation field which distorts the spots. On the other hand, we notice that, when taking into account the local orientations resulting from the 6DTV reconstruction, the intensity distributions in the spots are closer to those observed experimentally than those from the 3D reconstruction. This captures the main trends of the real orientation field. Of course this does not prevent a direct comparison with another modality such as EBSD as shown later (see Fig. 7 >). 5.2. DCT analysis 5.2.1. Grain reference orientation deviation fields As the orientation field is directly available from the 6DTV reconstruction (in the form of a Rodrigues vector for each voxel), the grain reference orientation deviation (GROD) field was computed with respect to the average orientation in each grain for each load step. Fig. 7 > displays such fields for each load step in the slice corresponding to the re-polished EBSD face. As we deal with volume data, it is also possible to generate 3D visualizations of the GROD field for selected grains and their neighborhood [see Fig. 7 >(d)]. Qualitatively, we notice that, initially, the misorientation in each grain is negligible, confirming that the microstructure can be considered deformation-free in the reference state. In addition, a consistent evolution of the GROD field with loading is observed: in the intermediate load state (e = 0.7%) the field is heterogeneous with clusters of higher activity and it homogenizes in most grains at the final load state (e = 1.1%). The DCT data were quantitatively validated by comparison with the GROD field in the re-polished EBSD on a few grains of interest. The activity is overall on the same order of magnitude in most of the grains. Also patterns are visually similar. However, we observe that, in the present case, DCT is not able to reconstruct close to the grain boundary where most of the misorientation takes place, especially close to triple junctions. 5.2.2. Statistical analysis In addition to visualization of the deformation field, statistical data analysis can be carried out to plot the mosaicity evolution in a given grain [Fig. 8 >(a)] or the mean misorientation distribution evolution in the entire microstructure [Fig. 8 >(b)]. In agreement with the visual GROD observations, these quantities vary qualitatively as expected and can be used directly to compare with full-field crystal plasticity simulations. 6. Discussion We observed that DCT acquisition in the undeformed state reconstructed with the 6DTV algorithm is able to reconstruct a reliable 3D grain map which compares well with the EBSD measurement in the bulk. We also saw that the grain shape obtained is not perfect near grain boundaries. In the present case, this is mainly attributed to the thickness of the scintillator selected at Psiche for the present experiment (50 mm thickness). Because the diffracted beams are not incident perpendicular on the scintillator, the extra thickness resulted in slightly blurred spots which impedes the ability of the detector to resolve the exact spot shapes. Also a few small grains are missed in the reconstruction process due to the selected segmentation thresholds. Indeed, with a large box beam acquisition, a compromise needs to be found between separation of diffraction spots and the precision of the segmentation. However, since the number of missing grains in the undeformed configuration is very limited and involves only the smallest grains, this is assumed to have negligible influence on the representativity of the microstructure. As a result, after dilation within the absorption mask, the grain map can be considered to be a reliable digital twin and used as input for subsequent full-field simulations. Regarding the reconstruction of deformed volumes, the majority of the grains (77%) are still reconstructed after 1% strain, but an increasing number of grains are lost as deformation proceeds. This is caused by the increase of orientation spread in the grains which leads to diffraction spot overlap. This diffraction spot overlap and the drop of diffraction signal at the periphery of a grain are detrimental to fine quantitative analysis of the plasticity close to the grain boundaries, where most of the lattice misorientation accumulates. This effect can be counter-balanced to some extent by reducing the height of the illuminated sample volume. In the present study, we used slit heights from 278 mm to 220 mm for the different deformation states, corresponding to 1800 and 1400 illuminated grains per acquisition, respectively. On the other hand, the observed evolution of the misorientation field in the central region of the grains remains consistent and compares well with EBSD data. The orientation data from these regions may be used for qualitative analysis of plastic activity. The extended (box) beam illumination used in DCT is the fastest technique to map thousands of grains, with isotropic spatial resolution in three dimensions. This is a major advantage when trying to produce a statistically representative analysis of microstructural events, especially as it gives direct non-destructive access to volume information of entire neighborhoods of grains. In addition, as mentioned in Section 2, a post mortem DCT scan used as ground truth for the Psiche data validation has been performed at the ESRF ID11 beamline in a sub-region with optimized acquisition conditions for performance comparison [a DOI has been assigned to the raw data in the ESRF repository: (Joste et al., 2025 >)]. Using a scan height of 60 mm and a high-resolution detector system (10 mm free-standing LuAG screen), the user can significantly improve the accuracy of the grain shape and orientation field reconstruction (Fig. 9 >). We emphasize that the further degradation of the diffraction signal at higher levels of deformation can be handled using slice beam illumination and a forward modeling strategy (Suter et al., 2006 >). Regarding the GROD analysis and more specifically the comparison between DCT and EBSD data, many reasons can be invoked to explain the observed differences. Generally speaking, 6D-DCT is a mathematical optimization technique, trying to solve an under-determined, inverse problem using regularization techniques. This makes it inherently challenging (with respect to scanning techniques) to achieve similar accurate values. Especially close to grain boundaries (regions of intense modifications: discontinuities, dislocation accumulation, precipitates, defects), the diffraction signal is diffuse and less intense (limited volume). In other words, the signal-to-noise ratio is locally degraded. As a result this signal contribution is less likely to be taken into account. Moreover, the 6D-DCT framework only considers changes in crystal orientation and neglects elastic distortion of the crystal lattice. More advanced reconstruction techniques considering these additional degrees of freedom are still under development (Shen et al., 2020 >; Reischig & Ludwig, 2020 >). For the present experiment, the thickness of the scintillator limits our ability to capture information close to grain boundaries. A thinner scintillator would improve the acquisition but at the cost of a longer scan time or a reduced signal-to-noise ratio. In the end, this is a parameter that can be adjusted within the available hardware to tune the compromise of precision versus acquisition speed. In addition, the level of deformation is also clearly a limiting factor. As mentioned, reducing the quantity of grains in the field of view allows reconstruction at larger deformation simply by decreasing the chance of spot overlap. The material choice can affect the performance when presenting a particular texture, annealing twins or precipitates at grain boundaries. Hence, all these reasons may yield non-reconstructed regions in the final volume. Numerical dilation based on orientation similarity is usually carried out to suppress them, particularly when material simulations are needed. In the present case, one may introduce a simple precision metric describing how well the DCT is able to capture the microstructure, using the ratio of voxels assigned to a grain over the total number of voxels in the illuminated part of the specimen. Using this metric, we found 81% in the initial configuration (step 0), 79% for step 1 and this value drops to 53% for step 2. Meanwhile, the ff-3DXRD data are expected to bring additional information on local deformation mechanisms. For instance it can be processed using the ImageD11 software ) which allows the user to index the grains (crystal mean orientation) within the illuminated volume. Further refinement of the diffraction peak positions allows the measurement of the mean elastic strain tensor in each grain. The average stress tensor for each grain can then be obtained using linear elasticity and the elastic constants of the material. The mean values obtained can be compared with simulation results and will be presented in another paper. This will allow us to take full advantage of the multimodal nature of the present experiment which facilitates the collection of richer datasets concurrently, as done on beamline 1-ID at the APS (Lienert et al., 2011 >) or at CHESS (Nygren et al., 2020a >). In the present paper, the experiment was conducted step by step but in principle it can be performed continuously as the Bulky stress rig allows movement of the cross head very slowly (50 nm s-1); this would require the acquisition of near-field and far-field modalities simultaneously as mentioned previously. On other beamlines where the setup comprises a diffractometer, such as ID11 and ID06 at ESRF, other modalities become available during the experiment such as topo-tomography (Ludwig et al., 2001 >, 2007 >; Proudhon et al., 2018 >; Stinville et al., 2022 >), scanning 3DXRD (Bonnin et al., 2014 >; Hayashi et al., 2019 >; Wright et al., 2020 >; Henningsson et al., 2020 >) or DFXM (Simons et al., 2015 >). The ability to zoom in on a given grain or grain environment to observe at higher resolution will be key in future work to elucidate plasticity and fracture mechanisms based on a quantitative analysis of slip bands, geometrically necessary dislocation densities and intragranular elastic strain fields. Furthermore a crystal plasticity simulation (finite element or FFT) can be performed on real microstructures imaged by the DCT. Ultimately, the experimental data can be directly compared with the results of the simulations on a voxel-by-voxel basis (i.e. lattice orientation, elastic strain tensor). Overall, this approach represents a promising route towards the improvement of mechanical modeling. 7. Conclusions We have demonstrated the feasibility of an in situ multimodal X-ray experimental setup on the Psiche beamline at Soleil applied to the study of polycrystal plasticity on a commercially pure titanium grade 2 material. This experimental apparatus enables the acquisition of DCT, PCT and ff-3DRXD modalities while keeping the sample installed on the test rig during a tensile test. We have successfully reconstructed grain maps from the DCT images at every load step by taking advantage of the DCT reconstruction code developed at the ESRF. The local rotation field obtained has been compared with post mortem EBSD measurements and showed good agreement. Despite a number of limitations in grains shape accuracy, the resulting digital microstructures allow consistent qualitative and statistical analysis of the orientation spread evolution in each grain as deformation proceeds. As an immediate benefit, this work demonstrates the possible use of a combination of near-field (DCT) and far-field (3DXRD) modalities for the user community at the SOLEIL synchrotron beamline. The help of the workshop at the Centre des Materiaux is acknowledged for machining the parts to upgrade Bulky. Jean-Pierre Deslandes at Soleil is thanked for designing the beam stop holder. Figure 1 Overview of the multimodal experimental setup on the Psiche beamline at Soleil. Figure 2 In situ test rig Bulky for DCT acquisition. (a) 3D sketch front overview of Bulky with the modified grip system compatible with DCT. (b) Bulky installed on Psiche tomograph in the DCT acquisition configuration. Figure 3 In situ experimental overview: (a) specimen geometry showing the different acquisition zones and loading curve, (b) near-field images integrated over 5deg showing the diffraction spots acquired during DCT scans at each step (the insets show a zoomed-in view of a given location), (c) ff-3DXRD single frame image and zoom on a given detector zone showing 1deg integrated images at each load step (logarithmic scale has been applied). Figure 4 Visualization of DCT reconstructions in the undeformed configuration (IPF color coding along the z axis): (a) PCT reconstruction of the sample gauge length showing the DCT superimposed, (b) repolished EBSD map of the area corresponding to the DCT scan, (c) DCT SIRT reconstruction, (d) DCT 6DTV reconstruction; columns (c) and (d) show from top to bottom the full volume, the volume cropped as repolished and the slice corresponding to the EBSD map. Figure 5 DCT reconstruction during in situ tensile test (IPF color coding along the tensile axis): (a) raw reconstructions of each loading step restricted to the common zone, (b) DCT slices corresponding to the repolished EBSD slice, (c) digital twin of the undeformed state after numerical cleaning and dilation. Figure 6 Illustration of the improved reconstruction quality using the local orientation field: experimental spots recorded on the detector for grain 44 are compared with simulated spots from the SIRT reconstruction (second row) and from the 6DTV reconstruction (third row), all images are plotted using arbitrary units and the same scale. Figure 7 Study and validation of the GROD field from DCT. (a) IPF-Z DCT slices and corresponding repolished EBSD face, (b) GROD field slices evolution with load, (c) quantitative comparison between DCT and EBSD fields in the final deformation state, (d) 3D Paraview visualization of GROD field evolution for a grain and its environment. 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PMC10000804 | J Synchrotron Radiat J Synchrotron Radiat J. Synchrotron Rad. Journal of Synchrotron Radiation 0909-0495 1600-5775 International Union of Crystallography 36891841 yi5135 10.1107/S1600577522012073 JSYRES S1600577522012073 Research Papers Versatile modulators for laser-based FEL seeding at SwissFEL Laser-based FEL seeding at SwissFEL Calvi Marco a* Liang Xiaoyang ab Ferrari Eugenio cd Alarcon Arturo c Prat Eduard c Reiche Sven c Schmidt Thomas a Voulot Didier c Zhang Kai a Ganter Romain c a Photon Science Division, Paul Scherrer Institute, 5232 Villigen, Switzerland b Institute of Biomedical Engineering, ETH Zurich, 8092 Zurich, Switzerland c Large Research Facility, Paul Scherrer Institute, 5232 Villigen, Switzerland d M Division, Deutsches Elektronen-Synchrotron, Notkestrasse 85, 22607 Hamburg, Germany Yabashi M. Editor RIKEN SPring-8 Center, Japan Correspondence e-mail: [email protected] 01 3 2023 01 2 2023 01 2 2023 30 Pt 2 s230200 276283 14 10 2022 22 12 2022 (c) Marco Calvi et al. 2023 2023 This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-BY) Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited. A full version of this article is available from Crystallography Journals Online. Two modulators have been designed, assembled, magnetically tested and installed at SwissFEL in order to implement laser-based seeding at the soft X-ray beamline Athos. The Paul Scherrer Institute is implementing laser-based seeding in the soft X-ray beamline (Athos) of its free-electron laser, SwissFEL, to enhance the temporal and spectral properties of the delivered photon pulses. This technique requires, among other components, two identical modulators for coupling the electron beam with an external laser with a wavelength range between 260 and 1600 nm. The design, magnetic measurements results, alignment, operation and also details of the novel and exotic magnetic configuration of the prototype are described. soft X-ray FEL EEHG insertion devices H2020 European Research Council810451 This work has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme project HERO grant agreement No. 810451. pmc1. Introduction X-ray free-electron lasers (FELs) are revolutionary research instruments that allow the study of matter on spatial and time scales of atomic processes (McNeil & Thompson, 2010 >; Pellegrini et al., 2016 >; Bostedt et al., 2016 >). Most X-ray FEL facilities are based on the self-amplified spontaneous emission (SASE) process (Kondratenko & Saldin, 1980 >; Bonifacio et al., 1984 >), which starts from the electrons' shot noise and offers limited temporal coherence. A way to improve the longitudinal coherence of the FEL radiation is to start the FEL process with a coherent seed signal. This can be achieved with the self-seeding mechanism (Feldhaus et al., 1997 >), where the seed signal is monochromated SASE radiation produced in a first stage. A better option is to use an external laser for the seed signal. To reach shorter FEL wavelengths approaching the X-ray regime, laser-based advanced schemes employing magnetic chicanes and modulators like high-gain harmonic generation (Yu, 1991 >) and echo-enabled harmonic generation (EEHG) (Stupakov, 2009 >; Rebernik Ribic et al., 2019 >) are required. Laser-based seeding is preferred since it offers higher stability and synchronization to an external source. SwissFEL is the X-ray FEL facility at PSI in Villigen, Switzerland. Its hard X-ray beamline, Aramis, has been under regular user operation since 2019 (Prat et al., 2020 >). The soft X-ray beamline of SwissFEL, Athos, started user operation in 2022 > (Abela et al., 2019 >). Athos has an innovative design with flexible undulator modules (Liang et al., 2021 >) and compact integrated chicanes (CHICs) (Prat et al., 2016 >), offering an unprecedented control over several FEL properties such as the polarization and the peak power. The modulators presented in this article are used in a two-stage research program Hidden Entangled and Resonating Order (HERO) (Aeppli et al., 2020 >) to manipulate the driving electron beam with an external laser signal for a better control of the temporal and spectral characteristics of the Athos FEL pulse. The first stage consists of one optical laser, one modulator and a magnetic chicane. This can be used to produce trains of attosecond pulses locked to the external laser following the ESASE scheme (Zholents, 2005 >; Duris et al., 2021 >) or from energy modulation and taper (Saldin et al., 2006 >). The installation is completed by adding a second, larger chicane and a second identical modulator to enable the operation of EEHG to obtain full control on the coherence properties of the FEL pulse. The design of the modulator supports the exploration of new operation modes beyond ESASE/energy modulation and standard EEHG. Combined with a very short laser pulse, a strong taper in the modulator field gives an ESASE current modulation with gradually growing time separation between the current spikes, supporting superradiant operation with the fresh slice technique (Tanaka et al., 2016 >) down to even single-cycle operation of the soft X-ray beamline Athos (Tanaka, 2015 >). Also, by tuning half the modulator to the primary external laser wavelength and the other half to its second harmonic, the energy modulation can be tailored for a more efficient ESASE, HGHG or EEHG process (Hemsing & Xiang, 2013 >). Finally, the sub-period configuration enhances the performance of self-modulation for a single attosecond pulse (MacArthur et al., 2019 >). 2. Design To satisfy the above requirements, the modulators were designed with great operational flexibility, where the strength of each pole can be tuned individually, making it unique also compared with the work of Tanaka et al. (2021 >) where exclusively the strength of each period can be adjusted independently. The choice of the magnetic field amplitude was based on the period length, lu = 0.2 m, imposed by the mechanical frame of the existing CHICs and the resonance condition of the modulator, where g is the Lorentz factor, K the deflection parameter defined as m and e the mass and the charge of the electron and the equivalent magnetic field amplitude which shall be evaluated using the Fourier series as presented by Calvi et al. (2018 >). This is especially important for this modulator where the field profile, B y (z), is far from sinusoidal and thus the maximum amplitude does not represent well the resonance condition. Given the design electron beam energy interval between 2.75 and 3.25 GeV1, K shall range between 12 and 36 to cover the wavelength scale between 260 and 1600 nm. Each of the two modulators consists of 16 full poles and two half poles, one in each of the two extremes as shown in Fig. 1 >. Its magnetic structure contains only permanent magnets arranged in a modified Halbach array (Halbach, 1983 >), see Fig. 2 >, where an additional pair of magnets horizontally magnetized enhances the on-axis field. On the one hand, the absence of ferromagnetic poles reduces the non-linearity of the system (the field could be in good approximation regarded as the superposition of the individual pole contributions) and thus simplifies the operation, while on the other hand increases the stray fields and the forces between poles (especially along the beam axis) and reduces the maximum K-value. Each magnetic pole can be independently displaced vertically with a servo motor and precisely positioned by means of an absolute angular encoder, which allows for novel magnetic configuration discussed in SS4. This flexibility requires high reliability of the individual components because of the large number of motors and encoders: if one pole does not move to the target position or worse becomes stuck, this can compromise the transport of the beam and the operation of the entire downstream beamline. Advanced control software has been developed as well to avoid the manual operation of the individual degree of freedom which can generate unnecessary beam losses. In the following subsections, the magnetic and mechanical designs of the two modulators are presented with particular care to their technical implementation but without the details of the simulation studies. 2.1. Magnetic design The building block of the magnetic structure is the half pole as presented in the top part of Fig. 2 >. It consists of four NdFeB (remanence, B r = 1.2 T) magnets: one magnetized along the beam axis (1, z-axis), one vertically (2, y-axis) and two horizontally (3, 4, x-axis) with opposite directions. To build the modulator, this unit must be assembled in two distinct configurations, where each magnet of one assembly has the opposite magnetization direction than in the other one. In the bottom part of Fig. 2 > the beginning of the modulator magnetic structure is presented using the two blocks and applying only a rotation around the y-axis. This results in a half pole followed by alternating full poles, for simplicity referred as 1/2, 1, 1 configuration. The choice of the half pole units was driven as well by the requested compatibility with the existing CHICs. They could be assembled in the future using these new components, thus reducing the field integral errors generated by the presence of the iron pole implemented in their present design. The magnetic optimization was performed using the software RADIA (Chubar et al., 1998 >) and their final geometry is shown in the production drawings of Fig. 3 >. It is worth noticing that the maximum field amplitude is not achieved when magnets 1 and 2 have the same length along the z-axis, as is the case in a pure Halbach structure, because of the presence of the side magnets 3 and 4. 2.2. Mechanical design The poles are fixed on the modulator frame by means of mechanical clamps on the 45deg short face of the magnets (Fig. 3 >). To minimize the forces required to keep them in place, the two half pole units are glued together to overtake the repulsive forces and consequently reducing the torque required on the fixing screws. Each period of the modulator is an individual mechanical unit with an independent closed frame equipped with two drive systems to change the distance between the upper and the lower poles (this distance is called the gap) from 11 to 90 mm. At the minimum gap a hard stop system is installed and carefully calibrated to avoid colliding with the vacuum chamber. This unit is presented in Fig. 4 >, where the four motors, the absolute rotary encoders and chain driven gearboxes (ratio 3) are visible. While assembling one of these units, the attractive force (mainly along the z-axis) between the positive and negative poles reaches about 1000 N and requires a reinforced guiding system with respect to the one originally planned for the CHIC (where the forces are lower and repulsive) to avoid a collision between the magnets. These forces vanish when several units are set together because of the periodic nature of the modulator: one pole is attracted both by its left and right neighbour resulting in zero net longitudinal force. Nevertheless, this is not the case at the extremes where this symmetry is broken, neither if one pole is accidentally or purposely (see SS4.3) moved out of the magnetic axis. In both these cases, the system can be safely operated without risks of collision thanks to the double vertical guiding system implemented on each of the four poles. However, there are residual longitudinal displacements up to 0.2 mm when extreme configurations are reached (single pole fully out and the remaining fully in at minimum gap). During the magnetic optimization the closed frame of the individual units can be open from one side (preferentially at open gap, 90 mm) to access the magnets and applying, if required, mechanical shims on top and/or on the side of the magnets. This was used for tuning the vertical orbit when spurious horizontal field components (ideally all zero) were accumulating and giving rise to a net vertical displacement of the electron beam. 3. Magnetic measurements The magnetic field was measured and calibrated with a Senis Hall probe (Popovic et al., 2007 >), measuring the three components of the magnetic field on-axis. The first field integrals (Clarke, 2004 >) were measured with the moving wire to avoid systematic errors due to the unavoidable Hall probe calibration uncertainty (in particular between the positive and negative branches of the V B curves) and by the residual Hall planar effect, especially while measuring the x-component. 3.1. Alignment The magnetic field profile along the beam axis was measured at a fixed gap of 14 mm for different vertical (y) positions around the nominal beam axis height. To align all poles with respect to each other along a straight line, the parameter k n , hereafter called the local K (Pfluger et al., 1999 >), is defined as the magnetic field integral between two nearest zeros, z n (zeros of the magnetic field), as follows, where for k 1 and k 18 the zeros value shall be chosen according to the extremes of the measurement region: z 1 as the starting position and z 19 as the end position. In Fig. 5 > the relative variation, dk n /k, as a function of the vertical position, before and after the alignment corrections are presented for all poles: the vertex of the parabola, y 0, represents the centre of the magnetic field and it is used as the reference for the alignment. 3.2. Calibration The magnetic profile of the HERO modulator consists of a half pole followed by full poles which yields a beam orbit wiggling off-axis at a distance that depends on the electron beam energy and field strength, i.e. the K-value. Having an even number of full poles, this configuration produces an output offset (no systematic net kick) of about the size of the wiggling amplitude, thus changing the input axis and leaking dispersion. For all those previous reasons, we decided to optimize the orbit on the alternative scheme consisting of 1/4, 3/4, 1 pole strength. This approach works as expected only for ideal magnetic poles, well spatially localized, but for real poles (especially those in the ends of the modulator which produce long tails) it shall be modified as following: 1/4+e, 3/4+e, 1. Estimating the value of e is not trivial and it depends on the gap. A practical approach is to feedback its value on the results of the measurements as follows, where De is the correction which requires a suitable choice of e.2 The first step of this optimization consists of measuring the profile for several gaps while all poles are set at the same gap. Secondly, estimating the local K-value, k n , as defined in equation (3). In the second measurement a new set of gaps shall be estimated using the new value of k n (g) following this algorithm: estimate k(g) as the average local K between k 3 and k 15; evaluate the new set of gaps solving the following set of equations, k 1(g) = k/4, k 2(g) = 3k/4, k 3(g) = k,..., k 16(g) = k, k 17(g) = 3k/4, k 18(g) = k/4. This procedure shall be iterated usually twice to obtain a stable result where only offsets (and not kicks) are left. Now, it is possible to refine these results using equation (3) and iterating with the following set of modified equations: k1(g) = (1/4 + e)k, k 2(g) = (3/4 + e)k, k 3(g) = k,..., k 16(g) = k, k 17(g) = (3/4 + e)k, k 18(g) = (1/4 + e)k. Depending on the choice of the parameter e, this procedure could require more or less iterations. In Fig. 6 >(a), the results of this optimization are reported for the gap of the individual poles, g n , as a function of the actual (average) K-value. For compatibility with the literature and the standard naming at the Athos beamline the previous curves are inverted and fitted with an exponential function, and the coefficients stored in the control system database for the operation. 4. Advanced operational modes Using equation (5) it is possible to operate the modulator in its standard mode: it is sufficient to set a K-value and calculate back 18 gaps, g n . This allows an operational range of 8.0 < K < 36.0 corresponding to a wavelength range between 95.4 nm and 1875.0 nm for a 3.0 GeV electron beam. It is nevertheless possible to operate down to 15.9 nm (K = 3) with the penalty of an offset in the electron beam orbit at the end of the modulator, since first g 1 and g 18 and then g 2 and g 17 reach the 90 mm limit. In Fig. 7 > the orbit for several K values is presented, starting from the bottom with the one calculated out of the strongest field amplitude using the formula Residual upstream kicks are left over at very low K which generates offsets below 50 mm which could have been further minimized with more optimization iterations but they are nevertheless cancelled during the operation with beam by the orbit feedback. 4.1. Taper operation To operate the modulator with a linear taper, it is sufficient to set the individual poles at their value as follows, The magnetic measurements were performed for different taper values (a) and indicate that for a strong taper value there might be a small but non-negligible exit angle, see Fig. 8 >. This is generated around the entrance and can be corrected using the upstream correction steerers: we suggest for easy operation to use the orbit feedback which will naturally correct it. In this mode, the operation is limited by the minimum gap of g 1 and the maximum gap of g 18. For an optimum straight orbit, the taper shall be started from the very beginning (K 1) up to the last pole. Reverse taper, if required, can be applied as well in a very similar way. 4.2. Triple period length One additional mode identified as potentially useful for the operation of this modulator is the triple period with which much longer resonance wavelengths are possible, above 4 mm. It can be achieved keeping the poles (1, 4, 7, 10, 13, 16, 17, 18) at the maximum gap (90 mm, off) and the remaining ones closed at the strength required. This mode reduces the number of equivalent resonance poles to three and generates a beam horizontal offset with an unavoidable non-zero dispersion as clearly visible in Fig. 9 >. Both these parameters have to be carefully considered when operating the modulator in this exotic configuration. 4.3. Double K operation: K A K B The most advanced mode available and magnetically tested of this modulator consists of splitting the available poles into two families, with different K-values, K A > K B . To keep a straight orbit, the K-values cannot be set with a sharp transition, but a smooth connection between the two sides needs to be organized as follows, where t is the pole number where the first modulator finishes and t + 1 where the second starts. This mode was initially designed to detune pole by pole the modulator without losing the electron beam and for this purpose it was optimized with K B set at its minimum possible value, 13.0. An example of this configuration is presented in Fig. 10 >, where the magnetically measured orbit is presented for a quasi-continuous transition (from bottom to top, 14 resonant poles, 11, 10, 9 down to zero) between the modulator A and B. Nevertheless, it is possible to set arbitrary values of both K A and K B but this approach was not extensively validated in the magnetic laboratory and will require extra care when commissioning it with beam. Before concluding, it is important to remark that equation (8) is valid for 3 < t < 15; for the extreme cases of t = 3 and t = 15, the value of 3/4K A and 3/4K B , respectively, are not correct because there are only three poles left and the only sequence which gives both zero for the first and second field integral is 1/2, 1, 1/2. But since that specific pole is already a hidden 3/4, to transform it into a half the correct coefficient is 2/3 in front of K A when t = 3 and for K B when t = 15. Here, for the sake of clarity the full matrix (13 x 18) of the coefficients is shown in Fig. 11 > using the nominal value of the coefficients, which already give reasonably straight orbits. To improve the small but non-negligible kick between the two modulators, at position n = t + 2 the coefficient (indicated with a + sign) has to be reduced by about a percentage: the measured value was saved in lookup tables for the operation. 5. Conclusions and outlooks The two HERO modulators have been designed, assembled, magnetically tested and installed in the SwissFEL tunnel (Fig. 12 >). The large degrees of freedom, made possible by the compact and modular design, have been used to implement advanced operational modes, such as the K A K B mode which to our knowledge has never been investigated in previous projects. The second modulator (the one most downstream) has been very recently tested as well with beam at the SwissFEL where it demonstrated a net energy transfer from an external laser tuned at 800 nm. The full setup will be tested with beam in 2023. A preliminary assessment of micro-bunching instability excludes that the residual field of the modulator can spoil the beam for normal SASE operation; it would require a significantly longer beamline to accumulate an energy modulation from the micro-bunching. On the contrary, wavelengths longer than 100 nm should be excited to not produce coherent emission and self-modulation from the current spike of the Athos beam tail, thus a K value lower than 8.6 (at 3.15 GeV, the new reference electron beam energy of Athos) shall be set when the modulator is not in operation. The magnetic assessment of these two prototypes is very satisfactory but the system could be further improved by changing the design of the poles to compensate magnetically the longitudinal forces. This shall reduce from one side the requirement on the vertical guiding system and improve the position accuracy of the poles especially along the beam direction. This upgrade could be done as well to compensate the vertical forces even if they were not at all a concern. If required, stronger fields are achievable by substituting the vertical magnet (the one magnetized along the y-axis) by a CoFe pole. Differently from the CHIC configuration, where the central pole splits to introduce horizontal offsets, in the modulator configuration the non-linearity introduced by the iron pole can be harnessed to produce no net field integral (spurious kick). The authors would like to thank the technical staff of the Insertion Device group, the engineers and technical help from the PSI central workshop for the assembly of these modules. Figure 1 Functional view of the modulator with its nine CHIC modules with 18 independently movable magnetic poles. The black solid line is an example of the measured magnetic field profile, By. Figure 2 Details of the half pole magnet assembly in its xz-plane projection. The two polarities are presented on the left and on the right with which is possible to build the full structure. Figure 3 Detail of the production drawings of the north half-pole with the standard indexing and dimensions in millimetres. At the bottom is the aluminium (Al) support on top of which the four magnets are glued. Figure 4 One magnetic period representing the mechanical unit of the modulator. In yellow are the magnets coated with TiN, on the right are the motors equipped with encoder and driving electronics, on the top is the gearbox made with a rubber chain, with a ratio of 1:3. Figure 5 (Left) Relative variation of the local K, k n , for each pole, as a function of the vertical position before correction. (Right) The same quantity after corrections. On the vertical scale an offset is added between each pole to better distinguish their individual characteristics. Figure 6 On the left in red: g 1 and g 18 (on the right: K 1 and K 18); in blue: g 2 and g 17 (K 2 and K 17); in black: g 3 to g 16 (K 3 to K 16). On the left side, the data are as-measured; on the right side, the data have been rearranged and fitted for the operation. Figure 7 The orbit within the modulator for the operational K range between 8 (top) and 36 (bottom). The orbit is estimated for an electron beam energy of 3.0 GeV. Figure 8 The orbit in the modulator around the maximum K value for different linear taper settings. Figure 9 The orbit within the modulator for several gaps when operated in the triple period length mode. Note that the vertical division scale has been increased to 1 mm because of the larger amplitude of this configuration. Figure 10 The orbit in the modulator for several possible arrangement of the K A K B configuration, starting from the bottom with only K A . In blue is the number of resonant poles at K A and in red that at K B . Figure 11 The full matrix (13 x 18) of the coefficients; for simplicity's sake K A and K B are indicated with A and B, respectively - see text. To improve the small but non-negligible kick between the two modulators, at position n = t + 2 the coefficient (indicated with a + sign) has to be reduced by about a percentage: the measured value was saved in lookup tables for the operation. Figure 12 The second modulator downstream in the SwissFEL tunnel. This unit was already tested with an electron beam of about 3 GeV. 1 From the design and procurement phase of the two modulators, the extraction energy from the main linac was increased from 3.0 GeV to 3.15 GeV, shifting the tuning range from 2.9 to 3.4 GeV. With these new parameters a higher maximum K of 38 should have been specified to cover all possible options. 2 e was evaluated experimentally measuring the double integral IIB for two closed values of e with the following formula: e = (e2 - e1)/(IIB2 - IIB1). 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PMC10000805 | J Synchrotron Radiat J Synchrotron Radiat J. Synchrotron Rad. Journal of Synchrotron Radiation 0909-0495 1600-5775 International Union of Crystallography 36891846 gy5037 10.1107/S1600577522011614 JSYRES S1600577522011614 Research Papers Total scattering measurements at the Australian Synchrotron Powder Diffraction beamline: capabilities and limitations Total scattering measurements at ANSTO D'Angelo Anita M. a* Brand Helen E. A. a Mitchell Valerie D. a Hamilton Jessica L. a Oldfield Daniel b Liu Amelia C. Y. c Gu Qinfen a* a Australian Synchrotron, Australian Nuclear Science and Technology Organisation (ANSTO), 800 Blackburn Road, Clayton, Victoria 3168, Australia b Australian Nuclear Science and Technology Organisation (ANSTO), New Illawarra Road, Lucas Heights, New South Wales 2234, Australia c School of Physics and Astronomy, Monash University, Wellington Road, Clayton, Victoria 3800, Australia Bergamaschi A. Editor Paul Scherrer Institut, Switzerland Correspondence e-mail: [email protected], [email protected] 01 3 2023 13 1 2023 13 1 2023 30 Pt 2 s230200 327339 29 4 2022 02 12 2022 (c) Anita M. D'Angelo et al. 2023 2023 This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-BY) Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited. A full version of this article is available from Crystallography Journals Online. Total scattering data can be obtained on the PD beamline although there are constraints to the capabilities offered including longer acquisition times and necessary dilution of highly absorbing samples. This work details the considerations needed to successfully carry out total scattering studies on this bending-magnet beamline, which has not been specifically designed for total scattering experiments. This study describes the capabilities and limitations of carrying out total scattering experiments on the Powder Diffraction (PD) beamline at the Australian Synchrotron, ANSTO. A maximum instrument momentum transfer of 19 A-1 can be achieved if the data are collected at 21 keV. The results detail how the pair distribution function (PDF) is affected by Q max, absorption and counting time duration at the PD beamline, and refined structural parameters exemplify how the PDF is affected by these parameters. There are considerations when performing total scattering experiments at the PD beamline, including (1) samples need to be stable during data collection, (2) highly absorbing samples with a mR > 1 always require dilution and (3) only correlation length differences >0.35 A may be resolved. A case study comparing the PDF atom-atom correlation lengths with EXAFS-derived radial distances of Ni and Pt nanocrystals is also presented, which shows good agreement between the two techniques. The results here can be used as a guide for researchers considering total scattering experiments at the PD beamline or similarly setup beamlines. pair distribution function powder X-ray diffraction Mythen-II detector total scattering Australian Research CouncilFT180100594 LE0454166 This research used equipment funded by the Australian Research Council (ARC) (grant No. LE0454166). ACYL acknowledges support from the ARC (grant No. FT180100594). The authors are grateful to the group of Professor Shizhang Qiao for the synthesis of the nanoparticle samples. pmc1. Introduction In a typical powder diffraction experiment from a crystalline specimen, where only the Bragg reflections are studied, only information on the average long-range order of atomic structures is available. However, further insight into the functional properties may be obtained through understanding the local structure by analysing both the sharp Bragg and the diffuse scattering from disorder via total scattering analysis. Both Bragg and diffuse scattering are considered in total scattering experiments and the pair distribution function (PDF) is obtained through Fourier transform of the total scattering data. A PDF shows the probability of finding an atom at distance r from a specified atom. The technique has applications for nanoparticles, disordered materials, non-periodic materials such as amorphous glasses, as well as systems where the local structure is different to the average structure. PDF measurements can provide information on a medium-length scale and are complementary to other techniques such as extended X-ray absorption fine structure (EXAFS). EXAFS provides local information up to the first three atomic shells or ~6 A as the signal attenuates dramatically when the distance between the neighbouring and absorbing atom increases. In contrast, a PDF can provide information beyond 1000 A depending on the instrument resolution in reciprocal space. EXAFS is used to target the local structure of a specific element whereas PDF provides local structure information from all atom pairs. However, this means pairs close to each other can be hard to resolve using PDF. Structural details that can be examined include (1) the direct atom pair separation length from peak positions, (2) the degree of disorder of the atom pair from the peak width, (3) static and dynamic disorder, (4) the phases present in mixed-element systems using differential PDFs, (5) details about specific atom-atom correlations via partial PDFs and (6) coordination number from the integrated peak intensity if the data are on an absolute scale (Takeshi & Billinge, 2012 >). High counting statistics are needed as both the Bragg and the diffuse scattering contribute to the PDF. The atomic X-ray form factor decreases with increasing Q, which contributes to a decrease in Bragg peak intensity and non-uniform counting statistics. The S(Q) is obtained by subtracting self-scattering and normalizing the coherent scattering intensity per atom I coh(Q), according to equation (1) where Q is the magnitude of the scattering vector, ci is the atomic concentration and fi is the X-ray form factor for atom type i (Farrow & Billinge, 2009 >; Billinge & Farrow, 2013 >). The reduced structure function F(Q) is determined from S(Q). A Fourier transform is applied to the normalized total scattering data to generate the reduced PDF G(r), where r0 is the number density, g(r) is the PDF, Q is the magnitude of the wavevector transfer, S(Q) is the structure function and r is the distance between two atoms in the sample [equation (2)] (Farrow & Billinge, 2009 >). Note there are many different total scattering correlation functions, of which their definition also varies (Keen, 2001 >), High-energy X-rays are generally used in PDF experiments to increase the momentum transfer and real space resolution. For example, experiments have been carried out using a resolution of 0.126 A for 28-ID-1 at the National Synchrotron Light Source II, and 0.0898 A for the General Materials Diffractometer (GEM) at ISIS Neutron Source of Rutherford Appleton Laboratory (Frandsen et al., 2019 >). A disadvantage of lower real space resolution is that correlation lengths that are similar cannot be resolved. This is because the limits of the G(r) function are determined by the Q-range. As an example, the Zn--Se and Zn--Te bonds in ZnSe0.5Te0.5 cannot be distinguished at Q max = 17 A-1 as the bond length difference is 0.14 A, whereas they are clearly visible at Q max = 40 A-1 (Proffen et al., 2003 >). A high Q-range can typically be achieved in two ways: (1) increasing the beam energy if using a flat-panel 2D detector, or (2) increasing the 2th angle using a point or 1D detector if the maximum energy is limited. PDFs can also be generated using neutron or electron sources. PDFs generated from electrons (ePDF) allow for smaller samples to be analysed and data with higher spatial resolution can be obtained (McBride & Cockayne, 2003 >, 2007 >). Neutron PDF experiments are commonly carried out at spallation sources including ORNL, ISIS, J-PARC and the European Spallation Source. Reactors such as D4 at Institut Laue-Langevin can also generate short-wavelength neutrons (i.e. 0.35 A and covering 1.5 to 140deg 2th) with a hot source that makes them suitable for total scattering studies (Fischer et al., 2002 >). Synchrotron hard X-ray sources are generally preferred over laboratory X-ray sources due to the higher energy and higher flux available. The energy used on dedicated PDF beamlines is typically >60 keV, i.e. 76.6 keV at I15-1 at Diamond Light Source, 86.7 keV at 11-ID-B at the Advanced Photon Source (APS) and up to 117 keV at 28-ID-1 at Brookhaven National Laboratory. However, reasonable values of Q max can be achieved using lower-energy X-rays such as those generated in the laboratory using Mo Ka (17.4 keV) (Rantanen et al., 2019 >; Galliez et al., 2014 >) and Ag Ka (22.1 keV) (Wu et al., 2018 >; Bueken et al., 2017 >) radiation. Dykhne et al. (2011 >) detailed a comparison of PDF data generated from laboratory X-ray sources for pharmaceutical applications. Furthermore, PDF studies have also been carried out on lower-energy beamlines such as DanMAX at MAX IV with a maximum energy of 35 keV. PDF studies at the X-ray Diffraction and Spectroscopy (XDS) beamline at the Brazilian Synchrotron Light Laboratory (LNLS) have been carried out at 20 keV with Q max = 20 A-1 using a Mythen-II detector by collecting between 1deg and 165deg 2th, and the results were compared with PDF studies carried out at the 11-ID-B PDF beamline at the APS (Saleta et al., 2017 >). In their work the authors reported that XDS can obtain high-quality PDF data and is preferential to study r regions >30 A in comparison with the APS. This was attributed to a smaller dampening factor calculated from XDS compared with APS. Despite this, studies carried out at lower energies may also only report qualitative results due to the limited resolution achieved. The Australian Synchrotron is a third-generation light source that operates at 3 GeV and 200 mA during user operations. The PD beamline is optimized to carry out in situ studies in a broad range of fields including energy, condensed matter science, materials engineering and environmental science. It is located on a bending magnet with a magnetic field of 1.3 T and a critical energy of 7.78 keV. After several years of user operations, the PD beamline optics were upgraded and simplified to better meet the needs of the user community. In the original beamline design the double-crystal monochromator (DCM) had both a flat Si(111) and an Si(311) flat/sagittally bent crystal pair. The mirrors were also designed with multiple stripes for optimal reflectivity over the full energy range of the beamline (4-30 keV). The recent upgrade removed the Si(311) crystal pair and redesigned the crystal mounting for added stability as well as upgrading the mirrors to add horizontal focusing. The vertical collimating mirror (VCM) and vertically focusing mirror (VFM) were replaced with a single-stripe Rh-coated flat and toroid mirror that provided additional flux through the ability to horizontally focus the beam to ~0.8 mm x 0.6 mm in the first endstation. The useable range of energies on the beamline is now 8-22 keV as the reflectivity of the Rh mirror coating drops off substantially above 22 keV. The PD beamline consistently receives requests to carry out total scattering experiments for various materials including battery electrodes, piezoelectrics and coordination framework materials. Early in user operations, studies were carried out by Haverkamp & Wallwork (2009 >) to assess the feasibly of PDF measurements at the PD beamline using the Mythen-II detector at 21 keV. In this work the need for a wide Q-range, fluorescence elimination and removal of the detector gaps between the detector modules (as per standard beamline procedure) were highlighted. The Mythen-II can achieve Q max = 40 A-1 if data are measured over 155deg 2th at 40 keV (Bergamaschi et al., 2010 >). However, it is important to note that the Mythen-II detector efficiency at 5-10 keV is 85% and decreases to ~25% at 20 keV for a 300 mm silicon wafer thickness that is used at the PD beamline. The efficiency could be improved by increasing the thickness of the silicon wafer. The Mythen-II has been used in PDF studies at the Materials Science Beamline (Swiss Light Source) with Q max = 23 A-1 (Cerny et al., 2009 >) and Q max = 28 A-1 (Saha et al., 2021 >). These recent beamline upgrades have prompted us to provide an update of the current PDF capabilities at the PD beamline. The aim of this study was to benchmark total scattering measurements carried out at the beamline. It demonstrates what can be offered to our user community through exploration of variations in Q max, absorption and counting time. A case study comparing the PDF atom-atom correlation lengths with EXAFS-derived radial distances is also presented. 2. Experimental 2.1. Materials Ni powder (99.8%), Cu powder (10 mm spheroidal, 99%) and SiO2 nanopowder (10-20 nm, 99.5%) were purchased from Sigma-Aldrich. Ni, NiPt (1:1), dealloyed NiPt (1:1) and Pt metal nanoparticles (20 wt.%) supported on carbon were synthesized via the methods described by Wang et al. (2018 >). 2.2. Powder diffraction Diffraction data were collected at the PD beamline at the Australian Synchrotron, ANSTO, using a Mythen-II detector over 124deg 2th using four detector positions (Bergamaschi et al., 2010 >). Samples were packed into 0.3 mm borosilicate capillaries and rotated during data collection. The Mythen-II at the PD beamline has 16 modules that cover 80deg 2th and each module has an angular range of 4.83deg with a physical separations of 0.17deg. Patterns were obtained at starting angles of 2deg 2th (position 1), 2.5deg 2th (position 2), 51.5deg 2th (position 3) and 52deg 2th (position 4). The in-house-developed program PDViPeR was used to normalize and splice the four patterns and remove the gaps in the data. The wavelength, zero error and instrument contribution to the peak profile were determined using the line position and line shape standard NIST LaB6 660b. The refined wavelength was 0.58907 (1) A for the nanoparticle and Cu sample collections, and 0.58926 (1) A for the Ni powder. The instrument momentum transfer achievable by measuring 124deg 2th was 19 A-1. An empty capillary was used as the background for the Ni and Cu powder. An SiO2-filled capillary was used for the Cu mixed with SiO2 samples. A capillary filled with carbon was used as the background for the metal nanoparticles so a PDF of the metal nanocrystal without a carbon contribution could be extracted. xPDFsuite was used to generate the S(Q), F(Q) and G(r) functions (Yang et al., 2014 >) using Q max = 18 A-1. This software uses an ad hoc approach to correct and normalize the measured data before transformation to the PDF (Juhas et al., 2013 >). The background scale was optimized to ensure that S(Q) oscillates around 1. Q min = 0.5 A-1, r min = 0.01 and r step = 0.01 were used for all data. Q broad = 0.0017 (1) and Q damp = 0.0035 (1) were determined from refining a NIST Si 640d standard in a 0.3 mm capillary between 1 A and 2000 A in TOPAS (version 6; Coelho, 2018a >,b >). Rietveld refinement and modelling of PDF patterns were carried out in TOPAS (version 6), and PDFs for NiPt, dealloyed NiPt and Pt were modelled up to 15 A using a numerical spherical shape function (Usher et al., 2018 >). 2.3. Transmission electron microscopy Transmission electron microscopy (TEM) images for Ni were obtained on a JEOL 2200FS TEM at 200 kV. Images for the NiPt, dealloyed NitPt and Pt nanoparticles were obtained using a double-corrected FEI Titan 3 FEGTEM operated at 300 kV in scanning transmission electron microscopy (STEM) mode with a probe convergence semi-angle of 15 mrad. Bright-field (BF) and high-angle annular dark-field (HAADF) images were collected. Samples were prepared by adding a small quantity of the powder to ethanol and dropping 3 ml of the solution onto a Cu holey carbon Quantifoil grid then allowing to dry. Particle counting was carried out using ImageJ 1.53k via watershed image processing of the HAADF images. The average diameter of the nanoparticles was determined by fitting a Gaussian distribution to a scatter plot of the binned counts plotted as a function of particle size. 2.4. X-ray absorption spectroscopy Samples were measured using X-ray absorption spectroscopy (XAS) at the XAS beamline at the Australian Synchrotron, ANSTO. XAS spectra were recorded at the platinum L nickel K-absorption edges in fluorescence mode at 4 K in a helium atmosphere using a 100-element solid-state HP-Ge detector (Canberra/Mirion, France). The excitation energy was selected using an Si(111) DCM, which was calibrated at the Pt L Ni K-absorption edges using inline metal foils (the first maximum of the first derivative was at 11562.76 eV and 8331.49 eV for Pt and Ni, respectively). The samples were diluted to 1000 p.p.m. in cellulose and pressed into 7 mm pellets which were then loaded into Perspex holders and sealed with Kapton tape. The corresponding reference foils for Pt and Ni were analysed simultaneously with the appropriate samples. X-ray absorption fine structure (XAFS) scans were performed with a count time of 2 s for each energy step in the pre-edge and X-ray absorption near-edge spectroscopy (XANES) region, with 10 eV steps in the pre-edge region of the XAS spectra, and 0.25 eV steps in the XANES region. In the EXAFS region, spectra were collected in steps of 0.035k to a maximum of 20k, with count time increasing linearly from 2 s up to 10 s at the end of the EXAFS range. Data were pre-processed using the in-house program Sakura (Kappen et al., 2015 >) and the Demeter package for normalization and EXAFS fitting (Ravel & Newville, 2005 >). The R-factors of the EXAFS fits obtained and presented in Section 3.5.2 were 0.005 and 0.004 for Ni and Pt, respectively. 3. Results and discussion 3.1. Instrument characterization The effect of the instrumental resolution on the PDF was investigated using NIST Si 640d with a certified lattice parameter of 5.43123 (8) A. Peaks dampen at high G(r) owing to changes in the effective angular resolution (DQ/Q) across Q in reciprocal space. The amplitude of the PDF peaks will dampen less at high r for high reciprocal space resolution instruments such as the PD beamline, which is optimized for high-resolution studies with an energy resolution of DE/E = 7.4 x 10-4 at 15 keV. The Mythen-II detector also provides an intrinsic angular resolution up to 0.004deg with a 50 mm strip pitch (Haverkamp & Wallwork, 2009 >; Gozzo et al., 2010 >). The intrinsic angular resolution is greater for the PD beamline compared with those beamlines that are optimized for PDF studies because of the beamline optics and the use of the Mythen-II instead of a 2D detector, The Gaussian dampening envelope was determined by equation (3), where S(r) is the r-dependent scale factor, sQ is the width of the Gaussian determined by fitting the PDF (Q damp) to 2000 A and r is the distance between two atoms in the sample. Fig. S2 of the supporting information shows the r-dependent scale factor up to 3000 A. The degree of dampening is due to the Q resolution of the instrument and is advantageous when information of the medium-range atomic structure is required. For example, through modelling the crystalline contribution in the 40-60 A region, correlations from nano-crystalline components up to 40 A were extracted from cement data measured on the 11-ID-B beamline at the APS (White et al., 2015 >). Work by Saleta et al. (2017 >) compared PDF data obtained on a Mythen detector at XDS at the LNLS with data obtained on 11-ID-B at the APS and showed that the PDF peaks attenuate quicker in the APS data. The LNLS PDF data were considered higher quality at higher r whereas the APS data were considered higher quality at lower r. The main advantage of the total scattering setup at the PD beamline is the lower dampening compared with typical high-energy PDF beamlines. To investigate the effect of sample volume on the effective angular resolution in both real and reciprocal space, NIST Si 640d data were collected in both a 0.3 mm and a 1 mm capillary. Individual peaks were fitted with a pseudo-Voight and the full width at half-maximum (FWHM) determined. As expected, Fig. S3(a) shows that the FWHM increases as 2th increases and the FWHM is larger for a 1 mm capillary compared with a 0.3 mm capillary. The FWHM of the Si(111) reflection was 0.0072 (1)deg measured in a 0.3 mm capillary and 0.0142 (1)deg in a 1 mm capillary. The effective angular resolution is lower for a larger sample volume as scattering extends over a greater angular range. The broadening contribution from a 0.3 mm capillary can span 0.006deg versus 0.019deg for a 1 mm capillary at a 762 mm sample-to-detector distance (Bergamaschi et al., 2010 >; Gozzo et al., 2010 >). Work by Scarlett et al. (2011 >) has also shown sample displacement artefacts in the diffraction data of minerals nucleating on the walls of 1 mm capillaries as the peaks shift from their ideal positions to higher and lower angles. These authors went on to formalize this observation in a sample displacement correction for this geometry. PDFs were generated from the Si collected in a 0.3 mm and 1 mm capillary to determine the effect that capillary size and effective angular resolution have on the peak FWHM across r. The peak width is convoluted with an instrument broadening term in real space and can be described using equation (4) if the Q-dependent broadening is linear, where s0 is the FWHM of a peak, d is the Gaussian broadening term (Q broad) and s q is the FWHM of a peak convoluted with the broadening term (Thorpe et al., 2002 >). The s q of the first peak at 2.35 A was used to determine s0 and establish the FWHM as a function of r from equation (4). Fig. S3(b) shows that the FWHM increases as r increases and is larger for the 1 mm capillary. At low r there is only a small difference in the peak widths (0.04 A at r = 50 A) between the capillary sizes and this difference increases due to peak broadening effects across Q, In this work, the lattice parameters, atomic displacement parameters (ADPs) and scale factor were calculated in 'box-car' refinements using 10 A windows and moving the centre of the 'box' by 5 A. The dampening and broadening parameters were set to zero as this allowed us to determine how these parameters change across r. Lattice parameters were determined with and without correcting for instrumental effects on the peak profile and zero offset. In both, the lattice parameter was consistent across r [Fig. 1 >(a)]. The lattice parameters obtained by fitting the PDF without corrections were lower than the value of 5.4311 (1) A from the Rietveld refinement, although they were comparable. Fig. S1 shows the fit to the NIST Si 640d standard in reciprocal space. The lattice parameter was 5.4302 (1) A by refining between 1 A and 100 A, and at low (5 A) and high (200 A) r was ~0.02% or 0.001 A lower than the lattice parameter calculated from the Rietveld refinement of the Bragg peaks. This difference between the lattice parameter calculated from the PDF and Bragg peaks can be attributed to a zero offset and peak asymmetry in reciprocal space. The PDF was then generated and corrected for the peak profile described by fundamental parameters and zero offset using TOPAS (version 7; Coelho, 2020 >, 2021 >). The corrected lattice parameter was 5.4307 (1) A by refining between 1 A and 100 A and at low (5 A) and high (200 A) r was <0.01% or <0.001 A lower than the lattice parameter calculated from Rietveld refinement of the Bragg peaks. The Bragg peaks in the data from the PD beamline can have peak asymmetry that attributed to to axial divergence that can be modelled using a circles function via a fundamental parameters approach (Cheary & Coelho, 1992 >). In both real and reciprocal space, the peak position is determined from a delta function convoluted with line shape. The line shape has instrument and sample contributions. If an asymmetric line shape is not considered in the model then the intensity centre position can be different from the 'ideal' peak position obtained from the delta function only (Jeong et al., 2005 >). Larger differences between the lattice parameter calculated from real and reciprocal space data can then be observed, as shown in this work. Asymmetric peak shapes can also cause r-dependent peak shifts, a reduction in the ability to fit peaks at high r (shown by a higher R wp) and changes to dampening behaviour (Olds et al., 2018 >; Jeong et al., 2005 >). The PDF-derived lattice parameter in this work shows good agreement with the Rietveld-derived lattice parameter once the data were corrected for peak profile and zero offset effects. Consequently, PDFs should be corrected for instrumental effects to minimize unwanted effects in the PDF. There was an increase in the ADP across r showing an increase in peak width attributed to r-dependent peak-broadening [Fig. 1 >(b)]. A decrease in scale factor at high r may also indicate dampening of the peaks (Qiu et al., 2004 >) [Fig. S4(a)]. The ADP was 0.567 (2) A2 determined from Rietveld refinement and comparable to the 0.553 (1) A2 from the PDF by refining the data between 1 A and 100 A [Fig. 1 >(b)]. Fig. S4(b) also shows the change in R wp across r. Both dampening and broadening of the peaks due to the instrument can be accounted for using a standard and the values determined applied to samples during analysis. 3.2. Effect of Q-range An energy of 21 keV is typically used for measurements at the PD beamline as flux dops off significantly above 22 keV. Ideally, all samples are measured at the highest available energy to increase the momentum transfer, i.e. to obtain a high Q max and low Q min. The ability to resolve two peaks in a PDF is primarily determined by the Q range. Q min = 0.37 A-1 (d 17 A) can be reached if the data are measured from 2deg 2th at 21 keV. Angles lower than 2deg 2th cannot be accessed if also measuring to the higher angles needed for total scattering experiments, as scattering from our current beam stop is observed at higher angles (typically >60deg 2th). However, for reciprocal space measurements, data from 1deg 2th (d 33 A) are routinely collected for materials with large unit cells by positioning the beam stop to reduce the small angle scattering and not cut the background intensity. The PD beamline is considering modifying the beam stop to reach lower angles while also avoiding backscatter. There is a trade-off between accessing low and high angles in the beamline setup with our current beam stop, so for total scattering experiments Q min = 0.5 A-1 should be used to generate the PDFs. Measuring samples at an energy lower than 21 keV was considered in this work if samples contain elements that will have significant fluorescence when measured at 21 keV. The Mythen-II detector is sensitive to fluorescence, so the contribution of a fluorescence background to the measured data is minimized if the energy selected is below the absorption edge or greater than 6 keV above the absorption edge (Bergamaschi et al., 2010 >). However, using a lower energy will obviously reduce the achievable Q max and decrease the Q max used, and hence cause an increase in peak-broadening, decrease in peak height and increase in the intensity of termination ripples (Qiu et al., 2004 >). The extent to which the loss in real space resolution and variation in the calculated structural parameters increases was investigated for experiments where lower energies are considered. To investigate this, PDFs of an Ni powder sample were generated with Q max = 14 A-1, 15 A-1, 16 A-1, 17 A-1 and 18 A-1. The instrument parameters were also refined for each Q max. Fig. 2 >(a) shows that there is little to no observable difference between PDFs with Q max = 17 A-1 and 18 A-1. There are three peaks between 7.7 A and 8.7 A in both of these PDFs, and the loss in resolution is obvious when Q max <= 16 A-1 as there are only two peaks visible in this region. The loss in resolution is also highlighted by the peak at 3.5 A for Q max = 14 A-1 where the peak and termination ripples cannot be distinguished from each other. Resolution in real space can be estimated by dr = 2p/Q max (Petkov, 2012 >) so the resolution achieved using Q max = 18 A-1 is 0.35 A, and for Q max = 16 A-1 it is 0.39 A. This shows that only differences between correlation lengths that are >0.35 A can be resolved at the PD beamline. Changes in the lattice parameter and ADP were quantitatively evaluated by fitting the PDF data between r = 1 A and r = 30 A. The refined parameters are presented in Table S1. The lattice parameters for Q max = 14-18 A-1 are comparable and the ADPs increased when Q max decreased. Peaks appear visibly broader at 2.5 A and 4.3 A for Q max = 14 A-1 compared with Q max = 18 A-1 [Fig. 2 >(b)]. Larger ADPs are obtained from broader peaks as they indicate atomic motion or static disorder. Correlations in the PDF broaden due to these effects as well as termination of the function at Q max. This is due to convolution of G(r) with a broadening function, sin(Q maxDr)/Dr. A low Q max resulted in the highest discrepancy between the ADPs of an experimental and a calculated PDF (Toby & Egami, 1992 >). Termination broadening can be reduced and is minimal if a high enough Q max is used. Toby & Egami (1992 >) report that the errors in a PDF are minimal when Q max > 30 A-1. Qiu et al. (2004 >) showed from neutron PDF data of Pb that the peak intensity of the next-nearest neighbour at 3.48 A does not increase further above Q max = 37 A-1 as the resolution is no longer measurement-limited. However, it is important to note that this is material-dependent. Consequently, the ADPs determined here are larger at lower Q max due to peak-broadening from termination of the PDF. Although PDFs can be generated with a lower Q max, the structural parameters for some materials may not be able to be determined owing to decreasing accuracy of the calculated parameters. Only qualitative data and relative changes between samples may be useable in this case. Users of the PD beamline will need to consider whether the achievable real space resolution is adequate to resolve the correlations of interest in their materials. 3.3. Absorption and the PDF X-ray absorption effects are not generally an issue on higher-energy beamlines and those specifically designed for total scattering experiments. However, absorption of a sample needs to be considered when carrying out experiments on the PD beamline. For example, at 21 keV, CeO2 in a 0.3 mm capillary has mR = 1.59, compared with mR = 0.52 that can be achieved on other beamlines that reach 60 keV. Data quality is lower for highly absorbing samples in Debye-Scherrer geometry as the Bragg intensity is dependent on the diffraction angle and scattering may not be from the entire sample volume (Von Dreele & Rodriguez-Carvajal, 2008 >). Any absorption contribution needs to be removed from the measured intensity in a total scattering experiment prior to Fourier transform. S(Q) needs a multiplicative correction applied to remove absorption effects that dampen the intensity at high Q (Peterson et al., 2003 >). Using the ad hoc method of data correction in this work relies on the input of data without absorption effects (Juhas et al., 2013 >). Although minor absorption can be corrected using standard data analysis programs, the significant peak intensity reduction observed in a highly absorbing sample often means meaningful results cannot be obtained. Absorption can be minimized by employing a smaller internal diameter capillary or diluting the sample with a known material to reduce the quantity of highly absorbing material. An alternative method used to reduce mR of highly absorbing samples by the XRD1 beamline (5.5-14 keV) at Laboratorio Nacional de Luz Sincrotron involved fixing a thin layer of the material onto the outside of a capillary rather than filling the capillary (Carvalho et al., 2017 >). Generally, diluting with an amorphous material such as amorphous SiO2 to obtain mR < 1 is recommended as sample preparation is more straightforward and mR can be decreased further than is achievable using a smaller internal diameter capillary. However, the addition of amorphous diluents will contribute to the PDF so the measured background should account for this by being an equivalent capillary filled with dilutant. The effect of absorption on the resultant PDF pattern was investigated using powdered Cu (undiluted) with mR 2.27 (packing density = 5.28 g cm-3) and Cu (diluted), which was mixed with amorphous SiO2 to reduce the absorption so that mR 0.9 (Cu + SiO2). A capillary filled with amorphous SiO2 was removed as the background. A Rietveld refinement was carried out on the diluted and undiluted Cu using a Pearson VII to describe the peak shape. The structural parameters are presented in Table S2. The refined ADP for the undiluted sample (0.296 A2) was lower than that determined for the diluted Cu (0.517 A2) when the ADPs were refined. However, when the ADP obtained for the diluted sample was used for the undiluted Cu and fixed [Fig. 3 >(b)], the structural model of the undiluted Cu predicted the high-th reflections were lower intensity. The low-th reflections in the undiluted Cu are lower in intensity due to absorption, so the model fits these while underestimating the intensity of the high-angle peaks. Relative absorption decreases as th increases as the high-angle reflections can diffract from the surface and reduce absorption (Cullity, 1978 >). These results show that absorption is greater for low-angle reflections as they have a longer pathlength through the sample. Figs. 3 >(c) and 3 >(d) show there are additional artefacts in S(Q) and F(Q) due to poor background subtraction when the absorption contribution is not removed. Peterson et al. (2003 >) showed that a sine wave can be introduced into the S(Q) from a poorly corrected background. The artefacts manifest as a curve in the baseline of these functions and propagate into the PDF. The increased noise is also higher in the undiluted sample as shown in Fig. 3 >(d). This is likely from lower counting statistics as more photons are absorbed by the sample so fewer are reaching the detector. The scattering from the empty capillary shows the characteristic broad amorphous peak at 7-9deg 2th [Fig. 3 >(a)] like the scattering of the diluted Cu. The undiluted Cu pattern does not exhibit this broad capillary scattering peak and the model does not appear to fit the data well at low r. This indicates an increase in the error of the low-r peak intensities. Figs. S5(a) and S5(b) show that the fit to the undiluted Cu PDF is poorer compared with the Cu diluted with SiO2 as demonstrated by difference plot and higher R wp. The difference in fit quality also has a clear effect on the calculated ADP as shown in Table S3. Users of the PD beamline will need to dilute highly absorbing samples to minimize absorption and improve the quality of their PDF. 3.4. Counting time duration Data for the Ni particles were collected for 10-900 s to understand the effect of counting time on acceptable counting duration for stable samples. Note that counting time is highly sample-dependent but is generally longer than is required for reciprocal space measurements. It is well known that a longer measurement time will lead to better counting statistics, so the optimum counting time needs to be balanced with an acceptable counting time duration. This is also important at large-scale facilities where the total beam time is limited. F(Q) shows that the noise at higher Q decreases as counting time increases from a reduction in statistical error [Fig. 4 >(a)], as expected. The resultant PDFs in Fig. 4 >(b) show that the intensity of the ripples relative to the peak intensity decreased for the PDFs obtained with the higher counting time of 900 s compared with 10 s. Most noticeable is the lower-quality PDF obtained when collecting for only 10 s. The peaks at higher r cannot be differentiated from the ripples in the background and these prominent ripples contribute to the correlation peak shape. The first correlation at 2.5 A is asymmetric due to the contribution of large ripples to the peak profile. Interpreting a PDF peak profile with poor counting statistics is problematic as these peaks are convoluted with a sine function that cannot always be modelled well and this will affect the refined parameter values. Consequently, a low counting time affects the quality of the PDF and the calculated structural data. The lattice parameters determined from modelling the PDFs were comparable for all counting times (Table S4). However, the PDF collected for 900 s has the lowest R wp. These results also exemplify that counting times are sample dependent. R wp was lower for Ni (Table S1) powder than for the Ni particles when all were collected for 240 s. The total number of Ni atoms per sample volume interacting with the beam is lower for the Ni particles (20 wt%) due to the presence of carbon. Hence counting times for supported nanoparticle samples can be longer if the scattering from the particles is weak from dilution by the support, the particles are a few nanometres in diameter and they are dispersed. Longer counting times that ensure good counting statistics above 90deg 2th may also result in backscatter contributing to the peak intensities in reciprocal space. It is important to ensure that the collected data are free from backscattering as this contribution can undesirably contribute to the PDF, hence contaminating the data. A compromise is needed between the optimum acquisition time and data quality, so it is suggested that an acquisition time of 600 s is acceptable for similar samples. 3.5. Nanoparticle case study Bimetallic Pt-based nanoparticles have applications as electrocatalysts for formic acid and ethanol electro-oxidation and oxygen reduction. Nanoparticles such as NiPt are reported to have superior performance compared with monometallic nanoparticles due to synergistic effects between their elements as well as typically exhibiting a reduction in surface poisoning (Zhang et al., 2018 >). The PDFs of a series of metal particles, Ni, NiPt, dealloyed NiPt and Pt were obtained to establish the data quality for nanomaterials at the PD beamline. The PDF crystallite sizes were compared with those obtained from TEM and the first three correlation lengths were compared with those calculated using XAS. 3.5.1. Crystallite size distribution and morphology The crystallite size distribution and morphology were obtained for the NiPt, dealloyed NiPt and Pt nanoparticles using STEM. The HAADF signal in STEM mode is proportional to the atomic number so measuring the crystallite size is facilitated using STEM. Both NiPt and dealloyed NiPt possess round and facetted single crystallites as shown in Figs. 5 >(b) and 5 >(c), whereas the morphology of Pt appears more variable [Fig. 5 >(d)]. Images of Ni were obtained using BF-TEM and show that the particles are significantly larger than the NiPt, dealloyed NiPt and Pt nanoparticles [Fig. 5 >(a)]. These differences in crystallite size are reflected in the reciprocal and real space diffraction data. Fig. 8 shows that the Ni powder diffraction pattern has sharp Bragg peaks while the nanoparticles have broad peaks. The NiPt and dealloyed NiPt nanoparticles were also slightly larger than Pt as shown in Table 1 >. The particle diameter histograms are presented in Fig. S6, and additional STEM images are presented in Fig. S7. 3.5.2. Local environment from XANES and EXAFS A comparison with the structural parameters determined from PDF was established for the supported particles. The EXAFS data from Ni and Pt were fit using the predicted scattering of the neat face-centred cubic structures. The plotted results of the fit are given in Fig. 6 >, which show good agreement out to 5 A and the third shell of atomic distances. The determined radial distances between the absorbing atom and the indicated shell are given in Table 2 >. EXAFS fit parameters of the Pt and Ni nanoparticle analysis are presented in Table S5. A comparison between the EXAFS radial distances and the PDF correlation length is discussed in Section 3.5.3. The Ni phase and Pt phase can be probed by examining their respective edges. The PtNi nanoparticles were analysed at both edges and compared with the Ni and Pt nanoparticles. The spectra in Fourier transform (R) space are presented in Fig. 7 >(a) and the radial distance functions are presented in Fig. S8. It can be qualitatively observed that the Pt phase in the NiPt and dealloyed NiPt nanoparticles exists predominately as the neat metal, closely resembling the spectra for Pt. Both NiPt and dealloyed NiPt can be fit reasonably well with only scattering from Pt-Pt and Pt-O in the first shell without significant contribution from Ni. This would suggest that the nanoparticles contain a relatively pure Pt phase. This is further corroborated with a XANES analysis, in which the absorption edge is identical between the Pt nanoparticles and the NiPt and dealloyed NiPt [Fig. 7 >(b)]. In contrast, the Ni phase of the NiPt and dealloyed NiPt differ significantly from the pure nanoparticles and cannot be fit with only Ni-Ni and Ni-O scattering paths. The XANES analysis likewise shows a significant decrease in the pre-edge feature present in the Ni nanoparticles, behaviour which is associated with NiPt alloy formation [Fig. 7 >(c)]. This suggests that the Ni phase is not pure and has Pt-Ni alloy characteristics (Chen et al., 2016 >). 3.5.3. Correlation lengths from PDFs Nanometre-sized crystallites have broad peaks as the diffracted intensity is spread further from the reciprocal lattice points. To minimize line broadening, crystallites are recommended to be 1-5 mm to maintain a good power average (McCusker et al., 1999 >). Fig. 8 >(a) shows that only the Ni possesses relatively narrow Bragg peaks whereas the NiPt, dealloyed NiPt and Pt have broader peaks due to the size of the crystallites. The PDFs of Ni and NiPt, dealloyed NiPt, and Pt nanoparticles are shown in Fig. 8 >(b). The larger crystallite size and long-range atomic order of Ni are reflected in the PDF as correlations do not attenuate like that observed for NiPt, dealloyed NiPt and Pt. The correlations for the nanoparticles dampen by ~15 A due to their finite crystallite size. The structures of all the nanoparticles from the PDFs were refined using the space group Fm 3 m (No. 225) and the occupancy in the 4a sites were split in the NiPt and dealloyed NiPt models, i.e. the occupancy for Ni was 0.5 and Pt was 0.5. The ADP of each Ni and Pt site in the NiPt and dealloyed NiPt model were constrained. All nanoparticles were corrected for finite crystallite size using a numerical spherical shape function (Usher et al., 2018 >). Fits to the PDFs are shown in Fig. S9 and structural details in Table S6. Microstrain, structural defects and effects from the surface atom geometry which are often observed in nanomaterials may be present in these samples (Bertolotti et al., 2018 >). There was no improvement to the fit when tetrahedron-shaped functions were tested and the TEM data show that the crystallites for NiPt and dealloyed NiPt were round and facetted. The size of the Pt nanoparticle sample was 1.9 nm, which corresponds well with the average particle size of 2.0 nm from TEM (Table 3 >). The values obtained from the PDF for PtNi (2.2 nm) and dealloyed PtNi (2.4 nm) are lower than the 3.1 nm obtained for both samples using TEM. A limitation of TEM in the particle-size distribution is that the number of particles sampled is orders of magnitude lower than the number that are sampled using X-ray diffraction. Particle sizes are obtained by measuring the diameter from a 1D image so the length along other axes is absent and may be a different length if the particle shape is irregular. However, accurate particle sizes from modelling diffraction data are also compromised if the fit quality is poor. A correct model and reasonable starting parameters are needed for least-squares minimization refinements from existing knowledge of the sample. The difference between the particle sizes from the PDF and TEM observed in this work are proposed to be caused by the difference between how the particle size is determined using both techniques. The first three correlation lengths were determined from the refined Ni and Pt structural models. For Ni these were 2.48 A, 3.54 A and 4.31 A, and for Pt they were 2.76 A, 3.91 A and 4.78 A. Both the Ni and Pt correlation lengths agree well with the values obtained using EXAFS. Correlations for the NiPt and dealloyed NiPt nanoparticles are at larger r than the corresponding peaks for Ni, and smaller r than the corresponding peaks for Pt. This is due to the combination of the two elements with different atomic sizes in the samples. The correlations at 3.83 A and 3.84 A for the NiPt and dealloyed NiPt are at positions closer to those of the Pt (3.91 A) rather than Ni (3.54 A). This implies that the structure is closer to Pt, rather than Ni, and both NiPt and dealloyed NiPt are not a 50:50 random alloy. This conclusion is corroborated by EXAFS. We conclude that accurate correlation lengths can be determined from the total scattering data measured on the PD beamline. 4. Considerations Total scattering data can be obtained on the PD beamline, although there are constraints to the capabilities offered. These include: (i) Measurements require stable samples and longer acquisition times. Collection times on the PD beamline can be longer than at dedicated higher flux X-ray PDF beamlines. Data should be collected over 124deg 2th using the Mythen-II positioned at four different starting angles. It takes ~2 min to physically move from 2.5deg 2th (position 2) to 51.5deg 2th (position 3, see Section 2.1) as the diffractometer moves the Mythen-II at 0.4deg per second. This excludes the time it takes to measure the data. In the nanoparticle samples reported here, the total measurement time is ~18 min (240 s per position for four positions plus 124 s to move the detector). In comparison, I15-1 at Diamond Light Source has in situ battery sample environments than can collect PDF data in 2 min (Diaz-Lopez et al., 2020 >). This makes non-ambient/in situ PDF measurements at the PD beamline more challenging as the conditions must remain stable throughout the whole data collection. (ii) Dilution is essential for highly absorbing samples. Artefacts will be observed in PDFs of highly absorbing samples that are not diluted. This work shows that there are significant effects on the resultant PDF for highly absorbing samples, with the most noticeable effect on the calculated ADPs. Absorption causes a reduction in low-angle Bragg peak intensities and inaccuracies in background subtraction. It is essential that highly absorbing samples are always diluted so that mR < 1. Users at the PD beamline should calculate mR of their samples at 21 keV if they are planning a total scattering experiment and dilute if necessary. (iii) Correlation differences >0.35 A can be resolved. Correlation lengths that differ by >0.35 A can be resolved due to the maximum momentum transfer of the PD beamline. Users need to consider whether the real space resolution offered by the PD beamline at 21 keV and an angular range of 124deg 2th is appropriate for their experiment. 5. Summary In this work we explored the capabilities of carrying out total scattering experiments on the PD beamline, including investigations into the maximum resolution (Q max) absorption effects and counting time. The maximum momentum transfer is 19 A-1 if the data are collected at 21 keV. Systems typically challenging to analyse using the Bragg peaks can be measured on the PD beamline via total scattering experiments. However, the PDF capability of the beamline can be further developed. Future upgrades planned for the PD beamline include a Mythen-III detector that will reduce data collection time and improve data quality. It includes an increase in angular range (150deg 2th) that will achieve a higher momentum transfer of 21 A-1 if the data are collected at 22 keV for PDF studies. The Mythen-II requires two patterns to be collected, which are spliced together and normalized to remove gaps in the data from physical gaps between the modules. The measurement time will be halved using the Mythen-III as data without gaps between the modules will be obtained in a single pattern. These results detail the total scattering capability offered and should be used as a guide for those considering and designing total scattering experiments at the PD beamline. Supplementary Material Figures S1 to S9 and Tables S1 to S6. DOI: 10.1107/S1600577522011614/gy5037sup1.pdf The authors acknowledge the use of the instruments and scientific and technical assistance at the Monash Centre for Electron Microscopy, a node of Microscopy Australia. We also thank the Australian Synchrotron scientific advisory panel for their ongoing contribution to the development of the Powder Diffraction beamline. Figure 1 Data obtained from NIST Si 640d show (a) the Bragg peak-derived lattice parameters are comparable and the PDF-derived value does not significantly change across r, where the circles are the lattice parameters corrected for peak shape and zero offset, and the triangles are the lattice parameter without correcting for peak shape and zero offset; and (b) the ADP increases across r due to peak broadening. The blue dashed lines indicate the lattice parameter and ADP obtained from Rietveld refinement. Figure 2 Data for an Ni sample show the (a) difference in resolution for Q max = 14 A-1 to 18 A-1, and (b) loss of resolution observed for Q max = 14 A-1 compared with Q max = 18 A-1. In (a) the dotted plot is the measured data, and the line (black) shows the calculated model. Figure 3 Error is introduced into the PDF if highly absorbing samples are not diluted. (a) Comparison in baseline between the empty borosilicate capillary, SiO2, diluted (Cu + SiO2) and undiluted Cu; (b) the diffracted intensities for undiluted Cu at high angles are stronger than what the structure model for Cu would predict; (c) S(Q); and (d) F(Q) showing the difference in background subtraction of Cu. Figure 4 Data for the Ni particles showing (a) noise in F(Q) at higher Q decreases as the counting time increases, and (b) how the increase in statistical error manifests in G(r). For (b), the dotted plot is the measured data and the line (black) shows the refined model. Figure 5 (a) Ni consists of large crystallites as shown in the BF-TEM compared with (b) NiPt, (c) dealloyed NiPt and (d) Pt nanoparticles from the STEM-HAADF images. Figure 6 Radial distances determined using EXAFS for (a) Ni and (b) Pt, and the corresponding k-plots for (c) Ni and (d) Pt show good agreement with the PDF-determined correlation lengths. The dotted (blue) spectra are the measured data and the line (red) spectra is the fitted model. Figure 7 (a) EXAFS Pt-edge comparison of the NiPt and dealloyed NiPt nanoparticles, and XANES plots of (b) Pt and (c) Ni. Figure 8 (a) Diffraction patterns and (b) PDFs of the Ni, NiPt, dealloyed NiPt and Pt nanoparticles. Table 1 Crystallite size of the nanoparticles determined from STEM Sample Size (nm) Number of particles NiPt 3.1 (1) 532 Dealloyed NiPt 3.1 (1) 310 Pt 2.0 (1) 452 Table 2 EXAFS radial distances of Ni and Pt Here R is the radial distance and s2 is the Debye-Waller factor. Ni-Ni Pt-Pt R (A) s2 (A2) R (A) s2 (A2) First shell 2.48 +- 0.01 0.002 2.77 +- 0.01 0.003 Second shell 3.51 +- 0.01 0.003 3.92 +- 0.01 0.004 Third shell 4.32 +- 0.01 0.004 4.80 +- 0.01 0.007 Table 3 Crystallite size of the nanoparticles determined from PDF PtNi Dealloyed PtNi Pt Sphere length (nm) 2.2 (1) 2.4 (1) 1.9 (1) References Bergamaschi, A., Cervellino, A., Dinapoli, R., Gozzo, F., Henrich, B., Johnson, I., Kraft, P., Mozzanica, A., Schmitt, B. & Shi, X. (2010). J. Synchrotron Rad. 17, 653-668. Bertolotti, F., Moscheni, D., Guagliardi, A. & Masciocchi, N. (2018). Eur. J. Inorg. Chem. 2018, 3789-3803. Billinge, S. J. L. & Farrow, C. L. (2013). J. Phys. Condens. Matter, 25, 454202. Bueken, B., Van Velthoven, N., Willhammar, T., Stassin, T., Stassen, I., Keen, D. A., Baron, G. V., Denayer, J. F. M., Ameloot, R., Bals, S., De Vos, D. & Bennett, T. D. (2017). Chem. Sci. 8, 3939-3948. Carvalho, A. M. G., Nunes, R. S. & Coelho, A. A. (2017). Powder Diffr. 32, 10-14. 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PMC10000806 | J Synchrotron Radiat J Synchrotron Radiat J. Synchrotron Rad. Journal of Synchrotron Radiation 0909-0495 1600-5775 International Union of Crystallography 36891862 ay5611 10.1107/S1600577523000395 JSYRES S1600577523000395 Beamlines The soft X-ray and XUV split-and-delay unit at beamlines FL23/24 at FLASH2 Split-and-delay unit at FLASH2 Dreimann Matthias a* Wahlert Frank b Eckermann Dennis a Rosenthal Felix a Roling Sebastian b Reiker Tobias a Kuhlmann Marion c Toleikis Sven c Brachmanski Maciej c Treusch Rolf c Plonjes Elke c Siemer Bjorn b Zacharias Helmut a a Center for Soft Nanoscience, Westfalische Wilhelms-Universitat Munster, Busso-Peus-Str. 10, 48149 Munster, Germany b Physikalisches Institut, Westfalische Wilhelms-Universitat Munster, Wilhelm-Klemm-Str. 10, 48149 Munster, Germany c Deutsches Elektronen-Synchrotron DESY, Notkestr. 85, 22607 Hamburg, Germany Amemiya Y. Editor University of Tokyo, Japan Correspondence e-mail: [email protected] 01 3 2023 15 2 2023 15 2 2023 30 Pt 2 s230200 479489 21 11 2022 16 1 2023 (c) Matthias Dreimann et al. 2023 2023 This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-BY) Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited. A full version of this article is available from Crystallography Journals Online. The properties of the recently installed split-and-delay unit at beamlines FL23 and FL24 at FLASH2 are presented. Its operational range, performance parameters and results of a first experiment are described. A split-and-delay unit for the extreme ultraviolet and soft X-ray spectral regions has been built which enables time-resolved experiments at beamlines FL23 and FL24 at the Free-electron LASer in Hamburg (FLASH). Geometric wavefront splitting at a sharp edge of a beam splitting mirror is applied to split the incoming soft X-ray pulse into two beams. Ni and Pt coatings at grazing incidence angles have been chosen in order to cover the whole spectral range of FLASH2 and beyond, up to hn = 1800 eV. In the variable beam path with a grazing incidence angle of thd = 1.8deg, the total transmission (T) ranges are of the order of 0.48 < T < 0.84 for hn < 100 eV and T > 0.50 for 100 eV < hn < 650 eV with the Ni coating, and T > 0.06 for hn < 1800 eV for the Pt coating. For a fixed beam path with a grazing incidence angle of thf = 1.3deg, a transmission of T > 0.61 with the Ni coating and T > 0.23 with a Pt coating is achieved. Soft X-ray pump/soft X-ray probe experiments are possible within a delay range of -5 ps < Dt < +18 ps with a nominal time resolution of t r = 66 as and a measured timing jitter of t j = 121 +- 2 as. First experiments with the split-and-delay unit determined the averaged coherence time of FLASH2 to be tc = 1.75 fs at l = 8 nm, measured at a purposely reduced coherence of the free-electron laser. time-resolved pump-probe XUV soft X-rays free-electron laser Bundesministerium fur Bildung und Forschung05K13PM2 05K16PM1 05K19PM1 Westfalische Wilhelms-Universitat MunsterWestfalische Wilhelms-Universitat MunsterWestfalische Wilhelms-Universitat MunsterThe SDU at FL23 and FL24 at FLASH2 has been funded by the Bundesministerium fur Bildung und Forschung (grant No. 05K13PM2 to Westfalische Wilhelms-Universitat Munster; grant No. 05K16PM1 to Westfalische Wilhelms-Universitat Munster; grant No. 05K19PM1 to Westfalische Wilhelms-Universitat Munster). pmc1. Introduction Time-resolved pump/probe experiments in the extreme ultraviolet (XUV) and X-ray spectral regions performed with free-electron lasers (FELs) contribute to a fundamental understanding of the femtosecond dynamics of high-energetic processes of a large variety of physical systems. The requirements to perform such experiments are demanding and the key parameter to access the dynamics under investigation is the time evolution of the system. In the past many experiments have been carried out to examine the dynamics in atoms (Radcliffe et al., 2012 >; Ding et al., 2019 >), molecules (Krikunova et al., 2012a >; Schnorr et al., 2014 >; Liekhus-Schmaltz et al., 2015 >; Lehmann et al., 2016 >; Hollstein et al., 2019 >; Rebholz et al., 2021 >), clusters (Krikunova et al., 2012b >; Ferguson et al., 2016 >; Oelze et al., 2017 >; Asmussen et al., 2022 >), hot dense matter (Zastrau et al., 2014 >) and condensed matter systems (Inoue et al., 2016 >; Pardini et al., 2018 >; Roseker et al., 2018 >; Vinko et al., 2020 >; Liu et al., 2021 >). Results on time-resolved imaging (Gunther et al., 2011 >; Rath et al., 2014 >), spectro-temporal characterization of XUV pulses (Ding et al., 2021 >) and stimulated X-ray emission (Kroll et al., 2020 >) have also been reported. In recent years the technique of two-color pump/probe experiments was utilized to reveal the non-equilibrium dynamics in diamond (Inoue et al., 2016 >) and magnetite (Pontius et al., 2018 >). The investigation of the temporal dynamic of systems is key to the fundamental understanding of its underlying physics. Special set-ups offer the possibility to provide two XUV beams directly within the FEL with a limited temporal delay range up to 1 ps (Allaria et al., 2013 >; Hara et al., 2013 >; Marinelli et al., 2015 >). A different approach is the use of a split-and-delay unit (SDU). These devices provide a large temporal delay range combined with a high temporal stability (Sorgenfrei et al., 2010 >; Wostmann et al., 2013 >; Osaka et al., 2016 >; Lu et al., 2018 >). Until 2020 at FLASH2 only one SDU was permanently installed, integrated within the reaction microscope endstation (REMI) at beamline FL26 which specializes in atomic, molecular and optical physics and is provided by the Max Planck Institute for Nuclear Physics (Schmid et al., 2019 >; Meister et al., 2020 >). It offers time-resolved experiments with a temporal delay of +-2500 fs and delivers a high total transmission of T > 0.7 for photon energies below hn < 150 eV. Currently FLASH2 offers FEL beams with photon energies in the fundamental up to hn 300 eV, and in the future up to hn 540 eV as planned in the FLASH2020+ project (Ronsch-Schulenburg et al., 2019 >). Taking the third harmonic of the radiation into account, X-ray pump/X-ray probe experiments even at a photon energy of hn = 1620 eV can then be performed. With the commissioning of the Free-Electron LASer in Hamburg 2 (FLASH2) the use of variable-gap undulators was introduced (Faatz et al., 2016 >), enabling new experimental possibilities for users, i.e. the flexibility to change the photon energy of the FEL beam within a few seconds (Faatz et al., 2017 >). Further, operating the undulators in two groups allows two almost independently choosable, distinct photon energies to be generated (Schneidmiller et al., 2019 >). Future sub-femtosecond pulses (Beye et al., 2020 >) are of particular importance to elucidate the electronic dynamics associated with inner-shell excitations. To fully explore the new possibilities requires an instrument which allows the aforementioned features of FLASH2 to be combined with the possibility to perform time-resolved experiments. To enable such experiments with high flexibility, an SDU has been built and permanently implemented at FLASH2, as described in this paper. It is located in the Kai Siegbahn hall, with the nominal distance of its first mirror to the source being 61 m. It serves beamline FL24 with focused and unfocused open ports as well as beamline FL23 to perform time-resolved pump/probe experiments in the XUV and soft X-ray regions. A major feature of the presented SDU is that, in principle, it does not affect the FEL beam except in the introduced delay. It further allows the use of the SDU in combination with other instruments of the beamline, e.g. optical pump beam, and the users' own analysing devices at the open port, e.g. spectrometers. 2. Concept and design of the SDU 2.1. Optical set-up The SDU was designed with the demand to cover the whole envisioned spectral region of FLASH2 with a reasonable transmission. As SDUs based on amplitude splitting suffer from high absorption within the utilized materials in the XUV and soft X-ray regime (Haga et al., 1998 >; Hilbert et al., 2013 >), the SDU presented here relies on geometric wavefront splitting at a sharp edge of a beam splitting mirror at grazing incidence angles. The conceptual design is shown in Fig. 1 >. The first mirror (BS), as the beam splitter, separates the top part of the beam (green) from the lower part (red) by reflecting it downwards [see upper-left inset in Fig. 1 >(a)]. The lower part of the FEL beam propagates unaffected below BS into the fixed beam path. It is reflected by the mirrors Ni1-Ni4 at a grazing incidence angle of thf = 1.3deg in the horizontal plane, as shown in Fig. 1 >. The mirrors are arranged in a symmetrical order, thus after passing the SDU the beam leaves the SDU collinear to its original direction. The beam is directed onto the experiment passing close to the cutting edge of the recombining mirror (RC). The nominal distance between Ni1 and Ni2 (and between Ni3 and Ni4) is l f = 2075 mm. The nominal distance between Ni2 and Ni3 is 554 mm. The downward reflected beam is directed into the variable delay path which has been designed at a grazing incidence angle of thv = 1.8deg. As shown in Fig. 1 >(b), this beam is reflected at the mirrors DL1, DL2 and RC. As indicated by the orange arrows, the positions of DL1 and DL2 can be adjusted to set the distance between BS/DL1 and DL2/RC in the range l v = 700-2450 mm. The beam is reflected on the lower edge of RC toward the experiment. RC acts as the counterpart of BS recombining both beams except for a narrow gap between them - see right-hand inset in Fig. 1 >(a). As the beam is split horizontally, both partial beams will be separated vertically in the far field. This enables the SDU to be combined with other techniques, e.g. spectrometry, analyzing both beams simultaneously after the interaction. On a spectrometer detector the beams are separated in the vertical plane while the respective signals can be resolved in the horizontal plane. The SDU is moved into the beam for operation. Via the vertical positioning of the optical bench the splitting ratio of both partial beams is set. The distance between the cutting edge of BS and the cutting edge of RC amounts to d total = 5400 mm. This long distance is a consequence of the requested delay range of up to +18 ps for a grazing angle of thv = 1.8deg. As the mirrors DL1 and DL2 can be moved along the beam path, a change of the distance l v leads to a change of the optical path length in the variable beam path. The difference in time delay between both beams Dt can be calculated as where c is the speed of light, l v the distance between BS and DL1 at a glancing angle of thv, and l f the distance between Ni1 and Ni2 at a glancing angle thf. With the aforementioned values a nominal absolute time delay of Dt v = 9.22-32.25 ps in the variable delay path and Dt f = 14.25 ps in the fixed delay path with respect to the direct beam is added. This results in a delay range of -5 ps < Dt < +18 ps for the present SDU. For Dt < 0 ps, the ray from the variable beam path hits the experiment first, pumping the sample under investigation, and accordingly the ray from the fixed beam path acts as the probe. For Dt > 0 ps the beam from the fixed beam path is used to pump the sample and the beam from the variable beam path probes the sample under investigation. For an increase of the total transmission at high photon energies (hn > 800 eV) the beam paths can be switched to Pt-coated mirrors by moving the whole optical bench with all optical components horizontally and under vacuum - see Fig. 1 >(c). In this set-up the mirrors Pt1-Pt4 (light blue) form the fixed delay path (blue), again under thf = 1.3deg glancing angle. For the variable delay paths the Ni and Pt coatings are evaporated side by side on the same set of mirrors (BS, DL1, DL2, RC). Therefore, the variable delay path with Pt-coated section of the delay mirrors (magenta) lies parallel to the delay path with the Ni-coated section (green). This results in a total of 12 mirrors providing four beam paths with four reflections each. Fig. 2 > shows the calculated transmissions in the individual beam paths based on CXRO (Henke et al., 1993 >) for a horizontal linear polarization of the FEL beam. The variable beam path with Ni coatings shows a total transmission of T Ni,v > 0.50 up to photon energies of hn = 650 eV. This covers the future maximum photon energy of hn = 540 eV reachable by FLASH2 in the fundamental (Ronsch-Schulenburg et al., 2019 >). Due to its shallower grazing incidence angle of thf = 1.3deg, the total transmission of the fixed beam path of T Ni,f > 0.60 is noticeably higher in this spectral region. Furthermore, the total transmission up to hn = 800 eV for the respective beam paths amounts still to T Ni,f > 0.45 and T Ni,v > 0.30. Approaching the Ni L-edge at hn = 852.7 eV, the total transmission decreases noticeably. To complement the performance of the SDU at high photon energies, a second Pt-coated beam path is implemented. The Pt-coated mirrors cover the energy region 800 eV < hn < 1800 eV, providing a transmission of T Pt,v > 0.13 and T Pt,f > 0.29 for photon energies of hn < 1500 eV. For higher photon energies, at least T Pt,v > 0.06 in the variable beam path is achieved which is sufficient to provide a probe beam in most cases. The total transmission in the fixed delay path is T Pt,f = 0.23 at hn = 1800 eV. Given this fact, even odd harmonics of the fundamental FEL beam can be transmitted with reasonable intensity in soft X-ray pump/soft X-ray probe experiments from the XUV beyond the soft X-ray region. Additionally, the Pt-coated mirrors offer a second possibility for an increased reflectivity for photon energies below hn 150 eV. Experimentally the total transmission of the Ni coating was measured to T v = 0.47 and T f = 0.54 at hn = 124 eV. This somewhat lower transmission than calculated originates from losses at the beam splitter edge, e.g. optical refraction, which has not been taken into account in the calculations. As the presented total transmission of each beam path covers a broad spectral range, the scope of application of the SDU is not restricted to pump/probe experiments with only one photon energy. With a two-color operation of the FEL (Schneidmiller et al., 2019 >) and proper selection of thin filters in the respective beam paths, two-color pump/probe experiments are possible where the optical pump and optical probe do not share the same photon energy. The mirror sizes were designed for the estimated divergences presented in 2013 (Kuhlmann & Plonjes, 2013 >). Based on this paper, for a photon energy of hn = 150 eV the calculated spot size for a Gaussian profile at the entrance of the SDU amounts to Ks = 8 mm (6s) (3 mm FWHM). Given the geometrical design, the effective aperture of the SDU is limited by the size of the mirrors which are given in Table 1 >. The dimensions of the Ni mirrors in the fixed delay beam path are 380 mm x 25 mm and for the Pt mirrors are 250 mm x 25 mm. In the fixed delay paths the nominal horizontal clear aperture amounts to 8.4 mm for the Ni-coated mirrors and 5.4 mm for the Pt-coated mirrors. For all mirrors of the fixed delay paths the nominal vertical clear aperture amounts to 20 mm. Notice that just the lower half of the vertical clear aperture of the mirrors is used in the fixed delay path. As indicated in Fig. 3 >, the 6s footprint of a FEL beam at hn >= 150 eV is included in the mirror surface. In the 2s footprint the included peak-to-valley value amounts to Ds < 2.5 nm at 150 eV. For lower photon energies the 6s beam diameter surpasses the aperture of the mirrors and the beam cannot be covered by the mirrors entirely. Given the mirrors in the variable beam path with a size of 250 mm x 50 mm, a nominal vertical clear aperture of 7.5 mm follows. When cutting the beam in the middle the BS mirror includes nearly the whole top half 6s-footprint of this beam. According to the assumed divergences this holds true down to hn = 70 eV. The nominal horizontal aperture of the variable beam path is given by the width of the applied coatings of the mirror which is 30 mm for the Ni coating and 12 mm for the Pt coating, and thus entails the entire beam over the whole spectral range. The X-ray mirrors were manufactured and characterized by Carl Zeiss SMT GmbH before application of the coatings. The reflective coatings were applied by the Fraunhofer Institute for Material and Beam Technology IWS. An exemplary height profile of the Ni1 mirror is shown in Fig. 3 >. For this mirror, the radius of curvature was determined by a Carl Zeiss D100 Direct measuring interferometer to over 500 km along the tangential mirror axis with a slope error below 0.032 arcsec (r.m.s.). In the sagittal plane the slope error amounts to 0.038 arcsec (r.m.s.). As can be depicted from Fig. 3 >, the maximal peak-to-valley value is 8.1 nm. The roughness has been measured by a DI Nanoscope D3100M to below 0.3 nm (r.m.s.) and confirms the excellent quality of the mirrors for applications with XUV and soft X-ray beams under grazing angles. The other Ni-coated mirrors show a similar profile with tangential slope errors of below 0.03 arcsec (r.m.s.). The tangential and sagittal slope of the variable beam path mirrors and the Pt-coated mirrors is below 0.02 arcsec (r.m.s.) and 0.04 arcsec (r.m.s.), respectively. The minimum radius of these mirrors is over 300 km and the maximal peak-to-valley value is below 7.1 nm. The beam splitting edge of the mirrors BS and RC were cut at 10 mm from the end of the respective mirror, reducing the clear aperture of these mirrors. 2.2. Technical realization A technical drawing of the SDU is shown in Fig. 4 >. All parts are made, as far as feasible, out of stainless steel to avoid strain within the SDU which may be caused by temperature driven expansions. The optical bench (1) consists of a single sheet of stainless steel with a thickness of 4 mm. The upper part of the optical bench is connected via metal plates (2), increasing the stiffness and serving as platforms for the mirror holders of BS, RC and for the fixed beam path. The mechanical contact of the optical bench has been decoupled from the vacuum chamber in order to avoid any bending of the optical bench when the vacuum chamber is evacuated. Bellows (3) allow the optical bench to be moved vertically and horizontally as a whole. The optical bench rests on top of two granite blocks which are decoupled from vibrations of the ground floor by vibration isolating damping elements. There is no additional treatment of anti-vibrational mechanisms of the floor of the beamline and the SDU. An improved vibrational isolation can be achieved in future by the insertion of active damping elements. The mirrors are attached on mirror holders (4) which allow an adjustment of the pitch and roll angles with high accuracy (Noll, 2003 >; Wostmann et al., 2013 >). A gimbal mount is realized by braces with flexible joints which face towards the center of the mirrors' surface. Hence, all three pivot points are located within the mirror surface with a low parasitic movement of the mirror. Every mirror holder is equipped with two UHV-compatible stepper motors with gear units which allow an in situ movement of the pitch and roll axes with respect to the beam. 200 motor steps correspond to one revolution of the motor, which in turn deflects the pitch angle by 25 mrad and results in a lateral movement of 625 mm of the unfocused beam at the sample position at a distance of 25 m. The two last mirrors of every beam path (Ni4, Pt4 and RC) are dedicated to adjusting the position and overlap of the partial beams at the experiment. The mirrors DL1, DL2, RC, Ni4 and Pt4 are equipped with piezoelectric actuators. This allows the pitch angle to be set at a nominal accuracy of dth = 38 nrad in the range Dth = 1250 mrad. The yaw axis around the surface normal is of minor importance and was therefore aligned manually before installation of the SDU via fine-thread screws. All movable parts are equipped with limit switches to prevent accidental damage to any parts. The variable mirrors DL1 and DL2 [see (5) in Fig. 4 >] are both placed on sleds which move on guiding rails (6) along the variable beam path. The guiding rails are connected to the optical bench via corner profiles. Every sled is driven individually, but identically, by a stepper motor (7) via a ball screw drive (8). The positions of the variable mirrors are measured by optical encoders with a nominal division of dl v,Enc = 5 mm of the encoder marker, corresponding to a temporal resolution of dt = 66 as. While moving, the pitch angle of the mirrors is measured via an optical interferometer (9; Attocube, IDS3010), and is automatically corrected in a closed feedback loop via the piezoelectric actuators. As both delay mirrors are moved independently on guiding rails over a distance of Dl v = 1.75 m, mechanical inaccuracies are inevitable. These arise from reproducible errors from not perfectly linear guiding rails as well as non-reproducible errors like mechanical inaccuracies of the utilized bearings. A laser was applied parallel to the delay stage at the height of the central mirror surface of the respective delay mirror. The mirror was replaced by a CCD camera (Basler, acA645-gm100) with a pixel size of 9.9 mm which measured the height deviation of the sled when moving along the delay stage. As these measurements were performed in a cleanroom tent under ambient flow conditions the fluctuations were noticeable. The averaged height deviation does not exceed Dh = 8 mm and Dh = 25 mm for the delay stages of DL1 and DL2, respectively. As the angular deviation has a greater impact on the stability of the system, the angular deviation has been measured over the delay range by the optical interferometer. The results are shown in Fig. 5 >. The peak-to-valley value for DL1 shows a maximum deviation of Dth = 92 mrad and for DL2 of Dth = 111 mrad. Since both maximum deviations are within the provided correction range of the closed-feedback interferometric system of Dth = 1250 mrad, those errors are corrected automatically with a precision of dth = 0.66 mrad (r.m.s.) (red lines in Fig. 5 >) by the piezoelectric actuators. As can be seen in equation (1), the accuracy of the delay setting directly depends on the precise positioning of the sleds. Therefore, the reproducibility of the motorized delay stage was verified by the use of the optical encoder and the interferometer. The motorized delay stage was moved for a certain amount of motor steps and the position of the sled was simultaneously and independently measured by the optical encoder and the optical interferometer. For 20000 motor steps, corresponding to a distance of Dl M = 5 mm or a nominal delay of Dt = 66 fs, the optical encoder measured a distance of Dl E = 4.998 +- 0.003 mm while the interferometer measured a distance of Dl I = 5.000 +- 0.001 mm. For a movement of 200000 motor steps, corresponding to a nominal distance of Dl = 50 mm or Dt = 660 fs, the optical encoder measured a real distance of Dl E = 49.987 +- 0.003 mm while the interferometer specified Dl I = 49.989 +- 0.003 mm. In both measurements the mirror travel distances determined by the external devices of optical encoder and interferometer are in good agreement with each other. The relative difference between the encoder position and the interferometer position amounts to 0.04% or dt = 26 +- 40 as in this example. In every single case, the differences of the optical encoder and the interferometer system did not exceed the nominal resolution of the optical encoder of dl v,Enc = 5 mm. Therefore, the mechanical realization ensures a temporal resolution of better than dt = 66 as. The optical bench is placed into a vacuum chamber with a total length of 6 m which is pumped by three ion-getter pumps (Agilent, Vacion Plus 500 Diode, 500 l s-1 each) to maintain a vibration-free vacuum at about 5.0 x 10-9 mbar. To reach the working pressure of the ion getter pumps, a turbomolecular pump (Pfeiffer Vacuum, HiPace700, 685 l s-1) is installed which is mechanically isolated from the vacuum chamber, and is turned off after pump down to avoid vibrations. 2.3. Beam manipulation and diagnostic The intensity ratio of the two partial beams can be chosen according to the needs of the experiment by moving the optical bench in the vertical direction. In Fig. 6 >(a) for a nominal splitting ratio of 63:37 the fluctuations of this ratio are shown on a shot-to-shot basis for 500 shots taken at 1 Hz. Fig. 6 >(b) shows a histogram of these fluctuations. In this particular case the splitting ratio is varying by 0.029 (r.m.s.) and +-0.033 (FWHM), respectively. These fluctuations arise in particular from the pointing of the FEL beam leading to a varying splitting of the footprint at the beam splitting edge. In situ single-shot intensity measurements are important for pump/probe experiments. To enable this, the mirrors Ni2 and Pt2 are equipped with microchannel plate detectors which measure the emitted photoelectrons of the mirror surface with intra-bunch single-pulse resolution (so-called PATIM) - see Fig. 4 > (10). With the intensity data provided by both gas monitor detectors (GMDs) in the hall and in the tunnel (Tiedtke et al., 2008 >), this allows the proportion of the beam intensity passing the fixed delay beam path to be calculated. In Fig. 7 > a calibration measurement taken at a wavelength of l = 8.5 nm is depicted. This shows the pulse energy deviation of the signal with respect to the GMD located in the hall. Over the scanned pulse energy range up to E pulse = 50 mJ an accuracy of about dE pulse 4 mJ (r.m.s.) with respect to the GMD hall is achieved. As reference, the pulse energy deviations of the GMD detector in the tunnel are shown in red, which show a pulse energy deviation of <2 mJ (r.m.s.). Within every beam path, a retractable blocking stage - see Fig. 4 > (11) - is available which allows the individual beam path to be blocked to check beam positions separately. The blocking stages can be equipped with filter windows in order to separate distinct photon energies of the FEL, e.g. the fundamental from a harmonic. With this option, the SDU enables time-resolved two-color pump/probe experiments. Furthermore, the filter stage of the delay path provides a Ce:YAG screen for beam position monitoring. Another imaging stage is located right behind the SDU in a distinct vacuum chamber. With this imaging stage the recombined partial beams can be imaged via a Ce:YAG screen. Additionally, this monitoring chamber provides another stage with interchangeable filters. 3. Performance 3.1. Basic alignment A prealignment was carried out with an optical laser at l = 660 nm with a reduced coherence length of the order of 300 mm (Schafter + Kirchhoff, 51nano-FCM) to show the linearity of the delay stage. A special feature of this optical laser is that it exhibits additional coherence signals at temporal intervals of Dt = 6.6 ps, 11.5 ps, 18.1 ps, 24.7 ps and 29.6 ps. The SDU was inserted partially into the optical beam leaving one part unaffected. In this case, two coherence measurements were examined simultaneously, see Fig. 8 >: one interference measurement of the partial beams of the fixed delay path and the variable beam path (black circles), and one interference measurement of the direct beam and the variable delayed beam (red circles). The known temporal distances of the coherence signals are assigned to the lateral positions of the delay stage leading to a linearity calibration of the delay stage. The resulting slope gives a ratio of 1 ps per (2.95 +- 0.02) x 105 stepper motor steps. This corresponds to a reflection angle of th = 1.83deg which is in the design range of the reflection angle of thv = 1.8deg. Given the mechanical constraints, a delay range of -5 < Dt < +18 ps is achieved. According to the slope the difference between the fixed delay path and the direct beam amounts to 14.3 ps. Bypassing the fixed delay path gives the opportunity to extend the achievable delay range up to 33 ps. 3.2. Temporal stability As the temporal jitter is the temporal deviation of the given delay between both beams, interference fringes offer an opportunity to evaluate this characteristic of the SDU. A change of time delay between both beams results in a movement of the observed interference fringes. Considering only one local point of the whole interference pattern this movement corresponds to a change of intensity I Exp at this local point. By evaluating the intensity at this point at a nominally fixed delay position the actual time delay between both partial beams can be obtained. With the minimum and maximum intensity of the interference fringes (I max, I min) the time delay dt can be calculated by It shall be emphasized here that, due to the ambiguity of the arccos function, the time delay can only be examined unambiguously for values of I min < I Exp < I max. Therefore, it is reasonable to measure between two interference fringe maxima and to use a laser with a long wavelength, because that enlarges the measurable delay jitter. An optical laser with a wavelength of l = 633 nm was used to create suitable interference patterns. The interference pattern was measured with a camera (Basler, acA720-290gm) at a distance of L = 24 m from the RC. As the detector is placed perpendicular to the main propagation direction of the beams, the impact of small vibrational movements of the camera on the measured interference fringes is negligible. The data were taken at a rate of 80 Hz. The temporal jitter of the SDU is shown in Fig. 9 >. The resulting time delays of the data are binned in count rates by intervals of 10 as and shown as a histogram. A Gaussian profile is fitted to the histogram, indicating a timing jitter of t j = 121 +- 2 as (FWHM). In Fig. 10 >, the fast Fourier transform of the temporal jitter is shown. A main resonance of the optical bench at 11 Hz is observed, probably resulting from ground floor vibrations. 3.3. Pointing stability To measure the pointing stability of the SDU, both partial FEL beams at l = 10 nm were set on separate positions on an XUV-sensitive CCD camera (Princeton Instruments, PIXIS-XO:2KB). After passing the Ni-coated beam path, the spot positions were recorded on a shot-to-shot basis over 500 shots at a distance of L = 24 m. The beam size was reduced by inserting an aperture with a diameter of 2 mm at a distance of 13 m in front of the SDU. The resulting angular pointing stability is shown in Fig. 11 >. The pointing stability of the fixed beam path amounts to du f,horizontal = 6.5 mrad (FWHM) in the horizontal plane and du f,vertical = 2.8 mrad (FWHM) in the vertical plane. The pointing stability of the variable delay path in the horizontal plane is du v,horizontal = 4.3 mrad (FWHM) while in the vertical plane it shows du v,vertical = 9.7 mrad (FWHM). The difference in the pointing stability in the respective pitch and roll axis of the mirror mounts is attributed to the reversed angular orientation of the mirror mounts in these beam paths. The variable beam path is affected more by vibrations in the vertical direction introduced from the ground floor into the SDU. The footprint of an unfocused beam at the sample position is of the order of 10 mm and the displacement from pointing of the variable beam path at this position amounts to 0.2 mm (FWHM) in the vertical direction. The overlap between both partial beams of the SDU is thus maintained. The pointing of the FEL in this set-up was determined to 1.5 mrad. As a comparison, an angular pointing measurement before decoupling the SDU from external vibrations was taken. In this measurement the pointing stability is lowered to du f,horizontal = 13.2 mrad (FWHM) and du f,vertical = 3.5 mrad (FWHM) in the fixed beam path and is decreased slightly to du f,vertical = 8.0 mrad (FWHM) in the variable delay path while no significant change was measured for du v,horizontal = 4.3 mrad (FWHM). 4. Characterization of FEL pulses 4.1. Visibility measurements When commissioning the SDU, both partial beams of the FEL operating at l = 8 nm were overlapped directly on an XUV-sensitive CCD camera and thereby created interference fringes. At short wavelengths a small crossing angle O between both partial beams is necessary to resolve the arising interference patterns. Therefore, the camera was placed at a distance of L = 24 m from the RC mirror. The visibility was derived from these interference fringes by When increasing the temporal delay between both beams, the visibility is reduced due to the finite temporal coherence time of the FEL beam. From the visibility measurements the coherence time can be determined as the half width at half-maximum (HWHM) of the temporal visibility distribution of a beam. Typically, the coherence time of an FEL beam lies in the order of some femtoseconds, allowing the coherence time of the FEL pulse to be determined by means of the SDU. Likewise, a quantification of the zero-delay position with femtosecond accuracy is enabled with this set-up. The FEL was slightly detuned on purpose leading to a significantly shorter coherence time which allows the performance of the SDU to be characterized more precisely. An exemplary interference pattern taken at l = 8 nm and Dt = 0 fs is shown in Fig. 12 >(a). Well defined interference fringes were obtained which can be distinguished from the diffraction patterns of the beam-cutting BS mirror by moving the pointing of the fixed delay beam path (bottom semicircle) slightly aside, which then leads to tilted interference fringes in the interference pattern. From the spatial overlap q of both beams, the width of the interference fringes w, and the distance of the CCD camera to the RC mirror, the crossing angle O and the gap g between both beams at the position of the RC can be calculated. At a fringe spacing of w = 270 mm and an overlap of q = 500 mm, O = 30 mrad and g = 200 mm follow, indicating a narrow gap between both beams at the RC. For the presented shot the visibility evaluated in the area marked by the red rectangle amounts to V = 0.8. By changing the delay consecutively in steps of 0.5 fs and taking 25 measurements at each delay step, the delay distribution of the visibility follows, depicted in Fig. 12 >(b). A Gaussian distribution was fitted to the data yielding an average coherence time of tc = 1.75 +- 0.04 fs (HWHM). The comparatively low average visibility V = 0.74 +- 0.08 at Dt = 0 fs is attributed to the reduced spatial coherence due to the detuning of the FEL. The obtained coherence time shows that with the SDU a temporal resolution in the sub-femtosecond timescale can be achieved as already shown by the jitter data obtained via visible light. 4.2. Focused beams and wavefront Beamline users typically request focused beams to perform their experiments. With the Kirkpatrick-Baez (KB) optics installed at the end of beamline FL24, both beams at l = 15 nm were focused to a distance of 3.1 m from the KB optics. An XUV Hartmann wavefront sensor was illuminated by the diverging light behind the focus position (Keitel et al., 2016 >). From the pattern obtained, the wavefront at the focal point was reconstructed for the beams of the fixed delay beam path and variable delay beam path individually. The result of this wavefront backpropagation calculation is shown in Fig. 13 >. The achieved spot size for the beam of the variable delay beam path amounts to 19 mm (FWHM) and 14 mm (FWHM) in the horizontal and vertical directions, respectively. For the fixed delay beam path the backpropagation calculation yields a spot size of 7 mm (FWHM) and 11 mm (FWHM) in the horizontal and vertical directions. 5. Summary A split-and-delay unit has been built for FLASH2 in the Kai Siegbahn hall and serves beamlines FL23 and FL24. The device allows users to perform pump/probe experiments with XUV and soft X-ray beams with a temporal delay of -5 ps to +18 ps at a sub-femtosecond temporal resolution. With the possibility to choose between Ni-coated mirrors and Pt-coated mirrors, the full fundamental spectral range of FLASH2 is covered as well as harmonics up to a photon energy of hn = 1800 eV. The temporal jitter between both partial beams was measured via interferences at l = 633 nm and amounts to t j = 121 +- 2 as (FWHM). This is close to the nominal delay resolution of dt = 66 as provided by the encoder and dt = 13 as provided by the nominal resolution of the stepper motors. At a splitting ratio of 63:37 a splitting ratio fluctuation of the order of 0.029 (r.m.s.) and +-0.033 (FWHM) has been measured. The angular pointing stability including the pointing error of the FEL amounts to du f,horizontal = 6.5 mrad (FWHM), du f,vertical = 2.8 mrad (FWHM) for the fixed delay path and du v,horizontal = 4.3 mrad (FWHM), du v,vertical = 9.7 mrad (FWHM) for the variable delay path. In the fixed delay paths the PATIM is installed to measure the relative intensity within the beam path on a shot-to-shot basis even for pulse trains. Additionally, with implemented filter stages a variety of two-color pump/probe experiments is possible. The functionality of the SDU is demonstrated by visibility measurements in order to examine the coherence properties of a FEL beam with reduced temporal coherence. In this case the measured coherence time was tc = 1.75 +- 0.04 fs, revealing the outstanding opportunities of the SDU to perform pump/probe experiments in the XUV and soft X-ray region with sub-femtosecond time resolution. With the KB optics at the end of beamline FL24, the beams were focused. The beam of the variable delay path was focused to a spot size of 19 mm (FWHM) and 14 mm (FWHM) in the horizontal and vertical directions, respectively, and for the beam of the fixed delay beam path a spot size of 7 mm (FWHM) and 11 mm (FWHM), respectively, was achieved. The support of the DESY groups FS-FLASH and FS-BT in particular is gratefully acknowledged. The authors thank Carl Zeiss SMT for the characterization of the X-ray mirrors. Open access funding enabled and organized by Projekt DEAL. Figure 1 Schematic drawing of the SDU showing FEL beams utilizing the Ni-coated mirrors (orange). The initially top section of the beam (green) is reflected by the beam splitter mirror (BS) downwards into the variable delay path (gray mirrors). The mirrors DL1 and DL2 can be moved along the beam path as indicated by the orange arrows and induce a temporal delay into this beam. The initially lower section of the FEL beam (red) passes the BS unreflected (see top inset) and propagates in the fixed delay path (Ni1-Ni4, orange mirrors). Both beams are recombined at the recombining mirror (RC, see lower inset). A small gap between both beam parts remains after leaving the SDU. The optical bench [gray frame, in (c)] can be moved horizontally to change the positioning from the the Pt-coated mirrors Pt1-Pt4 (light blue). In the variable delay path the Ni and Pt coatings are evaporated side-by-side on the mirrors BS, DL1, DL2 and RC. Figure 2 Calculated total transmission of the mirror coatings used in the different beam paths based on CXRO (Henke et al., 1993 >), assuming a FEL beam p-polarized in the horizontal plane. The grazing incidence angle thv = 1.8deg is associated with the variable beam path, and a grazing incidence angle of thf = 1.3deg is applied in the fixed beam path. Note that only the same type of mirror coating [Ni (red and green) or Pt (blue and magenta)] can be chosen simultaneously. Figure 3 Surface profile of the Ni1 mirror. Characterized is the clear aperture of 370 mm x 20 mm. The typical profile results in a concave radius of above 500 km and a slope error of 0.032 arcsec (r.m.s.) tangential and 0.038 arcsec (r.m.s.) sagittal. The 2s and 6s footprints of a FEL beam at 150 eV are drawn for reference according to Kuhlmann & Plonjes (2013 >). Figure 4 Mechanical structure of the SDU. (1) Optical bench, (2) base metal plates, (3) moving base with vacuum bellows, (4) mirror mountings, (5) DL1 and DL2 at -5 ps, 0 ps and +18 ps positions, (6) guiding rails, (7) UHV-compatible stepper motors of the delay stage, (8) ball screw drives, (9) two beam interferometer, (10) relative pulse intensity measurement device, (11) retractable blocking stages with filters, (12) beam splitter BS, (13) recombination mirror RC. Figure 5 Slope deviation of the mirrors (a) DL1 and (b) DL2 over the delay range as measured by the interferometric system. The peak-to-valley value of the slope deviation amounts to Dth = 80 mrad for DL1 and Dth = 165 mrad for DL2 over the whole delay path. The angular deviation of the beam is compensated by the closed feedback interferometer system acting on the mirrors, yielding a correction precision of dth = 0.66 mrad (r.m.s.) (red line, see text). Figure 6 (a) Measured splitting ratio variations for an average splitting ratio of 63:37 over 500 shots, taken at a rate of 1 Hz. (b) Splitting ratio variation of both partial beams. The splitting ratio variation amounts to +-0.033 (FWHM) and 0.029 (r.m.s.), respectively. Figure 7 Calibration measurement of the relative pulse intensity measurement device (PATIM detector) at a wavelength of l = 8.5 nm on a shot-to-shot basis with respect to the GMD in the hall. Over the considered pulse energy range, the absolute energy deviation is dE pulse 4 mJ (r.m.s.). The pulse energy measured by the tunnel GMD is simultaneously obtained and given as a reference with dE pulse < 2 mJ. Figure 8 Linearity of the delay stage as measured by a laser with reduced coherence time. The black circles refer to an interference of the two beams in the fixed delay path and variable delay path. The red circles refer to the measured interferences between the beam of the variable delay path and a beam passing directly through the SDU. The additional delay obtained by bypassing the fixed delay path amounts to 14.3 ps. The covered delay range of the SDU spans between the crossed markers, allowing delays between -5 ps and 18 ps with the conventional set-up and up to 32 ps when bypassing the fixed delay path. The fit results in a slope of (2.95 +- 0.02) x 105 steps of the stepper motor for 1 ps. In the inset the residuals of the slopes are shown, which amounts to 6 fs. The error is given by the coherence length of the individual coherence signals. Figure 9 Jitter histogram of the SDU derived from interference measurements with visible light at 633 nm. A Gaussian profile with a width t j = 121 +- 2 as (FWHM) has been fitted to the data. 2000 data points were taken with a sample period of 80 Hz, i.e. over a measurement time of 25 s. Figure 10 Fast Fourier transform of the interference measurements with visible light. The SDU with dampers shows a prominent resonance at 11 Hz arising from the incoupling of ground vibrations from the hall. Figure 11 Pointing stability of both Ni-coated beam paths in the horizontal and vertical axis. In the fixed delay path a pointing stability of du f,horizontal = 6.5 mrad (FWHM) and du f,vertical = 2.8 mrad (FWHM) was measured. For the variable delay path it amounts to du v,horizontal = 4.3 mrad (FWHM) and du v,vertical = 9.7 mrad (FWHM). Figure 12 (a) Interference pattern taken at l = 8 nm and Dt = 0 fs. The visibility in the region marked by the red rectangle amounts to V = 0.8. (b) Temporal visibility distribution of the FEL beam at l = 8 nm. Every point represents the average of 25 individual interference patterns taken at one delay. The resulting coherence time is tc = 1.75 +- 0.04 fs. The average visibility in the maximum was determined to V = 0.74 +- 0.08. Figure 13 Beams in the focus position taken from backpropagation calculations with the Hartmann wavefront sensor at l = 15 nm. The diameter of the spot size in the variable delay beam path (a) amounts to 19 mm (FWHM) and 14 mm (FWHM) at a focal length of 3.1 m and in the fixed delay beam path (b) amounts to 7 mm (FWHM) and 11 mm (FWHM) at a focal length of 3.2 m in the horizontal and vertical directions, respectively. Table 1 Nominal apertures of the mirrors of the SDU for the incoming beam The clear aperture takes into account the existence of a 5 mm not specified rim at the edges of the optical surfaces. 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PMC10000807 | J Synchrotron Radiat J Synchrotron Radiat J. Synchrotron Rad. Journal of Synchrotron Radiation 0909-0495 1600-5775 International Union of Crystallography 36891844 ye5028 10.1107/S1600577523000528 JSYRES S1600577523000528 Research Papers Adaptive vibration control method for double-crystal monochromator base on VMD and FxNLMS Adaptive vibration control method for DCMs Bai Yang ab Gong Xuepeng a* Lu Qipeng a* Song Yuan a Zhu Wanqian c Xue Song c Wang Dazhuang a Peng Zhongqi a Zhang Zhen a a Changchun Institute of Optics, Fine Mechanics and Physics, Chinese Academy of Sciences, Changchun 130033, People's Republic of China b University of Chinese Academy of Sciences, Beijing 100049, People's Republic of China c Shanghai Advanced Research Institute, Chinese Academy of Sciences, Shanghai 201204, People's Republic of China Bhattacharyya D. Editor Bhabha Atomic Research Centre, India Correspondence e-mail: [email protected], [email protected] 01 3 2023 20 2 2023 20 2 2023 30 Pt 2 s230200 308318 07 8 2022 21 1 2023 (c) Yang Bai et al. 2023 2023 This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-BY) Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited. A full version of this article is available from Crystallography Journals Online. With the performance of synchrotron radiation sources increasing significantly, higher requirements have been placed on the stability of double-crystal monochromators (DCMs). Using traditional passive vibration control techniques, such as optimized structures, elastic damping and vibration-absorbing materials, is unlikely to meet the future requirements of DCMs. The proposed method is the first attempt of an adaptive filtering algorithm based on variational modal decomposition in the field of DCMs development, which is an advancement for the development of high-performance DCMs at synchrotron radiation facilities. Double-crystal monochromators (DCMs) are one of the most critical optical devices in beamlines at synchrotron sources, directly affecting the quality of the beam energy and position. As the performance of synchrotron light sources continues to improve, higher demands are placed on the stability of DCMs. This paper proposes a novel adaptive vibration control method combining variational modal decomposition (VMD) and filter-x normalized least mean squares (FxNLMS), ensuring DCM stability under random engineering disturbance. Firstly, the sample entropy of the vibration signal is selected as the fitness function, and the number of modal components k and the penalty factor a are optimized by a genetic algorithm. Subsequently, the vibration signal is decomposed into band frequencies that do not overlap with each other. Eventually, each band signal is individually governed by the FxNLMS controller. Numerical results have demonstrated that the proposed adaptive vibration control method has high convergence accuracy and excellent vibration suppression performance. Furthermore, the effectiveness of the vibration control method has been verified with actual measured vibration signals of the DCM. active vibration control double-crystal monochromator FxNLMS algorithm variational modal decomposition synchrotron radiation National Natural Science Foundation of China62104224 Yuan SongNational Natural Science Foundation of China61974142 Xuepeng GongThe work is supported by National Natural Science Foundation of China (Nos. 61974142, 62104224) and 'Xu-Guang' Talent Program of Changchun Institute of Optics, Fine Mechanics and Physics (CIOMP), China, Chinese Academy of Sciences (CAS) (E01672Y6Q0). pmc1. Introduction Nowadays, the large scientific research platform based on synchrotron radiation provides advanced experimental technology tools for many disciplines, such as life science, physics, chemistry, biology, medicine, materials, archeology, etc. (Jin et al., 2017 >). Double-crystal monochromators (DCMs) are one of the key optical instruments on hard X-ray beamlines at synchrotron radiation facilities, separating the hard X-rays from the synchrotron radiation source. As the performance of synchrotron radiation sources significantly increases, higher requirements are placed on the stability of DCMs. Using traditional passive vibration control techniques, such as optimized structures, elastic damping and vibration-absorbing materials, is unlikely to meet the future requirements of DCMs (Yamazaki et al., 2013 >; Baker et al., 2013 >; Wu et al., 2021a >,b >). Therefore, it is necessary to design and implement efficient active vibration control techniques to further improve the stability performance of DCMs. The least-mean-square (LMS) algorithm is widely used in active vibration control (Sun et al., 2007 >), and requires no precise mathematical model of the controlled object and can adjust the filter parameters adaptively based on system input and output response; it also has a simple structure and can be easily implemented, and has strong approximation capability for linear systems. However, when the input vector is relatively large, the LMS algorithm encounters the problem of noise amplification. The normalized LMS (NLMS) algorithm conquers this problem by normalizing the adaptive step size. The filter-x normalized least-mean-square (FxNLMS) algorithm based on adaptive filtering technology has been applied in the field of active vibration control due to its excellent convergence accuracy, powerful adaptive capability and robustness (Fallah & Moetakef-Imani, 2019 >; Yi et al., 2019 >). The classical FxNLMS algorithm is illustrated in Fig. 1 > The computational equation of the FxNLMS algorithm is shown in Table 1 >. Empirical mode decomposition (EMD) is flawed by end effects, modal aliasing and over-enveloping; the short-time Fourier transform (STFT) processing signal cannot obtain high time-frequency resolution in the meantime; the local mean decomposition (LMD) also yields a false product function component (Wu & Huang, 2009) >; Bao et al., 2020 >). To solve the above problem, the variational modal decomposition (VMD) algorithm was proposed by Dragomiretskiy & Zosso (2014 >). VMD screens the intrinsic mode function (IMF) components in the form of solving the optimal solution of the variational problem (Dragomiretskiy & Zosso, 2014 >). This method continuously iterates to search for the most suitable solution and adaptively achieves effective signal decomposition. Constructing and solving the constrained variational model to decompose the signal involves techniques such as the Wiener filter, Hilbert transform and frequency mixing. It is applied in mechanical fault diagnosis since it adopts a non-recursive conceptual framework. For instance, Wang et al. (2015 >) proposed a method to analyze faults by friction factor and applied variational model decomposition analysis, which is known to be more effective in diagnosis by comparison. Dey et al. (2015 >) combined VMD and Teager energy operators for fault diagnosis. The VMD algorithm makes the decomposition results stable by constructing the variational problem. However, VMD suffers from the deficiency that the decomposition effect is strongly influenced by the number of modal components k and the penalty factor a (Ram & Mohanty, 2017 >). When using the VMD method to process the vibration signal, the values of the number of modal components k and the penalty factor a are set empirically before the calculation. Therefore, the combination of [k, a] parameters of VMD is subject to artificial factors that can lead to under-decomposition of the decomposition results. The value of the penalty factor a is important to ensure the accuracy of the VMD algorithm when reconstructing the signal. If the initial value of a is not set properly, the VMD algorithm will decompose the overlapping modal signals or the center frequency will be unstable, which may lead to incorrect decomposition of the algorithm and failure to obtain optimal resolution. Li et al. (2017 >) proposed an independent VMD method that found optimal modes by peak search and similarity principle. Wang et al. (2018 >) used the energy difference of the decomposed signal as a criterion to determine the preset modal parameters. The genetic algorithm (GA) is an optimization algorithm that simulates natural biological selection and genetic evolution. The algorithm consists of three genetic operators: selection, crossover and mutation. The GA can solve nonlinear problems quickly, efficiently and rapidly on a global scale (Singh & Harshit, 2014 >). Bian (2017 >) proposed a VMD method based on the GA to optimize the number of modal components k and the penalty factor a. Based on the above introduction, this paper proposes a novel adaptive vibration control method based on VMD and FxNLMS to ensure the stability of ultra-precision optical instruments under random engineering disturbance. Since the VMD algorithm has a better de-correlation capability, VMD is implemented into the adaptive filtering algorithm. The extraction properties of VMD coefficients are used to decompose the input signal in the multi-scale space, reducing the dynamic spectral range of the adaptive filter's auto-correlated array of input vectors. Consequently, the convergence speed and stability of the FxNLMS algorithm are improved. The primary process of this method is as follows: firstly, VMD is proposed to decompose the vibration signals into IMFs; then, the sample entropy of the vibration signal is selected as the fitness function, and the number of IMFs and penalty factor are optimized with GA; eventually, each IMF is controlled individually by the FxNLMS controller. Simulation results have demonstrated that the convergence accuracy and vibration suppression performance of the proposed method is much better than for the FxNLMS algorithm. Furthermore, the effectiveness of the method was verified with actual measured vibration signals. 2. The proposed algorithm 2.1. GA-VMD basic principle The VMD process is essentially the solution process of the variational problem, which involves three critical concepts: classical Wiener filtering, Hilbert transform and frequency mixing (Dragomiretskiy & Zosso, 2014 >). The basic idea of the VMD algorithm is to search for a new way to adapt the signal decomposition process, which turns the decomposition process into a new constrained variational problem that can be decomposed. The VMD algorithm abandons the principle of refinement sieving of the modal signal in EMD. In this paper, the sample entropy is considered as the fitness function of the GA to optimize modal components k and the penalty factor a. The specific process is as follows. For each modal function, the Hilbert transform is applied to the function to create a linear operator which can obtain the resolved spectral signal, expressed by Mixing of all modal analysis signals is given by The constrained equation for the constructed variational model is given by (Dragomiretskiy & Zosso, 2014 >) where d(t) is the Dirichlet function; * is the convolution operation; {u k } = {u 1,..., u k } is the set of IMFs obtained after the VMD decomposition of the modes; and {o k } = {o1,..., o k } is the combination of the component center frequencies. Introducing the penalty factor a and Lagrangian multiplier l(t), which transforms the constrained variational problem into the unconstrained variational problem, the extended Lagrangian is expressed by where a is the penalty factor and l is the Lagrangian multiplier. The variational problem is addressed by the alternate direction method of multipliers (ADMM) (Nocedal & Wright, 2006 >) and the optimal solution is obtained by updating , and l n+1. is denoted by where n is the iteration. The Parseval Fourier isometric transformation is utilized for equation (5), and the frequency domain range expression is given by Equation (6) is converted into the form of the non-negative frequency interval integral, and then is given by The optimal solution of the problem can be obtained as Base on a similar scheme, the central frequency problem is transformed to the frequency domain, From equation (9), the updated formula of the center frequency is given by where is equivalent to the Wiener filtering result of the current residual [ ]. The VMD algorithm is continuously updated in the frequency domain, after which the Fourier inversion is performed to obtain the results in the time domain. The practical procedure is illustrated as follows: (1) Initialize , , and n = 0. (2) According to equations (8) and (10), update {u k }, {o k }. (3) Update the Lagrangian multiplier l, which is given by (4) The above iterations continue until convergence. The deterministic conditions is The sample entropy method is less dependent on data length and more resistant to interference, which has been widely applied in vibration signal research. The sample entropy of the vibration signal is selected as the fitness function of the GA, which is applied to determine whether the individual modal function components and penalty factors meet the decomposition requirements. The vibration signal X i (n) is a time series of length , i = 1, 2,...N. The vectors constructing X i (n) are , ,..., , given by where m is the vector length. The maximum value of the absolute value of the element-specific difference of two vectors is given by For individual X m (i), the difference between X m (i) and X m (j) is calculated to be less than that to the quantity j (1 <= j <= N - m, j i) of parameter v, defined as When the dimension is m + 1, the difference between X m+1(i) and X m+1(j) is calculated to be less than to the quantity j (1 <= j <= N - m, j i) of parameter v, defined as B (m)(v) and A (m)(v) are the m point probability and m + 1 point probability of being able to match the two sequences of elements under the similarity tolerance v. The sample entropy of this time series is defined as The dimension m is generally taken as m = 1-2; v = 0.1 std 0.25 std (where std is standard deviation of the data). The sample entropy is selected as the fitness function of the GA to adaptably obtain the [k, a] optimal parameter combination of the VMD, which realizes the adaptive determination of modal components k and the penalty factor a of the vibration signal for an ultra-precision optical instrument. A schematic diagram of VMD parameter optimization by GA is shown in Fig. 2 >. 2.2. Active vibration control algorithm A schematic diagram of the adaptive vibration control method based on VMD and FxNLMS is shown in Fig. 3 >. Actual vibration signals are constantly subject to baseline drift (trend term of the signal) in the acquisition process caused by environmental interference. Since the correctness of the vibration signal analysis result is directly related to the trend term, it is necessary to pre-process the vibration signal to eliminate the tendency term. The Savitzky-Golay filter is a polynomial-based least-squares fitting filter method (Schafer, 2011 >), which has the advantage of following the variation of the baseline drift (Krishnan & Seelamantula, 2013 >). The specific process of adaptive vibration control method is as follows: The vibration signal is ; the function is set to be The square sum of x k with error is According to the extreme value condition of the least-squares method, the first-order partial derivative and second-order partial derivative of P with respect to a i are 0, given by According to equation (22), m + 1 coefficients a j can be established. The signal with the trend term removed is given by The input signal is decomposed by GA-VMD (VMD optimized by GA) into a series of signals with different frequency bands . As shown in Fig. 3 >, the output signal of the ith filter is given by The error signal is given by According to the Lagrangian optimality principle, the updated equation is given by where a is the leakage factor (0 <= a <= 1); the leakage factor is introduced to restrict the power of the actuator to reduce nonlinear distortion. b is the convergence factor (0 <= b <= 2). In practice, the iteration step is large when is excessively small. For double-precision floating-point inputs, c is 2.22044604925031341 x 10-16; for single-precision floating-point inputs, c is 1.192092896 x 10-7; for fixed-point input, c is 0 (Madisetti & Williams, 1999 >; Akhtar et al., 2004 >). 2.3. Numerical simulation In order to verify the effectiveness of the proposed adaptive vibration control method, numerical simulations were performed to demonstrate the accuracy of the method. The expression of the input simulation signal x(t) is where Fig. 4 > shows comparison results of the vibration control under the harmonic superimposed signals. Figs. 4 >(a) and Fig. 4 >(b) show the input simulation signal decomposition IMFs time domain and frequency domain spectra. Three-fold harmonic, five-fold harmonic, seven-fold harmonic, nine-fold harmonic and fundamental harmonic components are carefully separated from mixed harmonic signal with peaks of 150 Hz, 250 Hz, 350 Hz, 450 Hz and 50 Hz. Obviously, the GA optimized VMD algorithm has been made more simplified and robust. Fig. 4 >(c) shows the convergence results of the FxNLMS algorithm within 1 second, which decreases the amplitude by 66.85%. Meanwhile, the proposed method vibration suppression performance achieves 99.95%. 3. Case validations To further validate the performance advantages of the proposed adaptive vibration control method over the FxNLMS algorithm, in addition to the simulated mixed signals in Section 2.3, experiments on the measured vibration signals generated by ultra-precise optical instruments (DCMs at synchrotron facilities) are reported in this section. A DCM field vibration measurement diagram is shown in Fig. 5 >; the measurement sensor parameters are shown in Table 2 >. 3.1. Case 1 The actual measured signal of the DCM at Bragg@16 keV operating mode is shown in Fig. 6 >. The measured vibration signal is decomposed into different frequency bands with the variable modal decomposition algorithm optimized by the GA, as shown in Fig. 7 >. An optimization diagram of the GA is shown in Fig. 8 >. Fig. 9 > shows the vibration suppression performance of the FxNLMS algorithm and proposed method at Bragg@16 keV operating mode. Fig. 10 > shows the vibration signal's RMS values under the FxNLMS algorithm and proposed method at Bragg@16 keV. From Figs. 9 >(a) and 10 >(a), it can be seen that the proposed adaptive vibration control method has a significant advantage over the FxNLMS algorithm in terms of vibration suppression in the pitch direction at Bragg@16 keV. The FxNLMS algorithm decreased the angular displacement in the pitch direction by 16.89%, while the proposed method reduced the angular displacement in the pitch direction by 84.73%. Similarly, from Figs. 9 >(c) and 10 >(b), the FxNLMS algorithm decreased the angular displacement in the roll direction by 54.08%, while the proposed method reduced the angular displacement in the roll direction by 88.74%. From Figs. 9 >(b) and 9 >(d), in the frequency range 0-150 Hz, the proposed method shows a ~10 dB decrease in the pitch direction and ~20 dB reduction in the roll direction. Consequently, the proposed method has a satisfactory vibration suppression performance at low frequencies. On the contrary, the FxNLMS algorithm has weak vibration damping ability at low frequencies. 3.2. Case 2 The actual measured signal of the DCM at 5-30deg uniform scanning operating mode is shown in Fig. 11 >. The measured vibration signal is decomposed into different frequency bands with the variable modal decomposition algorithm optimized by GA, as shown in Fig. 12 >. The optimization diagram of the GA is shown in Fig. 13 >. Fig. 14 > shows the vibration suppression performance of the FxNLMS algorithm and proposed method at 5-30deg uniform scanning operating mode; Fig. 15 > shows the vibration signal's RMS values under the FxNLMS algorithm and proposed method at 5-30deg uniform scanning. It can be visualized from Figs. 14 >(a) and 15 >(a) that both the FxNLMS algorithm and the proposed method have vibration suppression effects. Compared with the FxNLMS algorithm, the proposed adaptive vibration control method decreases the angular displacement in the roll direction by 85.03%. From Figs. 14 >(b) and 14 >(d), in the frequency range 0-150 Hz, the proposed method shows ~40 dB decrease in the pitch direction and ~10 dB reduction shown in the roll direction. Consequently, the proposed method has a satisfactory vibration suppression performance at low frequencies. On the contrary, the FxNLMS algorithm has weak vibration damping ability at low frequencies. In particular, it can been seen from Fig. 14 >(c) that the FxNLMS algorithm suffers from control failure and vibration amplification in the pitch direction in the time range 35-50 s. Therefore, it can be seen that the stability and adaptive capability of the proposed adaptive control method has been relatively prominent. 4. Conclusion This paper presents a novel method for adaptive vibration control based on VMD and the FxNLMS algorithm for DCMs at synchrotron radiation facilities. The VMD is optimized by a GA, and then a separate controller is designed for each decomposition signal based on the FxNLMS algorithm. The results show that the proposed adaptive vibration control method is superior to the conventional FxNLMS algorithm in terms of vibration suppression and convergence rate. Moreover, the process has a significant computational weight, which can be considered in the future to optimize the filter structure. This work should be of great significance for solving the stability of DCMs in the future. In future work, the application of the proposed method in practical beamline engineering will be promoted. Figure 1 Block diagram of the FxNLMS algorithm. Figure 2 Schematic diagram of VMD parameter optimization by the GA. Figure 3 Schematic diagram of the adaptive vibration control method base on VMD and FxNLMS. Figure 4 Numerical simulation results: (a) time domain IMFs; (b) frequency domain IMFs; (c) control results. Figure 5 DCM field vibration measurement diagram. Figure 6 Actual measured signal of the DCM at Bragg@16 keV: (a) pitch direction; (b) roll direction. Figure 7 Decomposed signal and spectrum: (a) time-domain results in the pitch direction; (b) frequency-domain (0-500 Hz) results in the pitch direction; (c) time-domain results in the roll direction; (d) frequency-domain (0-500 Hz) results in the roll direction. Figure 8 Optimization diagram of the genetic algorithm: (a) pitch; (b) roll. Figure 9 Vibration suppression performance comparison results at Bragg@16 keV: (a) time-domain results in the pitch direction; (b) frequency-domain (0-500 Hz) results in the pitch direction; (c) time-domain results in the roll direction; (d) frequency-domain (0-500 Hz) results in the roll direction. Figure 10 Vibration signal RMS results at Bragg@16 keV: (a) pitch direction; (b) roll direction. Figure 11 Actual measured signal of the DCM at 5-30deg uniform scanning: (a) pitch direction; (b) roll direction. Figure 12 Decomposed signal and spectrum: (a) time-domain results in the pitch direction; (b) frequency-domain (0-500 Hz) results in the pitch direction; (c) time-domain results in the roll direction; (d) frequency-domain (0-500 Hz) results in the roll direction. Figure 13 Optimization diagram of the genetic algorithm. Figure 14 Vibration suppression performance comparison results at 5-30deg uniform scanning: (a) time-domain results in the pitch direction; (b) frequency-domain (0-500 Hz) results in the pitch direction; (c) time-domain results in the roll direction; (d) frequency-domain (0-500 Hz) results in the roll direction. Figure 15 Vibration signal RMS value results at 5-30deg uniform scanning: (a) pitch direction; (b) roll direction. Table 1 Computational expressions of the FxNLMS algorithm Interference signal x(n) Primary path P(z) Primary path response d(n) = p T (n)x(n) Secondary path S(z) Estimation of secondary path Finite impulse response filter W(z) Filter output signal y(n) = w T x(n) Anti-vibration signal y'(n) = s T (n)y(n) Secondary path filtering signal Residual signal update function (Madisetti, 2009 >) e(n) = d(n) + y'(n) = + Adaptive learning rate m(n) Table 2 Measurement sensor parameters Brand Germany / attocube Model IDS3010 Probe type IDSHI1010632 Resolution 1 nm Repeatability (vacuum state) 2 nm Bandwidth 10 MHz References Akhtar, M. T., Abe, M. & Kawamata, M. (2004). 2004 47th Midwest Symposium on Circuits and Systems (MWSCAS'04), 25-28 July 2004, Hiroshima, Japan. Baker, R., Barrett, R., Clavel, C., Dabin, Y., Eybert-Berard, L., Mairs, T., Marion, P., Mattenet, M., Zhang, L., Baboulin, D. & Guillemin, J. (2013). J. Phys. Conf. Ser. 425, 052015. Bao, W., Tu, X. & Li, F. (2020). J. Vib. Meas. 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PMC10000808 | J Synchrotron Radiat J Synchrotron Radiat J. Synchrotron Rad. Journal of Synchrotron Radiation 0909-0495 1600-5775 International Union of Crystallography 36891856 yi5128 10.1107/S1600577523000826 JSYRES S1600577523000826 Research Papers In situ wet pharmaceutical granulation captured using synchrotron radiation based dynamic micro-CT Wet pharmaceutical granulation captured by dynamic micro-CT Ding Xiao Fan a Zeinali Danalou Sima b Zhang Lifeng b* Zhu Ning abc* a Division of Biomedical Engineering, University of Saskatchewan, 57 Campus Drive, Saskatoon, SK, S7N 5A9, Canada b Department of Chemical and Biological Engineering, University of Saskatchewan, 57 Campus Drive, Saskatoon, SK, S7N 5A9, Canada c Canadian Light Source Inc., 44 Innovation Blvd, Saskatoon, SK, S7N 2V3, Canada Yabashi M. Editor RIKEN SPring-8 Center, Japan Correspondence e-mail: [email protected], [email protected] 01 3 2023 17 2 2023 17 2 2023 30 Pt 2 s230200 430439 08 4 2022 30 1 2023 (c) Xiao Fan Ding et al. 2023 2023 This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-BY) Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited. A full version of this article is available from Crystallography Journals Online. This article outlines the procedure for performing synchrotron radiation based dynamic micro-computed tomography of wet granulation of pharmaceutical powders and subsequent methods for quantitative analysis. Synchrotron radiation based dynamic micro-computed tomography (micro-CT) is a powerful technique available at synchrotron light sources for investigating evolving microstructures. Wet granulation is the most widely used method of producing pharmaceutical granules, precursors to products like capsules and tablets. Granule microstructures are known to influence product performance, so this is an area for potential application of dynamic CT. Here, lactose monohydrate (LMH) was used as a representative powder to demonstrate dynamic CT capabilities. Wet granulation of LMH has been observed to occur on the order of several seconds, which is too fast for lab-based CT scanners to capture the changing internal structures. The superior X-ray photon flux from synchrotron light sources makes sub-second data acquisition possible and well suited for analysis of the wet-granulation process. Moreover, synchrotron radiation based imaging is non-destructive, does not require altering the sample in any way, and can enhance image contrast with phase-retrieval algorithms. Dynamic CT can bring insights to wet granulation, an area of research previously only studied via 2D and/or ex situ techniques. Through efficient data-processing strategies, dynamic CT can provide quantitative analysis of how the internal microstructure of an LMH granule evolves during the earliest moments of wet granulation. Here, the results revealed granule consolidation, the evolving porosity, and the influence of aggregates on granule porosity. dynamic CT in situ micro-CT time-resolved imaging wet granulation Natural Sciences and Engineering Research Council of CanadaRGPIN-2019-06007 Canada Foundation for InnovationNational Research Council CanadaCanadian Institutes of Health ResearchUniversity of SaskatchewanThis work was supported by NSERC Discovery grants. All of the research described in this article was performed at the Canadian Light Source, a national research facility of the University of Saskatchewan, which is supported by the Canada Foundation for Innovation (CFI), the Natural Sciences and Engineering Research Council (NSERC), the National Research Council (NRC), the Canadian Institutes of Health Research (CIHR), the Government of Saskatchewan, and the University of Saskatchewan. pmc1. Introduction Synchrotron radiation micro-computed tomography (micro-CT) has developed towards sub-second data acquisition (DAQ) speeds so that temporally evolving internal structures can be investigated non-destructively (Garcia-Moreno et al., 2021 >; Marone et al., 2017 >; Mokso et al., 2011 >; Villanova et al., 2017 >). This capability, sometimes referred to as dynamic CT (Dewanckele et al., 2020 >), is built upon developments over the past 20 years towards fast tomography, in situ imaging and time-resolved imaging (Bernard et al., 2005 >; Chen-Wiegart et al., 2012 >; Eggert et al., 2014 >; Lame et al., 2003 >). An area of research that could benefit from dynamic CT is wet granulation of pharmaceutical powders. Wet granulation is the most widespread mode of granulation in capsule and tablet production. This process involves a liquid binder sprayed onto a pharmaceutical powder bed where countless droplets interact with the powders (Narang & Badawy, 2019 >; Shanmugam, 2015 >). A simplified model is used for wet-granulation research where a single liquid droplet is released onto the powders, which forms a single granule. This so-called 'single-drop impact method' is suitable for studying controlled granulation (Emady et al., 2011 >; Hapgood et al., 2003 >). The earliest stages of wet granulation are called wetting and nucleation (Iveson et al., 2001 >). The granule's internal structure that develops during wetting and nucleation can influence the final granule properties, which in turn can influence pharmaceutical product performance (Ban & Goodwin, 2017 >; Emady et al., 2011 >; Li et al., 2012 >; Poutiainen et al., 2011 >). Although internal structures are an area of interest, they are rarely investigated because the granule's opaque appearance prevents direct observation (Li et al., 2019 >). Previous wet-granulation studies have been more focused on external structures and formation mechanisms (Emady et al., 2011 >, 2013 >; Gao et al., 2018 >, 2020 >; Iveson et al., 2001 >; Li et al., 2019 >, 2021 >). There have been lab-based micro-CT studies on granule internal structures, but these have focused on the final granule product and not on in situ granulation (Crean et al., 2010 >; Davis et al., 2017 >; Farber et al., 2003 >; Matsui et al., 2019 >). The X-ray flux from lab-based scanners is not sufficient to capture in situ wet granulation (Li et al., 2019 >). Although there have been in situ studies that used 2D projections to observe the powder and liquid interactions, these cannot provide any quantitative analysis of the 3D changes inside the granule (Li et al., 2019 >, 2021 >). Dynamic CT is a time-resolved 3D imaging technique that is non-destructive and does not require altering of the sample itself. Furthermore, as a synchrotron radiation based technique, image contrast can be enhanced with phase-retrieval algorithms (Paganin et al., 2002 >). Dynamic CT can bring new insights to the in situ wet-granulation process in 3D, which has previously only been studied via 2D and/or 3D ex situ imaging techniques. A basic principle among CT techniques is to acquire 2D X-ray projections of the sample around a central axis. To avoid motion artefacts, the sample's structure is static during DAQ (Elliott & Dover, 1982 >; Iniewski, 2009 >; Romans, 2018 >). Dynamic CT keeps the conventions of acquiring projections around a central axis, but this time the sample's structure is changing. To avoid motion artefacts during dynamic CT, there is a condition that the rate of change of the sample's microstructure be sufficiently slow, i.e. within one detector pixel in the time for one scan (Maire et al., 2016 >). To successfully scan a fast-deforming sample, even faster DAQ speed is required (Mokso et al., 2010 >). A research issue in dynamic CT is that the raw data are enormous, often upwards of 100 GB. For each raw dataset, it may be possible to perform CT reconstruction from tens of thousands of time points using a so-called 'sliding-window' reconstruction (Garcia-Moreno et al., 2019 >). Processing multiple datasets can quickly become a heavy task without a strategy at hand. Therefore, three data-processing strategies are introduced in this article for dealing with dynamic CT data, specifically for wet granulation. These strategies incorporate analysis methods used in studying pharmaceutical granules to enhance sliding-window reconstruction for efficient analysis of temporally evolving granules. The objective of this article is to use dynamic CT to directly capture and quantify the changing internal structures during wet granulation. In addition, this article quantifies the loss in spatial resolution when reducing the number of projections acquired per CT (proj/CT), an often-cited compromise in dynamic CT literature (Garcia-Moreno et al., 2018 >; Marone et al., 2020 >). The experimental and data-processing procedures presented allowed for novel 3D in situ investigation of the changing internal structure, chiefly the porosity, during the earliest moments of wet granulation. The success of this article serves as a framework and guide towards future investigations with dynamic CT in pharmaceutical and chemical engineering research. 2. Materials and methods 2.1. Wet-granulation experiment The wet granulation of lactose monohydrate (LMH) as a representative pharmaceutical material was scanned using dynamic CT. The wet-granulation experiment was performed in situ during the scan by releasing a 15 ml droplet of deionized water positioned 2.5 cm above the powder bed. The droplet was released by a dispenser system which consisted of a micropipette connected to a timed syringe pump. The timed system allowed sufficient time for the researchers to safely exit the experimental hutch, start the sample rotation stage, and take control of the imaging system before the droplet would be released from the pipette. A schematic diagram of inside the experimental hutch is shown in Fig. 1 >. An additional scan raised the drop height to 15 cm because an increased drop height has been observed to change the mechanism by which the granule forms (Emady et al., 2011 >; Li et al., 2019 >). The pharmaceutical samples were loaded into cylinder containers (Fisher Scientific International Inc., USA) and mounted onto an air-bearing rotation stage (Aerotech Inc., USA). Each dynamic CT scan involved one cylinder at a time. The distance from the X-ray source to the sample was 26 m and the distance from the sample to the detector was 50 cm. The sample-to-detector distance is one of the parameters for phase-retrieval algorithms to enhance image contrast (Paganin et al., 2002 >; Wilkins et al., 1996 >). DAQ involved several manual steps. The undisturbed powder bed was first put into free rotation at a constant speed. The high-speed camera was turned on and began collecting projections in free-running mode. The camera's internal memory allowed for 20 s of recording before the earliest projections were overwritten. Once the droplet dispenser system was turned on, it was closely monitored. To ensure that the earliest data would not be overwritten, the camera was stopped at ~18 s after the droplet was released. This mode of data collection has been called 'buffer mode' (Maire et al., 2016 >). 2.2. Synchrotron dynamic CT setup Dynamic CT scans of in situ wet granulation were performed at the 05B1-1 beamline at the Canadian Light Source. The X-ray source was a white beam filtered through 0.8 mm of aluminium filter. The peak energy was around 20 keV. Indirect detection was used consisting of an AA-40 beam monitor (Hamamatsu Photonics K.K., Japan) attached to a 200 mm-thick LuAG scintillator. Images were captured by a PCO.DIMAX HS4 camera (PCO AG, Germany). The effective pixel size of the detector was 5.5 mm. Each dynamic CT raw dataset consisted of a continuous sequence of 20000 projections that were collected on the fly. The DAQ speed was 500 projections per 180deg rotation of the sample, with no delay between projections. The field of view (FOV) for each projection was 11 mm x 4.4 mm. The exposure time was 1 ms. The exposure time was an optimized choice found by trial and testing. If a shorter exposure was used, the reduced signal would result in poorer signal-to-noise ratio; whereas, if a longer exposure time was used, the temporal resolution would be slower and therefore compromise capturing in situ wet granulation. The frame rate of the high-speed camera was calculated by taking the reciprocal of the exposure time because each X-ray projection acquired is synonymous with a frame captured by the camera. The frame rate of this study comes to 1000 frames s-1. The rotation speed of the sample was calculated from the frame rate and the number of proj/CT, as demonstrated in equation (1). The rotation speed of this study was 360deg s-1, 2.3. Spatial resolution test The number of projections, N projections, for a p rotation of the sample can be calculated from the distance extending from the centre of rotation, radius (R) and the pixel size (p) (Joseph & Schulz, 1980 >; Kak & Slaney, 2001 >), For the FOV width of 11 mm used in this study, N projections 3000 by equation (2). However, only 500 projections were acquired during actual in situ experiments. The choice to reduce the number of proj/CT rotation for faster DAQ speed is an often-cited compromise (Garcia-Moreno et al., 2018 >; Marone et al., 2020 >). A bar pattern phantom (QRM GmbH, Germany) was scanned at 3000, 500 and 100 proj/CT to demonstrate the deterioration in spatial resolution with fewer projections. The spatial resolution for each scan of the phantom was calculated from the modulation transfer function (MTF) at discrete spatial frequencies (Sharma et al., 2010 >). The resolution at the 10% MTF is considered the limit of human vision (Ghani et al., 2016 >; Langner et al., 2009 >; Sun et al., 2022 >). 2.4. Dynamic CT data-processing strategies Dynamic CT data were acquired while wet granulation was performed in situ. CT reconstruction could be performed with any 500 consecutive projections because the sample was continuously rotating during DAQ. The intrinsic temporal resolution of each CT was 500 ms. The time interval between any two CTs is equivalent to the time elapsed between each CT's first projection. This mode of CT reconstruction has been called sliding-window reconstruction (Rasche et al., 1995 >; Zanette et al., 2012 >). The three data-processing strategies that were used are shown in Fig. 2 > for dynamic CT of wet granulation. Strategy A outlines examining transverse cross-sectional views, which allows for fast processing time at small time intervals for local variations in porosity at different vertical positions of the granule. Strategy B follows 3D analysis of the granule's overall porous structure. Strategy C examines longitudinal cross-sectional views to capture the vertical movement of liquid onto/through the powder bed with quantitative analysis in 3D. Time-zero was defined as the time when the droplet impacted on the powder bed and the wetting part of wet granulation began. Time-zero varied from scan to scan and needed to be manually determined from each raw dataset because DAQ was manually started and stopped. 2.5. Software used Original Python scripts were written to organize raw data according to the strategies of Fig. 2 >. CT reconstruction was performed with UFO-KIT based software (Farago et al., 2022 >; Paganin et al., 2002 >; Vogelgesang et al., 2016 >). ImageJ (Rasband, 2012 >) was used for post-processing, such as reorienting the region of interest (ROI), thresholding, and binarizing images to measure physical features of the granules. Dragonfly (Objects Research Systems Inc., Canada) was used for 3D rendering (ORS, 2021 >). MATLAB (The MathWorks Inc., USA) was used to create graphical results. 3. Results and discussion 3.1. Spatial resolution in dynamic CT It is known that the resultant granules of similar materials have pores that are 10 to 100 mm in diameter (Farber et al., 2003 >). Therefore, a spatial resolution of 10 mm was the minimum visible requirement of the imaging system. Visually, the spatial resolution at 3000 proj/CT, Fig. 3 >(a), was less noisy than the spatial resolution at 500 proj/CT, Fig. 3 >(b). In both, the 100 line-pairs per millimetre (Lp mm-1) patterns were partially resolved and the 67 Lp mm-1 patterns were fully resolved. At 100 proj/CT, shown in Fig. 3 >(c), the 100 Lp mm-1 patterns became indistinguishable from noise. Quantitatively from Fig. 3 >(d), the spatial resolutions of the imaging system at 3000, 500 and 100 proj/CT with 95% confidence were 10.2 +- 0.5, 11.1 +- 0.2 and 14.4 +- 2.4 mm, respectively. This shows that using 500 proj/CT can visualize the majority of pores. There were two reasons for reducing the number of projections from 3000 to 500. The first was that the focus of this study was the porous structure within the granule. Since it was known that most pores are larger than 10 mm, using 500 proj/CT could reliably produce satisfactory results. The shape of the granule could be identified from the characteristic gap that forms by droplet impact, which spans 30-40 mm (Li et al., 2019 >). The second reason was that the number of projections directly affects the rotation speed, i.e. a 3000 proj/CT scan must physically rotate slower than 500 proj/CT for the same exposure time. The deterioration in spatial resolution with fewer projections was demonstrated on granule data, as shown in Fig. 4 >. There is potential to use a DAQ speed of 300 or 250 proj/CT because the spatial resolution is still sufficient to distinguish and measure the pores within the granule. At 100 proj/CT is the limit where noise prevents accurate measurement of the pores, but the granule shape could still clearly be seen by the gap. At 50 proj/CT, the image has become too noisy to accurately measure the granule shape. For dynamic CT to expand into more diverse granulation experiments, the capability and potential of using fewer projections and faster DAQ speeds need to be understood. There are several factors which may influence the rate of granulation, and situations that require faster DAQ speeds are within the realm of possibilities. The results in Fig. 4 > are a step towards mature dynamic CT applications to wet granulation. 3.2. Evolving granule cross sections Strategy A followed temporally evolving transverse cross-sectional views of the granule with a time interval of 100 ms. The gap formed between the granule and the powder bed upon impact of the water droplet was used to segment the granule in each cross section, as shown in Fig. 5 >. Over the 20 s observed, the gap between the granule and the powder bed expanded as the granule contracted, so the polygon drawn in Fig. 5 >(c) to segment the granule could be reliably reused at other time points. Cross-sectional volumes at different vertical positions of a granule and associated porosities over time are plotted in Figs. 6 >(c) and 6 >(d). The cross sections were in the upper, middle and lower regions of the granule, which serve as representative conditions and overall changes of their respective regions (Gao et al., 2018 >). The volumes and porosities were calculated by first binarizing the images and summing the pixels, which constituted the granule area and the void area within the granule (pores). The porosity is the percentage of void volume to total volume. The cross-sectional volume and porosity trended in opposite directions, which reflected liquid binder dissipating throughout the granule. The greater volume at a lower position of the granule is because LMH granules become imbedded in the powder bed after wet granulation. Furthermore, because the upper layer was directly exposed to air, evaporation could have affected the faster rate of change in porosity. This suggests that there is local variation in the rate at which pores form, i.e. closer to the surface or closer to the powder bed. This variation could be explored by applying dynamic CT and strategy A to more diverse pharmaceutical materials and experimental conditions. The sliding-window reconstruction produces a smoothing effect at time intervals under the intrinsic temporal resolution due to overlapping data points (Rasche et al., 1995 >; Zanette et al., 2011 >, 2012 >). For this study, the evolution of pores as shown in Figs. 6 >(c) and 6 >(d) was linear enough, R 2 ranging from 0.90 to 0.98 (where R 2 relates to goodness of fit), that such smoothing did not influence observations. The trajectory of evolving porosity did not differ from when analysed using time intervals where the smoothing was absent. 3.3. Evolving porosity in the granule Strategy B was used for analysing the evolution of porosity and pores after the granule has consolidated. Segmenting the granule from the surrounding powders followed the procedure shown in Fig. 5 > but performed for every slice. A volume within the granule was extracted and analysed to be representative of the internal structures of the granule (Gao et al., 2018 >). 3D renderings of the granule, surrounding powders and representative cylinder are shown in Figs. 7 >(a)-7 >(c). The pores within the representative volume at four time points are shown in Figs. 7 >(d)-7 >(g). A plot of the evolving granule porosity at a 100 ms time interval is shown in Fig. 8 >. Like that carried out in Fig. 7 >, a representative volume was extracted from the granule to calculate the porosity. With 95% confidence bounds, the linear fit of evolution of porosity has an R 2 of 0.90. The goodness of fit shows confidence that the evolution in porosity in the LMH granule during the first 15 s of wet granulation is linear. The changing porosity, pore count, and total and mean volumes are listed in Table 1 >. The mean pore volume was of the order of 10-5 mm3, whereas the total pore volume was of the order of 10-1 mm3. The number of pores may decrease with time, while the total pore volume increased. This suggests that smaller pores over time have consolidated into larger pores. The porosity progressed from 2 to 9% over the observed time points. The results from Fig. 8 > and Table 1 > may not have captured the evolution of pores until the end. Since this study has revealed that the evolution of pores is possibly a linear process, time-lapse imaging could be considered as a follow up. Time-lapse imaging would not allow analysis using time intervals but it would allow for scanning longer processes. Furthermore, each dynamic CT scan was followed by ~25 minutes of readout time during which another scan cannot be taken. Given the observed linearity in the evolution of pores thus far, imaging in step-and-shoot mode or writing raw data directly to disk could be considered (Kak & Slaney, 2001 >). These imaging options would eliminate the long readout time and be suitable for granulation experiments with even slower dynamics than the LMH granulation shown in this study. Conversely, granulation studies with faster dynamics would be harder to deal with. The evolution of pores in the LMH was relatively slow compared with the DAQ speed, which satisfied the condition that the dynamics be within one detector pixel per scan. Should the evolution of pores be faster, there would be more intrusive motion artefacts. In such cases, approximation models are required to relax the one detector pixel condition (Nikitin et al., 2019 >; Ruhlandt et al., 2017 >). 3.4. Droplet-powder interactions Strategy C captured wet granulation in longitudinal cross-sectional views and whole volumes for quantitative analysis at a 100 ms time interval. The longitudinal cross sections showed vertical movement that would not be obvious in transverse cross sections. There were two experiments where this was the case. In one, the droplet descent into the powder bed was clearly visible and measured. In the other, the initial powder bed was inhomogeneous and contained large aggregates. The liquid dissipating through the aggregate was captured and the variations in granule porosity quantified. The progression of a droplet descending when the drop height was raised to 15 cm is shown in Fig. 9 >. It was observed that the rate of granulation became significantly slower when releasing a droplet from 15 cm. The average speed of a droplet descending into the powder bed was 1.27 +- 0.28 mm s-1 when the drop height was 2.5 cm. At a 15 cm drop height, the droplet descended at a speed of 0.074 mm s-1. This slower rate may be a consequence of different granulation mechanisms, which occur at different drop heights. An LMH granule forming in an inhomogeneous powder bed is shown in Fig. 10 >. The liquid travelling through an aggregate is visible from the change in grey value in the first few seconds. After the granule consolidated at around 2.5 s, two regions of differing porosity emerged. Two ROIs labelled 'a' and 'b' were extracted as representative volumes for regions outside and inside the aggregate, respectively. The evolving porosities of the two ROIs are shown in Fig. 11 >. The regions inside and outside of the aggregate not only possessed different porosities but also possessed different rates of increasing porosity. The porosity inside the aggregate, ROI 'b', was consistent over time as the microstructure was well established earlier on in the time sequence. The porosity outside of the aggregate, ROI 'a', showed a steep increase and then levelling off. This resembles a logarithmic growth curve and was fitted as such. The presence of an aggregate increased the rate at which the liquid binder dissipated in the first 16 s of wet granulation before levelling off as the main driving force behind porosity changed from granulation to evaporation. Inhomogeneous powder beds are a separate area of interest for pharmaceutical research (Liu et al., 2013 >; Oka et al., 2015 >). The results of Fig. 11 > show potential for dynamic CT to explore the influence of inhomogeneous powder beds and the presence of aggregates. The porosity curves shown in Fig. 11 >, and similarly in Figs. 8 > and 6 >, are not straight curves depicting the evolving porosity. While it would be ideal to be able to simply track how granule porosity changes over time in an unbroken line, it remains a challenge. The oscillations and variability could be the result of several factors, i.e. imaging, physical or chemical. Wet granulation is a system of loose particles disrupted by a liquid droplet. To perform dynamic CT on wet granulation in situ requires accelerating an already complex system to a fast rotation speed. It may even be possible that the granule is experiencing some motion on the submicrometre scale, considering there are no obvious motion artefacts in the data. There is visible space between the granule and the surrounding powder bed, as shown in the supporting videos. At present, dynamic CT with the imaging parameters in this article has shed light on how porosity evolves during the earliest moments of wet granulation, but it has not revealed the full story. With further enthusiasm in studying the relationship between granule microstructure and dissolution (Ansari & Stepanek, 2008 >), powder mixing (Oka et al., 2015 >), and granule drying (Li et al., 2022 >), each phenomenon may present their own post-processing challenges when investigated with dynamic CT. 4. Conclusions Dynamic CT was used to capture and quantify the evolving porosity of LMH granules. This was carried out non-destructively and without alterations to the pharmaceutical material. The enormous raw dataset from dynamic CT can be difficult to process so data-processing strategies were useful when processing dynamic CT of wet granulation. Strategy A analysed temporally evolving transverse cross sections at different regions of the granule. Strategy B followed 3D renders of the granule for quantitative analysis of granule porosity over time. Strategy C captured the droplet-powder interactions and the influence of large aggregates on granule porosity. Since the porous structure of granules is known to influence pharmaceutical product performance, the success of quantifying wet granulation of a common material like LMH is a critical first step towards investigation of more diverse pharmaceutical formulations and experimental conditions. The analytical approaches with dynamic CT presented in this article can serve as a framework to bring new insights to pharmaceutical granulation research. Supplementary Material Click here for additional data file. Supporting video sc2-upper. DOI: 10.1107/S1600577523000826/yi5128sup1.mp4 Click here for additional data file. Supporting video sc2-middle. DOI: 10.1107/S1600577523000826/yi5128sup2.mp4 Click here for additional data file. Supporting video sc2-lower. DOI: 10.1107/S1600577523000826/yi5128sup3.mp4 Click here for additional data file. Supporting video sc11-XZ. DOI: 10.1107/S1600577523000826/yi5128sup4.mp4 Click here for additional data file. Supporting video sc4-YZ. DOI: 10.1107/S1600577523000826/yi5128sup5.mp4 We acknowledge Marcus Lau and Myra Martel from the University of Saskatchewan for preparing the LMH powders. We also acknowledge Dr Sergey Gasilov for the support of dynamic CT technique development. Figure 1 The experimental setup for performing in situ wet granulation. The sample on top of the rotation stage was positioned between the X-ray source and detector and below the droplet dispenser system. Figure 2 A schematic drawing of three data-processing strategies for organizing the raw data before CT reconstruction. As an illustration, the time points are not indicative of any scan. (a) Transverse cross-sectional views to capture the temporally evolving granule at different regions of the granule (strategy A). (b) The whole volume and 3D analysis of evolving porosity inside the granule (strategy B). (c) Longitudinal cross-sectional views extracted from whole volumes to capture processes with vertical movement (strategy C). Figure 3 CTs of bar patterns with 3000, 500 and 100 proj/CT are shown in (a), (b) and (c), respectively. The bar patterns increase in spatial frequency from 33 to 200 Lp mm-1. The fit of the MTF from discrete spatial frequencies and the spatial resolution at the 10% MTF are shown in (d). Figure 4 A demonstration of deterioration in spatial resolution with fewer and fewer proj/CT on an image of a granule. The scale bar represents 1 mm. Figure 5 A flowchart beginning with the CT image to segment out the granule and pores. (a, b) Starting with the CT image, a threshold was applied to create a binarized mask. (c) A polygon was drawn using the gap between the granule and powder bed to segment the granule. (d, e) From the segmented granule, two masks, one including the pores and the other without, were made. (f) A mask of the pores alone was generated with the exclusive pixel information. Figure 6 Three equidistant cross-section volumes in the upper, middle and lower sections of an LMH granule were chosen and are shown in a 3D model, with the orthogonal view shown in (a) and the front view shown in (b). The cross-sectional volumes and porosities are plotted in (c) and (d), respectively. Supporting videos of each cross section are provided and are titled 'sc2-upper', 'sc2-middle', and 'sc2-lower'. Figure 7 Three-dimensional renders of an LMH granule after 15 s into the wet-granulation process are coloured in blue and the surrounding powder bed is coloured in grey in (a) and (b). A cylinder representation of the granule's internal structure is coloured in red and is shown in (c). Three-dimensional renders of the internal pores at four time points are shown in (d)-(g). The colour bar represents the volume of the pores in mm3. The black scale bar is 1 mm in every image. Figure 8 The evolving porosity of an LMH granule with 95% confidence bounds. The fit of the porosity data shows a linear trend that has an R 2 of 0.90. Analysed using a 100 ms time interval, the data points are separated by 100 ms. Figure 9 Longitudinal cross sections capturing the droplet descending slowly. The scale bar represents 1 mm. A supporting video of this process titled 'sc11-XZ' is provided. Figure 10 Longitudinal cross sections at various time points capturing droplet interaction with an aggregate, outlined in red, and the resulting granule with regions of differing porosity. Two ROIs are outlined in blue and labelled 'a' and 'b'. The scale bar represents 1 mm. A supporting video of this process titled 'sc4-YZ' is provided. Figure 11 The evolving porosity of ROIs 'a' and 'b' with 95% confidence bounds. Fits 'a' and 'b' are the fitted curves to ROIs 'a' and 'b', respectively. The curves show how, in the presence of an aggregate, the resulting granule can have variations in porosity. The data points are separated by 100 ms. 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PMC10000809 | J Synchrotron Radiat J Synchrotron Radiat J. Synchrotron Rad. Journal of Synchrotron Radiation 0909-0495 1600-5775 International Union of Crystallography 36891859 rv5170 10.1107/S1600577522011791 JSYRES S1600577522011791 Beamlines Assessing the prospect of XAFS experiments of metalloproteins under in vivo conditions at Indus-2 synchrotron facility, India In vivo XAFS of metalloproteins at Indus-2 Lahiri Debdutta a* Agrawal Richa b Chandravanshi Khileshwari c Rajput Parasmani c Agrawal Ankur c Dwivedi Ashutosh c Makde Ravindra D. c Jha S. N. c Garg Nandini ad a High Pressure and Synchrotron Radiation Physics, Bhabha Atomic Research Centre, Mumbai 400085, India b Department of Biochemistry and Molecular Biology, University of Chicago, 929 E 57th Street, Chicago, IL 60637, USA c Beamline Development and Application Section, Bhabha Atomic Research Centre, Mumbai 400085, India d Homi Bhabha National Institute, Training School Complex, Anushaktinagar, Mumbai 400094, India Strange R. W. Editor University of Essex, United Kingdom Correspondence e-mail: [email protected] 01 3 2023 13 1 2023 13 1 2023 30 Pt 2 s230200 449456 01 9 2022 10 12 2022 (c) Debdutta Lahiri et al. 2023 2023 This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-BY) Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited. A full version of this article is available from Crystallography Journals Online. The feasibility of X-ray absorption fine-structure (XAFS) experiments of ultra-dilute metalloprotein solutions at Indus-2 is evaluated. The feasibility of X-ray absorption fine-structure (XAFS) experiments of ultra-dilute metalloproteins under in vivo conditions (T = 300 K, pH = 7) at the BL-9 bending-magnet beamline (Indus-2) is reported, using as an example analogous synthetic Zn (0.1 mM) M1dr solution. The (Zn K-edge) XAFS of M1dr solution was measured with a four-element silicon drift detector. The first-shell fit was tested and found to be robust against statistical noise, generating reliable nearest-neighbor bond results. The results are found to be invariant between physiological and non-physiological conditions, which confirms the robust coordination chemistry of Zn with important biological implications. The scope of improving spectral quality for accommodation of higher-shell analysis is addressed. XAFS metalloproteins pmc1. Introduction Metalloproteins (MPs) represent one of the most diverse classes of proteins, with the intrinsic metal atoms providing catalytic, regulatory or structural roles critical to protein function ). They are at the heart of diverse biological processes related to disease propagation, e.g. gene regulation, protein matrix degradation, antibiotic resistance. Therefore, research of MPs understandably occupies center stage in the contemporary battle against diseases, with the aim of understanding the origin of the diseases, the functioning of drugs, drug resistance and discovery of new drugs (Cho et al., 2017 >). The intricate nature of biochemical reactions in living cells demands high specificity, which is defined by the geometrical and chemical precision of the metal-binding with amino acid residues of the protein. Thus, determination of the coordination chemistry of the metal is critical to the understanding of MP functioning. The structural aspect of this problem is solved (within 1.2 A resolution) for MP crystals at low temperature (i.e. under non-physiological conditions) by employing synchrotron-based X-ray diffraction crystallography (XRD) (Yamamoto et al., 2017 >; Petrova & Podjarny, 2004 >; Shi, 2014 >). However, the scope of crystallography is fundamentally limited due to its insensitivity to chemical state and amorphous structure. This precludes chemical speciation of metals and structural determination of MPs under real in vivo conditions (e.g. solution form at room temperature). Besides, practical problems with XRD emanate from (i) the reliance upon high-quality crystals that are difficult to fabricate and (ii) the inability to monitor the real-time chemical state of the metal that could be susceptible to synchrotron radiation (Weik et al., 2000 >; Corbett et al., 2007 >; O'Neill et al., 2002 >). These limitations of XRD necessitate alternative techniques with sensitivity to amorphous structure and chemical state, both of which are met by X-ray absorption fine structure (XAFS) (Koningsberger & Prins, 1988 >). X-ray absorption spectroscopy measures the absorption of X-rays in materials as a function of incident X-ray energy (Koningsberger & Prins, 1988 >). The atom of interest is excited by tuning the X-ray energy to its binding edge (E 0), which makes this technique element-specific. XAFS is based on interference between the ejected photoelectron and its backscattered counterpart (by neighboring atoms). Since the coherence of the electron waves underlines the interference phenomenon, XAFS information is localized within the coherence length of the electron (l 10 A). This essentially eliminates long-range-order dependence and means the technique is sensitive to amorphous structure. Near-neighbor species [Z (+-5)], coordination number [N (+-1)], radial distance [R (+-0.01 A)] and disorder [s2 (+-0.001 A2)] information are retrieved from XAFS analysis (Lahiri, 2008 >). X-ray absorption near-edge structure (XANES) is the portion of the XAFS spectrum near an absorption edge, that is sensitive to the chemical state and coordination geometry through modulations of (i) the edge energy (E 0) increases with higher oxidation state (Pantelouris et al., 1995 >), (ii) the intensity of the first post-edge peak or 'white-line' (Brown et al., 1977 >) - proportional to the oxidation state, and (iii) the pre-edge peak intensity - sensitive to the coordination-symmetry controlled pd orbital hybridization (Shishido et al., 2009 >). Thus, XAFS is collectively capable of reconstructing the metal-ligand coordination chemistry of MPs (also under in vivo conditions) that has inspired its integration into the MPs problem (Ascone et al., 2005 >; Shi et al., 2011 >; Cotelesage et al., 2012a >,b >; Strange et al., 2005 >). India is home to endemic diseases (e.g. malaria, tuberculosis, hepatitis), which involve MPs (Goldberg et al., 1990 >; Goncalves et al., 2017 >; Chim et al., 2014 >; Tellinghuisen et al., 2004 >). Research of the relevant MPs is therefore a prime scientific mandate of the Government of India. A protein crystallography beamline (Kumar et al., 2016 >) has been commissioned at the Indus-2 (2.5 GeV) synchrotron facility in India ) to this effect. Recognizing the parallel importance of XAFS for MPs, it was proposed to be initiated at the existing bending-magnet-based XAFS beamline BL-9 ). Bio-XAFS experiments are amongst the most challenging (Ortega et al., 2012 >), due to the inherent limitations of low metal concentration (mM) (Ranieri-Raggi et al., 2003 >), large disorder and susceptibility to radiation damage (Weik et al., 2000 >; Corbett et al., 2007 >; O'Neill et al., 2002 >). This mandates advanced supportive technologies, e.g. high photon flux (Fischetti et al., 2004 >; Gauthier et al., 1999 >; Cotelesage et al., 2012a >,b >; Proux et al., 2005 >; Adachi et al., 2001 >) and efficient fluorescence detectors (Cramer et al., 1988 >) for high signal statistics, fast scanning monochromators for short exposure time (Khalid et al., 2011 >) and cryo-cooling for arresting radiation damage (Ramanan et al., 2015 >). These experimental facilities are moderately satisfied at BL-9, e.g. flux 1011 photons s-1, four-element silicon VORTEX detector (Barkan et al., 2003 >), QEXAFS (Poswal et al., 2016 >) and cryo-cooling (Ramanan et al., 2015 >), which encouraged XAFS measurements of Cu protein (powder) at this beamline (Dutta Gupta et al., 2021 >). Following the first successful experiments, we undertook a realistic assessment of the scope of XAFS of MPs under in vivo conditions at BL-9, e.g. in solution form, down to ultra-dilute concentrations. This task includes actual XAFS measurement of ultra-dilute MP solution at BL-9 and evaluation of spectral quality, reliability of results, the scope of advanced analysis and technological suggestions for improvement. In this paper, we present such evaluation with the example of Zn K-edge XAFS of analogous synthetic Zn (0.1 mM) M1dr solution at room temperature (Agrawal et al., 2019 >). M1 is a protease from Deinococcus radiodurans (Uniprot ID: Q9RVZ5) - radioresistant bacterium. M1dr belongs to the M1 Zn metalloprotease family whose sequence homologs are involved in tumor growth, angiogenesis, hormone regulation, immune cell development and Huntington's disease (Kelly et al., 1997 >). Structural pre-determination for the crystal counterpart of M1dr (with XRD) (Agrawal et al., 2019 >) provided the reference for the XAFS reliability test [Figs. 1 >(a), 1 >(b)]. XRD of an M1dr crystal (T = 77 K) revealed a tetrahedral configuration of Zn, forming bonds with (N, O) bridging atoms of His322, His326 and Glu345 [Fig. 1 >(b)]. The choice of Zn protein (vis-a-vis other metals) was inspired by a few factors: (i) its ubiquitous biological importance, since Zn is the second most abundant transition metal in organisms and the only metal present in all enzyme classes (Krezel & Maret, 2016 >; Maret, 2013 >) - therefore, XAFS evaluation for any one Zn MP (e.g. M1dr) would potentially represent a wide range of MP systems based on Zn; (ii) XAFS assumes particular importance as the only probe for Zn-proteins, since Zn2+ is inaccessible to other spectroscopic techniques due to its filled 3d level (Penner-Hahn, 2005 >); (iii) from a practical perspective, Zn-protein serves as a good benchmark for XAFS feasibility tests because of prior extensive XAFS investigations and structural cataloging (Bobyr et al., 2012 >; Dent et al., 1990 >; Feiters et al., 2003 >; Murphy et al., 1997 >; Tierney & Schenk, 2014 >; Kleifield et al., 2001 >; Amiss & Gurman, 1999 >; Meyer-Klaucke et al., 1999a >; Giachini et al., 2007 >, 2010 >; Clark-Baldwin et al., 1998 >; Christianson, 1991 >; Pace & Weerapana, 2014 >; Laitaoja et al., 2013 >); (iv) K-edge XANES is a good marker for XANES calibration due to the fixed and stable (against radiation) Zn2+ state (Giachini et al., 2010 >; Penner-Hahn, 2005 >; Al-Ebraheem et al., 2010 >; Castorina et al., 2019 >). Our experiments were statistically challenged by one-order-of-magnitude lower metal concentration and the unavailability of a standard multi-element germanium detector (Bobyr et al., 2012 >; Dent et al., 1990 >; Feiters et al., 2003 >; Murphy et al., 1997 >; Tierney & Schenk, 2014 >; Kleifield et al., 2001 >; Amiss & Gurman, 1999 >; Meyer-Klaucke et al., 1999a >,b >; Giachini et al., 2007 >, 2010 >; Clark-Baldwin et al., 1998 >; Christianson, 1991 >; Pace & Weerapana, 2014 >; Laitaoja et al., 2013 >). Nonetheless, we undertook this challenge with the understanding that a feasibility test under the worst experimental conditions warrants foolproof credibility. We met the challenge with strategies such as (a) a large sample area by injecting solution inside an (X-ray transparent) Kapton bag ); (b) XAFS measurement in fluorescence mode with a four-element silicon VORTEX detector (equipped with fast electronics) (Barkan et al., 2003 >); (c) shielding of the detector from stray photons; (d) minimization of the sample-detector distance, and (e) iterative data collection on fresh solutions, to guard against radiation damage. No radiation damage was shown between successive scans, as the Zn2+ state remained stable between XANES scans. Our strategies generated reproducible Zn K-edge XAFS spectra up to DE = +400 eV past the edge (k = 11 A-1). Although spectral range and quality are statistically compromised (as speculated), the Fourier transform of the XAFS spectra over k = 2.5-10 A-1 generated a reproducible first-shell peak over R = 0.8-2 A and permitted reliable first-shell analysis. XAFS analysis reproduced coordination and bond-length (and distribution) results of XRD (Agrawal et al., 2019 >), within intrinsic analytic uncertainty. Negligible evolution of the coordination chemistry of M1dr between low-temperature crystal (T = 77 K, pH 5.5) (Agrawal et al., 2019 >) and in vivo (T = 300 K, pH 7) conditions demonstrates robustness of Zn--(O/N) bonds. This robustness resembles the behavior of four-domain proteins of the M1 family and accounts for efficient substrate binding in the absence of the C-domain (Agrawal et al., 2019 >). Thus, novel perspectives of M1dr are unraveled by this experiment. Our success confirms the feasibility of XAFS of MPs solution at Indus-2 BL-9, down to ultra-dilute concentrations. Since the beamline is capable of delivering X-rays in the energy range 5-20 keV, a plethora of metals (Z = 23-42; >=53) can be probed, covering a wide range of MPs (and organometallics in general). XANES and first-shell results can be reliably obtained for these MPs that would provide information on the chemical state of the metal, identity of ligand groups, (N/O):S coordination ratio and geometric distortion (Bobyr et al., 2012 >; Dent et al., 1990 >; Feiters et al., 2003 >; Amiss & Gurman, 1999 >; Meyer-Klaucke et al., 1999a >,b >; Giachini et al., 2010 >; Clark-Baldwin et al., 1998 >; Christianson, 1991 >; Pace & Weerapana, 2014 >; Laitaoja et al., 2013 >; Smolentsev et al., 2005 >; Longa et al., 1999 >; Vlasenko et al., 1999 >; Sagi et al., 1999 >; Katsikini et al., 2009 >; Bertoncini et al., 1999 >). This information can adequately address diverse biological problems such as disease-marking, binding properties, protein aggregation, multi-site heterogeneity, mutation and cellular catalysis (Smolentsev et al., 2005 >; Longa et al., 1999 >; Vlasenko et al., 1999 >; Sagi et al., 1999 >; Katsikini et al., 2009 >; Meyer-Klaucke et al., 1999a >; Bertoncini et al., 1999 >). The prospect of improving the spectral quality of XAFS, to accommodate higher-shell-based novel scientific problems (Giachini et al., 2007 >; Kleifield et al., 2001 >; Murphy et al., 1997 >; Tierney & Schenk, 2014 >), is addressed. This work should inspire in vivo XAFS experiments of MPs at beamlines with modest facilities like ours. 2. Experimental details 2.1. Sample preparation M1dr protein was expressed in Rosetta(DE3)pLysS E.coli expression host and purified from cell lysate through Ni-NTA chromatography using 50 mM phosphate buffer pH 7 (Agrawal et al., 2019 >). Purified protein was stored at -80degC with 20% glycerol v/v (Fig. 2 >). This was mixed with 0.1 mM ZnCl2 externally and spun at 12000 rpm for 10 min, prior to XAFS experiment. The solution was injected into a large Kapton bag and sealed for XAFS measurement. 2.2. Experimental setup for XAFS A schematic layout and photograph of BL-9 are depicted in Figs. 3 >(a) and 3 >(b). The beamline is designed to deliver monochromatic X-rays of energy ~5-20 keV and flux ~1011 photons s-1 at the sample position. A Si(111) double-crystal monochromator, consisting of a water-cooled first crystal and horizontally focusing second crystal, was aligned for monochromatic X-rays around the Zn K-edge (9.659 keV). Higher harmonics were rejected and the beam vertically collimated by a Rh-coated meridional cylindrical pre-mirror. The final spot size at the sample position was approximately 1 mm (H) x 0.2 mm (V). For reference, XAFS for Zn foil and ZnO powder were measured in transmission with gas-filled ion chambers. Mixtures of helium/nitrogen and nitrogen/argon gases were respectively filled in incident and transmission ion chambers. XAFS for Zn foil was used for energy calibration of the monochromator. XAFS of M1dr solution (inside the Kapton bag) was measured in fluorescence mode, due to the dilute metal content ). A gas-filled ion chamber and silicon drift detector (SDD) were employed for monitoring the intensities of the incident and (Zn ) fluorescence photons, respectively. The SDD was mounted on a (remote-controlled) motorized xyz stage and adequately shielded against stray photons. 2.3. Fluorescence detector The choice of SDD played a key role in the improvement of the XAFS data quality. A single-element SDD (active area = 50 mm x 50 mm, collimated area = 30 mm x 30 mm) was initially employed but its statistical inefficiency due to low input count-rate (ICR 106) and high dead-time generated poor signal. This problem was overcome with the installation of an efficiently designed four-element SDD, that was geometrically and electronically adapted for high-quality signal ). Four sensors of the SDD (each active area = 40 mm x 40 mm, collimated area = 30 mm x 30 mm) are located on the surface of a (virtual) sphere, centered at the sample. This geometry generates equal solid angles for the four sensors, so that they are uniformly illuminated and the total solid angle of the detector is increased fourfold. This leads to a fourfold increase of the fluorescence photon collection efficiency. A digital pulse processor of the single-element SDD was replaced by a four-channel Xspress-3 readout with high ICR (= 3.5 x 106 counts s-1) and 20% dead-time ), which enabled handling of 12-14 times higher photon flux. These upgrades jointly promoted the efficient utilization of beam flux. The readout has been integrated with a data acquisition system and GUI developed to automatically configure detector parameters (e.g. acquisition time, calibration factor) through an EPICS-LabVIEW interface. 2.4. Data collection XAFS spectra were acquired in steps of (i) 10 eV (1 s) over the pre-edge, (ii) 0.5 eV (1 s) over the XANES and (iii) 0.05 A-1 (15 s) over the EXAFS regions (Kane et al., 2014 >). Iterations were limited to (x3) scans due to time constraints. [Several diagnostic tests were exercised to pre-determine the optimal experimental setup. These included evaluation of data quality for various concentrations of Zn samples, sample holders (Kapton bag vis-a-vis cuvet) and detectors (single vis-a-vis four-element SSD).] 3. Results and discussions 3.1. XANES Zn K-edge XAFS data m(E) were processed using ATHENA software (Ravel & Newville, 2005 >). Datasets for M1dr solution were reproducible, within statistical fluctuations. The signal-to-noise ratio could be ideally improved with 10-15 scans. However, the number of iterations was limited to x3 in our case, due to time constraints. The average of the x3 datasets was smoothened by the interpolative smoothing algorithm of ATHENA with three iterations. Fig. 4 > displays normalized Zn K-edge XANES spectra for standards [Zn foil (Zn0), ZnO powder (Zn2+)] and M1dr solution. An overplot of the smoothed and original dataset for M1dr (Fig. 4 >) rules out the scope of data distortion, as far as XANES and first-shell EXAFS analysis are concerned. Henceforth, the smoothened dataset was used for analysis. XANES data were analyzed for (i) edge energy (E 0) and (ii) white-line intensity. (i) The edge energy (E 0) was defined at the half-point of the rising edge of the absorption curve. XANES spectra of the standards demonstrate a positive shift of E 0: 9659 eV (Zn) - 9659.9 eV (ZnO), consistent with increasing oxidation state. For M1dr, E 0 = 9659.9 eV coincides with E 0 for ZnO. [In principle, E 0 can also be defined at the point of inflection of XANES derivative spectra. In this work, normalized XANES spectra (rather than derivative) are presented to enable calibration of Zn ligand coordination with white-line intensity (Penner-Hahn, 2005 >; Al-Ebraheem et al., 2010 >; Castorina et al., 2019 >).] (ii) White-line (WL) features (A, B) represent the probability of 1s - 4p electronic transitions (Koningsberger & Prins, 1988 >). The WL is significantly pronounced from Zn to ZnO, consistent with the increased availability of empty p states due to lower electron content. XANES features beyond A, B are distinct between Zn and ZnO. XANES peaks (A, B, C, D) for M1dr resemble the peaks of ZnO with respect to the centroid and relative intensity (except for overall broadening due to disorder). Thus, both E 0 and WL jointly confirm the Zn2+ oxidation state for M1dr. Besides the oxidation state, the WLs for Zn MPs are reportedly sensitive to ligand coordination (N) via the density of states (Penner-Hahn, 2005 >; Al-Ebraheem et al., 2010 >; Castorina et al., 2019 >). Standardized correlation between WL and N sets the criterion: WL < 1.5 = N = 4. By this criterion, WL = 1.35 for M1dr (magnified in the inset of Fig. 4 >) unambiguously confirms N = 4, consistent with the XRD model (Agrawal et al., 2019 >). We remark that the conventional pre-edge peak for tetrahedral geometry (corresponding to the s - d transition) is absent in Zn K-edge XANES, since the s - d transition is forbidden for Zn2+ due to the fully occupied d-shell of Zn2+. 3.2. EXAFS Normalized XAFS data of Fig. 4 > were background-subtracted to derive the XAFS oscillations kh(k) shown in Fig. 5 >(a). kh(k) for M1dr solution decays fast, consistent with the presence of large disorder and the absence of high-Z backscattering neighbors. Raw k 3kh(k) for M1dr are presented in the inset of Fig. 5 >(a), for comparison of the spectral quality with reported data of Zn proteins (Meyer-Klaucke et al., 1999a >; Dent et al., 1990 >; Giachini et al., 2007 >, 2010 >; Murphy et al., 1997 >; Shi et al., 2011 >; Bobyr et al., 2012 >; Feiters et al., 2003 >; Clark-Baldwin et al., 1998 >; Amiss & Gurman, 1999 >). Raw k 3kh(k) for M1dr are dominated by noise beyond k = 8 A-1 whereas the reported spectra retain good quality up to k 11 A-1. This disparity may be attributed to the relative efficiencies of the four-element SDD (vis-a-vis the multi-element germanium detectors employed in the reported experiments). Fourier transforms |kh(R)| of raw and smoothed data for M1dr (over the transformation range k = 2.5-10 -1) are presented in Fig. 5 >(b). They are similar over R = 0.8-2 A, confirming that the first shell is negligibly contaminated by noise. This is consistent with the fact that XAFS oscillations of low-Z neighbors decay fast with increasing k. Thus, the first shell for M1dr may be concluded to be reasonably robust against noise. In contrast, higher-shell features of raw and smoothed |kh(R)| are rendered irreproducible by noise. It is impractical to attempt quantitative fitting of the higher shell for such a (noisy) dataset. Therefore, analysis was henceforth focused on the first-shell fit of (smoothed) kh(k), using the FEFFIT program (Ravel & Newville, 2005 >). The reference first-shell structure for M1dr in Fig. 1 >(b) is derived from XRD (Agrawal et al., 2019 >): Zn-O (x2), R = 1.9 A; Zn-N (x1), R = 2.0 A; Zn-N (x1), R = 2.1 A. The reliability of the XAFS fit results will be ultimately tested against this distribution. XAFS fitting of this distribution presents two complications, described in the following paragraphs. (i) Intrinsic deviation of XAFS results from geometric distribution. The geometric equivalent of the above distribution is N = 4 with mean bond-length R = 1.975 A and bond-length distribution s2 = 0.009 A2. In principle, XAFS is expected to reproduce these values. However, these values may not be reproduced in reality, since XAFS is essentially an interference phenomenon. Scattering contributions (kh i ) for closely spaced bond-lengths (as in the above distribution) can be slightly out of phase and partially cancel each other, so that the net spectra khtot is of lower amplitude and phase-shifted. This represents lower effective coordination and/or shifted mean bond-length (Lahiri et al., 2014 >), relative to the geometric distribution. This defines the intrinsic uncertainty of XAFS results, which has to be taken into account for meaningful comparison of XAFS and XRD results. We theoretically pre-estimated this mismatch for the atomic distribution of M1dr via (a) simulation of kh i for the crystallographic distribution, by exercising the 'NOFIT' handle of the FEFFIT program; (b) generation of a synthetic dataset khtot and (c) fitting of khtot with N ZnO, R ZnO and variables (assuming that O, N have similar backscattering factors). Fit results (R ZnO = 1.94 A, = 0.007 A2) deviated slightly from the geometric equivalent (R ZnO = 1.975 A, = 0.009 A2). The deviations (|DR| = 0.035 A, |Ds2| = 0.002 A2) thus define the intrinsic uncertainty of XAFS results for M1dr. (ii) Degeneracy of models, e.g. single-neighbor type (O/N) vis-a-vis both neighbor types (O + N). This problem arises due to similar backscattering factors of O and N. In principle, the degeneracy could be resolved by exploiting large bond-length differences (e.g. Zn--O < Zn--N). For small bond-length differences (like for M1dr), first-shell fitting (by itself) is unable to resolve the degeneracy (Giachini et al., 2007 >; Dent et al., 1990 >; Clark-Baldwin et al., 1998 >). {The degeneracy can be reduced with higher-shell XAFS analysis. For example, Zn--O and Zn--N bonds could form distinct angles with second-shell atoms: Zn--O--O and Zn--N--O. Such angular disparity can be exploited to resolve the degeneracy, e.g. through determination of angles with multiple-scattering-based XAFS fitting (Haskel, 1998 >). In our case, the scope of such analysis is precluded by the domination of noise at higher k [see inset of Fig. 5 >(a)].} We proceeded with first-shell XAFS analysis of M1dr with the following understandings. The fitting was designed for a single ZnO path of coordination N, mean bond-length R and spread s2. XAFS for the first shell of M1dr [kh(q), Fig. 5 >(c)] was filtered out from the whole spectrum of Fig. 5 >(a) by back-transforming kh(R) over R = 0.8-2 A. The presence of beats in kh(q) indeed confirms the presence of closely spaced multiple bond-lengths, consistent with the crystallographic model of M1dr. A phase derivative method (Piamonteze et al., 2005 >) was employed to obtain an independent estimate of the bond-length split (D) from the phase ph(q) of the XAFS [inset of Fig. 5 >(c)]. The inflection position k b (~11.5 A-1) of ph(q) is related to D (= p/2k b); k b 11.5 A-1 = D 0.13 A, which is close to the crystallographic standard deviation of bond lengths. We remark that, as the transform range of kh(k) (k = 2.5-10 A-1) bypasses k b, the spatial resolution is reduced to the extent that split ZnO peaks become indistinguishable in kh(R) of Fig. 5 >(b). This warranted first-shell fitting with a single ZnO path. A (smoothened) XAFS dataset for M1dr was fit over k = 2.5-10 A-1, R = 0.8-2 A. The strategy of a simultaneous fit for k w=0-2-weighted transforms was adopted, in order to decouple correlations between variables [(N, s2), (R, DE 0)] and minimize uncertainties in fit results [DE 0 = energy correction, relative to edge position (E 0)]. The contribution of the background was corrected by exercising the 'bkg' option of the FEFFIT program. = 0.87 was pre-determined by fitting XAFS for reference Zn foil with the constraint N ZnZn = 12 (Kelly et al., 2009 >). Preliminary (N, R, s2) fit results were refined by constraining N = 4 (consistent with XRD), leading to the best-fit results: R = 1.953 (1) A, s2 = 0.0093 (1) A2; R-factor = 0.001. [A comparison of experimental and fit spectra is shown in Fig. 5 >(b).] Thus, XAFS reproduced the crystallographic results (R ZnO = 1.975 A, = 0.009 A2) within (pre-determined) intrinsic uncertainties (DR = +-0.035 A, Ds2 = +-0.002 A2). Since the crystallographic results were obtained at T = 77 K (Agrawal et al., 2019 >), this implies that the coordination chemistry of Zn in M1dr is robust and varies negligibly from T = 77 K to 300 K. The role of thermal disorder is concluded to be minimal. This observation unravels a novel perspective of M1dr. M1dr is a Zn metallopeptidase of the M1 Merops family. It is unique in the sense that it is the only two-domain protein amongst the four-domain M1 family peptidases characterized so far (Agrawal et al., 2019 >). The reported high-resolution XRD structure for this protein corresponded to non-physiological conditions: 0.2-0.25 M ammonium formate, 0.1 M bis-tris and 20-27% polyethylene glycol 3350 at pH 5.5 (Agrawal et al., 2019 >). In contrast, XAFS of M1dr was measured for pH 7.0, i.e. under physiological conditions. The similarity of bond lengths and coordination between XRD and XAFS essentially represents invariance of the Zn coordination chemistry between the two pH conditions. This characteristic is identical to the four-domain proteins of the M1 family. (Zn coordination remains invariant for proteins of different compositions in the M1 peptidase family.) We can therefore conclude a resemblance of M1dr with the four-domain proteins. Strong coordination chemistry may be responsible for the (observed) efficient substrate-binding for M1dr in the absence of the C-domain. 3.3. Scope of improvement XANES and first-shell EXAFS analysis of an ultra-dilute MP solution at beamline BL-9 has been tested to be reliable and feasible in this work. First-shell EXAFS provides information on the metal-ligand unit within a radius R 2 A (e.g. ligand identity, molecular composition and configuration), with implications for disease-marking, binding properties, protein aggregation, multi-site heterogeneity, mutation and cellular catalysis (Smolentsev et al., 2005 >; Longa et al., 1999 >; Vlasenko et al., 1999 >; Sagi et al., 1999 >; Katsikini et al., 2009 >; Meyer-Klaucke et al., 1999b >; Bertoncini et al., 1999 >). However, interesting science exists beyond the metal-ligand unit, i.e. at higher shells (HS) (R > 2 A). For example, electron-spin transport for regulation of chemical reactions and switching behavior is determined by the inter-unit coupling geometry (Giachini et al., 2007 >; Kleifield et al., 2001 >; Murphy et al., 1997 >; Tierney & Schenk, 2014 >). Future XAFS experiments of MPs at BL-9 will be designed for the accommodation of such advanced problems. Since HS structural information is contained in the high-frequency component of kh(k), it is ultra-sensitive to noise. Success of HS analysis of MPs would therefore mandate high signal statistics. Since our diagnostic tests demonstrated a significant improvement of the signal between four-element SDDs, we conclude that the incident photon flux is sufficient and the statistical problem is related to detection inefficiency. Therefore, employment of a highly efficient multi-element germanium detector at BL-9 can be expected to generate the required statistics for HS analysis. We plan to incorporate micro-focusing and a multi-element germanium detector in the next phase of beamline upgradation. 4. Conclusion We have successfully measured Zn K-edge XAFS of analogous synthetic Zn (0.1 mM) M1dr solution under in vivo conditions at bending-magnet-based beamline BL-9 of Indus-2. Despite a one order-of-magnitude lower metal concentration and the unavailability of a multi-element germanium detector (used in standard XAFS experiments of MPs), we obtained a sufficiently fair spectral quality for reliable first-shell analysis, with strategies such as large sample area, four-element SDD and fast electronics. XAFS results reproduced the Zn+2(O/N)4 coordination chemistry of the M1dr crystal at T = 77 K. This confirmed the feasibility of XAFS of ultra-dilute metalloprotein solutions at BL-9 with the present facilities. Deployment of a standard multi-element Ge detector in the future would significantly enhance the capabilities of this beamline and extend the scope of such work. Figure 1 (a) Crystal structure of M1dr at 77 K. (b) Zn-binding sub-unit of M1dr. Figure 2 SDS gel image of M1dr Zn-NTA purification. M = protein marker (numbers shown in kDa), (U)I = (Un)introduced, W = cell lysate, S = supernatant after spin, P = pellet after spin, F = flow through after Zn binding, B = bound M1 protein. Figure 3 (a) Schematic outline (adapted from the RRCAT website) and (b) photograph of the XAFS beamline BL-9 at Indus-2. Figure 4 Normalized Zn K-edge XANES spectra for Zn foil and ZnO powder standards and for M1dr solution. Datasets are shifted relative to each other for clarity. Raw (black dotted) and smoothened (pink solid) datasets for M1dr are overplotted. Inset: magnified image of the white line for M1dr. White-line intensity = 1.35 suggests N = 4 coordination of Zn. Figure 5 (a) Zn K-edge XAFS oscillations kh(k) for Zn foil and ZnO powder standards and for M1dr solution at room temperature, laterally shifted relative to each other for clarity. Raw (black dotted) and smoothened (pink solid) datasets for M1dr are overplotted. Inset: k 3kh(k) for M1dr highlights noise over the higher k-region. 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PMC10000810 | J Synchrotron Radiat J Synchrotron Radiat J. Synchrotron Rad. Journal of Synchrotron Radiation 0909-0495 1600-5775 International Union of Crystallography 36891855 tv5040 10.1107/S1600577523000772 JSYRES S1600577523000772 Research Papers Low-density tissue scaffold imaging by synchrotron radiation propagation-based imaging computed tomography with helical acquisition mode Low-density tissue scaffolds imaging by SR-PBI-HCT Duan Xiaoman a Li Naitao a Cooper David M. L. b Ding Xiao Fan a Chen Xiongbiao ac* Zhu Ning ade* a Division of Biomedical Engineering, College of Engineering, University of Saskatchewan, Saskatoon, SK S7N 5A9, Canada b Department of Anatomy, Physiology and Pharmacology, College of Medicine, University of Saskatchewan, Saskatoon, SK S7N 5A9, Canada c Department of Mechanical Engineering, College of Engineering, University of Saskatchewan, Saskatoon, SK S7N 5A9, Canada d Department of Chemical and Biological Engineering, College of Engineering, University of Saskatchewan, Saskatoon, SK S7N 5A9, Canada e Canadian Light Source, Saskatoon, SK S7N 2V3, Canada Stevenson A. Editor Australian Synchrotron, Australia Correspondence e-mail: [email protected], [email protected] 01 3 2023 16 2 2023 16 2 2023 30 Pt 2 s230200 417429 28 4 2022 29 1 2023 (c) Xiaoman Duan et al. 2023 2023 This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-BY) Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited. A full version of this article is available from Crystallography Journals Online. This work shows that combining helical acquisition mode with synchrotron radiation propagation-based imaging computed tomography is a powerful tool for tissue engineering applications with image qualities of high contrast, low noise level and, most importantly, fewer ring artifacts. Visualization of low-density tissue scaffolds made from hydrogels is important yet challenging in tissue engineering and regenerative medicine (TERM). For this, synchrotron radiation propagation-based imaging computed tomography (SR-PBI-CT) has great potential, but is limited due to the ring artifacts commonly observed in SR-PBI-CT images. To address this issue, this study focuses on the integration of SR-PBI-CT and helical acquisition mode (i.e. SR-PBI-HCT) to visualize hydrogel scaffolds. The influence of key imaging parameters on the image quality of hydrogel scaffolds was investigated, including the helical pitch (p), photon energy (E) and the number of acquisition projections per rotation/revolution (N p), and, on this basis, those parameters were optimized to improve image quality and to reduce noise level and artifacts. The results illustrate that SR-PBI-HCT imaging shows impressive advantages in avoiding ring artifacts with p = 1.5, E = 30 keV and N p = 500 for the visualization of hydrogel scaffolds in vitro. Furthermore, the results also demonstrate that hydrogel scaffolds can be visualized using SR-PBI-HCT with good contrast while at a low radiation dose, i.e. 342 mGy (voxel size of 26 mm, suitable for in vivo imaging). This paper presents a systematic study on hydrogel scaffold imaging using SR-PBI-HCT and the results reveal that SR-PBI-HCT is a powerful tool for visualizing and characterizing low-density scaffolds with a high image quality in vitro. This work represents a significant advance toward the non-invasive in vivo visualization and characterization of hydrogel scaffolds at a suitable radiation dose. tissue hydrogel scaffolds propagation-based imaging helical acquisition mode ring artifact removal Natural Sciences and Engineering Research Council of CanadaRGPIN 06007-2019 RGPIN 06396-2019 The following funding is acknowledged: Natural Sciences and Engineering Research Council of Canada (grant nos. RGPIN 06007-2019; RGPIN 06396-2019). Experimental research described in this paper was performed at the Canadian Light Source, which is a national research facility supported by the Canada Foundation for Innovation (CFI), NSERC, National Research Council (NRC), Canadian Institutes of Health Research (CIHR), Government of Saskatchewan and the University of Saskatchewan. pmc1. Introduction In scaffold-based tissue engineering and regenerative medicine (TERM), scaffolds made from biomaterials are used to support and facilitate cell growth and tissue regeneration, as well as transport nutrients and metabolic wastes (Chen, 2019 >). For this, scaffolds should possess appropriate architectural, mechanical and biological properties to mimic those of native tissues or organs. As such, visualization and/or characterization of scaffold properties via in vitro and/or in vivo studies are essential to TERM applications (Duan et al., 2021 >). In scaffold-based TERM, hydrogels are the most commonly used biomaterials for scaffolds with water-swollen crosslinked polymeric networks (Nezhad-Mokhtari et al., 2019 >). Typically, hydrogels have similar density to the surrounding environment (e.g. water for in vitro study or soft tissue after implantation for in vivo study). To visualize and characterize hydrogel scaffolds in TERM, synchrotron radiation computed tomography (SR-CT) imaging holds promise for both in vitro and in vivo applications (Duan et al., 2021 >; Zhu et al., 2011 >; Ning et al., 2018 >, 2021 >; Olubamiji et al., 2014 >; Naghieh & Chen, 2021 >; Izadifar, Babyn, Kelly et al., 2017 >; Izadifar, Babyn, Chapman et al., 2017 >; Olubamiji et al., 2016 >, 2017 >; You et al., 2016 >; Bawolin & Chen, 2016 >). For in vitro scaffold visualization, SR-CT is non-destructive/non-invasive imaging which means scaffolds can be imaged without the need for sectioning and other processes. As such, it allows accurate capture of scaffold structures in longitudinal studies of the same scaffolds. It is noted that, among various in vitro imaging techniques, scanning electron microscopy (SEM) (Bartos et al., 2018 >; Stachewicz et al., 2019 >; Vitas et al., 2019 >) has been commonly used for morphology and pore/surface structure of the scaffolds with the wide magnification range 10-500 000x (Zhu et al., 2021 >), but the approach involves destructive sample preparation. Such destructive processes may change the structure (e.g. pore size) of scaffolds, leading to inaccurate results and findings. Confocal laser scanning microscopy (CLSM) (Bagherzadeh et al., 2013 >; Phipps et al., 2012 >) is another commonly used technique for tissue scaffold visualization, which enables collection of data in 3D but is limited to the penetration depth range 200-300 mm (Parrilli et al., 2014 >). CLSM has been commonly used for quantitative biological analysis and can provide complementary information on tissue scaffolds to SR-CT imaging (morphology). For in vivo scaffold visualization, SR-CT has the merits of high spatial resolution and high contrast, as well as a relatively fast scan speed. Magnetic resonance imaging (MRI) (Mueller et al., 2021 >; Chen et al., 2020 >; Kotecha et al., 2017 >), ultrasound imaging (UI), photoacoustic (PA) imaging (Teodori et al., 2017 >) and optical coherence tomography (OCT) (Chen et al., 2011 >; Wang et al., 2018 >) can visualize scaffolds non-destructively and non-invasively. However, MRI requires a long scan time to achieve a high spatial resolution [e.g. about 100 h for 100 mm (Edlow et al., 2019 >)], whereas SR-CT needs only seconds to minutes to achieve a much higher resolution [e.g. lower than 10 mm at dose rates suitable for in vivo applications (Harrison et al., 2022 >)]. Hydrogel imaging with a long-time scan may change some of the properties of a hydrogel, leading to motion artifacts. High-resolution imaging (<100 mm) of UI/PA requires high-frequency scanning, which may heat up scanned scaffolds. Besides, for in vivo UI, the presence of bone can greatly limit the resolution. OCT can achieve a high resolution, but its penetration depth is a shortcoming compared with SR-CT, particularly for full scaffold imaging in vivo in animal models (e.g. rats). Compared with conventional absorption contrast CT imaging using a conventional (polychromatic) X-ray tube as a source, monochromatic SR-CT can obtain both absorption contrast and phase contrast. The latter can achieve a higher contrast for low-density scaffolds, especially when combined with phase retrieval (PhR). It is noted that SR-CT is a broad class of techniques that includes interferometer CT (Miao et al., 2016 >), edge-illumination CT (Hagen et al., 2014 >; Momose et al., 2003 >), diffraction-enhanced imaging CT [SR-DEI-CT (Chapman et al., 1997 >)], analyzer-based imaging [SR-ABI-CT (Wernick et al., 2003 >)] and propagation-based imaging CT [SR-PBI-CT (Suzuki et al., 2002 >)]. SR-PBI-CT has the great advantage of simple implementation and fast acquisition compared with SR-DEI-CT/SR-ABI-CT (Izadifar et al., 2016 >). As such, it has great potential for clinical studies (Fedon et al., 2018 >; Longo et al., 2014 >; Castelli et al., 2011 >) and in vivo animal imaging (Taba et al., 2018 >). Therefore, this work involved the application of SR-PBI-CT to hydrogel scaffold imaging. Despite many advantages, SR-PBI-CT image quality for low-density scaffolds still requires improvement. Ring artifacts, for example, are an issue for SR-PBI-CT (Pelt & Parkinson, 2018 >) due to systematic errors or defects on the scintillator, monochromator or filters. Such artifacts usually make it difficult to process and analyze images using existing methods for visualizing and charactering samples. This is a particular concern for imaging low-density scaffolds due to their relatively low contrast. Methods have been developed to reduce ring-like artifacts, including pre-processing and post-processing algorithms, but these approaches suffer from various limitations in practical application. For example, low-pass Fourier filtering (Raven, 1998 >) poses the risk of introducing additional artifacts in the reconstructed background. The Sarepy sorting wind method (Vo et al., 2018 >) usually has limited ring artifact removal efficacy for noisy images and for images with unresponsive stripes which result from dead pixels on the detector and/or damaged areas of the scintillator. In addition, these algorithms involve manual hyperparameter selection (e.g. the size of convolution kernel or window size which are defined by the user). Different values will produce different outcomes and it usually takes a long time to determine the optimal value. Besides, for different regions of interest (ROIs), the hyperparameter values need to be finely tuned for optimal artifact removal and therefore subjectivity is a limitation. These limitations raise a great need to address the issue of ring artifacts if SR-PBI-CT is to realize its larger potential for imaging of low-density targets, including hydrogel scaffolds. This paper explores the integration of SR-PBI-CT with helical acquisition mode (hereafter SR-PBI-HCT) to address the ring artifact issues which negatively impact hydrogel scaffold visualization. Helical acquisition mode has been widely used for clinical CT, but is still relatively novel when combined with SR-PBI-CT. To the best of our knowledge, the SR-PBI-HCT technique, though reported previously (Pelt & Parkinson, 2018 >), has not yet been used in the visualization and characterization of scaffolds for tissue engineering applications. To optimize image quality of hydrogel scaffolds with SR-PBI-HCT, we studied the impact of helical pitch (p, definition given in Section 2.2), X-ray photon energy (E) and the number of acquisition projections per revolution (360deg) (N p). Although some of these have been discussed previously (Oliva et al., 2020 >; Taba et al., 2019 >; Nesterets et al., 2018 >) for regular SR-PBI-CT imaging, optimal parameters for SR-PBI-HCT remain unclear. Furthermore, we evaluated the image quality of hydrogel scaffolds using SR-PBI-HCT at a low-level radiation dose (i.e. 342 mGy, which is acceptable for in vivo imaging). Our present study, though carried out in vitro, is thus intended to serve as a step towards 3D non-invasive in vivo characterization for TERM applications using hydrogel scaffolds. 2. Materials and methods 2.1. Scaffold preparation In this work, 4% w/v aqueous alginate made from medium-viscosity alginate powder (alginic acid sodium salt from brown algae, CAS 9005-38-3, Sigma-Aldrich) and mixed material solutions (3% w/v alginate with 1% w/v gelatin, made from gelatin powder from porcine skin, G1890, Sigma) were prepared. The preparation process was similar to the procedure developed in our previous work (Ning et al., 2021 >). The prepared solutions were then magnetically stirred at room temperature overnight, or until thoroughly mixed. The crosslinking solution was a calcium chloride dihydrate (CaCl2*2H2O, CAS 10035-04-8, Sigma-Aldrich) solution at 50 mM concentration, with 0.1% w/v polyethyleneimine (PEI, J61270, Alfa Aesar) solution used as the solvent. The surfaces of 12-well plates were coated with 3 ml of the same PEI solution and were left in an incubator at 37degC overnight. The PEI coating was used to ensure that the scaffolds did not stick to the well plate once printing and crosslinking were complete. The next day, the PEI solution coated on printed plates was replaced with 3-4 ml of the crosslinking solution. The syringes containing different solutions were loaded into the bioprinter and attached to the printing arm. An envisionTEC 4th Generation 3D-Bioplotter Manufacturer Series - an extrusion based (pneumatic) bioprinter - was utilized. Scaffolds were fabricated with dimensions 10 mm x 10 mm x 5 mm with a strand diameter of 500 mm following a grid pattern [Fig. 1 >(a)] with inter-strand distances of 1 mm and 1.5 mm. These were kept in containers in a fridge at 4degC for 2 days. 2.2. Synchrotron imaging system setup The SR-PBI-HCT imaging experiments were performed at the Biomedical Imaging and Therapy (BMIT) 05ID-2 beamline (Wysokinski et al., 2007 >) of the Canadian Light Source. On this beamline, the double-crystal Si(111) monochromator can produce photon energies of 25-140 keV. Fig. 2 > displays a schematic diagram illustrating the SR-PBI-HCT and SR-PBI-CT imaging setups. The essence of PBI is propagation with distance [i.e. distance from sample to detector (SDD)], which can turn phase distortions into interference fringes and produce large contrast values at the edges of structures. All scans were performed at SDD = 1.5 m, a distance that can provide satisfactory contrast and spatial resolution for hydrogel scaffold imaging with a reconstructed voxel size of 13 mm based on a previous study by our group (Section S1 of the supporting information). For parallel beam geometry, SR-PBI-HCT scanning usually involves a horizontal rotation range over 360deg for achieving the full imaging of the ROI (Section S2 of the supporting information), whereing SR-PBI-CT scanning takes place with a rotation of exactly 180deg. The adjustment of photon flux can be achieved through placing neutral density filters (NDFs) with particular thickness in the beam. The detector, with a pixel size of 13 mm and an image depth of 16 bit, consists of a beamline monitor (AA60 HAMAMATSU, Japan) with a scintillator [LuAg500, lutetium Lu3Al5O12 garnet (LuAG) doped by the luminescent Ce3+, thickness of 500 mm] converting X-rays to visible light, an optic system and a complementary metal-oxide semiconductor (CMOS) digital camera (ORCA Flash 4.0). The active areas of the detector for SR-PBI-CT and SR-PBI-HCT imaging are 716 x 2048 pixels (9.31 mm x 26.624 mm) and 200 x 2048 pixels (2.6 mm x 26.624 mm), respectively. As discussed, p is a parameter for SR-PBI-HCT imaging, which can be defined as where s v is the vertical speed (mm s-1) of the rotation stage, t is the required time (s) for a full rotation/revolution and h FOV is the height of the field of view (FOV) (mm) (i.e. the height of the active detector). In addition, the radiation dose was measured using a calibrated ionization chamber (PTW 31010, Freiburg, Germany). The dose rate (Gy s-1) was measured and then the surface entry radiation dose D (Gy) using SR-PBI-HCT was calculated by where Dt is the exposure time (s) for each projection and N p/p is the effective projection number per pitch. Since SR-PBI-CT only involves half a revolution (i.e. 180deg) and there is no pitch, the effective projection number is given by N p/2. Given the fact that the beam flux may not be exactly uniform (especially for SR-PBI-CT, as seen later in Fig. 5) and that the surface entry dose rate was measured at a position in front of the geometric center of the detector in our study, the dose rate measured is approximately its maximum value and the dose evaluated from equation (2) is the approximate maximum surface entry dose. 2.3. Imaging of hydrogel scaffolds with SR-PBI-HCT Table 1 > displays the detailed experimental imaging conditions for five different test groups. The image quality of hydrogel scaffolds using both SR-PBI-HCT and SR-PBI-CT was first evaluated. In addition, in order to determine the spatial resolution of SR-PBI-HCT and SR-PBI-CT, a 3D bar pattern phantom (5 mm x 5 mm, QRM, Mohrendorf, Germany) was also imaged. Then, we conducted three parameter test experiments using SR-PBI-HCT, i.e. varied p (1.3, 1.5, 1.7, 1.9 and 2.5), E (30 keV, 40 keV and 50 keV) and N p (500, 1000 and 3000), and examined their corresponding effects on hydrogel scaffold image quality qualitatively and quantitatively. Due to the limited active detector height, multiple rotations (i.e. three rotations for p = 1.3, 1.5, 1.7, 1.9, and two rotations for p = 2.5) were taken for imaging the scaffolds when using SR-PBI-HCT. In the end, we scanned the hydrogel scaffold using SR-PBI-HCT at a low-level radiation dose (i.e. 342 mGy) to evaluate the feasibility of SR-PBI-HCT for in vivo imaging. 2.4. Image processing and evaluation metrics In order to apply existing image reconstruction and post-processing algorithms tailored to SR-PBI-CT, the projections obtained using SR-PBI-HCT needed to be converted to virtual projections acquired by SR-PBI-CT (Pelt & Parkinson, 2018 >; Fu et al., 2014 >). The steps to form virtual projections in our study are presented schematically in Fig. 3 > and the Python script is available on GitHub: Assuming the rotation stage is moved downwards, this conversion process can be expressed by where i and i' are the row indexes, j and j' are the column indexes, and k and k' are projection indexes. I i, j, k and denote the gray value of the SR-PBI-HCT projection and the gray value of the converted virtual SR-PBI-CT projection at the (i, j, k) and (i', j', k') positions, respectively; w is the linear interpolation weight. Values i and i' are i = 1, 2,..., M, where and i' = 1, 2,..., M', and M' = . N R is the number of rotations and d is the pixel size [s v, t and h FOV have the same definitions as in equation (1)]. Let be the vertical movement speed (units of pixels per projection), then the vertical translation for the kth projection will be kv (unit of pixels). The relationship between i' and i can be expressed by Given the fact that there is no displacement/translation in the horizonal direction for SR-PBI-HCT scanning, we have where mod(...) denotes the modulo operator and N is the total column number of the SR-PBI-HCT projection. The above equation indicates that the column index needs to be horizontally flipped if is odd. k and k' are the projection indexes and k = 0, 1,..., N R N p - 1 (corresponding angle range from 0 to 360N Rdeg) while k' = 0, 1,..., N p/2 - 1 (corresponding angle range from 0 to 180deg). The relationship between k and k' is given by w in equation (3) is the linear interpolation weight and can be calculated by In the present study, we noticed that, if p < 2, there were some redundant data (i.e. same rows appearing in different projections) for the SR-PBI-HCT projections during conversion, which were discarded. Before the projection conversion from SR-PBI-HCT to virtual SR-PBI-HCT, the background (i.e. flat and dark) corrections were first completed. Then, the transport of intensity equation (TIE) (Paganin et al., 2002 >), a popular PhR algorithm, was performed on each projection. The d/b value (2000 for all cases) can be calculated approximately (Thompson & Vaughan, 2001 >). The open-source software package Ultra-Fast-Online (UFO) was used to perform PhR (i.e. TIE) on the projections and the CT reconstruction [filtered-backprojection (FBP) algorithm] (Vogelgesang et al., 2016 >). In addition, after PhR, images obtained using SR-PBI-CT were also processed with common ring artifact removal methods, i.e. low-pass Fourier filtering (Raven, 1998 >) and the Sarepy sorting wind method (Vo et al., 2018 >). For low-pass Fourier filtering, the essence of ring artifact removal is to filter the vertical stripes (i.e. ring artifact areas) in the 2D frequency domain, and the key parameters that need to be tuned are the horizontal and vertical sigma, i.e. 10 and 1 in our work, respectively. For the Sarepy sorting wind method, the essence of ring artifact removal is to employ a median filter (along the horizontal direction) to remove vertical stripes, where the first step is to retrieve the response of each pixel by sorting intensities along each column of a sinogram. The key parameters to be tuned are the window size and the signal-to-noise ratio (SNR). The window size indicates the median filter size and, in our study, a value of 10 (unit of pixels) was selected. The SNR parameter controls the sensitivity of the stripe detection and a value of SNR from 1.1 to 3.0 is recommended. To compare image quality across the different imaging conditions, both objective estimation of quantitative evaluation metrics and subjective evaluation (e.g. image perception and cognition) were employed. Quantitative evaluation metrics include SNR and contrast-to-noise ratio (CNR) (Yao et al., 2019 >), which are formulated as I E and s denote the mean gray value and the standard deviation of the ROI, respectively. The foreground represents the ROI including objective hydrogel scaffolds and the background represents the ROI excluding objective samples. In addition to SNR and CNR, which mainly focus on the image noise level, the modulation transfer function (MTF) (Fujita et al., 1992 >) of the 3D QRM bar pattern phantom CT images and Fourier shell correlation (FSC) (Van Heel & Schatz, 2005 >) were also calculated for the spatial resolution analysis. FSC is used to estimate the correlation coefficient between Fourier shells of two 3D reconstructions computed from two independent datasets. In our study, two mutually independent datasets were generated by scanning scaffolds with double projection numbers per rotation and using the odd projections for one reconstruction and the even projections for a second reconstruction. 3. Results and discussion 3.1. Comparison of SR-PBI-HCT and SR-PBI-CT The reconstructed hydrogel scaffold (4% w/v alginate) images using SR-PBI-HCT and SR-PBI-CT are shown in Fig. 4 >. From the results [Figs. 4 >(a1)-4 >(d1)], the SR-PBI-HCT image does not show obvious artifacts whereas the SR-PBI-CT image presents serious ring artifacts. Such artifacts disrupt the continuity of the strand grayscale values in the image and thus impair the capacity to accurately visualize/analyze strand properties. Although these artifacts can be removed somewhat using image-processing algorithms, there are still some obvious remaining artifacts. The gray value profiles across strands [Figs. 4 >(a2)-4 >(d2)], as indicated by the red lines in Figs. 4 >(a1)-4 >(d1), were also visualized, providing the quantitative contrast information. SR-PBI-HCT can cause helical artifacts due to the nature of its helical acquisition. Compared with ring artifacts, helical artifacts spread over larger regions of the volume and thus have reduced effects. In this work, we applied a linear interpolation in the conversion process from SR-PBI-HCT projections to virtual SR-PBI-CT projections to lessen the effect of defects (i.e. the cause of artifacts). As a result, SR-PBI-HCT has significantly reduced artifacts compared with SR-PBI-CT. Theoretically, the use of a large-area detector and/or increased pitch would help to further reduce the helical artifacts because these can spread artifacts over a larger region of the volume. However, such settings may lead to other issues, for example, additional artifacts/noise problems if using a large-area detector (see Fig. 5 > and related discussion), and streaking artifacts if using a pitch of more than 2 (see Fig. 8 and related discussion). As such, the imaging settings become complicated, particularly for the case of imaging live animals where the radiation dose is critical; as such, a trade-off has to be made between the radiation dose and imaging quality. In addition, we noticed that the results from SR-PBI-HCT have higher values of SNR and CNR than those of SR-PBI-CT (even for areas without ring artifacts). One of the reasons behind this could be the different intensity/flux distribution ranges in the vertical direction due to different active detector height (i.e. 2.6 mm for SR-PBI-HCT and 9.31 mm for SR-PBI-CT). Fig. 5 > shows the flat-field images and their vertical gray value profiles (i.e. flux distribution) obtained using SR-PBI-HCT and SR-PBI-CT. From Figs. 5 >(a1) and 5 >(b1), SR-PBI-HCT only uses the central X-ray beam so that the overall intensity is stronger and the distribution is more uniform compared with the beam received by a larger detector in SR-PBI-CT. Specifically, for SR-PBI-HCT, the vertical X-ray flux intensity drop is 11.43% (from center to sides), which is much smaller than the drop of 53.77% for SR-PBI-CT. Although the scanned scaffold has a smaller height (5 mm) than the active height of the detector (9.31 mm), the intensity differences still negatively affect the image quality (i.e. increased noise level in the relatively low-flux areas). This effect will become more severe with low-dose imaging for in vivo imaging. Generally, the smaller detector in SR-PBI-HCT overcomes the limitation of beam height for some cases and is also beneficial to control radiation dose distribution for in vivo imaging. Additionally, the small detector in SR-PBI-HCT reduces the possibility of including damaged pixels, thereby decreasing the possibility of introducing additional unexpected artifacts. Although objects can also be imaged with SR-PBI-CT using a small detector via multiple view scanning (by moving the sample longitudinally for the next scan), the intensity distributions are less uniform because overlapping only occurs at the margins between different views. The overlapping in SR-PBI-CT also increases the radiation dose compared with continuous scan mode in SR-PBI-HCT. Fig. 6 > displays the reconstructed results of the 3D QRM bar pattern phantom from SR-PBI-HCT and SR-PBI-CT for the analysis of spatial resolution. None of the PhR and ring artifact removal algorithms were applied to avoid introducing impacts on spatial resolution. From Figs. 6 >(a2) and 6 >(b2), the line pairs at the center region of the phantom show comparable spatial resolution reflected by the similarly discernible line pairs between SR-PBI-HCT and SR-PBI-CT. However, due to the effect of ring artifacts, we notice that there are obvious structural distortions on the line pairs for SR-PBI-CT, as indicated by the red arrows. The curves in Figs. 6 >(a3) and 6 >(b3) show the relationship between the MTF amplitude and spatial resolution [measured as line-pairs per millimetre (Lps mm-1)], as well as the corresponding cutoff resolution (10% MTF, i.e. smallest resolvable object) of 17.2 Lps mm-1 (i.e. linewidth: 29.07 mm) for SR-PBI-CT and 17.6 Lps mm-1 (i.e. linewidth: 28.41 mm) for SR-PBI-HCT. SR-PBI-HCT shows a slightly higher spatial resolution than SR-PBI-CT and the main reason may be the disruption of ring artifacts on SR-PBI-CT images. By combining the results from Figs. 4 > and 6 >, SR-PBI-HCT shows promise of addressing the ring artifacts, while having no loss in spatial resolution. 3.2. Influence of helical pitch of SR-PBI-HCT The effect of p on hydrogel scaffold images is shown in Fig. 7 >. Overall, there is little visible effect on reconstructed 3D results of p [Figs. 7 >(a1)-7(d1)] with the range 1.3-1.9, and all the strands within the assessed ROIs are visibly recognizable [Figs. 7 >(a2)-7(d2)]. The gray value profiles of strands for all cases are very close to each other [Figs. 7 >(a3)-7(d3)]. Nevertheless, one can still observe the slight artifacts on images obtained with p = 1.3, as indicated by the red arrow [Fig. 7 >(a2)]. These artifacts are diminished for images with p = 1.5, 1.7 and 1.9. This is because higher p actually disperses the ring artifacts in a larger vertical region. As a result, the artifacts assigned to each SR-PBI-HCT slice will be reduced. In addition, SNR and CNR are improved when p increases from 1.3 to 1.5, but are then reduced when p increases from 1.5 to 1.9. The analysis of 3D spatial resolution [Figs. 7 >(a3)-7(d3)] with FSC also produces similar results. The Fourier image resolution (FIRE) is the reciprocal of spatial frequency at the intersection of the smoothed FSC curve with a correlation threshold of 1/7 (i.e. 0.14). One possible reason could be, for a reconstructed slice, the illumination range of X-rays (i.e. the total angle covered by the flux when samples are rotating) decreases with increasing p. A small illumination range usually produces worse image quality (Hayes et al., 2021 >). In summary, p = 1.5 can produce higher image quality because it balances the effects from ring artifact dispersion and illumination range. For a parallel beam geometry system, it should be realized that, when p > 2.0, overlapping between adjacent rotations will be incomplete, and this will lead to streaking artifacts, as shown in Fig. 8 > (p = 2.5). 3.3. Influence of photon energy on SR-PBI-HCT The effect of E on hydrogel scaffold images using SR-PBI-HCT is shown in Fig. 9 >. The main differences associated with X-ray energies are the variations in image contrast, which generally involve the combination of phase contrast and absorption contrast in synchrotron radiation imaging. In the typical X-ray photon energy range 10-140 keV, the absorption coefficient scales as 1/E 3 (Als-Nielsen & McMorrow, 2011 >) before or after the absorption edge. A lower E has a higher absorption coefficient and therefore is less penetrating. When E = 40 keV and 50 keV, the X-ray photons penetrate with less absorption, resulting in little difference in image contrast (Fig. 9 >). Thus, the strands are hard to identify for E = 40 keV and 50 keV [Figs. 9 >(b2) and 9 >(c2)] and the edges of strands are not clear [Figs. 9 >(b1) and 9 >(c1), as indicated by the red arrows]. Besides, 3D spatial resolution analysis with FSC shows that both 40 keV and 50 keV have much larger values of FIRE than 30 keV [Figs. 9 >(b1) and 9 >(c1)]. Consequently, to obtain scaffold images with higher SNR and CNR using SR-PBI-HCT, 30 keV is optimal compared with 40 keV and 50 keV. Theoretically, with E < 30 keV, imaged objects have a higher absorption coefficient and therefore result in higher contrast; however, for our scaffolds, imaging in a tube with 50 ml water (in vitro, simulating in vivo imaging conditions for scaffolds implanted in a rat leg for nerve tissue regeneration), E < 30 keV results in higher noise level and higher radiation dose because a larger proportion of photons are absorbed. 3.4. Influence of projection numbers of SR-PBI-HCT The effect of N p on hydrogel scaffold images is shown in Fig. 10 >. For N p = 500, 1000 and 3000, hydrogel scaffolds present sufficient image contrast for segmentation and 3D volume rendering display [Figs. 10 >(a1)-10 >(c1)]. Although the noise is increased for N p = 500 and 1000 compared with N p = 3000, strands can still be clearly identified [Figs. 10 >(a2)-10 >(c2)], which is also supported by quantitative analysis with the value of FIRE [Figs. 10 >(a4)-10 >(c4)]. The radiation dose for N p = 500 is lower than for the two other cases (see Table 1). Therefore, N p = 500 is the optimum when considering image quality and radiation dose. 3.5. Low-dose imaging Although the image quality is satisfactory with E = 30 keV and N p = 500, the 17.67 Gy radiation dose is high for in vivo live-animal imaging. We tested the feasibility of SR-PBI-HCT for hydrogel scaffold imaging within an acceptable dose range [~500 mGy for rat protocols (Pratt et al., 2014 >)]. Specifically, a scaffold imaging experiment with 2 x 2 binning mode and N p = 250 was also conducted. In addition, an NDF with a thickness of 80 mm was used to reduce the photon flux. The measured radiation dose was 54.72 mGy s-1. Results (Fig. 11 >) show that scaffolds can still be identified and segmented from the background (i.e. water) while the radiation dose (~342 mGy) remains suitable for in vivo animal imaging. This study reveals that SR-PBI-HCT imaging, though lacking in vivo results currently, is promising for in vivo TERM applications. 4. Conclusions In TERM, visualization of low-density tissue scaffolds following their implantation is crucial, yet challenging. In this paper, we present a study on the integration of helical acquisition mode with SR-PBI-CT to non-invasively and non-destructively visualize/characterize low-density scaffolds. The results demonstrate the improvement of contrast and the significant advantage of avoiding ring artifacts without introducing additional artifacts. We also showed that p = 1.5, E = 30 keV and N p = 500 were suited for hydrogel scaffold imaging in vitro using the current SR-PBI-HCT configuration of BMIT. In addition, with 2 x 2 binning mode and N p = 250, the SR-PBI-HCT can produce satisfactory results while the radiation dose (~342 mGy, voxel size of 26 mm) remains suitable for in vivo animal imaging. The results obtained in this study reveal that the SR-PBI-HCT imaging method is a powerful tool for visualizing and characterizing hydrogel scaffolds in terms of image quality and radiation dose, forming a solid base for in vivo 3D non-invasive characterization in TERM. Supplementary Material S1: why 1.5 m was used for SR-PBI-CT/HCT imaging. S2: details of the required scanning angle for SR-PBI-HCT. DOI: 10.1107/S1600577523000772/tv5040sup1.pdf We acknowledge Dr Sergey Gasilov for his contribution to the development of the SR-PBI-HCT technique. Figure 1 Alginate scaffold: (a) designed scaffold structure and (b) printed scaffolds. Figure 2 Schematic of the SR-PBI-HCT and SR-PBI-CT imaging setups. Figure 3 Schematic of virtual projection stitching of SR-PBI-CT from SR-PBI-HCT projections. I i,j,k and are the gray value of the SR-PBI-HCT projection and the gray value of the converted virtual SR-PBI-CT projection at the positions (i, j, k) and (i', j', k'), respectively. i and i' are the row indexes, j and j' are the column indexes, and k and k' are the projection indexes. is the linear interpolation result of I i, j, k and I i+1,j,k with a weight of 1 - w and w. The virtual SR-PBI-CT projection at an index of k' is transformed from a sequence of SR-PBI-HCT projections at index of k, k + N p/2,..., k + (2N R - 1)N p/2 with an interval of N p/2. The total row M of the SR-PBI-HCT projection is while the total row M' of the virtual SR-PBI-CT projection is . mod(*,*) denotes the modulo operator. Figure 4 Comparison of SR-PBI-HCT and SR-PBI-CT (gray scale: 0-255); (a1) SR-PBI-HCT image, (b1) SR-PBI-CT image, (c1) SR-PBI-CT image with low-pass Fourier filtering and (d1) SR-PBI-CT image with Sarepy sorting. (a2)-(d2) corresponding gray value profiles of ROIs at red line positions. Figure 5 X-ray flux distribution comparison between SR-PBI-HCT and SR-PBI-CT. Flat images of SR-PBI-HCT (a1) and SR-PBI-CT (b1). Gray value profiles of the flat images (shown by the red lines) of SR-PBI-HCT (a2) and SR-PBI-CT (b2). Figure 6 Reconstructed 3D QRM bar pattern phantom using SR-PBI-CT and SR-PBI-HCT (gray scale: 0-255); (a1) SR-PBI-CT image and (b1) SR-PBI-HCT image and the corresponding (a2) SR-PBI-CT and (b2) SR-PBI-HCT ROIs enlarged from the yellow squares in (a1) and (b1); (a3)-(b3) corresponding MTF amplitudes with cutoff resolutions shown. Figure 7 Effect of helical pitches on hydrogel scaffold images using SR-PBI-HCT with pitches of 1.3, 1.5, 1.7 and 1.9; (a1)-(d1) reconstructed 3D results; (a2)-(d2) corresponding 2D images; (a3)-(d3) corresponding gray value profiles measured at the red line positions in (a2)-(d2); (a4)-(d4) 3D spatial resolution analyses with FSC, smoothed FSC and FIRE. Figure 8 Hydrogel scaffold images with a pitch of 2.5. (a1) Reconstructed slice; (a2) ROI [yellow square in (a1)]; (a3) example of converted virtual projections (gray regions are missing information between adjacent rotations). Figure 9 Effect of photon energies on hydrogel scaffold images using SR-PBI-HCT with 30 keV, 40 keV and 50 keV; (a1)-(c1) reconstructed 3D results; (a2)-(c2) corresponding ROI images; (a3)-(c3) corresponding gray value profiles measured at the red line positions in (a2)-(c2); (a4)-(c4) 3D spatial resolution analysis with FSC, smoothed FSC and FIRE. Figure 10 Effect of projection numbers on hydrogel scaffold images using SR-PBI-HCT with N p = 500, 1000 and 3000; (a1)-(c1) reconstructed 3D results; (a2)-(c2) corresponding ROI images; (a3)-(c3) corresponding gray value profiles measured at the red line positions in (a2)-(c2); (a4)-(c4) 3D spatial resolution analysis with FSC, smoothed FSC and FIRE. Figure 11 Reconstruction results of a hydrogel scaffold using SR-PBI-HCT with N p = 250 (2 x 2 binning): (a1) reconstructed 3D result; (a2) corresponding ROI image; (a3) corresponding gray value profile measured at the red line positions in (a2); (a4) 3D spatial resolution analysis with FSC, smoothed FSC and FIRE. Table 1 Experimental details in terms of imaging conditions for five different group tests Group Scan mode Imaged object Energy (keV) Projection number (rev-1) Binning mode Exposure time (ms per projection) Helical pitch Measured dose (Gy) No. 1: SR-PBI-CT versus SR-PBI-HCT SR-PBI-CT Hydrogel scaffold (4% w/v alginate) 30 3000 1 x 1 57.00 - 79.51 3D QRM bar pattern phantom SR-PBI-HCT Hydrogel scaffold (4% w/v alginate) 30 3000 1 x 1 57.00 1.3 122.33 3D QRM bar pattern phantom No. 2: Helical pitch comparison SR-PBI-HCT Hydrogel scaffold (3% w/v alginate with 1% w/v gelatin) 30 3000 1 x 1 57.00 1.3 122.33 1.5 106.02 1.7 93.54 1.9 83.70 2.5 63.61 No. 3: Photon energy comparison SR-PBI-HCT Hydrogel scaffolds (4% w/v alginate) 30 3000 1 x 1 57.00 1.5 106.02 40 14.40 48.38 50 10.82 31.16 No. 4: Projection number per revolution comparison SR-PBI-HCT Hydrogel scaffold (3% w/v alginate with 1% w/v gelatin) 30 500 1 x 1 57.00 1.5 17.67 1000 35.34 3000 106.02 No. 5: Low-dose imaging SR-PBI-HCT Hydrogel scaffold (3% w/v alginate with 1% w/v gelatin) 30 250 2 x 2 25.00 1.5 0.34 References Als-Nielsen, J. & McMorrow, D. 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PMC10000811 | J Synchrotron Radiat J Synchrotron Radiat J. Synchrotron Rad. Journal of Synchrotron Radiation 0909-0495 1600-5775 International Union of Crystallography 36891857 fv5157 10.1107/S1600577522011523 JSYRES S1600577522011523 Short Communications Beam-induced redox chemistry in iron oxide nanoparticle dispersions at ESRF-EBS Beam-induced redox chemistry at ESRF-EBS Thoma Sabrina L. J. a Zobel Mirijam a* a Institute of Crystallography, RWTH Aachen University, Jagerstrasse 17-19, Aachen, 52066 Nordrhein-Westfalen, Germany Favre-Nicolin V. Editor ESRF and Universite Grenoble Alpes, France Correspondence e-mail: [email protected] 01 3 2023 13 1 2023 13 1 2023 30 Pt 2 s230200 440444 18 8 2022 30 11 2022 (c) Thoma and Zobel 2023 2023 This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-BY) Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited. A full version of this article is available from Crystallography Journals Online. With the increased brilliance at the European Research Facility-Extremely Brilliant Source (ESRF-EBS), a beam-induced reduction of non-stochiometric iron oxide nanoparticles (almost maghemite composition) to magnetite was observed in a mixture of ethanol and water with low ethanol concentration. The storage ring upgrade of the European Synchrotron Radiation Facility makes ESRF-EBS the most brilliant high-energy fourth-generation light source, enabling in situ studies with unprecedented time resolution. While radiation damage is commonly associated with degradation of organic matter such as ionic liquids or polymers in the synchrotron beam, this study clearly shows that highly brilliant X-ray beams readily induce structural changes and beam damage in inorganic matter, too. Here, the reduction of Fe3+ to Fe2+ in iron oxide nanoparticles by radicals in the brilliant ESRF-EBS beam, not observed before the upgrade, is reported. Radicals are created due to radiolysis of an EtOH-H2O mixture with low EtOH concentration (~6 vol%). In light of extended irradiation times during insitu experiments in, for example, battery and catalysis research, beam-induced redox chemistry needs to be understood for proper interpretation of insitu data. beam-induced radiolysis radiation damage on inorganic materials ESRF-EBS European Synchrotron Radiation FacilityCH5509 European Synchrotron Radiation FacilityCH6070 The following funding is acknowledged: European Synchrotron Radiation Facility (grant No. CH5509); European Synchrotron Radiation Facility (grant No. CH6070). pmc1. Introduction During the past few decades the brilliance of modern synchrotron light sources has increased a lot (Raimondi, 2016 >) and mankind is constantly producing orders of magnitudes more photons in smaller and smaller cross sections (Bras et al., 2021 >). After the Extremely Brilliant Source (EBS) upgrade, the European Synchrotron Radiation Facility (ESRF) now produces an X-ray beam being ~100 times more brilliant than before (Raimondi, 2016 >). Increase in brilliance comes with a vast increase in experimental possibilities, that enable us to capture faster events than ever (Bras et al., 2021 >) and to achieve the same time resolution on weakly scattering amorphous samples as before on crystalline ones (Vaughan et al., 2020 >). Yet, unprecedented photon flux comes with higher risk of radiation damage (Bras et al., 2021 >). In macromolecular crystallography, radiation damage is a big issue that the community has been aware of for many years, constantly working on predicting damage thresholds and developing methods on how to prevent it (Bras et al., 2021 >; Fourme et al., 2012 >; Garman, 2010 >; Holton, 2009 >). Further, also in other diffraction (Vaselabadi et al., 2016 >; Hopkins & Thorne, 2016 >; Neuhold et al., 2012 >) as well as imaging (Lai et al., 2013 >; Wang et al., 2009 >; Beetz & Jacobsen, 2003 >) and spectroscopy (Rightor et al., 1997 >; Chidambaram et al., 2001 >) experiments, X-ray radiation damage was observed on radiation-sensitive, mostly organic, materials and studied for future obviation. On the contrary, inorganic solid materials are not expected to be affected by radiation damage in X-ray scattering experiments, since very high radiation doses [MGy; 1 Grey (Gy) = 1 J kg-1] are needed for beam-induced changes like the creation of interstitial-hole Frenkel-pairs, excited electronic states, phase transitions and induced crystallization (Bras et al., 2021 >; Tiliks et al., 1991 >). Yet, recently, organometal halide perovskites, applied in solar cells, were found to suffer from X-ray induced electronic degradation, namely a decrease of the X-ray beam-induced current (XBIC), even though the elemental composition in X-ray fluorescence (XRF) with common operando synchrotron nanoprobe conditions was not found to change (Stuckelberger et al., 2020 >). Such a material also exhibited a crystalline lead halide degradation phase, caused by iodine migration from the perovskite, in a scanning nano X-ray diffraction experiment (Ferrer Orri et al., 2022 >). Further, radiation damage under X-ray exposure was studied for inorganic materials (metal alloy, oxide and semiconducting) with X-ray photon spectroscopy (Astley et al., 2022 >). Changes in the binding energies were identified for all three materials, but due to different reasons, for example thermal expansion of the metal alloy. Here, we report on beam-induced changes in redox chemistry in iron oxide and associated changes in lattice parameters, which we observed while measuring total scattering on iron oxide nanoparticles (IONPs) dispersed in ethanol-water (EtOH-H2O) mixture with low ethanol (~6 vol%) concentration at ID31 ESRF-EBS with high-energy X-rays (65 keV). Redox chemistry is at the heart of the working principle of electrochemical and various catalytic processes. The number of in situ and operando experiments in these fields has risen rapidly because of the unprecedented time-resolution, enabling insight into structural details of, for instance, heterogeneous catalysts dispersed in liquid electrolytes in fuel cells (Chattot et al., 2021 >; Martens et al., 2021 >). Given the Fe3+ - Fe2+ beam-induced reduction in IONPs within only 12 s of exposure, what does happen to nanostructured, often multivalent, metal oxides exposed over hours during operando experiments? Many battery materials consist of spinel oxides, thus similar to our IONPs (Choi & Manthiram, 2006 >; Koga et al., 2013 >; Manthiram, 2020 >), and we want to create awareness that charge-discharge processes of batteries could similarly be affected due to radiolysis of the surrounding organics. Further, nanomaterial research and general solvation effects on solid-liquid interfaces may be induced in the future (Roy et al., 2021 >; Christensen et al., 2021 >; Aalling-Frederiksen et al., 2021 >). Besides direct impact of the radicals on the solid state structure, solvated radicals can massively impact chemical reactions, for instance operando catalysis experiments. Since materials with excited states behave chemically different, we suppose that increased reaction rates (Bras et al., 2021 >) or unforeseen influences of radiolysis products at solid-liquid or liquid-gas interfaces (Le Caer, 2011 >; Bras et al., 2021 >) cannot be excluded. Hence, reaction kinetics of the experiment may be altered in comparison to without beam. 2. Data collection and data treatment Total scattering measurements were performed at ID15-A at ESRF before the EBS upgrade and at ID31 at ESRF after the EBS upgrade, for IONPs in EtOH-H2O mixture with low EtOH concentration, IONPs in water (only ID31) (for information regarding sample preparation see Section S1 of the supporting information) and water, taken in 1 mm Kapton capillaries. Both beamlines are equipped with a PILATUS3 X CdTe 2M detector (253.7 mm x 288.8 mm sensitive area, 172 mm x 172 mm pixel size). For data processing and treatment, we used the following: for masking Fit2D and for calibration pyFAI-calib2 (Ashiotis et al., 2015 >), for radial integration xpdtools ), for pair distribution function (PDF) processing PDFgetX3 (Juhas et al., 2013 >), for PDF modelling DiffPy-CMI (Juhas et al., 2015 >) and PDFgui (Farrow et al., 2007 >), and for fitting of I(Q) data IgorPro by WaveMetrics. 2.1. Details on data collection on ID15A - pre-EBS upgrade Data were taken with an energy of 68 keV (0.1823 A) and a beam size of about 120 mm x 120 mm [vertical x horizontal (v x h), being a little bit smaller in the vertical] at ID15A before the upgrade. The photon flux on the samples was estimated to be 5 x 1010 to 1 x 1011 photons s-1 (see Section S2 of the supporting information). Ten scans of 25 s each were taken on every sample resulting in a total radiation dose of ~195-390 kGy (see Section S2 of the supporting information for calculation). NIST chromium(III) oxide standard was used for distance calibration and determination of instrumental resolution (Q damp = 0.0181 A-1; Q broad = 0.0185 A-1). 2.2. Details on data collection on ID31 - after-EBS upgrade Data were taken with an energy of 65 keV (0.1907 A) and a beam size of about 100 mm x 300 mm (v x h, +-50 mm) at ID31 after the upgrade. The photon flux on the samples was 1 x 1014 photons s-1. Ten scans of 6 s each were taken on every sample resulting in a total radiation dose of ~40000 kGy (see Section S2 of the supporting information for calculation). NIST cerium(IV) oxide standard was used for distance calibration and determination of instrumental resolution (Q damp = 0.0159 A-1; Q broad = 0.0119 A-1). 3. Results and discussion Performing total scattering on IONPs of 7 and 15 nm in diameter, dispersed in an EtOH-H2O mixture with low EtOH concentration (6 vol%) at ESRF-EBS, we observed shifts of the Bragg peaks over the total time of exposure of 60 s, whereby the biggest shift was observed between the first and second scan between 6 and 12 s. Initially, the IONPs feature an inverse spinel structure with a composition close to maghemite (see Section S3 of the supporting information). Fig. 1 >(a) shows the diffraction pattern of such an IONP dispersion in the Q-range of 1-5 A-1 in comparison with that of water. The diffraction patterns are similar, yet the first sharp diffraction peak (FSDP) of the dispersion is broadened and slightly shifted to lower Q-values due to the low amount of EtOH (~6 vol%; for more information about the EtOH content see Section S4 of the supporting information). The Bragg peaks of the IONPs are visible on top of the broad diffraction from the solvent. In Fig. 1 >(b) the ten scans taken successively at ID31 after the EBS upgrade are shown in the Q-range of the (333) and (440) Bragg reflexes between 3.7 and 4.5 A-1, for one exemplary dispersion of IONPs in EtOH-H2O mixture with low EtOH concentration (ID31-1-EtOH-H2O). Evidently, both peaks shift to lower Q-values after the first scan. This becomes even clearer in the inset which shows the (440) Bragg reflex for scan 1, 3 and 10 only, corrected for the water background. The Bragg peaks of the same IONPs dispersed in solely water (ID31-1-H2O) did not shift - see Fig. 1 >(c). Further, we compared these data with data of samples prepared the same way taken at ID15-A ESRF before the EBS upgrade, thus with lower photon flux per irradiated volume, and consequently resulting in lower total radiation dose despite the smaller beam size and longer exposure time ( cf. Section S2 of the supporting information). In this case, too, no peak shift of the IONP Bragg peaks over time could be observed - see Fig. 1 >(d) (shown for ID15-A-1-EtOH-H2O). We proved the reproducibility of the shift of Bragg peaks in EtOH-H2O mixtures after the upgrade (case 1) and its lack in only H2O after the upgrade (case 2) and the lack in EtOH-H2O mixtures before the upgrade (case 3), for several samples each. In order to quantify the shift and make sure it is bigger than the uncertainty in instrumental resolution, three scans (1, 3 and 10) of two samples of each of the three cases were investigated in more detail. For those scans a Gaussian function was fitted to the (440) reflex in I(Q) data (see Section S5 of the supporting information). Since a shift to lower Q-values is associated with a lattice expansion, this lattice expansion was also confirmed and quantified by PDF fits on the differential-PDFs (d-PDF, IONP dispersion minus water background) (see Section S6 of the supporting information). The results of both evaluations are listed in Table 1 > in comparison with the total applied radiation dose, clearly showing the correlation of radiation dose and Bragg peak shift. When water is irradiated with ionizing radiation, like radioactive nuclei, beams of charged particles and X-rays with a photon energy >100 eV, radiolysis of water takes place (Le Caer, 2011 >; Zhang et al., 2012 >). Thereby, many different species are formed, such as hydrated electrons, dihydrogen and hydrogen peroxide molecules, oxonium and hydroxyl ions, as well as hydroxyl radicals and hydrogen atoms. Hydrated electrons and hydrogen are strong reducing agents, which are known to readily reduce dissolved metal ions to their lower oxidation state, in contrast to the hydroxyl radical, which is oxidative (Le Caer, 2011 >). Alcohols are known to be scavengers for the oxidative hydroxyl radical (Simic et al., 1969 >; Zhang et al., 2012 >; Yamaguchi et al., 2016 >). By abstraction of hydrogen from the alcohol, the oxidative hydroxyl radical is scavenged by the alcohol, and thereby organic reducing radicals are evolving (Yamaguchi et al., 2016 >; Zhang et al., 2012 >). Moreover, upon radiation with X-rays, ethanol can also be oxidized to acetaldehyde via the production of two electrons and two protons (Yamaguchi et al., 2016 >). Consequently, in a mixture of water and alcohol a reducing environment for chemical reactions is created upon irradiation with ionizing radiation. For instance, graphene oxide can be reduced to graphene in such a reducing medium with comparable alcohol content (Zhang et al., 2012 >). Hence, we are convinced that in our case the non-stochiometric (mostly Fe3+containing) IONPs are reduced to magnetite. Magnetite crystallizes in the inverse spinel structure, with forming a cubic close packing, Fe3+ ions occupying the tetrahedral sites and a 1:1 mixture of Fe2+ and Fe3+ ions on the octahedral sites (Cervellino et al., 2014 >). Magnetite nanoparticles stored in air are fully or partially oxidized to maghemite over time, by creation of vacancies on the octahedral sites accompanied by a lattice contraction (Cervellino et al., 2014 >; Sidhu et al., 1977 >). In maghemite (g-Fe2O3) all of the initial one-third of Fe2+ was oxidized to Fe3+, and thus the number of vacancies is maximal. These vacancies can either be randomly distributed on the octahedral sites or vacancy ordering can exist, lowering the symmetry of the structure (Cervellino et al., 2014 >). In our case in the synchrotron beam, in the reducing medium created upon irradiation with the highly brilliant X-rays, the opposite reaction is taking place: parts of the Fe3+ ions are reduced to Fe2+ whereby the vacancies are filled again, and the lattice is expanding, due to the bigger ionic radius of Fe2+ in comparison with Fe3+ (Sidhu et al., 1977 >). Since no other iron source besides the IONPs is present, this reduction will most likely be accompanied by the release of oxygen from the crystal structure for providing charge balance. For full conversion from maghemite to magnetite the release would amount to ~11% of the initial oxygen content of the IONPs. Oxygen present at the IONP surface and in the dispersion could then also be involved in reactions with the evolving radicals. The process is schematically shown in Fig. 2 >. This assumption matches the experimental observations very well, since at first the Bragg peaks shift from first to second scan, strongly. Then, an equilibrium state seems to be reached, matching the fact that all vacancies are filled at some point. Further, the radiation dose per second (see Section S2 of the supporting information) in our experiments has been higher than the total radiation doses reported in studies which aimed at synthesizing inorganic nanoparticles via radiolysis by deliberate exposure of metal ion precursor solutions to strong ionizing radiation (Cubova & Cuba, 2020 >). We exclude that the expansion is thermal expansion for two reasons: in the case of thermal expansion the IONPs in water only should be likewise influenced considering similar thermal conductivity; further, the increase of the lattice parameter of ~0.03 A would be caused at temperatures way above the boiling point of water (Bayer, 1972 >; Petric & Ling, 2007 >; Levy et al., 2004 >), but no evaporation of the solvent is observed in the I(Q) scans. 4. Conclusion In summary, we have observed beam-induced structural changes in iron oxide nanoparticles created by radiolysis during total scattering experiments on nanoparticle dispersions in an EtOH-H2O mixture with low EtOH concentration. The iron oxide nanoparticles were reduced from an almost maghemite (mainly Fe3+ containing) composition to magnetite in the highly brilliant synchrotron beam at ID31, ESRF-EBS. This is, to our knowledge, the first showcase of beam-induced damage in inorganic solids after the ESRF upgrade. Given its nature in redox chemistry and evolving radicals in solution, we expect this study to be highly relevant to a range of fields including in situ studies in catalysis and energy applications such as fuel cell or battery research. Time-dependent sample-specific changes need to be questioned in light of possibly radiolysis-induced redox chemistry. 5. Related literature The following references, not cited in the main body of the paper, have been cited in the supporting information: Bondaz et al. (2020 >); Caruntu et al. (2004 >); Cooper et al. (2020 >); Greaves (1983 >); Qu et al. (2011 >); Thoma et al. (2019 >). Supplementary Material Sections S1 to S6, including Tables S1 to S5 and Figures S1 to S4. DOI: 10.1107/S1600577522011523/fv5157sup1.pdf We gratefully acknowledge beam time at the European Synchrotron Radiation Facility granted from proposals CH5509 and CH6070. We thank our local contacts from beamline ID15-A and ID31, Gavin Vaughan and Marta Mirolo, for the assistance in operating the beamlines, as well as Jakub Drnec, Veijo Honkimaki and Andreas Magerl for discussions. Open access funding enabled and organized by Projekt DEAL. Figure 1 (a) I(Q) data in the Q-range of the FSDP for water and IONPs dispersed in EtOH-H2O mixture with low EtOH concentration, showing that the FSDP is slightly shifted for the mixture. (b) Evolution of I(Q) scans of IONPs in EtOH-H2O mixture with low EtOH concentration taken over time at ID31 after the EBS upgrade between 3.7 and 4.5 A-1 showing that the Bragg reflexes in this region shift to lower Q-values after the first scan. The inset shows scans 1, 3 and 10 in the region of one of those Bragg reflexes corrected for the water background to point out the shift. (c) I(Q) scans for the same IONPs just dissolved in pure H2O for the same Q-range. Scans were taken with the same acquisition time, beam size and with the same flux as data in panel (b). No shift can be observed as pointed out in the inset showing the same Bragg reflex corrected for water background as inset of panel (b). (d) I(Q) scans of IONPs in EtOH-H2O mixture with low EtOH concentration prepared the same way as in panel (b) but measured at ID15-A before the EBS upgrade with lower photon flux on the sample. No shift can be observed; see also inset. Figure 2 Change of the crystalline structure of the IONPs dispersed in an EtOH-H2O mixture with low EtOH concentration upon irradiation with highly brilliant X-rays. Illustrations of the unit cells have been made using VESTA (Momma & Izumi, 2011 >). Black balls represent oxygen atoms, blue balls represent iron atoms, vacancies are illustrated by partial filling and the red and grey polyhedra illustrate [FeO6] octahedral and [FeO4] tetrahedral units, respectively. IONPs before radiation with the X-ray beam are non-stochiometric, but mostly containing Fe3+ (see Section S3 of the supporting information), crystallized in inverse spinel structure (here described with unit cell for simplicity), therefore possessing vacancies on the octahedral sites (left side). Induced by the irradiation with X-rays, both oxidative and reducing radicals are created by radiolysis of water. Due to the presence of EtOH, the oxidative radical is scavenged, a reducing atmosphere is created and Fe3+ is reduced to Fe2+, which fills the vacancies and leads to expansion of the crystal lattice due to its bigger ionic radius (unit cell on the right side). Table 1 Shifts in the (440) reflex and expansion of lattice parameter (from d-PDF fit) in comparison with estimated radiation dose for the three different types of investigated samples Note that for PDF data no error is provided and therefore also the lattice expansion calculated from the PDF fits does not possess an uncertainty. Therefore, the error propagation of the shift from the (440) reflex is provided. See the supporting information for details on how the tabulated values were determined. Sample Shift (440) (%) Expansion in a (%) Radiation dose (kGy) ID31-1-EtOH-H2O -0.36 +- 0.06 0.34 40000 ID31-2-EtOH-H2O -0.53 +- 0.05 0.47 40000 ID31-1-H2O -0.00 +- 0.03 0.00 40000 ID31-2-H2O 0.00 +- 0.07 0.00 40000 ID15-A-1-EtOH-H2O -0.13 +- 0.06 + 195-390 ID15-A-2-EtOH-H2O -0.02 +- 0.04 0.00 195-390 + Data quality for the PDF fit too bad. References Aalling-Frederiksen, O., Juelsholt, M., Anker, A. S. & Jensen, K. M. O. (2021). Nanoscale, 13, 8087-8097. 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PMC10000812 | J Synchrotron Radiat J Synchrotron Radiat J. Synchrotron Rad. Journal of Synchrotron Radiation 0909-0495 1600-5775 International Union of Crystallography 36891843 ve5166 10.1107/S1600577522011997 JSYRES S1600577522011997 Research Papers Development of an insertion device selectively operational as a helical/figure-8 undulator Helical/figure-8 undulator insertion device Tanaka Takashi a* Seike Takamitsu b Kagamihata Akihiro b Aoyagi Hideki b Kai Tomoya b Sano Mutsumi b Takahashi Sunao a Oura Masaki a a RIKEN SPring-8 Center, Koto 1-1-1, Sayo, Hyogo 679-5148, Japan b Japan Synchrotron Radiation Research Institute, Koto 1-1-1, Sayo, Hyogo 679-5198, Japan Svensson S. Editor Uppsala University, Sweden Correspondence e-mail: [email protected] 01 3 2023 13 1 2023 13 1 2023 30 Pt 2 s230200 301307 20 11 2022 19 12 2022 (c) Takashi Tanaka et al. 2023 2023 This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-BY) Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited. A full version of this article is available from Crystallography Journals Online. An insertion device capable of switching the operation mode between helical and figure-8 undulators has been developed. An insertion device capable of switching the operation mode between helical and figure-8 undulators, and thus referred to as a helical-8 undulator, has been developed. It has the advantage that the on-axis heat load can be kept low regardless of the polarization state, even when a high K value is required to lower the fundamental photon energy. This is in contrast to conventional undulators in which the on-axis heat load tends to be significantly high to generate linearly polarized radiation with a high K value, and optical elements can be seriously damaged. The principle of operation, specification and light source performance of the developed helical-8 undulator are presented together with further options to enhance its capability. undulator polarization low heat load pmc1. Introduction Controlling the polarization state of synchrotron radiation (SR) is one of the most important subjects in the development of insertion devices (IDs). The most straightforward way is to employ an elliptically polarized undulator (EPU), whose magnetic field is given in a general form as with k u = 2p/lu, where lu denotes the undulator period, B x,y are the horizontal and vertical components of the magnetic field vector, z denotes the longitudinal coordinate, and B x0,y0 are the peak values of B x,y . The fundamental wavelength l1 of undulator radiation is then defined as with being the so-called (horizontal and vertical) deflection parameters or K values, where g is the relative electron energy, e and m are the charge and rest mass of an electron, and c is the speed of light. It is well known that the spectrum of undulator radiation is quasi-monochromatic at the wavelengths of l1/n, where n is a positive integer referred to as a harmonic order. To control the polarization state flexibly, B x0 and B y0 should be independently tuned, with the magnet configuration satisfying the boundary condition of accelerator operation. For this purpose, a number of concepts have been proposed, and EPUs based on these concepts have been put into practical use (Yamamoto et al., 1989 >; Elleaume, 1994 >; Sasaki et al., 1993 >; Hara et al., 1998 >; Bahrdt et al., 2001 >; Schmidt & Calvi, 2018 >). The EPU works as a linear undulator to generate linearly polarized radiation (LPR) when B x0 B y0 = 0 (B x0 = 0 or B y0 = 0), while it works as a helical undulator to generate circularly polarized radiation (CPR) when |B x0| = |B y0|; more specifically, it can generate horizontally polarized radiation (HPR), vertically polarized radiation (VPR), left-handed CPR, and right-handed CPR, by satisfying the conditions B x0 = 0, B y0 = 0, B x0 = B y0 and B x0 = - B y0, respectively. Now let us turn to a well known issue regarding the operation of linear undulators: when the fundamental energy needs to be rather low, and thus relatively high K values are required (high-K condition), the radiation power coming from high-order harmonics becomes much higher than that from the fundamental radiation. This can potentially result in serious damage to optical elements such as mirrors and monochromators, and thus the heat load should be reduced by limiting the angular acceptance of optical elements usually by closing the aperture of the slit in the front-end section, which results in a significant loss of available photon flux. It should be stressed that this problem in the linear undulators is in contrast to the helical undulators, in which no high-order harmonics are contained in the radiation emitted on-axis, and thus the on-axis heat load is much lower. The figure-8 undulator is an ID proposed to solve the above problem in the linear undulators under the high-K conditions (Tanaka & Kitamura, 1995 >), which generates a magnetic field given as meaning that the period of the horizontal field is twice that of the vertical one; then the electron trajectory projected onto the transverse plane forms a 'figure of eight', and most of the radiation power coming from high-order harmonics diverges off-axis, keeping the linearly polarized fundamental radiation concentrated on-axis. As a result, the on-axis heat load is kept low even under the high-K conditions and is much lower than that of the conventional linear undulators. The figure-8 undulator has another advantage that the 0.5th-harmonic radiation, which is vertically polarized and is halved in energy, is available as well as the horizontally polarized fundamental radiation. Although the maximum photon flux is relatively lower than that of the fundamental radiation (depending on the ratio K x /K y ), it offers a simple scheme to switch the polarization state; to be specific, HPR or VPR are available at the same photon energy just by tuning the gap of the undulator. Figure-8 undulators have been constructed and installed at SPring-8 (Tanaka et al., 1998 >) and ELETTRA (Diviacco et al., 2002 >), as IDs for soft X-ray and vacuum ultraviolet beamlines, respectively, and have been successfully operated for more than two decades. One disadvantage of the figure-8 undulators developed above is that CPR is not available. In 2011, we proposed a new undulator concept (Tanaka & Kitamura, 2011a >) to overcome this difficulty, which offers a scheme to switch the operation mode between helical/figure-8 undulators, and thus is referred to as a 'helical-8 undulator'. Recently, we have built an insertion device based on this concept to generate LPR and CPR with a low on-axis heat load and installed at SPring-8. The purpose of this paper is to report on its design, specification and performance. 2. Principle of operation The magnet configuration of the helical-8 undulator is similar to that of a helical undulator composed of six Halbach undulator arrays as illustrated in Fig. 1 >, where the horizontal and vertical magnetic fields are independently generated by the side arrays (A-D) and central arrays (E, F), respectively. The helicity of CPR can be switched by moving the side arrays along the longitudinal axis by half a period. Helical undulators with this configuration have been constructed and installed at SPring-8 and several SR facilities before. The mechanism to switch the operation mode in the helical-8 undulator is based on a composite-period undulator (CPU) scheme, which was originally proposed to expand the wavelength tunability of X-ray free-electron lasers (Tanaka & Kitamura, 2011b >). The magnetic array in this scheme is modified from a conventional Halbach array to generate a magnetic field given as meaning that it is composed of two different periods, where B 1 and B 2 denote the peak values of the magnetic fields with the period of lu and 2lu. In the following discussions, the former and latter are referred to as the fundamental and double-period components, respectively. Fig. 2 >(a) shows a schematic drawing of an undulator array to generate such a magnetic field. The yellow arrows indicate the magnetization vectors of individual magnet blocks, which are decomposed into two components indicated by blue and red arrows as shown nearby; the former and latter form the fundamental and double-period components, respectively. Now let us consider the case when the top and bottom arrays are shifted by DZ along the longitudinal axis towards opposite directions; it is easy to show that the magnetic field reduces to and then we have which means that the period of this undulator array can be switched between lu and 2lu. As a result, the wavelength tunability can be significantly expanded compared with what is available with conventional undulators. It should be noted that the configuration shown in Fig. 2 >(a) is not efficient in terms of attainable magnetic field strengths. As is well known, the peak field of the Halbach array is proportional to a geometrical factor given as where M is the number of magnet blocks per period, and the magnetization vector of a specific magnet block is 2p/M rotated with respect to adjacent ones (Halbach, 1983 >). Although larger M results in a higher peak field, M = 4 is generally chosen, because G(4) 0.9 is usually acceptable. It is easy to understand that M is effectively 2 for both of the fundamental and double-period components in the configuration shown in Fig. 2 >(a). Recalling G(2) 0.63, the peak field attainable with this configuration is about 30% lower than what is usually available. From a practical point of view, the peak field of the double-period component (B 2) can be lower than that of the fundamental one (B 1); to be specific, B 2 can be as low as B 1/2 to have the same K values. In such a case, we can enhance B 1 at the expense of B 2; an example is illustrated in Fig. 2 >(b), where M = 4 is attained in the fundamental component, but the Halbach condition is violated in the double-period component, which eventually results in lower B 2. Fig. 2 >(c) is another example to proceed this idea further, where the double-period component is generated by tilting the vertically magnetized blocks in the original Halbach configuration. Fig. 2 >(d) shows an alternative configuration, where the phase relation between the fundamental and double-period components is shifted by 90deg. In this case, we have which are identical to equations (1) and (5), or the horizontal magnetic fields of the helical and figure-8 undulators. Now it is obvious that the undulator configuration shown in Fig. 1 >, with the side arrays A-D equipped with the CPU scheme explained in Fig. 2 >(d), works as a helical-8 undulator and can be selectively operated as a helical or a figure-8 undulator. It is worth noting that the relation between B 1 and B 2 depends on the angle th indicated in Fig. 2 >(d), which is referred to as a CPU angle in the following discussions. 3. Specifications and performance evaluation Based on the concept described in the preceding section, we developed a helical-8 undulator with lu of 120 mm and specifications summarized in Table 1 >, as an ID for the soft X-ray beamline BL17SU at SPring-8. The design and specification of the developed helical-8 undulator, which is referred to as HEU120 in this paper, are reported in this section, together with the light source performance experimentally demonstrated. Note that numerical studies presented in the following have been carried out using the numerical codes RADIA (Chubar et al., 1998 >) and SPECTRA (Tanaka, 2021 >). Fig. 3 >(a) shows a photograph of the mechanical frame of HEU120 to hold the magnetic arrays and allow for the vertical motion to open and close the gap. Fig. 3 >(b) shows the bottom side of the magnetic arrays (C, D and F) mounted on a common girder of the mechanical frame; the side arrays (C and D) are equipped with a mechanical function to allow for the phase motion, or the longitudinal shift to switch the operation mode. Phase conditions for respective operation modes are illustrated in Figs. 4 >(a)-4(c); for example, Fig. 4(a) shows the arrangement for the helical mode, where the side arrays generate the fundamental horizontal field without the double-period component. To facilitate the following discussions, let DZ A,B,C,D be the longitudinal shifts of the side arrays A, B, C and D, with the origins (DZ A,B,C,D = 0) being defined as the positions in this condition. By shifting the side arrays in the same direction by -lu/2 (DZ A,B,C,D = -60 mm) as shown in Fig. 4 >(b), the polarity of the horizontal field is flipped and the helicity of CPR is switched. For convenience, the former/latter modes are defined as the CW/CCW (clockwise/counter-clockwise) helical modes. Fig. 4 >(c) shows the arrangement for the figure-8 mode, where the (A, C) and (B, D) arrays are shifted by lu/4 towards opposite directions (DZ A,C = -DZ B,D = -30 mm) to generate the double-period horizontal field without the fundamental component. Note that magnet blocks located in the center of the side arrays are painted yellow to clarify the positions of the side arrays in respective operation modes. The light source performance of the helical-8 undulator is strongly dependent on the ratio K x /K y ; let k h and k l be K x /K y in the helical and figure-8 modes, respectively. It is obvious that the optimum condition in the helical mode is k h = 1, while that in the figure-8 mode depends on a number of conditions such as the acceptable heat load, tunable range, photon flux and degree of polarization. Considering various boundary conditions and other factors besides the optimum conditions mentioned above, the dimensions of the magnet blocks have been determined as shown in Fig. 5 >, where we have three important points to be addressed. First, the magnet block length (20 mm) of the central array is slightly shorter than lu/4 = 30 mm; the extra (10 mm-long) empty spaces are spent by mechanical clamps to fix the magnet blocks onto the common girder. Second, thanks to the relatively wide (30 mm) magnet block of the central array, the magnetic field is sufficiently uniform along the horizontal axis so that the dynamic multipole effect can be neglected. In other words, we do not have to apply special schemes such as multiwire coils to correct the dynamic multipole, as is often required for operation of EPUs. Third, the central array is slightly shifted up/downward with respect to the side array to weaken the vertical field and satisfy the condition k h 1. Besides the dimensions of the magnet blocks described above, the CPU angle th should be optimized according to the light source performance. As an example, we numerically evaluated the expected performance of HEU120 available in the figure-8 mode for three different values of th, i.e. th = 15deg, 45deg and 75deg, which correspond to k l = 0.30, 0.96 and 1.45, respectively, The parameters used in the calculations are summarized in Table 2 >. Note that the fundamental photon energy is 500 eV, and the angular acceptance is four times the angular divergence of the photon beam. Figs. 6 >(a) and 6 >(b) show the light source performances available in the figure-8 mode with the 0.5th-harmonic and fundamental radiation, respectively, where the photon flux (solid lines) and Stokes parameter S 1/S 0 (dashed lines) representing the polarization property are plotted. Because of the large angular acceptance, the photon flux reaches maximum at the photon energy slightly detuned to the lower side, which is common to undulator radiation. The above numerical process was repeated to evaluate the effect of the CPU angle on the light source performance, as summarized in Figs. 7 >(a)-7(d); the K value ratio k l (a), heat load of radiation (b), photon flux (c) and degree of polarization defined as |S 1/S 0| (d) are plotted as a function of the CPU angle. Note that the flux and polarization available at the detuned energy for the 0.5th-harmonic/fundamental radiation (VPR/HPR) are plotted in red/blue lines. As th increases, the heat load is drastically reduced, which is the most important advantage of the figure-8 undulator. In addition, the photon flux of the 0.5th-harmonic radiation increases as well; however, the performance of the fundamental radiation degrades as th. This means that we have to compromise in choosing the value of th, and we have finally chosen th = 45deg focusing on the flux of the 0.5th-harmonic radiation and reduction of the heat load. From a technical point of view, this was a reasonable choice because we have already had several experiences of manufacturing magnet blocks with the 45deg inclined easy axis of magnetization (Bizen et al., 2018 >; Tanaka & Kagamihata, 2021 >). Figs. 8 >(a) and 8 >(b) show the horizontal and vertical magnetic field distributions, respectively, measured at the minimum gap of 20 mm for the three operation modes. As shown in Fig. 8 >(a), the phase and period of the horizontal field change accordingly depending on the operation mode. Note that the vertical field does not depend on the operation mode and thus only one result is shown in Fig. 8 >(b). To evaluate the trajectories for the three operation modes, the field distributions shown in Figs. 8 >(a) and 8 >(b) are integrated twice; the results are plotted in Figs. 8 >(c)-8(e) in terms of the position of an 8 GeV electron projected on the transverse plane, where we find a typical trajectory specific to each operation mode, suggesting that HEU120 works as expected. Fig. 9 > shows the quality of the magnetic field of HEU120 in the three operation modes, where the phase errors evaluated from the field distributions shown in Figs. 8 >(a) and 8 >(b) are plotted as a function of the longitudinal coordinate, together with their RMS (root mean square) values. It is worth noting that only the 0.5th-harmonic and fundamental radiation are needed in HEU120 (applications using high-order harmonics are not supposed), and thus the RMS phase errors around 4deg are sufficiently low; in other words, it is reasonable to say that the quality of HEU120 is close to ideal. Having verified the magnetic performance, HEU120 has been installed in the SPring-8 storage ring as an ID for BL17SU. To characterize the radiation and evaluate its performance quickly, we measured the spatial profiles of radiation in the three operation modes, by inserting an alumina fluorescent screen at the front-end section 19 m from the source; note that the beam current was reduced to 0.8 mA from the nominal one (100 mA), and a 1 mm-thick copper plate was inserted in front of the fluorescent screen to avoid saturation. The measurement results are shown in Fig. 10 > (top), together with the spatial power density calculated with the measured field distributions (bottom). Due to several factors such as absorption by the copper plate and quantum efficiency of the fluorescent screen, the measured profile does not necessarily represent the radiation power density; even so, we find a relatively good agreement between the measured and calculated results. 4. Outlook The experimental results shown in the preceding section strongly suggest that HEU120 has a sufficiently good performance as an ID to generate LPR and CPR with a low on-axis heat load. In other words, the helical-8 undulator concept, i.e. the six-array configuration equipped with the CPU scheme, works fine to switch the operation mode between the helical and figure-8 undulators. Nevertheless, we have to mention that HEU120 developed in this work has a disadvantage that the photon flux of the 0.5th-harmonic radiation (VPR) is rather lower than that of the fundamental radiation (HPR). This is not the case for the conventional EPUs, where the photon flux available for VPR is equivalent to that for HPR. One idea to solve this problem is to apply the CPU scheme to the central arrays E and F as well as the side arrays, so that the vertical magnetic field can be switched according to equation (11) as well as the horizontal one. Then, another operation mode referred to as a 'vertical figure-8 mode' is available, in which the magnetic field is given as Because the above formulas are obtained by swapping the horizontal and vertical magnetic fields of the figure-8 undulator given in equations (5) and (6), the fundamental radiation is vertically polarized in this mode. Furthermore, 'inclined' linear polarization is available as reported in the previous paper (Tanaka & Kitamura, 2011a >), as well as the HPR and VPR. Note that the structure of the mechanical frame becomes more complicated to realize the above scheme, because all of the six arrays should be shifted along the longitudinal axis independently; it is worth mentioning, however, that such an ID to allow for the phase motion of six arrays has been constructed before (Tsuchiya et al., 2016 >). Introducing the vertical figure-8 mode mentioned above simultaneously brings an advantage in the design of the helical-8 undulator, in particular the choice of the CPU angle th. As explained before using Fig. 7 >, we have two points to take care of: the photon flux of the 0.5th-harmonic radiation and reduction of the heat load. It is obvious that the former is no longer important if the vertical figure-8 mode is available. Thus, th can be lower than 45deg to improve the light source performance of the fundamental radiation, assuming that the resultant higher heat load is acceptable, and the technical issue in manufacturing the magnet blocks with an inclined easy axis can be solved. For example, th = 30deg is a reasonable choice as is obvious from Fig. 7 >. Finally, let us compare the helical-8 undulator with another solution known as an 'APPLE-Knot' undulator (Zhang et al., 2020 >). Although a higher degree of polarization is available in the linear polarization modes, the APPLE-Knot undulator cannot be operated as a purely helical undulator; instead, it generates a specially designed magnetic field to produce CPR. As a result, the photon flux of CPR available with the helical-8 undulator is expected to be much higher than that with the APPLE-Knot one. 5. Summary We have presented the development of the helical-8 undulator, a new type of ID to generate LPR and CPR with a low on-axis heat load, by switching the operation mode between the helical and figure-8 ones. The light source performance experimentally demonstrated, together with the options to enhance its capability, definitely indicates that the helical-8 undulator can be a powerful candidate for IDs that need to control the polarization states under the high-K conditions. The authors thank Dr Soutome of RIKEN SPring-8 Center for evaluating the effects of HEU120 on the accelerator operation in SPring-8. Figure 1 Schematic drawing of a helical undulator composed of six Halbach arrays. The definition of the coordinate system in this paper is shown for reference. Figure 2 Four different undulator configurations to generate the composite period magnetic fields. Blue and red lines indicate the magnetic field distributions for the fundamental and double-period components, respectively. Figure 3 Photograph of HEU120 under construction: (a) mechanical frame and (b) magnetic arrays C, D and F. Figure 4 Operation modes of HEU120: (a) CW helical, (b) CCW helical and (c) figure-8 modes. Figure 5 Dimensions (in mm) of magnet blocks in HEU120: (a) top view and (b) cross-sectional view. Figure 6 Comparison of light source performances between three different CPU angles: (a) 0.5th-harmonic and (b) fundamental radiation. Solid and dashed lines show the photon flux and Stokes parameter, respectively. Figure 7 Light source performances plotted as a function of the CPU angle th: (a) K value ratio k l , (b) heat load, (c) photon flux and (d) polarization. Figure 8 Results of the magnetic field measurement at the gap of 20 mm: (a) horizontal and (b) vertical field distributions, and (c-e) projections of electron trajectories (second field integral) evaluated for the three operation modes. Figure 9 Phase errors evaluated from the measured field distributions for the three operation modes. Figure 10 Top: profiles of SR measured by inserting a fluorescent screen. Bottom: radiation power density calculated with the measured field distributions. Table 1 Specifications of HEU120 installed at SPring-8 BL17SU Magnetic period 120 mm Total length 3600 mm CPU angle (th) 45deg Minimum gap 20 mm Maximum K y 4.76 Maximum K x 4.55 (helical) 3.53 (figure-8) Minimum photon energy 223 eV (helical) 272 eV (figure-8) Table 2 Parameters assumed in the numerical study to evaluate the light source performance of HEU120 Electron energy 8 GeV Beam current 100 mA Natural emittance 2.4 nm rad Coupling constant 0.02% Energy spread 0.0011 Betatron functions (x, y) 31.2 m, 5 m Dispersion functions (x, y) 0.146 m, 0 m Angular acceptance 0.1 mrad x 0.08 mrad References Bahrdt, J., Frentrup, W., Gaupp, A., Scheer, M., Gudat, W., Ingold, G. & Sasaki, S. (2001). Nucl. Instrum. Methods Phys. Res. A, 467-468, 21-29. Bizen, T., Kinjo, R. & Tanaka, T. (2018). Phys. Rev. Lett. 121, 124801. Chubar, O., Elleaume, P. & Chavanne, J. (1998). J. Synchrotron Rad. 5, 481-484. Diviacco, B., Bracco, R., Millo, D. & Zangrando, D. (2002). 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PMC10000813 | J Synchrotron Radiat J Synchrotron Radiat J. Synchrotron Rad. Journal of Synchrotron Radiation 0909-0495 1600-5775 International Union of Crystallography 36891854 ok5087 10.1107/S1600577523000747 JSYRES S1600577523000747 Research Papers Evaluation of quantitative synchrotron radiation micro-X-ray fluorescence in rice grain Evaluation of quantitative SR-mXRF in rice grain Limmer Matt A. a Webb Samuel M. b Seyfferth Angelia L. a* a Department of Plant and Soil Sciences, University of Delaware, Newark, DE 19716, USA b Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Menlo Park, CA 94025, USA Kvashnina K. Editor ESRF - The European Synchrotron, France Correspondence e-mail: [email protected] 01 3 2023 15 2 2023 15 2 2023 30 Pt 2 s230200 407416 10 12 2022 27 1 2023 (c) Matt A. Limmer et al. 2023 2023 This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-BY) Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited. A full version of this article is available from Crystallography Journals Online. This work demonstrates which elements are the most easily quantifiable by synchrotron radiation for microprobe X-ray fluorescence (SR-mXRF) imaging in order to understand elemental distributions in plant tissues, with a focus on metals and metalloids in rice grain. This work provides SR-mXRF users with data to understand which elements can be reliably quantified and guidance on how to consider the limitations of the technique to most effectively interpret such data. Concentrations of nutrients and contaminants in rice grain affect human health, specifically through the localization and chemical form of elements. Methods to spatially quantify the concentration and speciation of elements are needed to protect human health and characterize elemental homeostasis in plants. Here, an evaluation was carried out using quantitative synchrotron radiation microprobe X-ray fluorescence (SR-mXRF) imaging by comparing average rice grain concentrations of As, Cu, K, Mn, P, S and Zn measured with rice grain concentrations from acid digestion and ICP-MS analysis for 50 grain samples. Better agreement was found between the two methods for high-Z elements. Regression fits between the two methods allowed quantitative concentration maps of the measured elements. These maps revealed that most elements were concentrated in the bran, although S and Zn permeated into the endosperm. Arsenic was highest in the ovular vascular trace (OVT), with concentrations approaching 100 mg kg-1 in the OVT of a grain from a rice plant grown in As-contaminated soil. Quantitative SR-mXRF is a useful approach for comparison across multiple studies but requires careful consideration of sample preparation and beamline characteristics. arsenic metal(loid)s rice bran rice grain synchrotron radiation microprobe X-ray fluorescence SR-mXRF National Science Foundation, Directorate for Engineering1930806 Angelia L SeyfferthMatt A LimmerNational Institute of Food and AgricultureNIFA-2018-67019-27796 Angelia L SeyfferthUse of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences (contract no. DE-AC02-76SF00515). This research was also funded by the US National Science Foundation, Directorate for Engineering (grant no. 1930806 awarded to ALS and MAL) and the US National Institute of Food and Agriculture (grant no. NIFA-2018-67019-27796 awarded to ALS). pmc1. Introduction The concentration, speciation and localization of elements in plant tissues have implications for both plant survival and consumers of plants. Understanding the localization of toxic and nutrient elements can provide mechanistic insight into plant tolerance and homeostasis (Conn & Gilliham, 2010 >). Therefore, measurements of elemental species and/or localization must also be quantitative. To quantify concentrations of elements, plant parts are typically homogenized, digested and the liquid digest quantitatively analyzed. Obtaining a spatially finer understanding of elemental concentrations via digestion is thus limited to the resolution of dissection, which becomes time-consuming or impractical at smaller scales. Mechanistic understanding requires measurement of the concentrations and/or flows of elements at the scale of the governing phenomena, which may range from tissue to sub-cellular (i.e. millimetre to nanometre). Thus, methods to quantitatively measure the concentration distribution are needed to advance our mechanistic understanding of elemental homeostasis and cycling. Several techniques exist for determining the localization of elements and nutrients in plants; each has advantages and disadvantages. Laser-ablation coupled with inductively coupled mass spectrometry (LA-ICP-MS) allows for quantitative elemental mapping in plant tissues at 10-100 mm resolution, depending on the spot size and sensitivity that the system can achieve. However, quantification can suffer from matrix effects requiring matrix-matched standards, isotope dilution or calibration with doped gels (Becker et al., 2008 >; Pozebon et al., 2017 >; Bauer et al., 2019 >; Pan et al., 2022 >). High-resolution secondary ion mass spectrometry (SIMS, e.g. nanoSIMS) can quantify a wide variety of elements at low concentrations and at sub-cellular spatial resolution (50 nm), but accuracy is affected by the matrix, and few nanoSIMS instruments currently exist (Moore et al., 2012 >). NanoSIMS also has the advantage of resolving isotopes and mapping light elements such as carbon (de Samber et al., 2020 >). Synchrotron radiation microprobe X-ray fluorescence (SR-mXRF) imaging is a widely used, non-destructive technique to measure elemental localization in plant tissues at the micrometre (SR-mXRF) to nanometre (SR-nanoXRF) scale (Kopittke et al., 2014 >; Punshon et al., 2009 >; Seyfferth et al., 2011 >; Zhao et al., 2014 >). Elements investigated in plants by SR-mXRF include As, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Ni, P, S, Se, Si, Tl and Zn, typically in the mg kg-1 range (Punshon et al., 2009 >). A major advantage of SR-mXRF over LA-ICP-MS or nanoSIMS is that it can be combined with microprobe X-ray absorption spectroscopy (mXAS) or X-ray absorbance near-edge structure (XANES) imaging to provide elemental speciation, which is particularly important for elements that exist in various forms. However, for heavier elements, SR-mXRF may be less sensitive and accurate than LA-ICP-MS, especially when the incident X-ray beam cannot reach the K-edge of the desired element (e.g. tungsten and cadmium), requiring the use of the M-edge that yield fewer fluorescent X-rays and may overlap with fluorescent X-rays from the K-edge of lighter elements (VanderSchee et al., 2020 >). Additionally, despite SR-mXRF being a powerful tool for answering questions of elemental localization and speciation, it is generally considered qualitative or semi-quantitative. To date, there have been few attempts to evaluate the quantitative robustness of this technique. Quantitative SR-mXRF is seldom attempted, but quantitative bulk XRF with benchtop units has been reported. Portable or benchtop energy-dispersive XRF (ED-XRF) instruments have been used to quantify Zn, Fe, Ca and Cd in rice grain, and good correlations have been found between ED-XRF and ICP-OES (Paltridge et al., 2012 >; Guild & Stangoulis, 2022 >; Taleon et al., 2020 >; Li et al., 2018 >; Yada et al., 2006 >), suggesting that quantitative SR-mXRF is possible for rice grain. Quantifying elements in any type of sample by SR-mXRF generally requires either thickness-matched standards or, more commonly, standards in a different matrix, such as a thin film. Using standard thin films yields areal concentrations, which are simpler but less informative and less comparable with bulk measurements. Additionally, areal concentrations are problematic for non-flat samples, samples thick enough to attenuate the emitted fluorescence for the element of interest and samples of variable composition, resulting in semi-quantitative measurements. Although not reported in plants, fully quantitative SR-mXRF has been successful in other matrices. In an early method development paper, Mavrogenes et al. (1995 >) quantified Sr content in fluid inclusions in quartz, determining a method detection limit of ~2000 p.p.m. Wang et al. (2010 >) quantified Ca, Cu, Fe and Zn in a mouse brain using certified reference materials and normalizing by Compton scattering. Iron concentrations in human brains measured by SR-mXRF were comparable with ICP-MS measurements (Zheng, Nichol et al., 2013 >) by quantifying with Fe thin films and correcting for fluorescence attenuation with sample thickness (Hopp et al., 2010 >). Numerous researchers have used SR-mXRF to investigate the localization of As species in rice (Oryza sativa L.) because of concerns surrounding human exposure via consumption. Unlike hyperaccumulators, where As levels are sufficiently high to overcome detection limitations, levels of As in rice grain are low (~0.2 mg kg-1). However, the various As species present in the environment and the numerous As-plant interactions make SR-mXRF an ideal tool for this system. Beginning in the soil, SR-mXRF has been used to demonstrate As and Fe co-localization in Fe-rich root plaques (Kramar et al., 2017 >; Neumann et al., 2017 >; Seyfferth et al., 2010 >, 2011 >; Smith et al., 2009 >; Yamaguchi et al., 2014 >; Limmer et al., 2021 >). SR-mXRF has also been used to study interactions of As with rice roots. Kopittke et al. (2014 >) collected XANES spectra at each SR-mXRF pixel of hydroponic rice roots and found that arsenate was reduced to As(III)-thiol complexes as the As(V) moved into the root. Seyfferth et al. (2017 >, 2021 >) reported As accumulation in lateral root junctions and used SR-mXRF at multiple incident beam energies to parameterize maps of arsenite and arsenate. In above-ground tissues, SR-mXRF has been used to investigate the elemental distribution in rice leaves (Wu et al., 2016 >; Zheng et al., 2011 >), internodes (Moore et al., 2014 >; Smith et al., 2009 >; Yamaoka et al., 2010 >; Zheng et al., 2011 >) and nodes (Chen et al., 2015 >; Moore et al., 2014 >; Yamaguchi et al., 2012 >; Zheng et al., 2011 >). Of most importance for human health, SR-mXRF has been used to examine As and other elements in rice grain. Carey et al. (2011 >, 2012 >) performed SR-mXRF microtomography on rice grains exposed to different forms of As and Se. Lombi et al. (2009 >) and Kyriacou et al. (2014 >) analyzed 70 mm-thick grains for As, Cu, Fe, Mn and/or Zn. Lu et al. (2013 >) analyzed 200 mm-thick rice grains and germinating rice grains to identify elemental remobilization during germination. Meharg et al. (2008 >) and Muehe et al. (2019 >) imaged grains split in half, while Zheng et al. (2011 >) imaged whole grains during development to identify As localization. Zheng et al. (2013 >) measured As localization in hydroponically grown plants using hand sections of grains divided longitudinally in half or cross-sections from the middle third of the grain. Though these reports provide information about elemental localization at varying levels of detail, quantification is relative, and differences in sample preparation and beamline characteristics can hinder comparisons between studies. This study aimed to determine the accuracy of SR-mXRF in quantifying the concentration of elements in rice grain. To test this, thin sections were quantified and averaged by SR-mXRF and compared with bulk concentrations measured by ICP-MS after acid digestion. Fluorescence signals were integrated by two different methods to examine their effect on quantification. We also determined the reproducibility of the SR-mXRF method and generated quantitative SR-mXRF maps of elements in rice grains. Method detection limits were estimated, and application considerations were discussed. We report that SR-mXRF can be performed quantitatively in rice grain for Zn, Cu, As and Mn. 2. Materials and methods 2.1. Rice grain Unpolished rice grain (i.e. brown rice) was obtained from 33 unique treatment combinations from several different hydroponic, pot and field studies. Hydroponic studies included experiments with and without arsenic added to the media (Limmer, Wise et al., 2018 >; Limmer & Seyfferth, 2020 >), while other media constituents were generally held constant. Growth chamber pot studies included soils either moderately contaminated with As [16 mg kg-1 soil As (Seyfferth et al., 2016 >)] or spiked with arsenic [25 mg kg-1 As (Teasley et al., 2017 >)] and amended with different Si-rich materials. A field study with Si-rich amendments with soil As at background levels (5 mg kg-1 As) in Delaware was also included (Limmer, Mann et al., 2018 >; Limmer & Seyfferth, 2021 >). Rice varieties included three long grains - 'IR66', 'Jefferson' and 'Lemont' - and one medium grain - 'M206'. All grains were mature and air-dried. 2.1.1. Rice grain ICP-MS analysis Bulk elemental analysis of the dehusked, unpolished rice grain followed previously published methodology (Seyfferth et al., 2016 >). For each treatment combination, ~400 mg of finely ground rice grain was digested in 7 ml of trace metal grade concentrated nitric acid via microwave digestion (Mars 6 Express, CEM Corporation). The vessels were ramped to 200degC over 20 min and held for 10 min. The digested solutions were diluted to 4% acid and analyzed with ICP-MS (Agilent 7500cx) operating in He collision mode. Certified rice flour (NIST 1568a) and acid blanks were included with the digestion. Recovery of the reference material was acceptable for As (103-110%), Cu (79%), Fe (75-84%), K (88-91%), Mg (77-80%), Mn (92%), P (86-87%), S (88-92%) and Zn (88%) (n = 2). 2.1.2. Rice grain thin sections For SR-mXRF measurements, 30 mm-thick grain sections were prepared. Individual grains (4-6) were first embedded in EPO-TEK 301-2FL epoxy, taking care to orient the grain for ease of sectioning. Typically, three grains were placed vertically (for cross-sections) into a small piece of foam, while two grains were laid on their side (for longitudinal sections). For samples from pot studies (soil or hydroponic), each grain on the slide came from the same plant, while grains on the same slide from the field site were from the same treatment combination. Thin sections were prepared by Spectrum Petrographics Inc. (Vancouver, WA, USA) under low-oxygen conditions. Rice grains were confined to a monolayer on a quartz slide and processed using universally standard thin sectioning methods, including surface preparation, mounting to quartz, cutoff, grinding, lapping and polishing. Section types included cross-sections through the center of the grain and longitudinal sections through the center of the grain. 2.1.3. Rice grain SR-mXRF measurements SR-mXRF measurements were conducted on 50 rice grains at Stanford Synchrotron Radiation Lightsource (SSRL) using beamline 10-2 in two separate experiments. The beamline was equipped with a 30 pole, 1.45 Tesla wiggler insertion device and a double-crystal Si(111) monochromator. A pinhole aperture created a spot size of 25 mm. The sample was rastered across the beam using a step size of 25 mm and a dwell time of 300 ms per pixel. The incident X-ray energy was 13 keV, and fluorescence photons were collected with a single-element Vortex detector 45deg from the sample (90deg from the incident beam). Fluorescence signals were integrated using two different methods. First, regions of interest (ROIs) in the fluorescence spectrum were centered on the Ka emission energy, and counts were accumulated in each bin at the beamline (As, Ca, Cl, Cu, Fe, K, Mn, P, S, Si and Zn). This method cannot separate overlapping fluorescent emission lines and thus could be subject to error, particularly for lighter elements where overlapping emission lines are more common. Second, fluorescence spectra were fit during data post-processing at each pixel using the PyMCA module within the Microprobe Analysis ToolKit [SMAK version 1.4 (Webb et al., 2011 >; Sole et al., 2007 >)]. Additional elements not previously included in the ROIs were identified and included (e.g. Ar present in the atmosphere). Fluorescence spectral fitting can also deconvolve overlapping elements and account for other potential artifacts, such as pile-up (Sole et al., 2007 >). For XRF calibration, thin films with known elemental concentrations (Micromatter, Surrey, BC, Canada) were measured during each experiment while keeping all setup and detector parameters constant. Each film contained 20-75 mg cm-2 of a single compound vacuum deposited onto a 6 mm-thick mylar film. After data collection, samples were post-processed using SMAK. Elevated concentrations of Cl in the epoxy allowed for the demarcation of the background from the grain. The fluorescence signal at each pixel was normalized by the incident beam intensity, and the average intensities of elements in the background were subtracted for each element. Normalized intensities from the standard thin films were used to quantify the sample intensities using a one-point calibration. Standard thin films and samples were kept at the same distance from the detector to account for signal attenuation in the air. Areal concentrations were converted to mass concentrations using an assumed density of 1.35 g cm-3 for rice grain and corrected for X-ray attenuation in the material assuming rice grain was similar in composition to 'soft tissue' (a preset material in SMAK). For comparison with ICP-MS data, elemental concentrations were averaged within the grain. For K and Cu, thin films were not analyzed during one of the experiments, so n = 29, whereas, for all other elements, n = 50. To create quantitative maps for elements of interest, the quantitative SR-mXRF data were corrected by the regression line between SR-mXRF and ICP-MS data. In addition, Gaussian blurring was used to lightly smooth the image with a standard deviation of 0.8 in a neighborhood of 3 pixels. The image was also stretched in the x direction to correct for the sample being held at 45deg relative to the incident beam and the detector. 2.1.4. Statistical analysis All regression analyses were performed using SAS 9.4 and PROC REG. Binary flags were included for grain orientation and synchrotron experiments. The experiment flag was never significant and was removed from the models, indicating that the elemental calibration could account for variability in beamline characteristics between experiments. Residuals did not show evidence of heteroscedasticity or non-normality. SR-mXRF method detection limits (MDLs) were estimated following the approach of Twining et al. (2003 >). Fluorescence counts, a discrete random event, follow a Poisson distribution. For simplicity, the background area and sample area were considered equivalent, where C MDL is the estimated MDL (mg kg-1), o is the fluorescence yield as calculated from the standard (counts s-1)/(mg cm-2), x s is the thickness of the sample (cm), t is the dwell time (s pixel-1), p is the number of pixels in the sample, r is the sample density (kg cm-3), a is a dimensionless attenuation factor to correct for sample thickness and n b is the estimated number of counts from the background. Additional details of the derivation are provided in the supporting information. 3. Results 3.1. Agreement between SR-mXRF and ICP-MS The agreement between average SR-mXRF and ICP-MS grain concentrations was minimally affected by grain orientation and fluorescence integration method (Table S1 of the supporting information). Linear models with ICP-MS grain concentration, grain orientation and their interaction found that grain orientation p-values remained >0.1 for all elements tested, and the interaction between grain orientation and ICP-MS grain concentration was insignificant for most elements (Table S1 of the supporting information). Thus, the optimal model for SR-mXRF was a simple linear regression with ICP-MS concentration as the descriptor. Comparing fluorescence integration methods, neither p values nor model fit statistics appreciably differed between methods for most elements, with only sulfur exhibiting differences in distribution (Fig. S1 of the supporting information) and moderate differences in fit slope (Table S1). Because minimal differences were observed between fluorescence integration methods, the simpler and less time-consuming ROI method was used throughout. Rice grain concentrations measured by SR-mXRF and ICP-MS were more strongly in agreement for higher-Z elements than for lower-Z elements (Fig. 1 >). Copper and zinc exhibited the highest agreement between SR-mXRF and ICP-MS, with slopes not significantly different than unity. Arsenic and manganese also showed a significant correlation between SR-mXRF and ICP-MS and a high level of accuracy (As slope = 0.46, Mn slope = 0.58). Potassium and sulfur showed a weak correlation between SR-mXRF and ICP-MS data (R 2 < 0.1 or p > 0.1). Both iron and phosphorous did not show a correlation between the methods (Fig. S2 of the supporting information). Calcium was observed in SR-mXRF measurements, but poor ICP-MS recovery (12%) prevented comparison. Silicon was also observed in SR-mXRF measurements, but its concentration was not certified in the reference material, and its presence in the quartz slide hindered quantification. 3.2. Reproducibility The use of multiple grains placed in a single thin section allowed for method reproducibility to be tested on a subset of samples. Of the 33 unique treatment combinations, multiple grains (2-4) were analyzed for 12 treatment combinations. For each, the relative standard deviation (RSD) was used to measure reproducibility. Note that this also includes any variability between grains from an individual plant, making these biological replicates rather than analytical replicates. The overall average RSD for the elements of interest was 29%. Most elements were more reproducible than this, apart from P and S (Fig. 2 >). Separating by section type (longitudinal versus cross-section) revealed minimal differences in reproducibility except for P and S, in which cross-sectioned grains showed improved reproducibility compared with longitudinally sectioned grains. The maximum RSD never exceeded 100% for these biological replicates and stayed within 50% for As, Fe, K and Mn (combined data). 3.3. Method detection limits Estimated MDLs were generally less than 1 mg kg-1 and were lower for high-Z elements (Table S2). Increasing the number of pixels in the scan slightly decreased the MDLs. Dwell time was held constant to avoid affecting the MDL. Except for As, all average grain concentrations were more than one order of magnitude above their corresponding MDL. For As, the median sample was one order of magnitude greater than the MDL, and seven samples were less than the MDL. 3.4. Quantitative SR-mXRF maps Using the calibrated SR-mXRF data (i.e. Fig. 1 >), quantitative concentration maps were generated for selected grains. Fig. 3 > shows a cross-section of a grain obtained from a field site with low levels of soil As [~5 mg kg-1 (Limmer, Mann et al., 2018 >)]. Several elements were localized in the ovular vascular trace (OVT), stylar vascular trace (SVT) and/or the bran layer. Arsenic, manganese and zinc were highly concentrated in the OVT, with concentrations of ~7, 500 and 100 mg kg-1, respectively. In this grain, only Zn, and to some extent As, showed substantial accumulation in the endosperm. A cross-section of another grain from the same treatment, but with the section taken through the embryo, showed a similar accumulation of most elements in the bran (Fig. S3). However, the OVT and SVT were not apparent, and K, Mn, S and Zn accumulated in the embryo. Interestingly, concentrations of elements in the embryo (Fig. S3) were of the same order of magnitude as concentrations in the OVT in Fig. 3 >. In contrast to these grains grown under low-background As, Fig. 4 > shows a cross-section from a grain exposed to high soil As (~24 mg kg-1) in a pot study (Teasley et al., 2017 >). The deformed grain had localized concentrations of elements similar to those from the low-As treatment, except for As. Arsenic in the OVT was ~20 mg kg-1, approximately 3x the concentration of As in the OVT of the low-As grain (Fig. 3 >). Concentration maps for longitudinally sectioned grains show variable concentrations of elements depending on whether the section was included the OVT. For example, Figs. 5 > and S4 show one grain where the OVT was included in the section, resulting in high concentrations of As, Mn and Zn relative to other grains where the OVT was not evident. Note that As was not uniformly concentrated throughout the OVT, with a hot spot approaching 100 mg kg-1 As. 4. Discussion 4.1. Agreement between SR-mXRF and ICP-MS At this hard X-ray beamline, the agreement between SR-mXRF and ICP-MS concentrations was better for high-Z elements than low-Z elements. Fig. 6 > shows the relationship between the SR-mXRF calibration coefficient (i.e. the fluorescence yield) and the slope of the SR-mXRF/ICP-MS fit line. Elements with larger calibration coefficients had slopes close to the ideal value of unity (i.e. Mn, Cu and Zn). Arsenic was a notable exception, likely arising from the concentrations being close to the SR-mXRF MDL. Iron (not shown) would be expected to perform well, with a calibration coefficient of ~0.25 counts mg-1 cm-2. However, SR-mXRF drastically overestimated the concentration of Fe in the grain (Fig. S2), an artifact we believe resulted from Fe contamination during the sectioning process. Lighter elements, such as S and K, had small SR-mXRF calibration coefficients and slopes much below unity. Phosphorous performed particularly poorly (Fig. S2) with a slope near zero (Fig. 6 >), suggesting either P was not detectable or that the 3D distribution of P could not be captured with 2D sections. Most elements had intercepts with confidence intervals that included zero, indicating adequate agreement at low concentrations, with the accuracy limited by the SR-mXRF MDL. For elements with slopes near unity and intercepts near zero, quantitative SR-mXRF values could be used directly without adjustment. For other elements, calibration coefficients were necessary to generate accurate quantitative maps. The poor performance of SR-mXRF for S and P likely arose from several factors. First, the beamline operating at 13 keV was not ideal for low-Z elements, as shown by the small calibration coefficients. With increasing energy far above the edge, the X-ray absorption cross-section decreases, decreasing the fluorescence yield. Low-Z elements are also intrinsically less efficient producers of fluorescent X-rays due to their higher yield of auger electrons, further decreasing fluorescence yield (Hubbell et al., 1994 >). Additionally, the sample matrix and air strongly attenuate the fluorescent X-rays emitted from these elements. As assumed here, the matrix for fluorescence attenuation correction may be incorrect or not homogeneous. SR-mXRF is typically explicitly used to investigate spatial variability in elemental composition, so assumptions of compositional uniformity can be problematic. This is most problematic when the fluorescence energy of one element is close to (and above) the excitation edge of other elements and when these elements are co-localized at high concentrations (Fendorf & Sparks, 1996 >). This may explain the poor performance of K (due to S and Cl), S (due to Si and P) and P (due to Si and Mg) and highlights the need for fluorescence attenuation corrections that vary in space and/or adaptively adjust with measured elemental intensities. Finally, errors can arise from 3D heterogeneities not captured in 2D thin sections, as some of these elements are present in the embryo at substantially high concentrations. Thus, using quantitative SR-mXRF is likely to perform better for high-Z elements with higher fluorescence yields, lower attenuation by air, fewer strongly absorbing elements (e.g. within 1 keV) and at beamlines designed for the element(s) of interest. Sample-specific factors, such as the 3D heterogeneity and the colocalization of similar elements, will also strongly affect the success of quantitative SR-mXRF. 4.2. Elemental distribution in rice grains The false-color images presented here agree with the literature that many elements primarily accumulate in the bran layer of rice, including Ca, Cu, K, Fe, Mn and P (Kyriacou et al., 2014 >; Lombi et al., 2009 >; Lu et al., 2013 >; Meharg et al., 2008 >; Sakai et al., 2015 >). Others have also shown Fe, Mn and inorganic As accumulation in the OVT (Carey et al., 2011 >). Here we found that As, Mn and Zn most strongly accumulated in the OVT. Previous work has shown that both S and Zn are concentrated in the bran layer but can also slightly penetrate the endosperm to varying degrees (Kyriacou et al., 2014 >; Lombi et al., 2009 >; Meharg et al., 2008 >; Sakai et al., 2015 >). In this work, we observed grains where S was strongly localized to the bran (e.g. Fig. 4 >) and grains where S penetrated the endosperm ( e.g. Fig. S4), suggesting the extent of S localization may vary. Of the elements we quantified, Zn was most able to penetrate the endosperm, although much of the Zn remained in the bran. Additionally, minor longitudinal heterogeneity appears in Zn concentrations in the endosperm, with more Zn penetrating the endosperm near the awn (Fig. 5 >). As previously reported, Cd and Ni are homogeneous throughout the grain, although there are limited observations of these elements due to low concentrations in the grain and the high energy needed to excite the K-edge for Cd (Meharg et al., 2008 >); thus, Cd may be better suited for LA-ICP-MS mapping. Lu et al. (2013 >) found Ca, Fe, K, Mn and Zn accumulated in the embryo, with Fe and Ca mainly in the scutellum and Zn mainly in the plumule and radicle. We observed Cu, K, Mn, S and Zn in the embryo but not As. Importantly, the distribution of elements can also be affected by elemental speciation. There are several reports of such effects for As and Se, with the organic forms dispersed throughout the grain while the inorganic forms accumulate in the bran and OVT (Carey et al., 2012 >; Zheng et al., 2011 >; Zheng, Li et al., 2013 >; Limmer & Seyfferth, 2022 >). Quantitative SR-mXRF can theoretically separate species when species can be identified by differences in their XANES spectra, but we are unaware of any such reports that quantitatively resolve these species. Of all the rice plant parts, elemental concentrations and distribution in grain most directly affect human health. Grain concentration and speciation of toxic elements, such as arsenic, have health-based limits due to exposure risks (Meharg et al., 2009 >). Conversely, Fe and Zn deficiencies affect billions of people globally, resulting in efforts to increase grain concentrations of these essential elements (Slamet-Loedin et al., 2015 >). In both cases, human exposure depends on the extent of elemental localization in the bran and the extent of polishing. For Fe and Zn, chelation with P in the form of phytic acid strongly reduces bioavailability to consumers (Perera et al., 2018 >). Thus, quantitative elemental maps could provide colocalization information and molar ratios of metal:phytic acid. Additionally, because trace elements are a minor fraction of osmolytes in the phloem transported to filling grains, the grain concentration of such elements is governed by the phloem concentration of each element (Zhang et al., 2007 >). Collectively, methods to quantify the localization of elements in grain could inform studies of the regulation of elemental homeostasis in various plant tissues and aid in protecting human health. 4.3. Application considerations The application of quantitative SR-mXRF involves several considerations, many of which are also of interest for qualitative SR-mXRF and SR-nanoXRF [see Donner et al. (2013 >) for a comprehensive discussion of qualitative SR-mXRF considerations]. Fundamental facility considerations include the suitability of the beamline, particularly factors such as the incident beam energy, the size of the incident beam relative to the size of the features of interest and the available detector(s). Elements of interest must have edges (ideally K-edges for most elements) below the incident beam energy available, although sensitivity will decrease as the energy increases farther from the edge. The size of the incident beam must be smaller than the size of the features of interest but large enough to map the desired area in a reasonable amount of time. Finally, the detector must be sensitive enough at the desired energy to measure the low concentrations of the desired element. An He sample chamber is likely to be necessary for low-Z elements to minimize fluorescent X-ray attenuation. Method detection limits, while valuable, are a function of the aforementioned factors and factors discussed below, limiting their direct application to other situations. Regardless, the MDLs in Table S2 and the literature (e.g. Mihucz et al., 2010 >) can provide a first-order approximation in other settings. Performing quantitative SR-XRF at finer spatial resolution (e.g. SR-nanoXRF) is likely to become increasingly difficult as the volume probed by the X-ray becomes more heterogenous at the nano-scale. This is perhaps best addressed by making very thin sections [e.g. 2 mm (de Samber et al., 2020 >)] to minimize the attenuation of fluorescent X-rays by the matrix. Sample preparation is also an important consideration. Because heterogeneous samples are of most interest in SR-mXRF and 2D SR-mXRF sections are most frequently analyzed, determining how to reduce a 3D sample to a 2D sample is crucial. Ideally, the sample should be homogeneous across the third dimension, allowing a 2D thin section to be made. If 3D information is needed, microtomography or confocal SR-mXRF may be better alternatives. Rice grains, except for the embryo, are largely homogeneous along the length of the grain, making cross-sections ideal 2D thin sections. Thus, grain cross-sections can map elemental changes from the bran into the endosperm but cannot provide elemental information along the length of the grain. Furthermore, cross-sections are generally symmetric across the OVT, thereby potentially further minimizing the amount of sample to scan. Determining the optimum sample thickness depends on several factors. Although thicker samples increase fluorescence for high-Z elements, minimal improvement occurs for low-Z elements. For example, in a 30 mm-thick rice grain section, the S fluorescence is only representative of a 10 mm-thick sample due to fluorescence attenuation within the sample. Additionally, high-Z elements in exceptionally thick samples, such as a whole rice grain (e.g. Zheng et al., 2011 >), will complicate interpretation as the 3D sample is projected onto a 2D plane, combining signals from the bran and the endosperm. Even sections of moderate thickness (i.e. 1 mm) can result in blurred images due to heterogeneity in depth (Carey et al., 2010 >). Thick samples also complicate the comparison of low-Z and high-Z elements when samples are not homogeneous with depth, as the fluorescence signal is practically a surface measurement for low-Z elements and a depth-integrated measurement for high-Z elements. An additional consideration is the number of replicate samples to analyze. Because beam time is limited, analyzing replicate samples is unfortunately not often a priority. Thus, conclusions about an entire population may be based on a single rice grain. Analysis of replicate grains here showed that the RSD of mean grain elemental concentrations averaged ~30% but was considerably higher for P and S, which are low-Z elements that are better investigated using a different beamline and/or different experimental parameters (e.g. He atmosphere). However, this RSD does not consider changes in the localization of elements, only the average concentration in a grain, and thus may be a liberal estimate of the variation between replicate samples. Care should also be taken in selecting samples to examine in detail. For example, if several grains are scanned coarsely for a trace element, and the grain with the highest fluorescence is studied at a finer resolution (i.e. 'hot spot' selection), this grain is likely not representative of the population. Given the results of this work, the application of quantitative SR-mXRF to other plant parts seems possible. However, more care must be taken when working with hydrated samples by either collecting data very quickly (e.g. with a Maia detector) or under cryogenic conditions (Castillo-Michel et al., 2017 >) to avoid sample distortion during dehydration. Additionally, plant parts must be large enough to enable the measurement of bulk concentrations for validation. In rice, the nodes are an area of interest because of the heterogeneity and the high concentrations of metals sequestered (Yamaji & Ma, 2014 >). Qualitative SR-mXRF has already been performed in the nodes (Chen et al., 2015 >; Moore et al., 2014 >; Yamaguchi et al., 2012 >; Zheng et al., 2011 >), although quantitative SR-mXRF may be complicated by the 3D heterogeneity of the nodes. Nevertheless, quantitative SR-mXRF would be a powerful tool in the nodes, allowing measurement of sequestered metal(loid) and sulfur (e.g. phytochelatins) concentrations in this critical plant organ. Using quantitative SR-mXRF in other plant organs and plant parts will require validation through comparison with other accurate, quantitative techniques. Supplementary Material Calibration statistics, derivation of method detection limit, comparison of fluorescence integration methods, additional elemental correlations and additional quantitative elemental maps. DOI: 10.1107/S1600577523000747/ok5087sup1.pdf Figure 1 Simple linear regression fits between ICP-MS and SR-mXRF average elemental concentrations (a) As, (b) Cu, (c) K, (d) Mn, (e) S and (f) Zn in rice grains. The shaded region indicates the 95% confidence interval for the best-fit line. The dashed lines indicate the 95% prediction interval for the best-fit line. For K and Cu, n = 29; for all other elements, n = 50. Figure 2 Reproducibility of SR-mXRF measurements of replicate sample scans for grains sectioned longitudinally, cross-sectionally or combining the two sectioning types. The combined values include replicates across and within section types. Note that replicate scans have biological variability as each scan is from a different grain from an individual plant. Error bars denote the maximum and minimum values measured (n = 12 for combined, n = 8 for longitudinal and cross-section). Missing bars indicate that no data were available. Figure 3 Quantitative SR-mXRF elemental concentrations in the cross-section of a rice grain grown in soil with low levels of As (~5 mg kg-1). Scale bar denotes 300 mm. OVT: ovular vascular trace; SVT: stylar vascular trace. Figure 4 Quantitative SR-mXRF elemental concentrations in the cross-section of a rice grain grown in soil with elevated levels of As (~24 mg kg-1). Scale bar denotes 300 mm. EM: embryo. Figure 5 Quantitative SR-mXRF elemental concentrations in the longitudinal section of rice grains grown in soil with elevated levels of As (~24 mg kg-1). Scale bar denotes 1 mm. 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PMC10000814 | Cancers (Basel) Cancers (Basel) cancers Cancers 2072-6694 MDPI 10.3390/cancers15051449 cancers-15-01449 Editorial The Heterogeneous Complexity of Myeloid Neoplasm: Multi-Level Approaches to Study the Disease Awada Hussein Visconte Valeria * Department of Translational Hematology and Oncology Research, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH 44106, USA * Correspondence: [email protected]; Tel.: +1-216-445-6895 24 2 2023 3 2023 15 5 144919 2 2023 22 2 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). pmcMyeloid neoplasms (MNs) include a spectrum of bone marrow malignancies that result from the clonal expansion and arrest of differentiation of myeloid progenitor cells . MNs account for around 25% of all hematological malignancies and typically arise in older patients who are in their seventh or eight decade as they accumulate genetic alterations throughout their lifetime . Perturbation of normal genetic and epigenetic regulation is mostly due to the disruption of key cellular processes such as self-renewal, proliferation, and differentiation . The rise of population aging and exposure to environmental carcinogens have been attributed to triggering bone marrow insults . Myelodysplastic syndromes (MDS), acute myeloid leukemia (AML) and myeloproliferative neoplasm (MPN) remain the most frequently encountered MNs and hence are the subject of the foremost research efforts as well. However, despite the mortality risks of these diseases, their courses are highly variable in terms of response to therapies and survival, which range from weeks to several years. While novel clinical markers were long considered in the past to better characterize these malignancies, the heterogeneous genetic nature of these disorders has left us with more questions. Therefore, seeking explanations behind the underlying pathogenetic processes driving clonal trajectories has prompted us to shift our focus more toward the understanding of the landscape of genetic aberrations instigating these malignancies . Technologies to detect genetic defects have been largely used over the years, spanning from the gold standard karyotype analysis and fluorescence in situ hybridization to plasmid cloning and sequencing, single nucleotide polymorphism (SNP) arrays, comparative genomic hybridization arrays (CGH) and classical bidirectional sequencing . The latter has been replaced by large-volume DNA segment amplified sequencing via next-generation sequencing (NGS), either through whole exome and genome or targeted deep sequencing . The introduction of RNA sequencing (RNA-seq) via NGS further added new dimensions by which sequencing techniques detect polymorphisms spanning from differential expression or alternative splicing (AS). By converting extracted messenger RNAs (mRNA) into complementary DNA (cDNA), RNA-seq provides more accurate differential quantification of highly or lowly expressed genes while examining the function of cellular transcriptome through changes in gene expression, AS, or isoforms . However, RNA-seq may obscure small yet significant differences between individual cell subsets when large amounts of cells are sequenced. Such differences may be critical in the early processes of clonal evolution of myeloid disorders into more aggressive diseases, disease relapse, as well as the assessment of minimal residual disease . Further integration of single-cell DNA sequencing (scDNA-seq) to study the methylome complements scRNA-seq by uncovering methylation patterns influencing the levels of expression detected by scRNA-seq in leukemia cells at single-cell level. Further insights on gene regulatory landscapes may also be inferred by the assay for transposase-accessible chromatin using ATAC-seq which isolates and quantifies non-coding DNA regions. Hence, it provides a wider view of possible targetable active genomic areas of transcriptional influence and subsequent myeloid disease evolution . Diagnostic NGS mutational panels are today an integral part of the management of myeloid disorders as they have been included in diagnostic tests. Along with the techniques mentioned above, diagnostic NGS has yielded the identification of several classes of leukemogenic drivers. Among others, the most relevant include epigenetic DNA methylation regulators (e.g., ASXL1, DNMT3A, EZH2, IDH1, IDH2, TET2), tumor suppressors (e.g., TP53), signaling pathways activators (e.g., FLT3, KRAS, NRAS, JAK2), transcription factors (e.g., CEBPA, ETV6, RUNX1), splicing factors (e.g., SF3B1, SRSF2, U2AF1) as well as shuttling proteins (e.g., NPM1) . The hallmark of unmasking these leukemogenic mutations is to determine the unique genetic profile dictating the course and prognosis of each patient. Indeed, molecular mutations set the groundwork for improved classifications as reflected in the 2022 European Leukemia Net (ELN) recommendations which have further incorporated mutations in BCOR, EZH2, SF3B1, SRSF2, STAG2, U2AF1 and ZRSR2 genes into its adverse risk category of AML on top of their 2017's recommendations for inclusion of ASXL1, RUNX1 and TP53 . Moreover, the International Prognostic Scoring System (IPSS) and its revised version (IPSS-R) are now falling in favor of the molecularly upgraded IPSS-M which incorporates the lesions in 31 genes of confirmed independent impact on MDS prognosis . The new mutation system divides MDS patients into six survival strata . Similarly, a molecular version of the chronic myelomonocytic leukemia (CMML)-specific prognostic scoring system (CPSS-Mol) entailing ASXL1, NRAS, RUNX1 and SETBP1 mutations provides better clinical insights than the original CPSS . Unveiling these mutations further serves as the cornerstone for new differential hematological entities, including benign conditions such as clonal hematopoiesis of indeterminate potential (CHIP) and clonal cytopenia of undermined significance (CCUS). However, perhaps the biggest clinical benefit of understanding the genetics and epigenetics of MNs pertains to identifying targetable mutations whose inhibition may improve disease outcomes. As more and more mutations are discovered, targeted panels are further expanding and thus leave us with more options for precision target therapy. Indeed, the identification of IDH1 and IDH2 as targetable mutations led to the introduction of the IDH1 inhibitors, Enasidenib and Ivosidenib, and the IDH2 inhibitor, Olutasidenib, which are now approved in full effect for refractory/relapsed MN . As the use of modern genomic sequencing techniques has become the tested workhorse in characterizing new myeloid disease diagnoses, further advancement is still seeking to understand molecular interactions while also minimizing the role of human bias. In this context, the application of machine learning (ML) algorithms is becoming one approach to attempt simplification of complexity. As opposed to supervised learning, unsupervised learning prevents human bias and hence may contribute to increasing the accuracy of analyses by eliminating unnoticed errors perpetrated in prioritizing unrepresentative datasets, effect measurements and reporting . Traditional unsupervised Artificial Intelligence (AI) techniques, especially Deep Learning (DL) algorithms, allow the integration of molecular signatures for disease subclassification, prognostication, and prediction of treatment response. More importantly, it allows the integration of multi-omics in one model . This is the case of novel studies performed in our laboratory by combining genomic and transcriptomic data of MNs . For example, DL techniques succeeded in the novel subcategorization of newly diagnosed AML patients regardless of their primary or secondary subtypes into four clusters of invariant molecular features and unique prognoses . In this multicenter study of 6,788 AML patients, the unsupervised analysis resulted in 97% cross-validation accuracy, far superior to the 74% accuracy yielded when applying standard supervised analysis attempting to use molecular patterns to predict traditional path-morphologic classifications . Similar unsupervised learning approaches have identified 14 distinct molecular clusters of clinical heterogeneity in MDS . Each of these clusters has its unique pathobiological associations, treatment responses, and prognosis . ML approaches are also capable of unmasking the morphological consequences of specific molecular signatures while also tracing the evolutionary origins of MNs . In another study, 1079 MDS patient specimens were sequenced in order to define an association between molecular profiles and bone marrow morphologic characteristics and clinical traits . Five unique morphological profiles with distinct clinical features were identified, among which profile 1 was mostly high-risk MDS while the low-risk disease predominant profiles 2, 3, 4 and 5 were characterized by pancytopenia, monocytosis, megakaryocytosis and erythroid dysplasia, respectively . In turn, the low-risk MDS group was classified into eight genetic groups, which served as the basis for subsequent geno-morphologic combinations . Six geno-morphologic signature associations were yielded and hence improved our understanding of the impact of genetic alterations on clinic-morphologic traits . So far, the diagnosis of MN fairly relies on bone marrow studies, yet these studies are subject to inter-observer variability bias as the diagnosis might be challenging in certain scenarios, especially in patients with pancytopenia or minimal dysplasia . Radakovich et al. instead developed an ML model for MN diagnosis based on genomic and clinical data only; thus, it can be used to empower diagnostic decision-making in cases of uncertainty. In this international multicenter study, a gradient-boosted DL strategy was adopted as the geno-clinical features of 2697 patients with MDS or one of the MPN disorders were used to train their model . The final model retained 15 geno-clinical variables, including JAK2, KRAS, and SF3B1 status, and then proved to accurately differentiate MDS, MPN subclasses, as well as benign conditions like CHIP, CCUS and Idiopathic cytopenia of undetermined significance with AUROC of 0.951 and 0.926 for the test and training cohorts, respectively . Moreover, ML techniques may be helpful in developing personalized models that predict treatment response and thus aid in personalizing treatment selection according to each patient's characteristics. Along with this line of research, Nazha et al. screened the genomic architecture of a cohort of 433 MDS patients with varying responses to the hypomethylating agents (HMA) azacitidine and decitabine . By utilizing an unbiased ML recommender system, mutational signatures composed of two to three mutations in ASXL1, BCOR, EZH2, NF1, RUNX1, SRSF2 and TET2, were identified as an association with HMA resistance . The result had an accuracy rate of 87% and 93% in the training and validation cohorts, respectively . Such finding is further enforced by the estimation of around 30% of HMA-treated MDS patients had at least one of the signatures, and thus using ML techniques could have accurately predicted HMA resistance and the need to consider other therapeutic options . Despite its proven utility, the implementation of AI methods in the daily clinical setting certainly remains challenging. It requires physicians to understand AI basics, as well as institutions to be capable of implementing its networks in their medical records. The latter brings about several hurdles, including the need for electronic types of medical records in order for AI to easily recall the large volumes of patient data that are necessary for its proper functioning. In addition, having the appropriate logistics, such as experienced data scientists, dedicated clinician-scientists, availability of the required technological tools, the volume and adequacy of data used to develop AI models, as well as clinically meaningful needs and standards for AI development are all prerequisites for its successful implementation in clinics. Author Contributions H.A., V.V. Conceptualization and writing; All authors have read and agreed to the published version of the manuscript. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Integration of genomic and transcriptomic features using machine learning. Schematic representation of the possible combination of results derived from techniques used to study gene variations and RNA profiles including chromatin analysis. The figure was created using BioRender.com. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. References 1. Murati A. Brecqueville M. Devillier R. Mozziconacci M.J. Gelsi-Boyer V. Birnbaum D. Myeloid malignancies: Mutations, models and management BMC Cancer 2012 12 304 10.1186/1471-2407-12-304 22823977 2. Smith A. Howell D. Patmore R. Jack A. Roman E. 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PMC10000815 | Int J Environ Res Public Health Int J Environ Res Public Health ijerph International Journal of Environmental Research and Public Health 1661-7827 1660-4601 MDPI 10.3390/ijerph20053795 ijerph-20-03795 Article Stigma, Sociodemographic Factors, and Clinical Factors Associated with Psychological Distress among COVID-19 Survivors during the Convalescence Period: A Multi-Centre Study in Malaysia Abdul Taib Nur Iwana Conceptualization Investigation Writing - original draft Writing - review & editing 1 Nik Jaafar Nik Ruzyanei Conceptualization Formal analysis Writing - review & editing Supervision 2* Azman Nazirah Conceptualization Formal analysis Investigation Writing - original draft 2 Leong Bin Abdullah Mohammad Farris Iman Conceptualization Formal analysis Writing - review & editing Visualization 3 Mohamad Kamal Nurul Ain Conceptualization 2 Baharudin Azlin Conceptualization Project administration 2 Bin Abdullah Muhammad Najib Project administration 4 Chidambaram Suresh Kumar Project administration 5 Adlan Alif Investigation 5 Tan Loong Hui Investigation 4 Tamilselvam Satya Investigation 4 Mohamed Said Mohd Shahrir Project administration 6 Abd Samad Anuar Project administration 7 Binti Dollah Siti Nordiana Project administration 8 Tchounwou Paul B. Academic Editor 1 Department of Psychiatry, Faculty of Medicine and Health Sciences, University Malaysia Sarawak, Kota Samarahan 94300, Malaysia 2 Department of Psychiatry, Faculty of Medicine, University Kebangsaan Malaysia, Kuala Lumpur 56000, Malaysia 3 Department of Community Health, Advanced Medical and Dental Institute, Universiti Sains Malaysia, Kepala Batas 13200, Malaysia 4 Department of Psychiatry & Mental Health, Hospital Sungai Buloh, Ministry of Health Malaysia, Sungai Buloh 47000, Malaysia 5 Department of Medicine, Hospital Sungai Buloh, Ministry of Health Malaysia, Sungai Buloh 47000, Malaysia 6 Department of Medicine, Faculty of Medicine, University Kebangsaan Malaysia, Kuala Lumpur 56000, Malaysia 7 Health Technology Assessment Section, Medical Development Division, Ministry of Health Malaysia, Putrajaya 62590, Malaysia 8 Department of Psychiatry, Hospital Angkatan Tentera Tuanku Mizan, Kuala Lumpur 53300, Malaysia * Correspondence: [email protected] 21 2 2023 3 2023 20 5 379508 12 2022 09 2 2023 15 2 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). High rates of psychological distress among COVID-19 survivors and stigmatisation have been reported in both early and late convalescence. This study aimed to compare the severity of psychological distress and to determine the associations among sociodemographic and clinical characteristics, stigma, and psychological distress among COVID-19 survivors across two different cohorts at two different time points. Data were collected cross-sectionally in two groups at one month and six months post-hospitalisation among COVID-19 patient from three hospitals in Malaysia. This study assessed psychological distress and the level of stigma using the Kessler Screening Scale for Psychological Distress (K6) and the Explanatory Model Interview Catalogue (EMIC) stigma scale, respectively. At one month after discharge, significantly lower psychological distress was found among retirees (B = -2.207, 95% confidence interval [95% CI] = -4.139 to -0.068, p = 0.034), those who received up to primary education (B = -2.474, 95% CI = -4.500 to -0.521, p = 0.014), and those who had an income of more than RM 10,000 per month (B = -1.576, 95% CI = -2.714 to -0.505, p = 0.006). Moreover, those with a history of psychiatric illness [one month: (B = 6.363, 95% CI = 2.599 to 9.676, p = 0.002), six months: (B = 2.887, CI = 0.469-6.437, p = 0.038)] and sought counselling services [one month: (B = 1.737, 95% CI = 0.385 to 3.117, p = 0.016), six months: (B = 1.480, CI = 0.173-2.618, p = 0.032)] had a significantly higher severity of psychological distress at one month and six months after discharge from the hospital. The perceived stigma of being infected with COVID-19 contributed to greater severity of psychological distress. (B = 0.197, CI = 0.089-0.300, p = 0.002). Different factors may affect psychological distress at different periods of convalescence after a COVID-19 infection. A persistent stigma contributed to psychological distress later in the convalescence period. COVID-19 survivors psychological distress perceived stigma post-hospitalisation This research received no external funding. pmc1. Introduction Malaysia reported their first COVID-19 case in January 2020, which was transmitted from international travelers. In March 2020, a more massive outbreak occurred from an annual mass religious assembly in Kuala Lumpur, and the public was advised to practise social distancing. The Government of Malaysia implemented the first lockdown via a Movement Control Order (MCO) nationwide following a drastic increment in COVID-19 cases beginning on 18 March 2020. In Malaysia, moderate to very high levels of psychological distress as a result of the COVID-19 pandemic were similarly reported . Anxiety, particularly, was reported to be severe among university students, Malaysian women who were Malays and pregnant suffered from a loss of income during the pandemic . Another study in a neighbouring country, Myanmar, reported more psychological distress among those who were self-employed and older than 45 years old . Multiple studies have shown that the rate of psychological distress among COVID-19 survivors in early convalescence was high, and a high level of post-traumatic stress was seen especially among those who were symptomatic . A systematic review and meta-analysis found that a high prevalence of depression, anxiety, insomnia, and PTSD was documented among COVID-19 survivor regardless of gender, group, or region . These psychological complications were precipitated by a lack of control among infected people, job losses, wage losses, and uncertainty about the future . Both female gender and the persistence of symptoms were risk factors in developing psychological distress among COVID-19 survivors 9 months after discharge . Moreover, a study among COVID-19 survivors in the Philippines found that the prevalence of anxiety and depression 8 weeks after discharge were significantly reduced . However, the available evidence regarding long-term psychological distress among COVID-19 survivors is still insufficient. It has been shown that people who were impacted financially due to COVID-19 and drank alcohol in the past four weeks were more likely to have higher levels of psychological distress . A systematic review showed that, among health care workers during the pandemic, factors such as a younger age, the female gender, and a low monthly household income were associated with psychological distress . Similarly, some sociodemographic factors were found to be associated with psychological distress after being infected with COVID-19, such as age, gender, employment status, and perceived stigma . Stigmatisation is not uncommon during disease outbreaks. The stigma during the COVID-19 pandemic was mainly associated with those who were infected with it, those at risk of being infected, such as healthcare workers, and those from a particular race and groups that were linked to the initial spread of the illness . The fear of a disease with an unknown cause may lead to stigmatisation. Among the Malaysian public, a study found that higher levels of psychological distress were associated with higher levels of fear of COVID-19 . A local study among healthcare workers found that higher cautious attitudes towards COVID-19 significantly predicted higher anxiety scores . This was further explored in another study which revealed that the "fear of COVID-19" and "stress of COVID-19" were associated with psychological distress among health care workers . Multiple studies also concluded that the fear associated with the pandemic, containment measures, high numbers of people infected, and deaths were associated with high prevalence rates of psychological distress across populations . The lack of knowledge regarding COVID-19, as it was a relatively new disease, may have had a large contribution in the development of the stigma during the pandemic. Fear of the infection among the public led to negative feelings, such as anxiety, anger, resentment, hostility, and disgust. This led to social rejection and the discrimination of people who were being labelled. Similar occurrences of stigma were reported in different countries around the world during the pandemic . However, the data were still limited in exploring the association between stigma and psychological distress among COVID-19 survivors. Studies have shown that more severe symptoms of anxiety and stress were reported among those with chronic diseases and a history of medical and/or psychiatric illnesses . This may be due to postponement, inaccessibility to medical services and treatment, and sensitivity to external stressors associated with the pandemic . Moreover, psychiatric ill health at follow-up was found to be associated with persistent physical symptoms, such as breathlessness and myalgia. Multiple studies reported significant psychiatric morbidity, such as anxiety and depression after 6 months of acute infection and hospitalisation for COVID-19 . Coping style has also been reported to be associated with psychological distress during the COVID-19 pandemic. Those with a positive coping style during the pandemic may have promoted emotional well-being, whereas those with a negative coping style showed higher levels of psychological distress . One strategy to cope with psychological distress is to seek psychological assistance, which can be in the form of counselling . A study among college students in China during the pandemic found that those who sought counselling had higher fear, depression, and trauma scores compared to those who did not seek counselling. They also showed that experience with seeking psychological help, as well as perceived mental health, could effectively predict psychological help-seeking behaviour . Most of these studies were conducted among the public; thus, data were limited regarding counselling-seeking behaviour among COVID-19 survivors. At the time of writing, the peak of the COVID-19 pandemic had passed, and normal living had resumed in many countries. However, the recurrence of a similar pandemic in the future is possible, as many new emerging pathogens have been identified that could be a potential public health threat . The negative impact of the COVID-19 pandemic on mental health indicated that we were not fully prepared to cope with its negative consequences. This study aimed to compare the severity of psychological distress among COVID-19-infected survivors and to determine the associations among sociodemographic and clinical characteristics, stigma, and psychological distress among survivors across two different cohorts at two different time points (one month and six months post-hospitalisation). To our current knowledge, this is the first study conducted among COVID-19 survivors in Malaysia to investigate psychological and psychosocial impact, particularly exploring stigma. Understanding more about the different factors affecting mental health among COVID-19 survivors can provide a basis for appropriate intervention to support our patients and to prevent detrimental effects in future pandemics. 2. Materials and Methods 2.1. Study Design and Respondents This study recruited COVID-19 patients following their discharge from three centres, i.e., Hospital Sungai Buloh (a designated COVID-19 government hospital in Malaysia), Hospital Canselor Tuanku Muhriz (HCTM) (a public teaching hospital) and the Agro Exposition Park Serdang (MAEPS) Quarantine Centre. Data were collected cross-sectionally at two different points, i.e., the first group at one month post-hospitalisation (between April 2020 and October 2020) and the second group at six months post-hospitalisation (between November 2020 and July 2021). Cases were recruited during spikes in COVID-19 cases in Malaysia during March 2020 and October 2020 until January 2021. Subjects were recruited via consecutive sampling during both study periods. The inclusion criteria included: (1) those who had been diagnosed with COVID-19 and had a positive COVID-19 Polymerase Chain Reaction (PCR) test, (2) those who were discharged from the three designated centres, (3) those who were 18 years old and above, and (4) those who were able to read and write in the English or Malay languages. Moreover, patients were excluded from the study if they were: (1) medically or mentally unstable or (2) non-Malaysians. All discharged COVID-19 patients who met all the study's selection criteria were invited to participate in the study via electronic mail, phone call, or text message. Before signing an informed consent form, respondents who voluntarily agreed to participate were informed about the study's procedures, purposes, participation benefits and risks, and assurance of anonymity, as well as their right to withdraw from the study at any point in time. Those who provided consent were asked to complete the questionnaires using an online survey platform, Google Forms. Data cleaning was performed to remove duplicate responses. The sample size was estimated by using the G*Power 3.1.9.7 sample size calculator for estimating the sample size of two independent means, where type I error (a) = 0.05, Power (1 - b) = 0.8, allocation ratio = 1:1, and effect size = 0.35 (with reference to a study on the psychological impact of COVID-19 on COVID-19 survivors in China ). The estimated sample size needed was 244 subjects (inclusive of 20% dropout), whereby 122 subjects were needed per group/cohort. 2.2. Data Collection and Measures Subjects were identified and recruited consecutively from the discharge registries of COVID-19 patients for the study period from the three centres. Selected patients were contacted and informed regarding the study. Respondents who consented to participate were divided according to their discharge dates into either the one-month or six-months post-hospitalisation groups. All respondents were administered a sociodemographic and clinical characteristics questionnaire, the Malay version of the Kessler Screening Scale for Psychological Distress (K6), and the Malay version of the Explanatory Model Interview Catalogue stigma scale (EMIC) during their assessments. The outcome variable of this study was the level of psychological distress. It was measured using the Kessler Screening Scale for Psychological Distress (K6). The K6 assesses distress based on questions about anxiety and depressive symptoms that a person has experienced in the most recent 4-week period. It is a six-item self-rated psychological screening instrument developed by Kessler et al. (2002) . The Malay version of the K6 was validated by Tiong et al. (2018) with good reliability and validity and exhibited good internal consistency with a Cronbach's a of 0.859. It registered a sensitivity and specificity of 78.1% and 75.8%, respectively . The Explanatory Model Interview Catalogue stigma scale (EMIC) is a 15-item self-rated scale originally designed to measure stigma among patients with leprosy. Higher scores indicate higher levels of perceived stigma. It was shown to be a valid and reliable tool in assessing stigma among recovered patients with COVID-19, with acceptable internal consistency with a Cronbach's a of 0.79 . The adaptation of the Malay version for the EMIC scale was performed by two independent bilingual native Malay-speaking language professionals, and the backwards translation into English language was carried out by another two bilingual native English-speaking language professionals who had not seen the original English version of the EMIC. Then, the translated and back-translated copies of the EMIC were examined by a panel of experts before a draft of the Malay version of the EMIC was constructed. The draft of the Malay version of the EMIC was administered to 20 COVID-19 survivors to screen for semantic quality, comprehensibility, redundancy of words and sentences, and duration of administration. Following feedback from the interviews of the 20 COVID-19 survivors, the semantic quality, comprehensibility, redundancy of words and sentences, and duration of administration were acceptable, and hence, no further amendments were made by the panel of experts. The Malay version of the EMIC was then administered to the COVID-19 survivors in this study for the validation study. The EMIC-SS-M reported an acceptable internal consistency with a Cronbach's a of 0.727, and its domains reported an acceptable Cronbach's a ranging from 0.708 to 0.795. EFA and CFA confirmed that the EMIC-SS-M consisted of 15 items in 4 domains . Data on gender, age, ethnicity, employment and income, marital status, education status, previous medical or psychiatric illness, and counselling-seeking behaviour were recorded. The response to gender was reported in two groups: male or female. Age was recorded as a continuous variable. Ethnicity was recorded as either Malay or non-Malay. Employment status was recorded in three groups: retired, unemployed/housewife/students, and employed. Monthly income was reported in three groups: those who earned less than RM 5000/month, those who earned between RM 5000 to RM 10,000/month, and those who earned more than RM 10,000/month. Marital status was assessed in two groups: married and single/divorced/separated. Education status was reported in three groups: those who obtained up to primary, secondary, and tertiary education. Responses to history of medical and psychiatric illnesses and counselling seeking behaviour were recorded either as "Yes" or "No". 2.3. Statistical Analysis All data were analysed using the Statistical Package for Social Sciences, version 26 (SPSS 26; SPSS Inc., Chicago, IL, USA). Descriptive statistics for socio-demographic and clinical characteristics, as well as the total EMIC score and total K6 score, were reported. All categorical variables (gender, age, ethnicity, employment and income, marital status, education status, previous medical or psychiatric illness, and counselling seeking behaviour) were reported with a frequency and percentage. Moreover, all continuous variables (K6 and EMIC scores) were not normally distributed (p > 0.05 under the Kolgomorov-Smirnov test) and were thus reported with the median and interquartile range (IQR). The difference in sociodemographic and clinical characteristics between the one-month and post six-months post-hospitalisation groups of COVID-19 survivors were evaluated with Pearson's chi-squared test, whereas the difference in total K6 and EMIC scores between the two groups were evaluated using the Mann-Whitney U test. The associations between socio-demographic and clinical variable, the EMIC scores (independent variables), and the K6 scores (dependent variable) in the two groups at different periods after discharge were measured using a multivariate general linear model with bootstrapping with 2000 replications. The statistical significance was p < 0.05, and all p values were two-sided. 2.4. Ethics This study was approved by the Medical Research Committee of the Universiti Kebangsaan Malaysia Medical Centre (UKM PPI/111/8/JEP-2020-352) and the Medical Research and Ethics Committee of the Ministry of Health Malaysia (NMRR-20-1288-55105), and it abides by the regulations of the 1964 Declaration of Helsinki and its subsequent amendments. 3. Results 3.1. Respondent Characteristics A total of 371 respondents were enrolled in the study, with 219 in the post one-month hospitalisation group and 152 in the post six-months hospitalisation group. All respondents' socio-demographic and clinical characteristics are summarised in Table 1. In the context of socio-demographic characteristics, the two groups of COVID-19 survivors differed in employment status and monthly income. Those in the one-month post-hospitalisation group consisted of more of those who were unemployed/housewifes/students (39.7%) compared with more employed respondents in those in the six-months post-hospitalisation group (75.7%; p < 0.001). Those in the one-month post-hospitalisation group consisted of a larger proportion of people with a monthly income between RM 5000 to RM 10,000/month (84.0%) compared with a larger proportion of people with a monthly income of less than RM 5000 in those in the six-months post-hospitalisation group (62.5%; p < 0.001). In terms of clinical characteristics, only counselling-seeking behaviour registered a significant difference between the two groups of COVID-19 survivors, whereby the proportion of respondents who sought counselling services was less in the one-month post-hospitalisation group (31.4%) compared with those in the six-months post-hospitalisation group (41.4%; p = 0.031). In addition, the level of perceived stigma was significantly higher in the one-month post-hospitalisation group (median = 11.0, IQR = 9.0) compared with that of the six-months post-hospitalisation group (median = 5.5, IQR = 8.0; p < 0.001). No significant difference in the level of psychological distress was noted between the two groups of COVID-19 survivors. 3.2. Associations among Demographic and Clinical Characteristics, Perceived Stigma, and Psychological Distress among COVID-19 Survivors One Month after Discharge The associations between the demographic and clinical characteristics, perceived stigma, and severity of psychological distress among COVID-19 survivors one month after discharge are presented in Table 2. The multivariate general linear model indicated that employment status, monthly income, education status, history of psychiatric illnesses, and counselling-seeking behaviour were significantly associated with psychological distress among COVID-19 survivors after one month of being discharged from the hospital. It was found that significantly lower psychological distress was documented in those who retired (B = -2.207, 95% confidence interval [95% CI] = -4.139 to -0.068, p = 0.034) compared with those who were employed, in those who only received up to primary education (B = -2.474, 95% CI = -4.500 to -0.521, p = 0.014) compared with those with tertiary education, and those with an income of more than RM 10,000 per month (B = -1.576, 95% CI = -2.714 to -0.505, p = 0.006) compared with those with an income of RM 5000 to 10,000 per month, respectively. In contrast, those with a history of psychiatric illnesses (B = 6.363, 95% CI = 2.599 to 9.676, p = 0.002) compared to with those without a history, as well as those who sought counselling services (B = 1.737, 95% CI = 0.385 to 3.117, p = 0.016) compared with those who did not, had a significantly higher severity of psychological distress. Although a higher perceived stigma was documented in this group, it was not significantly associated with psychological distress (p = 0.487). 3.3. Associations among Demographic and Clinical Characteristics, Perceived Stigma, and Psychological Distress in COVID-19 Survivors Six Months after Discharge At six-months after being discharged from the hospital, significantly higher psychological distress was documented in respondents who had a history of psychiatric illness (B = 2.887, CI = 0.469-6.437, p = 0.038) compared with those without a history and in those who exhibited counselling-seeking behaviour (B = 1.480, CI = 0.173-2.618, p = 0.032) compared with those who did not seek counselling services, respectively. In contrast, a higher degree of perceived stigma contributed to the greater severity of psychological distress among COVID-19 survivors after six months of discharge (B = 0.197, CI = 0.089-0.300, p = 0.002). The associations among socio-demographic and clinical characteristics, perceived stigma, and the severity of psychological distress among COVID-19 survivors six months after discharge from the hospital are illustrated in Table 3. 4. Discussion This study compares the severity of psychological distress and the associations among socio-demographic and clinical characteristics, perceived stigma, and severity of psychological distress among COVID-19 survivors at one month and six months post-hospitalisation. We found that there was no difference in the severity of psychological distress among COVID-19 survivors between the one month and six months groups. Our findings depict that COVID-19 survivors may have had persistent and long-term distress even after they recovered from the acute infection. This finding is consistent with a study of COVID-19 survivors conducted in another country, whereby the authors found that, after 9 months of being infected by COVID-19, about 19% of patients still reported psychological distress . However, different factors were found to be associated with psychological distress at these different time points after discharge. At one month after discharge, those with a higher income were associated with less distress. This is supported by other studies, which have reported that individuals with the lowest socio-economic status were the most vulnerable to psychological distress . As the COVID-19 pandemic negatively impacted the economy through multiple restrictions that brought businesses and jobs to a standstill and possibilities of job retrenchments, those with a lower income were affected more, as they had less savings to prepare for these threats. Those with a higher income may have had more financial security in the face of these economic difficulties. This study also found that retired patients and those with primary school education had less distress at one month after discharge. There is accumulating evidence of retirement had positive effects on mental health, likely because of relief from stressful work, increased leisure time, increased physical activity, longer sleep duration, and fewer sleep difficulties . Those with primary school education were found to have lower distress, which could be explained by lower levels of insight into the significance of emotional symptoms leading to less-reported psychological symptoms . However, as denoted by our study, these socio-demographic characteristics (monthly income, employment status, and education level) were no longer protective against psychological distress after being labelled as COVID-19 survivors as time progressed. Hence, psychological or psychosocial factors may have played a role in maintaining the severity of psychological distress as time progressed after one was labelled as a COVID-19 survivor, as COVID-19 survivors in our study did not exhibit depreciation in the severity of psychological distress when we compared the COVID-19 survivors at one month and six months post-hospitalisation. At both one and six months post-hospitalisation, higher psychological distress was reported in those with a history of psychiatric illness, and they exhibited counselling-seeking behaviour. This is supported by previous studies, which have pinpointed that the pandemic negatively affected those with mental illness, leading to a higher rate of relapse . Psychological distress is a positive predictor of attitude towards seeking counseling, especially in those who were mental-health literate . This finding is also echoed by another study in China among college students, which revealed that those who sought counselling had higher fear, depression, and trauma scores compared to those who did not seek counselling . The experience of seeking psychological help and perceived mental health can effectively predict psychological help-seeking behaviour . We found that the level of perceived stigma was higher in the earlier recovery period. This could be explained by insufficient knowledge and contradictory information about COVID-19, as it was a relatively new disease at that time. Stigma were also perpetuated by anxiety of getting infected and regarding the use of protective measures, with much misinformation in social media during the initial phase of the pandemic. The process of stigmatisation involves four main components, which are (1) labelling which personal characteristics are signaled or noticed as conveying important differences, e.g., being infected with COVID-19; (2) stereotyping which differences are linked to undesirable characteristics, e.g., those who were infected were not disciplined and not adherent to the preventive measures; thus, they were seen as having a bad attitude; (3) separating the normal group and the labelled group; and (4) discrimination of the labelled group through devaluation, rejection, and exclusion from the community . In order to adequately combat stigma, Thornicroft identified three core problems where anti-stigma measures can be directed . The core problems consist of problems of knowledge (ignorance), attitudes (prejudice), and behaviour (discrimination) . Reductions in stigma over time could be due to continuous efforts by governmental and non-governmental bodies in the country and globally to provide correct information on COVID-19. This reduced the damaging effects of the infodemic during COVID-19 and increased empathy towards population groups at risk of stigmatisation. Our results also show that a persistent stigma contributed to psychological distress at the later phase of recovery. The association of a higher stigma with higher psychological distress has been consistently shown in multiple studies in the context of various medical conditions, such as schizophrenia, obesity, and infertility . Despite the overall level of stigma being lower for those in the six-months post-hospitalisation group, a more persistent level of stigma contributed significantly to psychological distress in this period. Information overload regarding the dangers of COVID-19 may instill fear, which may also lead to a stigma. This may explain the persistent stigma despite many educational efforts regarding the infection. The findings of our study should be interpreted in consideration of several limitations. Firstly, the respondents' characteristics in our study were derived from three centres, not the whole of Malaysia's COVID-19 patient population, therefore restricting the generalisability of our findings. Secondly, the demographic parameters of the two groups of respondents were not sufficiently matched, limiting the comparison between these groups. Third, our respondents were representative of two cross-sectional cohorts despite comparing COVID-19 survivors sampled at two different post-COVID-19 infection time points. As a result, the researchers were unable to confirm the associated factors reported in this study as causal. Furthermore, there were potential selection and response biases, as we only included those who were medically stable upon discharge from the hospital via an online data collection method which may have excluded patients who were not technologically savvy. Furthermore, data collection after six months may have led to recall bias, as it involved retrospective data and could be a negatively selected cohort that included more of those who were unemployed and those with counselling-seeking behaviour, as employed respondents may have been too busy to participate. Finally, we did not include other possible factors that may have contributed to psychological distress, such as coping methods and social support, which may be confounding factors in this study. There is still limited literature on assessing changes in stigma levels across time and their association with stress. Therefore, we recommend that future longitudinal cohort studies are conducted to confirm our findings, including assessments of coping strategies, social support, and changes in stigma over time with their association with stress. Despite these limitations, this study is the first to determine the psychological distress factors, including stigma, among COVID-19 patients in Malaysia. Based on our findings, we included a few recommendations which could enhance the mental well-being of infected survivors during an infection pandemic. Because our findings indicate that a lower socioeconomic status may worsen psychological distress, financial assistance should be provided for those infected survivors who struggle with financial constraints. As those who were employed fared worse regarding mental health status compared with those who were retired, the government should emphasise screening for work-related stress among the working class and offer sufficient online counselling services to help them curb work-stress. Efficient and sufficient mental health services such as telemedicine (online mental health consultation during movement lockdown) and regular mental health follow-up services for those with a history of psychiatric illness should also be implemented to ensure that the mental health of this group of COVID-19 survivors is safeguarded. The perceived stigma among COVID-19 survivors should also be curbed effectively. In a systematic review of 24 studies on strategies for reductions in stigma on diseases, psychoeducation was the most common strategy and was effectively used to counter stigma . 5. Conclusions From this study, we conclude that different factors may affect psychological distress at different periods of convalescence after a COVID-19 infection. COVID-19 does not discriminate against who it infects, and various factors, as highlighted in this study, change its mental health outcome, such as monthly income, employment status, education status, history of psychiatric illness, and counselling-seeking behaviour. Furthermore, persistent stigma contribute to psychological distress, and this suggests the need to apply psychosocial intervention to curb psychosocial issues among COVID-19 survivors, such as regular mental health screenings at primary care for those at risk and providing sufficient mental health services, such as rapid referrals to specialist centres, psychiatric consultations via face-to-face methods, or regular teleconsultation. This study provides valuable data for clinicians regarding the need to screen for perceived stigma among COVID-19 survivors, such as exploring how they view themselves with illnesses (i.e., low self-esteem or guilt) during follow-up consultations, and the need to provide psychosocial interventions, such as psychoeducation, to combat stigmatising perceptions. Social work assistance should be sought to curb factors that worsen psychological distress, such as financial status, work-related stress, those with a history of psychiatric illness, and the perceived stigma of being infected with COVID-19. Currently, COVID-19 survivors in Malaysia undergo follow-up in their primary care clinic after discharge. Unfortunately, these consultations commonly focus on physical symptoms, whereas mental health symptoms appear to be overlooked. If detected, these symptoms are usually self-reported by patients instead of being detected by the clinician through active screening. Our findings not only emphasise the need to actively screen mental health symptoms but also suggest the need for interventions such as psychoeducation, psychiatric referrals, or telemedicine psychiatric consultations that many psychiatric centres provided following the COVID-19 pandemic. Acknowledgments We would like to express gratitude to all patients and staff from the Hospital Canselor Tuanku Mukhriz, Hospital Sungai Buloh, and MAEPS Quarantine Centre who were involved in this study. We would also like to thank the Director General of Health Malaysia for his permission to publish this article. Author Contributions Conceptualisation, N.I.A.T., N.R.N.J., N.A., N.A.M.K. and M.F.I.L.B.A.; methodology, N.I.A.T., N.R.N.J. and M.F.I.L.B.A.; formal analysis, M.F.I.L.B.A.; investigation, N.I.A.T., N.A., A.A., L.H.T., M.S.M.S. and S.T.; data curation, N.I.A.T., N.R.N.J., N.A. and M.F.I.L.B.A.; writing--original draft preparation, N.I.A.T.; writing--review and editing, N.I.A.T., N.R.N.J. and M.F.I.L.B.A.; supervision, N.I.A.T. and M.F.I.L.B.A.; project administration, A.B., M.N.B.A., S.K.C., A.A.S. and S.N.B.D. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement This study was approved by the Medical Research Committee of the Universiti Kebangsaan Malaysia Medical Centre (UKM PPI/111/8/JEP-2020-352) and the Medical Research and Ethics Committee of the Ministry of Health Malaysia (NMRR-20-1288-55105), and it abides by the regulations of the 1964 Declaration of Helsinki and its subsequent amendments. Informed Consent Statement Informed consent was obtained from all subjects involved in this study. Data Availability Statement The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy and confidentiality concerns. Conflicts of Interest The authors declare no conflict of interest. ijerph-20-03795-t001_Table 1 Table 1 Sociodemographic and clinical characteristics of participants. Variables Subjects One Month after Discharge Subjects Six Months after Discharge p-Value Number of Participants (n = 219) Percentage (%) Number of Participants (n = 219) Percentage (%) Age 32.0 # 22.0 $ 34.0 # 19.0 $ 0.329 Gender: Female 80 36.5 54 35.5 Male 139 63.5 98 64.5 0.930 Ethnicity: Malay 168 76.7 125 82.2 Non-Malay 51 23.3 27 17.8 0.248 Employment status: Retired 14 6.4 10 6.6 Unemployed/housewife/student 87 39.7 27 17.8 Employed 118 53.9 115 75.7 <0.001 * Monthly household income: <RM 5000 0 0.0 95 62.5 RM 5000-RM 10,000 184 84.0 43 28.3 >RM 10,000 35 16.0 14 9.2 <0.001 * Marital status: Married 98 55.3 83 54.6 Single/divorced/separated 121 44.7 69 45.4 0.062 Education status: Primary education 9 4.1 8 5.3 Secondary education 60 27.4 50 32.9 Tertiary education 150 68.5 94 61.8 0.411 History of psychiatric illness: No 211 96.3 146 96.1 Yes 8 3.7 6 3.9 0.884 History of medical illness: No 169 77.2 113 74.3 Yes 50 22.8 39 25.7 0.531 Counselling-seeking behaviour: No 148 67.6 86 56.6 0.031 * Yes 71 31.4 66 43.4 Median total EMIC score 11.0 # 9.0 $ 5.5 # 8.0 $ <0.001 * Median total K6 score 9.0 # 7.0 $ 10.0 # 5.0 $ 0.272 * Statistical significance at p < 0.05, # = median, $ = interquartile range. ijerph-20-03795-t002_Table 2 Table 2 Multivariate general regression model with bootstrapping with 2000 replications for socio-demographic and clinical characteristics, perceived stigma, and severity of psychological distress among COVID-19 patients after one month of being discharged from the hospital. Variables B BCa 95% Confidence Interval Standard Error p-Value Lower Upper Age -0.033 -0.084 0.021 0.025 0.170 Gender: Male Reference Female 0.629 -0.588 1.815 0.563 0.248 Ethnicity: Non-Malay Reference Malay -0.092 -1.351 1.274 0.631 0.874 Employment status: Employed Reference Retired -2.207 -4.139 -0.068 1.090 0.034 * Unemployed/housewife/student -0.751 -1.839 0.284 0.606 0.218 Monthly household income: RM 5000-RM 10,000 Reference >RM 10,000 -1.576 -2.714 -0.505 0.556 0.006 * Marital status: Married Reference Single/divorced/separated 0.326 -0.990 1.689 0.703 0.647 Education status: Tertiary education Reference Secondary education -0.516 -1.850 1.046 0.679 0.481 Primary education -2.474 -4.500 -0.521 1.027 0.014 * History of psychiatric illness: No Reference Yes 6.363 2.599 9.676 1.865 0.002 * History of medical illness: No Reference Yes 0.127 -1.215 1.407 0.628 0.832 Counselling-seeking behaviour: No Reference Yes 1.737 0.385 3.117 0.648 0.016 * Total EMIC score -0.028 -0.113 0.046 0.041 0.487 * Statistical significance at p < 0.05. ijerph-20-03795-t003_Table 3 Table 3 Multivariate general regression model with bootstrapping with 2000 replications for socio-demographic and clinical characteristics, perceived stigma, and severity of psychological distress among COVID-19 patients after six months of being discharged from the hospital. Variables B BCa 95% Confidence Interval Standard Error p-Value Lower Upper Age -0.032 -0.126 0.060 0.037 0.395 Gender: Male Reference Female -0.079 -1.524 1.269 0.698 0.918 Ethnicity: Non-Malay Reference Malay -0.273 -2.269 1.769 0.964 0.808 Employment status: Employed Reference Retired 0.071 -2.830 4.694 1.891 0.960 Unemployed/housewife/student -0.610 -2.025 1.069 0.755 0.431 Monthly household income: <RM 5000 Reference RM 5000-RM 10,000 0.808 -1.799 3.053 1.390 0.549 >RM 10,000 0.451 -2.297 2.877 1.372 0.731 Marital status: Married Reference Single/divorced/separated 0.326 -1.415 2.317 0.925 0.659 Education status: Tertiary education Reference Secondary education -0.516 -2.025 1.069 0.755 0.431 Primary education -2.474 -2.830 4.694 1.891 0.960 History of psychiatric illness: No Reference Yes 2.887 0.469 6.437 1.523 0.038 * History of medical illness: No Reference Yes 1.136 0.801 2.895 0.827 0.188 Counselling-seeking behaviour: No Reference Yes 1.480 0.173 2.618 0.681 0.032 * Total EMIC score 0.197 0.089 0.300 0.051 0.002 * * Statistical significance at p < 0.05. 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PMC10000816 | Age as a breast cancer (BC) prognostic factor remains debatable. Several studies have investigated clinicopathological features at different ages, but few make an age group direct comparison. The European Society of Breast Cancer Specialists quality indicators (EUSOMA-QIs) allow a standardized quality assurance of BC diagnosis, treatment, and follow-up. Our objective was to compare clinicopathological features, compliance to EUSOMA-QIs and BC outcomes in three age groups (<=45 years, 46-69 years, and >=70 years). Data from 1580 patients with staged 0-IV BC from 2015 to 2019 were analyzed. The minimum standard and desirable target on 19 mandatory and 7 recommended QIs were studied. The 5-year relapse rate, overall survival (OS), and BC-specific survival (BCSS) were also evaluated. No meaningful differences in TNM staging and molecular subtyping classification between age groups were found. On the contrary, disparities in QIs compliance were observed: 73.1% in <=45 years and 46-69 years women vs. 54% in older patients. No differences in loco-regional or distant progression were observed between age groups. Nevertheless, lower OS was found in older patients due to concurrent non-oncological causes. After survival curves adjustment, we underscored evidence of undertreatment impacting BCSS in >=70 years women. Despite a unique exception--more invasive G3 tumors in younger patients--no age-specific differences in BC biology impacting outcome were found. Although increased noncompliance in older women, no outcome correlation was observed with QIs noncompliance in any age group. Clinicopathological features and differences in multimodal treatment (not the chronological age) are predictors of lower BCSS. breast neoplasms neoplasms staging age groups aged quality indicators health care combined modality therapy outcome assessment health care prognosis undertreatment National Funds through FCT--Fundacao para a Ciencia e a Tecnologia, I.P.UIDB/4255/2020 Associated LaboratoryLA/P/0053/2020 FCT-supported fundsUIDB/00051/2020 UIDP/00051/2020 The article publication was supported by National Funds through FCT--Fundacao para a Ciencia e a Tecnologia, I.P., within CINTESIS, R & D Unit (reference UIDB/4255/2020) and within the scope of the project RISE, Associated Laboratory (reference LA/P/0053/2020). ASB, acknowledges FCT-supported funds from UnIC@RISE (UIDB/00051/2020 & UIDP/00051/2020). pmc1. Introduction Age as a prognostic factor in breast cancer (BC) remains debatable due to the lack of standardized comparative studies on the impact of multimodal treatment on age groups . A relative consensus exists on the better prognosis of BC due to more favorable and indolent tumor biology in older women . Consequently, the trend toward de-escalating therapy proceeds, often without a routine comprehensive geriatric evaluation to guide personalized multidisciplinary team (MDT) decision-making . Irrespective of age, a guiding principle of BC treatment is to consider patients' BC cure probability to minimize a preventable cause of death . Survival is the key-measure of cancer treatment effectiveness . The EUROCARE-5 BC sub-analysis showed significant differences in cancer survival between 29 European countries and worse outcomes in older patients . Cancer registries should provide an estimate of population-based cancer incidence, and survival . Ideally, a central anonymized online European database would enable standardized comparability of cancer staging, treatments, and outcomes. The European Society of Breast Cancer Specialists (EUSOMA) certification process includes a set of quality indicators (QIs) that certified breast units should follow, enabling a standardized audit of BC diagnosis, treatment, and follow-up . QIs are useful strategic tools that enhance the quality assurance of clinical practice . However, they do not currently include verifiable outcome measurements to draw inferences on survival or age as a prognostic factor. The latest EUSOMA position paper update emphasizes the likely correlation between undertreatment and worse BC outcome in elderly patients. Non-penalizing compliance and flexible QIs structure to allow a personalized therapeutic approach in older adults are discussed but not formally assumed . The current aging population and the socioeconomic impact of the associated higher cancer incidence strengthens the ethical commitment and clinical challenge of treating older women with BC through well-supported evidence . Despite heterogeneous physiology and competing causes of mortality, recent studies have shown the negative impact of omitting multimodal BC treatment in older adults . This single-center study was based on the patient cohort at the Breast Center of S. Joao University Hospital (BC-CHUSJ), certified by EUSOMA since 2017. The aims were to compare clinicopathological features, compliance to EUSOMA QIs and related survival outcomes in three pre-specified age groups (<=45 years, 46-69 years, and >=70 years) from 2015 to 2019. 2. Materials and Methods 2.1. Study Design A benchmarking audit with international standards is an ethical imperative to strive for good clinical care . Our voluntary candidacy for EUSOMA certification, obtained in 2017, provides access to useful mandatory yearly monitoring of our daily work. The EUSOMA central database (eusomaDB) enabled quantifying QIs compliance, performing data analysis and benchmarking, and creating an opportunity for clinical research. This observational study sought to contribute to answering the age-specific issue raised in a previous study whether the poorer outcome in older patients results from a minimalist therapeutic attitude, noted by lower QIs compliance, or whether it results from tumor (and patient) characteristics that are difficult to compare . We analyzed the clinicopathological features and compliance of QIs in three different age groups over a 5-year period from 2015 to 2019. 2.2. Study Population All patients with a newly diagnosed in-situ or invasive BC for 5 years (2015-2019) were included. Age groups were categorized as <=45 years, 46-69 years, and >=70 years according to the epidemiological profile of the population-based screening in Portugal. Patients with recurrent disease or patients who had only partial treatment at our center were excluded. 2.3. Data Collection The primary outcome was to evaluate the impact of clinicopathological features and compliance to EUSOMA QIs on survival by age group. We retrospectively analyzed anonymized data from our patients in the eusomaDB. Hospital ethics committee approval to prospectively collect patients' data in the eusomaDB was obtained (CES 93-16). The clinicopathological and immunohistochemistry (IHC) features were recorded according to EUSOMA standards. No gene expression studies for therapeutic decisions were considered. The QIs were selected from the latest 2017 update of the EUSOMA working group . From the 17 QIs main groups, we selected all the 19 mandatory and 7 recommended indicators: 9 on diagnosis, 12 on surgery and loco-regional treatment, and 5 QIs systemic-treatment-related. No QIs on staging, counselling, follow-up, and rehabilitation were considered. The QIs minimum standard and desirable target completeness were recorded in the three age groups. The completeness of registry data has been certified annually by our data-manager and validated centrally by eusomaDB. As QIs for outcome measurements are lacking, we also analyzed the relapse rate, overall survival (OS), and BC-specific survival (BCSS). Patients were followed up until 31 December 2020. The median follow-up was 2.57 years (95% CI [1.30; 4.01]). 2.4. Statistical Analysis QIs were computed for each age group using a proportional test to assess the QI performance. For continuous variables, normality was assessed by visual inspection of the data distribution, supported by QQ-plot analysis. If normally distributed, the results were summarized by the mean and standard deviation; otherwise, the median and interquartile range were described. Absolute (number) and relative (%) frequencies were reported for categorical variables. The Pearson kh2 test and Fisher's exact tests were used to assess the differences between the independent categorical variables across the pre-defined groups. The Kruskal-Wallis rank-sum test was used to evaluate statistically significant differences between the medians of the three age groups. Survival analyses were performed using Cox proportional hazards models and the Kaplan-Meier method. A competitive risk analysis was performed to untangle BC from all-cause mortality. Internal validation was performed using bootstrap to assess the Cox models' robustness regarding the events (death from all causes and BC). In brief, the models were run 1000 times with replacement (with event stratification), and the concordance index was gauged. All statistical analyses were performed using the survminer (version 0.4.9) and survival (version 3.2.13) packages, using R language (v.4.1.2) . p-values < 0.05 were considered statistically significant. 3. Results Patient characteristics are described in Table 1. A total of 1580 patients staged 0-IV were analyzed. A normal age distribution was observed [Figure S1], with the age extremes being proportional (19.6% were <=45 years and 21.8% were >=70 years). One-third of <=45 years and 46-69 years women were overweight or obese, contrasting with 21.5% in >=70 years women. The proportion of 46-69 years women referred from population-based screening was 30.6%. Slightly lower physical examination accuracy was observed for the detection of malignancy in this age group. More multicentric/multifocal lesions were identified at younger ages, probably due to the more widespread use of magnetic resonance imaging (MRI). The median tumor size was 17 mm in 46-69 years women, compared to 20 mm in the other two age groups. Axillary staging was statistically different: positive axilla was more prevalent in age extremes (29.0% in <=45 years and 21.0% in >=70 years). No meaningful difference was observed in the proportions of 0-III TNM stages between age groups. There was a lower prevalence of stage IV at diagnosis in the youngest women (1.0% in <=45 years versus 3.6% in 46-69 years and 3.5% in >=70 years). Invasive grade 3 (G3) tumors were significantly more in <=45 years women (53.3%) than in 46-69 years (35.1%) or >=70 years (26.0%) women. The proliferation activity index (Ki67) was selectively studied in invasive G2 tumors; however, it was not performed in most cases, namely in 86.0% of older women. The proportion of luminal tumors was identical, apart from a lower prevalence of luminal-B tumors in >=70 years women (15.0% in <=45 years versus 8.0% in >=70 years), as well as the triple negative (TN) tumors distribution. Concerning HER2 overexpression, we found a significant difference between age groups: 21.0% in <=45 years, 16.0% in 46-69 years, and 10.0% (and 5.8% not assessed) in >=70 years women. Surgery, with a predominance of breast conservative surgery, was the first treatment option in all age groups. Most significant was the difference in the omission of surgery: 17.4% in older women, 3.0% in 46-69 years, and 0.6% in <=45 years. The proportion of younger women who began their treatment with neoadjuvant chemotherapy (37.4%) was identical to that of older women who started with endocrine therapy (32.3%). Adjuvant chemotherapy or radiotherapy was significantly less undertaken in older patients (12.8% and 47.7%, respectively). To emphasize the Table 1 contingency cross-table significance values that are driving the observed significances, we provide in Table S3, the adjusted standardized residuals. The QIs compliance is given in Table 2. Of the 19 selected mandatory QIs, we reached threshold requirements in 9 QIs in all three age groups, in 3 of them with 100% completeness (in cancers with a pre-operative diagnosis, in cancers discussed by a multidisciplinary team, and in HER2 invasive cancers treated with neo-adjuvant chemotherapy plus trastuzumab). In contrast, we did not achieve the minimum requirement in mandatory 4 QIs in any age group (in pathological and IHC characterization of invasive and non-invasive cancers and pN+ invasive cancers receiving post-mastectomy radiotherapy). Regarding the recommended QIs, we achieved 100% completeness in invasive cancers' clinical and imagiological axillary staging in all age groups. Still, we failed to meet the minimum standard in 2 QIs, in the waiting time until first treatment and in immediate reconstruction after mastectomy. The QIs compliance was 73.1% in the youngest and 46-69 years women, and 54.0% in the older patients. In the <=45 years group, we attained the desired target in 11 of 19 mandatory QIs and 2 of 7 recommended QIs. The minimum standard was reached additionally in 3 mandatory QIs and 3 recommended QIs. In the 46-69 years group, the desirable target was achieved in 13 mandatory QIs and in 2 recommended QIs and the minimum standard was reached in 4 QIs (1 mandatory and 3 recommended QIs). In the >=70 years group, we reached the desired target in 8 obligatory QIs and 2 recommended QIs and the minimum standard in another 3 mandatory QIs and 1 recommended QI. The 5-year loco-regional relapse showed no disparities between age groups. The same was found for distant progression with no significant difference in the distribution of sites of metastatic disease between younger or older patients [Table S1]. We observed a considerable difference in >=70 years women's 5-year OS . The higher mortality found in older patients was mainly explained by concurrent non-oncological causes of death [Figure S2] and faded when considering the BCSS . Survival differences according to age are mainly explained by differences in tumor grade (but not tumor size), axillary stage and combined multimodal treatment [Figure 2]. 4. Discussion Chronological age should not be a determinant of BC prognosis or treatment . This is well-defined in the youngest women thanks to several consensus meetings, but despite similar experts' efforts, a therapeutic stigma on advanced age persists in daily practice nowadays . The postulated consensus that advanced age is associated with a more favorable tumor biology (more luminal tumors, fewer TN or HER2 tumors, lower proliferative rates) that allows less intensive treatment is contradicted by the observed poorer outcome of BC in older patients . Although several studies have investigated clinicopathological features at different ages, few made an age group direct comparison . Our cohort showed no statistically significant age pattern disparities when compared to other studies . As reported in previous studies, the median tumor size corresponded to a cT1 stage in all age groups . More relevant was the significant difference regarding multicentric/multifocal lesions more frequently found at younger ages. Contrary to several studies , on TNM pathologic staging and molecular subtypes, we found no significant differences between age groups. There were three exceptions in younger patients: a more prevalent positive axilla, a lower prevalence of stage IV at diagnosis, and a higher prevalence of luminal-B tumors. However, a more aggressive biology of BC in younger patients was only significantly observed in one pathological feature: a higher prevalence of invasive G3 tumors. Likewise , no significant differences were found between age groups regarding hormonal status. The same was observed concerning the proportion of TN tumors. As for HER2-positive tumors, a difference was seemingly uncovered, though inconclusive, since HER2 over-expression was not studied in 5.8% of older patients. Like Ki67 (not performed in 85.9% of >=70 years), this incomplete IHC study regarding HER2 status in older adults discloses a potential preconceived advanced age intention-not-to-treat . Considering genetic assessment, there was inevitably an age bias, as more younger patients were studied, and more genetic mutations were identified in <=45 years women. Even so, two BRCA mutations were identified in >=70 years patients. International guidelines, such as the EUSOMA QIs, should be regarded as recommendations for excellence in clinical care . The obtained EUSOMA certification with annual monitoring of their QIs helped to improve our quality control. The QIs also validated our cohort conclusions about diagnostic and therapeutic options. Some disparities in the QIs compliance were observed between age groups. We corroborate the reported difficulty of covering every case on QIs since, over time, some patients needed a non-standard approach which was not easy to audit. As in the previously cited study devoted to the variation in compliance to EUSOMA QIs by age , we noted a significant lower compliance to QIs in older patients. This did not occur in the other two age groups. Concerning the BC diagnosis (initial clinical and imagiological study, axillary staging, and pre-operative needle-biopsy confirmation of cancer), we reached the mandatory QIs target recommendations (with a minor exception in younger patients). We did not match the QIs minimum requirements in any age group in the characterization of in situ or invasive disease. Still, the pathological requirements in those two QIs substantially changed during our study period. From the recommended diagnostic QIs, we achieved the minimum requirements for the MRI study and the genetic counseling referral apart from older women. The most astounding of our audit, which demanded corrective action, was the noncompliance in any age group of the non-obligatory <=6 weeks' waiting time to start treatment. We reached almost complete QIs compliance regarding surgical treatment in all age groups with two exceptions: the omission of sentinel node biopsy in nine older women with cN0 invasive BC and not having reached the immediate reconstruction after mastectomy QIs target, most evidently in older women. Regarding radiation therapy, noncompliance in three mandatory QIs was observed in >=70 years women. The minimum requirements for post-mastectomy radiotherapy were not achieved in all age groups. Endocrine therapy was appropriate in all age groups, but existing QIs were not discriminative for chemotherapy in luminal tumors. Noncompliance of mandatory QIs regarding systemic treatment of TN tumors was observed in older patients. Otherwise, in the HER2-positive tumors we reached the two QIs target recommendations for all age groups. In summary, our MDT discussion, surgical treatment (except for immediate reconstruction), endocrine therapy, and anti-HER2 therapy were above the mandatory thresholds in all age groups. However, this was not observed for adjuvant radiotherapy in pN+ disease or adjuvant chemotherapy in TN tumors, mainly for >=70 years women . Nonetheless, a possible correlation between EUSOMA QIs noncompliance and possible undertreatment in older patients could not be concluded, because neither the former is sufficiently discriminatory in monitoring BC best practices and outcomes , or includes age-specific standards, such as geriatric covariates, nor is the latter consensually defined in the literature . The EUSOMA QIs assess compliance of mandatory variables to adequate BC diagnosis and treatment but are not helpful as a tool for predicting an objective outcome, such as the 5-year BCSS. Without irrefutable evidence , and despite the observed differences in multimodal treatment, the clinical outcome data reported in our cohort showed no significant difference in 5-year loco-regional relapse or distant progression between the three age groups. Nevertheless, a significant difference was found regarding vital status, and concurrent non-oncological causes of death could not be the only explanation for the higher mortality in older patients . After adjusting survival curves for the Cox hazard proportional model, we underscored evidence of undertreatment impacting BCSS in >=70 years women [Figure 2 and Figure S2 and Table S2]. As previously reported , the older age subgroup was being comparatively less treated. A higher proportion of omission of surgery and a less frequent option for adjuvant chemotherapy or radiotherapy was observed with a deleterious impact on survival [Figure 2]. Multivariable analysis showed no significant difference regarding age group (HR 0.87, p = 0.698), but evidence of undertreatment impacting survival: surgery (HR 0.11, p < 0.001) and adjuvant radiotherapy (HR 0.37, p = 0.004) allow a reduction of BC-specific mortality risk. Furthermore, an analysis of the competing risks [Figure S2] showed that younger women die more from cancer. In older women, there is no difference between causes of death in the first two years. Nonetheless, they die more from non-oncological causes after that period, reversing the trend observed in younger women. This underscores the significance of scrutinizing BCSS in addition to OS, and considering, through appropriate multimodal treatment, BC as a preventable cause of mortality in older patients. Finally, Cox model robustness was assessed using bootstrapping with replacement. One thousand runs were performed for each event (death from all causes and BC), and the concordance index (C-index over 0.8) suggests robust models for both events [Figure S3]. 5. Conclusions Our study sought to contribute to demystifying the common misconception of age-specific differences in BC biology impacting outcomes, which we did not observe in daily practice. Despite a unique exception--a higher prevalence of invasive G3 tumors in <=45 years patients--a more aggressive biology was not observed at younger ages nor the contrary in older women. Although there was increased noncompliance in older women, no outcome correlation was observed with QIs noncompliance in any age group. Chronological age should not be considered a prognostic factor in BC, as clinicopathological features and multimodal treatment differences are the main predictors of lower BCSS in older patients. For this age subgroup, earlier diagnosis, and personalized treatment--supported not by a frailty demand (to de-escalate therapy) but by a multidimensional geriatric assessment to pursue biological age and socio-family circumstance--are demanded. Supplementary Materials The following supporting information can be downloaded at: Figure S1: Histogram and descriptive statistics of age groups; Figure S2: Competitive risk curves of death from all causes and death from breast cancer; Figure S3: Internal validation using bootstrap to assess the Cox models' robustness; Table S1: Patient's prognosis stratified by age group; Table S2: Cox proportional hazards progression model. Table S3: Adjusted Standardized Residuals Pearson's chi-squared. Click here for additional data file. Author Contributions Conceptualization, F.O., A.S.B. and B.P.; methodology, F.O., A.S.B. and B.P.; software, A.S.B.; validation, A.S.B. and B.P.; formal analysis, A.S.B. and B.P.; investigation, F.O., A.S.B. and B.P.; resources, F.O., A.S.B. and B.P.; data curation, F.O.; writing--original draft preparation, F.O., A.S.B. and B.P.; writing--review and editing, F.O., I.A. and J.L.F.; visualization, F.O., A.S.B., B.P., I.A. and J.L.F.; supervision, J.L.F.; project administration, F.O.; funding acquisition, F.O. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The study was conducted following the Declaration of Helsinki, and S. Joao University Hospital ethics committee approval to prospectively collect patients data in eusomaDB was obtained (CES 93-16). Informed Consent Statement Patient written consent was waived due to this study being based on a retrospective analysis of anonymized data from Breast Center of S. Joao University Hospital patients in eusomaDB. Data Availability Statement All research data was archived in the hospital's database and can be provided, if requested, to the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 All-cause mortality and BC-specific mortality by age group (* p < 0.05, *** p < 0.001). Figure 2 Adjusted Cox proportional hazard regression model for all-cause mortality and BC-specific mortality (* p < 0.05, ** p < 0.01, *** p < 0.001). cancers-15-01446-t001_Table 1 Table 1 Cohort clinicopathological characteristics stratified by age group. Characteristic N <=45 y, N = 310 1 46-69 y, N = 926 1 >=70 y, N = 344 1 p-Value 2 Sex 1580 0.6 Woman 310 (100%) 923 (99.7%) 344 (100%) Man 0 (0%) 3 (0.3%) 0 (0%) Body Mass Index (BMI) 1580 <0.001 Not evaluated 91 (29.4%) 421 (45.5%) 235 (68.3%) <18.5-24.9 133 (42.9%) 186 (20.0%) 35 (10.2%) 25.0-29.9 59 (19.0%) 199 (21.5%) 39 (11.3%) >=30.0 27 (8.7%) 120 (13.0%) 35 (10.2%) Referral from screening programme 1580 3 (1.0%) 283 (30.6%) 15 (4.4%) <0.001 Clinical examination/Suspicious of malignancy (yes) 1580 258 (83.2%) 614 (66.3%) 274 (79.7%) <0.001 Side location of the lesion 1580 0.5 Left 158 (51.0%) 497 (53.7%) 173 (50.3%) Right 152 (49.0%) 429 (46.3%) 171 (49.7%) Disease extent by imaging or clinical examination 1580 <0.001 Localized 259 (83.5%) 837 (90.4%) 324 (94.2%) Multicentric/Multifocal 51 (16.5%) 89 (9.6%) 20 (5.8%) Magnetic Resonance Imaging (RMI) (yes) 1580 225 (72.6%) 375 (40.5%) 51 (14.8%) <0.001 Median Tumor size by imaging or physical examination [mm, (IQR)] 1551 20 (15, 32) 17 (11, 27) 20 (12, 30) <0.001 Axillary staging (including only invasive tumors) 1415 0.001 Negative 204 (71.1%) 664 (81.0%) 248 (79.2%) Positive 83 (28.9%) 151 (19.0%) 65 (20.8%) TNM stage 1580 <0.001 0 22 (7.1%) 107 (11.6%) 28 (8.1%) I 125 (40.3%) 483 (52.1%) 154 (44.8%) II 143 (46.1%) 288 (31.1%) 136 (39.5%) III 17 (5.5%) 15 (1.6%) 14 (4.1%) IV 3 (1.0%) 33 (3.6%) 12 (3.5%) Invasive histological type at biopsy (including invasive and microinvasive tumors) 1423 0.2 Ductal/No Special Type (NST) 240 (83.3%) 658 (80.3%) 239 (75.6%) Lobular 23 (8.0%) 82 (10.0%) 35 (11.1%) Other 25 (8.7%) 79 (9.7%) 42 (13.3%) Final pathology 1580 <0.001 In situ 22 (7.1%) 107 (11.5%) 28 (8.1%) Invasive (including microinvasive) 236 (76.1%) 762 (82.3%) 314 (91.3%) Invasive at biopsy only with pathological complete response 52 (16.8%) 57 (6.2%) 2 (0.6%) Modified Bloom-Richardson Grade (including only invasive tumors) 1415 <0.001 G1 41 (14.3%) 209 (25.6%) 91 (29.0%) G2 93 (32.4%) 320 (39.3%) 142 (45.4%) G3 153 (53.3%) 286 (35.1%) 80 (25.6%) Lymphovascular invasion (including operated invasive tumors) 1283 <0.001 No 154 (65.8%) 610 (81.0%) 250 (84.5%) Yes 80 (34.2%) 143 (19.0%) 46 (15.5%) Oestrogen receptor status (including only invasive tumors) 1415 0.044 Not performed 1 (0.3%) 0 (0%) 0 (0%) Negative 63 (22.0%) 135 (16.6%) 48 (15.3%) Positive 223 (77.7%) 680 (83.4%) 265 (84.7%) Progesterone receptor status (including only invasive tumors) 1415 0.4 Not performed 0 (0%) 6 (0.7%) 4 (1.3%) Negative 89 (31.0%) 258 (31.7%) 92 (29.4%) Positive 198 (69.0%) 551 (67.6%) 217 (69.3%) HER2 overexpression (including only invasive tumors) 1415 <0.001 Not Performed 0 (0%) 0 (0%) 18 (5.8%) Negative 226 (78.7%) 685 (84.0%) 263 (84.0%) Positive 61 (21.3%) 130 (16.0%) 32 (10.2%) Proliferation activity index (Ki67) in invasive G2 tumors 555 <0.001 Not performed 57 (61.3%) 203 (63.4%) 122 (85.9%) <5% 4 (4.3%) 21 (6.6%) 4 (2.8%) 5-30% 23 (24.7%) 82 (25.6%) 13 (9.2%) >30% 9 (9.7%) 14 (4.4%) 3 (2.1%) Molecular subtyping (including only invasive tumours) 1415 <0.001 Luminal A-like 182 (63.4%) 585 (71.8%) 224 (71.5%) Luminal B-like 43 (15.0%) 94 (11.5%) 26 (8.3%) HER2 missing 0 (0%) 0 (0%) 18 (5.8%) HER2 positive 20 (7.0%) 43 (5.3%) 11 (3.5%) Triple Negative 42 (14.6%) 93 (11.4%) 34 (10.9%) BRCA1 + BRCA2 1580 <0.001 No genetic assessment 123 (39.7%) 784 (84.7%) 334 (97.1%) Negative 173 (55.8%) 129 (13.9%) 8 (2.3%) Positive 14 (4.5%) 13 (1.4%) 2 (0.6%) First treatment 1580 <0.001 Surgery 190 (61.3%) 717 (77.4%) 210 (61.0%) Neoadjuvant chemotherapy 116 (37.4%) 169 (18.3%) 16 (4.7%) Primary endocrine therapy 4 (1.3%) 37 (4.0%) 111 (32.3%) Support treatment/Surveillance/ Patient refusal 0 (0%) 3 (0.3%) 7 (2.0%) Surgery 1580 <0.001 Breast conservative surgery 170 (54.9%) 655 (70.8%) 194 (56.4%) Mastectomy 138 (44.5%) 243 (26.2%) 90 (26.2%) No surgery 2 (0.6%) 28 (3.0%) 60 (17.4%) Endocrine therapy (yes) 1580 237 (76.5%) 721 (77.9%) 271 (78.8%) 0.8 Chemotherapy (yes) 1415 96 (33.4 %) 260 (31.9%) 40 (12.8%) <0.001 Radiotherapy (yes) 1580 211 (68.1%) 687 (74.2%) 164 (47.7%) <0.001 1 N (%); Median (IQR). 2 Fisher's exact test for count data with simulated p-value (based on 2000 replicates); Pearson's chi-squared test; Kruskal-Wallis rank sum test. cancers-15-01446-t002_Table 2 Table 2 EUSOMA Quality Indicators compliance by age group. <=45 Years 46-69 Years >=70 Years Evidence Mand/Recom Min. Req. (%) Target (%) Cases/ Total Result (%) 95% I.C. Cases/Total Result (%) 95% I.C. Cases/Total Result (%) 95% I.C. 1 Cancers who underwent pre-operative physical examination, mammography/ultrasound of both breasts and axillae III Mandatory >90 >95 289/310 93.2 (89.7; 95.7) 895/926 96.7 (95.2; 97.7) 328/344 95.3 (92.4; 97.2) 3a Invasive cancers who underwent axillary staging by ultrasound +/- FNA/CNB III Recommended 85 95 287/287 100 (98.4; 100) 815/815 100 (99.4; 100) 313/313 100 (98.5; 100) 3b Cancers (invasive or in situ) with a pre-operative confirmed diagnosis (B5 or C5) III Mandatory 85 90 310/310 100 (98.4; 100) 926/926 100 (99.5; 100) 344/344 100 (98.6; 100) 4a Invasive cancers with histological type, grading, ER/HER2, pN, margins, vascular invasion & size recorded II Mandatory >95 >98 243/288 84.4 (79.5; 88.3) 748/819 91.3 (89.1; 93.1) 245/316 77.5 (72.4; 81.9) 4b Non-invasive cancers with histological pattern, grading, size, margins & ER recorded II Mandatory >95 >98 17/22 77.3 (54.2; 91.3) 65/107 60.7 (50.8; 69.9) 12/28 42.9 (25.0; 62.6) 5 Waiting time <= 6 weeks between the date of first diagnostic examination (mammogram/ultrasound) and surgery/other treatment IV Recommended 80 90 194/310 62.6 (56.9; 67.9) 476/926 51.4 (48.1; 54.7) 202/344 58.7 (51.3; 62.0) 6a Cancers examined preoperatively by MRI (excluding PST) IV Recommended 10 NA 102/164 62.2 (54.3; 69.5) 193/638 30.3 (26.7; 34.0) 38/294 12.9 (9.4; 17.4) 6b Cancers treated with PST undergoing MRI IV Recommended 60 90 107/123 87.0 (79.4; 92.2) 155/177 87.6 (81.6; 91.9) 10/19 52.6 (29.5; 74.8) 7 Cancers refered for genetic counselling IV Recommended 10 NA 187/310 60.3 (54.6; 65.8) 142/926 15.3 (13.1; 17.9) 10/344 2.9 (1.5; 5.5) 8 Cancers discussed pre and postoperatively by a MDT III Mandatory 90 99 310/310 100 (98.5; 100) 926/926 100 (99.5; 100) 344/344 100 (98.6; 100) 9a Invasive cancers receiving just 1 operation (excl. reconstruction) II Mandatory 80 90 276/288 95.8 (92.6; 97.7) 784/819 95.7 (94.0; 97.0) 307/316 97.2 (94.5; 98.6) 9b DCIS receiving just 1 operation (excl. reconstruction) II Mandatory 70 90 20/22 90.9 (69.4; 98.4) 87/99 87.9 (79.4; 93.3) 25/28 89.3 (70.6; 97.2) 9c Immediate reconstruction after mastectomy III Recommended 40 40 54/138 39.1 (31.0; 47.8) 79/243 32.5 (26.7; 38.8) 4/90 4.4 (1.4; 11.6) 10a M0 invasive cancers receiving postoperative RT after BCT I Mandatory 90 95 145/154 94.2 (88.9; 97.1) 542/565 95.9 (93.9; 97.3) 129/160 80.6 (73.5; 86.4) 10b Cancers >= pN2a+ receiving post-mastectomy RT I Mandatory 90 95 6/7 85.7 (42.0; 99.2) 11/18 61.1 (36.1; 81.7) 1/4 25.0 (1.3; 78.1) 10c Cancers pN1 receiving post-mastectomy RT I Mandatory 70 85 18/35 51.4 (34.3; 68.3) 37/60 61.7 (48.2; 73.6) 6/19 31.6 (13.6; 56.5) 11a Invasive cancers cN0 who underwent SLNB only (excluding PST) I Mandatory 90 95 146/147 99.3 (95.7; 100) 560/563 99.5 (98.3; 99.9) 73/82 89.0 (79.7; 94.5) 11b No more than 5 nodes excised in invasive cancers who underwent SLNB I Recommended 90 95 184/188 97.9 (94.3; 99.3) 573/590 97.1 (95.3; 98.3) 170/175 97.1 (93.1; 98.9) 11c Invasive cancers <= 3cm (incl. DCIS component) who underwent BCT (BRCA patients excluded) I Mandatory 70 85 80/105 76.2 (66.7; 83.7) 412/473 87.1 (83.7; 89.9) 119/146 81.5 (74.1; 87.3) 11d Non-invasive cancers <= 2cm treated with BCT II Mandatory 80 90 3/6 50.0 (18.8; 81.2) 46/48 95.8 (84.6; 99.3) 14/17 82.4 (55.8; 95.3) 11e DCIS who do not undergo axillary clearance II Mandatory 97 99 20/20 100 (80.0; 100) 102/103 99.0 (93.9; 99.9) 23/24 95.8 (76.9; 99.8) 12 Endocrine sensitive invasive cancers receiving HT I Mandatory 85 90 217/223 97.3 (94.0; 98.9) 662/680 97.4 (95.8; 98.4) 259/263 98.5 (95.9; 99.5) 13a Invasive cancers ER negative (T > 1cm or N+) receiving adjuvant CT I Mandatory 85 95 59/60 98.3 (89.9; 99.9) 117/121 96.7 (91.2;98.9) 27/44 61.4 (45.5; 75.3) 13b Invasive cancers HER2 positive (T > 1cm or N+) treated with adjuvant CT who received adjuvant trastuzumab I Mandatory 85 95 9/9 100 (62.9; 100) 45/47 95.7 (84.3; 99.3) 10/10 100 (65.5; 100) 13c Invasive cancers HER2 positive treated with neo-adjuvant CT who received neo-adjuvant trastuzumab I Mandatory 90 95 29/29 100 (85.4; 100) 44/44 100 (90.0; 100) 2/2 100 (19.8; 100) 13d Inflammatory breast cancer who received neo-adjuvant CT II Mandatory 90 95 3/3 100 (31.0; 100) 6/7 85.7 (42.0; 99.2) 1/1 100 (5.5; 100) Diagnostic Qis; Loco-regional treatment Qis; Systemic treatment Qis; Mandatory requirements Qis; Recommended requirements Qis; Minimum standard Qis; Desirable target Qis; QIs noncompliance. 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PMC10000817 | Cells Cells cells Cells 2073-4409 MDPI 10.3390/cells12050734 cells-12-00734 Article Hypergravity Increases Blood-Brain Barrier Permeability to Fluorescent Dextran and Antisense Oligonucleotide in Mice Dubayle David Conceptualization Methodology Software Formal analysis Investigation Resources Data curation Writing - original draft Writing - review & editing Supervision Project administration Funding acquisition 1* Vanden-Bossche Arnaud Methodology Resources Writing - review & editing 2 Peixoto Tom Validation Formal analysis Investigation Data curation Writing - review & editing 3 Morel Jean-Luc Methodology Validation Formal analysis Resources Data curation Writing - original draft Writing - review & editing Supervision Project administration Funding acquisition 34* Mariggio Maria A. Academic Editor 1 CNRS, INCC, UMR 8002, Universite Paris Cite, F-75006 Paris, France 2 INSERM, SAINBIOSE U1059, Mines Saint-Etienne, Universite Jean Monnet Saint-Etienne, F-42023 Saint-Etienne, France 3 University Bordeaux, CNRS, INCIA, UMR 5287, F-33000 Bordeaux, France 4 University Bordeaux, CNRS, IMN, UMR 5293, F-33000 Bordeaux, France * Correspondence: [email protected] (D.D.); [email protected] (J.-L.M.) 24 2 2023 3 2023 12 5 73426 1 2023 13 2 2023 16 2 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). The earliest effect of spaceflight is an alteration in vestibular function due to microgravity. Hypergravity exposure induced by centrifugation is also able to provoke motion sickness. The blood-brain barrier (BBB) is the crucial interface between the vascular system and the brain to ensure efficient neuronal activity. We developed experimental protocols of hypergravity on C57Bl/6JRJ mice to induce motion sickness and reveal its effects on the BBB. Mice were centrifuged at 2x g for 24 h. Fluorescent dextrans with different sizes (40, 70 and 150 kDa) and fluorescent antisense oligonucleotides (AS) were injected into mice retro-orbitally. The presence of fluorescent molecules was revealed by epifluorescence and confocal microscopies in brain slices. Gene expression was evaluated by RT-qPCR from brain extracts. Only the 70 kDa dextran and AS were detected in the parenchyma of several brain regions, suggesting an alteration in the BBB. Moreover, Ctnnd1, Gja4 and Actn1 were upregulated, whereas Jup, Tjp2, Gja1, Actn2, Actn4, Cdh2 and Ocln genes were downregulated, specifically suggesting a dysregulation in the tight junctions of endothelial cells forming the BBB. Our results confirm the alteration in the BBB after a short period of hypergravity exposure. blood-brain barrier permeability centrifugation hypergravity dextran antisense oligonucleotide CNES (Centre national d'etudes spatiales4800001060 4800001082 4800001144 4800001182 CNRS (Centre national de la recherche scientifique)Universite Paris CiteUniversite de BordeauxFundings came from CNES (Centre national d'etudes spatiales, grants #4800001060, 4800001082, 4800001144 and 4800001182); CNRS (Centre national de la recherche scientifique); Universite Paris Cite; Universite de Bordeaux. pmc1. Introduction Astronauts are exposed to successive phases of hypergravity phases during the takeoff and landing of spaceflights and due to microgravity in space. The most important and earliest reported symptom, related to days 1-3 of the spaceflight, is space motion sickness due to vestibular dysfunction . The fluid shift is the shift in the distribution of human body fluids due to microgravity exposure. It was proposed to be responsible for space motion sickness. Several ground devices and protocols were rapidly developed to reproduce this phenomenon, such as centrifugation and parabolic flights . Moreover, the decreases in plasma volume and cardiac performance, and the increase in intracranial blood pressure participate in vascular deterioration, as recently reviewed . Furthermore, the alterations in gravity induce cardiovascular adaptations via the modifications in endothelial and smooth muscle vascular cell functions . It is noticeable that the effects of centrifugation are only partially described in humans . Like during microgravity exposure, hypergravity exposure, from 1.5 to 5 g, affects the vestibular functions and modifies gene expression in the brain and cognitive performances . The use of hypergravity by centrifugation is required to qualify the biological effects of space motion sickness. Likewise, centrifugation, close to 2x g, is also proposed as a countermeasure against the deleterious effects of microgravity seen in humans . Therefore, before the exposure of humans to centrifugation, it is important to study its biological impacts. The cerebral blood vessels are crucial in brain functions regarding oxygen supply and exchanges of nutrients and wastes. The endothelial cells of brain capillaries are organized to form the blood-brain barrier (BBB), assuming the fine-tuning of these exchanges to maintain brain homeostasis . The efficacy of the BBB is regulated by the nychthemeral rhythms . BBB alterations are clearly implicated in stroke and neurodegenerative disorders . Gravity changes are able to modify endothelial cell functions . Many in vitro models have been developed to reproduce the BBB , and experiments that exposed endothelial cells to gravity modifications revealed miscellaneous results, as reviewed . Depending on hypergravity levels from 3 g to 20 g, endothelial cells modify their gene expression, angiogenesis, cytoskeleton architecture and tube formation . Moreover, in devices that reproduce the barrier function, the effects of short-term exposure to hypergravity remain unclear. In fact, exposure (2 g and 4 g) increases the barrier efficacy, shown by resistance measurements of the endothelial cell culture , whereas a higher level (10 g) decreases it, as shown by the increase in fluorescent molecules passing through the culture monolayer . The effects of hypergravity on the capacity of endothelial cells to form a barrier in vitro are insufficient to interpret the modifications in the BBB observed in vivo. More information should be collected in vivo. In mice exposed to hypergravity at 2 g for 24 h, we measured the transit through the BBB of different fluorescent molecules with different sizes, such as dextrans and antisense oligonucleotides (AS). We also investigated the regulation of expression of genes involved in junctions between endothelial cells. 2. Materials and Methods 2.1. Animals and Centrifugation In accordance with the principles of the European community, the experimental protocols were validated by the local ethics committee (CEEA-Loire, APAFIS #38819), the animal welfare committee of PLEXAN (PLateforme d'EXperimentations et d'ANalyses, Faculty of Medicine, Universite Jean Monnet, Saint Etienne, France, agreement ndeg42-18-0801) and the French Ministry of Research. In this study, 86 male C57BL/6JRJ mice (8 weeks old, 22.5 +- 0.1 g, Janvier Labs, France) were used. The animals were housed (3 mice per cage) in standard conditions (22 degC, humidity 55%; 12 h/12 h day/night cycle; unlimited access to food and water). They were familiarized with the centrifugation room the week before the experiments and monitored by video in the centrifuge. In order to expose all the animals to the same environmental conditions, the mice were centrifuged at 2x g for 24 h, and the control mice in normogravity at 1 g for 24 h were placed simultaneously in the experimental room. The centrifugation protocol was detailed in our previous publication. 2.2. In Vivo Injection of Antisense Oligonucleotide and Dextrans All the fluorescent molecules were diluted in saline solution (sodium chloride 9 g/L) and retro-orbitally injected in the blood, under isoflurane anesthesia (5%). In our hands, this route of administration is safer (more rapid, efficient and reproducible) than other routes of i.v. administration. Sham mice were injected with vehicle solution. Mice received only one injection with one fluorescent tracer. Phosphorothioate antisense oligonucleotide directed against angiopoietin-2 (Angpt2, named AS, GCG-TTA-GAC-ATG-TAG-GG, 6084.9 g/mol, Eurogentec) was coupled to 5-carboxyfluorescein (excitation: 492 nm, blue light; emission: 518 nm, green light) and injected (18 mg/Kg). Fluorescein isothiocyanate-dextrans D40, D70 and D150 (FD40-100MG, FD70S-100MG, FD150S-1G, respectively, Sigma-Aldrich, St. Louis, MI, USA) were solubilized in vehicle (2x g/100 mL to be injected retro-orbitally at 150 mg/Kg, near 200 mL/mouse). Fluorescein isothiocyanate-dextrans were maximally excited at 490 nm (blue) and maximally emitted at 525 nm (green). 2.3. Collection of Biological Samples Mice were randomly killed by lethal intraperitoneal injection of sodium pentobarbital (Euthasol, 175 mg/Kg, i.p.), within 2 h after stopping the centrifuge. Before intracardiac perfusion, a catheter was introduced in the right atrium, and blood samples were collected and placed in microtubes. Finally, mice were perfused intracardiacally (5 mL/min) with 30 mL of phosphate-buffered saline (0.01 M PBS, pH: 7.4) to discard blood cells and residual fluorescence of the injected tracers into vessel lumen. This step was followed by 30 mL formalin solution (10%, Merck, HT501128) to fix the tissues. Brains and the left lobe of livers were dissected and post-fixed for 24 h in a formalin solution at room temperature, placed for 48 h in a 30% sucrose-PBS solution at 4 degC and cryopreserved before being sliced. 2.4. Corticosterone Assay The microtubes containing blood samples were centrifuged (10 min at 2000x g) and 20 mL of serum was collected. Some serum samples were excluded due to hemolysis. The others (n = 60) were used for corticosterone assay (ELISA kit, K014, Arbor Assays, Ann Arbor, MI, USA), following the protocol of the supplier. 2.5. Histology Using a freezing microtome (frigomobil, Reichert-Jung), coronal sections of the brain (40 mm thick) were made. Olfactory bulbs were removed, and 3.2 mm after beginning the rostro-caudal slicing, the new slices were collected and placed individually in 48-well plates. To ensure reproducibility, we anatomically selected three similar brain slices for each mouse. Using a binocular device, the slices corresponding to interaural 1.98 mm; Begma -1.82 mm of the Atlas of the mouse brain in stereotaxic coordinates were retained. Indeed, the medial habenular nuclei and mammillothalamic tract were anatomical landmarks, as well as the form and volume of the hippocampus. In the same manner, the left lobes of the liver were sliced (40 mm), and three slices per mouse were mounted. All the floating sections were incubated for 10 min in DAPI (4',6-diamidino-2-phenylindole, 1:250,000, Interchim, Mannheim, Germany) and rinsed twice in PBS (10 and 20 min, respectively). Finally, they were mounted on glass slides (Superfrost) with a handmade medium based on Mowiol. All slices were DAPI-labeled and mounted on the same day. Slices presenting red blood cells in capillaries in ROI were excluded to reduce experimental bias . 2.6. Image Acquisition The fluorescence of labeled brain slices was observed by confocal microscopy (SP5, Leica Microsystems, Wetzlar, Germany) and the slide scanner Nanozoomer (2.0 HT, Hamamatsu Photonics, Shizuoka Prefecture, Japan). The Nanozoomer contains a fluorescence imaging module using objective UPS APO 20X NA 0.75 combined with an additional lens 1.75X. Virtual slides were acquired with a TDI-3 CCD camera. The fluorescent acquisitions were conducted with a mercury lamp (LX2000 200W--Hamamatsu Photonics, Massy, France), and the set of filters adapted for DAPI and FITC/FAM fluorescence were usable for both fluorescein isothiocyanate-dextrans and 5-carboxyfluorescein antisense oligonucleotide. The DAPI labeling, revealing the double strain of DNA in the cell nuclei, was used for the automated focus required for Nanozoomer imaging. To reduce bias, all images (slices from control and centrifuged mice) were performed randomly in one batch. To localize antisense oligonucleotides in the brain and liver tissues, some images were acquired with SP5 confocal microscope. In this case, fluorescent molecules were excited with the 488 nm line of Argon laser and all acquisition parameters were kept constant. 2.7. Fluorescence Analyses Several types of fluorescence analyses were double-blindly performed on Nanozoomer images. To evaluate the intensity level of fluorescence, the ndpi files generated by Nanozoomer were converted into tiff images with NDPI software (version 2). The tiff files were opened with Fiji software 2.9.0, and the intensity levels were measured in regions of interest , dorsal thalamic nuclei (THAL) and the retrosplenial and ectorhinal cortices (DCx and LCx, respectively) on both hemispheres of the three slices. No filter settings were applied to the images and we checked that the images did not have any saturated dots. The mean of fluorescence was calculated for each mouse and reported in the statistical analysis. A similar analysis was performed in three liver slices. Five ROI were randomly placed on each slice. Moreover, the image analysis of fluorescent spots was performed with QuPath directly on the ndpi files. The software is able to identify and localize fluorescent spots. We empirically determined parameters to segregate fluorescent spots in brain slices from 5 mice (control and centrifuged mice) and we applied these parameters to the project containing the entire sample. The parameters were: pixel size 0.5 mm, background radius 30 mm, median filter radius 0, sigma 1, minimum area 5 mm2, maximal area 1000 mm2 and threshold 7. The collected data were attributed to experimental groups (2 g vs. 1 g) and compared statistically. The analyses, reported, were performed on the ROI anatomically defined as HPC (hippocampus), THAL (grouping all medio-dorsal and lateral thalamic nuclei), DCx (containing retrosplenial cortices), SoCx (containing somatosensorial cortices) and PirCx (containing piriform cortices). A similar analysis was performed on the left lobe liver slices. 2.8. Gene Expression by RT-qPCR For this experiment, 16 mice were used (8 were exposed to 2 g and 8 to 1 g, as described before). They were anesthetized with isoflurane 5% and decapitated, and the brains were directly frozen and stored at -80 degC. Hippocampus were dissected on ice and placed in 2 mL tubes containing 500 mL of Tri-reagent (MRCgene) and 10 ceramic beads (diameter 1.5 mm). Samples were mashed in a Beadbug6 shaker (Benchmark, 3 cycles, level of speed 4350 and 60 s time). RNA was isolated, following the instruction of the protocol elaborated by MRCgene. The concentration of RNA was measured with Nanodrop (Thermoscientific, Waltham, MA, USA) and adjusted close to 100 ng/mL. The cDNA was produced with the RT-i-script gDNA clear cDNA synthesis kit (Bio-Rad's reference 1725035), using 100 ng of RNA and following the protocol from the supplier. The qPCR was performed using the endothelial cell contacts by junction M96 (predesigned for use with SYBR green; Bio-Rad's plate reference 10029202) and the Sso-advanced universal SYBR green PCR kit (Bio-Rad's reference 1725275). The qPCR was performed with CFX96 thermocycler (Bio-Rad). Samples were allocated randomly in plates, and some of them were tested twice to verify the quality of the experiment. The validation of Hprt and Gapdh as reference genes was evaluated with CFX Maestro software (Bio-Rad). The analysis of gene expression was performed on Actb, Actg1, Actn1, Actn2, Actn4, Cdh2, Cdh5, Cldn1, Cldn3, Cldn5, Ctnna1, Ctnnb1, Ctnnd1, Dsp, F11r, Gja1, Gja4, Gja5, Jam2, Jup, Ocln, Tjp1, Tjp2 and Vim. The threshold of the regulation by hypergravity on gene expression was chosen at 1.5. To discuss the RT-qPCR results, we checked the brain localization, cell types expressing genes and function of proteins encoded by these genes in endothelial cells using databases: and (accessed on 26 January 2023). 2.9. Statistical Analysis The data were statistically compared using paired t-tests, non-parametric Mann-Whitney test, or two-way ANOVA with post hoc comparisons when applicable. The normogravity (1 g) is the control condition. The software used was GraphPad Prism V9, which calculated the p value as the probability of observing two identical conditions. If p < 0.05, the two compared conditions were considered statistically different. 3. Results 3.1. Effects of Centrifugation on Mice The body weight gain, expressed as the difference in weight in a 24 h period , is the difference in body weight measured before and after exposure to centrifugation (2x g) or control conditions (1 g). As expected, the exposure to centrifugation induced a decrease in body weight . More precisely, the decrease in body weight was similar in mice injected with saline solution (Sham) and solution containing fluorescent antisense oligonucleotide directed against angiopoietin-2 (AS) . The decrease in body weight gain due to centrifugation was similarly observed in mice injected with dextrans . The effects observed in mice injected with AS or dextrans (D40, D70 and D150) were similar . In conclusion, the injection of fluorescent tracers did not influence the effect of centrifugation on body weight gain. To explain the decrease in body weight, we also measured the food and water consumption. As shown in Figure 1D and 1E, the comparison of food and water consumption, respectively, during the day before the centrifugation with the consumption during the 24 h of centrifugation exposure showed that both food and water consumption specifically decreased in the group exposed to the centrifugation (two-way ANOVA, time x gravity p = 0.0001 for both parameters). The stress was evaluated by the concentration of corticosterone in plasma. The comparison between 1 g and 2 g conditions, including all the samples, did not reveal a variation in corticosterone concentration . We also separately analyzed the corticosterone concentration in each experimental group. In mice injected with saline solution (Sham), AS, D40, D70 and D150, the centrifugation had no significant effect on the corticosterone concentration . In conclusion, centrifugation at 2x g did not modify the plasma concentration of corticosterone in mice injected with fluorescent tracers. 3.2. Effects of Centrifugation on Extravasation of Fluorescent Dextrans in Brain The extravasation of dextrans through the BBB were firstly evaluated by the analysis of fluorescence intensities of several brain areas. To minimize local variations, we performed all analyses on slices containing similar anatomical landmarks. The regions of interest were distributed in different cerebral areas . The centrifugation was not able to modify the fluorescent levels in THAL , HPC and LCx . In DCx, the hypergravity can increase the level of fluorescence only in D70 . The differences in D70 fluorescence across brain sections are illustrated in Figure 3E. More marked fluorescence diffusion is observed in the DCx of 2 g-exposed mice. In conclusion, these sets of data analysis suggested that centrifugation significantly increased the presence of D70 in DCx. 3.3. Effects of Centrifugation on Extravasation of Fluorescent AS in Liver We tested the ability of hypergravity to promote the passage of a molecule that can be captured by liver parenchyma cells. To test this hypothesis, we injected mice with fluorescent antisense oligonucleotides and compared the 1 g and 2 g conditions. The same quantification methods used for dextrans were applied on images obtained with Nanozoomer . A significant increase in fluorescence in liver parenchyma was revealed in mice exposed to hypergravity . Moreover, the number of areas containing fluorescence evaluated with QuPath was higher in 2 g in comparison with 1 g . With confocal microscopy, the presence of AS was qualitatively revealed as spots of fluorescence close to vessel walls in the liver parenchyma. Taken together, these results strongly suggest that hypergravity increased the AS extravasation in the liver parenchyma. 3.4. Effects of Centrifugation on Extravasation of Fluorescent AS in Brain The qualitative analysis of images obtained with Nanozoomer and confocal SP5 showed fluorescent spots in the brain parenchyma only in slices from 2 g-exposed mice . The confocal images also revealed that these fluorescent spots were more localized in the brain parenchyma close to the vessel walls . The quantitative analyses of images from Nanozoomer showed an increase in fluorescence level in HPC and DCx due to hypergravity exposure . The analysis with QuPath software was used to segregate fluorescent areas from the background in several brain regions using the same filtering parameters in both 1 g and 2 g conditions. The analyses confirmed that the exposure to hypergravity increased the number of fluorescent spots in HPC and DCx, but not in THAL . Moreover, it also revealed an increase in the number of fluorescent spots in SoCx and PirCx . In conclusion, our data suggest that hypergravity induced a BBB leakage able to increase the presence of AS in brain parenchyma. 3.5. Effects of Centrifugation on Expression of Genes Involved in Endothelial Cells Interactions Using Hprt and Gapdh as reference genes, the RT-qPCR analysis of the expression of genes involved in the regulation of endothelial cells interactions revealed that Gja4, Ctnnd1 and Actn1 were upregulated. Cdh2 was downregulated more than 2-fold and Ocln, Actn2, Jup, Actn4, Tjp2 and Gja1 were downregulated between 1. 2-fold . The expressions of Actb, Actg1, Cdh5, Cldn1, Cldn5, Ctnna1, Ctnnd1, Dsp, F11r, Gja5, Jam2, Tjp1 and Vim were considered not altered (less than 1.5-fold modification), and Cldn3 appeared not expressed. The names, functions and cell types expressing these genes are summarized in Table 1 and supplementary Table S1. 4. Discussion In the present study, our results suggest that hypergravity induces an increase in BBB permeability, allowing the passage of antisense oligonucleotides as well as dextran from blood to brain parenchyma. Moreover, the RT-qPCR experiments suggested an alteration in the expression of genes involved in endothelial cell junctions. In a ground model of hindlimb unloaded animals without or in combination with radiation , as well as during spaceflight , the BBB was altered, suggesting that vestibular regulations were involved. As reviewed recently, the increase in gravity by centrifugation modifies vestibular function and induces motion sickness . Our experiments confirm the decrease in body weight generated by centrifugation . It is linked with the decrease in food intake , and probably linked to vestibular impairments . Hypergravity exposure at 2 g increases the corticosterone concentration when it is measured during the first hour of exposure . The increase in the hypergravity level can transiently increase the plasma corticosterone level . Nevertheless, as our data showed, after 24 h of weak exposure at 2 g, the corticosterone levels were not altered in the first hour following the stop of the centrifuge . The stress induced by the centrifugation is controversial and probably depends on the design of the centrifuge and experimental procedure with animals . Moreover, our data showed a large spread of individual values of corticosterone concentration, confirming other studies . In motion sickness, the relationship between brain and intestinal functions were known and clearly demonstrated, including microgravity and hypergravity models . The most probable link is hypophagia. In mice and rats, the decrease in food intake was observed at the beginning of the 2 g exposure (first two days) and depended on the vestibular organ . The hypophagia could have several causes, including: 1. modifications in microbiota that can decrease the gastric acid synthesis , 2. metabolism dysregulation, such as decreases in leptin and insulin plasma concentrations and 3. modifications in the expression of the starvation-induced genes . Moreover, the serotonin pathways are involved in this phenomenon . In conclusion, our results also confirm that the hypophagia induced a decrease in body weight. This is more related to the hypergravity and not related to an increase in corticosterone levels . Fluorescent polysaccharides such as dextrans are safe at low concentrations, available in sizes from 3 to 2000 kDa. They can be used to study BBB permeability and to determine the size of a BBB leak . After 24 h exposure to 2 g hypergravity, our results demonstrated that 70 kDa dextran can be exported in cortex parenchyma, but not 40 or 150 kDa dextran. The lower molecular weight dextrans, the faster they are excreted. In fact, in less than one hour, dextrans between 30 and 40 kDa were excreted in urine, whereas the 62 kDa dextran was always present in the blood circulation and not highly present in urine . This suggests that after 24 h, the 40 kDa dextran would be excreted. Thereby, the BBB leak required more than one hour of hypergravity exposure, confirming our previous data showing that short exposure (1-9 min at 5 g) was not efficient in destabilizing the BBB . Because we cannot exclude an alteration in urine excretion of dextran in the hypergravity context, our data should be completed by the evaluation of the kinetics of dextran excretion in centrifuged mice. Because of the molecule shape, 150 kDa dextran was unable to flow from the circulation to the tissues in physiological conditions . Our results showed that the BBB leak is not sufficient for 150 kDa dextran extravasation, suggesting that this leak was not comparable to the BBB disruption induced by stroke or acute hypertension . In our previous study , the extravasation of IgG (around 150 kDa) was measured, suggesting that the nature of the molecule is also a crucial parameter. Moreover, our data showing the extravasation of antisense oligonucleotide in the cortex and hippocampus confirm that the BBB properties depend on the brain areas and the chemical nature of the markers . In conclusion, our results showed an increase in the transfer of fluorescent molecules from blood to tissues, suggesting a global modification in effluxes due to hypergravity. To assess the alterations in the BBB in centrifuged mice, we focused the molecular investigation on gene expression using a set of primers targeting consensual genes involved in BBB efficacy. As reviewed recently , all of the proteins encoded by the genes studied here are involved in the scaffoldings required to maintain endothelial cell interactions to create the BBB, as well as in the initiation of angiogenesis and/or vascular repair. The database queries concerning the expression level in non-neuronal cells of the brain indicated that the proteins encoded by studied genes are also expressed in endothelial cells, but not exclusively (Supplementary Table S2). As expected, the modifications in gene expression are related to the durations of centrifugation and the levels of hypergravity, as suggested by the comparison between this current study and the RNAseq performed previously on the same device and the same mouse strains . Moreover, the regulation of gene expression is not comparable to acute and chronic stress (Supplementary Table S3). Globally, the observed modifications could be interpreted as a specific dysregulation of gene expression that can alter the turnover and replacement of proteins involved in BBB efficacy as observed in BBB disruption models such as stroke, middle cerebral artery occlusion or hypoxia. 5. Conclusions This work suggests that the modification in gravity, which is accompanied by a modification in the vestibular functions, leads to an alteration in the BBB via a modification in the expression of genes which code the proteins in the junctions between the endothelial cells. As now studied, an alteration in the BBB, and not its destruction, allows the passage of molecules defined by their sizes and their chemical natures. Our work insists on this point; an alteration in the BBB is characterized according to the means of study, i.e., markers and measurement methods. This can be considered in two antagonistic ways, either as a minimally invasive physical means of crossing the BBB by molecules of therapeutic interest or, on the contrary, as something deleterious that can be found in the pathology of alterations in vestibular functions during spaceflight. The most important limit of this study is that the RT-qPCR was performed on RNA extracted from whole brain, and the query of hipposeq.janelia.org indicated that we cannot exclude the alteration in molecular scaffolding of synapses also implicating these genes. Finally, our study can be considered an extension of studies relating to the effectiveness of molecules to modulate the passage across the BBB. In a hypergravity context, but also in other models of alteration in vestibular functions, the transduction pathways involved in alterations in the BBB should also be investigated. For example, the angiopoietin-2 pathway is crucial for endothelial cell disassembly , and GPCR internalization in endothelial cells should be considered in the context of centrifugation. The last topic that we can investigate is the effects of gravity modulation on angiogenesis, which is required to renovate the endothelium and form new brain capillaries. In fact, experiments on cultured endothelial cells have suggested that hypergravity reduces their capacity to form tubes and alters their responses to angiogenic factors . In centrifugation as well as during parabolic flights, the in vivo responses to angiogenic factors have not yet been investigated. Moreover, it has been shown that during the takeoff and landing of a space module (BION-M 1), hypergravity induces cardiovascular changes . More experiments should be conducted to precise how these cardiovascular changes can modify the structure of the BBB and neurovascular unit functions. To restore physiological functions after spaceflight or bed-rest in humans, a daily sequence of short exposure to centrifugation close to 2x g has been proposed. It is crucial to verify if this protocol has any effect on the BBB. Recently, biomarkers of BBB alteration have been listed , and their investigation in the spaceflight context should be performed. Finally, centrifugation can be considered to potentiate vectorization and should be used to investigate cell functions with antisense oligonucleotides in pathophysiological contexts. Acknowledgments We thank Laurence Vico for the access to SAINBIOSE U1059 facilities during centrifugation campaigns; Laurence Vico and Mathieu Beraneck for helpful discussions; Anne Prevot for reading the MS. The Nanozoomer imaging was conducted in the Bordeaux Imaging Center, a service unit of the CNRS-INSERM-Bordeaux University, member of the national infrastructure France BioImaging supported by the French National Research Agency (ANR-10-INBS-04). We thank Ghislaine Roux (PLEXAN) for technical assistance and breeding management. The results here have been presented in the Federation of European Neuroscience Societies congress, Paris 2022, and the German Neuroscience Society Meeting, Gottingen 2023. Supplementary Materials The following supporting information can be downloaded at: Comparisons between the RTqPCR data and queries of databases are organized in Supplementary Tables S1, S2 and S3. Table S1: List of genes modulated by 2 g, Table S2: Cell types expressing genes modified by 2 g exposure and Table S3: Comparison of gene expressions with database from . Click here for additional data file. Author Contributions Conceptualization, J.-L.M. and D.D.; methodology, J.-L.M., A.V.-B. and D.D.; validation, J.-L.M., D.D. and T.P.; formal analysis, J.-L.M., D.D. and T.P.; investigation, J.-L.M., D.D. and T.P.; resources, J.-L.M. and D.D.; data curation, J.-L.M., D.D. and T.P.; writing--original draft preparation, J.-L.M. and D.D.; writing--review and editing, J.-L.M. and D.D.; supervision, and project administration J.-L.M. and D.D.; funding acquisition, J.-L.M. and D.D. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement In accordance with the principles of the European community. The animal study protocol was approved by the Ethics Committee (CEEA-Loire) and the animal welfare committee of the University Jean Monnet, Saint Etienne, France, and the French Ministry of Research (protocol #11385, 10/10/2017). Protocols applied to mice were performed in PLEXAN, samples were treated and analyzed in INCC, UMR 8002 and INCIA, UMR 5287. Informed Consent Statement Not applicable. Data Availability Statement Data are available upon request from the corresponding authors. Conflicts of Interest The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript or in the decision to publish the results. Figure 1 Variation in body weight and food and water consumption in normogravity and hypergravity. (A) Variation in body weight (D weight on 24 h expressed in grams) in 24 h in all mice exposed to both normogravity (1 g, circle) and hypergravity (2 g, point) conditions (n = 35 per condition). (B) Variation in body weight of mice injected with saline solution (Sham, n = 3 per condition, circle) and antisense oligonucleotide (AS, n = 9 per condition, point). (C) Variation in body weight of mice injected with antisense oligonucleotide (AS, n = 9 per condition), dextrans D40 (n = 6 per condition), D70 (n = 9 per condition) and D150 (n = 8 per condition). p values < 0.05 are precise. (D) Food and (E) water consumption during the 24 h; before the centrifugation (before) and after the period of centrifugation (after) in both 2x g and in 1 g groups. The consumption of food and water are expressed in g per cage, each cage containing three mice (n = 7 cages per group). Figure 2 Effects of centrifugation on corticosterone concentration. (A) Corticosterone concentration (in pg/mL) in both normogravity (1 g, circle) and hypergravity (2 g, point) conditions (n = 30 per conditions). (B) Corticosterone concentration (in pg/mL) in both normogravity (1 g) and hypergravity (2 g) conditions in Sham-, AS-, D40-, D150-injected mice (n = 6 per conditions). Figure 3 Effects of centrifugation on extravasation of fluorescent dextrans in brain. Measures were performed on Nanozoomer images from (A) thalamus (THAL), (B) hippocampus (HPC), (C) dorsal (DCx) and (D) lateral (LCx) cortices. Fluorescence levels were used as an index of extravasation. They were expressed as grey level (arbitrary unit, A.U.) per surface (mm2) of the D40 (n = 11), D70 (n = 25) and D150 (n = 22), measured in both normogravity (1 g, circle) and hypergravity (2 g, point) conditions. Red circles on schema of the brain slices represented the ROI used for the analyses, and the p values < 0.05 are indicated. (E) Typical images obtained with Nanozoomer of brain slice from D70-injected mice exposed to normogravity (1 g, left) and hypergravity (2 g, right). The right panel is a magnification of the left one. Red circles indicate the ROI used for fluorescence measurements. Scale bar represents 1 mm. Figure 4 Effects of centrifugation on extravasation of fluorescent AS in liver. (A) Typical images of liver slices from mice exposed to normogravity (1 g) and hypergravity (2 g) obtained with Nanozoomer (upper panel) and confocal SP5 (lower panels, with two different magnifications). Red arrows indicate AS fluorescent spots. (B) The fluorescent level, expressed as grey level (A.U.) per surface (mm2) of the AS in liver (n = 24), was used as an index of extravasation. Red circles on schema of the liver slices represented the ROI used for the analysis. (C) Density of fluorescent spots (number of spots per mm2) used as an index of AS inclusion into the liver tissue. The analysis was performed in the whole slice (blue shape) and red circles on the schema indicated the localization of spots. The 1 g and 2 g conditions are associated with white and black points, respectively, and the p values < 0.05 are indicated. Figure 5 Effects of centrifugation on extravasation of fluorescent AS in brain. (A) Typical images of brain slices from mice exposed to normogravity (1 g, upper panel, left) and hypergravity (2 g, upper panel, right) obtained with Nanozoomer. Red arrow indicates fluorescent spots. A focus on the hippocampus area of the brain slice from mice exposed to hypergravity (2 g, lower panel) is illustrated by confocal SP5 images obtained at two different magnifications (right panel was an enlargement of the image on the left; corresponding to the red rectangle). (B) Fluorescence level expressed in grey level (A.U.) per surface (mm2) of the AS (1 g, n = 12 vs. 2 g, n = 12) in thalamus (THAL), hippocampus (HPC), dorsal (DCx) and lateral (LCx) cortices used as an index of extravasation. Red circles on schema of the brain slices represent the ROI used for the analyses; the 1 g and 2 g conditions are associated with white circle and black points, respectively, and the p values < 0.05 are indicated. Figure 6 Density of fluorescent spots in brain. (A) The brain regions used for the analysis were delimited by red shapes on Nanozoomer image (1 g on the left and 2 g on the right). These red ROI were placed on the thalamus (THAL), hippocampus (HPC), dorsal cortex (DCx), somatosensorial cortex (SoCx) and piriform cortex (PirCx). The white arrows indicate some spots with high levels of fluorescence. (B) Density of fluorescent spots (number of spots per mm2) was used as an index of AS diffusion in the brain parenchyma. The 1 g and 2 g conditions are associated with white circles and black points, respectively, and the p values < 0.05 are indicated. Figure 7 Relative expression of genes involved in endothelial cell contacts. The gene expressions in 1 g and 2 g were compared using Hprt and Gapdh as reference genes (in inset). The upregulated and downregulated genes are indicated in red and green, respectively. Genes indicated in black are considered not affected. cells-12-00734-t001_Table 1 Table 1 Relative expression and functions of genes modulated by 2 g. Gene Name Relative exp. # Function of the Genes Actn1 Actinin a1 2.24 - Adherens-type junctions, binding actin to the membrane. Actn2 * Actinin a2 -1.67 - Adherens-type junctions, binding actin to the membrane. Actn4 Actinin a4 -1.78 Probably involved in vesicular trafficking. Cdh2 Cadherin 2 -2.61 - Ca2+-dependent cell adhesion molecule; development of the nervous system; may regulate dendritic spine density. Ctnnd1 Catenin d1 3.46 - Key regulator of cell-cell adhesion; regulates gene transcription and activity of Rho family GTPases and downstream cytoskeletal dynamics. Gja1 Gap junction protein a1 -1.58 - Gap junctions, intercellular channels for the diffusion of low molecular weight materials; major protein of gap junctions in the heart with crucial role in the synchronized contraction of the heart. Gja4 Gap junction protein a4 2.00 - Gap junctions, intercellular channels for the diffusion of low molecular weight materials, element of the connexons. Jup Junction plakoglobulin -1.52 - Major cytoplasmic protein integrated in both desmosomes and intermediate junctions; forms complexes with cadherins and desmosomal cadherins. Ocln Occludin -1.54 - Membrane protein required for formation and (cytokine-induced) regulation of the tight junction paracellular permeability barrier; induces adhesion when expressed in cells lacking tight junctions. 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PMC10000818 | Foods Foods foods Foods 2304-8158 MDPI 10.3390/foods12050981 foods-12-00981 Article Colloidal Nanoparticles Isolated from Duck Soup Exhibit Antioxidant Effect on Macrophages and Enterocytes Xu Ligen Data curation Writing - original draft 12 Duan Mingcai Data curation 12 Cai Zhaoxia 3 Zeng Tao Data curation 2 Sun Yangying Data curation 1 Cheng Shuang Writing - review & editing 1 Xia Qiang Writing - review & editing 1 Zhou Changyu Writing - review & editing 1 He Jun Conceptualization Resources Writing - review & editing 14* Lu Lizhi Conceptualization Resources Writing - review & editing 2* Pan Daodong Writing - review & editing 1 Patra Jayanta Kumar Academic Editor 1 Key Laboratory of Animal Protein Food Deep Processing Technology of Zhejiang Province, College of Food and Pharmaceutical Sciences, Ningbo University, Ningbo 315832, China 2 Institute of Animal Husbandry and Veterinary Science, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China 3 Hubei Hongshan Laboratory, National Research and Development Center for Egg Processing, College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China 4 Zhejiang-Malaysia Joint Research Laboratory for Agricultural Product Processing and Nutrition, Ningbo University, Ningbo 315832, China * Correspondence: [email protected] (J.H.); [email protected] (L.L.) 25 2 2023 3 2023 12 5 98126 12 2022 07 2 2023 17 2 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). Food-derived colloidal nanoparticles (CNPs) have been found in many food cooking processes, and their specific effects on human health need to be further explored. Here, we report on the successful isolation of CNPs from duck soup. The hydrodynamic diameters of the obtained CNPs were 255.23 +- 12.77 nm, which comprised lipids (51.2%), protein (30.8%), and carbohydrates (7.9%). As indicated by the tests of free radical scavenging and ferric reducing capacities, the CNPs possessed remarkable antioxidant activity. Macrophages and enterocytes are essential for intestinal homeostasis. Therefore, RAW 264.7 and Caco-2 were applied to establish an oxidative stress model to investigate the antioxidant characteristics of the CNPs. The results showed that the CNPs from duck soup could be engulfed by these two cell lines, and could significantly alleviate 2,2'-Azobis(2-methylpropionamidine) dihydrochloride (AAPH)-induced oxidative damage. It indicates that the intake of duck soup is beneficial for intestinal health. These data contribute to revealing the underlying functional mechanism of Chinese traditional duck soup and the development of food-derived functional components. duck soup colloidal nanoparticles RAW 264.7 Caco-2 antioxidant effect Fundamental Research Funds for the Provincial Universities of ZhejiangZhejiang Science and Technology Major Program on Agricultural New Variety Breeding2021C02068-10 Modern Agricultural Technical Foundation of ChinaCARS-42-25 National Key R&D Program of China2021YFD2100103 This work has been promoted by the Fundamental Research Funds for the Provincial Universities of Zhejiang, Zhejiang Science and Technology Major Program on Agricultural New Variety Breeding (No. 2021C02068-10), the Modern Agricultural Technical Foundation of China (CARS-42-25), and the National Key R&D Program of China (2021YFD2100103). pmc1. Introduction Food-grade nanoparticles, such as nanoemulsions and liposomes, have been successfully developed with excellent stability and efficacy . Soups can produce colloidal nanoparticles (CNPs) in the case of self-assembly, which have been observed in soups made with clams , Chinese medicine , and pig bones . Nutrients migrate from food raw materials to water, and then form self-assembling particles between molecules that interact with each other covalently and noncovalently during heating, ranging in size from the nanometer to the micron scale. Soups have rich flavors and complex components, and the CNPs in them change the degree of digestion and the absorption of nutrients in raw materials , whereas further research is needed to investigate the ingestion and functioning of these nanoparticles. Macrophages are immune cells that serve a variety of purposes. They are widely distributed throughout the body and serve as significant study subjects for cellular phagocytosis, cellular immunity, and molecular immunology. Macrophages have a strategic role in intestinal homeostasis and intestinal physiology . RAW 264.7 is considered to be one of the best models of macrophages, and it has several uses in the study of inflammation, immunity, apoptosis, and tumor research . The colonic mucosal epithelium is the fulcrum that maintains intestinal homeostasis, and these barrier-forming cells can precisely control redox signaling and thus avoid tissue damage . The interactions between food and intestine have been studied using the Caco-2 cell model, which is well adapted for this purpose . Oxidative stress is caused by the imbalance between oxidation and antioxidation in biological systems. It usually leads to the excess accumulation of reactive oxygen species (ROS) and induces the damage of cellular components and cell apoptosis . A large number of studies on oxidative stress and anti-inflammatory processes have conducted out in Caco-2 cells or RAW 264.7 cells, confirming the representativeness of such cell models . Therefore, studying the interactions of food-derived CNPs with both macrophages and enterocytes should be appropriate for revealing the effects of CNPs on intestinal health. Duck meat, as a quality and nutritious meat resource, is becoming more and more well-liked by consumers worldwide, especially in Asia . In China, old duck is often used as the raw material for duck soup, and is it believed to have a curative effect on inflammation. The aged ducks (500 days of age) were found to have a significant antioxidant capacity, with abundant metabolites . However, the CNPs in duck soup, and their biological effects on intestine have not been characterized yet. To further understand the bioactivity of duck soup, the CNPs were isolated from duck soup, and their interactions with Caco-2 cells and RAW 264.7 cells were investigated to reveal the antioxidant effects of these CNPs on the intestinal tract. Therefore, this work should expand our knowledge of the biological function mechanism of food soup, and contribute to the development of gastrointestinal protection. 2. Materials and Methods 2.1. Materials All the analytical-grade reagents used were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China), including sodium dihydrogen phosphate, glucose, sodium chloride, sodium hydroxide, and disodium hydrogen phosphate. Triglyceride kits, BCA protein assay kits, total antioxidant capacity colorimetric (T-AOC) assay kits (FRAP method), 2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS), cell counting kit-8 (CCK-8), and 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) were provided by Sangon Biotech Co., Ltd. (Shanghai, China). The following products were purchased from Sigma-Aldrich Co., Ltd. (Shanghai, China): Hoechst 33342 staining solution, dimethyl sulfoxide (DMSO), DiBAC4(3) staining solution, 2,2'-Azobis(2-methylpropionamidine) dihydrochloride (AAPH), and penicillin-streptomycin (100x, Sterile). The following products were obtained from Invitrogen Co., Ltd. (Carlsbad, CA, USA): Fetal bovine serum (FBS), 25% Pancreatin + Ethylene Diamine Tetraacetic Acid (EDTA), Dulbecco's modified minimal essential medium (DMEM), MitoSOX Red staining solution, phosphate buffered saline (PBS), Hank's balanced salt solution (HBSS), and minimal essential medium (MEM). The RAW 264.7 and Caco-2 cell lines were procured from the BeNa Culture Collection (Su Zhou, China). The HiPrep 16/60 Sephacryl S-500HR (1.0 x 120 cm) column was purchased from General Electric Company (Fairfield, CT, USA). Cell culture flasks, 96-well plates (black transparent flat bottom), and 24-well plates were purchased from Corning Company (Corning, NY, USA). 2.2. The Preparation of Duck Soup Fresh 600-day-old Sheldrake carcasses (Huaying, Xinyang, China) were purchased. The duck breast meat was cut into square pieces with a side length of about 2 cm, blanched in boiling water to remove blood, and then washed on the surface in clean water. Meat pieces were cooked in deionized water (meat/water, w/v, 1:3) for 3 h at 100 degC, and heated with an induction cooker (300 W), during which the water loss caused by cooking was supplemented according to the liquid level. The duck soup was filtered twice with eight layers of cotton gauze to remove solids, and then stored at -40 degC for future use . 2.3. Separation of the CNPs from the Duck Soup The CNPs of duck soup were separated according to a reported method, with some modifications . The duck soup was filtered through a 0.45 mm filter membrane after being centrifuged at 400x g for 10 min. Four milliliters of duck soup supernatant were separated using a pre-equilibrated chromatographic column equipped with AKTA avant150 (General Electric Company, Fairfield, Connecticut). The concentration of phosphate buffer was adjusted to 0.02 M. The flow rate was 1 mL/min. The automatic collector was set at 4 mL/tube, and the UV monitor was set at 280 nm. The eluent at each stage isolated from the column was labeled as Fn (n for the scientific count), depending on the peak time. Each Fn (1 mL) was gently injected into the sample pool of dynamic light scattering (DLS) (Malvern, UK) at 25 degC for measurement. The viscosity and refractive index (RI) were 0.8872 and 1.330, respectively . The CNPs were selected from the eluents based on the polymer dispersity index (PDI), the hydrodynamic diameter, light scattering, and the z-potential. Each determination was repeated three times. 2.4. The Morphologies of the CNPs The CNPs were dripped onto the copper net covered with Formvar films; the excess solution was slowly wiped off with filter paper along the edge of the copper mesh, and dripped with 1% uranyl acetate for dyeing. Morphologies were observed under a transmission electron microscope (TEM) at 80 kV. 2.5. Major Compositions Analysis of the CNPs The major compositions (lipids, proteins, and carbohydrates) of the obtained CNPs were detected . A protein detection was performed according to the method of BCA protein quantitative kits. An anthrone-sulfuric acid test was used to assess the number of polysaccharides in CNPs. According to the method provided by the kits, the content of triglyceride in the sample was measured using the GPO-PAP enzyme assay. The absorbance measurement was conducted with a multifunctional microplate reader (Infinite 200 PRO, TECAN, Switzerland). All of the above indexes were tested 3 times. 2.6. Determination of Antioxidant Activities The ABTS free radical scavenging capacity of CNPs was evaluated based on a published method, with slight modifications . The ABTS powder was weighed and prepared to 7 mM, and the potassium persulfate reagent was weighed and prepared to 140 mM. Then, 5 mL of ABTS solution was mixed with 88 mL potassium persulfate solution and placed away from light for 12-16 h. The mixture was then diluted 50 times, with distilled water as the ABTS+ reserve solution. The ABTS+ reserve solution (200 mL) and the sample solution (50 mL) were absorbed and added into the enzyme-labeled plate. The absorbance was measured at 734 nm after standing for 10 min in a dark environment at room temperature, and the measurement was repeated 3 times. The reported method was used to test the ferric reducing antioxidant power (FRAP) of the CNPs . The 100 mM FeSO4*7H2O solution was diluted with deionized water to 0.15, 0.30, 0.60, 0.90, 1.20, and 1.50 mM as the standard for calibration curves. Samples (5 mL) and FeSO4*7H2O standard (5 mL) were added into 96-well plates in equal quantities, then FRAP solution reagent (5 mL) was added into each well and incubated at 37 degC for 5 min, and distilled water (5 mL) was used as the control. Finally, absorbance was measured at the wavelength of 593 nm, and test temperature was set at 37 degC, and the measurement was repeated 3 times. The DPPH radical scavenging ability of CNPs was tested, the published method was slightly modified (the ratio of DPPH solution to sample solution was 1:1, 100 mL) . The 0.1 mM DPPH solution (100 mL) and sample solution (100 mL) were absorbed, then added to 96-well plates and mixed. After being kept in dark for 30 min, the plate was placed on the enzyme label analyzer, the absorbance was recorded at 517 nm, and the measurement was repeated 3 times. 2.7. Toxicity Test of the CNPs on Cells Using the CCK-8 kit, the toxicity of CNPs to Raw 264.7 and Caco-2 cells was investigated. The cells (100 mL, 5 x 104 cell/well) were inoculated into 96-well plates and incubated overnight in incubators (37 degC, 5% CO2). Each well of the cells was incubated for 24 h with 100 mL of CNPs at different concentrations, and mixed with 10 mL of CCK-8 solution for 4 h. The absorbance values were measured at 450 nm, and the cell viability was calculated by referring to Gao et al. . Each determination was repeated three times. 2.8. Observation of the Uptake of CNPs by Raw 264.7 Cells and Caco-2 Cells Nile red (1 mg/mL) was mixed with CNPs (1 mg/mL) and incubated for one hour at 40 degC. The filtrate was extracted via centrifugation at 4000x g for 5 min. The retained particles were washed with HBSS and centrifuged, and repeated several times until no red fluorescence was observed in the filtrate. The remaining particles were re-suspended in HBSS for use. The cell suspension was inoculated at 5 x 104 cell/well in 24-well plates, and incubated overnight (37 degC, 5% CO2). Then, the medium was removed and HBSS was used to wash the cells twice. The cells were fixed with 4% paraformaldehyde and stained with Hoechst 33342 (1-10 mg/mL). The Hoechst 33342-stained cells and Nile Red-tagged colloidal particles were mixed and incubated for 3 h. Fluorescence was observed via inversed fluorescent microscope (IX-53, Olympus, Japan). The excitation and emission wavelengths of Nile red were 549 nm and 628 nm, respectively. The excitation and emission wavelengths of Hoechst 33342 were 346 and 460 nm, respectively. The instrument provided software for observation under a unified background, and the whole experiment was carried out three times in a dark environment . 2.9. Detection of Cell Membrane Potential and Mitochondrial Superoxide DiBAC4 (3) staining solution (5 mm) and Mito-sox Red staining solution (2.5 mm) were applied for the determination of cell membrane potential and mitochondrial superoxide, respectively, using HBSS as the solvent. The procedures were as follows: 200 mL of 5 x 104 cell/well cells were seeded in a black 96-microwell plate and cultured overnight in an incubator (37 degC, 5% CO2). The staining solution was added to each well at a dosage of 100 mL, and the excess staining solution was cleared after a certain period of incubation (30 min for DiBAC4 (3) and 10 min for Mito-Sox Red). Then, 100 mL of various concentrations (100 mg/mL, 500 mg/mL, and 1000 mg/mL) of the CNPs, and HBSS (control) were added, and then 50 mL AAPH (6.4 mm) was added and incubated for 30 min. Finally, 510 nm and 580 nm were chosen as the excitation and emission wavelengths, respectively, and the fluorescence intensity was observed under an inverted fluorescence microscope. Each determination was repeated three times. 2.10. Statistical Analysis The data were presented as mean +- standard deviation. Statistical differences were examined via a one-way analysis of variance (ANOVA) combined with Duncan multiple comparison. The significance level was set at p < 0.05. Graphs were performed by Origin 2019 (Origin Lab, Northampton, MA, USA). 3. Results and Discussion 3.1. Isolation and Properties of the CNPs Three eluents, F1, F2, and F3, were separated and collected in the range of 100 to 160 min, among which F1 in the range of 100 to 120 min had a stronger signal of light scattering intensity than other eluents . As shown in Table 1, the average hydrodynamic diameters of F1, F2, and F3 were 255 nm, 220 nm, and 147 nm, respectively, and the light scattering intensity in F1 was roughly three times greater than in F2. It has been indicated that larger particle sizes of CNPs are more efficiently phagocytosed by macrophages . The minimum PDI of F1 indicated a narrow sample size distribution, while the particles in F2 and F3 may not have a uniform size. The maximum negative z-potential of F3 indicated a greater ionic bond interaction with the chromatographic gel, resulting in a delay in separation. The TEM micrograph of F1 confirmed that the particles contained in F1 had a uniform spheroid shape . Thus, the representative F1 was chosen to learn the nano-functional properties of the CNPs from duck soup. 3.2. Major Components and Antioxidant Activities of CNPs The CNPs obtained after 3 h of continuous simmering of the duck soup had a lipids content of 51.2%, followed by 30.8% of proteins and 7.9% of sugars (Table 2). It has been found that the constituent proteins in the particles are mainly associated with antioxidant activity , and that the second most abundant protein in CNPs offers the possibility of antioxidant activity. Protein extracts from duck meat have been shown to have a good ability in antioxidant and free radical scavenging . The comparison revealed that the composition of CNPs in the duck soup was similar to that of porcine bone soup , but significantly different from that of freshwater clam , which contained 60% carbohydrates. This may be due to the differential compositions between clams and duck meat, indicating that the formation of CNPs in the soup should be closely associated with the ingredients of raw materials. It is common practice to utilize spectrophotometric techniques to assess food antioxidant potential, including the determinations of ABTS and DPPH, both of which involve the scavenging of free radicals . Another method monitoring the iron ion reducing capacity is expressed as FRAP, and a high FRAP value indicates a stronger antioxidant activity . As the CNP concentration increased, the antioxidant capacity showed an increasing trend in Figure 2, demonstrating that CNPs had a powerful antioxidant capability, but the effect of the high concentration of CNPs on cells needs to be explored. 3.3. Cytotoxicities of CNPs Some nanoparticles that are used as food additives are toxic to Caco-2 cells, disrupting the cell tight junction permeability barrier and exacerbating the intestinal barrier injury inflammatory response caused by oxidative stress . Similarly, it has been found that SiO2 CNPs have cytotoxic effects on macrophages at high concentrations . However, self-assembled nanoparticles derived from porcine bone and freshwater clam had a protective effect on cells . There was no significant difference in cell activity between the CNP treatments and the control, indicating that CNPs had no significant toxicity to Caco-2 cells or Raw 264.7 cells , and could significantly promote the growth and proliferation of these cells when the concentration was 50-300 mg/mL. However, when the concentration of CNPs gradually increased; as can be seen from Figure 3, the cell activity showed a trend of decline. 3.4. Interactions of Caco-2 Cells and Raw 264.7 Cells with CNPs Nile red is a lipophilic fluorescent dye that can be used for CNPs containing abundant lipids, and its reliability has been widely verified . As observed in Figure 4, after incubation, almost every cell nucleus was wrapped in the CNPs, and all regions of the cell except the nuclear region emitted a red fluorescence, indicating that the CNPs were not only attached to the cytoplasmic membrane, but also engulfed by the cells. The obtained CNPs from duck soup have been proven with significant antioxidant capacity, and here, their absorption by cells through the endocytic pathway implies the potential to improve the antioxidant capacities of cells. 3.5. Determination of Membrane Potential and Mitochondrial Superoxide Content in Cells Oxidative stress is caused by an imbalance between reactive oxygen species (ROS) production and the antioxidant capacities of cells, which is a cellular state that is characterized by an excessive production of ROS . ROS are produced by aerobic cells during metabolism, and the overproduction of ROS can cause cellular damage to intestinal epithelial cells . Therefore, the body's antioxidant system needs exogenous antioxidants to effectively avoid the occurrence of oxidative stress. According to several reports, the reduction in oxidative stress prevents intestinal barrier deterioration and lowers inflammatory reactions inside the gut . AAPH is a free radical initiator that can release hydroxyl radicals upon the stimulation of cells, thus causing oxidative stress and some damage to cell membranes. High concentrations of AAPH can severely damage cells, causing oxidative stress and further activating uncoupling proteins on the mitochondria, leading to a decrease in mitochondrial respiration rate and thus reducing intracellular free radical levels . In the absence of AAPH-induced cell damage (AAPH-), the fluorescence intensity of the groups in Caco-2 cells with additional CNPs was comparable to that of the control group, with no discernible differences based on the green fluorescence of DiBAC4(3) in Figure 5A. The relative fluorescence units (RFU) of Caco-2 cells were found to be much lower than those of the control group when the concentration of CNPs was 1000 mg/mL, as shown in Figure 5B. In Raw 264.7 cells, it was noticed that the fluorescence intensities of the groups with additional CNPs did not change substantially from that of the control group, based on the green fluorescence of DiBAC4(3) in Figure 5C. Moreover, the RFU of the groups added with various concentrations of CNPs did not differ noticeably from the control group in Figure 5D. When the AAPH inducer was added to the cells, as shown in Figure 5 (AAPH+), the green fluorescence in Caco-2 cells and Raw 264.7 cells was extinguished, while a decrease in RFU could be observed. However, the fluorescence was significantly restored by the addition of CNPs, probably due to the alleviation of cellular damage caused by AAPH radicals, which significantly restored the cellular membrane potential and thus counteracted the hyperpolarized state of the cellular membrane caused by extracellular hydrogen peroxide radicals. For Caco-2 cells, the presence of AAPH has been reported to cause an increase in cell permeability, which can be reduced by CNPs . CNPs can also protect the macrophage cytoplasm and membrane from AAPH-induced oxidative damage . Therefore, it can be inferred that CNPs in the appropriate concentration range extracted from duck soup would rather protect than damage the membranes of Caco-2 and Raw 264.7 cells under oxidative stress. The mitochondrion is the main site of ROS production in cells, and also the target organ of cellular oxidative stress damage . Mito-Sox Red is a specific fluorescent indicator for the detection of reactive oxygen ROS levels, and its fluorescence intensity is proportional to the ROS concentration. As shown in Figure 6A,C, there was no difference in the red fluorescence when Caco-2 cells and Raw 264.7 cells ingested the CNPs (AAPH-). As shown in Figure 6B,D, the fluorescence intensities of Caco-2 cells and Raw 264.7 cells were not significantly different from those of the control group, indicating that the CNPs in duck soup had almost no effect on mitochondrial reactive oxygen radicals. When the cells were subjected to AAPH radical-induced damage (AAPH+), as observed in Figure 6A,C, the fluorescence of Caco-2 cells and Raw 264.7 cells almost disappeared, and the strong fluorescence could hardly be seen under the microscope, which indicated that AAPH radicals could resist the oxygen respiration in mitochondria and the production of ROS. When CNPs were added to co-incubate with the cells, the red fluorescence in the cells was significantly restored compared to the control group, counteracting some of the inhibition of mitochondrial ROS by AAPH and increasing the production of ROS, indicating that CNPs could relieve the oxidative stress of cells. In Figure 6B, different concentrations of CNPs could restore the intracellular fluorescence intensity of Caco-2 cells compared to the control group, except for 1000 mg/mL of CNPs, which had no such effect. As shown in Figure 6D, compared with the AAPH group, different concentrations of CNPs significantly increased the intracellular fluorescence intensity of Raw 264.7 cells and promoted ROS proliferation in mitochondria. It was speculated that when the concentration of CNPs exceeds 1000 mg/mL, it might cause toxic damage to the cells, which in turn impaired the mitochondrial function, which was consistent with the detection of the effects of CNPs on the cell membrane potential. The experimental results showed that 100 mg/mL and 500 mg/mL CNPs could effectively maintain mitochondrial oxygen respiration and shield cells from the oxidative harm brought on by hydrogen peroxide radicals. Interestingly, duck meat is considered to have a pyretolysis effect on the body in Chinese folk and Chinese medicine, and duck soup is highly popular . Further investigations revealed that the consumption of duck meat reduced energy metabolism in rats . In this study, CNPs extracted from duck soup benefited the growth of macrophages and intestinal epithelial cells, and had the effect of alleviating the oxidative stress of the cells, which has implications for explaining the potential antioxidant benefits of duck meat and soup. It is worth mentioning that further tests should be carried out in mice to validate the function of CNPs in duck soup. 4. Conclusions In conclusion, this study successfully extracted bioactive colloidal nanoparticles from duck soup, verifying their antioxidant activity. In a suitable concentration range, the CNPs were able to interact directly with RAW 264.7 cells and Caco-2 cells, and alleviate their cellular damage when exposed to oxidative stress. This study will contribute to the extraction and application of food-derived CNPs for better efficacy, and promote new attempts at nanotechnology in the food field. Author Contributions Conceptualization, J.H. and L.L.; data curation, L.X., M.D., T.Z. and Y.S.; writing--original draft preparation, L.X.; resources, J.H. and L.L.; writing--review and editing, Z.C., S.C., Q.X., C.Z., J.H., L.L. and D.P. All authors have read and agreed to the published version of the manuscript. Data Availability Statement Data used for all findings in this study are available upon request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Chromatographic isolation and TEM of CNPs. (A) Isolation of the CNPs with a combination of chromatography and DLS. The solid line indicates the UV absorbance, and the dashed line indicates the light scattering intensity. F1, F2, and F3 are the three eluents collected at different periods, and are indicated by green arrows, and the range of F1 is marked by red lines. (B) TEM micrograph of the CNPs. Figure 2 Antioxidant activities of the CNPs in vitro under different concentration conditions. A: Measured using ABTS method, expressed as free radical scavenging activity (%). B: Measured using FRAP analysis, expressed as Fe2+ equivalent. C: Measured using DPPH method, expressed as free radical scavenging activity (%). Figure 3 Cytotoxicities of the CNPs under different concentration conditions. (A) Cytotoxicities of CNPs on Caco-2 cells. (B) Cytotoxicities of CNPs on Raw 264.7 cells. Different superscript letters indicate significant differences between CNPs with different concentrations at the same time point (p < 0.05). Figure 4 Observation of direct effects of Caco-2 cells and Raw 264.7 on the CNPs. The two images on the left are panoramic views, while the two images on the right are partial views. The nuclei were stained blue, while the CNPs were stained red. After incubation, the CNPs wrapped around the nucleus, as indicated by the green arrow. Figure 5 Determination of membrane potential in Caco-2 cells and Raw 264.7 cells. The effect of the CNPs at different concentrations on cell membrane potential in Caco-2 cells and Raw 264.7 cells are shown in (A) and (B), and in (C) and (D), respectively; different superscript letters indicate significant differences (p < 0.05). Figure 6 Determination of mitochondrial superoxide content in Caco-2 cells and Raw 264.7 cells. The effect of the CNPs at different concentrations on cell mitochondrial superoxide in Caco-2 cells and Raw 264.7 cells are shown in (A) and (B), and in (C) and (D), respectively; different superscript letters indicate significant differences (p < 0.05). foods-12-00981-t001_Table 1 Table 1 Colloidal properties of F1, F2, and F3. Hydrodynamic Diameter (nm) Derived Count Rate (kcps) PDI z-Potential mV F1 255.23 +- 12.77 a 467.00 +- 29.4 a 0.183 +- 0.017 c -4.35 +- 0.86 b F2 220.25 +- 7.45 b 158.20 +- 1.30 b 0.517 +- 0.011 a -5.15 +- 2.40 b F3 147.40 +- 17.14 c 127.76 +- 7.92 b 0.493 +- 0.002 b -11.44 +- 2.45 a The letters a, b, and c indicate significant differences (p < 0.05). PDI, polymer dispersity index. foods-12-00981-t002_Table 2 Table 2 Major compositions of the CNPs. CNPs Lipids Proteins Carbohydrates (mg/mL) 511.7 +- 41.8 307.9 +- 20.8 78.8 +- 5.9 % 51.2 30.8 7.9 %: Relative content of each component in the CNPs. CNPs, colloidal nanoparticles. 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PMC10000819 | Foods Foods foods Foods 2304-8158 MDPI 10.3390/foods12050998 foods-12-00998 Article Hierarchical Effects of Lactic Fermentation and Grain Germination on the Microbial and Metabolomic Profile of Rye Doughs Mancino Walter Formal analysis Investigation Writing - original draft Writing - review & editing 1+ Carnevali Paola Conceptualization Methodology Writing - original draft Writing - review & editing 2+ Terzi Valeria Conceptualization Methodology Investigation Writing - original draft Writing - review & editing 3 Perez Pascual Garcia 14 Zhang Leilei Methodology Data curation Writing - original draft Writing - review & editing 1 Giuberti Gianluca Conceptualization Methodology Writing - original draft Writing - review & editing 1 Morelli Lorenzo Conceptualization Writing - review & editing Supervision 1 Patrone Vania Conceptualization Writing - original draft Writing - review & editing Supervision 1* Lucini Luigi Conceptualization Methodology Writing - review & editing Supervision 1 Ibrahim Salam A. Academic Editor 1 Department for Sustainable Food Process (DiSTAS), Universita Cattolica del Sacro Cuore, 29122 Piacenza, Italy 2 R&D Food Microbiology & Molecular Biology Research Barilla G. e R. Fratelli S.p.A., 43122 Parma, Italy 3 Council for Agricultural Research and Economics, Research Centre for Genomics and Bioinformatics, 29017 Fiorenzuola d'Arda, Italy 4 Nutrition and Bromatology Group, Analytical and Food Chemistry Department, Faculty of Food Science and Technology, Universidade de Vigo, 32004 Ourense, Spain * Correspondence: [email protected] + These authors contributed equally to this work. 27 2 2023 3 2023 12 5 99823 12 2022 21 2 2023 22 2 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). A multi-omics approach was adopted to investigate the impact of lactic acid fermentation and seed germination on the composition and physicochemical properties of rye doughs. Doughs were prepared with either native or germinated rye flour and fermented with Saccharomyces cerevisiae, combined or not with a sourdough starter including Limosilactobacillus fermentum, Weissella confusa and Weissella cibaria. LAB fermentation significantly increased total titrable acidity and dough rise regardless of the flour used. Targeted metagenomics revealed a strong impact of germination on the bacterial community profile of sprouted rye flour. Doughs made with germinated rye displayed higher levels of Latilactobacillus curvatus, while native rye doughs were associated with higher proportions of Lactoplantibacillus plantarum. The oligosaccharide profile of rye doughs indicated a lower carbohydrate content in native doughs as compared to the sprouted counterparts. Mixed fermentation promoted a consistent decrease in both monosaccharides and low-polymerization degree (PD)-oligosaccharides, but not in high-PD carbohydrates. Untargeted metabolomic analysis showed that native and germinated rye doughs differed in the relative abundance of phenolic compounds, terpenoids, and phospholipids. Sourdough fermentation promoted the accumulation of terpenoids, phenolic compounds and proteinogenic and non-proteinogenic amino acids. Present findings offer an integrated perspective on rye dough as a multi-constituent system and on cereal-sourced bioactive compounds potentially affecting the functional properties of derived food products. LAB yeast fermentation rye metagenomics dough metabolomics germination Barilla G. e R. F.lli SpaComune di Cremona, Camera di Commercio di Cremona, Provincia di Cremona, Istituto Gregorio XIVEuropean UnionThis work was supported financially by Barilla G. e R. F.lli Spa and by Comune di Cremona, Camera di Commercio di Cremona, Provincia di Cremona, Istituto Gregorio XIV within the framework of the project "CREMONA AGRI-FOOD LAB: realizzazione e sviluppo di un sistema integrato di ricerca in ambito agri-food". PGP acknowledges the "Margarita Salas" grant supported by the European Union through the "NextGenerationEU" program from the University of Vigo. pmc1. Introduction Rye bread is one of the most consumed cereal-based foods in northern Europe, China, and North America . In these regions, rye (Secale cereale L.) is a valuable crop because of its resistance towards cold temperatures and northern climates . From a nutritional perspective, rye flour is gaining attention for its health-promoting potential considering its hypocholesterolemic, anti-diabetic, anti-inflammatory, and cardioprotective properties . Whole grain rye is characterized by a high content of dietary fibers, such as arabinoxylans and cellulose, and bioactive compounds with antioxidant properties, such as polyphenols . Driven by consumer demand for sustainable and healthier products, the utilization of rye in cereal-based, functional foods has been widely explored . In this context, seed germination or sprouting has gained popularity in cereal processing as an effective practice to improve grains' nutritional quality and functional value. Sprouting involves the activation of endogenous hydrolytic enzymes that results in augmented digestibility of cereal proteins and starch . Moreover, it increases the bioavailability of simple sugars, amino acids, phenolic compounds, minerals, and certain vitamins . Likewise, sourdough fermentation is a traditional process that has been shown to affect different attributes of baked goods due to metabolic activities of indigenous yeasts and lactic acid bacteria (LAB). In addition, LAB can positively affect the nutritional value of fermented cereals by increasing the content of bioactive compounds, vitamins, and minerals, and decreasing the amount of anti-nutritional factors . Lactic fermentation has emerged as a promising alternative to improve gut health, preventing digestion-related issues such as gluten sensitivity, and playing a role in the detoxification of common food mycotoxins . Previous research had shown that both germination of grains before fermentation and the type of fermentation markedly affected the structure and the potential bioactive properties of rye constituents . However, to the best of our knowledge, no comprehensive multi-omics study has yet been made to evaluate the impact of seed sprouting and lactic acid fermentation on rye dough composition and quality. Consequently, we took advantage of targeted metagenomics to assess the evolution of the inoculated starters and their overall impact on bacterial ecology. Untargeted metabolomics analyses were applied to uncover microbial contribution to the biochemical profile of grain doughs. We sought to unravel dynamic relationships between microorganisms and food matrix components and identify potential markers of functional capacity important for developing value-added rye products. The use of advanced metagenomics techniques can be important to verify and validate the ecological success of certain starters, which can be interesting for large-spectrum industrial productions . In this context, this approach may be also of interest from an industrial perspective to gain further insight into the effect of traditional technologies such as germination and fermentation on rye flour microbial, chemical, and technological multiple changes aiming to encourage the production of newly rye-based wholesome ingredients and related food products. Finally, the study of oligosaccharide profiles as a function of fermentation, can give a fundamental contribution to understanding how the substrates are modified and which carbohydrate components are present within the dough system . 2. Materials and Methods 2.1. Microbial Strains and Growth Conditions Limosibacillus fermentum UC3641, Weissella confusa UC4052, and Weissella cibaria UC405, previously isolated from sorghum sourdough , were used as LAB starters. The strains were grown in MRS medium in anaerobic conditions, using a jar and the anaerocult P reagent (Merck, Germany) at 37 degC. Cultures were harvested by centrifugation (4000x g x 10 min), washed twice with sterile saline solution (0.9% NaCl), and re-suspended in 5 mL of water. Saccharomyces cerevisiae was a commercial, compressed fresh baker's yeast (Lessafre, Marcq-en-Baroeul, France), reintegrated in water before use. 2.2. Germination of Rye Flour and Micro-Malting Process Unprocessed commercial rye grains (SU Bendix winter rye) were subjected to a preliminary sieving step. Kernel size fractions between 2 and 2.5 mm were obtained using an Octagon 200 test sieve shaker (Endecotts Ltd., London, UK). Malting was performed on 100 g sieved seed batches with an Automatic Micromalting System (Phoenix Biosystems, Adelaide, SA, Australia) . The following malting cycle (144 h in total) was applied: 15-min wash at room temperature; 7-h and 15-min steep at 15 degC; 8-h rest at 19 degC; 9-h steep at 15 degC; 6-h germination at 19 degC; 30-min steep at 15 degC; 88-h and 30-min germination at 15 degC; 7-h kilning from 30 to 40 degC; 6-h kilning from 40 to 60 degC; 6-h and 30-min kilning from 60 to 70 degC; 4-h and 30-min kilning from 70 to 80 degC; and 30-min kilning at 25 degC. The chemical composition of native rye flour was: dry matter (DM): 90.2 g/100 g; total starch: 59.0 g/100 g DM; crude protein: 9.3 g/100 g DM; total dietary fiber: 17.6 g/100 g DM, free glucose: 0.28 g/100 g DM. The chemical composition of sprouted rye flour was: DM: 93.1 g/100 g; total starch: 49.3 g/100 g DM; crude protein: 11.1 g/100 g DM; total dietary fiber: 24.1 g/100 g DM, free glucose: 5.6 g/100 g DM. 2.3. Dough Preparation and Chemical-Physical Characterization In brief, 250 g of native or sprouted rye flours were mixed with 250 mL of tap water and 1% (w/v) of NaCl. The kneading process was preformed through a commercial kneading machine (IMETEC ZERO-GLU KM 1500, Tenacta Group Spa, Azzano S. Paolo, Italy), initially without inoculum, at machine speed 1 for 2 min. As for fermentation, three different experimental conditions were tested: (i) S. cerevisiae fermentation, in which S. cerevisiae was inoculated at a final concentration of 2% (w/w), SC; (ii) mixed fermentation, in which a mix of the three LAB strains (109 CFU/mL) plus S. cerevisiae (2% w/w) was added to the dough, LAB + SC; and (iii) spontaneous fermentation, where non-inoculated doughs were prepared by replacing the inoculum with an equal volume of plain water, considered as the control. The initial concentration of LAB in each inoculated dough was between 1.89-3.00 x 107 cfu/g, as expected, whereas the initial concentration of S. cerevisiae in inoculated doughs was 1.30-1.68x106 cfu/g. After inoculation, the kneading process was carried out for 5 min at machine speed 3. All doughs were maturated for 24 h; for the mixed fermentation, an initial maturation with only inoculated LAB was performed for 8 h (30 degC and 60% relative humidity), after which yeast was added and the dough was left to leaven for the remaining 16 h (35 degC and 65% relative humidity). The same temperature and humidity conditions were applied for SC and control. Dough samples were prepared in duplicate and fermentations were repeated twice. All the doughs were analyzed for pH (pH meter Hanna Edge), total titratable acidity (TTA) , water activity (aw) (Aqualab Serie 4; Steroglass, Perugia, Italy), dough rise, total yeast, and LAB count. Dough rise was determined as follows: after mixing, 20 g of the dough was transferred into a graduated cylinder. The height of the dough was measured before and after fermentation, and the dough increase in volume was calculated as previously reported . Yeast and LAB counts were performed in duplicate on YPD agar supplemented with chloramphenicol and MRS agar with 1% of cycloheximide, respectively. 2.4. Extraction of DNA and Full-Length 16S Metagenomics Analysis Microbial cells were harvested from doughs as previously described . Total DNA was extracted from bacterial pellets using the Fast DNA Spin Kit for Soil (MP biomedicals, Irvin, CA, USA) according to the protocol supplied. DNA quantity was determined by a Qubit fluorimeter (Life Technologies, Carlsbad, CA, USA), while DNA integrity was checked through 0.8% agarose gel electrophoresis. Samples were prepared according to the guidelines for preparing SMRTbell template for sequencing on the PacBio Sequel I System (PacBio, Menlo Park, CA, USA) at Macrogen (Seoul, Republic of Korea). The library was constructed with SMRTbell(r) Express Template Prep Kit 2.0 including 27F-1492R primers along with barcode according to the manufacturer's instructions (Pacific Biosciences, Menlo Park, CA, USA); library purification was carried out using Ampure(r) PB bead (Pacific Biosciences, Menlo Park, CA, USA). Purified SMRTbell library from the pooled and barcoded samples was sequenced on a single PacBio Sequel cell using a SMRT Cell 1M v3 Tray. The resulting data were processed using the rDNATools pipeline . Ambiguous reads and short reads (<1199 bp) were filtered out, and extra-long tails were trimmed according to the target size of the bacterial 16S rRNA gene. Chimeras were identified and removed using the software Vsearch v2.14.2 using the default settings. Then, a distance matrix was generated using the Unweighted/Weighted UniFrac distance, and reads were clustered using the average neighbor method. Operational Taxonomy Unit (OTU) picking was based on the de novo method; sequences that shared over 99% similarity were assigned to a single OTU. Taxonomic assignment of OTUs was obtained using QIIME2 bioinformatic pipeline v 2022.2 , which provides functionality for working with and visualizing taxonomic annotations of features. OTUs were aligned with the representative sequence of the NCBI 16S Database with a similarity cutoff of 97% for species differentiation. 2.5. Oligosaccharide Semi-Quantification of Rye Doughs The oligosaccharide semi-quantification of the different rye doughs was performed by high-performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) approach. All samples were subjected to a previous extraction, in which 1 g of sample was mixed with 10 mL of deionized water, and the resulting mixture was homogenized by a high-speed rotor (Polytron PT 1600-E) for 1 min and centrifuged at 8000x g for 10 min at 4 degC (Eppendorf 5810R, Hamburg, Germany). The supernatants were collected, syringe filtered (0.22 mm pore size), and transferred into vials for the subsequent analysis. The experimental conditions applied for HPAEC-PAD analysis were previously described . The equipment employed consisted of a Dionex ICS-5000+ (Thermo Fisher Scientific, Waltham, MA, USA), containing an electrochemical cell with a gold working electrode combined with a pH-Ag/AgCl reference electrode as detection system. The chromatographic separation was achieved through a Dionex CarboPac PA200 column (3 x 250 mm) coupled to a guard column (3 x 50 mm), as the stationary phase (both purchased from Thermo Scientific), which provides a high-resolution separation of monosaccharides and linear oligosaccharides. The mobile phase consisted in a binary solvent system that included 100 mM NaOH (eluent A) and 1 M sodium acetate in 100 mM NaOH (eluent B). The experimental runs presented a total time of 75 min, the flow rate was adjusted at 0.4 mL min-1 and temperature for both column and detector compartments were set at 27 degC, following a multi-gradient elution system: 0-10 min, 98% A; 10-35 min, 55% A; 65-75 min, 98% A. The amperometric detector was set in terms of several potentials and durations as follows: E1 = 0.10 V (t1 = 0.40 s); E2 = -2.00 V (t2 = 0.01 s); E3 = 0.60 V (t3 = 0.01 s); E4 = -0.10 V (t4 = 0.06 s). The semi-quantification of oligosaccharides was achieved according to their polymerization degree (PD), with respect to individualized standards that are representative of three different well-separated structural classes: monosaccharides, low-PD oligosaccharides, and high-PD oligosaccharides. Thus, xylose was applied as the reference standard for monosaccharides (y = 3.8348x + 1.007, R2 = 0.9918), arabinotriose was selected as the reference standard for low-PD oligosaccharides (y = 3.7960x + 1.656, R2 = 0.9878), and arabinooctaose was chosen as the reference standard for high-PD oligosaccharides (y = 0.9963x + 0.8871, R2 = 0.9842). The results were expressed as carbohydrate content in mg g-1 of sourdough. The standards and reagents employed for oligosaccharide profiling were purchased from Sigma Aldrich(r), Darmstadt, Germany (xylose) and Megazyme(r), Bray, Ireland (arabinotriose and arabinooctaose). 2.6. Untargeted Metabolomic Profiling of Rye Doughs The untargeted metabolomic profiling of the different rye doughs was obtained through an ultra-high performance liquid chromatography instrument (Agilent 1200 series), presenting a binary pump and JetStream electrospray source, coupled to a quadrupole time-of-flight mass spectrometer (UHPLC/QTOF-MS; Agilent iFunnel 6550). Before the analytical determination, 1 g of each rye doughs was mixed with 10 mL of the extraction solvent MeOH:H2O:HCOOH (80:19.9:0.1, v/v/v) and the extraction was performed under the same conditions described earlier . All analytical conditions were set as described elsewhere . Briefly, the injection volume was 6 mL, the chromatographic separation was achieved through an Agilent Zorbax Eclipse Plus C18 column (50 x 2.1 mm, 1.8 mm), applying an AcN:H2O binary mobile phase with a gradient elution: 6%-94% organic phase for a 33 min run, with a flow rate of 200 mL min-1. The analytical conditions for the QTOF performance were employed as follow: N2 as sheath gas with a flow of 10 L min-1 at 350 degC, drying gas was applied with a flow of 8 L min-1 at 330 degC, nebulizer pressure was set at 60 psi, nozzle voltage at 300 V, and capillary voltage at 3.5 kV. The mass spectrometer was adjusted in positive polarity and SCAN mode, with a detection range of 100-1200 m/z, with a nominal resolution of 40,000 FWHM. Moreover, quality control pooled samples were obtained and further analyzed through data-dependent MS/MS mode (12 precursors per cycle at 1 Hz, 50-1000 m/z, positive polarity), applying different collision energies: 10, 20 and 40 eV. The acquired raw data were later processed by the Agilent MassHunter Profinder v.10.0 using the "find-by-formula" algorithm, through mass and retention time alignments (5-ppm and 0.05 min tolerance, respectively). The annotation of the obtained chemical features was based on their accurate mass and isotopic patterns, given by the exact masses, relative abundance and m/z spacing, using the FooDB database (available at foodb.ca) to achieve their identification. Data reduction was achieved by applying the "filter-by-frequency" feature, exclusively retaining the features observed in all the replicates within the same treatment. As a result, the untargeted compound annotation obtained was in compliance with identification level 2 provided by the COSMOS Metabolomics Standard Initiative (putatively annotated compounds). To improve the confidence in the compound annotation raw data were processed by MS-DIAL software (v. 4.90), achieving the identification of chemical features through MS-MS spectral data, according to mass accuracy data, mass isotopic patterns, and spectral alignment matching. The parameters set for identification were: retention time range, 1-32 min; mass range, m/z 80-1200; mass tolerance, 0.05 Da. For data reduction, a filter step was performed, removing the identities that were not acquired in, at least the 80% of replicates. Finally, a score cut-off of 80% was selected to retain those compounds with the highest identification fidelity according to MS2 level. All chemicals used for extraction and chromatographic equipment were LC-MS grade, purchased from VWR (Milan, Italy). 2.7. Statistics A two-way analysis of variance (ANOVA) was performed to analyze the effect of starter and germination on dough general parameters using GraphPad Prism version 8.0.0 for Windows (GraphPad Software, San Diego, CA, USA). Metagenomic data were processed using the QIIME2 v.2020.2 platform for diversity analysis of dough microbial communities. Observed species, Chao1, Shannon and Gini-Simpson indices were calculated to assess within sample diversity; sequencing depth was characterized by Good's coverage. Weighted and unweighted Unifrac distances were calculated, and Principal Coordinate Analysis (PCoA) was performed on the distance matrices to visualize community variation across samples. The OTU table was uploaded to the Microbiomeanalyst server for compositional profiling and comparative analysis, using 10% prevalence in samples for the low count filter, the default settings for other filtering and total sum scaling for data normalization . To test for significance in differential bacterial taxa abundance according to starter and flour, respectively, the algorithm DeSeq2 was used . The name of the samples was as follows: nDLY (native rye-Dough-LAB + SC); sDLY (sprouted rye-Dough-LAB + SC); nDY (native rye-Dough-SC); sDY (sprouted rye-Dough SC); nDCTR (native rye-Dough-Control); sDCTR (sprouted rye-Dough-Control); nF (native rye-Flour); sF (sprouted rye-Flour). For both oligosaccharide and untargeted metabolomics profiling approaches, all sourdough samples were extracted in triplicate, and two technical determinations were carried out for each replicate (n = 6). The results for carbohydrate content of rye sourdoughs were statistically analyzed by one-way ANOVA followed by Duncan's post hoc test, setting a significance value of a = 0.05, using the software SPSS 25 (IBM). The Agilent Mass Profile Professional v. 15.1 software analyzed the metabolomics data as previously indicated compounds were filtered by abundance, log2-transformed, and normalized at the 75th percentile . The abundance of each compound was further baselined against the median abundance among all samples. Afterwards, an unsupervised multivariate hierarchical cluster analysis (HCA) was performed to evaluate the similarities and dissimilarities of all factors as a function of their metabolic profile (Euclidean distance, Ward's linkage rule). Later, a Chemical Similarity Enrichment Analysis for Metabolomics (ChemRICH, available at chemrich.fiehnlab.ucdavis.edu) was performed to define the chemical composition of sourdoughs due to the addition of LAB. To that aim, compounds were filtered on Volcano plot and only the compounds showing a significantly different accumulation between treatments (p < 0.05) and with fold-change values > 2 were considered . Finally, a supervised multivariate orthogonal projection to latent structures discriminant analysis (OPLS-DA) was carried out by the SIMCA v. 16.0.2 software (Umetrics). Model quality was evaluated according to goodness-of-fit parameters (R2X and R2Y), and goodness-of-prediction parameter (Q2Y). OPLS-DA predictive models were further statistically validated by cross-validation ANOVA (CV-ANOVA), and model overfitting was discarded through the development of permutation tests (n = 100). Such approach was followed by a variable importance in projection (VIP) analysis, providing insight into the compounds exhibiting the highest influence on the discrimination between treatments, known as VIP markers, according to their given VIP score . 3. Results 3.1. General Parameters of Rye Doughs Key technological parameters of the different doughs after fermentation are presented in Table 1. Rye doughs fermented with LAB + yeast showed total LAB counts reaching approximately 10 log CFU/g after 24-h fermentation; this value significantly exceeded the numbers of total LAB in either SC or control rye doughs, suggesting the actual growth of inoculated LAB starters. No significant difference in final LAB abundance was found between native and sprouted rye flour. A similar trend was observed for total yeast counts. Before fermentation, the pH was 6.23 +- 0.04 for doughs made with native flour and 5.88 +- 0.07 for doughs with sprouted rye flour, respectively. As expected, the addition of LAB starters caused a decrease in the pH value to about 4 both in sprouted and native rye doughs, as compared to either SC or control. However, the difference between final pH values was significant only when comparing LAB + SC vs. SC (p = 0.002) in sprouted rye samples. Consistently, the application of LAB starters significantly increased total acid concentration in rye doughs as compared to either yeast or control (Table 1). A two-way ANOVA revealed that there was a significant interaction between the effects of starter and germination as concerns TTA value (p-value interaction < 0.0001). Notably, the TTA level of LAB + SC doughs made with sprouted rye flour was higher than that of the corresponding doughs produced from native rye flour (2.08 +- 0.05 vs. 0.82 +- 0.06, p-value < 0.0001). The inclusion of LAB as a starter for rye flour fermentation displayed no significant impact on dough aw. Significant differences were observed for yeast (alone or in combination with LAB) versus control in native rye flour doughs (p = 0.0004 and p-value = 0.006, respectively). Concerning dough volume, LAB seemed to contribute to dough rise during leavening markedly. The volume increase was higher for LAB + SC vs. SC or control regardless of the type of rye flour used; differences were all statistically significant but for LAB + SC vs. SC in native rye dough (220% +- 18.87% vs. 143.3% +- 4.71%; p-value = 0.01). Analogously to aw, statistical analysis indicated that the type of fermentation had the same effect in sprouted and native rye doughs (p-value = 0.22). 3.2. Diversity of Bacterial Communities in Rye Doughs PacBio SMRT sequencing of the complete bacterial 16S rRNA gene resulted in 167,207 total filtered high-quality reads, with numbers ranging from 4,188 to 16,844 reads per sample (mean 10,450 reads). Clustering to 99% similarity yielded 5,657 distinct OTUs; the mean number of OTUs per sample was 353 (range 58-609; Table 2). To assess sample diversity, different indexes were calculated including Chao1, Shannon, and Gini-Simpson (Table 2). No significant differences were found among dough samples in any alpha diversity indexes. As expected, flours displayed the highest bacterial diversity among all tested samples, suggesting that fermentation exerted a selection pressure on the community structure of the dough microbiota (Table 2). Overall, native rye flour and doughs tended to have a lower bacterial richness as compared to their counterparts obtained from sprouted rye. Good's coverage ranged from 97% to 99% suggesting that a high percentage of the total species was represented in each sample (Table 2). Beta diversity, based on an unweighted UniFrac distance matrix, highlighted three distinct bacterial clusters in the PCoA plot . Differences in microbial composition between the samples allowed a clear discrimination between flour samples and dough samples obtained by mixed fermentation, respectively. A further group, including both spontaneously fermented doughs and doughs produced by S. cerevisiae fermentation, could be identified. 3.3. Bacterial Taxonomic Composition of Dough Samples More than 93% of the sequences obtained by the PacBio SMRT sequencing were associated with known taxa; unclassified reads were 6.5% of the total sequences. Weissella, Limosillactobacillus, Salmonella, Latilactobacillus, and Microcoleus were the dominant genera in the metagenomic dataset, with an average abundance of 28.48%, 19.89%, 13.86%, 12.53%, and 6.36% of the total reads, respectively. At species level, reads were assigned to 56 different taxa, and the species with an average greater than 0.25% were 12 taxa . The bacterial community composition of flours differed between sprouted and native rye. Salmonella enterica represented more than 65% of bacterial microbiota in sprouted rye flour followed by Microcoleus anatoxicus, Delftia acidovorans, and others . Conversely, the most abundant bacterial species in native rye flour was the cyanobacterium Microcoleus anatoxicus, accounting for about 43% of the total sequences; less common species included Pantoea agglomerans and Cutibacterium acnes. Our results indicated that, sprouted rye doughs had greater uniformity in the bacterial community structure for all the 3 fermentation conditions than the native rye counterparts. L. fermentum and W. confusa/cibaria were the only bacterial species detected in dough samples inoculated with the lactic acid starter. As expected, the two closely related Weissella species were indistinguishable using 16S rRNA gene sequencing. L. fermentum sequences greatly outnumbered those ones classified under the species pair W. confusa/W. cibaria , reaching 93.56 +- 4.73% in native rye doughs, and 65.26 +- 0.27% in sprouted rye doughs. Yeast-leavened doughs were enriched in cereal-sourced LAB with a different species composition based on the rye flour used. Latilactobacillus curvatus was dominant (relative abundance > 89%) in sprouted rye doughs, while Lacticaseibacillus paracasei and Latilactobacillus graminis were found at low relative abundance. Native rye doughs harbored a bacterial community consisting of Lactoplantibacillus plantarum (with an average of 42.6%) followed by Lacticaseibacillus paracasei (relative abundance > 16%); Lactobacillus curvatus was detected with a relative abundance over 4%, while W. confusa/cibaria accounted for 38.5% of total sequences in nDY . Control doughs resulted in a microbiota that differed from that of other doughs and was broadly dominated by indigenous W. confusa/cibaria strains. 3.4. Differential Analysis of Discriminant Species between Dough Samples Differential analysis of the abundance of microbial species revealed several features that varied significantly according to either starter or germination. When considering the impact of starter, L. fermentum was higher in LAB + SC samples compared to either SC or control samples, regardless of rye germination . As for native rye doughs , control samples were enriched in W. confusa/cibaria and P. agglomerans with respect to native doughs inoculated with LAB and yeast. Moreover, the species L. plantarum and L. paracasei were more abundant in yeast-leavened samples than doughs obtained by mixed fermentation, or native spontaneous fermentation samples. Among sprouted rye doughs, spontaneously fermented samples displayed significantly higher levels of W. confusa/cibaria and S. enterica than those observed in SC samples and in LAB + SC samples, respectively. Notably, the inoculation S. cerevisiae alone in sprouted rye samples was associated with higher proportions of endogenous LAB species including L. curvatus, L. graminis and L. paracasei. In fact, these species resulted in being higher in abundance in SC samples with respect to both LAB + SC samples and control samples. As regards the comparison between sprouted and native rye flours, L. curvatus was significantly higher in sprouted rye doughs as compared to native rye doughs (p-value = 0.045). Conversely, L. plantarum was significantly higher in native rye samples to sprouted samples (p-value = 0.045). 3.5. Oligosaccharide Semi-Quantification of Rye Sourdoughs The oligosaccharide semi-quantification of rye flour doughs is shown in Figure 4. In general, germination played a critical role on carbohydrate compositions since native rye doughs exhibited a lower carbohydrate content to the sprouted counterparts. In parallel, adding fermentation starters played a significant role on the carbohydrate content of rye doughs . Thus, for native rye doughs, SC-mediated fermentation promoted a significant decrease of both monosaccharides and low-PD oligosaccharides that were further significantly decreased in the LAB + SC fermentation by whereas high-PD carbohydrates were not affected . In total, the combination of LAB with SC led to a harsh carbohydrate content reduction of 55.5% with respect to control. In contrast, the fermentation of sprouted rye doughs caused only a significant decrease in the carbohydrate content in the case of mixed fermentation, as SC fermentation did not promote any significant difference in comparison to control . As a result, concerning total carbohydrate content, the LAB + SC treatment led to a 22.8% decrease with respect to control, suggesting a lower impact of fermentation than that observed for native-derived dough. Concerning the different carbohydrates, the high-PD oligosaccharides content was not affected by the type of fermentation, whereas the contents of both monosaccharides and low-PD oligosaccharides was significantly decreased after the LAB + SC combined fermentation . 3.6. Untargeted UHPLC/QTOF-MS Metabolomic Profile of Rye Doughs Rye doughs were subjected to metabolic profiling via UHPLC/QTOF-MS, providing 1909 annotated chemical features (Table S1). From these annotated compounds, 158 were identified according to their MS2 spectral features (Table S2). The effect of grain germination and the addition of starters on the metabolic profile of rye doughs were evaluated by an unsupervised multivariate hierarchical cluster analysis (HCA), and the results are displayed in Figure 5. Among the factors involved in this study, germination was the most prevalent factor affecting the metabolome of samples since it ruled the establishment of two major clusters. Secondarily, within both clusters, fermentation starters played a significant role, providing three subclusters according to the different experimental conditions involved in dough production: non-inoculated, SC, and LAB + SC . Due to the heterogeneous metabolic profile of fermented rye doughs, an additional supervised multivariate orthogonal projection to latent structures discriminant analysis (OPLS-DA) was performed. It was followed by a variable importance in projection (VIP) analysis, with the aim of discriminating the effect of germination and starters on the metabolome of these matrices, providing insight on the metabolic markers mostly involved in such discrimination (VIP markers). The Figure 6 shows the OPLS-DA models and the proportion of VIP markers according to their chemical class for the discrimination between germination conditions , fermentation starters on native rye-derived doughs , and fermentative starters on sprouted rye-derived doughs . Moreover, the full list of VIP markers associated with all models, together with their VIP score, logFC values, and chemical class are provided in Tables S4-S6, respectively. In all cases, the obtained OPLS-DA models showed high-quality parameters in terms of goodness-of-fit, given by R2X and R2Y coefficients, and predictability, given by the Q2Y coefficient . Concerning germination, the OPLS-DA model spotted a definitive role of this factor on the metabolome of rye doughs, indicating a clear discrimination between native sprouted rye-derived doughs . Phenolic compounds, terpenoids, and phospholipids predominantly represent the VIP markers provided for the discrimination between native and sprouted-derived doughs. In general, and regarding the logFC values (Table S3), sprouted rye-derived doughs exhibited an enhanced accumulation of phenolic compounds, including flavonoids like mulberrin, zapotinin, and isoferreirin (logFC = 13.5), and phenolic acids, mostly represented by spermidine esters, and resorcinols. In the case of terpenoids, triterpenoid acids like 3-benzoyloxy-6-oxo-12-ursen-28-oic acid and 2, 3, 23-triacetylsericic acid (log FC = 13.5) were accumulated in sprouted rye-derived doughs (Table S3). To a lesser extent, amino acids like His and Phe derivatives and oligopeptides, as well as glucosinolates were found to be differentially up accumulated in sprouted rye doughs, providing evidence on the metabolic richness of this matrix. Conversely, the accumulation of lipid metabolites did not follow a clear pattern. Metabolites like docosan-1-ol, phosphatidylethanolamines, and 3-hydroxy-9-hexadecenoylcarnitine exhibited a decrease in native rye doughs (log FC = TM9.4; Table S3), whereas saturated fatty acids 22-hydroxydocosanoate and 10-hydroxymyristic acid methyl ester and some di-glycerides were mostly measured in sprouted rye doughs (log FC = 13.5; Table S3). As germination played such a discriminant effect on the metabolome of rye sourdoughs, two additional OPLS models were performed to evaluate the impact of starters on either native rye or sprouted rye doughs . In both cases, a clear discrimination between SC-fermented doughs and LAB + SC-fermented doughs was obtained, and phenolic compounds, terpenoids, and lipid metabolites were mostly identified as VIP markers . Firstly, in the case of native rye doughs, the inoculation of LAB promoted a general up-regulation of the metabolome, since 54% of VIP markers possessed logFC = 8.6, and only 17% of markers were found to be down-accumulated as compared to SC-fermented doughs (Table S4). Thus, terpenoids were generally accumulated, ranging from triterpenoids and sesquiterpenoids to monoterpenoids, including sterols and carotenoids (Table S4). Likewise, phenolic compounds were all up-accumulated due to LAB addition, involving flavonoids, phenolic acids like p-coumaroyl derivatives, and spermine and putrescine esters, stilbenes, coumarins and lignans (Table S4). In parallel, amino acids were also selected as VIP markers accumulated in LAB + SC fermented doughs, represented by both proteinogenic amino acids, such as Gln (logFC = 3.3), Gly (logFC = 3.4), and Cys derivatives (log FC = 8.6), and non-proteinogenic amino acids, like ornithine (logFC = 2.0; Table S4). Peptides were found to be accumulated as a result of LAB inclusion (log FC = 2.3-8.6; Table S4) as well, suggesting an intense proteolytic activity. In contrast, lipid metabolites showed an unclear pattern of accumulation between LAB + SC fermentation and SC fermentation, as given by log FC values. Notably, lysophospholipids were mainly accumulated in LAB + SC-fermented doughs (log FC = 2.0-8.6), whereas fatty acids were heterogeneously detected (Table S4). A similar trend was observed for the inclusion of LAB as fermentation starters on the metabolome of sprouted rye doughs, as indicated by the corresponding OPLS-DA . A metabolic stimulation was shown by the inclusion of LAB, with 76% of VIP markers up regulated in LAB + SC-fermented matrices to those fermented exclusively with SC (Table S5). Again, phenolic compounds constituted the class with the highest contribution to VIP markers, followed by terpenoids and lipid metabolites . Considering phenolic compounds, lignans were the compounds presenting the highest accumulation (log FC = 10.0 for schidigeragenin B and clusin, Table S5), together with ferulic, caffeic acid esters (log FC = 10.0), whereas flavonoids presented much lower log FC values (Table S5). Considering terpenoids, LAB inclusion promoted the accumulation of high-isoprene subunits terpenoids, including steroids, triterpenoids and sesquiterpenoids (log FC = 10.0, Table S5), whereas the accumulation of diterpenoids was reduced (log FC < -3.3). In parallel, in the case of amino acids and peptides, the accumulation and down-accumulation did not follow a clear pattern. While saturated fatty acids, especially octadecanoic acids (log FC = -12.8), and sulfur-containing compounds, like 1-methoxyspirobrassinin (log FC = -12.8), were harshly down accumulated upon the addition of sourdough LAB starters, lysophospholipids were found accumulated upon the inclusion of LAB (log FC = 3.7-10.0; Table S5). 4. Discussion This study explored the microbial, chemical, and technological profiles of rye doughs made with either native or sprouted flour and fermented with S.cerevisiae in combination or not with selected LAB starters. Two complementary approaches were applied to assess the metabolic profiling of rye doughs after fermentation. Firstly, the carbohydrate profile of rye doughs was assessed, and results indicate that grain germination and LAB fermentation played a significant role in the composition of these constituents. The carbohydrate content of sprouted rye doughs was significantly higher than that of native rye doughs, due to the induction of hydrolytic enzymes during seed germination, which includes amylases, pentosanases and glucanases . Due to hydrolytic activity, insoluble fiber is mainly converted into soluble sugars, such as monosaccharides, that were spotted as the major sugar constituents of rye doughs in this work. Notably, VIP analysis indicated that characteristic oligosaccharides of sprouted rye doughs were maltopentaose and maltotetraose, functional maltodextrins potentially involved in glycemic control response and enterocyte differentiation . Considering the fermentation starters, including LAB promoted a significant decrease in carbohydrate content in terms of monosaccharides and low-PD oligosaccharides. This can be explained considering the heterofermentative metabolism of sourdough LAB, which relies on the activity of a wide range of catabolic enzymes . Analysis of technological parameters revealed important features of experimental doughs connected to the evolution of bacterial ecology during fermentation and the interplay between starter inoculation and germination. As expected, the application of sourdough LAB starters led to a substantial reduction of pH, especially in comparison to yeast-leavened doughs, as a result of LAB extensive exploitation of carbohydrates for organic acids biosynthesis . Indeed, the germination-related enzymatic breakdown of carbohydrates into simple sugars can boost fermentative metabolism by sourdough LAB resulting in the accumulation of organic acids . Interestingly, LAB + SC fermentation was also associated to a greater dough rise as compared to either SC or control. Heterofermentative LAB activity can affect dough leavening through the production of CO2 . In mixed LAB + SC samples, metagenomics analysis highlighted a strong dominance of inoculated L. fermentum over Weissella strains at the end of the fermentation. On the other hand, endogenous W. confusa/cibaria was the predominant taxon in control samples. The latter finding is not surprising since several studies identified Weissella alone, or in combination with other LAB, as the dominant bacterial genus in rye sourdough after 24 h fermentation which may include or not refreshments . Indeed, the ecological fitness of sourdough microorganisms is largely dependent on the interplay between strain-specific traits and process conditions including temperature, pH, dough hydration, fermentation time, and type of flour . All these parameters can contribute to affect the growth rate of organisms, their competitiveness in sourdough fermentation and eventually their impact on product quality. It is thus presumably to suppose that a long fermentation at elevated temperature (i.e., 35 degC) as applied in the present study selected for L. fermentum owing to the thermophilic behavior and high acid resistance of this Limosilactobacillus species . Consistent with this hypothesis, the cultivable microbiota of sourdoughs fermented at 37 degC was constituted by L. fermentum strains exclusively . Notably, the VIP analysis on the metabolomics profile of doughs revealed that in both native and sprouted rye doughs there was an accumulation of mannitol when LAB were added as starters. Conversion of fructose to mannitol by heterofermentative LAB has been reported in sourdough fermentations . Consistent with this, L. fermentum UC3641 has in its genome two Open Reading Frames (ORFs) encoding for a NAD(P)H-dependent mannitol/alcohol dehydrogenase . In addition to oligosaccharide semi-quantification, a metabolomics approach was employed to investigate the overall effect of germination and fermentation on the metabolome of rye sourdoughs. The unsupervised HCA analysis of doughs revealed that germination of rye grains played a major role on the metabolic profile than fermentation, which was also supported by the results from the OPLS-DA models. Germination has been previously assessed as a physiological process in which phytohormones can play a critical role on the development and metabolome of rye grains, which may affect further processing, including fermentation . Furthermore, germinated grains show a high biosynthetic potential and promote the activity of hydrolytic enzymes that lead to structural modifications , which could reflect in greater accessibility or diversity of fermentative bacteria. We assessed the presence of LAB species that could be differentially associated to either native or sprouted rye flour regardless of the fermentation conditions. Metagenomic data revealed that the species L. plantarum was typical of the microbiota of native rye doughs, whereas L. curvatus was significantly higher in doughs made with sprouted rye. As for L. plantarum, this species is known to metabolize a wide range of different carbohydrates of varying complexity, thanks to its rich repertoire of lytic enzymes . Furthermore, among the significant compounds responsible for the discrimination between sprouted-derived rye doughs obtained from OPLS-DA model, several compounds were spotted as VIP markers, especially primary metabolites as amino acids, peptides, and lipid metabolites. Concerning phenolics, the accumulation of flavonoids and phenolic acids was mostly modulated by fermentation, with yeast as the sole fermenting agent or in combination with LAB, which agrees with the previous study by Katina et al. who reported increased levels of phenolic compounds after fermentation, especially in germinated rye. In parallel, sourdough fermentation contributed to increase significantly the content of total phenolic compounds, especially phenolic acids and alkylresorcinols, because of the pH reduction caused during fermentation . Such compounds were found in this work as discriminant metabolites associated to LAB fermentation, as it is the case of feruloyl, caffeoyl, and coumaroyl derivatives, thus being in line with the results provided by other authors . Ferulic and p-coumaric acids are the most prevalent phenolics attributed to rye, reaching a proportion of about the 95% of total phenolic compounds . Notably, all the LAB starters used in our experimental conditions presented in their genomes ORFs encoding for esterases, phenolic acid decarboxylases and phenolic acid transferases , suggesting the possible involvement of these enzymes in the conversion of p-coumaric acid and ferulic acid in their esterified derivatives. It is important to note that phenolic acids have been previously reported in their esterified forms with diverse biogenic amines as reflected by our results with spermine, spermidine, and putrescine. Moreover, the same authors reported an increase in the accumulation of flavonoids due to mixed fermentation, agreeing with present findings. Overall, the polyphenols enrichment associated with LAB + SC fermentation may suggest an enhancement of the nutritional value of rye doughs, given the biological activities of these compounds as multifaceted bioactive compounds, acting as antioxidant, anti-inflammatory, antitumor and antimicrobial agents, among other health-promoting properties . In the case of terpenoids, little is known about the effect of fermentation on biosynthesis of these compounds in rye sourdough that were widely identified during the current research as triterpenoids. Nevertheless, the presence of terpenoids may improve the shelf life and safety of these matrices, due to their associated antibacterial properties . Concerning protein-derived metabolites, both germination and fermentation played a critical role on the catabolites determined in rye doughs, which agrees with the existing literature. Germination can increase the total proteolytic activity in rye whereas acidification mediated by both LAB and yeasts in sourdough fermentation triggers cereal protease activity by shifting the dough pH to the optimum of aspartic proteases, which represent the major proteases in rye and wheat . Even more important for proteolysis is the activity of strain-dependent intracellular peptidases of sourdough lactobacilli, which enhances the accumulation of amino acids in fermented doughs providing key sources of nitrogen for yeast growth . Thus, all these factors contribute to the plethora of free amino acids and peptides spotted in this work. The amount and type of peptides and amino acids occurring in cereal doughs mostly account for the overall quality of bread in that many of these compounds act as taste-active components and flavor precursors. However, as a result of the proteolytic activity of sourdough starters, non-proteinogenic amino acids, like citrulline or ornithine, were previously spotted as well as in this case. L. fermentum is among the Lactobacillus species that can convert arginine to ornithine via the arginine-deiminase (ADI) pathway . Notably both native and sprouted rye doughs fermented by sourdough LAB were enriched in g-glutamyl dipeptides such as g-glutamylglutamic acid and g-l-glutamyl-l-pipecolic acid in the present study. Besides being naturally presented in certain foods, the synthesis of g-glutamyl dipeptides may occur in fermented foods via microbial g-glutamyl transpeptidases and g-glutamyl cysteine synthetases. Formation of g-glutamyl dipeptides in sourdough fermented by Limosilactobacillus reuteri was attributed to strain-specific biosynthetic capabilities and consistently improved the sensory attributes of the resulting bread . Lipid metabolites play a minor role on the composition of rye sourdoughs motivated by the low-fat content of rye flour . According to our results, the accumulation of lipid metabolites did not show a clear pattern, regardless of the germination and fermentation conditions, with the exception of lysophospholipids, which were harshly accumulated as a result of mixed fermentation. This finding could be explained by the activity of hydrolytic enzymes, such as lipases and phospholipases acting on tryacylglycerides, which were found to be heterogeneously accumulated during fermentation. These enzymes could be sourced from rye flour as well as sourdough starter microorganisms. Nevertheless, the results reported by Koistinen et al. on the untargeted metabolomic profile of rye sourdoughs indicate that fermentation promoted the accumulation of phosphatidylcholines, whereas oxidized fatty acids were found to be down-accumulated. Remarkably, in the present study, a higher level of hydroxy fatty acids was detected in doughs fermented with sourdough LAB as compared to S. cerevisiae alone. It is known that hydratases by sourdough lactobacilli can convert oleic acid, linoleic acid, and linolenic acid to hydroxy fatty acids. 5. Conclusions The results of the present study provide a comprehensive view of multiple compositional changes induced by germination and lactic fermentation in cereal flour, which may have implications for the nutritional value, sensory attributes, and functional characteristics of rye bakery products. Fermentation by selected sourdough lactic acid bacteria in addition to baker's yeast resulted in lower levels of simple sugars and increased levels of mannitol in the dough system, and could thus represent a relevant strategy to reduce sugar in baked goods. Grain germination promoted the accumulation of maltooligosaccharides, a class of molecules displaying several potential biological capabilities. Overall, the combination of rye germination with the combined fermentation of S. cerevisiae and LAB promoted the accumulation of nutritionally important metabolites, such as polyphenols, terpenoids, hydroxy fatty acids, and peptides, which also contribute to the enhancement of the technological and sensorial properties associated with rye flour. Indeed, the integrated information provided by metagenomics and untargeted metabolomics offered new insights into the impact of processing technologies on dough quality, which can guide the design and development of novel, health-promoting rye foods. Supplementary Materials The following supporting information can be downloaded at: Figure S1: Automated system for malting rye grains, Table S1: Dataset of annotated compounds by UHPLC/QTOF-MS analysis, Table S2: Dataset of annotated compounds by UHPLC/QTOF-MS analysis, according to their MS2 spectral features, Table S3: List of VIP markers associated with the OPLS-DA model discriminating between germination conditions of rye doughs (sprouted vs. native), Table S4: List of VIP markers associated with the OPLS-DA model discriminating between starters of native rye doughs (SC+LAB vs. SC), Table S5: List of VIP markers associated with the OPLS-DA model discriminating between starters of sprouted rye doughs (SC+LAB vs. SC). Click here for additional data file. Author Contributions Conceptualization, P.C., V.T., G.G., L.M., L.L. and V.P.; Data curation, P.G.P. and L.Z.; Formal analysis, W.M.; Investigation, W.M., V.T. and P.G.P.; Methodology, P.C., V.T., P.G.P., L.Z., G.G. and L.L.; Supervision, L.M., L.L. and V.P.; Writing--original draft, W.M., P.C., V.T., P.G.P., L.Z., G.G. and V.P.; Writing--review and editing, W.M., P.C., V.T., P.G.P., L.Z., G.G., L.M., L.L. and V.P. All authors have read and agreed to the published version of the manuscript. Data Availability Statement Raw sequences of full 16S rRNA gene profiling are accessible through SRA study accession number PRJNA850138, with biosample accession numbers from SAMN29160520 to SAMN29160535. Conflicts of Interest Paola Carnevali is an employee in Barilla G. e R. Fratelli S.p.A. The company provided the commericial rye flour that has been exploited for experimental analysis. Figure 1 Beta-diversity analysis of rye dough and flour samples. PCoA plot based on unweighted UniFrac distances of microbial communities among all samples. Solid symbols represent doughs made with native rye flour (squares) and sprouted rye flour (circles); empty symbols represent native rye flour (squares) and sprouted rye flour (circles). Dough samples were colored according to the starter used for fermentation: blue = LAB + SC, green = SC, pink = no inoculated starter. Figure 2 Relative abundance of bacterial species found in dough and flour samples. The bar plot represents the top 15 most abundant taxa among all samples identified to the species level. "Others" refers to merged species that individually showed a relative abundance below 0.25%. "Unclassified" are bacterial taxa not identified at the phylum level. The results are expressed as average of replicates for each type of dough. In the x-axis are reported the name of the samples as follow: sD, sprouted rye dough; nD, native rye dough; s_F, sprouted rye flour; n_F, native rye flour, CTR, dough control; LAB + SC, dough fermented with LAB + SC; SC, dough fermented with SC. Figure 3 Differential abundance analysis of bacterial species among rye doughs. Box and whiskers plot indicate the proportion of differentially abundant taxa between starters used for fermentation in native rye doughs (A) and in sprouted rye doughs (B), and between native and sprouted rye doughs (C). Only significant species (FDR < 0.05) detected by DESeq2 are reported in the figure. Boxes represent the minimum and maximum of abundance values of replicates for each condition; the line in the box is the median. Figure 4 HPAEC-PAD carbohydrate content of rye sourdoughs. Carbohydrate content of native rye doughs (A). Carbohydrate content of sprouted rye doughs (B). Results are expressed as carbohydrate content (mg g-1) referred as xylose, arabinotriose, and arabinoctaose equivalents for monosaccharides, low-polymerization degree oligosaccharides (low-PD), and high-polymerization degree oligosaccharides (high-PD) contents, respectively (B). Vertical bars indicate standard deviation (n = 6). Different letters indicate statistically significant differences (p < 0.05) among treatments. Figure 5 Unsupervised hierarchical cluster analysis on the untargeted metabolic profiling of rye dough. The fold change values, represented by a color range, for each compound were calculated with respect to the median of all samples, and further used to obtain a fold change-based heatmap, according to Ward's algorithm (Euclidean distance). The factors involved in clustering were NATIVE and SPROUTED, for native and sprouted-derived rye sourdough, respectively; and control, SC, and LAB + SC, for unfermented, yeast-fermented and yeast and LAB-combined fermented rye sourdough, respectively. Figure 6 Orthogonal projection to latent structures discriminant analysis and variable importance in projection analysis of rye doughs. The OPLS models were combined with the proportion of VIP markers on the discrimination of metabolomic effects caused by germination conditions (A,B), fermentation starters on native rye doughs (C,D), fermentation starters on sprouted rye doughs (E,F). foods-12-00998-t001_Table 1 Table 1 Rye dough parameters measured after 24 h of maturation. Parameter Rye flour Starter p-Value Control SC LAB + SC Starter Germination Starter x Germination pH Native 4.19 +- 0.11 a 4.57 +- 0.36 b 3.97 +- 0.10 a 0.0005 0.005 0.06 Sprouted 4.57 +- 0.10 a 5.33 +- 0.01 b 4.03 +- 0.01 a TTA (%) Native 0.39 +- 0.01 a 0.42 +- 0.03 a 0.82 +- 0.06 bA <0.0001 <0.0001 <0.0001 Sprouted 0.50 +- 0.02 a 0.56 +- 0.08 a 2.08 +- 0.05 bB aw Native 1.02 +- 0.002 a 0.94 +- 0.02 b 0.95 +- 0.01 b 0.0001 0.044 0.026 Sprouted 0.99 +- 0.01 a 0.94 +- 0.01 b 0.96 +- 0.01 ab Dough rise (%) Native 70 +- 4.24 a 143 +- 4.95 b 220 +- 18.38 c <0.0001 0.22 0.06 Sprouted 90 +- 4.24 a 130 +- 4.24 ab 180 +- 28.30 b Total LAB (log CFU/g) Native 9.16 +- 0.04 a 6.70 +- 0.09 bA 10.32 +- 0.38 c <0.0001 0.008 0.09 Sprouted 8.94 +- 0.01 a 5.29 +- 0.01 bB 9.53 +- 0.12 c Total yeast (log CFU/g) Native 6.27 +- 0.01 a 8.33 +- 0.10 b 8.24 +- 0.17 b <0.0001 0.56 0.05 Sprouted 5.47 +- 0.66 a 8.73 +- 0.03 b 8.34 +- 0.01 b Data are mean values +- SD. Statistical analysis was carried out using two-way ANOVA followed by Tukey's post hoc test. Different superscript letters (a, b, c) in a row indicate significant differences (p < 0.05) between starter types within each flour group; different superscript letters (A, B) in a column indicate significant differences (p < 0.05) between native and sprouted rye flour within each starter type group. foods-12-00998-t002_Table 2 Table 2 Number of OTUs, alpha diversity indexes, and Good's coverage of sequenced samples. Samples * OTUs Chao1 Shannon Gini-Simpson Good's Coverage nDLAB + SC1 217 386.5 0.733 0.132 0.991 nDLAB + SC2 348 485.8 1.486 0.296 0.987 nDSC1 194 230.1 2.414 0.610 0.993 nDSC2 236 271.3 3.108 0.696 0.984 nControl1 58 100.0 0.093 0.013 0.998 nControl2 415 449.0 3.995 0.751 0.993 sDLAB + SC1 466 602.5 2.579 0.595 0.981 sDLAB + SC2 454 565.8 2.624 0.600 0.981 sDSC1 327 438.0 1.712 0.278 0.988 sDSC2 417 533.4 2.587 0.444 0.982 sControl1 227 312.1 0.989 0.173 0.993 sControl2 305 430.4 1.199 0.205 0.989 nF1 284 300.8 3.211 0.740 0.998 nF2 504 541.1 4.947 0.884 0.996 sF1 596 737.5 5.014 0.841 0.974 sF2 609 799.0 5.028 0.794 0.967 * nDLAB + SC: native rye-dough-LAB + SC; sDLAB + SC: sprouted rye-dough-LAB + SC; nDSC: native rye-dough-SC; sDSC: sprouted rye-dough-SC; nDControl: native rye-dough-control; sDControl: sprouted rye-dough-control; nF: native rye-flour; sF: sprouted rye-flour. 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PMC10000820 | Healthcare (Basel) Healthcare (Basel) healthcare Healthcare 2227-9032 MDPI 10.3390/healthcare11050711 healthcare-11-00711 Article Maximum Heart Lactate Threshold-Based Low-Volume High-Intensity Interval Training Prescriptions Provide Similar Health Benefits in Metabolic Syndrome Patients Reljic Dejan Conceptualization Validation Formal analysis Investigation Writing - original draft Supervision Project administration Funding acquisition 123* Frenk Fabienne Investigation Data curation 12 Herrmann Hans Joachim Validation Writing - review & editing Funding acquisition 123 Neurath Markus Friedrich Writing - review & editing 13 Zopf Yurdagul Validation Writing - review & editing Project administration Funding acquisition 123 Oliveira Rafael Academic Editor Brito Joao Paulo Academic Editor 1 Department of Medicine 1, University Hospital Erlangen, Friedrich-Alexander University Erlangen-Nurnberg, 91054 Erlangen, Germany 2 Hector-Center for Nutrition, Exercise and Sports, Department of Medicine 1, University Hospital Erlangen, Friedrich-Alexander University Erlangen-Nurnberg, 91054 Erlangen, Germany 3 German Center Immunotherapy (DZI), University Hospital Erlangen, Friedrich-Alexander University Erlangen-Nurnberg, 91054 Erlangen, Germany * Correspondence: [email protected]; Tel.: +49-9131-85-45220 28 2 2023 3 2023 11 5 71113 1 2023 23 2 2023 25 2 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). Exercise is an integral part of metabolic syndrome (MetS) treatment. Recently, low-volume high-intensity interval training (LOW-HIIT) has emerged as a time-efficient approach to improving cardiometabolic health. Intensity prescriptions for LOW-HIIT are typically based on maximum heart rate (HRmax) percentages. However, HRmax determination requires maximal effort during exercise testing, which may not always be feasible/safe for MetS patients. This trial compared the effects of a 12-week LOW-HIIT program based on: (a) HRmax (HIIT-HR), or (b) submaximal lactate threshold (HIIT-LT), on cardiometabolic health and quality of life (QoL) in MetS patients. Seventy-five patients were randomized to HIIT-HR (5 x 1 min at 80-95% HRmax), HIIT-LT (5 x 1 min at 95-105% LT) groups, both performed twice weekly on cycle ergometers, or a control group (CON). All patients received nutritional weight loss consultation. All groups reduced their body weight (HIIT-HR: -3.9 kg, p < 0.001; HTT-LT: -5.6 kg, p < 0.001; CON: -2.6 kg, p = 0.003). The HIIT-HR and HIIT-LT groups similarly, improved their maximal oxygen uptake (+3.6 and +3.7 mL/kg/min, p < 0.001), glycohemoglobin (-0.2%, p = 0.005, and -0.3%, p < 0.001), homeostasis model assessment index (-1.3 units, p = 0.005, and -1.0 units, p = 0.014), MetS z-score (-1.9 and -2.5 units, p < 0.001) and QoL (+10 points, p = 0.029, and +11 points, p = 0.002), while the CON did not experience changes in these variables. We conclude that HIIT-LT is a viable alternative to HIIT-HR for patients who are not able/willing to undergo maximal exercise testing. obesity cardiometabolic health quality of life interval training exercise prescription cardiorespiratory fitness maximal oxygen uptake lactate heart rate glycemic control H.W. and J. Hector FoundationMED1710 Manfred Roth FoundationResearch Foundation for Medicine at the University Hospital Erlangen.This study has been supported by the H.W. and J. Hector Foundation (funding number: MED1710), the Manfred Roth Foundation and Research Foundation for Medicine at the University Hospital Erlangen. pmc1. Introduction The metabolic syndrome (MetS) is a pathology defined by the presence of several cardiometabolic disorders, including obesity (in particular excess abdominal fat), hypertension, dyslipidemia, hyperglycemia and insulin resistance . The occurrence of MetS has significantly risen worldwide during the past decades , with the latest estimates suggesting that globally, ~13% to ~31% of adults are affected . Recently, it has been reported that COVID-19 pandemic-related measures like quarantines, social distancing and lockdowns have further contributed to the spread of MetS . This trend is alarming because MetS is associated with an increased risk of several serious secondary diseases, such as cardiovascular disease , different cancers , all-cause mortality and diminished quality of life (QoL) . Additionally, recent observations indicate that excess body weight and the existence of cardiometabolic risk factors constitute an increased risk of developing a critical or lethal disease progression following a COVID-19 infection . Therefore, effective therapeutic measures for MetS treatment are probably more urgent now than ever before. Dietary adaptations, particularly a reduction in caloric intake, and an increase in physical activity are cornerstones in obesity and MetS treatment . While caloric intake restriction is of paramount importance to achieve weight loss , it has been demonstrated that physical activity independently lowers the risk of developing several chronic health conditions and premature death, regardless of body mass index (BMI) . It has been suggested that the level of cardiorespiratory fitness (CRF), objectified by the determination of maximal oxygen uptake (VO2max), is a major outcome for predicting cardiovascular and all-cause mortality, more significant than other well-established health risk factors like obesity, elevated blood pressure or nicotine abuse . Despite a plethora of evidence on the wide range of health benefits associated with regular exercise, a large part of the global population , particularly obese individuals , do not meet the minimum physical activity guidelines of 75 min of vigorous-intensity or 150 min of moderate-intensity aerobic activity per week . Over the last decade, surveys have consistently shown that time constraints are among the most frequently reported obstacles to regular exercise, both in the general population as well as in clinical cohorts . Thus, in the past few years, there has been an increasing scientific interest in designing and evaluating less time-consuming exercise approaches for preventing and treating chronic health conditions . In this regard, low-volume high-intensity interval training (LOW-HIIT) has appeared as an innovative exercise modality to elicit comparable or even greater improvements in CRF and cardiometabolic outcomes in comparison to traditional continuous endurance training . By definition, LOW-HIIT is a particular subtype of interval training involving a total duration of <=10 min of intense interval bouts of >=80% of maximum heart rate (HRmax), embedded in an overall exercise session of <=30 min (when initial warm-up, recovery between intervals, and cool-down are added up) . Recent research from our laboratory and other researchers has demonstrated that LOW-HIIT can effectively improve several cardiometabolic risk factors as well as subjective measures, such as QoL, in obese MetS patients. Prescriptions for physical exercise are typically based on four main components: frequency, intensity, time, and type of exercise, also referred to as the FIIT principle . Among these, intensity is considered the most important element of the physiological responses to exercise . As with other cardiovascular training types, exercise intensity for LOW-HIIT is most commonly prescribed based on percentages of HRmax. For a rough estimation of exercise intensity, HRmax can be calculated using different formulas , most frequently via the "220--age" equation . However, due to high interindividual variability in heart rate (HR) values , it is rather recommended to directly measure HRmax during an exhaustive exercise test in order to obtain more precise results. Although our own studies and data from other research groups indicate that guideline-based cardiopulmonary exercise testing (CPET) is generally safe and tolerable in clinical settings, maximal exhaustion may be contraindicated in certain patient populations. Furthermore, in some individuals, true HRmax may not reached due to peripheral muscular fatigue or a lack of motivation. Alternatively, exercise intensity can be prescribed using specific physiological thresholds based on ventilatory or blood lactate responses during incremental exercise. Ventilatory (VT) and lactate thresholds (LT) reflect specific submaximal metabolic inflection points of respiratory variables and blood lactate concentration . Ventilatory thresholds and LT have been traditionally used to design training programs and predict performance in endurance and team sports athletes , but threshold-based exercise intensity prescription is also an interesting approach in clinical settings because it does not require maximal effort, making it potentially safer for high-risk patients. Additionally, it has been reported that threshold-based intensity prescriptions for traditional endurance training regimens were superior in improving VO2max in sedentary healthy individuals and cardiometabolic risk factors in MetS patients , when compared to intensity prescriptions based on percentages of HRmax. In this context, endurance training programs involving exercise intensities at or above the LT were found to be particularly effective in lowering blood pressure in type 2 diabetic patients and visceral fat in obese women with MetS . Furthermore, it has been suggested that threshold-based training seems to be related with a lower instance of non-responders to exercise compared to training programs based on maximum values . However, to our knowledge, no research has yet been undertaken to compare the effects of a LOW-HIIT program using LT-based exercise intensity prescriptions on cardiometabolic health status in a clinical setting. Thus, the main objective of this investigation was to compare the effects of a 12-week LOW-HIIT intervention either prescribed based on: (a) percentages of HRmax (HIIT-HR) or, (b) LT (HIIT-LT), on various cardiometabolic health indices and QoL in a cohort of obese patients diagnosed with MetS. We hypothesized that both LOW-HIIT protocols would improve cardiometabolic health status and QoL compared with a physically inactive control group but, on the basis of previous research using traditional endurance training regimens , we expected HIIT-LT to provide superior improvements than HIIT-HR. 2. Materials and Methods 2.1. Study Design The present investigation was a sub-group analysis of a larger clinical trial examining the impact of various exercise modalities on multiple health outcomes in MetS patients. Other parts of this research project have been previously published elsewhere . The present sub-study presents previously unpublished data from the HIIT-LT group and a sub-sample of the HIIT-HR and a non-exercising control group (CON) that additionally received capillary blood sampling during the CPET for the measurement of lactate concentrations. In the overall trial, patients were allocated at random to different interval training protocols that were performed 2 times per week for a duration of 12 weeks or to the CON. All patients were provided with standard care nutritional consultation to support their weight reduction. Analogous to the main trial, the key outcome of this investigation was VO2max. Further outcomes of interest were cardiometabolic risk indices, body composition and QoL. The sample size determination and randomization process applied in the main trial were reported elsewhere . Briefly, sample size calculation was based on the previous work of Reljic et al. , indicating a large effect of LOW-HIIT on VO2max (d = 0.97), that resulted in an estimated number of 16 patients per group to yield a statistical power of 95%. To account for dropouts, the aim was to recruit at least 25 patients for randomization into each group. Randomization was preceded by stratification according to VO2max, gender, age and BMI using the software MinimPy version 3.0 to reduce the heterogeneity of patients' main characteristics between groups. The randomization was conducted by a co-worker not engaged in the acquisition and analysis of the data. All patients were fully informed about the objectives and procedures of the study, which conformed to the Helsinki Declaration, and gave their written consent before being included in the study. The study protocol was approved by the Medical Ethical Committee of the Friedrich-Alexander University Erlangen-Nurnberg (approval number: 210_17B) and registered at ClinicalTrials.gov (ID-number: NCT03306069). 2.2. Patients Patients were recruited via flyers that were posted in medical practices and newspaper advertisements. In a first step, all interested persons were screened for eligibility by phone call or personal visit. The inclusion criteria were: age >=18 years, a self-reported mainly physically inactive lifestyle as defined previously and clinical diagnosis of MetS as classified by the International Diabetes Federation . Criteria for exclusion were: pregnancy, clinical diagnosis of heart disease, oncological diseases, substantial musculoskeletal disorders or other major health limitations that may constitute contraindications to safe participation in exercise. All patients agreed not to change their usual lifestyle habits, apart from the study intervention. Patients were required to attend at least 75% of the scheduled 24 LOW-HIIT sessions to be included in the final analysis. 2.3. Health Examinations One week before starting the intervention, patients received the baseline examination, including several standardized assessments and measurements as described in detail below. The second examination was conducted during the week following the termination of the LOW-HIIT intervention with a minimum 3-day interval between the last training session. Patients were instructed to appear overnight-fasted, to abstain from alcohol and to avoid strenuous physical activities for at least 24 h prior to each examination. If patients were required to take medication, care was taken to ensure that it was taken at the same time of day for both examinations. Prior to the second examination and regularly during the intervention period, patients were asked whether there had been any changes in the type of medication or dosage taken. The post-intervention examinations were scheduled at a similar daytime (08:00-08:30 a.m.) to minimize potential circadian bias and lasted approximately 2-3 h for each patient. All measurements were carried out under stable and standardized laboratory conditions (temperature: 22-24 degC, and humidity: 30-50%) within the examination rooms of the Hector-Center for Nutrition, Exercise and Sports at the University Hospital Erlangen and in the standardized order described below. During the examinations, the patients were dressed in their casual clothes, with an exception for the anthropometric measurements, which were performed in underwear without shoes, and the CPET, for which the patients wore a sport dress or comparable clothing. All examinations were performed by a team of highly experienced personnel, consisting of two study nurses (>5 years of work experience) and an exercise physiologist and physician (>10 years of work experience) who were assisted by two medical students. All the staff involved in the data collection were blinded to the assignment of the patient's group. 2.3.1. Hydration Testing After arriving at the research center, patients were first asked to provide a urine sample for a routine screening for urinary tract infections, kidney disorders and diabetes, and for measuring urine specific gravity (USG). Urine sample analyses were conducted within 30 min of collection using Multistix(r) 10 SG dipsticks (Siemens HealthCare, Erlangen, Germany). 2.3.2. Determination of Blood Pressure and Resting Heart Rate Following urine collection, patients entered a quiet experimental room and after 5 min rest, resting HR (HRrest) and blood pressure were recorded with an automatic upper arm blood pressure monitor (M5 professional, Omron, Mannheim, Germany) . According to recent guidelines , systolic (SBP) and diastolic (DBP) blood pressure were measured twice at both upper arms at intervals of 60 s and the average value from the side with the higher blood pressure was recorded. In addition, mean arterial blood pressure (MAB) was estimated according to the following formula :MAB = DBP + (1/3 [SBP - DBP]). 2.3.3. Blood Collection After blood pressure measurements, patients remained in the sitting position and venous blood samples were taken from the antecubital area. The blood collection tubes (Sarstedt, Nurmbrecht, Germany) were immediately further prepared and forwarded to the central laboratory of the University Hospital Erlangen for measurement of the serum concentrations of glucose, triglycerides, total cholesterol, low-density (LDL) and high-density lipoprotein cholesterol (HDL) using a photometrical determination method (Clinical Chemistry Analyzer AU700 or AU5800, Beckman Coulter, Brea, CA, USA), glycated hemoglobin A1c (HbA1c) using turbidimetric immunoassays (COBAS Integra 400, Roche Diagnostics, Mannheim, Germany) and insulin using a chemiluminescence assay (Liaison XL, DiaSorin, Saluggia, Italy). The homeostasis model assessment index (HOMA-index) was calculated according to the following formula :Homeostasis model assessment-index = (insulin x glucose)/405. 2.3.4. Anthropometric Measurements For standardization reasons, patients were asked again to empty their bladder, if necessary, before the measurements. Anthropometric evaluation included measurement of body weight and determination of body composition. More specifically, body weight, fat mass (FM), body fat percentage (FM%), fat free mass (FFM) and total body water (TBW) were determined using a multi-frequency segmental bioelectrical impedance analysis device (seca mBCA 515, Seca, Hamburg, Germany) with confirmed validity . Patients' waist circumference was measured in the upright position with a flexible tape (Seca, Hamburg, Germany) to the nearest millimeter, at the approximate midpoint between the last touchable rib and the upper iliac crest, as previously described . 2.3.5. Determination of the Metabolic Syndrome Severity Score Metabolic syndrome severity was assessed according to the MetS z-score. The score was calculated using sex-specific equations based on HDL, triglycerides, glucose, waist circumference and MAB, as previously suggested :Males: [(40 - HDL)/9.0] + [(triglycerides - 150)/81.0] + [(glucose - 100)/11.3] + [(waist circumference-102)/7.7] + [(MAB - 100)/9.1] Females: [(50 - HDL)/14.1] + [(triglycerides - 150)/81.0] + [(glucose - 100)/11.3] + [(waist circumference - 88)/9.0] + [(MAB - 100)/9.1] 2.3.6. Cardiopulmonary Exercise Testing Cardiopulmonary exercise testing was performed on a stationary electronically braked cycle ergometer (Corival cpet, Lode, Groningen, The Netherlands) using two different standard exercise protocols . Both protocols commenced with a brief familiarization period, followed by measurements from the resting 12-lead electrocardiogram (ECG, custo cardio 110, custo med, Ottobrunn, Germany), blood pressure and respiratory variables. Subsequently, the HIIT-HR and the CON performed a continuously incrementing ramp protocol, beginning at a workload of 50 W and then increasing by 1 W every 5 s (females) and 1 W every 4 s (males), respectively. Using this approach, maximal exertion was typically achieved within 8-12 min, as generally recommended for ramp protocols . The HIIT-LT group performed a step incremental test, with a starting workload of 50 W, followed by a stepwise increase in the load by 25 W (females) and 30 W (males), respectively, every 3 min, as recommended to quantify the LT in untrained individuals . With the step incremental test, maximum exertion was typically achieved within 10-14 min. Both protocols were performed with a constant cadence ranging between 60-80 rpm until volitional exhaustion. During all CPET, exercise ECG was permanently monitored (custo cardio 110, custo med, Ottobrunn, Germany) and blood pressure was measured every 2 min with a standard cuff sphygmomanometer (ERKA, Bad Tolz, Germany). An open-circuit breath-by-breath spiroergometric system (Metalyzer 3B-R3, Cortex Biophysik, Leipzig, Germany) was used to continuously measure oxygen uptake (VO2) and carbon dioxide output (VCO2). At rest, immediately after termination of the exercise and at the 1st, 3rd and 5th min of recovery, 20 mL of capillary blood was sampled from the hyperemized earlobe to measure blood lactate concentrations. In the step incremental test (HIIT-LT group), capillary blood samples were additionally drawn within the last 20 s of each workload stage in order to determine the LT. Blood samples were immediately placed in collection tubes containing a hemolyzing solution, and subsequently measured in our laboratory using an enzymatic-amperometric method (LabTrend, BST Bio Sensor Technology, Berlin, Germany). Upon termination of the exercise, perceived exertion was requested from each patient using the 6-20 Borg scale . Patients had to fulfill a minimum of two of the following criteria in order to assume that maximum exertion had been achieved: a plateau in VO2, reaching a peak respiratory exchange ratio (RERmax) of >=1.1, a peak blood lactate level of >=8.0 mmol/L, an age predicted HRmax of >=90% (according to the equation: 220--age) and a perceived exertion value of >=19 on the Borg scale . 2.3.7. Determination of Lactate and Ventilatory Thresholds The lactate threshold was defined at the workload when blood lactate concentration had reached >=4 mmol/L, as first established by Mader et al. and later justified by Heck et al. . Since then, the 4 mmol/L LT is also widely referred to as the "onset of lactate accumulation" (OBLA), and broadly used in exercise physiology and practice for performance diagnostics and training prescription . Although it is clear that lactate accumulation does not occur suddenly at a sharp point but rather continuously in a transition zone , it is well accepted that the LT frequently corresponds with a blood lactate level of ~4 mmol/L in untrained individuals . Moreover, the fixed 4 mmol/L LT was found to have high reproducibility and predictability in cycling endurance performance and to be useful in prescribing exercise intensity for MetS patients . Determination of HR (HRLT) and workload (WLT) at the 4 mmol/L LT was performed by applying the software Winlactat version 5.5.2.9 (Mesics, Munster, Germany). First (VT1) and second (VT2, also termed the respiratory compensation point, RCP) ventilatory thresholds were determined independently through visual inspection by two investigators from plots of VCO2 and VO2 (the V-slope method) . In case of any discrepancy, a consensus was achieved by discussion. Heart rate and workload (WVT1 and WVT2) at both VTs were identified using an automated software (MetaSoft Studio, Cortex Biophysik, Leipzig, Germany). 2.4. Assessment of Self-Reported Quality of Life Self-reported QoL was measured with the validated EuroQol Group questionnaire (EQ-5D-5L) . The questionnaire consists of the simple EQ visual analogue scale (VAS) ranging from 0-100 (higher ratings imply better QoL) and the EQ-5D index, composed of 5 sub-categories (mobility, self-care, usual activities, pain/discomfort, anxiety/depression, each categorized into 5 severity levels). The values of the 5 sub-categories are transformed into a single variable, with a score of 1.0 representing perfect subjective health and a score of 0 representing the poorest possible health status, respectively . The questionnaires were completed by the patients in a separate waiting lounge at the Hector-Center for Nutrition, Exercise and Sports. Any questions or uncertainties about the questionnaire could be resolved immediately with the investigators. 2.5. Monitoring of Daily Nutrition and Nutritional Counseling Before study enrolment and during the final intervention week, patients were instructed to track their daily food intake over a duration of 3 successive days before each of the two examinations, with the help of a standardized 24 h nutrition protocol (Freiburger Ernahrungsprotokoll; Nutri-Science, Freiburg, Germany). After delivery, the protocols were evaluated by a registered dietitian using the software PRODI 6 expert (Nutri-Science, Freiburg, Germany). In addition, patients' resting metabolic rate (RMR) was estimated using the Harris-Benedict equation , as follows:Males: RMR (kcal/day) = 66.5 + 13.8 x weight (kg) + 5.0 x size (cm]) - 6.8 x age (years) Females: RMR (kcal/day) = 655 + 9.6 x weight (kg) + 1.8 x size (cm) - 4.7 x age (years) Based on the food record analysis, anthropometric data and the estimated RMR, patients received individual consultation during a personal conversation with a dietitian to support their weight loss. The dietary recommendations were made in accordance with the current obesity treatment guidelines, targeting a daily calorie reduction of 500 kcal . Furthermore, patients were advised to consume at least 1.0 g/kg of protein per day to counteract a loss of muscle mass during caloric restriction, as previously recommended . After the consultation, patients were provided with handouts, including recipes and nutrient lists, to increase adherence and to support them in the home-based implementation of the nutritional recommendations. 2.6. LOW-HIIT Protocols Patients allocated to the exercise groups performed 2 supervised sessions per week of LOW-HIIT on electronically braked cycle ergometers (Corival cpet, Lode, Groningen, The Netherlands) in our exercise center with a minimum of 1 day recovery between sessions for a total of 12 weeks (24 sessions in total). In order to maximize adherence, patients had the option to schedule their sessions individually during the exercise center's opening hours. The structure of the LOW-HIIT intervention was in accordance with the protocol introduced by Reljic et al. . In brief, the protocol commenced with a short low-intensity warm-up period of 2 min. Subsequently, patients performed 5 vigorous interval bouts of 1 min duration (by accelerating the cadence and/or increasing the ergometer watt load) divided by 1 min recovery phases. After the fifth interval bout, the protocol concluded with a cool-down of 3 min duration at low intensity, corresponding to an accumulated total duration of 14 min/session. In the HIIT-HR group, patients were instructed to reach a minimum exercise intensity of 80-85% HRmax during the intervals for the first 4 weeks. The target intensity during intervals was progressively increased as follows: week 5-8: 85-90% HRmax and week 9-12: 90-95% HRmax. In the HIIT-LT group, the initial minimum exercise intensity to be achieved during intervals was set at a HR corresponding to 95-100% of the LT for the first 4 weeks and then elevated to a HR corresponding to 100-105% of the LT (week 5-12). Patients were equipped with a chest strap HR monitor (Acentas, Horgertshausen, Germany) in every exercise session, allowing them to follow their HR in real-time on a screen. The HR responses were recorded in every session and later analyzed using the software Heart Rate Monitoring Team System (Acentas, Horgertshausen, Germany). Average power output and energy expenditure were recorded from the cycle ergometer's digital displays after each session. Certified physiotherapists monitored every single session to ensure that the imposed level of exercise intensity was reached. 2.7. Statistical Analysis A priori sample size calculation was conducted using the software G*Power (Heinrich-Heine-University Dusseldorf, Dusseldorf, Germany). Data analyses were performed using the software package SPSS version 24.0 software (IBM Corp., Armonk, NY, USA). Initially, data normality was analyzed using the Shapiro-Wilk test. If the data were normally distributed, a 2 x 2 repeated measures ANOVA was conducted to examine the data for both the main effects (group and time) and interaction effects (group x time). In case of significant results, Holm-Sidak post hoc tests for multiple comparisons were performed. Significant main effects of time were followed by separate post hoc paired t-tests for each group. Levene's test was utilized to check and verify the homogeneity of variance. If no normal distribution of data was present, log or square root transformation was conducted and the respective statistical analyses were performed with the transformed data. If this procedure did not improve the data heterogeneity (i.e., HbA1c, serum insulin concentration, HOMA-index and EQ-5D index), non-parametric tests were used for analysis, including the Friedman two-way analysis of variance by ranks, post hoc Dunn's Bonferroni tests for group comparisons and Wilcoxon's tests for within-group comparisons. Effect sizes were evaluated using partial eta-squared (ep2) for the ANOVAs and Kendall's coefficient of concordance (W) for the Friedman tests, respectively, and rated as small (0.01-0.05), medium (0.06-0.13) and large (>=0.14) for ep2, and small (<=0.10), medium (>=0.30), and large (>=0.50) for W . For all analyses, the significance level was set at p < 0.05. Data are shown as means +- standard deviation (SD) and pre-/post-intervention changes of the outcome values are reported with 95% confidence intervals (95% CI). 3. Results 3.1. Study Flow Fifty patients (25 each for the HIIT-HR and CON groups) were randomly selected from a larger cohort of the main trial and agreed to additional blood draws during the CPET for the determination of lactate concentrations. Thirty-seven patients were additionally screened for eligibility to be included in the HIIT-LT group until 25 eligible patients were enrolled, resulting in a total sample of 75 patients (25 per group). Seventeen patients dropped out during the study (HIIT-HR = 5, HIIT-LT = 5 and CON = 7). The reasons for dropout are depictured in Figure 1. Consequently, the final analysis involved the data of 58 patients (HIIT-HR = 20, HIIT-LT = 20 and CON = 18). At the baseline, the three groups did not differ significantly in the primary outcome of VO2max and the other main outcomes of interest. Moreover, we did not detect any significant gender effects and therefore, the data of females and males were jointly evaluated in all analyses. Compliance with the LOW-HIIT protocols (the number of scheduled vs. completed exercise sessions) was noticeably high in both exercise groups with 96 +- 6% in the HIIT-HR group and 94 +- 8% in the HIIT-LT group. 3.2. Training Data and Adverse Events The HRLT at the baseline examination corresponded to 94 +- 4% of the HRmax in the HIIT-LT group. The average peak HR recorded during each interval bout over the 12 weeks corresponded to 93 +- 7% of the HRmax in the HIIT-HR group and 96 +- 3% of the HRmax in the HIIT-LT group, respectively, verifying that the target exercise intensity was successfully reached in both groups. The mean session HR (including the warm-up and cool-down phase) corresponded to 79 +- 6% of the HRmax in the HIIT-HR group, and 82 +- 5% of the HRmax in the HIIT-LT group, respectively. The average peak HR reached during the single intervals and the average session HR were not significantly different between both groups (Table 1). Furthermore, there were no significant differences between both groups in the average power output and energy expenditure per session, with average values of 99.7 +- 24.6 W, 550 +- 149 kilojoules (kJ) and 8.5 +- 2.3 kJ/FFM in the HIIT-HR group, and 105.8 +- 33.2 W, 549 +- 164 kJ and 8.5 +- 2.3 kJ/FFM in the HIIT-LT group, respectively. There were no adverse events observed that were related to the LOW-HIIT. 3.3. Hydration Status and Anthropometric Data During both examinations, the USG values were within the normal ranges for all patients, without significant group differences. There were main effects of time for body weight (p < 0.001, e2 = 0.54), BMI (p < 0.001, e2 = 0.54), FM (p < 0.001, e2 = 0.42), FM% (p < 0.001, e2 = 0.29), FFM (p = 0.003, e2 = 0.15), TBW (p < 0.001, e2 = 0.21) and waist circumference (p < 0.001, e2 = 0.56). Furthermore, there was a group-by-time interaction for waist circumference (p < 0.001, e2 = 0.25) and trend toward an interaction effect for FM (p = 0.055, e2 = 0.10) All groups significantly reduced their body weight (HIIT-HR: -3.9 kg, 95% CI: -5.5 to -2.3 kg, p < 0.001; HIIT-LT: -5.6 kg, 95% CI: -7.8 to -3.4 kg, p < 0.001; CON: -2.6 kg, 95% CI: -4.2 to -1.0 kg, p = 0.003). The quantity of weight loss was not significantly different between the three groups (p = 0.064), but compared to the exercise groups, the CON did not significantly reduce FM and waist circumference. Compared to the CON, the decrease in waist circumference was larger in the HIIT-HR (-7 cm, 95% CI: -9 to -1 cm, p = 0.010) and HIIT-LT (-8 cm, 95% CI: -11 to -3 cm, p < 0.001) groups. Table 2 displays all group specific pre-/post-intervention anthropometric variables. 3.4. Nutrition Data There was a main effect of time for energy (p = 0.024, e2 = 0.09) and fat intake (p = 0.005, e2 = 0.14). Post hoc tests indicated that the reduction in energy and fat intake per day only reached statistical significance in the CON (-410 kcal, 95% CI: -747 to -72 kcal, p = 0.020, and -30 g, 95% CI: -46 to -8 g, p = 0.007, respectively), however, there were no significant differences in daily calorie reduction between the groups. post-intervention, there were no significant group differences in nutritional intake (Table 3). 3.5. Cardiopulmonary Exercise Testing Data During both CPET examinations, all patients fulfilled at least two maximal exertion criteria . In all three groups, there were no significant differences in post-intervention resting lactate concentrations (HIIT-HR: 1.2 +- 0.3 and 1.1 +- 0.2 mmol/L, HIIT-LT: 1.1 +- 0.2 and 1.0 +- 0.3 mmol/L, CON: 1.2 +- 0.3 and 1.2 +- 0.2 mmol/L), maximal lactate levels (HIIT-HR: 7.5 +- 1.7 and 7.7 +- 2.1 mmol/L, HIIT-LT: 7.9 +- 1.5 and 7.9 +- 2.6 mmol/L, CON: 7.9 +- 1.8 and 7.5 +- 1.8 mmol/L) RERmax (HIIT-HR: 1.03 +- 0.1 and 1.04 +- 0.1, HIIT-LT: 1.02 +- 0.1 and 1.03 +- 0.1, CON: 1.01 +- 0.1 and 1.01 +- 0.1) and HRmax (HIIT-HR: 158 +- 17 and 160 +- 18 b/min, HIIT-LT: 156 +- 21 and 158 +- 19 b/min, CON: 158 +- 21 and 157 +- 21 b/min), indicating that maximal exhaustion levels were similar during both examinations. In the HIIT-LT group, there was a high agreement between WLT and WVT2 (p < 0.001, r = 0.78). A main effect of time and group-by-time interaction was found for relative (p < 0.001, e2 = 0.50, and p < 0.001, e2 = 0.39, respectively) and absolute VO2max (p < 0.001, e2 = 0.32, and p < 0.001, e2 = 0.42, respectively), relative (p < 0.001, e2 = 0.67, and p < 0.001, e2 = 0.48, respectively) and absolute Wmax (p < 0.001, e2 = 0.66, and p < 0.001, e2 = 0.62, respectively), WVT1 (p < 0.001, e2 = 0.64, and p < 0.001, e2 = 0.58, respectively) and WVT2 (p = 0.026, e2 = 0.10, and p < 0.001, e2 = 0.39, respectively). Additionally, there was a main effect of time for HRVT1 (p = 0.002, e2 = 0.21) and HRVT2 (p < 0.001, e2 = 0.26). The HIIT-HR and HIIT-LT groups showed similar improvements in relative VO2max (3.6 mL/kg/min, 95% CI: 2.5 to 4.7 mL/kg/min, p < 0.001, and 3.7 mL/kg/min, 95% CI: 2.3 to 5.0 mL/kg/min, p < 0.001), absolute VO2max (301 mL/min, 95% CI: 194 to 409 mL/min, p < 0.001, and 257 mL/min, 95% CI: 154 to 360 mL/min, p < 0.001), relative Wmax (0.3 W/kg, 95% CI: 0.2 to 0.4 W/kg, p < 0.001, and 0.3 W/kg, 95% CI: 0.2 to 0.4 W/kg, p < 0.001), absolute Wmax (25 W, 95% CI: 20 to 30 W, p < 0.001, and 26 W, 95% CI: 20 to 31 W, p < 0.001), WVT1 (30 W, 95% CI: 24 to 35 W, p < 0.001, and 30 W, 95% CI: 22 to 38 W, p < 0.001) and WVT2 (17 W, 95% CI: 5 to 30 W, p = 0.012, and 22 W, 95% CI: 12 to 32 W, p < 0.001). In the HIIT-LT group, there was a significant increase in WLT (12 W, 95% CI: 3 to 22 W, p = 0.012). None of these outcomes improved in the CON. By contrast, absolute VO2max (-214 mL/min, 95% CI: -221 to -8 mL/min, p = 0.037) and WVT (-17 W, 95% CI: -28 to -7 W, p = 0.003) decreased from post-intervention. Compared to the CON, the HIIT-HR and HIIT-LT groups exhibited significantly greater increases in relative VO2max (4.0 mL/kg/min, 95% CI: 2.0 to 5.9 mL/kg/min, p < 0.001, and 4.1 mL/kg/min, 95% CI: 2.1 to 5.9 mL/kg/min, p < 0.001), absolute VO2max (415 mL/min, 95% CI: 237 to 594 mL/min, p < 0.001, and 371 mL/min, 95% CI: 196 to 547 mL/min, p < 0.001), relative Wmax (0.3 W/kg, 95% CI: 0.2 to 0.4 W/kg, p < 0.001, and 0.3 W/kg, 95% CI: 0.2 to 0.4 W/kg, p < 0.001), absolute Wmax (30 W, 95% CI: 21 to 38 W, p < 0.001, and 30 W, 95% CI: 21 to 39 W, p < 0.001), WVT1 (32 W, 95% CI: 12 to 53 W, p < 0.001, and 40 W, 95% CI: 19 to 60 W, p < 0.001) and WVT2 (35 W, 95% CI: 16 to 54 W, p < 0.001, and 40 W, 95% CI: 20 to 59 W, p < 0.001). Pre-/post-intervention CPET outcomes for each group are shown in Table 4. 3.6. Cardiometabolic Data A group-by-time interaction was found for SBP (p < 0.001, e2 = 0.29), DBP (p < 0.001, e2 = 0.26), MAB (p < 0.001, e2 = 0.33) and the MetS z-score (p < 0.001, e2 = 0.30). Additionally, a main effect of time was observed for HRrest (p < 0.001, e2 = 0.33), SBP (p < 0.001, e2 = 0.43), DBP (p < 0.001, e2 = 0.40), MAB (p < 0.001, e2 = 0.48), HbA1c levels (p < 0.001, W = 0.23), serum insulin concentration (p < 0.001, W = 0.25), HOMA-index (p < 0.001, W = 0.28) and the MetS z-score (p < 0.001 e2 = 0.61). Post hoc tests showed that both in the HIIT-HR and HIIT-LT groups, there were significant reductions in HRrest (-6 b/min, 95% CI: -9 to -2 b/min, p = 0.006, and -6 b/min, 95% CI: -7 to -3 b/min, p < 0.001), SBP (-11 mmHg, 95% CI: -15 to -7 mmHg, p < 0.001, and -13 mmHg, 95% CI: -16 to -9 mmHg, p < 0.001), DBP (-8 mmHg, 95% CI: -11 to -4 mmHg, p < 0.001, and -10 mmHg, 95% CI: -13 to -7 mmHg, p < 0.001), MAB (-9 mmHg, 95% CI: -12 to -6 mmHg, p < 0.001, and -11 mmHg, 95% CI: -14 to -8 mmHg, p < 0.001), HbA1c levels (-0.2%, 95% CI: -0.4 to -0.1%, p = 0.012, and -0.3%, 95% CI: -0.4 to -0.2%, p < 0.001), serum insulin concentrations (-5 mU/mL, 95% CI: -8 to -1 mU/mL, p = 0.007, and -3 mU/mL, 95% CI: -8 to -2 mU/mL, p = 0.019), HOMA-index (-1.3 units, 95% CI: -2.4 to -0.2 units, p = 0.005, and -1.0 units, 95% CI: -2.1 to -0.2 units, p = 0.014) and the MetS z-score (-1.9 units, 95% CI: -2.6 to -1.8 units, p < 0.001, and -2.5 units, 95% CI: -3.0 to -2.0 units, p < 0.001). No significant changes occurred in the CON. Compared to the CON, the HIIT-HR and HIIT-LT groups showed significantly greater pre-/post-intervention reductions in SBP (-12 mmHg, 95% CI: -19 to -4 mmHg, p < 0.001, and -13 mmHg, 95% CI: -20 to -5 mmHg, p < 0.001), DBP (-8 mmHg, 95% CI: -13 to -2 mmHg, p = 0.007, and -10 mmHg, 95% CI: -16 to -4 mmHg, p < 0.001), MAB (-9 mmHg, 95% CI: -15 to -4 mmHg, p < 0.001, and -11 mmHg, 95% CI: -17 to -6 mmHg, p < 0.001) and MetS z-score (-1.6 units, 95% CI: -2.6 to -0.5 units, p < 0.001, and -2.2 units, 95% CI: -3.2 to -1.1 units, p < 0.001). Pre-/post-intervention cardiometabolic variables for each group are shown in Table 5. 3.7. Self-Reported Quality of Life Data A main effect of time was detected for EQ-VAS (p < 0.001, e2 = 0.24) and the EQ-5D index (p = 0.031, W = 0.08). Both the HIIT-HR and HIIT-LT groups experienced a pre-/post-intervention increase in EQ-VAS (10 points, 95% CI: 1 to 18 points, p = 0.029, and 11 points, 95% CI: 5 to 17 points, p = 0.002), whereas no significant changes were recorded in the CON (Table 6). 4. Discussion Exercise intensity is a crucial--if not the most pivotal--variable in exercise prescription . Intensity prescriptions for (LOW-)HIIT programs are typically based on percentages of the HRmax, which, however, may be associated with several limitations in clinical populations. Given the rising popularity of LOW-HIIT in prevention programs and clinical exercise interventions, it is timely to investigate the viability of alternative approaches for exercise intensity prescriptions in individuals, where determination of the HRmax may not be feasible. To our knowledge, this investigation was the first to compare the effects of a LOW-HIIT intervention based on either the HRmax or the submaximal LT in obese patients with MetS. The major result was that the HIIT-HR and HIIT-LT produced similar improvements in key cardiometabolic outcomes and self-reported QoL after a period of 12 weeks. The finding that the two LOW-HIIT protocols had similar beneficial effects on cardiometabolic health and QoL was in contrast to our hypothesis based on some previous research, reporting that threshold-based exercise intensity prescriptions are superior to relative percent concepts in improving various cardiometabolic outcomes . When analyzing the training data (average HR and power output), however, it becomes evident that the physiological demands were comparable between both LOW-HIIT protocols. Furthermore, compliance with both protocols was similarly very high (HIIT-HR: 96 +- 6%, and HIIT-LT: 94 +- 8%) and thus, it is plausible that both protocols yielded similar benefits. In this context, it is noteworthy that 4 mmol/L LT data for obese MetS patients have rarely been described in the literature. We found that the HRLT corresponded to 94 +- 4% of the HRmax in our patients, which is in accordance with the well-established 3-phase model introduced by Skinner et al. , illustrating that the HR at the 4 mmol/L LT typically exceeds 90% of the HRmax. When comparing both LOW-HIIT protocols, it is notable that all patients in our study were able and willing to reach maximal exertion during the CPET. Thus, in general, if patients are physically able and no symptoms occur during exercise that would require premature termination, we recommend that CPET should be performed until exhaustion in order to acquire maximum performance data and to use the established criteria to verify that maximum exertion has been reached . However, it is an important practical result of this investigation that exercise intensity prescription for the LOW-HIIT protocols can also be feasibly generated using a submaximal exercise test until the LT is reached, which may constitute a viable approach if maximal CPET is contraindicated or patients are not able/motivated to exercise until exertion. Both LOW-HIIT protocols induced improvements in several health-related outcomes that can be considered clinically meaningful. First, patients involved in the LOW-HIIT improved VO2max by ~3.7 mL/kg/min. The importance of CRF for health and longevity has been well-established in decades of research . It has been reported, for example, that each VO2max increase by 1 mL/kg/min is associated with a 9% risk decrease in overall mortality . Recent large-scale research verified these findings, demonstrating that each 3.5 mL/kg/min improvement in CRF is related to a decreased risk of premature death due to cardiovascular disease and cancer each by 15% . Second, the reduction in MetS z-score indicates an improvement in overall MetS severity, which was mainly related to reductions in blood pressure (-12 mmHg SBP/-9 mmHg DBP, on average) and waist circumference (-8 cm, on average). Large prospective cohort studies have indicated a reduced risk of coronary heart disease by 22% and stroke by 41%, respectively, per each -10 mmHg SBP/-5 mmHg DBP decrease and an 8% reduction in all-cause mortality per -5 cm decrease in waist circumference . Additionally, both LOW-HIIT protocols had beneficial effects on glucose metabolism as indicated by significant reductions in the HbA1c levels, fasting insulin and HOMA-index. Improvements in these outcomes have been associated with improved cardiometabolic health and a lower risk of colorectal cancer , for example. Third, self-reported QoL improved in response to both LOW-HIIT protocols. The mean pre-intervention EQ-VAS scores were markedly lower in our patient cohort than the values reported for the general population , which is in line with data from other researchers indicating a relationship between MetS and a diminished QoL . The marked post-intervention improvement in EQ-VAS following LOW-HIIT underscores the well-established association between physical activity , CRF levels and enhanced QoL. Taken together, these findings highlight the pleiotropic effects of exercise on a broad range of important health markers and support the "exercise is medicine" message . Although we clearly recommend that patients who are willing and capable of being more physically active should be encouraged to perform higher volumes of exercise in order maximize the health benefits, our observations provide further evidence that even very small doses of targeted exercise can provide meaningful improvements in the physiological and psychological outcomes. The three groups achieved an average weight loss of ~3.5% within the 12-week study period, which is in accordance with most lifestyle-intervention programs for obesity . It is noteworthy that the relative weight loss amounts tended to be greater in the two exercise groups compared to the CON, but the total difference did not reach statistical significance (exercise groups vs. CON, p = 0.066). This finding is not surprising as the three groups did not significantly differ in the amount of caloric reduction, which is the key component to achieve a negative energy balance and to reduce body weight . Although there is evidence that (LOW-)HIIT, compared to traditional continuous endurance training, may have different (more pronounced) effects on some physiological factors associated with weight loss, including higher excess post-exercise oxygen consumption , stronger post-exercise suppression of appetite perception or greater changes in concentrations of distinctive gut hormones and leptin , our results suggest that the extremely low volume of exercise applied in the present study did not have a substantial impact on the daily overall energy balance. Thus, when it comes to pure weight loss, higher-volume exercise modalities with greater energy expenditure (e.g., longer-lasting endurance exercise or HIIT involving more and/or longer intervals) may be more effective compared to our very low-volume HIIT protocol. However, in agreement with previous reports, it is too short-sighted to define a successful obesity treatment solely in terms of pure weight loss because it is more important to improve the CRF and other cardiometabolic health outcomes than to strictly follow anthropometric measures to improve morbidity and longevity . In this regard, we observed substantial differences between the patients allocated to the CON and those performing LOW-HIIT, with only the "exercisers" achieving significant improvements in cardiometabolic health and QoL, despite similar weight loss. Finally, we note some potential limitations to this investigation. Firstly, we note that all patients received standard care nutritional counseling in addition to the LOW-HIIT, which may represent a confounding variable for the observed pre-/post intervention changes. However, we do not feel that the nutritional modification had any meaningful effect on the major research question of this study (HIIT-HR vs. HIIT-LT) because both groups received the same counseling and there were no significant differences in the nutritional intake between the HIIT-HR and HIIT-LT groups. Nevertheless, it cannot be completely ruled out that potential between-group variations in nutrition might have affected the results to some extent. Secondly, we are well aware that numerous LT as well as VT concepts exist and one can argue why we used the fixed 4 mmol/L LT to prescribe the exercise intensity to the HIIT-LT group. Specific reasons for selecting the 4 mmol/L LT are given in the methodology section, but we highlight that it was the major aim of this study to compare the effects of LOW-HIIT prescriptions based on maximal versus submaximal exercise parameters and not to investigate which threshold concept might be the best for obese MetS patients. Nevertheless, we do not rule out that another threshold concept/exercise prescription approach may have achieved even better results or might even have been superior to the HRmax-based prescription method. Future research may wish to explore this important question. Moreover, further research is necessary to investigate whether the findings obtained by this specific cohort of obese MetS patients may be transferred to other (clinical) populations. Lastly, it must be considered that all the examinations and the LOW-HIIT intervention were carried out in a well-controlled clinical environment. Thus, it remains to be elucidated to which degree our findings can be applied to non-clinical settings. 5. Conclusions The HIIT-HR and HIIT-LT induced similar improvements in cardiometabolic health and QoL in obese MetS patients. Thus, the practical take-home message for clinicians and exercise physiologists who wish to implement LOW-HIIT in clinical populations, is that exercise intensity can feasibly and effectively be prescribed using a submaximal LT-based exercise test if patients are not willing or able to perform maximal CPET. Acknowledgments We would like to thank Alisia Gerl, Melanie Klaussner, Maike Tobschall and Kerstin Weidlich for supervising and instructing the exercise sessions. We would also like to thank Kathinka Faustka and Julia Kratzer for their assistance in data collection. We are especially grateful to all patients for their willingness to participate in this study. Parts of this manuscript are used in the medical dissertation of co-author Fabienne Frenk. We acknowledge financial support by Deutsche Forschungsgemeinschaft and Friedrich-Alexander-Universitat Erlangen-Nurnberg within the funding programme "Open Access Publication Funding". Author Contributions Conceptualization, D.R.; methodology, D.R.; validation, D.R., H.J.H. and Y.Z.; formal analysis, D.R.; investigation, D.R. and F.F.; data curation, F.F.; writing--original draft preparation, D.R.; writing--review and editing, H.J.H., M.F.N. and Y.Z.; supervision, D.R.; project administration, D.R. and Y.Z.; funding acquisition, D.R., H.J.H. and Y.Z. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Medical Ethical Committee of the Friedrich-Alexander University Erlangen-Nurnberg (approval number: 210_17B). Informed Consent Statement Informed consent was obtained from all subjects involved in the study. Data Availability Statement The datasets generated and analyzed during the current study are not publicly available but are available from the corresponding author on reasonable request. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Study flow chart. healthcare-11-00711-t001_Table 1 Table 1 Average heart rate during intervals and during the whole exercise session 1. Variable HIIT-HR HIIT-LT Week 1-4 Whole session (%HRmax) 79.1 +- 5.7 81.8 +- 4.8 Intervals (%HRmax) 92.6 +- 6.0 95.5 +- 3.4 Week 5-8 Whole session (%HRmax) 78.2 +- 7.7 81.6 +- 5.4 Intervals (%HRmax) 92.3 +- 7.4 95.4 +- 4.1 Week 9-12 Whole session (%HRmax) 78.0 +- 7.3 82.0 +- 6.1 Intervals (%HRmax) 92.7 +- 8.2 95.6 +- 3.7 Values are presented as mean +- SD. HRmax = maximum heart rate. 1 including warm up, intervals, recovery periods and cool-down. healthcare-11-00711-t002_Table 2 Table 2 Anthropometric and hydration variables. Variable HIIT-HR (n = 20) HIIT-LT (n = 20) CON (n = 18) Pre Post Pre Post Pre Post Weight (kg) 117.1 +- 30.3 113.2 +- 29.3 c 117.5 +- 24.5 111.9 +- 24.4 c 110.7 +- 21.8 108.1 +- 23.2 b BMI (kg/m2) 38.0 +- 7.7 36.8 +- 7.5 c 39.5 +- 8.2 37.5 +- 8.2 c 37.3 +- 5.1 36.4 +- 5.7 b FM (kg) 50.0 +- 17.6 46.3 +- 16.4 c 52.5 +- 17.4 47.8 +- 18.2 c 49.8 +- 11.4 48.3 +- 13.7 FM (%) 42.3 +- 8.2 40.5 +- 8.5 c 44.0 +- 7.0 41.8 +- 7.9 b 45.0 +- 6.2 44.5 +- 7.1 FFM (kg) 67.1 +- 17.0 67.1 +- 18.0 65.1 +- 11.4 64.1 +- 11.1 60.9 +- 14.3 59.8 +- 13.8 b TBW (L) 50.0 +- 13.0 49.8 +- 13.3 48.7 +- 8.4 47.8 +- 8.1 a 45.9 +- 10.3 44.9 +- 10.0 c USG (mg/dL) 1025 +- 12 1022 +- 11 1026 +- 14 1023 +- 12 1024 +- 10 1023 +- 10 Waist (cm) 116.8 +- 21.3 110.2 +- 18.1 c 119.4 +- 13.6 111.0 +- 15.0 c 113.3 +- 13.4 111.8 +- 15.4 Values are presented as mean +- SD. BMI = body mass index, FM = fat mass, FFM = skeletal muscle mass, TBW = total body water, USG = urine specific gravity. a (p < 0.05), b (p < 0.01), c (p < 0.001): significantly different compared to pre-intervention. healthcare-11-00711-t003_Table 3 Table 3 Nutritional intake. Variable HIIT-HR (n = 20) HIIT-LT (n = 20) CON (n = 18) Pre Post Pre Post Pre Post Energy (kcal/d) 2320 +- 669 2111 +- 875 2359 +- 1204 1966 +- 790 2206 +- 606 1796 +- 620 a Protein (g/d) 96.8 +- 42.4 96.9 +- 37.9 107.3 +- 95.7 97.1 +- 60.9 94.0 +- 26.0 86.2 +- 30.5 Protein (g/kg/d) 0.9 +- 0.3 0.9 +- 0.3 1.0 +- 0.5 0.9 +- 0.6 0.9 +- 0.3 0.9 +- 0.3 Fat (g/d) 95.4 +- 39.4 83.3 +- 39.1 86.7 +- 41.3 76.7 +- 41.2 93.9 +- 32.1 66.6 +- 26.2 b Fat (g/kg/d) 0.9 +- 0.3 0.8 +- 0.3 0.8 +- 0.3 0.7 +- 0.4 0.9 +- 0.3 0.7 +- 0.3 a CHO (g/d) 211.3 +- 50.1 220.6 +- 106.5 233.7 +- 104.8 186.7 +- 73.8 212.2 +- 73.0 183.9 +- 83.6 CHO (g/kg/d) 1.9 +- 0.6 2.0 +- 0.7 2.1 +- 1.0 1.7 +- 0.7 2.0 +- 0.8 1.8 +- 0.9 Fiber (g/d) 24.3 +- 9.4 23.7 +- 12.0 21.1 +- 9.0 23.4 +- 11.4 22.8 +- 13.4 22.0 +- 8.8 Values are presented as mean +- SD. CHO = carbohydrates. a (p < 0.05), b (p < 0.01): significantly different compared to pre-intervention. healthcare-11-00711-t004_Table 4 Table 4 Cardiopulmonary exercise testing variables. Variable HIIT-HR (n = 20) HIIT-LT (n = 20) CON (n = 18) Pre Post Pre Post Pre Post VO2max (mL/kg/min) 21.6 +- 4.8 25.2 +- 5.3 c 22.0 +- 6.9 25.7 +- 7.4 c 21.6 +- 7.0 21.2 +- 7.4 VO2max (L/min) 2.4 +- 0.6 2.7 +- 0.7 c 2.5 +- 0.6 2.8 +- 0.7 c 2.4 +- 0.8 2.2 +- 0.8 a Wmax (W/kg) 1.4 +- 0.4 1.7 +- 0.4 c 1.4 +- 0.5 1.7 +- 0.6 c 1.4 +- 0.5 1.4 +- 0.5 Wmax (W) 156.6 +- 42.0 181.3 +- 38.8 c 156.7 +- 45.1 182.2 +- 49.6 c 153.3 +- 57.7 148.5 +- 53.7 WVT1 (W) 58.0 +- 25.3 87.3 +- 24.0 c 63.4 +- 26.2 93.1 +- 30.2 c 62.2 +- 33.8 57.1 +- 28.6 WVT2 (W) 125.2 +- 35.8 142.4 +- 33.4 a 132.6 +- 35.3 154.6 +- 38.7 c 136.0 +- 41.6 119.2 +- 41.0 b WLT (W) * --- --- 138.9 +- 38.8 151.3 +- 46.7 a --- --- Values are presented as mean +- SD. VO2max = maximal oxygen uptake, Wmax = maximal power output, WVT1, WVT2 and WLT = power output at ventilatory threshold 1, power output at ventilatory threshold 2 and power output lactate threshold, respectively. a (p < 0.05), b (p < 0.01), c (p < 0.001): significantly different compared to pre-intervention. * only determined in the HIIT-LT group. healthcare-11-00711-t005_Table 5 Table 5 Cardiometabolic variables. Variable HIIT-HR (n = 20) HIIT-LT (n = 20) CON (n = 18) Pre Post Pre Post Pre Post HRrest (b/min) 75.7 +- 9.8 70.3 +- 8.0 b 74.6 +- 12.4 69.4 +- 12.3 c 77.3 +- 8.8 74.7 +- 10.0 SBP (mmHg) 143.3 +- 13.1 132.0 +- 11.6 c 140.0 +- 13.6 127.4 +- 13.4 c 137.5 +- 10.9 137.8 +- 7.8 DBP (mmHg) 93.5 +- 7.8 86.0 +- 7.0 c 88.6 +- 9.7 78.4 +- 7.4 c 88.3 +- 8.9 88.4 +- 7.6 MAB (mmHg) 110.1 +- 8.3 101.2 +- 7.2 c 105.7 +- 10.2 94.8 +- 7.8 c 104.8 +- 8.8 105.0 +- 6.4 Glucose (mg/dL) 103.0 +- 18.6 101.0 +- 13.2 104.9 +- 14.1 99.8 +- 15.0 94.1 +- 17.4 92.6 +- 14.3 HbA1c (%) 5.7 +- 0.5 5.5 +- 0.4 b 5.7 +- 0.4 5.4 +- 0.4 c 5.6 +- 0.9 5.6 +- 0.7 Triglycerides (mg/dL) 135.2 +- 59.2 130.0 +- 35.0 116.5 +- 45.1 119.4 +- 55.4 120.0 +- 90.0 119.1 +- 62.4 Cholesterol (mg/dL) 219.3 +- 35.1 215.3 +- 36.2 208.8 +- 36.4 211.5 +- 34.4 218.0 +- 37.2 217.0 +- 31.1 LDL (mg/dL) 147.8 +- 28.8 143.9 +- 26.8 140.7 +- 28.0 141.2 +- 29.3 148.7 +- 29.9 148.2 +- 22.6 HDL (mg/dL) 49.2 +- 10.0 49.4 +- 11.2 47.0 +- 9.4 49.8 +- 12.0 55.1 +- 12.3 53.0 +- 12.1 Insulin (mU/mL) 18.3 +- 11.7 12.7 +- 9.3 b 19.7 +- 10.8 17.3 +- 12.8 a 18.2 +- 12.6 15.9 +- 8.0 HOMA-index 4.8 +- 3.6 3.4 +- 2.1 b 5.2 +- 3.0 4.2 +- 3.0 a 4.4 +- 3.6 3.8 +- 2.4 MetS z-score 3.3 +- 4.0 1.4 +- 3.0 c 3.3 +- 2.6 0.8 +- 3.0 c 2.0 +- 2.8 1.5 +- 3.1 Values are presented as mean +- SD. HRrest = resting heart rate, SBP = systolic blood pressure, DBP = diastolic blood pressure, MAB = mean arterial blood pressure, HbA1c = glycated hemoglobin A1c, LDL = low-density lipoprotein cholesterol, HDL = high-density lipoprotein cholesterol, HOMA = homeostasis model assessment, MetS = metabolic syndrome. a (p < 0.05), b (p < 0.01), c (p < 0.001): significantly different compared to pre-intervention. healthcare-11-00711-t006_Table 6 Table 6 Quality of life variables. Variable HIIT-HR (n = 20) HIIT-LT (n = 20) CON (n = 18) Pre Post Pre Post Pre Post EQ-VAS 63.0 +- 15.3 72.6 +- 21.1 a 63.9 +- 15.7 74.7 +- 17.2 b 60.4 +- 25.1 65.7 +- 28.3 EQ-5D index 0.84 +- 0.16 0.86 +- 0.20 0.85 +- 0.12 0.88 +- 0.14 0.87 +- 0.14 0.86 +- 0.19 Values are presented as mean +- SD. BMI = body mass index, FM = fat mass, FFM = skeletal muscle mass, TBW = total body water. a (p < 0.05), b (p < 0.01): significantly different compared to pre-intervention. 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PMC10000823 | Healthcare (Basel) Healthcare (Basel) healthcare Healthcare 2227-9032 MDPI 10.3390/healthcare11050638 healthcare-11-00638 Article Healthcare Resource Utilization in Patients with Newly Diagnosed Atrial Fibrillation: A Global Analysis from the GARFIELD-AF Registry Mantovani Lorenzo G. Conceptualization Formal analysis Writing - original draft 12* Cozzolino Paolo Writing - review & editing 1+ Ferrara Pietro Writing - review & editing 12+ Virdone Saverio Methodology Data curation 3 Camm A. John Writing - review & editing Supervision 4 Verheugt Freek W. A. Writing - review & editing 5 Bassand Jean-Pierre Writing - review & editing 36 Turpie Alexander G. G. Writing - review & editing 7 Hacke Werner Writing - review & editing 8 Kayani Gloria Funding acquisition 3 Goldhaber Samuel Z. Writing - review & editing 9 Goto Shinya Writing - review & editing 10 Pieper Karen S. 3 Gersh Bernard J. Writing - review & editing 11 Fox Keith A. A. 12 Haas Sylvia Writing - review & editing 13 van Eickels Martin 14 Kakkar Ajay K. 3 on behalf of the GARFIELD-AF Investigators++ Giordano Raffaele Academic Editor 1 Center for Public Health Research, University of Milan-Bicocca, 20900 Monza, Italy 2 Laboratory of Public Health, Istituto Auxologico Italiano--IRCCS, 20165 Milan, Italy 3 Thrombosis Research Institute, London SW3 6LR, UK 4 Cardiovascular Clinical Academic Group, St. George's University of London, London SW17 0RE, UK 5 Onze Lieve Vrouwe Gasthuis (OLVG), 1091 AC Amsterdam, The Netherlands 6 Department of Cardiology, University of Besancon, 25030 Besancon, France 7 Faculty of Health Sciences, McMaster University, Hamilton, ON L8S 4L8, Canada 8 Department of Neurology, Ruprecht-Karls-University of Heidelberg, 69120 Heidelberg, Germany 9 Division of Cardiovascular Medicine, Harvard Medical School, Boston, MA 02115, USA 10 Department of Medicine (Cardiology), Tokai University School of Medicine, Isehara 259-1193, Japan 11 Department of Cardiovascular Medicine, Mayo Clinic College of Medicine and Science, Rochester, NY 55905, USA 12 Centre for Cardiovascular Science, University of Edinburgh, Edinburgh EH16 4TJ, UK 13 Formerly Department of Medicine, Technical University of Munich, 81675 Munich, Germany 14 Bayer AG, 13353 Berlin, Germany * Correspondence: [email protected] + These authors contributed equally to this work. ++ The full list of GARFIELD-AF investigators is provided in the supplemental materials. 21 2 2023 3 2023 11 5 63825 1 2023 13 2 2023 17 2 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). The management of atrial fibrillation (AF), the most common sustained arrhythmia, impacts healthcare resource utilization (HCRU). This study aims to estimate global resource use in AF patients, using the GARFIELD-AF registry. A prospective cohort study was conducted to characterize HCRU in AF patients enrolled in sequential cohorts from 2012 to 2016 in 35 countries. Components of HCRU studied were hospital admissions, outpatient care visits, and diagnostic and interventional procedures occurring during follow-up. AF-related HCRU was reported as the percentage of patients demonstrating at least one event and was quantified as rate-per-patient-per-year (PPPY) over time. A total of 49,574 patients was analyzed, having an overall median follow-up of 719 days. Almost all patients (99.5%) had at least one outpatient care visit, while hospital admissions were the second most frequent medical contact, with similar proportions in North America (37.5%) and Europe (37.2%), and slightly higher in the other GARFIELD-AF countries (42.0%; namely Australia, Egypt, and South Africa). Asia and Latin America showed lower percentages of hospitalizations, outpatient care visits, and diagnostic and interventional procedures. Analyses of GARFIELD-AF highlighted the vast AF-related HCRU, underlying significant geographical differences in the type, quantity, and frequency of AF-related HCRU. These differences were likely attributable to health service availability and differing models of care. atrial fibrillation healthcare resource utilization inpatient care outpatient care the Thrombosis Research Institute (London, UK)This work was supported by the Thrombosis Research Institute (London, UK). pmc1. Introduction Atrial fibrillation (AF) is the most common arrhythmia and, with its progressively increasing prevalence, impacts public health and healthcare resource utilization (HCRU) . AF affects approximately 37.5-million adults worldwide with about 400 new cases per 1-million inhabitants diagnosed annually . AF patients are at increased risk for stroke and suffer an increase in morbidity and mortality . AF's association with hypercholesterolemia, diabetes mellitus, arterial hypertension, chronic kidney disease (CKD), dementia, obesity, and sleep apnea may confer a negative prognosis . AF's association with healthcare resource utilization (HCRU) presents a large economic burden . AF is estimated to account for more than 1% of total healthcare expenditures in high-income countries, mostly attributable to hospitalization . Other resource use and cost contributors include medical visits, emergency room (ER) admissions, and diagnostic and interventional procedures often required by AF patients (e.g., electrocardiography, laboratory tests, cardioversion, catheter ablation, etc.) . Several studies have evaluated the multiple aspects of AF, including its HCRU burden, studied according to specific contexts, settings, or treatment options . The objective of this study was to characterize the global HCRU in AF patients within the Global Anticoagulant Registry in the FIELD-AF (GARFIELD-AF). The GARFIELD-AF registry defines a non-interventional, observational study that characterized a global population of non-valvular AF patients. This multicenter global registry documented patients' and sub-populations' baseline characteristics, treatment strategies, and outcome measures by including five prospective cohorts of adult subjects who were newly diagnosed with non-valvular AF (diagnosed within the previous six weeks before enrolment) and having at least one additional risk factor for stroke. GARFIELD-AF also included a validation cohort of retrospective patients diagnosed with non-valvular AF between 6 and 24 months prior to enrolment . 2. Materials and Methods 2.1. Study Design and Data Source A prospective cohort design was used to characterize resource utilization associated with the care of AF patients. The study investigated the GARFIELD-AF registry, an observational worldwide registry that prospectively and consecutively enrolled sequential cohorts of 52,167 newly diagnosed AF patients at risk of stroke from December 2009 to August 2016 in 35 countries. Eligible patients were aged 18 years or older, enrolled consecutively into five cohorts (representing seven years of enrollment, from 2010 to 2016) including ~10,000 participants each; the additional retrospective cohort (GARFIELD-AF Cohort 1) was excluded. Participants with a follow-up period of less than three months were excluded from the analysis. Data were extracted from the final study database lock (June 2019) in 2020. The GARFIELD-AF study design has been reported elsewhere . Baseline patient characteristics--including demographic information, clinical conditions, risk stratification, and antithrombotic treatment--were collected at inclusion in the registry . Risk stratification was documented through CHA2DS2-VASc (congestive heart failure, hypertension, age >= 75 years [doubled], diabetes, stroke [doubled], vascular disease, age 65-74 years, and sex category [female]). Follow-up data on treatments and outcomes were collected at four monthly intervals up to 24 months. GARFIELD-AF data were captured using an electronic case report form (eCRF) designed by Dendrite Clinical Systems Ltd. (Henley-on-Thames, UK). Oversight of operations and data management were performed by the coordinating center (Thrombosis Research Institute, London, UK). The study is registered at ClinicalTrials.gov (unique identifier: NCT01090362). Patients were selected from multiple healthcare settings and were registered by the identifying clinician registered using the eCRF. Data were collected from five clinical sources associated with the patient (i.e., hospital, emergency department, anticoagulation clinic, stroke unit, and office-based settings such as general or family practitioners, cardiologists, and internists) through a review of patient notes and clinical records . Data on HCRU and changes in medication treatment were stored in a dedicated follow-up and events dataset. 2.2. Outcomes Measures and Definitions HCRU in AF patients was evaluated focusing on medical contacts and is reported as the proportion and frequency of at least one event (besides the recruitment visit, which was excluded from the analysis). Events included in the analysis of HCRU studied were those linked to AF and its sequalae and collected during follow-up visits as per study protocol and according to standardized outcome definitions . Studied HCRU items include hospital admissions, outpatient hospital attendance, ER admissions, family doctor visits, stroke unit admissions and office-based specialist visits, and diagnostic and interventional procedures occurring during the follow-up period. General practitioner visits, office-based specialist visits, and hospital-based outpatient visits were grouped as "outpatient care visits," to adequately compare information from different countries and settings. Diagnostic and interventional procedures covered all those derived from follow-up events, including those specific to AF (such as electrical cardioversion and ablation), methods for pulmonary embolism diagnosis (e.g., computed tomography scan, magnetic resonance imaging scan, and invasive angiography) and interventions required for cardiovascular diseases (including percutaneous coronary intervention [PCI] bare metal stent, PCI drug eluting stent, PCI balloon angioplasty, coronary artery bypass graft, valve replacement, pacemaker, and carotid stent). Data on medication use were not included in this study, as this has been evaluated in previous analyses of the GARFIELD-AF registry . For the purpose of this analysis, patients were divided into two groups according to the enrolment cohorts, which allowed to account for possible differences in HCRU over the whole study period. Group A included participants recruited into GARFIELD-AF Cohorts 2 and 3 from 2010 to 2013; Group B included those in Cohorts 4 to 6 from 2013 to 2016. In particular, we split patients into two 3-year timeslots since, by Cohort 3, the non-Vitamin K antagonist oral anticoagulants (NOAC) were approved in most of the countries included in the GARFIELD-AF registry. In addition, there has been an increased use in newly diagnosed patients with AF receiving guideline-recommended treatment . The 35 countries within the registry were grouped by geographical region, according to the classification provided by the GARFIELD-AF dataset used: Asia (China, India, Japan, Korea, Singapore, Thailand, Turkey, and United Arab Emirates), Europe (Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Hungary, Italy, The Netherlands, Norway, Poland, Russia, Spain, Sweden, Switzerland, Ukraine, and United Kingdom), Latin America (Argentina, Brazil, Chile, and Mexico), North America (Canada and United States), and other GARFIELD-AF countries (Australia, Egypt, and South Africa, henceforth defined as "others"). 2.3. Statistical Analysis Continuous variables were described with mean and median as central tendency measures, while standard deviation (SD) and interquartile range (IQR) were described as dispersion measures. Categorical variables have been presented using frequency and percentage. The Student's t test was used to assess differences between continuous variables, and the Chi square (kh2) or Fisher's exact tests were used when needed to assess differences between categories. AF-related HCRU was reported as the percentage of patients having at least one event included in the analyses and quantified as rate-per-patient-per-year (PPPY). Region-specific HCRU rates were subsequently compared using a Poisson regression model, which was adjusted for possible known confounders and modifiers collected in the registry, such as sex, age at enrolment, type of AF (i.e., (i) paroxysmal: AF that lasts less than 7 days and resolves spontaneously or with intervention; (ii) persistent: AF episode that continues for more than 7 days, irrespective of whether the episode was terminated by cardioversion or if it self-terminated; (iii) permanent: when AF is accepted by the patient (and physician) and a rate-control strategy is needed; or (iv) new onset--unclassified), AF therapy (i.e., antiplatelet [AP], alone or in combination with Vitamin K antagonists [VKA] or NOAC), comorbidities, prior transient ischemic attack, prior bleeding, CHA2DS2-VASc score, country income level (i.e., high, upper-middle, or lower-middle), and healthcare system payer (i.e., single payer, universal public insurance, public-private insurance, or private insurance). Results are expressed as incidence rate ratios (IRR) with 95% confidence intervals (95% CI). All p-values were two-sided, with values <0.05 considered statistically significant. Analyses were performed using STATA statistical software version 13.1 . 3. Results 3.1. Baseline Sample Characteristics This study involved a total of 49,574 patients, with an overall median follow-up period of 719 days (IQR, 597-730). The cohort was mainly constituted of subjects enrolled in Europe (56.5%) and Asia (28.4%), with a median age of 71 years (IQR, 63-78). More than half of the participants were men (55.7%). Prior bleeding was reported in 2.5%; transient ischemic attack in 4.4%; diabetes in 22.4%. A complete overview of patient demographics and clinical characteristics, according to region, is listed in Table 1. Differences according to the cohorts of enrollment are presented in Table 2. The two cohort groups differed in almost all the baseline clinical characteristics. 3.2. Healthcare Resource Utilization The vast majority of patients (99.5%) had at least one outpatient care visit, excluding the enrollment medical contact. Hospitalization was the second-most frequent medical contact, with almost one-third (30.4%) of patients having at least one hospital visit. Higher proportions of patients with more than one hospitalization were observed in North America (37.5%), Europe (37.2%), and others (42.0%). Of these, stroke unit admissions accounted for around 1% in all groups. Higher numbers of procedures and ER admissions were registered in North America with, respectively, 25.1% and 31.0% patients. A lower proportion of patients with at least one ER admission was seen in Asia (8.1%), and a lower proportion of ER procedures was recorded in Latin America (7.5%). Table 3 reports the number of GARFIELD-AF participants with at least one HCRU event. The cohort groups' PPPYs results aggregated by region are presented in Figure 1 and Figure 2. Outpatient care visits were the most frequent event in both groups (i.e., participants enrolled between 2010 and 2013 and those enrolled from 2013 to 2016). Large variations in the type of other medical contacts were observed across regions and between the two cohort groups. In Group A (GARFIELD-AF Cohorts 2 and 3), patients with higher PPPY rates for procedures and hospitalizations were in Europe and others, while lesser values for both events were seen in Asia. In the latter, the lowest PPPY rate for ER visits was also registered. Patients in Europe showed lower PPPY rates for outpatient visits . Overall, AF-related HCRU trends in Group B (GARFIELD-AF Cohorts 4 to 6) mirrored those in Group A, with narrower regional differences as compared with the previous . Compared with Europe, patients in North America showed higher HRCU rates for all medical contacts, and those in Latin America and Asia showed lower ones. 4. Discussion In this real-world observational study, we combined global data from the GARFIELD-AF registry on the estimated HCRU in AF patients and compared it among regions and over time. Our findings highlighted the extensive resources utilized in almost 50,000 subjects from 35 countries worldwide. Important disparities still exist in their utilization among patients in the five regions after controlling for various confounders, such as patients' characteristics and clinical status, as well as societal aspects (for instance, country income level and healthcare system payer). All HCRU components showed an overlapping pattern across the five regions in the two study groups, but frequencies changed across cohorts. In particular, the analyses highlighted narrower regional differences in the second period along with the differences shown between the two groups as compared with the first one. Overall, these changes may indicate an increasingly progressive concordance with evidence-based guidelines for patients newly diagnosed with AF across countries, mirroring trends seen in previous GARFIELD-AF research. It appears that clinical practice and treatment of AF patients has become more uniform over time, likely due to a wider use of NOACs and specific AF procedures, such as electrical cardioversion and ablation . As regards to the regional distribution of AF-related HCRU, a primary reason for these differences may be the availability of services and the differing models of healthcare and AF-care organization, beyond differences in healthcare system and payer . In certain settings, gate-keeping systems--such as an initial visit to a general practitioner for access to specialist care or the presence of transitional care facilities--influenced the patient's use of services. A previous analysis conducted in Latin American countries included in the GARFIELD-AF registry has suggested inadequate management of AF patients, with therapy underuse attributable to physician choice, difficulties in accessing healthcare, adverse economic conditions, and lower educational levels . Additionally, access to primary and cardiology care in rural communities may be a recurring challenge for older and disabled AF adults, resulting in gaps in access to health services . Another factor influencing HCRU variations across regions is inconsistent patient demographics, particularly the population age structure. These differing age structures may persist also after appropriate confounder correction . Thus, greater numbers and frequencies of medical contacts may be at least partly attributed to the larger proportion of elderly people in some countries. Similarly, AF epidemiological metrics should be considered in the interpretation of our results. Although global rates are relatively stable, higher and more premature mortality due to AF was shown in middle-income countries . In contrast, a lower risk of death in Asia and Europe compared with other regions is a common observation, likely linked to the highly protective healthcare system and easier access to services in these regions . Living in North America or Latin America was instead associated with a higher risk of early death . A bias toward lower reported medical contacts may exist in countries where such services lack or are underused, resulting in a suboptimal level of care. When analyzing the type of healthcare contacts, it is worth noting that hospitalizations account for higher HCRU rates. Drivers for urgent and elective hospitalization in AF patients have been extensively described in the literature, and include cardiovascular and non-traditional risk factors, as well as considerable rates of readmission, particularly in comorbid, higher CHA2DS2VASc score, and post-ablated AF patients . Overall, inpatient care is the main determinant of healthcare costs associated with AF. Thus, further research is needed to develop specific effective transitional and integrated care interventions . In summary, although marked differences in resource use for AF patient care were observed worldwide, using the expansive GARFIELD-AF registry, our findings suggest that AF substantially contributes to resource consumption with a subsequent important impact on healthcare expenditure worldwide . The management of AF is complex, and convergence towards guideline-directed care is crucial to maximize patient's benefit from tailored treatment options. Yet, implanting integrated AF care models has been proven to reduce disease and resource burden of AF . In this sense, our findings may serve as actionable indicators of novel value-based organizational approaches to support changes in the management of AF. This paper has a number of strengths and weaknesses. The design features of GARFIELD-AF registry include the random selection of sites and the enrolment of patients without exclusion according to comorbidities or treatment that ensures, respectively, the representativeness of the national care settings and population aimed to study, thus providing reliable estimates of research outcomes. Despite these strengths, this research should be interpreted in the context of its limitations. The study did not consider other possible unmeasured confounders, which may influence HCRU in AF patients. However, we included those mainly associated with the outcomes, and the use of robust statistical analysis allowed us to balance factors potentially correlated to such confounders. The reported burden of resource consuming was quantified excluding medication use, which was previously characterized in other GARFIELD-AF studies . The differences in healthcare systems and organization across the countries included in the GARFIELD-AF registry may reflect variability in types, amounts, and patterns of HCRU events. 5. Conclusions Within the GARFIELD-AF registry, a vast amount of HCRU was documented in AF patients from 35 countries worldwide. Important geographical differences exist in the type, quantity, and frequency of HCRU in patients with AF. Changes in AF care and variable adherence to evidence-based guidelines determined different patterns of HCRU, with a trend toward convergence of clinical practices over time. Acknowledgments We thank the physicians, nurses, and patients involved in the GARFIELD-AF registry. Medical writing support was provided by Rebecca Watkin, Thrombosis Research Institute (London, UK). SAS programming support was provided by Madhusudana Rao (Thrombosis Research Institute, London, UK). Supplementary Materials The following are available online at Section S1, Complete list of GARFIELD-AF investigators. Section S2, Appendix to Results: Table S1: Interventional procedures included in the analysis. Click here for additional data file. Author Contributions Conceptualization, L.G.M.; methodology, L.G.M. and P.C.; software, P.C.; validation, L.G.M., P.C., P.F., S.V., A.J.C., F.W.A.V., J.-P.B., A.G.G.T., W.H., G.K., S.Z.G., S.G., K.S.P., B.J.G., K.A.A.F., S.H., M.v.E. and A.K.K.; formal analysis, P.C. and P.F.; investigation, L.G.M. and P.F.; data curation, P.C.; writing--original draft preparation, P.F.; writing--review and editing, L.G.M., P.C., P.F., S.V., A.J.C., F.W.A.V., J.-P.B., A.G.G.T., W.H., G.K., S.Z.G., S.G., K.S.P., B.J.G., K.A.A.F., S.H., M.v.E. and A.K.K.; visualization, P.F.; supervision, A.J.C. and K.S.P.; funding acquisition, G.K. and A.K.K. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The GARFIELD-AF registry was conducted in accordance with the principles of the Declaration of Helsinki, local regulatory requirements, and the International Conference on Harmonization-Good Pharmacoepidemiologic and Clinical Practice guidelines. Independent ethics committee and hospital-based institutional review board approvals were obtained. Informed Consent Statement Written informed consent was obtained from all subjects involved in the study. Data Availability Statement Data are available on reasonable request. The data underlying this article will be shared on reasonable request from Karen S. Pieper ([email protected]). Conflicts of Interest L.G.M. received grants and personal fees from Bayer AG, Daiichi Sankyo, Pfizer and Boehringer Ingelheim. A.J.C. received institutional grants and personal fees from Bayer, Boehringer Ingelheim, Pfizer/BMS, Daiichi Sankyo; F.W.A.V. received grants from Bayer Healthcare; personal fees from Bayer Healthcare, BMS/Pfizer, DaiichiSankyo, and Boehringer-Ingelheim; J.P.B. received personal fees from the Thrombosis Research Institute; A.G.G.T. received personal fees from Bayer Healthcare, Janssen Pharmaceutical Research & Development, and Portola; W.H. personal fees from Bayer AG; S.Z.G. received research support from Boehringer-Ingelheim, BMS, BTG EKOS, Daiichi, Janssen, NHLBI, and the Thrombosis Research Institute; S.Z.G. also served as a consultant for Agile, Bayer, Boehringer-Ingelheim, BMS, Daiichi, Janssen and Zafgen; S.G. received personal fees from Thrombosis Research Institute and the American Heart Association, as well as grants from Sanofi, Pfizer, Ono, Bristol Myer Squibb, the Vehicle Racing Commemorative Foundation and Nakatani Foundation for Advancement of Measuring Technologies in Biomedical Engineering; K.S.P. serves as consultant for Thrombosis Research Institute, AstraZeneca, and Bayer; B.J.G. is a data safety monitoring board member at Mount Sinai St Luke's, Boston Scientific Corporation, St Jude Medical Inc., Janssen Research & Development LLC, Thrombosis Research Institute, Duke Clinical Research Institute, Duke University, Kowa Research Institute Inc, Cardiovascular Research Foundation, and Medtronic; he also received general consulting for Janssen Scientific Affairs, Xenon Pharmaceuticals and Sirtex Medical Limited; K.A.A.F. has received grants and personal fees from Bayer/Janssen and AstraZeneca and personal fees from Sanofi/Regeneron and Verseon; S.H. received personal fees from Aspen, Bayer, BMS, Daiichi-Sankyo, Portola, Sanofi; M.v.E. is an employee of Bayer AG; A.K.K. received research support from Bayer AG and Sanofi, and personal fees from Bayer AG, Janssen, Pfizer, Sanofi, Verseon and Anthos Therapeutics; P.C., P.F., S.V. and G.K. declare no conflicts of interest. Figure 1 Regression-adjusted per-patient-per-year rates of healthcare resource utilization in patients with atrial fibrillation in cohort Group A (GARFIELD-AF Cohorts 1 and 2) by region. Abbreviations: IRR, incidence rate ratio; 95% CI, 95% confidence interval; ER, emergency room. Figure 2 Regression-adjusted per-patient-per-year rates of healthcare resource utilization in patients with atrial fibrillation in cohort Group B (GARFIELD-AF Cohorts 4 to 6) by region. Abbreviations: IRR, incidence rate ratio; 95% CI, 95% confidence interval; ER, emergency room. healthcare-11-00638-t001_Table 1 Table 1 Patient demographics and clinical characteristics. Total Asia Europe Latin America North America Other GARFIELD-AF Countries N (%) 49,574 (100.00) 14,059 (28.36) 27,987 (56.45) 4004 (8.08) 1554 (3.13) 1970 (3.97) Cohort Group A (C2-3) 16,459 (33.20) 3711 (26.40) 10,449 (37.34) 1390 (34.72) 331 (21.30) 578 (29.34) Group B (C4-6) 33,115 (66.80) 10,348 (73.60) 17,538 (62.66) 2614 (65.28) 1223 (78.70) 1392 (70.66) Sex Male 27,586 (55.65) 8251 (58.69) 15,293 (54.64) 2106 (52.60) 849 (54.63) 1087 (55.18) Female 21,988 (44.35) 5808 (41.31) 12,694 (45.36) 1898 (47.40) 705 (45.37) 883 (44.82) Age Mean (SD) 69.62 (11.48) 67.54 (12.00) 70.73 (10.90) 69.77 (11.92) 71.05 (11.84) 67.32 (12.04) Median (IQR) 71 (63-78) 69 (60-76) 72 (64-79) 71 (63-78.5) 72 (64-80) 68 (60-76) Comorbid conditions Diabetes No 38,458 (77.58) 10,903 (77.55) 21,975 (78.52) 3011 (75.20) 1143 (73.55) 1426 (72.39) Yes 11,116 (22.42) 3156 (22.45) 6012 (21.48) 993 (24.80) 411 (26.45) 544 (27.61) Chronic kidney disease No 22,753 (45.90) 7941 (56.48) 11,055 (39.50) 1876 (46.85) 926 (59.59) 955 (48.48) I 9994 (20.16) 2702 (19.22) 6034 (21.56) 809 (20.20) 164 (10.55) 285 (14.47) II 5896 (11.89) 1020 (7.26) 4314 (15.41) 192 (4.80) 112 (7.21) 258 (13.10) III/IV 4918 (9.92) 960 (6.83) 3387 (12.10) 241 (6.02) 125 (8.04) 205 (10.41) V 226 (0.46) 108 (0.77) 74 (0.26) 26 (0.65) 11 (0.71) 7 (0.36) Unknown 5786 (11.67) 1328 (9.45) 3123 (11.16) 859 (21.45) 216 (13.90) 260 (13.20) Missing 1 (0.00) 0 (0.00) 0 (0.00) 1 (0.02) 0 (0.00) 0 (0.00) Hypercholesterolemia No 27,841 (56.16) 9751 (69.36) 14,317 (51.16) 2327 (58.12) 623 (40.09) 823 (41.78) Yes 20,257 (40.86) 4020 (28.59) 12,778 (45.66) 1468 (36.66) 904 (58.17) 1087 (55.18) Unknown 1476 (2.98) 288 (2.05) 892 (3.19) 209 (5.22) 27 (1.74) 60 (3.05) Cirrhosis No 48,647 (98.13) 13,784 (98.04) 27,541 (98.41) 3912 (97.7) 1505 (96.85) 1905 (96.70) Yes 272 (0.55) 92 (0.65) 134 (0.48) 14 (0.35) 14 (0.90) 18 (0.91) Unknown 655 (1.32) 183 (1.30) 312 (1.11) 78 (1.95) 35 (2.25) 47 (2.39) Congestive Heart Failure No 39,688 (80.06) 11,274 (80.19) 22,237 (79.45) 3200 (79.92) 1323 (85.14) 1654 (83.96) Yes 9886 (19.94) 2785 (19.81) 5750 (20.55) 804 (20.08) 231 (14.86) 316 (16.04) Vascular Disease No 42,209 (85.14) 12,400 (88.20) 23,553 (84.16) 3425 (85.54) 1285 (82.69) 1546 (78.48) Yes 7365 (14.86) 1659 (11.80) 4434 (15.84) 579 (14.46) 269 (17.31) 424 (21.52) Type of atrial fibrillation Permanent 6285 (12.68) 1195 (8.50) 4193 (14.98) 643 (16.06) 35 (2.25) 219 (11.12) Persistent 7427 (14.98) 2438 (17.34) 4104 (14.66) 597 (14.91) 97 (6.24) 191 (9.70) Paroxysmal 13,739 (27.71) 5169 (36.77) 6933 (24.77) 1050 (26.22) 332 (21.36) 255 (12.94) New onset (unclassified) 22,123 (44.63) 5257 (37.39) 12,757 (45.58) 1714 (42.81) 1090 (70.14) 1305 (66.24) Stroke prophylaxis AP or none 15,933 (32.14) 6013 (42.77) 7431 (26.55) 1484 (37.06) 500 (32.18) 505 (25.63) VKA +- AP 19,352 (39.04) 4140 (29.45) 12,396 (44.29) 1580 (39.46) 341 (21.94) 895 (45.43) NOACs +- AP 13,598 (27.43) 3752 (26.69) 7733 (27.63) 874 (21.83) 694 (44.66) 545 (27.66) Unknown 691 (1.39) 154 (1.10) 427 (1.53) 66 (1.65) 19 (1.22) 25 (1.27) History of bleeding No 48,157 (97.14) 13,723 (97.61) 27,230 (97.3) 3841 (95.93) 1470 (94.59) 1893 (96.09) Yes 1237 (2.50) 230 (1.64) 712 (2.54) 158 (3.95) 72 (4.63) 65 (3.30) Unknown 180 (0.36) 106 (0.75) 45 (0.16) 5 (0.12) 12 (0.77) 12 (0.61) Prior transient ischemic attack No 47,138 (95.09) 13,631 (96.96) 26,460 (94.54) 3815 (95.28) 1457 (93.76) 1775 (90.10) Yes 2183 (4.40) 302 (2.15) 1451 (5.18) 173 (4.32) 81 (5.21) 176 (8.93) Unknown 253 (0.51) 126 (0.90) 76 (0.27) 16 (0.40) 16 (1.03) 19 (0.96) CHA2DS2-VASc score 0 1366 (2.76) 684 (4.87) 470 (1.68) 115 (2.87) 46 (2.96) 51 (2.59) 1 6072 (12.25) 2300 (16.36) 2904 (10.38) 442 (11.04) 175 (11.26) 251 (12.74) 2 9950 (20.07) 3116 (22.16) 5428 (19.39) 728 (18.18) 286 (18.40) 392 (19.90) 3 11,954 (24.11) 3181 (22.63) 7010 (25.05) 933 (23.30) 366 (23.55) 464 (23.55) 4 10,837 (21.86) 2584 (18.38) 6481 (23.16) 982 (24.53) 366 (23.55) 424 (21.52) 5 5624 (11.34) 1356 (9.65) 3399 (12.14) 470 (11.74) 188 (12.10) 211 (10.71) 6-9 3771 (7.61) 838 (5.96) 2295 (8.20) 334 (8.34) 127 (8.17) 177 (8.98) Abbreviations: C, enrolment cohort; SD, standard deviation; IQR, interquartile range; AP, antiplatelet therapy; VKA, vitamin K antagonists; NOAC, non-vitamin K antagonist oral anticoagulants. healthcare-11-00638-t002_Table 2 Table 2 Differences in patient characteristics according to cohort groups. Cohort Group A Cohort Group B Total p-Value N (%) 16,459 (33.20) 33,115 (66.80) 49,574 (100.0) Sex 0.316 Male 9211 (55.96) 18,375 (55.49) 27,586 (55.65) Female 7248 (44.04) 14,740 (44.51) 21,988 (44.35) Age 0.139 Mean (SD) 69.73 (11.40) 69.57 (11.52) 69.62 (11.48) Median (IQR) 71 (63-78) 71 (62-78) 71 (63-78) Comorbid conditions Diabetes 0.247 No 12,819 (77.88) 25,639 (77.42) 38,458 (77.58) Yes 3640 (22.12) 7476 (22.58) 11,116 (22.42) Chronic kidney disease * <0.001 None 93 (0.57) 22,660 (68.43) 22,753 (45.90) I 8099 (49.21) 1895 (5.72) 9994 (20.16) II 2434 (14.79) 3462 (10.45) 5896 (11.89) III/IV 1629 (9.90) 3289 (9.93) 4918 (9.92) V 70 (0.43) 156 (0.47) 226 (0.46) Unknown 4134 (25.12) 1652 (4.99) 5786 (11.67) Missing 0 (0.00) 1 (0.00) 1 (0.00) Hypercholesterolemia <0.001 No 9765 (59.33) 18,076 (54.59) 27,841 (56.16) Yes 6689 (40.64) 13,568 (40.97) 20,257 (40.86) Unknown 5 (0.03) 1471 (4.44) 1476 (2.98) Cirrhosis <0.001 No 16,365 (99.43) 32,282 (97.48) 48,647 (98.13) Yes 83 (0.50) 189 (0.57) 272 (0.55) Unknown 11 (0.07) 644 (1.94) 655 (1.32) Congestive Heart Failure 0.001 No 13,041 (79.23) 26,647 (80.47) 39,688 (80.06) Yes 3418 (20.77) 6468 (19.53) 9886 (19.94) Vascular Disease 0.345 No 14,049 (85.36) 28,160 (85.04) 42,209 (85.14) Yes 2410 (14.64) 4955 (14.96) 7365 (14.86) Type of atrial fibrillation <0.001 Permanent 2160 (13.12) 4125 (12.46) 6285 (12.68) Persistent 2556 (15.53) 4871 (14.71) 7427 (14.98) Paroxysmal 4199 (25.51) 9540 (28.81) 13,739 (27.71) New onset (unclassified) 7544 (45.84) 14,579 (44.03) 22,123 (44.63) Stroke prophylaxis <0.001 VKA +- AP 8161 (49.58) 11,191 (33.79) 19,352 (39.04) NOACs +- AP 1781 (10.82) 11,817 (35.68) 13,598 (27.43) None +- AP 6232 (37.86) 9701 (29.29) 15,933 (32.14) Unknown 285 (1.73) 406 (1.23) 691 (1.39) History of bleeding <0.001 No 15,979 (97.08) 32,178 (97.17) 48,157 (97.14) Yes 472 (2.87) 765 (2.31) 1237 (2.50) Unknown 8 (0.05) 172 (0.52) 180 (0.36) Prior transient ischemic attack <0.001 No 15,582 (94.67) 31,556 (95.29) 47,138 (95.09) Yes 870 (5.29) 1313 (3.96) 2183 (4.40) Unknown 7 (0.04) 246 (0.74) 253 (0.51) CHA2DS2-VASc score <0.001 0 386 (2.35) 980 (2.96) 1366 (2.76) 1 1951 (11.85) 4121 (12.44) 6072 (12.25) 2 3218 (19.55) 6732 (20.33) 9950 (20.07) 3 3975 (24.15) 7979 (24.09) 11,954 (24.11) 4 3610 (21.93) 7227 (21.82) 10,837 (21.86) 5 1947 (11.83) 3677 (11.10) 5624 (11.34) 6-9 1372 (8.34) 2399 (7.24) 3771 (7.61) * Major discrepancies in rates of chronic kidney disease between groups may be attributable to changes in disease definition that occurred after the enrollment of GARFIELD-AF Cohort 2. Abbreviations: SD, standard deviation; IQR, interquartile range; AP, antiplatelet therapy; VKA, Vitamin K antagonists; NOAC, non-Vitamin K antagonist oral anticoagulants. healthcare-11-00638-t003_Table 3 Table 3 Patients with at least one medical contact. HCRU Events * (N [%]) Total Asia Europe Latin America North America Other GARFIELD-AF Countries Hospitalizations 15,046 (30.35) 2617 (18.61) 10,412 (37.20) 607 (15.16) 582 (37.45) 828 (42.03) ER admissions 8129 (16.40) 1137 (8.09) 5637 (20.14) 434 (10.84) 482 (31.02) 439 (22.28) Procedures ^ 7167 (14.46) 1540 (10.95) 4527 (16.18) 302 (7.54) 390 (25.10) 408 (20.71) * Outpatient care visits were not reported since all patients had at least one. ^ Type and number of interventional procedures included in the analysis are detailed in Table S1 (supplementary materials). Abbreviations: HCRU, healthcare resource utilization; ER, emergency room. 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PMC10000824 | Foods Foods foods Foods 2304-8158 MDPI 10.3390/foods12051001 foods-12-01001 Article Physicochemical Parameters, Antioxidant Capacity, and Antimicrobial Activity of Honeys from Tropical Forests of Colombia: Apis mellifera and Melipona eburnea Zapata-Vahos Isabel Cristina Conceptualization Methodology Investigation Writing - original draft Writing - review & editing 1* Henao-Rojas Juan Camilo Software Writing - review & editing 2* Yepes-Betancur Diana Paola Writing - review & editing 3 Marin-Henao Daniela Methodology Writing - original draft 4 Giraldo Sanchez Carlos Eduardo Software Investigation 5 Calvo-Cardona Samir Julian Software Investigation 6 David Dorely Methodology Investigation Writing - original draft Writing - review & editing 7 Quijano-Abril Mario Conceptualization Methodology Investigation Writing - original draft 4 Yang En-Cheng Academic Editor Chen Yue-Wen Academic Editor Peng Chi-Chung Academic Editor Sung I-Hsin Academic Editor Wu Ming-Cheng Academic Editor Nai Yu-Shin Academic Editor Gregorc Ales Academic Editor 1 Facultad de Ciencias de la Salud, Grupo de Investigacion Atencion Primaria en Salud, Universidad Catolica de Oriente, Rionegro 054040, Colombia 2 Corporacion Colombiana de Investigacion Agropecuaria-Agrosavia, Centro de Investigacion La Selva, Kilometro 7, Via a Las Palmas, Vereda Llanogrande, Rionegro 054048, Colombia 3 Servicio Nacional de Aprendizaje, SENA, Grupo de Investigacion en Innovacion y Agroindustria, Centro de la Innovacion, la Agroindustria y la Aviacion, Cra 48 # 49-62, Rionegro 054040, Colombia 4 Grupo de Investigacion en Estudios Floristicos, Facultad de Ingenierias, Herbario, Universidad Catolica de Oriente, Rionegro 054040, Colombia 5 Grupo de Investigacion Sanidad Vegetal, Facultad de Ciencias Agropecuarias, Universidad Catolica de Oriente, Rionegro 054040, Colombia 6 Zootecnista, PhD Data Plus CIC, Pereira 660008, Colombia 7 Tecnoparque Nodo Rionegro, Centro de la Innovacion La Agroindustria y la Aviacion, Servicio Nacional de Aprendizaje--SENA, Rionegro 054040, Colombia * Correspondence: [email protected] (I.C.Z.-V.); [email protected] (J.C.H.-R.) 27 2 2023 3 2023 12 5 100109 12 2022 04 1 2023 07 1 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). Honey is a functional food used worldwide and recognized for its multiple health benefits. In the present study, the physicochemical and antioxidant properties of honey produced by two species of bees (Melipona eburnea and Apis mellifera) in two seasons were evaluated. In addition, the antimicrobial activity of honey against three bacterial strains was studied. The quality of honey analyzed by LDA (linear discriminant analysis) showed four clusters mediated by the interaction, the bee species, and the collection season resulting from a multivariate function of discrimination. The physicochemical properties of the honey produced by A. mellifera met the requirements of the Codex Alimentarius, while the M. eburnea honey had moisture values outside the established ranges of the Codex. Antioxidant activity was higher in the honey of A. mellifera, and both kinds of honey showed inhibitory activity against S. typhimurium ATCC 14028 and L. monocytogenes ATCC 9118. E. coli ATCC 25922 showed resistance to the analyzed honey. bee honey antioxidant potential inhibitory activity physicochemical property Universidad Catolica de Oriente201844 This research was funded by Universidad Catolica de Oriente, grant number 201844. pmc1. Introduction Honey is a sweet and viscous fluid produced by bees, which is one of the oldest traditional medicines and has had different uses in the culinary industry as a flavoring and sweetener. In addition, multiple beneficial health effects have been reported, such as treatments for burns, wounds, ulcers, gastrointestinal disorders, respiratory diseases, and cancer . Some of these benefits are attributed to bactericidal and antioxidant honey activity . The antioxidant and antibacterial activity of honey may be due to the presence of enzymes, such as glucose oxidase and catalase, as well as compounds, such as phenolic acids, flavonoids, organic acids, etc. . Honey also contains macronutrients; carbohydrates represented as sugars and proteins, vitamins, minerals, and others . The composition of honey varies according to the botanical origin as the main sources where the bees collected the nectar possibly being monofloral or polyfloral . Another variation of honey could be due to the species of bees that produces it. Apis mellifera is a bee introduced from Europe. It belongs to the order Hymenoptera and family Apidae, and Melipona eburnea is a native South American stingless bee. It belongs to the same order and family Meliponinae . However, few studies have compared the characteristics of the honey produced by these two species. The quality of honey may vary depending on the species that produce it. Several parameters, such as moisture content, pH, acidity, organic acids content, and 5- (5-HMF) content determine the quality of honey . Some research suggests the quality and physicochemical characteristics of honey could be better when hives are established at the limits of conserved natural forests in contrast to being located in areas close to urban centers . The quality and other parameters of honey could be influenced by season . In Colombia, beekeepers have not yet managed to take advantage of the high potential of the honey market due to the poor characterization and differentiation of their products and the high levels of counterfeiting with sugar . Due to the megadiversity of flora that exists in the forests of Colombia , there is currently no representative sampling that allows us to elucidate the influence of its physicochemical characteristics. The objective of this work was to characterize and compare the physicochemical properties and antioxidant and antimicrobiological activity of the honey produced by two species of bees, one native (M. eburnea) and the other introduced (A. mellifera) in the forests of the western slope of Magdalena in Colombia, which is considered a diversity hotspot. 2. Materials and Methods 2.1. Study Area The study was carried out with 24 hives (12 from Apis mellifera and 12 from Melipona eburnea) established in two municipalities of eastern Antioquia, located in the very humid premontane forest life zone (El Carmen de Viboral and San Carlos, Antioquia, Colombia) . The selected municipalities are characterized by their current beekeeping production as a strategy for the generation of economic resources for communities in post-conflict times as well as forest conservation and management. 2.2. Obtaining the Samples To have a better representation in terms of flowering peaks, climatic season, and harvest time, samplings were carried out at two times of the year (dry season and rainy season) . For both A. mellifera and M. eburnea, the honey was obtained directly from the hives; collections involved between 15 and 20 mL of immature honey. The M. eburnea honey was collected using sterile 20 mL syringes where the samples of both kinds of honey were subsequently filtered and stored in plastic containers marked with the hive code and the date of collection. Nine (9) experimental replicates were taken for each possible interaction between bee species, season, and municipality, while each experimental variable was measured in triplicate in the laboratory for each real sample. 2.3. Physicochemical Characterization The physicochemical parameters of the honey of the two bee species were evaluated following the described methods in Codex Alimentarius and official analytic methods of the AOAC . The moisture content (% w/w) was determined by the refractometric method using a refractometer (ATAGO), and it was calculated using the Wedmore table. The pH values were measured with a pH meter (Jenway) using a solution of 10 g of honey in 75 mL of distilled water. Free and lactonic acidity were determined potentiometrically by titration, adding NaOH (0.05 mol/L) until reaching pH of 8.3 and 8.5, respectively (mg equivalent of acid. Kg-1). The ashes (%) were analyzed by muffle incineration, achieving constant weight. Electrical conductivity was measured according to the method proposed by the International Honey Commission of 2009 (mS/cm). The color was measured by the Bianchi method combining the color parameters determined in mm of Pfund with the absorbance of samples at a given wavelength. 2.3.1. Diastase and HMF Activity Diastase activity was determined using the AOAC 958.09 method. A buffered mixture of soluble starch and a honey solution was used in a water bath at (40 +- 1 degC) for the time required to reach a specified endpoint (determined spectrophotometrically) until reaching an absorbance value (0.235) at 660 nm (Diastase Number, DN). 2.3.2. HMF Activity The HMF content determination was based on the method by Zappala et al. (2005). Amounts of 3.5 g of honey samples were diluted up to 5 mL with distilled water, filtered on 0.45 mm filter, and immediately injected in a UHPLC (UltiMateTM 3000 high-performance liquid ultrachromatography equipment) equipped with a diode array detector. The HPLC column was a Raptor C18, 2.7 mm, 150 x 3 mm. The HPLC conditions were the following: isocratic mobile phase, 90% water at 10% methanol; flow rate, 0.4 mL/min; and injection volume, 10 mL. All the solvents were HPLC grade (Merck, Milan, Italy). The wavelength range was 220-660 nm, and the chromatograms were monitored at 283 nm . 2.3.3. Sugars Three types of sugar were analyzed, fructose, glucose, and sucrose, for the two kinds of honey produced by the two species of bees. UHPLC UltiMateTM 3000 high-performance liquid ultrachromatography equipment was used. A total of 3.5 g of honey was weighed and diluted with distilled water to 5 mL; then, 18 mL was taken and made up to 25 mL in water for an acetonitrile mixture (50:50) and passed through 0.22 mm syringe filters. A CAD aerosol charge detector was used with an acetonitrile solution. Water (75:25) as a mobile phase and a column Asahipak 5 mm NH2p-50 4E 100a of 250 x 4.6 mm and a flow of 1 mL/min was used. The oven temperature was 30 degC. The values are expressed in percentages (%). 2.4. Determination of Total Phenols Content The determination of phenols was carried out by the Folin-Ciocalteu colorimetric method . In a reaction tube, 50 mL of the honey dilution, 425 mL of distilled water, and 125 mL of the Folin-Ciocalteu reagent were added. This was stirred and then allowed to stand for 6 min. Subsequently, 400 mL of 7.1% Na2CO3 was added. After 1 h in the dark, absorbance was read at 760 nm. A standard curve was constructed using gallic acid as a standard. The analyses were carried out in triplicate, and the results were expressed as mg equivalent of gallic acid/100 g of honey. 2.5. Antioxidant Capacity Analysis 2.5.1. DPPH Free Radical Trapping Activity The Brand-Williams method was used with some modifications . In a test tube, 10 mL of the honey dilution and 990 mL of a DPPH solution were added. As a reference, the same amount of DPPH and 10 mL of the sample solvent (water) were used. The antioxidant capacity was evaluated using the decrease in absorbance after 30 min of reaction at a wavelength of 517 nm. The percentage of radical inhibition was calculated, and the results were expressed as TEAC (Trolox equivalent antioxidant capacity) values (mmol of Trolox/100 g of honey) by constructing a standard curve, using Trolox as an antioxidant. The analyses were carried out in triplicate. 2.5.2. ABTS Free-Radical-Trapping Activity Evaluation of the antioxidant capacity by the ABTS+ cationic radical method was used . The radical was activated by a reaction between ABTS with potassium persulfate. In a test tube, 10 mL of the honey dilution and 990 mL of an ABTS solution standard at 0.7 of absorbance were added. As a reference, the same amount of ABTS and 10 mL of buffer 7.4 were used. The ability of the samples to trap the ABTS radical was evaluated using the decrease in absorbance after 30 min of reaction and at 732 nm of wavelength. The results were expressed as TEAC values by constructing a standard curve using Trolox(r) as an antioxidant. 2.5.3. FRAP (Reducing Capacity) FRAP was carried out according to the method of Benzie and Strain . A volume of 50 mL of honey was mixed with 950 mL of solution FRAP (A solution of TPTZ, FeCl3, and acetate buffer pH 3.6). After 30 min, absorbance data were measured at a wavelength of 593 nm. The reference curve was constructed using ascorbic acid. The activities of the samples were expressed as AEAC (antioxidant capacity in ascorbic acid equivalents: mg of ascorbic acid/100 g of honey). 2.6. Antimicrobial Activity The antimicrobial activities of the different kinds of honey were determined using the diffusion methodology in three bacterial strains, Escherichia coli ATCC 25922, Salmonella typhimurium ATCC 14028, Listeria monocytogenes ATCC 19118, which were selected for their relationships with spoiled or contaminated food. This methodology makes it possible to measure the area of inhibition of the two kinds of honey. The microorganisms were reactivated 24 h before on TSA agar (trypticase soy agar) seeded by the exhaustion method and incubated at 37 degC. They were then inoculated in BHI (brain heart infusion) until reaching turbidity equivalent to the concentration of 0.5 of the McFarland standard. This solution was massively seeded on the surface of the Muller Hinton agar using a sterile swab and making a horizontal sweep along the entire surface. The process was repeated three times. Rotating the petri dish 60deg, 0.5 mm discs were impregnated with each of the kinds of honey, and they were deposited in the culture medium in an equidistant and random manner. Three repetitions of each of the evaluated honey were made and a solution of ciprofloxacin (160 mg/mL) was used as a positive control . 2.7. Statistic Analysis Determination of Data Normality Criteria Descriptive statistical analyses were obtained for each of the variables evaluated, and hypothesis tests were also applied to meet the assumptions of normality and homoscedasticity using the Shapiro-Wilk and Bartlett tests, respectively . Additionally, the multivariate normality test was performed, calculating Royston's H index . This was done both in the original data and in the normalized matrices, confirming the effectiveness of the process. Linear Discriminant Analysis (LDA) and Multiple Range Tests To determine the influence of the factors on the response variables, given the impossibility of performing an analysis of variance due to the non-parametric nature of the data, a linear discriminant analysis was carried out using all possible combinations of the controlled factors as discriminant factors (honey collection season, bee species, and their interaction). To perform this analysis, it was necessary to transform the data through a standardization process using the Z-score methodology reported by Kappal , establishing linear polynomial relationships between all the variables studied to differentiate between categorical groups that were previously defined as well as their interactions. In this case, to establish which physicochemical, functional, and microbiological variables of the kinds of honey had discriminant weight, the Lambda-Wilks parameter was used , which was chosen to verify that it should not be greater than 0.05. Subsequently, each of the clusters formed for each variable was compared using the non-parametric Kruskal-Wallis test (p < 0.05). It is important to highlight that this second stage of analysis should have been carried out with the original database (un-normalized) to preserve the dimensionality and interpretation of the measurements. Principal Component Analysis (PCA) and Correlation Analysis To reduce the dimensionality and determine the variables that contribute the most to the general variability of the honey population studied, a principal component analysis (PCA) was used for the variables associated with the quality of the honey of each species of bee, which allows observing the behavior of the variables characterized as a whole for each type of honey . In addition, the correlation coefficient matrix (r) was constructed with Spearman method using the Factoextra and corrplot libraries . All the aforementioned analyses were carried out using the statistical software, Statgraphics Centurion XVI.II, in the company of the free software, R, and its R Studio complement. 3. Results and Discussion 3.1. Influence of the Bee Species and the Time of Collection on the Quality Characteristics and Biological Activities of the Two Kinds of Honey Produced in Tropical Forests of Colombia 3.1.1. Physical-Chemical Analysis Figure 2 shows the proposed linear discriminant model represents 96.95 % of the total variability of the sampled population (LD1:54.31%, LD2: 42.64%). In addition, it is possible to observe the formation of clusters according to the species of bees from which the honey comes and the honey collection season. There are four clearly defined clusters, which confirms that honey quality is mediated by the interaction, the bee species, and the collection season resulting from a multivariate function of discrimination . On the other hand, the municipality did not have an appreciable discriminating capacity. These clustering results are like those obtained by Conti , although they found the grouping based on the quality characteristics of the honey according to their locality of production and could not demonstrate a statistical relationship of such clustering. It is important to highlight the variables with the greatest discriminating power between the honey of two species of bees and in two collecting seasons are: D-fructose, D-glucose, D-sucrose, ABTS radical uptake capacity, total polyphenolic compounds, electrical conductivity, and free acidity. The multiple range test of Kruskall-Wallis p-values for the characteristics in the honey produced by the two species of bees during the two collect seasons are shown in Table 1. The color variable did not show normal behavior, and the Kruskal-Wallis test did not show significant effects between the honey produced by A. mellifera and M. eburnea bees (p >= 0.05), and there were also no differences by season. According to the Pfund scale to classify the color of honey , the honey produced by A. mellifera and M. eburnea correspond to light amber in the dry season (72 and 60.83 mm PFund, respectively) and extra light amber in the rainy season (48.21 and 49.69 mm PFund, respectively). Similarly, other researchers have found variations in the color of honey are related to the floral source and the collection season since the heat could exert a darkening action; for example, Melaku and Tefera found A. mellifera honey is darker in the May to June harvest season (equivalent to a dry season) than the honey harvested in the October to November season (equivalent to a relatively rainy season). In the honey stingless bee (Tetragonula species), a Pfund average of 67 +- 19 mm, which classifies it in the color range of light amber , was reported. The honey of the two species fluctuated between 0.15 and 0.26% ash. There were no significant effects for the ash content in the honey produced by the studied species (p >= 0.05) nor by season. The ash contents of honey were higher than those reported by Melaku and Tefera , who found an average ash content of 0.12 +- 0.12% in A. mellifera honey samples, while the honey from different species of Mellipona was reported with values higher than those found in the present study . According to Nanda et al. , mineral content of honey is highly dependent on the types of flowers used by bees for nectar. The species A. mellifera had moisture values lower than those reported by M. eburnea (p <= 0.05); this variable showed no variation by season. The moisture content of honey is an important factor reflected in its shelf life since honey with high moisture content (above 20 %) can be susceptible to fermentation during storage . Likewise, honey with a moisture content between 18 and 20% is considered mature and stable . The moisture content of the honey fluctuated between 19.8 and 26.9%, which may be related to the high relative humidity of the municipalities where the study was carried out, which has also been reported by Adgaba et al. who suggested high-moisture content of honey from humid regions can be related to the difficulties of bees to evaporate moisture from honey against high relative humidity in the air. The pH of honey from A. mellifera had significant differences according to the season (p <= 0.05), but not between species. The pH values in the honey were between 3.55 and 4.48, which is similar to the average pH value of 3.02-4.16 found in a study from A. mellifera honey in Eastern Amhara Region, Ethiopia . The free acidity level of honey samples ranged from 36.12 to 49.46 meq acid. Kg-1 and did not present statistically significant differences between the species nor the temporality (p >= 0.05), whereas lactonic acidity in honey showed differences mediated by season but not by bee species. In the same way as our study, the honey produced by M. favosa also found a high average free acidity of 50.6 meq acid. Kg-1, indicating the presence of higher amounts of weak acids, such as organic acids with low ionization . According to Nascimento et al. and Apriceno et al. , honey acidity depends on its content of organic acids, particularly the gluconic acid that results from the spontaneous hydrolysis of glucone-d-lactone enzymatically formed by glucose. Therefore, the total acidity in honey must be evaluated as the sum of free and lactone acidities; likewise, the lactonic acidity is considered the reserve of acidity when the honey becomes alkaline . Regarding the HMF analysis, significant differences were observed mainly for the rainy collection season between the two species of bees. The values found are low considering the limits established by the Codex Alimentarius (less than 80 mg/Kg) , which further suggests the freshness of the honey. Other researchers have reported values higher than those found in the present study; for example, Hoxha et al. found HMF values between 12.61 and 663.58 mg/kg in local and imported brand honey available in different markets. Likewise, in 21 Italian honey samples, the HMF values ranged from 3.35 to 43.21 mg/kg . The 5-Hydroxymethylfurfural (HMF) is a cyclic aldehyde formed during the decomposition of fructose and glucose. However, factors, such as acidic conditions, high temperature, high water content, and metallic containers, can influence their formation; likewise, it can be produced during food processing through the Maillard reaction or extended storage . Consequently, High HMF levels generally provide an indication of overheating, storage in poor conditions, or aging of the honey. In addition, HMF can be converted to 5-sulfoxymethylfurfural (SMF), a genotoxic compound in in vivo conditions, which represents a risk to the health of consumers . The diastase activity values for the honey produced by the species A. mellifera were 12 and 60 diastase numbers for the dry and rainy collection seasons, respectively, while the honey produced by the M. eburnea did not show activity (results are not included in the table). Diastase activity in honey is related to its freshness and heat treatment; this activity also can vary depending on the floral sources used by bees. In honey regulatory standards, it has been established that diastase activity should not be less than eight diastase number (DN) units, where 1 DN unit hydrolyses 1 mL of 1 % starch solution using 1 g of honey for 1 h at 40 degC . In monofloral and polyfloral honey of A. mellifera from different regions of Brazil, similar values of diastase activity have been reported, varying between 7.15 and 57.69 DN . In other studies, very low diastase activity has also been found in honey from the Melipona bee genus, which could be part of its nature ; this suggests this parameter should not be indicative of the quality of honey from M. eburnea. Glucose and fructose presented statistically significant differences by species and by season (p <= 0.05); values in the dry season were higher than in the rainy season. Regarding sucrose, there were differences between A. mellifera in the dry season and M. eburnea in the rainy season, presenting a high consideration in the values obtained in A. mellifera in the rainy season and M. eburnea in the dry season. The soluble solids (degBrix) did not appear different between A. mellifera and M. eburnea in the rainy collection season, while different for A. mellifera in the dry season (p >= 0.05). The sugar values reported in this research showed similar behavior to those shown by Nascimento et al. who reported fructose can represent 36% and glucose 31%. Honey crystallization depends on the fructose-to-glucose ratio (F/G) since glucose is less soluble in water than fructose, and higher values of 1.14 of the fructose/glucose ratio indicate honey tends to crystallize easily . In this study, this relationship varied between 1.17 and 1.19 for M. eburnea and A. mellifera from 1.18 to 1.25 according to the collected season (dry and rainy, respectively). The average sucrose content of honey samples ranged from 0.19 to 2.43%, which is slightly lower than that reported by Melaku et al. . In addition, a low honey sucrose concentration could be related to it being fully converted into glucose and fructose by the action of invertase enzyme . The electrical conductivity did not present differences between the values obtained by the season from A. mellifera and M. eburnea, but there were differences in the electrical conductivity of honey from M. eburnea in the dry and rainy season (152 and 126.8 mS/cm, respectively). The values in the present study are lower than those shown by who reported values between 173 to 927.33 mS/cm in multi-floral honey produced in some regions of Algeria. Honey electrical conductivity is strongly dependent on the concentrations of mineral salts and organic acids, and this parameter could show variability according to the floral origin and is a factor integrated into the international standards of honey for the discrimination of honeydew and honey blossom . The results of the physicochemical parameters for the two classes of honey are within those established by the Codex Alimentarius : ashes less than 0.6%, HMF less than 80 mg/Kg, the sum of fructose and glucose greater than 45 g. 100 g-1, sucrose less than 5 g. 100 g-1, and diastase greater than 8 Schade units. However, parameters, such as humidity and acidity, were higher than the values required by the standard (20% and 40 meq acid. Kg-1, respectively) for the honey produced by the species M. eburnea. This indicates the honey produced by species without a sting might have a shorter shelf life due to a tendency for fermentation. Studies, such as , suggest a normative basis for honey produced by stingless bees, showing higher values in acidity and humidity compared to honey produced by A. mellifera. This is reaffirmed by studies in Brazil . The differences in the physicochemical parameters presented between the dry and rainy seasons could be explained by environmental factors, nectar moisture content, time of year, place of collection, soil composition, and degree of maturity of the honey . Likewise, it is important to note not all the plant species selected by the bees showed constant blooms throughout the year; therefore, the resource was not always the same . 3.1.2. Biological Analysis Table 1 shows the results of antioxidant activity and total phenol compounds. Low DPPH radical-scavenging activity was found for both A. Mellifera and M. eburnea, showing a higher antioxidant capacity for A. mellifera honey (p <= 0.05). In addition, differences were found according to the harvest rainy season between the two species. On the other hand, ABTS radical-scavenging activity presented differences between species in the dry season. The honey produced by A. mellifera showed a greater reducing capacity by the FRAP method than the honey produced by M. eburnea (p <= 0.05). This variable did not show significant differences between the honey collection seasons. Honey from A. Mellifera had a higher content of total phenols compared to honey from M. eburnea but did not show differences for the collection season. Considering that antioxidants act via multiple mechanisms depending on the reaction system or the radical or oxidant source, the antioxidant activity was measured by three methods (ABTS, DPPH, and FRAP) . The results obtained in this study are similar to those obtained by Nascimento who report a range between 26.0 and 100.0 mg GAE100 g-1 for monofloral and multi-floral honey produced by A. mellifera in Brazil. Alvarez Suarez et al. reported a value of 54.30 mg GAE. 100 g-1 for polyfloral honey. The phenol content in honey is an important parameter that not only determines the quality but also its biological potential , mainly as antioxidant activity . The amount of phenols found in the present analysis provides added value to the honey produced in this type of forest ecosystem. On the other hand, the ability to trap free radicals of the honey (studied here by DPPH and ABTS) was lower compared to other studies carried out in Mozambique on commercial honey that had values of 19.17 and 160.01 mmol of Trolox 100 g-1 for DPPH . Bodo et al. found ABTS values of 100 mmol of Trolox. 100 g-1 in multi-floral honey from Hungary. This adds up to an extraordinary number of biological properties attributed to honey since it has anti-inflammatory, antibacterial, antiviral, and anticancer effects. Some compounds found in honey and related to health effects are polyphenols compounds, such as vanillic acid, caffeic acid, ellagic acid, syringic acid, ferulic acid, p-cumaric acid, benzoic acid, and others; flavonoids, such as apigenin, quercetin, pinocembrin, chrysin, galangin, and kaempferol; and antioxidants, such as tocopherols, ascorbic acid, superoxide dismutase, and catalase . The inhibition capacity of honey against some microbial agents, such as S. typhimurium and L. monocytogenes, can be seen in Table 1. The results relating to the inhibition capacity of the honey against the bacterium, E. coli ATCC 25922, suggest resistance of the strain against the evaluated samples. The results obtained showed both species, A. mellifera and M. eburnea, exhibited inhibition capacity for the bacteria, S. typhimurium and L. monocytogenes. The last one showed a greater inhibition halo and presented a statistical significance between A. Mellifera in the dry and rainy seasons (p <= 0.05); M. eburnea did not exhibit differences. S. typhimurium did not present differences between the species or collection season (p >= 0.05) The results of antimicrobial activity for the two types of honey in this study showed there is no activity against the E. coli ATCC 25922 strain that is consistent with that reported by Aguilera et al. in A. Mellifera honey. In contrast, found antimicrobial activity against the E. coli strain ATCC 31617 for the angel bee species (Tetragonisca angustula). The two kinds of honey in the present study showed inhibitory activity against L. monocytogenes and S. typhimurium strains. Similar studies found an inhibitory effect of multi-floral honey against L. monocytogenes 1/2B and S. typhimurium NRRLE 4463 at 50 and 75%, respectively . Some authors attribute the antibacterial activity of honey to the presence of hydrogen peroxide resulting from the activity of catalase and glucose oxidase as well as the presence of phenols compounds, phenolic acids, flavonoids, ascorbic acid, organic acids, methylglyoxal, and bee protein defensin 1 . The variation in antimicrobial activities of honey mainly depends on the flower types, environmental conditions, geographical location of the floral sources, peroxide activity, and non-peroxide mechanisms that result in different bioactive compounds . 3.2. Principal Components Analysis The results of the principal component analysis (PCA) are consolidated in Figure 3, where two independent data structures are shown given the grouping between the two bee species, obtaining a representation of the variability of the samples of 78.75 and 82.18% for A. mellifera and M. eburnea, respectively . Figure 3a shows in the case of A. mellifera, the main component one is associated with the mineral content of honey, antioxidant capacity, and total polyphenol content. Component 2 is mainly associated with the degBrix content and humidity. Additionally, it can be observed the variables that contribute in greater proportion to the variability both in factors associated with quality and in their biological activities of A. mellifera honey are D(+)-saccharose, degBrix, HMF, humidity, and inhibitory capacity of Listeria monocytogenes. These results are like those found by Conti et al. where it is shown that in a multivariate way, humidity and sugar content accompanied by some minerals, such as magnesium and potassium, are quality variables that contribute a greater proportion to the variability of South American unifloral and multi-floral honey, which is directly related to the characteristics of the polem minerals that contain the local floral species. Regarding the PCA obtained for the honey collected from M. eburnea , it is observed that component one is highly related to the DPPH radical-trapping activity, pH, and electrical conductivity, these variables being the ones that contribute most to the variability of the samples. For its part, component two is related to the inhibitory activity of Listeria monocytogenes and the scavenging capacity of ABTS radicals. For this species, the variables that contribute in greater proportion to the variability of quality are related to pH, electrical conductivity, and the capacity to capture radicals DPPH and ABTS accompanied by the content of total polyphenols. This indicates high interspecific variability in the functional properties of the honey produced by stingless bees, which could be explained by the incipient research on quality improvement regarding the homogeneity, stability, and biochemical properties of the honey produced by stingless bees, accompanied by its lack of control for its sale and incipient regulations in force worldwide, as reported by Braghini et al. . Additionally, some probable correlations are appreciated given the closeness of the vectors for both kinds of honey, a hypothesis that was confirmed by the analysis shown in the following section. 3.3. Correlations Analysis The analysis of independent correlations between the variables studied in A. Mellifera and M. eburnea honey are shown in Figure 4a,b, respectively. In the case of honey produced by A. Mellifera, free acidity had a significant direct relationship with lactonic acidity and ash. It is like Mulugeta et al. , and free acid had a negative correlation with Brix and Sucrose. In M. eburnea, this variable was directly related to HMF, moisture, lactonic acidity, and ABTS and negatively related to DPPH, Brix, and pH. The last one was according to other authors. It is due to the organic acid, such as gluconic acid, that increases ion H+ and decreases pH . Regarding color, honey obtained from A. mellifera had positive relationships with TPC, FRAP, minerals, HMF, free acidity, and lactonic acidity and a negative relationship with sucrose. Honey from M. eburnea had positive relationships with TPC, minerals, glucose, and pH and negative correlations with sucrose, electrical conductivity, and S. typhimurium inhibition. Some authors report a high correlation between color, FRAP, and phenols. It could be explained by the color of honey being influenced by pigments, phenols, minerals, HMF, and products of the Maillard reaction, and they present antioxidant activity . Sucrose concentration for A. mellifera showed positive relationships with fructose and pH and negative relationships with ABTS, free acidity, TPC, lactonic acidity, color, and Listeria inhibition. For M. eburnea honey, sucrose had only positive correlations with Brix and negative correlations with TPC, ABTS, color and glucose, and free acidity. Electrical conductivity showed positive correlations with fructose and HMF. Negative correlations are presented with DPPH and lactonic acidity. The electrical conductivity of M. eburnea showed positive correlations with free acidity and HMF. Negative correlations were found with DPPH, pH, TPC, ABTS, color, ash, and FRAP. The measurement of electrical conductivity depends on the free acid of the honey. The higher the acid content, the higher the resulting conductivity. This is in agreement with Mulugeta et al. . In the case of the S. typhimurium inhibition variable for A. Mellifera, it did not show statistically significant relationships with any measured variable, and in the case of M. eburnea honey, it showed negative correlations with color and Brix. The ABTS variable for A. Mellifera honey showed positive correlations with FRAP, TPC, and Listeria inhibition and negative correlations with sucrose, fructose, and pH. Regarding ABTS correlations for M. eburnea honey, it showed positive correlations with TPC, ash, moisture, lactonic acidity, free acidity, and HMF and negative correlations with electrical conductivity, sucrose, fructose, and L. monocytogenes inhibition. There was high variability in the results and a high correlation of phenols with FRAP, ABTS, and DPPH, which explains why the antioxidant activity was due to the presence of secondary metabolites, such as phenolic compounds. These phenolic compounds come from nectar sources, which varied throughout the evaluated seasons, as well as the diversity of plants visited by bees . Results showed higher levels of these phytochemicals could produce a more potent antibacterial effect against L. monocytogenes. Some authors showed there is a high correlation between phenolic compounds, such as quercetin, rutin, and chlorogenic acid, with antibacterial activity, showing greater inhibition in Gram-positive bacteria than in Gram-negative bacteria . 4. Conclusions Bee species and the collection season influence the quality characteristics of the different kinds of honey produced by A. mellifera and M. eburnea in tropical forests of Colombia. The honey produced by A. mellifera complies with the Codex Alimentarius parameters, and those produced by M. eburnea have some parameters out of range; thus, this study proposes the construction of a different standard for honey produced by stingless bees than for native bees. In addition, the antioxidant activity had higher values in honey from A. mellifera. Honey inhibited the growth of the two strains, L. monocytogenes and S. typhimurium. The inhibition halo was greater in L. monocytogenes. The establishment of hives for productive purposes inside these forests can be an important strategy due to the high diversity of resources for bees as well as the frequency of their availability. Later studies focusing on the characterization of this type of phenol can elucidate the chemical diversity of this type of honey and its biological potential. Acknowledgments The authors thank the Vice-Rectory for Research and Graduate Studies of the Universidad Catolica de Oriente for funding the activities carried out for this work, and the Colombian Agricultural Research Corporation--Agrosavia and Servicio Nacional de Aprendizaje--SENA for the time dedicated by the researchers and the use of the assets inherent in the preparation of this document. Author Contributions Conceptualization, I.C.Z.-V., J.C.H.-R. and M.Q.-A.; methodology, I.C.Z.-V., D.P.Y.-B., C.E.G.S., D.D. and M.Q.-A.; software, J.C.H.-R., S.J.C.-C. and C.E.G.S.; investigation, I.C.Z.-V., D.M.-H., C.E.G.S., S.J.C.-C., D.D. and M.Q.-A.; formal analysis, J.C.H.-R.; writing--original draft preparation, I.C.Z.-V., J.C.H.-R., D.P.Y.-B., D.M.-H., D.D. and M.Q.-A.; writing--review and editing, I.C.Z.-V., J.C.H.-R., D.P.Y.-B. and D.D. All authors have read and agreed to the published version of the manuscript. Data Availability Statement Not applicable. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Location of sampled hives, morphological illustrations, and botanical characteristics of the honeys produced by A. mellifera and M. eburnea reported in tropical forests of Colombia. Botanical characterization of honey adapted from . Figure 2 Linear discriminant analysis for honey produced by Apis mellifera and Melipona eburnea in humid and dry harvesting seasons of tropical Colombian forest. (a) Discriminant weight of the variables associated with quality. Larger size of the squares and more color intensity indicates higher discriminant weight. (b) Cluster plot showing clustering of honey by the interaction of bee species and honey harvesting season. Figure 3 Principal component analysis of variables associated with the quality of honey produced by (a) Apis mellifera and (b) Melipona eburnea in Colombian forest. Figure 4 Spearman's correlation analysis between the variables associated with the quality of honey produced by (a) Apis mellifera and (b) Melipona eburnea in tropical Colombian forest. he presence of color indicates statistically significant relationships (p < 0.05), and the intensity of red or blue color indicates the magnitude of the correlation coefficient, whether it is an inversely proportional or directly proportional relationship, respectively. foods-12-01001-t001_Table 1 Table 1 Comparison of multiple ranges of Kruskall-Wallis for variables associated with the quality of honey produced by Apis mellifera and Melipona eburnea in the dry and rainy seasons in the Colombian Andean region. Different letters indicate significant differences between treatments according to the Kruskall-Wallis test. Variable A. mellifera x Dry A. mellifera x Rainy M. eburnea x Dry M. eburnea x Rainy p-Value KW Test Color (mm PFund) 72 +- 21.2 a 48.21 +- 24.8 a 60.83 +- 21.7 a 49.69 +- 23.2 a 0.186 Ash (%) 0.2 +- 0.09 a 0.26 +- 0.14 a 0.155 +- 0.02 a 0.15 +- 0.1 a 0.079 Moisture (%) 22.68 +- 2.9 a 19.8 +- 2.6 a 25.44 +- 1.7 b 26.94 +- 2.2 b 0.000 pH 4.15 +- 0.38 b 3.55 +- 0.23 a 4.48 +- 0.8 ab 3.67 +- 0.24 a 0.000 Free acidity (meq acid. Kg-1) 42.14 +- 7.4 a 46.47 +- 7.01 a 36.12 +- 27.3 a 49.46 +- 25.2 a 0.786 Lactonic acidity (meq acid. Kg-1) 0 +- 0.01 a 0.2 +- 0.06 b 0 +- 0.02 a 0.5 +- 0.05 b 0.000 HMF (mg/Kg) 4.82 +- 2.46 ab 7.42 +- 2.19 b 4.34 +- 2.7 ab 3.8 +- 3.3 a 0.032 % D-(+) Glucose 30.11 +- 2.41 b 22.19 +- 4.34 a 27.94 +- 1.37 b 20.7 +- 3.45 a 0.000 % D-(-) Fructose 36.63 +- 2.20 d 27.74 +- 1.31 c 32.94 +- 1.95 b 24.63 +- 2.52 a 0.000 % D(+)-Saccharose 2.73 +- 0.69 b 0.9 +- 0.84 ab 0.42 +- 0.4 ab 0.19 +- 0.16 a 0.000 degBrix 77.5 +- 1.94 b 77.8 +- 2.70 a 69.5 +- 2.23 ab 71.95 +- 1.63 a 0.007 Electric conductivity (mS/cm) 144.5 +- 21.89 ab 134.4 +- 12.12 a 152 +- 60.85 b 126.80 +- 15.40 a 0.007 DPPH (mmol Equiv TX/100 g) 0.76 +- 0.24 bc 1.05 +- 0.23 c 0.05 +- 0.26 ab 0.095 +- 0.02 a 0.000 ABTS TEAC (mmol Equiv TX/100 g) 4.12 +- 2.10 b 9.97 +- 5.05 b 0.36 +- 1.38 a 2.25 +- 1.6 ab 0.000 FRAP (mg Equiv ascorbic acid/100 g) 73.22 +- 28.50 b 117.46 +- 29.99 b 15.61 +- 14.25 ab 10.79 +- 2.69 a 0.000 TPC (mg Equiv GAE/100 g) 49.2 +- 6.94 b 62.39 +- 12.23 b 25.69 +- 9.44 a 32.89 +- 13.14 a 0.001 S. typhimurium inhibition (mm) 8.71 +- 0.85 a 8.66 +- 0.71 a 8.69 +- 1.03 a 8.29 +- 2.84 a 0.470 Listeria inhibition (mm) 10.49 +- 7.76 a 26.03 +- 4.91 b 25.56 +- 4.60 ab 23.26 +- 6.28 ab 0.015 Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). 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PMC10000825 | Foods Foods foods Foods 2304-8158 MDPI 10.3390/foods12051057 foods-12-01057 Review Sustainable and Bio-Based Food Packaging: A Review on Past and Current Design Innovations Versino Florencia 12* Ortega Florencia 13 Monroy Yuliana 1 Rivero Sandra 13 Lopez Olivia Valeria 4 Garcia Maria Alejandra 13 Petronilho Silvia Academic Editor Martinez Mario M. Academic Editor 1 Centro de Investigacion y Desarrollo en Criotecnologia de Alimentos (CIDCA), UNLP-CONICET-CICPBA, 47 y 116, La Plata 1900, Argentina 2 Facultad de Ingenieria, Universidad Nacional de La Plata (UNLP), 47 y 115, La Plata 1900, Argentina 3 Facultad de Ciencias Exactas, Universidad Nacional de La Plata (UNLP), 47 y 115, La Plata 1900, Argentina 4 Planta Piloto de Ingenieria Quimica (PLAPIQUI), UNS-CONICET, Camino La Carrindanga km.7, Bahia Blanca 8000, Argentina * Correspondence: [email protected] 02 3 2023 3 2023 12 5 105731 12 2022 14 2 2023 21 2 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). Food loss and waste occur for many reasons, from crop processing to household leftovers. Even though some waste generation is unavoidable, a considerable amount is due to supply chain inefficiencies and damage during transport and handling. Packaging design and materials innovations represent real opportunities to reduce food waste within the supply chain. Besides, changes in people's lifestyles have increased the demand for high-quality, fresh, minimally processed, and ready-to-eat food products with extended shelf-life, that need to meet strict and constantly renewed food safety regulations. In this regard, accurate monitoring of food quality and spoilage is necessary to diminish both health hazards and food waste. Thus, this work provides an overview of the most recent advances in the investigation and development of food packaging materials and design with the aim to improve food chain sustainability. Enhanced barrier and surface properties as well as active materials for food conservation are reviewed. Likewise, the function, importance, current availability, and future trends of intelligent and smart packaging systems are presented, especially considering biobased sensor development by 3D printing technology. In addition, driving factors affecting fully biobased packaging design and materials development and production are discussed, considering byproducts and waste minimization and revalorization, recyclability, biodegradability, and other possible ends-of-life and their impact on product/package system sustainability. barrier properties active material intelligent packaging circular economy biobased inks and dyes bioadhesives bioplastic agri-food by-products ANPCyT, ArgentinaPICT 2019-03380 PICT 2019-2827 PICT 2018-3285 CONICET, ArgentinaPIP 2022-00493CO UNS and CONICET, ArgentinaPGI 24/ZQ18 Projects PICT 2019-03380: PICT 2019-2827, and PICT 2018-3285 (ANPCyT, Argentina); PGI 24/ZQ18 (UNS and CONICET, Argentina), and PIP 2022-00493CO (CONICET, Argentina), and Drs. Florencia Ortega and Yuliana Monroy Post-Doctoral Fellowship CONICET (CONICET, Argentina). pmc1. Introduction Food packaging comprises items, such as containers, cups, tableware, straws, bags, wraps, and boxes, that protect or contain food. Within a growing urbanized population food packaging is needed for the transport, storage, and consumption of food products. By 2050 the world's population is estimated to reach 9.7 billion, two-thirds of which will be living in urban areas with the consequent increase in food requirements and changes in food-consumption patterns . People living in cities tend to lead more hectic lifestyles, spending little time buying or preparing food, preferring processed food to fresh foods, and purchasing mainly in supermarkets or convenience stores to save time. Such patterns are clearly evidenced nowadays in high-income countries, though small shops and traditional grocery stores are going out of business and being replaced by supermarkets even in developing countries, thus leading to lager supply chains strictly dependent on packaged food . A large portion of food is lost along the supply chain due to damage caused by different environmental factors, such as moisture, oxidation, thermal degradation, or microbial contamination . On average, 30% of the edible part of the global food production is lost or wasted in the supply chain . Thus, several research and policies have focused on reducing such losses . Bishop et al. reported that, only considering UK households, over 2 million tons of fruit and vegetables waste are expected to be generated every year by 2030, along with 105.7 kilo tons of plastic waste from the packaging in which is purchased . Therefore, not only does an increase in food demands entail greater land-use requirements, soil degradation, biodiversity loss, eutrophication, freshwater scarcity, energy resources depletion, and overall climate change by the agri-food industry, but also more plastic-waste pollution from agriculture and food packaging. Furthermore, a large portion of food packaging is discarded with food waste, making it difficult and expensive to separate in waste management systems, ending up in less preferred waste treatments and incurring an environmental burden across both food and plastic value chains . Most food packaging is directly disposed of after use (95%) and more than a third do not enter the recollection systems . In May 2018, the European Commission approved a directive by which single-use plastics were prohibited or at least restricted, intending to reduce plastic waste pollution of seas and oceans . The set of approved standards comprises requirements for product labeling, waste collection, and public awareness regarding the environmental problem and responsible consumption. Since then, a slight reduction in fossil-based plastic and a mild increase in biobased plastic and post-consumer recycled plastic production has been reported in Europe, though the overall global plastic production has continued to grow (reaching 390.7 million tons in 2021) . Aiming to develop more sustainable food packaging, some significant efforts have been focused on biobased and/or biodegradable materials, especially bioplastics, paper, and cardboard. Alternative cellulose sources to wood have been studied aiming at tree and biodiversity conservation in forests and rainforest ecosystems . Plant-based bioplastics, both biodegradable (such as poly-lactic acid (PLA), polyhydroxybutyrate (PHB), or biopolymer-based plastics) and non-degradable (biobased polyolefins: e.g., bio-PE, bio-PP, and bio-PET), are currently being commercialized and agri-food industry by-products and waste are being studied as raw materials to minimize agriculture environmental impact and land-use . However, enhanced recyclability capacity, retrieval quantity, and good separation through waste management systems are crucial for non-biodegradable bioplastics to be effectively recycled through conventional recycling infrastructure and technology . Compostable bioplastics may be preferred to non-degradable plastics for single-use applications, such as food packaging and foodservice ware, when reutilization or reduction is not possible and recyclability is limited . Nonetheless, even for this type of material, adequate waste stream management and industrial composting facilities are needed, since compostability will only occur under specific conditions . Several studies have been conducted on the environmental impact of biobased materials over fossil conventional plastic packaging , yet no general result has been reported on which is the more sustainable alternative. The overall environmental impact of the package depends on the packed food product, distribution logistic and distances, clean energy resources, available waste management systems, re-use frequency, recyclability and/or biodegradability, end-of-life scenario selected for the packaging material, and overall cost of production, use, and disposal . Regarding the further reduction of food waste and losses, two main strategies are followed. On the one hand, there is agri-food industry by-products reduction and revalorization as feedstock for biomaterials and active compounds recovery, aiming to reduce by-products treatment costs and overall environmental impact . On the other hand, the development of active and intelligent packaging to prevent food spoilage and increase food safety is being performed . Extended shelf-life increases the chances of the food to be eaten with preserved nutritional value and reduced toxicity risk. Besides, the use of sensors and new identification technologies may provide better detection of food degradation markers to optimize food distribution and conservation and thus reduce food loss . The food packaging industry is a thriving market that plays a key role in the modern economy. Food packaging design is crucial to guarantee food safety throughout the supply chain, optimize storage and transportation, and facilitate the communication of product information to retailers and customers . Approximately 9% of the vast amount of research reported on food packaging (41,602 publications from 1960-2022) focuses on packaging design. More sustainable food packaging design aims for minimum environmental burden at the end-of-life of the food product and package. Consequently, research and innovation in food packaging are growing fast. The literature review within the last decade indicates an accelerated increase in annual publications involving sustainable food packaging (19-35% annual increase) together with active and intelligent food packaging (13-33% annual increase). Among food, packaging paper is the second (34%) most used and studied material after plastic (37% both rigid and flexible) . A sustained increase in work related to innovations and innovative food packaging is observed, with an incipient but fast increase in studies on biobased, especially bioplastic food packaging, and active and intelligent packaging for food, both showing a remarkable growth in the last couple of years . Such tendencies evidence the work towards developing environmentally friendly and sustainable food packaging material focusing on: (i) enhancing biopolymers performance through physical, chemical, and enzymatic modification and composite formulation, (ii) developing natural-based nanocomposites for active and intelligent sustainable materials, (iii) synthesizing new biobased and biodegradable materials, (iv) scaling up bioplastic processes, (v) searching for new renewable sources, especially undervalued raw materials, and (vi) improving materials reutilization and recycling capacity . Novel packaging systems are aimed not only at ensuring food safety and traceability, but have also gained great importance for building more sustainable food chains, reducing food losses and waste, as well as the overall packaging environmental impact. In previous works, a recompilation of the latest research and development on biobased packaging materials and composites form agri-food waste and byproduct was conducted, with a special focus on materials properties and processing technologies for greener production . Thus, this review gathers the advances reported in the last decade in the research and development on packaging materials and design aiming to improve food chain sustainability within a circular economy paradigm. Biobased materials with enhanced barrier and surface properties, and specific activity for food conservation, such as antimicrobial or antifog properties, were reviewed. An overview on recent advances in intelligent packaging development, especially biobased sensor development by 3D printing technology, is provided. A further assessment of fully biobased packaging design and materials, considering waste minimization and revalorization, recyclability, biodegradability, and compatibility of the packaged food products with current waste management technologies is given. 2. Sustainable Food Packaging Design Considering that over 80% of the environmental impact of a product is determined at the design stage, design plays a key role in its sustainability . In food packaging, an efficient design can increase the sustainability of the packaging and the food product it contains by minimizing both food and packaging waste at their end-of-life. However, ensuring food preservation accounts for a larger portion of the overall life-cycle environmental impact of the packed food product. On average, the packaging is estimated to comprise only 10% of the energy input required for a single person's weekly food consumption and can ensure that the residual 90% is not wasted throughout the supply chain . The relative environmental impact that packaging has on a food product depends on the food type, mainly on its perishability, cost, and overall emissions and resources depletion in production. For example, greenhouse gasses (GHGs) emission from dairy products and meats is greater than those from fruits and vegetables by 13-18%, largely exceeding GHGs in packaging manufacturing and end-of-life . Therefore, focus on minimizing food waste for animal food products would yield greater benefits to the system, while emphasis should be placed on reducing the impact of the packaging of fruit and vegetables . Life cycle assessment (LCA) of both the food product and the preferred packaging system should be evaluated in each case for better decision-making, considering real available waste management systems and possible optimized alternatives. Consequently, apart from the protection of the product, designing the most effective and sustainable packaging is a complex process involving numerous sectors implicated in the whole supply chain, including the intended market . To minimize the environmental impact of the packaging system, it is important to consider its capacity to contain, protect, and preserve the product as to extend its shelf-life and guarantee food safety, but it should also be adequately sized, easy-to-open and easy-to-empty, and with clearly accessible information to prevent food from being wasted. In addition, the packaging materials should meet the desired mechanical and barrier properties remaining as light-weighted as possible, food-safe, ideally reusable or recyclable, and disposed of with minimum to zero pollution. Several research efforts have focused on enhancing materials barrier properties for sustainable food packaging (See Section 3). Most of the design decisions are aimed to reduce packaging and food waste, and all of them indirectly have an economic impact. A large portion of domestically wasted food could be because the packaging does not meet consumer needs, such as packages that are too large or difficult to empty . For instance, easily emptied packaging systems reduce both product waste and cost per unit and make cleansing easier for better recyclability of the packaging material, therefore lowering water use for this matter. There are various research and novel start-ups commercializing easy emptying packaging coating, especially biobased and food-safe materials . In this regard, LiquidGlide(r), a spin-off from the Varanasi Lab at MIT (Massachusetts Institute of Technology), has launched a novel, biobased, durable, superhydrophobic material for packaging applications. Additional packaging attributes that can influence food waste comprise resealability, easy-to-open, grip, portioning or dosage, and communication of food safety/freshness information . Innovations in intelligent and smart food packaging (See Section 4.2 and Section 5.2) aim to facilitate detection, recording, and communication of food quality and safety to overcome problems in expiration date labeling lack of uniformity within the global market that can lead to confusion in the actual state of the product . Unclear or wrong date labeling may therefore cause potential food intoxication in consumers or unnecessary disposal of still fresh products, increasing human health risks and food waste. Nonetheless, further emphasis on the LCA need of these packaging/product systems is required to assure net environmental benefits, focusing on intelligent and smart systems cost, reusability, and recyclability . It is important to identify the packaging functions that influence food waste to achieve more efficient packaging systems. Good mechanical resistance helps protect the product from damage due to vibrations during transport on rough roads and compression during storage as provides protection from contamination to guarantee food safety . Besides, damaged products may also be rejected by customers. A real understanding of the natural characteristics and shelf-life of the food is needed to select the best packaging system. Verghese et al. reported significant annual losses of bananas in Australia partially because of improper packaging, which could be significantly overcome by replacing reusable plastic crates with more stable corrugated carton crates and clear bags for fruit neck protection and further in situ retailer owned ripening rooms . Similarly, a novel non-toxic biobased aloe vera coating on papaya fruit has proven to delay ripening and significantly extend their shelf-life . Materials with barrier properties have been widely studied to reduce food perishability (See Section 3). Depending on the food matrix characteristics, the diffusion or transmission of different substances, e.g., oxygen, carbon dioxide, ethylene, nitrogen, water vapor, and volatile molecules, microorganisms, light, or heat will need to be controlled to avoid undesired chemical and physical reactions in the food product that could lead to unpleasant odors or flavors, or even spoilage. Multi-layered packaging systems have the potential to provide barrier properties to multiple undesired or hazardous compounds simultaneously or provide the barrier materials with the mechanical or thermal resistance needed, and reversely. Currently, multi-layered packaging is widely used in the food and beverage industry, such as juice and milk cartons, usually consisting of 75% paperboard, 20% of plastic (mostly PE), and 5% of aluminum foil . Despite their potential for increasing food shelf-life and mostly entailing less material to achieve the same functionality as mono-materials, their recyclability is rather difficult . Palombini et al. studied the sustainability of packaging used for organic food in Brazil, showing that the dominant features in poor or non-recyclable polymer packaging were metallization and the use of opaque materials, polymer blends, the presence of adhesives for labeling or multi-layered packaging, and printed labeling over the packaging . Better sorting of disposed multi-layer packaging would be required for high-quality recycled materials, though these are currently found in different recycling pathways, hampering their recyclability. Kiaser et al. clearly overview the two main available technologies for multi-layered packaging recycling: compatibilization methods (for polymer-based packaging) and component separation by dissolution-reprecipitation technique or delamination for independent recycling of each component when possible . The major limitations of these methods are that: (i) common compatibilization does not seem to be feasible for post-consumer packaging due to fluctuation in composition and the need for more rigorous labeling and sorting of packaging waste stream; (ii) even though the dissolution-reprecipitation method could be available for the recycling of existing materials in the near future, it is a very high energy demand process; (iii) systematic delamination could be an ecologically and economically more sustainable solution if separation and adequate sorting of the delaminated layers are possible . In this regard, further investigation of the optimization of multi-layered packaging recycling routes is needed. Alternatively, fully biodegradable multi-layer packaging systems designed to be composted at their end-of-life could probably reduce food waste and at the same time both avoid problems in recyclability and reduce the environmental impact of this type of food packaging. Yet, more research is needed considering LCA of the proposed materials, contemplating that a large proportion of disposed packaging labeled as compostable or biodegradable is not correctly treated. Misdirection occurs either because these materials are thrown away without proper classification or do not even enter the urban waste treatment streams, e.g., non-biodegradable plastic packaging, or are rejected from recycling plant due to a lack of available processing technologies or a shortage in volume needed for cost effective recycling, or are separated in composting facilities by mistake to avoid compost contamination or because there is no adequate separation technology to tell one type of plastic from another when the material is mixed with other biowaste . Furthermore, novel composite and nanocomposite materials have also been studied to enhance materials barrier properties for food packaging. A focus on more sustainable materials fully biobased and biodegradable especially aiming to use by-products and waste from the agri-food industry is extensively reported in the literature review (See Section 3 and Section 5.1). For instance, Bascon-Villegas et al. recently published a work on composite PLA and polybutylene adipate-co-terephthalate (PBAT) with lignocellulose nanofibers obtained from wheat straw . Similarly, Silva dos Santos et al. formulated a coating paperboard packaging based on chitosan, palmitic acid, and active carbon that provides increased fat and moisture barrier for paper packaging preserving biodegradability and recyclability . Besides, many new active properties can be imparted to packaging materials to extend food shelf-life . For example, Manfredi et al. highlighted that milk waste savings within the fresh milk supply chain by the application of an antimicrobial coating on regular multi-layer paperboard packaging is greater than the impact generated by the coating production . Active packaging contains compounds are able to preserve the organoleptic or sensory characteristics to ensure the food product's quality. Of particular interest are active packaging materials containing natural antioxidants and antimicrobials, which not only extend the shelf-life of packaged products by preventing rancidity reactions, but also prevent the growth of foodborne pathogens . Biobased nanocomposites have been studied for innovation in sensors for the better detection of food degradation markers and thus a reduction in food loss. In this regard, active and intelligent packaging is currently receiving great interest, although they are still being researched and are not widely commercialized. A thorough revision of recent research on these materials has been conducted in the following Section 4 and Section 5.2. The development of advanced methods for recycling and remanufacturing should be considered in designing new circular and sustainable systems. Some key aspects related include recyclable and non-hazardous materials; efficient logistic and transportation systems; energy and resource efficient technologies; and product design aimed at recyclability, remanufacturability, and reusability . Even though the goal of reuse and recycling is entailed within the circular economy model, this is poorly achieved for food packaging since it is difficult to guarantee the quality and safety of materials for food contact from the available waste management systems. Currently, reuse is only viable for refillable and cleanable containers (e.g., glass bottles, stainless-steel containers) . In the long term, chemical safety of recycled food packaging could be achieved by replacing unsafe substances from materials design, avoiding their entrance to the recycling stream. However, a period of 10-30 years is estimated for a significant reduction in contaminants levels . In the short term, clear labeling of packaging materials, consumers' education to identify more sustainable packaging alternatives and more efficient safety and sorting systems within the waste management and recycling systems could contribute to a more sustainable use of food packaging . Besides, sustainable recycling systems need to be cost effective in terms of the selected recycling technology, material quality of recycled products (depending on the type and conditions of secondary raw materials recollected from waste stream), availability (in terms of economy of scale), and recollection and sorting cost . Facilitating recyclability has not only environmental and economic benefits, but also a social contribution, considering that dry waste sorting and selling constitute the income source for low-income workers in some countries . In addition, packaging design can considerably affect the logistics in transportation, handling, and storage throughout the supply chain . Such decisions have direct implications in distribution times and product preservation. However, the best packaging systems depends on the frequency of reuse, transportation routes and distances, and end-of-life treatment . As regards resources reduction, water and energy saving technologies, by-products and biowaste revalorization are being achieved by the development of new biobased packaging materials and novel heat insulating materials that can help reduce energy in preserving thermosensitive foods as well as reducing their spoilage rate . To reduce the environmental impact of biodegradable and compostable bioplastics, the preferred route of disposal and treatment is composting. However, most packaging materials nowadays reported as compostable (according to current legislation) require specific composting conditions (thermophilic: ~58 degC) to be fully and safely composted that are only feasible in industrial composting facilities . Therefore, compostable bioplastic-based food packaging faces some obstacles to fulfill its purpose. A large volume of bioplastics does not enter composting systems due to the lack of industrial composting facilities or the poor sorting of these materials. Waste misdirection results from improper labeling, workers inexperience with biodegradable plastics treatment, a lack of appropriate sorting technologies in sorting facilities, or a lack of consumer education on waste separation or environmental protection. When biodegradable plastics wrongly end up in landfills, they can decompose anaerobically producing methane, a more harmful GHG than CO2, hindering their sustainability . By occasional littering or waste mistreatment, bioplastics can also enter water streams and marine environments in which they are not able to biodegrade or can degrade, posing a hazard to life in such ecosystems. Research has been conducted to prove biodegradable materials' ecotoxicity . Zimmermann et al. reported that many bioplastics available on the market are just as toxic as conventional plastics, presenting clear bioluminescence inhibition of Aliivibrio fischeri . Wang et al. thoroughly revised the available studies on plastics degradation in marine environments, highlighting that only a few works consider the toxicity of degradation products, including material fragments or debris and additives, which should not be toxic . In this regard, the chemical safety of materials can be further achieved by bottom-up green chemistry design in the development of new biobased and biodegradable materials. On this matter, ecotoxicity assays of new biodegradable materials research is being conducted, especially after compost or soil degradation, usually showing promising results . Furthermore, non-biodegradable and fossil-based plastics, biodegradable bioplastics can break into small particles, producing microplastic pollution and affecting different species of plant and animals adversely . This points out the necessity of an in-depth understanding of the environmental degradability performance of available and upcoming biodegradable plastics. In product-packaging design, it is also important to consider and understand the consumers perception and acceptance of the packaging. Some authors imply that consumers, tend to have a poor understanding of the benefits of packaging and mostly a negative image of its role to preserve food, e.g., removing the package prematurely to let the food "breathe" . On the contrary, other research findings indicate that consumers recognize the importance of packaging plays in food safety and quality in relation to the information that it provides and show a pro-environmental consciousness . A study carried out by Harpen et al. using an immersive 3D virtual supermarket environment revealed that customers tend to prefer unpacked fruits and vegetables . Herbes et al. indicated that consumers mostly focus on the end-of-life stage of packaging and are mostly unaware of the negative impacts of packaging in the upstream and middle-stream supply chain . Consumers' desire to pay for green or sustainable products grows as environmental awareness rises, yet the higher price of these products limits sales to a niche market . Consequently, future research should contemplate consumers involved in packaging alternatives design as well as cost reduction for the increased accessibility of novel sustainable food packaging. A systemic focus on understanding the relationship between packaging, products, supply chain networks, and physical processes is required in an era of multi-product physical supply chains that have a global footprint . Padhi et al. identified sustainable design and development products, strategic commercialization, technology optimization, and sustainable product returns and recycling as the most important sustainable supply chain processes . The conception of a new eco-design approach gives place to further innovation in packaging and products with a critical rethinking of the system and new perspectives towards sustainable production. From the literature review, some basic principles for sustainable design can be outlined :A focus on real demand and problems and try to find solutions with social, environmental, and economic benefits altogether. A shift in the design dynamics from the application requirements up, thinking in terms of functions and services rather than the product itself. Consideration of realistic and updated life-cycle and process thinking, having an integrated view of the supply chain, thus taking into account the product manufacturing, distribution, consumption and end-of-life. Inclusion of users, stakeholders, and different experts in the design process as much as possible. Research and innovation need to be grounded on justifiable priorities within the available time frame and scope of the project. A holistic understanding of the supply chain drivers, barriers, and opportunities is required in view of sustainable long-lasting packaging-product systems designed towards a circular economy. 3. Strategies to Improve Barrier and Hydrophobicity Properties for Food Packaging With the main premise of preserving the food matrix quality, packaging industries and the scientific sector are striving to develop new materials to minimize deteriorative changes mainly due to physical and chemical modifications experienced by foods during their distribution and storage . Food packaging is considered an integral part of the preservation system and, therefore, the package provides a barrier between the food matrix and the external environment, protecting it against physical, chemical, and biological damage . To control the chemical and physical reactions in the matrix, packaging material must be able to act as a barrier to external factors that affect the food quality, such as the gaseous atmosphere, water activity, light, and temperature. In this sense, the barrier properties of a material are related to the protection of the food matrix inside the package by controlling mass transfer . The controlled transference of diverse compounds, such as gasses, water vapor, and volatile molecules, is relevant to minimize the reactions that cause food degradation by creating a favorable atmosphere around the packed product . Food stability is characterized by its chemically unstable nature and hence requires to be protected from spoilage, lipid deterioration, and microbial contamination. Therefore, polymeric materials must prevent the penetration of compounds from the surrounding environment to guarantee a "high barrier" . A schematic representation of how packaging protects food from external agents, such as gaseous compounds, water vapor, UV radiation, and microorganisms, is shown in Figure 2. In this regard, some examples of alternatives to develop materials with enhanced barrier properties are also addressed. As it is well known that the most common degradation processes of packaged foods are caused by oxygen, such as lipid oxidation, microorganism growth, enzymatic browning reactions, and vitamin loss, among others. According to Zabihzadeh-Khajavi et al. controlling the oxygen permeability of the packaging system can extend the shelf-life of the packed food . Ethylene, on the other hand, is considered the aging hormone of plants since it is responsible for the growth and ripening of fruits . A very high concentration of ethylene around the food can accelerate its decay. Therefore, the elimination of this gas from the packaging or the control of its permeability is relevant to increase the shelf-life of fresh products . While carbon dioxide is one of the most employed components in the gas mixtures for modified atmosphere packaging (MAP) due to its antibacterial properties , this gas can prevent the microbial growth of fresh meat, cheese, or baked goods, and minimize the respiration rate of fruits and vegetables . Moreover, moisture content alters the nutritional and organoleptic properties and safety of food products. The reaction rates of lipid oxidation, microbial growth, and browning are altered when the food moisture content changes. Effective packaging can play a major role in maintaining product moisture to extend the shelf-life of food. Given the importance of quantifying the barrier properties, mainly to gasses and water vapor, it is relevant to use normalized or standardized methods that allow the comparison with other materials. As it was stressed by Baschetti and Minelli, numerous methodologies have been employed to study the permeability of different gasses and volatile compounds through polymeric matrices under diverse experimental conditions . The most important international standards are American Standards and Table 1 summarizes the ASTM norms for the determination of gasses and water vapor permeability in polymers for food packaging . Another important barrier property of food packaging is its capacity to block UV (200-400 nm) and visible (400-700 nm) radiation. The exposure of food products to UV light (natural or artificial) throughout their useful life (harvesting, elaboration, storage, distribution, sale, and preparation by consumers) can induce photo-oxidation and photo-degradation reactions that affect the quality of food . Many photosensitive food components (proteins, vitamins, pigments, fats, and oils, etc.) are the substrates for these reactions in which free radicals and oxygen reactive compounds are produced, inducing food deterioration by the development of undesirable flavors and aromas, color loss, and a decrease of the nutritional value . As an alternative to avoid photodegradation, different methodologies can be used, such as reflective layers, as well as the coating or incorporation of organic and inorganic absorbers to the polymeric matrices . On the other hand, the study of the UV barrier capacity of food packages is relevant if the products, once packaged, will be submitted to microbial decontamination by light-based technologies, such as UV radiation. According to Cassar et al., this methodology can reduce the food microbial charge but also leads to product deterioration or the generation of undesirable residues . Regarding visible radiation, transparent packaging allows consumers to visualize the packaged product and highlight some of the food organoleptic characteristics that condition the acceptability of the product. For this reason, opacity is a relevant optical property that conditions the quality of packaging films . In accordance with other authors, it is relevant to find multifunctional high-performance barrier materials considering several issues, such as optical and mechanical properties, renewability, and bio-friendliness, to develop food packaging that assures food quality and fits all consumers' necessities . 3.1. Enhanced Gas and Water Vapor Barrier Properties The permeation of low molecular weight gasses/vapors through films is measured by the ability of the polymer matrix to absorb and diffuse the penetrant. The main mechanisms that affect mass-transfer of substances across packaging materials are diffusivity, solubility, and permeability, which are closely related to the composition and structure of the polymer matrix . Factors, such as the shape and size of the permeant, morphology, crystallinity, and chain orientation of the polymer, influence the diffusion and solubility coefficient. Other variables that modify the matter transfer coefficient are related to the processing methods, which can alter polymeric chain configuration or induce crystallization/orientation, including polymer blending, multi-layer coextrusion, casting, or applied nanotechnology . The enhancement in the barrier properties in nanocomposite materials is attributed to the more tortuous path created by the presence of different nanofillers. This fact is explained considering that the nanofillers force the low molecular weight molecules to adopt particular tortuous pathways, producing a significant lag time. A higher aspect ratio of fillers compels the permeating gas molecule to follow a more tortuous path, leading to improved barrier properties . Furthermore, gas diffusion through materials is also controlled by the crystal structure domains. It is generally assumed that ordered crystalline domains should act as an effective barrier to the diffusion of gasses and small molecules, making the amorphous phase the only pathway available for permeation. Moreover, penetrants cannot sorb in crystalline domains because their solubility coefficients are lower compared to those of their amorphous counterparts . To achieve a better barrier performance, the assembly of nanoparticles to obtain nanocomposites constitute a strategy to improve barrier properties. Organic nanoparticles, such as carbon nanotubes, nanocrystals of cellulose or starch, and inorganic nanoparticles, such as nanoclays or montmorillonite (MMT), can physically and chemically interact with polymeric matrices to induce stronger and reinforced structures, enhancing both the barrier and mechanical properties . MMT nano-clays can enhance the polymer barrier performance because of their exfoliated structures, small particle size, high aspect ratio, and exceptionally large surface areas that enlarge the tortuous path for small diffusing molecules . Thus, the nanomaterials can be applied to improve the performance of conventional materials due to their particle size and their large surface area . Other approaches have been tested to regulate the transport of water/gasses through the packaging, e.g., the use of different polymer mixtures, polymer crosslinking by chemical reactions to narrow the intermediate chains, assembly of micro and nanoparticles to obtain composites, nanostructured matrices formed by an electrodynamic process, and coatings or multi-layer films to design packaging materials with high water/gas barrier properties . In this regard, multi-layer packaging constitutes an emerging technique that integrates the characteristics of different polymers or layers to create a package with enhanced performance in terms of properties . These materials have improved properties as compared to typical single-layer films, exhibit high-barrier to water vapor and gasses, such as oxygen, carbon dioxide, and aromatics, as well as high mechanical strength and good sealing capacity . Multi-layer films can be prepared by different methods, such as melt coextrusion, film-forming solution molded by casting, and compression molding of sandwich structures . In the design of multi-layer film packaging different layers can be assembled. In this sense, to enhance the techno-functional properties of polymers, an inner barrier layer film usually consists of polymers with higher oxygen barrier properties, while polymers with higher water vapor barrier and resistance from a mechanical point of view act as an outer layer. The barrier layer is in direct contact with the external environment, acting as a barrier to substances that cause the degradation of packaged food, such as moisture, oxygen, and microorganisms . According to Alias et al., multi-layered films based on biomass combined with synthetic biodegradable polymer films obtained from natural monomers showed better transparency, water solubility, and mechanical properties than single-layer films . Meanwhile, multi-layer films based on biomass sources demonstrate better barrier properties in terms of water vapor permeability and offer advanced properties in terms of oxygen permeability, still exhibiting a significant improvement in terms of physical and mechanical properties . In addition, to achieve high-performance biodegradable multi-layer films with tailor-made properties, knowledge of their microstructure and film processing steps is required. The challenge is to improve the protective barrier of the individual layers and therefore optimize the design of the matrices, so current research must be redirected in this way . It is pertinent to emphasize that the most important factor is the adhesion between layers to combine different layers that improve the barrier protection of the individual layers and thus optimize the resulting system. In this sense, research considering the scale, interaction, and architecture of structural layers in multi-layer matrices and their influences on barrier capacity performance is hardly reported in the literature. The layer-by-layer assembly generates a stratified structure with a significantly enhanced gas barrier and mechanical or even optical properties due to confinement and/or interfacial effects. Layer-by-layer assembly is based on different interactions, such as electrostatic attraction, hydrogen bonding, hydrophobic attraction, or entanglement between polymeric chains in neighboring layers. The interactions that are established depend on the characteristics of the polymers that make up the multi-layer system. Particularly, for some semi-crystalline polymers, the confinement generated by the stratified structure leads to a specific orientation of the crystals, nanoparticles, or compounds, inducing an increase in the tortuosity for diffusion . 3.2. Enhanced Hydrophobicity Many biodegradable and renewable biopolymers are hydrophilic since they are conformed by polar molecules. This inherent hydrophilicity often results in moisture absorption that leads to the deterioration of the mechanical properties of the material and affects its dimensional stability, both undesirable effects for packaging applications . To reduce the hydrophilicity of biodegradable and natural polymers, several methodologies can be carried out. One of these alternatives is to modify the chemical structure of the biopolymers to improve their hydrophobicity. According to this, Wang et al. stressed that chemical modifications of starch may be carried out to achieve adequate physicochemical characteristics by blocking or adding functional groups, improving the hydrophobicity of starch-based materials . For instance, Petronilho et al. have worked on hydrophobic starch-based films by transesterification with sunflower oil in alkaline medium, showing increased water resistance (lower solubility and water vapor transmission, and higher contact angles) . In the case of cellulose, for instance, chemical modification by silylation, esterification, amidation, and grafting are some of the reactions that can be used to obtain more hydrophobic cellulose . Plasma treatment is another methodology usually used to increase the hydrophobicity of natural polymers. Pankaj et al. highlighted plasma treatment as a novel, more environmentally friendly technology to obtain more hydrophobic starch derivatives . The effects of plasma occur on the surface of the material without altering the properties of the bulk involving the generation of reactive species, such as ions, radicals, electrons, photons, and other excited species. Generally, the plasma induces different reactions, such as surface cleansing, removal of organic contaminants, degradation (etching), cross-linking of polymer chains, and modification of the functional groups present on the surface. The different physical and chemical changes that the surface experiences depend on the gas used to generate the plasma . Goiana et al. studied how a dielectric barrier discharge (DBD) plasma treatment affects the hydrophilicity, WVP, and mechanical properties of corn starch-based films . The authors concluded that this treatment produces more hydrophobic starch materials with improved mechanical performance, equally interesting properties for food packaging. Following this idea, the use of cold plasma treatment increases the surface roughness of biopolymer films . The magnitude of the effect on the film's roughness depends on some factors, such as power supply (voltage), exposure time, and uniformity of the exposed energy of the plasma species onto the film surface . Among the effects that induce cold plasma treatment, the reduction of the contact angle between the film's surface and the water drops can be mentioned. Despite plasma treatments representing an effective method to reduce the hydrophilic character of natural polymers, they can reduce the biodegradability of materials . A more innovative method to enhance the biopolymers hydrophobicity is based on the reduction of the contact angle between water droplets and the material surface considering its topographical features. Therefore, materials with a specific surface topography can include air compartments that hold water droplets, minimizing their contact with the material surface. In this regard, bioinspired hierarchical surfaces are very interesting mostly because of their wetting behavior properties . For example, lotus leaves and rose petals present superhydrophobic surfaces with different wetting behaviors . Two methodologies can be employed to obtain materials with the hierarchical structures of lotus leaves or rose petals: directly molding from hierarchical templates or growing nanowires or nanorods onto designed microstructures . Following the first methodology, Luis et al. proposed a method to mimic the lotus leaf surface to fabricate zein-based films . The authors demonstrated that films produced using the lotus negative template presented lotus-leaf-like rugosities, resulting in very hydrophobic surfaces. Likewise, de Oliveira Gama et al. "decorated" the surfaces of films based on thermoplastic starch (TPS) and blends of TPS/low-density polyethylene (LDPE) to improve their hydrophilic properties . These authors employed nanocomponents to decorate the materials surfaces and they demonstrated the feasibility of creating topographical patterns to generate hydrophobic and even superhydrophobic features that can be useful in minimizing the water absorption. Other techniques, such as electrospinning, have been used in the design of superhydrophobic nanostructure fibers and surfaces. Pardo-Figuerez et al. developed an innovative PLA and nanostructured silica (SiO2) superhydrophobic multi-layer material with PET by electrospinning and electrospraying . Moreover, Zhang et al. prepared a fully biobased thermo-resistant edible super-hydrophobic coating from coffee lignin and beeswax with a promising application for food packaging of food that require high-temperature sterilization . Likewise, fully biobased materials from food industry waste with enhanced hydrophobic properties were developed and studied by Goncalves et al. . The authors used recovered starch, oil, and wax from potato chips manufacturing to produce starch-based films with enhanced flexibility and wettability with tailored roughness. For a further and thorough overview of the most recent studies on the application of superhydrophobic coatings and surfaces for sustainable food packaging, the authors recommend the work of Ruzi et al. . 3.3. Enhanced UV-Light Barrier Characteristics A strategy to enhance UV-light barrier properties includes the assembly of light stabilizers into the film matrix to deactivate the reactive degradation elements, preventing their occurrence by consuming the products responsible for initiating the deterioration reactions and hindering free radicals spread. Aluminum foil represents the best material for UV-visible light blocking, but it poses some disadvantages related to its recyclability, high cost, and non-transparency. In this sense, the smart selection and application of UV absorbers to functionalize materials for specific functions constitute a promising strategy . Inorganic materials are based mainly on the metal oxide particles, such as titanium dioxide (TiO2), cerium oxide (CeO2), iron oxide (Fe2O3), zirconium dioxide (ZrO2), and zinc oxide (ZnO), that impart enhanced light barrier properties by scattering the incident light . However, there are concerns regarding the diffusion of metal nanoparticles from film packaging to the food matrix and to the human body after ingestion. For this reason, the European Union legislation (Commission Regulation, 2011 (EU) Ndeg 10/2011 on Plastic Materials and Articles Intended to Come into Contact with Food) established the type and quantity of additives in materials intended to remain in direct contact with food, though further advances in research are needed to find safe alternatives . According to Song et al., inorganic nanoparticles can easily agglomerate, having a negative influence on the barrier UV performance of matrices . This fact limited their large-scale industrial production and consequently the practical applications of UV-blocking films. In contrast, organic UV-blocking alternatives are identified as phenolic-type UV-absorbers which exhibit good photostability, because the compounds can absorb light energy and make it less harmful, and are typically involved with hydrogen bonds from either O-H-O bridges or O-H-N bridges . Organic adsorbents are generally based on triazine, benzotriazole, benzophenone, anthranilates, dibenzoylmethane, and light amine stabilizers . However, the dangers of organic absorbents for human health restrict their use by promoting the search for green alternatives. In this sense, molecules, such as flavonoids or natural compounds that act as green radiation absorbers (such as tannins, kombucha tea, lignin, grape syrup, pine extract, and others), are interesting substitutes to metal nanoparticles and organic absorbents, blocking radiation in a broad range of wavelengths and decreasing the risks associated with potential migration to the food matrices . Another alternative to improve UV-light barrier characteristics is the design of a hybrid material, defined as a material that comprises different systems. In this sense, hybrid UV-blocking absorbers can result from the combination of organic UV absorbers and inorganic matrices or from the integration of inorganic UV-blocking absorbers with organic polymers . 4. Active and Intelligent Food Packaging The increasing demand for fresh, healthy, and long-shelf-life foods require innovations in packaging design. In this sense, new, intelligent, and smart packaging able to sense and communicate information from the packaged food product has been developed. Before moving forward, it is necessary to clarify three terms that are sometimes used interchangeably: active, intelligent, and smart packaging . Active materials are specifically designed to interact with the food or its surrounding environment, modifying their composition or characteristics to preserve the organoleptic or sensory characteristics of the product to ensure its quality for longer periods of time. Antimicrobials, antioxidants, flavor and gas scavengers, and light blockers are some examples of active substances usually used in food packaging . Intelligent packaging materials, on the other hand, are aimed to sense changes within the food package and provide information about the status of the food inside . Although limited to detecting and communicating, intelligent packaging can enhance security, safety, and convenience, providing real-time quantitative information on package integrity and food freshness, maturity, or contamination . Finally, smart packaging results from the combination of both intelligent and active packaging technology, though it is sometimes used indistinctly to refer to either one or the other . However, food packaging systems with a single function of freshness keeping or monitoring may not be able to meet all practical needs . The global market for active and intelligent packaging materials presents a growing tendency with a forecasted CARG of 5.5% from 2021 to 2030, estimated to reach $38.66 billion by 2030 , although the pandemic context has probably affected the development of this market. 4.1. Active Materials and Packaging Systems Active containers, commonly used in food packaging, are those that contain a substance capable of preserving the organoleptic or sensory characteristics of a product to ensure its quality. Of particular interest are active packaging containing natural antioxidants and antimicrobials that not only extend the shelf-life of packaged products by preventing rancidity reactions, but also prevent the growth of foodborne pathogens . The EU Commission defined active compounds as any substance or device that can extend the shelf-life or maintain/improve the packaging environment . According to European Regulation No 450/2009, the active agent can be an individual substance or a combination of substances . These compounds are included in the packaging materials formulations and exert specific functions, such as releasing or absorbing CO2, O2, ethylene, odors, flavors, antioxidants, and antimicrobials. Recently, Atta et al., Amin et al., and Pandey et al. extensively reviewed biodegradable active packaging materials for food applications . Active materials can be categorized by active agent or function. There are numerous types of substances used in food packaging that can act as active agents. Thus, a classification based on the purpose of the active packaging proves clearer and more useful in terms of application. However, many active compounds can have more than one function, making their classification complex. Active packaging materials containing food-approved synthetic active compounds were first developed. Among the synthetic antimicrobials, active films that include potassium sorbate, benzoate, and propionate in their formulation stand out . Synthetic antioxidants, e.g., butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA), have been also included in both polymeric and biodegradable film formulations and were used as active packaging to prevent lipid oxidation in foods . Biobased materials have great potential to replace conventional synthetic ones for packaging applications . The use of natural additives in food packaging formulations not only protects them from the environment, but also provides protection against microbial contamination and agents that stimulate oxidative rancidity reactions, among others, reducing the need of additives in the food matrix . Some of the polymers and natural compounds can be obtained from industrial agri-food waste and their use would add value to this waste and keep it in circulation. Of particular interest is the use of chitosan or its derivatives due to intrinsic antimicrobial activity against a broad spectrum of microorganisms . This biodegradable polymer derives from chitin that is mainly obtained from waste from the fishing industry, such as crustacean exoskeletons . Likewise, by-products and waste from fruit and vegetable processing are an important source of bioactive compounds with high nutritional and functional value, such as vitamins, minerals, antioxidants, and antimicrobial compounds, although they are often discarded or derived for animal feed . The essential oils (EOs) that can be obtained from these sources have been widely studied as additives for the development of active food packaging, mainly due to their antioxidant and antimicrobial capacity and their GRAS (generally recognized as safe) character . Therefore, the use of active compounds derived from agricultural by-products not only contributes to the recovery of these compounds with specific activities, but also generates added value for them. For example, Bof et al. have developed and characterized active biodegradable films based on corn starch and chitosan with the addition of lemon essential oil (LEO) and grapefruit seed extracts (GSE) . The inclusion of these active compounds, which are by-products or derivatives of citrus processing residues, did not affect the mechanical properties of the material and provided antimicrobial capacity on contact. Similarly, Kanmani and Rhim developed antimicrobial active films with GSE in a carrageenan matrix, with additional UV-barrier capacity, particularly important for UV-sensitive food packaging . Moreover, Bof et al. have demonstrated that the biodegradable films based on corn starch and chitosan and the active film containing GSE reduced the post-harvest weight loss, without the incidence of rottenness, of packed blueberries during their refrigerated storage in comparison to clamshell PET containers . Besides, their performance under real conditions of transport and commercialization was evaluated, also considering the costs and possible scaling of the process. Additionally, several EOs have been included in film formulations to impart antioxidant properties, such as rosemary extracts, tea polyphenols, eugenol, oregano, thyme extracts, red propolis, and green coffee oil extracts . Thus, EOs exhibited combined and synergic actions since they can confer both antimicrobial and antioxidant capacities. This effect was demonstrated for gelatin films containing red propolis extract which exhibited both antioxidant capacity and inhibited the growth of S. aureus, L. monocytogenes, S. enteriditis, and E. Coli . Similarly, chitosan films loaded with eugenol were also effective in the inhibition of S. aureus and E. Coli and presented antioxidant capacity . Other natural compounds, e.g., a-tocopherol, ascorbic acid, lycopene, gallic acid and resveratrol, with proven antioxidant capacity have also been incorporated in polymer matrices to functionalize them . Considering their action mechanism, antioxidant compounds can also be used as oxygen scavengers . These compounds can be incorporated into the package as sachets, stickers, or embedded into the polymer matrix of the food packaging. In addition, different strategies have been promoted to modulate the release kinetics of the active compound and thus ensure the effect of the active compound incorporated. Among them, encapsulation stands out, a technique of particular interest in the case of thermolabile compounds that will be incorporated into matrices that require processing at high temperatures. Likewise, novel methods have been developed for the design of micro and nano-systems that transport antioxidant and antimicrobial active ingredients. These structures allow the controlled release of the active components for different purposes . The protection of active compounds by electrospinning or electrospraying are novel techniques with the potential to be scaled up . Spray drying is a widely used technological tool to encapsulate active compounds due to its low cost and because it allows the continuous production of large amounts of product in short periods of time. However, it requires careful control of the operating conditions to avoid losses of the active compounds during the process . In recent years, research reporting the formulation of active biomaterials, including metallic nanoparticles obtained by green synthesis techniques, has increased, highlighting Ag and Cu nanoparticles and ZnO nanorods . In this regard, Ortega et al. have synthesized Ag nanoparticles by reduction with maltose in situ of the filmogenic suspension of starch-based biomaterials, characterizing the materials obtained and demonstrating their antimicrobial capacity . They have also obtained nanocomposite materials based on corn starch by adding silver nanoparticles synthesized with reducing compounds present in lemon juice. These materials also exhibited the ability to inhibit the growth of fungi, yeasts, and bacteria . Likewise, nanoparticles, e.g., those of TiO2 and montmorillonite, as well as zeolites, are efficient as ethylene scavengers and were useful to reduce fruit decay and maintain quality of fruits during postharvest storage . Zeolites have also been used as CO2 scavengers in food packaging . Nonetheless, even though progress has been made in nanoparticle migration studies and their cytotoxicity, it is still necessary to advance the framework legislation that regulates the use of nanocomposite active materials for their extensive use in contact with food . Progress is still required in the design and large-scale manufacture of these active biomaterials for use in the food industry. Besides, the involved production costs should be considered since some biobased materials still require process optimization and the discovery of alternative raw materials for their production. Innovative research is being conducted on the preparation of composite materials with natural and synthetic additives using emerging technologies such as 3D printing, which could further lead to improved functionality of the obtained materials (See Section 5.2). Future research in the field should be devoted to improving controlled release kinetics to obtain continuous release throughout the shelf-life of packaged food. 4.2. Intelligent and Smart Food Packaging Intelligent packaging can non-destructively and in situ monitor the food quality, detect storage conditions, expiration date, safety diagnostics, monitor microbial growth, and determine the freshness of food . One aspect of this technology is based on the use of interactive indicators, dye-based compounds that allow the evaluation of the chemical and microbiological quality of foods. Temperature changes during storage, oxygen concentration, or microbial activity can be monitored in real-time. According to Moradi et al., there are three main types of commercially available, intelligent, and smart packaging: time-temperature, freshness, and gas-release indicators . Those of freshness include acid-base indicators based on pigments whose colors are affected by the pH of the environment . Recently, special attention has been paid to the use of natural colorimetric indicators sensitive to changes in pH, including anthocyanins from different vegetable sources . Sensors are also used in the design of intelligent packaging, some of which can be obtained by 3D printing . To this end, nanomaterial-based functional ink formulations with innovative electronic designs and architectures printed on packaging materials are used to achieve various functionalities, such as radio frequency communication, time-temperature indicators, gas sensors, and freshness indicators, among others. New concepts, such as integrated printed radio frequency antennas; printed temperature sensors; and printed supercapacitors, using electronic inks, are still under development . Nowadays, several intelligent systems are commercially available . These can be classified into three types: sensors, indicators, and data carriers . A sensor is a device that responds to physical or chemical stimuli measuring it or simply detecting it by some sort of signal emission. Specific sensors named biosensors have been developed to detect hazardous substances, such as foodborne pathogens and allergens, residual antibiotics, or pesticides . These sensors can detect, record, and convey information relevant to biological systems. Most of the commercial biosensors are a combination of antibody-based receptor and optical transducer . For example, Food Sentinel System(r) developed by Sire Technologies Inc. (Crowthorne, UK) is an antibody-based biosensor where a membrane with immobilized antibodies is used as a part of the barcode that acts as the sensor. As the pathogens interact with antibodies, a localized dark bar is formed which renders the barcode unreadable. ToxinGuard(r) developed by Toxin Alert (Mississauga, ON, Canada) is another system where antibodies are printed on polyethylene packaging material. The interaction between pathogens and antibodies results in the production of a fluorescent signal that indicates a pathogenic attack. Besides, gas sensors are employed for the detection of gaseous analytes, e.g., oxygen, water vapor, carbon dioxide, ethylene, etc., inside the package. Papkovsky and Dimitriev described optical oxygen sensors based on the principle of quenching or luminescence upon gaseous analyte contact . The use of pH-sensitive dyes, such as methyl red and curcumin, in starch-PVA films for the detection of basic volatile amine released from rotten meat and fish have been reported . On the other hand, indicators can determine the presence or concentration of another substance, or the reaction between two or more substances, by giving characteristic changes in color. Among them, freshness indicators provide information about the product quality by determining the chemical changes resulting from the microbial growth within the product. For instance, COX Technologies (Louisville, KY, USA) markets FreshTag(r) which are colorimetric labels that indicate the production of volatile amines by fish and shellfish during storage . Besides, the SensorQ(r) indicator produced from a polymer matrix containing a solution of the bromocresol green dye was used to monitor meat and poultry freshness . Likewise, time-temperature indicators (TTIs) are labels that provide a visual indication of the temperature abuse of perishable products, mostly frozen foods, during distribution and storage from the point of production to that of consumption. The operating principle of these devices is mainly based on different reactions between two or more substances triggered or accelerated by time and temperature variations that result in (usually irreversible) color changes of the indicator . TTIs are based on diffusion, enzymatic, microbial, or polymer-based systems, several of which are commercially available. Diffusion-based indicators rely on either phase-transition or rheology and diffusion rate dependence of colored substances with temperature. For example, Khairunnisa et al. studied different vegetable oils and their combination to tailor their viscosity and the activation energy of the TTIs . The 3M Company (Maplewood, MN, USA) has commercialized diffusion-based TTIs by the trade name 3M Monitor Mark(r) and Freshness Check(r) for a wide range of exposure temperatures and times. Kim et al. used isopropyl palmitate on a microporous polymer structure over a colored layer, as the fatty acid ester fills the pores the reflection index of the material decreases letting the lower layer show. Indicating that the length of the TTI device influences its application range. Enzymatic TTIs use the enzymatic hydrolysis reaction of the substrate, which is sensitive to temperature and or pH variations that can usually be identified by color change of the matrix . Similarly, microbial TTIs use metabolites produced by microorganisms, namely yeast and lactic bacteria, under certain time and temperature conditions, generating pH changes, indicated by halochromic compounds . These types of biosensors are being studied due to their sensitivity, safety and wide conditions range . For instance, novel TTIs using laccase , lipase , and glucose oxidase have been successfully developed. An example of commercial enzymatic TTI is VITSAB(r), which is based on color change resulting from a pH drop due to controlled enzymatic hydrolysis of a lipid substrate . Non-enzymatic browning reactions, such Maillard reactions, have been studied and modeled to tailor the reaction activation energy by varying the reactive concentrations or pH conditions of the TTI systems . In addition, one of the most studied TTIs systems is based on polymerization reactions, typically poly-diacetylene (PDA) based. PDAs present thermochromism, among other chromisms, changing from blue to red upon stimulation with various sensing applications . Polymer-based TTIs systems under the trademark Lifelines Freshness Monitor(r) are commercialized by Lifelines Technology Inc. (Leicester, MA, USA). Recently, Suppakul et al. have developed a diffusion based TTI temperature sensitive device by combining this simple technology with a thermochromic polydiacetylene/silica nanocomposite . Likewise, FreshCode(r) (Varcode Ltd., Chicago, IL, USA) and Tempix(r) (Tempix AB, Gavle, Sweden) are based on barcodes printed with fading inks that disappear due to temperature abuse . These inks are colored compounds that produce transparent or lightly colored products by reaction, whose kinetics are regulated by time and temperature, directly indicating changes of food shelf life . Besides, the integrity indicator Ageless Eye(r) developed by Mitsubishi Gas Chemical Company (Tokyo, Japan) is an oxygen indicator tablet that turns pink when the oxygen concentration is less than 0.01% and blue when it goes beyond 0.5% . Research and development in this area are constant, even more so than those related to commercially available alternatives. The use of active and Intelligent packaging introduced new concepts and agents that required a clear updated regulatory framework on food contact materials. In fact, the lack of such a legal frame for many years led to reluctance by food packaging manufacturers to take on novel smart packaging systems which were not fully covered by the current legislation on food contact materials . Regulations vary among countries, though they are mainly based on substance migration limits and toxicological properties . The European approach is based on the theory that all materials should be explicitly cleared accordingly to listed hazardous substances, while in the United States substances that are not likely to cause any health problem are directly cleared (or deemed not to require regulation) on the basis of chemical or toxicological data extrapolation beneath minimal dietary exposure . In addition to complying with current regulations, packaging manufacturers must also consider potential human health effects from the possible migration of contaminants, especially for intelligent devices that are placed in direct contact with food. Intelligent devices must also be properly labeled to increase consumer confidence in the safety of packaged foods as well as in the use of these technologies. Finally, radio-frequency identification (RFID) tags provide the ability to identify, control and manage goods through the supply chain and have been successfully applied for this purpose. These are more advanced, reliable, and efficient than the conventional barcode tags for food traceability . RFID tags for monitoring temperature, relative humidity, pressure, pH, and light exposure of the products are already available in the market which aid in enhancing food quality and safety. For example, DogboneTM, which detects and measures temperature and moisture levels in the environment, and CircusTM Tamper Loop, an NFC RFID tag that informs about authentication reordering, expiration, care, and label integration, were developed by Avery Dennison Smartrac (USA). Besides, this company developed the sensor AD-327 FCC(r), which informs about supply chain management, inventory, and logistics . According to a 2019 report by IDTechEx, the RFID market was $11.6 billion and will grow to $13 billion by 2022, which reflects the potential of this technology . Although, the design of intelligent systems based on RFID technology still faces many challenges to overcome. Among them, the aspects related to the food safety, cost, reading range, multi-tag collision, multi-parameter sensors, recycling problems, security, and privacy of the RFID system should be solved . Cost is an important factor to consider in intelligent and smart packaging development. If the cost is high in comparison to the product, a price rise could imply fewer sales and a consequent increase in product waste. Likewise, investments in these technologies may be too high from both economic and environmental perspectives . Therefore, new, low-cost materials and methods for sensors, indicators, and data carriers are sought . In this regard, 3D and 4D printing of biobased composite materials plays a key role, which may constitute the object of future research and development. Intelligent packaging and collaboration in data sharing and information transparency may facilitate more synchronized supply chains with greater visibility and traceability, which can help reduce inventory and prevent food from going bad in stock by more precise demand forecast and sales data . It is also imperative to study and control any possible negative effects of intelligent packaging on recycling systems. Bibi et al. remarked upon the need for recycling programs for RFID sensor tags . Hence, materials used should be one of the most important considerations with the aim for low environmental impact elements. In this regard, bio-based smart packaging is a potential option that combines sustainable production and real-time monitoring of food quality to guarantee health safety, prevent food loss, and provide both economic and environmental benefits . Table 2 summarizes recent novel biobased active materials and intelligent systems innovations as well as their applications in food packaging. 5. Biobased Packaging One of the applications with the greatest impact on the growing demand for bioplastics is for containers and packaging, particularly biodegradable materials in food packaging. Before the COVID-19 pandemic, many multinationals, such as Danone, Coca-Cola, and Nestle, had committed to adopting bioplastics in their packaging . In this sense, there are numerous investigations and developments of biodegradable and biobased materials due to their potential lower environmental impact. Numerous works have been reported and an extensive literature revision has been conducted by various researchers in the field of material sciences and the packaging industry . A comparison of different biomass sources for bioplastics and biopolymers along with various processing technologies discussion can be found among available literature . The latest works focus on new biomass sources to reduce bioplastics cost and climate impact due to extensive land use . In view of more sustainable packaging systems, fully biobased packages are sought, considering all parts. Therefore, herein, a thorough overview of biobased adhesives, inks, and dyes for food packaging is presented. 5.1. Bioadhesives for Food Packaging The term adhesive describes a formulation that can hold two or more specimens together. In other words, an adhesive can join materials by surface bonding (adhesion), with a bond possessing adequate internal strength (cohesion) . The materials which are to be joined are called adherents or substrates and the process of attaching one adherent to another is called bonding. The adhesives include a wide range of materials with very different specifications that produce adhesion through different mechanisms depending on the characteristics of the substrate. The effectiveness of the adhesive bonding capacity depends on several factors, which are intermolecular forces in the adhesive, wettability of the adhesive in the adherent, types of chemical bonds, functional groups involved, type of interface between the adhesive and the adherent, type of substrate, and surface tension generated . Likewise, Dohr and Hirn reported that the strength of the adhesive bond depends on various physical properties of both the substrate and the adhesive . Both the way the adhesive is applied and its chemical composition determine the possible applications. Other factors, such as the dispersion of the adhesive over the substrate and the contact area, which in turn have a great impact on the adhesive forces that can develop, are also relevant . Although in former times adhesives were prepared from natural sources, synthetic formulations of adhesive are nowadays based on thermoset resins: phenol-formaldehyde, an alkaline catalytic salt (PF), urea-formaldehyde, an acid catalytic salt (UF), melamine-formaldehyde (MF), and polymeric diphenylmethane diisocyanate resin (pMDI) . These synthetic adhesives cause health problems during the manufacture process. For instance, formaldehyde was declared a carcinogen by the World Health Organization (WHO) in 2004. Similarly, the International Agency for Research on Cancer (IARC) reclassified formaldehyde as carcinogen category 1 in 2004 . In addition, a comparative increase in price and the foreseen lack of availability for petroleum-based synthetic adhesives have prompted the development of green derivatives from economic and renewable resources. Biobased adhesives are formulations based on natural raw materials, which are not derived from mineral or fossil sources. This term includes adhesives formulated with biopolymers obtained from plants, animals, and natural gums . In the search to develop bio-based adhesives with properties similar to traditional formulations, proteins, tannins, lignin, and polysaccharides are eco-compatible biopolymers that could fit these requirements . Their use is mainly limited to paper, cardboard, aluminum foil, and wood for construction applications. These adhesives develop stickiness quickly but exhibit low-strength properties. Most are soluble in water and use water as a solvent agent. They are supplied as liquids or dry powders to mix with water, though some are dispersions . Figure 4b shows a classification of adhesives according to the source of the polymers from which they are prepared, as it was reported by Ebnesajjad and Landrock . The global market for adhesives and sealants is growing rapidly and is expected to be worth $85.8 billion by 2026, with packaging being one of the most relevant applications . Adhesives in packaging industries are critical to the structure of most paper and paperboard packaging. In this regard, paper-based materials have been broadly applied as packaging material for food products. From a production point of view, adhesive selection can significantly affect process line efficiency and production performance . Adhesives are commercialized in many shapes and types, and the choice will be determined by the substrates on which the adhesive for bonding will be applied, the machinery used in the process, and other factors, such as potential requirements for food-safe materials . The types of adhesives used in the paper industry are water-based adhesives, both synthetic and biopolymer-based (starch, cellulose derivatives, proteins), solvent-based adhesives (polyurethane and acrylic base), and 100% solid adhesives, such as heat-sealing adhesive and hot melts. Gadhave and Gadhave reported the development of a water-based heat-expandable adhesive that has thermally insulating properties and has been used in protective food packaging . Protein-based adhesives (animal or vegetable) were the first polymers to be used in ancient times as adhesives in contact with the skin due to their biocompatibility and biodegradability properties. Likewise, protein-based adhesive formulations have been developed as substitutes for formaldehyde resins, particularly urea-formaldehyde resins in applications for construction material adhesives and paper and coating manufacturing . There is numerous research on adhesives to bond wood made from tannins , lignin , cellulose derivatives and modified starches , and vegetable proteins, such as casein , gluten , and soy proteins . Adhesives based on animal protein can be formulated from collagen, gelatin, or casein. Collagen is prepared from different parts of animal bodies, such as skins or bones, and gelatin is derived from the denaturation of collagen. The reversible gel-sol conversion by water absorption is an important property for the application of these adhesives. Casein is obtained from cow's milk by acid precipitation using different acid media, which can result in diverse properties depending on the resulting molecular structures. The primary structure of this protein can be chemically modified to achieve different rheological behaviors . Research related to the development of biobased composite adhesives for applications in food packaging is still incipient. Fatty acids derived from vegetable oils were used as the base for monomers that once polymerized form pressure-sensitive adhesives . Heinrich, Wang et al., Wu et al., Mahieu et al., and Ding et al. have reported the use of blends based on starches and proteins from different sources for green bioadhesives preparation . Due to the high number of polar groups in both proteins and polysaccharides, the hydrophilicity of composite systems is often the biggest obstacle to overcome in adhesive formulations . For proteins, this includes making functional groups available for crosslinking, for instance, by tertiary and quaternary structure modification. Chemical modifications can be made in the structure of the polysaccharides and proteins (starch, cellulose, gelatin, soy proteins, among others) to improve the crosslinking of the matrix using crosslinking agents. Kumar et al. have reported that the use of crosslinked starch with citric acid results in materials with good mechanical performance . These properties are explained and revealed from a structural point of view by the interactions established between the carboxyl groups of citric acid and the hydroxyl groups in starch. On the other hand, Olomo informed that adhesives chemically treated using HCl as a cassava starch gelatinization modifier were of higher quality than those modified in presence of NaOH . While native starch contains only hydroxyl groups and is limited in scope, chemically modified starch shows superior water resistance properties for adhesive applications . Other modifications to prepare biobased adhesives with improved water resistance may involve esterification, transesterification, alkylation, acetylation, succinylation, or enzymatic reactions. The use of starch as a raw material in the manufacture of adhesives has the advantages of its renewability, biodegradability, and availability, in addition to its low cost and non-toxicity . Cassava (Manihot esculenta) is produced mainly in the tropical and subtropical regions of Africa, South America, and Asia. The high starch content of cassava and its higher proportion of amylopectin, compared to other starch sources, make it an important source of biopolymers to be used in the development of biobased adhesives. It can be employed as native starch, but it can be modified by different means to improve its properties of consistency (viscosity) . Cassava starch as a base to produce adhesives has many notable characteristics, including high paste viscosity, high paste clarity, and high freeze-thaw stability . Besides, Li et al. worked on improving the adhesion-to-fibers and film properties of corn starch by starch sulfo-itaconation for a better application in warp sizing . Biobased adhesives based on colloidal solutions in general dry slowly and therefore require a very long setting time. Bioadhesives containing starch, dextrin, and/or casein are primarily used in labeling applications, while starch-based formulations are also widely used in the obtention of corrugated boards . Starch-based adhesives are mainly composed of water, starch, sodium hydroxide, borax or boric acid, and other additives, such as preservatives, adhesion enhancers and defoamers, among others . Most starch-derived adhesives are used in the paper and textile industries as bonding agents and gluing materials. Corrugated cardboard is produced by the adhesion of a grooved layer of paper to another flat layer. To join them, a two-phase adhesive is usually employed: a liquid and a solid phase containing a mixture of starch and sodium hydroxide or native starch and borax, respectively. Developing biobased adhesive formulations for bonding different substrates, such as corrugated cardboard paper, glass, among others, to propose applications in the formation of film packaging and in the labeling process, constitute a great challenge. Currently, most containers that are in contact with food have adhesive in their structure. Adhesive formulations can be found in three different forms . It can form the structure of food packaging by combination with different materials (usually polymers, paper, cardboard, or glass), commonly known as laminate or multi-layer packaging. Moreover, the adhesive can help to provide the geometry of the container (box sealing) or can be used for labeling. The most common way to find adhesives on food packaging is of the first type; where the adhesive is applied on the total surface of the materials or substrates, joining different materials and forming multi-layer materials. Examples of practical applications are flexible film lamination, paper-film combinations, cardboard-film, aluminum-film, cardboard-aluminum, cardboard-aluminum-plastic, rigid multi-layer systems based on plastic, sacks, bags, among others. For such applications, the adhesive industry uses a wide variety of raw materials and formulations, combining different compounds to form special types of biobased adhesives. There is currently a growing push in the packaging and adhesive industry to improve the sustainability of processes and products. Therefore, the general trend in the adhesive industry for use in food packaging is a reduction in the use of solvents as well as molecular weight components of the adhesive that could more easily migrate from the package to the food. The use of biobased adhesives in the labeling of glass containers is an interesting alternative, since for this purpose there are few studies that contemplate the use of bioadhesives. The removal of the label-adhesive using only water is a desirable characteristic not only from an operational point of view, but also from an economic and environmental level. Easily removable labels would facilitate the sorting of recyclable waste packaging and therefore increase the recyclability of the whole package and reduce water and energy use. In addition, compared to the common industry practice for label removal using diluted NaOH solutions, water is a low-cost natural resource that, most importantly, could be reused, ultimately requiring simple effluent treatments before discharge. Regarding the legal background surrounding adhesives intended to be used for food contact applications, normally, the adhesives are used to stick together packaging materials and not intended for direct food contact. However, the adhesives as components of the packaging material might contribute to the migration of constituents into the food matrix. Adhesives, as well as food contact materials, are regulated according to EU Framework Regulation (EC) No 1935/2004. Plastic materials and articles are additionally regulated by a specific measure, Regulation (EU) No 10/20115 on plastics and therefore harmonized at EU level. This regulation establishes, among other requirements, a list of authorized compounds. It is pertinent to emphasize that adhesives do not yet have such specific harmonized legislation. Alternatively, reference is made to the opinions of the European Authority focusing on aspects of Food Safety, Council of Europe resolutions, national legislation, and even non-European legislation for risk assessments . The Commission Regulation (EC) No. 2023/2006, recommends procedures to assure the safety of adhesives for food contact applications, particularly on Good Manufacturing Practices for adhesives. Yet, there is currently no legal obligation for adhesive manufacturers to provide a declaration of conformity with Regulation (EC) No 1935/2004. However, if the adhesive falls under the Plastics Regulation, the adhesive manufacturer shall provide the specific information to enable the adhesive user to ensure compliance of substances with migration potential . According to the specifications reported by Romero Zaliz et al., adhesives for returnable glass containers must be formulated with raw materials that are included in the positive lists of the FDA and the Argentine Food Code (Mercosur) for use in Food Containers and Equipment in Contact with Food (Argentine Food Code Chapter IV, FDA 21 CFR 175.105) . For this reason, there is a high interest in the development of new strategies to produce new renewable materials that totally or partially replace petroleum-derived reagents, resulting in innovative products with special functionality, less toxicity, high biocompatibility, and/or biodegradability . Using natural resources or bio-based materials as adhesive raw materials could help future societies become less dependent on hazardous chemicals, volatile organic compounds, and petroleum-derived chemicals; in addition to promoting safer working conditions. Consumer trends toward green products are prompting plastics industries to investigate more benign alternatives to petroleum-based polymers. Moreover, the recent classification of formaldehyde as a harmful substance has accelerated the investigation of more ecological and renewable alternatives, such as protein-based adhesives, to avoid harmful emissions both during production and during the lifetime. Furthermore, the use of bio-renewable or waste raw materials helps to reduce the carbon footprint, aligned with the current circular economy framework. As an added benefit, the inherent biodegradability of renewable materials, e.g., starch, polyhydroxyalkanoates, or cellulose, is often higher than that of synthetic materials, e.g., polypropylene and polyethylene. The shift towards more ecofriendly alternatives has manifested itself in the adhesives industry first through the gradual change from solvent adhesives to water-based or high-solid content adhesives, and now by the renewed interest in the design of biobased adhesives. Nonetheless, it is convenient to modify the formulations based on natural polymers, such as polysaccharides and proteins, to improve their rheological properties, their adhesion capacity, and their mechanical resistance when applied for the bonding of different substrates to be used in food packaging. Consequently, composite adhesive formulations obtained from chemically modified biopolymers constitute an innovative proposal to overcome such difficulties. Further research on the selection and compatibility of biobased adhesive formulations is essential since the adhesive characteristics presented will depend as well on the type of substrates applied to. Additionally, progress is still required in relation to the legislation that frames the adhesives that can be used in the manufacture of containers in contact with food. 5.2. Biobased Inks and Dyes in the Food Industry The use of dyes and pigments dates back to 3500 BC, when the civilizations used natural extracts for coloring. Then, in 1856, the first synthetic dye was created by Perkin and a significant number of dyes were quickly discovered thereafter and adopted by industries . Nowadays, the most used colorants (dyes, inks, and pigments) by industries are organic molecules derived from petrochemicals and other chemicals that are causing important environmental damage. According to The Synthetic Dye and Pigment Global Market Report 2022, the global synthetic dye and pigment market grew from $54.54 billion in 2021 to $59.82 billion in 2022 and is expected to grow to $79.45 billion in 2026 . People are more inclined to be nature-friendly and health-conscious, which has created a revolution in research and development in eco-friendly and non-toxic colorants, pushing dyes and pigments manufacturers to shift back to natural dyes. Several countries have imposed a ban on the import of synthetic dyes. For example, the use of azo dyes is banned in India owing to their environmental and health impacts. In general, environmental considerations are becoming vital factors during the selection of consumer products. Natural dyes are applied in several areas because they have some special properties, e.g., soothing color, are biodegradable, non-hazardous, non-carcinogenic, and present antimicrobial resistance, among others . In relation to the food industry, colorants are widely used since, in many products, they are a highly valued attribute by consumers. They are currently being extended to food packaging technologies by applying colorimetric indicators or sensors to exhibit color changes with variations in pH, temperature, and gas for control of the food's quality. In the last five years, especially in food packaging, the research of natural dyes and inks has increased according to the Scopus database (November 2022), although it is not a field that has been extensively studied. Natural colorants can be classified according to their hue (red, yellow, brown, blue, purple, black, green, and orange), their origin (vegetal, animal, bacterial, fungal, etc.) or their chemical structure as follows: flavonoid derivatives (anthocyanins), isoprenoid derivatives (carotenoids), nitrogen heterocyclic derivatives (betalains) (Table 3). According to Iqbal and Ansari, these natural products present the advantages of being eco-friendly, biodegradable, renewable, not health hazardous or non-toxic; can be obtained from waste biomass, and in some cases present antibacterial and/or UV protective properties . In contrast, natural dyes are expensive because of limited available sources, they are difficult to produce, and the reproducibility of shades is hard to control, among others. Colorants are widely used in the food industry as an essential ingredient since in some products the color is a relevant characteristic. Viera et al. established that color is responsible for 62-90% of the consumers' acceptability . There is a growing worldwide concern for food quality and safety in the modern era, and given the availability of natural colorants, their use in the food industry is increasing. In this sense, there has been extensive research on the use of natural colorants in food products . Another use of natural colors is associated with 3D printing technology (additive manufacturing), since this technology can increase the acceptability of certain foods because of the change in the form and presentation, yielding a more attractive product for consumers. Inks for 3D food printing are classified based on their ease of use, nutrition-related components (protein, carbohydrate, fat, fiber), and functional compounds, such as vitamins and antioxidants, that can be incorporated through pigments or natural inks derived from fruits and vegetables extracts. Besides, the additives applied to edible inks play an important role in improving the flow behavior, sedimentation, and lubrication properties of the material to be printed . In this sense, there are some publications regarding the use of fruits and vegetables extracts to obtain more attractive presentations for kids. Lee et al. used spinach powder and xanthan gum to print spinach dispersions and Derossi et al. printed fruit snacks using banana, beans, mushrooms, and lemon juice, while Qiu et al. investigated the 3D printing of apple and edible rose blends as a dysphagia food . Escalante-Aburto et al. suggest that the production of 3D food printing must be considered a zero-waste technique to reduce the environmental impact . Thus, the development of bioinks should be focused on using low-carbon and low-water footprint food ingredients, leading to the introduction of a new market for novel and edible composites. Bioinks should be sold as a packaged ingredient that is inserted into the printer to obtain 3D-printed foods. The packaging material must be recyclable or biodegradable and innocuous to fit in with the sustainable technology and food safety concepts. On the other hand, in 2013, Skylar Tibbits put forward the term 4D printing in his TED talk based on 3D printing . Such 4D printing is based on smart materials and includes a fourth dimension: time, in addition to 3D spatial coordinates. Thus, 4D printing enables the shape, properties, or function of the 3D-printed products to change over time under environmental stimuli, such as temperature, concentration differences, water, pH, or light . Color changes represent one of the most common applications of 4D printing food. He et al. studied the spontaneous color change induced by pH in ready-to-eat 4D foods with anthocyanin-rich purple sweet potatoes, while Ghazal et al. aimed to investigate changes in colors and flavors of 3D-printed healthy food products in response to an external or internal pH stimulus . For the formulation, a combination of red cabbage juice, vanillin powder, potato starch, and different fruit juices was used. The changes in color, texture, flavor, and taste induced by the stimulus were determined, revealing that the color of the 3D-printed product changed from blue (control sample) to red, purple, violet, blue-green, and green-yellow colors when sprayed with pH solutions of different pH (2-10). In addition, dried 4D samples exhibited color and anthocyanin stability when stored at room temperature for three weeks. Likewise, Chen et al. discussed the possibility of using microwaves to stimulate the color change of 3D-printed curcumin lotus root gel . Based on traditional 3D printing technology, this work uses microwaves as the stimulus to obtain the color change of printed products and provides a new method for producing colorful and attractive food through 4D printing. Another use for 4D printed materials could be as quality sensors or indicators for food packaging. The obtention and use of natural inks or dyes provide an advantage for food packaging. Because of their low molecular weights, coloring agents, photo-initiators, solvents, and oils may migrate from printing inks, favoring natural dyes over synthetic dyes. Studies to obtain natural dyes from beet, red beet, potato, red onion, quince, black carrot, and hibiscus to use them in areas where contact with the human body is frequent, such as textile, tissue engineering, and food packaging, are being carried out . In addition, it is known that these plant extracts used as inks present antimicrobial activity, and for this reason, they are preferred for active and intelligent packaging development. There are many studies concerning the use of natural dyes or inks in the development of intelligent packaging such as films or 4D printing sensors . In this sense, Alizadeh-Sani et al. have critically reviewed pH-sensitive smart packaging films based on natural colorants for the monitoring of food quality and safety . Meanwhile, Tracey et al. released an advanced 3D printing approach to intelligent food packaging . Among the smart packages, those containing TTIs are the most studied. As mentioned in Section 4.2, these indicators adhere to the surface of the packaging and produce an irreversible color change in response to environmental conditions. Rachmelia and Imawan developed the TTI label using black corn extract and chitosan matrix. In this study, anthocyanin was obtained from black corn extract, while chitosan was used as a matrix . The use of non-toxic ingredients was aimed at making the TTI label safe to apply in packaging products. The label's color was observed over time at temperatures of 10, 25, and 40 degC. The label changed color from purple, to blue, to yellow being the fastest color changes at higher temperatures (40 degC) and slowest at low temperatures (10 degC). Furthermore, Mataragas et al. developed a microbial TTI based on the violacein (a microbial violet pigment produced by Janthinobacterium sp.) formation for monitoring the shelf-life of minced beef-vacuum-packed cooked meat products. When the temperature varied from 0 to 15 degC, the color changed from purple to violet . Other materials with a similar operation are pH and gas indicators, which due to these factors play an important role being indicative of food quality, shelf-life, microbial growth rate, and food deterioration . Erna et al. obtained curcumin/rice starch films for sensitive detection of hypoxanthine in chicken and fish meat, since the deprotonation of curcumin occurs when the indicator is exposed to a pH level of 9 or above, where the indicator's color changes from yellow to a reddish-brown or wine-red color ; while Boccalon et al. developed potato starch composite films containing red onion skin extract as intelligent pH indicators for food packaging . The films were tested by monitoring their color changes when applied to meat and milk storage. According to Tracey et al., highly sensitive, self-indicating, multifunctional smart components using biocompatible, nontoxic materials via lower cost intelligent packaging systems and devices can be developed by 3D printing in comparison to conventional fabrication methods . Zhou et al. used coaxial 3D printing followed by ionic crosslinking to create fruit freshness keeping and visual monitoring labels with high pH sensitivity and effective shelf-life extension capability . Cellulose nanofibers (CNF)-based ink with blueberry anthocyanin was used to create the shell of fibers, exhibiting high formability and print fidelity as well as sensitive visual pH responsiveness for freshness monitoring. Chitosan containing 1-methylcyclopropene (1-MCP), an ethylene receptor inhibitor with good freshness-keeping performance and no toxicity, was loaded into the hollow microchannels of the fibers. The 1-MCP was trapped by the electrostatic effect of chitosan and CNF exhibited a sustained release behavior . Finally, the 3D-printed labels prolonged the shelf-life of litchis by approximately six days. On the other hand, Li et al. developed an interlayer with chitosan, mulberry anthocyanin as a natural dye, and lemongrass essential oils as an antibacterial agent and antioxidant using a 3D printer, and cassava starch as a protective layer to form indicator films . These were used to monitor the quality, freshness, and preservation of cold meats by observing the color changes of the indicators. When chilled pork spoiled, the color of the indicator films changed from red to gray-blue (RGB), and the RGB tone values could be analyzed by a smartphone application to determine pork freshness . The use of smart packaging based on natural colorants and biodegradable films is intended as an attractive alternative for food packaging due to their low or nontoxicity, eco-friendliness, easy preparation, biodegradability, availability, renewability, and pollution-free properties. The main challenges of natural inks and biodegradable food packaging are related to the improvement of their functionality at reasonable costs. Likewise, progress in the assessment of their safety is needed. According to Bautista et al., this agrees with the global challenges of the packaging industry, some of the most important being: (1) increase sustainability of manufacturing processes, (2) improve the recyclability of materials, and (3) improve performance and functionality. In addition, the printing of labels with bioinks or the use of smart inks, including natural dyes, in the design of freshness indicator devices is a field that is still in its infancy with great potential . foods-12-01057-t003_Table 3 Table 3 Classification of natural dyes according to their chemical structure and source. Color Classification Source Example Reference Yellow/ Orange/ Red Curcumin Plant/Vegetable Turmeric (Curcuma longa L.) Carotenoids Plant/Vegetable Carrot (Daucus carota L.), Annatto (Bixa orellana), Tomato (Solanum lycopersicum), Paprika (Capsicum annuum L.), petals of marigold (Tagetes erecta L.) Aryl carotenoids Microorganisms Brevibacterium linens, Streptomyces mediolani, Mycobacterium aurum Red/Pink Betalains Plant/Vegetable Beetroot (Beta vulgaris L.), Opuntia lasiacantha Carminic acid Microorganisms Cochineal (Dactylopius coccus) Anthocyanins Plant/Vegetable Hibiscus rosa sinensis flowers Blue/ Purple Anthocyanins Plant/Vegetable Grapes (Vitus labruscana L.), red cabbage (Brassica oleracea var. capitata f. rubra), cherry (Prunus cerasus), blueberry (Vaccinium sect. Cyanococcus), red onion skin (Allium cepa L.), Beetroot (Beta vulgaris L.) Tyrian purple (6,6'-dibromoindigo) Animal Mollusks Bolinus brandaris Ultramarine Blue Mineral Lapis lazuli Green Chlorophylls Plant/Vegetable Spinach (Spinacia oleracea), kiwi pomace (Actinidiaceae), green beans (Phaseolus vulgaris), grass, alfalfa (Medicago sativa) Terre-Verte (Green Earth) Mineral Mixture of hydrosilicates of Fe, Mg, Al, and K (gluconite and celadenite) but other minerals are likely to be present Malachite Copper carbonate hydroxide 6. Conclusions Changes in lifestyle have directly influenced the type of food consumed, as well as consumption habits, which in turn has generated the need for the food industry to develop new containers and packaging. Today, practically any food product is marketed packaged, not only to contain the food, but also to protect it throughout the entire production chain, until it reaches the point of sale or consumption. Hence, the way we produce and consume food has substantial environmental, social, and economic impacts, requiring sustainable solutions for proper and efficient land use, better food preservation technologies during processing and packaging, and novel transport, distribution, and marketing systems to guarantee that these costs are well exceeded by benefits. Food packaging fulfills very important functions for food preservation, protecting it from external agents, preventing physical, chemical, and/or microbiological contamination, as well as its possible adulteration. In this way, they fulfill the function of a barrier against the environment that surrounds them, protecting food from humidity, oxidation, UV radiation, and microorganisms. New packaging technologies aim to further extend the shelf-life of food products by active systems that can slow the natural oxidation process or avoid microbial growth. Intelligent and smart packaging can further monitor food quality and spoilage to guarantee food safety for the consumers and prevent food losses due to improper conservation or transportation, or inefficient logistics and marketing. Investments in packaging have the potential to reduce overall environmental impacts associated with food production, distribution, and consumption, aiming to minimize food waste. However, systemic LCA approaches to determine the optimum product/package combination are needed. Effective assessment requires updated qualitative and quantitative information. Specialized sustainability agents within the industry, academia, and government are needed to develop a sustainability culture. Further efforts should also be made to raise awareness and educate food and packaging supply chain stakeholders and consumers on the role of packaging in extending food shelf-life and the opportunities to use it more effectively. Such opportunities entail: enhanced protection and larger shelf-life for fresh food products with potential tailored solutions; recovery of surplus and unsalable produce from farms to rescue supply chains; revalorization and use of agri-food industry byproducts and waste for packaging materials production; proper size (or portioning), function (i.e., easy-to-open, easy-to-empty, fit-for-purpose), reusable and/or recyclable food packaging design; new packaging materials and technologies to extend shelf-life (i.e., active, intelligent and smart packaging); unified labeling regulation for better use-by or best-before date indications for manufacturers, retailers, and consumers to avoid confusion regarding productions dates and prevent the unnecessary disposal of food; and intelligent packaging and data sharing to create more synchronized supply chains to reduce excess or out-of-date stock. A gap exists between product/packaging system design, materials supply, manufacturing and commercialization, and the return flow from recyclable materials that enter the waste management stream, which hinders circularity. To close the cycle, a holistic understanding of the supply chain components, their opportunities, and limitations is required for transitioning sustainable production systems towards a circular economy. Acknowledgments The authors would like to thank the National Council of Research in Argentina (CONICET), the National Agency for the Promotion of Science and Technology of Argentina (ANPCyT), the National University of the South (UNS) in Bahia Blanca and the National University of La Plata (UNLP). Author Contributions F.V., F.O., Y.M., S.R., O.V.L. and M.A.G. contributed to the study conceptualization, visualization, and design, as well as literature search and data analysis. A first draft of the manuscript was jointly written and commented on previous versions of the manuscript by F.V., F.O., Y.M., S.R., O.V.L. and M.A.G. All authors have read and agreed to the published version of the manuscript. Data Availability Statement Data sharing is not applicable--no new data generated. Conflicts of Interest The authors declare no conflict of interest. Commercial products and manufacturers' names listed in this review are not the only ones that have been commercialized and developed in the world. The authors are not related to any of the commercial products/manufacturers that are referenced in this review. Figure 1 Total number of publications reported in literature in the last ten years on food packaging for specific keywords. Literature review carried out on Scopus search engine of bibliographic databases. Figure 2 Barrier function of food packaging and hazardous agents for food safety present in the environment, along with some examples of enhanced barrier materials technologies. Figure 3 Classification of active, intelligent, and smart packaging, considering main properties, applications, interactions, and some examples. Figure 4 (a) Schematic representation of the function of adhesives in food packaging on different substrates; (b) Classification of adhesives according to the source of the polymers. Figure 5 Areas of application of natural inks and dyes. foods-12-01057-t001_Table 1 Table 1 ASTM standard methods for the determination of gasses and water vapor permeability in polymeric food packaging. Norm Method Permeant ASTM D 1434 Manometric/volumetric All gasses ASTM D 7709 Gravimetric Water vapor ASTM E 3985 Dynamic with electrochemical sensor O2 ASTM E 96 Gravimetric Water vapor ASTM E 2945 Static cells with analytic technique All gasses ASTM F 3136 Accumulation method with optical sensor O2 ASTM F 1249 IR sensor Water vapor ASTM F 2622 Dynamic with sensor O2 ASTM F 3299 Coulometric P2O5 sensor Water vapor ASTM F 2476 Dynamic with IR sensor CO2 foods-12-01057-t002_Table 2 Table 2 Active and intelligent biodegradable films and their food applications. Active Biodegradable Films Biopolymer Active Compound Food Application References Soy protein isolate Montmorillonite (0.5%wt) + clove essential oil (0.5%v) Bluefin tuna filets Chitosan and Corn starch Lemon essential oil (1-3%wt) and grapefruit seed extract (1-3%wt) Blueberries conservation simulating transport and commercial conditions Corn starch Green synthesized AgNPs in situ (143 ppm) Cheese preservation Tapioca starch Grape pomace extracts (8% v/v) and cellulose nanocrystals (10% v/v) Ready to eat chicken meats PLA Commercial nanoparticles: TiO2 (3%wt); (2%wt) nano-TiO2 + (1%wt) nano-Ag Cottage cheese preservation Curdlan + PVA Thyme essential oil (1-2%wt) Chilled pork meat preservation Whey protein Oregano and garlic essential oils (2%wt) Kasar cheese Chitosan-Cassava TPS bilayer films Oregano and or cinnamon leaf essential oils (0.25%wt) Sliced pork meat Gelatin/Gellan gum Red radish anthocyanins 5, 10, 15, and 20 mg/100 mL Milk and fish quality Zein Laurel or rosemary leaves extracts (1-10%) Cheese slices Chitosan Propionic acid Pastry dough Intelligent Biodegradable Films Function Intelligent System and Innovative Characteristic Food Application References Temperature sensor A passive RFID tag modified with a copper-doped ionic liquid Fresh products, for the identification of cold chain failures Temperature abuse indicator Au nanoparticles included in alginate hydrogel. Biobased, food safe, cost-effective, time sensible Fresh products Thermal insulation Commercial pale-yellow carnauba wax. Biobased and biodegradable insulator Beverages pH-based freshness indicator Biodegradable films containing anthocyanins from different sources. Real-time monitoring of food freshness. Fresh products: cheese, yogurt, fish, pork, shrimp, and beef pH sensitive Natural compounds showing color changes with pH in biodegradable films Fish and seafood products CO2 detector Labels containing natural (anthocyanins) or commercial (bromothymol blue and tetrabutyl-ammonium) dyes. Fermented products such as kimchi Oxygen indicator UV-light activated oxygen sensitive biobased film with methyl blue indicator Suggested for food products packed in modified atmosphere Hydrogen sulfide indicator Biobased films containing silver nanoparticles (detect up to 0.81 mmole H2S) or ferrous sulfate (detect 100 ppm H2S) and had a fast response (3 min). Meat and meat products. 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PMC10000826 | Foods Foods foods Foods 2304-8158 MDPI 10.3390/foods12051064 foods-12-01064 Article Yeast Strain Influences the Hop-Derived Sensory Properties and Volatile Composition of Beer Kumar Ashly Methodology Formal analysis Investigation Data curation Writing - original draft + Warburton Andrea Methodology Formal analysis Data curation Writing - original draft Writing - review & editing Visualization Silcock Patrick Conceptualization Methodology Formal analysis Resources Writing - review & editing Visualization Supervision Funding acquisition Bremer Phil J. Conceptualization Methodology Resources Writing - review & editing Supervision Funding acquisition Eyres Graham T. Conceptualization Methodology Formal analysis Resources Writing - review & editing Visualization Supervision Project administration Funding acquisition * Camara Jose Sousa Academic Editor Cozzolino Rosaria Academic Editor Department of Food Science, University of Otago, P.O. Box 56, Dunedin 9054, New Zealand * Correspondence: [email protected] + In memory of our friend, colleague and beer enthusiast, Ashly Kumar, who tragically passed away on 9 September 2022. 02 3 2023 3 2023 12 5 106401 2 2023 23 2 2023 28 2 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). The perception of hop-derived flavour in beer is not well understood, particularly regarding the effect that different yeast strains and fermentation parameters have on perceived hop aroma and the mechanisms responsible for these changes. To evaluate the influence of yeast strain on the sensory properties and volatile composition of beer, a standard wort, late-hopped with New Zealand Motueka hops (5 g*L-1), was fermented with one of twelve yeast strains under constant conditions (temperature and yeast inoculation rate). The bottled beers were evaluated using a free sorting sensory methodology, and their volatile organic compounds (VOC) were assessed using gas chromatography mass spectrometry (GC/MS) with headspace solid-phase microextraction (SPME) sampling. Beer fermented with SafLager W-34/70 yeast was associated with a hoppy flavour attribute, whereas WY1272 and OTA79 beers were sulfury, and WY1272 was also metallic. WB06 and WLP730 beers were perceived to be spicy, with WB06 beer also perceived as estery, whereas VIN13 beer was sour, and the WLP001 beer was astringent. Beers fermented using the twelve yeast strains had clearly distinct VOC profiles. Beer made with WLP730, OTA29, SPH, and WB06 yeasts had the highest 4-vinylguaiacol levels, which contributed to their spicy attribute. Beer made with W3470 had high levels of nerol, geraniol, and citronellol, which supported its sensory characterisation as being 'hoppy'. This research has illustrated the important role that yeast strain has on modulating hop flavour in beer. beer fermentation hops yeast strains terpene biotransformation sorting task Emerson's BreweryA.K. received the JP Dufour Scholarship from Emerson's Brewery to support this research. pmc1. Introduction The perception of flavour and the quality of beer is dependent on the raw materials used and the variety of reactions the happen during wort production and fermentation. Flavour generation reactions during fermentation vary depending on the nature of the ingredients, including the yeast strain used, and the fermentation conditions . Yeasts are essential in beer production where the yeast strain influences the type of beer produced and its flavour profile due to the metabolites generated during fermentation . Yeasts also alter the perception of hop flavour in beer by biotransforming hop flavour precursors during fermentation, with different yeast strains producing beers with differing concentrations of hop-related volatile organic compounds (VOC) . Hop flavour in beer is dependent on complex physical, chemical, and biological changes that happen during wort production and fermentation. Hops (Humulus lupulus L.) are responsible for the characteristic bitterness of beer that counteracts the sweetness from malt and conveys drinkability to beer. Volatile compounds derived from hop essential oil are responsible for a variety of aroma attributes, particularly when hops are added late in the boil (late hopping) or added during the fermentation or maturation stages of production (dry hopping). The perception of hop flavour also changes during fermentation due to modification of hop compounds by yeast during fermentation (biotransformation reactions) . These yeast-hop interactions generate a diverse range of hop flavours in beer including resinous, fruity, citrusy, floral, and spicy attributes . Despite a large quantity of research on the composition of hop essential oil, much is unknown about the mechanisms by which hops contribute to beer flavour . Important chemical components in hop essential oils are terpene hydrocarbons (including monoterpenes such as b-myrcene, sesquiterpenes such as a-humulene and b-caryophyllene), oxygenated compounds (alcohols, aldehydes, ketones, and esters, which may be terpene derivatives), and sulfur compounds . Terpenoids are a diverse class of compounds derived from isopentenyl pyrophosphate precursors, which generate a range of aroma and flavour characters . The biotransformation of monoterpenes by yeast during fermentation has been reported by King and Dickinson , where yeast activity converts monoterpene alcohols such as geraniol and linalool to a range of other terpenoid products via either isomerisation (e.g., converting geraniol to nerol), reduction (e.g., to citronellol), or esterification . Biotransformation of hop-derived compounds by different yeast strains has been shown to impact on the VOCs profile in beer and has been speculated to also impact on the perceived flavour of beer, although comprehensive studies have not been reported in the literature. The free-sort method has been demonstrated to be an effective rapid sensory evaluation technique to describe beer characteristics . Free sorting aims to measure the degree of similarity between samples by sorting the samples into groups according to their similarities and differences. The inclusion of an additional descriptive step enables the sensory characterisation of the samples . The aim of this study was to examine the influence of different yeast strains on the volatile composition and the sensory properties of the resulting beers. A standard wort, late-hopped with New Zealand Motueka hops, was fermented using one of 12 different yeast strains, including a selection of beer and wine yeasts, to obtain a diverse range of sensory characteristics. Sensory analysis was carried out using a free sorting task methodology with a panel of 14 assessors, including trained sensory panelists, volunteers, and brewing professionals. The VOC composition of the beers was assessed using gas chromatography mass spectrometry (GC/MS) with headspace solid-phase microextraction (HS-SPME) sampling. 2. Materials and Methods 2.1. Brewing and Fermentation Yeast strains (Table 1) were propagated in sterile (autoclaved) malt medium (350 mL, 10% w/v) for 20 h at 20 degC. Fermentis SafAle US-05, SafAle WB-06, SafAle BE-256, SafLager W-34/70, and SafLager S-23 yeasts were provided by Fermentis (Lille, France); White Labs California Ale WLP001 (San Diego, CA, USA) was purchased from Homebrew West (Auckland, New Zealand); Anchor VIN13 hybridised wine yeast and Exotics SPH wine yeast (Anchor Oenology) were supplied by Scott Laboratories (Petaluma, CA, USA). OTA29 and White Labs Chardonnay White Wine Yeast WLP730 were propagated from agar slopes from the University of Otago yeast collection according to the protocol described below (Section 2.2). OTA79 (University of Otago yeast collection) and Wyeast American Ale II WY strain1272 (Hood River, OR, USA) were supplied as a slurry by The Emerson's Brewing Company (Dunedin, New Zealand). Wort was produced at The Emerson's Brewing Company with an original gravity of 10degP at an efficiency of 80%. Milled malt (194 kg) was added into the mash vessel and mixed with hot water (582 L) to achieve a strike temperature of 50 degC together with CaCl2 (100 g), lactic acid (150 mL), and b-glucanase (100 mL). A temperature programmed mash with rests at 50 degC (15 min) and 67 degC (45 min) was utilised to achieve saccharification before increasing the temperature to 75 degC for mash out. The sweet wort was recirculated in the lauter tun for 15 min for clarification before transfer to the kettle for wort boiling. The pre-boil gravity and pH of the wort sample were determined to be 1.038 and 5.33, respectively. The wort was boiled for 60 min with antifoam and bittering hops (Simcoe; 580 g) to achieve 20 IBU. Kettle finings (koppafloc; 100 g) and magiFood (100 g) were added after 45 min to reduce haze in the final beer. After 60 min, the boil was stopped, and Motueka hops (6000 g total; dose rate of 5 g*L-1) were added for a 5-min steep. The wort was transferred into a whirlpool vessel for 10 min for clarification to remove spent hops and trub. Wort was cooled to 18 degC using a heat exchanger during transfer to a holding tank with in-line oxygenation using O2 at 50 L*min-1 (pressure = 4 psi). From the final wort volume (1200 L), 12 x 10 L aliquots were added into sterile 12 L fermenters. Each wort (10 L) was inoculated with one of the 12 propagated yeasts to achieve a target pitch rate of 1 x 107 viable cells*mL-1. Fermentation took place at 20 degC for 9 days until a constant gravity was achieved in all fermenters. The beer was then matured and clarified at 4 degC for 9 days prior to bottling. Beer samples were bottled into 330-mL brown glass bottles with the addition of 1.5 g sucrose to carbonate the beer to 2.0 volumes CO2 by holding the samples in an incubator at 20 degC for 14 days. 2.2. Yeast Preparation Yeast cultures were propagated based on the origin of each yeast strain (Table 1), before calculating the required yeast volumes to the fermenter for each yeast strain to achieve the target pitch rate. 2.2.1. Rehydration from Dry Yeast To initially determine cell density per gram, 1 g of each dry yeast was rehydrated in filtered water (100 mL) (boiled and cooled to room temperature) and stirred for 10 min using a magnetic stirrer. Cell viability (initial cell count) was estimated to determine the amount of yeast suspension required to obtain 1 x 107 cells*mL-1 (for a 10 L ferment). For final pitching, dry yeast (11.5 g) was added to a malt solution (10degP, 1 L), incubated for 24-48 h at 20 degC, and revived 48 h prior to yeast enumeration and pitching (Section 2.2.5). 2.2.2. Propagation of Cultures from Agar Slopes (Otago Culture Collection) Yeast strains on agar slopes in the Otago culture collection required several propagation steps to obtain sufficient numbers for the target pitch rate. Yeasts recovered from agar slopes were inoculated into duplicate 10-mL autoclaved malt solution (10% w/v) in 20-mL Universal vials using a sterile loop and incubated for 24 h at 20 degC. These cultures (two x 10-mL volumes) were added to 180 mL of sterile malt medium (10% w/v) in a 500-mL Schott bottle and propagated for 24-48 h at 20 degC. The resulting yeast suspension was added to 2800 mL of autoclaved malt medium (10% w/v; 10degP) in a 5-L conical flask. The starter culture was stirred for 24-48 h at 20 degC with a magnetic stir bar, prior to yeast enumeration and pitching (Section 2.2.5). 2.2.3. Commercial Slurry Fresh liquid yeast slurry (250 mL) was provided by Emerson's from their yeast propagation tanks. The slurry was added into 1 L (in a 3-L conical flask) of fresh brewery wort at 10degP provided by the brewery and stirred for 24-48 h at 20 degC with a magnetic stir bar, prior to yeast enumeration and pitching (Section 2.2.5). 2.2.4. Commercial Yeast Slurry A vial of White Labs WLP-001 California Ale(r) yeast was purchased commercially and propagated by adding the vial to 1 L (in a 3-L conical flask) of fresh brewery wort (10degP, 1 L) and stirring for 24-48 h at 20 degC with a magnetic stir bar prior to yeast enumeration and pitching (Section 2.2.5). 2.2.5. Yeast Pitching Approximately 24 h prior to pitching, each propagated yeast culture was centrifuged (3000 rpm for 10 min at 20 degC) in 1-L bottles (Nalgene 3120-1000 Centrifuge bottle), the supernatant was discarded, Emerson's fresh brewery wort (10degP, 200 mL) was added to the slurry, and the suspension was agitated (200 rpm, 60 min) to resuspend the yeast. Cell numbers were estimated, and the volume of yeast slurry required to achieve the target inoculation rate of 1 x 107 viable cells /mL in 10 L wort was calculated. The number of yeast cells in each starter culture (2.2.1-2.2.4) was estimated using an Oculyze BB 1.0 (Oculyze GmbH, Hochschulring, Germany) with methylene blue (MB) as a stain (1:1 ratio). A microscopic slide (200-mL sample chamber; Grafelfing, Germany) was prepared and analysed under 400x magnification to calculate cell numbers, budding cell values, and culture viability using the cloud-based platform . Five pictures were taken of the most appropriate dilution, and the percentage cell viability (>90% was obtained) and mean yeast numbers per mL (million cells per mL) were estimated . 2.3. Temperature of Fermentation S. cerevisiae was the most common (8/12) yeast used in this study (Table 1), along with one S. cerevisiae hybrid, two S. pastorianus strains, and one S. bayanus strain. The S. cerevisiae stains typically produce an ale-style beer; S. pastorianus and S. bayanus primarily produce lager-style beers . The use of different yeast strains posed a question of which temperature should be used for fermentation, as this would likely impact on yeast growth rates, fermentation time, and VOC production . Despite lager yeast strains being typically fermented at 8-15 degC and ale yeast strains at 14-20 degC, it was decided to remove temperature as an experimental variable and carry out all fermentations at 20 degC. 2.4. Analysis of Beer Samples Using Free Sorting Sensory Methodology Prior to study commencement, ethics was approved by the University of Otago Human Ethics Committee (Reference 18/154). A total of 14 panelists completed the free sorting task, with eight from the University of Otago Department of Food Science sensory panel, four brewing professionals/expert beer tasters from Emerson's Brewery, and two Department of Food Science postgraduate students. The free sorting task was completed over five sessions of 2 h each with two initial training sessions used to familiarise the panelists with the sensory space of the beer samples and the free sorting task itself, followed by three formal evaluation sessions. Session one included a taste identification test using five sample solutions (sucrose, citric acid, caffeine, sodium chloride, and alum) followed by a descriptive test where four beer samples from the twelve experimental samples were evaluated. The beers were presented in pairs, and panelists were asked to comment on the sample's aroma, appearance, flavour, and mouthfeel, as well as their overall impression of the difference between the two beers. In session two, the sorting task protocol was explained, and a mock sorting task was carried out to familiarise the panelists with the sorting task methodology using six of the twelve beer samples. The formal evaluation sessions were completed during the remaining three sessions. In each evaluation session, the panelists received a tray of all twelve beer samples presented in a balanced order according to a Williams Latin Square design. Beer samples (40 mL) were served at 10 degC +- 2 degC in 200-mL lidded plastic cups identified with random 3-digit codes. The panelists were instructed to smell and taste the samples in the order presented and sort them into groups based on their similarities of sensory attributes. The panelists were instructed to sort the samples into any number of groups, provided that a minimum of two groups and a maximum of 11 groups were formed. A group could contain up to 11 samples if preferred, and a panelist could choose any criteria (sensory attributes) to sort the samples. Panelists also separately recorded individual descriptions on each sample. Retasting was allowed for confirmation of groupings. Once the samples were sorted into groups, the panelists were asked to record the characteristic sensory attributes of each group. The groups and sensory attributes were recorded using Compusense Cloud (Compusense Inc., Guelph, ON, Canada) on Apple iPads (Apple Inc., Cupertino, CA, USA). Filtered water, plain crackers, and sliced carrots were provided as palate cleansers between samples. Maximum alcohol consumption for a panelist at each evaluation session was equivalent to a maximum of 1.50 standard drinks. Panelists were provided with food after each session. 2.5. Analysis of Beer VOC Using Headspace Solid Phase Microextraction and Gas Chromatography Mass Spectrometry The VOC profiles of the beer samples were measured using gas chromatography mass spectrometry (GC/MS) coupled with headspace solid-phase microextraction (HS-SPME). Aliquots of each beer (8 mL) were combined with 2.5 g analytical grade sodium chloride (NaCl; BDH Laboratory Supplies, England) in 20-mL headspace vials and capped with PTFE-lined silicon septa screw caps. Blank samples were prepared with deionised water (8 mL) and NaCl (2.5 g). Each sample was incubated at 40 degC for 5 min with agitation, followed by SPME extraction for 30 min at 40 degC using a multipurpose sampler (Agilent PAL3 RSI 85 Autosampler; Palo Alto, CA, USA). Analysis was completed with an Agilent 6890 N gas chromatograph connected to an Agilent 5975 VL mass spectrometer (MSD) with triple axis detector (Agilent Technologies, USA). Helium was used as the carrier gas in constant flow mode at a rate of 1.0 mL*min-1. Separation of analytes was achieved using a Zebron ZB-Wax column (60 m, 0.32-mm i.d., 0.5-mm film thickness; Phenomenex, Torrance, CA, USA). Samples were desorbed in the inlet at 240 degC for 5 min in the splitless mode. The initial oven temperature was 50 degC for 5 min, then heated at 5 degC.min-1 to 210 degC, followed by 10 degC.min-1 to 240 degC, and held for 5 min. The MSD was operated in electron impact (EI) ionisation mode at 70 eV with an ion source temperature of 230 degC with a scan range of m/z 29-300. Analyses were completed in quadruplicate (samples, n = 12; blanks, n = 4). To prevent order effects, samples were analysed according to a modified Williams Latin Square design. 2.6. Data Analysis 2.6.1. Analysis of Sensory Data The sorting task data, groups, and sensory attributes were exported from Compusense for all evaluation sessions. Due to the free-sort methodology enabling panelists to use their own vocabulary, textual preprocessing was required. This included correcting spelling, standardising word endings, combining synonyms, and selecting key words . Multiple researchers confirmed synonymy of attributes and final selection of key words, with any terms used in less than three groups removed from further analysis. The data were initially analysed using the Factorial Approach for Sorting Task data (FAST) , which applies multiple correspondence analysis (MCA) to the group and attributes data and projects the data onto a two-dimensional map of the samples and attributes and evaluates significant associations between the samples and attributes. A contingency table of the attributes was generated (Appendix A, Table A1) and used to evaluate the relationship between the sensory and analytical data with the application of multiple factor analysis for contingency tables (MFACT) . 2.6.2. Analysis of GC-MS Data Exported GC-MS data was analysed using PARADISe (PARAFAC2 based Deconvolution and Identification System), version 3.87 . This resulted in a table of relative peak area for each detected compound for all samples. Retention indices (RIs) were determined using cubic spline interpolation after running a C9-C30 saturated alkane standard using the same GC temperature program. VOCs were regarded as "unknown" if the mass-spectra match value was below 700 or the calculated RI did not match the reported RI. The relative peak area table was analysed by one-way analysis of variance (ANOVA) with a level of confidence of 95% (Appendix A, Table A2) followed by Tukey post-hoc testing to identify significant groupings. 2.6.3. Analysis of the Relationship between the Sensory and GC-MS Data Due to the ability of multiple factor analysis (MFA) to analyse multiple data sets of variables collected from the same set of samples, it is particularly useful when investigating the relationship between different experimental measures . MFA produces sample and attribute projections that represent the similarities between the samples and between the different data sets of variables. The extension of MFA to include contingency tables (MFACT) allows for the investigation of relationships between the sensory characterisation completed using the free-sort method and volatile analysis using GC-MS. Due to the use of contingency data for the sensory attributes, means of the four replicates of each sample were calculated for the volatile data, which were unit scaled as part of the MFACT analysis. Only the VOCs determined to be significantly different across the beer samples were included in the MFACT. All data analysis was completed using R version 3.5.3 and the RStudio IDE with the tidyverse suite of packages , plus additional packages agricolae , SensoMineR , and FactoMineR . 3. Results and Discussion 3.1. Summary of Beer Groups Formed In the sorting task, panelists created between three and ten groups from the twelve beer samples with four, five, and six groups being most common. The groups most frequently contained two beers , with one sample per group being the next most frequent, showing that the sensory attributes of the beers were being perceived by the panelists as being distinctly different. 3.2. Representation of Beers and Sensory Attributes A total of 96 distinct attributes were generated by the panelists, which were refined through textual processing to 41. Hoppy was the most commonly used attribute to describe the twelve beers, followed by fruity, sulfury, bitter, floral, citrus, green/grassy, spicy, sweet, and honey . From the FAST analysis, hoppy was significantly (p < 0.05) associated with beer W3470 (Table 2) while sulfury was significantly (p < 0.05) associated with samples WY1272 and OTA79. WY1272 was also significantly associated with metallic. WB06 was significantly associated with the terms spicy and estery while WLP730 was significantly associated with spicy. The use of sour was significantly associated with the VIN13 beer sample while astringent was significantly associated with the WLP001 beer. No terms were significantly associated with US05, BE256, S23, OTA29, or SPH. 3.3. Representation of Similarity Co-Occurrence Matrix of Beers The co-occurrence matrix reflects the perceived similarity of the different beers (Table 3). The most similar beers were WB06 and WLP730, associated together 17 times (40.5%); WLP730 and SPH, associated 15 times (35.7%); WB06 and SPH, associated 13 times (31.0%); S23 and WY1272, associated 13 times (31.0%); and US05 was associated with WY1272 and OTA29 12 times each (28.6%). The least similar beers were OTA79 and WLP730, which were not grouped together at all. Samples were grouped alone a total of 75 times, with each sample grouped alone between three and nine times (Table 3). BE256 and VIN13 were most frequently grouped alone nine times (21.4%) while S23 was alone only three times (7.1%), indicating it was perceived as more similar to the other samples. 3.4. Relationship of Beer VOCs with Sensory Attributes The twelve beers were projected similarly in the FAST and MFACT, so to prevent duplication, only the MFACT is presented to illustrate the relationships between the twelve beers and their sensory attributes and VOC profile . Factors 1 (21.82%), 2 (14.32%), and 3 (12.50%) in the MFACT show that the beers were distributed in all dimensions with a total explained variance for these three factors of 48.64%. Factor 1 separated beers WB06, OTA29, SPH, and WLP730 from the other beers . This placement was consistent with the co-occurrence similarity matrix where beer WLP730 was grouped 17 times with WB06 and 15 times with SPH, and WB06 and SPH were placed together 13 times (Table 3). The sensory attributes with the highest F1 positive loadings were spicy, corn, phenolic, and estery, while those with the highest F1 negative loadings were bitter, citrus, hoppy, and tropical fruit. This has been reflected in the contingency table (Appendix A, Table A1) where spicy had the highest frequency of use with WB06, WLP730, and SPH; corn and phenolic were associated more frequently with these four beers than any of the others, and estery was most frequently used with WB06. The FAST analysis also reflected this, where spicy was significantly associated with WLP730 and WB06, which was also significantly associated with estery (Table 2). In contrast, citrus and tropical fruit were never used to describe WB06, and although hoppy was used to describe all beers, it was used most frequently with, and was significantly associated to, W3470 while it was used least frequently with WB06. The VOCs with the highest positive loadings on F1 were 4-vinylguaiacol (172; spicy, clove, phenolic), hexanoic acid (149; cheesy), dimethyl sulfide (2; sulfurous, onion, sweet corn, vegetable), 1-propanol (12; alcoholic, earthy, fermented), ethyl hexanoate (36; sweet, fruity, banana, estery), and propyl hexanoate (54; fruity) . In WLP730, OTA29, SPH, and WB06 beers , 4-vinylguaiacol was significantly higher. It is produced from malt-derived ferulic acid by heat and/or enzyme decarboxylation, with most conversion (60-90%) attributed to yeast activity. However, not all yeast strains expressed the POF+ (phenolic off-flavour) gene that allowed synthesis of 4-vinylguaiacol from ferulic acid . Hence, the other eight beers only contained trace amounts of 4-vinylguaiacol, as the POF+ gene is not present in the majority of commercial ale and lager yeast strains. The volatile analysis showed that ethyl hexanoate (36) was significantly higher in beers OTA29 and WB06 . The WB06 beer also showed the highest abundance of propyl hexanoate (54), which corresponds to the literature where WB06 is known to produce estery, fruity, and phenolic flavours . On F1, there was good agreement between those VOCs with large positive loadings and the sensory attributes used to describe the beers. For example, spicy and phenolic attributes were associated with higher abundance of 4-vinylguaiacol, estery was associated with ethyl hexanoate and other esters, and higher levels of DMS were associated with a corn attribute. The VOCs with the largest negative loadings on F1 were nerol (142; lemon, fruity), geraniol (147; floral, rose, fruity, citrus), ethyl dihydrocinnamate (153; rose, honey, fruity), b-ocimene (38; green, tropical, floral), citral (134; citrus, green, herbal), and citronellol (138; floral, fruity, citrus) . This reflects the sensory attributes that were also negatively loaded on F1 (bitter, citrus, hoppy, and tropical fruit) and with the citrusy and resinous characteristics consistent with higher levels of the terpene compounds. For example, geraniol (147) was significantly higher in WLP001 and US05 compared to WB06, OTA29, SPH, and WLP730 , and a similar pattern was found with nerol being lowest in WLP730 and OTA29 . The separation of beers on F2 was largely due to WLP001, US05, and WB06 with negative loadings and OTA29, OTA79, and WY1272 with positive loadings . The main drivers in sensory characteristics were the significant association of astringency with WLP001 while OTA79 and WY1271 were significantly associated with sulfury (Table 2). Other negatively loaded sensory attributes included estery, acidic, and alcoholic while hoppy, musty, wine-like, and lemony were positively loaded. The most important positively loaded VOCs on F2 were 2-ethylhexyl acetate (67; earthy, herbal, dirty), octyl acetate (84; green, earthy, citrus), hexyl acetate (42; fruity, green apple), (E)-3-hexen-1-ol acetate (53; green, fruity, unripe banana, earthy), geranyl acetate (137; floral, green, citrus, winey), heptyl acetate (64; green, fruity, citrus), nerol acetate (129; floral, fruity, citrus, tropical), 2-phenylethyl acetate (146; floral, rose, honey), and 3-methylbutyl acetate (isoamyl acetate; 21; sweet, fruity, banana). Acetate esters are important flavour compounds in beer and are often present at relatively high concentrations . The presence of these VOCs relates to the positively loaded sensory attributes and relative positioning of the WLP001 and OTA29 beers, which had the lowest and highest abundance of acetate esters, respectively, as illustrated by the abundance isoamyl acetate and 2-phenylethyl acetate . Factor 3 separated beer BE256 from beers WY1272 and WLP730 . This separation was due to the sensory attributes metallic and astringent (negative loading) followed by flat, malty, and rancid (positive loadings) . The FAST analysis identified that metallic was significantly associated with WY1272, which is driving this separation. The VOCs loaded negatively on F3 were 2-methyl-3-heptanone (24; fruity, green, leafy), 4-methyl-2-pentanone (8; solvent, green, fruity, dairy), 2-methylbutyl 2-methylpropanoate (32; fruity, tropical, banana), 2-nonanol (91; waxy, musty, fruity), and 6-methyl-5-hepten-2-one (57; green, musty fruity). In contrast, butyl 9-decenoate (156), 3-methylbutyl octanoate (116; sweet, fruity, green), 2-methylpropyl octanoate (97; fruity, green, floral), and ethyl nonanoate (94; fruity, waxy, tropical) had positive loadings. 3.5. Terpenoid Compounds Present in Beers The primary aim of this study was to evaluate the impact of yeast strain on hop flavour characteristics in beer and to gain an understanding of what VOC in the beers may be responsible for these differences. The relative abundance of the monoterpene alcohols and monoterpene esters in the twelve beers was investigated to see whether any trends existed across the twelve yeast strains. Hop terpenoids form part of the essential oil component found in hops that contributes aroma-active compounds to beer. Hop terpenoids in beer originate from hops added during the brewing process, which may be modified by biotransformation reactions by yeast during fermentation . The monoterpene alcohols geraniol (147), nerol (142), citronellol (138), and linalool (95) are important aroma-active terpenoids found in beer. Geraniol was significantly higher in WLP001 and US05 compared to WB06, OTA29, SPH, and WLP730 beers . The highest nerol concentration was found in the WLP001, WY1272, and US05 beers, and the lowest concentration occurred in the WLP730 and OTA29 beers . Nerol contributed a rose-like flavour, and geraniol contributed a rose-like, floral, and citrusy flavour in beer . Although the abundance of geraniol and nerol tended to be highest in the ale S. cerevisiae yeast strains, WB06 (S. cerevisiae yeast strain) had the fourth lowest abundance of geraniol and nerol, with two S. pastorianus strains producing beers with higher levels. King and Dickinson suggested that the extent of monoterpene transformation is strain specific, rather than related to either S. cerevisiae or S. pastorianus strains. Citronellol is a yeast biotransformation product of geraniol typically found at low levels in hop essential oil, but at much greater abundance in beer. The abundance of citronellol followed a similar pattern to nerol . Linalool, one of the most frequently occurring and abundant terpene alcohols in hops, is a product of the oxidation of myrcene, and it is found in hop essential oil. Linalool has been identified as an important odour-active compound in lager beer . In the current study, the level of linalool did not significantly differ between the twelve beer samples. This means that although it may have contributed to a generic hop flavour, it did not appear to be an important compound in yeast-specific biotransformations, at least in beers late-hopped with New Zealand Motueka hops. Citronellol acetate (117) was found at the highest abundance in WY1272, significantly higher than all other beer samples other than OTA79 , showing a different pattern compared to citronellol. Nerol acetate and geranyl acetate illustrated similar patterns, with the highest abundance in WY1272 and OTA79. Citronellol acetate was reported to contribute a floral, fruity, pear, and apple character, and nerol acetate contributed a floral and green flavour to beer . Citronellol acetate is not naturally found in hops, which suggests the production of terpene esters during fermentation by yeast esterase activity. Citronellol acetate is formed either by acetylation of citronellol, after reduction from geraniol, or by reduction from geranyl acetate . It is known that monoterpene acetate esters of terpenes are formed in beer during fermentation and are particularly expressed in high concentration in beers that are late-hopped. King and Dickinson postulated that terpenoid ester formation occurs with lager yeast strains but not in ale strains, although only two strains for each of S. cerevisiae and S. bayanus were investigated for that study. In contrast, the current experiment, supported by a study conducted by Richter, Eyres, Silcock, and Bremer , showed that ale yeast strains can form higher amounts of citronellol acetate, geranyl acetate, and nerol acetate than the selected S. bayanus strains. It was also observed that the beers with high levels of the yeast-derived ethyl acetate and higher alcohol acetate esters (e.g., isoamyl acetate, 2-phenylethyl acetate) did not necessarily have the highest levels of terpene acetate esters. This is likely because the formation of the terpene acetate esters requires the presence of abundant monoterpene alcohols, in addition to alcohol acetyltransferase (AATase) activity and acetyl CoA. In contrast, US05 and WLP001, which had amongst the highest levels of geraniol and nerol but the lowest levels of the higher alcohol acetate esters, had the lowest levels of citronellol acetate and nerol acetate. This is probably due to reduced AATase activity rather than reduced acetyl-CoA availability, as yeast growth was not excessive . 4. Conclusions The sensory characteristics and VOC profiles of beers fermented by twelve different yeast strains under the same conditions differed considerably, with links being evident between the presence of specific volatile compounds and perceived sensory attributes. Beers high in 4-vinylguaiacol were perceived to be spicy and clove-like, the abundance of dimethyl sulfide was associated with a corn character, and beers with higher concentrations of acetate and ethyl esters were perceived to be fruity and estery. Biotransformation of terpenes involved a complex series of reaction pathways, which led to distinct patterns of hop-derived terpenoid compounds in the twelve beers as a function of yeast strain. The levels of monoterpene alcohols (geraniol, nerol, and citronellol) varied across the twelve samples, although there were no differences in the abundance of linalool. The terpene acetate esters detected varied between the twelve beers, but their occurrence did not correlate with the abundance of fermentation esters or monoterpene alcohols. To understand the generation of these biotransformation products in beer, further studies need to be conducted under model conditions where the precursors can be closely controlled, to further understand the biosynthetic reactions. Finally, understanding how yeast strain and fermentation factors influence hop aroma in beer will help the brewing industry to better understand how to control beer flavour to meet consumer demands. Acknowledgments This research was conducted as part of the MSc thesis of A.K. . The authors thank Emerson's Brewery for the JP Dufour Scholarship and Jamie Scrimgeour for supporting the research (2018-2019), as well as the Department of Food Science technicians, Michelle Leus, Michelle Petrie, and Ian Ross, for technical assistance. Author Contributions Conceptualisation, G.T.E., P.S., and P.J.B.; methodology, A.K., A.W., G.T.E., P.S., and P.J.B.; formal analysis, A.K., A.W., P.S., and G.T.E.; investigation, A.K.; resources, G.T.E., P.S., and P.J.B.; data curation, A.K. and A.W.; writing--original draft preparation, A.K. and A.W.; writing--review and editing, G.T.E., P.S., P.J.B., and A.W.; visualisation, A.K., G.T.E., P.S., and A.W.; supervision, G.T.E., P.S., and P.J.B.; project administration, G.T.E.; funding acquisition, G.T.E., P.S., and P.J.B. With the exception of A.K. All authors have read and agreed to the published version of the manuscript. Data Availability Statement The data presented in this study are available on request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Appendix A foods-12-01064-t0A1_Table A1 Table A1 Frequency of use of distinct descriptors with twelve beer samples. Attribute US 05 WLP 001 W 3470 S 23 WB 06 WLP 730 OTA 29 OTA 79 WY 1272 VIN 13 BE 256 SPH Total Hoppy 7 6 12 3 2 3 8 11 7 6 6 4 75 Fruity 5 2 6 6 3 5 5 4 8 4 4 2 54 Sulfury 4 1 2 5 3 1 2 12 9 6 4 2 51 Bitter 3 8 7 7 2 3 1 5 5 1 5 2 49 Floral 6 5 2 3 5 3 6 2 3 4 1 4 44 Citrus 5 8 1 6 0 2 2 2 3 6 6 2 43 Green/ Grassy 2 3 4 5 2 2 7 0 6 0 2 6 39 Spicy 1 4 0 1 7 8 2 2 0 1 0 6 32 Sweet 5 2 0 3 5 1 3 2 1 4 3 2 31 Honey 4 3 2 6 2 1 1 1 3 4 2 1 30 Sour 0 2 4 0 3 3 0 2 4 6 1 2 27 Phenolic 2 1 1 2 4 5 4 1 0 2 1 4 27 Woody 2 3 1 2 1 2 4 1 2 2 1 2 23 Estery 3 3 0 1 6 4 2 0 2 0 0 2 23 Malty 1 0 3 4 1 1 1 1 1 0 3 3 19 Lemony 0 0 4 1 1 3 0 1 2 4 1 1 18 Yeasty 2 1 1 0 3 0 1 4 0 2 1 2 17 Solvent-Like 3 2 2 0 2 1 1 2 0 1 2 0 16 Acidic 2 2 3 1 3 2 0 0 1 1 0 1 16 Fragrant 1 1 2 0 2 3 2 1 0 0 2 1 15 Creamy 2 2 0 0 1 1 2 2 1 0 0 3 14 Banana 3 2 0 1 1 0 2 1 1 0 0 3 14 Apple/Pear 1 1 0 2 1 1 1 1 1 3 1 1 14 Caramel 2 2 0 0 0 0 2 2 0 3 2 1 14 Corn 0 1 0 0 3 3 2 0 0 1 1 3 14 Tropical Fruit 1 1 2 3 0 0 1 0 1 2 2 0 13 Light 1 0 1 2 0 2 0 0 2 2 3 0 13 Alcoholic 1 2 3 1 1 1 1 1 0 0 0 1 12 Wine-Like 2 0 1 0 0 0 2 1 2 2 2 0 12 Resinous 0 3 2 1 0 1 1 2 0 0 2 0 12 Flat 0 0 1 3 0 0 1 1 0 0 2 2 10 Grainy 0 1 0 1 1 2 0 0 1 0 2 2 10 Cooked Vegetable 1 1 1 0 1 1 0 1 0 0 3 1 10 Characterless 0 0 1 2 0 0 1 0 1 1 2 1 9 Soapy 1 1 0 1 0 1 1 0 1 2 0 1 9 Rancid 1 0 1 0 1 0 0 0 0 1 2 2 8 Astringent 0 3 0 0 1 2 0 0 0 0 1 0 7 Musty 1 0 0 0 0 0 1 2 1 0 2 0 7 Metallic 1 1 0 0 0 1 0 0 3 1 0 0 7 Stale 0 0 0 0 1 0 0 1 0 1 1 0 4 Burnt 0 0 0 0 1 0 0 0 0 1 1 0 3 Total 76 78 70 73 70 69 70 70 72 74 74 70 866 foods-12-01064-t0A2_Table A2 Table A2 Volatile organic compounds identified in twelve beer samples. No. Volatile Compound RI (calc) RI (lit) Match CAS No. p-Value 1 acetaldehyde 713 702 852 75-07-0 0.000 2 dimethyl sulfide 760 754 948 75-18-3 0.000 3 ethyl acetate 896 888 944 141-78-6 0.000 4 ethanol 937 932 935 64-17-5 0.296 5 ethyl propanoate 963 953 793 105-37-3 0.000 6 ethyl 2-methylpropanoate 972 961 780 97-62-1 0.000 7 n-propyl acetate 981 973 942 109-60-4 0.000 8 4-methyl-2-pentanone 1014 1010 891 108-10-1 0.016 9 2-methylpropyl acetate 1019 1012 952 110-19-0 0.000 10 3-methyl-2-pentanone 1025 1019 731 565-61-7 0.000 11 a-pinene 1031 1028 840 80-56-8 0.251 12 1-propanol 1038 1036 938 71-23-8 0.000 13 ethyl butanoate 1042 1035 941 105-54-4 0.000 14 5-methyl-3-hexanone 1082 1036 746 623-56-3 0.334 15 dimethyl disulfide 1083 1077 738 624-92-0 0.000 16 2-methylpropyl propanoate 1085 1079 708 540-42-1 0.002 17 2-methyl-1-propanol 1088 1092 949 78-83-1 0.000 18 2-methylpropyl 2-methylpropanoate 1094 1090 940 97-85-8 0.000 19 1-(1-ethoxyethoxy)-pentane 1107 1098 924 13442-89-2 0.206 20 b-pinene 1115 1112 741 127-91-3 0.020 21 3-methyl-1-butyl acetate 1126 1122 951 123-92-2 0.000 22 ethyl pentanoate 1138 1134 877 539-82-2 0.050 23 5-methyl-2-hexanone 1145 1156 873 110-12-3 0.000 24 2-methyl-3-heptanone 1154 1179 844 13019-20-0 0.001 25 b-myrcene 1167 1161 959 123-35-3 0.079 26 a-phellandrene 1167 1167 863 99-83-2 0.105 27 2-methylpropyl 2-methylbutanoate 1178 1179 827 2445-67-2 0.026 28 2-heptanone 1186 1182 871 110-43-0 0.000 29 a-terpinene 1188 1180 897 99-86-5 0.346 30 2-methylbutyl propanoate 1191 1197 907 2438-20-2 0.000 31 ethyl 4-methylpentanoate 1192 1190 755 25415-67-2 0.000 32 2-methylbutyl 2-methylpropanoate 1197 1199 976 2445-69-4 0.006 33 2-methyl-1-butanol 1201 1208 945 137-32-6 0.000 34 3-methyl-1-butanol 1203 1209 960 123-51-3 0.254 35 b-phellandrene 1218 1211 920 555-10-2 0.940 36 ethyl hexanoate 1236 1233 978 123-66-0 0.000 37 (2R,5R)-2-methyl-5-(prop-1-en-2-yl)-2-vinyltetrahydrofuran 1248 1243 779 54750-70-8 0.164 38 b-ocimene 1255 1250 883 13877-91-3 0.001 39 unknown 1259 - - - 0.000 40 styrene 1267 1261 962 100-42-5 0.000 41 4-pentenyl butanoate 1271 - 726 30563-31-6 0.004 42 hexyl acetate 1274 1272 923 142-92-7 0.000 43 bicyclo [4.2.0]oct-1-ene, 7-exo- - 836 - 0.579 44 ethyl 5-hexenoate 1280 1271 761 - 0.000 45 2-methylbutyl 2-methylbutanoate 1282 1284 935 2445-78-5 0.752 46 ethyl 5-methylhexanoate 1288 - 922 10236-10-9 0.000 47 a-terpinolene 1292 1283 873 586-62-9 0.137 48 1,2,4-trimethyl-benzene 1293 1282 709 95-63-6 0.000 49 ethyl (Z)-3-hexenoate 1293 1292 752 64187-83-3 0.000 50 2-methylbutyl 3-methylbutanoate 1297 1299 825 2445-77-4 0.285 51 ethyl (E)-3-hexenoate 1302 1289 912 26553-46-8 0.018 52 unknown ester 1316 - - - 0.198 53 (E)-3-hexenyl acetate 1319 1306 769 3681-82-1 0.000 54 propyl hexanoate 1320 1316 762 626-77-7 0.000 55 ethyl heptanoate 1335 1331 943 106-30-9 0.000 56 methyl 4-methylenehexanoate 1340 1345 817 73805-48-8 0.000 57 6-methyl-5-hepten-2-one 1342 1338 916 110-93-0 0.001 58 1-hexanol 1346 1355 909 111-27-3 0.355 59 ethyl 2-hexenoate 1349 1340 821 1552-67-6 0.000 60 unknown 1351 - - - 0.000 61 2-methylpropyl hexanoate 1354 1350 754 105-79-3 0.000 62 rose oxide 1360 1350 895 16409-43-1 0.000 63 3-ethoxy-1-propanol 1373 1373 853 111-35-3 0.000 64 heptyl acetate 1374 1377 905 112-06-1 0.000 65 verbenyl ethyl ether 1376 1377 722 80581-06-2 0.992 66 hop ether 1380 1360 900 344294-72-0 0.032 67 2-ethylhexyl acetate 1384 1420 895 103-09-3 0.000 68 unknown 1387 - - - 0.001 69 unknown 1391 - 784 - 0.006 70 2-nonanone 1394 1390 943 821-55-6 0.000 71 2-isobutenyl-4-vinyl-tetrahydrofuran 1400 - 732 - 0.146 72 (E)-4-hexenyl butanoate 1404 1478 749 - 0.000 73 1,3-dimethyl-1-cyclohexene 1405 - 787 2808-76-6 0.000 74 2-octanol 1410 1412 777 123-96-6 0.000 75 3-(4-methyl-3-pentenyl)-furan 1425 1429 911 539-52-6 0.225 76 ethyl octanoate 1439 1435 941 106-32-1 0.000 77 1-octen-3-ol 1442 1450 768 3391-86-4 0.976 78 (Z)-linalool oxide 1446 1444 800 5989-33-3 0.928 79 1-heptanol 1448 1453 878 111-70-6 0.000 80 6-methyl-5-hepten-2-ol 1455 1465 774 1569-60-4 0.064 81 3-methylbutyl hexanoate 1461 1451 884 2198-61-0 0.000 82 acetic acid 1464 1449 938 64-19-7 0.000 83 3-(2,6,6-trimethyl-1-cyclohexen-1-yl)-2-propenal 1465 - 744 4951-40-0 0.000 84 octyl acetate 1476 1474 903 112-14-1 0.000 85 4-tert-pentylcyclohexene 1477 - 717 51874-62-5 0.000 86 nerol oxide 1479 1469 827 1786-08-9 0.556 87 ethyl 7-octenoate 1487 1478 925 35194-38-8 0.000 88 2-decanone 1499 1494 706 693-54-9 0.000 89 unknown 1502 - 704 - 0.001 90 decanal 1505 1498 748 112-31-2 0.000 91 2-nonanol 1510 1521 822 628-99-9 0.000 92 2-acetylfuran 1517 1499 755 1192-62-7 0.008 93 propyl octanoate 1521 1510 935 624-13-5 0.000 94 ethyl nonanoate 1537 1531 914 123-29-5 0.009 95 linalool 1540 1547 975 78-70-6 0.820 96 1-octanol 1550 1557 932 111-87-5 0.000 97 2-methylpropyl octanoate 1553 1548 944 5461-06-3 0.000 98 (E)-p-2-menthen-1-ol 1567 1571 902 29803-81-4 0.213 99 ethyl 3-nonenoate 1570 - 832 91213-30-8 0.068 100 methyl 4,4-dimethyl-3-oxopentanoate 1577 - 744 - 0.828 101 isopulegol 1578 1571 805 89-79-2 0.669 102 2-methylpropanoic acid 1582 1570 910 79-31-2 0.000 103 fenchol 1589 1582 846 1632-73-1 0.776 104 myrcenol 1603 1585 867 543-39-5 0.758 105 2-undecanone 1604 1598 781 112-12-9 0.000 106 2-decanol 1609 1601 764 1120-06-5 0.016 107 terpinen-4-ol 1611 1602 911 562-74-3 0.953 108 cis-verbenol 1613 1663 781 1845-30-3 0.501 109 caryophyllene 1624 1595 827 87-44-5 0.155 110 citronellol formate 1627 1660 732 105-85-1 0.000 111 (Z)-p-2-menthen-1-ol 1631 1638 901 29803-82-5 0.440 112 ethyl decanoate 1641 1638 946 110-38-3 0.000 113 2,6-dimethyl-5-hepten-1-ol 1647 1654 730 4234-93-9 0.022 114 4-(1-methylethyl)-cyclohexanol 1649 1667 879 4621-04-9 0.003 115 1-nonanol 1652 1660 875 143-08-8 0.000 116 3-methylbutyl octanoate 1660 1658 920 2035-99-6 0.001 117 citronellol acetate 1663 1660 948 150-84-5 0.000 118 ethyl trans-4-decenoate 1668 1676 891 76649-16-6 0.062 119 ipsdienol 1674 1631 876 35628-00-3 0.025 120 decyl acetate 1681 1680 868 112-17-4 0.000 121 3-methylbutanoic acid 1683 1666 766 503-74-2 0.874 122 ethyl 9-decenoate 1694 1694 917 67233-91-4 0.000 123 humulene 1698 1667 905 6753-98-6 0.411 124 methyl geranate 1703 1686 892 2349-14-6 0.403 125 2-undecanol 1709 1717 861 1653-30-1 0.431 126 endo-borneol 1713 1702 917 507-70-0 0.986 127 3-(methylthio)-1-propanol 1722 1719 903 505-10-2 0.000 128 propyl decanoate 1724 1724 817 30673-60-0 0.000 129 nerol acetate 1728 1724 898 141-12-8 0.000 130 unknown 1734 - - - 0.001 131 unknown 1737 - - - 0.000 132 2,6-dimethyl-1,5,7-octatrien-3-ol 1740 - 886 29414-56-0 0.067 133 ethyl undecanoate 1741 1739 746 627-90-7 0.003 134 citral 1743 1718 764 5392-40-5 0.000 135 cis-piperitol 1750 1758 780 16721-38-3 0.557 136 1-decanol 1755 1760 879 112-30-1 0.000 137 geranyl acetate 1758 1752 911 105-87-3 0.000 138 citronellol 1760 1765 944 106-22-9 0.000 139 7-methyl-3-methylene-6-octen-1-ol 1784 1800 827 13066-51-8 0.000 140 methyl perillate 1789 - 772 26460-67-3 0.000 141 ethyl 10-undecenoate 1795 - 869 692-86-4 0.000 142 nerol 1798 1797 940 106-25-2 0.000 143 ethyl benzeneacetate 1799 1783 735 101-97-3 0.046 144 myrtenol 1802 1797 753 515-00-4 0.216 145 17-octadecynoic acid 1810 - 792 34450-18-5 0.000 146 2-phenylethyl acetate 1831 1813 787 103-45-7 0.000 147 geraniol 1842 1847 962 106-24-1 0.000 148 ethyl dodecanoate 1844 1841 951 106-33-2 0.000 149 hexanoic acid 1858 1846 977 142-62-1 0.001 150 3-methylbutyl pentadecanoate 1863 1863 827 2306-91-4 0.000 151 benzyl alcohol 1886 1870 754 100-51-6 0.014 152 (Z)-ethyl pentadec-9-enoate 1898 - 799 56219-09-1 0.000 153 ethyl dihydrocinnamate 1901 1893 884 2021-28-5 0.000 154 bicyclo [2.1.1]hexane-1-carboxylic acid, 5,5- - 756 3753-38-6 0.000 155 p-menth-1(7)-en-9-ol 1914 1889 836 29548-16-1 0.000 156 butyl 9-decenoate 1918 1874 731 0-00-0 0.001 157 phenylethyl alcohol 1924 1906 944 60-12-8 0.136 158 2-ethyl-hexanoic acid 1961 1960 852 149-57-5 0.923 159 heptanoic acid 1969 1950 722 111-14-8 0.255 160 b-phenylethyl butanoate 1980 1958 785 103-52-6 0.000 161 2-acetylpyrrole 1985 1973 728 1072-83-9 0.484 162 cis-1,3,5-trimethyl-cyclohexane 1996 - 664 1795-27-3 0.473 163 p-mentha-1,8-dien-7-ol 2016 2016 756 536-59-4 0.439 164 unknown acid 2028 - - - 0.328 165 (E)-nerolidol 2034 2042 922 40716-66-3 0.066 166 ethyl tetradecanoate 2048 2049 866 124-06-1 0.036 167 g-nonalactone 2056 2024 885 104-61-0 0.972 168 octanoic acid 2068 2060 930 124-07-2 0.000 169 nonanoic acid 2183 2171 746 112-05-0 0.000 170 2-phenylethyl hexanoate 2188 2162 716 6290-37-5 0.000 171 epicubebol 2190 2169 752 38230-60-3 0.307 172 4-vinylguaiacol 2214 2188 916 7786-61-0 0.000 173 ethyl hexadecanoate 2253 2251 790 628-97-7 0.062 174 ethyl 9-hexadecenoate 2283 2281 873 54546-22-4 0.218 175 n-decanoic acid 2287 2276 953 334-48-5 0.000 176 9-decenoic acid 2353 2341 931 14436-32-9 0.000 177 neric acid 2363 2366 751 4613-38-1 0.989 Figure 1 (a) Frequency of the number of groups formed by the panelists during the free sorting task (14 panelists x 3 sessions); (b) frequency of the number of beers sorted into each group; (c) frequency of the ten most commonly used attributes. Figure 2 Separation of twelve beer samples (a,c) with sensory attributes (blue triangles) and VOCs (red asterisks) (b,d) on the MFACT plot on Factors 1-3. Labelled VOC numbers correspond to important contributors; refer to Appendix A, Table A2 for VOC identification. Figure 3 Relative abundance (deconvoluted peak area) of VOC in twelve beer samples. (a) 4-vinylguaiacol, (b) ethyl hexanoate, (c) dimethyl sulfide, (d) geraniol, (e) nerol, (f) isoamyl acetate, (g) 2-phenylethyl acetate, (h) citronellol, (i) linalool, (j) citronellol acetate, (k) nerol acetate, and (l) geranyl acetate. Values are plotted as means +- standard deviation. In each graph, samples sharing the same letter are not significantly different at p < 0.05. Note the different y-axis values. foods-12-01064-t001_Table 1 Table 1 Yeast characteristics and pitching rates. Yeast Strain 1 Supplier Species Pitch Rate 2 Yeast Type SafAle US-05 (US05) Fermentis S. cerevisiae 1 x 107 cells*mL-1 Dry SafAle BE-256 (BE256) Fermentis S. cerevisiae 9.58 x 106 cells*mL-1,* Dry SafAle WB-06 (WB06) Fermentis S. cerevisiae 1 x 107 cells*mL-1 Dry SafLager W-34/70 (W3470) Fermentis S. pastorianus 1 x 107 cells*mL-1 Dry SafLager S-23 (S23) Fermentis S. pastorianus 1 x 107 cells*mL-1 Dry VIN13 hybridisedwine yeast (VIN13) Anchor Oenology S. cerevisiae hybrid 2.50 x 106 cells*mL-1,* Dry Exotics SPH wine yeast (SPH) Anchor Oenology S. cerevisiae 5.47 x 106 cells*mL-1,* Dry Chardonnay white wine yeast WLP730 White Labs S. cerevisiae 1 x 107 cells*mL-1 Agar slope OTA29 University of Otago yeast collection S. bayanus 1 x 107 cells*mL-1 Agar slope California ale WLP001 White Labs S. cerevisiae 4.14 x 106 cells*mL-1,* Liquid OTA79 University of Otago yeast collection S. cerevisiae 2.54 x 106 cells*mL-1,* Slurry American ale II strain 1272 (WY1272) Wyeast S. cerevisiae 3.18 x 106 cells*mL-1,* Slurry 1 All fermentations were held at 20 degC. 2 Target pitch rate was 1 x 107 cells*mL-1. * Under pitched yeast below the target pitch rate. foods-12-01064-t002_Table 2 Table 2 Significant association of beer samples to sensory attributes using FAST analysis. Beer Descriptor Intern (%) 1 Global (%) 2 p-Value W3470 Hoppy 16.67 8.66 0.03 OTA79 Sulfury 14.39 5.89 0.001 WY1272 Sulfury 12.33 5.89 0.04 Metallic 4.11 0.81 0.03 WLP730 Spicy 11.42 3.70 0.01 WB06 Spicy 10.14 3.70 0.02 Estery 8.70 2.66 0.01 VIN13 Sour 8.33 3.12 0.04 WLP001 Astringent 3.85 0.81 0.04 1 Frequency of use of the descriptor with the sample as a percentage of all descriptors used for the sample. 2 Frequency of use of the descriptor across all samples as a percentage of the total number of descriptors used across all samples and evaluations. foods-12-01064-t003_Table 3 Table 3 Co-occurrences of beer samples in the sorting task. Samples WY 1272 W3470 S23 BE256 OTA79 WLP 001 US05 VIN13 SPH OTA29 WLP30 WB06 Alone WY1272 7 13 10 8 6 12 11 3 10 5 2 5 W3470 7 10 6 10 3 11 3 6 8 6 3 5 S23 13 10 11 4 6 7 8 2 7 4 5 3 BE256 10 6 11 5 4 7 8 3 7 3 4 9 OTA79 8 10 4 5 6 6 8 5 10 0 5 8 WLP001 6 3 6 4 6 9 4 11 7 7 5 8 US05 12 11 7 7 6 9 1 5 12 5 4 4 VIN13 11 3 8 8 8 4 1 3 4 7 6 9 SPH 3 6 2 3 5 11 5 3 11 15 13 7 OTA29 10 8 8 7 10 7 12 4 11 7 10 5 WLP730 5 6 4 3 0 7 5 7 15 7 17 4 WB06 2 3 5 4 5 5 4 6 13 10 17 8 Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. References 1. Bamforth C.W. Current perspectives on the role of enzymes in brewing J. Cereal Sci. 2009 50 353 357 10.1016/j.jcs.2009.03.001 2. Liu S.Q. 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PMC10000827 | Diagnostics (Basel) Diagnostics (Basel) diagnostics Diagnostics 2075-4418 MDPI 10.3390/diagnostics13050837 diagnostics-13-00837 Review Solving STODS--Surgical Temporary Ocular Discomfort Syndrome Hirabayashi Matthew T. 1 Barnett Brad P. 2* Han Jae-Ho Academic Editor Zambito Ylenia Academic Editor 1 Department of Ophthalmology, University of Missouri School of Medicine, 1 Hospital Dr, Columbia, MO 65212, USA 2 California LASIK & Eye, 1111 Exposition Blvd. Bldg. 200, Ste. 2000, Sacramento, CA 95815, USA * Correspondence: [email protected] 22 2 2023 3 2023 13 5 83707 12 2022 02 1 2023 04 1 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). The term STODS (Surgical Temporary Ocular Discomfort Syndrome) has been coined to describe the ocular surface perturbations induced by surgery. As one of the most important refractive elements of the eye, Guided Ocular Surface and Lid Disease (GOLD) optimization is fundamental to success in achieving refractive outcomes and mitigating STODS. Effective GOLD optimization and the prevention/treatment of STODS requires an understanding of the molecular, cellular, and anatomic factors that influence ocular surface microenvironment and the associated perturbations induced by surgical intervention. By reviewing the current understanding of STODS etiologies, we will attempt to outline a rationale for a tailored GOLD optimization depending on the ocular surgical insult. With a bench-to-bedside approach, we will highlight clinical examples of effective GOLD perioperative optimization that can mitigate STODS' deleterious effect on preoperative imaging and postoperative healing. LASIK Keratopathy dry eye STODS LALEX SMILE This research received no external funding. pmc1. Introduction Maintaining a healthy ocular microenvironment is requisite for tear-film stability and good vision . In the wake of ocular surgery, all patients develop, in varying degrees, Surgical Temporary Ocular Discomfort Syndrome (STODS). To combat STODS, a molecular, cellular, and anatomic understanding of the ocular perturbations resulting from STODS is requisite. Armed exclusively with preservative-containing artificial tears, soap, and heat, numerous doctors and patients alike have found ocular surface microenvironment optimization to be an elusive goal. STODS is a term popularized through the Refractive Surgery Alliance (accessed on 6 December 2022)). Here, we use STODS to describe the temporary disturbance to the ocular surface following ocular surgeries involving incisions (manual or laser-assisted) to the cornea. The importance of STODS is its distinction from "dry eye disease". The proposed draft for LASIK Patient Labeling Recommendations from the United States Food and Drug Administration also lists moderate dry eye symptoms as a relative contraindication for treatment, so confidently navigating preoperative ocular surface abnormality, optimization, and postoperative STODS will only become of even greater importance for refractive surgeons in years to come . STODS is likely due to corneal nerve plexus transection and attenuated by other factors including up-regulation of inflammatory mediators. Corneal nerve fiber bundles are known to decrease significantly after procedures like LASIK but substantially return by one year postoperatively; hence the temporary nature of the condition . A stable, healthy tear film not only maximizes the quality and accuracy of preoperative measurements for surgical planning but also provides the greatest postoperative vision . To achieve and maintain refractive targets, a careful coordination between optometrist, ophthalmologist, and patient is necessary. Effective co-management for the mitigation of STODS requires a proactive approach. This coordinated approach, or Guided Ocular Surface & Lid Disease (GOLD) optimization, is critical for achieving refractive targets and keeping patients 20/Happy. Surveillance for preoperative signs in the asymptomatic is of the utmost importance. Ensuring a stable tear film with non-invasive tear breakup time (NITBUT) and assessing glandular health with meibography is critical . As we learned from the Prospective Health Assessment of Cataract Patients' Ocular Surface (PHACO), 80% of patients have clinically significant ocular surface disease prior to surgery but only 22% of these patients carry a diagnosis of dry eye disease (DED) . While the importance of GOLD optimization and effective co-management for STODS mitigation is a growing topic of discussion, it remains a new topic with limited literature on what approaches are most efficacious or efficient. Although it is always important to consider optimizing the ocular surface for all patients to improve their vision, it is of critical importance before cataract surgery. Cataract surgery is considered a form of refractive surgery with patients expecting excellent postoperative results with clear and stable vision regardless of the type of intraocular lens (IOL). In this paper, we will discuss how health of the ocular surface is assessed, what molecular changes refractive surgery induces, and how targeted therapies aim to enhance GOLD optimization and prevent, as well as treat, STODS should it occur. This review, with a few case studies, attempts to take a bench-to-bedside approach to solve and effectively combat STODS and attenuate concomitant disruption of ocular surgery to provide the greatest chance for symptom minimization. 2. The Importance of Preoperative Optimization First, patients should be counseled that an optimized ocular surface provides the greatest probably of the most accurate preoperative measurements possible, and thus, the most accurate outcome possible. This applies to keratometry, corneal tomography or topography, and biometry as well as current manifest refraction. Without a regular and stable ocular surface before surgery, patients should understand they might not be able to realize their full visual potential. Rapid tear breakup time (TBUT), punctate epithelial erosions, or low tear lakes with desiccation affect the reliability, reproducibility, and accuracy of the preoperative measurements . This therefore increases the possibility of a surgeon making an inappropriate IOL recommendation or selecting an incorrect IOL power leading to suboptimal visual outcomes for patients. A suboptimal ocular surface can have a profound impact on topography measurement . Multifocal and extended depth of focus (EDOF) presbyopia-correcting IOLs provide the greatest change for spectacle independence following cataract surgery but are exquisitely sensitive to ocular surface disease with more variability and dissatisfaction with vision than monofocal patients . These patients should be specifically counseled that GOLD optimization will remain paramount after surgery for the best possible outcome. 3. Key Elements to Preoperative Evaluation Whether patient is an outside referral or established in the practice, the surgeon must evaluation their corneal health preoperatively. There are components to the ocular surface exam with some clues evident when entering the room. Excessive or frequent blinking, skin manifestations of rosacea, blepharitis, incomplete blink, lagophthalmos, or eye rubbing are often evident from across the room. Elucidating details regarding contact lens wear, ocular comfort, or symptoms of dry eye or blepharitis also help in guiding both the exam and final IOL recommendation. Classic clues for ocular surface abnormality include anterior blepharitis, meibomian gland dysfunction, punctate corneal staining, anterior basement membrane corneal dystrophy, or Salzman's nodules, many of which are common in the cataractous population . For a more objective analysis of the ocular surface, corneal tomography/topography shows fluctuating and significant irregularity . A traditional way to assess the corneal tear film is tear breakup time (TBUT). This is an "invasive" assessment that involves placing fluorescein in the tear film and timing its evaporation, which is considered normal if >10 s . New methods such as NITBUT have been gaining popularity and typically involve video topography . Examples include the CA-800 (Topcon, Tokyo, Japan), TearCheck (ESW Vision, Houdan, France), and Keratograph 5M (Oculus, Wetzlar, Germany). Results are comparable to traditional TBUT but may be more repeatable and reliable since they are a noninvasive test . Importantly, it can be incorporated with a dropless preoperative evaluation that will not interfere with topography. As many groups use an anesthetic/fluorescein solution for applanation tonometry and TBUT, assessment of corneal sensation with an esthesiometer is precluded. As corneal sensation may play a central role in STODS, it is important to assess corneal sensation both postoperatively. For this reason, devices such as iCare (Vantaa, Finland), that do not require anesthetic for use, may be ideal in the refractive surgery setting. 4. Molecular Changes in Ocular Surface Abnormality and during Refractive Surgery Striving for GOLD optimization and the prevention/treatment of STODS requires an understanding of the molecular factors that influence ocular surface changes during surgery. Several mechanisms have been proposed and studied. Cataract surgery alone leads to ocular surface changes and dry eye syndrome through several mechanisms that disrupt tear film stability . Corneal nerve destruction during wound creation, triggering the inflammatory cycle, goblet cell loss, and meibomian gland dysfunction have all been reported after cataract surgery . Ocular surface inflammation appears to play a dominate role over tear secretion . Longer operative times, light or heat from the microscope, use of a lid speculum, and the severity of intraocular inflammation can all impact the postoperative ocular surface . Misuse of postoperative drops is also among the major contributors to STODS . One of the best studied theories involves a cycle of ocular surface inflammation comprised of both soluble and cellular mediators . For example, patients with and without Sjogren's syndrome appear to have identical T-cell activation and infiltration with upregulation of CD3, CD4, CD8, CD11a, and HLA-DR, the latter two markers specific to lymphocyte activation . Additionally, increased inflammatory cytokines such as interleukin 1 (IL-1) and upregulation of matrix metalloproteinases (MMPs) have been demonstrated in the tear film of patients with dry eye symptoms and ocular surface diseases . Other responses to ocular surface stress include hyperosmolarity and increase in MMPs mediated by intracellular pathways including mitogen-activated protein (MAP) kinase and inflammatory cytokines. This results in a cycle where hyperosmolarity then induces inflammation of limbal epithelial cells by further upregulating inflammatory cytokines . The degree to which patients have these various perturbations at baseline and their susceptibility to such perturbations has underlying genetic causes . Rosacea and elevated levels of MMPs are an area ripe for exploring genetic underpinnings that can contribute to STODS. It has been reported that 80% of persons with rosacea have concurrent MGD. Though poorly understood, early research suggests gene-gene and gene-environment interactions are central to rosacea's developments. Whether through inherited genes or through epigenetic modifications that occur through environmental influence, understanding how genes impact STODS is critical . It does appear that certain ocular insults result in chronic ocular surface disease. So instead of STODS, a patient develops Surgical Chronic Ocular Discomfort Syndrome or SCODS. The genetic risk factors as well as epigenetic modifications that occur in the setting of ocular surgery are areas requiring further research. For example, currently we have no evidence to support the idea that epigenetic modifications occurring in longstanding STODS may contribute to the development of SCODS. Alternatively, the insult may cause purely neurogenic and inflammatory changes that are better blunted by certain genetic predispositions in lieu of epigenetic modifications. Additionally, dysregulation of the balance between proteases and protease-inhibitors has been observed in ocular surface disease. These include MMPs but also cathepsins and plasminogen activators (and their relevant inhibitors) . MMPs, serine proteases, and cysteine proteases are all shown to be upregulated in ocular surface disease and all play a role in protease-activated receptor (PAR) inflammatory signaling . The concept of the eye biome has increased in popularity recently with evidence for a distinct microbiome in those with dry eye disease compared to healthy individuals . As expected, those with a blepharitis component have increased prevalence of Streptophyta, Corynebacterium, and Enhydrobacter . Meibomian gland dysfunction has been associated with increased bacteria on the ocular surface and an increase in bacterial load has been associated with decreased goblet cell density . In this way, alterations in the ocular microbiome have major effects on tear film stability. Since the most optimal therapy would involve tailoring the treatment to the pathophysiology, certain surgical techniques induce unique changes. Following Photorefractive Keratectomy (PRK), new neurites emerge from the severed nerve endings into the epithelial-stroma interface as early as the first week after surgery . There is around 85-95% sensitivity recovery after PRK by 3 months that is directly related to the intensity of the laser application . Laser In-Situ Keratomileusis (LASIK) on the other hand severs both stromal and sub-basal nerves during flap creation with direct ablation of the stromal nerve plexus . There is typically under 10% of the sub-basal nerves remaining after LASIK with evidence for both continued regression after surgery since the nerves are unable to connect with the flap leading to significantly reduced nerve density up to 5 years postoperatively . Laser-Assisted Lenticule Extraction (LALEX) shows superior postoperative corneal sensitivity compared to LASIK, likely because the nerves outside the lenticule area remain untouched . Femtosecond lasers used for cataract surgery (e.g., capsulotomies) have also been associated with a dose-dependent induction of cell-induced inflammation . The use of the femtosecond laser in LASIK surgery does not appear to alter corneal sensitivity or dry eye outcomes compared to LASIK alone. In fact, femtosecond flaps may have superior postoperative tear film stability compared to mechanically created flaps . Cell-mediated inflammation still plays a major role in ocular surface disease following PRK, LASIK, and LALEX . The emergence of Microinvasive Glaucoma Surgeries (MIGS) and the "MIGS Revolution" has allowed for the intervention of glaucoma earlier in the disease course and is often paired with intraocular surgery often with the goal to reduce the medication burden and lessen ocular surface side effects of topical glaucoma therapy . There is currently limited literature concerning unique variations of inflammation when cataract surgery is performed with MIGS but there is no clear evidence that MIGS increase inflammation postoperatively when performed correctly. Although it is important to consider glaucomatous eyes can have a unique profile of inflammatory cytokines . Thus, it is clear that ocular surface and dry eye disease is a complex entity with delicate interplay between inflammatory mediators, tear film integrity, and the native microbiome and these all must be considered when attempting to treat patient symptoms. 5. Methods for Preop Ocular Surface Optimization Lifestyle changes, although challenging to introduce, are important to consider for GOLD optimization. Discussing the impact of environmental factors with patients such as overhead fans, placement of vents, and any other source of blowing air that would contribute to tear desiccation is critical. For people with incomplete lid closure and/or Continue Positive Airway Pressure (CPAP) use, nighttime ointment or occlusive mask during sleep is critical. Prolonged reading or binge-watching media will also exacerbate symptoms of dry eye . It may be best at these early stages of treatment to remind patients that the surgery discussion must wait until the ocular surface improves and accurate measurements are possible. The most pervasive first-line treatment for dry eye symptoms are artificial tears or ocular lubricants which generally result in a ~25% improvement of symptoms . The issue is, artificial tears, although soothing even when preservative free, are not wholly benign and do not effectively restore ocular surface perturbations. Other than the immediate symptomatic relief they provide, they also have a "dilution" effect of the inflammatory mediators. Starting newly diagnosed patients on artificial tears QID and placing collagen punctal plugs such as the DuraPlug (Katena, Denville, NJ, USA) that will dissolve after 3-4 months are reasonable first-line treatment options for mild disease before and after surgery. While there is some evidence for the efficacy of lid scrubs and warm compresses, these methods do not appear to alter lipid layer thickness or tear interferometry . Additionally, perhaps it is not the best initial step in therapy to apply soaps that may further disrupt the biome, mechanical scrubbing, or heat to tissue that is already inflamed. For primarily lid margin disease or blepharitis, especially with Demodex, hypochlorous acid-containing lid scrubs such as Avenova(r) (NovaBay Pharmaceuticals, Emeryville, CA, USA) or OCuSOFT (OCuSOFT Inc., Richmond, TX, USA) can provide effective relief . Lotilaner Ophthalmic Solution has also demonstrated efficacy specifically for Demodex treatment . Warm compresses are particularly useful for blepharitis because since the meibomian gland is a holocrine gland, it is similar in concept to skin exfoliation in that expression can regenerate atrophic glands . Devices such as LipiFlow(r) (Johnson & Johnson, New Brunswick, NJ, USA) or TearCare(r) system (Sight Sciences, Menlo Park, CA, USA) are more technologically sophisticated than warm compresses but use the same principle of heat to express meibomian glands with the goal of resetting the microenvironment . A similar method for addressing meibomian gland disease is Intense Pulsed Light (IPL) therapy. Light is converted to heat that ablates vessels and restricts the inflammatory mediators to the gland structures and has proven effective for abating symptoms of dry eye . The E-EYE IRPL(r) (ESWIN, Houdan, France) was the first registered and medically certified IPL for the DED & MGD in 50+ countries outside of the U.S. Now available in the U.S. along with Optilight(r) (Lumenis, Yokneam, Israel), we have IPL devices with proven efficacious protocols for optimizing ocular surface disease. To address more of the root causes of ocular surface disease, topical anti-inflammatories have been growing in popularity. Lifitegrast ophthalmic solution (Xiidra(r), Novartis AG Pharma, Basel, Switzerland) addresses this by binding to lymphocyte function-associated antigen-1 (LFA-1) to prevent the inflammatory cascade with good efficacy in treating symptoms . A T-Cell suppressant regimen such as Xiidra(r) BID, a topical steroid such as Eysuvis(r) (Alcon, Fort Worth, TX, USA) QDAY prior to surgery are reasonable approaches to address the inflammatory component. Similarly, topical cyclosporine A including cyclosporine ophthalmic solution 0.09% (Cequa, Sun, Princeton, NJ, USA) and cyclosporine ophthalmic solution 0.05% (Restasis(r), AbbVie, Chicago, IL, USA) as well as new generic 0.05% cyclopsporine formulations, inhibit T-cell activation and production of inflammatory cytokines by inhibition of calcineurin . A regimen such as two months of Cequa(r) BID and Eysuvis(r) (Alcon, Fort Worth, TX, USA) QDAY with potential erythromycin ointment nightly for MMP-9 inhibition is another reasonable way to approach GOLD optimization from the inflammatory perspective before surgery. The use of topical corticosteroids has been proven effective for the signs and symptoms of ocular surface disease . Strategic use of these inflammatory mediators must be considered with other therapies. For example, full punctual occlusion with lifitegrast results in new tears diluting the lifitegrast until it dissociates and makes the LFA-1 accessible to enable T-Cell ocular surface invasion. While tetracyclines do reduce bacterial flora, they also have been shown to inhibit lipase activity, MMP-9 levels, and inflammatory cytokines . A short course of doxycycline would also be a reasonable approach before ocular surgery. Lastly, while autologous serum tears derived from blood samples theoretically have the benefit of containing other biochemical factors to mimic an individual's tear film more closely, they are difficult to obtain and evidence for efficacy is limited in the long term . An alternative to the previously mentioned autologous serum tears is a hypoosmolar ophthalmic solution with a "patented formula" of human placental-derived biomaterials from amnionic fluid such as Regener-Eyes(r) (Regener-Eyes(r), Palm Harbor, FL, USA) which is typically dosed 1-4 times per day for symptoms of dry eye . An immune-privileged site, amnionic fluid contains several factors to promote cell growth and regeneration of collagen . Cell-free amnionic fluid derivatives have also proven effective in not only cases of corneal wound healing but also for symptoms of keratoconjunctivitis sicca . Another approach is to place a few drops of these solutions into an ocular surface drug depot system such as Hyper-CLTM (EyeYon Medical, Ness Ziona, Israel). This theoretically protects the ocular surface from the mechanical action of blinking and increases the ocular surface contact time with the solution. Nevertheless, for applications that involve optimizing corneal topography/tomography, the presence of a contact lens that may induce astigmatism may not be the ideal approach. To address reducing MMPs further, low-dose oral options such as doxycycline 40 mg (Oracea(r), Galderma, Lausanne, Switzerland) or doxycycline hyclate 20mg (Periostat(r), Galderma, Lausanne, Switzerland) provide improvement of meibomian gland dysfunction without disrupting the microbiome of the eye . Erythromycin or even postoperative antibiotic therapy may disrupt this biome further so these options with minimal antibiotic properties or favoring intracameral antibiotics over a course of topicals may prevent STODS and help optimize the ocular surface. Similarly, the "pro-biotic" concept is relatively new to ophthalmology but treatments such as HydroEye(r) (Spring, TX, USA) provide a proprietary blend of Gamma-linolenic acid (GLA), omega 3 fatty acids (EPA and DHA), antioxidants, and vitamins to support the tear film. The ingredients have demonstrated success and are another reasonable option both before surgery for GOLD optimization and postoperatively for the prevention of STODS . Another benefit of oral preparations is patient acceptance. They are comfortable with taking a natural option long-term and appreciate that the anti-inflammatory effects can also have systemic benefits. These are also excellent options as therapy by an alternate route may reduce the future need to add more topical medications. Another "minimally invasive" option is varenicline nasal spray (TyryvaTM, Oyster Point Pharma, Princeton, NJ, USA) which has shown excellent efficacy and speed to clinical effect . Neuroinflammation of the corneal nerves, trigeminal ganglion, and trigeminal brainstem complex have been associated with dry eye changes so this provides a promising new approach especially since it is not another topical therapy . A month or two of this therapy is reasonable before surgery and can easily be continued after as well. A step past the amniotic fluid-derived therapies is amnionic membrane grafts (AMG) which are composed of epithelial cells, their basement membrane, and a matrix of connective tissue all bathed in amnionic fluid containing numerous anti-inflammatory, immunomodulatory cytokines, and growth factors . Cryopreserved AMG has shown promise in regeneration of corneal nerve and acceleration of ocular surface recovery in cases of dry eye . Cryopreserved AMG can also be placed under a generic bandage contact lens or even a Kontur Contact Lens (Kontur, Hercules, CA, USA) for easy application . Unlike dehydration, lyophilization is a gentler process that is less likely to denature proteins and thus, once rehydrated, results in more therapeutic potential . Lyophilized autologous serum tears have demonstrated equivalent efficacy to fresh samples . Lyophilized AMGs have been successfully used in pterygium surgery and are likely reasonable for other ocular surface disease applications as well . Either lyophilized or cryopreserved AMGs are reasonable intermediate steps under contact lenses but additional studies, especially molecular analysis, will be necessary to tease out details if one is superior to the other. Neox(r) Flo (BioTissue, Miami, FL, USA) is another tissue product containing both amnionic membrane and umbilical cord (AM/UC) that has already demonstrated efficacy to assist in wound healing . Demonstrated ophthalmic applications are currently limited but it will likely provide a promising option when used under a Kontur or bandage contact lens. Experiments are underway to compare NEOX(r) FLO to Prokera(r) to determine the utility for optimizing the ocular surface prior to biometry. Both this product as well as a new lyophilized amniotic membrane graft, Xcellereyes(r) (Oculus Biologics, Willowbrook IL, USA) may be superior to Prokera(r) for preoperative optimization as the retaining ring of the Prokera(r) can ride up onto cornea, inducing astigmatism . The Prokera(r) line of products can also leave a residue on the ocular surface when first removed. This paradoxically worsens the NITBUT and accordingly the quality of topography . Logistically, due to this residue and the potential astigmatism induced by the retaining ring, same day Prokera(r) removal followed by biometry is non-ideal. In our current protocol, we stagger topography and biometry three days after Prokera(r) removal to mitigate these potential confounders . Dehydrated AMGs under contact lenses do not leave a similar residue and the use of a large diameter contact lens such as a Kontur may be less likely to induce astigmatism and effectively retain therapeutic factors on the ocular surface . Experiments are underway to better understand if substitution of a lyophilized product under a contact lens may provide the superior therapeutic benefit of Prokera(r) compared to dehydrated AMG without the residue deposition and astigmatism induction by the Prokera(r). 6. Intraoperative Management As discussed earlier, preserving the ocular microbiome may play a major role in managing ocular surface disease. Intraocular methods such as the newly described "Shimada Technique" employs 0.25% Povidone-Iodine washes during cataract surgery to incredible antimicrobial effect . This technique, or intracameral antibiotics at the conclusion of surgery, may remove the need for postoperative antibiotics that continue to disrupt the ocular microbiome for the week following surgery . Faster return to normal flora may equate to faster return to a healthy ocular surface. Use of dispersive viscoelastic such as OcuCoat(r) (Bausch + Lomb, Laval, QC, Canada) on the ocular surface instead of a balanced saline solution may also prevent intraoperative drying and epithelial damage. For laser-based surgeries, several of the previously mentioned considerations can further guide laser selection. Eyes with significant ocular surface disease may experience less STODS with PRK or LALEX over LASIK, for example. Additionally, femtosecond-assisted cataract surgery should be kept to the minimal necessary power. This concept of "low-energy" LALEX has gained popularity recently as technology continues to advance . While LASIK flap thickness or hinge position have not demonstrated an effect on ocular surface symptoms, there is evidence that smaller flap diameters do create less nerve transection, and accordingly less ocular surface disease . Thus, the ratio between the corneal and flap diameter is another consideration surgeons can use to minimize the risk of STODS. Another consideration is the demonstrated symptom profile of femtosecond created flaps compared to mechanically created flaps . 7. Postoperative Management of STODS Any laser or cataract surgery can result in postoperative disruption to the tear film and cause Surgical Temporary Ocular Discomfort Syndrome (STODS) . Staying ahead of the inflammatory cascade using the methods mentioned above is likely the best way to prevent STODS, but there are ways to manage it should it occur. When it does occur, it is also essential to carefully pair the appropriate therapeutic target with the underlying pathophysiology of the cause. Many of the previously mentioned therapies can be continued into the postoperative period, especially those that have routes other than application onto the ocular surface such as HydroEye(r) or TyryvaTM. As mentioned before, avoiding postoperative antibiotics may lead to faster restoration of the native ocular microbiome. Similarly, avoiding postoperative corticosteroid drops likely benefit the ocular surface by reducing exposure to preservatives. Products like the dexamethasone eluting punctal plug Dextenza(r) (Ocular Therapeutix, Inc., Bedford, MA, USA) or subconjunctival triamcinolone can spare the ocular surface after surgery and provide effective control of intraocular inflammation . Of the therapies that can or should be continued, cylcosporine 0.05% has been shown to improve visual outcomes after multifocal IOL implantation . As referenced earlier, laser-based surgeries, including femtosecond-assisted cataract surgery, exert unique changes on the ocular surface (Table 1). In addition to cell-mediated inflammation, the destruction of corneal nerves and incited inflammation from Femto, LASIK, PRK laser-based surgeries implies these patients may not only benefit from medications with cellular anti-inflammatory mechanisms, but also therapies that assist with corneal nerve regeneration. Examples of this would include the amnion-derived bio-tissues discussed earlier such as NEOX(r) FLO (Table 2). 8. Conclusions Recognizing the signs of ocular surface disease is paramount in any surgical workup. This should be followed by an earnest discussion about the importance of GOLD Optimization for obtaining optimal measurements and thus optimal outcomes. Part of this discussion with patients is ensuring they understand the major role they play in their own outcome. Patient compliance is a known issue with dry eye treatment and the degree to which they comply with the treatment options surgeons suggest will directly correlate to their outcome . Investing in their eyes, and their vision, is a lifetime commitment that starts before ocular surgery. Many new treatments for STODS and general dry eye symptoms are constantly developed so this remains an active and innovative area of research. Patents already exist for ways to regulate blood flow to restore a more normal oxygen status to dysfunctional meibomian glands . Understanding that there's no definitive cure for dry eye and the patient will never be able to abandon caring for their ocular surface is perhaps the best way to ensure happy patients and happy surgeons. In particular we must understand that although STODS may be temporary, if left untreated it can create a chronic inflammation or Surgical Chronic Ocular Disease Syndrome (SCODS). The effective refractive surgeon will identify the various intrinsic factors such as genetic propensity, ocular surface disease prior to surgical intervention, and various anatomical factors that may increase the risk of STODS to SCODS conversion. They will not be flat-footed and reactionary, but instead, proactive in mitigating STODS at the gate and ensuring it never becomes SCODS. Author Contributions M.T.H. and B.P.B. conceived and wrote the manuscript. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement Not applicable. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Neurotrophic Etiologies for Surgical Temporary Ocular Discomfort Syndrome (STODS) with a variety of corneal surgical interventions. Transection of nerve occurs in a varying amount with the variety of corneal surgeries as compared to cataract surgery without femtosecond arcs. When femtosecond arcs are employed, the potential for greater nerve transection and thus STODS develops. Similarly, LASIK transects more corneal nerves than LASIK; therefore one would expect greater STODS in the former surgery. Fortunately, by one year, all corneal surgeries are followed by robust nerve regrowth to nearly baseline levels. Figure 2 Non-invasive preoperative assessment with CA-800 (Topcon) Tear Film Analyzer. (A) Placido disk non-invasive tear breakup time (NITBUT) provides an objective measure of tear film stability (B). Through use of meibography (C) to demonstrate to patients contributing factors to tear film instability NITBUT with Zernike analysis of the resulting astigmatism, spherical aberration, coma, and higher order aberrations (D) to convey to patients the visual impact of tear film instability, clinicians can help patients better understand the impact of ocular surface disease even prior to surgery. Moreover, this analysis helps gauge IOL suitability and the likelihood of quality preoperative topography. A NITBUT of 1 s, for example, will undeniably result in poor topography as a Pentacam is captured over 2 s. Figure 3 The impact of tear film stability on corneal topography. Sequential same day (A,B) Scheimpflug image in the setting of unstable tear film demonstrating a difference (C) of 23.5 diopters on the axis of astigmatism (D). In addition to varying magnitude and axis of astigmatism, tear film instability also results in increased wavefront error (E). Repeat topography after GOLD optimization reveals a more regular astigmatism with less same day variability (F,G). The best fit topography prior to GOLD optimization (A) was compared to (F) which revealed a 0.8D increase in magnitude in astigmatism (H,I) as well as a decrease in wavefront error (J). Figure 4 Mean visual acuity with four multifocal IOLs after the induction of different values of positive cylinder. Mean visual acuity (A) and patient satisfaction scores (B) with four multifocal IOLs after the induction of different values of positive cylinder (green 1/4 very satisfied; yellow 1/4 moderately satisfied; orange 1/4 not satisfied; red 1/4 not at all satisfied). Values are reported as median, with range in brackets . Figure 5 Prokera for preoperative STODS mitigation (A) demonstrates a view at the slit-lamp of standard Prokera placement (Note: even in best case scenario retaining ring presses on cornea). (B) Prokera has ridden up on cornea therapy inducing artifactual astigmatism. When immediately removed, Prokera leaves a film on the cornea (C) that interferes with the mires and paradoxically results in a worsened NITBUT (D) as compared to prior to insertion (E). Figure 6 Guided Ocular Surface & Lid Disease (GOLD) Co-management algorithm. Incorporating aspects of the ASCRS algorithm one proposed GOLD co-management protocol involves first probing for signs with inflammatory for MMP-9 measurement (relatively low cost and placeable in every optometric office with a CLIA waiver). Symptoms can be established as well with SPEED II. Once uncovering ocular surface disease, the referring optometrist could first start the patient on a T-cell suppressant regimen such as BID cequa and QDAY Eysuvis. Patient would then first see the surgeon in 2-4 weeks after beginning the regimen. If tear film analysis revealed continued tear film instability and significant glandular disease, the surgeon would place a Prokera and refer back to the optometrist for meibomain gland optimization. Through interventions such as IPL and heated expression, the tear film can be stabilized as measured and help restore tear film stability. Once fully optimized, the stability of the tear film and meibomian gland health can be confirmed prior to Biometry and Topography to guide surgery. Figure 7 Speculum-Free Insertion of Amniotic Membrane Graft Under Kontur--Through use of a Kontur large diameter contact lens, an antibiotic drop, and an amniotic graft (A), a self-retaining amniotic membrane graft without a retaining ring can be created. A drop of antibiotic is placed inside the Kontur (B), and then the non-hydrated amniotic membrane is grasped with a pair of jeweler forceps (C). The amniotic membrane is placed with one edge touching the antibiotic fluid in the Kontur and the amniotic membrane sequentially adheres to the contact lens through hydrostatic attraction, similar to the application of a screen protector (D). Next an antibiotic drop is instilled in the eye (E) and then the patient is told to look down and pull down on their lower lid (F). The clinician then pulls up on the lower lid and places the Kontur with adhered AMG (G). Once on the eye (H) the patient is told to look down thereby centering the lens and AMG over the cornea (I). Next the patient and clinician release their fingers from the lids (J). Figure 8 Solving STODS, a graphical summary. The ocular surface microenvironment undergoes unique perturbations from each surgical insult. A variety of therapeutics and interventions can be utilized to blunt the impact of STODS on the ocular surface. diagnostics-13-00837-t001_Table 1 Table 1 Summary of Unique Pathways to Surgical Temporary Ocular Discomfort Syndrome (STODS) Following Different Ocular Surgeries. Ocular Surgery Mechanism Potential Therapeutic Targets Cataract Surgery Along Cataract Surgery + MIGS Ocular Surface Inflammation; corneal nerve loss; microbiome disruption TYRVAYATM--Spares surface CEQUA(r), RESTASIS(r) HydroEye(r) Regener-Eyes(r) PRK LASIK Corneal nerve loss, worst with LASIK NEOX FLO AMG TYRVAYATM--Spares surface CEQUA(r), RESTASIS(r) LALEX Corneal nerve loss, least of all laser-based refractive surgeries Regener-Eyes(r) TYRVAYATM--Spares surface CEQUA(r), RESTASIS(r) FLACS Ocular Surface Inflammation, correlated with energy TYRVAYATM--Spares surface CEQUA(r), RESTASIS(r) HydroEye(r) Regener-Eyes(r) diagnostics-13-00837-t002_Table 2 Table 2 Strategies, Mechanisms, and Option of Various Treatments for Ocular Surface Optimization and Surgical Temporary Ocular Discomfort Syndrome (STODS). Class Mechanism Examples Duration Special Uses/Comments Lifestyle Changes Preventing tear desiccation Fan placement, avoiding prolonged reading Indefinitely This should become a permanent lifestyle change for all patients Increasing Tear Films Tear replacement; inflammatory marker dilution REFRESH TEARS(r) (Abbvie, Chicago, IL, USA) QID, 2-4 months pre-op Frequent use of unpreserved tears can worsen symptoms Punctal Occlusion DuraPlug 2-4 months pre-op Avoid using with some anti-inflammatories, diluting them in the tear film reduces effect Hypchlorous Acid Scrubs Lotilaner Ophthalmic Solution Meibomian exfoliation, mechanical debridement Avenova(r), OCuSOFT 2-4 months pre-op For predominantly blepharitis or Demodex symptoms Warming Heat-based meibomian expression Warm Compresses IPL BID 2 months pre-op Careful applying heat and debridement to inflamed tissue Heat-based meibomian expression LipiFlow(r), TearCare(r) 2 months pre-op More regulated and gentle on eyelids Pro-Biotics Tear film support HydroEye(r) 2 months pre-op Surface sparing, easy to continue post-op Anti-inflammatories Corticosteroid Prednisolone Acetate BID 2 months pre-op Monitor for pressure response LFA-1 Xiidra(r) BID 2 months pre-op Paired with Prednisolone Acetate BID Calcineurin inhibition CEQUA(r), RESTASIS(r), BID 2 months pre-op Paired with Prednisolone Acetate QDAY Verenicline TYRVAYATM BID 1 month pre-op Nasal spray; spares ocular surface; easy to continue post-op IPL OptiLight 4 section pre-op Can repeated post-op MMP Inhibition Doxycycline ORACEA(r), Periostat(r) 1-2 weeks pre-op Minimal antibacterial properties so microbiome preservation Advanced Tear Substitutes Autologous Serum Tears Serum Tears 2 months pre-op Theoretically contain more patient-specific bio-compatible materials Amnionic-Based Therapies Amnionic Fluid-Based Tears Regener-Eyes(r) QID 2 months pre-op Can be used under drug depot system (Hyper-CLTM) Cryopreserved or Lyophilized Amnionic Membrane Grafts AMG Post-Op Can be placed under contact lens; consider for post-laser STODS Amnionic and Placental derived NEOX FLO Post-Op Can be placed under contact lens; consider for post-laser STODS Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). 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PMC10000828 | Foods Foods foods Foods 2304-8158 MDPI 10.3390/foods12051036 foods-12-01036 Article Emerging Perspectives of Blockchains in Food Supply Chain Traceability Based on Patent Analysis Mastilovic Jasna Conceptualization Data curation Writing - review & editing 1 Kukolj Dragan Conceptualization Software Data curation Writing - original draft Supervision 23 Kevresan Zarko Conceptualization Data curation Writing - original draft Writing - review & editing 4* Ostojic Gordana Conceptualization Supervision 3 Kovac Renata Writing - review & editing 4* Deric Marina Writing - review & editing 4 Samek Dragana Ubiparip Writing - review & editing 4 Zhang Min Academic Editor 1 Institute BioSense, University of Novi Sad, 21000 Novi Sad, Serbia 2 Institute for Artificial Intelligence Research and Development of Serbia, University of Novi Sad, 21000 Novi Sad, Serbia 3 Centre for Identification Technologies, Faculty of Technical Sciences, University of Novi Sad, 21000 Novi Sad, Serbia 4 Institute of Food Technology, University of Novi Sad, 21000 Novi Sad, Serbia * Correspondence: [email protected] (Z.K.); [email protected] (R.K.) 28 2 2023 3 2023 12 5 103625 1 2023 18 2 2023 20 2 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). In the field of blockchain (BC) technology application in the food supply chain (FSC), a patent portfolio is collected, described, and analyzed using Latent Dirichlet Allocation (LDA) modeling, with the aim of obtaining insight into technology trends in this emerging and promising field. A patent portfolio consisting of 82 documents was extracted from patent databases using PatSnap software. The analysis of latent topics using LDA indicates that inventions related to the application of BCs in FSCs are patented in four key areas: (A) BC-supported tracing and tracking in FSCs; (B) devices and methods supporting application of BCs in FSCs; (C) combining BCs and other ICT technologies in FSC; and (D) BC-supported trading in FSCs. Patenting of BC technology applications in FSCs started during the second decade of the 21st century. Consequently, patent forward citation has been relatively low, while the family size confirms that application of BCs in FSCs is not yet widely accepted. A significant increase in the number of patent applications was registered after 2019, indicating that the number of potential users in FSCs is expected to grow over time. The largest numbers of patents originate from China, India, and the US. food chain traceability blockchain application database patent portfolio Latent Dirichlet Allocation topic modeling food safety food authenticity Ministry of Education, Science and Technological Development of the Republic of Serbia451-03-68/2022-14/200222 451-03-68/2022-14/200325 451-03-68/2022-14/200 Secretariat of Higher Education and Research of Vojvodina Province142-451-2406/2022-01/01 This research was funded by the Ministry of Education, Science and Technological Development of the Republic of Serbia (under Grants: 451-03-68/2022-14/200222; 451-03-68/2022-14/200325; 451-03-68/2022-14/200) and the project Blockchain platform for support of certification of added value agricultural products funded by Secretariat of Higher Education and Research of Vojvodina Province under Grant 142-451-2406/2022-01/01. pmc1. Introduction The food supply chain (FSC)is comprised of all the stages through which food products are delivered to consumers, from agricultural production, through storage, processing, distribution, and further toward food consumption, is a complex system which includes different operators, resources, and processes . Economic performance, as well as the quality and safety of food products delivered to consumers through FSCs, depends on inputs from all FSC stakeholders: farmers, warehouses, processors, logistics, distributors, wholesalers, and many others . With the development of more complex FSCs, tracing and tracking of food from raw materials to the very products delivered to the consumer, with disruptions, fragmentation, poor product traceability, improper product flows, food contamination, and food recall, have become the prevailing issue of FSCs, demanding much effort from all stakeholders, and coming into the focus of both consumers and policymakers . Common to all supply chains is that they could benefit from implementing traceability , especially in reverse supply chains when unused, faulty, or damaged products are returned to the manufacturer in an attempt to improve quality . The weakness of all supply chains, including supply chains of pharmaceutical products, courier express, luxury goods, consumer electronics, manufactured goods, automobiles, textile, wood, dangerous goods, and particularly agri-food products, is that transparent information is not currently offered to the final consumer as part of supply chain management . As a technological concept that evolved from the first cryptocurrency, blockchains (BCs) are increasingly entering into many areas of economics . A BC is a database consisting of encrypted blocks of records in a distributed public ledger of digital events shared among participants of a chain which enables future verification of all executed transactions. It is globally used as a robust tool for supporting financial systems, but its use in other fields of economics is being widely considered, particularly regarding tracking in the supply chains, resulting in its expansion . BC technology facilitates security, privacy transparency, decentralization, and immutability , thus enabling trustful, secure, and authenticated systems of logistics and information exchange to be established in supply networks . Insight into the capabilities of blockchain-based traceability solutions come from the academic literature, in which it is pointed out that both tracking and tracing via BC applications contribute to better transparency in supply chains and are thus gaining in popularity, particularly in food and pharmaceutical supply chains. The conclusion, however, is that, in spite of numerous applications reported in the literature, most of them are conceptual in nature, and real implementations of BC-based traceability solutions are very rare . Thus, during the past few years, the application of BC technology in FSCs has come into the focus of numerous researchers. BC technology is proposed as a solution for tracing in FSCs, regarding their safety, immutability, transparency, and scalability , with the ability to follow a product along its lifecycle , including internal traceability in food processing and enhanced mechanisms for control and response to food recalls in the FSC . Wang et al. emphasize that application of BC technology contributes to FSC digitalization and disintermediation with extended visibility, traceability, and improved data security, while Balamurugan et al. state that blockchain technology may support the identification of unsafe or fake foodstuffs, in order to block further access to them. When proposing a framework for traceability within FSCs and addressing the key challenges for FSC stakeholders, Baralla et al. emphasized BCs, in combination with IoT, as technologies of choice for the development of such a system. An easy and simple methodology for integration of BC technology in FSCs that allows traceability and provides the consumers with sufficient information to make informed purchasing decisions was proposed by Bettin-Diaz et al. based on best practices engineering, technology, and marketing. BC technology has also been proposed as a solution for the protection of an increasing number of IoT-oriented applications , product-selling platforms, on-demand service platforms , and cloud computing solutions developed for, or applied in, the FSC. Advantages brought into FSCs through the introduction of BC technology are widely discussed in academic literature; ensuring the integrity and privacy of datasets even when they are released to the public , counterfeit of hardware and software faults, security issues during communication, system management difficulties , internet stability and security complications , data security accountability, protection, neutrality, and efficiency of processes are just a few examples. However, the fact that diverse barriers, preventing the expansion of BC technology application in FSCs , still exist is widely elaborated in the academic literature . Patents can be used as a detailed source of both quantitative and qualitative technological information and are thus widely used for monitoring technology trends and industry competitiveness . Reviews based on the analysis of patent portfolios have been published in different technological fields, such as, for example, the application of carbon nanotubes , battery technologies for electric mobility , telecommunications , artificial intelligence in the automotive industry , multi-compartment refrigerators , new space missions , and other emerging or promising technologies. Among numerous methodologies for the exploration of technology trends based on patent data and topic modeling, a method based on the use of hierarchical probabilistic models for uncovering patterns of words has been proven to be among the most effective in discovering and classifying hidden topics behind patent documentation . In the present study, we sought to gain insight into the technology trends in the field of BC technology application in FSCs via collection of patent portfolio, which was analyzed to determine application trends and distributions and then subjected to topic modeling to identify latent topics and relate them to development trends and opportunities in FSCs. 2. Materials and Methods Review Approach Extraction of the patent portfolio from patent databases was performed using the commercial software platform PatSnap . Both active granted patents and pending patent applications were extracted. Patent title, abstract, and body text were searched using the following terms: ((blockchain OR "block chain" OR blockchains OR "block chains") AND (traceability) AND ("food supply chain" OR "food value chain" OR "agri-food chain" OR "food supply chains")). Extracted patent attributes included application number, title, classification code, priority and application date, abstract, family count, legal status, and the number of backward and forward citations . A patent portfolio of 82 documents was created, among which 26% were active patents, 70% were pending patent applications in different phases, and 4% of documents were undetermined. The patent application trends and the application authority structure was analyzed for the created patent portfolio. For the interpretation of the obtained unstructured document collections, topic modelling based on Latent Dirichlet Allocation (LDA) was performed . The documents were parsed prior to data processing. The text in abstracts were pre-processed in the following manner:- Removal of punctuations, digits, words, and tags shorter than four characters - Conversion of letters to lowercase - Filtering of words not contributing to the meaning of the phrase (stop words) - Reduction of words to their roots using the Porter stemming algorithm The pre-processed abstract texts were used to group patents in the portfolio into technology topics using Latent Dirichlet Allocation modeling (LDA) . In LDA, documents are viewed as random mixtures of different topics, where each topic is characterized according to the distribution of words. The topic distributions in documents share the common Dirichlet prior a, and the word distributions of topics share the common Dirichlet prior e. Given the parameters a and e for document d, parameter thd of a multinomial distribution over K topics is constructed from Dirichlet distribution thd|a. Similarly, for topic k, parameter bk of a multinomial distribution over all words is derived from Dirichlet distribution bk|e. The generative process of the LDA method is given in Table 1. The number of topics embodied in the patent dataset were selected based on evaluation of the predictive likelihood of models with a different number of topics and constructing the predictive likelihood curve . Bearing in mind the rather small number of patent documents in the portfolio, the first point where the curve changed direction (4 topics) was selected as the optimal number of topics. Based on the LDA distribution results of documents from patent portfolios across four latent topics (i.e., technology fields), the probabilities of participation of each topic in the considered documents were obtained as values ranging from 0 to 1 . Each document represents a probabilistic mixture of topics generated from the patent collection contents, and the patents in the portfolio were each assigned the topic with the highest calculated probability. Principal component analysis was used to visualize the distribution of patents from the portfolio across LDA topics. As LDA does not provide topic labels, identified latent topics were initially labeled as topics A, B, C, and D. To reveal the logical content and name, the identified latent topics content of patents in each topic were manually checked, the most frequent terms associated with each topic by LDA were taken into consideration, the most frequent patent classification codes were analyzed, and we proposed titles that best described each topic. We manually classified the most frequent words, identified by LDA as nouns, verbs, adverbs, and other words, to enable understanding of the common features for each topic. The International Patent Classification (IPC) system was used, given it is the most frequently used system to hierarchically organize the structure of technical fields and technological areas of a patent. The patent, depending on the technical broadness of the covered invention, may have several IPC codes assigned to it, and all assigned IPC codes were taken into consideration in the analysis. The outcome of the LDA was used to draw a dynamic picture of the development of BC technologies application in FSCs, with analysis of geographical distribution, and identification of the most valuable patent applications was based on patent forward citations and patent family size . In the cited works, influential patents having dominant technology or high applicability are distinguished and, accordingly, were used as an indicator of a patent's technological significance. A simple patent family representing a set of patents applied in various countries to protect a single invention, besides representing the importance of technology, also reveals the width of its potential market . A larger patent family is indicative of the attractiveness of the protected invention or that the technology is more innovative. Obtained results were used as the base for identifying the potential drivers and paths in future development of BC applications in FSCs. 3. Results 3.1. Patent Application Trends The annual count of published patent applications of a patent portfolio in the field of BC application in FSCs shows that the first patents in this field were filled after 2015, while significant growth of patent applications started from 2019, and the number of applications increased exponentially from this year onwards. Considering that application of the BC technology actually began at the end of the first decade of this century, such a trend is expected and understandable. It can be concluded that the growth in the practical application of BCs in FSCs is still in the initial phase, with significant growth expected in upcoming years. An increase in the application of BCs in patents related to FSCs from 2019 is in line with the results of Nan et al. , whose results indicate a significant increase in BC-based patent applications in the US and China since 2017. In the geographic distribution of application authorities of the observed patent portfolio , it is quite interesting that the leading countries with the highest shares of patents are China (38% of the total number of patent applications) and India (28% of the total number of patent applications), while the United States, with 20% of the total number of patent applications, is only in third place. The remaining 21% of patents are registered elsewhere in the world, with the European Patent Office and Australia taking both 7% of initially filed applications and the rest of the world claiming the remaining, only 7%. 3.2. Modeling of Portfolio Technology Space Based on the LDA Based on the LDA, patents from the portfolio were found to be distributed across four latent topics. The first step in the analysis of obtained results was the visualization of relations among formed topic clusters in a scatter plot presented in the factorial plane using PCA . It is obvious that patents from the analyzed patent portfolio are distributed in four mutually distinguished topics. Topic A, placed in the lower right quadrant of the factorial plane, is distinguished from the other three topics in relation to being the first, most influential dimension of principal component analysis, explaining 42.21% of the total variability. Topic C, positioned in the lower left quadrant, is quite similar to topic A, in terms of being the second PCA dimension, while topic D, and particularly topic B, are differentiated from topics A and C in that this PCA dimension explains 35.83% of the total variability. Analysis of the content of identified latent topics was performed based on analysis of the most frequent words revealed from the LDA, and the most frequent IPC codes attributed to the patents. Frequencies of occurrence of the most frequent words in abstracts of patent applications, allocated to four topics , provide a step forward in understanding the underlying trends in issues of patenting activities in which hidden topics occur. The most frequent words revealed for each topic from LDA were further classified into three groups of words and are presented in Figure 6 with different colors. The first group of words are verbs, which most probably indicate which operation, action, or activity in FSCs are being addressed in the patents (colored in green). The second group is nouns, which most probably indicate the subject or object in FSCs targeted by the patent (colored in dark gray). The third group of words are revealed as the most frequent and most common for all topics, i.e., the words from the patent database search pattern, or the adverbs that have not been taken into consideration in the analysis of issues targeted within topic clusters of the patent portfolio. Verbs and nouns identified as the most frequent in each topic are further systematized in Table 2, in which data used for revelation of hidden topics are presented. The second step forward toward understanding the underlying trends regarding the occurrence of issues that are latent topics of patenting activities, was the analysis of the most frequently assigned IPC codes within topics (Table 1). Table 1 also contains explanations of the most frequent IPC subgroups in the portfolio, together with the frequency of their appearance in the patents assigned to each hidden topic. However, within all topics, the most frequently assigned IPC codes were, as expected based on patent portfolio search criteria, with the IPC group G06Q, described as "Data processing systems or methods, specially adapted for administrative, commercial, financial, managerial, supervisory or forecasting purposes; systems or methods specially adapted for administrative, commercial, financial, managerial, supervisory or forecasting purposes, not otherwise provided for". Further analysis was directed toward understanding the focus of each of the four identified hidden topics in the analyzed patent portfolio. Besides data from Table 2, including the most common words in the abstracts and the most frequently assigned IPC codes, the analysis also included an in-depth review of the patents assigned to each topic by LDA, which we performed manually. Topic A: The IPC code most frequently assigned to patents on topic A is G06Q10 (Data processing systems: administration, management), followed by G06Q30 (Data processing systems: commerce), and G06Q50 (Systems or methods specially adapted for a specific business sector) (Table 2). This indicates that classified patents, as being on topic A, are related to data processing systems that support administration, management, and commerce in a specific business sector, which, in this study, is the food sector. The structure of the words most frequently occurring in topic A (Table 2) points out that the patents assigned to topic A are related to tracing and tracking of quality and safety of food products and production processes on their way to consumers. Patents on topic A are mainly oriented to the technical application of blockchain technology (CN114511333A, CN114722117A, US20220198446A1), in combination with the different labeling solutions of QR code (CN105868995), two-dimensional labels (CN108681843A, US20220230134A1), dot-matrix labels (US20200279365A1), DNA tags (US20190285602A1), RFID (CN208400175U, US20220230134A1), NFC (CN105868995A), and different configurations (EP2374017B1). The technical part of topic A includes patents describing the collection of data (IN202011040078A, EP3891501A4). Patents within topic A also include blockchain applications for food safety data collection and tracing (CN114722117A) in different industry sectors, such as the grain and oil sectors (CN114386992A) or the meat industry, with the application of networks of sensors for data collection (CN105868995A), including monitoring from stable to table (CN208077495U), and structuring of data according to HACCP (CN109657996A) principles. Some patents involve decision-making modules that, based on input data, can estimate "pollution score" (US11282023B2) or even recall food products from the market (US20220215353A1). Some patents on topic A are dedicated to the provision of information to consumers, including information about products collected in the food chain (WO2022140465A1, US20210366586A1) or information about prepared meals (EP3899831A1). Based on the provided information, Topic A is labeled as BC-supported tracing and tracking in FSCs. Topic B: The IPC codes most frequently assigned to patents from on Topic B (Table 2) are G06Q10 (Data processing systems: administration, management), G06F16 (Digital computing or data processing equipment or methods, specially adapted for specific applications), and G06Q50 (Systems or methods specially adapted for a specific business sector). Based on this information, it is obvious that, for topic B, the patents related to data processing systems supporting administration and management in FSCs with digital data processing equipment, systems, or methods are included. The structure of words most frequently used in the patent abstract, in the case of patents assigned topic B, indicates that, in these patents, the issues of connecting, detecting, improving, predicting, or supporting of operations in FSCs are treated on the level of the enterprise, storage, cabinet, meal, or cell (Table 2). Based on this information, it seems that patents assigned topic B are most probably related to novel equipment, methods, or systems through which detection, prediction, improvement, or other operations at different levels in FSCs is achieved. Revision of patents on topic B reveals that these patents focus on the description of new apparatus, devices, or methods that can be used for different purposes in different phases of a FSC. New devices and methods described in topic B are: pathogen sampling device (CN114190325A), conversion conveying device (CN216970928U), quality safety sampling and inspection device (CN113552297A), temperature and humidity detection alarm device (CN110542452A), food supply chain hazard prediction method and device (CN113378383A), and improvement of Byzantine consensus method based on food supply chain (CN112330238A). Some patents also deal with new devices for food distribution (CN206451226U, CN103956001B). Based on the provided information, topic B is labeled as devices and methods supporting BC application in FSCs. Topic C: The IPC codes most frequently assigned to patents on topic C are G06Q10 (Data processing systems: administration, management), G06Q50 (Systems or methods specially adapted for a specific business sector), and G06Q30 (Commerce, e.g., shopping or e-commerce) (Table 2). In patents on topic C, the issue of data processing systems and methods in FSCs is upgraded with commercial aspects. The structure of the words most frequently used in patent abstracts, in the case of patents assigned to topic C, indicates that, in these patents, issues of the utilization of novel technologies, such as artificial intelligence, IoT, sensor networks, and other digital solutions for securing, managing, accessing, or learning in agriculture on the level of a farmer, farm, or storage are the focus (Table 2). This information points out that patents assigned to topic C are most probably related to a combination of BCs with other innovative ICT solutions, such as artificial intelligence, sensor networks, Internet of Things, and others. Indeed, analysis of the patent portfolio for topic C reveals that, besides the application of BCs in FSCs, common to patents within this topic, is the use of other novel technologies to improve the use of blockchains in food applications, including IoT (AU2021107272A4, IN201911048905A, IN202121045069A, AU2021106931A4) or a combination of artificial intelligence with IoT (AU2021103789A4, IN202241021982A) for the achievement of diverse management goals, such as, for example, to develop new apparatuses for detecting quality and shelf life of processed seafood (IN201941005285A) or Smart Public Food Distribution Management Systems (IN202241000876A). Based on the provided information, Topic C is labeled as combining BC and other ICT technologies in FSCs. Topic D: The IPC codes most frequently assigned to patents on topic D are G06Q20 (Payment architectures, schemes, or protocols), G06Q30 (Commerce, e.g., shopping or e-commerce) and G06Q10 (Data processing systems: administration, management) (Table 2). According to such assignment of IPC codes, patents on topic D focus more on blockchain application in supporting commercial aspects of FSCs, payment protocols, and e-commerce. The structure of the words most frequently used in the patent abstract, in the case of patents assigned to topic D, confirms this statement, as they indicate that, in these patents, the issues of e-commerce and online shopping and purchasing, as well as the issues of transactions, delivery, packaging, and distribution processes for fresh commodity and other units are the focus (Table 2). Analysis of patents on topic D confirms that they include distribution of food, including intelligent shopping baskets (CN113077314A), purchases incented by donations (US17/323427), end-to-end food delivery systems (US17/249142), platforms for distribution and shopping on-line (CN202111208284.X), and fresh food e-commerce supply chains (CN202111336297.5). Based on the provided information, topic D is labeled as BC-supported trading in FSCs. 3.3. The Most Valuable Patents or Applications within Topics For the studied patent portfolio, forward citations and patent family sums, as well as the sum of these indicators, were analyzed . Because patenting of BC-based inventions intended for FSCs started relatively recently , the value indicators for most patents is consequently quite low. In the case of forward citation, such a result is expected, as there has not been sufficient time for this indicator to grow. On the other hand, low values of family size confirm that patents related to the application of blockchains in FSCs do not yet address the wider market. In order to gain insight into the potentially most influential patents from the portfolio, and particularly into their distribution among topics revealed from LDA, 16 patents with the highest scores of observed value indicators are presented (Table 3) by identified topics. The most outstanding patent regarding value indicators (CA2776577) is on topic C, to which patents combining blockchain and other ICT technologies in FSCs are assigned. This patent presents a system and method for establishing an agricultural pedigree for at least one agricultural product. The highest number of patents with relatively high-value indicators are on topic A, to which patents related to BC-supported tracing and tracking in FSCs are assigned. Such an outcome is quite understandable, since traceability in the FSC is required in food-related regulations worldwide. 3.4. Distribution of Topics by Country of Origin Leading countries holding patents denoted as being topic A, which deals with BC-supported tracing and tracking in FSCs, are the US and China, while a significantly smaller number of patents on this topic have been filed in India, the EU, and other countries . Regarding topic B, China represented 77% of patents, which implies that this country takes the leading role in the development of novel devices and methods supporting the application of BCs in FSCs . The highest number of patents on topic C, related to combining BC and other ICT technologies in FSCs, was submitted in India with 71% of the total number of patent applications. A significant number of patent applications from this topic originate in Australia (18%), while only 11% come from the rest of the world . The highest share of patents on topic D, which deals with BC-supported online trading in FSCs, comes from China (45%), closely followed by the US (33%) . 4. Discussion 4.1. Patenting Trends and Patenting Landscape The first fact confirmed in the present investigation is that patenting activities in the field of application of BCs in FSCs were initiated quite recently, and the number of granted patents is very low. The number of applications started to increase during the last four years, while the value of the patents, in terms of forward citations, is quite low for the majority of patent applications. An exponential increasing trend in BC-related patenting in general started in 2017 and reached 14072 patents in 2021, from 231 in 2016 . Patent analysis in other areas shows similar trends, but with differences, in terms of patents targeting different fields of application. For example, BCs on 5G patent portfolios consisted of over 1000 public and over 120 approved patents in the periods 2014 to 2020 , while, for BC and energy, a study from 2020 revealed 24 approved and 295 public patents . Additionally, the low number of patents with high value in terms of family size points out the absence in global spread of the developed solutions. These observations are aligned with findings from the academic literature, stating that, despite the enormous potential for large-scale improvements in FSCs through the utilization of BCs , its adoption in FSCs is still in the introductory phase , and successful projects and sector-specific studies of BCs in FSCs are still scarce . The situation of BC technology application in other supply chains might be a good indicator of probable development in FSCs. Patent life cycle analysis results indicate that patents related to BCs based on 5G peaked in 2018, followed by a bottleneck period beginning in 2019 . Patenting in the cluster of smart contract applications, privacy preserving and intellectual property, certificate issuance and verification, tokenization, virtual reality gaming, and interoperability is also in a stage of maturity . Such findings indicate that similar fast development might also be expected in the case of BC application in FSCs. However, governance and broader partnership issues required for successful, sustainable applications have still not been solved , and BC technology, although demonstrated as a good solution in some FSCs, is not yet ready for mass acceptance. This situation is the consequence of different barriers associated with the implementation of BC technology in FSCs, which are still present in practice. These barriers include technological, organizational human resources-related, and economic barriers . The barriers and challenges are diverse and related to human capital, such as gaps in understanding , lack of skills and training , and an insufficient number of trained members in the workforce . Barriers are related also to technical issues in application , including scalability , and development and adoption of the most appropriate BC structures for each FSC . Economic barriers include a lack of opportunities for further research , availability of funds and clarity on economic gains , and incentivization , as well as policy barriers, such as lack of regulations . Worth mentioning also are those barriers related to the interest of stakeholders in preserving established hierarchies and their unpreparedness to accept increased obligations related to fraud prevention, as well as waste, losses, and environmental impact reduction . Another interesting observation is related to the countries that have been depicted as leaders in patent activities in the field of BC application in FSCs. Namely, the present analysis highlighted India and China as the countries with the highest number of patents dealing with the above-mentioned issue. The identified situation regarding geographical distribution of patents dealing with BC in FSCs is somewhat different from that of the general distribution of patents related to BCs. In 2021, the largest share of BC-related patents originated from China (36%), followed by the US (26%), while India represented only 2% of patents . When it comes to China, one of the possible explanations for the expansion in this area, and quite possibly in related areas, might be an adoption of the country's strategic development, which will ensure its independence in the IT domain from foreign technologies . Regarding India, the patents are primarily focused on applying BCs in combination with other internet technologies, which can be attributed to the rapid development of the IT sector in that country and its orientation toward export . 4.2. Application Areas It might be interesting to analyze the drivers that resulted in the development of paths, represented by LDA-identified topics, in development and patenting in the field of application of BCs in FSCs. The most frequently assigned IPC to the patents related to BC application in FSCs was G06Q, for all identified latent topics. However, the analysis of 335 patents related to BC technology in general awarded in the US, as of 2020 , revealed that the most frequents IPC codes were H04L (transmission of digital information, e.g., telegraphic communication) present in 84% of patents, followed by G06F (electric digital data processing) present in 53% of patents, while G06Q (data processing systems or methods, specially adapted for administrative, commercial, financial, managerial, supervisory, or forecasting purposes) was assigned to 33% of analyzed patents. This observation points out the somewhat specific focus of patents related to BCs in FSCs. BC technology is characterized by quite wide possibilities for application in different fields. Backman et al. identified that, beside financial transactions in cryptocurrencies, integrity verification in insurance, data management in human resources, privacy and security in secure storage, reputation of education, governance of e-voting, integrity of IoT business, electronic health records, and, finally, business industry supply chain are also prospective fields for BC application. For BCs in FSCs as the subfield, the latest research by Zhao et al. points out, based on findings from the academic literature, that applications have been developed in four main directions comprising, in addition to traceability, manufacturing, water management, and information security. In the present research, we confirm that the most developed field of BC application in FSCs is related to traceability enhancement. Considering that traceability represents a legal obligation in FSCs, it is quite understandable that the largest number of patent applications is registered in the domain of application of BC technology for tracing and tracking along FCSs . Achieving internal traceability within a company involved in FSCs requires extensive engagement of human resources, as well as continuous record-keeping. Those processes can be significantly facilitated by applying contemporary digital technologies . In terms of external traceability along the FSC, the issue of trust and confidence in records is circulating between companies in the FSC . Thus, BC technology represents a logical solution, since it is designed to overcome such problems. It also reduces the level of human engagement while increasing the level of confidence, not only among FSC members, but also among consumers. Another important element that additionally simplifies the process of data collection within FSCs are devices and instruments that enable direct upload of measured or registered data into digital databases (EP3520350B1). The application of such equipment and its coupling with BCs, as a technology-enabling reliable collection and storage of original data, represents a prerequisite for further progress in this area. These circumstances have logically contributed to the development of technical solutions and their patenting in the field of development of devices and methods which support the application of BCs in FSCs. There are numerous platforms, IoT applications, patents using artificial intelligence, and sensor networks that have been developed to enable communication and facilitate specific operations and transactions in FSCs . These systems use or generate a significant quantity of important information. Confidence in and reliability of these data emerge as an issue, representing in many cases an obstacle for their acceptance and application on a large scale . In this case also, BCs represent an ideal solution and, thus, a driving force for patentable innovation in this domain. The authors of also agree that it is important that the introduction of blockchain technology in FSCs is not limited to this single technology, but rather to a whole stack of new technologies, including sensing technologies, big data analytics, artificial intelligence, Internet of Things robotics, digital twins, cyber-physical systems, 3D printing, and others. As the patents on topic C are mainly dealing with the implementation of IoT and other novel technologies, it could be assumed that development IT (development of mobile networks and accessibility of satellite communication) can be used to overcome remote agricultural production, but also remote consumption in Australia, while development of IT communications in India allows for overcoming the problem of missing conventional infrastructure. Finally, online trade is becoming part of the daily activities in many economic sectors, including food , and the application of BCs as a technology supporting online trade solutions is, therefore, another tenable field of innovation and patenting. Renda agrees that benefits of introducing BC technology in FSCs may address much wider issues related to food security and safety and human health in general, reduction of food waste and losses, animal welfare and climate change , as well as the development and digitalization of rural areas . 4.3. Further Prospects The structure of the analyzed patent portfolio reflects realistic needs for improvements in FSCs, as well as the developmental needs related to the enabling of BC applications to address these needs. The increasing trend in the number of patents related to the application of BCs in FSCs during the recent few years indicates the possibility for increased application of BCs in FSCs. The progress of BC applications in FSCs in the future also depends on the implementation of measures aimed to remove identified barriers for the application of BCs in FSCs. Regulations introducing BCs as a technology to support traceability in FSCs, as well as regulatory arrangements related to this issue, would undoubtedly support the process of introduction of BCs in FSCs. Another direction toward widening of the application of BCs in FSC might be the identification of stakeholders in FSCs motivated to voluntarily adopt the implementation of BCs as a tool for improvement of their market position. Some of the early potential adopters of BC technology in FSCs are depicted in the academic literature, and they can be divided into two groups. The first group represents the producers of added-value food, motivated to improve the traceability of their products by using BCs, since it will enable them to more easily secure confidence in origin or other quality trade which separates their product from other similar products and provides all the necessary data to prove it more easily. This group of producers will benefit from consumers' confidence in the origin and quality of their products. Therefore, this group of users has a relatively high probability of spontaneous implementation of BC solutions. Representative users from this group are producers of organic products , halal food , and traditional food . This observation is supported, also, through the fact that almost half of the initiatives and projects registered in the field of BC application in FSCs is targeting food integrity. On the other hand, the main goal for the application of BCs in the second group is to secure food safety and traceability in complex FSCs. Some stakeholders will implement BC technology to prevent or detect fraud and trust issues, as in the case of pork and mango in supermarkets , while others will improve traceability to further improve quality. Examples of the latter are dairy production , edible oil production and distribution , and beef production . The fact that BC technology is attracting the attention of numerous stakeholders is also confirmed through the existence of many projects and initiatives that aim to contribute to the establishment of a trusted environment, are the building of more sustainable, more transparent, and more integrated FSCs. Based on the analysis of projects and initiatives related to BCs in FSCs targeting FSC issues, besides the dominant issue of food integrity (50% of identified initiatives), there were also issues of supervision and management of FSCs (14.5% of identified initiatives), waste reduction and environmental awareness (10%), support of small farmers (16%), food safety (6%), and food security (4%) . To strengthen the further application of BCs in FSCs, it is necessary to clearly emphasize the contribution of this technology to the economic performance of FSC stakeholders and FSCs as a whole. Additional funding of research targeting technical barriers and obstacles, as well as the development of educational and promotional strategies, might also contribute to further growth of BC application in FSCs. Finally, the number of potential users in FSCs is expected to grow over time, as the development of BC technology provides numerous benefits that will become more significant than the mere mandatory effect of its application. A most recent study providing content-based analysis of the academic literature revealed that BC application in the agrifood sector is a relatively recent topic that started to gain traction in 2018, when the research community started intensively dealing with this issue, with more than 75% of identified related records in the academic literature being published in 2020 and 2021. The identified sharp increase in the number of patents and patent applications related to BC application in FSCs is a reflection of vigorous research activities, resulting in ready-to-implement solutions in this field. The obtained results will support research community members in making decisions related to the development of high-readiness level products in the field of BC application in FSCs. The main limitation related to present research is the lag period of patents, which has certainly contributed to incomplete results within the past year. The second limitation is related to the fact that patent application does not necessarily result in an approved patent, which was taken into consideration, but, due to the low number of approved patents, they had to be taken into account for this analysis. However, the clearly identified increasing trend in the number of patents suggests that these limitations most probably do not affect the obtained results to a great extent. However, the development of BC applications in FSCs is closely related to integration with other information and communication technologies, in particular IoT. Finding that combination of BC technology with other information and communication technology, and IoT in particular, has been adopted for the improvement of FSCs was already emphasized in . 5. Conclusions BCs have great potential for application in FSCs. Numerous research activities have focused on the possibility of applying BCs in FSCs, but, as a consequence of numerous barriers, this technology is not yet widely applied in FSCs. The process of patenting in the field of application of BC technologies in FSCs started relatively recently, and the highest number of patents originate from China, India, and the US. The analysis of latent topics using LDA indicates that inventions related to the application of BCs in FSCs are patented in four key areas: (A) BC-supported tracing and tracking in FSCs; (B) devices and methods supporting the application of BCs in FSCs; (C) combining BCs and other ICT technologies in FSCs; and (D) BC-supported trading in FSCs. The countries of the patent applications differ significantly by topic, with China leading, in the case of BC-supported tracing and tracking (A), development of devices and methods supporting the application of BCs in FSCs (B), and BC-supported trading in FSCs (D), while, in the case of combining BCs and other ICT technologies in FSCs (C), the dominant share of patents is from India. In the case of the US, patent activities are significant in the case of BC-supported tracing and tracking (A) and BC-supported trading in FSCs (D). Based on the indicators, the value of patents were relatively low, which, in the case of patent forward citation, can be attributed to the short time that has elapsed since patent application, while, in the case of family size, it confirms that the application of blockchains in FSCs has not yet been widely accepted, and it is still rather a matter of solving case-level challenges. The most influential are patents on the topic area related to BC-supported tracing and tracking in FSCs, which can be attributed to the fact that traceability in FSCs is a legal obligation for all entities involved in food supply, which further increases the need to simplify the extensive work of food tracing and tracking and make it more reliable by applying new IT solutions. The exponential growth in the number of patent applications in the field of BC technology application in FSCs suggest that a significant increase in the adoption of this technology can be expected in the near future. Development and implementation of measures for the mitigation of barriers related to the introduction of BC technology in FSCs would undoubtedly accelerate this process. Author Contributions Conceptualization and Experimental Design: J.M., D.K., Z.K. and G.O.; Statistical analyses: D.K.; Results analysis and Data curation: J.M., D.K. and Z.K.; Writing--Original Draft Preparation: Z.K. and D.K.; Writing--Review and Editing: Z.K., J.M., R.K., M.D. and D.U.S.; Supervision: D.K. and G.O. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement The data presented in this study are available on request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Screenshot of an example of a relational table for collected patent documents. Figure 2 Predictive likelihood of models with different number of topics. Figure 3 Example of topics heat map resulting from the LDA Analysis. Figure 4 Patent portfolio publication trend (A) and authorities of the portfolio applications (B). Figure 5 PCA-based distribution of patents in four LDA topics in the factorial plane. Figure 6 Frequencies of occurrence of the twenty most frequent keywords by topics. Figure 7 Assessment of the value of patents in the analyzed patent portfolio. Figure 8 Distribution of patents within topics by country of origin. foods-12-01036-t001_Table 1 Table 1 Pseudocode of LDA generative process. choose a multinomial distribution thd for document d (d {1,..., M}) from a Dirichlet distribution with parameter a choose a multinomial distribution bk for topic k (k {1,..., K}) from a Dirichlet distribution with parameter e for each word wn (n {1,..., Nd }) in document d select a topic Zn from thd select a word wn from bZk,n foods-12-01036-t002_Table 2 Table 2 Input data for latent topic patent portfolio analysis and labeling. Hidden Topic Topic Label (Proposed by Authors) Words among the 20 Most Common Words Three Most Frequent IPCs Verbs Nouns WIPO IPC (Frequency) Description of IPC A BC SUPPORTED TRACING AND TRACKING IN FSC inform trace track code provide generate relate receive manage configure traceability quality safety process product consumer G06Q10 (19) Data processing systems: administration, management G06Q30 (11) Data processing systems: commerce G06Q50 (7) Systems or methods specially adapted for a specific business sector B DEVICES AND METHODS SUPPORTING BC APPLICATION IN FSC connect detect comprise improve predict support arrange cabinet storage enterprise cell meal G06Q10 (5) Data processing systems: administration, management G06F16 (5) Digital computing or data processing equipment or methods, specially adapted for specific applications G06Q50 (4) Systems or methods specially adapted for a specific business sector C COMBINING BC AND OTHER ICT TECHNOLOGIES IN FSC secure manage access learn technology artificial intelligence IoT base network sensor digit farmer agriculture quality storage farm G06Q10 (25) Data processing systems: administration, management G06Q50 (11) Systems or methods specially adapted for a specific business sector G06Q30 (9) Commerce, e.g. shopping or e-commerce D BC SUPPORTED TRADING IN FSC shop purchase transact deliver distribute process interact store package fresh commodity unit field online e-commerce G06Q20 (9) Payment architectures, schemes or protocols G06Q30 (8) Commerce, e.g. shopping or e-commerce G06Q10 (5) Data processing systems: administration, management foods-12-01036-t003_Table 3 Table 3 The 16 potentially most influential patents from the observed patent portfolio by identified topics. Application Number Title Topic Family count Citation Family + citation Topic A: BC SUPPORTED TRACING AND TRACKING IN FSC EP2008875543 High-reliability product/activity tracking system A 10 24 34 US16/386147 DNA Based Bar Code for Improved Food Traceability A 6 19 25 US17/159123 Food Supply Tracking, Verification, and Feedback System A 8 14 22 AU2020203178 A Machine Type Communication System or Device for Recording Supply Chain Information on a Distributed Ledger in a Peer to Peer Network A 4 14 18 CN201811587331.4 Food tracing and query analysis system and method based on a HACCP system A 1 15 16 EP2019892440 System, device, and process for tracking product A 6 9 15 US17/257503 Enterprise Consumer Safety System A 2 9 11 CN201610175359.1 Meat food supply chain tracing method based on RFID, QRCode and NFC A 2 9 11 US15/378124 Supply chain tracking of farm produce and crops A 2 8 10 US16/875011 Dot-matrix product information encoding for food traceability A 5 3 8 TOPIC B: DEVICES AND METHODS SUPPORTING APPLICATION OF BC IN FSC CN201410151778.2 Internet fast food distribution cabinet B 2 12 14 CN202010543066.0 Fresh food supply chain knowledge graph construction method based on semi-structured data B 1 9 10 TOPIC C: COMBINING BC AND OTHER ICT TECHNOLOGIES IN FSC CA2776577 A system and method establishing an agricultural pedigree for at least one agricultural product C 13 90 103 EP2019823567 Systems and methods for permission blockchain infrastructure with fine-grained access control and confidentiality-preserving publish/subscribe messaging C 6 10 16 US16/978312 Non-specific, wireless detection of electrically or magnetically labeled bacteria and/or virus C 4 3 7 TOPIC D: BC SUPPORTED TRADING IN FSC EP2017761847 System and method for determining or monitoring a process variable in an automation plant D 6 22 28 Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). 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PMC10000829 | Breast cancer represents one of the most common tumor histologies. To date, based on the specific histotype, different therapeutic strategies, including immunotherapies, capable of prolonging survival are used. More recently, the astonishing results that were obtained from CAR-T cell therapy in haematological neoplasms led to the application of this new therapeutic strategy in solid tumors as well. Our article will deal with chimeric antigen receptor-based immunotherapy (CAR-T cell and CAR-M therapy) in breast cancer. chimeric antigen receptor (CAR) macrophages T cells immunotherapy breast cancer This research received no external funding. pmc1. Introduction Breast cancer (BC) is the most common female cancer worldwide. According to Globocan, it is the number one diagnosed cancer with an estimated 2.3 million new cases (11.7%) globally in 2020, and is the fifth leading cause of cancer mortality . The BC incidence is increasing, especially in highly developed countries where screening strategies help to reduce cancer mortality while in poor-developing countries, the BC incidence remains low but the mortality rate is still higher . However, in advanced countries, the diagnosis of de novo metastatic BC still represents approximately 3% to 6% of new BC diagnoses and has not declined despite the wide diffusion of mammography screening . BC is a very heterogeneous disease, clinically distinguished into several subtypes according to the expression of hormone receptors (HRs) and the human epidermal growth factor receptor 2 (HER-2) status: luminal BC, HER-2 positive BC, and triple-negative BC (TNBC). HR and HER-2 are the targets for numerous specific treatments in early and advanced settings. TNBC is defined by the lack of expression of HR and HER-2 and accounts for approximately 15% of all BCs . TNBC has long been considered a major unmet need due to its aggressive behavior and poor prognosis related to its deficiency of specific therapeutic targets. These tumors tend to relapse early and rapidly metastasize in the lungs, liver, and central nervous system, determining a worse survival . For these reasons, chemotherapy is still a cornerstone in the treatment of this BC subtype. Immunotherapy with checkpoint inhibitors was shown to be effective in TNBC, especially in the case of programmed death 1 (PD-1) expression in tumor tissue . Furthermore, TNBC has the highest tumor mutational burden (TMB) among all BC subtypes . A high mutational level can lead to the production of tumor "neoantigens" which could be recognized by antigen-presenting cells in the tumoral microenvironment (TME) enhancing antitumor immune response . Even if BC has always been considered a poorly immunogenic tumor, TN and HER2+ subtypes show considerable immune infiltration. As a demonstration, tumor-infiltrating lymphocytes (TILs) are frequently present in TN and HER2+ tumor samples and are associated with good prognosis and are predictive of immunotherapy efficacy . Otherwise high immune infiltration has a completely different effect in the luminal subtypes and lobular BC, suggesting a bad prognosis . Especially for TNBC, the intrinsic molecular characteristics (determined by mRNA profiles, gene expression, and proteomics) can distinguish different intrinsic subtypes of TNBCs defined as basal-like 1 or 2, luminal androgen receptor, and mesenchymal tumors . Each intrinsic subtype is associated with an individual TME, shaped by the molecular features and genomic signatures of cancer cells . The quality of immune cells and distribution in the tumoral tissue is also important in TNBC, distinguishing between "cold tumors" and "inflamed tumors" and inflammation areas in the stromal tissue, margins, or fully inflamed tumoral tissue . Generally, immune-rich early TNBCs have less clonal heterogeneity, somatic mutation, and a minor expression of neoantigens but a high expression of TILs, CD8 + T cells, or memory T cells . Conversely, metastatic sites seem more heterogeneous and immunodepleted with fewer TILs, CD8 + T cells, or dendritic cells, a low TMB, and increased clonal diversity . In the metastatic environment, there is instead a greater presence of metastasis-associated macrophages (MAMs) with a pro-tumoral phenotype that is able to increase immune escape strategies and cancer diffusion . Tumor-mediated immune suppression is a real issue responsible for acquired resistance to active immunotherapy (ICIs) . Targeting the immune system with a combination of different targets, especially in advanced BC, will become a valuable therapeutic strategy to achieve the best survival results. This strategy aims to convert non-responders to responder patients, maintain an achievable lasting response and overcome acquired immune resistance. Interfering with immune evasion, promoting the antitumor phenotype of immune cells, or enhancing antitumor immunity are the expected goals . Many early-phase clinical trials are ongoing in several solid tumors (including BC patients) with new active compounds targeting macrophages or neutrophils . New emerging treatments in solid tumors are now being used in immunotherapy after the incredible results that have been achieved in the onco-hematology field. Among these is adoptive cell therapy (ACT), that exploits TILs or T cells genetically engineered to express modified T-cell receptors (TCR) or chimeric antigen receptors (CAR). CAR-based therapies with T cells or natural killer (NK) cells are promising as potential practice-changing effectors in BC, especially for tumors with poor targetable antigens (like TNBC), even if there are still significant limitations depending on the resistance of an unfavorable TME and side effects . The presence of an extracellular matrix (ECM) in the tumor stroma constitutes a physical barrier to the transfer of CAR-T cells. Macrophages engineered with CAR (CAR-M) may overcome this barrier by producing metalloproteinases and can enhance the antitumor effect thanks to antigen-specific phagocytosis . In our review, we give an overview of the potentiality of CAR-based therapies in BC. 2. CAR Molecule Structure A CAR is an artificial fusion protein that is composed of an extracellular antigen binding domain which includes an antigen recognition domain, for example, a mAb-derived single chain variable fragment (scFv) that is involved in the binding between the T cell and a tumor-associated antigen (TAA) . A hinge region is linked with the scFv, providing CAR flexibility; its length can be modified to optimize the distance between CAR-T cells and targeted cancer cells and ameliorate the signal transduction process . Moreover, a transmembrane domain is involved in intracellular signal transmission pathways. For this purpose, this region includes both costimulatory and signaling domains (e.g., CD3z, also called CD247) responsible for CAR-T cell activation . 3. The CAR-T Cell Generations Ameliorating CAR vectors can improve the safety and efficacy of CAR-T-cell therapy . For this purpose, several CAR generations have been conceived, and currently, the fifth generation is already being tested . The principal differences among the CAR generations consist of specific costimulatory molecules. The first generation contains only the CD3z signaling end domain, whose linking with the extracellular scFv modifies and activates T cells . However, due to its short survival time and incomplete T-cell activation, it was necessary to conceive a second and third generation of CARs, which include one or two additional costimulatory molecules (respectively), such as CD27, CD28, 41BB, ICOS, and OX-40. These molecules increase the cells' persistence and cytolytic capacity . T cells redirected for universal cytokine-mediated killing (TRUCKs) or "armored CAR-Ts" represent the fourth generation of CARs, which includes a nuclear factor of the activated T cells (NFAT) domain . The domain promotes cytokine secretion, mainly interleukin (IL)-12, IL-15, and the granulocyte-macrophage colony-stimulating factor (GM-CSF), which aims at modulating the anti-tumor microenvironment. In fact, armored CAR-Ts carry out a simultaneous antitumor activity directed to both tumor cells expressing and not expressing the CAR-targeting antigen. The other advantage of this strategy is determined by the local release of IL-12, with a lower risk of systemic toxicity related to the cytokine secretion . Such CAR-Ts can be tested for targeting TNBC-related antigens. In recent years, a fifth generation of CARs has been developed. It contains a fragment of IL-2 receptor b (IL-2Rb), which induces the secretion of Janus kinases (JAKs) and signal transducer and activator of transcription (STAT)-3/5 . Such novel CAR generation aims to avoid terminal phenotypic differentiation of effector cells; consequently, fifth generation CAR is able to promote their expansion in vitro, and their persistent cytotoxicity in vivo . 4. Targets for CAR-T Cell Therapy in BC The development of CARs has led to searching for new targets for cancer therapy, especially for histologies without ERBB2 and HR expression , such as TNBC . All studies testing potential targets for CAR-T cell therapy in BC tumors are shown in Table 1. Abbreviations: CAR: chimeric antigen receptor; CEA: carcinoembryonic antigen; EpCAM: epithelial cell adhesion molecule; FR: folate receptor; HER2: human epidermal growth factor receptor 2; MUC1: Mucin1; PRLR: prolactin receptor; ROR1: receptor tyrosine kinase-like orphan receptor; TEM8: tumor endothelial marker 8; TNBC: triple-negative breast cancer; VEGFR1: vascular endothelial growth factor receptor 1. 4.1. Integrins In our research, we paid attention to the membrane receptors to identify a specific target for CAR molecules. In this context, Integrins represent a potential target for CARs because of their proven involvement in cell proliferation and metastatization and because of their high expression in BC . In particular, CAR molecules targeting avb3-integrin were conceived and tested, showing their cytolytic activity against different tumors in vitro, including MDA-MB-231 TNBC cell lines. After testing these molecules in vivo, some complete responses were reported in mice that were affected by metastatic melanoma . Moreover, as reported by some studies, this therapy demonstrated selective cytotoxicity against avb3-expressing cell lines without involving normal cells . Therefore, based on what has been reported, avb3CAR-T cell therapy seems promising and deserves further study to verify its efficacy in BCs. 4.2. Mesothelin Another potential target for CAR molecule development is Mesothelin, a tumor differentiation glycoprotein that is involved in cell adhesion, which is normally expressed on the mesothelial cells but is overexpressed in several solid neoplasms including TNBC . Its activity in oncogenesis through different cell signaling pathways such as MAPK, PI3K, and NF-kB has been reported . Since its overexpression has been reported in 67% of TNBCs, with a limited expression in normal breast cells , Mesothelin represents an appealing target for CAR molecule development. In this regard, Hu et al., evaluating the Mesothelin expression on three TNBC cell lines, such as MDA-MB-231, BT-549, and Hs578T, reported that only BT-549 cells expressed the molecule. Then, the authors produced second-generation mesothelin-redirected CAR-Ts and tested it in vitro and in vivo. It is noteworthy that the researchers disrupted the gene locus of PD-1 in T cells before CAR transgene insertion. Their CAR-Ts demonstrated an interesting increase in antitumor activity and cytokine secretion against PD-L1-expressing tumor cells in culture . This is probably due to the high expression of PD-L1 in TNBC cells , suggesting a potential use of these CAR-Ts in order to overcome the suppressive effects of PD-1/PD-L1 axis in BCs . Thanks to these interesting data, some clinical trials have been developed and are currently underway, with the aim of evaluating the activity of CAR molecules in TNBCs. In particular, a Phase I clinical trial (NCT02792114) is evaluating the safety and tolerability of Mesothelin-redirected CAR-Ts in metastatic/advanced Mesothelin-expressing BCs, including TNBCs. Another Phase I/II clinical trial (NCT02414269) is testing second generation Mesothelin-redirected CAR-Ts in different tumors, including BC. Moreover, two other clinical studies (NCT02580747 and NCT01355965, the latter only in Mesothelioma) have been completed but no official data have been published yet. 4.3. TEM8 Some studies have demonstrated that the endothelium of several neoplasms often overexpresses an integrin-like protein called tumor endothelial marker 8 (TEM8), also known as ANTXR1, and is usually expressed during endothelial cell development but rarely in adults . As evidence of this, an elevated expression of TEM8 was found both in invasive/relapsed BC and in several BC cell lines , so the upregulation of this molecule could represent a potential target for CAR-T cell development . In this regard, a single dose of a specific L2CAR-T cell therapy, derived from the L2 Monoclonal Antibody (Mab) against TEM8, showed a complete response against TNBC in vitro and significant cancer reduction in vivo and TNBC xenografts . For these reasons, TEM8 represents a promising target for CAR-T cell therapy against TNBC. 4.4. MUC1 In TNBC, MUC1 represents a highly selective overexpressed target . It is a glycosylated transmembrane molecule with altered epithelia . It produces mucin, which protects cells against pathogens . Tumor cells overexpress MUC1, activating the intracellular pathways involved in cancer proliferation . Jiang et al.'s recent cohort study, involving more than 5800 BC patients, demonstrated the predictive role of MUC1 and its correlation with a poor prognosis . In particular, neoplasms overexpress a hypo-glycosylated variant of MUC1, also known as tumor-specific MUC1 (tMUC1) which exposes new epitopes for the immune system . For this purpose, antibodies specifically binding to tMUC1 have been developed and tested . One of these molecules, the so-called TAB004, served as a reference point for creating a CAR molecule containing its extracellular scFv. The derived CAR-T cells, known as MUC28z CAR-T cells demonstrated their efficacy in heightening the expression of both leukocyte activation markers and cytokines in vitro. These effects led to significant cell lysis in vitro and a reduction in cancer cell growth in vivo . Recently, a new CAR molecule targeting tMUC1, known as huMNC2-CAR44, has been activated in a clinical trial recruiting 69 (HER2-positive, HER2-negative, triple-negative) BC patients; the estimated study completion date is 15 January 2035 (NCT04020575). Another Phase I study is currently testing the safety, tolerability, feasibility, and preliminary efficacy of the administration of CAR-T cells targeting tMUC1 in 112 patients with advanced tMUC1-positive solid tumors (including BC) and multiple myeloma. The estimated study completion date is 31 October 2036 (NCT04025216). 4.5. ROR1 Receptor tyrosine kinase-like orphan receptor (ROR)1 is a highly expressed molecule during embryogenesis but not in adults. BC cells highly express ROR1, especially in cases with a poor prognosis; ROR1 overexpression was found in some TNBC cell lines (e.g., in MDA-MB-231) but not others . ROR1-based CAR-T cell therapy was also shown to induce MDA-MB-231 apoptosis in tumor models through significant IL-2 and IFNg production . A Phase I trial is testing the ROR1-specific CAR-T cells' efficacy in 60 subjects with hematological and solid tumors, including triple negative BC. Patients will be followed up for approximately 15 years after study completion. The estimated study completion date is 1 December 2023 (NCT02706392). The first study results were recently published, suggesting a better CAR-T cell therapy efficacy from adding Oxaliplatin to the lymphodepletion regimen . Another Chinese Phase I study is currently recruiting 40 patients with advanced solid tumors (including BC) to investigate the efficacy of TILs and CAR-TILs against several molecular targets, including ROR1, MUC1, HER-2, Mesothelin, PSCA, EGFR, GD1, GPC3, Lewis-Y, AXL, Claudin18.2/6, and B7-H3. The estimated study completion date is 1 January 2035 (NCT04842812). 4.6. Natural Killer Group 2, Member D Ligand (NKG2DL) Under certain pathological conditions, both innate and adaptive immune cells (including CD8+ and some CD4+ T cells, NK cells, gd T cells) express a type II transmembrane protein, called natural killer group 2, member D (NKG2D) , which in turn contribute to enhancing cytotoxicity and production of cytokines by effector cells and promoting their proliferation and survival. Moreover, NKG2D can cooperate with other receptors (including TCR in T cells or NKp46 in NK cells) by acting as a costimulator for their responses . In tumor cells, including TNBC cells, an upregulation of stress-induced ligands has frequently been reported; NKG2D can naturally recognize these ligands so it has been considered as a potential target for immunotherapy in several studies. CAR molecules that were obtained by fusion of the full-length NKG2D with the CD3z cytoplasmic region together with endogenous DAP10 costimulation, were demonstrated to react with NKG2DL-expressing tumor cells through cytokine and chemokine production, thus enhancing cytotoxicity . These results were also confirmed by in vivo studies . More recently, NKG2DL-redirected CAR-Ts were tested by Han et al. in TNBC cell lines and TNBC mouse models . In this case, NKG2DL-redirected CAR-Ts were obtained by the fusion between the extracellular domain of human NKG2D and the TCR CD3z alone or co-stimulatory domains, such as 4-1BB or CD27. The authors demonstrated that the elevated expression of CD25 and the presence of IL-2 were required to promote CAR-T expansion in vitro in the absence of any costimulatory domains. Moreover, NKG2DL-redirected CAR-Ts were able to recognize and kill TNBC NKG2DL-expressing MDA-MB-231 and MDA-MB-468 cell lines . Based on these results, a Phase I clinical trial (NCT04107142) evaluated the safety and tolerability of NKG2DL-redirected CAR-T cells in patients with various solid neoplasms including TNBC, but, to date, no results have been reported yet. 4.7. Chondroitin Sulfate Proteoglycan 4 (CSPG4) CSPG4 is a hyperglycosylated transmembrane protein with a low expression in normal tissues and hyperexpressed in several tumor types, including TNBC. It has been suggested that CSPG4 is involved in the neuronal network regulation and epidermal stem cells homeostasis . Second-generation CSPG4-redirected CAR-Ts were tested in various CSPG4-expressing cell lines (including SENMA, UACC-812, CLB, MDA-MB-231, MILL, PHI, and PCI-30), and demonstrated a significant capacity in cell growth suppression . The same results were obtained in preclinical mouse models of several human tumors (including BCs). In another study, second-generation CSPG4-redirected CAR-Ts using murine-based scFvs reported target antigen-dependent cytotoxicity and cytokine secretion against several tumor (including BC) cell lines . 4.8. EpCAM Epithelial cell adhesion molecule (EpCAM) represents a well-known molecule whose expression has been related to poor prognosis and tumor metastatization . Several treatment strategies targeting EpCAM have shown benefits for different tumor types. Currently, a Chinese clinical trial is recruiting patients with nasopharynx neoplasms and BC, which aims to evaluate the safety of the engineered CAR-T cells recognizing EpCAM. These molecules were developed through lentiviral transduction of the third generation of CAR genes. Different patient cohorts will receive the experimental treatment in a dose-escalating manner; the estimated study completion date was set for July 2022 (NCT02915445). 4.9. Intercellular Adhesion Molecule-1 (ICAM-1) ICAM-1 is a transmembrane protein that is involved in white blood cell diapedesis. It is overexpressed on the surface of many cancer cells, including TNBC cells . ICAM-1 plays a role in tumor growth, invasion, and metastasis . In order to avoid CAR-T-related cytotoxicity in normal cells, Park et al. generated CAR-Ts with micromolar (instead of nanomolar) affinity, and demonstrated that these ICAM-1-redirected CAR-Ts were more efficacious and safe than their higher affinity homologs . More recently, the same results were confirmed in preclinical models . 4.10. HER-2 BC with HER2 overexpression represents the tumor subgroup for which CAR-T cells have been designed . Several clinical trials are currently ongoing to test CAR molecules targeting HER-2. One of them is a Phase I trial, testing the safety and preliminary therapeutic efficacy of CCT303-406 cells in 15 patients with HER-2-positive stage IV solid tumors (that have failed standard treatment of relapsed or difficult-to-treat), including BC. The estimated study completion date is April 1, 2023 (NCT04511871). An American, multicenter, Phase I/II trial is currently ongoing, recruiting 220 patients with HER-2-positive tumors, including BC, to evaluate the safety, tolerability, and clinical activity of HER2-specific dual-switch CAR-T cells, BPX-603, administered with rimiducid. The estimated study completion date is January 2, 2025 (NCT04650451). Another American dose-escalation study is being conducted at the City of Hope Medical Center (Duarte, CA) to investigate the side effects and the best dose of HER2-CAR-T cells in treating patients with BC metastasized to the brain or leptomeninges. For this purpose, 39 patients will receive HER2-CAR-T cells via intraventricular administration over five minutes once weekly for three doses, which could be implemented at the principal investigator's discretion. Patients will be followed up at the end of treatment at 4 weeks, 3, 6, 8, 10, and 12 months, and then for up to 15 years. The estimated study completion date is 31 August 2023 (NCT03696030). A third American Phase I trial is currently studying the safety and efficacy of combining HER2-specific CAR-T cells with an intra-tumor injection of CAdVEC, an oncolytic adenovirus designed to help the immune system activation against cancer. Our trial is recruiting 45 patients with HER-2 positive tumors. The estimated study completion date is 30 December 2038 (NCT03740256). However, although most patients did not have significant complications. In some cases, the non-specificity of HER-2 expression between tumor cells and healthy cells can lead to serious side effects; a case of cardiopulmonary failure from excessive T-cell activation has been reported . Therefore, to avoid drawbacks related to the non-tumor-specificity of the marker, the research sought to deepen its understanding of the receptor to evaluate whether it was possible to find a more specific variant in BC. In this regard, a potential antigen is p95HER2, a truncated version of HER2 was found in 40% of HER2-positive BCs. This variant is more tumor-specific than the constitutive form since it was not found in normal breast cells. p95HER2 has already been evaluated as a target for a bispecific antibody against cancer cells in vitro and in vivo without significant side effects. Due to the encouraging results reported, this variant could, therefore, be a future target for developing CAR-based therapies . 4.11. VEGF Vascular endothelial growth factor receptor (VEGFR)1 is a Tyrosin-kinase receptor that is involved in the migration and survival of hematopoietic stem cells, and its overexpression is related to the process of BC metastatization . Therefore, VEGFR1 represents a potential candidate for immunotherapy. To date, VEGFR1 was tested as a part of a VEGFR1-CD3 bispecific antibody and demonstrated promising results against MDA-MB-231 and MDA-MB-435 TNBC cell lines. These results warrant further studies on VEGFR1 activity, for example as a target for CAR-based therapies . Moreover, because the main functions of the normal endothelial cells are VEGFR2-dependent , VEGFR1 inhibition could prevent endothelial toxicity. At present, this is only a hypothesis, so further investigation is needed. 4.12. c-MET Hepatocyte growth factor receptor, also called c-Met, is a cell-membrane protein tyrosine kinase that is expressed in several types of solid neoplasms, including BC . Onartuzumab, an anti-c-Met monoclonal antibody, has been administered in patients with metastatic, solid tumors . Tchou et al. tested c-Met as a potential target for CAR-T cell therapy; for this purpose, the scFv of the CD19 binding domain of a CD19-CAR molecule was substituted for that of onartuzumab, and then its effectiveness against BC cells was confirmed in vitro and in vivo . Subsequently, the new c-Met CAR-T cells were administered through a single intratumoral mRNA injection in a BC patient cohort (NCT01837602). The injections were well tolerated, and no significant drug-related adverse events were reported. Moreover, analyzing tumor specimens (four TNBC and two ER+ HER2-negative BCs) in which the CAR-T cells were injected, there was wide tumor necrosis at the injection site and macrophage infiltrates within the necrotic areas . 4.13. AXL AXL, a receptor of the TAM tyrosine kinase receptor family, and its high-affinity ligand, called growth arrest-specific protein 6 (GAS6), are involved in cancer cell expansion, metastasization, and survival; moreover, AXL low expression in adult normal cells and its overexpression in several tumor types (including BC) and some cell lines (including MDA-MB-231 ) make AXL a potential target for CAR molecule development . Wei et al. developed AXL-redirected CAR-Ts using an AXL-specific scFv; these CAR-Ts were tested in AXL-expressing TNBC cell line MDA-MB-231, and demonstrated an antigen-dependent cytotoxicity and cytokine production; these results were confirmed in an in vivo evaluation in MDA-MB-231-established xenograft models . Other researchers generated AXL-redirected CAR-Ts with a constitutively activated IL-7 receptor (C7R); they demonstrated a significant tumor cell killing capacity, which was more efficacious than by using conventional AXL-redirected CAR-Ts, in TNBC MDA-MB-231 and MDA-MB-468 cell lines. This improvement was probably due to the co-expression of C7R, which helped to prolong survival and reduce rates of tumor relapse . However, further investigations are required to confirm these results. 4.14. Disialoganglioside GD2 GD2 is a surface protein that is normally expressed only in peripheral nociceptors, neurons, and melanocytes; consequently, GD2 expression has been demonstrated in neuroectoderm-derived tumors, such as melanomas and neuroblasomas . Its cell-type restriction render GD2 a potential target for CAR molecule development. In fact, GD2 has been studied mainly as a target for treatments against neuroblastoma . However, more recently, Seitz et al. used the scFv derived from anti-GD2 mAb dinutuximab to produce GD2-redirected CAR-Ts. The researchers evaluated the GD2 expression in several TNBC cell lines, demonstrating a very low expression in MDA-MB-231, whereas Hs578T and BT-549 uniformly expressed GD2. However, in an in vitro assay, these CAR-Ts did not demonstrate any specific tumor cell killing activity towards MDA-MB-231, whereas they induced specific cytotoxicity and cytokine production upon co-cultivation with the Hs578T and BT-549 cell lines . To date, one study is currently testing the feasibility, safety, and efficacy of multiple fourth generation CAR-T cells targeting Her2, GD2, and CD44v6 surface antigen in BC (NCT04430595), but no results have been published yet. 4.15. PRLR In mammals, prolactin is an important hormone for milk secretion and mammary tissue growth by binding with the prolactin receptor (PRLR) in the breast glands . PRLR is overexpressed in some BC histotypes, especially in the MDA-MB-231 TNBC cell line and even more in T47DHER2+ and SKBR-3 cell lines . This correlation between PRLR and HER2 expression could led to the development of a CAR-based therapy targeting PRLR against BC. However, in a combination of these two targets, a bispecific antibody cytotoxicity was reported, mainly caused by the combinatory inhibition of the two rather than the effect of T-mediated cytotoxicity . Although the results seem promising, future studies on CAR-based therapies targeting PRLR must avoid causing cross-toxicity of other organs expressing the same receptor, such as the prostate, liver, etc. . 4.16. CEA CEA is a well-known tumor marker that is expressed in several solid neoplasms . In normal cells, only a small amount of CEA is expressed in the cell membrane, especially toward the cell cavity, under physiological conditions to avoid recognition by CAR-T cells targeting CEA. To date, a Phase I-II study is recruiting 40 patients with different solid tumors to test the efficacy and safety, recommended dose, and infusion plan of CEA-targeted CAR-T cells therapy. The estimated study completion date is 30 April 2023 (NCT04348643). 4.17. CD44v6 CD44 variant domain 6 (CD44v6), a CD44 family member, has demonstrated its role in tumorigenesis, tumor cell invasion, and metastasis. In normal tissue, its presence is reported only on epithelial and hematopoietic cell subgroups, especially during embryogenesis and hematopoiesis . In contrast, it is expressed in multiple squamous cell carcinomas, in a proportion of adenocarcinomas of differing origin, a proportion of lymphoma and melanoma, so it represents an attractive target for cancer therapy. A Phase I-II clinical trial is currently investigating the feasibility, safety, and efficacy of CD44v6 CAR-T cell therapy in 100 patients with several tumors, including BC. Understanding more about 4SCAR-CD44v6 T cell functions is another objective of this trial. The estimated study completion date is 31 December 2023 (NCT04427449). 4.18. Trophoblast Cell-Surface Antigen 2 (TROP2) TROP2 is a transmembrane protein that is expressed on human trophoblast cell surface and is often present in several epithelial tumor types (including TNBC), in which it is associated with poor prognosis . Zhao et al. developed and tested in vitro (against gastric cancer cell lines) and in vivo bispecific PD-L1-redirected CAR-Ts . The authors reported a higher antitumor activity with bispecific CAR-Ts compared with monospecific CAR-Ts. In spite of CAR-T-mediated TROP2 targeting in TNBC not being comprehensively investigated, the results obtained in gastric cancer cell lines warrant further investigation in other tumor types, including TNBC. 4.19. Epidermal Growth Factor Receptor (EGFR) EGFR is a transmembrane glycoprotein belonging to the ERBB receptor tyrosine kinase family, that is involved in tumor growth and metastatization . Its overexpression has been reported in several tumor types, including TNBC. Li et al. produced EGFR-redirected CAR-Ts using the non-viral piggy Bac transposon system, and demonstrated their antitumor activity firstly in human lung tumor xenografts and then in a phase I clinical trial (NCT03182816) against non-small cell lung tumors . With regards to TNBCs, Liu et al. tested EGFR-redirected CAR-T antitumor activity in vitro and in vivo, reporting EGFR overexpression in several tumor cell lines, such as Hs578T, MDA-MB-468, and MDA-MB-231, respectively. These CAR-Ts demonstrated antitumor activity and cytokine secretion in these cell lines Recently, Xia et al. generated third-generation EGFR-redirected CAR-Ts, which demonstrated antitumor activity, specific cytokine production. Moreover, the authors found that T cell activation markers (such as CD25 and CD69) were upregulated in case of co-cultivation with EGFR-positive TNBC cell lines . However, more preclinical and clinical studies are needed to confirm these findings. Several clinical trials (NCT03182816, NCT02873390, NCT02862028, and NCT03170141), are testing the effects of EGFR-redirected CAR-Ts in regards to the production of anti-CTLA-4, anti-PD-1, or anti-PD-L1 antibodies in EGFR-positive solid tumors . With regards to TNBC, two studies are ongoing. The first one (NCT05341492) is evaluating the safety and efficacy of EGFR/B7H3 CAR-T cell therapy in EGFR/B7H3-positive advanced solid cancers (including TNBC), and the second one is testing the anti-tumor activities and safety profiles of CAR-EGFR-TGFbR-KO T cell therapy in previously treated advanced EGFR positive solid tumors (including TNBC) However, no results regarding TNBC patients have yet been officially reported 4.20. Prostate-Specific Membrane Antigen (PSMA) Although at first glance it does not appear to be related to BC, it has recently been demonstrated that this molecule is present in circulating breast cancer cells, and is related to a worse prognosis . Although PSMA is expressed in normal prostate and is upregulated in prostate tumors, it is not prostate cancer restricted. In TNBC, PSMA is currently under evaluation as a target for CAR molecule development. In an ongoing, open label, Phase I trial, an anti-PSMA/CD70 bi-specific CAR-T cell therapy has been tested in several cancer types (including TNBC) expressing PSMA or CD70, another potential tumor target, that is overexpressed in many cancer types and scarcely expressed in normal tissue (NCT05437341). A second Phase I trial testing the feasibility, safety, and efficacy of anti-GD2/PSMA bi-specific CAR-T cell therapy in patients with GD2 and PSMA-positive tumors (including TNBC), is currently ongoing (NCT05437315). Moreover, in China, a third Phase I trial is currently verifying the feasibility, safety, and efficacy of PSMA-specific CAR-T cell therapy in patients with PSMA positive neoplasms, including TNBC (NCT04429451). To date, no results have been published yet. 4.21. Folate Receptor Alpha In normal cells, the DNA synthesis pathway is efficient in the presence of folate, which is conducted inside the cell through a suitable receptor called the folate receptor (FRa). FRa is often overexpressed in BC, especially in TNBC and that correlates with poor clinical outcomes . FRa-CAR-T cells were demonstrated to target FRa + TNBCs and to reduce tumor growth in MDA-MB-231 tumor xenograft . To limit toxicity, Lanitis et al. designed a trans-signaling CAR with two different signaling domains (CD3z and CD28) located in two different CARs and one T cell to link with mesothelin and FRa in tumor cells. In these conditions, the activation occurs only in the case of simultaneous antigen linkage, and that in turn may activate T cell activity . Therefore, FRa could become a potential target for immunotherapy in BC. 5. Overcoming the CAR- Problems in Solid Tumors: Macrophage-Based Cell Therapeutics To date, CAR-T cell therapy has shown efficacy against hematological neoplasms, but conversely, its results against solid tumors were disappointing. The reason for this difference probably lies in the presence of a TME around solid tumors, which modulates the immune response against malignant cells, preventing the penetration of CAR-T cells . To overcome the obstacle constituted by the TME, CAR molecules were conceived, taking inspiration from gamma-delta (gd) T and natural killer (NK) cells for their specific biological features. Indeed, these cells can identify a wide range of tumor-associated antigens (TAAs) independently of the major histocompatibility complex (MHC) , with a consequently lower impact in terms of immune-mediated toxicity . However, problems in the expansion process of these immune cells limit their application in clinical practice . On the other hand, the phenomenon of "T cell exhaustion" represents a widely known and not a fully resolved topic . Therefore, it was necessary to look for other strategies, for example, by exploring other immune cells . In this context, macrophages seem to be an interesting option for immunotherapy development. Indeed, they present a wide range of immune activities, including their role as antigen-presenting cells which may modulate adaptive immune responses, phagocytosis, and pro-inflammatory cytokine secretion . Moreover, they constitute a considerable percentage of immune cells within the TME of solid tumors, which recruit peripheral blood monocytes and then promote their differentiation into tumor-associated macrophages (TAMs) . TAMs are often reported as both M1 (pro-inflammatory) and M2 (anti-inflammatory) phenotypes, but a higher M2 concentration is more frequently associated with a poor prognosis . Their presence is considered crucial for TME regulation, especially regarding stimulation of tumor growth, angiogenesis, and metastatization . Moreover, it was reported that TAMs have a role in cytotoxic lymphocyte recruitment in the TME . In light of all of this, targeting TAMs has become the goal of numerous immunological approaches, such as TAM depletion, repolarization, or inhibition of TAM-secreted suppressive molecules . Moreover, instead of directly targeting TAMs, different studies have evaluated the role of macrophages in cancer therapy. For example, antibody-dependent cellular phagocytosis is an interesting strategy (ADCP) . It uses antibodies against specific tumor-associated antigens via the Fab region, which are internalized through the binding of Fc receptors (such as CD16a or CD32a) on macrophages. Moreover, macrophages may stimulate phagocytosis through these receptors and other surface molecules, including Mac1 or LRP1, whose intracellular mechanism of action is similar to that played by CD16a and CD32a. Indeed, their cytoplasmic region is rich in tyrosine-based activation motifs, which can activate MAPK and PI3K/AKT signaling pathways, with a consequential phagocytosis process against cancer cells . Bispecific antibodies targeting different TAAs or macrophage receptors is another option . Engaging phagocytosis checkpoint inhibitors, such as CD47, could enhance phagocytosis mechanisms by blocking the macrophage-inactivating signals . However, using antibodies is not so simple; to date, some challenges must be overcome before they are clinically developed. Firstly, macrophages expose, on their extracellular membrane, the inhibitory FC receptor (FcgRIIb), which counteracts cell activation. Secondly, Mab-therapy cannot discriminate between antitumoral and/or protumoral TAMs . For these reasons, another option is adoptive cell therapy based on ex vivo genetically engineered CAR macrophages (CAR-Ms). Among the different types of CAR molecules, second and third-generation CARs are preferred because of their capacity to potentiate phagocytic activation signals . CAR-Ms provide some advantages with regards to T cells: (1) a lower risk of GVHD, which allows CAR production in advance for "on-demand" use; and (2) a significant production of MMPs, which allows macrophages to degrade ECMs and, consequently, get close to the tumor cells . However, some problems are still unresolved: (1) Although their efficacy and safety profile have been reported in animal studies, in humans, it is still unclear; and (2) the use of viral transfection in CAR gene transfer could promote insertions with an unforeseeable impact on treatment. In this context, the CRISPR/Cas9 genome targeting system could represent a valuable option to overcome this problem . Moreover, regenerative medicine could represent a potential strategy for limiting the high cost of CAR therapy by providing a sustainable source of CAR-Ms. Delivering CARs to induced pluripotent stem cell (iPSC)-derived macrophages may extend CAR-M cell therapy to a larger-patient population. In a recent study, iPSC-derived CAR-Ms reduced tumor growth by activating phagocytosis in leukemia, ovarian, and pancreatic cancer cell lines. Moreover, the same results were reported in vivo in an ovarian cancer mouse model . 6. CAR-M in Solid Tumors and BC To date, several researchers have attempted to employ CAR-M against solid tumors and BC. Different CAR-phagocytes (CAR-P) have been designed to guide macrophages against specific targets. In particular, CAR-P expressing the FcRv or Megf10 intracellular region was shown to stimulate phagocytosis of TAA by the TCR-CD3z-mediated recruitment of SYK kinase. Usually, complete phagocytosis is uncommon, suggesting that the CAR-P macrophage link with target cells is insufficient to obtain that. In this context, it is worth remembering that the PI3K signal pathway demonstrated its involvement in target internalization and phagocytosis enhancement in macrophages . For this reason, a "tandem" CAR (CAR-P tandem) has been conceived by connecting the PI3K p85 subunit with CAR-P-FcRv. This molecule demonstrated an increase in the phagocytic activity of CAR-P, especially in terms of whole-cell phagocytosis . CAR-147 is a CAR molecule that is composed of a single-strand antibody fragment targeting HER2, a murine hinge region of IghG1, and a trans-membrane and intracellular domain of mice-derived CD147. Co-culturing CD147 with HER2 + human BC cells led to an intense MMP expression, demonstrating the capacity of CAR-147 to target HER2 and effectively promote MMP production in macrophages. Indeed, CAR-147 macrophages were shown to increase the amount of T cells close to tumor cells compared with those in tumors that were treated with controlled macrophages, demonstrating their potential to destroy the extracellular matrix into tumors. Moreover, CAR-147 macrophages have shown an antitumor effect by increasing IL-12 and IFNg levels in tumor tissue . An intravenous CAR-147 injection significantly inhibited cancer growth in 4T1 BC mouse models, but the same was not shown in vitro. Recently, at the University of Pennsylvania, an adenovirus-induced CAR-M composed of an anti-HER2 CAR and the CD3z intracellular domain was designed, demonstrating in vitro its specificity in terms of antigen-specific phagocytosis against HER2-positive tumor cells. A single injection of anti-HER2 CAR-M was shown to reduce tumor load and prolong survival in mice. It was also able to transform M2 macrophages into M1 macrophages, stimulate an inflammatory TME and promote anti-tumor cytotoxicity. In addition, HER2 CAR-M may produce epitope diffusion, which could become another solution for avoiding tumor immune escape . Another study combined an anti-HER2 CAR with transduced primary human CD14+ peripheral blood monocyte-derived macrophages. These CAR-Ms promoted phagocytosis of the HER2+ ovarian cancer cell line SKOV3 in a dose-dependent manner. The authors further demonstrated that macrophage transduction is unaffected by the anticancer effect since their transduction with a control CAR lacked antitumor activity . Moreover, in vivo, the SKOV3 tumor burden in NOD-SCID mice was considerably lower in the cases that were treated with primary human anti-HER2 CAR-Ms. The authors also demonstrated that CAR-Ms survived and resisted the immunosuppressive cytokines that were secreted by the TME. On the contrary, CAR-Ms secreted pro-inflammatory cytokines, determining a macrophage conversion from an M2 to an M1 phenotype, and consequently transforming TME into a proinflammatory environment. Furthermore, a combination of donor-derived T cells with CAR-Ms increased the antitumor response in vivo . Pierini et al. demonstrated that the infusion of murine-derived anti-HER2 CAR-Ms determined an inhibition of tumor growth, a prolongation of overall survival, and an increase of CD4+ and CD8+ T cells, NK cells, and dendritic cells in the TME. The authors also reported that CAR-Ms have a critical role in regulating the TME through the upregulation of MHC I/II expression on the cancer cells . 7. Clinical Applications of CAR-M Strategy against BC and Other Solid Tumors Until December 2022, three clinical trials have evaluated a CAR-M-based strategy in solid tumors, two of which achieved FDA approval (Table 2). The first one (a Phase I clinical trial) tested CT-0508 (CARISMA Therapeutics Inc., Philadelphia, PA, USA), a therapy consisting of anti-HER2 CAR macrophages infused in 18 patients with relapsed/refractory HER2 over-expression tumors. The study evaluated the effects of adenovirus transduction CAR-M. The estimated study completion date is February 2023 (NCT04660929). The second trial tested MCY-M11 (MaxCyte Inc., Gaithersburg, MD, USA), consisting of mRNA-targeted PBMCs (not only CAR-M) which express mesothelin-CAR, in patients with relapsed/refractory ovarian cancer and peritoneal mesothelioma (NCT03608618). A third trial (CARMA-2101), not yet recruiting, will be conducted at the Centre Oscar Lambret (Lille, France). This observational study aims to determine the antitumor activity of new CAR-Ms in 100 BC patients' derived organoids. In particular, researchers will test the CAR-M activity against organoids that are derived from HER2-negative, HER2 low, and HER2-positive BC, and then they will compare the activity of CAR-Ms and non-modified macrophages. The estimated study completion date is 1 September 2023 (NCT05007379). 8. Conclusions Recently, the remarkable advances reported in the field of immunotherapy have profoundly changed our approach toward many types of cancer. By gaining a better understanding of the role immune cells play in tumor progression mechanisms, it was possible to develop both drugs directed against specific immunological targets and forms of immune cell-based therapy, such as CAR technologies that led to the creation of CAR-T cells that are also effective in the clinical setting (especially in the field of haematological malignancies). To date, several problems significantly limit the application of CAR-T cells especially toward solid tumors. Due to this, further studies of CAR-M in tumor therapy are interesting because of the known adaptability of these immune cells to solid tumors. Indeed, the first results have shown that CAR-M is very promising in the fight against cancer; preclinical data have confirmed their efficacy (in terms of tumor phagocytosis and growth inhibition both in vitro and in vivo) and also in several solid tumors, including TNBC. The latter represents a heterogeneous BC subtype that is usually resistant to standard therapies. However, its immunogenic nature led to favorable clinical benefits from the new immune checkpoint inhibitors, such as atezolizumab, recently approved by the FDA in combination with nab-paclitaxel against metastatic TNBC . With regards to CAR-based therapy in TNBC, this is an emerging field, whose improvement depends on discovering the suitable and targetable TAAs, mostly in preclinical and early clinical stages. In our article, we discussed different novel CAR-based target antigens evaluated against BC. A lot of them have only been tested in "in vitro" and "in vivo" studies, and a small part of them were also evaluated in humans, as summarized in Table 1 and Table 2. Furthermore, scientific research is discovering other potential targets, such as specific embryonic antigen-4, which was evaluated in "in vitro" and "in vivo" studies in BC patients, but with less data to date in comparison with the aforementioned targets . Therefore, we still have little clinical data to judge the effective validity of these new therapeutic approaches against BC (and not only), which need further studies. Due to this shortage of relevant data on small series, there are still many unanswered questions. For example, is CAR-based (M or T cell) therapy more effective than standard treatments against TNBC? Is it enough to consider a mono-immunotherapy, or should it be combined with other strategies, perhaps even with other types of immunotherapy (e.g., with immune checkpoint inhibitors)? Regarding the latter question, it appears that combination regimens may lead to better efficacy, especially in terms of overcoming the TME. Indeed, in a recent study that was conducted in immunocompetent mouse models of HER2+ solid tumors (including BC), a combination of anti-PD-1 with HER2 CAR-M cell therapy demonstrated better OS and tumor control than monotherapy strategies . In this regard, other strategies were evaluated. For example, cancer-associated fibroblasts (CAF)-targeting and monocyte-eliminating agents were tested with the aim to enhance CAR-T antitumor effects in TNBC also . It seems clear that the success of CAR-based therapy in BC will depend on the ability to select the best antigens to be used as a basis for the development of more effective, and at the same time more manageable and less toxic CAR molecules. In the near future, it is hoped that some more data can be obtained from ongoing studies. Acknowledgments This work was partly supported thanks to the contribution of Ricerca Corrente by the Italian Ministry of Health within the research line L2 (Innovative therapies, Phase I-III clinical trials). Figure 1 has been created with Biorender.com (accessed on 28 February 2023)). Author Contributions Conceptualization, G.S.; methodology, G.S.; writing--original draft preparation, G.S. and C.G.; review and editing, G.S., C.G., M.P., S.B., C.C., C.L., L.R., G.M. and U.D.G.; supervision, U.D.G.; All authors have read and agreed to the published version of the manuscript. Conflicts of Interest The authors declare no conflict of interest. Figure 1 The image summarizes all the molecules that are currently being evaluated as potential targets for CAR development in BC. cancers-15-01597-t001_Table 1 Table 1 Trials testing the potential targets for CAR-T cell therapy in BC. Target Expression in Healthy Tissue Role Drug Agent Trial Status avb3-integrin Platelets, macrophages, dendritic cells, activated endothelial cells Cell proliferation, adhesion, metastatization, angiogenesis only preclinical data TEM8 Endothelium Endothelial cell development L2CAR-T only preclinical data AXL Bone marrow stroma and myeloid cells Tumor expansion, metastasization and survival AXL-CAR-Tcells only preclinical data CSPG4 Oligodendrocyte progenitor cells neuronal network regulation and epidermal stem cells homeostasis -T cells only preclinical data TROP-2 Epithelial tissue Invasiveness only preclinical data GD2 Neuroectoderm Cell signal transduction modulation Her2, GD2, and CD44v6 NCT04430595 ongoing ICAM-1 Endothelial cells and immune cells Migration, invasiveness Only preclinical data Mesothelin Mesothelial cells Cell adhesion Anti-mesothelin CAR-T cells NCT01355965 NCT02580747 NCT02414269 NCT02792114 completed unknown ongoing ongoing MUC1 Epithelial tissue Production of mucin huMNC2-CAR44 CAR T-TnMUC1 TILs/CAR-TILs targeting multiple antigens * NCT04020575 NCT04025216 NCT04842812 ongoing ongoing ongoing ROR1 Embryogenic tissue Cell survival and differentiation in embryogenesis ROR1-CAR-T TILs/CAR-TILs targeting multiple antigens * NCT02706392 NCT04842812 ongoing ongoing EpCAM Epithelial tissue Cell adhesion EpCAM CAR-T NCT02915445 ongoing EGFR Epithelial tissue Cell survival, proliferation EGFR/B7H3 CAR-T, TGFbR-KO CAR-EGFR T Cells NCT05341492, NCT04976218 ongoing ongoing HER-2 Epithelial tissue (in particular breast, skin, and gastrointestinal, respiratory, reproductive, urinary tracts) Cell proliferation, differentiation, and survival CCT303-406 CAR-T BPX-603 CAR-T HER-2 CAR-T HER-2 CAR-T + CAdVEC ** TILs/CAR-TILs targeting multiple antigens * NCT04511871 NCT04650451 NCT03696030 NCT03740256 NCT04842812 ongoing ongoing ongoing ongoing ongoing NKG2D Innate and adaptive immune cells Cytotoxicity and secretion of cytokines NKG2DL CAR-T cells NCT04107142 unknown CEA Epithelia (in particular enteric tissue) and embryogenic tissue Cell migration, proliferation, and survival CEA CAR-T cells NCT04348643 ongoing CD44v6 Epithelial tissue and hematopoietic cells Cell survival, proliferation, migration 4SCAR-CD44v6 T-cell NCT04427449 ongoing PSMA Endothelium, prostate Angiogenesis and immune modulation bi-4SCAR PSMA/CD70 Tcells, bi-4SCAR GD2/PSMA T cells, 4SCAR-PSMA T cells NCT05437341 NCT05437315 NCT04429451 ongoing ongoing ongoing FRa Epithelial tissue (mammary ducts, lungs, kidneys the choroid plexus) Cell growth and survival only preclinical data c-MET Epithelial tissue Cell differentiation, proliferation, migration, angiogenesis, and epithelial-mesenchymal transition cMet RNA cells NCT01837602 completed Abbreviations: AXL = tyrosine-protein kinase receptor UFO; CAR-T = chimeric antigen receptor T cells; CD44v6 = CD44 variant domain 6; CEA = carcinoembryonic antigen; c-MET = tyrosine-protein kinase Met; CSPG4 = chondroitin sulfate proteoglycan-4; EGFR = epidermal growth factor receptor; EpCAM = epithelial cell adhesion molecule; FRa= folate receptor alpha; GD2 = Disialoganglioside; ICAM-1 = Intercellular Adhesion Molecule-1; MUC1 = Mucin1; NKG2D = natural killer group 2, member D ligand; PSMA = prostate-specific membrane antigen; ROR1 =Receptor tyrosine kinase-like orphan receptor; TEM8 = tumor endothelial marker; TILs = tumor-infiltrating lymphocytes; TROP-2 = trophoblast cell-surface antigen 2. * TILs and CAR-TILs targeting HER2, Mesothelin, PSCA, MUC1, Lewis-Y, GPC3, AXL, EGFR, Claudin18.2/6, ROR1, GD1, or B7-H3. ** CAdVEC is an oncolytic adenovirus designed to help the immune system, including HER2-specific CAR-T cells, react to the tumor. cancers-15-01597-t002_Table 2 Table 2 Trials on CAR-M in BC and other solid tumors. Target Drug Agent Trial Status HER-2 CT-0508 NCT04660929 Recruiting Mesothelin Intraperitoneal MCY-M11 and cyclophosphamide NCT03608618 Terminated (no results posted yet) HER-2 HER-2 CAR-M NCT05007379 Not yet recruiting Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. |
PMC10000830 | Cells Cells cells Cells 2073-4409 MDPI 10.3390/cells12050820 cells-12-00820 Article Increased Radiation Sensitivity in Patients with Phelan-McDermid Syndrome Jesse Sarah Conceptualization 1 Kuhlmann Lukas Investigation 2 Hildebrand Laura S. Writing - review & editing 2 Magelssen Henriette Writing - review & editing 3 Schmaus Martina Writing - review & editing 4 Timmermann Beate Writing - review & editing 5 Andres Stephanie Writing - review & editing 6 Fietkau Rainer Writing - review & editing 2 Distel Luitpold V. Writing - original draft 2* Wang San Ming Academic Editor Cheung Edwin Chong Wing Academic Editor Shao Ningyi Academic Editor 1 Department of Neurology, Ulm University, 89081 Ulm, Germany 2 Department of Radiation Biology, Erlangen University, 91054 Erlangen, Germany 3 Department of Oncology, Oslo University Hospital (The Norwegian Radium Hospital), 0424 Oslo, Norway 4 Department of Radiotherapy and Radiation Oncology, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany 5 Clinic for Particle Therapy at WPE, University Hospital Essen, 45147 Essen, Germany 6 Medicover Munchen Ost MVZ, Humangenetik, 81667 Munich, Germany * Correspondence: [email protected] 06 3 2023 3 2023 12 5 82016 2 2023 03 3 2023 03 3 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). Phelan-McDermid syndrome is an inherited global developmental disorder commonly associated with autism spectrum disorder. Due to a significantly increased radiosensitivity, measured before the start of radiotherapy of a rhabdoid tumor in a child with Phelan-McDermid syndrome, the question arose whether other patients with this syndrome also have increased radiosensitivity. For this purpose, the radiation sensitivity of blood lymphocytes after irradiation with 2Gray was examined using the G0 three-color fluorescence in situ hybridization assay in a cohort of 20 patients with Phelan-McDermid syndrome from blood samples. The results were compared to healthy volunteers, breast cancer patients and rectal cancer patients. Independent of age and gender, all but two patients with Phelan-McDermid syndrome showed significantly increased radiosensitivity, with an average of 0.653 breaks per metaphase. These results correlated neither with the individual genetic findings nor with the individual clinical course, nor with the respective clinical severity of the disease. In our pilot study, we saw a significantly increased radiosensitivity in lymphocytes from patients with Phelan-McDermid syndrome, so pronounced that a dose reduction would be recommended if radiotherapy had to be performed. Ultimately, the question arises as to the interpretation of these data. There does not appear to be an increased risk of tumors in these patients, since tumors are rare overall. The question, therefore, arose as to whether our results could possibly be the basis for processes, such as aging/preaging, or, in this context, neurodegeneration. There are no data on this so far, but this issue should be pursued in further fundamentally based studies in order to better understand the pathophysiology of the syndrome. Phelan McDermid syndrome SHANK3 deficiency radiation sensitivity chromosomal aberrations atypical teratoid rhabdoid tumor Deutsche ForschungsgemeinschaftFriedrich-Alexander-Universitat Erlangen-NurnbergWe acknowledge financial support by Deutsche Forschungsgemeinschaft and Friedrich-Alexander-Universitat Erlangen-Nurnberg within the funding programme "Open Access Publication Funding". pmc1. Introduction Autism spectrum disorders (ASDs) are characterized by deficits in social interaction, limitations in communication and repetitive, stereotyped behaviors and special interests . A prevalence of 0.9-1.1% for autism spectrum disorders can be assumed . Based on twin and family studies, the heritability of autism spectrum disorders is around 40% . Copy-number variations or de novo mutations in the SHANK genes occur in around 1% of all patients with ASD , so that these are among the most common ASD-associated genes, with SHANK3 accounting for the majority at 0.73% . SHANK3 stands for SH3 domain and ankyrin repeat-containing protein and is located on the long arm of chromosome 22, encoding structural proteins of the postsynapse of excitatory neurons . Deletions or pathogenic variants of SHANK3 lead to the clinical picture of Phelan-McDermid syndrome (PMS, OMIM#606232), which can be considered representative of syndromic autism as a monogenetic form of ASD . PMS is characterized by global developmental delay with motor hypotonia, expressive and receptive language development delay, cognitive impairment and other neuropsychiatric comorbidities, such as mood disorders, epilepsy, regression, schizophrenia and autism spectrum disorder . The latter is of clinical and developmental relevance in up to 70% of these patients . The clinical variability is wide, a circumstance that has not yet been conclusively explained pathophysiologically, although there are first indications of epigenetic changes on chromosome 22 . The genetic changes underlying the syndrome also vary greatly and include point mutations, deletions, inversions and translocations affecting SHANK3 on chromosome 22q13.3 . By now, patients with genetic aberrations in the chromosomal region 22q13 without SHANK3 involvement are also known to show a similar phenotype so that the nomenclature of the syndrome was recently adjusted to PMS-SHANK3 related or PMS-SHANK3 unrelated . The SHANK3 gene product has no known linkage to DNA repair, cell cycle control or cell death control. It is thought that perturbations in these functions may lead to increased radiation sensitivity. A gene located near SHANK3 and associated with radiation sensitivity is the PPP6R2 gene, which encodes protein phosphatase 6 . It is also located on 22q13.3, 300kb upstream of SHANK3. Another gene that is certainly associated with radiation sensitivity is XRCC6, positioned at 22q13.2, encoding the KU70 protein. This protein is important for the initiation of DNA double-strand break repair . Even more distant (22q11.23) is the SMARCB1 gene, in which pathogenic germline variants are associated with the occurrence of atypical teratoid/rhabdoid tumors that are more common in PMS patients . Radiation sensitivity refers to both stochastic and deterministic risk . On the one hand, this means an increased risk of cancer susceptibility even at low doses of ionizing radiation (IR), and at higher doses, as in radiotherapy, an increased risk of adverse therapeutic effects. Radiation sensitivity already varies considerably in healthy individuals but can be greatly increased by a factor of 2-4 in genetic syndromes, such as ataxia-telangiectasia or Nijmegen breakage syndrome . There are many other autosomal recessive disorders in which only limited increased radiation sensitivity occurs. These are, e.g., Fanconi anemia, Ligase IV, Bloom syndrome, Werner syndrome, Xeroderma pigmentosum and Cockayne syndrome . Lymphocytes and the analysis of chromosomal aberrations after ex vivo irradiation are particularly suitable for testing individual radiation sensitivity . The main reason why lymphocytes are particularly suitable is that they are in the G0 cell cycle phase and are, therefore, uniformly sensitive to radiation. In addition, they then pass through the entire cell cycle control system before the metaphases are then analyzed to determine radiation sensitivity. In an affected individual with Phelan-McDermid syndrome suffering from an atypical teratoid/rhabdoid tumor (AT/RT) WHO grade 4, individual radiation sensitivity was analyzed prior to the start of radiation therapy and was significantly increased compared with the control cohort. Therefore, this pilot study was undertaken with the question of whether increased radiation sensitivity also exists in other patients with Phelan-McDermid syndrome. 2. Materials and Methods The blood of 20 PMS patients was compared with that of 591 patients in a control cohort and used to study radiation sensitivity. Thus, 9 of the PMS patients were female and 11 were male. The mean age was 9.8 years with a range of 3.5 years to 31.8 years. The patients varied widely with respect to disease burden, with impairments in receptive and expressive communication, motor skills and cognition (Table 1). Eight of them suffered from autism and five from epilepsy. The severity of disease was scored according to the clinical categorization in Table 1. High values represent a high clinical burden of disease. The highest possible score for the most severe clinical impairment is 14. PMS patients were compared with 218 healthy subjects, 226 patients with rectal cancer and 147 patients with breast cancer. The healthy subjects were 57.3% female and had a mean age of 50.4 years, the rectal cancer cohort was 28.3% female/63.2 years, and the breast cancer cohort was 100% female/57.3 years. Due to the young age of PMS patients, subgroups were formed with healthy subjects younger than 30 years, resulting in 51 subjects, of whom 58.8% were female and the average age was 25.6 years. Cancer patients are older, so a higher age of 45 years was used to form a young group. Patients with rectal cancer were 50% female and had a mean age of 35.5 years; breast cancer patients were 100% female and had a mean age of 39.1 years. All blood samples were taken before the start of radiotherapy. Some of the data have already been published . All patients and healthy individuals gave their written informed consent for the scientific processing of their material and data. The ethics committee of the University Hospital Erlangen approved the study, including the use of the individual patient data. Approval of the Ethics Committee 21_19 B. The G0 three-color fluorescence in situ hybridization (FISH) assay was used to determine the radiation sensitivity of patients using blood lymphocytes. For this purpose, 9 mL of blood was drawn from each patient and half of the blood was irradiated with 2 Gray (Gy) of a 6 MeV linear accelerator (Versa HD, Elekta, Stockholm, Sweden). The other half remained unirradiated to determine the background of chromosomal aberrations. Lymphocytes were stimulated by phytohemagglutinin to undergo the cell cycle and, 47 h later, cells were arrested in metaphase by colcemid. Lymphocytes were prepared on slides and chromosomes were hybridized by probes for chromosomes #1, #2 and #4. Images were acquired via a Zeiss fluorescence microscope (Axioplan Z2, Zeiss, Gottingen, Germany) and an automatic metaphase finder (Metasystems, Metapher 4, Altlussheim, Germany). Chromosomal aberrations in the three chromosomes studied were analyzed using image analysis software (Biomas, Erlangen, Germany). The number of possible DNA breaks representing the different chromosomal aberrations was scored according to Savage and Simpson . Acentrics were counted as one break, dicentrics, translocations and rings as two breaks and insertions as three breaks. Complex aberrations were scored according to how many DNA double-strand breaks would have been necessary for their formation. An average value of breaks per metaphase (B/M) was derived from at least 200 metaphases from unirradiated and 150 metaphases from 2 Gy irradiated samples . The SPSS Statistics 28 program (IBM, Armonk, NY, USA) was used for analyses and statistical work. Statistical significance was calculated with the t test for cohorts n > 30 and with the Mann-Whitney U test for groups with n <= 30. Tests were always performed two-sided. Charts were generated using Excel (Microsoft Corporation, Redmond, WA, USA) and Prism (GraphPad Software, San Diego, CA, USA). 3. Results 3.1. A Case of a Phelan-McDermid Syndrome Patient with an Atypical Teratoid/Rhabdoid Tumor A 6-year-and-8-month-old boy was diagnosed with an atypical teratoid/rhabdoid tumor (AT/RT), WHO grade 4, in the fourth ventricle/aqueduct in 2018. The boy was known to have Phelan-McDermid syndrome (PMS). A 3.2Mb deletion was diagnosed in the terminal end of chromosome 22 in region 22q13. The boy's symptoms included unspecified intellectual disability, impaired speech and sleep problems. A change in health status was characterized by stiffness and reclusiveness, suspected epilepsy and focal activity on electroencephalogram. The tumor was diagnosed by MRI. It was resected, with no residual tumor visible. Chemotherapy was administered due to EU-RHAB V5 2016 protocol, consisting of three cycles of doxorubicin (Dox), ICE (Ifosfamide, Carboplatinum, Etoposide) and VCA (Vincristine, Cyclophosphamide, Actinomycin) and an intraventricular injection of methotrexate for 5 months . No evidence of tumor was found on MRI examinations performed at regular intervals. Proton radiotherapy was originally planned according to the EURHAB protocol with 30 x 1.8 Gy single fraction dose up to a total dose of 54 Gy . The therapy was started immediately due to the aggressiveness of the tumor. In parallel, radiation sensitivity testing was performed by irradiation of whole blood in G0 ex vivo and chromosome aberration analysis by 3-color fluorescence in situ hybridization . A significantly increased rate of chromosomal aberrations of 0.74 breaks per mitosis was found. Due to these results, it was decided to decrease both the fraction dose and the total dose. After 11 fractions had already been applied with 1.8 Gy per fraction, the single dose was reduced to 1.4 Gy per fraction and given for a further 8 fractions. Therefore, the total dose was reduced to 31 Gy. Since then, MRIs have been performed on a regular basis. Until the last MRI in June 2022, no progression of disease nor any late effects occurred. In July, the mother reported that the boy was doing well, with delayed development due to PMS. 3.2. Individual Radiation Sensitivity of 20 Patients with Phelan-McDermid Syndrome Because of the increased radiation sensitivity of the index patient, we were interested to know if other individuals with PMS were similarly more radiosensitive. Therefore, we studied the radiation sensitivity of 11 males and 9 females with PMS with an average age of 9.8 years (Table 1). The PMS cohort with an average radiation sensitivity of 0.653 B/M had a clearly increased radiation sensitivity compared to cohorts consisting of healthy individuals with an average radiation sensitivity of 0.417 B/M, patients suffering from rectal cancer with an average radiation sensitivity of 0.434 B/M and breast cancer with an average radiation sensitivity of 0.489 B/M. Because of the young age of the PMS cohort, additional subgroups of these cohorts were formed, consisting of healthy individuals younger than 30 years (mean radiation sensitivity 0.377 B/M), rectal cancer patients (0.476 B/M) and breast cancer patients (0.562 B/M) younger than 45 years (Table 2). Background aberrations in lymphocytes among the PMS patients (0.0131 B/M) were very low, with the exception of one individual . The PMS background aberrations were also clearly lower compared to the young cohorts with cancer (healthy 0.0182; rectal cancer 0.0549; breast cancer 0.0996 B/M p < 0.017). However, there was no difference to the healthy group (p = 0.213) . The chromosomal aberrations induced by 2 Gy after background withdrawal displayed for the whole cohorts (p < 0.001), as well as for the young cohorts (p < 0.041), that the PMS cohort had a strongly increased radiation sensitivity, with an average B/M of 0.653. Only 2 of the PMS patients had no increased radiation sensitivity by our definition, and in 17 of the 20, the radiation sensitivity was increased to a value where dose reduction would be recommended in the event of radiotherapy . This is particularly noteworthy, as nearly all PMS individuals had increased radiation sensitivity. This is in contrast to the other cohorts, where only a few individuals had an increased radiation sensitivity, whereas the majority still had average radiation sensitivity. 3.3. Mutation Type and Radiation Sensitivity Next, we were interested in whether the locations and the type of the genetic aberrations are associated with the increased radiation sensitivity. There were six frameshift mutations, one nonsense mutation and one intragenic deletion within the SHANK3 gene (0.634 B/M) . All variants were located in exon 20 of SHANK3 and there was no difference in radiation sensitivity in these cases. Next, we studied the association of increased radiation sensitivity in the seven subjects carrying a large deletion, including the SHANK3 gene, four subjects with relatively small deletions, not including the SHANK3 gene, and one subject with a ring chromosome. These patients had a radiation sensitivity of 0.666 B/M . We did not find any difference in the radiation sensitivity of the subjects with a deletion compared to subjects with pathogenic variants in the SHANK3 gene. The ring chromosome had a very high radiation sensitivity (0.8 B/M), while the relatively small (mean 128 kb) terminal deletions not encompassing the SHANK3 gene had a slightly lower mean (0.619 B/M) compared to the larger (mean 4.2Mb) deletions encompassing the SHANK3 gene with a higher mean (0.668 B/M) (p = 0.257) . Next, we studied whether a high burden of symptoms was related to the increase in radiation sensitivity. The individuals had markedly different limitations in terms of impaired receptive and expressive communication, motor skills, cognitive, autistic disorders, epilepsy and gastrointestinal disorders (Table 1). We formed a score with eight items (Table 1), with a maximum of 14 points representing the highest burden. The median score was 6 points, with a range from 0 to 12 points. Patients with a high disease burden above the median had an average radiation sensitivity of 0.686 B/M compared with patients with a lower disease burden of 0.620 B/M, but without a clear difference (p = 0.290) . Additionally, we were interested in whether the genetic variants were related to the disease burden score we used . We could only find a tendency towards a higher disease burden in the group that was not related to SHANK3 (p = 0.170), with the limitation that the groups are very small. The patient with the ring chromosome had the highest disease burden score, which also had significantly increased radiation sensitivity. 4. Discussion The PMS cohort had massively increased radiation sensitivity compared to the three control cohorts. A peculiarity compared to the studied cancer cohorts is that almost all individuals with PMS are affected by this clearly increased radiation sensitivity. Even though the studied group of 20 individuals was not very large, it must be assumed that a large proportion of patients with PMS have increased radiation sensitivity. To our knowledge, this is the first study of PMS patients for individual radiation sensitivity. We found no difference in patients, regardless of whether the syndrome was classified as SHANK3-related or SHANK3 non-related. It is remarkable that PMS patients with SHANK3 non-relation are only slightly less sensitive to radiation than patients with SHANK3 relation. This study was prompted by a patient with an atypical teratoid/rhabdoid tumor whose individual radiation sensitivity was significantly elevated at 0.74 B/M. The proton therapy dose was, therefore, reduced from a total dose of 54 Gy to 31 Gy to avoid late effects in normal tissue . Four years after radiotherapy, the patient was still free of disease, despite the radiation dose being significantly lowered. This is particularly noteworthy, as this tumor is highly aggressive, often leading to tumor recurrence or progression . The successful treatment with relatively low doses could indicate that a reduction in the dose was adequate. So far, no late adverse events have occurred. Contrast this with the case of a 12-month-old girl with PMS who developed AT/RT. She was treated with surgery, induction chemotherapy and moderate radiotherapy with 1.64 Gy single doses up to a total dose of 45.9 Gy, followed by three cycles of chemotherapy. Over the next year and a half, MRI revealed diffuse volume loss in both the cerebral hemispheres and brainstem, myelomalacia in the cervical spinal cord and multiple foci of diffusion restriction, consistent with progression of brainstem necrosis. Symptoms included acute right-sided paralysis, global hypotonia, verbalization arrest and development of acute encephalopathy and dysautonomia, with intermittent bradycardia, hypotension and hypopnea . This indicates a PMS patient who developed a very severe side effect from a moderate dose of IR. This is in line with our observation that patients with PMS have significantly increased radiation sensitivity. The radiation sensitivity of the PMS cohort was compared with that of healthy subjects and cancer patients and with the same cohorts but limited to young subjects. The reason for this is that radiation sensitivity can change with age . There are two opposing effects here. First, the DNA damage response becomes weaker with age, leading to a slight increase in radiation sensitivity with age . The opposite is true in cancer patients, where younger subjects often suffer from genetic alterations that lead to early onset of cancer and increased radiation sensitivity at the same time . The young patients in our breast cancer cohort were significantly more radiosensitive compared to the overall cohort, whereas the PMS patients were still significantly more radiosensitive on average. We equate, here, the chromosomal aberrations induced by 2 Gy IR as a measure of radiation sensitivity. It has been shown for a long time that chromosomal aberrations are best suited for this purpose. We used a three-color FISH approach because the three largest chromosomes can be used to check about 22% of the total DNA . In contrast to commonly used conventional cytogenetics, in addition to dicentric and acentric aberrations, FISH can effectively find aberrations, such as insertions and translocations . FISH with more than 3 chromosomes and up to 24 chromosomes is increasingly difficult to score and offers no advantage in reliability of prediction from radiation sensitivity over the three-color FISH . In the studied PMS individuals, the average radiation sensitivity was increased by 56.6% compared to healthy individuals and by 73.2% compared to the young healthy cohort. If one does not want to make the connection with radiation sensitivity directly, it is nevertheless the case that PMS individuals have a 56% to 73% increased mutation rate in the chromosomes. Nevertheless, a question arises about the pathophysiological cause of the detected increased radiation sensitivity. It does not appear to be only dependent on SHANK3, as no association of genotype with radiation sensitivity was seen, keeping in mind that the groups studied are relatively small. Epigenetic changes may also play a causal role here, which is certainly interesting to follow up. Another question according to the clinical relevance of the data arises. In patients with Phelan-McDermid Syndrome, the development of cancers seems not markedly increased compared to other radiation sensitive diseases, such as Li Fraumeni syndrome, Fanconi anemia, Ataxia telangiectasia and Nijmegen breakage syndrome. Tumors that occur in PMS typically include rhabdoid tumors, as in our index patient and neurofibromas involving the NF2 gene in patients with PMS and ring chromosome . Atypical teratoid/rhabdoid tumors arise from pathogenic mutations in SMARCB1 that are also located on chromosome 22q (22q11.2) . The atypical teratoid/rhabdoid tumor is frequently described in patients with PMS , and there may be a link between PMS and downregulation of genes on chromosome 22 through epigenetic changes . Based on the current data in the literature on the incidence of tumors in Phelan-McDermid syndrome, the increased radiation sensitivity does not appear to be associated with an increased tumor tendency. It remains to be debated whether the increased radiation sensitivity may lead to premature aging of the disease, starting early in development, as Braak described for Alzheimer's disease, since we already detect a significantly increased radiation sensitivity in young children in our cohort . Indications for this could be the description that some patients with PMS develop a dementia syndrome in the course of their lives . Systematic studies on this are still lacking but would be exciting. Another explanation could be that selective radiation sensitivity occurs in PMS, which is relatively specifically susceptible to ionizing radiation. Other predisposition syndromes, such as BRCA1-/BRCA2-related tumor predisposition syndrome, Fanconi anemia and Li Fraumeni syndromes, are sensitive to both IR and chemicals, as they interfere with different repair pathways that are also responsible for chemicals. In contrast, PMS could interfere with the repair of lesions specifically caused by the relatively rare environmental IR. In addition, a specifically increased incidence of IR-induced chromosomal aberrations could also be caused by PMS, so that chromosomal aberrations are clustered and other mutations are not correspondingly frequent. This could then mean that the radiation sensitivity is not quite as high as here from the measurements. It would, therefore, be important to study radiation sensitivity in PMS using other methods and on other cells such as fibroblasts. We suspected that larger deletions could lead to both a higher disease burden and increased radiation sensitivity . However, there is only a slight tendency for this association. To date, several studies have found correlations between deletion size and phenotype but results are conflicting. Two recent large cohort studies also addressed genotype and phenotype correlations . The latter is the largest study to date, in terms of the number of individuals with PMS, with 210 participants (including 21 individuals with a SHANK3 variant) from Spain. The authors observed that there was a positive correlation of deletion size with brain MRI findings, ear abnormalities and toe syndactyly, while abnormal behavior correlated negatively. Interestingly, individuals with large deletions were more likely to have macrocephaly, while small deletions were associated with microcephaly. A French study subdivided 22q13.3 deletions into two classes: Class I included small deletions containing SHANK3, to which the authors added those with SHANK3 pathogenic variants; Class II contained all other (larger) deletions located on chromosome 22q. Class I individuals had a less pronounced delay in development and higher cognitive abilities but were more prone to regression of skills and psychiatric disorders. Class II individuals more often had abnormalities of the kidneys, eyes and spine. Ataxia and muscular hypotonia were also more common. 5. Conclusions Although our pilot study revealed very interesting results with increased radiation sensitivity in patients with PMS, the results should be interpreted with caution due to the small number of patients and the arbitrary determination of our clinical score. It would be interesting to reproduce the results in a larger group of patients with PMS. It can certainly be concluded that recommendations should be given to help analyze radiation sensitivity in patients with PMS when planning any radiation therapy. In addition, the results of this pilot study suggest that a reduction in the radiation dose must be considered in PMS and discussed with affected families. Author Contributions S.J.: conceptualization, methodology, formal analysis, writing (original draft), writing (review and editing) and validation. L.K.: formal analysis, perform experiments, writing (review and editing). L.S.H.: formal analysis, perform experiments, writing (review and editing). H.M.: writing (original draft), writing (review and editing), validation and visualization. M.S.: conceptualization, writing (original draft), writing (review and editing), validation and visualization. M.S.: writing (review and editing), validation and visualization. B.T.: writing (original draft), writing (review and editing) S.A.: formal analysis, writing (original draft), writing (review and editing), validation and visualization. R.F.: conceptualization, provide infrastructure, writing (review and editing). L.V.D.: conceptualization, methodology, formal analysis, writing (original draft), writing (review and editing), validation and visualization. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The ethics committee of the University Hospital Erlangen approved the study, including the use of the individual patient data. Approval of the Ethics Committee 21_19 B. Informed Consent Statement Written informed consent was obtained from all patients or their authorized representatives. Patients consented to blood collection and use of clinical data. Data Availability Statement All data are available in the main text. Further data can be obtained from the corresponding author on request. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Case of a 6-year-and-8-month-old boy and radiation sensitivity testing. (A) Timeline since tumor diagnosis. (B) Proton therapy dose plan for the atypical teratoid/rhabdoid tumor (B) in a transverse plane and (C) a sagittal plane. (D) Timeline of radiation sensitivity assay. (E) A metaphase with the three stained chromosome pairs without aberrations. Red-stained chromosome #1, green-stained chromosome #2 and yellow-stained chromosome #4. (F) A metaphase with an insertion of a fragment of chromosome #2 into chromosome #1 and a dicentric chromosome consisting of chromosome #2 and an unstained chromosome. The yellow arrows point to the chromosomal aberrations. (G) The same image with DAPI staining shows that both damaged chromosomes are dicentric. The yellow arrows point to the centromeres. Figure 2 Radiation sensitivity testing in 20 subjects with Phelan-McDermid syndrome. Chromosomal aberrations in ex vivo irradiated lymphocytes from 20 individuals with Phelan-McDermid syndrome compared with a cohort of healthy individuals, patients with rectal cancer and breast cancer. (A) Background aberrations of all ages from 9 to 91 years and (B) of young healthy individuals younger than or equal to 30 years and cancer patients younger than or equal to 45 years. Chromosomal aberrations after ex vivo irradiation with 2 Gy IR and subtraction of background aberrations give (C) radiation sensitivity of the whole cohort or (D) young individuals. B/M = Breaks per metaphase. The dashed blue line indicates individuals with increased radiation sensitivity and the solid red line indicates individuals with significantly increased radiation sensitivity. Figure 3 Location of pathogenic variants in the SHANK3 gene in eight patients with PMS and associated radiation sensitivity expressed as breaks per metaphase. The location of the variants in the SHANK3 gene of eight individuals with PMS is indicated by blue rings. The height indicates the associated radiation sensitivity (fs = frameshift mutation and Ter = termination mutation). The mutation marked by an asterisk is a microdeletion in the SHANK3 gene with unknown location. The dashed blue line indicates individuals with increased radiation sensitivity and the solid red line indicates individuals with significantly increased radiation sensitivity. Figure 4 Deletion regions of chromosome 22 in 12 PMS patients and their radiation sensitivity expressed as breaks per metaphase. The entire chromosome and a subsection (red box) of chromosome 22 are indicated. The location of the SHANK3 gene is indicated as a short red line. The blue lines indicate the length of deletions in 11 individuals. The upper closed line indicates an individual with a ring chromosome. Radiation sensitivity of individuals is indicated in the right column. The dashed blue line indicates individuals with increased radiation sensitivity, and the solid red line indicates individuals with significantly increased radiation sensitivity. Loss of function of the SMARCB1 gene (red arrow) and its product, the tumor suppressor protein INI1, is associated with tumor incidence and, in particular, with atypical teratoid/rhabdoid tumors. Figure 5 Association of pathogenic variant locations and burden of disease with radiation sensitivity. (A) Radiation sensitivity associated with PMS pathogenic variants within the gene compared with deletions in the terminal region of chromosome 22q, including the ring chromosome and ring chromosome compared with deletions, not including SHANK3. (B) A score of 14 points for the most severe disease burden was formed from all clinical characteristics. The low group had <=6.0 points and the high group had > 6.0 points. (C) Pathogenic variants within the SHANK3 gene compared with deletions in the terminal region of chromosome 22q (unrelated or associated with the SHANK3 gene) and their association with a PMS disease burden score. B/M = Breaks per metaphase. The dashed blue line indicates individuals with increased radiation sensitivity and the solid red line indicates individuals with significantly increased radiation sensitivity. The dashed black line indicates the median PMS disease burden score. chr. = chromosome. cells-12-00820-t001_Table 1 Table 1 Clinical characteristics and symptoms of 20 patients with Phelan-McDermid syndrome. A disease burden score was built from these symptoms to assess disease burden. The possible scores for the various clinical symptoms are given, with a high number of points representing the most severe disease burden in each case. An additional point could be given for further symptoms. Variable Phelan McDermid Syndrome n = 20 (%) Score (pnts) sex (male; female) 11 (55%); 9 (45%) - age (range) 9.8 (3.5-31.8) - impaired communication receptive (minimal; light; medium; heavy) 7 (35%); 5 (25%); 5 (25%); 3 (15%) 3 impaired communication expressive (minimal; light; medium; heavy) 1 (5%); 9 (45%); 7 (35%); 3 (15%) 3 motor skills (normal; mild hypotonia; moderate-severe hypotonia; fine motor disturbance) 1 (5%); 13 (65%); 2 (10%); 4 (20%) 3 cognition (not tested; below average intelligence level) 14 (70%); 6 (30%) 1 autism (no; yes; no data) 11 (55%); 8 (40%); 1 (5%) 1 epilepsy (no; yes; no data) 14 (70%); 5 (25%); 1 (5%) 1 complaints of the gastrointestinal tract (no; yes; no data) 17 (85%); 2 (10%); 1 (5%) 1 cells-12-00820-t002_Table 2 Table 2 Clinical characteristics of control cohorts. Cohort (n) Variable Total Cohort Subgroup "Young" Patients Healthy individuals n = 218 sex (male/female) 93 (42.7%)/125 (57.3%) 21 (41.2%)/30 (58.8%) age (range) 50.4 (9-81) 25.6 (9-30) Rectal cancer n = 226 sex (male/female) 162 (71.7%)/64 (28.3%) 9 (50%)/9 (50%) age (range) 63.2 (23-87) 35.5 (23-45) Primary tumor (T1/T2/T3/T4) 7 (3%)/28 (12.2%)/146 (64.8%)/45 (20.1%) 1 (3%)/3 (15.2%)/12 (66.7%)/3 (15.2%) Regional lymph nodes (N0/N1) 86 (38.2%)/140 (0%) 6 (33.3%)/12 (66.7%) Distant metastasis (M0/M1) 185 (81.8%)/41 (18.2%) 15 (81.8%)/3 (18.2%) Breast cancer n = 147 sex (female) 147 (100%) 27 (100%) age (range) 57.3 (28-91) 39.1 (28-45) cells-12-00820-t003_Table 3 Table 3 Radiation sensitivity in the different cohorts. 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PMC10000831 | Int J Environ Res Public Health Int J Environ Res Public Health ijerph International Journal of Environmental Research and Public Health 1661-7827 1660-4601 MDPI 10.3390/ijerph20053800 ijerph-20-03800 Article Impact of 3D Printing on the Overall Project Success of Residential Construction Projects Using Structural Equation Modelling Waqar Ahsan 1* Othman Idris 1 Pomares Juan Carlos 2 Otero Marta Academic Editor 1 Department of Civil & Environmental Engineering, University Technology PETRONAS, Seri Iskandar 32610, Malaysia 2 Civil Engineering Department, University of Alicante, 03690 Alicante, Spain * Correspondence: [email protected] or [email protected] 21 2 2023 3 2023 20 5 380027 1 2023 17 2 2023 18 2 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). After a decade of research and development, 3D printing is now an established technique in the construction sector, complete with its own set of accepted standards. The use of 3D printing in construction might potentially improve the outcome of the project as a whole. However, traditional strategies are often used in the residential construction industry in Malaysia, which causes serious public safety and health issues along with a negative impact on the environment. In the context of project management, overall project success (OPS) has five dimensions, such as cost, time, quality, safety, and environment. Understanding the role of 3D printing in relation to OPS dimensions in Malaysian residential construction projects would allow construction professionals to adopt 3D printing more easily. The aim of the study was to find the impact of 3D construction printing on OPS while considering the implications for all five dimensions. Fifteen professionals were interviewed to first evaluate and summarise the impact factors of 3D printing using the current literature. Then, a pilot survey was conducted, and the results were checked using exploratory factor analysis (EFA). The feasibility of 3D printing in the building sector was investigated by surveying industry experts. Partial least squares structural equation modelling was used to investigate and validate the fundamental structure and linkages between 3D printing and OPS (PLS-SEM). A strong correlation was found between 3D printing in residential projects and OPS. Highly positive implications are indicated by the environmental and safety dimensions of OPS. Malaysian decision-makers may look to the outcomes of introducing 3D printing into the residential construction industry as a modern method for increasing environmental sustainability, public health and safety, reducing cost and time, and increasing the quality of construction work. With this study's findings in hand, construction engineering management in Malaysia's residential building sector might benefit from a deeper understanding of how 3D printing is used for improving environmental compliance, public health and safety, and project scope. 3D printing overall project success (ops) public health environmental protection residential construction in Malaysia This research received no external funding. pmc1. Introduction A construction project's success is determined by five essential elements that must be present in the finished result for it to be considered a success. These essential elements are cost reduction, time management, improvement in public health and safety, and environmental sustainability . Cost, time and quality (CTQ) are commonly indicated as scope dimensions. Construction project success is critically dependent on improving economic, environmental, and social sustainability. It is the reason that existing studies advocated focusing on cost, environmental compliance and improvement in public well-being . It is also one of the major reasons why the construction industry is always improving and new technologies are being adopted. The construction sector has been driven to embrace modern procedures and tools such as digital fabrication to keep up with the demands of current architectural design for flexibility, complexity, high performance, intricacy, customisation of material, and technology . Tay et al. and Yang et al. argued that inefficient production methods in the residential construction sector have been advocated for replacement by the use of automation in the construction industry. A shift to this digital architecture paradigm is expected to have far-reaching positive effects on the quality of human-made structures. Consequently, it is incumbent upon architects to design fully automated production methods that promote ideals such as equity, sustainability, democracy, diversity, and inclusion . Subrin et al. and Tobi et al. stated that understanding the shifts brought about by technological developments in the architectural sector may increase the efficacy of architectural education, stimulate novel strategies for design and construction, and influence the course of future research. Safety has always remained a significant challenge for the construction industry, as it can be compromised if a sustainable contribution is not made through modern technologies . Hundreds of workers die worldwide, even on a residential scale, because of ignorance of safety standards and certain limitations of traditional construction methods adopted . In accordance with Antreas and Piromalis , this is the reason that makes the residential construction sector significant enough to be improved with advanced technologies. In addition to manufacturing, 3D printing has several important applications in various fields, as stated by Kantaros and Kondiah et al. . In regenerative medicine, 3D printing is used to create biocompatible scaffolds that can support the growth of new tissues and organs, offering new treatment options for patients with injuries or diseases . In the aerospace industry, 3D printing is used to manufacture lightweight parts with complex geometries, improving fuel efficiency and reducing emissions. Other applications of 3D printing include creating prosthetic limbs, architectural models, and customised dental implants . In three-dimensional printing, the material is deposited in layers using a computer model as a blueprint. Software, hardware, and raw materials are the foundations of 3D printing. The first 3D printer was invented in 1983 by Charles W., and it has since become one of the fastest-growing industries . A century ago, this technology was exceedingly difficult to operate and pricey. Three-dimensional printers have grown increasingly ubiquitous in industrial settings and have progressively found their way into daily life . According to E. Ali and Korolev et al. , when talking about 3D printing's applications in manufacturing, the phrases "3D printing" and "additive manufacturing" are sometimes used interchangeably. When 3D printing technology advances, we will be able to use it to fabricate dwellings. Complex structures may be constructed more sustainably and with less material waste if this method is used. In addition to its various benefits, 3D printing may be included at any stage of the design process, from the creation of preliminary idea drawings to the construction of fully realised houses. Yang et al. stated that building using a 3D printer eliminates the need for mould support and removal, increasing material efficiency and reducing concrete resource waste . Time frame for building: Due to the enhanced efficiency of 3D printing technology compared to traditional techniques, the construction time may be cut in half, and the payback period may be reduced. Three-dimensional construction printing technology has also shown positive implications regarding maintaining safety on residential construction sites. During the printing process, little noise, dust, and harsh vibrations are produced. Buchanan and Gardner and Elfatah argued that the computer-controlled, layer-by-layer construction method helps minimise room for human error and ensures a superior final product . As 3D printing can be tailored to specific requirements, stockpiles can be minimised; single-piece production eliminates the need for assembly, and the time between printing and delivery is drastically cut down . Beneficial in extreme crises Markin et al., Ji et al. and Holt et al. found that the phrase "extreme environment" is sometimes used to denote that traditional manufacturing processes are no longer applicable and that human activity is impeded when addressing an environment defined by harsh conditions, such as weightlessness or sub-zero temperatures. Three-dimensional printing has the potential to completely alter the construction industry. It is true that 3D printing might significantly cut down on construction expenses, but the expensive cost of printers is a major downside. Hossain et al. and Jawad et al. argued that the intricacy of 3D printing software necessitates the use of trained professionals. According to the available data, the electricity consumption of 3D printers is much higher than that of conventional methods, by a factor of 100 . The high price of the technology is one of the challenges that 3D printing offers to the construction sector. It is unclear if 3D printing will lead to price increases or decreases. El-Sayegh et al., Koc et al. and Shahrubudin et al. stated that the costs associated with switching to 3D printing from more traditional methods of production fall into two main categories. Time is the first factor to consider. A better-quality surface finish requires more time and effort to produce, increasing the related labour cost . The optimization cost is a secondary criterion. Costs will increase due to the additional time and effort required for optimization, which might lead to a more complex construction than is required . However, materials, labour, and infrastructure are the three most obvious expenditures from the perspective of the entire cost structure of a building. With its automated process, 3D printing will reduce construction costs by decreasing the necessity for, and price of, human labour , as the 3D printing system generates an easier and simpler construction with limited human labour. The construction safety risk reduces, so it is no longer necessary to implement collective or individual-level safety measures. Serious accidents involving major injuries, such as falling from heights, can also be avoided with the use of 3D construction printing . Ultimately, it shows positive implications for public health and safety. The use of 3D printers in the building industry is not new, and it is only expected to increase in popularity. Iuliano et al. and Tahmasebinia et al. stated that, due to the rapidity of the projects and the lower cost, an additive building has the potential to disrupt the traditional construction business . Corporate activities that have negative effects on the environment and society are coming under closer scrutiny, so technology improvements such as 3D printing are quite welcome. Business enterprises have an essential role in modern society, and one aspect of this role is to conduct themselves ethically. Cascone et al. and Garcia-Alvarado et al. argued that with help from the government and numerous partners, 3D printing is likely to keep improving, allowing for faster and cheaper house building in Malaysia. There is a chance that in the near future, the residential sector of Malaysia may adopt a prefabrication or on-site 3D printing method for buildings that would dramatically cut carbon emissions . Historical data reveal that architects and the construction industry are willing to adopt and improve upon this innovation . Many authors have produced overviews of this technology and its consequences and uses in construction, and the number of these publications is growing. Shahzad et al., Mascarenhas and Makhoul, and Kazemian and Khoshnevis stated that, while these analyses cover a wide range of topics, they often zero in on one facet of technology and how it is used. However, the present level of study lacks the systematisation necessary to offer a full overview of the technology's uses and influence in the residential building sector of developing nations such as Malaysia. Despite the lack of information, further study is needed to determine how 3D printing affects new home construction in Malaysia. It has been observed that the lack of understanding of 3D printing among Malaysian residential building stakeholders prevents its use in ensuring overall project success (OPS) as measured by cost, time, quality, public health safety and environment. Despite this basic familiarity, research shows that practitioners require a deeper understanding of the implementation notion at hand. According to Besklubova et al., Elias Ali and Pan et al. , while this is an obvious need for the residential construction industry, earlier research on residential 3D building in Malaysia has not made the connection to OPS. A project is regarded as successful if it is finished on schedule, under budget, and to a high enough quality standard as determined by OPS. The risk of 3D construction printing as a new construction technology has been accompanied by a lack of awareness of the more real impact factors that significantly influence the OPS of residential building projects in Malaysia. In Malaysia, where efforts are being made to secure the long-term profitability of smaller-scale building projects, the results of this research are crucial to the growth of the residential construction sector. Since 3D printing has already been shown to be a technology that brings high sustainability to the construction industry, businesses and people in the residential building projects in Malaysia would do well to educate themselves on the current impact elements of adopting the technology. With that in mind, the goal of this research is to examine how 3D printing can influence the OPS of Malaysia's residential construction sector. The purpose of this research was to examine the link between 3D printing and operating profit margins in Malaysian residential construction projects using partial least square (PLS) modelling. 2. Research Model Development 2.1. 3D Printing Measurements Concrete 3D printers are widely used in the modern construction technique known as 3D printing. Multiple academic articles are required on the method to evaluate its structure and methodology . The use of 3D printing in the building industry has the potential to increase sustainability in various ways. There are five main advantages of using 3D printing in construction, such as, "Function", "Sustainability", "Standardization", "Creativity" and "Credibility". These are, perhaps, the most far-reaching implications of 3D printing for the construction sector. Table 1 shows the 3D printing impact factors that are identified as being related to project success. 2.2. Cost, Time, Quality, Safety, Environment Measurements A completed building project is now seen as par for the course. This highlights the need for establishing common ground amongst the project's customers, designers, and consultants over the definition of success. Principles of project management state that timely, cost-effective, and high-quality project completion, along with public health and environmental protection, are the five most important reflective indicators of a construction project's success. The variables that affect a construction project's safety and completion on schedule are listed in Table 2. 2.3. Connection between 3D Printing and Residential OPS The overall success of a project is dependent on five important aspects, including cost, time, quality, environment, and safety precautions. If these five conditions are met, every construction project may be deemed a success; hence, the OPS is based on effective time management, regulated cash flow, quality assurance, environmental preservation, and public safety . It is because of the fact that the construction project's success should provide benefit to society, the environment, and the economy . It has been investigated whether or not 3D printing may be used to expedite mundane but necessary building operations. Before declaring the endeavour a success, however, it was necessary to weigh the numerous implications of 3D printing. Three-dimensional printing's application to the building trades will determine the project's ultimate success. E. Ali, Korolev et al. and Tan conclude that advancements in 3D printing of buildings will allow for a higher probability of success in the long run. These days, projects are more complex and need novel approaches that use the synergy of those involved to ensure the project's success. Elfatah, Lee et al. and Marchment et al. stated that conflicts may be avoided and participants' expectations for the project's success can be bolstered by using 3D printing in construction to resolve and manage their future concerns. According to El-Sayegh et al., Florea et al. and Hossain et al. , in terms of overall project success and project completion, the impact of 3D construction printing on employees and businesses is different from what has been witnessed in the literature thus far. OPS becomes therefore certain when any construction project does not result in any causing injury to workers, increased cost than planned budget, impacted the timeline, reduced quality, and affected the environment. This research is significant because it will contribute to our understanding of 3D building printing and its impact on OPS by shedding light on how these technologies are really put to use. To the best of our knowledge, this is also one of the first empirical studies performed in Malaysia. In this study, 3D printing is an independent variable and OPS is an independent variable. The main hypothesis is, H1: There is a significant relationship between 3D printing and OPS. The paper goes into depth on the impact that different 3D printing methods had on OPS in the Malaysian manufacturing industry, including the planning and implementation stages. The value of a study is shown when it provides theoretical insight into the phenomena being examined, in this case, 3D building printing in a new nationwide environment. The hypothesis of this study relative to the connection between 3D construction printing and residential OPS is given in Figure 1. 3. Research Approach The first stage in developing a theory is to devise a research approach. Ringle et al., The first stage in developing a theory is to devise a research approach. Ringle et al., Savalei and Tarka stated that, in reality, the theoretical model is only a synopsis of a literature review tailored to the issue at hand, which is then utilised to generate preliminary hypotheses. The three steps of the conceptual modelling method are (1) establishing the model's components, (2) categorising the constructs, and (3) documenting their relationships. Structural equation modelling (SEM) is a statistical technique that allows researchers to test complex theoretical models that involve multiple interrelated variables. SEM is a form of regression analysis that includes both observed and latent (unobserved) variables, and it can be used to analyse both cross-sectional and longitudinal data . The technique allows researchers to test and refine theoretical models, estimate and test direct and indirect effects between variables, and assess the fit of the model to the data. The methodology flowchart is presented in Figure 2. Fifteen specialists in the Malaysian residential building industry were contacted to validate and classify the 3D construction impact factors according to Table 1. Zyphur and Pierides stated that it has been shown from prior research that there are just a handful of worldwide drivers impacting 3D printing acceptance and regulatory focus. Therefore, this study included a pair of questionnaires. Before using measuring equipment in the main research, it is standard practice to perform a pilot study to ensure its accuracy. Primary research is the next stage to test this notion. 3.1. Pilot Study The pilot study employed exploratory factor analysis (EFA) to analyse the results of a single survey given to a random selection of Malaysian residential construction firms. Between 150 and 300 samples are required for EFA analysis . According to Igolkina and Meshcheryakov, Shirgaokar and Larsson et al. , it is advisable to limit the number of variables in a factor evaluation to between 20 and 50, since any more than that makes it hard to offer a granular breakdown of the factors. This is because a larger sample size enabled the researchers to use fewer variables. The population size was determined according to the most advanced and locally active residential construction companies (40) and the number of employees in each company (10). This gave a population size of 400, from which the sample size of 200 was determined in accordance with randomised sampling and a 5% margin of error. 3.2. Main Questionnaire The results of the research led to the creation of a standardised cross-sectional survey. The activity categories were revised once the first interviews and EFA evaluation were completed. The Malaysian state of Perak served as the study's location. In order to assess the impact of 3D printing on the construction industry and residential OPS issues, we solicited responses from a large pool of industry professionals via a primary survey. The four main sections of this questionnaire were: (1) the respondent's demographic profile; (2) the impact factors of 3D construction printing (Table 1); (3) the overall project characteristics (time, cost, quality, safety, and environment); and (4) open-ended questions to solicit suggestions for additional activities deemed essential by respondents. Figure 3 shows the distribution of the survey to the three key stakeholders: contractors, consultants, and clients. Subspecialties of these careers include those of electrical engineers, architects, quantity surveyor specialists, civil engineers, and mechanical engineers. The demographic profile of the participants involved in the study is shown in Figure 3. From an education perspective, 11% had a diploma, 41% had a bachelor's degree, 35% had a master's degree, 6% had a PhD, and the remaining 9% had other types of education. From an organisation perspective, there were 64% contractors, 12% consultants, and 24% client developers. From a current working position perspective, 35% were managers, 22% were design engineers, 23% were site engineers, 14% were senior managers, and only 3% were directors. It is observed that 12% of respondents were not familiar, 56% were familiar, 22% were moderately familiar, and only 10% were totally familiar with 3D printing technology in the construction industry. From a profession perspective, 64% were civil engineers, 14% were architects, 12 were quantity surveyors, 6% were electrical engineers, and only 4% were mechanical engineers. From an experience perspective, 13% of participants had less than 5 years, 36% had 5 to 10 years, 33% had 10 to 15 years, 11% had 15 to 20 years, and 7% had greater than 20 years of experience. 3.3. Analytical Approach To explore how 3D printing in construction affects the success of building projects, four models from the literature were analysed and compared to the best alternative generated by utilising 3D printing to establish a simulation model for successful construction development. Multiple linear regression, system dynamics, structural equation modelling, and artificial neural networks. The link between non-observed variables has barred the regression equation from being applied. This substantially limits the use of the regression equation. The system's dynamics could not be exploited as there was no temporal link in the data (i.e., the information is not time-related) (i.e., the information is not time-related). According to Savalei and Tarka , the fundamental purpose of the research is to analyse how 3D printing of a home building may be utilised to accomplish OPS, and the ANN is undoubtedly a predictive advanced model that does not completely fit the sort of data included in the study. The SEM technique may be used to describe the connection between as many unobservable and observable components as the study's scope permits. SEM is an efficient approach for controlling typographical mistakes . To establish the association between 3D printing in home building and OPS, a model was constructed using SEM in this study. Despite the broad use of hypothesis testing methodologies, SEM has become a well-established non-experimental scientific methodology. Ringle et al. Savalei and Wang and Rhemtulla have shown this tactic's efficacy over time. According to Larsson et al., Igolkina and Meshcheryakov and Shirgaokar , as an added bonus, SEM is widely recognised and used as a credible resource for data collection and analysis in the social sciences because of its apparent usefulness in the building industry. Activities within the 3D printing impact factors and OPS were studied using the PLS model, which incorporates both formative and reflecting components, to determine the nature of the interaction between them. The measurement model applying PLS explains the relationships between the idea of 3D construction printing and the observed indicators. 4. Results An EFA was performed to explore the factor structure of 23 important 3D printing influence components. The link was constructed by combining previously established factors of factorability. The Kaiser-Meyer-Olkin (KMO) test is often used to determine whether or not missing linkages between variables are negligible. Ringle et al., Wang and Rhemtulla and Zyphur and Pierides stated that the lowest possible value of the KMO index for effective factor analysis is 0.6. In addition to the non-identity matrix construction, the sphericity test of the correlation matrix proposed by Bartlett allows for its construction in the identity matrix form. Assuming Bartlett's sphericity test succeeds, only then can we have faith in the factor analysis (p < 0.05). Both the KMO sample sufficiency measure (0.544) and Bartlett's test seem to be statistically significant. Although variables with fair values (0.3) do not precisely match the factor solution, the initial communalities (estimates of variance when all contributing factors are included) are consistent with the null hypothesis. Based on what we know now, it seems that prehistoric societies had great prosperity. There was no negative loading factor. In the EFA, five of the 20 variables had eigenvalues greater than 1. The five components and their eigenvalues explained 59.418 percent of the total variance. Four critical components, 3DP.PR1, 3DP.PR2, 3DP.PR3, and 3DP.PR4, made up the last component of inventiveness. Due to their large cross-loadings and loadings below 0.6, the variables 3DP.PR2 and 3DP.PR4 were removed from the analysis; similarly, the variable 3DP.PR2 was not permitted to go to the standardisation phase. Thus, Table 3 provides a rundown of the top five candidates for extraction components in light of 3D printing theory. The statistical validity of the EFA-retrieved variables was assessed. According to Hayward et al. and Savalei , Cronbach's alpha values over 0.6 are considered sufficient for newly created measures, with 0.7 being the norm and 0.8 reflecting exceptional reliability. High reliability is statistically indicated by a Cronbach's alpha score over 0.7. All average set correlations were greater than 0.3, indicating statistical stability among the objects' internal variables. 4.1. Measurement Model (First Order Construct) Evaluation of a measurement model includes estimating indicator consistency, combination reliability, extracted average variance (AVE), and discriminant validity. In accordance with PLS standards, this study employs a suitable weighting scheme, data measure, maximum iterations, abort criteria, and starting weights . Figure 4 is an example of a structural equation model (SEM) addressing the hypothesis indicated in Figure 1. Based on the current literature, Table 1 and Table 2 define and classify the many 3D printing and OPS aspects of the idea. In accordance with Fraserhealth, Savalei and Wang and Rhemtulla , to increase the composite's reliability and AVE, the threshold should be increased if eliminating indications with external loadings between 0.40 and 0.65 on the scale considerably improves the reliability and AVE. The variables assessing environmental stress with values below 0.5 were ruled out as insufficient to fulfil this criterion and, hence, were not further investigated. Table 4 shows that all constructs and their factors pass this test, and Figure 4 provides the external loadings for each variable in the simple measurement model. Loading factors greater than 0.6 were found for all of the external loads for items pertaining to the "Function", "Sustainability", "Standardization", "Creativity", and "Credibility" constructs, with the exception of 3DP.PS4 and 3DP.PR3, which had loadings of 0.569 and 0.573, respectively. However, the last construct, credibility, was excluded from the analysis because factor 3DP.PR1 was the only factor in the construct and that is not feasible in terms of construct modelling. Further strong relationships were found among all other constructs, indicating high composite reliability, Cronbach's alpha and Ave. As can be seen in Table 4, all approved models had CRs greater than 0.70, indicating their reliability. When evaluating the convergent validity of model designs, AVE values over 0.50 are often tolerated. A concept has discriminant validity if it can be separated from other, comparable concepts by independent, external criteria. The construct's selective validity demonstrates how its uniqueness enables it to catch occurrences that are poorly captured by other model conceptions. The cross-loading criterion, the criterion of a ratio of correlations between heterotraits and monotraits (HTMT), and the criterion of independence are the three methods available for verifying discriminant validity. Ringle et al. found that quantitative evidence for discriminative validity might be found by contrasting the square root of the AVE for each construct with correlations between any two constructs. The maximum allowable correlation between latent variables is the AVE's square root. The results, shown in Table 5, demonstrate the discriminant validity of the measurement model. Assuming that the constructs were reliably assessed, the HTMT method offers a unique way to assess the discriminative validity of variance-based SEMs by studying the correlation between two constructs. In this study, the HTMT model was used to evaluate the discriminant's efficacy. An HTMT value of 0.85-0.90 will do to show the difference between the two ideas. A score of less than 0.90 on the HTMT indicates conceptual similarity between the model structures, whereas a score of less than 0.85 indicates conceptual dissimilarity. All of the HTMT readings for the buildings under study are summarised in Table 6. Therefore, the ideas have enough discriminant validity. This study also used cross-loading criteria to prove discriminatory validity. According to Igolkina and Meshcheryakov and Zyphur and Pierides , the indicator loading on a particular latent construct is compared to the loading on all other latent constructs in a specific row to see whether there should be any discrepancy. So, their structures must have more indicator loading than standard structures. Table 7 shows that the indicator loads of all latent constructs exceed their cross-loadings on all other constructs (by row). The results show that most structures only have one dimension. 4.2. Measurement Model (Second Order Construct) Since the primary variables were all second-order latent factors, the bootstrap approach was used to assess the importance of each first-order latent variable. The 3D printing implementation project had a formative aspect, whereas the home-based OPS was more reflective. High levels of correlation between variables from various formative measuring methods are not usually immediately apparent. Jak and Cheung and Zyphur and Pierides stated that there is a high potential for collinearity because of the close connection between the constituent elements. By quantifying the worth of 3D printing's impact, we were able to examine the collinearity between the construct's formative components. Internal VIF values were used to probe collinearity issues while handling reflective-formative second-order construct types. The components of the function stage, the sustainability stage, the creative stage, and the standardisation stage stood out, contributing to a relatively high standard route coefficient for 3D printing operations as indicated in Figure 5. From Table 8, it was found that the creativity construct had the highest outer loading b of 0.495. For sustainability, function, and standardization, the outer loadings are 0.463, 0.259, and 0.211, respectively. The path significance values for all of the construction were less than 0.05 indicating high acceptability of results. The VIF values obtained were less than 3.5, indicating that each factor is contributing independently towards forming a higher-order construct. The formative second-order construct, therefore, contributed significantly to 3D printing in construction. Table 9 shows the bootstrapping statistics of reflective second-order constructs. Cost and environment have indicated the highest path coefficients of 0.789 and 0.741. For quality, safety, and time, the path coefficients were 0.719, 377, and 0.451, respectively. The evidence of significant and effective outcomes of reflective constructs indicates acceptable statistics regarding the impact of 3D printing in residential OPS. 4.3. Structural Model (Path Analysis) Path analysis is an effective method for researchers interested in linear regression. Path analysis is the standard technique for scientific and social management. In addition, path analysis is the primary technique for investigating all intricate interrelationships concurrently. Much of the work in SEM analysis is performed with the use of a structural equation model . The structural model may be used to analyse the interrelationships between the variables. Creating a structural equation model was the next crucial stage of the SEM analysis. According to Igolkina and Meshcheryakov and Shirgaokar , if the relationships between the variables are known, a structural model may be employed. The structural model provides a more thorough explanation of the connections between the various variables. The information demonstrates the connection between endogenous (inside) and exogenous (outside) variables. When assessing the aforementioned structural model, the overall model fit is of utmost importance, followed by the size, direction, and relevance of the estimated parameters. The research that used SEM to test the theory is shown in Figure 1. We utilised PLS-SEM to examine the impact of 3D printing adoption on OPS-related aspects based on the research framework provided by this model. Bootstrapping helped us evaluate the model's null hypothesis and its significance . By randomly resampling the original dataset, the bootstrapping method creates new samples of the same size as the original samples. How reliable the data is, how much room for error there is in the predicted route coefficients, and what degree of significance we can be sure of using. As the Table 10 shows, 3D printing has a major effect on OPS at residential construction projects. The study indicated that 3D printing had positive and statistically significant benefits on productivity in residential construction. 4.3.1. Explanatory Power of the Structural Model The results indicate not just strong covert validity and discriminant validity in the evaluative model but also high levels of reliability at the item level. According to Igolkina and Meshcheryakov and Savalei , to evaluate a model's ability to explain data, one approach is to determine what percentage of the total variance in the dependent variable can be attributed to the model. Multiple R2 correlations between dependent variables inside a model may be determined using PLS. In PLS, the R2 value is said to be the same as in traditional regression. According to Shirgaokar and Zyphur and Pierides , the coefficient of determination (R2) is a statistical indicator of complete randomness. It was the independent factors inside the dependent variable that provided the reason for this. A higher R2 value indicates a more robust structural model. Table 11 displays the results of studies on regression. With an updated R2 of 0.9, we see that the exogenous latent variable (3D printing in construction) can explain 90% of the variation in project performance, making it the most important dependent variable in our model. Larsson et al. and Tarka stated that whether or not an independent construct has a significant impact on the dependent constructs may be inferred by analysing the change in R2 after eliminating the construct from the model. The effect size (f2) measurement is therefore carried out in accordance with Equation (1) . A high effect size of 1.33 was observed. The computation of the effect size is, (1) Effect Size=f2=R2include-R2exclude1-2R2exclude f2=0.02 -Moderate;f2=0.15 -Medium;f2=0.35 -High 4.3.2. Predictive Relevance of the Structural Model Predictive validity assessment is an essential part of any structural model. The blind method was used to verify the redundancy estimates obtained by cross-validation for each dependent variable. Statistical analysis of the data revealed that the Q2 scores had a predictive value (0.919 for project performance) above zero, suggesting that the independent construct had predictive relevance for the dependent construct under consideration. Table 12 shows that Q2 is greater than 0. It is reasonable to assume that the model has a very good predictive ability. 4.3.3. Analysis of Performance Matrix Importance PLS-SEM provides empirical evidence for the relative significance of an independent variable in explaining the dependent variable. According to Igolkina and Meshcheryakov , in deciding amongst these potential managerial measures, it is crucial to consider both their relevance and their performance. By analysing the model's overall effects (importance), as well as the mean value for latent variable scales, key areas for improving management operations (or the model's focus) may be identified (performance). The success of the 3D printing in this investigation relied on importance-performance map analysis (IPMA). Table 13 presents the overall performance and importance of latent exogenous variables in 3D printing in construction. 5. Discussion Success rates on Malaysian residential projects can be greatly improved by incorporating 3D printing. There is a good basis for investigating connections between the models thanks to the statistical data gathered by testing the models using the modified SEM approach. The function formative construct includes 3DP.PF1: "Making customized residential structures available to the wider market", 3DP.PF2: "Simpler and more efficient installation", 3DP.PF3: "Increased rate of construction efficiency", 3DP.PF4: "Accuracy that is far higher than before", and 3DP.PF5: "Quick and easy prototypes". Three-dimensional printing has been discovered to be a simpler and more efficient installation method in Malaysia's residential sector. It is advantage number two in providing effective outcomes for the residential sector of Malaysia, as ultimately it can help to save a lot of time and reduce costs. El-Sayegh et al. and Yang et al. also indicate a similar behaviour where 3D printing is found to be significant for housing projects in terms of effective installation. According to Kazemian and Khoshnevis and Poluektova , the other factors have a moderately similar impact on Malaysia's housing industry. The most important factor from a functional standpoint is the customization of residential structures, which is consistent with current research, which classifies 3D construction printing as the most highly customizable technology currently available in the world for the construction industry. The different behaviour is observed in the sense of an overall combination of factors, because ultimately it is indicated that the function construct contributes significantly to the 3D printing impact, with customisation and efficient installation being the most notable factors. The sustainability formative construct includes, 3DP.PS1: "Enables the design and construction of environmentally responsible buildings", 3DP.PS2: "The recycling of trash into a new product", 3DP.PS3: "The printing technique will reduce waste, minimizing production/environmental construction's effect", 3DP.PS5: "Enable and provide advanced healthcare", and 3DP.PS6: "Reduced human impact". The sustainability construct clearly shows that 3D construction printing will have a positive impact on the environment of the residential construction sector while also being critical in terms of making the residential sector sustainable. The observed indication of environmental sustainability is in accordance with Bergeron et al. and Mascarenhas and Makhoul , where it is regarded as the main advantage of adopting 3D construction printing in the housing industry. It is also a notable factor from the outcomes that the human impact is being reduced with the help of 3D construction printing, which is effectively identified by M. H. Ali et al. and Z. Xu et al. , where 3D construction printing is found to be more error-free and reliable in terms of promoting sustainability in the housing industry. The primary distinction in this regard is the overall sustainability perspective, in which recycling is also identified as important for Malaysia's residential sector mobile and is also indicating a positive impact on worker health and safety outcomes. The standardisation formative construct includes, 3DP.PD1: "Fewer logistical procedures and waste", 3DP.PD2: "A shorter supply chain and a faster design cycle", 3DP.PD3: "Reduce human mistakes", and 3DP.PD4: "Evaluate ideas generated during brainstorming considering the intended outcomes". It is indicated by the standardization construct that there is a significant improvement in the reduction of human mistakes while adopting 3D construction printing in the residential construction sector of Malaysia. The indication is strong in the sense of M. H. Ali et al. and Han et al. , where it is highly regarded as a prominent technology that can minimise errors in construction as a significant level of automation is involved. The observed behaviour is also indicating that it is also important in terms of generating new ideas in construction, which is truly evident from Adaloudis and Bonnin Roca in other parts of the world where 3D printing is found to be highly innovative for model housing architecture. The outcomes provide a unique way of understanding the impact of 3D printing in the residential construction sector of Malaysia, which is different in the sense of an effective combination of factors promoting a shorter supply chain, error reduction, and improved idea generation. The creativity formative construct includes, 3DP.PC1: "Provide Innovative solutions", 3DP.PC2: "Modelling architectural construction", 3DP.PC3: "Permits more geometric flexibility when designing buildings that would not be achievable otherwise", and 3DP.PC4: "Flexible design and brand improvement". The flexible design and brand improvement discount are highly significant for the residential construction sector of Malaysia, which is critical in the sense of Aghimien et al. and Skoratko and Katzer where 3D construction printing is already regarded as a prominent technology for the housing sector. The ranking of factors indicated in the creativity construct is entirely different in the sense of Abdalla et al., Adaloudis and Bonnin Roca, and Sun et al. , which is unique in the context of Malaysia's residential sector and their efforts to promote the adoption of 3D construction printing. It is also indicated by creatively constructing that, with the help of 3D construction printing, the possible applications of improving geometric flexibility and implementing innovative solutions become very high. The observed behaviour is in accordance with the grassing research while also indicating differentiation, which is fully linked with the different states of the Malaysian residential construction sector. The time reflective construct includes, T1: "On time project delivery", T2: "Timely project delivery involving variations", and T3: "Timely availability of resources needed for project completion". It is evident that 3D printing is directly improving the on-time project delivery aspect of ops, which shatters the argument provided by existing research in which time is regarded as one of the critical factors. The indication is also so strong in terms of promoting the timely availability of resources required for project delivery, and the study is providing effective insights in accordance with Bedarf et al., Plarre et al., and Lam et al. on 3D printing in the housing industry. The impact condition is completely different from Besklubova et al. and Ting et al. , in which time was regarded as important, but project delivery is most critical for Malaysian residential construction projects if they adopt 3D construction printing in the future. It is a strong indication that 3D printing for construction will improve the project timeline. The cost reflective construct includes, C1: "Profit margin improvement", C2: "Cash flow enhancement", and C3: "Reduction in variable costs". It is found that profit margin improvement is the ultimate benefit of adopting 3D construction printing, and with respect to Khoshnevis, Pan et al., and Shahzad et al. , it is also found to be reliant on making the construction project successful. The cost of production is already prone to being one of the critical advantages of 3D construction printing, as is evident from the outcomes of this study. It is in accordance with the concept that there is any improvement in sustainability with the help of cost improvement, while it is different only in the sense of global research, where the Malaysian residential construction sector is giving more prominence to profit margin improvement. The observed behaviour is indicating significant implications for the residential construction sector in Malaysia, where it will be necessary to adopt 3D construction printing in terms of cost reduction and promoting OPS. The quality reflective construct includes, Q1: "All specifications are met", Q2: "All resources are available for required quality delivery", and Q3: "Delivering project with compliance to equipment and raw material quality". The availability of resources is critically linked with the quality delivery of overall projects involving 3D construction printing, and this is in accordance with existing research in terms of identifying the quality parameters as critical outcomes of implementing 3D construction printing in the housing industry. It is also an effective indication of different research outcomes because, in the Malaysian residential construction sector, there is more importance given by participants to resource management, which is totally linked with implementing 3D construction printing in accordance with Bedarf et al. and Hoffmann et al. . This behaviour is attributable to the concept of overall compliance with raw material quality as well as maintaining the quality delivery of projects, which is in accordance with existing research indicating successful outcomes of projects. The environmental protection reflective construct includes, E1: "Sustainable logistics with reduced wastage of materials", E2: "Environmental protection objectives and standards satisfied", and E3: "Reduced energy consumption with a reduction in net embodied carbon". The reduction in waste generation of materials with sustainable logistics is identified to be the highest significant sub-factor that is critically improving the environment and ultimately providing benefits to overall project success. It is the reason that the positive implications of 3D construction printing are linked with maximizing the environmental benefits. Further, when the environmental standards are met in the residential project, it ultimately improves the rate of success and also allows the 3D printing technology to be adopted as a new technology for further development with environmental protection. Similar is the case with the reduction in energy consumption, as the project completed by 3D construction printing will have benefits for the environment and ultimately reduce embodied carbon. As indicated by Cascone et al. and Gomez et al. , significant positive implications exist in terms of improving the environment with the help of 3D construction printing. The different results observed from the analysis where a reduction in waste of materials can be regarded as a critical factor that will ultimately improve environment protection. It is especially relevant in the residential construction environment of Malaysia, where a significant challenge has always been to reduce the waste of construction activities, which is ultimately susceptible to the effective relationship between 3D construction printing and the environmental production aspect of overall project success . The public health and safety reflective construct includes, S1: "Sustainable physical well-being of workers", S2: "Increased security and safety, less dependency on human resources", and S3: "Effective public health hazards management on worksite with technology". It is the unique quality of 3D construction printing observed from analysis that helps reduce human resource dependency, which ultimately improves safety on the work site. Na et al., Kazemain et al., and Faham et al. have shown significant evidence of increasing public health safety when automation is involved, and a similar effect is observed in the case of adopting theory-based construction printing. That is indicated in terms of improving the sustainable physical well-being of workers as well as providing effective public health hazard management on the job side with automatic technology. However, different behaviour is observed, indicating the greater importance of increasing worker security and safety with the help of 3D construction printing. Positive implications are observed as 3D construction printing contributes significantly to public health and safety aspects of overall project success and can be highly appropriate for residential construction environments in Malaysia. All five dimensions of the overall project success have contributed significantly to indicating the possible benefits of using 3D construction printing in the residential construction sector of Malaysia. The cost-time benefits are clearly indicated, which ultimately maximise the success rate of projects while ensuring further implications for increasing safety for workers and maintaining efficient public health standards. Furthermore, an incorrect implication was observed with regard to the environment, as a result of which construction 3D printing ultimately helps to reduce waste, which supports overall project success. It is the reason that 3D construction printing is positively linked with increasing public health and environmental compliance. For the practical improvement of public health and the production of the environment on residential construction project work sites, it is important that 3D construction printing is adopted by the use of the latest technology. Even at a smaller scale, 3D construction printing can revamp the overall residential construction and sea of Malaysia, which can ultimately contribute to reducing the frequency of injuries and allowing the workers to ultimately protect the environment, which is one of the critical hazards contributing to global climate change. The creativity aspects are more prominent in the analysis, which indicates possible ways of innovation that can further improve the efficiency of technology in maintaining overall project success in residential construction projects in Malaysia. 6. Conclusions To verify the links between 3D printing and OPS (cost, time, quality, public health safety, and environmental protection) in Malaysian home construction, a PLS-SEM study was conducted. Specialists in the building trade provided the evidence necessary to verify the direct and indirect channels as essential to the structural model. Furthermore, both direct and indirect correlations between activity components and covariates were confirmed. Based on the findings, 3D printing may be used to save costs and boost the success rate of any project. Three-dimensional printing improves Malaysian house construction by reducing time and cost. Furthermore, 3D printing is vital for housing developments. Additional factors may impact Malaysia's housing market in the future, but 3D construction printing will always help to improve all scope dimensions of a project. Residential building modification is functionally important. Three-dimensional printing is the most adaptable building method, according to a study. Customisation and successful installation cause varying behaviour. The sustainability build shows that 3D construction printing will favourably impact the home building sector's environment. According to a study, 3D printing in houses promotes environmental sustainability. Three-dimensional building printing reduces human impact and promotes sustainability in the housing industry, according to research. Sustainability-wise, recycling is important for Malaysia's mobile home sector and worker health and safety. Standardisation in Malaysia's house-building sector eliminates human mistakes. According to research, it may decrease construction errors via automation. Three-dimensional printing is revolutionising model home design in different parts of the world, as shown by the observed behaviour. The findings provide a new way to measure 3D printing's impact on Malaysia's homebuilding industry, which encourages a shorter supply chain, mistake reduction, and idea creation. Flexible design and improvement are significant for Malaysia's residential building industry, as 3D printing is a prominent housing technology. Unique in Malaysia's home industry, the creative construct ranks components by worldwide demand to stimulate 3D building printing. Three-dimensional printing may boost geometric flexibility and generate unique solutions. It may be enhanced by cutting expenses; however, research suggests that the Malaysian residential construction market emphasises profit margins. Three-dimensional printing will be required to cut costs and enhance OPS in Malaysia's homebuilding industry. The availability of resources affects the quality of 3D construction printing projects, which is consistent with past research stressing quality criteria as critical consequences of adopting 3D printing in the housing industry. In Malaysia's residential building business, resource management is linked to 3D printing. The behaviour is related to compliance with raw material quality and quality project delivery, which is consistent with excellent project outcomes. It has been noted by academics that the methods and tactics for implementing 3D printing have been understudied. While many have looked at the effects of 3D printing on OPS, most have neglected to address the true causes of this condition. The present research fills a gap in the existing literature by examining the relationship between 3D printing and OPS. The primary gain from this research is a stronger foundation for engineering project management in the residential construction industry in Malaysia, specifically in one chosen sector. Second, this research paves the way for more research by demonstrating the tangible benefits to all dimensions of OPS brought about by the widespread use of 3D printing. This new information may encourage further study of 3D-printed residential developments. And lastly, despite Malaysia's standing as a developed nation with strong development in construction, most 3D printing legislation and plans have not moved beyond the planning phases. Three-dimensional printing has gained popularity in Malaysia as a rapid and cost-effective manufacturing technology. However, its widespread use raises concerns about its potential impact on public safety and the environment. One of the major limitations of 3D printing is the lack of regulations and standards governing the production and use of 3D-printed products, which can pose risks to public safety. The use of low-quality or untested materials in 3D printing can result in weak, defective, or hazardous products that can harm users. In addition, the disposal of 3D-printed waste, which can be non-biodegradable and contain toxic materials, poses a threat to the environment. Another limitation is the energy consumption associated with 3D printing, which can contribute to carbon emissions and climate change. Overall, the adoption of 3D printing in Malaysia should be accompanied by proper regulations and guidelines to ensure public safety and minimise its impact on the environment. No studies have looked at the outcomes of employing 3D printing in the OPS, and there is a gap in research on the issue in the Malaysian residential construction industry. This kind of study is crucial to the development and general use of 3D printing in the field. This study provides the groundwork for 3D printing to become widely used in the Malaysian construction industry. Project practitioners, such as building owners and contractors, may benefit from the article's guidance since it explains how they can utilise 3D printing to improve the public health safety, environment, time, cost, and quality of their projects. This study has the potential to impact the success of future residential projects by assisting project stakeholders in accepting and executing 3D printing with a focus on OPS at every stage of the project's existence. Author Contributions Conceptualization, A.W. and I.O.; methodology, A.W.; software, A.W.; validation, A.W., I.O. and J.C.P.; formal analysis, A.W.; investigation, A.W.; resources, I.O.; data curation, J.C.P.; writing--original draft preparation, A.W.; writing--review and editing, I.O.; visualization, J.C.P.; supervision, J.C.P.; project administration, I.O. All authors have read and agreed to the published version of the manuscript. Informed Consent Statement Informed consent was obtained from all subjects involved in the study. Data Availability Statement The data are not publicly available due to privacy restrictions. Conflicts of Interest The authors declare no conflict of interest. Abbreviations 3DP 3D printing OPS Overall project success SEM Structure equation modelling IPMA Importance-performance map analysis ANN Artificial neural network EFA Exploratory factor analysis Figure 1 Hypothesised relation between 3D printing and OPS (time, cost, quality, public health and safety and environment). Figure 2 Research methodology flowchart. Figure 3 Demographic profile. Figure 4 Structural model with path coefficients. Figure 5 Bootstrapping analysis showing outer path indicating loading and p-values and inner weights with p-values. ijerph-20-03800-t001_Table 1 Table 1 Three-dimensional printing impact factors evident from existing literature. 3DP Phases Assigned Code Activities References Function 3DP.PF1 Making customised residential structures available to the wider market 3DP.PF2 Simpler and more efficient installation 3DP.PF3 Increased rate of construction efficiency 3DP.PF4 Accuracy that is far higher than before 3DP.PF5 Quick and easy prototypes Sustainability 3DP.PS1 Enables the design and construction of environmentally responsible buildings 3DP.PS2 The recycling of trash into a new product 3DP.PS3 The printing technique will reduce waste, minimising production/environmental construction's effect 3DP.PS4 Reduce the usage of formwork 3DP.PS5 Enable and provide advanced healthcare 3DP.PS6 Reduced human impact Standardisation 3DP.PD1 Fewer logistical procedures and waste 3DP.PD2 A shorter supply chain and a faster design cycle 3DP.PD3 Reduce human mistakes 3DP.PD4 Evaluate ideas generated during brainstorming considering the intended outcomes Creativity 3DP.PC1 Provide innovative solutions 3DP.PC2 Modelling architectural construction 3DP.PC3 Permits more geometric flexibility when designing buildings that would not be achievable otherwise 3DP.PC4 Flexible design and brand improvement Credibility 3DP.PR1 Increased value in global market 3DP.PR2 Bonding for accuracy and complexity 3DP.PR3 Supports unusual and green designs 3DP.PR4 Integration with other components ijerph-20-03800-t002_Table 2 Table 2 Factors leading to overall project success. OPS Factors Code Sub-Factors References Time management T1 On time project delivery T2 Timely project delivery involving variations T3 Timely availability of resources needed for project completion Cost management C1 Profit margin improvement C2 Cash flow enhancement C3 Reduction in variable costs Quality management Q1 All specifications are met Q2 All resources are available for required quality delivery Q3 Delivering project with compliance to equipment and raw material quality Public health and safety S1 Sustainable physical well-being of workers S2 Increased security and safety, less dependency on human resources S3 Effective public health hazards management on worksite with technology Environmental protection E1 Sustainable logistics with reduced wastage of materials E2 Environmental protection objectives and standards satisfied E3 Reduced energy consumption with reduction in net embodied carbon ijerph-20-03800-t003_Table 3 Table 3 Rotated component matrix with reliability statistics. Variables Component Cronbach's Alpha 1 2 3 4 5 3DP.PF1 0.872 0.894 3DP.PF2 0.842 3DP.PF3 0.770 3DP.PF4 0.749 3DP.PF5 0.736 3DP.PS1 0.784 0.836 3DP.PS2 0.750 3DP.PS3 0.707 3DP.PS4 0.699 3DP.PS5 0.654 3DP.PS6 0.649 3DP.PC1 0.864 0.910 3DP.PC2 0.861 3DP.PC3 0.802 3DP.PC4 0.781 3DP.PD4 0.842 0.806 3DP.PD1 0.753 3DP.PD3 0.720 3DP.PR3 0.649 0.701 3DP.PR1 0.617 Eigen value 3.917 3.411 3.389 2.592 2.139 %Variance 15.066 13.121 13.034 9.969 8.228 Extraction Method: principal component analysis. Rotation method: Varimax with Kaiser normalization. Variables 3DP.PR2, 3DP.PR4 and 3DP.PD2 deleted from EFA because of cross loading and loading less than 0.6. ijerph-20-03800-t004_Table 4 Table 4 Construct initial loadings, modified loadings, and reliability statistics. Construct Assigned Code Initial Loadings Modified Loadings Cronbach's Alpha Composite Reliability AVE Function 3DP.PF1 0.924 0.924 0.896 0.924 0.71 3DP.PF2 0.902 0.902 - - - 3DP.PF3 0.778 0.778 - - - 3DP.PF4 0.819 0.819 - - - 3DP.PF5 0.780 0.780 - - - Sustainability 3DP.PS1 0.816 0.811 0.821 0.875 0.584 3DP.PS2 0.755 0.766 - - - 3DP.PS3 0.744 0.737 - - - 3DP.PS4 0.564 Deleted - - - 3DP.PS5 0.699 0.704 - - - 3DP.PS6 0.801 0.796 - - - Standardisation 3DP.PD1 0.825 0.825 0.806 0.885 0.719 3DP.PD3 0.867 0.867 - - - 3DP.PD4 0.853 0.853 - - - Creativity 3DP.PC1 0.883 0.883 - - - 3DP.PC2 0.881 0.881 - - - 3DP.PC3 0.890 0.890 - - - 3DP.PC4 0.893 0.893 - - - Credibility 3DP.PR1 0.867 Excluded - - - 3DP.PR3 0.573 Deleted - - - Cost C1 0.911 0.911 0.873 0.922 0.798 C2 0.850 0.850 - - - C3 0.918 0.918 - - - Time T1 0.907 0.907 0.834 0.900 0.755 T2 0.865 0.865 - - - T3 0.822 0.822 - - - Quality Q1 0.725 0.725 0.701 0.829 0.626 Q2 0.856 0.856 - - - Q3 0.774 0.774 - - - Safety S1 0.628 0.628 0.699 0.776 0.638 S2 0.806 0.806 - - - S3 0.756 0.756 - - - Environment E1 0.792 0.792 0.692 0.752 0.575 E2 0.634 0.634 - - - E3 0.697 0.697 - - - ijerph-20-03800-t005_Table 5 Table 5 Correlations of latent variables and discriminant validity. Cost Creativity Environment Function Quality Safety Standardization Sustainability Time Cost 0.893 Creativity 0.788 0.887 Environment 0.728 0.703 0.711 Function 0.353 0.331 0.701 0.843 Quality 0.205 0.197 0.483 0.399 0.787 Safety 0.28 0.282 0.354 0.397 0.356 0.734 Standardisation 0.252 0.253 0.326 0.378 0.257 0.563 0.848 Sustainability 0.19 0.184 0.561 0.444 0.752 0.33 0.258 0.764 Time 0.336 0.316 0.707 0.777 0.378 0.381 0.383 0.429 0.865 ijerph-20-03800-t006_Table 6 Table 6 HTMT statistics. Cost Creativity Environment Function Quality Safety Standardization Sustainability Time Cost Creativity 0.107 Environment 0.699 0.641 Function 0.39 0.355 0.092 Quality 0.281 0.26 0.66 0.505 Safety 0.384 0.379 0.643 0.518 0.547 Standardisation 0.298 0.292 0.498 0.439 0.33 0.716 Sustainability 0.233 0.218 0.731 0.518 0.266 0.462 0.308 Time 0.38 0.347 0.144 0.125 0.489 0.51 0.46 0.514 ijerph-20-03800-t007_Table 7 Table 7 Statistically determined cross-loadings for discriminant validity. Cost Creativity Environment Function Quality Safety Standardisation Sustainability Time C1 0.911 0.238 0.792 0.375 0.197 0.233 0.245 0.195 0.359 C2 0.85 0.216 0.51 0.197 0.129 0.276 0.238 0.118 0.184 C3 0.918 0.245 0.645 0.363 0.217 0.244 0.197 0.192 0.347 3DP.PC1 0.238 0.883 0.792 0.375 0.197 0.233 0.245 0.195 0.359 3DP.PC2 0.216 0.881 0.645 0.363 0.217 0.244 0.197 0.192 0.347 3DP.PC3 0.245 0.89 0.51 0.197 0.129 0.276 0.238 0.118 0.184 3DP.PC4 0.815 0.893 0.519 0.215 0.144 0.251 0.216 0.139 0.209 E1 0.711 0.671 0.883 0.375 0.197 0.233 0.245 0.195 0.359 E2 0.144 0.139 0.634 0.438 0.599 0.172 0.115 0.555 0.436 E3 0.276 0.259 0.697 0.619 0.331 0.392 0.356 0.377 0.665 3DP.PF1 0.351 0.331 0.621 0.924 0.372 0.347 0.339 0.406 0.907 3DP.PF2 0.361 0.339 0.6 0.902 0.373 0.388 0.33 0.398 0.847 3DP.PF3 0.253 0.234 0.525 0.778 0.332 0.295 0.27 0.364 0.667 3DP.PF4 0.276 0.259 0.697 0.819 0.331 0.392 0.356 0.377 0.765 3DP.PF5 0.226 0.211 0.506 0.778 0.264 0.233 0.297 0.32 0.722 Q1 0.045 0.036 0.357 0.318 0.725 0.19 0.195 0.262 0.305 Q2 0.194 0.183 0.398 0.334 0.856 0.29 0.254 0.182 0.319 Q3 0.244 0.245 0.386 0.289 0.774 0.363 0.145 0.257 0.267 S1 0.24 0.237 0.295 0.391 0.286 0.628 0.625 0.222 0.383 S2 0.181 0.187 0.247 0.238 0.278 0.806 0.152 0.262 0.219 S3 0.175 0.178 0.211 0.195 0.188 0.756 0.118 0.182 0.188 3DP.PD1 0.24 0.237 0.295 0.391 0.286 0.628 0.825 0.257 0.383 3DP.PD3 0.23 0.219 0.308 0.296 0.202 0.408 0.867 0.222 0.303 3DP.PD4 0.16 0.179 0.217 0.262 0.151 0.367 0.853 0.166 0.277 3DP.PS1 0.045 0.036 0.357 0.318 0.262 0.19 0.195 0.816 0.305 3DP.PS2 0.144 0.139 0.634 0.438 0.182 0.172 0.115 0.755 0.436 3DP.PS3 0.194 0.183 0.398 0.334 0.257 0.29 0.254 0.744 0.319 3DP.PS5 0.244 0.245 0.386 0.289 0.222 0.363 0.145 0.699 0.267 3DP.PS6 0.095 0.095 0.373 0.316 0.262 0.238 0.262 0.801 0.311 T1 0.351 0.331 0.621 0.924 0.182 0.347 0.339 0.406 0.907 T2 0.276 0.259 0.697 0.819 0.331 0.392 0.356 0.377 0.865 T3 0.226 0.211 0.506 0.778 0.264 0.233 0.297 0.32 0.822 ijerph-20-03800-t008_Table 8 Table 8 Bootstrapping statistics for formative constructs. Path b SE t-Values p-Values VIF Creativity - 3D printing 0.495 0.034 15.344 <0.001 1.148 Function - 3D printing 0.259 0.032 9.431 <0.001 1.453 Standardization - 3D printing 0.211 0.024 9.298 <0.001 1.202 Sustainability - 3D printing 0.463 0.033 12.933 <0.001 1.262 ijerph-20-03800-t009_Table 9 Table 9 Bootstrapping statistics for reflective constructs. Path b SE t-Values p-Values OPS - cost 0.789 0.036 22.640 <0.001 OPS - quality 0.719 0.059 11.658 <0.001 OPS - time 0.451 0.054 8.258 <0.001 OPS - safety 0.377 0.058 6.483 <0.001 OPS - environment 0.741 0.031 23.804 <0.001 ijerph-20-03800-t010_Table 10 Table 10 Path validation between 3D printing and OPS. Path b SE t-Values p-Values 3D printing - OPS 0.92 0.006 48.3 <0.001 ijerph-20-03800-t011_Table 11 Table 11 R-square determination. Endogenous Latent Variable R2 Adjusted R2 Explained Size Project success 0.9 0.9 Highly predictive ijerph-20-03800-t012_Table 12 Table 12 Predictive relevance. 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PMC10000832 | Diagnostics (Basel) Diagnostics (Basel) diagnostics Diagnostics 2075-4418 MDPI 10.3390/diagnostics13050892 diagnostics-13-00892 Review Artificial Intelligence, Augmented Reality, and Virtual Reality Advances and Applications in Interventional Radiology von Ende Elizabeth Ryan Sean Crain Matthew A. Makary Mina S. * Khandoker Ahsan Academic Editor Division of Vascular and Interventional Radiology, Department of Radiology, The Ohio State University Wexner Medical Center, Columbus, OH 43210, USA * Correspondence: [email protected] 27 2 2023 3 2023 13 5 89209 1 2023 12 2 2023 23 2 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). Artificial intelligence (AI) uses computer algorithms to process and interpret data as well as perform tasks, while continuously redefining itself. Machine learning, a subset of AI, is based on reverse training in which evaluation and extraction of data occur from exposure to labeled examples. AI is capable of using neural networks to extract more complex, high-level data, even from unlabeled data sets, and better emulate, or even exceed, the human brain. Advances in AI have and will continue to revolutionize medicine, especially the field of radiology. Compared to the field of interventional radiology, AI innovations in the field of diagnostic radiology are more widely understood and used, although still with significant potential and growth on the horizon. Additionally, AI is closely related and often incorporated into the technology and programming of augmented reality, virtual reality, and radiogenomic innovations which have the potential to enhance the efficiency and accuracy of radiological diagnoses and treatment planning. There are many barriers that limit the applications of artificial intelligence applications into the clinical practice and dynamic procedures of interventional radiology. Despite these barriers to implementation, artificial intelligence in IR continues to advance and the continued development of machine learning and deep learning places interventional radiology in a unique position for exponential growth. This review describes the current and possible future applications of artificial intelligence, radiogenomics, and augmented and virtual reality in interventional radiology while also describing the challenges and limitations that must be addressed before these applications can be fully implemented into common clinical practice. interventional radiology artificial intelligence machine learning deep learning radiogenomics This research received no external funding. pmc1. Introduction Artificial intelligence (AI) is the development of computer algorithms to process and interpret data as well as perform tasks with partial or complete autonomy, while continuously refining its logic and decision making. Only with the more recent development of powerful computational hardware capable of collecting, storing, and processing large amounts of data has the field of AI become relevant to radiology. Specifically, the field of interventional radiology (IR) is in a unique position to benefit from advances in AI to not only improve image processing, but also guide and predict outcomes of their minimally invasive procedures. First officially introduced in the 1950's, the growth of AI began with the introduction of artificial neural networks (ANN), an idea inspired by biologic neural networks in which the passage of information occurs via inputs and outputs from adjacent neurons. Since its introduction, there has been further progression into computational learning models, which include machine learning (ML) and deep learning (DL) . ML is based upon "reverse training", in which its education occurs through exposure to specific, labeled data . DL is a specialized subset of machine learning built from multilayered artificial neural networks (ANN) for use in more complex, higher-level tasks . An ANN is a computational model that includes multiple levels of learning algorithms, or input and output 'neurons', and if one of these layers involves a convolutional filter, then it is classified as a convolutional neural network (CNN). With the incorporation of neural networks, DL can automatically discern information from large sets of unlabeled data by training a CNN with numerous neural layers, between input and output, that contribute to the plasticity of the DL . This allows DL to better emulate human intelligence, reasoning, and learning . These algorithms can even identify specific characteristics of pathology that are beyond human discernibility. However, because of the need to train its neural networks, DL currently has limited applications in fields like interventional radiology where the case data is limited and often highly variable. A constraint of AI's current dependence on neural networks is the reliance on data-rich domains to train algorithms. The field of diagnostic radiology is optimal for such training as it is a unique data-rich specialty that has progressed rapidly in the modern age of technology. AI has already been successfully implemented in several areas of diagnostic radiology which has been shown to improve efficiency and patient outcomes when used in conjunction with trained radiologists. AI has already been successfully used to assess brain perfusion in acute strokes, delineate brain tumors, and protocol radiological studies. Although many of these listed examples are considered diagnostic radiology specific, there can be substantial overlap between diagnostic radiology and interventional radiology. The rapidly growing field of research called "Radiogenomics," a close relative of AI, combines ML and DL image processing with clinical, histological, and pathological data, in an attempt to correlate precise imaging patterns with pathologic and/or histologic subtypes . Though centered around its ability to extract complex data from medical images, the data obtained will help tailor patient-specific IR treatments. The applications for AI in IR continue to increase with advancing modern technology and the evolving healthcare landscape. AI has the ability to further revolutionize healthcare, specifically for IR, through precision diagnosis, customized treatment plans, and real-time procedural support. Furthermore, although not a direct form of AI, similar fields such as augmented reality (AR) and virtual reality (VR) stand to improve physician education and training, improve patient understanding, and enhance procedural guidance as well as reduce risk and procedural complications. The purpose of this review is to highlight the evolving applications of AI in IR, utilizing the previously described techniques, in the pre-procedural, intra-procedural, and post-procedural settings to improve patient selection, treatment planning and execution, procedural training, intraprocedural augmentation, and treatment follow-up. While AI has many uses, the complexity of the pre-procedural, intra-procedural, and post-procedural applications of AI in IR has presented several challenges and ethical dilemmas that have limited its integration when compared to fields like diagnostic radiology. We further explore current limitations to the progress of AI in IR, and ethical considerations that arise during the adoption of these nascent technologies. As AI has the potential to become more integral to the everyday workflow of both diagnostic and interventional radiologists, it is crucial to understand its various applications and limitations. 2. AI Applications Applications of AI in IR can be divided into pre-procedural, intra-procedural, and post-procedural categories, as summarized in Table 1. Pre-procedural applications include, but are not limited to, patient selection as well as the utility of radiogenomics, AR, and VR. Intra-procedural applications include, but are not limited to, procedural guidance and radiation exposure. Post-procedural applications are tailored to the evaluation of procedural outcomes and follow-up. 2.1. Pre-Procedural Applications 2.1.1. Patient Selection Patient selection is crucial for a treatment's effectiveness, and therefore, the ability to determine which therapies will be most effective for which patients is essential. A multidisciplinary approach to treatment is a key facet of IR, as numerous treatments are decided after multidisciplinary conferences and tumor board discussions, as well as in-depth risk-benefit reviews. AI models have the potential to aid in optimal patient selection by impartially assessing risk and predicting the potential outcomes of therapy . A reliable method for predicting the benefit of treatment prior to its completion would be a significant advancement in the field. For example, Morshid et al. (2019) created an algorithm to predict the response of hepatocellular carcinoma (HCC) following transcatheter arterial chemoembolization which outperformed traditional systems . Similarly, Daye et al. (2019) demonstrated the use of ML in the evaluation of pre-ablation CT texture patterns to predict post-treatment local progression following tumor ablation for adrenal metastases with an accuracy of approximately 95% . By predicting which patients will have better responses to different treatments, interventionalists will be able to protect patients from the adverse effects of ultimately ineffective treatments and efficiently delegate limited treatment resources to patients with a greater likelihood of response. Furthermore, the creation of algorithms to produce a summarized report of pertinent patient-specific information would not only be more efficient in daily practice but would also likely reduce human errors . Incorporation would assist providers in making the most thorough and accurate therapeutic decisions for their patients . Similar algorithms have been proposed for safety screening, a useful tool for example in pre-procedural analysis prior to MRI-guided procedures or in patients with contrast allergies . 2.1.2. Radiogenomics The emerging field of radiogenomics combines medical imaging and molecular pathology, as shown in Figure 2, to improve diagnosis, prognosis, and treatment outcomes . There is a new realization that medical imaging contains a significant amount of "untapped" clinically relevant data that was not previously understood . The ability to foresee an outcome or benefit of treatment prior to performing it is a major challenge in interventional radiology. However, the adoption of DL has the potential to mitigate this challenge . If accurate diagnoses are possible without the need for tissue sampling, such is the case for HCC diagnosis on MRI, it would decrease unnecessary procedures, leading to decreased patient risk and a decrease in hospital cost . Furthermore, the development of radiogenomics could be crucial to IR and its role in the treatment of oncology patients, such as those with HCC, renal cell carcinoma (RCC), colorectal cancer (CRC) with metastases to the liver, and lung cancer patients . For example, radiogenomic studies have demonstrated potential in the correlation of HCC gene patterns with aggressive imaging features on CT, such as infiltration or microvascular invasion. As these are indicative of a poor prognosis, it would be crucial to detect, or, at a minimum, suggest these findings on initial imaging in order for the proper treatment option to be chosen as quickly as possible . Additionally, radiogenomic studies have been performed on RCC indicating associations between CT imaging features with tumor mutations and therefore clinical outcome . As there is evidence demonstrating loss of certain mutations with increased aggressiveness of RCC tumors and worse survival rates, radiogenomic-based triage tools would be helpful for determining whether RCC patients would benefit most from surgery or IR intervention with thermal ablation . Overall, these applications have the potential to more accurately prognosticate and predict a patient's response to a particular treatment, creating a more tailored and specific treatment approach. These augment the role of interventional radiologists as clinicians who partake in the treatment plan of patients, rather than strictly proceduralists. It is important as clinicians to establish accurate prognoses, as early as possible, and determine which patients would benefit most from specific treatments while decreasing patient risk, radiation exposure, and hospital cost as much as possible. 2.1.3. Augmented Reality and Virtual Reality Advances in AR allow operating physicians the ability to visualize procedures and determine their desired approach in the pre-procedural setting via 3D image rendering and manipulation . Incorporation into clinical practice would enable visualization of difficult anatomy and/or improved procedural technique without added risk to patients . For example, the degree of atherosclerotic plaque and its potential effect on wires and catheters can be determined preoperatively rather than intraoperatively . This may not only improve the efficiency and performance of a procedure, but potentially decreases radiation to the patient and operator. Furthermore, development of VR simulations could allow patients a pre-procedural virtual experience of the procedure. Although this would only be speculative, these simulations may improve a patient's understanding of their procedure, thereby improving informed consent. 2.2. Intra-Procedural Applications 2.2.1. Procedural Guidance and Support AI has the potential to assist in and improve procedures in a variety of methods integral to IR, such as image fusion, catheter positioning and probe trajectory, vessel analysis or information regarding the availability of angiography suite supplies . The most developed intraprocedural applications of DL techniques to date have been in the synthesis of pre-procedural 3D anatomic data fused onto 2D real-time fluoroscopic images for improved guidance during procedures. The ability to fuse pre-procedural images onto 2D fluoroscopic images allows for real-time feedback and thus advanced precision during biopsies and ablations. For this process, matching artificial intelligence software is incorporated into virtual and augmented reality to perform automatic landmark recognition through fiducial markers and motion compensation . More recently, this technique has been applied to vascular procedures, such as the angiographic localization of a bleed . Additionally, DL methods being studied for use in tumor ablation therapy include optimization of probe trajectory and selection of energy settings to maximize tumor treatment while simultaneously minimizing injury to adjacent tissue . Another area of potential improvement for IR procedures includes the generation of digital subtraction angiography (DSA), a method of subtracting a mask image from the real-time angiogram. This technique requires patient cooperation as patient motion causes misregistration artifacts. DL algorithms utilizing generative adversarial networks for the creation of DSA images from a single live image without mask data acquisition, such as those suggested by Gao et al. (2019) would circumvent these issues of artifact . Due to the numerous vascular interventions performed by IR providers, vessel analysis is also an optimal area for AI development. A presentation by Molony et al. at the Transcatheter Cardiovascular Therapeutics 2018 annual meeting demonstrated the ability of ML and IVUS to perform vessel analysis in cardiology procedures . The use of IVUS in interventional radiology procedures is not a novel idea, and therefore these ML methods would be easily transferable to vascular analysis and post-treatment evaluation for IR procedures . Vessel analysis with AI has similarly been studied by Cho et al. (2019) through the development of an AI algorithm capable of estimating real-time fractional flow reserve in coronary angiography, a process also easily transferable to IR procedures for peripheral arterial disease . Lastly, AI may demonstrate procedural support by providing information regarding supply stock availability . Currently, this information is amassed either beforehand or by other team members, which is not only time consuming but also introduces unnecessary errors. However, the introduction of touchless devices such as eye-tracking systems or voice-driven smart assistants in the IR suite could alleviate some of these issues . Furthermore, voice recognition and gesture-capture camera systems have been studied for various actions such as turning on and off operating room machinery or operating technology while in the IR suite . This would reduce the time and personnel needed to perform these tasks. Augmented reality embedded in lead glasses has also been evaluated to display important information to the operator while scrubbed into cases . These have also been evaluated as smart assistants to help make suggestions intraoperatively on things like sheath size and deployment of different stents which may be time saving but also advantageous to the novice provider . Additionally, the use of smart assistants could be beneficial for cost analysis, as a greater knowledge of device costs could lead to more cost-effective decision making intra-operatively . 2.2.2. Radiation Exposure Intraprocedural radiation exposure has the potential for substantial reduction with the utility of AI. For example, it has already been evaluated in endoscopy with AI-equipped fluoroscopy that reduces radiation exposure by 38% via ultrafast collimation . The incorporation of AR, such as multi-modality image fusion via superimposition of pre-procedural 3D anatomic data onto 2D fluoroscopic images for improved guidance, as well as the use of adversarial networks for the creation of DSA images without the acquisition of mask images, would each individually and cumulatively decrease the necessary images obtained during a procedure and therefore the amount of radiation to the patient . Furthermore, AI algorithms by Zimmermann et al. (2020) utilizing mobile eye-tracking glasses determined the amount of avoidable radiation per procedure was approximately 11 min. This is the amount of time the x-ray was on while the operator was not looking at the fluoroscopy screen . Similarly, Bang et al. (2020) demonstrated significantly lower radiation to both the patient and operating personnel with the use of AI enabled fluoroscopy systems vs. traditional systems . These applications are crucial to both patients and operators. Many of the patients in IR undergo frequent procedures for maintenance, such as routine nephrostomy or biliary drain exchanges and routine fistulography and intervention for patients with dialysis access. Therefore, even a small decrease in radiation for each procedure will generate an even larger cumulative decrease over time (7, 8). Likewise, interventional radiologists and technologists perform numerous procedures on a daily basis, and therefore small decreases in radiation for each procedure produces a much larger cumulative decrease in their total lifetime radiation exposure (7, 8). 2.3. Post-Procedural Applications Treatment Evaluation and Follow-Up Rapid, accurate, and objective assessment of the outcomes of IR procedures is critical. Having a clear understanding of these post-procedural outcomes will improve treatment predictions and future clinical decisions . These outcomes can further be compiled into longitudinal studies that depend on systematic, objective, and reliable assessments throughout the research program. Finally, to make the results of the longitudinal studies generalizable, standardized objective outcome measures are necessary for multi-site clinical treatment research programs. While diagnostic radiology studies have demonstrated the utility of AI to improve the accuracy, objectivity, and timing of imaging analyses, there have been limited published applications on IR post-procedural outcomes research . An example of where AI applications have been used successfully in IR involves the use of a decision tree, more specifically a Random Forest, in which relationships can be made from complex data sets . This has been used successfully in IR to predict pneumothorax following CT-guided lung biopsy, in-hospital mortality following transjugular intrahepatic portosystemic shunt, and length of hospital stay following uterine artery embolization . These applications were possible due to the availability of large volumes of patient-specific demographics and clinical data in the electronic health records . Based on these applications, it would therefore be feasible for similar methods to predict other relevant and actionable clinical outcomes, such as the development of acute kidney injury (AKI) following intraprocedural contrast usage . Within the realm of interventional oncology, in order to develop a more valid and reliable assessment of Response Evaluation Criteria in Solid Tumors (RECIST) following chemotherapy, which depends on a reader's measurement of tumor volume, Kidd et al. (2022) validated a fully automated Convolutional Neural Network (CNN) to calculate tumor size and treatment response . This DL model could be applied more reliably and objectively to assess the outcomes of interventional radiology procedures, such as liver metastases, than traditional expert based RECIST. Comparably, Dohan et al. (2020) demonstrated the ability of AI to predict overall survival and identification of "good responders" more accurately than RECIST in the evaluation of colorectal liver metastases . Similarly, in order to develop a more objective, standardized, and rapid assessment of mechanical thrombectomy outcomes in the treatment of acute ischemic stroke, Nielsen et al. (2021) designed a DL method to determine scores of thrombolysis in cerebral infarction (TICI) . This artificial intelligence algorithm facilitates a more rapid, accurate, and reliable outcome, which can be used to develop more meaningful and effective management plans and prognoses as well as incorporate the findings into a larger longitudinal and multi-site research program. Likewise, Saillard et al. (2020) developed DL algorithms based on digitized histological slides to build models for predicting the survival of patients after hepatocellular carcinoma resection, a paradigm that can also be used following interventional radiology procedures, such as resections and ablations, to investigate the benefits of adjuvant systematic therapies . These clinical studies illustrate the potential benefits of using AI to measure outcomes following IR procedures. Interventional oncology stands to benefit significantly as the growth of AI in post-procedural follow-up continues to allow for more specific and tailored treatment of oncology patients. Further research is clearly needed to apply the growing body of DL methods being developed for imaging analyses to assessments in IR post-procedural evaluation. 3. Training and Education Advances in ML combined with VR simulation programs create new methods of teaching and preparation, allowing trainees the ability to practice procedural skills in a simulated environment . Currently, there are already orthopedic surgical simulations being used in training, created from patient-specific anatomic modelling data from cross-sectional imaging and manual image segmentation . Related simulations have been developed for IR education and training. A unique aspect of IR training encompasses the development of spatial and cognitive awareness, tactile sensation and motor techniques that are required to operate IR equipment efficiently and successfully . As the conventional training approach of "see one, do one, teach one" is replaced with "see many before doing many," trainees have less hands-on experience than ever before . The estimated 10,000 h of practice required to attain an experienced level of expertise becomes more difficult to accomplish in today's training programs . Inadequate proficiency leads to higher complication rates or operator errors, longer procedural times, and increased radiation to patients and operators . Conversely, the implementation of VR simulation systems in education programs could counteract this predicament to provide trainees with sufficient hours of experience. Further, as case mix varies across institutions, IR physicians may possess very different skill sets based on their training environments . Simulation databases could help expose trainees to a wider case variety. The incorporation of VR simulation systems coupled with standard teaching methods would ensure optimal training in a safe and effective environment, with the added benefit of reduced procedure times and operator errors . ML and VR can improve IR education and procedural proficiency in both a national and international context. Through VR, more interventionalists can be trained in areas that have limited training programs, educators, and resources. The same data sets used to train the AI program could be utilized as education cases with standardized reports as the answer key. Assisting in the training of interventional radiologists worldwide would also serve to bolster the number of diverse cases and data sets. More diverse international cases would also ensure that the AI program does not become inherently biased to the anatomy and pathology of a single group of patients. 4. Limitations In IR, there are different logistical and ethical obstacles that impede the implementation of AI into practice, as shown in Table 2. From a logistical perspective, the obstacles to AI implementation include small datasets relative to diagnostic radiology, standardization of AI learning, variations in patient anatomy and pathology, and difficulty incorporating and coordinating new technology into established healthcare systems . A substantial number of standardized cases is required to build the foundation for an AI neural network. As IR is a relatively newer field of medicine, there are fewer established cases available to train the network. Establishing a sufficient repository of cases will require cooperation and data sharing between different healthcare systems, both nationally and potentially internationally . That cooperation in itself is difficult to achieve given corporate competition and proprietary interests and could even act as a potential risk to violating patient privacy . If cases are being contributed from different institutions, this will inevitably create inconsistencies in protocoling, procedure approach, reporting language, and subjective assessments of severity. Preventing inconsistencies requires standardization of practice across institutions and the establishment of a common lexicon . Even if this was feasible, it would also require the creation of a central quality control agency to oversee this multifaceted project and ensure that this standardization was being upheld . Currently, the regulation of AI in healthcare is subjective and poorly delineated across health systems and national governances . Diagnostic radiology is a data-rich specialty whose progression in the modern age of technology has enabled it to combat AI's neural networks' reliance on data-rich domains to train their algorithms. However, in comparison to diagnostic radiology, interventional radiology is a relatively newer field with fewer total cases and a smaller network of physicians collecting new data. To overcome the limitation of attaining large quantities of high-quality data sets, interventional radiology could utilize techniques developed by neuroradiology researchers working to improve AI brain tumor delineation. These researchers have created data augmentation techniques that improve the generalization capabilities of deep neural networks by generating synthetic training examples. Data augmentation categories include elastic transformations, affine image transformations, pixel-level transformations, and various approaches for generating artificial data. A disadvantage of affine transformations in brain tumor AI training is that it can produce correlated images and generate anatomically incorrect examples . Recent innovative research has also investigated building algorithms that generate artificial images, for example based on tumor growth models, that can be followed as a separate modality by other techniques to ensure the correctness of such phantom/artificial images given that they were found to still produce valid tumor characteristics . If techniques similar to the Batch Adjusted Network Gradients (BANG) were modified for IR, they could allow for more representative and extensive training data as well as augmenting cases in real-time to improve the robustness of the deep learning program in previously imperfect examples . Once the AI system is established, it may be difficult to ensure that it is performing optimally which could silently and detrimentally affect patient care. A complex multifaceted AI system that has unclear mechanical rationale and limits can be prone to debugging errors and requires frequent iterative feedback to ensure it is learning correctly . Therefore, for AI to be applicable in radiology it not only needs to be able to process images correctly, but it also must have a separate functional self-monitoring system that ensures the quality of its results . IR is rapidly evolving from a technological perspective, and it may be difficult to integrate AI systems into the constantly progressing equipment and software used for procedures and data analysis. For AI systems to provide benefits in a clinical/procedural setting, they must function seamlessly with both old and modern imaging scanners and software. A further complication of this integration is that, within a single health system, there are various technologies likely designed by different companies in different countries, each of which could potentially be incompatible with AI processing. In addition to technological barriers to AI integration into clinical practices, hospital staff and their ability to adapt to new technology can also be a barrier. Both academic and private practice hospital staff come from a variety of backgrounds, and some may be limited by their ability to operate new AI technology or their desire to disrupt the current flow of their established clinical practice. Many physicians operate at a high level and may believe that new AI technology will only serve to disrupt their process or make errors that could harm their patients . Private practices especially may be more likely to distrust AI software as it could detrimentally affect their immediate productivity and compensation. Furthermore, many IR private practices do not perform the extremely complex vascular procedures that AI and augmented reality have the highest potential to improve, which makes the technology less desirable. Like with any new technology, implementing AI tools into an established practice is an inherently time and resource consuming process that may risk being poorly received by staff. The intraprocedural implementation of AI into IR has its own set of limitations. During procedures, interventional radiologists use their finely-honed technical expertise along with split second decision making to ensure success. As with all technology, there is a risk of technical difficulties or system failure that causes the program to freeze or severely delays output functions. If an AI program cannot keep up with physicians, then it cannot be relied upon which will drastically reduce its procedural applicability . At the end of the day, it is the interventional radiologist who will need to make decisions based on the input from the AI system and make patient care decisions . Furthermore, there is wide variety in normal patient anatomy. The ability of AI to distinguish the variations in normal vs. pathological could be a major challenge. Differences in size, ethnicity, gender, age, and congenital anomalies can greatly alter the landscape of a patient for interventional procedures and imaging. The ability for AI programs to tolerate this variability is unknown. A significant barrier to the implementation of VR simulation systems is the significant cost of such a large technological investment. Although a reasonable concern, future studies may suggest overall cost-saving . The cost of simulation devices, although substantial, could more substantially reduce the cost of procedural complications and prolonged hospital stays from inexperienced operator complications. Although slightly different, simulation-based central venous catheter (CVC) courses significantly reduced CVC-related infections and hospital costs . Moreover, considering the financial burden and potential patient benefit, it may be beneficial for training programs to share the financial burden with other departments, such as cardiology and vascular surgery . Overall, at this time, there are significant advancements being made in AI applications in the medical setting, particularly in diagnostic radiology. While there are many limitations and fewer widely implemented AI applications currently in use in the field of interventional radiology, there are many prospective applications that will develop as technology progresses and a greater understanding of AI is achieved. 5. Ethical Considerations Beyond the technological, economical, and biological obstacles to AI implementation in interventional radiology, there are also ethical dilemmas to consider. For example, when considering the variability of patient anatomy across the globe, no AI program data set can be adequately trained for all variations of both normal anatomy and overlapping disease presentations. Inadequate training and poor differentiation can lead to misdiagnosis and procedural complications when using AI . The prevalence of different diseases as well as pretest and posttest probabilities also vary between patient populations. With different populations and pathologies running the risk of being underrepresented in the reference data sets, this creates a risk of a breach of justice in medical ethics. For AI to be used in a responsible and ethical manner, there needs to be a coordinated effort to prioritize human rights and freedoms, including privacy, dignity, and safety . Radiologists and AI system programmers will need to altruistically advocate for patient care and ignore monetary influences. Other ethical considerations include patient privacy, patient safety, and the responsibility of physicians utilizing AI assistance . To efficiently amass enough standardized cases to train the AI program, it will be crucial for healthcare systems to share patient information since no single center will see sufficient case volume and variety. Patients will have to provide informed consent to share their private medical records with medical and corporate entities, and these groups will have to ensure that this information is protected and not misused . When seeking to improve the efficiency of radiology services, it is important to prioritize patient safety over procedural and diagnostic interpretive speed. Physicians serve as advocates for their patients and thus must enact high standards for AI programs to protect patients from adverse consequences. Interventional radiologists must oversee and intervene if AI assisted procedures or imaging study reads are causing errors such as missing ischemic strokes or incorrectly mapping vessels. As part of patient safety, physicians are directly responsible for the proper management of their patients . However, this relationship could be muddled by the introduction of AI. If AI programs are allowed to automate patient scheduling, image analysis, and post-procedural follow-up and prognosis, then the accountability of the associated radiologist becomes unclear . 6. Conclusions Numerous, impactful areas of IR stand to benefit greatly from the incorporation of AI. Integration of these techniques would not only benefit procedural planning and performance as well as treatment follow-up, it is also poised to improve patient experience, decrease radiation exposure to both the patient and operators, and potentially decrease hospital costs and adverse events. The benefits of AI in IR are far-reaching and can help on an individual patient level by improving scheduling and the efficacy of minimally invasive procedures, but also on an international level by optimizing global radiology education. Multiple studies have already demonstrated the positive impact of AI integration in the IR setting, and the capabilities are only getting broader with advancing medical imaging technology and more comprehensive prognostic models. There are definite limitations that must be overcome and ethical considerations which must be taken into consideration before the wide breadth of AI applications is demonstrated in daily practice. However, continued enthusiasm as well as research and data collection are key to unlocking the potential AI applications in IR. Author Contributions Conceptualization, E.v.E. and M.S.M.; Literature search and data curation, E.v.E., S.R. and M.A.C.; Manuscript writing--original draft preparation, E.v.E., S.R. and M.A.C.; Manuscript writing--review and editing, E.v.E. and M.S.M.; Supervision, M.S.M. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement Not applicable. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Schematic outlining the relationship between AI, ML, and DL, as well as aspects that make up each of them individually. Figure 2 Schematic diagram outlining the aspects of radiogenomics. diagnostics-13-00892-t001_Table 1 Table 1 Summary of pre-procedural, intra-procedural and post-procedural AI applications with associated examples and references. AI Applications Example Reference Pre-Procedural Applications Safety-screening Algorithms useful in prescreening of patient charts Gurgitano et al. Patient Selection Patient selection using DL-based predictive models to categorize patients as responders and non-responders. Pre-procedural virtual experience of their upcoming procedure Augmented Reality Visualization of difficult anatomy Virtual Reality New method for teaching and training Radiogenomics Combining ML and DL image algorithms with molecular pathology to improve preprocedural diagnosis, prognosis and outcome. Moussa et al. Patient Selection Algorithms designed to predict the response of HCC to TACE prior to the procedure Morshid et al. Intra-procedural Applications Image Fusion Fusion of 3D anatomic data onto 2D fluoroscopic images for advanced precision during biopsies/ablations and for angiographic localization of bleeding Gurgitano et al. Smart-Assistant Devices Augmented reality embedded lead glasses capable of displaying useful/relevant information to the operator while he/she is scrubbed into a case Iezzi et al. Voice-recognition and gesture-capture camera systems for operating IR suite machinery. Cost Effectiveness Smart assistance capable of analyzing device cost prior to use Ablation Probe Trajectory DL algorithms for optimization of probe trajectory in tumor ablations to maximize tumor treatment while minimizing injury to adjacent structures D'Amore et al. Radiation Exposure Decreased radiation using AI enabled fluoroscopy systems Bang et al. Radiation Exposure Mobile eye-tracking glasses for estimation of avoidable radiation per procedure Zimmermann et al. Post-procedural Applications Treatment Follow-Up Fully automated CNN to calculate tumor size and treatment response Kidd et al. Treatment Follow-Up Algorithms designed to predict overall survival as well as categorization of "good responders" and "bad responders" following treatment Dohan et al. diagnostics-13-00892-t002_Table 2 Table 2 Limitations and ethical considerations associated with the implementation of AI into IR. Limitations/Challenges Ensuring optimal AI learning Small datasets for AI training Standardization of IR practice Procedural applicability and incorporation of new technology into an established healthcare system Variations in patient anatomy and pathology Currently there are fewer suitable uses for AI in IR compared to diagnostic radiology Ethical Considerations Conflicts of interest between AI developers and radiologists Effort to prioritize human rights and freedoms such as privacy, dignity and safety Disruption of the direct responsibility between physicians and their patients Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. References 1. Gurgitano M. Angileri S.A. 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PMC10000833 | Healthcare (Basel) Healthcare (Basel) healthcare Healthcare 2227-9032 MDPI 10.3390/healthcare11050773 healthcare-11-00773 Article A Single-Center, Randomized Controlled Trial to Test the Efficacy of Nurse-Led Motivational Interviewing for Enhancing Self-Care in Adults with Heart Failure Dellafiore Federica Conceptualization Investigation Resources 1 Ghizzardi Greta Conceptualization Methodology Investigation Writing - original draft 2 Vellone Ercole Conceptualization Writing - review & editing Supervision 23 Magon Arianna Methodology Software Validation Investigation 4 Conte Gianluca Validation Investigation Data curation Visualization 4 Baroni Irene Conceptualization Methodology Writing - original draft 4 De Angeli Giada Investigation Resources Data curation 4 Vangone Ida Investigation Resources Data curation 1 Russo Sara Investigation Resources Data curation 5 Stievano Alessandro Methodology Resources Supervision Project administration 67 Arrigoni Cristina Conceptualization Methodology Visualization Supervision 1 Caruso Rosario Conceptualization Methodology Software Formal analysis Writing - review & editing Funding acquisition 48* Yee Chow Susan Ka Academic Editor 1 Department of Public Health, Experimental and Forensic Medicine, Section of Hygiene, University of Pavia, 27100 Pavia, Italy 2 Department of Biomedicine and Prevention, University of Rome Tor Vergata, 00133 Rome, Italy 3 Department of Nursing and Obstetrics, Wroclaw Medical University, 50-368 Wroclaw, Poland 4 Health Professions Research and Development Unit, IRCCS Policlinico San Donato, 20097 San Donato Milanese, Italy 5 Nursing Degree Course, Section Istituti Clinici di Pavia e Vigevano S.p.a., University of Pavia, 27100 Pavia, Italy 6 Department of Clinical and Experimental Medicine, University of Messina, 98122 Messina, Italy 7 Centre of Excellence for Nursing Scholarship, OPI, 00136 Rome, Italy 8 Department of Biomedical Sciences for Health, University of Milan, 20133 Milan, Italy * Correspondence: [email protected] or [email protected]; Tel.: +39-0252774940 06 3 2023 3 2023 11 5 77301 2 2023 21 2 2023 05 3 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). Background: The role of nurse-led motivational interviewing (MI) in improving self-care among patients with heart failure (HF) is promising, even if it still requires further empirical evidence to determine its efficacy. For this reason, this study tested its efficacy in enhancing self-care maintenance (primary endpoint), self-care management, and self-care confidence after three months from enrollment in adults with HF compared to usual care, and assessed changes in self-care over follow-up times (3, 6, 9, and 12 months). Methods: A single-center, randomized, controlled, parallel-group, superiority study with two experimental arms and a control group was performed. Allocation was in a 1:1:1 ratio between intervention groups and control. Results: MI was effective in improving self-care maintenance after three months when it was performed only for patients (arm 1) and for the patients-caregivers dyad (arm 2) (respectively, Cohen's d = 0.92, p-value < 0.001; Cohen's d = 0.68, p-value < 0.001). These effects were stable over the one-year follow-up. No effects were observed concerning self-care management, while MI moderately influenced self-care confidence. Conclusions: This study supported the adoption of nurse-led MI in the clinical management of adults with HF. education heart failure motivational interviewing randomized clinical trial self-care Italian Ministry of HealthThis research was partially supported by 'Ricerca Corrente' funding from the Italian Ministry of Health to IRCCS Policlinico San Donato. pmc1. Introduction Heart failure (HF) is a major public health concern worldwide, affecting approximately 1-2% of the global adult population . HF is a clinical syndrome caused by several potential underlying etiologies and characterized by key symptoms such as dyspnea, ankle swelling, exhaustion, and clinical signs (e.g., peripheral edema) . HF is associated with poorer quality of life, increased hospitalization rates, more health-related costs, and decreased overall survival in patients . It also places health-related challenges on the well-being of informal caregivers, because it is associated with a reduced quality of life and health-related issues . Patients with HF need to adhere to the recommended medication regimen and pay special attention to dietary sodium and liquids restrictions, exercise regimen, body condition monitoring, behaviors and mood control, accurate symptom detection, therapy impact evaluation, and other self-care behaviors . These demands are often mismatched from the required self-care practices, as the self-care behaviors of adults with HF were extensively described as mainly inadequate . Self-care is the decision-making process that includes behaviors that help maintain heart failure stability (self-care maintenance), allow patients to perceive symptoms (self-care monitoring), and manage signs and symptoms (self-care management) . Self-care maintenance includes exercising (e.g., brisk walking), avoiding getting sick, medical adherence, and dietary and liquids adherence. Self-care monitoring is based on promptly recognizing the cardinal HF symptoms and signs (e.g., gaining weight, dyspnea, peripheral edema). Self-care management reflects patients' knowledge and health literacy in decision-making when symptoms and/or signs occur. Overall, self-care behaviors are positively influenced by the patient's perception of adequately performing demanding self-care behaviors (self-care confidence) . Among the strategies to sustain adequate self-care in patients with HF, motivational interviewing (MI) showed promising results . By exploring and resolving ambivalence, MI, a goal-directed and patient-centered counseling technique, assists individuals in improving their health-related behaviors . The essential components of MI include showing empathy, creating discrepancies in the perceptions derived from interpreting the gap between expected behaviors and unhealthy performed ones, refraining from disagreements, promoting self-efficacy, and sustaining a shared strategy . Individual psychosocial behavioral interventions utilizing MI showed improved medication adherence and high levels of participant satisfaction in several chronic conditions . Recent studies show that nurse-led MI is safe and effective in pursuing behavioral changes in patients with chronic conditions because nurses are healthcare professionals who work closely with the patient's needs, beliefs, and behaviors, and are able to detect misconceptions regarding clinical aspects . A recent meta-analysis of nine experimental studies shows that MI has moderate effects on enhancing self-care confidence and self-care management and large effects on improving self-care maintenance . Despite this important synthesis of evidence, the authors stated that more empirical and experimental research is still required to corroborate the efficacy of MI on self-care in patients with HF because of the current heterogeneity in the several included populations and the poor adoption of clinical trials measuring self-care with theory-grounded self-report scales . In other words, more randomized controlled trials are required to close the gap of evidence that currently undermines the generalizability and transferability of the efficacy of MI in managing HF . For this reason, this randomized clinical trial (RCT) aimed (a) to test the efficacy of nurse-led MI in enhancing self-care maintenance (primary endpoint), self-care management, and self-care confidence after three months from enrollment in adults with HF compared to usual care, and (b) assess changes in self-care over follow-up times (3, 6, 9, and 12 months). 2. Materials and Methods 2.1. Design This was a single-center, randomized, controlled, parallel-group, superiority study with two experimental arms and a control group. Allocation was based on a 1:1:1 ratio between intervention groups and control. The ClinicalTrial.gov identifier is NCT05595655. This study was approved by the Ethical Committee of San Raffaele Hospital (approval #74/INT). 2.2. Study Setting This study enrolled ambulatory patients in the Heart Failure Clinic of the IRCCS Policlinico San Donato in northern Italy. The focus of care ranges from prenatal diagnosis to rehabilitation, from newborns to the very elderly; the medical-nursing staff is specialized in several areas of cardiology, heart surgery, vascular surgery, and anesthesia with a high focus on clinical research . IRCCS Policlinico San Donato is a reference center for cardiovascular diseases . 2.3. Participants Participants were patients with HF who did not practice adequate self-care and their caregivers. Patients met the requirements for participation if they met the following criteria: (a) had a diagnosis of HF classified as New York Heart Association (NYHA) class II-IV; (b) had evidence of inadequate self-care determined by a score of 0, 1, or 2 on at least two items of the self-care maintenance or self-care management scales of the Self-Care of HF Index v.6.2 (SCHFI v.6.2) ; (c) were willing to sign the informed consent to be enrolled; and (d) with age >= 18 years. Patients who had a myocardial infarction during the previous three months and/or had severe cognitive impairment with a six-item Screener score between 0 and 4 and/or residing in a nursing home where self-care was not required or had an informal caregiver who did not wish to be involved in the study were all excluded from the study. Informal caregivers were eligible to be enrolled if the patients confirmed them as the principal caregivers. Both were not eligible to be enrolled if either the patient or the caregiver refused to participate in the trial in the baseline period; however, if one participant left the study after enrollment, the other one was allowed to continue. Eligible dyads were enrolled after having received a clinician invitation letter stating the aim of the study and the procedure. 2.4. Experimental Arms A trained nurse with experience in educating patients with HF delivered MI. Four registered nurses were trained to participate in a 32 h training course on MI and 8 h refresh training on evidence-based care regarding HF. The registered nurses were females; two of them had a Master of Science in nursing, one was a doctoral student (PhD student) in nursing science, and one had a bachelor's degree. The nurses' average age was 28.75 years (standard deviation, SD = 5.12; range: 24-36). They had 5.75 years of work experience in cardiology (SD = 4.35; ranges, 2-12). The intervention included face-to-face nurse-led MI interventions that lasted around 30 min. The first MI had to be performed within 2 months from enrolment and followed by four other MI interventions at 3, 6, 9, and 12 months performed by the same interventionist. To strengthen the intervention and to sustain adherence to the protocol, the nurse who performed the MI contacted the patients via telephone three times during the first two months after MI. This scheduled approach for delivering MI 5 times during the study has never been tested in previous studies . In arm 1, the MI was delivered only to patients; in arm 2, MI was delivered simultaneously to the dyad patient and caregiver. Participants enrolled in the experimental arms (arms 1 and 2) received MI interventions as an add-on approach to the standard of care. 2.5. Standard of Care and Control Group Standard of care included clinical visits in the outpatient settings every 6 to 12 months, depending on the severity of the patients' HF conditions and their specific clinical pathways. Education in the standard of care was based on discussions with patients about relevant materials geared toward HF self-care. Patients in the control group received standard of care only. 2.6. Procedures A research assistant (outcome assessor) screened the patients using the SCHFI v.6.2 and the six-item Screener following the study protocol after patients and caregivers gave their consent. After the eligibility screening, when a patient was eligible, the protocol-required questionnaires were administrated to both patients and caregivers. They received questionnaires individually at baseline and at each follow-up, and they were not permitted to work together to complete the questionnaires. At 3, 6, 9, and 12 months after enrollment, follow-up data were collected via telephone. The outcome assessor was kept blind regarding the research arms at both the baseline and all follow-up points. Interventionists and participants were not blind to the study arm. 2.7. Randomization A web-based system generated the randomization sequence, assigning participants in a 1:1:1 ratio to either the intervention or control group using a simple randomized process. Allocation sequences were accomplished using computer-generated algorithms that were made available after the trial. The interventionists were not informed of the allocation sequence. The randomization process started after the site employees (study nurses) entered the patients' information into the database (RedCap). Each randomization number was generated and sent by a study nurse to the interventionist (a trained nurse who performed the MI), who was not the professional who had to assess the outcomes. Each participant's enrollment and follow-ups were always communicated to the trial coordinator. 2.8. Measurements The measurements for patients were socio-demographic and clinical characteristics. Socio-demographics were sex (male, female); age (years); marital status (single, married, divorced, widower); education (high schools or higher, lower than high schools); employment (active worker, retired); income (more than necessary to live, the necessary to live, and not the necessary to live). Clinical characteristics were NYHA class (II, II, IV functional class), Charlson comorbidity index (CCI, score) , ejection fraction (HFpEF = preserved ejection fraction; HFmrEF = midrange ejection fraction; HFrEF = reduced ejection fraction), time with HF (months), BMI (kg/m2), Montreal cognitive assessment (MoCA) (score) . The outcomes of this study were the self-care maintenance scores measured using SCHFI v.6.2 . Outcomes The SCHFI v.6.2 was used to assess the score of self-care maintenance at baseline, after 3 months (primary endpoint), and over the follow-up times. The SCHFI v.6.2 also allowed researchers to measure secondary outcomes: self-care management and self-care confidence at baseline and over the follow-up times. Each score has a range of 0 to 100. Higher scores indicate better self-care. Only if a patient had previously reported experiencing HF symptoms, such as dyspnea, did they have to fill out the self-care management scale. A score of less than 70 on each domain denoted adequate self-care. 2.9. Sample Size The pooled mean of self-care maintenance described using the SCHFI v.6.2 in two previous descriptive studies performed in northern Italy was 53.55, with a pooled standard deviation of 18.98 . Previous studies showed that MI could improve the mean of self-care scores in patients with HF by increasing the mean scores with a delta (D) ranging from 6 to 15 (pooled mean D = 10.95) . Therefore, 49 patients per arm were required to reject the two-tailed null hypothesis of equal mean scores between the study arms with a power of 80%. A sensitivity analysis considering slights variations in the D and accounting for 20-25% of attrition as per similar research showed that a total of 180 +- 6 participants was necessary to preserve enough power (80%) to detect significant mean differences between the experimental arms and the control group (60 +- 2 participants per arm). 2.10. Treatment Fidelity The trial coordinator evaluated treatment fidelity by randomly applying an evaluation of the performed MI in arms 1 and 2 using the Motivational Interviewing Treatment Integrity (MITI) Scale . The MI interventions were all audio-recorded, and the MITI was used to randomly evaluate 4 MI interventions per arm at each time point. The scores ranged between 2 and 5, and the median of the assessments in both arms was 3, indicating an ideal technical quality score. 2.11. Timeline Enrollment required approximately 36 months (from May 2017 to May 2020; the study ended with the last follow-up in May 2021) to avoid overwhelming the activities of the involved staff in the study (i.e., four interventionists, a trial coordinator, two outcome assessors, a study nurse, a data manager, the principal investigator, and the co-investigators). The study was conducted at a cardiovascular hub center that remained operational during the COVID-19 pandemic waves. As a result, the researchers were able to conclude the study during the pandemic by leveraging the center's ongoing interactions with heart failure patients. Figure 1 shows the patient flow. 2.12. Statistical Analysis All data were analyzed by using an intention-to-treat approach. Categorical variables were described in terms of absolute and relative frequencies. Interval and continuous variables were evaluated for normality using the Shapiro-Wilk test, and data with a normal distribution are presented using the mean and standard deviation (SD). The median and interquartile ranges (IQR) were used to summarize non-normally distributed data. Baseline characteristics were compared between arms to determine if they were equal. Missing scores in the outcomes were 12%, 10%, and 11% (respectively, the extent of the missingness in arms 1, 2, and 3) in each arm, which were imputed by employing multiple imputations based on random effects models after having assessed that the missing mechanisms (missing in relation to time and study arm) and patterns (monotone missingness based on sensitivity analysis) supported the missing at random (MAR) assumptions. The delta (D) of the self-care scores was calculated at each follow-up period by subtracting the baseline self-care score (T0) to determine the changes in self-care scores during follow-up times (T1, T2, T3, and T4). As the primary endpoint was a significant improvement in arms 1 and 2 of self-care maintenance scores over the control group, a two-sample t-test was employed to compare the delta of self-care score in arms 1 and 2 versus the control arm 3, under the assumptions of the central limit theorem . A similar approach was performed for each follow-up time and the secondary outcomes (self-care management and self-care confidence). Precisely, the t-test effect size estimates were computed using d statistics for independent t-tests (Cohen's d), where d values lower than 0.5 indicated small effects, between 0.5 and 0.8 moderate effects, and greater than 0.8 large effects . In addition to this approach, data on the primary and secondary outcomes at each follow-up time were summarized in adequate (scores equal to or greater than 70) or inadequate (scores lower than 70) and compared (arm 1 vs. arm 3; arm 2 vs. arm 3) using chi-square test or Fisher's exact test when appropriate. Mixed models for repeated measures were used to analyze changes across time (from baseline to T4) in primary and secondary outcomes, following the strategy of a previous study . As a dependent variable, these models included the outcome scores available from T0 to T4 for each patient in the study arm. By having included a random intercept in the models, the inter-dependence between self-care maintenance, management, and confidence on the same subject was addressed. The randomization arm (nominal variable) was included in the models as an independent variable, along with the baseline characteristics (i.e., age, sex, income, NYHA, CCI score, MoCA, time since diagnosis, ejection fraction, and self-care confidence). Furthermore, the slopes derived from the models were compared between arms 1 and 2 versus the slopes of arm 3 for each outcome. The significance level was set at 0.05 in all tests, and analytics were performed using Stata Statistical Software: Release 17 (StataCorp. 2021; College Station, TX, USA: StataCorp LLC). 3. Results 3.1. Participants' Characteristics Patients' baseline characteristics, stratified and compared by arm, are shown in Table 1. No differences are found in relation to the baseline characteristics. The majority of patients were females (r in arms 1, 2, and 3: 51.1%, 55.0%, and 52.5%, respectively) as well as the majority of caregivers (in arms 1, 2, and 3: 73.4%, 69.5%, and 74.0%, respectively). In arms 1, 2, and 3, patients reported mean ages of 68.39 (SD = 12.14), 69.44 (SD = 6.71), and 71.08 (SD = 12.95), respectively. In arms 1, 2, and 3, caregivers reported mean ages of 56.28 (SD = 9.12), 59.44 (SD = 11.10), and 58.17 (SD = 9.08), respectively. In arms 1, 2, and 3, most of caregivers were married: 57.1%, 63.2%, and 61.50%, respectively; for patients, 54.1%, 45.0%, and 37.7%, respectively. Most patients and caregivers reported an educational status lower than high schools: for patients in arms 1, 2, and 3, 72.1%, 75.0%, and 73.8%, respectively; for caregivers, 59.1%, 63.7%, and 62.9%, respectively. Specifically, regarding patients, most of them answered that they have the necessary income to live. The median (IQR) time with HF was approximately 4 years in the three arms. The median (IQR) BMI indicated values within normal scores. Overall, most patients were in NYHA II class, with two comorbidities, an HF with preserved ejection fraction (HFprEF) and inadequate self-care maintenance and management scores. 3.2. Self-Care Maintenance (Primary Endpoint), Management, and Confidence at the First Follow-Up (T1, 3 Months) The increase in the self-care maintenance scores (primary endpoint) from baseline to T1 (3 months after enrolment) is higher in arms 1 and 2 compared to arm 3 . In arms 1, 2, and 3, the mean D indicating an increase in the self-care maintenance score is 12.84 (SD = 11.50), 10.81 (SD = 13.05), and 2.78 (SD = 10.33), respectively, indicating a large effect size in the D between arm 1 and arm 3 (Cohen's d = 0.92, p-value < 0.001), and moderate effect size in the D between arm 2 and arm 3 (Cohen's d = 068, p-value < 0.001). Regarding self-care management scores, no differences are found between arm 1 and arm 2 versus arm 3 (see Table 2). Conversely, regarding self-care confidence scores, only the increased scores observed in arm 2 are significantly higher than those in arm 3, with a moderate effect size (Cohen's d = 058, p-value = 0.002). The comparisons of the dichotomized scores into adequate (scores >= 70) and inadequate (scores < 70) do not show significant differences for each outcome (see Table 2). 3.3. Changes in Self-Care Maintenance, Management, and Confidence over Follow-Up Times The description of self-care maintenance, self-care management, and self-care confidence scores over time are reported in Figure 2 and Table 2. In relation to self-care maintenance, we generally find stability since 1 year (T4) of the effects detected at T1 (after 3 months). No differences are found in relation to self-care management scores over time. Conversely, regarding self-care confidence, at T1 and T2, moderate-small improvements are observed in arm 2 compared to arm 3; in arm 1, self-care confidence shows small-moderate improvements at T3 and T4 (see Table 2). The trends over time (from baseline to T4) derived from the mixed models in self-care maintenance, self-care management, and self-care confidence scale scores are shown in Figure 3. Regarding the self-care maintenance slopes, arm 1 and arm 2 versus arm 3 show significant differences (p-values = 0.038; p-values = 0.047, respectively). No differences are found concerning self-care management scores (p-values = 0.398; p-values = 0.447, respectively). Regarding self-care confidence, only the comparison between trends of arm 2 and 3 show significant differences (p-values = 0.031). These trends are confirmed when the mixed models are adjusted for age, sex, income, NYHA, CCI score, MoCA, time since diagnosis, ejection fraction, and baseline self-care confidence. 4. Discussion This study demonstrated that nurse-led MI performed using a scheduled approach (every three months over one year) was effective in improving self-care maintenance with stable effects over the follow-up times. The scheduled approach used to deliver MI in this study is a significant innovation, since no previous randomized controlled trials have utilized a similar approach . This approach allows for a more structured and consistent delivery of motivational interviewing to participants, which may enhance its effectiveness. In this study, nurse-led MI also improved self-care confidence, with some differences when the intervention was performed only for patients (arm 1) or for the dyads of patients and caregivers (arm 2). Overall, the results derived from this RCT corroborate previous evidence , adding additional insights regarding five main aspects: (a) the nurse-led MI performed with scheduled recurrences over time likely produces stable effects in improving self-care maintenance over time; (b) the characteristics of HF (e.g., NYHA class or ejection fraction) seem to play a non-significant role on influencing the efficacy of MI in improving self-care maintenance over time; (c) the effects of MI performed only for patients seemed to be more stable over the effects showed by performing MI to the dyads in a different way from the effects shown in a previous study ; (d) the role of MI in improving self-care management remains unclear; (e) self-care confidence seems positively influenced by MI. The efficacy of nurse-led MI on self-care maintenance has important clinical implications because it means that aspects such as treatment adherence, which are highly problematic among patients with HF, might be susceptible to significant improvements when trained nurses employ MI in clinical practice. It is not surprising to find that the nurse-led MI effectively leads patients toward behavioral change . In this regard, the key features of MI, such as adopting open-ended questions, affirmation of patients' strengths, adopting reflective listening, and summarizing key points of the discussion, have the potential to be effective in patients with several clinical conditions, from individuals with HFprEF to patients with HFrEF. The more stable effects on improving self-care maintenance shown in arm 1 over arm 2 may be explained by the nature of the training performed by the interventionists, which was mainly focused on the elements of MI per se and a brief refresh about evidence-based care for patients with HF rather than focusing on providing the skills to manage the complexity of the dyadic relationships during the MI. In other words, it is reasonable that interventionists found it easier to perform the MI only for patients rather than simultaneously managing the dyad as required in arm 2. In this regard, we have to acknowledge that a previous multicentric RCT found that the effects of MI performed for the patient-caregiver dyad were larger than the MI performed only for patients . From a theoretical perspective, if we consider the contribution of caregivers to the self-care practices of patients with HF , the MI performed for the dyad should be the best option. However, the evidence from this study points out that delivering MI to the dyad should be based on different training from the one designed only to provide skills for delivering MI-based interventions because the complexity of the dyadic relationship should be included in educating the interventionists. Among self-care behaviors, self-care management practices seem to be less susceptible to changes than self-care maintenance. This aspect is theoretically explainable by the role of several aspects that determine self-care management, such as disease-specific knowledge and, broadly speaking, health literacy . In fact, self-care management reflects different individual-level characteristics into actions, from values, beliefs, knowledge, and so on, to behaviors that reflect a decision-making process triggered by the detection of signs and/or symptoms . Considering these aspects, it is reasonable to think that self-care management requires complex and multiple interventions to be modified (e.g., psychosocial interventions combined with knowledge-based education and MI). Therefore, complex and multiple interventions should aim to affect the main determinants of self-care management rather than self-care management per se. Self-care confidence is also susceptible to improvement after MI interventions. Considering that self-care confidence is one of the strongest predictors of self-care behaviors , this result might have interesting clinical implications because improving self-care confidence may trigger virtuous circles to improve several other health-related outcomes. The differences emerging between the two experimental arms of this study (i.e., arm 1 shows effects after six months, while arm 2 shows brief-term effects) require more investigations with future studies and might reflect the complexity of managing MI in a dyadic setting. This study has several limitations. First, the single-center design limits the generalizability of the results. Second, the self-report scale used to assess primary and secondary outcomes (SCHFI v6.2) was the best option when the protocol of this RCT was written; however, it is currently outdated because the new SCHFI v7.2 is psychometrically more robust and allows researchers to assess self-care monitoring. Third, patient attrition over the trial was considerably large (19.3% at T4); this aspect requires further mitigation strategies in future studies and a more robust approach to ensure patient adherence to the protocol. Four, the poor focus of the educational course for educating the interventionists regarding managing the complexity of the dyadic relationships between patients and caregivers might be considered a source of bias, especially in interpreting the effects of arm 2. Finally, it is important to interpret the stability of the effects observed in relation to self-care maintenance with caution, given the repeated MI in the experimental procedure. While the results of this study suggest that the repeated MI approach may produce stable effects over a one-year follow-up period, it is important to consider that individual patients may respond differently to repeated interventions. Therefore, the generalizability of the findings to all patients with heart failure should be approached with caution. 5. Conclusions Nurse-led MI shows efficacy in improving self-care maintenance in patients with HF over a one-year follow-up. This RCT confirms previous evidence and supports the adoption of nurse-led MI in the clinical management of HF. Future research should corroborate this evidence in specific subgroups to enhance the external validity of this intervention and should explore the effects of nurse-led MI on clinical outcomes. Acknowledgments The authors express their gratitude to all of the patients and their caregivers who agreed to be enrolled in the study. Author Contributions F.D.: conceptualization, methodology, data curation, writing--original draft, project administration, investigation. G.G.: data curation, writing--original draft, project administration, investigation, visualization. E.V.: supervision, validation, writing--review and editing. A.M.: methodology, data curation, writing--original draft, project administration, investigation. G.C.: methodology, data curation, project administration, investigation. I.B.: methodology, data curation, project administration, investigation. G.D.A.: data curation, project administration, investigation. I.V.: data curation, project administration, investigation. S.R.: data curation, project administration, investigation. A.S.: supervision, validation, writing--review and editing. C.A.: supervision, validation, writing--review and editing. R.C.: conceptualization, methodology, formal analysis, supervision, funding acquisition, writing--review and editing. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethical Committee of San Raffaele Hospital (approval #74/INT). Informed Consent Statement Written informed consent was obtained from all subjects involved in the study. Data Availability Statement Data are available from the corresponding author upon reasonable request. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Patient flow. Figure 2 Self-care scores by arms over follow-up times. Figure 3 Model-based self-care maintenance, self-care management, and self-care confidence scores by follow-up times. healthcare-11-00773-t001_Table 1 Table 1 Patients' characteristics at baseline (n = 182). Arm 1: MI Only for Patients (n = 61) Arm 2: MI for Patients and Caregivers (n = 60) Arm 3: Usual Care for Patients and Caregivers (n = 61) p-Value n % n % n % Variables Sex Males 33 54.1 33 55.0 32 52.5 0.960 Age Years (mean; SD) 68.39 12.14 69.44 6.71 71.08 12.95 0.403 Marital status Single 8 13.1 10 16.7 9 14.8 0.374 Married 33 54.1 27 45.0 23 37.7 Divorced 8 13.1 10 16.7 7 11.5 Widower 12 19.7 13 21.7 22 36.1 Education High schools or higher 17 27.9 15 25.0 16 26.2 0.937 Employment Retired 50 82.0 48 80.0 45 73.8 0.423 Income More than necessary to live 6 9.8 8 13.3 10 16.4 0.264 The necessary to live 46 75.4 44 73.4 49 80.3 Not necessary to live 9 14.8 8 13.3 2 3.3 Time with HF Months (median; IQR) 48.00 24.00-70.00 47.50 26.75-60.00 46.50 27.00-67.00 0.315 BMI Kg/m2 (median; IQR) 24.36 22.00-27.92 25.00 22.75-26.5 25.71 22.00-28.31 0.585 MoCA Score (median; IQR) 28.00 18.00-30.00 25.00 19.00-29.00 25.00 21.00-28.00 0.456 NYHA Class II 36 59.00 38 63.3 42 68.9 0.595 III 21 34.4 18 30.0 18 29.5 IV 4 6.6 4 6.7 1 1.6 CCI Score (median; IQR) 2 2.0-5.0 2 2.0-4.0 2 1.0-4.0 0.877 EF HFpEF 31 50.8 32 53.3 34 55.7 0.855 HFmrEF 14 23.0 10 16.7 13 21.3 HFrEF 16 26.2 18 30.0 14 23.0 Self-care maintenance Inadequate (score < 70) 54 88.5 53 88.3 57 93.4 0.564 Self-care management Y= Inadequate (score < 70) 47 95.9 51 98.1 34 94.40 0.657 Self-care confidence Inadequate (score < 70) 44 72.1 48 80.00 52 85.2 0.200 Self-care maintenance Score (0-100) (median; IQR) 46.66 29.56-63.33 53.33 35.83-63.33 46.66 36.67-56.66 0.162 Self-care management Y= Score (0-100) (median; IQR) 41.00 29.00-55.25 41.28 25.00-57.50 45.11 28.75-55.00 0.566 Self-care confidence Score (0-100) (median; IQR) 50.04 45.56-71.20 52.41 37.82-67.24 51.00 38.92-66.72 0.687 Legend: MI = motivational interviewing; SD = standard deviation; CCI = Charlson comorbidity index; MoCA = Montreal cognitive assessment; NYHA = New York Heart Association; EF = ejection fraction; HFpEF = preserved ejection fraction (left ventricular ejection fraction >= 50%); HFmrEF = midrange ejection fraction (left ventricular ejection fraction that ranges from 40 to 49%); HFrEF = reduced ejection fraction (left ventricular ejection fraction < 40%); IQR = interquartile range. Y= In arms 1, 2, and 3, patients with symptoms who filled this part of the SCHIFI were, respectively, 49, 52, and 36 because patients without recent experiences of signs and symptoms were not asked to fill the questions regarding self-care management. healthcare-11-00773-t002_Table 2 Table 2 Self-care changes and frequencies of patients adequate in self-care during follow-up, and comparisons between experimental arms (arms 1 and 2) and control group. Arm 1: MI Only for Patients (n = 61) Arm 2: MI for Patients and Caregivers (n = 60) Arm 3: Usual Care for Patients and Caregivers (n = 61) Arm 1 vs. Arm 3 Cohen's d (p-Value) Arm 2 vs. Arm 3 Cohen's d (p-Value) N Mean SD Mean SD Mean SD D in Self-care maintenance scores T1 182 12.84 11.50 10.81 13.05 2.78 10.33 0.92 (<0.001) 0.68 (<0.001) T2 182 14.60 11.92 10.39 12.01 3.34 11.57 0.96 (<0.001) 0.60 (<0.001) T3 182 17.31 14.71 11.23 13.68 2.73 14.59 0.99 (<0.001) 0.60 (0.001) T4 182 16.37 17.95 7.82 13.89 2.02 11.91 0.94 (<0.001) 0.45 (0.015) D in Self-care management scores T1 114 14.26 19.29 11.50 28.61 10.12 23.06 0.20 (0.788) 0.05(0.586) T2 107 11.65 20.41 8.51 20.26 9.4 17.88 0.12 (0.672) 0.05 (0.427) T3 108 11.12 18.64 7.79 20.40 9.2 16.72 0.11 (0.663) 0.07 (0.382) T4 101 11.50 18.16 12.04 19.10 12.57 23.36 0.05 (0.421) 0.03 (0.462) D in Self-care confidence scores T1 182 7.11 14.22 9.04 6.84 3.06 12.84 0.31 (0.101) 0.58 (0.002) T2 182 7.40 16.06 8.36 9.78 1.79 17.54 0.24 (0.183) 0.37 (0.043) T3 182 10.08 17.71 9.69 9.07 3.75 16.55 0.37 (0.044) 0.32 (0.079) T4 182 12.13 13.47 12.56 10.13 3.60 18.55 0.53 (0.004) 0.60 (0.001) Arm 1 vs. Arm 3 (p-Value) Arm 2 vs. Arm 3 (p-Value) Patients adequate in self-care maintenance (scores >= 70) N % N % N % T1 19 31.1 26 43.3 4 6.6 1.000 0.626 T2 21 34.4 19 31.7 4 6.6 0.134 0.297 T3 29 47.5 21 35.0 14 23.0 0.313 0.751 T4 23 37.7 23 38.3 19 31.3 0.507 0.872 Patients adequate in self-care management (scores >= 70) N % N % N % T1 14 30.4 11 21.6 6 14.3 1.000 0.295 T2 11 24.4 10 20.0 3 8.6 0.342 0.545 T3 11 25.6 8 17.0 5 12.8 0.454 0.269 T4 7 15.9 10 22.7 7 21.2 0.342 0.330 Patients adequate in self-care confidence (scores >= 70) N % N % N % T1 19 31.1 17 28.3 10 16.4 0.261 0.003 T2 18 29.5 19 31.7 9 14.8 0.015 0.023 T3 20 32.8 21 35.0 12 19.7 0.046 0.011 T4 27 44.3 23 38.3 14 23.0 <0.001 <0.001 Legend: SD = standard deviation; p-values in bold are <than a (5%). 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PMC10000834 | Healthcare (Basel) Healthcare (Basel) healthcare Healthcare 2227-9032 MDPI 10.3390/healthcare11050643 healthcare-11-00643 Article Functional Improvement and Satisfaction with a Wearable Hip Exoskeleton in Community-Living Adults Kim Jihye Conceptualization Data curation Formal analysis Investigation Resources Software Visualization Writing - original draft 1+ Chun Hyelim Conceptualization Data curation Formal analysis Investigation Resources Software Visualization Writing - original draft 1+ Lee Su-Hyun Conceptualization Formal analysis Validation Writing - review & editing 1 Lee Hwang-Jae Methodology Supervision Validation Writing - review & editing 2* Kim Yun-Hee Funding acquisition Methodology Supervision Validation Writing - review & editing 13* Giansanti Daniele Academic Editor Chen Tin-Chih Toly Academic Editor 1 Center for Prevention and Rehabilitation, Samsung Medical Center, Department of Physical and Rehabilitation Medicine, Heart Vascular Stroke Institute, Sungkyunkwan University School of Medicine, Seoul 06351, Republic of Korea 2 Robot Business Team, Samsung Electronics, Suwon 16499, Republic of Korea 3 Department of Health Sciences and Technology, Department of Medical Device Management and Research, Department of Digital Health, SAIHST, Sungkyunkwan University, Seoul 06351, Republic of Korea * Correspondence: [email protected] (H.-J.L.); [email protected] or [email protected] (Y.-H.K.); Tel.: +82-2-3410-2824 (ext. 2818) (Y.-H.K.) + These authors contributed equally to this work. 22 2 2023 3 2023 11 5 64309 11 2022 18 2 2023 21 2 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). Demand for wearable devices and supportive technology is growing as these devices have the potential to enhance physical function and quality of life in users. The purpose of this study was to investigate usability and satisfaction after performing functional and gait exercise with a wearable hip exoskeleton in community-living adults. A total of 225 adults residing in the local community participated in this study. All participants performed 40 min of exercise once with a wearable hip exoskeleton in various environments. The EX1, which functions as a wearable hip exoskeleton, was used. Physical function was assessed before and after exercise with the EX1. After completing exercise with the EX1, the usability and satisfaction questionnaires were evaluated. Gait speed, timed up and go test (TUG), and four square step test (FSST) showed statistically significant improvements after exercise with the EX1 in both groups (p < 0.05). In the 6 min walking test (6MWT), a significant increase was observed in the middle-aged group (p < 0.05). In the short physical performance battery (SPPB), there was a significant improvement in the old-aged group (p < 0.05). On the other hand, positive results in usability and satisfaction were noticed in both groups. These results demonstrate that a single session of exercise with the EX1 was effective in improving physical performance of both old-aged adults, with positive feedback from most of the participants. exoskeleton device gait robot personal satisfaction physical fitness Samsung ElectronicsPHO021239 This study was supported by Samsung Electronics (PHO021239). pmc1. Introduction Physical activity, which is associated with quality of life, decreases with age due to loss of muscle mass and strength , cardiac and respiratory disease , and other factors. Decreased physical activity during the aging process leads to metabolic disorders and other chronic diseases, such as cancer, diabetes, and cerebrovascular and cardiovascular diseases . Regular physical activity such as walking, cycling, dancing, and running leads to positive physiological changes. Regular physical activity improves autonomic balance, bone density, capillary density, muscle fiber size, neuromuscular coordination, stroke volume, blood coagulation, and inflammation . Moreover, these physiological changes result in decreased depression, weight gain, fractures, injurious falls, osteoporosis, and mortality, as well as increased physical and cognitive function . A previous study reported that regular physical activity could increase the life expectancy of the world's population . Walking is one of the easiest regular physical activities. Walking is the cheapest and easiest activity for health promotion and consumes larger amounts of energy than other daily sports activities. Gait function refers to the mobility required for daily life performance, which can predict clinical conditions in various aspects . In previous studies, inactive adults experienced 3-8% muscle loss over 10 years with decreased metabolic rate and fat accumulation during rest , but walking exercise improved walking economy and functionality , muscle size and quality , functional balance, and reaction time , which are related to gait quality. Recently, functional exercise has been carried out under various conditions, such as applying a load, using props, or combining various conditions. Resistance gait exercise enhances balance and gait parameters and decreases the fear of falling . A previous study showed that, after resistance gait exercise using an underwater treadmill, gait parameters including step length, velocity, and cadence were significantly increased . In addition, an assistive therapeutic exercise program is effective in improving preferred gait speed . Robot technology in the healthcare field is being actively used in clinical practice as it has been developed in the surgical and rehabilitation medical fields. However, in recent years, the technological advances in daily assistive robots have gone beyond their limited use for patients in the medical field . Wearable exoskeletons are being actively developed to assist and strengthen physical functions for not only disabled, but also non-disabled people. A wearable exoskeleton helps to improve gait by assisting with voluntary movement of the lower extremity joints during gait . Furthermore, gait exercise with a wearable hip exoskeleton improves cardiopulmonary function. In a previous study, gait training with an assisted-exoskeleton robot required less oxygen consumption than a home exercise program of self-paced overground walking without a robot at the same speed in the elderly . The EX1, developed by Samsung Electronics (Suwon, Republic of Korea), is a personalized robot with a light weight of 2.1 kg that is worn on the hip joints. In our previous study in the elderly, there were statistically significant improvements in gait speed, excessive muscle activity, respiration, and metabolic energy during gait with the EX1 compared to walking without it . Demand for wearable devices and supportive technology is growing because these devices have potential to enhance physical functions and quality of life of users. Furthermore, among the factors to be considered when using a wearable robot, user stability and fit are important. Previous studies on the EX1 investigated the effect on physical function rather than usability and satisfaction with the wearable robot. Thus, wearable devices need to be evaluated by actual users in terms of wearability . This study was designed to investigate the usability, satisfaction, and physical function from a single session of functional and gait exercise with the EX1 in community-living old-aged adults. 2. Materials and Methods 2.1. Study Participants The participants were recruited from local community residents who use silver town or welfare centers. Two-hundred and twenty-five adults who met the following inclusion criteria were enrolled in this study: no history of central nervous system disease and age between 40 and 84 years. Subjects with uncontrolled severe high blood pressure or diabetes, history of uncontrolled cardiovascular disease, severe dizziness that might lead to a fall, and cognitive disorders that hinder the ability to understand or comply with study instructions were excluded. General characteristics of the subjects are summarized in Table 1. The study protocol was approved by the Institutional Review Board of Samsung Medical Center, Seoul, Korea (No. 2021-04-058), and informed consent was provided by all subjects before participating in the study. 2.2. Experimental Protocol This study protocol was designed as a single group, and all participants performed a single session of exercise with EX1. All participants received 40 min of exercise with the EX1: 20 min of functional exercise including sit-to-stand and balance exercise and 20 min of gait exercise including stair climbing and over-ground and incline walking with the assist and resistance modes of EX1. To confirm the effect of a single session of exercise with EX1, physical functions measured by the 10 m walking test (10MWT), timed up and go test (TUG), four square step test (FSST), 6 min walking test (6MWT), and short physical performance battery (SPPB) were evaluated before and after the exercise program. All outcome measures were performed without the EX1. After completing a single session of exercise with the EX1, the usability and satisfaction questionnaire for EX1 were evaluated . 2.3. Wearable Hip Exoskeleton (EX1) The EX1, which was developed by Samsung Electronics, is a minimized exoskeleton worn on the hip joints. It is very lightweight and user-customizable, weighing approximately 2.1 kg . EX1 can provide assistive or resistive torque forces around both hip joints for both extension and flexion direction during gait as needed, and it promotes physical function in daily life. EX1 consists of a pair of actuators that generate force on the left and right hip joints, a hip brace on the waist, a pair of thigh frames, and a thigh belt . 2.4. Measurement Tools 2.4.1. Physical Function Evaluation To measure physical function, 10MWT, TUG, FSST, 6MWT, and SPPB were performed. The 10MWT is the most effective method for predicting falls and evaluating gait ability by measuring gait speed, and it has high test-retest reliability in healthy adults ( inter-tester reliability (ICC) = 0.93-0.91) . For measurement, subjects were asked to walk a total of 15 m at a comfortable speed. The time it took to walk 10 m was measured, excluding the initial 2.5 m acceleration and the final 2.5 m deceleration, and the result in seconds was converted into speed (m/s). Measurements were repeated twice, and the average value was used. To evaluate the dynamic balance ability among physical functions, TUG and FSST were measured. The TUG test is used as a standard test method in clinical practice as a representative test method for measuring gait ability as well as dynamic balance ability of elderly and brain injury patients, and it has high ICC in the elderly (ICC = 0.92-0.99) . The FSST is a method of evaluating dynamic balance and movement ability. It measures the time it takes to walk forward, backward, and sideways over a low obstacle as fast as subjects can, and it has high test-retest reliability (ICC = 0.87) . To evaluate walking endurance, 6MWT was measured. The 6MWT is a representative method for measuring gait aerobics and endurance in the elderly and patients with cardiopulmonary disease. To measure gait endurance, subjects are asked to walk a set trajectory for 6 min, and the total walking distance is measured. It has excellent test-retest reliability for the elderly (ICC = 0.95) . SPPB is a test that consists of walking speed, sit-to-stand on a chair, and balance tests. It has been used as a predictive tool for dysfunction and can be helpful in functional monitoring in the elderly. The score ranges from a minimum of 0 points to a maximum of 12 points. SPPB has been shown to have validity as a predictor of fall risk and mortality and has excellent test-retest reliability for the elderly (ICC = 0.91) . 2.4.2. The Usability and Satisfaction Questionnaire As there was no appropriate usability and satisfaction questionnaire for evaluating the wearable hip exoskeleton, the questionnaire was developed and applied through previous research and consultation with experts (Table 2). A questionnaire was developed by setting usability and satisfaction evaluation areas. The safety questionnaire is one of the most important parts to determine whether EX1 can be used safely. The operability question is a factor to realize the function of the gait robot. The questionnaire includes operational convenience, effectiveness, and efficiency. 2.5. Data Preprocessing and Statistical Analysis All data were analyzed with SPSS version 22.0 program (IBM, Armonk, NY, USA). Results were calculated as mean and standard deviation values. Statistical significance levels for all measurements were set as p < 0.05. Physical function evaluation, usability, and satisfaction tests were analyzed by dividing the age into two subgroups: a middle-aged group 40 to 64 years old and an old-aged group 65 to 84 years old. To evaluate the feasibility of a single session with the EX1, paired t-tests were used to compare outcome measures between post-exercise. To evaluate the usability and satisfaction questionnaire, frequency analysis was used. In addition, the usability and satisfaction tests with standardized b coefficients in linear regression analysis were used to investigate the relationship between physical function and questionnaire. 3. Results 3.1. Physical Function In the 10MWT, TUG, and FSST, significant increases after a single exercise session with the EX1 were observed in both groups (p < 0.05) . In the 6MWT, there was a statistically significant improvement only in the middle-aged group (p < 0.05). In the SPPB, there was a significant improvement in the old-aged group (p < 0.05) but not in the middle-aged group. 3.2. Usability and Satisfaction of the EX1 In the safety domain (involving items such as risk of falls and control of risk factors), after using the EX1, positive responses were confirmed in both groups. The responses were mostly positive in the satisfaction domain, but there were some items with difference by age group. The middle-aged group showed more positive responses than the old-aged group in the easiness of use, usefulness, and perception of the EX1. In addition, the middle-aged group showed more positive responses than the old-aged group in the questionnaire on whether the assist and resist mode of the EX1 helped with gait exercises. Though the old-aged group tended to prefer the assist mode, the middle-aged group tended to prefer the resistance mode of the EX1 . 3.3. Regression Analysis between General Characteristics, Usability, and Satisfaction As a result of regression analysis of 128 cases with a combination of 16 dependent variables and 8 independent variables, there were 15 significant results (Y = aX + b) (Table 2). The dependent variable (Y) consisted of a total of 16 items related to safety (4 items) and satisfaction (12 items), and the independent variable (X) consisted of a total of 8 items: age, gender, height, weight, body mass index (BMI), health status (2 items; health conditions, level of activity), and experience of fall. The younger the user, the easier they tend to think the EX1 was to use, they tend to think that the assist mode of the EX1 helps most with gait, and they tend to be willing to continue using the EX1. If the users think they have better health conditions, they tend to think the EX1 was easy to use. If the user is more active, they think that they control the risk posed by the EX1 (Supplementary Table S1). 3.4. Regression Analysis between Usability, Satisfaction, and Physical Function As a result of regression analysis of 240 cases by combining 10 dependent variables and 24 independent variables, there were 25 significant results (Y = aX + b) (Table 2). The dependent variable (Y) consisted of a total of 10 items regarding functional evaluation baseline (5 items) and difference of functional evaluation between baseline and after single-session exercise with the EX1 (5 items). The independent variable (X) consisted of a total of 24 items related to age, gender, height, weight, body mass index (BMI), health status (2 items; health condition, level of activity), experience of fall, safety (4 items), and satisfaction (12 items). If the users thought they have better health conditions, they tended to have a faster FSST and TUG. If the users considered themselves active, they tended to have faster gait speed, and walk farther in 6 min. If the users had experience of falling, they tended to have a lower SPPB, slower gait speed, and walk less in 6 min; after gait exercise with the EX1, these users tended to have a higher SPPB than before exercise. If the users thought there was no risk of falling when they were turning or leaning forward while wearing the robot, after gait exercise with the EX1, they tended to have a lower SPPB. If the users thought the robot was easy to use, they tend to have a higher SPPB, and faster TUG; after gait exercise with the EX1, these users tended to have a faster FSST than before exercise (Supplementary Table S2). 4. Discussion Our study demonstrates that a single session of exercise with the EX1 improved physical functions. In addition, the positive results of the EX1 were confirmed by conducting a usability and satisfaction survey after exercise with the EX1. In this study, statistically significant improvements in gait speed, balance ability, and gait endurance were confirmed through a single session of exercise with the EX1. The results of this study suggest that a single session of exercise with the EX1 has several key advantages for physical function and efficiency. Functional exercise that is effective for improving gait function includes gait exercise at various speeds and directions, treadmill gait, and stair climbing. In addition, the resistance and assistance of the EX1 can be adjusted based on individual physical ability. Therefore, it improves physical function gradually by controlling the intensity and duration of the exercise. To determine gait quality, we used objective, sensitive, and powerful measurement tools that measure gait performance, dynamic balance ability, and gait endurance . In our study, there was a statistically significant improvement in gait speed. Previous studies have shown that lower gait speed is associated with age, education, and particularly modifiable factors such as impairment of activities of daily life, physical inactivity, and cardiovascular disease . Gait speed is a clinical indicator related to survival rate and predicts functional ability in the elderly . In a previous study, it was reported that gait speed increased by 0.1 m/s as the survival rate increased . These results indicate the importance of staying active and healthy for old-aged people. Balance ability, an essential factor in gait ability, is the ability to maintain balance against numerical movement and external stimuli, which reduces muscle weakness due to decreased physical activity and is highly associated with risk of fall due to dynamic balance ability during gait . Physical changes due to aging lead to a decrease in gait function by reducing balance, and it is highly related to the incidence of injuries in the elderly . In our study, a statistically significant improvement was shown in dynamic balance ability through a single session of exercise with the EX1. Falling is a serious problem that threatens the health of the elderly and can lead to premature death due to physical damage, psychological dysfunction, and onset of various diseases . Preventing falls and improving body function require planned and consistent exercise. Decreased physical activity leads to muscle strength weakness, which causes reduced balance ability. Balance training is an essential element in an exercise program because it is highly correlated with risk of falls according to dynamic balance ability while walking. Gait endurance is also one of the major factors influencing risk of falls in the elderly. It is known that the weaker the walking endurance, the higher the likelihood of a fall in the elderly. In our study, endurance improved immediately after a single session of exercise with the EX1 in both age groups, but a significant result was found only in the middle-aged group. Endurance is increased through long-term gait training , but in our study, the time was insufficient to derive significant results with a single session of exercise with the EX1. However, although it was not significant after a single session of exercise, walking distance for 6 min improved, indicating the potential for positive results with long-term exercise. The SPPB is a fast and useful measurement tool for predicting falls . In our study, the SPPB improved immediately in the old-aged group after a single session of exercise with the EX1, but no significant result was shown due to the ceiling effect in the middle-aged group. This study was performed to confirm the usability and satisfaction of the EX1. Considering user physical condition and environment, it was divided into user questionnaire evaluation and user function evaluation, and the safety, operability, and satisfaction of the EX1 were evaluated. If an expert or designer conducts a usability evaluation while listening to users' opinions, it is possible to understand the users' needs, inconveniences, strengths, and expectations in a complex way. Regression analysis showed a difference in the experience feedback for the EX1 by age or physical function. However, there were no implications for usability, perceptions, or satisfaction with general characteristics other than age. As the results of the questionnaire included usability and satisfaction, both old-aged groups had positive experience feedback for the EX1. Among them, more positive results were shown in the middle-aged group than the old-aged group in a few items. We think that these results were caused by middle-aged people being more open-minded and faster to learn new technologies than the elderly. In the questionnaire, both the assist and resistance modes of the EX1 helped with gait, with strongly positive answers in the middle-aged group compared to the old-aged group. In addition, in a questionnaire on the preference between the assist and resistance modes of the EX1, the old-aged group preferred the assist mode, but the middle-aged group preferred the resistance mode. These results indicated that the elderly, who have a decline in gait ability, prefer gait assist, but middle-aged people who need gait training prefer the resist mode of the EX1. In regression analysis between general characteristics and questionnaire, users tended to think that using the EX1 would have no negative social perceptions if they thought they had lower health conditions. Younger users tend to have positive opinions of the EX1. Although there might be perceptions that exercise using robots is applied only to patients or the elderly , this study confirmed that healthy people had positive perceptions of exercise with robots. Therefore, we think that the EX1 can provide a meaningful exercise program not only for the elderly, but also for young people. In regression analysis between questionnaire and physical function, people who had experienced a fall within the last 6 months had better balance ability and physical function after a single session of exercise with the EX1. It was confirmed that the higher the satisfaction with the function of the robot, the better the physical endurance and dynamic balance. We think that such exercise will bring positive results when applied to people with reduced balance ability, which is a risk factor for falls. 5. Conclusions This study demonstrated that a single session of exercise with the EX1 in old-aged persons improved physical performance, including gait and balance, and received positive feedback. A newly developed wearable hip exoskeleton, the EX1, is a potentially useful exercise device for improving gait and physical function not only in the elderly, but also in middle-aged people. In this study, we emphasized short-term usability and satisfaction evaluation of the EX1 for healthy subjects. In future studies, the long-term effects of the EX1, not only in healthy subjects but also in persons with impaired physical function, must be addressed. Additionally, it is necessary to conduct more structured studies and repeat pre-post experiments by repeating specific training protocols in scheduled programs. Supplementary Materials The following supporting information can be downloaded at: Table S1: Regression analysis between general characteristics, usability, and satisfaction; Table S2: Regression analysis between usability, satisfaction, and physical function. Click here for additional data file. Author Contributions Conceptualization, J.K., H.C. and S.-H.L.; methodology, H.-J.L. and Y.-H.K.; software, J.K. and H.C.; validation, S.-H.L., H.-J.L. and Y.-H.K.; formal analysis, J.K., H.C. and S.-H.L.; investigation, J.K., H.C. and S.-H.L.; resources, J.K. and H.C.; data curation, J.K. and H.C.; writing--original draft preparation, J.K. and H.C.; writing--review and editing, S.-H.L., H.-J.L. and Y.-H.K.; visualization, J.K. and H.C.; supervision, H.-J.L. and Y.-H.K.; project administration, Y.-H.K.; funding acquisition, Y.-H.K. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement All participants recruited through Samsung Medical Center provided informed consent before participating in the present study. This study protocol was approved by the ethics committee of the Samsung Medical Center Institutional Review Board (No. 2021-04-058). Informed Consent Statement Informed consent was obtained from all subjects involved in the study. Written informed consent has been obtained from the participants to publish this paper. Data Availability Statement The data used and/or analyzed during the current study are available from the corresponding author on request. Conflicts of Interest The authors declare no conflict of interest. Abbreviations 10MWT: 10 m walking test, TUG: timed up and go, FSST: four square step test, 6MWT: 6 min walking test, SPPB: short physical performance battery, ICC: inter-tester reliability Figure 1 Experimental protocol. Figure 2 EX1 developed by Samsung Electronics. Figure 3 Changes in physical function. (a) 10 m walking test; (b) timed up and go test; (c) four square step test; (d) 6 minute walking test; (e) short physical performance battery (* p < 0.05). Figure 4 Responses to the questionnaire about usability and satisfaction of the EX1. The items of the usability and satisfaction evaluation include the safety of wearing, the risk of falling, the ease of operation, the feeling of weight, the fit, and the noise of the EX1. Refer to Table 2 for the usability and satisfaction questionnaire. healthcare-11-00643-t001_Table 1 Table 1 General characteristics of participants. Characteristics Total (N = 225) Middle-Aged (n = 75) Old-Aged (n = 150) Gender (male/female) 80/145 31/44 49/101 Age (year) 67.52 (10.99) a 74.31 (4.79) 53.96 (6.30) Height (cm) 161.53 (8.26) 159.85 (8.85) 164.88 (7.32) Weight (kg) 61.76 (10.58) 59.93 (9.32) 65.43 (11.98) BMI (kg/m2) 23.59 (3.06) 23.42 (2.92) 23.94 (3.33) MMSE-K 28.16 (1.82) 27.95 (1.93) 28.57 (1.49) GDS-15 3.71 (3.32) 4.1 (3.43) 2.93 (2.99) FES-K 99.32 (3.30) 99.07 (3.92) 99.81 (1.24) EQ-5D 0.90 (0.08) 0.89 (0.92) 0.93 (0.46) a Mean (SD), middle-aged group = 40-64 years, old-aged group = 65-84 years; BMI = body mass index; MMSE-K = Mini-Mental State Examination--Korean; GDS-15 = Geriatric Depression Scale-15; FES-K = Fall Efficacy Scale--Korean; EQ-5D = EuroQol-5 dimension. healthcare-11-00643-t002_Table 2 Table 2 The usability and satisfaction questionnaire of the EX1. Domain No. Item Strongly Disagree Disagree Neutral Agree Strongly Agree Safety 1 Did you have any risk of falling when turning or leaning forward while using the EX1? 1 2 3 4 5 Safety 2 Did you have a risk of fall or injury while using the EX1? 1 2 3 4 5 Safety 3 Do you think you can control the risks posed by EX1 yourself? 1 2 3 4 5 Safety 4 Did you fear falling while walking with the EX1? 1 2 3 4 5 Satisfaction 1 Was the EX1 easy to use? 1 2 3 4 5 Satisfaction 2 Do you think people with healthy people can also use the EX1? 1 2 3 4 5 Satisfaction 3 Were you not shamed for using EX1? 1 2 3 4 5 Satisfaction 4 Do you think the EX1 will have negative social perceptions? 1 2 3 4 5 Satisfaction 5 Was the weight of the EX1 appropriate for walking? 1 2 3 4 5 Satisfaction 6 Was the EX1 comfortable to wear? 1 2 3 4 5 Satisfaction 7 Were you satisfied with the material of the EX1? 1 2 3 4 5 Satisfaction 8 Do you think the assist mode of the EX1 helps with gait? 1 2 3 4 5 Satisfaction 9 Do you think the resistance mode of the EX1 helps with gait exercises? 1 2 3 4 5 Satisfaction 10 Was there any disturbance caused by noise when using the EX1? 1 2 3 4 5 Satisfaction 11 Are you satisfied with the color and design of the EX1? 1 2 3 4 5 Satisfaction 12 Are you willing to continue using the EX1? 1 2 3 4 5 Satisfaction 13 Between assist and resistance modes of EX1, which was more helpful? 1 Resistance mode 2 Assist mode Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). 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PMC10000835 | Healthcare (Basel) Healthcare (Basel) healthcare Healthcare 2227-9032 MDPI 10.3390/healthcare11050751 healthcare-11-00751 Article Exploration of Sex and Age-Based Associations in Clinical Characteristics, Predictors of Severity, and Duration of Stay among COVID-19 Patients at the University Hospital of Saudi Arabia Assiri Rasha Assad Conceptualization Methodology Writing - review & editing Project administration 1 Bepari Asmatanzeem Conceptualization Formal analysis Data curation Writing - original draft Project administration 1* Patel Waseemoddin Conceptualization Formal analysis Data curation Writing - review & editing 2 Hussain Syed Arif Conceptualization Methodology Writing - review & editing 34 Niazi Shaik Kalimulla Formal analysis Data curation Writing - review & editing 5 Alshangiti Asma Data curation Writing - review & editing Visualization 6 Alshangiti Safia Ali Data curation Writing - review & editing Visualization 6 Cordero Mary Anne Wong Formal analysis Writing - review & editing Funding acquisition 1 Sheereen Shazima Data curation Writing - review & editing Visualization 7 Sharma Manoj Academic Editor Batra Kavita Academic Editor 1 Department of Basic Health Sciences, College of Medicine, Princess Nourah bint Abdulrahman University, Riyadh 11671, Saudi Arabia 2 Department of Pediatrics, King Abdullah bin Abdulaziz University Hospital, Riyadh 11564, Saudi Arabia 3 Respiratory Care Department, College of Applied Sciences, Almaarefa University, Dariyah, Riyadh 13713, Saudi Arabia 4 Department of Internal Medicine, King Abdullah bin Abdulaziz University Hospital, Riyadh 11564, Saudi Arabia 5 Department of Preparatory Health Sciences, Riyadh Elm University, Riyadh 12611, Saudi Arabia 6 College of Medicine, Princess Nourah bint Abdulrahman University, Riyadh 11671, Saudi Arabia 7 Department of Pathology, Manipal Academy of Higher Education, Mangalore 576104, India * Correspondence: [email protected] 03 3 2023 3 2023 11 5 75112 1 2023 25 2 2023 26 2 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). COVID-19 infection has a spectrum of variable clinical severity between populations because of their characteristic demographic features, co-morbidities, and immune system reactions. This pandemic tested the healthcare system's preparedness, which depends on predictors of severity and factors related to the duration of hospital stays. Therefore, we carried out a single-center, retrospective cohort study in a tertiary academic hospital to investigate these clinical features and predictors of severe disease and study the different factors that affect hospital stay. We utilized medical records from March 2020 to July 2021, which included 443 confirmed (positive RT-PCR) cases. The data were explained using descriptive statistics and analyzed via multivariate models. Among the patients, 65.4% were female and 34.5% were male, with a mean age of 45.7 years (SD +- 17.2). We presented seven age groups with ranges of 10 years and noticed that patients aged 30-39 years old comprised 23.02% of the records, while patients aged 70 and above comprised 10%. Nearly 47% were diagnosed as having mild, 25% as moderate, 18% as asymptomatic, and 11% as having a severe case of COVID-19 disease. Diabetes was the most common co-morbidity factor in 27.6% of patients, followed by hypertension (26.4%). Our population's predictors of severity included pneumonia, identified on a chest X-ray, and co-morbid conditions such as cardiovascular disease, stroke, ICU stay, and mechanical ventilation. The median length of hospital stay was six days. It was significantly longer in patients with a severe disease and who were administered systemic intravenous steroids. An empirical assessment of various clinical parameters could assist in effectively measuring the disease progression and follow-up with patients. COVID-19 clinical features severity age group co-morbidities Saudi Arabia hospital stay health systems Princess Nourah bint Abdulrahman University, Riyadh, Saudi ArabiaPNURSP2023R147 This research was funded by Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2023R147), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia. pmc1. Introduction Coronavirus disease 2019 (COVID-19), defined by the World Health Organization (WHO), is a highly transmittable acute respiratory disease caused by a novel strain of coronavirus, severe acute respiratory syndrome coronavirus 2 (nCoV-2019 or SARS-CoV-2), which belongs to the Coronaviridae family and caused the recent viral outbreak . Its first case was identified in Wuhan, China, in December 2019, and within a short period of time, it had crossed most borders . It spread to more than 200 countries, resulting in the first-ever pandemic caused by a coronavirus. As a result, WHO announced the outbreak of COVID-19 to be a "public health emergency of international consideration" on January 30 2020 . In Saudi Arabia, the largest country in the Arabian Peninsula, the first confirmed case of COVID-19 was documented on March 2 2020 . Saudi Arabia has a well-established healthcare system with 497 hospitals (287 are MOH hospitals), 99,617 total physicians, and 22.6 beds per 10,000 people. Among the MOH hospitals, 65 are accredited by the Central Board of Accreditation for Healthcare Institutions (CBAHI), established by the Ministry of Health . In addition, several private and public hospitals have international accreditations, such as the Joint Commission International (JCI), which is accredited by the International Society for Quality in Health Care (ISQua), which serves as a safety collaborating center designated by the World Health Organization . Nearly 75 Saudi hospitals are JCI-accredited . Saudi authorities prepared public and private institutions to deal with the pandemic with strategic preparedness and a COVID-19 response plan, in line with the WHO operational planning guidelines to support the country. The authorities launched a governance system constituted of responsible committees to continuously monitor national and international updates, trace contacts, screen the population, increase awareness, and take appropriate actions to arrest the spread of this disease. Restrictions on social and religious gatherings, travel, and businesses were set ahead of the first 100 confirmed COVID-19 cases . Mass screening programs were carried out in three stages. The first stage focused on screening individuals in highly populated districts through field tests in 807 locations; the second stage was facilitated through the Mawid app self-assessment tool, which classifies users as high-risk. The low-risk group was the targeted population and was screened in designated primary care centers. The third stage was screening suspected COVID-19 cases with no symptoms at specialized drive-through testing centers, so-called takkad centers. The Hajj pilgrimage for 2020 was scaled down to confine participants, and no cases of COVID-19 were detected among pilgrims. As of 30 March 2020, the Saudi health authority announced that COVID-19 treatment is free for all citizens and residents. The country preserved all primary health services and immunization programs and supported all COVID-19 drugs and vaccine proposals . According to the WHO, as of November 1, 2022, globally, over 600 million confirmed cases of COVID-19, including more than six million deaths, were reported; Saudi Arabia accounted for less than a million cases and around nine thousand deaths . Usually, coronavirus causes a mild flu-like infection. However, like the severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), which caused severe outbreaks worldwide in the last decade, COVID-19 causes a spectrum of clinical features ranging from asymptomatic cases and mild upper respiratory infections to life-threatening pneumonia . COVID-19 usually presents as a fever, cough, fatigue, and shortness of breath. Less frequent manifestations include a sore throat, chest pain, headache, diarrhea, and loss of taste or smell. Severe pneumonia can cause acute respiratory distress syndrome (ARDS), demanding respiratory support and extended care in intensive care, which can lead to life-threatening complications, multiple organ dysfunction syndrome, and death . Despite the reports showing that SARS-CoV-2 centrally manifested as a respiratory infection, new data demonstrated that it must be considered a systemic disease infecting multiple systems, such as the gastrointestinal, immune, hematopoietic, cardiovascular, and respiratory systems . Contemporary studies have documented that the clinical characteristics of the disease are variable between populations because of their distinctive demographic characteristics and co-morbidities, specifically correlating with the severe form of the disease compared to the milder form . However, few studies have concentrated on discrepancies between sex and age clusters for clinical characteristics and severity among COVID-19 patients for an extended period in the early infection waves in Saudi Arabia. Therefore, recognizing these variations is often helpful in identifying the progression to a severe form of the disease and possibly optimizing COVID-19 case management . In addition, this awareness helps in formulating preventive measures, recognizing the effect of COVID-19 on hospital capacity, and improving patient bed capacity and health systems through risk stratification. King Abdullah bin Abdulaziz University Hospital (KAAUH), located in the capital city of Saudi Arabia, Riyadh--the region with the highest population--and one of the mainstream hospitals of this region, is a COVID-19 testing and vaccination center. It is on the Princess Nourah bint Abdulrahman University (PNU) campus, which has 20 colleges and 121 academic programs. It is a 400-bed, designated academic MOH hospital accredited by CBAHI and JCI; it is, therefore, one of the few hospitals that have national and international accreditations reflecting the commitment to the maintenance of high-quality health services and a compelling center for research documentation. Nearly 450 COVID-19 cases from March 2020 to July 2021 were treated at this hospital. During this period, the country faced its highest peak of cases, and most citizens and residents were not vaccinated. Therefore, we aimed to conduct a retrospective cohort study, investigating the differences in clinical variables and analyzing predictors of severity and factors that influence the duration of hospital stay among COVID-19-admitted cases. As far as our knowledge, to date, very few studies have targeted a more extended period for recruiting admitted COVID-19 patients (one and a half years) that focused on diverse age groups and sex-based differences for clinical characteristics, co-morbidities, severity, and duration of stay in Saudi Arabia. 1.1. Objectives of the Study 1.1.1. Aim of the Study The study's goal was to evaluate the severity of diverse symptoms and signs that developed in patients with COVID-19 disease and analyze variations in different clinical factors with age clusters and sex in confirmed COVID-19 patients admitted to a university hospital in Saudi Arabia retrospectively. 1.1.2. Specific Objectives To assess the severity of different symptoms and signs of COVID-19 disease that developed in patients. To investigate the differences in clinical variables with age, sex, and different co-morbidities. To compare the outcome of COVID-19 patients by severity levels. To assess the predictors of severity and duration of hospital stay (DoHS). 2. Materials and Methods 2.1. Study Design and Setting This study was conducted as per the guidelines from STROBE (strengthening the reporting of observational studies in epidemiology) . This was a single-center, non-interventional, non-exhaustive, retrospective cohort study, conducted at King Abdullah bin Abdulaziz University Hospital (KAAUH), Riyadh, Riyadh Province, Saudi Arabia. KAAUH is a referral hospital with nearly 400 beds and one of the designated COVID-19 hospitals and COVID-19 vaccination centers in Saudi Arabia (KSA). 2.2. Population We included all patients with confirmed COVID-19 infection who were admitted to King Abdullah bin Abdulaziz University Hospital (KAAUH) between March 2020 and July 2021. Confirmed case of COVID-19 was defined as positive for SARS-CoV-2 virus through real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay on nasopharyngeal swab specimens. On admission, RT-PCR nasopharyngeal swabs were sent to all patients with clinical suspicion of COVID-19. The result was reported as either positive or negative and was available within 24 h. Therefore, we included 443 cases with positive RT-PCR results; cases with negative RT-PCR and incomplete clinical data were excluded. 2.3. Institutional Ethical Approval The study was approved by Institutional Ethical and Review Board (PNU IRB and KAAUH IRBN), Riyadh, Saudi Arabia (PNU IRB number 21-0352, dated 16 September 2021, KAAUH IRB number RO2021-P-019, dated 3 October 2021). 2.4. Data Collection A convenient sampling method for recruiting records from COVID-19 patients was used. Health Information Management (HIM) office provided Medical Record Numbers (MRN) of these confirmed COVID-19 patients admitted to Internal Medicine, Pulmonology, and Critical Care departments. Electronic medical record data were obtained using institutional software (TrakCare) and entered into the data collection form for detailed review. Data collected included: demographic characteristics (age, sex); co-morbidities, such as diabetes, hypertension, obesity, asthma, chronic obstructive pulmonary disease (COPD), chronic kidney disease (CKD), cardiovascular disease, thyroid disease, obesity, and immunosuppression; relevant clinical features (fever, cough, breathlessness, chest pain, loss of taste or smell, headache, myalgia, loose stools, respiratory rate, oxygen saturation (SpO2), chest X-ray); severity; and outcome (discharge or death), as displayed in Table 1. 2.5. Data Management and Analysis Plan Data were analyzed using the JMP SAS statistical software. They were described through descriptive statistics, such as frequency, percentages, measures of central tendency, and proportions, and were analyzed via inferential statistical tests, such as chi-square test to assess categorical variables and multivariate models. Odds ratios (ORs) and their corresponding 95% CIs were calculated, and logistic regression analysis was applied to adjust for confounders of the association between different variables and case fatality of COVID-19. The values were compared at 0.05 level of significance to test the results of the study for the corresponding degrees of freedom. 3. Results 3.1. Age and Sex A total of 443 patients were included in the analysis; 65.4% of them were female and 34.5% were male, with a mean age of 45.7 years (SD +- 17.2). Of these, we presented seven age groups with ranges of 10 years and noticed that patients aged 30-39 years comprised 23.02% of the total, followed by patients aged 18-29 years (19.86%), 40-49 years (18.96%), 60-69 years (14.67%), and 50-59 years (12.87%), as depicted in Figure 1. Nearly 7.5 % of patients were in the range of 70-79 years and 3.2% were 80 and above. This lower number of patients aged 70 and above reflects the country's expected life expectancy at birth of 75 years. 3.2. Clinical Characteristics of COVID-19 among Study Participants Table 2 shows the frequency distribution of a variety of symptoms seen in our study participants on admission. The most common clinical manifestation was a fever (55.76%), followed by a cough (53.27%), dyspnea or shortness of breath (37.92%), headache (22.35%), diarrhea (20.32%), and myalgia (19.41%). A further analysis was conducted among age clusters and sex-based differences. 3.3. A. Different Age Groups Table 3 displays the spectrum of clinical characteristics among the seven age clusters of the cohort. Fever, cough, and dyspnea were found to have a higher incidence in the age groups 60-69, 50-59, and 40-49 years and were statistically significant. Moreover, nearly 65% of "70-79 years" and "80 and above" patients reported these symptoms. Ageusia and abdominal pain rates were found to be comparatively higher in younger age groups, and were statistically significant; however, they remained low (around 2%), as seen in Table 3. 3.4. B. Sex-Based Differences A higher percentage of males complained of a fever (p = 0.0103), a cough, dyspnea, ageusia, and arthralgia, while headache, myalgia, abdominal pain, and anosmia were seen comparatively more in females, but were not statistically significant except for fever, as displayed in Table 3. 3.5. Co-Morbidities in the Study Participants In our study, 46.28% of patients were obese. Diabetes was the most common co-morbidity found in 27.6% of patients, followed by hypertension (26.4%) and asthma or other respiratory disorders (14%), as depicted in Figure 2. 3.6. A. Different Age Groups Of the patients that were obese, nearly 10% belonged to the 30-39 years group, followed by the 40-49 years group, which comprised 9% of them. Hypertension (8.8%), diabetes (7.67%), and cardiovascular diseases, such as congestive heart failure and hypothyroidism (p = 0.0481 *), were higher in incidence among the age group 60-69 years and were statistically significant (p < 0.0001 ****). It was also seen that diabetes and HTN were notably more common in ages 70-79 years (4.74%, 5.42%), 50-59 years (5.65%, 4.5%), and 40-49 years (4.3%, 4.5%). While asthma and other respiratory disorders were comparatively higher in the younger age group of 30-39 years, as seen in Figure 3 and Table 4. A nominal logistic fit analysis was used to adjust for covariates of age and sex. 3.7. B. Sex-Based Differences A higher percentage of males suffered HTN (p = 0.0119) and cardiovascular disease (p = 0.0467 *), while obesity (0.0467 *), hypothyroidism (0.0448 *), and a vitamin D deficiency (0.0235 *) had comparatively higher rates in females and were statistically significant, as displayed in Table 4. 3.8. The Severity of COVID-19 Nearly 47% of patients were diagnosed as having mild, 25% as moderate, 18% as asymptomatic, and 11% as having severe COVID-19 disease, as depicted in Figure 4. Pneumonia on chest X-rays was seen in 48% of patients, while 18.08% required oxygen support on arrival, 14.22% were transferred to the ICU, and 4.07% needed intubation. There was a statistically significant difference between different age groups and sexes, as seen in Table 5. A higher percentage of elderly patients needed oxygen support on arrival, were admitted to the ICU, and required intubation. For further analysis, the four severity groups were narrowed down into two groups, 'mild disease' with asymptomatic-mild disease patients together, and 'severe disease' comprising moderate-severe disease. Table 6A-C represent the association found among various clinical parameters: age, sex, and interventions opted by the severity of the disease. 3.9. Predictors of Severity and Duration of Hospital Stay (DoHS) A multivariate logistic regression analysis indicated that the patient's respiratory need for O2 on arrival, the presence of pneumonia on chest X-ray, admission to the ICU, and existing cardiovascular disease (MI, CHF, IHD) and stroke were significant predictors for the severity (Table 7). Figure 5 displays the leverage plots of independent predictors with the duration of stay in the hospital. Table 8 shows the duration of hospital stay (DoHS) in days for the patients, with a maximum of 118, an average of 9.72 +- 14.2 (SD), and a median of 6 days. A comparison of the factors showed that patients with severe symptoms were associated with a more prolonged DoHS than those with mild-to-moderate symptoms (mean 32 and 5.5 days, estimate = 3.29, p < 0.0018, respectively). The results also indicated that patients admitted or transferred to the ICU were associated with a significantly longer DoHS (mean 29.26 and 6.4 days, estimate = 6.41, p < 0.0001) than patients in the general isolation ward. Similarly, patients on mechanical ventilation, CVD, or stroke, were associated with a longer DoHS than those without these disorders (estimate = 5.65, p = 0.0011; 2.19, p = 0.04; and 5.6 days, p = 0.02, respectively). We also discovered that patients who received systemic intravenous steroids had a longer DoHS (estimate = 1.75, p = 0.02). 3.10. The Outcome of COVID-19 Patients with Severity Level, Age, and Sex Nearly 97.5% of the patients were cured and discharged, while 2.5% succumbed to the disease. As displayed in Table 9 and Figure 6, this 2.5% comprised elderly males diagnosed with severe disease on admission. 4. Discussion In our study, we explained the different clinical factors, co-morbidities, and severity of 443 documented patients with COVID-19 at KAAUH, a CBAHI-accredited MOH hospital in Riyadh, Saudi Arabia, along with predictors of severity and characteristics associated with more extended hospital stays. Hospital accreditation generates a positive impact on most patient safety indicators and thereby is one of the driving forces towards improving quality healthcare in KSA. The Riyadh region is the most COVID-19-infected province of Saudi Arabia, according to the MOH-launched "COVID-19 Statistics E-Platform" . One of the limitations of the previous studies is the main focus on midlife adults and older adults aged 60 and above. However, further subgroup analysis of elderly patients is present in very few studies; thereby, exploring the age-related clinical features of COVID-19 across all age groups is a matter of interest for study. Another typical limitation in earlier research works was the insufficient or missed information about the healthcare accreditation standards of the hospitals studied. In our study, the early midlife age range of 30-39 years comprised one-fourth of our patients, followed by younger patients who comprised one-fifth (19.86%), while the remaining one-fourth were 40-49 years (18.96%) and 50-59 years (12.87%), as depicted in Figure 1. The previous reports showed similar results: the middle-aged "40 to 60 years" were the most commonly infected group . While only one-tenth of patients were in the ranges of 70-79 years and 80 and above. However, individuals of all age groups can be infected by the virus . Nearly two-thirds of the patients were female (65.4%) and the remaining one-third (35%) were male, with a mean age of 45.7 years (SD, +-17.2). However, a single-arm meta-analysis indicated that men represent a significantly higher percentage of COVID-19 patients at 60% (95% CI [0.54, 0.65]) , and other previous studies have also reported a significantly higher number of men having the infection compared to women . In addition, previous studies have recorded more men than women infected with other coronavirus infections, such as SARS-CoV and MERS-CoV . The disproportionate number of female hospitalizations in the present study can be due to the greater input of female patients from the affiliated women's university (PNU). Our findings indicated that a fever (55.76%), a cough (53.27%), and dyspnea (shortness of breath, 37.92%) were the most frequent symptoms in COVID-19 patients, which is in accordance with multiple earlier studies . These symptoms were more commonly found in older patients of 60-69 years old, 70-79 years old, or 80 years old and above, which were statistically significant compared to younger groups . Nearly one-fourth of patients complained of headache (22.35%), diarrhea (20.32%), or myalgia (19.41%). It was noted that ageusia and abdominal pain were statistically higher in younger age groups than in older patients; however, their incidence was low (around 2%). Research evidence indicates that the disorder's complete clinical characteristics are nonspecific and unclear, as the associated manifestations range from mild to severe, and resulting in death in several patients . There were no differences between the sexes for most symptoms among the COVID-19 patients, except for the fever, which was higher in males than females (p = 0.0103, Table 3). Previous studies have consistently noted poorer outcomes in men in terms of morbidity and mortality, confirming the male sex as an independent risk aspect for COVID-19 . The more robust innate and adaptive immune response in females can be attributed to numerous factors, though primarily to estrogen being immune strengthening as opposed to testosterone being immune suppressing . It was found that diabetes was the most common co-morbidity (27.6%) seen in the admitted COVID-19 patients of our study, followed by hypertension (26.4%), which is comparable to the prevalence in the general population. Our findings revealed that, among different age clusters, many 60-69-years-old patients infected by SARS-CoV-2 had chronic underlying diseases, including hypertension (8.8%), diabetes (7.67%), and cardiovascular diseases such as congestive heart failure (p < 0.0001 ****) and hypothyroidism (p = 0.0481 *), which were statistically noteworthy, as depicted in Table 4 and Figure 3. It was also seen that diabetes and HTN were notably more common in ages "70-79" years (4.74%, 5.42%), 50-59 years (5.65%, 4.5%), and 40-49 years (4.3%, 4.5%). This agrees with an earlier report in which COVID-19 patients were documented to have co-morbid disorders . In addition, research data show that patients aged 60 years old and above are at a higher risk than children, who are less likely to have an infection or show mild to asymptomatic infections . In addition, our study revealed that 60-70% of patients in the "70-79 years" and "80 and above" ranges were suffering from diabetes or hypertension, and nearly 55% of each group were obese. The augmented risk of acquiring severe COVID-19 complications in older people with co-morbid conditions such as diabetes or hypertension has been well documented in the literature . Previous studies from the United States, Italy, and China have noted that the diabetic population is at a more prominent risk for disease complications and infection susceptibility . The most extensive study of COVID-19 cases (72,314 cases) from China showed a higher incidence of mortality among patients with diabetes and COVID-19 (2.3% without diabetes vs. 7.3% with the disease) . In contrast, the occurrence of asthma and other respiratory disorders was comparatively higher in the younger age group of 30-39 years . Therefore, identifying host risk characteristics associated with severe COVID-19 infections may allow the design of specific approaches to prevent and treat the disease . There were differences among the sexes in co-morbidities among our cohort of admitted COVID-19-positive patients. It was seen that a higher percentage of males suffered HTN (p = 0.0119 *) and cardiovascular disease (p = 0.0467 *), while obesity (0.0467 *), hypothyroidism (0.0448 *), and vitamin D deficiencies (0.0235 *) were comparatively higher in females and were statistically significant, as displayed in Table 4. The lower vitamin D level has been invariably associated with an augmented risk of upper respiratory tract infections and pneumonia, secondary to weak immune systems and raised inflammatory cytokines . Research studies also link vitamin D depletion to poor COVID-19 prognosis and mortality . We found that 47% of the patients were diagnosed as having mild, 25% as moderate, 18% as asymptomatic, and 11% as having severe COVID-19, as depicted in Figure 5. Nearly 15% of the patients were transferred to the ICU, which is sensitive to understanding the severity of the disease, wherein 50% of them (7% of the total, p < 0.0001 ****) were elderly patients, followed by the 40-49 years age group (3.4% of the total), which was found to be statistically significant compared to the younger age group, as displayed in Table 5. Additionally, more males (18.95%, p = 0.0417 *) were admitted to the ICU. Similar findings were reported in a systematic review and meta-analysis comprising seven studies (1813 COVID-19 cases with a more significant proportion of male patients), demonstrating that patients admitted to the ICU were older (mean age = 62.4 years) compared with non-ICU patients (mean age = 46 years). Additionally, they found that 1591 primarily older male patients with co-morbid diseases admitted to ICUs had moderate to severe ARDS . In our study, pneumonia discovered on chest X-rays was seen in 48% of patients, 18.08% required oxygen support on arrival, and 4.07% required intubation. In our study, oxygen on arrival was required more in the age group of 60-69 years, followed by 40-49 years, 50-59 years, and 70-79 years (p < 0.0001 ****), and was managed with systemic steroids (11.29%, p < 0.0001 ****). Our study revealed that more patients aged 70-79 years and 60-69 years required intubation, which was statistically significant compared to younger patients (p = 0.002). Therefore, the exploration of age-related effects on COVID-19 severity helps us progress strategies required for developing the healthcare system in COVID-19-treating hospitals. In addition, more males (18.95%, p = 0.0417 *) were admitted to ICU, and there was a statistical difference between the sexes. A higher percentage of males were given oxygen support on arrival (p = 0.0081 *) and treated with systemic steroids (p = 0.0294 *), while no difference in sexes was found for intubation. Similar findings were reported by Chen et al., who proposed that nCoV-2019 is more likely to infect elderly adult men with chronic co-morbidities due to these patients' more vulnerable immune functions . Furthermore, the analysis of two groups of mild and severe COVID-19 disease showed that a fever, a cough, and dyspnea were more common in severe disease cases. At the same time, diarrhea and ageusia were more common in mild disease cases (Table 6A-C). We used a multiple logistic regression and found that pneumonia discovered by chest X-rays and co-morbid conditions, such as cardiovascular disease, stroke, an ICU stay, and mechanical ventilation, were predictors of severity depicting statistically significant differences (Table 7). Guan et al. documented that patients with severe COVID-19 disease were older than those with non-severe disease, and any co-morbidity was more expected among patients with severe disease than those with non-severe COVID-19 disease . Another study documented similar findings, wherein it was seen that older age and hypertension were independently associated with severe disease at admission . The median period of hospital stay in our study was six days. A similar result was reported by Alwafi et al. (range: 0-55 days) . Alghamdi et al. presented that the DoHS in Saudi Arabia among COVID-19 patients ranged from 4 to 15.6 days . We found that it was significantly longer in patients with severe disease who needed oxygen support or mechanical ventilation, as expected, and also that a longer stay occurred in patients with CVD or stroke who were administered systemic intravenous steroids, as depicted in Figure 6 and Table 8. Wang et al. reported similar results, showing that clinical severity was firmly correlated to the period of stay (p < 0.01) and that a longer DoHS was associated with patients with admission to a provincial hospital, 45 years of age or older, and severe illness . Our analysis' mortality rate for COVID-19 was 2.5%, proximate to the 3.4% reported in the literature . Previous studies found that the prevailing fatality rate of the disease was in the range of 3% to 14% . In severe cases, the virus causes alveolar damage, leading to advanced respiratory failure, and causing death . In our study, the mortality rate was relatively high among the elderly (p = 0.0006 *) , and patients with pneumonia on chest X-ray (p < 0.0001 ****), which resembles previous findings. Research evidence demonstrates that the aged and those with underlying chronic disorders produce severe and lethal respiratory failure because of alveolar injury from the virus . In our study, the age groups of 70-79 years and 80 and above were significantly associated with in-hospital mortality. In addition, our study revealed that nearly 55.67% of patients aged "70-79 years" and the majority (78.5%) of patients aged "80 and above" were suffering from severe disease at the time of admission, and nearly 15% of each group succumbed to the disease. This was compared to another study conducted by Abolfoutoh et al., wherein mortality was high among patients aged 70 and above . A meta-analysis, including studies from different countries--fifty from China, three from the USA, and one each from Germany, Iran, Italy, Singapore, South Korea, and the UK--revealed that patients aged 70 years and above have a higher infection threat, severity, and mortality risk compared with patients younger than 70 years . Moreover, ICU patients were more likely to receive prolonged treatment and mechanical ventilation. No independent mortality association was observed in the present study with any co-morbidities . However, established risk factors such as CVD, lung infiltrates, and stroke were noted to be substantial risk factors for severe COVID-19 in our study. It nonetheless suggests that the increased risk for worse outcomes is the accumulative effect of clustering with cardiometabolic multimorbidity or chronic diseases, which increases complications . Most of these characteristics have been connected to advancing acute respiratory distress syndrome (ARDS), secondary to SARS-CoV and MERS-CoV . All these risk factors, together with DM and HTN, lead to worsening pre-existing chronic inflammation, progressing to cytokine storm and prompt impairment of the endothelial function if left untreated . Additionally, previous studies indicated that HTN, renal failure, and CVD raised the death risk in COVID-19 cases . Furthermore, ACE inhibitors as antihypertensive are linked between hypertension and COVID-19 severity as ACE2 serves a role in SARS infections . Table 10 summarizes the important highlights of the current study in comparison to the available literature. In comparing the death rate of COVID-19 to that of other coronaviruses, the available data demonstrate that COVID-19 infection resulted in a lower mortality rate than that documented for SARS (9.60%) and MERS (34.4%) . Thus, comprehensive analyses of the pathogenic and virulence mechanisms of SARS-CoV, SARS-CoV-2, and MERS-CoV are required to demonstrate these deviations. We acknowledge some limitations. Our findings are confined to the accuracy of medical record-keeping, given its retrospective design. In addition, the single-centric nature of the investigation limits the generalization of results to the general population as the study sample is limited to 1 hospital among 497 hospitals (of which 65 are accredited by the Ministry of Health). Therefore, the study sample is not fully representative of COVID-19 patients admitted to hospitals in Saudi Arabia during the study period, which directs future research toward multicentric studies and huge sample sizes. Despite these limitations, the results of the present study are strong and add significance to the literature on COVID-19 patients within the Arab region, as it is a study performed in a hospital with international accreditation in the capital city of KSA, in a region with the highest COVID-19 infection rate, is longer than one and a half years in duration, thoroughly describes hospitalized patients during earlier waves, and differentiates clinical characteristics based on sex, seven different age clusters from "18-29 years" to "80 and above", severity and its predictors, duration of hospital stay, and outcomes. 5. Conclusions In summary, this study, conducted in a CBAHI-accredited MOH hospital in Riyadh, Saudi Arabia, discovered that COVID-19 patients were most likely to present with a mild fever, cough, and shortness of breath on admission. There was an increased disease severity rate in elderly male patients aged "70 and above" who were transferred to the ICU, showed pneumonia on a chest X-ray, required intubation, and had a higher incidence of various co-morbidities, such as hypertension and cardiovascular diseases. In contrast, younger female patients suffered from vitamin D deficiency, obesity, and hypothyroidism. Moreover, the older age groups of 70-79 years and 80 years and above were significantly associated with in-hospital mortality. The hospital's preparedness and quality of care were reflected in the lower mortality rate and the average length of hospital stay. More extensive epidemiologic studies and randomized trials covering multiple institutions are needed to determine a more accurate in-hospital severity and mortality rate in the country. Therefore, age and sex must be considered when estimating the clinical findings, severity, and mortality of COVID-19. This may improve the management of potentially severe COVID-19 patients by ensuring appropriate resource allocations and putting forward preventative measures. Acknowledgments We thank Amel Fayed, Department of Clinical Sciences, College of Medicine, Princess Nourah bint Abdulrahman University, Riyadh, for statistical advice in this study. Author Contributions Conceptualization, R.A.A., A.B., W.P. and S.A.H.; Data curation, A.B., W.P., S.K.N., A.A., S.A.A. and S.S.; Formal analysis, A.B., W.P., S.K.N. and M.A.W.C.; Funding acquisition, M.A.W.C.; Methodology, R.A.A. and S.A.H.; Project administration, R.A.A. and A.B.; Visualization, A.A., S.A.A. and S.S.; Writing--original draft, A.B.; Writing--review and editing, R.A.A., W.P., S.A.H., S.K.N., A.A., S.A.A., M.A.W.C. and S.S. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The study was approved by Institutional Ethical and Review Board (PNU IRB and KAAUH IRBN), Riyadh, Saudi Arabia (PNU IRB number 21-0352, dated 16 September 2021, KAAUH IRB number RO2021-P-019, dated 3 October 2021). Informed Consent Statement Not applicable. Data Availability Statement Data that underlie the results reported in this article, after deidentification, protocol, and statistical analysis will be available on request. Researchers should provide a methodologically sound proposal. Conflicts of Interest The authors declare that there is no conflict of interest of this study. Figure 1 Age-wise distribution of patients of the cohort. Figure 2 Descriptive statistics of different co-morbidities in the cohort. Figure 3 Age-wise distribution of co-morbidities in COVID-19 admitted patients. Figure 4 Descriptive statistics of severity of COVID-19 among study participants. Figure 5 Leverage plots of independent predictors with duration of stay in hospital. Figure 6 Mosaic plot displaying contingency analysis of outcome by (A) age groups, (B) sex, and (C) severity of disease. healthcare-11-00751-t001_Table 1 Table 1 Classification of severity of COVID-19. Asymptomatic Mild COVID-19 Moderate COVID-19 Severe COVID-19 Severe Cases Critical Cases COVID-19 PCR + ve, no symptoms Symptoms (fever, cough, myalgia), RR < 24, SPo2 > 94% in room air, no pneumonia Symptoms with shortness of breath, RR 24-30, SPo2 90-94 in room air, pneumonia Pneumonia plus any one of the following: RR > 30, SPo2 < 90 in room air, severe respiratory distress, requiring respiratory support ARDS, respiratory failure requiring ventilation support, sepsis, septic shock, MODS healthcare-11-00751-t002_Table 2 Table 2 Clinical characteristics displayed by the study participants. Clinical Characteristics N Percentage Fever 247 55.76% Cough 236 53.27% Dyspnea 168 37.92% Headache 99 22.35% Nausea/Vomiting 76 17.16% Myalgia 86 19.41% Diarrhea 90 20.32% Ageusia 28 6.32% Abdominal pain 36 8.13% Arthralgia 11 2.48% Anosmia 15 3.39% healthcare-11-00751-t003_Table 3 Table 3 Association between clinical symptoms with age clusters and sex (N = 443). p < 0.05 (*), p < 0.01 (**), p < 0.0001 (****). Symptoms Age Groups (Years) Sex 18-29 30-39 40-49 50-59 60-69 70-79 80 and Above p-Value Female % Male % p-Value Fever <0.0001 **** 0.0103 * Yes 8.80% 9.03% 11.51% 9.03% 10.38% 4.97% 2.03% 51.38% 64.05% No 11.06% 14.00% 7.45% 3.84% 4.29% 2.48% 1.13% 48.62% 35.95% Cough <0.0001 **** 0.3679 Yes 8.35% 8.35% 10.84% 9.48% 9.48% 4.97% 1.81% 51.72% 56.21% No 11.51% 14.67% 8.13% 3.39% 5.19% 2.48% 1.35% 48.28% 43.79% Dyspnea <0.0001 **** 0.4136 Yes 3.61% 6.09% 7.45% 7.45% 8.80% 3.16% 1.35% 36.55% 40.52% No 16.25% 16.93% 11.51% 5.42% 5.87% 4.29% 1.81% 63.45% 59.48% Headache 0.09 0.1329 Yes 6.09% 5.42% 3.39% 3.16% 3.39% 0.68% 0.23% 24.48% 18.30% No 13.77% 17.61% 15.58% 9.71% 11.29% 6.77% 2.93% 75.52% 81.70% Nausea/Vomiting 0.058 0.8424 Yes 3.61% 2.48% 3.84% 2.93% 3.61% 0.45% 0.23% 16.90% 17.65% No 16.25% 20.54% 15.12% 9.93% 11.06% 7.00% 2.93% 83.10% 82.35% Myalgia 0.438 0.2299 Yes 2.48% 4.06% 4.06% 3.39% 3.39% 1.58% 0.45% 21.03% 16.34% No 17.38% 18.96% 14.90% 9.48% 11.29% 5.87% 2.71% 78.97% 83.66% Diarrhoea 0.180 0.9834 Yes 4.06% 3.16% 5.64% 3.16% 2.71% 1.13% 0.45% 20.34% 20.26% No 15.80% 19.86% 13.32% 9.71% 11.96% 6.32% 2.71% 79.66% 79.74% Ageusia 0.006 ** 0.3459 Yes 1.58% 2.26% 2.03% 0.23% 0.00% 0.23% 0.00% 5.52% 7.84% No 18.28% 20.77% 16.93% 12.64% 14.67% 7.22% 3.16% 94.48% 92.16% Abdominal pain 0.025 * 0.0937 Yes 2.48% 1.81% 2.03% 0.00% 1.13% 0.23% 0.45% 9.66% 5.23% No 17.38% 21.22% 16.93% 12.87% 13.54% 7.22% 2.71% 90.34% 94.77% Anosmia 0.212 0.5056 Yes 0.45% 1.58% 0.90% 0.23% 0.23% 0.00% 0.00% 3.79% 2.61% No 19.41% 21.44% 18.06% 12.64% 14.45% 7.45% 3.16% 96.21% 97.39% Arthralgia 0.550 0.1697 Yes 0.45% 0.90% 0.23% 0.23% 0.68% 0.00% 0.00% 1.72% 3.92% No 19.41% 22.12% 18.74% 12.64% 14.00% 7.45% 3.16% 98.28% 96.08% healthcare-11-00751-t004_Table 4 Table 4 Association between co-morbidities with age clusters and sex (N = 443). p < 0.05 (*), p < 0.0001 (****). Co-Morbidities Age Groups (Years) Sex 18-29 30-39 40-49 50-59 60-69 70-79 80 and Above p-Value Female % Male % p-Value Diabetes <0.0001 **** Yes 1.13% 1.81% 4.51% 5.64% 7.67% 4.74% 2.03% 25.17% 32.03% 0.1272 No 18.74% 21.22% 14.45% 7.22% 7.00% 2.71% 1.13% 74.83% 67.97% Hypertension <0.0001 **** 0.0119 * Yes 0.68% 0.68% 4.29% 4.51% 8.80% 5.42% 2.26% 22.76% 33.99% No 19.19% 22.35% 14.67% 8.35% 5.87% 2.03% 0.90% 77.24% 66.01% Asthma and any other respiratory disorders 0.1663 0.3063 Yes 2.48% 4.97% 2.71% 1.35% 1.35% 0.45% 0.68% 12.76% 16.34% No 17.38% 18.06% 16.25% 11.51% 13.32% 7.00% 2.48% 87.24% 83.66% Obesity 0.102 0.0467 * Yes 6.55% 10.16% 9.26% 7.45% 8.13% 4.06% 0.68% 52.76% 33.99% No 13.32% 12.87% 9.71% 5.42% 6.55% 3.39% 2.48% 47.24% 66.01% CVD <0.0001 **** 0.0467 * Yes 0.00% 0.45% 1.13% 1.13% 2.48% 2.26% 0.68% 6.21% 11.76% No 19.86% 22.57% 17.83% 11.74% 12.19% 5.19% 2.48% 93.79% 88.24% Renal disease 0.6263 0.1933 Yes 0.45% 0.23% 0.23% 0.45% 0.45% 0.00% 0.23% 1.38% 3.27% No 19.41% 22.80% 18.74% 12.42% 14.22% 7.45% 2.93% 98.62% 96.73% Stroke 0.2038 0.3252 Yes 0.23% 0.00% 0.00% 0.23% 0.45% 0.23% 0.23% 1.72% 0.65% No 19.64% 23.02% 18.96% 12.64% 14.22% 7.22% 2.93% 98.28% 99.35% Vitamin D deficiency 0.2782 0.0235 * Yes 0.23% 0.45% 0.00% 0.00% 0.23% 0.45% 0.00% 2.07% 0.00% No 19.64% 22.57% 18.96% 12.87% 14.45% 7.00% 3.16% 97.93% 100.00% Hematological disorders 0.7098 0.3623 Yes 1.13% 0.45% 0.45% 0.45% 0.23% 0.23% 0.00% 3.45% 1.96% No 18.74% 22.57% 18.51% 12.42% 14.45% 7.22% 3.16% 96.55% 98.04% Hypothyroidism 0.0481 * 0.0448 * Yes 0.45% 1.81% 2.48% 1.58% 2.03% 0.23% 0.23% 10.69% 5.23% No 19.41% 21.22% 16.48% 11.29% 12.64% 7.22% 2.93% 89.31% 94.77% Hyperthyroidism 0.7658 0.357 Yes 0.00% 0.00% 0.23% 0.00% 0.00% 0.00% 0.00% 0.34% 0.00% No 19.86% 23.02% 18.74% 12.87% 14.67% 7.45% 3.16% 99.66% 100.00% healthcare-11-00751-t005_Table 5 Table 5 Association between interventions by age groups and sexes (N = 443). p < 0.05 (*), p < 0.01 (**), p < 0.0001 (****). Parameters Age Groups (Years) Sex 18-29 30-39 40-49 50-59 60-69 70-79 80 and Above p-Value Female % Male % p-Value Required O2 on arrival <0.0001 **** 0.0081 * Yes 1.13% 1.35% 4.06% 2.71% 4.97% 2.48% 1.35% 14.48% 24.84% No 18.74% 21.67% 14.90% 10.16% 9.71% 4.97% 1.81% 85.52% 75.16% Systemic IV steroids <0.0001 **** 0.0294 * Yes 1.35% 3.39% 4.97% 4.74% 6.32% 3.16% 1.81% 22.41% 32.03% No 18.51% 19.64% 14.00% 8.13% 8.35% 4.29% 1.35% 77.59% 67.97% Transferred to ICU <0.0001 **** 0.0417 * Yes 0.90% 0.90% 3.39% 2.26% 3.61% 2.48% 0.68% 11.72% 18.95% No 18.96% 22.12% 15.58% 10.61% 11.06% 4.97% 2.48% 88.28% 81.05% Required intubation 0.0024 ** 0.0609 Yes 0.00% 0.23% 0.90% 0.68% 1.13% 1.13% 0.00% 2.76% 6.58% No 19.91% 22.85% 17.87% 12.22% 13.57% 6.33% 3.17% 97.24% 93.42% Home isolation 2.26% 3.61% 3.16% 0.23% 0.68% 0.45% 0.00% 0.0037 ** 11.38% 8.50% 0.3373 healthcare-11-00751-t006_Table 6 Table 6 A: Association analysis of clinical characteristics by COVID-19 severity (N = 443). p < 0.05 (*), p < 0.01 (**), p < 0.0001 (****); B: Association analysis of COVID-19 severity by age groups and sex (N = 443). p < 0.01 (**), p < 0.0001 (****); C: Association analysis of interventions and chest X-ray with the severity of the disease (N = 443). p < 0.05 (*), p < 0.0001 (****). A Clinical Parameters Severity of Disease Mild % Severe % p-Value I. Symptoms Fever 45.1 75.16 <0.0001 **** Cough 41.26 75.16 <0.0001 **** Dyspnea 24.83 61.78 <0.0001 **** Headache 24.48 18.47 0.1424 Nausea/Vomiting 15.73 19.75 0.2879 Myalgia 17.83 22.29 0.2597 Diarrhea 17.13 26.11 0.0264 * Ageusia 8.39 2.55 0.0096 ** Abdominal pain 9.09 6.37 0.3075 Arthralgia 3.15 1.27 0.2016 Anosmia 3.5 3.18 0.8616 II. Co-morbidities Diabetes 21.51 40.4 <0.0001 **** Hypertension 45.22 54.78 <0.0001 **** Asthma and any other RS disorders 12.54 16.56 0.2567 Obesity 44.8 50.33 0.2729 Cardiovascular disease (MI, CHF, others) 3.94 15.23 <0.0001 **** Renal disease 2.15 1.99 0.9095 Cancer/leukemia 0.00% 0.00% Stroke 1.08 1.99 0.4523 Vitamin D deficiency 1.79 0.66 0.3114 Hematological disorders 3.58 0.66 0.067 Hypothyroidism 7.53 10.6 0.2851 Hyperthyroidism 0.36 0.00% 0.352 B Mild Severe p -Value I. Age Groups (Years) 18-29 27.27% (78) 6.37% (10) <0.0001 **** 30-39 29.37% (84) 11.46% (18) 40-49 16.78% (48) 22.93% (46) 50-59 10.49% (30) 17.20% (27) 60-69 9.79% (28) 23.57% (37) 70-79 5.24% (15) 11.46% (18) 80 and Above 1.05% (3) 7.01% (11) II. Gender Female 70.98% 55.41% 0.0011 ** Male 29.02% 44.59% C Parameters Severity of Disease Mild % Severe % p -Value Home isolation 12.59 6.37 0.0336 * Required O2 on arrival 3.5 44.59 <0.0001 **** Transferred to ICU 0.7 38.85 <0.0001 **** Required intubation 0.35 10.9 <0.0001 **** Chest X-ray: pneumonia 23.78 92.99 <0.0001 **** Systemic IV steroids 11.89 50.96 <0.0001 **** Systemic oral steroids 22.93 6.64 <0.0001 **** healthcare-11-00751-t007_Table 7 Table 7 Multiple regression results of predictors of severe COVID-19 (N = 443). p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****). Level1 Level2 Odds Ratio Lower 95% Upper 95% Prob > Chisq Sex Male Female 1.49 0.74 2.98 0.26 Required O2 on arrival Yes No 7.89 2.64 23.60 0.0002 *** Transferred to ICU Yes No 26.01 3.52 192.32 0.0014 ** Diabetes Yes No 0.88 0.39 1.96 0.75 Hypertension Yes No 0.79 0.33 1.86 0.58 Asthma and any other respiratory disorders Yes No 2.40 0.95 6.06 0.06 CVD (MI, CHF, others) Yes No 3.37 1.04 10.89 0.0425 * Renal disease Yes No 0.14 0.01 4.13 0.26 Stroke Yes No 20.52 1.29 326.53 0.0324 * Hematological disorders Yes No 0.21 0.01 5.62 0.35 Hypothyroidism Yes No 1.15 0.37 3.56 0.80 Hyperthyroidism Yes No 0.00 0.00 1.00 Chest X-ray: Pneumonia Yes No 27.90 11.63 66.96 <0.0001 **** healthcare-11-00751-t008_Table 8 Table 8 Independent factors associated with the length of hospital stay obtained using the standard least squares method. p < 0.05 (*), p < 0.01 (**), p < 0.001 (***); p < 0.0001 (****). Duration of Hospital Stay Standard Least Squares Method Estimates Term Mean Std ERROR Estimate Std Error t Ratio Prob > |t| Duration of hospital stay (days): maximum = 118, minimum = 1, mean = 9.72 +- 14.2 (SD), median = 6 Intercept 24.493488 7.154958 3.42 0.0007 *** Systemic IV steroids No 6.5915 7.1781791 -1.756811 0.755274 -2.33 0.0205 * Yes 18.7281 7.2112043 Reference Transferred to ICU No 6.4316 7.2047820 -6.417144 1.196474 -5.36 <0.0001 **** Yes 29.8871 7.3034964 Reference Required intubation No 8.3420 7.1128093 -5.656731 1.717231 -3.29 0.0011 ** Yes 42.2222 7.5955612 Reference Cardiovascular disease (MI, CHF, others) No 9.5074 7.0680036 2.1945388 1.082989 2.03 0.0434 * Yes 12.1389 7.4010742 Reference Stroke No 9.5092 6.9696932 -5.621868 2.442903 -2.30 0.0219 * Yes 25.1667 8.1083768 Reference Severity of disease Asymptomatic 5.5385 7.4129636 -3.295884 1.507726 -2.19 0.0294 * Mild 5.5577 7.2660448 -3.364786 1.06918 -3.15 0.0018 ** Moderate 11.2455 7.2969359 -1.7349 1.191134 -1.46 0.1460 Severe 32.0000 7.2273743 Reference healthcare-11-00751-t009_Table 9 Table 9 Contingency table of outcome by age groups. p < 0.01 (**). Outcome 18-29 30-39 40-49 50-59 60-69 70-79 80 and Above Total p-Value Cured and discharged 85 99 80 55 60 28 12 419 0.0013 ** 100% 100% 98.77% 98.21% 95.24% 87.50% 85.71% Death 0 0 1 1 3 4 2 11 0% 0% 1.23% 1.79% 4.76% 12.50% 14.29% Total 85 99 81 56 63 32 14 430 healthcare-11-00751-t010_Table 10 Table 10 Summary of main results and important highlights of current study in comparison to previous studies. Parameters Main Results of Current Study Results of Previous Studies New Contributions/Important Highlights of Current Study Clinical characteristics Fever Most frequent symptoms Similar results seen Statistically more commonly in older patients of age groups 60-69 years, 70-79 years, and 80 years old and more compared to younger groups. Cough Dyspnea Ageusia Other symptoms found Nonspecific symptoms Statistically higher in younger age groups than in older patients. Abdominal pain Co-morbidities Diabetes Most frequent co-morbidities Diabetes and HTN were the most common co-morbid conditions a. More 60-69 years older patients had statistically higher HTN, DM, CHF, and hypothyroidism. b. Diabetes and HTN were common in ages 70-79 years, 50-59 years, and 40-49 years. In contrast, asthma and other respiratory disorders were comparatively higher in the younger age group of 30-39 years. c. More males suffered HTN and cardiovascular disease, while obesity, hypothyroidism, and vitamin D deficiencies were comparatively higher in females. Hypertension Asthma/respiratory disorders Predictors of severity Pneumonia on chest X-ray Statistically significant differences found for pneumonia, CVD, stroke, ICU, and mechanical ventilation Older age, male sex, and presence of co-morbidities associated with severe disease at admission a. The age groups of 70-79 years and 60-69 years required intubation, which was statistically significant compared to younger patients. b. A higher percentage of males were given oxygen support on arrival and treated with systemic steroids. c. Age groups 70-79 years and 80 and above were significantly associated with in-hospital mortality. d. Nearly 55.67% of "70-79 years" and the majority of "80 and above" were suffering severe disease at the time of admission, and nearly 15% of each group succumbed to the disease. Co-morbid conditions such as CVD, stroke ICU stay Mechanical ventilation Length of hospital stay The median period of hospital stay was six days Similar results seen It was significantly longer in patients with severe disease who needed oxygen support or mechanical ventilation, as expected, and also longer in patients with CVD or stroke and administered systemic intravenous steroids. 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PMC10000837 | Healthcare (Basel) Healthcare (Basel) healthcare Healthcare 2227-9032 MDPI 10.3390/healthcare11050717 healthcare-11-00717 Article Initial Development of a Patient Reported Experience Measure for Older Adults Attending the Emergency Department: Part I--Interviews with Service Users Graham Blair 1* Smith Jason E. 2 Nelmes Pam 1 Squire Rosalyn 2 Latour Jos M. 13 Giansanti Daniele Academic Editor 1 School of Nursing and Midwifery, Faculty of Health, University of Plymouth, Plymouth PL4 8AA, UK 2 Department of Emergency Medicine, University Hospitals Plymouth NHS Trust, Plymouth PL6 8DH, UK 3 School of Nursing, Midwifery and Paramedicine, Faculty of Health Sciences, Curtin University, Perth 6102, Australia * Correspondence: [email protected] 28 2 2023 3 2023 11 5 71717 1 2023 22 2 2023 25 2 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). Older adults are a major Emergency Department (ED) user group who may be especially vulnerable to the consequences of crowding and sub-optimal care. Patient experience is a critical component of high-quality ED care and has previously been conceptualised using a framework focusing on patients' needs. This study aimed to explore the experiences of older adults attending the ED in relation to the existing needs-based framework. Semi-structured interviews were conducted during an emergency care episode with 24 participants aged over 65 years in a United Kingdom ED with an annual census ~100,000. Questions exploring patient experiences of care confirmed that meeting the communication, care, waiting, physical, and environmental needs were prominent determinants of experience for older adults. A further analytical theme emerged which did not align to the existing framework, focused on 'team attitudes and values'. This study builds on existing knowledge relating to the experience of older adults in the ED. In addition, data will also contribute to the generation of candidate items for the development of a patient reported experience measure for older adults attending the ED. emergency medicine emergency department aged patient experience communication University of PlymouthRoyal College of Emergency MedicineBG received a doctoral research fellowship grant from the Royal College of Emergency Medicine to support the development of PREM-ED 65 (October 2017). The APC was funded by the University of Plymouth. pmc1. Introduction Older adults aged over 65 years are a major user group of Emergency Departments (EDs), comprising over 29% of attendances in a recent UK retrospective cohort study . In many countries, the number of older adults attending the ED is increasing above predictions based on population size alone. Contributors may include increasing comorbidity, gaps in primary healthcare, and increasing numbers of repeated ED attendances among frail older adults . Older adults are more likely to present to the ED with high acuity conditions , yet atypical presentations and non-specific symptomatology are also more common. This clinical complexity may contribute to increased healthcare costs and ED resource utilisation . Furthermore, older adults more frequently suffer from background comorbidities, long-term conditions, and have more nursing care requirements . For all of these reasons, older adults encounter above-average ED length-of-stay (LOS) and are at increased odds of requiring hospital admission . In addition to the range of challenges posed by older adults, wider demand for emergency care is being encountered internationally . Many systems have failed to keep pace with this demand. As a result, ED crowding is now a significant public health concern, responsible for an estimated 4000 excess deaths in the UK alone during 2020-2021 . Older adults fare unfavourably when treated within a pressured emergency care system and are significantly more likely than the general population to suffer 30-day mortality following a protracted ED LOS. The reasons for this are likely to be complex, but delayed medication administration, poor continuity of care, increased risk of nosocomial infections, and circadian disruption resulting from sleep deprivation have all been postulated . The proportion of over 65s is projected to double in most nations before 2050 . Consequently, the demand on ED services from older adults is likely to reflect this trend. To meet the needs of older adults as a predominant ED user group and reduce the risks that they may encounter when accessing care, there is a pressing requirement to ensure that the ED environment and care processes are thoroughly considered and fit for purpose. Effective and meaningful measurement of the quality of emergency care is essential to enable comparison between different settings and drive improvements in clinical outcomes, patient experience, and safety. In many healthcare systems, this continues to be achieved using process-centred performance metrics. For example, recently introduced ED performance standards in England include timely ambulance handover, time-to-triage assessment, and ED LOS . Whereas some performance metrics--such as time-based targets--have been demonstrated to lead to meaningful improvements in some aspects of care , a limitation is that they may fail to effectively capture outcomes of care that matter most to patients. To this end, the International Federation for Emergency Medicine (IFEM) recommends the adoption of both Patient Reported Outcome Measures (PROMs) and Patient Reported Experience Measures (PREMs) within their recently updated framework on quality and safety in emergency medicine . Patient experience is recognised within prominent definitions of quality of care and is associated with improved clinical outcomes and patient safety . However, meaningful evaluation of patient experience can be challenging. For example, surveys are frequently undermined by poor response rate and issues with face validity and reliability . Furthermore, generic surveys may not identify specific vulnerabilities in care processes from the patients' perspective, representing a missed opportunity to effect change and improve services . Patient reported experience measures (PREMs) are psychometrically validated questionnaires that are directly reported by patients and aim to provide standardised evaluation of individual experiences of care . To ensure content validity, PREMs should be developed in conjunction with patients and care providers to capture aspects of care that are important . Several PREMs relating to ED care have been developed, although limitations of these instruments include uncertain validity, reliability, and responsiveness . No instrument has yet been developed to specifically measure the experience of older adults, aged 65 years and above, in the ED. The overall aim of our project is to develop and validate a PREM to address the unmet need of an instrument for older adults in the ED, known as the Patient Reported Experience Measure for patients attending the Emergency Department aged over 65 (PREM-ED 65). From the outset, the development process for PREM-ED 65 is designed to ensure the final instrument meets the internationally accepted COnsensus-based Standards for the selection of health Measurement INstruments (COSMIN) guidelines . PREM development is planned using a step-wise mixed methods approach. This is summarised in Table 1. In order to derive suitable candidate questionnaire items, an initial systematic review of patient experiences in the ED has been conducted. This has resulted in a conceptual framework to guide understanding of patient experience in the ED. This framework is based around the needs of ED patients and includes five analytical themes: communication needs, emotional needs, care needs, waiting needs, and physical/environmental needs . The next step of the PREM-ED 65 development process is to consider how the five analytical themes are experienced by older adults attending the ED and health professionals responsible for delivering emergency care. We conducted a two-part qualitative study aiming to explore the experiences of older adults attending the ED (Part I), and the experiences of emergency department healthcare professionals (see Part II--Focus Groups with Professional Caregivers). Part I aims to explore older adults' experiences of an ED visit in relation to the pre-established conceptual framework and to determine if any additional analytical themes emerge. 2. Materials and Methods We adopted a qualitative design using semi-structured interviews conducted with older adults aged >= 65 years during their ED visit. This study is reported following the COnsolidated criteria for REporting Qualitative research (COREQ) checklist . Ethical approval was prospectively obtained from the UK Health Research Authority (18/LO/1194) and institutional approval from the University of Plymouth (17/18-973). 2.1. Research Team and Reflexivity Interviews were conducted by a male-identifying researcher (BG) who is an academic emergency physician and research fellow with prior experience and formal training in qualitative research methods. Two female-identifying clinical academic nurses (PN, RS) assisted in the transcription and initial coding of data. A male-identifying professor in emergency medicine (JES) and male-identifying clinical nursing professor (JML) with extensive experience of quantitative and qualitative research in acute care settings were involved in data analysis. Principles of rigour and trustworthiness for qualitative research were applied . Researchers considered their own clinical experiences and the need to exclude these during the analysis and interpretation of findings. The first author maintained reflexive notes and discussed perceptions with co-researchers during the study. No relationship was established with participants prior to study commencement. Standard information was issued to all participants prior to recruitment and consent. Participants were told the research's purpose was to inform PREM development as part of the lead researcher's PhD study. 2.2. Theoretical Framework The interviews were deductive, informed by the overarching definition of quality of care proposed by Darzi . This definition encompasses three domains: patient experience, clinical effectiveness, and patient safety. Although our interviews focus on capturing the experiences of people in ED, the use of these three domains also allows for the exploration of clinical expectations and perceptions of safety in the ED, which are known to be related to experience. The three domains of the quality-of-care definition were used to formulate the interview questions in the interview guide. 2.3. Participant Selection Inclusion criteria were adults aged >= 65 years attending the ED. Patients who lacked mental capacity to give informed consent , who were too unwell to participate or required immediate life-saving treatment ('Category 1' triage category), did not speak English, or were in police/prison custody were excluded from the study. A purposive sampling strategy was used to encourage recruitment of a representative cross-section of patients attending the ED. Patients were sampled based on the presence of either traumatic injury or medical illness, age group, clinical frailty score and acuity (Table 2). A sampling matrix was used to support the inclusion of patients from each sampling category. Twenty-four patients were recruited during daytime hours (0800-1800) between September 2018 and April 2019. In addition, a single patient was recruited but then withdrawn prior to the interview occurring, due to them being transferred away from the ED. The computerised ED administration system was used to screen potentially eligible patients. Once the patient's attending clinician indicated that treatment was complete, the clinician was approached and asked to give their assent for the patient to be invited to participate. Eligible patients were then approached by the lead researcher who presented them with verbal information about the study, and a written patient information sheet. Sampling was conducted until the researchers were satisfied that sufficient data had been collected through the interview process to reach 'data saturation'. This suggests the collection of further data is unlikely to add value . Based on insights from the literature, it was estimated that between 20 and 30 participants would be needed . Patients who were approached but did not wish to participate were not recorded and not recruited into the study. 2.4. Setting Interviews were held within a single ED in the South West of England (Annual Census ~100,000/annum). As a regional major trauma centre, the ED receives patients from urban and rural settings within a wide geographical catchment (population ~1.65 million), and notably has a higher-than-average proportion of attendances from older adults. 'In situ' interviews, conducted within the ED during an acute care episode, were selected as the preferred approach to maximise ecological validity whilst minimising recall bias. A range of clinical areas within the ED were utilised for interviews and included bedspaces, ambulatory spaces, relatives' rooms, and the attached short-stay clinical decision unit. Where a patient was identified for interview but was transferred to an inpatient setting before an interview within the ED was possible, the study protocol allowed for interviews to take place on the receiving inpatient ward, provided this was clinically appropriate and within 24 h of admission. 2.5. Data Collection The interviewer used an interview guide (Table 3). Three questions were posed to all participants, each exploring one of the three domains of Darzi's original definition of quality of care . Prompts were prospectively developed from our understanding of the existing literature and were suggested as part of the question guide, although the interviewer could deviate beyond these if discussion deemed it necessary. Interviews were audio recorded using a digital voice recorder with noise cancelling technology and a dual lapel microphone to ensure clarity. Interviews were transcribed verbatim, and a proportion of transcripts were cross-checked to ensure accuracy. Additional field notes were taken to capture appropriate non-verbal, paralinguistic communication, where appropriate. 2.6. Data Analysis Transcripts were uploaded into NVivo Version 12 (QSR International, Massachusetts, 2012), a software programme used for qualitative analysis. Framework analysis following a mixed inductive-deductive approach was adopted, following the seven steps described by Gale et al. . The first two steps of this approach are transcription and familiarisation. For this, two members of the research team (BG, RS) took responsibility for transcription, and worked collaboratively to cross-check each other's work. This ensured both accuracy of transcription and familiarity with the interview content. The third step is coding. Interview transcripts were selected and open coded using an inductive approach. A modified approach to step four--developing an analytical framework--was adopted. Rather than develop a new framework, we adopted our pre-existing needs-based framework . As such, for step five--applying the analytical framework--we switched to a deductive approach to index codes under the exisitng analytical themes based on 'best fit'. For the final two steps--charting and interpreting data--all researchers met to review data, summarise findings and identify illustrative quotations. Where data was deemed not compatible with existing themes, these were discussed between the researchers and either a pre-existing theme was agreed, or a new theme was formulated and agreed. 2.7. Presentation of Findings Presentation of findings includes description of the study participants; coding and emerging themes, including the frequency (prevalence) of statements aligned to each theme; and detailed discussion of findings by analytical theme, supported by illustrative quotations. 3. Findings 3.1. Description of the Study Participants Twenty-four participants were recruited and completed the interviews. Mean age was 74 years (range 65-91 years). A larger proportion of participants were female (62.5% versus 37.5% male). Almost all patients declared at least one long-term condition or co-morbidity (95.8%). Patients had a range of education levels ranging from no formal qualifications to professional qualifications. Participants were recruited from across the acuity spectrum and included a majority of 'very urgent' (Category 2) and 'urgent' (Category 3) presentations (35% and 54%, respectively). Two-thirds of patients (66.6%) presented with a non-traumatic medical complaint. Most patients had lower or moderate levels of frailty as assessed by the clinical frailty score (mean CFS = 2.6 out of 9, range 1-6). All but a single patient lived in his or her own accommodation (Table 4). An overview of individual participant characteristics can be found in Electronic Supplementary Material (Table S1). 3.2. Coding and Emerging Themes Framework analysis of transcripts was conducted, and statements assigned to an existing analytical theme where appropriate. Five hundred statements were identified which were directly aligned to experience. Of these, 452 statements were organised under one of the five existing analytical themes within the established conceptual framework. These were most prevalent around waiting needs (146 statements), followed by statements related to 'communication needs' (125 statements). 'Emotional' and 'physical/environmental' needs were evenly distributed (67 and 66 statements, respectively). Statements related to 'care needs' were slightly less prevalent (48 statements). During the framework analysis an additional analytical theme emerged, relating to Attitudes and Values of the Team. This was initially identified within the interviews data, to accommodate 59 unique statements relating to patients' perceptions of the ED team members, teamwork, and professionalism. The data identified several new sub-themes were also identified. These are presented in Table 5 and include Social Communication (under communication needs), Reassurance (under emotional needs), and Waiting Experiences (under waiting needs). Selected statements within this report are presented with reference to the study participant number (Pn). 3.3. Presentation of Findings 3.3.1. Communication Needs The theme of Communication Needs encompasses statements that relate to patient-provider communication. These are divided into inter-personal communication, which consists of components of experience typically featured within a healthcare consultation, informational communication, which consists of the giving and receiving of information (for example, discharge instructions), and the new subtheme social communication, which consists of components of communication such as conversation not formally considered within consultation frameworks or models. Interpersonal Communication The interviews confirmed that patients placed immense value on inter-personal communication with care providers and the wider ED team. Patients preferred for staff to display a communication style that was calm, unrushed, and polite: Well.....they spoke to you in a good way.... they sounded as if they were interested and they weren't rushing... they seemed genuinely polite (P13) Patients were able to detect when staff communicated with them in a way that encouraged them to feel valued as individuals, and this positively affected their perceptions of care: They made you feel that you were an individual kind of thing. They made you feel....you know, that you were on a level with any any dignitary paying a lot of money (P18) Most patients confirmed that staff introduced themselves by name and role, and this was viewed as beneficial and could help demystify roles, especially in instances where the patient was unfamiliar with the ED environment or had not attended before. However, a succession of staff introductions could be confusing where patients were presented with multiple new members of staff in a short space of time, as was reported by one patient: I find it very confusing I mean there were three or four nurses that came in and told me their names and then the consultant came in and said good morning my name is blur blur and before he started on me he disappeared and I haven't seen him again! (P4) Similarly, patients also wanted to know the role of staff members, particularly when they were administering a task. Patients understood that staff had a job to do including tasks that would potentially be unpleasant or uncomfortable. A friendly attitude meant that patients readily accepted such experiences, which might otherwise be aversive. Patients were sometimes cognisant of conversations happening around them in the clinical environment. In such instances, they wanted to be active participants in discussions relating to their care rather than passive listeners. Being talked about without the opportunity to contribute to discussions during a clinical handover was perceived as undermining by one participant: I'm not stupid... they're talking about bed 'G4'...I'm G4! I'm not stupid, it's not rocket science. Weird. Just weird... I'd like to be in on the conversation, rather than just hearing in the distance. (P17) Some of the older participants reported that communication with medical staff had evolved compared to their historical experiences. Demonstrating politeness and offering explanations as part of the ED consultation positively affected patients' experiences: cos no matter what you ask them, they're there they speak to you politely and they don't dismiss you. The doctors explain everything to you which they never did years ago. So you know exactly what's going on and you know exactly what's wrong with you. (P24) In the olden days if you came into hospital everything was kept secret (laughs) if you know what I mean. But today it's quite relevant to let people know what's going to happen and what's going on. (P05) Sensory problems and impairments are frequent amongst the older adult population. Recognition of a hearing impairment and adaptation of communication strategy was essential in facilitating a positive experience for one participant but prevented communication of a test result for another. Regarding providing information, patients valued understandable and appropriately detailed explanations. Repetition of questioning was also noted as a common feature of the ED consult, which could adversely affect experience. When explanations were provided which were too advanced for a patient to fully understand, particularly where this occurred without checking understanding, this could result in some frustration as expressed by one participant: Information needs to be broken down to suit your average patient. You know, we're not all nurses or doctors. And I've had this for years, people have come along and said this and that and I've not understood it. It doesn't work all the time for me. (P20) Although, reflecting the local population, participants were predominantly White British, the importance of cultural and language recognition was noted by one patient who had a Welsh background: I got on with one particular nurse cos she was Welsh ... she spoke in welsh (P14) Informational Communication In addition to valuing interpersonal aspects of communication, patients had a great desire to be kept informed of their situation and the progress of their ED journey. Although verbal informational communication was considered most frequently by participants, other methods of delivery including written information and multi-media delivery--for example, using 'on screen' presentations in the waiting area was also desirable. Positive experiences of informational communication also included the progress of tests and investigations, and communication of 'next steps' during physical procedures: The doctor came in this morning and told me about my blood test ... I've got to go down for an X-ray and I've got to go for scans. (P05) Social Communication Although not routinely considered as a part of consultation or communication skills frameworks, social communication was mentioned by some patients as important to their experience and is therefore considered within this synthesis. Patients expected staff to be friendly towards them and use positive body language such as smiling: It's nice to have a smiling face and just to be sociable and polite and I hope I'm the same to them. (P07) The use of humour by staff was seen as very welcome by some patients. Despite this, one patient who was previously a senior nurse remarked that the use of humour should adhere to professional boundaries and that using colloquial terms of endearment, rather than addressing patients by their name, was inappropriate: Not being too familiar, you know ... [I don't like] being called 'babe'. (P02) 3.3.2. Emotional Needs In the original framework, the theme Emotional Needs was divided into sub-themes encompassing coping with uncertainty, recognition of suffering and empowerment. Following framework analysis, an additional sub-theme of reassurance was added. Coping with Uncertainty Perceptions of uncertainty were mentioned by several study participants and could be a fundamental emotion associated with attending the ED, particularly if not familiar with the setting previously. Uncertainty also elicited feelings of vulnerability when patients were unsure of outstanding investigation or treatment plans: "I was told 'do this and take your dressing gown off and put a nightdress on and we will do an ECG' or something like that, and I thought what's an ECG, you know?... it didn't worry me, but I was concerned what they were going to do!" (P06) Conversely, it was recognised that perceptions of uncertainty could be effectively managed by simple interventions such as keeping patients informed of the next stages of care. Even where patients were accepting of uncertainty, they still desired basic information, such as whether they would be staying overnight: I did ask someone if I was stopping overnight, cos nobody had said, perhaps they didn't know but you know, sometimes it's helpful. (P13) Recognition of Suffering Recognition of suffering was addressed by several patients. This extends beyond pain, to include recognition and attention to other forms of suffering and distress. One patient, who felt her suffering was not recognised in ED, was able to give an example of an experience in oncology: ... with Oncology, immediately when you went in there was a member of staff with you. Whether it was a ward assistant of whether it was a nurse, somebody was with you, talking through your problems, how you felt and so you felt hum, you felt loved and comforted whereas I don't [in ED] (P11) Empowerment Empowerment is defined by the European Patient Forum as "any process that helps people gain control over their own lives, and increases their capacity to act on issues that they themselves define as important" . Staff provided empowerment to patients by making them feel like individuals, legitimising their reasons for attendance: Yes I feel like my concerns have been taken seriously, yes I do. (P12) Although some older adults were appreciative of having appropriate say in their care decisions, others recognised the potential importance of following clinical advice, for example, surrounding a decision to admit to hospital: It's no good talking to medical staff and completely ignoring what they have to tell you, and if they advise that I should be over night, because they want to find out why I've gone down twice ... it's an obvious answer isn't it. (P19) On occasion the environment of the ED could be physically disempowering. One patient reflected humorously on her experience of being attached to monitoring equipment, likening this to 'being chained up like Houdini' (INT_14). Some patients expressed fear of judgement from staff, which could be disempowering and affect their ability to make decisions: You can see them thinking 'how the fuck did he manage to do that?!' (laughs) ... do you know what I mean? And you feel as though you're being judged as a village idiot. (P17) Provision of Reassurance During analysis, several statements relating to the provision of reassurance were encountered. Many patients viewed the provision of reassurance as a key positive determinant of their experience, and a sense of reassurance was often conveyed through good patient-provider communication: [The staff are] quite happy, they introduce themselves, they sit down ... they talk to you as a human being. That they reassure you. That's quite nice. (P04) Reassurance could also be provided as an active process; for example, by the positive actions of staff, showing thoroughness and diligence, or in one case, giving passive reassurance that material property was safe: 3.3.3. Care Needs The third analytical theme, Care Needs is sub-divided into symptom relief and procedural care. Symptom Relief With regards to symptom relief, pain management was central to achieving a good experience. Patients made clear that they expected pain assessment and the provision of analgesia early on in their ED stay as a priority: Keeping the pain at bay, really, is the big thing (P16) Staff met patients care needs by maintaining comfort during potentially painful procedures, for example IV cannulation. Explanation of procedures was reported to be extremely helpful, including effects on physical dignity: I had to take my bra off you see, and when [the nurse] was putting the pads on I said I'm sorry.... He said 'don't worry about that--I won't be worried about that!' (chuckle) He was very kind about that ... I think when you get a bit older you get a bit embarrassed. (P03) Procedural Care Procedural care is defined as care delivered during medical or nursing procedures. Competence was valued highly in relation to this sub-theme. Patients were perceptive of when they were being seen by a junior member of staff and whilst they were happy to be attended by trainees, desired to have the attention of more senior clinicians. One patient, who attended following a therapeutic excess of paracetamol was a critical observer of the doctor who made an antidote drug calculation. Even so, the friendly nature of the encounter mitigated any negative effect on overall experience: Well I think he was struggling to calculate whether the amount of paracetamol taken was too much, I think he really struggled with the calculation (Interviewer: Did That affect your experience?) No, not really because he was so nice. He came to me later on and apologised and said ' I'm so sorry for what you've gone through today' and that was so nice... yeah, so that was OK. (P22) 3.3.4. Waiting Needs The theme Waiting Needs was subcategorised into the comfort (associated with waiting), impact of crowding and a new subtheme, waiting experience: Comfort Whilst Waiting Waiting could be uncomfortable and witnessing other patients' suffering distressing. However, these negative aspects of the experience could be mitigated through accepting shared experiences, resulting in camaraderie amongst patients: "I think there was a bit of ... you know ... the patients forming a group, and the doctors and nursing staff forming a group. We were all in the same boat." (P22) There was an awareness of the breadth of acuity presenting to the ED ("....you're dealing with the serious to ridiculous, aren't you?! (P11), that patients underwent triage, and that having a lower triage category could necessitate a longer wait. However, patients wanted to have accurate information about waiting times, which were not always provided: "... they were giving answers they thought we wanted to hear, for example, ' somebody is coming'... well... somebody wasn't coming, and that was an issue! Somebody was coming 2 h later!(laughs) ... I think I'd rather be told the real state of things." (P11) Impact of Crowding The waiting room could be cramped, and being near other unwell patients was intimidating and upsetting for some. Patients frequently reported physical discomfort whilst waiting, which was due to the metal chairs and gurneys in the department: The seats... oh the seats were dreadful! Someone was lying on the floor in preference to sitting on the chairs because they were in such a lot of pain! (P22) Providing basic comfort measures was a positive determinant of experience; even providing blankets improved the experience for some patients. Conversely, ambient temperature was problematic for some patients, who reported thirst and headaches because of heat and inability to access refreshments: I was cold...and I had a blanket brought to me straight away. I thought it was lovely. (P04) It would have been nice to have water. It's warm in here. I've got a bit of a headache now and I think it's just the heat. (P21) Being cared for in corridor spaces could provoke significant anxiety for patients, who perceived this experience as unsafe and undignified: Just the thought of having to wait in the corridor ... just waiting there. No, I didn't like that. Because everybody's walking by you and they're looking at you as if to say 'what's wrong with her'?! (P04) Waiting Experience Study participants recognised the necessity of waiting for their care to be initiated and were understanding and accommodating of the need to wait. Even so, negative consequences of waiting were reported and extended beyond boredom and frustration. On occasion, participants found the waiting experience to be intimidating, particularly when in close confines with other patients and those who were acutely unwell. Patients expected waiting to feature as a part of their ED experience, and were tolerant of the need to wait, even where this was prolonged: Waiting is part of life's rich tapestry isn't it? .... You put up with it. (P20) 3.3.5. Physical and Environmental Needs The theme Physical and Environmental Needs describes how the physical environment of the ED influences patient experience. This includes the provision of fundamental needs such as refreshments. Interaction with the environment formed an important determinant experience for many patients and included the subtheme comfort (associated with physical needs). The presence of clear signage to the department and reception was noted to be absent, which was a negative determinant of experience. Cleanliness and hygiene of the ED environment was a crucial factor in the experience of patients: [The environment] has got to be clean, otherwise you get all of the bugs, don't you? (P03) There were several aspects of the ED physical environment which could be negative determinants of experience. For example, one patient remarked that the environment of the waiting area--including the presence of others who were intoxicated, violent or agitated--could be unsettling and frightening: No. Just that ... people screaming ... you hear it in here ... men, screaming. Alcoholics who want a drink. And that's upsetting when you're trying to go to sleep. And you don't feel safe then because you think are they going to come around here, you know. (P04) Background noise could be problematic, particularly for patients with pre-existing hearing difficulties. Monitor alarms are a constant presence in some parts of the ED, and study participants experienced this as noise pollution. ".... all the buzzers and beeps... it is like having a train at the bottom of your garden." (P23) Comfort (Associated with Physical Needs) In terms of basic needs provision, patients appreciated the provision of refreshments and noticed when these were not offered: "When we came in [to ED] we were offered tea, and it went so much more quickly" (P01) 3.3.6. Attitudes and Values of the Team As many statements related to perceptions of teamwork and staff attitudes and professionalism, a new analytical theme, labelled 'Attitudes and Values of the Team', and was added to the existing framework. The two subthemes associated with this theme are perceptions of teamwork and staff behaviours. Perceptions of Teamwork Patients were active observers of team-based processes, and took reassurance from witnessing effective communication between different team members: Yeah ... when you're on a trolley you tend to watch. And what I noticed was how they were talking and passing information. And I thought that was brilliant. They knew exactly where to go, you could see it. (P04) Staff Attitudes and Professionalism Patients expected professionalism from the wider team, and had a sense of the staff members working together for them as an individual patient: I felt that everyone was part of a team that had one aim in focus which was to look after the patient which was me. (P17) Values that patients perceived staff exhibited during care episodes that contributed to a positive experience included kindness, politeness and an approachability were reported: "Everybody's kindness and professionalism stood out today. (Interviewer: And what is it that gives you that impression of kindness?) ... well, it's staff being attentive and ... the fact that I've asked questions and the staff have always answered politely without being harassed. I really feel like I can approach them." (P09) However, some patients did find the number of team members and different roles confusing: There is so many people doing so many different jobs, each with their own coloured uniform, and you just wonder what they were doing. (P22) Regarding continuity of care, some patients were aware of team shift changeovers and felt a burden when establishing a rapport with a new team. Shift changeovers during a phase of care could increase their sense of patients' vulnerability: "...knowing that when staff finish their shift ... that they are going...that they are passing you over, you lose that continuity. I know they all work as a team... but [as a patient] you may have to re-establish something emotionally [with the new team]" (P09) 4. Discussion This study aimed to understand the experiences of older adults in the ED. Framework analysis using a combined inductive-deductive approach confirms the conceptual validity of a 'needs based' framework amongst older adults in a UK ED setting. Statements were notably prevalent around waiting needs and 'communication needs'. There was also sufficient quality and quantity of statements to confirm the presence of the three remaining analytical themes. It should be noted that reporting of prevalence of themes within qualitative research is controversial and that the number of statements related to a theme is not necessarily proportional to its significance . However, in our case, presenting frequencies of statements related to the themes provides important assurance that data has been explored in its entirety. Accordingly, this ensures data used to inform item generation for PREM-ED 65 accurately reflects the full breadth of patient experiences reported in the interviews . In addition to the existing five analytical themes, a new descriptive theme emerged, describing the role of staff professionalism and teamwork in contributing to the patient experience. This is supported by the previous literature suggesting that patients are direct observers of team-based processes and that observation of constructive teamwork is a positive determinant of patient experience in the ED. Patients in our study have provided narratives that will contribute to the further development of PREM-ED 65. This study builds on the body of the existing literature emphasising patients' desire to have their basic human needs and comfort addressed during emergency care episodes . If aiming to optimise patient experience, the provision of humanistic, holistic care should be considered an important caring aspect of the ED alongside the clinical objectives. Specifically, in our study, participants also expected their dignity and privacy to be respected wherever possible. Including these factors in a PREM is desirable from the evidence of our patient interviews and findings. Facilitators of a positive patient experience that were identified included personable communication, the provision of timely information relating to the progress of clinical assessment/onward disposition, and measures to promote both physical and emotional comfort. Although it is useful to conceptualise these facilitators as discrete elements of patient experience--for example, when identifying a focus for service improvement--they may not be mutually exclusive and can overlap. For example, when looking at facilitators related to the analytical theme of social communication, providers' use of humour may extend beyond a purely social function to promoting the development of trustful patient-provider relationships . Hence, in this case, social communication may have a role in meeting both communication and emotional needs. The potential for themes to interact and overlap should be considered when applying the original needs-based framework to the real-world setting. Although the focus of participants' discussion focused on their perceptions of relational aspects of care, as opposed to technical care elements, the need for prompt pain relief and symptom control was a common topic within our study participants. This is also recognised in the literature, for example in a prospective observational study of pain management in the ED by Van Zanden and colleagues where 43.7% of patients arriving in the ED desired pain relief, and the provision of pain relief was associated with higher satisfaction. Pain was also highlighted in a recent qualitative study exploring patients' experiences in an Australian ED . Many patients in this study described pain as a memorable aspect of their ED visit. In contrast to the positive experience, patients who do not receive timely pain medication had negative ED experiences, as confirmed elsewhere in the literature. Our interviews suggest that both the length of wait and care delivered whilst waiting for medical assessment and treatment form an important determinant of older adults' experience in the ED. Indeed, waiting is ubiquitously associated with accessing emergency care, and the literature suggests that patients often expect a long ED waiting time . In the UK setting, older patients wait longer, and have a prolonged stay in the ED compared to younger patients . However, there may be some international variation in wait times experienced by older adults . As such, it is important that a PREM aimed at older adults examines the experience of waiting, which may give locally relevant insights into where improvement would be beneficial. Strengths and Limitations This study represents a unique attempt to interview patients 'in situ', within the ED, during their stay. Ecological validity is a concept originally described in the social sciences following recognition that experimental conditions must mimic the 'real world' to promote external validity . Importantly, ecological validity may also be impaired if interviews and surveys are conducted away from the setting of interest . To this end, in situ interviews maximise ecological validity whilst also minimising recall bias, as patients are reporting experiences from 'in the moment', as they are lived. The effect of recall bias may be especially significant where the time spent in the ED itself is short; in this situation, self-reported perceptions of a care episode are likely to be affected by subsequent admission to other hospital departments. The available literature also suggests that recall bias may be more pronounced in older adults, who have been noted to recall events more positively in hindsight . Conversely, potential limitations of the 'in situ' approach may include concerns about privacy, confidentiality, and the effect of disclosing information on care. To mitigate against these potential effects, the informed consent procedure included explicitly informing participants that information would not be shared with caregivers. Furthermore, interviews were conducted in private settings within the ED, wherever possible. Another limitation of our study is that it did not include older patients meeting the Rockwood criteria of 'very frail'. However, a recent qualitative study was conducted across three EDs in the UK and specifically recruiting older adults with frailty (Rockwood CFS > 5). The findings of this study derived some similar themes, including information and communication in the ED, time waiting in the ED, and environment/personal comfort . The general experience of the researchers towards the 'in situ' approach to interviewing patients in the ED is a positive one. However, those utilising this approach in the future may consider pre-arranging a private space, away from the immediate clinical area, in which to conduct the actual interviews. This may optimise interviewer-interviewee communication and enhance comfort for both parties. Strategies to promote representativeness of the sample, including recruitment of 'hard to reach' groups, should also be considered. In our experience, recruitment of very frail older adults was difficult. This is also reported in the literature, which suggests recruitment of this group may be improved by considering when to approach very frail participants (e.g., following a period of rest), building personal rapport when explaining the project, giving them more time to consider participation, and--with their agreement--discussing with, or indeed involving, relatives and trusted friends . Although the time required to implement these strategies may seem at odds with 'in situ' interviews conducted during an ED stay, many older adults currently experience protracted waits for admission, increasing the potential relevance and feasibility in the current context . Finally, it should be noted that patient interviews were conducted prior to, and were therefore not influenced by, the COVID-19 pandemic. In summary, these findings build on our previous conceptual framework. This confirms face validity amongst an older adult population attending the ED. In qualitative research, triangulation refers to the use of multiple methods to develop a more comprehensive understanding of phenomena . Data from this study will be triangulated with both the existing literature and an accompanying focus groups study with healthcare professionals. (Part II) This will yield a comprehensive list of candidate items for inclusion in PREM-ED 65+. Subsequently, shortlisting and prioritisation of candidate items for inclusion in the final instrument is planned using a nominal groups technique. This will involve a range of stakeholders, including older adults and their carers . The fourth and final step of development will then consist of psychometric field testing of the draft instrument. The anticipated result will be an instrument which meaningfully and usefully measures patient experience for older adults attending the ED. 5. Conclusions Older adults are a significant and growing ED user group, both within the UK and internationally. Understanding their experiences is essential to ensuring the design and provision of ED services to meet their specific needs. This study utilised 'insitu' interviews carefully conducted immediately following emergency care to gain real time insights into patient experiences and needs. Findings from this study confirm that older adults' experiences of ED care can be categorised using a pre-existing 'needs-based' conceptual framework, although several new subthemes and an analytic theme emerged, which were not previously identified within a systematic review. Aside from providing discrete insight into the lived experience of older adults attending an ED, data from this study will inform a comprehensive list of items for inclusion, in a patient reported experience measure, named PREM-ED 65+. Acknowledgments The authors thank the study participants for their time and valuable information shared during the interviews. Supplementary Materials The following supporting information can be downloaded at: Table S1: Table of Individual Study Participant Characteristics. Click here for additional data file. Author Contributions Conceptualisation, B.G., J.E.S. and J.M.L.; methodology, B.G., J.E.S. and J.M.L.; software, B.G.; validation, B.G., P.N. and R.S.; formal analysis, B.G., P.N. and R.S.; investigation, J.E.S. and J.M.L.; resources, B.G.; data curation, B.G.; writing--original draft preparation, B.G. and J.M.L.; writing--review and editing, J.E.S., P.N. and R.S.; supervision, J.E.S. and J.M.L.; project administration, B.G.; funding acquisition, B.G. and J.E.S. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement This study received approval from the UK Health Research Authority (18/LO/1194) and institutional approval from the University of Plymouth (17/18-973). Informed Consent Statement Informed consent was obtained from all subjects involved in the study. Data Availability Statement The data presented in this study are available on request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. healthcare-11-00717-t001_Table 1 Table 1 Stepwise mixed methods approach to PREM ED 65+ development (note the current study is within step 2). Step 1: Conceptualising Patient Experience in the ED Systematic Review * Qualitative meta-synthesis * Derivation of conceptual framework of patient experience of ED care Step 2: Understanding experiences specific to older adults in the ED Qualitative study * Part I: Interviews with patients aged over 65 years * Part II: Focus groups with ED staff (professional caregivers) Step 3: Generation and Prioritisation of Candidate Items Consensus Setting (Nominal Groups Technique) * Generation of initial candidate items from existing data (Steps 1 and 2) * Generation and prioritisation of candidate items Step 4: Psychometric Field Testing Administration of draft PREM to patients * Confirmation of structural validity and reliability healthcare-11-00717-t002_Table 2 Table 2 Purposive Sampling Categories. Gender Male Female Age 65-84 years (old age) 85+ years (very old age) Presentation Type Primary medical complaint Primary traumatic injury Acuity Australasian Triage Category * Triage Category 1-3 (Higher Acuity) * Triage Category 4-5 (Lower Acuity) Frailty Clinical Frailty Scale (CFS) * CFS 1-3 (Lower Frailty) * CFS 4-6 (Moderate Frailty) * CFS 7-9 (Severe Frailty) healthcare-11-00717-t003_Table 3 Table 3 Interview Guide. 1. What do you feel has affected your experience of visiting the A&E Department today? a. likes/dislikes, areas for improvement, communication, emotional needs, technical competence of staff, waiting experience? 2. What did you expect from your A&E visit today? a. Understanding, Reassurance, Medication, Other symptomatic relief, onward care/referral to services? 3. How safe have you felt during your time in A&E today? a. Feelings of security and vulnerability, experience of mistakes/mishaps, medication safety, ability to speak up? healthcare-11-00717-t004_Table 4 Table 4 Summary of participant characteristics. N (%) Gender Female 15 (62.5) Male 9 (37.5) Age 65-74 years 12 (50) 75-84 years 10 (41.7) 84 years and above 2 (8.3) Highest level of Education Primary 10 (41.6) Secondary/Vocational 7 (29.2) Post-secondary (e.g., degree) 5 (20.8) Acuity (Australian Triage Scale) 2-3 (Very Urgent/Urgent) 9 (62.5) 4 (Lower Acuity) 15 (37.5) Presentation Type Medical Illness 16 (66.6) Traumatic Injury 8 (44.4) Frailty (Clinical Frailty Scale) 1-3 (Lower Frailty) 15 (62.5) 4-6 (Moderate Frailty) 9 (37.5) 7-9 (Higher Frailty) 0 healthcare-11-00717-t005_Table 5 Table 5 Analytical Themes and Sub-Themes. Analytical Theme Existing Sub-Theme New Sub-Theme Communication Needs Interpersonal Communication Informational Communication Social Communication Emotional Needs Acknowledging Uncertainty Recognising Suffering Providing Empowerment Reassurance Care Needs Symptom Relief Procedural Care Waiting Needs Impact of Crowding Comfort 1 (associated with waiting) Waiting experience Physical/Environmental Needs Comfort 1 (associated with physical needs) Attitudes and Values of the Team (new) - Perceptions of teamwork Staff attitudes and professionalism 1 For the purposes of the conceptual model, 'comfort' is considered a single concept; however, comfort associated with waiting and comfort associated with physical needs are considered under the respective analytical theme. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). 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PMC10000839 | During radiation therapy (RT) of glioblastoma, daily MRI with combination MRI-linear accelerator (MRI-Linac) systems has demonstrated significant anatomic changes, including evolving post-surgical cavity shrinkage. Cognitive function RT for brain tumors is correlated with radiation doses to healthy brain structures, especially the hippocampi. Therefore, this study investigates whether adaptive planning to the shrinking target could reduce normal brain RT dose with the goal of improving post-RT function. We evaluated 10 glioblastoma patients previously treated on a 0.35T MRI-Linac with a prescription of 60 Gy delivered in 30 fractions over six weeks without adaptation ("static plan") with concurrent temozolomide chemotherapy. Six weekly plans were created per patient. Reductions in the radiation dose to uninvolved hippocampi (maximum and mean) and brain (mean) were observed for weekly adaptive plans. The dose (Gy) to the hippocampi for static vs. weekly adaptive plans were, respectively: max 21 +- 13.7 vs. 15.2 +- 8.2 (p = 0.003) and mean 12.5 +- 6.7 vs. 8.4 +- 4.0 (p = 0.036). The mean brain dose was 20.6 +- 6.0 for static planning vs. 18.7 +- 6.8 for weekly adaptive planning (p = 0.005). Weekly adaptive re-planning has the potential to spare the brain and hippocampi from high-dose radiation, possibly reducing the neurocognitive side effects of RT for eligible patients. radiotherapy glioblastoma resection cavity hippocampi cognitive function dose reduction National Institutes of Health (NIH)R37CA262510 National Cancer Institute (NCI)K12CA226330 University of Miami Sylvester Comprehensive Cancer CenterResearch reported in this publication was supported by the National Institutes of Health (NIH)/National Cancer Institute (NCI) under award numbers R37CA262510 and K12CA226330. Additional support was provided by the University of Miami Sylvester Comprehensive Cancer Center. pmc1. Introduction Glioblastoma is the most common primary brain cancer worldwide and is frequently fatal. The standard of care for glioblastoma is maximal safe surgical resection followed by daily radiation and chemotherapy for 6 weeks, and then continued chemotherapy with temozolomide for 6 months . Brain tumors and their treatment (surgery, chemotherapy, and radiation) can affect the patient in multiple ways. For example, cognitive decline can be a delayed side effect of radiation therapy, which can include attention problems, memory, and the speed of processing information. Approximately 30% to 50% of brain tumor patients present with cognitive decline symptoms 6 months after radiation therapy (RT) . Studies suggest that the irradiation of the hippocampus and healthy brain tissue is correlated to post-treatment cognitive dysfunction . Hybrid MRI-linear accelerator (MRI-Linac) systems have gained in popularity since their initial 510(k) marketing authorization in 2012 . Such systems offer improved soft tissue delineation over cone beam computed tomography (CBCT)-guided RT . A previous study has shown the possibility of the daily tracking of glioblastoma resection cavity (RC) volumes during RT . RC volumes play a significant role in the treatment planning process, as RT guidelines for glioblastoma include the treatment of the RC plus additional margins . Changes to the RC volume have consequences for the RT dose distribution in structures surrounding the RC. The standard of care for glioblastoma RT is based on one plan after the surgical resection of the tumor, and is usually generated about a week or more prior to the RT start date. However, the daily MRI-Linac treatments have permitted the visualization of significant anatomical changes during RT, including evolving RC shrinkage . Here, we demonstrate that RC shrinkage can be adapted on the MRI-Linac platform with savings in seemingly normal brain structure doses. 2. Materials and Methods 2.1. Daily Set up Images Daily set-up images were acquired before treatment delivery on a 0.35-T MRI-Linac (ViewRay MRIdian, Cleveland, OH, USA). Patients were immobilized in a custom thermoplastic mask, and they were imaged with a vendor-supplied head and neck anterior flexible coil and torso posterior flexible coil wrapped around the thermoplastic mask and baseplate (total 11 channels). Images were acquired using a balanced steady-state free procession pulse sequence (bSSFP), characterized by its short acquisition time and high signal-to-noise ratio. Images acquired with this pulse sequence combine T1/T2 contrast behavior ; however, at the high flip angles used here the images are predominantly T2-weighted as we have previously demonstrated . The set-up MRI scans used for this study were acquired with 1.5 x 1.5 x 1.5 mm voxel dimension, TR/TE = 3.35/1.45 ms, flip angle = 60 degrees and bandwidth = 536 Hz/pixel. The field-of-view for the set-up scans was chosen to fit the entire head and the acquisition time was 172 s. 2.2. Patient Data Base and Analysis Ten patients were selected from a prospective non-interventional study of 0.35-T MRI-Linac glioblastoma RT. Thirty-six patients were evaluated for possible inclusion in this study. Of the 36 patients analyzed, 26 had a gross total or near-total resection defined as resection cavity only on MRI (no enhancing tumor lesion) or resection cavity with small tumor lesion and/or a postoperative change of less than 20 mL. Near total resection or gross total resection was defined by an attending radiation oncologist after evaluating the surgery note from the neurosurgeon, the radiology notes from the radiologist, and the post-surgery imaging. Of those 26 patients, 13 had cavity shrinkage during RT, defined as any volume decrease with a volume reduction range from the planning of the MRI RC to the beginning of Week 5 of treatment MRI RC of: -0.88 mL to -12.47 mL. Of those 13 patients, 3 were excluded from analysis due to the location of the tumor involving both hippocampi. Daily T2-weighted TRUFI bSSFP treatment set-up MRI scans from the MRI-Linac were transferred to radiation oncology software (MIM Software, Cleveland, OH, USA) where RC, planning tumor volume (2 cm clinical target volume expansion within brain followed by 3 mm expansion of RC), brain, and organs at risk (OARs) were contoured on the planning scan and last day of every treatment week (Fractions 5, 10, 15, 20, 25). These contours were reviewed by a senior radiation oncologist. Figure 1 shows the structures that were contoured for most patients. Constraints for brainstem, optic structures, eyes, and lenses are typically included on modern cooperative group protocols for brain radiotherapy, as excess dose can cause vision loss, cranial neuropathies, or paralysis . We also include hippocampi, as emerging data suggest that hippocampal doses are correlated to neurocognitive function after radiotherapy , and lacrimal glands, as excess doses can cause permanent xeropthalmia . Fractionated RT plans were created for every patient, with a prescription of 60 Gy to the planning target volume (PTV). The PTV is the sum of the clinical target volume (CTV), a 2 cm expansion from the resection cavity within the normal brain confined by anatomic barriers such as dura, plus 3 mm for daily setup error. Treatment was delivered over a period of six weeks (5 days per week, 2 Gy per day). These plans that were not adapted are denoted as static plans. Diagnostic and MRI-Linac MRIs and computed tomography (CT) scans taken pre-RT were used to plan the individual static plans for the MRI-Linac planning system . The dose distribution to organs at risk and the brain were recorded. For the adaptive plans, MRI-Linac set-up scans from fractions 5, 10, 15, 20, and 25 (the last days of each week of treatment) were used to calculate treatment plans for the following five fractions of treatment. Contours were created for each fraction to account for changes in RCs and shifts in OARs. For consistency, constraints on each weekly plan were kept constant (including the threshold and importance values). Each plan was normalized to 95% coverage at prescription dose to the PTV. The weekly plans were divided in dosage, prescribing 10 Gy per week for six weeks, summing a total of 60 Gy . The adaptive plans were then transferred to MIM software. After fusing all plans to the pre-RT scan as a reference, a summation of dose was performed. The accumulated dose was then recorded for OARs and healthy brain. Dose values to the hippocampus recorded from non-adaptive plans and adaptive plans were compared using a paired t-test, and the statistical analysis was conducted in Minitab. We considered p values < 0.05 to be statistically significant. 3. Results The mean and max doses for the static and adaptive plans for the uninvolved hippocampi and brain (Table 1) and a summary of statistical values (Table 2) are shown. For every patient, there is a dose reduction to the hippocampus and brain tissue, with dose to healthy tissue trending downwards and isodose lines freeing the hippocampi as the weeks progressed (Table 1). Only in patients 3 and 5 did the dose to OARs not change significantly during treatment due to cavity position superior in the brain, far from the hippocampi. The difference between the hippocampus mean (p = 0.003) and max (p = 0.036) and brain mean (p = 0.005) for the static and adaptive plans all reached statistical significance . From the 10 patients evaluated, 9 also showed dose reductions to optic structures (mean static plan 96.7 Gy, mean adaptive plan 84.5 Gy, p = 0.044) and brainstem (mean static plan 18.21, mean adaptive plan 14.5, p = 0.041). 4. Discussion MRI-guided RT can reduce doses to healthy tissue by adapting treatment plans to changes during RT . Brain tumor resection cavities are clearly visible on hybrid MRI-RT systems without exogenous contrast, and the cavities of a significant percentage of patients with gross or near total resection (36% of patients analyzed) shrink during treatment. This study demonstrates that weekly adaptive re-planning in glioblastoma can lower the dose to healthy brain tissue by adapting to shrinking resection cavities. Adaptive RT might improve glioblastoma RT toxicity as hippocampal RT doses have been correlated with worsened neurocognitive outcomes, while sparing the hippocampi from irradiation has been correlated with improved neurocognitive outcomes . Studies have shown that increasing the hippocampal dose correlates with declines in neurocognitive outcomes , and sparing the hippocampi from doses above 55 Gy improves neurocognitive outcomes. Similarly, for normal-appearing uninvolved brain, radiation doses beyond 35 Gy cause cortical atrophy on serial imaging that may lead to decreased neurocognition . Furthermore, doses as low as 10 Gy disrupt white matter connectivity . Another technique that limits brain RT dose is proton therapy . A randomized trial of protons vs. photons in glioblastoma for improved brain sparing reduced grade 2 toxicities and high-grade lymphopenia , which might also be possible with other brain-sparing techniques such as this one. Adaptation as demonstrated here should not negatively impact tumor control, as tumor cells would be expected to shift with the normal-appearing brain towards the shrinking resection cavity. As an alternative to hybrid MRI-RT systems, standalone MRI during RT has been proposed to reduce boost margins during RT to reduce the volume of brain treated in efforts to limit toxicity. This has been shown to improve target coverage and reduce the dose to the normal brain . One study has even suggested that such an adaptive strategy could improve patient overall survival . The current work expands on these existing studies to demonstrate feasibility on an integrated MR-Linac system with integrated adaptive radiotherapy capabilities, and demonstrates improvement in hippocampal sparing, which could improve patient quality of life. The main limitation for the integration of standalone MRI during RT in clinical practice is the cost, availability, and coordination of standalone MRIs for patients during RT as well as the extra effort for re-simulation and planning. Hybrid MRI-RT systems essentially nullify these concerns since they allow for treatment and MRI in the same session and include an adaptive online radiotherapy workflow . To this end, the UNITED trial (NCT04726397) uses a hybrid MRI-RT system with weekly adaptation to intact disease and cavity based on T1 post-contrast MRI with 5 mm CTV margins. In this study, we propose weekly adaptation on non-contrast T2-weighted or FLAIR images, as the US Food and Drug Administration has advised to "minimize repeated [gadolinium contrast] imaging studies when possible, particularly closely spaced MRI studies ". In the case of rapid tumor recurrence during RT, an expansion of edema on T2 or FLAIR would be visible around the resection cavity, and this could trigger gadolinium contrast administration in select patients. 5. Conclusions The weekly adaptive MRI-guided re-planning of shrinking glioblastoma resection cavities has the potential to spare brain and hippocampi from high-dose radiation, reducing RT toxicity. Clinical trials of MRI-guided adaptive RT should measure its impact on neurocognition and other toxicities. Acknowledgments I: Beatriz Guevara, am grateful to all of those with whom I have had the pleasure to work during this project. Without the help of Jenniffer Donoso, who provided administrative support, William Amestoy, who provided the guidance to learn to use the planning systems, Kyle Padgett, who provided guidance during the methodology of this article, this project would not have been possible. Author Contributions Conceptualization, D.M., K.C. and E.A.M.; methodology, B.G. and K.B.; validation, B.G. and E.A.M.; formal analysis, B.G.; investigation, B.G., K.C., D.M. and A.V.; resources, G.A.A., M.I.D.L.F., A.V., J.M., A.B., J.C.F. and E.A.M.; data curation, K.C.; writing--original draft preparation, B.G.; writing--review and editing, K.C. and E.A.M.; visualization, B.G.; supervision, E.A.M.; project administration, A.V. and E.A.M.; funding acquisition, E.A.M. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional Review Board of Sylvester Comprehensive Cancer Center at the University of Miami Miller School of Medicine (protocol code 20190678, date of approval 9 December 2019, protocol code 20160817, date of approval 17 April 2017). Informed Consent Statement Informed consent was obtained from all subjects involved in the study. Data Availability Statement The data presented in this study will be submitted to The Cancer Imaging Archive pending acceptance of this manuscript. Conflicts of Interest Mellon is the principal investigator of NCI Academic-Industrial Partnership grant R37CA262510, and as part of this and other work receives research support from ViewRay, Inc. He is also a consultant for ViewRay, Inc and receives travel funding from ViewRay. Mellon receives no personal funds from and holds no interests in ViewRay, Inc. There was no role for ViewRay, Inc. or any other sources outside of the University of Miami, in the collection, analysis, or interpretation of data; writing of data; or decision to publish the results. There are no other conflict of interest. Figure 1 Contoured structures on the MRI scan: RC (red), PTV (green), Brainstem (yellow), right hippocampus (red), left hippocampus (pink), optic nerves (magenta), eyes (magenta), lenses (blue, green), lacrimal glands (blue, green). Figure 2 (A): Flow chart for static plan (60 Gy prescription to the PTV). (B): Flow chart for Adaptive planning (10 Gy prescription to the PTV with a total of 6 different plans and accumulated dose of 60 Gy). W1-W6 stands for Week 1-Week 6 of RT. Figure 3 (A): Resection cavity shrinkage visualized on MRI-Linac: T2-weighted bSSFP treatment set-up scans This shows the PTV and RC volume trending downwards as the weeks progressed. Week 1: Volume of RC is 3.0 mL and mean dose to brain is 3.3 Gy. Week 2: Volume of RC is 1.3 mL and dose to brain is 2.7 Gy. Week 3: Volume of RC is 0.8 mL and dose to brain is 2.7 Gy. Week 4: Volume of RC is 0.6 mL and dose to brain is 2.5 Gy. Week 6: Volume of RC is 0.5 mL and dose to brain is 2.5 Gy. (B): Example adaptive replanning comparing the dose distribution for the first week of treatment (left) and the dose distribution for the sixth week (right). Isodose lines are shown in color surrounding the PTV (area shaded in blue). Both left and right hippocampi are contoured, shown in pink (left) and red (right). During the first week of treatment, the 4 Gy isodose line (light blue) completely covers the right hippocampus. Comparing the 4 Gy isodose line between the two scans, at Week 6, this line has reduced radius sparing the right hippocampus from radiation dose. The dose also decreases to healthy tissue surrounding the PTV outside of the hippocampi. Figure 4 Static plan vs. adaptive re-planning (bars represent standard deviation). * Indicates statistical significance between the static and adaptive plans. cancers-15-01555-t001_Table 1 Table 1 Static vs. adaptive plans for all patients. Mean and maximum (max) dose to hippocampus and brain. All numbers shown are in units of Gy. PATIENT # SUM HIPPOCAMPUS BRAIN UNINVOLVED HIPPOCAMPUS Static Plan Adaptive Plan Static Plan Adaptive Plan mean max mean max mean mean 1 23.8 44.4 11.8 19.9 23.0 18.5 both 2 12.2 16.8 8.9 11.2 14.3 9.7 left 3 2.4 3.7 2.0 2.9 13.0 11.2 both 4 11.0 13.7 7.6 12.1 26.4 26.0 left 5 4.6 9.6 3.9 6.9 19.5 17.9 both 6 10.4 17.0 7.2 15.6 19.4 15.9 right 7 22.8 29.2 16.4 21.5 22.7 22.4 both 8 13.1 43.6 9.5 32.4 21.3 19.2 both 9 12.3 16.2 8.7 15.1 14.3 13.7 both 10 12.9 15.9 8.4 14.5 32.5 32.1 right cancers-15-01555-t002_Table 2 Table 2 Statistical data for static and adaptive plans. All numbers shown are in units of Gy. Sum hippocampus refers to the accumulated dose between the left and right hippocampi (involved hippocampi excluded). STATIC PLAN (SP) ADAPTIVE RE-PLANNING STDEV SP STDEV AP p-VALUES Hippocampus mean 12.5 8.4 6.7 3.96 0.003 Hippocampus max 21 15.2 13.7 8.19 0.036 Brain mean 20.6 18.7 6.0 6.81 0.005 Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. |
PMC10000840 | Diagnostics (Basel) Diagnostics (Basel) diagnostics Diagnostics 2075-4418 MDPI 10.3390/diagnostics13050874 diagnostics-13-00874 Article The Tomographic Study and the Phenotype of Wormian Bones Al Kaissi Ali 12* Ryabykh Sergey 2 Ben Chehida Farid 3 Al Kaissi Hamza 4 Kircher Susanne Gerit 5 Stransky Martin J. 67 Grill Franz 1 Ottaviani Giulia Academic Editor 1 Pediatric Department, Orthopedic Hospital of Speising, 1130 Vienna, Austria 2 National Medical Research Center for Traumatology and Orthopedics n.a. G.A. Ilizarov, 640014 Kurgan, Russia 3 Ibn Zohr Diagnostic Institute of Radiology, Tunis 1003, Tunisia 4 Surgical Outpatient Clinic of Landesklinikum Hospital, 3400 Klosterneuburg, Austria 5 Center of Pathobiochemistry and Genetics, Medical University of Vienna, 1090 Wien, Austria 6 Department of Neurology, Yale School of Medicine (USA), New Haven, CT 06510, USA 7 Policlinic at Narodni, 110 00 Prague, Czech Republic * Correspondence: [email protected] 24 2 2023 3 2023 13 5 87426 1 2023 12 2 2023 21 2 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). Background: We describe patients who were recognized via conventional radiographs of the skull as manifesting wormian bones. Wormian bones are not a specific diagnostic entity and can be seen in variable forms of syndromic disorders. Materials and Methods: Seven children and three adults (of 10-28 years) were seen and diagnosed in our departments. The principal complaints for the pediatric and adult group were: ligamentous hyperlaxity, a history of delayed walking and occasional fractures, which later in life started to manifest a constellation of neurological symptoms such as nystagmus, persistent headache, and apnea. Conventional radiographs were the first traditional tools used to recognize wormian bones. We performed 3D reconstruction CT scans to further understand the precise etiology and the nature of these wormian bones and attempted to connect them with a broad spectrum of unpleasant clinical presentations. Our group of patients was consistent with the phenotypic and genotypic diagnoses of osteogenesis imperfecta type I and type IV as well as patients with multicentric carpotarsal osteolysis syndrome. Results: Three-dimensional reconstruction CT scan of the skulls confirmed that these worm-like phenotypes are in fact stemmed from the progressive softening of the sutures. The overall phenotype of the melted sutures is akin to overly stretched pastry. The most concerning sutures in this pathological process are the lambdoid. The overstretching of the lambdoid sutures was responsible for the development of sub-clinical basilar impression/invagination. Patients with certain forms of skeletal dysplasia such as osteogenesis imperfecta type I and IV manifested the heterozygous mutation of COL1A1/COLA2, shown as typical overstretching of the sutures. Similarly, patients with multicentric carpotarsal osteolysis syndrome with a heterozygous missense mutation of MAFB also manifested the phenotype of overly stretched pastry along the skull sutures. Conclusion: What we encountered via 3D reconstruction CT scan in our group of patients was entirely different than the traditional description that can be found in all relevant literature of the last decades. The worm-like phenomenon is in fact a pathological sequel occurring as a result of a progressive softening of the sutures, which results in the overstretching of the lambdoid sutures, a pathological process roughly similar to an overly stretched soft pastry. This softening is totally connected to the weight of the cerebrum (the occipital lobe of the cerebrum). The lambdoid sutures represent the weight-bearing zone of the skull. When these joints are loose and soft, they adversely alter the anatomical structures of the skull and lead to a highly hazardous derangement of the craniocervical junction. The latter causes the pathological upward invasion of the dens into the brain stem, leading to the development of morbid/mortal basilar impression/invagination. wormian bones radiology tomography phenotype overly stretched pastry bulging of the occipital lobe of the cerebrum basilar invagination This research received no external funding. pmc1. Introduction The traditional definition of wormian bones is small bones that are often found within the sutures and fontanelles of the skull. Some instances are often considered to be a simple anatomical variant. Previous studies concluded that around 8%-15% of the population has at least one wormian bone . In patients with significant pathologies, there are least ten wormian bones larger than around 6 mm x 4 mm arranged in a mosaic-type pattern. The skull itself is composed of several flat bones that fuse together after birth. These sites of fusion are the bony sutures in which wormian bones most commonly occur . Wormian bones are more commonly seen in patients with several types of bone dysplasia. Osteogenesis imperfecta is the most common type of bone dysplasia, in which wormian bones represent one of the main diagnostic features. Wormian bones can be also encountered in a long list of heritable syndromes . Weissbach et al. in two occasions described fetal wormian bones and their clinical outcomes in prenatally diagnosed fetuses . Osteogenesis imperfecta (OI) is a clinically and genetically heterogeneous group of heritable disorders of the connective tissue characterized by reduced bone mass (osteopenia) with associated bone fragility. The resulting skeletal manifestations are due to a generalized deficiency in the development of both membranous and endochondral bone and include markedly thin calvarium with delayed closure of the fontanelles and the sutures and excessive wormian bone formation. OI type I is a purely qualitative collagen defect, whereas OI of type II, III, and IV are qualitative and quantitative alterations in collagen synthesis . Type I osteogenesis imperfecta (OMIM 166200) is the commonest form of osteogenesis imperfecta and is inherited as an autosomal dominant condition. Affected individuals may have blue sclerae with a tendency to fractures of the long bones, although healing occurs without deformity. Cells from individuals with type I (OI) secrete about half the normal amount of type I procollagen. More than 95% of individuals with OI are heterozygous for mutations in either of the two type I collagen genes, COL1A1 and COL1A2. The majority of mutations associated with OI occur in the type I collagen encoding genes and give rise to an autosomal dominant form of the disease. Novel mutations in seven other genes involved in collagen assembly and processing and in two genes involved in cellular differentiation have recently been associated with autosomal recessive forms of OI . Osteogenesis imperfecta type IV (OMIM 166220) is similar to type I, but the sclerae might be normal and mild short stature with limb bowing is a feature. There have been extensive studies regarding the true nature of wormian bones and why they occur. Silence and Glorieux described wormian bones in association with osteogenesis imperfecta type I and IV. Platybasia and basilar impression/basilar invagination may occur because of bone softening in patients with osteogenesis imperfecta . Semler et al. discussed wormian bones in osteogenesis imperfecta in correlation with phenotypic and genotypic characteristics . Sykes et al. found linkage to the COL1A2 locus in eight pedigrees . Lund et al. suggested that type I mutations were common, and that parental mosaicism was an explanation for some recurrences . The connection between wormian bones and osteogenesis imperfecta has been known for decades. Cremin et al. studied the association between wormian bones and osteogenesis imperfecta, and they introduced the idea of "significance". They considered wormian bones to be "significant" when there were more than ten of them and especially when they were arranged in a mosaic-like pattern. Based on this study, they suggested that the presence of significant wormian bones could be a strong argument in favor of osteogenesis imperfecta. Based on conventional radiographic interpretations, they concluded that 88% of 81 patients with a diagnosis of osteogenesis imperfecta had a significant number of wormian bones (more than ten). They confirmed that significant wormian bones were not only found in osteogenesis imperfecta but also in other bone dysplasias . Multicentric carpotarsal osteolysis syndrome (MCTO) (OMIM 166300) is a serious bone disorder characterized by bilateral wrist and ankle joint deformities in connection with carpal and tarsal bone osteolysis and progressive nephropathy. The early clinical phenotype can be confused with juvenile idiopathic arthritis. MAFB mutations (also known as V-maf musculoaponeurotic fibrosarcoma oncogene homolog B) have been identified in all MCTO patients. MAFB, a single-exon gene, is located at 20q12 and encodes the MAFB protein, which functions in the activation and differentiation of osteoclasts and development of podocyte foot processes in the renal system. MAFB is a negative regulator of receptor activator of nuclear factor kB ligand--(RANKL--receptor activator of nuclear factor kappa-B ligand) mediated osteoclast differentiation . Reduced MAFB expression leads to osteoclast activity, resulting in osteolysis predominantly in the carpal and tarsal bones . 2. Materials and Methods The study protocol was approved by the Ethics Committee of the (Ilizarov Scientific Research Institute, No. 4(50)/13.12.2016, Kurgan, Russia). Informed consents were obtained from the patients' guardians. Seven children and three adults (of 10-28 years) were enrolled in this study. We fully documented these children through detailed clinical and radiological phenotypic characterizations at the clinic of orthogenetics (osteogenetische ambulanz) in the orthopedic Hospital of Speising (Pediatric Department) and through the scientific collaboration of the first author with Ilizarov Center, Kurgan, Russia, collaboration with the Ibn Zohr Institute of Diagnostic radiology in Tunis and scientific collaboration with Policlinic at Narodni, Prague, Czech Republic. This study was conducted based on confirmed diagnosis via phenotype and genotype of a group of children and adults and was carried out between 2016 and 2022. We subdivided our patients in accordance with the phenotypic and genotypic diagnostic process. 3. Results 3.1. Patients with Osteogenesis Imperfecta Type I Seven unrelated patients (age range from 10-15 years) were diagnosed with osteogenesis imperfecta type I. All demonstrated early natural history of ligamentous hyperlaxity and dentin abnormalities. A lateral skull radiograph of a 10-year-old boy with OI type I found frontal bossing and the abundance of a worm-like phenotype over lambdoid sutures (arrow); note the odontoid hypoplasia (arrow head), atlanto-axial instability and a hypoplastic posterior arch of the atlas. His genotype indicated mutation COL1A2 p.G1078D. Figure 1a, the sagittal reformatted CT scan of the cranium of a 12-year-old boy with OI type I shows massive downward thinning of the occipital area (downward bulging of the occipital lobe of the cerebrum) in connection with progressive softening of the fragile lambdoid sutures (arrow) . He manifested mutation in COL1A1, showing c.3233 G>A. Interestingly, one child of 15 years with no history of fractures was a client of our department because of post-adulthood scoliosis (Cobbs angle of 28deg). Surprisingly, he started to complain from bouts of headache, nystagmus, and nocturnal apnea. These symptoms were neglected by other institutes and were treated on symptomatic grounds. Sagittal CT scan of the cranium of this 15-year-old boy with OI type I found bulging of the odontoid process of 6 mm above Chamberlain's line, signifying basilar impression (arrow). Note the redundancy and the downward bulging of the occipital area (occipital lobe of the cerebrum) in connection with progressive thinning of the lambdoid sutures (arrow head). His genotype demonstrated mutation in COL1A2, pGly634Asp . Patients with Osteogenesis Imperfecta Type IVB Three adult patients of 18, 23, and 28 years all received the diagnosis of OI type IVB. The natural histories of these patients were almost similar. All had short stature, large head, and a history of occasional but not frequent fractures. Interestingly, bowing of the long bones or any other frequent abnormalities did not manifest, as usually seen in patients with OI type IV. All manifested signs of subclinical basilar impression but nevertheless at different ages. The 18-year-old-girl started to experience sudden bouts of intractable headaches associated with nystagmus. She mentioned progressive deterioration in her educational achievement because of headache and poor concentration. The lateral skull radiograph displayed a massive downward redundancy of the occipital area (the occipital part of the skull that contains the occipital cerebrum looks like a bag of worms; arrow) in connection with progressive softening of the lambdoid area. The downward bulging of the lambdoid was obviously overwhelmed with abundant wormian-like bones. Chamberlain's line displayed 6 mm bulging of the dens into the brain stem. The severity of the upward bulging of the odontoid could be easily assessed via the semi-translation of the atlas onto the occiput (arrow), which resulted in atlanto-axial dislocation. Her genotype demonstrated homozygous mutation in COL1A2 (p.G322S) . 3D reconstruction CT scan superior view of a 23-year-old male patient with OI type IVB demonstrated massive bulging of the occiput, causing tremendous disfigurement of the cranial anatomical structures . The 3D reconstruction CT scan of the skull of a 28-year-old patient with OI type IVB demonstrated massive bulging of the occipital area secondary to progressive softening and thinning of the occipital bones, causing a severe bulge over the lambdoid sutures (note the overly stretched pastry-like phenotype), leading to downward shifting of the skull with subsequent upwards ascending of the dens into the brain stem. His genotype exhibited a carrier of the heterozygote mutation in COL1A2, NM_000089.3c.1801>A,p.(Gly601 Ser.) . The 3D sagittal CT scan of the craniocervical junction displayed immense thinning of the occiput (arrow) and downward bulging around the borders of the lambdoid sutures. Pathological upward bulging of the dens 8 mm above Chamberlain's line caused the development of subclinical basilar invagination . 4. Multicentric Carpotarsal Osteolysis Syndrome Episodes of headaches and dizziness were the most bothersome symptomatology in an 8-year-old girl. She was diagnosed with multicentric carpotarsal osteolysis syndrome with a heterozygous mutation of the MAFB gene. Her mutation in MAFB demonstrated heterozygous mutation (c.176>T p.Pro59Leu). Neurological examination disclosed dyslexia, alternating strabismus, general hypotonia, and lower limb dysmetria. We started our skeletal survey to further understand the reason behind her unpleasant symptoms. The lateral skull radiograph indicated an 8-year-old girl with multicentric carpotarsal osteolysis syndrome showing multiple wormian bones in the lambdoid sutures and prominence of the occipital bone associated with progressive thinning protuberance of the squamous part. The soft bones of the skull base may have allowed for progressive infolding of the dysplastic clivus and pathological ascending of the dens into the brain stem. Combination with platybasia is a predisposing factor for basilar impression that can lead to severe distortion of the spinal cord and the anterior brain stem . The 3D reconstruction CT scan of the lambdoid sutures displayed downward redundancy of the lambdoid sutures in a pattern similar to a stretched pastry, which was interpreted via radiology as wormian bones. Note the stretched-pastry-like phenotype (arrows) . . One of our patients was a 10-year-old girl with craniofacial features of (long occipital region, microstomia, and dental malocclusion), arched palate, short and wide distal parts of digits, carpo-tarsal osteolysis, osteoporosis, history of clavicular fracture, and ligamentous hyperlaxity. Her clinical phenotype was consistent with multicentric carpotarsal osteolysis syndrome, her genotype demonstrated heterozygous missense mutations in the MAFB gene (c.184A>C p.Thr62Pro). A 3D reconstruction CT scan was organized to further understand the nature of these wormian bones. The 3D reconstruction CT scan found progressive separation of the lambdoid sutures, giving the appearance of a stretched-pastry-like appearance (arrow head denotes progressive softening and melting of the sutures, in comparison to the normal coronal suture-arrow) . Sagittal 3DCT of the craniocervical junction found upward bulging of the dens of more than 5.8 mm above Chamberlain's line *. The overall picture is of upward invasion of the dens into the brain stem . We summarized the actual phenotype of the wormian bones via enlarging the 3D reconstruction CT scans of some of the aforementioned patients to illustrate clearly through a 3D reconstruction CT scan of the lambdoid sutures a clear delineation of the typical phenotype of wormian bones. All manifest an overly stretched pastry-like phenotype (arrows) . 5. Discussion In this paper, we focused our efforts on understanding the factual origin of what wormian bones are. We performed extensive conventional and tomographic studies on our current patients. These results are somehow different from what has so far been published in regard to "wormian bones". The tomographic phenotype of these worm-like lesions in fact is a pathological process of a progressive softening of the lambdoid sutures. The lambdoid sutures are the weight-bearing zone of the skull, and it is well known that the cerebrum is considered the largest and the heaviest part of the brain, consisting of more than 85% of the total brain weight . Thereby, the overload of the cerebrum on the weight-bearing zone of the skull (namely the fragile lambdoid sutures) can result in outward and downward bulging of the posterior lower part of the occiput (specifically around the occipital lobe of the cerebrum). Proper assessment of the evolution and the pathological outcome of these wormian bones is imperative (particularly along the lambdoid sutures). The lambdoid sutures are designed to be the weight-bearing joint of the skull in comparison with the other sutures. Over the last century, several theories appeared in an attempt to detect the etiology behind the development of wormian bones. Riveiro and Von Tschudi postulated that wormian bones occur in connection with a mechanical reason. They suggested that wormian bones are man-made artificial deformities induced in the skulls of children through cranial deformation by adopting mechanical measures . The mechanical measures as described by Riveiro and Von Tschudi are based on the fact that the skulls of infants are soft and liable to mal-manipulation and therefore can be molded in accordance with tribal cultural practices. They believed that wormian bones can occur as a result of this deliberate molding. The phenomena of skull molding in infancy received additional studies . However, the theory of Riveiro and Von Tschudi has been refuted by Ell-Najjar and Dawson . Others stated that wormian bones may develop in connection with progressive cerebral expansion, and this would explain why they are found in higher numbers in patients with hydrocephalus . Several other studies attempted to discuss the etiology of wormian bones. Some authors explained wormian bones as a manifestation seen in children born with congenital neurological deficits such as hydrocephalus, craniosynostosis, cerebral palsy, and so forth . We reviewed the aforementioned papers with great care. Unfortunately, none of the results emerged from genuine recognition of wormian bones. In fact, we encountered a noticeable issue with different authors who, for instance, were confused and mixed up the multi-sutural appearances of the skull, particularly with progressive enlargement of the skull as seen in children with hydrocephalus. We published several papers regarding children with craniosynostosis and all were documented via 3D reconstruction CT scan, though none exhibited wormian bones. Marti et al. used 3D reconstruction CT scans in order to explain wormian bones. They attributed the reason behind wormian bones as a manifestation of plagiocephaly. They assumed that the cranial asymmetry of the skull leads to pressure exerted on the infant cranial bones, which results in the development of wormian bones. They based their hypothesis on the CT scan images of their group of 12 children with plagiocephaly; the mean number of wormian bones they encountered was (2.33 W bones/CT) . We examined the CT scan images of Marti et al. and we disagree with their conclusion. The images of Marti et al. are of normal sutures rather than genuine wormian bones. The CT scans of Marti et al. are of children with persistent openings of the anterior and posterior fontanels. These occur in children with certain forms of syndromic and non-syndromic disorders. Wormian bones are usually much more abundant and can never exhibit a tiny or a solitary worm-like phenotype. Multicentric carpotarsal osteolysis is a condition that is characterized by progressive osteolysis of the carpal and tarsal bones and progressive nephropathy, resulting in hypertension and renal failure in early adult life. The tubular bones involved in the foci of osteolysis are said to have a "sucked candy" appearance. Previous reports have described carpotarsal osteolysis associated with peripheral corneal clouding and pulmonary stenosis . Beals and Bird described the association of corneal clouding with osteolysis but without nephropathy . Malecha et al. similarly focused on carpotarsal osteolysis associated with bilateral corneal clouding in patients described as being diagnosed with idiopathic multicentric osteolysis . Basilar impression/invagination is a serious deformity of the craniocervical junction. It may lead to a life-threatening situation because of stenosis of the foramen magnum and compression of the medulla oblongata. Sudden death or severe neurological deficits due to brain stem compression, particularly in patients with sudden unstable head-neck movements or jarring can occur . This is a type of vertical atlanto-axial instability, or type 2 according to Goel's classification. There is no dislocation of the C2 dentoideum into the vertebral canal . 6. Conclusions The weight of the cerebrum exerts a direct load on the loose and soft boney junction/joints of the occiput as presented by the lambdoid sutures. The reconstruction CT scans of our current patients exhibited typical softening and melting of the lambdoid sutures. This overall process is somehow similar to an overly stretched soft pastry. The risk of progressive softening of the lambdoid is not restricted to the distorted anatomy of the cranium, but also includes the total derangement of the craniocervical junction. Basilar impression/invagination is a serious complication of the craniocervical junction. It may lead to a life-threatening situation because of stenosis of the foramen magnum and compression of the medulla oblongata. Sudden death or severe neurological deficits due to brain stem compression can occur. Pediatric spine surgeons should ensure regular follow-ups if neurological deficits worsen. In this case, decompression operation might be an option. The term wormian is not just a term given to skulls; in fact, it is a more complicated pathological process along the lambdoid sutures occurring because of the progressive softening of the lambdoid sutures, which are considered the weight-bearing zone of the skull /brain and specifically the occipital lobe of the cerebrum. Acknowledgments We wish to thank the collaboration and the consent of the families and the guardians of the patients. Author Contributions A.A.K. conceptualization and methodology; software and validation, A.A.K. and S.R.; formal analysis, A.A.K., S.R., F.G. and S.G.K.; investigations, S.G.K.; data curation, A.A.K., F.B.C., H.A.K. and F.G.; writing--original draft preparation, A.A.K. and M.J.S.; writing--review and editing, F.G.; visualization, A.A.K.; supervision, A.A.K. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The study protocol was approved by Ethics Committee of the (Ilizarov Scientific Research Institute, No. 4(50)/13.12.2016, Kurgan, Russia). Informed consents were obtained from the patients' guardians. Informed Consent Statement Signed consent forms were obtained from the patients' guardians. Conflicts of Interest The authors declare no conflict of interest. Figure 1 (a-c) Lateral skull radiograph of the 10-year-old boy with OI type I showing frontal bossing and abundance of worm-like phenotype over lambdoid sutures (arrow); note the odontoid hypoplasia (arrow head), atlanto-axial instability and hypoplastic posterior arch of the atlas. His genotype exhibited mutation COL1A2 p.G1078D (a). Sagittal reformatted CT scan of the cranium of a 12-year-old boy with OI type I showing massive downward thinning of the occipital area (downward bulging of the occipital lobe of the cerebrum) in connection with progressive softening of the fragile lambdoid sutures (arrow). He manifested mutation in COL1A1, showing c.3233 G>A (b). Sagittal CT scan of the cranium of a 15-year-old boy with OI type I showing bulging of the odontoid process of 6 mm above Chamberlain's line, signifying basilar impression (arrow). Note the redundancy and downward bulging of the occipital area (occipital lobe of the cerebrum) in connection with progressive thinning/softening of the lambdoid sutures, arrow head). His genotype demonstrated mutation in COL1A2,pGly634Asp (c). Figure 2 (a,b) Lateral skull radiograph of an 18-year-old girl with OI type IV B showing a massive downward redundancy of the occipital area along the lambdoid sutures (the occipital part of the skull that contains the occipital cerebrum looks like a bag of worms, arrow) in connection with progressive softening of the lambdoid area. The latter was obviously overwhelmed with an abundancy of wormian-like bones. Chamberlain's line indicates 6 mm bulging of the odontoid into the brain stem. The severity of the upward bulging of the dens can be easily assessed via the semi-translation of the atlas onto the occiput (arrow), which resulted in atlanto-axial dislocation. Her genotype demonstrated homozygous mutation in COL1A2 (p.G322S) (a). Three-dimensional reconstruction CT scan superior view of a 23-year-old male patient with OI type IVB showing massive bulging of the occiput, causing tremendous disfigurement of the cranial anatomical structures. His genotype demonstrated mutation in COL1A2 c.2827G>A (b). Figure 3 (a,b) Three-dimensional reconstruction CT scan of the skull of a 28-year-old patient with OI type IVB showing the massive bulging of the occipital area secondary to progressive softening and thinning of the occipital bones, causing a severe bulge over the lambdoid sutures (note the overly stretched pastry-like phenotype--arrow) leading to downward shifting of the skull with subsequent upwards ascending of the dens into the brain stem. His genotype exhibited a carrier of the heterozygote mutation of COL1A2, NM_000089.3c.1801>A,p.(Gly601 Ser.) (a). Three-dimensional sagittal CT scan of the craniocervical junction showing immense thinning of the occiput (arrow) and downward bulging around the borders of the lambdoid sutures. Pathological upward bulging of the dens 8 mm above Chamberlain's line causing the development of subclinical basilar invagination (b). Figure 4 (a,b) Lateral skull radiograph showing an 8-year-old girl with multicentric carpotarsal osteolysis syndrome showing multiple wormian bones in the lambdoid sutures and prominence of the occipital bone associated with progressive thinning protuberance of the squamous part. The soft bones of the skull base might allow for progressive infolding of the dysplastic clivus and translocation of the odontoid into the posterior fossa. Combination with platybasia is a predisposing factor for basilar impression and can lead to severe distortion of the spinal cord and the anterior brain stem (a). Three-dimensional reconstruction CT scan of the lambdoid sutures showing downward redundancy of the lambdoid sutures in a pattern similar to a stretched pastry, which was interpreted via radiology as wormian bones. Note the stretched-pastry-like phenotype (arrows) (b). Figure 5 (a,b) Three-dimensional reconstruction CT scan of a 10-year-old girl with multicentric was organized to further understand the nature of these wormian bones. Three-dimensional reconstruction CT scan shows progressive separation of the lambdoid sutures, giving an overly stretched-pastry-like appearance (arrow head denotes progressive softening and melting of the sutures, in comparison to the normal coronal suture-arrow) (a) Sagittal 3DCT of the craniocervical junction shows Chamberlain line (which joins the hard palate to the posterior lip of the foramen magnum). The odontoid process length is greater than 5.8 mm. The overall picture is of dolicho-odontoid with posterior inclination (b) * Chamberlain line: a line connecting the back of the hard palate with the opisthion. Seen on a lateral view of the craniocervical junction via conventional radiograph, but with much better visualization and localization via 3D sagittal CT scan of the craniocervical junction has been applied. The importance lies in diagnosing basilar impression or invagination, particularly when the tip of the dens is more than 3 mm above the line. Figure 6 (a-c) We summarized the actual phenotype of the wormian bones via enlarging the 3D reconstruction CT scans of some of the aforementioned patients to illustrate clearly through 3D reconstruction CT scan of the lambdoid sutures a clearly delineation of the typical phenotype of wormian bones. All manifest an overly stretched pastry-like phenotype (arrows). 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PMC10000841 | Background: Pharmacogenetics is a personalized medicine tool that aims to optimize treatments by adapting them to each individual's genetics, maximizing their efficacy while minimizing their toxicity. Infants with cancer are especially vulnerable, and their co-morbidities have vital repercussions. The study of their pharmacogenetics is new in this clinical field. Methods: A unicentric, ambispective study of a cohort of infants receiving chemotherapy (from January 2007 to August 2019). The genotypes of 64 patients under 18 months of age were correlated with severe drug toxicities and survival. A pharmacogenetics panel was configured based on PharmGKB, drug labels, and international experts' consortiums. Results: Associations between SNPs and hematological toxicity were found. Most meaningful were: MTHFR rs1801131 GT increasing the anemia risk (OR 1.73); rs1517114 GC, XPC rs2228001 GT, increasing neutropenia risk (OR 1.50 and 4.63); ABCB1 rs1045642 AG, TNFRSF11B rs2073618 GG, CYP2B6 rs4802101 TC and SOD2 rs4880 GG increasing thrombocytopenia risk (OR 1.70, 1.77, 1.70, 1.73, respectively). Regarding survival, MTHFR rs1801133 GG, TNFRSF11B rs2073618 GG, XPC rs2228001 GT, CYP3A4 rs2740574 CT, CDA rs3215400 del.del, and SLC01B1 rs4149015 GA were associated with lower overall survival probabilities (HR 3.12, 1.84, 1.68, 2.92, 1.90, and 3.96, respectively). Lastly, for event-free survival, SLC19A1 rs1051266 TT and CDA rs3215400 del.del increased the relapse probability (HR 1.61 and 2.19, respectively). Conclusions: This pharmacogenetic study is a pioneer in dealing with infants under 18 months of age. Further studies are needed to confirm the utility of the findings in this work to be used as predictive genetic biomarkers of toxicity and therapeutic efficacy in the infant population. If confirmed, their use in therapeutic decisions could improve the quality of life and prognosis of these patients. pharmacogenetics SNP (single nucleotide polymorphism) chemotherapy infant toxicity therapeutic efficacy overall survival event-free survival anemia neutropenia thrombocytopenia Fundacion Mutua MadrilenaAsociacion Pablo UgarteAsociacion Esperanza y SonrisaThis research was funded by the XIII convocatoria de Ayudas a la Investigacion Cientifica en Salud de la Fundacion Mutua Madrilena, Asociacion Pablo Ugarte-APU, and Asociacion Esperanza y Sonrisa. The L.S. contract is financed by the "Infraestructura de Medicina de Precision asociada a la Ciencia y Tecnologia" (IMPaCT, IMP/00009). The G.G.O. contract is financed by Asociacion Pablo Ugarte-APU. pmc1. Introduction The definition of "infant" in oncology is not homogeneous and varies depending on the collaborative group and the criteria employed (pharmacokinetic characteristics, tumor biology, therapeutic approaches, etc.). Although cancer in children older than 18 months can be considered a rare disease (or a group of rare diseases) due to its relatively low population incidence, cancer in the first stage of life is still rare. Oncological diseases in children under 12 months of age account for 11.4% of the total (about 100 cases per year in Spain), and only 15% of pediatric cancer is diagnosed in the first 18 months of life . Global efficacy of pharmacologic treatments ranges from 25 to 60%, with chemotherapy achieving the lowest result . Regarding toxicity, drug side effects represent the fourth cause of mortality in the USA and the sixth in the EU , besides being responsible for 30% of hospitalizations of patients over 55 years and provoking higher expenses than chronic conditions such as diabetes mellitus or cardiovascular diseases . Specifically, in pediatric patients, the incidence of drug-related toxicity in hospitalized patients reaches 9.5% , with an average prevalence of 24% . This is especially relevant in infants due to the severity of their side effects that could compromise their lives and the lack of studies involving the drug response (particularly with chemotherapeutic agents) in this population. In this work, we evaluate for the first time the role of individual genetic variability in under-18-month-old infants' responses to chemotherapy drugs. The medical findings of the adult population cannot be directly extrapolated to pediatrics due to the latter's singularity at different levels. Infants present pharmacokinetic and pharmacodynamic differential features affecting drug management that must be considered and studied in detail: absorption differences (gastric pH variations, digestive emptying, absorption surface, quality of gut flora, muscle flow and its proportion, corneal and epidermal thickness); distribution differences (body composition, with more water and less fatty and muscular tissue that alters the management of hydrophobic/hydrophilic substances and the fraction of free drug, less restrictive hematoencephalic barrier that predisposes to neurotoxicity); differences in drug metabolism (variable maturity of important enzymatic complexes as CYP450); differences in excretion (lower kidney mass/blood flow, lower glomerular filtration, and alterations in tubule secretion) . So, it becomes greatly important to perform pharmacogenetic studies in children, and particularly in infants, as the rules of the game change and we need to understand the different genes and SNPs that will be of relevance here differently than in adults. In order to understand their relationship and maximize treatment efficacy while minimizing toxicity and long-term sequelae in this population, we evaluated potential associations between single nucleotide polymorphisms located in pharmacogenes and response to chemotherapeutic drugs in a cohort of 64 infants under 18 months of age. 2. Materials and Methods 2.1. Study Design Ambispective observational study of correlation between genetic variants and clinical parameters in patients up to 18 months-old, diagnosed with oncological disease and having received or receiving chemotherapy at Hospital Universitario y Politecnico La Fe (Valencia, Spain). Retrospective recruitment comprises biobanked samples from January 2007 to September 2016, and prospective recruitment from October 2016 to August 2019. Clinical and analytical data (neoplasm, chemotherapy and other drugs, hematological support, clinical and analytical toxicity, epidemiological and diagnostic data, survival) from all the genotyped patients were available in the electronic or paper medical records . Data were abstracted from patients' medical records by two independent pediatric oncologists. Before genetic analysis, informed consent was obtained for each patient. Ethical approval was obtained from the Comite de Etica de Investigacion Clinica del Hospital Universitario y Politecnico La Fe de Valencia, on 7 September 2016. 2.2. Clinical Parameters 2.2.1. Drugs and Therapeutic Schemes Protocols employed, chemotherapy administered in each cycle, and their doses are cited. Both the absolute and relative to body surface or weight doses were annotated (recommended based on age and protocol). Doses from each cycle were registered (Supplementary Materials, Table S1). If there was a difference between theoretical (according to protocol) and actually administered doses, this was marked in blue (Table S1) if it was due to treatment toxicity in previous cycles. Those patients who received transfusion support, omeprazole, and azole antifungals (possible enzymatic inducers/inhibitors) were annotated for the statistical analyses. 2.2.2. Clinical and Analytical Toxicity. Survival Data Antineoplastic drug-derived toxicities were grouped as qualitative variables (grades 0-5). The grade of toxicity assigned to each group was the maximum degree observed for the patient in all the specific toxicity categories within the group following the CTCAE 4.0 (Common Terminology Criteria for Adverse Events 4.0) classification scale. We evaluated the hematologic, infectious, digestive, renal, neurologic, respiratory, allergic, dermatologic, otologic, and ophthalmologic organic toxicities during the period of time corresponding to induction in solid tumors and previous to maintenance therapy or hematopoietic progenitor transplantation in the leukemia patients. For survival data, overall survival and event-free survival were recorded from diagnosis to the last registered visit or exitus, ranging from 43 to 6.429 days. Data were censored to correctly perform the statistical analyses. 2.3. Genetic Variants 2.3.1. SNPs Selection The pharmacogenetics panel configuration (Supplementary Materials, Table S2) was based on PharmGKB database information, databases of scientific articles on this topic published from 1990 to the present (PubMed, MEDLINE, Cochrane Central Register), and journals in the pediatrics, oncology, and hematology fields. 2.3.2. Samples and DNA Extraction Sample collection: 2-5 mL of peripheral blood per patient was obtained from the Pediatric Oncohematology Department Biobank (this belongs to the national Biobanks Network of the Instituto de Salud Carlos III). It has the required license, reference PT13/0010/0026) or by direct extraction from the patient after informed consent obtention. It was kept frozen (at -20 degC) in EDTA solution. DNA extraction: The "Nucleospin Blood" (Macherey Nagel, Duren, Germany) mini kit was used following the manufacturer's instructions. DNA was eluted in TE (Tris-EDTA) and preserved upon its genotyping. DNA quality control: the final concentration and purity of DNA were evaluated by spectrophotometry with a NanoDrop 2000 (NanoDrop Technologies Inc., Wilmington, DE, USA). 2.3.3. Genotyping Panel for genotyping was designed to include 62 polymorphisms that presented potential implication in patients' treatment efficacy or drugs toxicity based on the bibliographic search performed (design named VIP-Onco). We could obtain suitable samples from 64 patients, and their genotyping was carried out at Centro Nacional de Genotipado (CEGEN, Santiago de Compostela, Spain) by mass spectrometry, employing a MassArray Analyzer (Agena Bioscience, San Diego, CA, USA). 2.4. Statistics The statistical analyses were performed in collaboration with the Biostatistics and Data Science Unit at the Instituto de Investigacion Sanitaria La Fe (Valencia, Spain). The correlation of polymorphisms with severe toxicity or overall survival (OS) and event-free survival (EFS) was calculated with logistic regression and Cox regression, respectively, both penalized with Elastic Net. The association of clinical outcomes with pharmacogene variants was evaluated with R software (version 4.1.0) by fitting logistic or Cox regression models penalized with elastic net (alpha: 0.5; lambda: min; 500 iterations). Elastic net is a type of regularized regression that combines the strengths of Lasso and Ridge regression. It uses variable selection like Lasso and deals with multicollinearity like Ridge. It assigns coefficients only to variables deemed important for explaining the response variable, reducing the risk of overfitting in datasets with more variables than observations, which is the case in this study (and "omic" studies in general) that have a much larger number of variables included than the number of samples (patients) analyzed. Using elastic net as a predictive model-building method does not give confidence intervals or p-values, as these are typical of hypothesis-contrast studies, which are not suitable as they are not able to include all the covariates and confounding factors that we are able to include with the current method . The models included as covariates for each patient all the SNPs included in the panel, age at diagnosis, type of tumor, drugs received, and the clinical data described in Section 2.2. 3. Results 3.1. Descriptive Data 3.1.1. Chemotherapy Drugs Administered Altogether, 24 chemotherapeutic drugs from 37 different protocols (Supplemental Materials, Table S3) were registered. Considering the 64 genotyped patients, 305 chemotherapy cycles were included (a mean of 4.6 cycles/patient). 3.1.2. Clinical and Analytical Toxicities Hematologic, digestive, and infectious toxicities were the most frequently registered. The toxicities with a severity degree over or equal to 3 in the CTCAE 4.0 scale and an incidence greater than 5 cases were: among clinical parameters, fever, neutropenia, and oral mucositis; among analytical parameters, anemia, neutropenia, thrombocytopenia, and hypertransaminasemia (increase of AST, ALT, and GGT). Respiratory toxicity was not frequent (3-7% incidence) but severe in all cases (>=3). Death directly attributable to drug toxicity was not found in the group. 3.1.3. Genotyping The polymorphisms analyzed in our cohort and the frequency of the variants are shown in Supplementary Material, Tables S2 and S4. 3.2. Correlation between Genetic Polymorphisms and Toxicity Only those clinical variables present in five or more patients with severe toxicity (>=3 on CTCAE 4.0) were included in the correlation study. Thus, the analyzed variables were anemia (43 patients), neutropenia (52 patients), thrombocytopenia (30 patients), hypertransaminasemia (ALT, 15 patients; AST, 15 patients; GGT, 10 patients), neutropenic fever (26 patients), and oral mucositis (7 patients). After elastic net penalized logistic regression, associations were found (Table 1) between severe anemia and 12 polymorphisms (7 with protective effect and 5 with increased risk) of 12 genes and severe neutropenia and 16 polymorphisms (10 with protective effect and 6 with increased risk) of 16 genes. Severe thrombocytopenia was also associated with 18 genetic polymorphisms in 16 genes (9 with a protective effect and 9 with an increased risk). 3.3. Correlation between Polymorphisms and Survival 3.3.1. Overall Survival (OS) Logistic regression evidenced associations between overall survival and 26 genetic polymorphisms belonging to 21 different genes. Among these, 14 are factors of poorer prognosis (increased probability of death during follow-up or lower overall survival: HR higher than 1), whereas the other 12 polymorphisms imply a better prognosis (HR lower than 1) (Table 2). 3.3.2. Event-Free Survival Associations (Table 2) between event-free survival and 13 polymorphisms in 13 genes were observed. Seven polymorphisms represent a poorer prognosis and increased probability of relapse (HR higher than 1), whereas the other six were associated with a lower risk of disease progression (HR lower than 1). 4. Discussion Whereas most research in the field of cancer chemotherapy efficacy and toxicity has focused on adults, the current paper focuses on an orphan group, infants under 18 months old, using a specially designed pharmacogenetics panel. Particular infant pharmacokinetics and pharmacogenetic characteristics have been considered. The genetic association between SNP variants and therapeutic response, efficacy, and toxicity in oncologic infant patients during a 12-year follow-up at the tertiary Hospital Universitario y Politecnico La Fe (Valencia, Spain) has been evaluated. Given the nature of the data (62 genetic polymorphisms and 37 types of clinical toxicities vs. 64 patients), which include many more variants than observations, advanced statistical analyses were required, and thus, logistic regression with an elastic net penalty was employed. This statistical method offers robust results. For associations between genetic variants and survival, Cox regression was employed because it considers the temporal evolution. Associations have been found between 57 variants of 38 SNPs in 32 different genes and the incidence of severe toxicity (anemia, neutropenia, and thrombocytopenia) and survival (overall and event-free). The genes associated encode proteins that transport or metabolize the drugs administered (7 and 17 genes, respectively), have a role in DNA repair and/or control of tumor suppression (5 genes), or other cell functions (4 genes). The SNPs that showed stronger association with any of the clinical parameters evaluated (toxicities and survival) are marked in bold-type in Table 1 and Table 2 and are summarized based on the function of the proteins encoded in Table 3. These biological functions are drug transporters or metabolizers (7 and 17 genes, respectively); DNA repair and/or control of tumor suppression (5 genes); and other functions (4 genes). Our results show variants that increase the risk of hematological toxicities and variants that decrease this risk, showing a protective effect. The same happens with prognosis, in terms of overall survival and relapse. For clarity, we will only include in this discussion the findings that increase the risk of toxicity, death, or relapse. 4.1. Hematological Toxicities In our hands, patients carrying rs1801131 GT in MTHFR showed the highest increased risk (OR 1.73) of anemia amongst all the SNPs analyzed, which is consistent with other published works, including pediatric oncology scenarios . Regarding neutropenia, we found two SNPs with the highest risk: C8orf34 rs1517114 GC (OR 1.50) and XPC rs2228001 GT (OR 4.63). The second is discussed in the following paragraph, and regarding the first, it has been reported as a predictor of severe diarrhea in irinotecan-treated patients , but as far as we know, this is the first work describing increased neutropenia risk. Genetic variants that increase the activity of enzymes responsible for metabolizing drugs that require activation could increase the toxicity of the drug. This occurs with CYP2B6 and cyclophosphamide . Our study reinforces that rs4802101 TC increases the activity of CYP2B6 and favors the transformation of cyclophosphamide into 4-hydroxy-cyclophosphamide, which is converted to the phosphorous mustard (antineoplastic metabolite) and acrolein, the metabolite that is especially toxic for hematopoietic tissue and urinary apparatus, responsible for very common hemorrhagic cystitis, and in our hands showing an increased risk of thrombocytopenia (OR 1.70). AG at rs1045642 in the ABCB1 gene has shown a 1.70 OR for thrombocytopenia. This gene encodes glycoprotein-G, a very much studied efflux pump in the field of pharmacogenetics. In the literature, the A allele seems to increase thrombocytopenia risk in the context of pediatric leukemia . SOD2 encodes superoxide dismutase 2, a mitochondrial enzyme responsible for diminishing reactive oxygen species and involved in the metabolism of many chemotherapeutic drugs, such as cisplatin, asparaginase, methotrexate, and cyclophosphamide. Variant rs4880 GG has been previously related to ototoxicity , but instead we have found an association with thrombocytopenia (OR 1.73). Platelets, in the same manner as cochlear cells, are very vulnerable to free radicals. If the GG variant is causing a decrease in activity of the SOD2 enzyme, the accumulation of ROS could be toxic for the platelets, promoting the observed toxicity. The effects of TNRSF11B are discussed in the following paragraph. 4.2. Survival Establishing a relationship between a polymorphism and prognosis is usually much more complex, since multiple biases and confounding factors interfere. However, we have emphasized those results with stronger associations after employing models that are really stringent. Some of the HR values obtained are quite high, especially considering that we are dealing with a small sample size, so we have to keep in mind that this is an initial pilot study and the results need to be validated in larger populations. Even so, some of our results are supported by previous studies, and of special interest in our group is the relationship between MTHFR rs1801133 GG and overall survival. In a previous publication from our group , with a totally different cohort of neuroblastoma patients, we already found that this variant was correlated with poorer overall survival in both normal and amplified MYCN patients. It is not clear yet if these results are through a direct effect of the enzyme on the chemotherapeutic drugs, or related to DNA methylation and or synthesis, or a combination of all, but recent literature find similar results in different adult and pediatric cancers . Our findings regarding TNFRSF11B rs2073618 GG seem to be new in the context of cancer survival. We found 1.84 HR but also 1.77 OR regarding thrombocytopenia. This gene has been extensively studied in the context of rheumatoid arthritis and osteoporosis, but also with aromatase inhibitors in the context of breast cancer. Being part of the TNF receptor family, we could hypothesize that this signaling pathway could be altered in patients bearing this variant, with potential effects on their immune system tumor surveillance. TNFR2 directly promotes the proliferation of some kinds of tumor cells and, by activating immunosuppressive cells, it supports immune escape and tumor development . Regarding XPC, we found decreased overall survival in patients rs2228001 GT (HR 1.68) (also in TT to a lesser extent) and also a remarkably high odds ratio for neutropenia probability (OR 4.63). The literature supports our findings relating the observed effects of platinum compounds on toxicity but also on prognosis in different types of cancer . These two effects could in fact be a consequence of each other: if the T allele is really promoting neutropenia, we could expect that the clinician's response would be a delay in the following chemotherapy cycles and/or drug dose reduction. This "actuation," meaning a decrease in the therapeutic effect, could be the cause of the decreased survival and not the genetic variant itself, but this hypothesis must be confirmed. CYP3A4 also showed a variant with a remarkable HR (rs2740574 CT, 2.92). The CYP3A4 enzyme metabolizes doxorubicin, vinblastine, and vincristine, among other drugs, and the T allele in this SNP promotes increased transcription of the gene , a higher drug clearance rate, and lower exposure to the drug, which could explain a lower therapeutic effect. The highest HR found in our study, related to overall survival, belongs to rs4149015 GA in SLC01B1 impaired transport of many substrates, including methotrexate and irinotecan . This would mean a lower entry into hepatocytes and therefore a reduced clearance of these drugs, which could lead to toxicity and a higher risk of death. Last but not least, we have two more genes associated with event-free survival. The first is CDA, where rs3215400 del.del has been found to be associated with overall survival; it showed 2.19 HR for the first and 2.92 for the later. The encoded enzyme, cytidine deaminase, is directly involved in the inactivation of citarabine, and the selected variant implies a deficient activity. Our patients carrying this variant also showed more neutropenia (OR 1.20). All these effects are in agreement with previously published results both in pediatric and adult populations . Our last relevant SNP is located in SLC19A1, rs1051266. TT showed a 1.61 HR for event-free survival. This gene encodes RFC-1, or folate transporter protein 1, named for its role in the transport of folate and drugs such as methotrexate (administered to 26.6% of our patients). TT carriers have a worse prognosis, in agreement with the literature, in pediatric leukemia patients and with children and young adults with osteosarcoma, where higher levels of methotrexate are found in the systemic circulation . In our hands, fewer SNPs were found to be associated with EFS compared to those associated with OS. We don't have a certain explanation for this, but it is true that the patients without any relapse events were 42, while the patients alive were 51 at the end of the follow-up, so we had a larger sample size for the OS analyses, and this could increase the probability of finding associations. If the associations proposed in this pilot study could be validated in future, larger studies, then the goal would be to use the information in clinical practice. To do so, the inclusion of a high-quality pharmacogenetics report in the electronic medical records seems mandatory as a tool for helping clinicians prescribe in a pharmacogenetics-guided manner. Of course, pediatric oncologists would need to evaluate this new piece of information together with all the rest of the data in order to make a therapeutic decision. In our opinion, only the variants with a higher level of evidence should be included in these clinical pharmacogenetic analyses (those included in drug labels, those included in a relevant clinical implementation guideline, those considered Clinical Annotations Level 1 in PharmGKB, and lastly, those that are not yet in the three previous sources but do have supporting evidence in the literature of a relevant effect on efficacy/toxicity). 5. Conclusions The results of the present work encourage further studies in order to confirm the findings. Increasing scientific contributions support the use of pharmacogenetics as a tool to guide the optimization of treatments, regarding both toxicity and the pharmacological response of patients. Pediatric oncologists are becoming aware that these strategies could make a difference in patients' clinical management, especially in vulnerable populations. Confirming the findings of this pilot study could bring some of the proposed SNPs to a level of clinical evidence high enough to include them in the guidelines for chemotherapeutic drug use in infants, a population with special vulnerability. The present work is a pilot study, and it has several limitations that we need to keep in mind to prevent unreasonable expectations: It has a small sample size regarding statistics (although a large one if we take into account that the patients included are very rare); it mixes together patients with different tumor types (although the tumor type is included as a covariate to decipher its weight); and the clinical data were retrieved retrospectively from medical records, which is never as accurate as having a prospective design (we tried to guarantee the maximum accuracy with a well-designed clinical data sheet and two different pediatric oncologists collecting the data). We find two steps to confirm these findings: First, a pilot ambispective study very similar to the one presented here, but multicentric at the national or, better yet, international level, to confirm the associations of the SNPs and clinical variants presented here in a larger cohort. The chemotherapy protocols are common in Europe, and this would definitely be an advantage, and we could also start recording long-term follow-up of the patients (cancer survivorship programs, e.g.). After this, with the confirmed SNPs, a more ambitious study could be proposed, acting prospectively, modifying the therapy according to the validated genetic variants in the patients, without altering the drug label specifications of use, and therefore not involving a clinical trial but a real-world quaternary prevention study. Acknowledgments We want to thank Desire Ramal, from the Unidad de Oncologia Pediatrica La Fe, for her constant support. Supplementary Materials The following supporting information can be downloaded at: Table S1. Chemotherapy Doses; Table S2. Genotyping panel; Table S3. Protocols; Table S4. Genotype in variants associated with clinical outcomes. Click here for additional data file. Author Contributions Conceptualization, M.J.H. and A.C.; methodology, M.J.H. and P.B.; validation, A.U., G.G.O. and L.S.; formal analysis, D.H.; investigation, A.J.-R., M.d.M.A., J.B. and P.G.; resources, M.J.H., P.B. and A.C.; data curation, A.U, G.G.O. and P.G.; writing--original draft preparation, A.U. and L.S.; writing--review and editing, S.F.A. and M.J.H.; supervision, M.J.H., A.C. and S.F.A.; project administration, M.J.H. and A.C.; funding acquisition, M.J.H. and A.C. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board (or Ethics Committee) at CEIm Hospital Universitario y Politecnico La Fe (protocol code 2016/150 and date of approval: 7 September 2016). Informed Consent Statement Informed consent for publishing the results of the patients' analyses is not requested in this type of study, as the data are anonymized and no personal images or other identification information is retrieved. All patients received and signed an Informed Consent for the Ethics Committee before participating in the study. Data Availability Statement All the data generated in the study is included here. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Patients' inclusion diagram. Patients recruited and those finally included in the study after applying exclusion criteria such as the absence of clinical data or biological data (a blood sample for genotyping). Figure 2 The most common toxicities observed in the study patients during the follow-up period, as well as their severity level according to the CTCAE 4.0 classification. The proportion of patients with anemia, neutropenia, thrombocytopenia, liver enzyme elevations, oral mucositis, and neutropenic fever is shown. The toxicity is graded in five categories according to the CTCAE 4.0 Classification, without any patient achieving grade 5 toxicity. CTAE: Common Terminology Criteria for Adverse Events. ALT: alanine aminotransferase; AST: aspartate aminotransferase; GGT: gamma-glutamyl transferase. cancers-15-01424-t001_Table 1 Table 1 Polymorphisms associated with toxicities. Those with protective effects are highlighted in green, while those with increased risk are highlighted in red. Panel 1-A includes results of anemia, Panel 1-B neutropenia and Panel 1-C, the results of thrombocytopenia. Bold-type values are those over 1.50 or under 0.66. 1-A ANEMIA GENE SNP VARIANT ODDS RATIO ATIC rs16853826 GG 1.14 MTHFR rs1801131 GT 1.73 DPYD rs1801158 TC 1.08 TNFRSF11B rs2073618 GG 1.42 UGT1A rs4124874 TT 1.13 ABCB1 rs1045642 GG 0.91 ITPA rs1127354 CC 0.91 FCGRA1 rs1801274 AG 0.81 ESR1 rs2234693 TT 0.85 XRCC1 rs25487 TT 0.87 CDA rs3215400 del.C 0.86 ATIC rs4673993 TT 0.73 1-B NEUTROPENIA GENE SNP VARIANT ODDS RATIO C8ORF34 rs1517114 GC 1.50 MTHFR rs1801133 GA 1.02 XPC rs2228001 GT 4.63 CDA rs3215400 del.del 1.20 ABCC2 rs3740066 TC 1.03 CYP2C19 rs4244285 AG 1.15 ABCB1 rs1045642 GG 0.57 ERCC1 rs11615 GG 0.80 C8ORF34 rs1517114 GG 0.85 ATIC rs16853826 GA 0.52 FCGR2A rs1801274 GG 0.88 MTRR rs1801394 GG 0.75 XRCC1 rs25487 TT 0.93 CDA rs3215400 del.C 0.78 MTR rs3768142 TT 0.70 FCGR3A rs396991 CA 0.82 CYP2B6 rs4802101 TT 0.85 1-C THROMBOCYTOPENIA GENE SNP VARIANT ODDS RATIO ABCB1 rs1045642 AG 1.70 C8ORF34 rs1517114 GC 1.45 MTHFR rs1801131 GT 1.41 MTHFR rs1801133 GG 1.32 MTRR rs1801394 AG 1.06 TNFRSF11B rs2073618 GG 1.77 XPC rs2228001 GT 1.40 CYP2B6 rs4802101 TC 1.70 SOD2 rs4880 GG 1.73 MTHFR rs1801131 TT 0.79 ATIC rs16853826 GA 0.75 ESR1 rs2234693 TT 0.77 CDA rs3215400 del.C 0.79 FCGR3A rs396991 CC 0.93 ATIC rs4673993 TT 0.73 SLC22A1 rs683369 CG 0.74 ABCC2 rs8187710 GG 0.38 cancers-15-01424-t002_Table 2 Table 2 Polymorphisms associated with survival variation. The associations of SNP variants with protective effects are shown in green, and those with an increased probability of a lower survival rate are shown in red. Panel 2-A shows the results according to overall survival, and Panel 2-B shows the results according to event-free survival. Bold-type values are those over 1.50 or under 0.66. 2-A GLOBAL SURVIVAL GENE SNP VARIANT HAZARD RATIO ATIC rs16853826 GG 1.22 SLC19A1 rs1051266 TT 1.06 MTHFR rs1801133 GG 3.12 MTRR rs1801394 AG 1.32 TNFRSF11B rs2073618 GG 1.84 XPC rs2228001 GT 1.68 XPC rs2228001 TT 1.10 ENOSF1 rs2612091 CT 1.05 CYP3A4 rs2740574 CT 2.92 ERCC1 rs3212986 CC 1.19 CDA rs3215400 del.del 1.90 SLC01B1 rs4149015 GA 3.96 CYP2C19 rs4244285 AG 1.09 SOD2 rs4880 AG 1.34 SLC19A1 rs1051266 CT 0.70 TP53 rs1042522 GG 0.09 TP53 rs1042522 CG 0.19 ITPA rs1127354 CC 0.59 NQO1 rs1800566 GG 0.56 MTRR rs1801394 GG 0.41 SLC22A2 rs316019 CC 0.25 MTR rs3768142 GT 0.95 FCGR3A rs396991 CC 0.48 UGT1A rs4124874 GT 0.77 SLC01B1 rs4149015 GG 0.26 FOLH1 rs61886492 GG 0.15 2-B EVENT-FREE SURVIVAL GENE SNP VARIANT HAZARD RATIO SLC19A1 rs1051266 TT 1.61 UMP rs1801019 CG 1.05 XRCC1 rs25487 TT 1.39 ENOSF1 rs2612091 CT 1.08 SLC22A2 rs316019 CC 1.16 CDA rs3215400 del.del 2.19 CYP3A5 rs776746 CT 1.28 TP53 rs1042522 CG 0.85 SLC01B1 rs11045879 TT 0.72 ITPA rs1127354 CC 0.67 CYP3A4 rs2740574 TT 0.59 SLC01B1 rs4149056 TT 0.87 ABCC2 rs8187710 GG 0.63 cancers-15-01424-t003_Table 3 Table 3 Classification of the genes where the more relevant associations between SNPs and toxicity/survival have been found, according to the main function of the protein (transport, metabolism, DNA reparation, tumor suppression, or other biological functions). SLC: Solute carrier family; ABC: ATP-binding cassette family; EFS: event-free survival; OS: overall survival; Tbp: thrombocytopenia; An: anemia; Np: neutropenia. TRANSPORT PROTEINS Solute Carrier Family (SLC) GENE SNP VARIANT EFFECT SLC01B1 rs4149015 GA-GG OS SLC19A1 rs1051266 TT EFS SLC22A2 rs316019 CC OS ATP-binding cassette family (ABC) GENE SNP VARIANT EFFECT ABCB1 rs1045642 AG, GG Tbp, Np ABCC2 rs8187710 GG Tbp, EFS METABOLIC ENZYMES Phase I GENE SNP VARIANT EFFECT CYP2B6 rs4802101 TC Tbp CYP3A4 rs2740574 TT, CT EFS, OS FOLH1 rs61886492 GG OS NQO1 rs1800566 GG OS SOD2 rs4880 GG Tbp CDA rs3215400 del.del EFS, OS ITPA rs1127354 CC OS Phase II MTHFR rs1801131 GT An rs1801133 GG OS MTRR rs1801394 GG-AG OS ATIC rs16853826 GA Np DNA REPAIR GENE/TUMOR SUPPRESSOR GENE GENE SNP VARIANT EFFECT TP53 rs1042522 CG-GG OS XPC rs2228001 GT Np, OS C8ORF34 rs1517114 GC Np GENES WITH OTHER FUNCTIONS GENE SNP VARIANT EFFECT TNFRSF11B rs2073618 GG Tbp, OS FCGR3A rs396991 CC OS Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). 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PMC10000842 | Chemical, physical, and infectious agents may induce carcinogenesis, and in the latter case, viruses are involved in most cases. The occurrence of virus-induced carcinogenesis is a complex process caused by an interaction across multiple genes, mainly depending by the type of the virus. Molecular mechanisms at the basis of viral carcinogenesis, mainly suggest the involvement of a dysregulation of the cell cycle. Among the virus-inducing carcinogenesis, Epstein Barr Virus (EBV) plays a major role in the development of both hematological and oncological malignancies and importantly, several lines of evidence demonstrated that nasopharyngeal carcinoma (NPC) is consistently associated with EBV infection. Cancerogenesis in NPC may be induced by the activation of different EBV "oncoproteins" which are produced during the so called "latency phase" of EBV in the host cells. Moreover, EBV presence in NPC does affect the tumor microenvironment (TME) leading to a strongly immunosuppressed status. Translational implications of the above-mentioned statements are that EBV-infected NPC cells can express proteins potentially recognized by immune cells in order to elicit a host immune response (tumor associated antigens). Three immunotherapeutic approaches have been implemented for the treatment of NPC including active, adoptive immunotherapy, and modulation of immune regulatory molecules by use of the so-called checkpoint inhibitors. In this review, we will highlight the role of EBV infection in NPC development and analyze its possible implications on therapy strategies. Epstein Barr Virus nasopharyngeal carcinoma tumor associated antigens immunotherapy tumor microenvironment check-point inhibitors This research received no external funding. pmc1. Background Carcinogenesis is a multistep process that consists of the transformation of healthy cells into cancer cells. This process goes through three different phases: initiation, promotion, and progression. In the first stage, a genetic mutation predisposes to the development of cancer. During the second stage, the accumulation of other genetic mutations allows the cells to acquire a cancerous phenotype. Finally, in the progression phase, the further accumulation of mutations results in genetic heterogeneity as well as the acquisition of a more aggressive phenotype and the development of cellular polyclonality . Chemical, physical, and infectious agents may induce carcinogenesis. Viruses are the major causes of infectious-induced carcinogenesis . The occurrence of virus-induced carcinogenesis is a complex process caused by an interaction across multiple genes, mainly depending by the type of the virus. Mechanisms underlying virus-induced carcinogenesis are not clearly established. Molecular mechanisms mainly suggest the involvement of a dysregulation of the cell cycle . Among the virus-inducing carcinogenesis, Epstein Barr Virus (EBV) plays a major role in the development of both hematological and epithelial malignancies . EBV belongs to the gamma-herpesviridiae family and it is the causative agent of infectious mononucleosis. Several lines of evidence demonstrated that nasopharyngeal carcinoma (NPC) is consistently associated with EBV infection. Specifically, about 95% of nonkeratinizing and undifferentiated NPCs are associated with EBV although, like other solid tumors, genetic susceptibility and environmental factors also contribute to their development. EBV is found in both malignant and premalignant lesions of nasopharynx . In addition, numerous studies reported an elevated anti-EBV antibodies titers and the presence of EBV DNA in nearly all endemic undifferentiated variant of NPC . Although EBV efficiently infects and induces growth transformation of epithelial cells in vivo, its ability are much less efficient in vitro . To enter host cells, EBV uses a glycoprotein named Gp350/20, which in turn interacts with the complement receptor 2 (CR2/CD21). The latter is a membrane-associated protein that plays a key role in B lymphocyte function. Nevertheless, CD21 is also expressed by epithelial cells of both nasopharynx and oropharynx . Once infected host cells, EBV rapidly replicates and gets out of the infected cells causing their lysis (lytic phase). Nevertheless, EBV can also enter a dormant phase, named "latency". During this phase, characterized by a partial expression of the viral antigens, three forms of latency (namely "type I, type II, and type III" latency) can be distinguished based on the number of the expressed antigens. Neoplastic transformation induced by EBV is most likely caused by the latent phase rather than the lytic phase. During the latent phase, EBV is not silent and multiple viral genes are activated. Some of these genes can promote cancerogenesis in presence of host cell DNA mutations. Products of these viral genes are proteins as well as macromolecules, which play an important role in the permanence of EBV in host cells as well as in the transmission of viral DNA into daughter cells during mitosis . As a result, these macromolecules and especially those with a protein structure are considered as viral oncoproteins and, even more, as tumor-associated antigens (TAAs) in the host infected cells. TAAs are major players for eliciting a host immune response. In this review, we will highlight the role of EBV infection in NPC development and analyze its possible implications on therapy strategies. 2. EBV-Induced Carcinogenesis in NPC During EBV latent infection phase, expression of the full viral genome is restricted and few viral genes are expressed, leading the virus to escape to its recognition and destruction by the host immune system as well as to remain silent within the host cell. As we mentioned before, there are three forms of latency expressed by EBV-infected cells, each one characterized by a unique expression of EBV-associated proteins and RNAs. Type I latency is associated with the expression of one viral antigen, namely EBNA1 (EBV nuclear antigen-1); type II latency by the expression of EBNA1, LMP1/2 (latent membrane protein) and EBERs (EBV-encoded small RNAs). Lastly, type III latency is associated with the greatest number of viral antigens, namely EBNA 1, 2, and 3, EBNA-LP, LMP-1 and 2, and EBERs . Type II latency is acknowledged to be associated with Hodgkin's lymphoma and NPC . According to some lines of evidence , following a first phase of EBV infection of peripheral blood B-lymphocytes, EBV reactivates in B-lymphocytes and especially in those present in the tonsillar Waldeyer's ring. Infected B -lymphocytes release several virions. As a result, virus directly sheds from lymphocytes into epithelial cells of nasopharynx, establishing a latent infection, which can lead to cancerogenesis. Cancerogenesis may be induced by the activation of different EBV "oncoproteins" such as EBNA1, LMP1, and LMP2. EBNA1 binds to the origin of replication of the viral genome and enables the viral episome to segregate with the host chromosomes during mitosis. EBNA1 can also lower the nuclear levels of P53, leading to an impaired DNA repair and genetic instability . P53, better known as "the guardian of genome" plays a crucial role in regulating cell cycle and promoting DNA-damage repair. In case of DNA-damage, P53 blocks the cell cycle and stimulates DNA-damage repair and when the damage cannot be repaired, P53 stimulates apoptosis, thus preventing DNA damage from being perpetrated on daughter cells. When P53 is down regulated, DNA-mutations could be acquired by cells inducing carcinogenesis . LMP-1, like other oncogenes, represents a key transforming protein. It inhibits the differentiation of epithelial cells, stimulating their growth by promotion of cell cycle progression. Furthermore, LMP-1 also stimulates the expression of the epidermal growth factor receptor (EGFR), promoting cell growth through activation of the MAP-Kinase pathway . In addition, in epithelial cells, LMP1 inhibits P53-mediated induction of apoptosis, and more importantly, induces the sensibilization of lymphocytes to TGF-beta, dampening the immune response against cancer cells . Lastly, two isoforms of LMP2 exist, namely LMP2A and 2B. Both are able to initiate oncogenic transformation in EBV-infected cells through the activation of protumorigenic signaling pathways such as promotion of cancer cell division and migration by activation of the Ras/PI3K/Akt and the b-catenin/Wnt pathways by LMP2A . In summary, it can be stated that EBV viral oncoproteins have the ability to induce carcinogenesis although they need further alterations in both oncogenes and tumor suppressor genes of infected cells. 3. Impact of EBV-Infection on the Tumor-Microenvironment of NPC The tumor microenvironment (TME) is the region surrounding the tumor cells. It includes stromal cells such as fibroblasts, endothelial cells, and immune cells as well as non-cellular components such as the extracellular matrix. On the other hand, the tumor immune microenvironment (TIME) refers only to the immune component of the TME and does not include the stromal and extracellular components . Both immunostimulating and immunosuppressive cells are present in TIME; the former stimulates an antitumor immune response, the latter inhibits the antitumor immune response and promotes tumor progression. Composition of the TIME strongly depends on the cytokines production during cancer progression. First, cancer cells stimulate an innate and aspecific response mediated by IL-1, IL-6, and TNF-alpha production; this kind of response mainly stimulates macrophages and neutrophil granulocytes to attack the tumor cells. Nevertheless, this aspecific response is not efficient enough at fighting cancer cells. Therefore, then, mainly due to dendritic cells engulfing and exposing TAAs, an adaptive immune response characterized by cytokines production from helper T-lymphocytes is induced. If "the game is conducted" by the TH1 helper lymphocytes (inflammatory), a robust anti-cancer immune response develops and cytokines such as IL-12, IL-2, and IFN-gamma mainly stimulate cytotoxic T-lymphocytes (CTLs) and natural killer cells (NKs) . On the other hand, if the mix of cytokines produced (mainly by cancer cells) stimulates the generation of T-helpers other than TH1, for example TH2, Th17, and/or T-Regs (regulatory), the result will be a particularly immunosuppressed TIME, namely rich in T-Regs, M1 macrophages, and myeloid-derived suppressor cells (MDSCs). Overall, in squamous cell carcinoma of the head and neck (SCCHN), TIME mainly contains immunosuppressive cells such as T-regulatory lymphocytes (T-Regs), M1 macrophages, and myeloid-derived suppressor cells (MDSCs). In contrast, a very low number of immunostimulanting cells such as TH1 helper lymphocytes, CTLs, and NKs are present. In NPC, TIME also exhibits mainly an immunosuppressive infiltration and, even more, EBV-positive NPC displays a higher immunosuppressive TIME as compared to that of EBV negative NPC . In NPC, besides the above-mentioned immunosuppressive cells, TIME also contains two unique cell subtypes, namely LAMP3 dendritic cells and M1-M2 macrophages. LAMP3 dendritic cells express on their membrane both cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed death 1 ligand (PD-L1). Their interaction by CTLA-4 on Tregs and PD-L1 by CTLs induces down-regulation of antigen processing/presentation and induction of energy, respectively . M1-M2 macrophages are cells displaying an intermediate phenotype between tumor-suppressing M1 and tumor-promoting M2 subtypes. M1-M2 macrophages produce a high quantity of inflammatory cytokines such as IL-1, IL-6, and TNF-alpha that in turn stimulate the further recruitment of other macrophages and concomitantly increase the aspecific response in site of the adaptive anti-cancer immune response. From a molecular viewpoint, production of these cytokines is mediate by the constitutive activation of NF-kB signaling (prompted by LMP1 oncoprotein) . The final result is a stimulation of chronic and non-specific inflammation, mainly characterized by the prevalence of macrophages and neutrophil granulocytes instead of dendritic cells. This results in a reduced presentation of TAAs by dendritic cells to naive T lymphocytes and ultimately in a lower production of CTLs and NKs. 4. Translational Implications EBV-infected NPC cells can express proteins potentially recognized by immune cells in order to elicit a host immune response. In addition, NPC cells express numerous immune regulatory molecules such as PD-L1, CD40, CD70, CD80, and CD86 which modulate T-cell activity . Lastly, viral antigen expression by EBV infection can also induce the expression of TAAs. On this basis, three immunotherapeutic approaches have been implemented for the treatment of NPC including active and adoptive immunotherapy and modulation of immune regulatory molecules. Active immunotherapy consists in delivering specific TAAs in the form of therapeutic vaccines with the aim to enhance their recognition on cancer cells by the immune system and ultimately the elimination of malignant cells. Adoptive immunotherapy aims to directly activate effector cells such as CTLs and NKs . 4.1. Active Immunotherapy It consists in administering therapeutic vaccines to patients affected by NPC. Two strategies can be adopted including administration of EBV-associated antigens and administration of adequately "loaded" or "stimulated" dendritic cells (DCs) with associated EBV antigens. DCs are the most efficient antigen presenting cells (APCs) able to present EBV antigens to CTLs. In different pre-clinical reports, authors have shown that DCs are able to expand a functional population of CD8+ T-cells specific to EBV-antigens . One of the first therapeutic anti-EBV vaccine has employed EBV-pulsed DCs. Lin et al. treated 16 patients with recurrent chemo-refractory NPC with four injections at weekly intervals of a vaccine consisting of autologous DCs. Autologous monocyte-derived dendritic cells were cultured from patients with advanced NPC, matured with cytokine, pulsed with HLA-A1101-, A2402-, and B40011-restricted epitope peptides from EBV-LMP2 and subsequently injected into inguinal lymph nodes. Specific CTL responses were elicited or boosted in 9 patients receiving the vaccine, achieving partial tumor reduction in 2 patients . Based on these results, further trials have been implemented with the aim to improve the entity of the elicited specific T-cell mediated response. One approach utilized the transfection of autologous DCs with viral vectors containing EBV antigens. The results demonstrated an increased targeted T-cell proliferation of both CD4+ and CD8+ T-cells, but no significant improvement of clinical responses . In an alternative approach, virus encoding EBV-antigens were directly administered. This modified vaccinia virus Ankara (MVA) expressing a fusion gene of the CD4+ T-cell epitope-rich domain of EBNA1 and a full-length LMP2 was administered to 18 patients with advanced NPC, in the contest of a phase I trial. The drug was well tolerated and it induced differentiation and functional diversification of responsive T-cells specific for EBNA1 and LMP2 . Phase II clinical trials assessing the rate of clinical responses of this type of vaccine are ongoing. Further vaccines were also made using EBV antigens (EBNA1, LMP2, and LMP1) fused together or with adjuvant proteins (heat shock proteins) , having the purpose of eliciting a specific TH1 and CTLs response. However, no data about the obtained clinical responses have been reported. 4.2. Adoptive Immunotherapy This strategy consists in "ex vivo" stimulation of autologous CD8+ T cells followed by intravenous reinfusion of the obtained mature-CTLs into the patient. Straathof et al. carried out a phase I dose escalation clinical trial including ten patients with advanced-chemorefractory NPC treated with autologous EBV-restricted CD8+ lymphocytes. Patient peripheral blood mononuclear cells (PBMCs) were stimulated with autologous irradiated EBV lymphoblastoid cells (LCLs). This process aimed to enhance the expression of EBV antigens. PBMCs were then co-cultured with EBV-specific CTLs to allow their maturation. Lastly, CTLs were reinfused in the patients. The results of the trial demonstrated that the injection was well-tolerated. As a result, a phase II trial was implemented; adding further 13 patients to patients already included in the phase I trial . An overall survival (OS) of 87% and 70% at 2-years, respectively, as well as a good response rate was observed. Generation of CTLs from peripheral blood is difficult, expensive, and time consuming. In fact, the first step consists in the maturation of PBMCs into mature CTLs and this requires their stimulation with LCLs and interleukin-2; to obtain the latter, further expensive procedures are required. Maturation of CTLs may not occur and if it does, it takes time. As a result, in order to implement an effective adoptive immunotherapy some authors also tried to isolate CTLs from the TME, utilizing tumor-infiltrating lymphocytes (TILs). Li et al. reported the results of a trial where 20 patients with locally advanced NPC were treated with a combination of chemotherapy and adoptive TIL-based immunotherapy. In particular, 20 patients received a single-dose of TIL infusion following concomitant chemoradiation (cisplatin plus curative-intent radiation therapy). As result, nineteen of 20 patients experienced an objective antitumor response (overall response rate: 95%), and 18 patients displayed disease-free survival longer than 12 months. The combination therapy showed a good response rate and a good efficacy; however a clear benefit from immunotherapy was not quantifiable since patients were also treated with chemo-radiotherapy. 4.3. Immune Checkpoint Inhibitors TIME of NPC, especially those who are EBV related, is strongly immunosuppressed due to the special pattern of cytokines produced by both immune and tumor cells . On the other hand, NPC cells express numerous immune regulatory molecules (defined as immune checkpoints) which can be targeted. On this basis, it has been hypothesized that the use of checkpoint inhibitors can achieve therapeutic benefits in patients with EBV-related NPC. As we mentioned before, both immune checkpoints CTLA-4 and PD-L1 exert an inhibitory signal on lymphocytes (especially on Treg and CTLs). Checkpoint inhibitors act by removing these inhibitory brakes, leading to stimulation of lymphocytes and in turn, tumor-cells destruction reported was near to 90%. Median PFS rate at 12-month was about 60% . Subsequently, Yang et al. carried out a phase III randomized trial, comparing administration of cisplatin-gemcitabine to that of the combination of camrelizumab and cisplatin-gemcitabine, in patients with platinum-sensitive recurrent/metastatic NPC. PFS, main endpoint of the study, was significantly longer in the camrelizumab group (median 9.7 months) as compared to chemotherapy alone (median 6.9 months, hazard ratio 0.54, p = 0.0002) . Both ORR and toxicity did not significantly differ between the two treatment arms. Toripalimab is a humanized IgG4K monoclonal antibody targeting PD-1. Toripalimab has shown encouraging results in phase I and II trials, both alone and in combination with cisplatin-gemcitabine in platinum-sensitive patients with advanced NPC . In an international, double-blind, randomized, placebo-controlled phase III trial, Mai et al. compared the administration of cisplatin-gemcitabine to that of the combination of toripalimab and cisplatin-gemcitabine in patients with recurrent/metastatic platinum-sensitive NPC . A significant improvement in PFS was reported in the toripalimab arm compared to chemotherapy alone. Median PFS was 11.7 and 8.0 months for toripalimab plus chemotherapy and chemotherapy alone, respectively (hazard ratio = 0.52, p = 0.0003). In addition, the ORR was significantly higher in the toripalimab arm (77.4%) than in the placebo arm (66.4%), (p = 0.0335). A subgroup analysis, in the context of the same trial, revealed that the subgroup of patients with PD-L1 positive tumors benefited more from immunotherapy combination as compared to those affected by PD-L1 negative tumors in terms of DFS. 5. Current Therapeutic Guidelines in NPC and Future Perspectives The correlation between EBV infection and NPC development is well established; however, the molecular mechanisms underlying this type of correlation still need to be better defined. Data available from the literature, mostly extrapolated from carcinogenicity studies on B-lymphocytes, demonstrated that some oncoproteins are involved. These oncoproteins are LMP-1, LMP-2, and EBNA1. The latter, as foreign antigens, are potentially able to elicit a specific host immune response against infected tumor cells (transformed epithelial cells of the nasopharynx). 5.1. Current Guidelines in NPC Most NPCs are diagnosed as locally advanced disease, and in this case, the standard treatment is concurrent or sequential chemo-radiotherapy . About half of the patients treated with upfront chemo-radiotherapy experience disease recurrence. The prognosis of the latter is poor and the therapeutic possibilities are fewer. In patients not amenable to surgery or re-irradiation at the site of recurrence, systemic therapy is the gold standard. When choosing systemic therapy, it is strictly necessary to distinguish two categories of patients, namely platinum-sensitive and platinum-refractory patients. The first category includes those who have never undergone chemotherapy (chemo-naive) or those who experience a relapse more than 6 months after the last platinum-containing therapy. The second category includes those who have experienced disease progression/relapse within 6 months of their last platinum treatment . In patients considered platinum-sensitive, the gold standard is platinum-based polychemotherapy, and in particular "doublets-containing platinum, namely cDDP-5fluorouracil, cDDP-docetaxel, cDDP-gemcitabine, and CBDCA-paclitaxel. The scheme of choice, based on data from randomized trials, is the cDDP-gemcitabine doublet . In platinum-refractory patients, the therapy of choice is monochemotherapy with taxanes (docetaxel or paclitaxel alone), while nivolumab can be used as an alternative . Figure 4 shows a decision algorithm in a patient diagnosed with NPC. 5.2. Future Perspectives Different therapeutic approaches have been aimed at eliciting an adaptive immune response toward EBV oncoproteins utilizing an active or an adoptive immunotherapy. Both therapeutic approaches have not obtained clear and incontrovertible results, so far. As TIME in NPC is highly immunosuppressed and expressed checkpoint molecules, the use of checkpoint inhibitors has been evaluated in several clinical studies (as reported in Table 1). The results obtained were particularly encouraging, so much so that a PD-1 inhibitor (nivolumab) is already used in clinical practice while two other drugs with the same mechanism of action (camrelizumab and toripalimab) have given encouraging results in phase III studies. These drugs will probably soon obtain an approval from the regulatory authorities. In the near future, the combination of checkpoint inhibitors and active and/or adoptive immunotherapy should be implemented. Author Contributions F.P., A.O., F.S., A.P., R.F., M.C. (Michele Caraglia), M.C. (Marco Cascella), F.L., R.A.R., G.S., M.P., M.L.M., A.P., M.M., M.F., F.C., G.D.V.S. and F.I. written and revised the manuscript. All authors have read and agreed to the published version of the manuscript. Data Availability Statement Data are reported in the manuscript and at link Conflicts of Interest The author declares no conflict of interest. Figure 1 There are three forms of latency expressed by EBV-infected cells, each one characterized by a unique expression of EBV-associated proteins and RNAs. Figure 2 Differences between TME and TIME. Figure 3 Check-point inhibitors act by removing the so-called "inhibitory brakes", namely stimuli elicited through particular ligands (CTLA-4 and PD-1 for example) which lead cytotoxic T lymphocytes (CTLs) to anergy. The molecules best known and used in clinical practice are CTLA-4 inhibitors (ipilimumab) and PD-1 inhibitors (nivolumab, pembrolizumab, camtelizumab, and toripalimab). Figure 4 Current therapy options in NPC overall. cancers-15-01626-t001_Table 1 Table 1 Clinical trials with check-point inhibitors enrolling patients with recurrent/metastatic NPC. Trial/Year Phase Setting Drugs ORR PFS OS KEYNOTE-028 2017 I/II R/M P-ref Pembrolizumab 26% 6 months NA CheckMate 358 2017 I/II R/M P-ref Nivolumab 16% NA NA NCT03097939 2020 II R/M P-ref Nivolumab + Ipilimumab 30% 5 months NA CAPTAIN 2020 II R/M P-ref Camrelizumab 28% 4 months 17 months NCI-9742 2018 II R/M P-ref Nivolumab 21% 3 months 17 months POLARIS-02 2020 II R/M P-ref Toripalimab 21% 2 months 17 months NCT026059672 2020 II R/M P-ref Spartalizumab vs. Standard therapy 17% 35% 2 months 7 months 25 months 16 months KEYNOTE-122 2022 III R/M P-ref Pembrolizumab vs. standard therapy 21% 23% 4 months 6 months 18 months 18 months JUPITER-02 2022 III R/M P-sen Toriplaimab + GC Placebo + GC 79% 67% 21 months 8 months NR NR CAPTAIN-1s 2022 III R/M P-sen Camrelizumab + GC Placebo + GC 87% 81% 11 months 7 months NR NR RATIONALE 2022 III R/M P-sen Tislelizumab + GC Placebo + GC 70% 55% 9 months 7 months NR NR Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. |
PMC10000843 | Diagnostics (Basel) Diagnostics (Basel) diagnostics Diagnostics 2075-4418 MDPI 10.3390/diagnostics13050914 diagnostics-13-00914 Review Amide Proton Transfer-Chemical Exchange Saturation Transfer Imaging of Intracranial Brain Tumors and Tumor-like Lesions: Our Experience and a Review Koike Hirofumi 1* Morikawa Minoru 2 Ishimaru Hideki 2 Ideguchi Reiko 3 Uetani Masataka 1 Miyoshi Mitsuharu 4 Imperiale Alessio Academic Editor 1 Department of Radiology, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan 2 Department of Radiology, Nagasaki University Hospital, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan 3 Department of Radioisotope Medicine, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki 852-8588, Japan 4 MR Application and Workflow, GE Healthcare Japan, Hino 191-0065, Japan * Correspondence: [email protected] 28 2 2023 3 2023 13 5 91413 2 2023 23 2 2023 27 2 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). Chemical exchange saturation transfer (CEST) is a molecular magnetic resonance imaging (MRI) method that can generate image contrast based on the proton exchange between labeled protons in solutes and free, bulk water protons. Amide proton transfer (APT) imaging is the most frequently reported amide-proton-based CEST technique. It generates image contrast by reflecting the associations of mobile proteins and peptides resonating at 3.5 ppm downfield from water. Although the origin of the APT signal intensity in tumors is unclear, previous studies have suggested that the APT signal intensity is increased in brain tumors due to the increased mobile protein concentrations in malignant cells in association with an increased cellularity. High-grade tumors, which demonstrate a higher proliferation than low-grade tumors, have higher densities and numbers of cells (and higher concentrations of intracellular proteins and peptides) than low-grade tumors. APT-CEST imaging studies suggest that the APT-CEST signal intensity can be used to help differentiate between benign and malignant tumors and high-grade gliomas and low-grade gliomas as well as estimate the nature of lesions. In this review, we summarize the current applications and findings of the APT-CEST imaging of various brain tumors and tumor-like lesions. We report that APT-CEST imaging can provide additional information on intracranial brain tumors and tumor-like lesions compared to the information provided by conventional MRI methods, and that it can help indicate the nature of lesions, differentiate between benign and malignant lesions, and determine therapeutic effects. Future research could initiate or improve the lesion-specific clinical applicability of APT-CEST imaging for meningioma embolization, lipoma, leukoencephalopathy, tuberous sclerosis complex, progressive multifocal leukoencephalopathy, and hippocampal sclerosis. magnetic resonance imaging amide proton transfer imaging brain tumor tumor like lesion This research received no external funding. pmc1. Introduction Chemical exchange saturation transfer (CEST) is an important molecular magnetic resonance imaging (MRI) technique that can generate image contrast based on the proton exchange between labeled protons in solutes and free, bulk water protons . Amide proton transfer (APT) imaging is the most frequently reported amide-proton-based CEST technique. It generates image contrast by reflecting the associations of mobile proteins and peptides resonating at 3.5 ppm downfield from water . APT-CEST does not require an exogenous contrast and is safe in patients with renal failure or with an intolerance to contrast media. The technique has shown a potential clinical utility for differentiating radiation necrosis from tumor recurrence or progression and for differentiating between high-grade and low-grade glioma . Although the origin of the APT signal intensity (SI) in tumors is unclear, previous studies have suggested that the APT SI is increased in brain tumors because of increased mobile protein concentrations in malignant cells in association with increased cellularity . Previous reports reached a consensus view that the APT SI is higher in highly malignant tumors than in less-malignant tumors and is also higher in malignant tumors than in non-tumor lesions such as radiation necrosis. However, there are few reports on the APT SI of benign tumors and tumor-like lesions including demyelinating disease. We believe that the APT-CEST technique is suitable for application in a wider ranging field, including benign tumors and tumor-like lesions such as demyelinating disease. Therefore, our aim was to review the current applications and findings of the APT-CEST imaging of various brain tumors and tumor-like lesions to help differentiate between benign and malignant tumors and to estimate the nature of lesions. 2. Mechanism of APT CEST Imaging The APT effect is measured as a reduction in bulk water intensity due to the chemical exchange of water protons with labeled backbone amide protons of endogenous mobile proteins and peptides in tissue . Thus, specific molecular information is obtained indirectly through the bulk water signal. Such labeling is accomplished using selective radiofrequency (RF) irradiation at the MR frequency of the backbone amide protons, which are 3.5 ppm downfield of the water resonance, causing saturation that is transferred to water protons . 3. MRI Protocol All patients were examined with a 3-T MRI system (SignaTM Architect, GE Healthcare, Milwaukee, WI, USA) equipped with a 48 channel receiver array coil. APT-CEST images were acquired before contrast administration using single-shot, fast spin echo sequences with the following parameters: field of view = 220 x 220 mm; matrix = 128 x 128; spatial resolution = 1.7 x 1.7 mm; slice thickness = 8.0 mm; repetition time/echo time (TR/TE) = 3000/26.6 ms; and number of slices = 1. Twenty-nine saturation frequency offsets from -7.0 to 7.0 ppm in increments of 0.5 ppm were used to attain a sufficient signal-to-noise ratio within the clinical time frame. The APT imaging consisted of RF saturation (one pulse with a duration of 2000 ms) and an average B1 radiofrequency field equivalent to a continuous RF power level of 2.0 mT (microtesla). The water frequency shift due to field inhomogeneity was measured in a separate image acquired using the water saturation shift referencing method with 11 offset frequencies ranging from -1.875 to 1.875 ppm at intervals of 0.375 ppm. One reference image was acquired without a saturation RF pulse, resulting in a full Z-spectrum within the offset range. The water saturation shift reference image was acquired with a TR/TE of 3000/26.6 ms, an RF saturation amplitude of 0.5 mT, and a total duration of 2000 ms with a continuous wave. The total acquisition time for both the APT and water-saturation shift reference images was 2 min 9 s. 4. APT-CEST Image Processing The APT data were analyzed in MATLAB (MathWorks, Inc., Natick, MA, USA). The magnetization transfer ratio asymmetry (MTRasym) was also calculated. According to the shift-corrected data, the MTRasym values at +-3.5 ppm with respect to water frequency were calculated as follows :MTRasym (+3.5ppm)=Ssat(-3.5ppm)-Ssat(+3.5ppm)S0, where Ssat is the SI with selective imaging and S0 is the SI in the absence of RF for imaging SI normalization. B0 inhomogeneity was corrected on a pixel-by-pixel basis with a water saturation reference map. 5. APT-CEST Imaging of Malignant Tumor 5.1. Oligodendroglioma Oligodendroglial tumors are diffuse, infiltrating gliomas that most commonly arise in the frontal lobe. They have a peak incidence in the fourth or fifth decade of life. They are the second most common intracranial glial tumors and are mainly composed of World Health Organization (WHO) grade II and III oligodendrogliomas . 5.2. Diffuse Astrocytoma The 2016 WHO classification of diffuse astrocytic tumors indicates three grades with different aggressiveness . Although diffuse astrocytoma (WHO grade II) is a relatively slow-growing tumor with a median survival time of 5-8 years, it has a high recurrence rate due to its diffuse infiltration of brain tissue and inherent malignant potential to transform into a high-grade astrocytoma such as anaplastic astrocytoma or secondary glioblastoma . 5.3. Glioblastoma Glioblastoma (GBM) is the most common malignant brain tumor in adults. With a designation of WHO Grade IV, it is also the most lethal primary brain malignancy, with a median survival time of only 15 months . APT-CEST imaging shows an iso-intensity or mild punctate hyperintensity in low-grade gliomas which is significantly lower than what is seen in high-grade gliomas . There are also significant differences in APT SI between grade II and III glioma and grade III and IV glioma . In our case, the APT SI was higher in GBM than in oligodendroglioma and diffuse astrocytoma. 5.4. Malignant Lymphoma Intracranial lymphoma may present as essentially primary central nervous system (CNS) B-cell non-Hodgkin lymphoma (B-cell PCNSL), metastatic CNS lymphoma, intravascular lymphomatosis, T-cell PCNSL, and intracranial Hodgkin's lymphoma . Primary central nervous system lymphoma is responsible for 1-2% of all central nervous system tumors. It encompasses lymphoma exclusively involving the brain, spinal cord, eyes, meninges, and cranial nerves . Primary central nervous system lymphomas show more homogeneous APT SI than high-grade gliomas. The maximum APT SI in primary central nervous system lymphomas was lower than in high-grade gliomas . In our case, B cell lymphoma showed a high APT SI . 5.5. Brain Metastasis Brain metastases (BMs) are associated with a poor prognosis irrespective of the primary tumor they originate from. Lung cancer, breast cancer, and melanoma are the most common causes of BM, accounting for 67-80% of cases . A previous report showed that APT SIs in perilesional tissue in GBM were significantly lower in the solitary brain metastases . Moreover, one report showed that the APT SI of solitary BMs was lower than that of enhanced areas of GBM . In our case, the margin of BMs showed a relatively high APT SI . 6. APT-CEST Imaging of Meningioma Meningioma is the most frequently diagnosed primary brain tumor in adults . Meningiomas are categorized into three WHO grades with 15 histological subtypes, indicating heterogenous clinical and molecular genetic characteristics . As most meningiomas are benign and categorized as WHO Grade I with a slow-growing behavior, most require no immediate treatment. However, some subtypes corresponding to WHO grades II and III are associated with a higher risk of recurrence and shorter survival times. One study investigated the feasibility of APT-CEST imaging for differentiating benign from atypical meningiomas , while another study investigated the feasibility of APT-CEST imaging for differentiating growing meningiomas from non-growing meningiomas . Preoperative transcatheter arterial embolization (TAE) of meningiomas with polyvinyl alcohol microparticles has often been performed and is considered a safe, efficient, and cost-effective method with few complications . However, to our knowledge, there are no reports of APT-CEST imaging in connection with the embolization of a meningioma. In our case, the APT SI of an atypical meningioma decreased after TAE . 7. APT-CEST Imaging of Benign Lesion 7.1. Sphenoid Sinus Mucocele Paranasal sinus mucocele is defined as the accumulation and retention of mucoid secretion within the sinus, leading to the thinning, distension, and erosion of its bony walls. Sphenoid sinus mucocele is pathologically benign and comprises 1-2% of all paranasal sinus mucoceles . To the best of our knowledge, there are no reports of APT-CEST imaging of sphenoid sinus mucocele. However, this lesion is not a tumor, and a low APT SI is expected. In our case, sphenoid sinus mucocele showed a low APT SI . 7.2. Solitary Fibrous Tumor The solitary fibrous tumor (SFT) is a new combined entity for grade I-III soft-tissue tumors. It was introduced in the 2016 World Health Organization classification of tumors of the central nervous system . In our case, this tumor was pathologically diagnosed as grade I after surgery. There are no reports on APT-CEST imaging of SFT. However, this lesion is a benign tumor, and we expected that its APT SI would be lower than that of a malignant tumor because SFT typically shows low proliferation. In our case, SFT showed a low APT SI . 7.3. Schwannoma Schwannoma is a benign tumor and develops from the Schwann sheath. The cranial nerve that is most often affected (in 90% of cases) is the vestibulocochlear nerve (cranial nerve VIII), followed by the trigeminal nerve (cranial nerve V). Acoustic schwannomas grow into the cerebellopontine angle, displacing the brainstem and cerebellum. In most cases, these originate from within the internal auditory canal, the dilation of which is an early radiological sign of tumor growth . This lesion is a benign tumor, and we expected that the APT SI would be lower than that of malignant tumor because the proliferation ability of schwannoma is expected to be low. However, schwannoma showed a high APT SI in our case . A recent study showed that the APT SI in schwannomas with high SI on a T2-weighted sequence was higher than that of meningiomas . 7.4. Lipoma Intracranial lipomas are rare growths that represent less than 0.1% of all brain tumors. Most intracranial lipomas are asymptomatic and are found incidentally on imaging while assessing other conditions . There are no reports on the APT-CEST imaging of a lipoma. This lesion is a benign tumor, and we expected the APT SI of intracranial lipoma would be lower than that of malignant tumors because the proliferation ability of lipomas is expected to be low. In our case, the suspected lipoma showed a low APT SI . 8. APT-CEST Imaging of Demyelinating Disease and Tumor-like Lesion 8.1. Radiation Necrosis The occurrence and extent of radiation necrosis depends on the age of the patient receiving radiotherapy and the lesion volume . A previous study reported that the APT SI of a glioma was higher than that of radiation necrosis . In our case, radiation necrosis showed a low APT SI . 8.2. Leukoencephalopathy Toxic leukoencephalopathy is characterized by progressive damage to the white matter. Its causes include a wide spectrum of diseases that may injure and cause structural alteration to the white matter. The insults may be due to toxic metabolites secondary to chemotherapy or immunosuppressive therapy, environmental, or infectious in origin . There are no reports on APT-CEST imaging of leukoencephalopathy. However, this lesion is not a tumor, and we expected the APT SI to be low because the proliferation ability of leukoencephalopathy is expected to be low. In our case, suspected leukoencephalopathy showed a low APT SI . 8.3. Tuberous Sclerosis Complex Tuberous sclerosis complex (TSC) is an autosomal dominant disorder with high clinical variability. It shows various features on brain imaging, including subependymal nodules, radial bands, cortical hamartomas, and subependymal giant cell astrocytomas . In our case, this lesion was suspected to be cortical hamartoma. There are no reports on APT-CEST imaging of TSC and hamartoma. The lesions are benign, and we expected that the APT SI would be lower than that of malignant tumor because the proliferation ability of hamartoma is expected to be low. In our case, the suspected hamartoma showed a low APT SI . 8.4. Progressive Multifocal Leukoencephalopathy Progressive multifocal leukoencephalopathy (PML) is a rare but often fatal brain disease caused by the reactivation of the polyomavirus JC (JCV) . PML almost exclusively affects immunocompromised patients such as those with HIV/AIDS . In systemic lupus erythematosus, risk factors for CNS infection include disease activity, current and cumulative corticosteroid dose, and the use of other immunosuppressants There are no reports on APT-CEST imaging of PML. However, this lesion is not a tumor, and we expected the APT SI would be low because the proliferation ability of PML is expected to be low. In our case, PML showed a low APT SI . 8.5. Hippocampal Sclerosis Hippocampal sclerosis (HS) is the most common histopathologic abnormality found in adults with drug-resistant temporal lobe epilepsy . There are no reports on APT-CEST imaging of HS. However, this lesion is not a tumor, and we expected the APT SI to be low because the proliferation ability of HS is expected to be low. In our case, PML showed a low APT SI . 9. Discussion Recent studies on gliomas have demonstrated a positive correlation of hte APT SI with the cell proliferation index . These results suggest that high-grade tumors, which demonstrate a higher proliferation, have higher densities and numbers of cells (and higher concentrations of intracellular proteins and peptides) than low-grade tumors. The concentration of mobile proteins and peptides per cell may increase with the grade of glioma. In the study of Togao et al., tumors with necrosis showed a higher APT SI than those without necrosis. Although it was difficult to confirm whether this was a direct relationship, highly concentrated mobile proteins and peptides in the extracellular space, such as microscopic necrosis or fluid collection in the microcystic space, might also increase the APT SI in tumors. The alternation in tissue pH might affectthe APT SI. Our case also showed that high-grade tumors tended to have a higher APT SI than low-grade tumors, and that demyelinating disease and tumor-like lesions tend to have a low APT SI. TAE is a standard pre-operative procedure for meningiomas aimed at reducing intra-operative bleeding and facilitating dissection. In our case of atypical meningioma after TAE, contrast-enhanced, T1-weighted sequences showed no apparent change to the meningioma, but APT-CEST imaging showed a decrease in the SI . After the operation in our case, a part of the tumor showed necrosis and ischemic change upon pathological analysis. APT-CEST imaging may be used as an indicator of therapeutic effects on tumors. Moreover, APT-CEST imaging does not involve the injection of a contrast agent and can be used in patients with renal failure and those who show adverse reactions to contrast media. There have been few reports on APT-CEST imaging of benign lesions. In our case, they tended to show low APT SIs. However, schwannoma showed a high APT SI consistent with T2 hyperintensity. The area showing T2 hyperintensity suggests a cystic or heterogeneous appearance. Small schwannomas are usually homogeneous and are histologically composed of Antoni type A pattern, while heterogeneous and cystic schwannomas are larger and include Antoni B pattern or a mix of type A and B patterns . Therefore, this area may show higher proliferation and a high APT SI, and one study showed a higher APT SI in schwannomas with a high SI on a T2-weighted sequence compared to that of meningiomas . Zhang et al. indicated that APT imaging may have some value in the determination of malignant brain tumor boundaries, but there is no doubt that this approach can be used in the differential diagnosis between gliomas and meningiomas . Similarly, the non-contrast region of GBM also showed a high APT SI, suggesting that a liquid containing high concentrations of intracellular proteins and peptides. However, sinus mucocele shows low a APT SI because although this lesion may contain viscous fluid, there are less intracellular proteins and peptides. However, the number of cases examined was small, and further studies are needed. In addition, because the evaluations described in this study were made based on a visual assessment, quantitative evaluation is also necessary. 10. Conclusions APT-CEST imaging can provide additional information on intracranial brain tumors and tumor-like lesions over the information provided by conventional MRI methods. This which may help indicate the nature of lesions, differentiate between benign and malignant lesions, and determine therapeutic effects. Future research could initiate or improve the lesion-specific clinical applicability of APT-CEST imaging for meningioma embolization, lipoma, leukoencephalopathy, TSC, PML, and HS. For these purposes, we may have to answer specific research questions and suggest guidelines to allow the development of APT-CEST imaging to reach its full potential. Author Contributions H.K., data curation, formal analysis, supervision, validation, visualization, and writing--original draft; M.M. (Minoru Morikawa), conceptualization, funding acquisition, methodology, project administration, writing--review and editing, and software; H.I., data curation; R.I.; data curation; M.U.; funding acquisition; project administration, M.M. (Mitsuharu Miyoshi). All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement This study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board of the Nagasaki University Hospital (The date of approval (13 December 2019) and the project identification code (19081913-2)), which waived the need for written informed consent from the patients. Data Availability Statement The datasets during this study are not publicly available but are available from the corresponding author on reasonable request. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Mechanism of APT CEST imaging. The APT effect is measured as a reduction in bulk water intensity due to chemical exchange of water protons with labeled backbone amide protons at 3.5 ppm. Figure 2 Images from a 35-year-old woman with an oligodendroglioma. (a) Three-dimensional, contrast-enhanced, T1-weighted sequence shows ill definition and faint enhancement of an oligodendroglioma in the left temporal lobe (white arrow). (b) APT-CEST sequence (axial section) shows wide low SI (white arrow) and parts with relatively high SI (red arrow), consistent with oligodendroglioma. Figure 3 Images from a 41-year-old man with diffuse astrocytoma post-operation. (a) Three-dimensional, contrast-enhanced, T1-weighted sequence shows ring enhancement in the right cerebellar hemisphere (white arrow). Local recurrence or malignant transformation is suspected. (b) APT-CEST sequence (axial section) shows relatively high SI in the center of a ring of enhancement (white arrow). Figure 4 Images from a 77-year-old man with a GBM. (a) Three-dimensional, contrast-enhanced, T1-weighted sequence shows ring enhancement of a GBM in the left deep periventricular white matter and adjacent splenium of corpus callosum (white arrow). (b) APT-CEST sequence (axial section) shows high SI consistent with GBM (white arrow) and extremely high SI consistent with a poor contrast area (red arrow). Figure 5 Images from a 72-year-old woman with a GBM after operation and radiotherapy. (a) Three-dimensional, contrast-enhanced, T1-weighted sequence shows enhancement at the marginal dead space of the surgical site in the right temporal lobe (white arrow). Local recurrence is suspected according to the clinical course. (b) APT-CEST sequence (axial section) shows relatively high SI consistent with the enhancement (white arrow). Figure 6 Images from a 68-year-old man with diffuse large B cell lymphoma. (a) Three-dimensional, contrast-enhanced, T1-weighted sequence shows an intense enhancement intraventricular mass (white arrow). (b) APT-CEST sequence (axial section) shows a high SI throughout the intraventricular malignant lymphoma (white arrow). Figure 7 Images from a 70-year-old man with brain metastasis from esophageal cancer. (a) Three-dimensional, contrast-enhanced, T1-weighted sequence shows ring enhancement of brain metastasis in the left temporal lobe (white arrow). (b) APT-CEST sequence (axial section) shows relatively high SI at the margin of the brain metastasis (white arrow). Figure 8 Images from a 64-year-old woman with an atypical meningioma before and after transcatheter arterial embolization (TAE). (a) Three-dimensional, contrast-enhanced, T1-weighted sequence shows atypical falcine meningioma involving superior sagittal sinus before TAE (white arrow). (b) Three-dimensional, contrast-enhanced, T1-weighted sequence shows atypical falcine meningioma involving superior sagittal sinus after TAE (white arrow). There was no apparent change in the atypical meningioma compared with pre-TAE imaging. (c) APT-CEST sequence (axial section) shows high SI consistent with meningioma before TAE (white arrow). (d) APT-CEST sequence (axial section) shows a decrease in SI in the left side of the atypical meningioma after TAE (white arrow). However, color bar and range for (c,d) were slightly different. Figure 9 Images from a 74-year-old woman with a non-growing intracranial meningioma. (a) Three-dimensional, contrast-enhanced, T1-weighted sequence shows a convexity meningioma in the left temporal lobe (white arrow). (b) APT-CEST sequence (axial section) shows low SI consistent with meningioma (white arrow). Figure 10 Images from a 77-year-old woman with a growing intracranial meningioma. (a) Three-dimensional, contrast-enhanced, T1-weighted sequence shows a dural attached lesion in the right posterior fossa (white arrow). (b) APT-CEST sequence (axial section) shows relatively high SI consistent with meningioma (white arrow). Figure 11 Images from a 67-year-old woman with a growing meningioma post operation. (a,b) Three-dimensional, contrast-enhanced, T1-weighted sequence shows partially increasing cerebellopontine angle meningioma (white arrow). (c) APT-CEST sequence (axial section) shows relatively high SI consistent with the increasing part of the tumor (white arrow). Figure 12 Images from a 38-year-old man with sphenoid sinus mucocele. (a) T2-weighted sequence (coronal section) shows a sphenoid sinus mucocele (white arrow). (b) APT-CEST sequence (axial section) shows low SI consistent with sphenoid sinus mucocele (white arrow). Figure 13 Images from a 46-year-old woman with a solitary fibrous tumor. (a) Three-dimensional, contrast-enhanced, T1-weighted sequence shows enhancement of a solitary fibrous tumor in the right cerebellopontine angle (white arrow). (b) APT-CEST sequence (axial section) shows low SI consistent with solitary fibrous tumor (white arrow). Figure 14 Images from a 38-year-old woman with a schwannoma. (a) T2-weighted sequence (axial section) shows a schwannoma in the right cerebellopontine angle with uneven high SI (white arrow). (b) APT-CEST sequence (axial section) shows high SI consistent with schwannoma (white arrow). Figure 15 Images from a 24-year-old woman with a suspected lipoma. (a) T1-weighted sequence (axial section) shows a suspected lipoma in the left cerebellopontine angle with high SI (white arrow). (b) APT-CEST sequence (axial section) shows low SI consistent with suspected lipoma (white arrow). Figure 16 Images from a 52-year-old man with suspected radiation necrosis after irradiation for nasopharyngeal cancer. (a) Three-dimensional, contrast-enhanced, T1-weighted sequence shows multiple sparse enhancements in the bilateral temporal lobe (white arrow). Radiation necrosis is suspected according to the clinical course. (b) APT-CEST sequence (axial section) shows low SI consistent with the enhancement in the bilateral temporal lobe (white arrow). Figure 17 Images from a 70-year-old man with suspected radiation necrosis after operation and irradiation for brain metastasis from esophageal cancer. (a) Three-dimensional, contrast-enhanced, T1-weighted sequence shows multiple sparse enhancements in the left temporal lobe (white arrow). Radiation necrosis is suspected according to the clinical course. (b) APT-CEST sequence (axial section) shows low SI consistent with the enhancement in the left temporal lobe (white arrow). Figure 18 Images from a 50-year-old man with methotrexate-induced leukoencephalopathy. (a) Fluid-attenuated inversion recovery (FLAIR) sequence (axial section) shows low SI in periventricular white matter (white arrow). Leukoencephalopathy is suspected according to the clinical course. (b) APT-CEST sequence (axial section) shows low SI consistent with a leukoencephalopathy lesion (white arrow). Figure 19 Images from a 4-year-old girl with tuberous sclerosis complex. (a) FLAIR sequence (axial section) shows slightly high SI in the left frontal lobe (white arrow). This lesion was suspected to be a cortical hamartoma. (b) APT-CEST sequence (axial section) shows low SI consistent with suspected cortical hamartoma (white arrow). Figure 20 Images from a 43-year-old woman under treatment with an immunosuppressant drug for systemic lupus erythematosus who presented with progressive multifocal leukoencephalopathy (PML). (a) FLAIR sequence (axial section) shows PML with high SI in the right cerebral hemisphere (white arrow). (b) APT-CEST sequence (axial section) shows low SI consistent with PML (white arrow). Figure 21 Images from a 36-year-old woman with hippocampal sclerosis. (a) FLAIR sequence (coronal section) shows left hippocampal sclerosis with high SI (white arrow). (b) APT-CEST sequence (coronal section) shows low SI consistent with hippocampal sclerosis (white arrow). There is no difference between the left and right hippocampal SI. 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PMC10000844 | Int J Environ Res Public Health Int J Environ Res Public Health ijerph International Journal of Environmental Research and Public Health 1661-7827 1660-4601 MDPI 10.3390/ijerph20053804 ijerph-20-03804 Article The Role of the COVID-19 Crisis in Shaping Urban Planning for Improved Public Health: A Triangulated Study Ferhati Koudoua Conceptualization Methodology Validation Formal analysis Investigation Writing - original draft Writing - review & editing Visualization Project administration 1 Chouguiat Belmallem Saliha Conceptualization Validation Formal analysis Visualization Supervision 2 Burlea-Schiopoiu Adriana Validation Investigation Writing - review & editing Visualization Supervision 3* Tadano Yara de Souza Academic Editor Godoi Ricardo Henrique Moreton Academic Editor Tchounwou Paul B. Academic Editor 1 AVMF Research Lab, Department of Project Management, Faculty of Architecture and Urbanism, Constantine 3 University, El Khroub 25000, Algeria 2 Department of Project Management, Faculty of Architecture and Urbanism, Constantine 3 University, El Khroub 25000, Algeria 3 Department of Management, Marketing, Business Administration, Faculty of Economics and Business Administration, University of Craiova, 200585 Craiova, Romania * Correspondence: [email protected] 21 2 2023 3 2023 20 5 380413 1 2023 16 2 2023 18 2 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). This paper aims to assess the impact of the COVID-19 pandemic on the link between urban planning practices and public health. A triangulated study was conducted to gain a comprehensive understanding of the topic. The first phase consisted of semi-structured interviews with health and urban planning experts, which were analyzed with the aid of Artificial Intelligence tools. The second phase involved an on-site investigation in the city of Algiers, including a survey, site visits, and a thorough analysis of the master plan for land use and urban planning. The findings emphasize the critical importance of a comprehensive health-centric approach to city design, improved governance and management practices, community involvement, and political commitment to prioritize health in urban planning. Furthermore, the results proved a strong correlation between prioritizing public health in urban planning practices and residents' satisfaction with the city's response to the COVID-19 pandemic. In conclusion, it is necessary to consider public health as a priority in urban planning practices and as a need for all stakeholders to work towards a healthier and more equitable urban environment. urban practices public health COVID-19 triangulated study policy This research received no external funding. pmc1. Introduction The increasing rate of urbanization has raised concerns about urban areas' environmental and health conditions. The challenges emphasize the need for safe distancing and precautionary measures in cities during and after the COVID-19 pandemic. A review of the existing literature and empirical evidence helps in understanding the impact of the COVID-19 pandemic on the relationship between public health and urban planning practices and guides the consideration of human health in city planning after the pandemic . More information is needed to connect urban planning and public health in light of the consequences of COVID-19 in order to ensure adequate environmental conditions and create healthy cities, which has been an ongoing effort for nearly 30 years based on past research and practices, including sanitary surveys, park planning, and urban environment investigations . A straightforward guide that outlines the factors impacting public health is necessary for policymakers and urban planners to understand the connection between shared urban planning and public health aspects . The consideration of environmental and health factors as determinants of public health by all stakeholders has become crucial , especially in light of the COVID-19 pandemic. However, there need to be more explicit references in the literature and in the policy that addresses this topic. The impact of the pandemic on city health and well-being has been studied, and it has been found that changes in health and well-being were negative, especially in crowded neighborhoods . The results show that urban planning practices need to be revised in the current circumstances, leading to changes in city management rules and urban planning, particularly in Algeria. The pandemic has accelerated the shift towards remote work and online learning, significantly increasing sedentary behavior and screen time, which can negatively affect mental and physical health. Additionally, with the outbreak of COVID-19, cities worldwide have faced significant disruptions to daily life as governments implemented measures to slow the spread of the virus. These measures included the closure of schools, offices, and businesses, the cancellation of cultural events such as concerts and tradeshows, and bans on gatherings , which showed the importance of access to green and outdoor spaces for physical activity and mental well-being after passing this challenging era. Three years after the COVID-19 pandemic, it is important to study the long-term impact of urban planning practices on public health. The pandemic has changed our understanding of what makes a city healthy; the focus is on access to healthcare facilities, clean air, and water, and adequate public spaces. Understanding how the pandemic has altered our perception of a healthy city is essential. Conducting a study on the impact of post-COVID-19 urban planning practices on public health can provide valuable insights into how urban design can promote healthy behaviors and reduce health disparities. This paper raises the challenge of comprehending the effect of the pandemic on urban planning behavior and the relationship between urban planning practices and public health to offer clear guidance to engineers and policymakers. This study is innovative in its examination of the context of northeastern Algeria and its focus on identifying opportunities for improvement in urban spaces through the evaluation of green spaces, sewage treatment technologies, and the role of urban planners in promoting healthy cities. It bridges the gap between macro health-oriented strategies and actual managerial practices by examining the practical implementation of policies to enhance public health. 2. Theoretical Framework and Hypotheses Development To understand how urban public health and urban planning might be affected by COVID-19 consequences, we need to have a clear understanding of the main elements in direct contact with the individual's health, namely their daily activities, macro agendas affecting their lifestyle, and also the rudiments that shape the environment surrounding these individuals. Every urban element, idea, or practice has associated health issues . According to Lazuardi et al. , there is a close relationship between health and urban planning; they highlight the role of public health indicators in cities in improving citizens' psychological conditions. Giacoman and colleagues found that urban planning practices such as increasing access to green spaces and promoting active transportation can positively affect public health outcomes, such as reducing rates of obesity and improving mental health. Braubach et al. and EL helou suggest that certain urban design elements, such as mixed land use and pedestrian-friendly streets, can improve public health outcomes, such as reducing rates of obesity and promoting physical activity. Cain and colleagues support their study that urban design practices that encourage walking and biking, such as providing safe and accessible pedestrian and bike infrastructure, can increase physical activity levels and reduce rates of obesity and other chronic diseases. In addition, Rowe and colleagues found that some aspects of the built environment, such as access to green spaces and social cohesion, can positively affect mental health outcomes, such as reducing rates of depression and anxiety. Many researchers agree that there are different levels of influence, explained as follows: First, at the sanitary level, the rapid and dispersed urban growth in 19th-century cities due to fast industrialization practices and lack of epidemiological considerations in urban planning led to unhealthy living conditions and the importance of considering the impact of architectural spaces on public health ; the environment of the urban periphery can greatly impact the state of public health , which does not consider the illnesses' special requirements. The characteristics of the urban space are linked to negative health outcomes, such as health issues, disability, mental illness, and even mortality . In addition, evidence shows that air and noise pollution from traffic, poor sanitary systems, and residential exposure to high traffic are shown to have negative health effects, including asthma, lung development, allergies, sleep disturbance, children's cognitive development, and increased risk of hypertension and coronary heart disease . Based on the above considerations, it is hypothesized that: Hypothesis (H1): Implementing urban planning practices that promote improved sanitary systems after COVID-19 significantly positively impacts public health outcomes. The second level of influence on public health in urban environments is related to governance and the decision-making processes in urban planning. The design and planning of neighborhoods and cities can significantly impact residents' mental and physical well-being. Jutraz and Kukec suggest that neighborhoods can impact individual health by shaping behaviors and limiting resources, with proper facilities and resources such as parks and recreational centers promoting healthy behaviors and improving public health outcomes. The availability of green spaces can also impact public health. For example, industrialization and technological advancements in the 19th and 20th centuries negatively impacted green space availability, which led to a decline in urban green spaces and negatively affected water quality and quantity . Furthermore, modern planning practices have often ignored the importance of greenery in cities , but recent studies suggest that access to green spaces can positively impact mental and physical health . Based on these considerations, it is hypothesized that: Hypothesis (H2): Implementing urban planning practices that prioritize residents' mental and physical well-being post-COVID-19 significantly impacts public health outcomes. Policy implications in urban planning could be observed and evaluated in programs and initiatives provided through the last decade . The Algerian government has shown a commitment to sustainable development over the years. Moreover, the government commitment was solidified in the aftermath of the United Nations Conference on Environment and Development in 1992, which emphasized the importance of sustainable policy orientations that prioritize the well-being of citizens and their harmonious relationship with nature . The high council for the Environment and Sustainable Development was established in 1994 to oversee environmental protection and sustainable development efforts . However, this council faced criticism for its lack of efficiency . In 2002, the National Plan of Action for the Environment and Sustainable Development was created to improve the quality of the environment and urban life quality. The decree ndeg03-10 of 2003 established the fundamental principles and regulations of environmental management and encouraged public participation in environmental protection efforts . In 2006, the Town Orientation Law was approved by the parliament to establish specific provisions for a sustainable development policy, and an international competition was launched to create a sustainable urban master plan for Algiers. The winning project, "Making Algiers a Green Metropolis of the Mediterranean", will be implemented in 2035. In the regulation context, in response to the critical environmental issues resulting from the intensive development strategy since the post-independence period, the Algerian government has developed a National Environmental Strategy. This strategy involves the creation of effective policies for the protection, enhancement, and regulation of environmental-related usage at various scales and in different management contexts, including land management, waste management, natural resource management, and energy control through the implementation of various laws and regulations, such as the 1983 Law on Environmental Protection, the 1987 Law on Territorial Planning, the 1990 Law on Land orientation, the 2001 Law on Sustainable Territorial Development, the 2002 Law on Coastal Protection and Valorization, the 2003 Law on Environmental Protection in the Context of Sustainable Development, and the 2004 Law on Major Risk Prevention, as well as laws on energy control and the promotion of renewable energy . Finally, we hypothesized that: Hypothesis (H3): Policy initiatives and programs that support sustainable development and improve the urban environment significantly impact public health outcomes. Based on the previous theoretical framework study , discussing the different elements and issues related to urban planning that might affect public health and the development of the three research hypotheses, we propose in Figure 1 the following study model: 3. Materials and Methods A triangulated study that employs a mixed methodology approach was conducted in two phases. In the first phase, qualitative methods, including semi-structured interviews, were used to gain an in-depth understanding of the relationship between urban planning and public health after COVID-19, as viewed by health and urban planning professionals. The second phase involved a combination of quantitative and qualitative methods, including a survey, site visits, and document analysis to supplement the findings of the first phase. The triangulated study is a research strategy that employs multiple data sources, methods, theories, and/or researchers to address a research question, enhance the validity and credibility of the findings, and reduce the presence of any research biases in this paper. 3.1. Planning and Carrying out the Interview There were three main phases for conducting the interview: the planning, the actual carrying out, and the analysis of the empirical material . In the interview planning process, we had to make sure that the method was suitable to the study's objectives and that it could answer the research question. According to the participatory design processes and the evaluation and action research, the choice of research methods is always linked to the scope and objective of the study . Since the primary study objective is to understand and have an impact on relations based on professional experience, local practices, or human constructions of the meaning of public and urban planning practices, the qualitative interview should be considered . After ensuring that an in-depth interview is a suitable method for the research, it is time to ask the important questions: who, where, and what should be the topics to be included in the interview? It is essential to pick suitable informants when the research methodology is based on personal records . The choice of participants for the semi-structured interviews was based on the criteria of expertise and relevance to the research topic. Managers, doctors, and engineers from urban planning and health administration were selected as participants because they have professional experience and knowledge in the areas of urban planning, public health, and governance and are therefore well-suited to provide valuable insight and perspectives on the northeastern part of Algeria: 09 wilayas, as shown in Figure 2. The nine wilayas studied in the research showcase a diverse socio-economic profile, reflecting Algeria's cultural and geographic richness. Algiers serves as the political and economic center, while Tizi Ouzou is a significant city in the Kabylie region known for its coastal forests. Constantine is the capital of eastern Algeria, and a commercial hub such as Annaba boasts large steel, food, and port industries. Despite having well-developed healthcare systems, these metropoles faced significant health challenges during the pandemic, including overcrowding and stress on healthcare systems. Guelma, Setif, Skikda, and Jijel are mixed-activity wilayas with a smaller population featuring agriculture and industry. Finally, El Taref is a smaller coastal city with a lower population size, agricultural economy, and cultural heritage. Before the pandemic, these cities faced complex public health issues such as air and water pollution and insufficient healthcare facilities and staff. The sampling phase for the interviews was guided by the Hagaman and Wutich method , which recommended around 20 participants to identify new themes and saturate categories; ultimately, 12 interviews were conducted due to the non-availability of some participants. We designed the semi-structured interviews based on the themes from the theoretical framework: sanitary system, spaces management, and policy implementation-related questions (25 questions), enabling openly valid and reliable answers (Appendix A). The interviews were done in person and via an online platform (Google meet and zoom) between 7 September 2022 and 19 December 2022, depending on the ability of the participants and authors. The study conducted in-depth qualitative interviews with 12 participants, with varying lengths between 36 and 48 min; anonymity was ensured and consent was obtained before discussing public health, urban managerial planning strategies, and potential impacts. The thematic analysis of the categorized questions was conducted using Generative Pre-trained Transformer (GBT) to identify patterns and trends in the interview data, which generated three main themes:Sanitary system and epidemiology. Healthy city, management, and green spaces implementation. Health integration in urban planning policy. The themes were analyzed to extract qualitative data. 3.2. On-Site Investigations The methodology involved a mixed-method approach. We used a combination of a survey, site visits, and an Urban Development Master Plan (PDAU) analysis. A questionnaire (Appendix B) was designed using a 5-point Likert scale based on the theoretical framework and the semi-structured interview perspectives to conduct the survey. In addition to the survey, we conducted a site visit to investigate the main declared initiatives and to observe ongoing projects and initiatives. Then we analyzed Algiers city PDAU horizon 2035 to understand the urban planning practices from all perspectives. The survey: Sampling and case study: The respondents were the citizens of Algiers, the capital and largest city of Algeria, which had undergone significant changes in its urban conditions before and during the COVID-19 pandemic. Pre-pandemic, Algiers was characterized by a rapidly growing population, an estimated 4.5 million residents in 2020, and a bustling commercial center. However, like many large cities, Algiers faced challenges related to the lack of appropriate accessible public spaces, sewage and water pollution problems, air pollution due to of the high industrial concentration and the considerable number of car users in the center of the city, and overcrowding in certain areas. During the pandemic, the city saw a significant decrease in population mobility and economic activity, with measures such as lockdowns and social distancing regulations implemented to slow the spread of the virus. A random sampling method was used to select 200 participants from the population, and we identified 112 valid and complete questionnaire responses. The sample was chosen to be representative of the population in terms of demographic characteristics such as age, gender, occupation, and education level. Data collection: The survey was administered in person to the selected participants. We made sure to explain the purpose of the survey and ensure that the participants understood the questions before they began answering. Data analysis: A categorical data analysis was conducted to examine the relationship between variables. The analysis included the use of cross-tabulations, the chi-squared test of independence, and Kendall's Tau B correlation analysis. The cross-tabulation, also known as contingency tables, was used to determine the variables' frequency distribution and test the relationship between two categorical variables. The chi-squared test of independence was used to determine whether the frequency distribution of one categorical variable is independent of the frequency distribution of another categorical variable. Finally, Kendall's Tau B correlation analysis was used to determine the association between two ordinal categorical variables. This method allowed us to examine the relationship between different variables in each hypothesis to provide insight into their association and dependence. b. Site visits: In order to validate the findings from the survey and to investigate the main declared initiatives and ongoing projects in Algiers that reflect the COVID-19 effect on urban behaviors in the city, we conducted a series of site visits. To summarize the findings of our on-site visits, we employed a statistical approach by calculating the mean completion percentages for each initiative. This was achieved by summing the completion percentages and dividing them by the number of initiatives, as detailed in Table 4. Our estimation of the progress of each initiative was based on a combination of our judgment and observed documents, where we observed the ongoing activities and their progress, and then conducted informal interviews with government officials and members of civil society to gather additional information about the status of initiatives and projects' execution; we also analyzed available data and documents when needed. These sources of information were used to estimate the completion percentages for each initiative and record them in our findings. c. Document analysis: In this step, we analyzed the main urban planning tool for Algiers: PDAU horizon 2035 . We examined and categorized the content from the presentation and components, pressures, opportunities, risks, and emerging problems, and synthesized the key points in a SWOT matrix used for identifying and analyzing an initiative's strengths, weaknesses, opportunities, and threats. 4. Results 4.1. The Semi-Structured Interview For the purpose of analysis, a code was assigned to each of the twelve interviewees as follows: I#1, I#2, I#3, I#4, I#5, I#6, I#7, I#8, I#9, I#10, I#11, and I#12. In Figure 3, the variables of age and experience are presented on the Y axis (in years), with interviewees I#1 to I#12 shown on the X axis. The age range varied between 32 to 61 years, with a mean age of 45.83, and most of the interviewees were in the age range between 37 and 52. The experience variable ranged from 7 to 38, with a mean of 18.58. Similarly, when we look at the frequency of the experience variable, most interviewees had between 11 and 24 years of experience, with only one interviewee at each level of experience. Males (9 males and 3 females) were predominant in the gender distribution among the interviewees because of the specificity of the field of activity. This data provides a general overview of the demographic characteristics of the interviewees and can be used to understand the background and qualifications of the interviewees. In this phase, 12 interview answers were analyzed to investigate the perceptions of urban planners, managers, and public health professionals on the impact of managerial urban planning practices on public health. The results were grouped into three main themes. 4.1.1. Sanitary System: Sewage, Toxification, Trash, and Epidemiology Theme The results from the analysis of the interviews from the first theme are presented and interpreted in Table 1. 4.1.2. Healthy City, Management, and Governance Theme The results from the analysis of the interviews from the theme management, governance, and leadership skills for the identification of the healthy city concept are presented and interpreted in Table 2. 4.1.3. Health Integration in Urban Planning Policy Theme The results from the analysis of the interviews from the third theme, integrating health concepts in the urban planning process, are presented and interpreted in Table 3. 4.2. On-Site Investigation The demographic analysis of the sample of 112 survey respondents provides insight into the characteristics of the population surveyed. The gender distribution of the respondents is notably skewed, with 67% identifying as male and 33% identifying as female. The respondents' ages range from 36-55 years old, and the preponderant are those between 36-45 years old (28.6%). 4.2.1. The Survey Regarding occupation, the sample comprises 47.3% of respondents working in the public sector, 15.2% are from the private sector, 16.1% are retired, 6.3% are self-employed, and 15.2% are students. Regarding educational attainment, the sample is relatively well-educated, with 55.4% of respondents holding a university degree, 13.4% having post-graduate education, 20.5% having a high school education, and 10.7% having less than a high school education. Categorical data analysis: Before performing categorical data analysis in SPSS, we ensured that the data met specific requirements. Firstly, the data should be in a format appropriate for the tests. This typically means that the data should be in a categorical format; in our case, it is ordinal. Additionally, it is important to check for missing data and ensure no missing values in the analyzed variables. The test results on satisfaction with the city's response to COVID-19 with the city's public health system accessible to all residents. The case processing summary shows no missing data, with all 112 respondents included in the analysis. The distribution of responses for both variables, with the chi-square tests indicating a statistically significant association between the two variables (p < 0.001) through Pearson Chi-Square, Likelihood Ratio, and Linear-by-Linear Association. The symmetric measures of Kendall's tau-b and Spearman Correlation were used to examine the ordinal association between the two variables and both measures yielded high values of 0.698 and 0.772, respectively, which are statistically significant (p < 0.001) and indicate a strong ordinal association between the two variables. The data suggest a statistically significant and robust association between city residents' satisfaction with the city's response to COVID-19 and their perceptions of the city's public health system's accessibility and preparation for future epidemics. The test results addressed satisfaction with the city's response to COVID-19 and with the city's public health system's preparation for future epidemics. The crosstab table shows the count of responses for satisfaction with the city's response to COVID-19 and the city's public health system preparation for future epidemics. The chi-square tests indicate a significant association between these two variables (with a p-value less than 0.001). The symmetric measures (Kendall's tau-b, Spearman Correlation, and Pearson's R) also show a strong positive correlation between the two variables, with values of 0.741; 0.816; and 0.789, respectively. These results suggest that as the city's public health system prepares for future epidemics, satisfaction with the city's response to COVID-19 also increases. For the second research hypothesis, the results of the tests addressed satisfaction with the city's efforts to promote a healthy lifestyle with the city's management level for green spaces. The first set of results is a crosstabulation and chi-square analysis examining the relationship between satisfaction with the city's response to the COVID-19 pandemic and the city's public health system's preparation for future epidemics. The chi-square tests indicate a statistically significant association between the two variables (p < 0.001), with the largest expected count being 8 and the smallest expected count being 1.13. Additionally, the symmetric measures (Kendall's tau-b, Spearman correlation, and Pearson's R) all indicate a strong correlation between the two variables, with Kendall's tau-b, spearman correlation, and Pearson's R coefficient of 0.741; 0.816; and 0.789, respectively. The second set of results is a crosstabulation and chi-square analysis examining the relationship between satisfaction with the city's efforts to promote a healthy lifestyle, the city's management level for green spaces, and the importance of green space accessibility. The chi-square tests indicate that there is a statistically significant association between the two variables (p < 0.001), with the largest expected count being 47 and the smallest expected count being 0.64. Additionally, the symmetric measures (Kendall's tau-b, Spearman correlation, and Pearson's R) all indicate a strong correlation between the two variables, with Kendall's tau-b, spearman correlation, and Pearson's R coefficient of 0.836; 0.882; and 0.916, respectively. The crosstab shows the counts of responses for the two variables "satisfaction with the city's efforts to promote a healthy lifestyle" and "the importance of green spaces accessibility" on a 5-point Likert scale. The chi-square tests indicate a significant association between the two variables, as the p-values are less than 0.001 for all three tests (Pearson Chi-Square, Likelihood Ratio, Linear-by-Linear Association). The symmetric measures section provides information on the strength and direction of the association between the two variables, with all three measures (Kendall's tau-b, Spearman Correlation, Pearson's R) showing a strong positive association (values ranging from 0.699 to 0.804) and significant p-values (less than 0.001). The N of valid cases is 112. Overall, the results suggest that there is a significant positive association between satisfaction with the city's efforts to promote a healthy lifestyle and the importance of green space accessibility. Results for statistical analysis of the third hypothesis show the frequency of responses for the two variables being studied: "satisfaction with health and safety measures implemented in the city after COVID-19" and "the city's urban planning policies level promoting health and well-being". The chi-square tests determine if there is a significant association between the two variables. The Pearson Chi-Square, Likelihood Ratio, and Linear-by-Linear Association tests were significant, with p-values less than 0.001. The symmetric measures are used to quantify the strength of the association between the two variables. The ordinal-by-ordinal measures, Kendall's tau-b, and Spearman Correlation indicate a strong association between the two variables, with values of 0.811 and 0.862, respectively. The interval-by-interval measure, Pearson's R, also indicates a strong association with a value of 0.844. The results of the following two variables show a significant association between satisfaction with the health and safety measures implemented in the city after COVID-19 and the effectiveness of management addressing public health concerns related to COVID-19. Moreover, the p-values are <0.001 for all three chi-square tests (Pearson Chi-Square, Likelihood Ratio, Linear-by-Linear Association) and all three symmetric measures (Kendall's tau-b, Spearman Correlation, and Pearson's R). The chi-square tests and symmetric measures provide measures of the strength and direction of the association between the two variables. For example, Kendall's tau-b, Spearman Correlation, and Pearson's R coefficient are 0.889; 0.912; and 0.908, respectively, all of which are close to 1 and indicate a strong positive association between the two variables. 4.2.2. On-Site Visit The data concerning the public health situation in the city of Algiers after COVID-19 were collected from the interviews and revealed ongoing efforts in various domains, including sanitary systems and epidemiology, water quality, toxic exposure risk, waste management, efficient land utilization, and green space administration. To validate these findings, a series of site visits were conducted, including the examination of VRD (Roads and various networks) plans, observations of construction sites, systematic analysis of potable water, assessments of pipeline conditions during precipitation, interactions with corporations and citizens, and inspections of public parks. The key initiatives spotted are presented in Figure 4. The findings indicate that the efforts to prevent sewage mishaps and maintain the sewage network are underway and currently stand at 40% completion, which included operational measures to clean sewer systems, address malfunctioning systems, and repair existing breakdowns. The quality of water was found to comply with established standards. Upgrading outdated pipelines and the maintenance of access points is ongoing at 60% completion. The measures implemented include cleaning and maintaining 696,751 gutters and stormwater channels, essential for proper water drainage, and removing over 400,000 tons of mud and waste. The mitigation of toxic exposure risk is underway at 30%. The collection and disposal of waste are being handled by a private company and the government and are currently at 40% completion. The incorporation of new technologies in waste management is underway at 30%. Additionally, the transformation of the Oued S'mar public landfill into a public garden has been fully completed. Furthermore, the renovation and creation of green spaces across all districts of Algiers are in progress and stand at 20% completion, although we found gaps in the availability and accessibility of green spaces in different parts of the city. Finally, the Bainam forest tree plantation initiative is underway at 90% completion after its severe degradation over the last ten years due to several reasons such as fires and natural causes. When discussing with citizens during our investigation, we also observed the launch of awareness campaigns in partnership with members of civil society, which focus on the role of citizens in preserving the environment and enhancing public health conditions. As a result, on average, 58% of the areas inspected have been found to validate the initial findings. The detailed steps of the investigation are presented in Table 4. 4.2.3. Document Analysis: PDAU Horizon 2035 A rigorous diagnostic method, accompanied by continuous communication with local authorities and sectoral institutions, was ensured to deeply understand the territory and its specific realities for making this document analysis that englobes all the parts of the PDAU for the identification of the conditions, options, and principles established by the new territorial management tool that should guide and structure the development of the territory of the wilaya of Algiers for the next 12 years. We synthesized the main strengths, opportunities, weaknesses, and threats from the analysis in a SWOT matrix as shown in Figure 5 below. Since independence, Algiers has faced a demographic explosion which resulted in infrastructure construction without regard for coherence, sustainability, or public health considerations. To address this issue, the Algerian government has decided to develop a strategic plan for the city (in 2016) to become a flagship city, safe for its residents, and competitive for economic agents, with good sustainable urban governance. Therefore, the PDAU analysis is structured as follows:Presentation and Components: The master plan of the PDAU in Algiers has six pillars that form the foundation for the future of Algiers, intending to become a reference in the Mediterranean and the world:Economic Development, Competitiveness, and Employment. Opening the city to the World and Internationalization. Territorial Cohesion, Social Cohesion, and Housing. Environment, Protection, and Enhancement. Territorial Model. Governance. These pillars are materialized through 82 key projects corresponding to concrete intervention proposals and provide substance to the territorial model proposed in the master plan. The key projects include the Port of Algiers, the Logistics Corridor of Ezzouar and Bab Ezzouar, the Hussein Dey/Mohammedia Seashore, the Central Station of Algiers, the Complementary Network of Industrial and Service Activities, the Faculty of Medicine, the Faculty of Law, the Douera Stadium, the participation of the private sector in residential production, the improvement of conditions for businesses and the banking sector, the revitalization and commercial upgrading of Algiers, the revitalization of urban core areas and agricultural activity, El Harrach Park and El Hamiz Park, the regional energy network of Algiers, the regional communication systems, e-wilaya strategy, the management of public space, and several ports and universities. The Pressures: The report analyzes the current pressures faced by Algeria. The economy relies heavily on tertiary activities and is struggling with a 20% unemployment rate and a growing informal economy. The country is also facing an economic crisis and a slow population growth rate, with 70% of its population under the age of 40, and an increasing demand for housing, equipment, and infrastructure. Housing conditions are precarious, with informal housing being reconstructed, and there is a new migration phenomenon due to factors such as climate change, economic challenges, and geopolitical issues. Urbanization is a significant challenge, with a 94% urbanization rate and a shortage of urbanized land leading to food insecurity. The country also faces water stress, with only 160 L per capita per day, and energy security risks, with an average of 3150 kWh per household per year. Finally, climate change poses significant risks, including a temperature increase of +2deg by 2030, a sea level rise of +16 cm, a decrease in rainfall of -10-15%, increased drought, and erosion. Problems: Algeria is facing urbanization issues, excessive consumption of natural resources, degradation of the environment, and transportation problems. The Algerian economy faces structural weaknesses such as a lack of diversification and integration into the global economy, dependence on international markets (hydrocarbons and agriculture), low human resources qualifications, technological innovation lag, and a lack of infrastructure to support economic activities. The challenges of sustainable development, based on the three pillars of efficient economic development, social equity, and environmental sustainability, are constantly being postponed due to various pressures. Opportunities: However, with its excellent geographical location, Alger enjoys a rich cultural and natural heritage. The country and its capital are increasingly attracting foreign direct investment, and with its rich natural resources (energy and minerals) and economic stability, Algeria has a favorable industrial potential supported by public investment in infrastructure to support economic activity. The youth of its population provide Algeria with a promising future and a good foundation for economic dynamism, provided it is trained, educated, and qualified. Therefore, it is important to prioritize sustainable development through the new economic, environmental, and social models stated in various national, regional, and sectoral plans to ensure a sustainable future. The various communities and sectors need to use all the tools developed at the highest level to address the existing problems in resource protection, environmental protection, and territorial planning. Agriculture is a valuable economic resource that should be preserved and developed sustainably, considering the climate and soil richness. Technological innovations should be introduced in agricultural production and processing to improve the profitability of the sector and the utilization of its resources. The export potential of agricultural products opens up prospects for this sector, especially for specific products with natural comparative advantages and growing demand. Urbanization operations should maintain strong agricultural dynamics, mainly when they spread to fertile areas of the territory. Although the equipment networks are generally well organized in terms of quantity and territory, they still need to be reorganized to better respond to social needs and the aspirations of the population and allow the emergence of a polycentric urban system. The road and highway network, well established in much of the wilaya of Alger territory, is overgrowing due to a vast construction and expansion program. Mobility: The public transportation network is a crucial aspect of the mobility sector, including different modes, their new paths, redirections, and requirements that have been highlighted in the PDAU as a crucial tool for territorial management and provides a vision for the city's mobility plans involving stakeholders: first highways and expressways--equipped with characteristics to ensure optimal mobility and safety; main arteries--which form the leading urban network, complementing the first level. On these arteries, the traffic is mostly passing, and the first function is to serve the main generators and development poles of the region; secondary arteries have a similar function to the previous level but with less importance from a geographical point of view; and collector streets that locally important roads at the commune level. In complement to the road network, the mobility system envisages a three-level parking subsystem: park-and-ride lots, street parking (paid and non-paid), and off-street parking--accompanying parking lots. Finally, the mobility system considers the soft mode networks, i.e., the pedestrian and bike path networks. Risks: The plan considered managing earthquakes and reducing seismic risk in the region, focusing primarily on the seismic vulnerability of buildings. Measures to improve seismic resistance in new construction, renovation of existing buildings to increase their resistance, restrictions on construction in high-risk areas, and preparation for earthquakes by securing unstable equipment, are all part of the plan. Additional studies may be conducted for high-risk seismic areas to further understand the risks and determine appropriate technical solutions, as mentioned in the document. The plan also includes measures to improve infrastructure, such as increasing open spaces and improving road connectivity. In addition, the plan provides guidelines for reducing the risk of landslides and falling rocks. The laws related to urban planning and development in Algeria, such as Law No. 90.29 and Law No. 04-05, also address these issues and identify high-risk areas and implement measures to mitigate the risks. Highlights and Critics: From the observed urban planning practices expressed in the document, it is evident that hard work has been dedicated to the document's execution, which included many positive points in fields of land use, sustainable resources alternatives implementation, and mega projects planning, but some points present a weakness in the plan's strategies. First, we are continuing with the existing urban development plans (POS--land use plan), while we should be transitioning to more comprehensive urban development projects (Projets Urbains de Developpement). Second, we are limiting our focus to the wilaya, while the metropolitan area is already a functional entity and the Mediterranean network is becoming more important. Third, we are neglecting external pressures such as the climate, geopolitics, and resources, which significantly impact development. Fourth, we are comparing ourselves to other countries, while we should first focus on internal benchmarks between the different communes in Algiers. Finally, while the PDAU covers various aspects of seismic risk mitigation, it does not include any considerations for pandemics or other similar health crises. In today's world, where pandemics like COVID-19 have profoundly impacted communities, it is important to include public health considerations in any risk mitigation plan. Failure results from a limited response to such crises and leaves communities vulnerable. 5. Discussion The first theme of healthy cities and sanitary systems encompasses the issues of sewage, toxic materials, waste management, and epidemiology, which are of crucial importance in urban planning practices. The opinions and insights shared by the interviewees shed light on the current state of the sewage systems, industrial zones, and the associated risks to public health. The consensus among the interviewees is that there is a need for collaboration between the conservation and public health sectors to address these issues and to maintain public health and cleanliness in cities. While some interviewees believe that the risk of exposure to toxic materials and chemical mixtures in urban environments is relatively low, others consider it a significant cause of concern. In addition, the rapid development of industrial zones in some areas of Algeria presents difficulties for control agencies monitoring toxic waste data, thus emphasizing the importance of a well-functioning waste management system to ensure public health. In addition, the interviewees emphasize the significance of preventive strategies in urban planning, particularly in light of the ongoing pandemic. They advocate for a shift in traditional planning approaches towards a more holistic, health-centric approach to city design to ensure public health and safety in urban areas. The importance of staying informed about new waste management technologies and their impact on public health is also noted. In conclusion, the insights shared by the interviewees provide evidence for the critical role of the sanitary system in promoting public health in urban areas. Furthermore, their views highlight the need for a concerted effort between various stakeholders and the government to address these pressing issues. Examining the interviewees' viewpoints about the impact of urban planning practices on public health reveals several salient themes. Firstly, there is a perception of a disconnect between overarching health-oriented strategies and their implementation at the city level, requiring improved governance and management practices to bridge this gap. Secondly, the interviewees recognize the importance of collaboration between various sectors to achieve the goals of healthy cities, highlighting the need for clear plans, budgets, and a continuous approach. Additionally, the qualifications and training of healthy urban planners emerged as a point of contention among the interviewees, with some perceiving a need for sustainability and green city expertise. In contrast, others acknowledged the vital role of qualified managers and urbanists in achieving healthy city objectives. The interviewees also emphasized the crucial role of community involvement in realizing healthy city goals, highlighting successful examples in Algeria, where cooperation and collaboration between the government and citizens have been instrumental in advancing the healthy city concept. In conclusion, the collective views of the interviewees demonstrate the significance of urban planning practices that prioritize residents' mental and physical well-being in achieving improved public health outcomes. In addition, the need for competent governance, effective managerial decisions, and community involvement is emphasized, further underlining the importance of prioritizing health in urban planning. The participants highlighted the importance of taking a comprehensive approach to improving the health and well-being of urban communities. Moreover, they acknowledge the current limitations in urban planning, and the need for increased efforts to address the identified gaps in urban health. Also, they emphasized the need for a shared vision among stakeholders, policy decision-makers, and partners to achieve a healthier and more equitable urban environment. As a result, they advocate for establishing healthy urban planning as a norm and the need for evidence-based leadership and political commitment to bring about change in urban planning practices. Furthermore, they discuss the significance of identifying urban health problems and the need for appropriate actions to address them. Finally, they recognize the importance of innovative technologies and nature-based solutions in promoting public health, and suggest that programs and initiatives aimed at sustainable development will positively impact public health. In conclusion, the participants provide valuable insights into the need for a holistic and proactive approach to urban planning that prioritizes public health. Furthermore, the emphasis on the role of nature-based solutions, innovative technologies, and sustainable development highlights the potential for positive change in the urban environment. The findings provide a snapshot of the socioeconomic profile of the respondents. The gender distribution shows a slight skew towards males, while the age distribution highlights a significant presence of individuals aged between 36 and 55. The occupation distribution highlights a majority of respondents working in the public sector, with a notable presence of students and self-employed. Furthermore, the educational attainment of the sample reveals a relatively high level of education among respondents, with the majority holding a university degree. The results from the first hypothesis-related variables provide strong evidence that implementing urban planning practices that promote improved sanitary systems has a significant positive impact on public health outcomes, as measured by residents' satisfaction with the city's response to the COVID-19 pandemic. Through various statistical tests, we have determined a statistically significant and strong association between residents' perceptions of the city's public health system's accessibility and preparation for future epidemics and their satisfaction with the city's response to the COVID-19 pandemic. This highlights the importance of effective public health measures in fostering positive perceptions of government response during times of crisis. The city's public health system's preparation for future epidemics is critical in ensuring that residents feel satisfied with the city's response to the COVID-19 pandemic. Our results suggest that the city's public health system preparation after COVID-19 for future epidemics positively impacts the public health outcome. The results from the second hypothesis-related variables, including chi-square tests and symmetric measures, indicate a statistically significant and strong association between satisfaction with the city's efforts to promote a healthy lifestyle, the city's management level for green spaces, and the importance of green space accessibility. These findings suggest that the city's efforts to promote a healthy lifestyle, such as through accessible green spaces, significantly impact residents' perceptions of their overall well-being. This highlights the crucial role that urban planning practices can play in fostering positive public health outcomes, particularly in the context of the ongoing COVID-19 pandemic and its impact on mental and physical health, which provides strong empirical evidence supporting the research, which posits that the implementation of urban planning practices that prioritize residents' mental and physical well-being post-COVID-19 has a significant positive impact on public health outcomes. The results from the third hypothesis-related variables provide evidence of a significant positive association between policy initiatives and programs that support sustainable development and improve the urban environment and their impact on public health outcomes. The use of chi-square tests and symmetric measures such as Kendall's tau-b, Spearman correlation, and Pearson's R all indicate a strong positive correlation between the studied variables. Specifically, the analysis shows a strong association between satisfaction with health and safety measures implemented in the city after COVID-19 and the city's urban planning policies' level of promoting health and well-being, as well as between satisfaction with health and safety measures implemented in the city after COVID-19 and the effectiveness of management in addressing public health concerns related to COVID-19. These findings suggest that cities prioritizing sustainable development and improving the urban environment through policy initiatives and programs positively impact public health outcomes, particularly during the COVID-19 pandemic. The site visits investigation aimed to examine the public health situation in Algiers and the urban planning practices after the COVID-19 pandemic, as declared in the semi-structured interviews' responses from two perspectives:Sanitary System and Epidemiology: Results revealed ongoing efforts in various domains of sanitary systems and epidemiology from many levels, such as the efforts to prevent sewage mishaps and maintain the sewage network, including operational measures to clean sewer systems, address malfunctioning systems, repairing existing breakdowns, enhancing the quality of potable water, upgrading the outdated pipelines, and maintaining its access points. The mitigation of toxic exposure risk is also ongoing in the city's industrial zone. Waste collection and disposal are being handled by a private company and the government incorporating new technologies and sorting strategies, which supports that there is a positive effect of the pandemic on strengthening the link between urban planning and public health in Algiers. Healthy City, Management, and Green Spaces Implementation: The results suggest that efforts to improve and maintain green spaces are increasing. Nevertheless, disparities in the availability and accessibility of green spaces were identified across the city. The Bainam forest tree plantation initiative, which had suffered severe degradation in the past decade due to fires and natural conditions, is progressing well and is nearly complete. The launch of awareness campaigns in partnership with civil society, aimed at raising awareness about the role of citizens in preserving the environment and improving public health, was noted during the investigation. Furthermore, the transformation of the Oued S'mar public landfill into a public garden has been completed. These findings highlight the positive impact of the COVID-19 pandemic on the relationship between city management and public health outcomes. The COVID-19 pandemic has shaped new behaviors in urban planning to promote public health. The study's findings indicate ongoing efforts in various domains to improve public health conditions and address the challenges posed by the pandemic, even if it is still at the modest progress rate of 58% of the required degree, which sheds light on the gap between the macro health-oriented strategies and actual managerial practices and the need to change its strategies. The document analysis of the PDAU (Master Plan of Algiers) highlighted the various challenges and opportunities faced by the city in its efforts to achieve sustainable urban planning practices. The city is grappling with issues such as urbanization, excessive consumption of natural resources, degradation of the environment, transportation problems, and economic structural weaknesses, such as a lack of diversification and infrastructure. Despite these challenges, the city has several advantages: its favorable geographical location, rich cultural and natural heritage, rich natural resources, and a young, dynamic population. The Master Plan of Algiers seeks to address these challenges through six pillars, including economic development, territorial cohesion, environment, territorial model, and governance, and through 82 key projects to improve the city's infrastructure, housing, and public health. The SWOT matrix helped us to identify the strengths, weaknesses, opportunities, and threats of the urban planning document, which gave us a clear understanding of the actual current urban planning practices and allowed us to locate public health considerations in the city's Physical Development and Urbanism plan (PDAU). The city must prioritize sustainable models, including economic, environmental, and social models, and preserve and develop its agricultural sector. The urbanization of Algiers must be carefully planned so as not to compromise its agricultural dynamics; additionally, it should aim to respond to the needs and aspirations of its population. According to the investigation done after the classification of the interview's findings, we found that while the relevant laws, regulations, and plans related to sustainable development and environmental protection, such as the National Plan of Action for the Environment and Sustainable Development and the Town Orientation Law, were in place, their implementation and enforcement on-site were not always consistent. On the other hand, a new policy announced by the Algerian Ministry of Environment and Renewable Energy at the end of December 2022 has initiated, in conjunction with the healthcare sector, the creation of a report on the implementation of the Arab Strategy for Health and Environment 2017-2030. In urban planning practices, it was observed that the importance of waste management was considered; however, a need for implementation and enforcement was identified. Moreover, we found that the formation and qualification of healthy city urban planners faced some difficulties as some things could have been improved in terms of sustainability and green city knowledge among the local urban administration. The investigation on the implementation of the significant decisions, initiatives, and policies for enhancing public health after COVID-19 in Algeria revealed that current urban planning practices require improvement to align better with initiatives and policies aimed at promoting sustainable development and environmental protection. On-site visits revealed positive progress in certain areas, such as transforming a public landfill into a public garden. Still, only 58% of the defined initiatives and decisions were applicable. The study also highlighted the recent policy announced by the Ministry of Environment and Renewable Energy, which aims to create a report on the implementation of the Arab Strategy for Health and Environment and a new regulation for the management, protection, and development of green spaces. In order to summarize and clarify the findings from all the discussion parts, we gathered in the table below all the study phases that contributed to either supporting or rejecting the research hypotheses (Table 5). 6. Conclusions The relationship between urban planning practices and public health is crucial in shaping individuals' well-being in urban spaces. The findings of this study, obtained through semi-structured interviews and a survey, emphasized the role of the COVID-19 pandemic in the shaping of a comprehensive, health-centric approach to city design, improved governance and management practices, community involvement, and political commitment to prioritize health in urban planning. The results showed a strong correlation between implementing urban planning practices prioritizing public health and residents' satisfaction with the city's response to the COVID-19 pandemic. The need for a proactive, preventive approach to addressing public health and environmental issues was highlighted rather than it being a reactive one. It is imperative that policymakers invest in advanced and effective sewage treatment technologies, upgrade existing sewage infrastructure, incorporate health considerations into urban planning policies, and promote sustainable development and eco-friendly practices. In addition, the availability and accessibility of green spaces and the implementation of healthy-urban planning concepts in universities should also be prioritized. The findings emphasize the critical importance of sanitary systems, healthy city management and governance, and health integration in urban planning policy. In addition, the need for stakeholders to work together towards a healthier and more equitable urban environment was also emphasized. These insights highlight the significance of considering public health as a priority in urban planning practices and the need for a comprehensive long-term approach that considers the needs of both individuals and communities. In conclusion, urban planning practices are vital in promoting public health and ensuring the well-being of individuals living in urban spaces. Therefore, all stakeholders, including policymakers, city planners, and the general public, must work towards achieving a healthier and more equitable urban environment. Author Contributions Conceptualization, K.F.; methodology, K.F.; validation, K.F., S.C.B. and A.B.-S.; formal analysis, K.F., S.C.B. and A.B.-S.; investigation, K.F.; data curation, K.F.; writing--original draft preparation, K.F.; writing--review and editing, K.F. and A.B.-S.; visualization, K.F. and A.B.-S.; supervision, K.F., S.C.B. and A.B.-S. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement Some or all data, models, or codes that support the findings of this study are available from the corresponding authors upon reasonable request. Conflicts of Interest The authors declare no conflict of interest. Appendix A. The Interview Guide Introduction: Welcome. This interview is a part of our research study titled "From Sanitary Systems to Policy Implications: Understanding the Impact of Urban Planning Practices on Public Health Post COVID-19". The study aims to understand the impact of urban planning practices on public health, focusing on northeastern Algeria and explicitly addressing the impact of the sanitary system, urban green spaces, and policy on public health outcomes. Our goal is to provide clear guidance for engineers and policymakers to adjust current management practices to improve public health outcomes, especially in the COVID-19 framework. We will be asking you a series of questions about your professional experience and familiarity with healthy urban planning concepts, as well as specific themes related to sanitary systems, urban green spaces, and the role of policy for healthy cities. Participation in this study is greatly appreciated, and all information provided will be kept anonymous and confidential. However, this interview will be recorded for research purposes. Questions: Can you tell me your age and occupation? How many years of experience in urban planning/ Public health practices? How familiar are you with the concept of healthy urban planning? Can you describe your professional experience and background concerning urban and public planning practices? Sanitary System: 5. What are some of the current challenges or problems you have encountered regarding your city's sewage system, toxicity, trash, or epidemiology? 6. Can you explain the current condition of the sewage system, collection, paths, and treatment in your city? 7. How are sanitary system problems identified in your city? 8. What are the problems faced and suggested solutions for better public health circumstances in the city? 9. How do you see the management and governance of the city impacting public health and well-being? 10. Can you say something about the gap between macro health-oriented strategies and basic managerial techniques in the city? 11. How do fragmentation issues between different sectors affect the promotion of a healthy city? Urban Green Spaces: 12. How does the formation and qualification of healthy urban planners impact the implementation of new healthy city concepts in urbanism curriculums? 13. How important do you believe public community involvement is in achieving healthy city goals? 14. Can you say something about the availability and accessibility of urban green spaces in your city? 15. How do urban green spaces impact citizens' mental and physical health? Policy: 16. How do you identify urban health problems in your city? 17. How do you integrate health concepts into the urban planning process? 18. How should urban planning policies be changed to promote healthier cities, especially after COVID-19? 19. Can you explain the current state of urban planning policy from a healthy perspective? 20. How do you involve other stakeholders in this process? 21. How do you ensure sustainable development is achieved through this process? 22. How do you measure the success of this process within your position? 23. What are the most important initiatives and policies in your wilaya concerning healthy urban planning? 24. What are some of the barriers you have encountered in implementing healthy urban planning? 25. What are some of your suggestions for future development in healthy urban planning? That was all. Thank you for your time and assistance in this important study. Appendix B. The Questionnaire Thank you for taking the time to participate in this survey. This questionnaire aims to assess the applicability of initiatives and decisions related to public health, urban planning, and management in the city of Algiers after the COVID-19 pandemic. Your participation is entirely voluntary and confidential. Your answers will be used for research purposes only and kept strictly confidential. The survey consists of 18 questions divided into three parts: demographic information, sanitary system and epidemiology; healthy city, management, and green spaces implementation; and health integration in urban planning policy. Please note that your participation in this survey is entirely voluntary and confidential. The questionnaire is designed as a Likert scale, where 5 represents "strongly agree", 4 represents "agree", 3 represents "neutral", 2 represents "disagree", and 1 represents "strongly disagree". Please answer all questions as honestly and accurately as possible. Your input is greatly appreciated. Demographic Information: Gender:Male Female Age:Specify Occupation:Student Public Sector Employee Private Sector Employee Self-Employed Retired Education Level:Less than High School High School University Post-Graduate Part 1: Sanitary System and Epidemiology 5. How satisfied are you with the city's response to the COVID-19 pandemic?Strongly dissatisfied Dissatisfied Neutral Satisfied Strongly satisfied 6. How well do you think the city's public health system is prepared for future epidemics?Strongly unprepared Unprepared Neutral Prepared Strongly prepared 7. Do you think the city's public health system is accessible to all residents?Strongly disagree Disagree Neutral Agree Strongly agree Part 2: Healthy City, Management, and Green Spaces Implementation 8. How satisfied are you with the city's efforts to promote a healthy lifestyle?Strongly dissatisfied Dissatisfied Neutral Satisfied Strongly satisfied 9. How well do you think the city's management of green spaces is?Poorly Somewhat poorly Neutral Somewhat well Well 10. How important are the city's accessible green spaces?Not at all important Somewhat unimportant Neutral Somewhat important Very important Part 3: Health Integration in Urban Planning Policy 11. How well do the city's urban planning policies promote health and well-being?Poorly Somewhat poorly Neutral Somewhat well Well 12. How satisfied are you with the city's efforts to integrate health into urban planning decisions?Strongly dissatisfied Dissatisfied Neutral Satisfied Strongly satisfied 13. How satisfied are you with the overall health and safety measures implemented in Algiers after COVID-19?Strongly Disagree Disagree Neutral Agree Strongly Agree 14. How effective do you believe the city's management has been in addressing public health concerns related to COVID-19?Strongly Disagree Disagree Neutral Agree Strongly Agree Figure 1 Model of the study. Source: authors. Figure 2 Study location map. Source: authors. Figure 3 Age and experience. Source: authors. Figure 4 Key initiatives after COVID-19 in Algiers. Source: authors. Figure 5 SWOT matrix for PDAU horizon 2035. Source: authors. ijerph-20-03804-t001_Table 1 Table 1 Theme 1: sanitary elements affecting public health in urban space--results interpretation. Sub-Theme Main Declarations Interpretation Proper sewage system I#9: "It is the responsibility of the conservation and public health sectors to prevent disasters linked to sewage overflowing or pollution". I#1: "SEAAL (Algiers water and sanitation company) is doing quite good work in collecting and purification". I#6: "Things become complicated from early December to late march. It is the rain and floods season, and because of the old, degraded condition of pipes and maintenance holes, specifically in the ancient part of the city... it always requires interventions to solve it; rains bring persistent urban challenges". Interviewees emphasize the need for joint efforts between conservation and public health sectors to prevent sewage-linked disasters and pollution. They praise the work of SEAAL in the collection and purification process and shed light on the challenges during the rain and floods season, particularly in the ancient part, due to the old and degraded condition of pipes and maintenance holes. Risk of toxic contamination in cities I#10: "I believe that the risk of exposure to toxic materials and chemical mixtures in the urban environment on the local level is relatively low". I#2: "Unfortunately, the fast-going act of the industrial zones of the northern wilayas of Algeria turns the mission of checking toxic waste data for control agencies even more difficult". Interviewees discuss the issue of exposure to toxic materials and chemicals in the urban environment. They noted that the rapid development of industrial zones in the northern region of Algeria makes it difficult for control agencies to monitor toxic waste data. Waste management practices in urban space I#7: "On the local level, the waste management field used to suffer from the lack of availability of human and material resources, which lasted until four years ago when the municipal council took on the responsibility of signing a contract with a private company (Extranet) along with the government capabilities for a better household waste collecting and disposal operating: double rounds, better efficiency, and recycling strategy". I#12: "we understand the emphasis of waste management for a healthy city, and we aim to stay updated to the emerging waste management technologies and their associated effects on the public health". The interviewees highlight the importance of waste management in ensuring public health in urban areas. They emphasize the role of the local government and private sector in improving waste collection and disposal through the use of efficient systems and recycling strategies, and stress the importance of staying informed about new waste management technologies and their impact on public health. Both quotes showcase a commitment to preventative strategies in urban planning to address public health concerns. Experience gained from the COVID-19 pandemic in urban planning I#6: "we learned from the COVID-19 pandemic that education alone cannot fix the problem, neither can medicine... we now have chronic diseases associated with the habitable environment, just like respiratory diseases, obesity, and others, so we need to keep working on the same path to achieve a city that can fight pandemics and infectious illnesses and be an aid to the medical profession to make their mission easier and less complicated". I#11: "COVID-19 pandemic demonstrated to us how venerable we can be and how non-prepared our cities are to face such a circumstance, but we took a precious experience from the catastrophe in what concerns crisis management and pandemics fighting from punctual levels such as hospitals and healthcare facilities". The interviewees underline the importance of implementing innovative and holistic preventive strategies in urban planning to address public health concerns and address the vulnerability exposed by the COVID-19 pandemic. Additionally, they discuss the valuable experience gained from the pandemic in crisis management and fighting pandemics at the local level, including hospitals and healthcare facilities. New preventive strategy in urban planning I#1: "The COVID-19 pandemic has highlighted the need for innovative preventive strategies in urban planning to ensure public health and safety in our cities". I#5: "The new normal has made it imperative for urban planners to reconsider traditional planning approaches and embrace a holistic, health-centric approach to city design". The interviewees emphasize the importance of innovative solutions to ensure public health and safety in urban areas after the pandemic. However, the interviewees also acknowledge the need for a shift in traditional planning approaches towards a more holistic, health-centric approach to city design to reflect the new normal. ijerph-20-03804-t002_Table 2 Table 2 Theme 2: management, governance, and leadership skills for the identification of the healthy city concept--results and interpretation. Sub-Theme Main Declarations Interpretation Managerial practices I#1: "A key factor in the success of the healthy city movement is good city governance and right managerial decisions". I#5: "Healthy urban planning is always related to good city governance and optimum managerial choices, along with taking into consideration the realistic factors affecting the optimistic aspirations". The interviewees emphasized the importance of effective city governance and sound decision-making in creating a healthy urban environment. They highlight the relationship between healthy urban planning and good governance, emphasizing that realistic factors must be considered to achieve positive outcomes. Gap between macro health-oriented strategies and basic managerial techniques in the city I#7: "The challenge in bridging the gap between macro health-oriented strategies and basic managerial techniques in the city lies in the lack of integration between the two approaches". I#9: "The discrepancy between macro health-oriented strategies and basic managerial techniques in the wilaya highlights the need for a more comprehensive and coordinated approach to urban planning and public health". The interviewees highlight the challenge of aligning macro health strategies with local urban planning practices and emphasize the need for a more integrated approach to improve urban public health outcomes. They underline the importance of harmonious coordination between health-oriented strategies and managerial techniques in urban planning. Fragmentation issues between different sectors I#2: "It is clear that there is a problem of fragmentation when it comes to collaboration between the two sectors to achieve the desired aims of healthy cities". I#12: "To achieve the healthy city goals, many efforts should be granted, and official collaborations of vertical levels of governance are needed. The efforts must include clear plans, budgets, and continuity mindset while applying them". The interviewees suggest a disconnection between macro-level health strategies and the city's practical, day-to-day, management techniques. This disconnection leads to a fragmentation in collaboration between the two sectors, which hinders the achievement of the goal of a healthy city. To overcome this challenge, it is necessary to implement an official collaboration between different levels of governance, with clear plans, budgets, and a continuous mindset in implementing these plans. formation and qualification of healthy urban planners I#9: "I am afraid we still lack the right expertise and qualification in sustainability and green cities at the level of local urban administrations, which represents a fence limiting the good practices here". I#3: "The concept of healthy urban planning is already emerging in our universities... fresh graduates are well formed in what concerns the public health implementation in urban planning phases as they proved this in several projects in the last three years either as newly recruited engineers or even as trainees". I#5: "The good qualification of managers and urbanists in terms of healthy planning is a key factor in achieving the healthy city objectives from the managerial level". The interviewees had mixed views about the challenge of creating a harmonious relationship between macro health-oriented strategies and local managerial techniques in urban planning due to the need for more integration between the two approaches. They expressed the need for increased expertise and qualification in sustainability and green city practices among local urban administrators. On the other hand, healthy urban planning is gaining traction in universities, with fresh graduates well-equipped to implement public health in urban planning projects. Therefore, the qualification of urban planners in healthy planning is crucial in achieving healthy city objectives. Implication of public community in the healthy city goals I#8: "The healthy city project is applied to many cities in the north, middle, and even in the Sahara area of Algeria with some varieties in geographical and environmental elements as well as the nature of the urban texture in each city.... We mention one of some successful examples, Tizi Ouzou, which received the healthiest city reward twice this year and last year. However, it would not be possible without the union of efforts between the government and citizens of the city". I#4: "Regarding implementing the healthy city concept and common efforts between community and government, the sustainable healthy concept has not yet been applied correctly. First, there needs to be a degree of awareness. Then the citizens should already feel a certain level of healthiness in their city to initiate the enhancement process". The interviewees highlight the challenge of integrating macro health-oriented strategies with basic managerial techniques in cities and the need to integrate the two approaches. Tizi Ouzou is mentioned as a successful example of a healthy city, highlighting the importance of the union of efforts between the government and citizens. Implementing the healthy city concept requires awareness and a sense of healthiness within the city's community to initiate the enhancement process. Source: Authors. ijerph-20-03804-t003_Table 3 Table 3 Theme 3: integrating health concepts in the urban planning process--results and interpretation sub-theme. Main Declarations Interpretation The current state of urban planning from a healthy perspective I#10: "We need to admit that we are still far from the ideal healthy city requirements for the moment... it is obvious that the attempt to improve the situation on both levels is ongoing". The interviewee highlighted that the challenge of implementing healthy urban planning lies in the lack of integration between macro health-oriented strategies and basic managerial techniques in the city. Urban health problems' identification I#7: "We need to identify the illness first to take actions that can improve health in the urban space. The identification process could be rigorous and expensive in both time and budget, but an absolute urgency to solve the existing issues and avoid future complications". The interviewee mentioned that the challenge in implementing healthy urban planning is due to the disconnect between high-level health strategies and practical management techniques in the city. Need for change in planning and policy I#3: "To develop a shared prevision for a healthier and more equitable urban place, we need a serious commitment from the different stakeholders, partners, and policy decisions" I#3: "Organizational attempts are needed to establish healthy urban as a norm for planning practices". I#12: "Building environment and public health professions require some more proof of leadership and political commitment" (I#12) I#5: "We need to start planning our urban habitable space more healthily, especially roads, maybe implementing more walkable roads near habitats with special design that encourages citizens to walk more frequently". Changes are needed in urban planning and policy-making to develop a healthier and more equitable urban place. Therefore, to achieve a healthier and fairer urban environment, there must be a strong commitment from all stakeholders and policymakers, including efforts to establish healthy urban planning as a norm, leadership and political support from environment and health professionals, and a focus on designing walkable roads to encourage healthy habits. Source: Authors. ijerph-20-03804-t004_Table 4 Table 4 On-site investigation steps. Theme Sub-Theme Initiative/Decision Location in Algiers Type of Investigation Status and Percentage Sanitary system and epidemiology Sewage and wastewater Prevention of disasters linked to sewage overflowing or pollution by maintaining the sewage system. Cheraga Kouba Ben Aknoun Casbah Beb El Oued Checking the VRD plans Observation In progress: 40% ONA (National Sanitation Office): wastewater treatment plant under construction as part of a multi-year national action plan. Dar El Beida Construction site observation Project manager declaration In progress: 20% Quality of water SEAAL: improvement of the quality of water produced and distributed at the level of the Wilayas of Algiers. Kouba checking the results of periodic analyzes of the drinking water (pH, total dissolved solids, heavy metals, presence of bacteria and virus's tests) Compliant with standards: 100% Rain and floods season Interventions to solve old, degraded pipes and maintenance holes in ancient part of the city. Casbah Cheraga Draria Hussein Dey Observation of pipes in a rainy day In progress: 60% Risk of exposure to toxic materials Minimizing the risk of exposure to toxic materials and chemical mixtures in urban environment on local level. Reghaia lake Oued El Harrach Rouiba Visits to 16 company, 5 of them are suspended until acquiring purification stations for toxic and dangerous liquids. In progress: 30% Healthy city, management, and green spaces implementation. Better use of land Transforming Oued S'mar public landfill into a public garden Oued S'mar Observation of a 45 Ha public garden for relaxation Done 100% Green spaces management Renovation of green spaces in all the districts of Algiers Cheraga Draria Hussein Dey Beb El Oued Kouba Mouhamadia Observation In progress: 20% Bainam forest tree plantation initiative Bainam Observation In progress: 90% Waste management Waste collecting and disposal operating with contract with private company and government capabilities Beb El Oued Casbah Citizen's confirmation Observation In progress: 60% Using new technologies in collection Cheraga Draria Hussein Dey Beb El Oued Kouba Citizen's confirmation Observation In progress: 30% Source: authors. ijerph-20-03804-t005_Table 5 Table 5 Hypotheses supporting status according to study phases. Source: authors. Hypothesis Interviews Survey PDAU Analysis Site Visits Hypothesis 1 Supported Supported No contribution Supported Hypothesis 2 Supported Supported Supported Supported Hypothesis 3 Supported Supported Supported No contribution Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. References 1. Afrin S. Chowdhury F.J. Rahman M. COVID-19 Pandemic: Rethinking Strategies for Resilient Urban Design, Perceptions, and Planning Front. Sustain. Cities 2021 3 10.3389/frsc.2021.668263 2. Tonne C. Adair L. Adlakha D. Anguelovski I. Belesova K. Berger M. Brelsford C. Dadvand P. Dimitrova A. Giles-Corti B. Defining pathways to healthy sustainable urban development Environ. 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PMC10000845 | Cells Cells cells Cells 2073-4409 MDPI 10.3390/cells12050692 cells-12-00692 Article Mitochondrial Metabolomics of Sym1-Depleted Yeast Cells Revealed Them to Be Lysine Auxotroph Lagies Simon Methodology Formal analysis Data curation Writing - original draft Writing - review & editing Visualization 12+ Pan Daqiang Conceptualization Methodology Formal analysis Investigation Data curation Writing - original draft Writing - review & editing Visualization 13+ Mohl Daniel A. Writing - review & editing Visualization 12 Plattner Dietmar A. Writing - review & editing Supervision Funding acquisition 2 Gentle Ian E. Writing - review & editing 4 Kammerer Bernd Conceptualization Writing - review & editing Supervision Project administration Funding acquisition 1256* Vascotto Carlo Academic Editor Tikkanen Ritva Academic Editor 1 Core Competence Metabolomics, Hilde-Mangold-Haus, University of Freiburg, 79104 Freiburg, Germany 2 Institute of Organic Chemistry, University of Freiburg, 79104 Freiburg, Germany 3 Institute of Pharmaceutical Science, University of Freiburg, 79104 Freiburg, Germany 4 Institute of Medical Microbiology and Hygiene, Medical Center-University of Freiburg, Faculty of Medicine, University of Freiburg, 79104 Freiburg, Germany 5 BIOSS Centre for Biological Signalling Studies, University of Freiburg, 79104 Freiburg, Germany 6 Spemann Graduate School of Biology and Medicine (SGBM), University of Freiburg, 79104 Freiburg, Germany * Correspondence: [email protected] + These authors contributed equally to this work. 22 2 2023 3 2023 12 5 69222 12 2022 10 1 2023 17 2 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). Metabolomics has expanded from cellular to subcellular level to elucidate subcellular compartmentalization. By applying isolated mitochondria to metabolome analysis, the hallmark of mitochondrial metabolites has been unraveled, showing compartment-specific distribution and regulation of metabolites. This method was employed in this work to study a mitochondrial inner membrane protein Sym1, whose human ortholog MPV17 is related to mitochondria DNA depletion syndrome. Gas chromatography-mass spectrometry-based metabolic profiling was combined with targeted liquid chromatography-mass spectrometry analysis to cover more metabolites. Furthermore, we applied a workflow employing ultra-high performance liquid chromatography-quadrupole time of flight mass spectrometry with a powerful chemometrics platform, focusing on only significantly changed metabolites. This workflow highly reduced the complexity of acquired data without losing metabolites of interest. Consequently, forty-one novel metabolites were identified in addition to the combined method, of which two metabolites, 4-guanidinobutanal and 4-guanidinobutanoate, were identified for the first time in Saccharomyces cerevisiae. With compartment-specific metabolomics, we identified sym1D cells as lysine auxotroph. The highly reduced carbamoyl-aspartate and orotic acid indicate a potential role of the mitochondrial inner membrane protein Sym1 in pyrimidine metabolism. compartment-specific metabolomics mitochondria yeast sym1 lysine auxotroph mitochondrial DNA depletion syndrome MDDS European Research Council (ERC)648235 University of FreiburgThis work was supported by the European Research Council (ERC) Consolidator Grant 648235. We acknowledge support by the Open Access Publication Fund of the University of Freiburg. pmc1. Introduction Subcellular compartmentalization allows the spatial distribution of metabolites among different cell organelles so that individual metabolites can be involved in multiple biological processes simultaneously. Although compartment-specific metabolite distribution was shown over four decades ago , it has been for a long time a challenge to get further insights into the activity of subcellular metabolites. To elucidate subcellular compartmentalization, isolated mitochondria were investigated using metabolomic analyses , highlighting the extension of metabolomics from cellular to subcellular level. Consequently, the barrier of compartmentalization could be overcome by showing compartment-specific metabolite distribution and regulation upon genetic or environmental change . Yeast Saccharomyces cerevisiae SYM1 is an ortholog of human MPV17, whose mutations cause mitochondrial DNA depletion syndrome (MDDS) . Ninety-eight pathogenic variants of MPV17 have been reported till the end of 2017, presenting mostly with hepatic and neurologic manifestations . However, the function of the protein MPV17 is still undefined. Several biological models, including mice, yeast, and zebrafish, have been applied to modulate the function of MPV17 , showing various abnormal phenotypes. While MPV17-deficient humans developed liver disease , Mpv17-deficient mice suffered from renal failure . Mpv17-depleted mice showed deoxynucleotide insufficiency in liver mitochondria . Furthermore, impaired folate-mediated one-carbon metabolism was observed in mitochondria of HeLa cells with reduced MPV17 expression , presenting depleted mitochondrial dTMP pools. Therefore, MPV17 was predicted to be involved in the transport of dTMP from the cytosol to the mitochondria . This is similar to a yeast model investigated before, proposing the yeast ortholog Sym1 as a transporter for TCA cycle intermediates . Moreover, Sym1 is reported to be a channel-forming protein that maintains structural and functional stability as well as membrane potential . Recently, Sym1 was presented to be part of a high molecular weight complex with undefined function , leaving more questions to be explained. Liquid chromatography (LC)-quadrupole time-of-flight (QTOF) mass spectrometry-based metabolomics has been widely applied in plant phytochemical analysis , nutrition science , and cancer biomarker discovery . Although QTOF can supply accurate masses of analytes, it is still a challenge to identify metabolites. Furthermore, the ability to detect thousands of mass features highly increases the data complexity . Importantly, LC-QTOF analysis complements well with untargeted GC-MS profiling, since its analytes do not have to be volatile and it does not have strict molecular weight restrictions. On the other hand, more compounds can be ionized by electron ionization and the following fragmentation reactions yield a high amount of fragments. Numerous fragment ions in combination with system independent retention indices obtained by GC make the identification process more robust. Therefore, we applied mitochondrial metabolomics in this work to study the mitochondrial inner membrane protein Sym1 with an unknown function using GC-MS profiling, targeted LC-MS, and a U(H)PLC-QTOF/MS-based workflow focusing on only significantly changed metabolites. Consequently, fifty-one metabolites were identified as significantly changed, of which forty-one were novel compared to the combined method. With the help of a powerful chemometrics platform, this workflow highlights a strategy in identifying altered metabolites within a highly complex data matrix. 2. Materials and Methods 2.1. Yeast Culture and Mitochondria Isolation Saccharomyces cerevisiae strains sym1D and the corresponding wildtype (WT) BY4741 were obtained from Euroscarf. The detailed cell culture condition and the isolation of mitochondria have been described before . 2.2. Phenotype Test of Sym1D Cells Cultures of sym1D and WT were grown in liquid synthetic galactose medium (SC medium) (0.67% [w/v] yeast nitrogen base without amino acids (Becton, Dickinson and Company, Sparks, MD, USA), 0.77 g/L SC amino acids (MP Biomedicals, Irvine, CA, USA), 2% [w/v] galactose (Sigma-Aldrich, Taufkirchen, Germany)) overnight at 23 degC to exponential phase before 1 OD600 unit of each culture was taken. The cells were pelleted at room temperature at 4000 rpm for 5 min. After removal of the culture medium, the cells were washed and resuspended in 1 mL sterile dH2O, with which a tenfold serial dilution including five concentrations was made. A quantity of 3 mL of each concentration was plated on agar plates (SC medium + 3% agar (Sigma-Aldrich)) with or without a specific metabolite. 2.3. Combined GC-MS Profiling and Targeted LC-MS Details of the combination of GC-MS profiling and the targeted analysis of amino acids and pyrimidine metabolism intermediates are provided in our previous work . The raw data of GC-MS and the combination with LC-MS are displayed in Supplementary Table S1. MetaboAnalyst 5.0 was used for statistical analyses, including auto-scaling, principal component analysis, ANOVA followed by false-discovery rate correction, as well as heat map generation . 2.4. U(H)PLC-QTOF Workflow As shown in Figure 1, this workflow starts with a full-scan of WT and mutants using a U(H)PLC-QTOF instrument (6545, Agilent Technologies, Waldbronn, Germany). After data processing with Mass Profinder (Agilent Technologies) and Mass Profiler Professional (Agilent Technologies), precursor lists were generated including significantly changed metabolites. These precursor lists were applied to targeted MS/MS analyses to obtain the spectral information of the interested metabolites. Metabolites were identified by searching the spectra in the supplied library (Agilent Technologies) as well as from the literature. Briefly, the dried sample pellets were resuspended in 100 mL of water before 5 mL of each was injected onto a HILIC-Z column (100 x 2.1 mm, Agilent Technologies) with the flow rate set to 0.6 mL/min. Buffer A was 10 mM ammonium formate in water while buffer B was 10 mM ammonium formate in 10/90 water/acetonitrile (LC-MS grade, Carl Roth, Karlsruhe, Germany). The following gradient was used: 100% B to 75% B in 10 min, 75% B to 60% B till 12 min, 60% B till 15 min, 60% B to 100% B till 17 min, post run 5 min. The mass spectrometer was operated in full-scan mode. All samples were analyzed in technical four replicates so that statistical analysis could be made. The data sets of WT and mutants were processed using Mass Profinder for peak picking and alignment with a 10-ppm mass accuracy and 0.25-retention time window before statistical analysis was done by Mass Profiler Professional. The significantly changed features were exported as precursor lists including accurate mass and retention time for further targeted MS/MS analyses. All chromatographic settings remained the same except that the mass spectrometer was operated in targeted MS/MS mode with the exported precursor lists. After the analysis with the inclusion lists for each sample, the data files were processed with MassHunter Qualitative Analysis (Agilent Technologies) by using Compound Discovery with Find by Targeted MS/MS. The acquired mass spectrum was searched in the supplied METLIN mass spectral library and the literature. 3. Results 3.1. GC-MS-Based Metabolic Profiling Combined with Targeted LC-MS To evaluate if the deletion of SYM1 causes compartment-specific metabolic alterations, the isolated mitochondria and cytoplasm were subjected to compartment-specific metabolomic analysis using combined GC-MS profiling and targeted LC-MS analysis. The annotated metabolites with their normalized intensities are displayed in Supplementary Table S1. As expected, the cytoplasm and mitochondria of sym1D cells showed different metabolic patterns. By analyzing the data using PCA, all groups can be discriminated from each other. As seen in Figure 2 (left), PC1 discriminates cytoplasm (Cyto) from mitochondria (Mito), while PC2 discriminates sym1D from WT. Interestingly, the shift between the two cytoplasm groups is greater than that between the mitochondria groups. To present how every single metabolite contributes to the discrimination of groups in the PCA score plot, a PCA loading plot was applied. The individual loading values are displayed in Supplementary Table S2. Loading 1 shows how the metabolites differentiated the compartments, highlighting a group of metabolites, including several fatty acids and steroid intermediates. The metabolites in loading 2 differentiate sym1D from WT, among which cytidine, ornithine, citrulline, gluconic acid, orotic acid, and carbamoyl-aspartate contributed the most. To present the detailed alteration of significantly changed metabolites among groups (results of statistical analyses are displayed in Supplementary Table S3), a heat map is shown in Figure 3. The metabolites are mainly clustered into two groups, including metabolites with either higher abundance in mitochondria or cytoplasm. Subclusters can be observed in each group, presenting different regulation patterns of metabolites. According to ANOVA, lysine was the top significantly changed metabolite. Interestingly, lysine biosynthesis intermediates, 2-aminoadipate (2-AAA) and saccharopine, were accumulated. We thus focused on lysine metabolism in the following experiments. 3.2. Sym1D Cells Are Lysine Auxotroph While lysine was significantly reduced in sym1D cells, the intermediates for lysine biosynthesis, 2-AAA and saccharopine, were accumulated as depicted in Figure 4. To examine the function of lysine, a growth test was investigated. Surprisingly, sym1D cells could not grow without lysine at any temperature tested (19, 23, and 30 degC), as shown in Figure 5. As a control, they could grow on both plates of synthetic medium with full amino acids (SCGal) and without arginine (SCGal-Arg). While the depletion of arginine maintained the growth with slight repression, the depletion of lysine repressed the growth completely. Interestingly, the fungal 2-aminoadipate pathway for lysine biosynthesis takes place in mitochondria for the first four steps and in the cytosol for the last four steps . De novo biosynthesis of lysine begins with a-ketoglutarate in mitochondria. The intermediate a-ketoadipate is exported to the cytosol before the synthesis continues for four further steps to form lysine. Therefore, it is possible that any step of this synthesis pathway was inhibited due to the deletion of SYM1. However, the addition of either intermediate for lysine biosynthesis or lysine could not rescue the growth defect of sym1D cells as shown in Supplementary Figure S1. 3.3. Establishment of a Workflow for Detection of Unknown Metabolites An LC-QTOF-based workflow was developed with the focus on significantly altered metabolites caused by the deletion of SYM1, supplying a strategy to reduce the data complexity. Four replicates of Mito and Cyto of WT or sym1D cells were analyzed in full scan mode. The average mass or retention time (RT) deviation was calculated based on the mass or RT deviation of all compounds. The average RT deviations of four replicates of Mito and Cyto were 0.00973 and 0.012 min, respectively, indicating a highly stable RT. The average mass deviations were 0.891 and 0.915 ppm, respectively, satisfying the requirement for the workflow. Due to different matrix effects, Mito and Cyto were processed separately. An example of a total ion chromatogram (TIC) with extracted chromatograms of compounds is displayed in Figure 6, showing good separation of compounds through HILIC-based chromatography. After the targeted analysis of metabolites provided in the MS-inclusion list (Supplementary Table S4), the metabolites were identified through a library search. As shown in Figure 7, the measured spectra of the target with an accurate m/z of 175.1192 and an RT of 8.03 min was compared with the spectra in the library, identifying this compound as arginine. Through library search of all the targets, 27 out of 34 and 12 out of 27 compounds were found as putative metabolites in Mito of WT and sym1D cells, respectively, whereas 47 out of 62 and 39 out of 58 compounds were found as putative metabolites in Cyto of WT and sym1D cells, respectively, as summarized in Table 1. Seven and eight metabolites could be identified through an automatic library search in Mito of WT and sym1D cells, respectively, whereas 24 and 21 could be identified in Cyto of WT and sym1D cells, respectively. However, the rest of the putative metabolites could not be identified due to the absence of spectral information, suggesting a further extension of the library. The identified metabolites are listed in Table 2, covering amino acids and related metabolites, peptides, nucleotides, and lipids. The reduced arginine and increased citrulline, together with reduced orotic acid, correlate with the acquired data using combined methods, indicating reproducible results across different analytical techniques. Except for those metabolites that were identified already, forty-one novel metabolites could be revealed including two metabolites from the arginine metabolism pathway, 4-guanidinobutanal and 4-guanidinobutanoate; modified nucleobase and nucleoside, 1-methylguanine, 5-methyl cytosine, and 5-methylcytidine; phospholipids; modified amino acids; and tri-peptides. 4. Discussion Compartment-specific metabolomics provides new opportunities in research of sub-cellular metabolism. In that way, metabolic alterations in mitochondria could be observed, which could otherwise be overlooked in whole cell analyses. The isolation process of mitochondria on the other hand could potentially influence the metabolome itself. Nowadays, different isolation protocols are available which are adequate to metabolomics and for which a sustained metabolic pattern was shown . In particular, our applied methodology had been shown to have intact membrane integrity, and metabolic patterns derived from well-characterized mutants had been conserved . This method was employed to study the mitochondrial inner membrane protein Sym1 without a defined function. A combination of GC-MS profiling with targeted LC-MS has increased the coverage of metabolites. Importantly, specific metabolite distributions, for example, for fructose-6-phosphate or sedoheptulose-7-phosphate were observed matching those observed in the literature . Our study found differentially regulated lysine metabolism and identified sym1D cells as lysine auxotroph. While the depletion of arginine maintained the growth with slight repression, the depletion of lysine repressed the growth completely. Generally, yeast cells can synthesize all twenty amino acids . The knock-out of Sym1 protein makes lysine an essential amino acid, indicating that lysine biosynthesis is severely impaired. Lysine biosynthesis intermediates, 2-aminoadipate (2-AAA) and saccharopine, were accumulated, indicating an impaired lysine biosynthesis or degradation. Remarkably, accumulating saccharopine levels caused by mutations of its degrading enzyme resulted in a functional disruption of mitochondria in Caenorhabditis elegans . Furthermore, genetically caused saccharopine accumulation in mice also resulted in mitochondrial dysfunction, especially apparent in the liver. Further, that liver dysfunction was lethal . This is particularly interesting since all reported patients with MDDS caused by mutations in MPV17, the human ortholog of SYM1, presented with liver dysfunction and 91% of them with liver failure . Our results might suggest a role of lysine metabolism in human MDDS, which should be addressed in future studies. Another consequence of the loss of the Sym1 protein was the reduction of pyrimidine biosynthesis intermediates, carbamoyl-aspartate and orotic acid. It is worth noticing that this strain does not contain the gene URA3, resulting in an interrupted UMP biosynthesis. Consequently, the two intermediates are not expected to be observed in both WT and the mutant. Thus, the occurrence and reduction of the two precursors may indicate that they are not only precursors for UMP but also play important roles in cellular function that need to be elucidated. To discover potential novel metabolites associated with this protein, a workflow was applied focusing on only significantly changed metabolites in sym1D cells in comparison to WT. This workflow is a useful strategy to reduce the data complexity while interesting targets can be discovered. Consequently, several novel metabolites could be identified including 4-guanidinobutanal and 4-guanidinobutanoate, originating from an alternative arginine degradation pathway . Although there is little research done on these two metabolites in S. cerevisiae, their existence and reduction in the sym1D mutant indicate potential biological roles for cellular stress tolerance. The reduction of arginine and increase of citrulline in sym1D cells showed a similar effect as the heat stress-induced remodeling of arginine metabolism in WT cells . This might indicate that sym1D cells were under cellular stress despite the normal growth temperature, at least at the metabolite level. Consequently, sym1D cells cannot manage to survive at 37 degC. However, it is still not clear how Sym1 is involved in cellular stress management. In future works, the compartment-specific metabolomics workflow should be applied to sym1D cells under different stress conditions, such as heat-stress, ethanol stress, or osmotic stress. Those results could then be correlated to non-metabolic stress markers, such as heat shock proteins. Although several peptides were significantly changed in sym1D cells, no hypothesis for any biological functions can be made before the origin of the detected peptides is identified. They could be either breakdown products of proteins or free cellular peptides that may have biological functions. Except for glutamyl-cysteine, known as the precursor for glutathione, the other identified dipeptides and tripeptides do not have known biological roles. One further concern is the origin of these peptides. Although altered peptide levels have been identified in patients with liver disease or non-small-cell lung cancer , the origin or function of these peptides was not identified. Therefore, further investigation should be made to find out the origin and the biological function of oligopeptides in cells or patients, which could help in understanding their roles in diseases and cellular mechanisms. By applying whole cell experiment as described in , the existence of free peptides in cells can be examined. Therefore, further experiments are required. Nevertheless, the volume of databases is key for this workflow to identify unknown metabolites. Overall, compartment-specific metabolomics is a useful tool for identifying metabolic dysregulations in subcellular compartments. Still, it is worth noting that the metabolic network is far more complex to expect a single alteration as the response to the loss of a specific protein. Acknowledgments Work included in this study has also been performed in partial fulfillment of the requirements for the doctoral thesis of DP at the University of Freiburg. We would like to thank Nils Wiedemann from the University of Freiburg for co-supervision of the student DP and his financial support. Additionally, we would like to acknowledge Nils Wiedemann from the University of Freiburg for supplying the yeast strains and his discussion about this manuscript. Supplementary Materials The following are available online at Figure S1: Phenotype test of WT and sym1D cells with addition of lysine in various concentrations (right) and intermediates of lysine biosynthesis (left). Table S1: Metabolite intensity table, Table S2: PCA loadings, Table S3: Results of statistical analysis, Table S4: Inclusion list for MSMS analysis. Click here for additional data file. Author Contributions Conceptualization, D.P. and B.K.; Data curation, S.L. and D.P.; Formal analysis, S.L. and D.P.; Funding acquisition, D.A.P. and B.K.; Methodology, S.L. and D.P.; Project administration, B.K.; Supervision, D.A.P. and B.K.; Visualization, S.L., D.P. and D.A.M.; Writing-original draft, S.L. and D.P.; Writing-review & editing, D.P., S.L., D.A.M., D.A.P., I.E.G. and B.K. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement Data are displayed in the Supplementary Material. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Overview of the U(H)PLC-QTOF based workflow. This workflow aims to identify significantly changed metabolites between two groups. Figure 2 Principal Component analysis (PCA) score plot (left) and its loading plot (right). PC1 discriminates mitochondria (Mito) from the cytoplasm (Cyto), while PC2 discriminates sym1D from WT. The PCA loading plot shows how the individual metabolites contributes to the discrimination of groups. For readability, closely spaced metabolites only discriminating component 1 were not labeled but blue-rimmed. These were present in the C-cluster: 3-phosphoglyceric acid, alanine, AMP, aspartate, citrate, glutamate, Mannose-6P, phosphoenolpyruvate, threonine, trehalose, tryptophan, tyrosine, and UMP; and in the M-cluster: 1-monopalmitin, 1-octadecanol, 5-methyluridine, adenine, dCytidine, eicosanoic acid, ergosterol, glucose, hexacosanoic acid, lauric acid, maltose, methylphosphate, myo-inositol, myristic acid, NAD+, O-phosphocolamine, and pyroglutamate. N = 3. Figure 3 Heat map of significantly altered metabolites in mitochondria (Mito) and cytoplasm (Cyto), isolated from BY4741 (WT) and sym1D cells. Lysine biosynthesis intermediates, 2-aminoadipate and saccharopine, and lysine are highlighted in bold style with asterisk. Auto-scaled z-scores are displayed. Samples and metabolites were clustered with Ward's method and Euclidean distance measure. N = 3. Figure 4 Accumulation of lysine biosynthesis intermediates, 2-aminoadipate (2-AAA) and saccharopine, and reduction of lysine in cytoplasm of WT (black) and sym1D (orange) cells. Y-Axes show normalized intensities of each metabolite in arbitrary units. n.d.: not detected, n = 3, error bars indicate standard deviation. Figure 5 Growth test of BY4741 and sym1D on SCGal plates at 19, 23, and 30 degC for five days. BY4741 and sym1D were incubated on SCGal plate with full amino acids (left), without arginine (middle), and without lysine (right). Figure 6 An example of a total ion chromatogram (TIC) with extracted chromatograms of compounds. Figure 7 Identification of arginine through library search. The identification of metabolites was done by searching library with mass spectral information. cells-12-00692-t001_Table 1 Table 1 The number of found and identified metabolites according to the inclusion list. Mito Cyto Targets Putative Identified Targets Putative Identified WT 34 27 7 62 47 24 sym1D 27 12 8 58 39 21 cells-12-00692-t002_Table 2 Table 2 Through library search, identified metabolites that are significantly (p-value < 0.001) reduced or increased (FC > 3) in Mito and Cyto of sym1D cells. Mito Reduced Increased Arginine 13E/Z-Docosenamide Gln Lys 5-Methylcytidine GPEtn(18:1(6Z)/18:1(6Z))[U] 5-Methylcytosine Hypoxanthine Asn Asp Orotic acid Gln Ser Met PC(10:0/18:1(9Z)) Glutamine Sorbitol Frag Gln Ser Glu Cyto Reduced Increased 1-Methylguanine 5-Methylcytidine 3-Hydroxy-DL-kynurenine 5-Methylcytosine 4-Guanidinobutanal Ala Glu 4-Guanidinobutanoate Citrulline 4-Methylene-L-glutamine Glu Cys Arginine Cys Met Tyr Arg Gly Glu Thr Glu Asn Ser Glutamine Asn Val Ser His Gln Gln Gly Tyr Ile Tyr Thr Guanosine Ser Gln Methyl-myo-inositol or glucoside Ser Glu N-Acetyl-L-glutamate 5-semialdehyde Ser Glu Na-Acetyl-L-arginine Thr Ala Asp PC Val Asp Asp PC Isobutylglycine Phe Ile Ala Queuine PS Pyroglutamic acid Thiamine Thr Asn Thr-Arg Val Gln Val Thr Val Trp Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). 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PMC10000846 | Foods Foods foods Foods 2304-8158 MDPI 10.3390/foods12051076 foods-12-01076 Article Does Nepal Have the Agriculture to Feed Its Population with a Sustainable Diet? Evidence from the Perspective of Human-Land Relationship Liu Ying Conceptualization Methodology Software Validation Writing - original draft 1 Yang Yanzhao Writing - review & editing Supervision Funding acquisition 123* Zhang Chao Conceptualization Investigation 4 Xiao Chiwei Writing - review & editing 1 Song Xinzhe Visualization 1 Lavelli Vera Academic Editor 1 Institute of Geographic Sciences and Natural Resources Research, Chinese Academy of Sciences, Beijing 100101, China 2 College of Resources and Environment, University of Chinese Academy of Sciences, Beijing 100049, China 3 Key Laboratory of Carrying Capacity Assessment for Resource and Environment, Ministry of Natural Resources, Beijing 101149, China 4 Faculty of Geography, Yunnan Normal University, Kunming 650500, China * Correspondence: [email protected] 02 3 2023 3 2023 12 5 107623 12 2022 28 2 2023 28 2 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). Nepal is one of the least developed countries in the world, with more than 80% of the population engaged in agricultural production and more than two-fifths of the population still living below the poverty line. Ensuring food security has always been a key national policy in Nepal. Using a nutrient conversion model and an improved resource carrying capacity model as well as statistical data and household questionnaires, an analysis framework for food supply balance is developed in this study, which quantitatively analyzes the balance of food supply and demand in Nepal from the perspectives of food and calories during the period 2000-2020. Nepal's agricultural production and consumption have increased significantly, and the diet has been relatively stable over the past two decades. The diet structure is stable and homogeneous, with plant products occupying the absolute position in overall dietary consumption. The supply of food and calories varies widely from region to region. Although the increasing supply level at the national scale can meet the needs of the current population, the food self-sufficiency level cannot meet the needs of the local population development at the county level due to the influence of population, geographical location, and land resources. We found that the agricultural environment in Nepal is fragile. The government can improve agricultural production capacity by adjusting the agricultural structure, improving the efficiency of agricultural resources, improving the cross-regional flow of agricultural products, and improving international food trade channels. The food supply and demand balance framework provided a reference for achieving balance between the supply and demand of food and calories in a resource-carrying land and provides a scientific basis for Nepal to achieve zero hunger under the framework of the Sustainable Development Goals. Furthermore, development of policies in order to increase agricultural productivity will be critical for improving food security in agricultural countries such as Nepal. supply-demand balance land carrying capacity household questionnaires sustainable diet Nepal Strategic Priority Research Program of the Chinese Academy of SciencesXDA20010201 XDA28060302 This research was funded by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA20010201)/ (XDA28060302). pmc1. Introduction Almost one-fifth of the global population lives in South Asia, where most countries have high population density and relatively low rates of economic growth . Agriculture has always had an important economic position in Nepal. In order to feed their rapidly growing population, agricultural resources are under enormous pressure in the region, and the food supply cannot meet the needs of population development . As a result, food insecurity, linked to poverty, persists . A large number of studies have confirmed the importance of food insecurity in South Asia . Poverty is considered to be an important contributor to, and a key consequence of, hunger and food insecurity. In particular, the number of poor people can be used to measure the state of food security in a country . Food security has long been defined as the supply of food that meets the daily needs of all people, and the most important element is the supply and access to food . By the 1996 World Food Summit, the definition had expanded to "people having physical and economic access to safe and nutritious food to meet their dietary needs for an active and healthy life" (2020). As one of the key factors in promoting economic development, food security plays an integral role in maintaining social stability and achieving national autonomy, especially in developing countries . Indeed, agricultural environments in most South Asian countries are highly vulnerable to human or natural factors such as extreme weather events, natural disasters, resource constraints, labor shortages, and armed conflicts . Since the soaring food prices in 2008, how to provide food to the world in 2050 also has been a concern of scholars (FAO) . What is more, since 2019, the world has been affected by the COVID-19 pandemic, food supply disruptions, and lack of income; the number of hungry people has also increased; and food insecurity in developing countries has continued to increase, thus exposing the vulnerability of the world's external food supply . Ensuring food security and maintaining sustainable agricultural development are increasingly focus of scholars . In general, the factors affecting food security can be measured from two different aspects, one is the analysis of food availability, access, and use from the individual and/or micro level and the other is the analysis of food supply and demand at the national and/or regional scale . Rapid population growth has led to a steady increase in the demand for food, which is even more pronounced in developing countries where food production may not be able to meet the demand for food based on existing arable land, due to environmental problems, urbanization, and increasing water scarcity . Therefore, many scholars have conducted studies on food security from the perspective of food supply and demand . Food supply refers to the quantity, variety, and quality of food that a unit can consume at will . Among them, the balance between food supply and demand means that the amount of food supply can meet the basic needs of regional population development . Food supply and demand balance research focuses on trend and forecast analysis. Some studies analyze the relationship between regional food production and food demand and find that the lag between them leads to an imbalance between food supply and demand . There are also studies about the relationship between food supply and demand and food price fluctuations, analyzing the trend of food price changes and making suggestions for ensuring food security . Researchers analyze the current situation and future trend of food supply and demand balance through different perspectives, such as food availability, diet structure, food circulation and trade, arable land potential, and farmers' behavior . To date, research methods related to the balance between food supply and demand are mainly qualitative or quantitative analysis from the aspect of variety, total quantity, or region. The main methods used include: (1) using the analytical hierarchy process, comprehensive index system, entropy method, and other relative numerical index system evaluation methods for agricultural resource analysis ; (2) using the absolute numerical index methods such as the water pollutant concentration, the original ecological food carrying capacity, the ecological footprint, the minimum arable land area per capita, and the population carrying capacity to analyze and evaluate the supply level and the spatio-temporal characteristics of agricultural products ; and (3) based on the comprehensive index system methods such as dietary nutrition supply standards and food demand forecasting models, the regional balance of food supply and demand, the nutritional structure of the residents' diets, and the resource and environmental effects of food consumption were analyzed . Nepal is a landlocked developing country in South Asia dominated by an agricultural economy, which generates about one-third of its gross domestic product (GDP). As far as the authors know, the relatively stagnant performance of Nepal's agricultural sector is mainly due to the country's vulnerability to man-made and natural disasters, severe climate change, and limited land/production resources resulting in low crop yields and post-harvest losses . The 2011 Nepal Demographic and Health Survey by the Population Division Ministry of Health and Population, Government of Nepal, found that despite high rates of poverty reduction due to factors such as urbanization and rising income from migrant exodus, Nepal continues to suffer from severe malnutrition, food insecurity, and poverty . In particular, the Government of Nepal has prioritized nutritious and sufficient food throughout the year as one of the fundamental rights of its citizens in the constitution and through the Right to Food and Food Sovereignty Act . More importantly, in line with its commitment to the Global Sustainable Development Goals (SDGs), Nepal has also prioritized food and nutrition security as a key sector dedicated to eradicating hunger and malnutrition, increasing agricultural productivity, and ensuring sustainable food production systems. Land is the basis of food production. With the growing contradictions between land, food, and population, the question of whether land production capacity and food demand can be balanced has increasingly become a focus of research . Among them, obtaining the number of people that existing land resources can support has become a key element in analyzing the balance between food supply and demand. The research mainly focuses on the issues of internal food supply in Nepal and analyzes the changing characteristics of food supply and demand balance in Nepal from 2000 to 2020. Combining with the Food Balance Sheet of FAO, using the nutrient conversion model and improved resource carrying capacity model, the spatial and temporal variation characteristics of food supply and demand levels in Nepal are analyzed from the national, regional, and county scales. The study has two objectives: (1) to establish a framework of food supply and demand balance, examining the food supply and demand situation in Nepal from both the basic human needs and comprehensive demand aspects; (2) to assist the Nepalese government in identifying regional agricultural development differences and formulating more appropriate regional agricultural development policies according to local conditions. 2. Materials and Methods This study is based on data on the production and consumption of agricultural products in Nepal. According to the two standards of the basic cereal supply level (basic demand) and the calorie consumption level (comprehensive demand) and combined with the concept of carrying capacity of land resources, a model of land carrying capacity (LCC) was constructed to measure the level of food supply and demand in Nepal . Among them, the actual calorie supply is calculated taking into account different types of food use, waste, and edible proportions. The analysis is carried out according to different research scales, i.e., from country and geographical region to county. 2.1. Data Sources The structure of agricultural production in Nepal is relatively simple, and thus agricultural products are mainly divided into plant and animal products in this study. Vegetal products are divided into eight categories, including grains, roots, sugar crops, oil crops, pulses, spices, vegetables, and fruits. Animal products are divided into three categories, namely meat, eggs, and milk. Agricultural production data are mainly from the Food and Agriculture Organization of the United Nations (FAO) website (FAO; accessed on 10 September 2022), the Food and Agriculture section in the Statistical Yearbook Nepal (2013-2019), and the Statistical Information on Nepal Agriculture (2019/20) . Specific sources of data on agricultural products are shown in Appendix A. It should be noted that this study used the same statistical range as FAO. Data on food consumption were obtained from "Food supply (kcal/capita/day)" section of the FAO food balance sheet, and the questionnaire on food consumption of residents from Nepal. Based on the understanding of the residents' food intake, the Nepalese Residents' Food Consumption Questionnaire conducts preliminary research of the contents of the questionnaire by sorting the relevant literature and summarizing the previous research experiences and following the principles of scientificity, systematicity, independence, and comparability. After the design, experts from Nepal were invited to discuss and form the final survey questionnaire. The questionnaire mainly includes the following basic contents, i.e., family residence, number of family members and age structure, and main food types, as well as the type and quantity of food intake for three meals a day. A questionnaire survey was conducted in Nepal in December 2021. A total of 200 households' food consumption was counted, and the validity of the questionnaire reached 100%. Data obtained from the questionnaire were analyzed using Reliability analysis and Factor analysis in IBM SPSS software, and the number of family members, caloric intake of rice, vegetables, meat, eggs, fish, and milk were selected for further analysis. The results showed that the reliability (0.769) and validity (0.779) passed the test, and the quality of the questionnaire results was high, which proved that the results were usable. National population data are from Area and Population section of the Nepal Statistical Yearbook and FAO. However, the Central Bureau of Statistics of Nepal conducts a national census every ten years, and the results of the 2020 census have not been released. Therefore, county-level population data in 2020 comes from WorldPop accessed on 15 September 2022). The grid data were divided into counties via the county vector boundary using the ArcGIS (10.x) software. According to the total population of the country in 2020, the county-by-county conversion is conducted in equal proportions. This ensures that the sum of the population of the counties is equal to the total population of the country. The three major ecological divisions of Nepal are mapped with reference to Nepal: A Country Study and are combined with district administrative divisions. 2020 land use data from GlobeLand30 dataset accessed on 30 September 2022). Land area data are from the Statistical Yearbook Nepal 2019 . 2.2. Food-Calorie Conversion Model Foods varies by calorie content, and a consistent measurement of food supply and demand levels is realized using the food-calorie conversion model:Energy=EixCaloriei where Energy is the calorie supply level; Ei is the i-th category of food (Table 1); and Caloriei is the calorie contained in the i-th category of food. To estimate calorie intake on the consumption side, based on food production as a data source, a calorie supply model was constructed by comprehensively considering food use (conversion factor), food waste (waste coefficient), and food edibility (intake part coefficient). The model has matching parameters for each food (Table 1). Table 1 provides food-calorie conversion parameters for major categories of food. Food calorie data from FAO Statistics accessed on 6 September 2022) matched 180 identified food items. The food conversion factor, intake part coefficient, and waste coefficient in distribution and consumption are mainly from Global Food Losses and Food Waste . Referring to the World Bank's classification of national income groups, the corresponding parameters are selected according to the level of economic development and the geographical region where Nepal is located. For detailed parameters of other foods, please see the Appendix A and Appendix B of the paper. 2.3. Definitions of Food and Calorie Demand Levels in Nepal There are differences in recommended nutrient intake standards for different populations and different living standards. According to the human caloric requirements in the relevant FAO and WHO reports, the caloric requirements of an adult individual weighing 70 kg are 2450-2730 kcal/day . The Lancet Health Commission recommends a caloric intake of 2500 kcal/day. Ceren and his colleagues believe that global calorie demand has been increasing over the past 50 years, reaching 2370 kcal/person/day . This study determined food and calorie requirements for Nepal based on food supply data from the FAO Food Balance Sheet. In particular, only primary agricultural products are considered in terms of food supply, while aquatic products are not included. It has been calculated that per capita food consumption in Nepal in the past five years is 403 kg, and calorie intake per capita is 2425 kcal. Therefore, the cereal per capita and calorie standards were 400 kg/year and 2400 kcal/day, respectively. 2.4. Food Demand-Supply Balance Model: Land Carrying Capacity Model Combined with the concept of carrying capacity of land resources, the LCC model was constructed and improved. It can measure the number of people in Nepal that can be sustainably supported by regional arable land resources under a given level of food consumption. The model is calculated according to two aspects of meeting the basic and comprehensive demands of human survival. Model results are used to measure the state of regional food supply and demand. The LCC and LCCI was analyzed in relation to cereal demand and calorie requirement based on the characteristics of food production in Nepal using the following model:LCC=LCCc=G/400LCCE=E/2400 LCCI=LCCcI=P/LCCcLCCEI=P/LCCE where LCCc is LCC estimated against cereal demand; LCCE is LCC estimated against calorie requirement; G is cereal production; E is calorie supply; P is current population size; LCCI is LCC index; LCCcI is estimated relative to cereal demand and measures the degree of cereal supply-demand balance; and LCCEI is estimated relative to calorie requirement and measures the degree of calorie supply-demand balance. LCCI values are classified into three levels and six ratings (Table 2) to describe the food demand-supply balance level. 3. Study Area 3.1. Geographical Environment in Nepal Nepal is a landlocked country and borders China to the north, and India to the south, east and west. It is commonly divided into three ecological divisions, namely the Mountain belt, the Hill belt, and the Terai belt, according to decreasing height above sea level (asl) . The Mountain region is mainly north of the northern mountain region above 4000 m asl and represents the central part of the Himalayas. The Hill region is located in the southern part of the mountains, mostly between 1000 and 4000 m asl. The Tarai region is a lowland tropical and subtropical zone with flat alluvial land extending along the Nepal-India border, parallel to the mountains, and there is a fertile, humid, and flat agricultural strip. As shown in Figure 3, forest and cropland are the main land use types in the country. Cropland is mainly distributed in the western and southern regions, and forest land and grassland primarily in the northern and eastern regions. 3.2. Agricultural Environment Two-thirds of Nepal's population are engaged in agriculture, contributing approximately a quarter of the national GDP. Due to its flat lands, rivers, and fertile soil, most of the agriculture takes place in the Terai area. It can be seen from Figure 4 that since 2000, the cereal production and per land cereal production in Nepal are constantly increasing. Furthermore, the increase in agricultural production technology and conditions of agricultural production have been constantly improving in Nepal. However, with economic development and population growth, cities have expanded unrestrictedly in recent decades. Since 2001, the per capita arable land in Nepal is decreasing. Notably, this puts downward pressure on the future food supply in the country. Among the vegetal products in Nepal, the main cereal crops are rice, maize, and wheat. From 2000 to 2020, rice production accounted for more than 50% of cereal production, showing a downward trend. The proportion of maize yield was generally stable between 20% and 30%, and the overall growth trend was high. In contrast, wheat production is relatively low and remained below 20% until 2007, but after 2007 the share of wheat production increased. Overall, the structure of cereal crops in Nepal is relatively stable . Among other vegetal products, the production of sugar, oilseeds, pulses, fruits, and vegetables also showed an increasing trend, with the highest growth recorded in vegetables (1.66 times) and potatoes (1.65 times) . Livestock in Nepal are mainly poultry, cattle, sheep, and pigs. Among them, the number of poultry keeps increasing rapidly (3.36 times). The number of cattle and sheep are growing slowly, and the number of pigs is the least. The production of meat, eggs, and milk in Nepal shows an increasing trend. Among them, milk production was the highest and grew the fastest, from 120.30 tons to 245.53 tons. Egg production is the lowest but also continues to increase . 4. Results 4.1. Food Consumption and Calorie Supply 4.1.1. Food Consumption and Calorie Supply Based on FAO Data Figure 6 is a calorie conversion based on FAO food supply data for 2000 and 2020. Over the past 20 years, Nepal's diet structure has been relatively stable, with grains as the main source of calories, accounting for more than 60%. Food consumption is dominated by plant-based products, with the top five being grains, vegetable oils, roots, beans, and sugars. Eggs accounted for the least consumption in 2020. Grains occupy an absolute position in the dietary consumption as a whole, and the nutritional intake is relatively simple . 4.1.2. Food Consumption and Calorie Supply Based on the Questionnaire Through interviews and exchanges with the Nepalese residents, we learned about some dietary habits and the main types of food. Nepal has a unique eating habit, usually two meals a day. However, according to the results of the questionnaire, most urban families have adjusted to three meals. Breakfast is relatively simple, mostly milk tea and biscuits. Dinner is usually taken seriously, usually rice with some lentils or curry rice. Rice is one of the main foods in Nepal. By sorting and summarizing the results of the questionnaire, the research objects are mainly the young and middle-aged population, and the population aged 19-59 makes up about 69.85%. The average daily calorie intake per person in 200 households is 2046 kcal, of which the top three are grains with 1287.49 kcal, meat consumption with 267.56 kcal and beans with 116.46 kcal. Among the main types of food, milk tea and rice are the main foods, accounting for more than 90% . The obtained results after calorie conversion show that the food consumption structure of Nepalese residents is mainly rice, which accounts for 62.91%. This is consistent with the statistical results published by FAO . Meat, vegetables, pulses, and milk are relatively small. The structure of the diet of the Nepalese residents is very simple. Milk tea is the main breakfast, and rice is the main food at noon and in the evening. Grains occupy an absolute position in the dietary consumption as a whole, and the nutritional intake is relatively simple. There is little difference between urban and rural areas. 4.2. Spatio-Temporal Characteristics of the Cereal Supply and Demand Balance According to the Nepal's cereal consumption in the 2015-2020, the per capita cereal demand in Nepal is 400 kg. This section mainly analyzes the relationship between cereal supply and demand in Nepal from 2000 to 2020 from country to geographical region to county. (1) Cereal supply and demand balance at the national scale Figure 8a shows that the carrying capacity of land resources (or LCC) in Nepal maintained a fluctuating increase from 2000 to 2020, but the cereal supply population was consistently lower than the actual population. In 2000, the cereal carrying capacity was 17.79 million people, and the total population of the country was 23.94 million people. The level of cereal supply was far lower than the actual demand of the country. However, with the continuous increase in cereal supply, the carrying population also increases, and the carrying capacity of land resources is significantly increased. By 2020, the gap between the food supply population and the country's actual population will be significantly narrowed . Additionally, the average carrying capacity continued to increase . As a result, the national level of cereal supply has been improving since 2000. According to the classification of LCCI values , it can also be seen that the national cereal carrying capacity of Nepal is continuously improving. After 2018, it shifted from a deficit to a critical balance. Since 2018, the country has entered a state of tight balance of cereal supply, and the relationship between people and cereal continues to improve. Notably, the level of food supply at the national level has improved significantly. (2) Cereal supply and demand balance in the geographical regions Figure 10 shows the supply and demand of cereals in the three geographical regions (i.e., the Mountain belt, the Hill belt, and the Terai belt) in Nepal in 2020. The Mountain region has the lowest carrying capacity with only 1.42 million people, and the Tarai region has the highest carrying capacity of 15.47 million people. The cereal carrying capacity level gradually improved from north to south according to the topographical distribution. Specifically, the Mountain region has harsh climatic conditions, human living environment, and agricultural production conditions, and the entire region has only 1.93 million inhabitants. As a result, the region has the weakest carrying capacity, and food supply and demand are in a state of shortage. Compared to the Mountain region, the Hill region has showed some improvement. The gap between the carrying population and the actual population is the smallest. There are 13.08 million people in the region where the capital is located, accounting for nearly half of Nepal. The current cereal supply cannot meet the population development in the region and is in a state of deficiency. The Tarai region is the main arable land area in Nepal and has excellent conditions for agricultural production, so the food supply is the most abundant, which can meet the needs of about 15.47 million people and is in a state of balance surplus. (3) Cereal supply and demand balance in the counties Next, the cereal supply and demand balance in the counties had obvious regional regularities . The carrying capacity sequentially decreases from the northeast to the southwest. The counties with high carrying capacity are concentrated in the Tarai region, among which Jhapa, Morang, and Kailali have a carrying population of more than 1 million people. The counties with the lowest carrying capacity are concentrated in the Mountains region, such as Manang, Mustang, and Humla with a carrying population of less than 0.03 million people. It can be seen in Figure 11b that the carrying status of Nepalese counties varies greatly. In particular, the carrying index of the capital Kathmandu is 16.92, which is in a state of severe deficiency. The current level of cereal supply can only support 0.15 million people, which is completely unable to meet the local demand of 2.52 million people. In addition, the carrying index of Humla, Bhaktapur, Lalitpur, and other counties is all greater than 2.5, which is in a state of severe deficiency. The gap between the carrying population and the actual population is the smallest in the Hill region, and it is the area with the largest number of counties (14 in total) in the balanced state, especially Jhapa, Bardiya, and Kanchanpur. 4.3. Spatio-Temporal Variations of the Calories Supply and Demand Balance According to the standard of 2400 kcal per capita calorie intake of the Nepalese residents, the change in the calorie supply and demand from country and geographical region to county in the period 2000-2020 was calculated. The number of people who can be satisfied is expressed through the population carrying capacity, and then the relationship of supply and demand and its changes are analyzed in terms of the carrying status. (1) Calorie supply and demand balance at the national level The research results in Figure 12a show that, from 2000 to 2020, the carrying capacity of land resources in Nepal has generally maintained a fluctuating increase, and the gap between the carrying population and the actual population is decreasing. Except in 2016, the carrying population exceeded the actual population after 2012. In 2000, the LCC was 22.60 million, slightly lower than the national total population of 23.94 million. By 2020, the LCC will reach 29.78 million people, which is higher than the national 29.13 million. During 2000-2020, the average carrying capacity level also continued to increase . Therefore, the level of calorie supply in Nepal is relatively good, ranging from a critical balance to a surplus balance , and has been in a state of balance in the last 20 years. The calorie supply meets the needs of local population development. (2) Calorie supply and demand balance in the geographical regions Figure 14 shows the calorie supply and demand in the three geographical regions (i.e., the Mountain belt, the Hill belt, and the Terai belt) in Nepal in 2020, and the results are similar to the cereal supply and demand. The Mountain region has the lowest carrying capacity with only 0.51 million people, while the Tarai region has the highest carrying capacity of 17.40 million people. The calorie carrying capacity gradually improved from north to south in accordance with the topographical distribution. Specifically, the caloric supply is similar to the level of cereal supply, and the Mountain region is still the worst due to the harsh natural conditions and is in a state of deficiency. Compared to the Mountain region, the Hill region has some improvement. The gap between the carrying population and the actual population is the smallest and has increased in relation to the level of cereal supply. The Tarai region is the main arable land area in Nepal, and the conditions of agricultural production are superior, so the level of calories supply is the highest and reaches 17.40 million people, which is in a surplus state overall . Therefore, the supply of calories is much better than that of cereals, but it is still a deficiency in mountainous areas, which may be related to the conditions of local agricultural production and the economic development level. (3) Calorie supply and demand balance in the counties It can be seen from Figure 15a that the changes in calorie-based carrying capacity are similar to grains, and it is also similar to the variation in carrying capacity of geographical divisions, with obvious regional laws. The carrying capacity decreases sequentially from the northeast to the southwest. The counties with high carrying capacity are concentrated in the Telai Plain, among which Jhapa, Morang, Kailali, and Bara have a carrying population of more than 1 million people. The counties with the lowest carrying capacity are concentrated in the northern mountainous region. For example, Manang, Mustang, Dolpa, and Mugu have a carrying population of less than 0.02 million people. Figure 15b shows that the carrying capacity of Nepal's counties vary widely. The bearing index of the capital Kathmandu is 12.64, which is in a state of severe deficiency. The current level of food supply can only carry 0.20 million people, which is completely unable to meet the local demand of 2.50 million people. In addition, the carrying capacity index of Humla, Bhaktapur, Lalitpur, Kalikot, and other counties are all greater than 2.0, which is in a state of severe deficiency. Combined with the results of cereal supply and demand, the variation law of carrying capacity in Nepal's counties is relatively consistent, and the spatial distribution is consistent. 5. Discussion 5.1. Characteristics and Impact of Internal Agricultural Production on Food Supply and Demand Although the cereal supply and demand balance and the calorie supply and demand balance in Nepal are constantly improving since the 21st century, the level of cereal supply to meet basic human needs is still a long way off. In contrast, a comprehensive supply based on calories can meet the development needs of local population. Therefore, the current food supply can meet the human needs as a whole in Nepal, but there is still a need to continuously improve the level of cereal supply. Spatially, cultivated land resources are mainly concentrated in the Terai Plain region of Nepal, and the spatial variation in agricultural production capacity is large. By 2020, cultivated land will account for only about 28% of the total land resources in the country, and the cultivated land resources are tight. At the same time, under the influence of population growth and the continuous decline of cultivated land, the cultivated land area per capita is also declining, resulting in an increasingly fragile arable land base for agricultural production, which will have a greater impact on agricultural production, especially food supply . In addition, there is a large gap in the level of land resources production in the counties. The basic conditions of agricultural production in the Terai region and the Hill region are good, and the land production capacity is strong, so the pressure on the food supply is relatively low. However, the Mountain region have been affected by natural conditions such as topography and climate, and the food supply is in a state of deficiency. The growth of food production depends not only on the expansion of planted areas but also on investments in agricultural science and technology, such as good varieties, pesticides, agricultural machinery, and the use of irrigation technology, which require large capital investments . At present, the investment of Nepal government in agriculture is insufficient, and the agricultural infrastructure lags behind, which greatly limits the development of agricultural production . 5.2. Changes in Food Supply and Demand under an Open System With population growth, economic development, urban expansion, geopolitical cooperation, and integration in Nepal, the supply and demand relations of agricultural products face increasingly complex domestic and foreign situations, and food security must consider domestic supply and international trade. Therefore, the international agricultural market has become an important factor that must be considered to ensure food security objectives. From Nepal's own supply, its food production cannot meet consumer demand, and by considering food imports and exports combined with the conversion factor, part of the food must be imported. Thus, our study further discusses the supply and demand balance of Nepal's agricultural products in an open system. Food supply in the open state is expressed by net imports in domestic production. The data originate from the domestic yield, import quantity, and export volume of the main foods in the balance table of FAO. Figure 16a shows the change in cereal carrying capacity, and we can clearly see that the open system increases Nepal's cereal carrying capacity before 2010, i.e., from deficiency to severe deficiency. After 2010, the LCCI became small, and it gradually increased the national food carrying capacity. Therefore, moderate food imports are conducive to balanced food, which is conducive to national food security. The supply of own calories can meet the needs of domestic residents, and being under the open system is more favorable for the Nepal's calories supply and demand balance, especially after 2010; i.e., the balanced surplus is turned into a complete surplus. Therefore, international trade is obviously helpful for improving the supply and demand balance of Nepalese agricultural products after 2010. Additionally, moderate import of agricultural products is conducive to national supply and demand balance, and the national development of agricultural products can be appropriately encouraged to ensure domestic food supply and relieve population pressure. It is worth noting that only the basic foods are considered in this study, but food trade is a complex network that requires further exploration of the impacts of imports and exports on the domestic food supply. For example, individual food types should be analyzed, and in-depth exploration of Nepalese countries in the open system should be performed in order to provide scientific management recommendations for the import and export of domestic food. 6. Conclusions Starting with the premise of single internal food supply, a research framework for regional food supply-demand balance was constructed from the perspectives of land resource utilization, agricultural production characteristics, and the food consumption level and structure. Based on the concept of LCC, this study analyzes and compares the temporal and spatial changes in the food supply and demand balance at the national, geographical, and county scales in Nepal from 2000 to 2020, under the aspects of basic human needs and comprehensive needs. At the same time, changes in food supply and demand in Nepal under the open trade system were discussed. Lastly, this study briefly explored other factors affecting food supply-demand when only considering internal supply and the changes in food supply-demand under the influence of open international trade. Several key findings emerge from our analysis. First, the production and consumption of agricultural products in Nepal have increased significantly in quantity since 2000. This reflects an increase in agricultural productivity in Nepal and somewhat improved living standards. However, the residents' nutritional intake is relatively simple, and cereals as the main source of calories account for more than 60%. Second, although the levels of food and calorie supply have fluctuated over the past two decades, the basic level of food supply is far below the actual national demands. Among them, the level of food supply in the plains is the best in the last two decades. In addition, Nepal needs to increase food production to meet the growing needs of its population, and our experiments show that global trade flows of food can greatly improve Nepal's food supply and demand. Several important policy implications emerge from our findings. First, it is necessary to ensure the quantity and quality of cultivated land, and improving the quality of cultivated land can directly improve the carrying capacity of land. At the same time, the conclusions of this study can also help the government to understand the development differences between countries and regions in order to increase financial support for areas with weak agricultural development. Second, it is necessary to optimize the food planting structure and improve the nutritional intake status of the residents. We found that Nepal is still on a high grain diet with a serious lack of fat intake. In the future, it is necessary to improve the non-cereal supply capacity and increase the proportion of meat, eggs, and milk supply in the future. In order to achieve the optimization of the food consumption structure, the improvement of the nutrient intake level and the diversification of the nutrient intake structure must be performed. Third, the Nepalese government should focus on improving the level of agricultural cultivation, such as promoting international cooperation in agriculture, increasing funding support for agricultural technology, and improving the management level of crop planting. Of course, appropriate food trade policies can also effectively alleviate the pressure of population development. This study sheds light on the balance of food supply and demand in Nepal. However, it still has some limitations: First, there are many deficiencies in data, such as the lack of long-term series of sub-county agricultural data. The household consumption questionnaire investigates only some primary foods and cannot well reflect the food consumption of the whole household. Second, research methods can be further improved. In this study, LCC is used to characterize the balance of food supply and demand, while multi-index analysis and footprint method can be used in future studies. Third, our study only considered primary agricultural products, which may have somewhat affected the accuracy of the results. In this regard, the research team will employ other useful methods to collect the necessary data and information to support further research. Author Contributions Conceptualization, Y.L. and C.Z.; Methodology, Y.L.; Software, Y.L.; Formal analysis, Y.L.; Writing--original draft, Y.L.; Investigation, C.Z.; Writing--Review and Editing, C.X. and Y.Y.; Visualization, X.S.; Supervision, Y.Y.; Funding acquisition, Y.Y. All authors have read and agreed to the published version of the manuscript. Data Availability Statement The data presented in this study are available request to authors. Conflicts of Interest There are no conflict of interest to declare. Appendix A. The Specific Agricultural Product Data Sources Agricultural Products Classification Vegetal Products Grains Maize, Paddy, Wheat, Millet, Barley, Buckwheat Potato / Sugarcane / Oilseed / Pulses Lentil, Chickpea, Pigeonpea, Blackram, Horsegram, Soyabean Spices Large Cardamom, Ginger, Garlic, Turmeric, Dry Chilli Vegetables Cauliflower, Broccoli, Cabbage, Tomato, Tree tomato, Broad Leaf Mustard, Carrot, Capsicum, Chilli, Peas, French Beans, Broad Beans, Asparagus Beans, Cowpea, Asparagus, Okra, Brinjal, Onion, Cucumber, Pumpkin, Squash, Bitter Gourd, Pointed Gourd, Sponge Gourd, Ridge Gourd, Snake Gourd, Bottle Gourd, Ash Gourd, Balsam Gourd, Kakari, Kundru, Chayote, Watermelon, Drumsticks, Fennel Leaf, Coriander Leaf, Cress, Amaranthus, Fenugreek Leaf, Swisschard, Lettuce, Spinach, Yam, Elephant Foot Yam, Colocasia Fruits Sweet Orange, lime/lemon, Mandarin, Apple, Pear, Walnut, Peach, Plum, Apricot, Persimmon, Pomegranate, Hog Plum, Kiwi, Mango, Banana, Guava, Papaya, Jackfruit, Pineapple, Litchi, Arecanut, Coconut Animal Products Meat Buff, Mutton, Chevon, Chicken, Duck meat Egg Hen egg, Duck egg Milk / Appendix B. Food-Calorie Conversion Parameters for All Specific Foods Major Food Specific Foods Calories (kcal/100 g) Conversion Factor Intake Part Coefficient Waste Coefficient Cereals T1 330 0.78 0.76 0.92 T2 340 0.78 0.76 0.92 T3 356 0.78 0.76 0.92 T4 340 0.78 0.76 0.92 T5 280 0.9 0.87 0.92 T6 334 0.78 0.76 0.92 Potatoes T7 67 0.82 0.80 0.76 T8 91 0.82 0.80 0.76 Sugar cane T9 70 0.77 0.76 0.88 T10 30 0.77 0.76 0.88 T11 390 0.77 0.76 0.88 Beans T12 343 0.9 0.89 0.89 T13 358 0.9 0.89 0.89 T14 346 0.9 0.89 0.89 T15 335 0.9 0.89 0.89 T16 50 0.77 0.72 0.58 T17 343 0.9 0.89 0.89 Pulses T18 340 0.9 0.89 0.89 T19 245 0.5 0.50 0.89 T20 315 0.5 0.50 0.89 T21 252 0.5 0.50 0.89 T22 291 0.5 0.50 0.89 T23 355 0.5 0.50 0.89 T24 262 0.5 0.50 0.89 T25 289 0.5 0.50 0.89 T26 184 0.9 0.89 0.89 T27 414 0.9 0.89 0.89 T28 711 0.9 0.89 0.89 Vegetables T29 37 0.77 0.72 0.58 T30 22 0.77 0.72 0.58 T31 23 0.77 0.72 0.58 Fruits T32 48 0.77 0.72 0.58 T33 43 0.77 0.72 0.58 T34 26 0.77 0.72 0.58 T35 45 0.77 0.72 0.58 T36 52 0.77 0.72 0.58 T37 41 0.77 0.72 0.58 T38 16 0.77 0.72 0.58 T39 15 0.77 0.72 0.58 T40 34 0.77 0.72 0.58 T41 26 0.77 0.72 0.58 Spices T42 337 0.77 0.72 0.58 T43 47 0.5 0.50 0.89 T44 40 0.77 0.72 0.58 Oilseed T45 387 0.9 0.89 0.89 T46 0 0.9 0.90 1.00 T47 158 0.9 - 1.00 Meat T48 77 1 0.96 0.84 T49 174 1 0.96 0.84 T50 119 1 0.96 0.84 T51 122 1 0.96 0.84 T52 291 1 0.96 0.84 T53 326 1 0.96 0.84 T54 123 1 0.96 0.84 Milk T55 97 1 0.99 0.87 T56 73 1 0.99 0.87 T57 61 1 0.99 0.87 T58 69 1 0.99 0.87 T59 94 0.9 0.89 0.87 Egg T60 139 0.9 0.89 0.87 Note: All food calorie conversion factors are referenced in this table, but this does not mean that all foods in this table are involved. (Source: accessed on 6 September 2022. The food conversion factor, intake part coefficient, and waste coefficient in distribution and consumption are mainly from Global Food Losses and Food Waste ). Figure 1 Study framework. Figure 2 Location and three major ecological divisions of Nepal. Source: Nepal: A Country Study . Figure 3 Maps showing land cover types (2020) in Nepal. Source: GlobeLand30 dataset accessed on 30 September 2022). Figure 4 Changes in (a) arable land and per capita level, (b) cereal production and per capita level, and (c) cereal production per land in Nepal from 1990 to 2020. Figure 5 Changes in the production of (a) cereal products, (b) other vegetal products, and (c) animal products in Nepal from 1990 to 2020. Figure 6 Calorie supply in Nepal in 2000 and 2020 based on FAO data. Figure 7 Calorie supply in (a) Nepal and (b) urban and rural residents in 2020 based on household questionnaire. Figure 8 National level of cereal supply in Nepal from 2000 to 2020: (a) cereal carrying capacity and real population; and (b) average carrying capacity. Figure 9 National level of cereal supply and demand from 2000 to 2020. Figure 10 Results of changes in food supply in three geographical regions of Nepal in 2020. Figure 11 Spatial pattern of (a) LCC and (b) cereal supply-demand status of counties in Nepal in 2020. Figure 12 National level of calorie supply in Nepal from 2000 to 2020: (a) calories carrying capacity and real population; and (b) calorie carrying capacity per land. Figure 13 National level of calorie supply and demand from 2000 to 2020. Figure 14 Results of changes in calorie supply in the three geographical regions of Nepal in 2020. Figure 15 Spatial pattern of (a) land carrying capacity and (b) calorie supply-demand status of counties in Nepal in 2020. Figure 16 The balance of (a) cereal and (b) calorie supply and demand in Nepal from 2000 to 2019 according to open and closed system. foods-12-01076-t001_Table 1 Table 1 Food-calorie conversion parameters for the main food categories. Food Calories (kcal/100 g) Conversion Factor Intake Part Coefficient Waste Coefficient Maize 356 0.78 0.76 0.92 Millet 340 0.78 0.76 0.92 Rice 280 0.9 0.87 0.92 Wheat 334 0.78 0.76 0.92 Potato 67 0.82 0.80 0.76 Sugarcane 30 0.77 0.76 0.88 Pulses 340 0.9 0.89 0.89 Fruits 45 0.77 0.72 0.58 Vegetables 22 0.77 0.72 0.58 Spices 337 0.77 0.72 0.58 Oilseeds 387 0.9 0.89 0.89 Meat 176 1 0.96 0.84 Milk 61 1 0.99 0.87 Egg 139 0.9 0.89 0.87 foods-12-01076-t002_Table 2 Table 2 The evaluation criteria based on LCCI for food demand-supply balance. 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PMC10000847 | Cells Cells cells Cells 2073-4409 MDPI 10.3390/cells12050801 cells-12-00801 Communication Tubule-Derived Follistatin Is Increased in the Urine of Rats with Renal Ischemia and Reflects the Severity of Acute Tubular Damage Nagayama Izumi Takayanagi Kaori Hasegawa Hajime Maeshima Akito * Tikkanen Ritva Academic Editor Department of Nephrology and Hypertension, Saitama Medical Center, Saitama Medical University, Kawagoe 350-8550, Japan * Correspondence: [email protected]; Tel.: +81-049-228-3481 04 3 2023 3 2023 12 5 80130 1 2023 01 3 2023 02 3 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). Activin A, a member of the TGF-beta superfamily, is a negative regulator of tubular regeneration after renal ischemia. Activin action is controlled by an endogenous antagonist, follistatin. However, the role of follistatin in the kidney is not fully understood. In the present study, we examined the expression and localization of follistatin in normal and ischemic rat kidneys and measured urinary follistatin in rats with renal ischemia to assess whether urinary follistatin could serve as a biomarker for acute kidney injury. Using vascular clamps, renal ischemia was induced for 45 min in 8-week-old male Wistar rats. In normal kidneys, follistatin was localized in distal tubules of the cortex. In contrast, in ischemic kidneys, follistatin was localized in distal tubules of both the cortex and outer medulla. Follistatin mRNA was mainly present in the descending limb of Henle of the outer medulla in normal kidneys but was upregulated in the descending limb of Henle of both the outer and inner medulla after renal ischemia. Urinary follistatin, which was undetectable in normal rats, was significantly increased in ischemic rats and peaked 24 h after reperfusion. There was no correlation between urinary follistatin and serum follistatin. Urinary follistatin levels were increased according to ischemic duration and were significantly correlated with the follistatin-positive area as well as the acute tubular damage area. These results suggest that follistatin normally produced by renal tubules increases and becomes detectable in urine after renal ischemia. Urinary follistatin might be useful to assess the severity of acute tubular damage. follistatin acute kidney injury ischemia reperfusion Grants-in-Aid for Scientific Research (C)17K09686 20K08596 Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT)40835344 This research was funded by Grants-in-Aid for Scientific Research (C) (grant numbers, 17K09686, 20K08596) and Young Scientists (B) (grant number 40835344) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT). pmc1. Introduction Activins are pleiotropic factors belonging to the transforming growth factor-beta (TGF-beta) superfamily and regulate cell growth and differentiation in many organs . The expression of activin A is undetectable in normal kidneys but is upregulated in tubular cells of the kidney after renal ischemia in both mice and rats . Activin A inhibits tubular regeneration by modulating the expression of Pax-2, a critical factor for kidney development, after renal ischemia . Activin A significantly inhibits cell growth and induces apoptosis in cultured proximal tubular cells (LLC-PK1) . Furthermore, activin A produced by tubular cells activates renal interstitial fibroblasts in a paracrine manner during the fibrotic processes of the kidney . These data suggest that activin A functions as a negative regulator of tubular regeneration after kidney injury as well as a potent activator of renal fibrosis . In various tissues, local activin A effects are modulated by its endogenous antagonist follistatin . Follistatin was first isolated from ovarian follicular fluid as a specific inhibitor of follicle stimulating hormone . Through neutralization of activin A, follistatin participates in various processes such as cell growth, development, and differentiation . For example, previous studies demonstrated that follistatin administration attenuates ischemic diseases including myocardial and hepatic ischemia-reperfusion injury (IRI) . In the kidney, exogenous administration of recombinant follistatin has been reported to reduce renal damage by blockade of endogenous activin A effects in models of renal ischemia/reperfusion , unilateral ureteral obstruction , and cisplatin nephropathy . A previous report showed that follistatin is ubiquitously expressed in rat tissues including the kidney . However, the precise distribution of follistatin in normal and damaged kidneys is not fully known. To address this issue, we examined the localization of follistatin mRNA and protein in normal and ischemic rat kidneys and assessed whether follistatin is detectable in urine of rats with renal ischemia in the present study. 2. Materials and Methods 2.1. Experimental Protocols Wistar rats (male, 8-week-old) were obtained from Japan Charles River (Yokohama, Japan). Rats under specific pathogen-free "SPF" conditions were provided with autoclaved food and sterile water ad libitum. Renal ischemic injury was induced in rats as described previously . Briefly, under general anesthesia with 1.5% isoflurane, renal ischemia was induced by clamping both renal arteries for 45 min using a non-traumatic vascular clamp. Core body temperature was maintained at 37 degC by placing the rats on a homoeothermic table. After the ischemia periods, the clamps were removed, and reperfusion of the kidneys was confirmed visually. The rats were subsequently sacrificed after various post-ischemic periods (n = 6 in each group). The kidneys were removed and frozen for RNA extraction or fixed in 10% formalin for routine paraffin embedding and sectioning for histologic analysis. Urine was collected from individual rats housed in metabolic cages. After centrifugation at 10,000 rpm for 5 min, all urine samples were stored at -80 degC until the day of analysis. Sham operations (n = 8) were performed in a similar manner, except for clamping of the renal arteries. Blood samples were obtained at the time of death, and serum samples were maintained at -20 degC until measurements. All animal procedures were approved by and conducted in accordance with the Fundamental Guidelines of the Ethics Review Committee for Animal Experimentation of Saitama Medical University (approval number 3117). 2.2. Measurement of Renal Function Urinary or serum creatinine and blood urea nitrogen (BUN) levels were measured by a standard method at Oriental Kobo Life Science Laboratory (Nagahama, Japan). 2.3. Immunohistochemical Analysis Immunostaining was performed using the DAKO EnVision labelled polymer system (Agilent Technologies, Inc, Santa Clara, CA, USA). Briefly, paraffin-embedded sections (3 mm-thick) were deparaffinized, hydrated according to standard methods; soaked in protein block serum-free solution (Dako); and incubated with primary antibody overnight at 4 degC. After washing with Tris-buffered saline containing 0.1% Tween 20 (TBS-T), sections were incubated with a peroxidase-conjugated secondary antibody followed by diaminobenzidine and then counterstained with hematoxylin. Following staining, the slides were photographed under a light microscope BX-61 (Olympus Optical Co., Ltd., Tokyo, Japan) and analyzed. For the immunostaining control, the primary antibody was replaced with PBS, which did not show positive signals, confirming specificity. Quantification of follistatin-positive areas was performed by measurement of positive areas in five randomly selected fields of the kidney at x100 magnification using ImageJ software (National Institutes of Health, Bethesda, MD, USA). The primary antibodies used in this study are as follows: mouse anti-follistatin antibody (60060-1-ig; Proteintech, Rosemont, IL, USA), rabbit anti-inhibin bA antibody (ab97705; Abcam, Cambridge, UK), rabbit anti-Na-Cl co-transporter (NCC) antibody (ab3553; Abcam), mouse anti-aquaporin (AQP)-1 antibody (sc-25287), mouse (AQP)-2 antibody (sc-515770), mouse anti-megalin (G-9) antibody (sc-515750; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-uromodulin antibody (HPA043420; Sigma-Aldrich, St. Louis, MO, USA), goat anti-kidney injury molecule (KIM)-1 antibody (AF3689), and goat anti-neutrophil gelatinase-associated lipocalin (NGAL) antibody (AF1857; R&D Systems, Minneapolis, MN, USA). 2.4. Histological Examination Semi-quantitative analysis of the tubule damage area was performed as described previously . 2.5. ELISA Urinary and serum rat follistatin (LS-F21531; LSBio, Seattle, WA, USA), urinary rat KIM-1 (DY3689), urinary rat NGAL (DY3508), and urinary rat L-FABP (DY1565; R&D Systems) were measured by ELISA according to the manufacturer's instructions. 2.6. In Situ Hybridization In situ hybridization (ISH) was performed using an ISH Reagent Kit from Genostaff Co., Ltd. (Tokyo, Japan) and a DIG Nucleic Acid Detection Kit from Roche (Basel, Switzerland). Probes for in situ hybridization were obtained from Genostaff Co., Ltd. (Tokyo, Japan). Sections were deparaffinized, rehydrated, and fixed in 4% paraformaldehyde (PFA) in PBS at room temperature (RT) for 10 min. After digestion with proteinase K (5 mg/mL) at 37 degC for 10 min, sections were postfixed in 4% PFA in PBS at RT for 15 min. Hybridization was performed with sense or antisense probes (0.5 ng/mL) at 58 degC for 16 h. Slides were then rinsed once in G-Wash at 60 degC for 10 min, 50% formamide in G-Wash at 60 degC for 10 min, washed twice in G-Wash at 60 degC for 20 min, and washed twice at 0.1x G-Wash at 60 degC for 20 min. Slides were incubated in 1x blocking solution for 60 min at RT and then incubated in a 1:5000 diluted solution of AP-conjugated anti-digoxigenin antibody for 60 min. After washing, the signals were detected with nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyphosphate. 2.7. Real-Time PCR Kidneys were homogenized using a microhomogenizer and total RNA was extracted with ISOGEN (Nippon Gene, Toyama, Japan). First-strand cDNA was prepared using SuperScript III First-strand (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Real-time PCR was performed using the StepOne Plus Real-time PCR System (Applied Biosystems, Foster City, CA, USA). Reactions included 5 mL of a SYBR Green Real-time PCR Master Mix (TOYOBO, Osaka, Japan), 0.5 mL of 3' primer, 0.5 mL of 5' primer, and 1 mL of cDNA. Samples were incubated at 50 degC for 2 min, then at 95 degC for 2 min, followed by 49 cycles of 15 s at 95 degC, 15 s at 58 degC, and 60 s at 72 degC. The expression of each gene was quantified in separate tubes with the following primers: rat follistatin (154 bp) sense 5'-AAAACCTACCGCAACGAATG-3', antisense 5'-AGGCATTATTGGTCTGATCC-3'; and rat GAPDH (130 bp) sense, 5'-CTACCCACGGCAAGTTCAAT-3%', antisense 5'-TACTCAGCACCAGCATCACC-3'. Values were presented as relative expression normalized to GAPDH. 2.8. Statistical Analysis Statistical analyses were performed using GraphPad Prism 8.3 (GraphPad software, San Diego, CA, USA). The Shapiro-Wilk test was used for testing normality. For two group comparisons, Student's t-test was used for normally distributed data and the Mann-Whitney test or Wilcoxon test was used for skewed data. The Kruskal-Wallis and Dunn's multiple comparison tests were used to compare the means of more than two variables. Spearman's rank correlation coefficient was used to analyze the correlations. Values of p < 0.05 were considered significant. 3. Results 3.1. Localization of Follistatin in Normal and Ischemic Rat Kidneys We first examined the localization of follistatin in normal kidneys by immunostaining. Follistatin was present in renal tubules of the cortex . Immunostaining using serial sections showed that follistatin-positive renal tubules were negative for AQP1 and megalin but positive for uromodulin , NCC , and AQP2 . These data suggest that follistatin was localized in distal tubules and collecting ducts of cortex in normal rat kidneys. We next examined the localization of follistatin in ischemic kidneys. Acute kidney injury was induced in rats by renal ischemia for 45 min. Low magnification images revealed that the follistatin-positive area increased transiently in the kidney after renal ischemia . Quantitative analysis demonstrated that the follistatin-positive area was significantly increased in the kidney after renal ischemia . Follistatin, which was detected in the cortex of normal kidneys, was increased in ischemic kidneys at 24 h after reperfusion . Follistatin was detected in renal tubules not only in the cortex but also in the outer and inner medulla of ischemic kidneys . Immunostaining using serial sections revealed that follistatin-positive tubules in the cortex were negative for megalin . In contrast, follistatin-positive thin tubular cells in the outer medulla were positive for uromodulin and negative for AQP1 . Thin tubular cells expressing follistatin in the inner medulla were negative for AQP2 in ischemic kidneys . These data suggest that follistatin was increased in distal tubules of the outer medulla as well as in the loop of Henle of the inner medulla after renal ischemia. We then compared the localization of follistatin and acute kidney injury (AKI) biomarkers such as KIM-1 and NGAL in ischemic kidneys. Expression of both KIM-1 and NGAL was detected in tubular cells of ischemic kidneys . Immunostaining using serial sections revealed that follistatin was not co-localized with KIM-1 or NGAL in ischemic kidneys at 24 h after reperfusion . Follistatin is a local modulator of activin A effects in various organs. We also compared the localization of follistatin and activin A in ischemic kidneys and found that follistatin and activin A were not co-localized . 3.2. Expression and Localization of Follistatin mRNA in Normal and Ischemic Rat Kidneys Next, we assessed the distribution of follistatin mRNA in normal kidneys by in situ hybridization. Follistatin mRNA was observed in the outer medulla of normal rat kidneys . Hybridization signals were detected in nuclei of renal tubules, but not in the glomeruli. A control experiment with a sense probe revealed no hybridization signal . A combination of in situ hybridization and immunostaining showed that follistatin mRNA was not expressed in renal tubules positive for uromodulin but was co-localized in thin tubular cells positive for AQP1 in the outer medulla , suggesting that follistatin mRNA was expressed in the descending loop of Henle of the outer medulla in normal rat kidneys. We next assessed the localization of follistatin mRNA in ischemic kidneys at 24 h after reperfusion. Follistatin mRNA was observed in tubular cells negative for uromodulin in the outer medulla of both normal and ischemic kidneys . Follistatin mRNA was distributed in thin tubular cells of ischemic kidneys, which were negative for AQP2 , suggesting that the expression of follistatin mRNA was upregulated in the descending loop of Henle in the inner medulla after renal ischemia. We also measured the mRNA expression level of follistatin in the kidneys after renal ischemia by real-time PCR. There were no significant differences in the expression level of follistatin between normal and ischemic kidneys . 3.3. Urinary Follistatin Level in Rats with Renal Ischemia We further tested if urinary follistatin became detectable in rats with AKI. Renal ischemia was induced in rats and urinary follistatin levels were measured at the indicated time-points after reperfusion by ELISA. No follistatin was found in the urine of normal rats. In contrast, urinary follistatin became detectable at 12 h, peaked at 24 h after reperfusion, and decreased thereafter, which reflected serum creatinine level . The molecular weight of follistatin ranges from 31 to 39 kDa, raising the possibility that circulating follistatin is increased, filtered by the glomerulus, and then excreted into the urine after renal ischemia. To test this possibility, we measured serum follistatin levels 24 h after reperfusion, the time-point at which urinary follistatin peaked after renal ischemia. Urinary follistatin 24 h after renal ischemia was significantly higher than that at 0 h after renal ischemia . Serum follistatin was undetectable in both normal and ischemic rats , suggesting that urinary follistatin originates from the kidney rather than the blood. We then compared the time course of changes in urinary follistatin with other AKI biomarkers. Urinary NGAL was elevated at 3 h, peaked at 24 h after reperfusion, and decreased thereafter . Urinary L-FABP was elevated at 3 h and rapidly decreased thereafter. Urinary follistatin was detectable at 12 h and peaked at 24 h after reperfusion, which reflected the results for urinary KIM-1 . We also analyzed the correlation of urinary follistatin levels with urinary NGAL and urinary KIM-1 at 24 h after reperfusion. Urinary follistatin was significantly correlated with urinary KIM-1 but not urinary NGAL . 3.4. Correlation of Urinary Follistatin with Severity of Acute Tubular Damage in Ischemic Rats Lastly, we investigated the correlation between urinary follistatin and the severity of acute kidney damage. Renal ischemia for 15, 30, and 45 min was induced in rats, and urine, kidney tissues, and serum were collected at 24 h after reperfusion. Both serum creatinine and BUN were significantly increased in rats with renal ischemia for 30 and 45 min but not 15 min. Urinary follistatin significantly increased in rats with renal ischemia for 30 and 45 min but not 15 min, which was similar to the results for urinary KIM-1 . Urinary NGAL was elevated in rats with renal ischemia for 45 min but not 15 and 30 min . We also examined the expression of follistatin in kidneys with renal ischemia for the indicated periods at 24 h after reperfusion by immunostaining. In addition to the cortex, follistatin was strongly detected in the medulla of kidneys after renal ischemia for 30 and 45 min but not 15 min . Quantitative analysis demonstrated that the follistatin-positive area as well as ATN area were significantly increased in the kidney with renal ischemia for 30 and 45 min. Urinary follistatin was significantly correlated with both the follistatin-positive area and ATN area , suggesting that urinary follistatin reflects the severity of acute tubular damage. 4. Discussion In the present study, we observed the difference in the distribution between follistatin mRNA and protein in the kidney. In normal kidneys, follistatin mRNA was localized in the descending limb of Henle in the outer medulla , and follistatin protein was mainly distributed in distal tubules of the cortex, which is downstream of its mRNA production site . Follistatin has a heparin-binding site and binds to heparan sulfate proteoglycan , a key component of the basement membrane of renal tubules . In addition, heparinase treatment resulted in significant suppression of follistatin binding to the cell surface . Taken together, it is likely that follistatin mRNA is translated into protein in the descending limb of Henle, followed by the secretion of follistatin protein into the lumen, which is sequestered by binding to heparan sulfate on the cytoplasmic surface of the distal tubules . We demonstrated that urinary follistatin was increased in rats with renal ischemia . Serum follistatin was undetectable in both normal and ischemic rats , suggesting that urinary follistatin originates from free follistatin produced by renal tubules. Considering that heparan sulfate is shed and discharged into the urine under AKI , it is possible that free follistatin and heparan-sulfate-bound follistatin increased in the urine after renal ischemia. Further study will be required to clarify this issue. We previously demonstrated a transient decrease in follistatin mRNA expression in the kidney after renal ischemia by Northern blot analysis . In contrast, we found the upregulated expression of follistatin mRNA in the descending loop of Henle in the inner medulla after renal ischemia by in situ hybridization . Real-time PCR showed that there was no significant decrease in follistatin mRNA expression after renal ischemia . This discrepancy might be attributed to the difference of age of rats. Wistar rats of 200 g body weight (estimated age, 6 weeks old) was used in previous study . In contrast, 8-week-old rats were used in this study. The difference of hybridization probe sequence might also affect the sensitivity of in situ hybridization, leading to the difference of signal distribution. Previous studies demonstrated that follistatin plays a protective role under a variety of stresses . The expression of follistatin is regulated transcriptionally or post-transcriptionally. The degradation rate of follistatin mRNA is also strictly controlled. Sequence analysis of the mouse follistatin promoter identified several consensus binding sites for transcription factors such as CREB, Sp1, AP-1, AP-2, Tcf, and Brachyury-T . Stress triggers the binding of a transcription factor such as nuclear factor erythroid 2-related factor 2 (Nrf2) to the follistatin promoter and thereby activates follistatin transcription . The WNT/b-catenin , MAPK-ERK-CREB , cAMP-dependent protein kinase (PKA), or MEK kinase (MEKK) pathways are also involved in follistatin regulation. Follistatin is also important for cellular energy homeostasis and cell survival under glucose deprivation, which inhibits follistatin mRNA decay and thereby upregulates follistatin expression . Considering that activin A stimulates follistatin mRNA expression , it is possible that mRNA expression of follistatin was increased by activin A in ischemic kidneys. However, this is unlikely because localization of activin A did not overlap with follistatin in ischemic kidneys . In the present study, both follistatin mRNA and protein were upregulated in the inner medulla of ischemic kidneys . A previous report showed that the thin limb of Henle in the inner medulla of the kidney became pimonidazole positive after renal ischemia , suggesting that the inner medulla is a target of hypoxic injury. If hypoxia is a potent inducer of follistatin expression in ischemic kidneys, urinary follistatin might be useful as a senser of renal hypoxia. AKI is a common but complicated disorder associated with increased mortality and morbidity . AKI is associated with progression to advanced chronic kidney disease. The diagnosis of AKI is usually based on increases in serum creatinine levels; however, it is less sensitive and specific for detection of acute decline of renal function in AKI. To overcome this problem, several AKI biomarkers, including NGAL, KIM-1, IL-18, L-FABP, and TIMP-2*IGFBP-7, have been identified to predict the onset of AKI, AKI severity, and renal prognosis of AKI in critically ill patients . In the present study, we showed that the timing of elevation of urinary follistatin after renal ischemia was later than that of urinary NGAL or urinary L-FABP . Urinary follistatin increases after renal ischemia in parallel with serum creatinine level , suggesting that urinary follistatin is not suitable as an early diagnostic marker for AKI. On the other hand, we provide evidence for a significant association between urinary follistatin and AKI severity . Urinary follistatin levels were increased according to the duration of ischemia and was significantly correlated with follistatin-positive area as well as acute tubular damage area. These data suggest that urinary follistatin could be helpful for assessing the severity of acute kidney damage in the clinical setting. The combination of urinary follistatin with standard clinical parameters and/or established AKI biomarkers will be advantageous for monitoring of the stage of tubular recovery and assessment of the appropriate timing of intervention for patients with AKI. Acknowledgments We would like to thank Saori Nakahashi and Hitomi Hirate for their expert technical assistance. We also thank the lab members for their helpful comments. Author Contributions A.M. designed the study; I.N. and K.T. carried out the experiments; I.N. analyzed the data; I.N. and A.M. created the figures; I.N., H.H. and A.M. drafted and revised the paper. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The study was approved by the Institutional Ethics Committee of Saitama Medical University (approval number 3117, approval date 15 March 2021). Informed Consent Statement Not applicable. Data Availability Statement The datasets used and analyzed in the current study are available from the corresponding author upon reasonable request. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Localization of the follistatin protein in normal rat kidneys. (a) Localization of follistatin (brown) in normal rat kidneys was examined by immunostaining. Cortex, CO; outer medulla, OM; inner medulla, IM. Bar = 0.2 mm. (b-f) Identification of follistatin-positive tubules in rat normal kidneys using serial sections. Aquaporin 1, AQP1; Na-Cl co-transporter, NCC; aquaporin 2, AQP2. Asterisks indicate identical tubules. Bar = 50 mm. Figure 2 Localization of the follistatin protein in kidneys after renal ischemia. (a) Localization of follistatin (brown) in kidneys after renal ischemia was examined by immunostaining (left panel). Bar = 2 mm. Quantitative analysis of follistatin-positive area (right panel). Five randomly selected fields of the kidney were assessed at x100 magnification. The follistatin-positive area was measured by ImageJ. Values are the means +- S.E. (n = 6-8). * p < 0.05, ** p < 0.01 vs. 0 h (b,c) Localization of the follistatin protein in normal and ischemic kidneys. The kidneys were collected at 0 h and 24 h after reperfusion. Ischemia reperfusion, IR. Bar = 0.2 mm in (b), 50 mm in (c). Cortex, CO; outer medulla, OM; inner medulla, IM. Bar = 50 mm. (d) Localization of follistatin protein and megalin, aquaporin 1 (AQP1), uromodulin (UMOD), or aquaporin 2 (AQP2) in ischemic kidneys was evaluated using serial sections. Cortex, CO; outer medulla, OM; inner medulla, IM. Bar = 50 mm. (e) Localization of follistatin, neutrophil gelatinase-associated lipocalin (NGAL), and kidney injury molecule-1 (KIM-1) in ischemic kidneys was evaluated using serial sections. Bar = 0.2 mm (upper panels), 50 mm (lower panels). (f) Localization of follistatin and activin A in ischemic kidneys was evaluated using serial sections. Bar = 50 mm. Asterisks indicate identical tubules. Figure 3 Expression and localization of follistatin mRNA in normal and ischemic rat kidneys. (a) Localization of follistatin mRNA in normal kidneys was examined by in situ hybridization. Hybridization signals are shown as blue color. Bar = 0.2 mm (left panel), 50 mm (right panels). (b) Follistatin mRNA (blue) and uromodulin (brown) in normal kidneys was evaluated by double staining. Cortex, CO; outer medulla, OM; inner medulla, IM. Bar = 0.2 mm (left panel), 50 mm (right panels). (c) Localization of follistatin mRNA and aquaporin 1 (AQP1) in normal rat kidneys was evaluated using serial sections. Asterisks indicate identical tubules. (d) Follistatin mRNA (blue) and uromodulin (brown) in normal and ischemic kidneys at 24 after reperfusion was evaluated by double staining. Cortex, CO; outer medulla, OM. (e) Follistatin mRNA (blue) and aquaporin 2 (AQP2, brown) in normal and ischemic kidneys was evaluated by double staining. Inner medulla, IM. Bar = 50 mm in (c-e). (f) Quantitative analysis of follistatin mRNA expression by real-time PCR. Values (relative expression ratio to GAPDH) are means +- S.E. (n = 5-8). Figure 4 Urinary follistatin levels significantly increased after renal ischemia. (a) Time course changes in urinary follistatin and serum creatinine levels after reperfusion (n = 8). (b) Urinary and serum follistatin levels in normal and renal ischemia (45 min) rats were measured by ELISA. Values are means +- S.E. (n = 8). *** p < 0.001; N.S., not significant. (c) Time course changes in urinary follistatin, urinary NGAL, KIM-1, and L-FABP after reperfusion. (d,e) Correlations between urinary follistatin and urinary NGAL (d) or urinary KIM-1 (e) at 24 h after reperfusion. Renal ischemia (45 min) was performed, and urine was collected 24 h after reperfusion. Figure 5 Correlation between urinary follistatin and severity of kidney damage. (a,b) Serum creatinine and BUN in rats with renal ischemia at 24 h after reperfusion. N.S., not significant, * p < 0.05, *** p < 0.001 vs. 0 h. (c-e) Urinary follistatin, urinary NGAL, and urinary KIM-1 in rats with renal ischemia for the indicated periods at 24 h after reperfusion. N.S., not significant, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. 0 h. (f) Localization of follistatin (brown) in kidneys after renal ischemia for 15, 30, and 45 min was examined by immunostaining. Bar = 2 mm. (g) Quantitative analysis of follistatin-positive area. Five randomly selected fields of the kidney were assessed at x100 magnification. The follistatin-positive area was measured by ImageJ 1.53a. Values are the means +- S.E. (n = 5-6). N.S., not significant, * p < 0.05 vs. 0 h. (h) Semiquantitative analysis of the histological changes induced by renal ischemia. The ATN area was calculated as described in Section 2. N.S., not significant, * p < 0.05, ** p < 0.01 vs. 0 h. (i,j). Correlation between urinary follistatin and follistatin-positive area (i) or ATN area (j) at 24 h after reperfusion. Figure 6 Possible mechanism of follistatin excretion into urine after renal ischemia. Aquaporin 1, AQP1; aquaporin 2, AQP2; Na-Cl co-transporter, NCC. 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PMC10000848 | Diagnostics (Basel) Diagnostics (Basel) diagnostics Diagnostics 2075-4418 MDPI 10.3390/diagnostics13050868 diagnostics-13-00868 Review Similarities in Pathogenetic Mechanisms Underlying the Bidirectional Relationship between Endometriosis and Pelvic Inflammatory Disease Kobayashi Hiroshi 12 Vitagliano Amerigo Academic Editor 1 Department of Gynecology and Reproductive Medicine, Ms.Clinic MayOne, Kashihara 634-0813, Japan; [email protected] 2 Department of Obstetrics and Gynecology, Nara Medical University, Kashihara 634-8522, Japan 24 2 2023 3 2023 13 5 86806 2 2023 15 2 2023 22 2 2023 (c) 2023 by the author. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). Background: Endometriosis is a common inflammatory disease characterized by the presence of endometrial cells outside of the uterine cavity. Endometriosis affects 10% of women of reproductive age and significantly reduces their quality of life as a result of chronic pelvic pain and infertility. Biologic mechanisms, including persistent inflammation, immune dysfunction, and epigenetic modifications, have been proposed as the pathogenesis of endometriosis. In addition, endometriosis can potentially be associated with an increased risk of pelvic inflammatory disease (PID). Changes in the vaginal microbiota associated with bacterial vaginosis (BV) result in PID or a severe form of abscess formation, tubo-ovarian abscess (TOA). This review aims to summarize the pathophysiology of endometriosis and PID and to discuss whether endometriosis may predispose to PID and vice versa. Methods: Papers published between 2000 and 2022 in the PubMed and Google Scholar databases were included. Results: Available evidence supports that women with endometriosis are at increased risk of comorbid PID and vice versa, supporting that endometriosis and PID are likely to coexist. There is a bidirectional relationship between endometriosis and PID that shares a similar pathophysiology, which includes the distorted anatomy favorable to bacteria proliferation, hemorrhage from endometriotic lesions, alterations to the reproductive tract microbiome, and impaired immune response modulated by aberrant epigenetic processes. However, whether endometriosis predisposes to PID or vice versa has not been identified. Conclusions: This review summarizes our current understanding of the pathogenesis of endometriosis and PID and discusses the similarities between them. endometriosis immune response microbiota pelvic inflammatory disease tubo-ovarian abscess This research received no external funding. pmc1. Introduction Endometriosis is an estrogen-dependent inflammatory disease that is characterized by the growth of endometrial glands and stroma located outside of the uterine cavity . This disease can be classified into three variants: ovarian endometrioma, superficial peritoneal disease, and deep infiltrating endometriosis. Of these, ovarian endometrioma is a common disease. Endometriosis affects approximately 10% of reproductive-aged women and is accompanied by pelvic pain and infertility. Endometriosis is associated with a wide array of diseases, such as ovarian cancer, breast cancer, autoimmune diseases, cardiovascular disease, and asthma, compromising the quality of life . Additionally, women with endometriosis have been reported to be at an increased risk of pelvic inflammatory disease (PID) . PID is a common inflammatory disease and is characterized by an infection of the upper genital tract (e.g., the uterus, fallopian tubes, ovaries, and pelvic peritoneum) . PID is often believed to be a sexually transmitted disease, but bacterial vaginosis (BV) is also associated with an increased risk of PID . Ovarian abscesses (OA) and tubo-ovarian abscesses (TOA) are serious complications of PID and prove difficult to treat due to resistance to treatments and several complications . They often affect the female reproductive tract and other adjacent pelvic organs or structures, leading to adverse reproductive outcomes . long-term complications and sequelae include infertility, ectopic pregnancy, chronic pelvic pain, and recurrent infections due to dense pelvic adhesions and macroscopic anatomic and morphological abnormalities . The available clinical and epidemiological evidence supports that PID and TOA occur more frequently in women with endometriosis than in those without endometriosis ; however, it is unclear if women with endometriosis are more prone to PID or vice versa. This review aims to investigate the mechanisms underpinning the bidirectional relationship between endometriosis and PID and to discuss their similarities. Our current understanding of anatomical, morphological, microbiological, immunological, and epigenetic alterations would provide new insights into a potential association between the two diseases. 2. Materials and Methods 2.1. Search Strategy and Selection Criteria A computerized literature search was performed to identify relevant studies in English. The PubMed and Google Scholar electronic databases were searched for studies published between January 2000 and October 2022. The search terms included endometriosis, immune response, microbiota, pelvic inflammatory disease, and tubo-ovarian abscess. In the search strategy, these keywords were combined with the Boolean operators AND and OR, as described in Table 1. Inclusion criteria included the publication of original studies and reference lists in review articles. The references of each article were searched to identify potentially relevant studies. Figure 1 shows the first identification phase which includes records identified through a database search. Terms in the titles and abstracts were searched during the first screening. During the second screening, duplicates were removed, and titles and abstracts were read to remove inappropriate papers. The final eligibility phase included full-text articles for analysis after excluding those wherein detailed data could not be extracted. 2.2. Selection of Studies The literature search on PubMed and Google Scholar provided 554 records . After removing unsuitable (titles and abstracts irrelevant to the topic) and duplicate articles, we obtained 103 records, of which 82 were excluded, and 21 met the selection criteria. This figure shows the number of articles identified by keyword combinations and the number of records identified through database searching, records after duplicate removal, records screened, removal of inappropriate articles by reading full-text articles, and full-text articles assessed for eligibility. 3. Results 3.1. A Bidirectional Relationship between Endometriosis and PID 3.1.1. PID PID is defined as an infection of the upper genital tract, including endometritis, salpingitis, peritonitis, OA, and TOA . Approximately one-third of patients with PID manifest TOA . The common bacterial organisms isolated and identified were sexually transmitted organisms (e.g., Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma genitalium, and Trichomonas vaginalis); anaerobic and aerobic organisms associated with BV (e.g., Atopobium vaginae, Mycoplasma genitalium, Gardnerella vaginalis, Prevotella species, Sneathia, and Megasphaera); and organisms isolated from the enteric or respiratory tracts (e.g., Bacteroides, Escherichia coli, aerobic Streptococcus, or Haemophilus influenza) . In some cases, it can be difficult to identify the causative pathogens of PID. The pathogenic bacteria frequently isolated from clinical specimens in women with TOA, a severe form of PID, are facultative anaerobic bacteria (e.g., Escherichia coli and Enterococcus spp) and obligate anaerobic bacteria (e.g., Bacteroides fragilis) . Uncommon pathogens from other body sites can also reach the ovaries through the bloodstream . In addition, TOA in women with ovarian endometrioma are frequently polymicrobial, complicated by sexually transmitted organisms (e.g., Neisseria gonorrhoeae and Chlamydia trachomatis) and obligate anaerobic bacteria (e.g., Bifidobacterium, Bacteroides, Eubacterium, and Clostridium) . TOA infections can also be caused by a multitude of different microbes from BV (e.g., Mycoplasma genitalium, Gardnerella vaginalis) and normal intestinal commensal organisms (e.g., Ruminococcus gnavus, a Gram-positive anaerobe) . The development of TOA in women with ovarian endometrioma has been reported to be associated with lower genital tract infections and endometriotic cyst rupture . Therefore, a mixed infection caused by numerous BV-associated bacterial organisms that can infect the upper genital tract may predispose women with ovarian endometrioma to abscess formation . Much of the literature discusses ovarian endometrioma and PID, but very little focus has been placed on peritoneal endometriosis. Therefore, it is unknown whether peritoneal endometriosis is associated with PID. A wide array of pathogenetic mechanisms and risk factors have been proposed for the development and progression of PID or TOA: young age (<25 years), the size of the abscess >6 cm, multiple sexual partners, frequent partner turnover, a previous history of PID, a previous history of intrauterine device (IUD) insertion, a previous laparotomy, assisted reproductive technology, oocyte retrieval, structural genital anomalies, drug consumption, cigarette smoking, diabetes, and some immune deficiency diseases . However, it is rarely (4%) reported to occur in virgins, even at a young age . A comprehensive review of the literature identified a study including 16 virgins in 2019 . TOA in virgins is often secondary to associated comorbidities, such as congenital malformations of the female genitourinary tract, obstructed hemivagina with a renal anomaly, bowel malrotation, complicated appendicitis, bacteremia caused by wound sepsis or dental procedures, and prior abdominopelvic surgery . Thirteen (81%) of 16 virgins had these relevant comorbidities . Therefore, these comorbidities, including genitourinary tract anomalies, can predispose virgins to TOA. 3.1.2. Are Women Having PID Prone to Endometriosis? This subsection focuses on the prevalence or incidence of endometriosis in women with PID to investigate whether PID may predispose women to endometriosis. In a recent study, 81 (69.2%) of 117 patients with PID had TOA, 59 (72.8%) of whom were complicated with ovarian endometrioma, indicating that about half (50.4%) of PID patients experienced concurrent endometriosis . Furthermore, Elizur et al. reported that concurrent endometriosis was diagnosed in 21 (14.2%) of 148 patients with PID or TOA . In addition, women with PID had a 4-fold increased risk of developing endometriosis compared to women without PID or the general population , respectively. Therefore, endometriosis is common in patients with PID or TOA, with incidences ranging from 14.2% to 72.8%. Available evidence supports that patients with PID are at increased risk of comorbid endometriosis. 3.1.3. Are Women Having Endometriosis Prone to PID? Next, the author summarizes whether patients with endometriosis are at increased risk of comorbid PID. In a retrospective cohort study, Grammatikakis et al. identified 21 (2.9%) patients with PID among 720 women who underwent surgery for ovarian endometrioma, suggesting that the prevalence of PID in women with endometriosis is significantly higher than in the general population . These patients had an average age of 31 years (range: 21-39 years), no laterality of the ovary, and a mean diameter of the endometriotic cyst of 3.5 cm . Furthermore, a recent retrospective study also found that 196 (63%) of 311 patients who underwent surgery for PID had a history of endometriosis, demonstrating that patients with PID experience a higher prevalence of endometriosis compared to the general population or the population in tertiary care . Similar results have been obtained in several studies. Women with endometriosis are more likely to develop TOA than women without endometriosis, with risk factors being younger age and more advanced stage . The potential risks for developing TOA in women with ovarian endometrioma were reproductive tract infections and the spontaneous rupture of endometriotic cysts . Furthermore, the rate of recurrent PID in endometriosis patients with TOA was as high as 21.4%, indicating that endometriosis is an independent risk factor for PID recurrence . Coexisting endometriosis was the potentially important risk factor that influences TOA postoperative recurrence . In addition, pelvic abscess formation following oocyte retrieval during an in vitro fertilization (IVF) cycle is a rare complication (0.1-0.4%) but is known to be at increased risk in patients with endometriosis . In contrast, one retrospective study showed that oocyte retrieval was not linked to the development of TOA in women with endometriosis . Despite some inconsistent evidence in the literature, much data support the association between IVF treatment and TOA risk. The treatment strategies for PID patients with endometriosis are essentially the same as those for women without endometriosis . However, the treatment of PID in patients with endometriosis is often complicated by refractory to antibiotic treatment, increased need for surgery, multiple intraoperative and postoperative complications, and prolonged hospitalization . Overall, an increasing body of evidence suggests that endometriosis may be associated with an increased risk of incidence, severity, and recurrence of PID, as well as difficulty in treatment selection. Taken together, TOA can be caused by a complex combination of comorbid endometriosis, surgical procedures associated with IVF treatment, and treatment resistance. 3.2. A Pathophysiological Mechanism Coupling Endometriosis and PID In the previous section, I showed that endometriosis and PID are likely to coexist. This section summarizes why women with endometriosis are at increased risk for PID, with a particular focus on the distortions in the morphology of the pelvic organs (i.e., anatomical distortions), the periodic bleeding of ectopic lesions, alterations of the reproductive tract microbiota, and impaired immune systems. 3.2.1. Distortion of the Pelvic Anatomy Favoring Bacteria Proliferation Several potential causes support the increased risk of PID in women with endometriosis, one of which may be the distorted anatomy favoring bacteria proliferation. Endometriosis is characterized by abnormal activation of inflammation, macrophage infiltration, and angiogenesis that leads to surrounding tissue adhesion and fibrosis formation, thereby distorting the pelvic organs . PID typically causes anatomical distortions of the ovaries, fallopian tubes, and retroperitoneum through inflammation caused by bacterial infection, resulting in a frozen pelvis. Despite clinical differences, endometriosis and PID share some common characteristics, such as inflammation, adhesions, and distortion of the pelvic organs. It is well known that a history of various intrauterine manipulations and pelvic surgery or retrograde menstruation caused by congenital obstructive abnormalities predisposes to PID or endometriosis, respectively . The distorted anatomy of the pelvic organs provides an optimal environment for bacteria and may favor the growth of diverse bacteria. Therefore, similarities in morphological changes in an anatomical structure may be associated with an increased risk of PID in women with endometriosis. 3.2.2. Nutrients from Old Blood in Endometriotic Lesions The second cause that explains the risk may be old blood within the endometriotic cyst or in the peritoneal cavity. Lactic acid produced by Lactobacillus acidifies the vagina, thereby providing a prompt defense against infections of pathogenic bacteria . The menstrual blood increases the pH of the vagina and uterine cavity negatively, which affects commensal microbiota and is conducive to pathogenic bacterial proliferation, contributing to an increase in the risk of bacterial infection . An increase in vaginal fluid pH is known to be associated with BV . Furthermore, degenerated old blood within endometriotic cysts and retrograde menstrual blood that enters the peritoneal cavity are suitable culture mediums for bacteria and can promote the growth of pathogenic microorganisms. Blood supplies the bacteria with oxygen, nutrients, and energy substrates such as glucose, lactate, and proteins. In fact, blood agar plates are used in the clinical laboratory for the identification of bacteria . Various bacteria such as Escherichia coli, Staphylococcus aureus, Streptococcus pyogenes, and Streptococcus pneumoniae form colonies on blood agar plates . Therefore, degenerated old blood in women with endometriosis favors the growth of pathogenic bacteria. Periodic bleeding in ectopic lesions creates a nutrient-rich environment that favors the growth of diverse bacteria and may facilitate the spread of infection . Taken together, these findings suggest that women with endometriosis are more prone to infections and more likely to develop PID than normal individuals or women without endometriosis. In contrast, menstrual blood contributes to the defense against an array of microorganisms transported by sperm . Menstrual blood discharge is rich in hemocyanins, bactericidal peptides generated from hemoglobin . Hemocidins exhibit potent antimicrobial activity toward some strains of bacteria, especially Gram-negative bacteria . However, normal menstrual discharge and degenerated old blood in endometriotic cysts may differ in their physiological properties. It is not known whether hemocyanins in endometriotic cyst fluid are sufficient to stop bacterial growth. 3.2.3. Alterations of the Gut and Reproductive Tract Microbiome Thirdly, alterations in the reproductive tract microbiomes in patients with endometriosis may be intricately linked with the development of PID or TOA . Microbiota is the diverse collection of vital microorganisms that live in symbiosis with humans . They are found in the skin, mouth, gastric, gut, and female reproductive tract. The commensal microbiota regulates inflammatory, immune, and metabolic functions, which constitute a protective barrier against pathogen infections and eliminate foreign intruders or pathogenic bacteria . In fact, the hygiene hypothesis states that commensal bacteria promote early protection against inflammatory diseases and allergies . On the other hand, alteration of the composition and function of the microbiota, i.e., dysbiosis, is linked with various conditions, such as inflammatory bowel disease, allergies, autoimmunity, reproductive disorders, and cancer through the stimulation of innate immune responses and the induction of several inflammatory cascades . The microbiota of women with endometriosis is characterized by reduced Lactobacillus abundance in the lower genital tract and diverse flora of facultative and anaerobic organism populations, such as Gardnerella, Streptococcus, Enterococci, Escherichia coli, and Mollicutes in the upper genital tract, peritoneum, and endometriotic lesions . Pathogenic organisms associated with BV ascend from the bottom to the top of the reproductive tract and may predispose to PID and severe forms of abscess formation. Therefore, exposure to infection caused by pathogenic microorganisms in the lower and upper reproductive tract and the peritoneal cavity is a hallmark of endometriosis . In addition to abnormalities in the reproductive tract microbiota, dysbiosis in the gut microbiota has been identified in animal models, such as mice and nonhuman primates (e.g., rhesus monkeys), and women with endometriosis . There is evidence from animal models showing that the transplantation of endometrial fragments increases specific gut microbes, such as obligatory anaerobic bacteria (e.g., Ruminococcaceae, Bifidobacterium, and Parasutterella generae) . Therefore, endometriotic lesions cause changes in the gut and reproductive tract microbial abundance, composition, and diversity, promoting the further growth of pathogenic bacteria . Indeed, dysbiosis of the reproductive tract microbiota in patients with BV or PID has been reported to lead to multiple detrimental reproductive outcomes, including infertility . Today it is well known that patients with endometriosis are constantly exposed to inflammation. Increased numbers of Escherichia coli and higher levels of bacterial endotoxin are observed in the menstrual blood of women with endometriosis compared with control women, leading to the Toll-like receptor 4 (TLR-4)-mediated proliferation of endometriotic lesions . Lipopolysaccharide (LPS), a cell-wall component (endotoxin) produced by Gram-negative bacteria, activates nuclear factor kappa B (NF-kB) and its downstream target, cyclooxygenase-2 (Cox-2) . Prostaglandin E2 (PGE2), a downstream product of Cox-2, generates the inflammatory microenvironment. The PGE2 level in the menstrual blood of women with endometriosis has been reported to be 2-3 times higher than in serum or peritoneal fluid . PGE2 mediates bacterial growth in women with endometriosis via inflammatory responses and proinflammatory cytokine production . PGE2 is also known to inhibit the process of bacterial killing by macrophages to attenuate antibacterial immunity . In addition, Noh et al. reported that Ureaplasma urealyticum infection is implicated in the development of pelvic endometriosis via the TLR-2 signaling pathway in the in vivo mouse model . This animal study showed that bacterial infection can accelerate the progression of endometriosis. Taken together, the reproductive tract microbial abundance, composition, and diversity are hallmarks of endometriosis, resulting in a decrease in beneficial microbes and an increase in harmful microbes . This microbiota dysbiosis induces pelvic inflammation and bacterial growth possibly through the TLR-mediated activation of NF-kB and macrophage dysfunction, leading to the development and progression of PID and, ultimately, TOA. Although it is currently unknown whether endometriosis induces changes in microbial diversity and composition or vice versa, endometriosis and PID are closely related pathophysiologically. 3.2.4. Impaired Immune Systems Fourthly, the strong association between endometriosis and PID suggests that they may share a common biological basis in pathogenesis. This subsection focuses on the aberrant activation of the immune system between endometriosis and PID and discusses whether endometriosis predisposes to PID. Natural killer cells, macrophages, mast cells, neutrophils, dendritic cells, and Tregs have been identified in the endometrium as immune cells associated with physiological processes, such as the menstrual cycle . The vital role of these immune cells includes endometrial protection, decidualization, embryo implantation, placentation, the process of repeated tissue breakdown, repair, and regeneration during the menstrual cycle, and scavenging menstrual debris . There is evidence that different types of immune cells have aberrant functions in women with endometriosis; alterations to and the complex interplay of innate and adaptive immune cells, i.e., the dysregulation of the immune system, have been implicated in the pathogenesis of endometriosis . A broad range of alterations in immune cell numbers, distributions, and functions (e.g., increased number and activation of peritoneal and endometrial macrophage phenotypes, altered dynamics in T-cell reactivity and NK cytotoxicity, dysregulation of effector functions in the T lymphocytes, an imbalance in T helper cell subsets (Th1/Th2/Th17), and an increased proportion of Treg cells) and changes in localized and systemic inflammatory mediator profiles have been reported in the serum, peritoneal fluids, and eutopic and ectopic endometrium of women with endometriosis . Treg cells in the peritoneal cavity are elevated in women with advanced endometriosis, allowing ectopic endometriotic cell implantation, survival, proliferation, and progression . Treg cells also contribute to immune evasion, causing further persistent inflammation via the decreased clearance of ectopic endometrial cells . On the other hand, women with sexually transmitted organisms-induced inflammation, a typical case of PID, are characterized by a disproportionate influx of innate immune cells and impaired innate immunity (e.g., the activation of macrophages and their phenotypes) and alterations of T-cell activation pathways (e.g., T-cell-mediated production of interferon-g, altered dynamics in T-cell reactivity and NK cytotoxicity) . The development of such PID is associated with immune dysfunction by innate lymphoid cells. The aberrant activation of innate lymphoid cells is also involved in the progression of endometriotic lesions . Therefore, an already existing endometriosis with altered immune cell profiles, imbalances in immune cell function, and compromised immunosurveillance could be involved in the development and pathogenesis of PID . We believe that a pathophysiological similarity between these two diseases is the defective immune system, such as disruption of the beneficial symbiosis between host and commensal microbes and incomplete defense against invading pathogenic microbes. 3.2.5. The Immune Landscape Modulated by Epigenetic Factors Finally, this subsection briefly summarizes whether the epigenetic modifications may predispose women with endometriosis toward PID or vice versa. The immune function across endometriosis is largely modulated by selective epigenetic reprogramming. There are fundamental changes in gene expression through aberrant DNA methylation, histone modifications, and the altered expression of non-coding microRNAs in the eutopic endometrium in patients with endometriosis compared to a normal healthy endometrium . Endometriosis is characterized by complex epigenetic modifications that alter the T-cell landscape . Epigenetic modifications play critical roles in the development, activation, and differentiation of CD4+ and CD8+ T cells, the maintenance of Treg cells to ensure the stable expression of Foxp3, and the memory formation of NK cells . Treg cells may modulate the local host-defense mechanism against endometriotic cell proliferation and bacterial invasion . Consequently, epigenetic modifications induce alterations in the immune cell function, number, populations, and phenotypes, promoting the development of endometriosis and bacterial infection. An impaired immune defense in patients with concurrent endometriosis can lead to the exacerbation of PID, even influencing the development of TOA. Furthermore, it is well known that the hormonal landscape in endometriosis is modulated by epigenetic modifications, leading to estrogen dependence and progesterone resistance . Given that progesterone suppresses endometrial inflammation via the downregulation of inflammatory cytokine and chemokine transcripts, epigenetic modifications to progesterone receptors and their targets result in a proinflammatory phenotype . In addition, an increased ratio of ERb-to-ERa due to ERb hypomethylation represents a molecular signature of inflammation . Endometriosis-specific epigenetic alterations create a beneficial environment for successful bacterial growth and spread through both aberrant immune function and altered sex steroid hormone responsiveness . Persistent inflammation due to the elevation of proinflammatory cytokines and disturbed cytokine profiles is thought to be an important trigger of PID exacerbations . Epigenetic modifications help to explain why women with endometriosis are more prone to PID. Conversely, an infection can also trigger epigenetic changes that influence the development of endometriosis . For example, sepsis-associated epigenetic modifications have been reported to be associated with enhanced host-pathogen interaction, impaired macrophage function, prolonged immunosuppression, exaggerated inflammation, impaired mitochondrial energy metabolism, and deficient wound healing . An epigenetic link between endometriosis and PID has been suggested, but research is still in its infancy. 4. Discussion This review summarizes the pathophysiological mechanisms that explain the bidirectional relationship between endometriosis and PID and discusses their similarities. Endometriosis and PID are common inflammatory disorders. The accurate and early detection of PID is crucial for the selection of optimal care and better prognosis because endometriosis with TOA is often a difficult-to-treat disorder . Some studies revealed that endometriosis is associated with an increased risk of incidence, severity, complications, treatment failure, and recurrence of PID or TOA . Conversely, patients with PID are more likely to develop endometriosis, with a survey of the National Health Insurance Research Database showing that individuals with PID had a 3-fold increased risk of developing endometriosis compared with an age-matched control group including patients without PID . Although studies are still limited in number, endometriosis predisposes women to PID and vice versa, with these two conditions influencing each other. A causal relationship between endometriosis and PID remains unclear, but both diseases are likely to coexist. Therefore, this article reviews our current understanding of the shared pathogenesis of endometriosis and PID and highlights the following similarities: (1) the distorted anatomy favorable to bacteria proliferation, (2) periodic hemorrhage of the endometriotic focus, (3) alterations to the gut and reproductive tract microbiome, and (4) impairment of the immune response modulated by epigenetic factors. The author first focuses on the anatomical similarities between endometriosis and PID. In more advanced stages, both diseases commonly cause severe adhesions and fibrosis . Adhesions secondary to inflammation associated with endometriosis readily facilitate the spread of pathogens to the pelvic cavity. The similarities in the dramatic anatomical changes within the pelvic cavity are thought to be the reason why endometriosis often coexists with PID or TOA . The second pathophysiological mechanism may be the bloody content of the endometrioma or in the peritoneal cavity, which favors bacterial growth. Pooled blood contains nutritional components that promote bacterial growth, suggesting that endometriosis is prone to PID, particularly TOA . Surgical procedures (e.g., IVF treatments) in such settings also contribute to infection and abscess formation. Thirdly, the altered microbial abundance, composition, diversity, and function in the reproductive tract of women with endometriosis may be associated with the development and pathogenesis of PID . Such dysbiosis has negative impacts on immune function including disruptions to the immune pathway, aberrant expressions of immune mediators, attenuated immunosurveillance, and disruption to the immune defense system, all of which may facilitate the development of endometriosis . Over time, this immune dysregulation can progress into a chronic state of inflammation and reduced pathogen resistance, which may drive the vicious cycle of PID onset and progression. Finally, such immune dysfunction can be mediated by endometriosis-specific epigenetic modifications, altering the Treg cell abundance, affecting defense mechanisms to combat pathogens, and increasing the risk of PID, and ultimately, TOA . Figure 2 illustrates the pathophysiological mechanisms underlying the bidirectional relationship between endometriosis and PID. There are at least two possibilities of a potential model: (1) ascending genital tract infections with exogenous pathogenic bacteria may cause TOA via the secondary infection of endometriotic cysts and (2) microbiota dysbiosis associated with endometriosis may trigger the development of TOA . The former implies that endometriosis is incidentally found in PID, while the latter suggests that endometriosis may predispose to PID. However, at present, there is no direct evidence to prove a bidirectional relationship between them. In the latest article published in January 2023, Kitaya and Yasuo suggested substantial commonality between endometriosis and chronic endometritis in terms of immunological, inflammatory, and infectious aspects and discussed a novel antibiotic strategy for the prevention and treatment of endometriosis. Recent advances in omics technologies may greatly contribute to the elucidation of the mechanistic underpinnings of the spectrum of endometriosis, including endometritis and pelvic inflammation. Taken together, clinicians should always be aware that endometriosis may have pelvic inflammation as an underlying molecular mechanism and vice versa to provide the best care for both diseases. In conclusion, this review summarizes that endometriosis and PID are likely to coexist and that PID, especially TOA, is a progressive disease caused by a complex combination of anatomical and environmental factors (e.g., an increase in opportunistic pathogens), immunological, epigenetic factors (e.g., altered dynamics in T-cell reactivity), surgical procedures (e.g., IVF treatment), and the presence of coexisting endometriosis. In particular, the dysbiosis of reproductive tract microbiota may predispose women with endometriosis to PID through a broad range of immune dysfunction, including the reduced capability of eliminating pathogenic bacteria. Acknowledgments Figure 2 was created by Toyomi Kobayashi (Ms.Clinic MayOne, Nara, Japan; (accessed on 6 February 2023). Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement No new data were created. Conflicts of Interest The authors declare no conflict of interest. Figure 1 The number of articles identified by searching for keyword combinations. Figure 2 Pathophysiological mechanisms underlying the bidirectional relationship between endometriosis and PID. (A) Ascending genital tract infection causes PID, followed by secondary infection of endometriotic cysts, and ultimately TOA. (B) Endometriosis-associated microbiota dysbiosis induces pathogenic bacterial growth and pelvic inflammation, leading to secondary infections in endometriotic cysts and then the development of TOA. Bold arrows indicate the first trigger of infection, dotted arrows indicate the second infection. diagnostics-13-00868-t001_Table 1 Table 1 The search strategy. Search Mode The Keyword and Search Term Combinations Search term 1 endometriosis OR endometrioma OR ovarian endometrioma Search term 2 pelvic inflammatory disease Search term 3 immune response OR immune system OR innate OR adaptive Search term 4 microbiota OR microbiome OR commensal OR pathogenic Search term 5 tubo-ovarian abscess OR ovarian abscess Search Search term 1 AND Search term 2 Search term 1 AND Search term 2 AND Search term 3 Search term 1 AND Search term 2 AND Search term 4 Search term 1 AND Search term 2 AND Search term 5 Search term 1 AND Search term 3 AND Search term 4 Search term 2 AND Search term 3 AND Search term 5 Search term 1 AND Search term 5 Search term 2 AND Search term 5 Search term 1 AND Search term 2 AND Search term 5 Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). 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PMC10000849 | Diagnostics (Basel) Diagnostics (Basel) diagnostics Diagnostics 2075-4418 MDPI 10.3390/diagnostics13050988 diagnostics-13-00988 Article Outpatient Hysteroscopic Polypectomy--A Retrospective Study Comparing Rigid and Semirigid Office Hysteroscopes Chiofalo Benito Conceptualization Writing - original draft 1* Calandra Mauro Writing - original draft 1 Bruno Valentina Formal analysis 1 Tarantino Vincenzo Data curation 2 Esposito Giovanni Data curation 2 Vizza Enrico Supervision 1 Corrado Giacomo Writing - review & editing 2 Scambia Giovanni Supervision 2 Catena Ursula Writing - review & editing 2 Pavlik Edward J. Academic Editor Vitagliano Amerigo Academic Editor 1 Gynecologic Oncology Unit, Department of Experimental Clinical Oncology, IRCCS Regina Elena National Cancer Institute, 00128 Rome, Italy 2 Division of Gynecologic Oncology, Department of Woman, Child and Public Health, Fondazione Policlinico Universitario A. Gemelli-IRCCS, Catholic University of Sacred Heart, 00168 Rome, Italy * Correspondence: [email protected]; Tel.: +39-06-5266-2433 05 3 2023 3 2023 13 5 98831 10 2022 22 2 2023 01 3 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). Endometrial polyps are one of the most common pathological conditions in gynecology. Hysteroscopy is the gold standard for the diagnosis and treatment of endometrial polyps. The purpose of this multicenter, retrospective study was to compare patients' pain perception during an operative hysteroscopic endometrial polypectomy in an outpatient setting with two different hysteroscopes (rigid and semirigid) and to identify some clinical and intraoperative characteristics that are related to worsening pain during the procedure. We included women that underwent, at the same time as an diagnostic hysteroscopy, the complete removal of an endometrial polyp (using the see-and-treat strategy) without any kind of analgesia. A total of 166 patients were enrolled, of which 102 patients underwent a polypectomy with a semirigid hysteroscope and 64 patients underwent the procedure with a rigid hysteroscope. No differences were found during the diagnostic step; on the contrary, after the operative procedure, a statistically significant greater degree of pain was reported when the semirigid hysteroscope was used. Cervical stenosis and menopausal status were risk factors for pain both in the diagnostic step and in the operative one. Our results confirm that operative hysteroscopic endometrial polypectomy in an outpatient setting is an effective, safe, and well-tolerated procedure and indicate that it might be better tolerated if a rigid rather than semirigid instrument is used. endometrial polyps outpatient hysteroscopy operative hysteroscopy VAS score This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. pmc1. Introduction Hysteroscopy is the best method for the direct evaluation of the endometrial cavity of patients with a broad spectrum of gynecological pathologies, such as abnormal uterine bleeding, endometrial polyps, submucous myomas, infertility, mullerian abnormalities, endometrial hyperplasia, and cancer. Endometrial polyps are among the most common pathological conditions in gynecology; their prevalence is estimated between 10% and 40% in symptomatic women and up to 12% in asymptomatic women . Usually, the symptomatology related to endometrial polyps is abnormal uterine bleeding, vaginal bleeding during menopause, and infertility . The risk of the cancerization of endometrial polyps and, consequently, the need for resection do not appear to be related to a polyp's size; however, the risk of cancer seems to be higher in patients presenting uterine bleeding, aged > 60 years, with diabetes mellitus, with hypertension, and who use tamoxifen . Nowadays, hysteroscopy represents the gold standard not only for diagnosis but even for the treatment of intra-uterine pathologies, which can be performed in outpatient or inpatient settings . The advantages of the outpatient procedure are evident, given that it does not require cervical dilatation, an operating room, or hospitalization, thereby minimizing costs. On the contrary, the disadvantages are represented by the pain experienced during the procedure and by the lack of widespread surgical skills among the operators in this kind of surgery. The "see and treat" strategy, applied in the outpatient setting, perfectly integrates the operative step with the diagnostic procedure . This has been enabled by the introduction of innovative miniaturized instruments (5 or 7 Fr), scopes with working channels and continuous flow systems, and intrauterine morcellator devices . These instruments allow an operator to quickly and effectively perform many hysteroscopic procedures without increasing the patient's discomfort . Operative outpatient procedures, also called "office hysteroscopies", are usually performed without any medication; in fact, there is no high-quality evidence that using local anesthetics, nonsteroidal anti-inflammatory drugs, or other medical approaches, either when compared amongst themselves or with a placebo, improves a patient's compliance or reduces their pain scores during hysteroscopy . It has been proven that the outpatient procedure is also less expensive in terms of direct and indirect costs when compared with an inpatient hysteroscopy, for which the latter is usually performed in an operative room under general or local anesthesia . Polypectomy is the most common hysteroscopic operative procedure worldwide and many studies have proven its safety and efficacy both in general and in an outpatient setting . The procedure can be performed with rigid and semirigid hysteroscopes, which are equipped with a working channel for the introduction of cold operative instruments and/or miniaturized radiofrequency devices. The choice of how to perform the procedure must account for different factors, such as the operator's skills, patient compliance, the number and size of polyps, and the quality of the hysteroscopic imagery; furthermore, the correct choice of instrumentation is very important . Due to the lack of similar studies in the literature, the aim of the present study was to compare two different methods of performing an outpatient hysteroscopic polypectomy with a rigid and a semirigid hysteroscope to test the safety and effectiveness of both procedures and establish which of the two treatments is superior. 2. Materials and Methods This article reports the results of a multicenter, retrospective pilot study. The study was approved by the local Ethical Committee of the IRCCS "Regina Elena" National Cancer Institute of Rome (promotion center) and of the Fondazione Policlinico Universitario A. Gemelli--IRCCS of Rome (participant center) (RS n. 1778/22, DIPUSVSP-PD-01-231). The design, analysis, interpretation of data, drafting, and revision of the study followed the Helsinki Declaration's Committee on Publication Ethics guidelines (accessed on 1 September 2022)), and the reporting of the study was conducted using observational, routinely collected health data statements , which are available through the Enhancing the Quality and Transparency of Health Research Network website (www.equator-network.org (accessed on 1 September 2022)). The study was unadvertised, and no remuneration was offered to participants. Each enrolled patient was informed about aims and procedures and signed an informed consent to facilitate data collection for research purposes. The first goal of this study was to compare operative outcomes and patients' pain perceptions with respect to outpatient operative hysteroscopic endometrial polypectomy among two different groups of patients treated with two different hysteroscopes (rigid and semirigid) to assess which technique is more effective and less painful; the second goal was to identify clinical and intraoperative characteristics related to increased pain during the procedure. 2.1. Patients: Inclusion and Exclusion Criteria We retrospectively and consecutively included all women with hysteroscopic diagnosis of endometrial polyp that underwent, at the same time of the diagnostic hysteroscopy, an outpatient operative procedure for the complete removal of the polyp (using the see-and-treat strategy) with the two different hysteroscopes mentioned earlier from March 2021 to July 2022. The study took place at the IRCCS "Regina Elena" National Cancer Institute of Rome (promoting center) and at the CLASS Hysteroscopy Center of Fondazione Policlinico Universitario A. Gemelli--IRCCS of Rome (participant center). We excluded patients with multiple polyps and other concomitant intrauterine pathologies requiring an additional procedure, patients with final histological diagnosis of myoma and endometrial carcinoma, patients that postponed the procedure, patients treated with instruments other than those under study, and patients that underwent procedures under any type of analgesia and those performed by trainees or unexperienced surgeons. 2.2. Treatments & Instrumentation Patients were divided into two groups: the first group underwent polypectomy with a semirigid office hysteroscope (Gynecare Versascope, GYN group), and the second group underwent the procedure using a rigid office hysteroscope (Storz 5 mm Bettocchi hysteroscope, BETT group). In the present study, the choice of the instrument depended on physician's preferences. Gynecare VersascopeTM is a 3.2 mm semirigid hysteroscope; it has a 1.9 mm optic (fiber optic) with a 0deg viewing angle and a single-use outer sheath equipped with irrigation and suction channels and an additional expandable plastic channel for the insertion of 7 or 5 Fr operative instruments . Bettocchi continuous-flow is an outpatient operating hysteroscope (Karl Storz SE & Co.Kg, Tuttlingen, Germany); it consists of a 2.9 mm optic (lens optic) with a 30deg hole-oblique range of vision, an inner operating sheath for irrigation that is 4.3 mm in diameter, and an outer sheath for fluid aspiration that is 5 mm in diameter . In both cases, the procedure was carried out without the use of any kind of analgesia or premedication, using a vaginoscopic approach, performing the so-called "no touch" technique, and using 5 Fr instruments (bipolar electrodes, scissors, and grasping forceps) for the operative procedure. For uterine distension, a sterile saline solution was used, with filling pressure of up to 100 mmHg, which was maintained using a manual or an automated pressure delivery system. 2.3. Patients' Evaluation We evaluated the following clinical parameters in both groups: age, menopause, and number of attempts, and type of delivery. post-procedural parameters evaluated were stenosis of the cervical canal (based on the operator's subjective impression, which was overcome solely by the rotation of the instrument), post-operative complications, and the size (largest diameter) and histologic type of the removed lesions. Individual pain sensation at two different times during the exam was recorded using a 10 cm visual analogue scale (VAS); each patient was asked to describe the intensity of pain during the hysteroscopy, after the diagnostic phase, and after the operative procedure. 2.4. Statistical Analysis Kolmogorov-Smirnov test was used to analyze data distribution. Data were expressed as mean +- standard deviation (SD) and median +- range when appropriate. Mann-Whitney test or t-test were used for the analysis of continuous variables; additionally, Mann-Whitney test was used to compare VAS scores for all different groups between diagnostic and operative hysteroscopic steps since data were found to be non-parametric. t-test was used to confirm that the two groups were homogeneous in terms of clinical characteristics, since data were normally distributed, together with the Fisher's exact test, which was used in the analysis of contingency tables for categorical data. Spearman correlation test was used to verify any correlation between polyps' size and VAS 2. Significance was set at p <= 0.05. Statistical analyses were performed using GraphPad Prism ver. 9.0.0 (GraphPad Software, San Diego, CA, USA). Post hoc power analysis was conducted to estimate the power of our sample size in order to detect meaningful differences between groups in the operative phase. The model by Noether et al. was applied . 3. Results A total of 166 patients were enrolled from March 2021 to July 2022. Of these, 102 underwent a polypectomy with a semirigid hysteroscope (GYN group) in the promotion center, and 64 underwent the procedure with a rigid hysteroscope (BETT group) in the participant center. All the procedures were carried out by two surgeons experienced in hysteroscopy, namely, B.C. and U.C. Both groups were homogenous in terms of clinical parameters, the number of patients with an intra-procedural finding of severe cervical stenosis, and the size and histologic type of the polyps; furthermore, all the removed lesions were pedunculated, not sessile (Table 1). No post-operative complications were detected in the two groups. With regard to the VAS scores during the procedure, no differences were found after the diagnostic step (GYN median 0, range 0-3; BETT median 0, range 0-8); on the contrary, after the operative procedure, a statistically significant greater pain degree of was reported in the GYN group (GYN median 1, range 0-7; BETT median 0, range 0-8) . After the data were processed to search for correlations between high VAS scores and clinical parameters or intraoperative findings, no correlation was found between VAS score and patients that underwent previous vaginal deliveries vs. those without previous vaginal delivery (p = 0.2 in the diagnostic step; p = 0.3 in the operative step) and previous cesarean section vs. those without previous cesarean section (p = 0.3 in the diagnostic step; p = 0.7 in the operative step). Conversely, cervical stenosis and menopause status were identified as statistically significant risk factors for pain both in the diagnostic step and in the operative one. Regarding the polyp size and histological type of the removed lesions, no correlations were found with VAS score during the operative step (r = 0,14; p = 0.07; p = 0.17) (Table 2). Following post hoc power analysis, our sample resulted in a 37.6% level of power for detecting statistical differences between comparators, with an alpha error of 0.05. 4. Discussion Hysteroscopy represents the gold standard for the diagnosis and treatment of intrauterine pathologies, and it should be considered for the treatment of endometrial polyps whenever possible . Several types of hysteroscopes with different technological capabilities are currently available, and it is widely known that the smaller the diameter of the endoscope, the lower the degree of pain perceived; however, other factors must play a role in the genesis of pain during hysteroscopy. The use of mini-hysteroscopes (3.3 mm with diagnostic sheath) seems to reduce the level of pelvic pain during this procedure . Our study aimed to investigate pain perception during an office-based polypectomy among two groups of patients treated with a rigid and a semirigid hysteroscope; the results have shown that in the diagnostic phase, the procedure is painless and well-tolerated when using both rigid and semirigid hysteroscopes, each with two different diameters (3.6 vs. 5 mm). With regard to the operative procedure, the data show that the women who had a polypectomy with the rigid hysteroscope (BETT group) experienced less pain when compared with the GYN group. The results regarding the GYN group, although higher than the BETT group, are still acceptable, and are similar to other data obtained by a previous study, wherein a Gynecare Versascope was used to perform outpatient hysteroscopies, and the mean intraoperative VAS score during the procedure was 1.2 . The reason for such a difference could be related to the semirigid nature of the working channel of the Gynecare hysteroscope; indeed, the introduction of the operative instrument inside the expandable plastic channel provokes a sudden increase in the diameter of the hysteroscope; moreover, the position of the channel is uncontrollable due to its flexibility. With the rigid hysteroscope, thanks to its 30deg degree optical range, lateral movements of the instruments during the procedure can be avoided. This theory, although it cannot be adequately proven, can explain the increase in pain in the GYN group during the operative step. Our secondary aim was to identify clinical characteristics and intraoperative parameters related to increased pain during the procedure. Our findings suggest that the correlation of pain with menopause and cervical stenosis was statistically significant; indeed, it is reasonable to think that cervical stenosis is more frequent in menopausal patients, for whom even partial synechiae render the transit of the instrument less comfortable and, therefore, more painful. These findings were confirmed both in the diagnostic and operative step, independently of the type of instrumentation used. Furthermore, no correlations were found between the pain score and diameter of the lesion or the histologic type of the removed polyps. Our personal experience suggests that the surgical complexity is independent of the nature of the polyps, and it is likely that several factors play a role in this regard. The data regarding pain scores and polyp sizes are conflicting in the literature; however, few studies have considered the correlation with the histologic type . There are conflicting data regarding the correlations between menopause, speculum placement, and a lack of previous vaginal delivery; according to a previous study, they seem to be associated with pain occurrence and intensity . On the contrary, a Hungarian study found did not find any differences in the level of pain felt by the patients with different parity and menopausal statuses . The majority of patients enrolled in the present study tolerated the outpatient procedure with low pain levels; this choice made it possible for them to receive a safe, efficient, and effective assessment and treatment, which avoided the disadvantages of a general anesthesia and hospital staying. In a randomized trial, a significant patient preference for office-based hysteroscopy was reported, offering faster recovery times compared to an inpatient operative hysteroscopy and, of no small degree of importance, minimized procedural costs . Conversely, other authors have observed that an office hysteroscopic polypectomy may be associated with a higher risk of failed or incomplete polyp removal . In our centers, more than 90% of hysteroscopic polypectomies are performed in an outpatient setting, and, in the present study, all the procedures were successfully completed, with no residual polyp fragments. The anxiety of patients undergoing hysteroscopy is mainly caused by the fear of feeling pain during the procedure; it is also associated with increased and persistent postoperative pain, and it seems to reduce patient satisfaction . Obviously, it is very important to provide a patient with comfort and a friendly atmosphere while trying to medicalize the procedure as little as possible by using the vaginoscopic technique . 'No-touch' vaginoscopic hysteroscopy is significantly faster to perform than the traditional technique and it is less painful, thereby reducing the likelihood of inducing a vasovagal reaction . Communication and patient education have been proven to be effective tools with which to reduce preoperative anxiety; preoperative anxiety is reduced by the ability of doctors to answer patients' questions, which also increases patients' satisfaction . In an outpatient setting, accurate counselling should be considered an essential part of the procedure, with the objective of achieving pain and anxiety reduction . In this study, the patients were welcomed in both participating centers in dedicated clinics, with the presence of nurses adequately trained to support each woman. During the procedure, the surgeons provided support to the patient, involving her in the procedure and explaining the different steps of the procedure and any abnormalities, if present. These findings may have contributed to the very low pain levels reported during the hysteroscopic procedure. Unsurprisingly, in the sample used in this study, even in cases where the VAS score was high, no vaso-vagal reactions occurred, and the use of atropine was not necessary in any case. In a randomized perspective trial that evaluated the acceptance and feasibility of rigid hysteroscopes versus the flexible kind, the level of discomfort caused by the introduction of a rigid instrument was higher than that of the flexible one, even though rigid hysteroscopes provide superior optical qualities . Although prospective and randomized, this study was limited to diagnostic hysteroscopy; it did not evaluate the execution of an operative procedure, which is the more painful phase of hysteroscopy, according to our data. To the best of our knowledge, this is the first study that compares two different hysteroscopes for operative hysteroscopic polypectomy in an outpatient setting. In the literature, there are no studies that are similar to the ours; therefore, it is difficult, in a field that has been invaded by new, different optics and instruments over the last 10 years, each with different technologies, to find studies that can constitute a valid form of comparison. Our data are not robust enough to draw any definite conclusions; however, despite the limitations of a retrospective series with low statistical power, our study showed that an operative hysteroscopic endometrial polypectomy in an outpatient setting with different types of instruments seems to be an effective, safe, and well-tolerated procedure. Moreover, it may be better tolerated if a rigid rather than a semirigid instrument is used. To clarify this last point, further studies and prospective randomized trials are required to establish the best way to perform endometrial polypectomy, while also considering surgeons' and patients' preferences. Due to the advent of new technologies such as uterine morcellators, well-designed prospective studies are expected to be able to determine the best surgical techniques with which to perform outpatient endometrial polypectomy . Author Contributions Conceptualization, B.C.; writing--original draft preparation, B.C. and M.C.; data collection V.T. and G.E.; writing--review and editing, U.C. and G.C.; statistical analysis, V.B.; supervision, E.V. and G.S. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The study was approved by the local Ethical Committee of the IRCCS "Regina Elena" National Cancer Institute of Rome (promotion center) and of the Fondazione Policlinico Universitario A. Gemelli--IRCCS of Rome (participant center) (RS n. 1778/22, DIPUSVSP-PD-01-231). Informed Consent Statement Informed consent for research purposes and for data processing was obtained from all subjects involved in the study. Data Availability Statement The data that support the findings of this study are available from the corresponding author, upon reasonable request. Conflicts of Interest The authors have no relevant financial or non-financial interests to disclose. Figure 1 VAS score in the two groups during the diagnostic and the operative steps. **** Mann-Whitney test: p < 0.0001. diagnostics-13-00988-t001_Table 1 Table 1 Clinical, intra-, and post-operative characteristics of enrolled patients. BETT GYN p Value Total number 64 102 N.A. Age (mean +- S.D.) 52.5 +- 14.2 51.5 +- 12.4 ns (* p = 0.6) Parity (n pregnancies) (mean +- S.D.) 1.4 +- 1.3 1.5 +- 1.5 ns (* p = 0.7) Cervical stenosis (%) 9.4 19.6 ns (SS p = 0.08) Vaginal deliveries (%) 48.4 54.9 ns (SS p = 0.4) Cesarean sections (%) 18.7 10.8 ns (SS p = 0.2) Menopausal status (%) 48.4 50 ns (SS p = 0.9) Polyp's largest diameter (median (range)) 15 (5-40) 10 (5-30) ns (^ p = 0.06) Polyp's histologic type (FGP vs. HP) 74.6% vs. 25.4% 83.3% vs. 16.7% ns (SS p = 0.17) * t-test was used for comparisons between groups in terms of age and parity since data were normally distributed. ^ Mann-Whitney test was used for comparisons between groups in terms of polyp size. SS Fisher's exact test was used for comparisons between groups to assess differences in the categorical variables between the two groups. Significance was set at p <= 0.05. N.A--not applicable; BETT--rigid hysteroscope group; GYN--semirigid hysteroscope group; FGP--fibroglandular polyp; HP--hyperplastic polyp; ns--not significant. diagnostics-13-00988-t002_Table 2 Table 2 Correlation of VAS score with patients' clinical and intra-operative characteristics. VAS in the Diagnostic Step p Value VAS In the Operative Step p Value No Cervical Stenosis (n = 140) Cervical Stenosis (n = 26) No Cervical Stenosis (n = 140) Cervical Stenosis (n = 26) Median (range) 0 (0-5) 1 (0-8) p < 0.0001 0 (0-6) 3 (0-8) p < 0.0001 Fertile status (n = 84) Menopause (n = 82) Fertile status (n = 84) Menopause (n = 82) Median (range) 0 (0-5) 0 (0-8) p < 0.05 0 (0-5) 1 (0-8) p < 0.05 - - FGP (n = 133) HP (n = 33) Median (range) - - 1 (0-8) 0 (0-7) p < 0.65 VAS--visual analogue scale; FGP--fibroglandular polyp; HP--hyperplastic polyp. 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PMC10000850 | Foods Foods foods Foods 2304-8158 MDPI 10.3390/foods12050917 foods-12-00917 Article Perceptions of Food among College Students in the Field of Food Science: A Food Sustainability Approach de Moraes Prata Gaspar Maria Clara 12+ Soar Claudia Investigation Data curation Writing - original draft Writing - review & editing 3+ Aguilera Mari Conceptualization Investigation Data curation Writing - original draft Writing - review & editing 456 Gomez Maria Clara Conceptualization Investigation Data curation Writing - original draft Writing - review & editing 1 Celorio-Sarda Ricard Investigation Data curation Writing - original draft Writing - review & editing 278 Comas-Baste Oriol Investigation Data curation Writing - original draft Writing - review & editing 278* Larrea-Killinger Cristina Conceptualization Investigation Data curation Writing - original draft Writing - review & editing Supervision 129 Vidal-Carou M. Carmen Conceptualization Investigation Writing - original draft Writing - review & editing Supervision 278 Zandstra Elizabeth H. Academic Editor 1 Departament d'Antropologia Social, Facultat de Geografia i Historia, Universitat de Barcelona (UB), Carrer de Montalegre 6, 08001 Barcelona, Spain 2 Institut de Recerca en Nutricio i Seguretat Alimentaria (INSA-UB), Universitat de Barcelona, Av. Prat de la Riba 171, 08921 Santa Coloma de Gramenet, Spain 3 Nutrition Post-Graduate Program, Department of Nutrition, Federal University of Santa Catarina, 88040-900 Florianopolis, Brazil 4 Departament de Cognicio, Desenvolupament i Psicologia de l'Educacio, Seccio Cognicio, Facultat de Psicologia, Universitat de Barcelona (UB), Passeig de la Vall d'Hebron 171, 08035 Barcelona, Spain 5 Institut de Neurociencies (UBNeuro), Universitat de Barcelona (UB), Passeig de la Vall d'Hebron 171, 08035 Barcelona, Spain 6 NeuroDevelop eHealth Lab, eHealth Center, Universitat Oberta de Catalunya (UOC), Rambla de Poble Nou 156, 08018 Barcelona, Spain 7 Departament de Nutricio, Ciencies de l'Alimentacio i Gastronomia, Facultat de Farmacia i Ciencies de l'Alimentacio, Campus de l'Alimentacio de Torribera, Universitat de Barcelona (UB), Av. Prat de la Riba 171, 08921 Santa Coloma de Gramenet, Spain 8 Xarxa d'Innovacio Alimentaria (XIA), C/Baldiri Reixac 4, 08028 Barcelona, Spain 9 Anthropology of Crises and Contemporary Transformations (CRITS), Departament d'Antropologia Social, Facultat de Geografia i Historia, Universitat de Barcelona (UB), Carrer de Montalegre 6, 08001 Barcelona, Spain * Correspondence: [email protected] + These authors contributed equally to this work and share first authorship. 21 2 2023 3 2023 12 5 91720 12 2022 10 2 2023 18 2 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). The complex concept of food sustainability has become crucial in all spheres of life. Dietitians, food scientists, and technologists are in a unique position to promote sustainability in food systems. However, the perceptions of food sustainability among food science professionals and college students are under-researched, particularly in Spain. The aim of this study was therefore to analyze perceptions related to food and to food sustainability in a sample of Human Nutrition and Dietetics (HND) and Food Science and Technology (FST) students in Barcelona (Spain). An exploratory and descriptive cross-sectional study was carried out using qualitative and quantitative methodology and convenience sampling. Two focus groups and an online questionnaire were conducted (300 participants completed the survey, 151 from HND and 149 from FST). Although the students expressed concern about food sustainability, their dietary choices were primarily associated with or influenced by taste/pleasure and health/nutrition. The issue of sustainability seemed more internalized by women than men, whereas the generalized conception of a sustainable diet was essentially based on environmental aspects, with socioeconomic dimensions largely overlooked. The concept of sustainability should be promoted among food science students in all its multidimensionality, and actions need to be implemented that bring sustainability closer to students' social practices, which should be incorporated into all university education and that is taught by professors duly trained in the subject. sustainable diet food perception eating practices dietetic food technology college students Catedra d'Alimentacio Saludable i Sostenible UB-Danone.This research was funded by the Catedra d'Alimentacio Saludable i Sostenible UB-Danone. pmc1. Introduction Food sustainability is a broad, multidimensional, and complex concept that is difficult to define . It attempts to reconcile public health and ecological discourses but also involves the economic, social, and cultural dimensions of food . According to the Food and Agriculture Organization (FAO), "sustainable diets are those diets with low environmental impacts which contribute to food and nutrition security and to healthy life for present and future generations. Sustainable diets are protective and respectful of biodiversity and ecosystems, culturally acceptable, accessible, economically fair and affordable; nutritionally adequate, safe and healthy; while optimizing natural and human resources" . Sustainability has become a key concept in all spheres of social, cultural, economic, and political life . On a global level, the definition of the Sustainable Development Goals in 2015 by the United Nations, followed by the presentation of the European Green Deal in 2019, made sustainability the central axis of strategies aimed at improving human living conditions and the environment. In this context, food systems have been increasingly associated with environmental, economic, and social impacts that directly affect the sustainability of the planet . Public and private agents have developed actions and multiplied recommendations to integrate sustainable consumption into daily life . Due to its direct links with human and environmental health, sustainability has also become inseparable from discourses on healthy eating. In the 1980s, Gussow and Clancy (1986) highlighted the importance of the connection between nutrition, health, and the environment. More recently, this link was also evidenced in a report issued by the EAT-Lancet Commission . Social perceptions, which are based on subjective and socially elaborated knowledge, influence social practices. Although there are official definitions and institutional recommendations regarding sustainability , individuals perceive this concept in multiple ways . Food professionals, such as dietitians, food scientists, and technologists, are in a unique position to influence sustainability at different stages of the food chain (i.e., from production to consumption) by developing production techniques and/or promoting sustainable food practices . Although in recent years more and more studies have explored perceptions of food sustainability among lay individuals , little research has addressed this issue in professionals from the field of food and nutrition . In a study of US dietitians, Hawkins et al. (2015) reported that over 45% of survey participants agreed that climate change is an important issue and should be considered in practice-related behaviors. However, it was found that only 8% of dietitian workplaces provided funding for diet-related climate change mitigation activities. A survey of American nutrition and dietetics programs showed that 68% of educators were interested in sustainability education techniques, but felt inadequately prepared to put them in practice . Undergraduate students are at a crucial stage of professional training. As a young population, they are potentially open to absorbing new trends and constitute a highly suitable collective to receive training aimed at fostering sustainability , especially students enrolled in the field of food science . Burkhart et al. (2020) observed that Australian dietetic students were familiar with and concerned about sustainability, but only in a superficial way. To the best of our knowledge, despite the importance of sustainability for professionals working in food and nutrition, very few studies have hitherto analyzed the social perceptions of sustainability-related issues among professionals or students in this field. Most of the available research has assessed the level of familiarity with the concept of sustainability or the importance attached to it among dietetic professionals or college students, mainly in an Anglo-Saxon context. In Spain, the perceptions of sustainability among these groups have been little explored. Understanding social perceptions among food science professionals or college students is fundamental to improve their academic training, promote a more critical perspective of the food system, and ultimately promote sustainability . In this context, the aim of this study was to analyze the perceptions of food and food sustainability among college students of Human Nutrition and Dietetics (HND) and Food Science and Technology (FST) in Barcelona (Spain). 2. Materials and Methods 2.1. Setting and Sample An exploratory and descriptive cross-sectional study aiming to analyze perceptions of food among college students using both qualitative and quantitative methodology was carried out between May 2020 and September 2021 by an interdisciplinary team. The study was conducted with a convenience sample of male and female college students enrolled in any of the four years of bachelor's degrees in HND and FST at the University of Barcelona (UB), a reference institution in these fields in Spain. According to the UB, the HND bachelor's degree trains professionals capable of developing activities aimed at feeding the individual or groups of individuals to attend to their physiological or pathological needs, taking into account the principles of health protection and promotion, disease prevention, and dietary and nutritional treatment. On the other hand, the training in FST is essential for the growth, improvement, and diversification of the food industry, which must respond to new needs, concerns, and conveniences of today's society in the food field. No exclusion criteria were established with respect to the age of the participants, place of residence, or nationality. To characterize the sample, data were collected about educational level, year of education, gender, age, municipality of residence, type of cohabitation, work activity or internship, parental level of education, and average monthly household income. 2.2. Data Production and Analysis A fully structured questionnaire was developed specifically for the study (see Supplementary Material) based on data obtained in the qualitative research phase, as well as from previous studies on the food perceptions of HND college students and/or dietitians and the general population . The qualitative phase was based on focus groups, a useful technique in the exploratory stages of investigation that collects data through group discussions on a specific topic proposed by the researcher . The resulting insights serve as a resource for understanding social representations and deepening research questions . Two focus groups were conducted with a total of 13 students (11 females): five from HND and eight from FST. Participants were recruited by sending an email announcing the project to all students enrolled in these courses, and all those who responded positively participated in the focus groups. Due to restrictions related to the COVID-19 pandemic, the two focus groups were held online in October/November 2020 via the Zoom platform. The participants were equipped with a microphone and a video camera. The 90-minute focus groups were led by an anthropologist specializing in food studies and qualitative techniques, and observed by a student with a master's degree in anthropology and ethnography. The focus group discussions were guided by an interview script specifically drafted for this study, which was composed of open questions about perceptions related to food. The resulting data were later incorporated into the instrument used in the quantitative phase to improve accuracy and constituted a complementary part of the overall results. The focus group discussions were recorded with the consent of the participants and were transcribed verbatim. The discussions were analyzed on a thematic basis by coding the content in analytical categories defined according to the objectives of the study and the emerging discourses: perceptions of healthy eating/food, meanings of food, trust/distrust of food, food sustainability, culinary activity, food choices, vegetarianism, and changes in perception while studying the degrees. Two researchers independently read the transcripts to identify the categories, the results were compared, and the final categories were determined after discussion between the researchers. Finally, the coding and systematization of the data was carried out using the qualitative data analysis software Atlas-Ti (version 8. Visual Qualitative Data Analysis. 2017). Based on the insights obtained from the qualitative stage, a questionnaire was developed. The questionnaire was sent for review to 20 experts from the fields of nutrition (12), statistics (1), anthropology (5), and sociology of food (2), and adjustments were made based on their suggestions regarding question clarity, relevance, and pertinence. A pilot test was carried out with 30 students who were invited to comment on the questionnaire after completing it. The final questionnaire contained 31 multiple choice or Likert scale questions (including those for the sample characterization) addressing the perceptions related to food, healthy eating, and sustainable issues. In addition, it contained an open-ended question: "Which word do you associate with the concept of "food"? (Indicate one word only)." For the analysis of perceptions regarding food sustainability, five questions were selected from the questionnaire: besides a free-association task and self-assessment of diet, they were focused on aspects influencing food choices, food concerns, and what contributes to a sustainable diet. The questionnaire was administered online between April and May 2021 via the Survey Monkey website. A total of 385 responses were obtained, 85 of which were excluded because they were incomplete, leaving 300 complete responses (78.0%). For the analysis, third-year students were grouped together, resulting in three groups (1st-, 2nd/ 4th-year students). All data derived from the questionnaire were entered into SPSS v. 24 for statistical analysis. Descriptive results were expressed as means and standard deviations or frequencies according to the nature of the data. Textual data collected with the free-association task were pre-processed to reduce data dispersion and synonyms, multi-words were identified, and verbs were reduced to infinitives. The final corpus, which included 42 words, was codified into analytical categories defined by the authors. Statistical results were obtained by comparing the distribution of response frequencies using the chi-square statistic, which was considered significant when <0.05. 3. Results 3.1. Participant Profile Table 1 summarizes the general characteristics of the college students who completed the questionnaire. Out of the 300 participants, 151 were studying HND and 149 FST, representing 47.0% and 45.0% of the total enrollments in the two bachelor's degrees, respectively. All the participants were students of the University of Barcelona, and all lived in Catalonia (Spain). The overall mean age was 21.25 (+-3.16) years (21.76 +- 3.80 for the HND students and 20.73 +- 2.24 for the FST students). Most of the informants were female (80.3%) (kh2 = 11.53, p = 0.030). The monthly household income of about 50% of the sample was 2000-4999 euros. The parents of 57.4% of the participants had received at least higher education. In total, 59.3% of the participants were not in employment, and the proportions differed between the two degrees (kh2 = 8.17, p = 0.010). For example, a higher percentage of the HND students reported doing an internship. 3.2. Food Perceptions and Choice In the free-association task, no words from the semantic field of sustainability were associated with "food". The most-cited words were related to pleasure, nutrition, and health . The qualitative phase of the study confirmed this dual perception of eating--an act with a physiological/nutritional dimension that also confers pleasure: "The first thing is to get nourishment, nutrition, but then also to enjoy the food as such, uh... to share it, as a means of social connection" (Carlos, HND). The categorization of words into analytical categories revealed that while the category "food" was the most frequent among the FST students (36.9% FST versus 19.3% HND, kh2 = 11.66, p = 0.001), the "hedonic and gustatory" dimension was the most recurring among the HND students (41.3% versus 28.9% in FST, kh2 = 4.90, p = 0.026) (Table 2). No significant differences were observed in student perceptions according to degree year or gender. The students assessed their eating practices by indicating levels of agreement (somewhat or totally agree) with different diet typologies. The option "healthy eating" was the most prevalent (90.0%), being statistically higher in the HND students (kh2 = 9.88, p = 0.021). This was followed by "pleasant and convivial" (89.3%) and "Mediterranean" (86.3%), the latter being statistically higher among the FST students (kh2 = 9.77, p = 0.044). Between 40% and 50% of the students agreed (somewhat or totally) with the dietary patterns related to sustainability: "ecological and natural", and "responsible and socially committed". Moreover, 26.0% of the students described their diet as vegetarian/vegan (somewhat or totally), which may be associated with a greater awareness of the sustainability concept, as observed in the focus groups: "I don't eat meat and when I avoid certain foods, I do it because of that, because of sustainability or the origin, things like that. (...) I started when I was very young because of the animals, the little animals. Now, it's because of sustainability and, to some extent, inertia" (Amanda, HND). "Sustainability, that's why I started and why I continue [to avoid eating meat]. In other words, that's why I'm not very strict, because I understand that maybe [eating] a small amount of meat won't change the current situation of the meat industry. But it's true that I limit it a lot and I try to make those around me aware of the carbon footprint of meat consumption" (Elena, FST). No statistical differences were found in the self-perception of dietary practices according to degree year. However, among the advanced HND students, the tendency to describe their diet as "ecological and natural", "pleasant and convivial", or "gourmet" was lower compared to first-year students, while agreement with "light" as a descriptor increased. Among the advanced FST students, there was a decline in the choice of "pleasant and convivial", and "responsible and socially committed". Female participants were statistically more likely to describe their diets as "light" (kh2 = 15.25, p = 0.004) and "vegetarian/vegan" (kh2 = 17.26, p = 0.002) . Regarding the aspects that most influenced food choices, "pleasure and taste" and "nutritional composition" were the most important (Table 3). Taste and pleasure were also the most frequently highlighted aspects in the focus groups in several cases alongside other elements: "I think I'm a mixture of everything because I'm influenced by the taste something has. Obviously, [I'm aware of] the impact it's going to have on the environment, but I'm also very much a person who takes whatever happens to be in the fridge, especially as I live with my parents. And as my grandparents were farmers, they have a garden and we have a lot of products from my grandparents' garden, so I eat a lot of what's in season ... so I think the factor that most determines what I eat is whether it's in season or not" (Gemma, HND). "I'm trying to cut down on calories a little, I try not to eat too many carbohydrates and I also try to eat as little processed food as possible (...) this seems obvious to me, for health reasons" (Samanta, FST). "Ecology, environment and animal welfare" was the third selected aspect, but with much less frequency. However, according to some participants in the focus groups, food and the quality of food may also be chosen with the aim of reducing the environmental impact. Several strategies may be implemented, considering the type of food (especially animal products), the season, the type of production (whether it is organic or a genetically modified organism, the degree of naturalness), and its origin, mainly if it is local: "I try not to eat genetically modified food, I make sure it's organic, or at least that there are no genetically modified organisms or excessive pesticides, herbicides, that it's not agribusiness" (Amanda, HND). "I try to have a little bit of sustainable food. I also try to look at where everything comes from" (Samanta, FST) "I try not to consume [genetically modified organisms], and I go to markets and look for local products" (Alba, FST) Pleasure and taste preferences were the most important aspects when choosing food for the first-year HND students, while nutritional composition became more important in later years. Furthermore, a statistical difference was observed for "state of mind": 4.3% of the first-year students chose this option, but it was irrelevant in the decisions of the second-, third-, and fourth-year students (kh2 = 9.09, p = 0.011). Additionally, the time of availability gained more influence (1.1% in 1st year and 11.6% in 4th year) (kh2 = 9.57, p = 0.008). Similar trends were found among the FST students, for whom nutritional composition became more important in later years (kh2 = 9.83, p = 0.009). "Pleasure and taste" and "nutritional composition" were the aspects that most influenced the food choices of both female and male students, whereas "ecology, environment and animal welfare" was statistically more important among women (Table 3). 3.3. Food Concerns The food-related aspects that most concerned the students, according to the frequency of "worried" and "very worried" responses, were plastic use (78.0%), food waste (77.7%), and fish contamination (74.0%), the latter being statistically higher among the FST students (kh2 = 17.40, p = 0.002). Those of least concern were fattening (23.0%), genetically modified products (25.3%), and additives (colorants, preservatives, and flavorings) (32.0%), the latter being statistically higher among the HND students (kh2 = 12.05, p = 0.017) . Additionally, compared to the HND students, the FST students showed statistically higher levels of concern for hygienic conditions outside of home (kh2 = 15.28, p = 0.004), hygienic conditions at home (kh2 = 10.86, p = 0.028), contamination by bacteria (kh2 = 17.26, p = 0.002), concentration of pollutants such as mercury and dioxins (kh2 = 14.30, p = 0.006), mad cow disease (BSE) (kh2 = 14.30, p = 0.006), animal cloning (kh2 = 12.76, p = 0.012), and allergic reactions (kh2 = 9.48, p = 0.050). Conversely, the HND students showed statistically higher levels of concern for obesity (kh2 = 10.57, p = 0.032). The analysis revealed a decreasing level of concern in the more advanced students of the HND degree for most of the aspects and a transformation in concerns, although only two differences were significant. There was a reduction in concern about the concentration of pollutants: in the first year, 74.5% of the students were worried or very worried, as opposed to 41.9% of the fourth-year students (kh2 = 17.95, p = 0.022). Concern about additives used in food or beverages also decreased: 57.5% of the first-year students were worried or very worried, compared to only 18.6% from the fourth year (kh2 = 22.07, p = 0.005). Similarities were observed between the HND and FST students, but the trends in the latter were less linear. It is notable that the issue of additives was frequently mentioned by students in the focus groups, especially by the FST students, who described becoming less concerned after beginning their academic training: "The issue of additives has changed a lot, because I used to be one of those people who went to supermarkets and saw a product with additives and others without additives, and bought the one without additives, but now I understand that if they are there, it's for a reason. And not only that they are there for a reason, but that sometimes it is better that they are there than not" (Eric, FST). "Before I started the degree, I regarded it as something more negative, and [now] I understand that it's very regulated, that we have a lot of food legislation and we know that in the European Union, EFSA is regulating it" (Elena, FST). Women showed higher levels of concern for almost all aspects analyzed . Their main cause for concern was the use of plastics and plastic packaging (82.1%, sum of "concerned" and "very concerned"), which was statistically higher than in men (kh2 = 17.89, p < 0.001), followed by food waste (77.5%) and animal welfare (75.1%), also statistically higher (kh2 = 10.98, p = 0.027), and chronic non-communicable diseases (75.1%). In the case of animal welfare, the results corroborated the data obtained in the focus groups. The main issue of concern for men was food waste (77.6%), followed by contamination by bacteria, the concentration of contaminants such as mercury and dioxins, contamination in fish (all 70.7%), and unhygienic conditions at home (65.5%). Statistical differences, always with higher indices for women, were also found for hygienic conditions outside home (restaurants, shops, etc.) (kh2 = 10.56, p = 0.032) and animal cloning (kh2 = 10.83, p = 0.028). 3.4. Perceptions of a Sustainable Diet The three most important aspects for a diet to achieve sustainability were judged to be, in order of importance, not wasting food, consuming Km0 or local products, and consuming fresh and seasonal products. Most of the other elements were chosen by less than 5% of the students of either degree (Table 4). Using biodegradable or compostable materials was selected significantly more frequently by the FST students. In the HND degree, statistically significant differences were found in the conceptions of a sustainable diet among students of different years. New students attributed more importance to consuming organic products (9.2%, kh2 = 27.51, p < 0.001), while this aspect almost disappeared among the second-, third-, and fourth-year students (<0.5%). Additionally, there was an increase in the importance attached to "not wasting food" (17.0%, 24.0%, 26.4%, kh2 = 9.01, p = 0.011) among the more advanced students. No significant differences were observed between the years in the FST students. Male and female participants shared similar conceptions of what is necessary to achieve a sustainable diet. However, following a Mediterranean diet was significantly more important for male participants, as was following a vegetarian diet and/or reducing the consumption of animal products. This difference was notable, as female students attached more value to the principles of a vegetarian diet and were more against animal products in all other questions of the questionnaire. 4. Discussion Understanding social perceptions of sustainability is critical for guiding a transition towards more sustainable diets . Although studies on perceptions of food sustainability among young people and university students have been carried out , to the best of our knowledge, this is the first among college students in the field of food science in Spain. This population is of great importance due to their potentially influential role in food sustainability practices in the future . Based on their skills, curriculum, and involvement in food and nutrition, they may act as promoters of climate change mitigation and other strategies vital for planetary health . Food technologists, for example, can promote more sustainable practices along the whole food chain to produce food in a more sustainable way. Dietitians can make food recommendations taking into account the principles of sustainability, such as advising the consumption of fresh, seasonal, and local products, as well as promoting the use of methods to reduce food waste. Regarding the participant characteristics, the average age was similar to that of other studies with university students . Focusing on a sample of younger individuals provides insights into the construction of knowledge, and access is gained to environments where certain trends circulate. Notably, this generation of students grew up in the context of increasing environmental, social, and ethical awareness of issues related to food and the climate crisis . A statistical difference between genders was expected, as HND students are predominantly women . 4.1. Social Perceptions Related to Food and Sustainability Understanding perceptions of food and food choices is complex because of the diversity of factors involved, but also crucial, considering the impact of food on sustainability . The free-association task revealed that the students mainly associated food with hedonic and nutritional dimensions. When describing their own eating practices and what influences their food choices, the students attached far more importance to pleasure, nutrition, and health than aspects related to sustainability. In the qualitative phase of the study, almost all participants directly or indirectly mentioned taste and/or food preferences as the main aspects conditioning their dietary practices. Compared with the FST students, the HND students were more likely to define food using words related to pleasure and taste, and they also regarded their diet as healthier. Additionally, the food choices of the HND students were more influenced by "disease prevention and health effects". These differences may be associated with the profile of each bachelor's degree, as the HND students receive training more "holistic", less technical, but also more focused on health and healthy eating issues. The central role of pleasure and taste in the students' relationship with food is not surprising, as the same perception is found among the general Spanish population and those who cook in Spanish households , and fundamentally guides food choices in Spain . These results also corroborate data obtained on a European level, which reveal that Europeans prioritize taste, food safety, and cost over sustainability . However, it is noteworthy that pleasure and taste seemed to become less important for more advanced students, indicating that their food perceptions may have been changed by their training. In the contemporary context of the medicalization of food , the association between food, health, and nutrition is becoming increasingly internalized by the general population in different contexts , especially among individuals from privileged social classes and women . As these groups comprise the majority of the study sample, the observed association of food with health and nutrition could be expected. Available data indicate that the overall Spanish population attaches a similar importance to these aspects rather than those associated with sustainability . In the case of food science students, the relationship between food and nutrition/health may be even stronger given their academic curriculum. A study of Brazilian, Spanish, and French dietitians reported that their training increased concern for health and nutrition . In the present study, the qualitative and quantitative analysis revealed a similar transformation in students as they progressed in their degrees. A low percentage of students signaled that the effects on body shape were a dietary choice criterion, and gaining weight was the element that least concerned the participants. In the focus groups, only one female participant revealed that she dieted with the aim of changing her body. This result is perhaps surprising, considering that young people, especially females, are more likely to suffer body image dissatisfaction and go on slimming diets . This tendency may be higher among HND students, who are reported to associate their profession with a thin body model , and to be at higher risk of eating disorders . The main food-related concerns expressed by the students were issues associated with sustainability, such as the use of plastic and plastic packaging, food waste, and contamination of fish. Therefore, while sustainability was not prominently associated with dietary practices or given as a reason for food choices, it was the subject of a high level of concern when considered a separate category. These results indicate a discrepancy between concerns and practices, the latter being more influenced by aspects such as pleasure and taste. Other studies have also shown that environmental worries are not necessarily translated into more sustainable practices . These data may suggest that sustainability, as a dimension of food practices or a significant factor in the relationship with food, is in transition and is not yet a hegemonic discourse among the study participants. The concerns expressed by the students were also observed in a Eurobarometer survey in which contamination of fish, meat, or dairy products featured prominently. However, other concerns raised by European and Spanish populations were not important for the student cohort, such as the use of pesticides on fruits and vegetables, the use of additives, or the presence of antibiotics and hormones . Although both degrees studied by the participants were in the field of food science, they differed in the type of training and professional activity involved. This was reflected in the higher concern shown by FST students for hygiene and contamination issues, whereas HND students attached more importance to chronic diseases and obesity. The results of this study indicate considerable consensus on what constitutes a sustainable diet. Among students of both degrees, regardless of gender, the aspects most frequently chosen were "not wasting food", "consuming Km0 or local products," and "consuming fresh and seasonal products", all of which receive extensive coverage in the Spanish media. Sustainability was essentially associated with environmental issues, with few students considering aspects related to social and economic dimensions, such as "consuming fair trade products" or "buying products directly from the producer". These results corroborate those of other studies in various countries, including Spain , which report that common perceptions of a sustainable diet do not encompass the complexity and multidimensionality of the concept , as defined by the FAO. Compared with the first-year students, the more advanced students did not show a significantly more holistic conceptualization of food sustainability. Certain trends were observed; for example, fourth-year students attached less importance to consuming organic food and were more concerned about food waste but showed little awareness of the relevance of sociocultural aspects of sustainability. In a study in Australia, Burkhart et al. (2020) analyzed the level of familiarity of nutrition students with concepts related to food sustainability and found that they were largely unaware of its association with social development, economic resilience, and cross-cutting issues. The results were considered unsurprising, given the priority assigned by the media to environmental aspects of sustainability and the lack of emphasis placed by nutrition training on social, economic, and political issues. Therefore, the promotion of a more global, critical, and complex view of sustainability in this field would be desirable. In the present study, the aspects the students most associated with a sustainable diet are in line with the conceptions held by the general population and are the result of contemporary phenomena (e.g., industrialization, urbanization, globalization) that generate certain perceptions and concerns regarding food . The element considered most important was "not wasting food", which increased in significance for more advanced HND students. Food waste is gaining prominence in political, media, and academic discourse as a key issue in the contemporary food system . Indeed, reducing food waste constitutes one of the targets of the United Nations Sustainable Development Goals (Goal 12). In Spain, both the Spanish state and the autonomous community of Catalonia have passed laws in the last two years aimed at reducing food waste by 50% throughout the food chain by 2030. Although people in Spain are making efforts to apply measures to reduce food waste , three out of four Spanish households in 2020 were still wasting food to some extent . Verdugo et al. (2020) analyzed food waste among Spanish university students and found that it corresponds to 14.5% of the food on a plate. In the present study, the food science students at least showed awareness of the problem. Therefore, even though there may be discrepancies between the norms/perceptions held by this population and their actual practices , the results could be regarded as promising in terms of achieving waste reduction targets in the future. The second most important aspect of a sustainable diet was judged to be "consuming Km0 or local products". In different cultural contexts, including Spain, local products are being increasingly valued, and are associated with trust, good quality, and health . The TNS Sofres survey (2014) shows that in Spain, the three elements that matter the most in consumer evaluation of food quality are, in order of importance, the origin and place of production, product appearance, and place of purchase. The growing interest in local products is a reaction to the transformations wrought by food modernity, marked by an industrial food system that weakens the links between food and territory and between consumers and food . "Eating local" is an attempt to return to the traditional ways and know-how that individuals are afraid of losing in a globalized society , and a means of rediscovering a sense of security regarding modern food . Interestingly, in their perceptions of what is needed to eat sustainably, the students placed a great deal of importance on where the product comes from, but this aspect was weakly associated with their food choices, confirming the discrepancy between perceptions/norms and practices. Finally, the third most important aspect associated with a sustainable diet was "consuming fresh and seasonal products". As pointed out by other studies, these two characteristics may also be associated with the valuation of proximity and natural and artisanal products, as opposed to what is perceived as industrialized, transformed, chemical, toxic, and coming from a distant and unknown territory . Food freshness is valued by the Spanish population as an attribute of food quality and a healthy diet . A Eurobarometer survey of the public perception of food risks found that freshness is the most important food concern among Spanish consumers . According to Garcia-Gonzalez et al. (2020) , an "abundance of fresh products" is the factor the Spanish most relate with a sustainable diet. 4.2. Social Perceptions According to Gender Gender analysis revealed that the topic of food sustainability may be more internalized by women than by men. Female participants were statistically more likely to describe their diet as vegetarian/vegan and they had a greater tendency to regard their dietary practice as responsible and socially committed or ecological and natural. The analysis of the aspects influencing food choices also showed that "ecology, environment, and animal welfare" was more important for women than men. In the analysis of food-related concerns, women expressed higher levels of concern for most of the proposed elements, including those directly associated with sustainability. In almost all the questions in the questionnaire and in the focus group discussions, women were more appreciative of vegetarian diets and concerned about animal welfare, in agreement with other studies that report a higher prevalence of vegetarianism in the female collective . In Spain, more than two out of three vegetarians are female . One of the multiple motivations for vegetarianism is the impact food has on the environment . Russel et al. (2021) described vegetarianism as having a positive influence on sustainability attitudes and behaviors, including a preference for foods grown with sustainable agricultural practices. In a study of dietitians, those who were vegetarian were more likely to be involved in activities that promote climate change mitigation . This differential attitude between female and male participants may be related to historically constructed gender roles. From childhood, through the socialization process, men, and women incorporate different norms, values, and roles that shape their female and male identities, as well as their actions in society . Empathy and caring, especially through food, are values that are more associated with femininity. Women also have a greater tendency to seek information about food and nutrition and to undertake more diets . Furthermore, there is a "gendered" division of food arising from socially constructed gender roles: meat, especially red meat, is associated with masculinity, whereas vegetables are associated with femininity . Therefore, women are more likely to be concerned about aspects related to the environment, animal welfare, and health, as found in the present study. Garcia-Gonzalez et al. (2020) also verified that Spanish women attach more importance than men to the sustainability of the food they buy. Finally, it is noteworthy that although men and women differed in their perceptions of sustainability issues, a more homogeneous discourse emerged regarding the actions required for a sustainable diet, revealing a generalized perception and strong consensus on this topic. Despite the relevance of this work, especially in Spain, some methodological limitations should be emphasized. First, this study was carried out with students from a single academic institution in the Barcelona region, so it would be of interest to expand the sample to include other institutions and geographical contexts. Likewise, the convenience sampling of the research may entail a risk of bias because the participants may share a certain profile or interests related to the topic. Also, the qualitative phase of the study was based only on two focus groups, and more in-depth data could have been obtained with semi-structured interviews. The authors should discuss the results and how they can be interpreted from the perspective of previous studies and of the working hypotheses. The findings and their implications should be discussed in the broadest context possible. Future research directions may also be highlighted. 5. Conclusions This is the first study to analyze perceptions related to food sustainability among college students in the field of food science in Spain. Understanding social perceptions among this group is crucial to produce knowledge that can be applied to improve their academic training, to foster a critical perspective of the food system, and to promote sustainability. Although the students expressed concern about sustainability-related issues, at least in their conceptions, their eating practices were mainly associated with or influenced by taste/pleasure, health, and nutrition. Gender differences were identified, showing that the topic of food sustainability may be more internalized by women than men. Regarding the question of what constitutes a sustainable diet, the generalized view of the student population, regardless of the degree studied or gender, was that sustainability is primarily associated with environmental aspects (not wasting food, consuming Km0 or local products, and consuming fresh and seasonal products), with social and economic dimensions occupying a minor role. Awareness of food sustainability issues was not significantly higher among the more advanced students compared to those in the 1st year, indicating their perceptions had not been changed by training. Therefore, as academic background influences professional practices, there is a need to promote the concept of sustainability in all its complexity and multidimensionality among food science students. This lack of holistic conceptualization also calls for the development of actions that bring sustainability closer to the social practices of students. Academic training is a privileged space for the implementation of strategies that pursue these objectives. Given that many of the students are likely to find employment in different sectors of the food system, food sustainability should be discussed during their training in a more holistic, transdisciplinary, and intersectoral way. To adequately address food sustainability in university curricula, the teaching staff also require continuous training . Additionally, building more synergies with social science disciplines would allow students to incorporate a more complex view of diet and sustainability. Finally, initiatives that bring academic practices closer to sectors outside the university, especially non-profit organizations or public policies, would be of great interest. Acknowledgments The authors thank Laura Arciniegas for her support in different stages of the research. Supplementary Materials The following supporting information can be downloaded at: Questionnaire designed for the study on the food perceptions among college students of Human Nutrition and Dietetics and Food Science and Technology. Click here for additional data file. Author Contributions M.C.d.M.P.G., M.A., M.C.G., C.L.-K. and M.C.V.-C.: conceptualization. M.C.d.M.P.G., C.S., M.A., M.C.G., R.C.-S., O.C.-B., C.L.-K. and M.C.V.-C.: investigation. M.C.d.M.P.G., C.S., M.A., M.C.G., R.C.-S., O.C.-B. and C.L.-K.: data analysis. M.C.d.M.P.G., C.S., M.A., M.C.G., R.C.-S., O.C.-B., C.L.-K. and M.C.V.-C.: writing--original draft preparation. M.C.d.M.P.G., C.S., M.A., M.C.G., R.C.-S., O.C.-B., C.L.-K. and M.C.V.-C.: writing--review and editing. C.L.-K. and M.C.V.-C.: supervision. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The study was conducted in accordance with the Declaration of Helsinki and was approved by the Bioethics Commission of the University of Barcelona, protocol number IRBOC003099 (2020). Informed Consent Statement Informed consent was obtained from all subjects involved in the study. Data Availability Statement Not applicable. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Word cloud based on associations with food. Larger font means higher frequency of mentions. Different colors here are used for better clarity of the different words, they don't follow a specific pattern. Figure 2 Self-assessment of eating practices: the level of agreement with different diet typologies among college students pursuing bachelor's degrees in Human Nutrition and Dietetics (HND) and Food Science and Technology (FST), showing the distribution (%) of responses. Figure 3 The level of agreement with different dietary typologies in the student dietary self-assessment according to gender, showing the distribution (%) of responses. Figure 4 The level of concern for different food-related issues among college students pursuing bachelor's degrees in Human Nutrition and Dietetics (HND) and Food Science and Technology (FST), showing the distribution (%) of responses. Figure 5 The level of concern for different food-related issues among college students pursuing bachelor's degrees in Human Nutrition and Dietetics (HND) and Food Science and Technology (FST) according to gender, showing the distribution (%) of responses. foods-12-00917-t001_Table 1 Table 1 General characteristics of the college students enrolled in bachelor's degrees in Human Nutrition and Dietetics (HND) and Food Science and Technology (FST). Total (n = 300) n (%) HND (n = 151) n (%) FST (n = 149) n (%) Year of training 1st 96 (32.0%) 47 (31.1%) 49 (32.9%) 2nd/3rd 120 (40.0%) 61 (40.4%) 59 (39.6%) 4th 84 (28.0%) 43 (28.5%) 41 (27.5%) Gender Female 241 (80.3%) 132 (87.4%) 109 (73.2%) Male 58 (19.3%) 18 (11.9%) 40 (26.8%) Other 1 (0.3%) 1 (0.7%) 0 Household income <999 EUR/month 22 (7.3%) 15 (9.9%) 7 (4.7%) 1.000-1.999 EUR/month 100 (33.3%) 38 (25.2%) 62 (41.6%) 2.000-4.999 EUR/month 158 (52.7%) 85 (53.3%) 73 (49.0%) >5.000 EUR/month 20 (6.7%) 13 (8.6%) 7 (4.7%) Parental level of education Elementary school 9 (3.0%) 7 (4.6%) 2 (1.3%) Secondary school 31 (10.3%) 14 (9.3%) 17 (11.4%) Sixth form 88 (29.3%) 46 (30.5%) 42 (28.2%) Bachelor's degree 140 (46.7%) 70 (46.4%) 70 (47.0%) Master's degree 32 (10.7%) 14 (9.3%) 18 (12.1%) Employment situation Employee 92 (30.7%) 53 (35.1%) 39 (26.2%) Internships 30 (10.0%) 20 (13.2%) 10 (6.7%) Unemployed 178 (59.3%) 78 (51.7%) 100 (67.1%) foods-12-00917-t002_Table 2 Table 2 Frequency (%) of word categories associated with "food" by the college students enrolled in bachelor's degrees in Human Nutrition and Dietetics (HND) and Food Science and Technology (FST) through the free-association task. * Indicates statistically significant differences in responses between collectives. Total Bachelor's Degree Gender NHD (%) FST (%) kh2 p Value Female (%) Male (%) kh2 p Value Food (e.g., eat, food product) 28.1 19.3 36.9 11.66 0.001 * 28.7 25.9 0.177 0.674 Hedonic and gustatory dimension (e.g., pleasure, tasty, enjoy, happiness) 35.1 41.3 28.9 4.91 0.026 * 34.6 37.9 0.250 0.617 Health 5.7 8.0 3.4 2.95 0.085 6.3 3.4 0.672 0.412 Nutrition/nutrients (e.g., nutrition, energy, nutritious) 13.7 12.0 15.4 0.78 0.375 12.9 17.2 0.757 0.384 Vital aspect/need (e.g., hunger, need, survival) 12.7 12.7 12.8 0 0.964 12.1 13.8 0.134 0.715 Other (e.g., stress, habit) 4.7 6.7 2.7 2.61 0.105 5.4 1.7 1.41 0.235 foods-12-00917-t003_Table 3 Table 3 Aspects influencing food choices of college students pursuing bachelor's degrees in Human Nutrition and Dietetics (HND) and Food Science and Technology (FST), showing the distribution of responses. * Indicates statistically significant differences in responses between collectives. Aspects Influencing Food Choices Total (%) Bachelor's Degree Gender HND (%) FST (%) kh2 p Value Female (%) Male (%) kh2 p Value Nutritional composition of foods 24.5 26.8 22.1 2.62 0.105 23.2 30.2 3.60 0.058 Eating/preparation facility 8.0 7.3 8.7 0.463 0.496 7.5 10.3 1.15 0.284 Time of availability 5.5 6.0 5.0 0.263 0.608 6.2 2.6 2.52 0.112 Price 6.7 7.3 6.0 0.402 0.526 6.0 9.5 1.94 0.164 Concern for body image 5.2 3.6 6.7 3.05 0.081 4.4 7.8 2.40 0.122 Ecology, environment, and animal welfare 8.4 7.3 9.4 0.963 0.327 9.8 2.6 6.89 0.009 * Place of provenance/origin 3.4 4.0 2.7 0.801 0.371 3.7 1.7 1.21 0.271 Pleasure and taste preference 25.9 26.5 25.2 0.210 0.647 26.3 24.1 0.366 0.545 Disease prevention/health effects 5.2 7.0 3.4 4.19 0.041 * 5.6 3.4 0.933 0.334 State of mind 4.5 1.3 7.7 14.97 0.000 * 3.9 6.0 1.03 0.310 Composition of foodstuffs with respect to chemical additives (preservatives, etc.) 3.0 3.0 3.0 0.001 0.977 3.3 1.7 0.841 0.359 Observation: Participants could choose two response options. foods-12-00917-t004_Table 4 Table 4 Perceptions of what constitutes a sustainable diet among college students pursuing bachelor's degrees in Human Nutrition and Dietetics (HND) and Food Science and Technology (FST) according to gender and showing the distribution (%) of responses. * Indicates statistically significant differences in responses between collectives. Aspects That Constitute a Sustainable Diet Total (%) Bachelor's Degree Gender HND (%) FST (%) kh2 p Value Female (%) Male (%) kh2 p Value Consuming Km0 or proximity products 18.9 20.1 17.7 1.60 0.205 19.2 17.8 0.341 0.559 Consuming organic products 4.3 3.1 5.4 3.17 0.075 4.0 5.2 0.512 0.474 Not wasting food 22.5 22.5 22.4 0.006 0.936 21.7 25.3 2.44 0.119 Following a Mediterranean Diet 1.45 1.1 1.8 0.766 0.381 1.0 2.9 3.97 0.046 * Using biodegradable or compostable materials 9.0 7.3 10.7 4.08 0.043 * 8.9 9.8 0.180 0.672 Following a vegetarian diet and/or reducing consumption of animal products 10.0 11.3 8.7 2.06 0.151 1.1 5.7 5.66 0.017 * Consuming fair trade products 2.4 2.2 2.5 0.067 0.796 2.6 1.1 1.41 0.235 Reducing the consumption of industrial products 5.0 5.1 4.9 0.013 0.910 5.0 5.2 0.012 0.912 Being part of a consumer group/consumer cooperative 0.2 0.4 0 1.99 0.159 0.3 0 0.485 0.486 Shopping in the neighborhood market or stores 6.1 5.7 6.5 0.525 0.615 5.7 8.0 1.58 0.209 Growing/producing your own food 1.8 1.5 2.0 0.293 0.588 2.1 0.6 1.87 0.172 Buying products directly from the producer 1.7 1.8 1.6 0.057 0.812 1.7 1.7 0.004 0.952 Consuming fresh and seasonal products 16.9 17.9 15.9 1.08 0.299 16.9 16.7 0.007 0.932 Observation: Participants could choose three response options. 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PMC10000851 | Healthcare (Basel) Healthcare (Basel) healthcare Healthcare 2227-9032 MDPI 10.3390/healthcare11050665 healthcare-11-00665 Case Report Effect of Three Weeks of High-Intensity, Long-Term Preoperative Rehabilitation for Esophageal Cancer Patients with Stroke Sequelae Who Were Considered Unfit for Surgery Due to Low Activity: A Case Report Kinoshita Tokio 12 Nishimura Yukihide 3* Zaiki Rikito 12 Yasuoka Yoshinori 12 Umemoto Yasunori 1 Koike Yumi 12 Kawanishi Makoto 12 Tajima Fumihiro 1 1 Department of Rehabilitation Medicine, Wakayama Medical University, Wakayama 641-8509, Japan 2 Division of Rehabilitation, Wakayama Medical University Hospital, Wakayama 641-8510, Japan 3 Department of Rehabilitation Medicine, Iwate Medical University, Shiwa-gun 028-3639, Japan * Correspondence: [email protected] 24 2 2023 3 2023 11 5 66526 1 2023 09 2 2023 21 2 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). Treatment of esophageal cancer is based on tumor-node-metastasis (TNM) classification, and surgical treatment is chosen based on the patient's ability to tolerate surgery. Surgical endurance partly depends on activity status, with performance status (PS) generally used as an indicator. This report describes a 72-year-old man with lower esophageal cancer and an 8-year history of severe left hemiplegia. He had sequelae of cerebral infarction and a TNM classification of T3, N1, and M0, and was judged ineligible for surgery because his PS was grade three; he underwent preoperative rehabilitation with hospitalization for 3 weeks. He had been able to walk with a cane in the past, but once he was diagnosed with esophageal cancer, he began using a wheelchair and was dependent on assistance from his family in his daily life. Rehabilitation consisted of strength training, aerobic exercise, gait training, and activities of daily living (ADL) training for 5 h a day, according to the patient's condition. After 3 weeks of rehabilitation, his ADL ability and PS improved sufficiently for surgical indication. No complications occurred postoperatively, and he was discharged when his ADL ability was higher than that before preoperative rehabilitation. This case provides valuable information for the rehabilitation of patients with inactive esophageal cancer. esophageal cancer preoperative rehabilitation postoperative complications performance status chronic cerebrovascular disease This research received no external funding. pmc1. Introduction Treatment plans for patients with esophageal cancer are determined in accordance with the Japanese guidelines based on tumor-node-metastasis (TNM) classification . In cases of category 3 tumors of the TNM classification, the indication for a treatment plan is determined based on the presence or absence of surgical tolerance in our hospital. Although a clear standard for surgical tolerance has not been established, it is determined based on activity status in addition to cardiopulmonary, renal, liver function, and glucose tolerance. Performance status (PS) is a commonly used index for evaluating activity status , and PS 0-2 (the patient is ambulatory and capable of self-care but unable to perform work activities and is active more than 50% of waking hours) is considered desirable for surgical indications, in terms of decreasing the occurrence of postoperative complications . However, if the amount of activity is lower than that of PS 3 (capable of only limited self-care and confined to a bed or chair for more than 50% of waking hours), chemoradiotherapy and palliative symptomatic therapy are often chosen instead of surgery (Table 1). The reported incidence of postoperative pulmonary complications associated with esophagectomy ranges from 30-60% , and has been shown to increase mortality, prolong hospital stay, and present additional medical costs . While numerous studies have shown that preoperative rehabilitation helps prevent postoperative complications , it is also important for improving and maintaining patients' activities of daily living (ADL) and activity before deciding on a treatment plan and proceeding with the treatment. Patients with cerebrovascular disorders suffer from a wide range of sequelae, including higher brain dysfunction, motor paralysis, and sensory impairment, depending on the site of brain damage . This results in a long-term decline in ADL ability and activity . Preoperative rehabilitation for patients who are not eligible for surgery due to low activity tolerance, or for patients with esophageal cancer complicated by cerebrovascular disease, has not yet been reported. This case report describes a patient with esophageal cancer with sequelae of cerebral infarction and a TNM classification of T3, N1, and M0, who was determined to not be eligible for surgery because his PS was 3. Instead, he underwent preoperative rehabilitation with hospitalization for 3 weeks. This is a detailed description of the patient's rehabilitation until his PS improved and surgery became possible, as well as the course of his rehabilitation until discharge after surgery. 2. Case Report The patient was a 72-year-old man with esophageal cancer. He visited his primary care physician complaining of esophageal transit disturbance, and was diagnosed with lower esophageal cancer by upper gastrointestinal endoscopy and computed tomography (TNM classification: T3, N1, M0). The patient had a cerebral infarction 8 years prior, with left hemiplegia as an aftereffect. However, he was able to walk independently with the use of a cane and could perform ADLs independently. After being discharged from the hospital, the patient did not undergo outpatient rehabilitation or exercise on his own in order to prevent the deterioration of physical function. Thereafter, his activity gradually declined with age, and at the time of his diagnosis of esophageal cancer, he used a gait aid and a lower limb brace to walk around at home and a wheelchair for outdoor mobility. The patient was not in the habit of performing exercise and spent > 50% of the day in bed. The patient's PS was 3 at the time of diagnosis, and after a comprehensive review of his operative tolerance, surgery was not indicated. However, the gastroenterological surgeon consulted with the Department of Rehabilitation about whether PS could be improved through rehabilitation, and the Department of Rehabilitation began to consider it. At the time of the examination in the Department of Rehabilitation, the Glasgow Coma scale was E4V5M6 and the patient's verbal communication was good. A cranial nerve examination revealed mild drooping of the left angle of the mouth due to facial nerve palsy. The National Institutes of Health Stroke Scale results were as follows: 1 point for "Level of consciousness commands," 4 points for "Motor arm", 3 points for "Motor leg", and 1 point for "Sensory," for a total of 10 points . The Manual Muscle Test (right/left) results were as follows: deltoid 5/0, biceps 5/0, extensor carpi radialis longus 5/0, triceps 5/0, flexor digitorum profundus 5/0, abductor pollicis minor 5/0, iliopsoas 4/2, quadriceps 4/3, tibialis anterior 4/0, extensor digitorum longus 4/0, and triceps muscle of calf 4/0 . Range of motion was -5deg in the left knee joint during extension and 5deg in the left ankle joint during dorsiflexion, with no obvious restrictions in the other joints. Sensory examination revealed mild blunting of the superficial and deep senses. However, no new acute neurological deficits appeared in the patient. In addition, PS, functional independence measure (FIM), cardiopulmonary exercise testing (CPET), the 6-Minute Walk Test (6 MWT), the 10-m walking test (10 MWT), hand-held dynamometer (HHD) assessments, and body weight were also evaluated. The FIM is an independent rating scale for ADLs, consisting of 13 motor and 5 cognitive items, each of which is rated on a scale of 1 to 7 points, with higher scores indicating better ADL ability. The maximum score is 91 for motor items and 35 for cognitive items, with a maximum total score of 126 . CPET is a well-established test for exercise tolerance that analyzes exhaled gases . In this case, measurements were taken with a bicycle ergometer exercise load and an AERO AE300S (MINATO MEDICAL SCIENCE CO., LTD.) instrument. Exhaled gas analysis was performed using the breath-by-breath method, and peak VO2 max was measured. The 6 MWT measured a 6-min walking distance with maximal effort, as indicated by the American Thoracic Society . For the 10 MWT, the shortest time to walk 10 m was measured three times, and the average value was calculated . For the 6 MWT and 10 MWT, the patient walked with a cane while wearing a lower limb orthosis. To determine maximum knee-joint extension muscle strength on the left and right sides, HHD was measured three times using m-tas F-1 (ANIMA Co., Tokyo, Japan) and the average value was calculated . The patient was given light assistance due to the risk of falling. Results obtained at the time of examination in the Department of Rehabilitation are shown in Table 2 and Table 3. The PS was 3, the total FIM score was 97 points, the motor score was 62 points, and the cognitive score was 35 points. The ADLs that showed particularly low ability were lower body dressing, toileting, bed-to-chair transfer, toilet transfer, shower transfer, locomotion, stairs, and other movements that mainly affected lower extremity function. VO2 max in CPET was 821 mL/min and 14.1 mL/kg/min; 6 MWT was 105 m; and 10 MWT was 33.2 s. Lower limb muscle strength in the HHD assessment was 80 N in the right leg and 42 N in the left leg. The patient's PS was thought to have declined due to the loss of muscle strength in the healthy side of the lower limb and the impaired mobility caused by severe left hemiplegia. However, since the patient had previously been able to walk with a cane, the paralyzed leg was probably strongly affected by muscle weakness due to disuse, which was also apparent in the healthy leg muscles; therefore, it was judged that there was sufficient room for improvement through rehabilitation, and rehabilitation was started on the same day. The main objective of rehabilitation was to improve PS by increasing muscle strength in both lower limbs and improving ADL. In addition, since the patient's VO2 max was 14.1 mL/kg/min, which is low enough for a concerning high risk of postoperative complications, we aimed to improve VO2 max as much as possible before surgery. This hospital provides high-intensity, long-duration rehabilitation therapy tailored to the patient's general condition . After admission, patients undergo approximately 2 h and 30 min of preoperative rehabilitation twice a day: once in the morning and once in the afternoon. The preoperative rehabilitation period lasts 22 days and includes 44 sessions of rehabilitation. During the initial rehabilitation, we use a pamphlet prepared by the Department of Rehabilitation Medicine to instruct the patient on deep diaphragmatic breathing, efficient coughing and huffing with intense contraction of the abdominal muscles, as well as the rehabilitation process from preoperative to postoperative discharge . Muscle strengthening exercises include squats (300 times/session), calf raises (100 times/session), and step ascents and descents using a 20-cm step (100 times/session) . Aerobic exercise consists of bicycle ergometer exercise (30 min/session) and hand ergometer exercise (20 min/session). The exercise load is set at 70-80% load using the heart rate (HR) reserve method, with peak HR measured by CPET . The physical therapist mainly directs repetitive gait training, stair climbing, and ADL training. Muscle strength training and aerobic exercises are also supervised by the physical therapist, but most of these are performed by independent trainers. The results of the 22-day preoperative rehabilitation are shown in Table 2 and Table 3. The FIM showed improvement in all items that had previously been declining, with a total score of 117. In addition, the patient became independent when walking with a cane and in all personal activities in the hospital. The patient's PS improved to 2 due to his increased independence in ADLs in the hospital. CPET results showed an increase in VO2 max of 959 mL/min and 16.6 mL/kg/min. Walking ability tests were performed without assistance from a physical therapist, as the patient was walking independently with a cane and wearing a brace. Consequently, his 6 MWT distance extended to 125 m, and his 10 MWT time decreased to 30.9 s. The HHD results also increased to 120 N and 82 N on the right and left sides, respectively. Due to this improvement in physical function, the indication for surgery was reconsidered at a preoperative conference of the Gastrointestinal Surgery Department. Therefore, the patient was judged to be operable, and surgery was subsequently performed. The surgical techniques performed were: thoracoscopic subtotal esophagectomy, thoracoscopic gastric tube creation, two-region lymphatic dissection, and reconstruction of the posterior sternal pathway gastric tube. The operation time was 8 h and 21 min, and the operative blood loss was 45 mL. The postoperative diagnosis was esophageal squamous cell carcinoma (T3, N1, M0, and stage III). Postoperatively, the patient was admitted to the intensive care unit for management. On the first postoperative day (POD), at 8:00 a.m. the patient was extubated from the ventilator and at 11:00 a.m. transferred to the general ward. Mobilization was performed at 11:30 a.m., and gait training began with assistance in cooperation with a nurse. Supplemental oxygen therapy was administered using a mask at a flow rate of 10 L/min. The patient had a right chest drain, a cervical drain, and a J-tube. . The patient was instructed to spend as much time as possible (6 h/day) in a chair-sitting position outside of daytime rehabilitation . After POD 5, the training location was moved from the ward to the rehabilitation room. While training in the rehabilitation room, the frequency and distance of gait training increased appropriately. Low-intensity muscle strength training was initiated. Supplemental oxygen therapy was discontinued for POD 7. Bicycle ergometer exercises were added to the training program , and the muscle strength training load was increased. The patient was eventually able to walk outdoors unassisted and was discharged home on POD 26. The results of the evaluation at discharge are shown in Table 2 and Table 3. At discharge, the patient had a PS of 2 and a total FIM score of 117, and he maintained an ADL ability equivalent to that of the day before surgery. CPET results were VO2 max of 980 mL/min and 17.8 mL/kg/min; 6 MWT was 128 m; 10 MWT was 26 s, showing improvement in comparison to his results on the day before surgery. There was no dysarthria or dysphagia after the CVA onset. Postoperatively, there was no hoarseness or obvious dysphagia; a jelly diet was started on POD 11. On POD 15, the diet was changed to a soft, bite-sized diet. In addition, there were no adverse events during rehabilitation from the preoperative period to discharge, and no postoperative complications occurred. After being discharged from the hospital, the patient was followed up by a gastroenterological surgeon once a month; there has been no recurrence, and the patient is living independently. In accordance with the local legislations and institutional requirements, ethical review and approval were not required for this case report. In order to publish any potentially identifiable images or data included in this article, a written informed consent was obtained from the patient and his family. This study conforms to all case report guidelines and reports the required information accordingly. 3. Discussion Most of previous reports on preoperative rehabilitation for patients undergoing esophageal cancer surgery have focused on preventing postoperative respiratory complications, atelectasis, and shortening hospital stay duration . This case report is the first to detail the preoperative-to-postoperative course and physical training of a patient with esophageal cancer. This patient was deemed ineligible for surgery due to a gradual decline in PS over the course of 8 years after a stroke, but became eligible for surgery after 3 weeks of intensive preoperative rehabilitation and was then discharged home. A previous study reported a preoperative rehabilitation program for the prevention of pulmonary complications consisting of respiratory rehabilitation, including respiratory muscle and thoracic stretching to increase pulmonary compliance, deep breathing and abdominal breathing training, sputum training, muscle strength training using weights, and 20 min of bicycle ergometer exercise, for approximately 60 min per day, respectively . In other reports, training consisted of respiratory rehabilitation, upper and lower limb and abdominal muscle strength training, and 15 min of ergometer exercise, for 40-60 min per day, respectively . Akiyama et al. employed 20-30 min of ergometer exercise at 60-70% of maximum HR, and 20 sets of 10-15 squatting exercises twice a day for 7 days . Since rehabilitation in this case aimed to improve ADL and PS, the patient was instructed to perform 300 squats, 100 calf raises, 100 step ascents and descents, 30 min of lower limb ergometer exercise at 70-80% of HR reserve, 20 min of upper limb ergometer exercise, and one set of ADL training, including gait training, in the morning and afternoon, for a total of approximately 5 h per day of long, high-intensity rehabilitation. Previous studies on the effects of rehabilitation on patients with chronic cerebrovascular disease have shown that 60 min of physical therapy, including gait training 4-5 times a week for 4 weeks, increases muscle strength in both paralyzed and nonparalyzed limbs . Endurance exercise, balance training, and resistance exercise were performed for 60 min, three times a week for 19 weeks, and improved walking ability, walking speed, 6-min walking distance, and muscle strength, especially in the paralyzed side of the lower limb . In a meta-analysis of acute, subacute, and chronic stroke patients, peak VO2, peak workload, walking speed, and walking endurance increased with only 20-40 min of endurance exercise 3-5 times per week for 10 weeks . Zaiki et al. found significant improvement in FIM in patients with chronic cerebrovascular disease who performed 75 min of rehabilitation twice a week for 1 year but no improvement in patients who performed only 50 min of rehabilitation, suggesting the importance of rehabilitation duration . Although the duration of preoperative rehabilitation was 3 weeks in this report, which was shorter than in previous studies, it is possible that the longer daily rehabilitation time and addition of high-intensity loads improved ADL earlier. In addition, the repetitive training for gait and mobility impairments directed by the physical therapist was thought to have contributed significantly to the improvement of ADL ability and PS. A review by Guinan et al. indicated that early mobilization after surgery in patients with esophageal cancer is important for enhancing recovery after surgery to maximize the increase in lung function, prevent gastroesophageal reflux, and prevent postoperative complications such as atelectasis due to basal ventilation/perfusion matching. Many reports also recommend frequent early postoperative activity starting on POD 1, with a wide variety of specific recommendations, but most agree that these activities should include sitting out of bed for at least 2 h and short walks (10 m/100-200 feet). Haines et al. reported that the type of surgical incision and time to early mobilization were independently associated with the occurrence of postoperative pulmonary complications and that patients who could not get out of bed on POD 1 were 3.0 times (95% confidence interval 1.2-8.0) more likely to have such complications. In addition, preoperative VO2 max correlated strongly with survival in patients with esophageal and gastric cancer , and preoperative peak VO2 was found to be higher in patients without postoperative complications than in those with postoperative complications . In this study, the patient was discharged home without any postoperative complications, and his ADL ability post-discharge has improved compared to that preoperatively; in addition, he can move around independently and perform personal activities without the assistance of his family. The patient was trained to walk more than 50 m with assistance the day after surgery with the assistance of nurses and was encouraged to sit in a chair for 6 h/day after walking to reduce the amount of time he spent lying in bed. Moreover, the patient's VO2 max was improved by preoperative rehabilitation, which may have helped prevent complications. In addition to the preoperative improvement in ADL ability, the patient's general condition did not deteriorate, and he did not experience any complications; this was due to the implementation of muscle strength training and aerobic exercises, as well as the gradual increase in exercise load, which enabled discharge home with better ADL ability than before preoperative rehabilitation had begun. This study reported one case only. The cerebrovascular disease patients have a wide range of sequelae and a varying disease severity; thus, it is difficult to generalize the content and course of rehabilitation before and after surgery. In some cases, two-stage surgery may be indicated. However, this report describes a patient with esophageal cancer who was judged to be ineligible for surgery due to low activity and tolerance for surgery but became operable after 3 weeks of intensive preoperative rehabilitation. The content and course of rehabilitation in this case may provide valuable information for patients with esophageal cancer and low activity. 4. Conclusions In some cases of esophageal cancer, providing 3 weeks of high-intensity prolonged preoperative rehabilitation to patients that are ineligible for surgery can improve ADL and PS to the point of surgical indication. Therefore, cancer patients who are judged to be inoperable owing to low activity may benefit from intensive rehabilitation in a hospital before deciding on a treatment plan. Acknowledgments The authors thank the nursing staff in the our hospital for supporting rehabilitation therapy. Author Contributions All listed authors have made a substantial, direct, and intellectual contribution to the work. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Ethical review and approval were not required for this case report in accordance with the local legislation and institutional requirements. Informed Consent Statement In order to publish any potentially identifiable images or data included in this article, a written informed consent was obtained from the patient and his family. This study conforms to all case report guidelines and reports the required information accordingly. Data Availability Statement The authors' raw data supporting the conclusions of this article will be made available upon reasonable request. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Preoperative rehabilitation. (A) Preoperative rehabilitation guidance was provided using a pamphlet prepared by the Department of Rehabilitation. (B) Muscle-strengthening exercises included climbing steps and stairs. (C) Cardiopulmonary exercise testing. Figure 2 Postoperative rehabilitation. (A) Walking down the hallway for the first time at 11:30 am after discharge from the intensive care unit (postoperative day 1). (B) The patient was instructed to spend as much time as possible during the day in a sitting-chair position (6 h/day) outside of rehabilitation (postoperative day 1). (C) The patient resumes bicycle ergometer exercise while on continuous infusion with an enterostomy (postoperative day 7). (D) The patient is able to walk outdoors unassisted before discharge (postoperative day 26). healthcare-11-00665-t001_Table 1 Table 1 Performance Status Scales. Score Description 0 Fully active, able to carry on all pre-disease performance without restriction. 1 Restricted in physically strenuous activity, but ambulatory and capable of performing a light or sedentary work (e.g., light housework or office work). 2 Ambulatory and capable of all selfcare but unable to carry out any work activities. Up and about more than 50% of waking hours. 3 Capable of only limited selfcare, confined to a bed or a chair for more than 50% of their waking hours. 4 Completely disabled. Cannot carry out any selfcare. Totally confined to a bed or a chair. This table was modified by the authors, based on a reference (in Japanese). healthcare-11-00665-t002_Table 2 Table 2 Medical records of patients in the present case study. On Admission Day before Surgery At Discharge PS 3 2 2 Total FIM 97 117 117 Motor subscale 62 82 82 Cognition subscale 35 35 35 Body weight (kg) 58.0 57.5 55.0 CPET VO2 max (mL/min) 821.0 959.0 980.0 VO2 max (mL/Kg/min) 14.1 16.6 17.8 6 MWT (m) 105.0 125.0 128.0 10 MWT (s) 33.2 30.9 26.0 HHD Right (N) 80.0 120.0 120.0 Left (N) 42.0 82.0 82.0 PS, Performance Status; FIM, Functional Independence Measure; CPET, Cardiopulmonary Exercise Testing; 6 MWT, 6-min walk test; 10 MWT, 10-m walk test; HHD, Hand-held dynamometer. healthcare-11-00665-t003_Table 3 Table 3 Changes in FIM during each period. On Admission Day before Surgery At Discharge FIM of the total 97 117 117 Motor subscale 62 82.0 82.0 Eating 6 7 7 Grooming 5 7 7 Bathing 5 6 6 Upper body dressing 5 6 6 Lower body dressing 4 6 6 Toileting 4 6 6 Bladder management 7 7 7 Bowel management 7 7 7 Bed to chair transfer 4 6 6 Toilet transfer 4 6 6 Shower transfer 4 6 6 Locomotion (ambulatory or wheelchair level) 4 6 6 Stairs 3 6 6 Cognition subscale 35 35 35 Cognitive comprehension 7 7 7 Expression 7 7 7 Social interaction 7 7 7 Problem solving 7 7 7 Memory 7 7 7 FIM, functional independence measure. FIM tallies 18 items for daily living, which are graded on a 7-point scale: 1, total assistance; 2, maximal assistance; 3, moderate assistance; 4, minimal contact assistance; 5, supervision or set-up; 6, modified independence; and 7, complete independence. The full score is 126 points, with a minimum score of 18 points. The higher the score, the more independent the patient is. 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PMC10000852 | Int J Environ Res Public Health Int J Environ Res Public Health ijerph International Journal of Environmental Research and Public Health 1661-7827 1660-4601 MDPI 10.3390/ijerph20053807 ijerph-20-03807 Article Research and Public Interest in Mindfulness in the COVID-19 and Post-COVID-19 Era: A Bibliometric and Google Trends Analysis Kwon Chan-Young Tchounwou Paul B. Academic Editor Department of Oriental Neuropsychiatry, Dong-Eui University College of Korean Medicine, 52-57, Yangjeong-ro, Busanjin-gu, Busan 47227, Republic of Korea; [email protected] 21 2 2023 3 2023 20 5 380717 1 2023 20 2 2023 20 2 2023 (c) 2023 by the author. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). Public and research interest in mindfulness has been growing, and the Coronavirus disease 2019 (COVID-19) pandemic seems to have accelerated this growth. This study was conducted to investigate the public and research interest in mindfulness in the context of COVID-19. The term 'Mindfulness' was searched in Google Trends, and data were collected from December 2004 to November 2022. The relationship between the relative search volume (RSV) of 'Mindfulness' and that of related topics was analyzed, and 'Top related topics and queries' for the search term 'Mindfulness' were investigated. For bibliometric analysis, a search was conducted in the Web of Science database. Keyword co-occurrence analysis was conducted, and a two-dimensional keyword map was constructed using VOSviewer software. Overall, the RSV of 'Mindfulness' increased slightly. The RSVs of 'Mindfulness' and 'Antidepressants' showed an overall significant positive correlation (r = 0.485) but a statistically significant negative correlation during the COVID-19 era (-0.470). Articles on mindfulness in the context of COVID-19 were closely related to depression, anxiety, stress, and mental health. Four clusters of articles were identified, including 'mindfulness', 'COVID-19', 'anxiety and depression', and 'mental health'. These findings may provide insights into potential areas of interest and identify ongoing trends in this field. mindfulness COVID-19 bibliometric analysis VOSviewer Google trends This research received no external funding. pmc1. Introduction Mindfulness, defined by Jon Kabat-Zinn as "paying attention in a particular way: on purpose, in the present moment, and non-judgmentally" , has been reported to be associated with a variety of psychological benefits . Mindfulness training has also been reported to benefit individuals with physical illnesses such as chronic pain, common cold, psoriasis, irritable bowel syndrome, and diabetes . According to an analysis of the 2012 National Health Interview Survey, 2.5% of American adults practice mindfulness meditation in their lifetime, which represents 5.7 million people . Structured mindfulness training programs such as mindfulness-based stress reduction and mindfulness-based cognitive therapy (MBCT) have contributed to the popularization of mindfulness . The popularity of mindfulness in modern society is related to increased stress and anxiety in modern society, as well as a growing public interest in well-being . The popularity of mindfulness is extending not only to individuals, but also to businesses and organizations (e.g., corporate mindfulness) . In addition, mindfulness training has recently become more popular in the general population, especially in the form of smartphone applications, in conjunction with information and communication technology (ICT) . Coronavirus disease 2019 (COVID-19) has highlighted the importance of mental health worldwide since the end of 2019. According to a comprehensive systematic review of the prevalence of mental health problems during the COVID-19 pandemic, the overall pooled prevalence of depression, anxiety, distress, and insomnia reached 31%, 32%, 41%, and 38%, respectively, among all types of target populations (e.g., general population, medical staffs, students, patients with COVID-19, and patients with other clinical conditions) . Importantly, mental health problems associated with the pandemic are not only limited to COVID-19 patients but also affect the general population, including vulnerable groups such as healthcare workers and people with chronic conditions . Moreover, recent observational studies suggest the long-term mental health sequelae of COVID-19, including post-traumatic stress disorder (PTSD), anxiety, and depression . Accordingly, in the era of COVID-19, mental health is considered to be a high public health priority . In the context of COVID-19, ICT has become increasingly popular as a coping tool during the pandemic . Advanced ICT services such as telepresence service, remote monitoring service, virtual visit, and environmental disinfection are designed to meet the needs of this pandemic and help individuals and clinicians . In addition, mindfulness-based approaches are considered to be beneficial in mitigating the long-term negative impact of the pandemic on human mental health . This is because mindfulness training cultivates an individual's ability to cope with the various and widespread stresses experienced in life, and this ability is needed in the COVID-19 or post-COVID-19 era . Additionally, mindfulness has the potential to be involved in addressing collective mental health challenges due to the COVID-19 pandemic, not only at the individual level, but also by engaging in social processes with collective mindfulness . Encouragingly, mindfulness combined with ICT has played a role, especially in improving mental health, during the COVID-19 pandemic . Moreover, some countries, such as South Korea, have been offering mind-body modalities, including mindfulness, through telemedicine services to the general population and individuals with COVID-19 during the pandemic . Bibliometric analysis is now firmly established as a scientific specialty, and new trends in topics of interest, competing groups, and possibilities for cooperation can be identified through the analysis . In other words, bibliometric analysis explores research topics of interest, uncovers and provides an overview of research trends, and promotes related research . Google Trends can be used to analyze health trends and measure public interest in topics of interest . Analysis of this database is also being utilized in other academic areas, not limited to health, including social science . This approach can be used to better understand public health behavior through big data analysis; thus, mindfulness during the COVID-19 pandemic may be an appropriate topic for analysis. In addition, in terms of big data utilization, Google Trends analysis has the potential to be used not only for simple monitoring but also for forecasting . Mindfulness, either by itself or in combination with ICT, is gaining popularity in the context of COVID-19, and this is not limited to the medical field. Therefore, investigating both research and public interest in mindfulness in the era of COVID-19 may help establish health policies and research directions in this field. The purpose of this study was to investigate the public and research interest in mindfulness in the context of COVID-19 by Google Trends and bibliometric analysis, respectively. The COVID-19 pandemic has put pressure on the public to regard health as a global public good , and public interest and dissemination of information in the public have become an important basis for health policy in the era of COVID-19 . Although the bibliometric analysis technique provides information limited to a pictorial view of the relevant fields, associated keywords, prominent authors, institutions, citing patterns, and global cooperation among authors, it is considered valuable as a tool to support future research directions and, thereby, future R&D decision-making . Therefore, investigating the public interest, as well as the research interest in mindfulness through this study, will provide a perspective on the value and research direction of mindfulness from the perspective of public mental health in the future. In particular, filling the gap between public interest and research interest will provide an important research direction for establishing policies to manage public mental health in the COVID-19 and post-COVID-19 era. 2. Materials and Methods 2.1. Google Trends Analysis 2.1.1. Data Sources Google Trends is a service that aggregates search terms used on Google's search engine, the results of which are anonymized and categorized. When comparing two or more search terms in this service, the relative search popularity can be compared within a range of 0-100 points (i.e., relative search volume [RSV]). An RSV of 100 means the highest number of searches during a given period, and an RSV of less than 100 is calculated as the proportion of the highest number of searches. For example, an RSV of 50 means half the number of searches compared to an RSV of 100. 2.1.2. Analytical Strategy Since Google Trends provides data from 2004 and this analysis was conducted in December 2022, the search period was December 2004 to November 2022, a total of 18 years. In addition to analyzing the entire period, the period was subdivided into 6 periods of 3 years each (i.e., Period 1: December 2004-November 2007; Period 2: December 2007-November 2010; Period 3: December 2010-November 2013; Period 4: December 2013-November 2016; Period 5: December 2016-November 2019; Period 6: December 2019-November 2022). The reason why the period was subdivided into three years is because Period 6 was a three-year period related to COVID-19. Specifically, as Period 6 is 3 years from the time severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was discovered, this analysis was able to examine changes in public interest in mindfulness before and after the COVID-19 era. Thus, the 3-year subdivision was expected to be useful for examining public interest in mindfulness related to COVID-19. To further investigate the longitudinal variation in public interest in mindfulness, four related topics were searched, including 'Mental health', 'Psychotherapy', 'Psychoactive substance', and 'Antidepressants'. In the case of 'Mindfulness', it was searched as a search term because it was not classified as a topic in Google Trends. The RSV trend lines of 'Mindfulness' and the four related topics were calculated for the entire period, and the degree of change was quantified based on the slope value. In addition, Pearson's correlation coefficient was calculated to analyze the correlation between the RSVs of the search results. The correlation coefficient was tested for statistical power with a 2-tailed test, and p < 0.05 was considered statistically significant. Finally, 'Top related topics' and 'Top related queries' for the search term 'Mindfulness' were investigated for each period (1st to 6th period), and the popularity of this search term according to the region was also investigated. The data collected from Google Trends were downloaded from the web page as csv format files, and statistical analysis and visualization were performed using Microsoft Excel 365 (Microsoft Corporation, Redmond, WA, USA) and SPSS version 18 (SPSS Inc., Chicago, IL, USA). Specifically, in the calculation of Pearson's correlation coefficient, RSVs of the search results were regarded as variables in the software SPSS. The correlation coefficient was calculated to identify the linear correlation between these variables, and the type was selected as Pearson. 2.2. Bibliometric Analysis 2.2.1. Data Sources The search database for bibliometric analysis was the Science Citation Index Expanded database and Social Sciences Citation Index database of the Web of Science Core Collection. The search terms used were determined by referring to previously published bibliometric studies in this field . Specifically, COVID-19 and related terms were searched in the title search field, and mindfulness was searched in the topic (i.e., title, abstract, or keywords) search field. In addition, the literature type was limited to "Article". The search queries were as follows: (TI = (COVID-19) OR TI = (Coronavirus disease 2019) OR TI = (COVID-2019) OR TI = (2019-nCoV) OR TI = (nCov-2019) OR TI = (SARS-CoV) OR TI = (Severe acute respiratory syndrome coronavirus 2) OR TI = (Novel Coronavirus)) AND (TS = (mindfulness*) OR TS = (mindful*)) AND DT = (Article). The search date was 10 January 2023, and there was no limitation on time span and language. The bibliographic information retrieved from the web page was downloaded as a "plain text" file in txt format with the option of "full record with cited references". No duplications were found among the documents retrieved. A total of 435 articles were included. The bibliographic data were extracted as full records with cited references. 2.2.2. Analytical Strategy For bibliometric analysis, VOSviewer version 1.6.18 (Centre for Science and Technology Studies, Leiden University, Leiden, The Netherlands) , a software that analyzes bibliometric data and visualizes networks, was used. This freely available software allows for easier understanding and analysis of network data by providing a visual representation of the complex linking structure of bibliographies. Additionally, the software helps identify key articles, authors, and institutions related to specific keywords and topics. The plain text file downloaded from the Web of Science Core Collection were opened and analyzed in this software. To understand research trends in this field, the most frequently cited articles were selected from the retrieved articles. The year of publication, study design, related clinical topics, target population, journal of publication, and total number of citations of the selected articles were analyzed. In addition, the number of publications in this field according to the country was analyzed. To investigate important research topics in this field and their changes over time, keyword co-occurrence analysis and overlay visualization were conducted. For the keyword co-occurrence analysis, a two-dimensional keyword map was constructed using VOSviewer software. The unit of analysis was 'all keywords', the counting method was 'full counting', and the minimum number of occurrences of a keyword was 'five'. In this map, the size of the node reflects the frequency of the keyword, and the weight of the connection line indicates the number of articles in which keywords co-occur. Clustering was performed at a resolution of 1.00 with a minimum cluster size of 40, and individual clusters were differentiated by colors including red, blue, yellow, and green. The two-dimensional keyword map was converted and presented using the overlay visualization function, through which changes in research on this topic according to the timeline of COVID-19 were visualized. Moreover, cooperation pattern analysis analyzed the current state of collaboration in mindfulness research in the context of COVID-19 among authors, institutions, and countries. Clustering was performed at a resolution of 1.00 with a minimum cluster size of 10. The connection strength between nodes was calculated as the total link strength (TLS). TLS indicates how closely a node is connected to other nodes, and the higher the value, the stronger the link. 3. Results 3.1. Trends of Public Interest in Mindfulness Based on the R2 values, the most suitable trend line models were adopted, and their slopes were examined for the five RSV trajectories (Table S1). Only the trend line for 'Mindfulness' was adopted as linear given that its search popularity has steadily increased. However, the search popularity of the other four topics slightly decreased over time before increasing again. Overall, 'Mental health' had the highest RSV in all sub-periods. The RSV for this term showed a gradually decreasing trend since 2004, which was the lowest from 2010 to 2013; subsequently, it was greatly increased until November 2022. The RSVs of the remaining four topics showed a slight and gradual increase until November 2022. Visually, public interest in 'Mindfulness' and the other four topics in the context of COVID-19 showed the sharpest increase in 'Mental health' . Correlation analysis of the five RSV trajectories was conducted. Throughout the entire period, the RSVs of four topics except for 'Mindfulness' showed a statistically significant positive correlation with each other (r = 0.344 to 0.834; all p < 0.001). This trend was also consistently observed in the sub-periods (Table S2). However, the correlation between the RSV trajectory of 'Mindfulness' and that of other topics was different. Specifically, throughout the entire period (i.e., December 2004 to November 2022), the RSV trajectory of 'Mindfulness' showed a statistically significant negative correlation with that of 'Psychotherapy' (r = -0.432, p < 0.001). In addition, in the 6th period (i.e., December 2019 to November 2022), the period after the discovery of SARS-CoV-2, the RSV trajectory of 'Mindfulness' showed a statistically significant negative correlation with that of 'Antidepressants' (r = -0.470, p = 0.004). In other words, a significant negative correlation between public interests in 'Mindfulness' and that of 'Antidepressants', which had not been previously discovered, was found in the COVID-19 era (Table 1). Throughout the entire period, 'meditation' and 'mindfulness meditation' were the queries most related to the search term 'Mindfulness'. There were no new related queries found in the 6th period. However, notably, 'youtube' appeared as a related query since the 4th period. Furthermore, throughout the entire period, 'Meditation' was the topic most related to the search term 'Mindfulness'. 'Psychological stress' was a related topic until the 3rd period; however, 'Anxiety' was one of the top five related topics in the 6th period. In other words, since the middle of 2010, public interest in mindfulness contents via YouTube has increased, and in the era of COVID-19, anxiety, a clinical symptom along with mindfulness, has begun to receive attention as related topics to mindfulness (Table 2). 3.2. Trends of Research Interest in Mindfulness 3.2.1. Frequently Cited Articles The top 10 most frequently cited articles were examined. Three studies (3/10, 30%) directly mentioned mindfulness in the title. Six of the studies (6/10, 60%) were cross-sectional studies. One of these studies (1/10, 10%) was an intervention study, a before-after study in which mindfulness meditation was provided as an intervention for female teachers. The clinical topic of one study was considered not directly related to mindfulness because it was about operational directives. Of the remaining nine studies, there were four studies (4/9, 44.44%) on psychological stress (including psychological flexibility), one study (1/9, 11.11%) on occupational burnout and PTSD, one study (1/9, 11.11%) on job insecurity and emotional exhaustion, and one study (1/9, 11.11%) on wellness. The remaining two studies (2/9, 22.22%) were about three or more mental health-related aspects. The target population of five studies (55.56%) was limited to specific occupational groups. Among these studies, healthcare workers including clinicians were common in two studies (2/9, 22.22%) , and restaurant workers , college students , and teachers were the target population in one study each (1/9, 11.11%). Of the 435 studies retrieved, 308 studies were cited once or more (a total of 4390 citations). Among them, the total number of citations of the top 10 most frequently cited articles was 1377 (31.37%) (Table 3). 3.2.2. Publications of Articles Among the 435 articles retrieved, 339 articles (77.93%) were published in five countries. According to the number of publications, the top five countries were the United States (n = 149, 34.25%), China (n = 74, 17.01%), UK (n = 51, 11.72%), Italy (n = 34, 7.82%), and Spain (n = 31, 7.13%). In particular, articles published in the United States and China accounted for more than 50% of the total retrieved articles . Among the 435 articles retrieved, 123 articles (28.28%) were published in five journals. According to the number of publications, the top five journals were International Journal of Environmental Research and Public Health (n = 48, 11.03%), Frontiers in Psychology (n = 38, 8.74%), Frontiers in Psychiatry (n = 16, 3.68%), Mindfulness (n = 11, 2.53%), and Frontiers in Public Health and PLoS ONE (n = 10, 2.30%, each) . 3.2.3. Keyword Co-Occurrence Analysis A total of 182 keywords were found in five or more articles, and the top five most common keywords were 'COVID-19' (n = 255, 58.62%), 'mindfulness' (n = 226, 51.95%), 'depression' (n = 113, 25.98%), 'anxiety' (n = 113, 25.98%), and 'stress' (n = 111, 25.52%). The keyword co-occurrence network was divided into four clusters. Cluster 1 (red) was related to 'mindfulness' and showed connectivity across the searched keywords. The keywords unique to Cluster 1 included 'burnout', 'nurses', 'satisfaction', 'emotional regulation', 'life', and 'benefits'. Cluster 2 (blue) was related to 'COVID-19' and showed connectivity across the searched keywords. The keywords unique to Cluster 2 included 'pandemic', 'coronavirus', 'telehealth', 'health', and 'care'. 'Stress' belonged to Cluster 2; however, it was a large node and had a topology close to Cluster 1. Cluster 3 (yellow) was related to 'mental health' and showed a topology closer to Cluster 1. On the other hand, Cluster 4 (green) was related to 'anxiety and depression' and showed a topology closer to Cluster 2. The keywords unique to Cluster 3 included 'youth', 'self-compassion', and 'scale'. The keywords unique to Cluster 4 included 'disorder', 'psychological distress', 'therapy', and 'psychometric properties' . In the keyword map visualized with the overlay visualization function, there was no noticeable difference between each keyword according to the timeline of COVID-19, but 'mental health' seemed to be a relatively recent research trend, compared to other important nodes such as 'mindfulness', 'COVID-19', and 'stress' . 3.2.4. Cooperation Pattern Analysis The cooperation pattern of authors on mindfulness research was analyzed. The cooperation pattern network was based the authors of at least two documents on mindfulness. A total of 141 authors were included in the network, but the clusters were not connected to each other. The size of the largest set of connected authors was 13. In other words, it suggests that cooperation among authors on mindfulness research in the context of COVID-19 is insufficient. The top five authors with the strongest link strength were all Spanish researchers, including Miquel Bennasar-Veny (University of the Balearic Islands, Spain) (TLS: 25), Mauro Garcia Toro (University of the Balearic Islands, Spain) (TLS: 25), Pablo Alonso coello (Iberoamerican Cochrane Centre, Spain) (TLS: 23), Javier Garcia Campayo (University of Zaragoza, Spain) (TLS: 23), and Elena Gervilla (University of the Balearic Islands, Spain) (TLS: 23) . The cooperation pattern of organizations on this topic was also sporadic. A total of 178 organizations of at least two documents were included in the network, and the size of the largest set of connected organizations was 95. The top five institutions with the strongest link strength were mostly United States organizations, including Johns Hopkins University (United States) (TLS: 13), Rutgers University (United States) (TLS: 13), University of Manitoba (Canada) (TLS: 13), University of Nottingham (England) (TLS: 13), and Harvard Medical School (United States) (TLS: 12) . Finally, a total of 45 countries of at least two documents were included in the network, and all nodes were connected. The top five countries with the strongest link strength were United States (TLS: 101), England (TLS: 93), China (TLS: 56), Canada (TLS: 48), and Italy (TLS: 46). Three clusters were found in this network. The largest Cluster 1 (red) has the core node of the United States, the second largest Cluster 2 (green) has the core node of Spain, and the smallest Cluster 3 (blue) has the core node of France . 4. Discussion This study aimed to examine the trends of public and research interest in mindfulness in the context of COVID-19. Through Google Trends analysis, public interest in mindfulness was monitored and quantified, and through bibliographic analysis, research interest in mindfulness was analyzed and visualized. 4.1. Trends of Public Interest in Mindfulness Google Trend analysis revealed that the overall RSV of 'Mental health', a topic potentially related to mindfulness, was increased over time. In contrast, the RSVs of 'Mindfulness', 'Psychotherapy', 'Psychoactive substance', and 'Antidepressants' were only increased slightly. The study period (December 2004 to November 2022) was subdivided into 6 periods of 3 years, and the 6th period was 3 years from the discovery of SARS-CoV-2 in December 2019. This study aimed to investigate the change in public interest in mindfulness related to COVID-19 by focusing on the difference between indicators analyzed in the 6th period and the previous periods. Notable correlations between the RSV of the search term 'Mindfulness' and that of the topics 'Psychotherapy' and 'Antidepressants' were observed. Specifically, the RSVs of 'Mindfulness' and 'Psychotherapy' showed an overall significant negative correlation (r = -0.432) but a statistically significant positive correlation in the 4th (r = 0.541) and 5th (r = 0.739) periods. This finding may be explained by the popularization of mindfulness-based psychotherapy, such as MBCT; however, there is no evidence to confirm this possibility. Nevertheless, it is interesting that the search volume of MBCT in MEDLINE on PubMed and the RSV of MBCT in Google Trends were increased during this period. The RSVs of 'Mindfulness' and 'Antidepressants' showed an overall significant positive correlation (r = 0.485) but a statistically significant negative correlation in the 6th period (r = -0.470). As the current study does not suggest causality for the associations found and does not rule out contingency, only some assumptions can be made about these associations. Mindfulness-based interventions (MBIs) have been shown to help improve several psychiatric disorders, including depressive disorders . Accordingly, MBIs have been considered as a promising adjuvant option for depression treatment in clinical settings . However, a recent meta-analysis of 30 randomized controlled trials found that a standardized MBI (MBCT) could have beneficial effects similar to those of cognitive behavioral therapy in the treatment of depression . In addition, MBIs combined with ICT have been reported to be effective in improving mental health, including depression, anxiety, and stress, in the context of the COVID-19 pandemic . These findings suggest that MBIs may be regarded as an alternative rather than an adjuvant to antidepressants in the treatment of depression. In addition to the accumulated clinical evidence on MBIs, media and publicity may have contributed to the public perception of mindfulness . The difficulty in accessing mental health providers due to the COVID-19 pandemic may also explain the findings . Based on the analysis results of the top related queries and topics for the search term 'Mindfulness', there were no new related queries in the COVID-19 era (i.e., 6th period). However, although psychological stress was popular as a topic related to mindfulness before the COVID-19 era (i.e., 1st to 5th period), anxiety appeared to be a popular related topic in the COVID-19 era. A meta-analysis of 43 community-based studies found that the rates of anxiety in the general population could be more than three times higher during the COVID-19 pandemic , making anxiety a major mental health problem associated with COVID-19. Based on the top related queries and topics of the current study, mindfulness may have initially gained popularity as a management method for psychological stress. However, lately, especially in the era of COVID-19, it is possible that mindfulness is gaining popularity for managing psychiatric symptoms such as anxiety. 4.2. Trends of Research Interest in Mindfulness The 10 most popular articles were identified among the 435 articles retrieved, and the majority of studies (60%) were cross-sectional studies. On the other hand, only one intervention study was included as the most popular articles. Excluding one study not directly related to mindfulness, the majority of studies (55.56%) were on stress-related conditions, including psychological stress and PTSD. In addition, the majority of them (55.56%) targeted specific occupational groups, such as healthcare workers, restaurant workers, college students, and teachers. These findings can be interpreted as indicating that intervention studies of MBIs have not yet had a significant impact on this research field of mindfulness in the era of COVID-19 and may further suggest a potential gap with public interest in mindfulness (e.g., managing psychiatric symptoms such as anxiety). Articles in this field were published the most in the United States and China (51.26%). According to the results of the cooperation pattern analysis, the United States and China were establishing the largest cluster of the cooperation pattern of countries on mindfulness research topics. However, the cooperation between authors and institutions in this field was not international, but sporadically. The largest network of cooperation among authors was centered in Spain, and that of cooperation between institutions was centered on institutions located in the United States. However, many countries share a common mental health burden from COVID-19 , and cooperation to address it is encouraged. Consistently, a recent bibliometric analysis of mindfulness research pointed to geographic inequalities in research in this field and highlighted the need for more collaboration . Although mindfulness is considered to have originated in oriental religious practices, MBIs have demonstrated their mental health benefits in a variety of populations in different countries, so it is worth researching mindfulness as a resource to improve human mental health in the COVID-19 or post-COVID-19 period . For example, a panel of psychiatrists from 15 countries, through the Delphi consensus, proposed a protocol for telemental health care during this pandemic, and MBI was considered its first-line intervention . According to keyword co-occurrence analysis, the most common keywords in the included studies were 'COVID-19', 'mindfulness', 'depression', 'anxiety', and 'stress'. Clustering identified four clusters in the constructed keyword co-occurrence network, and each cluster was formed around 'mindfulness', 'COVID-19', 'mental health', and 'anxiety and depression'. 'Mindfulness' and 'COVID-19' accounted for the largest and most widespread nodes, showing relevance in almost all domains. The 'Stress' node, classified as a cluster of COVID-19, was of significant size, and it was considered to be closely related to mindfulness based on its topology. On the other hand, the 'mental health' and 'anxiety and depression' clusters were smaller than those of 'mindfulness' and 'COVID-19'. The 'mental health' cluster had a topology closer to that of 'mindfulness', and the 'anxiety and depression' cluster had a topology closer to that of 'COVID-19'. Analysis of the related nodes demonstrated that the 'mental health' cluster included the non-clinical population and some psychological elements that may be associated with mindfulness, such as self-compassion. The 'anxiety and depression' cluster tended to include clinical elements such as disorder, psychological distress, and therapy. Articles on mindfulness in the context of COVID-19 were closely related to depression, anxiety, stress, and mental health. The findings are consistent with the positive association between daily SARS-CoV-2 infection rates and the major depressive disorder and anxiety disorder prevalence observed during the COVID-19 pandemic . In addition, according to the clusters, studies with more clinical relevance may focus on anxiety or depression in the context of COVID-19, and studies on self-care for non-clinical or sub-clinical populations may focus on other aspects of mental health in the context of mindfulness. 4.3. Implications on the Public and Research Fields This study analyzed both public and research interest in mindfulness in the context of COVID-19. The results suggest a growing public interest in managing psychiatric symptoms during COVID-19, with mindfulness as an intervention and possibly an alternative to antidepressants, along with a growing public interest in mental health. However, most of the studies that had the most important impact in this field were cross-sectional studies, and intervention studies using MBI were lacking. In addition, despite the emphasis on global efforts to address the mental health burden in the COVID-19 era , research in this field showed a lack of multi-national cooperation. Encouragingly, however, our keyword co-occurrence analysis found anxiety and depression as important clinical elements related to mindfulness. The implications of this study highlight resolving the gap between the public interest and research interests found, and further interventional, ideally multi-national, studies of MBIs on anxiety or depression in the context of COVID-19 may be encouraged. Our Google Trends analysis also found an increased public interest in mindfulness contents via YouTube since the middle of 2010. Some studies found a critical link between mental health and YouTube content during the COVID-19 pandemic . Nevertheless, according to our bibliographic analysis, studies in this field do not seem to consider online platforms such as YouTube as an important research topic in the context of mindfulness research. Therefore, to fill this gap, an attempt to investigate the use of MBIs via online platforms including YouTube in the public and demonstrate their effectiveness and safety on mental health of individuals could be encouraged. 4.4. Strengths and Limitations This study has the strength of suggesting current views and future research directions in this field by combining and analyzing bibliometrics and Google Trends data. In particular, the proposed future research directions are based on bridging the gap between public and research interest in mindfulness in the context of COVID-19. Given that public interest and dissemination of information to the public are an important basis for health policy , and that strategies to maintain the integrity of the mental health of individuals should be implemented at the social level in the era of COVID-19 , this study has public health relevance. Perhaps the methodology attempted in this study can be used as a reference for researchers to investigate public interest and research interest in a specific topic in the future. Even in that case, the interpretation of the results should focus on discovering the gap between the two interest types (i.e., public and research interest), discussing the importance of filling the gap, and providing a solution strategy to fill the gap. However, some limitations should be acknowledged. First, due to the nature of Google Trends and bibliometric analysis, the causality of the findings cannot be explored, and the possibility of discovery due to chance cannot be ruled out. In addition, as Google Trends can be influenced by a country's Internet penetration and the relative popularity of Internet search engines, it may be premature to interpret the findings from this study as indicative of a global public interest in mindfulness. Second, as the search term 'Mindfulness' was searched in English in Google Trends, the findings may only be meaningful in countries where the term is searched in English. In addition, there was no topic for 'Mindfulness' in the Google Trends system, limiting comparability with other topics. Third, as the search database for bibliometric analysis in this study was limited to the Web of Science, it is possible that some related articles were missed. 5. Conclusions This study conducted Google Trends and bibliometric analysis to investigate public and research interest in mindfulness related to COVID-19. As a result, the RSV for 'Mindfulness' showed a slight increase over the entire period. In particular, from December 2019 to November 2022, the three years belonging to the COVID-19 period, the RSV trajectory of 'Mindfulness' showed a statistically significant negative correlation with that of 'Antidepressants'. The majority of most popular articles in this field were cross-sectional studies. It was found that most studies in this field were published in the United States and China. However, the cooperation between authors and institutions in this field was sporadically. In a network map, four clusters were identified (i.e., 'mindfulness', 'COVID-19', 'anxiety and depression', and 'mental health') in the studies. However, one of the important limitations of this study is that our methodology does not prove the potential causality of the findings. According to the findings and the limitations, further interventional, ideally multi-national, studies of MBIs on anxiety or depression in the context of COVID-19 may be encouraged. Additionally, research investigating the public use of MBIs via online platforms such as YouTube, and verifying its effectiveness and safety, may also be encouraged. The findings may provide insights into potential areas of interest and identify ongoing trends in this field. Additionally, the findings of this study may be referenced by policy makers responsible for future R&D decision-making on the mental health burden of COVID-19. Supplementary Materials The following supporting information can be downloaded at: Table S1: Formulas of trend lines adopted based on the R2 value for each search term; Table S2: Pearson's correlation between the five search terms (Mindfulness, Mental health, Psychotherapy, Psychoactive substance, and Antidepressants) for the entire period (December 2004 to November 2022); Figure S1: Number of search results for MBCT on PubMed (from 1982 to 2022); Figure S2: Relative search volume of MBCT in Google Trends (from 2004 to 2022, monthly). Click here for additional data file. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement The data used to support the findings of this study are included within the article. Conflicts of Interest The author declares no conflict of interest. Figure 1 Relative search volume of 'Mindfulness', 'Mental health', 'Psychotherapy', 'Psychoactive substance', and 'Antidepressants' (2004 to 2022, monthly) in Google Trends. Note. The y-axis represents the relative search score and is distributed between 0 and 100 points. The x-axis represents the period. Figure 2 (a) Countries and (b) Journals with more than five articles published on mindfulness during the study period. Figure 3 (a) Keyword co-occurrence network graph and (b) Overlay visualization network graph of mindfulness research topics in the COVID-19 era. Note. Red cluster: mindfulness; Blue cluster: COVID-19; Yellow cluster: mental health; Green cluster: anxiety and depression. Figure 4 The cooperation pattern of (a) authors, (b) organizations, and (c) countries on mindfulness research topics in the COVID-19 era. ijerph-20-03807-t001_Table 1 Table 1 Pearson's correlation between the search term 'Mindfulness' and four other search terms over time. Correlation Mental Health Psychotherapy Psychoactive Substance Antidepressants Mindfulness Total 0.412 ** -0.432 ** 0.055 0.485 ** P1 0.011 -0.142 0.069 -0.115 P2 -0.030 0.120 0.332 * 0.430 ** P3 0.306 -0.033 -0.230 -0.214 P4 0.695 ** 0.541 ** 0.223 0.527 ** P5 0.781 ** 0.739 ** 0.559 ** 0.688 ** P6 -0.235 -0.056 -0.015 -0.470 ** Note. * indicates p < 0.05; ** indicates p < 0.01 (two-tailed test). P1 = Period 1: December 2004-November 2007; P2 = Period 2: December 2007-November 2010; P3 = Period 3: December 2010-November 2013; P4 = Period 4: December 2013-November 2016; P5 = Period 5: December 2016-November 2019; P6 = Period 6: December 2019-November 2022. ijerph-20-03807-t002_Table 2 Table 2 Top queries and topics related to the search term 'Mindfulness' over time. Related Queries P1 P2 P3 P4 P5 P6 Top 1 meditation meditation meditation meditation mindfulness mindfulness meditation mindfulness meditation Top 2 mindfulness meditation mindfulness meditation mindfulness meditation meditation meditation meditation Top 3 mindfulness therapy mindfulness therapy stress mindfulness training mindfulness youtube what is mindfulness Top 4 mindfulness stress reduction mindfulness stress training mindfulness stress kids mindfulness kids mindfulness Top 5 mindfulness training mindfulness mindfulness therapy youtube mindfulness what is mindfulness mindfulness meaning Related Topics P1 P2 P3 P4 P5 P6 Top 1 Meditation Meditation Meditation Meditation Meditation Meditation Top 2 Therapy Therapy Therapy Exercise Sati Sati Top 3 Mindfulness-based stress reduction Training Training Training Course Exercise Top 4 Psychological stress Mindfulness-based stress reduction Mindfulness-based stress reduction Course Exercise Therapy Top 5 Training Psychological stress Psychological stress Therapy Training Anxiety Note. P1 = Period 1: December 2004-November 2007; P2 = Period 2: December 2007-November 2010; P3 = Period 3: December 2010-November 2013; P4 = Period 4: December 2013-November 2016; P5 = Period 5: December 2016-November 2019; P6 = Period 6: December 2019-November 2022. ijerph-20-03807-t003_Table 3 Table 3 Top 10 most frequently cited articles. Title Publication Year Study Design Relevant Clinical Topics (Target Population) Journal of Publication Total Citation Stress and parenting during the global COVID-19 pandemic 2020 Cross-sectional study Psychological stress (parents with a child under the age of 18 years) Child Abuse and Neglect 485 Surgery in COVID-19 patients: operational directives 2020 Commentary Others: operational directives (not specific) World Journal of Emergency Surgery 183 Mindfulness, Age and Gender as Protective Factors Against Psychological Distress During COVID-19 Pandemic 2020 Cross-sectional study Psychological stress (general population) Frontiers in Psychology 125 Occupational burnout syndrome and post-traumatic stress among healthcare professionals during the novel coronavirus disease 2019 (COVID-19) pandemic 2020 Commentary Occupational burnout syndrome and PTSD (healthcare professionals) Best Practice and Research Clinical Anesthesiology 114 Do mindfulness and perceived organizational support work? Fear of COVID-19 on restaurant frontline employees' job insecurity and emotional exhaustion 2021 Literature review and cross-sectional study Job insecurity and emotional exhaustion (restaurant frontline employees) International Journal of Hospitality Management 99 Clinician Wellness During the COVID-19 Pandemic: Extraordinary Times and Unusual Challenges for the Allergist/Immunologist 2020 Commentary Wellness (clinicians) The Journal of Allergy and Clinical Immunology: In Practice 90 The moderating roles of psychological flexibility and inflexibility on the mental health impacts of COVID-19 pandemic and lockdown in Italy 2020 Cross-sectional study Psychological flexibility and inflexibility (general population) Journal of Contextual Behavioral Science 77 Psychological flexibility and inflexibility as sources of resiliency and risk during a pandemic: Modeling the cascade of COVID-19 stress on family systems with a contextual behavioral science lens 2020 Cross-sectional study Psychological flexibility and inflexibility (general population) Journal of Contextual Behavioral Science 72 Psychiatric symptoms, risk, and protective factors among university students in quarantine during the COVID-19 pandemic in China 2021 Cross-sectional study Psychiatric symptoms including anxiety, depression, and traumatic stress (university students) Globalization and Health 68 Positive Impact of Mindfulness Meditation on Mental Health of Female Teachers during the COVID-19 Outbreak in Italy 2020 Before-after study Mindfulness skills, empathy, personality profiles, interoceptive awareness, psychological well-being, emotional distress, and burnout levels (female teachers) International Journal of Environmental Research and Public Health 64 Abbreviations. 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PMC10000853 | Healthcare (Basel) Healthcare (Basel) healthcare Healthcare 2227-9032 MDPI 10.3390/healthcare11050764 healthcare-11-00764 Article Patterns and Trends in Pharmacological Treatment for Outpatients with Postherpetic Neuralgia in Six Major Areas of China, 2015-2019 Han Gang Conceptualization Investigation Resources 12+ Han Yun Software Formal analysis Investigation Writing - original draft Visualization 123+ Yu Lingyan Methodology Validation 24 Zhao Yuhua Conceptualization Writing - review & editing Supervision Project administration 5* Yu Zhenwei Methodology Software Data curation Writing - review & editing Supervision Project administration 12* Botturi Andrea Academic Editor 1 Sir Run Run Shaw Hospital, College of Medicine, Zhejiang University, Hangzhou 310016, China 2 Research Center for Clinical Pharmacy, Zhejiang University, Hangzhou 310058, China 3 College of Pharmaceutical Science, Zhejiang University, Hangzhou 310058, China 4 Second Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310009, China 5 Affiliated Xiaoshan Hospital, Hangzhou Normal University, Hangzhou 311202, China * Correspondence: [email protected] (Y.Z.); [email protected] (Z.Y.) + These authors contributed equally to this work. 06 3 2023 3 2023 11 5 76412 12 2022 23 2 2023 03 3 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). The aim of this study was to assess the patterns and trends of pharmacological treatment for outpatients with postherpetic neuralgia (PHN) in China in the period 2015-2019. Prescription data for outpatients with PHN were extracted from the database of the Hospital Prescription Analysis Program of China according to the inclusion criteria. The trends in yearly prescriptions and corresponding costs were analyzed and stratified by drug class and specific drugs. A total of 19,196 prescriptions from 49 hospitals in 6 major regions of China were included for analysis. The yearly prescriptions increased from 2534 in 2015 to 5676 in 2019 (p = 0.027), and the corresponding expenditures increased from CNY 898,618 in 2015 to CNY 2,466,238 in 2019 (p = 0.027). Gabapentin and pregabalin are the most commonly used drugs for PHN, and more than 30% of these two drugs were combined with mecobalamin. Opioids were the second most frequently prescribed drug class, and oxycodone accounted for the largest share of the cost. Topical drugs and TCAs are rarely used. The frequent use of pregabalin and gabapentin was in accordance with current guidelines; however, the use of oxycodone raised concerns about rationality and economic burden. The results of this study may benefit the allocation of medical resources and management for PHN in China and other countries. gabapentin pregabalin oxycodone prescription cost This research received no external funding. pmc1. Introduction Postherpetic neuralgia (PHN) is a chronic complication of herpes zoster (HZ) caused by damage to peripheral nerve tissue during the onset of herpes zoster, which is defined as obvious pain 3 months after herpes zoster . PHN is often described as burning pain, tingling or itching, and its pain score is always >=4 on a 10-point visual analog scale . The burning and allodynia pain of PHN in the thoracolumbar region are more intensive, while the tingling and numbness of PHN in the face are more intense . Approximately 20% of herpes zoster patients develop PHN . In the United States, the total incidence rate of PHN is 57.5 cases per 100,000 person-years . A study on herpes zoster and its complications in China reported that the incidence rate of PHN was 0.48 per 1000 person-years . The pain caused by PHN is often unbearable, which impairs the work of many employees. This is equivalent to an annual indirect loss of CNY 28,025 (USD 4221), which not only leads to the loss of personal wages but also has a wider economic impact on society as a whole through productivity loss . There are many treatment options for PHN, mainly including the most widely used pharmacological therapy and nonpharmacological methods such as nerve blocks, neuromodulation and nerve stimulation . In the field of pharmacological treatment, some anticonvulsants, antidepressants, opioids and local therapeutic drugs have been proven to be able to relieve pain. Among the drug recommendations in many countries, gabapentin, pregabalin and TCAs are often used as first-line drugs, while topical drugs and opioids are also often suggested in treatment . At present, we have known that many drugs, such as gabapentin and pregabalin, are widely used in clinical treatment . However, side effects of PHN drugs are common, and some drugs, especially opioids, have a potential risk of addiction . Currently, little is known about the status of PHN drug application in China. Considering the increasing prevalence of herps zoster in China , we designed this cross-sectional study to analyze the patterns and trends of PHN drug use, as well as its costs. 2. Materials and Methods 2.1. Study Design and Ethics This study was a retrospective prescription-based cross-sectional study, and informed consent was waived as part of the approval. Ethical approval was obtained from the Ethics Committee of Run Run Shaw Hospital, College of Medicine, Zhejiang University (Reference Number KEYAN20210924-33). 2.2. Data Source and Prescription Inclusion Prescription data were derived from the widely used database of the Hospital Prescription Analysis Cooperative Project of China for pharmaco-epidemic studies . The database was initiated in 2003, and the following items of prescription were included in the database: prescription code, date of prescription issued, sex and age of patient, department of physician, hospital code, drug generic name, strength, price and cost of drug, diagnosis. Prescriptions for patients with a diagnosis of PHN were extracted, and those meeting the following criteria were included for analysis: (1) prescriptions written from 2015 to 2019; (2) prescriptions from hospitals situated in 6 major regions of China (Beijing, Shanghai, Hangzhou, Guangzhou, Chengdu and Tianjin) and participated in the program continuously; (3) prescriptions for adult outpatients (age > 18 years) diagnosed with PHN. Prescriptions with missing values were excluded from the analysis. 2.3. Analysis The prescriptions for patients with PHN were represented by the number of corresponding prescriptions per year. The annual cost was the sum of the total cost of PHN patients' prescriptions. It should be noted that inflation factor or discount rate was not considered either. The trends in annual prescriptions and expenditures were analyzed, and further stratified and illustrated by drug class and specific drugs. The PHN-treated drugs were classified to analyzed as follows: (1) anticonvulsants, including gabapentin, pregabalin, carbamazepine, oxcarbazepine, lamotrigine, valproic acid, topiramate and other analogs; (2) antidepressants, including tricyclic antidepressants (TCAs) and other serotonin (5-HT) and norepinephrine (NE) reuptake inhibitors; (3) opioids, compound preparations containing opioids that are also classified as opioids; and (4) topical drugs, including capsaicin, lidocaine, flurbiprofen and diclofenac . The trends in prescription numbers and costs for overall and individual drugs were assessed using the Mann-Kendall test. The trends in percentages were assessed using the log-linear test. The Wilcoxon signed rank test was used for the difference between the male and female prescription percentages. The average proportion and standard deviation of the combined use of gabapentin and pregabalin in five years were calculated. The trend package in R (version 4.2.1) software was used for statistical analysis. The statistical significance was set as p < 0.05. 3. Results 3.1. Demographic Characteristics of Patients and Overall Trends A total of 19,196 prescriptions were included in this study. Detailed demographic characterizations of patients with PHN prescriptions are shown in Table 1. The percentages of prescriptions for females were slightly higher (p = 0.043), and the proportion did not significantly change during the study period (p = 0.198). The yearly prescriptions and expenditures are shown in Figure 1A. The yearly prescriptions increased from 2534 in 2015 to 5676 in 2019 (p = 0.027), and the corresponding expenditures increased from CNY 898,618 in 2015 to CNY 2,466,238 in 2019 (p = 0.027). 3.2. Trends in Prescriptions and Cost of Drug Class and Specific Drug The yearly total prescriptions for four major classes of PHN drugs--anticonvulsants, antidepressants, opioids and topical drugs--increased during the study period , and detailed prescription numbers are listed in Table 2. Anticonvulsants were the most frequently prescribed drug class, followed by opioids. Antidepressants and topical drugs were rarely used. Table 3 shows the costs and percentage of specific drugs. There was a certain difference between the trend in expenditure and the trend in prescriptions. The total costs of opioids were always higher than those of anticonvulsants, which had most prescriptions . Gabapentin and pregabalin were the most frequently used drugs. Prescriptions of pregabalin increased rapidly (p = 0.002), with the largest increase of 379% in 2018. Regarding second-line opioid drugs, the proportion of oxycodone prescriptions was large and continuously increasing (p = 0.031). For antidepressants, the number of prescriptions of traditional TCA amitriptyline was greater than the others. The topical drugs were mainly lidocaine and capsaicin. In anticonvulsants, the costs of pregabalin also increased. The average proportion of oxycodone costs per year was approximately 21.3% of the total costs. This was the drug with the largest proportion of the annual amount, and the proportion was stable (p = 0.220). Among antidepressants, the total costs of duloxetine and venlafaxine were higher, while the total costs of amitriptyline were lower. 3.3. Trends in Combination of Drugs Gabapentin and pregabalin, as the first-line choice drugs, were combined with other drugs . Mecobalamin was the drug most commonly used in combinations. On average, 36.7% of gabapentin prescriptions jointly used mecobalamin, as well as 30.0% of pregabalin prescriptions. 4. Discussion This is the first study to analyze the patterns and trends in pharmacological treatment for outpatients with PHN in China. The yearly prescriptions and costs of PHN drugs have been increasing. Two anticonvulsant drugs--gabapentin and pregabalin--were the most commonly used drugs, which was in line with current practice guidelines. At the same time, we also found that oxycodone in opioids was used in large quantities and costs in a large proportion, which might be an unreasonable use in the treatment of PHN. The percentages of antidepressants and topical drugs were relatively low, in both prescriptions and corresponding costs. Regarding the combination of gabapentin and pregabalin, we unexpectedly found that mecobalamin was used more frequently. The prescriptions for patients with PHN increased during the study period. In China, 7.26% of herpes zoster patients have PHN, and the incidence rate in women is slightly higher than that of men (7.45% vs. 7.03%) , which is consistent with the results of our study. In addition, the number of people diagnosed with herpes zoster continued to increase from 2015 to 2019 . Therefore, the increase in the number of people diagnosed with PHN might be related to the progressive rise of prescriptions for PHN. At present, the treatment of PHN is based on symptom control, and many studies have proven that antiviral drugs for herpes zoster have no significant effect on PHN or its prevention. Therefore, the treatment of PHN usually follows the principle of neuralgia treatment . In the Chinese guidelines, the first-line drugs include pregabalin and gabapentin, TCAs (such as amitriptyline, etc.) and 5% lidocaine patches, and the second-line drugs include opioids . The first-line treatment of PHN in the United States includes TCAs, gabapentin and pregabalin, and a topical lidocaine 5% patch. Opioids and capsaicin patches are recommended as second-line or third-line therapeutic drugs . The French guidelines for neuralgia regard TCAs and other serotonin-norepinephrine reuptake inhibitors (duloxetine and venlafaxine, etc.), and gabapentin as first-line drugs for the treatment of neuralgia, with pregabalin, weak opioid tramadol and capsaicin patches recommended as second-line drugs, and other powerful opioids as third-line drugs . According to the Canadian Pain Society consent statement, gabapentin, TCAs and serotonin-norepinephrine reuptake inhibitors are first-line drugs for the treatment of neuropathic pain. Opioids are recommended as second-line drugs, while cannabinoids are newly recommended as second-line drugs . In general, gabapentin, pregabalin and TCAs are often used as first-line drugs, while opioids are not the first choice for PHN. Thus, the use of most frequently prescribed anticonvulsant, mainly gabapentin and pregabalin, was in accordance with current guidelines and evidences. Regional differences in drug use were not significant in this study. Gabapentin and pregabalin, which are used most, are voltage-gated cation channel regulators . Daily doses of 1800 mg to 3600 mg of gabapentin can provide patients with effective pain relief levels . The general dosage of pregabalin for the treatment of PHN is between 75 mg and 600 mg per day, which is taken two to three times per day . In our study, the treatment time of gabapentin single prescription is generally about 16 days, and the single oral dose is between 100 mg and 1500 mg, two to four times a day. The treatment time of pregabalin in a single prescription varies greatly, and the single oral dose is between 75 mg and 300 mg, one to three times a day. It is similar to the recommended dosage. The use of pregabalin had a significant increase during the study period, and its prescriptions exceeded gabapentin in 2018. Gabapentin and pregabalin are recognized as drugs with good relief effects on PHN . The increase in the use of pregabalin may be due to the following reasons. The first is the difference in the results of drug action. Omar et al.'s study on the difference between pregabalin and gabapentin initially showed that pregabalin was better at alleviating pain, while gabapentin had better effects on anxiety, insomnia and fatigue symptoms . Previous studies have confirmed that pregabalin is highly effective and safe for patients with PHN in China . Additionally, the widespread use of gabapentin and pregabalin calls special focus to the effective management of its use, as these drugs also have side effects. An overdose of gabapentin and pregabalin will produce euphoric effects and can lead to delirium . Compared with pregabalin, the abuse of gabapentin is a growing trend. A British survey found that the proportion of lifetime gabapentin abuse was 1.1%, compared with 0.5% in pregabalin . Another reason for this may be related to the expiration of the patent. According to the database of the China Pharmaceutical Industry Information Center, the patent protection date of pregabalin expired in 2018. Although the brand of pregabalin used by patients has not changed in the past five years, the expiration of the patent has increased the attention it has received in wider society, especially for PHN patients, medical institutions and related pharmaceutical companies. More prescribers realize that the role of pregabalin in PHN may be better than that of gabapentin, so they are more willing to prescribe pregabalin. Other anticonvulsant drugs, such as oxcarbazepine, have proven to have no better therapeutic effect than both gabapentin and pregabalin on neuralgia, and their use is rare . Opioids are widely used in pain control, and oxycodone is the drug with the largest number of prescriptions in the current study. Oxycodone is a semisynthetic k-opioid receptor agonist with a wide range of applications . Some studies have also shown that the use of oxycodone is not completely beneficial to the treatment of PHN . A study by Gaskell et al. on oxycodone in the treatment of neuralgia suggested that there was no reported result within the scope of their study that can prove that oxycodone has substantial benefit results, such as the overall impression of clinical changes in the treatment of neuralgia . Thus, although opioids were recommended as second-line treatment for PHN, oxycodone was not recommended, or only a very weak recommendation. However, oxycodone has the advantages of long duration of action and no histamine release or ceiling effect compared with other opioids, so it is still used frequently . Compared with the status of other countries, the study by Gudin et al. found that 21.6% of PHN patients received opioids as initial treatment for PHN in the United States, while among the other first-line treatment methods of PHN, gabapentin was 15.1%, pregabalin was 3.3% and TCAs were 2.5%, which proved that excessive use of opioids was common . Opioids are prone to cause peripheral nerve injury, which leads to increased noxious hypersensitivity, various adverse reactions and drug interactions . It can also be seen from the conclusion that the cost of oxycodone accounts for a large proportion of overall expenditure and its spending has been sustained at a high level over the five years period of the study. Therefore, the widespread use of oxycodone has raised concerns about rationality and the economic burden on patients. For this phenomenon, the relevant departments should maintain a high degree of vigilance and remind prescribers to reduce or limit the use of related addictive drugs if necessary. Prescribers should evaluate the pain degree of patients before using drugs, and relevant departments can set different indicators of analgesic use for different pain levels, so as to re-evaluate whether opioid analgesics should be used to manage the PHN. The use of antidepressants is far lower than that of anticonvulsants and opioids, which reflects physician behavior and patient preference. Antidepressants have a certain relieving effect on PHN . However, TCAs such as amitriptyline cannot achieve satisfactory effects for all people in the treatment of PHN pain . Another reason for the infrequent use of TCAs is the adverse effects of TCAs. They may cause nausea, headache, constipation and other negative effects that patients are unwilling to bear . At present, there are also experiments proving that the combination of amitriptyline and other analgesic drugs, such as pregabalin, may have a better effect . Topical drug use did not change much in our study range, although many clinical trials have confirmed that local drug use has a certain therapeutic effect on PHN and has fewer adverse effects . In the combination of drugs, we found that the frequency of mecobalamin, which does not belong to main treatment drugs, was high. Mecobalamin is a vitamin medicine and is the activated form of vitamin B12. A few studies shows that it not only has a good therapeutic effect on PHN, but can also relieve peripheral polynomialism, entrapment neuropathy and glossopharyngeal neuropathy . A study showed that in four trials including 383 participants, the scores of the pain numerical scale in the vitamin B12 group decreased faster, compared with the placebo group. Vitamin B12 can improve the quality of life of patients with PHN and significantly reduce the number of patients using analgesics . The combined use of mecobalamin seems to be justified and reasonable, but more relevant studies are also needed in the future to confirm the safety of its use and its impact on patients. There are also several limitations to our study. First, the severity of PHN and clinical outcome were not measured and matched with the prescription. If the patient's pain degree is included in future studies, hierarchical statistics on the drugs used could be better gathered. Second, the rationale of drug use was not assessed, due to the large number of prescriptions. Other comorbidities may cause some deflection among the statistical results. Although all patients with prescriptions are diagnosed with PHN, they also contain many drugs that might not be used to treat PHN. Finally, sampling bias may exist: although prescriptions were from many hospitals located in representative areas of China, primary care or non-hospital-based outpatient prescriptions are not included in our study. Therefore, in future research plans, the study of the correlation between the pain degree of patients and the corresponding drug selection should be included, and include more patient disease information, making it possible to better analyze the rationality of drug use. 5. Conclusions The status and trends of pharmacological treatment for outpatients with PHN in China during a five-year period were analyzed in this study, and the yearly prescriptions and corresponding costs were both found to have increased. Gabapentin and pregabalin were the most frequently used drugs for PHN, which is in accordance with current practice guidelines. Among them, the use and cost of pregabalin showed a significant increasing trend. Oxycodone, as an opioid drug with a strong analgesic effect, had the third most yearly prescriptions but took the largest share of cost, which raised concerns about the rationality of its use and economic burden for PHN patients. The discovery of mecobalamin as the most commonly used drug may be due to its beneficial effect on peripheral nerves, but more research is still needed to study its mechanism of action on PHN in future. The percentages of antidepressants and dermal drugs were relatively low, which reflected physicians' behavior and patients' preferences in China. The results of this study indicate that the relevant departments and prescribers should attach great importance to the use of drugs in the treatment of PHN, especially with regard to the use of addictive drugs. Our study may benefit the allocation of medical resources and management for PHN in China, as well as other countries. Acknowledgments The authors would like to thank the Hospital Prescription Analysis Cooperative Project of China for collecting and providing data. Author Contributions Conceptualization, G.H., Y.Z.; data curation, Z.Y.; formal analysis, Y.H.; investigation, G.H., Y.H.; methodology, Z.Y., L.Y.; project administration, Z.Y., Y.Z.; resources, G.H.; software, Y.H., Z.Y.; supervision, Z.Y., Y.Z.; validation, L.Y.; visualization, Y.H.; writing--original draft, Y.H.; writing--review & editing, Y.Z., Z.Y. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of Sir Run Run Shaw Hospital, College of Medicine, Zhejiang University (KEYAN20210924-33, date of approval, 24 September 2021). According to the requirements of national legislation and institutions, this study does not require written informed consent to participate. Data Availability Statement The original contributions in the research are included in the articles, and further inquiries can be directed to the corresponding authors. Conflicts of Interest The authors state that the study was conducted in the absence of any potential conflicts of interest with regard to business or financial relationships. Figure 1 Prescription trends of PHN in six regions of China. (A) Annual number of patients and costs; (B) total prescriptions of the four classes of drug trends; (C) total costs of the four classes of drug trends; (D) average ratios of five years in the combination of gabapentin (left) and pregabalin (right). The data are expressed as the mean +- SD of 5 years. healthcare-11-00764-t001_Table 1 Table 1 Demographic characteristics of the study sample. 2015 2016 2017 2018 2019 P1 P2 Age (year) <=45 204 (8.1%) 258 (8.4%) 231 (7.3%) 333 (7.6%) 404 (7.9%) 0.086 0.424 46-65 910 (36.1%) 1135 (37.1%) 1166 (37.0%) 1600 (36.5%) 1883 (36.7%) 0.027 0.970 66-80 1009 (40.1%) 1221 (39.9%) 1280 (40.6%) 1766 (40.3%) 2035 (39.7%) 0.027 0.432 >=81 396 (15.7%) 449 (14.7%) 478 (15.2%) 681 (15.5%) 807 (15.7%) 0.027 0.711 Sex Male 1212 (48.1%) 1418 (46.3%) 1539 (48.8%) 2160 (49.3%) 2554 (49.8%) 0.027 0.198 Female 1307 (51.9%) 1645 (53.7%) 1616 (51.2%) 2220 (50.7%) 2575 (50.2%) 0.086 0.115 Note: Data were presented as numbers (percentage%); P1: p value for trends in number in yearly prescriptions, assessed by the Mann-Kendall test; (P): P2: p value for trend in portion of prescriptions, assessed by the log-linear test. healthcare-11-00764-t002_Table 2 Table 2 Number of yearly prescriptions for specific drugs from 2015 to 2019. Drug Class Name 2015 2016 2017 2018 2019 P1 P2 Anticonvulsant Gabapentin 1053 (41.6%) 1405 (45.9%) 1492 (46.3%) 1792 (38.1%) 1651 (29.1%) 0.086 0.177 Pregabalin 95 (3.7%) 108 (3.5%) 177 (5.5%) 848 (18.1%) 1413 (24.9%) 0.027 0.007 Oxcarbazepine 25 (1.0%) 27 (0.9%) 21 (0.7%) 22 (0.5%) 36 (0.6%) 0.807 0.020 Carbamazepine 36 (1.4%) 29 (0.9%) 35 (1.1%) 30 (0.6%) 28 (0.5%) 0.221 0.036 Opioid Oxycodone 208 (8.2%) 342 (11.2%) 322 (10.0%) 586 (12.5%) 790 (13.9%) 0.086 0.031 Tramadol 260 (10.3%) 354 (11.6%) 363 (11.3%) 443 (9.4%) 472 (8.3%) 0.027 0.184 Codeine 157 (6.2%) 178 (5.8%) 223 (6.9%) 269 (5.7%) 204 (3.6%) 0.221 0.235 Morphine 14 (0.6%) 35 (1.1%) 54 (1.7%) 218 (4.6%) 208 (3.7%) 0.086 0.070 Antidepressant Amitriptyline 108 (4.3%) 133 (4.3%) 96 (3.0%) 144 (3.1%) 93 (1.6%) 0.807 0.035 Doxepin 57 (2.2%) 44 (1.4%) 44 (1.4%) 39 (0.8%) 37 (0.7%) 0.043 0.007 Duloxetine 33 (1.3%) 46 (1.5%) 51 (1.6%) 91 (1.9%) 159 (2.8%) 0.027 0.013 Venlafaxine 7 (0.3%) 16 (0.5%) 19 (0.6%) 22 (0.5%) 16 (0.3%) 0.312 0.270 Topical formulation Lidocaine 62 (2.4%) 61 (2.0%) 65 (2.0%) 109 (2.3%) 145 (2.6%) 0.086 0.462 Capsaicin 59 (2.3%) 67 (2.2%) 87 (2.7%) 59 (1.3%) 55 (1.0%) 0.613 0.166 Flurbiprofen 18 (0.7%) 35 (1.1%) 34 (1.1%) 68 (1.4%) 61 (1.1%) 0.221 0.222 Diclofenac Sodium 42 (1.7%) 33 (1.1%) 35 (1.1%) 22 (0.5%) 21 (0.4%) 0.086 0.009 Note: Data were presented as numbers (percentage%); Percentage refers to the ratio of total PHN prescriptions in the current year; All drugs include their compound preparations; P1: p value for trends in number in yearly prescriptions, assessed by the Mann-Kendall test; P2: p value for trend in portion of prescriptions, assessed by the log-linear test. healthcare-11-00764-t003_Table 3 Table 3 Yearly cost of specific drugs from 2015 to 2019. Drug Class Name 2015 2016 2017 2018 2019 P1 P2 Anticonvulsant Gabapentin 155,505 (17.3%) 159,436 (16.2%) 151,208 (15.2%) 180,982 (8.8%) 195,228 (7.9%) 0.221 0.027 Pregabalin 27,771 (3.1%) 32,793 (3.3%) 59,028 (5.9%) 290,640 (14.1%) 512,669 (20.8%) 0.027 0.002 Oxcarbazepine 4357 (0.5%) 4900 (0.5%) 3098 (0.3%) 3774 (0.2%) 6248 (0.3%) 0.807 0.068 Carbamazepine 1267 (0.1%) 944 (0.1%) 1299 (0.1%) 1149 (0.1%) 1278 (0.1%) 0.807 0.091 Opioid Oxycodone 170,930 (19.0%) 204,129 (20.7%) 179,090 (18.0%) 526,434 (25.5%) 569,162 (23.1%) 0.086 0.220 Tramadol 40,023 (4.5%) 41,393 (4.2%) 37,204 (3.7%) 47,173 (2.3%) 53,565 (2.2%) 0.221 0.015 Codeine 11,925 (1.3%) 13,011 (1.3%) 12,002 (1.2%) 17,951 (0.9%) 14,457 (0.6%) 0.221 0.040 Morphine 10,735 (1.2%) 18,246 (1.9%) 30,230 (3.0%) 90,688 (4.4%) 73,661 (3.0%) 0.086 0.161 Antidepressant Duloxetine 7801 (0.9%) 10,319 (1.0%) 11,132 (1.1%) 18,926 (0.9%) 31,881 (1.3%) 0.027 0.215 Venlafaxine 1282 (0.1%) 2302 (0.2%) 2760 (0.3%) 3498 (0.2%) 3590 (0.1%) 0.027 0.996 Amitriptyline 785 (0.1%) 1113 (0.1%) 745 (0.1%) 1261 (0.1%) 1296 (0.1%) 0.221 0.084 Doxepin 147 (<0.1%) 172 (<0.1%) 266 (<0.1%) 135(<0.1%) 114 (<0.1%) 0.462 0.669 Topical formulation Flurbiprofen 10,396 (1.2%) 11,686 (1.2%) 6887 (0.7%) 14,361 (0.7%) 10,129 (0.4%) 1.000 0.022 Lidocaine 4590 (0.5%) 2918 (0.3%) 3922 (0.4%) 7673 (0.4%) 15,481 (0.6%) 0.221 0.442 Capsaicin 3888 (0.4%) 3868 (0.4%) 5069 (0.5%) 3551 (0.2%) 3413 (0.1%) 0.221 0.188 Diclofenac Sodium 2217 (0.2%) 1490 (0.2%) 1358 (0.1%) 868 (<0.1%) 826 (<0.1%) 0.027 0.008 Note: Data were presented as costs (percentage%); percentage refers to the ratio of total PHN prescriptions' costs in the current year; all drugs include their compound preparations; P1: p value for trends in costs in yearly prescriptions, assessed by the Mann-Kendall test; P2: p value for trend in portion of prescriptions' costs, assessed by the log-linear test. 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PMC10000854 | Defects in DNA repair pathways can lead to genomic instability in multiple tumor types, which contributes to tumor immunogenicity. Inhibition of DNA damage response (DDR) has been reported to increase tumor susceptibility to anticancer immunotherapy. However, the interplay between DDR and the immune signaling pathways remains unclear. In this review, we will discuss how a deficiency in DDR affects anti-tumor immunity, highlighting the cGAS-STING axis as an important link. We will also review the clinical trials that combine DDR inhibition and immune-oncology treatments. A better understanding of these pathways will help exploit cancer immunotherapy and DDR pathways to improve treatment outcomes for various cancers. DNA damage response cancer therapy immunotherapy cell death biomarker tumor microenvironment DNA repair cell death Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences2021-RC310-013 the CAMS Innovation Fund for Medical Sciences2021-I2M-1-067 the National Natural Science Foundation of China82272757 Beijing Hope Run Special Fund of Cancer Foundation of ChinaLC2021R02 This study was supported by the Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences to M.D (2021-RC310-013), the CAMS Innovation Fund for Medical Sciences to M.D (2021-I2M-1-067), the National Natural Science Foundation of China to M.D (82272757) and Beijing Hope Run Special Fund of Cancer Foundation of China to M.D (LC2021R02). pmc1. Introduction Cancer has become a leading cause of death in many countries and is still a major public health problem worldwide . The classical and primary therapies are surgery, radiotherapy, and chemotherapy. Along with a better understanding of the molecular biology of the tumor cells, molecularly targeted therapies are designed to inhibit a target that is abnormal in malignant tissues when compared with normal tissues . In comparison, most target drugs have shown limited efficacy against solid tumors, largely due to the fact that tumors frequently develop resistance to these therapies . In recent years, immunotherapy has had remarkable clinical success, including immune checkpoint blockade (ICB) and adoptive cell therapy. The antibodies targeting programmed cell death 1 (PD1), PD1 ligand 1 (PDL1), and cytotoxic T-lymphocyte-associated protein 4 (CTLA4) as ICBs have been approved for broad application to treat solid tumors . Anti-PD therapy dominates ICB therapies and has been shown to be superior to anti-CTLA4 therapy in a wide variety of tumors . However, the response rate of anti-PD therapy alone is usually only 20% in advanced-stage cancers, and adaptive immune resistance mechanisms also help cancer cells to escape attacks by the immune system. Thus, combining immunotherapy with other approaches to improve the anti-tumor effect is reasonable. Researchers have proposed the promising approach of utilizing DNA repair deficiency to enhance anti-tumor immunity . The DNA damage response (DDR) is essential for maintaining genomic stability by repairing different types of DNA damage . Cancer cells with high underlying levels of DNA damage are more dependent on DDR for survival when compared to normal cells . Deficiencies in DDR result in the accumulation of DNA damage and enhance immunogenicity in tumors. Numerous studies have identified that DNA damage agents modify systemic immune functions . In addition, clinical data show that a loss of mismatch repair could be a predictive biomarker for ICB response . Thus, combining DDR network inhibitors with immunotherapy attracts more attention to clinical testing. Here, we review the mechanism of DDR and discuss its interactions with anti-tumor immunotherapy. We also present the clinical implications of DDR, including combination with immune-oncology treatment in clinical trials and immune response prediction as a biomarker. Finally, we evaluate the opportunities and development of DDR-immunotherapy combinations in anticancer therapies. 2. DNA Damage and Repair Pathway DNA injuries occur as a result of intrinsic or extrinsic agents and can include modifications to bases and sugars, double-strand breaks (SSBs, DSBs), DNA-protein crosslinks, and base-free sites . While some specific DNA lesions can lead to mutations that cause cancer, the main consequence of DNA injuries is the threat they pose to DNA integrity and stability . To prevent accumulated DNA lesions from causing irreversible harm, cells initiate DDR, which senses the DNA damage, signals its presence, and mediates its repair. DDR kinases, including DNA-dependent protein kinase (DNA-PK), ataxia telangiectasia mutated (ATM), and ataxia telangiectasia and Rad3-related (ATR), are activated at DNA lesions, which then mediate cell cycle arrest and DNA repair . In the cell cycle arrest pathway, ATM and DNA-PK are mainly activated by DSBs, while ATR is activated by SSBs. These kinases phosphorylate downstream cell cycle checkpoint kinases. The active CHK1 and CHK2 then phosphorylate p53, CDC25, and WEE1, which increases the expression of p21 (p53), inhibits CDK activity and leads to cell cycle arrest at G1/S and G2/M transition (CDC25 and WEE1) . In addition, the molecular pathways of primary DNA repair mechanisms that function in common types of DNA damage are introduced below . 2.1. Base Damage Repair Base excision repair (BER): Base damage occurs when chemical bonds within the DNA molecule are formed abnormally. BER can remove a single damaged base. At the beginning of BER, a series of lesion-specific DNA glycosylases remove the damaged base by cleaving the N-glycosidic bond linking the base to its corresponding deoxyribose . Apurinic/apyrimidinic endonuclease 1 (APE1) and poly ADP-ribose polymerase 1 (PARP1) can sense and bind to the damage site. This catalyzes poly ADP-ribosylation (PAR) and some other protein substrates, which allows for the recruitment of repair proteins. The next synthesis/ligation step of BER is divided into two sub-pathways--short-patch and long-patch . In short-patch BER, the polymerase beta (Pol b) fills the generated gap with the correct nucleotide . The successive ligation of the DNA ends demands either DNA ligase I (LIG1) or the complex of DNA ligase III (LIG3) and X-ray repair cross-complementing protein 1 (XRCC1). In long-patch BER, proliferating cell nuclear antigen (PCNA), replication factor-C (RFC), flap endonuclease-1 (FEN1), Pol d/e, and LIG1 are included . 2.2. Bulky Base Damage Repair Nucleotide excision repair (NER): This pathway removes bulky lesions, which involves removing the damaged base and several adjacent nucleotides . The significant lesions initiating NER are pyrimidine dimers, such as cyclobutene pyrimidine dimers (CPD), and 6-4 photo-products induced by ultraviolet -DNA intra-strand crosslinks . In the recognition step, there are two different pathways, termed global genome NER (GG-NER) and transcription-coupled NER (TC-NER), whose recognition factor is the XPC/HR23B/CEN2 (XP complementation group C/Rad23 homolog B/Centrin-2) protein complex and CSA/B (Cockayne syndrome A and B, displacing the stalled RNA polymerase II), respectively . The following excision and polymerization steps are all the same. XPB and XPD orchestrate the asymmetric unwinding of the DNA helix, accompanied by XPA and RPA binding to the damaged region. Then, the structure-specific endonucleases XPG and XPF/ERCC1 lead to nucleotide excision. Lastly, the resulting gap is resynthesized by Pol d/e and sealed by LIG1 . Mismatch repair (MMR): This corrects mis-incorporated bases and strand crosslinks that occur during DNA replication. Defective MMR (dMMR) causes microsatellite instability (MSI) and an increased mutation frequency, which increases the risk of certain cancers such as Lynch syndrome and colon cancer. The MLH/MSH/PMS gene family plays a critical role in MMR . The MSH2-MSH6 heterodimer preferentially recognizes base-base mismatches and small insertion/deletion loops (IDLs), while the MSH2-MSH3 heterodimer recognizes larger IDLs. MLH1 and PMS2, which contain the primary endonuclease activity (~90%), facilitate downstream events. The degradation of the error-containing strand is performed by Exo1 . Then, polymerized DNA (synthesized by Pol d), accompanied by PCNA and RPA, resynthesizes a vast gap, and LIG1 or LIG4 seals the remaining nick . Inter-strand crosslink (ICL) repair: ICLs are a form of DNA damage in which two complementary DNA strands are covalently linked. To resolve ICLs, Fanconi Anemia (FA) proteins are primarily involved during the S phase of the cell cycle . FANCM and its interacting partners (FAAP24 and MFH) recognize the lesions and recruit the FA core complex and UBE2T/FANC, to monoubiquitinate the ID2 complex (FANCI and FANCD2 heterodimer) . Then, the monoubiquitinated central complex activates FANCP/SLX4-FANCQ/XPF to unhook ICLs, generating different types of lesions. These ICL-associated lesions are repaired by other DNA repair pathways, including translesion synthesis (TLS) and homologous recombination (HR) . 2.3. Translesion Synthesis TLS repair: TLS, an DNA damage tolerance mechanism, uses specialized DNA Pols to bypass DNA damage or fill single-strand DNA (ssDNA) gaps by inserting and/or extending nucleotides . It can be error-prone or error-free. Two models have been proposed to explain TLS: the Pol switching model and the gap-filling model . In the former, the inserter TLS enzyme (usually a Pol h, Pol i, or Pol j), which incorporates a nucleotide opposite the DNA lesion, is replaced by extender TLS enzyme (usually Pol z (REV3 and REV7), in some cases by Pol j) . The Rev1-Pol z complex is the most efficient among TLS Pols , initiated by monoubiquitinated PCNA . In the latter, TLS polymerases (Rev1, Rev3, etc.) repair ssDNA to protect cells from replication stress, though the exact order of events is still unknown . The TLS pathway has also been implicated in other DDRs, including HR, NER, and non-homologous end joining (NHEJ) . 2.4. SSB and DSB Break Repair SSB repair (SSBR): SSBs arise either directly or indirectly (e.g., during BER of base damage) . Therefore, SSBR shares several enzymatic steps with the BER pathway. In the long-patch SSBR pathway, SSBs are detected by PARP1, following end processing by APE1/PNKP (poly-nucleotide kinase 30-phosphate)/APTX (aprataxin). Next, FEN1 removes the damaged termini, following which Pol b and LIG1 repair the gap . Different from this, APE1 recognizes the lesion and LIG3 catalyzes ligation in the short-patch SSBR pathway, while TDP1 (tyrosyl-DNA phosphodiesterase 1) executes the end-processing function in the TOP1-SSB pathway . DSBs repair: The main processes are HR, single-strand annealing (SSA), classical NHEJ (cNHEJ), and alternative end joining (A-EJ) . HR repair is mostly error-free and only happens during the S phase and subsequent G2/M phases . Firstly, the Mre11-Rad50-Nbs1 (MRN) complex senses DSBs and stably recruits ATM , which can phosphorylate itself and downstream cellular targets, including MDC1. Then, RNF8 recognizes MDC1 and promotes the ubiquitylation of histone H1 . RNF168 recognizes ubiquitylated H1 and recruits BRCA1 and 53BP1 to mediate the HR and NHEJ pathways, respectively . In the next step, CtIP, Exo1, and BRCA1 are implicated in the DNA end resection. The emergent ssDNA protected by replication protein A (RPA), which BRCA2 displaces, invades duplex DNA molecules through the assistance of RAD51 and BRCA1-BARD1-PALB2. With sister chromatid DNA as a template, DNA Pol d/e chiefly mediates the nascent strand synthesis , while the SSA pathway directly joins two homologous 3' ssDNA ends after extensive DNA end resection and RPA displacement, requiring RAD52, XPF-ERCC1 and LIG1 . NHEJ does not require template DNA for repair, which distinguishes it from HR. It is an error-prone means of repair which can operate throughout the cell cycle. The Ku heterodimer (Ku70 and Ku80 subunits) is needed to recognize DSB termini . Then, DNA-PK is recruited by binding Ku80 . Finally, the XRCC4-XLF, Pol m, and LIG4 complex joins the DNA ends together to complete the damage repair . When the key NHEJ components are lacking, the A-EJ pathway, also known as microhomology-mediated end joining, is enhanced in the DDR . It requires PARP1 and Pol th (encoded by POLQ) to elicit the re-joining of the two DNA ends by using very short homologous sequences (2-20 bp). Due to the synthetic lethal relationship between HR and the A-EJ pathway, Pol th is a novel druggable target for cancer therapy . 3. The Interplay between DDR Deficiency and Immune Response Figure 2 depicts the complex interaction between DDR deficiency and immune response. 3.1. Innate Immune Response Genome instability is the hallmark of all forms of cancer , providing opportunities for intervention due to weak genome maintenance. DDR deficiency enhances genetic instability and imperfections , thus increasing endogenous nucleus-derived DNA generation in the cancer cell cytoplasm, which elicits an innate immune response. 3.1.1. Cytosolic DNA Generation The formation of cytosolic DNA includes cytosolic nucleosome-free DNA fragments, cytosolic chromatin fragments (CCF), and micronuclei (MN) , derived from nuclear DNA, mitochondrial DNA, or even extracellular nucleosomes as a result of DNA damage . However, the molecular mechanisms of cytosolic DNA accumulation are still under exploration. Defects in the DDR pathway cause replication forks to stall or collapse, leading to loss of chromosomal integrity maintenance and generating DNA fragments. For instance, a defect in MLH1 in the MMR system leads to a loss of regulation of Exo1. This causes unrestrained DNA end resection, leading to increased formation of ssDNA. Ultimately, this leads to chromosomal abnormalities and the release of nuclear DNA into the cytoplasm . Similarly, MRE11 excessively degrades unprotected newly replicated genomes following RAD51 or BRCA2 dysfunction, resulting in increased fragmentation of nascent DNA . On the other hand, the depletion of SAMHD1, which promotes the degradation of nascent DNA by stimulating the exonuclease activity of MRE11, leads to the release of ssDNA fragments . This suggests a double-edged sword characteristic. In PARP-dependent DNA repair pathways, DNA structure-specific endonuclease MUS81 (a member of the XPF family) cleaves aberrant DNA structures at sites of stalled replication forks to preserve genome integrity . Replication stress and unrepaired dsDNA also contribute to chromosomal instability . Deletion of the interferon-stimulated gene (ISG15), which plays critical roles in the DDR to modulate p53 signaling and error-free DNA replication, was associated with CCF formation . Interestingly, BLM RecQ-like helicase limits ISG induction to prevent genome instability . Meanwhile, chromosomal instability leads to a preponderance of MN , which also results in the persistence of unrepaired DSBs during mitosis . Mitochondrial DNA is also part of cytosolic DNA. Activation of intrinsic BAK and BAX-mediated apoptosis leads to the appearance of the BAK/BAX macropores, which allow the inner mitochondrial membrane to herniate into the cytosol, carrying matrix components, including the mtDNA . Aberrant mtDNA packaging can also promote its escape into the cytosol, such as the loss of function of TFAM, an mtDNA packaging protein, which can elicit moderate mtDNA stress . It is generally accepted that mtDNA activates DNA sensors upon its release into the cytoplasm . To prevent host cytosolic DNA from accumulating and being recognized by DNA sensors, DNases degrade DNA molecules to maintain homeostatic conditions. For instance, DNase II rapidly degrades DNA derived from pathogens or apoptotic cells within endolysosomes , and three prime repair exonuclease (Trex1), a major DNA-specific 3'-5' exonuclease in mammalian cells, degrades endogenous retroviruses and byproducts of DNA replication . 3.1.2. Cytosolic Nucleic Acid Sensing Pathway Pattern recognition receptors (PRRs), which include pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), detect cytosolic DNA and trigger innate immunity. When pathogenic nucleic acids are detected, the DNA sensor transduces a signal to the nucleus to produce proinflammatory cytokines. Among the downstream signaling for innate immune response, the cGAS-STING-IFN (cyclic GMP-AMP synthase, stimulator of interferon genes, interferon) pathway has been demonstrated to play an important role . Defects in SWI/SNF subunits, including PBRM1, ARID1A, and SMARCA4, lead to replication stress and accumulation of cytosolic DNA, which facilitates cGAS-STING pathway activation following DNA damage . Recognition of ruptured micronuclei or chromatin fragments by cGAS links genome instability to the innate immune response . Upon the binding of cytosolic DNA, cGAS, as one of the most significant PRRs, catalyzes the synthesis of cyclic-dinucleotide 2'3'-cGAMP (cGAMP), which binds to STING at which point STING translocates from the endoplasmic reticulum to the Golgi apparatus, activating a variety of downstream signaling molecules . It recruits and activates tank-binding kinase 1 (TBK1), which in turn phosphorylates STING to activate the interferon regulatory factor 3 (IRF3). Then, IRF3 translocases to the nucleus to induce ISGs and type 1 IFN (IFN-I) expression . In parallel, it also activates IKK, which triggers the nuclear factor kB (NF-kB) signaling pathway to produce IFN-I, ISG, and proinflammatory cytokines, such as tumor necrosis factor (TNF)-a, interleukin (IL)-1b and IL-6 . Considerable evidence now suggests that IFN-induced immune responses are crucial for cancer immunotherapy. The produced IFNs, binding to the heterodimer type I IFN receptors (IFNAR1/IFNAR2), activate the JAK/STAT signaling pathway on the dendritic cells (DCs) to produce ISGs and proinflammatory cytokines, such as IFNg and IP-10 (CXCL10), which influence adaptive immunity. IFNs also regulate the maturation, migration, and activation of various innate and adaptive immune cells, such as natural killer (NK) cells, macrophages, plasma B cells, CD8+ cytotoxic, and CD4+ helper T cells . Moreover, cGAS also localizes to the nucleus, where it plays a role in regulating the DDR. When in the nucleus, cGAS is recruited to dsDNA and interacts with PARP1 to suppress HR progression . It also acts as a decelerator of DNA replication forks to suppress replication-associated DNA damage . However, nucleosomes have a higher binding affinity for cGAS than dsDNA, but they have significantly lower potency for activating cGAS . The above suggests a complex connection between cytoplasmic and nuclear functions of cGAS in DDR-immunity interplay. There are several other cytosolic DNA sensors that regulate type I IFNs and cytokine production, including DDX41 , DDX60, IFNg-inducible protein 16 (IFI16) , DNA-PK, and MRE11, which converge on STING . Meanwhile, DNA-dependent activators of IFN-regulatory factors (DAI) directly trigger TBK1 activation . In addition, absent in melanoma (AIM2)-like receptors, sensing dsDNA activates the ASC/Caspase1 inflammasome pathway to produce IL-1b instead . There are also other pathways in which RNA polymerase III synthesizes 5'-PPP RNA from the AT-rich dsDNA or RNA: DNA hybrid, which induces IFN-b through the RIG-I (retinoic acid-induced gene I)- MAVS (mitochondrial antiviral signaling) pathway . 3.1.3. Other Mechanisms Furthermore, DDR activation prevents tumor cells from evading immunosurveillance of NK cells and/or CD8(+) T cells by shedding membrane ligands (through poorly understood mechanisms). Stimulation of ATR or ATM--major DNA damage checkpoints--can upregulate the ligands, which activate NKG2D receptors to alert the innate immune system . Likewise, inhibition of DNA damage pathway components can also prevent the upregulation of major histocompatibility complex class I-related molecules A and B (MICA and MICB), which serve as membrane ligands . Cellular senescence could be triggered by DNA damage, causing mammalian cells to enter an irreversible growth arrest that prevents abnormal cells from proliferating. This process is dependent on DDR regulators such as ATM/ATR, as well as the p53/p16 (INK4a) dependent pathway . One key feature of senescence is the senescence-associated secretory phenotype (SASP), which involves the expression and secretion of various proinflammatory cytokines and chemokines. These secreted factors can stimulate the immune system and promote chronic inflammation either directly or indirectly, offering potential therapeutic opportunities . 3.2. Adaptive Immune Response 3.2.1. Influence Tumor Antigenicity Defects in DNA repair may increase the number of neoantigens in the tumor. For instance, low BER/SSBR gene expression leads to high neoantigen production, which enables a higher probability of recognition by the immune system . The number of neoantigens is directly proportional to the number of non-synonymous mutations, which would be increased by a deficiency of multiple DNA repair pathways, including MMR, POLE/POLD1 (encoding the catalytic and proofreading subunits of Pol e and Pol d), and HR . Studies have shown that neoantigen-reactive T cells may be a key factor in the effectiveness of immunotherapy, particularly in tumors with a high tumor mutational burden (TMB). Cancer-associated antigens, including neoantigens derived from genetic alterations, are presented to CD8+ T cells through the major histocompatibility complex (MHC) on DCs, and professional antigen-presenting cells (APCs). However, most neoantigens are usually not recognized by the immune system, so identifying highly tumor-specific antigens is crucial for the development of personalized immunotherapy . Recent technological advances allow new strategies to emerge in predicting, identifying, and validating neoantigens, with the ultimate goal of creating personalized vaccines for cancer treatment . Additionally, tumors with high TMB resulting from dysfunction in the DDR process may have better clinical outcomes when treated with ICBs such as CTLA4 and PD1 in certain types of cancer . This has been retrospectively validated in patients with advanced lung cancer, gastrointestinal carcinomas, ovarian cancer, skin melanomas, and glioma , suggesting that increased neoantigen burden is a predictive factor for a better outcome when using ICBs . On the other hand, tumor aneuploidy, which is derived from chromosome instability, also provides an independent prognostic value as a biomarker . A higher aneuploidy score is associated with poor prognosis among patients with lower-TMB (<80th percentile) tumors treated with immunotherapy and non-small cell lung cancer (NSCLC) treated with radiotherapy and ICB . Now, aneuploidy has been determined to affect immune cell action against the tumor adversely. However, the mechanisms underlying this observation are not well understood, with one proposed explanation being that most tumors with extensive aneuploidy often have fewer infiltrating immune cells . 3.2.2. Immune Checkpoint Interaction DDR defects have been shown to modulate the expression of immune checkpoints and other co-stimulatory molecules. PDL1 is one of the hot spots for immune checkpoint blockade, with links to DDR defects. Specifically, tumoral PDL1 expression is more common in dMMR cancers relative to MMR-intact tumors, which have been identified in colorectal and endometrial carcinomas . Nevertheless, the loss of MMR proteins seems to be less correlated with tumoral PDL1 expression in breast carcinoma, where MMR gene mutations are less common . PDL1 is primarily induced by IFNg through the JAK1/JAK2-STAT1/STAT2/STAT3-IRF1 axis . This pathway is activated by innate immunity in response to damaged DNA . PARP inhibitors (PARPi) have been shown to potentiate IFN-g-induced PDL1 expression in NSCLC cell lines and pancreatic cancer . PDL1 upregulation, mediated by DNA damage signaling , has been linked to ATM/ATR-CHK1 pathway activation in BRCA2-depleted cells, for example , or the cGAS-STING-TBK1-IRF3 pathway . Furthermore, the greater release of DAMPs from excessive DNA damage promoted by DDR deficiency could also upregulate PDL1 expression in the neighboring surviving tumor cells, due to the TLR4/MyD88/TRIF signaling mediated by HMGB1 . Expression of PDL1 in tumors can serve as a potent mechanism for potentially immunogenic tumors to escape from host immune responses by negatively regulating T-cell antigen receptor signaling by binding PD1 . Finally, blockading PDL1-PD1 binding may result in the remission of advanced-stage cancer, although it does not necessarily mean that PDL1+ tumors have higher response rates . On the other hand, intracellular PDL1 can protect the mRNA of NBS1, BRCA1, and other DNA damage-related genes from degradation, thereby increasing cellular resistance to DNA damage . Moreover, the expression of a co-stimulatory molecule related to DDR has implications for the immune system, as it is required to activate CD8+ T cells . Co-stimulatory B7-1/B7-2 signals on antigen-presenting cells, which interact with CD28 molecules on the T-cell surface, may induce clonal expansion and activation of cytotoxic T cells (CTLs). Increasing CD8(+) CD28(-) T-cell apoptosis compared to CD8(+) CD28(+) T cells is correlated with an impaired DDR following treatment with etoposide, a topoisomerase II inhibitor . Similarly, CTLA4 can exacerbate the DDR and induces T-cell apoptosis . 3.2.3. Induce Immunogenic Cell Death Fas ligand (FasL/CD95L), triggering apoptotic cell death following ligation to Fas (CD95/APO-1), helps to maintain tumor cells in a state of immune privilege by inducing apoptosis of anti-tumor immune effector cells . Therefore, FasL in tumor cells may decrease lymphocyte infiltration, reduce anti-tumor immunity in vivo and promote tumor development . Conversely, Fas expression in various human cancer cells enhances the anti-tumor efficiency of CD8+ T or NK cells. In human colon cancer cohorts, Fas expression has been strongly correlated with dMMR and MSI-high (MSI-H) tumors, and it also induced senescence caused by chronic DNA damage . 3.2.4. Role in Immunogenic Diversity DDR kinases activated by purposeful genotoxic insults can regulate cell type-specific processes: variable gene segment recombination (VDJ), class-switch recombination (CSR), and somatic hypermutation (SHM) . These processes are required for the normal development and function of immune responses , in which programmed DNA damage occurs at a specific site . Multiple components of the DDR pathway are involved with these intermediates. For instance, DNA-PK, XLF4, SHLD1, and LIG4 participate in RAG-induced (in VDJ) or AID-initiated (in CSR) DSBs repair . During SHM, error-prone non-canonical BER and/or MMR help to diversify mutations in the variable region of immunoglobulin genes to create high-affinity antibodies . DNA repair is critical for antibody diversification and influences the development of the adaptive immune system . Disturbances in the balance between enzymatic mutagenesis and DNA repair are at the basis of lymphoid malignancies . This raises the intriguing possibility that therapeutic agents that target DDR proteins may be used to manipulate immune responses. 4. Combining DDR Inhibition and Immunotherapy 4.1. Potential Mechanism and Clinical Implication Tumor immunotherapy, including ICB and adoptive cell transfer, can manipulate specific components of the immune system to reverse immunity suppression and target various cancers. PD1/PDL1 inhibitors and CTLA4 inhibitors have shown encouraging therapeutic effects in these approaches . Nevertheless, only a minority of cancer patients respond to ICB in the clinic. Even among dMMR/MSI-H mCRC (metastatic colorectal cancer) patients for whom PD1 blockade is a guideline-recommended, first-line treatment option, response rates range between 30% and 50% . These data suggest the existence of intrinsic resistance mechanisms, which are often contingent on the tumor microenvironment (TME) . As a consequence, the development of novel therapeutic designs, as well as the discovery of biomarkers, are currently areas of intense research activity . Combination regimens of traditional DNA-damaging approaches, such as chemotherapy drugs and radiotherapy, have been shown to enhance immunity by increasing antigens to stimulate T-cell-mediated immunity and modulating certain aspects of the immunosuppressive milieu . Moreover, there is evidence to suggest that lower DDR factor expression in tumors may be associated with a better response to anticancer immunity, implying substantial potential benefits from DNA repair inhibitors . Thus, there is considerable interest in combining ICB with DDR inhibition (DDRi), in order to enhance genomic instability and immunotherapy activity and potentially achieve additional anti-tumor responses . 4.2. Treatment Strategies for Combining DDR Targets DDR kinase inhibitors, such as those targeting PARP, ATM, ATR, DNA-PK, CHK1/2, BER, and WEE1, have been tested in clinical trials as a way to kill tumor cells, as cancer cells are more sensitive to compromised repair systems compared to normal cells (Table 1) . With the expectation that the combination of DDRis with ICBs will show high potency, multiple studies exploring this combination are ongoing (Table 2). An archetypal example is PARPi, which have shown significant therapeutic efficacy in BRCA-deficient cancers by blocking BRCA-independent DNA repair in ovarian and breast cancer . However, PARPi have only improved progression-free survival without reaching statistical significance in cancer-specific mortality in patients with germline BRCA mutations . ICB has been proposed to optimize these clinical outcomes. In the BRCA1(-) tumor model, CTLA4 blockades combined with PARPi induce protective anti-tumor immunity and significant survival benefit by locally inducing anti-tumor immunity and increasing levels of IFNg . Accumulating evidence has also suggested that olaparib, a type of PARPi, triggers robust local and systemic anti-tumor immunity through a STING-dependent anti-tumor immune response independent of BRCA deficiency. This response can be further augmented by combining olaparib with PD1 blockade . The clinical results of combining PARPi with an ICB, such as in advanced triple-negative breast cancer and advanced or metastatic non-small cell lung cancer , support further research on using this strategy in various cancers. Moreover, in PARP inhibitor-resistant cancers, PARG inhibitors may impair cancer cell survival by suppressing replication fork progression and show comparable killing ability . This offers the potential for combining PARG inhibitors with ICB. Although other DDRis are being evaluated as monotherapies or in combination with cytotoxic or molecularly targeted agents in solid tumors, only a few early-phase trials currently focus on combining them with ICB . Clinical trials with AZD6738 , an ATR inhibitor, and AZD1775 (NCT02617277), a WEE1 inhibitor, individually as well as in combination with durvalumab in patients with advanced cancers, are currently ongoing. New DDRis are being developed, such as WRN inhibitors, which have shown promising synthetic lethal interaction with MSI tumors . As dMMR cancers are exceptionally responsive to ICB , the viability of WRN inhibition plus ICB deserves further exploration. 4.3. DDR-Related Biomarkers for Predicting Immune Response As our understanding of the relationship between DDR and immune responses continues to grow, it is expected that additional DDR-related biomarkers will be identified to predict a patient's response more accurately to immunotherapy. Several clinical trials have demonstrated that dMMR/MSI-H is significantly associated with long-term responses to immunotherapy and better prognosis in colorectal and non-colorectal malignancies treated with ICBs. Compared to chemotherapy, pembrolizumab has fewer treatment-related adverse events without compromising overall survival, supporting it as an efficacious first-line therapy . In practice, pembrolizumab (anti-PD1) has been approved for dMMR/MSI-H refractory or metastatic solid tumors, and nivolumab (anti-PD1) for dMMR/MSI-H CRC . One plausible hypothesis is that dMMR contributes to high TMB, though the specific mechanisms remain unclear . TMB also has emerged as a promising biomarker of immunotherapy response across multiple cancer types. A high TMB may be a biomarker for identifying patients who will benefit from ICBs, irrespective of PDL1 expression level . In many cases, TMB is a more reliable predictive marker for PD1 and PDL1 blockade immunotherapy response than PD1 or PDL1 expression; for example, the presence of ten or more mut/Mb was associated with improved response and prolonged progression-free survival, irrespective of tumor PDL1 expression in NSCLC . Though higher TMB has been reported frequently in tumors with deleterious DDR gene alterations, mutations in different types of DDR pathways do not always exhibit high mutational load. In addition, clinical outcomes among patients with low TMB tumors are heterogeneous, with TMB status showing no ability to predict ICB-response in melanoma patients . Recently, DDR scores quantifying the tumor signature of DDR pathways in tumors have provided new insights for guiding immunotherapeutic strategies. This is because DDR scores are not just closely associated with TMB and genome alteration, but also provide information regarding real-time DNA repair function . There is evidence that patients with low DDR pathway signature scores might not benefit from a monoclonal anti-PD1 therapy, making these scores potentially useful for predicting treatment response in tumor tissues . Similarly, studies have found that patients with high DDR scores have significantly higher survival rates after receiving ICBs compared to those with low DDR scores, while the reverse is true for traditional treatments . Furthermore, tumor aneuploidy has been found to predict prognosis independently among patients with lower TMB (<80th percentile) tumors treated with immunotherapy . 5. Conclusions and Future Prospects It has been reported that DDRi can enhance immune signaling within the TME and complement neoantigens. However, different forms of DDR defects may have varying effects on tumor immunogenicity. DDRi may not generate sufficient neoantigens in tumors with low neoantigen burden to stimulate an immune response. Meanwhile, it can also be challenging to reduce the immune-suppressive effects of DDRi. For example, PARPis and ICB have not produced dramatic responses in patients with HR-deficient high-grade serous ovarian cancer, as PARPis can mediate immune resistance and tumor progression by upregulating VEGF-A. This has led to the development of combination therapies using PARPis, ICB, and bevacizumab (anti-VEGF) . Combining DDRi with ICB, radiotherapy, or chemotherapy may also be a promising means for achieving a favorable balance between immunogenicity and TME. DNA-PK inhibitors are being studied in combination with radiation and ICB in clinical trials (NCT04068194, NCT03724890). It is important to pay careful attention to specific therapeutic approaches for combination treatments. Optimizing the dose and schedule of DDRi agents may allow for increased tumor damage while sparing normal tissue by taking advantage of the differences in DDR and immune response between cancer and normal cells. The order in which combination drugs are administered and the line of therapy should also be considered. It is important to consider the toxicities of combination treatments versus monotherapy, as these can limit the development of combination therapies. Some DDR members are broad-spectrum and are necessary for maintaining homeostasis in normal tissues, which means that severe adverse events may occur when combined with ICBs. This is also the reason why many DDRis are eliminated in preclinical or phase I clinical trials. To optimize the use of combination therapies involving DDRis and ICBs, more specific and sensitive biomarkers are needed to identify the most suitable patient population and predict treatment outcomes. DDR scores are likely to be important predictive factors, but the definition of DDR deficiency genes varies across different tumor types. It is controversial as to which mutated genes (distinguished as heterozygous or homozygous, germline or somatic) should be used to characterize DDR status in tumors. Under conditions of active anti-tumor immunity, DDR scores have been found to positively correlate with immune-related biomarkers, such as the number of T cells (such as CD4+ activated memory cells, CD8+ cells), T-cell receptor repertoire, PDL1 expression, and broad immune infiltrate. Thus, integrating immune biomarkers into the DDR score may improve its predictive ability. In this review, we discuss the classical mechanism of DDR and its interplay with the immune system. We also present a compilation of studies on the combination of DDRi and ICBs for various cancer types, with the goal of inspiring new ideas for improving the efficacy of anti-tumor therapies and sparking innovation. While combination therapy has achieved impressive results in the clinic, increasing the success rate of treatment remains a challenge, and the rate of failure is still relatively high. A deeper understanding of the role of DDR in the immune system will be crucial for the design of future clinical trials. Author Contributions All authors (Y.X., S.N., M.D.) contributed to the writing, reviewing and editing of the manuscript. All authors have read and agreed to the published version of the manuscript. Conflicts of Interest The authors declare no conflict of interest. Figure 1 The molecular pathways of DNA damage repair. (a) BER can remove a single damaged base and is divided into two sub-pathways: short-patch (SP-BER) and long-patch (LP-BER). (b) For bulky base damage, NER removes the damaged base and several adjacent nucleotides, through global genome NER (GG-NER) and transcription-coupled NER (TC-NER); MMR corrects mis-incorporated bases and strand crosslinks; ICL repair, also known as FA pathway, resolves the covalently linked DNA strands. (c) TLS repair uses specialized DNA Pols to bypass DNA damage or fill single-strand DNA gaps by inserting and/or extending nucleotides, via Pol switching model and the gap-filling model. (d) SSBR repair shares most enzymatic steps with BER pathway; the main repair processes for DSB are HR, SSA, cNHEJ, and A-EJ. (A-EJ: alternative end joining; BER: base excision repair; cNHEJ: classic non-homologous end joining; DSB: double-strand break; HR: homologous recombination; ICL: inter-strand crosslink; MMR: the Mismatch Repair; NER: nucleotide excision repair; NHEJ: non-homologous end joining; SSBR: single-strand break repair; TLS: Trans lesion synthesis). Figure 2 The interaction between DNA damage response (DDR) deficiency and immune response. The cytosolic DNA fragments, derived from nuclear, mitochondrial, or even extracellular DNA, are detected by sensors, including cGAS, DDX41, DDX60, IFNg-inducible protein 16 (IFI16), DNA-PK, MRE11, and RNA polymerase III (Pol III). The cGAS-STING-IFN pathway plays an important role in innate immune response, which activates interferon regulatory factor 3 (IRF3) and nuclear factor kB (NF-kB) to produce IFN, ISG, and other cytokines, such as tumor necrosis factor (TNF)-a, interleukin (IL)-1b and IL-6. In other pathways, RNA polymerase III synthesizes 5'-PPP RNA from the AT-rich dsDNA or RNA: DNA hybrid to induce IFN-b through the RIG-I (retinoic acid-induced gene I)- MAVS (mitochondrial antiviral signaling) pathway. The produced IFNs activate JAK/STAT signaling pathway on the dendritic cells (DCs) to produce ISGs and proinflammatory cytokines and it also regulates many innate and adaptive immune cells, such as natural killer (NK) cells, CD8+ cytotoxic and CD4+ helper T cells. DDR deficiency also increases neoantigen and senescence-associated secretory phenotype (SASP) production. DDR process dysfunction can regulate ligand expression, including programmed cell death ligand 1 (PD-L1), Fas, CD80, and major histocompatibility complex class I-related molecules A and B (MICA and MICB) to alert the innate and adaptive immune systems. The potential molecular mechanisms of combining DDR target and immunotherapy are shown in the text box: (1) DDR deficiency increases cytosolic DNA generation, which elicits an innate immune response; (2) DDR activation prevents tumor cells from evading immunosurveillance by NK cells and/or CD8(+) T cells by shedding membrane ligands; (3) DNA damage triggers cellular senescence and promotes chronic inflammation; (4) DDR deficiency increases the number of neoantigens of the tumor, which enables a higher probability of recognition by the immune system; (5) DDR modulates the expression of immune checkpoints and other co-stimulatory molecules to lead immune escape; (6) DDR is critical for antibody diversification and influences the development of the adaptive immune system. cancers-15-01619-t001_Table 1 Table 1 DDR inhibitors in clinical trials. Target Drug Tumor Type PARP Olaparib, Niraparib, Veliparib, Rucaparib TNBC, SCLC, NSCLC, ovarian cancer, bladder cancer, prostate, colorectal cancers, pancreatic cancer, advanced solid tumors ATM AZD-1390, M-4076, XRD-0394, AZD0156, M3541 Solid tumors ATR Berzosertib, Ceralasertib, RP-3500, ART-0380, ATRN-119, M-4344, M-1774, Elimusertib Ovarian, Advanced NSCLC, SCLC, Gynae or neuroendocrine, HNSCC, relapsed CLL, PLL, B-cell lymphomas DNA-PK M3814, AZD-7648, CC-115, BR2002, BR101801 GBM, HNSCC, prostate, ES, CLL MEK1/2 Selumetinib, Binimetinib, Cobinetinib, Trametinib Melanoma, colorectal cancer, NF1-associated neurofibroma WEE1 Adavosertib, ZN-c3, IMP7068, SY-4835, Debio0123 Endometrial serous carcinoma, osteosarcoma, solid tumor, NSCLC, gastric carcinoma, AML, other myeloid malignancies CHK1/2 MK8776, LY2603618, CCT245737, LY2606368 NSCLC, refractory SCLC, relapsed AML, relapsed lymphoma, pancreatic carcinoma, ovarian, breast, prostate, pediatric solid tumors BER TRC102 GBM, lymphoma, hematologic malignancies, NSCLC Abbreviation: AML, acute myeloid leukemia; CLL, chronic lymphocytic leukemia; DDRi, DNA Damage Response inhibition; DLT, dose-limiting toxicity; ES, Ewing sarcoma; GBM, glioblastoma multiforme; HNSCC, head, and neck squamous cell carcinoma; ICB, immune checkpoint blockade; NSCLC, non-small cell lung carcinoma; ORR, overall response rate; OS, overall survival; PFS, progression-free survival; PLL, prolymphocytic leukemia; SCLC, small cell lung carcinoma; TNBC, triple-negative breast cancer. cancers-15-01619-t002_Table 2 Table 2 Clinical Trials Using immune checkpoint blockade (ICB) + DNA Damage Response inhibition (DDRi). (accessed on 1 November 2022)). ICBs DDRi Study Identifier (Status) Tumor Type Phase Primary Endpoint Pembrolizumab Niraparib NCT02657889 (completed) Ovarian, advanced TNBC I/II DLTs, ORR NCT04475939 (recruiting) Advanced or metastatic NSCLC III 3-year PFS, 5-year OS NCT03308942 (completed) NSCLC II ORR Olaparib NCT04191135 (not recruiting) TNBC II/III PFS NCT04548752 (recruiting) Inherited BRCA-mutation pancreatic cancer II PFS NCT02861573 (recruiting) Metastatic castration-resistant prostate cancer Ib/II ORR Nivolumab Rucaparib NCT03338790 (not recruiting) Metastatic castration-resistant prostate cancer I ORR NCT03522246 (not recruiting) Ovarian cancer III 7-year PFS NCT03572478 (terminated) Prostate or endometrial cancer Ib/II DLTs Veliparib NCT02944396 (completed) Metastatic or advanced NSCLC II PFS Camrelizumab (SHR-1210) Apatinib NCT03394287 (completed) Advanced TNBC II ORR Tislelizumab (BGB-A317) Pamiparib (BGB-290) NCT02660034 (completed) Advanced solid tumors I AEs Dostarlimab (TSR-042) Niraparib NCT03307785 (not recruiting) Solid tumor I/II DLTs NCT03602859 (not recruiting) III or IV nonmucinous epithelial ovarian cancer III 5-year PFS Durvalumab Olaparib NCT03167619 (completed) Advanced TNBC II 1-year PFS NCT02734004 (not recruiting) Ovarian, breast, SCLC, gastric cancers I/II Disease control rate NCT02546661 (not recruiting) Muscle-invasive bladder cancer I AEs NCT03459846 (not recruiting) Urinary bladder neoplasms II PFS NCT02484404 (recruiting) Recurrent ovarian cancer I/II ORR NCT03334617 (recruiting) Advanced NSCLC II ORR Durvalumab Olaparib NCT03534492 (completed) Resectable urothelial bladder cancer II Pathological complete response rate Durvalumab+ Tremelimumab Olaparib NCT02953457 (not recruiting) Recurrent or refractory ovarian, fallopian tube, or primary peritoneal cancer with BRCA1 or BRCA2 mutation II DLTs Durvalumab Olaparib NCT03851614 (not recruiting) Mismatch repair proficient colorectal cancer, pancreatic adenocarcinoma, leiomyosarcoma II Changes in genomic and immune biomarkers Ceralasertib NCT02264678 (recruiting) Advanced Solid Tumors I/II AEs Olaparib NCT02484404 (recruiting) Advanced Solid Tumors and Advanced or Recurrent Ovarian, TN BC, Lung, Prostate, and Colorectal Cancers I/II ORR Adavosertib (AZD1775) NCT02617277 (not recruiting) Advanced solid tumors I DLTs Ipilimumab or Nivolumab Niraparib NCT03404960 (not recruiting) Pancreatic adenocarcinoma I/II PFS Avelumab Talazoparib NCT03330405 (not recruiting) Locally advanced (primary or recurrent) or metastatic solid tumors Ib/II DLTs NCT03565991. (not recruiting) BRCA1/2 or ATM alterations tumor II Confirmed Objective Response NCT03637491 (terminated) Locally advanced or metastatic RAS-mutant solid tumors Ib/II DLTs NCT03642132 (completed) Ovarian cancer III PFS NCT03964532 (not recruiting) Advanced breast cancer I/II AEs Atezolizumab Olaparib NCT02849496 (not recruiting) Mutant Non-HER2-positive breast cancer II PFS Rucaparib NCT03101280 (completed) Advanced gynecologic cancers, TNBC IB AEs Niraparib NCT03598270 (not recruiting) Recurrent ovarian cancer III PFS Tremelimumab Olaparib NCT02571725 (not recruiting) BRCA-deficient ovarian cancer I/II DLT Abbreviation: ICBs, immune checkpoint blocks; DDRi, DNA damage response inhibitors; DLTs, dose-limiting toxicities; ORR, overall response rate; TNBC, triple-negative breast cancer; NSCLC, non-small cell lung cancer; PFS, Progression-Free Survival; AEs, adverse events. 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PMC10000855 | Healthcare (Basel) Healthcare (Basel) healthcare Healthcare 2227-9032 MDPI 10.3390/healthcare11050742 healthcare-11-00742 Article The Price to Pay for Being Yourself: Experiences of Microaggressions among Non-Binary and Genderqueer (NBGQ) Youth Arijs Quinn Conceptualization Methodology Formal analysis Investigation Resources Data curation Writing - original draft Writing - review & editing 1* Burgwal Aisa Conceptualization Methodology Resources Supervision Project administration 1 Van Wiele Jara Resources Supervision 1 Motmans Joz Supervision Project administration Funding acquisition 2 Dettori Marco Academic Editor 1 Transgender Infopunt, Ghent University Hospital, 9000 Ghent, Belgium 2 Center for Sexology and Gender, Ghent University Hospital, 9000 Ghent, Belgium * Correspondence: [email protected] 03 3 2023 3 2023 11 5 74206 1 2023 26 2 2023 01 3 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). This study explores the experiences of NBGQ youth with microaggressions. It investigates the types of microaggressions they face and their subsequent needs and coping mechanisms and the impacts on their lives. Semi-structured interviews with ten NBGQ youth in Belgium were conducted and analyzed using a thematic approach. The results showed that experiences of microaggressions were centered around denial. The most common ways to cope were finding acceptance from (queer) friends and therapists, engaging in a conversation with the aggressor, and rationalizing and empathizing with the aggressor, leading to self-blame and normalization of the experiences. Microaggressions were experienced as exhausting, which influenced the extent to which the NBGQ individuals wanted to explain themselves to others. Furthermore, the study shows an interaction between microaggressions and gender expression, in which gender expression is seen as a motive for microaggressions and microaggressions have an impact on the gender expression of NBGQ youth. NBGQ non-binary genderqueer microaggressions gender expression Department of Equal Opportunities of the Agency for Internal Administration of the Flemish GovernmentABB/GKII/GK/2020/002 This research was funded by Department of Equal Opportunities of the Agency for Internal Administration of the Flemish Government, project scope statement number ABB/GKII/GK/2020/002. pmc1. Introduction A transgender* person is a person who does not identify with their assigned sex at birth, and the term is used to describe people who do not identify as cisgender . Transgender* includes binary (trans man/woman) and non-binary or genderqueer identities. The terms "trans" or "transgender" often get used in a confusing manner to signify trans men and women, while the transgender* umbrella includes a diversity of identities, such as non-binary, genderqueer, agender, genderfluid, polygender, and more . When "trans" is used in research to mean trans men and women or the general trans* umbrella is used, it is often not clear what is being referred to. For this reason, we use the term "trans*", in which the asterisk signifies that it is used as an umbrella term to emphasize inclusion of a range of gender identities . Non-binary and genderqueer identities comprise multiple gender identities that are not (merely or completely) male or female and fall outside the gender binary . Richards et al. state that people with non-binary or genderqueer identities may identify as both male and female at one time, as different genders at different times, or as no gender at all, or they may dispute the very idea of there being only two genders. Non-binary and genderqueer are used as umbrella terms for various identities, such as pangender (a person whose gender identity reflects multiple gender identities ), agender (a person who experiences a lack of gender or does not feel any connection to masculinity or femininity ), or genderfluid (people who experience their gender identity as shifting and changing ). This list is not exhaustive but gives some examples of what a non-binary or genderqueer identity can mean. For this research, non-binary and genderqueer were defined as people who do not identify as (only or entirely) a man or woman. This is based on self-identification. To talk about identities under the trans* umbrella that are not binary, we use the term "non-binary and genderqueer" (NBGQ) (except when referring to the work of other authors; in this case, their own terminology is used). While "genderqueer" is associated with political statements against the gender system and "non-binary" is a more apolitical statement of identity, both have been used as umbrella terms to include a variety of gender identities . In this paper, we chose to use both terms simultaneously, using the abbreviation NBGQ to emphasize the variety of gender identities and to avoid valuing one term over another. This abbreviation has been increasingly used in research over recent years . According to the review by Zhang et al. , the proportions of people identifying as (binary) transgender are 0.3-0.5% among adults and 1.2-2.7% among children and adolescents. Including gender diversity, the proportions increase to 0.5-4.5% among adults and 2.5 to 8.4% among children and adolescents, which indicates a rise in the proportion of trans* and gender-diverse people . Additionally, an online survey indicates a growing proportion of young trans* people (14-25 years old) identifying as non-binary . In Belgium, the estimate for the proportion of gender-incongruent (=binary trans) people is 0.6-0.7% based on a representative population sample . Additionally, Van Caenegem et al. estimated the proportion of gender-ambivalent (=NBGQ) people to be 1.9-2.2%. In a Dutch study, 0.8-1.1% of people were reported to be gender-incongruent, while 3.2-4.6% of people were reported to be gender-ambivalent . A study by the European Union Agency for Fundamental Rights , using a probability sample, estimated that 23% of European trans* people identify as non-binary, 9% as genderqueer, 11% as genderfluid, 7% as agender, and 1% as polygender. Another probability sample from Belgium in a study by Motmans et al. estimated the amount of trans* people who identify as non-binary to be 25%. Lastly, in the Canadian census, among 30.5 million Canadians, 0.33% self-identified as transgender or non-binary . 1.1. Trans* People and Violence Experiences Among European and Belgian lesbian, gay, bisexual, trans, and intersex people (LGBTI) (various umbrella terms are used to refer to lesbian, gay, bisexual, trans, and intersex people; in this article, multiple abbreviations are used, such as LGBT, LGBTI, LGBTI+, LGBTQ, and LGBTQIA+, as they were used by the original authors), 11% and 14%, respectively, have been attacked physically or sexually in the past 5 years . For transgender people, these proportions amount to 17% in the EU and 19% in Belgium . Up to 48% of transgender people have faced harassment and 50% in Belgium, which is the highest proportion among all subgroups . Belgium has the second highest numbers of reported cases of people suffering non-verbal in-person harassment due to being LGBTI in the EU (28%), with a higher percentage (35%) for trans people . Although trans men and women's experiences with violence have been widely documented, NBGQ people's experiences with violence have not gained much attention. Most existing research focuses on binary transgender people alone, on comparisons between binary and NBGQ transgender people, or on the general trans* population, making it difficult to ascertain the specific experiences and needs of non-binary and gender-diverse people . Additionally, Aparicio-Garcia et al. indicate that trans* people experience more violence than non-trans* people, with non-binary and gender-diverse people facing the worst outcomes and the highest numbers of traumatic experiences . In Belgium, 5-9% of NBGQ people were found to have had experiences of physical or sexual violence in the year prior to the study due to being LGBTI . The more subtle the experiences were, the more NBGQ people had experienced them: 23-32% of NBGQ people in Belgium experienced verbal in-person harassment and 22-49% non-verbal in-person harassment in the previous year due to being LGBTI . Subtle forms occurred more often: 33-36% of the NBGQ people who had experienced offensive gestures and 21% who had experienced insults or name-calling had encountered these experiences more than six times in the previous year . Additionally, Motmans et al. indicate that most transgender people face verbal or psychological violence (80%), while a smaller proportion face sexual (32%), physical (27%), and material (18%) violence. However, research often focuses on physical and sexual violence, while verbal or psychological violence remain underexplored, possibly because they are harder to grasp or because people may not categorize these acts as "violent enough". 1.2. Microaggressions In this context, this research focused on more subtle verbal and psychological forms of violence. These experiences are often categorized under the term "microaggressions", a concept that originated in racial discrimination research. Racial microaggressions were defined as verbal and physical racial attacks and covert behavioral and verbal comments that demean a person's racial identity or invalidate minorities' thoughts and experienced realities . The concept has evolved over time and recently it has been applied to LGBT people . Microaggressions refer to subtle, covert, everyday experiences of discrimination against marginalized groups that can be conscious or unconscious and can include behaviors and comments--both verbal and psychological--that invalidate someone's identity or thoughts . However, research on LGBT people and microaggressions has been focused on workplace settings and experiences of genderqueer people and trans men and women, or genderqueer people and the general LGBTQ population have often been mixed . A literature review by Nadal et al. on microaggressions and the LGBTQ community found no publications on genderqueer or gender-nonconforming people and microaggressions. Most studies were undertaken on microaggressions and LGB people, the general LGBTQ population, and the general trans* population . It is thus hard to deduce NBGQ people's specific experiences and needs; therefore, a study on their experiences of microaggression is needed. Three forms of microaggressions exist: micro-assaults (overt verbal or nonverbal insults and behaviors), micro-insults (statements or actions that demean a person's identity), and microinvalidations (negating or nullifying minority groups' psychological thoughts, feelings, or experiential reality) . In addition, Nadal et al. identified 12 themes in the experiences of microaggressions among trans* people: using transphobic or incorrectly gendered terminology, the assumption of a universal transgender experience, exoticization, discomfort around/disapproval of transgender experience, endorsement of gender-normative and binary culture or behavior, denial of the existence of transphobia, assumptions of pathology or abnormality, physical threats, denial of individual transphobia, denial of bodily privacy, familiar microaggressions, and systemic microaggressions regarding the use of public restrooms, the criminal justice system, healthcare, and identification documents. For the purpose of this research, we defined microaggressions as forms of psychological and verbal violence manifesting as the more subtle and day-to-day experiences of violence, such as (but not limited to) misgendering, deadnaming, denial of identity, making someone feel ashamed of or bad about themselves, and inappropriate remarks or jokes. Due to the nature of microaggressions as being subtle and covert, not everyone may identify these occurrences as microaggressions. While these occurrences can be objectively defined as microaggressions, the subjective experience of these occurrences as microaggressions is different to different people. In this research, we only included microaggressions that were defined as such by participants; however, this does not mean that certain occurrences that are not defined as such could not still be categorized as microaggressions. 1.3. Additional Challenges of NBGQ People NBGQ people face additional challenges when dealing with microaggression. For example, the research by Kiekens et al. indicate a differences in the experiences of microaggressions among sexual minority and gender minority groups, with gender minority youth experiencing more familial microaggressions, invalidation of LGBTQ identity, and threatening behaviors. 1.3.1. Misgendering, Deadnaming, and Authenticity A typical form of microaggression against trans* people is minimalizing or denying the person's experiences, such as misgendering or deadnaming . Stating that the person is not a real man, woman, or NBGQ person counts as psychological abuse . The research by Pulice-Farrow et al. indicates differences in the experiences of binary trans and NBGQ people: binary trans people are misgendered more by being referred to with their gender assigned at birth while NBGQ people are misgendered more using binary language. The research by Pulice-Farrow et al. also indicates that challenging authenticity differs between binary and NBGQ identities: binary trans people are questioned about not being real men or women, while NBGQ people are questioned about being real trans* people. Additionally, gender minority youth are more likely to experience invalidation of their identities or familial microaggressions . 1.3.2. The Role of Gender Nonconformity in Experiences with Violence Another factor is that NBGQ people are hardly able to pass, which means to be perceived and recognized as the gender they identify with, as there is "nothing to pass as" . It is almost impossible for them to present in such a way that they will definitely not be misgendered . More visible forms of "being different" often lead to more stressful situations and discrimination . Pulice-Farrow et al. indicate that microaggressions change according to the transitions one has undergone, and Gordon and Meyer suggest that gender nonconformity is an incentive for prejudice and discrimination. Furthermore, Klemmer et al. find that gender-nonconforming students are at higher risk of experiencing school violence than gender-conforming students, regardless of their gender identity or sexual orientation, which is in line with research by D'Haese et al. . Additionally, NBGQ people experience less validation and more social pressure to conform to the norm . Butler states that gender norms are inherently violent as they have regulatory power that shapes how people can and should look and behave. Passing indeed often intersects with safety concerns, with self-presentation being weighed against maximal safety . Furthermore, avoidance behavior is an coping strategy often applied among trans* people . The study by the FRA shows that, in Belgium, 5-14% of NBGQ people always and 12-41% often avoid certain locations for fear of being assaulted, threatened, or harassed. Additionally, NBGQ people avoid being open in public spaces (37-74%), around their family (18-33%), at the workplace (35-41%), at school (6-19%), and at bars or restaurants (18-32%) for fear of being assaulted, threatened, or harassed. Higher numbers of NBGQ people are not open about their identity with anyone in their family (25-45%) than trans men (16%) or trans women (1%) . These findings indicate that gender expression, rather than sexual or gender identity itself, plays a role in the experience of violence. This may be related to the cisnormativity that is present in society. Collier and Daniel describe cisnormativity as "the seemingly natural and ahistorical assumption of cisgender identities that structures institutions and interactions and results in the erasure of gender-variant experiences and realities". Non-binary people are often seen as a threat to gender structures since they challenge the notion of gender as a fixed binary fact rooted in biology . Among trans* people, experiencing violence is related to openness about their gender identity in everyday life (among family, friends, neighbors, at work, or when using health services), while for other LGBTI groups, the prevalence of violence remains the same regardless of openness . Therefore, experiences of microaggressions need to gain focus, as well as how gender expression and nonconformity influence and interact with such experiences. 1.3.3. Epistemic Exploitation Another form of violence inflicted on minority groups is epistemic exploitation. Toole coined the term "epistemic exploitation", which she uses to refer to epistemic oppression in which marginalized knowers are expected to educate dominantly situated knowers about their oppression. She argues that this act is exploitative because it requires emotional and cognitive labor that is unrecognized and uncompensated and it places an unfair burden on those who are already marginalized. In this way, Toole states, mental energy is diverted away from people's own projects, goals, and interests in the service of someone else. Fiani and Han have researched activism and educating others as a final stage of identity development. They find that most NBGQ people report that assuming an educator role requires demanding emotional labor, and often they prefer people to refer to the Internet instead . 1.4. Situation of NBGQ Youth According to Arnett , the impact of stress from negative day-to-day experiences among minorities is especially pronounced for adolescents and young adults between 18-25 years of age since they often transition from living at home to living independently. Specifically, for NBGQ youth, the separation from their parents is a crucial step in the development of their gender identity and their being able to accept themselves independently of their family's expectations . In line with this, research on racial minorities and adolescence shows that young people are at increased risk of exposure to microaggressions because they begin spending their free time outside their home and in public spaces . This--combined with the fact that the mean age of initial consciousness of being non-binary is 15.6, while the mean age of coming out is 25.8 --means that young NBGQ people may experience more microaggressions from their close environment, since they often have not come out yet. Adolescents and young adults between 18 and 24 years old experience the most physical and sexual violence within the trans* population in the EU and have the highest rate of non-verbal in-person harassment due to being LGBTI in Belgium . Lastly, an increasing number of young trans* people identify as non-binary . For example, Statistics Canada shows that proportions of trans* and NBGQ people are three to seven times higher for people born between 1981 and 2006 than for people born in 1980 or earlier. Therefore, this research focused on the experiences of NBGQ youth, who have been defined by studies as adolescents and young adults and are operationalized for this study as people aged 18-25. 1.5. Impact Microaggressions can have a significant impact on NBGQ people's lives. Most trans* people in Belgium who experience violence (57%) state that this had an impact on one or more areas, such as psychological problems or fear of going outside . Carroll labels the stress of living in a hostile environment with daily microaggressions "mundane extreme environmental stress". This type of stress is experienced day-to-day, influences the psyche and world view, and is environmentally induced. According to the minority stress model, psychosocial stressors such as violence have negative effects on minority groups' lives . The type of stress that microaggressions based on gender identity elicit is extra strong, since it is out of one's control. The impact of daily stress depends strongly on resources, networks, and coping strategies, which can lower the negative effect . Therefore, it is important to explore whether NBGQ youth seek help after experiences with violence and what their needs in this regard are. According to attribution theory, attribution processes influence the outcomes of violent experiences . Whether NBGQ people attribute the experience to themselves or to discrimination plays a significant role in the effects violence has on their wellbeing. This theory distinguishes between behavioral and categorical self-blame, respectively, when blaming oneself for something one did or for who one is, with the latter having more negative effects on wellbeing. Since gender identity is about who one is and gender expression about what one does, it is interesting to explore whether these processes play a role in the experience of violence among non-binary people. The research by Jones et al. shows that a lack of validation, which microaggressions may elicit, leads to worse mental health and quality of life among non-binary and transgender people. Sue also highlights that more covert and difficult-to-identify microaggressions often have a stronger impact and a higher emotional toll than more overt encounters and posits that unintentional, covert forms of microaggressions may be extra dangerous because they often go unseen and are more pervasive. Furthermore, a difference may exist between binary and NBGQ trans people in terms of the resources required to cope with negative experiences: non-binary people report community support and creative outlets as being more important than binary trans people . 1.6. The Belgian Context The increasing attention paid to legal gender registration procedures makes Belgium an interesting place for this research. Additionally, in Belgium, only 51.3% of NBGQ people live according to their gender identity and 33.3% of NBGQ people are never or seldom approached and treated according to their gender identity . Attitudes towards NBGQ people are not entirely supportive: 42% of Belgians do not support adding a third option as a gender marker on official documents . Microaggressions are recurrent: regarding people who have experienced threatening gestures or inappropriate staring in the past year, Belgium has the lowest amount of NBGQ people in Europe who report experiencing this only once (as opposed to multiple times) . This indicates that NBGQ people's situation is especially challenging. In conclusion, while changes are occurring at the policy level to include NBGQ people, the population feels divided, which may lead NBGQ people to live in a not-so-supportive environment where microaggressions may be common. Various data about experiences of verbal in-person harassment in Belgium can be found, as described in Section 1.1. However, specific data about microaggressions among non-binary people in Belgium are not available. 1.7. Research Goals This research aimed to provide insight into the experiences of NBGQ youth (18-25 years old) with microaggressions; the subtle and day-to-day experiences of verbal and psychological violence they face; and the subsequent needs, coping mechanisms, and impacts on their lives. To do this, we focused on distinct aspects of the topic. First, we aimed to show the experiences of NBGQ youth with microaggressions in general and, specifically, those related to being NBGQ (misgendering, deadnaming, claiming inauthenticity, and epistemic exploitation). Secondly, we explored how gender expression and nonconformity interact with these experiences. Lastly, we studied the needs NBGQ have when confronted with microaggressions and how they cope with these experiences. 2. Materials and Methods 2.1. Participants Ten participants were interviewed. Seven identified as non-binary, one as genderfluid, and two did not label their identity. The youngest participant was 18 years old and the oldest was 24 years old. All participants were born in Belgium and were white, although one participant had a parent who migrated to Belgium. Nine participants lived in Flanders and one in Brussels. Seven participants had higher education or were studying in a higher education establishment. Five participants were students, three were unemployed, and two had jobs. Lastly, four participants lived alone or with a supporting partner or friends, two participants lived in student residences, one participant lived in a government facility, and three participants lived with their parents. 2.2. Procedure The research was approved as part of the overarching study "Genoeg-Enough-Assez" by the Medical Ethics Committee of the Ghent University Hospital. As the goal was to gauge people's subjective experiences, a qualitative approach was chosen. Data were collected using semi-structured interviews to explore experiences of microaggressions among NBGQ youth. This method provided focus and ensured complete and in-depth answers while leaving room for flexible input from participants. Semi-structured interviews were conducted using a topic list, which is described in Section 2.3. Participants were recruited through the survey in the study "Genoeg-Enough-Assez". The survey was promoted through posters, flyers, and social media in LGBTI+ and non-LGBTI+ contexts. At the end of the survey, people had the option to leave their contact information if they wished to be invited for an interview. Anyone living in Belgium for at least two years and identifying with an LGBTI+ label could participate, regardless of whether they had experiences of violence. Among the people 18 to 25 years old who responded to the question "How would you describe your gender identity at the moment" by ticking the box "gender diverse (genderqueer, non-binary, agender, genderfluid)" and decided to leave their contact information, a random selection was used to decide who would be contacted and invited. They were sent an e-mail with an invitation to the interview, and one reminder was sent if a participant did not reply. Thirteen participants were contacted; three did not respond to the invitation, which made the non-response level relatively low. In the end, ten participants were recruited, reaching the level of saturation. The participants were all unknown to the researchers. Participants were invited for the interview at a quiet location of their choice. This location was generally in their hometown at a separate room booked in an LGBTQ+ space (such as a rainbow house), youth center, or university building, although one interview took place at a bar and two interviews took place in a park. The interviews were carried out by the first author. Only the participant and interviewer were present. Before the interview, the interviewer introduced themselves and shared their gender identity, affiliation, and why they were undertaking the research with the participant. The background and objectives of the research were explained, along with which topics the interview would contain. We defined microaggressions to them as we described in the introduction. They then read and signed an informed consent form. They were asked to give permission for an audio recording of the interview to be produced and the interviewer explained how the data would be treated and that their anonymity would be assured. All participants gave their consent to record, so all the interviews were audio-recorded. The interviews took approximately one hour. Due to the recording, no notes were taken during the interview. Participants received a debriefing after the interview with contact information for resources they could reach out to if they wanted to talk about their experiences. Participants did not receive an incentive to participate in this study. After the interview, recordings were saved at a secure location under an alphabetical code. Then, the recordings of the interviews were transcribed in full, leaving out personal details participants shared that could lead to the possibility of them being identified through the data. The data were pseudonymized and transcriptions were saved under a different alphabetical code. Once the transcription was finished, the audio recording was deleted. Names used in Section 3 are fictional. Transcripts were not returned to participants for comment or correction. 2.3. Material An interview guide was used to provide a structure for the semi-structured interviews. This guide included questions regarding the overarching violence study, as well as additional questions for this specific research. The topic list for the overarching study included questions about general experiences of violence, reporting violence, help-seeking, and attitudes about official instances. This was constructed to fit the goals and framework of the overarching study in collaboration with the guidance committee. It was then adapted to explicitly include experiences of microaggressions by applying the same questions to experiences of microaggressions instead of violence as a whole, and questions regarding the interaction with gender expression were added. This was approved by the supervisors. 2.4. Data Analysis The transcriptions were analyzed using Braun and Clarke's thematic analysis . This is a method for identifying, analyzing, and reporting patterns and themes within data . A theme is a patterned response that captures something important about the data in relation to the research question . This analysis is a suitable approach for research questions that aim to explore people's experiences. A semantic approach was used as opposed to a latent approach to reduce the subjectivity of the researcher's judgment and because the interest of this study was more related to people's stated experiences than their assumptions. The thematic analysis was carried out by the first author using the six steps proposed by Braun and Clarke :The data were transcribed and, during this process, initial notes or remarks were written down; Initial codes were generated using NVivo software. During this step, overarching codes were assigned deductively based on the research questions, such as "types of microaggressions", "reaction", "motive", "impact", "needs", "coping", and "gender expression". Then, within these categories, descriptive codes were assigned inductively from the data to segments that were relevant to the research topic; Connections between codes were sought and codes were sorted into potential themes with the help of a visual thematic map; Themes were reviewed. Minor themes that did not have enough data to support them were scrapped, and themes were joined or broken down according to what seemed appropriate. This was reviewed by reading through the data segments within one theme to verify whether they formed a coherent pattern, and the dataset was considered as a whole to make sure the themes accurately represented the data; Then, the themes were defined, and we specified what each theme brought to the general analysis and in what ways they related to each other; Lastly, segments of the data were selected to represent and provide evidence for each theme and to explain how the themes answered the research questions. As thematic analysis is interpretative and relies on the researcher's judgment, it was important for the first author, who conducted the interviews and analyzed the data, to reflect on their positionality as a researcher and how their perception may have influenced results. They are a young NBGQ person, a master student at the time of the study, and barely older than the age group they were researching, which had multiple consequences. On the one hand, talking to an NBGQ person may have allowed the participants to feel safer, to be more open, and to talk more freely. Several participants indicated that this gave them the feeling they did not have to explain their identity and that they were talking to someone who could understand their experiences. However, it was important to be mindful that this may also have led to participants not explaining their experiences in as detailed a manner as they would to someone that they thought did not understand. To avoid this, it was important to not make assumptions and to keep asking questions. Furthermore, the researcher has their own experiences with NBGQ microaggressions and gender expression and their own needs and coping mechanisms. It was crucial to be aware that their individual experiences were personal, did not speak for all NBGQ experiences, and were not generalizable. Therefore, it was important that, during the interviews and analysis, they did not steer or interpret people's words to reflect their own thoughts and experience. This was an active, conscious process of which they needed to be aware. 3. Results Thematic analysis resulted in the identification of themes and subthemes. The identified themes and subthemes are presented in two main sections: (i) experiences of microaggressions; (ii) and impact, needs, and coping. 3.1. Experiences of Microaggressions Among the experiences of microaggressions, three themes were identified: (i) types of microaggressions; (ii) perceived motive; and (iii) reaction. An overview of the themes and subthemes identified in this part is shown in Table 1. 3.1.1. Types of Microaggressions There were eight subthemes identified in this first theme: (i) abusive language; (ii) jokes; (iii) offense; (iv) sexualizing; (v) online; (vi) misgendering and deadnaming; (vii) epistemic exploitation; (viii) denial as a central theme. Abusive Language Commonly used abusive language included swear words. The most common way to insult participants based on their gender expression was through slurs referring to "being gay". Abusive language involved claiming that NBGQ people were attention seekers, that they were not able to choose, or that they had to choose between being a man or a woman. Finley stated that a student in their class explained "non-binary" to other people as "it's attention seeking people who don't know what to do with their identity". Participants reported aggressors making transphobic comments or trying to make them feel uncomfortable; for example, by asking inappropriate questions. Finley said that their care provider asked them whether they "are a boy or a girl?". After Finley said that they are non-binary, the care provider responded: "No but do you have a vagina?". Two participants were told that they had mental problems and should be hospitalized. Ali even stated that people were animalizing them by making barking noises when they passed by. Participants were blamed for the microaggressions they experienced. Ellis' mother told them multiple times that "if you get hit because of how you dress, you can't count on my support". Aggressors told participants they did not belong at the current location and that they felt uncomfortable around queer people. This happened especially at gendered toilets but also bars and youth homes. Jokes Participants frequently experienced jokes about gender diversity or were made fun of. Alex's identity "was often seen as a joke or something funny". After Sam shared their gender identity to their teacher, the teacher joked that "then from now on, I am Napoleon". Finley also mentioned memes on the internet that ridiculed gender diversity in general. Offense Some participants experienced more offensive forms of microaggressions, such as being threatened, shouted at, catcalled, and harassed. Furthermore, they experienced stalking, disapproving looks, and being avoided because of how they looked. Max shared that "I felt really attacked when I was walking on the street with my partner, someone walked towards us and started shouting things". Jules stated that, while with their partner, "two men came to us who started pushing our heads together saying kiss for us, kiss for us, but we were sitting next to the water so I was really scared that they would kill us then so that's the only time I feared for my life". Sexualizing Four participants reported being sexualized. Max mentioned being queerbaited, which they described as being flirted with because they looked queer but then being rejected when they got closer. This also included receiving sexualized looks and hand signs because of how they were dressed and aggressors making sex noises. Someone asked Finley, "if I wanted to bang him because I was a guy but not exactly, so he could experiment without really doing it with a real guy". Online Alex mentioned that the media can be violent: "Maybe one more thing I consider to be violence is some news articles (...) they always hit me hard, they feel like violence, how they write, and they are using misinformation (...) or they write that these are scientific facts while they are not, and I find it annoying because I know other people will read it and maybe they will not know what's going on and just believe it". Participants highlighted the negative consequences of media and the Internet; for example, reading about the bad experiences of other people. However, the positive aspects of the media and Internet were also mentioned, such as meaningful online friendships that helped them cope. Participants also mentioned a sensation of living in a social media bubble. Charlie mentioned that, "it (what they find on the internet) is targeted, so I will see it (positive queer messages), but people who say oh pronouns, why do we have to put that in our e-mail are not gonna see that because they will block it, so yeah, that's my own social media bubble". Alex said: "there was an article, and I thought it was shared to call out a transphobic opinion in the media, so I went to look at the comments to see that other people were also calling it transphobic and then it turned out that I was in the Belgian channel instead of the trans* friendly channel, I just felt safe for a moment and then it turned out I was not in the safe place that I thought I was". Misgendering and Deadnaming Deadnaming and misgendering were common experiences. Deadnaming was most commonly experienced with family who did not put in effort or accept the person's name. Deadnaming went hand in hand with misgendering. Luca said: "my family doesn't accept it or doesn't get it and especially the pronouns they and them, they don't know them, they also don't wanna try using them, so they don't call me by my name, and they also don't use my pronouns". Misgendering happened frequently and in multiple contexts: from family or friends who were not accepting or misgendering out of habit to fleeting contacts who categorized quickly. Participants had mixed feelings about misgendering. On the one hand, they felt it denied their identity and that it was confirmation they did not pass. Luca said: "it's like you're not being seen by your family, who should be close to you". On the other hand, they often did not react to misgendering, as they understood it was a habit, and it was not the most important part of their identity. Jules shared that, "the whole debate is being reduced to pronouns alone while it is so much more than that, that's something that I talk about a lot with my parents, that of everything, I think that if they get pronouns wrong, I know I've been their child for many years and they have always addressed me in a certain way so pronouns are the last step of my gender identity". Epistemic Exploitation Participants mentioned that they were expected to educate others on their identity and issues. To be a "walking search engine", as Sam phrased it, was experienced as tiring. Sam stated that, "it is really tiring to keep proving, to prove yourself as who you are and to keep explaining the same things. I have even considered creating a PowerPoint or a small leaflet that explains everything and just give it to people". Denial as a Central Theme Many microaggressions centered around denial. Participants reported that aggressors denied the existence of NBGQ identities and adhered to binary divisions by stating that only two genders exist or that people who claim to be NBGQ were seeking attention. Finley said that a care provider recommended that they go through a "complete transition" even if they did not want this, which reflects a binary viewpoint. Six participants said that aggressors had engaged in discussion about their gender identity, denied their gender identity, or denied them as a person. Sam mentioned that, after explaining their identity to their parent for hours, the parent said that, "she will still remain my daughter". Finley claimed that, because of their gender expression, their parent made comments about their gender identity not being believable. Ellis was even denied as a person: after coming out, their partner told them multiple times that they "had no soul". Another form was denial of information about gender diversity; aggressors claimed information to be wrong and inaccurate. Robin mentioned the use of adherence to linguistic correctness to endorse misgendering. In discussions with their friends, "the bottom line usually is that it's not linguistically correct, ok but what is most important, whether someone feels validated and accepted and also just comfortable, or linguistic correctness". Lastly, participants felt denied when aggressors claimed they did not understand how someone could be gender diverse. While talking about mastectomy, Robin's friend stated that "they couldn't understand at all why a woman would ever do that". 3.1.2. Perceived Motive The second theme consisted of two subthemes; namely, (i) gender expression and (ii) other motives. Gender Expression All participants referred to gender expression as a perceived motive. Jules experienced more microaggressions after their gender expression changed, and many said they were wearing certain clothing, had just changed their hair, or were looking particularly masculine or feminine when microaggressions occurred. On the one hand, a more queer gender expression or a gender expression typical for the gender opposite to the sex assigned at birth elicited more abusive language, sexualization, and offensiveness. On the other hand, a gender expression that was in line with expectations for the sex assigned at birth elicited more denial and misgendering. This puts NBGQ youth in a position where, no matter what they do, they may be faced with microaggressions. Alex stated that, "this was one of the first times that I had dressed differently than I normally dress, and this made that, it was clear that my clothes had an impact on how people look at me in a sexual way", while Robin stated that, "my family and my friends have a certain impression of me and they see a woman and then they stick with that until you actively say well actually not". Other Motives Participants referred to intentional motives to do harm. These included the aggressor being transphobic and/or not accepting the participant's identity. Other motives were that aggressors seemed to get a kick out of it, were inciting each other, or wanted to act tough. According to some, aggressors claimed it was too much effort to not commit microaggressions, such as misgendering and deadnaming. However, only a minority thought that microaggressions were committed on purpose. More than half of the participants perceived microaggressions as unintentional. They stated that microaggressions, especially misgendering, occurred out of habit, and people did not realize that they were hurting them. Max said: "parents are causing so much violence, but unintended, you know, that, I think often that parents don't realize what they're doing and yet they cause so much damage, just by saying something simple or by not thinking about it, they can really hurt me a lot". Furthermore, incomprehension or having wrong or not enough information were perceived motives. Finley said that microaggressions, such as inappropriate questions, occurred to them because "they thought oh that's an opportunity to learn". 3.1.3. Reaction Three subthemes were identified within the third theme: (i) conversation with aggressor; (ii) no reaction; and (iii) dependent on context. Conversation with Aggressor Almost all participants engaged in a conversation with the aggressor, which did not always have the desired effect. Ellis said: "I have told them that I am non-binary, and I don't want to be addressed as she/her but they keep doing it anyway" and Charlie stated that, "when I meet people in real I try to talk about it but it doesn't always go as well when I, sometimes I try and if I don't get good reactions I'm discouraged to continue trying". Robin said that people sometimes reacted well in the conversation about misgendering but continued to commit microaggressions anyway: "I tried to explain it, and she accepts it, that I correct her, but using it herself is just, she accepts when other people do it and that's fine but using the words herself is still a problem". Sometimes, aggressors did not react well, as stated by Robin: "that they have a certain opinion about something, and I think that opinion is wrong, or that information is missing, then I find it important I share that information, but then we're like arguing, or fighting". Microaggressions were related to acceptance of gender identity. Max said they "understand that it happens but I just think they sometimes are just not doing their best or something, to accept their child, neighbor or pupil". Some participants said that they tried to understand why the aggressor had done what they did or put the blame on themselves. Max stated: "I don't know where people come from or what they have experienced, maybe they have experienced something horrible, it's possible, but I don't know that and then I just blame myself, like I shouldn't have walked there at that hour, I shouldn't have worn that, I shouldn't have gone out there wearing this, with those people, and then yeah, I really just blame myself". Three participants consoled the aggressor because the aggressor made them feel guilty. Sam said: "she puts herself usually in a position of the victim saying oh but I'm your mother and I'm trying and oh you're so rude to me but then yeah, I just try to think ok that's my mom, I'm not gonna be able to change that I think". No Reaction Many participants did not react to microaggressions. They alternated between having a conversation with the aggressor and not reacting or shifted from reacting to microaggressions to not reacting. Max said: "usually I reply and then you see in their face that they didn't realize, and it helps for a while but then, it doesn't have any long-term effects and then well, I try to still do it but after a while I just stop". Many reported having no energy or feeling too despondent to react. Max said they, "tried for a while to react, to ask every time oh is it about me and then you notice they stop but after a while it's just annoying and I had to give up because there is no point and now, I feel that it really asks a lot of energy". They either blocked off the aggressions, assumed that what happened was normal, looked for distractions, or decided to let people make mistakes. They felt sadness, cried, and hid their pain. Half of the participants said they left the location. When the relationship with the aggressor was already complicated, participants did not react to not complicate things. Six participants said they did not react because they did not want to explain themselves or educate others. Charlie said: "I have to give an explanation for sure, that's a requirement, that I can convince them, and I find it so annoying, I just don't wanna do that". Max said: "I don't think it's my responsibility to educate them because I have the feeling I have done that with so many people already, and they could also just do it themselves" and that "suddenly my uncle shouts at me and says explain the difference between gender and sex, but you know, I know that but why am I being asked to explain that to the whole family and I was just, it was well intended, but it is just incredibly annoying, like I have to teach you but I don't want to, and if you would just ask if I want to explain I can say no but he just told me to explain, and I did, but it is just so annoying to me". Another reason to not react was fearing the aggressor's reaction or out of safety concerns. They also wanted to avoid a fight or wanted to not appear too extreme. Finley said they, "usually didn't react because he scared me, he was a really bad person, so I was a little scared of the consequences if I stood up". Some participants did not see the severity of the situation at first. Charlie said: "I think I didn't realize at that moment, maybe even the severity of it, at that moment I just thought, I think I was just laughing with it actually". Dependent on Context Whether they reacted depended on the location. Robin said, "I usually don't react unless I am at an LGBT space". It was also a result of safety considerations. As Max said, "if it feels bad, I just freeze, then I just, then I just leave, I'm not gonna react to that, I will just take care of my own safety but if I know it's something small or if it is people that I know I will talk back to them". Their reaction also depended on whether the aggressor was known or unknown to the person. The effects it had were divided: some reacted more easily when experiencing aggression from a familiar person (such as Max), others when experiencing aggression from a stranger. Sam said: "it hurts me less when the people are unknown to me, I still take it with me but when I don't know the person, I get angry more easily and I try to explain more, while if I know the person and I'm close to them, well I close myself off, then I don't know so well what to say". 3.2. Impact, Needs, and Coping The second part of the analysis centered on the impact and subsequent needs and coping mechanisms resulting from being confronted with microaggressions. Three subthemes were identified: (i) impact; (ii) needs; (iii) and coping. An overview of identified themes and subthemes can be found in Table 2. 3.2.1. Impact Seven subthemes were identified under the impact theme: (i) gender expression; (ii) fear; (iii) wellbeing; (iv) others; (v) world view; (vi) actions; and (vii) evolution. Gender Expression Almost all participants felt inhibited in their gender expression because of microaggressions that occurred or the threat of microaggressions (e.g., experiences of others). Luca said: "the first weeks and months I thought ah yeah it's clearly not ok that I look like this or that I behave like this, so it has, it has inhibited me in that way". When asked whether microaggressions had impacted their gender expression, most participants said they had not. However, while talking about gender expression and microaggressions, most participants shared stories of consciously adapting their gender expression. Most participants adapted their gender expression because of experiences with microaggressions. Some adapted their gender expression to appear more queer to confuse people or to be identified as queer. Sam said they "pay a lot of attention to clothes, what you wear, sometimes I also avoid makeup because for me it isn't something masculine or feminine but sometimes, I avoid it because I don't want people to see me as a woman that day". However, most adapted their gender expression to appear less queer, especially when alone in public. They did this to not have to explain themselves, as this was too exhausting. Jules stated that they, "know it (the gender expression) has an effect and I think that for example I wear more unrevealing clothes to avoid things, or because otherwise it's too exhausting". Other reasons to limit expression were to enjoy the protection of passing, because they were discouraged by others, to protect other queer people who were with them, or to avoid suffering. Max said: "I am not 100% happy with it (how they express), of course not, I would like to see it differently, but it's, I'm not gonna be annoying about it because then I would, I would have to come out again and I just wanna spare myself of that feeling". Another reason was to appear less queer out of fear of being regarded as inferior, of not being accepted, or of the consequences, such as physical violence or being outed. Two participants indicated that microaggressions did not change how they expressed their identities, but it made them more aware of their expression. Four participants felt like microaggressions were the price to pay for expressing themselves the way they did. They did not adapt their expression but saw microaggression as something that happened when they appeared as nonconforming. Jules said about gender expression that, "you notice that you have to face the consequences", and that it is, "a little bit the price you have to pay almost, something you just accept". Whether participants chose to adapt their expression depended on the environment (e.g., in a supportive environment or online they were more likely to express themselves), whether they felt comfortable with the people, and whether they were around queer people. Finley explained that, "It makes me not free to present myself how I want in public, I have to be in a very good day to present in an androgynous way if I want to be kept at peace I just go with a sweatshirt and jeans and try to, I can't really be invisible but try to keep it not really out and proud". Fear Participants were afraid to come out or to be open about themselves. Sam experienced, "fear to come out of the closet, actually I still have a lot of fear to be rejected when I say that I am non-binary, I mostly notice with family member I'm very scared to say that and, yeah because even if I do, then there's all these microaggressions". Participants reported fear of being alone in public and of physical violence. Charlie reported, "specific moments that you realize that damn if I would be wearing a skirt here, then it would not have ended well, while I would've wanted to wear that". Finley explained that, "sometimes in places I can get scared because I see a lot of old guys or when I go in a public bathroom I always feel out of place and uncomfortable and there's still a lot of situations where I don't really feel safe". Sam feared to take medical and legal steps in transition and Ali feared being kicked out of the house. Wellbeing Many participants reported that microaggression impacted their mental health through frustrations, insecurities, doubts, low self-esteem, exhaustion, anger, sadness, and suicidal thoughts. Jules said: "using swear words or misgendering are so-called microaggressions because ok, it doesn't threaten you in your physical integrity or something, but I think that when you also look at suicide numbers that in that way it's more than just a microaggression because it keeps contributing to low self-esteem for example". To some, experiencing microaggressions raised awareness that this could actually happen to them. Finley stated that, "it just keeps confusing me that I was in that situation, it feels a little unreal and it's weird and its, it's a situation that I heard about online and I heard people talking about it, it's, it's weird when I think about it". Misgendering and deadnaming, in particular, led to dysphoric feelings, and participants felt their identities were being denied. They also felt inhibited in their identity development. Luca said: "it had the effect on me that I stopped myself to be open about my gender identity to my environment, and to myself" and "I really felt like, like it became clear once again that I shouldn't be honest about my gender identity, because it wouldn't be accepted". Ali mentioned that, "I find it hard to express myself to my parents, let alone in public, and it got so far that I stopped acting manly and that I forced myself to start acting more feminine again". They also started doubting their gender identity: "first I thought, am I just kidding myself, like am I being serious with myself but yeah, I know that I am still searching so then I sometimes got remarks like oh you're just confused, you know". Max even said: "if the right attitudes would've been there, it would've been just easy to, then I would've been able to be non-binary, and that's not the case now" and "it's just that, I would just like it if, if it would be like that (that NBGQ identities were accepted by society) but it's not the case, and I will not mourn it, it's, it's ok, well it's not nice but it's just ok, I feel like I am being who I am enough to enjoy it but then later I can find more depth (in their identity)". Microaggressions elicited feelings of denial. Sam said: "in that moment it was like, well like a piece of my identity was just thrown off the table that was not nice at all because I didn't feel acknowledged in my identity or in my being, so mostly that, and I remember very well that I, well it was a mix of frustrations and tears and sadness and I thought to myself how could people think like that, and it's weird, mostly that, and it's not nice". Participants hid their identity more and took decisions between openness and safety. Robin said: "when I notice that someone really has a more aggressive appearance than I will never share my pronouns, then I will just pretend to be a woman completely, I'd rather be misgendered than to get into trouble I think". Some stated they were repressing these experiences, such as Max, who said they "have the feeling that I repressed a lot of things that happened because I really, ok it's not worth remembering, it's not worth talking about it, I don't wanna deal with it". Others Participants became more distant or less trusting of others. Max stated that, "I feel like, that I still carry these experiences with me, that they are all things that made just a little bit more distant to people, a little bit more careful, and I don't like that evolution". Some reported breaking ties with people, relationship problems, or fear of commitment. Max reported that, "I think that my trust has been broken, like the ability to have relationships has suffered". Furthermore, they were worried about other queer people's wellbeing. Max said: "I can handle it pretty well because I've been dealing with it (microaggressions) for four years now so it hurts me less, but I would mind a lot if someone else would encounter it that doesn't have as thick of a skin as I do, that's the problem". The impact of microaggressions depended on the aggressor. Finley said that microaggressions from a parent "really hurt and influenced me for a moment and yeah I took them very personal because he was from my family, and he was supposed to be close to me". World View Microaggressions impacted participants' world views: they lost faith in humanity or did not understand why people would treat them this way. Robin stated that they felt like they were dragged out of their safe bubble: "I have a very good group of friends and I can be myself with my friends because a few of my friends are LGBT themselves and for you it's the most normal thing in the world, but when you read these comments, you realize again that it's not the most normal thing in the world for everyone and then you get pulled out of it, you're hit with reality and that resonates with you, like how is that possible, that it's still a problem for many". Actions Actions that participants undertook after having experienced microaggressions included avoidance and being vigilant in public spaces. Some reported not going to certain places alone or avoiding certain gender expressions. Charlie avoided unknown places and people. A few participants also said they had been warned by their families to be careful. Participants also compared their experiences to those of others to minimize their experiences. They normalized how microaggressions happened to them and learnt to cope with them. They often blamed themselves. Jules said: "we, as a community, describe many things that are actually more than a microaggression as just a microaggression". Furthermore, Max stated that, "I encounter many microaggressions but then I hear from friends that they experience more and then I think, is what I encounter this little, it's like, something weird that, I think it's something psychological that you, you will minimize your own violence if other people, what they encountered is bigger and I don't know why that is, but it's, you have to like prove that it's real or something". Sam learnt to handle microaggressions better: "I have learnt, I don't know, it's always a reality check when these things happen and then I learn that ok, when it happens again, I can react to it better this way or another way, they're learning moments in a way but it's not nice at all". Evolution Participants did not initially see the severity of the aggressions or even felt euphoric (e.g., when being misgendered as the gender opposite what was assigned at birth). However, they later realized the severity, which resulted in them being more careful or experiencing feelings of exhaustion. Some participants also indicated that over time they felt less shame and it evolved for the better. Jules said: "the first times that people addressed me as sir or like ah you can't enter the women's toilet it was first a feeling of euphoria, like I tricked you, while now I notice that it's shifting to a form of microaggression for me and how I experience it, initially I liked it because it helped me in my search while now it's just very tiring". 3.2.2. Needs There were two subthemes identified in this theme; namely, (i) validation and (ii) others. Validation Many participants experienced the need for validation: to be accepted, have their identity confirmed, be seen as an NBGQ person, and feel understood. Jules highlighted the need to accept themselves. Luca said: "I just needed someone in my environment that said that it was fucked up and that I was allowed to look the way I look and that it doesn't have to mean necessarily that I'm a man or a woman". Others Some needed others to seek protection (Charlie: "I thought it was very important, that he, even though I didn't know the guy, that he could say ah they can't mess with us, if someone would do something to harm us, yeah I thought that was very important") or to not feel alone. Max said: "sometimes I need to complain to someone who knows me well, who is queer themselves, that's nice, that can help and just the feeling that I'm not the only one who experiences this, that's a nice feeling". The most common need was to talk about the experiences to the aggressor or to a therapist or professional. All participants felt the need to talk to friends. One recurring theme was talking to people (friends or therapists) who were queer or had an affiliation with queerness. They wanted to talk to people who were aware of trans* issues--and to queer friends or therapists specifically--so that they did not have to explain their reality. Jules explained that talking to queer friends helped because, "they are people who experience the same or something, and then you have many ways to talk about it, you can laugh about it, or also share tips, I don't know, it's very different than if I would share the story with my parents". Luca said: "I would like it so much to have a queer therapist, because you, you just have a certain something that you just understand" and "I think what can help is to get in touch with someone that is similar to you, that when I look for help, I can find someone who talks to me who is also queer or also trans, that would be nice". 3.2.3. Coping Within this theme, three subthemes were identified: (i) talking; (ii) not coping; and (iii) internal. Talking The main coping strategy was talking. Two participants needed conversations with their aggressors to cope. Others talked to people who were close to them: parents, partner, and, especially, (online) friends. Ali stated that this helped because, "you get that support, of which you think wow do I even deserve this and then well, you create a nice connection with this person and you know that if something is happening I can share it, and reverse too, and it just feels good, you know that you can be yourself completely with the gender identity you have, you can be yourself completely and can express yourself to this person, and that, it just feels so good". Six participants wanted to talk to queer friends as they understood the situation and struggles better. Finley explained that having queer friends was important because of "the shared experience that they have, there are people who have similar experiences to mine and can relate and that makes a lot of things easier of course" and that "it's really different, we can relate on other levels than I related with other friends, I didn't lose contact with them but it's, it's different in some ways". Talking to queer friends related to seeking validation. Sam said: "you recognize each other's stories, so it gives a feeling of connection and it's very nice to be heard, you get the recognition that you need in that moment, and so it gets easier to let it (microaggressions) go or put them aside". However, participants also mentioned that they did not always share negative experiences with queer friends because "when I am together with other queer people, I'd rather do fun stuff than not fun stuff" (Max). Almost all participants talked about microaggressions with their therapists. They did not seek help for these experiences but talked about them with therapists they were already seeing (although Max explained that experiences of microaggression could not be seen separately from the other issues they were facing). Participants indicated a risk of experiencing microaggressions if therapists were not specialized. Jules said: "I could share things about my identity and then they would just say oh that's interesting, that's an interesting world you live in, like, my life isn't a circus you know". Others said that when therapists were not educated, they were unable to help: "therapists I've had are actually examples of people that actually, to whom I had to explain what I experienced instead of that they gave me tools to work with" and "the way they were not prepared to answer my needs was actually really, yeah, really poignant" (Jules). They wanted a therapist who had knowledge about queer issues or was queer themselves because they did not want to explain their identity to the therapist. Max said: "it has been a tough search because I was looking for someone who understands what being queer is because I don't want to have to explain and I was looking for someone who, well I was really just looking for someone who would understand me, and it didn't really work out". Talking was useful as it helped the participants to find acceptance and to have the feeling that they were not alone. As talking was a prominent need, it seemed that this was often fulfilled. Not Coping One participant was unable to cope with the experiences as they were in an unsafe environment at home. Various participants repressed these experiences. In particular, it was challenging for them as young people to cope with these experiences, as some lived with their parents or were tied to a certain school environment. Finley said: "it (microaggressions) was coming from pretty much everywhere and I was not leaving it". For some, the only way to cope with microaggressions was to leave the unsafe environment. Ali said: "I told the teachers what I thought in a friendly way, saying look It's a pity that it went this way and that I was not accepted by the students and the teachers, and then I just left" and "it's a lot easier, now that I'm not at school anymore, it (microaggressions) has diminished a lot, it has gotten a lot better since I left the school". Finley said: "now it's better because I just don't see him (the main aggressor) anymore, I took distance, it's better". Internal Strategies to cope within themselves were attempts to not take it personally and to rationalize and relativize. Some withdrew within themselves to cope. Charlie said: "it (microaggressions) didn't feel aimed at me, or at least I don't take it that way, well actually I think I also just really did my best to not take it that way". Max described that, "I don't know, then I just take it in or something and then, it's like something disgusting that you eat and then just digest I think, I don't bother with it, it's just swallowing for a moment and then you continue" and stated that they, "go through what happened, if necessary, recording a voice message or something to just make clear what just happened, like that are the worse parts and the less bad parts". Robin said they, "understand it, that it's new or unknown to people, it's starting, now it's starting to be talked about, it's starting to appear in the media, which is amazing but a lot of people don't know it so I understand that for many people it's not really known and that they are not just gonna start doing it or actively thinking about it themselves if it's not applicable to them, so in this way I can put it into perspective". 4. Discussion This research aimed to gain insights into the experiences of NBGQ youth (18-25 years old) with microaggressions and into the subsequent needs, coping mechanisms, and impacts on their lives. Secondly, we wanted to explore how gender expression and nonconformity interact with these experiences. Participants' experiences of microaggressions seemed to be centered around feelings of denial and rejection. Common needs were to be validated, accepted, and seen as a NBGQ person and to talk about these experiences with the aggressor or with (queer) friends or therapists. Many engaged in conversations with the aggressors to educate them, although this was a tiring experience and NBGQ often felt too exhausted to do this. Other ways of coping were to try to understand why these experiences had happened or to normalize them. Gender expression interacted with microaggressions, as it was a perceived motive for the microaggressions, and microaggressions also had an impact on gender expression. Often, NBGQ youth adapted their gender expression to either look more or less queer depending on their needs, and if they did not, they thought of microaggressions as the price to pay to express themselves. Figure 1 shows how the themes brought forward by participants regarding their experiences with microaggressions, their needs and coping mechanisms, and the impacts on their lives and gender expression may relate. Many experiences were centered around denial: denial of NBGQ identities, of the person's gender identity, or of them as a person. This was a recurring theme among the different types of microaggressions and closely related to misgendering, which was reported by all participants and can be seen as invalidating and not acknowledging a person's gender identity. Participants reported feeling denied in their identity and fearing being open about their gender identity and in their gender expression. They felt that many microaggressions denied and rejected their existence and experience and they felt they were not accepted by people. This led to these NBGQ individuals experiencing a fear of rejection and had a direct impact on wellbeing. This was in line with previous research that found that denying trans* people's identity and experiences, misgendering, and deadnaming are common experiences , and that invalidation of identity is common among trans* youth . Although the participants encountered microaggressions that centered around them not being seen or validated as NBGQ people, the findings from the study by Pulice-Farrow et al. , which showed that NBGQ people are often doubted with regard to being trans*, were not reflected here. However, this may have been because microaggressions were mostly committed by people who were not queer and may not have had ideas about what being trans* means. In this study, we did not explore if and to what extent participants identified as trans*, so it is possible that not being seen as trans* was not an issue to them as opposed to not being seen as NBGQ. This study broadens insights regarding the impact of microaggressions. Encountering microaggressions had impacts on the lives of young NBGQ people, who experienced more fear, expressed more vigilance, and employed adaptive behaviors when in public (such as not being alone, adapting expression, avoiding places). This shows that microaggressions are a strong psychosocial stressor, in line with Meyer's minority stress model . Participants had three main ways of coping. The most important was talking about these experiences, especially with queer (or at least accepting) friends or specialized therapists. Participants indicated that talking to queer people was easier and made them feel understood. Young NBGQ people seemed to be seeking this validation that was denied to them through microaggressions from (queer or accepting) friends who would give them the acceptance they needed. Many stated that they had needs for validation, to be accepted, to have their identity confirmed by others, and to be seen as an NBGQ person. However, there is a risk of encountering microaggressions when talking to an unspecialized therapist. A second strategy to handle microaggressions was engaging in a conversation with the aggressor. Participants seemed to weigh the importance of explaining how something was hurtful or educating people against the exhaustion of continually explaining their reality. Most participants engaged in conversations with the aggressors but reported that they did not have the desired effect: aggressors did not react well or the effect lasted for a short time. Many participants ended up not reacting because it was too tiring to keep explaining their identity or situation. In turn, the need to explain and to educate others about their identity was seen as a form of violence, and the occurrence of microaggressions and engaging in conversation with the aggressor may be a loop of microaggressions and exhaustion for NBGQ youth. This is in line with epistemic exploitation theory . Participants experienced having to educate others or having to explain themselves as a form of microaggression. In accordance with what Toole suggests, participants explained that educating others took mental energy and emotional labor. These findings are also in line with the study by Fiani and Han that found that non-binary people prefer not to assume the role of educator. It seems that, to avoid this, they turn to queer friends and queer/specialized therapists in order to not have to explain themselves and to get out of this loop by receiving validation. Furthermore, to avoid having to explain or educate, participants often adapted their gender expression. Another strategy was to rationalize and to try to understand why people commit microaggressions. Participants put the events in perspective and tried to empathize with the aggressor, for whom NBGQ identities were a new topic they had little information about. Some participants even blamed themselves for the microaggressions occurring to them. This may have been especially the case because denial of the identities of young NBGQ people and other microaggressions often occur among people who are close to them, such as family or friends, or people who have a significant impact on their lives, such as teachers or health providers. This reflects the findings in the study by Kiekens et al. that gender minority participants have many experiences of familial microaggressions. In line with attribution theory , these coping strategies had an impact on participants' wellbeing. However, whether young NBGQ people blame themselves for what they do (how they dress, where they walk) or who they are (their identity) should be investigated with more detail in further research, as this influences mental health outcomes . It is striking that many participants perceived microaggressions as unintended, committed because people do not understand NBGQ identities or lack information. It can be questioned whether this is merely a perception or a coping mechanism, such as attempting to not take microaggressions personally. Participants said they took the blame for microaggressions and normalized that they happened. They may not want to see people who are so close or important to them as "bad people". In this light, it can be discussed whether these "unintentional" microaggressions are as unintended as people claim them to be. All but one participant had conversations with the aggressor, but microaggressions kept occurring to them. This makes the boundary between, for example, misgendering as a habit and not wanting to put effort into stopping it hard to distinguish. The last strategy adopted was using gender expression. Most participants adapted their gender expression. One option was that they adapted their expression to send the message to people that they are queer. This way, they could try to nonverbally explain themselves as a NBGQ person to possible aggressors. This was in line with the study by Fiani and Han , who found that passing is challenging. The other option was to adapt their expression to be less visibly queer to avoid microaggressions. This was partly due to the weighing of safety concerns, as also found in the study by Fiani and Han . However, an additional consideration of expression versus exhaustion (because of having to explain and encountering microaggressions) was found. Participants weighed whether they were too exhausted to deal with microaggressions or not. If participants did not adapt their gender expression, they experienced microaggressions as the price they had to pay for expressing themselves, which in turn can contribute to normalizing microaggressions. It indeed seems that gender nonconformity is an incentive and that visible forms of "being different" lead to more stressful situations, as research has suggested. Gender nonconformity was perceived as an incentive for microaggressions, which is in line with findings that gender nonconformity is an incentive for general violence . While the findings of the FRA show that, for trans* people, openness about gender identity is related to the amount of violence they experience, this study found that microaggressions people experience also influence their openness about their gender identity. 5. Conclusions This study broadens research on microaggressions with a trans* and NBGQ lens. Specifically, it provides an in-depth look at the experiences of microaggressions of a more specific subgroup rather than looking at microaggressions and the LGBTQ or trans* community as a whole . This research combined different aspects of research on microaggressions, gender expression, and epistemic exploitation and tried to find how they are interconnected. The study found that experiences of microaggressions among NBGQ youth were centered around denial of their identity or NBGQ identities in general. They felt rejected in their existence and experience and felt unaccepted by people. To cope, they talked to queer or accepting friends or therapists to seek validation. They also tried engaging in conversations with the aggressor, although this did not have the desired result. The young NBGQ people weighed the need to educate and explain to the aggressor against educating being experienced as tiring and exhausting. Another strategy was to rationalize and try to understand the aggressor. This led to the young NBGQ people perceiving microaggressions as unintentional and engaging in self-blame or normalization. Lastly, microaggressions and gender expression interact. On the one hand, the young NBGQ people adapted their gender expression to appear more queer to "explain" their identity to people; on the other hand, they adapted their gender expression to appear less queer to avoid rejection. The importance of expressing themselves was weighed against the exhaustion of dealing with microaggressions. When NBGQ youth did not adapt their gender expression, they experienced microaggressions as "the price to pay" for expressing themselves. One limitation of this study was that it had a relatively small sample size, and it is not possible to generalize these results. However, it gives indications of which aspects play a role and what is important to NBGQ youth when it comes to microaggressions. These different aspects and how they influence each other can be further researched. As this research was explorative, further studies can research this more specifically and in detail. The topics found in this explorative study can serve as a guide for questionnaires or interview guides in further studies. While focusing on a small age category may give more reliable results about this specific group, this study cannot speak for the experiences of all NBGQ people, and research will need to map out experiences of older NBGQ people. Another limitation is that a certain profile of NBGQ youth was overrepresented. All participants were white and able-bodied, and most reported they had studied or were studying. All of them were open about their identity to at least one person. Almost all mentioned having a network of queer friends, online or in real life, with whom they could be themselves. As this is a useful coping mechanism, it would be valuable to explore how NBGQ people who are not out or do not have a network of queer friends cope and how they can be assisted. As this was an initial exploration of young NBGQ people's experiences with microaggressions, further research is needed to confirm every aspect of how these experiences relate in detail. For example, a future study could investigate how the relation with the aggressor impacts the experience of microaggressions, and if this is related to NBGQ people experiencing microaggressions as unintentional or empathizing with the aggressor. Furthermore, the impact of microaggressions through the media and the Internet could be studied, as this aspect could only be briefly touched upon here. Though it is more challenging, it may be interesting to not only look at perceived motives but to study the attitudes and experiences of the friends and family of NBGQ people with whom they have these conversations. It's important to note that participants categorized microaggressions mostly as unintentional. As Sue posits that unintentional forms of microaggressions can be dangerous as they go unseen (participants often did not realize the severity at first), and they are more pervasive, future research should focus on this aspect in more depth. Lastly, as one prominent need of NBGQ people is to talk to a therapist who is queer or specialized in queer issues, one recommendation is that efforts must be made to ensure the availability and accessibility of specialized therapists. Author Contributions Conceptualization, Q.A. and A.B.; Methodology, Q.A. and A.B.; Formal analysis, Q.A.; Investigation, Q.A.; Resources, Q.A., A.B. and J.V.W.; Data curation, Q.A.; Writing--original draft, Q.A.; Writing--review & editing, Q.A.; Supervision, A.B., J.V.W. and J.M.; Project administration, A.B. and J.M.; Funding acquisition, J.M. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The study was conducted in accordance with the Declaration of Helsinki, and approved by the Medical Ethics Committee of Ghent University Hospital (protocol code BC-10233 E02 approved on 28 December 2021). Informed Consent Statement Informed consent was obtained from all subjects involved in the study. Written informed consent has been obtained from the patient(s) to publish this paper. Data Availability Statement The data presented in this study are available on request from the corresponding author. The data are not publicly available to ensure anonymity for the participants. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Experiences of microaggressions among NGBQ youth. This figure summarizes the main experiences of NBGQ youth with microaggressions and how different elements of these experiences relate. healthcare-11-00742-t001_Table 1 Table 1 Experiences of microaggressions: themes and subthemes. Themes Subthemes Types of microaggressions Abusive language Jokes Offense Sexualizing Online Misgendering and deadnaming Epistemic exploitation Denial as a central theme Perceived motive Gender expression Other motives Reaction Conversation with aggressor No reaction Dependent on context healthcare-11-00742-t002_Table 2 Table 2 Impact, needs, and coping: themes and subthemes. Themes Subthemes Impact Gender expression Fear Wellbeing Others World view Actions Evolution Needs Validation Others Coping Talking Not coping Internal Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. References 1. Anderson V.N. What does transgender mean to you? 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PMC10000856 | Foods Foods foods Foods 2304-8158 MDPI 10.3390/foods12050920 foods-12-00920 Article Fermented Wheat Germ Alleviates Depression-like Behavior in Rats with Chronic and Unpredictable Mild Stress Hu Zheyuan Conceptualization Methodology Software Writing - original draft 1 Zhao Penghui Writing - review & editing 1 Liao Aimei Data curation 1 Pan Long Data curation 1 Zhang Jie Visualization 1 Dong Yuqi Visualization 1 Huang Jihong Writing - review & editing Supervision Project administration Funding acquisition 123* He Weiwei Writing - review & editing Funding acquisition 4* Ou Xingqi Formal analysis 5 Gonzalez-Manzano Susana Academic Editor 1 School of Biological Engineering, Henan University of Technology, Zhengzhou 450001, China 2 School of Food and Pharmacy, Xuchang University, Xuchang 461000, China 3 State Key Laboratory of Crop Stress Adaptation and Improvement, College of Agriculture, Henan University, Kaifeng 475004, China 4 Key Laboratory of Micro-Nano Materials for Energy Storage and Conversion of Henan Province, Institute of Surface Micro and Nano Materials, College of Chemical and Materials Engineering, Xuchang University, Xuchang 461000, China 5 College of Life Science and Technology, Henan Institute of Science and Technology, Xinxiang 453003, China * Correspondence: [email protected] or [email protected] (J.H.); [email protected] (W.H.); Tel./Fax: +86-0371-67756843 (J.H.) 22 2 2023 3 2023 12 5 92011 1 2023 09 2 2023 15 2 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). Depression is a chronic mental illness with devastating effects on a person's physical and mental health. Studies have reported that food fermentation with probiotics can enrich the nutritional values of food and produce functional microorganisms that can alleviate depression and anxiety. Wheat germ is an inexpensive raw material that is rich in bioactive ingredients. For example, gamma-aminobutyric acid (GABA) is reported to have antidepressant effects. Several studies concluded that Lactobacillus plantarum is a GABA-producing bacteria and can alleviate depression. Herein, fermented wheat germs (FWGs) were used to treat stress-induced depression. FWG was prepared by fermenting wheat germs with Lactobacillus plantarum. The chronic unpredictable mild stress (CUMS) model was established in rats, and these rats were treated with FWG for four weeks to evaluate the effects of FWG in relieving depression. In addition, the study also analyzed the potential anti-depressive mechanism of FWG based on behavioral changes, physiological and biochemical index changes, and intestinal flora changes in depressed rats. The results demonstrated that FWG improved depression-like behaviors and increased neurotransmitter levels in the hippocampus of CUMS model rats. In addition, FWG effectively altered the gut microbiota structure and remodeled the gut microbiota in CUMS rats, restored neurotransmitter levels in depressed rats through the brain-gut axis, and restored amino acid metabolic functions. In conclusion, we suggest that FWG has antidepressant effects, and its potential mechanism may act by restoring the disordered brain-gut axis. FWG depression behavior neurotransmitters intestinal microbes Major Science and Technology Projects for Public Welfare of Henan Province201300110300 Innovation Demonstration Special Project of Henan Province201111110100 Henan Provincial Key Science&Technology Special Project22110011070 Central Government Guides the Local Science and Technology Development Special FundZ20221341069 Zhongyuan Scholars of Henan Province in China192101510004 Zhongyuan Scholar Workstation Funded ProjectZYGZZ2020015 214400510015 Key Project Foundation of Natural Science Research of Universities of Henan Province in China20A550004 Program for Zhongyuan Leading Talents of Science and Technology Innovation in Henan Province204200510016 This study was obtained by Major Science and Technology Projects for Public Welfare of Henan Province (201300110300), Innovation Demonstration Special Project of Henan Province (201111110100), Henan Provincial Key Science&Technology Special Project (22110011070), Central Government Guides the Local Science and Technology Development Special Fund (Z20221341069), Zhongyuan Scholars of Henan Province in China (No. 192101510004), Zhongyuan Scholar Workstation Funded Project(ZYGZZ2020015, 214400510015), Key Project Foundation of Natural Science Research of Universities of Henan Province in China (20A550004), and the Program for Zhongyuan Leading Talents of Science and Technology Innovation in Henan Province (204200510016). pmc1. Introduction Depression is a chronic mental illness that negatively affects a person's physical and mental health. Recurrent episodes of depression reduce the life expectancy and quality of life of an individual and enhance suicidal tendencies . The global estimate of people suffering from depression is more than 350 million and is expected to increase due to the increased stress in life. Depression is anticipated to top the total global burden of disease by 2030 , thus prioritizing the development of a treatment for depression. Standard pharmacological regimens, such as antidepressants, have limited efficacy, significant adverse effects, and rapid resistance , which might be attributed to the hitherto unclear pathogenesis of depression. Recently, brain-gut axis disorders, caused by dysbiosis of gut flora, have emerged as the main pathogenesis pathway of depression. Restoration of the gut microbiota in depressed patients can alleviate their depressive symptoms . Fermented probiotic foods are emerging as healthy therapeutics that are associated with restoring the gut microbiota in depressive patients. Gut symbionts are found in gut-brain signaling, immunological homeostasis, and hormone regulation, and are known to reduce depression-related symptoms by regulating brain function . Likewise, probiotics modulate the interaction between the gut and brain, thereby managing the effects of depression . According to several studies, probiotic fermentation of raw materials can result in enriched nutritious products. These products can facilitate the proliferation of good gut bacteria and bioactive ingredients that can reduce depression and anxiety . Wheat germs are a rich and inexpensive source of bioactive ingredients, containing high-quality proteins, amino acids, fats, vitamins, minerals, and other components . Moreover, g-aminobutyric acid (GABA), also found in wheat germs, has reported antidepressive effects . The findings of several studies revealed that Lactobacillus plantarum is a GABA-producing bacteria that can alleviate depression . Fermented wheat germs (FWGs) are obtained from the fermentation of wheat germs by Lactobacillus plantarum and comprise probiotics, prebiotics, protein, short-chain fatty acids, and GABA. Although there are a number of studies on probiotics for depression relief, there are still fewer studies on probiotic fermented foods for depression relief, especially Lactobacillus plantarum-FWG for depression relief, which has not been demonstrated by any other method before. In addition, most of the drugs developed for depression relief are based on the monoamine hypothesis using chemical methods, which usually have some side-effects on human health. In contrast, the study of Lactobacillus plantarum-FWG for depression alleviation adopts in a novel way a popular probiotic fermented food health therapy, in recent years, to develop new antidepressant functional foods that are healthy, green, and safe for the human body, providing a new direction for clinical treatment of depression. Finally, this study can fill the gap of fermented cereal products in the field of depression alleviation and provide new ideas for the development of cereal foods in the field of nutritional foods and functional foods. Herein, this study investigated the anti-depressive effects and potential mechanisms of Lactobacillus plantarum-FWG in depressed rats . With a CUMS rat model, the animal behaviors, neurotransmitter index, and gut microbes were evaluated to verify the therapeutic efficacy of FWG to alleviate depression. 2. Materials and Methods 2.1. Materials Lactobacillus plantarum (M618) was preserved in our laboratory. Bainong 207 defatted wheat germ powder was purchased from Henan Kunhua Biotechnology Co. (Anyang, China). Date palm GABA-positive compound tablets were obtained from MCOK BIOTECHNOLOGY (USA). The elevated plus maze and GABA content kit (plant source) were supplied by Zhengzhou Yixinyuan Instruments (Zhengzhou, China). 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), and acetylcholine (ACH) assay kits were obtained from Equipment Co. Elabscience Biotechnology Co. GABA assay kits were supplied from Wuhan Fine Biotech Co. (Wuhan, China). The ZQZY-98 oscillating incubator was purchased from Shanghai Zhichu Instruments Co. (Shanghai, China). The digital display PHS-3C laboratory PH meter was purchased from Hefei Zhuoer Instrument Co. (Hefei, China). The XFH-40MA stainless-steel autoclave was purchased from Zhejiang Xinfeng Medical Instrument Co. (Shaoxing, China). The YSW-CB-V vertical purification bench was purchased from Shenzhen Yongshengwang Industrial Co., Ltd. (Shenzhen, China). The TG16-WS desktop high-speed centrifuge was purchased from Changsha Xiangyi Centrifuge Instrument Co. (Changsha, China). The FDU-21 freeze dryer was purchased from Tokyo RIKEN, Wako, Japan. The SynergyHTX multifunctional enzyme labeler was purchased from BioTek, Winooski, VT, USA. 2.2. Preparation of FWG The Lactobacillus plantarum was cultured in De Man Rogosa Sharpe(MRS) liquid media at 37 degC for 24 h to obtain a seed solution. The number of active bacteria in the seed solution of Lactobacillus plantarum was determined to be about 2 x 108 CFU/mL by the dilution coating plate method. After drawing on Wu's study as well as the results of single-factor (Lactobacillus plantarum inoculum, fermentation time, ratio of material, pH) experiments and response surface experiments in the pre-laboratory, FWG preparation conditions were determined with the goal of optimizing the GABA content in FWG. Wheat germ powder and 50 mmol/L of acetic acid-sodium acetate buffer salt were mixed in a ratio of 1:7, shaken well, and adjusted to pH 4.45 with NaOH/HCl. They were then sterilized using an autoclave at 105 degC for 30 min and cooled to room temperature, and 3.82% of the weight of wheat germ powder was inoculated with Lactobacillus plantarum seed solution (mL/g) in the ultra-clean table and mixed evenly. This was followed by fermentation in a shaker (37 degC, 100 rpm/min) for 24 h. After fermentation, the fermentation broth was centrifuged at 5000 r/min for 15 min to obtain FWG. The FWG was stored at 4 degC and subsequently poured into several culture media plates (FWG, about 1/3 of each plate). Each plate was covered with cling film and dense and small holes were made on the surface of the cling film with a toothpick; then, the plates were placed in a refrigerator at -80 degC overnight to ensure that the FWG had been frozen into a solid form. Finally, the FWG was lyophilized according to the freeze dryer procedure. The lyophilized powder was analyzed for GABA content, using a GABA content kit. The results are displayed in Table 1. Excess lyophilized powder was subsequently stored in a -80 degC refrigerator. 2.3. Animals Male SD rats weighing 170-190 g (SCXK(Yu)2019-0002) were obtained from the Henan Huaxing Experimental Animal Farm in China. Rats were kept in a room with a humidity of 40-60% and a temperature of 22-26 degC with water and meals readily available . The animals were treated according to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. The Henan University of Technology ethics committee permitted the animal experiments conducted in this study. 2.4. CUMS Procedure and Treatment Six groups were formed with seven male SD rats in each: blank control (no stress), depression model (CUMS), positive control (GABA), low-dose FWG, medium-dose FWG, and high-dose FWG. A week before the experiments, the rats were acclimatized and fed with 1% sucrose water. Following acclimatization feeding, the rats were weighed and evaluated for 1% sucrose preference. The rats were then subjected to seven stressors over four weeks, except for the blank control group. The stressors included day and night reversal, inclined cage at 45deg (24 h), water fasting (24 h), wet bedding (400 mL of water spread onto the bedding for 10 h), tail clamping (1 min), swimming (4 degC water for 5 min), and strobing (300 times/min for 12 h) . The animals were randomly given a stressor per day, and the same stressor was not delivered for three successive days. Their body weights were measured weekly. The elevated plus maze experiment (EPM), forced swimming test (FST), open field test (OFT), and sugar preference test (SPT) were conducted four weeks after concluding the modeling phase to examine the anxiety-depression-like behavior of the rats. The behavioral findings of the remaining groups were compared to the blank control group to determine the efficiency of the model. If the model responded poorly, the modeling period was further extended in two-week intervals (with a maximum of two months) until the optimum model response. The CUMS group continued with the stressors for another four weeks, while the other groups stopped all stress stimulation. The following doses were used for gavage treatment intervention over four weeks: blank control group (normal saline 7 mL/kg*d), depression model group (normal saline 7 mL/kg*d), positive control group (26 mg/kg*d), low-dose FWG group (43 mg GABA/kg*d), medium-dose FWG group (43 mg GABA/kg*d), and high-dose FWG group (60 mg GABA/kg*d). The above experimental procedures are illustrated in Figure 1b. 2.5. Elevated plus Maze Experiment EPM is an unconditioned reflex model that uses the animal's exploratory nature in a different environment and the fear of an elevated open arm to create a conflicting state for the anxiolytic assessment of drugs. The rat was positioned in the middle of the maze with its head facing the open arm. Each experimental animal was placed in the same position thereafter, while the camera monitor was turned on to record the following indexes within five minutes: frequency of entries in the open arm (OE) and the closed arm (CE). The frequency with which the rat's two forepaws fully enter into the corresponding arm was the criterion for determining the OE and CE frequencies . Open-arm dwell time (OT) and closed-arm dwell time (CT) were measured in seconds. The feces of the rats were removed after each test and the maze was cleaned with 75% ethanol to mask the smell. The first and second elevated plus maze experiments were performed after the modeling and treatment interventions, respectively. 2.6. Forced Swimming Experiment The behavioral test used in this experiment was developed by Porsolt et al. in 1977 for the evaluation of behavioral despair . The experiment was split into two parts. The first part was pre-swimming, in which rats were confined in a transparent cylindrical container (water depth of 25-30 cm, water temperature 23 +- 1 degC) to swim for 15 min. The rats were then blown dry after pre-swimming and returned to their original cages. Experimental water was changed after each experiment to prevent external effects on subsequent experiments. The rats were placed in the pool again for a 6 min "test swim" after 24 h from the pre-swimming test and monitored. The duration of the static state of rats (e.g., body slightly curled, only nostrils exposed to maintain breathing, forepaws stopped digging, and hind paws occasionally paddled) during the last four minutes was observed. The static state is an animal response to abandon the thought of fleeing after it is unable to escape, which depicts a desperate predicament and behavioral despair. The first and second forced swimming experiments were performed after the modeling and treatment interventions, respectively. 2.7. Open Field Test (OFT) The OFT was performed to study the spontaneous activity and exploratory behavior of rats . The rats were placed in a 1 m x 1 m x 0.4 m observation box with black walls and a white floor, and red and blue lines separating the bottom surface into 16 equal squares (i.e., four red squares in the center and 12 blue squares in the periphery). Rats were put in the middle of the observation box to evaluate their horizontal and vertical activity scores. Within five minutes, the center region residence time, the number of squares traversed, and the frequency of upright times were collected . To avoid interference from previous experiments, the experimental apparatus was washed with 75% ethanol after each experiment. The first and second OFTs were conducted after modeling and treatment interventions. 2.8. Sugar Preference Test (SPT) The rats were trained with 1% sucrose water during laboratory acclimatization: Two bottles of 1% sucrose water were given for 24 h and then switched to two bottles of water for 24 h for the rats to acclimate to the sucrose water intake . Following a 12 h fast, the first SPTwas performed. The rats were fed in respective cages, and two water bottles of similar shape were placed in each cage, 1% sucrose water and pure water. The 24 h experiment was conducted and the bottle positions were switched every 12 h . Following that, both bottles were removed and weighed to determine each rat's rate of sucrose preference. Sugar water preference rate = [sucrose water consumed/(sucrose water consumed + pure water expended)] 100%. The second and third sugar preference experiments were performed according to the above procedures after four weeks of modeling and treatment intervention. 2.9. Animal Handling and Tissue Dissection The rats were fasted for 24 h after the behavioral test and deeply anesthetized with ether. Blood was collected from the eyeballs, and the rats were executed by severing their heads . The rats were dissected on ice, and their heart, liver, spleen, lungs, kidneys, and cecum contents were removed, as well as the intact brain tissue, from which the brain, cerebellum, and cortex were separated. The aforementioned organs and tissues were preserved in liquid nitrogen for fast freezing and then in a -80 degrees Celsius refrigerator until usage. 2.10. Detection of Neurotransmitters The rat hippocampus was dissected, weighed, and placed in a sterilized homogenization tube by reviewing the data combined, performing ELISA, and adding pre-chilled PBS buffer (in 2 additions) and Phenylmethylsulfonyl fluoride (PMSF) protease inhibitors at a working concentration of 1 nm/mL at a ratio of 1:9. The homogenization tube was rotated for 1-2 min to produce a 10% tissue homogenate. The levels of 5-hydroxytryptamine, 5-hydroxyindoleacetic acid, acetylcholine, and GABA in the hippocampus tissue homogenate supernatant were measured by the double antibody sandwich method . 2.11. 16S rRNA Sequence Analysis of the Gut Microbiota To obtain microbial genomic DNA from samples of rat cecum contents, we employed the E.Z.N.A.(r) Soil DNA Kit (Omega Bio-Tek, Norcross, GA, USA). A 1% agarose gel was utilized to investigate DNA extracts, and a NanoDrop 2000 UV-Vis spectrophotometer (Thermo Scientific, Wilmington, DE, USA) was used for measuring DNA concentration and purity. The thermal cycle PCR machine (Gene Amp 9700, ABI, USA) amplified the highly variable V3-V4 regions of the bacterial 16S rRNA gene 27 times with primers 338F (5'-ACTCCTACGGGAGGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'). The PCR reaction system was composed of 4 mL of 5x TransStart FastPfu buffer,10 ng of DNA template, 2 mL of 2.5 mM dNTPs, 0.8 mL of each upper and lower primer (5 mM), 0.4 mL of TransStart FastPfu DNA polymerase, and topped up to 20 mL with H2O. PCR reaction was carried out three times. The final PCR products were purified, quantified, and sequenced using Illumina's MiseqPE300 platform. For microbiome analysis, raw sequences were quality-controlled using fastp (version 0.20.0) and spliced using FLASH (version 1.2.7) . Chimeras were removed using UPARSE (version 7.1) . Operable taxonomic units (OTUs) were constructed, and each sequence was classified and annotated using the RDP classifier (version 2.2), with a 70% comparison threshold set against the SILVA 16S rRNA database (version 138) . The richness and diversity of samples were assessed using diversity analysis, and the results were subjected to the Wilcoxon rank sum test between the exponential groups. The principal coordinate analysis (PCoA) distance visualization approach was employed to investigate b-diversity in the microbial community. To investigate the variations in species composition across groups, colony bar plots at the family level and Wilcoxon rank-sum tests of substantially different colonies were utilized. PICRUSt2 predicted the effect of FWG on fecal microbiota function in CUMS rats. 2.12. Statistical Analysis Experimental data on body weight, behavioral assessments, and neurotransmitter levels within the hippocampus were presented as mean +- standard error (SEM) of at least three independent experimental data samples. One-way ANOVA was used for statistical analysis. GraphPad Prism version 8.0 was used to run one-way ANOVA between groups (GraphPad software, Inc., La Jolla, CA, USA). In addition, ANOSIM analysis was performed using QIIME scripts. The Wilcoxon rank sum test was based on R's stats package (version 3.3.1) and the Python scipy module. 3. Results 3.1. GABA Content in FWG Lyophilized Powder In order to ensure that the GABA content in FWG lyophilized powder would not exceed the detection range of the kit when using the kit, different masses of FWG lyophilized powder were dissolved in 1 mL of water (mg/mL) for the determination, and the results in Table 1 showed that 10 mg of FWG lyophilized powder dissolved in 1 mL of water gave the best detection results, and the GABA content in FWG lyophilized powder was 19,797.54 +- 2719.322 (mg/g). 3.2. Amelioration of CUMS-Induced Depression-like Behavior in Rats by FWG The influence of FWG on the weight of CUMS rats is depicted in Figure 2a. Before the test, there was no appreciable difference in the weight of rats in all groups. Rats stimulated by CUMS exhibited considerably lower body mass indexes than the control group after four weeks of CUMS modeling. The body weight indices of CUMS rats improved dramatically after four weeks of FWG administration. There was no significant difference between the high-dose FWG group and the blank control group. The anxiolytic effect of drugs is usually assessed in the elevated plus maze experiment by OE and OT metrics, which are negatively correlated with anxiety. As demonstrated in Figure 2b, the OE of rats in the CUMS group was noticeably lower when compared to the control group (**** p < 0.0001). On the contrary, four weeks of treatment intervention restored the OE in the low-, medium-, and high-dose FWG groups to the level of the control group (N.S, # p > 0.05). Among these, the difference in the OE and OT of the high-dose FWG group was the largest as compared to the control group (**** p < 0.0001), thus indicating that FWG could improve the anxiety of depressed rats. FST was designed to assess the level of despair in CUMS rats in a static state, and the study revealed that CUMS rats had a longer swimming immobility time and more pronounced despair . As depicted in Figure 2c, CUMS rats had significantly more immobility time as compared to healthy controls (#### p < 0.0001). Following four weeks of intervention, the immobility time of rats in the high-dose FWG groups decreased (**** p < 0.0001) and returned to a normal level (N.S), thus indicating that the high-dose FWG could improve the despair in depressed rats. The number of traversal squares indicated the motor ability and exploratory behavior of rats, and the depressed rats have a lower number of traversed lattices. According to Figure 2d, the number of traversing lattices was appreciably lesser in CUMS rats than in the blank control group (#### p< 0.0001). After four weeks of FWG treatment, the low-, medium-, and high-dose FWG groups reported an increase in the frequency of traversing lattices in comparison to the CUMS model group. However, only the high-dose FWG group reported statistical significance (*** p < 0.001). Thus, a high-dose FWG treatment could improve the motor activity and independent exploratory behavior of depressed rats. A lack of pleasure is a critical depressive trait that is assessed mainly by the percentage of sugar water ingested in the SPT . In Figure 2e, the percentage of sugar water intake in each group of rats was not significantly different (N.S, p > 0.05). CUMS rats had a significantly lower percentage of sugar water intake than the blank controls (#### p < 0.0001). After four weeks of treatment intervention, rats in the low-, medium-, and high-dose FWG groups reported a significantly higher percentage of sugar water consumption than the CUMS model group (**** p < 0.0001, ## p < 0.001), indicating that FWG could improve the pleasure deficit in CUMS rats, but not fully restore them to normal levels. 3.3. Modulation of Neurotransmitters in the Hippocampus of CUMS Rats by FWG The levels of 5-HT, 5-HIAA, Ach, and GABA in the hippocampus of CUMS rats were significantly reduced in comparison with the normal control (## p < 0.01) . The low-dose FWG group exhibited a substantial increase in 5-HT and 5-HIAA levels after four weeks of treatment intervention as compared with the CUMS model group (* p < 0.05), which returned to normal levels that were comparable to the blank control group (N.S, # p > 0.05). The recovery of the other two neurotransmitters displayed an upward trend, but it was not significantly different. The medium-dose FWG group reported a significant increase in the levels of 5-HT, 5-HIAA, Ach, and GABA as compared with the CUMS model group (* p < 0.05, ** p < 0.01). The concentrations of 5-HT, 5-HIAA, and Ach were all restored to levels comparable to the control group (N.S, # p > 0.05), while GABA did not return to the normal level (# p < 0.05). In comparison to the model set, the high-dose FWG group exhibited slightly higher levels of 5-HT,5-HIAA, Ach, and GABA than the blank control group (* p < 0.05, **** p < 0.0001, # p > 0.05). Figure 2 and Figure 3 demonstrate that the CUMS model was successfully established, and FWG had a similar impact on depression-like behaviors and neurotransmitter levels in the hippocampus of depressed rats. Furthermore, different doses of FWG had different degrees of improvement in depression, and the combined behavioral improvement results and neurotransmitter restoration effects highlighted that a high dose of FWG exhibited the strongest effect. 3.4. Analysis of Gut Microbe Diversity The dilution curves in Figure 4a,b reached a plateau, indicating that the sequencing depth and coverage of the samples could be further analyzed. The a-diversity is reflected by community richness (Sobs index), while the species diversity of the samples is reflected by the community diversity index (Shannon index). There was a significant decrease in both species richness and diversity in the CUMS model group as compared with the blank control group (p < 0.05) , and both species richness and diversity increased after treatment intervention with GABA and FWG (p > 0.05). To differentiate samples with different microbial communities, we employed PCoA analysis based on b-diversity analysis. The rats in the CUMS model group had significantly different fecal microbiota than rats in the control group, and the fecal microbiotas of the GABA and FWG groups were similar to that of the blank control . The results of an analysis of similarity (ANOSIM), based on PCoA scores, indicated a statistically remarkable divergence between all four groups . 3.5. Analysis of Intestinal Flora Composition at Different Classification Levels Differences in the species composition of gut microorganisms at the family level in different groups of samples are depicted in Figure 5. The model group had a higher abundance of both Lactobacillaceae and Erysipelotrichaceae and a lower abundance of Peptostreptococcaceae, Lachnospiraceae, and Oscillospiraceae than the other groups . To determine the significance of their differences in different groups, the aforementioned families were treated to a multigroup rank sum test analysis. Figure 5b,c indicate that the abundance of Lactobacillaceae and Erysipelotrichaceae in the model set was considerably greater than in the blank control group (p < 0.01). After the treatment with GABA or FWG, the abundance of Lactobacillaceae decreased significantly until the level reached that of the blank control group (p > 0.05), while the abundance of Erysipelotrichaceae only decreased slightly. The abundance of Peptostreptococcaceae was lower in the model group in comparison to the blank control group (p > 0.05). Likewise, treatment with GABA or FWG significantly increased the abundance of Peptostreptococcaceae until the level reached that of the blank control group (p > 0.05) . Meanwhile, the abundance of Lachnospiraceae was lower in the model group than in the blank control group (p < 0.01). After treatment with GABA or WFG, the abundance of Lachnospiraceae increased significantly until the level reached that of the blank control group (p > 0.05) . The abundance of Oscillospiraceae was significantly lower in the model group relative to the control group, but the difference was not statistically significant (p > 0.05). The abundance of Oscillospiraceae increased significantly after GABA or FWG treatment until the level reached that of the blank control group (p > 0.05) . Figure 6 demonstrates that FWG regulated the composition of the gut microbiota at the genus level. The abundance of Lactobacillus and Marvinbryantia in the CUMS model group was higher than those in all the other groups, while the abundance of unclassified_f__Lachnospiraceae and Romboutsia was lower than those in all the other groups . The Kruskal-Wallis H test was performed on the above genera, and the boxplot results are displayed in Figure 6b-e. The abundance of Lactobacillus was significantly higher in the model group than in the blank control group (p < 0.01) . After treatment with GABA or FWG, the abundance of Lactobacillus significantly decreased until the level of the blank control group (p > 0.05). Likewise, the abundance of Romboutsia and unclassified_f__Lachnospiraceae in the model group was significantly lower than that of the normal control (p < 0.05, p < 0.01) . After treatment with the FWG intervention, the abundance of Romboutsia and unclassified_f__Lachnospiraceae returned to levels that were close to that of the blank control group. The CUMS model group reported an increased abundance of Marvinbryantia compared to the control group (p > 0.05) . After treatment with FWG, its abundance decreased to levels similar to the blank control group (p > 0.05). 3.6. Correlation Analysis between Gut Microbiota and Neurotransmitters The analysis revealed 28 strong correlations between neurotransmitters and specific gut microflora . Among them, Ach was significantly and positively correlated with Romboutsia, g__norank_f__Eubacterium_coprostanoligenes_group, and NK4A214_group, while it was significantly and negatively correlated with Lactobacillus. Besides that, 5-HIAA was positively correlated with Romboutsia and negatively correlated with Lactobacillus. GABA and norank_f__Lachnospiraceae reported a significant positive correlation. 3.7. Impact of FWG on Gut Microbiota Function The analysis identified 29 KEGG pathways, with significant differences in KEGG Path Level 2 for each group of rats . Among them, the carbohydrate and amino acid metabolic pathways were considered to be dominant, and depressed rats were usually characterized by a dysfunctional amino acid metabolism . Hence, a rank sum test was performed on the amino acid metabolic pathways for each group of rats . The results revealed that the CUMS model group had lower levels of amino acid metabolism than the normal group did (p < 0.01), implying dysfunctional amino acid metabolism. After treatment with GABA or FWG, the level of amino acid metabolism greatly improved and returned to levels near the blank control group (p > 0.05). The results of the KEGG Module for each group of rats were subjected to a rank sum test to further explore amino acid metabolism in depression . The results demonstrated that there were 14 differential modules of amino acid metabolic functions among the groups of rats, including M00525, M00017, M00026, M00570, M00023, M00609, M00846, M00015, M00845, M00028, M00025, M00024, M00135, and M00038 (Table 2). The biosynthetic functions of lysine and proline were upregulated, while those of tryptophan, GABA, glutamate, isoleucine, and histidine were decreased in the CUMS model group. After GABA or FWG intervention, the effects were reversed. 4. Discussion FWG improved the depression-like behaviors of CUMS rats in the current study, including improved weight loss, increased percentage of OT, decreased despair, improved mobility, and alleviated pleasure deficit symptoms. These findings are consistent with the results from previous studies, involving fermented American red ginseng (ARG) and fermented GABA oolong tea . Neurotransmitter deficiency is the root cause of depression. Most antidepressants function by enhancing monoamine neurotransmitters in the brain . However, these drugs have their limitations and side-effects in clinical practice. We evaluated the levels of 5-HT, 5-HIAA, Ach, and GABA in the hippocampus tissue to study the effect of FWG on neurotransmitter release. FWG was observed to increase the levels of 5-HT, 5-HIAA, Ach, and GABA in rat hippocampus tissues, which is consistent with earlier research . Hence, FWG is generally recognized as safe (GRAS) and may be a potential alternative to treat depression-like behavior without side-effects. According to emerging evidence, depression development may be linked to the gut flora . Thus, we postulated that the anti-depressive effect of FWG might be correlated with intestinal microbiota and gut microbial function. The a-diversity data revealed that there was a remarkable decrease in both species' richness and diversity in the CUMS model group in comparison with the blank control group (p < 0.05). Moreover, there was a general upward trend in both species' richness and diversity after therapeutic intervention with GABA or FWG (p > 0.05). The PCoA results revealed that the intestinal flora of the CUMS model group differed significantly from that of the blank control group, validating the effects of chronic stress on the gut microbiota. The intestinal flora distribution in FGW rats was similar to that of the blank control group, suggesting that FWG intervention could change the structure of the intestinal flora. These findings were consistent with a recent study that discovered a noticeable change in the microbiota of depressed individuals after GABA-rich fermented milk intake . Our findings are also congruent with previous research that found significant changes in the gut flora composition in rats with irritable bowel syndrome and depressed mice, which were fed with a barley and soybean fermentation mixture . These results suggested that the anti-depressive effects of FWG might be mediated by the intestinal flora. We observed that the abundance of Lactobacillaceae and Erysipelotrichaceae increased in the rat fecal microbiota of the CUMS model group. In addition, there was a lower abundance of Peptostreptococcaceae, Lachnospiraceae, and Oscillospiraceae in the CUMS model group than in the blank control group. There was also an increase in the abundance of Lactobacillus and Marvinella in the rat fecal microbiota of the CUMS model group, and a significant decrease in the abundance of Romboutsia and unclassified _f__Lachnospiraceae in the CUMS model group. However, the Lactobacillus strain might damage the host's neurological function and lead to dysbiosis . In some studies, the abundance of the highly immunogenic and inflammation-related Erysipelotrichaceae significantly increased in depressed patients, as well as those with IBS and neurodegenerative diseases . Furthermore, there is a correlation between IBS and depression . The study discovered that patients with depression had remarkably lower levels of Peptostresptococcaceae in their gut than healthy controls . In a separate study, the abundance of Peptostreptococcaceae was lower in the CUMS model than in the control group . From the above findings, we inferred that Peptostreptococcaceae is a group of depression-associated bacteria. On the contrary, the abundance of Lachnospiraceae can affect the metabolism of short-chain fatty acids, intestinal permeability, and neurotransmitter secretion , and its abundance was significantly reduced in CUMS rats . The unclassified _f__Lachnospiraceae belonged to the Lachnospiraceae family, and a study on the intestinal flora of Chinese children with autism spectrum disorders (ASDs) reported that the abundance of unclassified _f__Lachnospiraceae was significantly reduced in ASD patients . In summary, we concluded that Lachnospiraceae might be an important marker for identifying neurological diseases, especially depression. According to a study on the gut microbiota of depressed people, the abundance of Oscillospiraceae was reduced . In a study on fish oil improving depression-like behavior and intestinal flora dysbiosis in chronically mildly stressed rats, CMS increased the abundance of Marvinbryantia . In contrast, a reduced abundance of Romboutsia in the intestinal flora of patients had been reported in studies on neurological diseases, such as Alzheimer's disease and neurodegenerative disorders . It could be observed that FWG downregulated the abundance of Lactobacillaceae, Erysipelotrichaceae, Lactobacillus, and Marvinbryantia, while increasing the abundance of Peptostreptococcaceae, Lachnospiraceae, Oscillospiraceae, Romboutsia, and unclassified__f__Lachnospiraceae . Taken together, our findings and the corroborating evidence from the literature uncovered a relationship between changes in the intestinal flora and depression-like symptoms, as well as the efficacy of FWG treatment in restoring the intestinal microbiota in CUMS rats. Furthermore, the results of our Spearman correlation analysis revealed an important role of gut microbiota in alleviating depression. A strong correlation between neurotransmitter levels and gut microbiota genus was also established, thus providing strong support for the brain-gut axis theory. PICRUSt2 functional prediction data disclosed that the model group had dysfunctional amino acid metabolism as compared to the blank control group, with increased biosynthesis of lysine and proline and decreased biosynthesis of tryptophan, GABA, glutamate, isoleucine, and histidine. GABA or FWG intervention decreased the biosynthetic functions of lysine and proline and increased the biosynthetic functions of tryptophan, GABA, glutamate, and isoleucine. According to several studies, the genes related to lysine biosynthesis were significantly elevated in the fecal flora of depressed rats . Hence, proline is significantly and positively correlated with the severity of depression. In addition, proline supplementation was reported to aggravate depression and microbial translocation in mice , as the accumulation of proline disrupted GABA production, glutamate release, and synaptic transmission. On the other hand, tryptophan affects the body's mood, sleep quality, and appetite, and tryptophan levels were significantly reduced in depressed patients . GABA, a naturally occurring non-protein amino acid, is an essential inhibitory neurotransmitter in the mammalian central nervous system. Studies have reported that GABA affects the monoamine levels in the brain, such that GABA deficiency triggers emotions such as anxiety, restlessness, fatigue, and depression . Likewise, GABA supplementation alleviated sleep disturbance in mice . Glutamate, an acidic amino acid, is a key excitatory neurotransmitter in the central nervous system, and its dysregulation might be a major cause of depression . Low glutamate levels manifest as decreased excitability, resulting in a low mental state, depression, tiredness, and sleeplessness. Isoleucine aids in blood sugar regulation, and its deficiency can cause fatigue, melancholy, disorientation, and irritability. In summary, we speculated that FWG can alleviate depressive symptoms in CUMS rats by restoring the metabolic balance of lysine, proline, tryptophan, GABA, glutamate, and isoleucine. It should be noted that the functional analysis of the intestinal flora in this study was performed by 16S rRNA sequencing technology to sequence and analyze very small DNA fragments in DNA extracts of the sample gut microbiota. Further mathematical computations were performed based on the sequencing data to predict the metabolic pathways. As a result, these data should be regarded as guidelines, and not as substantial evidence. With a rich database of whole-genome sequences of intestinal bacteria and a thorough annotation of their metabolic pathways, a precise understanding of intestinal microbial functions can be established. The involvement of lysine, proline, tryptophan, GABA, glutamate, and isoleucine in this study justified the requirement of future investigations, whereby untargeted metabolomics can be used to analyze more relevant metabolomics in cecum content samples. Overall, our findings are consistent with previous research, indicating that CUMS resulted in gut microbiota dysregulation and that FWG could alleviate depressive symptoms. More research is required to fully elucidate the mechanisms of FWG in relieving depression and other neurological conditions. 5. Conclusions In this study, we demonstrated that FWG improved depression-like behavior and increased neurotransmitter levels in the hippocampus of CUMS model rats. Our findings provided support for the role of intestinal microbiota in brain health and the anti-depressive effects of FWG in the gut-microbiota-brain axis. In particular, FWG could effectively change the intestinal microbiota structure of CUMS rats, remodel the intestinal microbiota, restore the neurotransmitter levels in depressed rats through the brain-gut axis, and recover amino acid metabolism functions. In conclusion, we believe that FWG has antidepressant potential. Our findings provide new ideas for the clinical treatment of depression and theoretical support for the development of safe novel antidepressant functional foods. Acknowledgments I would like to thank my supervisors, Huang and He, for providing financial support and guidance on the experiment and paper writing. Author Contributions Z.H.: Experiment Design, Conceptualization, Methodology, Software, Writing--Original Draft Preparation. J.H.: Funding Acquisition, Writing--Reviewing and Editing, Supervision, Project Administration. W.H.: Funding Acquisition, Writing--Review and Editing. P.Z.: Article Revision and Editing. A.L. and L.P.: Data Management and Organization. J.Z. and Y.D.: Visualization. X.O.: Formal Analysis. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The animals were treated according to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. The Henan University of Technology ethics committee permitted the animal experiments conducted in this study (protocol code:P20210918-1). Data Availability Statement The datasets generated for this study are available on request to the corresponding author. Conflicts of Interest The authors declared no conflict of interest. Figure 1 Experimental program overall design diagram (a) Schematic illustration for rational design of preparation of FWG for treatment of depression in rat by modulating gut microbes (Drawn by Figdraw). (b) Schematic diagram of a phased protocol for animal experiments. (FWG, Fermented wheat germs; CUMS, chronic unpredictable mild stress; 5-HT, 5-hydroxytryptamine; 5-HIAA, 5-hydroxyindoleacetic acid; ACH, acetylcholine; GABA, g-aminobutyric acid; SPT, sucrose preference test; EMP, elevated plus maze experiment; FST, Forced swimming experiment; OFT, open field test.). Figure 2 Effects of fermented wheat germ on the behavior of depressed rats. (a) Effect of FWG on body weight in CUMS rats. (b) Time to open arm as a percentage of total time (OT%). (c) Effect of fermented wheat germ on the immobility time of depressed rats in FST. (d) Number of lattices traversed by rats in OFT. (e) Effect of fermented wheat germ on sucrose preference in SPT. (** p < 0.01, *** p < 0.001, and **** p < 0.0001 indicate an appreciable difference compared to the model group. ## p < 0.01, ### p < 0.001, and #### p < 0.0001 represent that the difference was appreciable in contrast with the normal control group). Figure 3 Effect of fermented wheat germ on neurotransmitters in the hippocampus of depressed rats. (a) Hippocampus 5-HT concentration. (b) Hippocampus 5-HIAA concentration. (c) Hippocampus ACH concentration. (d) Hippocampus GABA concentration. (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 indicate an appreciable difference compared to the model group. # p < 0.05, ## p < 0.01, and #### p < 0.0001 represent that the difference was appreciable in contrast with the normal control group). Figure 4 Analysis of gut microbe diversity. (a) Sample dilution curve based on Sobs index. (b) Sample dilution curve based on Shannon index. (c) Wilcoxon rank-sum test for Sobs index of differences between index groups. (d) Wilcoxon rank-sum test for Shannon index of differences between index groups. (e) PCoA analysis based on beta diversity analysis. (f) The results of an analysis of similarity (ANOSIM) based on PCoA. (* p < 0.05 indicates a statistically significant difference.) Figure 5 Differences in the species composition of gut microorganisms at the family level in different groups of samples. (a) Community bar-pot analysis at the family level. (b) Wilcoxon rank-sum test post hoc plots for Lactobacillaceae. (c) Wilcoxon rank-sum test post hoc plots for Erysipelotrichaceae. (d) Wilcoxon rank-sum test post hoc plots for Peptostreptococcaceae. (e) Wilcoxon rank-sum test post hoc plots for Lachnospiraceae. (f) Wilcoxon rank-sum test post hoc plots for Oscillospiraceae. (* p < 0.05, ** p < 0.01, *** p < 0.001 indicates a statistically significant difference.) Figure 6 FWG regulated the composition of the gut microbiota at the genus level. (a) Community bar-pot analysis at the genus level. (b) Kruskal-Wallis H test box line graph for Lactobacillus. (c) Kruskal-Wallis H test box line graph for Romboutsia. (d) Kruskal-Wallis H test box line graph for unclassified_f__Lachnospiraceae. (e) Kruskal-Wallis H test box line graph for Marvinbryantia. (* p < 0.05, ** p < 0.01, *** p < 0.001 indicates a statistically significant difference.) Figure 7 Correlation analysis between gut microbiota and neurotransmitters. (* p < 0.05, ** p < 0.01, *** p < 0.001 indicates a statistically significant difference.) Figure 8 Effect of FWG on fecal microbiota function. (a) Differential pathways on KEGG Path Level2 in various groups of rats. (b) Wilcoxon rank -sum test of each group of rats in the amino acid metabolic pathway. (c,d) Differences between groups of rats related to the function of amino acid metabolism Module. (* p < 0.05, ** p < 0.01, *** p < 0.001 indicates a statistically significant difference.) foods-12-00920-t001_Table 1 Table 1 GABA content in FWG. Mass of FWG Dissolved in 1 mL of Water (mg/mL) Absorbance (Measuring Tube) Absorbance (Control Tube) GABA Content in FWG (mg/g) 1 mg 0.218 0.209 1 mg 0.229 0.225 1 mg 0.237 0.224 10 mg 0.308 0.211 19,078.58 10 mg 0.328 0.239 17,509.94 10 mg 0.341 0.225 22,804.1 100 mg 0.967 0.187 100 mg 0.869 0.212 100 mg 0.947 0.175 Notes: GABA content (mg/mL) = 1960.8 x (A + 0.0003), where A = measuring tube absorbance - control tube absorbance. A value of A hovering around 0 requires an increase in sample mass. If the absorbance of the measuring tube is greater than 0.6, the sample needs to be diluted. foods-12-00920-t002_Table 2 Table 2 Annotation of Modules related to amino acid metabolism. Modules Number Module Description Information M00525 Lysine biosynthesis, acetyl-DAP pathway M00017 Methionine biosynthesis, aspartate >= homoserine M00026 Histidine biosynthesis, PRPP >= histidine M00570 Isoleucine biosynthesis, threonine >= 2-oxobutanoate M00023 Tryptophan biosynthesis, chorismite >= tryptophan M00609 Cysteine biosynthesis, methionine >= cysteine M00846 Siroheme biosynthesis, glutamate >= siroheme M00015 Proline biosynthesis, glutamate >= proline M00845 Arginine biosynthesis, glutamate >= acetylcitrulline M00028 Ornithine biosynthesis, glutamate >= ornithine M00025 Tyrosine biosynthesis, chorismite >= tyrosine M00024 Phenylalanine biosynthesis M00135 GABA biosynthesis, eukaryotes, putrescine >= GABA M00038 Tryptophan metabolism, tryptophan>= kynurenine Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). 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PMC10000857 | Healthcare (Basel) Healthcare (Basel) healthcare Healthcare 2227-9032 MDPI 10.3390/healthcare11050687 healthcare-11-00687 Article Living with Epilepsy in Adolescence in Italy: Psychological and Behavioral Impact Turner Katherine 1* La Briola Francesca 1 Vignoli Aglaia 12 Zambrelli Elena 1 Chiesa Valentina 1 Fongoni Laura 1 Baldi Olivia 1 Canevini Maria Paola 12 Giansanti Daniele Academic Editor 1 Epilepsy Center, Childhood and Adolescence Neuropsychiatry Unit, ASST Santi Paolo e Carlo, San Paolo Hospital, 20142 Milan, Italy 2 Department of Health Sciences, University of Milan, 20142 Milan, Italy * Correspondence: [email protected]; Fax: +39-0250323159 25 2 2023 3 2023 11 5 68712 12 2022 10 2 2023 22 2 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). Background: People with epilepsy have a higher prevalence of behavioral and neuropsychiatric comorbidities compared to the general population and those with other chronic medical conditions, although the underlying clinical features remain unclear. The goal of the current study was to characterize behavioral profiles of adolescents with epilepsy, assess the presence of psychopathological disorders, and investigate the reciprocal interactions among epilepsy, psychological functioning, and their main clinical variables. Methods: Sixty-three adolescents with epilepsy were consecutively recruited at the Epilepsy Center, Childhood and Adolescence Neuropsychiatry Unit of Santi Paolo e Carlo hospital in Milan (five of them were excluded) and assessed with a specific questionnaire for psychopathology in adolescence, such as the Questionnaire for the Assessment of Psychopathology in Adolescence (Q-PAD). Q-PAD results were then correlated with the main clinical data. Results: 55.2% (32/58) of patients presented at least one emotional disturbance. Body dissatisfaction, anxiety, interpersonal conflicts, family problems, uncertainty about the future, and self-esteem/well-being disorders were frequently reported. Gender and poor control of seizures are associated with specific emotional features (p < 0.05). Conclusions: These findings highlight the importance of screening for emotional distress, recognition of the impairments, and provision of adequate treatment and follow-up. A pathological score on the Q-PAD should always require the clinician to investigate the presence of behavioral disorders and comorbidities in adolescents with epilepsy. epilepsy adolescence behavioral disorders psychiatric comorbidities This research received no external funding. pmc1. Introduction Adolescents with epilepsy are at increased risk of developing psychological and behavioral disorders compared to the general population and those with other chronic medical conditions . The International League Against Epilepsy (ILAE) has identified that the prevalence of neuropsychiatric disorders in children with epilepsy is around 35-50%, and over 50% presented an intellectual disability (ID) . Several investigations have reported that up to 50% of subjects go undetected and untreated. Mood, anxiety, and attention-deficit hyperactivity disorder (ADHD) occur most frequently in adolescents with epilepsy . Neuropsychiatric symptomatology in pediatric epilepsy has been associated with different hypotheses. Firstly, the unpredictable nature of seizures could influence psychological development, thereby fostering psychiatric disorders. Moreover, coexisting neurological comorbidities may contribute to the development of psychological distress. Several authors have found that ID is the most common neuropsychiatric manifestation in children with epilepsy (30-40%) . Of note, anti-seizure medications may cause cognitive and behavioral side effects. Furthermore, epileptogenic circuits could be linked with modifications in brain structure and function, possibly resulting, through many different mechanisms, in neuropsychiatric manifestations . Behavioral disturbances can precede epilepsy onset or may be present in the early phase; indeed, the association between epilepsy and behavioral difficulties can be bidirectional . Some authors have suggested that depressed mood and anxiety may precede the onset of epilepsy and act as independent risk factors for the development of unprovoked seizures . The worldwide pandemic caused by the SARS-CoV-2 virus was associated with an increased risk of psychiatric sequelae in the general population . Furthermore, during the pandemic, a significant number of patients with epilepsy experienced worsening psychological symptoms and an increase in seizure frequency . Within this perspective, in the present study, we investigated the psychological and behavioral sequelae experienced by a group of adolescents with epilepsy using a specific questionnaire for psychopathology in adolescence, the Assessment of Psychopathology in Adolescence (Q-PAD). We also investigated whether the clinical characteristics of epilepsy, such as epilepsy type, epilepsy onset, seizure frequency, anti-seizure medications, age, intelligence quotient (IQ), and gender interfere with emotional well-being. 2. Materials and Methods 2.1. Participants Patients with a diagnosis of epilepsy were consecutively recruited and prospectively evaluated within this study. The inclusion criteria included: (1) diagnosis of epilepsy; (2) aged between 14 and 19 years old; and (3) fluency in the Italian language. The exclusion criteria were: (1) intellectual disability; (2) major progressive neurological pathologies; (3) history of psychiatric disorders (diagnosis according to DSM-5); and (4) receiving medications other than antiepileptic drugs. We included patients with a total IQ under the normal range but with a normal General Ability Index (GAI). It is well known that patients with epilepsy have more difficulty in working memory and processing speed, so the GAI could be considered a more accurate representation of their intelligence. The diagnosis of epilepsy was based on the ILAE classification . Seizure types were classified according to clinical semiology provided by description from a witness or, when possible, by electroencephalogram (EEG) or video-EEG recording of the episode . Patients were considered seizure-free if they were without clinical seizures for at least 1 year, using the last clinical visit documenting seizure status as the end point of follow-up. Refractory epilepsy was defined as uncontrolled seizures after at least two first-line anticonvulsant medication trials . Intelligence assessments were performed according to best practice standards using the Wechsler Intelligence Scale for Children (WISC-III or WISC-IV) or Wechsler Adult Intelligence Scale (WAIS-R or WAIS-IV). For all patients, intelligence assessments were selected according to best practice standards. We classified patient IQ (intelligence quotient) as follows: normal IQ; borderline intellectual functioning (BIF); and mild, moderate, severe or profound ID according to Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5) criteria . The sample included 63 adolescents attending the Epilepsy Center, Childhood and Adolescence Neuropsychiatry Unit of Santi Paolo e Carlo Hospital, University of Milan, Milan. Two adolescents refused to participate in the study. We enrolled our patients over a span of two years (2019-2020). All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. The Ethics Committee of San Paolo Hospital reviewed and approved the study protocol. All patients and their caregivers provided their informed written consent before being enrolled in the study. 2.2. Questionnaire The Questionnaire for the Assessment of Psychopathology in Adolescence (Q-PAD) is a self-administered instrument to assess psychopathological domains described in the scientific literature regarding adolescents aged from 14 to 19 years. The questionnaire contains 81 items in a Likert-type scale with values from 1 to 4, formulated in a language that is familiar according to the linguistic uses of adolescents. The scores of each item are added up for each scale and then converted to percentile values. Therefore, the output of the Q-PAD consists of eight scores referring to the following main psychological areas: body dissatisfaction, anxiety, depression, substance abuse, interpersonal conflicts, family problems, uncertainty about the future, and psychosocial risks. A further ninth domain, called self-esteem and well-being, concerns well-being and adjustment and is assessed in positive terms. The questionnaire has been validated on a sample of 1454 Italian adolescents (Internal Consistency: 0.83, Test-Retest Reliability: 0.84) . 2.3. Data Analysis All statistical analyses were performed using the Statistical Package for Social Sciences (SPSS) version 27.0 for Windows. Qualitative data were reported as absolute frequencies and percentages. Quantitative data were expressed as means and standard deviations (SDs). Demographics and clinical variables of adolescents were compared with the Mann-Whitney U test or Student's t-Test for independent samples, depending on the distribution. The normality of the data and homogeneity of variances were tested by the Shapiro-Wilk test and Levene's test, respectively. Significance was set at a p-value of 0.05. We compared the questionnaires by dividing the participants according to the type of epilepsy, epilepsy onset, frequency of seizures, controlled seizures vs. uncontrolled, anti-seizure medications, age (age 14-15 years vs. 16-19 years), IQ, and gender. 3. Results 3.1. Patient Characteristics and Clinical Variables Sixty-three patients (mean age of 17.24 +- 2.05 years, range: 14-19 years; 19 [32.8%] males, 39 [67.2%] females) were consecutively enrolled in the study; five of them had to be excluded for the following reasons: two showed moderate-severe ID, and three patients were not Italian native speakers (two Hispanic and one Arabic). Demographic and clinical data were collected from the patients' medical charts. Forty (69.0%) patients had focal epilepsy (23 [39.7%] focal epilepsy of unknown etiology, 17 [29.3%] structural focal epilepsy); 18 patients (31.0%) had generalized genetic epilepsy (GGE). The mean age of epilepsy onset was 10.05 years (+-5.1), and the mean duration since diagnosis was 8.07 years (+-5.82). Characteristics of patients are presented in Table 1. 3.2. Psychological Variables The Q-PAD findings suggest the presence of at least one neuropsychiatric disturbance in 55.2% (32/58) of the adolescents with epilepsy. Our sample had a clinical range of body dissatisfaction (19.0%), anxiety (19.0%), depression (6.9%), substance abuse (1.7%), interpersonal conflicts (22.4%), family problems (25.9%), and uncertainly about the future (22.4%). Moreover, concerning self-esteem/well-being, 31.0% had a normal range, 20.7% borderline values and 48.3% showed a clinical range . Statistically significant differences were found considering age as a covariate: older adolescents (age 16-19 years) showed more interpersonal conflicts (mean = 63.5 +- 26.86 vs. mean = 42.89 +- 30.0) and uncertainty about the future (mean = 55.77 +- 29.27 vs. mean = 37.39 +- 27.16) than younger patients (age 13-15 years; p = 0.016, p = 0.022 respectively). 3.3. Clinical Variables x Psychological Variables We evaluated the association between some of the clinical variables and the psychological constructs. The type and the onset of epilepsy had no impact on Q-PAD measures. However, we found a statistically significant difference between adolescents with controlled seizures vs. uncontrolled seizures on anxiety, depression, interpersonal conflicts, family problems, and psychosocial risks . In relation to treatment (monotherapy vs. polytherapy) and IQ (Normal IQ vs. Borderline Intellectual Functioning), we found no differences in the psychological dimensions investigated (p > 0.05). Finally, with respect to gender, we found no differences in the psychological dimensions investigated apart from depression; girls tended to be more depressed (mean = 54.74 +- 27.77) than boys (mean = 37.89 +- 24.39, p = 0. 027). 4. Discussion Although most studies on epilepsy and psychological variables have gathered information from parents and teachers, an interesting aspect of our work was to focus on the patient's point of view. As a general result, our data showed that adolescents with epilepsy have a high rate of neuropsychiatric disturbances (55.2%, 32/58), in accordance with the literature . We found a clinical range of body dissatisfaction (19.0%), anxiety (19.0%), depression (6.9%), substance abuse (1.7%), interpersonal conflicts (22.4%), family problems (25.9%), uncertainly about the future (22.4%), and low self-esteem/well-being (48.3%). These findings are thus in accordance with a previous meta-analysis by Lin and colleagues, showing that subjects with epilepsy are at increased risk for neuropsychiatric disorders, such as internalizing behaviors (anxiety, mood and social disorders) prevailing over externalizing functioning (aggressive outbursts or conduct disorders) . While these manifestations may be considered consequences of epilepsy, increasing evidence suggests that these disturbances might precede the onset of epilepsy and seizures. Therefore, epilepsy and neuropsychiatric issues may have a bidirectional correlation, sharing a common underlying pathogenesis . Psychiatric disorders, in particular mood and anxiety disorders, may be a reaction to psychosocial obstacles, lower quality of life, and perceived stigmatization by parents . Providing patients, especially those with drug-resistant seizures, with psychological support to orientate their psychological patterns to efficacious ones, improve the overall quality of life and emotional well-being, and reduce fatigue, should be seriously considered. Such cognitive-behavioral programs could help adolescents to adopt more effective ways of coping with their clinical conditions and experience easier psychosocial adjustment . Moreover, we found that older adolescents (aged from 16 to 19 years) have more interpersonal conflicts and uncertainty about the future compared to younger adolescents (aged from 14 to 15 years); this result is consistent with previous studies . Considering the clinical variables, our results showed that the type of epilepsy, anti-seizure medication, epilepsy onset, epilepsy duration, and IQ do not influence the answers to the questionnaire. The scores on the Q-PAD were markedly higher in people with refractory epilepsy, which represents, therefore, one of the major risk factors for poor mental health in childhood. Adolescents with active epilepsy are at higher risk for anxiety, mood disorders, interpersonal conflicts, and family problems and have higher psychosocial risk. All these outcomes should be closely followed up to identify a possible pathway away from stigma and loneliness as early as possible . Some authors have suggested that higher rates of anxiety and mood disorders are associated with the occurrence of seizures in public places . Davies et al. reported that both uncomplicated and complicated epilepsy groups showed a substantial increase in emotional and behavioral disorders. However, only the complicated epilepsy group (identified as having additional neurological problems or severe learning difficulties) was associated with a markedly increased rate of hyperactive and pervasive developmental disorders . Adolescence is characterized by physical, psychological, and emotional changes. Young people may feel intense emotions, fear for the future, low self-esteem, and difficulties with peers. Epilepsy has an important psychological and social impact on adolescents at this critical time of life . The fact that mental health disturbances are much more commonly associated with epilepsy than with other chronic pathologies, such as diabetes or asthma, indicates that the psychological consequences of epilepsy are not an inevitable result of a chronic and potentially life-threatening disease that requires daily therapy; neurological pathology and social stigma are likely to be key risk factors . It is well known that female patients may suffer from mood disorders 2-3 times more often than males; our data are in accordance with the findings that the female gender had an increased incidence of depressive disorders compared to males . The early identification and appropriate management of these disturbances should translate into better seizure control, fewer anti-seizure medications and side effects, improved quality of life, reduced costs of healthcare delivery, and better outcomes for society as a whole . The COVID-19 epidemic increased the prevalence of mental health problems such as depression, anxiety, and sleep disorders in people with epilepsy . During the unprecedented lockdown in Italy, many patients with chronic conditions lost their regular follow-up programs; for this reason, it is crucial to monitor the impact of COVID-19 on this vulnerable cohort . This research has an important strength: to our knowledge, this is the first work to investigate the influence of behavioral disorders in adolescents with epilepsy by focusing on the patient's point of view and perspective. Our work has some limitations to disclose. Firstly, the study was conducted in a single center, reflecting the practice style of our tertiary Epilepsy Center, and the subjects were referred to our epilepsy center for pharmacoresistant epilepsy. Secondly, we did not have any controls with another chronic pathology to verify whether they demonstrated similar psychiatric profiles. Thirdly, the study was questionnaire-based, and an in-depth clinical psychiatric interview with a specific focus on psychological/psychiatric problems was missing, in particular concerning the assessment of disease severity. 5. Conclusions Adolescents with epilepsy exhibit specific psychobiological profiles. Future research is warranted to evaluate the implications from both a research perspective (the genes and circuits implicated) and from a clinical perspective. Our results encourage physicians to always explore psychological issues in adolescents with epilepsy and suggest potential areas of intervention. In conclusion, the psychological assessment of adolescents affected by epilepsy should be integrated with clinical practice to promote early diagnosis and management, considering the higher prevalence of behavioral disturbances in this population. Acknowledgments We are grateful to all patients and families who participated in the study. Author Contributions K.T. formulated the idea of the study; F.L.B., A.V., E.Z., V.C. and M.P.C. recruited patients for the study; K.T. analyzed data; K.T., F.L.B., L.F., O.B. and M.P.C. interpreted data; K.T. drafted the paper. All authors revised the draft versions, read, and approved the final manuscript. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of San Paolo Hospital of Milano (ADO-EPI--13). Informed Consent Statement Informed consent was obtained from all subjects and caregivers involved in the study. Data Availability Statement The data presented in this study are available upon request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Distribution of adolescents with epilepsy on the Q-PAD. Figure legend: distribution of Q-PAD scores (minimum score, first (left) quartile, median, third (right) quartile, and maximum score), deg outlier value. Abbreviations: Q-PAD: Questionnaire for the Assessment of Psychopathology in Adolescence. Figure 2 Q-PAD scores for adolescents with uncontrolled seizures vs. controlled seizures. Figure legend: Q-PAD scores of adolescents with uncontrolled seizures compared to patients with controlled seizures (minimum score, first (lower) quartile, median, third (upper) quartile, and maximum score). * Significant scores. deg Outlier value. Abbreviations: Q-PAD: Questionnaire for the Assessment of Psychopathology in Adolescence. healthcare-11-00687-t001_Table 1 Table 1 Demographic and clinical characteristics. Variable N = 58 Age (years) Mean 17.24 SD 2.05 Range 14-19 Younger Adolescents (14-15 years) 18 (31.0%) Older Adolescents (16-19 years) 40 (69.0%) Gender Male 19 (32.8%) Female 39 (67.2%) Education (years) Mean 10.36 SD 2.13 Range 7-13 Type of epilepsy Focal Epilepsy Unknown 23 (39.7%) Structural 17 (29.3%) Generalized Genetic Epilepsy 18 (31.0%) Epilepsy onset (years) Mean 10.05 SD 5.1 Range 0-18 Epilepsy duration (years) Mean 8.07 SD 5.82 Range 0-28 Anti-seizure Medications Monotherapy 26 (44.8%) Polytherapy 23 (39.7%) No anti-seizure Medications 9 (15.5%) Seizure frequency Daily 3 (5.2%) Weekly 5 (8.6%) Monthly 4 (6.9%) Sporadic 15 (25.9%) None 31 (53.4%) Intelligence assessment Total IQ Mean 86.50 SD 23.69 Verbal IQ Mean 80.66 SD 25.67 Performance IQ Mean 82.90 SD 20.80 Verbal Comprehension Mean 100.33 SD 14.54 Perceptual Reasoning Mean 113.56 SD 12.32 Working Memory Mean 96.89 SD 15.10 Processing Speed Mean 87.78 SD 14.63 Abbreviations: SD: standard deviation; IQ: intelligence quotient. healthcare-11-00687-t002_Table 2 Table 2 Q-PAD scores. Q-PAD Scores Variable N = 58 Body dissatisfaction Mean 51.59 SD 28.87 Range 9-99 Normal range 47 (81.03%) Clinical range 11 (18.97%) Anxiety Mean 57.16 SD 26.84 Range 5-97 Normal range 47 (81.03%) Clinical range 11 (18.97%) Depression Mean 49.22 SD 27.67 Range 10-99 Normal range 54 (93.10%) Clinical range 4 (6.90%) Substance abuse Mean 34.47 SD 21.85 Range 10-95 Normal range 57 (98.28%) Clinical range 1 (1.72%) Interpersonal conflicts Mean 57.10 SD 29.23 Range 5-99 Normal range 45 (77.6%) Clinical range 13 (22.4%) Family problems Mean 59.71 SD 25.67 Range 2-99 Normal range 43 (74.1%) Clinical range 15 (25.9%) Uncertainty about the future Mean 50.07 SD 29.66 Range 10-99 Normal range 45 (77.6%) Clinical range 13 (22.4%) Psychosocial risk Mean 16.44 SD 19.49 Min-Max 1-80 Normal range 58 (100%) Self-esteem/well-being Mean 48.58 SD 27.88 Range 1-99 Normal range 18 (31.6%) Borderline 12 (21.0%) Clinical range 27 (47.4%) Abbreviations: SD: standard deviation; Q-PAD: Questionnaire for the Assessment of Psychopathology in Adolescence. 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PMC10000858 | Diagnostics (Basel) Diagnostics (Basel) diagnostics Diagnostics 2075-4418 MDPI 10.3390/diagnostics13050888 diagnostics-13-00888 Review Update on Lean Body Mass Diagnostic Assessment in Critical Illness De Rosa Silvia 12 Umbrello Michele 3 Pelosi Paolo 45 Battaglini Denise 4* Zhang Zhongheng Academic Editor 1 Centre for Medical Sciences--CISMed, University of Trento, Via S. Maria Maddalena 1, 38122 Trento, Italy 2 Anesthesia and Intensive Care, Santa Chiara Regional Hospital, APSS, 38123 Trento, Italy 3 S.C. Anestesia e Rianimazione II, Ospedale San Carlo Borromeo, ASST dei Santi Paolo e Carlo, 20142 Milano, Italy 4 IRCCS Ospedale Policlinico San Martino, 16132 Genova, Italy 5 Dipartimento di Scienze Chirurgiche e Diagnostiche Integrate, Universita degli Studi di Genova, 16132 Genova, Italy * Correspondence: [email protected] 26 2 2023 3 2023 13 5 88802 2 2023 23 2 2023 24 2 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). Acute critical illnesses can alter vital functions with profound biological, biochemical, metabolic, and functional modifications. Despite etiology, patient's nutritional status is pivotal to guide metabolic support. The assessment of nutritional status remains complex and not completely elucidated. Loss of lean body mass is a clear marker of malnutrition; however, the question of how to investigate it still remains unanswered. Several tools have been implemented to measure lean body mass, including a computed tomography scan, ultrasound, and bioelectrical impedance analysis, although such methods unfortunately require validation. A lack of uniform bedside measurement tools could impact the nutrition outcome. Metabolic assessment, nutritional status, and nutritional risk have a pivotal role in critical care. Therefore, knowledge about the methods used to assess lean body mass in critical illnesses is increasingly required. The aim of the present review is to update the scientific evidence regarding lean body mass diagnostic assessment in critical illness to provide the diagnostic key points for metabolic and nutritional support. critical care lean body mass muscle mass assessment CT scan bioelectrical impedance analysis electromyography musculoskeletal ultrasound This research received no external funding. pmc1. Introduction After a critical illness, survivors who have been admitted to an intensive care unit (ICU) often present with reduced physical function. This could be one of the long-term effects of acute skeletal muscle atrophy and neuromuscular weakness acquired during critical illness as a result of extended bed rest, systemic inflammation, and bioenergetic failure . Intensive care-acquired weakness (ICUAW) is a 'clinically detected weakness in critically ill patients in whom there is no plausible etiology other than critical illness' . ICUAW is a substantial contributor to long-term disability in survivors of critical illness. Patients with ICUAW are then classified according to those with critical illness polyneuropathy (CIP), critical illness myopathy (CIM), or critical illness neuromyopathy (CINM). In addition, a further subclassification (histologically) for CIM includes cachectic myopathy, thick filament myopathy, and necrotizing myopathy . Despite the influence of ICUAW on functional outcome being clearly established, the literature is scarce regarding muscle mass status upon admission to ICU. Data on pre-admission muscle status in ICU patients are currently available only for certain chronic disease groups . The quantification of pre-ICU muscle mass in larger ICU populations and the determination of whether it may be a factor in post-ICU functional impairment are key questions that have only been partially solved . Lean body mass (LBM) represents non-adipose tissue mass, excluding any additional mass from sudden changes in water content . Several tools, including computed tomography (CT), ultrasound (US) imaging, and bioelectrical impedance analysis (BIA) are used to measure lean body mass. Particularly, US and abdominal CT scans are new emerging tools for body composition assessment in ICU patients, although further validation of these techniques in the ICU population is still needed. However, the effectiveness of muscle mass monitoring is helpful in guiding adequate nutritional support during the acute critical illness, recovery phase, and rehabilitation periods. In addition, a lack of uniform bedside monitoring tools could impact the nutrition outcome. Metabolic assessment has a pivotal role in critical care, and understanding the patients' nutritional status and risk is crucial . Several methods used to monitor LBM are becoming increasingly used in the ICU, and knowledge about their advantages and limitations is essential. The aim of the present review is to update the scientific evidence regarding emerging imaging techniques for LBM diagnostic assessment in critical illnesses to provide diagnostic key points for metabolic and nutritional support. 1.1. Computed Tomography Assessment of Nutritional Status and Lean Body Mass The amount and quality of skeletal muscles can be assessed using cross-sectional imaging modalities such as computed tomography (CT). Increasing evidence has demonstrated the prognostic value of the area and the quality of skeletal muscle measured using CT as biomarkers of sarcopenia and frailty. Analysis of the muscle cross-sectional area (CSA) on a single cross-sectional image at the level of the third vertebra (L3) is an accurate surrogate of whole-body muscle mass . The determination of circumferential skeletal muscle area or psoas muscle area, both typically obtained at lumbar vertebral levels, is the most common approach. In addition, this region contains visceral, subcutaneous, and intermuscular adipose tissue, psoas and paraspinal muscles, transversus abdominus, external and internal oblique abdominals, and rectus abdominus. De Marco et al. assessed abdominal CT images in a cohort of healthy patients to define the normal reference values and age-associated down-trend for CT muscle parameters at L4 in a healthy population. The lower reference range for the psoas wall muscle area was <22.0 cm2 in males and <11.1 cm2 in females, and, for the abdominal wall muscle area, it was <112.2 cm2 in males and <75.6 cm2 in females. There was a graded decline observed among older compared to younger adults (especially >=60 years of age). Toledo et al. , in their observational study, found that sarcopenia was a risk factor in lower 30-day survival, higher hospital mortality, and higher complications in critically ill patients. Moreover, there was a low correlation between sarcopenia and body mass index, whereas Looijaard et al. found that low skeletal muscle quality at ICU admission, assessed using CT scan, was independently associated with higher 6-month mortality in mechanically ventilated patients . In critical care, sarcopenic obesity is prevalent but scarcely investigated. Severe muscle depletion or sarcopenia is one of the most common complications of acute and chronic illnesses. In addition, the CT analysis was not biased by the fluid overload that frequently presents in critically ill patients . In a critical care setting, a CT scan could be useful for analyzing sarcopenia, sarcopenic obesity, and myosteatosis using the third lumbar vertebrae skeletal muscle . Despite being a relatively new measure tool in critical illnesses, in chronic diseases, the use of a CT scan to analyze sarcopenia was well investigated. Joppa et al. showed that the prevalence of sarcopenic obesity is 2.5 times higher in chronic obstructive pulmonary disease, associated with worse physical performance and higher systemic inflammatory burden . Although CT is considered a gold standard method used to assess body composition , its use in critically ill patients is limited despite the advocation of the substantial depletion of skeletal muscle-standardized approaches for determining muscularity. Implementation of this technique could offer several advantages for the care of critically ill patients, although newer methods with easier bedside availability are emerging, as explained below. 1.2. Bioelectrical Impedance Analysis In critical care, real-time knowledge of body composition (fat, muscle, bone, and water) is advantageous for personalization and clinical optimization in terms of nutrition, fluids, and medication dosing adjustment. Bioelectrical impedance analysis (BIA) is a safe, quick, and inexpensive technique for the assessment of body composition . BIA analyzers inject an alternating sinusoidal electric current through active electrodes and register resistance and reactance through recording electrodes . This method estimates body fat and muscle mass, whereby a weak electric current flows through the body and the voltage is measured to calculate the impedance (resistance) of the body. Based on the principle that body water is stored in muscle, a person with more muscle mass has a higher probability of having more body water, which leads to lower impedance. The BIA technique requires the operator to place active electrodes in the right side on conventional metacarpal and metatarsal lines and record electrodes in standard positions at wrist and ankle. The measuring of the phase angle (PA) or the "classic" bioelectrical impedance vector analysis ("classic" BIVA) have emerged as alternative techniques to overcome the limitations of BIA, basing their main strength on the use of raw impedance parameters . Particularly, BIVA has been assessed in its valuation of the impact of hyperhydration on ICU mortality in critically ill patients . However, experience with BIVA-guided fluid management in the ICU is limited. The substantial difference lies in the fact that the BIA method processes the measurements made through software, returning estimated values of the parameters listed above. Instead, the BIVA method, in addition to processing the parameters found in equations, includes the parameters in a biavector graph. Despite these several advantages, some limitations can influence the accuracy, including instrument-related factors (i.e., electrodes quality), technician-related factors ( intra-operator variability), subject-related factors (i.e., supine position with each limb slightly away from the body, after an overnight fast, and once the bladder is emptied), and environment-related factors (i.e., environmental temperature) . Loojaard et al., in a prospective observational study enrolling 110 critically ill patients, compared the bioelectrical impedance analysis of (BIA)- and CT-derived muscle mass to determine whether BIA identified the patients with a low skeletal muscle area on the CT scan and to determine the relation between the raw BIA and raw CT measurements. BIA identified critically ill patients with a low skeletal muscle area on the CT scan, as defined by previously found cutoffs, and the BIA-derived low phase angle corresponded to low CT-derived skeletal muscle area and density . Nakanishi et al. investigated muscle mass monitoring capacity using BIA and an ultrasound through an assessment of fluid balance. The authors found that muscle mass monitoring using BIA was complicated by the fluid shift and could not monitor the change of muscle mass in critically ill patients, although muscle mass assessment at one point moderately correlated with ultrasound and CT. In contrast, the use of ultrasound-to monitor progressive muscle atrophy was used throughout the ICU stay, without the influence of a fluid shift. Despite a correlation being found between raw impedance parameters, fluid ratios, overhydration, and the adverse outcome of critical illness, cutoff and reference values remain elusive. BIA-derived muscle mass could be a promising biomarker for sarcopenia, correlating well with CT-analysis. Its use is still limited in critical care, however observational data are encouraging, inviting the implementation of this technique. 1.3. Musculoskeletal Ultrasound Ultrasounds are increasingly being used to assess changes in muscle size and quality over time . The advantages of this include the high axial resolution, low procedural risks, absence of ionizing radiation, and ease of use, even early in the course of disease. Below, we provide an update on the most common techniques used to assess muscle ultrasound in critical care. 1.3.1. Respiratory Muscles Ultrasound Various insults, including invasive mechanical ventilation, sepsis, electrolytes disbalance, critical illness polyneuropathy, and myopathy can contribute to the contractile dysfunction of respiratory muscles. Up to 40% of patients admitted to the ICU are invasively and mechanically ventilated, and the use of a controlled passive ventilator modality has been associated with a reduction in breathing and diaphragm contractility, thus causing ventilator-induced diaphragm dysfunction (VIDD) . Moreover, spontaneous breathing and assisted ventilatory modalities have been associated with potential diaphragm atrophy or damage to the myofibers, depending on the stage of the disease in which they were applied (i.e., spontaneous breathing resulted in myofibers damage if allowed too early during severe lung injury) . The diaphragm contributes to 60-70% of the respiratory workload. Therefore, diaphragmatic dysfunction, defined as a loss of diaphragm force-generating capacity specifically associated with the use of mechanical ventilation, is a possible determinant of respiratory failure in critically ill patients . Diaphragmatic dysfunction is more common in patients who are ventilated for longer but can also be observed even after relatively short periods of mechanical ventilation . The assessment of intercostal muscle thickness has been recently proposed to complete the evaluation of respiratory muscular dysfunction . The monitoring of respiratory muscle function in ICU patients is still an uncommon practice, although recent techniques for assessing readiness for weaning from the ventilator, respiratory muscle function, and strength have been applied . Among these techniques, ultrasound imaging has increased in popularity since it is a rapid, accurate, and repeatable tool, which allows for optimal diagnostic accuracy and is easily available at the bedside . However, ICU clinicians are still poorly trained to use ultrasounds for evaluating the function of the respiratory muscles . The effective use of an ultrasound for examining the respiratory muscles requires the study of the diaphragm and the accessory inspiratory (parasternal, external intercostal, scalene, and sternocleidomastoid) and expiratory muscles (transversus abdominis muscle, internal and external oblique muscle) . The measuring of diaphragmatic function includes an assessment of thickness, thickening, and displacement. Diaphragmatic Thickness Diaphragmatic thickness represents a measure of diaphragm size obtained in the zone of opposition . Technically speaking, a B-mode ultrasound is used with a 7.5-10 MHz linear probe placed in parallel between the 8-10 intercostal spaces in the mid-axillary line in the area of opposition, either during tidal breathing or a maximal inspiratory effort, to assess diaphragmatic thickness . The structures encountered when using an ultrasound beam include skin and soft tissues, intercostal muscles (hypoechogenic), parietal and visceral pleurae (hyperechogenic), the diaphragm (hypoechogenic), and parietal and visceral peritoneum (hyperechogenic) . A normal thickness measured at end-expiration in healthy volunteers is around 1.1-1.4 mm in women and 1.3-1.9 mm in men . In general, a value of 1.73-2.19 mm at end-expiration is considered normal . In invasively and mechanically ventilated patients, diaphragmatic ultrasound was recently validated, showing a high reproducibility of right hemidiaphragm thickness, with a good correlation with diaphragm electrical activity . Thickness of the diaphragm is a reliable measure of weaning from the ventilator, showing a similar performance to other weaning indexes . Diaphragmatic Thickening Diaphragmatic thickening represents the contraction of the muscle during breathing to quantify the magnitude of the respiratory effort . Diaphragm excursion is usually assessed both via M-mode ultrasonography. An advantage of M-mode is that it visualizes the movement of the diaphragm over time and provides an accurate measurement of diaphragmatic displacement over a respiratory cycle . Using the B-mode setting with a higher-frequency (>10 MHz) linear probe, the thickening fraction is calculated as a fraction of the difference between thickness at end inspiration and end expiration: (end-inspiratory thickness - end-expiratory thickness) divided by the end-expiratory thickness x 100 . Excursion positively correlates with lung inspiratory volumes and is higher during forced inspiratory breathing . The diaphragm appears thicker in the upright position compared to the supine position . Several definitions and thresholds are available, making univocal interpretation difficult. Abnormal thickening has been defined as a fraction of less than 20% or a tidal excursion of less than 10 mm . The percentage of thickening during normal breathing is around 30% and 35% on both sides in healthy men and women , while around 11% in mechanically ventilated subjects . Some authors have reported a mean thickening fraction of 20% during tidal breathing, without significant difference between right or left hemidiaphragm in mechanically ventilated patients. However, the assessment of bilateral measures is difficult and poorly reproducible, and the right diaphragm is easier to investigate . Another important variable is the ratio of the thickness between the two sides of the diaphragm. A normal ratio is set between 0.7 and 1.5 in healthy men and 0.6 and 1.6 in healthy women; moreover, values far from the threshold are representative of an imbalance between the two hemidiaphragms . A meta-analysis in 1071 patients found that the diaphragm thickening fraction is highly specific and that diaphragmatic excursion is highly sensitive to the weaning outcome, with possible variability across different ICU populations . Diaphragmatic ultrasonography demonstrated good sensitivity and specificity in predicting reintubation within 48 h from weaning . Another meta-analysis and additional recent studies have confirmed diaphragmatic dysfunction as a predictor of weaning outcome . The cutoffs more associated with weaning failure were 11-14 mm in excursion and 30-36% in thickening . Chien et al. suggested the combination of a diaphragmatic ultrasound and echocardiography to assess weaning prediction . This was also confirmed by Silva et al. in patients undergoing a spontaneous breathing trial in pressure support mode and by Haji et al. in their T-tube trial . Another approach is the combination of the rapid shallow breathing index (RSBI) with ultrasonography. This approach demonstrated that the RSBI seems to be more accurate in the prediction of successful weaning when used alone . According to the available evidence, the use of diaphragmatic ultrasounds seems promising in the weaning phase and to diagnose diaphragmatic dysfunction. Despite this, the limitations of an ultrasound should be always considered, including the need for training, inter-operator variability, and patient characteristics. Diaphragmatic Displacement Diaphragmatic displacement is useful for investigating the cyclic caudal movement of the diaphragm during the respiratory cycle and can be visualized through M-mode ultrasonography using a convex probe with a frequency of 3.5-5 MHz . This technique allows one to assess diaphragm displacement, the speed of contraction, and inspiratory and total respiratory cycle timings. A diaphragm excursion of 49 mm is considered normal in spontaneously breathing subjects . During non-invasive ventilation, diaphragmatic excursion was recently considered to be a potential predictor of NIV response, showing a good sensitivity and specificity for a value of 1.37 cm . In patients with severe COVID-19, diaphragmatic excursion at hospital admission can accurately predict the need for ventilatory support and mortality . Intercostal Muscles Thickness The external intercostal muscles extend from the tubercles of the ribs dorsally to the costochondral junctions ventrally, with fibers oriented obliquely. External intercostal muscles are activated during inspiration, whereas internal intercostal muscles are activated during the expiratory phase. De Troyer et al. showed that the third dorsal external intercostal is usually activated in the early phase of inspiration , and that the amount of the activation of the external intercostal muscles during basal breathing was associated with the degree of their mechanical response . Among external intercostal muscles, the parasternal seem to be those with more inspiratory action on the lungs despite a having lower pressure-generating ability than the other external intercostal muscles . Interestingly, Sampson et al. demonstrated that chest wall deformation during breathing depends on the coordination of inspiratory intercostal muscles, being parasternal and not necessarily the most prominent . In summary, during breathing, external intercostal muscles contract, generating torque more generated toward the lower rib than the upper rib, thus raising the ribs as a final effect. On the contrary, internal intercostals muscles contract, generating torque to the lower ribs . The evaluation of intercostal muscles with ultrasound has been recently introduced. The parasternal intercostal muscles can be investigated using a 10-15 MHz, linear transducer in M-mode, placed 3-5 cm laterally to the sternum, in the sagittal plane, between the second and the third rib . The patient is placed 20 degC head up and the pleural line is observed in B-mode as a "bat sign", just above the parasternal intercostal muscle with the three biconcave layers, as well as the two linear hyperechoic membranes from the anterior and posterior ribs. Thickness is measured between the fascial borders as hyperechogenic structures. During inspiration, increased thickness is observed, and the rib cage is moved cranially and anteriorly. Thickening fraction is calculated as follow = [(End insp thickness - End exp thickness)/End exp thickness x 100] . In a human setting, whether the usefulness of diaphragmatic ultrasounds for measuring is questioned or not, the literature on intercostal muscles is scarce. Nakanishi et al. showed that both the diaphragm and intercostal muscles become atrophic and lose thickness following excessive inspiratory support . Dres et al. showed that the parasternal intercostal muscle-thickening fraction was associated with a failed spontaneous breathing trial in patients who were mechanically ventilated. Patients with diaphragmatic dysfunction showed a fraction exceeding 8%, while 10% was predictive of weaning failure . Umbrello et al. reported that patients without diaphragmatic dysfunction showed a higher diaphragm (>30%) and lower parasternal intercostal thickening fraction (<5%) compared to patients with diaphragm dysfunction . Yoshida et al. proposed the evaluation of intercostal muscle thickness via ultrasound at rest and during maximal breathing, showing a significant increase in the thickness of the intercostal muscle in the first [from mean (standard deviation) of 1.97 (0.66) to 2.51 (0.94)], second [from 2.17 (0.83) to 2.62 (0.95)], third [from 2.65 (1.13) to 3.19 (1.24)], fourth [from 2.79 (0.86) to 3.55 (1.02)], and sixth [from 2.52 (0.52) to 2.80 (0.66)] intercostal spaces of the anterior portions . The usefulness of assessing the ultrasound thickness of intercostal muscles is still to be determined. However, evidence is encouraging, suggesting that a low parasternal intercostal thickening fraction may reflect low inspiratory effort, whereas low or high levels may reflect elevated inspiratory work by extra-diaphragmatic muscles, depending on the mechanical respiratory support provided by the ventilator . 1.3.2. Limb Muscles The rectus femoris is the most commonly investigated muscle, likely because it is easy to identify and analyze with a single image, and because it is considered a functionally important muscle for the performance of daily living and, at the same time, is subject to significant wasting during bedrest and illness, more than muscles of the upper limbs . This technique allows for the assessment of both muscle mass (thickness or cross-sectional area--CSA) and quality (echodensity), as well as an estimation of the muscle's force-generating capacity (the pennation angle). Rectus femoris ultrasound is generally performed using a high-frequency linear transducer array probe (8-12 MHz), using the B-mode setting. Briefly, patients are studied in the semirecumbent position with extended knees; the probe is placed on the anterior part of the thigh, at 2/3 of an imaginary line connecting the anterior superior iliac spine and the midpoint of the proximal border of the patella. A mark can be drawn on the skin to increase the reproducibility of the subsequent measurements. The transducer is oriented transverse to the longitudinal axis of the thigh at a 90deg angle; the probe is coated with water-soluble transmission gel to increase the acoustic contact, and care is taken to reduce the pressure on the tissues and the consequent distortion of the image as much as possible. Typical values of quadricep thickness and rectus femoris CSA in healthy volunteers have been reported to be 2.6 cm and between 4.53 and 8.68 cm2 , respectively. On the other side, in critically ill patients, average values at ICU admission have ranged between 0.98 and 2.23 cm for quadricep thickness and from 2.26 to 4.42 cm2 for rectus femoris CSA . Information about muscle composition can be gathered using the quantification of muscle echodensity, which is calculated by performing a grey-scale analysis of image pixels, using standard software for image editing. This process has been shown to correlate with bioptic findings as it reflects the muscle composition: an increased echogenicity represents a more homogenous muscle . Quantification of muscle echodensity requires exporting the muscle ultrasound scan as a digital image file for subsequent, offline computer analysis, and the absolute value of density of the image critically depends on the settings with which the image was acquired with. Changes in quadricep muscles' echodensity have been associated with negative outcomes . Eventually, muscle architecture can be described using the pennation angle, i.e., the angle of the insertion of muscle fibers into their aponeurosis, which provides information about muscle strength: the larger the pennation angle, the more contractile material is present, and thus the higher the capacity to produce force . The rectus femoris pennation angle is measured using the same method and in the same position of muscle area and thickness; a longitudinal view is obtained by rotating the probe parallel to either the lateral or medial head of the muscle. Few studies have investigated the pennation angle in critically ill subjects; in healthy subjects, the average pennation angle of the rectus femoris has been reported to range from 8.76 +- 1.78 to 17.5 +- 3.9deg . In critically ill patients at ICU admission, the pennation angle was 10.8 +- 2.6deg , and an angle <4.4deg was found to be associated with a worse outcome . It is well known how limb muscle size, structure, and function deteriorate during the course of ICU stay, i.e., by approximately 3% per day in the first week of ICU stay ; sonographic findings of reduced rectus femoris CSA have been found to be associated with poor clinical outcomes . Using a CT scan as the reference method to define low muscle mass, a cutoff value of <2 cm for the thickness of the quadriceps' muscle layer thickness and a rectus femoris CSA of <4.7 cm2 had an AUC of 0.84 and 0.76, respectively . However, several limitations have to be carefully considered when using specific cutoffs for muscle US, specifically, the lack of external validation in the majority of the studies, and the lack of standardization for the site of measurements. The use of US to measure muscle mass has consistently been found to be reliable in several investigations. Puthucheary et al. reported an excellent coefficient of determination (R2) of 0.97 for the measurements of rectus femoris CSA using two blinded independent raters. Grimm et al. evaluated the reproducibility of muscle echogenicity, showing excellent inter- (0.915) and intra-rater (0.972) coefficients. The ultrasound measurement of the rectus femoris size was recently validated through a comparison with a CT-derived skeletal muscle area at L3 level in a prospective observational trial on 200 non-critically ill patients; a combined score of the ultrasound measurement, together with sex, height, and weight, predicted muscle mass with an R2 of 0.74 . In 15 critically ill subjects, the interrater reliability of the rectus femoris CSA had an intra-class correlation coefficient between 0.87 and 0.9; feasibility, defined as the percentage of measurements that were obtainable, ranged from 75% to 100% . A study of muscle US image acquisition by physical therapists and students found an average intraclass correlation coefficient for all rates of 0.903, indicating excellent reliability of image acquisition regardless of the level of experience of the operator, severity of patient illness, or patient setting . Another longitudinal, validation study assessed the inter-observer reliability of muscle ultrasound in 29 long-stayer, critically ill subjects; the authors evaluated two measurement sites: at the midpoint or at two-thirds of the length between the anterior superior iliac spine and the upper border of the patella. inter-observer reliability ICC scores were 0.74 and 0.76 at the "midpoint" and 0.83 and 0.81 at the "two-thirds" site, respectively, showing that the method is reproducible, with a higher reliability at the two-third site . Previous studies showed a negative correlation between muscle size and quality and ICU length of stay or mortality . More recent studies have confirmed these findings. For example, a 3-week follow-up analysis of rectus femoris CSA in ICU trauma patients showed that 100% of participants experienced severe muscle mass loss, and 45% of rectus femoris muscle mass was lost by day 20, together with a progressive increase in echogenicity score . A recent single-center observational study in 35 young trauma patients staying in the ICU for at least 7 days found that the rectus femoris cross-sectional area, mid-arm circumference, and calf circumference were reduced rapidly during the first week of the ICU stay, whereas the relationship between muscle loss and the clinical outcome was less defined . With the aim of investigating the degree of change in rectus femoris muscle size over time, Wu et al. prospectively enrolled 284 critically ill subjects; the authors confirmed an average daily muscle atrophy rate of about 1%, with the highest reduction occurring in the third and fourth weeks of stay; daily atrophy rates were approximately three-times higher in women than in men, and protective factors of muscle atrophy included higher BMI and lower initial muscle size . An observational study on 74 critically ill subjects analyzed the time course of rectus femoris muscle thickness on the first, third, and seventh days of ICU stay; the muscle size was reduced by 15% on average over the first week, and a greater reduction was associated with worse clinical outcomes . The association between US-assessed muscle mass and muscle strength generation was evaluated in an observational investigation on 37 septic patients: the authors found a significant association between a decrease in rectus femoris CSA between the second day of stay and ICU discharge and a lower handgrip strength at hospital discharge, suggesting the significant clinical impact of muscle wasting . A recent longitudinal investigation confirmed how severe and critical COVID-19 patients showed a 30% reduction in the rectus femoris cross-sectional area, with an average 16.8% increase in echodensity from days 1 to 10 . A strong correlation between increased echodensity and inflammation was confirmed in several muscle biopsy studies . Several recent investigations have focused on the factors associated with muscle wasting: Lee et al. conducted a prospective observational study to determine the association between baseline quadricep muscle status, premorbid functional status, and 60-day mortality in 90 patients. The authors found that every 1% loss in quadricep muscle thickness over the first week of critical illness was associated with 5% higher odds of 60-day mortality; moreover, a higher nutrition risk, sarcopenia, and frailty at baseline was associated with lower baseline muscle size and higher 60-day mortality, suggesting a complex relationship between premorbid functional status, muscle mass, and outcome . Mukhopadhyay et al. demonstrated that a higher nutritional risk assessment at admission is associated with a higher subsequent muscle reduction, allowing them to identify patients at risk of muscle loss . A recent observational study in a sample of critically ill, COVID-19 patients found an average 30% reduction in rectus femoris CSA over the first week of ICU stay, with a significantly higher reduction in non-survivors, together with a significant increase in muscle echodensity over the first week, again of a higher extent in non-survivors. Interestingly, the change in rectus femoris area was related to the cumulative protein deficit over the first week of ICU stay, suggesting that changes in muscle size and quality are related to the outcome of critically ill patients and are influenced by nutritional management strategies . In a longitudinal investigation, Yanagi et al. found that, among 72 critically ill patients staying in the ICU for >2 days, low quadricep muscle mass at ICU discharge was associated with a low muscle function, as assessed using the Medical Research Council sum score, and was associated with an almost four-times higher 1-year mortality, highlighting the utility of muscle mass measurements to identify high-risk patients and suggesting the use of muscle mass as a relevant patient-centered outcome . Muscle ultrasound has also been shown to be an early predictor of physical disability: in a prospective investigation of 41 critically ill patients admitted to the ICU for respiratory failure or sepsis, the change in rectus femoris CSA over the first week of ICU stay was a strong predictor of muscle weakness at hospital discharge . In another investigation, a loss in pennation angle was observed during the first week of ICU stay, and such loss predicted the subsequent development of ICU-acquired weakness in a sample of 50 critically ill and mechanically ventilated subjects . A potential advantage of muscle ultrasound, as compared with other methods for the assessment of lean body mass, such as bioelectrical impedance, is that it can be less dependent on body hydration state. To test this hypothesis, da Silva Passos et al. recently published the results of a prospective cohort study. The authors compared the findings of a BIA-derived phase angle with rectus femoris CSA and found that only rectus femoris CSA was a significant predictor of mortality in a sample of 160 mechanically ventilated and critically ill subjects . The authors described the relationship between rectus femoris CSA and quadricep muscle thickness, with volitional measures of strength and function at 7 days after the ICU admission of 29 patients with sepsis. The authors showed an expected decrease in both in rectus femoris CSA and thickness (23.2% and 17.9%, respectively), while only the rate of change per day of CSA was correlated with muscle strength on day 7. Similarly, Puthucheary et al. showed that thickness measurements significantly underestimate ICU muscle wasting compared with rectus femoris CSA . In summary, the literature seems consistent in the utility of rectus femoris ultrasound (especially in the cross-sectional area at the lower third of the thigh) to track the loss of muscle mass and possibly muscle function and as a marker of the severity of an illness and possible negative outcomes. However, so far, no unanimous cutoffs to define sarcopenia or an increased risk of mortality have convincingly been reported, and the relationship of muscle ultrasound with nutritional or physical intervention is yet to be demonstrated. Part of this uncertainty strictly depends on the variability in probe settings, and a shared, standardized protocol is needed. 1.4. Electromiography The mainstay of the prevention of ICUAW is the minimization of the risk factors assessed in prospective studies that unfortunately assessed subsets of ICU patients (severe sepsis, multiorgan failure, prolonged mechanical ventilation (greater than ~7 days), or those receiving high doses of corticosteroids). When a group of patients with severe sepsis, multiorgan failure, or prolonged mechanical ventilation have undergone full electrophysiological and histological investigation, CIP and CIM have been found to significantly overlap . Although the clinical assessment of muscle weakness using the Medical Research Council (MRC) score can quantify strength impairment, electromyography (EMG) remains the hallmark in diagnosing and differentiating ICUAW types . Li et al. assessed peripheral nerve biopsies from the sural nerve from ICU patients and found a reduction in the sodium channel subtype Nav1.6 on the sural nerve . This may potentially explain why its dysfunction affects neurological functions across all systems of the body during critical illness . In a critical care setting, the inability to wean from ventilator support is common a symptom of the ICUAW method, and diaphragmatic EMG remains technically challenging within the ICU setting. An examination of the phrenic nerve (prolonged latencies or decreased motor unit action potentials) and diaphragm EMG (pattern of fibrillations and positive sharp waves or reduced number of motor unit potentials) can assist with diagnosis . However, several factors can limit an EMG examination's effectiveness and influence the results. A lack of patient cooperation and interactions with other electronic devices may obscure both nerve conduction and EMG results, providing technically inadequate studies for diagnosis. In addition, very often, patients presenting with anasarca and hypothermia can alter the amplitude and velocity of recordings in nerve conduction studies. The gold standard for the diagnosis of critical illness neuropathy remains electrodiagnostic testing, which includes nerve conduction studies and needle electromyography . The evaluation of profound weakness in the ICU setting should be implemented, and electrodiagnostic testing is an essential tool that can direct the clinical team in determining further management . 2. Nutritional Outcome and Metabolic Assessment Acute critical illnesses can determine the significant deterioration of one or more vital functions with profound biological, biochemical, metabolic, and functional modifications. The entity of this stress response mainly depends on the severity of the clinical state and its duration. Whether the cause of critical illness is trauma, septic or surgical stress, or a patient's nutritional status and nutritional risk could guide metabolic support . Nutritional status upon admission remains complex, and it is not clear whether the long-term physical limitations after critical care are attributable to the impairments acquired during ICU stay, or to preadmission functional impairment caused by chronic disease or general frailty . It might be difficult to provide appropriate nutritional support to critically ill patients due to prolonged fasting periods, delivery hurdles brought on by insulin resistance, and gastrointestinal dysfunction. There is not much data to support nutrition advice, especially when it comes from outcomes such as muscle mass, strength, and function. The loss of LBM is a clear marker of malnutrition. However, only a few RCTs of nutrition therapies in critical illness included an endpoint of muscle mass, strength, or function . Despite increasing evidence, there is still no study which has confirmed that nutrition interventions are useful in enhancing any muscle strength or function in critically ill patients. Therefore, the use of LBM to guide nutritional assessment and therapy in critical illnesses is still highly debated. 3. Future Directions and Implications An innovative tool to potentially assess muscle mass is the use of near-infrared spectroscopy (NIRS) technology. In the near-infrared spectrum (700-1100 nm), photons are capable of several centimetres of tissue penetration, before being absorbed by metalloproteinases such as hemoglobin, myoglobin, and mitochondrial cytochrome oxidase. Briefly, NIRS yields values of tissue oxygen saturation of hemoglobin (StO2), which represents spatially integrated information from arterioles, capillaries, and venules, and provides information about oxidative metabolism and the intramuscular matching between O2 delivery and utilization, that can provide the link between catabolism, inactivity, and the loss of lean body mass. In a proof-of-concept physiological investigation involving 26 healthy participants, NIRS was found to be a reliable tool to investigate skeletal muscle oxidative capacity . In healthy subjects, even a short (10-day) period of horizontal bed rest was shown to impair in vivo oxidative function during exercise, as assessed using NIRS, and muscle catabolic processes induced by inactivity were demonstrated to be less energy consuming than anabolic ones . Since the penetrability of light into tissues is proportional to its wavelength, NIRS has also been suggested to be able to assess the depth of subcutaneous tissue. In 93 healthy subjects, NIRS was found to be significantly associated with lean body mass . Similar findings were reported for non-critically ill and hospitalized patients, using dual-energy X-ray absorptiometry as the comparator for lean body mass . For the assessment of metabolic bone illnesses such osteoporosis, sarcopenia, and obesity, dual-energy X-ray absorptiometry is currently considered as one of the most adaptable imaging modalities. However, in a critical care setting, the use of this tool is still infrequent, despite several reports in non-critically ill patients revealing its utility in detecting changes in muscle mass. Reference standard values at X-ray absorptiometry in all age categories were investigated by Imboden et al., who found that men had a higher mean lean mass than women (60.8 kg vs. 42.3 kg). From the youngest to oldest age categories, the mean lean mass significantly declined for both men and women. Men's mean lean mass was shown to fall with increased age, but women's mean lean mass remained stable until the fifth decade, at which point it began to decline . Potential innovations in the bedside assessment of muscle mass by means of ultrasonography include shear wave elastography, superb microvascular imaging, and contrast-enhanced ultrasound. The first is a technique used to measure tissue stiffness because of a disease, which is based on the generation of shear waves determined by the displacement of tissues induced by the force of a focused ultrasound beam; superb microvascular imaging is an innovative technique designed for imaging microvascularization that cannot detect using a color Doppler, allowing the visualization of low-velocity and microvascular flow. The latter involves the administration of intravenous contrast agents such as gas-filled microbubbles to better visualize organs and blood vessels. A recent observational investigation compared the findings of these techniques between critically ill patients diagnosed with ICU-acquired weakness and healthy controls and identified specific features of the muscle of critically ill subjects, shedding a light on these innovative imaging modalities . A recent validation study investigated the reliability and reproducibility of shear-wave elastography muscle measurements in critically ill patients; the authors found how inter-operator reproducibility and intra-operator reliability were well above 0.9 . In 130 non-critically ill patients, lower leg shear-wave velocities were compared between sarcopenic and non-sarcopenic subjects: velocities in the sarcopenia group were significantly smaller than in the healthy control group and were positively correlated with an appendicular skeletal muscle mass index and grip strength . Recently, the measurement of temporalis muscle thickness was proposed as a measure of muscle wasting in neuro-critically ill patients. In an observational trial, a decrease in rectus femoris size, as assessed via ultrasound, was paralleled by the ultrasound measurements of temporalis muscle thinning; moreover, the finding was also confirmed by CT-based measurements of the temporalis muscle size and lay the foundation for the assessment of the temporal changes of other muscle groups in critically ill subjects . The measuring and tracking of LBM have important implications: LBM assessment may provide crucial chances to identify critically ill patients at high nutritional risk at an early stage and to direct and evaluate metabolic care after ICU admission. Bioelectrical impedance analysis, musculoskeletal ultrasound, and CT-scan analysis are becoming popular in ICU . The strengths and limitations of these various methodologies must be taken into consideration when interpreting the results. Exciting new research in this field has focused on the quantity as well as the quality of lean body mass, thus providing information on the infiltration of adipose tissue and intramuscular glycogen storage. Lean body mass measurement techniques are continuously being improved to increase their usefulness at the bedside, providing useful tools to clinicians to direct metabolic assistance . 4. Conclusions Mechanical ventilation and critical illnesses predispose reduced muscles' strength, prolonging ICU duration and complications. Several tools (including computed tomography, bioelectrical impendence, ultrasound, electromyography, near-infrared spectroscopy, and dual-energy X-ray absorptiometry) are currently being used to assess lean body mass; however, their use in critical illnesses is still poor. Future research is warranted to better address the utility of these tools in critical care. Author Contributions Conceptualization, S.D.R., M.U. and D.B.; investigation, S.D.R., M.U. and D.B.; writing--original draft preparation, S.D.R., M.U. and D.B.; writing--review and editing, S.D.R., M.U., P.P. and D.B.; D.B. supervision. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement Not applicable. Conflicts of Interest The authors declare no conflict of interest. Figure 1 A cross-sectional computed tomography (CT) image at the third lumbar vertebra (L3) showing skeletal muscle segments. Skeletal muscle segmented in green color. Figure 2 Ultrasonographic assessment of diaphragm thickness and thickening. Ultrasonographic assessment of diaphragmatic thickness visualizing the normal diaphragm in the zone of opposition using a 7.5-10 MHz linear probe. View in B-mode and M-mode of the diaphragmatic thickness. In both modalities, the probe is placed parallel to an intercostal space between the 8th and the 10th spaces. Diaphragmatic thickness is measured at end inspiration and end expiration, and the thickening fraction is calculated according to the formula = (EIT - EET)/EET x 100. Figure 3 Ultrasonographic assessment of diaphragm displacement during inspiration and expiration. Ultrasonographic view of the normal diaphragm in the region of the liver dome in M-modes during inspiration and expiration. This technique uses a 3.5-5 MHz convex probe placed between the midclavicular and anterior axillary lines, directed cranially, medially, and dorsally in the region of the liver dome. This technique allows one to measure diaphragm displacement, contraction speed, inspiratory time, and total respiratory timing. Figure 4 Ultrasonographic assessment of the quadriceps muscle. Left panel: the image shows the standardized level of the ultrasound scan of the lower limb; in the supine position, the linear probe is placed on the anterior part of the thigh, at 2/3 of an imaginary line connecting the anterior superior iliac spine (ASIS) and the midpoint of the proximal border of the patella, with the probe perpendicular to the muscle. Upper right panel: the figure shows a transverse scan of the quadricep muscle, which is composed of three vastus muscles (medialis, intermedius, and lateralis) and the rectus femoris; the red dashed line represents the rectus femoris cross-sectional area, the double-arrow line depicts the quadriceps muscle layer thickness. Lower middle panel: the picture shows a longitudinal scan of the quadricep muscle; the pennation angle is measured at the intercept of the fascicular path (dashed line) to the lower aponeurosis (solid) line. Lower right panel: the diagram shows the grayscale histogram in a transverse axis of the rectus femoris. VI vastus intermedius, VM vastus medialis, VL vastus lateralis. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). 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