Detection of HTLV-1 proviral DNA in cell-free DNA: Potential for non-invasive monitoring of Adult T cell leukaemia/lymphoma using liquid biopsy?
Fragmented genomic DNA is constitutively released from dying cells into interstitial fluid in healthy tissue. In cancer, this so-called 'cell-free' DNA (cfDNA) released from dying malignant cells encodes cancer-associated mutations. Thus, minimally invasive sampling of cfDNA in blood plasma can be used to diagnose, characterise and longitudinally monitor solid tumours at remote sites in the body. ~5% of carriers of Human T cell leukaemia virus type 1 (HTLV-1) develop Adult T cell leukaemia/lymphoma (ATL), and a similar percentage develop an inflammatory CNS disease, HTLV-1 associated myelopathy (HAM). In both ATL and HAM, high frequencies of HTLV-1 infected cells are present in the affected tissue: each carrying an integrated DNA copy of the provirus. We hypothesised that turnover of infected cells results in the release of HTLV-1 proviruses in cfDNA, and that analysis of cfDNA from infected cells in HTLV-1 carriers might contain clinically useful information pertaining to inaccessible sites in the body- e.g. for early detection of primary or relapsing localised lymphoma type ATL. To evaluate the feasibility of this approach, we tested for HTLV-1 proviruses in blood plasma cfDNA. CfDNA (from blood plasma) and genomic DNA (gDNA, from peripheral blood mononuclear cells, PBMC) was isolated from blood from 6 uninfected controls, 24 asymptomatic carriers (AC), 21 patients with HAM and 25 patients with ATL. Proviral (HTLV-1 Tax) and human genomic DNA (the beta globin gene, HBB) targets were quantified by qPCR using primer pairs optimised for fragmented DNA. Pure, high quality cfDNA was successfully extracted from blood plasma of all study participants. When compared with uninfected controls, HTLV-1 carriers had higher concentrations of cfDNA circulating in their blood plasma. Patients with ATL who were not in remission had the highest levels of blood plasma cfDNA in any group studied. HTLV-1 proviral DNA was detected in 60/70 samples obtained from HTLV-1 carriers. The proviral load (percentage of cells carrying proviruses) was approximately tenfold lower in plasma cfDNA than in PBMC genomic DNA, and there was a strong correlation between the proviral load in cfDNA and PBMC genomic DNA in HTLV-1 carriers that did not have ATL. cfDNA samples in which proviruses were undetectable also had very low proviral load in PBMC genomic DNA. Finally, detection of proviruses in cfDNA of patients with ATL was predictive of clinical status: patients with evolving disease had higher than expected total amount of proviruses detectable in plasma cfDNA. We demonstrated that (1) HTLV-1 infection is associated with increased levels of blood plasma cfDNA, (2) proviral DNA is released into blood plasma cfDNA in HTLV-1 carriers and (3) proviral burden in cfDNA correlates with clinical status, raising the possibility of developing assays of cfDNA for clinical use in HTLV-1 carriers.


Vitamin D supplementation: less controversy, more guidance needed.
Vitamin D is a secosteroid hormone with multiple functions that extend beyond the regulation of intestinal calcium absorption. In recent years, the publication of research articles investigating associations between vitamin D status and health has reached an all-time high, and an increase in supplementation studies has followed. Given the pleiotropic effects of vitamin D, the scientific focus has gone beyond its known classic benefits on skeletal health to include diabetes and cardiovascular, neurological, respiratory, renal, and liver diseases, yet numerous conflicting findings continue to emerge. This review presents some examples of recent work within the context of controversies surrounding vitamin D and highlights key factors that should be considered when designing vitamin D supplementation regimens.


Dietary Factors and Female Breast Cancer Risk: A Prospective Cohort Study.
Breast cancer is the leading cause of cancer in females and has become a major global health priority. This prospective cohort study investigated the association of dietary factors, including food items and dietary habits, with the risk of breast cancer in Korean women. Study participants were women aged 30 years or older, recruited from the National Cancer Center in South Korea between August 2002 and May 2007. They were followed until December 2014 using the Korea Central Cancer Registry to identify breast cancer cases. Among 5046 non-pre-diagnosed cancer participants, 72 breast cancer cases were prospectively identified. Participants with breast cancer had a significantly higher educational level (college or higher: 58.3% vs. 39.5%, p = 0.01), were more likely to have ever smoked (22.2% vs. 7.8%, p < 0.001), and were more likely to have a history of benign breast tumors (10% vs. 4%, p = 0.02) than non-cases. Consumption of grilled meat conferred a significantly higher risk of breast cancer in all women (hazard ratio (HR) 1.77, 95% confidence interval (CI) 1.09-2.85) and in postmenopausal women (HR 3.06, 95% CI 1.31-7.15). High-cholesterol food intake was associated with a higher risk in all women (HR 1.69, 95% CI 1.01-2.82). Irregular meal intake was associated with an elevated risk in all women (HR 2.19, 95% CI 1.20-3.98, p for trend = 0.01) and in premenopausal women (HR 2.35, 95% CI 1.13-4.91, p for trend = 0.03). Our findings suggest that grilled meat and high-cholesterol food intake and irregular eating habits may be associated with a higher risk of breast cancer. Further studies with longer follow-up periods that include information on portion size, hormone receptor status, carcinogen levels in grilled meat, and a classification of foods by source are required.


QUASI: a novel method for simultaneous superposition of multiple flexible ligands and virtual screening using partial similarity.
The structure of many receptors is unknown, and only information about diverse ligands binding to them is available. A new method is presented for the superposition of such ligands, derivation of putative receptor site models and utilization of the models for screening of compound databases. In order to generate a receptor model, the similarity of all ligands is optimized simultaneously taking into account conformational flexibility and also the possibility that the ligands can bind to different regions of the site and only partially overlap. Ligand similarity is defined with respect to a receptor site model serving as a common reference frame. The receptor model is dynamic and coevolves with the ligand alignment until an optimal self-consistent superposition is achieved. When ligand conformational flexibility is permitted, different superposition models are possible and consistent with the data. Clustering of the superposition solutions is used to obtain diverse models. When the models are used to screen a database of compounds, high enrichments are obtained, comparable to those obtained in docking studies.


Hepatoprotective and Antioxidant Activities of Desmodium Triquetrum DC.
The hepatoprotective and antioxidant activities of ethanol extract of Desmodium triquetrum DC leaf were investigated against carbon tetrachloride (1 ml/kg i.p) induced hepatic damage in rats at a dose of 200 mg/kg body weight p.o. The test extract significantly (P<0.05) reduced the elevated levels of serum transaminases, alkaline phosphatase, bilirubin and reversed the antioxidant enzyme and non-enzyme levels. It dose dependently inhibited thiobarbuturic acid induced lipid peroxidation in vitro (IC(50)=59.9 μg/ml). Histopathological studies provided supportive evidence for biochemical analysis. Silymarin (25 mg/kg) is a known hepatoprotective drug used as a reference drug. The results indicated that D. triquetrum has potent hepatoprotective and antioxidant activity that may be due to the presence of flavonoids in the plant.


Prolonged therapy with the anticonvulsant carbamazepine leads to increased plasma clearance of fentanyl.
Fentanyl is a potent analgesic that accounts for an increasing number of overdose deaths in the United States. This study tested whether altered pharmacokinetics plays a pivotal role in the increased fentanyl dose requirements in patients receiving the enzyme-inducing anticonvulsant, carbamazepine. Neurosurgical patients receiving carbamazepine for >6 weeks (N = 11) or no carbamazepine (N = 6, controls) received a single bolus dose of fentanyl (200 μg) intravenously. Plasma was collected before and for up to 9 h after the bolus. Fentanyl concentrations were measured using liquid chromatography-mass spectrometry. Pharmacokinetic variables were derived from plasma concentration-time curves best fitted to a two-compartment model. Fentanyl clearance was significantly higher in the carbamazepine group compared to controls (mean ± SD: 20.1 ± 6.8 vs 13.2 ± 4.8 ml/min per kg, P < 0.05), and area under the plasma concentration curve (AUC) was significantly lower (150 ± 65 vs 233 ± 70 ng/ml × min, P < 0.02). Volume of distribution was larger in the carbamazepine group, but the difference was not statistically significant (5.4 ± 3.1 vs 3.6 ± 1.2 l/kg, P > 0.15). The terminal elimination half-life did not differ between the two groups. Chronic carbamazepine therapy leads to increased fentanyl clearance and decreased AUC, which may result in decreased duration of therapeutic plasma concentrations of fentanyl and an increased dose requirement. Assuming that carbamazepine does not change fentanyl pharmacodynamics, patients on chronic carbamazepine therapy may require more frequent or higher fentanyl doses to maintain therapeutic plasma concentrations.


Enhanced Pharmacokinetic Bioanalysis of Antibody-drug Conjugates using Hybrid Immunoaffinity Capture and Microflow LC-MS/MS.
The increasing complexity and diversity of antibody-drug conjugates (ADCs) have led to a need for comprehensive and informative bioanalytical methods to enhance pharmacokinetic (PK) understanding. This study aimed to evaluate the feasibility of a hybrid immunoaffinity (IA) capture microflow LC-MS/MS (μLC-MS/MS) method for ADC analysis, utilizing a minimal sample volume for PK assessments in a preclinical study. A robust workflow was established for the quantitative analysis of ADCs by the implementation of solid-phase extraction (SPE) and semi-automation in µLC-MS/MS. Utilizing the µLC-MS/MS approach in conjunction with 1 µL of ADC-dosed mouse plasma sample volume, standard curves of two representative surrogate peptides for total antibody (heavy chain, HC) and intact antibody (light chain, LC) ranged from 1.00 ng/mL (LLOQ) to 5000 ng/mL with correlation coefficients (r2) values of > 0.99. The linear range of the standard curve for payload as a surrogate for the concentration of total ADC was from 0.5 ng/mL (LLOQ) to 2000 ng/mL with high accuracy and precision (< 10% CV at all concentrations). Moreover, a high correlation of concentrations of total antibody between two assay approaches (µLC-MS and ELISA) was achieved with less than 20% difference at all time points, indicating that the two methods are comparable in quantitation of total antibody in plasma samples. The µLC-MS platform demonstrated a greater dynamic range, sensitivity, robustness, and good reproducibility. These findings demonstrated that the cost-effective µLC-MS method can reduce reagent consumption and minimize the use of mice plasma samples while providing more comprehensive information about ADCs being analyzed, including the total antibody, intact antibody, and total ADC.


Non-human primate fetal kidney transcriptome analysis indicates mammalian target of rapamycin (mTOR) is a central nutrient-responsive pathway.
Developmental programming is defined as the process by which gene-environment interaction in the developing organism leads to permanent changes in phenotype and function. Numerous reports of maternal nutrient restriction during pregnancy demonstrate altered renal development. Typically this alteration manifests as a reduction in the total number of glomeruli in the mature kidney of the offspring, and suggests that predisposition to develop chronic renal disease may include an in utero origin. In a previous study, we defined the transcriptome in the kidney from fetuses of control (CON, fed ad libitum) and nutrient-restricted (NR, fed 70% of CON starting at 0.16 gestation (G)) pregnancies at half-way through gestation (0.5G), and established transcriptome and morphological changes in NR kidneys compared to CON. One goal of the present study was to use transcriptome data from fetal kidneys of CON and NR mothers at 0.5G with histological data to identify the molecular mechanisms that may regulate renal development. A second goal was to identify mechanisms by which NR elicits its affect on fetal baboon kidney. We have used an end-of-pathway gene expression analysis to prioritize and identify key pathways regulating the 0.5G kidney phenotype in response NR. From these data we have determined that the mammalian target of rapamycin (mTOR) signalling pathway is central to this phenotype.


[Energy dissipation in the brown fatty tissue of warm-blooded animals].
The uncoupling mechanism of respiration and phosphorylation in brown adipose tissue as well as factors affecting this process are discussed. An assumption has been suggested and substantiated experimentally that the uncoupling of respiration and phosphorylation in liver mitochondria caused by vitamin K alimentary deficit may have mechanism similar to the unique uncoupling mechanism of brown fat mitochondria.


Total evidence: molecules, morphology, and the phylogenetics of cichlid fishes.
We present a most comprehensive phylogenetic analysis of the family Cichlidae. New data analyzed include mitochondrial 16S rRNA sequences and two nuclear loci (Tmo-M27 and Tmo-4C4) for a large taxonomic sampling with emphasis on South American species. We also incorporate a published morphological data set for a total evidence analysis. Character congruence among mitochondrial (74 taxa) and nuclear data (50 taxa) was high. However, partition-homogeneity tests suggest significant heterogeneity among molecular and morphological data. In agreement with results obtained from molecular data alone, total evidence analysis (1,460 characters for 34 taxa) supports a robust phylogenetic hypothesis for the family Cichlidae that is congruent with drift-vicariance events associated with the fragmentation of Gondwana. Our analyses confirm the placement of Malagasy/Indian cichlids as the most basal lineages, with a sister-group relationship to the monophyletic African and Neotropical clades. Total evidence suggests that the controversial African genus Heterochromis is at the base of the African radiation. Among more than 50 Neotropical genera analyzed, Retroculus is identified as the basal taxon, with successive branching of Cichla, Astronotus, geophagines (including crenicichlines) + chaetobranchines, and cichlasomines + heroines. Relative rate tests applied to mitochondrial DNA suggest significantly higher rates of genetic variation in Neotropical than in African taxa, and both mitochondrial and nuclear sequences show that rate heterogeneity among Neotropical lineages is confined to the geophagine cichlids.


Capture of the circulating Plasmodium falciparum biomarker HRP2 in a multiplexed format, via a wearable skin patch.
Herein we demonstrate the use of a wearable device that can selectively capture two distinct circulating protein biomarkers (recombinant P. falciparum rPfHRP2 and total IgG) from the intradermal fluid of live mice in situ, for subsequent detection in vitro. The device comprises a microprojection array that, when applied to the skin, penetrates the outer skin layers to interface directly with intradermal fluid. Because of the complexity of the biological fluid being sampled, we investigated the effects of solution conditions on the attachment of capture antibodies, to optimize the assay detection limit both in vitro and on live mice. For detection of the target antigen diluted in 20% serum, immobilization conditions favoring high antibody surface density (low pH, low ionic strength) resulted in 100-fold greater sensitivity in comparison to standard conditions, yielding a detection limit equivalent to the plate enzyme-linked immunosorbent assay (ELISA). We also show that blocking the device surface to reduce nonspecific adsorption of target analyte and host proteins does not significantly change sensitivity. After injecting mice with rPfHRP2 via the tail vein, we compared analyte levels in both plasma and skin biopsies (cross-sectional area same as the microprojection array), observing that skin samples contained the equivalent of ∼8 μL of analyte-containing plasma. We then applied the arrays to mice, showing that surfaces coated with a high density of antibodies captured a significant amount of the rPfHRP2 target while the standard surface showed no capture in comparison to the negative control. Next, we applied a triplex device to both control and rPfHRP2-treated mice, simultaneously capturing rPfHRP2 and total IgG (as a positive control for skin penetration) in comparison to a negative control device. We conclude that such devices can be used to capture clinically relevant, circulating protein biomarkers of infectious disease via the skin, with potential applications as a minimally invasive and lab-free biomarker detection platform.


Detection methods for microRNAs in clinic practice.
MicroRNAs (miRNA) are short non-coding RNA molecules that regulate gene expression. miRNAs profiles are specific for cell lineages and tissues, and their changes reflect pathological processes. This fact introduces the possibility of their use in diagnostics. The application of miRNAs in diagnostics is critically dependent on the establishment of miRNA profiles that can discriminate patients from normal healthy individuals with good sensitivity and specificity and on the development of methods for their accurate and high-throughput quantification. In this review, we present an overview of some of the different techniques and methods currently used to detect miRNAs. We focus on methods that can be employed in routine clinic diagnostics indicating their advantages as well as their shortcomings, with special attention being paid to the most innovative ones. Since disease-specific miRNAs can be found in blood serum, we also present emerging methods for the detection of circulating miRNAs as a way of fast, reliable and non-invasive diagnostic.


WHY DO IMMIGRANT YOUTH WHO NEVER ENROLL IN U.S. SCHOOLS MATTER? AN EXAMINATION OF SCHOOL ENROLLMENT AMONG MEXICANS AND NON-HISPANIC WHITES.
Using data from the 2000 Public Use Sample of the U.S. Census, this research examines how estimates of school enrollment and school-work patterns among Mexican-origin adolescents are affected by including or excluding young immigrants who never enrolled in U.S. schools. The analysis demonstrates that a non-trivial share of adolescents who were born in Mexico almost certainly never enrolled in U.S. schools; these youth most likely migrated to the United States for work. Excluding these adolescents from analyses substantially reduces gaps in school enrollment between Mexicans and Whites and between native and foreign-born Mexicans. Excluding never-enrolled immigrant youth also changes the relationship between duration of U.S. residence and idleness among Mexican immigrant youth, revealing that additional years of residence in the United States increase the likelihood of being out of school and not working compared to in school and not working. Overall, inferences about the level of school enrollment and intra-ethnic differences in school enrollment by duration of residence depend on how those who are likely to have never enrolled in U.S. schools are treated. Inferences about interethnic differences also are affected, although they are somewhat less sensitive to this issue.


Social Behavior as a Transdiagnostic Marker of Resilience.
The recent shift from psychopathology to resilience and from diagnosis to functioning requires the construction of transdiagnostic markers of adaptation. This review describes a model of resilience that is based on the neurobiology of affiliation and the initial condition of mammals that mature in the context of the mother's body and social behavior. The model proposes three tenets of resilience-plasticity, sociality, and meaning-and argues that coordinated social behavior stands at the core sustaining resilience. Two lines in the maturation of coordinated social behavior are charted, across animal evolution and throughout human development, culminating in the mature human reciprocity of empathy, mutuality, and perspective-taking. Cumulative evidence across ages and clinical conditions and based on our behavioral coding system demonstrates that social reciprocity, defined by plasticity at the individual, dyadic, and group levels, denotes resilience, whereas the two poles of disengagement/avoidance and intrusion/rigidity characterize specific psychopathologies, each with a distinct behavioral signature. Attention to developmentally sensitive markers and to the dimension of meaning in human sociality may open new, behavior-based pathways to resilience.


Diverse domain architectures of CheA histidine kinase, a central component of bacterial and archaeal chemosensory systems.
Chemosensory systems in bacteria and archaea are complex, multi-protein pathways that enable rapid cellular responses to environmental changes. The CheA histidine kinase is a central component of chemosensory systems. In contrast to other histidine kinases, it lacks a sensor (input) domain and utilizes dedicated chemoreceptors for sensing. CheA is a multi-domain protein; in model organisms as diverse as Escherichia coli and Bacillus subtilis, it contains five single-copy domains. Deviations from this canonical domain architecture have been reported, however, a broad genome-wide analysis of CheA diversity is lacking. Here, we present results of a genomic survey of CheA domain composition carried out using an unbiased set of thousands of CheA sequences from bacteria and archaea. We found that four out of five canonical CheA domains comprise a minimal functional unit (core domains), as they are present in all surveyed CheA homologs. The most common deviations from a classical five-domain CheA architecture are the lack of a P2/CheY-binding domain, which is missing from more than a half of CheA homologs and the acquisition of a response regulator receiver (CheY-like) domain, which is present in ~35% of CheA homologs. We also document other deviations from classical CheA architecture, including bipartite CheA proteins, domain duplications and fusions, and reveal that phylogenetically defined CheA classes have pre-dominant domain architectures. This study lays a foundation for a better classification of CheA homologs and identifies targets for experimental investigations.


CAUSAL INFERENCE FOR CONTINUOUS-TIME PROCESSES WHEN COVARIATES ARE OBSERVED ONLY AT DISCRETE TIMES.
Most of the work on the structural nested model and g-estimation for causal inference in longitudinal data assumes a discrete-time underlying data generating process. However, in some observational studies, it is more reasonable to assume that the data are generated from a continuous-time process and are only observable at discrete time points. When these circumstances arise, the sequential randomization assumption in the observed discrete-time data, which is essential in justifying discrete-time g-estimation, may not be reasonable. Under a deterministic model, we discuss other useful assumptions that guarantee the consistency of discrete-time g-estimation. In more general cases, when those assumptions are violated, we propose a controlling-the-future method that performs at least as well as g-estimation in most scenarios and which provides consistent estimation in some cases where g-estimation is severely inconsistent. We apply the methods discussed in this paper to simulated data, as well as to a data set collected following a massive flood in Bangladesh, estimating the effect of diarrhea on children's height. Results from different methods are compared in both simulation and the real application.


Modeling the relationship between estimated fungicide use and disease-associated yield losses of soybean in the United States II: Seed-applied fungicides vs seedling diseases.
Use of seed-applied fungicides has become commonplace in the United States soybean production systems. Although fungicides have the potential to protect seed/seedlings from critical early stage diseases such as damping-off and root/stem rots, results from previous studies are not consistent in terms of seed-applied fungicide's ability to mitigate yield losses. In the current study, the relationship between estimated soybean production losses due to seedling diseases and estimated seed-applied fungicide use was investigated using annual data from 28 soybean growing states in the U.S. over the period of 2006 to 2014. National, regional (northern and southern U.S.), state, and temporal scale trends were explored using mixed effects version of the regression analysis. Mixed modeling allowed computing generalized R2 values for conditional (R2GLMM(c); contains fixed and random effects) and marginal (R2GLMM(m); contains only fixed effects) models. Similar analyses were conducted to investigate how soybean production was related to fungicide use. National and regional scale modeling revealed that R2GLMM(c) values were significantly larger compared to R2GLMM(m) values, meaning fungicide use had limited utility in explaining the national/regional scale variation of yield loss and production. The state scale analysis revealed the usefulness of seed-applied fungicides to mitigate seedling diseases-associated soybean yield losses in Illinois, Indiana, North Carolina, and Ohio. Further, fungicide use positively influenced the soybean production and yield in Illinois and South Dakota. Taken together, use of seed-applied fungicide did not appear to be beneficial to many of the states. Our findings corroborate the observations made by a number of scientists through field scale seed-applied fungicide trials across the U.S and reiterate the importance of need base-use of seed-applied fungicides rather than being a routine practice in soybean production systems.


Effect of prophylactic intraocular pressure-lowering medication (brinzolamide) on intraocular pressure after ranibizumab intravitreal injection: A case-control study.
To observe the effect of prophylactic intraocular pressure (IOP)-lowering medication (brinzolamide) on IOP after ranibizumab intravitreal injections (IVIs). This prospective case-control study included 352 eyes from 352 patients (1 eye per patient) who were treated with ranibizumab intravitreal injection and divided randomly into two groups. Two hundred and three patients in control group only received the ranibizumab IVI, but 149 patients in case group received one drop of prophylactic intraocular brinzolamide preinjection. The IOP was measured by noncontact tonometer before injection, at 10, 30, 120 min and 1 day after injection in a sitting position. The mean IOP measured before injection, at 10, 30, 120 min and 1 day after injection individually were 15.79 ± 2.21 mmHg, 19.33 ± 4.86 mmHg, 16.64 ± 2.93 mmHg, 16.17 ± 3.13 mmHg, and 15.07 ± 2.55 mmHg in case group and were 15.82 ± 2.57 mmHg, 21.34 ± 5.88 mmHg, 18.17 ± 4.06 mmHg, 17.59 ± 4.42 mmHg, and15.48 ± 2.92 mmHg in control group. Comparing two groups, the mean increase on IOP was statistically significant at 10, 30, 120 min postinjection (P < 0.05). IVI of ranibizumab causes a considerable short-term transient rise on IOP in most patients. The effect of prophylactic IOP-lowering medication on IOP after IVIs can be statistically significant from 10 min to 2 h after IVIs.


Does Pemetrexed Work in Targetable, Nonsquamous Non-Small-Cell Lung Cancer? A Narrative Review.
Pemetrexed is currently mainly considered for the treatment of advanced nonsquamous non-small-cell lung cancer (NSCLC) negative for gene mutations/rearrangements (wild-type disease (WTD)). This narrative review aimed to highlight the role of pemetrexed in the treatment of onco-driven nonsquamous advanced NSCLC by reviewing published clinical studies. For epidermal growth factor receptor (EGFR) mutations, patient survival following first-line pemetrexed-platinum was longer than for WTD. Later-line pemetrexed-based treatment after tyrosine kinase inhibitor (TKI) failure provided greater benefits than non-pemetrexed regimens. First- and later-line pemetrexed-based therapy also provided survival benefits in patients with anaplastic lymphoma kinase (ALK) or ROS proto-oncogene 1 (ROS1) rearrangements. In patients with rearranged during transfection (RET) proto-oncogene rearrangements, survival with pemetrexed was similar to that in ALK- and ROS1-positive patients and longer than that in patients with Kirsten rat sarcoma (KRAS) virus proto-oncogene mutations or WTD, although the available studies were limited. For Erb-b2 receptor tyrosine kinase 2 (ERRB2) mutations, first-line pemetrexed showed outcomes similar to those for EGFR and KRAS alterations. Data on pemetrexed in patients with KRAS mutations or MNNG HOS-transforming (MET) expression were limited. Pemetrexed could be an option for first- and second-line treatment for TKI failure in nonsquamous advanced NSCLC with select targetable driver mutations.


Regulating DNA-Hybridization Using a Chemically Fueled Reaction Cycle.
Molecular machines, such as ATPases or motor proteins, couple the catalysis of a chemical reaction, most commonly hydrolysis of nucleotide triphosphates, to their conformational change. In essence, they continuously convert a chemical fuel to drive their motion. An outstanding goal of nanotechnology remains to synthesize a nanomachine with similar functions, precision, and speed. The field of DNA nanotechnology has given rise to the engineering precision required for such a device. Simultaneously, the field of systems chemistry developed fast chemical reaction cycles that convert fuel to change the function of molecules. In this work, we thus combined a chemical reaction cycle with the precision of DNA nanotechnology to yield kinetic control over the conformational state of a DNA hairpin. Future work on such systems will result in out-of-equilibrium DNA nanodevices with precise functions.


A Mouse Model to Study the Pathogenesis of γ-herpesviral Infections in Germinal Center B Cells.
CD30-positive germinal center (GC)-derived B cell lymphomas are frequently linked to Epstein-Barr Virus (EBV) infection. However, a suitable animal model for the investigation of the interplay between γ-herpesvirus and host cells in B cell pathogenesis is currently lacking. Here, we present a novel in vivo model enabling the analysis of genetically modified viruses in combination with genetically modified GC B cells. As a murine γ-herpesvirus, we used MHV-68 closely mirroring the biology of EBV. Our key finding was that Cre-mediated recombination can be successfully induced by an MHV-68 infection in GC B cells from Cγ1-Cre mice allowing for deletion or activation of loxP-flanked cellular genes. The implementation of PrimeFlow RNA assay for MHV-68 demonstrated the enrichment of MHV-68 in GC and isotype-switched B cells. As illustrations of virus and cellular modifications, we inserted the EBV gene LMP2A into the MHV-68 genome and induced constitutively active CD30-signaling in GC B cells through MHV-68 infections, respectively. While the LMP2A-expressing MHV-68 behaved similarly to wildtype MHV-68, virally induced constitutively active CD30-signaling in GC B cells led to the expansion of a pre-plasmablastic population. The findings underscore the potential of our novel tools to address crucial questions about the interaction between herpesviral infections and deregulated cellular gene-expression in future studies.


Laparoscopic adrenalectomy: preoperative data, surgical technique and clinical outcomes.
laparoscopic adrenalectomy has become the standard treatment for adrenal lesions. The better clinical outcoms of laparoscopic technique are valid for treatment of small benign masses (< 5-6 cm), instead there are still open questions in literature regarding the correct management of larger lesions (> 6 cm) or in case of potentially malignant adrenal tumors. The aim of this study is to evaluate the outcomes of laparoscopic adrenalectomy in a referral surgical department for endocrine surgery. at the University Hospital Policlinico "P. Giaccone" of Palermo between January 2010 and December 2017 we performed a total of 81 laparoscopic adrenalectomy. We created a retrospective database with analysis of patients data, morphologic and hormonal characteristics of adrenal lesions, surgical procedures and postoperative results with histological diagnosis and complications. Mean size of adrenal neoplasm was 7,5 cm (range 1.5 to 18 cm). The mean operative time was 145 min (range 75-240). In statistical analysis lenght of surgery was correlated to the lesion diameter (p < 0.05) but not with pre-operative features or histological results. 5 intraoperative complications occurred. Among these patients 4 presented bleeding and 1 a diaphagmatic lesion. No conversion to open surgery was necessary and no intraoperative blood transfusion were required. Mean estimated blood loss was 95 ml (range 50-350). There was no capsular disruption during adrenal dissection. Mean length of hospital stay was 3.7 days (range 3-6 days). Laparoscopic adrenalectomy is a safe procedure with low rate of morbidity. An accurate preoperative radiological examination is fundamental to obtain a stringent patients selection. The lesion diameter is related to longer operative time and appeares as the main predictive parameter of intraoperative complications but these results are not statistically significant. On the other side secreting adrenal tumors require more attention in operative management without increased rate of postoperative complications.


Validation of the Japanese translation of the Swallowing Disturbance Questionnaire in Parkinson's disease patients.
The Swallowing Disturbance Questionnaire (SDQ) was developed as a self-rated screening tool for dysphagia in patients with Parkinson's disease (PD). We developed the Japanese version of this questionnaire (SDQ-J), according to the cross-cultural adaptation guidelines, and examined its reliability. Subjects were 61 Japanese patients with PD (mean age, 67.0 ± 9.2 years) who answered the SDQ-J before undergoing videofluoroscopic examination of swallowing (VF). We compared the findings of the questionnaire with the patients' aspiration status during VF. Cronbach's alpha coefficient for the 15 questions of the SDQ-J was 0.84. According to the SDQ-J, 15 patients (24.6%) were diagnosed with dysphagia, while 9 patients (14.8%) aspirated liquid during VF. The sensitivity and specificity of the SDQ-J in predicting aspiration were 77.8 and 84.6%, respectively; therefore, the SDQ-J significantly predicted aspiration during VF (P < 0.01). The positive predictive value (PPV) and negative predictive value (NPV) for the SDQ-J were 0.46 and 0.96, respectively. The SDQ-J appears to be a reliable and useful screening tool for Japanese PD patients with aspiration. As the NPV was higher than the PPV in the SDQ-J, this questionnaire could potentially be used for early identification of severe dysphagia in patients with PD.


Evaluation of collateral ligament injuries of the metacarpophalangeal joints with magnetic resonance imaging and magnetic resonance arthrography.
Injuries of the collateral ligaments of the metacarpophalangeal (MCP) joints of the hand require accurate diagnosis and prompt treatment to prevent loss of function. The most common and well-known collateral ligament injury is injury to the ulnar collateral ligament of the first MCP joint of the hand, often referred to as a gamekeeper injury. Although less common, injuries involving the collateral ligaments of the second through fifth digits and the radial collateral ligament of the first digit also occur. The diagnosis of collateral ligament injury is often made clinically, however, magnetic resonance imaging and magnetic resonance arthrography of the MCP joints can be very useful in the evaluation of severity of the injury. This provides important information for the hand surgeon and aids in treatment planning. Imaging of the collateral ligaments requires small fields of view and several technical considerations can provide improved image quality. These factors will be discussed and illustrated in this article.


A Tutorial on Evaluating the Time-Varying Discrimination Accuracy of Survival Models Used in Dynamic Decision Making.
Many medical decisions involve the use of dynamic information collected on individual patients toward predicting likely transitions in their future health status. If accurate predictions are developed, then a prognostic model can identify patients at greatest risk for future adverse events and may be used clinically to define populations appropriate for targeted intervention. In practice, a prognostic model is often used to guide decisions at multiple time points over the course of disease, and classification performance (i.e., sensitivity and specificity) for distinguishing high-risk v. low-risk individuals may vary over time as an individual's disease status and prognostic information change. In this tutorial, we detail contemporary statistical methods that can characterize the time-varying accuracy of prognostic survival models when used for dynamic decision making. Although statistical methods for evaluating prognostic models with simple binary outcomes are well established, methods appropriate for survival outcomes are less well known and require time-dependent extensions of sensitivity and specificity to fully characterize longitudinal biomarkers or models. The methods we review are particularly important in that they allow for appropriate handling of censored outcomes commonly encountered with event time data. We highlight the importance of determining whether clinical interest is in predicting cumulative (or prevalent) cases over a fixed future time interval v. predicting incident cases over a range of follow-up times and whether patient information is static or updated over time. We discuss implementation of time-dependent receiver operating characteristic approaches using relevant R statistical software packages. The statistical summaries are illustrated using a liver prognostic model to guide transplantation in primary biliary cirrhosis.


Landscape analysis of constraint satisfaction problems.
We discuss an analysis of constraint satisfaction problems, such as sphere packing, K-SAT, and graph coloring, in terms of an effective energy landscape. Several intriguing geometrical properties of the solution space become in this light familiar in terms of the well-studied ones of rugged (glassy) energy landscapes. A benchmark algorithm naturally suggested by this construction finds solutions in polynomial time up to a point beyond the clustering and in some cases even the thermodynamic transitions. This point has a simple geometric meaning and can be in principle determined with standard statistical mechanical methods, thus pushing the analytic bound up to which problems are guaranteed to be easy. We illustrate this for the graph 3- and 4-coloring problem. For packing problems the present discussion allows to better characterize the J-point, proposed as a systematic definition of random close packing, and to place it in the context of other theories of glasses.


Kondo screening cloud around a quantum dot: large-scale numerical results.
Measurements of the persistent current in a ring containing a quantum dot would afford a unique opportunity to finally detect the elusive Kondo screening cloud. We present the first large-scale numerical results on this controversial subject using exact diagonalization and density matrix renormalization group (RG). These extremely challenging numerical calculations confirm RG arguments for weak to strong coupling crossover with varying ring length and give results on the universal scaling functions. We also study, analytically and numerically, the important and surprising effects of particle-hole symmetry breaking.


LinguAPP: An m-Health Application for Teledentistry Diagnostics.
An Android/iOS application for low-cost mobile devices to aid in dental diagnosis through questionnaire and photos is presented in this paper. The main purposes of our app lie in the ease of use even for nonexperienced users, in the limited hardware requirements that allow a wide diffusion, and in the possibility to modify the questionnaire for different pathologies. This tool was developed in about a month at the beginning of the COVID-19 (SARS-CoV-2) pandemic and is still in use in Italy to allow support to patients without going to the hospital, if not strictly necessary.


Trends in all-cause mortality among patients with chronic myeloid leukemia: a Surveillance, Epidemiology, and End Results database analysis.
Outcomes for patients with chronic myeloid leukemia (CML) have improved after the advent of tyrosine kinase inhibitors (TKIs), which target the BCR/ABL fusion gene product. Nonetheless, differences in survival persist between age groups. The authors performed a retrospective cohort study using the Surveillance, Epidemiology, and End Results (SEER) database to assess 5-year overall survival (OS) in various patient age groups. Patients who had a diagnosis of CML were identified using the SEER 19 registries database. Patients who were included had SEER diagnosis codes for CML not otherwise specified (code 9863) and BCR/ABL-positive CML (code 9875) diagnosed between January 2000 and December 2005. Patients were divided into cohorts based on age at diagnosis: ages 15 to 44 years, 45 to 64 years, 65 to 74 years, and 75 to 84 years. OS was estimated using the Kaplan-Meier method, and Cox regression was used to estimate predictors of patient survival. In total, 5138 patients with a new CML diagnosis were identified. Five-year OS improved for all patients between the years 2000 and 2005. Compared with patients who were diagnosed in 2000, 5-year survival improved among patients ages 15 to 44 years (hazard ratio [HR] for mortality, 0.424; P < .0001), ages 45 to 64 years (HR, 0.716; P = .0315), and ages 65 to 74 years (HR, 0.692; P = .0126); and patients ages 75 to 84 years had an increased 5-year OS rate from 19.2% in 2000 to 36.4% in 2005 (HR, 0.568; P < .0001). OS at 5 years improved among all patients, including those ages 75 to 84 years, a group with historically poor outcomes. However, older age retained an association with worse survival, suggesting opportunities for further progress.


COVID-2019 Pneumonia: Severity and distribution of lung changes observed on the initial chest X-ray as an indicator of final outcomes.
This study aimed to assess the correlation between the severity of the initial chest x-ray (CXR) abnormalities in patients with a confirmed diagnosis of COVID-19 and the final outcomes. This retrospective study was conducted at the Royal Hospital, Oman between mid-March and May 2020 and included patients who had been admitted with a confirmed diagnosis of COVID-19 and had a final outcome. Serial CXRs were identified and examined for presence, extent, distribution and progression pattern of radiological abnormalities. Each lung field was divided into three zones on each CXR and a score was allocated for each zone (0 is normal and 1-4 is mild-severe). The scores for all six zones per CXR examination were summed to provide a cumulative chest radiographic score (range: 0-24). A total of 64 patients were included; the majority were male (89.1%) and the mean age was 50.22 ± 14.86 years. The initial CXR was abnormal in 60 patients (93.8%). The most common finding was ground glass opacity (n = 58, 96.7%) followed by consolidation (n = 50, 83.3%). Most patients had bilateral (n = 51, 85.0%), multifocal (n = 57, 95.0%) and mixed central and peripheral (n = 36, 60.0%) lung abnormalities. The median score of initial CXR for deceased patients was significantly higher than recovered patients (17 versus 11; P = 0.009). Five CXR evolution patterns were identified: type I (initial radiograph deteriorates then improves), type II (fluctuate), type III (static), type IV (progressive deterioration) and type V (progressive improvement). A higher baseline CXR score is associated with higher mortality rate and poor prognosis in those with COVID-19 pneumonia.


[The slack observation of a public enemy: East and West German psychiatrist and psychotherapist Dietfried Müller-Hegemann in files of the East German state security (Stasi)].
This paper is the first to present and assess material gathered on a psychiatrist and psychotherapist by East German Intelligence Service Stasi, officially referred to as Ministry of National Security (Ministerium für Staatssicherheit, MfS), over the course of several decades.We have analysed all files on professor of psychiatry Dietfried Müller-Hegemann (1910-1989) preserved in the authority now responsible for preserving, studying and making all files of the former East German Secret Service accessible (BStU). The material gained from different authorities within the MfS and on a variety of topics showed significant differences in relevance, in terms of both their quality and their amount.In the 1950 s, Müller-Hegemann's career was willingly promoted by political leaders, while in the 1960 s these same authorities started to try to hinder it, until finally Müller-Hegemann emigrated to West Germany. Even though the material presented here substantiates how such influence was exerted in a single case only, it might also help to better understand procedures commonly applied by the MfS and political hierarchies. The files show that from the end of the 1950 s on leading health ministry staff and political hierarchies started categorising Müller-Hegemann as politically unreliable and as a 'public enemy' and trying to hinder his career. By contrast, it was not before after his 'defection' (emigration) in 1971 that he was first officially dealt with by the MfS.What the study of the material gained made clear is that despite a great power, this power of the Secret Service was indeed restricted. On the one hand, the MfS needed to rely on very subjective opinions of both colleagues and neighbours on the 'target'. On the other hand, in the case of Müller-Hegemann the MfS did not succeed in stopping all contact to his family left behind.Another result of our analysis is that intelligence operations on Müller-Hegemann were sporadic and relaxed and by no means systematic. Even though this is the result of a single cases study only, it might still cast some shade of doubt on findings in contemporary research saying that psychiatrists and psychotherapists stood in the special focus of the Secret Service's work.


Molecular interactions between fenoterol stereoisomers and derivatives and the β₂-adrenergic receptor binding site studied by docking and molecular dynamics simulations.
The β2 adrenergic receptor (β2-AR) has become a model system for studying the ligand recognition process and mechanism of the G protein coupled receptors activation. In the present study stereoisomers of fenoterol and some of its derivatives (N = 94 molecules) were used as molecular probes to identify differences in stereo-recognition interactions between β2-AR and structurally similar agonists. The present study aimed at determining the 3D molecular models of the fenoterol derivative-β2-AR complexes. Molecular models of β2-AR have been developed by using the crystal structure of the human β2-AR T4 lysozyme fusion protein with bound (S)-carazolol (PDB ID: 2RH1) and more recently reported structure of a nanobody-stabilized active state of the β2-AR with the bound full agonist BI-167107 (PDB ID: 3P0G). The docking procedure allowed us to study the similarities and differences in the recognition binding site(s) for tested ligands. The agonist molecules occupied the same binding region, between TM III, TM V, TM VI and TM VII. The residues identified by us during docking procedure (Ser203, Ser207, Asp113, Lys305, Asn312, Tyr308, Asp192) were experimentally indicated in functional and biophysical studies as being very important for the agonist-receptor interactions. Moreover, the additional space, an extension of the orthosteric pocket, was identified and described. Furthermore, the molecular dynamics simulations were used to study the molecular mechanism of interaction between ligands ((R,R')- and (S,S')-fenoterol) and β2-AR. Our research offers new insights into the ligand stereoselective interaction with one of the most important GPCR member. This study may also facilitate the design of improved selective medications, which can be used to treat, prevent and control heart failure symptoms.


Crucial role for Nox2 and sleep deprivation in aircraft noise-induced vascular and cerebral oxidative stress, inflammation, and gene regulation.
Aircraft noise causes endothelial dysfunction, oxidative stress, and inflammation. Transportation noise increases the incidence of coronary artery disease, hypertension, and stroke. The underlying mechanisms are not well understood. Herein, we investigated effects of phagocyte-type NADPH oxidase (Nox2) knockout and different noise protocols (around-the-clock, sleep/awake phase noise) on vascular and cerebral complications in mice. C57BL/6j and Nox2-/- (gp91phox-/-) mice were exposed to aircraft noise (maximum sound level of 85 dB(A), average sound pressure level of 72 dB(A)) around-the-clock or during sleep/awake phases for 1, 2, and 4 days. Adverse effects of around-the-clock noise on the vasculature and brain were mostly prevented by Nox2 deficiency. Around-the-clock aircraft noise of the mice caused the most pronounced vascular effects and dysregulation of Foxo3/circadian clock as revealed by next generation sequencing (NGS), suggesting impaired sleep quality in exposed mice. Accordingly, sleep but not awake phase noise caused increased blood pressure, endothelial dysfunction, increased markers of vascular/systemic oxidative stress, and inflammation. Noise also caused cerebral oxidative stress and inflammation, endothelial and neuronal nitric oxide synthase (e/nNOS) uncoupling, nNOS mRNA and protein down-regulation, and Nox2 activation. NGS revealed similarities in adverse gene regulation between around-the-clock and sleep phase noise. In patients with established coronary artery disease, night-time aircraft noise increased oxidative stress, and inflammation biomarkers in serum. Aircraft noise increases vascular and cerebral oxidative stress via Nox2. Sleep deprivation and/or fragmentation caused by noise triggers vascular dysfunction. Thus, preventive measures that reduce night-time aircraft noise are warranted.


Predictive performance of 'Diprifusor' TCI system in patients during upper abdominal surgery under propofol/fentanyl anesthesia.
To evaluate the predictive performance of 'Diprifusor' TCI (target-controlled infusion) system for its better application in clinical anesthesia. The predictive performance of a 'Diprifusor' TCI system was investigated in 27 Chinese patients (16 males and 11 females) during upper abdominal surgery under total intravenous anesthesia (TIVA) with propofol/fentanyl. Measured arterial propofol concentrations were compared with the values predicted by the TCI infusion system. Performance was determined by the median performance error (MDPE), the median absolute performance error (MDAPE), the divergence (the percentage change of the absolute PE with time), and the wobble (the median absolute deviation of each PE from the MDPE). The median (range) values of 14.9% (-21.6%-42.9%) for MDPE, 23.3% (6.9%-62.5%) for MDAPE, -1.9% h(-1) (-32.7%-23.0% h(-1)) for divergence, and 18.9% (4.2%-59.6%) for wobble were obtained from 227 samples from all patients. For the studied population, the PE did not increase with time but with increasing target propofol concentration, particularly following induction. conclusions: The control of depth of anaesthesia was good in all patients undergoing upper abdominal surgical operation and the predictive performance of the 'Diprifusor' target controlled infusion system was considered acceptable for clinical purposes. But the relatively bigger wobble showed that the pharmacokinetic model is not so suitable and requires improvement.


Comparison of Open, Laparoscopic, and Robotic Colectomies Using a Large National Database: Outcomes and Trends Related to Surgery Center Volume.
Previous studies have shown that high-volume centers and laparoscopic techniques improve outcomes of colectomy. These evidence-based measures have been slow to be accepted, and current trends are unknown. In addition, the current rates and outcomes of robotic surgery are unknown. The purpose of this study was to examine current national trends in the use of minimally invasive surgery and to evaluate hospital volume trends over time. This was a retrospective study. This study was conducted in a tertiary referral hospital. Using the National Inpatient Sample, we evaluated trends in patients undergoing elective open, laparoscopic, and robotic colectomies from 2009 to 2012. Patient and institutional characteristics were evaluated and outcomes compared between groups using multivariate hierarchical-logistic regression and nonparametric tests. The National Inpatient Sample includes patient and hospital demographics, admission and treating diagnoses, inpatient procedures, in-hospital mortality, length of hospital stay, hospital charges, and discharge status. In-hospital mortality and postoperative complications of surgery were measured. A total of 509,029 patients underwent elective colectomy from 2009 to 2012. Of those 266,263 (52.3%) were open, 235,080 (46.2%) laparoscopic, and 7686 (1.5%) robotic colectomies. The majority of minimal access surgery is still being performed at high-volume compared with low-volume centers (37.5% vs 28.0% and 44.0% vs 23.0%; p < 0.001). A total of 36% of colectomies were for cancer. The number of robotic colectomies has quadrupled from 702 in 2009 to 3390 (1.1%) in 2012. After adjustment, the rate of iatrogenic complications was higher for robotic surgery (OR = 1.73 (95% CI, 1.20-2.47)), and the median cost of robotic surgery was higher, at $15,649 (interquartile range, $11,840-$20,183) vs $12,071 (interquartile range, $9338-$16,203; p < 0.001 for laparoscopic). This study may be limited by selection bias by surgeons regarding the choice of patient management. In addition, there are limitations in the measures of disease severity and, because the database relies on billing codes, there may be inaccuracies such as underreporting. Our results show that the majority of colectomies in the United States are still performed open, although rates of laparoscopy continue to increase. There is a trend toward increased volume of laparoscopic procedures at specialty centers. The role of robotics is still being defined, in light of higher cost, lack of clinical benefit, and increased iatrogenic complications, albeit comparable overall complications, as compared with laparoscopic colectomy.


Synovial chondromatosis of the foot presenting with Lisfranc dislocation.
Primary synovial chondromatosis is rare in the foot. We report a case of synovial chondromatosis affecting multiple sites of the foot and causing bone erosions in a 44-year-old woman. Radiographs demonstrated erosions of multiple metatarsals including the tarsometatarsal joints, resulting in Lisfranc tarsometatarsal dislocation. Magnetic resonance imaging showed the widespread synovial proliferation and soft tissue masses affecting the foot and helped in arriving at a differential diagnosis and plan for needle biopsy. Diagnosis was made initially by needle biopsy under computed tomography guidance and was subsequently confirmed by histopathological assessment of the surgically excised synovial masses. To our knowledge, multifocal synovial chondromatosis causing Lisfranc dislocation in the foot has not been reported previously.


The impact of National Health Service Corps physicians in the lowering perinatal mortality rate in Dade County, Florida.
In some parts of Dade County, Florida, perinatal mortality rates have revealed serious problems in the delivery of health care to poor pregnant women. From 1982-1985, the reported perinatal mortality rates varied from 32-36 per 1000 live births, more than double the national average. Under the leadership of the Primary Health Care Consortium of Dade County (a federation of community health centers and other primary care providers), National Health Service Corps obstetricians and pediatricians served inner-city, medically needy patients as part of a coordinated perinatal plan from 1987-1989. Data on fetal and neonatal deaths, collected from census tracts adjacent to the community health centers, were used to study the impact of Corps obstetrician and pediatrician placement. The respective perinatal mortality rates were compared with those of 1986 as historic controls. Within a year, the overall perinatal mortality rate was reduced by 45%. As a result, an estimated 320 lives were saved between 1987-1989. This public health achievement represents a measurable impact due to assignment of National Health Service Corps physicians and can be used as a working model to reduce perinatal mortality in medically underserved communities in the United States.


Doctors' knowledge of tissue donation in children.
To find out whether doctors who work with children have a basic knowledge of tissue and organ donation which would allow them to initiate discussions with families. Anonymous self-completed questionnaire containing basic information about donation administered to 56 doctors working in paediatrics, emergency medicine and intensive care in one district general hospital. 89% doctors were aware that children could donate solid organs after death although 39.3% thought incorrectly that premature babies were eligible. Respondents were generally less knowledgeable about age limits for tissue (heart valve and eye), blood and bone marrow donation. The overall average score on the questionnaire was only 49.9%, this score did not vary with grade or paediatric training. Participants had poor knowledge about tissue and organ donation, which would make approaching families difficult. This study has identified an important training need for all doctors involved in the care of dying children.


Inhibition of Drp1 orchestrates the responsiveness of breast cancer cells to paclitaxel but insignificantly relieves paclitaxel-related ovarian damage in mice.
Chemoresistance and chemotherapy-related ovarian damage are well-reported in breast cancer (BC) young patients. Herein, the inhibition of the mitochondrial fission was invested to explore its chemosensitizing role in Paclitaxel (PTX)-resistant cells, and its ability to restore the ovarian integrity in mice receiving PTX or cisplatin chemotherapy. To establish these aims, PTX-resistance was generated in BC cells, which were treated with PTX in combination with Drp1 deficiency, via mdivi-1, or Drp1-specific siRNA transfection. Furthermore, the alterations in the ovarian structure and the endocrine-related hormones were explored in mice receiving repetitive doses of PTX or cisplatin. We found that combining PTX with mdivi-1 improved cell responsiveness to PTX, induced apoptosis- and autophagy-mediated cell death, and relieved cellular oxidative stress. Additionally, the expression of PCNA1 and cyclin B1 genes were downregulated, meanwhile, p53, p21, and mitochondrial fusion proteins (Mfu1&Mfu2) were increased. The in vivo investigations in mice demonstrated that PTX induced gonadotoxic damage similar to cisplatin, whereas dual treatment of mice with PTX+ mdivi-1 failed to restore their normal follicular count and the circulating levels of E2 and AMH hormones. These results suggested that combining Drp1 inhibition with PTX resensitized breast cancer cells to PTX but failed to offer enough protection against chemotherapy-related gonadotoxicity.


The characterization of amphibian nucleoplasmins yields new insight into their role in sperm chromatin remodeling.
Nucleoplasmin is a nuclear chaperone protein that has been shown to participate in the remodeling of sperm chromatin immediately after fertilization by displacing highly specialized sperm nuclear basic proteins (SNBPs), such as protamine (P type) and protamine-like (PL type) proteins, from the sperm chromatin and by the transfer of histone H2A-H2B. The presence of SNBPs of the histone type (H type) in some organisms (very similar to the histones found in somatic tissues) raises uncertainty about the need for a nucleoplasmin-mediated removal process in such cases and poses a very interesting question regarding the appearance and further differentiation of the sperm chromatin remodeling function of nucleoplasmin and the implicit relationship with SNBP diversity The amphibians represent an unique opportunity to address this issue as they contain genera with SNBPs representative of each of the three main types: Rana (H type); Xenopus (PL type) and Bufo (P type). In this work, the presence of nucleoplasmin in oocyte extracts from these three organisms has been assessed using Western Blotting. We have used mass spectrometry and cloning techniques to characterize the full-length cDNA sequences of Rana catesbeiana and Bufo marinus nucleoplasmin. Northern dot blot analysis shows that nucleoplasmin is mainly transcribed in the egg of the former species. Phylogenetic analysis of nucleoplasmin family members from various metazoans suggests that amphibian nucleoplasmins group closely with mammalian NPM2 proteins. We have shown that these organisms, in striking contrast to their SNBPs, all contain nucleoplasmins with very similar primary structures. This result has important implications as it suggests that nucleoplasmin's role in chromatin assembly during early zygote development could have been complemented by the acquisition of a new function of non-specifically removing SNBPs in sperm chromatin remodeling. This acquired function would have been strongly determined by the constraints imposed by the appearance and differentiation of SNBPs in the sperm.


A Modified P1 Moiety Enhances In Vitro Antiviral Activity against Various Multidrug-Resistant HIV-1 Variants and In Vitro Central Nervous System Penetration Properties of a Novel Nonpeptidic Protease Inhibitor, GRL-10413.
We report here that GRL-10413, a novel nonpeptidic HIV-1 protease inhibitor (PI) containing a modified P1 moiety and a hydroxyethylamine sulfonamide isostere, is highly active against laboratory HIV-1 strains and primary clinical isolates (50% effective concentration [EC50] of 0.00035 to 0.0018 μM), with minimal cytotoxicity (50% cytotoxic concentration [CC50] = 35.7 μM). GRL-10413 blocked the infectivity and replication of HIV-1NL4-3 variants selected by use of atazanavir, lopinavir, or amprenavir (APV) at concentrations of up to 5 μM (EC50 = 0.0021 to 0.0023 μM). GRL-10413 also maintained its strong antiviral activity against multidrug-resistant clinical HIV-1 variants isolated from patients who no longer responded to various antiviral regimens after long-term antiretroviral therapy. The development of resistance against GRL-10413 was significantly delayed compared to that against APV. In addition, GRL-10413 showed favorable central nervous system (CNS) penetration properties as assessed with an in vitro blood-brain barrier (BBB) reconstruction system. Analysis of the crystal structure of HIV-1 protease in complex with GRL-10413 demonstrated that the modified P1 moiety of GRL-10413 has a greater hydrophobic surface area and makes greater van der Waals contacts with active site amino acids of protease than in the case of darunavir. Moreover, the chlorine substituent in the P1 moiety interacts with protease in two distinct configurations. The present data demonstrate that GRL-10413 has desirable features for treating patients infected with wild-type and/or multidrug-resistant HIV-1 variants, with favorable CNS penetration capability, and that the newly modified P1 moiety may confer desirable features in designing novel anti-HIV-1 PIs.


O6-Methylguanine DNA lesions induce an intra-S-phase arrest from which cells exit into apoptosis governed by early and late multi-pathway signaling network activation.
The O(6)-methylguanine (O(6)MeG) DNA lesion is well known for its mutagenic, carcinogenic, and cytotoxic properties, and understanding how a cell processes such damage is of critical importance for improving current cancer therapy. Here we use human cells differing only in their O(6)MeG DNA methyltransferase (MGMT) or mismatch repair (MMR) status to explore the O(6)MeG/MMR-dependent molecular and cellular responses to treatment with the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). We find that O(6)MeG triggers MMR-dependent cell cycle perturbations in both the first and second cell cycle post treatment. At lower levels of damage, we show that a transient arrest in the second S-phase precedes survival and progression into subsequent cell cycles. However, at higher levels of damage, arrest in the second S-phase coincides with a cessation of DNA replication followed by initiation of apoptotic cell death. Further, we show that entry into apoptotic cell death is specifically from S-phase of the second cell cycle. Finally, we demonstrate the key role of an O(6)MeG/MMR-dependent multi-pathway, multi-time-scale signaling network activation, led by early ATM, H2AX, CHK1, and p53 phosphorylation and followed by greatly amplified late phosphorylation of the early pathway nodes along with activation of the CHK2 kinase and the stress-activated JNK kinase.


Linkage between melanocytic tumor development and early burst of Ret protein expression for tolerance induction in metallothionein-I/ret transgenic mouse lines.
We examined the basis of the all or none difference in inducing melanocytic tumor development among three transgenic mouse lines (304, 192 and 242) to which the same promoter-enhancer (metallothionein-I) and oncogene (ret) were introduced. We initially demonstrated that both skin melanosis and Ret protein expression in skin, thymus and brain first became detectable before or immediately after birth in the mice of the tumor developing lines (304 and 192), whereas they became detectable a few days after birth in the mice of the non-tumor developing line (242) by Western blotting and immunohistochemical analysis. Interestingly, the Ret protein expression in skin developed rapidly after birth as a burst with peak levels on 0.5-1.5 day newborns of lines 304 and 192 and on 7.0-7.5 day-old mice of line 242. The levels of autophosphorylation of Ret kinase in skin were, however, invariable among these three transgenic mouse lines. The mice of line 242, but not those of lines 192 and 304, responded to Ret protein immunization by increased antigen-dependent lymphocyte proliferation and T-cell-mediated tumor growth suppression in vitro. Furthermore, ret-transgenic mice of line 242, but not line 304, rejected the subcutaneously transplanted tumors that had originally developed in a mouse of line 304. These results suggest that whether oncogene product-specific-tolerance is established or not to antitumor immunity may be decided by the dynamics of ret oncogene expression before and after delivery and this is the primary factor determining development or non-development of melanoma.


A biorobotic model of the human larynx.
This work focuses on a physical model of the human larynx that replicates its main components and functions. The prototype reproduces the multilayer vocal folds and the ab/adduction movements. In particular, the vocal folds prototype is made with soft materials whose mechanical properties have been obtained to be similar to the natural tissue in terms of viscoelasticity. A computational model was used to study fluid-structure interaction between vocal folds and the airflow. This tool allowed us to make a comparison between theoretical and experimental results. Measurements were performed with this prototype in an experimental platform comprising a controlled air flow, pressure sensors and a high-speed camera for measuring vocal fold vibrations. Data included oscillation frequency at the onset pressure and glottal width. Results show that the combination between vocal fold geometry, mechanical properties and dimensions exhibits an oscillation frequency close to that of the human vocal fold. Moreover, computational results show a high correlation with the experimental one.


Increased serum midkine concentration as a possible tumor marker in patients with superficial esophageal cancer.
Midkine, a heparin-binding growth factor, is expressed in numerous cancer tissues and is reportedly elevated in patients with various neoplasms. The aim of this study was to evaluate the clinicopathological significance of serum midkine concentration (S-MK) in patients with superficial esophageal squamous cell carcinoma (SCC). Pretreatment S-MK was measured by enzyme-linked immunosorbent assay in 135 healthy controls, 16 patients with benign esophageal disease, and 60 patients with primary superficial esophageal squamous cell cancer (SESCC). All patients with SESCC underwent curative resection. The disease was staged according to TNM/UICC guidelines. Serum concentrations of carcinoembryonic antigen (CEA), squamous cell carcinoma antigen (SCC-Ag), and cytokeratin 19 fragment (CYFRA21-1) were also evaluated in the same populations. S-MK in patients with SESCC (388+/-411 pg/ml) was significantly higher than in benign esophageal disease or healthy controls (183+/-73 and 154+/-76 pg/ml, respectively). Using the mean + 2 standard deviations of healthy control S-MK (300 pg/ml) as the cut-off level, 50% of patients with esophageal SESCC were deemed positive. This S-MK positivity rate for detecting SESCC was significantly higher than for other tumor markers. Thus, S-MK may be useful as a tumor marker to detect SESCC.


Malignant transformation of murine fibroblasts by a human c-Ha-ras-1 oncogene does not require a functional epidermal growth factor receptor.
Although mutations in ras genes are thought to be important for the development of about 20% of human tumors, almost nothing is known about the way in which these mutations lead to cellular transformation. The known biochemical properties of the 21-kilodalton ras proteins suggest that they may behave as G proteins, regulating the proliferation of cells in response to growth factor stimulation of a receptor. Although the putative receptor(s) has not been identified, several lines of evidence, in particular the fact that rodent cell lines containing ras oncogenes produce transforming growth factor alpha, have suggested that the epidermal growth factor (EGF) receptor is involved in ras transformation. Here we show that murine fibroblasts with no EGF receptors can be transformed to a completely malignant phenotype with a mutated ras gene. It appears, therefore, that the EGF receptor is not required for ras-mediated transformation of these cells.


[The depressor effect of intracerebroventricular injection of BQ123 and endothelin (ET)--antiserum on hypertensive rats produced by aorta stenosis and hypersaline uptake].
An experimental model of hypertensive rat was made by abdominal aorta stenosis and hypersaline uptake. The blood pressure was significantly decreased by intracerebroventricular injection (i.c.v.) of BQ123 a potent antagonist of ET-1 receptor, in a dose-dependent manner. Negative chronotropic effect on the heart was produced by high dose of BQ123, while the depressor effect was produced by i.c.v. ET-antiserum. These results indicate that endothelin in CNS may play an important role in the pathological physiology of hypertension.


Ryanodine receptor type 1 gene mutations found in the Canadian malignant hyperthermia population.
Malignant hyperthermia (MH) is an autosomal dominant pharmacogenetic disorder that is manifested on exposure of susceptible individuals to halogenated anesthetics or succinylcholine. Since MH is associated primarily with mutations in the ryanodine receptor type 1 (RYR1) gene, the purpose of this study was to determine the distribution and frequency of MH causative RyR1 mutations in the Canadian MH susceptible (MHS) population. In this study, we screened a representative cohort of 36 unrelated Canadian MHS individuals for RYR1 mutations by sequencing complete RYR1 transcripts and selected regions of CACNA1S transcripts. We then analyzed the correlation between caffeine-halothane contracture test (CHCT) results and RYR1 genotypes within MH families. Eighty-six percent of patients had at least one RyR1 mutation (31 out of 36), five of which were unrelated individuals who were double-variant carriers. Fifteen of the 27 mutations identified in RYR1 were novel. Eight novel mutations, involving highly conserved amino acid residues, were predicted to be causal. Two of the mutations co-segregated with the MHS phenotype within two large independent families (a total of 79 individuals). Fourteen percent of MHS individuals (five out of 36) carried neither RYR1 nor known CACNA1S mutations. The distribution and frequency of MH causative RyR1 mutations in the Canadian MHS population are close to those of European MHS populations. Novel mutations described in this study will contribute to the worldwide pool of MH-associated mutations in the RYR1 gene, ultimately increasing the value of MH genetic diagnostic testing.


Cancer immunoprevention: a new approach to intercept cancer early.
Cancer immunoprevention refers to the modulation of the host immune response to control the initiation or development of cancer. The significant role of host immunity in early tumorigenesis has only recently been confirmed, as a better understanding of the mechanisms, molecules and cells involved in tumor immunology have been elucidated over the past two decades. Of utmost importance, preclinical and clinical evidences have demonstrated that early neoplastic cells (transformed cells that initiate cancer formation) express antigens that allow the immune system to distinguish them from normal cells. Furthermore, recognition of the aberrant cell by the immune cells activates a complex interaction of mutual modulation between the immune cells, the tumor and the tumor microenvironment that may result not only in inhibition but also promotion of cancer. The deepening understanding of cancer-related immunologic processes, properties, and components has spawned exploration of more rational, mechanism-based immunologic strategies (using vaccines, antibodies, and immune modulators) for cancer prevention. This introduction to the Cancer Prevention Research immunoprevention series will attempt to review the basics of the immune response modulation as a basis for potential application to cancer immunoprevention strategies with an emphasis on vaccines. Recognizing the fast-paced research in immune response modulation, the series will cover current understandings and future directions of cancer immunoprevention research.


Supplementation with aromatic amino acids improves leucine kinetics but not aromatic amino acid kinetics in infants with infection, severe malnutrition, and edema.
We investigated whether supplementation with an aromatic amino acid (AAA) cocktail consisting of 0.5 mmol each of phenylalanine, tryptophan, and tyrosine compared with isonitrogenous amounts of alanine (Ala) would improve measures of protein kinetics in 14 (8 with AAA, 6 Ala) children with edematous malnutrition (aged 6-24 mo) during the infected acute malnourished state. Supplementation started immediately after the baseline experiment, 2 d postadmission and continued to the end of the acute phase of treatment. The second (postsupplementation) experiment was done approximately 12 d postadmission. We measured leucine kinetics, phenylalanine and tyrosine fluxes, using an i.g. 8-h prime continuous infusion of (2)H(3)-leucine, and an i.v. 6-h prime continuous infusion of (13)C-leucine, (2)H(2)-tyrosine, and (2)H(5)-phenylalanine in the fed state. Leucine flux tended to be faster (P = 0.06) in the AAA group compared with Ala group after supplementation (mean difference +/- SEM): 22.6 +/- 10.9 micromol/(kg . h). The rate of leucine appearance from protein breakdown [28.1 +/- 9.4 micromol/(kg . h)] and the nonoxidative disposal of leucine [i.e., leucine to protein synthesis; 35.4 +/- 12.9 micromol/(kg . h)] were faster (P < 0.02) in the AAA group than in the Ala group. There was no significant effect of supplementation on leucine splanchnic metabolism, phenylalanine, and tyrosine fluxes. These findings are consistent with the hypothesis that the blunting of the protein catabolic response to infection in children with edematous malnutrition syndrome is due to limited availability of aromatic amino acids.


Ultrasound of the sentinel node in melanoma patients: echo-free island is a discriminatory morphologic feature for node positivity.
Unlike breast and thyroid cancer, the use of ultrasound (US)-guided fine needle aspiration cytology (FNAC) for preoperative staging is limited in melanoma. New US morphology criteria have shown that US-FNAC can correctly identify 50% of all involved sentinel nodes (SN) in melanoma patients before surgical excision. The aim of this study was to examine a new criterion: the echo-free island (EFI). A total of 1000 consecutively staged melanoma patients (Breslow thickness>1 or<1 mm, but ulcerated, Clark IV/V or regressed) scheduled for SN staging underwent preoperative US. US morphology items were assessed: peripheral perfusion, loss of central echoes, balloon shape, and EFI. FNAC was performed in case of suspicious and malignant US patterns. All patients proceeded to undergo an SN biopsy or direct completion lymph node dissection (CLND) (in the case of positive FNAC). In all, 57% of the patients were men. The mean/median Breslow thickness was 2.58/1.57 mm. The mean/median follow-up was 56/53 months. SN was positive in 21%. EFI information was available in 95.3%. EFI was seen in 40 patients (4%). EFI sensitivity was 10.8%, specificity was 97.6%, positive predictive value was 50%, and negative predictive value was 80.2%. EFI was significantly correlated to peripheral perfusion (67.5%). There was no correlation to balloon shape or loss of central echoes. Five-year melanoma-specific survival of patients with EFI was significantly worse: 80% versus 92% when absent. The EFI can be useful in the early detection of SN melanoma metastasis. It is an early sign of involvement and thus associated with a decreased survival.


Genetic variant of IRAK2 in the toll-like receptor signaling pathway and survival of non-small cell lung cancer.
The toll-like receptor (TLR) signaling pathway plays an important role in the innate immune responses and antigen-specific acquired immunity. Aberrant activation of the TLR pathway has a significant impact on carcinogenesis or tumor progression. Therefore, we hypothesize that genetic variants in the TLR signaling pathway genes are associated with overall survival (OS) of patients with non-small cell lung cancer (NSCLC). To test this hypothesis, we first performed Cox proportional hazards regression analysis to evaluate associations between genetic variants of 165 TLR signaling pathway genes and NSCLC OS using the genome-wide association study (GWAS) dataset from the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO). The results were further validated by the Harvard Lung Cancer Susceptibility GWAS dataset. Specifically, we identified IRAK2 rs779901 C > T as a predictor of NSCLC OS, with a variant-allele (T) attributed hazards ratio (HR) of 0.78 [95% confidence interval (CI) = 0.67-0.91, P = 0.001] in the PLCO dataset, 0.84 (0.72-0.98, 0.031) in the Harvard dataset, and 0.81 (0.73-0.90, 1.08x10-4 ) in the meta-analysis of these two GWAS datasets. In addition, the T allele was significantly associated with an increased mRNA expression level of IRAK2. Our findings suggest that IRAK2 rs779901 C > T may be a promising prognostic biomarker for NSCLC OS.


Diagnostic Utility of TLE1 (Transducer-Like Enhancer of Split 1) in Distinguishing Synovial Sarcoma from Mimicking Tumors.
Synovial sarcoma (SS) is a high-grade spindle cell tumor that accounts for 5% to 10% of soft tissue sarcomas. The majority originate from the deep intramuscular soft tissues of extremities with common sites including knee, ankle and feet. Immunohistochemical (IHC) stain TLE1 (transducer-like enhancer of split 1) is a potent diagnostic marker for distinguishing SS from mimicking tumors. The study was performed on 177 tumor cases, including 89 SS and 88 non-synovial sarcoma (N-SS) cases which were diagnosed at Department of Pathology and Laboratory Medicine, Aga Khan University Hospital, from July 2019 to June 2020. Hematoxylin and eosin (H&E) and IHC stained glass slides of these cases were reviewed. TLE1 expression was assessed based on the Remmele scoring system. Eighty-nine cases of SS and 88 cases of N-SS were included in the study. SS cases included 42 (47.2%) monophasic subtype, 6 (6.7%) biphasic subtype and 41 (46.1%) poorly differentiated subtype. Major tumor types in N-SS cases were 27 (30.7%) Ewing sarcoma (ES), 13 (14.8%) leiomyosarcoma, 10 (11.4%) undifferentiated sarcoma (US), 8 (9.1%) fibrosarcomatous dermatofibrosarcoma protuberans and 7 (8%) malignant peripheral nerve sheath tumor cases. Mean patients' age for SS cases was 26.14 years and for N-SS cases was 32.64 years. All 89 SS cases showed positive TLE1 expression. Out of 88 N-SS cases, 71 (80.7%) were TLE1 negative and 17 (19.3%) showed positive expression. This study shows that TLE1 is a very sensitive and relatively specific IHC marker for SS. TLE1 expression can be observed in other soft tissue sarcomas but diffuse strong TLE1 expression is highly specific for SS. The diagnosis should not solely rely on TLE1 expression and morphologic features but should include soft tissue specific lineage markers to avoid misdiagnosis.


Examination of a mutable system affecting the components of the segregation distorter meiotic drive system of Drosophila melanogaster.
Segregation distortion in Drosophila melanogaster is the result of an interaction between the genetic elements Sd, a Rsp sensitive to Sd, and an array of modifiers, that results in the death of sperm carrying Rsp. A stock (designated M-5; cn bw) has been constructed which has the property of inducing the partial loss of sensitivity from previously sensitive cn bw chromosomes, the partial loss of distorting ability from SD chromosomes, and a concomitant acquisition of modifiers on the X chromosome and possibly also on the autosomes. By several criteria the changes exhibited under the influence of M-5; cn bw are characteristic of the transposable-element systems which produce hybrid dysgenesis. In the first place, the magnitude of these effects depends on the nature of the crosses performed. The analogy is further strengthened by the observation that the changes induced by M-5; cn bw share other stigmata of Drosophila transposable-element systems, including high sterility among the progeny of outcrosses, and the production of chromosomal rearrangements. The possible relationship of this system to the P, I and hobo transposable element systems is discussed, as well as its bearing on aspects of the Segregation Distorter phenomenon which have yet to be explained.


Mortality risk of black women and white women with invasive breast cancer by hormone receptors, HER2, and p53 status.
Black women are more likely than white women to have an aggressive subtype of breast cancer that is associated with higher mortality and this may contribute to the observed black-white difference in mortality. However, few studies have investigated the black-white disparity in mortality risk stratified by breast cancer subtype, defined by estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) status. Furthermore, it is not known whether additional consideration of p53 protein status influences black-white differences in mortality risk observed when considering subtypes defined by ER, PR and HER2 status. Four biomarkers were assessed by immunohistochemistry in paraffin-embedded breast tumor tissue from 1,204 (523 black, 681 white) women with invasive breast cancer, aged 35-64 years at diagnosis, who accrued a median of 10 years' follow-up. Multivariable Cox proportional hazards regression models were fit to assess subtype-specific black-white differences in mortality risk. No black-white differences in mortality risk were observed for women with triple negative (ER-negative [ER-], PR-, and HER2-) subtype. However, older (50-64 years) black women had greater overall mortality risk than older white women if they had been diagnosed with luminal A (ER-positive [ER+] or PR+ plus HER2-) breast cancer (all-cause hazard ratio, HR, 1.88; 95% confidence interval, CI, 1.18 to 2.99; breast cancer-specific HR, 1.51; 95% CI, 0.83 to 2.74). This black-white difference among older women was further confined to those with luminal A/p53- tumors (all-cause HR, 2.22; 95% CI, 1.30 to 3.79; breast cancer-specific HR, 1.89; 95% CI, 0.93 to 3.86). Tests for homogeneity of race-specific HRs comparing luminal A to triple negative subtype and luminal A/p53- to luminal A/p53+ subtype did not achieve statistical significance, although statistical power was limited. Our findings suggest that the subtype-specific black-white difference in mortality risk occurs mainly among older women diagnosed with luminal A/p53- breast cancer, which is most likely treatable. These results further suggest that factors other than subtype may be relatively more important in explaining the increased mortality risk seen in older black women.


Metabolomic analysis of fibrotic mice combined with public RNA-Seq human lung data reveal potential diagnostic biomarker candidates for lung fibrosis.
Idiopathic pulmonary fibrosis (IPF) is a severe lung disease with poor survival that warrants early and precise diagnosis for timely therapeutic intervention. Despite accumulating genomic, transcriptomic, proteomic, and lipidomic data on IPF, evidence from water-soluble metabolomics is limited. To identify biomarkers for IPF from water-soluble metabolomic data, we measured the levels of various metabolites in bronchoalveolar lavage fluid (BALF) and serum samples from a bleomycin-induced murine pulmonary fibrotic model using gas chromatography/mass spectrometry. Thirty-two of 73 BALF metabolites and 29 of 74 serum metabolites were annotated. We observed that the levels of proline and methionine were higher in BALF but lower in serum than those in the control. Furthermore, analysis of public RNA-Seq data from the lungs of patients with IPF revealed that proline- and methionine-related genes were significantly upregulated compared to those in the lungs of healthy controls. These results suggest that proline and methionine may be potential biomarkers for IPF and may help to deepen our understanding of the pathophysiology of the disease. Based on our results, we propose a model capable of recapitulating the proline and methionine metabolism of fibrotic lungs, thereby providing better means for studying the disease and developing novel therapeutic strategies for IPF.


Diagnosis of Shashi-Pena Syndrome Caused by Chromosomal Rearrangement Using Nanopore Sequencing.
The aim of this study was to uncover the genetic cause of delayed psychomotor development and variable intellectual disability in a proband whose previous genetic analyses, including chromosome microarray and whole exome sequencing, had been negative. Long-read sequencing Oxford Nanopore Technology and RNA-seq analysis were performed on peripheral blood mononuclear cells. Genes with a fold change ≥ 1.5 and p ≤ 0.05 were identified as differentially expressed. Clinical examinations showed that the proband's features were similar to a rare autosomal-dominant neurodevelopmental syndrome, Shashi-Pena syndrome (MIM #617190). Karyotyping showed that a chromosomal balanced translocation t(2; 11) (p23; q23) was detected in the proband, her father, and her grandmother. Meanwhile, long-read sequencing identified 102 balanced translocations and 145 inversions affecting ASXL2 at an average of 15×. Combined with the family's RNA-seq results, the average mRNA expression of ASXL2 decreased in the patients. We identified a complex chromosomal rearrangement affecting ASXL2 as a pathogenic mechanism of Shashi-Pena syndrome in a Chinese family. This case study suggests that nanopore sequencing is suitable for pathogenic analysis of complex rearrangements, providing new avenues for the diagnosis of genetic diseases.


A sclerite-bearing stem group entoproct from the early Cambrian and its implications.
The Lophotrochozoa includes disparate tentacle-bearing sessile protostome animals, which apparently appeared in the Cambrian explosion, but lack an uncontested fossil record. Here we describe abundant well preserved material of Cotyledion tylodes Luo et Hu, 1999, from the Cambrian (Series 2) Chengjiang deposits, reinterpreted here as a stem-group entoproct. The entoproct affinity is supported by the sessile body plan and interior soft anatomy. The body consists of an upper calyx and a lower elongate stalk with a distal holdfast. The soft anatomy includes a U-shaped gut with a mouth and aboral anus ringed by retractable marginal tentacles. Cotyledion differs from extant entoprocts in being larger, and having the calyx and the stalk covered by numerous loosely-spaced external sclerites. The description of entoprocts from the Chengjiang biota traces the ancestry of yet another lophotrochozoan phylum back to the Cambrian radiation, and has important implications for the earliest evolution of lophotrochozoans.


Lymphoepithelioma-like carcinoma of the urinary bladder: a clinicopathologic study of 13 cases.
Lymphoepithelioma-like carcinoma (LELCA) of the urinary bladder is a rare variant of bladder cancer characterized by a malignant epithelial component densely infiltrated by lymphoid cells. It is characterized by indistinct cytoplasmic borders and a syncytial growth pattern. These neoplasms deserve recognition and attention, chiefly because they may be responsive to chemotherapy. We report on the clinicopathologic features of 13 cases of LELCA recorded since 1981. The chief complaint in all 13 patients was hematuria. Their ages ranged from 58 years to 82 years. All tumors were muscle invasive. A significant lymphocytic reaction was present in all of these tumors. There were three pure LELCA and six predominant LELCA with a concurrent transitional cell carcinoma (TCC). The remainder four cases had a focal LELCA component admixed with TCC. Immunohistochemistry showed LELCA to be reactive against epithelial membrane antigen and several cytokeratins (CKs; AE1/AE3, AE1, AE3, CK7, and CK8). CK20 and CD44v6 stained focally. The lymphocytic component was composed of a mixture of T and B cells intermingled with some dendritic cells and histiocytes. Latent membrane protein 1 (LMP1) immunostaining and in situ hybridization for Epstein-Barr virus were negative in all 13 cases. DNA ploidy of these tumors gave DNA histograms with diploid peaks (n=7) or non-diploid peaks (aneuploid or tetraploid; n=6). All patients with pure and 66% with predominant LELCA were alive, while all patients having focal LELCA died of disease. Our data suggest that pure and predominant LELCA of the bladder appear to be morphologically and clinically different from other bladder (undifferentiated and poorly differentiated conventional TCC) carcinomas and should be recognized as separate clinicopathological variants of TCC with heavy lymphocytic reaction relevant in patient management.


Lead Discovery of SARS-CoV-2 Main Protease Inhibitors through Covalent Docking-Based Virtual Screening.
During almost all 2020, coronavirus disease 2019 (COVID-19) pandemic has constituted the major risk for the worldwide health and economy, propelling unprecedented efforts to discover drugs for its prevention and cure. At the end of the year, these efforts have culminated with the approval of vaccines by the American Food and Drug Administration (FDA) and the European Medicines Agency (EMA) giving new hope for the future. On the other hand, clinical data underscore the urgent need for effective drugs to treat COVID-19 patients. In this work, we embarked on a virtual screening campaign against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Mpro chymotrypsin-like cysteine protease employing our in-house database of peptide and non-peptide ligands characterized by different types of warheads acting as Michael acceptors. To this end, we employed the AutoDock4 docking software customized to predict the formation of a covalent adduct with the target protein. In vitro verification of the inhibition properties of the most promising candidates allowed us to identify two new lead inhibitors that will deserve further optimization. From the computational point of view, this work demonstrates the predictive power of AutoDock4 and suggests its application for the in silico screening of large chemical libraries of potential covalent binders against the SARS-CoV-2 Mpro enzyme.


A two-step synthesis of α-keto vinyl carbinols from ketones.
Conjugate addition of lithium enolates onto terminal alkynyl- and allenyl-sulfoxides furnishes the corresponding allylic sulfoxides. The latter readily undergo a Mislow-Braverman-Evans rearrangement to yield the targeted α-keto vinyl carbinols. This two-step procedure does not require purification of the intermediates and constitutes the shortest approach to α-keto vinyl carbinols.


"I´m the one who has written this": reciprocity in writing courses for older adults in Norway.
Purpose: The aim of this article is to explore, theoretically and empirically, the reciprocity of care afforded by writing courses as community interventions for older adults. Methods: We narratively analyzed 209 excerpts of the anthology "I´m the one who has written this" written by teachers and participants of courses organized by the Church City Mission in Norway. Results: The reciprocity that appeared in the writing courses is grounded in the sense of vulnerability that both teachers and participants embraced, and that is experienced in three main relational movements that these writing courses convey: self-exploration, otherness and togetherness. In addition, the data suggests that these courses promote affective processing and existential meaning-making, motivation, as well as improvements of memory and attention. However, more research is needed to confirm these preliminary findings, and their possible effects in older adults with and without symptoms of dementia. Conclusion: Even though these writing courses for older adults are not explicitly therapeutic, they can have therapeutic effects, given the reciprocity afforded in these cultural community interventions. A theoretical exploration upon reciprocity in eldercare is hereby provided. These findings could shape improvements in aging and health care policies that are person-centered and focus on reciprocity.


Quantification of Macular Microvascular Changes in Retinitis Pigmentosa Using Optical Coherence Tomography Angiography.
To investigate macular structural and microvascular changes in retinitis pigmentosa (RP) eyes compared to age-matched controls using spectral domain optical coherence tomography (SD-OCT) and OCT angiography (OCTA). This was a cross-sectional study. The foveal and parafoveal thickness and extent of ellipsoid zone (EZ) disruption were measured on OCT. The foveal avascular zone (FAZ) area and retinal vessel density (VD) were automatically calculated for superficial (SCP) and deep capillary plexuses (DCP) and choriocapillaris using OCTA-integrated software. VD was assessed for the whole 6 × 6 mm image, foveal and parafoveal regions. Quantitative measures were compared between healthy and diseased eyes. The study included 30 eyes of 30 RP patients and 24 eyes of 24 controls. Mean age was 26.9±6.4 years for RP patients and 27.4±4.9 years for controls (p=0.76). Mean LogMAR visual acuity (VA) was 0.9±0.5 for RP patients and 0.05±0.05 for controls (p <0.001). Foveal and parafoveal thicknesses were significantly lower in RP cases as compared to the control group. EZ disruption was observed in RP cases only (869±211 µm). Mean FAZ area was significantly larger in RP eyes in both SCP and DCP. VD was significantly reduced in RP eyes at the level of SCP, DCP and choriocapillaris. VA, deep parafoveal VD, foveal, parafoveal and whole image choriocapillaris VD were negatively correlated with the extent of EZ disruption. We report OCTA findings in a relatively young cohort of RP patients. We demonstrated a reduction of retinal microvascular density in all studied layers on OCTA. We believe studying retinal vasculature in these patients is important, as a healthy blood supply is a prerequisite for the success of new cell-based therapies under trial for RP.