text
stringlengths 4
2.52M
| meta
dict |
|---|---|
During the last decade, we have experienced a difficult period with over 200 failed clinical trials and still no cure for Alzheimer's disease (AD). The scientific community has been critical regarding this outcome and a few potential explanations have been discussed. Firstly, the recruited populations have often been heterogeneous. Secondly, patients were demented and although mildly to moderately, most presented with irreversible brain damage, thus jeopardizing the potential effect of the drugs. Thirdly, we may have been aiming at the wrong targets, or at least we may have tried to simplify a multifactorial disease that may never be defeated with a magic bullet.
We need to review the evidence indicating both clinical and pathophysiological heterogeneity in AD. For instance, tangle pathology has long been thought to spread across the brain in a typical manner, initiating in the transentorhinal cortex, then spreading to the entorhinal and hippocampal areas, and finally extending to the lateral association cortex. This pattern would mostly underlie the typical amnestic presentation of AD. However, other subtypes have also been described histopathologically in which tau mainly affects the hippocampus (limbic-predominant AD) or predominantly occupies the association cortex (hippocampal-sparing AD) \[[@r1]\]. In this regard, the hippocampal-sparing subtype is more frequently related to non-amnestic presentations of AD, as well as to non-AD clinical diagnoses \[[@r1],[@r2]\]. This differential spread of tangle pathology can be reliably tracked in vivo with the help of magnetic resonance imaging (MRI) \[[@r2]\]. Recent studies have also identified a fourth AD subtype that displays minimal brain atrophy with similar disease severity as compared to the other subtypes \[[@r3]\].
In addition, postmortem data clearly shows that AD pathology rarely occurs in isolation \[[@r4]\]. Most AD patients harbor more than one pathology in the brain, with cerebrovascular disease being the most common coexisting pathology. Furthermore, the frequency of both cerebrovascular and Alzheimer's disease increases with age. However, in what way cerebrovascular disease and AD pathology act in synergy leading to downstream neurodegeneration and dementia is still unknown. Cerebral amyloid angiopathy (CAA), a form of cerebrovascular disease resulting from amyloid deposition in vessel walls, may be the link between these two frequently coexisting pathologies. It is interesting that anti-amyloid therapy has been reported to increase the incidence of microbleeds, potentially due to removal of amyloid through vessel walls. The big question is whether CAA is just a passenger on the AD train. How does CAA interact with amyloid and tau pathology? For instance, does CAA come in early on in disease pathogenesis by affecting the spread of neurofibrillary tangles across the brain? Or is CAA an event occurring in later stages, acting downstream to amyloid and tau pathology thus mostly contributing to neurodegeneration and brain atrophy? All these questions remain largely unanswered.
We recently conducted a comprehensive characterization of these AD subtypes in terms of cerebrovascular disease, including the amount and distribution of deep/lobar microbleeds and white matter hyperintensities, cortical superficial siderosis, perivascular spaces, lacunes, large brain infarction, and intracerebral hemorrhage \[[@r5]\]. We concluded that CAA seems to make a stronger contribution to hippocampal-sparing and minimal atrophy AD, whereas hypertensive arteriopathy, another form of cerebrovascular disease, may make a stronger contribution to typical and limbic-predominant AD. This study also revealed important mechanisms for minimal atrophy AD patients who harbor pathological levels of amyloid, tau, and neurodegeneration biomarkers in their cerebrospinal fluid, but who do not display overt brain atrophy. We speculate that due to low cognitive reserve in this subtype, neurodegeneration at the molecular level (i.e. cerebrospinal fluid) is sufficient to produce clinical symptoms of AD in the absence of neurodegeneration at the macrostructural level (i.e. MRI). Indeed, the finding of low cognitive reserve in minimal atrophy AD has been observed in several independent cohorts \[[@r5],[@r6]\]. Since we found that increased CAA is associated with lower cognitive performance in minimal atrophy AD \[[@r5]\], CAA in this subtype could contribute to lowering the threshold for the amount of AD pathology needed to produce cognitive impairment and dementia. Nonetheless, AD patients with minimal atrophy have progressed enough to display similar clinical severity compared to the other three subtypes. Of relevance, minimal atrophy AD patients are rather comparable in disease duration but they have slower cognitive decline over time \[[@r3]\]. These findings, together with the fact that they are usually younger, make minimal atrophy AD a good candidate for future intervention studies. Lower cognitive reserve leaves room for building up late-life cognitive resilience by increasing engagement in cognitive activities and exposure to leisure or physical activities. From a pharmacological perspective, it would be interesting to investigate how well the minimal atrophy subtype responds to acetylcholinesterase inhibitors. Previous studies have shown that patients with preserved medial temporal lobes respond better to that treatment \[[@r7]\]. The [Figure 1](#f1){ref-type="fig"} below shows how CAA and cognitive reserve may hypothetically modulate disease progression, which is thought to be primarily caused by the accumulative onslaught of amyloid, tau, and neurodegenerative pathology.
{#f1}
Evidence suggests that neurodegeneration can be expressed differently across different AD subtypes. Future research will also have to answer why amyloid pathology starts, what is triggering the cascade, and whether this differs in the different subtypes. Current data shows that dementia in AD is a downstream event that can be reached along different pathways. These different pathways may necessitate their own specific therapeutic strategies. The differences in biological factors (and modifiable life factors) between these AD subtypes may guide the potential targets for future trials. Recognizing the heterogeneity within AD implies opening the door to multifactorial intervention strategies. We believe that unraveling the heterogeneity within AD can promote personalized medicine approaches in the short term by guiding tailored cognitive interventions, and help in characterizing more homogeneous AD groups for drug discovery in the future. This may facilitate the tough endeavor of finding a cure for this disease.
**Funding:** This work was supported by the Swedish Foundation for Strategic Research (SSF), the Strategic Research Programme in Neuroscience at Karolinska Institutet (StratNeuro), the Swedish Research Council (VR), the Åke Wiberg foundation, Hjärnfonden, Alzheimerfonden, Demensfonden, Stiftelsen Olle Engkvist Byggmästare, and Birgitta och StenWesterberg
|
{
"pile_set_name": "PubMed Central"
}
|
Background {#Sec1}
==========
Acute kidney injury (AKI) is a common clinical complication among patients admitted to the intensive care unit (ICU). The incidence of AKI in the hospital and the ICU has been reported as 20--45% \[[@CR1], [@CR2]\]. AKI is an independent risk factor for morbidity and death among patients in the ICU and hospital \[[@CR3], [@CR4]\]. Increased morbidity and mortality rates due to AKI correlate with the severity of renal dysfunction \[[@CR5]--[@CR7]\]. AKI is also an independent risk factor for chronic kidney disease, end-stage renal disease, and increased hospital cost \[[@CR8]--[@CR13]\]. Growing evidence shows that kidney congestion is associated with higher risk of AKI in critically ill patients who have acutely decompensated heart failure or sepsis \[[@CR14]\]. Intra-abdominal hypertension, increased central venous pressure (CVP), and kidney mean perfusion pressure deficit have been used to indirectly predict kidney congestion and risk of AKI \[[@CR15]\].
Compartment syndrome, as a risk factor for AKI, is characterized by increased pressure within a confined body space, with potential to compromise its microcirculation. This pathophysiologic situation most commonly occurs in fascial compartments of the limbs and the abdomen \[[@CR16], [@CR17]\]. Depending on its extent and duration, intracompartmental hypertension can lead to impaired blood supply, neurologic deficit, and organ or tissue damage.
Intra-abdominal pressure is the steady-state pressure concealed within the abdominal cavity \[[@CR17], [@CR18]\]. Intra-abdominal pressure that exceeds 12 mm Hg in critically ill patients is considered a risk factor for AKI \[[@CR18]--[@CR25]\]. Among patients with acutely decompensated heart failure, intra-abdominal pressure of 8 mm Hg has been associated with AKI \[[@CR26], [@CR27]\]. The pathophysiologic relationship between intra-abdominal hypertension and AKI is explained by a decrease in the filtration gradient, which is the pressure difference across the glomerular basement membrane that drives filtration \[[@CR23]\].
To our knowledge, no noninvasive technology is available currently to measure kidney intracapsular pressure (KIP) in a clinical setting. Such a device potentially would assist clinicians to identify patients at risk for AKI from intraperitoneal hypertension and thus aid clinicians in the treatment of critically ill patients. Herein, we present results of a pilot animal study investigating the performance of a novel noninvasive tool that estimates KIP by measuring kidney elasticity. We hypothesize that KIP estimated by ultrasound surface wave elastography (USWE) would strongly correlate with directly measured KIP under different physiologic and pathologic states.
Methods {#Sec2}
=======
Experimental outline {#Sec3}
--------------------
Three healthy female pigs weighing 30--40 kg (from Manthei Hog Farm, Elk River, MN, USA) were purchased for use in this study. All animal husbandry and procedures were performed in accordance with the guidelines of the Mayo Clinic Institutional Animal Care and Use Committee (IACUC). The IACUC reviewed and approved this project (No. 130114).
All animals were sedated with short-acting injectable anesthetic (tiletamine/zolazepam 5 mg/kg) 60 minutes before the interventions. The animals were positioned supine on the operating table, and femoral arterial and central venous lines were placed under ultrasonography guidance for both monitoring and medication administration. The animals underwent endotracheal intubation and urinary indwelling catheterization. Next, they underwent bilateral flank incisions. The dissection was performed through the skin and subcutaneous tissue, avoiding injury to the peritoneum. A 22-gauge angio-catheter was positioned in each renal subcapsule under ultrasonography guidance and connected to pressure transducers. These catheters were flushed patent and without kinking. The skin was closed around the catheters with a subcuticular stitch. Bilateral KIP and bladder pressure, arterial pressure, and CVP were continuously monitored throughout each testing condition.
For evaluation of the correlation between the directly measured KIP and elastography in different physiologic and pathophysiologic states, the animals underwent individual study interventions, and pressures were documented. The interventions were chosen on the basis of their theoretical association with change in kidney hemodynamics and, therefore, in KIP. The KIP and elasticity were measured at baseline and after each individual intervention. A 20-minute period was given between each intervention to allow kidney hemodynamics to return to baseline. Table [1](#Tab1){ref-type="table"} summarizes the studied mechanical and pharmacologic interventions.Table 1Summary of pharmacologic and surgical interventionsInterventionDoseRationaleExpected change in KIPBaselineReference↔Perfusion with normal saline30 mL/kgKidney congestion↑Vasopressin infusion0.05 U/minKidney vasoconstriction↓Norepinephrine infusion0.1 mcg/kg per minKidney vasoconstriction↓Dopamine infusion2 mcg/kg per minKidney vasodilation↑Fenoldopam infusion0.1 mcg/kg per minKidney vasodilation↑PD^a^ fluid installation2 LIntra-abdominal HTN↑Ureter occlusionObstruction↑Renal vein occlusionKidney congestion↑Renal artery occlusionKidney ischemia↓*Abbreviations*: *HTN* hypertension, *KIP* kidney intracapsular pressure, *PD* peritoneal dialysis^a^PD0 is immediately after installation of PD fluid in the abdominal cavity; PD1 and PD2 are measurements recorded after 30 and 60 minutes, respectively, of PD fluid installation in the abdominal cavity
Following completion of all pharmacologic interventions, a peritoneal dialysis (PD) catheter was inserted in the low midline position. The fascia was closely approximated around the PD catheter to minimize variations in peritoneal pressure. Following PD catheter insertion, 2 L of peritoneal dialysate was instilled into the abdomen to simulate abdominal compartment syndrome. Intraperitoneal pressure measurements were repeated at installation and at 30 and 60 minutes after installation. Animals then underwent a midline laparotomy. The renal pedicles were exposed, and sequentially the ureter, renal vein, and renal artery were occluded with occlusive vascular clamps. Each occlusion step was sustained for 20 minutes. Following each step, direct renal subcapsular pressures were compared with USWE measurements. All animals were sacrificed at the conclusion of the acute study.
Principles of USWE {#Sec4}
------------------
Ultrasonography examination of the kidney can provide significant noninvasive information on the kidney and volume status of the patients who suffer from AKI \[[@CR28]\]. Knowing the size, echogenicity, and shape of the kidney or Doppler examination of renal artery flow and measurement of the resistive index (including intraparenchymal renal resistive index variation) can provide important information on the etiology of AKI \[[@CR29]--[@CR31]\]. USWE is a novel and noninvasive technique \[[@CR32], [@CR33]\] for measuring the viscoelastic properties of tissue. A small hand-held probe is used to produce local harmonic vibrations of the target tissue to generate propagation of transversal or shear waves. The shear wave speed is measured with an ultrasound beam that is generated by a transducer (Fig. [1](#Fig1){ref-type="fig"}). The surface wave examines the skin while the shear wave in the tissue layer examines the deep tissue. The measurements of wave speed and wave attenuation enable calculation of viscoelastic properties. With the use of 6.5-MHz probes, the tool is able to measure shear wave speeds with the depth of 45 mm. Using lower frequency probes (i.e., 5 MHz) has the potential to assess shear wave speeds in deeper tissues. A notable advantage of USWE is its ability to measure tissue viscosity and elasticity with high accuracy and precision; both properties may be associated with disease susceptibility \[[@CR34]\]. In addition, deep tissue layers can be analyzed to learn the contribution of various tissue types to disease manifestation. Further information on the USWE is provided in the [Appendix](#Sec9){ref-type="sec"}.Fig. 1Ultrasound surface wave elastography mechanics. A vibration is generated by a shaker on the skin. The surface and shear waves can be measured with the ultrasound probe
Scarring and edema can change the elasticity of each individual tissue. For the organs that reside close to the surface, palpation is one of the most common physical examination tools used to evaluate the tissue elasticity. For the tissues that are located internally, use of USWE has been proposed in the recent literature \[[@CR35]\]. Localized (i.e., neoplasm, infection, embolism, or infarction, or a combination) or diffuse (e.g., edema, inflammation, congestion, fibrosis) changes in elasticity can be detected in the kidney by elastography.
Statistical analysis {#Sec5}
--------------------
Categorical data were summarized as number and percentage; continuous variables as mean (SD) or median (IQR), as appropriate. Measurements were divided into a binary variable based on association with intra-abdominal hypertension simulated by the installation of PD fluids in the animal abdominal cavities (i.e., measurements with peritoneal fluid classified as the KIP hypertensive group vs. those without peritoneal fluid classified as the KIP normotensive group). We used linear regression models to evaluate the correlation between wave speed and KIP after adjustments for other hemodynamic variables and the binary variable of intra-abdominal hypertension.
Results {#Sec6}
=======
The study involved three pigs (six unique kidneys). Median and interquartile range (IQR) of monitored pressures were systolic blood pressure, 94 (88--100) mm Hg; diastolic blood pressure, 53 (50--59) mm Hg; KIP, 7 (7--10) mm Hg; bladder pressure, 7 (6--11) mm Hg; and intraperitoneal pressure, 7 (6--11) mm Hg. Median (IQR) of heart rate, temperature, and USWE wave speed were 116 (112--144) beats per minute, 36.7 °C (35.8--37 °C), and 2.01 (1.77--2.31) m/s, respectively. In order to acquire kidney images for this study, the average depth of the ultrasound (US) images was 40 mm, and the kidney surfaces were located at a depth of 25 mm. Figure [2](#Fig2){ref-type="fig"} shows all USWE wave speed measurements in the six kidneys.Fig. 2Wave speed changes of each pig, based on the intervention. PD0 is immediately after installation of peritoneal dialysis fluid in the abdominal cavity; PD30 and PD60 are measurements that are recorded after 30 and 60 minutes of peritoneal dialysis fluid installation in the abdominal cavity, respectively. AC, renal artery occlusion; BL, baseline; DP, dopamine infusion; FD, fenoldopam infusion; NE, norepinephrine infusion; PD, peritoneal dialysis; SL, saline infusion; UC, ureter occlusion; VC, renal vein occlusion; VS, vasopressin infusion
The mean (95% CI) difference between KIP and the intraperitoneal pressure was not significant (0.39 (− 0.9 to 0.2) mm Hg, *P* = .20). However, the KIP and intraperitoneal pressure differed significantly from bladder pressure (mean (95% CI) difference of bladder pressure vs. KIP was 3.8 (2.9--4.7) mm Hg, *P* \< .001; mean (95% C) difference of bladder pressure vs. intraperitoneal pressure was 3.4 (2.3--4.7) mm Hg, *P* \< .001).
KIP was significantly greater during the peritoneal dialysis (PD) fluid installation phase when it was compared with the other interventions (mean (95% CI), 3.7 (3.2--4.2) mm Hg; *P* \< .001). In the linear regression model, after adjustment for KIP hypertension, there was significant correlation between USWE wave speed and KIP (adjusted coefficient of determination (*r* ^*2*^) = 0.71; *P* \< .001). The estimate (95% CI) of the USWE wave speed for prediction of KIP stayed significant after adjustment for KIP hypertension (− 0.8 (− 1.4 to − 0.2) m/s; *P* = .008); systolic and diastolic blood pressure were not significant predictors of KIP. The variance inflation factor of the model was 1.01, which indicates no collinearity among variables in the model. In similar models for prediction of peritoneal and bladder pressures using USWE wave speed and after adjustment for KIP hypertension, *r* ^*2*^ was 0.72 (*P* \< .001) and 0.19 (*P* = .002), respectively. Figure [3](#Fig3){ref-type="fig"} shows the changes in KIP and USWE wave speed in each kidney included in the experiment.Fig. 3Overlay plots show correlation between wave speed and directly measured kidney intracapsular pressure (KIP). PD0 is immediately after installation of peritoneal dialysis fluid in the abdominal cavity; PD30 and PD60 are measurements that are recorded after 30 and 60 minutes of peritoneal dialysis fluid installation in the abdominal cavity, respectively. AC, renal artery occlusion; BL, baseline; DP, dopamine infusion; FD, fenoldopam infusion; NE, norepinephrine infusion; PD, peritoneal dialysis; SL, saline infusion; UC, ureter occlusion; VC, renal vein occlusion; VS, vasopressin infusion.
Discussion {#Sec7}
==========
In this pilot interventional swine model experiment, we identified significant correlation between USWE and KIP (*r* ^*2*^ = 0.71). Interestingly, although we found no significant difference between KIP and intraperitoneal pressure, bladder pressure was significantly greater than both KIP and peritoneal pressure measurements. Indeed, the *r* ^*2*^ for correlation between USWE and bladder pressure was lower (*r* ^*2*^ = 0.19) than the correlation of USWE wave speed and KIP (*r* ^*2*^ = 0.71) or peritoneal pressures (*r* ^*2*^ = 0.72). Apart from the installation of PD fluid in the abdominal cavity, which was associated with a significant increase in both KIP and intra-abdominal pressure, no other intervention significantly changed KIP or abdominal pressure.
The body of evidence is growing that increased KIP is associated with higher risk of AKI. Increased KIP could be caused by congestion or intra-abdominal hypertension. Rising KIP mitigates the differences between the glomerular filtration pressure and the proximal tubular pressure---filtration gradient. This effect, in turn, decreases the glomerular filtration rate (GFR) and therefore worsens kidney function \[[@CR23]\]. In addition, a significant increase in KIP would result in reduced renal blood flow and hence lower GFR \[[@CR36]\]. Yet, lymphatic drainage of the kidney interstitium is jeopardized in the clinical setting of KIP hypertension \[[@CR37]\].
In practice, the relationship between kidney congestion and AKI was described by Winton \[[@CR38]\] in a classic study in 1935. Winton reported decreasing urine production when CVP dramatically increased in a dog model. In a more recent study, the risk of AKI rose by 2% for each 1 cm H~2~O of increased CVP \[[@CR39]\]. Among patients with sepsis, increasing CVP was linearly associated with AKI \[[@CR40]\]. The potential importance of increased CVP has also been suggested among patients with acute decompensated heart failure, among whom CVP greater than 8 mm Hg is associated with worsening renal function, whereas interventions to decrease CVP could increase the chances of kidney recovery \[[@CR26], [@CR27]\]. These examples of congestive nephropathy are commonly seen among patients who undergo aggressive fluid resuscitation or have worsening heart failure and could become worse from progressive fluid overload \[[@CR41]\]. Currently, apart from using CVP as a surrogate for kidney congestion, no other test or tool allows clinicians to estimate KIP and identify patients at risk for congestive nephropathy.
The literature on the relationship between intra-abdominal hypertension and AKI is extensive. Sugrue et al. \[[@CR19]\] reported on 88 patients post laparotomy with complete intra-abdominal pressure monitoring through bladder pressure. The odds ratio (95% CI) of AKI among those with intra-abdominal pressure exceeding 20 mm Hg was 12.4 (3.8--41.7). In a follow-up study of post-emergent surgical patients, the same authors \[[@CR20]\] noted that the incidence of AKI was significantly associated with sepsis, age greater than 60 years, and intra-abdominal pressure greater than 18 mm Hg. Hering et al. \[[@CR42]\] demonstrated that mean (SD) intra-abdominal pressure in 16 mechanically ventilated patients increased from 12 (5) mm Hg to 14 (5) mm Hg (*P* \< .05) with prone positioning. In this small cohort, the authors reported a significant decrease in the renal fraction of cardiac output and renal vascular resistance index. However, they were not able to show any changes in effective renal blood flow, GFR, urine volume, and fractional excretion of sodium.
The role of intra-abdominal hypertension is well-recognized in several types of organ failure (i.e., splanchnic, respiratory, cardiovascular, and neurologic function) in addition to the kidney \[[@CR23]\]. International conference of experts on intra-abdominal hypertension and abdominal compartment syndrome recommended monitoring intra-abdominal pressure directly (using needle puncture) or indirectly (using a balloon catheter in the bladder, stomach, rectum, inferior vena cava, or uterus) \[[@CR18]\]. The aforementioned methods are all invasive and mostly inaccurate.
Physical examination is known to have poor sensitivity (40%) and thus is inaccurate for detection of intra-abdominal hypertension \[[@CR43]\]. Currently, measuring intravesical (bladder) pressure is considered the gold standard for intra-abdominal pressure measurement and monitoring \[[@CR43]\]. Almost all studies for validation of bladder pressure were done with patients who were undergoing anesthesia and paralysis for laparoscopy, which may not be a real representative of critically ill patients. Regardless, the reports of correlation between pressures measured through an intraperitoneal catheter versus measurements of bladder pressure do not have consistently high accuracy or reliability \[[@CR44]--[@CR46]\]. Importantly, in our small pilot study with a swine model, we found that bladder pressure was significantly greater than intraperitoneal pressure and KIP.
Because no noninvasive tool is available that allows clinicians to measure KIP hypertension, there is a critical need for a noninvasive device to accurately and reliably measure kidney compartmental pressure. To evaluate the potential performance of noninvasive USWE for estimation of KIP, we conducted this pilot study and noted excellent correlation between USWE and KIP or peritoneal pressure. Conversely, bladder pressure was not a reliable indicator of either KIP or peritoneal pressure. Our results may indicate that USWE has a potential role for the detection of intra-abdominal hypertension or congestive nephropathy, particularly in the clinical setting of aggressive volume resuscitation or acute decompensated heart failure.
Unlike other available tools, USWE is noninvasive, thus it could be potentially studied at the bedside to evaluate the natural history of wave speed changes during volume resuscitation in septic patients or fluid removal in patients with acute exacerbation of heart failure. Thus, USWE could potentially improve both diagnosis and fluid management among critically ill patients.
This pilot study has several limitations. The sample size for this study was small. Therefore, our findings need to be confirmed in a larger number of animals or in clinical investigations. Although installation of PD fluid increased pressures as expected, predicted changes in KIP did not occur following other interventions (Table [1](#Tab1){ref-type="table"}). This could be due to suboptimal dosages of drugs or a short timeline within and between each intervention, or both, in this pilot study.
Conclusion {#Sec8}
==========
In this swine model pilot study, we found that USWE is able to estimate KIP and intraperitoneal pressures accurately. We also demonstrated that bladder pressure does not appear to be an accurate surrogate for KIP and intra-abdominal pressure. Our results need to be confirmed in larger studies and then validated in human clinical investigations.
Appendix {#Sec9}
========
The dynamics of ultrasound surface wave elastography
Detection of wave propagation in skin and underlying tissue is guided by ultrasonography \[[@CR47], [@CR48]\]. This image clearly shows the skin, subcutaneous fat layer, and abdominal muscles. Tissue motion at a given location can be analyzed by cross-correlation of the ultrasound tracking beam through each specific location. For example, several points in the first muscle layer (indicated by yellow dots) were selected with image guidance; tissue motion was then measured at these points in response to local vibration (*excitation*) of the skin. The vibration is typically a continuous wave of 0.1 to 0.2 s with a frequency of 100 to 300 Hz. A high frame rate (approximately 2000 frames/s) detects tissue motion in response to the excitation. Tissue velocity is less than 1 mm/s, but the response signal is clear. Wave speed is measured by determining the change in wave phase at the other locations, relative to the first location (mean (SD) wave speed, 2.05 (0.21) m/s). Different tissue layers can be analyzed using 1 ultrasound surface wave elastography (USWE) measurement because the instrument captures all ultrasound data from the full depth. Thus, the wave speed in the skin is determined by analyzing ultrasound data directly from the skin. Wave speed is measured by the ultrasound tracking beams, and the measurement consequently is local and independent of the excitation source.
Implementation of USWE {#Sec10}
----------------------
For the use of commercial US machines for USWE measures, there is no need to change the US hardware, although the software should be modified \[[@CR49]\]. Ultrasound data start being collected after a function generator (FG33120A) submits digital pulses. Then, vibration synchronized signals are amplified and submitted to a handheld shaker. The US probe measures the vibration propagation speed in the skin and deeper tissues.
Clinical applications {#Sec11}
---------------------
Clinical applications of the USWE technique have involved measurement of biomechanical properties of skin in 30 healthy volunteers and 4 patients with systemic scleroderma \[[@CR34]\]. Both elasticity and viscosity were significantly greater in the systemic scleroderma group than the healthy group. It is, therefore, easy to discriminate fibrotic skin from healthy skin using viscoelasticity measurements. Because accurate measures of skin fibrosis disorders continue to be challenging, objective assessment of skin fibrosis using the USWE technique is a potentially valuable clinical tool that has received a quick review from physicians \[[@CR50]\]. USWE has been used to evaluate human lung stiffness \[[@CR51]\], myocardial tissues \[[@CR51]\], abdominal wall tension \[[@CR52]\], and biomechanical properties in wound healing and scar formation \[[@CR53]\]. Noninvasive measurement of carpal tunnel pressure was reported recently \[[@CR51]\] with analysis of changes of wave speed in the tendon. It showed that wave speed in the tendon increases linearly with carpal tunnel pressure.
AKI
: Acute kidney injury
CVP
: Central venous pressure
GFR
: Glomerular filtration rate
IACUC
: Institutional Animal Care and Use Committee
ICU
: Intensive care unit
IQR
: Interquartile range
KIP
: Kidney intracapsular pressure
PD
: Peritoneal dialysis
US
: Ultrasound
USWE
: Ultrasound surface wave elastography
KK is the recipient of the Mayo Clinic Department of Medicine Write-up and Publish grant, which provided 5 business days for completion of this manuscript.
Funding {#FPar1}
=======
This study was done without any support from a commercial entity. The Division of Nephrology and Hypertension at Mayo Clinic, Rochester, MN, USA provided a small grant (less than US\$10,000) for the laboratory expenses.
Availability of data and materials {#FPar2}
==================================
The dataset used and/or analyzed during the current study are available from the corresponding author on reasonable request.
KK participated in all stages of this study from the conception of the idea to the study conduct, statistical analysis, and manuscript preparation. SM, BA, JG, and SN provided guidance in technical aspects of the study and performed surgical procedures. SS and JL participated in manuscript preparation and final review. XZ provided expertise in the USWE and participated in the execution of the study and preparation of this manuscript. All authors read and approved the final manuscript.
Ethics approval {#FPar3}
===============
This study was reviewed and approved by the Mayo Clinic Institutional Animal Care and Use Committee (IACUC No. 130114). Informed Consent was not applicable as there was no human subject enrolled in this study.
Consent for publication {#FPar4}
=======================
Not applicable.
Competing interests {#FPar5}
===================
While XZ holds a patent on the USWS technology, he declared that he did not have any financial or non-financial competing interest in this study. The other authors declare that they have no competing interests.
Publisher's Note {#FPar6}
================
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
|
{
"pile_set_name": "PubMed Central"
}
|
Background {#Sec1}
==========
Polyhydroxyalkanoates (PHAs) are a family of microbial, biobased and biodegradable polyesters representing an attractive ecofriendly alternative to some fossil-based polymers. They are biosynthesized by various types of microorganisms in conditions of excess of available carbon and of a limited supply of one of the nutrients essential to bacterial cell growth \[[@CR1]\]. The biopolymers are synthesized by polymerization of 3-hydroxy fatty acids (3-OH FA) by a PHA synthase (PhaC), inside the bacterial cell in the form of granules that serve as carbon and energy storage compounds \[[@CR2]\]. PHA polymers are categorized into subclasses according to the side chain of their monomers: in particular, short-chain-length PHAs (scl-PHA with three to five carbon monomers) and the medium-chain-length PHAs (mcl-PHA with six to fourteen carbon monomers). Copolymers of scl- and mcl-PHA, block copolymers PHA and homopolymers PHA are named based on the monomer arrangements in the polymer chains \[[@CR3]\]. The properties of these biopolymers depend on their molar masses and their macromolecular architectures (the side chains of their monomers), \[[@CR3], [@CR4]\]. For example, the well-studied scl-PHA, P3HB (poly-3-hydroxybutyrate), has poor mechanical properties. Indeed, the isotactic and linear chains of P3HB result in the formation of large spherulites during crystallization, which may lead to a highly crystalline (60%) and consequently a brittle material. Besides, P3HB presents a poor processability with thermal input due to a high thermal sensitivity at a temperature higher than the melting temperature, with strong chains scissions. P3HB and PHBV (poly(3-hydroxybutyrate-*co*-3-hydroxyvalerate)) with a low HV content have not been largely utilized in biomedical areas due to the stiff and brittle nature. On the other hand, mcl-PHA properties ranges from rubber to tacky polymers with higher thermal resistance \[[@CR5], [@CR6]\]. Diverse products can thus be obtained from PHA including bioplastics, chemicals and feed supplements \[[@CR7]\]. Besides, different applications in medical and pharmaceutical industries can be considered \[[@CR8], [@CR9]\]. The main drawback to PHA usage is its high cost of production compared to conventional fossil-based polymers. Nonetheless, advances in PHA research have shown tremendous progresses in (i) finding alternative carbon sources for PHA production (ii) controlling PHA processing during production, and (iii) controlling PHA composition and molar mass \[[@CR10]--[@CR12]\].
Medium-chain-length PHAs are made up of monomer units with 6--14 carbon atoms and were first identified in *Pseudomonas putida* GPo1 \[[@CR13]\]. mcl-PHA are interesting as they can bear different functional groups in the side chains that can modify the physico-chemical and physical properties, offering a wide range of applications \[[@CR3]\]. Depending of the monomer composition, mcl-PHA have been demonstrated to have the elastomeric properties required for specific applications such as packaging materials or biomedical applications \[[@CR3], [@CR14]\]. Nevertheless, inconsistencies on their molar masses and their structures lead to variations of their thermo-mechanical properties, which are a strong drawback for their uses. Some organisms are naturally capable of synthesizing mcl-PHA and have been engineered in order to enhance polymer production or to produce mcl-PHA of interest \[[@CR15]--[@CR17]\]. Non-natural mcl-PHA producers have also been engineered such as yeast \[[@CR18]--[@CR21]\], bacteria \[[@CR16], [@CR22]--[@CR24]\], and plants \[[@CR23], [@CR25]\].
*Yarrowia lipolytica* is a GRAS organism (Generally Regarded As Safe) with large potentials in industrial biotechnology \[[@CR26]\]. This yeast is naturally capable of producing and accumulating large amount of lipids (more than 50% of its dry weight in large-scale fermentations \[[@CR27]\]). In regard to such potential, this yeast is becoming an organism of choice for the production of many compounds such as lipids \[[@CR27], [@CR28]\], proteins \[[@CR29]\] and biopolymers \[[@CR18]\]. It is naturally capable of growing on different substrates (sugars, oils, alkane, glycerol) and has been recently engineered to accept inexpensive carbon sources \[[@CR30], [@CR31]\]. *Y. lipolytica* was used to produce both PHA copolymer and homopolymer with an external fatty acid (FA) supply and the expression of the PhaC synthase from *Pseudomonas aeruginosa* \[[@CR18], [@CR19]\]. Haddouche et al. \[[@CR19]\] redirected the FA flux toward β-oxidation by deleting the neutral lipid synthesis pathway and by overexpressing the 2-enoyl-CoA hydratase domain of the MFE protein to enhance the synthesis of the 3-OH FA precursor. This led to an accumulation of PHA up to 7% of cell dry weight (CDW). However, the properties of the produced PHA were not investigated, likely because of its low accumulation level.
The production of tailor-made mcl-PHAs using diverse FA precursors could lead to different polymer properties for new materials. When using an heterologous organism, it is important to evaluate the resulting polymer with respect to the accumulation capacity, the yield, the polymer constituents, and the material properties. In this paper, we describe the production and the characterization of two mcl-PHA in *Y. lipolytica*, an homopolymer and a copolymer, highlighting the capacity of the engineered strain to accumulate high amount of polymer and giving evidence for the bioproduction of two different materials with distinct properties.
Methods {#Sec2}
=======
Strains construction {#Sec3}
--------------------
PhaC variants strains: the wild type version of PhaC was cloned on the JMP62 pTEF_URA3ex plasmid \[[@CR32]\]. Variants of the PhaC synthase were then generated by directed mutagenesis using the QuikChange II Site-Directed Mutagenesis Kit (Agilent), and the list of primers listed in Additional file [1](#MOESM1){ref-type="media"}: Table S4. After directed mutagenesis, the gene sequences were checked by Sanger sequencing services from GATC Biotech (Constance, Germany). After construction, all the plasmids were linearized and used to transform *Y. lipolytica* strain JMY_1877 \[[@CR33]\]. The yeast cells were made competent and transformed using Frozen-EZ Transformation kit (Zymo Research, Irvine, CA, USA). Yeast transformants were selected by auxotrophy on the adequate minimal medium and the presence of the genes of interest was checked by PCR. All the constructed strains used are described (Additional file [1](#MOESM1){ref-type="media"}: Table S1). ThYl_1166 was constructed by transformation of the strain JMY2333, a derivative of the strain JMY1915 \[[@CR19]\], with the plasmids JMP62 pTEF-PhaC_E130D S325T S477R-URA3ex and the plasmid JMP62 pTEF-CpPCT-LEU2ex. ThYl_657 was obtained by transformation of a the strain JMY2475 with the plasmid JMP62 pTEF-PhaC_E130D S325T S477R-URA3ex and the strain ThYl_1024 by transformation of the strain JMY2475 with the plasmid JMP62 4UAS-tef MfeC-LEU2ex \[[@CR34]\] and the plasmid JMP62 pTEF-PhaC_E130D S325T S477R-URA3ex.
Cultures for PHA production {#Sec4}
---------------------------
Pre-cultures were started from fresh colonies in 20 mL of the rich medium YPD (yeast extract 10 g/L, bactopeptone 10 g/L, glucose 10 g/L). Minimal medium YNB (glucose 40 g/L, YNB w/o AA 1.7 g/L, NH~4~Cl 5 g/L, phosphate buffer pH 6.8 50 mM) was used to grow cells for PHA production. Solutions of fatty acid methyl esters \[methyl myristate (mC14) and methyl laurate (mC12)\] were prepared at 20% (v/v) in H~2~O and Tergitol or tween. After 24 h and 48 h of culture, 2.5 g/L mC14 or mC12 was added. As exogenous medium chain fatty acids may be toxic to the cells at high concentration, mC14 was added sequentially and during the exponential growth phase rather than in one time at the beginning. After 5 days of culture, cells were harvested by centrifugation (500×*g* 10 min), washed once with 50% isopropanol (to remove excess fatty acids from the medium) and twice with H~2~O and stored at − 80 °C. Upon thawing, cells were resuspended in 100 mM Tris--HCl buffer at pH 8 with 0.5 mg/mL of zymolyase and lysed with a 28 °C incubation for 18 h with shaking. Cells were then freeze-dried until PHA extraction.
PHA extraction {#Sec5}
--------------
Polymer was extracted with chloroform from the dried cells using a Soxhlet apparatus. Typically, 50 mL of solvent were used to extract polymer from 1.5 g of dried cells and the filling and emptying cycles of the chamber were allowed to process 10 times before collection of the solvent containing the extracted materials. PHA in chloroform was then filtered using a 40 mL Graces' Reveleris^®^ cartridge and precipitated by addition of 1 volume of ethanol for 2 volumes of chloroform, followed by cooling at − 20 °C. After centrifugation, the supernatant was discarded, the PHA was dried on air and then used for further analysis.
NMR {#Sec6}
---
Polymer quantities were determined by NMR on a Bruker Avance II 500 spectrometer. The cells extracts were thoroughly dried, prior to being diluted in CDCl~3~ containing 1% TMS (internal standard) and transferred to 5 mm NMR tubes. NMR spectra were recorded at 298 K. Each NMR spectrum was acquired using an excitation flip angle of 30° at a radiofrequency field of 29.7 kHz, a relaxation delay of 10 s and 2 dummy scans. For each experiment, 16 scans were performed with a repetition delay of 6.5 s. PHA concentrations were determined by integration of the two specific AB double doublets at 2.55 ppm.
Transmethylation and GC/MS analysis {#Sec7}
-----------------------------------
PHA monomer composition was analyzed by transmethylation of the polymer in hot acid methanol \[[@CR35]\]. Briefly, 2 mL of a solution of methanol (with C17 FA prepared at 0.2 mg/mL as standard) with 2.5% sulfuric acid was added to the dried sample in addition to 1 mL of Toluene. Samples were heated at 80 °C for 3 h. Once the samples were cooled down, biphasic liquid extraction took place using 1.5 mL of 0.5 M NaCl and 1.5 mL hexane (containing mC20 FA at 0.1 mg/mL as internal standard). Analyses were performed on the organic phase with a gas chromatography coupled with mass spectrometry (GC--MS) *TRACE*™ 1310 equipped with the *TRACE*™ TR-5 column (Thermo-scientific). Standards of 3-OH fatty acids methyl esters were used to determine retention time and for relative quantification.
Size exclusion chromatography (SEC) {#Sec8}
-----------------------------------
The number average molar mass (*M*~n~), the mass average molar mass (*M*~w~) and the dispersity (*Đ*) of the PHA samples were determined by SEC, using a Malvern Instrument apparatus (Viscotek RImax). This device was equipped with a guard column 10 mm (8 μm) and three 300 mm columns (50, 150 and 500 Å). Refractive index (RI) and ultra-violet (UV) detectors were used. Tetrahydrofuran (THF) was used as the eluent at a flow rate of 1 mL/min. The apparatus was calibrated with linear polystyrene standard from 162 to 20,000 g/mol.
Elemental analysis {#Sec9}
------------------
Elementary analyses were performed on a ThermoFisher Scientific "Flash 2000" (US) device (absolute precision of 0.3%) with 1 mg sample burned up to 950 °C.
Thermogravimetric analysis (TGA) {#Sec10}
--------------------------------
Thermal degradations were studied by TGA. Measurements were conducted under nitrogen atmosphere (flow rate of 25 mL/min) using a Hi-Res TGA Q5000 apparatus from TA Instruments. Samples (1--3 mg) were heated from room temperature up to 700 °C at a rate of 20 °C/min.
Differential scanning calorimetry (DSC) {#Sec11}
---------------------------------------
Crystallization behavior of both PHAs was studied on a DSC Q 200 (TA Instruments). Samples of around 3 mg were heated from 25 to 180 °C at 10 °C/min under nitrogen flow (50 mL/min). This first scan was conducted in order to erase the thermal history of the bulk polymer. In ramp 2, the sample was cooled at 5 °C/min to − 70 °C and kept isothermal for 3 min. In ramp 3, the sample was once again heated at 10 °C/min to 180 °C. Ramp 3 was used for determination the glass transition temperature (T~g~), melting temperature (T~m~) and heat of fusion (ΔH~m~).
Uniaxial tensile tests {#Sec12}
----------------------
After a thermal treatment at 37 °C for 5 days (body temperature) of thin films (10 × 5 × 0.2 mm^3^) prepared by solvent casting, tensile tests were performed with a Discovery HR hybrid rheometer (TA instruments) at a crosshead displacement rate of 10 mm/min, at room temperature.
Results and discussion {#Sec13}
======================
Polymer accumulation depending on PhaC synthase variant expressed by the yeast {#Sec14}
------------------------------------------------------------------------------
Recent studies have demonstrated that some specific mutations in PhaC can enhance, alone or in combination, PHA polymer production and composition when expressed in bacteria \[[@CR36], [@CR37]\]. Consequently, we investigated the capacity of different PhaC synthase variants (Additional file [1](#MOESM1){ref-type="media"}: Table S1) to produce and accumulate PHA copolymer in *Y. lipolytica*. The strain JMY_1877 \[[@CR19]\] was used as a chassis strain to express the PhaC variants under the strong constitutive promoter pTEF and to produce PHA copolymer. PhaC was targeted to the peroxisome as this compartment is the site of fatty acid degradation by the β-oxidation pathway and therefore the site where 3-OH FAs, the substrates of PhaC, are synthesized as intermediates of the β-oxidation pathway. In this strain, lipid accumulation is abolished by the deletion of acyl-transferases in order to reduce fatty acids storage and improve their availability for PHA production.
The strains expressing the different PhaC variants were grown for PHA production as described in "[Methods](#Sec2){ref-type="sec"}" section with methyl myristate (mC14) as precursor. Cells were then harvested, the polymer extracted and the polymer accumulation and composition were measured (Additional file [1](#MOESM1){ref-type="media"}: Table S2). The first set of strains expressing PHA variants that we generated (ThYl_1475, 1479, 1480, 1481, 1485, 1487 and 1494) accumulated higher or equivalent levels of polymer than the ThYl_1475 strain expressing the wild-type PhaC. The most interesting variant, ThYl_1494, combined the four mutations E130D S325T S477R Q481M and led to 27% accumulation of polymer. The second set of variants generated (corresponding to the strains ThYl_1491, 1496 and 1498) combined the quadruple mutations and the mutations S482G, L484V and A547V respectively. No synergistic effect on polymer accumulation was observed: the strain ThYl_1491 showed similar performance than ThYl_1494 and the strains ThYl_1496 and ThYl_1498 lost their ability to improve polymer accumulation. Finally, we chose to use the quadruple variant PhaC E130D S325T S477R Q481M for downstream experiments.
NMR analysis and transmethylation followed by GC--MS analysis gave evidence for the production of a PHA composed of 3-OH FAs of different chain lengths, except for the strain ThYl_1496 in which no production occurred (Additional file [1](#MOESM1){ref-type="media"}: Table S2). In term of fatty acid composition, no major difference was observed among the PHA produced by the different strains tested. The composition is as follows: 26 to 30% of 3HO; 32 to 36% of 3HD; 25 to 29% of 3HDD and 9 to 13% of 3HTD.
mcl-PHA copolymer production {#Sec15}
----------------------------
Having identified a PhaC variant with a high capacity to accumulate PHA polymers in *Y. lipolytica*, we decided to investigate the physical properties of two polymers produced by this yeast: a copolymer and an homopolymer.
For the copolymer production, we used the strain ThYl_1166 (Additional file [1](#MOESM1){ref-type="media"}: Table S1). In this strain the MFE protein \[containing the two hydrogenase domains (domains A and B) and the single hydratase domain (domain C)\] is expressed under pPOX2, the strong fatty acid inducible promoter, to force fatty acid degradation. Consequently, this strain generates high amounts of 3-OH FAs, the substrates of PhaC synthase. This host successfully expressed the variant PhaC_E130D S325T S477R Q481M. ThYl_1166 was grown as described in "[Methods](#Sec2){ref-type="sec"}" section with mC14 FA. After 5 days, when the OD reached 25 and the glucose was completely consumed, we stopped the cultures. After polymer extraction, NMR and transmethylation analysis revealed that in these conditions, the strain ThYl_1166 accumulated 27% (g/g of DCW) of a mcl-PHA polymer composed of 29% of 3HO, 35% of 3HD, 26% of 3HDD and 10% of 3HTD (Table [1](#Tab1){ref-type="table"} and Additional file [1](#MOESM1){ref-type="media"}: Figure S1). These results are quite closed to the one observed for the PHA produced by strain ThYl_1491 suggesting that the difference in genetic pattern between ThYl_1166 (Q4, Δmfe1, pPOX-MFEABC, pTEF-PhaC_E130D S325T S477R Q481M-URA3ex, pTEF-CpPCT-Leu2ex) and ThYl_1491 (Q4, pTEF-PhaC_E130D S325T S477R Q481M S482G L484V-URA3ex, Leu2ex) did not affect PHA composition and accumulation.Table 1PHA accumulations and compositionsStrainSubstratePHA accumulation % (g/g)Fraction of 3OH FA (mmol)3HO3HD3HDD3HTDThYl_1166mC14 (5 g/L)27%29%35%26%10%ThYl_1024mC12 (5 g/L)28%ndnd\> 99%nd*nd* not detectable
mcl-PHA homopolymer {#Sec16}
-------------------
For the homopolymer production, only the C domain of the MFE protein (MFEC), encoding the 2-enoyl-CoA hydratase, essential to generate exclusively the 3-OH FA, substrates of PHA synthase was re-introduced into a Q4 Δ*mfe* strain expressing the quadruple variant of the PhaC synthase (E130D, S325T, S477R and Q481M). The two dehydrogenase domains of the MFE protein were not re-introduced in order to avoid further degradation of the 3-OH FAs. We obtained the strain ThYl_657 (Additional file [1](#MOESM1){ref-type="media"}: Table S1). To optimize the production, we also generated a strain overexpressing MFEC by adding in the genome, a second copy of the mfeC gene under the control of the strong promoter 4UASTEF. We obtained the strain ThYl_1024 (Additional file [1](#MOESM1){ref-type="media"}: Table S1). To evaluate the best substrate for homopolymer production, both strains ThYl_1024 and ThYl_657 were first grown in minimal medium with either methyl laurate (mC12) or mC14 as fatty acid precursors. After 5 days of culture, polymers were extracted and the polymers accumulations were estimated by NMR. The best accumulation level was obtained with the strain ThYl_1024 grown with mC12 with 15% (g/g) vs. less than 9% (g/g) for the others conditions (Additional file [1](#MOESM1){ref-type="media"}: Table S3). This result highlighted the interest to have a double copy of the mfeC gene to maximize the pool of the 3-OH FA precursor. Therefore, this combination of strain and fatty acid was used for a larger scale production.
ThYl_1024 was grown in 10 L of YNB supplemented with mC12 FA. After 5 days of culture and the addition of twice 2.5 g/L of mC12 the polymer was extracted and analyzed. In these conditions, ThYl_1024 strain accumulated 28% of PHA. NMR studies gave evidence of a homopolyester and GC analysis confirmed the production of a polymer composed of 3HDD chains (Additional file [1](#MOESM1){ref-type="media"}: Figure S2 and Table [1](#Tab1){ref-type="table"}).
This study clearly validates the optimization of the bioproduction of two types of polymers: by testing PhaC variants, we identified a PhaC enzyme variant (with E130D S325T S477R and Q481M mutations) with high performance in polymer accumulation in *Y. lipolytica.* We showed that the four mutations introduced within PhaC led to a fourfold improvement of polymer accumulation in the yeast with no impact on cell growth and polymer composition. It is quite surprising that the side chains of the 3-OH-FAs composing the PHA produced by the PhaC variants were not smaller than the ones composing the polymer produced by the wild-type PhaC. Indeed, some of the mutations tested were described to preferentially accommodate 3-hydroxy-butyric acid substrate (with four carbon atoms only) \[[@CR37], [@CR38]\]. Our results suggest that those mutations either improved the PhaC efficiency in the yeast with no change in substrate specificity or that in *Y. lipolytica*, small 3-OH FAs are more efficiently used by the MFE enzyme than by the PhaC enzyme. Then, we optimized the production of mcl-PHA homopolymer: the addition of a second copy of the 2 enoyl-CoA hydratase gene in *Y. lipolytica* genome, placed under a strong promoter, led to a 1.7-fold increase in polymer accumulation. Overall, the accumulation of both homopolymer and copolymer reached 28% (g/g) of CDW, corresponding to approximatively 2.9 g/L of culture. If we compare this result to the bioproduction of mcl-PHA in other heterologous organisms, our yeast system is more performant. In engineered *Saccharomyces cerevisiae*, mcl-PHA accumulation did not exceed 7% (g/g) of CDW \[[@CR39]\], in *Y. lipolytica*, mcl-PHA was produced only at 1 g/L of culture \[[@CR18]\] and in the bacteria *Escherichia coli*, mcl-PHA production was estimated to be 6% (g/g) of CDW \[[@CR24]\].
In our strategy, we took advantage of the synthesis of 3-OH FAs, as intermediates of the β-oxidation pathway, to produce PHA. Contrary to prokaryotic host in which mcl-PHA synthesis occurs in the cytoplasm, in yeast, the PHA synthesis is confined in the peroxisome where the β-oxidation takes place. This confinement presents a double benefit: it allows the concentration of the substrate (3-OH FA) and the enzyme (PHA synthase), enhancing the production of high molecular weight polymers. In addition, it allows a tight control of the monomeric composition. Indeed, the 3-OH FAs coming from the de novo fatty acid biosynthesis are located in the cytoplasm and cannot be used as substrate for polymer synthesis. Hence, the key interest of our strategy is to take advantage *Y. lipolytica* capacity to efficiently incorporate, activate FA and transport them to the peroxisome where they are degraded or incorporated into PHA.
Polymer characterizations {#Sec17}
-------------------------
The high accumulation rate of both PHA polymers in *Y. lipolytica* allowed us to purify the polymers and to carry out the analysis of their physical, thermal and mechanical properties.
### Molar masses and purity {#Sec18}
The number-average molar mass (M~n~), the mass-average molar mass (M~w~), and the dispersity (Ð) of the PHA samples were determined by size exclusion chromatography (SEC) using THF as eluent. The PHA copolymer showed values of M~n~ and M~w~ around 78,500 and 127,700 g/mol respectively with a dispersity of 1.6 while the PHA homopolymer showed higher M~n~ and M~w~ with values around 156,400 and 316,400 g/mol, respectively, and dispersity of 2 (Table [2](#Tab2){ref-type="table"}). These results indicate a narrow molar mass distribution and M~n~ values similar to commercial P3HB from e.g. Biocycle©, produced by microbial fermentation (M~n~ = 180,000 g/mol and Ð = 2.3, determined by SEC) \[[@CR40]\]. Elemental CHN analysis was carried out and showed that the amount of nitrogen, a good indicator of protein content and consequently of purification state, was below 0.5%. This indicates that the amount of residual impurities in the polymers is negligible (Table [2](#Tab2){ref-type="table"}). It is a key point since these impurities are well-known to catalyze the degradation of these biopolymers under thermal input.Table 2Results of the chains characterizations by SEC and elementary analysis of the PHAsSampleSECElementary analysisM~n~ (g/mol)M~w~ (g/mol)ÐC (%)H (%)N (%)PHA copolymer78,500127,7001.669.3 ± 0.110.4 ± 0.10.4 ± 0.03PHA homopolymer156,400316,4002.072.6 ± 0.210.9 ± 0.030
### Thermal properties of the mcl-PHAs {#Sec19}
Table [3](#Tab3){ref-type="table"} summarizes the thermal properties of PHAs determined from DSC analyses. As can be seen, the two PHAs presented significant differences in their thermal behaviors. For instance, the lower melting enthalpy (ΔH~m~) and melting point (T~m~), measured for the copolymer indicated a more amorphous behavior than the homopolymer. The glass transition and melting temperature (T~g~ and T~m~) of the PHA polymers were low and indicative of a flexible polymer at ambient temperature. These values are similar with the − 60 °C glass transition temperature and the 60 °C melting temperature of the polycaprolactone a widely used polymer in the field of biomedical application. In addition, although the crystallinity index of these materials could not be calculated since the enthalpies of the perfect PHA crystals are not available for these particular PHAs; a rough estimation of this value was obtained. We used the endothermic melting enthalpy determined by the second heating (ΔH~m~) of each PHA and the melting enthalpy (ΔH~m~^o^) of 100% crystalline P3HB according to equation X~c~ = ΔH~m~/ΔH~m~^o^, assuming 146 J/g as the melting enthalpy of 100% crystalline P3HB as cited in literature \[[@CR40]\]. From these results, it is possible to point out that the degree of crystallinity and melting temperature of both PHAs were significantly lower than the commercial P3HB that are 60% and 175 °C, respectively. Additionally, both PHAs presented two melting peaks, possibly due to different crystalline domains.Table 3Main thermal properties of the PHAsSampleDSC coolingDSC 2nd heatingT~c1~ (°C)T~c2~ (°C)T~c3~ (°C)ΔH~c~ (J/g)T~m1~ (°C)T~m2~ (°C)ΔH~m~ (J/g)Χ~c~ (%)T~g~ (°C)PHA copolymer− 9n.on.o8.319577.75.3− 39PHA homopolymer405.5n.o45.5457243.930.1n.o
### Thermogravimetric analysis of the mcl-PHAs {#Sec20}
Thermogravimetric analysis (TGA) was used to evaluate the thermal stability of the two PHA (Table [4](#Tab4){ref-type="table"}). Since some PHAs present a high thermal sensitivity such as the P3HB, with chains scissions and polymers degradations, this evaluation is major. The results showed that the polymers were thermally stable up to at least 200 °C and both displayed a single degradation step at a maximum degradation temperature of 225 and 253 °C for the PHA copolymer and the PHA homopolymer, respectively. Published studies reported the highest temperature of maximum degradation rate for a commercial P3HB homopolymer at around 288 °C \[[@CR40]\].Table 4Thermal degradation temperature at 2 wt% loss (T~d,2%~) and maximum degradation (T~d,max~) temperature of the PHAsSampleTGA (under nitrogen)T~d,2%~ (°C)T~d,max~ (°C)PHA copolymer204225PHA homopolymer226253
### Mechanical properties of the mcl-PHAs {#Sec21}
To evaluate the mechanical properties of these two polymers, uniaxial tensile test was performed (Table [5](#Tab5){ref-type="table"}, Additional file [1](#MOESM1){ref-type="media"}: Figure S3). The result showed that the PHA copolymer displayed an elastomeric behavior indicated by a low yield stress, low Young's modulus and high elongation at break values, in agreement with its low melting temperature and low crystallinity. In contrast, the PHA homopolymer showed thermoplastic elastomer properties with high tensile strength, high Young's modulus and elongation at break values corresponding to a semi-ductile behavior. For comparison, a brittle thermoplastic behavior has been reported for the commercial P3HB from Biocycle© with maximum elongation at break around 4 and 2%, tensile strength around 25 and 40 MPa and flexural modulus of around 3800 and 2200 MPa. The differences observed in the mechanical behavior between the PHAs and the commercial P3HB from Biocycle© agree with their thermal properties.Table 5Mechanical properties of PHAs under uniaxial tensile tests at room temperatureYield stress (MPa)Young's modulus (MPa)Elongation at break (%)PHA copolymer4.83 ± 0.650.019 ± 0.001755 ± 151PHA homopolymer11.45 ± 2.020.410 ± 0.080347 ± 51
Both polymers displayed interesting properties: the PHA copolymer is an elastomeric material with a low yield stress (4.83 MPa) and a high elongation at break (755%) due to its low melting temperature (two endothermic peaks at around 19 and 57 °C) and low crystallinity (5%). In contrast, the homopolymer has high tensile strength (11.45 MPa) and elongation at break values corresponding to a semi-ductile behavior (347%) due to its low melting temperature (two endothermic peaks at around 45 and 71 °C) and higher crystallinity (30%). Both polymers present lower crystallinity and higher elastomeric properties than P3HB which opens new perspectives of application for PHA.
To summarize the PHA synthesized are amorphous polymers with soft and elastomeric behaviors. Such properties are generally found for medium-chain-length PHAs. It is delicate to compare polymers that do not display equivalent molar masses and that have not been produced in the same conditions. However, we try to briefly confront our biosynthesized PHA to bacterial mcl-PHA previously characterized in the literature. For instance, the T~m~ and T~g~ values measured for our polymers are equivalent and consistent to the values for mcl-PHA produced by *P. putida* (around 60 °C and − 40 °C, respectively) \[[@CR41]--[@CR43]\]. A key-point in this comparison is the static mechanical behavior of our copolymer and, more particularly the elongation at break, higher than 750%. This is one of the highest values reported in the literature for more or less equivalent architectures. Such a behavior can impact directly on potential application areas.
These highly promising biosynthesized materials can find applications in distinct fields. The PHA homopolymer behaves like a soft thermoplastic with a high elongation and a low modulus such as e.g. some low-density polyethylene. Applications can be found for instance on biomedical areas or packaging where the biodegradation is a clear advantage to manage the end-of-life of the corresponding materials. PHA copolymer presents the typical mechanical behavior of a thermoplastic elastomer (TPE) such as some thermoplastic polyurethane (TPU), with uses in biomedical application, shoes, injected rubber gasket, or some seals. However, future studies regarding the mechanical behavior in static or dynamic tests need to be undertaken in connection with these potential functions. In general, the properties of these elastomeric and flexible polymers seem to be adequate for potential biomedical high added values purposes for soft tissues, such as heart valves, cardiac patches and other vascular applications, skin tissue engineering, wound healing and controlled drug release or delivery \[[@CR14]\]. However, bioresorbability in contact with living tissues, biocompatibility with in vivo tests and toxicity evaluation have first to be carried out.
### Future optimization and challenges {#Sec22}
The high price of PHA polymer stands as a major drawback for its industrialization. This is due to the use of expensive purified substrate such as glucose and the cost of downstream processes such as extraction and purification. To minimize the overall cost of production, it would be of interest to take advantage of the ability of *Y. lipolytica* to grow on cheaper substrates such as glycerol. Recently, a lot of efforts have been deployed to engineer this yeast to grow on biomass-derived substrates \[[@CR30], [@CR31]\]. In addition, optimization of PHA extraction from eukaryotic microorganism will have to be investigated. In the case of oleaginous organism, recent advances have been made to facilitate the process of lipid extraction and could be extended to PHA extraction \[[@CR44], [@CR45]\].
Conclusion {#Sec23}
==========
This study presents the optimization of the bioproduction of mcl-PHA polymers. We showed that metabolic engineering of the yeast *Y*. *lipolytica* lead to the production of two types of mcl-PHA in a heterologous microorganism at levels never reached before. This allowed us to fully investigate the biophysical properties of these polymers and to extrapolate on their potential applications. This work clearly describes a successful pluridisciplinary approach that opens interesting opportunities for producing custom made polymers and highlights the power of engineering to generate materials meeting the needs of society for a more sustainable future.
Additional file
===============
{#Sec24}
**Additional file 1: Table S1.** Strains used in this study. pTEF-PhaC: PhaC synthase from Pseudomonas aeruginosa targeted to peroxisome and expressed under the promoter pTEF; wt: wild type; pPOX2-MFE: MFE enzyme from *Y. lipolytica* expressed under the promoter pPOX2. MFEABC, the whole protein with its three catalytic domains is expressed; MFEC: only the domain C (encoding the 2 enoyl hydratase) is expressed. **Table S2.** polymer accumulation and composition in *Y. lipolytica* strains expressing different variants of phaC synthase. **Table S3.** homopolymer accumulation in *Y. lipolytica* strains expressing the MFEC domain and grown with different source of fatty acids as substrate. **Table S4.** Sequence of the primers used in directed site mutagenesis on PhaC. **Figure S1.** ^1^H NMR spectrum in CDCl~3~ with 1% TMS of the cellular extracts of strain ThYl 1166, grown in YNB supplemented with mC14 showing almost exclusively signals belonging to a mcl-PHA, and traces of free FA. **Figure S2.** ^1^H NMR spectrum in CDCl~3~ with 1% TMS of the cellular extracts of strain ThYl 1024, grown in YNB supplemented with C12 showing almost exclusively signals belonging to a homopolymer of 3HDD and traces of free FA. **Figure S3.** Stress-strain curves of synthesized PHAs stored at 37 °C during 5 days.
PHA
: polyhydroxyalkanoate
scl-PHA
: short-chain-length polyhydroxyalkanoate
mcl-PHA
: medium-chain-length polyhydroxyalkanoate
P3HB
: poly-3-hydroxybutyrate
FA
: fatty acid
3-OH FA
: 3-hydroxy fatty acid
3HO
: 3-hydroxyoctanoic acid
3HD
: 3-hydroxydecanoic acid
3HDD
: 3-hydroxydodecanoic acid
3TD
: 3-hydroxytetradecanoic acid
**Publisher\'s Note**
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Coraline Rigouin, Sophie Lajus and Connie Ocando contributed equally to this work
The authors thank the TWB Biotransformation culture platform where the polymer productions have been carried out. We also express our gratitude to MetaToul platform where RMN experiments were achieved. We finally thank the ICEO facility dedicated to enzyme screening and discovery, and part of the Integrated Screening Platform of Toulouse (PICT, IBiSA) for providing access to its analytical facilities.
SL, CR and CO designed the experiments, performed the experiments and analyzed the results; VB contributed to the experiments; JMN contributed to the experiments; CR, SL and CO wrote the paper with input from all authors. AM, LA and FB supervised the project. All authors read and approved the final manuscript.
Not applicable.
The datasets used during the current study are available from the corresponding author on reasonable request.
Not applicable.
Not applicable.
The authors declare that they have no competing interests.
|
{
"pile_set_name": "PubMed Central"
}
|
In crop breeding, most target traits are quantitative traits, also known as complex traits. It is crucial to determine the genetic architecture of complex traits to understand their biological mechanisms and to plan efficient genetic improvement strategies. To map quantitative trait loci (QTLs), appropriate data are required, including molecular markers and phenotypic traits from a well-designed experiment. The commonly used mapping populations in plants include F2, backcross (BC), double haploid (DH), and recombinant inbred (RI) populations. Of these, the F2 population provides the most abundant genetic information \[[@R1]\]. However, the F2 population is temporary, the individuals differ from each other in their genetic constitution, and they cannot be duplicated unless somatic cell cloning is applied. Thus, it is not possible in a QTL study to conduct multiple environmental experiments with an F2 population to analyze QTL × environment (QE) interactions. Furthermore, all individuals in different environments need to be genotyped, which is expensive in terms of time, labor, and money. The BC population shares the same features as the F2 population mentioned above; as a result of less segregation information for genotypes, additive effects cannot be distinguished from dominance effects, and some types of epistatic effects are also confounded \[[@R2]\]. To investigate QTL effects across different environments, immortal DH and RI populations have been developed and widely applied in QTL mapping for many species \[[@R3]--[@R7]\]. Since DH and RI populations are homozygous, their marker data can be used repeatedly with phenotypes of quantitative traits observed in different locations and years under various experimental designs. However, these populations cannot be used to analyze dominance effects and some types of dominance-related epistatic effects, which play important roles in hybrid heterosis. In the present study, we tested an experimental design including a permanent double backcross population derived from crossing DH or RI lines to both the homozygous parents. The advantage of such populations is that identical mapping populations can be replicated as necessary.
There remains a great challenge in methodology for analyzing epistasis and QE interactions, two principal genetic components of QTL effects. The importance of epistasis affecting complex quantitative traits has been well documented in numerous classical quantitative genetic studies \[[@R8],[@R9]\]. Several QTL studies have also indicated that epistasis is an important genetic basis for complex traits such as grain yield and its components, as well as heterosis and inbreeding depression \[[@R10]--[@R13]\]. The QE interaction is another important component for quantitative traits \[[@R14]--[@R20]\], but none of the previous methods has integrated these two parts into one unified mapping framework. Wang et al. \[[@R2]\] proposed a method based on a mixed linear model approach to tackle this problem for a DH or RI population; this method was later extended to a permanent F2 population \[[@R21]\]. However, those methods have some disadvantages in cofactor selection for controlling background genetic effects, false positive rates, and computational tractability. Therefore, a full-QTL model was proposed for systematically mapping QTLs underlying complex traits. This model was sufficiently flexible to include the effects of multiple QTLs, epistasis, and QE interactions \[[@R22]\].
Here, we propose a method under the framework of a mixed linear model based composite interval mapping (MCIM) for immortal double backcross populations. The proposed method uses the approach of the full QTL mapping model described by Yang et al. \[[@R22]\]. Monte Carlo simulations were carried out to assess the performance of the method.
1 Methods {#S1}
=========
1.1 Genetic model {#S2}
-----------------
The proposed genetic model is based on the mixed linear model. The mapping population consists of double back-cross populations derived from crossing DH or RI lines with both of the homozygous parents. The mapping population is used to map *s* segregating QTLs simultaneously with additive, dominance, and additive × additive epistatic effects, as well as their interaction effects with the environment, in which *t* pairs of QTLs are involved in epistatic interactions. The mixed linear model for the phenotypic value of the *k*th individual in the *h*th environment (*y~hk~*) can be expressed as follows (*h* = 1, 2, ..., *p; k* = 1, 2, ..., *n~h~*)
$$y_{hk} = \mu\sum\limits_{i}^{s}{a_{i}x_{A_{ki}}} + \sum\limits_{i}^{s}{d_{i}x_{D_{ki}}} + \sum\limits_{i,j \in (1,2,\cdots,s);i \neq j}^{t}{{aa}_{ij}x_{{AA}_{\mathit{kij}}}} + e_{h} + \sum\limits_{i}^{s}{{ae}_{hi}x_{A_{ki}}} + \sum\limits_{i}^{s}{{de}_{hi}x_{D_{ki}}} + \sum\limits_{i,j \in (1,2,\cdots,s);i \neq j}^{t}{\mathit{aae}_{\mathit{hij}}x_{{AA}_{\mathit{kij}}}} + \varepsilon_{hk},$$ where *μ* is the population mean; *a~i~* and *d~i~* are the fixed additive and dominance effects of *Q~i~* (*i*th QTL), respectively; *aa~ij~* is the additive × additive epistatic effect (fixed) between *Q~i~* and *Q~j~*. *x*~*A*~*ki*~~, *x*~*D*~*ki*~~, and *x*~*AA*~*kij*~~ are the coefficients of the above QTL effects that depend on the observed genotypes at the marker loci (let *M~i~*~−~ and *M~i+~* be two markers flanking *Q~i~* and *M~j~*~−~, *M~j+~* be markers flanking *Q~j~*, respectively) and the recombination frequencies (denoted by *r*~*M*~i−~*Q*~i~~, *r*~*Q*~*i*~*M*~i+~~ and *r*~*M*~j−~*Q*~j~~, *r*~*Q*~j~*M*~j+~~); *e~h~* is the random effect of the *h*th environment, $\left. e_{h} \right.\sim(0,\sigma_{E}^{2})$; *ae~hi~* is the random additive × environment interaction effect with coefficient *x*~*A*~*ki*~~, $\left. {ae}_{hi} \right.\sim(0,\sigma_{A_{i}E}^{2})$; *de~hi~* is the random dominance × environment interaction effect with coefficient *x*~*D*~*ki*~~, $\left. {de}_{hi} \right.\sim(0,\sigma_{D_{i}E}^{2})$; *aae~hij~* is the random additive-additive epistasis × environment interaction effect with coefficient *x*~*AA*~*kij*~~ (=*x*~*A*~*ki*~~ *x*~*A*~*kj*~~), $\left. \mathit{aae}_{\mathit{hij}} \right.\sim(0,\sigma_{{AA}_{ij}E}^{2})$; *ε~hk~* is the random residual effect, $\left. \varepsilon_{hk} \right.\sim(0,\sigma_{\varepsilon}^{2})$.
Model (1) can be expressed in the following matrix form for all the *n* individuals (*n*=*n*~1~+*n*~2~+...+*n~h~*): $$\begin{array}{l}
{\mathbf{y} = 1\mathbf{\mu} + \mathbf{X}_{A}\mathbf{b}_{A} + \mathbf{X}_{D}\mathbf{b}_{D} + \mathbf{X}_{AA}\mathbf{b}_{AA} + \mathbf{U}_{E}\mathbf{e}_{E} + \sum\limits_{k = 1}^{s}{\mathbf{U}_{A_{k}E}\mathbf{e}_{A_{k}E}} + \sum\limits_{k = 1}^{s}{\mathbf{U}_{D_{k}E}\mathbf{e}_{D_{k}E}} + \sum\limits_{h = 1}^{t}{\mathbf{U}_{{aa}_{h}E}\mathbf{e}_{{AA}_{h}E}} + \mathbf{e}_{\varepsilon}} \\
{= \mathbf{Xb} + \sum\limits_{u = 1}^{r + 1}{\left. \mathbf{U}_{u}\mathbf{e}_{u} \right.\sim N(\mathbf{Xb},\,\mathbf{V} = \sum\limits_{u = 1}^{r + 1}{\sigma_{u}^{2}\mathbf{U}_{u}\mathbf{R}_{u}\mathbf{U}_{u}^{T}})}} \\
\end{array}$$ where **y** is an *n*×1 vector of the observed phenotypic values, with *n* as the total number of individuals; **1** is an *n*×1 vector with all the elements=1; **b***~A~*=\[*a*~1~ *a*~2~···*a~s~*\]*^T^*, **b***~D~*=\[*d*~1~ *d*~2~···*d~s~*\]*^T^* and **b***~AA~*=\[*aa*~1~ *aa*~2~···*aa~t~*\]*^T^* are the parameter vectors for the fixed QTL effects with incidence matrixes **X***~A~*, **X***~D~* and **X***~AA~*, respectively; $\left. \mathbf{e}_{E} = {\lbrack e_{1}e_{2}\cdots e_{p}\rbrack}^{T} \right.\sim\left. (0,\sigma_{E}^{2}\mathbf{I}),\mathbf{e}_{A_{k}E} = {\lbrack{ae}_{k1}{ae}_{k2}\cdots{ae}_{kp}\rbrack}^{T} \right.\sim\left. (0,\sigma_{A_{k}E}^{2}\mathbf{R}_{A_{k}E}),\mathbf{e}_{D_{k}E} = {\lbrack{de}_{k1}{de}_{k2}\cdots{de}_{kp}\rbrack}^{T} \right.\sim\left. (0,\sigma_{D_{k}E}^{2}\mathbf{R}_{D_{k}E}),\mathbf{e}_{{AA}_{h}E} = {\lbrack\mathit{aae}_{h1}\mspace{7mu}\mathit{aae}_{h2}\cdots\mathit{aae}_{hp}\rbrack}^{T} \right.\sim(0,\sigma_{{AA}_{h}E}^{2}\mathbf{R}_{{AA}_{h}E})$ are random effect vectors with incidence matrixes **U***~E~*, **U**~*A*~*k*~*E*~, **U**~*D*~*k*~*E*~ and **U**~*AA*~*h*~*E*~, respectively; $\left. \mathbf{e}_{\varepsilon} \right.\sim(0,\sigma_{\varepsilon}^{2}\mathbf{I})$ is the random vector of residual effects; and **R***~u~* is an identity matrix (*u*=1, 2, ..., *r*+1) when there is no kinship between the original parents.
For any putative QTL, all the coefficients in model (2) are unknown; however, they can be substituted with their expectation inferred from the conditional probability of the QTL genotype given the two flanking markers. Let *p~ijk~* be the conditional probability for the QTL with *i* denoting the parental line (P*~i~*, *i*=1, 2), *j* denoting the genotype of flanking markers (*j*=1, 2, 3, 4), and *k* denoting the genotype of QTL (*k*=1, 2, 3). Conditional probabilities are calculated for the mixed population from two backcrosses between DH lines (or RI lines) and homozygous parents P~1~ and P~2~ ([Table 1](#T1){ref-type="table"}). Additionally, the coefficients of additive and dominance effects are 1 and −0.5 for genotype *Q~i~Q~i~*, 0 and 0.5 for *Q~i~q~i~*, −1 and −0.5 for *q~i~q~i~*, respectively. Thus the coefficients of the putative QTL effects in model (1) can be estimated by *x*~*A*~*k*~~=(*p~ij~*~1~−*p~ij~*~3~) and *x*~*D*~*k*~~=(*p~ij~*~2~−*p~ij~*~1~−*p~ij~*~3~)/2 for each progeny from the backcross between DH or RI lines and parent P*~i,~* and having the *j*th flanking marker genotype.
1.2 Strategy for detecting QTLs in the whole genome {#S3}
---------------------------------------------------
Prior to fitting model (1), the numbers and locations of QTLs with genetic main effects or QE interaction effects should first be identified based on a marker linkage map and phenotypic values. After obtaining these effects, a systematic mapping strategy \[[@R22]\] for complex traits was used in the present study to estimate various parameters of QTLs.
iii. Mapping QTLs through 1D genome scan. According to the composite interval mapping (CIM) approach \[[@R23]\], mapping multiple QTLs can be simplified to 1D searching along a chromosome. It can be achieved by testing a QTL in a certain genomic region with some selected marker intervals as cofactors to control the background genetic effects from other QTLs outside of the region. Suppose *c* marker intervals (*M*~1−~*M*~1+~, *M*~2−~*M*~2+~,..., *M~c~*~−~*M~c~*~+~,) were selected as QTL candidate intervals, the model to test the QTL in locus *i* can be expressed as follows: $$y_{hk} = \mu_{h} + a_{hi}x_{A_{ki}} + d_{hi}x_{D_{ki}} + \sum\limits_{l}{(\alpha_{hl}^{-}\xi_{kl}^{-} + \beta_{hl}^{-}\zeta_{kl}^{-} + \alpha_{hl}^{+}\xi_{kl}^{+} + \beta_{hl}^{+}\zeta_{kl}^{+})} + \varepsilon_{hk},$$
where *μ~h~* is the population mean in the *h*th environment; *a~hi~* and *d~hi~* are the additive and dominance effects of *Q~i~* in the *h*th environment, respectively; $\alpha_{hl}^{-}\,(\alpha_{hl}^{+})$ and $\beta_{nl}^{-}\,(\beta_{nl}^{+})$ are the additive and the dominance effects of the *l*th marker interval in the *h*th environment, respectively; $\xi_{kl}^{-}\,(\xi_{kl}^{+})$ takes the value of 1, 0, −1 for genotype *M~l~M~l~*, *M~l~m~l~*, *m~l~m~l~* respectively, while $\zeta_{kl}^{-}\,(\zeta_{kl}^{+})$ takes the value of −0.5, 0.5, −0.5 for these three marker genotypes; the remaining variables and parameters have the same definitions as those in model (1).
iv. Mapping epistasis through 2D genome scan. After the 1D genome scan is completed with *t* main effect QTLs identified, a 2D genome scan is conducted to search for all possible epistasis including the effects of *t* QTLs and the interaction effects from *f* pre-selected paired marker intervals as the genetic background control in the model. The model to test the significance of epistatic interactions between loci *i* and *j* can be written as follows: $$y_{hk} = \mu_{h} + {aa}_{\mathit{hij}}x_{A_{ki}}x_{A_{kj}} + \sum\limits_{s = 1}^{t}\left( {a_{hs}x_{A_{ks}} + d_{hs}x_{D_{ks}}} \right) + \sum\limits_{l}^{f}\left( {\alpha\alpha_{hl}^{A^{-}B^{-}}\xi_{kl}^{A^{-}}\xi_{kl}^{B^{-}} + \alpha\alpha_{hl}^{A^{+}B^{+}}\xi_{kl}^{A^{+}}\xi_{kl}^{B^{+}}} \right) + \varepsilon_{hk},$$
where $\alpha\alpha_{hl}^{A^{-}B^{-}}(\alpha\alpha_{hl}^{A^{+}B^{+}})$ is the interaction effect between the flanking markers of interval *I^A^* and *I^B^*, (*I*^A−^, *I*^B−^) and (*I^A^*^+^, *I^B^*^+^). All other variables and parameters have the same definitions as those in [equation (3)](#FD3){ref-type="disp-formula"}.
v. Hypothesis testing for significance of QTL effects.
Both the genetic models of (3) and (4) can be reformatted into the following general multivariate linear mixed model in matrix form: $$\mathbf{y} = \mathbf{W}_{\text{Q}}\mathbf{b}_{\text{Q}} + \mathbf{W}_{\text{B}}\mathbf{b}_{\text{B}} + \mathbf{\varepsilon},$$ where, **b**~Q~ is a tested vector of QTL parameters (consisting of additive, dominance, and epistatic effects) with coefficient matrix **W**~Q~, **b**~B~ is the vector consisting of the population mean and the background effects due to the significant QTLs and marker intervals with the corresponding coefficient matrix **W**~B~. The *F*-statistic based on the Henderson III method \[[@R24]\] is used to test the significance of QTL effects, which can be expressed as follows
$$F = \frac{\mathit{SSR}\left( \mathbf{b}_{\text{Q}} \mid \mathbf{b}_{\text{B}} \right)/\left( r_{\mathbf{W}} - r_{\mathbf{W}_{\text{B}}} \right)}{\mathit{SSE}/\left( n - r_{\mathbf{W}} \right)},$$ where *r*~w~ is the rank of the matrix **W** = (**W**~Q~⋮**W**~B~) , and *r*~**W**~*B*~~ is the rank of **W**~B~; *SSR*(**b**~Q~\|**b**~B~) is the extra regression sum of squares attributed to the **b**~Q~ given the **b**~B~ in the genetic model; *SSE* is the residual sum of squares of the model (5). Under the null hypothesis *H*~0~:**b**~Q~=0, the *F*-statistic follows the *F* distribution with (*r*~W~→*r*~W~B~~) and (*n*−*r*~w~)as the numerator and denominator degrees of freedom, respectively. For detailed information, refer to Yang et al. \[[@R22],[@R25]\]
Prior to scanning the genome for candidate QTLs by models (3) and (4), the candidate marker interval or paired marker intervals with significant effects (additive, dominance, or epistatic effects) as cofactors in models (3) and (4) must first be selected via the method of marker pair selection (MPS) \[[@R26]\]. The same searching procedure proposed by Yang et al. \[[@R22]\] was used in this study.
1.3 Genome wide threshold value and the estimation of QTL genetic effects {#S4}
-------------------------------------------------------------------------
We used the *F*-statistic based on the Henderson III method for a mixed linear model to determine the candidate marker intervals, as well as the QTLs with significant effects. The genome-wide threshold for the *F*-statistic was specified by the permutation procedure \[[@R27]\]. When the number of candidate QTLs and their positions in chromosome were obtained, the full model including effects of all candidate QTLs was constructed. The model selection procedure was adopted to further reduce the false positive rate of QTLs. Based on the result of model selection, the genetic effects of QTLs including QE interaction effects were estimated by the Bayesian method via Gibbs sampling \[[@R22]\].
2 Results {#S5}
=========
2.1 Simulation setting {#S6}
----------------------
A total of 200 Monte Carlo simulations for double back-cross populations with DH × P~1~ and DH × P~2~ progenies were conducted under three different environments to investigate the statistical properties of the proposed method in detecting QTL positions, estimating QTL effects, and predicting QE interactions. The size of mapping population was kept constant at 300 with three different ratios (1:1, 1:2, 1:5) of the number of DH × P~1~ population to the number of DH × P~2~ population, or equal ratios were used for the three populations with sizes of 180, 240, and 300. In all simulations, a genome of five chromosomes was constructed with 55 evenly distributed markers at a space of 10 cM. Suppose that 7 QTLs (denoted as Q1, Q2, Q3, Q4, Q5 Q6 and Q7), were involved in the genetic variation of a complex trait, of which 5 QTLs (Q1--Q5) had main genetic effects and 2 QTLs (Q6 and Q7) were pure epistatic QTLs. Five QTLs, Q1, Q3, Q5, Q6, and Q7, were involved in three pairs of epistatic effects denoted as EQ1 (Q1--Q6), EQ2 (Q3--Q5), and EQ3 (Q6--Q7), Only epistatic effects were set for EQ3. Q1 -- Q5 were located on chromosomes 1--5, and Q6, Q7 on chromosomes 4 and 5, respectively. The heritability of a simulated trait was assumed to be 60%. Detailed configurations of parameters for each QTL are shown in [Tables 2](#T2){ref-type="table"} and [3](#T3){ref-type="table"}.
2.2 The potential bias in QTL parameter estimation arising from ignoring epistasis {#S7}
----------------------------------------------------------------------------------
A simulation comparison between two QTL models (with and without inclusion of epistatic effects) was performed to investigate the impact of epistases on estimating QTL main/epistatic effects, QE interactions, and QTL positions. Model I included both main and epistatic effects, while Model II did not include epistasis. Two populations with or without epistasis were simulated. Both populations had a sample size of 300 (150:150). The above-mentioned factors led to a total of four different cases: epistases existed and were included in the model (Case I), epistases existed but were ignored in the model (Case II), epistases did not exist and were included in the model (Case III), and epistases did not exist and were not included in the model (Case IV).
The results of the simulation clearly show that the mixed linear model approach provided largely unbiased estimates of QTL positions, QTL main/epistatic effects, and QE interactions ([Tables 2](#T2){ref-type="table"} and [3](#T3){ref-type="table"}), with small standard deviations. The results also revealed that the power of detecting an individual QTL ranged from 93% to 100%, showing the high efficiency of the proposed method.
The results under four different cases revealed that the positions of QTLs with main effects could be precisely estimated whether the genetic model included the epistases or not. In the presence of epistasis, cases I and II both provided unbiased parameter estimates with minor differences for additive effects; similarly, when there were no QTL epistases, almost the same results were observed for the cases III and IV regardless of whether epistases were included in the model or not ([Table 2](#T2){ref-type="table"}). However, in the presence of epistasis, Case I tended to give much better estimates of dominance and interaction effects with the environment, compared with Case II ([Table 2](#T2){ref-type="table"}).
For estimating epistases, it should be noted that epistases can only be identified when they exist and when an epistatic model is used (Case I). In the other cases (Case II, Case III and Case IV), either using a model that omits epistasis (Case II and Case IV) or the absence of epistases (Case III and Case IV), will both result in undetectable epistatic effects. However, the results in [Table 3](#T3){ref-type="table"} revealed that epistatic parameters were well estimated by the epistatic model (Case I), although the statistical power was relatively lower than that of individual QTLs. Considering that the estimation accuracy of QTL parameters is affected by epistases, and that epistases are generally involved in genetic variation of quantitative trait, a model that includes epistasis is preferable for use in QTL studies.
2.3 Impact of heritability and population constitution on estimation of QTL parameters {#S8}
--------------------------------------------------------------------------------------
To investigate the impacts of heritability and population size on estimating QTL parameters, we conducted simulations with the several scenarios described in [Tables 4](#T4){ref-type="table"} and [5](#T5){ref-type="table"}. Model I with epistases (see Section 2.2) was used for three sample populations with different sizes and a constitution ratio of 1:1, that is; 180 (90:90), 240 (120:120), and 300 (150:150). The simulation results revealed that including epistases in the model was essential to reliably detect and quantify QTLs with the mixed linear model approach. The large heritability and population size could increase the statistical power and estimation precision of the QTL parameters. As shown in [Table 4](#T4){ref-type="table"}, the statistical power of every QTL with main genetic effects was nearly 100%, except for Q5 with relatively small heritability; this finding indicated that heritability is an important factor in QTL detection. In addition, as the population size increased to 240, most QTLs with main effects could be distinguished efficiently. For the epistatic interaction ([Table 5](#T5){ref-type="table"}), the statistical power and accuracy of epistatic parameters were improved with higher heritability or larger population sizes. Under all of the scenarios, had fewer type II errors than other two paired epistatic QTLs. Meanwhile, for each pair of epistatic QTLs, increased population size resulted in greater predictive power and more accurate estimates of parameters.
In addition to heritability and sample size, the population constitution may be another important factor affecting QTL mapping. Thus, using the same model as above (Model I), we performed another series of Monte Carlo simulations for mapping populations with the same size but three different constitutions, 300 (150:150), 300 (100:200), and 300 (50:250). As shown in [Tables 6](#T6){ref-type="table"} and [7](#T7){ref-type="table"}, the population with even proportions improved the accuracy of estimating the genetic effects of QTLs. In most cases, the estimates of parameters were slightly more accurate in the evenly distributed sample, as indicated by standard deviation, although the differences were not so clear ([Tables 6](#T6){ref-type="table"} and [7](#T7){ref-type="table"}). The false discovery rate also tended to increase with a more unevenly distributed population (0.0416, 0.0537 to the 0.0543 for three respective population constitutions; [Table 7](#T7){ref-type="table"}). In all cases, the power for the main QTL was higher than that for epistasis. Thus, a double backcross population with a large sample size and evenly distributed population is preferred and will enhance the accuracy of QTL analysis.
3 Discussion {#S9}
============
Backcrossing is a very commonly used breeding method. It is widely used in plant breeding to precisely improve the target trait by transferring the associated gene(s) from the donor parent to the recurrent parent, to control hybrid populations, and to overcome the barriers of distant crosses, and so on. Backcrossing is also a very useful design for dissecting the genetic mechanism of quantitative traits, and has been used in mapping QTLs for complex traits in crops and animals \[[@R28],[@R29]\]. Advanced backcrossing has been used for fine mapping of QTLs \[[@R30],[@R31]\]. However, when a traditional temporary BC population derived from crossing F~1~ with one of its parents is used to map QTLs for complex traits, it is impossible to obtain different phenotypes of each genotype in different environments. In the present study, we proposed mapping of QTLs based on an immortal double backcross population (that is, a population constructed by mating DH or RI lines with homozygous parents P1 and P2 separately). Because of the benefits of the identical genetic constitution of the mapping population and repeated multiple environment experiments, QTL and QE interactions can be analyzed accurately. Meanwhile, time and labor costs can be saved because the molecular genotype of each individual can be inferred from the molecular information of the parents and the DH or RI lines. Another advantage is that the double backcross population may improve the statistical power and precision of estimation, such as estimates of QTL positions, additive effects, and dominance effects, because of the increased population size and more abundant molecular genotypes at one locus or multiple loci. In QTL mapping, an F~2~ population is ideal for analyzing additive, dominance, and additive-additive epistasis of QTLs because it has richer genetic diversity than DH, BC, and RI populations. On the other hand, to overcome the shortcomings resulting from heterozygosity of individuals and the need to conduct repeated experiments, the permanent or immortal F~2~ (PF2 or IF2) populations derived from mating of DH or RI lines is proposed to mimic the F~2~ population. The double backcross population is also an excellent mimic of the F~2~ population, although it has fewer genotype types of multiple loci than the F~2~ population, and of the four types of epistases, only additive-additive epistasis can be analyzed. However, there are still many merits of the double backcross mapping population for QTL studies in crops and especially in animals.
Recent studies of QTL mapping in rice and maize have shown that dominance and epistasis, especially the additive × additive interaction, play a key role in contributing to heterosis \[[@R10],[@R32],[@R33]\]. In the conventional BC population, the additive and dominant effects can only be identified in a mixture, and the additive × additive interaction cannot be detected separately. The double backcross population proposed in the present study can be used to tackle this problem, and accurately and efficiently estimates additive, dominance, and additive × additive interaction effects. However, population structure should be taken into account when mapping QTLs in the double backcross population. A population with equal ratios of the two backcross populations has a more similar genetic structure to the F~2~ population, and should show greater statistical power and estimation accuracy compared with those of other populations. However, our simulation did not show large differences among three populations consisting of 150 and 150 lines, 100 and 200 lines, and 50 and 250 lines derived from crosses of DH × P~1~ and DH × P~2~. When there are extreme changes in proportions of the population, the double-backcross populations will become more similar to a traditional one-backcross population, and the coefficient matrix of the model will become singular; thus, some genetic effects of QTLs, such as dominance, cannot be analyzed. Thus, an evenly distributed population is preferable and may improve the precision of estimates and statistical power.
It is believed that epistatic interactions are important genetic buffers that provide functional redundancy for species to survive under adverse changes in their environment. As well, epistatic interactions could generate more phenotypic polymorphisms in response to natural and artificial selection \[[@R34]\]. So far, there is much research evidence on the role of epistatic interactions. However, to detect a pair of true positive epistatic QTLs usually requires a relatively large population. Actually, the size of the simulation population, 300, was large enough to detect epistasis under the simulation scenarios considered here. As shown in [Table 7](#T7){ref-type="table"}, irrespective of various population proportions, the false positive rates of epistasis detection are quite low, 0.04, 0.05, and 0.05 in the three surveyed cases. On the other hand, the simulation study suggests that if epistasis of the QTLs does contribute to a trait, it will affect the accuracy of estimated QTL parameters. When there is significant epistasis in agronomic traits, mapping QTLs with case II will lead to inferior estimates of the effects and positions. Therefore, the best strategy is to include epistatic effects in a QTL model, especially when some epistases are actually involved in genetic variation of complex traits.
This work was supported by the National Basic Research Program of China (2010CB126006 and 2011CB109306), the National Special Program for Breeding New Transgenic Variety (2008ZX08005-005), CNTC (110200701023), YNTC (08A05), and the National Institutes of Health (R01DA025095).
######
Conditional probabilities of QTL for two backcrosses populations from mating between DH (or RI) lines and parental lines (P~1~ and P~2~)
Backcross Types Marker genotypes Expected frequency[a)](#TFN1){ref-type="table-fn"}
-------------------------------- -------------------------------- ---------------------------------------------------- ------------------ --------------------------------------- --------------------------------------- --------------------------------------- ---------------------------------------
DH or RI×P~1~ *M~i~*~−~*M~i~*~−~*M~i+~M~i+~* *s*~1~*s*~2~/*s* *r*~1~*r*~2~/*s* 0 $\frac{1}{1 + 4r_{1}r_{2}}$ $\frac{4r_{1}r_{2}}{1 + 4r_{1}r_{2}}$ 0
*M~i~*~−~*M~i~*~−~*M~i+~M~i+~* *s*~1~*r*~2~/*r* *r*~1~*s*~2~/*r* 0 *r*~2~/(*r*~1~+*r*~2~) *r*~1~/(*r*~1~+*r*~2~) 0
*M~i~*~−~*m~i~*~−~*M~i+~M~i+~* *r*~1~*s*~2~/*r* *s*~1~*r*~2~/*r* 0 *r*~1~/(*r*~1~+*r*~2~) *r*~2~/(*r*~1~+*r*~2~) 0
*M~i~*~−~*m~i~*~−~*M~i+~m~i+~* *r*~1~*r*~2~/*s* *s*~1~*s*~2~/*s* 0 $\frac{4r_{1}r_{2}}{1 + 4r_{1}r_{2}}$ $\frac{1}{1 + 4r_{1}r_{2}}$ 0
DH or RI×P~2~ *M~i~*~−~*m~i~*~−~*M~i+~m~i+~* 0 *s*~1~*s*~2~/*s* *r*~1~*r*~2~/*s* 0 $\frac{1}{1 + 4r_{1}r_{2}}$ $\frac{4r_{1}r_{2}}{1 + 4r_{1}r_{2}}$
*M~i~*~−~*m~i~*~−~*m~i+~m~i+~* 0 *s*~1~*r*~2~/*r* *r*~1~*s*~2~/*r* 0 *r*~2~/(*r*~1~+*r*~2~) *r*~1~/(*r*~1~+*r*~2~)
*m~i~*~−~*m~i~*~−~*M~i+~m~i+~* 0 *r*~1~*s*~2~/*r* *s*~1~*r*~2~/*r* 0 *r*~1~/(*r*~1~+*r*~2~) *r*~2~/(*r*~1~+*r*~2~)
*m~i~*~−~*m~i~*~−~*m~i+~m~i+~* 0 *r*~1~*r*~2~/*s* *s*~1~*s*~2~/*s* 0 $\frac{4r_{1}r_{2}}{1 + 4r_{1}r_{2}}$ $\frac{1}{1 + 4r_{1}r_{2}}$
The putative **QTL** (*Q~i~*) and two flanking marker are ordered along the chromosome as *M~i~*~−~→*Q~i~*→*M~i+~*~,~ where the recombination rate between *M~i~*~−~ and *M~i+~* is denoted as *r*, the one between *M~i~*~−~ and *Q~i~* denoted as *r*~1~, and the one between *Q~i~* and *M~i+~* denoted as *r*~2~; *s*=1−*r*, *s*~1~=1−*r*~1~, and *s*~2~=1−*r*~2~.
######
Comparisons of the estimation of QTL parameter and power by models with and without epistases[a)](#TFN2){ref-type="table-fn"}
QTL Case Position *A* *D* *AE1* *AE2* *AE3* *DE1* *DE2* *DE3* Power
----- ------------- ------------- ------------- ------------- ------------- ------------- ------------- ------------- ------------- ------- -------
Q1 Expected 44.0 −2.78 −3.08 0.00 0.00 0.00 0.00 0.00 0.00
I 43.74(2.86) −2.67(0.31) −2.9(0.52) −0.00(0.78) −0.04(0.81) 0.05(0.84) −0.07(0.31) 0.00(0.18) 0.07(0.32) 98.0
II 43.72(2.87) −2.69(0.33) −1.49(1.62) −0.01(0.79) −0.04(0.80) 0.03(0.85) −1.04(1.12) 0.07(0.27) 0.97(1.03) 98.0
III 44.02(2.13) −2.69(0.35) −2.99(0.35) −0.00(0.84) −0.06(0.85) 0.04(0.87) −0.01(0.16) 0.00(0.16) 0.01(0.17) 100
IV 44.02(2.13) −2.69(0.33) −2.99(0.35) −0.00(0.84) −0.06(0.85) 0.04(0.87) −0.00(0.16) −0.00(0.14) 0.01(0.15) 100
Q2 Expected 75.0 0.00 2.50 −2.00 −0.70 2.70 0.00 0.00 0.00
I 75.36(2.75) 0.00(0.31) 2.36(0.32) −1.90(0.98) −0.65(1.01) 2.54(1.03) −0.01(0.16) 0.00(0.13) 0.00(0.15) 99.5
II 75.33(2.75) −0.03(0.33) 2.37(0.35) −1.86(0.97) −0.60(1.00) 2.58(1.05) −0.01(1.16) 0.00(0.12) 0.01(0.16) 99.5
III 75.33(2.48) 0.00(0.26) 2.34(0.35) −1.89(0.99) −0.64(0.98) 2.55(1.05) 0.00(0.14) 0.00(0.13) 0.00(0.14) 100
IV 75.33(2.48) 0.00(0.25) 2.34(0.34) −1.88(0.99) −0.63(0.98) 2.55(1.06) 0.00(0.15) 0.00(0.14) −0.00(0.14) 100
Q3 Expected 50.0 2.90 3.50 2.60 −1.30 −1.30 0.00 0.00 0.00
I 49.91(0.69) 2.53(0.46) 2.92(0.83) 2.56(0.88) −1.30(0.90) −1.18(0.86) 0.26(0.52) −0.01(0.23) −0.24(0.51) 99.5
II 49.91(0.69) 2.57(0.44) 2.12(1.43) 2.51(0.88) −1.31(0.91) −1.18(0.88) 0.88(0.96) −0.05(0.27) −0.84(0.91) 99.5
III 49.92(0.62) 2.53(0.43) 3.18(0.44) 2.57(0.88) −1.31(0.89) −1.18(0.86) −0.00(0.14) 0.01(0.15) −0.01(0.13) 100
IV 49.92(0.62) 2.53(0.43) 3.17(0.43) 2.57(0.88) −1.31(0.89) −1.18(0.86) −0.01(0.14) 0.01(0.15) −0.00(0.12) 100
Q4 Expected 73.0 −3.30 −1.90 0.00 0.00 0.00 −2.10 2.50 −0.40
I 74.20(2.85) −3.12(0.49) −1.73(0.41) 0.00(0.63) −0.03(0.69) 0.02(0.69) −1.91(0.44) 2.29(0.50) −0.37(0.34) 100
II 74.21(2.85) −3.12(0.50) −1.74(0.42) 0.00(0.62) −0.03(0.68) 0.02(0.68) −1.90(0.45) 2.29(0.51) −0.35(0.83) 100
III 73.59(2.20) −3.18(0.40) −1.79(0.32) 0.00(0.81) −0.05(0.87) 0.05(0.86) −1.93(0.37) 2.35(0.41) −0.42(0.29) 100
IV 73.59(2.20) −3.18(0.40) −1.79(0.31) 0.00(0.81) −0.05(0.87) 0.05(0.86) −1.93(0.37) 2.35(0.40) −0.42(0.29) 100
Q5 Expected 15.0 1.90 0.00 0.00 0.00 0.00 0.00 0.00 0.00
I 14.68(3.78) 1.82(0.28) −0.17(0.58) 0.00(0.75) 0.01(0.76) 0.01(0.77) 0.19(0.44) −0.03(0.22) −0.15(0.41) 93.0
II 14.72(3.79) 1.84(0.31) −0.96(1.03) −0.02(0.73) 0.01(0.76) 0.01(0.76) 0.83(0.91) −0.08(0.28) −0.73(0.83) 93.0
III 15.10(3.94) 1.82(0.25) 0.02(0.27) −0.01(0.81) −0.00(0.84) 0.02(0.86) 0.01(0.15) −0.02(0.16) 0.01(0.16) 92.5
IV 15.10(3.94) 1.82(0.25) 0.02(0.26) −0.01(0.81) −0.00(0.84) 0.01(0.86) 0.00(0.15) −0.02(0.14) 0.01(0.14) 92.5
Q1, Q2, Q3, Q4, and Q5 denote five QTLs with main effects; I, II, III, IV denote QTL models with or without inclusion of epistases when epistatic effects exist or do not exist, respectively; Expected stand for the true value of parameter; *A*, *D*, *AE*, *DE* are additive, dominance and their interaction effects with environment, numbers in parenthesis are their corresponding standard deviation of estimates. Power is probability of identifying QTLs in 200 simulations. Size of mapping population was 300, consisting of 150 lines of DH×P~1~ and 150 lines of DH×P~2~.
######
Estimate of parameter for paired epistatic QTLs and power by the model with epistases[a)](#TFN3){ref-type="table-fn"}
Epistatic QTLs QTL\_*i* QTL\_*j* *AA* *AAE1* *AAE2* *AAE3* Power
--------------------------------------------- -------------------------------------- ------------- ------------- ------------- ------------- -------- -------
EQ1(Q1--Q6) 44.0[b)](#TFN4){ref-type="table-fn"} 24.0 −3.09 2.52 −0.16 −2.36
43.75(2.85)[c)](#TFN5){ref-type="table-fn"} 23.08(2.96) −2.79(0.65) 2.17(0.70) −0.15(0.40) −2.03(0.68) 97.0
EQ2(Q3--Q5) 50.0 15.0 2.10 −2.10 0.20 1.90
49.90(0.72) 14.79(3.68) 2.05(0.55) −1.87(0.52) 0.12(0.39) 1.72(0.51) 74.5
EQ3(Q6--Q7) 24.0 79.0 −2.60 0.00 0.00 0.00
23.10(2.89) 79.37(3.22) −2.54(0.45) −0.01(0.24) −0.04(0.19) 0.05(0.28) 55.0
Three pairs of epistatic interaction, EQ1, EQ2, and EQ3, consist of Q1 and Q6, Q3 and Q5, and Q6 and Q7, respectively;
true value of parameter;
estimate of parameter and standard deviation (in parenthesis). *AA, AAE* are additive-additive epistases and their interaction effects with environment. Power is probability of identifying QTLs in 200 simulations. Size of mapping population is 300, consisting of 150 lines of DH×P~1~ and 150 lines of DH×P~2~.
######
Estimation of QTL effects and their interaction effects with environments under three different sample sizes
QTL (heritability %) Population Position *A* *D* *AE1* *AE2* *AE3* *DE1* *DE2* *DE3* Power
---------------------- ------------ --------------------------------------------- ------------- ------------- ------------- ------------- ------------- ------------- ------------- ------------- -------
Q1 (11.03) Expected 44.0[a)](#TFN6){ref-type="table-fn"} −2.78 −3.08 0.00 0.00 0.00 0.00 0.00 0.00
I 44.15(3.68)[b)](#TFN7){ref-type="table-fn"} −2.63(0.46) −2.49(1.09) −0.07(0.88) 0.10(0.93) −0.02(0.93) −0.33(0.67) 0.04(0.31) 0.30(0.64) 94
II 44.21(3.78) −2.66(0.37) −2.74(0.70) 0.00(0.81) 0.04(0.78) −0.03(0.83) −0.12(0.38) −0.01(0.20) 0.13(0.37) 99
III 43.83(3.06) −2.64(0.36) −2.88(0.55) 0.01(0.79) −0.05(0.82) 0.05(0.84) −0.06(0.27) −0.00(0.18) 0.06(0.29) 97.5
Q2 (7.68) Expected 75.0 0.00 2.45 −2.00 −0.70 2.70 0.00 0.00 0.00
I 75.17(3.44) 0.00(0.41) 2.41(0.44) −1.93(1.08) −0.51(1.06) 2.48(1.21) −0.03(0.18) 0.01(0.14) 0.02(0.18) 99
II 75.16(2.95) 0.01(0.34) 2.41(0.33) −1.88(0.99) −0.67(0.87) 2.58(1.09) −0.01(0.18) −0.00(0.16) 0.02(0.20) 99
III 75.61(2.77) 0.00(0.32) 2.36(0.33) −1.89(1.00) −0.66(1.03) 2.54(1.04) −0.01(0.16) 0.00(0.14) 0.01(0.15) 99
Q3 (16.53) Expected 50.0 2.90 3.52 2.60 −1.30 −1.30 0.00 0.00 0.00
I 49.92(1.48) 2.90(0.41) 2.89(0.96) 2.42(1.01) −1.14(0.98) −1.27(0.98) 0.54(0.77) −0.05(0.30) −0.48(0.70) 99.5
II 50.13(1.41) 2.52(0.52) 2.82(0.94) 2.51(0.94) −1.30(0.91) −1.16(0.90) 0.35(0.62) −0.05(0.25) −0.30(0.53) 100
III 49.95(0.65) 2.90(0.28) 3.26(0.64) 2.55(0.91) −1.32(0.92) −1.16(0.89) 0.28(0.54) −0.02(0.23) −0.26(0.52) 99.5
Q4 (12.65) Expected 73.0 −3.31 −1.87 0.00 0.00 0.00 −2.10 2.50 −0.40
I 73.82(3.50) −3.10(0.53) −1.72(0.49) −0.05(0.68) 0.11(0.73) −0.07(0.70) −1.93(0.55) 2.19(0.58) −0.24(0.52) 96
II 73.92(2.86) −3.19(0.37) −1.80(0.37) −0.02(0.67) −0.03(0.63) 0.04(0.69) −1.94(0.38) 2.28(0.44) −0.30(0.38) 100
III 74.18(2.97) −3.10(0.51) −1.72(0.41) −0.00(0.64) −0.03(0.71) 0.02(0.69) −1.89(0.46) 2.28(0.51) −0.36(0.34) 100
Q5 (2.36) Expected 15.0 1.92 0.00 0.00 0.00 0.00 0.00 0.00 0.00
I 14.86(5.15) 1.88(0.35) −0.43(0.83) −0.11(0.79) 0.08(0.80) 0.04(0.79) 0.53(0.81) −0.06(0.30) −0.47(0.71) 64
II 15.38(3.99) 1.82(0.33) −0.23(0.70) 0.02(0.77) −0.07(0.78) 0.06(0.77) 0.24(0.49) −0.01(0.25) −0.23(0.46) 92.5
III 14.54(3.90) 1.82(0.29) −0.16(0.61) −0.01(0.76) 0.02(0.78) 0.01(0.79) 0.20(0.45) −0.03(0.23) −0.17(0.43) 90.5
True values of QTL parameter;
estimates of QTL parameter and their standard deviation in parenthesis; *A, D, AE, DE* denote estimates of QTL additive, dominance effects and their interaction effects with environments, respectively; I, II and III represent three different population sizes; 180 (90 from DH×P~1~ and 90 from DH×P~2~), 240 (120 from DH×P~1~ and 120 from DH×P~2~) and 300 (150 from DH×P~1~ and 150 from DH×P~2~), respectively.
######
Estimation of epistatic effects and their interaction effects with environments under three different sample sizes
Epistatic QTL (heritability %) Population Position\_*i* Position\_*j* *AA* *AAE1* *AAE2* *AAE3* Power
-------------------------------- ------------ --------------------------------------------- --------------- ------------- ------------- ------------- ------------- -------
EQ1 (4.98) Expected 44.0[a)](#TFN8){ref-type="table-fn"} 24.0 −3.09 2.52 −0.16 −2.36
I 44.27(3.45)[b)](#TFN9){ref-type="table-fn"} 22.30(4.13) −2.93(0.74) 2.08(0.78) −0.21(0.59) −1.87(0.82) 64.5
II 44.50(3.39) 22.99(3.18) −2.78(0.70) 2.15(0.76) −0.19(0.41) −1.95(0.71) 90.5
III 43.82(2.96)[b)](#TFN9){ref-type="table-fn"} 23.07(3.03) −2.81(0.67) 2.15(0.73) −0.13(0.43) −2.02(0.69) 96.0
EQ2 (2.67) Expected 50.0 15.0 2.10 −2.10 0.20 1.90
I 50.22(1.07) 14.41(3.83) 2.33(0.76) −1.73(0.90) 0.13(0.55) 1.57(0.86) 27
II 50.10(1.19) 15.55(3.77) 2.16(0.54) −1.96(0.54) 0.23(0.41) 1.69(0.56) 63
III 49.96(0.58) 14.48(3.96) 2.07(0.58) −1.85(0.57) 0.13(0.38) 1.69(0.55) 72.5
EQ3 (2.10) Expected 24.0 79.0 −2.60 0.00 0.00 0.00
I 23.09(4.21) 79.94(3.32) −2.72(0.51) 0.05(0.25) 0.05(0.20) −0.10(0.22) 17.5
II 23.21(2.81) 79.34(3.43) −2.60(0.42) 0.03(0.29) −0.02(0.27) −0.01(0.23) 38
III 23.15(2.87) 79.44(3.33) −2.54(0.46) −0.02(0.23) −0.06(0.20) 0.07(0.27) 50.5
True values of QTL parameter;
estimates of QTL parameter and standard deviation in parenthesis; *AA* and *AAE* denote estimates of additive by additive effects and their interaction effects with environments, respectively; I, II and III have same definition as that in [Table 4](#T4){ref-type="table"}, with the false discovery rates of 0.103, 0.049, and 0.050, respectively.
######
Estimation of QTL effects under three different constitutions of mapping population
QTL (heritability %) Population Position *A* *D* *AE1* *AE2* *AE3* *DE1* *DE2* *DE3* Power
---------------------- ------------ ---------------------------------------------- ------------- ------------- ------------- ------------- ------------- ------------- ------------- ------------- -------
Q1 (11.03) Expected [a)](#TFN10){ref-type="table-fn"}44.0 −2.78 −3.08 0.00 0.00 0.00 0.00 0.00 0.00
I [b)](#TFN11){ref-type="table-fn"}43.83(3.06) −2.64(0.36) −2.88(0.55) 0.01(0.79) −0.05(0.82) 0.05(0.84) −0.06(0.27) −0.00(0.18) 0.06(0.29) 97.5
II 44.05(2.75) −2.63(0.43) −2.79(0.65) 0.04(0.90) −0.06(0.89) 0.02(0.93) −0.14(0.45) 0.03(0.30) 0.10(0.45) 100
III 44.13(2.18) −2.64(0.53) −2.91(0.67) −0.09(0.90) −0.02(0.85) 0.10(0.91) −0.11(0.64) 0.03(0.54) 0.08(0.60) 100
Q2 (7.68) Expected 75.0 0.00 2.45 −2.00 −0.70 2.70 0.00 0.00 0.00
I 75.61(2.77) 0.00(0.32) 2.36(0.33) −1.89(1.00) −0.66(1.03) 2.54(1.04) −0.01(0.16) 0.00(0.14) 0.01(0.15) 99
II 75.33(2.98) 0.02(0.32) 2.36(0.39) −1.79(1.04) −0.67(1.00) 2.49(1.08) −0.05(0.29) 0.01(0.31) 0.03(0.30) 99
III 75.14(2.71) −0.03(0.45) 2.34(0.49) −1.95(1.06) −0.63(1.02) 2.60(1.17) −0.05(0.65) 0.02(0.69) 0.03(0.68) 100
Q3 (16.53) Expected 50.0 2.90 3.52 2.60 −1.30 −1.30 0.00 0.00 0.00
I 49.95(0.65) 2.90(0.28) 3.26(0.64) 2.55(0.91) −1.32(0.92) −1.16(0.89) 0.28(0.54) −0.02(0.23) −0.26(0.52) 99.5
II 49.98(0.83) 2.93(0.29) 3.04(0.80) 2.65(0.97) −1.32(0.89) −1.28(0.92) 0.44(0.76) −0.04(0.33) −0.40(0.69) 100
III 50.00(0.77) 2.94(0.37) 2.89(0.91) 2.53(1.07) −1.25(0.98) −1.24(1.06) 0.68(1.05) −0.08(0.66) −0.59(1.00) 100
Q4 (12.65) Expected 73.0 −3.31 −1.87 0.00 0.00 0.00 −2.10 2.50 −0.40
I 74.18(2.97) −3.10(0.51) −1.72(0.41) −0.00(0.64) −0.03(0.71) 0.02(0.69) −1.89(0.46) 2.28(0.51) −0.36(0.34) 100
II 74.12(2.52) −3.15(0.48) −1.80(0.39) 0.02(0.75) 0.00(0.73) −0.04(0.72) −1.94(0.52) 2.30(0.56) −0.35(0.42) 100
III 74.38(2.12) −3.21(0.46) −1.84(0.49) −0.15(0.77) 0.05(0.82) 0.09(0.78) −1.87(0.69) 2.29(0.72) −0.39(0.64) 100
Q5 (2.36) Expected 15.0 1.92 0.00 0.00 0.00 0.00 0.00 0.00 0.00
I 14.54(3.90) 1.82(0.29) −0.16(0.61) −0.01(0.76) 0.02(0.78) 0.01(0.79) 0.20(0.45) −0.03(0.23) −0.17(0.43) 90.5
II 14.21(4.14) 1.86(0.29) −0.24(0.60) 0.09(0.81) 0.04(0.77) −0.12(0.79) 0.21(0.49) −0.04(0.27) −0.17(0.48) 74.5
III 15.52(4.89) 1.93(0.40) −0.06(0.62) −0.11(0.78) 0.03(0.97) 0.09(0.87) 0.32(0.78) 0.01(0.72) −0.33(0.79) 42.0
True values of QTL parameter;
estimates of QTL parameter and standard deviation in parenthesis; *A, D, AE, DE* denote the estimates of QTL additive, dominance effects and their interaction effects with environments, respectively; I,II and III represent three different constitutions of population, 300 (150 from DH×P~1~ and 150 from DH×P~2~), 300 (100 from DH×P~1~ and 200 from DH×P~2~) and 300 (50 DH×P~1~ and 250 from DH×P~2~), respectively.
######
Estimation of epistatic effects and their interaction effects with environments under three different constitutions of mapping population
Epistatic QTL (heritability %) Population Position\_*i* Positiona\_*j* *AA* *AAE1* *AAE2* *AAE3* Power
-------------------------------- ------------ ---------------------------------------------- ---------------- ------------- ------------- ------------- ------------- -------
EQ1 (4.98) Expected 44.0[a)](#TFN12){ref-type="table-fn"} 24.0 −3.09 2.52 −0.16 −2.36
I 43.82(2.96)[b)](#TFN13){ref-type="table-fn"} 23.07(3.03) −2.81(0.67) 2.15(0.73) −0.13(0.43) −2.02(0.69) 96.0
II 44.09(2.60) 22.89(3.35) −2.78(0.73) 2.11(0.75) −0.14(0.47) −1.96(0.72) 96.0
III 44.04(2.07) 23.23(3.19) −2.79(0.70) 2.03(0.87) −0.13(0.51) −1.91(0.83) 99.0
EQ2 (2.67) Expected 50.0 15.0 2.10 −2.10 0.20 1.90
I 49.96(0.58) 14.48(3.96) 2.07(0.58) −1.85(0.57) 0.13(0.38) 1.69(0.55) 72.5
II 49.93(0.97) 14.25(4.20) 1.98(0.48) −1.85(0.64) 0.21(0.44) 1.61(0.57) 56.5
III 49.94(0.82) 15.21(4.86) 2.07(0.61) −1.96(0.73) 0.25(0.58) 1.69(0.65) 34.0
EQ3 (2.10) Expected 24.0 79.0 −2.60 0.00 0.00 0.00
I 23.15(2.87) 79.44(3.33) −2.54(0.46) −0.02(0.23) −0.06(0.20) 0.07(0.27) 50.5
II 23.27(3.16) 80.22(3.53) −2.50(0.41) 0.06(0.31) −0.03(0.28) −0.04(0.21) 50.0
III 23.33(3.19) 79.62(3.30) −2.43(0.41) −0.01(0.22) −0.02(0.20) 0.03(0.24) 58.5
True values of the positions, additive-additive epistatic effects and their interaction with environment for paired epistatic QTLs;
estimate of parameter and standard deviation in parenthesis; *AA* and *AAE* denote estimates of additive by additive effects and their interaction effects with environments, respectively; I,II and III have same definition as that in [Table 6](#T6){ref-type="table"}, with false discovery rates of 0.0416, 0.0537, 0.0543, respectively.
|
{
"pile_set_name": "PubMed Central"
}
|
Introduction {#s1}
============
The stretch reflex is a neurophysiological response to the stimulation of muscle spindles. In the human triceps surae muscle group, the stretch reflex can be broadly divided into 3 components based on their onset latencies. The earliest response, the short latency reflex (SLR), is predominantly mediated by proprioceptive information from velocity-sensitive muscle spindle Ia afferents, but may also receive inputs from other sensory receptors [@pone.0023917-Burke1] and from the motor cortex [@pone.0023917-Zuur1]. During unconstrained human walking, SLR responses may not be naturally elicited in triceps surae muscles, but during the faster movement of running, the SLR has been observed in the gastrocnemius and soleus muscles as a short-lasting burst of activity superimposed on the pre-programmed activity that starts prior to ground contact [@pone.0023917-Dietz1], [@pone.0023917-Ishikawa1]. The size of the SLR dramatically decreases in the presence of ischemia, which is known to suppress Ia afferent activity [@pone.0023917-Dietz1], [@pone.0023917-Dietz2], confirming the major contribution of the Ia pathway to the SLR.
The precise function of the SLR during locomotion is a matter of ongoing study. It has been proposed on the basis of cat data that the SLR compensates for transient decreases in muscle stiffness in response to a joint perturbation [@pone.0023917-Nichols1], thereby minimising muscle yielding [@pone.0023917-Allum1]. In humans, evidence has been presented in support of this hypothesis in seated conditions [@pone.0023917-Allum1], [@pone.0023917-Allum2]. However, studies of SLR responses during the more complex task of human running have focused on the existence, timing and responsible neural pathways [@pone.0023917-Dietz1], [@pone.0023917-Ishikawa1], while the functional importance of the SLR is largely unexplored. In order to examine this issue during running, a method is required that can modify the size of the SLR. One potential method is high frequency Achilles tendon vibration, which decreases the efficacy of Ia afferent activity [@pone.0023917-Roll1], [@pone.0023917-Roll2]. Although muscle spindle type II and Golgi tendon organ (GTO) Ib afferents are also influenced by this method, they are much less sensitive to vibration than Ia afferents [@pone.0023917-Roll1], [@pone.0023917-Roll2], [@pone.0023917-Burke2], [@pone.0023917-Cordo1]. Tendon vibration decreases SLR amplitude in the human soleus muscle in response to a rapid dorsiflexion perturbation during standing [@pone.0023917-Bove1], [@pone.0023917-Nardone1], sitting [@pone.0023917-Allum1] and walking [@pone.0023917-Mazzaro1], and may thus be a suitable method of suppressing the SLR in running.
The purpose of this study was to examine triceps surae SLR function at different running speeds using Achilles tendon vibration to modify SLR size. Two hypotheses were tested: 1) High frequency Achilles tendon vibration would decrease SLR size in triceps surae muscles during running; 2) Assuming that the SLR is important for muscle stiffness regulation, a vibration-induced decrease in reflex amplitude, if sufficiently large, would lead to evidence of yielding at the ankle joint.
Materials and Methods {#s2}
=====================
Participants {#s2a}
------------
Ten healthy participants (7 males, 3 females; age 26±4 years; height 178±9 cm; body mass 71±12 kg) with no history of neurological, cognitive, metabolic, cardiovascular, pulmonary or lower limb musculoskeletal impairment volunteered to participate in this study. Prior to testing, participants were fully informed of the experimental procedures, and each participant provided written informed consent. The study was approved by the Griffith University Human Research ethics committee, and was performed in accordance with the Declaration of Helsinki.
Protocol {#s2b}
--------
Participants initially ran on an instrumented split-belt treadmill at a speed of 10 km/h for 2 minutes for familiarisation. Participants then ran at 7, 10, 12 and 15 km/h in a randomised order, with rest periods of 3--5 minutes between each speed to avoid fatigue. Two Achilles tendon vibration conditions were assessed at each speed: no vibration and 90 Hz vibration. Vibration was applied unilaterally to the right leg. Prior to data collection participants ran at each speed for a minimum of 30 s to enable adaptation to the speed. Subsequently, 30--40 s of data were collected for each vibration condition to allow meaningful data averaging. At each speed, the no vibration condition was always performed first to avoid after-effects of vibration that can last for several seconds [@pone.0023917-RibotCiscar1]. A minimum of 15--20 s elapsed between conditions. Participants were instructed to look forwards at all times to avoid possible effects of altered head and neck orientation on vestibulo/corticospinal-induced motoneurone excitability [@pone.0023917-Kennedy1] or neck proprioceptive input [@pone.0023917-Gottschall1].
Data collection and analysis procedures {#s2c}
---------------------------------------
### Electromyography (EMG) {#s2c1}
Surface EMG activity was recorded using bipolar surface electrodes (Duo-trode, Myotronics Inc; Australia) with an inter-electrode distance of 2 cm. Data were collected telemetrically (Noraxon Telemyo; AZ, USA) from the soleus (Sol), medial gastrocnemius (MG), lateral gastrocnemius (LG) and tibialis anterior (TA) muscles of the right leg at 1 kHz. EMG signals were band-pass filtered (10 Hz--500 Hz), rectified, low-pass filtered (40 Hz) and ensemble averaged to produce EMG profiles. Mean background EMG was quantified as the mean EMG throughout the stance phase.
To calculate SLR onset latencies, mean EMG was first calculated over the first 30 ms of the contact phase, i.e. prior to the commencement of the SLR which is approximately 40 ms at the earliest [@pone.0023917-Grey1], [@pone.0023917-Grey2], [@pone.0023917-Nardone2]. Onset latency was then determined as the first time point where this mean value was exceeded by 4 standard deviations. Visual inspection of all traces suggested that this procedure provided accurate and consistent latency estimates. SLR amplitude was defined as the difference between the level of activity at SLR onset and the size of the subsequent peak, thus taking into account differences in EMG activity at SLR onset between speeds [@pone.0023917-Dietz1], [@pone.0023917-Ishikawa1].
### Kinematics and ground reaction forces (GRFs) {#s2c2}
Kinematics of the pelvis and right leg were recorded using a 4 camera 3D motion analysis system (Vicon, Oxford Metrics; Oxford, UK) sampling at 100 Hz. Reflective markers were placed in accordance with the lower body model of Besier et al. [@pone.0023917-Besier1]. A marker was also placed on the vibrator to track displacement of the device relative to the ankle joint centre for each condition. Knee and ankle joint angles were determined from inverse kinematic analysis of the marker trajectories using Opensim software [@pone.0023917-Delp1]. GRFs were recorded separately from each leg at 1 kHz using 8 triaxial force sensors embedded in the split-belt treadmill (Bertec; OH, USA). At each speed and for each condition, steps were only included in the analysis if step duration was within ±5% of the averaged step duration for that condition, as determined from the treadmill GRF signals. This resulted in the inclusion of 32±2 steps per condition across all speeds. All reported EMG and GRF values were expressed relative to their respective peak values at 7 km/h.
### Ankle yielding {#s2c3}
Ankle and GRF difference curves were computed between the control and vibration mean traces over the time period 55--150 ms after ground contact, as this interval incorporates the expected time course of ankle yielding due to SLR depression [@pone.0023917-Allum1], [@pone.0023917-Stein1]. Ankle yielding was defined as a deviation in ankle trajectory between the control and vibration traces, and the amplitude and slope of this deviation were computed.
### Achilles Tendon vibration {#s2c4}
A servo-controlled custom-made vibrating motor (35×25 mm; 100 g) was attached over the Achilles tendon of the right leg using compressive tape designed to be sufficiently compliant to prevent blood flow occlusion but rigid enough to minimise movement. The motor was positioned approximately 3 cm proximal to the ankle joint. High frequency vibration was applied at 90 Hz as this frequency has been shown to produce optimal SLR depression [@pone.0023917-Nardone1], [@pone.0023917-Duysens1]. The motor was switched on approximately 5 s before data collection, remained on for the 30--40 s recording window, and was then switched off. Data from a typical participant running at 12 km/h are shown in [Figure 1](#pone-0023917-g001){ref-type="fig"}.
{#pone-0023917-g001}
Statistical analysis {#s2d}
--------------------
Two-way repeated measures ANOVA was used to assess the effects of vibration condition (no vibration, 90 Hz vibration) and locomotion speed (7, 10, 12, 15 km/h) on all outcome measures. Mauchly\'s test of sphericity was used to test the assumption of sphericity. Where this assumption was violated, Geisser-Greenhouse (GG) adjustments were used. Where significant main effects were observed, pair-wise comparisons were used to identify the location of differences between vibration conditions at each speed. For all tests, the minimum level of statistical significance was set at p\<0.05.
Results {#s3}
=======
Short latency reflex responses {#s3a}
------------------------------
Running speed had a significant main effect on SLR amplitude in all triceps surae muscles (soleus: F~2,\ 18~ = 1.36, p\<0.001; medial gastrocnemius: F~2,\ 18~ = 6.39, p\<0.001; lateral gastrocnemius: F~2,\ 18~ GG = 5.03, p\<0.01; [Figure 2A](#pone-0023917-g002){ref-type="fig"}). There was also a main effect of vibration on SLR amplitude in soleus (F~2,\ 18~ = 7.656, p\<0.05), LG (F~2,\ 18~ GG = 5.120, p\<0.05) and MG (F~2,\ 18~ = 8.434, p\<0.05; [Figure 2B](#pone-0023917-g002){ref-type="fig"}). The effects of these changes on ankle joint kinematics are shown in [Figure 2C](#pone-0023917-g002){ref-type="fig"}. Neither running speed nor vibration had a significant main effect on SLR latency in any of the examined muscles (soleus: F~2,\ 18~ = 0.97--1.05, p = 0.426--0.563; medial gastrocnemius: F~2,\ 18~ GG = 0.57--0.74, p = 0.651--0.701; lateral gastrocnemius: F~2,\ 18~ = 0.404--0.632, p = 0.558--0.753).
{#pone-0023917-g002}
Background muscle activation {#s3b}
----------------------------
During running at 7 km/h, average stance phase EMG decreased in Sol due to 90 Hz vibration (F~2,\ 18~ = 5.51, p = 0.017), but was not statistically altered by vibration at all other speeds (F~2,\ 18~ = 5.51, p = 0.100--0.938). Vibration had no statistically significant effects on average EMG in LG (F~2,\ 18~ = 1.09, p = 0.365), MG (F~2,\ 18~ = 0.10, p = 0.908) or the antagonist TA (F~2,\ 18~ GG = 1.06, p = 0.347). Average EMG was also computed in the period of stance that preceded the SLR response (approximately 0--40 ms, depending on SLR onset). No statistical differences were observed between conditions in Sol (F~2,\ 18~ = 7.70, p = 0.726), LG (F~2,\ 18~ GG = 1.39, p = 0.348) or MG (F~2,\ 18~ = 6.60, p = 0.405). Furthermore, no changes were evident in the TA muscle in the 0--40 ms time window (F~2,\ 18~ GG = 0.22, p = 0.685).
Global effects of vibration on kinematics and GRFs {#s3c}
--------------------------------------------------
Vibration had no statistically significant effects on any kinematic or GRF parameters when compared over the entire stance or step cycle. These included the range of ankle joint rotation during the stance phase (F~2,\ 18~ = 0.46, p = 0.645) and the entire step cycle (F~2,\ 18~ GG = 3.40, p = 0.100); ankle angle at ground contact (F~2,\ 18~ = 1.33, p = 0.300); range of knee joint rotation during stance (F~2,\ 18~ = 1.56, p = 0.257) and the entire step cycle (F~2,\ 18~ GG = 0.20, p = 0.694); knee angle at ground contact (F~2,\ 18~ = 1.37, p = 0.291); the area under the vertical GRF curve (F~2,\ 18~ = 4.60, p = 0.926) and peak vertical GRF (F~2,\ 18~ GG = 0.08, p = 0.830). Regardless of speed, the duration of the stance phase (F~2,\ 18~ = 3.63, p = 0.350) and the entire step cycle (F~2,\ 18~ GG = 1.28, p = 0.295) were both statistically unaffected by vibration.
Local effects of vibration on kinematics and GRFs {#s3d}
-------------------------------------------------
Vibration-induced depression of SLR amplitude in the triceps surae muscles led to ankle yielding starting approximately 60 ms after ground contact and lasting approximately 80 ms. There was a main effect of both vibration condition (F~2,\ 18~ = 3.128, p\<0.05) and running speed (F~2,\ 18~ = 2.847, p\<0.05) on the amplitude of ankle yielding. There was also a main effect of vibration condition (F~2,\ 18~ GG = 4.430, p\<0.05) and running speed (F~2,\ 18~ GG = 20.826, p\<0.01) on the velocity of ankle yielding. There was no main effect of vibration condition (F~2,\ 18~ = 0.338, p = 0.577) or running speed (F~2,\ 18~ = 2.113, p = 0.125) on yielding in the vertical GRF trace. Mean SLR and ankle yield data are shown in [Figure 2](#pone-0023917-g002){ref-type="fig"}.
Vibration efficacy {#s3e}
------------------
The mean range of displacement of the vibrating motor relative to the ankle joint centre ranged between 0.93±0.25 and 3.17±2.0 mm. Neither vibration condition (F~2,\ 18~ = 1.19, p = 0.345) nor gait speed (F~2,\ 18~ = 1.48, p = 0.141) significantly influenced the range of vibrator displacement. To determine whether the effects of vibration were altered over the course of a trial, the level of SLR depression due to vibration was compared between the first and last 5 steps of each 90 Hz vibration trial. Across all speeds there was no main effect in Sol (F~2,\ 18~ = 0.37, p = 0.812), LG (F~2,\ 18~ = 1.03, p = 0.528) or MG (F~2,\ 18~ = 0.78, p = 0.611).
Discussion {#s4}
==========
This study sought to examine the function of the short latency stretch reflex in human triceps surae muscles during running, using tendon vibration to modify the strength of the reflex responses. In support of our hypotheses, vibration produced clear decrements in triceps surae SLR size at all of the examined speeds. At running speeds between 7 and 12 km/h, where SLR amplitude was largest, vibration-induced depression of the SLR led to yielding at the ankle joint, suggesting that the SLR contributes to muscle stiffness regulation during running by minimising ankle yielding during the early contact phase. At the fastest running speed of 15 km/h, where the SLR was generally at its smallest, vibration still clearly decreased SLR size in all muscles, but ankle yielding was no longer evident. This suggests that the SLR plays a more important functional role at slow to intermediate running speeds than at faster speeds.
The origin of short latency stretch reflexes in running {#s4a}
-------------------------------------------------------
A fundamental issue in this study is whether the SLR responses observed here are genuine reflex responses or the result of a sudden increase in pre-programmed activity. In running, Dietz et al. [@pone.0023917-Dietz1] reported bursts of activity at latencies as early as 30--40 ms after ground contact in the medial gastrocnemius muscle. These responses were attributed to short latency reflexes since ischemia clearly suppressed the size of the EMG burst. In contrast, Ishikawa and Komi [@pone.0023917-Ishikawa1] reported considerably longer, speed-dependent onset latencies in the medial gastrocnemius but did not attempt to verify the reflexive nature of the responses. Our data using tendon vibration to suppress the EMG burst are consistent with the findings of Dietz et al. [@pone.0023917-Dietz1], suggesting that SLRs occur approximately 40 ms after ground contact in triceps surae muscles during running, and that their onset latencies are unaffected by speed.
The discrepancy in the literature regarding SLR latencies raises further questions concerning the stimulus for the SLR. Ishikawa and Komi [@pone.0023917-Ishikawa1] attributed the SLR to a rapid stretch of the muscle fascicles following foot contact, based on the observation that the time between fascicle stretch onset and SLR onset was constant at different running speeds. However, muscle fascicle stretch commenced 21--37 ms after ground contact. This would occur too late to trigger an SLR with a latency of 40 ms, as this value corresponds to the shortest possible SLR latency in these muscles [@pone.0023917-Nardone2]. The latencies observed here and by Dietz et al. [@pone.0023917-Dietz1] are consistent with the alternative view that the stimulus for the SLR is the propagation of mechanical vibration that results from foot-ground contact, and subsequently excites muscle spindles. In response to an Achilles tendon tap or foot sole vibration, SLRs are evoked in triceps surae muscles [@pone.0023917-Burke1], as well as in more proximal leg muscles not exposed to stretch [@pone.0023917-Duysens1], [@pone.0023917-Lance1]. The SLR can even be elicited during muscle shortening, further suggesting that the response is not triggered by fascicle stretch [@pone.0023917-Burke1], [@pone.0023917-Lance1], although this does not necessarily preclude an effect of fascicle stretch parameters on later reflex components. We attribute the stimulus of the SLR responses observed in this study to the transmission of mechanical vibration beginning at foot-ground contact, which can account for the consistent SLR latencies of approximately 40 ms observed here. A similar hypothesis has been proposed to account for SLR responses evoked during stumbling over an obstacle [@pone.0023917-Schillings1].
The functional importance of the SLR during running {#s4b}
---------------------------------------------------
When an isometrically contracting muscle is rapidly stretched, the amplitude of the resulting SLR is generally largest when intrinsic muscle stiffness is low, and thus muscle yielding is more likely to occur [@pone.0023917-Cronin1], [@pone.0023917-Sinkjaer1]. Conversely, as muscle force increases, intrinsic stiffness also increases, so the likelihood of muscle yielding in response to the same stretch stimulus decreases, resulting in a smaller SLR [@pone.0023917-Nichols1], [@pone.0023917-Allum1], [@pone.0023917-Sinkjaer1], [@pone.0023917-Hoffer1], [@pone.0023917-Nichols2]. Data from the present study provide support for this hypothesis in running, as vibration-induced depression of SLR responses coincided with high velocity ankle yielding at running speeds between 7 and 12 km/h, where SLR amplitude was largest. At the fastest running speed of 15 km/h, where intrinsic muscle stiffness would be expected to be higher, vibration still depressed the SLR in all muscles but ankle yielding was not observed. The SLR was also generally smallest at this speed. These findings suggest that the functional importance of the SLR declines at fast running speeds in this muscle group, as is the case at high force levels in isometric conditions.
Yielding at a joint may have significant functional implications during locomotion. For example, in cats, weakening of the ankle extensors by denervation of certain muscles leads to dramatic yielding at the ankle that cannot be immediately compensated for, resulting in severe disruption of the kinematic patterns at the ankle and knee [@pone.0023917-Pearson1], [@pone.0023917-Whelan1]. Yielding would also be expected to lengthen the muscle fascicles, which could alter the force-generating potential of the muscle and thus influence locomotor energetics. With regard to long-term implications, changes to the nervous system during development and after injury or training would require adaptation of reflex input in order to maintain optimal motor output and minimise yielding during locomotion [@pone.0023917-Whelan1].
Mechanisms of vibration-induced SLR depression during running {#s4c}
-------------------------------------------------------------
It is well established that the velocity-sensitive Ia pathway makes an important contribution to the SLR. Tendon vibration generally exerts its most potent effects on Ia afferents, which are more sensitive to vibration than type II or Ib fibres [@pone.0023917-Fallon1]. Accordingly, vibration led to clear suppression of SLR responses in this study. However, several other pathways may contribute to the SLR including Ib afferents, cutaneous receptors and mechanoreceptors in other muscles [@pone.0023917-Burke1], as well as a potential role of pre-programmed input from the motor cortex [@pone.0023917-Zuur1]. As the vibrating motor was switched on several seconds before data collection began, vibration may have suppressed ongoing activity from sensory receptors such as spindle type II and cutaneous afferents, which could in turn have modified the net Ia input to the motoneurones or the excitability of the motoneurones directly. Nonetheless, it is noteworthy that tizanidine (a selective group II afferent inhibitor) and lidocaine (a cutaneous afferent inhibitor), both of which require a longer time frame than vibration to take effect, do not influence mechanically evoked SLRs during locomotion [@pone.0023917-Grey1]. The relative contribution of each of the pathways contributing to the SLR may change at different running speeds and in different muscles. Therefore, the observed patterns of SLR modulation are not simply a reflection of changes in Ia afferent activity.
Methodological considerations {#s4d}
-----------------------------
In some participants we observed a transient fluctuation in vibration frequency of approximately ±5 Hz shortly after ground contact. Changes of such small magnitude are unlikely to affect vibration efficacy, as vibration frequencies between 70--100 Hz have been shown to decrease Ia-mediated responses [@pone.0023917-Allum1], [@pone.0023917-Roll2], [@pone.0023917-Courtine1]. Previous studies have shown that unilateral Achilles tendon vibration has no effects on joint displacements of the non-vibrated limb, suggesting that intra-limb coordination is unaffected by this paradigm, at least during walking [@pone.0023917-Verschueren1]. It should be noted that the amplitude of ankle yielding observed at running speeds between 7 and 12 km/h may have been substantially greater if a more potent Ia afferent blocking technique, such as ischemia, was used. We elected not to use this method because of its numerous limitations including the time required to induce ischemia, the limited time frame allowed for data collection and pain in the affected limb (see [@pone.0023917-Leukel1]). Using vibration we observed peak ankle yielding of 2.3°, which represents approximately 5% of the ankle range of motion during slow running. Allum et al. [@pone.0023917-Allum1] reported clearer evidence of yielding in seated conditions, although in their study vibration produced SLR decrements of up to 80%, which is much larger than the values obtained in this study. Yielding is also the resultant effect of changes in all plantar flexor muscles, several of which were not examined in this study. It is therefore likely that our data underestimate the extent of yielding that would occur in the absence of SLR activity, and thus the functional importance of the SLR.
Conclusions {#s4e}
-----------
During human running, SLR responses have long been known to occur in triceps surae muscles, but their functional relevance has not been determined in this context. The results of the present study showed that suppression of predominantly Ia afferent-mediated SLR responses using Achilles tendon vibration led to evidence of ankle yielding at slow to intermediate running speeds, but not at the fastest speed of 15 km/h. These results provide strong evidence for a role of the SLR in ankle stiffness regulation during the early contact phase of human running. In addition, our results suggest that the functional importance of the SLR in triceps surae muscles is speed-dependent, being greater at slow to intermediate running speeds than at faster speeds.
The authors gratefully acknowledge Professor Marco Schieppati and Professor Richard Nichols for critical evaluation of a previous version of this manuscript.
**Competing Interests:**The authors have declared that no competing interests exist.
**Funding:**NC was supported by a New Researcher Grant from Griffith University. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
[^1]: Conceived and designed the experiments: NJC CPC RSB. Performed the experiments: NJC CPC. Analyzed the data: NJC CPC. Wrote the paper: NJC CPC RSB.
|
{
"pile_set_name": "PubMed Central"
}
|
Introduction
============
Idiopathic central serous chorioretinopathy (ICSCR), first described by von Graefe in 1866, is characterized by idiopathic exudative detachment of the neurosensory retina in the posterior pole and one or more leaks from the retinal pigment epithelium. The precise pathophysiology of ICSCR is still unclear. Impairment in resorptive capacity and barrier function in retinal pigment epithelium was once considered to be an important contributing factor. However, Negi et al[@b1-tcrm-10-037],[@b2-tcrm-10-037] reported that simple dysfunction of retinal pigment epithelium could not lead to retinal detachment. Gass[@b3-tcrm-10-037] postulated that hyperpermeability of the choriocapillaris resulted in exudation of fluid into the subretinal space. Studies using indocyanine green angiography (ICGA) revealed that abnormalities of the choroidal vasculature, including hyperpermeability[@b4-tcrm-10-037]--[@b10-tcrm-10-037] and venous congestion,[@b10-tcrm-10-037]--[@b12-tcrm-10-037] may be the essential elements of ICSCR.[@b10-tcrm-10-037],[@b11-tcrm-10-037] Other studies[@b13-tcrm-10-037] using high-resolution ultrasonography have also shown hyperdynamic circulation within the choroid of eyes with ICSCR.[@b14-tcrm-10-037],[@b15-tcrm-10-037]
Focal laser photocoagulation is a classic treatment for acute ICSCR, and can shorten the duration by about 2 months.[@b16-tcrm-10-037] However, whether it would reduce recurrence rates is controversial. A prospective, randomized clinical trial[@b17-tcrm-10-037] found that the final recurrence rate in a laser photocoagulation group was unchanged. Recurrent attacks always lead to widespread decompensation of retinal pigment epithelium and permanent vision loss. Photodynamic therapy can decrease hyperpermeability in the choroid and reduce leakage from retinal pigment epithelium, hence reducing the recurrence rate.[@b18-tcrm-10-037]--[@b22-tcrm-10-037] Several studies[@b23-tcrm-10-037]--[@b25-tcrm-10-037] have reported that photodynamic therapy has a dose-dependent effect, and that reducing the dose of verteporfin could diminish the adverse effects in the retina and choroid. Zhao et al[@b26-tcrm-10-037] demonstrated that a 30% dose of verteporfin is safe and effective for acute ICSCR in Chinese patients.
The rapid development of imaging techniques has improved our understanding of the pathogenesis of ICSCR. Optical coherence tomography, which was invented in the late 1990s, has become an indispensible technique in evaluating and managing many ocular disorders. However, due to the signal loss occurring in the image path and the wavelength-dependent light scattering, conventional optical coherence tomography could not image the choroid clearly. Recently, Spaide et al[@b27-tcrm-10-037] described commercially available enhanced depth imaging spectral-domain optical coherence tomography (EDI-OCT) which enables quantitative measurement of choroidal thickness and topography[@b27-tcrm-10-037]--[@b32-tcrm-10-037] in various diseases, such as ICSCR,[@b33-tcrm-10-037] myopia,[@b34-tcrm-10-037] age-related macular degeneration,[@b28-tcrm-10-037],[@b35-tcrm-10-037] diabetes,[@b36-tcrm-10-037] and glaucoma.[@b37-tcrm-10-037]
Maruko et al[@b38-tcrm-10-037] reported that subfoveal choroidal thickness could be decreased using a half dose of photodynamic therapy in patients with ICSCR. In the current study, we performed EDI-OCT and ICGA in symptomatic eyes and fellow eyes of patients with acute ICSCR and evaluated the potential changes in subfoveal choroidal thickness after a 1/2 dose of photodynamic therapy in Chinese patients.
Materials and methods
=====================
Study design and patient recruitment
------------------------------------
This retrospective observational study was performed in consecutive patients diagnosed with unilateral acute ICSCR at the Department of Ophthalmology, Yellow River Hospital, Henan University of Science and Technology, Sanmenxia, Henan Province, People's Republic of China, from July 2011 to March 2013. The study received approval from the institutional review board of Yellow River Hospital (approval number 2011-07-03). Written informed consent was obtained from all patients, and the procedures used followed the tenets of the Declaration of Helsinki. For the comparative study, a group of healthy sex-matched and age-matched (±5 years) subjects were recruited from our students, hospital staff, and families.
Diagnosis of acute ICSCR
------------------------
All participants underwent the following clinical examination: best-corrected visual acuity, slit-lamp biomicroscopy, indirect ophthalmoscopy, fluorescein angiography, and ICGA (TRC-50DX/IMAGEnet 2000; Topcon Medical Systems Inc, Tokyo, Japan). Acute ICSCR was diagnosed if patients had: subretinal fluid involving the macula associated with idiopathic leaks from the retinal pigment epithelium seen on fluorescein angiography; subretinal fluid confirmed by optical coherence tomography; and persistent serous retinal detachment for less than 6 months without widespread decompensation of the retinal pigment epithelium.
Inclusion and exclusion criteria
--------------------------------
Inclusion criteria were: acute ICSCR; only a single eye with neurosensory retina or pigment epithelial detachment; age 20--70 years; and no spontaneous resolution after 3 months of conservative management. Exclusion criteria were: chronic ICSCR; a history of ocular disease, such as polypoidal choroidal vasculopathy, myopia, or age-related macular degeneration; a history of ocular surgery or photocoagulation; pregnancy; steroid use; and any other systemic disease.
Photodynamic therapy in acute ICSCR
-----------------------------------
The photodynamic therapy protocol was performed using a one third dose (2 mg/m^2^) of verteporfin (Visudine®; Novartis, Basel, Switzerland) to treat patients with acute ICSCR.[@b20-tcrm-10-037] After intravenous administration of the 1/3 dose of verteporfin for 10 minutes, we then used a 689 nm laser system (Carl Zeiss, Dublin, CA, USA) to deliver an activating laser (dose 50 J/cm^2^) to the fundus of each patient. The exposure time was 83 seconds. The laser spot size was the diameter of the hyperpermeability region of ICGA plus 500 μm.
Examination of subfoveal choroidal thickness
--------------------------------------------
Subfoveal choroidal thickness was evaluated in the patients with acute ICSCR at baseline and 3 days, one week, 4 weeks, and 12 weeks after photodynamic therapy. The choroid was observed by EDI-OCT (Spectralis HRA + OCT, Heidelberg Engineering, Heidelberg, Germany). The device was positioned close to the eye to obtain inverted images of the fundus, but the images were reinverted for display. Eye tracking was used during measurement. Seven sections, each containing 25 averaged raw scans, were obtained within a 5--30 degree rectangle centered on the fovea. Choroidal thickness (defined as the zonal area between the inner scleral surface and the outer retinal pigment epithelium surface) was measured subfoveally on the transfoveolar scan using the manufacturer's software (Heidelberg Eye Explorer version 1.6.1.0). Although the thickest section of the choroid might not be at the subfoveal region, in this study we measured the subfoveal choroidal thickness as an indicator to avoid selection bias. All measurements were performed independently by two experienced examiners. If the measurements differed by more than 15%, the examiners performed the measurement together twice.
Statistical analyses
--------------------
All data was analyzed by PASW statistics version 18.0 (SPSS Inc, Chicago, IL, USA). The independent *t*-test, paired *t*-test, or least significant difference *t*-test was used to evaluate subfoveal choroidal thickness. Fisher's Exact tests were used to compare the difference in other clinical data. Results were considered to be statistically significant at *P*-values of 0.05.
Results
=======
Baseline demographic characteristics
------------------------------------
A total of 63 patients were finally enrolled ie, 31 patients in the photodynamic therapy group and 32 patients in the control group. There was no statistically significant difference between the two groups with regard to mean age and sex (*P*=0.68 and *P*=0.78, respectively). The mean spherical equivalent refractive error in the photodynamic therapy group was −1.69±2.24 diopters compared with −2.54±1.96 diopters in the control group; the difference was statistically significant (*P*=0.02). The mean interval between onset of symptoms and treatment in the photodynamic therapy group was 45.74±22.85 weeks ([Table 1](#t1-tcrm-10-037){ref-type="table"}).
Characteristics of ICGA
-----------------------
Of the 64 eyes from the 32 patients in the control group, ten (15.63%) had hyperfluorescence defined as choroidal vascular hyperpermeability in the middle phase of ICGA and 54 eyes (84.37%) did not have hyperfluorescence.
In the photodynamic therapy group, before treatment, 30 (96.78%) symptomatic eyes and 23 (74.19%) fellow eyes from 31 patients showed a mottling hyperfluorescence in the middle phase of ICGA. Four weeks after treatment, we found that 20 (64.51%) symptomatic eyes showed hypofluorescence corresponding to the area of photodynamic therapy irradiation at the posterior pole. The rate of choroidal vascular hyperpermeability in the fellow eye remained unchanged. This phenomenon persisted throughout the study.
Changes in subfoveal choroidal thickness
----------------------------------------
The mean subfoveal choroid thickness changes at baseline and follow-up are summarized in [Tables 2](#t2-tcrm-10-037){ref-type="table"}, [3](#t3-tcrm-10-037){ref-type="table"} and [Figure 1](#f1-tcrm-10-037){ref-type="fig"}. At baseline, the mean subfoveal choroid thickness in symptomatic eyes was significantly greater than in fellow eyes (422±132 μm and 367±114 μm, respectively, *P*=0.036). Mean subfoveal choroid thickness in the fellow eyes was significantly greater than in the control group (367±114 μm and 274±53 μm, respectively, *P*\<0.001).
Based on the ICGA, the mean subfoveal choroid thickness of fellow eyes with hyperfluorescence was significantly greater than those without hyperpermeability (446±105 μm versus 279±115 μm, *P*\<0.001). When comparing the difference between subfoveal choroidal thickness in the fellow eyes without hyperfluorescence and that in the control group, we found a negative result (279±115 μm versus 274±53 μm, *P*=0.520).
After photodynamic therapy, the mean subfoveal choroidal thickness increased significantly from 422±132 μm at baseline to 478±163 μm at day 3 (*P*=0.022) and then decreased to 362±113 μm at week 4 (*P*\<0.001 versus baseline) and to 339±135 μm at week 12 (*P*\<0.001 versus baseline, [Figure 1](#f1-tcrm-10-037){ref-type="fig"}). During follow-up, an interesting case occurred in the photodynamic therapy group ([Figure 2](#f2-tcrm-10-037){ref-type="fig"}).
Absorption of subretinal fluid
------------------------------
At successive follow-ups, the subretinal fluid in 19 patients decreased significantly at 4 weeks after photodynamic therapy, but not at day 3 and week 1. After 12 weeks of follow-up, the subretinal fluid in 25 patients was already fully absorbed.
Safety
------
During follow-up, no adverse systemic or ocular events occurred.
Discussion
==========
In the current study, we used EDI-OCT to evaluate changes in subfoveal choroid thickness after photodynamic therapy in patients with acute ICSCR. Our findings support the view that photodynamic therapy can decrease choroidal vascular hyperpermeability and choroidal thickness. Our findings also showed that the subfoveal choroid in patients with acute ICSCR was thicker than in healthy subjects, with the subfoveal choroid in symptomatic eyes being significantly thicker than in the fellow eyes. To our knowledge, this is the first study using EDI-OCT to quantitatively assess changes in subfoveal choroidal thickness after a one third dose of photodynamic therapy in a relatively large sample of patients with acute ICSCR.
Subfoveal choroidal thickness in healthy subjects has been reported to be between 261 μm and 354 μm.[@b29-tcrm-10-037],[@b30-tcrm-10-037],[@b39-tcrm-10-037]--[@b42-tcrm-10-037] Our study showed that mean subfoveal choroidal thickness in the control group was 274 μm. The variation in results from the different studies may be attributable to variations in patient age, sex, axial length, and refractive condition, as well as the optical coherence tomography device used. Patient age[@b28-tcrm-10-037],[@b30-tcrm-10-037],[@b40-tcrm-10-037],[@b43-tcrm-10-037] has been recognized as the most influential factor in choroidal thickness. An EDI-OCT-based study undertaken by Margolis and Spaide[@b30-tcrm-10-037] reported mean decreases in choroidal thickness of 15.6 μm per year in normal eyes. Ikuno et al[@b40-tcrm-10-037] confirmed this finding. However, this theory might not suit for ICSCR patients. Imamura et al[@b33-tcrm-10-037] reported that choroidal thickness did not correlate with age in ICSCR patients (*r=*0.15, *P*=0.43). Axial length is another important influencing factor. Li et al[@b42-tcrm-10-037] reported that subfoveal choroidal thickness decreased by 58.2 μm per mm increase in axial length adjusted for age and sex. Esmaeelpour et al[@b44-tcrm-10-037] agreed with this view, and found that axial length was the most important determinant of choroidal thickness. Further, sex has been reported as another influential factor in choroidal thinning. In a multiple regression model,[@b42-tcrm-10-037] after adjustment for age and axial length, subfoveal choroidal thickness was on average 62 μm thicker in men than in women (95% confidence interval 21--104; *P*=0.0039). In contrast, Park and Oh[@b45-tcrm-10-037] analyzed the association between sex and choroidal thickness, and found that the mean subfoveal thickness in males was 9 μm thicker than in females, but the difference was not significantly different (*P*\>0.05). Moreover, an association between refractive condition and subfoveal choroidal thickness has also been reported. Ho et al[@b46-tcrm-10-037] suggested that subfoveal choroidal thickness decreased by 6.205 μm for each diopter in patients with myopia. However, ICSCR patients are often hyperopic. In the current study, the mean spherical equivalent refractive error in the photodynamic therapy group was −1.69±2.24 diopters compared with −2.54±1.96 diopters in the control group; this difference reached statistical significance (*P*=0.02). This might be one reason why the subfoveal choroid in patients with ICSCR is thicker than in the normal population.
Most acute ICSCR is actually bilateral, even if subretinal fluid is active in one eye. Choroidal vascular hyperpermeability might have a crucial role in ICSCR. In an ICGA study, Iida et al[@b10-tcrm-10-037] found that the incidence of choroidal vascular hyperpermeability was 62% in fellow eyes compared with 95% in symptomatic eyes. In the current study, 96.78% of symptomatic eyes and 74.19% of fellow eyes showed hyperfluorescence in the middle phase of ICGA in the photodynamic therapy group. This is similar to the findings of a study reported by Maruko et al.[@b47-tcrm-10-037] Choroidal vascular hyperpermeability is always accompanied by choroid thickening in ICSCR patients. Imamura et al[@b33-tcrm-10-037] reported that the choroid in patients with ICSCR was thicker bilaterally (including for symptomatic eyes and fellow eyes) than in normal eyes. In our study, the subfoveal choroid in symptomatic eyes was significantly thicker than in fellow eyes (*P*=0.036), and the subfoveal choroid in fellow eyes was significantly thicker than in age-matched and sex-matched control eyes (*P*\<0.001). The subfoveal choroid thickness of the fellow eyes with hyperfluorescence in ICGA was significantly higher than that in those without hyperpermeability (*P*\<0.001). When we compared the difference between subfoveal choroidal thickness in the fellow eyes without hyperfluorescence and the control eyes, we found a negative result (*P*=0.520). These findings indicate that choroidal vascular hyperpermeability plays an important role in pathophysiology of ICSCR.
Photodynamic therapy with verteporfin, which has an occlusive effect in the choriocapillary layers, could decrease choroidal vascular hyperpermeability and subsequently choroidal thickness.[@b18-tcrm-10-037],[@b21-tcrm-10-037] Schlotzer-Schrehardt et al[@b24-tcrm-10-037] and Schmidt-Erfurth et al[@b25-tcrm-10-037] reported that photodynamic therapy has a dose-dependent profile. Increasing the dose of verteporfin or laser irradiation would increase the risk of vascular damage or cytotoxicity to the retina and choroid. It is important to balance the maximum therapeutic effect and the minimum complications. Half-dose photodynamic therapy[@b38-tcrm-10-037] has been used widely to treat ICSCR, but might not be suitable for Chinese patients. Zhao et al[@b26-tcrm-10-037] used photodynamic therapy with various doses of verteporfin (70%, 60%, 50%, 40%, 30%, 20%, and 10% of the full dose) to manage ICSCR patients, and their results showed that 30% of the full dose of verteporfin was the lowest effective dose. In our study, following the recommendation of Zhao et al, we chose the 30% dose of verteporfin to manage our ICSCR patients. To our surprise, the mean choroidal thickness increased significantly from 422±132 μm at baseline to 478±163 μm at day 3. This transient increase in choroidal thickness may be a sign of increased exudation in the choroid. Eventually, after impairment of the choriocapillaris and vascular remodeling in the underlying choroid the choroidal thickness decreased to 362±113 μm at week 4 and to 339±135 μm at week 12. Accompanying the changes in choroidal thickness, at week 4, we found that 64.51% of symptomatic eyes showed hypofluorescence corresponding to the area of photodynamic therapy irradiation at the posterior pole. Maruko et al[@b38-tcrm-10-037] reported similar findings, ie, mean choroidal thickness increased from 389±106 μm at baseline to 462±124 μm (*P*=0.008) on day 2 after treatment, then reduced to 360±100 μm at week 1 and to 330±103 μm at week 4, with ICGA also showing decreased hyperpermeability Maruko et al[@b38-tcrm-10-037] used photodynamic therapy with a half dose (3 mg/m^2^) of verteporfin, which was higher than that (2 mg/m^2^) used in our study, but with similar efficacy Recently, a prospective, nonrandomized consecutive, open-label case series conducted by Uetani et al[@b48-tcrm-10-037] reported an opposite outcome. In that study, Uetani et al compared the effects of a half-dose of verteporfin with those of a one-third dose of verteporfin for ICSCR. In the half-dose photodynamic therapy group, choroidal thickness reduced significantly from baseline. However, in the 1/3 dose photodynamic therapy group, choroidal thickness decreased in two eyes in which subretinal fluid disappeared but did not change in the other eyes. However, only six patients were assigned to the photodynamic therapy group in that study, so the sample size was relatively small which might have led to bias.
Our retrospective study has some limitations. Acute ICSCR is well known to recur in some cases, so 3 months of follow-up seems to be short. A long-term study to evaluate the changes in subfoveal choroidal thickness and recurrence rate after photodynamic therapy is necessary. Additionally, the current EDI-OCT does not have automatic choroid segmentation software. Manual operation might lead to inaccuracy in some cases, so automatic choroid segmentation software is needed in future research. Moreover, the relationship between severity of hyperpermeability in ICGA and choroidal thickening should be investigated.
In conclusion, our findings revealed that: the subfoveal choroid in patients with acute ICSCR was thicker than in the normal population, with symptomatic eyes being significantly thicker than in fellow eyes; compared with half-dose or full-dose photodynamic therapy, a one-third dose of verteporfin could decrease choroidal vascular hyperpermeability and choroidal thickness in patients with acute ICSCR.
**Disclosure**
The authors report no conflicts of interests in this work.
{#f1-tcrm-10-037}
{#f2-tcrm-10-037}
######
Baseline demographic characteristics
PDT group (n=31) Control group (n=32) *P*-value
---------------------------------------------------------- ------------------ ---------------------- -----------
Age (years)
Mean ± SD 45.63±6.24 47.24± 5.9 0.68
Range 28--67 22--69
Sex
Male to female 26:5 27:5 0.78
Spherical equivalent refractive error (diopters)
Mean ± SD −1.69±2.24 −2.54±1.96 0.02
Range −3.50 to 2.75 −3.75 to 2.75
Interval between onset of symptoms and treatment (weeks)
Mean ± SD 45.74±22.85 -- --
Range 28--62 -- --
**Abbreviations:** PDT, photodynamic therapy; SD, standard deviation.
######
Subfoveal choroidal thickness at baseline
PDT group Control group (n=64)
----------- ----------- ---------------------- ---------- ---------
Mean ± SD 422±132 446±105 279±115 274±53
Range 136--642 331--642 136--372 97--335
**Notes:** Among-group comparison using Student's independent or paired *t*-tests. *P*-values\<0.05 are considered to be statistically significant.
**Abbreviations:** ICGA, indocyanine green angiography; PDT, photodynamic therapy; SD, standard deviation.
######
Subfoveal choroidal thickness during follow-up
PDT group (symptomatic eyes, n=31) PDT group (fellow eyes, n=31)
-------------------------------------- ------------------------------------ -------------------------------
Choroidal thickness at baseline (μm)
Mean ± SD 422±132 367±114
Range 136--642 113--573
Three days follow-up (μm)
Mean ± SD 478±163 371±123
Range 157--668 121--569
Four weeks follow-up (μm)
Mean ± SD 362±113 368±134
Range 112--574 108--581
Twelve weeks follow-up (μm)
Mean ± SD 339±135 368±121
Range 108--578 115--575
**Notes:** Among-group comparison using Student independent or paired *t*-tests. *P*-values\<0.05 were considered to be statistically significant.
**Abbreviations:** PDT, photodynamic therapy; SD, standard deviation.
[^1]: \#These authors contributed equally to this work
|
{
"pile_set_name": "PubMed Central"
}
|
Background {#Sec1}
==========
Gambling disorder is known to cause severe harm on both individual and societal levels (Langham et al. [@CR27]). Estimations of point prevalence of gambling disorder in adult populations vary between .2 and 2% globally (Lorains et al. [@CR29]; Grant et al. [@CR21]; Van Patten et al. [@CR51]). While the prevalence estimations have been stable during the past decade, the proportion with the most serious problems seems to increase, and it has been shown that minority groups and people with low socioeconomic status are at higher risk of developing gambling problems than the general population (Abbott et al. [@CR2]; Van Patten et al. [@CR51]). These trends illustrate the need of preventive actions in vulnerable groups, and development of effective treatments.
According to a systematic review, the prevalence of gambling problems in offender populations is remarkably high, with an average of about 33%, and a high degree of variability (5--73%, problem and pathological gambling combined) in the estimates, depending on differences in assessment methods, size and quality of the studies (Williams et al. [@CR56]). Despite this well known connection, and the fact that criminality is associated with a number of different psychiatric diagnoses, there are relatively few studies that focus on criminality and gambling. Both substance use disorders and antisocial personality disorder are highly overrepresented among offenders, and also connected to gambling disorder (Slutske et al. [@CR44]; Pietrzak and Petry [@CR39]; Lorains et al. [@CR29]). During the past four decades, a total of 37 studies focusing on prevalence of gambling disorder in offender populations have been performed; 27 articles with varying methods are included in the review by Williams and colleagues ([@CR56]), five more are summed in the literature part of a study by Zurhold et al. ([@CR59]), and another five have been published the last years (Kerber et al. [@CR25]; Turner et al. [@CR48]; May-Chahal et al. [@CR32]; Tessenyi and Kovacs [@CR46]; April and Weinstock [@CR9]). Only two of the mentioned studies include diagnostics based on the Diagnostic and Statistical Manual of Mental Disorders 4th Edition (DSM-IV) (American Psychiatric Association [@CR6]) criteria, in both cases in form of a self-report scale (Turner et al. [@CR49], [@CR48]). The South Oaks Gambling Screen is the most commonly used screening tool, but it is not sufficient for actual diagnoses, and has been criticized in various ways (Lesieur and Blume [@CR28]; Volberg [@CR53]). As far as the authors of this study know, there is no previous study on offenders performing full DSM diagnostics, including gambling disorder.
To summarize, previous studies indicate that gambling disorder is overrepresented among offenders, and that an increased knowledge about these complex connections is necessary for optimizing screening, possible treatment, and rehabilitation (Williams et al. [@CR56]). There is, however, a pronounced lack of gambling disorder research based on diagnostics through full DSM assessments (Zurhold et al. [@CR59]).
The primary aims of this study were to investigate the prevalence of, and association between, gambling disorder and other psychiatric diagnoses in a group of Swedish young, male violent offenders. Secondary aims were to compare the gambling and the non-gambling disorder groups concerning types of crimes and sociodemographic data.
Methods {#Sec2}
=======
Participants {#Sec3}
------------
The participants in this study originally took part in the Swedish research project DAABS (*the Development of Aggressive Antisocial Behavior Study*), which investigated a nationally representative cohort of violent offenders concerning mental and neurodevelopmental disorders (Wallinius et al. [@CR54]). They were all men aged 18--25 years and recruited while serving a sentence for violent (including hands-on sexual) crimes in the Western region of the Swedish Prison and Probation Service during the period March 2010--July 2012. Nine different prisons were involved, ranging from high to low security facilities, and all violent offenders in the age group (corresponding to a fifth of the national population) were asked to participate. Since there was only one women's prison in the defined area, no women were included due to power issues. Exclusion criteria were insufficient language skills (defined as the need of an interpreter for full participation) and short duration of stay at the current prison (≤ 4 weeks). Anonymous, basic information about the offenders who were excluded, or chose not to participate, was obtained and compared to the rest of the offenders to assess the representativeness of the sample.
Out of 421 imprisoned men of the right age and crime category, 23 were excluded due to insufficient language skills and 19 because of too short duration of prison stay. Of the remaining 379 clients, 109 declined participation leaving a total response rate of 71% (n = 270) among all of those who met the inclusion criteria. In six cases, there was not sufficient information from the clinical assessments to make a diagnostic evaluation about the presence of a gambling disorder, which yielded a group of 264 participants for this study.
In the group of men who declined participation (n = 109) there were no significant differences in mean age or type of index crime (general violent or sexual violent), compared to those who participated. However, in the smaller group that was excluded due to insufficient language skills, sexual violent crimes were overrepresented; 52% (n = 12) in comparison to 11% (n = 28) among the participants. In summary, the cohort of 264 men was considered representative for young Swedish male offenders convicted of these crimes.
Procedure {#Sec4}
=========
General Procedure {#Sec5}
-----------------
The eligible offenders received oral and written information about the study and were asked for informed consent. A small monetary compensation (200 SEK appr. USD25) was provided.
A preset protocol was followed, consisting of self-rating questionnaires, semi-structured diagnostic interviews and neuropsychological assessments. The questionnaires were completed by the participants before a full day of clinical assessments. The assessors were licensed psychologists with clinical experience from the field and specialized in the instruments used. All clinical data available from the Swedish Prison and Probation Service (i.e. prison medical records, detailed reports on previous living circumstances and history, and incidents during ongoing sanction) were read by the assessors and evaluated for quality.
Psychosocial and Criminal Background {#Sec6}
------------------------------------
Information on psychosocial background, e.g. marital status, ethnicity, family background, schooling and former contact with the healthcare system, were collected by the assessor from file information and structured interviews. The criminal backgrounds of the participants were also mapped out through information from files and structured interviews.
Diagnostic Assessments {#Sec7}
----------------------
Psychiatric assessments were based on the Structured Clinical Interview for DSM-IV Axis I Disorders (SCID-I) (First [@CR16]), lifetime clinical disorders, and the SCID-II (First et al. [@CR17]) for personality disorders. In order to assess the disorders not covered in the SCID (e.g. neurodevelopmental disorders and impulse control disorders, including gambling disorder/pathological gambling), an amendment, including a life-time DSM-IV symptom checklist of individual criteria or symptom definitions according to DSM-IV (American Psychiatric Association [@CR6]), was added to the structured interview protocol. Final diagnoses were based on all available information, provided by the clinical interview, medical and other records, self-rating questionnaires, neuropsychological assessments, and the clinical impression of the respondent during the 5--6 h assessment, and assigned in consensus between the clinical psychologist and a senior psychologist and researcher (EB or BH), according to the LEAD-principle (Spitzer [@CR45]), i.e. clinicians making decisions in consensus using all data available. Concerning the gambling disorder diagnoses, there was not much additional information available, and the definitive diagnoses were based on the structured DSM-IV symptom checklist (Billstedt et al. [@CR10]; Hofvander et al. [@CR24]).
Intellectual functioning was measured with the Wechsler Adult Intelligence Scales---third edition, (WAIS-III) (Wechsler [@CR55]). In this study, the General Ability Index (GAI) (Tulsky et al. [@CR47]) from WAIS-III was calculated. The Asperger Syndrome/high functioning autism Diagnostic Interview (ASDI) (Gillberg et al. [@CR19]) was used to assess signs of an autism spectrum disorder (ASD). The ASDI is a combined interview and observation schedule for clinical assessments. For participants potentially meeting diagnostic criteria for an ASD disorder, when possible, an in-depth autism spectrum examination was carried out, including either a "Diagnostic Interview for Social and Communication disorders" (DISCO) (Wing et al. [@CR57]), with parents/caregivers or an "Autism Diagnostic Observation Schedule" (ADOS) (Lord et al. [@CR30]), with the participant. For a thorough description of the diagnostic procedures see Billstedt et al. ([@CR10]) and Hofvander et al. ([@CR24]).
Substance Abuse {#Sec8}
---------------
The diagnostics in this study were performed in the middle of the transition from DSM-IV to DSM-5, where the two concepts "substance abuse" and "substance dependence" were merged into "substance use disorder" with three degrees of severity (American Psychiatric Association [@CR6], [@CR7]). Due to the extensive social problems and unconventional life situations of the participants, it was hard to assess the substance dependence criteria. Therefore, the PIs decided to use a limited set of criteria and in this study we report whether an individual fulfilled at least substance abuse diagnosis, based on the four DSM-IV substance abuse criteria, while a subject's substance abuse diagnosis may or may not have progressed to a dependence diagnosis for that specific substance.
Types of Crimes {#Sec9}
---------------
Information about types of crimes was collected from files and structured interviews (Wallinius et al. [@CR54]) and is reported as variables summarizing all reported (official or during interviews) crimes with a lifetime prevalence. Being convicted of a violent (including hands-on sexual) crime was an inclusion criteria for this study. All offenses were arranged in six groups: violent offences (i.e. murder/manslaughter, assault, unlawful threat, robbery, sexual offenses, and fire setting/arson), sexual offences, drug-related offences, property offences (i.e. theft, breaking and entering, and vandalism), traffic violations (i.e. driving under the influence, and driving without a license) and fraud. All categories include attempts as well as aggravated versions of the specific crimes listed above.
Statistical Analysis {#Sec10}
====================
All data were anonymized, coded and gathered in a database. The calculations were performed using IBM SPSS Statistics for Mac, version 23.0 (IBM, Armonk, NY). A *p* value \< .05 was considered statistically significant. All *p* values were two-tailed.
Initially, bivariate analyses were performed. Comparisons between groups (age) were assessed using the Student's *t* test. Dichotomous variables are presented as absolute and relative frequency and comparisons between groups were calculated with Fisher's Exact test. In order to restrain the false discovery rate among the 27 bivariate analyses of potential predictors in the multivariate model to .05 (i.e. the chance of any type I error among all of the 27 analyses) the Benjamini Hochberg (BH) correction procedure was performed on the *p* values from the analyses (Hochberg and Benjamini [@CR23]). The rate of missing data was low, ranging from 0 to 1.5%.
Secondly, we used logistic regression analysis to investigate the significant variables associated with gambling disorder. The limited number of participants with gambling disorder (n = 43) makes the risk of overfitting a potential issue given the large number of possible variables to include in the multivariate regression model. A general recommendation to minimize the risk of overfitting in logistic regression analysis is a maximum of one independent variable for every 8--10 cases of the dependent variable (Vittinghoff and McCulloch [@CR52]). We thus decided to limit the number of independent variables in the regression model to five, and we included the five variables with the lowest BH-corrected *p* values in the bivariate analyses. Missing data were handled by listwise deletion.
Ethical Considerations {#Sec11}
======================
Informed written consent was provided by all the offenders before participation. They were given the opportunity to receive feedback on the preliminary results of the assessment, together with the opportunity of referral to the prison psychiatrist for further assessment and possible treatment. The monetary reward was considered low enough not to influence the free consent. The study was approved by the Research Ethics Committee at Lund University (Register No. 2009/405).
Results {#Sec12}
=======
Gambling Disorder {#Sec13}
-----------------
The prevalence of gambling disorder in the cohort was 16% (n = 43). Under the assumption that this cohort is representative for young Swedish male violent offenders, this corresponds to a 95% confidence interval for the true proportion of gambling disorder in this population at 12.0--21.3%.
The percentages of participants with and without gambling disorder reporting each of the pathologic gambling criteria are shown in Table [1](#Tab1){ref-type="table"}.Table 1Frequency of diagnostic criteria for lifetime gambling disorderDiagnostic criterionProportion positive in gambling disorder group (%)Preoccupation with gambling81.4Needs to gamble with increasing amounts of money79.1Repeated unsuccessful efforts to control gambling58.1Withdrawal symptoms69.8Gambles as a way of escaping53.5Chasing losses81.4Lies to conceal the extent of involvement in gambling74.4Committed illegal acts to finance gambling51.2Jeopardized relationships, job etc.23.3Relies on others to provide money14.0
Sociodemographics {#Sec14}
-----------------
Sociodemographic variables are shown in Table [2](#Tab2){ref-type="table"}. There were no significant differences between the gambling disorder and the non-gambling disorder group concerning age, marital status, country of birth (Sweden or other), or employment. When it comes to elementary and middle school graduation, however, significantly fewer in the gambling disorder group had graduated in expected time (BH-adjusted *p* value, *p*~BH~ = .027).Table 2Sociodemographic data by occurence of gambling disorder, lifetimeTotal sampleGambling disorder groupNon gambling disorder group*p* value\*BH-adjusted *p* value\*\*Age, mean (years)22.322.622.3.281.380Married/living together (%)24.232.622.6.175.254Born in Sweden (%)73.572.173.8.851.884Not graduated elementary and middle school in expected age25.544.221.8**.004.027**Unemployed before arrest60.865.160.0.610.659Associations that remain significant after BH-adjustment are presented in bold text\*Fisher's exact test used for all categorical variables, Student's *t* test used for all numerical variables\*\*Benjamini--Hochberg adjusted *p* values using all 27 *p* values displayed in Tables [2](#Tab2){ref-type="table"} and [3](#Tab3){ref-type="table"}
Substance Abuse {#Sec15}
---------------
A total of 84.5% (n = 223) had at least one substance abuse diagnosis. There were significant differences between the groups considering cannabis abuse (*p*~BH~ = .043), cocaine abuse (*p*~BH~ \< .001) and anabolic steroids abuse (*p*~BH~ = .027), where the specific substance abuse diagnoses were more common in the gambling disorder group.
Other Psychiatric Diagnoses {#Sec16}
---------------------------
All psychiatric diagnoses, their percentages and *p* values from the bivariate analysis are shown in Table [3](#Tab3){ref-type="table"}.Table 3Psychiatric and substance abuse comorbidity by occurence of gambling disorder, lifetimeTotal sample, % (n)Gambling disorder group, % (n)Non gambling disorder group, % (n)*p* value\*BH-adjusted *p* value\*\*Mental retardation1.9 (5)7.0 (3)0.9 (2).032.096ADHD43.5 (114)53.5 (23)41.6 (91).179.254Autism spectrum disorders9.5 (25)0.0 (0)11.3 (25).019.086Conduct disorder79.2 (209)88.4 (38)77.4 (171).149.237Substance abuse (any)84.5 (223)93.0 (40)82.8 (183).109.226 Alcohol48.5 (128)53.5 (23)47.5 (105).508.596 Sedatives48.9 (128)65.1 (28)45.7 (100).029.096 Cannabis77.6 (204)93.0 (40)74.5 (164)**.008.043** Central stimulants48.9 (128)60.5 (26)46.6 (102).133.226 Cocaine40.8 (107)74.4 (32)34.2 (75)**\< .001\< .001** Hallucinogens34.0 (89)48.8 (21)31.1 (68).024.093 Anabolic steroids14.9 (39)30.2 (13)11.9 (26)**.004.027** Inhalants20.3 (53)16.3 (7)21.1 (46).540.608 GHB19.3 (51)30.2 (13)17.2 (38).058.157 Heroin33.8 (89)39.5 (17)32.7 (72).385.495 Opioid analgesics41.7 (110)53.5 (23)39.4 (87).093.209 Methadone, buprenorphine14.0 (37)9.3 (4)14.9 (33).472.580Psychotic disorders7.6 (20)7.0 (3)7.7 (17)1.001.00Affective disorders54.2 (143)65.1 (28)52.0 (115).134.226Anxiety disorders51.7 (136)62.8 (27)49.5 (109).134.226Eating disorders1.1 (3)4.7 (2)0.5 (1).070.172Antisocial personality disorder64.0 (169)83.7 (36)60.2 (133)**.003.027**Associations that remain significant after BH-adjustment are presented in bold text\*Fisher's exact test used for all categorical variables\*\*Benjamini--Hochberg adjusted *p* values using all 27 *p* values displayed in Tables [2](#Tab2){ref-type="table"} and [3](#Tab3){ref-type="table"}
The diagnosis of mental retardation was significantly more common in the gambling disorder group according to the initial analysis (*p* = .032) but after correction with the BH method, the *p* value was no longer significant due to the small sample size (*p*~BH~ = .096).
Antisocial personality disorder was significantly more common in the gambling disorder group in the bivariate analysis (*p*~BH~ = .027).
Regression Analysis {#Sec17}
-------------------
Five variables showed significant differences between the gambling disorder and non-gambling disorder groups in the bivariate analyses (see above and Tables [2](#Tab2){ref-type="table"}, [3](#Tab3){ref-type="table"}); elementary and middle school graduation in expected time, cannabis abuse, cocaine abuse, anabolic steroids abuse and antisocial personality disorder and were chosen for logistic regression analysis (see Table [4](#Tab4){ref-type="table"}). In this analysis, cannabis abuse, anabolic steroids abuse and antisocial personality disorder were not independently associated with gambling disorder. Gambling disorder was significantly more common among those who had not graduated elementary and middle school in expected time (AOR 2.79, CI 1.33--5.87, *p* = .007) and among those who had cocaine abuse (AOR 4.11, 1.75--9.63, *p* = .001). Nagelkerke R Square of the model was .219, χ^2^ = 36.2, with *p* \< .001. Table 4Logicistic regression on occurence of gambling disorderOR (95% CI)AOR (95% CI)*p* value\*Not graduated elementary and middle school in expected age2.85 (1.44--5.64)2.79 (1.33--5.87)**.007**Cannabis abuse4.55 (1.36--15.3)1.45 (0.35--6.05).607Cocaine abuse5.59 (2.67--11.7)4.11 (1.75--9.63)**.001**Anabolic steroids abuse3.22 (1.49--6.94)1.47 (0.62--3.46).381Antisocial personality disorder3.40 (1.45--7.99)1.88 (0.73--4.87).191Associations that remain significant in the multivariable regression model are presented in bold text\**p* values from the adjusted logistic regression analysis
Types of Crimes {#Sec18}
---------------
Types of crimes by occurrence of gambling disorder are shown in Table [5](#Tab5){ref-type="table"}. Initially there were significant associations between gambling disorder and drug-related (*p* = .022) and traffic violations (*p* = .036) but after a BH-correction for these five analyses, the associations were no longer significant (*p*~BH~ = .090 and *p*~BH~ = .090).Table 5Types of crimes by occurence of gambling disorder, lifetimeTotal sample, % (n)Gambling disorder group, % (n)Non gambling disorder group, % (n)*p* value\*BH-adjusted *p* value\*\*Violent offenses100 (264)100 (43)100 (231)N/ASexual offenses11.8 (31)11.6 (5)11.8 (26)1.001.00Drug-related offenses74.0 (194)88.4 (38)71.2 (156).022.090Property offenses87.9 (232)90.7 (39)87.3 (193).798.998Traffic violations64.6 (170)79.1 (34)68.1 (136).036.090Fraud25.9 (68)30.2 (13)25.0 (55).454.757\*Fisher's exact test used for all categorical variables\*\*Benjamini--Hochberg adjusted *p* values using the 5 *p* values displayed in this table
Discussion {#Sec19}
==========
Prevalence and the Need of Attention {#Sec20}
------------------------------------
The main finding of this study is the high prevalence of gambling disorder in violent offenders, comprehensively assessed with DSM-IV-based interviews, confirming the results from previous research (Williams et al. [@CR56]). This prevalence is about 40 times higher than in the general Swedish population. The large epidemiological study SWELOGS (Swedish Longitudinal Gambling Study) has shown that general gambling in Sweden appears to have reached a plateau (Romild et al. [@CR42]), but the proportion of problem gamblers in the population remains the same and gamblers with the most serious problems are even increasing.
The pronounced relationship between criminality and gambling disorder could form an incentive to pay more attention to gambling problems in the correctional system. Gambling has been reported as a usual, perhaps even normative, part of prison life (McEvoy and Spirgen [@CR33]) which likely impairs the chances of rehabilitation, and gambling severity is a significant predictor of criminal recidivism according to a study by April and colleagues (Lahn [@CR26]; April and Weinstock [@CR9]). The causal relations between gambling disorder and criminality are not fully understood (Turner et al. [@CR48]; May-Chahal et al. [@CR32]), but there is an obvious risk for criminal relapse when a person is in gambling debt, and this two-way connection motivates increased awareness and availability of gambling disorder treatment to people with a criminal lifestyle.
Concerning types of crimes, there were no significant differences between the gambling disorder and the non-gambling disorder group. However, this study---including only violent offenders---does confirm the picture that gambling disorder might be common among offenders regardless of type of crime, and not mainly connected with economic crime or property crime, which has also been suggested (Turner et al. [@CR49], [@CR48]; Cuadrado and Lieberman [@CR13]).
The frequency of each DSM gambling criterion in the gambling disorder group in this study is interesting and shows both similarities and differences compared to previous research. About 51% of the gambling disordered group reported having committed a criminal act to finance their gambling, which is similar to reports from Abbott and colleagues, Lahn and colleagues and the Williams' review (Abbott et al. [@CR1]; Lahn [@CR26]; Williams et al. [@CR56]). However, when compared to other gambling disordered groups, the offenders with gambling disorder in this study to a markedly less extent reported having jeopardized or lost a significant relationship, job etc. (23.3%) or relied financially on others because of gambling (14.0%) (Granero et al. [@CR20]; Christensen et al. [@CR12]). A possible explanation could be that this group of men is limited in their ability to perceive and assess consequences of their actions. Previous research has shown impaired risk assessment in offenders, which probably is connected to the high prevalence of antisocial personality disorder (Pachur et al. [@CR36]; May-Chahal et al. [@CR32]). Perhaps their relatively young age and complicated life situations could affect what kind of relationships they are in, and their appreciation of them. Pachur and colleagues suggest that enhanced thinking skills programs for offenders, aimed at reducing recidivism through changing attitudes, could be more successful with an increased focus on risk taking (Pachur et al. [@CR36]). Many criminal problem gamblers don't see their gambling as problematic at all (Lahn [@CR26]), which further emphasizes the need of cognitive behavioural interventions.
Early Problems and Detection {#Sec21}
----------------------------
We have also showed that, even if this group of convicted men has had a substantial amount of early onset problems with e.g. schooling and demonstrates a high prevalence of conduct disorder, the participants with gambling disorder stand out with significantly worse results concerning elementary and middle school graduation. This is interesting in the perspective of the development of early deviant behavior, and previous publications on the same population show parallel results; school adjustment problems (e.g. truancy, bullying and incomplete schooling) were the most distinct significant predictors of later aggressive behavior (measured as total score on Life History of Aggression) (Wallinius et al. [@CR54]). In line with this, an early criminal career has been associated with higher loss chasing in gambling according to a study by May-Chahal et al. ([@CR32]).
Swedish epidemiological research has shown that gambling problems are highly overrepresented among young, marginalized men (Abbott et al. [@CR2]) and it is probable that gambling problems and antisocial behavior develop simultaneously (Slutske et al. [@CR44]) and perhaps catalyze each other. A part of the longitudinal study on the Dunedin birth cohort concludes that gambling disorder in young adults has much in common with addictive disorders and other externalizing behaviors, from a personality perspective (Slutske et al. [@CR43]). Thus, young men with impulsive and delinquent behavior are clearly in the risk zone for gambling disorder and should be targets for preventive actions and treatment.
Psychiatric Comorbidity {#Sec22}
-----------------------
When it comes to the extremely high prevalence of psychiatric disorders in this cohort, it speaks for the need of competent psychiatric care for young offenders (Al-Rousan et al. [@CR5]). The criminal gamblers may be predisposed to gambling problems and form an interesting group from a biological point of view. They could represent a certain "antisocial pathway" to gambling disorder; characterized by low impulse control, high aggression levels and multiple drug use (Blaszczynski and Nower [@CR11]; Valleur et al. [@CR50]; Allami et al. [@CR4]). Gupta and colleagues suggest that the "pathways model" is applicable also for adolescents, confirmed by latent class analysis which showed a distinct impulsive and antisocial subgroup among the young problem gamblers (Gupta et al. [@CR22]). The antisocial gamblers often start at a young age (Valleur et al. [@CR50]; Allami et al. [@CR4]) and gambling may play a role in the evolvement of an antisocial lifestyle (Slutske et al. [@CR44]). According to various studies looking at gambling and personality, problem gambling could be considered part of a cluster of externalizing pathology. The personality profiles of pathological gamblers and substance abusers are often dominated by negative affect and unconscientious---and disagreeable disinhibition, a combination of traits also closely connected to antisociality and impulsivity (Maclaren et al. [@CR31]). It is believed that it is of great importance to pay more attention to the associations between both gambling disorder and substance use disorders, and criminal maintenance (McEvoy and Spirgen [@CR33]). There is an evident connection between criminality and gambling, and even though the chronology is complex, the research on this cohort enlightens the need of efforts early in life to affect the development of both social and psychiatric problems (Nilsson et al. [@CR35]). Further observation of this group of gamblers, through screening and the offering of treatment, would be necessary to evaluate the possible benefit of treatment interventions.
In the present study, we found significantly higher occurrence of antisocial personality disorder and three substance abuse diagnoses in the gambling disorder group; cannabis, anabolic steroids and cocaine. The variables that were independently associated with gambling disorder in the regression model were elementary and middle school graduation and cocaine abuse. Possible causal relations behind these findings are not possible to determine through this study, but our results enlighten the need of further studies.
The comorbidity between gambling disorder and substance use disorders has previously been demonstrated, and more serious alcohol problems have been shown to correlate with more severe gambling disorder and higher general dysfunction (Lorains et al. [@CR29]; Del Pino-Gutierrez et al. [@CR14]). Pietrzak and colleagues also demonstrated illicit drug use and severity of gambling problems to be indicators of antisocial personality disorder in treatment seeking gamblers (Pietrzak and Petry [@CR39]). The presence of substance abuse diagnoses was very high in this study, and enlightens the need of the offering of treatment to possibly decrease relapses in gambling disorder, and other addictive disorders, and criminality. There were no significant differences between groups considering alcohol abuse, but the prevalence is considerably high in the whole cohort; 48.5%, compared to approximately 4% in the general population (Andréasson [@CR8]). The significant overrepresentation of cocaine abuse in the gambling disorder group, still significant in the regression analysis, is interesting and might speak for a pronounced reward seeking behavior, together with problematic decision-making. Dufour and colleagues found an overrepresentation of problem gamblers among community cocaine users (Dufour et al. [@CR15]), and alterations in neural structure in specific parts of the orbitofrontal cortex---playing an important role for drive and responsibility---have been connected to both cocaine-use disorder and gambling disorder (Adinoff et al. [@CR3]). The field is complex; so far the studies are small and somewhat ambiguous, but there are interesting findings indicating the need of further analyses of addictive behaviors in this context (Adinoff et al. [@CR3]; Yip et al. [@CR58]). Our results show a connection between cocaine and gambling disorder in the studied group, even though a lot still remains unclear when it comes to the explaining pathobiology.
Limitations {#Sec23}
-----------
The study is limited by the relatively small size of the cohort, which may have lead to undetected type II errors. The exclusion of 23 participants due to language problems may have affected the results, since men born outside Sweden seem to be at higher risk for gambling problems (Abbott et al. [@CR2]). It cannot be precluded that the group of 109 men who declined participation in the study was different in any way considering prevalence of gambling disorder and other variables investigated. Analysis of the non-responders was limited to age and type of crime.
The cohort itself was defined by a number of factors that may limit the generalizability of the results because of selection biases. The age span was narrow (18--25 years), no females were included and all the participants were convicted of violent (including hands-on sexual) crimes. In addiction medicine research, maybe particularly concerning gambling disorder, it is important to underline that there are cross-cultural differences both within---and between countries. The current sample is a quite specific group in the Swedish population, which may of course limit the generalizability also to other similar populations (Medeiros et al. [@CR34]; Raylu and Oei [@CR41]).
Many parts of our data were selected retrospectively, hence a recurrent risk for recall bias. All diagnoses were based on professional assessments, and as always there are elements of subjectivity. The substance abuse diagnoses were comprehensively based on DSM-IV, but substance dependence criteria were not fully assessed. The gambling disorder diagnoses were based on a structured list of DSM-IV criteria, but no structured validated instrument was used, which may have affected the diagnostic validity of gambling disorder.
When performing multiple comparisons there is always a risk for type 1 errors. We handled this risk by using the BH method in which all *p* values from the bivariate analyses were adjusted to limit the total false discovery rate to 5%, prior to the regression analysis (Hochberg and Benjamini [@CR23]).
A main drawback with the cross-sectional design is the lack of time perspective, which also rules out the possibility to estimate risks and possible causal relationships.
Conclusions {#Sec24}
===========
In summary, this study investigated gambling disorder in a cohort of young men convicted for violent and/or sexual crimes and showed a high prevalence, 16%, when assessed with a structured DSM-based diagnostic instrument. The clinical implications could be divided in two sections; at first the direct need of illuminating gambling problems among offenders; both to provide treatment for the gambling problems themselves, but also as a conceivable way of diminishing the risk of criminal recidivism. Secondly, our findings emphasize the importance of early attention to gambling problems in vulnerable groups; here represented by young men with schooling problems and early-onset delinquent behavior.
This study was funded by the state-owned gambling monopoly of Sweden.
Conflict of interest {#FPar1}
====================
Carolina Widinghoff holds a researcher position at Lund University, Sweden, in collaboration with the Swedish state-owned gambling monopoly, Svenska Spel AB. Jonas Berge declares that he has no conflict of interest. Märta Wallinius declares that she has no conflict of interest. Eva Billstedt declares that she has no conflict of interest. Björn Hofvander declares that he has no conflict of interest. Anders Hakansson holds a researcher position at Lund University, Sweden, in collaboration with the Swedish state-owned gambling monopoly, Svenska Spel AB.
Ethical approval {#FPar2}
================
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
|
{
"pile_set_name": "PubMed Central"
}
|
1. Introduction {#sec1}
===============
Cancer is one of the main causes of death worldwide, claiming over six million people each year \[[@B1]\]. According to current estimates, cancer is 30--40% preventable over time with appropriate nutrition, regular physical activity, and avoidance of obesity \[[@B2]\].
Chemoprevention is a promising strategy which uses natural dietary compounds and/or synthetic substances to block, inhibit, reverse, or delay the process of carcinogenesis. Important preventive mechanisms include suppression of cell proliferation and apoptosis and modulation of epigenetic processes \[[@B3]--[@B6]\].
Many epidemiological studies suggest that diets particularly rich in fruits and vegetables have cancer preventive properties \[[@B7]--[@B9]\]. The beneficial effect of diets is attributable, at least in part, to polyphenols which have antitumour activities both in animal models and in humans \[[@B8]--[@B14]\]. Furthermore, several clinical trials testing the efficacy of natural compounds are underway, <http://www.clinicaltrials.gov/>; some of them are already concluded and demonstrate the chemopreventive activity of dietary polyphenols \[[@B15]--[@B17]\].
The current growing interest for dietary plants has led to a renewed attention for artichoke because of its high polyphenolic content. In the edible part, it mainly consists of hydroxycinnamic derivatives, in particular chlorogenic and dicaffeoylquinic acids. Our studies of bioavailability demonstrate that ferulic acid is one of the metabolites present in human plasma after an artichoke meal \[[@B18]\]. Preclinical reports indicate that ferulic acid has antiproliferative and chemopreventive activities*in vitro* and*in vivo* \[[@B19]--[@B21]\] and proposed the potential use of ferulic acid as an adjuvant agent during chemo- and/or radiotherapy \[[@B22], [@B23]\].
Our previous findings indicate that polyphenolic artichoke extracts (AEs) protected hepatocytes from oxidative stress and exhibited cancer chemopreventive properties, in part, by triggering apoptosis on human hepatoma cells Hep G2 \[[@B24]\] and on the human breast cancer cell line, MDA-MB231 \[[@B25]\].
Despite the growing scientific results regarding chemopreventive activities of natural dietary compounds \[[@B8]\], the cellular mechanisms underlying antitumour property of polyphenols are yet to be elucidated.
Cellular senescence, a state of cell cycle arrest, can be considered a relevant mechanism of tumour suppression \[[@B26]--[@B28]\]. Furthermore, emerging evidence has demonstrated that therapy-induced senescence is a critical mechanism through which many anticancer agents inhibit tumour progression \[[@B29]--[@B31]\]. Importantly, therapy-induced senescence can be achieved in administering agents at low doses. This approach can significantly reduce the side effects of conventional anticancer therapy, thus improving the quality of life for cancer patients \[[@B29], [@B30]\]. Innovative senescence therapies will be developed through improved knowledge of the molecular pathways controlling permanent growth arrest by specifically screening for senescence effectors.
Scientific evidence from*in vitro* studies indicates that the cancer prevention activity involved modulation of epigenetic processes. Epigenetics is defined as heritable changes in gene expression that are not accompanied by alterations in DNA sequence \[[@B32]\]. The main epigenetic processes are DNA methylation, histone modifications, and chromatin remodeling. Aberrant patterns of gene expression are key features of cancer and both genetic and epigenetic abnormalities are implicated in this molecular deregulation. In contrast to genetic modifications, epigenetic alterations are potentially reversible and strategies targeting the epigenome have been proposed for both cancer prevention and therapeutics \[[@B33]\].
Induction of premature senescence and modulation of epigenetic processes have been identified as relevant anticancer features of dietary polyphenolic compounds \[[@B34]\]. There are both*in vitro* and*in vivo*data showing that several bioactive food components can interfere with DNA methylation and histone modifications and affect the expression of genes involved in the carcinogenic process \[[@B35]\]. In particular, phenolic acids, including chlorogenic acid, have been shown to affect DNA methylation \[[@B36]\].
Since the induction of senescence requires moderate concentrations of anticancer agents and produces almost no side effects \[[@B37]\], we sought to investigate whether a low dose and chronic treatment of AEs could inhibit the growth of breast cancer cells through the induction of premature senescence. In the present study, we demonstrate that a moderate and chronic treatment of AEs causes a significant increase in senescence-associated *β*-galactosidase (SA-*β*-gal) detection and upregulation of p21^Cip1/Waf1^ and p16^INK4a^ protein expression in MDA-MB231 cells. Such a premature senescence is via ROS-mediated DNA damage and is associated with the modulation of epigenetic machinery. Altogether, these results highlight a significant contribution of senescence induction to AEs anticancer effects.
2. Materials and Methods {#sec2}
========================
2.1. Artichoke Extract Preparation {#sec2.1}
----------------------------------
The edible part (head) of fresh artichoke (*Cynara scolymus* L. cv Violetto di Provenza) buds was used for extract preparation and the analysis of polyphenols contained in the extracts was performed by HPLC as previously described \[[@B25]\].
2.2. Cell Lines and Cultured Conditions {#sec2.2}
---------------------------------------
Cell lines were maintained in a humidified incubator with 5% CO~2~ and 95% air at 37°C. HCT 116 cells, human colon carcinoma cell line, MDA-MB231, oestrogen receptor-negative breast cancer cells, HEY cells, human ovarian cell line, and K-562 cells, human erythromyeloblastoid leukaemia cell line (kindly supplied by Dr. Maurizio Fanciulli, Dr. Paola Nisticò, and Dr. Maria Giulia Rizzo, Regina Elena National Cancer Institute Rome) were grown in RPMI medium (Invitrogen Life Technologies, Milan, Italy) supplemented with 10% FBS, 10 IU/mL penicillin, and 10 *μ*g/mL streptomycin. GTL-16 cells, human gastric carcinoma cell line, DU 145 cells, human prostate carcinoma cell line, A549 cells, human lung carcinoma cell line, M14 cells, human melanoma cell line, U-373 MG cells, human astrocytic cell line, and Saos-2 cells, human osteosarcoma cell line (kindly supplied by Dr. Giovanni Blandino, Regina Elena National Cancer Institute, and Dr. Antonella Farsetti, CNR Rome, purchased from American Type Culture Collection, Rockville, MD, USA) were grown in D-MEM supplemented with 10% FBS, 10 IU/mL penicillin, and 10 *μ*g/mL streptomycin.
2.3. Reagents {#sec2.3}
-------------
Artichoke extracts were dissolved in PBS and 0.1% dimethylsulfoxide (Me~2~SO, Sigma-Aldrich, Milan, Italy). Paclitaxel (Ptx, Sigma-Aldrich) was dissolved in PBS. 5-Bromo-4-chloro-3-indolyl-*β*-d-galactopyranoside (X-gal) was purchased from IBI Scientific (Peosta IA, USA) and used as previously described \[[@B38]\]. N-Acetyl-cysteine (NAC, Sigma-Aldrich) was dissolved in PBS. Dihydroethidium (DHE, Molecular Probes-Invitrogen UK) was dissolved in Me~2~SO.
2.4. Cytotoxicity and Cell Proliferation Assays {#sec2.4}
-----------------------------------------------
Human cancer cells were seeded at density of 2.0 × 10^5^ in 6-well plates (Sarstedt, Numbrecht, Germany) in triplicate and cultured for 24 h before adding either the vehicle (0.1% Me~2~SO) or AEs. Cytotoxicity experiments were assessed in serum-free medium and cells were treated with AEs (200, 400, 600, and 800 *μ*M) for 24 h.
For the cell proliferation experiment, MDA-MB231 were seeded at a density of 5 × 10^3^ in 6-well plates in triplicate. After 24 h the cells were exposed to AEs (10 and 30 *μ*M) for 10 days. To determine the role of ROS in AEs-mediated senescence, cells were pretreated with 10 mM NAC for 2 h and then exposed to AEs (10 and 30 *μ*M) for 10 days.
Cells were harvested by trypsinization, stained with 0.4% trypan blue (Sigma-Aldrich), and counted using a haemocytometer.
2.5. Senescence-Associated *β*-Galactosidase (SA-*β*-gal) Assay {#sec2.5}
---------------------------------------------------------------
MDA-MB231 cells were seeded at a density of 5 × 10^3^ in 6-well plates in triplicate and cultured for 24 h before adding either the vehicle or AEs (10 and 30 *μ*M). After 10 days of a chronic treatment, with every 48 h renewal of medium and treatment, cultured cells were washed in PBS, fixed in 2% formaldehyde/0.2% glutaraldehyde for 15 minutes at room temperature. Cells were then washed and incubated with fresh SA-*β*-gal staining solution containing 1 mg/mL X-gal, 40 mM citric acid/sodium phosphate (pH 6.0), 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, and 2 mM MgCl~2~ for 16--18 h at 37°C. Blue-stained senescent cells were counted using a light microscopy (Olympus IX71-Olympus America, Center Valley, PA, USA) and the images were captured by digital camera (Olympus-Camedia C-5060).
2.6. Western-Blot Analysis {#sec2.6}
--------------------------
To prepare the whole-cell extract, cells were washed with PBS and suspended in ice cold RIPA lysis buffer (50 mM Tris-HCl pH 8, 1 mM EDTA, 150 mM NaCl, 1% NP40, 1% sodium deoxycholate, 0.1% SDS) with protease inhibitors. After 30 minutes of mixing at 4°C, the mixture was centrifuged (10,000 ×g) for 10 minutes and the supernatants were collected as whole-cell extracts. The protein content was determined with a protein assay reagent (Bio-Rad, Milan, Italy) using bovine serum albumin as a standard. An equal protein content of total lysates (25 *μ*g) from the control and from AEs treated samples was resolved on 10% SDS-PAGE with molecular weight markers (Bio-Rad). Proteins were then transferred to the PVDF membrane (EMD Millipore Billerica, MA, USA) and reacted with anti-p16^INK4a^ and anti-p21^Cip1/Waf1^ antibodies (Santa Cruz Biotechnology, CA, USA), acetylated-lysine polyclonal antibody (Cell Signalling Technology Inc., Danvers, MA, USA), and anti-actin antibody (Calbiochem-EMD Millipore Billerica, MA, USA) for protein normalization. The protein bands were revealed by chemiluminescence using an ECL detection kit (Amersham Bioscience, Cologno Monzese, Milan, Italy). Autoradiograms were quantified with ImageJ 1.43 software (National Institutes of Health, Bethesda, MD, USA).
2.7. Dot Blot Analysis of DNA 5-Methylcytosine and 5-mC Immunostaining {#sec2.7}
----------------------------------------------------------------------
MDA-MB231 cells were seeded at a density of 2.5 × 10^3^ in 6-well plates in triplicate and cultured for 24 h before adding either the vehicle or various concentrations of AEs (2.5, 5, 10, and 30 *μ*M) for 10 days and then harvested. Genomic DNA was isolated using the Wizard DNA purification kit (Promega, Madison, WI, USA) according to the manufacturer\'s instructions, transferred onto positively charged Hybond N+ membranes (Amersham Pharmacia Biotech, Piscataway, NJ, USA) using dot filtration apparatus and fixed by baking the membrane for 30 minutes at 80°C. After baking, membranes were incubated with 5 *μ*g/mL of 5-methylcytosine (5-mC) antibody (Calbiochem, San Diego, CA, USA) followed by incubation with a horseradish peroxidase-conjugated secondary antibody. Membranes were then treated with chemiluminescence detection reagents and exposed to Kodak autoradiograph films. The intensity of each dot was quantified with ImageJ 1.43 software.
For the immunostaining analysis, cells (1 × 10^5^) were cytospun using a Shandon Cytospin 3 (Thermo Scientific, Waltham, MA, USA) at 1000 rpm for 10 minutes and then processed for 5-mC immunostaining. Cells were permeabilized with 0.4% triton X-100 in PBS for 20 minutes. Cells were washed with PBS for 10 minutes and then blocked for 30 minutes with 3% preimmune goat serum in PBS. After 20 minutes of incubation with 3% H~2~O~2~ in order to quench endogenous peroxidase the cells were washed with PBS and incubated with 5-mC specific antibody (1 : 500 vol/vol) for 2 h. Cells were sequentially incubated with biotinylated prediluted antibody, horseradish peroxidase-conjugated streptavidin (UltraTek HRP, ScyTek Laboratories Inc, Logan, UT, USA) and AEC substrate (AEC substrate kit, ScyTek Laboratories Inc.) and counterstained with haematoxylin.
2.8. Intracellular ROS Detection {#sec2.8}
--------------------------------
MDA-MB231 cells were treated with either the vehicle or AEs (10 and 30 *μ*M). After 10 days, the oxidation-sensitive fluorescent probe DHE was used to assess the production of cytosolic superoxide anions. Briefly, at the end of exposure, cells were incubated with 5 *μ*M DHE for 40 minutes at 37°C in the dark and then rinsed twice with PBS. The cell-permeant DHE entered the cells, was oxidized by superoxide anions to form ethidium (ETH) which binds to DNA, and produced the fluorescent ETH-DNA. The fluorescent signals were obtained exciting the cultured cells at *λ* ~ex~ 300 nm and *λ* ~em~ 610 nm. Cells were visualized and counted by fluorescence microscope (Olympus IX71-Olympus America).
The role of ROS on AEs-treated cell proliferation was evaluated using the antioxidant NAC. Cells were preincubated with 10 mM NAC for 2 h and then exposed to AEs. After 10 days, cells were harvested by trypsinization, stained with 0.4% trypan blue, and counted using a haemocytometer.
2.9. Statistical Analysis {#sec2.9}
-------------------------
Statistical analyses were performed by Student\'s *t*-test using GraphPad 5.1 software. For all statistical tests, a two-tailed *p* value \<0.05 was considered significant. All data reported were verified at least in three independent experiments and expressed as mean ± SD.
3. Results {#sec3}
==========
3.1. Phenolic Composition of Artichoke Extracts {#sec3.1}
-----------------------------------------------
The artichoke extracts were found to contain monocaffeoylquinic acids (MCQA), dicaffeoylquinic acids (DCQA), and small amounts of a luteolin and an apigenin glycoside. The main phenolic components of the AEs found were chlorogenic acid and two dicaffeoylquinic acids (3,5-DCQA and 1,5-DCQA) at a ratio of about 1 : 1 : 1. The concentrations of chlorogenic acid, 3,5-DCQA, and 1,5-DCQA, determined by HPLC, as previously described \[[@B25]\], were found to be 725 ± 70, 738 ± 58, and 632 ± 48 mgL^−1^, respectively.
3.2. Effects of AEs on Human Cancer Cell Lines Viability {#sec3.2}
--------------------------------------------------------
We have previously reported that AEs exhibited cancer chemopreventive activities on a human hepatoma cell line, Hep G2 \[[@B24]\], and on a human breast cancer cell line, MDA-MB231 \[[@B25]\]. To investigate whether the antiproliferative activity of AEs could be extended to other tumours, we describe the effect of AEs on 10 cancer cell lines derived from various human tissues, as shown in [Table 1](#tab1){ref-type="table"}. This panel provides a way of presenting the cellular sensitivity or resistance at three levels of effect. After 24 h, 800 *μ*M AEs caused about 50% of death in prostate and melanoma cells; more than 50% of toxicity was detected in breast, ovary, lung, brain, bone, and leukaemia cells. Less than 50% of dead cells were observed in colon and gastric cancers. The more resistant cancer cells, such as gastric and colon, showed sensitivity to 1200 *μ*M AEs (unpublished results). These data indicated that high doses of AEs are cytotoxic for all tested cancer cell lines without any effect on untreated counterparts.
3.3. Low Doses of AEs Inhibit Breast Cancer Cell Growth via a Caspases-Independent Mechanism {#sec3.3}
--------------------------------------------------------------------------------------------
We have previously demonstrated \[[@B25]\] that high concentrations of AEs (from 200 to 800 *μ*M for 24 h) are able to activate an apoptotic program in MDA-MB231 to halt tumour progression. Since bioactive compounds concentrations required to induce apoptosis in tumour cell lines might not be reachable in target tissues, we asked whether AEs chronically administered at low and sublethal doses can affect the growth of tumour cells as well. To this purpose, we focused on MDA-MB231, which provide the scientific rationale for testing AEs as an antitumour agent against invasive and hormone resistant breast cancer phenotype. After 10-day treatment (from 2.5 *μ*M to 60 *μ*M) direct cell count assay indicated that the cellular growth was significantly inhibited by chronic exposure to low doses of AEs ([Figure 1(a)](#fig1){ref-type="fig"}). Treatments up to 30 *μ*M resulted in a relevant inhibition of cell proliferation in viable cells (about 90%); the highest concentration (60 *μ*M) induced a dramatic growth arrest and was slightly cytotoxic (about 70% viability). Based on these data, we focused on two noncytotoxic concentrations of AEs, namely 10 and 30 *μ*M, that modulate cell growth. Furthermore, we investigated whether these treatments caused a reduction in the number of living cells through the activation of caspases pathways. As shown in [Figure 1(b)](#fig1){ref-type="fig"}, in these experimental conditions protein expression analysis revealed that caspase-9 was not activated. Conversely, high concentration of AEs (400 *μ*M), used as positive control \[[@B25]\], triggers a significant activation/cleavage of caspase-9 after 24 h treatment. In addition, long term exposure to low concentrations of AEs did not activate caspase-8 (unpublished results). Altogether our results strongly suggested that low doses of AEs inhibit breast cancer cell growth via a caspases-independent mechanism.
3.4. AEs Induce Premature Senescence in Breast Cancer Cells {#sec3.4}
-----------------------------------------------------------
We investigated whether the cell growth arrest, in response to low doses of AEs, was caused by the induction of cellular senescence as demonstrated for chronic treatment of several polyphenols in cancer cells \[[@B39]--[@B42]\]. The detection of *β*-galactosidase positive cells reflects an increase in lysosomal mass in aging cells and it is widely regarded as a marker for senescence \[[@B43]\]. MDA-MB231 cells incubated without AEs showed no detectable SA-*β*-gal activity, whereas cells treated with 10 and 30 *μ*M AEs revealed a marked X-gal staining in a dose-dependent manner after 10 days, as depicted in [Figure 2](#fig2){ref-type="fig"}. Most of the SA-*β*-gal positive cells showed an enlarged and flattened morphology with increased volume and granularity that were consistent with cellular senescence status, as shown in magnification area of [Figure 2](#fig2){ref-type="fig"}.
To further investigate this antiproliferative mechanism, we examined the expression levels of p21^Cip1/Waf1^ and p16^INK4a^, two pivotal cell cycle regulators involved in cellular senescence \[[@B44], [@B45]\]. Western blotting data demonstrated that the expression levels of p21^Cip1/Waf1^ and p16^INK4a^ were significantly increased in a dose-depending manner in AEs-treated cells (Figures [3(a)](#fig3){ref-type="fig"} and [3(b)](#fig3){ref-type="fig"}). Optical density measurements were performed using ImageJ software to obtain quantitative values for the protein expressions. Altogether, these findings suggested that low doses and chronic exposure to AEs induced senescence in MDA-MB231 breast cancer cells.
3.5. Effect of AEs on Global Methylation Level in Breast Cancer Cells {#sec3.5}
---------------------------------------------------------------------
DNA hypermethylation is a major epigenetic modification that leads to silencing of tumour suppressor genes which may contribute to cancer development and progression \[[@B46]\]. Recent investigations suggest that some dietary phytochemicals may prevent cancer by modulating epigenetic processes \[[@B35], [@B47]\]. To examine whether chronic treatment has epigenetic effects on the DNA methylation level, cells were exposed to increasing concentrations of AEs, as shown in [Figure 4](#fig4){ref-type="fig"}. After 10 days, cells were harvested for immunocytostaining of DNA methylation using an antibody specific to 5-methylcytosine (5-mC). Treatment of AEs resulted in a reduced number of 5-mC-positive cells in a dose-dependent manner compared to untreated cells (Figures [4(a)](#fig4){ref-type="fig"} and [4(b)](#fig4){ref-type="fig"}).
To further verify the effect of AEs on DNA methylation, genomic DNA was isolated from cells and analysed by dot blot assay using anti-5mC antibody. As shown in [Figure 4(c)](#fig4){ref-type="fig"}, AEs treatment of MDA-MB231 cells significantly decreased the DNA global methylation level as quantified by densitometric values. These results demonstrated that AEs have a relevant effect on regulation of DNA methylation machinery.
3.6. Effect of AEs on Acetylation of Total Proteins on Human Breast Cancer Cells {#sec3.6}
--------------------------------------------------------------------------------
Protein acetylation of lysine residues is an important reversible modification controlling cellular protein expression \[[@B48]\]. We investigated the effect of AEs treatment on acetylation of total proteins in breast cancer cells. MDA-MB231 cells were cultured for 24 h before adding either the vehicle or various concentrations of AEs (10 and 30 *μ*M) for 10 days and then harvested. As shown in [Figure 5](#fig5){ref-type="fig"}, the level of protein acetylation is markedly increased in treated cells as indicated by reported densitometric values. These results provide evidence that long term exposure to low concentrations of AEs is associated with increased level of lysines acetylation of total proteins.
3.7. AEs Treatment Increases ROS Production in Breast Cancer Cells {#sec3.7}
------------------------------------------------------------------
Since reactive oxygen species (ROS) are well-known inducers of cellular senescence, we tested whether AEs increase oxidative stress in breast cancer cells. To accomplish this, cells were incubated with DHE which is an indicator of the presence of superoxide anion, key radical in ROS generation. As shown in Figures [6(a)](#fig6){ref-type="fig"} and [6(b)](#fig6){ref-type="fig"} AEs-treated cells, according to the increased number of bright red fluorescent cells, showed enhanced level of superoxide anions compared to control cells.
To determine the role of ROS in AEs-induced growth arrest, we sought to examine whether inhibition of ROS production by antioxidant NAC has any impact on senescence in breast cancer cells. As shown in [Figure 6(c)](#fig6){ref-type="fig"}, the presence of NAC significantly reduced the antiproliferative effect of 30 *μ*M AEs for long term exposure. Furthermore, NAC treatment decreased percentage of SA-*β*-gal positive cells (unpublished results). According to data in literature regarding the natural products modulation of ROS expression in senescence \[[@B39], [@B41], [@B49]\], our findings strongly support the hypothesis that an oxidative pathway is involved in AEs-induced growth arrest in breast cancer cells.
4. Discussion {#sec4}
=============
In this report, we provide evidence demonstrating that low doses and chronic AEs-treatments exert anticancer activity through induction of premature senescence in MDA-MB231, a triple negative and highly aggressive breast cancer cell line. Experimental data demonstrate that moderate doses of AEs inhibit the growth of breast cancer cells via a caspases-independent mechanism.
Several reports point to a crucial physiological role for cellular senescence in fighting tumorigenesis. Cellular aging is a state of cell cycle arrest induced at the end of the cellular life span or in response to agents causing oxidative stress and DNA damage. Moreover, senescence is regarded as an important mechanism either for fighting premalignant tumours and/or for inducing tumour regression \[[@B2]\] since it limits the proliferative capacity of cells.
Increasing evidence has demonstrated that many phytochemicals exert anticancer and chemopreventive activities through the induction of senescence growth arrest \[[@B39]--[@B42]\].
Beside cell cycle inhibitors, p21^Cip1/Waf1^ and p16^INK4a^ are identified as senescence markers \[[@B44], [@B45]\].
Both p21^Cip1/Waf1^ and p16^INK4a^ pathway induction can lead to the inhibition of pRb phosphorylation by inhibiting CDK2/cyclinE and CDK4/cyclinD complex, respectively. Furthermore, p21^Cip1/Waf1^ and p16^INK4a^ are likely to cooperate to keep pRb in a hypophosphorylated form during senescence \[[@B50]\]. According to data regarding the prosenescence property of a derivative of caffeic acid \[[@B51]\], we showed that low doses of AEs containing mono- and dicaffeoylquinic acids induce an increased number of SA-*β*-gal positive and flattened cells with enhanced expression of p21^Cip1/Waf1^ and p16^INK4a^.
High doses of AEs treatment induces cell death \[[@B25]\] in breast cancer as well as in other cell lines derived from various human cancers ([Table 1](#tab1){ref-type="table"}) whereas AEs chronically administered on MDA-MB231 exert antiproliferative activity via induction of a caspases-independent mechanism.
This is a significant finding since the major challenge for anticancer therapeutic strategy leading to apoptosis*in vitro* is that effective concentrations of an antitumour agent should be too high to be reachable*in vivo* \[[@B52]\].
Data in literature are consistent with our results since dietary polyphenols \[[@B42], [@B49], [@B53], [@B54]\] activate apoptotic machinery when used at high doses whereas low level treatments induce senescence in cancer cells.
The senescence program involves epigenetic processes such as DNA methylation, histone modifications, and chromatin remodeling. DNA hypermethylation is a major epigenetic mechanism in the silencing of tumour suppressor genes. Protein lysine acetylation is a posttranslational change that has long been known to play a prominent role in the regulation of gene expression via modulation of chromatin structure. This epigenetic process involves modification of histone and nonhistone proteins through acetyltransferase (HATs) and deacetylase (HDACs) activities. Abnormal DNA methylation and dysregulated histone acetylation are implicated in numerous reported diseases, including cancer \[[@B55]\]. In contrast to genetic modifications, epigenetic deregulation is potentially reversible and therapeutic strategies, targeting the epigenome, have been proposed for both cancer prevention and clinical treatment \[[@B56]\]. Many epigenetic modulators have been used in clinical trials (either completed or terminated) to treat human cancers (<http://www.clinicaltrials.gov/>). According to results of preclinical studies and clinical trials, epigenetic modulators, administered as monotherapy or in combination with conventional chemo- and radiotherapies, are potentially very useful \[[@B57]\].
Modulation of epigenetic processes has been identified as a relevant anticancer feature of many dietary polyphenolic compounds \[[@B33], [@B34], [@B58], [@B59]\]. There are both*in vitro* data and*in vivo*data showing that several bioactive food components can interfere with DNA methylation and histone modification affecting the expression of gene involved in the carcinogenic process \[[@B35]\]. Epigallocatechin-3-gallate (EGCG), the major polyphenolic constituent of green tea, has been demonstrated to inhibit DNA methyl transferase in several cancer cell lines, including MDA-MB231 cells \[[@B60]\]. This activity was associated with promoter demethylation and reactivation of p16^INK4a^. The positive clinical efficacy of EGCG in the treatment or prevention of chronic lymphocytic leukaemia \[[@B16]\] or prostate cancer \[[@B15]\] has been evaluated. A synergistic mixture of green tea plus capsicum has been demonstrated to have a chemopreventive effect versus different types of cancer in subjects testing positive for ENOX2, (ecto-nicotinamide adenine dinucleotide oxidase disulfide-thiol exchanger 2), which is ideally suited as a target for early diagnosis of cancer as well as for preventive intervention \[[@B17]\]. Curcumin has the potential to treat a wide variety of diseases including cancer \[[@B61]\]. Its epigenetic modulator activity has been a focus in clinical trials (terminated or completed). Phenol derivatives, including quercetin and resveratrol, have been shown to possess epigenetic properties through sirtuin activation \[[@B57]\]. Phenolic acids, including chlorogenic acid, the main component of AEs, have been shown to affect DNA methylation \[[@B36]\].
According to these data, we observed consistent DNA hypomethylation and increased total acetylation protein levels in AEs-treated MDA-MB231 cells.
It has been demonstrated that several anticancer polyphenolic compounds from fruit and vegetables induce tumour cellular growth arrest largely through the generation of ROS \[[@B62]\]. Low doses of resveratrol inhibit cell growth and enhance radiosensitization via the induction of ROS-mediated premature senescence in lung cancer cells \[[@B39], [@B49]\]. Sin et al. suggest that chronic treatment with 20(S)-ginsenoside Rg3, a compound extracted from ginseng, at a subapoptotic concentration caused senescence-like growth arrest and increased ROS production in human glioma cells \[[@B54]\]. Moreover, bisdemethoxycurcumin, a natural derivative of curcumin, suppresses human breast cancer cell proliferation by inducing oxidative stress senescence \[[@B41]\]. A relevant role of ROS was also demonstrated for the phenethyl isothiocyanate induction of apoptosis and senescence in tumours \[[@B53]\]. Altogether, these findings suggest the crucial role of ROS as effectors of polyphenol-induced prooxidant damage in cancer cells. Accordingly, we show that AEs-induced growth arrest is associated with increased ROS production, suggesting that AEs may induce senescence via an oxidative-mediated damage in breast cancer cells. To confirm this important contribution of ROS, we demonstrate that antioxidant NAC attenuates AEs proliferative effect on MDA-MB231 cells.
It is important to highlight that we have previously shown that AEs have a prooxidant activity on breast cancer cells \[[@B25]\] and an antioxidant effect on normal hepatocyte \[[@B24]\]. Given that aberrant redox system is frequently observed in many tumour cells \[[@B63]\], we hypothesize that AEs may selectively inhibit the growth of tumour cells with little or no toxicity on normal cells based on their differential redox status.
Conventional cancer therapy is traditionally based on the efficacy of cytotoxic treatments even though severe side effects on patients are a clinical relevant problem. An alternative strategy is the induction of cytostatis, which disables the proliferative capacity of cells without inducing cancer cell death. A promising cytostatic approach is prosenescence therapy which may provide a relevant growth inhibitory effect in both early and late stage cancers \[[@B29], [@B64]\]. Targeted prosenescence therapies may be of remarkable clinical interest since it might minimize toxicity and improve quality of life for cancer patients \[[@B29], [@B30]\]. Over the last few years, it has been demonstrated that several prosenescence polyphenols and natural compounds could represent a promising novel therapeutic approach for cancer intervention \[[@B39], [@B41], [@B42], [@B54], [@B65]\]. The suppressive role of senescence in cancer progression has promoted the idea that induced premature cell aging could be an alternative or a complement to conventional anticancer treatments.
In line with this, our present study demonstrates for the first time that AEs may induce ROS accumulation in MDA-MB231 breast cancer cells and modulate the p21^Cip1/Waf1^ and p16^INK4a^ pathways to cause a senescence-mediated tumour suppression. Our study adds a novel aspect of the underlying mechanisms of the anticancer properties of AEs. However, in order to provide a solid basis for evaluating the efficacy in human clinical trials, further studies are required on animal models. In particular, deep pharmacokinetic and metabolic studies of AEs are needed.
In conclusion, our findings propose dietary artichoke polyphenols as a very promising tool either for the management of cancer prevention in healthy, high risk breast cancer women or for the design of innovative, nonconventional, adjuvant therapies in cancer treatment.
The authors thank Dr. Marco Giorgio Paggi (Regina Elena National Cancer Institute) for his helpful comments and advice. The authors acknowledge Ms. Tania Merlino for her grammatical suggestions for the English paper. The Contract grant sponsor is Lega Italiana per la Lotta contro i Tumori (LILT), Contract Grant no. 08/12/C/73.
Conflict of Interests
=====================
The authors have declared no conflict of interests.
Authors\' Contribution
======================
Anna Maria Mileo, Stefania Miccadei, and Donato Di Venere conceived, designed, and performed the experiments. Claudia Abbruzzese analyzed the data. Stefania Miccadei and Anna Maria Mileo wrote the paper.
{#fig1}
{#fig2}
{#fig3}
{#fig4}
{#fig5}
{#fig6}
######
Cytotoxicity of AEs in human cancer cell lines.
AEs *μ*M
----------- ---------- ----- -------------- ----- ------------- ------ -------------- ----- -------------- -----
HCT 116 84.5 3.7 77.2 6.1 84.0 2.7 79.0 4.0 77.0 4.5
GLT-16 93.5 1.3 89.2 0.9 89.5 4.5 83.2^\*\*^ 2.7 69.7^\*\*\*^ 3.6
MDA-MB231 90.0 2.6 83.7 1.5 81.7^\*^ 1.0 43.2^\*\*\*^ 1.0 16.0^\*\*\*^ 1.4
HEY 91.7 2.2 91.0 3.6 90.0 2.1 73.5^\*^ 4.9 27.7^\*\*\*^ 1.7
DU 145 92.7 2.3 93.0 2.9 94.0 2.4 80.0^\*\*^ 3.4 51.0^\*\*\*^ 2.9
A549 86.5 5.9 86.2 5.5 82.5 11.2 70.0 2.0 28.3^\*\*^ 3.5
M14 88.2 5.6 83.0 2.9 77.7 10.9 74.3 2.5 39.6^\*\*^ 3.2
U-373 MG 93.7 3.7 95.0 1.4 90.2 6.3 91.5 1.7 16.5^\*\*\*^ 2.0
Saos-2 89.8 2.3 86.5 6.5 85.3 4.6 37.3^\*\*\*^ 6.9 18.3^\*\*\*^ 1.5
K-562 89.7 4.3 15.2^\*\*\*^ 4.3 2.7^\*\*\*^ 1.2 3.7^\*\*\*^ 1.5 1.5^\*\*\*^ 0.6
Ten tumour cell lines derived from various human tissues are treated with increasing concentrations of AEs (from 200 to 800 *μ*M) for 24 h. The results are the mean ± SD of at least three independent experiments. The statistical significance between groups was calculated using Student\'s *t*-test. Significant differences are indicated by asterisks.
GLT-16: ^\*\*^ *p* = 0.0092, ^\*\*\*^ *p* = 0.0009.
MDA-MB231: ^\*^ *p* = 0.0129, ^\*\*\*^ *p* \< 0.0001.
HEY: ^\*^ *p* = 0.0119, ^\*\*\*^ *p* \< 0.0001.
DU 145: ^\*\*^ *p* = 0.0041, ^\*\*\*^ *p* \< 0.0001.
A549: ^\*\*^ *p* = 0.0029.
M14: ^\*\*^ *p* = 0.0023.
U-373 MG: ^\*\*\*^ *p* = 0.0001.
Saos-2: ^\*\*\*^ *p* \< 0.0002.
K-562: ^\*\*\*^ *p* \< 0.0002.
[^1]: Academic Editor: Tullia Maraldi
|
{
"pile_set_name": "PubMed Central"
}
|
Introduction {#sec1}
============
Wildlife populations currently face a multitude of stressors that can adversely impact population dynamics, including habitat loss, disturbance from human activities and rapidly changing environments. Metabolic rate measurements (or other measures of energy expenditure) are integral in predicting the adverse effects of many of these stressors at both the individual and population levels. For example, they are a key and influential component of bioenergetic models ([@ref64]; [@ref9]), which are important in quantifying predator--prey interactions, mitigating human--wildlife conflicts and understanding the influence of prey availability on population dynamics ([@ref60]; [@ref59]; [@ref6]). In addition, anthropogenic disturbance and natural (and human induced) environmental variability often elicit behavioural responses ([@ref65]; [@ref61]), necessitating an understanding of the relationships between energy expenditure and specific behaviours.
Quantifying energy expenditure of free-ranging animals can be difficult, particularly for large carnivores that are wide-ranging and often logistically challenging to capture and handle. The use of doubly labelled water (DLW) remains one of the best techniques for estimating metabolic rates under natural conditions ([@ref44]; [@ref48]), but it can be infeasible for many species because it typically requires an animal to be captured twice within a period of days to weeks ([@ref51]). Metabolic rates of captive animals are thus increasingly used to fill the metabolic data gap and are particularly useful for the ability to isolate the costs of discrete activities and physiological or life history events that can then be applied to estimate the energy requirements of wild populations ([@ref62]; [@ref54]; [@ref45]). Captive studies cannot, however, mimic the complex behaviour exhibited by free-ranging animals. As a result, they have limited ability to provide insight into how metabolic rates reflect the collective influence of behaviour and life history events.
Adult female otariids are a tractable group for metabolic studies using DLW because their central-place foraging behaviour during lactation facilitates recapture within the measurement interval. Metabolic rates of free-ranging adult females have been quantified in 7 of the 14 extant species, revealing the high cost of existence for all but the two tropical species within this group ([@ref18]; [@ref3]; [@ref16], [@ref17]; [@ref57]; [@ref58]; [@ref42]). Several studies have found that diving behaviour influenced at-sea FMR ([@ref3]; [@ref16]; [@ref42]), namely dive depth and the percentage of time diving, but others have found no influence of these variables and no differences among individuals using different foraging strategies ([@ref17]; [@ref58]; [@ref34]; [@ref42]). Studies on captive animals have detected seasonal changes in metabolic rates, which may be related to intrinsic factors such as molting ([@ref62]; [@ref19]; [@ref40]). It is largely unknown, however, whether these same patterns are detectable in free-ranging individuals that exhibit complex behaviours and experience simultaneous physiological stressors.
The goal of this study was to investigate the role of intrinsic and extrinsic factors on at-sea FMR of adult female northern fur seals (*Callorhinus ursinus*). Specifically, we quantified the influence of season, diving behaviour and diet on at-sea FMR using DLW, animal-borne instruments and fatty acid (FA) analysis. We examined how these factors influenced foraging success to better understand the influence of variation in at-sea FMR on the energy available for offspring investment. The eastern stock of northern fur seals has declined by 66% since the 1970s, and while this decline was initially attributed to a female harvest and pelagic sealing, the decline from the 1990s onwards remains unexplained ([@ref56]). Changes in prey availability/distribution leading to reduced pup growth and survival is one hypothesis for the population decline. This hypothesis is primarily based on observations of contrasting pup growth rates (0.9 vs 1.9% day^−1^) and trip durations (5.8--8.9 vs 1.0--3.4 days) between St. Paul Island where the population is declining and Bogoslof Island where the population is increasing ([@ref5]; [@ref37], [@ref38]). Thus, in addition to elucidating the factors that affect energy expenditure, there is also a pressing conservation need to quantify metabolic rates and understand the trade-offs between energy expenditure and gain in this population.
{#f1}
Methods {#sec2}
=======
Sample collection {#sec3}
-----------------
Lactating northern fur seals were captured during the summer (July--August) and fall (September--October) of 1995 and 1996 at six rookeries on St. George and St. Paul Islands in Alaska, USA (*n =* 93, [Fig. 1](#f1){ref-type="fig"}, NMFS permit \# 837). Once captured in a net, seals were weighed to the nearest 0.1 kg with a Dyna-Link digital scale and physically restrained. Diazepam was administered intramuscularly to facilitate handling and reduce capture stress (0.15 ml, 10 IU ml^−1^). An initial blood sample was collected from an interdigital vein in the hind flipper to determine background isotope concentrations followed by an intraperitoneal injection of a pre-weighed dose of either 10% (55--61 g in 1995, 88--90 g in 1996) or 67% (12--20 g) sterile H~2~^18^O and 3 mL sterile tritiated water containing 1.0 mCi ^3^H. Satellite tags (ST-6, ST-10, Telonics, Mesa, AZ, USA), time-depth recorders (MK-3, MK-5, MK-6, Wildlife Computers, Redmond WA, USA) and VHF tags (ATS, Isanti, MN, USA) were attached to the dorsal pelage mid-back using a quick-setting epoxy (Devcon 5 Minute, Danvers, MA, USA), with each seal receiving variable tag combinations ([Table 1](#TB1){ref-type="table"}, [Fig. 2](#f2){ref-type="fig"}). The time-depth recorders (TDRs) had a resolution of 1 m and were programmed to record depth every 5 or 10 s. Tags were placed in succession along the dorsal midline, with the satellite tag placed first when applicable. Milk samples were collected via manual expression after an intramuscular injection of oxytocin (0.25 mL, 5 IU mL^−1^). Seals were held for approximately 3 h in a ventilated capture box to allow for isotope equilibration ([@ref13]). A final blood sample was collected before release to determine the equilibration and time zero isotope concentrations. Seals were recaptured after a single foraging trip to sea, with collection of mass, and blood and milk samples as described above. Seals were also given an enema upon recapture unless defecation occurred during handling to obtain hard parts for diet analysis (Supplemental Material). This resulted in diet estimates for a subset of seals, as enemas did not always result in a faecal sample.
######
At-sea field metabolic rates (FMR), foraging success and behavioural variables and the tag frontal surface area (FSA) by seal and season
Seal Season At-sea FMR (W kg^−1^) Mass change[^a^](#tblfn1){ref-type="table-fn"} (kg) Water influx (ml kg^−1^ day^−1^) Trip duration (days) \% dive Depth (m) FA cluster Tag FSA[^b^](#tblfn2){ref-type="table-fn"} (cm^2^)
------ -------- ----------------------- ----------------------------------------------------- ---------------------------------- ---------------------- ------------ ------------ ------------ ----------------------------------------------------
1 Fall 7.79 4.1 184.4 2.8 10.7 45.4 1 7.3
3 Fall 5.18 5.7 160.3 5.2 10.5 45.9 1 12.0
9 Summer 6.48 1.9 202.9 7.0 8.8 84.8 3 16.7
13 Both 6.14, 6.36 −0.2, 12.1 135.9, 140.9 5.8, 9.3 19.0, 12.5 52.1, 34.8 3 17.8
16 Fall 6.89 8.3 197.2 8.7 10.8 12.4 1 12.0
18 Both 8.80, 7.91 4.4, 10.9 138.7, 119.1 5.5, 6.3 14.5, 13.2 34.8, 24.4 3, 2 16.7
22 Fall 6.75 7.4 174.9 7.6 12.7 21.4 3 16.7
25 Summer 6.84 0.5 142.1 6.8 20.8 42.4 3 13.8
35 Fall 7.00 5.0 124.8 2.8 10.7 18.1 3 17.79
60 Fall 7.42 8.8 118.0 8.7 16.4 16.6 3 7.28
61 Fall 6.62 2.4 195.1 8.1 15.3 23.9 2 7.28
67 Fall 7.29 1.0 173.7 9.1 23.9 12.6 1 7.28
70 Fall 6.67 4.9 128.6 7.6 11.3 68.7 2 7.28
74 Fall 6.99 6.4 143.0 9.8 11.1 12.5 3 7.4
77 Fall 6.47 7.2 149.1 6.4 14.2 32.3 2 7.4
343 Both 7.51, 6.90 2.7, 4.6 173.0, 157.2 6.5, 4.8 7.8, 12.8 12.5, 52.6 1, 2 12.0, 7.3
344 Fall 6.46 3.7 183.7 7.2 12.1 11.8 1 12.0
345 Both 6.47, 7.01 3.8, 8.0 168.5, 128.6 7.1, 8.1 9.9, 16.8 7.6, 15.5 1, 3 7.3
349 Both 6.70, 6.90 5.5, 2.9 143.5, 119.9 8.6, 6.9 12.5, 13.8 29.2, 14.4 2, 3 16.5
350 Both 6.20, 7.08 3.7, 6.8 168.9, 201.6 5.7, 7.5 12.1, 18.1 36.2, 11.6 1 16.5
355 Both 7.09, 6.76 5.5, 5.2 181.9, 154.7 6.0, 6.2 9.7, 12.6 32.0, 18.9 2, 3 7.3
356 Summer 6.61 3.9 187.3 7.8 6.9 10.2 1 12.0
357 Fall 6.80 1.7 176.2 5.2 9.2 28.7 2 13.8
360 Both 7.44 3.4, 4.5 196.6, 154.3 3.9, 6.3 15.0, 13.0 38.5, 13.9 2, 3 12.0
361 Both 5.52, 7.61 2.9, 5.9 157.5, 204.8 5.6, 6.5 13.2, 15.4 42.1, 57.9 2 16.45
362 Both 5.50, 7.98 6.9, 4.0 254.1, 225.7 7.9, 7.7 5.7, 8.1 10.5, 8.0 1 7.3
367 Both 5.54, 7.06 6.5, 6.5 212.5, 217.4 6.7, 8.4 16.3, 9.8 11.9, 6.0 1 13.8
370 Both 5.93, 6.48 1.4, 3.7 194.5, 212.0 6.2, 8.0 20.4, 13.4 12.6, 9.2 1 7.3
371 Fall 7.30 5.3 110.8 6.9 15.3 22.3 2 13.8
374 Both 6.77, 8.69 0.0, 7.6 144.0, 141.3 5.0, 6.4 14.8, 10.6 14.5, 15.7 1, 3 7.3
375 Both 6.94, 9.68 4.2, 0.3 153.5, 126.7 6.1, 3.3 15.7, 8.1 17.6, 37.3 1, 2 16.5
376 Both 7.54, 7.55 2.4, 1.7 168.5, 166.6 4.7, 5.8 20.2, 12.8 26.0, 36.1 1, 2 12.0
380 Summer 6.26 5.5 174.0 6.8 16.2 12.8 1 16.5
Summer values are presented first where applicable
Adjusted mass change as described in text
The FSA of the anterior tag except where the footprint of the second tag exceeded that of the first. In these cases, the additional FSA was added. For all animals, the FSA of the VHF tag was also added because it was typically offset.
Blood samples were centrifuged to separate the serum component and stored at −20°C until analysis. Milk samples (0.25 mL) were placed in 2 mL of chloroform containing 0.01% butylated hydroxytoluene, and sample tubes were flushed with nitrogen; they were initially frozen at −20°C but were transferred to −80°C at the end of the field season.
Sample and data analysis {#sec4}
------------------------
Serum samples were analyzed for ^18^O by Metabolic Solutions (Nashua, NH, USA). The specific activity of ^3^H was determined in duplicate aliquots by scintillation spectrometry as described in [@ref15]. Total body water was derived from isotope dilution using ^18^O concentrations and the plateau (initial) and scaling (final) methods. We used the equation from [@ref52] to calculate CO~2~ production and a value of 23.9 kJ L^−1^ to convert CO~2~ to energy consumption. The resulting estimate of FMR integrates time spent onshore and at-sea. We estimated the at-sea component of FMR using the approach described in [@ref17]. Water influx was calculated using equations 5 and 6 in [@ref43], which is proxy for prey consumption that is also influenced by prey water content ([@ref13]).
Satellite locations during a foraging trip were selected between known departure and arrival times determined from VHF telemetry or TDRs. Poor-quality locations (ARGOS 'B' and 'Z') were removed, and the remaining locations were filtered based on a maximum transit rate of 3 m s^−1^ (R package *argosfilter*, [@ref22]). Filtered locations were used to estimate locations at hourly intervals with a time-correlated random walk model (R package *crawl*, [@ref35]). Dive data were analyzed with Wildlife Computers software (Zero-offset Correction, v1.22, and Dive Analysis, v 4.08). Model fits for the hourly location estimates were used to produce locations for every dive, which were then spatially joined with bathymetric (ETOPO2v2) and Bering Sea shelf water column temperature data (<https://www.afsc.noaa.gov/RACE/groundfish/survey_data/ebswater.htm>) to classify shelf or pelagic diving and the position of each dive with respect to the mixed layer depth (MLD). We calculated the mean maximum dive depth of dives \>3 m, percentage of time spent diving (\>3 m) while at sea and the percentage of locations that occurred in pelagic vs continental shelf habitat and above or below the MLD (only seals with satellite tags). Milk samples were analyzed for FA composition as described in Supplemental Material.
While these metrics are relatively simple descriptors of behaviour, we chose them for several key reasons. First, dive depth and the percentage of time spent diving at sea (considered a proxy for foraging effort; [@ref29]) have both previously been identified as factors influencing metabolic rates of otariids ([@ref3]; [@ref16]; [@ref28]; [@ref14]). These variables also integrate behaviour across the entire foraging trip and thus reflect the same timescale as the energetic measurement. In addition, several of these variables are generally believed to reflect consumption of two common prey species of northern fur seals; dive depth and position within the water column are associated with age-specific consumption of walleye pollock in continental shelf habitat, whereas squid consumption is largely associated with pelagic habitats ([@ref67]). While more sophisticated analyses or metrics exist for inferring foraging behaviour from contemporary telemetry and dive tags ([@ref12]), these approaches were largely unavailable to us for a variety of reasons or were inappropriate metrics for this species. For example, only about half of the study animals were wearing satellite tags, precluding the use of state-space models to assign behavioural states to each interpolated location. In addition, we were constrained to using the earliest versions of software available for the data and thus lacked the ability to extract and reanalyze the diving data to compute additional metrics, such as those derived from the entire dive profile ([@ref4]).
{#f2}
Statistical analyses {#sec5}
--------------------
Milk FA clusters were identified using an agglomerative hierarchical cluster analysis of a subset of 19 FAs that represented dietary FAs present in sufficient quantities (≥0.5%; [Table S1](#sup1){ref-type="supplementary-material"}; [@ref32]). We used a centred log ratio transformation, the squared Euclidean distance as a measure of similarity, and Ward's method for clustering ([@ref66]). The number of clusters was identified using a dynamic tree cutting algorithm ([@ref30]). We used a linear discriminant analysis to determine which FAs were most important in discriminating among clusters ([Fig. S1](#sup1){ref-type="supplementary-material"}). Each FA sample was then associated with the scat/enema sample and behavioural data representative of that foraging trip to understand how FA clusters reflected foraging behaviour ([Fig. S1](#sup1){ref-type="supplementary-material"}, Supplemental Material). To increase sample size, we used all milk samples collected in 1995 and 1996 (*n* = 291 trips). Similarly, clusters were described using diving (*n* = 191 trips) and tracking data (*n* = 99 trips) and scat/enema samples (*n =* 79 trips) from all seals instrumented in the study years.
Linear mixed effects models were used to examine the effect of season and foraging behaviour on at-sea FMR, with individual included as a random effect to account for repeated samples within the same year. We did not include year or island in the model because these two variables were confounded due to sample design and most useable measurements occurred in 1996 (see Results). Instrumentation has been shown to affect diving metabolic rates in captive northern fur seals ([@ref46]), presumably due to increases in hydrodynamic drag. To account for any potential effects of varying instrument size ([Fig. 2](#f2){ref-type="fig"}), we included the frontal surface area (FSA) of the tag(s) as a covariate. While FSA may not be the best representation of the drag an animal experiences ([@ref36]; [@ref39]), it has been used previously for this species ([@ref50]) and was the best metric we could reliably calculate given the dataset and the free-ranging nature of seals in our study. We calculated the total FSA (sum across all tags) and the effective FSA (the FSA of the anterior tag and any excess area by the second tag) to account for the fact that TDRs were placed directly behind the often larger satellite tag. We only present results using the effective FSA because the overall patterns were the same ([Table 1](#TB1){ref-type="table"}). Foraging behaviour variables included in the model were mean maximum dive depth, the percentage of time spent diving and the FA cluster of the milk sample collected at the time of recapture. The inclusion of FA cluster likely incorporated the variability associated with island or year, as spatial and temporal variability in diet are common within this population ([@ref2]; [@ref67]). We used model averaging of all candidate models with a cumulative sum weight of 95% to assess the effect of each variable on at-sea FMR. Variables were considered important when the 95% CI did not include zero. Model assumptions were assessed using residual and quantile plots of the full model.
We used a similar approach to determine the factors influencing foraging success, which we quantified using three variables: absolute mass gain, daily mass gain at sea, and water influx. We did not use a mixed effects approach for the two mass gain variables because the variance of the random effect in all models was estimated at exactly zero. Because seals spent variable amounts of time onshore, we adjusted mass to better reflect mass gain at sea using data on the fasting mass loss ashore during summer and fall from another fur seal species ([@ref24]). Explanatory variables included in the full model for each metric of foraging success included season, initial mass, trip duration, tag FSA, at-sea FMR and FA cluster. As above, we used model averaging to assess the effect of each variable on foraging success. All analyses were conducted using R v 3.4.1 (R Core Team 2017, <https://www.R-project.org/>). Means are presented ± SD.
Results {#sec6}
=======
Metabolic rates were obtained for all fur seals but a subset of measurements (*n* = 45) were excluded because isotope levels at recapture were too close to background to yield reliable estimates of energy expenditure. The final dataset consisted of 48 measurements from 33 seals that ranged in body mass from 26.0 to 47.6 kg ([Tables 1](#TB1){ref-type="table"}, [S2](#sup1){ref-type="supplementary-material"}, [Fig. 3](#f3){ref-type="fig"}). The average absolute and daily adjusted mass gains were 4.6 ± 2.7 kg (−0.2--12.1 kg) and 0.7 ± 0.4 kg day^−1^ (−0.03--1.8 kg day^−1^), respectively. Seals foraged in continental shelf and pelagic habitats up to 350 km from the colony ([Fig. 1A](#f1){ref-type="fig"}). They spent an average of 13.2 ± 3.8% (5.7--23.9%) of their time diving to depths \>3 m, with the vast majority of diving activity occurring at night (81.8 ± 12.2%). Seals dove to average maximum depths of 26.2 ± 17.4 m, with means for individual seals ranging from 6.0--84.8 m ([Table 1](#TB1){ref-type="table"}).
{#f3}
Foraging trips were assigned to one of four FA clusters, although none of the samples collected from DLW seals were classified into Cluster 4 ([Table 1](#TB1){ref-type="table"}, [Fig. S1](#sup1){ref-type="supplementary-material"}). Squid appeared to be the predominate prey species of seals with trips in Cluster 1, whereas variation in the proportion of age-zero pollock vs mature pollock appeared to distinguish seals in Cluster 2 and 3, respectively (Supplemental Material).
{#f4}
Individual models for at-sea FMR explained between 1.0--26% of the variability in the data ([Fig. 4](#f4){ref-type="fig"}, [Table S3](#sup1){ref-type="supplementary-material"}). Fur seals experienced a 7.2% increase in at-sea FMR from summer to fall and a 1.9% decrease in at-sea FMR for each additional day spent at sea ([Fig. 4](#f4){ref-type="fig"}). There was no effect of foraging behaviour or tag FSA on at-sea FMR. Total mass gain increased with trip duration and season, with seals gaining an additional 0.6 kg per additional foraging day and an average of 1.8 kg more during the fall. The daily rate of mass gain increased an average of 0.3 kg day^−1^ between summer and fall ([Fig. 5](#f5){ref-type="fig"}, [Tables S4--S5](#sup1){ref-type="supplementary-material"}). Water influx was not influenced by trip duration, but it varied among FA clusters. The highest estimates of water influx were for seals in FA Cluster 1, followed by FA Cluster 2 and lastly by FA Cluster 3 ([Fig. 5](#f5){ref-type="fig"}, [Table S6](#sup1){ref-type="supplementary-material"}), which is consistent with relative water content estimates of putative prey. Tag FSA was not important in any of the foraging metric models.
{#f5}
Discussion {#sec7}
==========
Metabolic rates collected from free-ranging animals provide insight into the collective influence of animal behaviour and physiology on energy expenditure. The life history and behaviour of female otariids lend themselves to metabolic studies, allowing us to quantify the influence of diving behaviour, diet and season on a species that has experienced an unexplained population decline since the 1990s. While data for this study were collected over two decades ago, our comparatively large sample size has nearly doubled the available metabolic data for this species. As such, our dataset contributes to our knowledge of the energetics of free-ranging mammals and is directly relevant to the conservation of northern fur seals.
Individual variation in time-activity budgets is an important driver of intraspecific variation in FMR since foraging is an energetically expensive activity ([@ref27]; [@ref1]; [@ref25]; [@ref63]). Despite this, we did not find a relationship between at-sea FMR and the relative time seals spent diving, a proxy for foraging effort. This is perhaps not surprising given that a relationship between at-sea FMR and relative time spent diving has been detected for Antarctic fur seals and New Zealand sea lions (but in opposite directions) and is apparently absent for other otariids ([@ref3]; [@ref16], [@ref17]; [@ref58]; [@ref42]). These contrasting relationships may be due to differences in the underlying mechanisms driving variation in the percentage of time spent diving, and how time at sea is allocated to other behaviours. Northern fur seals feed predominately at night; thus, a significant portion of their foraging trip is allocated towards other activities such as grooming, resting and transiting ([@ref7]). Considerable advancements in tagging technology have occurred since our study, which could be used to address the limitations of our study and better characterize the time-activity budgets during foraging trips. There have been several more recent studies on energy expenditure of northern fur seals that coupled accelerometers with metabolic measurements ([@ref33]; [@ref34]), but they were largely not focused on examining the factors influencing the rate of energy expenditure.
Seasonal changes in metabolic rates associated with the timing of molt have been measured in both terrestrial and marine species, with increases attributed to the added energetic cost of tissue generation and thermoregulation ([@ref20]; [@ref10]; [@ref31]; [@ref19]; [@ref40]). In northern fur seals, captive juveniles experienced a 50% (resting metabolic rate) and 16% (daily energy expenditure) increase during the fall compared with other seasons, which the authors hypothesized was due to the direct costs of molting ([@ref19]). The molt of adult female northern fur seals lasts approximately 15 weeks, with an average mid-date of molt of October 18--November 13 depending on age ([@ref47]). This timing corresponds to our sample collection dates, suggesting that at-sea metabolic rates may have been higher in the fall compared with summer because females were molting. The timing of this increase also coincides with seasonal increases in lactation costs ([@ref21]) that can result in increased foraging effort ([@ref29]), but this seems an unlikely explanation given we did not find any relationships between at-sea FMR and diving behaviour or foraging success.
Central-place foragers can compensate for higher energy demands by increasing foraging effort, trip duration or switching to energy rich prey, none of which are mutually exclusive ([@ref24]; [@ref49]; [@ref8]; [@ref41]; [@ref29]). Increases in trip duration across lactation appear to be a shared trait among many fur seal species, including northern fur seals ([@ref11]; [@ref23]; [@ref24]; [@ref26]); the average trip durations of seals in our study increased by a maximum of 0.3 and 0.85 days between summer and fall in 1995 and 1996, respectively. The decrease in at-sea FMR associated with longer trips is likely associated with changes in time-activity budgets ([@ref3]; [@ref33]); trip durations typically increase with travel distance from the colony, and thus, females may spend a greater percentage of time in transit or other behaviours on longer foraging trips. Seasonal changes in at-sea FMR did not affect any of the foraging success metrics, but the positive relationship between total mass gain and trip duration suggests the benefits associated with increasing trip duration appear to outweigh the metabolic costs associated with additional time spent at sea. The ability to increase trip duration to cope with higher energy demands is, however, not limitless, as females must balance trade-offs among trip duration, the fasting ability of the pup and the potential decline in milk energy delivery rate relative to time spent at sea ([@ref14]).
The time- and cost-intensive nature of metabolic studies using free-ranging animals often results in the energetic requirements of a species being quantified from a few studies, which is problematic if there is spatial or temporal variation in factors affecting metabolic rates. Dietary variation at multiple temporal and spatial scales is present within this population ([@ref2]; [@ref67]), and variable consumption of age-zero vs adult pollock has previously been attributed to inter-annual variation in at-sea FMR of northern fur seals ([@ref18]; [@ref14]). We found no evidence that FA cluster or dive depth had any influence on at-sea FMR, despite that FA clusters appeared to capture inter-island and temporal dietary variation related to variable consumption of squid, and age-zero and mature pollock. While the conclusions of [@ref14] were based on a relatively small number of animals with unknown foraging behaviour, they are consistent with findings by [@ref16] and [@ref28] that increases in dive depth were associated with reduced metabolic rates. Our study was conducted during the initial period of unknown population decline, and it may be that females had reached their metabolic ceiling, thus minimizing any diet-related effects, as has been suggested for Antarctic fur seals ([@ref14]). The metabolic rates reported here were similar to a 2011 study that found no effect of foraging strategies on at-sea FMR ([@ref34]), providing corroborating evidence that this population may currently have limited energetic flexibility to respond to future environmental changes. In a conceptual model of parental attendance, [@ref14] suggested that female otariids should first increase foraging effort (at-sea FMR) before increasing trip duration in response to changes in food availability because of the negative impact of longer trip durations on pup growth rates. Further investigation of the links between female foraging behaviour, pup growth and prey availability and the impacts on population dynamics is thus warranted, particularly given the contrasting pup growth rates and trip durations between islands experiencing divergent population trends.
Conclusion {#sec8}
----------
The eastern Pacific stock of northern fur seals has experienced an unexplained population decline since the 1990s, with pup production estimates in 2018 at their lowest in over 100 years ([@ref55]). There is thus a pressing need to understand the factor/s contributing to the decline, and in particular, the role that food limitation might play given that one of their primary prey resources during lactation is also the target species for the US largest fishery. Metabolic rate measurements are critical to the conservation of this species because they provide insight into how hard seals are working to find food and how energy needs might change in a temporally and spatially dynamic environment. They are also a key parameter in bioenergetic models that can be used to quantify population-level prey consumption, which is needed to better understand prey needs and potential overlap with fishing activities. In this study, we were able to nearly double the available metabolic data for this species, with data collection occurring during the initial population decline. We detected seasonal increases in at-sea FMR that coincided with the timing of the annual molt, but foraging behaviour appeared to have little impact on energy expenditure of northern fur seals. A large portion of the variation in at-sea FMR remained unexplained, which could be due to a combination of introduced variability associated with the method itself, other factors not considered here, or our inability to partition time-activity budgets into discrete behaviours. Future studies are thus warranted, particularly as the Bering Sea has experienced unprecedented warming with winter ice extent at record lows in the last 2 years that will likely have ecosystem-level impacts ([@ref53]). Despite study limitations, our data indicate that female northern fur seals appear to have reached a metabolic ceiling early in the current population decline, which may be a contributing factor to the lower pup growth rates observed in the Pribilof Islands compared with Bogoslof Island where fur seals have experienced healthy population growth. These high metabolic rates required a female to spend more of her energy gain on her own metabolic overhead, with no indication that individuals with higher metabolic rates were able to gain more mass while at sea without increasing their trip durations. While they do not provide direct support for the hypothesis that food limitation is contributing to the population decline, the results of our study provide indirect evidence that food limitation is likely reducing the amount of energy available a female has to invest in lactation with adverse consequences for reproductive success.
Supplementary Material
======================
######
Click here for additional data file.
This project would not have been possible with the assistance of numerous people, especially Rolf Ream, Rod Towell, Bruce Robson, Thomas Loughlin, George Antonelis, Tonya Zeppelin, Jason Baker, Mary Donohue, Nicole Horner, Katie Luxa, Dorian Houser, William Meyer, Sara Iverson, Alexander Boltnev, Robert Caruso, Jenn Jolly, Chris Gaburski, Sacha Vigneri and Gary Merculief.
Funding {#sec9}
=======
This work was supported by funding from the National Science Foundation (\#9500072) and NOAA (field component), and the Lenfest Ocean Program (\#A-03151, support for EAM).
|
{
"pile_set_name": "PubMed Central"
}
|
INTRODUCTION
============
Hemophilia is a hereditary disease of the mechanism of bleeding clinically manifested as a tendency to bleed which presents serious challenges in a dental practice.^[@B1]^
As inherited bleeding disorders account for 1 in 10,000 live birth and hemophilia represents the majority of such disorders, they are the priority group for dental care.^[@B2]^
Hemophilic children must be thought of as special patients. The bleeding tendency and fear of bleed may have a negative effect on preventive dental care of patients with hemophilia both at home and at dental appointments. Although there have been a number of studies regarding oral, surgical, and periodontal management of hemophilic patients, there are few studies regarding the oral health status in such patients.
Hemophilia A is the deficiency of factor VIII, it is most common, and hemophilia B also called the Christmas disease is the deficiency of factor IX, hemophilia B is affecting approximately 1 in 7,500 males and Von Willbrand disease. Hemophilia C, involving factor XI, is very rare but much milder than hemophilia C, involving factor XI, is very rare but much milder than hemophilia A or hemophilia B. Other factor defecies, such as those of factor II, V, and XII (1 case per 1 million) and factor VII (1 case per 500,000). Are rare and extremely un common.
As the age increase, the child\'s physical activity naturally increases, which results in more exposure to trauma, especially in hemophiliacs.
Oral health instructions create awareness of the need to return regularly for examination, professional prophylaxis, and treatment. Appropriate dental care is necessary for these individuals; however, the issue gains more importance as dental care affects the general health of hemophilic patients.^[@B3]^
The aim of the present study is to assess the oral hygiene status, the prevalence of dental caries, and treatment needs in children referred to the Hyderabad Hemophilic Society, Telangana state.
MATERIALS AND METHODOLOGY
=========================
Study Design
------------
A descriptive cross-sectional study was conducted at the Department of Pedodontics and Preventive Dentistry, Mamata Dental College, Khammam, Telangana, India.
The sampling procedure involved multistage stratified sampling,^[@B4]^ where the Hyderabad is divided into five strata, namely Hyderabad central, Hyd southern, northern, eastern, and western.
Eligible children were selected randomly from a list obtained from Hyderabad Hemophilic Society records. Age eligibility requires that the children fall into the appropriate age at the time of sampling.
Study Sample
------------
A total of 60 children, age ranging from 7 to 16 years, were selected for this study.
Research Methodology
--------------------
Data collection instrument included the number of data types and different data collection instruments. It consisted of retrospective data collection and concurrent data.
Study Questionnaire
-------------------
A questionnaire included questions about the presence of other hemophilic members of family, type of dietary habits, frequency of tooth brushing, education and economic level of parents, and parent\'s oral hygiene habits. The draft of the questionnaire was reviewed by the panel of experts which included faculty members from the Pedodontics and Preventive Dentistry, Public Health Dentistry.
Inclusion and Exclusion Criteria
--------------------------------
The children suffering from hemophilia were only included in the study. Other hematological disorders and systemic diseases were excluded.
Measurements of Oral Hygiene Status and Dental Caries
-----------------------------------------------------
Oral hygiene status was recorded by using OHI-S^[@B5]^ as reported by Grene and Vermillon.
Teeth affected by dental caries. The teeth restored/extracted as sequale of dental caries were assessed using decayed, missed, filled tooth (DEFT), DMFT^[@B6]^ for primary and permanent dentition respectively was recorded by using WHO criteria in 1997. Missing primary incisor teeth for children aged 7 years and older were considered exfoliated and were not included in the "m" component, as was missing primary canines or molars in children aged 9 or 10 years. When permanent teeth were missing, a history of the reason for tooth extraction was obtained to ensure that orthodontic extractions were not included in the "M" component.
Statistical Analysis
--------------------
Statistical analysis was performed using the Statistical Package of Social Science (version 21; SPSS Inc., Chicago, IL, USA). Frequency tables were computed. The results are presented in mean ± standard deviation and percentages. The observations were statistically analyzed using the Chi-square test and the Kruskal--Wallis test. *p* \< 0.05 was considered statically significant.
RESULTS
=======
[Table 1](#T1){ref-type="table"} shows that a total of 60 children were examined, children belong to the age group of 7--9 years were 21, 10--12 years were 20, and 13--16 years 19.
[Table 2](#T2){ref-type="table"} shows that a total of 93.90% of children require dental treatments. Children within the age of 7--9 years, 80.95% require preventive care, 33.3% fissure sealant, 23.80% extractions, 47.6% crowns and the age group of 10--12 years requires 100% restorations, 20% extractions, 65% pulp care, 7--9 years age group children require more dental interventions than compared to the 10--12 years and 13--16 years age group children.
[Table 3](#T3){ref-type="table"} shows that the difference between the two groups like DMFT and DEFT; the *p* value is 0.024; *p* \> 0.05, it is not statistically significant; the OHI-S between two groups, the *p* value is 0.043; *p* \> 0.05, it is not statistically significant.
[Table 4](#T4){ref-type="table"} shows that the mean DMFT/DEFT of 7--9 years was 4.76 and for 10--12 years was 3.75 and for 13--16 years was 3.11. The highest DMFT/DEFT scores were recorded in 7--9 years age group\'s children. The mean oral hygiene index simplified score was 1.28 in 7--9 years and 1.44 in 10--12 years and 1.87 in 13--16 years, respectively. The poor oral health status was recorded in the 13--16 years age group.
######
Sample distribution according to the age group
*Age group* *n*
-------------- -----
7--9 years 21
10--12 years 20
13--16 years 19
Total (*N*) 60
######
Treatments needs of study population according to the age group
*Treatment* *7--9 years* *10--12 years* *13--16 years*
----------------- -------------- ---------------- ----------------
Preventive care 17 (80.95%) 12 (60%) 8 (42.10%)
Fissure sealant 7 (33.3%) 5 (25%) 3 (15.78%)
Restoration 18 (85.7%) 20 (100%) 16 (83.1%)
Crown 10 (47.6%) 7 (35%) 8 (42.20%)
Pulp care 12 (57.14%) 13 (65%) 12 (63.15%)
Extraction 5 (23.80%) 4 (20%) 1 (5.26%)
######
Intra-analysis of mean DMFT and mean DEFT study population
*Sum of squares* *df* *Mean square* *F* *p value*
---------------- ------------------ -------- --------------- -------- -----------
DMFT 42.241 2 21.121 11.610 0.000
Between groups 103.69 57 1.819
Total 145.93 59
DEFT 138.44 2 28.087 28.087 0.000
Between groups 140.48 57
Total 278.93 59
DMFT + DEFT 28.051 14.026 3.970 3.970 0.024
Between groups 201.34 3.532
Total 229--240
OHI-S 3.705 1.85 3.314 3.314 0.043
Between groups 31.857 0.559
Total 35.562
*p* \> 0.005 is statically not significant
######
DMFT and DEFT group
*n* *Mean* *Standard deviation* *Standard error*
-------------- ----- -------- ---------------------- ------------------
DMFT + DEFT
7--9 years 21 4.76 2.343 0.511
10--12 years 20 3.75 1.743 0.390
13--16 years 19 3.11 1.370 0.314
Total 60 3.90 1.972 0.255
OHI-S
7--9 years 21 1.28 0.763 0.167
10--12 years 20 1.44 0.586 0.131
13--16 years 19 1.87 0.872 0.200
Total 60 1.52 0.776 0.100
*p* \> 0.005 is statistically not significant
[Table 5](#T5){ref-type="table"} shows that Out of 60 subjects 44 children were affected with dental caries. Overall prevalence of dental caries is 73.3% and patients showed poor oral hygiene status was 73.7%.
DISCUSSION
==========
The present study recorded the overall caries' prevalence as 73.3% with a mean DEFT and DMFT of 4.76 in the 7--9 years age group and 3.75 in the 10--12 years age group, and 3.11 in the 13--16 years age group. According to the WHO, the mean DMFT at the age of 12 years should not be more than 3.^[@B7]^
######
Prevalence of dental caries according to the age group
*Age group count % within age group* *DMFT and DEFT group*
-------------------------------------- ----------------------- -----------
7--9 years 3 (14.3) 18 (85.7)
10--12 years 6 (30.6) 14 (70.0)
12--16 years 7 (36.8) 12 (63.2)
Total 16 (26.7) 44 (73.3)
In a study, Boyd and Kinirons^[@B8]^ observed a slightly higher prevalence of dental caries in the primary dentition of hemophilic children compare to control. Similarly, in a study by Kabil et al.,^[@B9]^ DMFT and DEFT of hemophilic children were significantly higher than those of the non-hemophilic in Egypt.
Our results are in contrast with the study conducted by Sonbol et al.^[@B10]^ where significantly greater proportion of children with severe hemophilia were caries free compared with controls. Both the DMFS and DMFT were significantly greater in the controls compared with the hemophilic group. This may be due to the fact that there patients received dental care as dental department was situated next to the out patient hematology consulting room and the dental aspect of the service was considered as an integral part of the hematology visit.
In the present study, the oral hygiene index simplified was scored as poor in 13--16 years (1.87), 10--12 years (1.44), and 7--9 years (1.28) age group. These findings were similar to a study conducted in Poland where the oral hygiene index of hemophilic children was recorded as worse when compared to controls.^[@B11]^
Our results are in contrast with a study conducted by Zaliuniene et al.^[@B12]^ showing that the overall findings were better in deciduous dentitions, i.e., less overall caries experience and lower dental treatment needs were observed in children with hemophilia as compared to their healthy counterparts. However, there was a significant difference observed when permanent dentitions were compared between the hemophiliacs and controls.
In the present study, the mean DMFT of 4.76 in 7--9 years, 3.75 in 10--12 years, and 3.11 in the 13--16 years age group, which was not in agreement with the study conducted by Evangelista^[@B13]^ where the DMFT score was 0.9, which might be due to the early intervention of dental caries. The present study was not in accordance with a study by Rodrigues et al.^[@B14]^ who recorded a mean DEFT of 2.00.
In the present study, the crown requirement study was reported as the 7--9-year age group and 35% in the 10--12-year age group. The probable reason could be that as age increases, the prevalence of caries progression increases and the requirement of treatment needs increases, except for crowns, it decreased as age increases dental caries in mixed dentition, the spread of caries process is faster, so the requirement of crown is more in 7--9 years old children than in 10--12 years.
In the present study, the overall treatment required was 93.90%, in that 80.95% required preventive care, 33.33% fissure sealant, 100% surface fillings, 23.80% extraction, 63.15% pulp care, and 47.6% crown. The results of the present study were in accordance with the study reported by Sudhanshu et al.^[@B7]^
CONCLUSION
==========
Our data show that high dental caries was seen in 7--9 years old hemophilic children Hyderabad. The present study showed the oral hygiene status was poor and the dental caries, the prevalence was 73.3% and the 93.3% required treatment needs.
LIMITATIONS
===========
Further studies are recommended to better understand oral health of hemophilic children, and a longitudinal study with a large size is needed.
RECOMMENDATIONS
===============
Good oral health is essential to improve individual overall health and wellbeing. This overview points toward developing immediate and effective oral health promotional and interventional strategies to combat the diseases in this special group.
**Source of support:** Nil
**Conflict of interest:** None
|
{
"pile_set_name": "PubMed Central"
}
|
Background
==========
Within Sub-Saharan Africa during 2007 There were an estimated 22 million individuals living with HIV infection, 1.5 million HIV-related deaths, and 11.6 million children orphaned due to parental HIV infection \[[@B1]\]. A public health approach is clearly required to provide appropriate health care to meet the preventive, care and support, treatment, and bereavement needs associated with the epidemic. In terms of domains of interest for a public health approach, the endpoints must include both the patient and their informal carers and family. This is particularly true in resource limited settings where progressive disease compounds family-wide poverty, and where a limited health sector relies on informal caregivers to provide both inpatient and home care. Further, we must determine the effectiveness of interventions across the multiple dimensions of need that underpin the lived experience of disease, i.e. the physical (e.g. virological responses, symptoms and treatment side effects), psychological (e.g. anxiety and depression), social (family needs, food security and stigma) and spiritual needs (e.g. finding peace and comfort through spiritual care). These domains are inextricably linked, and arguably cannot be effectively addressed in isolation.
In 2003 the United States government (USG) funded a five-year, \$15 billion initiative to combat the global HIV/AIDS epidemic: the President\'s Emergency Plan for AIDS Relief (PEPFAR). The funds were allocated approximately as follows: treatment (55%), prevention (20%), assisting orphans and vulnerable children (10%) and care and support of individuals with HIV/AIDS (15%) for years 2003-2008. In 2008, PEPFAR was reauthorized for a further five years up to \$48 billion.
Evaluation of the effect of PEPFAR funding in its target countries has established that there has been a decrease in HIV-related deaths \[[@B2]\], and a reduction in the number of HIV positive births \[[@B3]\]. While the focus on increased access to treatment has achieved results, there has been a lack of evidence for the effectiveness of care and support on patient and family self-report outcomes such as quality of life, or on evaluation of the models of care being delivered. Lack of attention to these outcomes may undermine and diminish the gains brought by improved treatment access. Person-centred self report measures are essential as there is an increasing body of evidence that people living with HIV infection endure multiple distressing problems from the point of diagnosis and alongside treatment \[[@B4]\], including suicidal ideation \[[@B5]\], depression \[[@B6],[@B7]\], fatigue \[[@B8]\], poverty, malnutrition \[[@B9]\], pain \[[@B10]\] and other symptoms \[[@B11]\], poor access to symptom controlling drugs \[[@B12]\], spiritual distress \[[@B13]\] and information needs \[[@B14]\].
In order to evaluate funded programmes, and to facilitate evidence-based programming of the funding response, Public Health Evaluations (PHEs) have been commissioned in line with allocation of funding detailed above. The Care and Support PHE reported here has developed a multi-centred, two phase mixed-methods international study protocol. We report the protocol to provide access to the full study methods \[[@B15]\], and to offer examples of our responses to the methodological challenges of conducting longitudinal multi-methods and multidimensional research.
Methods/Design
==============
Aims and objectives
-------------------
This PHE has two aims. These are as follows. Aim 1) To describe the nature and scope of HIV care and support in two African countries, including the types of facilities available, clients seen, and availability of specific components of care \[Study Phase 1\]. Aim 2) To determine patient health outcomes over time and principle cost drivers \[Study Phase 2\].
The study objectives are as follows. 1) To undertake a cross-sectional survey of service configuration and activity by sampling 10% of the facilities being funded by PEPFAR to provide HIV care and support in Kenya and Uganda (Phase 1) in order to describe care currently provided. 2) To conduct patient focus group discussions at each of these (Phase 1) to determine care received. 3) To undertake a longitudinal prospective study of 1200 patients who are newly diagnosed with HIV or patients with HIV who present with a new problem attending PEPFAR care and support services. Data collection includes self-reported quality of life, core palliative outcomes and components of care received (Phase 2). 4) To conduct qualitative interviews with staff, patients and carers in order to explore and understand service issues and care provision in more depth (Phase 2). 5) To undertake document analysis to appraise the clinical care procedures at each facility (Phase 2). 6) To determine principle cost drivers including staff, overhead and laboratory costs (Phase 2).
Methods/Design
--------------
The study has a mixed methods design across two Phases.
The first Phase utilises survey methods, focus group discussions, document analysis and pharmacy checklist review among a randomly selected stratified sample of all HIV facilities receiving PEPFAR funds in Kenya and Uganda.
The second Phase at each site employs qualitative interviews with patients, family caregivers and staff, longitudinally applied outcome tools among patients in each of 12 facilities across Kenya and Uganda, and a costing study.
The study protocol is depicted in Figures [1](#F1){ref-type="fig"} and [2](#F2){ref-type="fig"}
{#F1}
{#F2}
Protocol development
--------------------
Kenya and Uganda are two of the 15 PEPFAR \'focus countries\'. They are both low-income countries with a high prevalence of HIV, and are countries with public health systems that may offer lessons for replication in other African countries.
The PEPFAR programme is country-wide in the two countries engaged in this PHE (and had been implemented for several years before the evaluation was commissioned. Therefore it was not possible to conduct a \'before and after\' study. One potential study design option was to compare outcomes at facilities receiving PEPFAR funding with those which did not, but there are few large facilities in the target countries which have never received PEPFAR funding, and they would have little motivation to participate. Little information exists regarding the quality of life of general populations, which could have been used as a comparison sample. Further, facilities did not have stated targets against which their performance could be compared.
A cross-sectional study would only be able to identify differences between facilities, which might be caused by population factors as well as variations in care and support delivery.
Therefore, a longitudinal cohort study design has been selected to allow the effect of care over time to be examined. This design offers the most feasible and robust option for evaluation of outcomes, although it is not possible to remove the effects of previous contact with PEPFAR Care and Support.
Patient self-reported health has been selected as the outcome of interest because care and support aims to improve quality of life, and could not be properly assessed without measuring this outcome. A mixed-methods design incorporating both quantitative and qualitative methods allows triangulation and greater understanding of the data and its context.
This evaluation of PEPFAR-funded care and support for HIV is led by King\'s College London (KCL, Principal Investigator), in collaboration with MEASURE Evaluation at the University of North Carolina (UNC), the African Palliative Care Association (APCA) and the Kenyan Hospice and Palliative Care Association (KEHPCA). The aims, methods and implementation of the evaluation have been planned and agreed in consultation with the members of United States Government (USG) Care and Support Technical Working Group, Kenyan and Ugandan PEPFAR Country Teams, and representatives of the Ministries of Health in Kenya and Uganda.
Ethical approval and procedures
-------------------------------
Ethical approval to undertake the study has been received from the Ugandan National Council for Science and Technology (UNCST, Ref SS 1964), the Kenyan Medical Research Institute (Ref KEMRI/RES/7/3/1) and the College Research Ethics Committee at KCL (Ref CREC/06/07-140). Subsequent tool changes following initial piloting have also been approved.
During longitudinal data collection, all questionnaires are stored separately from consent forms, in a locked filing cabinet at the facility. Upon completion of the study, anonymised questionnaires are taken from the facility to the APCA offices for storage in locked filing cabinets. These arrangements are in line with ethical guidance and the UK Data Protection Act 1998. Interview transcripts are stored without any identifying information and names etc are deleted from the transcript. Original digital recordings are deleted once transcribed. All study computers are password protected.
### Phase 1 methods
Sampling
--------
Of around 600 PEPFAR-funded HIV care facilities in each country, 60 have been selected for inclusion in the study (approximately 10% of PEPFAR-funded facilities). The inclusion criterion for eligibility in Phase 1 are that they receive PEPFAR funding to provide HIV care and support. The exclusion criteria are facilities that are paediatric-only or inaccessible (e.g. insecure, no road access). Facilities which do not meet the criteria above were replaced using the same random process (and replacements reported against the criteria).
According to routine monitoring patient numbers, the PEPFAR-funded care and support facilities include many smaller facilities. In order to capture a range of facility sizes within the study population, facilities were stratified by number of patients seen for HIV care in the previous financial year (according to national PEPFAR records) and divided into three strata (1 to 100, 101 to 500 and \>500 patients seen), resulting in unequal and calculable sampling fractions. Twenty facilities were randomly sampled within each of the strata for the study population.
Tool development & data collection procedure
--------------------------------------------
All tools were developed by a multidisciplinary team, including medical professionals, expert HIV researchers and programmers, in conjunction with USG Care and Support Technical Working Group and the PEPFAR country teams. All tools have been piloted in one large and one small Phase 1 facility in Uganda. These facilities were two of the 60 selected, and data from the pilot utilised in the final analyses. Following piloting, the wording and structure of the tools were modified and clarified.
Facilities are informed of the planned survey through the Ministry of Health (MOH) and invited to participate. Local African researchers attend each sampled site to collect data on a pre-arranged day. Data are recorded on two separate sets of identical forms. One set is left with the facility while the other is taken by the researchers for data entry.
Four data collection tools are used.
### Phase 1 data collection tool 1: Senior staff interview
The local researchers interview a group of senior staff, including facility managers and senior clinical staff, at each health facility to collect responses to closed and open-ended questions about patient numbers, infrastructure and staffing. This tool also includes a version of the Client Services Receipt Inventory (CSRI) (Beecham and Knapp 2001) adapted for the aims of this study and the HIV setting in Africa to identify services offered to patients with HIV. The CSRI asks if the facility offers various specific components of care under the four domains of care: clinical, psychological, spiritual, social and preventive. The tool is designed for use across the wide range of size and type of HIV care facilities funded by PEPFAR.
Researchers hold interviews with senior facility staff (approximately three per facility, each contributing to a single data collection form) to collect staff-reported information on facility structure, service delivery, care offered, and to ask their views about the services they offer. These staff members are also asked to provide blank service documents (including service aim, referral forms, assessment sheets and patient information sheets), where available.
### Phase 1 data collection tool 2: Facility document collection
In order to study the level of patient-level clinical information collection and management at each facility, the existence, format and language of specific clinical documents relating to care in the facility are recorded (service aim, inwards referral criteria, incoming/outgoing referral forms, patient charging, ART protocols, care protocols, first assessment sheets, ongoing contact assessment sheets, patient records, referral follow-up forms, stock control sheet, and information to patients). Blank example documents are taken, where available, for content analysis.
### Phase 1 data collection tool 3: Pharmacy review
Researchers record the level and place of drug stock for in-date and expired drugs separately, and if there had been previous stockouts (in-date drugs only) for various formulations of drugs commonly used in HIV care.
To complete the pharmacy review, researchers visit the pharmacy to review stocks and stock cards and complete the standard tables on current drug stocks, stockouts, stock levels and access. This is conducted with the assistance of the pharmacist (or dispenser or other staff who worked in the pharmacy).
### Phase 1 data collection tool 4: Patient focus group discussions
Researchers lead patient discussion groups using the semi-structured interview schedule. The focus group discussions (FGD) have two main aims: to act as a validation of the senior staff interview data relating to components of care offered, and to explore aspects relating to patients\' care (e.g. which components of care are valued and why, any problems in obtaining services).
FGDs are held with existing patients at each facility. Inclusion criteria are as follows: adult patients who have been under care for at least 6 weeks, who are known (by both the patient themselves and clinical staff) to be HIV positive and give informed consent to participate (following provision of an information sheet and consent form). Patients are purposively selected with the aim of obtaining a diverse group with respect to gender, age, disease stage and anti-retroviral (ARV) use.
Approximately five patients in each facility are invited to participate in the discussion group, led by the researcher. Researchers make notes on the responses to pre-specified questions on the interview schedule, and the FGD is digitally recorded as a back-up. During each FGD, demographic information is collected on participants\' gender, location (urban, rural or peri-urban), age and household size. Participants also state if they have accessed specific key components of care including daily cotrimoxazole (CTX), a mosquito bednet and nutritional counselling.
Data management and entry
-------------------------
Data are transferred from Phase 1 facilities immediately after collection. Quantitative data (i.e. closed questions from the senior staff interview and the pharmacy review) are double-entered by two different researchers, and validated, using EpiData v3.1. Errors in data entry and data recording are identified using consistency and logic checks, and followed-up by manual checking of questionnaires. Responses to open-ended questions and FGDs are entered into pre-formatted templates in MS Word 2003. Information from the record of documents available at the facility, and their content, are entered into tables in MS Word 2003 files.
### Phase 1 analysis plan
Senior staff interviews
-----------------------
Analysis is conducted using Stata v10 (quantitative) and NVivo v7 (open-ended questions). Frequency tables are generated for key responses, grouped by facility type where appropriate. A Spearman\'s rank test for correlation is conducted to test the reliability of routine data. Patient numbers are weighted to account for the stratified design used to select facilities. Thematic analysis of content is conducted on the responses to the open-ended questions \[[@B16]\]. The principal themes are organised into data categories and then agreed between two researchers.
Facility document analysis
--------------------------
To determine the availability of the various types of service documents, a matrix is developed to record the number of facilities who report having each document, and the number and percentage of facilities that provided examples for further analysis.
Where the percentage of facilities who provided examples of documents as a proportion of those who reported such documents existed is less than 20%, or where the absolute number of documents is five or fewer, no further analysis is undertaken. Researchers conduct telephone conversations with site representatives in these cases to determine the reason for non-provision.
In those instances where the percentage of facilities who provide examples of documents as a proportion of those who reported such documents existed was equal to or greater than 20%, content analysis is undertaken. Data are extracted to common tables, and frequencies described for the number of facilities reporting each type of recording sheet, whether a sample is obtained, the specific nature of the information in the document fields reported, and described according to facility type.
Pharmacy review
---------------
Analysis is conducted using Stata v10. Frequency tables are generated for each drug, grouped by facility type where appropriate. Data from the pharmacy review is compared with components of care offered according to the senior staff interview data in the CSRI.
Patient focus group discussions
-------------------------------
Information on FGD participants\' background and receipt of care items is entered into a predesigned table by the researchers, transferred into an Excel spreadsheet and then merged with the Stata database using a unique identifying variable. The care received by FGD participants is integrated with the facility staff reports of care offered.
Analysis of the FGDs text is conducted using NVivo v7. In the same way as for the open-ended questions in the senior staff interviews, thematic analysis of content is conducted on the notes from the FGDs. The principal themes are independently organised into data categories and then agreed between two researchers.
### Phase 2 Methods
Sampling procedure for Phase 2
------------------------------
Sampling health facilities
--------------------------
In Phase 1, the approximately 1200 facilities receiving PEPAR Care and Support funding in Kenya and Uganda were divided into three strata based on the number of patients treated in the past year, and 20 facilities selected at random from each stratum within each country. From these 60 facilities in each country, the largest six in each were selected to participate in Phase 2. The inclusion criteria for Phase 2, which applied in addition to those for Phase 1, were that facilities: recruited at least 30 new HIV patients a month; had sufficient staff with essential skills to conduct data collection; offered ongoing care and support to enable longitudinal data collection; had sufficient capacity to engage in the research.
The six largest facilities in each country were selected because they were the most likely to meet the inclusion criteria listed above, and to remain engaged in the longitudinal recruitment and data collection. Facilities were informed of the planned survey and invited to participate through the Ministries of Health in each country.
Sampling patients
-----------------
This Public Health Evaluation is not a trial, and does not aim to observe an expected effect. Therefore the Phase2 cohort study did not require statistically generated sample size calculation. The sample size of 100 patients per facility was selected based on the expected number of new patients registered per month with adequate follow-up data collection, and will be pooled at the country level.
Consecutive patients who meet the following inclusion criteria are approached for participation in the longitudinal study: 18 years of age or over; diagnosed HIV positive; patient knows of their diagnosis; has sufficient cognitive ability to answer the questions for the study; new to service or presenting with a new problem (social, psychological, spiritual or physical).
Participants give informed consent to participate following provision of an information sheet and consent form. These documents have been translated into local languages in Uganda (Kiswahili, Dholuo, Runyakitara and Luganda), and Kenya (Kiswahili, Dholuo). Information and consent form are read aloud by the health care worker for nonliterate prospective participants. Participants are reimbursed travel expenses to the facility of US\$5 per research interview visit. Each of the 12 facilities recruits 100 participants.
Data collection tools and procedure
-----------------------------------
### Qualitative interviews
At each facility, seven patients, three family caregivers and five staff are invited to interview. The purposive sampling strategy includes a variety of staff designations with direct patient contact. Eligible participants for the patient qualitative interviews are patients of the facility who have been diagnosed as HIV positive, over 18 years of age, been under care for at least six weeks, and are not involved in the longitudinal study. These patient participants are asked for consent to approach an identified adult informal carer (i.e. family member or friend who provides assistance/support).
Patient, carer and staff participants give informed consent to participate following provision of an information sheet and consent form, which is read aloud to the interviewee by the health care worker if the interviewee is non-literate.
The semi-structured Interview schedules are designed to gain greater understanding of service use, provision and experiences from the views of the patients, their informal caregivers and the facility staff. The principal themes of enquiry within the schedules for patients and carers are experience of facility care, choice of facility, the nature and content of clinical encounters, and principal needs. Within the interviews, the full range of medical, psychological, spiritual and social domains are addressed. Initial interview transcripts will be reviewed and the topic guide revised where needed to improve clarity of questions and fully explore key issues and any emerging themes. The interview schedules, information sheets and consent forms have been translated into local languages from the English versions twice, independently, by two local researchers. Each of these versions has been translated back to English by a third researcher, with any discrepancies discussed amongst the group and an agreed translation decided.
Qualitative interviews with staff members, patients and informal carers are conducted in private (usually in consulting rooms at the care facility) in local languages as indicated above, and digitally recorded.
One facility is located in a remote part of Uganda where the most widely spoken local language is nationally very rare, and spoken by none of the study researchers. Rather than exclude an entire ethnic group from this section of the evaluation, the patient and carer interviews are conducted by a member of facility staff trained in qualitative interviewing, with the researcher present.
### Longitudinal quantitative study
The data collection tools in the Phase 2 longitudinal study are four questionnaires, one of them (demography) used only once per participant and the others (validated tools) used four times at monthly intervals. The time points, each one month apart, are designated T0 (entry to the study), T1, T2 and T3. A \'patient pack\' has been created for data collection, consisting of all the tools bound in the order they should be used, with the pages colour coded by time point, and preceded by a log page to complete the dates of interviews and a front cover with the patient\'s ID number. For each facility, questionnaire packs have been prepared in two languages; English and a common local language. Translation procedure for the packs is the same as for qualitative interview schedules.
Basic demographic and medical details are collected using a brief questionnaire administered at T0 (recruitment to study). Four clinical questions are asked at T1 (one month later): World Health Organization (WHO) HIV disease staging, date and result of most recent CD4 test, and date of beginning ART. These items were moved from T0 to T1 because in piloting, health staff pointed out that the information would not be available to new participants at T0, and the response rate to these questions would be higher at T1.
The APCA African POS is an adapted version of the original POS, which was developed at KCL to address the multidimensional problems of patients with incurable progressive disease and subsequently adapted around the world \[[@B17],[@B18]\]. The African APCA POS was developed and validated in ten centres in six Sub-Saharan African countries in 2006 \[[@B19],[@B20]\]. Its ten items address the primary physical, psychological, emotional and spiritual concerns of patients and families and employs scoring methods appropriate for a range of literacy skills. The validation study demonstrated its properties included sensitivity to change, and it has high levels of patient and clinician acceptability. The ECOG is a clinician-rated single item measure of physical performance,, also at administered at all four time points \[[@B21]\]. Scores range from 0 (normal activity) to 4 (unable to get out of bed). The ECOG is the most widely used performance measure \[[@B22]\].
The MOS-HIV is a very widely used quality of life measure and has been culturally adapted to the Ugandan HIV setting \[[@B23],[@B24]\]. The 35 items address the domains of: role function; pain; physical functioning; cognitive functioning; overall health perception; mental health; vitality. The weighted subscores in these domains are then combined to produce two summary scores measuring physical health and mental health.
A version of the Client Services Receipt Inventory (CSRI) \[[@B25]\] has been adapted for the aims of this study and the HIV setting in Africa in order to collect information about services received by patients in the study. The CSRI records receipt of 52 components of physical, psychological, spiritual, social and preventive care, and whether they were received at the facility or from elsewhere.
With the exception of CD4 counts, data for the longitudinal quantitative study are self-reported by the participants, and recorded in the questionnaire packs provided by health care workers (HCWs) already employed at each facility.
HCWs are trained in the process of seeking informed consent and the completion of the questionnaires. A researcher maintains contact with each facility through regular visits including observing data collection, checking the use of appointment diaries and regular data entry, and delivering additional training as necessary.
Data collection (i.e. at recruitment (T0) and at three subsequent interviews a month apart) coincides with clinical appointments where possible. Once participants complete the longitudinal study, CD4 counts are extracted from patient records by the HCWs, or by the researchers themselves under the supervision of HCWs.
Piloting demonstrated that participants often could not remember the date and result of their last CD4 count. Accordingly, permission was granted by the ethics board overseeing the study to search patients\' records for CD4 counts. Therefore, researchers visit each facility and transcribe CD4 counts for the study participants from their file into a specially designed form which preserves anonymity while allowing records to be linked to participants. CD4 count is the only variable data obtained in this way. The decision to refer to patient records was taken because the researchers knew the information was collected by facilities, and participants had been informed of their result, but they simply could not remember the information.
One questionnaire pack contains all the data sheets for one individual. The pages are colour-coded to indicate time points. The front cover of the pack is blank apart from the patient\'s name. When the final time point is complete, this page is torn out and destroyed, making the data unidentifiable. The second page includes metadata logging the progress of data collection and management for each time point.
### Costing items
Because the provision of care is a complex area of investigation, there are potentially a number of cost components that could be accounted for. Due to the constraints of this large multi-methods study (of which economics are a component rather than the primary outcome of interest), the following key cost drivers are examined: labour (by staff type, staff salaries); medicines (ARVs, pain medicines, antibiotics, other) and their inventories (buffer stock); laboratory items (supplies and equipment); buildings (assigned market value or annual rent cost) and utilities; capital inputs (high end equipment and vehicles).
Because only the major cost drivers are included, the costs in this report are likely to be an underestimate of the real costs of providing care. Cost elements that may be significant, but which are not accounted for include: costs of developing training programmes, training of health providers, supervision, monitoring and evaluation including health information management, clinic administrative costs, drug and commodities management and maintenance and depreciation on capital assets.
As HIV care and support is provided in a clinical setting in which other non-HIV services are provided, it is necessary to estimate the proportion of some cost elements which are measured for all clinical services and attribute a share for HIV care and support. This is a common issue with the costing of services that are provided in an environment where several medical specialities are simultaneously provided separately at one facility. For labour costs, the proportion of time that staff are involved in providing care and support services is used to calculate the fraction of labour costs allocated to care and support.
Capital and building costs are allocated to HIV care and support using the proportion of all patients accounted for by HIV patients. Similarly, drug costs that are not ARVs and not for analgesia (opioids and non-opioids) are allocated to HIV care using the same proportion. Tools have been piloted and revised to maximise validity of the data collected, and for ease of data provision among facilities.
A data collection instrument was designed and tested to capture the identified cost elements in each of the Phase 2 facilities. Facility level data includes number of patients seen by staff category in a typical day, hours spent with HIV patients per week and hours worked per week by staff category; number of staff by category involved in HIV care; quantities of medications dispensed in the previous three months by drug type; numbers of laboratory tests conducted in the previous three months; information on physical buildings such as space and an equivalent in rental value of the space; utility costs per month including water, electricity, generator fuel, communications, waste disposal etc.; transport costs, fuel, costs of drivers, maintenance; clinical consumable costs per month (including gloves, syringes, cotton wool swabs, plasters, soap, sterilizing solution etc); amount spent on volunteer staff including training, travel reimbursements, payment in kind in previous three months.
Researchers gather the information required for the instrument with information provided by key informants at each facility. Key informants vary by facility but generally include the hospital administrator or manager, the accountant, clinicians, nurses and pharmacists. More than one site visit is undertaken in order to interview all relevant respondents and to complete the questionnaire.
Unit costs are required for some cost elements. These include staff (i.e. full salaries including allowances), prices of medicines and unit costs of laboratory tests. Salaries are obtained from each site and were average levels paid for each staff category. Drug costs are largely obtained from international sources such as the WHO and the *International Drug Price Indicator Guide*\[[@B26]\] while laboratory test costs are obtained from ATC \[[@B27]\].
Data management and entry
-------------------------
### Phase 2 Qualitative interviews
Interview recordings are transcribed verbatim into MS Word 2003 in the language in which they are conducted. If the interview is not conducted in English, two independent translations into English are then performed, either by the researchers or by experts from the Department of Linguistics at the local Institute of Psychology. A team of three then reconcile the two independent translations, referring back to the recorded interview if necessary, and agree upon a final version. For the three interviews conducted by a health worker, a team of three health workers at the facility translate the recordings into English, and a native-speaker linguist checks the transcript while listening to the tape. After the final translations are agreed, the recordings are destroyed.
A table containing demographic information on the participant is added to the beginning of each transcript. These data are extracted from interviews and subsequently entered into Excel tables.
### Phase 2 Longitudinal quantitative data
Immediately after collection, data are entered into a pre-designed EpiData v3.1 database with conditional checks for internal consistency. Data entry is conducted at the health care facility by an administrative staff member trained in the use of the tools and the database. When participants have completed the final data point, the completed data collection tools are transferred to the local central research office. There, the research staff conduct a second round of all data entry and validation of both rounds of data entry. Discrepancies identified are corrected by manual checking of questionnaires, and results revalidated until the two datasets are identical. The CD4 information from patient records is entered into a separate EpiData database and merged into the main dataset.
### Phase 2 Costing items
The data are entered into predesigned Excel spreadsheets. Analysis, including creation of graphs and linear regression, is conducted in Excel workbooks with data drawn together from the different sheets.
### Phase 2 Analysis plan
The three main sources of data (longitudinal quantitative study, qualitative interviews and costing) are analysed separately. Analysis plans for each data component are outlined below.
Qualitative interviews
----------------------
The interview verbatim transcripts are imported from Word into NVivo 7 for coding and analysis. Information on interviewees\' age, gender, household location, family size, profession (for staff), whether they were receiving ART (for patients) and relationship to patient (for carers) are extracted into an Excel table, subsequently imported into NVivo. Identifying information such as names of individuals or care facilities are removed from transcripts. Thematic analysis of content is conducted concurrently on the patient, carer and staff interviews to enable multiple perspectives on each coded theme.
Three coding frames are developed and subsequently combined into a single version applied to the transcripts for the remainder of the coding. One coding frame is developed in Uganda by the team of local researchers who had conducted the interviews, and a second by the Kenyan researchers. The third coding frame is developed at KCL in London. The intention is to explore cross-cultural differences and similarities in coding, and to generate a definitive coding frame that reflects local understanding of the data and scientific rigour.
In each three-person country research team, every researcher codes nine randomly selected interviews (three with a patient, three with a member of staff and two with a carer), creating hierarchical codes. The local team members agree on a coding frame by discussion, comparison and consensus. The three teams then meet and a unified coding frame is developed, combining the strengths of each country-level frame. Each code is reviewed for internal consistency and given an agreed definition to ensure it is applied using a standard meaning by each researcher. The researchers are trained in the use of NVivo 7 and in application of the new coding frame, which is applied to the entire dataset.
Quantitative longitudinal patient data
--------------------------------------
The primary outcomes are the physical and mental health summary scores derived from the MOS-HIV. Secondary outcomes are individual POS item scores, and a combined total POS score. Individual POS item scores are not expected to be parametric and are summarised using the median and inter-quartile range. A a p-value of 0.05 will be used to determine significance in all statistical tests except for the decision to include variables in multivariate analysis, when 0.1 will be used. Initial cross-sectional descriptive analysis of the data will be conducted first.
Time points will be compressed and recoded if necessary to make the most efficient use of the data. For example, if a participant is recorded to have completed T0, T2 and T3 but missed T1, then T1 is deleted from the record and T2 and T3 renumbered T1 and T2, to obtain a continuous series of three points.
Clinical variables (CD4 count, HIV disease stage, physical function) and demographic characteristics (age, gender, location of home, education, number of dependants) of participants will be described for the total sample and by country and facility. Variables relating to socioeconomic status will be incorporated into a principal components analysis to generate a single factor, and this will be split into quintiles of relative wealth\[[@B28]\].
For univariate analysis of demographic/clinical characteristics with primary outcomes at baseline, ANOVA will be used for ordinal variables and linear regression for continuous.
The 52 components of care recorded in the CSRI will be grouped into care themes according to the issues they address and the way in which they are delivered. Care themes are listed following.
Spiritual: visit by a religious leader, prayer with patients, and contact with traditional healer. Spiritual care is a distinct aspect of palliative care. Staff praying with patients, and a visit from a religious leader, are the most common types of spiritual care provided through health facilities. Many people with HIV visit traditional healers\[[@B29]\] and the care delivered by them fits the PEPFAR definition of spiritual care being sensitive to individual and community culture \[[@B30]\].
Counselling and advice: pre-and post-test counselling, adherence counselling, family planning counselling, patient HIV support groups, family counselling and psychiatric therapy. This theme comprises all \'talking therapies\'. It is sometimes difficult to distinguish counselling as listening and responding to the patient\'s worries and concerns from counselling as didactic imparting of information. VCT, for example, is a strategy of both prevention and care, assessed for its efficacy in reducing risk behaviour and HIV transmission\[[@B31]\].
Nursing: wound care and other nursing care.
Pain management: assessment of pain and provision of strong and weak opioids or non-opioid analgesics and treatment for neuropathic pain.
Pain is a common symptom in HIV \[[@B32]\] and all five components in this group are necessary for its relief. The WHO pain ladder \[[@B33]\] outlines the need for non-opioid and opioid analgesics until pain has been controlled. Neuropathic pain, which is particularly common in HIV \[[@B34]\] is caused by damage to nerves and does not respond to traditional pain medication.
Symptom management: treatment for anxiety/depression, nausea/vomiting, skin rash/itching, diarrhoea, laxatives, thrush, oral candidiasis, cryptococcus, other fungal infections, herpes, malaria and other opportunistic infections. The components in this theme were usually defined by the symptom treated, rather than the underlying cause or pathogen, because the cause of a symptom is often not known in HIV disease \[[@B35]\]. All these physical symptoms and conditions are common in HIV \[[@B35],[@B36]\].
Nutrition: food, multivitamins, nutritional advice, safe drinking water, therapeutic feeding for malnutrition. Poor nutrition comprises two problems: lack of macronutrients (wasting, malnutrition) and lack of micronutrients (vitamins and minerals). Both of these predispose individuals with HIV to infections and ill health. Lack of food is the most fundamental level of poverty.
Social: employment training/income generating activities (IGA), home help, household items, legal services, memory book work, loans/microfinance. The social group components were selected after advice from USG country mission teams. Memory book work was allocated to the social care group because it aims to reduce internalised stigma and improve relations between family members.
Prevention: prevention with positives, condoms, ITNs, infection control training, isoniazid for TB prophylaxis. This care group includes both components to protect the person with HIV from other infections, and components to prevent them from infecting others with HIV. Prevention with positives is the general name for a package of care designed to encourage behaviour change (condom use, reduction of partners, and revealing HIV status). Condoms prevent further infection and also protect the individual from other strains of HIV and from other STIs such as herpes. Insecticide-treated nets protect against malaria, which is more common and more aggressive in people with HIV\[[@B37]\], and the TB drug isoniazid can be used as a prophylactic for those at high risk of TB.
ART: ARVs and assessment of ARV treatment. Antiretroviral therapy includes regular assessment to observe signs of developing resistance, toxicity and side effects.
CTX: daily CTX. PEPFAR guidelines encourage daily CTX prophylaxis for everyone with HIV. It is a broad-spectrum antibiotic proven to reduce morbidity and mortality in people with HIV\[[@B38],[@B39]\]. At each interview participants are asked whether they had taken CTX on the previous day and whether they had been given daily prophylactic CTX in the last month. These answers are compared to test adherence.
TB: TB treatment. TB treatment is listed separately from treatment for other symptoms and infection, for two reasons. Firstly, it is the leading cause of death for people with HIV in Africa\[[@B40]\]. Secondly, the course of treatment lasts for four to six months, long after symptoms have resolved. The full course of treatment must be completed to prevent resistance and recurrence.
Baseline outcomes, demographic variables and care theme delivery will be compared between the facilities.
To test the hypothesis that participants who receive antiretroviral therapy or TB treatment at T1 (a month after recruitment) are likely to have more advanced disease, physical and mental health, and CD4 count, will be compared for recipients and non-recipients using t-tests. Physical and mental health outcomes will also be compared for those who attend with and without a carer, to test the hypothesis that carers are more likely to accompany those in poorer health.
The longitudinal outcome analysis will be conducted as follows. Mean and 95% confidence intervals for physical and mental health score are plotted over time as a graphical representation of change in health states.
Longitudinal multilevel modelling is an approach which makes the most efficient use of data collected over time. Unlike most statistical tests, it includes all time points at once, which both reduces the number of tests to be carried out (making false positive results less likely) and allows change to be modelled as a continuous effect. This means that rather than simply finding variables which are associated with any change in outcome, the magnitude of the change can also be considered.
A common problem in longitudinal studies of health outcomes is that patients with the worst health are the most likely to leave the study and be lost to follow-up, so that a comparison between the beginning and end of the study could find improved outcomes only because a proportion of those with poor health would not contribute to the later timepoint. Longitudinal analysis does not have this bias because all participants can be included whether they complete the study or not. Additionally, longitudinal analysis can reveal patterns over time which would not be identified using traditional methods.
The technique adopted in this study was multilevel mixed-effects linear regression, selected because it allows data to be clustered at two levels, by individual and by facility \[[@B41]\]. Many longitudinal studies suffer from the bias caused by the most unwell individuals being most likely to leave the study. To determine whether this bias was present, t-tests are used to compare the mean physical and mental health scores of those who completed all four observations with those who missed at least one. Traditional analysis of longitudinal data involves comparing the earliest observation with the last, so any difference in the scores of completers versus non-completers would bias the findings.
In addition, the same tests are used to compare the mean scores of those who only completed a single observation with those who completed two or more. This is to test the suitability of multi-level modelling, which is explained below. Multi-level modelling uses all data points except the first one, so anyone who only completed one observation would be excluded and it is necessary to test whether this would also cause a bias. For the same reason, mean change over time is reported as the mean of all individual score changes, rather than mean health score at one time point subtracted from mean health score at another.
Multilevel modelling is carried out using the Stata *xtmixed*command function. Outcomes are physical health score and mental health score, measured up to three times at monthly intervals. Baseline score is incorporated as a covariate and not as an outcome. Models included levels for facility and individual. The only random effect is time point (interview number, rather than actual time interval), which is allowed to have a random coefficient at the individual level. Other covariates are fixed. Demographic and care theme covariates are constant over time, receipt of ART and TB treatment varied. The default independent covariance structure is used. Variance and standard error of variance are reported for random-effects parameter estimates.
This analysis is repeated with the 20% of participants who had the lowest physical health score at T0, and with the 20% who had the lowest mental health score at T0, to determine whether the effect of improved outcomes over time extended to those in greatest need. For the APCA African POS, score distribution at each timepoint is tabulated for those who scored 0 (worst possible problem) on the items relating to pain and symptoms. This simple approach is adopted because a very few people scoring 0 on these items could introduce bias. The items for pain and symptoms are selected because people with complex, intractable problems in advanced disease may not experience improvement although average scores for the population increase.
The multilevel models will be repeated several times, each time with the addition of a single demographic covariate. Models will also be developed with ART receipt and TB treatment, which will vary over time.
Examining the relationship between health outcomes and care received is complicated by the potential bias that those in the worst health would probably receive the most care, whereas a lack of care could mean either no need of it, or lack of appropriate provision. To avoid this problem, a variable representing available care is required, which might have a closer association with health outcomes than the level of care individually received. Availability of care is defined as the percentage of individuals at a facility who receive care in a particular theme. Care themes are used rather than individual components in order to reduce the number of variables needed in the model and ensure stability. For example, the variable \'psychological care\', contains information on the percentage of people, per facility, who receive at least one component of psychological care at T1, T2 or T3. T0 is excluded because the model analyses change from T0 onwards.
Each of the care themes is included one by one in a univariate multilevel model as described above; with the difference that care theme has only twelve values, one for each facility, and does not change over time.
Following this, all individual-level and facility-level covariates associated with outcome at the 10% level in univariate analysis are taken forward into a multivariate model and eliminated in a downward stepwise procedure if the association is lost. It is usual to use 10% as the acceptance level with stepwise downward regression to avoid dismissing variables too early.
Costing study
-------------
The purpose of the costing survey is to develop understanding of the factors which influence per-patient costs, and to guide resource allocation, for example by observing economies of scale. Analysis is conducted using an Excel spreadsheet. Most data are collected in local currency (Kenyan and Ugandan shillings), and converted to US dollars at the current exchange rates. Since only twelve facilities are included and these facilities were not meant to be representative of all sites, all results are reported per facility without aggregation across the sample. The average costs per patient for one year of care and support are calculated using aggregated average costs per patient for each of the main components of care for a year.
Data integration
----------------
The final step of analysis is the integration of The Phase 1 and Phase 2 data are integrated to enable the outcome and cost data to be appraised in the light of the facility configuration data
Discussion
==========
This substantive study will provide novel data to inform the provision of HIV care and support programmes in Sub-Saharan Africa. The dual phases and mixed methods allow triangulation and integration of diverse types and sources of data to better determine patient outcomes and factors associated with patient and family experience of HIV care.
We believe that there are several elements to the design and implementation of this protocol that are original and offer methodological advantages. Firstly, the aims objectives and associated data collection tools have been informed by a large collaboration between practitioners, policy and programme designers, implementing partners, academics and Ministry of Health representatives across 3 continents. This has greatly improved the potential data demand and utilisation of the study. Second, all research staff involved in data collection and working with facilities are indigenous African researchers, and considerable capacity has been built through ongoing training and the conduct of this large study. Third, the novel mixed methods approach brings together an integrated mixture of outcome measurement, longitudinal evaluation, qualitative approaches, health economics, document analysis and drug availability review to evaluate HIV care and support. Fourth, the careful attention to multiprofessional inclusivity and consultation, and piloting of tools, has maximised the feasibility of this study where many logistical challenges are presented \[[@B42]\]. We have encountered ethnic and political violence and unrest, lack of available transport to some regions, and temporary closure of services to patients, although these challenges have been overcome. Fifth, we have paid considerable attention to data demand and utilisation, which has encouraged us to initiate processes of consultation and feedback at each stage of protocol design and to identify the data needs of our stakeholders. Sixth, we have been clear that this evaluation does not aim to compare outcomes between facilities or countries (e.g. to identify facilities performing less well), as it is important to encourage participation in this type of outcome evaluation without introducing objectives that may be interpreted as critical or competitive. We have generated a regular study newsletter to promote communication and information flow between research sites and partners. Seventh, we have also taken steps to reduce potential bias in the methods, for example by forward and back translation of tools and selection of person-centred outcome tools that have been locally validated. Our analytical procedures have attempted to identify bias commonly identified in this type of data for example by potential attrition of the sickest, and the use of longitudinal approaches that maximise use of the data. Error has been reduced through double entry of all quantitative data and careful translation of qualitative data. Lastly, we offer reimbursement to participants of \$5 for each research interview, which is felt to not be large enough to be seen as an unethical inducement to participate in the study, but is enough to comfortably cover costs of attending additional research interviews. The use of payments to take part in longitudinal research in low income countries is advocated, not least due to emerging evidence that a key reason for non-adherence to ART is inability to afford to attend HIV clinics \[[@B43]-[@B45]\].
Analysis is currently underway. We believe that the presentation of this protocol offers greater transparency in the aims, objectives and analysis of our study, and will permit greater focus on the reporting of our large dataset.
Competing interests
===================
The authors declare that they have no competing interests.
Authors\' contributions
=======================
The Principal Investigators are RH and IJH. All authors contributed to the design and protocol development. All authors have read and approved the final manuscript.
Pre-publication history
=======================
The pre-publication history for this paper can be accessed here:
<http://www.biomedcentral.com/1471-2458/10/584/prepub>
Acknowledgements
================
We are grateful to the United States Agency for International Development for funding this study under a sub-agreement GPO-A-00-03-00003-00, made under the authority provided to the University of North Carolina. The present study benefited from the participation of a wide range of partners, medical professionals, HIV specialists and palliative care researchers. The authors are grateful for the guidance provided by the United States Government Palliative Care Technical Working Group and to the Kenyan and Ugandan Country Teams. Finally we are grateful to the staff and patients at the participating facilities.
|
{
"pile_set_name": "PubMed Central"
}
|
Background
==========
Worldwide, colorectal cancer (CRC) affects millions of people and has a high morbidity and mortality rate \[[@b1-medscimonit-25-6872]\]. The American Cancer Society (ACS) estimated that approximately 101,420 new cases of CRC would be diagnosed by the year 2019 in the US, with an estimated mortality of approximately 51,020 people \[[@b2-medscimonit-25-6872]\]. In China, CRC is also considered to be a leading type of cancer \[[@b3-medscimonit-25-6872]\]. Standard treatment for early-stage CRC includes surgical resection, chemotherapy, and radiotherapy. However, due to the complexity of the pathogenesis of CRC, patient prognosis has remained unchanged in the past decades \[[@b4-medscimonit-25-6872]\]. The five-year survival rate of patients with advanced-stage CRC with metastatic disease is as low as 10% \[[@b3-medscimonit-25-6872]\]. The majority of patients with advanced-stage CRC die of metastases and tumor recurrence after surgery \[[@b1-medscimonit-25-6872],[@b5-medscimonit-25-6872]\]. Therefore, there remains an urgent need to continue to explore potential new treatment approaches for advanced-stage CRC.
It has been shown that less than 2% of the human genome encodes protein, and approximately 98% of the human transcriptome has no protein-encoding capacity, termed non-coding RNAs (ncRNAs) \[[@b6-medscimonit-25-6872]\]. Due to the lack of protein-encoding ability, ncRNAs were once considered to be 'noise' during genomic transcription \[[@b7-medscimonit-25-6872]\]. However, with advances in molecular research, ncRNAs have been shown to participate in several physiological processes, and abnormal expression of ncRNAs has been identified in human disease, including cancer, cardiovascular disease, and neurodegenerative disorders \[[@b8-medscimonit-25-6872]--[@b10-medscimonit-25-6872]\].
MicroRNAs (miRNAs) and long non-coding RNAs (lncRNAs), characterized by approximately 22 nucleotides (nt) and more than 200 nt, respectively, are the two main linear ncRNAs \[[@b11-medscimonit-25-6872]\]. Previous studies have shown that miRNAs and lncRNAs are involved in tumorigenesis in several human cancers, including CRC, breast cancer, and lung cancer, by modulating mRNA expression of oncogenes or tumor inhibitory genes \[[@b12-medscimonit-25-6872]--[@b15-medscimonit-25-6872]\]. Unlike miRNAs and lncRNAs, circular RNA (circRNA) is a novel ncRNA with circular structure and without the 5′ cap and 3′ poly-A tail \[[@b16-medscimonit-25-6872]\]. Recently, circRNA was also shown to be associated with tumorigenesis in several human cancers, including lung cancer, liver cancer, and oral cancer \[[@b17-medscimonit-25-6872]\]. However, the roles and underlying molecular mechanisms of circRNAs in CRC remain unknown.
Therefore, this study aimed to investigate the expression and effects of *Homo sapiens* (hsa)\_circ_0079993 of POLR2J4 and its effects in CRC tissues and cell lines.
Material and Methods
====================
Colorectal cancer (CRC) tissue samples and cell lines
-----------------------------------------------------
A total of 41 paired samples of colorectal cancer (CRC) tissue and adjacent normal colorectal tissue were obtained from patients who were diagnosed with CRC at Renmin Hospital of Wuhan University from 2013 to 2018. Written informed consent was provided by all the patients who participated in the study. The study was approved by the Ethics Committee of Renmin Hospital of Wuhan University. The normal colonic cell line, NCM460, and five human CRC cell lines HT29, SW480, DLD-1, HCT-116, and SW620 were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). All cells were maintained in RPMI-1640 medium (Hyclone, Logan, UT, USA) containing 10% fetal bovine serum (FBS), and 1% penicillin/streptomycin and incubated in an atmosphere containing 5% CO~2~ and 95% air at 37°C.
RNA extraction and quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
-----------------------------------------------------------------------------------------
Total RNAs of CRC tissue samples and cell lines were extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions. After determining the quality of the RNA with NanoDro2000c (Thermo Scientific, Waltham, MA, USA), 2 μg of extracted RNAs were used as the template to synthesis cDNA using the Bestar^TM^ qRT-PCR kit (\#2220) (DBI Bioscience, Ludwigshafen, Germany). RT-PCR was performed using Bestar^TM^ qPCR MasterMix (\#2043) (DBI Bioscience, Ludwigshafen, Germany) on an ABI 7500 system (ABI Biosystems, Foster City, CA, USA). The primer sequences used in this study are shown in [Table 1](#t1-medscimonit-25-6872){ref-type="table"}. The expression of circular RNA 0079993 (circ_0079993), POLR2J4, and CREB1 were normalized to GAPDH, and gene expression was quantified using the 2^−ΔΔCt^ method.
RNA transfection
----------------
Two small-interfering RNAs (siRNAs) against circ_0079993 (si-Circ\#1 and si-Circ\#2), mimics of miR-203a-3p.1, miR-450b-3p, miR-502-5p, and miR-1224-3p, and anti-miR-203a-3p.1, and the corresponding negative control (NC) for si-Circ\#1/si-Circ\#2 (si-NC) and miR-203a-3p.1 (miR-NC), were designed and obtained from GenePharma (Shanghai, China). All cell transfections were performed using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions.
Cell counting kit-8 (CCK-8) assay
---------------------------------
Cell viability of treated CRC cells was evaluated using the CCK-8 assay (Dojindo Molecular Technologies, Inc., Rockville, MD, USA), according to the manufacturer's instructions. Briefly, treated CRC cells were collected and seeded into 96-well plates at a density of 2×10^4^ cells/well, and cultured at 37°C for 24 h. Then, 10 μl of CCK-8 solution (Dojindo Molecular Technologies, Inc., Rockville, MD, USA) was added to each well and incubated at 37°C for a further 10 min. The absorbance values were detected at 450 nm.
Colony formation assay
----------------------
Cell proliferation of treated CRC cells was assessed using the colony formation assay. Briefly, treated CRC cells were plated into six-well plates at a concentration of 2,000 cells/well. Cells were cultured at 37°C for 14 days, followed by cell fixation using 4% paraformaldehyde and stained with Giemsa solution. The number of colonies was counted using microscopy.
*In vivo* assay of tumor growth
-------------------------------
*In vivo* tumor growth was assessed in 7-week-old male BALB/c mice. All animal studies were approved by the Institutional Animal Care and Use Committee of the Renmin Hospital of Wuhan University. Briefly, HCT116 cells that were stably transfected with small-interfering RNAs (siRNAs), si-NC or si-Circ\#1+\#2 were harvested and re-suspended with culture medium (1×10^5^ cells/ml). Then, 200 μL of HCT116 cell suspensions were subcutaneously injected into the left flank of the mice. Tumor volume was examined every 3 days until day 36 after cell inoculation. Tumor volumes were calculated from the measurements of length×width^2^×0.5.
Dual-luciferase reporter assay
------------------------------
To verify the interaction between circ_0079993 and miR-203a-3p.1 in HCT116 and SW620 cells, the wild type (WT) and mutant (Mut) fragments of circ_0079993 containing putative miR-203a-3p.1 binding sites were amplified and inserted into the pGL3 vector (Promega, Madison, WI, USA) to form circ_0079993-WT and circ_0079993-Mut recombinant plasmids. Then, HCT116 and SW620 cells (1×10^4^ cells/well) were plated into 96-well plates and maintained at 37°C overnight, followed by the co-transfection of miR-203a-3p.1 mimics and circ_0079993-WT or circ_0079993-Mut. The Firefly and Renilla luciferase activities of treated HCT116 and SW620 cells were detected with the Dual-Luciferase Assay System (Promega, Madison, WI, USA), and Renilla luciferase activity was normalized to the Firefly luciferase activity. The interaction between circ_0079993 and microRNAs, miR-450b-3p, miR-502-5p, miR-587 or miR-1224-3p, as well as CREB1 and miR-203a-3p.1, were also verified to be the same as circ_0079993 and miR-203a-3p.1.
DAVID online Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis
--------------------------------------------------------------------
The miRNAs with putative target sites with circ_0079993 were predicted using TargetScan 7.1 (*<http://www.targetscan.org/vert_71/>*). Biological pathways of the circ_0079993 target miRNAs underwent DAVID gene-annotation enrichment analysis (*<https://david.ncifcrf.gov>*).
Statistical analysis
--------------------
Data were presented as the mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) was performed using GraphPad Prism version 7 (GraphPad Prism Software, La Jolla, CA, USA) to compare the difference between groups. A P-value \<0.05 was considered to be statistically significant.
Results
=======
*Homo sapiens* circular RNA 0079993 (hsa_circ_0079993) of the POLR2J4 was highly expressed in colorectal cancer (CRC) and was correlated with poor prognosis
------------------------------------------------------------------------------------------------------------------------------------------------------------
Analysis of the genomic location and formation of circ_0079993 showed that circ_0079993 was the circularized product of the mRNA of POLR2J4, and its circular structure was verified by Sanger sequencing ([Figure 1A](#f1-medscimonit-25-6872){ref-type="fig"}). Analysis was undertaken of the expression of POLR2J4 in 275 samples of colorectal cancer (CRC) and 349 corresponding normal tissue samples based on The Cancer Genome Atlas (TCGA) data from the Gene Expression Profiling Interactive Analysis (GEPIA) website (*<https://bio.tools/gepia>*). The results showed that POLR2J4 expression was significantly lower in the CRC samples compared with the normal samples ([Figure 1B](#f1-medscimonit-25-6872){ref-type="fig"}). In the overall survival (OS) analysis, patients with CRC with high POLR2J4 expression had a worse prognosis than patients with low POLR2J4 expression ([Figure 1C](#f1-medscimonit-25-6872){ref-type="fig"}). These findings suggested that POLR2J4, the parental gene of circ_0079993, might play a role in the tumorigenesis of CRC.
To further investigate the role of circ_0079993 in CRC, its expression was studied by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) in 41 paired samples of CRC and adjacent normal colorectal tissues. The results showed that circ_0079993 expression was significantly upregulated in CRC tissues compared with adjacent normal tissues ([Figure 1D](#f1-medscimonit-25-6872){ref-type="fig"}). Compared with the non-metastatic CRC tissues, circ_0079993 expression was significantly increased in metastatic tissues ([Figure 1E](#f1-medscimonit-25-6872){ref-type="fig"}). Also, circ_0079993 expression in advanced-stage CRC (stage III/IV) was greater than in early-stage CRC (stage I/II) ([Figure 1F](#f1-medscimonit-25-6872){ref-type="fig"}).
Evaluation of circ_0079993 expression in CRC cell lines showed that when compared with the normal colonic cell line, NCM460, circ_0079993 expression was significantly upregulated in the five CRC cell lines, HT29, SW480, DLD-1, HCT-116, and SW620. The HCT-116 and SW620 cells showed the highest circ_0079993 expression ([Figure 1G](#f1-medscimonit-25-6872){ref-type="fig"}). Also, patients with CRC with high circ_0079993 expression showed significantly reduced overall survival (OS) rate when compared with patients with low circ_0079993 expression ([Figure 1H](#f1-medscimonit-25-6872){ref-type="fig"}). These results suggested that circ_0079993 might have a role in the pathogenesis of CRC.
Circ_0079993 knockdown inhibited CRC cell growth *in vitro* and *in vivo*
-------------------------------------------------------------------------
To assess the biological functions of circ_0079993 in CRC, we examined the effects of circ_0079993 knockdown on proliferation of CRC cells *in vitro* and *in vivo*. Two specific small-interfering RNAs (siRNAs) against circ_0079993 (si-Circ\#1, si-Circ\#2) were designed and synthesized to knockdown the expression of circ_0079993 in HCT116 and SW620 cells ([Figure 2A](#f2-medscimonit-25-6872){ref-type="fig"}). The knockdown efficiency of si-Circ\#1 and si-Circ\#2 were evaluated by qRT-PCR in HCT116 and SW620 cells ([Figure 2B, 2C](#f2-medscimonit-25-6872){ref-type="fig"}). The results from the CCK-8 assay showed significant inhibition of cell viability of HCT116 and SW620 cells transfected with si-Circ\#1 and si-Circ\#2 when compared with cells transfected with negative control siRNA (si-NC) ([Figure 2D, 2E](#f2-medscimonit-25-6872){ref-type="fig"}). In the colony formation assay, the number of colonies was significantly reduced in the HCT116 and SW620 cells transfected with si-Circ\#1 and si-Circ\#2 when compared with the si-NC group ([Figure 2F--2H](#f2-medscimonit-25-6872){ref-type="fig"}). The effects of circ_0079993 knockdown on tumor growth were assessed using an *in vivo* tumor growth assay. HCT116 cells stably transfected with si-Circ\#1, si-Circ\#2, or si-NC were inoculated subcutaneously into the left flank of nude mice. All mice developed xenograft tumors at the injection site, and tumor volume and weight were measured. Compared with the si-NC group, the tumor volume and weight were significantly reduced in the si-Circ\#1 and si-Circ\#2 groups ([Figure 2I--2K](#f2-medscimonit-25-6872){ref-type="fig"}).
Circ_0079993 sponged to multiple miRNAs in CRC cells
----------------------------------------------------
The target miRNAs of circ_0079993 were predicted using TargetScan version 7.2. The findings showed that circ_0079993 interacted with multiple miRNAs ([Table 2](#t2-medscimonit-25-6872){ref-type="table"}, [Figure 3A](#f3-medscimonit-25-6872){ref-type="fig"}). We selected four miRNAs, miR-203a-3p.1, miR-450b-3p, miR-502-5p, and miR-1224-3p for further study. In the dual-luciferase reporter assay, of the four miRNAs, miR-203a-3p.1 reduced the luciferase activity of HCT116 and SW620 cells driven by pGL3-Luc-circ_0079993 ([Figure 3B, 3C](#f3-medscimonit-25-6872){ref-type="fig"}). Analysis of the enrichment pathways of miR-203a-3p.1, miR-450b-3p, miR-502-5p, and miR-1224-3p was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) ([Figure 3D](#f3-medscimonit-25-6872){ref-type="fig"}). We also constructed the circRNA-miRNA-mRNA network of circ_0079993 within its four target miRNAs and found that the mRNA participated in multiple signaling pathways, including the adipocytokine signaling pathway, the cGMP-PKG signaling pathway, the mTOR signaling pathway, and the Ras signaling pathway ([Figure 3E](#f3-medscimonit-25-6872){ref-type="fig"}).
Circ_0079993 regulated the expression of CREB1, a target gene of miR-203a-3p.1, by sponging miR-203a-3p.1
---------------------------------------------------------------------------------------------------------
In the circRNA-miRNA-mRNA network of circ_0079993/miR-203a-3p.1, CREB1 was identified as a target gene of miR-203a-3p.1. Therefore, we then investigate the association between CREB1 and circ_0079993/miR-203a-3p.1. Relative circ_0079993 and CREB1 expression were detected in the miR-203a-3p.1 mimic treated HCT116 and SW620 cells using qRT-PCR. Compared with the NC mimics group, circ_0079993 expression in HCT116 and SW620 cells was not affected by miR-203a-3p.1 ([Figure 4A](#f4-medscimonit-25-6872){ref-type="fig"}). However, CREB1 expression of HCT116 and SW620 cells was significantly downregulated after transfection with miR-203a-3p.1 ([Figure 4B](#f4-medscimonit-25-6872){ref-type="fig"}). In the dual-luciferase reporter assay for miR-203a-3p.1 and circ_0079993, we found that the luciferase activity of HCT116 and SW620 cells was reduced by co-transfecting the cells with miR-203a-3p.1 and circ_0079993-WT ([Figure 4C, 4D](#f4-medscimonit-25-6872){ref-type="fig"}).
In the dual-luciferase reporter assay for miR-203a-3p.1 and CREB1, the luciferase activity of HCT116 and SW620 cells could only be reduced by co-transfecting the cells with miR-203a-3p.1 and CREB1-WT ([Figure 4E, 4F](#f4-medscimonit-25-6872){ref-type="fig"}). Also, miR-203a-3p.1 reduced the luciferase activity of HCT116 and SW620 cells transfected with pGL3-Luc-WT-CREB1-3′UTR. However, this effect could be blocked by the presence of circ_0079993 ([Figure 4G](#f4-medscimonit-25-6872){ref-type="fig"}). The expression of CREB1 was significantly decreased in miR-203a-3p.1 treated HCT116 and SW620 cells, and the downregulation induced by miR-203a-3p.1 of CREB1 was blocked by circ_0079993 ([Figure 4H](#f4-medscimonit-25-6872){ref-type="fig"}). These findings showed that circ_0079993 could regulate the expression of CREB1 by sponging miR-203-3p.1.
Anti-miR-203a-3p.1 reversed the inhibitory effects of circ_0079993 knockdown on the proliferation of CRC cells
--------------------------------------------------------------------------------------------------------------
Circ_0079993 was shown to act as a miRNA sponge for miR-203a-3p.1 in CRC cells. To determine whether miR-203a-3p.1 was involved in the biological effects of circ_0079993 in CRC, cell proliferation was assessed by the CCK-8 and colony formation assays when circ_0079993 was silenced in HCT116 and SW620 cells followed by treatment with anti-miR-203a-3p.1. The results from the CCK-8 assay showed that the inhibitory effects of si-Circ\#1+\#2 on cell viability of HCT116 and SW620 cells were reversed by anti-miR-203a-3p.1 ([Figure 5A, 5B](#f5-medscimonit-25-6872){ref-type="fig"}). Also, the findings from the colony formation assay showed that the inhibitory effects of si-Circ\#1+\#2 on colony formation of HCT116 and SW620 CRC cells were also abolished by miR-203a-3p.1 ([Figure 5C, 5D](#f5-medscimonit-25-6872){ref-type="fig"}). These findings indicated that circ_0079993 derived from POLR2J4 might be involved in the progression of CRC by releasing CREB1 from miR-203a-3p.1 and sponging miR-203a-3p.1 ([Figure 5E](#f5-medscimonit-25-6872){ref-type="fig"}).
Discussion
==========
Due to its widespread expression in human tissues and organs and complex biological functions, circular RNAs (circRNAs) have recently become an area of increasing research interest. Previous studies have shown that circRNA has several biological activities, including tumor progression \[[@b18-medscimonit-25-6872],[@b19-medscimonit-25-6872]\]. CircRNAs are expressed in specific tissues, which makes them promising diagnostic and therapeutic biomarkers in human cancers \[[@b20-medscimonit-25-6872]\]. However, the roles of circRNAs in colorectal cancer (CRC) have been poorly understood.
The findings from the present study showed that the expression of *Homo sapiens* circular RNA 0079993 (hsa_circ_0079993), a novel circRNA derived from the mRNA of the POLR2J4 gene, was significantly increased in the CRC tissues and cell lines, and was associated with a worse prognosis. *In vitro* and *in vivo* studies performed in this study showed that circ_0079993 knockdown significantly inhibited CRC cell proliferation. Also, circ_0079993 was shown to act as a sponge for multiple miRNAs, and miR-203a-3p.1 was shown to interact with circ_0079993 and CREB1 in CRC cells directly. Importantly, circ_0079993 indirectly regulated the expression of CREB1 in CRC cells by sponging miR-203a-3p.1, which also blocked the inhibitory effects of circ_0079993 knockdown on CRC cell proliferation. To the best of our knowledge, the present study was the first to report the expression, function and underlying mechanisms of circ_0079993 in human CRC.
The RNA polymerase II subunit J (POLR2J) family includes four members, POLR2J1, POLR2J2, POLR2J3, and POLR2J4. The POLR2J4 gene (31040 bp) is located at the 7p13 locus on the short arm of chromosome 7 and is one of the genes encoding variants of the hRPB11 subunit of *Homo sapiens* circular RNA 0079993 (hsa_circ_0079993) \[[@b21-medscimonit-25-6872]\]. To the best of our knowledge, no previous studies have shown an association between POLR2J4 and human cancer. Circ_0079993 from mRNA of POLR2J4 is a novel circRNA with unclear biological functions, and there have been no previously published studies.
According to the theory of competing endogenous RNA (ceRNA), circRNAs participate in the regulation of tumorigenesis of human cancers by acting as miRNAs sponges to affect corresponding mRNA expression \[[@b22-medscimonit-25-6872],[@b23-medscimonit-25-6872]\]. Sponging the specific miRNAs in the circRNA-miRNA-mRNA axis is considered to be an intrinsic function of circRNA \[[@b24-medscimonit-25-6872]\]. Therefore, we performed a bioinformatics analysis to construct the circRNA-miRNA-mRNA networks of circ_0079993. Circ_0079993 has been shown to interact with miR-203a-3p.1, miR-450b-3p, miR-502-5p, and miR-1224-3p, and miR-203a-3p.1 is associated with the progression of human tumors, including CRC \[[@b25-medscimonit-25-6872]\], which was consistant with our findings.
The cAMP response element binding protein 1 (CREB1) gene encodes a critical transcription agent that regulates multiple stress signals and growth factors \[[@b26-medscimonit-25-6872],[@b27-medscimonit-25-6872]\]. CREB1 has a key role in the initiation and progression of human tumors by acting as an oncogene \[[@b28-medscimonit-25-6872]\]. CREB1 regulates and is regulated by the expression of miRNAs, forming a feedback loop \[[@b29-medscimonit-25-6872]\], indicating a bidirectional interaction between CREB1 and miRNAs in human tumors. In this study, we demonstrated that miR-203a-3p.1 could negatively regulate CREB1 in CRC cells, and circ_0079993 could block these effects.
Conclusions
===========
The findings from this study showed that the *Homo sapiens* circular RNA 0079993 (hsa_circ_0079993), microRNA-203a-3p.1 (miR-203a-3p.1), and cAMP response element-binding protein (CREB1) axis had a role in the progression of colorectal cancer (CRC) cells *in vitro* and *in vivo*. The findings add to the understanding of the pathogenesis of CRC, but also may stimulate further studies to identify possible novel therapeutic targets for CRC. There may be further functional mechanisms for circ_0079993 in CRC, including protein encoding, which should be investigated further. It is likely that the circ_RNA regulation networks in the pathogenesis of CRC are highly complex and that further studies are needed to characterize these molecules.
The authors thank Dr. Fei Liao for cell culture, collection of samples, calculation of the data, and design of the figures and tables.
**Source of support:** Departmental sources
**Conflict of interest**
None.
{#f1-medscimonit-25-6872}
{#f2-medscimonit-25-6872}
{#f3-medscimonit-25-6872}
{#f4-medscimonit-25-6872}
{#f5-medscimonit-25-6872}
######
The primer sequences in this study.
Gene Primer sequences
---------------------------------------- ----------------------------------------
GAPDH Forward: 5′-TATGATGATATCAAGAGGGTAGT-3′
Reverse: 5′-TGTATCCAAACTCATTGTCATAC-3′
Hsa_circ_0079993 Forward: 5′-AAGATCGAGACACTGCCCTC-3′
Reverse: 5′-AGTGGTTTGCTGCCTCCTAT-3′
POLR2J4 Forward: 5′-AACATCAGGGGAGTTGGGAG-3′
Reverse: 5′-CTTGCACTTTGGGCTTAGCA-3′
CREB1 Forward: 5′-CTGCCTCTGGAGACGTACAA-3′
Reverse: 5′-CAAGCACTGCCACTCTGTTT-3′
######
The predicted microRNAs that interacted with hsa_circ_0079993 identified by the TargetScan algorithm.
miRBase ID Scores (site 1) Scores (site 2) miRBase ID Scores (site 1) Scores (site 2)
----------------- ----------------- ----------------- ----------------- ----------------- -----------------
hsa-miR-203 99 n/a hsa-miR-625 85 n/a
hsa-miR-1224-3p 95 n/a hsa-miR-1182 83 n/a
hsa-miR-769-3p 95 84 hsa-miR-615-3p 82 n/a
hsa-miR-502-5p 95 n/a hsa-miR-663b 82 n/a
hsa-miR-431 94 n/a hsa-miR-136 81 n/a
hsa-miR-450b-3p 94 83 hsa-miR-661 81 n/a
hsa-miR-149 93 66 hsa-miR-1257 80 n/a
hsa-miR-599 92 n/a hsa-miR-1278 80 n/a
hsa-miR-874 92 n/a hsa-miR-579 77 91
hsa-miR-1236 91 n/a hsa-miR-1200 75 n/a
hsa-miR-411 91 n/a hsa-miR-767-3p 75 n/a
hsa-miR-548k 91 n/a hsa-miR-330-5p 74 n/a
hsa-miR-614 91 n/a hsa-miR-646 73 n/a
hsa-miR-146b-3p 90 n/a hsa-miR-324-5p 72 n/a
hsa-miR-526b 90 n/a hsa-miR-513a-5p 72 n/a
hsa-miR-576-3p 88 n/a hsa-miR-1238 70 n/a
hsa-miR-1184 87 n/a hsa-miR-638 70 70
hsa-miR-498 87 n/a hsa-miR-942 68 n/a
hsa-miR-520g 86 n/a hsa-miR-326 66 n/a
hsa-miR-520h 86 n/a hsa-miR-516a-5p 47 n/a
hsa-miR-758 86 n/a hsa-miR-622 46 n/a
hsa-miR-924 86 91 hsa-miR-127-5p 41 n/a
[^1]: Study Design
[^2]: Data Collection
[^3]: Statistical Analysis
[^4]: Data Interpretation
[^5]: Manuscript Preparation
[^6]: Literature Search
[^7]: Funds Collection
|
{
"pile_set_name": "PubMed Central"
}
|
Introduction {#Sec1}
============
As estimated by Global Burden of Diseases (GBD 2016), stroke is the second leading cause of death^[@CR1]^. Subarachnoid hemorrhage (SAH) contributes to only 5% of the cases of stroke^[@CR2]^, but it occurs at a fairly young age and is associated with a high degree of mortality (in the range of 50%)^[@CR3]^. Thus SAH is a large burden on society. The progression of SAH includes early brain injury that appears the first 3 days after injury, followed by delayed cerebral ischemia (DCI) 3--4 days after injury, which reaches the highest incidence and severity 6--8 days after SAH^[@CR4]^. As vasospasm, microthrombosis, and neuroinflammation contribute greatly to the DCI and clinical prognosis, pharmacological approaches to treat this pathology have been focused on preventing vasospasm, microclot formation, and anti-inflammation for decades; however, to date, few agents have been found to exert beneficial effects on patient outcomes^[@CR5],[@CR6]^. The pathologies of SAH are caused by the extravasated blood presenting in the subarachnoid space (SAS). Thus a better understanding of extravasated blood clearance may help in the development of effective therapeutic approaches. When a SAH attack occurs, blood releases into the SAS, leading immediately to clot formation, which disappear within 2--3 days^[@CR7],[@CR8]^. To date, clot lysis and phagocytosis by macrophages and neutrophils were considered as ways to clear the extravasated blood and the subsequent clots in the SAS^[@CR9],[@CR10]^. However, the mechanism(s) by which extravasated blood is cleared is still unclear.
Recently, lymphatic vessels have been rediscovered and characterized in the meninges surrounding the central nervous system^[@CR11],[@CR12]^. These lymphatics are responsible for the drainage of cerebrospinal fluid (CSF) macromolecules and immune cells to the cervical lymph nodes (CLNs)^[@CR13],[@CR14]^. Meningeal lymphatics aid in the clearance of amyloid-beta in aged mice and transgenic mouse models of Alzheimer's disease, and the augmentation of meningeal lymphatic function alleviates age-associated cognitive impairment^[@CR13],[@CR15]--[@CR17]^. But in an experimental autoimmune encephalomyelitis (EAE) mouse model the ablation of meningeal lymphatics or inhibition of vascular endothelial growth factor receptor 3 (VEGFR3) diminished the EAE severity and the inflammatory response of brain-reactive T cells^[@CR14],[@CR18]^. Thus the meningeal lymphatics may play different roles in different neurological diseases. Embryonic mesenteric lymphatic vessels have recently been shown to clear extravascular red blood cells (RBCs) leaking from the adjacent developmental vascular remodeling, suggesting that the lymphatic system has the potential to clear the erythrocytes in interstitial fluid^[@CR19]^. But, as noted above, while meningeal lymphatics drain immune cells and macromolecules from CSF, it is still unclear whether they clear the extravasated blood after SAH.
Here we report that the extravasated blood is aggregated in CLNs, and the erythrocytes are accumulated in the lymphatics of CLNs inside and around meningeal lymphatic vessels for 4 h after SAH. We use CFSE (5-(and 6)-carboxyfluorescein diacetate succinimidyl ester) to label erythrocytes ex vivo and inject them into the cisterna magna and demonstrate that the labeled erythrocytes are also evident in the lymphatics of CLNs and meningeal lymphatics. When we ablate meningeal lymphatics by photoconverted visudyne, RBC drainage into CLNs is significantly reduced, while the neuroinflammation and the neurological deficits associated with SAH are exacerbated. We further block VEGFR3 and also observe a worse brain injury during SAH. We examine the structure and functional characteristics of meningeal lymphatics during neuroinflammation and find the lymph flow is augmented but lymphangiogenesis and expansion are not pronounced. Our findings demonstrate that meningeal lymphatics participate in the drainage of RBCs into CLNs during the very early stage of SAH and may be a potent target in the treatment of this pathology.
Results {#Sec2}
=======
RBCs in CSF are drained by the meningeal lymphatics to CLNs {#Sec3}
-----------------------------------------------------------
Tracers, proteins, and labeled T cells injected into the brain or CSF are found in CLNs, indicating the drainage function by meningeal lymphatics^[@CR14],[@CR20]^. To explore whether the erythrocytes released into the CSF, a condition called SAH, are drained to CLNs via the meningeal lymphatics, we injected autologous blood into the cisterna magna of mouse. Four hours later, we observed that the deep CLNs (dCLNs) and mandibular LNs were infused with blood, while no evidence of blood was observed in the saline-injected and control groups (Fig. [1a](#Fig1){ref-type="fig"}, Supplementary Fig. [1a, b](#MOESM1){ref-type="media"}). The data also suggest that the superficial parotid LNs, though located in the superficial anterior neck, did not drain the CSF erythrocytes, and there is no drainage of the axillary, the brachial, and the tracheobronchal LNs (Supplementary Fig. [1a](#MOESM1){ref-type="media"}). Next, we used anti-Ter119 antibody to label erythrocytes and anti-Lyve-1 antibody to visualize the lymphatics of CLNs. The number of Ter119-positive erythrocytes in the Lyve-1-positive lymphatic sinus was significantly greater in the SAH group when compared with the saline and control groups for both types of CLNs (for the comparison of dCLNs, *P* = 0.002, Con vs SAH, *P* = 0.0063, saline vs SAH, Fig. [1b, c](#Fig1){ref-type="fig"}; Supplementary Fig. [1c, d](#MOESM1){ref-type="media"}). We found that the Ter119-positive cells were distributed mainly in the Lyve-1-positive lymphatic sinus of CLNs.Fig. 1The erythrocytes in the cerebrospinal fluid after subarachnoid hemorrhage (SAH) were drained into deep cervical lymph nodes (dCLNs) via meningeal lymphatics.**a** Isolated dCLNs from mice 4 h after autologous blood (SAH) or saline injected into the cisterna magna (i.c.m.) or blank control group (Con). Scale bar, 2 mm. **b** Representative images of dCLNs isolated 4 h post-induction of the SAH, saline, and Con groups, stained for Lyve-1 (to image lymphatics, red) or Ter119 (to image erythrocytes, green). Regions of interest in the top images are shown below. White arrows, morphologically intact erythrocytes. Blue arrows, clusters of degraded membrane. Scale bars 200 μm (for top images) and 10 μm (for bottom images). *n* = 3 independent experiments. **c** The number of erythrocyte per mm^2^ in the Lyve-1-positive lymphatic sinus of dCLNs from the SAH, saline, and Con groups. Con/Saline; *n* = 12, SAH; *n* = 11 mice, pooled from 3 independent experiments. *P*(Con vs SAH) = 0.002; *P*(Saline vs SAH) = 0.0063. **d** Representative image of the meningeal lymphatics (green) on transverse sinus (TS) draining the erythrocytes (red) after 4 h of SAH, compared with the Con and saline groups. Regions of interest in the left images are shown on the right. White arrows, morphologically intact erythrocytes. Blue arrows, clusters of degraded membrane. Scale bar, 20 μm (the left and right images). *n* = 3 independent experiments. **e** Quantification of the number of erythrocytes per field accumulated into lymphatics on TS. Con/Saline; *n* = 13, SAH; *n* = 15 biologically independent animals, pooled from 3 independent experiments. *P*(Con vs SAH) \< 0.0001; *P*(Saline vs SAH) \< 0.0001. **f** The expression of classical lymphatic endothelial cell markers by the meningeal lymphatic vessels co-stained with erythrocytes. Representative images of podoplanin (PDPN, red) and Lyve-1 (gray) expressing vessels co-localized with erythrocytes (green) (top images). Representative images of Prox1 (green) and Lyve-1 (gray) expressing vessels co-localized with erythrocytes (red) (bottom images). Yellow arrow, erythrocytes inside the meningeal lymphatic vessels. Scale bar, 20 μm. *n* = 2 independent experiments. All data are presented as mean values ± SD; one-way ANOVA with Turkey's multiple-comparison test. \*\**P* \< 0.01, \*\*\*\**P* \< 0.0001. Source data are provided as a Source data file.
To determine whether the erythrocytes are drained to CLNs via the meningeal lymphatics, we isolated the meninges and stained the lymphatic vessels with antibodies. Erythrocytes were observed to accumulate around and inside of meningeal lymphatics at 4 h post-induction of SAH, while erythrocytes were not seen significantly entering into the lymphatics of the control and saline-injected groups (*P* \< 0.0001, Con vs SAH; *P* \< 0.0001, saline vs SAH; Fig. [1d, e](#Fig1){ref-type="fig"}). The corresponding orthogonal view (Supplementary Fig. [2](#MOESM1){ref-type="media"}) showed the erythrocytes co-localizing with Lyve-1-positive lymphatic endothelial cells, suggesting that these cells were drained via the meningeal lymphatic vessels. The identification of meningeal lymphatics was further confirmed by co-labeling them with other classical lymphatic endothelial cell markers including podoplanin (PDPN) and Prox1 with lyve-1, and the erythrocytes also showed accumulation into meningeal lymphatics (Fig. [1f](#Fig1){ref-type="fig"}). Morphologically intact erythrocytes and clusters of degraded membranes were seen at the same time in the lymphatics of CLNs and meninges (Fig. [1b, d](#Fig1){ref-type="fig"}), though it is not clear whether the erythrocytes were broken down before transportation by the lymphatics or after. But the data suggest that at least some erythrocytes were drained into CLNs before degradation.
To further confirm that the erythrocytes in meningeal lymphatics and CLNs were drained from exogenous injection, we labeled erythrocytes with CFSE in vitro and then injected them into the cisterna magna. Four hours later, the labeled erythrocytes also showed similar accumulation in the meningeal lymphatics (*P* \< 0.0001, Fig. [2a, b](#Fig2){ref-type="fig"}), dCLNs (*P* = 0.0006, Fig. [2c, e](#Fig2){ref-type="fig"}), and mandibular LNs (*P* = 0.0207, Fig. [2d, e](#Fig2){ref-type="fig"}), while the saline-treated group did not show any evidence of CFSE-labeled erythrocytes.Fig. 2Meningeal lymphatics drained erythrocytes labeled in vitro to cervical lymph nodes (CLNs).Erythrocytes (red blood cells (RBCs)) were labeled by CFSE (5-(and 6)-carboxyfluorescein diacetate succinimidyl ester) in vitro and then injected into cistern magna. **a** Representative images of meningeal lymphatics draining erythrocytes in CFSE at 4 h postinjection. White arrows, morphologically intact erythrocytes. Purple arrows, degraded membrane. Regions of interest in the left images are shown on the right. Scale bar, 20 μm (the left and right images). *n* = 2 independent experiments. **b** Quantification of the number of CFSE-labeled erythrocytes per field in meningeal lymphatics on TS (*n* = 12 mice per group, pooled from 2 independent experiments. *P* \< 0.0001). **c** Representative images of CFSE-labeled erythrocytes in the Lyve-1-positvie lymphatic sinus of dCLNs of mice at 4 h postinjection. Regions of interest in the top images are shown below. White arrows, morphologically intact erythrocytes. Purple arrows, clusters of degraded membrane. Scale bars, 200 μm (the top images) and 50 μm (the bottom images). **d** Representative images of CFSE-labeled erythrocytes in the Lyve-1-positive lymphatic sinus of mandibular LNs of mice at 4 h postinjection. White arrows, morphologically intact erythrocytes. Regions of interest in the top images are shown below. Purple arrows, clusters of degraded membrane. Scale bars, 200 μm (the top images) and 50 μm (the bottom images). *n* = 3 independent experiments. **e** Quantification of the density of CFSE-labeled erythrocytes per mm^2^ in Lyve-1-positive lymphatic sinus of dCLNs and mandibular LNs at 4 h postinjection (dCLN, Saline; *n* = 12, Erythrocytes in CFSE; *n* = 15 independent nodes, *P* = 0.0006; Mandibular LN, Saline; *n* = 12, Erythrocytes in CFSE; *n* = 23 nodes, pooled from 3 independent experiments. *P* = 0.0207). All data are presented as mean values ± SD; two-tailed unpaired Student's *t* test. \**P* \< 0.05, \*\*\**P* \< 0.001, \*\*\*\**P* \< 0.0001. Source data are provided as a Source data file.
Ablative meningeal lymphatics block RBC drainage to CLNs {#Sec4}
--------------------------------------------------------
To further verify whether meningeal lymphatic vessels serve as the route for the drainage of erythrocytes from the CSF into the CLNs, we ablated meningeal lymphatic vessels by injecting visudyne into the cisterna magna and photoconverted it by laser light. After 7 days of ablation, lymphatic coverage of the transverse sinus (*P* \< 0.0001, Con vs Laser + Visudyne; *P* \< 0.0001, Laser vs Laser + Visudyne; *P* \< 0.0001, Visudyne vs Laser + Visudyne; Fig. [3a, b](#Fig3){ref-type="fig"}) and the superior sagittal sinus were notably lower (*P* \< 0.0001, Con vs Laser+Visudyne; *P* = 0.0007, Laser vs Laser + Visudyne; *P* = 0.0003, Visudyne vs Laser + Visudyne; Fig. [3c](#Fig3){ref-type="fig"}). No difference in cerebral blood flow was observed between control mice and the Laser + Visudyne group (Supplementary Fig. [3a, b](#MOESM1){ref-type="media"}). The coverage of blood vasculature on the transverse sinus and superior sagittal sinus were not altered in the Visudyne-photoconverted group compared with Laser only or Visudyne only group (Supplementary Fig. [3c, d](#MOESM1){ref-type="media"}).Fig. 3Ablation of meningeal lymphatic vessels blocks drainage of the erythrocytes into dCLNs.**a** Representative images of meningeal lymphatics (labeled by Lyve-1, red) in the Con, Laser only, Visudyne only, and Laser plus Visudyne groups. Regions of interest in the top images are shown below. Scale bars, 1 mm (top images) and 200 μm (bottom images). **b** Quantification of the meningeal lymphatics coverage on the TS (*n* = 5 per group, *P*(Con vs Laser + Visudyne) \< 0.0001, *P*(Laser vs Laser + Visudyne) \< 0.0001, *P*(Visudyne vs Laser + Visudyne) \< 0.0001) and **c** superior sagittal sinus (SSS) from each group of mice (*n* = 5 per group, *P*(Con vs Laser + Visudyne) \< 0.0001, *P*(Laser vs Laser + Visudyne) = 0.0007, *P*(Visudyne vs Laser + Visudyne) = 0.0003). **d** Isolated dCLNs from mice 4 h after induction of SAH in the Laser (L + SAH), Visudyne (V + SAH), and Laser plus Visudyne (L + V + SAH) groups. Scale bar, 2 mm. **e** Representative images of dCLNs isolated 4 h post-induction of SAH and stained for the lymphatics (Lyve-1, red) and erythrocytes (Ter119, green) in the 3 groups of mice indicated. White arrows, morphologically intact erythrocytes. Blue arrows, clusters of degraded membrane. Regions of interest in the top images are shown below. Scale bars, 200 μm (the top images) and 10 μm (the bottom images). *n* = 2 independent experiments. **f** The number of erythrocyte per mm^2^ in Lyve-1-positive lymphatic sinus of dCLNs 4 h after SAH induction (L + SAH/L + V + SAH; *n* = 13, V + SAH; *n* = 12, pooled from 2 independent experiments. *P*(L + SAH vs L + V + SAH) = 0.0013, *P*(V + SAH vs L + V + SAH) = 0.006). **g** Representative images of the brain after 7 days of induction of SAH in the groups indicated. Arrows, blood clot in the brain. Scale bar, 1 mm. All data are presented as mean values ± SD, one-way ANOVA with Turkey's multiple-comparison test, \*\**P* \< 0.01, \*\*\**P* \< 0.001, \*\*\*\**P* \< 0.0001. Source data are provided as a Source data file.
On the seventh day after the ablation of the meningeal lymphatics, we induced SAH. After 4 h of SAH, blood infusion into the dCLNs and mandibular LNs were significantly lower in the mice with impaired meningeal lymphatics compared to the Laser only and the Visudyne only groups (Fig. [3d](#Fig3){ref-type="fig"}, Supplementary Fig. [3e, f](#MOESM1){ref-type="media"}). And the number of Ter119-labeled erythrocytes was markedly lower in the dCLNs (*P* = 0.0013, L + SAH vs L + V + SAH; *P* = 0.0060, V + SAH vs L + V + SAH; Fig. [3e, f](#Fig3){ref-type="fig"}) and mandibular LNs (Supplementary Fig. [3g, h](#MOESM1){ref-type="media"}) of the lymphatic-ablation group. In general, blood is released into the SAS when SAH occurs and is cleared within 2--3 days^[@CR7],[@CR8]^. But we found that clot clearance was blocked when the meningeal lymphatics were ablated as clots persisted in the brain until 7 days after the induction of SAH in the Laser + Visudyne group (Fig. [3g](#Fig3){ref-type="fig"}).
Ablation of meningeal lymphatics worsens SAH severity {#Sec5}
-----------------------------------------------------
Neuroinflammation is prominent in SAH, leading to cerebral cell damage and vasospasm^[@CR21]^. Microglia are resident brain macrophages, can be activated in SAH, and can display either classical pro-inflammatory phenotype or alternative anti-inflammatory phenotype polarization^[@CR22]^. We hypothesized that, after ablation of meningeal lymphatics, the microglia activation would be worsened owing to the prolonged exposure to erythrocytes degradants. We thus performed whole-brain flow cytometry to determine the ratio of CD16/32-posititive pro-inflammatory microglia and CD206-positive anti-inflammatory microglia. For these measurements, CD11b^+^CD45^low^ cell populations were gated as myeloid lineage cells including microglia, in which the CD16/32 was used as a marker for cells with pro-inflammatory phenotype and CD206 was used as a marker for cells with anti-inflammatory phenotype. The gating strategy is shown in Fig. [4a](#Fig4){ref-type="fig"}. Compared with SAH mice with intact lymphatics (SAH only, L + SAH and V + SAH group), SAH mice with lymphatic ablation (L + V + SAH group) showed a significantly greater percentage of CD16/32^+^CD206^−^ microglia (*P* \< 0.0001 SAH/L + SAH/V + SAH vs L + V + SAH, Fig. [4b, c](#Fig4){ref-type="fig"}) and a lower percentage of CD206^+^CD16/32^−^ microglia (*P* = 0.0146, SAH vs L + V + SAH; *P* \< 0.0001, L + SAH vs L + V + SAH Fig. [4b, d](#Fig4){ref-type="fig"}). When compared with the Laser + Visudyne group, the degree of activated microglia polarized to a pro-inflammatory phenotype was markedly greater after SAH as evidenced by an increased ratio of CD16/32^+^CD206^−^ subsets and decreased ratio of CD206^+^CD16/32^−^ cells. (*P* \< 0.0001, L + V vs L + V + SAH for CD16/32^+^CD206^−^ and *P* = 0.0086, L + V vs L + V + SAH for CD206^+^CD16/32^−^, Fig. [4c, d](#Fig4){ref-type="fig"}). The percentages of CD16/32 and CD206 double-positive cells, an intermediate state of polarization, and difference were only observed in the comparison between V + SAH and L + V + SAH groups (*P* = 0.025, Fig. [4e](#Fig4){ref-type="fig"}).Fig. 4Ablation of meningeal lymphatics aggravates the neuroinflammatory response, the defects of exploratory behavior, and short-term working memory in SAH.**a**--**d** Impaired meningeal lymphatics worsened the microglia activation in SAH. **a** Representative FACS plots showing gating strategy we used in flow cytometric analysis. Cell populations in the right dot plots defined as CD11b^+^CD45^low^ (microglia) were gated for further analysis. **b** Representative dot plots showing the ratio of CD16/32-positive subsets and CD206-positive subsets from different groups. **c** Quantification of CD16/32^+^CD206^−^ subsets of CD11b^+^CD45^low^ populations (*P*(SAH vs L + V + SAH) \< 0.0001, *P*(L + SAH vs L + V + SAH) \< 0.0001, *P*(V + SAH vs L + V + SAH) \< 0.0001, *P*(L + V vs L + V + SAH) \< 0.0001), **d** CD206^+^CD16/32^−^ subsets of CD11b^+^CD45^low^ populations (*P*(SAH vs L + V + SAH) = 0.0146, *P*(L + SAH vs L + V + SAH) \< 0.0001, *P*(L + V vs L + V + SAH) = 0.0086), and **e** CD16/32^+^CD206^+^ subsets of CD11b^+^CD45^low^ populations in different groups (*P*(V + SAH vs L +V + SAH) = 0.025). SAH/L + V + SAH; *n* = 14, L + SAH/V + SAH/L + V; *n* = 12, pooled from 2 independent experiments. **f**--**i** Ablation of meningeal lymphatics worsens the exploratory behavior and short-term working memory associated with SAH. **f** Percentage of time spent in the center area in the open field test (*P*(SAH vs L + V + SAH) = 0.0194, *P*(L + SAH vs L + V + SAH) = 0.0053, *P*(V + SAH vs L + V + SAH) = 0.009, *P*(L + V vs L + V + SAH) = 0.0138), **g** the number of entries into the center area in the open field test (*P*(SAH vs L + V + SAH) \< 0.0001, *P*(L + SAH vs L + V + SAH) = 0.0001, *P*(V + SAH vs L + V + SAH) = 0.0018, *P*(L + V vs L + V + SAH) = 0.0141), **h** percentage of time spent in the novel arm (NA) in the Y-maze test (*P*(SAH vs L + V + SAH) = 0.0006, *P*(L + SAH vs L + V + SAH) = 0.003, *P*(L + V vs L + V + SAH) = 0.0198), and **I** the number of entries into the NA in the Y-maze test in the groups of mice indicated (*P*(SAH vs L + V + SAH) \< 0.0001, *P*(L + SAH vs L + V + SAH) \< 0.0001, *P*(V + SAH vs L + V + SAH) = 0.0338, *P*(L + V vs L + V + SAH) = 0.0303). SAH; *n* = 22, L + SAH/V + SAH/L + V + SAH; *n* = 19, L + V; *n* = 15 mice, pooled from 2 independent experiments. All data are presented as mean values ± SD, one-way ANOVA with Turkey's multiple-comparison test, \**P* \< 0.05, \*\**P* \< 0.01, \*\*\**P* \< 0.001, \*\*\*\**P* \< 0.0001. NS, not significant. APC allophycocyanin, PE R-phycoerythrin, FITC fluorescein isothiocyanate. Source data are provided as a Source data file.
To assess the impact of the ablation of meningeal lymphatics on neurological function of the mice with SAH, we applied the open field test to evaluate the exploratory behavior of mice. SAH mice in the lymphatic-ablated group showed a significant decrease in the percentage of time spent in the center and the number of entrances into the center compared to the non-ablated SAH mice (*P* = 0.0194, SAH vs L + V + SAH, *P* = 0.0053, L + SAH vs L + V + SAH; *P* = 0.009, V + SAH vs L + V + SAH, Fig. [4f](#Fig4){ref-type="fig"}, *P* \< 0.0001, SAH vs L + V + SAH; *P* = 0.0001, L + SAH vs L + V + SAH; *P* = 0.0018, V + SAH vs L + V + SAH; Fig. [4g](#Fig4){ref-type="fig"}). We also used a Y-maze test to evaluate the short-term working memory of the SAH model. We found that the SAH mice with ablated meningeal lymphatics were more vulnerable than the mice with intact lymphatics, as demonstrated by the percentage of time spent in the novel arm (NA) (*P* = 0.0006, SAH vs L + V + SAH; *P* = 0.003, L + SAH vs L + V + SAH, Fig. [4h](#Fig4){ref-type="fig"}) and the number of entrances into the NA (*P* \< 0.0001, SAH vs L + V + SAH; *P* \< 0.0001, L + SAH vs L + V + SAH; *P* = 0.0338, V + SAH vs L + V + SAH, Fig. [4i](#Fig4){ref-type="fig"}). When compared with the mice only treated with photoconverted visudyne, the mice accompanied with SAH showed worse performance in both the open field (*P* = 0.0138, time spent in the center; *P* = 0.0141, number of entries into the center; L + V vs L + V + SAH, Fig. [4f, g](#Fig4){ref-type="fig"}) and the Y-maze test (*P* = 0.0198, time spent in the NA arm; *P* = 0.0303, number of entries into the NA arm, Fig. [4h, i](#Fig4){ref-type="fig"}).
Inhibition of VEGFR3 exacerbates SAH pathology {#Sec6}
----------------------------------------------
Meningeal lymphatics maintain the potential to grow or regress in adults^[@CR13],[@CR18]^. To further confirm the lack of integrity of meningeal lymphatics resulting in unfavorable outcomes in SAH, we inhibited VEGFR3, a tyrosine kinase receptor that promotes lymphangiogenesis, with MAZ51, a chemical inhibitor of VEGFR3 with proven effectiveness in inhibiting lymphangiogenesis^[@CR18]^. MAZ51 was given intraperitoneally for 30 days (Fig. [5a](#Fig5){ref-type="fig"}). As shown in Fig. [5b](#Fig5){ref-type="fig"}, meningeal lymphatics underwent regression after treatment with MAZ51, as evidenced by a reduction in the lyve-1-positive area in both the transverse and superior sagittal sinuses (Fig. [5c](#Fig5){ref-type="fig"}). We observed no change in cerebral blood flow (Supplementary Fig. [3a, b](#MOESM1){ref-type="media"}) and of CD31-positive blood vasculature area of the sinuses (Supplementary Fig. [3i--j](#MOESM1){ref-type="media"}).Fig. 5The blockage of VEGFR3 exacerbated the neuroinflammation and behavioral defects induced by SAH.**a** Scheme of the experiments in VEGFR3 tyrosine kinase inhibitor treatment experiments. Mice were treated with MAZ51 once a day for 30 days (5 days per week). After 7 days of SAH induction or sham treatment, mice were killed for analysis. **b** Representative images of meningeal lymphatics labeled by Lyve-1 (red) in the vehicle and MAZ51 treatment groups. Regions of interest in the top images are shown below. Scale bar, 1 mm (the top images) and 200 μm (the bottom images). **c** Quantification of the percentage of Lyve-1 coverage on TS and SSS. (Vehicle; *n* = 12, MAZ51; *n* = 13 mice. TS, *P* \< 0.0001, SSS, *P* \< 0.0001, two-tailed unpaired Student's *t* test). **d** Represen*t*ative FACS plots showing gating strategy in flow cytometric analysis. Cell populations characterized by CD11b^+^CD45^low^ (microglia) were gated for further analysis. **e** Representative dot plots showing the ratio of CD16/32-positive subsets and CD206-positive subsets. **f** Quantification of CD16/32^+^CD206^−^ subsets of CD11b^+^CD45^low^ populations (*P*(Vehicle + Sham vs Vehicle + SAH) = 0.0139, *P*(MAZ51 + Sham vs MAZ51 + SAH) \< 0.0001, *P*(Vehicle + SAH vs MAZ51 + SAH) \< 0.0001), **g** CD206^+^CD16/32^−^ subsets of CD11b^+^CD45^low^ populations (*P*(Vehicle + Sham vs MAZ51 + SAH) = 0.0233), and **h** CD16/32^+^CD206^+^ subsets of CD11b^+^CD45^low^ populations in different groups. Vehicle + Sham/Vehicle + SAH; *n* = 10, MAZ51 + Sham; *n* = 11, MAZ51 + SAH; *n* = 12 mice, pooled from 2 independent experiments. **i**--**l** VEGFR3 blockage worsened neurological performance of SAH. **i** Percentage of time spent in the center area in the open field test (*P*(Vehicle + Sham vs Vehicle + SAH) = 0.011, *P*(MAZ51 + Sham vs MAZ51 + SAH) \< 0.0001, *P*(Vehicle + SAH vs MAZ51 + SAH) = 0.0111), **j** the number of entries into the center area in the open field test (*P*(Vehicle + Sham vs Vehicle + SAH) = 0.0216, *P*(MAZ51 + Sham vs MAZ51 + SAH) = 0.0004), **k** percentage of time spent in the novel arm (NA) in the Y-maze test (*P*(Vehicle + Sham vs MAZ51 + SAH) = 0.0001, *P*(MAZ51 + Sham vs MAZ51 + SAH) = 0.0001), and **l** the number of entries into the NA in the Y-maze test in the groups of mice indicated (*P*(Vehicle + Sham vs Vehicle + SAH) = 0.0269, *P*(MAZ51 + Sham vs MAZ51 + SAH) = 0.0037, *P*(Vehicle + SAH vs MAZ51 + SAH) = 0.0221). Vehicle + Sham; *n* = 13, Vehicle + SAH/MAZ51 + Sham; *n* = 12, MAZ51 + SAH; *n* = 15 mice. All data are presented as mean values ± SD, one-way ANOVA with Turkey's multiple-comparison test (**f**--**l**), \**P* \< 0.05, \*\**P* \< 0.01, \*\*\**P* \< 0.001, \*\*\*\**P* \< 0.0001. NS, not significant. Source data are provided as a Source data file.
On the 30th day after treatment, vehicle- and MAZ51-treated mice were injected autologous blood or saline. Microglia activation was also detected by flow cytometry (the general gating strategy is shown in Fig. [5d](#Fig5){ref-type="fig"}). As our data show, the percentage of CD16/32^+^CD206^−^ pro-inflammatory microglia was increased after SAH induction (*P* = 0.0139, Vehicle + Sham vs Vehicle + SAH; *P* \< 0.0001, MAZ51 + Sham vs MAZ51 + SAH, Fig. [5e, f](#Fig5){ref-type="fig"}), but the proportion of activated microglia polarized to CD16/32^+^CD206^−^ phenotype were greater in the mice with meningeal lymphatic regression (*P* \< 0.0001, Vehicle + SAH vs MAZ51 + SAH, Fig. [5f](#Fig5){ref-type="fig"}). The percentage of CD206^+^CD16/32^−^ anti-inflammatory microglia was not markedly affected by SAH or MAZ51 induction but significantly reduced after SAH + MAZ51 treatment (*P* = 0.0233, MAZ51 + SAH vs Vehicle + Sham) (Fig. [5g](#Fig5){ref-type="fig"}). For the cell population characterized by CD16/32^+^CD206^+^, no statistical significance was observed (Fig. [5h](#Fig5){ref-type="fig"}). These results were consistent with the above findings, indicating that the deterioration of the meningeal lymphatic vessels leads to further aggravation of neuroinflammation caused by SAH.
To validate the regression of meningeal lymphatics resulting in worse neurological outcomes after SAH, we performed the behavioral tests on MAZ51-induced lymphatic regression mice. The exploratory behavior and short-term working memory were assessed by open field test and Y-maze test, respectively. Mice with degenerated meningeal lymphatics spent less time in the center after SAH (*P* = 0.0111, Vehicle + SAH vs MAZ51 + SAH, Fig. [5i](#Fig5){ref-type="fig"}) but did not show any difference in the number of entries into the center (Fig. [5j](#Fig5){ref-type="fig"}). The deficits of short-term working memory after SAH also worsened in mice treated with MAZ51, represented by less time spent in the NA (*P* = 0.0001, MAZ51 + Sham vs MAZ51 + SAH, Fig. [5k](#Fig5){ref-type="fig"}) and the number of entries into the NA (*P* = 0.0037, MAZ51 + Sham vs MAZ51 + SAH; *P* = 0.0221, Vehicle + SAH vs MAZ51 + SAH, Fig. [5l](#Fig5){ref-type="fig"}).
Characterization of meningeal lymphatics in SAH {#Sec7}
-----------------------------------------------
It has been reported that during acute inflammation, such as in intestinal inflammation, colitis, endocarditis, and rheumatoid arthritis, lymphatic flow and lymphangiogenesis are increased in the local peripheral lymphatic vessels, which is a mechanism for the body to reduce inflammation and edema^[@CR23]--[@CR28]^. To explore whether the neuroinflammation following SAH affects the function of the meningeal lymphatic vessels, we injected AF^488^-conjugated anti-Lyve-1 or fluorescent microbeads into the cisterna magna at day 7 after induction of SAH or saline injection. After 30 min, meninges were harvested and stained for Lyve-1 using AF^555^-conjugated secondary antibody. The percentage of meningeal lymphatics labeled by AF^488^-anti-Lyve-1 antibody (intracisterna magna (i.c.m.)) indicates the speed of meningeal lymphatic flow. A significantly greater percentage of meningeal lymphatics labeled by AF^488^ anti-Lyve-1 (i.c.m.) was seen in the SAH group vs the controls (*P* = 0.0122, Con vs SAH; *P* = 0.0303, Saline vs SAH; Fig. [6a--c](#Fig6){ref-type="fig"}), and all the lymphatics labeled by injected antibody were seen on the transverse sinus. A greater percentage of lymphatics labeled by AF^488^ anti-Lyve-1 (i.c.m.) also could be seen in dCLNs in the SAH group vs controls (*P* = 0.0183, Con vs SAH; *P* = 0.0181, saline vs SAH; Fig. [6d, e](#Fig6){ref-type="fig"}). Similarly, 2 h after fluorescent microbead injection, the bead coverage in dCLNs was significantly higher in the SAH group vs the controls (*P* = 0.0004, Con vs SAH; *P* = 0.0251, saline vs SAH; Fig. [6f--h](#Fig6){ref-type="fig"}). In addition, the higher contraction frequency of mandibular afferent lymphatic vessels was detected in the SAH group than that in the saline group (*P* = 0.0261, Fig. [6i, j](#Fig6){ref-type="fig"}).Fig. 6Meningeal lymphatic flow is augmented in the SAH mouse model.**a** Representative images of meninges and dCLNs stained for anti-Lyve-1 (AF^555^-conjugated secondary antibody, ex vivo) and AF^488^-conjugated anti-Lyve-1 (i.c.m.). Scale bars, 1 mm (for the meninges,); 200 μm (for the dCLNs). *n* = 2 independent experiments. **b** Quantification of area fraction (%) dividing the area of meningeal lymphatics labeled by AF^488^-conjugated Lyve-1 antibody (i.c.m.) by the area of meningeal lymphatics (Con; *n* = 10, Saline; *n* = 7, SAH; *n* = 6 mice. *P*(Con vs SAH) = 0.0122, *P*(Saline vs SAH) = 0.0303). **c** Quantification of meningeal lymphatic area of the TS. Con; *n* = 10, Saline; *n* = 7, SAH; *n* = 6 mice. **d** Quantification of area fraction (%) occupied by the area of dCLN lymphatics labeled by AF^488^-conjugated Lyve-1 antibody (i.c.m.) and the area of dCLNs. Con; *n* = 13, Saline; *n* = 11, SAH; *n* = 14 nodes, pooled from 2 independent experiments. *P*(Con vs SAH) = 0.0183, *P*(Saline vs SAH) = 0.0181). **e** Size of dCLNs in the Con, saline, and SAH groups. Con; *n* = 13, Saline; *n* = 11, SAH; *n* = 14 nodes, pooled from 2 independent experiments. **f** Representative images of exogenously injected fluorescent microbeads (1 μm in diameter, red) in the dCLNs of the control, saline injection, and autologous blood injection groups. Scale bar, 200 μm. *n* = 2 independent experiments. **g** The percentage of microbead coverage in the dCLNs. Con/SAH; *n* = 13, Saline; *n* = 9 nodes, pooled from 2 independent experiments. *P*(Con vs SAH) = 0.0004, *P*(Saline vs SAH) = 0.0251). **h** Size of dCLNs in the Con, saline, and SAH groups. Con/SAH; *n* = 13, Saline; *n* = 9 nodes, pooled from 2 independent experiments. **i** Representative images of ICG fluorescence of mandibular LNs and its afferent lymphatics at 30 min after ICG injection. Scale bar, 3 mm. Red circle, region of interest of lymphatic vessel for lymph flow frequency analysis. **j** Quantification of the number of the afferent lymphatic vessel contraction frequency (pulse per minute) (*n* = 6 mice per group, *P* = 0.0261, two-tailed unpaired Student's *t* test). All data are presented as mean values ± SD; one-way ANOVA with Turkey's multiple-comparison test (**b**--**e**, **g**, **h**), \**P* \< 0.05, \*\*\**P* \< 0.001. NS, not significant. Source data are provided as a Source data file.
To investigate whether the SAH process affects growth and expansion of the meningeal lymphatic vessels, we divided the meningeal lymphatics on the transverse sinus into eight different segments and measured the diameter and branching of each segment. Although we found significant dilation of vessel diameter (Fig. [7a, b](#Fig7){ref-type="fig"}) and a greater number of branches (Fig. [7a, c](#Fig7){ref-type="fig"}) in some segments in the SAH group, the lymphatic vessel area in the transverse sinus and the superior sagittal sinus did not change (Fig. [7d, e](#Fig7){ref-type="fig"}). Thus we conclude that lymphangiogenesis and lymphangiectasia of the meningeal lymphatics did not significantly alter at 7 days post-induction of SAH.Fig. 7The diameter, branching, and area of meningeal lymphatics do not change significantly after 7 days of SAH.**a** Representative images of meninges whole mounts stained for meningeal lymphatics (Lyve-1, green) from the indicated groups (4 mice per group). The lymphatics on left and right TS were divided into 8 segments (400 μm each), respectively. Regions of interest of each top image are shown in the row below (white asterisk, the branching of lymphatics). Scale bars, 1 mm (the top images) and 200 μm (the bottom images). **b** Quantification of lymphatic diameters in eight different segments. *X* axis represents 400--3200 μm distance to the junction of all sinuses. *n* = 4 mice per group. Left 800, *P*(Con vs SAH) = 0.002, *P*(Saline vs SAH) = 0.0067; Left 1200, *P*(Con vs SAH) = 0.0262, *P*(Saline vs SAH) = 0.0374; Right 2800, *P*(Con vs SAH) = 0.0005, *P*(Saline vs SAH) \< 0.0001. **c** Quantification of lymphatic branches in eight different segments. *X* axis represents 400--3200 μm distance to the junction of all sinuses. *n* = 4 mice per group. Left 1600, *P*(Saline vs SAH) = 0.0473; Left 400, *P*(Saline vs SAH) = 0.0287; Right 2800, *P*(Con vs SAH) = 0.0319, *P*(Saline vs SAH) = 0.0063; Right 3200, *P*(Saline vs SAH) = 0.0191. **d**, **e** The percentages of Lyve-1 coverage on the TS (**d**) and the SSS (**e**). *n* = 4 mice per group. All data are presented as mean values ± SD; two-way ANOVA (**b**, **c**) or one-way ANOVA (**d**, **e**) with Turkey's multiple-comparison test. \**P* \< 0.05, \*\**P* \< 0.01, \*\*\**P* \< 0.001, Con vs SAH, ^\#^*P* \< 0.05, ^\#\#^*P* \< 0.01, ^\#\#\#\#^*P* \< 0.0001, Saline vs SAH. NS, not significant. Source data are provided as a Source data file.
Discussion {#Sec8}
==========
SAH most commonly occurs due to the rupture of an aneurysm in the cerebral artery, releasing blood into the SAS and resulting in a series of early neurological complications and DCI. Understanding the process by which meningeal lymphatics drain fluid, macromolecules and immune cells from the CSF has shed a light on the pathogenesis of neurological diseases, including Alzheimer's disease, multiple sclerosis, and ischemic brain injury in past 5 years^[@CR13],[@CR14],[@CR16],[@CR18],[@CR29],[@CR30]^. However, whether the extravasated erythrocytes released into the CSF during SAH can be removed by meningeal lymphatics remains unclear. Here we show that the extravasated erythrocytes in the SAS are drained into dCLNs and mandibular LNs through the meningeal lymphatics and that the depletion of meningeal lymphatics blocks the clearance of extravasated blood.
During SAH, blood pours into the SAS, where erythrocytes break down and release hemoglobin (Hgb) and its products that contribute to brain injury^[@CR31],[@CR32]^. Previous studies report that extravasated erythrocytes and their degradation products in the SAS can be cleared via mechanisms of clot lysis or phagocytosis^[@CR7],[@CR9],[@CR10],[@CR33]^. With the upregulation of intercellular adhesion molecule-1 in cerebral blood vessel endothelial cells, macrophages and neutrophils enter the SAS and then phagocytose extravasated erythrocytes and Hgb^[@CR34]--[@CR36]^. It is proposed that macrophages and neutrophils are trapped in the SAS after phagocytosis of extravasated erythrocytes and Hgb, which then die and are degranulated within 2--4 days^[@CR33],[@CR35]^. However, the structure and function of meningeal lymphatics have been defined recently^[@CR11],[@CR12]^. Furthermore, microglia also play a role to clear in SAH by expressing heme oxygenase-1^[@CR37]^. Microglial activation and monocyte infiltration are observed 24 and 72 h after SAH, respectively^[@CR38]^. Here we show that erythrocytes can be detected in the CLNs (dCLNs and mandibular LNs) and meningeal lymphatics at 4 h post SAH, a time much early than macrophage/microglia-mediated clearance. These findings reveal that the extravasated erythrocytes can be drained into CLNs before being degraded into Hgb or phagocytosed by macrophages and neutrophils, at least in the very early stages of SAH (4 h after SAH in this study). Meanwhile, we found here that the depletion of meningeal lymphatics significantly blocked the drainage of extravasated erythrocytes into CLNs, demonstrating that meningeal lymphatics serve as the route of draining for erythrocytes into the CLNs. The blood clots were observed in the brain of SAH mice with ablated lymphatics indicating that the clot clearance may be affected by the reduced cerebral lymphatic drainage. As CSF flow is not unidirectional, ventricular blood presenting in some acute SAH is proposed that is refluxed from cisternal hemorrhage and not indicative of primary ventricle bleeding^[@CR39],[@CR40]^. Thus we considered that the clots on the pons and medulla in this study may result from the ventricular blood refluxed from SAS. There are several potential routes of extravasated blood clearance as discussed above. Different routes may participate in different phases of SAH, for example, before and after the degradation of erythrocytes. Great effort should be made in the future to clarify the kinetics and relative contribution of each of these pathways of extravasated erythrocyte clearance.
This study reveals a lymphatic route for clearing extravasated erythrocytes in SAH; however, the mechanism by which extravasated erythrocytes enter the meningeal lymphatics is still unclear. T cells and dendritic cells enter into and migrate through the meningeal lymphatics and peripheral lymphatic system via the C-C chemokine motif receptor 7--C-C chemokine motif ligand 21 pathway^[@CR14],[@CR18],[@CR41],[@CR42]^. Macromolecules can be endocytosed by brain lymphatic endothelial cells in zebrafish^[@CR43]^. Fluids and solutes diffuse into the peripheral lymphatic lumen due to the pressure difference between that of the interstitial fluid and the lumen^[@CR44]^, while chylomicron uptake into lacteals occurs by active transport vesicles through lymphatic endothelial cells^[@CR45],[@CR46]^. Little is known about the mechanism of extravasated erythrocytes entering meningeal lymphatics, but the known ways by which immune cells and macromolecules enter into the lymphatic lumen may provide some hints for future research.
Neuroinflammation that occurs as a result of SAH is caused by the accumulation of erythrocyte degradation products, including Hgb, methemoglobin, heme, and hemin in the SAS. Microglia are activated and accumulated in the brain accompanied by upregulation of inflammatory cytokines, including tumor necrosis factor-α, interleukin (IL)-1β, and IL-6. These inflammatory factors contribute to the neuronal cell death and secondary brain injury after SAH^[@CR38],[@CR47]--[@CR49]^. In this study, we found that microglial activation and polarization into a pro-inflammatory microglial cells in SAH are aggravated by the depletion of meningeal lymphatics. This exacerbation may have occurred because the extravasated erythrocytes were trapped in the SAS, thus increasing the accumulation of Hgb and its products and prolonging brain exposure to these degradation products. A previous study reported that the subarachnoid clot volume and spontaneous clearance rate are closely related to vasospasm^[@CR8]^. Thus promoting the clearance of subarachnoid erythrocytes via accelerating meningeal lymphatic flow may be a potential therapy for the pathologies associated with SAH.
The enhancement of lymphatic draining function, lymphangiogenesis, and lymphangiectasia are commonly observed in peripheral acute inflammation, including arthritis, bacterial keratitis, and colitis^[@CR50]--[@CR52]^, with the enhanced lymphatic flow and lymphangiogenesis reducing the local inflammation and edema. The overexpression of VEGF-C by viral infection or local injection has been shown to ameliorate, while the blockade of the VEGF-C/VEGFR-3 pathway exacerbates, inflammation^[@CR52]--[@CR56]^. Increasing meningeal lymphatic drainage was observed at day 7 of SAH in this study; however, lymphatic expansion and growth were not pronounced. These results are in consistent with previous reports that the morphology of meningeal lymphatics did not change in EAE-associated neuroinflammation^[@CR14],[@CR18]^. Meningeal lymphatics may have a limited growth capability when exposed to neuroinflammation. Further research is needed to determine whether modulating lymphangiogenesis by overexpression of VEGF-C or other means affects SAH induced-neuroinflammation.
In summary, we show that extravasated erythrocytes in the SAS are drained into CLNs through meningeal lymphatics during SAH. This study adds insight into the extravasated erythrocyte clearance pathway that occurs during the very early stages of SAH and provides a possible therapeutic avenue for its treatment, as well as possibly other types of intracranial hemorrhage.
Materials and methods {#Sec9}
=====================
Animals {#Sec10}
-------
Specific pathogen-free, C57BL/6 male mice (6--8 weeks old) were purchased from Shanghai Model Organisms Center. Mice were housed in the animal facility with controlled habituation and temperature, on 12-h light vs dark cycles, and fed with regular rodent's chow and sterilized tap water ad libitum. Mice were allowed to accommodate for 2 weeks before experimental procedures. All animal procedures were approved by Longhua Hospital - Animal Ethics Committee and were performed according to the Guiding Principles for the Care and Use of Laboratory Animals Approved by Animal Regulations of National Science and Technology Committee of China.
Induction of SAH {#Sec11}
----------------
An SAH model was established according to a previous publication^[@CR57]^. Briefly, 60 µl autologous blood was withdrawn from the right femoral artery after mice were anesthetized with ketamine hydrochloride (100 mg/kg, Fujian Gutian Pharma Co., Ltd, 1505223). The animal's head was fixed in a stereotactic frame (RWD), the posterior neck was incised, the posterior neck muscles were separated to access the cisterna magna, and 60 µl of autologous blood was injected at a low rate into this region. The needle was kept in place for 2 min to prevent backflow or CSF leakage. Sham-treated mice were similarly injected with 60 µl of saline. Then the mice were sutured and kept on a 37 °C heating pad (Thermo Plate) until entirely recovered from anesthetization. Mice in blank control group (Con group) were housed as usual and did not receive any additional treatment.
Behavioral analysis {#Sec12}
-------------------
The open field test was used to evaluate spontaneous activity and exploration behaviors. Mice moved freely in the box (60 cm × 60 cm × 25 cm) for 10 min, and then the distance traveled, the time spent in the center, and the number of entrances into the center area was recorded using the videotracking software EthoVision XT 12 (Noldus). Short-term memory was assessed by Y-maze test. The maze included the starting arm, the NA, and the other arm. Before the test, mice underwent a 5-min training period with a block of the NA in the maze. Two hours later, the NA was opened, and the mice were allowed to travel freely throughout the three arms, with the percentage of time spent in the NA and the number of entries into the NA in 5 min recorded by ANY-maze (Stoelting, America).
I.c.m. injection {#Sec13}
----------------
Mice were fixed in a stereotactic frame (RWD) after anesthetization with ketamine hydrochloride, and an incision was done along with separation of the posterior neck muscles to access the cisterna magna. Then 2 µl of fluorescent microbeads (Latex beads, amine-modified polystyrene, fluorescent red, Cat. No. L2778-1, Sigma) were injected into the cisterna magna at a rate of 0.5 µl/min or 5 µl of Alexa Fluor 488-conjugated anti-Lyve-1 antibody (AF488 anti-Lyve-1) (Cat. No. 53-0443-82, eBioscience) was injected into the cisterna magna at a speed of 1 µl/min. After injection, the needle was left in place for 2 min to prevent backflow and leakage. Then the mice were sutured and kept on a 37 °C heating pad until responsive. The AF^488^ Lyve-1 antibody was left to flow for 30 min and the fluorescent microbeads were left to flow for 2 h before the mice were killed.
Visudyne treatment {#Sec14}
------------------
To ablate the meningeal lymphatics, visudyne treatment was carried out according to a previous publication^[@CR14]^. Briefly, mice were anesthetized with ketamine hydrochloride and their heads were fixed in a stereotactic instrument. Visudyne (APExBIO, Cat. No. A8327) was reconstituted according to the manufacturer's instructions, and 5 µl was injected into the cisterna magna at a speed of 1 µl/min. Fifteen minutes later, a nonthermal 689-nm wavelength laser light (Changchun Laser Technology), with a dose of 50 J/cm^2^ and intensity of 600 mW/cm^2^, was applied on 5 different spots through the skull (the injection site, left and right transverse sinuses, the superior sagittal sinus, and the junction of all sinuses). For the Laser group, mice underwent the same procedures of laser treatment but without visudyne injection, and for the Visudyne group, mice were just given 5 µl of visudyne into the cisterna magna without laser treatment. During the laser procedure, the eyes of the mice were protected. Then the incision in the mice was sutured and the mice were kept on a 37 °C heating pad until entirely recovered from anesthetization.
VEGFR3 tyrosine kinase inhibitor administration {#Sec15}
-----------------------------------------------
MAZ51 (Cat. No. 676492, Merck Millipore) was dissolved in dimethyl sulfoxide and intraperitoneally injected at 10 mg/kg of body weight for 30 days (5 days per week). The control group was given the same volume of vehicle. On the 30th day, mice of both groups were divided into either an autologous blood injection or a saline injection group. On the seventh day after SAH induction, mice were killed for analysis.
Erythrocyte isolation and labeling {#Sec16}
----------------------------------
Whole blood was collected from mice from the right femoral artery after they were anesthetized, then 1:1 diluted with 2% fetal bovine serum (FBS)--phosphate-buffered saline (PBS), followed by centrifugation for 10 min (800 × *g*) without braking. The plasma and buffy coat layers were removed, the erythrocytes were collected from the bottom of tubes, and the cells were diluted into 10^6^ cells/ml, then CFSE (20 μM/ml, eBioscience, Cat. No. 65-0850-84) was added, followed by incubation for 10 min in 37 °C. After washing with 2% FBS--PBS, erythrocytes (about 10^6^ cells in 60 µl) were injected into the cisterna magma. The control group mice were injected with saline. Four hours after erythrocyte was injected, meninges, dCLNs, and mandibular LNs were harvested.
Flow cytometry {#Sec17}
--------------
Mice brains were dissected after transcardial perfusion by cold PBS, then minced into small pieces. Brain tissue was digested by collagenase A (1 mg/ml, Sigma Aldrich, Cat. No. 10103578001) for 30 min at 37 °C, then filtered by 70-μm nylon mesh cell strainers (BD bioscience). A cell suspension was made with 30% stock isotonic percoll (SIP) (GE, 17089109) and layered on the top of 70% SIP and then centrifuged at 500 × *g* at 25 °C for 30 min without braking. Cells were collected from the 70--30% SIP interphase and stained for live cells by Fixable Viability Dye eFluor^TM^ 780 (Cat. No. 65-0865-18, eBioscience), extracellular markers with the following antibodies at a 1:100 dilution: rat anti-CD11b fluorescein isothiocyanate (FITC)-conjugated antibody (11-0112-82, eBioscience), rat anti-CD45 PerCP-Cy5.5-conjugated antibody (45-0451-82, eBioscience), rat anti-CD16/32 allophycocyanin (APC)-conjugated antibody (558636, BD Bioscience) and intracellular marker rat anti-CD206 R-phycoerythrin (PE)-conjugated antibody (12-2061-80, eBioscience). The corresponding isotype control antibodies that were used are as follows: Rat IgG2b κ Isotype control FITC-conjugated antibody (11-4031-82, eBioscience) Rat IgG2a κ Isotype control PerCP-Cy5.5-conjugated antibody (45-4321-80, eBioscience), Rat IgG2b κ Isotype control PE-conjugated antibody (12-4031-82, eBioscience), and Rat IgG2b κ Isotype control APC-conjugated antibody (553991, BD Bioscience). Samples were tested and analyzed by Longzoe (Shanghai) Biotechnology Co., Ltd using BD Fortessa X20 and the FlowJo V10 software, and the company was blinded to the group allocations.
Laser speckle {#Sec18}
-------------
Mice were anesthetized by isoflurane, an incision was done along the midline to separate the skin of the skull, and RFLSI Pro+ laser speckle (RWD Life Science Co., Ltd) was used to detect mice cerebral blood flow. Laser speckle blood flow images were recorded and used to identify the regions of interest (ROIs). Within these ROIs, the mean blood flow index was calculated in real time.
In vivo imaging {#Sec19}
---------------
Mice were fixed in a stereotactic frame (RWD) after anesthetization with ketamine hydrochloride, an incision was performed, and the posterior neck muscles were separated to access the cisterna magna. Five µl of visudyne was injected into the cisterna magna at a speed of 1 µl/min, and the needle was kept in place for 2 min to avoid leakage. Control group mice were not injected with any solution. Fifteen minutes later, the distribution of visudyne was detected by KODAK In-Vivo Multispectral Imaging System FX using a 630-nm laser for excitation. Then mice were killed to acquire the skulls, and the visudyne distributions on the skull were also recorded.
Indocyanine green near-infrared (ICG-NIR) imaging {#Sec20}
-------------------------------------------------
ICG was dissolved in saline (2 mg/ml, Cat. No. 17478-701-02, Akorn). Mice from the SAH group (at 7 days post-surgery) and the saline-injected group were fixed in a stereotactic frame (RWD) after anesthetization, and cisterna magna was exposed. Five µl of ICG was injected into the cisterna magna (1 µl/min), and then the needle was left in place for 2 min to avoid leakage. ICG fluorescence of mandibular LNs and its afferent lymphatics were visualized by an IR laser (Changchun Laser Technology) and recorded continuously by an Olympus microscope (exposure times 200 ms) for 1 h. The images were analyzed using the Image J software. ROIs were identified in the afferent lymphatic vessel, and vessel contraction rate (pulse/min) was calculated to present the lymph flow function according to previous studies^[@CR20],[@CR58]^.
Tissue processing {#Sec21}
-----------------
dCLNs and mandibular LNs were harvested in a deep anesthesia condition, fixed in 4% paraformaldehyde (PFA) overnight, and then incubated serially in 10%, 20%, and 30% sucrose solutions for 3 days each. For immunofluorescence staining, CLNs were embedded in OCT, and 7-µm-thick sections were sliced by a cryostat (Leica, CM3050S). After transcardial perfusion with saline and 4% PFA for 15 min, the skullcap was harvested and fixed in 4% PFA overnight, and then the meninges were dissected from the skullcap.
Immunofluorescence {#Sec22}
------------------
For immunofluorescence, the whole mounts and sections were blocked by 0.3% PBST with 5% bovine serum albumin for 1 h at room temperature, then incubated with primary antibodies overnight at 4 °C. After washing with PBS three times for 15 min each, secondary antibodies were incubated for 2 h at room temperature. Finally, the whole mounts and sections were mounted with mounting medium with 4,6-diamidino-2-phenylindole (Cat. No. F6057, Sigma). The primary antibodies used in immunofluorescence included rabbit anti-Lyve-1 antibody (1:1000; Abcam, Cat. No. ab14917), rat anti-Ly76 \[Ter119\] antibody (1:500; Abcam, Cat. No. ab91113), rat anti-Ter119 PE-conjugated antibody (1:100; Cat. No. 12-5921-81, eBioscience), rat anti-Lyve-1 eFluor 660-conjugated antibody (1:200; 50-0443-80, eBioscience), hamster anti-PDPN antibody (1:200; Abcam, Cat. No. ab11936), rabbit anti-Prox1 antibody (1:100; AngioBio, Cat. No. 11-002P), and rat ant-CD31 antibody (Abcam, Cat. No. ab7388). The corresponding secondary antibodies were used as follows: Dylight^TM^ 488-labeled goat anti-rabbit antibody (1:200; KPL, Cat. No. 072-03-15-06), Dylight^TM^ 488-labeled goat anti-rat antibody (1:200; Cat. No. 072-03-16-06, KPL), Alexa Fluor 546 goat anti-hamster antibody (1:200, Invitrogen, Cat. No. A-21111), Alexa Fluor 488/555 goat anti-rat antibody (1:1000, Cell Signaling Technology, Cat. No. 4416S/4417S), and Alexa Fluor 555 goat anti-rabbit antibody (1:1000, Cell Signaling Technology, Cat. No. 4413S).
Image analysis {#Sec23}
--------------
For the whole-mount staining of meninges, images were acquired with an Olympus VS120 microscope and a 10× objective with 0.4 NA, or acquired with an Olympus FV1000 confocal microscope and a 40× objective with 0.95 NA, with a resolution of 1024 × 1024 pixels and a *z*-step of 4 µm. The exposure time and brightness/contrast of each image were applied equally across all images, and images were analyzed using the Image J (NIH) software. For the CLN sections, images were acquired with an Olympus VS120 microscope and a 20× objective with 0.75 NA.
The numbers of erythrocyte per mm^2^ in the Lyve-1-positive lymphatic sinus of CLNs were calculated, with only the erythrocytes with intact morphology included. The mean value of five sections of each CLN was used to make a plot graph. The numbers of erythrocytes in meningeal lymphatics per field were calculated, and four to five fields of each meninges were quantified to acquire the mean value. The percentage of meningeal lymphatics labeled by AF488 Lyve-1 antibody (i.c.m.) was defined by dividing the area of AF^488^ Lyve-1 antibody (i.c.m.) labeled by the area of meningeal lymphatics. The percentage of dCLN lymphatics labeled by AF^488^ Lyve-1 antibody (i.c.m.) was determined by dividing the area AF^488^ Lyve-1 antibody (i.c.m.) labeled per section by the area of the dCLN section. The mircobead coverage in dCLN was quantified by dividing the area of microbeads per section by the area of the dCLN section. Five to ten sections of each dCLN were quantified to acquire the mean value. Lymphatic ablation and lymphatic regression were measured by dividing the area of Lyve-1 labeled by the area of the sinus, and lymphatic coverage on transverse sinus and sagittal sinus was calculated separately. Percentage of blood vasculature coverage on sinuses was calculated by dividing the area of the CD31-positive vessels by the area of sinuses. Raw data were collected using the Microsoft Excel 2007 software.
Statistical analysis {#Sec24}
--------------------
Data were expressed as means ± SD, with differences between mean values determined by two-tailed unpaired Student's *t* test, one-way analysis of variance (ANOVA), or two-way ANOVA with Turkey's multiple-comparison test using the GraphPad Prism 6 Software. *P* values \< 0.05 were considered significant. The investigators responsible for data analysis were blinded to the group allocations.
Reporting summary {#Sec25}
-----------------
Further information on research design is available in the [Nature Research Reporting Summary](#MOESM2){ref-type="media"} linked to this article.
Supplementary information
=========================
{#Sec26}
Supplementary Information Reporting Summary Peer Review File
Source data {#Sec27}
===========
Source Data
**Peer review information** *Nature Communications* thanks the anonymous reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.
**Publisher's note** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary information
=========================
**Supplementary information** is available for this paper at 10.1038/s41467-020-16851-z.
We express gratitude to Fudan University for access to their confocal microscope and their mice behavioral analysis platforms. And thanks to Dr. Weian Zhang for the in vivo imaging and Professor Beihua Zhang for the laser speckle imaging. We are also grateful to Professor Baohua Tian for his help in the intact erythrocytes and the degradant differentiation. This work was sponsored by research grants from National Key R&D Program of China (2018YFC1704300 to Y.W.), National Natural Science Foundation (81822050 and 81920108032 to Q.L., 81904227 to Y.W.), Leading medical talents in Shanghai (2019LJ02 to Q.L.), Dawn plan of Shanghai Municipal Education Commission (19SG39 to Q.L.), the program for innovative research team of Ministry of Science and Technology of China (2015RA4002 to Y.W.), "Innovation Team" development projects (IRT1270 to Y.W.), Shanghai TCM Medical Center of Chronic Disease (2017ZZ01010 to Y.W.), Three Years Action to Accelerate the Development of Traditional Chinese Medicine Plan (ZY(2018-2020)-CCCX-3003 to Y.W.), and the program of Longhua Hospital (KY1932 to Y.W.).
J.C., L.W., H.X., L.X., Q.L., and Y.W. conceived and designed the study; J.C., L.W., S.C., and Z.G. performed the experiments; Y.Z. performed behavioral tests and data analysis; X.L. analyzed the data of blood flow index and counted the numbers of erythrocytes in CLNs and in meningeal lymphatics; Z.Z. analyzed the data of lymph flow frequency, counted the numbers of erythrocytes in CLNs, and calculated the percentage of meningeal lymphatics and blood vasculature coverage on sinus; C.W. counted the numbers of erythrocyte in CLNs and analyzed the area of AF^488^ Lyve-1 antibody (i.c.m.) labeled lymphatics and mircobead coverage (Y.Z., X.L., Z.Z., and C.W. were blinded to group allocations); J.C. and Q.L. drafted the manuscript; L.W., H.X., L.X., Q.L., and Y.W. revised the manuscript. All authors have approved the final version of the manuscript and have agreed to be accountable for all aspects of the work.
The raw data underlying Figs. [1c, e](#Fig1){ref-type="fig"}, [2b, e](#Fig2){ref-type="fig"}, [3b, c, f](#Fig3){ref-type="fig"}, [4c--i](#Fig4){ref-type="fig"}, [5c, f--l](#Fig5){ref-type="fig"}, [6b--e, g, h, j](#Fig6){ref-type="fig"}, [7b--e](#Fig7){ref-type="fig"} and Supplementary Figs. [1d](#MOESM4){ref-type="media"} and [3b, d, h, j](#MOESM4){ref-type="media"} are available via a source data file submitted with this manuscript. All other data are available from the corresponding authors upon reasonable request.
The authors declare no competing interests.
|
{
"pile_set_name": "PubMed Central"
}
|
Introduction {#s1}
============
Biological evolution is thought to have started with relatively simple, versatile, and multifunctional metabolites (Miller and Urey, [@B59],[@B60]). Adenosine was likely part of the "primordial soup" at the origin of life on Earth (Oro, [@B67]). Therefore it is not surprising that adenosine is an integral component of ATP, RNA (including poly-[A]{.ul} tails), NAD, and other compounds essential for basic biochemistry and mitochondrial bioenergetics. Glycine in turn is the most primitive amino acid, which has additional biochemical functions in carbon metabolism. It is highly likely that the biochemical and epigenetic control of genes through global biochemical regulation preceded the "invention" of transcription factors, which later assumed the role to fine-tune a preexisting primordial biochemistry-based regulatory system. For example, an energy crisis would lower ATP needed for RNA synthesis and increase adenosine, thereby promoting increased DNA methylation as will be discussed in this mini-review in more detail. Both processes would reduce gene transcription globally and conserve energy. Therefore, primordial regulatory networks acting on a global homeostatic level likely preceded the "invention" of gene specific mechanisms that require sophisticated control through transcription factors, which in turn are regulated by G protein coupled receptors (GPCRs) and protein kinase pathways. In contrast, therapy development conventionally starts with the pharmacology of drugs. For example, benzodiazepines were almost discovered by chance in 1957 leading to the subsequent characterization of the "benzodiazepine receptor" in the CNS in 1977. It turned out that the benzodiazepine binding site was in fact an integral part of the GABA~A~ receptor complex (Möhler and Okada, [@B62]). Drug-driven therapy development led to a major focus on "druggable" GPCRs, ion channels, and protein kinases, which still form the main-stay of CNS therapeutics today. Methods to exploit gene regulation therapeutically are still in its infancy and the therapeutic potential of epigenetic, biochemical, and metabolomic approaches constitutes a new frontier in therapy development. If the basis of the pyramid depicted in Figure [1](#F1){ref-type="fig"} is overlooked, it becomes obvious that a traditional pharmacological top-down treatment approach has limitations.
{#F1}
The Biochemistry of Epilepsy {#s2}
============================
In the following, I will focus on temporal lobe epilepsy (TLE), the most common form of epilepsy in adult patients, and the most thoroughly studied form of epilepsy in animal models, as well as on two key metabolites, adenosine and glycine, whose homeostasis is known to be affected in the epileptic brain. Adenosine, primarily through activation of adenosine A~1~ receptors, is an endogenous anticonvulsant and seizure terminator of the brain (Dragunow, [@B25]; During and Spencer, [@B27]) that also controls a wide range of cognitive and psychiatric phenotypes (Boison et al., [@B18]). In human surgical specimens, as well as in rodent models of TLE, overexpression of adenosine kinase (ADK) and resulting adenosine deficiency associate with astrogliosis and constitute a pathological hallmark of TLE (Riban et al., [@B70]; Gouder et al., [@B35]; Fedele et al., [@B30]; Boison, [@B10]; Li et al., [@B48], [@B47]; Aronica et al., [@B6], [@B5]). Consequently, therapeutic adenosine augmentation effectively suppresses seizures in a wide range of rodent models of epilepsy (Huber et al., [@B37]; Zuchora et al., [@B85]; Gouder et al., [@B34]; Anschel et al., [@B1]; Vianna et al., [@B79]; Li et al., [@B49], [@B48]; Wilz et al., [@B81]; Boison, [@B11], [@B12]; Boison and Stewart, [@B16]; Van Dycke et al., [@B77]). A focus of this mini-review however is the underappreciated biochemical adenosine receptor (AR) independent effects of adenosine that are tightly linked to the control of DNA methylation and that are under the control of ADK, an enzyme which also has a specific isoform expressed in the nucleus of cells (Boison, [@B14]). The cytoplasmic isoform of the enzyme is thought to regulate the homeostatic pool of adenosine thereby determining the level of AR activation (Boison and Aronica, [@B15]), whereas the nuclear isoform of the enzyme strongly affects DNA methylation status (Williams-Karnesky et al., [@B80]). Interestingly, ADK undergoes biphasic expression changes during epileptogenesis in modeled TLE (Gouder et al., [@B35]; Li et al., [@B48]; Boison, [@B14]), which form the basis of the ADK hypothesis of epileptogenesis: Acute insults to the brain such as traumatic brain injury (Clark et al., [@B21]), seizures (During and Spencer, [@B27]; Gouder et al., [@B35]), or a stroke (Pignataro et al., [@B68]) lead to an acute surge in adenosine associated with transient downregulation of ADK within the first 2 to 3 h after the injury (Gouder et al., [@B35]; Pignataro et al., [@B68]). This acute phase is followed by a "latent period" of epileptogenesis, which occurs during the first few days or weeks after an insult in rodent models, or weeks and months in humans. During this latent period, inflammatory processes are activated that lead to microglial and astroglial activation (Nabbout et al., [@B65]; Vezzani et al., [@B78]; Devinsky et al., [@B24]). Astrogliosis is associated with increases in ADK expression and consequential development of adenosine deficiency. We have previously shown that seizures originate in areas of astrogliosis with overexpression of ADK (Li et al., [@B48], [@B47]), that seizure onset during epileptogenesis temporally coincides with the emergence of astrogliosis and overexpressed ADK (Li et al., [@B46]), that overexpression of ADK as such is sufficient to generate partial seizures (Li et al., [@B46], [@B48]), and that overexpression of ADK triggers hypermethylation of DNA (Williams-Karnesky et al., [@B80]). Since therapeutic adenosine augmentation restores normal DNA methylation levels and prevents epilepsy progression long-term (Williams-Karnesky et al., [@B80]) increased ADK and increased DNA methylation status might form a vicious cycle implicated in the progression and maintenance of epilepsy. Therefore, dysregulation of ADK appears to play a significant role in the processes that turn a normal brain into an epileptic brain.
In the hippocampal formation, glycine can exert opposing effects that depend on the activation of presynaptic (Kubota et al., [@B45]; Winkelmann et al., [@B82]) vs. postsynaptic glycine receptors (GlyRs; Aroeira et al., [@B3]). It has recently been demonstrated (Chen et al., [@B20]) that low concentrations of glycine (10 μM) exert pro-convulsive effects, whereas higher glycine concentrations (100 μM) attenuate recurrent epileptiform discharges. The pro-convulsive actions of presynaptic GlyRs expressed on glutamatergic synapses (Winkelmann et al., [@B82]) is further supported by findings showing that the expression of edited GlyR encoding mRNAs are increased in the human epileptic hippocampus (Eichler et al., [@B28]) and that GlyR RNA editing regulates glycine affinity (Meier et al., [@B58]). These findings suggest that glycine homeostasis plays a crucial role in maintaining the balance between increased and decreased neuronal excitability. Hippocampal glycine is largely regulated by its reuptake transporter, glycine transporter 1 (GlyT1), found in both excitatory neurons and astrocytes (Tsai et al., [@B76]; Aragón and López-Corcuera, [@B2]; Cubelos et al., [@B23]; Eulenburg et al., [@B29]; Martina et al., [@B54]; Betz et al., [@B7]). Consequently, the genetic deletion of GlyT1 increased synaptic glycine availability (Gomeza et al., [@B33]). Engineered mice with a genetic deletion of GlyT1 in forebrain were characterized by a decrease in hippocampal glycine uptake, an increase in hippocampal NMDAR function, and a wide spectrum of pro-cognitive effects (Yee et al., [@B83]; Möhler et al., [@B64], [@B63]). Therefore, GlyT1 is considered a promising target for the treatment of cognitive symptoms in schizophrenia and several compounds have been tested in phase II and III clinical trials (Black et al., [@B9]; Singer et al., [@B73]; Möhler et al., [@B63]). A recent study (Shen et al., [@B72]) provided the first comprehensive analysis of GlyT1 dysregulation in chronic TLE. GlyT1 expression during epileptogenesis was characterized as a biphasic response with initial downregulation of GlyT1 after epileptogenesis-precipitating seizures followed by sustained pathological overexpression of GlyT1 in chronic epilepsy as demonstrated in two mechanistically different models of TLE in mice and rats (Shen et al., [@B72]). It was further demonstrated that human TLE is likewise associated with increased levels of GlyT1. Conversely, the pharmacological suppression of GlyT1 or the genetic ablation of GlyT1 in the hippocampus provided robust reduction of both acute as well as chronic seizure activity in three different model systems (Shen et al., [@B72]). Therefore, glycinergic regulation of network excitability is altered in epilepsy and GlyT1 presents a rational therapeutic target for the treatment of epilepsy. Pathological overexpression of GlyT1 in progressive epilepsy also implies altered interactions of GlyT1 with the transmethylation pathway (Figure [2](#F2){ref-type="fig"}), a novel hypothesis further discussed below.
{#F2}
The Epigenetics of Epilepsy {#s3}
===========================
The role of epigenetics in epilepsy development is a new and emerging research area (Garriga-Canut et al., [@B32]; Qureshi and Mehler, [@B69]; Kobow and Blümcke, [@B41]; Lubin, [@B51]; Henshall and Kobow, [@B36]). The fact that epigenetic changes might play a significant role at least in TLE is important because in contrast to genetic mutations, epigenetic changes are potentially reversible. The knowledge of epigenetic mechanisms implicated in the development of epilepsy provides a conceptual and mechanistic framework for the future development of epigenetic therapies tailored to prevent epilepsy (antiepileptogenic) or its progression (disease modifying). Currently used antiepileptic therapies fail to address the underlying causes of epilepsy and do not halt epileptogenesis (Löscher and Brandt, [@B50]). Epileptogenesis is characterized by a progressive increase in frequency and severity of spontaneous recurrent seizures (SRS). Several mechanisms are implicated in the epileptogenic cascade including neuro-inflammatory responses, neuronal cell loss, mossy fiber sprouting, aberrant connectivity, and gliosis coupled with adenosine dysfunction (Dudek et al., [@B26]; Seifert et al., [@B71]; Vezzani et al., [@B78]; Aronica et al., [@B4]; Boison, [@B13]). One potential unifying factor behind many of the pathological changes in epileptogenesis may be epigenetic modifications, which are likely further potentiated by epileptogenesis itself (Qureshi and Mehler, [@B69]; Kobow and Blümcke, [@B40]). Epigenetic modifications, which alter gene transcription without modifying the underlying DNA sequence, are plastic and can respond rapidly in response to environmental cues, an important endogenous mechanism for the control of gene expression. Changes in histone acetylation and methylation, as well as changes in DNA methylation have been shown to occur in mature cells in the central nervous system (CNS; Ma et al., [@B52]; Feng et al., [@B31]). Importantly, these changes occur regularly and rapidly. Even a single episode of neural synchronization exceeding 30 s in the hippocampus induces DNA methylation-dependent alterations in transcription of immediate-early genes and initiates a cascade of transcription factors contributing to long-term circuit alterations (Nelson et al., [@B66]). Therefore, epigenetic modifications offer new therapeutic alternatives to interfere with epileptogenesis.
The Methylation Hypothesis of Epileptogenesis {#s4}
=============================================
Although several epigenetic mechanisms such as histone modifications that involve the addition or removal of methyl or acetyl groups might be implicated in epileptogenesis (Henshall and Kobow, [@B36]), recent evidence points to a critical role of DNA methylation changes for the development and progression of epilepsy, which will be discussed in the following. Altered DNA methylation in the brain has been implicated in psychiatric, neurodegenerative, and neurological conditions, including epilepsy (Kobow et al., [@B42]; Ma et al., [@B52]; Feng et al., [@B31]; Martin and Wong, [@B53]; Masliah et al., [@B56]; Coppieters et al., [@B22]; Tremolizzo et al., [@B75]). The methylation hypothesis of epileptogenesis suggests that seizures by themselves can induce epigenetic modifications and thereby aggravate the epileptogenic condition (Kobow and Blümcke, [@B40]). Specifically, increased activity of DNA methylating enzymes as well as hypermethylation of DNA has been associated with the development of human and experimental epilepsy (Kobow et al., [@B42], [@B43]; Zhu et al., [@B84]; Williams-Karnesky et al., [@B80]; Miller-Delaney et al., [@B61]). Thus, interference with DNA methylation offers novel conceptual opportunities to prevent epilepsy.
Brain Homeostasis and the Control of DNA Methylation {#s5}
====================================================
DNA methylation status depends on the equilibrium of biochemical enzyme reactions that add methyl groups to cytidine groups in the DNA (5-methylcytidine, 5mC) catalyzed by DNA methyltransferases (DNMTs) and those that convert methyl groups to hydroxymethyl groups catalyzed by Ten-eleven translocation (TET) enzymes in preparation for active DNA demethylation. Here I will focus on those mechanisms that add methyl groups to DNA; those mechanisms are linked to the S-adenosylmethionine (SAM) dependent transmethylation pathway (Figure [2](#F2){ref-type="fig"}), which is under the control of adenosine and glycine regulated by ADK (Boison et al., [@B17]; Boison, [@B14]) and GlyT1 (Gomeza et al., [@B33]; Yee et al., [@B83]), respectively. DNA methylation requires the donation of a methyl group from SAM, a process that is facilitated by DNMTs. The resulting product, S-adenosylhomocysteine (SAH) is then further converted into adenosine and homocysteine (HCY) by SAH hydrolase (SAHH). Critically, the equilibrium constant of the SAHH enzyme lies in the direction of SAH formation (Kredich and Martin, [@B44]); therefore, the reaction will only proceed when adenosine and HCY are constantly removed (Kredich and Martin, [@B44]; Boison et al., [@B17]). If metabolic clearance of adenosine through ADK is impaired, SAH levels rise (Boison et al., [@B17]). SAH in turn inhibits DNMTs through product inhibition (James et al., [@B38]). Based on adenosine's role as obligatory end product of DNA methylation, we conclude that an increase in ADK and the resulting decrease in adenosine, as seen in chronic epilepsy (Li et al., [@B48]; Masino et al., [@B55]), would drive increased global DNA methylation in the brain. This process may be amplified, because adenosine is an inhibitor of ADK (Boison, [@B14]). Therapeutic adenosine augmentation may thus effectively reverse pathological DNA hypermethylation and thereby prevent epilepsy progression. The recent discovery of glycine-N-methyltransferase (GNMT) in the hippocampus (Carrasco et al., [@B19]) suggests that the availability of hippocampal glycine also controls the SAM-dependent transmethylation pathway by competing for methyl-groups. Increased GlyT1 as seen in chronic TLE (Shen et al., [@B72]) is expected to affect DNA methylation through interference with glycine homeostasis. Interestingly, the methylation of glycine leads to the formation of sarcosine, which is an endogenous inhibitor of GlyT1 (Javitt, [@B39]). Therapeutic glycine augmentation (e.g., via diet) may thus effectively divert methyl groups to the formation of sarcosine and thereby reduce: (i) pathological DNA hypermethylation; (ii) ameliorate the effects of pathologically overexpressed GlyT1; and (iii) prevent epilepsy progression.
Towards Epigenetic Therapies for Epilepsy Prevention {#s6}
====================================================
The antiepileptogenic potential of transient focal adenosine-delivery was tested in a rat model of systemic kainic acid (KA)-induced progressive TLE (Williams-Karnesky et al., [@B80]). Young male rats (130--150 g) received an acute dose of KA (12 mg/kg i.p.) to trigger status epilepticus (SE). Only rats that exhibited at least 3 h of acute convulsive SE were used further. Rats were subjected to continuous long-term monitoring to quantify seizure activity. Once rats had reached a stable rate of 3--4 SRS per week at 9 weeks post KA, the animals were randomized and each rat received bilateral intraventricular adenosine-releasing silk-implants, silk-only implants, or a corresponding sham treatment. Adenosine releasing implants were designed to transiently deliver a stable dose of 250 ng adenosine per brain ventricle per day restricted to 10 days of drug delivery (Szybala et al., [@B74]). Twenty four hours after the surgery, continuous video monitoring was initiated, maintained for 4 weeks, and resumed after a 4 week hiatus for an additional 4 weeks. In the control groups seizures continued to increase both in number and severity. In contrast, in recipients of adenosine-releasing implants, seizures were almost completely suppressed after polymer implantation. Remarkably, reduced seizure activity was maintained far beyond expiration of adenosine-release from the polymer for at least 12 weeks following implantation. Even at 12 weeks after implantation, seizure incidence was reduced by more than 70%. Importantly, and in line with prolonged reduction of seizures, mossy fiber sprouting at 21 weeks following KA was significantly attenuated in adenosine-treated rats compared to controls. In line with those profound antiepileptogenic effects, the transient delivery of adenosine restored normal DNA methylation status long-term. These data demonstrate that the transient delivery of adenosine is sufficient to restore normal DNA methylation status and to prevent epilepsy progression long-term (Williams-Karnesky et al., [@B80]).
Conclusions and Outlook {#s7}
=======================
The realization that a transient local dose of adenosine can have long-lasting antiepileptogenic effects based on shifting the transmethylation equilibrium through mass action may offer new therapeutic opportunities for small molecule ADK inhibitors. ADK inhibitors had been in pre-clinical development in the early 2000's for the indications of seizure management in chronic epilepsy, the control of chronic pain and robust anti-inflammatory effects in chronic conditions (McGaraughty et al., [@B57]). Although highly efficient in preclinical models, further drug development was halted due to unacceptable side effects related to chronic drug dosing (McGaraughty et al., [@B57]). The first generation of ADK inhibitors was designed to augment beneficial AR dependent effects of adenosine by raising extracellular levels of adenosine, which are at the same time responsible for wide-spread systemic adverse effects of those agents. Of note, patients with inborn global ADK deficiency develop hepatic encephalopathy and a wide range of neurological symptoms (Bjursell et al., [@B8]), however it is currently not known whether those symptoms are a primary cause of ADK deficiency in the brain, or secondary to hepatic encephalopathy. Early drug development efforts created a bias for the identification of agents with preferential action on the cytoplasmic isoform of ADK. ADK inhibitors with a higher selectivity for the nuclear isoform of ADK might capitalize on the epigenetic effects of adenosine while minimizing AR-mediated adverse effects. In addition, the short term use of ADK inhibitors, over days as opposed to chronic sustained drug dosing, might be acceptable if long-lasting epigenetically based therapeutic benefits can be achieved. Challenges for drug development remain. It needs to be determined whether new therapeutic agents can enter the brain and whether a higher level of selectivity for specific isoforms of ADK can be achieved. Due to the different distribution of nucleoside transporters within the brain there might be opportunities for the development of cell-type or isoform selective therapies. Glycine modifying therapies might constitute an alternative avenue to affect DNA methylation and potentially epileptogenesis. However, it needs to be determined how the adenosine and glycine systems interact on the epigenetic level. As discussed in this mini-review, understanding the biochemistry of epileptogenesis might light to the development of novel forms of therapeutic intervention.
Author Contributions {#s8}
====================
DB conceptualized and wrote the manuscript.
Conflict of Interest Statement {#s9}
==============================
The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The author's work is supported through grants from the National Institutes of Health (R01 NS084920, R21 NS088024, R01 MH083973) and through the Legacy Good Samaritan Hospital Foundations.
[^1]: Edited by: Jean-Marc Taymans, UMR1172, Jean-Pierre Aubert Research Center, France
[^2]: Reviewed by: Massimo Mantegazza, CNRS UMR7275 and University of Nice Sophia Antipolis, France; Rafal Kaminski, UCB Pharma, Belgium
|
{
"pile_set_name": "PubMed Central"
}
|
Introduction {#s1}
============
Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease, is a devastating adult-onset neurodegenerative disease with no cure and is fatal within 2 to 5 years after the disease onset. Phenotypically, it is characterized by progressive motor neuron loss resulting in muscle weakness, wasting, fasciculations, spasticity, and hyperreflexia. The vast majority of cases (∼90%) have no family history, while about 10% of patients have a genetic locus as causal entity for ALS. So far, mutations in *SOD1*, encoding for Cu/Zn superoxide dismutase, have been identified in 20% of familial ALS cases. Other frequent disease-causing genes include *C9ORF72*, TAR DNA-binding protein 43 (*TARDBP*), and fused in sarcoma/translocated in liposarcoma protein (*FUS/TLS*) [@pone.0070560-Andersen1]. Recently, it has been shown that large intermediate CAG repeat expansions in the ataxin-2 gene (*ATXN2*) contribute to almost 5% of the sporadic or familial ALS cases [@pone.0070560-Elden1]. Intermediate CAG repeats are now recognized as ALS13 associated locus (MIM183090). Based on several studies, it appears that CAG expansion in *ATXN2* might account for more familial ALS cases than *SOD1* mutations with an overall incidence of 2% [@pone.0070560-Elden1]--[@pone.0070560-VanDamme1]. SCA2 is another neurodegenerative disorder with no available cure exhibiting progressive cerebellar syndrome characterized by ataxic gait, cerebellar dysarthria, dysmetria, and dysdiadochokinesia.
Normal CAG length in *ATXN2* gene ranges from 13--31 repeats, with 22 CAG being most common. Alleles with ≥26 CAG are considered as large alleles [@pone.0070560-LaffitaMesa1]. While inconsistencies exist in the literature regarding the CAG range and nomenclature of the intermediate or indeterminate alleles, those with ≥27--33 CAG are associated with increased and specific risk for ALS [@pone.0070560-Elden1], [@pone.0070560-Lee1], [@pone.0070560-Gispert1].
Only few cases with 32--34 CAG expansions have been reported so far in SCA2 [@pone.0070560-Futamura1]--[@pone.0070560-Santos1]. Of note, none of the individuals with intermediate allele displayed typical clinical SCA2 picture. Therefore, starting point for SCA2 mutant range has been widely considered ≥34 CAG, while 37--75 CAG repeats are full penetrant [@pone.0070560-Pulst1] and massive expansions has been observed with severe disease starting in infants [@pone.0070560-Mao1], [@pone.0070560-Paciorkowski1].
First report relating ALS with intermediate alleles considered 27 repeats as the threshold [@pone.0070560-Elden1] while subsequently it has been refined to \>30 CAG [@pone.0070560-Lee1], [@pone.0070560-Gispert1]. However, in SCA2, these alleles are considered as large normal, which might be prone for intergenerational instability leading to full penetrant disease-causing alleles, but this instability has not been reported so far. Given the rarity of large and intermediate alleles (23--34 CAG), little is known about the origin of ALS-related alleles. Only one *de novo* mutation has been reported, but the data about the unstable behavior of the involved alleles were not revealed [@pone.0070560-VanDamme1]. Investigation in these patients would be important to understand events leading to genetic instability and for the accurate presymptomatic and prenatal diagnostics as well as genetic counseling.
Recently, we have shown that the highest worldwide concentration of large normal alleles (≥23 CAG) underlies the highest worldwide prevalence and incidence rates of SCA2 in Cuba. Based on this observation we postulated that *ATXN2* intermediate alleles with 32--35 CAG may evolve from large ANs with 23--31 CAG, explaining familial or sporadic cases associated either with SCA2, ALS, and Parkinsonism [@pone.0070560-VanDamme1]. In support of our postulates, we uncovered familial ALS cases resulting from large unstable alleles reaching ALS intermediate length and SCA2 pathogenic expansions at the *ATXN2* locus. In this study, we provide data on the global risk of intermediate *ATXN2* alleles for ALS.
Results {#s2}
=======
Clinical Phenotype of ALS with *de novo* CAG Expansions in Ataxin-2 Gene {#s2a}
------------------------------------------------------------------------
We identified a pedigree with three ALS (two fully developed ALS: III-13 and III-16; and one in early stages of ALS: III-6) cases segregating with intermediate *ATXN2* CAG repeat which originated from unexpanded allele ([Fig. 1](#pone-0070560-g001){ref-type="fig"}). Average age at onset in this family was 54.25±1.26 years and the survival was 25±21.28 months. Ataxia, slow saccades or other symptoms which would indicate co-morbidity of ALS and SCA2 were not found in these patients. Clinical records of two cases showed neither ataxic signs nor cerebellar anomalies excluding SCA2 presenting initially as motor neuron disease. Additional clinical information of these cases is presented in [Table 1](#pone-0070560-t001){ref-type="table"} and described below.
{#pone-0070560-g001}
10.1371/journal.pone.0070560.t001
###### Phenotype and *ATXN2* genotype for ALS case series.
{#pone-0070560-t001-1}
Case Age Clinical status Gender Ataxin-2 Genotype D12S1333 D12S1672 D12S1332
--------- ----- -------------------------------------- ------------- ------------------- ---------- ---------- ----------
I.1/I.2 -- Healthy Female/Male \[22/25\] \[251\] \[289\] \[190\]
NA NA NA
II.2 54 ALS[\*](#nt102){ref-type="table-fn"} Female \[22/≥27\] \[239\] \[289\] \[198\]
NA NA NA
II.7 76 Healthy Female 22/25 245 125 170
251 289 190
II.10 72 Healthy Female 22/25 239 125 170
257 289 190
II.11 67 Tremor Female 22/22 245 125 170
245 289 198
II.12 63 Healthy Female 22/25 231 125 170
251 289 190
III.6 57 ALS Female 22/31 231 129 170
239 289 198
III.9 59 Healthy Female 22/22 227 287 202
245 125 170
III.10 57 Healthy Male 22/25 245 125 170
257 289 190
III.11 50 Healthy Female 22/22 245 125 170
257 283 196
III.13 54 ALS Female 22/35 239 133 172
239 289 202
III.14 38 Healthy Female 22/22 245 125 170
231 289 202
III.16 54 ALS Male 22/32 231 129 170
239 289 198
IV.4 38 Healthy Female 22/22 239 133 172
245 287 196
IV.5 29 Healthy Female 22/22 239 133 172
245 185 202
Affected by history and genotype inferred from daugther.
De novo mutation causing disease. **EERC**: El Escorial Revised Classification.
The proband (III-16), appeared as a sporadic ALS case when assisted by consultants. This patient from pedigree A1 first experienced weakness and pain in the right heel at age of 53 years, progressing to paralysis of the leg, the left leg was also compromised within another month. MRI was indicated and misdiagnosis of lumbar radiculopathy was set. Treatment with non-steroidal anti-inflammatory drugs and Vitamin-B12 did not lead to an improvement. Four months later, upper right limb was started to be affected with wasting and weakness and within a month, it progressed to the left side. In all four extremities, hyperreflexia (+4) and moderate spasticity was present. Marked distal hypotrophy at first dorsal interosseum in both upper and lower limbs was observed with severe deterioration of first interosseum as well as in thenar and hypothenar eminences. Right hand adopted simian-like shape, requiring right-to-left hand change. About 6 months later, atrophy of arms, forearms, deltoid, and pectoralis muscles, accentuated pyramidalism featured by hyperreflexia, bilateral Hoffman and Troemmer signs predominating on the right side and Babinski reflex were evident. Sensation and coordination were normal. Both heels showed non-exhausting and exhausting clonus. Bulbar involvement was confirmed with remarked atrophy of the tongue edges, and tongue and palate fasciculations. Somatosensory evoked potentials of upper and lower limbs were normal. Electromyography (EMG) in muscles in all extremities (m. biceps brachii, gastrocnemius, tibialis, rectus femoris, pectoralis, spinalis) and the tongue showed diffused signs, widespread fasciculations, fibrillations and sharp-waves. At rest, there were spontaneous discharges of motor unit action potentials in interosseal muscles. On contrast, dropout of the number of motor units with increased firing rate was evident. In summary, EMG report was indicative of neurogenic changes with signs of active denervation and renervation, spontaneous fasciculations ([Fig. 2B](#pone-0070560-g002){ref-type="fig"}) and fibrillations in all four limbs and bulbar segments, reflecting acute and progressive denervation of the second motor neuron of the brain stem and the anterior horn of spinal cord and a definite diagnosis of a classical ALS was concluded. Magnetic resonance imaging showed generalized cortical atrophy (not shown). No cerebellar damage was evident on a recovered sagittal MRI image.
{#pone-0070560-g002}
Weakness in all the extremities progressed, and the patient subsequently developed dysarthria and dysphagia. Approximately 7 months later, the patient started to experience gait and later stance abnormalities and became quadriplegic, anarthric and aphagic. Neurological examination showed that patient was alert and oriented until his death. At this clinical stage he showed vasomotor disturbance congruent with Sudeck's atrophy. The entire clinical picture progressed several months until death caused by respiratory failure at age of 55 years. *ATXN2* CAG repeats length expansion was 22 and 32 CAG repeats ([Fig. 2A](#pone-0070560-g002){ref-type="fig"}, lane 14, [Table 1](#pone-0070560-t001){ref-type="table"}) with the longer allele belonging in the intermediate range associated with ALS.
The sister of proband's mother (II-2) lived between years 1933--1991 and started to experience similar symptoms at age of 54 years. The disease progressed to death within 4 years (at age of 58 years). According to the clinical records her first symptom was hoarseness progressing throughout∼15 years with pain (at age of 54 years) in the right heel and weakness in arms and legs. As she deteriorated, the course of her clinical picture occurred without dementia, cognitive or social dysfunction. A diagnosis of lumbar radiculopathy was proposed based on tomography performed in 1989. She subsequently developed dysarthria and dysphagia, was confined to wheelchair, and died due to respiratory failure. Although her genetic material was not available, a mutation near or beyond 31 CAG in *ATXN2* could be suspected, as her daughter's (III-6) genetic status of *ATXN2* is 22/31 CAG. Importantly, III-6 referred that, similarly to her mother, she has been suffering from progressive weakness and pain in lower limbs in last 7 months. Additionally, she complained about frequent cramps in the right arm and leg. At neurological examination, no signs of ataxia signs were found. She had motor neuron signs in all extremities and provoked fasciculations were evident in arms. Muscular strength in limbs was: superior limb was 4/5 distal, 4/5 proximal bilateral while in right inferior limb 4/5, 4/5 proximal bilateral and in left 4/5 distal. This clinical information is in agreement with early symptoms involving bombardment of motor neurons progressing toward typical ALS. In addition, her status highlights the possibility that the same genetic variants in *ATXN2* causing ALS in her mother might be the responsible for her clinical signs indicative for ALS. EMG was not performed in this patient due to her disapproval of this examination. While the definite ALS diagnosis in II-2 cannot be rule out, and was confounded in her time with lumbar radiculopathy, it seem clear that she presented a progressive neurodegenerative disorder involving motor neurons, and for her interviewed relatives this presentation resembled the picture of her first order nephews, our proband and III-13 (see below), both with a motor-neuron disease and where a hereditary pattern can be applied.
The examination of the patient III-13 (cousin of III-16) revealed weakness in the right leg and dysarthria lasting approximately 4 months. Similarly to the previous cases, the symptoms occurred at age of 54 years and led to death within 6 months. For about 3 months, the patient experienced muscle twitching in upper limbs and in m. gastrocnemius, as well as dysarthria, dysphagia and mild tongue atrophy. Neurological examination revealed spontaneous and provoked fasciculations in upper limbs, m. gastrocnemius and spinal muscles. Muscular strength in right upper limbs was 3/5 distal, 4/5 proximal bilateral while in right lower limb 3/5, 4/5 proximal bilateral and in left 4/5 distal. Both spatia interossea displayed hypotrophy with more profound picture on the right side and with marked deterioration in the first interosseum. Hyperreflexia (+3) and exhausting clonus in both heels was also observed. Babinski signs were discrete on the right side. Cutaneous sensibility was normal. On neuropsychological grounds cognitive and social function were preserved. Somatosensorial evoked potentials of upper and lower limbs were normal and MRI only showed generalized cortical atrophy and similarly to III-16 EMG of both upper and lower limb muscles (m. tibialis anterior, vastus medialis, gastrocnemius, first dorsal interosseous and biceps brachii) suggested denervation of second motor neuron compatible with ALS. On muscle contraction, decrease of recruitment pattern was evident, and in addition to motor neuron anomalies shown in III-16, a remarkably clear neurogenic EMG record was obtained in tongue all compatible with definite ALS. Genotype analysis revealed 22/35 CAG repeat size in *ATXN2* ([Table 1](#pone-0070560-t001){ref-type="table"}), which is in the pathogenic range for SCA2. Since the first consultation, the clinical picture worsened progressively until quadriplegia in the last 3 months and the patient's death at age of 55 years due to respiratory failure.
All these cases showed intermediate *ATXN2*≥31 CAG, which are scattered in 80 chromosomes from the general population (frequency 0.025, and see Supplementary Figure S2 in reference 6) but overrepresented only in the SCA2/New Mutation deriving sample [@pone.0070560-LaffitaMesa1]. In that study an isolated allele with 35 CAG was found with no connections to SCA2 founder families [@pone.0070560-LaffitaMesa1], and as far as we know, the carrier has not developed neurological signs. This patient is now 37 years old, 18 years below the average onset age of ALS individuals presented in this study.
Segregation Analysis of Intermediate-length *De Novo* Mutated Alleles {#s2b}
---------------------------------------------------------------------
DNA for genetic analysis was available from sib-ship pairs and revealed that at least three *de novo* mutations occurred. They have been proven in two patients (III-13 and III-16) and in one it was inferred (II-2) ([Fig. 1](#pone-0070560-g001){ref-type="fig"}). Female II-2 was the first person in the family with ALS symptoms and she was the first daughter of I-1 and I-2 deceased at age of 87 and 79 years, respectively, with no neurological disorders referred by their children ([Fig. 1](#pone-0070560-g001){ref-type="fig"}). They had eight other children ([Fig. 1](#pone-0070560-g001){ref-type="fig"}) with following *ATXN2* CAG repeat sizes: 22/25 (II-7), 25/30 (II-10), 22/22 (II-11) and 22/25 (II-12). The genetic material from II-2, II-3, II-4 and II-5 was not available. Neurological symptoms were observed only in one sibling (II-11) while the others appeared healthy despite advanced ages at examination (II-7, 63 years; II-10, 72 years; II-11, 67 years; II-12, 76 years). Siblings II-3, II-4 and II-5 were not examined as they live abroad. According to the family members, they are all healthy. Individual II-11 showed resting and kinetic tremor unrelated with ataxin-2 (normal genotype of 22/22 CAG) ([Fig. 1](#pone-0070560-g001){ref-type="fig"}) and different haplotypes in each allele ([Table 2](#pone-0070560-t002){ref-type="table"}). According to the segregation analysis of this pedigree, the most probable genotype in the parents was 22/25 and 22/30. As an evidence for parental genotype serves the fact that the alleles with 25 and 30 CAG had different STR haplotype in the healthy 72-years old daughter (II-10) who is also the mother of the proband III-16 ([Table 2](#pone-0070560-t002){ref-type="table"}). Moreover, 22/22 CAG and 22/25 CAG combinations were found in the other children (e.g. 22/25 in II-7, 22/22 in II-11, and 22/25 in II-12), suggesting the presence of a fourth allele in parents with 22 CAG repeats (for haplotypes see also [Table 2](#pone-0070560-t002){ref-type="table"} and [Fig. 3A](#pone-0070560-g003){ref-type="fig"}).
{#pone-0070560-g003}
10.1371/journal.pone.0070560.t002
###### Table. 2. Microsatellite haplotypes.
{#pone-0070560-t002-2}
No. *G*enotype Gender (Birth) Clinical status Age at onset/death Survival (mo) Site of onset, EERC
-------- ----------------------------------------- ---------------- ----------------- -------------------- --------------- -----------------------------------------
II-2 22/≥30[\*](#nt104){ref-type="table-fn"} Female (1933) Affected 54/58 48 (deceased) Spinal right (lower limb), ALS possible
III-6 22/31 Female (1955) Early symptoms 56, Alive -- --
III-16 22/32[\*](#nt104){ref-type="table-fn"} Male (1956) Affected 53/55 21 (deceased) Spinal right (lower limb), ALS definite
III-13 22/35[\*](#nt104){ref-type="table-fn"} Female (1957) Affected 54/55 6 (deceased) Spinal right (lower limb), ALS definite
NA: Not available, Inferred haplotype are bracketed,
sick by history.
One of the longer alleles (25 or 30 CAG) would segregate in II-2 as a *de novo* ≥27 CAG ALS mutation which was further transmitted to III-6 (22/31 CAG) and might be responsible for the early signs of ALS (transmissions T1 and T2 in [Fig. 4](#pone-0070560-g004){ref-type="fig"}). The allele with 30 CAG could be suggested as the possible source for *de novo* ALS cases with *ATXN2* CAG expansions. However, segregation analysis for the transmission T4 ([Fig. 4A](#pone-0070560-g004){ref-type="fig"}) conformed by II-7 (22/25 CAG) and III-13 (22/35 CAG) revealed that instead of the 30 CAG allele, the one with 25 CAG in II-7 (age 76 years) underwent a pathogenic expansion by 10 CAG and contributed to ALS onset at age of 54 years. This *de novo* mutated allele in III-13 crossed the threshold for ALS and reached the minimal repeat length for SCA2 (34 CAG) ([Fig. 4A](#pone-0070560-g004){ref-type="fig"}). The origin of these expansions from the 25 CAG allele with one CAA interruption was also confirmed in the unstable transmission T3, sib-ship pair of II-10 (25/30 CAG)/III-16 (22/32 CAG) ([Fig. 3B](#pone-0070560-g003){ref-type="fig"}, [Fig. 4A,B](#pone-0070560-g004){ref-type="fig"}) expanded by 8 repeats and was found associated with classical ALS phenotype described above. The contribution of proband's father to *ATXN2* mutagenesis is not likely as he was a carrier of 22/22 CAG alleles. One of these alleles was identified in proband with 8+4+8 CAG/CAA pattern. Despite the absence of genetic data in mother of III-6, we could identify the increase in CAG length by 6 repeats as compared with CAG in the aunts (II-7 and II-10) ([Fig. 4B](#pone-0070560-g004){ref-type="fig"}). Normal CAG expansions were found in two of her sisters (III-2 and III-7) with22/22 CAG genotype. In summary, the average for intergenerational instability in all described transmissions was 6.25±2.87 gained CAG units in alleles sized 25.5±1 CAG, resulting in *de novo* mutated alleles with ∼32CAG (transmission A in [Fig. 4A](#pone-0070560-g004){ref-type="fig"}) which is a greater CAG repeat than the threshold for ALS risk ≥30 CAG [@pone.0070560-Lee1], [@pone.0070560-Gispert1].
{#pone-0070560-g004}
Haplotype of Ataxin-2 Intermediate-length Polyglutamine Expansions {#s2c}
------------------------------------------------------------------
To clarify the mechanism of *de novo* CAG mutations, we performed SNIPs and STR haplotyping. The analysed markers span ∼550 kb and are lined up on chromosome 12q24.1 as follows: centromere-D12S1332-D12S1672-(CCG)nCCC-CAG-rs695871-rs695872-D12S1332-telomere ([Fig. 3A](#pone-0070560-g003){ref-type="fig"}). The mothers of patients III-13 and III-16 (II-7 and II-10) shared the truncated haplotype 6-4 (289 bp and 257 bp) for markers D12S1672 and D12S1332 embedding the 350 kb region including the SCA2 gene ([Fig. 3A,B](#pone-0070560-g003){ref-type="fig"}). For the D12S1333 telomeric marker at 20 kb, variation of 3 CA/GT dinucleotides was found (allele 4, 257 bp and allele 6, 251 bp), yielding closely related haplotype variants H1 (6-4-4) for II-7 and H′1 (6-4-6) for II-10.
As for the intergenerational transmissions, STR haplotype analysis revealed that all CAG instabilities involving parental haplotypes H1 or H′1 lead to significant changes in offsprings with the resulting haplotype H2: 4-4-10 in III-6, III-16 and H′2: 3-4-10 in III-13 ([Fig. 4A,B](#pone-0070560-g004){ref-type="fig"}). Further DNA haplotyping in III-6 showed that H2 variant was shared with III-16 and II-2.
The haplotypes in generation III are distinct from the original parental haplotypes regarding the microsatellites D12S1333 and D12S1332 but not the intragenic marker D12S1672. The more significant changes of STR were evident in the vicinity of SCA2 locus, for example D12S1333 decreased 9 and 6 CA/GT and D12S1332 increased 6 and 4 CA/GT dinucleotides in III-13, III-6 and III-16, respectively, while no changes were seen in D12S1672 marker ([Figure 4A,B](#pone-0070560-g004){ref-type="fig"}).
As for the intragenic SNIPs and the polymorphic CCG/CCC tract, we found C-C and (CCG)~2~CCC variants for SNIPs rs695871 and rs695872, respectively, as the haplotype co-segregating with the intermediate and the normal (25 CAG) alleles ([Fig. 4B](#pone-0070560-g004){ref-type="fig"}, [Table 2](#pone-0070560-t002){ref-type="table"}).
These results showed that the intragenic markers (SNIPs and D12S1672) are conserved but the flanking markers (D12S1332, D12S1333) are variable in the transmitted alleles, i.e.: 6-C-C-4-Intermediate CAG-(CCG~2~CCC)-6/4 and 3/4-C-C-4-Intermediate CAG-(CCG~2~CCC)-10, respectively.
Mechanism of De Novo Mutation: the Role of CAA Interruption Pattern {#s2d}
-------------------------------------------------------------------
Next, we analyzed CAA interruptions of the CAG tract by DNA sequencing. The interruption patterns were as follows: 16+8 (II-7 and II-10), 23+8 (III-16), 26+8 (III-13), and 8+8+4+8 (III-6) ([Fig. 4B](#pone-0070560-g004){ref-type="fig"}). This analysis suggested sequential lengthening of the 5′ repeat tract in the alleles with the concurrent absence of the most proximal CAA interruption. Lack of this interruption enhances the enlargement of CAG tract, but no instability was found in the (CCG)~2~CCC triplets flanking the CAG repeat ([Fig. 4B](#pone-0070560-g004){ref-type="fig"}).
Ataxin-2 Intermediate Alleles and ALS Risk {#s2e}
------------------------------------------
Genetic overlap between SCA2 and ALS has been reported [@pone.0070560-VanDamme1] without co-segregation of mutations in the most common ALS-related genes (*C9ORF72*, *TDP43*, *FUS*,*UBQLN2*,*ANG*,*OPTN*, *SPG11*, *PLEKHG5*, *VAPB*) in ALS cohorts with *ATXN2* intermediate-length ([Table S1](#pone.0070560.s001){ref-type="supplementary-material"}). We also found no segregation of *C9ORF72* hexanucleotide repeat expansion in our cases. Moreover, *de novo* mutations in our pedigree originated from the 25 CAG allele, which we consider as "large normal" [@pone.0070560-LaffitaMesa1] while other authors as intermediate ALS-risk alleles [@pone.0070560-Elden1].Therefore we investigated whether this allele, which is overrepresented in our population [@pone.0070560-LaffitaMesa1], could be considered as a risk factor for ALS. Meanwhile, we aimed to address the refinement of currently accepted threshold of intermediate *ATXN2* alleles as risk factors for ALS. In the first step of the meta-analysis we used 24 or 27 CAG as threshold for ALS risk ([Table 3](#pone-0070560-t003){ref-type="table"}). The combined risk was heterogeneous among 12 studies with χ^2^ = 29.97, df = 11, P = 0.0016. At this starting point we detected a higher prevalence of intermediate alleles in the ALS cases as compared with controls (OR = 1.23 95% CI 1.09--1.39). This modest value of association is also supported by the appearance of healthy controls with intermediate alleles and low risk for this range in some populations [@pone.0070560-Lahut1]. In the population study in Belgium and Netherlands, the contribution of the 30 CAG alleles to the ALS risk has been shown, although the threshold at 27 CAG was confirmed [@pone.0070560-VanDamme1] ([Table 3](#pone-0070560-t003){ref-type="table"}). To clarify this, we performed meta-analysis with CAG range between 24 and 27 repeats, and the heterogeneity of the risk alleles dropped (χ^2^ = 16.95, df = 11, P = 0.11) (data not shown) with no significant risk of the alleles in this range (OR = 1.02; 95% CI 0.91--1.16). These results point to \>27 triplet repeats length as one of the sources for heterogeneity seen in the first meta-analysis with threshold between 24 and 27 CAG. Furthermore, the data confirmed that the *de novo* mutations reported in this study originated from alleles with low or no risk for ALS with a propensity to cross the pathogenic range in further generations ([Fig. 5A](#pone-0070560-g005){ref-type="fig"}, [Table 3](#pone-0070560-t003){ref-type="table"} and [Fig. 6](#pone-0070560-g006){ref-type="fig"}).
{#pone-0070560-g005}
![General mechanisms for *ATXN2* gene *de novo* mutagenesis in the population.\
Two models can be proposed for explanation of *de novo* CAG expansions in *ATXN2*. Both involve loss of the CAA interruption in large alleles resulting in a minimal length of pure repeat within the CAG expansion. CAA interruptions break the CAG tract in discrete repeat arrays protecting it from instability [@pone.0070560-Ross1]. According to this study, the minimal length of the internal pure repeat leading to *de novo* mutations is 8 CAG.](pone.0070560.g006){#pone-0070560-g006}
10.1371/journal.pone.0070560.t003
###### Meta-analysis for case-controls series for ATXIN-2 polyQ intermediate length and ALS.
{#pone-0070560-t003-3}
Study, Population ALS cases/Controls (ratio) ATXN2 polyQcutoff, P-value[\*](#nt105){ref-type="table-fn"} PolyQ\>24/27OR (95%CI) 24≥PolyQ≤27OR (95%CI) PolyQ≥30OR (95%CI) PolyQ≥32OR (95%CI)
------------------------------------------------------------------------------- ---------------------------- ------------------------------------------------------------- ------------------------ ----------------------- -------------------- ---------------------
Elden et al., 2010, North America [@pone.0070560-Elden1]. 915/980 (0.93) ≥27, 3.6×10^−5^ 1.45 (1.24--1.70) 1.24 (0.99--1.55) 1.91 (1.67--2.19) 2.08 (1.99--2.18)
Lee et al., 2011, Northern Europe [@pone.0070560-Lee1]. 1294/679 (1.91) \>30, 6.2×10^−3^ 1.06 (0.90--1.25) 0.94 (0.75--1.16) 1.53 (1.48--1.58) 1.53 (1.48--1.58)
Van Damme et al., 2011, Belgium and theNetherlands [@pone.0070560-VanDamme1]. 1845/2002 (0.92) ≥32, 3.6×10^−2^ 0.96 (0.81--1.12) 0.91 (0.76--1.08) 1.49 (1.07--2.08) 2.09 (2.02--2.16)
Sorarú et al., 2011, Italy [@pone.0070560-Soraru1]. 247/256 (0.96) ≥24, 2.6×10^−2^ 1.54 (1.19--2.00) 1.47 (1.05--2.08) 1.60 (1.12--2.30) 2.06 (1.88--2.25)
Daoud et al., 2011, French andFrench Canadian [@pone.0070560-Daoud1]. 556/471 (1.18) ≥29, 2.4×10^--4^ 1.17 (0.96--1.42) 1.02 (0.79--1.33) 1.53 (1.22--1.93) 1.71 (1.43--2.04)
Corrado et al., 2011, Italy [@pone.0070560-Corrado1]. 232/395 (0.59) \>30, 8.9×10^−4^ 1.00 (0.61--1.65) 0.42 (0.15--1.19) 2.41 (1.82--3.19) 2.73 (2.46--3.03)
Ross et al., 2011, North America [@pone.0070560-Ross1]. 536/4877 (0.11) \>30, 1×10^−3^ 1.49 (1.07--2.06) 1.19 (0.82--1.74) 4.81 (2.89--8.01) 6.8 (4.25--10.87)
Chen et al., 2011, Chinese [@pone.0070560-Chen1]. 345/350 (0.99) ≥32, 4×10^−2^ 1.33 (0.98--1.81) 1.08 (0.67--1.74) 1.78 (1.35--2.34) --
Gispert et al., 2012, Europe [@pone.0070560-Gispert1]. 559/1369 (0.41) ≥30, 4×10^−3^ 0.93 (0.66--1.32) 0.75 (0.50--1.13) 2.43 (1.61--3.67) 2.77 (1.78--4.32)
Van Langenhove et al., 2012,Flanders-Belgian [@pone.0070560-VanLangenhove1]. 72/810 (0.09) ≥30, 1.2×10^−2^ 2.86 (1.43--5.73) 1.79 (0.70--4.57) 9.54 (5.19--17.55) 12.41 (9.93--15.51)
Gellera et al., 2012, Italy [@pone.0070560-Gellera1]. 658/551 (1.19) \>30, 1.4×10^−3^ 1.23 (1.02--1.50) 0.92 (0.68--1.24) 1.66 (1.38--2.00) 1.85 (1.75--1.95)
Lahut et al., 2012, Turkish [@pone.0070560-Lahut1]. 212/319 (0.66) \>30, 2.6×10^−2^ 1.26 (0.76--2.08) 0.83 (0.37--1.86) 2.53 (2.28--2.82) 2.53 (2.27--2.81)
As reported by authors in the original data.
When the meta-analysis was performed using repeat length threshold of ≥30 CAG, the ALS risk was estimated more accurately ([Fig. 5B](#pone-0070560-g005){ref-type="fig"}). We detected specific risks estimates in all populations ([Table 3](#pone-0070560-t003){ref-type="table"}) and a higher global risk (OR = 2.16; 95% CI 1.76--2.65, χ^2^ = 187.88, P = 0.000) representing a two-fold excess of intermediate alleles enriching ALS cases versus healthy control population. Excluding the most precise studies [@pone.0070560-Elden1], [@pone.0070560-Lee1], [@pone.0070560-Lahut1] ([Figure 5B](#pone-0070560-g005){ref-type="fig"}) and those with the most extreme odds ratios [@pone.0070560-VanLangenhove1], [@pone.0070560-Ross1] ([Table 3](#pone-0070560-t003){ref-type="table"}) resulted in homogenous relative risk \>1 (P = 0.12), which confidently input positive combined risk of 1.77; 95% CI 1.55--2.03 for intermediate allele in ALS patients versus controls. Although the risk for ALS was associated with the ≥30C AG alleles, it was higher in carriers of ≥32 CAG repeats ([Fig. 5C,F](#pone-0070560-g005){ref-type="fig"}) in all populations except Chinese [@pone.0070560-Chen1] with no alleles with ≥32 CAG. The combined risk was "significantly heterogeneous" (2.62; 95% CI 2.23--3.09, χ^2^ = 633.97, df = 10, P = 0.0000) and eight cases-control series showed odds ratio greater than 2 ([Table 3](#pone-0070560-t003){ref-type="table"}). In three populations, including USA [@pone.0070560-Lee1]; French Canadian [@pone.0070560-Daoud1] and European [@pone.0070560-Gispert1] controls with \>32 CAG were found. After independent exclusions of more precise risk estimates ([Fig. 5C](#pone-0070560-g005){ref-type="fig"}), such as Van Damme et al., 2011 (OR 2.70; 95% CI 2.20--3.29) [@pone.0070560-VanDamme1]; Lee et al., 2011(OR 2.73; 95% CI 2.33--3.20) [@pone.0070560-Lee1]; Elden et al., 2010 (OR 2.68; 95% CI 2.22--3.25) [@pone.0070560-Elden1]; and Gellera et al., 2012 (OR 2.71; 95% CI 2.26--3.27) [@pone.0070560-Gellera1] the direction and magnitude of risk effect was no longer modified. Similar approach with exclusion of the most influential case-control series (the above data and Ross et al., 2011 [@pone.0070560-Ross1], Van Langenhove et al., 2012 [@pone.0070560-VanLangenhove1]) resulted in significantly heterogeneous risk estimates (combined OR = 2.34; 95% CI 1.94--2.83, χ^2^ = 40.04, df = 4, P = 0.0000) for CAG ≥32 repeats associated with ALS risk across populations. Overall our meta-analysis confirmed that the more accurate estimates in all populations are achieved when alleles with ≥30 CAG are used as cut-off for intermediates and the effect is more robust when CAG tract expansion is close to the upper limit of the *ATXN2*intermediate length. Moreover, it supported our findings on the pathological nature of *de novo* mutations found in the family reported here.
Discussion {#s3}
==========
In SCA2, similarly to other triplet repeat disorders, the *de novo* mutations are thought to result from enlargement of the CAG repeat tract during father-to-child transmissions of pre-expanded alleles (23--30 CAG) to a pathogenic range of ≥34 CAG. Here we report three ALS cases coinciding with ≥30 CAG *ATXN2* alleles. For the first time, expansion of the normal repeat tract of ≤25 CAG, were shown to expand to a CAG repeat range causing or contributing to disease. *De novo* mutations in polyglutamine (polyQ)-related genes causing diseases have been reported in few SCA7 [@pone.0070560-Stevanin1]--[@pone.0070560-Mittal1], one SCA6 [@pone.0070560-Shizuka1] and a small number of Huntington's Disease cases [@pone.0070560-Almqvist1]. Regarding the *ATXN2* gene, there is no consistent evidence of novel mutations arising from non-pathogenic alleles (13--31 CAG). Schöls et al., [@pone.0070560-Schls1] interpreted as *de novo* mutation a father-to-daugther transmission with 34 CAG (currently recognized as pathogenic) expansion to 41 CAG in daugther presenting as a sporadic SCA2 case. The father died from cancer at age of 65 years without exhibiting ataxia. Alonso et al., [@pone.0070560-Alonso1] reported on a Mexican sib-ship pair where the parents (both with 22/22 CAG) appeared to transmit an intermediate-length allele with 33 CAG to a child with early SCA2 onset. Unfortunately, in the previous studies, neither STR nor SNPs haplotypes were shown preventing a clear conclusion on *de novo* mutations. Van Damme et al. [@pone.0070560-VanDamme1] suggested in a cases-control study a possible *de novo* origin for expansions of ≥32 CAG linked with sporadic ALS in one family, but no clear conclusion was made as DNA samples from siblings and parents were not available. In this study, we present three confirmed *de novo* mutations associated with ALS (transmissions T1, T3 and T4) in a single family. ALS in this family appeared first as sporadic but presented as familial following Mendelian pattern of inheritance. The familial form of ALS connected with intermediate *ATXN2* alleles has further been supported in a French-Canadian cohort showing a stronger association of alleles with more than 29 CAG in *ATXN2* with familial rather than sporadic ALS [@pone.0070560-Daoud1]. In our family, autosomal dominant inheritance pattern may be applied for the occurrence of ALS with *de novo ATXN2* expansions. The lacking of genetic heterogeneity with other prominent ALS genes ([Table S1](#pone.0070560.s001){ref-type="supplementary-material"}), the concomitant segregation of the intermediate and full CAG repeat expansion in our proband (III-13) and III-16 respectively (both individuals affected), and its absence in 80 chromosomes of the Cuban population satisfied the gold standard criteria pointing to a causative role. In two pedigrees, in which *SOD1*, *TARDBP*, *FUS*, and *ANG* mutations had been excluded, a co-segregation of the ALS phenotype with expanded *ATNX2* alleles has been reported [@pone.0070560-VanDamme1]. The CAG expansions were either in the SCA2-parkinsonism or classic cerebellar SCA2, which, together with the reported genetic overlap [@pone.0070560-VanDamme1] and without clinical heterogeneity or ALS phenotype modification [@pone.0070560-Corrado1], [@pone.0070560-VanDamme1], [@pone.0070560-Lahut1], [@pone.0070560-Daoud1], [@pone.0070560-Soraru1] support our hypothesis of CAG expansion in *ATXN2* being monogenic ALS cause (at least for longer intermediate CAG expansions). In addition, *de novo ATXN2* CAG expansions may also contribute to apparent sporadic cases as reported by Elden et al [@pone.0070560-Elden1]. This repeat behavior is supported by the meiotic and mitotic instability associated with large alleles [@pone.0070560-LaffitaMesa1]. All these observations may serve as a starting point for intergenerational risk estimations for CAG repeat instabilities in the large non-SCA2 expansions in *ATXN2* with ≥25 CAG.
Intermediate-length CAG expansions in *ATXN2* can lead to atypical SCA2 phenotype [@pone.0070560-Futamura1]--[@pone.0070560-CostanziPorrini1], however their association with ALS phenotype featured by shorter survival and onset at ∼54years of age helps to avoid misdiagnosis of ALS as SCA2 with motor neuron disease [@pone.0070560-Fischbeck1]. In our SCA2 population, ataxia combined with motor neuron phenotype has been noted [@pone.0070560-VelzquezPrez1], but such cases segregated within SCA2 pedigrees with cerebellar symptoms and a long disease duration excluding ALS. The intermediate *ATXN2* alleles associated either with familial or sporadic ALS arose as *de novo* CAG expansions unrelated to SCA2 families in our population.
Interestingly, one of the *de novo* mutations contributing to ALS crossed the pathogenic SCA2 threshold (35 CAG; T4 transmission) suggesting a similar mutagenic mechanism for both diseases ([Fig. 6](#pone-0070560-g006){ref-type="fig"}). The 35 CAG repeat expanded from a large normal allele which are overrepresented in Cuba [@pone.0070560-LaffitaMesa1] confirming their role as a source for new expansions.
The allele with 25 CAG repeat with only one CAA interruption in II-7 was transmitted as the 35 CAG expansion to III-13 ([Fig. 4A,B](#pone-0070560-g004){ref-type="fig"}). Similar allele would be the disease-causing mutation in II-2 and it is plausible to assume that in the preceding generation (grandparents and parents healthy), sequential loss of CAA in the 25 CAG allele resulted in CAG enlargements leading to ALS phenotype in successive generations (bottom model in [Fig. 6](#pone-0070560-g006){ref-type="fig"}).
Retrospective studies made in mortality archives between years 2001 and 2006 in Cuba showed low frequency of ALS, but Holguín province was third in the rating of death by ALS [@pone.0070560-Zaldivar1]. An introduction of a predisposed founder chromosome in this region would be proposed to contribute considerably to the ALS incidence in this region.
While the CAG sequence in SCA2 is pure, the ALS-related expansions are interrupted by CAA [@pone.0070560-Corrado1], [@pone.0070560-Choudhry1] suggesting different origins. Two internal CAG repeat structure either with single or three CAA interruptions in sporadic ALS case series was interpreted as multiple mechanisms for *ATXN2* repeat expansions [@pone.0070560-Ross1]. We observed a trend of pure CAG tracts gaining and removal of CAA interruptions in successive generations as shown in case II-10 (CAG 23+8) versus III-6 (CAG 8+8+4+8), respectively, indicating that both expansions share the same ancestor as previously proposed [@pone.0070560-Ramos1], and that mutational mechanism driving the removal or gaining CAA interruptions might occur concurrently.
Using microsatellites haplotype analysis, we found that the *de novo* mutations originate from the same ancestors although the extragenic microsatellites underwent some changes. On the other hand, we found SCA2 families in which the haplotype remained unchanged across generations [@pone.0070560-LaffitaMesa1], [@pone.0070560-Gispert2], [@pone.0070560-Hernandez1]. This suggests that there may be regions in the vicinity of *ATXN2* which can interact genetically and epigenetically [@pone.0070560-LaffitaMesa2] and delineate and/or contribute further to ALS phenotype. This hypothesis is in accordance with the study Lahut et al. [@pone.0070560-Lahut1].
Two intragenic SNPs were used for haplotype analysis: rs695871 (G/C) and rs695872 (T/C) in the first exon of *ATXN2* gene situated 177 and 106 bp, respectively downstream from the CAG tract. The G→C substitution results in Val to Leu change while the T to C polymorphism is silent (Arg residue).The haplotype variants are CC or GT [@pone.0070560-Ross1]. The CC haplotype was present in the 25 CAG allele, which expanded to intermediate CAG ([Fig. 3B](#pone-0070560-g003){ref-type="fig"}).This haplotype was confirmed also by the identification of the (CCG)~2~CCC sequence which is useful for haplotype discrimination [@pone.0070560-Mizushima1] and evolutionarily conserved in primates [@pone.0070560-Choudhry1]. The CC haplotype was connected with uninterrupted CAG tract and associated with intergenerational instability and ALS disease development in our three patients. This haplotype was reported in Indian population as enriched inSCA2 founders and in a subset of predisposed normal repeats, while the GT variant was found in the healthy population reducing the repeat sequence instabilities due to the presence of several CAA interruptions [@pone.0070560-Choudhry1].Taken together, these data suggest that the presence of the CC haplotype, loss of CAA interruptions and the repeat size are prerequisite for CAG tract instability while the GT variant appears to stabilize the repeat even in large normal and intermediate CAG expansions and to prevent the SCA2-related mutagenesis. Surprisingly, however, the ALS cases carrying GT variant with three CAA interruptions developed the disease earlier than those with CC variant and less interruptions. Moreover, the ALS patients with three interruptions had shorter CAG than patients with fewer CAA interruptions [@pone.0070560-Yu1].These observations suggest that besides the CAG repeat size, the discrete changes in *ATXN2* gene sequence probably affecting ataxin-2's physiological function may be a risk factors for ALS development and 'genetic hits' facilitating the disease.
The effect of (CCG)~2~CCC (encoding poly-proline tail varying from 1--4) on CAG instability and possibly ALS phenotype should be also considered as it may contribute to both to the CAG (in)stability increasing the number of continuous triplets folding as hairpin, and to the content of prolines residues which lowers the complexity of unstructured sequence regions in the N-terminal tail of ataxin-2 [@pone.0070560-Albrecht1].
Our meta-analysis further confirms the observation that the increased risk for ALS is specifically associated with long intermediate *ATXN2* repeats of ≥30 CAG and is greater with larger expansions. The heterogeneity observed in the combined risk is explained by longer intermediate alleles as the main factor. Pending other factors such as sample selection, gender ratio, SNPs haplotype or CAG calling approaches must be warranted and homogenized in further studies. Regarding this last technical point, the heterogeneity may reside in the range where authors don't use sequencing methods, but for ≥27 CAG, none heterogeneity is expected, and great part is driven for the risk associated with CAG size variation only.
High number of intermediate alleles with 27--31 CAG not associated with any phenotype [@pone.0070560-LaffitaMesa1] complicates the interpretation of the "ALS threshold". Only two out of 25 cases carrying intermediate CAG (32 and 33 CAG) had mild SCA2 phenotype and all intermediate alleles segregated within SCA2 pedigrees [@pone.0070560-LaffitaMesa1]. However, in a single family here, we showed three *de novo* mutated alleles penetrating with ALS disease. The question is what factors differentiate intermediate-length alleles segregating in SCA2 families from those associated with ALS? Our study point to a possible cis-acting factor, as STR haplotype changes were identified in all the *de novo* mutations.
In several back-to-back reports, *ATXN2* intermediate alleles associated with ALS have been defined differently across the populations, e.g. in the USA ≥27 CAG [@pone.0070560-Elden1], in Europe ≥30 CAG [@pone.0070560-Lee1], in Belgium and Netherland ≥32 CAG [@pone.0070560-VanDamme1], suggesting a range of at-risk alleles with 27--30 CAG and alleles causing the disease with ≥31 CAG. This view is corroborated by recent studies showing escalating effect for alleles sized with 27Q, 29Q, 31Q and 32Q in the accumulation of phosphorylated and truncated TDP-43 in response to cell stress [@pone.0070560-Hart1], [@pone.0070560-Hart2]. Both post-translational modifications of TDP-43 are hallmark features of ALS pathology [@pone.0070560-Neumann1].
In conclusion, we identified de novo CAG expansions in *ATXN2* causing ALS. The mutational mechanism involved the loss of CAA anchors in large normal alleles on a predisposed genetic background, leading to a subsequent CAG instability. Intermediate ATXN2-ALS alleles segregated as *de novo* mutations in families in the general population, highlighting the necessity for providing *ATXN2* genetic testing in ALS patients.
Methods {#s4}
=======
Patient's Resources and Clinical Characterization {#s4a}
-------------------------------------------------
First diagnosis of all patients was performed in the neuromuscular consultation at Clinical and Surgical Hospital Lucía Íñiquez Landín, Holguín, Cuba. Genetic status of the patients was not known at the time of first consultation. These individuals were enrolled in the research since they either met the revised El Escorial revisited criteria [@pone.0070560-Brooks1] for definite ALS or were related to ALS patients.
This study included a cohort of 17 individuals, four with familial ALS and 13 relatives. All studied subjects signed an informed consent form after being explained the purpose and methods of the study. All studies were approved by the review boards and ethics bureau of Center for Research and Rehabilitation of Hereditary in Holguin. Specifically, genealogical data were obtained for each family member and genetic DNA information was generated in regard to CAG expansions in the *ATXN2* gene aimed at determination of the founder allele.
The DNA testing of *ATXN2* was performed at the Department of Molecular Neurobiology of the National Center for the Research and Rehabilitation of the Hereditary Ataxias (CIRAH), Holguín. Since 1993, specialized database is available in this center enabling to confirm that there were no relationships between ALS cases and the 124 SCA2 families in the Cuban population. The genealogy database includes information from 124 multigenerational SCA2 families with more than 10.000 members across 15 generations, with names, demographic, genetic and clinical information.
Determination of *ATXN2* CAG Repeat Size and SNIPs Haplotype {#s4b}
------------------------------------------------------------
Peripheral blood leukocytes were extracted using EDTA as anticoagulant and genomic DNA was isolated using standard methods after informed consent was signed by patients or their guardians. In addition to SCA2, a complete set of polyQ diseases-related genes (SCA1, 3, 6, 7, 8, 17, HD) were analysed, with no remarkable findings. CAG repeat length in *ATXN2* was determined using Alfexpress II sequencing system and CAG fragments were separated with ReproGel™ high resolution system (GE Healthcare, Buckinghamshire, UK). PCR was performed as previously reported [@pone.0070560-LaffitaMesa1]. For detecting single nucleotide polymorphisms (SNPs), DNA was amplified with SCA2-FP3 and SCA2-RP3 primers [@pone.0070560-Ross1] and using PCR conditions as previously reported in Ramos et al., 2010 [@pone.0070560-Ramos1], using 1 unit of TopTaq polymerase per reaction (Qiagen, Mainz Germany). For Short Tandem Repeat (STR) sequencing, oligonucleotides used for fragment analysis were also used for sequence reactions. Gel extraction was performed using GFX™ pCR DNA and Gel Band Purification kit (GE Healthcare, Buckinghamshire, UK) and the sequencing reactions were developed on Beckman Coulter CEQ8000 automated sequencing equipment with GenomeLab 10.2 software (Beckman Coulter, Inc. Belgium) at IPK. For sequencing, samples were blinded for the IPK investigators. Reactions were also done in both sense using FP3-RP3, and SCA2A primers, resulting contigs were assembled and compared with the Reference Sequence NG_011572.1. ALS patients were not genotyped for SOD1 and FUS mutations, but we exclude the contribution of the pathogenic GGGGCC expansions of C9ORF72 by using both repeat-primed PCR assays [@pone.0070560-DeJesusHernandez1], [@pone.0070560-Renton1].
Short Tandem Repeat Haplotyping {#s4c}
-------------------------------
Three chromosome 12 microsatellite CA repeat markers were used to establish the haplotypes in this study. Markers D12S1333 and D12S1332, with approximate 200 kb telomeric and 350 kb centromeric location, respectively from the gene, and the third marker, Dl2S1672, is located in the first intron of the *ATXN2* gene. PCR reaction was followed by Gene Scan analysis for STR allele identification. Reactions were performed in 13 µl containing ∼ 10 mM Tris-HCl (pH 8), 1.5 mM MgCl~2~, 50 mM KCl, 10% DMSO, 250 mM dNTPs, 100 ng of each of the primers, 1.5 U Taq-polymerase, and ∼100 ng genomic DNA. The following primer pairs and annealing temperatures were used: Dl2S1332a-b Cy5-GCC AGG TAC AGT GGC TC/CTG GGA CCA CAG GTG TAG at 60°C, D12S l333a-b Cy5-TTC AGG TGG TAC AGC CGT/CAT CAG AAG GCT TCA TAG GAA T at 50°C and D12Sl672a-b Cy5-CAG AGG GAG ATT CCA TCC AA/CGG TTTGAC AAG TTTCGA GA at 50°C. STR lengths were determined by Alfexpress II sequencing system and the PCR fragments were separated with ReproGel high resolution system. Internal (100 and 300 bp) and external (50--500-step, 50 bp) Alfexpress ladders were used to determine the fragment size. Traces were analyzed using the software Allelelink according to the manufacturer's specifications.
Meta-analysis {#s4d}
-------------
Meta-analysis was performed using odds ratio as measure of overall ALS risk, and the data were retrieved from the literature. Data were tabulated and processed in the program EPIDAT 3.1 of the Pan-American Health Organization (PAHO). We searched the literature investigating the *ATXN2* intermediate alleles as risk factors for ALS and performed a meta-analysis when applicable of case--control association ([Table 3](#pone-0070560-t003){ref-type="table"}). Potentially useful studies were identified in review articles and through PubMed and Scopus, using the key words: ALS, ataxin-2 intermediate alleles and polyQ. The content of studies was also assessed manually to avoid redundant or unrelated data. We considered all studies published between 2010 until 2012. We avoided double counting using only the largest available published dataset from any study described in more than one published article. Given the heterogeneity in the nomenclature and thresholds of intermediate alleles we first considered threshold for intermediate alleles as originally authors reported in each work (i.e. 24, 27 and ≥30) (see also [Table 3](#pone-0070560-t003){ref-type="table"}).
Thresholds for 24, 27, 30, 32 CAG repeats used here as starting point in our meta-analysis, were in accordance to the current literature reporting, where these thresholds were previously tested either as statistical significant or functional in each study (for details about rationale for thresholds see [Text S1](#pone.0070560.s002){ref-type="supplementary-material"}). For discriminating specific thresholds we applied component analysis, first analyzing the risks beginning with ≥24/27 CAG, ≥30 CAG, then we eliminated the contribution of the last alleles and the last cut-off was ≥32 CAG. The Dersimonian and Laird's test was used to estimate the heterogeneity between relative risks in each metanalysis. When significant heterogeneity was absent, odds ratio and 95% confidence intervals (CIs) were calculated using a fixed-effects model while when significant heterogeneity was present, a random-effects model was used. Publication bias was investigated using funnels plots and both Begg's and Egger's tests. The significance level was set to P = 0.05.
We included 7, 471 ALS cases and 13,059 controls (ratio ∼1.75 control/cases) harboring 12 studies and including samples from North America, Europe, French-Canadian population, China, Italy, Belgium (Flanders), Germany and Turkey, published between 2010 and 2012 ([Table 3](#pone-0070560-t003){ref-type="table"}). The studies were identified by the search strategy described above, met the inclusion criteria, and contributed to the meta-analysis ([Table 3](#pone-0070560-t003){ref-type="table"}). Reports related with cases-controls studies in intermediate-length *ATXN2* CAG expansions and ALS in other languages than English were not found. There was no overlap in reporting ALS cases from the same country as authors did not specify whether samples were the same, which is not possible regarding that are different referral centers representing different regions. Lattante et al., 2012 [@pone.0070560-Lattante1] reported cases of sporadic ALS with *ATXN2* CAG expansions, but this study had no cases-controls design, therefore it was not included in our meta-analysis. Other two North-American studies [@pone.0070560-Yu1], [@pone.0070560-Lee2] were not included because were considered overlapping with Elden et al., 2010 [@pone.0070560-Elden1]. During the revision of the article two additional studies has been published recently, two original from Italy and China, but were not included and did not modify our metanalysis [@pone.0070560-Liu1], [@pone.0070560-Conforti1]. In the Chinese study, the repeat in *ATXN2* ranged between 30--35 CAG [@pone.0070560-Liu1].
Supporting Information {#s5}
======================
######
Major Mutations in ATXN2-ALS case series.
(XLSX)
######
Click here for additional data file.
######
Rational for polyQ thresholds used in Meta-Analysis.
(DOC)
######
Click here for additional data file.
######
Abstract in Japanese.
(DOCX)
######
Click here for additional data file.
We thank the reviewers for their thorough reading of the manuscript and their useful comments. We are indebted to the family A1 involved in this study for all their cooperation. Support of Dr. Ocilia Rodriguez is acknowledged. We would like to thank Dr. Rosa Rademaker, Patricia Brown and Mariely DeJesus-Hernandez from Mayo Clinic for providing the DNA positive control for *C9ORF72* expansion and primers for C9FTD/ALS hex-repeat expansion detection and for the technical assistance Acknowledgement belongs also to our colleagues from Neurobiology Department at CIRAH.
[^1]: **Competing Interests:**The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: JMLM. Performed the experiments: JMLM JMRP RMS YVM VK LLS JAVF MMG RRL. Analyzed the data: JMLM JMRP RMS YVM VK LLS JAVF MMG YGZ RRL. Contributed reagents/materials/analysis tools: JMLM PS LVP. Wrote the paper: JMLM POB MP PS LVP.
|
{
"pile_set_name": "PubMed Central"
}
|
1. Introduction {#s0005}
===============
The demand for diesel fuel continues to increase ([@bib6]). In response to these demands, and amid concerns over the environmental impact of fossil fuels, intensive research efforts have been made in developing renewable and sustainable methods for the production of diesel substitutes ([@bib26], [@bib28]). To date, biodiesel is the most extensively researched diesel fuel replacement ([@bib3]). Its synthesis traditionally involves chemical or mechanical extraction of oils from plant or algal sources followed by trans-esterification to yield either fatty acid methyl esters or ethyl esters. Innovations in microbial engineering have led to bioprocesses which obviate the need for a separate esterification step and potentially allow multiple industrial waste streams to be utilised ([@bib19], [@bib28]). The major downside to biodiesel or even precursor fuels such as the recently reported bisabolane ([@bib26]) is that they require energy intensive extraction procedures (*e.g.* usage of organic solvents for end-product isolation, high centrifugal forces for biomass recovery, physical methods for disruption of biomass) and/or further chemical modifications, and this can present a major economic barrier for the purpose of fuel commercialisation ([@bib7]).
Alcohols and alkanes are both highly attractive biofuel candidates as they do not require further chemical modification. In diesel engines, one particularly notorious issue with the use of alkanes is the formation of particulate matter. These carbonaceous particles, also known as soot, are attributed to incomplete combustion and have been shown in several studies to exacerbate respiratory illnesses and contribute to global warming ([@bib5], [@bib9]). In this regard, alcohols have generated a considerable amount of interest since their increased oxygenated content can significantly stimulate the completion of combustion and thereby lower the production of particulate matter.
Herein, we evaluate the fuel and physicochemical characteristics of saturated fatty alcohols and conclude that the C8 fatty alcohol, 1-octanol, is a highly attractive biofuel with diesel-like properties. Previously, 1-octanol had been synthesised by (i) the reversal of beta-oxidation ([@bib11]), (ii) rerouting branched-chain amino acid biosynthesis ([@bib23]) and (iii) extending the 1-butanol pathway ([@bib22]). In this study, we engineer a novel metabolic route for the production of 1-octanol in *Escherichia coli* and furthermore show that it can be naturally excreted into the media.
2. Materials and methods {#s0010}
========================
2.1. Strains and plasmids {#s0015}
-------------------------
*E. coli* BL21(DE3) was purchased from Novagen. The *E. coli* BL21(DE3)Δ*acrA* strain was constructed by P1 phage transduction, described previously (Datsenko and Wanner), using the BW25113Δ*acrA* strain (The Coli Genetic Stock Center, Yale, USA) as the donor strain. These two strains were engineered using genes encoding for *Mycobacterium marinum* carboxylic acid reductase (CAR), Sfp from *Bacillus subtilis*, *tes3* (thioesterase from *Anaerococcus tetradius* with accession no. EEI82564) and Ahr (an aldehyde reductase previously known as *yjgB*) from *E. coli*. The construct cloning approach was described previously ([@bib2], [@bib18]). The plasmids pET-TPC3 (encoding Tes3, Sfp and CAR (TPC3, [@bib2], [@bib18]), assembled as a synthetic operon ([@bib1]), and pCDF-Ahr~his~ (encoding Ahr with an N-terminal 6xHis-tag) were transformed into chemically competent *E. coli* BL21 (DE3) to generate the TPC3 Ahr strain.
2.2. *In vivo* production of fatty alcohols {#s0020}
-------------------------------------------
Strains were cultivated based on a slight modification of the method described previously ([@bib2]). LB, containing a 2% (v/v) overnight, LB-based inoculum (30 °C/150 rpm/\~18 h), was incubated at 30 °C/150 rpm until it had reached an OD600 of 0.5--0.7. Cells were harvested (17, 000*g*/1 min), washed twice with modified minimal medium ([@bib2]) prior to resuspension in the same medium. After addition of isopropyl β-[d]{.smallcaps}-1-thiogalactopyranoside (IPTG) at a final concentration of 20 μM, cells were incubated at 30 °C for 14 h. The cellular density was determined from absorbance measurements (attributed to light scattering) at 600 nm (OD~600 nm~) and glucose levels were quantified at 340 nm, based on the reduction of NAD^+^ by glucose-6-phosphate dehydrogenase. Yields were determined by expressing the total production of fatty alcohol (described in detail below) per gram of consumed glucose.
2.3. Fatty alcohol quantification {#s0025}
---------------------------------
For total fatty alcohol quantification, a 100 μl cell culture was mixed with 200 μl acetone (containing 0.2 mM 1-heptanol as the internal standard). For cellular and extracellular quantification of fatty alcohols, cell cultures were first separated into cellular and media fractions by centrifugation. The cellular fraction was resuspended in 100 μl of water prior to mixing with 200 μl acetone, while the media fraction was directly mixed with 200 μl acetone. All samples were centrifuged and the supernatant transferred to GC vials. Metabolite analysis was performed with an Agilent 7890A gas chromatograph equipped with a 5975 mass spectrometry detector, as described previously ([@bib2]). For 1-octanol and 1-hexanol identification, fragmentation patterns and retention times of the analytes were compared with the NIST mass spectral library and commercially available fatty alcohol standards. A standard curve for quantification was prepared with commercially sourced fatty alcohols. Data was normalised with respect to the internal standard and optical density at 600 nm. All given values are an average mean of measurements obtained from at least 6 independent cultures with error bars representing standard error.
3. Results {#s0030}
==========
3.1. A comparison of the fuel characteristics between fossil-derived diesel and saturated alcohols {#s0035}
--------------------------------------------------------------------------------------------------
Having recently demonstrated the *in vitro* production of a broad range of fatty alcohols based on the activities of CAR and AHR enzymes ([@bib2]), we surveyed the literature and compared the fuel characteristics of fossil-derived diesel and saturated fatty alcohols ranging in chain length from 2 to 12 carbons. Several fuel parameters relevant to diesel engines were considered, such as cetane number, viscosity and lubricity ([Table 1](#t0005){ref-type="table"}). Short-chain alcohols such as ethanol and butanol were found to have poor lubricity and high combustibility which would increase engine wear and stress. On the other hand, longer alcohols (\>10 carbon atoms long) exhibited high freezing points and extremely low vapour pressures which would render them ineffective in cold weather conditions. In between these two extremes, 1-octanol displayed an energy content, lubricity and viscosity similar to that of petroleum-derived diesel. We concluded that 1-octanol could potentially serve as a suitable fuel target.
3.2. Synthesis of 1-octanol {#s0040}
---------------------------
Given its fuel characteristics, we therefore set out to engineer a pathway for the production of 1-octanol. The previously engineered pathway, which resulted in the synthesis of longer chain fatty alcohols (C12-C16), consisted of *E. coli* acyl-ACP thioesterase TesA, *M. marinum* carboxylic acid reductase (CAR), *B. subtilis* phosphopantetheinyl transferase, (Sfp) and *E. coli* Ahr ([@bib2]). Since the chain length of the fatty alcohol was determined by the substrate specificity of TesA, this enzyme was therefore replaced with one that had previously been shown to generate the 8-carbon fatty acid precursor, octanoic acid ([@bib17]) ([Fig. 1](#f0005){ref-type="fig"}a). Induction of the pathway in the presence of IPTG for 14 hours led to the synthesis of 29(±4) 1-hexanol mg L^−1^ and 62(±3) 1-octanol mg L^−1^ with an average yield of 12(±0.4) mg of 1-octanol g^−1^ glucose and a productivity of 4.4 mg (±0.04) mg 1-octanol L^−1 ^h^−1^ ([Fig. 1](#f0005){ref-type="fig"}b; [Table 2](#t0010){ref-type="table"}). No other new metabolites were observed by gas chromatography--mass spectrometry (GC--MS) analysis.
3.3. Excretion of 1-octanol {#s0045}
---------------------------
We noted that during the early to late stationary phases of cell cultivation with TPC3-Ahr, a strong perfume-like fragrance emanated from the cell cultures which suggested the extracellular presence of product(s). After separation of the cells from the media, GC--MS analysis confirmed that 73±3% of the total 1-octanol in the culture was present in the media while the remainder was found to be localised within the cellular fraction ([Fig. 1](#f0005){ref-type="fig"}c). *E. coli* harbours a large range of efflux pumps ([@bib27]). Among them, the AcrAB--TolC complex is the most well-studied and has been implicated in tolerance to exogenously added solvents ([@bib30], [@bib34]). We constructed an Δ*acrA* deletion strain of *E. coli* BL21(DE3) in order to test whether AcrAB--TolC was responsible for the efflux of internally produced 1-octanol. The mutant strain displayed a \~3-fold decrease in excreted 1-octanol along with a similar drop in total 1-octanol production levels ([Fig. 1](#f0005){ref-type="fig"}c). The ratio of external to internal 1-octanol, however, was slightly lower for the Δ*acrA* deletion strain (2.0) compared to the wild-type strain (2.8). The marked reduction in both excretion and production suggested that the AcrAB--TolC complex is most likely responsible for the majority of 1-octanol efflux.
4. Discussion {#s0050}
=============
On account of its diesel-like characteristics, 1-octanol is an attractive biofuel target. In comparison to petrodiesel, its lower vapour pressure could reduce transportation and storage hazards, while its higher auto-ignition temperature could raise the air:fuel compression ratios and in turn improve fuel combustion efficiency. Real simulation tests with compression ignition engines have shown that 1-octanol can reduce particulate matter by as much as 20-fold in comparison to petrodiesel ([@bib16]). In the case of alternative fuels such as biodiesel, inefficient combustion can lead to the formation of hygroscopic by-products such as fatty acids, mono- and di-glycerides which can promote engine corrosion ([@bib3]). Furthermore, the cold flow properties of biodiesel are inferior to 1-octanol, (*e.g.* pour point; −9 °C *vs* −13.5 °C) making the latter a potentially better fuel to handle and operate under cold conditions, at least as a blending component.
In this study, we engineered a pathway that utilised precursors from the natural process of fatty acid synthesis. The octanoic acid precursor was generated by incorporating a fatty acyl-ACP thioesterase which was previously shown to have a preference for fatty acyl-ACP chains of 6--8 carbon atoms. Although the yield (62 mg L^−1^) was found to be \~2-fold lower than the [@bib11] study, the CAR-based platform required a considerably shorter cultivation period (14 h) for optimal production of 1-octanol ([Table 2](#t0010){ref-type="table"}). Thus, the productivity (4.4 mg1-octanol L^−1 ^h^−1^) exceeded previously obtained values of 2.1, 1.5 and 0.04 mg 1-octanol L^−1 ^h^−1^ ([@bib11], [@bib22], [@bib23]; [Table 2](#t0010){ref-type="table"}) without resorting to host engineering or advanced bioprocessing techniques. From a stoichiometric viewpoint, since 2 moles of glucose (MW=180) would be required for the synthesis of 1 mole of 1-octanol (MW=130), the maximum theoretical yield possible is 361 mg 1-octanol per gram glucose. Clearly then, the low yields obtained in this and previous studies indicate that there is still considerable room for metabolic improvements. We expect that optimisation of the process *via* fatty acid engineering ([@bib29]), modulated levels of efflux pumps ([@bib32]), regulated metabolic flux ([@bib10]) and *in situ* product removal techniques ([@bib4]) is likely to improve 1-octanol productivity, yields and titres even further.
A major advantage of biologically producing 1-octanol as an end-product lies in its propensity for extracellular localisation. This trait is advantageous for downstream purification of the end-product. In contrast, fuels such as biodiesel become confined to the internal cellular compartments due to their rather large structure and intracellular role as a carbon storage medium ([@bib19]). Energy-intensive extraction methods are therefore typically required to harvest, extract and isolate biodiesel from the host organism ([@bib7]). As suggested in this work, the extracellular localisation of 1-octanol is most likely facilitated by the AcrAB--TolC complex. The importance of TolC, though not AcrAB, for the internal production of free fatty acids (C8--C14) was recently observed in a study in which excretion unfortunately was not monitored ([@bib20]). Similarly, the over-expression of native *E. coli* transporters or transporter components have previously been found to stimulate either excretion ([@bib12]) or both excretion and production ([@bib32]) of native or non-native products. These studies on the role of TolC-dependent transporter complexes suggest that the elucidation of cause-and-effect may be complicated by their (1) overlapping substrate specificities and (2) complex sub-unit stoichiometry; such properties are likely to alter the dynamics of efflux in very subtle ways and make difficult the task of determining the efflux contribution of any single transporter complex. In addition, though not yet studied, it is possible that the overlapping susbstrate specificities may well serve complementary roles depending upon the physiological state of the cell.
Despite the complexity of these experimental systems, the simultaneous reduction in both excretion and total production of 1-octanol in the Δ*acrA* mutant, as well as previous work ([@bib20], [@bib32]), suggest that efflux pumps can have a substantial impact on the total flux of metabolic pathways that lead to a product with no obvious storage role within the cell. Studies are currently underway to shed further light on the correlation between excretion and production, as well as elucidating the entire complement of efflux pumps responsible for the excretion of fatty fuels of varying chain-lengths.
In summary, given the biological feasibility of its production, along with its natural excretion within the media and diesel-like fuel characteristics, we propose 1-octanol to be an attractive metabolic target for fuel-related applications.
This work was supported by the European Research Council under the European Union׳s Seventh Framework Programme (FP7/2007--2013)/Grant agreement 260661 (P.R.J.).
![Pathway engineering for the synthesis of 1-octanol. (a) Metabolic scheme depicting the pathway for the production of 1-octanol from glucose and fatty acids in *E. coli*: (1) fatty acid biosynthesis; (2) thioesterase releases free octanoate from FAS; (3) the CAR maturase, Sfp, prepares CAR~holo~ for catalysis; (4) CAR converts the free fatty acid to the corresponding aldehyde and (5) AHR reduces the aldehyde to the corresponding alcohol. (b) Example of GC--MS chromatogram showing the synthesis of 1-octanol. TPC3-Ahr strain was induced in modified minimal medium for 14 h, and analysed for fatty alcohols, as described previously ([@bib2]). (c) Distribution of 1-octanol in the two strains *E. coli* BL21(DE3)=WT, and *E. coli* BL21(DE3) Δ*acrA*=AcrA. Abbreviations: Glc, glucose; FAS, fatty acid synthesis; TES, thioesterase; reductase; CAR, carboxylic acid reductase; AHR, aldehyde reductase.](gr1){#f0005}
######
Fuel and physicochemical characteristics of petroleum-derived fuels and its potential substitutes.
**Energy content (MJ/L)** **Solubility**[a](#tbl1fna){ref-type="table-fn"}**(g/L)** **Cetane number**[b](#tbl1fnb){ref-type="table-fn"} **Lubricity**[c](#tbl1fnc){ref-type="table-fn"}**(μm corrected wear scar)** **Viscosity**[d](#tbl1fnd){ref-type="table-fn"}**cST** **Density**[a](#tbl1fna){ref-type="table-fn"} **Auto ignition temperature**[b](#tbl1fnb){ref-type="table-fn"}**(°C)** **Boiling point**[a](#tbl1fna){ref-type="table-fn"}**(°C)** **Flash point**[a](#tbl1fna){ref-type="table-fn"}**(°C)** **Vapour pressure**[a](#tbl1fna){ref-type="table-fn"}**(mmHg)** **Freezing point**[a](#tbl1fna){ref-type="table-fn"}**(°C)**
----------------------------------------------------------- --------------------------- ----------------------------------------------------------- ----------------------------------------------------- ----------------------------------------------------------------------------- -------------------------------------------------------- ----------------------------------------------- ------------------------------------------------------------------------- ------------------------------------------------------------- ----------------------------------------------------------- ----------------------------------------------------------------- --------------------------------------------------------------
Methanol 16 Miscible 2 1100 0.6\@40 °C 0.79 463 65 11 127 −98
Ethanol 19.6 Miscible 11 603 1.1\@40 °C 0.79 420 78 17 55 −114
1-Butanol 29.2 77 17 623 1.7\@40 °C 0.81 343 117 29 7 −90
1-Hexanol 31.7 7.9 23 534 2.9\@40 °C 0.81 285 158 59 1 −45
1-Octanol 33.7 0.59 39 404 4.4\@40 °C 0.83 270 195 81 0.08 −16
1-Decanol 34.6 \~0.04 50 406 6.5\@40 °C 0.83 255 233 108 \<0.1 6
1-Dodecanol 35.3 \~0.004 64 345 9.0\@40 °C 0.83 275 261 119 \<0.1 24
Hydrogenated bisabolene[e](#tbl1fne){ref-type="table-fn"} \~37 Immiscible 42 Unknown 2.91 0.82 Unknown 267 111 \<0.01 \<−78
Biodiesel 32.1 Immiscible 60 314 4--6\@40 °C 0.87 (avg) 177--330 315--350 100--170 \<1 −3 to −5
Petrodiesel[f](#tbl1fnf){ref-type="table-fn"} 40.3 Immiscible 45--50 315 1.8--5.8\@40 °C 0.84 (avg) 210 150--350 52--96 0.4 −12
Petroleum[g](#tbl1fng){ref-type="table-fn"} 32.1 Immiscible 13--17 711--1064 0.4--0.8\@20 °C 0.82 (avg) 246--280 27--225 −40 275--475 −60
[@bib501].
[@bib15].
[@bib33].
[@bib31].
[@bib26].
[@bib25].
[@bib21].
######
Comparison of engineered platforms for 1-octanol production in *E. coli*.
**1-Octanol producing system (strain name, mutations, genes induced)** **Titres of 1-octanol (mg L**^**−1**^**)** **Yield of 1-octanol**[a](#tbl2fna){ref-type="table-fn"}**(mg g**^**−1**^**glucose)** **Productivity (mg h**^**−1**^** L**^**−1**^**)** **Cultivation conditions** **Reference**
----------------------------------------------------------------------------------------- ---------------------------------------------------------------------------- --------------------------------------------------------------------------------------- --------------------------------------------------- ---------------------------- ---------------
BL21(DE3) 62 12 4.4 14 h, 30 °C, shake-flasks This work
*tes*, *sfp*, *car*, *ahr* M9-based medium with vitamins and metals[b](#tbl2fnb){ref-type="table-fn"}
MG1655 100 25 2.1 48 h, 37 °C, shake-flasks [@bib11]
*fadR ato*C(con) ∆*arcA* ∆*crp*::*crp*[c](#tbl2fnc){ref-type="table-fn"} ∆*adhE* ∆*pta* M9-based medium with vitamins and metals[b](#tbl2fnb){ref-type="table-fn"}
∆*frdA* ∆*fucO* ∆*yqhD* ∆*fadD fadBA betA*
BW25113 70 Not applicable 1.5 48 h, 30 °C, culture tubes [@bib22]
F^0^ \[*traD36*, *proAB*+, *lacI*^*q*^*Z*Δ*M15* (*tetR*)\]
Δ*ldhA* Δ*adhE* Δ*frdBC* Δ*pta* Terrific broth[b](#tbl2fnb){ref-type="table-fn"}
ATCC98082 (threonine overproducing strain) 15 Not given 0.04 48 h, 30 °C, shake-flasks [@bib23]
Δ*rhtA thrA*, *thrB*, *thrC*, *ilvA*, *leuA*,
*leuA*[b](#tbl2fnb){ref-type="table-fn"}*leuB*, *leuC*, *leuD*, *kivD*, *adh6* M9 medium with yeast extract[b](#tbl2fnb){ref-type="table-fn"}
Maximum possible theoretical yield is 361 mg 1-octanol per gram of glucose.
Supplemented with glucose.
Modified gene.
|
{
"pile_set_name": "PubMed Central"
}
|
Introduction
============
Common variable immunodeficiency (CVID) is a primary immunodeficiency disease characterized by a low concentration of serum immunoglobulins and an impaired antibody response to challenging antigens \[[@B1]\]. Although the pathophysiology of CVID is heterogeneous and largely unknown, several causes leading to an alteration of immunoglobulin concentrations in the blood have already been identified. These include a failure of B-cell maturation, including altered somatic hypermutation \[[@B2]\]; defective cell-membrane signaling \[[@B3]\]; T-cell abnormalities such as reduced expression of key membrane-expressed molecules (CD40 ligand, ICOS (inducible costimulator protein), L-selectin) \[[@B4]\]; impaired cytokine production \[[@B5]\]; and a reduced generation of antigen-specific memory T cells \[[@B6]\].
Whereas the antigen-presenting function of DCs has been reported to be normal in CVID \[[@B7],[@B8]\], their involvement in the origin of some CVID cannot be ruled out, as these cells are known to interact directly with B cells, to present antigen to T cells, and to produce cytokines implicated in B-cell differentiation \[[@B9]\]. Two major pathways of differentiation generating DCs are thought to exist, according to their membrane expression of the β-integrin CD11c \[[@B10]\]. Myeloid DCs (mDCs) include skin Langerhans\' cells and interstitial DCs and express CD11c at their surface. In contrast, plasmacytoid DCs (pDCs), which do not express CD11c, are CD123^+^. Recent data suggest a role for DCs in B-cell growth and differentiation, as the release by mDCs of soluble factors such as IL-12 and IL-6 and/or membrane molecules such as BAFF/APRIL induces the activation and the differentiation of normal B cells \[[@B11],[@B12]\]. In addition, the observation that pDCs directly induce the differentiation of plasma cells into immunoglobulin-secreting plasma cells suggests that pDCs are critically involved in humoral responses \[[@B13],[@B14]\]. Altogether, these new data prompted us to examine the blood distribution of DC subsets in 44 patients with CVID.
Materials and methods
=====================
Patient characteristics
-----------------------
Forty-four patients with CVID (17 to 77 years of age; 28 women and 17 men) were enrolled in this study after they had given their informed consent and following the approval of the local Ethics Committee. All patients were diagnosed as having CVID, on the basis of a specific medical history of recurrent bacterial infections associated with hypogammaglobulinemia (serum immunoglobulin (Ig)G and IgA and/or IgM at least two standard deviations below the normal mean) \[[@B15]\]. At the time of evaluation, none of the patients showed evidence of acute infection. As is frequently observed in CVID, 13 of the 45 patients had autoimmune diseases, 14 had splenomegaly, 5 had lymphoid hyperplasia, and 7 presented with a chronic granulomatous disease.
All the patients included in this study had blood CD19^+^CD3^-^B-lymphocyte counts above 1% of peripheral blood lymphocytes. The CVID patients were divided into two groups according to the detection of switched memory B cells (CD27^+^IgD^-^) as recently proposed by Warnatz and colleagues \[[@B16]\]. Group 1 (*n* = 22), comprising patients whose proportion of switched memory B cells was less than 0.4% of their total peripheral blood lymphocytes, was further subdivided according to whether they had increased (group 1a; *n* = 13) or normal (group 1b; *n* = 9) numbers of CD19^+^CD21^-^immature B cells. Group 2 (*n* = 15) comprised patients whose proportion of switched memory B cells was more than 0.4% of total peripheral blood lymphocytes. As Warnatz and colleagues excluded from their classification patients with granulomatous disease, we classified these patients in a distinct group (group 3; *n* = 7). Control patients were healthy Caucasian blood donors and health-care workers (*n* = 12, median 36 years; 8 women and 4 men); they were not matched with patients for sex or age.
Quantitation of blood DC precursors by flow cytometry
-----------------------------------------------------
DC subsets were measured using the DC kit purchased from BD Biosciences (Pont-de-Claix, France). Samples were analyzed on a FACSCalibur flow cytometer (BD Biosciences) and 10^6^white blood cells were acquired. Peripheral blood mDC and pDC subsets were defined by the concomitant lack of lineage markers, HLA-DR expression, and mutually exclusive membrane expression of CD11c or CD123, respectively. Absolute numbers of blood DC precursor subsets were calculated as percentage of white blood cells or expressed per ml of peripheral blood. Membrane expression of chemokine receptors was assessed by flow cytometry using a triple combination of monoclonal antibodies: BDCA2-FITC (Miltenyi Biotec, Paris, France); and HLA-DR-PerCp; and CCR2-PE or CCR5-PE or CCR7-PE or CXCR3-PE or CXCR4-PE (BD Biosciences).
Statistical analysis
--------------------
Median values found for blood cells were compared between groups using the nonparametric Mann--Whitney *U* test, with a level of significance of *P* = 0.05. The Spearman test was used to make correlations. The tests were performed with the statistical software Statistica Inc (Statsoft, Tucson, AZ, USA).
Results and discussion
======================
The median absolute count of circulating pDCs was significantly lower in CVID patients than in controls (*P* = 0.002). Although CVID patients had a lower mean mDC count, their median absolute count was not statistically significantly different from that of controls (*P* = 0.1) (Fig. [1](#F1){ref-type="fig"}).
However, when CVID patients were segregated in accordance with the Warnatz classification, the DC subset distributions were quite different between groups (Table [1](#T1){ref-type="table"}). Groups 1a and 3 had statistically significantly lower levels of circulating DCs of both types than controls, but this difference was far more dramatic concerning the pDC subset (*P* = 0.001 and *P* = 0.01 for mDCs and *P* = 0.00005 and 0.0003 for pDCs, respectively). In groups 1b and 2, blood pDCs and mDCs were not statistically different in comparison with those in controls.
When the various groups of CVID patients were compared with each other, the median absolute count of circulating mDCs was found to be significantly reduced in groups 1a and 3 vs group 2 (*P* = 0.009 and *P* = 0.02, respectively). Although the median values of mDCs were lower in group 1a than in group 1b, the difference did not reach statistical significance (*P* = 0.1). In contrast, CVID patients in groups 1a and 3 had significantly lower pDCs median counts than those in group 2 (*P* = 0.0005 and *P* = 0.001, respectively), and, to a lesser extent, in group 1b (*P* = 0.02 and *P* = 0.009). No differences were observed between group 1a and group 3.
Moreover, we found a significant inverse statistical correlation between the low peripheral pDC levels of 19 CVID patients and the expression of the chemokine receptor CCR7 (*R* = -2.31, *P* = 0.03, Spearman test). These 19 patients were followed in the Department of Internal Medicine at Haut-Lévêque Hospital, directed by JL Pellegrin and JF Viallard. They were chosen because blood samples could easily be taken, in consideration of the short time-period available to perform new experiments with chemokine receptors. Of these 19 patients, 5 belonged to group Ia, 5 to group Ib, 6 to group II and 3 to group III. No differences were observed between the different groups with regard to the expression of other chemokine receptors including CCR2, CCR5, CXCR3, and CXCR4. This observation is in agreement with the already published data concerning the CCL19- and CCL21-dependent migration of mature pDCs to lymph nodes \[[@B17]\].
The most salient feature of the present study concerns patients in group 1a, who were at higher risk of autoimmune diseases (10 of 13 patients) and splenomegaly (9 of 13), and in group 3, both groups exhibiting a marked decrease in their peripheral pDC levels. Patients in group 2, who did not develop such complications, were similar to healthy individuals.
From these preliminary observations we can only speculate on the diminished pDC counts observed in the patients of groups 1a and 3. The migratory properties of DCs seem to be altered, leading to sequestration in tissue and/or secondary lymphoid organs due to an alteration of chemokine receptor membrane expression and maturation. However, we cannot rule out a defect in their function, such as has been suggested regarding DCs derived *in vitro* from monocytes \[[@B18]\].
At least in some patients, hypogammaglobulinemia could rely on an alteration of the innate immunity, since pDCs seem to play a critical role in the generation of antibody responses: they have been shown *in vitro* to control the differentiation of activated B cells into plasma cells through the secretion of interferon α and β and of IL-6 \[[@B13]\]. B cells activated with pDCs preferentially secrete IgG over IgM, suggesting that pDCs may specifically target memory B cells, whereas mDCs, which can enhance B-cell proliferation and isotype switching toward IgA \[[@B19]\], as well as plasma cell differentiation \[[@B20]\], could induce the differentiation of naive B cells through the secretion of IL-12 and IL-6 \[[@B11]\]. Granulocyte-colony-stimulating factor and FLT3 ligand, through their ability to mobilize dendritic cells, may be a new and valuable therapeutic alternative for CVID patients.
Conclusion
==========
Our data provide evidence of a profound alteration in the distribution of subsets of DCs, especially pDCs, in the peripheral blood of CVID patients. Taken together, our results raise the possibility that innate immunity is involved in CVID pathogenesis.
Abbreviations
=============
CVID = common variable immunodeficiency; Ig = immunoglobulin; IL = interleukin; mDC = myeloid dendritic cell; pDC = plasmacytoid dendritic cell.
Competing interests
===================
The authors declare that they have no competing interests.
Authors\' contributions
=======================
JFV and FC were in charge of most of the CVID patients gathered in this study, performed the statistical analysis, analyzed the data, and contributed to the writing of the article. PB put together the data, supervised the laboratory examinations, analyzed the data, and contributed to the writing of the article. JFM analyzed the data, participated in the laboratory examinations, contributed to the editing of the article, and supervised the research group. JLP was, along with JFV, in charge of most of the CVID patients gathered in this study and contributed to the writing of the article. MA and FL were in charge of CVID patients and contributed to the writing and preparation of the article. All authors read and approved the final manuscript.
Acknowledgements
================
We are indebted to and would like to thank Ms N Berrie, Ms F Saussais, and Mr JC Carron for their skillful technical performance of flow cytometric analysis in the routine laboratory of the Bordeaux University Hospital.
We would also like to thank Gabriel Etienne, Philippe Morlat, and Maïté Longy-Boursier (Saint-André Hospital, Bordeaux), Didier Neau (Pellegrin Hospital, Bordeaux), Laurence Baillet (Haut-Lévêque Hospital, Pessac), Elisabeth Vidal (Dupuytren Hospital, Limoges) and Olivier Aumaître (Gabriel-Montpied Hospital, Clermont-Ferrand) who all were in charge of CVID patients.
Figures and Tables
==================
{#F1}
######
Absolute counts of peripheral blood DCs in controls and in patients with CVID
Dendritic cells per ml Healthy controls (*n* = 12) CVID patients^a^
--------------------------- ----------------------------- ------------------ --------------- --------------- ---------------
Myeloid DCs
Total (median) 14,361 8,432\*,^θ^ 13,475 15,267 5,568^Ψ,φ^
Percentiles (25th, 75th) 11,470, 16,693 1,428, 11,330 8,378, 16,143 9,462, 18,590 4,315, 11,100
Range 8,941--22,204 342--21,895 3,039--19,385 6,178--28,472 3,787--22,994
Plasmacytoid DCs
Total pDCs (median) 9,829 2,367\*,^λ,θ^ 3,647 8,980 1,403^Ψ,ξ,φ^
Percentiles (25th, 75th) 8,366, 16,555 353, 5,012 2,532, 11,590 4,096, 13,367 661, 2,448
Range 7,514--22,769 19--8,291 2,004--13,714 1,614--25,725 417--4,917
^a^Patients were grouped according to whether their proportion of switched memory B cells was \<0.4% (groups 1a, 1b) or \>0.4% (group 2) of total peripheral blood lymphocytes or they had granulomatous disease (group 3). Group 1 was subdivided according to whether numbers of CD19^+^CD21^-^immature B cells were increased (group 1a) or normal (group 1b). \**P* \< 0.001 vs controls;^θ^*P* \< 0.009 vs group 2; ^λ^*P* = 0.02 vs group 1b; ^Ψ^*P* \< 0.01 vs controls; ^ξ^*P* \< 0.009 vs group 1b; ^φ^*P* \< 0.02 vs group 2. Other comparisons between each population were not statistically significant. The nonparametric Mann--Whitney *U* test was used for comparisons. CVID, common variable immunodeficiency.
|
{
"pile_set_name": "PubMed Central"
}
|
Introduction {#s1}
============
The striatum is an important subcortical component of the basal ganglia-thalamo-cortical circuits. The rostral, medial, and ventral parts of the striatum are primarily connected to the ventral and medial prefrontal cortex, anterior cingulate cortex, and orbitofrontal cortex, whereas the dorsal striatum, including the head of caudate and part of rostral putamen, are connected to the dorsal and lateral prefrontal cortex (Haber et al., [@B26]; Haber and Knutson, [@B27]). These fiber connections provide anatomical support for dynamic and reciprocal signaling between the striatum and other brain regions, which underlie diverse psychological functions (Pauli et al., [@B51]). Indeed, five distinct striatal zones have been found to be linked to distinct brain functions: the anterior caudate for incentive behaviors and the evaluation of different actions, the posterior caudate for executive functions, the posterior putamen for sensorimotor processes, the anterior putamen for social and language-related functions, and the ventral striatum for the representation of stimulus value and related stimulus-driven motivational states (Pauli et al., [@B51]).
Studies have reported significant sex differences in reward-related processes and impulse control, which are sub-served by the striatal-cortical and striatal-subcortical circuits (Lighthall et al., [@B43]; Fattore, [@B17]). As one example, males and females differ in processing reward information and decision making. Studies found that the putamen showed a higher reward-related anticipatory response in males than in females (Dreher et al., [@B15]). Compared to females, males were faster and gained greater rewards in risky decision making, and they also showed increased activation in the dorsal striatum including the putamen (Lighthall et al., [@B43]). Sometimes even when the behaviors showed no sex differences, brain activations differed by sex. For example, when asked to reject immediate rewards in pursuit of a long-term goal, males showed a stronger reduction of activation than did females in the dorsal striatum, subgenual and pregenual anterior cingulate cortex, posterior orbitofrontal cortex, as well as more positive functional coupling between these regions (Diekhof et al., [@B11]). It seems that the striatal-frontal circuits are more frequently and strongly recruited during reward-related processing in males than in females. Based on the above results, we may also speculate that males made a greater effort (as shown in more positive functional coupling) to effectively suppress the stronger response (stronger reduction of activation) that were originally activated by immediate rewards.
Although researchers have discussed extensively sex differences in the structure and function of the human brain (Cahill, [@B8]; Gong et al., [@B23], [@B22]), specific sex differences within the striatum-connected structural circuits remain largely unknown mainly because of the limitations of earlier approaches to analyzing diffusion tensor imaging (DTI) data. Three approaches of analyzing DTI data have been developed: tract-based spatial statistics (TBSS), deterministic tracking, and probabilistic tractography. The TBSS method provides the most commonly used fractional anisotropy (FA) of fiber tracts, which quantifies directional strength of each voxel of the local tracts. The TBSS method does not directly quantify connections between brain regions (Smith et al., [@B55]). With the deterministic tracking method, seeds were placed in voxels with FA greater than a given threshold (e.g., 15) to include only white matter voxels, and then grown in both directions along the dominant diffusion orientation of voxels into fiber tracts or streamlines. Deterministic tracking has a limited capacity for resolving crossing fiber bundles, and consequently misses some fiber bundles for the lateral cortical regions (Mori and van Zijl, [@B45]). Probabilistic tractography builds up distributions on diffusion parameters at each voxel by using sampling techniques, and then samples from the distributions on voxel-wise principal diffusion directions, each time computing a streamline through these local samples to generate a probabilistic streamline or a sample. Probabilistic tractography is the preferred method because it is better at handling fiber crossings and image noise (Behrens et al., [@B4]), although it may lead to spurious connections (Parker and Alexander, [@B50]). Another drawback of probabilistic tractography is that it is quite time-consuming and computationally intensive. Therefore, few studies have used this method to study specific brain circuits, and even fewer on sex differences of specific brain circuits. Consequently, even though the anatomical connection pattern of the striatum has been revealed by animal studies, postmortem human studies, and *in vivo* human brain imaging studies of small samples (Haber et al., [@B26]; Leh et al., [@B39]; Haber and Knutson, [@B27]), and several studies of small samples have investigated the associations between striatum-projected fiber connection and behavioral and physiological measures (Cohen et al., [@B9]; Bohanna et al., [@B6]; Lei et al., [@B41]; Tziortzi et al., [@B59]), no study has examined sex differences in the striatum-projected structural connectivity.
The current study was designed to explore sex differences in anatomical connectivity of the striatum to cortical and subcortical regions. Following previous studies (Cohen et al., [@B9]; Lei et al., [@B41]), the striatum and nine target masks (see Figure [S1](#SM1){ref-type="supplementary-material"} for anatomical locations of these masks) for each hemisphere were created based on the automated anatomical labeling template (Tzourio-Mazoyer et al., [@B60]). The nine target regions included the medial orbitofrontal cortex (mOFC), lateral orbitofrontal cortex (lOFC), ventrolateral prefrontal cortex (vlPFC), dorsolateral prefrontal cortex (dlPFC), posterior cingulate cortex/retrosplenial cortex (PCC), rostral cingulate cortex (rostral CC), dorsal cingulate cortex (dorsal CC), hippocampus, and amygdala. Based on the previous studies showing that males relied on the striatal-frontal circuits to a greater extent than did females (Dreher et al., [@B15]; Diekhof et al., [@B11]; Lighthall et al., [@B43]), the current study hypothesized stronger fiber connection in males than in females between the striatum and the anterior cingulate cortex and orbitofrontal cortex.
Materials and methods {#s2}
=====================
Participants
------------
Fifty male and seventy nine female college students (mean age 20.10 years; range 19--22 years) were recruited from Beijing Normal University. All participants were right-handed Han Chinese with normal or corrected-to-normal vision. They self-reported having no history of neurological or psychiatric illnesses. They also passed the physical and clinical examinations for all freshmen administered by the University. Participants were scanned for diffusion tensor and high resolution 3D anatomical images. They all gave informed written consents and the study was approved by the Beijing Normal University Institutional Review Board.
Image acquisition
-----------------
Participants were scanned on a Siemens Trio 3T scanner with an eight-channel head coil in the Beijing Normal University Imaging Center for Brain Research. The diffusion-weighted data were acquired using a twice-refocused spin-echo EPI sequence with the following parameters: TR/TE = 7200/104 ms, 49 transverse slices, field-of-view = 230^\*^230 mm, matrix = 128^\*^128, slice thickness = 2.5 mm, 1 direction with *b*-value = 0 s/mm^2^, 64 directions with *b*-value = 1000 s/mm^2^. In addition, a high resolution 3D anatomical image was obtained using T1-weighted MP-RAGE sequence with the following parameters: TR/TE/FA = 2530/3.75 ms/7°, FOV = 220^\*^220 mm, matrix = 256^\*^256, slice thickness = 1 mm, 128 sagittal slices). Scanning lasted 18 min for each participant.
Image preprocessing
-------------------
Diffusion tensor images (DTI) were processed using the FMRIB\'s Diffusion Toolbox (FDT 2.0; Smith et al., [@B55]) from the FMRIB\'s Software Library (FSL, version 5.0.5; [www.fmrib.ox.ac.uk/fsl](http://www.fmrib.ox.ac.uk/fsl); Smith et al., [@B56]; Woolrich et al., [@B64]; Jenkinson et al., [@B34]). The standard preprocessing procedure, including correction of the diffusion data for eddy currents and head motion, fitting of diffusion tensor, and Bayesian estimation of diffusion parameters, was used for the probabilistic tractography of DTI data (Behrens et al., [@B5], [@B3]; Johansen-Berg et al., [@B37]). Bayesian estimation of diffusion parameters was conducted with a dual-fiber model allowing for crossing fibers by using the BedpostX program implemented in FMRIB\'s diffusion toolbox (Behrens et al., [@B5]). Detailed preprocessing steps were described in our previous study (Lei et al., [@B41]).
The non-diffusion-weighted images of all participants were spatially normalized into the Montreal Neurological Institute (MNI) standard space with FMRIB\'s Linear Image Registration Tool (FLIRT; Jenkinson et al., [@B33]; Greve and Fischl, [@B24]) and FMRIB\'s Nonlinear Image Registration Tool (FNIRT) by individual\'s high resolution T1-weighted structural image. The transformation matrix and the warp field from individual participants\' diffusion space to the MNI standard space, and the inversed transformation matrix and warp field from the MNI standard space to individual diffusion space were obtained through the normalization process. The matrix and warp field were then used, respectively, for the striatum and nine target brain regions in the MNI standard space warping into participants\' diffusion space, and for the normalization of the resulting tractography maps (scalar) into the MNI standard space. Visual inspection of normalized non-diffusion-weighted brain images was done to confirm that the registration was successful.
Seed brain region and target brain regions
------------------------------------------
One seed mask and nine target masks, including the striatum, mOFC, lOFC, dlPFC, vlPFC, rostral CC, dorsal CC, PCC, hippocampus, and amygdala, were created for each hemisphere based on the automated anatomical labeling template (Tzourio-Mazoyer et al., [@B60]) and previous studies (Cohen et al., [@B9]; Lei et al., [@B41]). Detailed information of these masks was presented in our previous study (Lei et al., [@B41]). Figure [S1](#SM1){ref-type="supplementary-material"} shows the anatomical locations of these brain masks for one hemisphere. All masks in the MNI standard space were transformed to individual diffusion space by using transformation matrix and warp field produced in the previous step and binarized. Volumes of these ten masks in individuals\' diffusion space were obtained. The total intracranial volume (ICV) was obtained from high resolution T1-weighted anatomical image by using the Freesurfer segmentation software package (<http://surfer.nmr.mgh.harvard.edu/>; Fischl et al., [@B19]).
Tractography and seed-based classification
------------------------------------------
Probabilistic tractography was performed from the striatum to the nine target regions in individuals\' diffusion space by PROBTRACKS program implemented in FMRIB\'s diffusion toolbox (Behrens et al., [@B5]). Five thousand tract-following samples were initiated in each voxel of the striatum, and were then tracked to the nine target regions, resulting in nine probabilistic maps of fiber connectivity, called tractography images. The value of each voxel in the tractography image represented the number of the tracking to the target region (connectivity between the voxel in the striatum to all voxels in the target region). Only voxels above the threshold of a minimum of 10 tracking samples per voxel were retained to assure true fiber connection after removing noise (Aron et al., [@B1]). The number of tracking samples of a given voxel to each target was then divided by the voxel\'s total tracking number to all regions to yield a proportional ratio. The resulting nine images were transformed back to the MNI standard space for group statistical analyses. The final spatial resolution was 1 mm^3^. All preprocessing was done separately for each hemisphere. Tractography images in the MNI standard space from two hemispheres were combined and used in general linear models to examine sex differences.
Statistical analysis
--------------------
Independent sample *t*-test in SPSS was used to examine sex differences in age, handedness, and ICV. Linear regression model with sex as a predictor and ICV as a covariate was used to examine sex differences in volumes of the ten brain regions.
Fiber connections between the striatum and nine target regions were analyzed using the general linear model with the tool "randomize" (Winkler et al., [@B63]). The permutation-based non-parametric test with the Threshold-Free Cluster Enhancement (TFCE) was used to perform group analysis (Nichols and Holmes, [@B47]; Smith and Nichols, [@B57]). This method has been highly recommended by the FSL group for research on finding statistically significant cluster-like structures. Nine preprocessed tractography images, each representing fiber connectivity between the striatum and one target region, were entered into nine general linear models, respectively. In order to exclude its potential confounding effect, the total intracranial volume (ICV) was entered into GLM as a covariate. Results were corrected for multiple comparisons with the TFCE algorithm (Smith and Nichols, [@B57]). We further extracted and plotted individual participants\' average fiber connection within the striatal regions that showed significant sex differences.
Results {#s3}
=======
Males were found to have larger ICV and larger volumes in all ten brain regions included in this study (Table [1](#T1){ref-type="table"}). Figure [1](#F1){ref-type="fig"} shows the group--averaged tractography map of the connections between the striatum (including the putamen and caudate) and nine target regions. Clear anterior-posterior, medial-lateral, and dorsal-ventral connectivity patterns were observed. The rostral CC, mOFC, and lOFC were found to mainly connect to the medial and ventral parts of the striatum; the dlPFC, vlPFC, and dorsal CC to the dorsal and lateral parts of the striatum; the amygdala and hippocampus to the posterior putamen; and the PCC to the posterior caudate.
######
**Descriptive statistics and sex differences**.
**Variables** **All (*****n*** = **129)** **Male (*****n*** = **50)** **Female (*****n*** = **79)** **Statistics**
-------------------------------------------------------------- ----------------------------- ----------------------------- ------------------------------- ---------------- ----------- ---------- --------- ----------
Age 20.36 0.84 20.52 0.84 20.25 0.83 −1.78 0.08
Handedness[^a^](#TN1){ref-type="table-fn"} 90.99 6.02 90.28 6.32 91.44 5.85 −0.1.07 0.29
**VOLUMES OF BRAIN REGIONS**[^b^](#TN2){ref-type="table-fn"}
dlPFC 101601.8 11576.5 107762.7 11174.7 97702.5 10086.9 −4.71 \<0.0001
vlPFC 28315.1 4851.6 30336.4 4809.9 27035.8 4451.5 −3.41 0.001
lOFC 32133.7 3217.6 33755.7 3277.2 31107.2 2736.5 −4.51 \<0.0001
mOFC 30932.9 3566.8 33020.1 3100.7 29611.9 3205.8 −5.27 \<0.0001
Rostral CC 16046.1 2607.6 17262.9 2613.4 15276.0 2306.0 −3.74 \<0.0001
Dorsal CC 25764.2 2483.0 27156.9 2375.5 24882.7 2131.2 −5.03 \<0.0001
PCC 42031.1 4901.4 44869.1 4624.3 40234.9 4190.3 −5.08 \<0.0001
Amygdala 2841.3 371.3 3042.7 369.2 2713.9 313.3 −5.09 \<0.0001
Hippocampus 11770.0 896.6 12343.5 929.0 11407.0 657.8 −6.03 \<0.0001
Striatum 23843.3 2325.4 25192.8 1930.8 22989.2 2150.8 −5.04 \<0.0001
ICV 1459216.8 231556.24 1543662.4 207768.4 1405770.2 231098.4 −3.43 0.001
*Determined using Edinburgh Inventory (Oldfield, [@B49]); Scores greater than 0 indicate right-handedness. A score of 100 indicates strong right-handedness*.
*Sex differences of the ten brain ROIs were analyzed with regression models with sex and ICV as predictors*.
*Two kinds of T statistics are shown: independent sample T statistics for age, handedness, and ICV; and T statistics for the regression coefficients of sex as a predictor in regression*.
*ICV, intracranial volume; dlPFC, the dorsolateral prefrontal cortex; vlPFC, the ventrolateral prefrontal cortex; mOFC, the medial orbitofrontal cortex; lOFC, the lateral orbitofrontal cortex; rostral CC, the rostral cingulate cortex; dorsal CC, the dorsal cingulate cortex; PCC, the posterior cingulate cortex/retrosplenial cortex*.
{#F1}
Overall, females and males showed similar patterns of fiber connection (Figures [S2](#SM2){ref-type="supplementary-material"}, [S3](#SM3){ref-type="supplementary-material"}). General linear model analysis nevertheless revealed significant sex differences in fiber connection between sub-regions of the striatum and five of the nine target regions (the hippocampus, rostral CC, lOFC, vlPFC, and dlPFC). As shown in Table [2](#T2){ref-type="table"}; Figures [2](#F2){ref-type="fig"}, [3](#F3){ref-type="fig"}, males showed greater fiber connection than did females between left ventral and medial caudate and the rostral CC, between the right ventrolateral putamen and the lOFC, between bilateral lateral putamen and the vlPFC, and between the left lateral caudate and the vlPFC, whereas females showed greater fiber connection between the right putamen and the hippocampus, between bilateral posterior caudate and the hippocampus, and between the right dorsolateral putamen and the dlPFC. These sex differences remained significant after controlling for the volumes of the striatum and the target region and ICV (see Table [S1](#SM4){ref-type="supplementary-material"}).
######
**Fiber connectivity that showed significant sex differences**.
**Fiber target regions** **Fiber-initiated regions in striatum** **Volume in striatum/mm^3^** **Tmax** **MNI coordinate**
-------------------------- ----------------------------------------- ------------------------------ ---------- --------------------
**MALES \> FEMALES**
Rostral CC Left caudate 755 5.307 −4, 13, −5
lOFC Right putamen 635 5.179 34, 3, 3
vlPFC Right putamen 3578 4.576 33, 4, −5
Left putamen 1130 4.218 −32, 0, −7
Right caudate 192 4.601 13, 12, 20
Right caudate 149 3.729 19, 6, 25
**MALES \< FEMALES**
dlPFC Right putamen 3667 3.949 31, 0, −8
Hippocampus Left putamen 840 3.653 −30, −12, −1
Right caudate 693 3.801 11, −1, 15
Left caudate 570 4.368 −14, −4, 17
*Rostral CC, the rostral cingulate cortex; lOFC, the lateral orbitofrontal cortex; vlPFC, the ventrolateral prefrontal cortex; dlPFC, the dorsolateral prefrontal cortex*.
{ref-type="fig"}. See Figure [1](#F1){ref-type="fig"} for abbreviations.](fncom-10-00100-g0002){#F2}
{ref-type="fig"}. See Figure [1](#F1){ref-type="fig"} for abbreviations.](fncom-10-00100-g0003){#F3}
Discussion {#s4}
==========
The current study explored sex differences in fiber connectivity between the striatum and nine target regions. Consistent with previous studies (Gur et al., [@B25]; Goldstein et al., [@B21]; Cosgrove et al., [@B10]), we found larger ICV and brain regions in males than females. Group--averaged tractography patterns of the striatum were also consistent with the results of previous studies in primates and humans (Lehericy et al., [@B40]; Haber et al., [@B26]; Draganski et al., [@B14]; Haber and Knutson, [@B27]), which confirmed the validity of the current tractography. Despite overall similarities between males and females, significant sex differences in striatal fiber connectivity were observed in the current study. Males showed stronger fiber connection between the left ventromedial caudate and rostral CC, between the right ventrolateral putamen and the lOFC, between bilateral putamen and the vlPFC, and between the right lateral caudate and the vlPFC, whereas females showed stronger fiber connection between the right dorsolateral putamen and the dlPFC, between the bilateral posterior caudate and the hippocampus, and between the left lateral putamen and the hippocampus.
Several lines of previous research had hinted at potential sex differences in the striatum-connected fiber sub-networks found in this study. First, TBSS studies found that, compared to females, males had higher white matter integrity in white matter regions and tracts that underlie striatal-frontal connection (Wakana et al., [@B61]; Lawes et al., [@B38]; Nowinski et al., [@B48]), such as bilateral superior corona radiate (Takao et al., [@B58]), bilateral internal capsule, and cingulate (Hsu et al., [@B29]). Second, functional coupling between the striatum and anterior cingulate cortex and prefrontal cortex has been found to be stronger in males than in females in reward-related processing and impulse inhibition (Lighthall et al., [@B43]; Fattore, [@B17]), suggesting potentially stronger fiber connection in males. As reviewed in the Introduction, males were found to be more likely to recruit striatal-frontal circuits for reward-related processes and impulse inhibition (Diekhof et al., [@B11]; Lighthall et al., [@B43]). Consistently, several studies implicated striatal-cingulate and striatal-prefrontal circuits for reward, emotion and cognitive control (Beckmann et al., [@B2]; Dixon and Christoff, [@B13]; Burton et al., [@B7]; Dixon, [@B12]; Jarbo and Verstynen, [@B32]; Porter et al., [@B52]; Morris et al., [@B46]).
In contrast to the stronger fiber connection between right putamen to the vlPFC in males, females showed stronger fiber connection between right putamen and the dlPFC. Both vlPFC and dlPFC are central for reward-related cognitive control (Dixon and Christoff, [@B13]; Dixon, [@B12]). The vlPFC has been found to represent associations between rules and outcomes that are signaled by the immediate environment, and the dlPFC has been found to represent the relationship between more complex/abstract rules for action and desired outcomes, and to play a critical role in pursuing future reward (Dixon and Christoff, [@B13]; Dixon, [@B12]). Thus, this sex difference within striatal-lateral prefrontal circuits may reflect sex-specific neuroanatomical correlates of reward-related cognitive control.
Females also showed stronger fiber connections between the putamen and the hippocampus and between the caudate to the hippocampus than did males. In animal studies and human brain imaging studies, the striatum-hippocampus balance has been implicated in compensation and competition between different memory and learning systems (e.g., the hippocampus system for declarative learning and memory, and the striatum system for procedural learning and memory; Ghiglieri et al., [@B20]). Because functional coupling between the striatum and the hippocampus is involved in episodic memory (Jiang et al., [@B35]), our finding of stronger fiber connection between the striatum and hippocampus in females than in males may provide a structural neural basis for the robust sex differences in episodic memory (for meta-analyses, see Wang, [@B62]; Hyde, [@B30]). Future studies should specifically examine this speculation.
The current study had several limitations that need to be discussed. First, we only used probabilistic tractography, which does not allow for the identification of afferent or efferent striatal pathway (Rushworth et al., [@B54]; Bohanna et al., [@B6]). Second, our participants were young college students with a narrow age range, so we could not examine age-related changes in the striatal circuitry. Third, we found significant sex differences in the striatum-projected fiber connections that may explain behavioral differences, but how these brain differences came about remains unknown. Some researchers (Herting et al., [@B28]; Lentini et al., [@B42]) have discussed biological factors (e.g., sex hormones) involved in sex differences in brain anatomy, whereas others (Joel, [@B36]; Fine et al., [@B18]; Miller and Halpern, [@B44]; Rippon et al., [@B53]) have speculated about the importance of social environments. Fourth, based on previous research, we speculated that our finding of sex differences in the striatum-projected connection could account for some sex differences in behavior, but this study did not directly test that possibility. The prefrontal neurons have been found to be significantly more spinous than those in the other lobes, indicating a higher ability of prefrontal neurons to integrate a large number of excitatory inputs (Elston, [@B16]; Jacobs et al., [@B31]). Although the current study found that anatomical striatal-prefrontal circuitries differ in male and female, it should be noted that functional stimulations within these striatal-prefrontal circuitries may not have the same patterns of sex differences. The relationships between brain structure and function, as well between brain and behaviors, are complex, so great caution is needed when linking significant sex differences in striatal-prefrontal fiber connectivity to sex differences in behaviors.
Conclusion {#s5}
==========
Using probabilistic tracking of diffusion tensor images, the current study found several significant sex differences in the fiber connection between the striatum and nine target regions after controlling for the ICV and the volumes of the striatum and target regions. These differences may help explain sex differences in relevant behaviors such as risky decision making, impulse inhibition, and memory.
Author contributions {#s6}
====================
CC, GX, and QD designed this research. XL and LB analyzed the data. XL, ZH, and CC wrote this paper.
Conflict of interest statement
------------------------------
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
This study was supported by the 111 Project of the Ministry of Education of China (B07008), the New Teacher Research Funds of Beijing Normal University (SKXJS2014024) and the National Science Foundation (31521023).
Supplementary material {#s7}
======================
The Supplementary Material for this article can be found online at: <http://journal.frontiersin.org/article/10.3389/fncom.2016.00100>
######
**Anatomical locations of the nine target regions in one hemisphere**. Amy, amygdala; Hipp, hippocampus; mOFC, the medial orbitofrontal cortex; rostral CC, the rostral cingulate cortex; dorsal CC, the dorsal cingulate cortex; PCC, the posterior cingulate cortex/retrosplenial cortex; lOFC, the lateral orbitofrontal cortex; vlPFC, the ventrolateral prefrontal cortex; dlPFC, the dorsolateral prefrontal cortex.
######
Click here for additional data file.
######
**Tracts between the striatum and each target region for males**. Only voxels with at least 5% target-ending tracts are displayed. Colors indicate proportion of target-specific tracts out of all tracts for a given voxel. See Figure [S1](#SM1){ref-type="supplementary-material"} for abbreviations.
######
Click here for additional data file.
######
**Tracts between the striatum and each target region for females**. Only voxels with at least 5% target-ending tracts are displayed. Colors indicate proportion of target-specific tracts out of all tracts for a given voxel. See Figure [S1](#SM1){ref-type="supplementary-material"} for abbreviations.
######
Click here for additional data file.
######
**Linear regression models for the nine fiber connections from Table [2](#T2){ref-type="table"}**.
######
Click here for additional data file.
DTI
: diffusion tensor imaging
mOFC
: the medial orbitofrontal cortex
lOFC
: the lateral orbitofrontal cortex
vlPFC
: the ventrolateral prefrontal cortex
dlPFC
: the dorsolateral prefrontal cortex
PCC
: the posterior cingulate cortex/retrosplenial cortex
dorsal CC
: the dorsal cingulate cortex
rostral CC
: the rostral cingulate cortex
Amy
: amygdala
Hipp
: hippocampus.
[^1]: Edited by: Pengsheng Zheng, IBM Thomas J. Watson Research Center, USA
[^2]: Reviewed by: Guy Elston, Centre for Cognitive Neuroscience, Australia; Chi-Hua Chen, University of California, San Diego, USA
[^3]: †These authors have contributed equally to this work and should be considered co-first authors.
|
{
"pile_set_name": "PubMed Central"
}
|
Introduction {#s1}
============
For stem cell characterization, understanding single cell level functional genomics has become increasingly important [@pone.0017455-Marson1], [@pone.0017455-Bianco1], [@pone.0017455-Takahashi1], [@pone.0017455-Zipori1], [@pone.0017455-Slack1], [@pone.0017455-Fan1], [@pone.0017455-Kurimoto1], [@pone.0017455-Svahn1]. As new regenerative therapies using tissue engineering [@pone.0017455-Bianco1] emerge, the existence of tissue-specific stem cells in adult organs has extensively been investigated in bone marrow, skin, heart, muscle, pancreas, lungs, and the nervous system. However, the characterization of differentiated progeny has been hampered by the lack of cell markers and low viability of the purified cells [@pone.0017455-Fabbri1]. For instance, single cell transplantation methods can significantly benefit from efficient cell isolation and handling techniques [@pone.0017455-Leong1]. Recent advances in mRNA amplification and cell sorting technologies offer insights into single cell genomics [@pone.0017455-Marson1], [@pone.0017455-Bianco1], [@pone.0017455-Takahashi1], [@pone.0017455-Zipori1], [@pone.0017455-Slack1], [@pone.0017455-Fan1], [@pone.0017455-Walker1]. However, single cell level genomic studies require amplification by a factor of a billion to reach detection levels from a few femtograms of mRNA present in a single cell. Therefore, to accurately profile single cell genes from a heterogeneous cell solutions or a tissue sample, it is essential to minimize RNA contamination from surrounding cells by enriching the fraction of the target cell type.
Cell pattering and encapsulation in droplets is a challenging and exciting field with multiple possible applications including tissue printing [@pone.0017455-Sun1], [@pone.0017455-Mironov1], cell sorting [@pone.0017455-Baret1], and cryobiology [@pone.0017455-Song1]. Several approaches have been developed to isolate single cells ([**Table 1**](#pone-0017455-t001){ref-type="table"}). The most common methods are microscale cell manipulation [@pone.0017455-Kvist1], serial dilutions of a culture or a co-culture of cells [@pone.0017455-Zhang1], laser capture microdissection (LCM) [@pone.0017455-Geigl1], and fluorescence-activated cell sorting (FACS) [@pone.0017455-Raghunathan1]. These methods have challenges with complexity, time consumption and inefficiencies in isolating cells that are contamination-free and viable. In addition, histological methods can damage mRNA both in frozen and paraffin-embedded sections [@pone.0017455-Farragher1]. Traditional FACS and LCM require large sample volumes (milliliters), and utilize expensive instruments used by skilled operators ([**Fig. 1a**](#pone-0017455-g001){ref-type="fig"}). FACS can sort cells, at a single cell level in nanoliter volumes. However, FACS does not pattern these cells. Recently, these traditional technologies have been modified and adapted towards microfluidics [@pone.0017455-Zeng1], [@pone.0017455-DiCarlo1], [@pone.0017455-Ino1], [@pone.0017455-White1], [@pone.0017455-Ottesen1], [@pone.0017455-Toriello1], [@pone.0017455-Love1]. Microengraving [@pone.0017455-Love1], [@pone.0017455-Lin1] has low complexity; it rapidly loads individual cells, and creates low mechanical stress during cell loading. However, it suffers from limited control over number of cells per well due to manual cell loading process. These new technologies have potential for single cell genomic analysis of target cells, *e.g.* stem cells and uncultured organisms [@pone.0017455-Walker1]. However, these capabilities arrive at a substantial cost in increased design complexity, development cost [@pone.0017455-Fu1]. Further, there are challenges to control the number of cells deposited to a predetermined location. The microfluidic systems are also great tools to handle and sort cells. Although the cell handling processes have been simplified in microfluidic systems, cell tracking for sorting on chip still requires peripheral setups followed by subsequent cell separation steps. As the heterogeneity of sample increases, the types of cells that need to be tracked in the sample also increase. The outcome is that the tracking system requires more complex peripheral setups and it can require high-end computerized controls which has an impact on scalability. A great example of such systems is best demonstrated by Quake and Hong [@pone.0017455-Fu1], [@pone.0017455-Hong1], [@pone.0017455-Pushkarev1], [@pone.0017455-Marcus1]. To address these challenges, we developed a simple, high-throughput platform for single cell isolation with direct access to patterned cells ([**Fig. 1b**](#pone-0017455-g001){ref-type="fig"}). The methodology is based on a "drop-on-demand" cell patterning technology that follows simple random sampling (SRS) [@pone.0017455-Ross1], [@pone.0017455-Sa1], [@pone.0017455-Grinstead1]. The system patterned an array of 10×10 droplets that encapsulated single cells from a heterogeneous cell mixture at a high-throughput of within 4 seconds. Subsequently, imaging systems with a wide field of view are available to monitor the patterned droplet array within a few seconds showing positions of target cells [@pone.0017455-Moon1]. Since the droplets are printed onto a glass surface, each encapsulated cell of interest can then be accessed freely.
{#pone-0017455-g001}
10.1371/journal.pone.0017455.t001
###### Performance comparison with conventional methods: isolating single cell\'s from a heterogeneous cell mixture.
{#pone-0017455-t001-1}
Categories FACS [@pone.0017455-Fu2] Microfluidics (μFACS [@pone.0017455-DiCarlo1], [@pone.0017455-Ino1], [@pone.0017455-White1], [@pone.0017455-Ottesen1], [@pone.0017455-Toriello1], [@pone.0017455-Marcy1]) Single cell droplet
------------------------------------------------- --------------------------- -------------------------- --------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ----------------------------------
Cell viability[\*](#nt101){ref-type="table-fn"} Overall process viability 80% 97% 97%
Platform Cell isolation method Electrostatic deflection Fluidics Droplet array
Throughput Parallel processing Low Medium High
Unit processing time ∼1 msec/cell ∼3 sec/cell ∼50 µsec/cell
Usage Heterogeneous sample Yes Yes [@pone.0017455-Fu2] Yes
Single cell accessibility Tube/dilution Closed channel/chamber Open-top nanoliter droplet array
Application Cell sorting Single cell PCR RNA analysis
\*Cell viability = post processing cell viability/reservoir cell viability.
In this paper, we experimentally and theoretically analyzed the cell encapsulation process in microdroplets. We analyzed the cell encapsulating droplets for cell viability and performed genomic analysis on the printed stem cells and compared them to the non-patterned controls. Here, we present the first time genomic analysis performed on cells patterned using cell printing indicating that the cells are genomically functional through the printing process compared to the controls. We used immunostaining prior to patterning to distinguish target cells within the heterogeneous population. Based on the optical images, each patterned droplet fell into one of the four categories: a droplet containing (i) a single target cell, (ii) no cells (empty droplet), (iii) a non-target cell, or (iv) multiple cells (of either the target and non-target cell type) ([**Fig. 2b**](#pone-0017455-g002){ref-type="fig"}). Then, we performed RNA extraction from cells within nanoliter droplets for genomic analysis. This permits high-throughput processing and reduction in time required for isolation of cells. Such novel methods can enable rapid biomarker studies in systems biology research [@pone.0017455-Megason1].
{#pone-0017455-g002}
Results and Discussion {#s2}
======================
We evaluated our drop-on-demand single cell patterning platform by measuring droplet volume, number of cells encapsulated per droplet for different cell loading concentrations and target cell content. Droplet volume was measured by two independent techniques: stroboscopic imaging in air and wetting contact angle measurements on a glass surface. Stable droplet generation at 25 Hz was achieved by employing a 34.4 kPa ejection pressure at 1.25 mPa·s viscosity. Under these conditions, the droplet diameter in air depended on the valve opening duration. The average droplet diameters in air were 220±9 µm, 245±12 µm, and 285±18 µm, corresponding to valve opening durations of 50 µs, 55 µs, and 60 µs, respectively. A 55 µs valve opening duration was chosen as the minimum value required to avoid clogging and formation of satellite droplets. We printed 50×50 array of droplets at a rate of 25 droplets per second. The array density was one droplet per mm^2^ with a 525±15 µm droplet spread diameter on the surface. This droplet spread diameter depended on the surface tension (28.3° contact angle on a glass slide) and droplet volume (7.6±0.6 nanoliter per droplet; see [**Materials and Methods**](#s3){ref-type="sec"}, *Drop-on-demand single cell patterning*).
Using these droplet generation parameters, we investigated the effect of the cell loading concentration on the patterned droplets. We obtained both experimental values and developed statistical models that describe the cell encapsulation process. Heterogeneous mixtures of mouse embryonic stem cells (mESCs) were prepared consisting of 10% to 50% stained target stem cells. The average number of target cells per droplet was calculated over 100 droplets (see [**Materials and Methods**](#s3){ref-type="sec"}, *Cell preparation and staining*). We tested cell loading concentrations ranging from a 0.5×10^5^ to 7.5×10^5^ cells/ml. Our theoretical and experimental results indicated that the 1.0×10^5^ cells/ml concentration was optimal to encapsulate single cells in droplets, since the resulting average number of cells per droplet and the standard deviation were 0.88±0.14 ([**Fig. 3a**](#pone-0017455-g003){ref-type="fig"}). The model (see **[Fig. S5a](#pone.0017455.s005){ref-type="supplementary-material"}** online) agrees with the experimental results [**Fig. 3a**](#pone-0017455-g003){ref-type="fig"}. These both indicate that the maximum single cell encapsulation event is likely to take place at the 1×10^5^ concentration. These statistics correspond to the case of single cell encapsulation within a droplet (with 36.8% probability), followed by other cases of an empty droplet ([**Fig. 3e**](#pone-0017455-g003){ref-type="fig"}, 36.8%), and a droplet containing two cells ([**Fig. 3e**](#pone-0017455-g003){ref-type="fig"}, 18.4%). Further, the number of cells per droplet was found to be independent of the target cell fraction ([**Fig. 3a**](#pone-0017455-g003){ref-type="fig"}, 10--50%) in the heterogeneous cell mixture. We observed that the standard deviation for the number of encapsulated cells per droplet was higher than ±1.5 cells when the cell loading concentrations exceeded 2.5×10^5^ cells/ml ([**Fig. 3b**](#pone-0017455-g003){ref-type="fig"}). However, the number of cells per droplet obtained by merging multiple droplets by ejection to the same location showed smaller standard deviation compared to ejecting a single droplet at a higher cell loading concentration (less than ±0.7 cells/droplet, [**Fig. 3b**](#pone-0017455-g003){ref-type="fig"}). This result is due to a small cell-to-droplet volume fraction of 1.7% using 7.6 nanoliter droplets. As we increase number of droplets, the sampling process fulfills the law of large numbers (LLN, n = 106, 90% confidence level and 15% tolerance), the cell encapsulation mechanism can be treated as a random cell sampling process from a large population of cells, *i.e.* Student\'s t-test and single factor analysis of variance (ANOVA). These statistical results as shown in [**Fig. 3**](#pone-0017455-g003){ref-type="fig"} indicate that our random cell encapsulation and patterning methods follow the central limit theorem (CLT) [@pone.0017455-Ross1], [@pone.0017455-Sa1], [@pone.0017455-Grinstead1]. This allows us to conclude that the average and standard deviation of sampled droplets can be used for statistical estimation of entire sample volume. This approach may provide a powerful tool for sampling single target cells in a high-throughput manner without searching the entire cell population in a reservoir.
{#pone-0017455-g003}
We investigated the viability of the cells patterned in droplets. The reservoir cell viability was 96.7±0.5% using a 10 µl volume at 7.5×10^5^ cells/ml. Following the 1.7 minutes of patterning process using a 19 µl sample, cell viability was observed to be 93.8±1.1% in a 50×50 array of cell encapsulating droplets. When the optimal cell concentration for single cell patterning (1.0×10^5^ cells/ml) was used, the patterned cell viability was 97.0±0.8%. The relatively higher patterning viability is due to reduced droplet packing density, which minimizes possible exposure to mechanical shear forces at the valve during cell encapsulation. Underlying this high viability was the low (1.7%) cell-to-droplet volume fraction we used during encapsulation process. Furthermore, we confirmed that patterned cell viability does not depend on the target cell concentration in the reservoir (see **[Fig. S4](#pone.0017455.s004){ref-type="supplementary-material"}** online).
Based on our statistical analysis (see [**Materials and Methods**](#s3){ref-type="sec"} *Statistical Modeling*), the single target cell encapsulation process followed Poisson distribution and matched with 90% confidence level and 15% tolerance. These results show that our system can be used to encapsulate single target cells from a heterogeneous solution that has 9±1% target cells by using only 10×10 array of droplets. This reduces time to reach to target cells without searching the entire sample volume in the ejection reservoir.
We validated that our patterning method produces droplet arrays that conform to CLT by comparing the fraction of target cells in the reservoir to the fraction in the patterned droplet array. The comparison is conducted using 10×10 droplet array subset randomly chosen from a 50×50 patterned droplet array. The fraction of target cells in the reservoir cell mixture (10%) was essentially mimicked (10±2.2%) in the patterned droplet subarrays ([**Fig. 3c**](#pone-0017455-g003){ref-type="fig"}). The other heterogeneous samples (20%, 30%, and 50%) agreed with this result. Therefore, evaluating a 10×10 droplet array allowed us to infer the target cell concentration in the reservoir. The fraction of empty droplets was also investigated with concentrations ranging from 1.0×10^5^ to 2.5×10^5^ cells/ml. Percentage of empty droplets determines the expected yield when searching for target cells (*i.e.*, cells to be isolated for further mRNA extraction and analysis, [**Fig. 3d**](#pone-0017455-g003){ref-type="fig"}). At a low cell concentration, 0.5×10^5^ cells/ml, the probability of patterning empty droplets was highest (72.8±12.8%). We choose 1.0×10^5^ cells/ml concentration to pattern single cell encapsulating droplets. The fraction of empty droplets at this concentration ranged from 32.9% to 48.3% over the range of cell mixture compositions studied. Despite these values, high-throughput target cell isolation was achieved with the automated rapid patterning capability.
The patterning process samples 10×10 of droplets (of total volume 0.76 µL) out of the total reservoir volume of 100 µL. While characterizing the statistics governing the patterned target cell droplets printed from a heterogeneous cell mixture, we observed agreement (0.9%∼7.0% difference) between the reservoir mixture composition and the composition observed in patterned subarrays for 10% to 50% target cell containing mixtures ([**Fig. 3e**](#pone-0017455-g003){ref-type="fig"}). Starting from a cell reservoir containing a 10% fraction of target cells, a target homogeneous droplet fraction of 9.5±1.4% was observed in a 10×10 droplet subarray at a reservoir cell density of 1.0×10^5^ cells/ml. Experimental results indicate that isolation of "a droplet containing a single target cell" within a 10×10 array is likely (at \>90% confidence level) for target cell fractions down to 1% ([**Fig. 3e**](#pone-0017455-g003){ref-type="fig"}). This lower limit of sampling fraction, 1% v/v, assumes the empty droplet percentage to be at the maximum value that we experimentally observed to be as 48.3% at 1.0×10^5^ cells/ml concentration. Moreover, when a droplet contained more than three cells, the fraction of droplets containing only target cells became zero in the 10×10 droplet subarray. We also experimentally observed the highest probability to pattern homogeneous droplets at the reservoir concentration of 1.0×10^5^ cells/ml. At the 1.0×10^5^ cells/ml reservoir concentration, the values for homogeneous droplet occurrences were 4.7±0.6%, 9.0±1.9%, 12.7±1.6%, and 21.6±5.2% for 10%, 20%, 30%, and 50% target cell concentrations, respectively ([**Fig. 3f**](#pone-0017455-g003){ref-type="fig"}). Our results demonstrate that higher single cell encapsulation probability yields higher homogeneity as dictated by Poisson distribution and shown by experimental results.
Finally, we evaluated the sensitivity and reproducibility of a functional genomic analysis of stem cell encapsulating droplets. For these evaluations, we used DNA microarrays to measure gene expression levels in RNA samples extracted from mouse embryonic stem cells obtained by using drop-on-demand. We compared the results with RNA extracted from a control pool of mESCs, which were isolated with serial dilution and manual pipetting. The total RNA quality from the droplet-based isolated cells was similar to that of the control when assessed with the Agilent bioanalyser using the 28s/18s ratio ([**Fig. 4a**](#pone-0017455-g004){ref-type="fig"}). This demonstrated that RNA remains intact throughout the patterning process. In a genome-wide analysis, the 1000 genes with the highest expression levels were measured on DNA microarrays for the printed and control cells. The reproducibility of the gene expression is illustrated by the scatter plots of the microarray measurements on replicate samples ([**Fig. 4b, c**](#pone-0017455-g004){ref-type="fig"}). Although the expression levels exhibited greater variability at the lower expression levels than the control samples, the median coefficient of variation (CV) across three replicates was 5% in both cases. Stem cell-related markers were utilized to assess whether the RNA obtained from the printed cell group provided useful biological information in comparison to the control samples. Altogether eleven stem cell markers including Kit and Notch1 were found in both the printed cells and non-printed control groups ([**Fig. 4d**](#pone-0017455-g004){ref-type="fig"}). These results indicate that we were able to successfully isolate and analyze mRNA from cell encapsulating droplets for functional genomic studies. (See [**Materials and Methods**](#s3){ref-type="sec"}, *Total RNA extraction and analysis*).
{#pone-0017455-g004}
There are also limitations on the drop-on-demand approach for single cell sorting compared to the microfluidic deterministic cell encapsulation approaches. As the heterogeneity of sample increases, the types of cells that need to be tracked in the sample also increase for microfluidic systems. The outcome is that the tracking system requires more complex peripheral setups and it can require high-end computerized controls which has an impact on scalability [@pone.0017455-Fu1], [@pone.0017455-Hong1], [@pone.0017455-Pushkarev1]. The drop-on-demand cell encapsulation approach presented here has a trade-off from a deterministic aspect, but on the other hand it offers lesser handling steps to sort and pattern cells, where cells might be affected by the conditions in the physical environment. For single cell encapsulation, microfluidic method is more deterministic and may provide a better control over cell encapsulation. Further, the deterministic approach offers an efficient system to handle minimum number of cells, i.e. single to ten cells. Second, microfluidic approach can be convenient in case an integrated on-chip experiment requires further cell handling steps after sorting such as on-chip polymerase chain reactions (PCR). Finally, throughput of drop-on-demand sorting process is limited by the parallel printing setup and the wide field of view imaging method.
We demonstrated a high-throughput drop-on-demand single cell isolation technology and presented how it fits to the statistical models. Our data revealed that the drop-on-demand single cell patterning platform can isolate viable target stem cells from a heterogeneous sample. In addition, this patterning approach can be adapted to generate multiple droplet arrays in parallel, further enhancing throughput. This work marks the first genomic analysis study on printed cells. Furthermore, we showed that functional genomic information, specifically mRNA expression levels, obtained from ejected cells was preserved throughout the entire process.
Materials and Methods {#s3}
=====================
Cell preparation and staining {#s3a}
-----------------------------
The work space is a HEPA filter equipped sterile hood (Cleanroom International, 13202). All materials were decontaminated with 70% ethanol and RNaseZAP® (Applied Biosystems) prior to introducing the cell solutions. Mouse embryonic stem cells (mESCs, wild type R1 cell line, ∼20% confluent) [@pone.0017455-Hwang1] were trypsinized (10×0.5 Trypsin-EDTA, Gibco, 15400) and passaged from Corning® flask (Corning, CLS3150) into a Falcon™ tube (BD, 352096). The cell solution was centrifuged at 1000 rpm for 3 minutes and cells were washed with DPBS (Dulbecco\'s phosphate buffered saline, Dulbecco, P1010) and resuspended in culture medium. The culture medium was prepared from 500 ml Knockout ES Media (Gibco, 11965-092), 50 ml ES FBS (Gibco, 10439-024), 1 ml 2-Mercaptoethanol (Sigma, P4333), 5 ml NEAA (Non-essential amino acid, Sigma, M7145), 5 ml L-Glutamin (Gibco, 25030), 2 µl LIF/ml (Leukemia inhibitory factor, Millipore, ESGRO®), and 5 ml Pen/Strep (Sigma, P4333). All components were passed through a sterile filter (500 ml Express Plus 0.22 µm membrane, Millipore, SCGPU05RE). A sample/aliquot of the cell solution was stained with 0.4% Trypan Blue solution (Invitrogen, 15250061) and counted with a hemacytometer (Hausser Scientific, 1483). Two sets of low (1×10^5^, 1.5×10^5^, and 2×10^5^ cells/ml) and high (2.5×10^5^, 5.0×10^5^, and 7.5×10^5^ cells/ml) cell concentrations were prepared. Using a molecular probes Live/Dead assay (Invitrogen, L-2334) for mammalian cells, pre- and post-ejection cell viabilities were recorded. Also, to make a heterogeneous cell mixture that contains both stained and non-stained cells, two samples of each concentration of cells were prepared and stored in separate test tubes. After staining with a Live/Dead assay and washing with DPBS to remove excessive staining solution, the stained and unstained cells were mixed to prepare 10% to 50% volume fraction heterogeneous solutions. These solutions served as models that represent different fractions of target cells. Pre-ejection cell viability was measured from samples taken directly from the cell solutions (see **[Fig. S4](#pone.0017455.s004){ref-type="supplementary-material"}** online). Post-ejection cell viability was measured from droplets ejected through the 150 µm valve orifice with a frequency ranging from 1 Hz to 1 kHz. The pulse duration was 55 µs to 65 µs, at 34.5 kPa of gas pressure.
Drop-on-demand single cell patterning {#s3b}
-------------------------------------
Drop-on-demand can generate droplets both with spatial control over position and temporal control over ejection. The single cell droplet ejector system consists of an automated xyz stage (NLS4 Series Precision Linear Stage, Newmark systems Inc.) and a sub-nanoliter dispensing valve (TechElan LLC, G100), which were synchronized with a control program, Labview™ (Labview, National Instruments Corporation) (see **[Fig. S1a](#pone.0017455.s001){ref-type="supplementary-material"}** online). The spatial resolution and repeatability of the stage are 0.13 µm and 5 µm, respectively. To image *in-situ* cell encapsulating droplets during the ejection process, a CCD camera (Edmund optics, EO-1312M) equipped with a 0.5× lens (INFINITUBE FM-200 (NT58-309, 2×) and a PL/FD-195 OBJ objective (NT59-115, 0.25×, Edmund optics) was combined with a synchronized light controller (S4000 strobe controller, Edmund optics) for stroboscopic illumination. The overall system, i.e. xyz stage, sub-nanoliter dispensing valve, and stroboscopic light controller, was synchronized and programmed by a PC-based DAQ control board (NI cDAQ-9172 and NI-9401, National Instruments). Pressure regulated nitrogen gas was connected to a syringe reservoir through an adapter cap (KDS503S6, Techni-Tool Inc.), and the syringe was connected to the sub nanoliter dispensing valve by tubing (Tygon® tube, Fisher scientific Inc.).
A heterogeneous sample comprising a mixture of cells and cell media solution was loaded into a 1 ml syringe reservoir and each droplet was printed at pre-determined positions on a glass surface or into a 96-well plate. The number of cells per droplet was primarily determined by the droplet size and the cell concentration in the printing solution. Prior to taking a 100 µL cell solution, the Falcon tube was vortexed. The reservoir was stirred also after loading samples to maintain an even cell distribution within the medium. High-frequency actuation (1 kHz) was used to eject the excess cell solution, after which the valve was washed out with DI water (∼60°C). We calibrated droplet size and maintained ejection stability to ensure that the orifice was free of cell debris and medium residues. Once this process was completed, 70% ethanol was ejected through the valve, leaving a small amount of ethanol in the syringe and valve until the next experiment. This ensured clean, repeatable, and reliable operation. The size of each droplet was determined by stroboscopic images during stable droplet formation. Adjustable ejection parameters were xyz stage speed, valve opening frequency and duration, and gas pressure. The valve opening time and gas pressure were regulated to control the droplet size and number of cells per droplet. A droplet size measurement was conducted both in air and on the glass substrate to provide feedback to the manual/automatic droplet size controller. Droplet stability was tested using 90 µs valve opening time and 34.5 kPa pressurized nitrogen gas. Stroboscopic images of ejected droplets were taken every second (see **[Fig. S2a](#pone.0017455.s002){ref-type="supplementary-material"}** online). If the ejector was not operated in a stable regime, satellite droplets were generated during ejection. Control over stable droplets without satellites was maintained at optimized ejection conditions, *i.e.* 64 µs valve opening time, 34.4 kPa pressurized nitrogen gas, and 25 Hz ejection frequency (see **[Fig. S2b](#pone.0017455.s002){ref-type="supplementary-material"}** online). The size of the ejected droplets was investigated in air and at the receiving surface of a slide glass substrate by ejecting 100 droplets. Three pressure and ejection conditions were used at 34.4 kPa of air pressure for pulse durations of 55 µs, 60 µs and 65 µs, yielding droplets of different sizes. As shown in [**Figure 2b**](#pone-0017455-g002){ref-type="fig"}, we ejected multiple droplets under same ejection conditions to investigate the droplet size uniformity. A liquid nitrogen bath was prepared by inserting a 60 mm (D)×15 mm (H) Petri dish inside a Styrofoam box, filling the dish and surrounding box area with liquid nitrogen, and placing the box 20 mm underneath the valve. A fluorescent microscope (Eclipse TE-2000 U, Nikon) equipped with a CCD camera (Spot RT-KE Mono, RT700, Diagnostic Instruments, Inc.) was paired with a software (Spot Advanced, Diagnostic Instruments, Inc.) to capture images of the frozen droplets at 10× and 20× magnification. Droplet diameters were obtained with the software by fitting circles around each droplet image (Spot Advanced). To measure the diameter of the ejected droplets on the surface (see **[Fig. S2c](#pone.0017455.s002){ref-type="supplementary-material"}** online), the same stroboscopic image setup was used after changing the lens to a 0.25× magnification tube (INFINITUBE FM-200 (NT58-309, 2×) and a PL/FD-390 OBJ objective (NT59-117, 0.125×), Edmund optics).
Droplet arrays (50×50 in size) were patterned at 25 Hz at 34.5 kPa of nitrogen gas pressure onto a 75 mm×50 mm glass microscope slide. The distance between the valve ejector and the slide was 1.5 mm. The drop-to-drop distance was determined to be larger than the droplet size to avoid overlapping patterns. The microscope slide was then placed inside a covered 100 mm (diameter)×15 mm (height) Petri dish. To avoid droplet evaporation while imaging, PBS surrounding the patterned droplet area was used to create a local humid environment. The droplet array was searched for target cells using a fluorescent microscope with an automatic stage (Axio observer equipped with Axio-Cam MRm, Carl Zeiss Micro-Imaging GmbH). Standard deviations were calculated from the average number of cells encapsulated per droplet on the patterned droplet array (see **[Fig. S3](#pone.0017455.s003){ref-type="supplementary-material"}** online).
Total RNA extraction and analysis {#s3c}
---------------------------------
To prevent contamination RNase AWAY® (Sigma Aldrich), a ribonuclease decontaminant, was ejected through the valve, followed by nuclease-free water (Applied Biosystems) in preparation for cell encapsulating droplet ejection. Then, the entire area surrounding the ejector system was sprayed and wiped with RNaseZAP®. Three rows in a 96-well plate were filled with 100 µL of RLT Buffer solution (QIAGEN, 1% mercaptoethanol) to lyse the ejected cells and to preserve the RNA. The 96-well plate was then placed underneath the ejector and one droplet was ejected into each of the first nine wells of the first row of wells. This procedure was repeated with additional four rows of the plate, where two, three, four and five droplets were ejected in each well resulting to a total of five groups of droplet samples, each group having an increasing droplet volume and increasing number of cells ranging from ten to fifty cells. For consistency of bioanalysis, before the RNA analysis each row was divided into three triplicates. Hence, the analysis was based on average 30, 60, 90, 120, and 150 cells for each triplicate for bioanalysis. To investigate nonprinted cells, 0.5 µl samples were drawn from the cell solution and pipetted into the three wells in each of the first three rows of the plate as controls. The controls had 1000 cells on average. Qiagen RNeasy Minikit (QIAGEN) was used to purify the total RNA. From the 96 well plate sample, triplicates were used to form single 300 µl samples, after which each sample was transferred into a QIAshredder column in a 2 ml collection tube. The lysate was homogenized by placing the tubes in a microcentrifuge for 2 minutes at 5000 rpm. Each flow-through was transferred to a fresh 1.5 ml eppendorf tube and 300 µl of 70% EtOH was added to each tube and mixed by pipetting. Each lysate solution was then applied to an RNeasy mini column in a 2 ml collection tube and spun for 15 seconds at 10,000×g, discarding the flow-through afterwards. A buffer, 350 µL, RW1 (QIAGEN, RNeasy Minikit) was added to each column, and the columns were centrifuged for 15 seconds at 10,000×g, discarding the flow-through afterwards. This step was repeated. Then the columns were transferred to new 2 ml catch tubes. Next, 500 µL of buffer RPE (QIAGEN, RNeasy Minikit) was pipetted into each RNeasy column, and the tubes were centrifuged for 15 seconds at 10,000×g (gravity), decanting the flow-through afterwards. Again, 500 µL of RPE buffer was pipetted into the tubes, and the columns were centrifuged for 15 seconds at 10,000×g and placed in new 2 ml catch tubes and spun at full speed for one minute to ensure that the membranes were dry. The columns were then transferred to new 1.5 ml collection tubes. The tubes were left at room temperature for one minute after pipetting 52 µL of ribonuclease-free water directly onto each membrane and then centrifuged for 1 minute at 10,000×g. The elution was repeated with the same volumes into the same tubes. The resulting eluant was speed vacuumed for 45 minutes at room temperature, and all samples were equilibrated to 15 µL. With the 2100 Expert® software, the isolated RNA was analyzed for quantity and quality using the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, using the RNA 6000 Pico Series II assay). All data is MIAME compliant and that the raw data has been deposited in a MIAME compliant database (GEO accession ID: GSE24330).
The total RNA was amplified with Ambion\'s Illumina TotalPrep RNA amplification kit (Ambion, Inc. Austin, TX). A brief description of the amplification/purification process is as follows: Reverse transcription to synthesize first strand cDNA. 2--3 ng sample (total RNA) is primed with the T7 oligo (dT) primer to synthesize cDNA containing a T7 promoter sequence. Second strand cDNA synthesis converts the single-stranded cDNA into a double-stranded DNA (dsDNA) template for transcription. The reaction employs DNA polymerase and RNase H to simultaneously degrade the RNA and synthesize second strand cDNA. Next, cDNA purification removes RNA, primers, enzymes, and salts that would inhibit *in vitro* transcription. *In vitro* transcription to synthesize cRNA generates multiple copies of biotinylated cRNA from the double-stranded cDNA templates. This is the amplification and labeling step. Then cRNA purification removes unincorporated NTPs, salts, enzymes, and inorganic phosphate. After purification and fragmentation, the cRNA is ready to use with direct hybridization array kits. For hybridization to arrays (AppliedMicroarrays CodeLink Mouse Whole Genome Bioarray), 10 µg of labeled cRNA was fragmented, denatured at 90°C for 5 min, and then hybridized at 37°C for 20 hours. The arrays were then washed in buffer at 47°C for 60 minutes. After washing, the arrays were incubated at room temperature for 30 minutes with a fluorophore (Streptavidin-Alexa Fluor 647, Invitrogen Eugene, OR), run through a series of 5 minute rinses, spun dry, then scanned on a GenePix 4000B scanner (Axon Instrument, Sunnyvale, CA). The Mouse Whole Genome Bioarrays contains 38,313 single 30-mer oligonucleotide probes. For the gene expression measurements were normalized to a median expression level of 1.0 across the array. 1000 genes with the highest expression levels "\>1" in the experimental samples was used for comparisons. Three Bioarrays were run for both the experimental and control samples to enable statistical analysis of the reproducibility of the gene expression measurements for the most abundant genes.
Statistical modeling {#s3d}
--------------------
Statistics is widely adapted to biological analysis. Number of cells used in experiments are large, and these experiments follow the law of large numbers (LLN) and sampling processes are typically random, i.e. simple random sampling (SRS) [@pone.0017455-Ross1], [@pone.0017455-Sa1], [@pone.0017455-Grinstead1]. In our statistical modeling, we described cell encapsulation as a random process with three random variables and their probability distribution functions (PDFs) where cells are encapsulated in droplets by a mechanical valve from a heterogeneous cell mixture. We followed three steps to model our target single cell encapsulation process. Based on experimental results, we (i) defined our system as a random system with random variables, (ii) checked whether each process was biased or un-biased, (iii) established statistical models with respect to each process and found parameters for the statistical models, e.g. mean (μ), variation (σ), and Poisson distribution parameter (λ), and (iv) evaluated overall system efficiency based on both CLT and SRS. Following these steps, we modeled our system as a random process (see **[Table S1](#pone.0017455.s006){ref-type="supplementary-material"} and [Fig. S5](#pone.0017455.s005){ref-type="supplementary-material"}** online).
Supporting Information {#s4}
======================
######
**Homogeneous single cell droplet sorting system.** A computerized xyz stage was synchronized with a pulse controller, Labview™. The automated stage positioned the substrate with 5 µm spatial resolution. Two ejectors and a 10× camera permitted *in-situ* imaging and lysing of the droplets. Schematic of droplet ejector showed cells flowing into the valve driven by a controlled air pressure pulse (34 kPa for 55 µs). A heterogeneous sample, mixture of cells and media solution, was loaded into a reservoir. Each droplet was placed at a predetermined position.
(TIF)
######
Click here for additional data file.
######
**Controlling droplet size.** Image and illustration of ejected droplet in air were shown. (**a**) Stroboscopic images of ejected droplets. When the valve opening time and the gas pressure is not optimized, we observed satellite droplets. The sequential images were taken every second. (**b**) Stable droplets, D~air~ = 245±12 µm (V≈7.7±1.2 nl), were generated during optimized ejection: *i.e.* 55 µs valve opening time, 34.4 kPa pressurized nitrogen gas, and 25 Hz ejection frequency. The droplet size was measured at 2.3 mm distance from the ejector. (**c**) Droplet size at bottom surface. After landing on the surface, the diameter of the ejected droplet was determined by the surface tension and droplet volume. H~droplet~ = 68±2 µm, D~droplet~ = 525±15 µm, (V≈7.6±0.6 nl). Scale bars are 1 mm in **a** and **b**, 100 µm in c, respectively.
(TIF)
######
Click here for additional data file.
######
**High throughput droplet patterning system, images of printed droplet array.** One hundred droplets were inspected for target homogeneous droplet sorting. (**a--d**) droplet array was generated under four different conditions, (**a**) 1×10^5^ cells/ml with 10% target cell concentration, (**b**) 1×10^5^ cells/ml with 50% target cell concentration, (**c**) 2×10^5^ cells/ml with 10% target cell concentration, (**d**) 2×10^5^ cells/ml with 50% target cell concentration. Scale bar, 1 mm.
(TIF)
######
Click here for additional data file.
######
**Pre- and post-patterning cell viability.** (**a**) Normalized cell viability was obtained comparing the two viabilities. Average and standard deviation of normalized viability were 96.4±0.8%, 97.9±2.3%, 97.2±2.2%, and 96.0±2.4% from 0.5×10^5^ to 2×10^5^ cells/ml of cell loading concentrations (n = 3 sets left y-axis reference). (**b**) Pre-patterning (flask) viabilities were measured based on 200 cells in a 10 µl sample volume. Post-print cell viabilities were obtained from 2500 droplets for each cell loading concentration, *i.e.* 70 cells at 0.5×10^5^ cells/ml and 575 cells at 2×10^5^ cells/ml. Overall mean and standard deviation of pre- and post-printing cell viabilities were 96.7±0.5% and 93.8±1.1%, respectively (right hand y-axis).
(TIF)
######
Click here for additional data file.
######
**Probability of encapsulating a single target cell in a droplet was presented by probability distribution functions, P(** ***X*** ** = ** ***X*** **~single\ target\ cell~).** (**a**) The number of homogeneous droplets was modeled using Poisson distribution in a random variable space, *i.e.* number of target cells. The model was verified using a coefficient, λ, and experimental results. (**b**) According to four different cell loading concentrations and target cell concentrations, 16 Poisson distributions resulting from 4 cases of concentration times 4 cases of target cell mixture concentrations and their coefficients, λ, were obtained comparing to experimental results. As cell loading density increases, target cell concentration, λ values increase, λ~max~ = 0.95 and λ~min~ = 0.03. Based on the analysis, statistical models for 1.0×105 cells/ml with 10% target cell mixture were based on λ = 0.05.
(TIF)
######
Click here for additional data file.
######
**Statistical modeling results for drop-on-demand target single cell encapsulation.** (**a**) Randomness of process was verified by three variables, number of samples (n), tolerance (ε), and confidence level (1−α) using an inequality^(\*\*)^. Following the law of large numbers (LLN), minimum sample number was decided as 100 droplets for 90% confidence level and 15% tolerance. This sampling volume of a droplet represented (0.76 µl = 10×10×7.6 nl) 0.76% of the total volume of the ejection reservoir (0.1 mL). (**b**) Random processes have different probability distribution functions in accordance with their parameters, *i.e.* number of cell containing droplets (*X~drop~*), number of cells in a droplet (*X~cell~*), number of target cells per droplet (*X~target\ cell~*), and number of single target cell containing droplets (*X~single\ target\ cell\ drop~*). These four different random variables are represented by three PDFs to statistically model the cell encapsulation process, i.e. binomial, Poisson, and normal distributions. We investigated probability values and parameters, λ, for each case with respect to the cell loading concentrations, cell volume fraction, and percentage of target cells. (**c**) In the case of simple random sampling (SRS) process, statistical characteristics of a small sampling volume could represent the characteristics of a large population based on the central limit theorem (CLT). In our experiments, the target cell fraction (*F* ~%~) shows same concentration as the reservoir concentration for 10% to 50% at 1.0×10^5^ cells/ml concentration (C~opt~) under conditions of 90% confidence level and 15% tolerance.
(DOCX)
######
Click here for additional data file.
Microarray analyses were performed using BRB ArrayTools developed by Dr. Richard Simon and BRB-Array Tools Development Team. We would like to thank Pei-Ann Lin as a high school student, as well as Dr. Can Erdonmez and Dr. Emre Kayaalp for their kind feedback.
**Competing Interests:**The authors have declared that no competing interests exist.
**Funding:**The authors would like to acknowledge NIH R21 EB007707, and the W.H. Coulter Foundation Young Investigation Award. This was also partially supported by RO1 A1081534, R21 AI087107, and Integration of Medicine and Innovative Technology (CIMIT) under U.S. Army Medical Research Acquisition Activity Cooperative Agreement, as well as made possible by a research grant that was awarded and administered by the U.S. Army Medical Research & Materiel Command (USAMRMC) and the Telemedicine & Advanced Technology Research Center (TATRC), at Fort Detrick, MD. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
[^1]: Conceived and designed the experiments: SM Y-GK ML L-LH UD. Performed the experiments: SM ML. Analyzed the data: SM Y-GK LD ML EH RJ L-LH UD. Contributed reagents/materials/analysis tools: SM ML. Wrote the paper: SM Y-GK LD ML EH RJ L-LH UD.
|
{
"pile_set_name": "PubMed Central"
}
|
Background
==========
There is great interest in understanding genetic factors related to variable expression of genes. Recently, several studies have shown the first evidence of heritability of mRNA between individuals \[[@B1]-[@B6]\]. By treating the expression phenotypes for each transcript (or probe) as a quantitative phenotype, a variance-components linkage analysis could be used \[[@B1]-[@B6]\]. The expectation is to detect linkage signals between the gene expression values and genomic regions. As pointed out by William et al. \[[@B6]\], several issues plague these studies, including the selection of informative expression values. Principal components (PC) is a dimension-reduction approach \[[@B7]\] and it has been shown to be a valuable tool in linkage analysis of correlated phenotypes \[[@B8]\]. Multivariate linkage analysis has been shown to be useful in identifying genomic regions with pleiotropic effect \[[@B9]\]. Given that the PC approach is another way to combine information from multiple phenotypes, it can be hypothesized that PC analysis might also be helpful in the identification of pleiotropic effects. Because of the large number of expression phenotypes in the Genetic Analysis Workshop 15 (GAW15) Problem 1 data set, we first selected the most genetically informative phenotypes based on those with high heritability estimates \[[@B10]\]. In this paper, we examine whether combinations of correlated expression phenotypes improved the linkage signals using PC and whether the PC results suggest new pleiotropic effects.
Methods
=======
Data
----
The GAW15 Problem 1 CEPH (Centre d\'Etude du Polymorphisme Humain) data consisted of 196 participants from 14 three-generation pedigrees with 14 individuals per family, 4 grandparents, 2 parents, and 8 offspring. Two hundred and seventy-six arrays including data on 3554 probe sets on the Affymetrix Human Focus Arrays were provided by GAW15. These probe sets had been selected as those with greatest inter-individual variability from a total of 8500 probe sets \[[@B2]\].
Selection of phenotype subsets
------------------------------
To increase the number of informative phenotypes, we excluded genes whose expression had little variation (standard deviation ≤0.3) and low call rates (absent calls \>90%) across samples; 3306 phenotypes (probe sets) remained. We further reduced the number by identifying those that were most likely to be genetic based on heritability estimates from a polygenic model, resulting in 45 phenotypes. Additional details of the selection process can be found in de Andrade et al. \[[@B10]\].
Principal components
--------------------
Principal-components (PC) analysis is a data reduction technique in which each component is a linear combination of the phenotypes, each PC describing as much variability of the phenotypes as possible \[[@B7]\]. Because the 45 phenotypes are on a common scale, the decompositions were made using the unscaled covariance matrix. The first ten PCs accounted for 84% of the variance in the 45 phenotypes; 14 components would have been required to explain 90% of the variance.
Genetic data
------------
For a subset of subjects, including founders, we observed a large number of missing genotypes. Recognizing that missing data can impact identity-by-descent (IBD) estimation \[[@B11]\] when there is linkage disequilibrium, we reduced the extent of linkage disequilibrium between single-nucleotide polymorphisms (SNPs) by removing SNPs with *r*^2^\> 0.30 using ldSelect \[[@B12]\]. Of the 2756 markers provided by GAW15, 2272 remained (mean spacing 1.2 cM). We then removed 2205 Mendelian inconsistencies (0.5% of matings/genotypes) primarily by removing the conflicting offspring genotypes. Multipoint IBD (MIBD) sharing among pairs of relatives was calculated using SIMWALK2 \[[@B13]\].
Quantitative trait linkage analysis
-----------------------------------
Prior to the linkage analysis, the 55 phenotypes (45 expression phenotype + 10 PCs) were normally transformed using the empirical normal quantile transformation \[[@B14]\], which has been shown to have increased power for variance-components analysis \[[@B15]\]. Variance-components linkage analyses were performed using the S-Plus/R library *multic*\[[@B16]\]. Sex was used as a covariate. We assessed the 55 phenotypes for evidence of linkage and considered \"strong\" linkage evidence as *p*\< 10^-9^, which is comparable to Table [1](#T1){ref-type="table"} in Morley et al. \[[@B2]\], and \"moderate\" linkage evidence as *p*\< 10^-4^for comparison with the single probe analyses. Finally, for the 45 expression phenotype models, we used a screening tool proposed by de Andrade et al. \[[@B17]\] to estimate bivariate linkage results. For those phenotypes that suggested strong bivariate linkage using the screening tool, bivariate linkage analysis was also performed \[[@B18]\].
######
Expression phenotypes with the strongest agreement and evidence of linkage for the Morley et al. \[2\], single-probe, and principal-components analyses
Morley et al. \[2\] Single probe Principal components
---------- -------- ------------- --------------------- -------------- ---------------------- ------- --- ------ ----------- -------
CH13L2 1p13.3 213060.s.at \<10^-10^ 11.6 \<10^-12^ 111.8 3 2.9 \<10^-3^ 112.2
ZP3 7q11 210910.at \<10^-9^ 13 \<10^-14^ 75.6 4 2.9 \<10^-3^ 64.3
PSPHL 7p11 205048.s.at \<10^-10^ 6.1 \<10^-7^ 64.3 4 2.9 \<10^-3^ 64.3
DDX17 22q13 208151x.at \<10^-9^ 5.9 \<10^-7^ 43.4 5 1.2 \<10^-2^ 47.7
UGT2B17 4q13 207245.at \-\-\-\-\-\-\-\-- 8.3 \<10^-9^ 62.3 2 7.1 \<10^-8^ 63.9
LRAP 5q15 219759.at \-\-\-\-\-\-\-\-- 8.1 \<10^-9^ 99 4 5.6 \<10^-6^ 97.7
HLA-DQB1 6p21.3 209480.at \-\-\-\-\-\-\-\-- 9 \<10^-10^ 36 1 10.7 \<10^-11^ 34.2
HLA-DPB1 6p21.3 201137.s.at \-\-\-\-\-\-\-\-- 8.8 \<10^-10^ 31.6 3 3.8 \<10^-4^ 38.3
The column \"LOD\" represents the maximum LOD scores in the chromosome where the genes are located with its respective *p*-values and positions in cM. The principal components results represent values at the same relative region as those found using the two other methods (single probe, Morley et al. \[2\]), even though the PC results are not composed of a single probe. The column \"PC\" lists the principal component that was used to find the maximum LOD score. The four bottom probes met our *p*-value \< 10^-9^criteria but did not appear on Morley\'s top 13 list. There were an additional 9 probes on Morley\'s list that were not in our top 45.
Results
=======
Figure [1](#F1){ref-type="fig"} shows the relative weighting for the first five PCs. For each component, the bar height represents the relative influence of a particular probe. Also included is the gene and gene location associated with each expression phenotype. The first PC is dominated by probe 209480.at, which has a relative weighting of 0.979. This first probe was the only component to show any increased linkage signal, changing from a LOD of 9.0 to 10.7. One possible reason for this increase is that the first PC is composed of two phenotypes (209480.at, 204769.s.at) that are associated with the HLA region on chromosome 6. Figure [2](#F2){ref-type="fig"} shows the linkage analysis for chromosome 6 using these two phenotypes. Separate lines are drawn for the two univariate analyses, the first PC, and the bivariate analysis using these probes.
{#F1}
{#F2}
Table [1](#T1){ref-type="table"} compares our results with those of Morley et al. \[[@B2]\]. They used the Affymetrix normalization method and their multipoint genome-wide linkage analysis was done using SIBPAL in S.A.G.E. \[[@B2]\]. Two of our six best linkage signals for the single phenotype analysis agreed with Morley\'s; four, including the HLA region identified using the PC approach, were not found by Morley. For the remaining nine top phenotypes identified by Morley et al., we were unable to compare the results because the specific phenotypes were not part of our final 45.
Additional review of all the bivariate estimates using the de Andrade screening approach showed only two new areas to investigate that would not have been previously flagged using a criteria of *p*\< 10^-9^. The *DDX17*gene signal, identified by Morley using probe 208151.x.at (22q13.1), was increased from *p*\< 10^-6^to *p*\< 10^-8^when used with probe 207598.x.at (*XRCC2*, 7q36) in a bivariate analysis. One area in chromosome 6 had a screening *p*-value of 10^-8^using probes 220386.s.at and 320.at but actual bivariate analysis yielded a *p*-value similar to the stronger of the phenotypes (10^-6^). Neither of these potential associations were strongly grouped in the first 10 PCs. The Dead/H Box 17 (*DDX17*) is a member of the DEAD box (asp-glu-ala-asp/his) protein family of RNA helicases that are involved in diverse cellular functions including mRNA splicing, ribosome assembly, translation initiation, mRNA stability, and cell growth and division, and *XRCC2*is essential for the efficient repair of DNA double-strand breaks by homologous recombination between sister chromatids.
Discussion
==========
We compared the total number of LOD scores greater than three across the genome for the first 10 PCs with the regions identified by each of the 45 phenotypes and found that in general, the PC analyses under-performed the single probe set analysis for known signals. The component that best reproduced the single probe set analysis (Component 1) was primarily composed of that probe set and also included another probe set that focused on that region. No new signals were detected using PCs despite the strong correlation between the probes. However, we observed a strong linkage signal on chromosome 6 in the HLA region when two probe sets from the HLA region were analyzed as bivariate traits. The two other increases in linkage signals from bivariate analysis that increased our list of interesting areas were not picked up using PCs.
Conclusion
==========
We observed that although PC has been suggested as a potentially useful screening tool for identifying genes linked to a cluster of highly correlated variables/phenotypes, it was not helpful in identifying linkage signals in this data set. Based on this analysis, other data reduction techniques should be investigated.
Competing interests
===================
The author(s) declare that they have no competing interests.
Acknowledgements
================
This research was partially funded by NIH grants R01HL71917 and CA94919.
This article has been published as part of *BMC Proceedings*Volume 1 Supplement 1, 2007: Genetic Analysis Workshop 15: Gene Expression Analysis and Approaches to Detecting Multiple Functional Loci. The full contents of the supplement are available online at <http://www.biomedcentral.com/1753-6561/1?issue=S1>.
|
{
"pile_set_name": "PubMed Central"
}
|
{#sp1 .49}
{#sp2 .50}
{#sp3 .51}
{#sp4 .52}
{#sp5 .53}
[^1]: Read before the South-Western Branch of the British Medical Association on October 7th, 1903.
|
{
"pile_set_name": "PubMed Central"
}
|
INTRODUCTION {#sec1}
============
Immunization of children with conjugate vaccines has proven to be a successful strategy to prevent infections caused by various encapsulated bacteria, such as Neisseria meningitidis and Haemophilus influenzae type b (Hib) ([@B1]), and has resulted in great decreases in the incidence of N. meningitidis serogroup C (MenC) and Hib disease in the world ([@B2], [@B3]). In addition to immune memory, persisting antibodies have been suggested to be a more appropriate correlate of long-term protection against disease ([@B4]). Circulating antibodies are particularly important for sustained protection against invasive MenC disease ([@B5]).
In Europe, MenC is the second most important cause of invasive meningococcal disease (IMD), after meningococcal serogroup B. In 2012, according to the European Centre for Disease Prevention and Control, 17% of IMD cases in Europe were caused by MenC ([@B6]). In addition, recent estimates from Spain (covering the period of 2005 to 2011), Germany (2002 to 2010), and Poland (2002 to 2011) found that MenC was responsible for 13.2%, 25.0%, and 36.6% of confirmed IMD cases, respectively ([@B7][@B8][@B9]).
The Hib-MenC-tetanus toxoid (TT) conjugate vaccine (Menitorix; GSK Vaccines) is a combination vaccine containing polyribosylribitol phosphate (PRP) from Hib and polysaccharide from MenC, each individually conjugated to TT as a carrier protein. The vaccine was developed to provide protection against Hib and MenC diseases, in a single injection, to infants and toddlers ([@B10]). Hib-MenC-TT offers an alternative vaccination schedule, compared with diphtheria-tetanus-acellular pertussis (DTPa)/Hib combined vaccines coadministered with MenC conjugate vaccines.
Hib-MenC-TT primary and booster vaccinations with different vaccination schedules and different combination vaccines were shown to be safe and immunogenic for infants and toddlers ([@B11][@B12][@B27]). Primary vaccination induced persistent antibodies against both antigens up to the second year of life ([@B12][@B13][@B15]). Persistence rates after booster vaccination varied in different studies ([@B22], [@B23], [@B27]), and there are no data available regarding antibody persistence after coadministration with a pneumococcal conjugate vaccine.
As mass vaccination schedules expand, there is a risk that adding more antigens to the schedule could lead to unexpected immune interferences ([@B28], [@B29]). If initial immune responses are not as robust as initially expected, then the antibody responses may not persist as long as protection is needed. The purpose of this extension study was to explore whether differences in initial antibody concentrations and titers for the coadministered antigens in the Hib-MenC and pneumococcal conjugate vaccines translated into differences in long-term persistence.
In the original randomized study, 1,548 healthy children were vaccinated with Hib-MenC-TT or a control MenC conjugate vaccine coadministered with a DTPa- or DTPa/Hib-containing vaccine and a pneumococcal conjugate vaccine. After primary (at 2, 4, and 6 months of age) and booster (at 11 to 18 months of age) vaccinations, immune responses were induced against all vaccine components ([@B16], [@B17]). In the current follow-up study, we evaluated the persistence of the immunogenicity of the Hib-MenC-TT conjugate vaccine up to 5 years after booster vaccination, with the evaluation of MenC antibody persistence as the primary objective. The persistence of antibodies to the pneumococcal conjugate vaccines administered in the study will be addressed in a separate publication.
MATERIALS AND METHODS {#sec2}
=====================
Ethical statement. {#sec2-1}
------------------
The antibody persistence study (ClinicalTrials.gov registration no. NCT00891176) was conducted in Germany, Poland, and Spain between May and December 2009 (year 2), May and November 2010 (year 3), and April and November 2012 (year 5). The protocol was approved by the following ethics committees: Comité Ético de Investigación Clínica del Hospital Universitario de Móstoles (Madrid, Spain), Comité Ético de Investigación Clínica del Hospital Clínico San Carlos (Madrid, Spain), Komisja Bioetyczna przy Okregowej Izbie Lekarskiej (Cracow, Poland), Ethik-Kommission der Bayerischen Landesarztekammer (Munich, Germany), Ethik-Kommission der Landesarztekammer Baden-Wurttemberg (Stuttgart, Germany), and Landesamt für Gesundheit und Soziales Geschaftss der Ethik-Kommission des Landes (Berlin, Germany). The study was conducted in accordance with the principles of good clinical practice and the Declaration of Helsinki, although some deviations in terms of study documentation and adherence to the study protocol were identified. Some additional good clinical practice deviations were noted at one of the study centers, which included the finding that annual reports and updates of protocol amendments and administrative changes had not been sent to regulatory authorities, as required by local law. After full investigation by an independent department and discussions with local regulatory agencies, however, it was determined that these findings did not have an impact on the safety of participants or on data integrity.
Study design and participants. {#sec2-2}
------------------------------
Written informed consent was obtained from each child\'s parent or guardian. Healthy children who had completed primary and booster vaccinations in a previous study (ClinicalTrials.gov registration no. NCT00334334/00463437) ([@B17]) and were in the blood sampling subset in the booster study and whose parents or legal guardians were considered by the investigators to be able to comply with study requirements were included in this open, long-term persistence study. Children were excluded from the persistence study if they had received a MenC, Hib, hepatitis B virus (HBV), or pneumococcal vaccine or had developed MenC, Hib, hepatitis B, or invasive pneumococcal disease since the booster vaccination or had received an immune-modifying drug in the previous 6 months, a blood product in the previous 3 months, or an investigational drug or vaccine in the 30 days before blood sampling. In the previous primary-booster vaccination study, exclusion criteria included vaccination against diphtheria, tetanus, pertussis, polio, MenC, Hib, hepatitis B, or Streptococcus pneumoniae. Immunization with vaccines (such as hepatitis B vaccine) when the first dose was given within the first 2 weeks of life was permitted, in accordance with national recommendations.
In the initial phase III, open, randomized, multicenter study ([@B17]), Hib-MenC-TT or a control MenC conjugate vaccine (MenC-cross-reacting material 197 \[CRM~197~\] \[Meningitec; Pfizer Inc.\] or MenC-TT \[NeisVac-C; Baxter Healthcare SA\]) was coadministered with a DTPa- or DTPa/Hib-containing vaccine (all manufactured by GSK Vaccines) and one of two pneumococcal conjugate vaccines, i.e., a 10-valent pneumococcal nontypeable Haemophilus influenzae protein D conjugate vaccine (PHiD-CV) (Synflorix; GSK Vaccines) or a 7-valent pneumococcal CRM~197~ conjugate vaccine (7vCRM) (Prevenar/Prevnar; Pfizer Inc.). Hib-MenC-TT, pneumococcal conjugate vaccines, and DTPa-containing vaccines were administered at 2, 4, and 6 months of age, with booster vaccination at 11 to 18 months of age ([@B17]). The MenC-TT and MenC-CRM~197~ vaccines were administered at 2 and 4 months of age (in Poland, to comply with national recommendations, a third dose was offered at approximately 7 months of age) and at 11 to 18 months of age.
There were four study groups: children were randomized (1:1:1:1) to receive Hib-MenC-TT plus either PHiD-CV (Hib-MenC + PHiD-CV group) or 7vCRM (Hib-MenC + 7vCRM group), MenC-CRM~197~ plus PHiD-CV (MenC-CRM group), or MenC-TT plus PHiD-CV (MenC-TT group) ([Table 1](#T1){ref-type="table"}). In Germany and Poland, groups administered Hib-MenC-TT were given DTPa-HBV-inactivated polio virus (IPV) vaccine (Infanrix penta/Pediarix; GSK Vaccines) and groups administered MenC conjugate vaccines were given DTPa-HBV-IPV/Hib (Infanrix hexa; GSK Vaccines) as booster vaccination. Because hepatitis B booster vaccination is not recommended in Spain, the Spanish Hib-MenC-TT groups received DTPa-IPV (Infanrix-IPV; GSK Vaccines) while the control groups received DTPa-IPV/Hib (Infanrix-IPV/Hib; GSK Vaccines) as booster vaccination. In Poland, children received a single dose of hepatitis B vaccine at birth, according to national recommendations.
######
Study groups[^*a*^](#T1F1){ref-type="table-fn"}
Group MenC vaccine Pneumococcal vaccine Coadministered vaccine
--------------------------------------------- --------------- ---------------------- ---------------------------------------------------------------------
Hib-MenC + PHiD-CV Hib-MenC-TT PHiD-CV DTPa-HBV-IPV or DTPa-IPV[^*b*^](#T1F2){ref-type="table-fn"}
Hib-MenC + 7vCRM Hib-MenC-TT 7vCRM DTPa-HBV-IPV or DTPa-IPV[^*b*^](#T1F2){ref-type="table-fn"}
MenC-CRM[^*c*^](#T1F3){ref-type="table-fn"} MenC-CRM~197~ PHiD-CV DTPa-HBV-IPV/Hib or DTPa-IPV/Hib[^*b*^](#T1F2){ref-type="table-fn"}
MenC-TT[^*c*^](#T1F3){ref-type="table-fn"} MenC-TT PHiD-CV DTPa-HBV-IPV/Hib or DTPa-IPV/Hib[^*b*^](#T1F2){ref-type="table-fn"}
The primary vaccination phase included doses at 2, 4, and 6 months. In the booster phase, vaccines were administered at 11 to 18 months.
Because hepatitis B booster vaccination is not recommended in Spain, the Spanish Hib-MenC-TT groups received DTPa-IPV and the control groups received DTPa-IPV/Hib as booster vaccination.
MenC-CRM~197~ and MenC-TT vaccines were administered at 2 and 4 months of age; in Poland, to comply with national recommendations, children were offered a third dose of MenC vaccines at ∼7 months of age.
Study objectives. {#sec2-3}
-----------------
The primary objective of this follow-up study was to evaluate the MenC antibody persistence after vaccination in all treatment groups that had received the Hib-MenC-TT conjugate vaccine, in terms of percentages of children with serum antibody levels above the assay cutoff value up to 60 months after booster vaccination (72 to 76 months of age). In this report, antibody persistence results are presented for blood samples obtained from children at approximately 3, 4, and 6 years of age. The secondary objectives included the evaluation of antibody persistence with respect to Hib capsular polysaccharide (anti-polyribosylribitol phosphate \[PRP\]) and hepatitis B surface antigen (anti-HBs) antibody concentrations.
Immunogenicity assessments. {#sec2-4}
---------------------------
Blood samples of approximately 5 ml were collected at 36 to 40 months of age (i.e., approximately 24 months after booster vaccination), 48 to 52 months of age (i.e., approximately 36 months after booster vaccination), and 72 to 76 months of age (i.e., approximately 60 months after booster vaccination). Antibodies against MenC at year 5 were measured by the serum bactericidal antibody (SBA) assay performed at Public Health England (PHE), using rabbit complement (i.e., rabbit SBA \[rSBA\]), with a cutoff value of 1:8 as an indication of protection ([@B30]). Earlier time points were tested with an in-house rSBA-MenC assay performed at GSK Vaccines ([@B16]), which prevents direct comparison of the year 5 results with the initial results from earlier study time points ([@B17]). Anti-PRP antibodies were assessed with an in-house enzyme-linked immunosorbent assay (ELISA) performed at GSK Vaccines, for which concentrations of ≥0.15 μg/ml and ≥1 μg/ml are considered indicative of short- and long-term protection, respectively ([@B31], [@B32]). Antibodies against hepatitis B were assessed at GSK Vaccines using a commercial chemiluminescence immunoassay (Centaur; Siemens Healthcare Diagnostics). Anti-HBs seroprotection was defined as an antibody concentration above 10 mIU/ml ([@B33]).
Safety assessments. {#sec2-5}
-------------------
Serious adverse events (SAEs) for the primary and booster phases of this study were reported earlier ([@B34]). In this study, SAEs that were assessed by the investigators as being potentially vaccine or study procedure related or due to a lack of vaccine efficacy were documented retrospectively, following booster vaccination, and were recorded at every visit/contact during the study. According to the definition, an SAE was any untoward medical occurrence that resulted in death, was life-threatening, required hospitalization, or resulted in disability/incapacity. Although it was unlikely that SAEs related to vaccination would occur so late after vaccination, this study was designed to ensure that any event that manifested only long after vaccination and that the investigator thought could have been related to the vaccination or any event that was related to a lack of vaccine efficacy during this long-term persistence phase and that fulfilled the definition of an SAE would be recorded.
Statistical analyses. {#sec2-6}
---------------------
Results are presented for the according-to-protocol cohorts for antibody persistence after booster vaccination, which included all eligible children who received the full primary and booster vaccination courses corresponding to their group, were compliant with the study procedures, and had available assay results. For each group, the antibody geometric mean concentrations (GMCs), the antibody geometric mean titers (GMTs), and the percentages of participants retaining antibody GMCs or GMTs above the predefined thresholds were calculated, with associated 95% confidence intervals (CIs).
Exploratory statistical analyses were performed in which potential differences between groups were defined when the asymptotic standardized 95% CI for the difference between the two groups in percentages of children with titers or concentrations above the proposed cutoff values did not contain the value 0 or when the 95% CI for the GMT/GMC ratios between groups did not contain the value 1. This analysis was performed using a one-way analysis of variance model based on the log~10~ transformation of the titers or concentrations, using the vaccine group and country as the only covariates. The results of all exploratory group comparisons should be interpreted with caution considering that there was no adjustment for multiplicity for these comparisons and the clinical relevance of the differences was not prespecified. The statistical analyses were performed using SAS software (version 9.22 on Windows; SAS Institute, Cary, NC, USA).
RESULTS {#sec3}
=======
Study participants. {#sec3-1}
-------------------
Of the 1,437 children who were part of the total enrolled cohort for the booster phase, 581, 561, and 539 were included in the total enrolled cohorts for persistence at 24, 36, and 60 months after booster vaccination, respectively. The according-to-protocol cohorts for antibody persistence included 571 children at 24 months after booster vaccination, 543 children at 36 months after booster vaccination, and 530 children at 60 months after booster vaccination. The reasons for exclusion from the according-to-protocol cohorts and the distributions for the study groups are presented in [Fig. 1](#F1){ref-type="fig"}.
{#F1}
Across the four vaccine groups, the mean ages of the children in the according-to-protocol cohorts for persistence were similar between groups and the majority of the children were of European descent ([Table 2](#T2){ref-type="table"}). The proportions of male and female subjects were similar across all groups, with a larger proportion of male subjects in the MenC-TT group and a larger proportion of female subjects in the Hib-MenC + PHiD-CV group. The gender and racial distributions in the according-to-protocol cohorts for persistence were consistent with the primary study ([@B17]).
######
Demographic characteristics of the study children (according-to-protocol cohorts for antibody persistence at 24 months \[year 2\], 36 months \[year 3\], and 60 months \[year 5\] after booster vaccination)
Parameter and time point Hib-MenC + PHiD-CV Hib-MenC + 7vCRM MenC-CRM MenC-TT
-------------------------- -------------------- ------------------ ---------- ------------ ----- ------------ ----- ------------
Age (mean ± SD) (mo)
Year 2 144 37.3 ± 1.2 140 37.3 ± 1.3 141 37.1 ± 1.2 146 37.2 ± 1.2
Year 3 133 49.2 ± 1.5 136 48.7 ± 1.2 136 48.8 ± 1.3 138 48.7 ± 1.1
Year 5 131 72.9 ± 1.0 134 72.9 ± 1.2 128 72.8 ± 1.0 137 72.9 ± 1.1
Female (%)
Year 2 144 57.6 140 50.7 141 50.4 146 44.5
Year 3 133 60.2 136 52.2 136 50.7 138 43.5
Year 5 131 58.8 134 50.7 128 49.2 137 46.0
European descent (%)
Year 2 144 95.1 140 100 141 95.7 146 98.6
Year 3 133 95.5 136 99.3 136 96.3 138 97.8
Year 5 131 96.2 134 99.3 128 96.9 137 97.8
*n*, total number of children in the group.
Immunogenicity against MenC. {#sec3-2}
----------------------------
Approximately 5 years after booster vaccination, the percentages of children with rSBA-MenC titers of ≥8, as measured by the PHE assay, were between 24.2% and 40.1% in all groups, with the highest value being observed in the MenC-TT group ([Table 3](#T3){ref-type="table"}). GMTs ranged from 7.2 in the MenC-CRM group to 11.9 in the MenC-TT group. At year 3, rSBA-MenC titers of ≥8, as measured by the GSK Vaccines assay, were retained by at least 72.4% of children in each group (see Table S1 in the supplemental material).
######
Serum bactericidal activity against MenC approximately 5 years after booster vaccination (according-to-protocol cohorts for antibody persistence at 60 months after booster vaccination)
-----------------------------------------------------------------------------------------------------------
Group *n*[^*a*^](#T3F1){ref-type="table-fn"} \% with rSBA-MenC\ rSBA-MenC GMT (95% CI)
titers of ≥8\
(95% CI)
-------------------- ---------------------------------------- -------------------- ------------------------
Hib-MenC + PHiD-CV 130 38.5 (30.1--47.4) 10.1 (7.9--12.9)
Hib-MenC + 7vCRM 134 25.4 (18.3--33.6) 8.5 (6.6--11.1)
MenC-CRM 128 24.2 (17.1--32.6) 7.2 (5.8--8.9)
MenC-TT 137 40.1 (31.9--48.9) 11.9 (8.9--16.0)
-----------------------------------------------------------------------------------------------------------
*n*, total number of children with available results.
Based on exploratory analyses, the percentages of children with rSBA-MenC titers of ≥8 at year 5 were higher in the MenC-TT and Hib-MenC + PHiD-CV groups than in the MenC-CRM group and in the MenC-TT group than in the Hib-MenC + 7vCRM group ([Table 4](#T4){ref-type="table"}). The rSBA-MenC GMT was higher in the MenC-TT group than in the MenC-CRM group. No difference between the MenC-TT and Hib-MenC + PHiD-CV groups was observed. Similar observations were made at year 3 (see Table S2 in the supplemental material).
######
Differences between groups (first group minus second group) in percentages of children with rSBA-MenC titers above the threshold and rSBA-MenC GMT ratios (first group to second group) approximately 5 years after booster vaccination (exploratory analyses; according-to-protocol cohorts for antibody persistence at 60 months after booster vaccination)[^*a*^](#T4F1){ref-type="table-fn"}
Group comparison Difference in proportions of children with rSBA-MenC titers of ≥8 (95% CI) rSBA-MenC GMT ratio (95% CI)
-------------------------------- ---------------------------------------------------------------------------- ------------------------------
MenC-CRM vs MenC-TT **−15.93 (−26.79 to −4.67)** **0.68 (0.47--0.96)**
MenC-CRM vs Hib-MenC + PHiD-CV **−14.24 (−25.26 to −2.92)** 0.73 (0.53--1.01)
MenC-CRM vs Hib-MenC + 7vCRM −1.15 (−11.62 to 9.39) 0.92 (0.67--1.28)
MenC-TT vs Hib-MenC + PHiD-CV 1.68 (−10.02 to 13.32) 1.15 (0.79--1.68)
MenC-TT vs Hib-MenC + 7vCRM **14.77 (3.59 to 25.62)** 1.38 (0.95--2.01)
For differences, 95% CIs not including 0 were regarded as indicating significant potential differences between groups. For GMT ratios, 95% CIs not including 1 indicated significant potential differences. Bold type indicates comparisons for which the exploratory analysis suggests a potentially significant difference.
Immunogenicity against Hib PRP. {#sec3-3}
-------------------------------
Approximately 5 years after booster vaccination, the percentages of children with anti-PRP concentrations of ≥0.15 μg/ml remained high in all groups (≥98.5%) ([Table 5](#T5){ref-type="table"}). Among all groups, at least 57.5% of children had anti-PRP concentrations of ≥1 μg/ml, with the highest percentages among the Hib-MenC-TT recipients (Hib-MenC + PHiD-CV and Hib-MenC + 7vCRM groups). Anti-PRP GMCs at year 5 ranged from 1.65 in the MenC-TT group to 2.95 in the Hib-MenC + PHiD-CV group.
######
Anti-PRP antibody persistence (according-to-protocol cohorts for antibody persistence at 60 months after booster vaccination)
Group and time point[^*a*^](#T5F1){ref-type="table-fn"} *n*[^*b*^](#T5F2){ref-type="table-fn"} \% with anti-PRP antibody levels of ≥0.15 μg/ml (95% CI) \% with anti-PRP antibody levels of ≥1 μg/ml (95% CI) Anti-PRP antibody GMC (95% CI) (μg/ml)
--------------------------------------------------------- ---------------------------------------- ---------------------------------------------------------- ------------------------------------------------------- ----------------------------------------
Hib-MenC + PHiD-CV
Postprimary 130 98.5 (94.6--99.8) 97.7 (93.4--99.5) 13.71 (11.11--16.93)
Postbooster M1 130 100 (97.2--100) 100 (97.2--100) 90.53 (75.73--108.23)
Postbooster M24 121 100 (97.0--100) 93.4 (87.4--97.1) 4.21 (3.41--5.21)
Postbooster M36 122 100 (97.0--100) 90.2 (83.4--94.8) 3.76 (3.03--4.66)
Postbooster M60 130 100 (97.2--100) 84.6 (77.2--90.3) 2.95 (2.40--3.62)
Hib-MenC + 7vCRM
Postprimary 132 100 (97.2--100) 97.0 (92.4--99.2) 10.70 (8.87--12.90)
Postbooster M1 130 100 (97.2--100) 100 (97.2--100) 65.65 (52.76--81.69)
Postbooster M24 125 100 (97.1--100) 84.8 (77.3--90.6) 3.77 (3.04--4.69)
Postbooster M36 125 99.2 (95.6--100) 79.2 (71.0--85.9) 2.80 (2.27--3.46)
Postbooster M60 132 100 (97.2--100) 75.8 (67.5--82.8) 2.56 (2.07--3.17)
MenC-CRM
Postprimary 127 98.4 (94.4--99.8) 86.6 (79.4--92.0) 4.23 (3.36--5.32)
Postbooster M1 127 100 (97.1--100) 99.2 (95.7--100) 33.49 (27.11--41.37)
Postbooster M24 118 100 (96.9--100) 78.0 (69.4--85.1) 2.51 (1.99--3.16)
Postbooster M36 123 99.2 (95.6--100) 67.5 (58.4--75.6) 1.94 (1.56--2.41)
Postbooster M60 127 99.2 (95.7--100) 66.9 (58.0--75.0) 1.66 (1.34--2.05)
MenC-TT
Postprimary 135 100 (97.3--100) 95.6 (90.6--98.4) 6.48 (5.48--7.67)
Postbooster M1 134 100 (97.3--100) 100 (97.3--100) 36.35 (30.22--43.73)
Postbooster M24 130 100 (97.2--100) 77.7 (69.6--84.5) 2.29 (1.84--2.85)
Postbooster M36 128 99.2 (95.7--100) 62.5 (53.5--70.9) 1.78 (1.42--2.24)
Postbooster M60 134 98.5 (94.7--99.8) 57.5 (48.6--66.0) 1.65 (1.30--2.09)
Postprimary, 1 month after the third dose at 6 months of age; postbooster M1, 1 month after booster vaccination; postbooster M24, approximately 24 months after booster vaccination; postbooster M36, approximately 36 months after booster vaccination; postbooster M60, approximately 60 months after booster vaccination.
*n*, total number of children with available results.
Based on exploratory analyses, there was no significant potential difference between groups in percentages of children achieving anti-PRP antibody concentrations of ≥0.15 μg/ml. Anti-PRP antibody GMCs were lower for the control MenC vaccine recipients than the Hib-MenC-TT groups, regardless of MenC vaccine type, and there was no difference in anti-PRP antibody GMCs between the two MenC vaccine groups ([Table 6](#T6){ref-type="table"}).
######
Differences between groups (first group minus second group) in percentages of children with anti-PRP antibody concentrations above the threshold and anti-PRP GMC ratios (first group to second group) approximately 5 years after booster vaccination (exploratory analyses; according-to-protocol cohorts for antibody persistence at 60 months after booster vaccination)[^*a*^](#T6F1){ref-type="table-fn"}
Group comparison Difference in proportions of children with anti-PRP antibody levels of ≥0.15 μg/ml (95% CI) Difference in proportions of children with anti-PRP antibody levels of ≥1 μg/ml (95% CI) Anti-PRP antibody GMC ratio (95% CI)
-------------------------------- --------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------ --------------------------------------
MenC-CRM vs MenC-TT 0.71 (−2.97 to 4.59) 9.47 (−2.34 to 20.99) 0.96 (0.70--1.31)
MenC-CRM vs Hib-MenC + PHiD-CV −0.79 (−4.34 to 2.11) **−17.69 (−27.93 to −7.31)** **0.54 (0.41--0.73)**
MenC-CRM vs Hib-MenC + 7vCRM −0.79 (−4.34 to 2.06) −8.83 (−19.76 to 2.20) **0.64 (0.47--0.87)**
MenC-TT vs Hib-MenC + PHiD-CV −1.49 (−5.29 to 1.41) **−27.15 (−37.34 to −16.49)** **0.56 (0.41--0.75)**
MenC-TT vs Hib-MenC + 7vCRM −1.49 (−5.29 to 1.37) **−18.29 (−29.18 to −6.99)** **0.65 (0.47--0.89)**
For differences, 95% CIs not including 0 were regarded as indicating significant potential differences between groups. For GMC ratios, 95% CIs not including 1 indicated significant potential differences. Bold type indicates comparisons for which the exploratory analysis suggests a potentially significant difference.
Immunogenicity against hepatitis B. {#sec3-4}
-----------------------------------
Considering the anti-HBs results determined with the chemiluminescence assay, at least 72.8% of children had antibody concentrations of ≥10 mIU/ml approximately 60 months after booster vaccination ([Table 7](#T7){ref-type="table"}). Anti-HBs antibody GMCs declined by 60 months after booster vaccination and were between 45.7 and 85.4 mIU/ml at 60 months after booster vaccination.
######
Anti-HBs antibody persistence at year 5 after booster vaccination, as assessed with a chemiluminescence assay (according-to-protocol cohorts for antibody persistence at 60 months after booster vaccination)[^*a*^](#T7F1){ref-type="table-fn"}
Group and time point[^*b*^](#T7F2){ref-type="table-fn"} *n*[^*c*^](#T7F3){ref-type="table-fn"} \% with anti-HBs antibody levels of ≥10 mIU/ml (95% CI) Anti-HBs antibody GMC (95% CI) (mIU/ml)
--------------------------------------------------------- ---------------------------------------- --------------------------------------------------------- -----------------------------------------
Hib-MenC + PHiD-CV
Postbooster M24 122 88.5 (81.5--93.6) 154.5 (106.8--223.5)
Postbooster M36 118 84.7 (77.0--90.7) 92.3 (63.8--133.6)
Postbooster M60 125 72.8 (64.1--80.4) 45.7 (32.2--64.8)
Hib-MenC + 7vCRM
Postbooster M24 119 93.3 (87.2--97.1) 218.9 (152.4--314.4)
Postbooster M36 119 89.9 (83.0--94.7) 142.5 (100.3--202.6)
Postbooster M60 128 84.4 (76.9--90.2) 85.4 (60.4--120.6)
MenC-CRM
Postbooster M24 118 92.4 (86.0--96.5) 211.1 (147.1--303.1)
Postbooster M36 120 86.7 (79.3--92.2) 130.8 (91.9--186.2)
Postbooster M60 122 82.0 (74.0--88.3) 66.7 (47.2--94.1)
MenC-TT
Postbooster M24 130 90.8 (84.4--95.1) 189.2 (134.2--266.7)
Postbooster M36 127 88.2 (81.3--93.2) 128.4 (91.3--180.6)
Postbooster M60 129 79.8 (71.9--86.4) 64.7 (46.4--90.2)
A booster vaccination for HBV was not given in Spain, in accordance with national recommendations. Data shown here include subjects from all countries combined.
Postbooster M24, approximately 24 months after booster vaccination; postbooster M36, approximately 36 months after booster vaccination; postbooster M60, approximately 60 months after booster vaccination.
*n*, total number of children with available results.
Safety. {#sec3-5}
-------
No SAEs considered by the investigators to be potentially vaccine or study procedure related or due to a lack of vaccine efficacy were reported following administration of the booster vaccination.
DISCUSSION {#sec4}
==========
This study reports the antibody persistence in healthy children up to 5 years after the Hib and MenC full vaccination course. At the time the study was conducted, children in the United Kingdom used to receive concomitant doses of Hib-MenC and pneumococcal conjugate vaccines as primary vaccinations. Our data are important in comparing persistence after booster vaccination, since children in the United Kingdom currently receive booster doses of Hib-MenC and pneumococcal conjugate vaccines simultaneously at 12 to 13 months of age ([@B35]).
For MenC, the percentages of children retaining seroprotective rSBA-MenC titers at year 5 after booster vaccination were 24.2%, 25.4%, 38.5%, and 40.1% in the MenC-CRM, Hib-MenC + 7vCRM, Hib-MenC + PHiD-CV, and MenC-TT groups, respectively. In contrast, retention of anti-PRP antibodies was nearly universal in all groups; at least 98.5% of children in each group were observed to have anti-PRP concentrations of ≥0.15 μg/ml at 5 years after booster vaccination.
The percentages of children with seroprotective rSBA-MenC titers of ≥8 were considerably lower than the rates of 72.4% to 96.4% reported 3 years after booster vaccination. The assays were performed in different laboratories with different procedures, however, and the results cannot be directly compared. It has been observed that the GSK Vaccines rSBA-MenC assay used previously has a higher sensitivity than the PHE rSBA-MenC assay used in the current follow-up study, especially for naturally acquired antibodies ([@B36]). In addition, the PHE laboratory uses a discontinuous method to interpolate titers from the standard curve, whereas GSK Vaccines uses a continuous method. The discontinuous method of titer calculation is known to result in lower titers than the continuous method.
The proportions of children with seroprotective rSBA-MenC titers of ≥8 in our study were consistent with the rates of 22% to 43% (measured with the same rSBA assay at PHE) reported in a previous study in the United Kingdom at 24 months after the administration of a Hib-MenC-TT booster dose to toddlers (12 to 14 months of age) ([@B19]). In that study, infants had been primed with two doses of MenC-CRM~197~ or MenC-TT at 2 and 3 or 2 and 4 months of age ([@B19]). In contrast, in a study in Spain in which the GSK Vaccines rSBA-MenC assay was used, greater percentages were observed more than 5 years after Hib-MenC-TT booster vaccination in toddlers, following Hib-MenC-TT or MenC-TT priming in infancy on a 2/4/6-month schedule (78% to 97%) ([@B23]). However, these results cannot be directly compared with the current study results, as different rSBA assay procedures were used.
The low MenC titers at 5 years after vaccination suggested that individuals may no longer be protected or contribute to herd immunity. The introduction of a MenC adolescent booster dose at ∼14 years of age was adopted in the United Kingdom in 2013, based on advice from the Joint Committee on Vaccination and Immunisation ([@B37]). The adolescent booster vaccination at 12 years of age was also incorporated into the vaccination calendar of all autonomous Spanish regions in 2014 ([@B7]).
The exploratory analyses revealed possible differences between groups in MenC antibody persistence. The percentages of children with rSBA-MenC titers of ≥8 at year 5 after booster vaccination were higher in the MenC-TT and Hib-MenC + PHiD-CV groups than in the MenC-CRM group and in the MenC-TT group than in the Hib-MenC + 7vCRM group. MenC GMTs were observed to be higher in the MenC-TT group than in the MenC-CRM group, which is consistent with previous observations suggesting that TT is a better carrier protein for MenC priming than is CRM~197~, in terms of seroprotection and GMTs ([@B19], [@B38]).
A clear effect of the coadministered pneumococcal conjugate vaccines could not be observed, since there were no significant potential differences in terms of MenC GMTs between PHiD-CV and 7vCRM recipients. Moreover, the control vaccines MenC-CRM~197~ and MenC-TT were coadministered only with PHiD-CV; no groups receiving these control MenC vaccines together with 7vCRM were included in this study.
High rates of seroprotection against the Hib antigen were observed in all groups. There was no significant potential difference between groups in the percentages of children with anti-PRP antibody concentrations of ≥0.15 μg/ml, and there was no decline in comparison with earlier time points. Comparison of anti-PRP antibody GMCs between groups showed better long-term persistence 5 years after booster vaccination for the Hib-MenC-TT recipients than for the DTPa/Hib recipients, regardless of the coadministered pneumococcal conjugate vaccine, which is consistent with a previous report ([@B23]). There was only a slight decrease in the year 5 anti-PRP antibody GMC values, compared to the previous time points (up to 3 years after booster vaccination). The high level of anti-PRP antibody persistence in all groups suggests that an additional Hib booster dose is not needed.
Infants and toddlers are routinely vaccinated against hepatitis B in many countries, with the expectation that such vaccinations will protect against infections that may be acquired years in the future. The purpose of assessing the hepatitis B antibody persistence was to explore the percentages of subjects retaining seroprotective antibody concentrations 5 years after vaccination with the various regimens. Our results showed that the anti-HBs seroprotection rates 5 years after booster vaccination ranged from 73% to 84% in all groups. These percentages appeared to be smaller than those at earlier time points (2 and 3 years after booster vaccination). Statistical comparisons were not performed, however, and the anti-HBs data from our study must be interpreted with caution.
In conclusion, 5 years after booster vaccination, protective anti-PRP antibody concentrations persisted in at least 98.5% of children in all groups, but protective rSBA-MenC antibody titers were shown to persist in only 24.2 to 40.1% of children, with the highest values in the Hib-MenC + PHiD-CV and MenC-TT groups. These results are consistent with previously reported Hib-MenC-TT vaccine data. No SAEs considered possibly related to vaccination were reported during the 5-year persistence phase of the study.
Supplementary Material
======================
###### Supplemental material
Supplemental material for this article may be found at <http://dx.doi.org/10.1128/CVI.00057-16>.
We thank the children and their families for participating in this trial and all investigators and their clinical teams for their contributions to this study. We also thank Melissa McNeely (XPE Pharma & Science, through GSK Vaccines) for publication management and Vasile Coman (XPE Pharma & Science) for drafting the manuscript.
D.K. and M.V.D.W. are employees of the GSK group of companies. Y.B. was an employee of the GSK group of companies and is a coinventor on a patent that, if granted, will be owned by the GSK group of companies. Y.B. and M.V.D.W. own stock options and stock grants in the GSK group of companies. J.C.T., J.B., R.K., and D.G. have no conflicts of interest.
All authors had full access to the data. J.C.T., J.B., R.K., D.G., D.K., and M.V.D.W. conceived and designed the experiments. J.C.T., J.B., R.K., and D.G. performed the experiments. D.K. and M.V.D.W. analyzed the data. J.C.T., J.B., R.K., D.G., D.K., Y.B., and M.V.D.W. contributed reagents, materials, and/or analysis tools. J.C.T., J.B., R.K., D.G., D.K., Y.B., and M.V.D.W. wrote the paper.
Menitorix, Infanrix-IPV, and Infanrix-IPV/Hib are trademarks of the GSK group of companies. Meningitec is a trademark of Nuron Biotech. NeisVac-C is a trademark of Pfizer. ADVIA Centaur is a trademark of Siemens Healthcare Diagnostics.
[^1]: **Citation** Tejedor JC, Brzostek J, Konior R, Grunert D, Kolhe D, Baine Y, Van Der Wielen M. 2016. Antibody persistence in young children 5 years after vaccination with a combined Haemophilus influenzae type b-Neisseria meningitidis serogroup C conjugate vaccine coadministered with diphtheria-tetanus-acellular pertussis-based and pneumococcal conjugate vaccines. Clin Vaccine Immunol 23:555--563. doi:[10.1128/CVI.00057-16](http://dx.doi.org/10.1128/CVI.00057-16).
|
{
"pile_set_name": "PubMed Central"
}
|
Introduction {#Sec1}
============
Emergency resuscitative thoracotomy (ERT) may serve as a lifesaving procedure for selected trauma patients presenting in extremis with pending or already witnessed cardiopulmonary collapse. Since the 1960s, the pendulum for resuscitative emergency thoracotomy has swung from conservative to a more aggressive approach, but the use, indications and risks are still debated \[[@CR1]--[@CR4]\]. Collective evidence and consensus have suggested that outcome is best for penetrating trauma patients with pending (or witnessed) cardiac arrest, with most favorable outcomes reported for patients with an isolated stab wound to the heart \[[@CR2], [@CR5]--[@CR7]\]. Notably, the majority of studies published stem from large-volume institutions in North America or South Africa, where penetrating trauma represents a predominating injury mechanism and for which surgeons and systems are readily trained to deal with these injuries \[[@CR2], [@CR8]\]. Contrary to the experience in regions with a high incidence of penetrating trauma, the predominant mechanism seen in the majority of European hospitals is related to blunt injuries, with even busy centers receiving far less than 10 % penetrating trauma. Blunt trauma victims are generally believed to have a poor outcome if cardiac arrest follows or circulatory collapse is pending. An ERT is believed to be rarely indicated in blunt trauma with cardiac arrest as outcomes are dismal in the vast majority \[[@CR2]\].
However, with the maturation of systems across Europe, an increasing reported series of resuscitative emergency thoracotomies from European populations have accumulated, even with successful experience from pre-hospital employment in one series \[[@CR9]\] and accumulating experience also from the recent wars \[[@CR10]\]. However, collectively, little is known about the outcome of resuscitative ET in the modern civilian European trauma populations besides anecdotal reports. Thus, we wanted to systematically review the reported experience and outcomes of ERT use in these civilian trauma populations and compare the results from the reported literature.
The aim of the study was to give a systematic overview of reported indications, outcomes and reports based on civilian European publications over the past decade. Secondly, we wanted to evaluate the standardized reported variables, factors and outcomes related to these reports.
Materials and methods {#Sec2}
=====================
Search methods and inclusions and exclusion criteria {#Sec3}
----------------------------------------------------
We performed a systematic literature search according to the PRISMA guidelines \[[@CR11]\] of the worldwide literature in PubMed/MEDLINE and EMBASE databases using the keywords and/or medical Subject headings (MeSH) terms "emergency thoracotomy", "emergency department thoracotomy", "resuscitative thoracotomy", "urgent thoracotomy", "Trauma", "Europe". The study was limited to the time period of January 2004 to December 2014 to present updated experiences and avoid dated reports from the past. Additional searches of other databases (EMBASE, etc.) were performed by a trained hospital librarian. The international database of prospectively registered systematic reviews (PROSPERO) was queried for potential planned or ongoing systematic reviews.
Titles and abstracts of studies were scrutinized for relevance. We included any report published in the English, German, or Scandinavian (Norwegian, Danish, Swedish) languages, and considered studies in French, Spanish or other European languages if sufficient information was obtained in English abstract form or, if further detailed information could be retrieved by contacting the authors.
The references of the identified fulltext articles were further hand-searched to retrieve additional studies. The 'grey literature' was searched using Google and Google Scholar to identify studies published outside the PubMed index, including the *European Journal of Trauma and Emergency Surgery*.
We excluded publications on the following criteria: case reports on only one or, only a couple of cases; reports of few cases (\<5 ERTs); reports from military medicine (e.g. the war in Afghanistan and Iraq); and, reports on pre-hospital resuscitative thoracotomy.
Based on small population samples, the high likelihood for heterogeneity and risk of bias in the included studies, we deferred formal meta-analysis and, rather, focused on the descriptive data and outcomes retrieved in the included reports.
Definitions used and data collected for each study {#Sec4}
--------------------------------------------------
There is no standard definition to Emergency Resuscitative Thoracotomy (ERT) in the literature and investigators use interchangeably the terms 'resuscitative thoracotomy', 'emergency thoracotomy', 'emergency department thoracotomy', 'urgent thoracotomy' and other combinations thereof, either focusing on the location of the procedure or the urgency of its indication. We scrutinized the definitions used in each paper as located either in emergency department or operating room, and as emergent or urgent in nature, and included papers that clearly reported on the procedure as part of the resuscitative or emergency management process of the trauma patient, either located in the emergency department (ED) or the operating room (OR). For consistent reading, we chose the term 'ERT' throughout the paper.
Demographic data such as geography, number of patients, age and gender were recorded for included studies.
Mechanism of injury (MOI) was defined as either blunt or penetrating. Location of major injury (LMOI) was limited to the major anatomic area of injury as cerebral, abdominal, thoracic, pelvic and/or multiple.
Injury severity was obtained from each study as reported by the injury severity score (ISS) \[[@CR12]\] and probability of survival (Ps) as per calculation by TRISS methodology \[[@CR13]\], if given.
*Survival* was obtained for each study for the entire cohort or, alternatively, for the subcohorts, as reported.
The following measures were further searched for in each paper:Indications for ERT, whether reported and, if yes, what type of indication reported (e.g. cardiac tamponade, shock, intraabdominal hemorrhage, etc.).Signs of life (SOL) if documented and how defined in each study.Vital signs, if documented and how defined.Neurological outcome, whether investigated and reported and, if yes, by what means reported (either as "good/poor") or by any formal score, such as Glasgow Outcome Score (GOS) \[[@CR14]\].
Data presentation and statistical analyses {#Sec5}
------------------------------------------
Data are presented in a descriptive manner and as reported in the respective studies. We assumed upfront that studies would be of a very heterogeneous nature and thus did not plan to perform a formal meta-analyses, as this would not be substantiated based on the very high risk of bias.
Results {#Sec6}
=======
The search result is shown in Fig. [1](#Fig1){ref-type="fig"}. For the study period, we found a total of 8 studies \[[@CR15]--[@CR22]\] that originated from Europe and for which the defined inclusion criteria were fulfilled. The study descriptives, patient demographics and main findings are reported in Table [1](#Tab1){ref-type="table"}. A further two publications were found from Norway and Ireland \[[@CR23], [@CR24]\]. Upon author contact, we were informed that 5 resuscitative emergency thoracotomies were performed, with no survivors in Tromsø, Norway. Contact with the group from Dublin failed. Due to a lack of detailed information from these sites, we could not include these studies in the collective presentation. A register study from Germany reported outcome on a subset of patients having emergency thoracotomy, but with no specific details to the group as such, and was thus excluded from the qualitative assessment \[[@CR25]\].Fig. 1PRISMA flowchart of included studies Table 1Studies reporting outcomes of emergency thoracotomy for trauma in EuropeReferencesStudy periodStudy design*N*Sex (M:F)Age (years)TRISS-score (Ps)ISS (median, range)MOI B:PSurvivors, *n* (%) B:PHudorovic, Croatia, Zagreb (2008) \[[@CR21]\]1995--2005R21n.r.n.r.n.r.n.r.0:210:10 (47.6 %)Søreide et al., Norway, Stavanger (2007) \[[@CR19]\]2001--2005R107:351 (range 21--77)4 % (0--90 %)35 (25--75)7:30:0 (0 %)Ferris et al., Scotland, Edinburgh (2008) \[[@CR22]\]2003--2005R6^a^n.r.n.r.n.r.n.r.0:60:1 (16.7 %)Pahle et al., Norway, Oslo (2010) \[[@CR20]\]2001--2007R10975:3430 (24--47)6 % (0.1--22 %)38 (26--50)82:2710:10 (18.3 %)Kandler et al., Denmark, Copenhagen, (2012) \[[@CR16]\]2000--2009R4440:432 years ± 14^b^44 ± 32 %^b^34 (IQR 25--48)16:285:21 (59.1 %)Van Waes et al., The Netherlands, Rotterdam (2012) \[[@CR17]\]2000--2011R5648:832 (IQR 25--41)n.r.25 (16--34)0:560:36 (64.3 %)Johannesdottir et al., Iceland, Reykjavik (2013) \[[@CR18]\]2005--2010R99:036 (range 20--76)85 % (1--96 %)29 (16--54)5:43:2 (55.6 %)Lustenberger et al., Switzerland, Zurich (2012) \[[@CR15]\]1996--2008R12192:2938 (range 16--84)n.r.41 (16--70)83:3831:32 (52.1 %)Data are reported as median with ranges unless otherwise specified*N* number, *M:F* male:female, *MOI* mechanism of injury, *ISS* Injury Severity Score, *B:P* blunt:penetrating, *n.r.* denotes not reported, *n.a.* not applicable, *R* retrospective, *IQR* interquartile range^a^Excluding 10 patients who underwent a procedure in the operating room due to lack of details^b^Reported as mean with standard deviations (mean ± SD)
The 8 studies reported a total number of 183 penetrating and 193 blunt resuscitative emergency thoracotomies, varying from 9 cases in Reykjavik, Iceland to 121 cases from Zurich, Switzerland. Among the included studies (Table [1](#Tab1){ref-type="table"}), four studies \[[@CR15]--[@CR17], [@CR20]\] accounted for 88 % of the patients included. For studies reporting gender, the male:female ratio (271 males, 78 females) was 3.5:1 (Table [1](#Tab1){ref-type="table"}). Mean age varied with more than two decades among studies, from lowest at 31 years to highest at 51 years.
Five studies reported the selected method of ET \[[@CR15]--[@CR18], [@CR20]\]. Four studies from Denmark \[[@CR16]\], Norway \[[@CR19]\], the Netherlands \[[@CR17]\] and Switzerland \[[@CR15]\] included both emergency department and operating room thoracotomies and stated these were done for resuscitative purposes. In the Edinburg study \[[@CR22]\], 6 patients had ERT in the ED and a further 10 in the OR. For the latter 10 procedures, no further descriptions were given and these 10 cases were thus excluded. Vital signs (systolic blood pressure, respiratory rate, pulse) were reported in 6 studies, and revised trauma score (RTS) in three studies from Iceland \[[@CR18]\], Norway \[[@CR19]\], and the Netherlands \[[@CR17]\].
MOI and LOMI {#Sec7}
------------
MOI is reported in Table [1](#Tab1){ref-type="table"} and Fig. [2](#Fig2){ref-type="fig"}. Studies reporting LOMI, or related location of severe injuries are depicted in Table [2](#Tab2){ref-type="table"}. Three studies reported specific injuries found during operation, without giving the LOMI \[[@CR15]--[@CR17]\].Fig. 2Breakdown of injury type, location and outcomes of the included patients in the identified studies. *EDT* Emergency department thoracotomies, *OR* operating room Table 2Reported LOMI for the included studiesReferences*N*CerebralThoracicAbdominalPelvicMultipleCroatia, Zagreb (2008) \[[@CR21]\]21n.r.21 (cardiac)n.r.n.r.(unknown)^a^Norway, Stavanger (2007) \[[@CR19]\]1014104Scotland, Edinburgh (2008) \[[@CR22]\]6--6------Norway, Oslo (2010) \[[@CR20]\]109n.r.n.r.n.r.n.r.n.r.Denmark, Copenhagen (2012) \[[@CR16]\]44n.r.n.r.n.r.n.r.n.r.Netherlands, Rotterdam (2012) \[[@CR17]\]56--56(10)10Iceland, Reykjavik (2013) \[[@CR18]\]9--6----3Switzerland, Zurich (2012) \[[@CR15]\]121^b^5011861n.r.n.r.^a^Associated extra-thoracic injuries were noted as a prognostically poor variable^b^Reported all injuries with AIS ≥3 for each body region
Indication for ERT {#Sec8}
------------------
All studies described an indication for ERT, but with variable degree of information and whether or not this was done according to a pre-stated institutional protocol. In the Croatian cohort, all were done for thoracic or suspected cardiac injuries, but with no other description available. The indication for each ERT that was performed was described in the other studies. The studies from Scotland and the Netherlands both included only penetrating injuries, of which the majority to the chest and with some form of physiological compromise. For blunt trauma, motor vehicle collisions and falls predominated, while stab wounds were more frequent than gunshots wounds for penetrating trauma (data not shown). Johannesdottir, Ferris and Soreide describe the indication as either suspected tamponade, cardiac arrest; and, witnessed loss of SOL or suspected exsanguination \[[@CR18], [@CR19], [@CR22]\].
Pahle \[[@CR20]\] stated the indications as either "unresponsive patient with penetrating injury who has shown SOL during transport or at the scene"; "exsanguinated patients without immediate response to fluid resuscitation"; and "obviously large abdominal bleeding and decreasing blood pressure with no response to fluid resuscitation before laparotomy", but with no further specification to the distribution in specific patients.
Kandler et al \[[@CR16]\] describe 'penetrating trauma with pulseless electric activity (PEA) within the last 5 min, unstable patients with ongoing intrathoracic bleeding, and as means of clamping the descending thoracic aorta as a step in the initial resuscitation', as indications for ERT \[[@CR16]\].
Van Waes et al. \[[@CR17]\] give the most detailed indication for ERT: (1) Loss of SOL on arrival ED, but present at scene; (2) failure to respond to resuscitation with SBP \<60 mmHg, or pericardial tamponade and SBP \<60 mmHg; (3) Hemothorax on chest X-ray and initial output \>1500 mL or ongoing output of \>200 mL/h for 2--4 h after insertion of the tube; (4) Hemothorax on chest X-ray, with \<1500 mL, but CTA findings prompting surgical intervention; (5) Massive air embolism.
Lustenberger et al. \[[@CR15]\] used the indication 'non-recordable blood pressure on ED admission, loss of SOL in the ED or immediately before hospital arrival and exsanguination from trauma without immediate response to fluid resuscitation'.
Prehospital factors and transport time {#Sec9}
--------------------------------------
Time variables were reported in different ways and for different time intervals across the studies. Hudorovic \[[@CR21]\] noted a difference in median prehospital transport time in survivors (median 150 min, range 15--180 min) compared to non-survivors (median 220 min, range 30--220 min). Pahle et al. \[[@CR20]\] did not find a significant difference in survivors and non-survivors, when assessing time from injury to arrival in ED; non-survivors arrived within a median of 40 min (IQR 18--84 min) compared to survivors who arrived in ED a median of 45 min (IQR 25--95 min) after injury (*P* = 0.477). The same non-significant findings occurred for those with penetrating injuries in the same study, with transport time to ED being almost reduced by half to 20 and 27 min for non-survivors and survivors, respectively \[[@CR20]\]. This time interval matched with the penetrating cohort from the Dutch group having a prehospital transport time at median 24 min (IQR 15--32) overall, but with significant difference in transport time between patients who went on to have ERT in the emergency department compared to the operating room (median 13 min (IQR 2--23) compared to 33 min (IQR 18--35; *P* = 0.006). Of note, the median time until actual ET was performed was 68 min (IQR 42--128) for the whole group; with less time passing to ERT in the ED (median 25 min (IQR 15--107)) compared to the operating room ERTs (median 79 (IQR 52--155) for a *P* = 0.037 \[[@CR17]\].
In the Swizz study \[[@CR15]\], the median time from ED admission to the start of ERT in the ED was 5 (range 2--17) min, and the median time from ED admission to the start of ERT in the OR was 25 (15--49) min.
Sign of life {#Sec10}
------------
Six out of 8 studies reported SOL \[[@CR15]--[@CR20]\] (Table [1](#Tab1){ref-type="table"}). According to one publication from Norway \[[@CR19]\], 7 of 10 (70 %) patients had SOL at scene, but only 4 out of 10 patients (40 %) had SOL in the ED. In the second study from Norway, 86 of 109 (79 %) had SOL at injury site, but SOL in the ED is not given \[[@CR20]\]. In Danish cohort, 19 of 21 (90.5 %) had SOL in ED \[[@CR16]\]. In the Dutch publication 55/56 (98 %) had prehospital SOL, and 50/56 (89 %) showed SOL in hospital \[[@CR17]\]. In the Iceland study, 6 of 9 (66.7 %) had SOL, but it is not described in detail if this was in ED. However, the 3 patients without SOL all died \[[@CR18]\]. In the Swizz study \[[@CR15]\], SOL is reported "at scene" 46/49 EDT (94 %), "en route" 40/49 (82 %) and "on admission" 33/49 (67 %) for those procedures that were performed in as EDT, but not mentioned for procedures performed in the OR. The Croatian report states "absence of SOL at the hospital was a herald of mortality", but mentions no distribution of SOL from their data \[[@CR26]\]. The Scottish study does not comment on SOL \[[@CR22]\].
Location of ERT {#Sec11}
---------------
Regarding the procedures performed in the OR, the Danish report \[[@CR16]\] stated that these were included if considered part of the immediate resuscitative process, but did not give any criteria for performing the ERT in the ED or OR. Similarly, the Swizz study stated that EDT was performed for patients in extremis, while those with pending shock or who were deemed to tolerate transport would be performed in the OR as the immediate resuscitative procedure \[[@CR15]\]. The same indications for ED and OR were noted in one Norwegian study \[[@CR19]\]. The Dutch group \[[@CR17]\] performed the procedure in ED if systolic blood pressure (SBP) was \<60 mmHg (50 % of cases), presence of cardiac tamponade or, loss of SOL in the ED (41 %). Procedures taken to the OR had SBP \>60 mmHg (but \<100 mgHg), initial chest output \>1500 mL blood, or ongoing chest tube output \>200 mL/h (together 50 % of cases in OR), pericardial tamponade with SBP \>60 mgHg (27 %). Most procedures were performed as an anterolateral thoracotomy, followed by sternotomy (Table [3](#Tab3){ref-type="table"}).Table 3Procedure type performed for ERTReferences*N*AnterolateralMedian sternotomyCombinedClam shellPosterolateralNorway, Stavanger (2007) \[[@CR19]\]1010 (100 %)n.r.n.r.n.r.n.r.Norway, Oslo (2010) \[[@CR20]\]10974 (68 %)10 (9 %)25 (23 %)----Denmark, Copenhagen (2012) \[[@CR16]\]44^a^33 (75 %)5 (11 %)4 (9 %)----The Netherlands, Rotterdam (2012) \[[@CR17]\]56^b^30 (54 %)22 (39 %)--4 (7 %)8 (14 %)Iceland, Reykjavik (2013) \[[@CR18]\]95 (56 %)2 (22 %)2 (22 %)----Switzerland, Zurich (2012) \[[@CR15]\]12181 (67 %)28 (23 %)8 (7 %)4 (3 %)--*n.r.* not reported^a^2 missing data for surgical approach^b^The authors state that total 64 incisions were performed
Survival {#Sec12}
--------
The collectively reported overall survival for all 376 ERTs is presented in Fig. [2](#Fig2){ref-type="fig"}, with breakdown in MOI and location of where performed (ED or OR) with survival rates. For blunt trauma survival was 25.4 % and for penetrating injuries 62.2 % (Fig. [2](#Fig2){ref-type="fig"}). Three out of the 5 articles reporting on blunt trauma ERT had survival rates above 10 % \[[@CR16], [@CR18], [@CR20]\], ranging from 12.2 % \[[@CR20]\] to 60.0 % \[[@CR18]\]. When strictly including only those ERTs designated as done in the ED and for blunt injury (*n* = 139), the survival was 12.9 % (*n* = 18).
As noted in Table [1](#Tab1){ref-type="table"}, only a few studies reported probability of survival and with considerable spread in the estimates.
Neurological outcome in survivors {#Sec13}
---------------------------------
Neurological outcome was reported in 5 of 8 studies \[[@CR15], [@CR17], [@CR18], [@CR20], [@CR26]\], most with favorable neurological long-term outcome in survivors, even in blunt trauma survivors. This was reported in both low volume and high volume studies. Among the 8 publications representing a total of 161 survivors after ERT none of the investigators reported neurological outcome using the Glasgow Outcome Scale (GOS) \[[@CR14]\] or similar objective measures. Rather, a qualitative designation as "poor" or "good" neurological outcome or 'neurologically intact' or 'without neurological impairment' was used in most studies. For 34 survivors, no statement on neurological status or outcome was made. Thus, outcome was available in 127 (78.9 %) of survivors, of which 86.6 % had a satisfactory or good neurological recovery. In 17 survivors, the outcome was designated as persistent neurological impairment or inability to live an independent life. One study \[[@CR26]\] reported neurological impairment in all survivors (*n* = 10) with none living independent lives after survival from ERT.
Discussion {#Sec14}
==========
This systematic review found 8 studies on ERT for trauma in European civilian trauma populations. Collectively, these accumulated survival rates are higher than previously reported in the literature---even for blunt trauma performed in the emergency department survival was 12.9 %. The findings are at variance with the perceived standards and outcome reported by major reports, reviews and guidelines previously \[[@CR2], [@CR5], [@CR27], [@CR28]\]. These studies report a much higher survival rate than previously reported and may indeed point to a less futile outcome for blunt trauma victims with pending cardiac arrest, witnessed loss of SOL and even who undergo cardiopulmonary resuscitation than previously argued for in most studies \[[@CR2]--[@CR4], [@CR27], [@CR28]\].
The role of ERT continues to stir considerable debate \[[@CR5]--[@CR7], [@CR27]--[@CR31]\]. Proponents have even suggested taking the procedure to the pre-hospital field for selected patients in extremis \[[@CR9], [@CR29]\]. While all agree that blunt mechanism is in principle associated with a disfavorable prognosis, there are some reports on favorable outcome in a few selected patients, thus arguing for a not completely nihilistic view even in those with blunt trauma \[[@CR28], [@CR31]\]. However, it is clear that more knowledge is needed in this area. Further, one may envision the indication of resuscitative emergency thoracotomy for trauma to change with the increasing availability of resuscitative endovascular balloon occlusion of the aorta (REBOA) technique \[[@CR32]\]. However, this is currently performed only in some specialized centers.
Moore et al. \[[@CR6]\] reported duration of prehospital CPR as a reliable means to establish futility for ERT. Notably, in a large German registry study of over 10,000 trauma patients, a limited number of 757 patients had documented prehospital cardiopulmonary resuscitation with on-scene or en route performed closed chest compression due to traumatic arrest \[[@CR25]\]. The rate of emergency thoracotomy in this cohort was 10.2 % (*n* = 77) for a reported survival rate of 13.0 % (95 % CI 5.5--20.5), including both penetrating and blunt trauma \[[@CR25]\]. From a registry perspective, this falls within the cumulatively reported range of 12.6 % survival for blunt injury ERT in this study (for those performed in ED alone).
The overall frequency by which lifesaving or hemostatic emergency surgery procedures are performed among injured patients is hard to tell from the worldwide literature. However, another updated German nationwide study of almost 13,000 trauma patients found immediate lifesaving procedures (performed during resuscitation) in 5.5 % of patients \[[@CR33]\]. Among the 713 patients that had interrupted resuscitation for an emergent procedure, this most frequently was done as a laparotomy (51 %), followed by craniotomy in 20 %, and only 10 % had thoracotomy (and 9.3 % had pelvic surgery) \[[@CR33]\]. In the German report, 70 procedures were emergency thoracotomies and lethality was 50 %. Notably, of the 70 ETs, 16 were for cardiac injury, of which only 5 survived (32 %) \[[@CR33]\]. In a study from Northern Norway (*n* = 142) \[[@CR23]\], \<3 % of all patients required hemostatic surgery on admission, and only 5 patients had an emergency thoracotomy, with no survivors. In another study from Dublin \[[@CR24]\], 5 % of penetrating trauma victims had a thoracotomy, although no further data was provided on indication nor on outcome in this report. However, these reports indicate that emergency salvage procedures are not commonly performed in European trauma populations \[[@CR23], [@CR24], [@CR33]\].
The number of relevant articles from European trauma hospitals on ERT is limited, as demonstrated by this systematic review. Our search only found 8 articles that fulfilled the criteria. Consequently, we cannot rule out a publication bias that may skew the results. Four studies accumulated 88 % of the entire cohort and may bias the results accordingly. However, the studies are from diverse geographic regions, with variable volume and difference in trauma systems. Also, as mentioned above, they match with experience reported from nationwide registry, such as the German Trauma registry database.
The report from Iceland on 60 % survival for blunt trauma included few (*n* = 5) patients. In contrast, Pahle and Lustenberger, respectively, had 82 blunt and 39 blunt cases, with 12.2 and 7.7 % blunt trauma survivors after ERT, respectively. This could point to a higher rate of survival in mature trauma system. Also, it may reflect an aggressive approach to emergency thoracotomy where one may entertain the possibility that some patients would have survived without being exposed to the ERT procedure. Accordingly, and as a flipside to that coin, hospitals with a more restrictive approach will have fewer survivors after ERT, in particular for blunt trauma. These nuances cannot be discerned from the available data, but certainly warrants future validation in other series before any firm conclusions can be made.
There are some limitations to this study that warrants mentioning. One is the selected reports available from a few centers of variable size and geography. While some reports stem from larger European urban areas such as Oslo, Copenhagen, Zurich and Rotterdam, there is a paucity of similar reports from larger, metropolitan regions in Europe where a large number of trauma patients are treated. As mentioned, a publication bias may thus exist. For example, little is known about ERT performed in major civilian trauma patients in the UK (London, Birmingham), Germany (Berlin, Munich), the Netherlands (Amsterdam, Utrecht), Spain (Barcelona, Madrid), Italy (Rome, Milan) or France (Paris, Marseilles), to mention but a few. It is thus not known if the findings from the selected studies are representative of Scandinavia, the British Isles or mainland Europe, as such. Further, there is considerable variation in the reports, with inconsistent data reporting among the studies. Of notice, all the reported studies were retrospective in design. Thus, use of other definitions, variable data records and non-recorded or missing data may introduce bias in the reported studies.
Despite these limitations, this is the first study to collectively review the experience and outcome of ERT in European civilian trauma patients in the 21st century. It calls into questions the somewhat nihilistic view expressed toward resuscitative emergency thoracotomy for blunt trauma. Further studies are warranted to explore the generalizability of the findings, in particular for blunt trauma.
Conclusions {#Sec15}
===========
In this collective, systematic review of European studies, half of procedures were for blunt trauma, with survivors after ERT in the ED collectively reported at 12.9 and 41.6 % for blunt and for penetrating injuries, respectively. There was no firm indication that neurological outcome were dismal, but further studies need to confirm these results, as considerable variation to previous collective reports exists. A protocolized, multicenter, prospective observational study should be launched to address these questions and arrive at better answers for correct indications and related outcomes of ERT in severely injured trauma patients.
We appreciate the time and guidance from our hospital librarian in helping out with literature search and manuscript retrieval in an early phase of the study.
Conflict of interest {#d30e1674}
====================
Jon Kristian Narvestad, Maryam Meskinfamfard, and Kjetil Søreide declare that they have no conflict of interest.
Ethical standard {#d30e1679}
================
This article does not contain any studies with human participants or animals performed by any of the authors.
|
{
"pile_set_name": "PubMed Central"
}
|
###### Strengths and limitations of this study
There is currently one systematic review on 'acupuncture and moxibustion' and 'chronic urticaria', published in 2009. Our review will assess the effectiveness and safety of acupuncture therapy for patients with chronic urticaria.Chronic urticaria is difficult to treat. The results of the systematic review could help clinicians make decisions about possible treatments for chronic urticaria.The different criteria for efficacy evaluation and forms of acupuncture therapies may cause significant heterogeneity in this review.
Introduction {#s1}
============
Description of the condition {#s1a}
----------------------------
Urticaria is a heterogeneous group of diseases involving the onset of pruritic wheals, angio-oedema or both.[@R1] Chronic urticaria is diagnosed when recurrent crops of urticaria continue for more than 6 weeks.[@R2] It is a common disease with 0.5--1% prevalence, and nearly 20% of people suffer from urticaria at least once during their lifetime.[@R5] Women suffer from urticaria nearly twice as often as men. Individuals between 20 and 40 years have a greater chance of suffering from chronic urticaria.[@R5]
Chronic urticaria negatively influences the patient\'s quality of life because of the itching or physical discomfort during outbreaks. The severity of urticaria varies between individuals. One study found that outbreaks lasted 6--10 weeks in 58% of respondents, while 12% of patients had outbreaks lasting 52 weeks per year. Patients suffering from chronic urticaria have their sleep affected an average of three times per week.[@R6] The disease also has a large impact on society because of its high direct and indirect healthcare costs.[@R2] [@R7]
Chronic urticaria can be divided into chronic spontaneous urticaria and chronic inducible urticaria. Depending on the underlying causes, which are varied and complex, chronic inducible urticaria is divided into physical urticaria (including symptomatic dermographism, cold-induced urticaria, pressure urticaria, solar urticaria, heat-induced urticaria and vibration-induced angio-oedema), cholinergic urticaria, contact urticaria or aquagenic urticaria. However, one patient may have several subtypes of urticaria at once. The most important diagnostic step of chronic urticaria includes a thorough history, physical examination and a ruling out of severe systemic disease. A thorough history should include most possible inducible factors or critical aspects of the nature of the patient\'s urticaria. There is no specific serum test for chronic urticaria. The autologous serum skin test may be used for non-specific autoantibodies against either IgE or the high-affinity IgE receptor,[@R8] and there are some specific provocations that may be used to test underlying causes.
The management of urticaria is aimed at alleviating symptoms. The first line includes elimination of the underlying causes and eliciting triggers. The second line includes symptomatic treatment with pharmacotherapy.[@R2] [@R9] [@R10] The first-line treatment is non-sedating antihistamines. If the first-line treatment is not effective after a maximum of 2 weeks, increasing the dosage up to fourfold is recommended. Second-line therapies should be added to the antihistamine treatment when patients do not respond to a fourfold increase in dosage. Short-term corticosteroids may be prescribed for exacerbations. No serious adverse events or death events were reported in studies according to some relevant studies when participants were taking H1-antihistamines.[@R11] The side effects of H1-antihistamines, which include headache, somnolence, fatigue, dry mouth, hay fever, allergies, etc, were reported in some studies.[@R12] [@R14]
Description of the intervention {#s1b}
-------------------------------
Acupuncture, which has been used for over 2000 years, is an important part of traditional Chinese medicine (TCM) and complementary and complementary medicine. Acupuncture therapy has very few adverse effects and is widely used for the treatment of urticaria in Asia.[@R15] [@R16] Many clinical studies have reported that acupuncture is effective for the treatment of urticaria.[@R15]
How the intervention might work {#s1c}
-------------------------------
In TCM, acupuncture is believed to function by regulating the balance of Qi circulation. In Western medicine, the mechanism of acupuncture is yet not clear. It is speculated that the potential mechanism of acupuncture for treating urticaria might be the suppression of the immunological reaction.[@R17] [@R18] Studies in animals and humans signified that acupuncture can attenuate histamine effect, modulate the function of the immune system and decrease the adhesion molecule.[@R19] [@R20]
Why it is important to do this review {#s1d}
-------------------------------------
So far, there is only one systematic review published in Chinese investigating acupuncture and chronic urticaria.[@R21] The previous review included 12 randomised controlled trials (RCTs), but only 4 of those studies strictly investigated the effectiveness of acupuncture. Additionally, the outcome assessments were not clearly or specifically described. Therefore, no definite conclusions on the effectiveness of acupuncture for chronic urticaria can be drawn. There have been at least four more RCTs reported in the past 5 years. Using a stricter search strategy, a more objective outcome evaluation and a rigorous review method, we expect that our systematic review will provide a more convincing conclusion.
Objectives {#s1e}
----------
To systematically evaluate the effectiveness and safety of acupuncture therapy for patients with chronic urticaria.
Methods and analysis {#s2}
====================
Inclusion criteria for study selection {#s2a}
--------------------------------------
### Types of studies {#s2a1}
All RCTs without restrictions on publication status will be included. Quasi-randomised trials will be excluded.
### Types of participants {#s2a2}
Patients with chronic urticaria, regardless of sex, age, race or educational and economic status, according to the following criteria: EAACI/GA2LEN/EDF/WAO guideline: definition, classification and diagnosis of urticaria.[@R1] [@R9]Chinese guidelines for the diagnosis and treatment of urticaria.[@R22] [@R23]
### Types of interventions {#s2a3}
Acupuncture is defined as the stimulation of acupuncture points by needles, including manual acupuncture, dermal needle, plum blossom needle, ear acupuncture, electroacupuncture or fire needle. Other stimulation methods including acupressure, moxibustion, laser acupuncture, pharmacoacupuncture, dry needling or transcutaneous electrical nerve stimulation will be excluded. Sham acupuncture includes sham acupuncture at selected acupoints, sham acupuncture at non-acupoints, needling at inappropriate acupoints, non-penetrating sham acupuncture and pseudo interventions).[@R24] The following treatment comparisons will be investigated.
Acupuncture compared with no treatment. Acupuncture compared with placebo or sham acupuncture.Acupuncture compared with other active therapies.Acupuncture in addition to active therapy compared with the same active therapy.
We will exclude RCTs in which one form of acupuncture was only compared with another form of acupuncture or a different type of TCM (eg, Chinese herbal medicine). We will assess the acupuncture treatment according to how the acupuncturists are trained and educated, their clinical experience, total numbers of acupuncture sessions, treatment duration and treatment frequency, etc.[@R25]
### Types of outcome measures {#s2a4}
#### Primary outcomes {#s2a4a}
Improvement in pruritus and wheals, using urticaria activity score (UAS)[@R2] [@R26] or other validated scales, at least after 2 weeks of treatment.
#### Secondary outcomes {#s2a4b}
Secondary outcomes include adverse events and quality of life using a health-related quality of life measure, including a generic, dermatology-specific or disease-specific instrument.[@R10] [@R27] The recurrence rate after at least 3 months of the treatment will also be evaluated.
Search methods for identification of studies {#s2b}
--------------------------------------------
### Electronic searches {#s2b1}
We will electronically search the following databases, regardless of publication status and languages: the Cochrane Central Register of Controlled Trials (CENTRAL), PubMed, EMBASE, the Web of Science, Traditional Chinese Medicine databases, China National Knowledge Infrastructure (CNKI), Chinese Biomedical Literature Database (CBM), Chinese Scientific Journal Database (VIP database) and Wan-Fang Database from their inception. The following search terms will be used: chronic urticaria, hives, nettle-rash, Fong-Tzen-Kwai, wind-rash-patch, acupuncture, manual acupuncture, filiform steel needle, electroacupuncture, fire needling, auricular acupuncture, ear acupuncture, dermal needle, abdominal acupuncture, pyonex and plum blossom needle. The equivalent search words will be used in the Chinese databases. A search strategy created according to the Cochrane handbook guidelines will be conducted in all the electronic databases.[@R28] The search strategy for PubMed is shown in [table 1](#BMJOPEN2015007704TB1){ref-type="table"}.
######
Search strategy used in PubMed
No Search items
---- --------------------------------
1 Randomised controlled trial.pt
2 Controlled clinical trial.pt
3 Randomised.ti,ab
4 Randomly.ti,ab
5 Placebo.ti,ab
6 Trial.ti,ab
7 Groups.ti,ab
8 1 or 2--7
9 Urticaria.Mesh
10 Chronic urticaria.ti,ab
11 hives.ti,ab
12 nettle-rash.ti,ab
13 Angioedema.ti,ab
14 Fong-Tzen-Kwai.ti.ab
15 wind-rash-patch.ti.ab
16 9 or 10-15
17 Acupuncture therapy. Mesh.
18 Acupuncture.ti,ab
19 Acupoints.ti,ab
20 Acupuncture\*.ti,ab
21 Body acupuncture.ti,ab
22 Scalp acupuncture.ti,ab
23 manual acupuncture.ti,ab
24 Auricular acupuncture.ti,ab
25 ear acupuncture.ti,ab
26 Electroacupuncture.ti,ab
27 Fire needling.ti,ab
28 dermal needle.ti,ab
29 plum blossom needle.ti,ab
30 Pyonex.ti,ab
31 Abdominal acupuncture.ti,ab
32 Filiform steel needle.ti,ab
33 17 or 18--32
34 8 and 16 and 33
### Searching other resources {#s2b2}
We will scan the reference lists of identified publications for additional trials. We will search PubMed, Turning Research Into Practice (TRIP) database and the Cochrane Library for existing systematic reviews possibly relevant to this systematic review to search their reference lists for additional trials. We will also search conference proceedings related to this topic.
Data collection and analysis {#s2c}
----------------------------
### Selection of studies {#s2c1}
We plan to conduct this systematic review between 1 January 2015 and 30 August 2015. All reviewers have undergone training to ensure a basic understanding of the background and purpose of the review. After electronic searching, the records will be uploaded to a database set up by EndNote software (V.X7). Records selected from other sources will also be moved to the same database. Two review authors (QY and ZQ) will independently screen the titles, abstracts and keywords of all retrieved studies and decide which trials meet the inclusion criteria. We will obtain the full text of all possibly relevant studies for further assessment. Excluded studies will be recorded with explanations. Any disagreements will be resolved by discussion between the two authors (QY and ZQ) and the third author (ZL) for arbitration when necessary. We will contact authors of trials for clarification when necessary. The study flow diagram is shown in [figure 1](#BMJOPEN2015007704F1){ref-type="fig"}.
{#BMJOPEN2015007704F1}
Data extraction and management {#s2d}
------------------------------
Two authors will independently extract data from the selected reports or studies and fill in the data extraction form. We will obtain data for general information, participants, methods, interventions, outcomes, results, adverse events, conflicts of interest, ethical approval and other information. We will contact the authors for further information when the reported data are not sufficient. Any disagreements will be resolved by discussion between the two authors, and further disagreements will be arbitrated by the third author (ZL).
Assessment of risk of bias in included studies {#s2e}
----------------------------------------------
Two authors (YY and XL) will assess the risk of bias with the Cochrane Collaboration\'s tool for risk of bias assessment for all included studies. We will evaluate the following domains for risk of bias: sequence generation, allocation sequence concealment, blinding of participants and personnel and outcome assessors, incomplete outcome data, selective outcome reporting and other sources of bias. The assessments will be classified into three levels: low risk, high risk and unclear risk.
Measures of treatment effect {#s2f}
----------------------------
We will use RevMan V.5.2 for data analysis and quantitative data synthesis. For continuous data, we will use the mean difference (MD) or standard MD (SMD) to measure the treatment effect with 95% CIs if no heterogeneity is found. If significant heterogeneity is detected, the random-effects model will be used. For dichotomous data, we will use a risk ratio (RR) with 95% CIs for analysis.
Unit of analysis issues {#s2g}
-----------------------
We will include data from parallel group-designed studies for meta-analysis. Only the first phase data will be considered in randomised cross-over trials. In these trials, participants are individually randomised to two intervention groups, and a single measurement for each outcome from each participant is collected and analysed.
Dealing with missing data {#s2h}
-------------------------
We will try to contact the first or corresponding authors of the included studies to retrieve missing or insufficient trial data. An intention-to-treat analysis will be performed, if possible (the analysis should include the data of all the participants in the groups to which they were originally randomly assigned), and a sensitivity analysis will be used to determine whether the results are inconsistent.
Assessment of heterogeneity {#s2i}
---------------------------
We will use the I^2^ statistic for quantifying inconsistencies among the included studies. When the I^2^ value is less than 50%, the study will not be considered to have heterogeneity. When the I^2^ value exceeds 50%, significant statistic heterogeneity exists among the trial and meta-analysis will not be performed. Subgroup analysis will be conducted to explore possible causes.
Assessment of reporting biases {#s2j}
------------------------------
We will use funnel plots to detect reporting biases and small-study effects. If more than 10 studies are included in the meta-analysis, we will conduct a test for funnel plot asymmetry using Egger\'s method.[@R29] We will include all eligible trials, regardless of their methodological quality.
Data synthesis {#s2k}
--------------
Data synthesis will be conducted with RevMan V.5.2 when a meta-analysis is allowed. The results will be expressed as RR for dichotomous data and SMD for continuous data. If the I^2^ test is less than 50%, the fixed-effects model will be used for data synthesis. If the I^2^ test is between 50% and 75%, the random-effects model will be conducted for data synthesis. If the I^2^ test is higher than 75%, we will investigate possible reasons from both clinical and methodological perspectives, and provide a descriptive analysis or conduct subgroup analysis.
Subgroup analysis {#s2l}
-----------------
There is no presubgroup plan. If data are available, factors like subgroups of the chronic urticaria and acupuncture methods will be taken into account. When significant heterogeneity exists, we will also conduct subgroup analysis if necessary.
Sensitivity analysis {#s2m}
--------------------
We will conduct sensitivity analysis to test the robustness of the primary decisions of the review process. The principal decision nodes conclude methodological quality, sample size and the effect of missing data. The meta-analysis will be repeated and studies of lower quality will be excluded. The result will be compared and discussed according to the results.
Grading the quality of evidence {#s2n}
-------------------------------
The quality of evidence for all outcomes will be judged using the Grading of Recommendations Assessment, Development and Evaluation working group methodology. The following domains will be assessed: risk of bias, consistency, directness, precision, publication bias and additional points. The assessments will be classified into four levels: high, moderate, low or very low.[@R30]
Ethics and dissemination {#s2o}
------------------------
A formal ethical approval is not necessary because the data that will be used are not individual and no privacy will be involved. The results will be disseminated through peer-reviewed publications or conference presentations.
Discussion {#s3}
==========
Studies have signified that acupuncture is effective for relieving the symptoms (mainly in pruritus and wheals) of chronic urticaria.[@R17] [@R31] Nevertheless, currently no systematic review related to acupuncture for chronic urticaria has been published in English. The evaluation of this systematic review will be divided into four sections: identification, study inclusion, data extraction and data synthesis. We hope that this review will give more convincing proof to assist clinicians during the decision-making process when dealing with chronic urticaria. This review has some potential limitations. Different types of acupuncture therapies and the efficacy evaluation criteria of urticaria in included trials may cause significant heterogeneity.
PRISMA-P Checklist of the protocol: Online supplementary appendix 1
The authors would like to thank Edanz Editing China for their editing work. They also thank Jing Zhou for her assistance to improve the paper.
**Contributors:** ZL is the guarantor. QY and ZL contributed to the conception of the study. The manuscript of the protocol was drafted by QY and revised by ZL. QY and ZQ will independently screen the potential studies and extract data from the included studies. YY and XL will assess the risk of bias and finish data synthesis. ZL will arbitrate any disagreements and ensure that no errors occur during the review. All authors read, provided feedback and approved the final manuscript.
**Competing interests:** None declared.
**Provenance and peer review:** Not commissioned; externally peer reviewed.
|
{
"pile_set_name": "PubMed Central"
}
|
Myosin VI is an ATP hydrolysis coupled motor protein involved in many cellular functions including endocytosis, protein secretion and the maintenance of both the Golgi morphology and stereocilia \[[@b1-11_47]\]. It is a unique myosin in that it moves to the minus end of an actin filament \[[@b2-11_47]\], while all other myosins move to the plus end \[[@b3-11_47]\]. Recently, we proposed that myosin VI moves using three types of steps: large and small forward steps (minus end directed), and backward steps (plus end directed) \[[@b4-11_47]--[@b6-11_47]\]. We introduced the adjacent binding state to explain how myosin VI generates the small steps and backward steps. In this state, both lever arms point the same direction to achieve an inter-head distance of less than 10 nm (parallel lever arms model; [Fig. 1A](#f1-11_47){ref-type="fig"}) \[[@b4-11_47],[@b5-11_47]\]. However, an alternative explanation of the adjacent binding state has the lever arm fold by the Lever Arm Extension (LAE) \[[@b7-11_47],[@b8-11_47]\], which is unique to myosin VI (LAE folding model; [Fig. 1B](#f1-11_47){ref-type="fig"}).
To clarify which model is correct, we constructed a myosin VI/V chimera in which the LAE was substituted for the IQ3-6 domains of the myosin V lever arm. This chimera should be able to generate small steps or backward steps by taking the adjacent binding state only according to the parallel lever arms model. We therefore measured chimera steps at the single molecule level by single molecule fluorescence imaging using total internal reflection fluorescence microscopy and found that the mutant generated small and backward steps like those of myosin VI. This result demonstrates folding of the LAE is not necessary and suggests the adjacent binding state occurs by directing both lever arms the same direction.
Materials and Methods
=====================
Preparation of protein samples
------------------------------
Human myosin Va cDNA (product ID: ORK07567), human myosin VI cDNA (product ID: ORK01080) and human calmodulin cDNA (product ID: ORK01403) were purchased from Kazusa DNA, Japan. To create myosin VI/V chimera constructs, myosin VI cDNA (1 to 830 amino acids) was introduced into pFastBac (Life Technologies) to make a baculovirus. This myosin VI fragment included the motor domain, the unique myosin VI insertion and the core of the IQ domain (see Results for definition). For biotin labeling, a HaloTag (Promega) fragment was attached at the N-terminal of myosin VI via the linker. To construct the myosin VI/V chimera, myosin V cDNA (807 to 1099 amino acids) was inserted after the above myosin VI sequence using the In-Fusion HD cloning kit (Clontech). This myosin V fragment included the lever arm domain just after the IQ2 motif and coiled-coil domain ([Fig. 2A, B](#f2-11_47){ref-type="fig"}). Furthermore, a GCN4 sequence, which ensures dimerization, and His6-tag, which enables affinity protein purification, were also inserted at the C-terminus ([Fig. 2A](#f2-11_47){ref-type="fig"}). Calmodulin was introduced into another pFastBac plasmid, and myosin VI/V chimeras were expressed using sf9 cells and two kinds of baculoviruses from the above pFastBac plasmids. For myosin VI expression and purification, myosin VI cDNA (1-1021) and calmodulin were introduced into a single pFastBac Dual plasmid (Life Technologies). A HaloTag was attached at the N-terminal of myosin VI, while GCN4, SNAP-Tag and His6-tag were attached at the C-terminal.
Protein purification was performed as previously described \[[@b4-11_47],[@b5-11_47]\]. During the purification, myosin VI/V chimeras or myosin VI were biotinylated at the HaloTag using a biotin-halo-ligand. Qdot585 streptavidin conjugates and biotinylated myosin VI/V chimeras or myosin VI were incubated as previously described \[[@b6-11_47]\] for single molecule fluorescence imaging.
Microscopy observation and analysis
-----------------------------------
All experiments were performed according to previously reported methods \[[@b6-11_47]\]. Briefly, a 10 μl volume microchamber was made by placing a small coverslip (18×18 mm, No. 1 Thickness, Matsunami, Japan) over a larger one (22×32 mm, No. 1 thickness, Matsunami) using double-sided adhesive tape (50 μm thickness). Next, 1.5 mg/ml actinin (Sigma-Aldrich) in assay buffer (AB: 20 mM HEPES-KOH pH 7.8, 25 mM KCl, 5 mM MgCl~2~ and 1 mM EGTA) was adsorbed onto the glass surface, followed by a 3 min incubation, a 20 μl AB wash, and finally an injection of 2 μg/ml non-fluorescent phalloidin labeled actin filament solution in AB into the chamber. After another 3 min incubation and 20 μl AB wash, 5 mg/ml α-casein in AB was injected into the chamber. After a 3 min incubation and 20 μl AB wash, motility buffer (MB: AB plus an oxygen scavenger system, ATP regeneration system, 20 μg/ml calmodulin and 100 μM ATP) mixed with Qdot585-labeled myosin VI/V chimeras was flowed into the chamber, and the chamber was sealed with nail polish. Qdot585-conjugated myosin VI/V chimeras were imaged using total internal reflection fluorescence microscopy (TIRFM), and the corresponding fluorescent images were captured with an EMCCD camera (Andor iXon).
Data analysis
=============
The center of a fluorescent spot in each frame was determined using a two-dimensional Gaussian fit according to a published method \[[@b9-11_47]\] using the custom written ImageJ plugin "PTA" (<https://github.com/arayoshipta/projectPTAj>). Steps were detected using a MATLAB program for Kerssemaker's chi-square based algorithm \[[@b10-11_47]\]. Distributions of steps were fitted to Gaussian functions using OriginLab, while dwell times and run lengths distributions were fitted to exponential decay functions.
Results
=======
Experimental setup
------------------
To clarify whether the folding of the myosin VI LAE is responsible for the adjacent binding state, we constructed myosin VI/V chimeras where amino acids 1--830 of myosin VI were followed by amino acids 807--1099 of myosin V ([Fig. 2A--D](#f2-11_47){ref-type="fig"}). Calmodulins bind to a myosin lever arm by mainly recognizing the IQ-motif of the consensus sequence of I(L/M)Q xxx RG(M) xxxR(K) amino acids with X being any amino acids sequence as reviewed \[[@b11-11_47],[@b12-11_47]\]. In myosin VI, the core amino acids sequence of the IQ motif (MQKTIRMWLCK) ends at 830 a.a., while the core amino acids sequence of the IQ2 motif (MQRYVRGYQAR) in myosin V ends at 806 a.a. Since in this study we used chimeras, the amino acids sequence between the IQ motif of myosin VI and the IQ3 motif of myosin V is the same as that between the IQ2 and IQ3 motif in myosin V, meaning that there exists no excessive or shortage of amino acids, which might otherwise compromise calmodulin binding capacity or rigidity of the lever arms. We evaluated the amount of bound calmodulin using SYPRO Red Protein Gel Stain and 4--15% acrylamide SDS-PAGE gels ([Supplementary Fig. S1](#s1-11_47){ref-type="supplementary-material"}). The ratio of calmodulin on myosin V to myosin VI/V chimera was 1:1.3, indicating that myosin VI/V chimera retain their binding capacity to calmodulin and have rigid lever arms like that of myosin V.
To visualize the stepping motion of the chimeras at the single molecular level, the N-terminus was labeled with Qdot585 using HaloTag, biotin-Halo ligand and streptavidin-coated Qdot585 ([Fig. 2E](#f2-11_47){ref-type="fig"}). Here, one of the two motor domains of myosin VI/V chimera was labeled with a Qdot585. Since the Qdot labeling of both myosin motor domains was a rare event \[[@b5-11_47]\], we could observe movement with single molecule resolution using TIRFM ([Fig. 3B](#f3-11_47){ref-type="fig"}).
Motile properties of myosin VI /V chimeras
------------------------------------------
Myosin VI/V chimeras generated processive steps that included three step types: large steps of 68 nm, small steps of 32 nm and backward steps of --34 nm ([Fig. 3B](#f3-11_47){ref-type="fig"} and [4](#f4-11_47){ref-type="fig"}; [Supplementary Movie S2](#s3-11_47){ref-type="supplementary-material"}). These steps were very similar to those previously reported for myosin VI: 76 nm, 41 nm and --40 nm ([Fig. 3A](#f3-11_47){ref-type="fig"}; [Supplementary Movie S1](#s2-11_47){ref-type="supplementary-material"}) \[[@b4-11_47]\]. Furthermore, since a single trace of myosin VI or myosin V/VI chimera contains all three step types, we could confirm that one of the two motor domains of the myosin VI/V chimera was labeled with a single Qdot and that the small and backward steps were not caused by Qdot-labeling of both motor domains from the same chimera or an aggregation of myosin VI/V chimeras.
The run length of the myosin VI/V chimera (81 nm; [Fig. 5A](#f5-11_47){ref-type="fig"}) was less than half the previously reported run length of myosin VI (\~200 nm) \[[@b4-11_47]\]. Processive movement in myosin is often considered the result of coordinating the ATP-hydrolysis cycle between the two motor domains via intramolecular strain \[[@b11-11_47]\]. We concluded that removing the LAE lowered the intramolecular strain and thus the run length too. However, a previous study examining chimera mutants of myosin V that retained the SAH (single alpha helix) domain, which is equivalent to the myosin VI LAE domain, from the dictyostelium myosin MyoM, had lower intramolecular strain because of decreased rigidness in the lever arm region \[[@b13-11_47]\]. The lower intramolecular strain seen in our myosin VI/V chimera despite a more rigid lever arm can be explained by the actomyosin geometry \[[@b14-11_47]\].
The rate constants of the large, small and backward steps of our chimera were 1.3 s^−1^, 2.2 s^−1^ and 1.3 s^−1^ at 100 μM ATP, respectively ([Fig. 5B--D](#f5-11_47){ref-type="fig"}), while those of myosin VI were about 3 s^−1^ for all step types at 100 μM ATP \[[@b6-11_47]\]. The decreased stepping rates for the myosin VI/V chimera might be explained by the reduced intramolecular strain. At 100 μM ATP, the rate limiting process for myosin VI stepping is ATP binding to the rear motor domain \[[@b15-11_47]\]. This binding is accelerated by intramolecular strain \[[@b16-11_47]\]. Therefore, since the intramolecular strain was decreased in our chimeras, we concluded it caused the ATP-binding rate in myosin VI/V chimera to decrease.
Discussion
==========
We previously reported that myosin VI can generate three kinds of steps (large, small and backward steps) by taking the adjacent binding state \[[@b4-11_47],[@b5-11_47]\]. We explained this state using the parallel lever arms model ([Fig. 1A](#f1-11_47){ref-type="fig"}). The model is based on measurements of the lever arm length during myosin VI stepping using gold nano particles and dark field imaging of the N-terminus position \[[@b4-11_47]\] and the simultaneous tracking of both the C-terminus and N-terminus positions using SHREC \[[@b5-11_47]\]. However, the adjacent binding state can also be explained by folding of the LAE ([Fig. 1B](#f1-11_47){ref-type="fig"}). We therefore prepared a myosin VI/V chimera mutant that substitutes the LAE with the IQ3-6 domains of myosin V ([Fig. 2](#f2-11_47){ref-type="fig"}) and performed single molecule fluorescence imaging, finding that our chimera can generate similar small and backward steps to those of myosin VI ([Fig. 3B](#f3-11_47){ref-type="fig"} and [Fig. 4](#f4-11_47){ref-type="fig"}). Unlike the LAE, the IQ3-6 is rigid, indicating the adjacent binding state is not the consequence of lever arm folding. Thus, we concluded the parallel lever arms model explains this state ([Fig. 3B](#f3-11_47){ref-type="fig"}).
The parallel lever arms model assumes the two lever arms point the same direction, which may help explain why the adjacent binding state is seen in myosin VI \[[@b4-11_47]\], but not in myosin V \[[@b9-11_47]\]. The lever arms in myosin VI are angled at about 180 degrees from each other \[[@b14-11_47]\], while in myosin V it is a much more modest 70 degrees ([Supplementary Fig. S2](#s1-11_47){ref-type="supplementary-material"}) \[[@b17-11_47],[@b18-11_47]\]. Because the 180 degree angle is attributed to the unique insertion of myosin VI \[[@b17-11_47]\], our chimera, which includes the insertion, should show the same angle. These differences may reflect differences in steric hindrance between the arms, as the adjacent binding state can only occur when the hindrance is low.
In a cell, myosin V, which cannot take the adjacent binding state, has been reported to function as a transporter. On the other hand, myosin VI, which can take the adjacent binding state, has been reported to function as a transporter and as an anchor. Since the adjacent binding state is unique to myosin VI, it may contribute to this second function. Indeed, the adjacent binding state can equally divide the external force onto both its motor domains. Part of the reason for this ability is that the lever arms are parallel in the adjacent binding state, so that both lever arms can take the postpower stroke state \[[@b4-11_47],[@b5-11_47]\]. It was reported using myosin V that the motor domain in the prepower stroke state spontaneously detaches from actin at a rate of 1 s^−1^ \[[@b19-11_47]\] and has weaker actomyosin binding than the postpower stroke state, which suggests the adjacent binding state would be ideal for anchoring function. One might worry that the ATP-binding rate for the postpower stroke state is higher than that for the prepower stroke state under no external load condition. However, an optical trapping study suggested that the ATP-binding rate dramatically slows when an external force is applied to the motor domains \[[@b20-11_47]\], meaning that the ATP-binding rate in the adjacent binding state is expected to decrease with external load.
Supplementary Information
=========================
K. I. is supported by a research fellowship from JSPS, Japan. We thank the members of QBiC for valuable discussions, R. Kawaguchi for technical help and P. Karagiannis (RIKEN, QBiC) for helpful discussions and comments on the manuscript. This work was supported by a Grant-in-Aid for JSPS Fellows 24-2619 (to K. I.) and a Grant-in-Aid for Young Scientists (B) 25840063 (to T. K.) from MEXT, Japan and RIKEN QBiC, Japan (to T. Y.).
{#f1-11_47}
{#f2-11_47}
{#f3-11_47}
{#f4-11_47}
{#f5-11_47}
|
{
"pile_set_name": "PubMed Central"
}
|
Clinicaltrials.gov identifier: NCT00993486
Haematopoietic stem cell transplantation (HSCT) using a haploidentical family donor offers the potential for cure to the approximate 20--55% of patients requiring transplant who do not have a suitable human leucocyte antigen (HLA)‐matched related or unrelated donor (Barker *et al*, [2010](#bjh15970-bib-0008){ref-type="ref"}; Petersdorf, [2010](#bjh15970-bib-0042){ref-type="ref"}; Ballen *et al*, [2012](#bjh15970-bib-0007){ref-type="ref"}; Pidala *et al*, [2013](#bjh15970-bib-0043){ref-type="ref"}; Ruggeri *et al*, [2015](#bjh15970-bib-0048){ref-type="ref"}; Sureda *et al*, [2015](#bjh15970-bib-0050){ref-type="ref"}). Although the feasibility of haploidentical stem cell transplantation has been questioned over the decades due to lethal graft‐versus‐host disease (GVHD), developments have led to a rapid increase in the use of haploidentical HSCT in recent years, challenging even matched unrelated donor as standard of care (Sureda *et al*, [2015](#bjh15970-bib-0050){ref-type="ref"}; Passweg *et al*, [2018](#bjh15970-bib-0039){ref-type="ref"}). Today\'s haploidentical transplants usually involve different *ex vivo* or *in vivo* T‐cell depletion strategies to avoid severe acute GVHD (Locatelli *et al*, [2017](#bjh15970-bib-0029){ref-type="ref"}; Zeiser & Blazar, [2017](#bjh15970-bib-0052){ref-type="ref"}; Al Malki *et al*, [2018](#bjh15970-bib-0001){ref-type="ref"}), a major complication of T‐replete allogeneic HSCT (Sureda *et al*, [2015](#bjh15970-bib-0050){ref-type="ref"}). Importantly, early trials of *ex vivo* T‐cell depletion could achieve complete engraftment without causing GVHD (Aversa *et al*, [1998](#bjh15970-bib-0006){ref-type="ref"}; Martelli & Aversa, [2016](#bjh15970-bib-0031){ref-type="ref"}). However, the reduction of T cells results in delayed immune reconstitution and may lead to lethal opportunistic infections and disease relapse in a high number of patients (Aversa *et al*, [1998](#bjh15970-bib-0006){ref-type="ref"}; Di Stasi *et al*, [2011](#bjh15970-bib-0020){ref-type="ref"}). Therefore, *ex vivo* strategies, such as selective graft depletion of alpha‐beta‐T cells or insertion of suicide genes to donor lymphocytes, have been developed to facilitate the transfer of haploidentical lymphocytes while diminishing the challenges of life‐threatening infections, GVHD, and relapse (Al Malki *et al*, [2018](#bjh15970-bib-0001){ref-type="ref"}). *In vivo* T‐cell depletion using cyclophosphamide is an extremely simple and effective approach to facilitating haploidentical transplantation, but it is also associated with the occurrence of graft failures and higher relapse rates after reduced‐intensity conditioning (Luznik *et al*, [2008](#bjh15970-bib-0030){ref-type="ref"}; Brunstein *et al*, [2011](#bjh15970-bib-0013){ref-type="ref"}; Ciurea *et al*, [2015](#bjh15970-bib-0018){ref-type="ref"}; Byrne & Savani, [2016](#bjh15970-bib-0014){ref-type="ref"}; Bashey *et al*, [2017](#bjh15970-bib-0009){ref-type="ref"}; McCurdy *et al*, [2018](#bjh15970-bib-0034){ref-type="ref"}). In addition, the long‐term patient outcomes following many of these strategies are currently unknown (Byrne & Savani, [2016](#bjh15970-bib-0014){ref-type="ref"}).
Donor T cells are a prerequisite of long‐lasting immunity to fight infections and relapse (Deol & Lum, [2010](#bjh15970-bib-0019){ref-type="ref"}). Attempts at prophylactic administration of untreated donor lymphocyte infusions at doses of 3 × 10^4^ CD3^+^ cells/kg or greater after T‐cell‐depleted haploidentical HSCT in the absence of relapsing cells almost always resulted in GVHD (Lewalle *et al*, [2003](#bjh15970-bib-0028){ref-type="ref"}). Therefore, several groups have worked on developing selective donor lymphocyte depletion strategies to remove host‐reacting donor T cells (Garderet *et al*, [1999](#bjh15970-bib-0022){ref-type="ref"}; Andre‐Schmutz *et al*, [2002](#bjh15970-bib-0004){ref-type="ref"}; Guimond *et al*, [2002](#bjh15970-bib-0025){ref-type="ref"}; Amrolia *et al*, [2006](#bjh15970-bib-0002){ref-type="ref"}; Mielke *et al*, [2007](#bjh15970-bib-0035){ref-type="ref"}; Bleakley *et al*, [2015](#bjh15970-bib-0012){ref-type="ref"}). We have taken advantage of the biological alterations that occur in donor cells upon exposure to patient cells, to selectively eliminate patient‐reactive T cells (Chen *et al*, [2002](#bjh15970-bib-0015){ref-type="ref"}; Guimond *et al*, [2002](#bjh15970-bib-0025){ref-type="ref"}; Mielke *et al*, [2008](#bjh15970-bib-0036){ref-type="ref"}). Indeed, T‐cell activation is associated with P‐glycoprotein pump inhibition, which in turn leads to intracellular accumulation of the rhodamine‐derived photosensitizer TH9402, a substrate of this pump. Alloreactive T cells with GVHD‐causing properties can then be eliminated from the donor T‐cell repertoire following exposure to visible light, whereas resting T lymphocytes capable of anti‐infection and anti‐tumour reactivity are preserved (Chen *et al*, [2002](#bjh15970-bib-0015){ref-type="ref"}; Guimond *et al*, [2002](#bjh15970-bib-0025){ref-type="ref"}; Mielke *et al*, [2008](#bjh15970-bib-0036){ref-type="ref"}; Perruccio *et al*, [2008](#bjh15970-bib-0041){ref-type="ref"}). This is a phase I clinical study to determine the maximum tolerated dose (MTD) and evaluate the safety of allodepleted T‐cell immunotherapy (ATIR101), administered without GVHD prophylaxis, in the context of CD34^+^‐selected haploidentical HSCT. Our data informed two phase 2 studies evaluating ATIR101 as an adjunctive infusion following T‐cell‐depleted haploidentical HSCT (Roy *et al*, [2018a](#bjh15970-bib-0045){ref-type="ref"}, [2018b](#bjh15970-bib-0046){ref-type="ref"}). A phase 3, randomized trial has also been initiated, comparing haploidentical HSCT + ATIR101 *versus* T‐cell replete haploidentical HSCT + post‐transplant cyclophosphamide. Herein, we also report the long‐term follow‐up (more than 8 years) of patients enrolled in this phase 1 study to show potential of ATIR101 to impact favourably on transplant‐related mortality (TRM), relapse and survival in the long‐term.
Materials and methods {#bjh15970-sec-0002}
=====================
Patients and donors {#bjh15970-sec-0003}
-------------------
Adults with high‐risk haematological malignancies without possibility of transplant from an HLA‐matched sibling donor, and lacking an 6/6 HLA‐A, B and DRB1 matched unrelated donor within 2--3 months, were enrolled into this phase I, single‐centre, dose‐ranging, open‐label study (NCT00993486; see Appendix [S1](#bjh15970-sup-0003){ref-type="supplementary-material"} for inclusion criteria). The objective was to determine the MTD and safety of ATIR101 in patients undergoing haploidentical peripheral blood HSCT with CD34^+^ cell selection. This study was conducted in accordance with the ethical principles of the Declaration of Helsinki and approved by the ethics committee of Hôpital Maisonneuve‐Rosemont. All patients and donors gave written informed consent. Patients meeting eligibility criteria were treated between January 2005 and August 2008 with an 8‐year median follow‐up of survivors.
Patient conditioning and transplant {#bjh15970-sec-0004}
-----------------------------------
Patients received myeloablative conditioning including total body irradiation (TBI) at a dose of 12 Gy, in six fractions of 2 Gy given twice daily over 3 days (starting 10 days prior to HSCT). The lungs were shielded to receive a maximum of 9 Gy. Thiotepa (5 mg/kg) was administered at 12‐h intervals on the day following TBI. Starting the next day (2 days after TBI), rabbit antithymocyte globulin 2·5 mg/kg/day (Thymoglobulin; Genzyme, Mississauga, Ontario, Canada) was infused over at least a 6‐h period for 5 days. Patients received methylprednisolone (1 mg/kg) intravenously twice daily on the antithymocyte globulin‐infusion days. Fludarabine was given at a dose of 40 mg/m^2^/day for 4 days starting 7 days prior to transplant. No immune suppressors were used after transplant. All donor peripheral blood CD34^+^ cells collected and isolated were infused on Day 0. Donor chimerism in lymphoid and myeloid compartments was measured at regular intervals before and after ATIR101 infusion (Appendix [S1](#bjh15970-sup-0003){ref-type="supplementary-material"}).
Manufacture of ATIR101 under good manufacturing practice conditions {#bjh15970-sec-0005}
-------------------------------------------------------------------
See Appendix [S1](#bjh15970-sup-0003){ref-type="supplementary-material"} for details of ATIR101 manufacturing. Photodepletion of host‐activated T cells in ATIR101 was evaluated immunophenotypically (CD25^+^ CD44^+^), and limiting dilution assays were used to calculate the frequencies of anti‐host and anti‐third party responding cytotoxic T‐lymphocyte precursors (CTLp), using previously described methods (Appendix [S1](#bjh15970-sup-0003){ref-type="supplementary-material"}) (Guimond *et al*, [2002](#bjh15970-bib-0025){ref-type="ref"}). Cells were cryopreserved until infusion 28--42 days after HSCT.
Determination of MTD {#bjh15970-sec-0006}
--------------------
Seven doses of ATIR101 were evaluated: 1 × 10^4^ (L1); 5 × 10^4^ (L2); 1·3 × 10^5^ (L3); 3·2 × 10^5^ (L4); 7·9 × 10^5^ (L5); 2 × 10^6^ (L6) and 5 × 10^6^ (L7) T cells/kg. MTD was defined *a priori* as the dose of T cells whereby dose‐limiting toxicity (DLT: grade III or IV acute GVHD within 30 days of ATIR101) would occur in 33% of patients. If the first L1 patient did not experience a DLT, the following 3 patients received the L2 dose; the study would be terminated if 2 patients at the L1 dose level were to experience a DLT (Appendix [S1](#bjh15970-sup-0003){ref-type="supplementary-material"}). After L1, 18 patients were to be treated at L2--L7 (in cohorts of 3) until MTD was determined. Acute GVHD and chronic GVHD (National Institutes of Health classification) were histologically graded as published (Glucksberg *et al*, [1974](#bjh15970-bib-0023){ref-type="ref"}; Filipovich *et al*, [2005](#bjh15970-bib-0021){ref-type="ref"}).
Secondary variables {#bjh15970-sec-0007}
-------------------
The secondary objective was to determine the safety of ATIR101. All adverse events (AEs) were monitored from HSCT until 30 days after ATIR101 infusion; serious adverse events (SAEs) were monitored throughout the study. Prophylaxis and monitoring for infections is described in Appendix [S1](#bjh15970-sup-0003){ref-type="supplementary-material"}.
Efficacy evaluations included immune reconstitution and relapse rate and, in addition to the pre‐planned analysis of overall survival (OS), TRM and relapse‐related mortality (RRM) have been exploratively analysed. TRM was defined as death due to causes other than disease relapse or progression. RRM was defined as death due to disease relapse or disease progression. Patient mononuclear cell populations pre and post transplantation were evaluated using flow cytometry (Appendix [S1](#bjh15970-sup-0003){ref-type="supplementary-material"}). See Appendix [S1](#bjh15970-sup-0003){ref-type="supplementary-material"} for a description of statistical analyses. Due to the small numbers of patients in each dose group, statistical comparisons of clinical outcomes between doses were not carried out.
Results {#bjh15970-sec-0008}
=======
Patient characteristics {#bjh15970-sec-0009}
-----------------------
Nineteen adult patients (median age: 54 years; range: 20--62) with high‐risk haematological malignancies underwent haploidentical HSCT followed by infusion of a single dose of ATIR101 at a median of 31 days after transplant (range 28--40; patient and disease characteristics are provided in Table [1](#bjh15970-tbl-0001){ref-type="table"}). At each dose level except for L1 (which included 1 patient as planned), 3 patients were given ATIR101. All patients except 1 at L7 received the planned ATIR101 dose. The majority of patients had high‐risk acute leukaemia or myelodysplastic syndrome (MDS). Importantly, 14 patients (74%) had active disease (including early relapse, partial remission or refractory disease) at time of HSCT and 10 patients (53%) had a disease history with ≥1 relapse. All donors were HLA‐A, ‐B, and ‐DR mismatched at 2--3/6 loci, except for 1 patient who received a single HLA‐DR mismatched graft.
######
Patient characteristics
Dose level Patient number Patient sex (age, years) Donor: sex, age (years), relationship to patient Diagnosis Disease status at HSCT HLA matches (A, B, DR), (*n*/6) CD34^+^ cell dose, ×10^6^ cells/kg CD3^+^ cell dose, ×10^4^ cells/kg Days from HSCT to ATIR101 infusion ATIR101 dose, T cells/kg
------------ ---------------- -------------------------- -------------------------------------------------- ------------------------------ -------------------------------------------- --------------------------------- ------------------------------------ ----------------------------------- ------------------------------------------------ --------------------------
L1 1 M (54) M, 50, brother CLL Refractory 4 8·27 1·09 32 1·0 × 10^4^
L2 2 F (57) F, 31, daughter AML Relapse 2 3 12·80 1·60 31 5·0 × 10^4^
L2 3 F (58) M, 27, son AML transformed from MDS CR1 4 12·40 2·00 33 5·0 × 10^4^
L2 4 M (59) M, 33, son MDS (RA) Refractory 3 7·40 0·97 28 5·0 × 10^4^
L3 5 M (40) F, 34, sister NHL relapsed post transplant CR3 5 5·93 1·12 34 1·3 × 10^5^
L3 6 M (58) M, 32, son AML transformed from MDS PR 3 13·70 0·65 28/312[†](#bjh15970-note-0003){ref-type="fn"} 1·3 × 10^5^
L3 7 F (52) F, 28, daughter AML Relapse 1 4 8·70 0·78 31 1·3 × 10^5^
L4 8 M (55) M, 58, brother Acute myelofibrosis Relapse 3 7·00 1·04 28/1294[†](#bjh15970-note-0003){ref-type="fn"} 3·2 × 10^5^
L4 9 M (21) M, 56, father AML Ref‐Rel 3 10·35 1·76 31 3·2 × 10^5^
L4 10 F (61) F, 35, daughter AML CR3 4 13·00 1·96 28 3·2 × 10^5^
L5 11 M (59) M, 22, son AML Relapse 1 3 13·90 1·43 40 7·9 × 10^5^
L5 12 F (20) M, 45, uncle Acute biphenotypic leukaemia Ref‐Rel 3 11·20 1·30 28 7·9 × 10^5^
L5 13 M (60) M, 34, son MDS: RAEB Untreated 4 12·35 1·40 28 7·9 × 10^5^
L6 14 F (38) M, 41, brother MDS: RAEB Refractory 3 15·19 0·42 28 2·0 × 10^6^
L6 15 M (37) M, 45, brother CML PR[‡](#bjh15970-note-0004){ref-type="fn"} 4 6·50 1·41 32 2·0 × 10^6^
L6 16 F (43) M, 39, brother AML CR1[§](#bjh15970-note-0005){ref-type="fn"} 3 9·59 0·75 28 2·0 × 10^6^
L7 17 F (54) F, 61, sister AML transformed from MDS CR1 4 8·11 1·75 34 2·6 × 10^6^
L7 18 M (44) M, 46, brother AML transformed from MDS Relapse 4 13·07 1·52 33 5·0 × 10^6^
L7 19 M (62) F, 38, daughter CLL PR 3 10·52 1·95 28 5·0 × 10^6^
AML, acute myeloid leukaemia; CLL, chronic lymphocytic leukaemia; CML, chronic myeloid leukaemia; CR, complete remission; F, female; HLA, human leucocyte antigen; HSCT, haematopoietic stem cell transplantation; M, male; MDS, myelodysplastic syndrome; NHL, non‐Hodgkin lymphoma relapsed after autologous stem cell transplantation; PR, partial remission; RA, refractory anaemia; RAEB, refractory anaemia with excess blasts; Ref‐Rel, refractory relapse.
Patient received a second ATIR101 infusion (same dose as first infusion).
After exposure to 3 tyrosine kinase inhibitors.
Molecular relapse.
John Wiley & Sons, Ltd
HSCT and engraftment {#bjh15970-sec-0010}
--------------------
After their conditioning regimen, patients received a median of 10·5 × 10^6^ immunomagnetically isolated CD34^+^ cells/kg (range: 5·9--15·2 × 10^6^) on Day 0, with a median graft T‐cell content of 1·4 × 10^4^ CD3^+^ cells/kg (range: 0·4--2·0 × 10^4^). Neutrophil (≥0·5 × 10^9^/l) and platelet (≥20 × 10^9^/l) engraftment were achieved rapidly at a median of 10 (range: 8--21) and 11 (range: 6--158) days, respectively. Complete (100%) and sustained donor chimerism in both myeloid and lymphoid lineages occurred in all patients within 3 weeks of transplantation, except for the L1 patient who showed 66% donor chimerism at Week 3 and died 1 month after the ATIR101 infusion due to multiple organ failure, secondary to post‐transplant lymphoproliferative disorder (Epstein--Barr virus \[EBV\] infection present at time of transplant). No patient experienced late graft rejection or failure.
Photodepletion of host‐reactive cells preserves functional T cells {#bjh15970-sec-0011}
------------------------------------------------------------------
The ATIR101 product was successfully generated for all patients. Photodepletion of activated T cells and preservation of resting T cells in the ATIR101 product were confirmed immunophenotypically for all products (Fig [1](#bjh15970-fig-0001){ref-type="fig"}A,B). Indeed, activated CD4^+^ and CD8^+^ T cells (CD25^+^ CD44^+^) were drastically reduced, whereas the majority of resting CD4^+^ and CD8^+^ T cells were preserved. In addition, photodepletion decreased donor CTLp directed against recipient cells by a mean of 1·14 log (93% elimination; Fig [1](#bjh15970-fig-0001){ref-type="fig"}C). In contrast, anti‐third party CTLp were preserved, indicating that ATIR101 T cells remained immunoreactive against other targets (*P* = 0·0008).
{#bjh15970-fig-0001}
Among CD4^+^ cells, photodepletion decreased the number of terminally differentiated effector memory cells (T~EMRA~:phenotype; Fig [2](#bjh15970-fig-0002){ref-type="fig"}D; *P* = 0·02), with a corresponding increase in central memory (T~CM~) cells (Fig [2](#bjh15970-fig-0002){ref-type="fig"}B; *P* = 0·009). In contrast, in CD8^+^ cells, a similar decrease in T~EMRA~ led to a relative increase in naïve cells (Fig [2](#bjh15970-fig-0002){ref-type="fig"}H,E, respectively; both *P* = 0·02).
{#bjh15970-fig-0002}
Safety and GVHD {#bjh15970-sec-0012}
---------------
Treatment‐emergent AEs in the first 30 days after ATIR101 infusion occurred in 17 patients, the most frequent being constipation (*n* = 6), pancytopenia (*n* = 4), pyrexia (*n* = 4), nausea (*n* = 3) and diarrhoea (*n* = 3). Besides GVHD, no treatment‐emergent AEs in this period were considered to be ATIR101 related. Overall, SAEs occurred in 17 patients, the most frequent being pyrexia (*n* = 10), multiple organ failure (*n* = 6), pancytopenia, pneumonia and recurrent leukaemia (all *n* = 4), and disease progression and sinusitis (both *n* = 3). None of the SAEs were considered to be related to the ATIR101 infusion.
Overall, 10 patients (53%) developed acute (grade I/II; *n* = 5) or chronic (extensive; *n* = 5) GVHD during the study. Five of the 10 patients with GVHD were in the 2 highest dose groups (L6 and L7), suggesting a higher probability of GVHD at higher ATIR101 doses. None of the cases of GVHD were considered to be an SAE.
The patient at ATIR101 dose L1 had grade I acute GVHD of the skin before ATIR101 infusion but no GVHD after ATIR101 administration. Therefore only 4 patients (1 each in L2 and L6; 2 in L7) developed acute GVHD post ATIR101 infusion; in these patients, this occurred late following treatment (21%; all grade II; median 102 days post HSCT). The clinical features and treatment of acute GVHD post ATIR101 are described in Table [SI](#bjh15970-sup-0003){ref-type="supplementary-material"}. Overall, 5 patients developed *de novo* chronic GVHD (highest severity, score 3, for skin rash in all 5 patients but without scleroderma) at a median of 144 days post HSCT (1 patient each at the L2, L4, L5, L6 and L7 doses).
Eight of the 10 patients developing GVHD responded well and rapidly to oral immunosuppressive treatment (median duration of immune suppressors was 6 months). Although 5 patients who died had GVHD at the time of death, GVHD was steroid‐sensitive in all patients, and has not been reported as the main cause of any deaths.
T‐cell recovery {#bjh15970-sec-0013}
---------------
Populations of T cells (CD3^+^, CD4^+^, CD8^+^) were measured by multiparameter flow cytometry in peripheral blood from ATIR101 infusion to 1 year post‐HSCT. Although T cells were low in the first months after HSCT, they started to recover 3--6 months after ATIR101 infusion (Fig [3](#bjh15970-fig-0003){ref-type="fig"}A). As expected, the recovery of CD4^+^ (helper T cells) and CD8^+^ (cytotoxic T cells) T‐cell subsets was comparable to that of CD3^+^ cells, interestingly with higher levels of CD4^+^ than CD8^+^ cells present at 12 months (Fig [3](#bjh15970-fig-0003){ref-type="fig"}B, C). Interestingly, patients also recovered B lymphocytes and NK cells rapidly after HSCT (Figure [S1](#bjh15970-sup-0001){ref-type="supplementary-material"}).
{#bjh15970-fig-0003}
Infections {#bjh15970-sec-0014}
----------
The proportion of patients with clinically significant infections and infections reported as SAEs, from the ATIR101 infusion until 1 year post‐HSCT, are shown in Fig [4](#bjh15970-fig-0004){ref-type="fig"}. The figure shows patients grouped according to dose cohorts: L1--L3 (*n* = 7), L4--L6 (*n* = 9), and L7 (*n* = 3). All of the patients in the lower‐dose cohorts (L1--L3) and the highest‐dose cohort (L7) had at least 1 clinically significant infection. Six of the 7 patients in cohorts L1--L3 had an infection reported as an SAE (85·7%; including an EBV infection in the patient receiving the L1 dose that was active at the time of transplantation and reported as an SAE post‐transplant). The high incidence of serious infections at these lower doses of ATIR101 is probably due to having insufficient levels of T cells. All 3 patients in the L7 cohort also had infections reported as SAEs in the first year post‐HSCT. Although 8 of the 9 patients (88.9%) in cohorts L4--L6 developed at least 1 clinically significant infection (Patient 16 in L6 had no clinically significant infections after ATIR101 infusion), no infections were reported as SAEs in this group.
{#bjh15970-fig-0004}
Eight patients were at risk for cytomegalovirus (CMV) infection, but only 3 developed CMV viraemia, which led to CMV disease in 2 cases. In 1 case, the patient had received a low ATIR101 dose (L2) and died of CMV disease. In contrast, the other patient with CMV reactivation had received a higher ATIR101 dose (L7) and responded to ganciclovir. Despite more than 90% of patients having a positive EBV serology at the time of transplant (data not shown), increasing EBV polymerase chain reaction (PCR) titres were noted in only 5 patients (26%). The first patient had received the lowest ATIR101 dose (1 × 10^4^ CD3^+^ cells/kg) and died shortly thereafter. Rising EBV titres were observed in 2 patients receiving ATIR101 as the only treatment for their EBV infection. In both patients, ATIR101 infusion at doses of 3 × 10^5^ or 2 × 10^6^ CD3^+^ cells/kg resulted in a sustained complete disappearance of EBV without the addition of any rituximab (Fig [5](#bjh15970-fig-0005){ref-type="fig"}A,B). The other 2 patients with EBV titres rising above 250 000 copies/ml received rituximab (Fig [5](#bjh15970-fig-0005){ref-type="fig"}C,D) (Reddy *et al*, [2011](#bjh15970-bib-0044){ref-type="ref"}). A single dose of rituximab was sufficient to control the viremia; in 1 of these 2 patients the elevated EBV PCR titre had already subsided at the time of rituximab administration (Fig [5](#bjh15970-fig-0005){ref-type="fig"}D). Although T cell levels were low in all of these patients' peripheral blood, CD3^+^, CD4^+^, and CD8^+^ cell levels increased in response to increasing EBV titres, suggesting a role for T‐cell expansion in the clearance of EBV infection.
{#bjh15970-fig-0005}
Long‐term patient outcomes {#bjh15970-sec-0015}
--------------------------
Twelve of the 19 patients in this very high‐risk population died during this long‐term follow‐up (8‐year OS 37%; Fig [6](#bjh15970-fig-0006){ref-type="fig"}A): 7 due to TRM (infections and other causes) and 5 due to relapse. In total, 8 patients relapsed after a median of 10·8 months following HSCT, with no cases of relapse occurring after 39·5 months through to more than 8 years of follow‐up. Only 1 of the patients with relapse was in complete remission (CR) at baseline (RRM at 30·8 months). Kaplan--Meier curves for OS, TRM, and RRM are shown in Fig [6](#bjh15970-fig-0006){ref-type="fig"}A,C,E.
{#bjh15970-fig-0006}
There was no case of TRM in the 9 patients of the L4--L6 cohorts; in contrast, all 3 patients in the L7 cohort died due to TRM. The lack of TRM in the L4--L6 cohort is in line with the trend for decreased infectious episodes in cohorts L4--L6, compared with L1--L3 and L7 (Fig [4](#bjh15970-fig-0004){ref-type="fig"}). To further explore any potential dose dependency, separate Kaplan--Meier curves for OS, TRM, and RRM were produced for the L1--L3 (*n* = 7), L4--L6 (*n* = 9) and the L7 (*n* = 3) cohorts (Fig [6](#bjh15970-fig-0006){ref-type="fig"}B,D,F). There was a markedly lower TRM and higher OS in the combined L4--L6 group compared with the L1--L3 and L7 groups from 12 months after HSCT onwards. Finally, estimated 8‐year RRM was 33% in L4--L6 compared with 58% in the L1--L3 group, and was lowest in the L7 dose group (0%). Overall, long‐term follow‐up demonstrated that 67% of patients in the L4--L6 dose range survived 8 years post‐transplant, compared with 14% in L1--L3 and 0% in L7.
MTD and determination of phase 2 dose {#bjh15970-sec-0016}
-------------------------------------
Although ATIR101 doses of up to 5 × 10^6^ CD3^+^ T cells/kg were administered in this study, MTD was not reached and no patient developed grade III/IV acute GVHD, despite the complete absence of GVHD immunoprophylaxis post‐transplant. As MTD was not reached, protocol Stage 3 to treat an additional 3 patients at the MTD was not completed (Appendix [S1](#bjh15970-sup-0003){ref-type="supplementary-material"}), and the appropriate dose for phase 2 was selected based on assessment of the doses evaluated.
Although RRM was lowest in the L7 dose group, all 3 of the patients in the L7 cohort experienced GVHD (2 grade II acute GVHD; 1 chronic GVHD), suggesting that increasing ATIR101 doses to 5·0 × 10^6^ CD3^+^ cells/kg potentially led to clinically significant GVHD and a poor outcome. Indeed these patients required immunosuppressive agents and developed serious infections (Fig [4](#bjh15970-fig-0004){ref-type="fig"}). Therefore, in light of the absence of serious infections, lower TRM and longer OS in the L4--L6 dose group, the L6 dose (2 × 10^6^ cells/kg) was selected for subsequent phase 2 studies.
Discussion {#bjh15970-sec-0017}
==========
This study evaluates a highly promising, well‐tolerated immunotherapeutic strategy allowing the delivery of high doses of *ex vivo* alloreactive‐depleted lymphocytes (ATIR101) from haploidentical donors early after T‐cell‐depleted transplant, in patients that did not receive post‐transplant immunosuppression as GVHD prophlaxis. Despite 5 × 10^6^ allodepleted cells/kg being infused, none of the patients in this study developed grade III/IV acute GVHD. There were no deaths due to GVHD or GVHD cases that were classified as an SAE, and most GVHD cases responded rapidly to treatment. No other ATIR101‐related serious or non‐serious AEs were reported. As well as demonstrating the feasibility of this approach, our data also highlight the potential for long‐term disease‐free survival (more than 8 years) following haploidentical HSCT with ATIR101.
The absence of grade III/IV acute GVHD is particularly compelling in view of the major histocompatibility complex (MHC) disparity between donor and recipient, the complete absence of post‐transplant immune prophylaxis for GVHD, and the administration of T‐cell doses more than 100‐fold higher than those previously reported to cause lethal GVHD with unmanipulated T cells (Aversa *et al*, [1998](#bjh15970-bib-0006){ref-type="ref"}). We believe that 3 factors contributed to the absence of grade III/IV acute GVHD or protracted chronic GVHD. First, our photodepletion process specifically eliminated more than 90% of both anti‐host activated cytotoxic precursors and CD4^+^ and CD8^+^ T cells. Second, photodepletion was previously shown to spare regulatory T cells, which may have a protective effect against GVHD (Mielke *et al*, [2008](#bjh15970-bib-0036){ref-type="ref"}; Bastien *et al*, [2010](#bjh15970-bib-0010){ref-type="ref"}, [2012](#bjh15970-bib-0011){ref-type="ref"}; Koreth *et al*, [2011](#bjh15970-bib-0027){ref-type="ref"}; Matsuoka *et al*, [2013](#bjh15970-bib-0033){ref-type="ref"}; Martelli *et al*, [2014](#bjh15970-bib-0032){ref-type="ref"}). Third, we speculate that our approach depletes both T cells reactive to HLAs encoded by the non‐shared haplotypes and T cells specific for the most immunogenic host minor histocompatibility antigens (MiHAs) presented by HLAs encoded by the shared haplotype (Perreault *et al*, [1998](#bjh15970-bib-0040){ref-type="ref"}). Unlike infusions containing T lymphocytes genetically modified to alter their function, such as cells engineered with the herpes simplex thymidine kinase or caspase 9 inducible suicide genes (Ciceri *et al*, [2009](#bjh15970-bib-0016){ref-type="ref"}; Zhou *et al*, [2015](#bjh15970-bib-0053){ref-type="ref"}), ATIR101 cells are not genetically modified. Use of suicide gene‐transduced T cells does not prevent the occurrence of GVHD and can result in lymphodepletion upon activation of the molecular switch in patients developing GVHD. By preventing the occurrence of severe GVHD, ATIR101 may preserve lymphocytes with beneficial graft‐versu‐leukaemia (GVL) and graft‐versus‐infection effects.
Importantly, patients engrafted rapidly (median 10--11 days) and demonstrated complete donor chimerism without any late graft failure or rejection, which represents a favourable engraftment profile in comparison to most umbilical cord blood and T‐replete haploidentical HSCT strategies (Luznik *et al*, [2008](#bjh15970-bib-0030){ref-type="ref"}; Brunstein *et al*, [2011](#bjh15970-bib-0013){ref-type="ref"}; Scaradavou *et al*, [2013](#bjh15970-bib-0049){ref-type="ref"}). All patients in cohorts L4--L6 remained free of serious infections at 1 year and the 8‐year TRM was 0% across these cohorts. For 3 of the 5 patients with rising EBV titres, EBV subsided following ATIR101 infusion as CD3^+^, CD4^+^ and CD8^+^ levels increased in response to infection. In general, post‐transplant T‐cell counts were initially low and started to increase 3--6 months after ATIR101 infusion. It has been reported that, in the absence of ATIR101, CD4^+^ T‐cell counts remain below 0·1 × 10^9^/l for as long as 10 months following CD34^+^ selected grafts (Aversa *et al*, [1998](#bjh15970-bib-0006){ref-type="ref"}). In addition, although CD4^+^ T‐cell counts usually reconstitute much later than CD8^+^ (Ogonek *et al*, [2016](#bjh15970-bib-0038){ref-type="ref"}), it was observed that CD4^+^ levels were higher than CD8^+^ levels at 12 months, possibly accounting for potential benefits of ATIR101 infusion. Interestingly, most CD4^+^ and CD8^+^ naïve T‐cells were preserved in ATIR101 and in patients post‐transplant (Fig [2](#bjh15970-fig-0002){ref-type="fig"} and Figure [S2](#bjh15970-sup-0002){ref-type="supplementary-material"}) without causing severe GVHD, suggesting that anti‐host reactive T cells within this cell subset form a minority of cells and were eliminated (Anderson *et al*, [2003](#bjh15970-bib-0003){ref-type="ref"}). The naive T‐cell repertoire, being approximately 100‐fold more diversified than the repertoire of memory T‐cells, (Arstila *et al*, [1999](#bjh15970-bib-0005){ref-type="ref"}) is positioned to respond to a wider range of pathogens than memory T cells (Yager *et al*, [2008](#bjh15970-bib-0051){ref-type="ref"}; Cicin‐Sain *et al*, [2010](#bjh15970-bib-0017){ref-type="ref"}).
Though this study was not aimed at assessing survival, it is noteworthy that the included patients had particularly advanced haematological malignancies and consisted predominantly of refractory or relapsed adult leukaemia patients. Ten of the 14 patients with acute leukaemia or MDS were not in CR at time of transplant (1 of the 4 patients in CR was in molecular relapse) and 1 patient had relapsed acute myelofibrosis. Relapse occurred in 8 patients during the study (median of 10·8 months following HSCT); however, only 1 of these patients was in CR at the time of transplant. In fact, ATIR101 afforded the 9 patients in the L4--L6 cohorts an 8‐year survival rate of 67%, despite 7 of these patients having active disease at baseline. Any GVL effect related to ATIR101 most probably reflects the fact that host lymphohaematopoietic cells are highly susceptible to immune elimination by donor cells (Mori *et al*, [1998](#bjh15970-bib-0037){ref-type="ref"}; Kolb *et al*, [2004](#bjh15970-bib-0026){ref-type="ref"}). Thus, it is most likely that *ex vivo* photodepletion of ATIR101 eliminates enough host‐reactive cells to reduce the risk of grade III/IV GVHD, while potentially preserving sufficient T‐cell numbers to trigger anti‐host lymphohaematopoietic cells' reaction and cause GVL. The fact that the *in vivo* milieu is a better immune‐stimulatory environment than our *ex vivo* culture system may thereby explain why, following T‐replete haploidentical HSCT, cyclophosphamide administered early post‐transplant may not only eliminate activated alloreactive T cells with the ability to cause GVHD but also deplete cells with GVL activity (Brunstein *et al*, [2011](#bjh15970-bib-0013){ref-type="ref"}; Grosso *et al*, [2011](#bjh15970-bib-0024){ref-type="ref"}; Ballen *et al*, [2012](#bjh15970-bib-0007){ref-type="ref"}). This factor, along with the requirement of post‐transplant immune suppression to prevent the development of lethal GVHD, may compromise GVL effects needed to control malignant disease (Luznik *et al*, [2008](#bjh15970-bib-0030){ref-type="ref"}; Brunstein *et al*, [2011](#bjh15970-bib-0013){ref-type="ref"}; Ciurea *et al*, [2015](#bjh15970-bib-0018){ref-type="ref"}; Rubio *et al*, [2015](#bjh15970-bib-0047){ref-type="ref"}). Thus, *ex vivo* cell processing may provide a uniquely favourable and controlled environment to generate 'selective' depletion of alloreactive cells and preserve cells mediating GVL.
Formally, the MTD for ATIR101 was not reached in this dose‐escalation study; however, doses above 2 × 10^6^ cells/kg might increase the risk of inducing GVHD, given that grade II acute GVHD was observed in 2 of the 3 patients in the highest dose cohort. As a result, the 2 × 10^6^ cells/kg (L6) dose was selected for evaluation in two phase II clinical trials of haploidentical HSCT with ATIR101 administration post transplant (NCT01794299; NCT02500550) (Roy *et al*, [2018a](#bjh15970-bib-0045){ref-type="ref"}, [2018b](#bjh15970-bib-0046){ref-type="ref"}). Importantly, results of the first phase II study in patients with acute lymphoblastic and myeloid leukaemia in remission (unpublished observations) are in line with the present study, with absence of grade III/IV acute GVHD in the first year following ATIR101 infusion (2 × 10^6^ cells/kg) in 23 patients and a 61% OS probability at 12 months post transplant (Roy *et al*, [2018a](#bjh15970-bib-0045){ref-type="ref"}). The 12‐month OS probability in a pooled analysis of 37 patients receiving a single dose of ATIR101 in the two phase 2 studies was 58% (Roy *et al*, [2018b](#bjh15970-bib-0046){ref-type="ref"}). The extensive duration of follow‐up for the present phase 1 study also highlights the potential for a durable benefit of ATIR101 treatment. In the absence of a clear standard between *in vivo versus ex vivo* T‐cell depletion methods, a large, phase III, randomized trial has also been initiated, comparing T‐cell‐depleted haploidentical HSCT + ATIR101 *versus* T‐cell replete haploidentical HSCT + post‐transplant cyclophosphamide (NCT02999854).
We demonstrate herein that photodepletion of anti‐host reactivity in the context of MHC‐mismatched cells offers a potential adjunctive treatment option allowing both anti‐infection and anti‐leukemia activity without promoting grade III/IV acute GVHD. Such an adjunct could be tested after *in vivo* allodepletion or other forms of haploidentical HSCT. The decreased toxicity afforded by the removal of post‐transplant immune suppression also has the potential to extend the upper age limit for transplantation and overcome comorbidities, further enabling potentially curative transplantation to more of the 20--55% of patients who do not currently have a suitable matched donor (Barker *et al*, [2010](#bjh15970-bib-0008){ref-type="ref"}; Petersdorf, [2010](#bjh15970-bib-0042){ref-type="ref"}; Ballen *et al*, [2012](#bjh15970-bib-0007){ref-type="ref"}; Pidala *et al*, [2013](#bjh15970-bib-0043){ref-type="ref"}; Sureda *et al*, [2015](#bjh15970-bib-0050){ref-type="ref"}).
Funding {#bjh15970-sec-0019}
=======
This study was supported by research funding from Kiadis Pharma and the Fonds de recherche du Québec -- Santé (FRQS)‐ThéCell. Joanne McGrail, a medical writer supported by funding from Kiadis Pharma, provided drafts and editorial assistance to the authors during preparation of this manuscript.
Author contributions {#bjh15970-sec-0020}
====================
D.C.R., S.L., S.C., G.S., T.K., L. Bernard, C.P. and J.R. designed the study. D.C.R., S.L., S.C., G.S., T.K., L. Busque, J‐S.D., I.A., L. Bernard, N.B. and J.R. contributed to the implementation of the clinical protocol. R.S.B., K.R., S.M., A.J.B., C.P. and D.C.R. contributed to the immune analyses and interpretation of the immune data. R.S.B. and M‐C.G. contributed to statistical analyses. D.C.R., A.J.B., S.M., K.R., C.P. and J.R. contributed to clinical data analysis and writing of the manuscript. All authors edited and approved the final manuscript.
Conflict of interest disclosure {#bjh15970-sec-0021}
===============================
D.C.R. is an author on a patent held by the Université de Montréal and Hôpital Maisonneuve‐Rosemont and has received research support from Kiadis Pharma. He has attended advisory boards by Novartis, BMS, Paladin and Fate Therapeutics. K.R. reports consulting fees during the conduct of the study. SM discloses consultancy outside the submitted work with Novartis and MSD (personal); speaker fees from Kiadis Pharma and Miltenyi Biotec (via institution), Jazz Pharma and Cellex (personal), and Celgene (personal and via institution); travel support from Kiadis Pharma and Miltenyi Biotec (via institution) and Celgene, MSD, Gilead, Cellex, DGHO and ISCT (other); research funding from Kiadis Pharma (other); EHA registration as a speaker (other). Outside the submitted work, L.B. has attended advisory boards for Novartis, Pfizer and BMS. Outside the submitted work, J.R. has received research grants from Celgene, Janssen and Sanofi Canada in the past 36 months and honoraria for conferences by Amgen, Sanofi, Celgene and Janssen Canada in the past 36 months. S.C. and J.R. declare royalties from ExCellThera outside the submitted work. The other authors declare no competing interests.
Supporting information
======================
######
**Fig S1.** Immune reconstitution of B and NK cells. Box and whisker plots of B lymphocytes and NK cells (A: CD19^+^; B: CD56^+^) from patients' peripheral blood after ATIR101 infusion and through to 1 year post‐HSCT.
######
Click here for additional data file.
######
**Fig S2.** Memory T cell recovery post‐transplant. Memory T cell subtypes: central memory (T~CM~, dark blue), effector memory (T~EM~, green), terminally differentiated effector (T~EMRA~, red) and T~NAIVE~ (pale blue) were evaluated within CD4 (left panels) and CD8 (right panels) T‐cells for patients administered ATIR101 at dose levels L2--L3, L4--L5 and L6--L7 over the first 36 weeks post‐ATIR101 infusion.
######
Click here for additional data file.
######
**Table SI**. Overview of acute GVHD occurring following administration of ATIR101.
**Appendix S1.** Supplementary methods.
######
Click here for additional data file.
The authors would like to thank Drs. Irwin Walker, Ronan Foley and Brian Leber from the Juravinski Cancer Center, Hamilton, Canada, for helpful discussions and patient care; Marieve Cossette for statistical analyses; Mireille Guérin and Jean‐Philippe Bastien for their contribution to cell assays; and the members of the Hôpital Maisonneuve‐Rosemont Cellular Therapy Laboratory, Apheresis and Stem Cell Transplantation Units as well as Radiation Oncology, Microbiology and other consultants for clinical trial support. We would like to thank Dominik Selleslag, Philippe Lewalle and Johan Maertens from AZ Sint‐Jan Brugge‐Oostende, Jules Bordet Institute, and the Catholic University of Leuven in Belgium, respectively, and Robert Negrin and Ginna Laport from Stanford University Medical Center, California, USA, for clinical trial support. In addition, we would like to thank A. John Barrett from the George Washington University, Washington DC, USA, as an originator of selective depletion and for support in data analysis and writing of the paper. Finally, we thank Edwin Wagena, Jeroen Rovers, Manfred Ruediger, Lisya Gerez and Andrew Sandler from Kiadis Pharma for their insightful comments and analyses.
|
{
"pile_set_name": "PubMed Central"
}
|
Introduction {#s1}
============
Hamstring muscle injuries (HMIs) present one of the greatest challenges for those working with athletes and are known to be the most common injuries in sports that involve high-speed running such as athletics,[@R1] cricket[@R2] and the various football codes.[@R3] Recurrence levels are high, ranging from 12% to 63%.[@R4] [@R9] The first month after return to play (RTP) is the highest risk time for recurrence,[@R4] [@R13] though the risk remains elevated for at least 12 months.[@R9] [@R14] [@R15] Despite a rapidly expanding body of the literature investigating HMI in recent decades,[@R16] there has been no significant reduction in HMI rates in professional sport[@R17] [@R18] and recurrences typically cost the athlete more playing time than primary injury.[@R10] [@R17] [@R19] HMIs are heterogeneous, making their prognosis difficult to predict[@R20] and there is no consensus on a single clinical or functional test or imaging investigation that provides strict criteria for safe RTP.[@R21]
A recent systematic review of risk factors for recurrent HMI determined that only five prospective studies fulfilled its inclusion criteria and concluded that no risk factor could be judged as having strong or moderate evidence ([box 1](#bx1){ref-type="boxed-text"}).[@R22] Box 1Risk factors for hamstring muscle injuryPrimary injuryIncreasing age[@R3] [@R9] [@R14] [@R23][@R28]Ethnicity---Black African or Caribbean[@R12] and Australian aboriginal[@R27]Previous injury---major knee injury (eg, ipsilateral anterior cruciate ligament reconstruction, independent of graft selection[@R27] [@R29] and history of osteitis pubis[@R27]Higher level of competition[@R12] [@R27]Later stages of football matches[@R5] [@R12] [@R30]Hamstring muscle strength imbalance profile[@R31]Recurrent injuryHistory of Hamstring muscle injury[@R3] [@R14] [@R15] [@R23] [@R26][@R28] [@R32] [@R33]Larger volume size of injury as measured on MRI[@R34]Grade 1 injury[@R10] [@R35]
Evidence for addressing modifiable risk factors includes hamstring muscle strengthening and effecting change in associated properties such as hamstring:quadriceps (H/Q) strength ratios and optimum angle of peak torque[@R36] for which reliability for the different measures varies.[@R37] [@R38]
There is little evidence regarding the efficacy of various treatments for HMI. Recent reviews of interventions for acute hamstring injury[@R39] [@R40] included only three randomised studies that included time to RTP as an outcome. No paper studied the same intervention. Consequently, clinicians are obliged to consider the evidence that is available in conjunction with their own clinical experience and apply it to the individual case in determining the particular treatment regime to be implemented.[@R41]
The aim of this paper was to use a clinical example to describe a treatment strategy for the management of recurrent hamstring injuries and examine the evidence for each intervention.
Clinical presentation {#s2}
=====================
A 26-year-old professional footballer first sustained an injury to his right hamstring when sprinting during a match. Clinically[@R42] and radiologically[@R43] he presented with a grade 2 biceps femoris musculotendinous junction strain ([figure 1](#BJSPORTS2012091400F1){ref-type="fig"}). Intermuscular haemorrhage tracking around the sciatic nerve was noted.
{#BJSPORTS2012091400F1}
Past injuries included left adductor strain 18 months prior; left soleus strain 18 months prior; left proximal hamstring tendinopathy 17 months prior; right knee grade 3 medial collateral ligament (MCL) tear 16 months prior; right Achilles reactive tendinopathy onset 8 months prior and left adductor strain 6 months prior.
Treatment included {#s2a}
------------------
RICE followed by soft tissue massage, stretching, core strengthening, progressive agility and neuromuscular control exercises, a graded running programme and an isolated hamstring strengthening programme with specific emphasis on eccentric exercises. He returned to full-team training (RTT) 21 days postinjury, RTP 30 days postinjury and then played the next three games without incident.
Two days after his third game, when the player was walking, he experienced a gradual onset of posterior thigh pain. Clinically, he presented with a grade 1 hamstring strain that was MRI negative. He RTT 7 days postinjury and RTP 16 days postinjury, playing a full game without incident. The player then went on holiday for a month which involved long periods of travel, sustained sitting and minimal exercise.
On returning to preseason training, he complained of 'tightness' in his right hamstring from the second day of training, worsening until day 5 when he was withdrawn from training reporting more intense discomfort after kicking a ball in shooting for goal. Clinically, he presented with a grade 1 hamstring injury that was confirmed on MRI ([figure 2](#BJSPORTS2012091400F2){ref-type="fig"}) to be more medial, towards the epimysium and not continuous with the scar tissue from the initial injury.
{#BJSPORTS2012091400F2}
Given the player\'s history of lower back pain, symptoms of 'tightness' on the right side and a feeling of general restriction throughout the lumbar spine, an epidural corticosteroid injection was performed at L4--5 followed by 2 days of complete rest from physical activity.
Postinjection the player reported a general improvement in his feeling of 'freedom' on the right side and felt able to swing his legs through fully into hip flexion when running for the first time since the onset of hamstring stiffness on return to preseason training. However, following a long drive (\>3 h) 5 days later he reported a return of his posterior thigh and lumbar spine restriction. He was treated similarly to the initial injury, RTT 10 days postinjury and RTP 21 days postinjury, playing a full preseason friendly game without incident.
Five days later he started another pre-season game. After 5 min, he sprinted with the ball and crossed the ball with a whipping action and immediately felt some tightness in his hamstring, without an associated feeling of tearing or pain, and was thereafter unable to sprint with confidence. He left the pitch and again presented clinically with a grade 1 injury. Subsequent MRI was reported as a small grade 2 injury ([figure 3](#BJSPORTS2012091400F3){ref-type="fig"}). This injury was again within the long head of biceps femoris muscle, at a similar level, but not continuous with the site of any previous injury.
{#BJSPORTS2012091400F3}
After this fourth episode the player underwent a more prolonged and intensive rehabilitation which included progressions based on the percentage of normal running activity in a game (accelerations, repeated efforts, total distance and high-speed distance). The strength programme had an even greater bias on eccentric hip and knee dominant hamstring strength exercises. He RTT 25 days postinjury and RTP 34 days postinjury as a substitute coming on late in the game. Despite extensively warming up prior to joining the play, after 2 min he accelerated at medium intensity and felt some hamstring pain. He played the few remaining minutes but was unable to sprint. Clinically, he presented with a grade 2 strain but MRI showed only a small myofascial tear ([figure 4](#BJSPORTS2012091400F4){ref-type="fig"}). The player had completed a full week\'s training prior to this game including repeated maximal sprinting and match play situations at full intensity.
{#BJSPORTS2012091400F4}
At this stage, the player had sustained five hamstring injuries in 5 months, none very severe clinically, all of which had undergone a routine yet comprehensive rehabilitation and graded RTP programme that had been successful with other players.
[Table 1](#BJSPORTS2012091400TB1){ref-type="table"} summarises the episodes of hamstring injury.
######
Summary of features of each hamstring injury episode
Clinical severity[@R42] Imaging severity[@R43] (and interventions)
--- ---- ------------------- ------------ ------------------------------------------- ------------------------- -------------------------------------------- --- ----------------------------------------------------------------------------------- ------------ --------- ---- ---- ---- --- --------------- ---
1 46 Sudden Match Sprint 2 15° 2 2 Long head 11.7 cm 21 30 6 5 3 3
2 49 Insidious Theme park Walking-2 days postgame 1 0° 1 0 N/A N/A 7 16 5 4 1 1
3 27 Sudden on gradual Training Kicking (shooting at goal) 1 0° 1 1 Long head 23.6 cm 10 21 7 4 N/A preseason 1
Day 1: epidural \#1
4 35 Gradual Match After long high-speed run 1 0° 1 2 Long head 27.9 cm 25 34 11 7 4 0
Day 17: Epidural \#2
5 Sudden Match Low speed run 2 min after 81 min on bench 1 0° 2 Myo-fascial tear Short head 28 cm 28 38 12 7 5
Day 5: lumbar routine, sitting and upright MRI commence aggressive neural sliders
Day 10: actovegin injections
Investigations {#s2b}
--------------
Between the fifth episode and RTP, a number of additional investigations were performed, including a standard lumbar and posterior thigh MRI; an upright lumbar MRI with standing, sitting and lumbar flexion views and nerve conduction studies, all of which were normal. Isokinetic assessment was conducted 22 days after the final episode using a Biodex 3. After familiarisation, the player completed five maximal efforts of concentric knee flexion and extension from 0° to 90° knee flexion at 60°/s. The results demonstrated right hamstring strength\>left (142 Nm vs 128 Nm, 10% asymmetry); right H/Q ratio\<left (0.57 vs 0.62) and right peak torque angle\>left (24° vs 23°).
The seven-part management plan {#s3}
==============================
The clinical challenge was to identify and address any possible factors that may have contributed to the recurrences, and to review and modify the rehabilitation programme accordingly. Potentially relevant risk factors and modifiable hamstring muscle properties are noted in [table 2](#BJSPORTS2012091400TB2){ref-type="table"}.
######
Currently proposed risk factors after episode 5
---------------------------------------------------- ---------------------------------------------------------------------------------------
Non-modifiable risk factors
History of HMI Yes: five episodes in 5 months
Age\>23/24 Yes: age 26
Black African/Caribbean ethnicity Yes
Past ACL/major knee injury history No: as per definition in study but Gd 3 MCL 18 months prior
Past osteitis pubis No: as per definition in study but bilateral Sportsman\'s hernia repair 8 years prior
Modifiable hamstring muscle properties
Bilateral hamstring strength asymmetry\>8% (REFS) No (injured side strength\>uninjured side)
Concentric H/Q ratio\<0.66 (REFS) Yes (0.58 vs 0.62)
Optimum angle (REFS) 23° (vs 24°)
---------------------------------------------------- ---------------------------------------------------------------------------------------
HMI, hamstring muscle injury.
It was decided early in this injury management to prolong the rehabilitation period to enable a higher volume of intervention and loading. Given the lack of evidence on known univariate risk factors, we investigated a range of other issues that the available evidence and our collective clinical experience suggested may be contributing to elevated risk of reinjury.
Biomechanical assessment and correction {#s3a}
---------------------------------------
A biomechanical assessment included review by a sports podiatrist. Significant findings included asymmetrical ankle joint dorsiflexion (L=7.5 cm vs R=9 cm on dorsiflexion lunge testing)[@R45] [@R46] and a positive Trendelenburg sign[@R47] on the right side with associated internal femoral rotation on treadmill gait analysis. Pelvic assessment was suggestive of a slight anterior rotation of the right ilium with associated functional leg length discrepancy (left approximately 5 mm longer than right).
The player was fitted with custom-made semirigid orthotics to address the 5 mm leg length discrepancy. They were worn in both trainers and football boots from the start of the rehabilitation programme. Specific hip abductor and lateral trunk strengthening exercises were increased into his programme.
Osteopathic manual therapy was started aiming at achieving a sustained resolution of the anterior tilt of the right ilium. Initial manipulation of the ilium was performed by an osteopathic specialist in Germany. Further sacroiliac joint (SIJ) manipulation was undertaken on a weekly basis and combined with specific stretches of the posterior chain. Other manipulations performed included right anterior astragalum (anterior-external position), anterior fibula head, internal hip, anterior iliacum and horizontal sacrum, T~11~-L~2~, T~7~-C~1~ level and first rib (right side) and occipital-C~1~-C~2~.
Neurodynamics {#s3b}
-------------
Although adverse neurodynamic signs were not objectively identified clinically or radiologically---SLR and slump test had at all times been consistently equal and within normal ranges as were repeated normal lumbar MRIs---the player continued to complain of symptoms consistent with a neural contribution to his presentation. Relevant complaints included prolonged sitting, especially while driving, causing numbness and aching in the right buttock and posterior thigh. The player also complained of a 'general restriction' in the right side during swing phase of high-speed running and kicking, and an asymmetrical feeling of heaviness on the right side when fatigued. These symptoms were addressed in two ways.
The first epidural (L4/5) resulted in an immediate resolution of these neural symptoms. However, the player felt that the effect wore off after 4--5 days which coincided with a long car journey in which the player had been sitting for 3 h. It is unclear whether the effect of the epidural had indeed worn off rapidly, or if it had been mechanically counteracted by sustaining the provocative sitting and flexed position. Due to the initial success of the epidural and unknown influence of the prolonged sitting and flexion on negating its effectiveness, a second epidural was performed between the fourth and fifth episodes, with a more prolonged rest period followed by strict instructions on avoiding any periods of prolonged sitting.
The initial rehabilitations included mobilising the neural structures within the posterior thigh via sliding techniques[@R48] complemented by mobilising and releasing areas along the course of the sciatic nerve. This included passive accessory mobilisation and manipulation of lower lumbar segments, manual release and stretching of the hip rotators, intermuscular mobilisation of the hamstring muscle group and mobilisation of the proximal tibio-fibular joint due to its close proximity to the common peroneal nerve.
As these techniques were components of the previous failed rehabilitations, a more aggressive neural mobilisation approach was started incorporating an SLR-biased technique sensitised with internal hip rotation, adduction and dorsiflexion. This was applied daily with a dosage of 3×10 repetitions.
Core stability/neuromuscular control/lumbar spine strengthening {#s3c}
---------------------------------------------------------------
The player\'s core stability programme was redesigned. The programme included a combination of global core type exercises; control exercises with an emphasis on transversus abdominis and internal oblique recruitment and reformer-based Pilates exercises with the aim of combining lumbopelvic control with dynamic recruitment of the hip extensors and hamstring muscles. The player continued to participate in the squad injury prevention programme, a multistation session conducted twice per week before training. These sessions comprise a circuit of proprioceptive, neuromuscular control, core stability/strength and a varied lower limb strength exercise.
Core strengthening was supplemented using Document Based Care (DBC; [www.dbc-clinic.com](www.dbc-clinic.com)) core strengthening machines which have been designed to isolate the specific muscles involved in trunk core stability through a series of specific loaded exercises incorporating the main global lumbar movements (extension, rotation, flexion and side flexion) while limiting movement and activity in the muscles around the hips and thoracic spine. Exercises on the DBC machines were completed every other day from week 2 of the rehabilitation.
Increase strength in hamstrings with eccentric-biased programme {#s3d}
---------------------------------------------------------------
Eccentric strengthening should form an essential part of any hamstring rehabilitation programme[@R19] [@R49] and the player was exposed to this early in all the rehabilitations. Initially, this was in the form of manual eccentric strengthening exercises completed prone on the plinth in three sets of six repetitions with maximal pain-free effort. From week 2, the player completed a 1-in-3 day-strength programme which included both hip and knee dominant hamstring exercises. The programme included double and single leg reverse dead lifts, Nordic hamstring exercises with variations of hip angle, high box step ups, YoYo flywheel hamstring exercises[@R50] and slide board hamstring curls in a bridge position.
In the final 'failed' rehabilitation, the player completed 11 strength sessions; in the latter rehabilitation the number of strength sessions was similar (12) with a similar volume of eccentric strength exercises. The significant difference was an increase in the number of exercises which focused on faster speed of contraction at longer hamstring muscle lengths such as drop lunges (hip dominant) and single leg cable sprinter kick outs (knee dominant).
Overload running programme {#s3e}
--------------------------
Hamstring rehabilitation protocols typically involve incremental progression of running speed with supplementary conditioning when appropriate. The total running and non-weight-bearing training load is usually progressed based on standard weekly training distances and high-speed running intensities. During the player\'s previous HMI, this type of running progression was administered, with the player RTT after completing various objective assessments.
Running load was significantly increased during this latter rehabilitation. The programme was designed to overload the player with high-speed running to provide greater exposure to excessive eccentric loads during this specific movement.[@R51] It comprised eight sets of 3 day cycles with consecutive days of running followed by a day of 'off legs' conditioning (pool training). The first of the two running days was progressed over the 24-day period based on both the maximum running speed and the player\'s individual training and match high-speed running and accelerations/deceleration requirements.
The second of the two running days was a submaximal running session based on the player\'s maximal aerobic speed (MAS).[@R52] This session (4×8×100 m at 120% MAS or 6 m/s) was, therefore, conducted eight times and this not only improved the player\'s aerobic running power but also served as a regular fitness test. As the session was standardised, close heart rate (HR) and Heart Rate Variability (HRV) monitoring determined the player\'s adaptation to this standard protocol during the rehabilitation.
[Table 3](#BJSPORTS2012091400TB3){ref-type="table"} demonstrates the programme.
######
Summary of the running overload programme
Cycle 1 2 3 4 5 6 7 8
------- ----------------- ----------------- ----------------- ----------------- ----------------- ----------------- ----------------- ------------------
Day 1 50% speed+match 60% speed+match 70% speed+match 80% speed+match 85% speed+match 90% speed+match 95% speed+match 100% speed+match
Day 2 MAS/assess MAS MAS MAS MAS MAS MAS MAS
Day 3 Bike Off Pool Off Bike Off Pool Off
MAS, maximal aerobic speed.
The programme was monitored using GPS tracking which provided data on variables including total distance, number of high-speed efforts, high-speed metres and accelerations/decelerations during training sessions.[@R53] In conjunction with video-based tracking of matches (Prozone), this provides an objective measure of each player\'s 'standard week' in terms of training and match loads. From this standard week incremental load percentages can be calculated.
Injection therapies {#s3f}
-------------------
The player consulted an international physician 5 days after the last episode. The physician concluded that this HMI was not serious and that abnormalities in the lumbar region were contributing to the reinjuries. He injected a number of sites with local anaesthetic followed by a mixture of Traumeel and Actovegin. Injections were performed in the lumbar regions centrally, over the facet joints and the iliolumbar ligaments bilaterally and the right SIJ. Three similar injections were administered into and adjacent to the HMI site.
Stretching/yoga/relaxation {#s3g}
--------------------------
The player started regular sessions of yoga-based stretching and relaxation with a qualified DRU yoga instructor. These consisted of DRU fascial warm-up techniques; intense hamstring and low back stretches; guided DRU relaxations and DRU breath training (techniques to improve lung capacity, stamina and to activate the relaxation response through the body). Sessions were undertaken for 60 min twice per week. The player soon reported subjective improvement in his hamstring freedom.
Outcome {#s4}
=======
The player RTT 33 days postinjury and was deemed available for selection in 39 days. He then RTP 42 days postinjury. He played 33 out of a possible 42 games until the end of the season. He missed seven games with a ruptured plantaris but did not miss any due to hamstring problems. He continued this treatment regime at maintenance level during this period.
Discussion {#s5}
==========
History and recurrence risk {#s5a}
---------------------------
There are a number of possible causative links between the player\'s previous injuries and this series of HMI. These included right knee grade 3 MCL rupture 16 months prior. Managed conservatively and despite causing no knee symptoms since RTP, persistent laxity may induce a subtle change in knee biomechanics during end-stage swing phase. It is possible that incomplete rehabilitation from the player\'s right adductor strain 18 months prior may increase load on the lateral hamstrings to compensate for medial weakness. However, the player has not had any adductor symptoms since the injury and his objective signs on preseason screening[@R54] [@R55] were within 'normal' ranges.[@R54] [@R55]
Right Achilles tendinopathy pain earlier in the season may have resulted in altered running mechanics and reduced plantar flexor power though this was not specifically measured. The player had proximal left hamstring pain 17 months prior reporting a similar feeling of 'restriction' during swing phase when high-speed running. Investigations of the lumbar spine at that time showed no abnormality and he was diagnosed with proximal hamstring tendinopathy. The symptoms of pain soon resolved, but a feeling of 'restriction' persisted for approximately 1 year.
Seven part management plan {#s6}
==========================
Seven interventions were performed following the fifth episode of HMI. There are varying degrees of evidence for these interventions.
Biomechanical assessment and correction {#s6a}
---------------------------------------
A significant association between functional leg length asymmetries of greater than 1.8 cm and approximately four times greater risk of HMI has been demonstrated in professional footballers.[@R56] Leg length asymmetries have also been associated with innominate bone rotation, anteriorly on the side of the shortened and posteriorly on the side of the lengthened limb[@R57]. SIJ dysfunction has also been reported to be associated with gluteal hip extensor weakness in patients with lower back pain, compared with patients with lower back pain without SIJ dysfunction.[@R58] Elite sprinters who subsequently sustained HMI also demonstrated reduced concentric hip extensor strength relative to uninjured sprinters.[@R59] These findings suggest that there may be a relationship between SIJ dysfunction, gluteal hip extensor weakness and HMI.
Despite treatment restoring the normal load transfer patterns on dynamic pelvic girdle testing during active straight leg raise in supine[@R60] and in weightbearing,[@R61] [@R62] the player consistently demonstrated a small leg length asymmetry. Despite this asymmetry being considerably less than 1.8 cm[@R56] and within normal variance and measurement error,[@R63] [@R64] given the circumstances of the player\'s recurrent injury history, custom-made orthotics were prescribed to correct this asymmetry. They were immediately well tolerated and remain a component of the player\'s overall management regimen even though their specific effect is incalculable.
The anatomical relationship between biceps femoris, which is also the most commonly injured of the hamstring muscles,[@R65] and SIJ stiffness has been demonstrated.[@R66] Several authors have also demonstrated the relationship between SIJ dysfunction and alterations in load transfer throughout the pelvis via leg asymmetries during walking,[@R70] altered muscle activation timing and strength including earlier hamstring activity onset during functional movements and compromised pelvic stability.[@R58] [@R71] [@R72] Subjects recovering from HMI have similarly been shown to demonstrate earlier hamstring activity onset when preparing for single leg stance,[@R73] suggesting a common trend of altered neuromuscular control between patients with SIJ dysfunction and hamstring injury.
However, it appears that this relationship between SIJ dysfunction and the hamstrings relates to muscle activity onset timing[@R58] rather than the muscle length as was proposed.[@R67] The hamstring length has also been shown not to be associated with the degree of lumbar lordosis or anterior pelvic tilt in several studies[@R74] despite excessive anterior pelvic tilt being suggested to be associated with HMI.[@R12] [@R78]
SIJ dysfunction has been reported in subjects with HMI[@R79] though the inter-rater reliability of many of the tests used is known to be poor[@R79] with almost all subjects including controls in these studies identified as having some level of dysfunction. While Cibulka *et al*[@R80] claimed that SIJ manipulation resulted in an increase in hamstring peak torque measured isokinetically, significant methodological flaws undermine the interpretation of this outcome[@R40] and further research is required to validate the role of SIJ manipulation in the treatment of the player with HMI.[@R71] It is also important to differentiate between studies diagnosing SIJ dysfunction and those based on identifying the SIJ as a pain source using a combination of manual pain provocation tests.[@R82]
Asymmetries in hip joint ROM have also been associated with SIJ dysfunction in patients with lower back pain[@R83] and pelvic innominate tilt has been reported to alter following SIJ manipulation.[@R83] Future research is proposed to investigate the effects of a pelvic belt on injured hamstring function.[@R84] Despite the current lack of clarity in the evidence, the theoretical basis for manual therapy to treat clinically diagnosed SIJ dysfunction justifies consideration in the management of the player with HMI.
Neurodynamics {#s6b}
-------------
Neural tension has been shown not to be a risk factor for HMI[@R28] in two prospective studies in Australian Rules football.[@R14] [@R25] However, its relationship with hamstring flexibility[@R48] [@R85] suggests that such 'sub-threshold' risk factors may combine to increase the recurrence risk. Neural tension has been shown to limit movement and increase stretch resistance when compared to hamstring flexibility measures without added neural tension with this attributed to the extensibility of the neural tissues.[@R85]
Adverse neural tension, defined as a positive slump test[@R86] reproducing the player\'s posterior thigh pain, subsequently reduced by cervical extension, was present in over half of a sample of rugby union players with a history of two or more clinically diagnosed grade 1 HMI in the past 2 years. This contrasted with no positive tests in age, gender and rugby position-matched control subjects.[@R87] Despite this there was no difference in hamstring flexibility between groups. The authors suggested that the proximity of the sciatic nerve to the hamstrings implicates scarring potentially compromising the normal mobility and nutrition of the sciatic nerve. This suggests that clinical signs of adverse neural tension, or more aptly 'neurodynamics' as proposed by Shacklock,[@R88] can persist beyond functional recovery.
Research has shown that including slump stretching in the rehabilitation of AFL players with clinically diagnosed grade 1 HMI and a positive slump test resulted in fewer missed matches prior to RTP.[@R89] Sciatic nerve slider exercises have also been shown to immediately increase lumbar flexion and slump test ROM in comparison to hamstring muscle stretching in isolation in a small pilot study of young footballers.[@R48]
Shacklock[@R88] suggests that the primary mechanical fault in the subject with positive neurodynamic tests may be one of 'reduced sliding (neural sliding dysfunction)' which implies a lack of mobility rather than excessive tension. Despite the player consistently demonstrating clinically acceptable symmetrical ranges and absence of symptoms on SLR and slump testing, his history and symptom of a 'lack of freedom' when running at high speed suggested a neural component to his presentation. Following the most recent HMI more aggressive and frequent neural mobilisation techniques were included in the player\'s management regimen, particularly immediately prior to training and playing.
The rate of MRI-negative scans for clinically diagnosed HMI ranges from 14% to 45%.[@R12] [@R17] [@R27] [@R35] [@R65] [@R90] This relatively common clinical presentation implies a referred posterior thigh pain subgroup of HMI.[@R27] The origin of the neural supply to the hamstring and posterior thigh implicates the lumbar spine and its neurodynamics to the lower limb as a potential source of pain referral and injury risk.
Orchard[@R93] proposed that subtle extraforaminal lumbosacral canal impingement of the L5 nerve root may be associated with the increasing age predisposition trend of hamstring and calf injuries in AFL players although injury rates of muscle groups supplied by nerve roots L1-4 show no propensity to increasing age.[@R94]
Several interventions attempted to address any possible contribution from the lumbar spine. Positive clinical experiences have been reported with imaging-guided corticosteroid injections to the region of the lumbosacral canal in athletes with recurrent hamstring problems, both as treatment for acute pain and a prophylactic intervention for high-risk players.[@R93] Epidural steroid injections have also been shown to be effective in achieving and sustaining rapid RTP in American footballers with acute lumbar disc herniation shown on MRI.[@R95]
While the effectiveness of such injections seems greatest in athletes with MRI-negative hamstring or lower limb muscle injury presentations, the frequency and minimal clinical and radiological grade of the HMI sustained by the player, in our opinion, warranted management with lumbar epidural steroid injection. This was undertaken following his third injury. On returning to running 4 days postinjection, during which time he had rested from all physical activity and avoided any sustained sitting, the player reported immediate and complete relief of his symptom of 'lack of freedom' during swing phase of running. However, an unavoidable long drive the following day corresponded to a return of his posterior thigh ache when sitting and during his next running session the day after he reported his 'lack of freedom' again. This sequence further indicated a mechanical relationship between his lumbar spine and running symptoms and hence the epidural injection was repeated at the next available opportunity.
Unilateral lumbar spine zygapophyseal joint mobilisations have been shown to immediately improve SLR ROM[@R96] which is a valid measure of lower limb mechanosensitivity.[@R97] Ankle dorsiflexion induces a different and earlier pattern of muscle activation and also significantly reduces SLR ROM.[@R97] Oscillatory unilateral lumbar spine mobilisation at L4-5 has also been shown to induce immediate side-specific changes in lower limb sympathetic nervous system (SNS) activity, specifically a sympatho-excitatory response.[@R98]
Although the optimal dose, grade and vertebral levels of lumbar manual therapy require further investigation and the mechanisms by which the increase in SLR ROM[@R96] and SNS stimulation[@R98] are unknown, this evidence suggests that beneficial effects from this intervention are possible. Such manual therapy has been an integral component of the player\'s pretraining and match routine throughout and since his rehabilitation and is reported to provide improved freedom of lumbar and lower limb mobility.
Core stability/neuromuscular control/lumbar spine strengthening {#s6c}
---------------------------------------------------------------
A progressive agility and trunk stabilisation programme significantly reduced the HMI recurrence rate at both 2 weeks and 12 months following RTP when compared with a stretching and isolated progressive hamstring strengthening programme.[@R99] Cameron *et al*[@R100] also showed that AFL players with poorer swing leg movement discrimination were more likely to subsequently suffer HMI and that a running technique programme achieved improvements in the same neuromuscular control measure.[@R101] Hamstring muscle stiffness, as measured by the passive knee extension test, was significantly reduced by a 4-week programme of two specific exercises performed twice per week.[@R102] A 'soccer-specific balance training programme' resulted in a significant reduction in HMI rates in a professional women\'s football team.[@R103]
Such research findings suggest that improvements in lumbopelvic control can reduce demands on the hamstrings, especially biceps femoris, and subsequently, injury. The player\'s ongoing programme incorporated all components of the programmes suggested to be effective in preventing reinjury and minimising demands on the hamstrings.
A localised approach to core strengthening with the utilisation of DBC machines[@R104] [@R105] complemented this programme. In conjunction with functional exercise, isolated strengthening of the trunk muscles was also undertaken with the goal of optimising the rate and gain in 'core strength'.
Increase strength in hamstrings with eccentric-biased programme {#s6d}
---------------------------------------------------------------
Eccentric exercise is the most researched means of achieving changes in hamstring muscle properties and reductions in HMI rates. While the 2010 Cochrane Collaboration systematic review on hamstring injury prevention[@R106] concluded that there was insufficient evidence to draw conclusions on the efficacy of interventions used for preventing hamstring injury in football, its inclusion criteria is perhaps too strict to dismiss the findings of excluded research from clinical practice. Several studies have investigated the effect of various forms of eccentric exercise on HMI rates in sport,[@R4] [@R14] [@R50] [@R107] strength[@R50] [@R110] and optimum angle measured on isokinetic equipment.[@R36] [@R112] The vast majority reported significant effects on their respective outcome measures.
Previous HMI has been associated with reduced eccentric hamstring strength and biceps femoris EMG activation towards full knee extension in athletes who have returned to training for their sport.[@R116] It has also been associated with shifting optimum angle towards greater angles of knee flexion compared with the uninjured leg,[@R113] [@R117] which is the opposite effect to eccentric training.[@R36] [@R112] Given that previous HMI is a strong risk factor for future injury,[@R3] [@R14] [@R15] [@R23] [@R26] [@R32] [@R33] altered muscle properties such as these may contribute to this risk.
Contrastingly, previous HMI has been shown to not significantly affect peak torque[@R113] [@R116] [@R117] or H/Q ratios.[@R113] [@R117] Similarly, eccentric exercise programmes have been shown not to significantly affect peak torque[@R112] [@R113] [@R115] or H/Q ratios,[@R112] [@R113] perhaps questioning their validity as risk factors for reinjury. Fatigue induced by a soccer-specific treadmill protocol and repeated eccentric isokinetic testing was shown to result in decreasing eccentric torque output, especially at higher testing speeds.[@R30] This suggests that the effects of previous injury, eccentric exercise and fatigue on hamstring muscle performance are contraction type, knee angle and parameter specific and it may be critical to work the muscle accordingly in rehabilitation. The weight of evidence indicates that the terminal swing phase represents the highest risk phase of the gait cycle during high-speed running[@R118] when it is known that the hamstrings are most active[@R119] and eccentrically as the knee is extending[@R123] while also at peak length.[@R123] This suggests that eccentric training, ideally at lengthened ranges, is the most indicated form of exercise in the rehabilitation and prevention of HMI.[@R19] [@R49]
Croisier[@R31] demonstrated in a large prospective study of professional footballers that preseason normalisation of strict strength imbalance criteria that included bilateral asymmetry and various H/Q ratios reduced injury rates to that of non-imbalanced players. The discriminatory benefits of eccentric testing were also demonstrated with over 30% of players identified as imbalanced exclusively on this basis. Unfortunately, the optimum angle was not included in this study. Similarly, the normalisation of isokinetically determined strength deficits in 17 of 18 athletes with recurrent HMI was associated with a zero recurrence rate in the subsequent 12 months.[@R124] These findings indicate that isokinetically determined imbalance criteria can prospectively identify players at higher risk of subsequent hamstring injury and that the normalisation of such criteria, irrespective of the form of exercise undertaken to achieve this, can significantly reduce hamstring injury rates.
Isokinetic testing was conducted after the completion of six strengthening sessions, 22 days post the latter injury and 8 and 16 days prior to RTT and RTP, respectively. While eccentric testing and mixed speed and contraction H/Q ratios have been reported to be more sensitive in detecting imbalances,[@R31] there is a low risk of discomfort[@R31] and injury.[@R125] In our setting, this risk was not justifiable. Concentric peak flexion torque was 10% greater in the injured leg though average eccentric peak torque reduction in athletes with a similar history of HMI has been shown to be more than double the concentric reduction.[@R124]
Optimum angles were almost symmetrical and preferable to those reported in uninjured subjects,[@R117] immediately after high-dose eccentric exercises[@R36] and after a preseason programme in uninjured footballers.[@R112] They were comparable to a similarly recurrently injured AFL player following an extensive eccentric-based rehabilitation programme.[@R113] However, the optimum angle has only been studied prospectively once thus far and there was no difference between the injured and uninjured groups of sprinters.[@R126]
Concentric H/Q ratios would indicate that the player remains predisposed to injury according to the thresholds reported in some studies,[@R100] [@R126] [@R127] but not others.[@R31] Based on the parameters that were measured and Crosier\'s criteria defining preseason muscle imbalance, the player would not be classed as imbalanced though he would have failed to meet the more strict normalisation criteria and was not subjected to eccentric testing.[@R31] [@R124]
Following the isokinetic test, the player completed a further six strengthening sessions, two high-speed running sessions and six team training sessions prior to RTP. While isokinetic testing was not repeated immediately prior to RTP, it is reasonable to expect that his isokinetic profile would have continued to improve as a result of this training. Continued optimisation of such factors remains a focus of the player\'s management indefinitely beyond his RTP.
Recent 'incontrovertible'[@R49] evidence demonstrated 'spectacularly good'[@R49] results in reducing both injury and recurrence rates in footballers with an eccentric Nordic hamstring exercise programme. New injuries and recurrences were reduced by 60% and 85%, respectively. The number needed to treat to prevent one injury (new or recurrent) was only 13 and to prevent one recurrence was only 3. No injuries were sustained during the exercise and there was no increase in the injury rate during the intervention period. The lack of significant decrease in the injury rate during the intervention period indicates that a critical volume of exercise needs to be reached before its effects are realised. However, once this volume is reached the effects were highly substantial. The authors suggest that the shift in optimum length, previously shown to be achieved by eccentric exercise and this particular exercise,[@R36] may be the mechanism by which such favourable effects were achieved.
The Nordic hamstring exercise also has its criticisms including being bilateral and thus potentially promoting existing asymmetries and that it is a single joint exercise, whereas biceps femoris which is the most commonly injured hamstring muscle[@R65] is biarticular.[@R128] It is also typically performed at slow velocity[@R4] [@R36] [@R109] [@R115] and at relatively short muscle lengths until subjects have improved sufficiently to be able to lower themselves to the ground. However, it has been shown to significantly and immediately shift optimum length[@R36] [@R112] [@R113] [@R117] and when performed in sufficient volume also dramatically reduce injury rates.[@R109] Unfortunately, the scheduling within a 6-week English Premier League preseason renders the application of this volume[@R109] of exercise very difficult to implement.
The need for eccentric training to be the foundation of muscle injury rehabilitation has already been outlined. However, it is important to note that in the elite sports setting, both in terms of striving to prevent HMI en masse, and within the context of injury rehabilitation, administering sufficient load to the hamstrings is a major challenge. In the context of an HMI rehabilitation which commonly average 3--4 weeks[@R8] the scheduling of optimal exercises and prescription of optimal dose are paramount. While most evidence for changing muscle properties revolves around the Nordic hamstring exercise, its limitations as outlined above demand that other exercises be prescribed for the player with acute unilateral injury. The programme should be progressed not just in terms of resistance and volume but also in velocity, muscle length at contraction and multiple joint motion. A range of such exercises have been outlined in previous studies[@R112] [@R113] [@R128] and include manual eccentrics in various positions, forward deceleration steps, box drops landing in squat or lunge, eccentric forward pulls, eccentric single and stiff leg dead lifts. Other exercises with similar goals include slideboard leg curls, hamstring catapults, sprinter eccentric leg curls and loaded hip bridges.
Pain and acute injury healing may contraindicate resistance exercise for the first 3--5 days and the player should also return to training and playing not in a fatigued state thus requiring recovery time. This can leave time for as few as 5--7 intensive strengthening sessions if conducted every other day should the player RTP on 3 weeks.
This player completed 11 strengthening sessions prior to the latter injury. Following this episode, he completed 12 sessions prior to RTP. While a similar number of sessions, the mechanism of injury and clinical presentation of this episode were less severe, enabling a more rapid progression to more functional, multijoint, higher speed and greater length at contraction exercises, such as the hip dominant drop lunges and knee dominant sprinter eccentric leg curls in single leg standing.
The player consistently produced greater force on the injured leg with isometric testing measured by a dynamometer in supine at 90° of hip and knee flexion[@R129] and in prone at 15° knee flexion during the latter stages of the latter rehabilitation.[@R15] [@R71] Askling\'s ballistic SLR test[@R130] was also devoid of pain and insecurity.
Several imaging studies[@R44] [@R65] [@R131] [@R132] have demonstrated pathological signs and biceps femoris atrophy and increased strain in a significant proportion of subjects up to 2 years beyond RTP. Given that recurrence risk remains elevated for up to a year and that the effects of eccentric exercise have also been shown to be temporary,[@R36] [@R114] it is imperative that the player continues his customised, varied eccentric programme indefinitely.
Overload running programme {#s6e}
--------------------------
Peak hamstring force has been shown to significantly increase with increasing running speed from 80% to 100% effort[@R118] and so running at high speed is an essential and the most functional component of late stage rehabilitation. By contrast, peak hamstring stretch does not change significantly with increases in speed from 80% to 100%[@R123] so a player should only increase speed above this level once full ROM has been restored. The player will only overcome their anxieties and fear of reinjury having successfully completed a sufficient volume of this unique task which is an inherent demand of most sports. AFL players demonstrated relatively poor performance in their first match on RTP[@R133] and it is reasonable to postulate this is associated with a lack of confidence with sprinting especially given the relationship between sprint distance and repetitions in successful football matches.[@R134] Sprinters have reported taking a median of 16, and up to 50 weeks, to feel that they have returned to preinjury levels of performance.[@R44] While in-house data trends and research[@R135] suggests that there is some risk in players completing high volumes of high-speed running, the risk in not performing a sufficient volume of such training contributing to reinjury upon returning to play may be even higher. Thus following the latter injury an overload approach was applied to the running component of the rehabilitation programme for three reasons: High-speed running provides functional eccentric loading.[@R19] [@R51]Expose the player and his hamstrings to substantially more running load than is required during a typical week then taper just prior to RTT. This was administered to achieve physiological and psychological benefits while recognising that the match situation and its associated sympathetic responses and injury risk being 15 times greater than training[@R11] cannot be readily simulated.The player\'s physical condition required improvement after repeated disruptions to training in recent months. The player\'s HR and HRV scores had been disproportionately high relative to his training load. Extra conditioning had been prescribed during this period however the player was still required to play matches and therefore the volume of conditioning had to be adjusted. A period of overload training provided an ideal opportunity to address this.
The results of this programme can be seen in [table 4](#BJSPORTS2012091400TB4){ref-type="table"}. The high-speed running volume was 49.8% higher in the overload rehabilitation programme than the previous rehabilitation programmes despite a moderate 16.8% increase in training time between the protocols.
######
Total training loads of the player\'s final 'overload' hamstring rehabilitation protocol compared with the previous 'standard' protocol
Sessions Duration (min) Dist (m) Average distance per minute High-speed distance (m) Average high speed distance per minute High-speed entries Average HR (bpm) Training load (RPE×min)
---------- ---------- ---------------- ---------- ----------------------------- ------------------------- ---------------------------------------- -------------------- ------------------ -------------------------
Standard 22 1130 83541 95.7 8950 8.94 996 136 5905
Overload 29 1320 106225 107.7 13409 14.5 1388 141 7245
The player was also not selected to play by the management when he was first declared available and so may have benefited from an extra week of team training prior to RTP.
Injection therapies {#s6f}
-------------------
Actovegin and Traumeel injections for treating muscle injuries are routine practice in German sports medicine. Actovegin is reported[@R136] to promote acceleration of muscle fibre synthesis in damaged muscle and de-toning of the hypertonic muscle bundle. Traumeel S, a homeopathic formulation, is alleged[@R137] to suppress the release of inflammatory mediators and stimulate the release of anti-inflammatory cytokines. There have been no controlled trials[@R138] into either substance for the treatment of HMI. Reduced HMI time to RTP was reportedly associated with Actovegin injections in professional footballers[@R139] though methodological flaws compromise the interpretation of outcomes of this pilot study.
However, there is not likely to be any harm in administering either substance.[@R140] While striving for the most comprehensive intervention and rehabilitation programme possible, the player consulted an acclaimed sports medicine physician and underwent a series of such injections. At the very least this had the psychological effect of ensuring the player felt he was provided with all interventions for which expert opinion suggests any benefit.
Stretching/yoga/relaxation {#s6g}
--------------------------
While evidence regarding hamstring flexibility as a risk factor for HMI remains ambiguous.[@R3] [@R14] [@R23] [@R25] [@R32] [@R126] [@R127] [@R141] higher stretching frequency during rehabilitation was associated with earlier restoration of AKE ROM and reduced time to RTP.[@R144] Soft tissue healing theory[@R145] also advocates stretching within the treatment regime to maximise scar extensibility.
Maximum hamstring muscle lengths are reached during terminal swing phase of high-speed running with increasing speed beyond 80% only affecting the moment and speed at which it occurs.[@R123] [@R146] Given that the moment of maximum length coincides with the highest risk phase of the gait cycle[@R118] [@R122] and that prior HMI increases mechanical strain at the proximal biceps MTJ even at relatively low eccentric loads,[@R132] it is essential that optimal tissue extensibility is restored.
Reduced flexibility into hip extension has also been implicated as a risk factor for HMI.[@R14] Given that the activation level of iliopsoas in the stance leg greatly increases the stretch of the hamstrings of the swing leg[@R118] and that it was during this moment in the running stride that our player complained of his 'lack of freedom' of his injured hamstring, several manual techniques were employed to optimise hip extension ROM on his contralateral leg. However, ROM on modified Thomas Test[@R147] was not significantly limited or asymmetrical at any stage during his various rehabilitations. Currently, we continue to stretch the player\'s hamstrings regularly, including from Thomas test position with the contralateral hip concurrently stretched into hip extension. However, we note that static hamstring stretching with the ankle held in plantar grade has been shown not to achieve a change in SLR ROM or neurodynamics.[@R48] [@R96]
Dru Yoga is shown to be effective in the treatment and management of low back pain and stress.[@R148] [@R149] Whether the relaxation or stretching is contributing the most benefit remains unknown.
Conclusion {#s7}
==========
We have presented a seven part intervention strategy for the management of recurrent HMI in one player. Whether any one of these interventions was the key factor in the success of this rehabilitation is impossible to determine. There are varying levels of evidence for every individual component of this treatment regimen. This gap between the evidence base and the reality of clinical practice and its variable outcomes intensifies the pressure on clinicians working in professional sport and invites the temptation of speculative interventions. The clinician is obliged to deliver interventions based on the evidence that is available in its varying levels and forms. This is true for the management of many injuries encountered in sports medicine and medicine generally. Our profession must acknowledge this inadequacy and pursue the evidence essential to overcome it.
###### What are the new findings?
- A new rehabilitation protocol is proposed for the management of recurrent hamstring injuries.
###### How might it impact on clinical practice in the near future?
- This paper will help clinicians in determining the rehabilitation protocol for a recurrent hamstring injury.
**Contributors:** PB coordinated the treatment programme and with AN was the main author of the paper. CM and DB were involved in the management of the patient and contributed to appropriate sections of the paper. AD was the radiologist involved in the management of the patient and provided the images.
**Competing interests:** None.
**Provenance and peer review:** Not commissioned; externally peer reviewed.
|
{
"pile_set_name": "PubMed Central"
}
|
Background {#Sec1}
==========
In cattle, ovarian follicular development is characterized by two or three consecutive follicular waves per estrous cycle. Each wave involves the recruitment of a cohort of follicles at the diameter of 3- to 4-mm. A selection phase follows, in which a single follicle continues its development and becomes the dominant follicle (DF), whereas subordinate follicles degenerate by atresia \[[@CR1]\]. The DF of the final wave of the estrous cycle pursues its growth and matures into an ovulatory follicle (OF) following the release of the preovulatory LH surge. The ovulatory process begins at the time when the endogenous preovulatory LH surge stimulates G protein-coupled LH/human chorionic gonadotropin receptors (LHCGR) located on granulosa and theca cells. Activation of the LHCGR induces a developmental program in different compartments of the DF to ensure the release of a competent oocyte via changes in formation/organization of hyaluronan-rich cumulus extracellular matrix followed by rupture of follicular wall, and by formation of the corpus luteum through differentiation of granulosa and theca cells into luteal cells. These processes are controlled by the expression of many genes that are either up- or downregulated in a temporally and spatially distinct fashion. The mechanisms ensuring the transition from a DF into an OF are not fully understood \[[@CR2]\]. This information is essential to advance our understanding of the cascade of events leading to ovulation and luteinization of the follicle, which may be further applied to enhance fertility.
Genes expressed in granulosa cells (GC) that control the growth of a bovine dominant or preovulatory follicle are rapidly downregulated as a consequence of post LH surge-mediated increases in intracellular signaling \[[@CR3], [@CR4]\]. In conjunction with the termination of specific gene expression in preovulatory follicles, LH/hCG induces expression of genes involved in ovulation and luteinization, as shown in rodents \[[@CR5]\] or in the bovine species \[[@CR4], [@CR6], [@CR7]\]. Most data on the temporal pattern of gene expression arising during ovulation have been obtained using RNA extracts from the immature eCG- and hCG-stimulated rodent ovaries. Moreover, the use of microarray in rodent or bovine studies are limited to genes present in the array, and results among bovine studies vary in relation to experimental design and treatment used \[[@CR4], [@CR6], [@CR7]\]. The objective of the present study was to identify candidate genes that are upregulated in GC of bovine preovulatory follicle in response to an ovulatory stimulus. The working hypothesis was that the transition from a DF into an OF results from transcriptional induction or upregulation of a subset of genes in GC. Gene expression was studied in GC because they represent an important compartment of the ovarian follicle involved in hormone synthesis and maturation of the oocyte \[[@CR2]\]. The GC were obtained from hCG-induced OF and growing DF collected during the first follicular wave of the bovine estrous cycle. Gene expression analysis was achieved by use of suppression subtractive hybridization (SSH) \[[@CR3], [@CR8]\] that results in enrichment of differentially expressed genes in OF, followed by the establishment of a GC subtracted cDNA library (OF-DF). The cDNA clones isolated from the subtracted library were validated for their differential expression pattern by cDNA macroarrays and characterized by sequencing. A subset of genes found to be differentially expressed by macroarrays were further validated by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) performed on independent follicles and corpus luteum obtained at different developmental stage. Also, we undertook the characterization of the differentially expressed mRNA of cysteine-rich secretory protein LCCL domain-containing 2 *(CRISPLD2)*, periostin (*POSTN)* and lethal (3) malignant brain tumor-like protein (*L3MBTL3)* full-length cDNAs since they were not experimentally characterized in the bovine species.
Methods {#Sec2}
=======
Experimental animal model and sample preparations {#Sec3}
-------------------------------------------------
Normal cycling crossbred heifers were synchronized with one injection of PGF~2α~ (25 mg, im; Lutalyse, Upjohn, Kalamazoo, MI) given in the presence of a corpus luteum (CL). Behavioral estrus was monitored at 12 h intervals, from 48 h to 96 h following the PGF~2α~ injection. From the time of PGF~2α~ injection until ovariectomy, ovarian follicular development was monitored by daily transrectal ultrasonography performed with a real-time linear scanning ultrasound diagnostic system (LS-300; Tokyo Keiki Co, Ltd., Tokyo, Japan) equipped with a 7.5-MHz transducer probe. At each examination, the diameter of the CL and individual follicles ≥ 4 mm were measured at their largest cross-sectional area using internal calipers. Following estrus synchronization by PGF~2α~, heifers were randomly assigned to the dominant follicle group (DF; *n* = 4) or the ovulatory hCG-induced follicle group (OF; n = 4). In the DF group, the ovary bearing the DF on the morning of day 5 of the estrous cycle (day 0 = day of estrus) was obtained by ovariectomy (via colpotomy). The DF was defined as \>8 mm by ultrasonographic measurement and growing while subordinate follicles were either static or regressing \[[@CR9]\]. The mean diameter of DF measured at the surface of the ovary was 10.4 ± 0.3 mm. The OF were obtained following an injection of 25 mg of PGF~2α~ (Lutalyse) on day 7 to induce luteolysis, thereby maintaining the development of the DF of the first follicular wave into a preovulatory follicle \[[@CR10]\]. An ovulatory dose of hCG (3000 IU, iv; APL, Ayerst Lab, Montréal, QC) was injected 36 h after the induction of luteolysis, and the ovary bearing the hCG-induced OF was collected by ovariectomy at 24 h after hCG injection. The mean diameter of OF was 12.9 ± 0.3 mm. Follicular fluid and mural GC with whole cumulus-oocyte complex were collected separately from individual DF or OF as described previously \[[@CR9]\]. Additionally, GC and follicular fluid were collected from 2 to 4 mm follicles that were obtained from slaughterhouse ovaries representing a total of three pools of 20 small follicles (SF). These experiments were approved by the Animal Ethics Committee of the Faculty of Veterinary Medicine of the Université de Montréal. Concentrations of progesterone (P~4~), estradiol-17β (E~2~) and their ratio (P~4~/E~2~) were analyzed by RIA of follicular fluid as previously described \[[@CR9]\]. The E~2~/P~4~ ratios were calculated for each sample: 1) 0.008, 0.06 and 0.01 for the three SF pools; 2) 17.3, 19.7, 14.3 and 63.4 for individual DF at day 5 (*n* = 4); and 3) 0.44, 0.87, 0.64 and 0.27 (n = 4) for individual OF. CL at day 5 of the estrous cycle were obtained by ovariectomy from cows following ultrasound monitoring of follicular development and estrus synchronization as described above. The CL were dissected from the ovarian stroma, frozen in liquid nitrogen, and then stored at −80 °C until RNA extraction. Total RNA was isolated from GC or CL by homogenization in lysis buffer (4 M guanidium isothiocyanate, 0.5% Na-N-laurylsarcosine, 25 mM NaCitrate, pH 7), and total RNA sedimented on a cesium chloride cushion by ultracentrifugation as previously described \[[@CR9]\]. The concentration of total RNA was quantified by measurement of optical density at 260 nm, and quality was evaluated by visualizing the 28S and 18S ribosomal bands following electrophoretic separation on a formaldehyde denaturing 1% agarose gel with ethidium bromide.
Suppression subtractive hybridization and differential hybridization screening {#Sec4}
------------------------------------------------------------------------------
To compare gene expression in GC collected from OF versus DF, the suppression subtractive hybridization (SSH) was performed as previously described \[[@CR8]\]. Identical amounts of total RNA (2 μg) from four OF or four DF were pooled within treatment groups to decrease inter animal variation. To generate sufficient amounts of double-stranded cDNA for an SSH experiment, both OF and DF cDNAs were amplified separately using the SMART PCR cDNA synthesis kit. One microgram of total RNA from each pooled group was reverse transcribed with an oligo-dT30 primer \[CDS: 5′-AAGCAGTGGTAACAACGCAGAGTACT(30)(A/C/G/T) (A/G/C)-3′\] and PowerScript (BD Biosciences Clontech) to generate the first strand cDNA. Second cDNA strands were produced with the SMART II 5′-anchored oligo and PCR-amplified for 15 cycles using Advantage 2 DNA polymerase (BD Biosciences Clontech). PCR-generated OF and DF cDNAs were digested with *Rsa*I to generate blunt-ended cDNA fragments \[from 0.2 to 2 kilobases (kb)\]. The OF cDNAs were subtracted against DF cDNAs (forward reaction: OF-DF) using PCR-Select cDNA subtraction technology (User manual: PT1117-1; BD Biosciences Clontech) \[[@CR8]\], and in a parallel experiment, the DF cDNAs were subtracted against the OF cDNAs (reverse reaction: DF-OF). The efficiency of subtraction was analyzed by comparing the abundance of cDNAs before and after subtraction by PCR using bovine gene-specific primers for a gene known to be induced by hCG, such as prostaglandin-endoperoxide synthase 2 (*PTGS2*), and a gene known to be downregulated by hCG, such as cytochrome P450, family 19, subfamily 1 (*CYP19A1*). PCR amplification was performed using Advantage 2 DNA polymerase (BD Biosciences Clontech), and 5-μl aliquots were removed following determined numbers of PCR cycles. The amplified products were resolved on a 2% agarose gel in TAE buffer (40 mM Tris-acetate, 1 mM EDTA, 0.5 μg/ml ethidium bromide). The difference in the number of cycles needed to generate an equal amount of the corresponding PCR product in subtracted and unsubtracted samples served to indicate the subtraction efficiency.
The subtracted cDNAs were cloned into the pT-Adv plasmid (BD Biosciences Clontech) to construct the OF-DF subtracted library and used to transform competent TOP10F' *Escherichia coli* as previously described \[[@CR11]\]. The subtracted OF-DF cDNA library (940 individual colonies) was used to establish macroarrays for differential screening following previously described methodologies \[[@CR8]\]. The insert of each cDNA clone was amplified in 96-well plates by PCR (28 cycles) using the PCR-nested primers 1 and 2R and AmpliTaq DNA polymerase (Roche Molecular Systems Inc., Laval, QC). To establish the cDNA macroarrays, an aliquot of each amplification product was denatured in 0.3 M NaOH with 5% bromophenol blue, and 10 μl were vacuum transferred with a 96-well dot-blot apparatus onto nylon membranes (Hybond-N^+^, GE Healthcare Life Sciences), which were then exposed to 150 mJ ultraviolet light (UV) to perform DNA cross-linking (Gs Gene Linker; Bio-Rad, Mississauga, ON). Control cDNAs (*CYP19A1, PTGS2*) were transferred onto the macroarrays. For each 96-well plate, two identical cDNA macroarray replicate membranes were generated. The OF-DF and DF-OF cDNA pools were used to generate complex hybridization probes for differential screening of macroarrays of the OF-DF cDNA library. Probes were obtained by performing the secondary nested PCR and were then purified (QIAquick PCR Purification Kit; Qiagen Inc., Mississauga, ON). To prevent nonspecific interaction of the probes to cDNAs on macroarrays during hybridization, the adaptors were removed by three successive digestions with *Afa*I, *Sma*I, and *Eag*I; the cDNA pools were again purified (QIAquick; PCR Purification Kit, Qiagen Inc.) and 100 ng were labeled with \[α^32^P\]-dCTP by random priming (Megaprime DNA Labeling System; GE Healthcare Life Sciences). The radioactive probes were purified (QIAquick Nucleotide Removal kit; Qiagen Inc.) and quantified using a beta counter. The hybridization and washing conditions of macroarrays were performed as previously described \[[@CR8]\]. Equal amounts of each heat-denatured cDNA probe (OF-DF, DF-OF) were used to hybridize each replicate of the OF-DF macroarray membrane. Following washing, membranes were exposed to a phosphor screen for 4 h and the images were digitized (Storm 840; GE Healthcare Life Sciences). The differentially hybridizing cDNA clones were characterized by DNA sequencing and their differential expression profiles were further validated by semiquantitative RT-PCR analysis from independent follicles obtained at different developmental stages.
Identification of differentially expressed cDNAs were obtained by sequencing. The cDNA clones identified as differentially expressed by the OF-DF subtracted probe were amplified by PCR for 15 cycles with the corresponding PCR-nested 1 and PCR-nested 2 oligos from the PCR product generated initially for the macroarrays. The PCR product was purified (Qiagen Inc.) and verified by agarose gel analysis for the presence of a single cDNA band before proceeding with sequencing. Sequencing reactions were performed on cDNA clones via the dideoxy sequencing method (Big Dye Terminator 3.0; ABI Prism, Applied BioSystem, PE, Branchburg, NJ) using the oligos PCR-Nested 1 or 2, and sequencing reactions were analyzed on an ABI Prism 310 sequencer (Applied Biosystems). Nucleic acid sequences were analyzed by BLAST (Basic Local Alignment Search Tool) against GenBank data banks.
Gene expression analysis {#Sec5}
------------------------
The cDNA clones that were identified as differentially expressed in the SSH differential screening experiment were used to compare their differential expression pattern in GC collected from follicles at different developmental stages and CL, using semiquantitative RT-PCR/Southern blot analysis (also called virtual Northern blot analysis; User manual: PT1117-1; BD Biosciences Clontech). To perform semiquantitative RT-PCR/Southern blot analysis, total RNA (1 μg) from GC (SF, DF, OF) or CL (D5) were reverse transcribed using SMART PCR cDNA synthesis technology (BD Biosciences Clontech, Mississauga, ON) as previously described \[[@CR8]\]. The cDNA products for each follicle or CL along with molecular weight standards (1 kb ladder, ϕX 174-RF/Hae III and l/Hind III; GE Healthcare Life Sciences) were separated on agarose gel, then transferred onto nylon membrane. Gene-specific probes derived from SSH cDNA fragments were generated by PCR (20 cycles) using the primers PCR Nested 1 and PCR-Nested 2R. Glyceraldehyde-3-phosphate dehydrogenase (*GAPDH*) probe was used as a constitutively expressed gene. Purified cDNA probes were labeled with \[α^32^P\]-dCTP as described above. Semiquantitative RT-PCR/Southern membranes were prehybridized, hybridized, and washed as described \[[@CR8]\]. Membranes were exposed to phosphor screen and the images were digitized (Storm 840; GE Healthcare Life Sciences).
Semiquantitative RT-PCR analysis was performed for genes that showed a weak signal by RT-PCR/Southern blot or to analyze differentially spliced transcripts \[[@CR8]\]. Gene-specific primers were designed into the open reading frame of the cDNA sequence and are described in Table [1](#Tab1){ref-type="table"}. SMART cDNAs were generated as described above, and 1 μl was used in a 25-μl PCR reaction using the Advantage 2 DNA polymerase. The number of PCR cycles were limited and optimized to fall in the linear range of amplification reaction for each gene to be analyzed. The PCR reactions were separated on a 2% TAE-agarose gel with ethidium bromide, PCR products were visualized by UV, and the images digitized. The digitized signals for each gene obtained either by semiquantitative RT-PCR/Southern blot or semiquantitative RT-PCR were analyzed by densitometry using ImageQuant software (GE Healthcare Life Sciences).Table 1Gene-specific primers used in SSH and semiquantitative RT-PCR analysisGenePrimer Sequence (5′-3′)^a^GenBank accession number*ADAMTS1*FwdCGATAAATGTGGCATCTGTGGAGNM_001101080RvAGCCCACACGACTTGGAACACTC*ADAMTS9*FwdGGAAATCCGTATTGGGAATGCTGNM_001206573RvCATCTAGCCTGCTGTACTTGGC*ARAF*FwdTCTAACAACATCTTCCTGCACGAGNM_001014964RvGGCAACTCATCAGCCTGGGTAC*CAPN2*FwdTGAGGGCTTCGAGGACTTCACTGNM_001103086RvGCTGTCACTGGTGAGTGTGTCAG*CRISPLD2*FwdCTATGGGATCCTGGACGACAGGAY369781RvCCTGCAAGTTCACTGCCTGACG*CYP19A1*FwdGTCCGAAGTTGTGCCTATTGCCAGCNM_174305RvCCTCCAGCCTGTCCAGATGCTTGG*FKBP4*FwdTCCATTCCTCGTGAATGAATGTCCNM_001034322RvTGAACCTTCAGCAACGTGCAGAG*FKBP5*FwdACTGGAGCAGGCTGCCATTGTCXM_615814RvCTTGAACATGTTGGCATAGATTCTG*GAPDH*FwdTGTTCCAGTATGATTCCACCCACGNM_001034034RvCTGTTGAAGTCGCAGGAGACAACC*KITLG1*FwdAGTAACCGTGTGACTGATGATGTGNM_174375RvAGAATGCTGGCAATGCTACGGCTG*KITLG2*FwdAGTAACCGTGTGACTGATGATGTGNM_174375RvCTAAGGGAGCTGGCTGCAACAG*L3MBTL3*FwdGTTATTACAGATGAGAGTGAGATGGAY437805RvACTTAAAGGACAGAAAGACGGC*L3MBTL3*FwdCTTACCTGGATGTGAAGAACATGGAY437805(3'end of ORF)RvCTGCAAGGTCTAAGACAGAGCTC*MMP1*FwdCTTGGACTTGCTCATTCTACTGACNM_174112RvGGCATCGATGCTCTTCACCGTTC*MRO*FwdGGTGAGTTCTGAAGTCATCCATGBC146146RvCCTGGTGTGCCGACCACCTTC*NUDT10*FwdGACAGGTGAGCTCTTTCACACTCNM_001035488RvGGAGTTATGTCTAGAGGCACAGTC*NUDT11*FwdGAACAGCAAAGATGTCCAGGATTGNM_001101296RvCACACACATGGTGCCTGGAGAG*P4HA3*FwdTTGGCAAGGTGGCCTATGACATGNM_001001598RvCTGGAGCAGCAGGTAAGGACTG*POSTN*FwdCCAAAGCCCACTGCCAGTTCTCAY445072RvGAAAGCCACTTTGATGTGAAGAATGAG*PSAP*FwdCTTCCTCCTGTACCCTCAGGACNM_174161RvGGGATGACGTTCTCTGAGACCAC*PTGS2*FwdGCATTCTTTGCCCAGCACTTCACCCNM_174445RvCTATCAGGATTAGCCTGCTTGTCTGG*RBP1*FwdCGGTCGACTTTACCGGGTACTGNM_001025343RvGTCATGTCACTCATTCCTAGAGAC*SDC4*FwdTCCGAGAAACTGAGGTCATCGACXM_584869RvGGTACACCAGCAGCAGCACGAG*TIMP1*FwdCGTCATCAGGGCCAAGTTCGTGNM_174471RvGCAAGGACTGCCAGGTGCACAG*TIMP2*FwdTGCAATGCAGACATAGTGATCAGGNM_174472RvCGCTTCTCTTGATGCAGGCGAAG*TNC*FwdCTATGTGCCCATTGCAGGAGGTGNM_001078026RvAACTTGGTGGTGATGGTTGAGCTC*USP53*FwdGAGAGCATACCCACCAGTCAGATGBC149007RvGTTACTTTGACATCAGGAGTAGAGTC*Fwd* foward primer, *Rv* Reverse primer^a^All primers were designed and validated by the authors
Characterization of bovine POSTN, CRISPLD2 and L3MBTL3 cDNAs {#Sec6}
------------------------------------------------------------
Isolation of full-length bovine periostin (*POSTN*), cysteine-rich secretory protein LCCL domain containing 2 (*CRISPLD2)* and lethal (3) malignant brain tumor-like protein (*L3MBTL3)* cDNAs was performed by screening a size-selected cDNA library for each of the cDNA as described \[[@CR11]\], since their respective full-length cDNAs were not experimentally characterized in the bovine species. Initially, the respective sizes of the full-length cDNA were estimated by performing a semiquantitative RT-PCR/Southern blot analysis with cDNA pool generated from hCG-stimulated GC and hybridized with radioactive probes for *POSTN*, *CRISPLD2* or *L3MBTL3* derived from the SSH screening experiment (Table [2](#Tab2){ref-type="table"}). Once the size of the full-length bovine *POSTN*, *CRISPLD2* and *L3MBTL3* cDNAs were determined, total SMART cDNAs from hCG-stimulated GC were size-fractionated by agarose gel electrophoresis, and cDNAs from 2.5 to 3.5 kb for *POSTN*, and from 4 to 6 kb for *CRISPLD2* and *L3MBTL3* were purified and used to construct a size-selected cDNA library based on the pDrive plasmid (Qiagen PCR cloning kit; Qiagen) that was then screened by radioactive hybridization as described \[[@CR11]\]. Positive *POSTN, CRISPLD2* and *L3MBTL3* hybridizing bacterial colonies were grown, their plasmid contents were isolated (QIA-prep, Qiagen), and the size of the cloned cDNA was analyzed following an *EcoR1* digestion and gel electrophoresis analysis. The cDNAs were sequenced via the dideoxy sequencing method (Big Dye Terminator 3.0; ABI Prism, Applied BioSystems, PE) that were analyzed on an ABI Prism 310 sequencer (Applied Biosystems). Nucleic acid sequences were analyzed by BLAST against GenBank data banks.Table 2Genes found to be differentially expressed in bovine granulosa cells of OF compared to DFGeneAccession\
Number^a^Freq^b^Accession Number^c^Identity\
(%)^d^E\
valueDescription^e^*ACTG1*EG5652813NM_001033618100%0BT Actin gamma 1*ADAMTS1*GR5089131NM_001101080100%2e^−126^BT ADAM metallopeptidase with thrombospondin type 1 motif 1*APP*EG5653351NM_001076796100%e^−107^BT Amyloid beta precursor protein*ARAF*EG5653274NM_001014964100%0BT A-Raf proto-oncogene. Serine/threonine kinase*ASB9*EG5653232AY43859599%4e^−179^BT Ankyrin repeat and SOCS box-containing 9*BIRC2*EG5652931XM_015466434100%2e^−67^BT Baculoviral inhibitor of apoptosis repeat containing 2*CAPN2*EG5653321NM_001103086100%8e^−57^BT Calpain 2, (m/II) large subunit*CAV1*EG56533820AY823915100%0BT Caveolin 1*CD83*EG5652651NM_00104659099%0BT CD83 molecule*CDC42SE2*EG5652711NM_00110253796%2e^−180^BT CDC42 small effector 2*CRISPLD2*EG56533316AY369781100%0BT Cysteine-rich secretory protein LCCL domain containing 2*CSRP3*EG5652841NM_00102468999%3e^−165^BT Cysteine and glycine-rich protein 3*CTSK*EG5653241NM_001034435100%5e^−168^BT Cathepsin K*DHTKD1*EG5653151NM_001205838100%3e^−170^BT Dehydrogenase E1 and transketolase domain containing 1*EGR1*EG5652681AY92430798%9e^−127^BT Early growth response 1*EIF4E3*EG5653111NM_00110230699%e^−179^BT Eukaryotic translation initiation factor 4E family member 3*FKBP5*EG5653256NM_001192862100%0BT FK506-binding protein 5*GEM*EG5652882NM_001083732100%0BT GTP binding protein overexpressed in skeletal muscle*GFPT2*EG5653185NM_00107688399%4e^−168^BT Glutamine-fructose-6-phosphate transaminase 2*GRIA3*EG5653203NM_00120605999%2e^−166^BT Glutamate ionotropic receptor AMPA type subunit 3*KIT*EG5653451XM_00520793799%7e^−110^BT KIT proto-oncogene receptor tyrosine kinase*KITLG*EG5652765NC_00730399%0BT KIT ligand*L3MBTL3*EG5653102AY437805100%7e^−73^BT Histone methyl-lysine binding protein*LRIG1*EG5653031XM_002696962100%2e^−84^BT Leucine-rich repeats and immunoglobulin-like domains 1*MAOA*EG5653444NM_18101499%2e^−109^BT Monoamine oxidase A*MMP1*GR5089143NM_174112100%8e^−83^BT Matrix metallopeptidase 1*MORF4L1*EG5653431NM_00103544898%4e^−38^BT Mortality factor 4 like 1*MRO*EG5652734XM_005224181100%1e^−95^BT Maestro*NT5E*EG5652991NM_174129100%0BT 5′-Nucleotidase, ecto*NUDT10*GR5089152NM_001035488100%2e^−138^BT Nudix hydrolase 10*NUDT11*EG5652723NM_001101296100%0BT Nudix hydrolase 11*P4HA3*EG5652692NM_001001598100%1e^−96^BT Propyl 4-hydroxylase subunit alpha 3*PAPSS2*EG5652831XM_58337099%0BT 3′-Phosphoadenosine 5′-phosphosulfate synthase 2*PIR*EG5652791NM_001102358100%1e^−101^BT Pirin (iron-binding nuclear protein)*PLA2G4A*EG5653051AY363688100%4e^−164^BT Phospholipase A2 group IVA*PLAT*EG5652948NM_17414699%0BT Plasminogen activator, tissue type*POSTN*EG56527533AY445072100%0BT Periostin*PSAP*EG5652782NM_17416199%8e^−89^BT Prosaposin*PTGES*EG5653421AY032727100%1e^−122^BT Prostaglandin E synthase*PTGS2*EG56528724AF031698100%0BT Prostaglandin-endoperoxide synthase 2*PTX3*EG5653172NM_00107625998%3e^−61^BT Pentraxin*RBP1*EG5652671NM_00102534399%5e^−101^BT Retinol binding protein 1*RGS2*EG5653464NM_001075596100%7e^−130^BT Regulator of G-protein signaling 2*RND3*EG5653001NM_001099104100%1e^−127^BT Rho family GTPase 3*SAT1*EG56534926NM_00103433399%0BT Spermidine/spermine N1-acetyltransferase 1*SDC4*EG5652893XM_58486999%1e^−136^BT Syndecan 4*SEH1L*EG5653401NM_00110308999%1e^−158^BT SEH1-like nucleoporin*SLC25A17*EG5652661NM_00104594899%0BT Solute carrier family 25 member 17*SOX4*EG5652851NC_00732499%8e^−173^BT SRY-box 4*SULT1A1*EG5653081NM_17752199%2e^−99^BT Sulfotransferase family 1A member 1*SULT1E1*GR5089162NM_17748899%4e^−60^BT Sulfotransferase family 1E member 1*TIMP1*GR5089173NM_174471100%2e^−90^BT TIMP metallopeptidase inhibitor 1*TIMP2*EG5653021NM_17447299%5e^−163^BT TIMP metallopeptidase inhibitor 2*TMEM176B*EG5653011NM_00109914599%3e^−165^BT Transmembrane protein 176B*TMSB4X*EG5652913NM_00111323199%5e^−173^BT Thymosin beta 4, X-linkedTMSB10EG5652641NM_174623100%2e^−131^BT Thymosin beta 10*TNC*EG5653262NM_00107802699%0BT Tenascin C*TNFAIP6*EG5653195NM_00100781399%0BT Tumor necrosis factor alpha-induced protein 6*USP53*EG5653041XM_015463685100%8e^−78^BT Ubiquitin specific peptidase 53*VNN2*EG5653372NM_001163920100%0BT Vanin 2ESTEG5653341AC_000158100%3e^−74^BT transcribed locus, chromosome 1ESTEG5653138AC_00015999%3e^−143^BT transcribed locus, chromosome 2 \[3'UTR of TNFAIP6\]ESTEG5653214AC_000159100%0BT transcribed locus, chromosome 2 \[intron 1 of TNFAIP6\]ESTEG5653392AC_00015999%0BT transcribed locus, chromosome 2 \[intron 1 of TNFAIP6\]ESTEG5653161AC_00015999%0BT transcribed locus, chromosome 2 \[intron 1 of TNFAIP6\]ESTEG5653412AC_00015999%4e^−70^BT transcribed locus, chromosome 2 \[3'UTR of TNFAIP6\]ESTEG5653361AC_00016199%0BT transcribed locus, chromosome 4 \[3'UTR of Ubiquitin-conjugated enzyme E2H; UBE2H\]ESTEG5652961AC_000163100%0BT transcribed locus, chromosome 6ESTEG5653071AC_00016798%4e^−106^BT transcribed locus, chromosome 10 \[intron 2 of Nidogen 2; NID2\]ESTEG5653281AC_00016999%0BT transcribed locus, chromosome 12 \[intron 1 of Glypican-6; GPC6\]ESTEG5652741AC_00016999%3e^−133^BT transcribed locus, chromosome 12ESTEG5652701AC_00017299%8e^−175^BT transcribed locus, chromosome 15 \[3'UTR of Rho GTPase activating protein 20; ARHGAP20\]ESTEG5653141AC_000179100%6e^−156^BT transcribed locus, chromosome 22 \[intron 28 of ADAM metallopeptidase with thrombospondin type 1 motif 9; ADAMTS9\]ESTEG5652951AC_000180100%0BT transcribed locus, chromosome 23 \[intron of runt related transcription factor 2; RUNX2\]ESTEG5653091AC_00018599%0BT transcribed locus, chromosome 28 \[3'UTR of Sterile alpha motif domain containing 8; SAMD8\]^a^GenBank accession numbers of differentially expressed bovine SSH cDNA clones^b^ *Freq* Frequency of cDNA clone identified from macroarray analyses of OF-DF subtracted library^c^Accession number of the best match found following nucleotide sequence comparison via BLAST search in GenBank^d^Identity (%) represents homology estimates of bovine SSH cDNA fragments with nucleotide sequences in GenBank via BLAST search^e^ *BT Bos taurus*, *EST* expressed sequence tag, *UTR* untranslated region
Statistical analysis {#Sec7}
--------------------
Gene-specific signals generated for semiquantitative RT-PCR/Southern blot and semiquantitative RT-PCR analyses were normalized with corresponding *GAPDH* signals for each sample. Homogeneity of variance between follicular groups and CL was verified by O'Brien and Brown-Forsythe tests. Corrected values of gene-specific mRNA levels were compared between follicular and CL groups by one-way ANOVA. When ANOVA indicated significant differences (*P* \< 0.05), multiple comparisons of individual means for SF, DF, OF and CL groups were compared by the Tukey-Kramer test (*P* \< 0.05). Pearson's correlation (*P* \< 0.05) analysis was used to compare amplicon levels for KITLG1 and KITLG2. Data were presented as least-square means ± SEM. Statistical analyses were performed using JMP software (SAS Institute, Inc.).
Results {#Sec8}
=======
Identification of differentially expressed genes by SSH {#Sec9}
-------------------------------------------------------
A cDNA library containing transcripts that are upregulated by hCG in GC was constructed by subtracting DF cDNAs from OF cDNAs (OF-DF). Reverse subtraction was also performed as a control and consisted of OF cDNAs subtracted from DF cDNAs (DF-OF). PCR amplification analysis was used to verify the efficiency of the subtraction procedure by comparing the expression of reference genes such as *PTGS2* in OF and *CYP19A1* in DF, before and after subtraction. In the OF sample, the *PTGS2* PCR product was observed after 18 cycles but was undetectable in the DF sample. In the OF-DF subtracted sample, the PCR-amplified *PTGS2* fragment was detected after 13 cycles, revealing that *PTGS2* cDNA had been efficiently enriched in the subtracted OF-DF sample when compared with unsubtracted OF sample, confirming the effectiveness of the subtraction (validation of subtraction is presented in a Additional file [1](#MOESM1){ref-type="media"}: Figure S1). Subtracted cDNAs were then cloned into plasmid vector to generate the OF-DF cDNA library. Differential hybridization screening was performed on the randomly selected bacterial colonies to eliminate false-positive clones. Colonies were spotted onto two identical sets of macroarrays, and the OF-DF or DF-OF subtracted cDNA preparations were used as probes to hybridize macroarrays. On each membrane, *PTGS2* and *CYP19A1* cDNAs were spotted as controls. Selection of differentially expressed cDNA clones was achieved by comparing signal intensities between the two macroarrays as defined by the following criteria. Positive clones hybridized with the OF-DF subtracted probes but not with the reverse subtracted probes, DF-OF. Representative differential screening results are illustrated in Additional file [1](#MOESM1){ref-type="media"}: Figure S2). Of the initial 940 clones, differential screening identified 279 positives as defined by the selection criteria based on comparing differential intensity of the signal. After visualization on agarose gels of PCR products derived from these clones followed by their sequencing, 261 clones generated sequencing results of adequate quality to be analyzed by BLAST against GenBank databases. Table [2](#Tab2){ref-type="table"} lists all the compared sequences with an example for each entry that was deposited in GenBank, as well as their frequency of identification by differential screening of the OF-DF library. This comparison revealed that 89.7% (234/261) corresponded to 60 non-redundant known genes, and 10.3% (27/261) corresponded to express sequence tags (EST). The BLAST analysis of these ESTs against the bovine genome revealed that 24 ESTs were associated with transcribed loci, either introns or untranslated regions (UTR) of mRNA, of defined genes as indicated in brackets for each EST (Table [2](#Tab2){ref-type="table"}) whereas three ESTs were considered novel transcribed loci.
Analysis of mRNA expression {#Sec10}
---------------------------
Genes identified by macroarray analysis as upregulated in GC following stimulation by hCG were further validated on the basis of their frequency of identification in the OF-DF subtracted library (Table [2](#Tab2){ref-type="table"}). Validation was performed initially by semiquantitative RT-PCR/Southern blot analysis using mRNA samples derived from GC collected from independent follicles at different developmental stages (SF, DF, OF) and CL obtained at Day 5 of the estrous cycle. For these analyses, mRNAs were reverse transcribed into cDNAs, separated on agarose gel, and transferred onto membranes that were then hybridized to cloned SSH cDNA fragments as described in Table [2](#Tab2){ref-type="table"}. Messenger RNA expression for tissue plasminogen activator (*PLAT*), early growth response 1 (*EGR1*), tumor necrosis factor alpha-induced protein 6 (*TNFAIP6*), regulator of G-protein signaling 2 (*RGS2*), spermidine/spermine N1-acetyltransferase 1 (*SAT1*), glutamine-fructose-6-phosphate transaminase 2 (*GFPT2*), KIT proto-oncogen receptor tyrosine kinase (*KIT*) and periostin (*POSTN*), showed a statistically significant increase or induction by hCG in GC of OF (Fig. [1](#Fig1){ref-type="fig"}). When mRNA signals observed for GC of OF were compared with that of DF, expression of *RGS2* and *POSTN* were induced, whereas for other genes, the increase was 32.8-fold for *PLAT*, 24.7-fold for *EGR1,* 7.9-fold for *TNFAIP6*, 18-fold for *SAT1*, 26.8-fold for *GFPT2* and 23.6-fold for *KIT*.Fig. 1Analysis of mRNA expression by semiquantitative RT-PCR/Southern blot. Total RNA was extracted from bovine GC collected from 2 to 4 mm small follicles (SF), dominant follicles (DF) at Day 5 of the estrous cycle, ovulatory follicles (OF) collected 24 h after injection of hCG, and corpora lutea (CL) from Day 5 of the estrous cycle, then used in mRNA expression analyses using semiquantitative RT-PCR/Southern blot analysis. *GAPDH* was used as a control gene and showed no significant difference in expression between groups. Gene-specific signals were normalized with corresponding *GAPDH* signals (1.8 kb) for each sample, and relative values are reported as percent of expression detected in OF. **a**) Expression of *PLAT* (2.5 kb) mRNAs was upregulated by 32.8-fold in OF compared with DF (*P* \< 0.0001); expression of *EGR1* mRNA (2.4 kb) was 24.7-fold higher in OF than in DF (*P* \< 0.0001); expression of *TNFAIP6* mRNA (1.6 kb) was upregulated by 7.9-fold in OF compared with DF (*P* \< 0.0001); and expression of *RGS2* mRNA (1.8 kb) was induced in OF compared with DF (*P* \< 0.0001); **b)** expression of *SAT1* (2.5 kb) mRNAs was upregulated by 18-fold in OF compared with DF (*P* \< 0.0001); expression of *GFPT2* mRNA (3 kb) was 26.8-fold higher in OF than in DF (*P* \< 0.0029); expression of *KIT* mRNA (5 kb) was upregulated by 23.6-fold in OF compared with DF (*P* \< 0.0001); and expression of *POSTN* mRNA (3.7 kb) was induced in OF compared with DF (P \< 0.0001). Probability values for each one-way ANOVA analysis are specified above in parentheses. Different letters denote samples that differed significantly (*P* \< 0.05) between group means for a specific gene. Data are presented as least-square means ± SEM, and the number of independent samples per group is indicated in parentheses
A semiquantitative RT-PCR assay was performed on selected genes described in Table [2](#Tab2){ref-type="table"} to confirm and compare their mRNA differential expression pattern in GC of hCG-induced OF. For this procedure, cDNAs were generated from total RNA, and the number of PCR cycles was optimized for each gene. All of the genes analyzed in this manner showed a statistically significant increase in hCG-treated GC of OF compared to that of DF (Fig. [2](#Fig2){ref-type="fig"}). Messenger RNA expression for KIT ligand (*KITLG1)* and isoform 2, *KITLG2*, each showed a 6.7-fold increase in GC of OF compared to that of DF (Fig. [2a](#Fig2){ref-type="fig"}). Intensity of amplicon signals obtained for *KITLG1* and *KITLG2* were highly correlated (*r* = 0.956). The FK506 binding protein 5 (*FKBP5*) mRNA was mainly observed in OF with a 8.6-fold increase in expression when compared to DF. Conversely, FK506 binding protein 4 (*FKBP4*) used as control, showed no difference in expression between follicular stages but was reduced by 2.8-fold in CL when compared to OF. Syndecan 4 (*SDC4*) transcript was mainly expressed in GC of OF wherein a 4.5-fold higher expression level was observed compared to DF. Calpain 2 (m/II) large subunit (*CAPN2*) transcript was detected in all groups and showed a 7.1-fold higher expression level in GC of OF compared to DF. The prolyl 4-hydroxylase subunit alpha 3 (*P4HA3*) mRNA was mainly observed in OF compared to other groups, and showed a 13.6-fold increase in OF compared to DF (Fig. [2b](#Fig2){ref-type="fig"}). Prosaposin (*PSAP*) mRNA was slightly increased by 1.9-fold in OF when compared to DF. Tenascin C (*TNC*) was induced in GC of OF when compared to DF and SF. ADAM metallopeptidase with thrombospondin type 1 motif 9 (*ADAMTS9*) found as an EST corresponding to intron 28 (Table [2](#Tab2){ref-type="table"}) was upregulated by 24.1-fold in OF compared to DF. Expression of the histone methyl-lysine binding protein (*L3MBLT3*) mRNA showed an increase of 7.5-fold in OF compared to DF whereas no expression was detected in CL (Fig. [2c](#Fig2){ref-type="fig"}). Cystein-rich secretory protein LCCL domain containing 2 (*CRISPLD2*) mRNA was induced in OF when compared to DF. A-Raf proto-oncogene serine/threonine kinase (*ARAF*) mRNA was upregulated by 7.2-fold in OF compared to DF, and maestro (*MRO*) mRNA was increased by 4-fold in OF compared to DF. TIMP metallopeptidase inhibitor 1 (*TIMP1)* was induced in GC of OF when compared to DF whereas TIMP metallopeptidase inhibitor 2 (*TIMP2*) was upregulated by only 1.5-fold (Fig. [2d](#Fig2){ref-type="fig"}). Matrix metallopeptidase 1 (*MMP1*) was induced in GC of OF when compared to SF and DF, and was reduced in CL. ADAM metallopeptidase with thrombospondin type 1 motif 1 (*ADAMTS1*) was upregulated by 18-fold in OF compared to DF. Expression of nudix hydroylase 10 (*NUDT10*) and nudix hydroxylase 11 (*NUDT11)* mRNAs where upregulated by 6.5-fold and 15.2-fold, respectively, reaching the lowest level in CL (Fig. [2](#Fig2){ref-type="fig"}e). Retinol-binding protein 1 (*RBP1*) mRNA was upregulated by 2.6-fold in OF, and ubiquitin specific peptidase 53 (*USP53*) was upregulated by 2.3-fold.Fig. 2Analysis of mRNA expression by semiquantitative RT-PCR. Total RNA was extracted from bovine GC collected from 2 to 4 mm small follicles (SF), dominant follicles (DF) at Day 5 of the estrous cycle, ovulatory follicles (OF) 24 h after injection of hCG (OF), and corpora lutea (CL) from Day 5 of the estrous cycle, then used in mRNA expression analyses using RT-PCR. *GAPDH* was used as a control gene and showed no significant differences in expression between groups. Gene-specific RT-PCR amplicons were normalized with corresponding *GAPDH* (727 bp) amplicons for each sample. **a)** *KITLG1* (587 bp) and *KITLG2* (496 bp) mRNA were shown to be upregulated each by 6.7-fold in OF compared with DF (P \< 0.0001); expression of *FKBP5* (450 bp) mRNA was increased by 8.6-fold in OF compared with DF (P \< 0.0001); *FKBP4* (416 bp) mRNA did not differ between follicular stages but was reduced by 2.8-fold in CL when compared to OF (P \< 0.0001); expression of *SDC4* (443 bp) mRNA was increased by 4.5-fold in OF compared to DF (*P* \< 0.0005); and expression of *CAPN2* (453 bp) mRNA was increased by 7.1-fold in OF compared to DF (*P* \< 0.0002); **b)** *P4HA3* (446 bp) mRNA was upregulated by 13.6-fold in OF compared to DF (P \< 0.0001); *PSAP* (356 bp) mRNA was increased by 1.9-fold (*P* \< 0.033); *TNC* (419 bp) mRNA was induced in OF compared to DF (P \< 0.0002); and expression *ADAMTS9* (454 bp) mRNA was increased by 24.1-fold in OF compared to DF (P \< 0.0001); **c)** *L3MBLT3* (1825 bp) mRNA was upregulated by 7.5-fold in OF compared to DF (P \< 0.0001); *CRISPLD2* (493 bp) mRNA was induced in OF compared to SF and DF (P \< 0.0001); *ARAF* (489 bp) mRNA was upregulated by 7.2-fold in OF compared to DF (P \< 0.0001); and *MRO* (474 bp) mRNA was increased by 4-fold in OF compared to DF (P \< 0.0001); **d)** *TIMP1* (487 bp) was induced in OF compared to DF (P \< 0.0001); *TIMP2* (477 bp) was increased by 1.5-fold in OF compared to DF (P \< 0.0001); *MMP1* (467 bp) was induced in OF compared to DF and SF (P \< 0.0001); and *ADAMTS1* (482 bp) was upregulated by 18-fold in OF compared to DF (P \< 0.0001); **e)** *NUDT10* (569 bp) was increased by 6.5-fold in OF compared to DF (P \< 0.0001); *NUDT11* (466 bp) was upregulated by 15.2 fold in OF compared to DF (P \< 0.0001); *RBP1* (444 bp) was increased by 2.6-fold in OF compared to DF (P \< 0.0001); *USP53* (435 bp) was upregulated by 2.3-fold in OF compared to DF (P \< 0.0001). Probability values for each oneway ANOVA analysis are specified above in parentheses. Different letters denote samples that differed significantly (P \< 0.05) between group means for a specific gene. Data are presented as least-square means ± SEM, and the number of independent samples per group is indicated in parentheses
Characterization of bovine POSTN, CRISPLD2 and L3MBTL3 cDNAs {#Sec11}
------------------------------------------------------------
Bovine full-length cDNAs for *POSTN, CRISPLD2* and *L3MBTL3* cDNAs were not previously experimentally characterized in the bovine species. Since only cDNA fragments were obtained from the screening of the OF-DF subtracted library and that potential alternatively spliced isoforms may be expressed, we undertook their characterization in GC to ascertain the identity of the OF-DF cDNA fragments. Bovine cDNA fragments for *POSTN*, *CRISPLD2* and *L3MBTL3* were cloned from the SSH screening of the OF-DF subtracted library as presented in Table [1](#Tab1){ref-type="table"}, and were used as probes to screen by hybridization size-selected cDNA generated from bovine GC that were collected 24 h following injection of hCG. The bovine *POSTN* cDNA consists of 3072 bp (Genbank: AY445072), and is composed of a 5′-UTR of 42 bp, an ORF of 2511 bp and a 3′-UTR of 519 bp containing two polyadenylation signals followed by a poly(A)^+^ tail. The coding region of bovine *POSTN* cDNA encodes a protein of 836 amino acids, with a theoretical molecular mass (Mr) of 93.1 kDa and an isoelectric point (pI) of 7. Amino acid homology analysis by PsiBlast revealed an overall identity level of 95% to human (NP_001129406). Bovine POSTN possesses a peptide signal (M^1^-A^21^) that may be cleaved between A^21^-N^22^, a cystein-rich domain called EMI domain (G^40^-A^94^), and four fasciclin domains (T^111^-T^231^, E^246^-P^367^, Q^381^-P^494^, R^508^-P^630^). Since five *POSTN* isoforms were reported in human, we verified by RT-PCR and sequencing if other *POSTN* isoforms were expressed in GC using primers targeting the entire ORF (Table [1](#Tab1){ref-type="table"}). A single full-length cDNA of 2511 pb was characterized.
The bovine *CRISPLD2* cDNA consists of 4020 bp (Genbank: AY369781), and is composed of a 5′-UTR of 210 bp, an ORF of 1488 bp, and a 3′-UTR of 2322 bp containing six AU-rich elements (ATTTA) as well as two polyadenylation signals followed by a poly(A)^+^ tail. The coding region of *CRISPLD2* cDNA encodes a protein of 496 amino acids, with a theoretical Mr. of 55.6 kDa, pI of 8.2, and an overall identity level of 83% to human (NP_113664) and 76% to mouse (NP_084485) proteins. Bovine CRISPLD2 protein possesses a peptide signal (M^1^-G^22^) that may be cleaved between G^22^-F^23^, a cystein-rich secretory protein, antigen 5, and pathogenesis-related protein (known as CAP domain; L^60^-Y^200^) and two Limulus factor C, Coch-5b2 and Lgl1 domains (LCCL-1: V^287^-F^378^; LCCL-2: L^388^-T^482^).
The bovine *L3MBTL3* cDNA characterized from GC consists of 3892 bp (Genbank: AY437805), and is composed of a 5′-UTR of 205 bp, an open reading frame (ORF) of 2322 bp and a 3′-UTR of 1570 bp containing three AU-rich elements (ATTTA) as well as two polyadenylation signals followed by a poly(A)^+^ tail. The coding region of bovine *L3MBTL3* cDNA encodes a protein of 773 amino acids, with a theoretical Mr. of 87.5 kDa, pI of 6, and an overall identity level of 96% to human (GenBank: NP_115814) and 87% to mouse (NP_766375) proteins. Since the bovine protein expressed in GC was missing the last seven amino acids at its carboxy-terminal end, corresponding to ^774^KNSHNEL^780^ when compared to human protein, we verified by RT-PCR and sequencing if other *L3MBTL3* isoforms would be expressed in GC by using primers targeting the 3′-end of the open reading frame (Table [1](#Tab1){ref-type="table"}). In parallel, RT-PCR analysis was performed using RNA extracted from a bovine endometrial epithelial and glandular cell line (Endo 8.3; \[[@CR12]\]. In GC, a single alternatively spliced transcript was observed coding for the 773 amino acids protein that we previously characterized using the size selected cDNA library screening technique. This truncated *L3MBTL3* isoform is referred as transcript 2 (GenBank: AY437805). Conversely, a single transcript was also observed in the endometrial cell line that corresponded to the complete protein of 780 amino acids with a theoretical Mr. of 88.3 kDa and pI of 6.1, referred as *L3MBTL3* transcript 1 (GenBank: KT881243). Bovine L3MBTL3 protein isoforms expressed in GC and the endometrial cell line possesses three malignant brain tumor (MBT) repeat domains (MBT-1: M^268^-K^336^; MBT-2: M^375^-Y^442^; MBT-3: M^479^-P^543^), C2HC zinc finger domain (G^557^-S^586^) and a sterile alpha motif domain (SAM; S^706^-K^770^).
Discussion {#Sec12}
==========
The preovulatory LH surge from the pituitary gland activates LH receptors on GC and theca cells, and induces a cascade of events in the dominant preovulatory follicle. They include the formation of the expanded hyaluronan-rich cumulus oophorus extracellular matrix (ECM), the release of a competent oocyte following the degradation/rupture of the follicular wall's ECM, and the differentiation of GC and theca cells into luteal cells that contribute to corpus luteum formation. These processes are controlled by the expression of several genes that are either up- or downregulated in a temporally and spatially distinct fashion. However, the molecular mechanisms regulating the developmental transition of a dominant preovulatory follicle into an ovulatory follicle are not fully understood \[[@CR2]\]. Identifying the precise subset of genes involved in these processes is essential to fully comprehend the cascade of events leading to ovulation and luteinization of the follicle, and will likely contribute to a better control of fertility.
We elected to use the SSH method for the identification of differentially expressed genes. This approach involved the screening by hybridization of the OF-DF subtracted GC cDNA library, which first allowed the selection of cDNAs based on differences in their signal intensities, and then their identification by DNA sequencing and further validation by semiquantitative RT-PCR. The genes upregulated in GC by hCG and identified by SSH in this study corroborate well those identified by microarray using a physiological model of endogenous LH release \[[@CR4]\]. Screening the OF-DF subtracted cDNA library allowed identification of genes such as ADAMTS1 \[[@CR13], [@CR14]\], EGR1 \[[@CR15]\], MMP1 \[[@CR16]\], PLAT \[[@CR16]--[@CR18]\], PTGES \[[@CR19]\], PTGS2 \[[@CR10]\], PTX3 \[[@CR4]\], TIMP1 \[[@CR16]\], TIMP2 \[[@CR20]\] and TNFAIP6 \[[@CR21]\] that had previously been shown to be differentially expressed in GC of bovine ovulatory follicles compared to that of dominant preovulatory follicles. The identification of these genes validates the physiological model and the analytical approach used herein. Moreover, from the list of genes identified in Table [2](#Tab2){ref-type="table"}, we also have previously investigated and confirmed the spatiotemporal induction of PLA2G4A \[[@CR22]\] and CAV1 \[[@CR23]\] at the mRNA and protein levels, and of VNN2 \[[@CR24]\] and RGS2 \[[@CR25]\] at the mRNA level, in GC collected at 0, 6, 12, 18, and 24 h post-hCG injection.
Of particular interest, FKBP5 and FKBP4 proteins, also known as FKBP51 and FKBP52, are nuclear receptor co-chaperones regulating cellular trafficking and activity of steroid hormone receptors such as the progesterone and glucocorticoid receptors. These co-chaperones compete for a common binding site on the heat shock protein 90 (Hsp90) that is complexed to steroid receptors. The level of FKBP4 and FKBP5 complexed to Hsp90 is determined by the abundance and affinity of each co-chaperone. FKBP5 is found in greater amount complexed to the progesterone receptor, as compared to FKBP4, and FKBP5 reduces, whereas FKBP4 increases, the binding affinity of the progesterone receptor toward its natural ligand \[[@CR26], [@CR27]\]. Interestingly, the presence of high levels of FKBP5 protein in various tissues of squirrel monkey primates has been identified as a potential cause of progesterone resistance in this species \[[@CR28]\]. In the present study, we show that the expression of FKBP4 mRNA in GC does not differ in follicles larger than 2 mm up to ovulation. FKBP4 may potentiate the action of the progesterone receptor in GC since knockout mice for FKBP4 have a reduced number of ovulation \[[@CR29]\]. Conversely, expression of FKBP5 mRNA increased in GC of hCG-induced ovulatory follicles. Since progesterone concentration increases in follicle following the LH/hCG preovulatory surge, and progesterone stimulates the transcription of FKBP5 \[[@CR30]\], the increase of FKBP5 expression in bovine GC following the LH/hCG surge may represent a short negative feedback loop involved in attenuating GC progesterone responsiveness. Likewise, FKBP5 as a scaffolding protein was recently shown to recruit the phosphatase PHLPP to facilitate dephosphorylation of AKT and its downstream target p38MAPKα (also known as MAPK14), which in turn decreases activation of the glucocorticoid receptor \[[@CR31]--[@CR33]\]. A similar mechanism may be associated with the progesterone receptor in GC after the LH/hCG surge. Indeed, it is known that phosphorylation of AKT/p38MAPKα is increased in GC by the activation of the EGFR following binding of the EGF-like proteins, epiregulin (EREG) and amphiregulin (AREG) \[[@CR34]\], and that EREG and AREG are induced in bovine GC after the LH/hCG surge \[[@CR35], [@CR36]\]. Such EGFR-dependent activation of the AKT/p38MAPKα pathway \[[@CR34], [@CR36]\] likely contributes to the phosphorylation and activation of the progesterone receptor \[[@CR37]\]. Based on these observations and our findings, we hypothesize that the FKBP5 increase in bovine GC following the preovulatory LH surge may limit the EGFR-dependent AKT/p38MAPKα phosphorylation, and thus reduce progesterone receptor phosphorylation and activation, thereby attenuating GC progesterone responsiveness during ovulation and initial luteinization. The demonstration of such regulatory mechanism will require further investigations.
In addition to the list genes whose expression and/or role in ovulation has been in part or clearly determined, we have reported the cDNA characterization and the increased expression of POSTN, CRISPLD2 and L3MBTL3 mRNA in hCG-induced OF, and have conceived their possible function in the ovulatory follicle from what is known in the literature. We found that *POSTN* mRNA was induced by hCG in GC of OF but was absent in DF, in keeping with findings by Christenson et al. \[[@CR4]\]. Only a single full-length *POSTN* mRNA was observed in hCG-stimulated bovine GC in the present study, as compared to the five *POSTN* isoforms identified in human (review: \[[@CR38]\]). The possibility that POSTN could undergo post-translational modifications in GC will require further investigation \[[@CR39]\]. POSTN is a secreted modular glycoprotein that interacts with various proteins of the ECM, growth factors and integrins. As an ECM protein, POSTN acts as an adhesive protein, binding to collagen I-V and fibronectin through its EMI domain, and to tenascin-C (TNC) through its FAS1 domains, to establish an interlaced ECM architecture. Thus, POSTN contributes to fibrillogenesis during normal tissue development, and tissue remodeling associated with various diseases, wound repair, inflammation and cancer \[[@CR38]\]. Interestingly, we observed a concomitant increase in *TNC* and *POSTN* mRNAs in GC at ovulation, a coordinated expression also shown to be induced by mechanical stress in fibroblasts \[[@CR40]\]. POSTN is known to interact with TNC, a complex disulfide-linked hexameric glycoprotein, to promote its incorporation to other ECM proteins such as collagen, fibronectin and heparan sulfate glycosaminoglycans \[[@CR41]\]. POSTN is also recognized as a matricellular protein, a class of ECM proteins that promote non-structural roles such as cell adhesion, migration, proliferation, differentiation and survival \[[@CR38], [@CR39]\]. For these functions, POSTN interacts with integrins, that are the major membrane receptors mediating adhesion to the ECM, which lead to intracellular activation of the PI3K/AKT and focal adhesion kinase signaling pathways \[[@CR38], [@CR42], [@CR43]\]. Such functions and activation could be present in GC since GC do express integrins \[[@CR44]\]. Lastly, the ability of POSTN to induce angiogenesis by increasing VEGF receptor (KDR) expression in endothelial cells \[[@CR43]\] may also be relevant to the marked neovascularization observed during the early stage of corpus luteum formation. Thus, the LH/hCG-dependent induction of *POSTN* mRNA in GC suggests its potential role as a key factor involved in various functions associated with ovulation, including GC survival, migration and luteinization, as well as angiogenesis and tissue remodeling required in the formation of the corpus luteum.
The role of CRISPLD2 in the female reproductive tract remains largely unexplored and, to our knowledge, this study is the first to document the hCG-dependent induction of *CRISPLD2* mRNA in GC of OF. CRISPLD2 is a member of the CAP (Cysteine-rich secretory proteins, Antigen 5, and Pathogenesis-related 1 proteins) superfamily of proteins, and is also known as CAPLD2. It is a secreted protein containing LCCL tandem domains that have been associated with multiple functions such as cell differentiation, migration, inflammation and immunity (review:\[[@CR45]\]). CRISPLD2 expression has been involved in lung morphogenesis \[[@CR46]\], kidney development \[[@CR47]\], and in craniofacial morphogenesis \[[@CR48]\]. Recently, it was shown to regulate proliferation, apoptosis, and migration of fetal lung fibroblasts, to contribute to mesenchymal-epithelial signaling, and to enhance wound repair \[[@CR46]\]. In the latter study, CRISPLD2 was associated with the abnormal expression of multiple extracellular matrix (ECM) genes that modulate lung development and repair. Since a parallel can be drawn with the ovulatory process in which multiple ECM proteins and ECM modifying enzymes are induced (Table [2](#Tab2){ref-type="table"}), we suggest that CRISPLD2 may interact with these proteins and modifying enzymes during ovulation. Interestingly, CRISPLD2 is regulated by progesterone (P4) and its receptor (PGR) in the uterus, its expression is high during decidualization, constitutive during pregnancy and is dysregulated in patients with endometriosis \[[@CR49]\]. A similar P4/PGR-dependent regulation of CRISPLD2 gene expression has been observed in rat granulosa cells \[[@CR50]\]. However, despite the fact that the bovine CRISPLD2 proximal promoter does not harbor a PGR response element, the effect could be indirect as PGR is known to enhance functional activity of SP1 and GC-rich regions \[[@CR51], [@CR52]\], which are present in the bovine proximal promoter. CRISPLD2 has also been identified as a potential inhibitory modulator of the inflammatory and immune response, since glucocorticoids and interleukin-1β (IL1-β) increase CRISPLD2 mRNA and protein expression in airway smooth muscle cells, and CRISPLD2 modulates the expression of IL1-β responsive inflammatory genes such as IL6 and IL8 \[[@CR53], [@CR54]\]. Considering that ovulation is an acute inflammatory reaction and that the LH surge induces IL1-β in GC (review: \[[@CR55]\]), another role for CRISPLD2 during ovulation may be to limit the inflammatory reaction. Thus, through its action on ECM proteins and modifying enzymes or the control of the inflammatory reaction, we propose that the induction of CRISPLD2 in GC of OF likely plays a key role in follicular rupture and ECM remodeling. Since CRISPLD2 knockout mice die during embryogenesis \[[@CR56]\], a tissue-specific conditional knockout approach may be more appropriate to ultimately document the significance of ovarian CRISPLD2 gene expression.
Expression of *L3MBTL3* mRNA in GC was 7.5-fold stronger in OF as compared to DF but this expression was extinguished in CL, suggesting a potential role for L3MBTL3 in LH surge/hCG-induced events, such as ovulation and initial luteinization. L3MBTL3 is known to interact with the polycomb group repressive complex 1 (PRC1) family of proteins that maintains genes in a transcriptionally repressive state by modification of the chromatin \[review: 57-58\]. L3MBTL3 localizes into foci of the DNA-rich regions of the nucleus, and is classified as a chromatin histone methyllysine reader protein \[[@CR57]\]. Methyllysine acts as docking site for specific reader proteins that alter chromatin structure to control various cellular processes. Proteins containing MBT domain such as L3MBTL3, which holds three MBT domains, are known to dimerize and to selectively recognize sequence specific mono and dimethyl-lysine residues within histone tails \[[@CR57], [@CR58]\]. This allows to target chromatin regulatory complexes, such as PRC1, to appropriate genomic loci, and has been functionally link to repression of gene expression. Moreover, the roles of MBT proteins have been associated with regulation of mitosis, tumor suppression, maintenance of cell identity and body pattern during development \[[@CR59], [@CR60]\]. In addition to the three MBT domains, L3MBTL3 harbors a SAM domain known to confer protein-protein interaction features and to act as a scaffolding protein for homo- or hetero-dimerization \[[@CR58], [@CR61]\]. Interestingly, we have shown in the present study that bovine *L3MBTL3* mRNA is alternatively spliced in GC, as compared to the complete mRNA expressed in bovine endometrial cells. To our knowledge, this is the first study to report an alternatively spliced isoform of *L3MBTL3* in mammalian species that results in a truncated open reading frame. The missing last seven amino acids, ^774^KNSHNEL^780^, of L3MBTL3 isoform 2 are located within the carboxy-terminal end following the SAM domain (^706^S-K^770^) known to confer protein/protein interactions \[[@CR61]\]. These last amino acids (^774^K-L^780^) are well conserved in all mammalian species, which likely indicates a fundamental function for these amino acids in the epigenetic control of gene expression. Taken together, we hypothesize that the increase of L3MBTL3 expression in GC of ovulatory follicles may be necessary to silence specific genes that would otherwise negatively impact ovulation and/or initiation of luteinization. Additional studies will be required to unravel the physiological role of L3MBTL3 in GC function, and to investigate the functional significance of the C-terminal truncated L3MBTL3 spliced variant.
Conclusions {#Sec13}
===========
In this study, we have identified several genes whose transcripts levels were increased following hCG injection. While the role of many of these genes is well documented, the exact function of genes such as FKBP5, POSTN, CRISPLD2, L3MBTL3 and others (Table [2](#Tab2){ref-type="table"}) in the ovulatory and luteinization processes remain to be fully explored.
Additional file {#Sec14}
===============
Additional file 1: Figure S1.Analyses of the cDNA subtraction efficiency. PCR analysis was performed on the indicated samples using *PTGS2* or *CYP19A1* specific primers, as described under Materials and Methods. PCR product aliquots were collected at increasing numbers of PCR cycles as indicated. The *PTGS2* DNA fragment (418 bp) was detected following 13 PCR cycles in the OF-DF subtracted sample but not until 18 PCR cycles in the corresponding unsubtracted OF sample. The *CYP19A1* DNA fragment (520 bp) was detected following 13 PCR cycles in the DF-OF subtracted sample but not until 18 PCR cycles in the corresponding unsubtracted DF sample. *PTGS2* or *CYP19A1* were not detected in the DF or OF samples, respectively. **Figure S2.** Representative differential screening results by macroarrays of the OF-DF cDNA library. PCR-amplified cDNA fragments (OF-DF) obtained by SSH were dot-blotted to generate two identical sets of membranes. A total of 940 individual cDNAs were dot-blotted. The macroarrays were then hybridized with two different probe set: subtracted OF-DF cDNAs (**A**), and reverse subtracted DF-OF cDNAs (**B**), as described under Materials and Methods. The two upper left hand dots for each membrane served as internal hybridization controls: A1 = *CYP19A1* (negative control) and A2 = *PTGS2* (positive control) for the OF-DF reaction. The cDNA clones that were found to be differentially expressed in the OF-DF membrane following comparison of hybridization signals among the corresponding spots of the two membranes were further characterized by sequencing. (PDF 275 kb)
CL
: Corpus luteum
DF
: Dominant follicles
eCG
: equine chorionic gonadotropin
ECM
: Extracellular matrix
GC
: Granulosa cells
hCG
: Human chorionic gonadotropin
LH
: Luteinizing hormone
OF
: Ovulatory follicles
OF-DF or DF-OF
: Granulosa cell subtracted cDNA library
SF
: Small follicles
SSH
: Suppression subtractive hybridization
**Electronic supplementary material**
The online version of this article (10.1186/s12958-017-0306-x) contains supplementary material, which is available to authorized users.
The authors would like to sincerely thank Isabelle Daneau for the sequencing reactions.
Funding {#FPar1}
=======
This work was supported by the Natural Sciences and Engineering Research Council of Canada Discovery grant \#104199 (JGL) and by internal funds from Université de Montréal (KN).
Availability of data and materials {#FPar2}
==================================
All data generated or analysed during this study are included in this published article.
JGL, MND, KN and JS participated in the design of the study and collect of samples. MND, VL, KN and JGL performed the laboratory work and data analyses. JGL, MND, KN, JS drafted the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate {#FPar3}
==========================================
The experiments were approved by the Animal Ethics Committee of the Faculty of Veterinary Medicine of the Université de Montréal.
Consent for publication {#FPar4}
=======================
Not applicable.
Competing interests {#FPar5}
===================
The authors declare that they have no competing interests.
Publisher's Note {#FPar6}
================
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
|
{
"pile_set_name": "PubMed Central"
}
|
Background
==========
Motile cilia are microscopic membrane-bound extensions of certain cells that are vital for the survival and reproduction of many eukaryotes. By beating in a regular pattern these evolutionarily-conserved organelles exert mechanical force; they thus play important roles in motility of small organisms and facilitate fluid movement across epithelial surfaces in complex multicellular animals. In addition to their role in fluid movement, motile cilia have recently been found to possess sensory functions in mammals, a feature previously thought to be restricted to non-motile \'primary\' cilia \[[@B1],[@B2]\]. Given that motile cilia are essential to physiology it is not surprising that defects in these organelles cause multiple human disorders \[[@B3]-[@B5]\]. Such ciliopathies include primary ciliary dyskinesia resulting in an inability to clear mucous and debris from airways \[[@B6]\], hydrocephalus caused by abnormal spinal fluid movement in the ventricles of the brain \[[@B7]\], and situs inversus (inversions of the normal left/right symmetry of organs) a consequence of altered nodal flow during embryogenesis \[[@B8],[@B9]\]. Motile cilia (and flagella, which are essentially long motile cilia) are also essential for the completion of the life cycles of various parasites of humans and animals. For example, they power locomotion of schistosome larvae (miracidia) enabling host-finding and thus infection of the snail intermediate host \[[@B10]\], and are required for migration of trypanosomes between gut and salivary glands in the tsetse fly vector \[[@B11]\], and for viability of the bloodstream trypanosome form \[[@B12]\].
Motile cilia (and flagella) are composed of nine microtubular doublets and, usually, two central mictrotubular singlets, comprising the axoneme; dynein arms and radial spokes associated with the axoneme generate and control axonemal bending and thus force generation. The regulation of ciliary beating has been the focus of much research \[[@B13]\], and proteomic studies including those on the model organism *Chlamydomonas reinhardtii*, have aimed to describe the repertoire of proteins present within eukaryotic cilia and flagella, or their component fractions \[[@B14]-[@B19]\]. The proteomic analysis of *Chlamydomonas*flagella revealed over 90 putative signal transduction proteins including kinases and phosphatases \[[@B14]\], some of which might be anchored to the axoneme \[[@B20]\], highlighting the importance of signalling and reversible protein phosphorylation in the function of motile cilia and flagella. This is further supported by the recent identification in this organelle of 32 flagella phosphoproteins with 126 *in vivo*phosphorylation sites \[[@B21]\]. Despite these advances, our understanding of kinase-mediated cell signalling mechanisms regulating ciliary motion is still rudimentary, being largely restricted to the roles of the cAMP-dependent protein kinase, protein kinase A (PKA) \[[@B13],[@B22]-[@B24]\], and protein kinase C (PKC) \[[@B13],[@B25]-[@B27]\].
The surface of the schistosome miracidium is almost entirely covered with numerous motile locomotory cilia which, in *Schistosoma mansoni*, emerge from 21 ciliated epidermal plates arranged in tiers \[[@B10]\]. The *S. mansoni*miracidium is fully developed in the egg when it is released in the faeces of the infected definitive (human) host; upon water contact the miracidium hatches and swims rapidly to locate a suitable intermediate freshwater snail-host. Miracidia movements respond rapidly to various environmental cues such as the presence of host-snail components \[[@B28]\] or salinity \[[@B29]\], suggesting that these motile cilia might possess sensory functions as is the case with *Paramecium*\[reviewed in \[[@B2]\]\]. While studying kinase-mediated cell signalling in *S. mansoni*during miracidia development we observed an unexpected event, namely that activation of p38 mitogen-activated protein kinase (p38 MAPK) attenuated miracidial swimming. This kinase, an orthologue of the yeast HOG kinase, participates in signalling cascades that regulate transcriptional responses to stress \[[@B30]\] as well as having other non-transcription factor targets such as cytosolic phospholipase A~2~\[[@B31]\]. Here we report findings that support a role for p38 MAPK in the regulation of ciliary motion of the multicellular eukaryote *S. mansoni*.
Results and Discussion
======================
Characterization of *S. mansoni* p38 MAPK
-----------------------------------------
As with other MAPKs, p38 MAPK has been highly conserved during metazoan evolution \[[@B32]\]. Recently, the draft genomes for *S. mansoni*and *Schistosoma japonicum*were published \[[@B33],[@B34]\] allowing *S. mansoni*p38 MAPK gene candidates to be identified and further assessed for similarity to p38 MAPKs from other organisms, including closely related schistosomes (Figure [1](#F1){ref-type="fig"}). For *S. mansoni*only a single putative p38 MAPK was identified and only partial cDNA reads were found; moreover, only one p38 MAPK was found in *S. japonicum*(Figure [1](#F1){ref-type="fig"}). Thus, in contrast to *D. melanogaster*and human which possess two and four p38 MAPKs respectively \[[@B32],[@B35],[@B36]\], it seems that schistosomes may possess one p38 MAPK orthologue only. Based on the partial sequence data (XP_002571000) spanning 74 amino acids, and supported by more complete data from *S. japonicum*, the identified *S. mansoni*p38 MAPK was most similar to p38α MAPK (MAPK 14) of humans, the most evolutionarily-conserved p38 MAPK \[[@B35]\]. In addition, there exist a number of putative exons on two separate scaffolds (Smp_scaff000038 and Smp_scaff06141) additional to the 74 amino acid fragment that, when translated, also match closely with the *S. japonicum*sequence; thus, we assume given the phylogenetic proximity between the two species that *S. mansoni*p38 MAPK is very similar to *S. japonicum*p38 MAPK. As for all other p38 MAPKs in the p38 subfamily, the dual phosphorylation site in the *S. mansoni*p38 MAPK activation loop reads Thr-Gly-Tyr (TGY)(Figure [1](#F1){ref-type="fig"}). The substrate binding site Ala-Thr-Arg-Trp (ATRW) is also conserved, as is the kinase interaction motif (KIM) docking site (Figure [1](#F1){ref-type="fig"}) which binds linear KIM sequences present in substrates and MAPK phosphatases \[[@B37]\]. Pair wise comparisons of the *S. mansoni*p38 MAPK fragment with corresponding sequences for other organisms revealed \~69-70% similarity with human, *Drosophila melanogaster*, *Caenorhabditis elegans*, or *Danio rerio*, and 86.5% with *S. japonicum*.
{#F1}
Anti-phospho p38 MAPK monoclonal antibodies were used in an attempt to detect phosphorylated p38 MAPK in *S. mansoni*. These antibodies, that bind p38 MAPK only when dually phosphorylated on Thr/Tyr of the TGY motif, have been used to detect phosphorylated p38 MAPK in many multicellular eukaryotes including *C. elegans*\[[@B38]\] and *D. melanogaster*\[[@B39]\]. Because phosphorylation on these residues results in activation of the enzyme, immunoreactivity directly correlates with p38 MAPK activity. The p38 MAPK amino acid sequence surrounding the TGY motif to which these antibodies are raised is highly-conserved between both *S. mansoni*and *S. japonicum*, and between *S. mansoni*and *C. elegans*, *D. melanogaster*, *D. rerio*and human (Figure [1](#F1){ref-type="fig"}). Western blotting of adult worm homogenates revealed that anti-phospho p38 MAPK antibodies recognized a single protein band with apparent molecular weight of approximately 42 kDa, essentially co-migrating with phosphorylated p38 MAPK from human astrocytoma (U251 MG) cells (Figure [2A](#F2){ref-type="fig"}). In marked contrast to adult worms, freshly-hatched swimming miracidia did not possess detectable levels of phosphorylated p38 MAPK (Figure [2A](#F2){ref-type="fig"}). A second antibody (anti-p38 MAPK) that detects p38 MAPK in vertebrates irrespective of phosphorylation state did not react with the *S. mansoni*protein precluding its use in this study.
{#F2}
A p38 MAPK immunoprecipitation kinase assay kit was next employed to confirm that the *S. mansoni*protein recognized by the anti-phospho p38 MAPK antibodies possessed p38 MAPK activity. Immunoprecipitates from adult worm homogenates phosphorylated the p38 MAPK substrate activating transcription factor 2 (ATF-2) (Figure [2B](#F2){ref-type="fig"}). The anti-inflammatory pyridinylimidazole compounds (SmithKline Beecham, SB) inhibit p38 MAPK with both activity of the phosphorylated enzyme and its autophosphorylation affected \[[@B40]\]. These inhibitors compete with ATP at the ATP binding site and exhibit no, or very weak inhibitory activity toward the closely related MAPKs, c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) \[[@B41]\]. SB 203580 inhibits both p38α and p38β MAPK and the interactions between the inhibitor and human kinases have been mapped using crystallography and amino acid substitution experiments \[[@B42],[@B43]\]. The crucial residues thought to be involved in this interaction are shown in Figure [1](#F1){ref-type="fig"}; although falling outside the *S. mansoni*sequence fragment, these residues are conserved in *S. japonicum*p38 MAPK. We thus postulated that SB 203580 would inhibit *S. mansoni*p38 MAPK. Pre-incubation of immunoprecipitates with SB 203580 prior to kinase assay reduced ATF-2 phosphorylation considerably when compared to immunoprecipitates incubated in dimethyl sulfoxide (DMSO) vehicle alone; inhibition appeared dose dependent with 5 μM SB 203580 almost completely attenuating ATF-2 phosphorylation (Figure [2B](#F2){ref-type="fig"}). Thus the antibody recognizes an *S. mansoni*protein with p38 MAPK-like activity that can be inhibited by the p38 MAPK inhibitor SB 203580.
Anisomycin is a known potent stimulator of p38 MAPK phosphorylation, and therefore activation, so we considered that this compound might activate p38 MAPK in freshly-hatched swimming miracidia that lacked the phosphorylated (activated) form of the enzyme (Figure [2A](#F2){ref-type="fig"}). Western blotting revealed that treatment of these miracidia with 20 μM anisomycin for 15 min induced considerable p38 MAPK phosphorylation when compared to controls, which appeared to increase after 30 min and was sustained over 60 min (Figure [2C](#F2){ref-type="fig"}).
The results of the above biochemical experiments, coupled with the bioinformatic analysis, are commensurate with the *S. mansoni*immunoreactive protein being a p38 MAPK orthologue.
Activated p38 MAPK is associated with *S. mansoni* cilia
--------------------------------------------------------
To determine the localization of activated p38 MAPK within *S. mansoni*miracidia, anti-phospho p38 MAPK antibodies and confocal laser scanning microscopy were used. Z-axis projections viewed in maximum pixel brightness mode revealed that freshly-hatched swimming miracidia possessed low levels of phosphorylated p38 MAPK, which is in agreement with western blotting analysis (Figures [2A](#F2){ref-type="fig"} and [2C](#F2){ref-type="fig"}). However where activation was evident, it appeared to be associated with the region occupied by the cilia (Figure [3C](#F3){ref-type="fig"}). Miracidia incubated with secondary antibodies alone possessed only background fluorescence (Figure [3B](#F3){ref-type="fig"}). When freshly-hatched swimming miracidia were incubated with 20 μM anisomycin for 30 min, a striking increase in p38 MAPK activation was observed (Figure [3G](#F3){ref-type="fig"}) whereas DMSO exposed (control) miracidia appeared similar to untreated miracidia (data not shown). Analysis of serial optical z-sections revealed activation largely at, or adjacent to, the ciliated surface of the parasite (e.g. Figure [3D](#F3){ref-type="fig"}). Fine z-sectioning and optical zooming revealed that activated p38 MAPK was associated with the shaft of the cilia (Figure [3E](#F3){ref-type="fig"}), further demonstrated through co-localization using anti-acetylated tubulin antibodies that permit visualization of these structures (Figure [3F](#F3){ref-type="fig"}). Immunoreactivity was present up to 6-7 μm from the tegument surface which correlates with the length (7-8 μm) of the cilia reported from transmission electron microscopy studies \[[@B10]\]. Staining for activated p38 MAPK was also observed in the tegument (Figures [3E](#F3){ref-type="fig"}); here the enzyme could either be associated with the base of the cilia, the microvilli (1 μm long \[[@B10]\]) that are known to exist among the cilia, the rootlet (2 μm long \[[@B10]\]) that is thought to provide support \[[@B44]\], or other structures. The semi-spherical terebratorium (anterior papilla) lacks cilia (Figure [3A](#F3){ref-type="fig"}) but possess filopodia-like sensory endings \[[@B10]\] thought to play a role in sensing the intermediate snail host. Although staining was observed at the terebratorium, it was noticeably less than that observed in the adjacent ciliated plates (Figure [3G](#F3){ref-type="fig"}).
{#F3}
While observing miracidia by immunofluorescence microscopy a solitary egg was found on the microscope slide, this egg had ruptured to allow hatching but fortunately still contained the miracidium which stained successfully with antibodies. Strikingly, and in contrast to swimming miracidia, the hatching miracidium possessed considerable p38 MAPK activity that *via*z-section analysis was found to be cilia-associated. Experiments were then designed in an attempt to re-capture this event and on several occasions miracidia were observed within ruptured eggs; such miracidia contained cilia-associated p38 MAPK activity considerably greater than that of freshly-hatched swimming miracidia (Figures [3H-K](#F3){ref-type="fig"}).
Although various kinases including PKA, casein kinase, adenylate kinase, cGMP-dependent protein kinase and a putative MAPK have been identified in proteomic screens of cilia/flagella e.g. \[[@B14],[@B15]\], to our knowledge p38 MAPK has not been detected by such methods. P38 MAPK has however recently been localized to the post-acrosomal region and upper flagellum mid-piece of human sperm by fluorescence microscopy \[[@B45]\]. Although sperm tails are classified as flagella, their regulation differs from motile cilia in some aspects; sperm also possess a number of unique accessory structures \[[@B19]\] making them somewhat distinct from other motile cilia.
Activation of p38 MAPK correlates with attenuation of cilia beat and swim velocity
----------------------------------------------------------------------------------
Because p38 MAPK was active in stationary miracidia within eggs but inactive in swimming miracidia, and because p38 MAPK localized to cilia, we next explored whether pharmacological activation or inhibition of p38 MAPK phenotypically affected ciliary function by performing swim velocity analyses. Miracidia were exposed to anisomycin or SB 203580 and their swim behaviour recorded by digital video microscopy (see additional file [1](#S1){ref-type="supplementary-material"}: Supplementary movie file). Determination of swim velocities revealed that freshly-hatched miracidia swam at speeds between 2.17 and 2.38 mm/s over 60 min (Figure [4A and B](#F4){ref-type="fig"}); DMSO treatment did not affect swim velocities over this duration (data not shown). However, anisomycin (20 μM) significantly attenuated miracidia swim velocity over time when compared to untreated (spring water) controls (*P*≤ 0.001) (Figure [4B](#F4){ref-type="fig"}); after 5 min and 30 min mean swim velocity was reduced by 25% to 1.70 mm/s (*P*≤ 0.01), and by 80% to 0.43 mm/s (*P*≤ 0.001), respectively. After 60 min anisomycin treatment swimming essentially ceased. On the other hand, SB 203580 (1 μM) significantly accelerated miracidia swim velocity (*P*≤ 0.001), particularly after 30 and 45 min treatment when it increased \~15 % (*P*≤ 0.001). Importantly, following 60 min anisomycin treatment, when spring water (to dilute the anisomycin concentration 10-fold) or SB 203580 (1 μM) were added, the miracidia started swimming displaying swim velocities of between 0.4 and 0.6 mm/s after 20 min (data not shown; see additional file [1](#S1){ref-type="supplementary-material"}: Supplementary movie file). That the effects of anisomycin could be reversed demonstrates that cessation of swimming was not due to larval death. Thus, the contrasting effects of SB 203580 and anisomycin on swim velocity and the finding that p38 MAPK localizes to the cilia are consistent with p38 MAPK playing a role in ciliary beat and thus miracidia swimming. Anisomycin is thought to indirectly activate p38 MAPK *via*cdc42/rac, MAPKKKs, MAPKK3 (MKK3) and MKK6 which ultimately phosphorylate p38 MAPK \[[@B46]\]. Importantly, orthologues of these pathway components have been identified in *S. mansoni*\[[@B33]\] suggesting that the mechanisms by which anisomycin exerts its effect on p38 MAPK are conserved between schistosomes and higher eukaryotes. P38 MAPK phosphatases have also been identified in *S. mansoni*\[[@B33]\], allowing rapid dephosphorylation of p38 MAPK when required. Although aspects of sperm flagella make them distinct from other motile cilia it is interesting that p38 MAPK inhibition by SB 203580 was recently shown to stimulate forward and hyperactivated motility of human sperm \[[@B45]\].
{#F4}
The mechanism by which p38 MAPK controls ciliary beat is not known. Active p38 MAPK could attenuate ciliary beat either by direct or indirect interactions involving phosphorylation of axonemal components or components within a ciliary signal transduction cascade, respectively. Studies with *C. reinhardtii*have demonstrated that phosphorylation and dephosphorylation control flagella motility \[[@B20]\] and have highlighted the complex nature of protein phosphorylation in this organelle \[[@B21]\]. Many potential kinase substrates exist that co-ordinate ciliary motility including the dyneins \[[@B47]\] and central pair kinesin KLP1 \[[@B48]\], but further characterisation of kinase substrates within cilia is needed.
Only a few other kinases have been reported to regulate cilia beat. PKA has been shown to attach to *Paramecium*ciliary axonemes, and strong evidence exists for PKA playing a positive role in *Paramecium*swimming and in controlling ciliary beat frequency of mammalian cilia \[[@B13],[@B22]-[@B24]\]. Interestingly, PKA has also been implicated in regulating the ciliary motion of *S. mansoni*miracidia \[[@B49]\]. While cGMP-dependent protein kinase (cGMP) has also been shown to positively regulate ciliary beat frequency \[[@B13],[@B50]\], like for p38 MAPK, PKC has been implicated in slowing ciliary beat \[[@B13],[@B25]-[@B27]\]. Clearly, more research is needed into the influence of kinase activities on ciliary movement. For example, ERK1/2 has recently been found to bind radial spoke protein 3 in mammals and regulate its interaction with PKA \[[@B51]\]; however, the extent to which ERK actually influences motility *via*this interaction warrants investigation.
Activation of p38 MAPK accelerates loss of cilia during larval transformation
-----------------------------------------------------------------------------
When miracidia penetrate a suitable intermediate snail host they shed their ciliated plates and develop into the next larval stage, the mother sporocyst. This transformation can also be performed *in vitro*\[[@B52]\] during which miracidia stop swimming before the plates are released. As p38 MAPK activation attenuates miracidia swim speed whereas inhibition accelerates it we explored the effects of anisomycin and SB 203580 on deciliation and thus swimming during miracidium-to-mother sporocyst transformation *in vitro*. DMSO did not affect the deciliation rate when compared to Chernin\'s balanced salt solution (CBSS) controls and none of the treatments affected the survival of the developing larvae (data not shown). Anisomycin accelerated the shedding of ciliary plates considerably (Figure [5B](#F5){ref-type="fig"}); after only 2 h transformation 52 % of parasites had stopped swimming having shed at least some cilia in contrast to only 18 % of parasites in the CBSS control group (P ≤ 0.001; Figure [5B](#F5){ref-type="fig"}). At this time point 20 % of anisomycin-treated larvae had shed all their ciliated plates compared to none in CBSS alone (data not shown). This effect of anisomycin persisted throughout larval transformation (Figure [5B](#F5){ref-type="fig"}). Although the effects of SB 203580 were somewhat less marked, at 21 h, 25 h and 29 h significantly more miracidia were observed swimming (with all cilia attached) than were present in the CBSS control group (Figure [5A](#F5){ref-type="fig"}). Thus, p38 MAPK activation appears to accelerate the initial rate of *S. mansoni*miracidium transformation by attenuating cilia-mediated swimming behaviour leading to early release of ciliated plates.
{#F5}
Conclusions
===========
Here biochemical, innunohistochemical and functional data are presented that are consistent with p38 MAPK playing an important part in the regulation of ciliary beat and thus swimming behaviour of the multicellular eukaryote, *S. mansoni*. The marked difference in p38 MAPK activation between un-hatched or stationary miracidia and actively swimming miracidia is striking. Localization of active p38 MAPK to both the cilium shaft and the tegument of stationary miracidia implies that p38 MAPK might play multiple parts in co-ordinating swim behaviour, including sensory roles as described/hypothesized for motile cilia in other organisms including parasites \[[@B1],[@B2],[@B53]\]. Given the conservation of both signalling processes and structure/function of motile cilia, we hypothesize that p38 MAPK might regulate ciliary beat frequency in a variety of metazoans. Thus our findings could have implications for studies into motility of other important multicellular eukaryotes including parasites of humans, and for research into various human ciliopathies.
Methods
=======
Sequence characterization of *S. mansoni* p38 MAPK
--------------------------------------------------
*S. mansoni*p38 MAPK gene candidates were identified from version 4.0 of the schistosome genome assembly by searching *S. mansoni*GeneDB hosted by the Wellcome Trust Sanger Institute, relying on the existing annotation \[[@B33],[@B54]\]. Although only partial cDNA reads were found, they were further assessed for similarity to p38 MAPKs from other organisms using the NCBI tBLASTx search tool \[[@B55]\], limited to bilateria (organism) and the nucleotide dataset. Protein sequences of candidates with matches to p38 MAPK genes were aligned to those of other organisms, including that for *S. japonicum*\[[@B34]\] using Geneious Pro 4.85 (Biomatters Ltd, Auckland, New Zealand) with Blosum62 cost matrix, threshold = 1 and default parameters. Pair wise identity scores were also obtained using Geneious Pro.
Isolation of *S. mansoni* miracidia and adult worms
---------------------------------------------------
Adult worms were recovered by portal perfusion of patent mice infected with *S. mansoni*(Belo Horizonte strain) and were immediately snap frozen in liquid nitrogen and stored at -80°C. Livers and spleens were then removed from the infected mice and *S. mansoni*eggs isolated; miracidia were then hatched from eggs for up to 2 h in natural spring water (Evian) and were collected under a dissecting microscope using a Pasteur pipette \[[@B52]\]. Miracidia were washed three times in spring water in a Stericup filter (0.45 μm PVDF membrane, Millipore, Watford, UK). The same filter was then used to concentrate the miracidia (to achieve approximately 10,000 miracidia/ml); enumeration of larvae was performed in aliquots under an inverted light microscope. Animal use received appropriate local ethical approval.
Pharmacological activation and inhibition of p38 MAPK
-----------------------------------------------------
The effect of the p38 MAPK activator anisomycin on p38 MAPK phosphorylation (activation) in *S. mansoni*was assessed by western blotting using anti-phospho p38 MAPK (Thr180/Tyr182) monoclonal antibodies (Cell Signalling Technology, New England Biolabs, Hitchin, UK) that recognize only the phosphorylated (activated) form of the enzyme. Freshly-hatched miracidia (\~900 per treatment) were incubated in anisomycin (20 μM in spring water) or spring water containing vehicle (0.02% (v/v) DMSO) for varying durations (15, 30 or 60 min) and then immediately placed on ice and proteins extracted by adding an appropriate volume of 5x SDS-PAGE sample buffer followed by brief homogenization. Samples were then boiled for 5 min and sonicated briefly. After cooling, protease and phosphatase inhibitors (Sigma, Poole, UK) were added at the manufacturer\'s recommended concentrations and samples stored at -20°C prior to electrophoresis.
*Schistosoma mansoni*protein samples were separated on 10% SDS-PAGE gels and were transferred to nitrocellulose using a semi-dry electrotransfer unit (Bio-Rad, Hemel Hempsted, UK). After staining with Ponceau S to confirm homogeneous transfer, membranes were blocked for 1 h in 5% (w/v) non-fat dried milk, and then incubated anti-phospho p38 MAPK monoclonal antibodies (1/1000 in tris-buffered saline/0.1% Tween-20 (TTBS) containing 1% (w/v) BSA) overnight at 4°C. Next, blots were washed in TTBS and incubated for 2 h at room temperature in horse-radish peroxidase-congugated secondary antibodies (Cell signalling Technology; 1/5000 in TTBS) before further washing and incubation in West Pico chemiluminescent substrate (Pierce, Tattenhall UK) for 5 min. Immunoreactive bands were then visualised using cooled CCD GeneGnome chemiluminescence imaging system (Syngene, Cambridge, UK). Equal loading of proteins on blots was checked by stripping blots for 3 h at room temperature with Restore western blot stripping buffer (Pierce), before briefly washing blots in TTBS and incubating blots with anti-actin antibodies (1:3000 in TTBS; Sigma). Human astrocytoma (U251 MG) cell lysates, used as positive control for detection of phosphorylated p38 MAPK, were kindly provided by Suzanne Newton (Kingston University).
To determine p38 MAPK activities of proteins immunoprecipitated using anti-phospho p38 MAPK antibodies, a non-radioactive p38 MAPK activity assay kit was used (Cell Signalling Technology). Ten adult worm pairs were homogenized in cell lysis buffer (250 μl), the lysate cleared by centrifugation at 13,000 rpm in a microfuge for 10 min at 4°C, and the supernatant recovered. Subsequently, 2 μl of immobilized anti-phospho p38 MAPK (Thr180/Tyr182) monoclonal antibodies were added to the lysate and samples were gently mixed overnight at 4°C. Subsequently, the immune complex was washed twice in cell lysis buffer and re-suspended in kinase buffer before 1 μl of ATP and 1 μl of ATF-2 fusion protein were added to start the kinase reaction. After 30 min the reaction was terminated by adding an appropriate volume of 5x SDS-PAGE sample buffer. The samples were then boiled, sonicated and processed for western blotting using anti-phospho ATF-2 primary antibodies. All buffers and reagents used were provided in the p38 MAPK assay kit. In parallel experiments, the p38 MAPK inhibitor SB 203580 (1 μM, 2 μM, or 5 μM) or vehicle (0.02 % (v/v) DMSO) was added to the immunoprecipitates 15 min prior to the start of the kinase assay.
Immunohistochemistry
--------------------
Freshly-hatched swimming *S. mansoni*miracidia were either fixed immediately in absolute acetone or were treated with anisomycin (20 μM), or vehicle (0.02 % (v/v) DMSO), for 30 min prior to fixing. In some experiments, miracidia were left to hatch from eggs for short durations (10 - 20 min) prior to fixing in absolute acetone; this was done in an attempt to recover miracidia in the process of hatching from the egg. All parasites were then stored at 4 °C. For further preparation acetone was removed and samples washed twice with phosphate buffered saline (PBS) before being permeabilized in 0.3% (v/v) Triton X-100 for 1 h and washed with PBS prior to blocking in 10% (v/v) goat serum (Invitrogen, Paisley, UK) for 1 h. After a further wash with PBS, parasites were incubated with anti-phospho p38 MAPK mAb (1:50 in 5% (w/v) BSA) for 3 days on a microfuge tube rotator. The parasites were then washed twice in PBS for 1 h each and incubated in Alexa fluor 488 secondary antibodies (1:500 in 5% (w/v) BSA) for 24 h in the dark, followed by a further wash in PBS for 1 h. To detect cilia, miracidia were also incubated as above in anti-acetylated tubulin mouse monoclonal antibodies (1:100 in 5% (w/v) BSA; Sigma, Poole, UK) and Alexa fluor 594 secondary antibodies. Next, parasites were placed on microscope slides, left to air dry prior to mounting in Vectashield (Vecta Laboratories, Peterborough, UK) anti-bleaching medium, and sealed with transparent nail polish. All incubations were carried out at room temperature and incubations and washes were done in 2 ml screw cap tubes. Miracidia and eggs were then visualized on a Leica TCS SP2 AOBS confocal laser scanning microscope using a 20x dry objective or 40x and 63x oil immersion objectives and images collected with Leica software. Since *S. mansoni*miracidia autofluoresce, the signal received for the negative controls (i.e. those incubated only with secondary antibodies) was reduced. This was achieved by reducing the power level of the photomultiplier tube, which was then kept constant for all observations.
Scanning Electron microscopy
----------------------------
For conventional scanning electron microscopy, acetone fixed miracidia or eggs were plated on individual coverslips and left to air dry, they were then placed in PBS for 4 h, dehydrated in ethanol, briefly soaked in hexamethyldisilazane and evaporated, and sputter-coated with gold-palladium. Specimens were then visualized on a Zeiss EVO50 scanning electron microscope.
Analysis of *S. mansoni* swim velocity
--------------------------------------
Freshly-hatched miracidia in spring water were divided into 200 μl aliquots and exposed to either SB 203580 (1 μM), anisomycin (20 μM), vehicle (DMSO, 0.02% (v/v)), or were left untreated. Each sample was then immediately placed into a small sterile Petri dish and the 200 μl droplet spread out using a pipette; care was taken to ensure that the size and spread of the droplet was consistent between experiments to minimize artefacts in measurement owing to the miracidia swimming out of the horizontal plane during recordings. Light influences considerably miracidia swimming behaviour, so light intensity and positioning also remained constant for all experiments which were performed at 27°C. Miracidia were videoed over 60 min. There were approximately 10 miracidia in each sample and at least 30 miracidia per treatment were analysed in three independent experiments. Visualization was achieved using an Olympus SZ4045 binocular dissecting microscope and avi-format video recordings were made using a JVC TK-1481 composite colour video camera linked to Studio Launcher Plus for Windows software. Digital videos were subsequently processed using the freely-available analysis software ImageJ \[[@B56]\] to determine swim path length of individual miracidia in 5s permitting swim velocities (mm/s) to be calculated at various time points after treatment.
Analysis of deciliation during larval transformation
----------------------------------------------------
Recovered eggs from schistosome-infected mice were hatched in spring water containing penicillin and streptomycin (100 units/ml each). Collected miracidia were then washed, and concentrated using Stericup filters, in sterile Chernin\'s balanced salt solution, pH 7.2, \[[@B57]\] containing glucose and trehalose and the same antibiotics (CBSS^+^). Approximately 1500 miracidia were placed onto individual wells of 6-well cell culture plates (Nunc, Loughborough, UK) and further 2 ml of either CBSS^+^, or CBSS^+^containing DMSO, SB 203580, or anisomycin (0.02% (v/v), 1 μM, and 20 μM final concentrations, respectively) added. The culture plates were then placed in a dark, humidified chamber in an incubator at 26°C. Three independent experiments were performed and media was not changed during larval development. At various time points during development (4h - 55 h), 30 parasites from each sample were randomly selected using an inverted microscope and the percentage of parasites retaining all of their ciliated plates was recorded. Larvae were determined as being alive if they displayed either swimming or contractile movements, or if flame-cell flickering was visible \[[@B52]\].
Statistical analysis
--------------------
Statistical analysis was performed using Minitab 15 Statistical Software; two sample t-tests or analysis of variance (ANOVA) were performed as appropriate.
Abbreviations
=============
ATF-2: activating transcription factor 2; CBSS: Chernin\'s balanced salt solution; DMSO: dimethyl sulfoxide; MAPK: mitogen-activated protein kinase; PBS: phosphate buffered saline; PKA: protein kinase A; PKC: protein kinase C; TGY: Thr-Gly-Tyr; TTBS: tween-tris buffered saline.
Authors\' contributions
=======================
MR designed and performed experiments and wrote the manuscript. DR supervised the project and modified the manuscript. AM conducted the bioinformatics (in conjunction with MR) and modified the manuscript. AW carried out the scanning electron microscopy, supervised the project, designed the experiments, and wrote the manuscript. All authors read and approved the final manuscript.
Supplementary Material
======================
###### Additional file 1
**Supplementary Movie File**. Combined example videos of miracidia in spring water (control) or SB 203580 (1 μM in spring water) for 60 min, anisomycin (20 μM in spring water) for 30 min or 60 min, or revived after anisomycin treatment (60 min anisomycin followed by 20 min in SB 203580 (1 μM)). Miracidia swim speed is increased slightly by SB203580 and is attenuated after 30 min anisomycin treatment; swimming stops after 60 min in anisomycin and is revived following subsequent incubation in SB203580.
######
Click here for file
Acknowledgements
================
We are indebted to Mike Anderson and Jayne King of the Natural History Museum (London) for the maintenance and passage of parasites. We would also like to thank Richard Giddens and Laura Grigis, Kingston University, for support with scanning electron microscopy.
|
{
"pile_set_name": "PubMed Central"
}
|
Recently, there has been a concerted effort to develop high-quality GaAs-based near-infrared diode lasers using the GaAsBi material system, and in particular to push their lasing wavelengths to the datacom/telecom wavelength range of 1.3--1.6 μm. The main motivation behind this effort is due to the interesting properties of the GaAsBi alloy, which is formed by substituting relatively small quantities of bismuth in place of arsenic in GaAs[@b1]. Incorporation of Bi in GaAs produces a strong reduction of the band gap, *E*~g~, (by up to \~80 meV per % Bi) due to valence band anti-crossing[@b1][@b2], while also strongly increasing the spin-orbit-splitting energy, Δ~SO~[@b3][@b4], between the top of the valence band and the spin-orbit-split-off band[@b4][@b5], something which does not occur in conventional semiconductor alloys. The unusual impact of Bi incorporation on the (In)GaAs material properties opens up a range of possibilities for practical applications in semiconductor lasers[@b5], photovoltaics[@b6], spintronics[@b3], photodiodes[@b7] and thermoelectrics[@b8]. Highly appealing for the development of semiconductor lasers is the possibility to grow GaAsBi laser structures such that Δ~SO~ \> E~g~ in the active region. This is highly significant as it promises to suppress the dominant efficiency-limiting loss processes in near-infrared lasers, namely Auger recombination, involving the generation of "hot" holes in the spin-orbit split-off band (the so-called "CHSH" process), and inter-valence band absorption (IVBA), where emitted photons are re-absorbed in the active region, thereby increasing the internal optical losses and negatively impacting upon the laser characteristics[@b9][@b10][@b11]. This Δ~SO~ \> E~g~ band structure is present in GaAsBi alloys containing \>10% Bi, at which composition the alloy band gap is close to 1.55 μm on a GaAs substrate providing a promising route to devices such as efficient 1.55 μm monolithic vertical cavity surface emitting lasers (VCSELs)[@b4][@b5]. This, combined with the aforementioned ability to engineer the band structure to eliminate the dominant Auger and IVBA loss mechanisms, makes GaAsBi alloys an attractive candidate material system for the development of highly efficient, uncooled GaAs-based lasers for applications in optical communications[@b9].
The first optically pumped GaAsBi based laser, consisting of a bulk-like 390 nm thick GaAs~0.975~Bi~0.025~ active layer containing 2.5% Bi grown using molecular beam epitaxy (MBE) was reported by Tominaga *et al*. and achieved optically pumped pulsed operation up to a temperature of 240 K[@b12]. This was followed by Ludewig *et al*. who demonstrated the first electrically pumped GaAsBi laser this time containing a quantum well (QW)-based active region[@b13]. This device, which was grown by metal-organic vapour phase epitaxy (MOVPE), consisted of a GaAs~0.978~Bi~0.022~ QW active region (containing 2.2% Bi), and electrically pumped pulsed operation was demonstrated at room temperature[@b13]. Since then the Bi composition in GaAsBi QW lasers has been increased up to 4.4% in MOVPE grown structures, although room temperature operation was not observed[@b14]. Using MBE growth, bulk-like GaAsBi optically pumped lasers with 5.9% Bi[@b15], and electrically pumped double heterostructure lasers with up to 4% Bi, have been demonstrated[@b16]. To overcome the challenges associated with MBE and MOVPE growth, a hybrid approach has been developed[@b17][@b18]. This hybrid growth method consists of growing the QW region using MBE (thereby allowing an increased Bi composition to be achieved in the active region), while the remainder of the laser structure is grown using MOVPE (thereby maintaining the capability for rapid high quality growth of the thick waveguide and cladding layers). Using this approach, GaAsBi/GaAs lasers consisting of three QWs containing \~6% Bi have been developed, and electrically pumped operation has been demonstrated at room temperature[@b18]. However, characterisation of these devices has revealed performance issues related to recombination via defect states[@b14]. This is in line with detailed theoretical investigations of the emission dynamics[@b19] and optical spectra in GaAsBi alloys, which have highlighted the strong role played by localised states (associated with Bi clustering) in determining the optical properties of bulk-like epitaxial layers. This demonstrates that despite the rapid progress that has been made in the development of this material system, there is a strong need for further improvement and optimisation of the growth and fabrication of GaAsBi materials and devices in order to realise their potential for practical applications.
A key aspect of efforts to continue the development of this material system is to develop a quantitative understanding of the impact of Bi incorporation on the properties and performance of existing devices, in order to enable the design and optimisation of longer-wavelength devices with increased Bi composition.
In addition to refinement of growth and fabrication processes, such optimisation requires a detailed understanding of the physical properties of the laser structures in order to develop improved device designs (including, e. g., specification of the QW thickness, alloy composition, strain and band offsets, as well as the number of QWs and the waveguide refractive index profile, etc.) to deliver the required laser characteristics (including high optical gain, output power and efficiency, and temperature stability).
In this report we present the first experimental measurements of the optical gain spectra of GaAsBi/(Al)GaAs QW lasers. We apply the segmented contact method[@b20] to directly measure the absorption, gain and spontaneous emission (SE) spectra of a series of multi-section devices, as well as to measure the internal (cavity) optical losses in GaAsBi laser structures containing \~2% Bi. We also present a theoretical analysis of the optical properties of these devices, using a model based upon a 12-band **k.p** Hamiltonian for GaAsBi and related alloys[@b21]. Initial theoretical investigations of the optical gain in dilute bismide alloys[@b22][@b23][@b24] have suffered from a lack of detailed information regarding the band structure of GaAsBi alloys. We have overcome these limitations by undertaking a series of detailed analyses of the electronic properties of GaAsBi alloys. On this basis we have directly parametrised the 12-band Hamiltonian of ref. [@b21] on the basis of (i) atomistic calculations of the alloy electronic structure, as well as (ii) detailed measurements of the electronic properties of GaAsBi QWs[@b25]. Based on this improved understanding of the GaAsBi band structure we have developed a theoretical model for dilute bismide QW lasers, which has been recently applied to elucidate the effects of Bi incorporation on the electronic and optical properties of ideal GaAs-based laser structures operating at wavelengths up to and including 1.55 μm[@b26]. Here, we apply this model to calculate the SE and optical gain spectra for a series of real GaAsBi laser structures, and compare the results of our calculations directly to experiment--the first such comparison between theory and experiment for this new class of semiconductor laser.
The experimental results we present provide the most detailed insight to date into the optical properties of QW lasers based on this novel material system. Furthermore, our calculations are in good, quantitative agreement with experiment, confirming our theoretical understanding of the unusual electronic and optical properties of GaAsBi laser structures and verifying the predictive capacity of our theoretical model for use in the design and optimisation of dilute bismide semiconductor lasers.
Samples and Experimental Methodology
====================================
A single quantum well (SQW) laser structure containing a GaAs~0.978~Bi~0.022~/AlGaAs QW was grown by MOVPE within Al~0.4~Ga~0.6~As cladding layers on an n-doped GaAs (001) substrate. A commercially available AIX 200-GFR reactor system with Pd-purified H~2~ as the carrier gas at a reduced reactor pressure of 50 mbar was used for the laser growth. The GaAsBi QW of the devices with 2.2% Bi was grown at 400 °C under pulsed precursor flow, as reported in ref. [@b13]. Part of the material was processed into Fabry-Perot lasers, as reported elsewhere[@b13][@b14][@b25]. To implement the segmented contact method a special contact mask was designed to fabricate top stripe contacts divided into 300 μm long sections with \~5 μm gaps between each section, as shown in [Fig. 1(a)](#f1){ref-type="fig"}. The mask contained stripes of different width to produce 20, 50 and 100 μm wide contacts. To form the metal contacts Pt/Au were deposited on the top GaAs:Zn *p*^+^ contact layer and an AuGe/Ni/Au-based contact was deposited on the *n*^+^-substrate backside. The sample was annealed at 400 °C for 30 seconds for low-ohmic contact formation. To avoid current spreading and short-circuiting between sections, the GaAs:Zn-contact layer was etched-off using the metal stripes as a mask.
A review of different experimental techniques for optical gain measurements in diode lasers, as well as detailed methodology and advantages of the segmented contact method, is given by Blood *et al*. in ref. [@b20]. The optical gain or modal gain, *G*, is defined as the fractional increase in energy of an optical mode per unit propagation length. It is proportional to the local material gain, *g*, with G = Γg, where Γ is the optical confinement factor defining the fractional overlap of the optical field intensity with the active QW layer(s). Denoting the total SE rate (per unit photon energy per unit area in the plane of the layer) by *I*~*spon*~, and assuming that *I*~*spon*~ is uniform along the contact stripe, the total amplified spontaneous emission (ASE) of the same polarisation emerging from a length *l* of pumped material per unit stripe width is
where *β* is the fraction of the SE coupled into the waveguide and *α*~*i*~ denotes the internal (cavity) optical losses[@b20]. Using [Eq. (1)](#eq1){ref-type="disp-formula"}, defining *C* as an extraction factor and taking into account reflection at the end facet with reflectivity *R*, the externally measured ASE spectrum can be written as
The [Eq. (1)](#eq1){ref-type="disp-formula"} can be solved analytically for two lengths *l* = *L* and *l* = *2L*, which together with [Eq. (2)](#eq2){ref-type="disp-formula"} gives the following expressions for the net modal gain (*G-α*~*i*~), and SE *I*~*spon*~[@b20].
By measuring ASE spectra from the end of a single pumped section of length *L*, *I*~*meas*~(*L*), and from two pumped sections of equal length, *I*~*meas*~(*2L*), and using [Eqs (3](#eq3){ref-type="disp-formula"}) and ([4](#eq4){ref-type="disp-formula"}), we analysed the net modal gain and SE spectra, respectively. Furthermore, the ASE spectra measured under pumping of only the first section at the end facet, *I*~*meas*~(*1*) can be used to determine the ASE spectrum from any other sections, assuming that all sections are identical. Therefore, the ASE spectrum measured under pumping of the second section from the end facet, *I*~*meas*~(*2*), with the first segment acting as a passive absorber, will be related to *I*~*meas*~(*1*) via the modal absorption, *A* (with *A* = *Γα*, where *α* is the material absorption of the gain medium), as *I*~*meas*~(*2*) = *I*~*meas*~(*1*)exp\[*−*(*A* + *α*~*i*~)*L*\][@b20]. From this the net modal absorption can be determined directly as
[Figure 1(b)](#f1){ref-type="fig"} shows an example of a cleaved bar which could be used both for laser characterisation (using solid stripe contacts) and investigation of the gain and absorption spectra (using segmented contacts). To suppress round-trip amplification for a single-pass measurement the end of the segmented contact was coated with a \~115 nm thick HfO~2~ layer using a Nordiko 2000 RF magnetron sputtering system, thereby providing anti-reflection coatings, which reduce the reflectivity of the cleaved facet to R \~ 1%. During the coating deposition the top part of the sample typically gets coated as well, making it difficult to form an electrical contact to the first section. As an additional measure to stop round-trip amplification, as well as to provide single-pass measurements, we used a sample consisting of 7 sections at the edge of the wafer, as shown in [Fig. 1(c)](#f1){ref-type="fig"}. In such a configuration at least 5 segments are acting as passive absorbers and the curved wafer edge provides negligible back reflection, thereby suppressing round-trip amplification.
An optical spectrum analyser and optical power meters were used to measure the ASE from the end facet, as well as the pure SE from the QW. The latter was collected from a small 100 μm diameter window milled in the substrate (*n*) contact[@b14][@b27]. To avoid the effects of GaAs substrate absorption on the measured SE from the GaAsBi QW[@b14][@b26], we also used a 20 μm × 50 μm window in the top (*p*) contact for the pure SE measurements. At low currents (up to 50 mA, or J = 330 A cm^−2^) electrical pumping was provided using a continuous wave (CW) source-meter unit, while at higher currents a pulsed voltage source was used to provide 500 ns long pulses at a frequency of 20 kHz, in order to avoid self-heating effects and to maximise the signal-to-noise ratio.
Theoretical Modelling
=====================
Due to the large differences in size and chemical properties between Bi atoms and the As atoms they replace, Bi acts as an isovalent impurity when incorporated into GaAs in dilute concentrations to form the GaAsBi alloy. It is this impurity-like behaviour of Bi atoms in GaAs and related III-V semiconductor matrices that gives rise to the unusual electronic properties of dilute bismide alloys, and significantly complicates the theoretical description of the material band structure[@b2][@b28].
We have previously developed an atomistic tight-binding model for GaAsBi alloys, which we have applied successfully to develop a quantitative understanding of the electronic, optical and spin properties of this unusual class of semiconductor materials[@b29][@b30][@b31][@b32]. Based on these analyses we demonstrated that substitutional Bi atoms form highly localised resonant impurity states in the GaAs valence band, as well as a range of bound states corresponding to pairs and larger clusters formed by Bi atoms located at second nearest-neighbour anion lattice sites relative to one another. Despite the extremely complicated valence band structure of a realistic, disordered GaAsBi alloy, further analyses we have undertaken have verified explicitly that the general features of the GaAsBi alloy band structure are determined in large part by the effects of the resonant states associated with isolated substitutional Bi atoms. In ref. [@b21] we showed in detail how these general electronic properties can be understood quantitatively in terms of a composition-dependent band-anticrossing interaction between these localised resonant impurity states, and the extended valence band edge states of the host matrix semiconductor.
Based on these insights, we have used a series of detailed supercell calculations to derive an extended 12-band **k.p** Hamiltonian for GaAsBi alloys[@b21][@b33]. The extended basis set of this Hamiltonian contains the spin-degenerate, zone-centre Bloch states of the GaAs host matrix conduction, heavy-hole, light-hole and spin-split-off-hole bands, in addition to the aforementioned Bi-related resonant impurity states. In ref. [@b25] we applied the 12-band model to the study of the band structure of GaAsBi QWs and, by comparison with the results of polarisation-resolved photo-voltage measurements, were able to extract information about and constrain the structural and band structure parameters of the QW and barrier materials, as well as the QW band offsets.
Combining the 12-band **k.p** Hamiltonian of ref. [@b21] with the constrained material parameters and band offsets of ref. [@b25] we have developed a theoretical model of the electronic and optical properties of dilute bismide QW lasers[@b26]. Our theoretical model is based on a numerically efficient reciprocal space (plane wave) approach to the calculation of the QW band structure, and includes electrostatic effects via self-consistent computation of the electronic structure with coupling to Poisson's equation for the charge density generated by the electron and hole populations (which are assumed to be independently in quasi-equilibrium). The model also explicitly incorporates key band structure effects in the computation of the electronic and optical properties, including: Bi-induced hybridisation and carrier localisation, temperature- and injection-dependent carrier spillout from the QW(s), band mixing and pseudomorphic strain. Full details of our theoretical model for GaAsBi lasers, as well as a detailed analysis of the impact of Bi incorporation on the material and device properties of GaAs-based dilute bismide semiconductor lasers, can be found in ref. [@b26].
We apply this theoretical model below to compute, analyse and compare to experiment, the SE and optical gain spectra of the GaAsBi SQW laser structures described in *the previous section*. To facilitate the analysis to be presented in *section* "*Results and Discussion*", we briefly discuss here the calculation of the material gain. Our calculation of the material gain spectrum for a given polarisation follows the approach of ref. [@b34], which consists of transforming the corresponding polarisation component of the SE rate according to
where Δ*F* is the quasi-Fermi level separation at temperature *T* in the presence of an injected carrier density *n*, *n*~r~ is the refractive index of the active region, and *r*~sp~ is the SE rate (calculated as described in ref. [@b26]). In this work we are concerned solely with TE-polarised gain spectra, and so *r*~sp~ is understood to represent only the TE-polarised component of the total SE rate (which gives rise to the leading factor of 3/2 in [Eq. (6)](#eq6){ref-type="disp-formula"}). This approach to calculating the material gain spectrum has the benefits that (i) it removes the anomalous absorption at energies below the active region band gap that can plague calculations undertaken using an energy- and crystal momentum-independent interband relaxation time (homogeneous spectral linewidth), and (ii) by definition, *g*(Δ*F*) = 0, so that the transparency point on the high energy side of the gain peak is maintained at the correct, thermodynamically-consistent energy. As we will see below, this latter characteristic of [Eq. (6)](#eq6){ref-type="disp-formula"} will enable us to associate a distinct carrier density *n* in the theoretical calculations with each current density *J* at which the gain spectrum was measured, and hence to compare the results of our theoretical calculations directly to the experimental gain spectra.
Results and Discussion
======================
We earlier reported on the characterisation and performance of Fabry-Perot lasers fabricated from these GaAs~0.978~Bi~0.022~/AlGaAs SQW laser structures[@b13][@b14][@b25]. We begin here by considering the modal absorption spectra, which were obtained by measuring ASE spectra (cf. [Eq. (2)](#eq2){ref-type="disp-formula"}) under pumping only the first, *I*~*meas*~(*1*) (equivalent to *I*~*meas*~(*L*) here), and then the second section, *I*~*meas*~(*2*), from the sample facet (cf. [Eq.(5)](#eq5){ref-type="disp-formula"}). These measurements, shown in [Fig. 2(a)](#f2){ref-type="fig"}, were carried out using a relatively low continuous current of 50 mA (corresponding to a current density J = 330 A cm^−2^), thereby avoiding self-heating effects and providing a low-noise signal compared to pulsed measurements. The modal absorption spectra measured in this manner are shown in [Fig. 2(b)](#f2){ref-type="fig"}, for a device fabricated from material located \~5--10 mm away from the wafer edge and for devices fabricated from material located \~2 mm away from the wafer edge.
Previous polarisation-resolved photo-voltage measurements we have undertaken on a series of GaAsBi laser structures having nominally identical Bi compositions of 2.2% revealed the presence of Bi compositional variations across a given wafer, where a variation of approximately 30 meV was observed in the QW band gap (corresponding in theoretical calculations to Bi compositional variations of up to ±0.4%)[@b25]. The measured absorption spectra presented in [Fig. 2(b)](#f2){ref-type="fig"} are consistent with these measurements: the absorption edge measured for the device fabricated from material located \~5--10 mm from the wafer edge is red-shifted by approximately 20 nm compared to the absorption edges measured for the devices fabricated from material located \~2 mm away from the wafer edge. As we will see in our discussion of the measured SE spectra for these devices below, this variation of the absorption edge across the wafer corresponds well to our previously calculated Bi compositional variations for laser structures containing \~2% Bi[@b25]. Furthermore, we note that the absorption edge depicted by the black line in [Fig. 2(b)](#f2){ref-type="fig"} is in good agreement with the previously measured SE peak and lasing wavelengths for Fabry-Perot lasers fabricated from the same GaAs~0.978~Bi~0.022~ QW material (932 and 946 nm respectively[@b13][@b14], denoted by arrows in [Fig. 2(b)](#f2){ref-type="fig"}).
The measured modal absorption of approximately 100 cm^−1^ close to 930 nm corresponds to a material absorption coefficient at this wavelength of 6025 cm^−1^, based on an optical confinement factor Γ = 1.66% calculated for a GaAs~0.978~Bi~0.022~ SQW laser structure[@b26]. The constant value of the net modal absorption (*A* + *α*~*i*~) at longer wavelengths below the QW band gap (\>1000 nm, with *A* = 0) enables us to extract the optical (cavity) losses. Based on the data shown in [Fig. 2(b)](#f2){ref-type="fig"} we find *α*~*i*~ = 10--15 cm^−1^ in the devices studied, with the larger values of *α*~*i*~corresponding to devices fabricated from material located closer to the wafer edge. We note that these optical losses are relatively small, indicating generally good optical quality of the fabricated waveguides.
Next, we turn our attention to the measured SE spectra from these devices, which can also be obtained using the segmented contact method (cf. [Eq. (4)](#eq4){ref-type="disp-formula"}), and compare the SE spectra measured using this approach to our previous measurements in which the SE was collected from a window milled in the substrate contact[@b14][@b26]. The advantage of using the segmented contact method to measure the SE from the end facet is that both the TE (in-plane) and TM (out-of-plane) polarised components of the SE can be collected, whereas TM-polarised light, which propagates along the laser cavity only, cannot be collected through a window milled in either the top (*p*) or substrate (*n*) contacts. Because of this, the integrated SE collected through a contact window can underestimate the radiative current density (*J*~rad~) in the device if there is appreciable TM-polarised emission. For comparative purposes, SE spectra were also measured from a window milled in the top contact in order to remove the effect of substrate (GaAs) absorption, which has been previously shown to encroach on the shorter wavelength (higher energy) SE[@b14][@b26]. [Figure 3(a)](#f3){ref-type="fig"} shows the SE spectra measured using these three approaches, for devices fabricated from material located \~5--10 mm away from the wafer edge, all of which we found to have the SE peak at the same wavelength. The measurements were performed at the threshold current density *J*~th~ \~ 1.6 kAcm^−2 ^[@b14].
The open grey triangles, closed green squares and open pink circles in [Fig. 3(a)](#f3){ref-type="fig"} show the SE spectra measured respectively from the top and substrate windows, and using the segmented contact method. Also shown in [Fig. 3(a)](#f3){ref-type="fig"} are a typical lasing spectrum (solid red line) and the measured substrate (GaAs) band edge transmission (dashed blue line). Examining the SE spectra measured using these three approaches we note that the SE measured from the substrate window is most affected by absorption in the (relatively thick) GaAs substrate, as highlighted by comparison with the GaAs band edge transmission. The SE measured from the top contact window does not undergo significant absorption, and hence is broader at shorter wavelengths and has a more symmetrical overall shape. The peak wavelengths of these two spectra are virtually identical, and are in good agreement with the calculated QW band gap of 1.323 eV (937 nm)--i.e. the transition energy between the lowest energy electron and highest energy hole states in the QW (*e*1-*hh*1, since the highest energy hole state is calculated to be heavy-hole-like at the QW Brillouin zone centre). Interestingly the SE spectrum measured using the segmented contact method, while virtually identical to the contact window measurements at the band edge (longer wavelengths), is significantly broader at shorter wavelengths. In particular, we observe a second peak in the SE spectrum obtained using [Eq. (4)](#eq4){ref-type="disp-formula"} at 905 nm, which is in good agreement with the calculated transition energy between *e*1 and the second-highest energy hole state (*lh*1, which is calculated to be predominantly light-hole-like at the QW Brillouin zone centre) 1.375 eV (902 nm). Based on this, as well as our calculations of the full SE spectra (to be discussed below), we therefore conclude that the excess high energy SE observed in the segmented contact measurements is attributable to TM-polarised radiative recombination of electrons occupying *e*1 conduction states with holes occupying *lh*1 valence states, which, as described above, is not possible to detect in measurements taken from a contact window.
Comparing the integrated SE for the spectra shown in [Fig. 3(a)](#f3){ref-type="fig"} we find that they are in the ratio 100:83:71, for measurements using the segmented contact method, as well as from the top and substrate contact windows, respectively. We note that this discrepancy in the ratios of the integrated SE, and the associated underestimation of *J*~rad~ from the window measurements, will tend to increase with increasing temperature due to broadening of the SE spectra as the distribution in energy of the carriers in the conduction and valence bands increases (leading to a larger portion of the high energy SE measured from the substrate contact window being absorbed by the GaAs substrate before it can be collected in the experiment). In ref. [@b14] we used a linear interpolation of low temperature data, where the contribution to *J*~rad~ from transitions involving *lh1* states as well as the effect of GaAs substrate absorption are negligible, to estimate *J*~rad~ at room temperature, which gave an approximately 45% greater value than that obtained using the integrated SE from the substrate window. Using the segmented contact approach, we confirm that the value of *J*~*rad*~ determined using the integrated SE measured from the substrate window is indeed underestimated by approximately 30% at room temperature due to the combined effects of GaAs substrate absorption and the undetected TM-polarised emission due to carrier recombination involving light-hole-like states. This is an important consideration for future SE measurements.
[Figure 3(b)](#f3){ref-type="fig"} shows the SE spectra measured at threshold using the segmented contact method for devices fabricated from material located away from the wafer edge (open red circles) and close to the wafer edge (open blue circles). Both spectra have similar overall shape with the main difference between them being the wavelength of the emission peak, which is approximately 20 nm shorter in the device fabricated from material located close to the wafer edge. We note that this shifted emission peak is consistent with the shift in the absorption edge across the wafer (cf. [Fig. 2(b)](#f2){ref-type="fig"}), as well as with the experimental data presented in ref. [@b25], and indicates that the Bi composition is slightly lower in the QW material at the wafer edge than closer to the centre of the wafer. In order to quantify this variation in Bi composition across the wafer, and to facilitate our theoretical analysis of the optical gain below, we have used the theoretical model described in *section* "*Theoretical Modelling*" to analyse the SE spectra shown in [Fig. 3(b)](#f3){ref-type="fig"}. Through our theoretical calculations we find that the measured SE peak wavelength of 910 nm at the wafer edge corresponds to a Bi composition of 1.8% in the QW (assuming in the theoretical calculations that the QW width is fixed at its nominal value of 6.4 nm[@b25]).
Next, by comparing the full calculated SE spectrum to the experimental data, we find that the spectral broadening is best described by a hyperbolic secant lineshape of the form , with a linewidth δ = 25 meV. We note that this lineshape function was previously found to describe well the SE spectra of 1.3 μm GaInNAs dilute nitride QW lasers, and that the best-fit linewidth of 17 meV at room temperature for the device of ref. [@b35] is similar to that obtained here. Using this lineshape and linewidth, the calculated SE spectrum for the wafer-edge device is shown in [Fig. 3(b)](#f3){ref-type="fig"} using a solid blue line. The theoretical calculation was also carried out at the threshold injection level, which was determined as the carrier density required to produce the TE-polarised threshold material gain of 1325 cm^−1^ (obtained using an effective index calculation to determine an optical confinement factor of 1.60% for a QW containing 1.8% Bi, and using the measured threshold current density of 1.6 kA cm^−2^ in similar devices[@b14]). Due to uncertainty in the absolute units and relative intensity of the SE measurements performed on different devices, we have normalised the calculated SE spectrum to its measured value at the SE peak in order to compare the theoretical and experimental data. We note that the measured and calculated SE spectra are in good overall agreement, with the spectral broadening observed in the experiment well described by a combination of the energy broadening of the electron and hole (quasi-Fermi) distribution functions at room temperature, as well as the aforementioned hyperbolic secant line broadening.
In order to quantify the Bi compositional variation across the wafer we have also calculated the SE spectrum for the device fabricated from material located away from the edge of the wafer. The calculation of the SE spectrum in this case proceeds as above, this time with the Bi composition being the only parameter allowed to vary. The result of this calculation is shown using a solid red line in [Fig. 3(b)](#f3){ref-type="fig"}. We find that the measured SE peak at 932 nm is well described in the theoretical calculations assuming that the QW contains the nominal Bi composition of 2.2%, and that the calculated SE spectrum at this composition is generally in good overall agreement with experiment for this device. Overall, these theoretical results suggest that the Bi composition in the QW is close to the nominal value of 2.2% across the central part of the wafer, but it is slightly reduced to 1.8% towards the wafer edges. The reduction of the Bi composition towards the wafer edge is possible if the temperature during growth is lower at the edges compared to the centre of the wafer; however, since the gas phase is optimized for 400 °C with relatively high V/III ratio a reduction of the Bi composition cannot be excluded. We recall that this variation in Bi composition is consistent with that determined previously via photo-voltage measurements performed on a series of MOVPE-grown laser structures having the same nominal Bi composition of 2.2%[@b25].
[Figure 4(a)](#f4){ref-type="fig"} shows exemplar ASE spectra measured from the wafer-edge device facet under pumping of a single section *I*~meas~(*L*) (solid blue line), and under pumping of two sections *I*~meas~(2*L*) (solid red line), at the threshold current density. By performing such measurements at different current densities and using [Eq. (3)](#eq3){ref-type="disp-formula"} we obtain the net modal gain spectra shown in [Fig. 4(b)](#f4){ref-type="fig"}, which were measured at current densities of 0.7, 1.4, 2.0 and 2.4 kAcm^−2^ (shown using black, blue, red and green open symbols, respectively). The measured gain spectra are relatively broad, with a full width at half maximum of approximately 100 meV at a current density of 2 kAcm^−2^, which is close to twice that observed for an InGaAs/GaAs SQW laser operating at a similar wavelength[@b36] and is most likely related to the strong Bi-induced inhomogeneous broadening that is characteristic of the optical spectra of GaAsBi alloys[@b30]. Based on the α~i~ = 15 cm^−1^ optical losses measured for the device fabricated from material located close to the wafer edge (cf. [Fig. 2(b)](#f2){ref-type="fig"}) the estimated peak modal gain at *J* = 2 kAcm^−2^ is *G*~peak~ = 24 cm^−1^. Using the calculated optical confinement factor of 1.60% for this GaAs~0.982~Bi~0.018~ SQW laser structure we estimate a peak material gain *g*~peak~ ≈ 1500 cm^−1^ at *J* = 2 kA cm^−2^, which agrees well with the calculated value of 1560 cm^−1^.
In order to compare our theoretical gain spectra to experiment we must determine the carrier density corresponding to each current density at which the gain spectra depicted by open symbols in [Fig. 4(b)](#f4){ref-type="fig"} were measured. In order to do this we recall from [Eq. (6)](#eq6){ref-type="disp-formula"} that the transparency point at which there is zero material/modal gain on the high energy side of the gain peak is given when the photon energy is equal to the quasi-Fermi level separation at a given level of injection. We therefore proceed by shifting the measured net modal gain spectra upwards by the α~i~ = 15 cm^−1^ optical losses in order to obtain the absolute modal gain *G* in the device. Next, we use the transparency points on each of these absolute modal gain spectra to extract the quasi-Fermi level separation Δ*F* corresponding to each current density in the experiment. Finally, using our theoretical model we calculate Δ*F* as a function of the injected carrier density *n* and, in doing so, determine the value of *n* which produces the extracted Δ*F* for each current density *J*. Following this procedure, we find that the current densities of 0.7, 1.4, 2.0 and 2.4 kA cm^−2^, at which the gain spectra were measured, correspond to quasi-Fermi level separations of 1.375, 1.409, 1.423 and 1.435 eV, which in turn correspond respectively to carrier densities in the theoretical calculations of 5.12, 7.11, 8.24 and 9.38 × 10^18^ cm^−3^. We then calculated the TE-polarised component of the SE rate at each of these carrier densities as outlined above, from which the theoretical net modal gain spectrum was determined at each carrier density as *Γg* − *α*~*i*~, with Γ = 1.60% and α~i~ = 15 cm^−1^, and where *g* is the TE-polarised material gain spectrum obtained from the SE rate using [Eq. (6)](#eq6){ref-type="disp-formula"}. The results of these calculations are shown in [Fig. 4(b)](#f4){ref-type="fig"} using, in order of increasing current/carrier density, black, blue, red and green solid lines. We note that the theoretical gain spectra shown using black, blue and red solid lines include *e*1-*hh*1 optical transitions only while the green solid line, corresponding to the highest current density of 2.4 kA cm^−2^, also includes *e*1-*lh*1 optical transitions. Including *e1-hh1* transitions only at the highest carrier density was found to underestimate the peak modal gain by \~10%, highlighting that TE-polarised optical recombination involving light-hole-like states becomes important at higher levels of injection. Overall, we see that the theoretical spectra are in good, quantitative agreement with experiment: the calculated magnitude of the net modal gain is in excellent agreement with that measured using the segmented contact method across the full investigated range of current densities (confirming the accuracy of the optical transition matrix elements derived within the framework of the 12-band **k.p** model[@b26]), and the overall shape of the experimental gain spectrum is well reproduced at each current/carrier density by our theoretical model.
From measurements of the gain spectra for several devices, fabricated from material located at the wafer edge, we present in [Fig. 5(a)](#f5){ref-type="fig"} the variation of the peak modal gain *G*~peak~ (closed symbols) as well as the threshold modal gain *G*~*th*~ (open symbols) as a function of current density, where the latter was determined as the sum of the cavity (*α*~*i*~) and mirror (*α*~*m*~) losses, *G*~th~ = *α*~*i*~ + *α*~*m*~, which is equal to the peak modal gain at threshold determined from the light output vs. current (L-I) characteristics for a series of Fabry-Perot devices with 20 μm wide contacts and cavity lengths *L*~c~ = 0.5--1 mm (shown in [Fig. 5(b)](#f5){ref-type="fig"}). As these Fabry-Perot devices were fabricated from the same part of the wafer as the devices for which the SE and gain spectra of [Fig. 4(a,b)](#f4){ref-type="fig"} were measured, it was assumed that they have the same *α*~*i*~ = 15 cm^−1^ optical losses; the facet (mirror) losses were calculated in each case as *α*~*m*~ = (*1/L*~*c*~)ln(*1/R*), where *R* is the facet reflectivity. Returning to [Fig. 5(a)](#f5){ref-type="fig"}, we note that the peak modal gain data obtained in this manner for the Fabry-Perot devices (open symbols) fit well with the overall trend observed for the multi-section devices upon which the segmented contact measurements were performed (closed symbols).
Based on the correspondence between the experimental current and theoretical carrier densities determined in our analysis of the gain measurements shown in [Fig. 4(b)](#f4){ref-type="fig"}, we have used our theoretical model to calculate the variation of *G*~peak~ with *J*. The results of this calculation are depicted by the solid black line in [Fig. 5(a)](#f5){ref-type="fig"}. We see that the theoretical calculation is again in good, quantitative agreement with the experimental data, confirming that the theoretical model is capable of describing the GaAsBi gain spectra across a wide range of current densities, as well as for a variety of multi-section and Fabry-Perot devices. The inset in [Fig. 5(a)](#f5){ref-type="fig"} presents a theoretical estimate of the optimum number of QWs, *N*~opt~, required to minimise the threshold current density *J*~th~ as a function of *L*~c~. This optimum number of QWs was calculated as *N*~opt~ = (α~i~ + α~m~)/*G*~0,~ where the modal gain *G*~0~ = 10.9 cm^−1^ corresponds to the maximum ratio *G*/*J* required to minimise *J*~th~[@b37]. *G*~*0*~ was determined as the point at which a line through *G* = 0 is tangent to a fit to the measured *G*~peak~ vs. *J* data[@b37]. Note that while this provides a reasonable estimate for low numbers of QWs it is generally less accurate for a higher number of QWs, due to non-uniform pumping and poorer hole transport. Notwithstanding this, the calculated variation of *N*~opt~ with *L*~c~ corresponds well to the results presented in ref. [@b14], in which we observed a reduction by close to a factor of two in the threshold current density per QW in going from an SQW device to one containing three QWs (for a fixed cavity length *L*~c~ = 1 mm).
Conclusion
==========
In this report we have presented the first experimental measurements of optical gain in dilute bismide semiconductor laser structures. We used the segmented contact method to study the optical properties of a series of GaAsBi SQW laser structures containing \~2% Bi. Using this approach we were able to directly measure the absorption, SE, and optical gain spectra, as well as to determine the optical (cavity) losses in the laser structures (the values of which we found to be in the range of 10--15 cm^−1^). In addition to confirming the well-known Bi-induced red-shift of the optical absorption and emission peak wavelengths, our experimental measurements also indicate that transitions involving higher energy light-hole-like valence states make appreciable contributions to the SE and optical gain at higher injection levels (*J* \> 2 kA cm^−2^).
In conjunction with these experimental measurements, we have also used theoretical calculations to facilitate our analysis of the laser devices. Beginning from a 12-band **k.p** model which has previously been constrained against detailed measurements of the electronic properties of GaAsBi QW laser structures, we analysed the SE from several devices. This enabled us to identify and quantify (i) Bi composition variations across the wafer from which the laser devices were fabricated, and (ii) Bi-induced inhomogeneous broadening of the optical spectra. Our calculations of the SE and optical gain spectra, as well as the peak modal gain as a function of current density, were found to be in good, quantitative agreement with experiment across a wide range of current densities for a range of multi-section and Fabry-Perot devices. This approach provides a powerful predictive capability for the design and optimisation of future bismide-based devices, including in particular highly efficient, cooler-free GaAs-based long-wavelength lasers.
Additional Information
======================
**How to cite this article**: Marko, I. P. *et al*. Optical gain in GaAsBi/GaAs quantum well diode lasers. *Sci. Rep*. **6**, 28863; doi: 10.1038/srep28863 (2016).
The authors acknowledge support from the Engineering and Physical Sciences Research Council, U.K. (project nos EP/H005587/01, EP/H050787/1 and EP/K029665/1), the European Union Framework programme (EU-FP7 project "BIANCHO" (FP7-257974) and the German Science Foundation (project nos DFG: VO805/4 and DFG: GRK1782). E.P.O'R. acknowledges support from Science Foundation Ireland (project no. 10/IN.1/I2994). Details of the data and how to request access are available from the University of Surrey publications repository at http://epubs.surrey.ac.uk/810996/ and from the University of Bristol research data repository at http://data.bris.ac.uk/.
**Author Contributions** I.P.M. and P.L. fabricated the laser devices, I.P.M. performed the experimental measurements, led the analysis of the experimental data and co-led the writing of the manuscript. C.A.B. performed the theoretical calculations, led the analysis of the theoretical data and co-led the writing of the manuscript. P.L. grew the laser structures. S.J., W.S., K.V., J.M.R., E.P.O'R. and S.J.S. secured the funding to support this study, supervised the experimental and theoretical work, contributed to the analysis of the experimental and theoretical data, and contributed to the preparation of the manuscript.
{#f1}
{ref-type="disp-formula"} for a series of different devices: a device fabricated from material located 5--10 mm from the wafer edge (cf. [Fig. 1(b)](#f1){ref-type="fig"}; solid black line), and devices fabricated from material located \~2 mm from the wafer edge (cf. [Fig. 1(c)](#f1){ref-type="fig"}; red, blue and green solid lines, respectively).](srep28863-f2){#f2}
{ref-type="disp-formula"}; open pink circles). The dashed blue line depicts the GaAs substrate transmission spectrum. (**b**) Measured (using the segmented contact method; open circles) and calculated (solid lines) SE spectra at threshold for a device fabricated from material located \~2 mm from the wafer edge (blue) and fabricated from material located 5--10 mm from the wafer edge (red).](srep28863-f3){#f3}
{ref-type="fig"}, under pumping of a single section, *I*~meas~(*L*) (solid blue line), and under pumping of two adjacent sections of the same length, *I*~meas~(2*L*) (solid red line). (**b**) Measured (using the segmented contact method, cf. [Eq. (3)](#eq3){ref-type="disp-formula"}; open symbols) and calculated (cf. [Eq. (6)](#eq6){ref-type="disp-formula"}; solid lines) net modal gain spectra for the same device, at injected current densities of *J* = 0.7, 1.4, 2.0 and 2.4 kA/cm^2^ (black, blue, red and green circles/lines respectively). The corresponding carrier densities in the theoretical calculations, determined as outlined in the text, are *n* = 5.12, 7.11, 8.24 and 9.38 × 10^18^ cm^−3^.](srep28863-f4){#f4}
{#f5}
|
{
"pile_set_name": "PubMed Central"
}
|
Data are available upon request to Dr. Slattery who may be contacted at <[email protected]>. Dr. Slattery will work with individuals who request data to obtain necessary data transfer agreements and MOU.
Introduction {#sec001}
============
MicroRNAs (miRNA) are non-coding RNAs that function as gene regulators \[[@pone.0162077.ref001]\]. As such, they provide a mechanism whereby genes involved in various signaling pathways can be regulated simultaneously \[[@pone.0162077.ref002]\]. Both aging and cellular senescence are thought to be regulated in part by miRNAs \[[@pone.0162077.ref003], [@pone.0162077.ref004]\]. It also has been suggested that miRNAs control the function of telomeres in cancer \[[@pone.0162077.ref005]\]. Telomeres, located at the end of chromosomes, shorten with every cell division; when they become too short the cell is unable to divide, leading to the induction of senescence and apoptosis, which are key features of normal cell function, but absent in tumor cells. Telomere length (TL) has been associated with colorectal cancer, with higher risk of disease for both long and short telomeres \[[@pone.0162077.ref006], [@pone.0162077.ref007]\]. Telomerase, a ribonucleoprotein enzyme that is essential for the replication of chromosome ends, is low or non-expressing in normal human somatic cells, but has been shown to be expressed in the majority of tumor cells \[[@pone.0162077.ref008]\]. Telomerase reverse transcriptase (TERT) is essential for maintenance of telomere length by protecting chromosome ends. TERT has been proposed to regulate miRNAs by regulation of miRNA biogenesis, with diminished primary miRNA expression in TERT-reduced cells \[[@pone.0162077.ref009]\], and has been shown to be associated with both TL and cancer \[[@pone.0162077.ref006], [@pone.0162077.ref010]--[@pone.0162077.ref014]\]. TERT is thought to be one of the most critical, if not the rate-limiting step, in production of telomerase activity \[[@pone.0162077.ref008], [@pone.0162077.ref015]\]. We have shown previously that *TERT* rs2853676 was associated with TL and that TL was associated with colorectal cancer \[[@pone.0162077.ref011]\]. Additionally *TERT*-CLPTM1L rs2853668 and *TERT* rs2736118 were associated with colorectal cancer risk in this study population \[[@pone.0162077.ref014]\].
In this paper we examine the associations between TL and miRNA expression in colorectal tissue. Additionally we test the hypothesis that variation in TERT influences miRNA expression levels. We focus on associations between miRNAs and *TERT* SNPs previously associated with colorectal cancer and examine miRNA expression in normal colonic mucosa tissue as well as the differential expression of miRNAs between carcinoma and normal colonic mucosa tissue.
Materials and Methods {#sec002}
=====================
Study population {#sec003}
----------------
The study population consisted of individuals previously enrolled in a study of Diet, Lifestyle, and Colon cancer at the University of Utah and the Kaiser Permanente Medical Research Program (KPMRP) \[[@pone.0162077.ref016]\] for whom both miRNA from carcinoma and adjacent normal colonic mucosa tissue were available. Study subjects included incident cases of colon cancer between the ages of 30 and 79 who were non-Hispanic white, Hispanic, or African American, and were able to provide a signed informed consent prior to participation in the study. The study was approved by the University of Utah Institutional Review Board for Human Subjects.
miRNA processing {#sec004}
----------------
RNA (miRNA) was extracted from formalin-fixed paraffin embedded tissues. We assessed slides and tumor blocks that were prepared over the duration of the study prior to the time of miRNA isolation to determine their suitability. The study pathologist reviewed slides to delineate carcinoma and normal colonic mucosa tissue. Cells were dissected from 1--4 sequential sections on aniline blue stained slides using an H&E slide for reference. Total RNA containing miRNA was extracted, isolated, and purified using the RecoverAll Total Nucleic Acid isolation kit (Ambion); RNA yields were determined using a NanoDrop spectrophotometer. 100 ng total RNA was labeled with Cy3 and hybridized to Agilent Human miRNA Microarray V19.0 and were scanned on an Agilent SureScan microarray scanner model G2600D. The Agilent Human microarray was generated using known miRNA sequence information compiled in the Sanger miRBase database v19.0. The microarray contains probes for 2006 unique human miRNAs. The miRNA array contains 60,000 unique human sequences and averages 30 replicates per probe sequence. Data were extracted from the scanned image using Agilent Feature Extract software v.11.5.1.1. Data were required to pass stringent QC parameters established by Agilent that included tests for excessive background fluorescence, excessive variation among probe sequence replicates on the array, and measures of the total gene signal on the array to assess low signal. If samples failed to meet quality standards for any of these parameters, the sample was re-labeled, hybridized to arrays, and scanned. If a sample failed QC assessment a second time the sample was deemed to be of poor quality and the individual was excluded from down-stream analysis. A 75^th^ percentile scaling was performed to normalize across arrays was done using preprocessCore \[[@pone.0162077.ref017]\] ([www.bioconductor.org](http://www.bioconductor.org/)) to minimize differences that could be attributed to the array, amount of RNA, location on array, or other factors that could erroneously influence expression measurements. Testing of both reliability and comparative validity were done \[[@pone.0162077.ref018]\]. We showed a 98% repeatability in the Agilent platform between repeat samples. Additionally, the Agilent platform had fairly good agreement with Nanostring results and 100% agreement in expression values and fold change with qPCR results \[[@pone.0162077.ref019]\].
RNA-Seq Sequencing Library Preparation and Data Processing {#sec005}
----------------------------------------------------------
Total RNA was available from 197 carcinoma and normal mucosa pairs. These samples were taken from the study subjects used for miRNA analysis and were extracted, isolated and purified in the same manner as previously described \[[@pone.0162077.ref020]\]. RNA library construction was done with the Illumina TruSeq Stranded Total RNA Sample Preparation Kit with Ribo-Zero. The samples were then fragmented and primed for cDNA synthesis, adapters were then ligated onto the cDNA, and the resulting samples were then amplified using PCR; the amplified library was then purified using Agencount AMPure XP beads. A more detailed description of the methods can be found in our previous work \[[@pone.0162077.ref021]\].
Sequencing was done using an Illumina TruSeq v3 single read flow cell and a 50 cycle single-read sequence run was performed on an Illumina HiSeq instrument. Reads were then aligned to a sequence database containing the human genome (build GRCh37/hg19, February 2009 from genome.ucsc.edu) and alignment was performed using novoalign v2.08.01. Python and a pysam library were used to calculate counts for each exon and UTR of the genes using a list of gene coordinates obtained from [http://genome.ucsc.edu](http://genome.ucsc.edu/). We dropped features that were not expressed in our data or for which the expression was missing for the majority of samples. A more detailed description of the methods can be found in our previous work \[[@pone.0162077.ref021]\].
Telomere Length (TL) Telomere-related Gene Analysis {#sec006}
---------------------------------------------------
TL was measured using a multiplexed quantitative PCR (qPCR) method previously described by Cawthon \[[@pone.0162077.ref022]\]. This method modified earlier qPCR methods in which telomere signals were measured separately from single copy gene signals in order to produce a T/S ratio corresponding to TL, which is proportional to the average telomere length in a cell. Therefore a larger T/S ratio represents a longer telomere length. The multiplexed PCR analysis uses a single dye and measures both the telomere signals and single copy gene signals in the same well. This is achieved by using CG clamps to stabilize the single copy gene giving it a higher melting point. The unique sequence is then amplified during a different cycle than the telomeric sequence, allowing for a single qPCR to determine the T/S ratio \[[@pone.0162077.ref006]\].
DNA was obtained from immortalized cell lines from study participants for the colon cancer study and from whole blood for the rectal cancer study. To assure the quality of telomere assay for these samples from different DNA origins we evaluated associations with TL separately for cases and controls from each study and as shown previous, results were similar for both study groups and we concluded that DNA source did not significantly alter TL length in this study. Two samples yielded a T/S ratio greater than three and were excluded. These samples were excluded because we concluded, through gel electrophoresis, that the DNA was degraded and therefore the T/S ratio was unreliable. We focused on Single Nucleotide Polymorphisms (SNPs) that were previously associated with TL and/or colon cancer \[[@pone.0162077.ref006], [@pone.0162077.ref014]\]. We analyzed *TERT*-*CLPTM1L* rs2853668 which is 5.1kb upstream of *TERT* and has been associated with colon cancer in GWAS \[[@pone.0162077.ref023]\]. We also test *TERT* rs2736118 and *TERT* rs2853676 with miRNA expression.
Statistical Analysis {#sec007}
--------------------
Our sample consisted of 1152 individuals with both SNP data and miRNA expression data; 363 individuals with both TL and miRNA expression data in the normal colonic mucosa; and 148 individuals who had both miRNA and mRNA data, and 176 cases in the colon cancer study and 184 cases in the rectal cancer study had both TL and SNP data.
Three major components of the analysis were conducted. First, we evaluated the linear associations between TL and those miRNAs for which at least 20% of the population had expression (N = 817 miRNA) by fitting a linear regression model to the log base 2 transformed expression levels; we adjusted for age and sex. P-values for each regression analysis were generated from the bootstrap method by creating a distribution of 1,000 β coefficients for each miRNA and evaluating H~0~: β = 0 vs. H~1~: β≠0 using the boot package in R. We adjusted for multiple comparisons using a false discovery rate (FDR) level of 0.05 \[[@pone.0162077.ref024]\]. We standardized the slopes generated from the overall dataset in order to compare the results across the miRNA.
Second, we assigned inheritance models for *TERT* SNPs based on previous findings for colorectal cancer risk using either dominant or recessive models \[[@pone.0162077.ref014]\]. We compared log base 2 transformed expression levels across the selected genotype models using the significance analysis of microarrays (SAM) technique implemented in the R package siggenes; p-values were based on 1,000 permutations. We utilized an FDR level of 0.05 to determine which *TERT* SNP/miRNA associations were significant after adjustment for multiple comparisons.
For both the TL/miRNA analysis and the TERT SNP/miRNA analysis, we evaluated miRNA expression in both normal colonic mucosa and for differentially expressed miRNAs (i.e. difference between carcinoma tissue and normal colonic mucosa). Looking at normal colonic mucosa allowed us to evaluate the effect of TL or SNP on overall miRNA expression. However, since an association with normal colonic mucosa could influence baseline level of the miRNA expressed and could potentially miss important changes in miRNA expression between carcinoma and normal colonic mucosa, we also evaluated miRNA differential expression. This allowed us to gain insight into regulation of miRNAs that could be more directly associated with cancer through their influence on carcinoma miRNA expression; by comparing to normal expression we were able to control for differences in tumor subsite.
One of the goals of the study was to identify non-telomere related mechanisms whereby *TERT* may be operating through altered miRNA expression levels. To accomplish this, we undertook the third part of the analysis, where we used miRTarBase ([www.http://mirtarbase.mbc.nctu.edu.tw/](http://www.http://mirtarbase.mbc.nctu.edu.tw/)) v6.0 (as of 09/15/2015) \[[@pone.0162077.ref025]\] to identify validated target genes for the significant miRNAs identified in our *TERT* SNP/miRNA analysis. We calculated the differential mRNA expression as the difference of the log base 2 of the RPKM (Reads per Kilobase per Million) for the carcinoma and normal mucosa tissues for the 4047 target genes identified by miRTarBase. We then evaluated the 6309 combinations of miRNAs with their respective mRNA target genes. We used the bootstrap method as described above to assess any linear relationship between the differential mRNA and miRNA expression levels, adjusting for age and sex.
QIAGEN's Ingenuity Pathway Analysis (IPA) (QIAGEN Redwood City, [www.qiagen.com/ingenuity](http://www.qiagen.com/ingenuity)) \[[@pone.0162077.ref026]\] was then used to evaluate the genes that were dysregulated in conjunction with the miRNAs. We considered direct relationships from the IPA knowledge base of genes only, limited to experimentally verified and mammalian results only, and considered all mutations, data sources, and tissues. We report canonical pathways that were significant enriched with these genes after adjustment for multiple comparisons.
Results {#sec008}
=======
The average age of study participants from the colon cancer study was slightly older than from the rectal cancer study ([Table 1](#pone.0162077.t001){ref-type="table"}). The distribution of age, sex, and AJCC stage was similar for those individuals included in the smaller TL dataset as with the larger SNP dataset.
10.1371/journal.pone.0162077.t001
###### Description of study population (includes cases only).
{#pone.0162077.t001g}
Colon Rectal
----------------------------------------------------------------------- -------- ------- -------- ------ ------
Telomere Length (TL)[^1^](#t001fn001){ref-type="table-fn"}(T/S ratio) 1.17 0.55 1.20 0.29
Age (years) 65.3 9.1 61.9 10.9
N \% N \%
Sex Male 380 55.7 280 59.6
Female 302 44.3 190 40.4
AJCC Stage 1 167 24.7 213 45.8
2 220 32.6 88 18.9
3 222 32.9 136 29.2
4 66 9.8 28 6.0
*TERT* (rs2736118)[^2^](#t001fn002){ref-type="table-fn"} AA 341 51.5 237 53.1
AG/GG 321 48.5 209 46.9
*TERT* (rs2853668) GG/GT 631 93.3 445 94.7
TT 45 6.7 25 5.3
*TERT* (rs2853676) CC 348 53.0 155 58.5
CT/TT 309 47.0 110 41.5
^1^ 179 individuals in the colon cancer study and 184 individuals in the rectal cancer study had TL, miRNA and SNP data.
^2^ 53 individuals with AA genotype had RNAseq data; 50 individuals with the AG/GG genotype had RNAseq; 148 individuals had both miRNA and mRNA (RNAseq) data.
Assessment of the linear relationship between TL and miRNA expression levels in for the 363 individuals with both TL and miRNA from normal colorectal mucosa showed that 34 miRNAs were associated with TL after adjustment for age and sex ([Table 2](#pone.0162077.t002){ref-type="table"}). Thirty-three of the miRNAs were directly associated with TL, in that TL increased as miRNA expression level increased. Only miR-487b was inversely associated with TL, showing lower levels of miR-487b expression with longer TL length.
10.1371/journal.pone.0162077.t002
###### Associations between TL (T/S Ratio) and miRNA expression levels adjusted for age (FDR\<0.05).
{#pone.0162077.t002g}
miRNA Mean Expression Beta Coefficient[^1^](#t002fn001){ref-type="table-fn"} P-value^2^ Q-Value
------------------- ----------------- -------------------------------------------------------- ------------ ----------
hsa-miR-1185-2-3p 6.81 0.17 \< .0001 \< .0001
hsa-miR-1207-5p 10.65 0.14 0.002 0.0481
hsa-miR-1226-5p 5.5 0.16 0.002 0.0481
hsa-miR-1229-5p 9.07 0.14 0.002 0.0481
hsa-miR-1247-3p 4.64 0.12 0.002 0.0481
hsa-miR-134 7.84 0.16 \< .0001 \< .0001
hsa-miR-3141 7.4 0.14 0.002 0.0481
hsa-miR-3158-5p 4.82 0.16 \< .0001 \< .0001
hsa-miR-3162-5p 11.19 0.15 0.002 0.0481
hsa-miR-3194-5p 7 0.14 0.002 0.0481
hsa-miR-3196 10.19 0.18 \< .0001 \< .0001
hsa-miR-3200-5p 4.75 0.17 0.002 0.0481
hsa-miR-3937 5.8 0.14 0.002 0.0481
hsa-miR-4253 5.29 0.18 \< .0001 \< .0001
hsa-miR-4459 14.38 0.15 \< .0001 \< .0001
hsa-miR-4496 5.74 0.17 \< .0001 \< .0001
hsa-miR-4497 11.57 0.17 0.002 0.0481
hsa-miR-4530 12.75 0.15 \< .0001 \< .0001
hsa-miR-4535 5.93 0.15 \< .0001 \< .0001
hsa-miR-4673 6.3 0.16 0.002 0.0481
hsa-miR-4689 6.64 0.16 0.002 0.0481
hsa-miR-4739 9.71 0.18 \< .0001 \< .0001
hsa-miR-487b 1.6 -0.17 \< .0001 \< .0001
hsa-miR-5006-5p 9.36 0.15 \< .0001 \< .0001
hsa-miR-5088 5.64 0.15 0.002 0.0481
hsa-miR-5195-3p 7.14 0.14 0.002 0.0481
hsa-miR-575 9.18 0.15 \< .0001 \< .0001
hsa-miR-601 5.31 0.15 \< .0001 \< .0001
hsa-miR-6076 7.84 0.14 0.002 0.0481
hsa-miR-662 5.32 0.16 0.002 0.0481
hsa-miR-718 6.59 0.18 \< .0001 \< .0001
hsa-miR-762 10.72 0.15 0.002 0.0481
hsa-miR-887 5.74 0.2 \< .0001 \< .0001
hsa-miR-939-5p 9.06 0.14 \< .0001 \< .0001
^1^Beta coefficients are from a linear regression of TL on miRNA expression after adjustment for age, sex, and study
Evaluation of differences in miRNA expression levels between carcinoma tissue and normal colorectal mucosa showed that *TERT* rs2736118 was associated with differential miRNA expression based on genotype ([Table 3](#pone.0162077.t003){ref-type="table"}). *TERT* rs2736118 was associated with 75 miRNAs with an FDR of \<0.05. Individuals with the AA (homozygous dominant) genotype were more likely to have greater miRNA expression in the tumor than in the normal colorectal mucosa (61% of the time) while individuals with the AG/GG genotypes were more likely to have greater miRNA expression in the normal colorectal mucosa (84% of the time). Neither *TERT-CLPTMIL* rs2853668 nor *TERT* rs2853676 significantly altered miRNA expression in normal colonic mucosa or miRNA expression between carcinoma and normal colorectal mucosa.
10.1371/journal.pone.0162077.t003
###### Associations between *TERT* rs2736118 and miRNA differential expression[^1^](#t003fn001){ref-type="table-fn"} with an FDR of \<0.05.
{#pone.0162077.t003g}
AA (N = 556) AG/GG (N = 508)
------------------- -------------- ----------------- --------- -------- -------- --------- --------
hsa-miR-1203 0.9040 1.2164 -0.3124 0.7861 1.3040 -0.5179 0.0044
hsa-miR-1237-5p 2.7048 2.8009 -0.0961 2.5824 2.8954 -0.313 0.0003
hsa-miR-125b-1-3p 3.3566 3.3173 0.0393 3.2864 3.3280 -0.0416 0.0069
hsa-miR-1266 0.9616 0.7167 0.2449 0.8296 0.7950 0.0345 0.0061
hsa-miR-1276 1.1104 1.0311 0.0793 0.9951 1.1146 -0.1195 0.0083
hsa-miR-1295b-3p 1.0382 1.0006 0.0376 0.9074 1.0876 -0.1803 0.0055
hsa-miR-1323 2.2143 2.1357 0.0786 2.0477 2.2264 -0.1788 0.0011
hsa-miR-1470 2.0574 2.3573 -0.2999 1.8649 2.4017 -0.5368 0.0008
hsa-miR-184 0.9277 0.9309 -0.0032 0.7507 1.0149 -0.2641 0.0011
hsa-miR-206 2.8032 3.1652 -0.3621 2.6350 3.2131 -0.5781 0.0109
hsa-miR-302c-5p 1.2712 0.9455 0.3257 1.1561 1.0997 0.0564 0.0001
hsa-miR-3122 0.8753 0.8425 0.0328 0.7107 0.8545 -0.1437 0.0151
hsa-miR-3131 4.0127 3.8855 0.1272 3.9049 3.9703 -0.0654 0.0001
hsa-miR-3150b-3p 1.0041 0.8180 0.1861 0.8778 0.8914 -0.0136 0.0100
hsa-miR-3161 3.8361 3.9214 -0.0852 3.8044 3.9553 -0.1509 0.0080
hsa-miR-3177-5p 1.9296 2.0896 -0.16 1.7230 2.1565 -0.4335 0.0003
hsa-miR-3181 0.7395 1.0470 -0.3075 0.6513 1.1449 -0.4936 0.0155
hsa-miR-3186-3p 0.7814 0.6846 0.0968 0.6631 0.7670 -0.1039 0.0046
hsa-miR-3189-5p 2.1073 2.1018 0.0055 2.0026 2.2128 -0.2102 0.0087
hsa-miR-339-3p 1.9875 1.8349 0.1527 1.8464 1.8830 -0.0366 0.0050
hsa-miR-34c-3p 2.3869 2.3467 0.0401 2.3206 2.4220 -0.1014 0.0152
hsa-miR-3615 0.7203 0.9008 -0.1804 0.6091 0.9742 -0.3651 0.0071
hsa-miR-3660 1.9127 1.9104 0.0022 1.7900 1.9813 -0.1913 0.0158
hsa-miR-3679-3p 0.4062 0.3906 0.0156 0.2972 0.4282 -0.131 0.0011
hsa-miR-3680-3p 2.9284 2.6281 0.3003 2.7893 2.6970 0.0923 0.0008
hsa-miR-378e 1.0146 0.8963 0.1183 0.8816 0.9639 -0.0824 0.0037
hsa-miR-3922-5p 0.6227 0.4820 0.1406 0.5307 0.5691 -0.0384 0.0031
hsa-miR-4300 0.8662 0.7173 0.1488 0.7436 0.7864 -0.0428 0.0022
hsa-miR-4303 0.8538 0.9330 -0.0792 0.7200 0.9955 -0.2755 0.0138
hsa-miR-4421 0.5460 0.7538 -0.2078 0.4893 0.8849 -0.3956 0.0113
hsa-miR-4436a 2.0374 1.9462 0.0911 1.9109 2.0796 -0.1688 0.0039
hsa-miR-4444 3.2323 2.9691 0.2632 3.0676 3.0396 0.028 0.0011
hsa-miR-4450 1.8216 1.6023 0.2192 1.7353 1.7504 -0.0151 0.0072
hsa-miR-4461 1.9050 1.7542 0.1507 1.7372 1.8575 -0.1203 0.0014
hsa-miR-4479 0.7581 0.7816 -0.0235 0.6689 0.8772 -0.2083 0.0158
hsa-miR-4489 3.5472 3.6088 -0.0616 3.4330 3.6676 -0.2347 0.0135
hsa-miR-4510 2.4625 2.5053 -0.0429 2.3292 2.5568 -0.2276 0.0064
hsa-miR-4519 3.0204 2.9033 0.1171 2.8279 2.9748 -0.1469 0.0004
hsa-miR-4522 3.4239 3.6634 -0.2395 3.2646 3.6946 -0.4300 0.0033
hsa-miR-4526 1.8260 1.8567 -0.0307 1.6271 1.8806 -0.2535 0.0013
hsa-miR-4638-5p 1.4805 1.5457 -0.0652 1.3145 1.6703 -0.3559 0.0014
hsa-miR-4654 1.3087 1.0013 0.3074 1.1610 1.1001 0.0609 0.0022
hsa-miR-4657 1.5101 1.0805 0.4295 1.3891 1.1962 0.1929 0.0032
hsa-miR-4659b-3p 1.7201 1.9028 -0.1827 1.5847 2.0117 -0.4271 0.0081
hsa-miR-4660 2.0659 1.8028 0.2631 1.9383 1.9375 0.0008 0.0043
hsa-miR-4674 2.5960 2.6555 -0.0594 2.4912 2.7185 -0.2273 0.0029
hsa-miR-4676-5p 1.0025 0.8863 0.1162 0.9258 1.0054 -0.0796 0.0127
hsa-miR-4682 0.7889 0.6533 0.1356 0.6994 0.7349 -0.0355 0.0114
hsa-miR-4684-3p 1.1204 1.1684 -0.048 1.0038 1.2391 -0.2353 0.0083
hsa-miR-4715-5p 1.6858 1.6206 0.0652 1.4839 1.7073 -0.2233 0.0007
hsa-miR-4717-3p 2.0039 1.7400 0.2639 1.7938 1.7920 0.0018 0.0001
hsa-miR-4731-3p 0.7447 0.6289 0.1158 0.6076 0.7042 -0.0966 0.0011
hsa-miR-4732-5p 4.1588 3.8034 0.3555 4.1413 3.9066 0.2347 0.0089
hsa-miR-4748 2.3375 2.2502 0.0874 2.1273 2.2557 -0.1285 0.0084
hsa-miR-500a-3p 1.5768 1.4419 0.1348 1.4121 1.5348 -0.1227 0.0006
hsa-miR-516b-5p 2.1095 2.0461 0.0634 1.9679 2.2117 -0.2438 0.0005
hsa-miR-518c-5p 0.7122 0.8026 -0.0904 0.6082 0.8813 -0.2731 0.0101
hsa-miR-5195-5p 2.8941 2.6472 0.2469 2.7678 2.7966 -0.0287 0.0020
hsa-miR-519e-5p 1.0583 1.0823 -0.024 0.9249 1.2156 -0.2907 0.0003
hsa-miR-520e 3.2490 3.6373 -0.3883 3.1534 3.7298 -0.5763 0.0068
hsa-miR-525-5p 0.8990 0.7384 0.1606 0.7517 0.8720 -0.1203 0.0000
hsa-miR-550a-5p 2.9657 3.0505 -0.0848 2.8758 3.0853 -0.2096 0.0097
hsa-miR-551b-5p 0.8574 0.6666 0.1908 0.7783 0.7348 0.0436 0.0109
hsa-miR-5572 2.7054 2.6641 0.0413 2.4570 2.6748 -0.2178 0.0010
hsa-miR-5584-5p 0.4473 0.4448 0.0024 0.3820 0.5262 -0.1442 0.0066
hsa-miR-566 1.7370 1.8000 -0.0631 1.6106 1.8912 -0.2806 0.0019
hsa-miR-583 2.7128 1.5976 1.1152 2.6492 1.7677 0.8814 0.0065
hsa-miR-616-3p 0.8733 0.5788 0.2945 0.8258 0.6369 0.1889 0.0157
hsa-miR-639 3.0031 3.1208 -0.1177 2.9192 3.1285 -0.2093 0.0072
hsa-miR-6509-5p 3.6249 3.6661 -0.0411 3.5866 3.7262 -0.1396 0.0104
hsa-miR-658 0.5168 0.6255 -0.1087 0.4250 0.6472 -0.2221 0.0055
hsa-miR-659-3p 3.4147 3.2518 0.1629 3.2092 3.3117 -0.1025 0.0003
hsa-miR-6716-5p 2.2443 2.1112 0.1331 2.1058 2.1845 -0.0787 0.0017
hsa-miR-6718-5p 2.9358 2.7755 0.1604 2.8682 2.8986 -0.0304 0.0008
hsa-miR-708-5p 3.1203 3.2337 -0.1133 3.0296 3.3207 -0.2911 0.0122
^1^Expression based on log2 transformed data
The 75 miRNAs dysregulated by *TERT* rs2736118 were associated with 4047 genes and 6309 miRNA/mRNA associations according to miRTarBase. To determine which of these genes might be associated with mRNA regulation in colonic tissue, we assessed each miRNA with its mRNA gene target in colonic carcinoma tissue and colonic normal mucosa using a linear regression. We identified 573 miRNA/mRNA associations p-value of \<0.05 prior to adjustment for multiple comparisons. Of these, 150 significant miRNA/mRNA target associations remained when the FDR was 0.05 and 270 miRNA/mRNA associations remained with the FDR of \<0.1 ([Table 4](#pone.0162077.t004){ref-type="table"}). The miRNAs and their targeted genes shown in [Table 4](#pone.0162077.t004){ref-type="table"} also indicate the direct of the association based on the beta coefficient. Of note is that the associations were limited to 13 miRNAs. All but five of these associations showed inverse associations between the miRNA and the mRNA.
10.1371/journal.pone.0162077.t004
###### Regression of differential miRNA expression and differential mRNA expression (FDR\<0.05).
{#pone.0162077.t004g}
miRNA Gene Beta FDR Q
----------------- ------------- ------- ---------
hsa-miR-3161 *WIZ* -0.24 0.006
hsa-miR-3161 *SLC25A40* -0.27 0.006
hsa-miR-3161 *EFNB2* -0.11 0.006
hsa-miR-3161 *TRIM5* -0.28 0.006
hsa-miR-3161 *PPIG* -0.24 0.006
hsa-miR-3161 *RELA* -0.2 0.006
hsa-miR-3161 *CSTF2T* -0.27 0.006
hsa-miR-3161 *SRGAP1* -0.31 0.006
hsa-miR-3161 *HMGN2* -0.26 0.006
hsa-miR-3161 *NUP50* -0.23 0.009
hsa-miR-3161 *TXNDC12* -0.25 0.009
hsa-miR-3161 *BCL2L11* -0.2 0.009
hsa-miR-3161 *RYBP* -0.25 0.009
hsa-miR-3161 *TMEM41A* -0.2 0.009
hsa-miR-3161 *PRDX3* -0.29 0.009
hsa-miR-3161 *WIPF2* -0.23 0.009
hsa-miR-3161 *TMEM30B* -0.31 0.009
hsa-miR-3161 *CTNND1* -0.22 0.009
hsa-miR-3161 *VMP1* -0.19 0.012
hsa-miR-3161 *TRIP11* -0.26 0.012
hsa-miR-3161 *INVS* -0.24 0.012
hsa-miR-3161 *GEMIN6* -0.17 0.012
hsa-miR-3161 *EOGT* -0.27 0.012
hsa-miR-3161 *UBXN2A* -0.2 0.012
hsa-miR-3161 *NUPL2* -0.18 0.016
hsa-miR-3161 *PCMTD1* -0.16 0.016
hsa-miR-3161 *TOM1L2* -0.21 0.016
hsa-miR-3161 *PTP4A1* -0.18 0.017
hsa-miR-3161 *QKI* -0.23 0.017
hsa-miR-3161 *SHFM1* -0.17 0.017
hsa-miR-3161 *THRB* -0.18 0.017
hsa-miR-3161 *SREK1* -0.21 0.017
hsa-miR-3161 *ACTBL2* 0.15 0.017
hsa-miR-3161 *CCDC43* -0.24 0.017
hsa-miR-3161 *PSMC2* -0.15 0.02
hsa-miR-3161 *ARMC10* -0.18 0.02
hsa-miR-3161 *SPOP* -0.27 0.023
hsa-miR-3161 *HIF1AN* -0.22 0.023
hsa-miR-3161 *ZNF706* -0.14 0.026
hsa-miR-3161 *ZNF100* -0.21 0.028
hsa-miR-3161 *MCTS1* -0.17 0.031
hsa-miR-3161 *ZNF431* -0.17 0.034
hsa-miR-3161 *ABHD2* -0.07 0.036
hsa-miR-3161 *PDIK1L* -0.21 0.036
hsa-miR-3161 *ELOVL5* -0.12 0.041
hsa-miR-3161 *BTC* -0.22 0.041
hsa-miR-3161 *NUP37* -0.09 0.045
hsa-miR-3161 *TPCN2* -0.19 0.045
hsa-miR-3161 *LRRC40* -0.25 0.048
hsa-miR-3161 *DSE* -0.19 0.052
hsa-miR-3161 *PPIL1* -0.14 0.055
hsa-miR-3161 *VSNL1* -0.19 0.057
hsa-miR-3161 *STARD7* -0.33 \< .001
hsa-miR-3161 *SRP54* -0.3 \< .001
hsa-miR-3161 *RBM3* -0.22 \< .001
hsa-miR-3161 *DGKH* -0.15 \< .001
hsa-miR-3161 *MON1B* -0.3 \< .001
hsa-miR-3161 *RPS6KB1* -0.32 \< .001
hsa-miR-3161 *EIF4G2* -0.24 \< .001
hsa-miR-3161 *TRMT5* -0.3 \< .001
hsa-miR-3161 *ZBED3* -0.27 \< .001
hsa-miR-3161 *SLC38A2* -0.24 \< .001
hsa-miR-3161 *SETDB2* -0.27 \< .001
hsa-miR-3161 *ABI2* -0.28 \< .001
hsa-miR-3161 *POU2F1* -0.27 \< .001
hsa-miR-3161 *ANP32E* -0.25 \< .001
hsa-miR-3161 *LBR* -0.27 \< .001
hsa-miR-3161 *MBNL1* -0.32 \< .001
hsa-miR-3161 *SREK1IP1* -0.24 \< .001
hsa-miR-3161 *CBR1* -0.25 \< .001
hsa-miR-3161 *DIP2A* -0.25 \< .001
hsa-miR-3161 *UBE2Q1* -0.3 \< .001
hsa-miR-3161 *SRSF2* -0.27 \< .001
hsa-miR-3161 *ZNF367* -0.21 \< .001
hsa-miR-3161 *NUDT21* -0.31 \< .001
hsa-miR-3161 *CDK1* -0.19 \< .001
hsa-miR-3161 *TOMM20* -0.26 \< .001
hsa-miR-3161 *NR2C2* -0.24 \< .001
hsa-miR-3161 *LRRC57* -0.23 \< .001
hsa-miR-3161 *NGRN* -0.25 \< .001
hsa-miR-3161 *PTAR1* -0.28 \< .001
hsa-miR-3177-5p *PRRG4* -0.18 \< .001
hsa-miR-3181 *FAM3C* -0.18 0.008
hsa-miR-3181 *ATP5E* -0.13 0.061
hsa-miR-3181 *KLHDC3* -0.17 0.061
hsa-miR-3615 *KHSRP* -0.17 0.008
hsa-miR-3615 *NIPA2* -0.25 0.008
hsa-miR-3615 *ZYG11B* -0.18 0.008
hsa-miR-3615 *HIST1H1B* -0.09 0.008
hsa-miR-3615 *PPP6R1* -0.2 0.011
hsa-miR-3615 *WNT4* -0.2 0.011
hsa-miR-3615 *CCNDBP1* -0.27 0.011
hsa-miR-3615 *CTC1* -0.21 0.011
hsa-miR-3615 *VASP* -0.19 0.013
hsa-miR-3615 *ARF6* -0.21 0.013
hsa-miR-3615 *LRCH3* -0.19 0.013
hsa-miR-3615 *ZBTB7A* -0.21 0.021
hsa-miR-3615 *SRM* -0.1 0.027
hsa-miR-3615 *OPA3* -0.21 0.042
hsa-miR-3615 *WDR12* -0.14 0.042
hsa-miR-3615 *SPRY1* -0.13 0.047
hsa-miR-3615 *ATP9A* -0.09 \< .001
hsa-miR-3615 *RPS8* -0.18 \< .001
hsa-miR-3615 *ALDOA* -0.22 \< .001
hsa-miR-3615 *COX20* -0.29 \< .001
hsa-miR-3615 *BMPR2* -0.22 \< .001
hsa-miR-378e *TGFB2* -0.14 0.052
hsa-miR-4510 *CDH7* 0.25 \< .001
hsa-miR-4638-5p *GINM1* -0.16 0.073
hsa-miR-4638-5p *OGFOD1* -0.15 0.073
hsa-miR-4638-5p *ALDH2* -0.17 0.073
hsa-miR-4638-5p *FEM1A* -0.19 0.073
hsa-miR-4638-5p *CNBP* -0.25 0.073
hsa-miR-4638-5p *RSBN1L* -0.17 0.073
hsa-miR-4638-5p *FAM120AOS* -0.19 0.073
hsa-miR-4638-5p *HMGB1* -0.15 0.073
hsa-miR-4638-5p *CAPZA2* -0.14 0.073
hsa-miR-4638-5p *PEX26* -0.2 0.073
hsa-miR-4638-5p *PI4K2B* -0.18 0.077
hsa-miR-4638-5p *RAB10* -0.14 0.077
hsa-miR-4638-5p *RRP36* -0.14 0.077
hsa-miR-4638-5p *OXA1L* -0.22 0.077
hsa-miR-4638-5p *ZBTB43* -0.21 0.077
hsa-miR-4638-5p *FGFR1OP2* -0.19 0.079
hsa-miR-4638-5p *HSD17B12* -0.14 0.079
hsa-miR-4638-5p *SGPP2* -0.22 0.079
hsa-miR-4638-5p *ERAP2* -0.24 0.079
hsa-miR-4638-5p *ZNF652* -0.14 0.079
hsa-miR-4638-5p *GABPB1* -0.19 0.081
hsa-miR-4638-5p *CIAO1* -0.1 0.081
hsa-miR-4638-5p *MTMR10* -0.15 0.081
hsa-miR-4638-5p *KCMF1* -0.12 0.081
hsa-miR-4638-5p *ZNF485* -0.17 0.081
hsa-miR-4638-5p *GNPNAT1* -0.16 0.087
hsa-miR-4638-5p *YIPF5* -0.16 0.087
hsa-miR-4638-5p *SNRPD1* -0.21 0.087
hsa-miR-4638-5p *TSPAN6* -0.12 0.092
hsa-miR-4638-5p *ZNF207* -0.09 0.092
hsa-miR-4638-5p *MRI1* -0.13 0.092
hsa-miR-4638-5p *MAPK1* -0.15 0.092
hsa-miR-4638-5p *RBM23* -0.13 0.092
hsa-miR-4638-5p *NUP93* -0.13 0.092
hsa-miR-4638-5p *RPL34* -0.11 0.092
hsa-miR-4638-5p *GNB4* -0.14 0.092
hsa-miR-4638-5p *RNF11* -0.16 0.092
hsa-miR-4638-5p *RAB9A* -0.18 0.092
hsa-miR-4638-5p *IRF1* -0.18 0.092
hsa-miR-4638-5p *TEP1* -0.13 0.092
hsa-miR-4638-5p *SCO1* -0.19 0.092
hsa-miR-4638-5p *CDK4* -0.09 0.092
hsa-miR-4638-5p *UGGT1* -0.13 0.092
hsa-miR-4638-5p *PRPF4* -0.17 0.092
hsa-miR-4638-5p *PNPT1* -0.11 0.092
hsa-miR-4638-5p *ENSA* -0.11 0.092
hsa-miR-4638-5p *CNKSR3* -0.14 0.092
hsa-miR-4638-5p *FOXK1* -0.09 0.092
hsa-miR-4638-5p *TRMT10B* -0.18 0.092
hsa-miR-4638-5p *MELK* -0.16 0.092
hsa-miR-4638-5p *ZNF561* -0.16 0.092
hsa-miR-4638-5p *KIAA1919* -0.13 0.092
hsa-miR-4638-5p *CCS* -0.14 0.092
hsa-miR-4638-5p *GK5* -0.13 0.092
hsa-miR-4638-5p *ZNF682* -0.18 0.092
hsa-miR-4638-5p *SF3B1* -0.08 0.096
hsa-miR-4638-5p *TNFAIP8L1* -0.12 0.096
hsa-miR-4638-5p *PSMB5* -0.14 0.097
hsa-miR-4638-5p *MCM4* -0.12 0.097
hsa-miR-4638-5p *SMG1* -0.1 0.097
hsa-miR-4638-5p *LRIG2* -0.13 0.097
hsa-miR-4638-5p *OPTN* -0.22 \< .001
hsa-miR-4638-5p *ZNF655* -0.17 \< .001
hsa-miR-4660 *CTBS* -0.2 \< .001
hsa-miR-4731-3p *HSP90AB1* -0.16 0.015
hsa-miR-4731-3p *IKZF4* -0.22 0.015
hsa-miR-4731-3p *SPTLC2* -0.24 0.023
hsa-miR-4731-3p *N4BP1* -0.24 0.023
hsa-miR-4731-3p *NRF1* -0.25 0.023
hsa-miR-4731-3p *SLC38A2* -0.21 0.023
hsa-miR-4731-3p *STAT5A* -0.2 0.027
hsa-miR-4731-3p *CHEK1* -0.22 0.027
hsa-miR-4731-3p *MFSD6* -0.34 0.027
hsa-miR-4731-3p *PPP2R5E* -0.25 0.027
hsa-miR-4731-3p *TMED10* -0.24 0.033
hsa-miR-4731-3p *ZNF678* -0.27 0.033
hsa-miR-4731-3p *HFE* -0.22 0.034
hsa-miR-4731-3p *TJP1* -0.25 0.034
hsa-miR-4731-3p *TFCP2* -0.17 0.034
hsa-miR-4731-3p *ZIC5* -0.2 0.034
hsa-miR-4731-3p *MYO10* -0.16 0.034
hsa-miR-4731-3p *ASB6* -0.21 0.034
hsa-miR-4731-3p *PIP4K2A* -0.17 0.034
hsa-miR-4731-3p *SRSF2* -0.21 0.034
hsa-miR-4731-3p *ZBTB40* -0.21 0.034
hsa-miR-4731-3p *TPM2* -0.24 0.034
hsa-miR-4731-3p *SRSF1* -0.19 0.038
hsa-miR-4731-3p *PTPN3* -0.21 0.041
hsa-miR-4731-3p *ANKS6* -0.22 0.041
hsa-miR-4731-3p *PTMA* -0.17 0.055
hsa-miR-4731-3p *SREK1* -0.19 0.058
hsa-miR-4731-3p *TRAM1* -0.17 0.059
hsa-miR-4731-3p *GALNT7* -0.18 0.059
hsa-miR-4731-3p *QKI* -0.16 0.059
hsa-miR-4731-3p *SKIL* -0.17 0.059
hsa-miR-4731-3p *GATAD2B* -0.22 0.059
hsa-miR-4731-3p *ANKRD40* -0.16 0.059
hsa-miR-4731-3p *TAF1D* -0.13 0.059
hsa-miR-4731-3p *VCPIP1* -0.19 0.059
hsa-miR-4731-3p *CCDC43* -0.17 0.059
hsa-miR-4731-3p *DIDO1* -0.17 0.06
hsa-miR-4731-3p *KCNC4* -0.18 0.06
hsa-miR-4731-3p *MAP3K9* -0.18 0.068
hsa-miR-4731-3p *EIF5A2* -0.19 0.068
hsa-miR-4731-3p *ATP9A* -0.13 0.074
hsa-miR-4731-3p *SHOC2* -0.21 0.074
hsa-miR-4731-3p *SLC1A4* -0.12 0.074
hsa-miR-4731-3p *GAN* -0.16 0.074
hsa-miR-4731-3p *SFXN5* -0.17 0.074
hsa-miR-4731-3p *ZNF432* -0.15 0.074
hsa-miR-4731-3p *ZNF585A* -0.1 0.076
hsa-miR-4731-3p *ACBD5* -0.3 \< .001
hsa-miR-4731-3p *SOS1* -0.28 \< .001
hsa-miR-4731-3p *FBXL5* -0.26 \< .001
hsa-miR-4731-3p *NDUFA5* -0.31 \< .001
hsa-miR-4731-3p *CLSTN3* -0.24 \< .001
hsa-miR-4731-3p *THYN1* -0.26 \< .001
hsa-miR-4731-3p *RABGEF1* -0.26 \< .001
hsa-miR-4731-3p *MYO6* -0.33 \< .001
hsa-miR-4731-3p *TLK1* -0.25 \< .001
hsa-miR-4731-3p *LYN* -0.24 \< .001
hsa-miR-500a-3p *C18orf25* -0.24 \< .001
hsa-miR-520e *CDIPT* -0.29 0.075
hsa-miR-520e *WWC1* -0.23 0.075
hsa-miR-520e *PDPK1* -0.3 0.075
hsa-miR-520e *ZNF12* -0.37 0.075
hsa-miR-520e *KBTBD6* -0.29 0.075
hsa-miR-520e *ZBTB33* -0.23 0.075
hsa-miR-520e *TSPAN6* -0.24 0.094
hsa-miR-520e *TFAP4* -0.22 0.094
hsa-miR-520e *SUGP1* -0.3 0.094
hsa-miR-520e *SERINC1* -0.22 0.094
hsa-miR-520e *NUDT3* -0.29 0.094
hsa-miR-520e *KCNJ8* -0.17 0.094
hsa-miR-520e *ZNFX1* -0.29 0.094
hsa-miR-520e *ABI2* -0.33 0.094
hsa-miR-520e *EFCAB11* -0.22 0.094
hsa-miR-520e *TM4SF5* -0.28 0.094
hsa-miR-520e *ZNF7* -0.24 0.094
hsa-miR-520e *HSPA13* -0.25 0.094
hsa-miR-520e *NEK8* -0.27 0.094
hsa-miR-520e *ITGA2* -0.29 0.094
hsa-miR-520e *FOXK1* -0.27 0.094
hsa-miR-520e *CYB5A* -0.23 0.094
hsa-miR-520e *BLCAP* -0.34 0.094
hsa-miR-520e *BSCL2* -0.26 0.094
hsa-miR-520e *KBTBD2* -0.35 0.094
hsa-miR-520e *ZMAT3* -0.17 0.094
hsa-miR-520e *ARL10* -0.23 0.094
hsa-miR-520e *CAPZA2* -0.31 0.094
hsa-miR-520e *ZNF805* -0.28 0.094
hsa-miR-520e *SLC35F6* -0.26 0.094
hsa-miR-520e *ZNF174* -0.37 \< .001
hsa-miR-520e *SLC6A4* -0.36 \< .001
hsa-miR-520e *BMP8B* -0.28 \< .001
hsa-miR-520e *ZNF426* -0.31 \< .001
hsa-miR-520e *ABHD15* -0.35 \< .001
hsa-miR-525-5p *KCNH1* 0.21 \< .001
hsa-miR-583 *NKD1* 0.08 0.068
hsa-miR-583 *SIGLEC9* -0.25 \< .001
hsa-miR-583 *KIF2C* 0.2 \< .001
hsa-miR-583 *PDZRN4* -0.25 \< .001
Using IPA to construct pathways enriched with the 270 genes with an FDR of \<0.1, we identified 14 pathways ([Fig 1](#pone.0162077.g001){ref-type="fig"}). Although less than 10% of genes in each pathway were included in our gene list, these pathways were significantly enriched by our genes. The enriched pathways included: neuregulin signaling, PTEN signaling, PI3K/AKT signaling, erythropoietin signaling, regulation of EIF4 and p706SK signaling, chronic myeloid leukemia signaling, tight junction signaling, telomerase signaling, aryl hydrocarbon receptor signaling, Rac signaling, molecular mechanisms of cancer, prolactin signaling NGF signaling, and FLT3 signaling in hematopoietic progenitor cells.
{#pone.0162077.g001}
Discussion {#sec009}
==========
These data suggest a direct association between miRNAs and TL as well as between *TERT* rs2736118 and miRNA differential expression between carcinoma tissue and normal colonic mucosa. While TERT expression is low in most somatic cells, endothelial cells represent an established exception and associations observed for differential expression between carcinoma and normal mucosa is a likely extension of that observation. Our results add support to the hypothesis that miRNAs are associated with TL and that genetic variation in telomere-related genes influence miRNA expression levels. Given the number of miRNAs altered by *TERT* rs2736118, our ability to show associations between altered miRNAs and their targeted mRNA, and the pathways represented by these target genes, we believe that our data support the hypothesis that *TERT* has functions beyond that of regulating TL. Focusing only on those miRNAs that were statistically associated with target mRNA expression in colon tissue, we identified 14 canonical pathways that were associated significantly with these *TERT*-regulated miRNAs after adjustment for multiple comparison, 13 of which were not associated with telomerase.
There have been few reports of miRNAs associated with TL that have looked at actual miRNA expression levels in conjunction with TL; our sample size is by far the largest study to date to examine miRNA expression with TL. One study with only two samples compared shortened telomeres to intact telomeres and showed that 47 miRNAs had over a twofold difference in expression levels \[[@pone.0162077.ref027]\]. MiR-155, miR-138, and miR-34a,b,c also have been hypothesized as being associated with TL \[[@pone.0162077.ref003], [@pone.0162077.ref005]\]; none of these miRNAs were associated in a linear fashion with TL in our study. The miRNAs associated with TL in our data for the most part increased TL with increased miRNA expression. Differences in association between our study and others may in part be from the methods of analysis. We assessed a linear association, while other studies have often used categorical variables and looked at either short or long TL, which could account for some of the differences observed.
A limitation of the study is that we are unable to confirm our observed associations between TL and miRNAs in bio-functional studies; we encourage others to do so. However, support for our findings come from several sources. First, we have previously shown that 26 of the 34 miRNAs associated with TL were differentially expressed between colorectal carcinoma and normal colonic mucosa and 28 of the 34 were dysregulated between carcinoma and adenomatous polyp tissue; three miRNAs were dysregulated between normal colonic mucosa and adenomatous polyp tissue \[[@pone.0162077.ref018]\]. Interestingly, the only miRNA that was directly associated with TL, miR-487b, was the only miRNA that was not associated with dysregulation between carcinoma, adenoma, and/or normal mucosa. Additionally, using bioinformatics tools to identify the target genes for the dysregulated miRNAs associated with TL, we observed that 12 of these miRNAs associated with TL target genes (summary in [Table 5](#pone.0162077.t005){ref-type="table"}) are components of the alternative lengthening of telomeres pathway (ALT) and Shelterin \[[@pone.0162077.ref028]\]. Shelterin is a conserved protein component on telomeres that serves as a functional framework of telomere chromatin pathways. ALT is a telomerase-independent pathway of lengthening telomeres in human cells. Given that there is no overlap between TERT SNP-associated miRNAs and TL-associated miRNAs, and that a large proportion of these miRNAs target genes in the ALT pathway, it is possible that miRNAs regulate TL through this alternative mechanism. As TL has been shown to be associated with CRC, the associations between TL and miRNA expression may unique clinical significance.
10.1371/journal.pone.0162077.t005
###### Summary of miRNAs related to telomere length and *TERT* rs2736118 differentially expressed in colorectal cancer.
{#pone.0162077.t005g}
Differentially Expressed:
------------------- --------------------------- --- --- ---------------------------------------------
Telomere Length
hsa-miR-1185-2-3p x
hsa-miR-1207-5p x x *CBX5*, *TERT*, *TP53*
hsa-miR-1226-5p x x *CBX5*, *RAD18*
hsa-miR-1229-5p x x * *
hsa-miR-1247-3p x *FANCA*, *RAD51L3-RFFL*, *SMC5*
hsa-miR-134 x x
hsa-miR-3141 x
hsa-miR-3158-5p x x
hsa-miR-3162-5p x x
hsa-miR-3194-5p x x
hsa-miR-3196 x x
hsa-miR-3200-5p x x
hsa-miR-3937 *FANCA*, *CBX5*
hsa-miR-4253 x * *
hsa-miR-4459 x x *TERF2*, *ERCC1*, *HSP90B1*, *SMC5*
hsa-miR-4496 x *RAD50*, *RAD51B*, *RAP1B*
hsa-miR-4497 x x *FANCA*
hsa-miR-4530 x x * *
hsa-miR-4535 x x * *
hsa-miR-4673 x x *XRCC3*
hsa-miR-4689 *CDKN1A*, *RAD18*, *RAD50*, *XRCC3*
hsa-miR-4739 x x
hsa-miR-487b
hsa-miR-5006-5p x x
hsa-miR-5088 x x
hsa-miR-5195-3p x x * *
hsa-miR-575 x x x *RAD51*
hsa-miR-601 x x * *
hsa-miR-6076 x x * *
hsa-miR-662 x x * *
hsa-miR-718 x x * *
hsa-miR-762 x x *CDK2*, *RAD54L2*
hsa-miR-887 x x * *
hsa-miR-939-5p x x *CDKN1A*
*TERT* rs2736118
hsa-miR-1203 x x
hsa-miR-1237-5p x
hsa-miR-125b-1-3p
hsa-miR-1266
hsa-miR-1276
hsa-miR-1295b-3p *WRN*
hsa-miR-1323 * *
hsa-miR-1470 x * *
hsa-miR-184 * *
hsa-miR-206 x x *HSP90B1*
hsa-miR-302c-5p *CBX4*
hsa-miR-3122 *PARP2*
hsa-miR-3131 * *
hsa-miR-3150b-3p *CDK2*, *CDKN1A*, *RAD18*, *RAD50*, *XRCC3*
hsa-miR-3161 x * *
hsa-miR-3177-5p x * *
hsa-miR-3181 x * *
hsa-miR-3186-3p * *
hsa-miR-3189-5p * *
hsa-miR-339-3p * *
hsa-miR-34c-3p * *
hsa-miR-3615 x * *
hsa-miR-3660 * *
hsa-miR-3679-3p x x * *
hsa-miR-3680-3p *CBX4*
hsa-miR-378e *RAP1B*
hsa-miR-3922-5p * *
hsa-miR-4300 * *
hsa-miR-4303 * *
hsa-miR-4421 x * *
hsa-miR-4436a * *
hsa-miR-4444 * *
hsa-miR-4450 * *
hsa-miR-4461 * *
hsa-miR-4479 x * *
hsa-miR-4489 * *
hsa-miR-4510 x x *TP53*, *MRE11A*
hsa-miR-4519 * *
hsa-miR-4522 x *PARP2*
hsa-miR-4526 * *
hsa-miR-4638-5p * *
hsa-miR-4654 * *
hsa-miR-4657 x x * *
hsa-miR-4659b-3p *CDKN1A*
hsa-miR-4660
hsa-miR-4674 x
hsa-miR-4676-5p *RAD51*
hsa-miR-4682 *CDKN1A*, *TP53*
hsa-miR-4684-3p *NR2F2*
hsa-miR-4715-5p *CBX4*
hsa-miR-4717-3p *WRN*
hsa-miR-4731-3p * *
hsa-miR-4732-5p x x * *
hsa-miR-4748 *WRN*
hsa-miR-500a-3p * *
hsa-miR-516b-5p * *
hsa-miR-518c-5p * *
hsa-miR-5195-5p * *
hsa-miR-519e-5p * *
hsa-miR-520e x x *CBX5*, *FANCA*, *NR2F2*
hsa-miR-525-5p * *
hsa-miR-550a-5p x *RAD51*
hsa-miR-551b-5p *ERCC1*
hsa-miR-5572 * *
hsa-miR-5584-5p *CDKN1A*, *RAD50*, *RAD54L2*
hsa-miR-566 *CBX5*
hsa-miR-583 x x *CBX5*, *CDKN1A*
hsa-miR-616-3p x x * *
hsa-miR-639 x *CDKN1A*
hsa-miR-6509-5p * *
hsa-miR-658 x *HSP90B1*, *SMC5*
hsa-miR-659-3p * *
hsa-miR-6716-5p *CBX5*
hsa-miR-6718-5p x x * *
hsa-miR-708-5p x *TOP2A*
^1^'T' signifies 'Tumor tissue', 'N' signifies 'Normal colonic mucosa', and 'AD' signifies 'Adenoma tissue'.
We also observed that genetic variation in *TERT*, which we have previously reported as being associated with TL and cancer \[[@pone.0162077.ref006], [@pone.0162077.ref014]\], was associated with miRNA expression levels. Many of these miRNAs have been shown to be dysregulated in CRC \[[@pone.0162077.ref018]\] and 26 of these miRNAs target ALT pathway genes ([Table 5](#pone.0162077.t005){ref-type="table"}). One of the pathways enriched by genes targeted by dysregulated miRNAs is 'telomerase signaling'. Since telomerase is generally not expressed in non-tumor somatic tissue but is expressed in the majority of tumors, it is logical that *TERT* rs2836118 was not associated with miRNA expression in normal colonic mucosa, but only in differential miRNA expression between carcinoma and normal colorectal mucosa. Telomerase is often reactivated by TERT upregulation, which gives tumor cells their immortal quality, and this is thought to be a key step in the adenoma-carcinoma transition in human tumorigenesis \[[@pone.0162077.ref029]\]. As stated previously, we identified miRNAs that were differentially expressed between normal colonic mucosa and adenomatous polyp tissues as well as between adenoma and carcinoma tissues, which show the changes in miRNA expression as tissue progresses from normal to adenomatous to cancerous \[[@pone.0162077.ref018]\]. Both hsa-miR-520e and hsa-miR-583 were differentially expressed between normal colonic mucosa and adenomatous polyp tissue \[[@pone.0162077.ref018]\]; in this study we show that they also regulate mRNA expression. These two miRNAs regulated 40 genes, including *ITGA2*, *BMP8B*, and *PDPK1*, which were found in numerous pathways in IPA, including PTEN Signaling, BMP signaling pathway, and Basal Cell Carcinoma Signaling. Hsa-miR-520e was associated with *CBX5*, *FANCA* and *NR2F2*, and hsa-miR-583 was associated with *CBX5* and *CDKN1A*, all of which are part of the ALT pathway. We also identified six miRNAs that were downregulated with the AG/GG (heterozygous-homozygous recessive) genotype of rs2736118 that we previously found were dysregulated between adenomatous and carcinoma tissue; these miRNAs were: hsa-miR-206, hsa-miR-4510, hsa-miR-4674, hsa-miR-639, hsa-miR-6718-5p, and hsa-miR-708-5p. This supports the hypothesis that TERT regulates miRNA expression and this influences the formation of adenomatous tissue and contributes to tumorigenesis.
The association of *TERT* rs2736118 genotype with miRNA expression levels may provide additional insight into pathways in which *TERT* is involved. We therefore examined genes and pathways regulated by the miRNAs that were influenced by *TERT* rs2736118. We observed that genetic variation in *TERT* was associated with dysregulation of 75 miRNAs, which in turn could influence translation and function of over 6000 genes. While many genes are regulated by these miRNAs, it is not clear which of these are related to colorectal cancer. Evaluation of over 6000 genes leads to a very non-specific assessment of potential functionality. To better focus on functionality we attempted to cluster genes together into what could be potentially a more common function and we also assessed seed regions. Using a cluster analysis approach, we still had over fifty pathways that also lacked specificity to colorectal cancer. Focusing on seed regions, we were left with 73 seed regions and did not minimize the potential number of genes that could be associated with *TERT*. Thus, we took an approach that focused only on those genes where the gene targeted by the miRNA was linearly associated with the corresponding mRNA expression in colorectal cancer. This reduced the number of genes and pathways considerably and was specific to colorectal cancer. However, while we gained specificity, we undoubtedly were limited in the target genes we assessed. We believe that the pathway information obtained is meaningful, but most certainly not complete. These data do however support the hypothesis that TERT has non-TL-related pathways \[[@pone.0162077.ref029], [@pone.0162077.ref030]\] that may be acting through altered miRNA expression.
In addition to its established role in telomere maintenance, it has been proposed that TERT operates through other pathways. Further examples of TERT involvement in non-TL related pathways include both NFκB complex and the Wnt/β-catenin pathways, which have been hypothesized as being telomerase-targeted pathways as both are pathways related to inflammation \[[@pone.0162077.ref005]\]; these pathways were both represented by our target genes in IPA although they are not shown in [Fig 1](#pone.0162077.g001){ref-type="fig"}. Most notably, the NFκB-signaling pathway activation has been suggested as being related to TERT \[[@pone.0162077.ref031]\]. TERT levels also have been associated with differential expression of genes involved in development/morphogenesis, signal transduction, and cell adhesion-signaling pathways \[[@pone.0162077.ref032], [@pone.0162077.ref033]\]. Our data also suggest that associations between *TERT* and colorectal cancer, possibly mediated by miRNAs, involve numerous pathways other than telomerase. These pathways involve tumor suppressor genes (such as PTEN signaling), apoptosis (PI3K/AKT signaling, NGF signaling, and FLT3 signaling), angiogenesis (Erythropoietin signaling), immune regulation and response (Prolactin signaling and Aryl Hydrocarbon Receptor), cell migration and proliferation (Neuregulin signaling), and growth factor activity (NGF signaling and EIF4 and p70S6K signaling). Another enriched pathway, Tight Junction Signaling, is important in cellular communication and facilities crosstalk across several of these signaling pathways including PI3K/AKT and PTEN \[[@pone.0162077.ref034]\].
Additionally, specific genes such as *STAT5A*, *MELK*, *MAPK1* and *MAP3K9*, *RELA* (part of the NFKB1 complex), *CDK1*, tumor necrosis factor genes, *IRF1*, and TGFβ-related genes were associated with miRNAs differentially expressed by *TERT* genotype. A number of translocations associated with cancer involve the Ig heavy chain locus on chromosome 14. For example t(8;14) brings c-MYC from chromosome 8 to the IgH gene locus causing an overexpression of c-MYC in Burkitt lymphoma. A t(18;14) translocation moving Bcl-2 from chromosome 18 to the IgH locus on 14 is involved in follicular lymphoma. Also CCND1 is involved in a t(11;14) translocation bringing cyclin d1 to the IgH resulting in cell cycle dysregulation in Mantle cell lymphoma. Studies suggest that these cancers are associated with telomere length \[[@pone.0162077.ref035]--[@pone.0162077.ref038]\] as well as with miRNA levels \[[@pone.0162077.ref039]\]. We show that *TERT* regulation of miRNAs alters Bcl-2 and an enriched pathway associated with the dysregulated genes is chronic myeloid leukemia, which has overlapping genes with some of the lymphomas previously studied. Our data support the hypothesis that TERT is involved in disease processes that include non-TL-related pathways.
One of the major strengths of our study was the availability of miRNA expression data, SNP data, and mRNA data on a large population. We have previously analyzed these SNPs with both TL and colon cancer, which helps facilitate the interpretation of our data. Additionally, we applied a FDR to guide our interpretation of meaningful results. Larger samples would have enabled us to split the sample and replicate our findings with a second data set. While we are unable to replicate these findings in a second data set, we encourage others with similar data to undertake such analysis. There are many available tools to assist in identifying verified mRNA targets for miRNA. In this study we used several bioinformatics tools to better understand function of these miRNAs, however all bioinformatics tools have limitations as to the extent of the knowledge and the specificity of the information to specific miRNAs. To have a more focused assessment of pathways, we restricted our bioinformatics assessment to only those targeted genes significantly associated with miRNA. Although functionality data of specific miRNA and TERT associations are not available or feasible from data available to us, we believe that the information provided gives direction for more laboratory-based studies that can directly test these associations.
Conclusions {#sec010}
===========
In conclusion, our data suggest that miRNAs are involved in regulating TL. *TERT* appears to influence carcinoma/normal mucosa differential expression. Given the number of miRNAs associated with TL, *TERT* rs2736118 and ALT genes, our data also support the hypothesis that telomere-related genes, in addition to affecting TL, impact non-TL-related functions that can importantly influence cancer risk.
The contents of this manuscript are solely the responsibility of the authors and do not necessarily represent the official view of the National Cancer Institute. We would like to acknowledge Dr. Bette Caan and the Kaiser Permanente Medical Research Program for sample contributions, Brett Milash and the Bioinformatics Shared Resource of the Huntsman Cancer Institute and University of Utah for miRNA and mRNA bioinformatics data processing, Sandra Edwards for the data collection and management, Wade Samowitz for slide review, and Erica Wolff and Michael Hoffman for miRNA processing.
CRC
: Colorectal cancer
FDR
: False discovery rate
IPA
: Ingenuity pathway analysis
FPMRP
: Kaiser Permanente Medical Research Program
mRNA
: messenger RNA
miRNA
: microRNA
PTEN
: Phosphate and tensin homolog
PI3K
: Phosphatidylinositide 3-kinases
AKT
: Protein kinase B
QC
: Quality Control
qPCR
: Quantitative polymerase chain reaction
RNA-seq
: RNA sequencing
SNP
: Single nucleotide polymorphism
TERT
: Telomerase reverse transcriptase
TL
: Telomere length
T/S ratio
: Telomere-to-single copy gene
[^1]: **Competing Interests:**The authors have declared that no competing interests exist.
[^2]: **Conceptualization:** MLS.**Data curation:** JH.**Formal analysis:** JH LM.**Funding acquisition:** MLS.**Investigation:** MLS RW.**Methodology:** MLS JH.**Project administration:** MLS.**Resources:** MLS.**Software:** JH.**Supervision:** MLS.**Validation:** JH LM.**Visualization:** MLS LM.**Writing -- original draft:** MLS AP LM.**Writing -- review & editing:** MLS JH AP LM.
|
{
"pile_set_name": "PubMed Central"
}
|
I[NTRODUCTION]{.smallcaps} {#sec1-1}
==========================
Colonic atresia (CA) is a rare cause of neonatal intestinal obstruction with an average of 1--2 reported cases per year in a tertiary care center worldwide. The incidence is approximately 1.8%--5% of intestinal atresia with an increased preponderance in males.\[[@ref1][@ref2][@ref3][@ref4][@ref5]\] Mortality and morbidity depends on early diagnosis and urgent management. It may be isolated or associated with other congenital anomalies such as malrotation, atresia of small bowel, cleft lip, Meckel\'s diverticulum, and Hirschsprung\'s disease (HD).\[[@ref1][@ref2][@ref3][@ref4][@ref5]\] Till date, there have been many studies but management of CA is challenging due to its rare and unusual presentation.
The purpose of this study was to highlight the rarity of this disorder and its associated anomalies and our objective was to review our experience in the management of CA with respect to staged surgery versus one step procedure for a better outcome of the disease.
S[UBJECTS AND]{.smallcaps} M[ETHODS]{.smallcaps} {#sec1-2}
================================================
The study was performed in the Department of Paediatric Surgery, Nil Ratan Sircar Medical College, Kolkata, India, from October 2013 to 2017. A total of 11 cases of CA were referred to this center, of which 9 cases were operated. Complete demographic data including age on presentation, sex, body weight, associated anomalies, and intraoperative findings including type of atresia, site of atresia, procedure performed colostomy or resection anastomosis, postoperative complications, hospital stay, and outcome were noted. Clinical examination was complete and routine blood investigations including complete hemogram, renal function test, liver function test, serology, straight X-ray abdomen, echocardiography, and contrast enema was done. Patients with severe sepsis or unfit for anesthesia were excluded from the study.
Age at presentation was considered at the time when the baby reached our referral center. Associated anomalies were noted by clinical examination, investigations, and intraoperative findings. Intraoperative finding of CA was classified according to the Grosfeld Classification: Type I atresia with a mucosal defect, Type II fibrous cord connecting the atretic ends, Type IIIa atresia with a V-shaped mesenteric gap defect, Type IIIb apple-peel deformity, and Type IV multiple atresia.\[[@ref6][@ref7]\]
All the neonates that reached our center were immediately resuscitated with intravenous fluid, antibiotics, and nasogastric suction. Electrolyte imbalance, metabolic status, and dehydration were corrected before operation. Out of 9 patients, 2 patients presented on the 4^th^ day with features of systemic inflammatory response syndrome and hence were excluded from contrast enema. While remaining 7 patients contrast enema was performed. Contrast enema revealed distal microcolon and the site of atresia as cutoff sign \[Figure [1c](#F1){ref-type="fig"} and [d](#F1){ref-type="fig"}\]. Plain abdominal X-ray revealed multiple air-fluid level and dilated bowel loops indicating a distal intestinal obstruction \[Figure [1a](#F1){ref-type="fig"} and [b](#F1){ref-type="fig"}\]. Echocardiography revealed associated cardiac anomalies.
{#F1}
After complete resuscitation, the patient was shifted to operation theater usually within 24 h of admission. Laparotomy was performed through right upper transverse incision. Out of 9 CA, six patients underwent initial double-barrel colostomy or ileostomy and biopsy from the distal colon and rectum was done and planned for definitive surgery after 4 weeks based on the biopsy report. Three cases underwent primary resection anastomosis and biopsy of the distal colon and rectum was done.\[[@ref8][@ref9][@ref10][@ref11]\] Biopsy was taken as full thickness up to 1 cm of the distal colon and resection up to 10 cm proximal dilated colon was done. Patency of the distal bowel was checked with normal saline. The time interval from reporting to our hospital to time of surgical intervention defined as time gap in hours was also noted \[[Table 1](#T1){ref-type="table"}\].
######
Management and outcome of colonic atresia
Postoperative patient was maintained on nasogastric suction, intravenous fluid, nutrition, and antibiotics. For those patients with a diverting stoma, feeding was started within 24 h. Patients with primary anastomosis feeding were delayed until there was passage of stool. The length of hospital was 12--27 days for those with primary anastomosis. Parenteral nutrition was added from day 2 for patient with primary anastomosis.
R[ESULTS]{.smallcaps} {#sec1-3}
=====================
A total of 11 cases were referred to our center over 4 years which were included in the study. Two cases of suspected CA based on radiological finding and clinical examination were excluded due to severe sepsis or unfit for anesthesia. Out of 9 cases, 6 were male and 3 were female (ratio 2:1). Clinical presentation was similar in all cases with characteristics radiological findings of distal microcolon and site of atresia in contrast enema and multiple air-fluid levels in plain abdominal X-ray. Mean age at presentation was 2 days and mean body weight was 2.1 kg \[[Table 2](#T2){ref-type="table"}\].
######
Demographic data of different types of colonic atresia
Associated anomalies are listed in \[[Table 2](#T2){ref-type="table"}\]. Most common findings were malrotation cardiac anomalies and HD. There were 3 cases of malrotation and 2 cases of HD and patient with atrial septal defect. Meckel\'s diverticulum was seen in 1 case \[[Figure 1d](#F1){ref-type="fig"}\] and unilateral cleft lip in 1 case. Operative findings and classification of atresia are shown in [Table 2](#T2){ref-type="table"}. Most common site of atresia in our series was in the transverse colon followed by ascending colon and sigmoid colon. Type IIIa was seen as the most common type of presentation. Type I atresia were seen in 2 cases and Type II atresia in 1 case \[[Table 2](#T2){ref-type="table"}\].
The decision of single-step procedure or initial stoma was made during the operation based on the patient condition and intraoperative findings. Three patients underwent primary resection and anastomosis after confirming the distal patency without any diverting stoma. Out of the remaining 6 patients, 5 underwent double-barrel colostomy and 1 underwent ileocolostomy irrespective of distal patency. Patient in which ileocolostomy was done had an ileal perforation which was detected intraoperative \[[Figure 2d](#F2){ref-type="fig"}\]. The mean time interval from reporting to the time of operation was 12.8 h.
{#F2}
Out of 9 cases, 2 cases showed the absence of ganglion cells with hypertrophied nerve bundles both in biopsy from the distal colon and rectum. Of these 2 cases, 1 had initial double-barrel colostomy and further underwent endorectal pull-through procedure (ERPT). Remaining 1 patient with initial resection anastomosis had anastomotic leak, underwent diverting colostomy but failed to survive due to sepsis. Closure of stoma was performed for patient with negative biopsy report after performing a distal colostogram. In one case with Type II sigmoid atresia, the distal rectum was inadequate for anastomosis and hence underwent ERPT procedure \[[Table 1](#T1){ref-type="table"}\].
Overall average hospital stay was 3.5 days for patients underwent initial colostomy and 21 days for patients underwent primary resection anastomosis. Postoperative complications included mainly wound dehiscence in 4 patients and anastomotic leak and sepsis in 2 patients \[[Table 1](#T1){ref-type="table"}\].
D[ISCUSSION]{.smallcaps} {#sec1-4}
========================
CA has been a rare disorder since Binninger first reported a case of CA in 1673. The first survivor was a case of sigmoid atresia reported in 1922 by Gaub.\[[@ref5][@ref6][@ref12]\] Since then, several studies have been published regarding type of atresia, associated anomalies, management with primary anastomosis, or staged procedure to reduce the mortality and morbidity. However, none could withstand a definitive guideline.
The cause of CA has been attributed to intrauterine mesenteric vascular accident secondary to thromboembolic event, volvulus, and strangulation of small bowel or lack of recanalization of the solid cord stage of intestinal development. CA as a result of choledochal cyst due to extrinsic mesenteric pressure effect has been reported earlier.\[[@ref12][@ref13][@ref14][@ref15]\] In our study, we did not find any intrauterine pressure effect as etiology to CA. CA may be an isolated anomaly or associated with other congenital anomalies association with malrotation, anorectal malformation, gastroschisis, HD, limb malformation, and choledochal cyst has been reported.\[[@ref1][@ref2][@ref3][@ref4][@ref5][@ref14][@ref15][@ref16][@ref19]\] In our study, we found association with malrotation in 3 cases, HD in 2 cases, unilateral cleft lip in 1, and cardiac anomaly 2 patient and Meckels diverticulum in 1 patient. Presence of anomalies alters overall mortality. In our study, we had one death due to postoperative anastomotic leak and sepsis and 1 neonate with associated HD and cardiac anomaly died secondary-to-postoperative sepsis.
Previous case series have reported Type III CA as the most common type and the right colon as the most common location.\[[@ref1][@ref2][@ref3][@ref4][@ref5][@ref6]\] The findings corroborated with our study, we found Type IIIa atresia in 6 cases (66.5%) and Type I in 2 cases (22.2%) and Type II in one case. Furthermore, most of the atresia was found proximal to splenic flexure \[[Figure 2](#F2){ref-type="fig"}\].
The diagnosis of CA has been dicey with patients without abdominal distension and without multiple air-fluid levels in X-ray. Moreover, delay in diagnosis increased the risk of mortality.\[[@ref1][@ref2][@ref15][@ref19]\] Hence, a high index of suspicion and contrast enema is essential for an early diagnosis of CA for patient who failed to pass meconium within 48 h. We performed contrast enema in seven patients with CA and found characteristics cutoff sign \[Figure [1c](#F1){ref-type="fig"} and [d](#F1){ref-type="fig"}\]. However, in four patients, this investigation was avoided due to sepsis and also their classical features of intestinal obstruction could be revealed clinically and in plain abdominal X-ray.
The time of presentation has been important for CA till the time of intervention. This has been highlighted in several series and case reports earlier.\[[@ref1][@ref2]\] In our study, we found two patients which presented on 6^th^ day and was in severe sepsis and unfit for anesthesia. Plain abdominal X-ray could only be obtained suggestive of distal intestinal obstruction. Average age of presentation was 2 days and mean interval for intervention was 12.8 h from time of presentation. Survival rate in our study was 77.7%.
Primary colo-colic anastomosis for atresia in the right colon and diverting colostomy for the left colon has been suggested by many studies earlier.\[[@ref1][@ref3][@ref4][@ref5][@ref16][@ref17][@ref18][@ref19][@ref20][@ref21][@ref22][@ref23][@ref24]\] Dalla Vecchia *et al*. reported null mortality in 21 CA and in 3 out of 21 with primary one-stage anastomosis approach to CA.\[[@ref15]\] Dassinger *et al*. found null mortality in 10 out of 12 cases with primary anastomosis.\[[@ref18]\] Karnak *et al*. reported failure in three cases of primary anastomosis.\[[@ref2]\] Both staged procedure and primary anastomosis was described by Pohlson *et al*.\[[@ref17]\] In our experience, we performed primary anastomosis in three cases and postoperative leak was found in two patients. These patients were later diagnosed as HD in one and another had leak probably due to poor nutritional status and sepsis or delayed return of bowel activity. Several reports of intestinal atresia stated that proximal dilated bowel lack interstitial cells of Cajal and abnormality of intestinal musculature due to dilation results in delayed return of bowel activity.\[[@ref25][@ref26]\] In our study, postoperative recovery in one patient with primary anastomosis was delayed by 21 days. However, patients with primary diverting stoma survived and were discharged early. Furthermore, there was no mortality seen in these patients who followed up for definitive surgery.
In our study, overall mortality was 22.2%. Etensel *et al*. in review of literature in 214 cases found an overall mortality rate 25.6% and in case of CA, HD must be ruled out.\[[@ref1]\] Mortality is also higher with delayed presentation and associated anomalies. Hence, we support the formal dictum as early presentation of CA within 72 h reduces the mortality rate by more than 50%. Park in his case report stated that association of CA and HD is a diagnostic challenge and attempt to primary anastomosis results in failure with leakage and sepsis.\[[@ref19]\] In our study, wound dehiscence was noted in four patients who may be probably due to prematurity and poor nutritional status.
C[ONCLUSION]{.smallcaps} {#sec1-5}
========================
CA is a very rare disorder that possesses a great challenge in diagnosis and management. Awareness for the condition is of immense importance. Contrast enema is a key to the diagnosis and must not be delayed where there is a doubt and the patient is stable. Associated anomalies must be given priority to avoid mortality. Staged procedure with initial colostomy and followed by definitive procedure is the preferred choice irrespective of location of atresia. All cases of CA must be biopsied from distal part of the colon and rectum to rule out HD as an associated anomaly. Further detailed studies are required based on histology and immunohistochemistry of intestine to decide the level of resection of the proximal dilated colon and distal colon to achieve early bowel activity and reduce the morbidity.
Financial support and sponsorship {#sec2-1}
---------------------------------
Nil.
Conflicts of interest {#sec2-2}
---------------------
There are no conflicts of interest.
|
{
"pile_set_name": "PubMed Central"
}
|
INTRODUCTION
============
Hypospadias is defined as an ectopic urethral meatus on the ventral aspect of the penis, scrotum, or perineum with or without ventral penile curvature, and estimates of its prevalance range from 3 to 8 cases per 1,000 male births \[[@B1]-[@B3]\]. Its etiology is incompletely understood. Hypospadias probably has a multifactorial origin that involves the actions of environmental factors and endocrine-active compounds against a genetic backdrop, although few molecular and mechanistic studies have been done \[[@B4],[@B5]\].
Activating transcription factor 3 (ATF3) is a member of the ATF/cAMP responsive element binding (CREB) family \[[@B6]\]. ATF3 may be involved in homeostasis, wound healing, cell adhesion, cancer cell invasion, apoptosis, and signaling pathways \[[@B7],[@B8]\]. Overexpression of ATF3 protein suppresses cell growth and slows the transition of cells from G1 to S phase. This evidence suggests that the ATF3 protein may play a negative role in cell cycle progression \[[@B9],[@B10]\]. Microarray results indicate that ATF3 is one of the genes that is upregulated in hypospadiac tissue \[[@B11],[@B12]\].
In this study, the role of expression of ATF3 protein was investigated in the etiology of hypospadias.
MATERIALS AND METHODS
=====================
Following institutional review board approval, informed consent was received from parents. Twenty-five males aged between 5 and 9 years (mean: 7.4 years) with hypospadias who were scheduled to undergo surgical repair were prospectively entered into the study from December 2008 to June 2009. Patients with an undescended testis, intersex condition, or known endocrine abnormalities; who had undergone testosterone replacement therapy preoperatively; or who had a family history of hypospadias or a maternal age higher than 35 at the time of birth were excluded from the study. The position of the urethral meatus was assessed according to the Ducket clasification by a single surgeon \[[@B13]\]. Excess prepucial tissue was obtained from ethnically comparable boys at the time of elective surgery for hypospadias. Controls consisted of 26 patients aged between 6 and 11 years (mean: 8.3 years) undergoing elective circumsicion. Tissue specimens were prepared for parafin-embedded or frozen sectioning. We included only cases from Turkey and controls living in the same area.
1. Antibodies and immunohistochemical techniques
------------------------------------------------
Skin specimens were evaluated for the expression of ATF3 protein by immunohistochemical staining by use of the standard protocol accompanying the ABC kit (Vector Laboratories, Inc., CA, USA) with overnight primary-antibody incubation with rabbit-anti-ATF3 (Santa Cruz Biotechnology, Inc., CA, USA) at a dilution of 1:400. Control sections were incubated without primary antibody. Specimens were examined under light microscopy with hemotoxylen dye by two independent pathologists blinded to the specimens.
Nuclear ATF3 immunoreactivity was considered abnormal when samples demonstrated more than 20% nuclear reactivity. If the immunoreactivity of the specimen was greater than 50%, it was recorded as strong intensity by the agreement of two observers.
2. Statistical analysis
-----------------------
Fisher\'s exact two-tailed tests were used to compare the frequency data of the two groups. Statistical significance was assigned at p\<0.05. To evaluate inter- and intraobserver agreement on immunohistochemical analysis, Cohen kappa statistical analysis was performed by using SPSS for Windows version 15.0 (SPSS Inc., Chicago, IL, USA).
RESULTS
=======
Forty-six patients were enrolled in this study. Five of the 25 hypospadiac patients were excluded because of a history of previous orchidopexy, testosterone replacement therapy, or diagnosed concomitant undescended testis. The location of the meatus in the hypospadiac patients is presented in [Table 1](#T1){ref-type="table"}. None of the hypospadiac patients had a proximally located meatus ([Table 1](#T1){ref-type="table"}).
ATF3 protein was localized in the nuclei of stromal cells and the vascular endothelium in the subcutaneous tissue of the prepuce. All of the patients had dorsal prepitual skin. Of the 46 specimens examined by immunohistochemistry, 16 (80%) of the 20 patients with hypospadias showed expression of ATF3 protein, and only 3 (11%) of the 26 patients undergoing circumcision stained positive (p\<0.05) ([Table 2](#T2){ref-type="table"}). Intra- and interobserver agreement was excellent with kappa (Cohen statistics) values of 0.88 and 0.80, respectively.
The immunoreactivity of expression of ATF3 protein in normal tissue and in a hypospadiac sample are shown in [Fig. 1](#F1){ref-type="fig"}. The staining intensities were 6.9 times as strong in patients in the hypospadiac group as in the control group (RR: 6.933, 95% CI: 2.3-11.2). The staining intensities were found to be similiar in patients with distal and middle hypospadias (p\>0.05) ([Table 3](#T3){ref-type="table"}). Strong staining intensities were found in 2 patients with a meatus located midshaft, whereas strong staining intensities were found in 2 patients with a distally located urethral meatus.
DISCUSSION
==========
The development of the male external genitalia is a complex process, comprising genetic programming, cell differentiation, hormonal signaling, enzyme activity, and tissue remodeling, which follows an orderly sequence, occurring in a time- and concentration-dependent manner \[[@B14]\]. Any disturbance in these processes can lead to hypospadias. The cause of hypospadias is largely unknown, however. Several etiologies for hypospadias have been suggested, including genetic, endocrine, and environmental factors \[[@B4],[@B5]\].
There are conflicting results in the literature about the association between ATF3 and hypospadias. The first human study to demonstrate a relationship between ATF3 and hypospadias was performed by Liu et al \[[@B15]\]. Immunohistochemical analysis of human foreskin demonstrated that 86% of the hypospadias samples were positive for the expression of ATF3, whereas only 13% of those from normal tissues were positive in their study. Beleza-Meireles et al analyzed DNA from 330 boys with nonsyndromic hypospadias \[[@B16]\]. ATF3 as a hormone-responsive gene has been shown to be related to the etiology of hypospadias. However, it is a well-known fact that most associations reported in genetic studies do not replicate across subsequent studies \[[@B17]\]. van der Zanden et al were unable to replicate the results of earlier studies \[[@B18]\]. Analyzed populations in mentioned studies were indeed dissimilar, and some of the populations even differed in ethnicity. We included only a Turkish population in this study.
This study demonstrated that ATF3 protein was overexpressed in an ethnically similiar hypospadiac pediatric age group. Conventional manual immunohistochemical methods were used because of cost, time, and space considerations to detect abnormal immunoreactivity. The cutoff point was chosen as 20% for abnormal immunoreactivity because sequential testing of different cutoffs from 0% to 95% in 5% increments revealed that 20% was the best cutoff for abnormal protein expression. The staining intensities appeared to be stronger in patients with more severe hypospadias than in those with mild hypospadias. All patients with a midshaft-located urethral meatus showed stronger intensities, whereas 3 of 18 distal hypospadiac patients showed strong intensities of ATF3 protein expression.
Most of the hypospadiac patients in this study were recorded as having a mild degree of hypospadias, and there were no patients with a proximally located meatus. The staining intensities were found to be stronger in patients in the hypospadiac group than in the control group. ATF3 overexpression may have been related to the severity of hypospadias in our study. None of the hypospadiac patients with a coronal or subcoranal meatus showed strong staining intensities. However, all of the hypospadiac patients with a midshaft-located meatus showed strong staining intensities.
There may be a difference in the etiology of hypospadias in different populations. A growing body of evidence suggests that the development of hypospadias has a two-hit etiology involving a genetic predisposition coupled with fetal exposure to an environmental disruptor \[[@B14]\]. Endocrine-related genes, namely androgen receptor gene (AR), encoding estrogen receptor 1 (ESR1) and ATF3, have been associated with hypospadias in previous studies \[[@B16],[@B19],[@B20]\]. However, the numbers of samples analyzed in these studies were relatively small. In a large study population, four single-nucleotide polymorphism genes including ATF3 were investigated by van der Zanden et al \[[@B18]\]. They were unable to show an association between hyospadias and ATF3. Differences in results between their and previous studies may also have been caused by disease heterogeneity due to differences in the criteria used to select the cases. In this study, we strictly excluded factors suspected to be of relevance in the etiology of hypospadias. Population stratification, which occurs when cases and controls are drawn from different subgroups that differ in disease prevalence and frequency of the genetic variant, may lead to spurious results. We selected only cases and controls living in the same area in Turkey to minimize the chance of population stratification.
The current study is not devoid of limitations. The power of our conclusion may be somewhat limited by the small study population. The clinical application of immunohistochemistry has been limited by discrepancies related to variability in the interpretation and stratification criteria and inconsistency in specimen handling and technical procedures. However, two blinded pathologists graded the blocks to minimize interpreter-related variability in this study. An automated immunostaining (ACIS) platform and quantitative imaging may also help to minimize technique-related variability, but the major drawback to this technology is the apparent lack of an agreed-on or clearly described standard ACIS scoring method.
CONCLUSIONS
===========
Our results indicate that ATF3 is up-regulated in the penile skin tissues of boys with hypospadias, which suggests a role for this transcription factor in the development of this abnormality.
We are greateful to Dr. Hamit Okur for providing excellent collaboration between our Department and the Department of Pediatric Surgery.
This work was supported by grants from the Health Ministry, Istanbul Goztepe Training Hospital Medical Research Foundation.
The authors have nothing to disclose.
{#F1}
######
Clinical features of all patients with hypospadias
######
Expression of ATF3 protein
ATF3: activating transcription factor 3, IHC: immunohistochemical
######
Expression of ATF3 protein in hypospadiac samples
ATF3: activating transcription factor 3, IHC: immunohistochemical
|
{
"pile_set_name": "PubMed Central"
}
|
Introduction
============
Primary hyperparathyroidism is the most common cause of hypercalcemia[@ref1] and the third most common endocrine disorder, with 100 000 new cases in the United States each year.[@ref2] Up to 2% of postmenopausal women could have this condition.[@ref3] [@ref4] Primary hyperparathyroidism is characterized by hypercalcemia with a high or insufficiently suppressed level of parathyroid hormone, and is caused by a solitary parathyroid adenoma in 85-90% of patients.[@ref5] [@ref6] Associated morbidities and costly sequelae include decreased bone mineral density, fractures, and kidney stones.[@ref7] [@ref8] However, little is known about risk factors for primary hyperparathyroidism.[@ref9] Monogenic disorders (such as multiple endocrine neoplasia I and II)[@ref10] account for fewer than 5% of cases,[@ref11] and neck irradiation also accounts for only a small fraction of cases.[@ref12]
Because the parathyroid adenoma of sporadic primary hyperparathyroidism is monoclonal,[@ref13] [@ref14] [@ref15] factors that chronically stimulate parathyroid hormone and increase the probability of a parathyroid cell undergoing a somatic mutation and subsequent clonal proliferation could increase the risk for primary hyperparathyroidism.[@ref11] [@ref14] [@ref16] Calcium intake is known to influence parathyroid hormone level[@ref17] [@ref18] [@ref19] [@ref20] [@ref21] [@ref22] and therefore could be important in the pathogenesis of primary hyperparathyroidism. However, no study to date has prospectively explored the relation between calcium intake and risk of developing primary hyperparathyroidism.
To examine the association between calcium intake and the risk of incident primary hyperparathyroidism, we conducted a prospective study of 58 354 women in the Nurses' Health Study I without history of primary hyperparathyroidism at baseline.
Methods
=======
Study population
----------------
The Nurses' Health Study I is an ongoing, prospective cohort study which began in 1976, enrolling 121 700 female registered nurses between 30 and 55 years of age and residing in 11 US states. The cohort is followed by using questionnaires mailed every two years that ask about lifestyle practices and newly diagnosed diseases. The average proportion of participants followed up has been more than 90%. The sample for our analysis was limited to the 58 354 women who answered either the 2006 or 2008 questionnaires, which included questions on lifetime history of primary hyperparathyroidism. The study protocol was reviewed and approved by the Brigham and Women's Hospital institutional review board.
Assessment of dietary intake
----------------------------
To assess the participants' diet, we used semiquantitative questionnaires on food frequency that asked about the average intake of more than 130 individual food items and 22 individual beverages during the previous year. The participants were asked to complete food frequency questionnaires in 1986, 1990, 1994, 1998, 2002, and 2006. Intake of specific dietary factors was computed from the reported frequency of consumption of each specified unit of food and from US Department of Agriculture data on the content of the relevant nutrient in specified portions. Nutrient values were adjusted for total energy intake to determine the nutrient composition of the diet independent of the total amount of food eaten. The food frequency questionnaire also asked about the use of calcium supplements, vitamin D supplements, and multivitamins. The intake of supplemental calcium, vitamin A, and vitamin D in multivitamins or in isolated form were determined by the brand, type, and frequency of reported use.
The food frequency questionnaire has been extensively validated.[@ref23] In a sample of 173 participants in the Nurses' Health Study I, nutrient intake reported on two food frequency questionnaires was compared with four seven-day records kept by the nurses who weighed and measured everything they ate or drank.[@ref24] The values for the nutrient data on the two food frequency questionnaires and the food diaries were highly correlated, and the degree of reproducibility was not modified by obesity or other personal characteristics. For example, correlation coefficients were 0.81 for skim or low fat milk and 0.94 for yogurt.[@ref24]
Assessment of non-dietary factors
---------------------------------
Age, body mass index, smoking status (never, past, current), physical activity (in metabolic equivalent task scores), history of diabetes, hypertension, diuretic use, menopausal status, and postmenopausal hormone use were ascertained from the biennial questionnaires. Self reported weight was highly reliable (*r*=0.97) among a subset of participants who underwent direct measurement of their weight.[@ref25] Physical activity reported on the questionnaires has been previously validated against physical activity diaries (*r*=0.79).[@ref26] Self reported hypertension[@ref27] and diabetes[@ref28] were previously validated in this cohort. Self reported age at menopause and type of menopause were previously validated in the Nurses' Health Study I and shown to be highly accurate.[@ref29] Race was self reported and categorized in this analysis as white and non-white.
Assessment of cases
-------------------
Participants were asked about a diagnosis of hyperparathyroidism on the 2006 and 2008 questionnaires. On the 2008 questionnaire, nurses were also asked about lifetime history of hyperparathyroidism. To identify primary (*v* secondary) hyperparathyroidism, we subsequently obtained the medical records of all participants who gave consent. We confirmed cases of primary hyperparathyroidism by pathology report of a resected adenoma or by elevated serum concentrations of calcium (≥10.6 mg/dL; 1 mg/dL=0.25 mmol/L) with high or insufficiently suppressed parathyroid hormone[@ref30] [@ref31] [@ref32] [@ref33] [@ref34] (≥50 pg/mL; 1 pg/mL=1 ng/L). The medical record confirmation rate was 75%. Cases were rejected after medical record review for a variety of reasons, most commonly incomplete data or identification of secondary hyperparathyroidism from vitamin D deficiency or renal insufficiency.
We included in the analysis only cases of primary hyperparathyroidism that were diagnosed during the 22 years between the date on which the 1986 questionnaire was returned and 31 May 2008. Participants with a history of primary hyperparathyroidism at baseline were excluded from the study.
Statistical analyses
--------------------
The study design was prospective; information on diet was collected before the diagnosis of primary hyperparathyroidism. For each participant, we counted person months of follow-up from the date on which the 1986 questionnaire was returned to the date on which primary hyperparathyroidism was diagnosed or death occurred, or 31 May 2008, whichever occurred first. Information on exposures of interest that was recorded in response to the 1986 questionnaire was updated on subsequent questionnaires. We allocated person time of follow-up according to exposure status at the start of each follow-up period. Dividing the cohort into five equal groups of nutrient intake allowed us to examine a wide range of nutrient intake while maintaining enough participants in the highest and lowest categories. If complete information on diet was missing at the start of a time period, the participant was excluded from that time period.
The relative risk---the incidence among women in a particular category of intake divided by the corresponding rate in the comparison category---was used as the measure of association. We used the Mantel extension test to evaluate linear trends across categories of calcium intake. We used a proportional hazards model to simultaneously adjust for several risk factors. The variables considered in these models were age; body mass index (\<22, 22-24.9, 25-29.9, and ≥30); race (white or non-white); physical activity level (divided into five equal groups of differing levels); alcohol intake (none, 0.1-4.9, 5-14.9, ≥15 g/day); use of thiazide or loop diuretics (yes or no); supplemental calcium intake (none, 1-500, \>500 mg/day); supplemental vitamin D intake (none, 1-400, \>400 IU/day); dietary intakes of calcium, vitamin D, vitamin A, and protein (total and animal protein intake); self reported diabetes; self reported hypertension; menopausal status; postmenopausal hormone use; and physical exam during the prior two years. Owing to the high correlation between the dietary intakes of calcium and phosphorus (*r*≥0.68 for all follow-up periods), we excluded phosphorus from the multivariable models. We calculated 95% confidence intervals for all relative risks. All P values were two tailed.
Results
=======
Dietary calcium intake
----------------------
During 1 475 978 person years of follow-up over a 22 year period, we confirmed 277 cases of incident primary hyperparathyroidism. Table 1[](#tbl1){ref-type="table"} shows the characteristics of the cohort, divided into five equal groups according to energy adjusted intake of dietary calcium in 1986. For our analyses, however, the updated dietary values were used for each time period. The mean daily intake of vitamin D, magnesium, total protein, animal protein, and vitamin A, and the physical activity level increased with increasing dietary calcium intake. The average daily alcohol intake and the percentage of current smokers decreased with increasing calcium intake. The percentage of nurses with self reported diabetes or hypertension or who were taking thiazide diuretics and the mean daily intake of supplemental calcium was similar across the five groups.
######
Age standardized baseline characteristics of women according to energy adjusted intake of dietary calcium in 1986
Dietary calcium intake
-------------------------------------------------------------- ------------------------ ----------- ----------- ----------- -----------
Age (years)\* 51.3 51.4 51.7 51.8 52.4
Body mass index 25.0 25.1 25.2 25.3 25.2
Physical activity (MET/week) 11.9 13.9 14.6 15.4 16.1
Dietary calcium intake (mg/day)† 431 564 672 811 1115
Calcium supplement intake (mg/day) 352 351 360 357 335
Calcium supplement use (% (No)) 55 (6351) 56 (6654) 58 (6833) 58 (6775) 55 (6370)
Total (dietary and supplemental) vitamin D intake (IU/day)† 254 287 322 366 465
Magnesium intake (mg/day)† 265 285 300 314 334
Total protein intake (gm/day)† 70.1 72.5 74.4 76.8 81.3
Animal protein intake (gm/day)† 50.3 51.7 53.5 56.2 62.1
Total (dietary and supplemental) vitamin A intake (mcg/day)† 1842 2051 2205 2329 2493
Alcohol intake (gm/day) 8.8 6.7 5.8 5.0 4.0
Smoking status (% (No))
Never smoker 43 (4955) 46 (5374) 46 (5458) 47 (5470) 50 (5760)
Past smoker 33 (3776) 34 (4068) 37 (4358) 37 (4375) 35 (4052)
Current smoker 24 (2758) 20 (2366) 17 (2002) 16 (1895) 15 (1686)
Self reported diabetes (% (No)) 2 (247) 2 (258) 2 (281) 3 (349) 3 (352)
Self reported hypertension (% (No)) 24 (2722) 22 (2620) 22 (2615) 23 (2648) 22 (2584)
Thiazide use (% (No))‡ 13 (1517) 12 (1392) 12 (1459) 12 (1450) 12 (1374)
Loop diuretic use (% (No))§ 1 (171) 2 (181) 2 (195) 1 (171) 2 (196)
Data are mean values, or the percentages (and numbers) of participants that are standardized to the age distribution of the study population. Study population divided into five equal groups according to intake of dietary calcium. MET=metabolic equivalent task scores.
\*Not adjusted by age.
†Energy adjusted.
‡From 1988.
§From 1994.
After adjusting for age, a higher intake of dietary calcium was associated with a reduced risk of primary hyperparathyroidism (table 2[](#tbl2){ref-type="table"}). The relative risk for women in the group with the highest intake of dietary calcium compared with women with the lowest intake was 0.61 (95% confidence interval 0.42 to 0.90, P=0.03 for trend).
######
Age adjusted and multivariable relative risks for incident primary hyperparathyroidism according to dietary and total calcium intake\*
Group 1 Group 2 Group 3 Group 4 Group 5 P for trend
--------------------------------------------- --------- --------------------- --------------------- --------------------- --------------------- -------------
Dietary calcium intake
Median within group (mg/day) 443 564 670 806 1070 ---
No of cases of primary hyperparathyroidism 69 57 57 50 44 ---
No of person years 290 985 296 872 298 068 297 109 292 944 ---
Age adjusted relative risk (95% CI) 1.0 0.81 (0.57 to 1.15) 0.80 (0.56 to 1.14) 0.70 (0.49 to 1.01) 0.61 (0.42 to 0.90) 0.03
Multivariable relative risk (95% CI)† 1.0 0.79 (0.55 to 1.13) 0.78 (0.54 to 1.11) 0.66 (0.45 to 0.98) 0.56 (0.37 to 0.86) 0.009
Total calcium intake
Median within group (mg/day) 522 737 999 1276 1794 ---
No of cases of primary hyperparathyroidism 86 61 43 42 45 ---
No of person years 289 554 294 850 297 322 298 933 295 321 ---
Age adjusted relative risk (95% CI) 1.0 0.69 (0.50 to 0.96) 0.48 (0.33 to 0.69) 0.45 (0.31 to 0.65) 0.48 (0.33 to 0.69) \<0.001
Multivariable relative risk (95% CI)‡ 1.0 0.64 (0.46 to 0.91) 0.42 (0.28 to 0.62) 0.39 (0.25 to 0.58) 0.41 (0.27 to 0.63) \<0.001
Population divided into five equal groups according to intake of calcium.
\*For illustrative purposes, medians within each group for intake of dietary and total calcium were derived from responses to the 1986 dietary questionnaire. However, the period specific medians were used for the 1986-2008 analysis. Relative risks are for the risk of primary hyperparathyroidism compared with the group that had the lowest intake of dietary or total calcium (that is, group 1).
†Multivariable model includes age, body mass index (categories: \<22, 22-24.9, 25-29.9, ≥30), race, smoking status (past, current, or never), calcium supplement intake, total vitamin D intake, dietary intakes of vitamin A and protein, alcohol intake, and diuretic use (thiazide or loop diuretic use).
‡Multivariable model includes age, body mass index (categories: \<22, 22-24.9, 25-29.9, ≥30), race, smoking status (past, current, or never), total vitamin D intake, dietary intakes of vitamin A and protein, alcohol intake (categories: none, 0.1-4.9, 5-14.9, ≥15 g/day), and diuretic use (thiazide or loop diuretic use).
After further adjusting for body mass index, race, smoking status, calcium supplement use, intake of vitamin D, dietary intake of vitamin A and protein, alcohol intake, and diuretic use, the adjusted relative risk for women in the group with the highest intake of dietary calcium compared with women with the lowest intake was 0.56 (95% confidence interval 0.37 to 0.86, P=0.009 for trend). The association between dietary calcium and risk of primary hyperparathyroidism was similar for calcium intakes not adjusted for energy.
Total calcium intake
--------------------
After adjusting for age, higher total daily intake of calcium (including dietary and supplemental calcium) was associated with a reduced risk of primary hyperparathyroidism (table 2). For women in the group with the highest intake of total calcium compared with women in the group with the lowest intake, the relative risk was 0.48 (95% confidence interval 0.33 to 0.69, P\<0.001 for trend), and the multivariable relative risk was 0.41 (0.27 to 0.63, P\<0.001 for trend).
Supplemental calcium intake
---------------------------
We also studied the relation between supplemental calcium intake and risk of primary hyperparathyroidism. Because of an insufficient number of cases of primary hyperparathyroidism among the different categories of supplemental calcium use in the earlier time periods, our analysis on the relation between supplemental calcium intake and primary hyperparathyroidism began in 1994 with follow-up until 2008. During 985 628 person years of follow-up over a 14 year period, we documented 257 cases of incident primary hyperparathyroidism.
Higher supplemental calcium intake was associated with a reduced risk of primary hyperparathyroidism (table 3[](#tbl3){ref-type="table"}). After adjusting for age, the relative risk for women taking more than 500 mg/day of calcium supplements compared with those not taking calcium supplements was 0.69 (95% confidence interval 0.50 to 0.94, P\<0.001 for trend). The multivariable relative risk for the same comparison, including adjustment for dietary calcium, was 0.41 (0.29 to 0.60, P\<0.001 for trend).
######
Age adjusted and multivariable relative risks for incident primary hyperparathyroidism according to supplemental calcium intake
Supplemental calcium intake (mg/day) P for trend
-------------------------------------------- -------------------------------------- --------------------- --------------------- ---------
No of cases of primary hyperparathyroidism 85 86 86 ---
No of person years\* 294 279 293 762 397 587 ---
Age adjusted relative risk (95% CI) 1.0 0.99 (0.73 to 1.34) 0.69 (0.50 to 0.94) \<0.001
Multivariable relative risk (95% CI)† 1.0 0.82 (0.59 to 1.15) 0.41 (0.29 to 0.60) \<0.001
\*Follow-up started in 1994 because of insufficient number of cases of primary hyperparathyroidism in participants taking supplemental calcium from 1984-93.
†Multivariable model includes: age, body mass index (categories: \<22, 22-24.9, 25-29.9, ≥30), race, smoking status (past, current, never), dietary calcium intake, total vitamin D intake, dietary intakes of vitamin A and protein, alcohol intake (categories: none, 0.1-4.9, 5-14.9, ≥15 g/day), and diuretic use (thiazide or loop diuretic use).
Additional analyses
-------------------
Since cases of primary hyperparathyroidism can be incidentally detected after routine measurement of serum chemistry panels,[@ref35] we performed analyses with additional adjustment for having a physical exam (yes or no) during the prior two years of each time period in our multivariable analysis. The association between increased calcium intake and lower risk of primary hyperparathyroidism was similar after adjusting for regular physical exams, and was similar in analyses restricted to women who had a physical exam during the prior two years of each time period.
Since the incidence of primary hyperparathyroidism seems to increase with age,[@ref36] [@ref37] we also performed stratified analysis by age (≥65 and \<65 years). The association between increased calcium intake and reduced risk of primary hyperparathyroidism was similar in older and younger women (P=0.21 for interaction); it was also similar after adjusting for menopausal status and use of postmenopausal hormone use. Since calcium absorption can depend on vitamin D status, we also performed analyses stratified by median total intake of vitamin D. The associations between higher calcium intake and decreased risk of primary hyperparathyroidism were similar among participants above and below this median level.
We also examined the association between cumulative calcium intake and risk of primary hyperparathyroidism, and the results were similar. Because we could not exclude the possibility that physicians stopped calcium supplements in participants with higher serum calcium concentrations shortly before primary hyperparathyroidism diagnosis, we also performed analyses with a four year lag between assessment of supplemental calcium use and primary hyperparathyroidism. In multivariable lag analyses, the relative risk of primary hyperparathyroidism was 0.79 (95% confidence interval 0.57 to 1.09) for women taking 1-500 mg/day of supplemental calcium and 0.71 (0.50 to 1.00) for those taking more than 500 mg/day of supplemental calcium, compared with women not taking supplemental calcium (P=0.12 for trend).
Discussion
==========
To our knowledge, we report results from the first prospective study of the relation between calcium intake and risk of primary hyperparathyroidism. In women, increased dietary and supplemental calcium intake was associated with a reduced risk for developing primary hyperparathyroidism, independent of age, body size, diet, and other factors.
Comparison with other studies
-----------------------------
Several studies have examined the effect of calcium intake in patients already diagnosed with primary hyperparathyroidism, with differing results.[@ref38] [@ref39] [@ref40] [@ref41] Insogna and colleagues[@ref39] found that calcium intake of 1000 mg/day compared with 400 mg/day suppressed the mean fasting level of parathyroid hormone by 19% among 18 study participants with primary hyperparathyroidism. Jorde and colleagues[@ref40] found that the level of parathyroid hormone decreased after four weeks in 17 of 24 participants with primary hyperparathyroidism who were given calcium supplementation. On the other hand, Locker and colleagues[@ref41] found no significant effect of dietary calcium intake on serum parathyroid hormone level among 71 participants already diagnosed with primary hyperparathyroidism. However, no study has prospectively examined the association between calcium intake and risk of developing primary hyperparathyroidism.
Limitations of the study
------------------------
There are several limitations to our study. Firstly, since our study population was female and almost entirely white, our findings are not necessarily generalizable to men or other races. Secondly, we cannot exclude selection bias. We only included cases confirmed by medical record review, and we could not obtain medical records for all women who self reported primary hyperparathyroidism. Thirdly, although the food frequency questionnaires have been well validated, calcium intake was not perfectly assessed in this study. However, because of the prospective design, any misclassification would be random with respect to case status, and therefore would probably underestimate the magnitude of the inverse association between calcium intake and risk of primary hyperparathyroidism. Fourthly, many cases of primary hyperparathyroidism may be asymptomatic and detected by routine blood work. However, in subanalyses restricted to women who had regular physical exams, the inverse relation between calcium intake and risk of developing primary hyperparathyroidism remained robust. Fifthly, since we conducted an observational study, there could be unknown confounders that we did not control for in our analysis. Finally, the magnitude of the association between higher supplemental calcium intake and lower risk of primary hyperparathyroidism was attenuated in lag analyses. Thus, we cannot exclude the possibility that some women with higher values of serum calcium on routine bloodwork were told to stop taking calcium supplements before their diagnosis of primary hyperparathyroidism.
Areas for future research
-------------------------
Calcium intake could have a role in the pathogenesis of primary hyperparathyroidism by influencing the production of parathyroid hormone.[@ref17] [@ref18] [@ref19] [@ref20] [@ref21] [@ref22] The monoclonal nature of the single parathyroid adenoma that causes the large majority of cases of primary hyperparathyroidism[@ref13] [@ref14] [@ref15] suggests a neoplasm that originates from single cells with a growth-conferring mutation. Because factors that cause parathyroid hyperplasia, such as lower calcium intake, increase the probability that a parathyroid cell will undergo a somatic mutation and subsequent clonal proliferation, such factors could increase the risk for developing primary hyperparathyroidism.[@ref11] [@ref14] [@ref16] The peak disease incidence occurring later in life also suggests that risk factors for primary hyperparathyroidism could be due to a chronic stimulus over time.[@ref42] Future research should examine other environmental and lifestyle risk factors that could chronically stimulate the parathyroid gland and thereby affect subsequent development of primary hyperparathyroidism.
### What is already known on this topic
1. Calcium intake is known to influence parathyroid hormone levels and therefore could be important in the pathogenesis of primary hyperparathyroidism
2. No study to date has prospectively explored the relation between calcium intake and risk of developing primary hyperparathyroidism
### What this study adds
1. Increased calcium intake, including both dietary and supplemental calcium, is independently associated with a reduced risk of developing primary hyperparathyroidism in women
We thank the participants in the Nurses' Health Study for their continuing cooperation. An abstract of this work was presented at the annual meeting of the American Society of Bone and Mineral Research on 19 September 2011.
Contributors: JP is the guarantor and takes responsibility for the integrity of the work as a whole, from inception to publication; contributed to the study conception and design, acquisition of data, analysis, interpretation of data, and drafting of the article; and approved the final version of the manuscript. GC contributed to the conception and design, interpretation of data, critical revisions of the article, and approved the final version of the manuscript. ET contributed to the conception and design, acquisition of data, interpretation of data, critical revisions of the article, and approved the final version of the manuscript.
Funding: This research was supported by the US National Institutes of Health grants DK084707, HL092947, DK91417, and CA087969.
Competing interests: All authors have completed the Unified Competing Interest form at [www.icmje.org/coi_disclosure.pdf](http://www.icmje.org/coi_disclosure.pdf) (available on request from the corresponding author) and declare: support from the US National Institutes of Health; no financial relationships with any organizations that might have an interest in the submitted work in the previous 3 years; no other relationships or activities that could appear to have influenced the submitted work.
Ethical approval: The institutional review board at the Brigham and Women's Hospital approved this study.
Data sharing: Requests for access to data, statistical code, questionnaires, and technical processes may be made by contacting the corresponding author at [jmpaik\@partners.org]([email protected]).
Cite this as: *BMJ* 2012;345:e6390
|
{
"pile_set_name": "PubMed Central"
}
|
Retraction {#S0001}
==========
We hereby inform to our readership of the retraction of the article *Emphysematous cystitis and emphysematous pyelitis: a clinically misleading association(doi:10.11604/pamj.2013.16.18.2505) by Mustapha Ahsaini, Amadou Kassogue, Mohammed Fadl Tazi, Anas Zaougui, Jalal Edine Elammari, Abdelhak Khallouk, Mohammed Jamal El Fassi and My Hassan Farih* of the Department of Urology, University Hospital, Center Hassan II, Fes, Morocco \[[@CIT0001]\]. From our investigation, it clearly appears that the majority of the contents of the article by Mustapha Ahsaini and al. published in PAMJ in September 17, 2013 was plagiarized from the article Emphysematous cystitis of the diabetic patient. 2009 Aug; 1(3): 114-116. PMCID: PMC3364639 by Affes Nejmeddine, Bahloul Atef, Dammak Youssef, Beyrouti Ramez, and Beyrouti Mohamed Issam, published in North American Journal of Medical sciences \[[@CIT0002]\]. Therefore, we withdraw this article of the medical literature in accordance with the guidelines and best editorial practices of the Committee on Publication Ethics. We apologize to our audience for this unfortunate situation.
|
{
"pile_set_name": "PubMed Central"
}
|
Multilingual abstracts {#Sec1}
======================
Please see Additional file [1](#MOESM1){ref-type="media"} for translations of the abstract into the five official working languages of the United Nations.
Background {#Sec2}
==========
In Burkina Faso, undernutrition, anaemia and diarrhoeal diseases are the leading causes of morbidity in children under the age of five. The most recent Demographic and Health Survey (DHS) of 2010 showed that 88% of children under five were anaemic, 35% were undernourished and 15% suffered from diarrhoea in the two weeks preceding the DHS \[[@CR1]\]. While DHS and national nutrition surveillance systems in Burkina Faso have routinely measured the height and weight of children under the age of five since the early 1990s, there is a lack of national anthropometric data for school-aged children (5--14 years) \[[@CR2]--[@CR4]\].
The determinants of children's nutritional status are multifactorial \[[@CR5]--[@CR7]\]. The direct causes of undernutrition in children are insufficient energy and nutrient intake, and recurrent infectious diseases (e.g. intestinal parasitic infection, malaria and diarrhoea) \[[@CR7]\]. Factors that affect children's nutritional status indirectly include a lack of access to clean water and improved sanitation, inadequate hygiene, a paucity of health education and, importantly, inappropriate agricultural practices and insufficiently healthy and diverse diets \[[@CR5]--[@CR9]\]. Low socio-economic and sanitary conditions prevail in Burkina Faso and together contribute to the burden of infectious diseases in children \[[@CR1], [@CR10], [@CR11]\], further compromising their nutritional status \[[@CR5]--[@CR9], [@CR12]\].
To address these challenges, research institutions and international development organisations are paying increased attention to enhancing synergies among agriculture, nutrition and health. The Sustainable Development Goals (SDGs) have recognised agriculture as a source of nutrition and well-being, as addressed in SDG 2: "End hunger, achieve food security and improved nutrition and promote sustainable agriculture" \[[@CR13]\]. Yet, there is a dearth of evidence to support the effect of agricultural and health interventions on improving children's nutritional status \[[@CR14], [@CR15]\]. To fill this research gap, a multi-country and multi-stakeholder project entitled "Vegetables go to School: improving nutrition through agricultural diversification" (VgtS), was developed to address schoolchildren's nutrition in an interdisciplinary way, through introducing school vegetable gardens and other school-based health, nutritional and environmental interventions. The VgtS project is active in five countries in Africa and Asia (Bhutan, Burkina Faso, Indonesia, Nepal and the Philippines), with the overall goal of improving schoolchildren's nutritional status \[[@CR16]\]. Under the VgtS project, two intervention studies were implemented in Burkina Faso and Nepal. These studies assessed schoolchildren's nutritional and health status at baseline and at 12 months follow-up, using a set of selected qualitative and quantitative indicators. The findings from these studies guided the development of complementary nutrition and water, sanitation and hygiene (WASH) interventions to operate alongside the school garden programme. Details of the study design and procedures have been described elsewhere \[[@CR16]\].
The Burkina Faso setting provided an opportunity to understand the complex interactions among agriculture, undernutrition, intestinal parasitic infections and WASH conditions. Agriculture is a major source of livelihoods in the country and inadequate WASH conditions are well known risk factors for both undernutrition and intestinal parasitic infections \[[@CR11], [@CR17]--[@CR20]\]. In this article, we report findings from a cross-sectional baseline survey carried out in Burkina Faso as part of the intervention component of the VgtS project.
Methods {#Sec3}
=======
Study area {#Sec4}
----------
We conducted a cross-sectional baseline study in February 2015. The schools participating in the VgtS project in Burkina Faso are located in the Plateau Central and the Centre-Ouest regions. The Plateau Central region is situated in the north-east, approximately 30--120 km from the capital, Ouagadougou. The Centre-Ouest region is located in the south-west, some 40--180 km from Ouagadougou (Fig. [1](#Fig1){ref-type="fig"}). The two regions are located in the semi-arid North-Sudanian zone, characterised by fields, bushes and scattered trees and a Sudano-Sahelien climate (a short wet and a long dry season, with annual precipitation of 600--1 000 mm).Fig. 1Study sites of the cross-sectional survey in Burkina Faso, February 2015
Sample size and sampling method {#Sec5}
-------------------------------
Our sample size calculation targeted the association between the prevalence of intestinal parasitic infection and the degree of risk among children, aged 8--14 years. We assumed a minimum prevalence of intestinal parasitic infections of 40%, with a coefficient of variation of 10% across schools and a proportion of high - risk children of 25%. We aimed for a power of 85% to detect a difference in infection rates (with *P* \< 0.05) between high- and low-risk children at eight schools, for a true odds ratio (*OR*) of at least 2. A Monte Carlo simulation (5 000 iterations) provided a minimal sample size of 400 children (i.e. 50 children per school). Eight of the 30 VgtS project schools in Burkina Faso were randomly selected to participate in the study \[[@CR16]\]. In each of the sampled schools, 55--60 children (boys and girls in ratio 1:1) were randomly selected; we assumed that the final sample size would be reduced by 15% due to non-response and missing data \[[@CR16]\]. The inclusion criteria for this study were: (i) schoolchildren between the ages of 8 and 14 years; (ii) parents/guardians of the children providing written informed consent; and (iii) children additionally providing oral assent.
Anthropometric survey {#Sec6}
---------------------
Trained field staff collected anthropometric measurements from the children, using a height measuring board and a digital scale (Seca 877; Seca, Germany) with a precision of 0.1 cm and 0.1 kg, respectively and adhering to standard procedures \[[@CR21]\]. Anthropometric indices were calculated in accordance with the World Health Organization (WHO) reference, using AnthroPlus (WHO; Geneva, Switzerland) \[[@CR22], [@CR23]\]. For children without an exact date of birth or whose age was unknown, school registration lists were consulted. If the exact month or date of birth was unavailable, anthropometric indices were calculated assuming 30 June (mid-year) as the child's date of birth. Three anthropometric indices --- height-for-age (HAZ, stunting), body mass index-for-age (BMIZ, thinness) and weight-for-age (WAZ, underweight) --- were expressed as differences from the median in z-scores. Children were classified as stunted, thin, or underweight if z-scores of HAZ, BMIZ and WAZ were less than - 2 standard deviations (SD) below the WHO reference median of the standard population. WAZ was only used for children aged 8--10 years, as reference data were not available for children over 10 years \[[@CR22], [@CR23]\]. Children were classified as overweight if BMIZ was above 1 SD. We considered children to be malnourished when classified as stunted, thin, underweight or overweight; undernourished children were those classified as stunted, thin or underweight. The categories of stunting, thinness and underweight are not mutually exclusive, as these conditions often overlap; an undernourished child can, for example, be classified as stunted and thin, concurrently.
Haemoglobin survey {#Sec7}
------------------
Haemoglobin (Hb) concentration was determined in finger-prick capillary blood samples, using a HemoCue portable device (HemoCue Hb 201 System; Ängelholm, Sweden) \[[@CR24]\]. Children were classified as mildly anaemic if Hb concentration was less than 11.5 g/dl for children aged 8--11 years and less than 12 g/dl for children aged 12--14 years. Children were classified as moderately and severely anaemic if Hb concentration was less than 11 g/dl and 8 g/dl, respectively \[[@CR25]\].
Parasitological survey {#Sec8}
----------------------
Children were asked to provide a fresh morning stool and a mid-morning post-exercise urine sample, collected on two consecutive days. Stool and urine samples were processed the same day by experienced laboratory technicians. From each stool, a single Kato-Katz thick smear was prepared for diagnosis of soil-transmitted helminths (*Ascaris lumbricoides*, hookworm and *Trichuris trichiura*), *Schistosoma mansoni* and other helminths. A formalin-ether concentration (FEC) technique was also performed on each sample to diagnose helminths and intestinal protozoa *(Blastocystis hominis*, *Chilomastix mesnili*, *Endolimax nana*, *Entamoeba coli*, *Entamoeba histolytica*/*E. dispar*, *Entamoeba hartmanni*, *Giardia intestinalis*, and *Iodamoeba bütschlii*) \[[@CR26], [@CR27]\]. Urine samples were examined for microhaematuria using reagent strips (Hemastix, Siemens Healthcare Diagnostics GmbH; Eschborn, Germany). A urine filtration technique was applied to detect the presence and number of *S. haematobium* eggs \[[@CR28]\]. Helminth infection intensity was calculated based on criteria established by the WHO \[[@CR29]\].
Questionnaire survey {#Sec9}
--------------------
Questionnaires were administered to children to determine their knowledge of nutrition and health and associated attitudes and practices (KAP) and to the caregivers to identify basic household socio-demographic and economic characteristics and WASH conditions. The KAP and household questionnaires were established according to international guidelines, using standardised questions amended by our research team \[[@CR1], [@CR30], [@CR31]\]. Both questionnaires were pre-tested in the study area in November 2014, with children and caregivers who did not subsequently participate in the survey (as part of a pilot study carried out in different schools and villages, far away from those schools selected for the present study). Final local adaptations were made prior to the start of the survey in February 2015.
Data entry and storage {#Sec10}
----------------------
Data were double-entered in Excel 2010 (Microsoft; Redmond, USA). After removing inconsistencies, the datasets were combined and the accuracy of the merged database was verified against the original data through random cross-checking. Data were transferred to and stored electronically on a secure and password-protected server at the Swiss Tropical and Public Health Institute (Swiss TPH; Basel, Switzerland).
Statistical analysis {#Sec11}
--------------------
Categorical variables were described by absolute and relative frequencies. Numerical variables were described by their mean and SD if they were normally distributed, and by their median and interquartile range, otherwise. To characterise household socioeconomic status, we conducted a factor analysis. A list of recorded household assets were included, which took into account the construction materials of the house wall, roof and floor \[[@CR32]\]. Four factors reflecting four different socioeconomic domains were retained, including; (i) housing wall materials; (ii) roof materials; (iii) floor materials; and (iv) main energy sources used.
To test for associations between undernutrition (including stunting, thinness and underweight) in children as an outcome variable and associated risk factors, we first conducted a univariable mixed logistic regression analysis with random intercepts at the level of the schools. We included random effects for schools in our logistic regression models, as outcomes might vary between schools due to local factors not accounted for in our models. Non-pathogenic, intestinal protozoa infections (*Trichomonas intestinalis* and *E. coli*) were excluded as potential risk factors for undernutrition in univariable and multivariable analysis. A new variable for hygiene behaviour was created using factor analysis with two conceptually similar categorical variables of: (i) mode of handwashing (e.g. handwashing with soap and water, with water only, with ash, and no handwashing); and (ii) handwashing frequency (before eating, after eating, after playing, and after defecation). Children were classified into one of three categories, reflecting poor, moderate or better hygiene behaviours.
Second, we used a multivariable mixed logistic regression model with random school intercepts and including the categorical exposure variables sex, age, project region and household socioeconomic status as additional independent variables. All other variables were added to the core model one by one, and those with a *P* \< 0.2 (using likelihood ratio test) were included in the final multivariable model. *OR*s were reported to compare relative odds, while differences and associations were considered as statistically significant if *P*-values were below 0.05, and indicating a trend if *P*-values were between 0.05 and 0.1.
Statistical analyses were performed with Stata version 13 (StataCorp; College Station, USA). Maps, including geographical coordinates of the schools, were established in ArcMap™ version 10 (Environmental System Research Institute; Redlands, USA) and with the Google Earth™ mapping software (<https://www.google.com/earth>).
Results {#Sec12}
=======
Study compliance and respondents' characteristics {#Sec13}
-------------------------------------------------
Overall, 455 schoolchildren from eight schools were enrolled in the study. Figure [2](#Fig2){ref-type="fig"} summarises study participation and compliance, from enrolment to the final sample included for statistical analysis.Fig. 2Participation in the different study groups of the cross-sectional survey in Burkina Faso, February 2015
Parasitological, anthropometric, Hb and KAP questionnaire data were linked by means of a unique identification code (ID). Erroneous ID codes or incomplete datasets with at least one of the parameters missing (e.g. anthropometrics, anaemia, urine and stool analyses, and child and household questionnaires) reduced the number of complete datasets from 455 to 424 children's records and 385 corresponding household records for subsequent analyses. For households with more than one participating child, one child was selected at random for analysis; hence, another 39 children were excluded and our final dataset comprised 385 children from 385 unique households.
The mean age of children interviewed was 11 years (SD 0.7 years, range: 8--14 years). The mean age of the children's caregivers interviewed was 45 years (SD 14.2 years, range: 20--95 years). Three-quarters of the children's caregivers had not received any formal education, whereas 59 (15.3%) attended primary school and the remaining 38 (9.9%) received at least a secondary level of education. Almost 90% of children's caregivers work in the agricultural sector. Respondents' demographic and economic characteristics are summarised in Table [1](#Tab1){ref-type="table"}.Table 1Characteristics of the study population in the Plateau Central and Centre-Ouest regions, Burkina Faso, February 2015Children's demographic characteristicsNumberPercentAge of children^a^Girls18848.8Boys19751.2Age group 1 (8--11 year)25165.2Age group 2 (12--14 years)13434.8Caregivers' demographic and educational characteristicsCaregivers' age^b^No formal schooling28874.8Primary education5915.3Secondary or higher education389.9Main occupation of head of householdAgriculture34489.4Merchant82.1Civil service92.3No employment20.5Others (housework or retirement)225.7Socioeconomic domainsRoof materialSimple (natural and baked clay)379.6Metal cover34890.4Wall materialSimple (natural clay)35993.3Baked or cemented clay266.7Floor materialSimple (clay, sand, mud, straw)25566.2Baked or cemented clay13033.8Energy usedSimple (charcoal, firewood)37697.7Electricity and gas92.3^a^ = mean age of 11.0 (±0.7) years^b^ = mean age of 45.0 (±14.2) years
Prevalence of malnutrition {#Sec14}
--------------------------
Table [2](#Tab2){ref-type="table"} shows the extent of malnutrition, stratified by anthropometric indicators, including age, sex and region. The prevalence of malnutrition and undernutrition in this study were high, at 37.1% and 35.1%, respectively. The prevalence of stunting was 29.4%, while 11.2% of the children were classified as thin. Three out of the 55 children under the age of 10 years were underweight, while eight children were classified as overweight.Table 2Prevalence of total and specific malnutrition indicators in schoolchildren, Burkina Faso, February 2015VariableMalnutrition \[*n* (%)\]Undernutrition \[*n* (%)\]Stunting^a^\
\[*n* (%)\]Thinness^a^\
\[*n* (%)\]Underweight^a^ \[*n* (%)\]Overweight^b^ \[*n* (%)\]Anaemia^c^ \[*n* (%)\]Sex Female (188)61 (32.5)57 (30.3)47 (25.0)24 (12.8)2 (1.1)4 (2.1)53 (28.2) Male (197)82 (41.6)78 (39.6)66 (33.5)19 (9.6)1 (0.5)4 (2.0)57 (28.9)Age group 8--11 year (251)69 (27.5)61 (24.3)47 (18.7)16 (6.4)3 (1.2)8 (3.2)55 (21.9) 12--14 years (134)74 (55.2)74 (55.2)66 (49.3)27 (20.2)*NA* ^d^0 (0)55 (41.0)Region Plateau Central (198)69 (34.9)64 (32.3)50 (25.3)19 (9.6)2 (1.0)5 (2.5)53 (26.8) Centre-Ouest (187)74 (39.6)71 (38.0)63 (33.7)24 (12.8)1 (0.5)3 (1.6)57 (30.5)Total143 (37.1)135 (35.1)113 (29.4)43 (11.2)3 (0.8)8 (2.1)110 (28.6)^a^ z-score \< − 2^b^ z-score \> 1^c^ The category of anaemia includes all children classified as anaemic (mild, moderate and severe) based on the concentrations of haemoglobin (Hb) determined in a finger prick blood sample. The cut-offs for anaemia are age-specific: Hb \<11.5 g/dl for children aged 8--11 years, and Hb \<12 g/dl for children aged 12--14 years^d^ *NA* not available
Intestinal parasitic and *Schistosoma* infections {#Sec15}
-------------------------------------------------
Table [3](#Tab3){ref-type="table"} shows differences in the prevalence of intestinal protozoa, faecal-oral transmitted helminths and *Schistosoma* infections in children, stratified by sex, age and region. We found that 86.2% of the children were infected with at least one intestinal parasite. Intestinal protozoa infections were highly prevalent (84.7%). *Entamoeba histolytica/E. dispar* was the predominant intestinal protozoon species (66.5%), followed by *E. coli* (37.4%), *G. intestinalis* (28.1%) and *T. intestinalis* (23.4%).Table 3Prevalence of helminths and intestinal protozoa infections in schoolchildren, Burkina Faso, February 2015VariableTrematodesTotal schistosomiasis^a^\
\[*n* (%)\]NematodesCestodesTotal faecal-oral transmitted helminths^c^\
\[*n* (%)\]ProtozoaTotal\
protozoa\
\[*n* (%)\]*S. haematobium* ^*a*^\
\[*n* (%)\]*S. mansoni* ^a^\
\[*n* (%)\]Hookworm\
\[*n* (%)\]*H. nana* ^b^\
\[*n* (%)\]*Entamoeba histolytica*/*E. dispar*\
\[*n* (%)\]*Entamoeba coli*\
\[*n* (%)\]*Giardia intestinalis*\
\[*n* (%)\]*Trichomonas intestinalis*\
\[*n* (%)\]*Balantidium coli*\
\[*n* (%)\]Sex Female (188)7 (3.7)0 (0)7 (3.7)0 (0)11 (5.9)11 (5.9)131 (69.7)67 (35.6)44 (23.4)39 (20.7)1 (0.5)161 (85.6) Male (197)8 (4.1)1 (0.5)9 (4.6)3 (1.5)14 (7.1)16 (8.1)^c^125 (63.5)77 (39.1)64 (32.5)51 (25.9)0 (0)165 (83.8)Age group 8--11 year (251)8 (3.2)0 (0)8 (3.2)2 (0.8)13 (5.2)15 (6.0)163 (64.9)93 (37.1)69 (27.5)51 (20.3)0 (0)209 (83.3) 12--14 years (134)7 (5.2)1 (0.8)8 (6.0)1 (0.8)12 (9.0)12 (9.0)^c^93 (69.4)51 (38.1)39 (29.1)39 (29.1)1 (0.8)117 (87.3)Region Plateau Central (198)8 (4.0)0 (0)8 (4.0)1 (0.5)5 (2.5)6 (3.0)110 (55.6)65 (32.8)49 (24.8)55 (27.8)0 (0)157 (79.3) Centre-Ouest (187)7 (3.7)1 (0.5)8 (4.3)2 (1.1)20 (10.7)21 (11.2)^c^146 (78.1)79 (42.3)59 (31.6)35 (18.7)1 (0.5)169 (90.4)Total (385)15 (3.9)1 (0.3)16 (4.2)3 (0.8)25 (6.5)27 (7.0)256 (66.5)144 (37.4)108 (28.1)90 (23.4)1 (0.3)326 (84.7)^a^ *Schistosoma haematobium*, *Schistosoma mansoni*^b^ *Hymenolepis nana*^c^ The category of total faecal-oral transmitted helminths includes children infected with hookworm and *Hymenolepis nana*. There is one child co-infected with hookworm and *Hymenolepis nana.*
Faecal-oral transmitted helminth infections were found in 7.0% of the children. *Hymenolepis nana* was the most frequently occurring species (6.5%). Only three children were infected with hookworm (0.8%). One child had a dual-species infection with hookworm and *H. nana*. Fifteen children were infected with *S. haematobium* (3.9%), while one child was infected with *S. mansoni* (0.3%).
Co-infections were common, affecting 32.5% of the children, whilst 15.6% and 4.7% suffered from triple and quadruplicate infections, respectively. Infections with *H. nana*, *S. haematobium,* hookworm and *S. mansoni* were of light intensity. The prevalence of intestinal protozoa and faecal-oral transmitted helminth infections differed significantly between schoolchildren in the Plateau Central region and those in Centre-Ouest (*P* \< 0.05).
Prevalence of anaemia {#Sec16}
---------------------
The mean Hb concentration was 12.3 g/dl (SD 0.7 g/dl). The prevalence of anaemia in our study sample was 28.6% (Table [2](#Tab2){ref-type="table"}). Few children were found to be severely anaemic (0.8%), while 11.2% were found to be moderately anaemic and 16.6% mildly anaemic.
Results from the questionnaire surveys {#Sec17}
--------------------------------------
Key results from children's nutrition and health KAP survey and from the household questionnaire are summarised in Table [4](#Tab4){ref-type="table"}. While 79.7% of the children reported using latrines at school for defecation, 22.1% reported washing their hands after defecation. Most children (87.8%) reported washing their hands before eating and 7.3% after playing. Four out of five (79.5%) children reported using soap and water to wash their hands. Combining the mode and frequency of handwashing, children were divided into one of three hygiene categories: 14.6% in the lower, 59.0% in the middle and 26.4% in the better hygiene category. Among the households participating in our survey, 55.3% did not own a latrine, while 23.1% had access to an improved latrine. The majority of children (82.1%) and 22.1% of their caregivers stated that they had never heard of malnutrition. Of the interviewed caregivers, 96.9% indicated that their participating child was breastfed.Table 4Key findings from children's nutrition and health KAP survey and household questionnaire in Burkina Faso, February 2015Children (*n* = 385)NumberPercentSelected KAP^a^ indicators:Handwashing^b^ Water only34489.4 Water and soap30679.5 With ash123.1 With mud10.3 Before eating33887.8 After eating5514.3 After playing287.3 After defecation8522.1 Do not wash hands164.2Hygiene behaviour^c^ Lower category (1)5614.6 Middle score (2)22759.0 Best category (3)10226.4Sanitary behaviour at school Using latrines at school30779.7 Open defecation (fields, bush)7118.5 Others (at home, teachers home)71.8Meals (day prior to the survey) Breakfast33085.7 Lunch35191.2 Dinner35893.0Nutritional knowledge Heard about malnutrition6917.9Households (*n* = 385)NumberPercentHousehold WASH^d^ characteristics Availability of soap (observational)11830.7Type of latrines used Flush toilet (i)00 VIP latrine^e^ (ii)143.6 Traditional pit latrine (iii)8321.6 EcoSan^f^ (iv)6015.6 Samplat latrine (v)153.9 No facilities/open defecation (vi)21355.3 Total improved^g^ (i, ii, iv, v)8923.1 Total unimproved^h^ (iii, vi)29676.9Nutritional knowledge and practices Heard about malnutrition30077.9 Participating child was breastfed37396.9^a^ Knowledge, attitudes and practices^b^ Multiple responses occurred for the variables characterising the mode (how) and frequency (when) of handwashing.^c^ A new variable for hygiene behaviour was created using factor analysis with two conceptually similar categorical variables of: (i) mode of handwashing (handwashing with water and soap, with water only, with ash, no handwashing); and (ii) its frequency (before eating, after eating, after playing, and after defecation). Children were classified into three categories with lower, middle and better hygiene behaviours.^d^ Water, sanitation and hygiene^e^ Ventilated improved pit (VIP) latrine is an improved type of pit latrine, which helps remove odours and prevent flies from breeding and escaping. Excreta are collected in a dry pit which has a vent pipe covered with a fly-proof screen at the top^f^ Ecological sanitation (EcoSan) toilets are linked to a closed system that does not need water. The toilet is based on the principle of safely recycling excreta resources to create a valuable resource for agriculture^g^ The total improved sanitation category includes sanitation facilities that hygienically separate human excreta from human contact. In our study, these were: (i) flush toilet, (ii) VIP latrine, (iv) EcoSan toilets, and (v) latrine with slab^h^ The total unimproved sanitation category in our study included: (iii) traditional pit latrines, and (vi) no facilities/open defecation)
Results from the logistic regression analysis {#Sec18}
---------------------------------------------
Table [5](#Tab5){ref-type="table"} provides an overview of the associations between undernutrition and all measured helminth and pathogenic intestinal protozoa infections, nutrition and health KAP, caregivers' socioeconomic characteristics and WASH conditions observed in univariable and multivariable regression analyses. The prevalence of undernutrition significantly differed between age groups, with the older age group (12--14 years) showing significantly higher odds of undernutrition (a*OR* = 3.45, 95%* CI* 2.12--5.62, *P* \< 0.001). Girls showed lower odds of being undernourished, but this association lacked statistical significance in the multivariable analysis. No significant association was observed between undernutrition and study region (*P* \> 0.05).Table 5Results from univariable and multivariable logistic regression analysis with undernutrition as outcomeUndernutrition\
*N* = 385 / *N*(cases) = 135Univariable logistic regression^a^Multivariable logistic regression^b^*OR*95% *CIP*a*OR*95%* CIP*SexMale1.00Female0.700.45--1.09**0.112**0.720.46--1.140.163Age group8--11 year1.0012--14 years3.572.20--5.78**\<0.001**3.452.12--5.62**\<0.001**RegionCentre-Ouest1.00Plateau Central0.890.35--2.270.804Multiple pathogenic parasites"yes" vs. "no"1.941.09--3.47**0.025**1.871.02--3.43**0.044**Intestinal pathogenic protozoa"yes" vs. "no"1.781.03--3.06**0.039**1.710.97--3.030.064*Hymenolepis nana*"yes" vs. "no"1.420.60--3.360.425*Schisotosoma haematobium*"yes" vs. "no"0.760.22--2.560.659*Giardia intestinalis*"yes" vs. "no"1.440.90--2.32**0.131**1.460.89--2.400.133*Entamoeba histolytica/E. dispar*"yes" vs. "no"1.390.85--2.25**0.187**1.410.85--2.340.184AnaemiaNo1.00Mild1.590.89--2.85**0.121**1.240.67--2.310.486Moderate^c^2.891.48--5.64**0.002**2.521.25--5.08**0.010**Middle score (2)1.00Hygiene^d^Lower category (1)1.150.59--2.250.676Best category (3)1.360.82--2.250.233Sanitary behaviour at schoolOpen defecation^e^1.00Using latrines at school0.970.48--1.950.922Others (at teachers')NaHousehold sanitary conditionsImproved latrines1.00No latrines/open defecation0.960.54--0.540.886Traditional latrine1.180.60--2.290.634Availability of soap"yes" vs. "no"1.140.70--1.840.599Child's eating habits (day prior to the survey)Breakfast"no vs. yes"^f^0.720.38--1.380.326Lunch"no vs. yes"^f^1.880.89--4.00**0.100**1.520.69--3.320.298Dinner"no vs. yes"^f^1.300.57--2.990.534Child "heard about malnutrition""no vs. yes"^f^1.110.64--1.950.709Caregiver "heard about malnutrition""no vs. yes"^f^1.140.67--1.940.618"Breastfed child""no vs. yes"^f^2.200.41--11.710.354Caregiver's educationNever went to school1.00Primary education1.300.71--2.370.390Secondary education0.870.40--1.890.716Caregiver's occupationAgriculture1.00Civil service0.350.04--3.010.341Merchant0.350.33--5.230.702Others^g^0.710.28--1.850.487^a^ *P*-value and odds ratio (*OR*) based on likelihood ratio test. In univariable logistic regression, the overall *P*-value of the models is indicated in bold letters^b^ *P*-value and adjusted (a) *OR* based on likelihood ratio test of the multivariable regression model. The mixed multivariable logistic regression model with random school intercepts included the categorical exposure variables sex, age group, socioeconomic domains and project region. All risk factors that had a *P*-value lower than 0.2 in the univariable analyses were included into the multivariable regression analysis (as indicated in the table)^c^ The category of moderate anaemia includes the severely anaemic children (*n* = 3)^d^ This variable was created with two conceptually similar categorical variables of: (i) mode of handwashing (handwashing with soap and water, with water only, with ash, no handwashing); and (ii) handwashing frequency (before eating, after eating, after playing, and after defecation) where multiple responses were possible. Children were classified into one of three categories, with lower, middle and better hygiene behaviours^e^ Open defecation includes the category of defaecating in the bush and behind the latrines^f^ The reference category for the *OR* is "yes" as compared to "no"^g^ 'Others' includes homemakers, retirees and unemployed people
Children infected with multiple pathogenic parasites and those with moderate - to - severe anaemia, were at significantly higher odds of being undernourished (a*OR* = 1.87, 95% *CI* 1.02--3.43, *P* = 0.044; and a*OR* = 2.52, 95% *CI* 1.25--5.08, *P* = 0.010, respectively).
Overall, children with better hygiene behaviours (third category) did not show lower odds for undernutrition than those in the middle or lower hygiene categories (*P* \> 0.5). Relying on traditional pit latrines or having no toilet facility at home was not associated with increased odds for undernutrition in children. Moreover, children who reported not having eaten lunch the day prior to the survey and children who were not breastfed showed higher odds of undernutrition, but these associations were not statistically significant (*P* \> 0.05). Neither the level of education of the children's caregivers nor their occupation showed any statistically significant association with undernutrition.
Discussion {#Sec19}
==========
This paper presents findings from a cross-sectional survey on the prevalence of undernutrition and associated risk factors among schoolchildren, aged 8--14 years, from eight schools in the Plateau Central and Centre-Ouest regions of Burkina Faso. We found that undernutrition was highly prevalent among the surveyed children. Approximately a third of the children were undernourished (35.1%).
According to a study conducted in Ouagadougou in 2008/09 for the WHO's "Nutrition Friendly School Initiative" (NFSI), the prevalence of stunting in schoolchildren (mean age of 11.5 years) was 8.8%, which is considerably lower than the prevalence of stunting among schoolchildren found in this study (29.4%) \[[@CR33]\]. The proportion of thinness in children in our study was 11.2%, which is, however, comparable with the 13.7% found in the NFSI study \[[@CR33]\]. Overweight children accounted for 2.1% of all children, with a higher incidence among children aged 8--11 years than among the older age group (3.2% vs. 0%), which is similar to the 2.3% reported in the NFSI study \[[@CR33]\].
While few children were classified as thin, a considerably higher proportion of children in our study were stunted. Thinness is often associated with short-term risk factors, like seasonal climatic variations (which cause food scarcity/shortages) and increased occurrence of illnesses \[[@CR34]\]. Our study was conducted in the post-harvest (mid-dry) season (February), before the commencement of the dry season (March-June) \[[@CR35]\], suggesting that the cause of undernutrition was mainly of a chronic nature, associated with long-term risk factors.
The findings from multivariable mixed logistic regression analyses demonstrated a considerably higher risk of undernutrition among children older than 12 years of age. These results are in accordance with other studies, showing a higher prevalence of stunting in older children in low-income countries in Asia and Africa \[[@CR36]--[@CR38]\]. Moreover, children with moderate and severe anaemia (combined category) and with multiple helminths and intestinal pathogenic protozoa infections ("multiple pathogenic parasites") showed significantly higher odds for undernutrition. Undernutrition and intestinal parasitic infections are intrinsically linked. While undernutrition and inadequate dietary intake lead to weight loss and weakened immunity and render a child more susceptible to infections, parasitic infections contribute to growth stunting by causing a vicious cycle of reduced food intake (loss of appetite), diarrhoea, malabsorption and/or increased nutrient wastage \[[@CR39]--[@CR41]\]. The observed association was statistically significant in our study, reinforcing evidence of the frequent coexistence of these conditions among children \[[@CR40]\]. Moreover, while anaemia contributed to higher odds of undernutrition among children in our study, the aetiology of anaemia is multifactorial and can result from nutritional deficiencies and parasitic infections, among other things, which have been closely connected to the nutritional status of African schoolchildren \[[@CR42]--[@CR45]\].
Our questionnaire survey revealed important inadequacies in nutrition- and health-related knowledge and practices, but no clear association between undernutrition and WASH conditions or nutritional and health KAPs.
Our study has three main limitations. First, the findings presented here cannot be generalised for all of Burkina Faso. Despite the random selection of schools with a sample size large enough for children in this age range, the results are only representative of two regions. Second, the anthropometric survey has certain limitations with respect to the inaccuracy of children's dates of birth. Indeed, we noted that a considerable number of children had their birthdays either on 31 December or on 1 January, according to the existing school records. Upon further probing in the interview, the children often did not know their exact date of birth. Hence, for these children, we took a mid-year point as the date of birth \[[@CR46]\]. Third, only one single Kato-Katz thick smear and FEC from two stool samples from two consecutive days were examined for each participant. Our results may therefore underestimate the true prevalence of parasitic infections, due to the low sensitivity of the Kato-Katz technique and urine concentration method \[[@CR47], [@CR48]\].
Despite these limitations, our findings highlight a number of important issues. First, undernutrition in schoolchildren in this part of Burkina Faso is highly prevalent. We therefore suggest giving greater attention to the overall nutritional status of school-aged children. So far, comprehensive population-based data, such as the DHS, focus on adolescents over the age of 15 years for sexual and reproductive health issues or on children under 5 years of age, as they are more vulnerable and prone to disease, illness and death \[[@CR1], [@CR49]--[@CR51]\]. Children under five are often the primary focus of strategies and actions to address malnutrition \[[@CR7], [@CR52], [@CR53]\]. Despite the increased odds of survival for children after the age of five (they generally have a lower prevalence of infections when compared to children under the age of five), school-aged children have increased nutritional needs to support the adolescent growth spurt, requiring diets rich in energy and micronutrients and sufficient in both quantity and quality \[[@CR54]\]. It is therefore crucial to address the nutritional needs of children in this age group to match their growth requirements \[[@CR55]\].
Second, the results of our study highlight the need for a more profound understanding of how helminths and other intestinal parasites mediate pathways to undernutrition. In particular, it is important to investigate other primary factors related to the burden of undernutrition among school-aged children, such as malaria and other parasitic infections, and the bioavailability and absorption of micronutrients so as to prevent long-term effects of undernutrition \[[@CR56]--[@CR58]\].
To address the factors underlying and contributing to schoolchildren's nutritional status, we support the growing recommendation from several agencies to enhance multidisciplinary strategies and programmes, including nutrition and WASH interventions for school-aged children, in order to ensure optimal health, growth and development continuing after the age of five \[[@CR59]--[@CR61]\]. Such measures should be reflected in the current development of targets and indicators for reaching SDG 2.
Conclusions {#Sec20}
===========
This study provides new insight into the burden of undernutrition and its risk factors among schoolchildren in Burkina Faso, a country that lacks data on the health of children, aged 8--14 years. Our study shows that undernutrition is highly prevalent in the eight schools of the Plateau Central and Centre-Ouest regions (32.3 and 38.0%, respectively) of Burkina Faso. We also observed that undernutrition, anaemia and parasitic infections were strongly associated. In view of these findings, concerted efforts are needed to address undernutrition and the associated risk factors among school-aged children. As part of the VgtS project, WASH, health education and nutritional interventions will be implemented with the goal of improving schoolchildren\'s health.
Additional file {#Sec21}
===============
Additional file 1:Multilingual abstracts in the five official working languages of the United Nations. (PDF 661 kb)
a*OR*
: Adjusted odds ratio
BMIZ
: Body mass index-for-age
*CI*
: Confidence interval
DHS
: Demographic and Health Survey
EKNZ
: Ethikkommission Nordwest- und Zentralschweiz
FEC
: Formalin-ether concentration
HAZ
: Height-for-age
Hb
: Haemoglobin
ID
: Identification code
IRSS
: Institute for Health Sciences Research
KAP
: Knowledge, attitudes and practices
NFSI
: Nutrition Friendly School Initiative
SD
: Standard deviation
SDGs
: Sustainable Development Goals
Swiss TPH
: Swiss Tropical and Public Health Institute
VgtS
: Vegetables go to School: improving nutrition through agricultural diversification
WASH
: Water, sanitation and hygiene
WAZ
: Weight-for-age
WHO
: World Health Organization
We would like to thank all of the study participants for their commitment, the national and district health authorities for their kind support and interest, and the team at the Institute for Health Sciences Research (IRSS) in Burkina Faso for their support and technical assistance during the field work and laboratory investigation. We also specifically thank our field team for their efforts in data collection and for their skilled stool and urine examination.
We appreciate the institutional involvement of the Ministry of Health in Burkina Faso. We are grateful to our project partners from the "Vegetables go to School" project; namely, the AVRDC-World Vegetable Centre (Shanua, Taiwan) and the University of Freiburg (Freiburg, Germany), for their valuable support. This study received financial support from the Swiss Agency for Development and Cooperation.
Funding {#FPar1}
=======
This work is part of the 'Vegetables go to School' research project (Collaborative Project); supported by the Swiss Agency for Development and Cooperation under grant agreement contract number 81024052 (project 7 F-08511.01). The funder had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript.
Availability of data and materials {#FPar2}
==================================
The dataset supporting the conclusions of this article will not be shared. The paper is written as part of the academic degree of a PhD and therefore the data will be used exclusively by the author.
Authors' contribution {#FPar3}
=====================
All listed authors contributed to the study design. SE, AMK and SD coordinated the field and laboratory work. TG supervised the laboratory technicians and assisted in data collection with BS. SE and AMK supervised the research assistants. SE performed the statistical analysis under the supervision of CS and drafted the manuscript. AMK, SD, PO, JG, AS, CS, JU and GC contributed to the interpretation of the data, manuscript writing and revisions. All authors read and approved the final manuscript.
Competing interests {#FPar4}
===================
The authors declare that they have no competing interests.
Ethics approval and consent to participate {#FPar5}
==========================================
The study protocol was approved by the "Ethikkommission Nordwest-und Zentralschweiz" in Switzerland (EKNZ, reference no. 2014--161) and by the "Comité d'Ethique pour la Recherche en Santé, Ministère de la Recherche Scientifique et de l'Innovation, et Ministère de la Santé" (reference no. 2015-02-026). The study is registered with the clinical trial registry ISRCTN (identifier: ISRCTN17968589). Community and school awareness-raising activities entailed holding two meetings in a classroom at each school; one four weeks prior to the study and one on the day of the study. The purpose of these meetings was to discuss the objectives, procedures, potential benefits and risks of the study with district educational authorities, school directors, teachers, parents and community representatives. Informed consent (via signature) was obtained from the child's parents or guardians. For illiterate parents/guardians, a fingerprint was obtained in the presence of a literate witness from the school (principal or teacher), whilst children assented orally. It was emphasised that participation was voluntary and that children could withdraw at any time without further obligation. All data records were anonymised, provided with a personal identifier and kept confidential.
Results were communicated to participants. Those found with mild or moderate anaemia (Hb \<11.5 g/dl for children aged 8--11 years and Hb \<12 g/dl for children aged 12--14 years) were referred to a local health centre and treated with iron supplements for 40 days, free of charge. Children found with severe anaemia (Hb \< 8 g/dl) and severely malnourished children were referred to a local health centre for further investigation, following national guidelines \[[@CR62], [@CR63]\]. Children infected with any kind of intestinal protozoa or helminth were treated according to national guidelines (i.e., a 15--50 mg/kg single dose of metronidazole for five consecutive days against intestinal protozoa infection, a triple dose of 400 mg albendazole against soil-transmitted helminth infections, a 40 mg/kg single dose praziquantel against schistosomiasis and four tablets of niclosamide of 500 mg in two doses for six consecutive days to treat *Hymenolepis nana*). Trained teachers, in collaboration with our research team, and local health personnel, with close involvement of the parents/guardians of infected children, administered anti-parasitic medications and carefully observed children for proper medication intake and adverse events. All treatments were provided free of charge.
|
{
"pile_set_name": "PubMed Central"
}
|
Introduction
============
Endometrial cancer is a malignant tumor in the female reproductive system with a high incidence rate, accounting for approximately 25% of all the female reproductive system tumors, and its incidence in developed countries (ranking fourth) is higher than that in developing countries (ranking seventh) ([@b1-ol-0-0-9250]--[@b2-ol-0-0-9250]). Endometrial cancer often occurs in perimenopausal and postmenopausal women ([@b3-ol-0-0-9250]), and a present investigation report has shown that the age of patients at the onset of endometrial cancer becomes smaller and smaller ([@b4-ol-0-0-9250]). Although the five-year survival rate of patients has improved with the development of medical technology, the prognosis of some endometrial cancer patients is poor, causing death ([@b5-ol-0-0-9250],[@b6-ol-0-0-9250]); thus, the early diagnosis of endometrial cancer patients is crucial. The mortality rate of patients with endometrial cancer in the advanced stage is much higher than that of patients in the early stage ([@b7-ol-0-0-9250]). Improving the specificity and sensitivity of the diagnosis of endometrial cancer patients is an important means to improve the survival rate of patients.
Ultrasound plays an extremely important role in the diagnosis of tumors. Ultrasound has many advantages such as noninvasiveness, high diagnostic sensitivity, no radioactivity and simple methods. It has been widely used in the diagnosis of tumors ([@b8-ol-0-0-9250],[@b9-ol-0-0-9250]). In addition, ultrasound can reduce the invasive examination for patients and risks of invasive operation for patients through its initial screening ([@b10-ol-0-0-9250]). Tumor abnormal protein (TAP) is a kind of abnormal glycoprotein and calmodulin complex expressed after the mutation of oncogenes and tumor suppressor genes in cells ([@b11-ol-0-0-9250]). It has been reported that abnormal glycoproteins are closely related to the occurrence, development, invasion, metastasis and prognosis of cancers ([@b12-ol-0-0-9250]). Therefore, the detection of TAP in serum can provide an important reference for the clinical diagnosis of tumors.
In the present study, 248 patients with suspected endometrial cancer who were admitted to the Gynecology Department of The Second People\'s Hospital of Liaocheng (Linqing, China) from September 2013 to September 2015 were selected, and the diagnostic accordance rate of TAP combined with vaginal ultrasound was analyzed.
Materials and methods
=====================
### Clinical data
A total of 248 women patients (average age of 41.6±11.6 years) with suspected endometrial cancer who were admitted to the Gynecology Department of The Second People\'s Hospital of Liaocheng from September 2013 to September 2015 were selected and randomly divided into the control (n=124) and the observation group (n=124). The control group received conventional ultrasound examination; the observation, underwent TAP combined with conventional ultrasound examination, and all patients were definitely diagnosed by hysteroscopy and diagnostic curettage examination. Patients without tumor history, without liver, kidney and other organ dysfunction, and without abnormal hemorrhage or coagulation dysfunction before operation were included. Patients with unqualified curettage specimens, receiving treatment before, with excessive large tumor mass or with other pulmonary or chest wall diseases were excluded. This study was approved by the Ethics Committee of the Second People\'s Hospital of Liaocheng, and patients or their family members signed informed consent.
### Instruments and methods
Hitachi Hivision Avivs color Doppler ultrasound diagnostic machine (Hitachi, Ltd., Tokyo, Japan) was applied for conventional ultrasound examination so as to observe patients\' tumor diameter, shape, edge status, internal echo information and whether there were space occupying lesions. At the same time, the fasting whole blood was collected from the included patients in the early morning, and TAP detection was conducted. Abnormal carbohydrate chain glycoprotein detection kits were purchased from Thermo Fisher Scientific, Inc., (Waltham, MA, USA), and the image analyzer for TAP detection was purchased from Zhejiang Aicor Medical Technology Co., Ltd. (Zhejiang, China).
### Statistical analysis
The experimental results were analyzed by using SPSS 22.0 software (IBM Corp., Armonk, NY, USA), and the analysis of variance followed by post hoc test (Least Significant Difference) was used for compari-sons among multiple groups. Count data were expressed as percentage and detected by χ^2^ test. The sensitivity and specificity of conventional ultrasound and TAP combined with conventional ultrasound in the diagnosis of endometrial cancer were calculated, respectively. T0he diagnostic values of conventional ultrasound and the combination of the two methods in the diagnosis of endometrial cancer were studied by using the receiver operating characteristic (ROC) curve. P\<0.05 was considered to indicate a statistically sgnificant difference.
Results
=======
### Basic data
In this study, 248 patients at an average age of 41.6±11.6 years with suspected endometrial cancer were included. After the pathological diagnosis by curettage, it was found that there were 60 cases of endometrial polyps, 96 of endometrial hyperplasia, 17 of uterine fibroids and 75 cases of endometrial cancer. In addition, the menopause data of patients were collected, in which there were 43 premenopausal patients (17.34%), confirming that the age of patients at the onset of endometrial cancer becomes smaller ([Table I](#tI-ol-0-0-9250){ref-type="table"}).
### Diagnostic results of the two programs
Of 248 patients receiving hysteroscopy and diagnostic curettage examination, there were 75 patients with early-stage endometrial cancer and 173 benign patients (including 60 patients with endometrial polyps, 96 with endometrial hyperplasia and 17 with uterine fibroids). The total diagnostic accordance rate of conventional ultrasound for endometrial lesions was 87.90% (n=218), and for early-stage endometrial carcinoma was 90.67% (n=68). For patients with endometrial polyps, the rate was 88.33% (n=53); with endometrial hyperplasia was 87.50% (n=84); with uterine fibroids was 76.47% (n=13). For TAP combined with vaginal ultrasound for endometrial lesions, the rate was 94.35% (n=234), and for early-stage endometrial cancer was 94.67% (n=71). For diagnosis for patients with endometrial polyps the rate was 95.00% (n=57), with endometrial hyperplasia it was 93.75% (n=90), and with uterine fibroids it was 94.12% (n=16); The diagnostic accordance rates of TAP combined with conventional ultrasound for endometrial lesions were higher than those of simple conventional ultrasound (P\<0.05) ([Table II](#tII-ol-0-0-9250){ref-type="table"}). Imaging manifestations of conventional ultrasound ([Table III](#tIII-ol-0-0-9250){ref-type="table"}).
### TAP detection results
The image analyzer for TAP detection was used to analyze the polarity of TAP detection results, which showed that the positive rate of TAP detection was 86.67% (n=65) in patients with endometrial cancer and 4.37% (n=8) with benign lesions; there was a significant difference between the two groups of patients (P\<0.05). Among the patients with benign lesions and positive TAP detection results, there were 5 cases of uterine fibroids, 2 of endometrial polyps and 1 of endometrial hyperplasia ([Fig. 1](#f1-ol-0-0-9250){ref-type="fig"}).
### Sensitivity and specificity of the two programs
The sensitivity and specificity of conventional ultrasound to endometrial cancer were 85.7 and 89.7%, respectively, and those of TAP combined with vaginal ultrasound to endometrial cancer were 90.0 and 92.8%, respectively. ROC analysis results revealed that the AUC of convention ultrasound in the diagnosis of endometrial cancer was 0.754 \[95% confidence interval (CI): 0.211--2.534\], and that of TAP combined with vaginal ultrasound in the diagnosis of endometrial cancer was 0. 814 (95% CI: 0.517--0.932); the difference between the two programs was significant (P=0.011) ([Fig. 2](#f2-ol-0-0-9250){ref-type="fig"}).
Discussion
==========
Prognoses of endometrial cancer in the advanced stage and some special cases of endometrial cancer are very poor, and their 5-year survival rates are relatively low, so the early diagnosis of patients with endometrial cancer is very necessary ([@b6-ol-0-0-9250],[@b7-ol-0-0-9250]). TAP is a type of complex that is expressed and released by tumor cells and can be found in the blood of tumor patients, which provides a very convenient specimen for the early screening of tumors ([@b13-ol-0-0-9250],[@b14-ol-0-0-9250]). TAP has become a hot research object for researchers, especially on the screening and early diagnosis of tumors, since it was discovered by the Union of Soviet Socialist Republics (USSR) scholars, Kostyantin, A. and Galakhin ([@b15-ol-0-0-9250],[@b16-ol-0-0-9250]). However, there are few diagnostic studies in patients with endometrial cancer in the early stage. This study was expected to provide a basis for clinical diagnosis of endometrial cancer in the early stage and improve the survival rate of patients.
In the present study, 248 patients with suspected endometrial cancer were included, and with other tumors were excluded. TAP combined with conventional ultrasound was used for diagnosis, whose results were compared with those of diagnostic curettage detection, so as to analyze the value of TAP combined with conventional ultrasound in the diagnosis of endometrial cancer in the early stage. The study\'s results showed that the diagnostic accordance rate of TAP for patients with endometrial cancer was 86.67%, and that of conventional ultrasound for patients with endometrial cancer was 90.67%, and that of the combination of the two was 94.35%; the diagnostic accordance rate of TAP and conventional ultrasound for patients with endometrial cancer were lower than that of the combination of the two (P\<0.05), indicating that TAP detection can improve the diagnostic effect of conventional ultrasound on endometrial cancer. In addition, ROC curve results also revealed that the combined diagnostic method had a high diagnostic value (AUC=0.814). The sensitivity of the TAP detection system is the expressed TAP when the number of tumor cells reaches 100,000 and above, but it takes about one year for the number of tumor cells to proliferate to 100,000 before the tumor has not formed a mass, which is extremely important for the treatment and survival of patients, especially of those with malignant tumors with fast development and poor prognosis ([@b17-ol-0-0-9250]--[@b20-ol-0-0-9250]). There are few researchers studying the effect of TAP detection in the diagnosis of endometrial cancer in the early stage, so the conclusions in this study still need to be further validated. This study also had its advantages: All patients with past tumor history were excluded to avoid the interference in TAP detection; a self-controlled test was conducted so that the interference of test samples was avoided. TAP is also used in many kinds of cancers, such as gastric, thyroid and colorectal cancer, and it is of positive significance in improving the accordance rate of tumor diagnosis ([@b21-ol-0-0-9250]--[@b22-ol-0-0-9250]). These indirectly confirmed the diagnostic value of TAP in the diagnosis of endometrial cancer. Benign endometrial lesions were also analyzed, which showed that approximately 20--40% of patients suffered from atypical endometrial hyperplasia before the onset of endometrial cancer. TAP combined with vaginal ultrasound also has a relatively diagnostic high accordance rate (93.75%) for endometrial hyperplasia, and among patients with TAP positive detection results, there was 1 case of endometrial hyperplasia. Due to the limitation of experimental time, the patients were not followed-up, and further investigation is needed for confirmation of the results.
In summary, TAP combined with transvaginal ultrasound in the diagnosis of early-stage endometrial cancer has a higher accuracy rate, and it is worth promoting and applying in clinical practice.
Not applicable.
Funding
=======
This study was supported by the Liaocheng Health Medical Letter \[2016\] No.3-146.
Availability of data and materials
==================================
All data generated or analyzed during this study are included in this published article.
Authors\' contributions
=======================
AM and DF designed the study. DF and FY collected and analyzed the patient data. AM prepared the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
==========================================
The study was approved by the Ethics Committee of The Second People\'s Hospital of Liaocheng (Linqing, China) and informed consent was signed by the patients or guardians.
Patient consent for publication
===============================
Not applicable.
Competing interests
===================
The authors declare that they have no competing interests.
{#f1-ol-0-0-9250}
![ROC curves of the two programs in the diagnosis of endometrial cancer. The AUC of convention ultrasound in the diagnosis of endometrial cancer is 0.754 \[95% CI: 0.211--2.534\], and that of TAP combined with vaginal ultrasound in the diagnosis of endometrial cancer is 0. 814 (95% CI: 0.517--0.932); the difference between the two programs is significant (P=0.011). ROC, receiver operating characteristic; AUC, area under the curve; TAP, tumor abnormal protein; CI, confidence interval.](ol-16-04-5186-g01){#f2-ol-0-0-9250}
######
Clinical data of patients.
Clinical data Control group (n=124)
----------------------------- -----------------------
Age (years) 41.6±11.6
Definite diagnostic results
Endometrial polyps 60
Endometrial hyperplasia 96
Uterine fibroids 17
Endometrial cancer 75
Nationality n (%)
Han 219 (88.31)
National minority 29 (11.69)
Place of residence n (%)
City 165 (66.53)
Countryside 83 (33.47)
Menopausal status n (%)
Premenopause 43 (17.34)
Postmenopause 205 (82.66)
######
Diagnostic accordance rates of TAP combined with conventional ultrasound and conventional ultrasound.
Rate Conventional ultrasound TAP combined with conventional ultrasound P-value
-------------------------------------------- ------------------------- ------------------------------------------- ---------
Total diagnostic accordance rate 218 (87.90) 234 (94.35) 0.031
Accordance rate of endometrial cancer 68 (90.67) 71 (94.67) 0.047
Accordance rate of endometrial hyperplasia 84 (87.50) 90 (93.75) 0.032
Accordance rate of endometrial polyps 53 (88.33) 57 (95.00) 0.024
Accordance rate of uterine fibroids 13 (76.47) 16 (94.12) 0.017
######
Imaging manifestations of conventional ultrasound.
Endometrial polyps Endometrial hyperplasia Uterine fibroids Endometrial cancer
--------------------------------------- ----------------------------------- ------------------------------------------- -------------------------------------------------- ------------------------------------------------------
Uterine volume No obvious increase No obvious change; increase in some parts Increased Obviously increased
Echo Hypoecho Equal echo Hyperecho Slightly enhanced Hypoecho; hyperecho in tiny minority Unevenly thickened
Intimal thickness change No significant Thickened Thickened Thickened
Lesion border Clear Clear uterine cavity line Relatively clear Rough surface; some unclear or even vanished borders
Lesion area liquidation/calcification Calcification in few lesion areas No liquidation/calcification Liquidation/calcification on in few lesion areas Liquidation in some lesion areas
[^1]: Contributed equally
|
{
"pile_set_name": "PubMed Central"
}
|
Background
==========
Molecular phylogenetics studies the hierarchical evolutionary relationships among species, or *taxa*, by means of molecular data such as DNA, RNA, amino acid or codon sequences. These relationships are usually described through a weighted tree, called a *phylogeny*, whose *leaves* represent the observed taxa, *internal vertices* represent the intermediate ancestors, *edges* represent the estimated evolutionary relationships, and *edge weights* represent measures of the similarity between pairs of taxa.
Accurately characterizing evolutionary relationships between organisms and their genomes is the basis of comparative genomic methods for interpreting meaning in sequence data, and for this reason the use of molecular phylogenetics has become widely used (and sometimes indispensable) in a multitude of research fields such as systematics, medical research, drug discovery, epidemiology, and population dynamics \[[@B3]\]. For example, the use of molecular phylogenetics was of considerable assistance in predicting the evolution of human influenza A \[[@B4]\], understanding the relationships between the virulence and the genetic evolution of HIV \[[@B5],[@B6]\], identifying emerging viruses as SARS \[[@B7]\], recreating and investigating ancestral proteins \[[@B8]\], designing neuropeptides causing smooth muscle contraction \[[@B9]\], and relating geographic patterns to macroevolutionary processes \[[@B10]\].
The literature on molecular phylogenetics proposes a number of criteria for selecting the phylogeny of a set $\mathcal{H}\,$of haplotypes extracted from *n* taxa from among plausible alternatives. The criteria can usually be quantified and expressed in terms of objective functions, giving rise to families of optimization problems whose general paradigm can be stated as follows \[[@B11]\]:
Problem 1
---------
-- *The Phylogeny Estimation Problem (PEP)*
$$\begin{array}{clc}
\textit{optimize} & {f\left( T \right)} & \\
{\qquad\mathit{s.t.}} & {g\left( \mathcal{H},T \right) = 1} & \\
& {T \in \mathcal{T},} & \\
\end{array}$$
where *T* a phylogeny of $\mathcal{H},$$\mathcal{T}\,$the set of all possible phylogenies of $\mathcal{H},$$\left. f:\mathcal{T}\rightarrow\mathbb{R} \right.$ a function modeling the selected criterion of phylogeny estimation, and $\left. g:\mathcal{H} \times \mathcal{T}\rightarrow\mathbb{R} \right.$ is a characteristic function equal to one if a phylogeny *T* is compatible (according to the selected criterion) for the set $\text{H.}$ A specific optimization problem is completely characterized by defining the functions *f* and *g*, and the phylogeny *T*^∗^ that optimizes *f* and satisfies *g* is referred to as *optimal*.
One of the classic criteria for phylogeny estimation is the *parsimony criterion*, which assumes that one taxon evolves from another by means of small changes and that the most plausible phylogeny will be that requiring the smallest number of changes. That evolution proceeds by small rather than smallest changes is due to the fact that the neighborhood of possible alleles that are selected at each instant of the life of a taxon is finite and, perhaps more important, that the selective forces acting on the taxon may not be constant throughout its evolution \[[@B12],[@B13]\]. Over the long term (periods of environmental change, including the intracellular environment), the accumulation of small changes will not generally correspond to the smallest possible set of changes consistent with the observed final sequences. Nevertheless, it is plausible to believe, at least for well-conserved molecular regions where mutations are reasonably rare and unlikely to have occurred repeatedly at any given variant locus, that the process of approximating small changes with smallest changes could provide a good approximation to the true evolutionary process of the observed set of taxa \[[@B14]\]. Such an assumption is likely to be reasonable, for example, in intraspecies phylogenetics, where few generations have elapsed since the observed taxa shared a common ancestor and thus the expected number of mutations per locus is much less than one. When such assumtions hold, a phylogeny of $\mathcal{H}\,$is defined to be optimal under the parsimony criterion if it satisfies the following requirements: (i) it has the shortest length, i.e., the minimum sum of the edge weights, and (ii) it is such that, for each pair of distinct haplotypes $h_{i},h_{j} \in \mathcal{H},$ the sum of the edge weights belonging to the path from *h*~*i*~ to *h*~*j*~ in *T*^∗^ is not smaller than the observed number of changes between *h*~*i*~ and *h*~*j*~\[[@B11]\]. The first condition imposes the assumption that the smallest number of mutations consistent with the observed sequences is a good approximation to the true accumulated set of mutations; the second condition correlates the edge weights to the observed data.
The parsimony assumption can be considered accurate in the limit of low mutation rates or short time scales, making it a reasonable model for situations such as analysis of intraspecies variation where little time is presumed to have elapsed since the existence of a common ancestor of all observed taxa. Maximum parsimony also remains valuable as a model for novel methodology development in phylogenetics because of its relative simplicity and amenity to theoretical analysis. Novel computational strategies, such as those developed in this paper, might therefore productively be developed and analyzed in the context of maximum parsimony before being extended to more complicated models of phylogenetics.
Finding the phylogeny that satisfies the parsimony criterion involves solving a specific version of the PEP, called the *Most Parsimonious Phylogeny Estimation Problem* (MPPEP). Some of the variants of the MPPEP, see e.g., \[[@B15],[@B16]\], can be solved in polynomial time, however, in the most general case, the problem is $\mathcal{N}\mathcal{P}$-hard \[[@B11],[@B17]\] and this fact has justified the development of a number of exact and approximate solution approaches, such those described in \[[@B11],[@B17],[@B18]\]. Some recent versions of the MPPEP, such as the Most Parsimonious Phylogeny Estimation Problem from SNP haplotypes (MPPEP-SNP) investigated in this article, play a fundamental role in providing predictions of practical use in several medical bioinformatics applications, such as disease association studies \[[@B19]\] or reconstruction of tumor phylogenies \[[@B20],[@B21]\]. In these contexts, it would be highly desirable to have the most accurate inferences possible for specific applications, but this in turn would imply to have algorithms able to exactly solve instances of such versions. As regards the MPPEP-SNP, the literature describes some (rare) circumstances for which it is possible to solve the problem in polynomial time (see Section Methods). Unfortunately, in the general case the MPPEP-SNP is $\mathcal{N}\mathcal{P}$-hard and solving provably to optimality therefore generally requires the use of exact approaches based on implicit enumeration algorithms, similar to the mixed integer programming strategies described in \[[@B1],[@B2],[@B22]\].
In this article, we explore the prospects for improving on the implicit enumeration strategy of \[[@B1],[@B2]\] using a novel problem formulation and a series of additional constraints to more precisely bound the solution space and accelerate implicit enumeration of possible optimal phylogenies. We present a formulation for the problem based on an adaptation of \[[@B23]\]'s mixed integer formulation for the Steiner tree problem extended with a number of preprocessing techniques and reduction rules to further decrease its size. We then show that it is possible to exploit the high symmetry inherent in most instances of the problem, through a series of strengthening valid inequalities, to eliminate redundant solutions and reduce the practical search space. We demonstrate through a series of empirical tests on real and artificial data that these novel insights into the symmetry of the problem often leads to significant reductions in the gap between the optimal solution and its non-integral linear programming bound relative to the prior art as well as often substantially faster processing of moderately hard problem instances. More generally, the work provides an indication of the conditions under which such an optimal enumeration approach is likely to be feasible, suggesting that these strategies are usable for relatively large numbers of taxa, although with stricter limits on numbers of variable sites. The work thus provides methodology suitable for provably optimal solution of some harder instances that resist all prior approaches. In future work, it may provide useful guidance for strategies and prospects of similar optimization methods for other variants of phylogeny inference.
Methods
=======
Notation and problem formulation
--------------------------------
Before proceeding, we shall introduce some notation and definitions that will prove useful throughout the article. The human genome is divided in 23 pairs of chromosomes, i.e., organized structures of DNA that contain many genes, regulatory elements and other nucleotide sequences. When a nucleotide site of a specific chromosome region shows a variability within a population of individuals then it is called a *Single Nucleotide Polymorphism* (SNP). Specifically, a site is considered a SNP if for a minority of the population a certain nucleotide is observed (called the minor allele) while for the rest of the population a different nucleotide is observed (the major allele). The minor allele, or *mutant type*\[[@B24]\], is generally encoded as '1', as opposed to the major allele, or *wild type*\[[@B24]\], generally encoded as '0'. A haplotype is a set of alleles, or more formally, a string of length *m* over an alphabet Σ = {0,1} \[[@B25]\].
Given a set $\mathcal{H}\,$of *n* haplotypes, denote $\mathcal{S} = \left\{ 1,2,\ldots,m \right\}$ as the set of alleles and *h*~*i*~(*s*), $s \in \mathcal{S}$, as the *s*-th allele of haplotype $h_{i} \in \mathcal{H}$. Given two distinct haplotypes $h_{i},h_{j} \in \mathcal{H},$ we denote $\mathcal{S}_{h_{i}h_{j}}$ as the subset of different alleles between *h*~*i*~ and *h*~*j*~, $\left. d_{h_{i}h_{j}} = \sum\limits_{s \in \mathcal{S}_{h_{i}h_{j}}} \middle| h_{i}\left( s \right) - h_{j}\left( s \right)| \right.$ as the *distance* between *h*~*i*~ and *h*~*j*~, and we say that *h*~*i*~ and *h*~*j*~ are *adjacent* if $d_{h_{i}h_{j}} = 1$. From a biological point of view, the adjacency between a pair of distinct haplotypes means that one of the two haplotypes evolved from the other by mutation of a specific SNP over time.
Consider a graph $G = \left( \mathcal{H},E \right)$ having a vertex for each haplotype in $\mathcal{H}\,$and an edge for each pair of adjacent haplotypes $h_{i},h_{j} \in \text{H.}$ Then, a *phylogenyT* of $\mathcal{H}\,$is a spanning tree of *G*, i.e., an acyclic subgraph of *G* in which a pair of vertices $h_{i},h_{j} \in \mathcal{H}$ is adjacent in *T* if $d_{h_{i}h_{j}} = 1$. It is worth noting that, according to the definition of the edge set *E*, in general a phylogeny of $\mathcal{H}\,$may not exist as the graph $G = \left( \mathcal{H},E \right)$ might not be connected. To always ensure the existence of a phylogeny for $\text{H,}$ we introduce the set $\mathcal{H}^{\prime}$ which consists of the minimum number of haplotypes that should be added to $\mathcal{H}\,$in such a way that, defined $\overline{\mathcal{H}} = \mathcal{H} \cup \mathcal{H}^{\prime}$ and $\overline{E} = \left\{ \left( h_{i},h_{j} \right):h_{i},h_{j} \in \overline{\mathcal{H}}\quad\text{and}\quad d_{h_{i}h_{j}} = 1 \right\}$, the graph $\overline{G} = \left( \overline{\mathcal{H}},\overline{E} \right)$ is connected. From a biological point of view, the set $\mathcal{H}^{\prime}$ represents the set of haplotypes that are ancestors of the observed ones but either had gone extinct or just were not observed in that sample (also called Steiner nodes).
Denote $\overline{T}$ as a phylogeny of $\overline{H}$, $\overline{E}\left( \overline{T} \right)$ as the edge set of $\overline{T}$, and $L\left( \overline{T} \right)$ as the *length* of the phylogeny $\overline{T}$, i.e., the sum of the distances $d_{h_{i}h_{j}}$, for all $\left( h_{i},h_{j} \right) \in \overline{E}\left( \overline{T} \right)$. Then, the problem of finding a phylogeny of $\mathcal{H}\,$that satisfies the parsimony criterion consists of solving the following optimization problem:
### Problem 2
The Most Parsimonious Phylogeny Estimation Problem from SNP haplotypes (MPPEP-SNP).Given a set $\mathcal{H}\,$of *n* haplotypes having *m* alleles each, find the minimum cardinality haplotype set $\mathcal{H}^{\prime}$ to be added to $\mathcal{H}\,$so that the phylogeny ${\overline{T}}^{\star}$ has minimum length.
If the haplotype set $\mathcal{H}^{\prime}$ is empty, i.e., if $G = \left( \mathcal{H},E \right)$ is connected, then MPPEP-SNP can be solved in polynomial time as the minimum spanning tree is a (optimal) solution to the MPPEP-SNP. Similarly, if the input haplotype set $\mathcal{H}\,$satisfies the *perfect phylogeny condition* i.e., the requirement that each allele changes only once throughout the optimal phylogeny (see \[[@B19]\]), then the MPPEP-SNP can be still solved in polynomial time \[[@B26]-[@B28]\]. Unfortunately, it is possible to prove that in the general case the MPPEP-SNP is $\mathcal{N}\mathcal{P}$-hard (see \[[@B1],[@B22]\]). In fact, the binary nature of the SNP haplotypes allows us to interpret a generic haplotype $h_{i} \in \mathcal{H}$ as a vertex of a *m*-dimensional unit hypercube, its *s*-th allele as the *s*-th coordinate of the vertex *h*~*i*~, and the set $\mathcal{H}^{\prime}$ as the set of Steiner vertices of the unit hypercube. Hence the MPPEP-SNP can be seen as particular case of the Steiner tree problem in a graph, a notorious $\mathcal{N}\mathcal{P}$-hard combinatorial optimization problem \[[@B29]\].
Finding the optimal solutions to the MPPEP-SNP is fundamental to validating the parsimony criterion, i.e., to verify whether, for a given instance of MPPEP-SNP, the results predicted by the criterion match the experimental ones. Unfortunately, the $\mathcal{N}\mathcal{P}$-hardness of the MPPEP-SNP limits the size of the instances analyzable to the optimum, which in turn affects the ability to validate the parsimony criterion, hence the practical utility of the predictions themselves. In order to address this concern, in the following section we shall develop an integer programming model able to provide optimal solutions to real instances of the MPPEP-SNP.
A mixed integer programming model for the MPPEP-SNP
---------------------------------------------------
Let $V = \left\{ 1,2,\ldots,n,n + 1,n + 2,\ldots,n + \middle| \mathcal{H}^{\prime} \middle| \right\}$ the set of potential vertices of a phylogeny $\overline{T}$ of $\mathcal{H}\,$and assume the convention to denote the *n* haplotypes in $\mathcal{H}\,$as the first *n* vertices in *V*. The first task in designing an integer programming model for the MPPEP-SNP that uses a polynomial-size number of variables consists of characterizing *V*, i.e., determining an upper and a lower bound on the cardinality of the set $\mathcal{H}^{\prime}$. In fact, observe that $\mathcal{H}^{\prime}$ contains potentially an exponential number of haplotypes, namely all vertices of the unit hypercube that belong to the set $\left\{ 0,1 \right\}^{m} \smallsetminus \mathcal{H}$. However, we can easily bound the cardinality of $\mathcal{H}^{\prime}$ by means of the following approach. Consider the complete graph $Ĝ = \left( \mathcal{H},Ê \right)$, where $Ê = \left\{ \left( h_{i},h_{j} \right)\;:h_{i},h_{j} \in \mathcal{H} \right\}$, and construct a minimum spanning tree $T_{Ĝ}$ of $\text{Ĝ.}$ Denote $E\left( T_{Ĝ} \right)$ as the set of edges (*h*~*i*~,*h*~*j*~) of $T_{Ĝ}$. Then, an upper bound UB on the overall number of Steiner vertices of the optimal phylogeny ${\overline{T}}^{\star}$ can be obtained by computing the sum
$$\begin{matrix}
{\mathit{UB} = \underset{(h_{i},h_{j}) \in E(T_{Ĝ})}{\sum}\left( d_{h_{i}h_{j}} - 1 \right).} & & & \\
\end{matrix}$$
Similarly, denote $L\left( T_{Ĝ} \right) = \sum\limits_{(h_{i},h_{j}) \in E(T_{Ĝ})}d_{h_{i}h_{j}}$, a lower bound LB on the overall number of Steiner vertices of ${\overline{T}}^{\star}$ can be obtained as \[[@B30],[@B31]\]:
$$\begin{matrix}
{\mathit{LB} = \left\lceil {\frac{L\left( T_{Ĝ} \right)}{2} - n + 1} \right\rceil.} & & & \\
\end{matrix}$$
Denote *u*~*i*~, *i* ∈ *V*, as a decision variable equal to 1 if the *i*-th vertex of *V* is considered in the optimal solution to the MPEPP-SNP and 0 otherwise; $x_{i}^{s}$ as a decision variable equal to 1 if in the optimal solution to the MPPEP-SNP the *s*-th coordinate of the vertex *u*~*i*~, *i* ∈ *V*, is 1 and 0 otherwise; $z_{\textit{ij}}^{s}$ as a decision variable equal to 1 if in the optimal solution to the MPPEP-SNP the pair of distinct vertices *i*,*j* ∈ *V* has a change at *s*-th coordinate, and 0 otherwise; and *y*~*ij*~ as a decision variable equal to 1 if the pair of distinct vertices *i*,*j*∈*V*is adjacent in the optimal solution to the MPPEP-SNP and 0 otherwise. Finally, let $V_{\mathcal{H}} = \left\{ 1,2,\ldots,n \right\}$, $V_{\mathcal{H}^{\prime}} = \left\{ n + 1,n + 2,\ldots,n + \mathit{UB} \right\}$, and *Q* = {1,2,...,*n* + *LB*}. Then, a valid formulation for the MPPEP-SNP is the following:
### Formulation 1
Basic Model
$$\begin{array}{llll}
\min & {\quad\underset{i,j \in V:i \neq j}{\sum}\underset{s \in \mathcal{S}}{\sum}z_{\textit{ij}}^{s}\qquad} & \quad & \qquad \\
\end{array}$$
$$\begin{array}{lll}
{s.t.} & {\quad\quad\quad x_{i}^{s} = h_{i}\left( s \right)\qquad} & {\mspace{216mu}\forall\quad s \in \mathcal{S},i \in \mathcal{H}\qquad} \\
\end{array}$$
$$\begin{array}{lll}
& {\quad\quad\quad\quad\quad x_{i}^{s} \leq u_{i}\qquad} & {\mspace{270mu}\forall\quad s \in \mathcal{S},i \in V\qquad} \\
\end{array}$$
$$\begin{array}{lll}
& {\mspace{85mu} z_{\textit{ij}}^{s} \geq + x_{i}^{s} - x_{j}^{s} + y_{\textit{ij}} - 1\qquad} & {\mspace{117mu}\forall\quad s \in \mathcal{S},i,j \in V:i \neq j\qquad} \\
\end{array}$$
$$\begin{array}{lll}
& {\mspace{85mu} z_{\textit{ij}}^{s} \geq - x_{i}^{s} + x_{j}^{s} + y_{\textit{ij}} - 1\qquad} & {\mspace{117mu}\forall\quad s \in \mathcal{S},i,j \in V:i \neq j\qquad} \\
\end{array}$$
$$\begin{array}{lll}
& {\mspace{81mu}\underset{s \in \mathcal{S}}{\sum}z_{\textit{ij}}^{s} = y_{\textit{ij}}\qquad} & {\mspace{225mu}\forall\quad i,j \in V:i \neq j\qquad} \\
\end{array}$$
$$\begin{array}{lll}
& {\mspace{81mu} y_{\textit{ij}} \leq u_{i}\qquad} & {\mspace{270mu}\forall\quad i,j \in V:i \neq j\qquad} \\
\end{array}$$
$$\begin{array}{lll}
& {\mspace{81mu} y_{\textit{ij}} \leq u_{j}\qquad} & {\mspace{270mu}\forall\quad i,j \in V:i \neq j\qquad} \\
\end{array}$$
$$\begin{array}{lll}
& {\mspace{45mu}\underset{j \in V:i \neq j}{\sum}y_{\textit{ij}} \geq u_{i}\qquad} & {\mspace{194mu}\forall\quad i \in V\qquad} \\
\end{array}$$
$$\begin{array}{lll}
& {\mspace{45mu}\underset{i,j \in C:i \neq j}{\sum}y_{\textit{ij}} \leq \underset{i \in C}{\sum}u_{i} - 1\qquad} & {\mspace{108mu}\forall\quad C \subset V:C \cap V_{\mathcal{H}} \neq \varnothing\qquad} \\
\end{array}$$
$$\begin{array}{llll}
& {\mspace{45mu}\underset{i,j \in V:i \neq j}{\sum}y_{\textit{ij}} = \underset{i \in V}{\sum}u_{i} - 1\qquad} & \quad & \qquad \\
\end{array}$$
$$\begin{array}{llll}
& {\mspace{72mu}\underset{i \in Q}{\sum}u_{i} = n + \mathit{LB}\qquad} & \quad & \qquad \\
\end{array}$$
$$\begin{array}{llll}
& {\mspace{72mu} u_{i}, x_{i}^{s}, z_{\textit{ij}}^{s},y_{\textit{ij}} \in \left\{ 0,1 \right\}.\qquad} & \quad & \qquad \\
\end{array}$$
The objective function (1a) aims at minimizing the length of the optimal phylogeny. Constraints (1b) impose that the coordinates of the first *n* vertices in *V* are exactly the ones of the input haplotype set $\text{H.}$ Constraints (1c) impose that the *s*-th coordinate of vertex *u*~*i*~, *i* ∈ *V*, can assume value 1 only if vertex *u*~*i*~ is considered in the optimal solution to the problem. Constraints (1d)-(1e) force variables $z_{\textit{ij}}^{s}$ to be one if in the optimal solution to the problem there exist a pair of adjacent vertices *ij* ∈ *V* having a different value at the *s*-th coordinate. Constraints (1f) impose that in an optimal solution to the problem two distinct vertices *ij* ∈ *V* can be adjacent only if $d_{h_{i}h_{j}} = 1$. Constraints (1g)-(1h) impose that in the optimal solution to the problem variable *y*~*ij*~ may assume value 1 only if both vertices *i* and *j* are considered. Vice versa, constraints (1i) impose that if in the optimal solution to the problem a vertex *u*~*i*~, *i* ∈ *V*, is considered then at least one variable *y*~*ij*~ must assume value 1. Constraints (1j) and (1k) impose the Generalized Subtour Elimination Constraints (GSEC) \[[@B23]\]. Finally, constraints (1l) impose that the first *n* + *LB*vertices in *V* have to be considered in the optimal solution to the problem.
Note that Formulation 1 can be easily extended to the case in which the haplotypes are represented by multi-character data, i.e., sequences over an alphabet *Σ* = {0,1,2,...,*γ*}. In fact, in such a case it is sufficient to replace each character *c* in the haplotype by a binary *γ* -vector *ν* such that the *s*-th coordinate of *ν* is equal to 1 if the character *c* is equal to *s*, *s* ∈ Σ, and 0 otherwise. For example, if the generic haplotype were, for example, the string 〈*AACGT*〉, then it could be represented as 〈1000 1000 0100 0010 0001〉.
Reducing model size
-------------------
Formulation 1 is characterized by a polynomial number of variables and an exponential number of constraints. Its implementation can be performed by means of standard branch-and-cut approaches that use GSEC separation oracles such as those described in \[[@B32]\].
It is worth noting that variables $x_{i}^{s}$ and $z_{\textit{ij}}^{s}$ can be relaxed in Formulation [1c](#F1){ref-type="fig"})-(1e) and the convexity constraint (1f) suffice to guarantee their integrality in any optimal solution to the problem. Moreover, Formulation 1 can be reduced in size by removing those variables that are redundant or whose value is known in the optimal solution to the problem. For example, variables *y*~*ij*~ can be removed from Formulation 1 as it is easy to realize that they are redundant. Similarly, all variables $z_{\textit{ij}}^{s}$ such that $i,j \in V_{\mathcal{H}}$ and *d*~*ij*~ \> 1 do not need to be defined as their value will be always zero for any $s \in \mathcal{S}$ and in any feasible solution to the problem. Variables *u*~*i*~, *i* ∈ *Q*, do not need to be declared as their value will be always 1 any feasible solution to the problem. Finally, variables $x_{i}^{s}$, $i \in V_{\mathcal{H}}$, can be removed as their value is univocally assigned by the input haplotype set $\text{H.}$ The reduction process can be further combined with the preprocessing strategies described in \[[@B1]\] to obtain even smaller formulations. Such strategies allow one to remove alleles from the input haplotype set $\mathcal{H}\,$without altering the optimal solution to the problem. For example, suppose that the haplotype set $\mathcal{H}\,$is such that there exists an allele $ŝ \in \mathcal{S}$ such that $h_{i}\left( ŝ \right) = 1$, for all $h_{i} \in \mathcal{H}$; then it is easy to realize that $ŝ\,$can be removed from $\mathcal{S}\,$as in any feasible solution to the problem the $\text{ŝ-th}$ coordinate of any vertex in the phylogeny would be characterized by having *xiŝ* = 1. A similar situation occurs when there exists an allele $ŝ \in \mathcal{S}$ such that $h_{i}\left( ŝ \right) = 0$, for all $h_{i} \in \mathcal{H}$. Analogously, suppose that the input haplotype set $\mathcal{H}\,$is characterized by *equal alleles*, i.e., by the existence of two alleles, say $ŝ_{1}$ and $ŝ_{2}$, such that $h_{i}\left( ŝ_{1} \right) = h_{i}\left( ŝ_{2} \right)$, for all $i \in \mathcal{S}$. Then it is easy to realize that if one removes one of the two alleles from $\text{S,}$ say $ŝ_{2}$, and multiplies the $ŝ_{1}$-th coordinate by 2 does not alter neither the structure nor the value of the optimal solution to the problem. Describing all the preprocessing techniques for shrinking the input haplotype set $\mathcal{H}\,$is beyond the scope of the present article. The interested reader will find a systematic discussion of this topic in \[[@B1]\].
By applying the previously cited reduction strategies to Formulation 1 and denoting $Ŝ\,$as the set of alleles resulting from the application of the preprocessing strategies described in \[[@B1]\], *w*^*s*^ as the number of alleles in $\mathcal{S}\,$equal to the *s*-th, $s \in Ŝ$, *Z* as the set for which variables $z_{\textit{ij}}^{s}$ are defined, *R* = {*n* + *LB* + 1,*n* + *LB* + 2,...,*n* + *UB*}, and $C_{\mathcal{H}} = \left\{ i \in C:i \in V_{\mathcal{H}} \right\}$, for any *C* ⊂ *V*, the following reduced formulation for the MPPEP-SNP can be obtained:
### Formulation 2
Reduced Model
$$\begin{array}{ll}
\min & {\underset{\begin{matrix}
{i,j \in V:} \\
{i,j \in Z} \\
\end{matrix}}{\sum}\;\underset{s \in Ŝ}{\sum}w^{s}z_{\textit{ij}}^{s}\qquad} \\
\end{array}$$
$$\begin{array}{llc}
{s.t.} & {\quad x_{i}^{s} \leq u_{i}\qquad} & {\mspace{230mu}\forall\quad s \in Ŝ,i \in R\qquad} \\
\end{array}$$
$$\begin{array}{ll}
{\underset{\begin{matrix}
{s^{\prime} \in Ŝ:} \\
{s^{\prime} \neq s} \\
\end{matrix}}{\sum}z_{\textit{ij}}^{s^{\prime}} + h_{i}\left( s \right) - x_{j}^{s} \leq 1\qquad} & {\mspace{99mu}\forall\quad s \in Ŝ,\quad i \in V_{\mathcal{H}},j \in V_{\mathcal{H}^{\prime}}\qquad} \\
\end{array}$$
$$\begin{array}{ll}
{\underset{\begin{matrix}
{s^{\prime} \in Ŝ:} \\
{s^{\prime} \neq s} \\
\end{matrix}}{\sum}z_{\textit{ij}}^{s^{\prime}} - h_{i}\left( s \right) + x_{j}^{s} \leq 1\qquad} & {\mspace{99mu}\forall\quad s \in Ŝ,\quad i \in V_{\mathcal{H}},j \in V_{\mathcal{H}^{\prime}}\qquad} \\
\end{array}$$
$$\begin{array}{ll}
{\underset{\begin{matrix}
{s^{\prime} \in Ŝ:} \\
{s^{\prime} \neq s} \\
\end{matrix}}{\sum}z_{\textit{ij}}^{s^{\prime}} + x_{i}^{s} - x_{j}^{s} \leq 1\qquad} & {\mspace{121mu}\forall\quad s \in Ŝ,\quad i,j \in V_{\mathcal{H}^{\prime}}:i,j \in Z\qquad} \\
\end{array}$$
$$\begin{array}{ll}
{\underset{\begin{matrix}
{s^{\prime} \in Ŝ:} \\
{s^{\prime} \neq s} \\
\end{matrix}}{\sum}z_{\textit{ij}}^{s^{\prime}} - x_{i}^{s} + x_{j}^{s} \leq 1\qquad} & {\mspace{122mu}\forall\quad s \in Ŝ,\quad i,j \in V_{\mathcal{H}^{\prime}}:i,j \in Z\qquad} \\
\end{array}$$
$$\begin{array}{ll}
{\underset{s \in Ŝ}{\sum}z_{\textit{ij}}^{s} \leq 1\qquad} & {\mspace{243mu}\forall\quad i,j \in V \smallsetminus R:i,j \in Z\qquad} \\
\end{array}$$
$$\begin{array}{l}
{\underset{s \in Ŝ}{\sum}z_{\textit{ij}}^{s} \leq u_{i}\mspace{252mu}\forall\quad i \in R,j \in V:i,j \in Z\qquad} \\
\end{array}$$
$$\begin{array}{l}
{\underset{s \in Ŝ}{\sum}z_{\textit{ij}}^{s} \leq u_{j}\mspace{252mu}\forall\quad j \in R,i \in V:i,j \in Z\qquad} \\
\end{array}$$
$$\begin{array}{l}
{\underset{\begin{matrix}
{j \in V:} \\
{j \in Z} \\
\end{matrix}}{\sum}\underset{s \in Ŝ}{\sum}z_{\textit{ij}}^{s} \geq 1\mspace{216mu}\forall\quad i \in Q\qquad} \\
\end{array}$$
$$\begin{array}{l}
{\underset{\begin{matrix}
{j \in V:} \\
{j \in Z} \\
\end{matrix}}{\sum}\underset{s \in Ŝ}{\sum}z_{\textit{ij}}^{s} \geq u_{i}\mspace{212mu}\forall\quad i \in R\qquad} \\
\end{array}$$
$$\begin{array}{cl}
\left. \underset{\begin{matrix}
{i,j \in C:} \\
{i,j \in Z} \\
\end{matrix}}{\sum}\underset{s \in Ŝ}{\sum}z_{\textit{ij}}^{s} \leq \underset{i \in C:i \in R}{\sum}u_{i} + \middle| C_{\mathcal{H}} \middle| - 1 \right. & \\
& {\forall\quad C \subset V:C \cap V_{\mathcal{H}} \neq \varnothing\qquad} \\
\end{array}$$
$$\begin{array}{l}
{\underset{\begin{array}{l}
{i,j \in V:} \\
{i,j \in Z} \\
\end{array}}{\sum}\underset{s \in Ŝ}{\sum}z_{\textit{ij}}^{s} = n + \mathit{LB} + \underset{i \in R}{\sum}u_{i} - 1\qquad} \\
\end{array}$$
$$\begin{array}{l}
{u_{i}, x_{i}^{s}, z_{\textit{ij}}^{s},y_{\textit{ij}} \in \left\{ 0,1 \right\}.\qquad} \\
\end{array}$$
Note that in Formulation 2 variables $x_{i}^{s}$ and $z_{\textit{ij}}^{s}$ cannot be relaxed anymore.
Strengthening valid inequalities
--------------------------------
By exploiting the integrality of variables *u*~*i*~, $x_{i}^{s}$, and $z_{\textit{ij}}^{s}$, a number of valid inequalities can be developed to strengthen Formulation 2.
### Proposition 1
Constraints
$$\begin{matrix}
{u_{i + 1} \leq u_{i}} & \quad & {\forall\quad i \in V \smallsetminus \left( Q \cup \left\{ n + \mathit{UB} \right\} \right)} & {} \\
\end{matrix}$$
are valid for Formulation 2.
### Proof
In a feasible solution to the problem variable *u*~*i*~, *i* ∈ *V*∖(*Q* ∪ {*n* + *UB*}), can assume only value 0 or 1. If *u*~*i*~ = 0, constraint (3) reduces to *u*~*i*\ +\ 1~ ≤ 0 which is trivially valid for Formulation 2. If *u*~*i*~ = 1, constraint (3) reduces to *u*~*i*\ +\ 1~ ≤ 1 which is again valid. □
Constraints (3) impose an ordering on the variables *u*~*i*~, *i* ∈ *R*, so that vertex *u*~*i*\ +\ 1~ can be considered in the optimal solution to the problem only if vertex *u*~*i*~ has been already considered.
### Proposition 2
Constraints
$$\begin{matrix}
{\underset{\begin{matrix}
{j \in V:} \\
{j \in Z} \\
\end{matrix}}{\sum}\;\underset{s \in Ŝ}{\sum}z_{\textit{ij}}^{s} \geq 2u_{i}} & \quad & {\forall\quad i \in R} & {} \\
\end{matrix}$$
are valid for Formulation 2.
### Proof
In a feasible solution to the problem a vertex *u*~*i*~, $i \in V_{\mathcal{H}^{\prime}}$, cannot be a terminal vertex. In fact, if such a condition held, a cheaper solution could be obtained by dropping *u*~*i*~ from ${\overline{T}}^{\star}$, contradicting the optimality of ${\overline{T}}^{\star}$ itself. Hence, the degree of any vertex in $V_{\mathcal{H}^{\prime}}$ must be at least 2. Now, in a feasible solution to the problem variables *u*~*i*~ ∈ {0,1}. If *u*~*i*~ = 0, constraint (4) reduces to
$$\underset{\begin{matrix}
{j \in V:} \\
{j \in Z} \\
\end{matrix}}{\sum}\underset{s \in Ŝ}{\sum}z_{\textit{ij}}^{s} \geq 0$$
which is trivially valid. Vice versa, if *u*~*i*~ = 1, constraint (4) reduces to
$$\underset{\begin{matrix}
{j \in V:} \\
{j \in Z} \\
\end{matrix}}{\sum}\underset{s \in Ŝ}{\sum}z_{\textit{ij}}^{s} \geq 2$$
which is again valid for the above arguments. □
### Proposition 3
Constraints
$$\begin{array}{lll}
+ & {x_{i}^{s_{2}} - x_{j}^{s_{2}} \leq 2\left( 1 - z_{\textit{ij}}^{s_{1}} \right) - \underset{\begin{array}{l}
{\quad s \in Ŝ:s \neq s_{1}} \\
\end{array}}{\sum}\quad z_{\textit{ij}}^{s}\qquad} & \qquad \\
& {\forall\quad s_{1},s_{2} \in Ŝ:s_{1} \neq s_{2},\quad i,j \in V_{\mathcal{H}^{\prime}}:i,j \in Z\qquad} & \\
\end{array}$$
$$\begin{array}{lll}
- & {x_{i}^{s_{2}} + x_{j}^{s_{2}} \leq 2\left( 1 - z_{\textit{ij}}^{s_{1}} \right) - \underset{\begin{array}{l}
{\quad s \in Ŝ:s \neq s_{1}} \\
\end{array}}{\sum}\quad z_{\textit{ij}}^{s}\qquad} & \qquad \\
& {\forall\quad s_{1},s_{2} \in Ŝ:s_{1} \neq s_{2},\quad i,j \in V_{\mathcal{H}^{\prime}}:i,j \in Z\qquad} & \\
\end{array}$$
are valid for Formulation 2.
### Proof
As observed in the previous proposition, in a feasible solution to the problem $\underset{s \in Ŝ}{\sum}z_{\textit{ij}}^{s}$, $i,j \in V_{\mathcal{H}^{\prime}}$, *i*,*j*∈*Z*, can assume only value 0 or 1. If $\underset{s \in Ŝ}{\sum}z_{\textit{ij}}^{s} = 0$, then constraint (5) (respectively constraint (6)) reduces to $+ x_{i}^{s_{2}} - x_{j}^{s_{2}} \leq 2$ (respectively $- x_{i}^{s_{2}} + x_{j}^{s_{2}} \leq 2$), which is trivially valid due to the integrality of variables $x_{i}^{s}$. If $\sum\limits_{s \in Ŝ}z_{\textit{ij}}^{s} = 1$, then either $\underset{\begin{matrix}
{s \in Ŝ:} \\
{s \neq s_{1}} \\
\end{matrix}}{\sum}z_{\textit{ij}}^{s} = 1$ or $z_{\textit{ij}}^{s_{1}} = 1$. If $\underset{\begin{matrix}
{s \in Ŝ:} \\
{s \neq s_{1}} \\
\end{matrix}}{\sum}z_{\textit{ij}}^{s} = 1$ then constraint (5), (respectively constraint (6)) reduces to $+ x_{i}^{s_{2}} - x_{j}^{s_{2}} \leq 1$ (respectively $- x_{i}^{s_{2}} + x_{j}^{s_{2}} \leq 1$), which is trivially valid. If $z_{\textit{ij}}^{s_{1}} = 1$ then constraint (5) (respectively constraint (6)) reduces to $+ x_{i}^{s_{2}} - x_{j}^{s_{2}} \leq 0$ (respectively $- x_{i}^{s_{2}} + x_{j}^{s_{2}} \leq 0$), which is again valid. □
Similar arguments can be used to prove the following proposition:
### Proposition 4
Constraints
$$\begin{array}{lll}
+ & {h_{i}\left( s_{2} \right) - x_{j}^{s_{2}} \leq 2\left( 1 - z_{\textit{ij}}^{s_{1}} \right) - \underset{\begin{array}{l}
{s \in Ŝ:} \\
{s \neq s_{1}} \\
\end{array}}{\sum}z_{\textit{ij}}^{s}\qquad} & \qquad \\
& {\forall\quad s_{1},s_{2} \in Ŝ:s_{1} \neq s_{2},\quad i \in V_{\mathcal{H}},\quad j \in V_{\mathcal{H}^{\prime}}\qquad} & \\
\end{array}$$
$$\begin{array}{lll}
- & {h_{i}\left( s_{2} \right) + x_{j}^{s_{2}} \leq 2\left( 1 - z_{\textit{ij}}^{s_{1}} \right) - \underset{\begin{array}{l}
{s \in Ŝ:} \\
{s \neq s_{1}} \\
\end{array}}{\sum}z_{\textit{ij}}^{s}\qquad} & \qquad \\
& {\forall\quad s_{1},s_{2} \in Ŝ:s_{1} \neq s_{2},\quad i \in V_{\mathcal{H}},\quad j \in V_{\mathcal{H}^{\prime}}\qquad} & \\
\end{array}$$
are valid for Formulation 2.
Given an input haplotype set $\mathcal{H}\,$and a pair of non-adjacent haplotypes *h*~*i*~ and *h*~*j*~, there exit multiple equivalent paths that may connect *h*~*i*~ and *h*~*j*~ in the unary hypercube. This characteristic constitutes the principal class of symmetries for the MPPEP-SNP and may lead to poor relaxations for the problem. For example, suppose that the input haplotype set $\mathcal{H}\,$is constituted by haplotypes *h*~1~ = 〈00〉 and *h*~2~ = 〈11〉. Then a possible solution to the instance may consist either of a star centered in haplotype *h*~3~ = 〈10〉 or a star centered in haplotype *h*~3~ = 〈01〉(see Figure [1](#F1){ref-type="fig"}). Note that both solutions are feasible and optimal for the specific instance. A possible strategy to break this class of symmetries consists of imposing the following constraints:
{#F1}
### Proposition 5
Constraints
$$\begin{array}{lll}
& {\underset{p = 1}{\overset{s}{\sum}}2^{s - p}x_{i}^{p} \leq \underset{p = 1}{\overset{s}{\sum}}2^{s - p}x_{i + 1}^{p}\qquad} & \qquad \\
& {\quad\forall\quad s \in Ŝ,i \in V_{\mathcal{H}^{\prime}} \smallsetminus R\qquad} & \\
\end{array}$$
$$\begin{array}{lll}
& {\underset{p = 1}{\overset{s}{\sum}}2^{s - p}x_{i}^{p} \leq \underset{p = 1}{\overset{s}{\sum}}2^{s - p}x_{i + 1}^{p} + \underset{p = 1}{\overset{s}{\sum}}2^{s - p}\left( 1 - u_{i + 1} \right)\qquad} & \qquad \\
& {\quad\forall\quad s \in Ŝ,i \in R \smallsetminus \left\{ n + \mathit{UB} \right\}\qquad} & \\
\end{array}$$
are valid for Formulation 2.
### Proof
The statement trivially follows from the integrality of variables $x_{i}^{s}$ and from constraints (2b). □
Constraints (9)-(10) impose an ordering on the coordinates of the vertices in $V_{\mathcal{H}^{\prime}}$ by means of the smallest big-M possible, i.e., a power of 2. Note that the distinction between constraints (9) and (10) is necessary, as in principle vertices in *R* may not be needed in the optimal solution to the problem.
### Proposition 6
Constraints
$$\begin{matrix}
{\underset{\begin{matrix}
{j \in V:} \\
{j \in Z} \\
\end{matrix}}{\sum}\;\underset{s \in Ŝ}{\sum}z_{\textit{ij}}^{s} \geq \underset{\begin{matrix}
{j \in V:} \\
{j \in Z} \\
\end{matrix}}{\sum}\;\underset{s \in Ŝ}{\sum}z_{(i + 1)j}^{s}} & & {\forall i \in V_{\mathcal{H}^{\prime}} \smallsetminus \left\{ n + \mathit{UB} \right\}} & \\
& & & {} \\
\end{matrix}$$
are valid for Formulation 2.
### Proof
In a feasible solution to the problem, the sum $\underset{s \in Ŝ}{\sum}z_{\textit{ij}}^{s}$, $i,j \in V_{\mathcal{H}^{\prime}}$, *i*,*j*∈*Z*, can assume only value 0 or 1. If $\underset{\begin{matrix}
{j \in V:} \\
{j \in Z} \\
\end{matrix}}{\sum}\;\underset{s \in Ŝ}{\sum}z_{(i + 1)j}^{s} = 0$, constraint (11) reduces to $\underset{\begin{matrix}
{j \in V:} \\
{j \in Z} \\
\end{matrix}}{\sum}\;\underset{s \in Ŝ}{\sum}z_{\textit{ij}}^{s} \geq 0$ which is trivially valid. Vice versa, If $\underset{\begin{matrix}
{j \in V:} \\
{j \in Z} \\
\end{matrix}}{\sum}\;\underset{s \in Ŝ}{\sum}z_{(i + 1)j}^{s} = 1$, constraint (11) reduces to $\underset{\begin{matrix}
{j \in V:} \\
{j \in Z} \\
\end{matrix}}{\sum}\;\underset{s \in Ŝ}{\sum}z_{\textit{ij}}^{s} \geq 1$ which is again valid due to Propositions 1 and 2. □
Proposition 6 forces vertices in $V_{\mathcal{H}^{\prime}}$ to be sorted according to a decreasing degree order. In this way, it is possible to avoid the occurrence of symmetric solutions to the problem differing just for the degree of the Steiner vertices (see e.g., Figure [2](#F2){ref-type="fig"}).
{#F2}
Let $Q_{3} = \left\{ i,j \in V_{\mathcal{H}}:d_{\textit{ij}} \geq 3 \right\}$ and *k* ∈ *V*, *k* ∉ *Q*~3~. Then the following proposition holds:
### Proposition 7
Constraints
$$\begin{matrix}
{\underset{s \in \mathcal{S}}{\sum}z_{\textit{ik}}^{s} + \underset{s \in \mathcal{S}}{\sum}z_{\textit{kj}}^{s} \leq 1} & \quad & {\forall\quad i,j \in Q_{3}} & {} \\
\end{matrix}$$
are valid for Formulation 2.
### Proof
In a feasible solution to the problem the path between two distinct haplotypes $h_{i},h_{j} \in \mathcal{H}$ cannot be shorter than the distance $d_{h_{i}h_{j}}$. Hence, if the distance between *h*~*i*~and *h*~*j*~is greater or equal to three, vertices *i* and *j* cannot be adjacent to a same vertex *k*, i.e., only one of the two sums $\sum\limits_{s \in \mathcal{S}}z_{\textit{ik}}^{s}$ or $\sum\limits_{s \in \mathcal{S}}z_{\textit{jk}}^{s}$ can be equal to 1. □
Note that if *k* ∈ *R* then (12) can be strengthened by replacing the right-hand-side by *u*~*k*~. Moreover, Proposition 7 can be generalized as follows. Consider the sets $Q_{d} = \left\{ i,j \in V_{\mathcal{H}}:d_{\textit{ij}} \geq d \right\}$, *d* ∈ {3,4,...,*m*}, *C* ⊂ *V* such that 2 ≤ \|*C*\| ≤ *d* − 1 and *C* ∩ *Q*~*d*~ = *∅*, and a path *p* that involves only vertices in *C*. Denote *p*~*k*~ the *k*-th vertex in *p*. Then the following proposition holds:
### Proposition 8
The family of constraints
$$\begin{matrix}
\left. \underset{s \in \mathcal{S}}{\sum}z_{\mathit{ip}_{1}}^{s}\; + \;\underset{k = 1}{\overset{|C| - 1}{\sum}}\underset{s \in \mathcal{S}}{\sum}z_{p_{k}p_{k + 1}}^{s}\; + \;\underset{s \in \mathcal{S}}{\sum}z_{p_{|C|j}}^{s}\; \leq \; \middle| C \right| & \quad & {\mspace{54mu}\forall\quad i,j \in K_{d},} & \\
& & & {} \\
\end{matrix}$$
called forbidden path constraints, are valid for Formulation 2.
### Proof
Similarly to Proposition 7, in a feasible solution to the problem the path *p* between two distinct haplotypes $h_{i},h_{j} \in \mathcal{H}$ cannot be shorter than the distance $d_{h_{i}h_{j}}$. Hence, if the distance between *h*~*i*~ and *h*~*j*~ is greater or equal to *d*, at most \|*C*\| vertices can belong to *p*. □
Experiments
===========
In this section we analyze the performance of our model to solve the MPPEP-SNP. Our experiments were motivated by a twofold reason, namely: to evaluate, with respect to Formulation 1, the benefits obtained by the removal of the redundant variables and by the inclusion of the valid inequalities previously described; and to allow the analysis of larger datasets with respect to the ones analyzable by means of \[[@B1]\]'s algorithm, currently the best known exact approach to solution of the MPPEP-SNP.
Similar to \[[@B1]\], we emphasize that the experiments aim simply to evaluate the runtime performance of our model for solving MPPEP-SNP. We neither attempt to study the efficiency of MPPEP-SNP for phylogeny estimation nor compare the accuracy of our algorithm to phylogeny estimation solvers that do not use the parsimony criterion. The reader interested in a systematic discussion about such issues is referred to \[[@B19],[@B33]\].
Implementation
--------------
We implemented Formulations 1 and 2 by means of Mosel 64 bit 3.2.0 of Xpress-MP, Optimizer version 22, running on a Pentium 4, 3.2 GHz, equipped with 2 GByte RAM and operating system Gentoo release 7 (kernel linux 2.6.17). In both formulations, we computed the overall solution time to solve a generic instance of the problem as the sum of the preprocessing time due to the implementation of \[[@B22]\]'s reduction rules plus the solution time taken by the Optimizer to find the optimal solution to the instance. In preliminary experiments, we observed that Formulation 2 has two main advantages with respect to Formulation 1, namely: it requires much less memory to load the model (at least 27% RAM less in the analyzed instances) and it is characterized by faster linear relaxations at each node of the search tree. As result, Formulation 2 allows potentially the analysis of larger instances than Formulation 1 and may be characterized by faster solution times. Hence, we preferred to use Formulation 2 in our experiments.
We considered two different implementations of Formulation 2, namely: the GESC-based Reduced Model (GSEC-RM) and the Flow-based Reduced Model (Flow- RM). The GESC-RM consists of Formulation 2 strengthened by the valid inequalities previously described. The Flow-RM consists of Formulation 2 strengthened by the valid inequalities and such that the GSEC are replaced by a multi-commodity flows. Specifically, by denoting $f_{\mathit{ij}}^{q}$ as a decision variable equal to one if there exists a flow from vertex 1 to vertex $q \in V_{\mathcal{H}}$ passing through edge $\left( {i,j}\in\overline{E} \right.$ and 0 otherwise, the Flow-RM can be obtained by replacing constraints [2l](#F2){ref-type="fig"}) with:
$$\begin{matrix}
{f_{\textit{ji}}^{q}\; + \; f_{\textit{ij}}^{q}\; \leq \;\underset{s \in \mathcal{S}}{\sum}z_{\textit{ij}}^{s}} & {\mspace{460mu}\mspace{460mu}} & {\forall\quad q\; \in \; V_{\mathcal{H}}:q\; \neq \; 1,\quad i,j\; \in \; V:i,j \in Z} & \\
& & & {} \\
\end{matrix}$$
$$\begin{matrix}
{\underset{i \in V:i \neq 1}{\sum}f_{1i}^{q} = 1} & {\mspace{460mu}\mspace{460mu}} & {\forall\quad q \in V_{\mathcal{H}}:q \neq 1} & {} \\
\end{matrix}$$
$$\begin{matrix}
{\underset{j \in V:i \neq j}{\sum}f_{\textit{ij}}^{q}\; - \;\underset{j \in V:i \neq j}{\sum}f_{\textit{ji}}^{q}\; = \; 0} & {\mspace{460mu}\mspace{460mu}} & {\forall\quad q\; \in \; V_{\mathcal{H}}:q\; \neq \; 1,\quad i \in V:i \notin \left\{ 1,q \right\}} & \\
& & & {} \\
\end{matrix}$$
$$\begin{matrix}
{\underset{i \in V:i \neq q}{\sum}f_{\textit{iq}}^{q} - \underset{i \in V:i \neq q}{\sum}f_{\textit{qi}}^{q} = 1} & {\mspace{460mu}\mspace{460mu}} & {\forall\quad q \in V_{\mathcal{H}}:q \neq 1} & {} \\
\end{matrix}$$
$$\begin{matrix}
{f_{\textit{ij}}^{q}\; \geq \; 0} & {\mspace{460mu}\mspace{460mu}} & {\forall\quad q\; \in \; V_{\mathcal{H}}:q\; \neq \; 1,\quad i,j\; \in \; V:i,j\; \in \;{Z.}} & \\
& & & {} \\
\end{matrix}$$
In preliminary experiments we observed that the Flow-RM outperforms the GESC-RM in terms of solution time. This fact is mainly due to the computational overhead introduced by the GSEC separation oracle which seems to be not compensated by the size of the analyzed instances. Hence, we did not consider the GESC-RM any further in our experiments.
During the runtime, we enabled the Xpress-MP automatic cuts and the Xpress-MP pre-solving strategy. Moreover, we also tested a number of generic primal heuristics for the Steiner tree problem to generate a first primal bound to the MPPEP-SNP (see, e.g., \[[@B34]\]). Unfortunately, in preliminary experiments we observed that the use of such heuristics interferes negatively with the Xpress Optimizer, by delaying the solution time of the analyzed instances. Hence, we disabled the used of the generic primal heuristics and enabled the use of the Xpress-MP primal heuristic instead. The source code of the algorithm can be downloaded at <http://homepages.ulb.ac.be/~dacatanz/Site/Software_files/iMPPEP.zip>.
Separation oracle for the forbidden path constraints
----------------------------------------------------
When using the Flow-RM, the valid inequalities (3)-(12) are loaded together with the reduced model. On the contrary, the valid inequalities (13) are dynamically generated by means of a separation oracle working as follows. Before loading the reduced model, we precompute the sets *Q*~*d*~, for all *d* ∈ {3,4,...,*m*}. Let $\left( \overline{u},\overline{x},\overline{z} \right)$ be the current fractional solution at a given node of the search tree and, for all *d* ∈ {3,4,...,*m*}, consider a pair of vertices *i*,*j*∈*Q*~*d*~. Then, the forbidden path constraints (13) are violated if there exists a path *p* having internal vertices in *C* ⊂ *V*, 2 ≤ \|*C*\| ≤ *d* − 1, *C* ∩ *Q*~*d*~ = *∅*, and such that
$$\begin{matrix}
\left. \underset{s \in \mathcal{S}}{\sum}{\overline{z}}_{\mathit{ip}_{1}}^{s} + \underset{k = 1}{\overset{|C| - 1}{\sum}}\underset{s \in \mathcal{S}}{\sum}{\overline{z}}_{p_{k}p_{k + 1}}^{s} + \underset{s \in \mathcal{S}}{\sum}{\overline{z}}_{p_{|C|j}}^{s} > \middle| C \middle| . \right. & & & {} \\
\end{matrix}$$
Note that searching for the most violated constraint (19) is in general $\mathcal{N}\mathcal{P}$-hard as it involves solving a longest path problem on the weighted graph ${\overline{G}}_{\overline{z}}^{V \smallsetminus Q_{d}}$, i.e., the graph $\overline{G}$ whose edges are weighted by variables $\overline{z}$ and whose vertex set is restricted to (*V*∖*Q*~*d*~) ∪ {*i*,*j*}. In order to have a fast separation oracle for the forbidden path constraints we do not solve exactly (19) but we use a heuristic approach instead. Specifically, we first sort edges of $\overline{E}$ in decreasing order according to their weights and we select two distinct vertices *v*~1~,*v*~2~ ∈ *V*∖*Q*~*d*~ such that edge (*v*~1~,*v*~2~) has the largest weight. Subsequently, we set *C* = {*v*~1~,*v*~2~}, mark *v*~1~ and *v*~2~ as visited, and build a simple path from vertex *i* to vertex *j* passing by *v*~1~ and *v*~2~. If *p* is such that (19) is satisfied then we add the constraint
$$\begin{matrix}
\left. \underset{s \in \mathcal{S}}{\sum}z_{\mathit{ip}_{1}}^{s} + \underset{k = 1}{\overset{|C| - 1}{\sum}}\underset{s \in \mathcal{S}}{\sum}z_{p_{k}p_{k + 1}}^{s} + \underset{s \in \mathcal{S}}{\sum}z_{p_{|C|j}}^{s} \leq \middle| C \right| & & & {} \\
\end{matrix}$$
to the formulation; otherwise, we select a different pair of vertices in *V*∖*Q*~*d*~ and iterate this procedure until either 10 distinct paths have been generated or all possible pairs of vertices in *V*∖*Q*~*d*~ have been selected. If all vertices have been selected but less than 10 distinct paths have been generated, then we select a larger subset of *V*∖*Q*~*d*~ (e.g., a triplet of vertices) and we iterate again the previous steps. It is easy to realize that this procedure may potentially generate all the possible paths violating (13). However, we stop the procedure after generating 10 paths or after considering subset *C* containing more than 5 vertices as we observed in preliminary experiments that this strategy provides the best trade-off between speed and tightness of the cut.
Branching strategies
--------------------
In preliminary experiments we observed that the standard branching strategy implemented in the Xpress-MP Optimizer is not appropriate for the problem as it is not able to exploit the dissimilarity of the weights *w*^*s*^ in the objective function. This inconveniently leads to formulations characterized by slow solution times. To improve this aspect we implemented a different strategy consisting of branching on the following constraints:
$$\begin{matrix}
{\underset{\begin{matrix}
{i,j \in V:} \\
{i,j \in Z} \\
\end{matrix}}{\sum}\; z_{\textit{ij}}^{s} \leq \alpha} & \quad & {\forall\quad s \in \mathcal{S}} & {} \\
\end{matrix}$$
or
$$\begin{matrix}
{\underset{\begin{matrix}
{i,j \in V:} \\
{i,j \in Z} \\
\end{matrix}}{\sum}\; z_{\textit{ij}}^{s} > \alpha,} & \quad & {\forall\quad s \in \mathcal{S}} & {} \\
\end{matrix}$$
where *α* ∈ {1,2,...,*q*} and $q = \min\left\{ \sum\limits_{k \in V_{\mathcal{H}}}h_{k}\left( s \right),n/2 \right\}$. Constraints (21)-(22) limit the number of changes along a phylogeny with respect to a given coordinate $s \in \mathcal{S}$ and tend to be more effective when the weights *w*^*s*^ are very dissimilar among them. This branching strategy can be implemented by introducing a decision variable
$$\begin{array}{lccc}
{\beta_{\alpha}^{s} = \left\{ \begin{array}{l}
{1\text{if\ the\ overall\ number\ of\ changes\ at\ coordinate}} \\
{\mspace{23mu} s \in \mathcal{S}\;\text{of}\;{\overline{T}}^{\star}\;\text{is\ equal\ to}\alpha} \\
{0\;\text{otherwise},} \\
\end{array} \right)} & & & \\
\end{array}$$
for all $s \in \mathcal{S}$ and *α* ∈ {1,2,...,*q*}, and by adding the following constraints
$$\begin{matrix}
{\underset{\begin{matrix}
{i,j \in V:} \\
{i,j \in Z} \\
\end{matrix}}{\sum}z_{\mathit{ij}}^{s} = \underset{\alpha = 1}{\overset{q}{\sum}}\beta_{\alpha}^{s}} & \quad & {\forall\quad s \in \mathcal{S}} & \\
{\mspace{63mu}\underset{\alpha = 1}{\overset{q}{\sum}}\beta_{\alpha}^{s} = 1} & \quad & {\forall\quad s \in \mathcal{S}.} & \\
\end{matrix}$$
We observed that even better runtime performance can be obtained by sorting the coordinates of the input haplotypes in decreasing way according to the weights *w*^*s*^ and by branching first on variables $\beta_{\alpha}^{s}$, then on variables *u*~*i*~, and subsequently on variables $x_{i}^{s}$ and finally on variables $z_{\mathit{ij}}^{s}$.
Performance analysis
--------------------
In order to obtain a measure of the performance of the Flow-RM, we compared \[[@B1]\]'s polynomial-size formulation versus the Flow-RM on \[[@B1]\]'s real instances of the MPPEP-SNP, namely: Human chromosome Y constituted by 150 haplotypes having 49 SNPs each; bacterial DNA constituted by 17 haplotypes having 1510 SNPs each; Chimpanzee mitochondrial DNA constituted by 24 haplotypes having 1041 SNPs each; Chimpanzee chromosome Y constituted by 24 haplotypes having 1041 SNPs each; and a set of four Human mitochondrial DNA from HapMap \[[@B35]\] constituted by 40 haplotypes having 52 SNPs each, 395 haplotypes having 830 SNPs each, 13 haplotypes having 390 SNPs each, and 44 haplotypes having 405 SNPs each, respectively. Such instances consist only of non-recombining data (Y chromosome, mitochondrial, and bacterial DNA) and can be downloaded at <http://homepages.ulb.ac.be/~dacatanz/Site/Software_files/iMPPEP.zip>.
Table [1](#T1){ref-type="table"} shows the results obtained by such comparison. Specifically, the fourth and fifth columns refer to the gaps (expressed in percentage) of the respective formulations, i.e., to the difference between the optimal value to a specific instance and the value of linear relaxation at the root node of the search tree, divided by the optimal value. The table shows that, excluding the cases in which the solution to a specific instance was trivially a minimum spanning tree (see e.g., Human chromosome Y, Chimpanzee mtDNA, and Chimpanzee chromosome Y), the Flow-RM is always characterized by (sometimes dramatically) smaller gaps. This fact derives on the one hand from the tightness of the Flow-RM with respect to \[[@B1]\]'s polynomial-size formulation and on the other hand from the efficiency of the strengthening valid inequalities previously described. The poor relaxations of their formulation led \[[@B1]\] to propose an alternative and faster exact approach to solution of the MPPEP-SNP based on the brute-force enumeration of all possible Steiner vertices necessary to solve a specific instance of the problem. To speed up the computation, the brute-force enumeration algorithm makes use of a set of reduction rules based on Buneman graph enumeration to decrease the number of Steiner vertices to be considered. Interestingly, despite the differences in terms of implementation language between the two programs (namely, Mosel for the Flow-RM and C++ for \[[@B1]\]'s brute-force enumeration algorithm), the Flow-RM proved to be competitive with \[[@B1]\]'s enumeration algorithm, being able to solve almost all the considered instances within 1 second computing time. Only in two cases, namely Human mtDNA 40×52 and Human mtDNA 395×830, the Flow-RM needed more than 5 minutes to find the corresponding optimal solutions. The deterioration of the runtime performance in those instances is mainly due to the overhead necessary to load the formulation (that in both cases is considerably bigger than in the other instances) and to an intensive use of the separation oracle for the forbidden path constraints. Possibly, a more thorough implementation of the separation oracle and the use of more performing languages (e.g., C++) could help in speeding up computations in those instances at least.
######
**Comparison between the gap of \[**\[[@B1]\]**\]'s polynomial size integer programming model for the MPPEP-SNP versus the gap of the flow-based reduced model and its strengthening valid inequalities**
**Dataset** **Haplotypes** **SNPs** **GAP (%)** **GAP (%)** **Optimum** **MST**
------------------------- ---------------- ---------- ------------- ------------- ------------- ---------
Human chromosome Y 150 49 0.00 0.00 16 yes
Bacterial mtDNA 17 1510 26.04 **20.83** 96 no
Chimpanzee mtDNA 24 1041 20.63 20.63 63 yes
Chimpanzee chromosome Y 15 98 0.00 0.00 99 yes
Human mtDNA 40 52 24.66 **1.37** 73 no
Human mtDNA 395 830 22.64 **7.55** 53 no
Human mtDNA 13 390 12.50 **6.25** 48 no
Human mtDNA 44 405 6.98 **4.65** 43 no
Interestingly, sometimes in real applications the number of haplotypes can be much bigger than the number of SNPs. Hence, it is important to test the ability of an exact algorithm to tackle instances of the MPPEP-SNP containing e.g., hundreds haplotypes. \[[@B1]\] observed that their brute force enumeration algorithm is able to tackle instances of the problem containing up to 270 haplotypes having up to 9 SNPs each within 12 hours computing time. Unfortunately, the authors also observed that their algorithm is unable to solve larger instances of the MPPEP-SNP, no matter the maximum runtime considered. In this context, the Flow-RM makes the difference, being able to tackle instances of the MPPEP-SNP having up to 300 haplotypes and 10 SNPs within 3 hours computing time. To show this result, we considered a number of random instances of the problem containing 100, 150, 200, 250, and 300 haplotypes, respectively. Fixing the number of haplotypes *n*∈{100,150,200,250,300}, we created an instance of the problem by generating at random *n* strings of length 10 over the alphabet *Σ*={0,1}. During the generation process, we randomly selected the number of SNPs equal to 1 in a given haplotype, and subsequently we randomly chose the sites of the haplotype to be set to 1. We iterated the instance generation process 10 times for a fixed value of *n*, obtaining eventually an overall number of 50 random instances of the MPPEP-SNP downloadable at <http://homepages.ulb.ac.be/~dacatanz/Site/Software_files/iMPPEP.zip>.
The results obtained in our experiments are shown in Table [2](#T2){ref-type="table"}. Specifically, the column "Time" refers to the solution time (expressed in seconds) necessary to solve exactly a specific instance of the MPPEP-SNP. Analogously, the column "Nodes" refers to the number of explored nodes in the search tree needed to solve exactly the instance. The table does not report on the performance of \[[@B1]\]'s enumeration algorithm, as their algorithm never found the optimal solution to the analyzed instances within the limit runtime of 3 hours. As a general trend, the table shows that the considered instances can be exactly solved within 1 hour computing time. The only exceptions are constituted by the 7th instance of the group 150×10, the 9th instance of the group 200×10, the 2th instance of the group 250×10, and 3th instance of the group 300×10which needed 8719.65, 4600.69, 7757.98, and 5371.05 seconds, respectively, to be solved. These instances are much more sparse than the others, are characterized by smaller reduction ratios, and tend to have more degenerate relaxations than the other instances. The presence of these factors might explain the loss of performance of the Flow-RM.
######
Performances of the Flow-RM on a set of random instances of the MPPEP-SNP
$\left| \mathcal{H} \right|\,$ **Instance** $\left| \mathcal{H} \right|\,$**post** **Time** **GAP (%)** **Nodes** $\left| \mathcal{H} \right|\,$ **Instance** $\left| \mathcal{H} \right|\,$**post** **Time** **GAP (%)** **Nodes**
-------------------------------- -------------- ---------------------------------------- ---------- ------------- ----------- -------------------------------- -------------- ---------------------------------------- ---------- ------------- -----------
100 1 57 520.05 0 1807 150 1 82 284.51 0 424
2 60 59.74 0 174 2 83 314.27 0.76 56
3 63 377.75 1.45 110 3 81 799.01 0 67
4 61 2491.62 3.81 3351 4 67 1809.26 2.66 6617
5 60 2918.09 4.63 2062 5 79 1001.14 2.29 187
6 57 349.54 1.59 264 6 74 1976.73 2.41 1071
7 65 258.53 1.90 85 7 73 8719.65 3.92 4814
8 58 293.97 0 1299 8 83 3497.73 2.17 421
9 62 862.48 2.85 540 9 72 1154.77 2.51 410
10 64 87.19 0 92 10 80 399.89 1.56 256
200 1 99 614.86 0 72 250 1 117 1155.41 0 197
2 99 1353.16 1.28 149 2 109 7757.98 1.72 1596
3 96 896.68 0.67 226 3 117 387.141 0.84 180
4 104 652.44 0.47 150 4 126 1267.77 0.51 114
5 96 382.83 0 56 5 116 188.188 0.84 162
6 106 2535.09 0.60 71 6 116 2311.61 1.14 685
7 100 233.50 0 21 7 116 1256.24 0 265
8 99 1650.17 0.96 79 8 124 67.556 0 528
9 87 4600.69 2.10 954 9 122 2000.77 0.53 107
10 102 2554.84 1.23 1965 10 111 1200.89 0.87 272
300 1 133 297.19 0 15
2 123 2753.53 0.39 68
3 142 5371.05 0 941
4 133 420.72 0 43
5 126 388.99 0 433
6 134 397.01 0 61
7 138 1173.65 0 1788
8 126 666.21 0 186
8 127 449.30 0.77 42
10 145 201.87 0 876
The results showed that the integrality gaps are usually very low, ranging from 0% to 4.63% and assuming in average a value about 1%, confirming once again the tightness of the Flow-RM and the efficiency of the strengthening valid inequalities.
Finally, we also tested the performance of the Flow-RM on a set of 5 HapMap Human mitochondrial DNA instances of the MPPEP-SNP that were not solvable by using \[[@B1]\]'s brute-force enumeration algorithm, namely: f1 constituted by 63 haplotypes having 16569 SNPs each, i2 constituted by 40 haplotypes having 977 SNPs each, k3 constituted by 100 haplotypes having 757 SNPs each, m4 constituted by 26 haplotypes having 48 SNPs each, and p5 constituted by 21 haplotypes having 16548 SNPs each. Such instances can be downloaded at the same address and consist only of non-recombining data (Y chromosome, mitochondrial, and bacterial DNA).
A part from m4, all the remaining instances gave rise to too large formulations (several hundreds Mbytes RAM) to be handled by the Xpress Optimizer. Hence, instead of analyzing entirely each instance we decomposed it into contiguous SNP blocks and analyzed the most difficult block separately. In more in detail, we define $\mathcal{H}_{r}$ to be the haplotype matrix obtained by the application of \[[@B1]\]'s reduction rules, we sorted the columns of $\mathcal{H}_{r}$ according to an increasing ordering of the weights *w*^*s*^, $s \in Ŝ$; subsequently, we considered the submatrices obtained by taking *k* contiguous SNPs (or *k*-block) in $\text{Ŝ,}$*k* ∈ {10,13,15}. We did not consider greater values for *k* as we observed that *k* = 15 was already a threshold after which the haplotype submatrix gave rise to too large formulations. For each *k*-block $\mathcal{B}\,$in $\mathcal{H}_{r}$ we considered the hamming distance $\left. d_{h_{i}h_{j}} = \sum\limits_{s \in \mathcal{B}} \middle| h_{i}\left( s \right) - h_{j}\left( s \right)| \right.$ between each pair of distinct haplotypes in $\mathcal{H}_{r}$, and chose the *k*-block maximizing the sum $\sum\limits_{h_{i},h_{j} \in \mathcal{H}_{r},h_{i} < h_{j}}d_{h_{i}h_{j}}$. Finally, we assumed three hours as maximum runtime per instance.
Table [3](#T3){ref-type="table"} shows the results obtained in our experiments. As for Table [2](#T2){ref-type="table"}, the columns "Time" and "Nodes" refer to the solution time (expressed in seconds) and to the number of nodes in the search tree necessary to solve exactly a specific instance of the MPPEP-SNP, respectively. In such a case, the values in the columns "Gap" refers to the gap between the best primal bound found within the limit time and the root relaxation and "Nodes" refers to the number of nodes explored in the tree search within the limit time.
######
Performances of the Flow-RM on a set of real instances of the MPPEP-SNP
**Dataset** **Haplotypes** **SNPs** **Block Size** **Time (sec.)** **GAP (%)** **Nodes** **MST Solution**
------------- ---------------- ---------- ---------------- ----------------- ------------- ----------- ------------------
10 1 \| \| yes
f1 63 16569 13 56 3.11 1 no
15 10286.1 26.92 773521 no
i2 40 977 10 781.85 20.00 37511 no
k3 100 757 10 150 7.65 353 no
13 588.38 14.29 11265 no
m4 26 48 10 5 5.88 109 no
p5 21 16548 10 22283.4 50.79 6125448 no
Table [3](#T3){ref-type="table"} shows that, apart from the instances f1 and m4, the Flow-RM was unable to exactly solve, within the limit time, the considered instances for values of *k* ∈ {13,15}. Specifically, The Flow-RM exactly solved in less than a minute the instance f1 when considering values of *k* ∈ {10,13} ; in 20 minutes the instance i2 when considering *k* = 10 ; in less than 3 minutes the instance k3 when considering *k* = 10; and the instance m4 in 5 seconds. In contrast, the Flow-RM was unable to solve the instance p5, regardless of the value of *k* considered. In fact, already when considering *k* = 10, the Xpress Optimizer took more than 12 hours to exactly solve the instance p5 and explored over 10 million nodes in the search tree. A more detailed analysis of the instance showed that, despite the presence of the strengthening valid inequalities, p5 is characterized by highly fractional relaxations. This fact implies the existence of equivalent optimal solutions to the instance that, on the one hand, delay the finding of a primal bound and, on the other hand, force the Optimizer to explore many more nodes in the tree search. This situation in more pronounced in p5 but also occurs in the instances i2 and k3. To improve the tightness of the formulation we tried to include in the Flow-RM also classical facets and strengthening valid inequalities for the Steiner tree problem in a graph (see \[[@B23],[@B36]-[@B38]\]). However, we did not observe any benefit from the inclusion. We suspect that the presence of highly fractional solutions to the problem could be caused both by poor lower bounds on the number of Steiner vertices considered in the Flow-RM and by the existence of a number of non trivial classes of symmetries still present in the problem. Investigating such issues warrants future research efforts.
In order to measure the performance of the model on multi-state character data we also considered \[[@B2]\] set of instances of the MPPEP-SNP. Specifically, we considered the following instances: a set of 41 sequences of O.rufipogon DNA (red rice) having 1043 sites each; 80 human mtDNA sequences having 245 sites each; 50 HIV-1 reverse transcriptase amino acid sequences having 176 sites each; a set of 500 sequences of mtDNA from the NCBI BLASTN best aligned taxa having 3000 sites each; a set of 500 sequences of mtDNA from the NCBI BLASTN best aligned taxa having 5000 sites each; and a set of 500 sequences of mtDNA from the NCBI BLASTN best aligned taxa having 10000 sites each. When running the same experiments described in \[[@B2]\] we registered a very poor performance for the Flow-RM, mainly due to the large dimension of the considered instances and the presence of symmetries despite the use of constraints (13)-(15). We observed that the combination of these two factors increased the runtime performance of the Flow-RM of 2-3 orders of magnitude with respect to \[[@B2]\] approach. However, we also observed that when performing \[[@B2]\]'s "window analysis" (i.e., when decomposing into blocks of 10 SNPs the input matrix) the Flow-RM performed better than \[[@B2]\]'s, being characterized by an average solution time of 8.27 seconds. This fact suggests that, when considering instances constituted by less than a dozen sites, an exact approach entirely based on integer programming may perform better than the implicit enumeration of the vertices of the generalized Buneman graph. Vice-versa, for larger instances the implicit enumeration of the vertices of the generalized Buneman graph appears more suitable.
Conclusion
==========
In this article we investigated the Most Parsimonious Phylogeny Estimation Problem from Single Nucleotide Polymorphism (SNP) haplotypes (MPPEP-SNP), a recent version of the phylogeny estimation problem that arises when input data consist of SNPs extracted from a given population of individuals. The MPPEP-SNP is $\mathcal{N}\mathcal{P}$-hard and this fact has justified the development of exact and approximate solution approaches such as those described in \[[@B1],[@B19],[@B22],[@B26]-[@B28]\]. We explored the prospects for improving on the strategy of \[[@B1],[@B2]\] using a novel problem formulation and a series of additional constraints to more precisely bound the solution space and accelerate implicit enumeration of possible optimal phylogenies. We present a formulation for the problem based on an adaptation of \[[@B23]\]'s mixed integer formulation for the Steiner tree problem extended with a number of preprocessing techniques and reduction rules to further decrease its size. We then show that it is possible to exploit the high symmetry inherent in most instances of the problem, through a series of strengthening valid inequalities, to eliminate redundant solutions and reduce the practical search space. We demonstrate through a series of empirical tests on real and artificial data that these novel insights into the symmetry of the problem often leads to significant reductions in the gap between the optimal solution and its non-integral linear programming bound relative to the prior art as well as often substantially faster processing of moderately hard problem instances. More generally, the work provides an indication of the conditions under which such an optimal enumeration approach is likely to be feasible, suggesting that these strategies are usable for relatively large numbers of taxa, although with stricter limits on numbers of variable sites. The work thus provides methodology suitable for provably optimal solution of some harder instances that resist all prior approaches. Our results may provide also useful guidance for strategies and prospects of similar optimization methods for other variants of phylogeny inference. In fact, if appropriately adapted, some of the results we presented here (e.g., symmetry breaking strategies) can be generalized with respect to other phylogenetic estimation criteria (e.g., the likelihood criterion) and provide important computational benefits.
Competing interests
===================
The authors declare that they have no competing interests.
Authors' contributions
======================
The authors equally contributed to conceive the work and write the article. DC implemented the algorithms and performed computations. All authors read and approved the final manuscript.
Acknowledgements
================
The first author acknowledges support from the Belgian National Fund for Scientific Research (FNRS) of which he is Chargé de Recherches, the Belgian American Educational Foundation (BAEF) of which he is honorary fellow, and the Carnegie Mellon University (CMU). Part of this work has been developed while Dr. Catanzaro was visiting the Tepper School of Business and the Department of Biological Sciences of the CMU during the academic years 2010-2011 and 2011-2012. This work was supported in part by U.S. National Institutes of Health awards 1R01CA140214 and 1R01AI076318.
|
{
"pile_set_name": "PubMed Central"
}
|
Introduction {#s1}
============
The sirtuin family is widely distributed from archaea and eubacteria to eukaryotes and seven different homologous proteins are found in the humans that show a central highly conserved region, defined as the catalytic core [@pone.0007350-Huhtiniemi1]. In comparison to other proteins of the family, Sirt-1 presents two large regions, i.e. the amino and carboxyl terminals, that are missing in all the other sirtuins. Sirt-1 is a NAD+-dependent deacetylase closely related to yeast *Sir2*, the first gene discovered in sirtuin family, which has NAD+ dependent class III histone deacetylase activity [@pone.0007350-Kim1]. Sites of phosphorylation and SUMOylation consensus were recently found also in the amino- and in carboxyl-terminal regions, and these were proposed having regulation and localization functions [@pone.0007350-Sasaki1].
Experimental data support the Sirt-1 implication in processes including chromatin remodelling, transcriptional silencing, chromosomal stability, cell cycle progression, apoptosis, autophagy, metabolism, growth suppression, inflammation, and stress response [@pone.0007350-Saunders1]. In fact, the Sirt-1 regulation activity occurs through the deacetylation reaction of various and different substrates such as p53 [@pone.0007350-Vaziri1], forkhead box class O (FOXO) transcription factors [@pone.0007350-Yang1], peroxisome proliferator activated receptor (PPAR)g co-activator 1a (PGC-1α) [@pone.0007350-Rodgers1], nuclear factor (NF)-kB and others, which are closely linked to some age-related diseases [@pone.0007350-Vaziri1]. Also Sirt-1 stimulates eNOS activity and increases the endothelial NO. Its inhibition in the endothelium of arteries inhibits endothelium dependent vasodilation and decreases bioavailable NO [@pone.0007350-Mattagajasingh1].
Moreover, it was seen that Sirt-1 is a negative modulator of adipogenesis by docking with the nuclear receptor co-repressor (NcoR) [@pone.0007350-Picard1] and induces a decrease of pro-inflammatory cytokine release [@pone.0007350-Rajendrasozhan1] and a promotion of carcinogenesis [@pone.0007350-Ghosh1] by the negative control of Nuclear factor-kB (NF-kB). Experimental data demonstrated that the inhibition of NF-kB in hepatocytes in vivo promotes hepatocarcinogenesis.
Kaeberlein *et al.* demonstrated firstly the anti-aging effects of *Sir2* showing that in *Saccharomyces cerevisiae* the integration of extra copies of *Sir2* extended lifespan up to 30% [@pone.0007350-Kaeberlein1]. Similar effects of *Sir2* were subsequently observed in *C. elegans* and *Drosophila melanogaster* [@pone.0007350-Wang1]--[@pone.0007350-Wood1]. In fact, the overexpression of *Sir-2* [@pone.0007350-Yamamoto1] increased lifespan up to 50% in *C. elegans* but in *Drosophila*, an extra copy of the *Sir2* gene extended lifespan in female and male by 29 and 18%, respectively.
Because Sirt-1 deacetylates non histone proteins, including various transcription factors, it is involved in the control of important biological mechanisms. Through its catalytic activity, it exhibits diversified functions in cell type-specific manner, which have pathophysiological implications in cancer, obesity, inflammation and neurodegenerative diseases [@pone.0007350-Saunders1], [@pone.0007350-DaliYoucef1]--[@pone.0007350-Haigis1]. Its modulation leads an increase in mitochondrial biogenesis, and an improvement of glucose metabolism in mitochondria but also in skeletal muscle and adipose tissues [@pone.0007350-Hollander1]. These evidences suggest that Sirt-1 could be a novel target to treat metabolic disorders such as type 2 diabetes.
Numerous experimental data [@pone.0007350-Kim1], [@pone.0007350-Milne1]--[@pone.0007350-Bemis1] have shown the modulation of the catalytic activity of Sirt-1 exerted through its allosteric effectors. Kim et al (2007) demonstrated that the nuclear protein AROS is an endogenous activator of Sirt-1 which increases its activity interacting with the allosteric site. Accordingly with the functional relevance of Sirt-1 activation, recent works focused on its interaction with small allosteric effectors [@pone.0007350-Milne1]--[@pone.0007350-Zhao1].
In particular, the phenolic compound, named resveratrol, is the first found allosteric activator, that has been reported to extend lifespan in *yeast* [@pone.0007350-Zhao1], *Caenorhabditis elegans*, *Drosophila* [@pone.0007350-Chang1] and rodents [@pone.0007350-Bemis1]. It improves the metabolism and glucose tolerance, as well as the overall physical performance in various stress tests [@pone.0007350-Rodgers1]. However recent efforts have identified new compounds significantly more potent than resveratrol [@pone.0007350-Milne1]--[@pone.0007350-Bemis1].
Even if the substrates and the effects of Sirt-1 activation have been well characterized, no structural information about its activation and regulation is known.
On the basis of the biological importance of this protein, we focused our attention to create a model of the whole structure of human Sirt-1 in order to understand its interaction with the allosteric effectors and to propose the molecular basis of its activation and regulation.
Results {#s2}
=======
Modelling of the catalytic core and allosteric site of Sirt-1 {#s2a}
-------------------------------------------------------------
The best model has been obtained for the catalytic core of human Sirt-1 using as template the structures of human Sirt-2 [@pone.0007350-Finnin1], Hst2 from Saccharomyces cerevisae [@pone.0007350-Zhao1] and Sir2-Af1 from Archaeoglobus flugidus [@pone.0007350-Chang1] is shown in [Figure 1](#pone-0007350-g001){ref-type="fig"}. This model has 94,9% residues in the most favoured regions and a Prosa Z-score of 8,7. These values, compared also with those of the template structures, indicate that a good quality model has been created.
{#pone-0007350-g001}
The catalytic core appears well structured in agreement with its low propensity to the disorder, as shown in [Figure 2](#pone-0007350-g002){ref-type="fig"}. Secondary structure predictions made by JPRED program agree with the structure of catalytic core obtained by comparative modeling ([Figure 3](#pone-0007350-g003){ref-type="fig"}). The core (from residue 244 to 498) is well structured. The model ([Figure 1](#pone-0007350-g001){ref-type="fig"}) shows the two structural domains typical of the sirtuin family of which the first one is a Rossman fold with the characteristic β-α-β motives of NAD proteins, and the second one is a smaller sub-domain where the zinc is located. At the interface between domains there is the binding site for NAD. The NAD molecule fits in a specific pocket constituted of a hydrophobic patch on the small sub-domain, and a hydrophilic patch on the large domain. In particular, the NAD molecule presents the adenine and ribose moieties inside the pocket, as described for other sirtuins [@pone.0007350-Chang1]--[@pone.0007350-Avalos1], but the nicotinamide group is near to the residues 269--295 of human Sirt-1. These residues (indicated with a white arrow in [Figure 1](#pone-0007350-g001){ref-type="fig"}) represent another conserved region that forms a flexible loop near the pocket. This loop was proposed as a "frontwall" [@pone.0007350-Avalos1] of the C-site, or a "ceiling"[@pone.0007350-Avalos1] on the pocket. Its flexible organization allows a structural rearrangement of the catalytic domain during the NAD binding, which seems essential for the catalysis. This central role is demonstrated by the orientation of this region in the crystallized sirtuins. In fact, the crystallized complexes with the NAD+ molecule show a same order orientation, but without NAD, this loop region shows a more poorly defined or disordered orientation [@pone.0007350-Zhao1], [@pone.0007350-Finnin1]. This suggests that the NAD+ binding influences the orientation of this flexible loop by triggering the assembly and disassembly of the C pocket [@pone.0007350-Avalos1] as well as the loop\'s orientation is a measure of the accessibility into the pocket for the nicotinamide or other small inhibitors [@pone.0007350-Huhtiniemi1].
{#pone-0007350-g002}
{#pone-0007350-g003}
The zinc ion is tetrahedrally coordinated by the thiol groups of four Cys residues (at positions 371, 374, 395, 398) which are folded into a single structural unit. This tetrad of cysteine residues are that is broadly conserved in the small sub-domain of all the sirtuin family [@pone.0007350-Finnin1]. and this small zinc-binding domain (see [Figure 1](#pone-0007350-g001){ref-type="fig"}) is thought to play a role in substrate-specific binding by the sirtuin proteins [@pone.0007350-Zhao2]. In the classic zinc finger, one zinc atom is bound to two cysteines and two histidines while in this case we have a Cys4-Zn. This structure is composed by about 30 residues included in a flexible structural environment that sterically favours the tetrahedrally coordination of the cysteines.
The allosteric site (from residue 181 to 243) is straddling between the N-terminal and the compact globular core of the protein and shows an all-alpha structure composed by four alpha helices in agreement with secondary structure and disorder predictions ([Figure 2b](#pone-0007350-g002){ref-type="fig"} and [3](#pone-0007350-g003){ref-type="fig"}).
Both the catalytic core and the allosteric site have compact and globular shapes according to the definition of globularity recently published [@pone.0007350-Costantini1] with a score of 5.1 and 4.3, respectively.
The two models were linked, as reported in [Methods](#s4){ref-type="sec"}, by a flexible loop and subjected to molecular dynamics simulation. The state of equilibrium was reached after 8 ns simulation. The structure remained very stable during the whole simulation time, as confirmed by all the indicators commonly used to analyse MD simulations ([Fig. 4A](#pone-0007350-g004){ref-type="fig"}). In particular, the Rossman fold, as well as the smaller domain and the four helices of the allosteric site were well conserved during the simulation. Only two flexible loops, i.e. the final loop of the allosteric site and the "frontwall" in the catalytic domain, presented some fluctuation. An evaluation of the distance between the allosteric site and the catalytic core was obtained in terms of center of mass distances along the trajectories. This distance decreased of 17 Å after 10 ns of simulation and was maintained constant during the last 5 ns of simulation.
{#pone-0007350-g004}
Modeling of the N-terminal and C-terminal regions {#s2b}
-------------------------------------------------
Various programs for predicting the secondary structure of globular proteins were unable to have a good consensus for the N-terminal and C-terminal segments of Sirt-1. They have differently predicted in amount and in sequence strings the scattered presence of non alfa and non beta regions in both terminal zones. This observation raised the suspect that the Sirt-1 could be an unordered protein and more appropriate algorithms were used. The N-terminal and C-terminal regions were found largely unordered by two specific structural tests. DISOPRED, a software devoted to the search of unordered regions predicted long segments in N-terminal region (1--150 and 160--182) as well as in C-terminal region (510--580, 585--640 and 680--740). Moreover, the Anchor program, in addition, was also able to predict the presence of definite protein binding regions [@pone.0007350-Meszaros1] in the unordered segments ([Figure 2](#pone-0007350-g002){ref-type="fig"} and [Table S1](#pone.0007350.s001){ref-type="supplementary-material"}). The N-terminal and C-terminal regions were modelled as reported in [Methods](#s4){ref-type="sec"} and resulted made of six very short helices inserted with seven short β-strands and of five short helices inserted with five short β-strands, respectively. The remaining large part of residues are in irregular conformations according to the predictions.
The two modelled regions were independently subjected to molecular dynamics simulations with the same protocol used for the allosteric and catalytic sites. The N-terminus reached a stable equilibrated state after 8 ns and the C-terminal after 4 ns of simulation, respectively. Both models looked very stable ([Fig. 4B and C](#pone-0007350-g004){ref-type="fig"}) and the secondary structures present in both models were well kept during the simulation time.
Complete model of Sirt-1 {#s2c}
------------------------
We created a whole model of the human Sirt-1 by comparing modelling using as templates the previously obtained models of N-terminal, allosteric site, catalytic site and C-terminal site. The model was minimized according to our recent papers [@pone.0007350-Paladino1].
The resulting model has 87.8% residues in the most favoured regions and a Prosa Z-score of -6.57.
The various structural parts that compose the global architecture of the protein are shown in [Figure 1](#pone-0007350-g001){ref-type="fig"}. The N-terminal and C-terminal regions are positioned behind the catalytic core, but the groove site of Sirt-1 is exposed to the solvent to receive its substrates. Distances among the centers of mass of catalytic core, N-terminal and C-terminal regions were evaluated. In details, the catalytic core-N-terminal distance resulted to be 38 Å, and that between the catalytic core and C-terminal of 48 Å. The catalytic core exchanges 2 hydrogen bonds and 8 salt bridges with the N-terminal site, and 5 hydrogen bonds and 13 salt bridges with the C-terminal site.
These values confirm that the three regions have distinct structural roles even if one can easily hypothesize that their very close relationships suggests an involvement in the functional activities of the Sirt-1. In fact, the allosteric site is positioned between the N- terminus and the catalytic core in the best position to regulate the enzymatic activity of Sirt-1. Moreover, the N-terminal and C-terminal sites are positioned in a region that does not prevent the catalytic core activity, and this confirms that they have a role of regulation and localization of Sirt-1.
AROS model {#s2d}
----------
AROS is the endogenous protein that is able to increase the Sirt-1 activity [@pone.0007350-Kim1], interacting with its allosteric site. Its model was obtained by fold recognition strategy and presents an all-alpha fold composed from 4 alpha helices in agreement with the secondary structure predictions made by JPred [@pone.0007350-Cuff1] ([Figure 3C](#pone-0007350-g003){ref-type="fig"}) and with the structural class prediction made by PRECLASSPRO server [@pone.0007350-Costantini2].
The AROS model was subjected to molecular dynamics simulations by using the protocol reported in the [Methods](#s4){ref-type="sec"}. A stable state was reached after 7 ns of simulation time and was kept constant up to the end of the dynamics ([Figure 4D](#pone-0007350-g004){ref-type="fig"}).
Sirt-1/AROS complex {#s2e}
-------------------
AROS is the endogenous protein that is able to increase the Sirt-1 activity [@pone.0007350-Kim1], interacting with its allosteric site. To investigate the best binding-groove of Sirt-1 for allosteric effectors we used the principal docking servers (i.e. PATCHDOCK, GRAMMX, CLUSPRO) to obtain a set of possible complexes. We firstly selected the complexes having AROS near to the allosteric site because experimental truncation data have suggested that some amino acids of the allosteric site are important for the binding of allosteric activators [@pone.0007350-Milne1]. It is worthy of note that in the most part of simulated complexes, AROS is placed in the region between the N-terminal site and the allosteric site ([Figure 5](#pone-0007350-g005){ref-type="fig"}). For a more detailed analysis, we selected two of the best complexes for each set, in terms of energy binding, interface ASA, number of atoms and residues at the interface, number of hydrogen bonds and salt bridges (see [Table 1](#pone-0007350-t001){ref-type="table"}). Moreover, we compared, in terms of physical-chemical and geometric properties, the exposed residues of Sirt-1 to the binding pocket in each complex to identify pharmacophore features. As shown in [Figure 6](#pone-0007350-g006){ref-type="fig"}, the binding groove presents basic residues at the top of the cavity, acidic residues at its bottom and hydrophobic residues as edges.
{#pone-0007350-g005}
{#pone-0007350-g006}
10.1371/journal.pone.0007350.t001
###### The selected complex obtained using CLUSPRO, PATCHDOCK and GRAMMX server.
{#pone-0007350-t001-1}
Complexes Binding free energy Interface ASA Interaction Residues H-bonds Salt Bridges
----------- --------------------- --------------- ---------------------- --------- --------------
**CL2** −9.96 1023.49 34 4 24
**CL10** −10.19 973.9 30 1 29
**PD7** −10.51 1231.39 34 7 39
**PD12** −8.92 1230.1 39 4 29
**GX2** −10.73 1471.8 48 5 11
**GX6** −12.65 1332.27 40 4 12
For each one are reported the value of: energy binding (Kcal/mol), interface ASA (Å^2^), number of atoms and residues at the interface, number of hydrogen bonds and salt bridge are reported.
The best Sirt-1/AROS complex was obtained using the CLUSPRO web server [@pone.0007350-Comeau1] that uses one of the best docking algorithm tested in CAPRI experiments with a success rate of about 71% [@pone.0007350-Comeau1]. The good quality of this complex is also suggested by the very low value of its binding free energy (−10.19 Kcal/mol), calculated by the DCOMPLEX server [@pone.0007350-Liu1]. For this complex, we have evaluated the interacting residues, the number of interchain H-bonds and salt bridges and the interface surface area ([Tables 2](#pone-0007350-t002){ref-type="table"} and [3](#pone-0007350-t003){ref-type="table"}). The AROS-Sirt-1 complex shows that AROS is located behind to the catalytic groove. This can be considered one of the most favourable structural site to increase the enzyme activity by conformational changes without penalizing the correct interaction between Sirt-1 and its substrates. In particular, AROS and Sirt-1 chains should form one H-bond and 6 salt bridges at their interaction surface. At the interface Sirt-1 also exposed four aromatic residues, one positively charged residue and five negatively charged residues. The interface region of AROS is composed by six and three positively and negatively charged residues, respectively. These data suggest that the predominant interaction between Sirt-1 and AROS is on electrostatic basis and that the four aromatic residues structurally closely in Sirt-1 might play an important role to favour the stacking interactions with other allosteric activators, as well as organic compounds.
10.1371/journal.pone.0007350.t002
###### Analysis of the best Sirt-1/AROS complex in terms of interface surface area (Å^2^), interchain H-bonds and number of interaction residues and salt bridges.
{#pone-0007350-t002-2}
Interface ASA Interchain H-bonds Interaction residues Salt bridges
------------ --------------- -------------------- ---------------------- --------------
**Sirt-1** 973.90 1 30 6
**AROS** 906.00 1 25 6
10.1371/journal.pone.0007350.t003
###### List of interaction residues between AROS and Sirt-1.
{#pone-0007350-t003-3}
Interaction residues
------------ --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
**Sirt-1** MET1; ALA2; **ASP3**; LEU7; **GLU161**; **ASP166**; SER169; HIS170; ALA171; SER172; SER173; SER174; **ASP175**; **[TRP176]{.ul}**; PRO184; **[TYR185]{.ul}**; **[PHE187]{.ul}**; VAL188; HIS191; LEU192; ILE194; GLY195; THR196; **ASP197**; THR219; **[TRP221]{.ul}**; GLN222; ILE223; **[TRP624]{.ul}**; **ARG627**; VAL628.
**AROS** **ASP24**; HIS25; LEU26; **ARG27**; **GLU42**; SER43; VAL44; GLN46; GLN47; ILE48; LEU49; **ARG50**; GLN51; **ARG53**; VAL62; **LYS64**; **LYS66**; **LYS69**; ALA70; THR73; VAL74; **GLU77**
The charged residues are evidenced in bold and those aromatic are also underlined.
Molecular dynamics of Sirt-1/AROS complex {#s2f}
-----------------------------------------
The Sirt-1/AROS complex was subjected to molecular dynamics simulations with the same protocol previously used. The complex reaches a stable equilibrated state after 7 ns of simulation and after that it remains stable. Even the secondary structures were kept enough stable during the simulation.
We have compared the conformation of AROS/Sirt-1 complex before and after the molecular simulation to verify how the mutual interactions changed. We inserted an acetyl-lysine and a NAD molecule in the AROS/Sirt-1 complex using as template the crystallographic structure of *Saccharomyces cerevisae* Sirt-1. Then, we focused our attention on the catalytic groove. We noted that, during the molecular dynamics, a conformational change of the protein occurs mainly in active site region. In details, the residues His 363, Asn 346, Ser 265, Gly 261 and Pro 447, indicated as important for its catalytic role [@pone.0007350-Khan1], are sterically changed (see [Figure S1](#pone.0007350.s004){ref-type="supplementary-material"}). Also Pro 447, that makes Van der Waals contacts with the aliphatic portion of the acetyl-lysine side chain and may affect the positioning of residues that contact the acetyl group, is more close to the Acetyllisine while four other residues (His 363, Asn 346, Ser 265, Gly 261) that affect the NAD orientation, approaches to the NAD.
Therefore these results could indicate that the molecular dynamics improves the interaction between Sirt-1 and AROS by some structural parameters (see [Table S2](#pone.0007350.s002){ref-type="supplementary-material"}) even if longer time scale will be used for studying this type of long range coupling.
Discussion {#s3}
==========
Human sirtuin-1, an oxidative stress-response and chromatin-silencing factor, is an unordered protein. About half of its sequence appears to be in a disordered state with long flexible poorly structured regions observed at N- and C-terminus. The modeling has also evidenced a large central globular part very well structured made by two close regions, the catalytic core and the allosteric site. Therefore, the protein can be considered composed by four different regions: N-terminal domain, allosteric site, catalytic core and C-terminal domain. The catalytic core is a central highly conserved structured region common to all the sirtuin family [@pone.0007350-Huhtiniemi1] which has the role of catalyzing the NAD^+^-dependent deacetylation reaction, involved in various nuclear events such as transcription, DNA replication, and DNA repair. The core is made by a well organized Rossman fold typical for NAD-dependent proteins. In details, the acetyl lysine substrate has been proposed to bind in a narrow channel that terminates near to the nicotinamide ribose. Moreover, the binding of acetyl-substrate is believed to mediate bending of the nicotinamide ring of NAD+ in a strained conformation also referred to productive NAD+ binding, promoting the cleavage of the ribosyl--nicotinamide bond [@pone.0007350-Zhao1], [@pone.0007350-Min1].
In this domain we find an atom of Zinc tetrahedrically coordinated with four cysteine residues also present in the other members of the sirtuin family. The allosteric site is a small structural domain made of four helices and interacting with the core. It is sited in a structural location where it can easily exert a control of the catalytic activity by conformational changes. Several articles report the presence of activators modulating the functional activity of the human sirtuin. One of the activators, the resveratrol, decreases the Michaelis constant of SIRT1 for both the acetylated substrate and NAD+, and increases cell survival by stimulating SIRT1-dependent deacetylation of p53 [@pone.0007350-Howitz1]. Moreover, a small nuclear protein, AROS (**A**ctive **R**egulator **O**f **S**irtuin), has been found to be the first known endogenous active modulator of SIRT1 which directly regulates SIRT1 function. AROS enhances SIRT1-mediated deacetylation of p53 both in vitro and in vivo, and it inhibits p53-mediated transcriptional activity. It is interesting to observe that AROS was unable to inactivate p53 when was used an AROS-binding-defective SIRT1 mutant. This clearly indicates a direct interaction of AROS with a specific site of Sirt-1. Our best model of AROS presents an all-alpha fold composed from 4 alpha helices. We found that AROS binds the same allosteric site of some small synthetic compounds (*not shown; manuscript in preparation*) proposed as putative therapeutics for the treatment of type 2 diabetes [@pone.0007350-Kim1], [@pone.0007350-Milne1].
The catalytic core and the allosteric site make a central structured domain (about from residues 181 to 498), compact and globular as assessed by various globularity indices.
Moreover Sirt-1 presents two long disordered structural segments at the terminal regions, missing in the other six homologous proteins belonging to the same family. The N-terminal region is 180 residues long and that C-terminal 249. These regions were predicted largely disordered by DISOPRED program that resulted one of the best algorithm for the accuracy of disorder prediction in CASP7 [@pone.0007350-Ward1]--[@pone.0007350-Bordoli1]. [Figure S2](#pone.0007350.s005){ref-type="supplementary-material"} shows the secondary structure of the best model of Sirt-1 calculated by DSSP algorithm that evaluates the Φ and ▒ dihedral angles of each residue and the backbone Hydrogen bonds. From this table as one can see both termini are characterized by numerous and large unstructured regions even if in the C-terminal more structured segments are presents.
Many proteins or protein domains show an intrinsic inability to form a well defined tertiary structure. This property is encoded in their sequence owing to the local depletion of typically buried amino acid residues as well as enrichment of typically exposed amino acid residues (about 40%) (see [Table S3](#pone.0007350.s003){ref-type="supplementary-material"}). Amino acid residues located in highly mobile regions of protein also possess the smallest volumes and molecular weights as well as the lowest hydrophobicities and the highest flexibility (see [Figures S3](#pone.0007350.s006){ref-type="supplementary-material"}, [S4](#pone.0007350.s007){ref-type="supplementary-material"}, [S5](#pone.0007350.s008){ref-type="supplementary-material"} and [S6](#pone.0007350.s009){ref-type="supplementary-material"}). Only in this way the protein can be so dynamically flexible to minimize unfavorable pairwise contacts.
In the last years it has become evident that there is a large number of proteins that do not require a stable structure even under physiological conditions in order to fulfil their biological role [@pone.0007350-Dyson1]--[@pone.0007350-Tompa1]. The importance of protein disorder is underlined by the abundance of partially or fully disordered proteins that are encoded in higher eukaryotic genomes [@pone.0007350-Ward1] and are involved in many important biological functions [@pone.0007350-Dyson1], which complement the functional repertoire of globular proteins [@pone.0007350-Xie1]. Disordered segments often act as flexible linkers between folded domains in multidomain proteins [@pone.0007350-Dyson1] and their function is often that of binding specifically other proteins, DNA or RNA through a process, termed coupled folding and binding, that involves a transition disordered-ordered with stable secondary and tertiary structural elements [@pone.0007350-Dyson1]. This "coupled binding and folding" confers several functional advantages in certain types of molecular interactions that often are essential for signaling processes. The high propensity of their residues to stay disordered makes these regions predominantly not structured and this can favour their putative functionality.
In fact, recent papers have shown the importance in Sirt-1 of various phosphorilations sites, nuclear localization signals (NLS) and nuclear export signals (NESs, amino acids ) that were mainly found in the terminal regions [@pone.0007350-Sasaki1], [@pone.0007350-Tanno1], [@pone.0007350-Ford1]. These authors suggested that these regions seem to be involved in regulating enzyme activity and have the peculiarity of being present only in Sirt-1. Moreover, this fact also suggests that C- and N-terminal regions of the human Sirt-1 might be involved in a more fine regulating role in order to exploit its biological mechanisms. In the disordered regions specific protein binding sites were identified by using Anchor program [@pone.0007350-Meszaros1]. The prediction of these binding sites is based on estimating the energy content in free and in the bound states, and identifying segments that are potentially sensitive to these changes. In particular, Anchor program has predicted in human Sirt-1 fourtheen disordered binding regions of which four located in the N-terminal domain, two in the allosteric site and other eight in the C-terminal domain ([Figure 2](#pone-0007350-g002){ref-type="fig"}-down and [Table S1](#pone.0007350.s001){ref-type="supplementary-material"}). The two disordered binding regions predicted in the allosteric site agree with those evidenced in Sirt-1/AROS complex (see [Table 3](#pone-0007350-t003){ref-type="table"}). All these data indicate that human Sirt-1 is an unordered protein and that its terminal domains can play different roles. The inherent flexibility of the two termini suggests that the protein has a malleable interface that can allow the binding of several partners or adopt different conformations, as manifested by its high binding capability.
Moreover the flexibility and the open structure of Sirt-1 termini could also favour the binding of phosphorylating proteins in order to activate regulation processes mediated by phoshorylation [@pone.0007350-Sasaki1]. In fact, very recently the presence of phosphorylation sites located in the amino- and in carboxyl-terminal regions were found [@pone.0007350-Sasaki1]. Therefore, in order to assess the ability of human Sirt-1 to be regulated by phorphorylation [@pone.0007350-Ford1] and if there are the structural features inducing the interaction with phosphorylating proteins, we have evaluated the solvent accessibility of each residue, suggested as a putative site of phosphorylation [@pone.0007350-Sasaki1]. Our data indicate that Ser14, Ser173, Ser538, Ser 539, Ser540, Thr 544 and Ser747 are largely exposed to the solvent ([Figure 7](#pone-0007350-g007){ref-type="fig"}) confirming that these residues have a high capability to be phosphorylated. If one considers the sequence position of Ser and Thr residues they are almost located in the N- and C- termini: Ser14, Ser26 and Ser27 are inserted in the segment 1--33 predicted by Anchor as a protein binding site. The residues Ser47 and Ser 159 are also included in the predicted segments 43--49 and 133--161, respectively, while the remaining Ser169, Ser173, Ser174, Thr196 and Thr 219 are very close to the above predicted segments. This suggests that these residues are actually involved in some way in the control of protein-protein binding and, as a consequence, in the control of the Sirt-1 function. The Sirt-1/AROS complex explains well this view by suggesting that the interaction between the two proteins occurs primarily through the involvement of the N-terminal segment with the achievement of greater compactness of the complex and the presence of changes that propagate to the active site in order to modulate the function. Work is in progress to simulate the presence of phosphate at various sites in the unordered regions in order to assess their structural or functional effects during the formation of the complex. The numerous and different structural features of unordered proteins such as conformational heterogeneity, secondary structural propensities, and tertiary contacts within disordered protein states can in principal generate many different interaction modes in proteins. Frequently these proteins mediate in signaling networks dynamic protein interactions that exhibit unusual binding characteristics, such as multisite dependence and ultrasensitivity. Such interactions are frequently modulated by phosphorylation, which requires disorder in the target protein both for optimal kinase accessibility and for subsequent accessibility of the binding motif [@pone.0007350-Lamming1], [@pone.0007350-Iakoucheva1]. Therefore this binding appears to require multiple sites to be phosphorylated, suggesting a binding mode in which multiple phosphoepitopes engage a single receptor site in dynamic equilibrium. These observations allows to hypothesize a binding mode in which the multiple sites found in Sirt-1 might engage the putative receptor sites of AROS (5 Ser and 4 Thr residues) in a dynamic equilibrium. Moreover, Sirt-1 contains numerous additional phosphorylation sites remote from the targeting regions, making their participation in the complex unlikely. It is possible, however, that they may serve as decoy sites that compete with the key binding sites for phosphorylation by the targeting kinase [@pone.0007350-Kim2].
{#pone-0007350-g007}
Finally, a detailed study of the binding patches between AROS and Sirt-1 has shown that in Sirt-1 there is a conserved pharmacophore pocket composed by basic residues at the top of the cavity, acidic residues at its bottom and hydrophobic residues as edges. Moreover we have focused our attention on the presence of five aromatic residues (i.e. Trp176, Tyr185, Phe187, Trp221, Trp624) in the pocket that could be involved in putative stacking-interactions. These data could explain the high affinity of the allosteric site for synthetic activators containing aromatic rings and hydrophobic anchor points. Investigations are in progress to study the Sirt-1 interaction also with small allosteric effectors that have been recently identified [@pone.0007350-Milne1].
Methods {#s4}
=======
Modelling of catalytic and allosteric sites of human Sirt-1 {#s4a}
-----------------------------------------------------------
The three-dimensional model of catalytic site of human Sirt-1 (UniProt code: Q96EB6, region 244--498) was performed by comparative modelling strategy using the template structures of human Sirt-2 (PDB code: 1J8F chain A) [@pone.0007350-Finnin1], Hst2 from Saccharomyces cerevisae (PDB code: 1Q1A chain A) [@pone.0007350-Zhao1] and Sir2-Af1 from Archaeoglobus flugidus (PDB code: 1M2G chain A) [@pone.0007350-Chang1] because the percentage of sequence identity between these proteins and human Sirt-1 was equal to 44%, 38% and 30%, respectively. Protein sequences were aligned with CLUSTALW [@pone.0007350-Thompson1]. The MODELLER9v5 program [@pone.0007350-Sali1] was used to build 10 full-atom models of catalytic site, we used the ProsaII program to check the fitness of the sequences relative to the obtained structures and to assign a scoring function, and the PROCHECK program [@pone.0007350-Laskowski1] to evaluate their stereochemical and structural packing quality. Secondary structures were assigned by the DSSP program [@pone.0007350-Kabsch1]. Secondary structure predictions were performed with Jpred [@pone.0007350-Paladino1] server but structural class predictions were made by using PRECLASSPRO server [@pone.0007350-Costantini2]. The globularity of the best model was evaluated according to our recent work [@pone.0007350-Costantini1].
Moreover, the three-dimensional model of allosteric site of human Sirt-1 region 181--243 was performed using the template structure of a hexokinase from Sulfolobus Tokodaii by comparative modelling (PDB code: 2E2N) [@pone.0007350-Nishimasu1]. As the sequence identity between this Sirt-1 region and the homologous template model was lower than 30% (i.e. 26%), we used a procedure strategy in agreement with the rules recently reviewed to improve the quality of the modelling results at low target-template sequence similarity [@pone.0007350-Dalton1]. Allosteric and catalytic sites have been connected by using the Builder module of Insight II and the related model was subjected to molecular dynamics simulations.
*Modelling of N-terminal and C-terminal regions of human Sirt-1* {#s4b}
----------------------------------------------------------------
The BLAST research [@pone.0007350-Altschul1] has found in databases only protein structures related to small sequences similar to the N-terminal and C-terminal regions of human Sirt-1 \[region 1--180 and 499--747, respectively\]. The prediction of unordered regions in the protein was made by Disopred server [@pone.0007350-Ward1]. Moreover, a prediction of protein binding sites in the unordered regions was made by Anchor program that identifies specific binding regions undergoing disorder-to-order transition using a general disorder prediction method IUPred based on the assumption that disordered proteins have a specific amino acid composition that does not allow the formation of a stable well-defined structure [@pone.0007350-Meszaros1]. The prediction of binding sites is based on estimating the energy content in free and in the bound states, and identifying segments that are potentially sensitive to these changes.
Therefore these regions were modelled by using a new approach combining fold recognition and comparative modelling. The preliminary models for N-terminal and C-terminal regions of Sirt-1 were obtained by fold recognition strategy using the Fugue and SAM-T06 servers [@pone.0007350-Shi1]--[@pone.0007350-Karplus1]. Then, these models with the crystallographic structures suggested by BLAST were used as template for applying the comparative modelling strategy in order to obtain the complete models for both regions. In details, we used as template the following structures deposited in PDB: 1W36 (577--625), 1QHZ (1--34) for the N-terminal region and 1TYC (76--101) for C-terminal region.
In the obtained models the loop regions were refined using the LOOPY module of Jackal package [@pone.0007350-Xiang1]. LOOPY appeared to yield the best results for loop modelling, with models that are on average of 2--8% better than those generated by other programs [@pone.0007350-Dalton1]. The models obtained for N-terminal and C-terminal regions of Sirt-1 were subjected to molecular dynamics simulations.
Molecular dynamics simulations {#s4c}
------------------------------
MD simulations were performed with GROMACS software package (v3.3.1) [@pone.0007350-VanDerSpoelDLindahl1]. Models of different Sirt-1 regions were put in cubic boxes filled with SPC216 water molecules and GROMOS43a1 was selected as force-field. In order to optimize the system, the models were previously subjected to energy minimization and position restraints cycles. The simulations were carried out with periodic boundary conditions by adding sodium ions in order to the net electrostatic charge of the system is zero. The bond lengths were constrained by the all atoms LINCS algorithm. Particle Mesh Ewald (PME) algorithm was used for the electrostatic interactions with a cut-off of 0.9 nm, according to recent papers [@pone.0007350-Paladino1]. Simulations were conducted at neutral pH where the tritable groups of His, Glu and Asp residues are unprotonated. All simulations were run for 15 ns at room temperature (300 K) coupling the system to an external bath. GROMACS routines were utilized to check the trajectories and the quality of the simulations.
Simulation of complete model of Sirt-1 {#s4d}
--------------------------------------
The whole model of human Sirt-1 was obtained by comparing modelling using as template the models obtained for N-terminal region, allosteric site, catalytic site, and C-terminal region and the same procedure and programs described above. This model was minimized by using 500 steps of energy minimization under conjugate gradient algorithm to optimize side chain conformations and avoid sterical clashes according to the commonly used procedure [@pone.0007350-Paladino1], [@pone.0007350-Costantini3]--[@pone.0007350-Costantini4].
Modelling and Simulation of Sirt-1/AROS complex {#s4e}
-----------------------------------------------
The three-dimensional model of AROS (UniProt code: Q86WX3, region 47--123) was performed by fold recognition strategy using the Fugue server [@pone.0007350-Shi1]. The model obtained for AROS was subjected for 15 ns to molecular dynamics simulations using the protocol reported above to assess its conformational stability.
To simulate the Sirt-1/AROS complex we used the docking web server CLUSPRO [@pone.0007350-Comeau1] that resulted the best docking program in CAPRI experiments with a success rate of about 71% [@pone.0007350-Comeau1]. The models were selected by evaluating some features and parameters. The "Protein--Protein Interaction Server" [@pone.0007350-Jones1] were used to identify the amino acids at the interface and to evaluate their solvent accessibility.
Moreover, the binding free energy between the different chains was calculated by using the DCOMPLEX program [@pone.0007350-Liu1].
Supporting Information {#s5}
======================
######
Regions predicted as protein binding sites by Anchor program
(0.03 MB DOC)
######
Click here for additional data file.
######
Analysis of interaction between AROS and Sirt-1 before and after MD
(0.03 MB DOC)
######
Click here for additional data file.
######
Amino acid composition of Sirt-1
(0.18 MB DOC)
######
Click here for additional data file.
######
Details of catalytic groove before and after molecular dynamics are shown. We reported in pink and green the carbon atoms related to Sirt-1 before and after dynamics but N, O and H atoms always in blue, red and white, respectively. The Acetil-lysine, NAD and Sirt-1 residues are evidenced with labels.
(0.19 MB DOC)
######
Click here for additional data file.
######
Secondary structure of the whole Sirt-1 structure assigned by DSSP program. The N-terminal and C-terminal region sequences are reported in green and blue, respectively. The helices and beta-strands are indicated in red and cyan, respectively.
(0.07 MB DOC)
######
Click here for additional data file.
######
Flexibility plot for Sirt-1 sequence. Ordinate reports the value of Hydrophobicity x Volume obtained with a shifting window of 5 according to Ragone et al. Protein Eng. 1989 2(7):497--504. Abscissa reports the residue position.
(0.09 MB DOC)
######
Click here for additional data file.
######
Average area buried. Lower values indicates higher exposures of residues. The graph shows that the residues in the globular part of the protein are in average more buried than the N and C termini. In particular, residues in the N-terminus are in average more exposed.
(0.03 MB DOC)
######
Click here for additional data file.
######
The average molecular weight of residues with a shifting window of 5. The graph shows that the compact globular core is made in average of high molecular weight residues while the N- and C- termini are made of low molecular weight residues and thus smaller residues are located in the more fluctuating or flexible structural regions. It is interesting to note the highly fluctuating values in the C-terminal region in agreement with the presence of more structured segments in respect to the N-terminal region.
(0.03 MB DOC)
######
Click here for additional data file.
######
Ramachandran Plot
(0.46 MB DOC)
######
Click here for additional data file.
**Competing Interests:**The authors have declared that no competing interests exist.
**Funding:**The funding is from the Institute in which this project was carried out. Funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. IA is supported by Doctorate in Computational Biology, CRISCEB, Second University of Naples, Naples, Italy.
[^1]: Conceived and designed the experiments: SC GC. Performed the experiments: IA. Analyzed the data: IA SC GC. Wrote the paper: IA SC GC.
|
{
"pile_set_name": "PubMed Central"
}
|
Introduction {#Sec1}
============
Chemical sensors using integrated photonics have attracted significant attention, due to their potential for environmental monitoring and high throughput screening for biomedical discovery^[@CR1]--[@CR5]^. Advanced technologies using absorbance, surface plasmon resonance (SPR), and fluorescence detection have been developed to achieve chip-scale optical sensing^[@CR6]--[@CR10]^. For instance, chemical sensors using optical micro-ring resonators with ppm-level detection have been demonstrated^[@CR11]--[@CR14]^. Meanwhile, SPR sensors using perfect absorbers or highly doped semiconductors are utilized for multispectral IR Spectroscopy and gas identification^[@CR15]--[@CR18]^. However, on-chip sensors capable of broadband mid-IR sensing have not yet been well developed^[@CR19],\ [@CR20]^. A mid-IR sensor is capable of providing label-free and real-time sensing applications because it overlaps with characteristic absorption bands of various organic and inorganic compounds^[@CR21],\ [@CR22]^. Prior studies utilized multilayer material platforms including Si-on-SiO~2~, Si-on-sapphire, and SiN~x~-on-SiO~2~, etc^[@CR23]--[@CR28]^. Though crystalline Si and amorphous SiN~x~ are transparent up to λ = 8 μm, SiO~2~ becomes opaque after λ = 3.7 µm and sapphire after λ = 4.5 µm, which exclude those platforms for mid-IR applications at longer wavelengths.
To overcome these challenges, we propose a monolithic a-Si on BTO platform for mid-IR integrated photonic devices and sensing applications because of following advantages. *i*. Both a-Si and BTO have a broad infrared transparent window up to λ = 7 μm, so a Si-on-BTO waveguide can be utilized to monitor characteristic absorption bands at longer wavelengths. *ii*. BTO has a lower refractive index **n** of 2.4 making it an ideal undercladding material for a Si waveguide with an index **n** of 3.5. The large difference of **n** between Si and the BTO ensures that the light is efficiently confined by the waveguide and consequently decreases the bending loss caused by waveguide curvatures. *iii*. Unlike other ferroelectrics, such as LiNbO~3~, BTO has the potential to be integrated on a Si wafer through various thin film deposition technologies, such as PLD, molecular beam epitaxy (MBE), and chemical vapor deposition (CVD), which enables the integration between functional oxides and Si photonics^[@CR29]--[@CR33]^. *iv*. BTO has high chemical stability and mechanical hardness making it capable for biochemical and toxic sensing under harsh environments. *v*. The restraint of the crystal lattice matching between the crystalline BTO layer and the upper Si device layer is relieved by the utilization of a-Si. For conventional silicon-on-insulator (SOI), it requires a crystalline Si film and involves sophisticated preparation processes, from oxygen ion beam implantation and high-temperature annealing to exfoliation. Alternatively, the proposed a-Si waveguide layer can be directly deposited and then patterned on the epitaxial BTO layer though a standard CMOS process.
In this work, we demonstrate a mid-IR Si-on-BTO waveguide for label-free chemical sensing. The waveguide structures and the waveguide mode profiles were designed and calculated by the two-dimensional finite difference method (FDM). In parallel, we prepared the epitaxial BTO thin film on LaAlO~3~ (LAO) (001) substrates by PLD and then develop the a-Si ridge waveguide on the BTO film through CMOS process. The waveguide structure and the compositions of the Si-BTO-LAO multilayer were characterized by a scanning electron microscope (SEM) equipped with energy-dispersive X-ray spectroscopy (EDX). Meanwhile, the waveguide mode profiles were recorded and analyzed at λ = 2.5 μm--3.2 μm. Selected organic solvents, heptane and methanol, were tested to evaluate the detection capability of our waveguide sensors. Chemical detection was accomplished by correlating the waveguide spectral attenuation with the characteristic absorption of the test solvents.
Results and Discussion {#Sec2}
======================
The fabricated mid-IR sensing device was inspected by a SEM with EDX. Figure [1(a) and (b)](#Fig1){ref-type="fig"} are the top and the side SEM images of a 10 μm wide a-Si on BTO waveguide. The Si layer has a well-defined ridge profile without bends or distortions found on the edge. No cracks or indents on the BTO film surface indicates that ion damage has not been introduced on the BTO film during the etching process. The sharp waveguide edges reduce the waveguide propagation loss caused by light scattering, which is critical to achieve a high signal-to-noise ratio and high sensitivity during waveguide sensing. Meanwhile, the cross-sectional image in Fig. [1(c)](#Fig1){ref-type="fig"} displays a clearly resolved interface between the top a-Si waveguide and the under-cladding BTO layer that confirms the lattice matching requirement between the Si and the crystalline BTO film relieved by using amorphous Si. The material composition of the monolithic Si-on-BTO platform was characterized by EDX using the emission lines of Si Kα at 1.74 keV and Ba Lα at 4.47 keV. The EDX scanning provides elemental distributions of the Si, Ba, Ti, and Al that are associated with the a-Si waveguide, BTO cladding layer, and LAO substrates, respectively. From the cross-sectional EDX scanning shown in Fig. [1(d)](#Fig1){ref-type="fig"}, the Si waveguide height is 1 μm and the BTO film thickness is 0.5 μm, while the LAO substrate is present underneath the device layers.Figure 1SEM images of a 10 μm wide Si-on-BTO waveguide captured from (**a**) the top, (**b**) the side, and (**c**) the cross-section of the device. A clear waveguide surface, smooth Si-BTO interface, and sharp waveguide edges were observed. (**d**) EDX element line scanning determined the structure and the composition of each device layer.
Numerical simulations of waveguide modes were calculated by using the two-dimensional finite difference method (FDM). For waveguide sensing application, it is critical to evaluate the optical fields of waveguide modes since the sensitivity of a waveguide sensor is determined by the interaction between its evanescent field and the molecules close to the waveguide surface. The structure utilized in our mode calculation was obtained from the SEM measurement, where the a-Si ridge is 1 μm tall and 10 μm wide and the BTO layer underneath the Si is 0.5 μm thick. The refractive index of a-Si, BTO and LAO were 3.5, 2.4 and 2.0, respectively. A 12 μm × 6 μm light source was chosen to excite the waveguide mode since its size is comparable to 9 μm core diameter of the mid-IR fiber used in the experiment. The intensity profiles corresponding to the TE and TM waveguide modes at λ = 2.5 μm, 2.85 μm, and 3.2 μm are depicted at Fig. [2(a)](#Fig2){ref-type="fig"}. Fundamental modes with similar ellipsoid intensity distribution are clearly resolved in the Si layer over λ = 2.5 μm to λ = 3.2 μm, while the evanescent fields in the air (z \> −0.5 µm) and inside the BTO layer (z \< −1.5 µm) increase as the mid-IR shifts to longer wavelengths. In addition, stronger evanescent fields are found in the TM mode profile comparing to that of the TE mode. Meanwhile, both the TE and the TM modes have relatively weak evanescent fields along the y directions (y \< −5 µm or y \> 5 µm) because the a-Si waveguide structure has a high y/z aspect ratio of 10/1. To better analyze the mode properties, Fig. [2(b and c)](#Fig2){ref-type="fig"} display one dimensional TE and TM polarized intensity profiles along the z-axis. The relative intensities confined in each layer, air, Si, BTO, and LAO, were calculated at λ = 2.5 μm, 2.85 μm, and 3.2 μm and the results are summarized in Table [1](#Tab1){ref-type="table"}. Both the TE and the TM modes expand their optical fields extensively into the upper air as well as the lower BTO layer. The relatively strong field found within the BTO layer is attributed to its high refractive index of **n** ~**BTO**~ = 2.4. Meanwhile, the optical intensity above the waveguide is found to be stronger for the TM mode comparing to that for the TE mode. For instance, at λ = 3.2 µm the TM mode has 10.10% intensity in the air that is 1.7 times higher than 6.01% for the TE mode. In addition, the evanescent fields increase as the wavelength increases from λ = 2.5 µm to 3.2 µm. These results indicate our waveguide sensor will exhibit a higher sensitivity when it operates with TM polarization light as well as at a longer wavelength. The preservation of the fundamental mode over a wide spectral range is another necessity to achieve high accuracy of waveguide sensing. An excitation of higher order modes will alter the mode profiles and vary the intensities of evanescent fields that consequently will lead to false signals upon spectrum scanning.Figure 2(**a**) The optical fields of the mid-IR waveguide were calculated at λ = 2.50 µm, 2.85 µm, and 3.20 µm. Fundamental modes with similar ellipsoid intensity distributions were resolved in the Si layer in all three wavelengths. (**b**) and (**c**) are the calculated 1D intensity profiles of the TE modes and the TM modes, along the z axis, respectively. Table 1The intensity distribution of a Si-on-BTO waveguide, which consists of up-cladding air, a-Si waveguide, BTO undercladding, and LAO substrate.PolarizationWavelength (µm)Intensity distribution in each layer (%)AirSiBTOLAOTE2.504.1988.447.070.322.855.0985.928.410.583.206.0183.369.680.95TM2.506.9381.8210.610.642.858.4977.5312.761.223.2010.1073.1014.772.07The distributions were calculated at two polarizations, TE and TM, and three different wavelengths of λ = 2.50 µm, 2.85 µm, and 3.20 µm.
In our experiments, methanol and heptane were selected as the analytes to evaluate the performance of our waveguide sensors due to their strong characterstic absorptions existing in the mid-IR regime. A light source with the TM polarization was utilized since TM light reveals a stronger evanescent field that will attribute to higher sensitivity. The wavelength of the probe light was sequentially scanned between λ = 2.5 µm and 3.2 µm because this spectrum regime overlaps with the absorption bands caused by -OH and -CH functional group while Si-on-BTO is transparent. The waveguide mode images were recorded before and after dropping the chemical analytes onto the waveguide surface. As shown in Fig. [3](#Fig3){ref-type="fig"}, without any chemicals present, a bright and sharp fundamental mode was observed through λ = 2.5 µm to 3.2 µm as expected from the simulation results. Upon dropping the heptane on the waveguide, the mode faded at λ = 3.0--3.2 µm due to the absorption caused by the -CH bond stretching. On the other hand, when methanol applies, drastic absorption appeared at λ = 2.8--2.9 µm that corresponds to the absorption due to the -OH bond stretching. Hence, our mid-IR sensor reveals distinct spectral attenuations when exposed to different chemicals, and the measured absorption results agree well with the previous studies from FTIR characterization. After the chemicals evaporate, we found that the mode profiles recovered and their intensities arrived back at the same levels just as before chemicals were applied. Thus, our mid-IR sensor is not only capable of accurate chemical identification, but also reusable.Figure 3The waveguide mode images were captured from λ = 2.5 µm to 3.2 µm with or without chemicals covering the waveguide. Fundamental modes were clearly observed over a broad spectral range. When heptane was presented, the mode disappeared at λ = 3.0 µm--3.2 µm corresponding to -CH absorption. For methanol, the mode vanished at λ = 2.8 µm--2.9 µm associating with -OH absorption. The mode intensities recovered when the analytes evaporated from the waveguide surface.
The real-time chemical detection was performed by reading the transient response of the mid-IR waveguide sensors. For heptane detection, the wavelength of the probe light was tuned to λ = 3.1 µm because it is within the -CH absorption band. The waveguide mode intensity upon adding the chemical is shown in Fig. [4(a)](#Fig4){ref-type="fig"}. Before t = 20 s the intensity was strong since there was no presence of any analyte. When the heptane was dropped on the waveguide surface at t = 20 s, the intensity decreased dramatically because the analyte, heptane, fully covered the mid-IR waveguide and absorbed its evanescent light. The waveguide intensity remained low until t = 50 s and then it started to recover because the heptane gradually evaporated. Eventually at t = 110 s the intensity reached its original level due to the heptane being completely left from the waveguide surface. A similar transient response was observed during the methanol sensing test shown in Fig. [4(b)](#Fig4){ref-type="fig"}. To track methanol, the light wavelength was shifted to 2.9 µm to match the characteristic -OH absorption. We found that the light intensity dropped at t = 25 s, which coincided with adding methanol onto the waveguide. After a while, the intensity recovered at t = 120 s indicating the analyte evaporated from the waveguide surface. Our time-resolved characterization demonstrates that the developed mid-IR sensor is suitable for *in-situ* monitoring of various chemical analytes with high accuracy.Figure 4Real-time detection of (**a**) heptane and (**b**) methanol using mid-IR waveguide sensors at λ = 3.1 µm and 2.9 µm, respectively. The mode intensity decreased when the analytes were dropped on the waveguide surface and then recovered when the analyte evaporated.
In summary, we demonstrated monolithic Si-on-BTO waveguides for label-free and real-time chemical detection. The epitaxial BTO thin film was prepared by pulsed laser deposition and it exhibited a broad spectral transparency from λ = 2.5 µm to 7 µm that enables our Si-on-BTO platform for mid-IR sensing application. SEM study revealed sharp waveguide side walls, as well as a smooth Si-BTO interface that reduced waveguide propagation loss, which is critical to achieve accurate chemical sensing. Heptane and methanol were then tested to exam the performance of the mid-IR waveguide sensor. Upon spectral scanning the waveguide modes showed strong intensity attenuation at λ = 3.0 µm--3.2 µm for heptane detection and λ = 2.8 µm--2.9 µm for methanol detection, corresponding to the characteristic -CH and -OH absorption bands, respectively. Furthermore, from real-time measurements, our mid-IR waveguide sensor can achieve *in-situ* chemical detection within milliseconds. The Si-on-BTO mid-IR waveguides provides a unique CMOS-compatible platform for label-free and high-throughput chemical screening.
Methods {#Sec3}
=======
Device fabrication {#Sec4}
------------------
Figure [5](#Fig5){ref-type="fig"} illustrates the fabrication process of the Si-on-BTO waveguide sensor. The BTO films were deposited on single-crystal LAO (001) substrates by PLD at 10 Hz with a KrF excimer pulsed laser (Lambda Physik, λ = 248 nm). The substrate temperature was maintained at 700 °C and the O~2~ partial pressure was 40 mTorr during the deposition. After deposition, the film was annealed at 600 °C in 200 Torr O~2~ for 1 hour and then cooled down to room temperature. A 1 µm thick a-Si thin film was then deposited on the BTO film by the plasma-enhanced chemical vapor deposition (PECVD) process. The precursor gas for the a-Si deposition was SiH~4~, and the deposition temperature was 200 °C. Since the Si film is amorphous, the restraint of lattice matching between the Si film and the crystalline BTO film was relieved, which enables the formation of a smooth interface between the BTO and the Si layers. The structure of the sensor waveguide was defined through photolithography, where a 50 nm thick Cr mask was deposited on the a-Si/BTO/LAO sample by electron beam evaporation and followed by the lift-off process. The waveguide structure was then transferred to the a-Si layer by reactive ion etching (RIE). SF~6~ was used for selective Si etching, so that it did not react with the BTO film, and therefore prevented the BTO surface from becoming rough due to ion damage. It is vital to have sharp Si waveguide facets as well as a smooth BTO-Si interface to reduce the scattering loss caused by surface roughness. Previous studies that utilize HF solution for BTO etching have shown undesired rough waveguide surfaces and edges. Finally, the Cr mask and the organic residue on the device surface were removed by a ceric ammonium nitrate solution and followed by oxygen plasma ashing.Figure 5The fabrication process of the mid-IR waveguide sensor using Si-on-BTO platform. The epitaxial BTO film was grown on a (001) LAO substrate by PLD and then followed by a-Si thin film deposition through PECVD. Using photolithography and lift-off, the waveguide structure was first defined by a Cr mask, and then transferred to the a-Si layer by RIE.
Optical and Sensing Characterization {#Sec5}
------------------------------------
To characterize the performance of our a-Si on BTO waveguides, a broad mid-IR test station was built and shown in Fig. [6](#Fig6){ref-type="fig"}. The light source is a pulsed laser with a wavelength tunable from λ = 2.4 μm to λ = 3.8 μm and the linewidth is of 3 cm^−1^. The laser has a pulse repetition rate of 150 kHz, a pulse duration of 10 nano seconds, and an average power of 150 mW. Using a reflective lens, the probe light was first collimated into a fluoride fiber that has a 9 μm core and 125 μm cladding, and then butt coupled into the waveguide. The core of the mid-IR fiber was lined up with the smoothly cleaved front facet of the Si-on-BTO waveguide as shown in Fig. [6(b)](#Fig6){ref-type="fig"}. The fine alignment between the optical fiber and the waveguide front facet was monitored by an upper microscope equipped with a long working distance 10x objective lens. The mid-IR light emitted from the waveguide end facet was focused by a barium fluoride biconvex lens with a 25 mm focal length and then imaged by a 640 × 512 pixel InSb camera cooled by liquid nitrogen. In the chemical sensing tests, 1 mL solution was dropped from a syringe onto the Mid-IR waveguide sensor with 1 cm^2^ surface area to ensure that the solution covers the entire waveguide array. The organic solvents include heptane and methanol (≥99.9%, Sigma-Aldrich). The chemical mixtures were prepared by weight percentages. During sensing experiments the temperature was maintained at 25 °C.Figure 6(**a**) Mid-IR test station to characterize the performance of our Si-on-BTO waveguide sensor. The probe light from a tunable pulsed laser (λ = 2.4 μm to 3.8 μm) was collimated into a mid-IR fiber using a reflective lens (RL) and then butt coupled into the waveguide. Meanwhile, the analytes was dropped onto the waveguide surface through a syringe. The mid-IR light emitted from the waveguide end facet were focused by a barium fluoride biconvex lens and then imaged by an InSb camera. (**b**) The core of the mid-IR fiber was lined up with the front facet of the Si waveguide. The fine alignment between the optical fiber and the waveguide was monitored by an upper microscope (MO).
Tiening Jin, Leigang Li, Bruce Zhang, Hao-Yu Greg Lin, Haiyan Wang and Pao Tai Lin contributed equally to this work.
**Publisher\'s note:** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
The authors gratefully acknowledge funding support provided by Texas A&M University (TAMU) and the Texas A&M Engineering Experiment Station (TEES). The authors thank M Square Laser for equipment contribution. Device fabrication and characterization were performed at AggieFab and the Materials Characterization Facility (MCF) at Texas A&M University and the Center for Nanoscale Systems (CNS) at Harvard University. L.L. and H.W. acknowledge the support of the thin film growth effort from the U.S. National Science Foundation (DMR-1643911).
T.J. performed the sensing measurement and SEM/EDX characterization. L.L. and B.Z. grew BTO thin film and measured x-ray diffraction. H.-Y.G. grew a-Si thin film. H.W. and P.T.L. supervised the research and reviewed the manuscript.
Competing Interests {#FPar1}
===================
The authors declare that they have no competing interests.
|
{
"pile_set_name": "PubMed Central"
}
|
1. Introduction {#s0005}
===============
East Coast fever (ECF), a fatal bovine disease, is caused by the apicomplexan parasite *Theileria parva* ([@b0245])*.* The disease kills approximately one million cattle every year in Africa and causes significant economic losses to small-hold farmers in eastern, central and southern Africa of approximately \$300 million annually (reviewed in [@b0235]). These estimates were derived several years ago and it is highly likely that the current losses due to ECF are much larger. Vaccination against ECF by an infection and treatment method (ITM) is available ([@b0280]). For control of ECF in southern Africa, except South Africa where vaccination is not permitted as it is free of ECF, ITM is performed with a single parasite isolate, and in parts of eastern Africa a composite ITM vaccine, the Muguga cocktail, is used ([@b0220]). Although ITM confers solid and long-lived immunity it has disadvantages in that it is an unstable and potentially lethal product, and animals vaccinated by the ITM protocol remain life-long asymptomatic carriers of the parasite that pose risks for spread of the disease. It also requires a liquid nitrogen cold chain for storage and oxytetracycline for co-treatment (reviewed in [@b0075]). The vaccine is expensive and laborious to produce, and it requires skilled personnel for delivery (reviewed in [@b0075]). Therefore, development of a subunit vaccine that is easier to produce and with minimal risks is necessary.
Although classified in the phylum Apicomplexa, aspects of the biology of *Theileria* are atypical of such organisms. For example, *Theileria* sporozoites are not motile, they have a less defined apical complex and host cell entry is not orientation-specific ([@b0320]). Sporozoites bind and enter bovine lymphocytes by a ''zippering'' process of the host and the sporozoite cell membranes ([@b0120]). After entry into the host cell, rhoptries/microspheres are discharged with a rapid escape of the sporozoite from the surrounding host cell membrane. Sporozoites differentiate to the schizont stage, which resides in the host cytoplasm surrounded by host cell microtubules that are seemingly nucleated by parasite molecules (reviewed in [@b0320]). Schizont-infected cells acquire a cancer-like phenotype and are the cause of disease ([@b0245]). Sporozoites also bind and enter macrophages/afferent lymph veiled cells by a zippering process, but these cells are not susceptible to transformation and sporozoites appear to only differentiate to an early schizont stage ([@b0325]). Several host cell signalling pathways that contribute to host cell transformation have been studied ([@b0090], [@b0070]). In addition, parasite molecules that are associated with the transformation process have been tentatively identified by taking advantage of comparative genomics and cancer biology ([@b0330], [@b0150], [@b0355]). A parasite-encoded prolyl isomerase has been recently identified as playing a key role in this complex host cell transformation process ([@b0215]).
There is good evidence for roles of *T. parva* sporozoite and schizont antigens as candidate vaccine antigens. Several schizont antigens are targets of cytotoxic T lymphocytes (CTLs) that lyse schizont-infected cells ([@b0140], [@b0135], [@b0145]) and two sporozoite antigens are targets of neutralising antibodies ([@b0085], [@b0310]). One of the latter antigens, p67, has been shown to consistently protect a proportion of immunised cattle against challenge with needle-injected sporozoites and from infected ticks in field trials (reviewed in [@b0235]). The p67-based vaccine might be improved by including additional antigens that can neutralise sporozoite infectivity.
As a first step towards improving our molecular understanding of the biology of *T. parva* we have undertaken a Multidimensional Protein Identification Technology (MudPIT) LC--MS/MS approach (reviewed in [@b0300]) to characterise the proteome of *T. parva* sporozoites purified by ion-exchange chromatography ([@b0230]). We have employed bioinformatic tools to analyse 2007 parasite proteins that we were able to detect and identified *T. parva* orthologs of *Plasmodium falciparum* invasion organelle proteins, calcium signalling proteins and surface proteins. Some of these may represent novel *T. parva* vaccine candidates.
2. Materials and methods {#s0010}
========================
2.1. Animals and sporozoite production {#s0015}
--------------------------------------
The procedure for sporozoites production has been previously described in detail ([@b0265]). Briefly, cattle aged 7--9 months, previously managed under a strict acaricide regime, were maintained for at least 2 months and tested for exposure to *T. parva*, *Theileria mutans*, *Babesia bigemina* and *Anaplasma marginale* using ELISA and, in certain instances, with PCR analysis ([@b0225], [@b0250]). The naïve cattle were then inoculated with *T. parva* stabilates of the Muguga isolate and infection of the cattle was assessed. A colony of *Rhipicephalus appendiculatus* nymphs, previously tested for viruses such as Bovine Viral Diarrhea virus (BVDV) and Bunya viruses, and demonstrated to be free from tick-borne haemoparasites ([@b0265]), was then applied to the infected cattle. The fully-fed nymphs were collected and incubated for 6 weeks at 24 °C to induce moulting into adults and infection rates assessed by microscopy as described before ([@b0045]). A total of 300 adult ticks infected with *T. parva* Muguga isolate (mean infection rate of 167 acini per tick) were allowed to feed on rabbits to induce sporozoite maturation. After 4 days, the tick salivary glands were dissected from the ticks. All animal procedures described in this article were approved by International Livestock Research Institute's (ILRI, (Kenya)), Institute Animal Care and Use Committee (IACUC File Number 2014.01).
2.2. Sporozoite purification {#s0020}
----------------------------
Dissected tick salivary glands were collected in a tube with 2 ml of cold PBS, transferred to a chilled glass hand homogenizer ('UNI-FORM' (England), 4--5 ml of homogenizer) and disrupted by gentle up, down and circular motion of the pestle until a uniform cloudy suspension was formed. The suspension was then centrifuged at 1000*g* at 4 °C for 5 min. The resulting supernatant containing sporozoites was applied to an 8 ml diethylaminoethyl cellulose (DE-52; Sigma--Aldrich (USA) Cat. No. D3764) column ([@b0230]). Flow-through fractions of 3.5 ml were collected in siliconised 10 ml conical bottomed glass tubes on ice. The sporozoite-rich cloudy fractions (numbers 2--5) were pooled and centrifuged at 12,000*g* at 4 °C for 5 min. The resulting sporozoite-rich small yellowish pellet was resuspended in SDS--PAGE sample buffer and heated at 100 °C for 5 min prior to fractionation on 10% one-dimensional SDS--PAGE and staining with Coomassie Blue.
2.3. In-gel trypsin digestion for LC--MS/MS analysis {#s0025}
----------------------------------------------------
The gel lane containing sporozoite material was excised and divided into four fractions of different size proteins (\>100 kDa, 100--55 kDa, 55--35 kDa and \<35 kDa). The gel pieces were de-stained overnight in 50% methanol, 5% acetic acid in water. After dehydration with acetonitrile, proteins were reduced with 10 mM DTT in 100 mM ammonium bicarbonate, and subsequently alkylated using 50 mM iodoacetamide in ammonium bicarbonate for 30 min at room temperature. Following washing of the gel pieces in 100 mM ammonium bicarbonate, proteolytic digestion was carried out with 100 ng of trypsin in 50 mM ammonium bicarbonate overnight at 37 °C. Tryptic peptides were extracted from each gel with 50% acetonitrile, 5% acetic acid in water, dried and re-suspended in 20 μl of 2% acetonitrile, 0.1% formic acid in water for LC--MS/MS analysis.
2.4. LC--MS/MS analysis {#s0030}
-----------------------
Peptides from each gel fraction were separated on an Ultimate 3000 RSLC nano System utilising a PepMap C18 column, 2 µm particle size, 75 µm × 50 cm (Thermo Scientific, USA) and subsequently analysed on a QExactive mass spectrometer (Thermo Scientific, USA). Peptides were introduced into the mass spectrometer using an EASY-Spray™ nano source at a flow rate of 250 nl/min at approximately 400--600 bars. The 15 most intense precursors were selected for MS/MS analysis using higher-energy collisional dissociation (HCD) fragmentation and all fragmented precursor ions were actively excluded from repeated MS/MS analysis for 27 s.
2.5. Data analysis {#s0035}
------------------
### 2.5.1. Protein identification and relative abundance {#s0040}
RNAseq data of the schizont stage of *T. parva* has recently been made available in GenBank (BioSample accession SAMN03981746) and used to improve the annotation of the genome (da Silva, pers. comm.). The format for open reading frame (ORF) locus tags in the re-annotated genome of the Muguga strain has been changed from TPXX_YYYY ([@b0130]) to TpMuguga_0Xg0YYYY, where X stands for the chromosome number and YYYY for the gene number. This study relied on the re-annotated genome and the new format for *T. parva* ORF locus tags has been adopted.
Raw MS/MS data files were analysed with Peaks software (Bioinformatics solutions) using a database containing all Uniprot database entries for *R. appendiculatus* and the re-annotated proteome of *T. parva* (a total of 16,969 protein entries with 4084 *T. parva* and 12,884 *R. appendiculatus* protein entries, July 2016). A peptide false discovery rate (FDR) of 1.2% was applied and only proteins with unique peptide identification were included in the final sporozoite proteome list.
In order to rank the identified proteins by their abundance in the sample, an Exponentially Modified Protein Abundance Index (emPAI) was calculated using Mascot (Matrix Science) ([@b0170]). EmPAI is the exponential form of protein abundance index (PAI) minus 1 (emPAI = 10^PAI^ − 1) and is proportional to protein content in a protein mixture ([@b0170]). PAI is calculated by dividing the number of observed peptides by the number of observable peptides per protein.
### 2.5.2. Classification of subcellular localization of the identified *T. parva* sporozoite proteins {#s0045}
Proteins were classified according to their putative localization in the sporozoite using TargetP ([@b0110]). Trans-membrane domains (TMDs) were predicted by TMHMM Server v. 2.0 ([@b0190]), signal peptides (SP) by SignalP 4.1 ([@b0270]) and glycosylphosphatidylinositol (GPI)-anchor signal by GPI-SOM ([@b0115]).
### 2.5.3. Classification of the identified *T. parva* sporozoite proteins by orthology {#s0050}
We clustered all sporozoite protein coding sequences of *T. parva* with the complete predicted proteome of *Plasmodium falciparum* into putative orthologous groups using the OrthoMCL standalone software Version 2.0.2 ([@b0205]). The blast step of OrthoMCL was performed using an E-value cut-off of 1e^−10^, and minimum 70% sequence identity over 75% sequence coverage. *Plasmodium falciparum* 3D7 release of 26-06-2017 sequence data was downloaded from PlasmoDB ([@b0015])
3. Results {#s0055}
==========
3.1. Sporozoite proteome determination and relative quantification of expressed sporozoite proteins {#s0060}
---------------------------------------------------------------------------------------------------
LC--MS/MS was used to identify proteins expressed in a sample of semi-purified *T. parva* sporozoites. A complete list of all the proteins identified in this study is provided in [Supplementary Table S1](#s0110){ref-type="sec"}. In total 4780 proteins were identified of which 2007 originated from *T. parva* and 2773 from *R. appendiculatus*. Most proteins (3847) were identified with two or more peptides, while 933 protein identifications were based on a single peptide match, which includes 296 *T. parva* proteins ([Supplementary Table S2](#s0110){ref-type="sec"}). Thus, there is higher confidence in the identification of 1711 expressed parasite proteins. Sequence coverage of over 85% was achieved for three proteins, glyceraldehyde 3-phosphate dehydrogenase NAD binding domain, histone H2B.1 and ribosomal protein S21e, all encoded by single copy genes. Protein TpMuguga_03g00168, annotated as a hypothetical protein, was identified by as many as 94 unique peptides.
The 2007 *T. parva* sporozoite proteins that were identified represent approximately 50% of the 4084 proteins predicted to be encoded by the re-annotated *T. parva* genome (da Silva, pers. comm). Of the 2007 proteins, 1287 were further ranked by calculation of relative abundance in the sample using emPAI values ([Supplementary Table S3](#s0110){ref-type="sec"}), a parameter that is useful for obtaining an overview of proteome profiles with a wide dynamic range of concentrations (30 fmol to 1.8 pmol/μl) ([@b0170]). The 20 most abundant *T. parva* sporozoite proteins are shown in [Table 1](#t0005){ref-type="table"}. Even within this data set, there is a large range in the abundance of parasite proteins with histones being the most abundant. Other housekeeping proteins, e.g., ribosomal proteins, heat shock protein 70, phosphoglycerate mutase and glyceraldehyde 3-phosphate dehydrogenase, feature in this list.Table 1Abundant *Theileria parva* sporozoite proteins. Proteins detected from whole cell lysates of sporozoites are ranked by relative abundance in the sample. Known antigens are in bold face.ORF locus tag[a](#tblfn1){ref-type="table-fn"}Annotation[b](#tblfn2){ref-type="table-fn"}Mass (Da)emPAI[c](#tblfn3){ref-type="table-fn"}TpMuguga_04g00404Histone H2B.112,0407228.34TpMuguga_04g00071Histone H2B13,682900.39TpMuguga_04g00675Histone H411,362457.35TpMuguga_04g00690Phosphoglycerate mutase 1 family29,221105.17TpMuguga_04g00050Ribosomal protein S1917,15099.38TpMuguga_03g00655Hypothetical protein17,29696.5TpMuguga_04g00036AhpC/TSA family22,12084.68TpMuguga_02g00487Ribosomal protein S6e25,55367.36TpMuguga_02g00903Actin42,27060.62TpMuguga_04g00322Histone H2A13,49860.26TpMuguga_03g00067Hypothetical protein11,57254.06TpMuguga_01g00726elF-Tu GTP binding domain49,77952.64TpMuguga_01g00541Hypothetical protein24,79652.36TpMuguga_04g00383GAPDH NAD binding domain36,88351.37**TpMuguga_01g00701RAP-170,44147.73**TpMuguga_02g00148Heat shock 70 kDa protein71,44542.22TpMuguga_04g00179RanBP1 domain38,98241.34TpMuguga_03g00700Hypothetical protein23,99540.00**TpMuguga_03g00287Sporozoite P67 surface antigen75,45339.16**TpMuguga_01g00067Acyl CoA binding protein10,04339.11[^1][^2][^3]
3.2. Identification of known *T. parva* antigens within the sporozoite proteome {#s0065}
-------------------------------------------------------------------------------
The major circumsporozoite surface protein of *T. parva*, p67, which is a target of sporozoite neutralising antibodies ([@b0095]), was identified among the 20 most abundant proteins ([Table 1](#t0005){ref-type="table"}). Additional antigens that had been previously characterised were also identified in this study, e.g., p104 ([@b0105]), p150 microneme/rhoptry proteins ([@b0335]) and p32, a merozoite antigen ([@b0340]). We searched the sporozoite proteome data ([Supplementary Table S1](#s0110){ref-type="sec"}) for characterised schizont CD8 T cell antigens ([@b0140], [@b0145]) and found evidence for expression of all known CTL antigens (reviewed in [@b0240]) in the sporozoite stage. In contrast to CD8 T cell antigens, CD4 T cell antigens still require better characterization. CD4 antigens are presented by intact schizont-infected cells ([@b0030]) as well as cell extracts ([@b0020]). Soluble cytosolic parasite antigens stimulatory to CD4 T cell clones with fractions ranging between 10 and 24 kDa ([@b0035]) have been described, but further analysis and identification of the specific constituent proteins is required. However, among the most abundant sporozoite proteins we identified a protein that is homologous to a *Theileria annulata* protein annotated as rhoptry associated protein-1 (RAP-1) ([Table 1](#t0005){ref-type="table"}). RAP-1 of *B. bigemina* has been shown to be a CD4 T cell antigen ([@b0025]), suggesting that the *T. parva* RAP-1 is a potential CD4 T cell antigen.
3.3. *Theileria parva* sporozoite proteins associated with apicomplexan invasion of host cells {#s0070}
----------------------------------------------------------------------------------------------
Sporozoite secretory organelle proteins are typically located in the micronemes, rhoptries and dense granules in *P. falciparum* (reviewed in [@b0210]). To determine whether these proteins are present in *T. parva* sporozoites, we clustered our identified *T. parva* sporozoite proteins, using OrthoMCL, into orthologous groups with all *P. falciparum* genes. A total of 1105 proteins of the 2007 *T. parva* proteins were defined as *P. falciparum* orthologs ([Supplementary Table S4](#s0110){ref-type="sec"}) and we searched this list for apical organelle proteins. We identified *T. parva* orthologs of eight rhoptry proteins, three microneme proteins and a dense granule protein ([Table 2](#t0010){ref-type="table"}). Featured in the list are orthologs of *P. falciparum* apical membrane antigen 1 (AMA-1), a microneme protein essential during host cell invasion ([@b0350]), and cell-traversal protein for ookinetes and sporozoites (CelTOS), a malarial antigen mediating host cell invasion ([@b0180]). AMA-1 is a leading malaria vaccine candidate ([@b0285]), and orthologs of it are also found in *Neospora caninum* and *Toxoplasma gondii* ([@b0375]).Table 2Invasion organelle proteins. Proteins detected from whole cell lysates of *Theileria parva* salivary gland sporozoites are aligned by orthology with *Plasmodium falciparum* sporozoite proteins.Orthologous groupOrganismORF locus tag [a](#tblfn4){ref-type="table-fn"}Gene name[b](#tblfn5){ref-type="table-fn"}Annotation (reference)*Rhoptry proteins*OG5_142870*P. falciparum*PF3D7_1452000PfRON2Rhoptry neck protein 2 ([@b0050])*T. parva*TpMuguga_01g00014--Hypothetical proteinOG5_153587*P. falciparum*PF3D7_0817700RON5Conserved *Plasmodium* protein, unknown function ([@b0060])*T. parva*TpMuguga_01g01161--Hypothetical proteinOG5_153563*P. falciparum*PF3D7_1347500ALBA4Conserved *Plasmodium* protein, unknown function ([@b0295])*T. parva*TpMuguga_02g00645--Hypothetical proteinOG5_128020*P. falciparum*PF3D7_0932300PfM18AAPM18 aspartyl aminopeptidase ([@b0200])*T. parva*TpMuguga_01g01150--aspartyl aminopeptidaseOG5_145111*P. falciparum*PF3D7_0906000RNaseIIRNB-like protein, putative ([@b0295])*T. parva*TpMuguga_01g00396--Hypothetical proteinOG5_141731*P. falciparum*PF3D7_1361800GACConserved *Plasmodium* protein, unknown function ([@b0295])*T. parva*TpMuguga_01g00092--Hypothetical proteinOG5_129273*P. falciparum*PF3D7_0814200ALBA1DNA/RNA-binding protein Alba 1([@b0210])*T. parva*TpMuguga_03g00655Hypothetical proteinOG5_154281*P. falciparum*PF3D7_1006200ALBA3DNA/RNA-binding protein Alba 3 ([@b0210])*T. parva*TpMuguga_03g00067Hypothetical protein
*Microneme proteins*OG5_147452*P. falciparum*PF3D7_1133400AMA1Apical membrane antigen 1, AMA1 ([@b0055])*T. parva*TpMuguga_01g00650--Apical membrane antigen 1*falciparum*OG5_171217*P. falciparum*PF3D7_1216600CelTOSCell-traversal protein for ookinetes and sporozoites, putative ([@b0180])*T. parva*TpMuguga_01g00232--Hypothetical proteinOG5_135185*P. falciparum*PF3D7_0212900--Leu/Phe-tRNA protein transferase, putative ([@b0210])*T. parva*TpMuguga_02g00627--Leucyl/phenylalanyl-tRNA protein transferase
*Dense granules*OG5_126706*P. falciparum*PF3D7_0818200--14-3-3 protein, putative ([@b0210])*T. parva*TpMuguga_02g00607Hypothetical protein[^4][^5]
In *T. parva*, mobilisation of Ca^2+^ is necessary for successful sporozoite invasion of bovine lymphocytes and reagents that prevent Ca^2+^ mobilisation significantly curb zippering and internalisation stages of the entry process ([@b0315]). [Table 3](#t0015){ref-type="table"} shows 13 proteins identified in *T. parva* clustered with the *Plasmodium* proteins that play a role in calcium signalling, including calmodulin, an intracellular target for Ca^2+^ activation that acts on proteins such as guanylyl cyclases, protein kinases and phosphatases to aid in signal transduction.Table 3Proteins involved with calcium signalling. Proteins detected from whole cell lysates of *Theileria parva* salivary gland sporozoites are aligned by orthology with *Plasmodium falciparum* sporozoite proteins.Orthologous GroupOrganismORF locus tag[a](#tblfn6){ref-type="table-fn"}Gene name[b](#tblfn7){ref-type="table-fn"}Annotation (reference)OG5_126800*P. falciparum*PF3D7_1434200CAMCalmodulin*T. parva*TpMuguga_02g00717--CalmodulinOG5_129380*P. falciparum*PF3D7_1027700PfCEN1Centrin-1 ([@b0185])*T. parva*TpMuguga_01g00227--CentrinOG5_152981*P. falciparum*PF3D7_1140000CACarbonic anhydrase ([@b0210])*T. parva*TpMuguga_02g00412--Hypothetical proteinOG5_126600*P. falciparum*PF3D7_0717500PfCDPK4Calcium-dependent protein kinase 4 ([@b0160])*T. parva*TpMuguga_01g01073--Calmodulin-domain protein kinase*P. falciparum*PF3D7_1337800CDPK5Calcium-dependent protein kinase, ([@b0100])*T. parva*TpMuguga_02g00399--Calcium-dependent protein kinaseOG5_131887*P. falciparum*PF3D7_1123100CDPK7Calcium-dependent protein kinase 7 ([@b0210])*T. parva*TpMuguga_04g00518--Protein tyrosine kinaseOG5_127599*P. falciparum*PF3D7_1436600PKGcGMP-dependent protein kinase ([@b0210])*T. parva*TpMuguga_03g00511Protein tyrosine kinaseOG5_133188*P. falciparum*PF3D7_1138400GCalphaGuanylyl cyclase ([@b0275])*T. parva*TpMuguga_02g00848--Guanylyl cyclaseOG5_141757*P. falciparum*PF3D7_1246400MTIPMyosin A tail domain interacting protein ([@b0175])*T. parva*TpMuguga_01g00513--Myosin light chainOG5_126577*P. falciparum*PF3D7_1329100MyoCMyosin C([@b0360])*T. parva*TpMuguga_01g00774--Myosin COG5_126674*P. falciparum*PF3D7_1211900ATPase4Non-SERCA-type Ca^2+^ --transporting P-ATPase ([@b0040])*P. falciparum*PF3D7_0106300ATP6Calcium-transporting ATPase, putative ([@b0040])*T. parva*TpMuguga_01g00720--P-type ATPase*T. parva*TpMuguga_02g00524--Calcium-transporting ATPaseOG5_127599*P. falciparum*PF3D7_0934800PKAccAMP-dependent protein kinase catalytic subunit ([@b0210])*T. parva*TpMuguga_02g00378--cAMP-dependent protein kinase[^6][^7]
3.4. Putative *T. parva* surface proteins within the sporozoite proteome {#s0075}
------------------------------------------------------------------------
We used two methods to search for novel putative surface proteins. The first method was to use bioinformatics tools to predict the presence of GPI anchors and SP in proteins expressed by sporozoites. Only 12 proteins of the 2007 sporozoite proteins were found in this category ([Table 4](#t0020){ref-type="table"}). The list includes TpMuguga_01g00939 (gp34), a protein that undergoes GPI modification when expressed in mammalian cells and a schizont surface protein ([@b0370]). Since there is no direct evidence for addition of GPI anchors to parasite expressed proteins, we searched for enzymes of the GPI biosynthesis pathway encoded by the *T. parva* genome. Eight genes are essential for GPI synthesis in *P. falciparum* ([@b0065]). We found homologs of all eight genes in *T. parva* (TpMuguga_04g00759, TpMuguga_04g00881, TpMuguga_04g00525, TpMuguga_01g00169, TpMuguga_02g00201, TpMuguga_03g00397, TpMuguga_03g00846 and TpMuguga_02g00741) and identified that the latter four are expressed in the sporozoite proteome. This suggests the existence of GPI anchored proteins in *T. parva*, although this remains to be formally proven.Table 4Potential sporozoite surface proteins. Proteins detected from whole cell lysates of *Theileria parva* salivary gland sporozoites predicted to contain a glycosylphosphatidylinositol anchor and either transmembrane domain or signal peptide.ORF locus tag[a](#tblfn8){ref-type="table-fn"}Annotation[b](#tblfn9){ref-type="table-fn"}Unique peptides[c](#tblfn10){ref-type="table-fn"}emPAI[d](#tblfn11){ref-type="table-fn"}TMD[e](#tblfn12){ref-type="table-fn"}GPI-anchor[f](#tblfn13){ref-type="table-fn"}SP[g](#tblfn14){ref-type="table-fn"}TpMuguga_01g00438Hypothetical protein341.01011TpMuguga_01g00939Hypothetical protein (gp34)123.65111TpMuguga_01g00939Hypothetical protein123.65111TpMuguga_01g00972Hypothetical protein40.45111TpMuguga_01g01056Merozoite antigen (p32)126.78011TpMuguga_02g00412Hypothetical protein50.3011TpMuguga_02g00538Hypothetical protein311.34111TpMuguga_02g00553Hypothetical protein62.95111TpMuguga_02g00950Hypothetical protein2\<0.05111TpMuguga_03g00287Sporozoite p67 surface antigen5339.16111TpMuguga_04g00437104 kDa microneme/rhoptry antigen (p104)6012.35111TpMuguga_04g00615Probable N-acetylglucosaminyl-phosphatidylinositol de-N-acetylase2\<0.05011[^8][^9][^10][^11][^12][^13][^14]
Secondly, we compiled from the literature a list of surface-exposed *P. falciparum* proteins and determined if orthologs exist within the *T. parva* sporozoite proteome. We identified orthologs of AMA-1 (TpMuguga_01g00650) and a hexose transporter (TpMuguga_01g01069) which were both identified by specific biotinylation of *P. falciparum* and *Plasmodium yoelii* surface proteins ([@b0210]).
3.5. *Theileria parva* proteins as candidate manipulators of host cell signalling leading to transformation {#s0080}
-----------------------------------------------------------------------------------------------------------
Lymphocyte transformation characterised by cancer-like cell phenotypes is solely dependent on viable schizonts and requires the schizont proteins to interact with and manipulate pathways regulating lymphocyte apoptosis, proliferation and gene expression ([@b0080], [@b0355]). We identified 17 proteins predicted to be manipulators of host cells ([@b0330]) in the sporozoite stage, suggesting that these proteins may be required immediately upon host cell entry. Transcripts for the genes encoding all 17 proteins are found in the RNAseq data from schizont-infected cells (see Section [2.5.1](#s0040){ref-type="sec"}), although at different levels ([Supplementary Table S5](#s0110){ref-type="sec"}). Among the identified proteins are members of two multigene families, the Sub-telomere-encoded Variable Secreted Protein (SVSP) gene family and the *T. annulata* schizont AT-hook (TashAT) gene family, or *T. parva* host nuclear (TpHN) gene family. TashAT proteins display a high degree of sequence conservation in their DNA-binding domains (AT-hook) in *T. annulata* but not in *T. parva* and contain an N-terminal signal sequence for transport across the parasite plasma membrane (reviewed in [@b0355]). TashAT family proteins are implicated in regulation of host gene expression and as accessory factors for parasite transcription ([@b0330]). Other families featured are DEAD-box RNA helicases with members implicated in alteration of RNA secondary structures and promotion of proto-oncogene c-myc expression (e.g., DDX6) ([@b0005]), thioredoxin/glutaredoxin family that are redox regulators and influence important signalling pathways such as nuclear factor kappa light chain enhancer of activated B cells (NF-kB) and activator protein-1 (AP1) pathways that are both activated in *Theileria*-transformed cells ([@b0330]). In addition, we identified *T. parva* Schizont-derived Cytoskeleton-binding Protein (TpSCOP), a protein that activates host NF-κB and mitogen-activated protein kinase (MAPK) pathways, leading to resistance to programmed host cell death ([@b0155]) and a peptidyl-prolyl isomerase, which has been demonstrated to interact with and lead to degradation of host ubiquitin ligase FBW7, thereby stabilising host c-JUN and promoting transformation of host cells ([@b0215]). Interestingly, the bulk schizont RNAseq data identified a high level of anti-sense transcription of the TpSCOP gene ([Supplementary Table S5](#s0110){ref-type="sec"}), suggesting that expression of this gene in the schizont stage may be under differential control. Resolution of the significance of this observation will require analysis of cell-cycle synchronised samples.
3.6. Sporozoite proteins involved in glycolysis and tricarboxylic acid (TCA) cycles {#s0085}
-----------------------------------------------------------------------------------
Proteins of the glycolytic pathway are conserved in most eukaryotic organisms and 10 genes encoding glycolytic enzymes have been found in *Theileria* genomes ([@b0130], [@b0255]). We identified expression of all the 10 enzymes of the glycolytic pathway in this study ([Fig. 1](#f0005){ref-type="fig"}) including lactate dehydrogenase (LDH), an enzyme that converts pyruvate, the last product of glycolysis, to lactate during low or no oxygen conditions. We also identified all the TCA cycle enzymes by MS ([Fig. 1](#f0005){ref-type="fig"}) with the exception that malate dehydrogenase is functionally replaced by malate-quinone oxidoreductase ([@b0130]), and was identified in the proteome data. Glutamate dehydrogenase was also identified in the proteome and glutamate has been suggested to be a supplementary intermediate for the TCA cycle ([@b0130]). The only functional link we have found between glycolysis and the TCA cycle is the presence of phosphoenolpyruvate carboxykinase. Neither glycerol kinase nor glycerol 3-phosphate dehydrogenase were identified, although *T. parva* encodes genes for both enzymes. These enzymes were identified with low levels in the schizont stage of *T. annulata* ([@b0365]).Fig. 1Most of *Theileria parva* proteins involved in glycolysis and tricarboxylic acid cycles were identified in this study. The glycolysis pathway is shown on the left and tricarboxylic acid cycle is shown on the right. Colour code for proteins: green, enzymes that were identified by MS; blue, enzymes encoded in the *T. parva* genome but not identified by MS; red, enzymes not encoded by the *T. parva* genome and not identified in this study. Enzymes identified by MS have open reading frame locus tags included in brackets (see below): HK, hexokinase (TpMuguga_01g00043); GPi, glucose 6-phosphate-isomerase (TpMuguga_03g00346); PFK, phosphofructokinase (TpMuguga_02g00577); FBP, fructose bisphosphatase; FBA, fructose bisphosphate aldolase (TpMuguga_01g00101); TPI, triosephosphate isomerase (TpMuguga_04g00464); Gly-3PDH, glycerol-3-phosphate dehydrogenase; Gly-K, glycerol kinase; GAPDH, glyceraldehyde phosphate dehydrogenase (TpMuguga_02g00858); PGK-1, phosphoglycerate kinase (TpMuguga_01g00965); PGM-1, phosphoglycerate mutase (TpMuguga_04g00690); ENO, enolase (TpMuguga_04g00700); PK, pyruvate kinase (TpMuguga_02g00134, TpMuguga_04g00607); LDH, lactate dehydrogenase (TpMuguga_01g01182); PC, pyruvate carboxylase; ACH, aconitate hydratase-1 (TpMuguga_01g01050); IDH, isocitrate dehydrogenase (TpMuguga_04g00620); OGDH, oxoglutarate dehydrogenase (TpMuguga_01g00262); SCL, succinyl coenzyme A ligase (TpMuguga_04g00660); SDH, succinate dehydrogenase (TpMuguga_01g00210); FH, fumarate hydratase-1 (TpMuguga_03g00078); MDH, malate dehydrogenase; MQOR, Malate: quinone oxidoreductase (TpMuguga_03g00758); CS, citrate synthase (TpMuguga_02g00666); PEPC, phosphoenolpyruvate carboxylase; PEPCK, phosphoenolpyruvate carboxykinase (TpMuguga_01g00495). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3.7. Tick proteins {#s0090}
------------------
Searching the Uniprot database entries for *R. appendiculatus* with the LC--MS/MS data led to identification of 2773 tick proteins in addition to the *T. parva* proteins. The complete list of all the identified tick proteins is presented in [Supplementary Table S1](#s0110){ref-type="sec"}, but a detailed analysis of vector proteins will be reported elsewhere.
4. Discussion {#s0095}
=============
In this study, we employed a DE-52 ion-exchange column purification method to semi-purify *T. parva* sporozoites from infected tick salivary glands. Although tick proteins constituted 58% (2773/4780) of the total number of proteins of all proteins identified by LC--MS/MS and the Peaks and Mascot software packages, we were able to identify 2007 *T. parva* proteins expressed in the sporozoite life-cycle stage. This represents ∼50% of the total predicted re-annotated *T. parva* proteome of 4084 proteins (see Section [2.5.1](#s0040){ref-type="sec"}). Approximately 40% of these proteins are annotated as hypothetical proteins. Hence, our data provides evidence that real genes encode these proteins, although their functions remain unknown.
Studies on the proteome of *P. falciparum* sporozoite stages have reported identification of ∼13% ([@b0195]), ∼19% ([@b0125]) and ∼36% ([@b0210]) of the 5524 annotated *P. falciparum* genes using materials purified by DEAE-cellulose chromatography only ([@b0125], [@b0195]), and on 17% w/v Accudenz cushion followed by DEAE-cellulose chromatography ([@b0210]). The range of vector contamination is an important component of such studies and varied depending on the method of parasite purification. [@b0195] reported 65--89% mosquito protein contamination in the samples analysed, while [@b0210] reported 60.1% mosquito proteins in the sample, but had to perform double purification, initially on a 17% w/v Accudenz cushion then followed by DEAE-cellulose chromatography, to reduce mosquito protein contamination to 29% ([@b0195], [@b0210]). The presence of mosquito material in the sample was not discussed by [@b0125]). We managed to achieve low levels of vector protein contamination with a single DE-52 column purification step.
Positive identification of proteins by MS confers confidence in their presence, especially when the proteins are identified by more than one unique peptide hit. Of the 2007 parasite proteins, 1708 were identified by more than one hit, providing a high degree of confidence in 85% of the data. For the purposes of our current analyses we have, however, reported on the entire data set of 2007 proteins. Failure to identify *T. parva* proteins does not directly imply their absence in the sample. Peptides from proteins with low abundance may fall below the detection limit or are simply not sequenced by the mass spectrometer due to limited dynamic range and/or speed of instrument acquisition. Furthermore, unknown, complex, or amino acid modifications leading to alteration of the peptide mass can only be detected in some cases by the Peaks software. Finally, trypsin digestion of proteins with low or irregular arginine/lysine content can lead to very short and very long tryptic peptides that fall out of the acquired mass range, and may therefore not be detected.
Identification of *T. parva* sporozoite proteins through MS has allowed us to assess the proteomic component of the mammalian-infective stage of the parasite. We have also calculated the emPAI using Mascot to estimate the relative abundance ranking of proteins in the sample, which revealed a very high range of abundance with histones ranked with an emPAI of 60--7200 for the different histone subunits to an emPAI index of 39 for the major circumsporozoite p67 protein. Subunits of two enzymes within the glycolytic pathway, phosphoglycerate mutase and glyceraldehyde 3-phosphate dehydrogenase, are among the 20 most abundant sporozoite proteins detected ([Table 1](#t0005){ref-type="table"}). A high abundance of histones is not unusual. [@b0210] reported histones as the third most abundant protein in *Plasmodium* sporozoite proteome after the circumsporozoite protein and mitochondrial ATP synthase, subunit beta ([@b0210]).
A number of known antigens were identified within the sporozoite proteome including p67, a leading vaccine antigen, and previously described components of secretory organelles (e.g., p104 and p150) ([Table 4](#t0020){ref-type="table"}). RAP-1, a rhoptry antigen protective against *P. falciparum* infection in *Saimiri* monkeys ([@b0290]), was also identified ([Table 1](#t0005){ref-type="table"}). Unexpectedly, the polymorphic immuno-dominant molecule (PIM), a well-known antigen ([@b0345]), was not identified in this study even though p150, an antigen that is immunologically cross-reactive with PIM ([@b0335]), was identified. The PIM protein sequence contains a large repetitive domain rich in glutamine and proline amino acid residues that is devoid of lysine and arginine residues ([@b0345]). An in silico digestion of PIM with trypsin leads to prediction of few detectable tryptic peptides (data not shown). This may explain why we did not detect it. All schizont proteins that have been identified as CTL antigens are also expressed in sporozoites (emPAI range of 0.76--31.69). Some of these antigens are encoded by housekeeping proteins (e.g., hsp90) and translation elongation initiation factor 1A, so expression by sporozoites of some CTL antigens is not unexpected. The finding that all the known CTL antigens are expressed by sporozoites raises the question of whether infection of dendritic cells by sporozoites results in direct presentation of antigen for priming CTL responses in vivo, provided the antigen load is sufficient.
A study on the proteome of *T. annulata* schizont stage, in which 798 proteins were identified, failed to identify any member of SVSP and TashAT families ([@b0365]). In contrast, we identified members of both families in the sporozoite stage ([Supplementary Table S5](#s0110){ref-type="sec"}). There is increasing evidence pointing to a role of these proteins in host-parasite interaction, for instance SVSP epitope-tagged TpMuguga_03g00882 protein expressed in mammalian cells was found to translocate into the host nucleus, pointing to potential role in transforming host cells ([@b0305]). In addition, we identified other predicted host manipulation proteins such as DEAD-box RNA helicases, peptidases and two proteins, namely a peptidyl prolyl-isomerase and TpSCOP, demonstrated to play roles in host manipulation by stabilising c-JUN and activation of NF-kB, respectively ([@b0330], [@b0155], [@b0215]). Host cell manipulation is known to occur at the schizont stage and the finding of these proteins in the sporozoite stage suggests early expression of these proteins may be required, or multi-functionality of the proteins.
Sporozoite invasion of lymphocytes begins with binding events that trigger the mobilisation of Ca^2+^ within the sporozoites ([@b0080]). The free intracellular Ca^2+^ can be utilised by calmodulin to remodel itself structurally so as to act on downstream effectors such as calcium-dependent protein kinases (CDPKs), which have been shown to be useful for parasite infectivity. We detected both calmodulin and CDPKs in this study ([Table 3](#t0015){ref-type="table"}). We also detected other calcium signalling proteins including guanylyl cyclases, cGMP-dependent protein kinases and cAMP-dependent protein kinases, all of which are important for downstream calcium signalling and have been shown to be essential for sporozoite invasion (reviewed in [@b0080]).
In motile apicomplexans such as *Plasmodium*, free calcium induces microneme secretion by sporozoites to aid in gliding motility and host invasion ([@b0210]). Although *T. parva* sporozoites are immotile ([@b0320]), we identified orthologs of *Plasmodium* microneme components, including AMA-1, CelTOS and Leu/Phe-tRNA protein transferase ([@b0055], [@b0180], [@b0210]). These proteins may be involved solely in invasion processes, together with the detected rhoptry proteins (PfRON2, Conserved *Plasmodium* protein, PfM18AAP and RNB-like protein) and dense granules protein (14-3-3 protein) ([@b0295], [@b0200], [@b0050], [@b0060], [@b0210]). Since most of these proteins are still classified as hypothetical in *T. parva*, they present an opportunity for further analysis in studies aiming to block parasite invasion of host cells.
In conclusion, these data establish the expression profile of *T. parva* sporozoite proteins, the only such profile available to date. One or more of the newly identified proteins, in particular putative surface proteins, may prove to be effective vaccine candidates and might be combined with the major surface protein p67 to induce broader anti-sporozoite immunity, and protection against ECF.
Appendix A. Supplementary data {#s0110}
==============================
Supplementary Table S1Supplementary Table S2Supplementary Table S3Supplementary Table S4Supplementary Table S5
We are grateful to Stephen Mwaura and Milton Owido (International Livestock Research Institute, Kenya) for providing the dissected tick salivary glands. We thank the CGIAR Research Program on Livestock and Fish, Kenya, the Norman Borlaug Commemorative Research Initiative, an initiative between the Feed the Future program of United States Agency for International Development, USA and United States Department of Agriculture-Agricultural Research Service, USA (58-5348-2-117F) and the Department for International Development of the United Kingdom and the Bill and Melinda Gates Foundation, USA (OPP1078791) for financial support. Mass spectrometry analysis was performed in the Target Discovery Institute Mass Spectrometry Laboratory, UK, led by Benedikt M. Kessler.
Supplementary data associated with this article can be found, in the online version, at [https://doi.org/10.1016/j.ijpara.2017.09.007](10.1016/j.ijpara.2017.09.007){#ir020}.
[^1]: Open reading frame (ORF) locus tag refers to a unique *T. parva* gene identifier.
[^2]: Hypothetical protein is of unknown function; AhpC/TSA, alkyl hydroperoxide reductase subunit C/ thiol specific antioxidant family; elF, elongation factor; RAP-1, homolog of a *Theileria annulata* protein annotated as Rhoptry-associated protein 1; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; NAD, Nicotinamide adenine dinucleotide; RanBP1, Ran binding protein 1; CoA, co-enzyme A.
[^3]: emPAI, relative protein abundance in sample.
[^4]: Open reading frame (ORF) locus tag refers to a unique *T. parva* or *P. falciparum* gene identifier.
[^5]: Gene name is as defined in *P. falciparum*.
[^6]: Open reading frame (ORF) locus tag refers to a unique *T. parva* or *P. falciparum* gene identifier.
[^7]: Gene name is as defined in *P. falciparum*.
[^8]: Open reading frame (ORF) locus tag refers to a unique *T. parva* gene identifier.
[^9]: Hypothetical protein is of unknown function(s).
[^10]: Unique peptides refers to the total number of peptides identified matching the protein sequence that do not match any other protein in the database searched.
[^11]: emPAI, relative protein abundance in sample.
[^12]: Predicted using the TMHMM Server v. 2.0 (<http://www.cbs.dtu.dk/services/TMHMM/>).
[^13]: Predicted using the GPI-SOM (<http://gpi.unibe.ch/>).
[^14]: Predicted using the SignalP 4.1 server (<http://www.cbs.dtu.dk/services/SignalP/>).
|
{
"pile_set_name": "PubMed Central"
}
|
H[untington]{.smallcaps} disease (HD)^1^ is one of eight neurodegenerative disorders associated with CAG expansion ([@B23]; [@B1]). Recently, new insights have been gained as to how CAG expansion might be associated with cell death. Intranuclear inclusions have been demonstrated both in vitro and in vivo in spinocerebellar ataxia type I ([@B26]), type III ([@B22]), Huntington disease ([@B2]; [@B7]; [@B8]; [@B17]), and dentatorubropallidoluysian atrophy (DRPLA) ([@B15]). These inclusions contain the respective gene products and only occur in the presence of an expanded polyglutamine tract.
We have previously shown that expression of a truncated form of huntingtin (up to amino acid 548) forms perinuclear aggregates ([@B17]). In contrast, a small huntingtin fragment corresponding to exon 1 encoding a protein product of \<20 kD and composed almost entirely of the polyglutamine tract clearly enters the nucleus and forms aggregates in vitro ([@B17]) and in vivo ([@B7]). To directly assess the relationship between protein size and nuclear import of huntingtin, we have created progressive truncations of huntingtin and assessed how the length of the protein influences its subcellular localization and aggregate formation.
In mammalian cells, the nucleus is enclosed by an envelope that is intimately associated with the endoplasmic reticulum. The nuclear envelope is punctured at intervals by nuclear pores that are the sites of exchange of molecules between the nucleus and the cytoplasm. Small proteins can diffuse freely through the nuclear pore whereas proteins \>40 kD generally have delayed transport ([@B13]). For proteins \>60 kD, passive diffusion is prevented and nuclear import is dependent on active transport ([@B12]; [@B21]; [@B30]; [@B9]).
In an effort to directly explore the relationship between the size of the protein and its subcellular localization, we have created a series of cDNA deletion constructs that express huntingtin proteins of different molecular weights. Additionally, we also have shown that even though wild-type huntingtin is recruited into aggregates ([@B17]), aggregates are able to form in the absence of the endogenous huntingtin. Second, the findings of this study reveal that huntingtin molecules of 47 kD and greater are not transported across the nuclear pore. By contrast, smaller NH~2~-terminal fragments of huntingtin can traverse the nuclear pore complex, which suggests that very small huntingtin fragments are transported into the nucleus mainly via passive diffusion.
The exact relationship between pathology and huntingtin aggregates in vivo is not clear. In an in vitro cell culture model we have demonstrated that the formation of perinuclear aggregates are associated with increased susceptibility to cell death ([@B17]). In the present study, we determine the influence of the length of the huntingtin protein on cell death. Here, we show that progressive truncations of huntingtin are correlated with an increasingly severe susceptibility to an apoptotic stress. The findings of intranuclear inclusions ([@B8]) associated with cytoplasmic perinuclear accumulation of huntingtin ([@B24]) in the neurons of patients with Huntington disease, together with findings of this paper, provide significant support for the toxic fragment model for the pathogenesis of HD ([@B11]; [@B28]), whereby proteolytic cleavage of huntingtin liberates an NH~2~-terminal fragment containing the glutamine tract that forms aggregates, which confer increased susceptibility to death from apoptotic stimuli.
Materials and Methods {#MaterialsMethods}
=====================
Vector Construction
-------------------
Expression constructs containing the full-length huntingtin cDNA (pRcCMV10366-15 and pRcCMV10366-128) or the first 1955 nucleotides of the huntingtin cDNA (pCI1955-15 and pCI1955-128) have been described previously ([@B11]; [@B17]). Site- directed mutagenesis was used to create a series of additional NH~2~-terminal truncations of huntingtin by introducing a translational termination codon at defined positions in the 1955-15 and 1955-128 constructs using the Transformer Mutagenesis kit (CLONTECH, Palo Alto, CA). Termination codons were inserted at nucleotide position 436, 771, 989, and 1597 as defined for huntingtin in GenBank/EMBL/DDBJ under accession number [L27350](L27350). The following mutagenesis primers were used: 436-15 and 436-128: 5′-CAGCAGCAGCAGCAACAGTGACCACCGCCGCCGCCG-3′; 771-15 and 771-128: 5′-GGCTGACGAATGACTCAACAAAG-3′; 989-15 and 989-128: 5′-GACCCGAAGAATGAGTCCAGGAG-3′; 1597-15 and 1597-128: 5′-GAACTTATAGCTTGAGGGGGTTCC-3′.
The selection primer for each mutagenesis was 5′-CATGGCTCGACACATGTTCAATATTG-3′. Authenticity of the mutagenized constructs was confirmed by DNA sequencing. Also, a huntingtin COOH-terminal construct, encoding amino acids 585--3,144, was constructed by standard subcloning as described ([@B29]).
Immunofluorescence and Western Blotting
---------------------------------------
Human embryonic kidney cells (HEK 293T) were grown on glass coverslips in DME (Gibco Laboratories, Grand Island, NY) with 10% FBS and antibiotics, in 5% CO~2~ at 37°C. The cells were transfected at 30% confluency with the calcium phosphate protocol by mixing Qiagen-prepared DNA (QIAGEN Inc., Chatsworth, CA) with 2.5 M CaCl~2~, and then incubating at room temperature for 10 min. 2× Hepes buffer (240 mM NaCl, 3.0 mM Na~2~HPO~4~, 100 mM Hepes, pH 7.05) was added to the DNA/calcium mixture, incubated at 37°C for 60 s, and then added to the cells. After 12--18 h, the media was removed, the cells were washed, and fresh media was added. At 36 h after transfection, the cells were treated with 35 μM tamoxifen (Sigma Chemical Co., St. Louis, MO) for 1 h, and then processed for immunofluorescence. The cells were washed with PBS, fixed in 4% paraformaldehyde/PBS solution for 20 min at room temperature, and then permeabilized in 0.5% Triton X-100/PBS for 5 min. After three PBS washes, the cells were incubated with anti-huntingtin antibody mAb 2166 (Chemicon International Inc., Temecula, CA) (1:2,500 dilution) or BKP1 (1:100 dilution) ([@B16]) in 0.4% BSA/PBS for 1 h at room temperature in a humidified container. The primary antibody was removed, the cells were washed, and secondary antibodies conjugated to Texas red or FITC were added at a 1:600--1:800 dilution for 30 min at room temperature. The cells were then washed again, and the coverslips were mounted onto slides with DAPI (4′,6′-diamidino-2-phenylindole; Sigma Chemical Co.) as a nuclear counterstain. Immunofluorescence was viewed using an Axioscope microscope (Carl Zeiss, Inc., Thornwood, NY), digitally captured with a CCD camera (Princeton Instrument Inc., Trenton, NJ) and the images were colorized and overlapped using the Eclipse software program (Empix Imaging Inc., Mississauga, ON). Intranuclear localization was determined by colocalization of huntingtin stain with the DAPI nucleus. The proportion of nuclear and cytoplasmic localization is presented as a percent of the total huntingtin-expressing cells. Appropriate control experiments were performed to determine the specificity of the antibody, including secondary antibody only and mock-transfected cells.
To determine the expression levels of truncated huntingtin, transfected 293T cells were harvested by gentle scraping 36 h after transfection. The cells were pelleted, washed with PBS, and then lysed. Equal amounts of protein were loaded onto standard 7.5% SDS-PAGE gels or 5--20% gradient SDS-PAGE gels, transferred onto polyvinylidene difluoride (PVDF) membrane, Western blotted using anti-huntingtin antibody BKP1 ([@B16]), and then detected using enhanced chemiluminescence (Amersham Corp., Arlington Heights, IL).
Size-exclusion Chromatography
-----------------------------
Size exclusion was performed using a 16/60-cm Sephacryl S-300 high resolution column calibrated with a HMW Gel Filtration Calibration Kit (Pharmacia Biotechnology Inc., Piscataway, NJ) using run buffer (5 mM MgCl~2~, 0.5 mM EDTA, 1 mM PMSF in 1× PBS buffer, pH 7.3) at a flow rate of 0.25 ml/min. The void volume was calculated to be 42 ml using Blue Dextran. A 10-cm plate of 293T cells was transfected with HD 1955-128 and harvested after tamoxifen treatment. The cells were washed with ice-cold PBS, suspended in 200 μl of 1× PBS, and then lysed using 1 ml of 18 mM CHAPS, 5 mM MgCl~2~, 0.5 mM EDTA in 1× PBS buffer, pH 7.3, containing complete protease inhibitor cocktail (Boehringer Mannheim Corp., Indianapolis, IN) for 30 min on ice. Fractions were collected in run buffer at 0.25 ml/min at 4°C.
Cell Viability Assays
---------------------
The viability of 293T cells expressing the huntingtin constructs was assessed by a modified MTT assay ([@B3]; [@B27]). The cells were seeded at a density of 5 × 10^4^ cells into 96-well plates and transfected with the huntingtin constructs or the control construct *lacZ* using the calcium phosphate method described above. At 48 h after transfection, a sublethal concentration of tamoxifen (35 μM) ([@B6]) was added to the cells for 3 h. For the modified MTT assay, tamoxifen-treated or untreated cells were incubated for 2 h in a 1:10 dilution of WST-1 reagent (Boehringer Mannheim Corp.) and release of formazan from mitochondria was quantified at 450 nm using an ELISA plate reader (Dynatech Laboratories, Chantilly, VA) ([@B19]; [@B3]). One-way analysis of variance and Newman-Keuls tests were used for statistical analysis. The transfection efficiency, measured by β-galactosidase staining and immunofluorescence, was ∼50%.
Transfection of Mouse Embryonic Stem Cells
------------------------------------------
Wild-type and HD^−/−^ embryonic stem (ES) cells were plated onto 25-mm cover glass coated with 0.1% gelatin in H~2~O in six-well plates (Falcon Plastics, Cockeysville, MD) at a density of 10^5^ cells per well. Cells were cultured in media as described previously ([@B18]) and transfected with 5 μg of DNA by lipofection using the cationic compound *N*-\[1-(2,3-Dioleoyloxy)propyl\]-*N*,*N*, *N* -trimethylammoniummethylsulfate (DOTAP; Boehringer Mannheim Corp.). The media was changed the following day, and 36 h after transfection cells were incubated in 35 μM tamoxifen for 90 min. Subsequently, cells were washed twice with PBS fixed with 4% paraformaldehyde and immunostained with mAb 2166 as described above.
Results {#Results}
=======
Small Fragments of Huntingtin Enter the Nucleus
-----------------------------------------------
We have demonstrated that huntingtin truncated within exon 1, composed of 40 amino acids and the polyglutamine tract, can enter the nucleus ([@B17]). Furthermore, intranuclear aggregates are formed in neurons from transgenic mice expressing truncated huntingtin containing exon 1 with an expanded polyglutamine tract ([@B7]). In contrast, full-length huntingtin or huntingtin proteins truncated at amino acid 548 (nucleotide 1955) (Fig. [1](#F1){ref-type="fig"} *a*) are not found within the nucleus and accumulate in the cytosol, particularly in the perinuclear region. To demarcate functional domains within huntingtin that may determine its cellular localization, we created a set of constructs expressing various regions of huntingtin and determined the subcellular localization of their expressed products via indirect immunofluorescence of transfected cells.
Subcellular localization of this series of truncated constructs (Fig. [1](#F1){ref-type="fig"} *a*) was evaluated in an in vitro model system that we have described previously ([@B17]). In this system, an apoptotic stress is induced in transiently transfected HEK 293T cells with sublethal levels (35 μM) of the apoptotic agent tamoxifen at 36--40 h after transfection. After 60 min of tamoxifen treatment, the cells are processed for indirect immunofluorescence. In 293T cells transfected with 1955 or full-length (10366) huntingtin cDNA constructs containing 128 glutamines, not treated with tamoxifen, huntingtin was diffusely expressed throughout the cytoplasm, with perinuclear aggregates present in only a low frequency of cells (∼1%). However, treatment of these cells with tamoxifen significantly increased the proportion of cells with perinuclear aggregates (Table [I](#TI){ref-type="table"}).
To assess how protein length alters the subcellular localization of huntingtin, HEK 293T cells were transiently transfected with the 436, 771, 989, 1597, 1955, full-length (10366), and COOH-terminal contructs (Table [I](#TI){ref-type="table"}; Fig. [1](#F1){ref-type="fig"} *a*), each containing 15 and 128 CAG repeats under the control of the human cytomegalovirus promoter. Protein products of ∼3, 16, 24, 47, 59, and 347 kD are expected from these respective constructs containing 15 repeats (Fig. [1](#F1){ref-type="fig"} *b*), whereas proteins with expanded polyglutamine tracts would migrate as larger protein products (Table [I](#TI){ref-type="table"}). Indirect immunofluorescence studies were performed before and after the addition of tamoxifen using anti-huntingtin antibodies mAb2166 or BKP1 that recognize the NH~2~ terminus of huntingtin. Both antibodies gave the same results. Nuclear localization was determined by colocalization with DAPI (Fig. [2](#F2){ref-type="fig"} *a*) or the nuclear protein p53 (data not shown).
After transfection of the 436, 771, and 989 constructs, aggregates were present for each construct with and without tamoxifen treatment at similar frequencies, in contrast to the transfection of 1955 and 10366 constructs that resulted in aggregates only after treatment with tamoxifen. These aggregates appear as large, irregularly shaped depositions of huntingtin (Fig. [2](#F2){ref-type="fig"} *a*). Size-exclusion chromatography demonstrated that the huntingtin forms stable high molecular weight complexes that do not denature in SDS-PAGE (Fig. [2](#F2){ref-type="fig"} *b*).
The subcellular localization of different huntingtin fragments varied according to the size of the protein product (Fig. [2](#F2){ref-type="fig"} *a*; Table [I](#TI){ref-type="table"}) and was similar with and without apoptotic stress. Protein products derived from the three smallest constructs (436, 771, and 989) expressing the most 40, 151, and 224 NH~2~-terminal residues of huntingtin were each able to enter the nucleus with both 15 and 128 glutamines. However, the frequency of nuclear entry varied among the constructs tested. For example, the localization of aggregates formed by expression of the 436-128 and 989-128 constructs was divided between the nuclear and perinuclear regions (Fig. [2](#F2){ref-type="fig"} *a*), whereas expression of the larger product (31 kD) from the 771-128 construct resulted in more cells with nuclear than perinuclear aggregates (Fig. [2](#F2){ref-type="fig"} *a*; Table [I](#TI){ref-type="table"}). In contrast, expression of the 1597, 1955, and 10366 constructs with 15 and 128 glutamines resulted in exclusive cytoplasmic localization of the gene products. Aggregates formed after transfection with 1597-128, 1955-128, and 10366-128 constructs were only observed in the perinuclear region (Fig. [2](#F2){ref-type="fig"} *a*; Table [I](#TI){ref-type="table"}). In addition, cells transfected with a construct containing the COOH-terminal region of huntingtin (amino acids 585--3,144) did not form aggregates, and the COOH-terminal portion of huntingtin was located diffusely throughout the cytoplasm before and after (Fig. [3](#F3){ref-type="fig"}) tamoxifen treatment.
The frequency of aggregates varied between constructs of different lengths containing the same number of polyglutamines. An increased frequency of aggregates was seen in cells expressing shorter huntingtin products than in cells transfected with the 1955-128 and 10366-128 constructs (Table [I](#TI){ref-type="table"}). HEK 293T cells were also transiently transfected with the same series of constructs expressing 15 glutamines (Table [I](#TI){ref-type="table"}) that did not form aggregates although the subcellular localization of the gene product was similar to that seen after expression of the constructs with 128 repeats (Fig. [4](#F4){ref-type="fig"}; Table [I](#TI){ref-type="table"}).
Aggregates Can Form in the Absence of Full-Length Endogenous Huntingtin
-----------------------------------------------------------------------
To further understand which factors contribute to the formation of aggregates, we determined whether endogenous huntingtin was required. Using immunofluorescent confocal microcopy and antibodies directed to epitopes within the NH~2~- and COOH-terminal portions of huntingtin ([@B14]; [@B17]), we have previously shown that aggregates containing truncated huntingtin comprise not only the NH~2~-terminal fragment, but also contain the COOH terminus of the endogenous protein that was not encoded by the transfected cDNA ([@B17]). The fact that COOH-terminal staining was present in aggregates in cells transfected with constructs expressing only truncated NH~2~-terminal huntingtin suggested that endogenous huntingtin was recruited into these aggregates. The question then arises whether full-length endogenous huntingtin is necessary for the formation of aggregates associated with expression of truncated mutant huntingtin.
HD^−/−^ ES cells are homozygous for a targeted mutation within exon 5 of the HD gene ([@B20]) and therefore lack full-length huntingtin expression. In ES cells, we are unable to detect a truncated huntingtin product and therefore if initially present, it is likely to be rapidly degraded in this cell system. Wild-type and HD^−/−^ ES cells were transfected with a truncated form of huntingtin (construct 1955) with 15 or 128 CAG repeats. After tamoxifen treatment, wild-type and HD^−/−^ ES cells transfected with constructs expressing huntingtin with 15 glutamines (1955) had no (wild-type) or very few (1% for HD^−/−^ ES) aggregates. In contrast, the frequency of aggregates was increased in both wild-type (3%) and HD^−/−^ ES cells (6.8%) transfected with huntingtin expressing 128 glutamines (1955-128) (Fig. [5](#F5){ref-type="fig"}). The aggregates were in the perinuclear region of the ES cells. These results clearly show that the presence of full-length endogenous huntingtin is not necessary or critical for formation of aggregates after transfection of truncated huntingtin with the expanded polyglutamine tract.
Increased Susceptibility to Cell Death Is Correlated with Aggregate Formation and Inversely Correlated with Length of Huntingtin
--------------------------------------------------------------------------------------------------------------------------------
We have previously demonstrated that expression of mutant huntingtin results in increased susceptibility to an apoptotic stress in transfected 293T cells induced by sublethal doses of tamoxifen ([@B17]). Furthermore, the 1955 constructs resulted in significantly more cell death than the full-length huntingtin constructs ([@B17]). To determine whether this observed difference represented a graded effect resulting from protein length, we assessed the change in viability of cells after expression of huntingtin of different lengths.
293T cells were transiently transfected with the series of huntingtin truncation and full-length constructs and the viability of the transfected cells was assessed by a modified MTT assay that measures mitochondrial function ([@B19]; [@B3]; [@B27]). Mock- and *LacZ*-transfected 293T cells treated with tamoxifen served as controls. In each assay, cells transfected with the truncated constructs exhibited significantly higher proportions of injury or death compared with cells that received full-length huntingtin containing the same polyglutamine length (*P* \< 0.01) (Fig. [6](#F6){ref-type="fig"}). Interestingly, this was seen even with proteins with 15 glutamines indicating that an NH~2~-terminal fragment of huntingtin may be proapoptotic even in the absence of polyglutamine expansion. However, measurement of cell viability indicated a polyglutamine repeat-dependent increase in cell death (*P* \< 0.01 for all constructs). The differences in toxicity are not due to differences in expression of these constructs in cells as revealed by Western blot (data not shown). Increased susceptibility to cell death for any of these huntingtin gene products were only seen in the presence of apoptotic stress (data not shown). Therefore, the susceptibility to an apoptotic stress is influenced by both the length of the huntingtin protein and the size of the polyglutamine tract.
Discussion {#Discussion}
==========
The factors determining the subcellular localization of huntingtin, and the relationship between its localization and cellular viability, are fundamental to understanding the pathogenesis of HD and may provide new therapeutic targets for delaying or preventing toxicity in vivo. Therefore, we performed experiments designed to investigate factors that may be influencing the nuclear transport of huntingtin. In particular, we wished to dissect how the length of the huntingtin protein was correlated with nuclear localization of huntingtin and its tendency to form aggregates. The findings of this study clearly and directly implicate protein length as a critical factor in influencing the subcellular localization of huntingtin. Whereas nuclear localization is crucially dependent on protein length, the likelihood of formation of aggregates appears to be directly related to the length of the polyglutamine tract.
The fact that these aggregates form even with a huntingtin fragment essentially composed of only 17 amino acids besides the 128-residue polyglutamine stretch is strong evidence for these aggregates forming by expanded polyglutamine tracts joining these molecules together. This also supports the in vitro data generated by assessing interaction between glutathione-S-transferase fusion proteins with expanded polyglutamine tracts ([@B25]).
Endogenous huntingtin is recruited into aggregates formed by a truncated form of the transfected protein ([@B17]). Here we show, using HD^−/−^ ES cells, that aggregates can form in the absence of full-length endogenous huntingtin, demonstrating that in this system, endogenous huntingtin is not necessary for formation of aggregates. Whereas extrapolation from this in vitro model using cell types not normally affected in HD to the in vivo situation must be done with caution, this may suggest that mutant huntingtin can form aggregates alone in vivo providing further evidence for a gain of function resulting from polyglutamine expansion.
Huntingtin derived from three constructs spanning up to 989 nucleotides (224 amino acids) were all seen partially or predominantly within the nucleus. These constructs would be expected to produce predicted proteins of \<40 kD. For example, the 989 construct containing 128 repeats would encode a protein of ∼39 kD. By contrast, mutant proteins of 60 kD or greater (derived from the 1597, 1955, and 10366 constructs) were observed in the perinuclear region in the cytosol with no nuclear localization. Furthermore, the expression of the COOH-terminal construct expressing a predicted product of 284 kD also produced a protein exclusively located diffusely throughout the cytoplasm. No aggregates were seen with this construct.
Proteins that traverse from the cytoplasm into the nucleus must go through the nuclear envelope and enter through the nuclear pore. Nuclear pore complexes are composed of many different polypeptides and have a large mass of \>100 MD. Transport across the nuclear pore may either be the result of passive diffusion or active transport ([@B30]; [@B21]; [@B13]). Small proteins, generally \<40 kD, can diffuse passively through the nuclear pore. For example, cytochrome c (13 kD) diffuses freely through the nuclear pores, whereas ovalbumin (43 kD) has a delayed transport, and BSA (66 kD) is completely prevented from passing through the nuclear pore ([@B13]). The relationship between size of huntingtin and its localization in the nucleus determined in this study is consistent with transport across the nuclear membrane being size related and resulting from passive diffusion across the nuclear membrane. Thus, a construct expressing a huntingtin product of ∼40 kD has some nuclear but predominantly perinuclear localization (construct 989), and huntingtin of predicted sizes of 60 kD or greater (derived from 1597, 1955, and 10366 constructs) are essentially excluded from the nucleus, similar to that seen with BSA ([@B13]).
However, size alone is not the only determinant of localization of the expressed product. For example, the gene product of the 771 construct was seen most frequently in the nucleus and seen more often than after expression of a smaller gene product from the 436 construct. This suggests that active transport may influence the localization of this protein.
Active import of proteins requires energy and also a nuclear localizing signal to which different cytosolic receptor complexes bind for transport ([@B30]; [@B21]). We have searched the NH~2~-terminal domain of huntingtin for a sequence motif suggestive of a nuclear localization signal (NLS) ([@B12]; [@B9]; [@B4]). Although no stringent consensus sequence for an NLS is defined, a basic hexapeptide sequence and a bipartite sequence are suggestive of this motif ([@B10]). Interestingly, within the first 548 amino acids of huntingtin, a single stretch of amino acids (90-PKKELSATKK-99) with some similarity to the NLS of the familial breast cancer BRCA-1 protein (PKKNRLRRKS) has been identified ([@B4]). Further analysis of the role of this sequence as a functional NLS is necessary in an effort to determine its role in nuclear transport.
We have previously shown that the expression of mutant huntingtin results in increased susceptibility to apoptotic stress in transfected 293T cells ([@B17]). This was initially shown in proteins derived from the 1955 compared with the full-length construct. In this study we have extended these findings and now show that constructs with 128 versus 15 repeats were more toxic to cells after apoptotic stress at all lengths of the protein. Furthermore, there is an increase in cell death with shorter constructs, again showing as postulated previously ([@B17]), that length of the protein is directly related to the susceptibility to cell death. Interestingly, expression of the truncated form of huntingtin even with a normal sized polyglutamine tract confers increased susceptibility to cell death from apoptotic stimuli demonstrating that this NH~2~-terminal sequence alone can be toxic to cells in vitro.
This is not inconsistent with the model for this disease whereby cleavage of wild-type huntingtin normally plays a role in influencing cellular viability. Disease could arise when a threshold is reached and inappropriate apoptosis occurs at least influenced by the presence of a truncated product containing an expanded polyglutamine tract. The margin between the effects at any one time between normal and mutant huntingtin may be narrow but cumulative over time resulting eventually in a disease with late onset.
These findings in this manuscript do not explain why each of these polyglutamine expansion diseases result in a selective pattern of neuronal loss, despite the fact that their gene products are expressed in similar regions in the central nervous system ([@B23]). One factor that may influence selectivity of neuronal loss is proteolytic cleavage of polyglutamine-containing proteins. For example, our own data suggest that atrophin-1, the androgen receptor, and huntingtin are each cleaved by different caspases with varying efficiencies ([@B29]) resulting in the formation of a truncated fragment containing the polyglutamine tract. Because the gene products have differential susceptibility to individual caspases, the particular pattern of cell loss for each disease could be due in part to the altered cellular or subcellular distribution of these particular enzymes, leading to altered interaction with their substrates.
These in vitro data, together with data generated by [@B17] and Cooper et al., (1998), are consistent with a model that proposes that a crucial step in the pathogenesis of this disorder is the progressive truncation of huntingtin resulting in altered localization, aggregate formation, and decreased cellular viability. This model is consistent with data generated from studies of brains from patients with HD which demonstrate both perinuclear accumulation of huntingtin and intranuclear inclusions ([@B8]; [@B24]) suggesting that this in vitro model may significantly recapitulate what occurs in vivo in animals and humans.
This work is supported by the Canadian Networks of Centres of Excellence (NCE, Genetics), Medical Research Council (MRC; Canada), and the Huntington Disease Society of America. A. Hackam is an MRC postdoctoral fellow. C.L. Wellington is an Alberta Heritage Foundation for Medical Research postdoctoral fellow. M.A. Kalchman is a MRC (Canada) predoctoral scholar. M.R. Hayden is an established investigator of the British Columbia Children\'s Hospital.
Address all correspondence to Dr. M.R. Hayden, Department of Medical Genetics, University of British Columbia, 416-2125 East Mall, NCE Bldg., Vancouver, BC V6T 1Z4. Tel.: (604) 822-9240. Fax: (604) 822-9238. E-mail: <[email protected]>
ES
: embryonic stem
HD
: Huntington disease
HEK
: human embryonic kidney
######
(*a*) Diagrammatic representation of the cDNA expression constructs used in this study. Bars represent huntingtin protein, with amino acids numbered according to GenBank/EMBL/DDBJ accession number [L27350](L27350). The locations of the polyglutamine tract (*Q*) are indicated. The names of the constructs, 436, 771, 989, 1597, 1955, and 10366 correspond to the nucleotide position of the translation termination codon. The amino acid length of each protein is also indicated at the right. (*b*) Western blot of huntingtin truncation constructs. HEK 293T cells were transfected with the 771, 989, 1597, 1955, and 10366 constructs, containing 15 glutamines. 36 h after transfection, cell lysates were prepared. Equal amounts of protein were loaded on a 5--20% SDS-PAGE gradient gel, and Western blotted using anti-huntingtin antibody BKP1, which recognizes the NH~2~ terminus. Multiple bands in the lanes represent different conformations of the protein or huntingtin multimers. The amino acid length (*aa*) of each expressed protein is also indicated.
######
(*a*) Immunofluorescence of 293T cells transfected with the 436, 771, 989, 1597, 1955, and 10366 constructs, each containing 128 glutamines. 36--40 h after transfection, the cells were processed for immunofluorescence using mAb 2166 (*red*) or BKP1 (*green*) to detect huntingtin. The cells were counterstained with the DNA stain DAPI (*blue*) to label the nucleus (the DAPI stain is not visible in the cells shown for 1597-128 and 10366-128). Huntingtin located in the nucleus appears pink as the red stain is overlaid with blue. Huntingtin from the 436, 771, and 989 constructs (*top row*) appears as large intranuclear aggregates. Huntingtin from the 1597, 1955, and 10366 constructs (*bottom row*) show perinuclear aggregates and diffuse cytoplasmic stain. (*b*) Size-exclusion chromatography of a lysate from 293T cells transfected with HD1955-128 and treated with tamoxifen. 1-ml fractions were collected at 4°C, and samples were immediately analyzed by reducing SDS-PAGE and Western blotting with BKP1 antibody. Large molecular weight complexes were not retained by the column and flowed through into the void volume. Huntingtin aggregates isolated in the void volume were observed as species that did not denature upon SDS-PAGE and did not exit the stacking gel (*V* ~o~ = 42 ml; lanes *2* and *3*). Under the same conditions, no aggregation was observed in the whole cell lysate (lane *1*) nor in the monomeric huntingtin fractions (*V* = 72 ml; lanes *4* and *5*). Bar in *a*: (*436-128*) 11.3 μm; (*771-128*) 5.31 μm; (*989-128*) 11.2 μm; (*1597-128*) 11.97 μm; (*1955-128*) 12 μm; (*10366-128*) 11.4 μm.
{#F3}
{#F4}
{#F5}
{#F6}
(*a*) Frequency of Aggregate Formation in Transfected Cells (%)
--------------------------------------------------------------------------------------------------- -- ----------- -- ----------- -- ----------- -- ------------ -- ------------ -- ---------------- -- ---------------
Without tamoxifen 45 81 70 4 0-1 0-1
*n* = 3 *n* = 1 *n* = 1 *n* = 2 *n* = 5 *n* = 5
With tamoxifen 43 92 78 13 27 9
*n* = 4 *n* = 3 *n* = 2 *n* = 2 *n* = 4 *n* = 4
(*b*) Subcellular Localization of Huntingtin Aggregates with 128 Polyglutamines in 293T Cells (%)
Construct 436-(128) 771-(128) 989-(128) 1597-(128) 1955-(128) 10336-(128)
Predicted protein size (kD) 18 31 39 61 73 362
Estimated[\*](#TFI-150){ref-type="table-fn"} size (kD) ND 68 75 77 115 530
Without tamoxifen
Nuclear 21 77 22 0 0 0
Cytoplasmic 79 23 78 100 0-1 0-1
No. of cells counted 288 176 66 427 \>1,000 \>1,000
*n* = 3 *n* = 3 *n* = 2 *n* = 2 *n* = 5 *n* = 5
With tamoxifen
Nuclear 36 71 38 0 0 0
Cytoplasmic 64 29 62 100 100 100
No. of cells counted 314 175 95 320 \>1,500 \>1,000
*n* = 4 *n* = 3 *n* = 2 *n* = 2 *n* = 5 *n* = 5
(*c*) Subcellular Localization of Huntingtin Protein with 15 Polyglutamines in 293T Cells
Length/size nucleotides 436-(15) 771-(15) 989-(15) 1597-(15) 1955-(15) 10366-(15) COOH terminus
Amino acids 1--40 1--151 1--224 1--427 1--548 1--3,144 585--3,144
Predicted protein size (kD) 3 16 24 47 59 347 284
Estimated size[\*](#TFI-150){ref-type="table-fn"} ND 40 43 72 80 370 280
Localization without tamoxifen (%)
Nuclear 21 86 11 0 0 0 0
Cytoplasmic 79 14 89 100 100 100 100
No. of cells counted 250 185 202 321 \>1,000 \>1,000 \>200
*n* = 2 *n* = 3 *n* = 2 *n* = 2 *n* = 5 *n* = 6 *n* = 1
Localization with tamoxifen (%)
Nuclear 57 82 21 0 0 0 0
Cytoplasmic 43 18 79 100 100 100 100
No. of cells counted 181 337 85 319 \>400 \>1,000 \>200
*n* = 2 *n* = 2 *n* = 2 *n* = 2 *n* = 5 *n* = 5 *n* = 2
Numbers in brackets indicate the polyglutamine length. *ND*, not detected.
Estimated by electrophoretic migration on Western blot. The polyglutamine tract causes aberrant migration.
|
{
"pile_set_name": "PubMed Central"
}
|
A new technique of antireflux plasty
**Bertrand Dore, Shwky El Abd, Adel Youssef, Saleh Mohalhel**
Department of Urology, Riyadh Central Hospital, Riyadh, Saudi Arabia
All the techniques described until now to correct vesico renal reflux include dissection of the distal ureter. Avoiding this, the original technique described in 1984 by Prof. Gil-Vernet is based on anatomical investigations of the lower ureter with its intrinsic muscular fibers, the transmural part and the function of waldeyer sheath in preventing the reflux.
It was done not only for cases with megatrigone and ectopic ureteric orifices but also for all grades of reflux. The advancement increases the length of intramural ureter and the mucosal roof of both orifices. Although this is a brief report for five cases only but a very good result with 18 months follow up was seen.
It seems a safe and easy method. Furthermore, with the new procedures of uretero-renoscopy this technique allows easy catheterization and ureteroscopy of both ureteric orifices in the future a situation which is very difficult after the classic when antireflux advancement procedure.
**Presented at the:** 4^th^ Saudi Urological Conference Riyadh Central Hospital 18 September 1986
Endoscopic submucosal teflon injection (STING): An alternative treatment of vesicoureteric reflux in children
**T. Merdad, V. DeBoe, L. Merchx, F. Keuppens**
Department of Urology and Pediatrics, Academic, Hospital of the Vrije Universiteit Brussels, Brussels, Belgium, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia
**Objectives**: Vesicoureteric reflux is a common urological disorder in children. Those who do not need prompt surgical treatment are managed conservatively, with the focus of preventing UTI, this treatment is suitable for approximately 70% of those patients. We used STING, which is a simple noninvasive treatment, for the other 30%.
**Materials and Methods**: 100 Refluxing units in 68 children were treated with STING with a maximum follow up of five years. Indications were high grade reflux (III-V), persistence or progression of reflux despite conservative therapy, breakthrough urinary infection despite antibiotics prophylaxis and bad antibiotic compliance.
**Results**: Reflux was cured with a success rate of 75% after a single injection and 96% after two injections. Best results were obtained in low grade reflux (I-II). 60-70% success rate was obtained in high grade reflux (III-V). The main advantage of STING over open reimplantations lies with its noninvasiveness, simplicity and possibility of being repeated in case of failure.
**Conclusion**: The technique of the endoscopic approach to treat reflux has been popularized by O'Donnell in 1984, because of the simplicity and efficacy reported by several authors. This technique has been the first choice of treatment options in the department of urology at the VUB University in Brussels.
**Presented at the:** 10^th^ Saudi Urology Conference King Fahad National Guard Hospital 26-28 November 1996
Immunologic changes in bacteriuric children with and without schistosomiasis
**Shawky El Abd**
Department of Urology, Riyadh Central Hospital, Riyadh, Saudi Arabia
The problem of UTI in children constitutes a worldwide problem that is furtherly augmented by bilharziasis in endemic areas. The work entailed assessment of the immunologic status in 30 infected children 20 of them had active schistosomiasis, compared to the control group. The efficiency of how antibilharzial drugs (Niridazol and Trichlorfone) plus their antibacterial and immunosuppressive effect was demonstrated. The antibiotic role was also observed.
The infested children were classified into four groups and the mean serum concentration of IgG, IgA and IgM were estimated by the immunodiffusion method. A follow up of these Igs two weeks after antibilharzial or antibacterial therapy was done.
**Presented at the:** 4^th^ Saudi Urological Conference Riyadh Central Hospital 18 September 1986
A study on urinary tract infections in children seen at KFU
**Khalid Al Umran**
Department of Paediatrics, King Faisal University, Al Khobar, Saudi Arabia
This is a retrospective study on patients with urinary tract infections (UTI) seen in King Fahd Hospital of the University (KFHU) during the five year period from February 1988 to January 1993. Data of patients with confirmed UTI were obtained from laboratory and medical records and analysed. Of the 212 children (ages 0-12) 104 were females and 108 were males.
Clinical diagnosis of UTI confirmed by microbiological assay and its consequences by radiological and laboratory investigations including renal functions tests.
Urine specimens from all patients were subjected to culture and organisms isolated from all patients. All isolates were gram negative bacilli predominantly Escherichia coli. Of the cultures a few yielded mixtures of gram negative bacilli and gram positive organisms. Antibiotic susceptibility tests on gram negative organisms revealed resistance to ampicillin cotrimaxazole and tetracycline but sensitivity generally to gentamicin, augmenting, nalidixic acid, nitrofurantoin and others.
The study presents a clinical and microbiologic profiles of UTI in paediatric age group as seen in our hospital, reviews the methods of diagnosis and management of such cases and recommends specific treatment by antibiotics.
**Presented at the:** 8^th^ Saudi Urological Conference King Fahd Military Medical Complex 9-10 November 1993
Urinary tract infection in children at K.F.M.M.C.: A four year audit
**S. Egail, I. Al Oraifi, A. Al Dayel, M. Ezzibdeh, K. Lubkiewicz, M. A. Babiker**
Department of Urology, King Fahd Military Medical Complex, Dhahran, Saudi Arabia
Urinary tract infection (UTI) is a common problem of childhood. We examined retrospectively the records of 192 patients who had confirmed UTI. Mean age of patients 12.8 months (range 0-120 months), and male to female ratio was 1:1.3.
122 (63.5%) children presented with febrile illness and 40 (20.85) had other symptoms. The commonest organism isolated from 243 cultures was E.coli (80.75), Klebsiella (9.9%), and other organisms (9.4%).
Diagnostic imaging studies, including KUB, ultrasound or IVU, were requested for 62 (32.3%) patients only. Abnormalities were detected in 27 patients.
As the results of this audit, we suggest radiological protocol for evaluation of all children with proven UTI.
**Presented at the:** 8^th^ Saudi Urological Conference King Fahd Military Medical Complex 9-10 November 1993
Urinary tract infection in children with posterior urethral valves and bilateral refluxing ureters
**C. Krishnappa, Akhileshwar Jha, Kuruvilla Zachariah, Zaid Roshdi, Mahmoud Murtaja, K. S. AbdulWahab**
Department of Urology, King Fahd Central Hospital, Gizan, Saudi Arabia
"25 cases of Posterior Urethral Valves and Bilateral refluxing ureters treated in KFCH for last 5 years". The age of the patient ranged from 1 day to 4 years. Clinical presentation were (a) UTI, b) retention of urine, (c) failure to thrive, (d) secondary effects of obstruction like prolapse of rectum and vesical calculi. 20 patients were treated with Urethroscopy and fulguration of valves. 10 cases who had grade III & IV vesica-ureteric reflux were also submitted to Anti-reflux procedure along with fulguration of post ureteral valve. Other 10 cases had grade I & II reflux along with post urethral valve were submitted only for excision of PUV. The second group of patient, when they were discharged from hospital, their RFT was normal (serum creatinine), their urine was sterile at the time of discharge. These children were followed up at 3 months interval time. Most of the patients during the follow up time itself developed UTI and rising serum creatinine level. These children were not on chemoprophylaxis at the time of follow up.
**Conclusion**: After the clinical trial, it is concluded that the chemoprophylaxis should be integral part of follow up till the definitive anti-reflux procedure is done. In persistent, refluxing cases even in Grade I & II, we recommend PUV valve excision as well as anti-reflux procedure in cases where it is cystoscopically proved, evidence for organic lesion at vesica-ureteric junction.
**Presented at the:** 8^th^ Saudi Urological Conference King Fahd Military Medical Complex 9-10 November 1993
Increasing prevalence of antimicrobial resistance among uropathogens causing urinary tract infections in children
**T. M. Gebriel, T. Ben-Hamida, M. Aboshkiwa**
Department of Medical Microbiology, Al-Fateh University of Medical Sciences, Tripoli, Libya
**Objective**: During childhood urinary tract infections considered an important cause of serious complications as chronic renal failure, guideline for the management of UTIs in children that recommended empiric therapy rely on the predictability of the agents causing the infections and knowledge of their antimicrobial susceptibility. In the present study, the frequencies of resistant uropathogens to the most commonly used antibiotics were retrospectively surveyed in Salah-Edden Hospital-Tripoli during the years 1992, 1995 and 1998.
**Results**: E.coli, Klebsiella pneumonaie and proteus sp. were the most common uropathogens, according for more than 70% of the total urine isolated studies. The prevalence of resistance among E.coli was more than 70% for ampicillin in each year studied. The prevalence of resistance to co-trimoxazole significantly rose from more than 49% in 1992 to more than 64% in 1998. Concerning Klebsiella pneumonaie the prevalence of resistance significantly rose from 0& to nalidixic acid; 0% to nitrofurantoin; 21% to augmentin and 32% to co-trimoxazole in 1992 to 16%; 16% 57% and 66% in 1998 respectively, while in case of proteus sp. the prevalence of resistance to co-trimoxazole significantly rose from 40% in 1992 to 100% in 1998.
**Conclusion**: There were statistically significant increase in the prevalence of antibiotic resistance from 1992 to 1998 among the most common uropathogens which leaves the physician with every few choices when treating UTIs without prior knowledge of the causative pathogens.
**Presented at the:** 13^th^ Saudi Urological Conference Riyadh Armed Forces Hospital 14-17 February 2000 (09-12 Dhu Al Qa'dah 1420)
Clean intermittent catheterization in Saudi children: Suggestion for a common protocol
**R. De Castro, K. A. Fouda Neel, A. M. Al Shammari, J. M. Abu Daia**
Department of Urology, Division of Pediatric Urology, King Faisal Specialists Hospital and Research Centre, King Khalid University Hospital, Department of Urology, Division of Pediatric Urology, King Fahd National Guard Hospital, Riyadh, Saudi Arabia
**Preface**: The introduction and popularization of method, practice and philosophy of Clean Intermittent Catheterization (CIC) of the urinary bladder in the treatment of bladder dysfunctions by Lapides in 1970 was one of the most import modern steps in the progress of urological science. This is particularly true for Pediatric Urology. In the treatment of severe urinary incontinence in children secondary to great urological malformations (extrophyepispadias complex and bilateral single ectopic ureter) and to neurogenic congenital bladder dysfunction\'s very few were possible before the CIC era and very much can be done now, thanks to this technique.
**Objective**: CIC is widely utilized in the Kingdom of Saudi Arabia, as well as in all the countries with a good level of sanitary organization, but the materials and the methods in use are not always the most appropriate. We should try to improve the health service for the patients on CIC in the entire Kingdom, contributing to the diffusion of the right methods of CIC and making available the best quality materials.
**Methods**: The Pediatric Urology Staff of three hospitals in Riyadh felt it was appropriate to pursue the mentioned aims. We started comparing our different experiences with children on CIC in the Kingdom and outside. We analyzed the materials our hospitals supplied to our outpatients on CIC, the stock in trade already available in our hospitals for impatient use and new wares available in the Kingdom from the pharmaceutical companies. We analyzed many different catheters and their cost.
**Results**: As a result of our investigations, we prepared a list of the best available article we believe should be supplied for CIC and a list of article which should not be used for this purpose.
**Conclusions**: We will present our agreement about the methods and materials for CIC in children and we would like to compare and discuss it openly to finalize a common protocol for CIC for Saudi children, hoping to avoid some mistakes and improve the quality of life of these patients.
**Presented at the:** 12^th^ Saudi Urology Conference Al Hada and Taif Armed Forces Hospitals Progam 23-25 February 1999 (7-9 Dhu Al Qa'dah 1419)
Extravesical ureteric reimplantation in children: Preliminary experience from KKUH
**S. M Soliman, K. N. Fouda**
Department of Urology, King Khalid University, Riyadh Saudi Arabia
**Background**: In the last decade there has been renewed interest in extravesical ureteric reimplantation (Extravesical detrusorrhaphy). This has been reported to be an effective technique for treating isolated VUR with avoiding the annoyances consequent to opening the bladder especially bladder spasms, yet its use especially in bilateral cases has been questioned because of a reported high incidence of voiding dysfunction thereafter. We reviewed our experience with extravesical ureteric reimplantation in the Paediatric Urological Service at KKUH in the last 2 years.
**Patients and Methods**: From 1998 through 2000, all patients with non-STINGable VUR who underwent extravesical ureteric reimplantation were reviewed. Patients were grouped into group A: undergoing unilateral reimplantation and group B: undergoing bilateral reimplantation. Preoperative voiding dysfunction was carefully searched for. Preoperative and postoperative radiological studies and signs of voiding dysfunction were reviewed and compared between both groups. Patients were judged to be catheter weaned when they were able to void at least half of their bladder volume. Those preoperative anticholinergic, CIC, or requiring concomitant open bladder surgery were excluded from the study.
**Results**: There were 11 patients with 16 refluxing units (8 boys, 3 girls, mean age 5.1 years), with a follow up range from 2--23 months: none showed preoperative signs of voiding dysfunction. Group A included 6 patients (7 refluxing units) one unit was nephrectomized due to nonfunction and group B included 5 patients (9 refluxing units). In both groups, none of the patients showed signs of remaining of D-novo VUR on postop VCUG. The average duration until patients were catheter weaned was 3 days in group A and none in this group developed any sign of voiding dysfunction on follow up. In group B, by contrast, it was 3.8 days and one developed significant daytime and night incontinence persisting 1 month after surgery.
**Conclusion**: Extraversical reimplantation is a highly successful technique in treating VUR, yet careful consideration should be given for cases with intended bilateral reimplantation, as this may be preceded by incomplete ability to empty the bladder and late voiding dysfunction, a complication that is seldom reported after intraversical reimplantation. Our preliminary experience with a limited number of patients suggests reverting to the "classical" intraversical techniques when it comes to reimplanting the ureters bilaterally and reserve the extraversical approach to unilateral cases.
**Presented at the:** 14^th^ Saudi Urological Conference King Fahd Military Medical Complex- Dhahran 13-15 February 2001 (19-21 Dhu Al Qa'dah 1421)
Endoscopic correction of complicated yesicoureteric refluxin children
**Khalid Fouda Neel**
Department of Surgery, Division of Urology, King Khalid University Hospital, Riyadh, Saudi Arabia
**Objective**: Endoscopic treatment of vesicoureteric reflux (VUR) is a widely used intervention with high success rate, low morbidity, short hospital stay and cost effective. Treatment of complicated VUR \[failed surgery, posterior urethral valves, neurogenic bladder, bladder dysfunction (NNBSD)\] is still challenging. Here we review our experience at King Khalid University Hospital, Riyadh, with VUR endoscopic correction using macroplastique® in complicated VUR.
**Materials and Methods**: 18 patients, 11 female and 7 male, were treated endoscopically to correct their VUR from 1998-2000. 9 of them had "complicated VUR" 3 with failed previous surgery for the treatment of primary VUR, 1 with PUV and 5 with NNBSD. Of the 9 patients with complicated VUR, 6 had bilateral VUR, 3 had unilateral VUR. Endoscopic correction of VUR was done for all patients and a follow up micturating cystourethrogram was requested 3-6 months after treatment and renal ultrasound one month after treatment.
**Results**: Of 9 patients with complicated VUR, 7 were available for follow up. Out of the 12 ureters treated and came for follow up, 10 showed complete resolution of VUR and 2 with same pre injection grade of reflux. None of the patients with persistent reflux had a 2^nd^ attempt yet. Only one patient had pyelonephritis post injection, none had obstructive changes and none had allergic reaction to the material.
**Conclusion**: Early result of endoscopic correction of VUR for complicated VUR is encouraging, with an 84% success rate and 15% complication rate. We strongly recommended this line of treatment, as the first option, for this difficult group of patients when surgical correction of VUR is decided.
**Presented at the:** 14^th^ Saudi Urological Conference King Fahd Military Medical Complex - Dhahran 13-15 February 2001 (19-21 Dhu Al Qa'dah 1421)
Variability of significance of nuclear medicine cystogram
**K. Fouda Neel, J. F. Schillinger, G. H. Kiruluta**
Division of Urology, King Khalid University Hospital, Riyadh,
Saudi Arabia, Department of Urology, Children\'s Hospital of Eastern Ontario, Ottawa, Ontario, Canada
Radionuclide cystography (RNC) is a widely used test in the diagnosis or as in indication of resolution, of vesicoureteric reflux (VUR). The recurrence (persistence) of reflux after one negative RNC has been previously reported. This prospective study further elaborates on this phenomenon.
85 patients with primary VUR, treated conservatively, between 1991 and 1996 having one negative RNC, were included in this study, 12 to 18 months after the negative study a repeat RNC was done. Two groups, those with and those without necurrence of reflux were identified and compared as to sex distribution, age of resolution, time between presentation and first negative RNC, change in grade between presentation and just before resolution, side of reflux at presentation and grade before resolution. The differences between both groups were statistically analyzed.
Of the 85 patients, 25 (29%) ha recurrence of reflux. Of these 18 (72%) patients had recurrence with grade two or greater and 15 (60%) of the 25 patients are still refluxing 12 months after the second positive RNC. There was no observed association between the two groups in any of the variables (p-values were greater than 0.05).
This study emphasizes the importance of a second negative RNC to indicate absence/resolution of reflux, as one negative RNC alone would have missed persistent reflux in 25 of 85 patients at one year and 15 of 85 patients at two years. Whether this is variability in the disease process or an unknown factor inherent in the test is unknown, in that there is no statistical difference in the parameters studied between the two groups.
**Presented at the:** 12^th^ Saudi Urology Conference Al Hada and Taif Armed Forces Hospitals Program 23-25 February 1999 (7-9 Dhu Al Qa'dah 1419)
Augmentation ureterocystoplasty with ipsilateral renal presrvation
**R. F. Talic**
Department of Surgery, King Khalid University Hospital, Riyadh, Saudi Arabia
**Introduction**: Evaluate the role of ureterocystoplasty with ipsilateral renal preservation in the management of patients with neuroversical dysfunction and impaired renal function who failed conservative medical treatment.
**Patients and Methods**: Five patients with neuroversical dysfunction (2 male and 3 female) with a mean age of 6.2 years were recruited in this ongoing study starting June 1996. All patients have vesicoureteric reflux (VUR), impaired and deteriorating renal function. Symptoms, renal function and radiological evaluation are recorded both pre and post operatively. Urodynamic assessment was included in the post operative follow up. Operative reconstruction involved augmentation cystoplasty using the distal ureter of one renal unit and transuretero-ureterostomy. Neureterostomy for correction of VUR in the contralateral kidney was required in 4 patients.
**Results**: All patients were followed up with an average of 12 months. All patients showed symptomatic improvement and were dry by day and night. Two patients continue to have positive urine cultures but no symptomatic urinary tract infections. Three patients have stable renal function, improvement was noted 2 patients. VUR was corrected in 3 patients and improved in 2 patients. Hydroureteronephrosis and obstruction improved in all patients.
**Conclusion**: Early results of this operative intervention are satisfactory and promising in the management of this difficult group of patients in terms of symptomatic improvement and stabilization of the renal function. The long-term effect on the renal function will require long-term follow up.
**Presented at the:** 11^th^ Saudi Urology Conference King Fahd Military Medical Complex -- Dhahran 24-26 February 1998 (27-29 Shawwal 1418)
Ureteroneocystostomy with and without the use of an intravesical catheter. A prospective randomized study
**A. Al Shammari, A. Bhatti, R. N. Kupchak, L. W. Mix, A. Decter, M. D. Leonard**
Department of Urology, Children\'s Hospital, University of Manitoba and University of Alberta, Canada, Department of Urology, King Fahad National Guard Hospital, Riyadh, Saudi Arabia
**Purpose**: To objectively assess postoperative pain, analgesic requirement, bladder spasms, nosocomial urinary tract infections, length of hospital stay and the outcome of "no catheter" intravesical ureteric reimplantation compared to intravesical ureteric reimplantation with a bladder cathter.
**Materials and Methods**: Over a period of one year, 33 patients (24F, 9M) aged 3-14 years with primary vesicoureteric reflux were randomly assigned to a "no catheter" group or to a "catheter" group after meeting study selection criteria. Two standard procedures of intravesical ureteric reimplantation were utilized with intra-operative low dose caudal anesthesia. All patients were given intravenous morphine for pain control postoperatively, either by a continuous intravenous infusion or by patient control analgesia. Postoperative pain was assessed by a patient driven pain scale and by interviewing patients and their parents at least twice a day as regards pain control/bladder spasms. Urine cultures were performed on the day of the surgery and 1^st^ and 3^rd^ postoperative days. Patients were followed at 6 weeks with a renal ultrasound and at three months with a voiding cystogram or nuclear cystogram. Postoperative analgesic requirements, occurrence of bladder spasms, urinary tract infection rate, length of hospital and the outcome are reported. Student\'s paire test was used for evaluation of statistical significance.
**Results**: There was no difference in sex distribution, mean age, mean body weight, or nosocomial urinary tract infection between the two groups The postoperative intravenous morphine requirement was significantly lower (p \< 0.05) in the "no catheter" group with a mean (+ 0.5) in the "catheter" group. Bladder spasms occurred in 80% of catheterized patients and only in 30% of non-catheterized patients. The length of hospital stay was significantly shorter (p \< 0.05) in the "no catheter" group with a mean (+ SEM) of 87.2 hours (+ 19.4) compared to 109.1 hours (+ 20.9) in the "catheter" group. All evaluated patients had stable upper tracts by renal ultrasound at 6 weeks and cure of their reflux by cystogram at three months, regardless of catheter status.
**Conclusion**: Intravesical ureteroneocystotomy without a catheter is safe and well tolerated. Patients with "no catheter" reimplantation have significantly lower postoperative intravenous analgesic requirements, shorter hospital stay and subjectively fewer bladder spasms than patients with catheter post-reimplantation do. "No catheter" reimplantation did not alter the expected outcome. We therefore recommended this technique as a safe and effective addition to the urologic armamentarium.
**Presented at the:** 11^th^ Saudi Urological Conference King Fahd Military Medical Complex -- Dhahran 24-26 February 1998 (27-29 Shawwal 1418)
STING for treatment of VUR
**Michael P. Leonard**
Department of Urology, Children\'s Hospital of Eastern Ontario, Ottawa, Ontario, Canada
This lecture will briefly review the history of the STING procedure. A profile of the commonly used materials for STING, along with their clinical results and safety concerns will be examined. Finally a review of Deflux® as it applies to STING will be carried out. This will culminate with the review of a series of Deflux® STING as carried out at CHEO. At the completion of the lecture, the following objectives will be reached:
An understanding of how the STING was developed and evolvedAn appreciation of the pros and cons of the varied materials used to effect the STINGCurrent clinical results with the use of Deflux® for STING, including the results of a series accumulated by the author.
**Presented at the:** 18^th^ Saudi Urological Conference King Abdulaziz University Hospital 20-23 February 2006 (21-24 Muharram 1427)
Endoscopic treatment of vesicoureteral reflux in children with subureteral injection of duflex: The alberta children\'s hospitals experience
**O. Bawazir, W. Hyndman**
Department of Surgery, Alberta Children Hospital, Calgary, Canada
**Purpose**: Vesicoureteral reflux is a risk for progressive renal damage associated with urinary tract infection. Mild to moderate reflux is routinely treated with long-term antibiotic prophylaxis to prevent recurrent infections and open surgical reimplantation for breakthrough infections despite antibiotic therapy. Endoscopic subureteral injection of implant material is a therapeutic alternative to long-term prophylaxis and open surgery. We present our experience with the use of subureteral injection of dextranomer/hyaluronic acid (Dx/HA) in Alberta Children Hospital, Calgary, Canada.
**Materials and Methods**: Between October 2003 and October 2004, 22 patients 7 months to 10 years old (mean age 2.6 years) underwent subureteral injection of Dx/HA for complex VUR at our institutions. Dx/HA was injected submucosally within the intramural ureter (modified STING) in most cases. Hydrodistension was use to open the ureter orifices. The average volume of injected material was measured for each ureter. Renal sonography was performed to determine if hydronephrosis was present. At 6 weeks fluoroscopic voiding cystourethrograms were used to evaluate for the presence of VUR.
**Results**: A total of 40 ureters were treated in 19 girls and 3 boys. Reflux was corrected in 16 patients out of 22 (73%), repeated injection required in 2 patients with low grade reflux which resulted in complete resolution. STING failed to correct reflux in 4 patients (18%) or 8 ureters (20%), which were managed by ureteral reimplantation.
**Conclusions**: Submucosal intraureteral implantation with Dx/HA corrected complex (STING) is a simple, safe and effective outpatient procedure for vesicouretral reflux. The use of the modified STING technique optimizes ureteral cooptation and improves the success rate.
**Presented at the:** 18^th^ Saudi Urological Conference King Abdulaziz University Hospital 20-23 February 2006 (21-24 Muharram 1427)
Comparative study between subureteral injection of dextronomer-hyaluronic acid copolymer and extravesical reimplantation in low grade VUR in children
**Osama Kamhawy**
Department of Urology, Faculty of Medicine, Zagazig University, Zagazig, Egypt
To compare the results of extravesical ureteral reimplantation to endoscopic subureteral injection of dextranomer hyaluronic acid co-polymer (Deflux, Q-Med Ab, Uppsola, Sweden) in the treatment of low grade vesicoureteral reflux in children, 20 children (30 ureters) underwent subureteral injection of the aforementioned material and 26 children (39 ureters) underwent extravesical ureteral reimplantation. Low grade reflux meant 1 to 3. Mean patient age at the time of surgery in the injection and reimplantation groups was 6 (range 2-13) and 5 (range 2-14) years respectively. The mean follow up period was 12 (range 7-29) and 14 (range 6-27) months respectively. Of the patients who underwent single injection, 80% were cured of reflux at 3 months and 83.3% were cured at the last follow up while in the reimplantation group 94.8% were cured at 3 months and improved to 97.4% at the last follow up. There was a significant difference in the cure rate between the two groups of the study at 3 months and last follow up. No complications were recorded in the injection group, while in the reimplantation group contralateral reflux was seen in 3 cases (23%) at 3 months postoperatively and persist in only one case (7.7%) at the last follow up, UTI was recorded in 3 cases (11.5%), and urine retention in 2 cases (7.7%).
We can conclude that in spite of lower success rate of injection than reimplantation, the lower morbidity, the ease of a second injection or reimplantation when indicated makes subureteral injection therapy a viable option in treating low grade vesicoureteral reflux in children when medical treatment fails.
**Presented at the:** 18^th^ Saudi Urological Conference King Abdulaziz University Hospital 20-23 February 2006 (21-24 Muharram 1427)
Are postoperative studies necessary after ureteral reimplantation? Study of single center
**Al Qahtani Saeed, Al Mudhin Fayez, A. Al Shammari**
Division of Urology, King Fahad National Guard Hospital, Riyadh, Saudi Arabia
**Purpose**: We evaluated the usefulness of postoperative imaging studies after ureteric reimplantation.
**Materials and Methods**: Records of 196 patients who had undergone simple or reconstructive ureteric reimplantation from 1994 to 2004 were reviewed retrospectively and 111 patients who fit the criterion. Study inclusion criterion was primary reflux with at least 1 year of postoperative follow up. Grades I to III reflux were defined as low and grades IV and V were defined as high. All patients were on prophylactic antibiotics. Evaluation included ultrasound in 6 weeks and a nuclear cystourethrogram 3 months postoperatively, and if reflux persisted the studies were repeated at 12 months.
**Results**: A total of 111 patients underwent simple or reconstructive ureteral reimplantation. At 3 months the procedure was successful in 90.1% of patients, and at 12 months the success rate increased to 98.2% of patients. There were 7 cases that were persistent refluxing and 4 with down grade at 3 months. At 12 months reflux resolved spontaneously in all of 11 patients and 2 needed redo. Those needed redo were high grade preoperatively with elimination syndrome. There was no statistically significant difference in the success rate at 1 year between high versus low grade reflux. Elimination syndrome significantly affects the success rates. Worsening hydronephrosis was noted in 0% at 6 weeks and at 12 months too.
**Conclusions**: Ureteral reimplantation is successful in treating vesicoureteral reflux. Postoperative cystourethrogram should be reserved for high grade reflux and those with elimination syndromes. Limiting these studies will help reduce patient discomfort and the cost of treatment.
**Presented at the:** 18^th^ Saudi Urological Conference King Abdulaziz University Hospital 20-23 February 2006 (21-24 Muharram 1427)
Can prophylactic antibiotics safely be discontinued in children with vesicoureteric reflux?
**Ahmed J. Al Sayyad, John G. Pike, Michael P. Leonard**
Division of Pediatric Urology, Children\'s Hospital of Eastern Ontario, University of Ottawa, Ottawa, Ontario, Canada
**Purpose**: To review the outcome of stopping prophylactic antibiotics in children with persistent VUR.
**Materials and Methods**: Retrospective review of patients with VUR off antibiotics over the past 12 years. Selection criteria: Children 4 years or older who were toilet trained, could verbalize and had a normal voiding pattern. Exclusion criteria: PUV, ectopic ureter, neurogenic bladder or severe voiding dysfunction. Outcome measures: Age of stopping antibiotics, duration on and off antibiotics, grade of reflux at the time of stopping antibiotics, the occurrence of UTIs and new renal scarring. All patients were followed with renal ultrasound.
**Results**: 78 patients \[67 F (85%), 11 M (14.1%)\] included. Bilateral VUR was present in 36 patients (46.2%), left VUR in 29 (37.2%) and right VUR in 13 (16.7%). Reflux grade ranged from I--III (I = 16.7%, II = 75.6%, and III = 7.7%). Mean age when taken off antibiotics was 5.74 years. Period on antibiotics prior to discontinuance ranged from 0--84 months, mean 26.2 months. Period off antibiotics ranged from 5--138 months, mean 37.7 months. 9 female patients (11.5%) developed UTI. 8 had cystitis (10.2%) and 1 had pyelonephritis (1.3%). Period off antibiotics in this group ranged from 5--60 months, mean 21.1 months. None of our patients, including those with UTI, developed new scarring of their kidneys by renal ultrasound.
**Conclusion**: Discontinuing prophylactic antibiotics in selected school age children is safe practice. The risk of significant upper tract infection is low, and the development of new renal scars unlikely.
**Presented at the:** 18^th^ Saudi Urological Conference King Abdulaziz University Hospital 20-23 February 2006 (21-24 Muharram 1427)
Do infants with low grade antenatal hydronephrosis benefit from screening for vesicoureteric reflux?
**M. Al Assiri, N. Al Said, J. P. Capolicchio, R. Jednak, P. Forbrs, J. L. Pipi Salle**
Department of Urology, The Montreal Children\'s Hospital, Montreal, Quebec, Canada
**Introduction**: The natural history of undiagnosed vesicoureteric reflux (VUR) in children with low grade antenatal hydronephrosis (LAHN); SFU\_\_-\_\_, is unknown, therefore screening infants with LAHN for VUR might be unjustified. We present a cohort of patients with LAHN who were managed without VCUG or long-term antibiotic prophylaxis and compare outcomes to similar group who were screened and given prophylaxis.
**Methods**: The charts of 262 consecutive children with LAHN seen from 1999-2001 were reviewed. Patients with ureteral dilation or other renal/bladder anomalies were excluded. Patients were subdivided into 2 groups: one group managed with prophylactic antibiotics and a screening cystogram (VCUG), while the other group was not. Urinary tract infection (UTI) was the main outcome measure.
**Results**: One hundred and forty-four with LAHN met our inclusion criteria, with a mean follow up of 20 months. Ninety-nine patients (80 males) underwent screening VCUG. Of those, 24 males (30%) were circumcised, 9 (9%) had VUR and were kept on prophylaxis. In addition, only 2 patients (2%) developed symptomatic UTI; both of them uncircumcised males without VUR. The other group; 45 patients (31 males), were managed without VCUG or prophylactic antibiotics. None developed UTI, 12/31 (39%) males were circumcised.
**Conclusion**: Our data suggest that patients with LAHN do not benefit from VCUG screening of prophylactic antibiotics. A prospective randomized study is required to confirm the long-term safety of this approach.
**Presented at the:** 17^th^ Saudi Urological Conference King Fahd Military Medical Complex 8-10 March 2005
Endoscopic treatment of vesicoureteral reflux with dextranomer/hyaluronic acid copolymer. Impact of voiding dysfunction on the overall treatment success
**Hisham El Shawaf**
Department of Urology, Ain Shams University, Cairo, Egypt
**Purpose**: Endoscopic injection of destranomer/hyaluronic acid (Dx/HA) copolymer has become an increasingly accepted treatment for primary vesicoureteral reflux (VUR) in children. Because some patients with VUR also experience voiding dysfunction, this prospective analysis was performed to assess the efficacy of that treatment for VUR of different etiologies together with evaluation of the impact of voiding dysfunction on endoscopic treatment outcome.
**Materials and Methods**: A total of 37 patients of both sex (male = 18 & female = 19) with VUR of different etiologies and grades (grade II to V), were included after failure of minimum course of 6 months of antibiotic prophylaxis and underwent endoscopic treatment with Dx/HA copolymer for VUR. Follow up consisted of pelviabdominal ultrasonography at one & 12 months visit and voiding cystourethrography 3, 6 and 12 months after injection. Of the 37 patients 9 were re-treated with dextranomer/hyaluronic acid copolymer because of persistent reflux (grade II or greater). The first implantation technique was recorded on videotaper. Voiding cystourethrography and micturition details were recorded at the 3 to 6 months follow up visit and compared with baseline measurements. Positive response was defined as reflux grade 0 or 1. As many as 2 repeat injections were offered to nonresponders, and those with persistent reflux (grade II or greater) were referred for open surgery. Clinical follow up was continued for a minimum of 1 year.
**Results**: The overall success rate was 75.7% (28/37) in all patients included in the study at 3 months follow up. Preoperative reflux according to the International Reflux Study Classification was grade II in 17 (39.5%), III in 15 (34.8%), IV in 10 (23.2%) and V in 1 (2.3%) ureteral renal units. We subsequently stratified our treatment results into 2 groups according to the etiology with 22 patients having primary VUR (affecting 25 renal units) and 15 patients with secondary VUR (affecting 18 renal units) of different etiologies. A positive response was observed in 81.8% of children with primary VUR, with 9.1% of patients requiring open surgery. While, among patients with secondary VUR the response rate was 66.6% and open surgery was performed in (13.3%). In all patients a positive response to treatment was sustained throughout the follow up period. The treatment was well tolerated, with no complications associated with the procedure. At baseline 15 patients had known controlled voiding dysfunction. Of the 9 patients (24.3%) who required re-treatment, the initial implant was correctly positioned in all patients included according to the videotape. Endoscopic observation at the time of re-treatment revealed that the implant was displaced in 5 patients, missed in one and remained correctly positioned in 3 patients. Of the retreated patients 2 had misdiagnosed voiding dysfunction, had not received any anticholinergic therapy and had a displaced implant.
**Conclusions**: Endoscopic treatment with Dx/HA copolymer appears to be an effective and well tolerated alternative to open surgery for first line treatment of VUR of either primary or secondary origin. Uncontrolled voiding dysfunction contributed to endoscopic treatment failure with dextranomer/hyaluronic acid copolymer because of implant displacement in this study.
Therefore, we suggest that patients with voiding dysfunction should be controlled with anticholinergics and/or micturition rehabilitation before endoscopic therapy.
**Presented at the:** 16^th^ Saudi Urological Conference King Faisal Specialist Hospital and Research Centre 2-4 March 2004 (11-13 Muharram 1425)
Can the "STING" change the paradigm of management in vesicoureteric reflux combined with pelviureteric junction obstruction?
**Sherif Soliman**
Department of Urology, King Khalid University Hospital, Riyadh, Saudi Arabia
**Objective**: To evaluate the possibility of one stage, double level correction in children with vesicoureteric reflux (VUR) combined with pelviureteric junction obstruction (PUJO).
**Patients and Methods**: Seven children (age range 10-48, median 15 months) having VUR (1 grade 3, 5 grade 4, 1 grade 5) along with established PUJ obstruction diagnosed by persistent hold up in washout diuretic renogram with a catheter in bladder, underwent simultaneous endoscopic correction of VUR by Dextranomer/Hyaluronic Acid Copolymer (DefluxÂÒ) with Anderson-Haynes dismembered pyeloplasty drained with a nephrostomy tube. Two patients had solitary functional renal units. In the first 3 patients a nephrostogram was performed 7-10 days postoperatively to confirm upper and lower tract potency before extubation. In the following, the nephrostomy tube was clamped on day 3 postoperative for 24 hours and if the child shows no evidence of obstruction or leakage it was removed. Patients were followed clinically and sonographically at 2, 4, 8 and 12 months. An MCUG was performed at 4 months to confirm or deny the disappearance of VUR.
**Results**: None of the patients had any evidence of obstruction or leakage. PUJO was successfully corrected in all patients as evidenced by reduction in their hydronephrosis. All but 1 boy (grade 5) had their VUR resolved. The latter needed a formal open reimplant after 2 redo injections to correct his VUR.
**Conclusion**: In children with VUR associating PUJO, endoscopic correction of the former can allow addressing the problem in one operative session away from the proposed effect of devascularizing the ureter if operated upon conventionally at its two poles simultaneously.
**Presented at the:** 17^th^ Saudi Urological Conference King Fahd Military Medical Complex 8-10 March 2005
Histopathological findings post dextranomer/hyaluronic acid implants in children with vesicoureteral reflux
**M. Al Mandil, A. Murphy, M. Souab, A. E. Khoury**
Department of Urology, The Hospital for Sick Children, Toronto, Ontario, Canada
**Aim of the Work**: Dextranomer/hyaluronic acid (Deflux) is being widely used as an implant for the treatment of vesicoureteral reflux as an alternative to other implants material. The literature is still lacking or deficient in documenting histopathological changes in humans after implants.
**Patients and Methods**: A retrospective review of patients undergoing ureteroneocystostomy after failed dextranomer/hyaluronic acid implants to capture demographic and clinical data of those patients. In addition, histopathological specimen review with further testing of excised ureteral segments looking at inflammatory changes of the surrounding tissue was conducted. Histopathological changes were correlated with the clinical findings looking at volume, duration of implant and degree of reflux.
**Results**: A total of 10 patients, (13 ureters) were eligible for the study. Mean age at implants 53 months, mean age at ureteroneocystostomy was 60 months. Mean time from implant injection to excision was 7 months. All ureters showed giant cell reaction with variable severity, all were encapsulated, all showed the presence of mast cells range (6-26 by HPF) and eosinophils range (1-30 by HPF). Atypia was not present in any specimen, marked to moderate fibrosis was present in all ureters. There was no correlation between the histopathological and clinical findings in regard to volume and degree of reflux. No sign of nuclear atypia or high turnover was noted.
**Conclusions**: Dextranomer/hyaluronic acid is stable and safe after 5 to 39 months of follow up of intra/subureteral injection. No signs of nuclear atypia or high nuclear turn over were noted. There is no correlation between volume of implant used and degree of inflammation.
**Presented at the:** 20^th^ Saudi Urological Conference King Fahad Hospital of the University -- Tabuk 18-20 March 2008
Double blind prospective randomized study to explore the effectiveness of prophylactic preoperative tolterodine on the success rate of endoscopic treatment of primary vesicoureteric reflux
**M. Salem, K. Fouda, H. Al Hazmi, A. Gomha**
Department of Urology, King Khalid University Hospital, King Saud University, Department of Surgery, Division of Pediatric Urology, Riyadh, Saudi Arabia
**Objective**: To study whether the prophylactic use of Tolterodine for patients with primary vesicoureteric reflux (VUR) and stable bladder can enhance the success rate of endoscopic treatment (ET) of VUR.
**Patients and Methods**: All patients with primary VUR, in need of ET, were divided to two groups: those with element of bladder dysfunction (Dysfunctional Voiding Symptoms Score DVSS \>6 in females and \>9 in males) and those with "stable bladder". The "stable bladder" group were randomized at their clinic follow up visit to two subgroups: subgroup A who were not started on Tolterodine, and subgroup B who were started on a prophylactic dose of Tolterodine one week before the procedure, and continued for a month after that. In the group with bladder dysfunction, classical management of the bladder was given, and the procedure was done on an improved bladder. Group one and subgroup A and B were compared and statistically analyzed.
**Results**: 44 patients (18 males and 26 females) with mean age of 5.2 (range 3-10) with VUR were included in the study. In group one (15 patients, 8 with bilateral VUR) had symptoms of bladder dysfunction, 14 (60%) out of 23 refluxing ureters showed complete resolution. In group two, 29 patients were randomized. In subgroup A (15 patients), 18 (83%) out of 24 ureters showed resolution of VUR, and in the second subgroup B (14 patients), 17 (74%) out of 23 refluxing ureters showed complete resolution. The overall success rate was 70% in all groups with 70 refluxing renal units. No statistical difference was found between the 3 groups in success rate (P0.464), nor between the two main groups (P0.254) nor between subgroup A and B (P0.365) in the success rate.
**Conclusion**: We did not find a close correlation between voiding dysfunction and the success rate of ET of VUR with the use of a prophylactic dose of Tolterodine in patients with stable bladder. It did not enhance the success rate of ET. Thus we cannot conclude that the assumed subclinical voiding dysfunction can affect the outcome of ET of VUR.
**Presented at the:** 21^st^ Saudi Urological Conference North West Armed Forces Hospital -- Tabuk 14-16 April 2009
|
{
"pile_set_name": "PubMed Central"
}
|
Background {#Sec1}
==========
Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) for pancreatic tumors has pooled sensitivity and specificity at 92% and 96%, respectively \[[@CR1]\], and it is an indispensable procedure for pancreatic cancer diagnosis. The main complications associated with EUS-FNA for pancreatic tumors is bleeding, pancreatitis, and post-procedural pain, among others, but the incidence rate is as low as 1.03%; therefore, EUS-FNA is considered a safe procedure \[[@CR2]\]. The incidence rate of peritoneal dissemination associated with puncture for pancreatic cancer was reported to be 16.3% after percutaneous puncture and 2.2% after EUS-FNA; the risk of peritoneal dissemination is lower in EUS-FNA than in percutaneous puncture \[[@CR3]\].
Needle tract seeding is a phenomenon in which tumor cells are found in the puncture route, and it is considered a subtype of peritoneal dissemination recurrence \[[@CR4]\]. To the best of our knowledge, however, only 18 cases (17 reports) of needle tract seeding associated with EUS-FNA for pancreatic cancer have been reported till date \[[@CR4]--[@CR20]\]. Therefore, it is necessary to accumulate a greater number of cases of needle tract seeding for a better understanding of the features. Herein, we reported two cases of needle tract seeding after EUS-FNA that were detected during surgery and diagnosed via partial gastrectomy.
Case presentation {#Sec2}
=================
Case 1 {#Sec3}
------
A 68-year-old woman with no relevant medical or family history was admitted to our hospital because of a persistent cough. On admission, her abdomen was not tender and no mass was detected. Computed tomography (CT) scan revealed inflammatory signs in the left lung field along with incidental inflammatory findings around the pancreas, because of which pancreatitis was suspected. Dynamic-enhanced CT revealed a 15-mm hypovascular tumor in the pancreatic body (Fig. [1](#Fig1){ref-type="fig"}a), and inflammatory findings around the pancreas lead to the suspicion that concomitant pancreatitis is associated with pancreatic cancer. Laboratory data showed elevation of tumor marker levels (CA19-9, 44 U/ml; DUPAN-2, 1300 U/ml; Span-1, 33.0 U/ml). Diffusion-weighted magnetic resonance image revealed high-signal intensity in pancreatic body tumor (Fig. [1](#Fig1){ref-type="fig"}b). Endoscopic ultrasonography (EUS) revealed a 14.7 × 8.5 mm hypoechoic tumor in the pancreatic body, and the tumor did not contact to the superior mesenteric artery (SMA) and portal vein (PV). EUS-FNA for the pancreatic tumor was performed (4 punctures using 22 G, 19 G, 20 G, and 20 G needles) via the trans-gastric approach, and no complications were noted (Fig. [1](#Fig1){ref-type="fig"}c). Cytology revealed adenocarcinoma (Fig. [1](#Fig1){ref-type="fig"}d). Based on the imaging findings, she was diagnosed as resectable pancreatic body cancer. She underwent distal pancreatectomy with splenectomy. However, during surgery, we noticed a small hard mass in the posterior gastric wall (Fig. [2](#Fig2){ref-type="fig"}a), and thus, we performed partial gastrectomy (Fig. [2](#Fig2){ref-type="fig"}b). The pathological findings of the specimen from partially resected stomach revealed adenocarcinoma cells which were linearly distributed in the gastric muscle layer; these findings indicated that the gastric tumor was needle tract seeding from pancreatic cancer due to EUS-FNA (Fig. [2](#Fig2){ref-type="fig"}c, d). The time from EUS-FNA to the detection of the gastric wall metastasis due to needle tract seeding was 25 days. The pathological findings of the main pancreatic tumor resulted in a diagnosis of invasive ductal carcinoma, pT1, pN1 (No.8a, 11p), and pM0 pStageIIB (UICC). She then underwent adjuvant chemotherapy with TS-1, but a CT scan revealed peritoneal dissemination after 6 months. Therefore, the chemotherapy regimen was changed from TS-1 to gemcitabine (GEM) + nab-paclitaxel; however, her condition was gradually worsened and she died due to peritoneal dissemination of pancreatic cancer 18 months after surgery.Fig. 1**a** Dynamic-enhanced computed tomography (portal phase) for case 1. A 15-mm hypovascular tumor was detected in the pancreatic body (arrow). **b** Diffusion-weighted magnetic resonance imaging. A hyperintense area can be observed in the pancreatic body tumor (arrow). **c** Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA). EUS-FNA was performed for the pancreatic tumor (4 punctures using 22 G, 19 G, 20 G, and 20 G needles) via the trans-gastric approach, without any complications. **d** Pathological findings of EUS-FNA. An adenocarcinoma can be observed (Papanicolaou staining)Fig. 2**a** Intraoperative findings for case 1. A small hard mass was detected in the posterior gastric wall, as indicated by the forceps. **b** Partial resection of the posterior gastric wall was performed. **c** Pathological findings. The specimen from the partially resected stomach showed that an adenocarcinoma was distributed linearly in the gastric muscle layer (arrow) (hematoxylin and eosin staining, loupe image). **d** Pathological findings. The findings of the gastric tumor were similar to those of the primary pancreatic cancer, indicating that gastric tumor was needle tract seeding from pancreatic cancer (hematoxylin and eosin staining)
Case2 {#Sec4}
-----
A 70-year-old man underwent CT scan as part of his follow-up for recurrence of basal cell carcinoma. Dynamic-enhanced CT scan incidentally revealed a 15-mm hypovascular mass 15 mm in size in the pancreatic body (Fig. [3](#Fig3){ref-type="fig"}a). He had no abdominal symptoms, and laboratory data showed no elevation in tumor marker levels. Positron emission tomography-CT (PET-CT) revealed abnormal accumulation of fluorine-18- deoxyglucose (FDG) in the pancreatic body, with a standardized uptake value of 3.74 (Fig. [3](#Fig3){ref-type="fig"}b); however, there was no abnormal accumulation of FDG in other parts of the body. EUS revealed a 15.2-mm hypoechoic tumor in the pancreatic body. Although his tumor was suspected to invade the splenic artery, the tumor did not invade the SMA or PV. EUS-FNA was performed (1 puncture using a 22 G needle) via the trans-gastric approach, and no complications occurred (Fig. [3](#Fig3){ref-type="fig"}c). Cytology revealed adenocarcinoma (Fig. [3](#Fig3){ref-type="fig"}d). He had small nodules in both the lungs, and it was difficult to differentiate them from lung metastasis of pancreatic cancer. Therefore, he underwent neoadjuvant chemoradiotherapy (50.4 Gy/28 Fr radiotherapy, and 2 cycles of chemotherapy: 600 mg/m^2^ GEM on days 8 and 22, and 60 mg/m^2^ TS-1 on days 1--21). After neoadjuvant chemoradiotherapy, his tumor marker levels were still within the normal ranges. The pancreatic tumor slightly shrunk, and small lung nodules showed no change. We suspected the lung nodules were not metastasis of the pancreatic cancer; therefore, he underwent radical antegrade modular pancreatosplenectomy procedure posterior (RAMPs posterior) \[[@CR21]\]. During surgery, we noticed a small hard mass in the posterior gastric wall (Fig. [4](#Fig4){ref-type="fig"}a), for which we performed partial gastrectomy (Fig. [4](#Fig4){ref-type="fig"}b). The resected specimen was diagnosed as needle tract seeding following EUS-FNA (Fig. [4](#Fig4){ref-type="fig"}c, d). The time from EUS-FNA to the detection of the gastric wall metastasis due to needle tract seeding was 113 days. At the end of the surgery, a small nodule was found in the mesenterium of the small intestine. We resected it, and on pathological examination, it was diagnosed as peritoneal dissemination. Pathological findings resulted in a diagnosis of invasive ductal carcinoma, pT2, pN0, and pM1 pStageIV (UICC). He received chemotherapy with only TS-1, as GEM could not be used owing to allergic reactions observed during neoadjuvant chemoradiotherapy. His condition is stable even after 18 months after surgery at the time of writing.Fig. 3**a** Dynamic-enhanced CT (portal phase) for case 2. A 15-mm hypovascular tumor in the pancreatic body (arrow). **b** Positron emission tomography-CT (PET-CT) findings. Abnormal accumulation of fluorine-18-deoxyglucose (standardized uptake value of 3.74) can be observed in the pancreatic body (arrow). **c** EUS-FNA findings. EUS-FNA was performed for the pancreatic tumor (1 puncture using 22 G, needle) via the trans-gastric approach, without any complications. **d** Pathological findings. EUS-FNA revealed an adenocarcinoma (Papanicolaou staining)Fig. 4**a** Intraoperative findings for case 2. A small hard mass was detected in the posterior gastric wall (arrow). **b** Partial resection of the posterior gastric wall was performed. **c** Pathological findings. Many abnormal luminal structures (adenocarcinoma) were confirmed in the resected gastric muscle layer (hematoxylin and eosin staining, loupe image). **d** Pathological findings. The findings of gastric tumor were similar to those of the primary pancreatic cancer, indicating that gastric tumor was a recurrence due to needle tract seeding from pancreatic cancer (hematoxylin and eosin staining)
Discussion and conclusions {#Sec5}
==========================
It has been reported that EUS-FNA for pancreatic tumor has pooled sensitivity and specificity, 92% and 96%, respectively \[[@CR1]\]. The main complications of EUS-FNA for pancreas tumor is bleeding, pancreatitis, post-procedural pain, and so on, but the incidence rate is as low as 1.03%; therefore, it is considered a safe procedure \[[@CR2]\]. Moreover, there was no significant difference in prognosis even when EUS-FNA was performed on the pancreatic body and tail cancer before surgery, and EUS-FNA is now an essential examination for the diagnosis of pancreatic tumors \[[@CR22], [@CR23]\]. However, in these reports, the type, stage, and resectability of pancreatic tumors were different and EUS-FNA has a risk of peritoneal dissemination, although its diagnosis due to FNA is difficult because pancreatic cancer itself often results in the development of peritoneal dissemination. Hence, the adverse effects of EUS-FNA may be ambiguous. In the future, the oncological safety of EUS-FNA should be reconsidered in limiting patients who undergo this procedure.
The first case of needle tract seeding after EUS-FNA in a patient with invasive ductal carcinoma derived from intraductal papillary mucinous neoplasm (IPMN) was first reported in 2003 \[[@CR5]\], then in 2005, needle tract seeding after EUS-FNA was reported in a patient with a common type of pancreatic adenocarcinoma \[[@CR6]\].In a search of the PubMed database and Ichushi (Japanese database) using the search term "\[(endoscopic ultrasound fine-needle aspiration) OR (EUS-FNA) AND (pancreatic cancer) OR (pancreatic adenocarcinoma) AND (needle tract seeding) OR (seeding)\]," till date, only 18 cases (17 reports) of needle tract seeding associated with EUS-FNA for pancreatic cancer have been reported, including invasive ductal carcinoma derived from intraductal papillary mucinous neoplasm (Table [1](#Tab1){ref-type="table"}) \[[@CR4]--[@CR20]\]. Regarding the tumor site, all the tumors were located in the pancreatic body or pancreatic tail, except for a case with pancreatic head cancer who did not undergo surgery, and two cases where there was no description. This was probably because the puncture route is included in the resection range for pancreatic head cancer. In 3 of 18 cases, including our 2 cases, needle tract seeding was detected during surgery. Therefore, intraoperative assessment for gastric wall metastasis is important as well as postoperative assessment, and if surgeon suspects gastric wall metastasis intraoperatively, partial gastrectomy should be performed without hesitation. In these reported cases, the median period until the gastric wall metastasis after EUS-FNA is 21 months, but it occurred only 10 days in the shortest case \[[@CR5]\]. As shown in our case 2, needle tract seeding after EUS-FNA cannot be controlled even after chemoradiotherapy.Table 1Reported cases of needle tract seeding after EUS-FNA for pancreatic tumorAuthorYearAgeSexLocation of pancreatic cancerTumor sizeFrequency of punctureEUS needleInitial treatmentStageDiscovery opportunityTime to recurrence (months)Recurrence tumor sizeTreatment for needle tract seeding1Hirooka200357MalePancreatic body20 mm322 GDistal pancreatectomy and partial gastrectomyT1N0M0Operative findings1MicroPartial gastrectomy2Paquin200565Malepancreatic tail22 mm522 GDistal pancreatectomyT1N0M0CT2150 mmChemotherapy3Ahmed201179MalePancreatic bodyUnknownSeveral timesUnknownCentral pancreatectomyT2N0M0PET-CT3945 mmTotal gastrectomy4Chong201155FemalePancreatic tail27 mm322 GDistal pancreatectomyT2N0M0PET-CT2640 mmNo indication of surgery5Katanuma201268FemalePancreatic body20 mm422 GDistal pancreatectomyT2N0M0EGDS22UnknownUnknown6Anderson201351MalePancreatic head50 mmUnknownUnknownChemoradiation therapyUnknownEGDS/EUS-FNAUnknown10 mmUnknown7Ngamruengphong201366MalePancreatic body/tailUnknown322 and 19 GSubtotal pancreatectomyUnknownEGDS/EUS27UnknownUnknown8Ngamruengphong201377FemalePancreatic tail40 mm319 GDistal pancreatectomy and partial gastrectomyUnknownEGD26UnknownUnknown9Sakurada201587FemalePancreatic body25 mmUnknown22 GDistal pancreatectomyT2N0M0Elevation of CA19-91920 mmPartial gastrectomy10Minaga201564FemalePancreatic body20 mm322 GDistal pancreatectomyT3N0M0Elevation of CA19-9812 mmPartial gastrectomy11Tomonari201578MalePancreatic body20 mm222 GDistal pancreatectomyT3N0M0EGDS2832 mmSubtotal gastrectomy12Kita201668FemalePancreatic bodyUnknown222 GIntensity-modulated radiation therapyUnknownPET-CT4UnknownUnknown13Yamabe201675MaleUnknown30 mmUnknown25 GChemotherapyUnknownCT/EUS-FNA324 mmChemotherapy14Minaga201672MalePancreatic body10 mmUnknownUnknownDistal pancreatectomyT1N0M0EGDs/EUS2430 mmGastrectomy15Iida201678FemaleUnknownUnknown322 GDistal pancreatectomyT3N0M0EGDS/PET-CT618 mmDistal gastrectomy16Yamauchi201667FemalePancreatic body25 mm119 GDistal pancreatectomyT3N0M0EGDS/EUS-FNA2328 mmPartial gastrectomy17Sakamoto201850MalePancreatic tail38 mm222 GDistal pancreatectomyT4N1M0EGDS2420 mmPartial gastrectomy18Matsumoto201850MalePancreatic body35 mm321 GDistal pancreatectomy and partial gastrectomyUnknownCT/EUS8UnknownPartial gastrectomy19Our case 1201968FemalePancreatic body15 mm422, 19, 20, and 20 GDistal pancreatectomy and partial gastrectomyT1N1M0Operative findings1MicroPartial gastrectomy20Our case 2201970MalePancreatic body34 mm122 GDistal pancreatectomy and partial gastrectomyT3N0M1Operative findings4MicroPartial gastrectomy*Abbreviations*: *CT* computed tomography, *PET-CT* positron emission tomography computed tomography, *EGDS* esophagogastroduodenoscopy, *EUS* endoscopic ultrasound, *EUS-FNA* endoscopic ultrasound-guided fine-needle aspiration
According to NCCN guidelines \[[@CR24]\] and clinical practice guidelines for pancreatic cancer 2016 from the Japanese Pancreas Society guidelines \[[@CR25]\], the treatment policy of pancreatic cancer varies according to the tumor resectability; surgery is the first treatment choice for resectable pancreatic cancer. For borderline resectable pancreatic cancer, it is a dominant opinion that neoadjuvant chemoradiotherapy is known to improve the prognosis, and for unresectable cases, chemotherapy is chosen. If we choose to perform chemotherapy for pancreatic cancer including preoperative treatment, it is necessary to differentiate it from other pancreatic tumors via EUS-FNA. However, whether EUS-FNA should be performed for all pancreatic tumors is controversial. Depending on the resectability and the localization of the tumor, it is necessary to consider the indications of EUS-FNA separately. For resectable pancreatic cancer that does not conflict with pancreatic cancer on the imaging studies, there may be a choice not to puncture the tumor. When EUS-FNA is performed for pancreatic body or tail cancer which is not included in the resection range, we should be aware of the risk of developing needle tract seeding in the gastric wall. In order to avoid needle tract seeding, biopsy needle with a covering sheath should be used \[[@CR26]\]. Although our institution had already used a biopsy needle with a covering sheath, needle tract seeding unfortunately developed in these two cases. Therefore, the other factors such as technical problem should be considered. To prevent needle tract seeding as much as possible, we recommend to avoid unnecessary EUS-FNA for resectable pancreatic body or tail cancer, when up-front surgery is planned. Actually, when we consider the cost of EUS-FNA and the selection of operative procedure for the patients in whom up-front surgery is planned, EUS-FNA has few benefits because EUS-FNA by itself does not influence the selection of the operative procedure and is costful. If EUS-FNA is performed, intraoperative and postoperative assessment is essential for gastric wall metastasis due to needle tract seeding. According to the report by Yamauchi et al., if gastric wall metastasis due to needle tract seeding is detected early, partial gastrectomy can control the disease \[[@CR4]\]. However, if the finding of gastric wall metastasis due to needle tract seeding is delayed, there is a report that lymph node metastasis has occurred \[[@CR7]\]. In addition, there is a report of recurrence after partial gastrectomy for gastric wall metastasis due to needle tract seeding \[[@CR18]\]; hence, post-operative assessment is important.
In conclusion, although EUS-FNA is a useful diagnostic tool, it may cause peritoneal dissemination and needle tract seeding at the puncture site. Therefore, physicians should decide its indication, especially for resectable pancreatic body or tail cancer, by taking the consideration of merit and demerit of EUS-FNA for each case.
**Publisher's Note**
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
We are grateful to Shuji Isaji, MD, PhD, Department of Hepatobiliary Pancreatic and Transplant Surgery, Mie University Graduate School of Medicine, for revising this article.
All authors were involved in the preparation of this manuscript. TM wrote the manuscript. KN, HY, ST, YN, and MO performed the endoscopic examination. TM, HN, YS, YH, KT, and MS performed the surgery. KK performed the pathological diagnosis. MS and SI revised the manuscript. All authors read and approved the final manuscript.
All authors do not have any funding.
The datasets obtained during the current study are available from the corresponding author on reasonable request.
Not applicable.
We obtained patients' consent for publication.
The authors declare that they have no competing interests.
|
{
"pile_set_name": "PubMed Central"
}
|
Introduction {#sec1-1}
============
As a fundamental element for life, the living systems developed numerous and different ways to sense, absorb and use the visible light. Recent advances in optobiology have demonstrated that light may be used for modulation and control of the biological function, in the production of biopharmaceuticals and in biomedicine \[**[@R1]**\].
Many studies demonstrated that the effects on cellular systems depend on the illumination parameters (the wavelength of light source, intensity, treatment timing, etc. For a better understanding of these processes, the effects of monochromatic light irradiation were largely studied.
A series of our previous studies revealed that irradiation with High Density Green Photons (HDGP) has complex pleiotropic effects on matter: modification of enzymes kinetics, (2-4) generation of a new local macromolecular architecture with a protective role toward subsequent aggressions, modification of transmembranar transport \[**[@R5]**-**[@R9]**\].
The other studies demonstrated even an in ovo cell proliferation under green light irradiations \[**[@R10]**,**[@R11]**\]. Various other effects of green light laser irradiation on different substrates were also described \[**[@R12]**,**[@R13]**\].
The purpose of the present paper is to reveal an alternative, and maybe new aspects concerning the irradiations of different cell cultures with high-density-green-photons (HDGP).
Material and methods {#sec1-2}
====================
**Cell cultures**
MEF and HUH7 cell line were cultivated in Dulbecco's modified Eagle' medium (DMEM) (Gibco, Life Technologies) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% antibiotic. Cells were seeded at different densities, in 3.5 cm Petri dishes, or 24 well plates, and incubated at 37˚C under a humidified atmosphere, with 5% CO2.
**Oxidative stress induction: Cell cultures treatment with hydrogen peroxide**
For the oxidative stress induction, cells were treated with different concentrations of Hydrogen peroxide in the presence (simultaneously or consecutive) or absence of green light irradiation for different durations; right after the treatment, the culture medium was changed and cells were incubated at 37˚C. Usually, cells were processed for subsequent experiments after 24,48 or 72 hours of incubation. During this incubation time, supplementary irradiations were performed.
**High-density green photon source and irradiation**
Cell cultures under different conditions were irradiated with a source of HDGP at different time points. As a source of HDGP, a pure monochromatic light emitting diodes (LED) was used (16V, 20W, 1000 lumens, EverRedTronics, E 20 WG 120 C) Diodes were mounted on ventilated copper radiators. A monochromatic green light with an absorption peak centered at λ-520nm was obtained, with intensities up to 140mW/cm2, spectral width of 10 nm. Time of cells irradiation varied between 1-60 min, at different intervals of time (e.g. 2x5 minutes /24hrs of incubation).
**RNA isolation**
The total RNA was prepared from cell lines by using Trireagent (Sigma, St. Louis, MO) according to manufacturer's instructions. The quantity and quality of the total RNA were assessed by spectrophotometry with Nano Drop 1000 (Thermo Scientific, Arlington, TX). Samples with a ratio 260/280 of 1.8-2.1 were used in the downstream analysis.
**cDNA synthesis and Quantitative PCR (qPCR)**
First cDNA was obtained from 2 μg of the total RNA by using High Capacity cDNA Archive Kit (ABI, Foster City, CA) in a total volume of 20μl. The final dilution of the samples was 2 ng/μl. The two-step relative quantification was performed on 7300 Real time PCR (ABI, Foster City, CA) by using hydrolysis probes. Then qPCR amplification was carried out in triplicate for each sample in a total volume of 25 μl in the following conditions: 95°C for 10 min, 95°C for 15 sec and 1 min at 60°C for 40 cycles. The level of each mRNA was normalized to reference gene hu18S (20x). Fold changes within tumoral tissues were determined compared with paired non-tumoral tissue. Data were analyzed with SDS 1.4 software using comparative Ct method \[2\^(-delta delta Ct)\]. The tested genes were super oxide dismutase (SOD1) and catalase (CAT). As an endogenous control, 18S gene was used.
**Cell proliferation assay**
Control and HDGP irradiated cells were incubated for a different period of time and cell proliferation was measured by two methods: using Alamar blue (Sigma --Aldrich) staining (assay cells were treated with Alamar Blue and after 1-3h incubation, the shift of color was determined spectrophotometrically) or by using the Tali® Image Cytometer (Life Technologies).
**Cell migration assay**
A scratch assay model \[**[@R14]**\] was used to determine cell migration. Briefly, cells were seeded at the same concentration in 3,5 cm Petri dishes. When cells reached confluence, scratches were made in cells monolayer with a sterile pipette tip. Irradiation of cells was performed: 4 times x 5min / 36hrs. At the beginning of the experiment, images were taken and at regular intervals, the closure of the scratch by migrating cells was microscopically monitorized. After 36hrs of incubations, the cells were stained with Coomassie blue and images were taken.
**Catalase activity**
The catalase activity was assessed by Catalase Activity assay kit (BioVision, CA). Samples and positive control were prepared in the same manner. Briefly, cells were homogenized with Assay Buffer, centrifuged at 10000g for 15 min at 4°C. Supernatant was collected for assay. The standard curve was drawn by using different concentrations of H2O2: 0, 2, 4, 6, 8 and 10 nmols.
**Statistical analysis**
All the statistical analyses were performed by using Graph Pad Prism 5.0 (GraphPad Software Inc, San Diego, CA). The comparisons between groups were performed by using nonparametric tests (Mann--Whitney U-test). Differences were considered significant if p-value was \<0.05.
Results {#sec1-3}
=======
**SOD gene expression is modified by HDGP action**
The results of the qPCR experiments are presented as an average of three experiments conducted in the same conditions. The control sample was used as calibrator for the analysis.
{#F1}
Hydrogen peroxide addition and/ or HDGP irradiation modify cellular SOD gene expression (**[Fig. 1](#F1){ref-type="fig"}**): in the hydrogen peroxide treated cells, the fold change of SOD determined by qPCR decreased compared with the control sample (fold change 0.7, p-value=0.009). The irradiated sample (IR) showed a greater increase in the fold change compared with the control sample (fold change 1.3, p-value=0.03) and also an increase of fold change compared to the hydrogen peroxide treated sample. In the sample which was simultaneously treated with H2O2 and irradiated with HDGP, the SOD gene expression is higher compared with the control sample (fold change 1.1, p-value=0.2), but smaller compared with the irradiated sample and increased compared to hydrogen peroxide sample.
**Catalase activity is modified by HDGP irradiation**
Compared with the control sample, the catalase activity was higher in treated hydrogen peroxide and in irradiated samples. In the sample simultaneously treated with H2O2 and irradiated with HDGP, the catalase activity was the highest determined among the studied samples (**[Fig. 2](#F2){ref-type="fig"}**).
{#F2}
**Proliferation assay**
A series of cell proliferation experiments was performed. A stimulation of the cell proliferation was observed in all the experiments; the degree of this stimulation was mainly the function of irradiation protocol. Irradiation for longer period of time (40-60 min) might induce an inhibition of proliferation for certain cell type (data not shown).
The simulative effects of HDGP irradiations (4x5 min/48 hrs incubation) on the proliferation of the two cell lines: MEF and HUH 7 are presented in **[Fig. 3](#F3){ref-type="fig"}**.
{#F3}
**MEFs migration rate is enhanced by exposure to HDGP**
To study the migration capacities of MEFs (controls and exposed to HDGP), a scratch model experiment was used: scratches ("wound gaps") were made in the cells monolayer and the "healing" of the gaps by cell migration and growth toward the center was monitored.
The microscope images were taken after 36 hrs. Comparing the images the capacity to close the gaps was evaluated and it was observed that the exposure to green light significantly activated the MEFs migration capacity (**[Fig. 4](#F4){ref-type="fig"}**).
{#F4}
Discussions and Conclusions {#sec1-4}
===========================
Our previous studies presented a series of new effects of green light irradiations on different substrates \[**[@R2]**-**[@R9]**\].
The mechanisms underlying the effects of visible light on cells are incompletely understood.
The present study demonstrated that high-density green photons (HDGP) irradiation stimulates cellular proliferation and migration. Mitochondria could be a possible site for the initial light effects: an increase of ATP production and consequently, stimulation and modulation in levels of growth factors, cytokines and other parameters. In turn, these effects led to an increased cell proliferation and migration with further modulations of cellular processes.
Our results concerning SOD gene expression and catalase activity indicate that HDGP irradiations also induce changes in intracellular antioxidant processes and consequently suggest possible antioxidant effects.
As a conclusion, we consider that all these cellular effects described, indicate that HDGP irradiation may have beneficial bio-medical applications, particularly in the field of regenerative medicine.
**Founding source:** This study was financially supported by the Romanian Program for Research, Development and Innovation, research grant PN ID 139/5.10.2011
[^1]: †The first two authors had an equal contribution in the article
|
{
"pile_set_name": "PubMed Central"
}
|
Introduction / objectives
=========================
Environmental contamination with nosocomial pathogens on the near--patient surfaces and equipment has been associated with its indirect transmission. Thorough, regularly scheduled cleaning and disinfection of the environment is essential to break this transmission. Environmental hygiene is a priority for all healthcare faciltiies. Unfortunately, despite regular cleaning following patient discharge with a multi-drug resistant organism, the environment may still harbor these organisms.
Methods
=======
A multidisciplinary team was formed to look into different ways to improve environment hygiene. The project was carried out in a 36-bedded medical ward from Jun to Dec 2010.The project looked into enhancement of the environmental cleaning by process re-design. The used of two bucket methods of cleaning the environment with micro-fibre cloths that were well soaked with Mikro Quart or phenolic instead of spraying the disinfectant on the cloths. The 3-days education workshops were tailored to the language and cultural needs of the staff. The topics covered "Highly Touch" surfaces, environmental hygiene and audit methods and finding. Effectiveness of the intervention was evaluated using a fluorescent marker, "Glo-germ" powder at random points in the ward. A checklist was used to monitor the trend of progress. The immediate feedback on the audit finding and action plan was developed for compliance failure to the staff and competency was monitored by direct observation.
Results
=======
The environmental audit showed an improvement from a median of 64% to 95% following the above intervention (*P*\< .001). However, no significant improvement in the nosocomial MRSA infection before and after intervention this could be the period is too short.
Conclusion
==========
The project intervention has spread hospital-wide and enhances using disposable cloths to reduce time in rinsing and prevent contamination.
Disclosure of interest
======================
None declared.
|
{
"pile_set_name": "PubMed Central"
}
|
Background
==========
Weight loss commonly occurs in patients with chronic obstructive pulmonary disease (COPD) \[[@B1]\]. In a subgroup of COPD patients, weight loss is suggested to follow a stepwise pattern related to acute disease exacerbations \[[@B2]\]. Recently, cytokine-mediated metabolic derangements have been considered to be among the candidates responsible for weight loss in COPD patients \[[@B3],[@B4]\]. It has been shown that tumor necrosis factor-α (TNF-α) circulating levels are increased in weight-losing COPD patients \[[@B3],[@B4]\]. COPD is characterized by a systemic inflammatory response that may be even more pronounced during an acute exacerbation of COPD \[[@B5]\].
Leptin, a protein synthesized by adipose tissue and encoded by the *ob gene*plays an important role in the energy balance and its circulating concentrations are proportional to the amount of fat mass (FM). Inappropriately increased leptin levels are thought to induce metabolic effects underlying anorexia and loss of body weight in chronic diseases including COPD \[[@B6],[@B7]\]. Administration of endotoxin or cytokines such as TNF-α or IL-1 produced a prompt and dose-dependent increase in serum leptin levels in both experimental animals \[[@B8]\] and humans \[[@B9]\]. In stable patients with emphysema, leptin was found to be positively related to plasma soluble TNF-receptor 55 \[[@B5]\]. The observed link between inflammatory cytokines and leptin led to the hypothesis that adipose tissue gene expression is regulated by inflammatory cytokines, which in turn could induce anorexia in acute or chronic inflammation.
Growth hormone (GH) is secreted by the anterior pituitary and mediates its major metabolic effects through activation of somatomedins, predominantly insulin-like growth factor I (IGF-I) \[[@B10]\]. In COPD, little is known about circulating GH or IGF-I concentrations. Some authors found a decrease in GH or IGF-I, others an increase \[[@B11]\]. IGF-I levels tended to be lower in stable and hospitalized COPD patients due to an exacerbation \[[@B12]\]. IGF-I mRNA levels were decreased in muscle biopsies from hospitalized patients due to an acute exacerbation of COPD \[[@B13]\]. In animal studies, recombinant IGF-I has been shown to ameliorate the protein catabolism and promote anabolism observed under hypoxic conditions \[[@B14]\]. However, daily administration of rGH increased lean body mass but did not improve muscle strength in COPD patients \[[@B15]\].
In our study, we measured cytokine (TNF-α, IL-1β, IL-6 and IL-8), leptin and IGF-I levels at the onset of a COPD exacerbation as well as 15 days later. The aim of this study was to elucidate two questions. Firstly, whether COPD exacerbations are accompanied by changes in circulating levels of leptin and IGF-I. Secondly, whether plasma IGF-I and leptin levels are related to those of cytokines as a possible reflection of the enhanced inflammatory status observed during a COPD exacerbation.
Methods
=======
Patients
--------
The patient group consisted of subjects consecutively admitted to our department due to an infectious exacerbation of COPD. COPD was defined according to GOLD criteria \[[@B16]\]. Irreversible chronic airflow obstruction was confirmed, i.e., \< 10% improvement in FEV1 expressed as percentage of predicted after inhalation of a β2-agonist. An infectious exacerbation of COPD was defined when at least two of the following three criteria were fulfilled: (a) recent increase in dyspnea (b) increased sputum volume and (c) sputum purulence, provided that one of the two criteria is purulent sputum \[[@B16]\].
All patients were current or ex-smokers. Patients with a medical history of comorbid diseases, such as diabetes mellitus, heart failure, lung cancer, collagen vascular disease or disturbances of thyroid function were excluded from the study. The final study group consisted of 52 patients (43 men, 9 women). The patients were treated with a standard protocol of medication consisting of nebulized short-acting β2-sympathicomimetics (salbutamol), inhaled anticholinergics (ipratropium bromide) and intravenously administered prednisolone. Prednisolone was given in the same dose (50 mg) for 7 days and then directly stopped, without tapering the dose. Antibiotic treatment was given to all COPD patients for 7 days. During hospitalization 38 of the 52 patients received supplemental oxygen therapy, according to measured arterial blood gases.
The control group consisted of 25 healthy age-matched adults (19 men, 6 women). They had no medical illnesses, had normal physical examinations, blood counts, chemistries and showed no symptoms or signs of infection at the time of the study.
All participants were informed in detail of the characteristics of the study and written consent was obtained. The study was approved by the ethics committee of our hospital.
Body composition
----------------
Body height was determined to the nearest 0.5 cm with subjects standing barefoot. Body weight was assessed to the nearest 0.1 kg by using a digital weighing chair while subjects wore light clothing and no shoes. Body mass index (BMI) was calculated according to the equation BMI = Weight (kg)/Height^2^(cm^2^). Fat mass (FM) was measured with bioelectric impedance analysis with the portable device OMRON BF 302, (OMRON Healthcare Europe B.V, Hoofddorp). The basic principles of bioelectric impedance analysis were described by Lukaski and colleagues \[[@B17]\].
Lung function
-------------
FEV1 and FVC were measured with standard spirometric techniques (SPIRO 232 MORGAN, Model 435 P.K MORGAN, Ltd. Gillingham, Kent, England). Arterial blood gases were obtained once in healthy individuals and twice in COPD patients (upon admission and after 15 days) with the subject in the sitting position and breathing ambient air. Lung function values were expressed as a percentage of predicted \[[@B18]\]. Arterial blood gases were analysed on a blood gas analyzer (Rapidlab 855, Bayer Healthcare LLC Diagnostics Division, NY 10591, USA).
Assessment of emphysema
-----------------------
The distinction between COPD patients with predominant emphysema and those with chronic bronchitis was based mainly on high-resolution computed tomography (HRCT). HRCT is a sensitive technique for the evaluation of the presence and severity of emphysema. In patients with emphysema the densitometric parameters differ substantially from the corresponding values in patients with chronic bronchitis and healthy control subjects. Five thin-section CT scans were obtained in each patient: two scans of the upper, two scans of the lower lung zones at 3 and 6 cm above and below the carina and one scan at the carina. The severity and extent of emphysema of each scan was visually scored on a four-point scale independently by two observers according to the direct observational method of Sakai \[[@B19]\]. For each of the lung sections, the score for the severity of emphysema was multiplied by the score for the extent and the resultant scores were subsequently summed to give a total HRCT score. Visual scores ranged from 0 (no emphysema) to 120 (severe emphysema). Patients with a score \< 30 were subtyped as chronic bronchitis and patients with a score ≥ 30 were subtyped as emphysema. According to this assessment the 52 patients with COPD were divided into 29 patients with chronic bronchitis and 23 patients with emphysema.
Collection and analysis of laboratory and inflammatory parameters
-----------------------------------------------------------------
Blood samples were collected in the morning, between 8.30 and 9.30 AM, when patients were in the fasting state for at least 10 h twice in COPD patients (on hospital admission-Day 1, and 15 days later- Day 15) and once in healthy individuals. For the measurement of TNF-α, IL-1β, IL-6, IL-8, leptin and IGF-I, blood was collected in plastic tubes containing ethylenediamine-tetraacetic acid (EDTA) and was centrifuged (1,000 × g for 10 min) within an hour. The supernatants were stored at -70°C until analysis. Cytokines (TNF-α, IL-1β, IL-6, IL-8) were measured with ELISA (BMS 223/3, BMS 224, BMS 213/2 and BMS 204, Bender MedSystems, MedSystems Diagnostics GmbH, Vienna, Austria Europe, respectively). Leptin and IGF-I were measured with radioimmunoassay (RIA, Active Human Leptin IRMA DSL -- 23100, Diagnostic Systems Laboratories, Inc., USA and IGF-I 100 T KIT -- Catalog No. 40-2100, Nichols Institute Diagnostics, USA, respectively). The lower detection limit of leptin was 0.10 ng/ml. The intra- and interassay variation coefficient were 4.9 and 6.6% respectively. The lower detection limit of IF-I was 0.06 ng/ml. The intra- and interassay coefficient were 3% and 9.8% respectively. All measured values for leptin and IGF-I were above the lower detection limit.
Statistical analysis
--------------------
Data were expressed as mean ± SD or as median (25--75 percentile) if they were not normally distributed. Statistical analysis was performed using nonparametric tests (Mann-Whitney U test for comparison between groups and Wilcoxon for comparison within groups). Dichotomized Δ was calculated by the formula Δ = D15-D1. Therefore ΔTNF-α = TNF-αD1-TNF-αD15, ΔLeptin = LeptinD1-LeptinD15 etc. The relations between continuous variables were evaluated with Spearman\'s rank correlation technique. Statistical significance was accepted at p \< 0.05. Data were analysed using SPSS (Statistical Package for the Social Sciences, version 14.0 for Windows, SPSS Inc, Chicago, IL).
Results
=======
Study group
-----------
The mean age of COPD patients was 65.8 ± 8.3 years and the control group 65.9 ± 9.6 years. Clinical characteristics of the COPD patients (on admission-Day 1) and the healthy controls are given in Table [1](#T1){ref-type="table"}.
######
Characteristics of the two groups
--------------------------------------------------------------------------------------------------
**COPD patients (n = 52)**\ **Healthy subjects (n = 25)**\ **p**
**43 M, 9 F**\ **19 M, 6 F**\
**(Mean ± SD)** **(Mean ± SD)**
----------------------- ----------------------------- -------------------------------- -----------
**Age, yr** 65,8 ± 8,3 65.9 ± 9.6 0.789
**Weight, kg** 71,1 ± 13,6 77.2 ± 10.1 **0.037**
**Height, cm** 166,48 ± 7,11 167.56 ± 6.91 0.669
**BMI, kg/m^2^** 25,6 ± 4,1 27.9 ± 4.1 **0.029**
**Fat mass, kg** 21,5 ± 6,6 25.9 ± 7.3 **0.011**
**Fat free mass, kg** 49,6 ± 9,0 51,2 ± 7,9 0.427
**% FM** 29,8 ± 5,1 31.6 ± 6.3 0.211
**FVC %Pred** 62,4 ± 14,3 92.6 ± 6.6 **0.000**
**FEV~1~%Pred** 44,1 ± 11,4 89.9 ± 8.0 **0.000**
**PaO~2~mmHg** 58,2 ± 12,5 87.6 ± 4.6 **0.000**
**PaCO~2~mmHg** 47,1 ± 10,8 39.2 ± 2.5 **0.000**
--------------------------------------------------------------------------------------------------
Patients with COPD had significantly lower body weight, BMI and fat mass than did the control subjects. The COPD patients had severe airflow limitation, decreased PaO~2~and increased PaCO~2~values. The control subjects had normal % FVC and % FEV1 on spirograms.
Course of the parameters during the exacerbation
------------------------------------------------
### Inflammatory parameters
Compared to their concentrations in the healthy subjects, TNF-α, IL-1β, IL-6 and IL-8 were significantly higher in the patients with COPD on D1. By Day 15 cytokine levels did not differ from those of healthy controls with the exception of IL-8. Although IL-8 levels were decreased on D15 in COPD patients, they still significantly differed from healthy subjects (Table [2](#T2){ref-type="table"}).
######
Concentrations of plasma cytokines, leptin and IGF-I in healthy controls compared to patients with COPD exacerbation on admission to the hospital (D1) and after two weeks (D15)
----------------------------------------------------------------------------------------------------------
**Markers**\ **COPD patients (D1)**\ **Healthy subjects**\ **COPD patients**\
**Median (25--75 percentile)** **(n = 52)** **(n = 25)** **(D15)**\
**(n = 52)**
-------------------------------- ------------------------- ----------------------- -----------------------
**TNF-α (pg/ml)** 63.9(46.7--84.5)\* †† 14 (9.2--15.8) 14.3 (10.5--19.9)
**IL-1β (pg/ml)** 12.5 (8.8--16.9) \* †† 3.6 (2.6--4) 3.8 (2.7--5.2)
**IL-6 (pg/ml)** 23 (17.7--44.6) \* †† 9.7 (7.5--12.3) 10.6 (6.5--14.9)
**IL-8 (pg/ml)** 39.8 (29.2--56.3) \* †† 13.7 (10--19) 18.5 (12.3--24) †
**Leptin (ng/ml)** 33.3 (16.7--45.5) \* †† 9.6 (4.5--13.7) 15.7 (8.5--26) †
**IGF-1 (ng/ml)** 59.7 (44.3--76) \* †† 133.2 (124--164.5) 96.6 (82.9--116.8) \*
**Leptin/% FM** 1.1 (0.6--1.55) \* †† 0.30 (0.16--0.43) 0.5 (0.26--0.94) \*
----------------------------------------------------------------------------------------------------------
Between healthy subjects and COPD patients \* p \< 0.01 † p \< 0.05
Between COPD patients D1 and D15 †† p \< 0.01
In COPD patients hospitalized due to an acute exacerbation, TNF-α, IL-1β, IL-6 and IL-8 levels on D1 were significantly higher compared to the levels measured on D15 (Table [2](#T2){ref-type="table"}).
When COPD patients who received oxygen therapy were compared to those who did not, no difference in cytokine levels was detected.
### Leptin
The levels of leptin in COPD patients with exacerbation were significantly higher on D1 and still on D15 compared to healthy subjects \[33.3 (16.7--45.5)\] ng/ml, p \< 0.0001 and \[15.7 (8.5--26)\] ng/ml, p = 0.022 respectively, versus \[9.6 (4.5--13.7)\] ng/ml). Leptin concentration decreased significantly from D1 to D15 of the COPD exacerbation (p \< 0.0001). After dividing leptin by %FM, the same pattern was seen. Thus, leptin/%FM was significantly elevated on D1 and nearly significantly increased on D15 compared to healthy subjects (p \<0.001 and p = 0.008 respectively).
### Insulin-like growth factor I (IGF-I)
Compared to its concentration in the healthy subjects \[133.2 (124--164.5)\] ng/ml), plasma IGF-I was significantly lower in COPD patients on D1 of the exacerbation and remained lower on D15 \[59.7 (44.3--76)\] ng/ml, p \<0.001 and \[96.6 (82.9--116.8)\] ng/ml, p \< 0.001 respectively versus healthy subjects). IGF-I levels were significantly increased from D1 to D15 throughout the exacerbation (p \< 0.001).
### Chronic bronchitis and emphysema
Characteristics of the study group stratified into the COPD subtypes (23 patients with emphysema and 29 patients with chronic bronchitis) are given in Table [3](#T3){ref-type="table"}.
######
Characteristics of patients with chronic bronchitis and emphysema
------------------------------------------------------------------------------
**Chronic bronchitis**\ **Emphysema**\ **p**
**(n = 29)**\ **(n = 23)**\
**25 M, 4 F** **18 M, 5 F**
----------------------- ------------------------- ---------------- -----------
**Age, yr** 68.9 ± 9.3 70.8 ± 7.0 0.524
**Weight, kg** 75.3 ± 14.1 65.9 ± 11.4 **0.022**
**Height, cm** 166.44 ± 7.10 166.52 ± 7.29 0.971
**BMI, kg/m^2^** 27.1 ± 4.2 23.7 ± 3.1 **0.003**
**% FM** 31.4 ± 4.5 27.9 ± 5.2 **0.047**
**Fat mass, kg** 23.7 ± 6.4 18.7 ± 5.7 **0.006**
**Fat free mass, kg** 51,5 ± 9,7 47,2 ± 7,7 0.058
**FVC %Pred** 65.7 ± 14.1 58.1 ± 13.8 **0.032**
**FEV~1~%Pred** 47.4 ± 11.2 39.2 ± 9.9 **0.005**
**PaO~2~mmHg** 59,4 ± 13,3 56,6 ± 11,6 0.256
**PaCO~2~mmHg** 49,6 ± 12,0 41,1 ± 9,5 0.276
------------------------------------------------------------------------------
Patients with emphysema were characterized by a significantly lower FVC and FEV1 compared to those with chronic bronchitis. Emphysematous patients had also a significantly lower BMI owing to a significantly lower FM (mean difference 5.04 kg). No difference was found in PaO~2~and PaCO~2~between patients with chronic bronchitis and emphysema.
No differences were seen in the concentrations of TNF-α, IL-1β, IL-6, IL-8, leptin and leptin/% FM between the two groups of patients on D1 and D15. On the contrary, IGF-I was significantly lower in emphysematous patients compared to patients with chronic bronchitis both on D1 and D15 \[49.5 (40.1--65.4)\] versus \[70.5 (48.6--78.5)\] ng/ml, p = 0.003 on D1 and \[84.7 (74.4--96.9)\] versus \[112.7 (92.4--124.1)\] ng/ml, p \< 0.001 on D15) (Table [4](#T4){ref-type="table"} and Table [5](#T5){ref-type="table"}, Figure [1](#F1){ref-type="fig"}).
{#F1}
######
Concentrations of plasma cytokines, leptin and IGF-I in patients with chronic bronchitis and emphysema on admission to the hospital (D1).
------------------------------------------------------------------------------------------
**Markers**\ **Chronic bronchitis**\ **Emphysema**\ **p**
**Median (25--75 percentile)** **(n = 29)** **(n = 23)**
-------------------------------- ------------------------- ------------------- -----------
**TNF-α (pg/ml)** 63.2 (45.5--85.7) 65 (47.2--80.2) 0.612
**IL-1β (pg/ml)** 12.6 (8.8--15.6) 12.3 (8.4--21.3) 0.941
**IL-6 (pg/ml)** 23.9 (19.7--40.4) 20.6 (15.9--52.6) 0.423
**IL-8 (pg/ml)** 39.7 (28.9--56.2) 42 (34.3--62.7) 0.574
**Leptin (ng/ml)** 37.7 (18.1--50.9) 32.5 (15.2--44.9) 0.214
**IGF-1 (ng/ml)** 70.5 (48.6--78.5) 49.5 (40.1--65.4) **0.003**
**Leptin/% FM** 1.1 (0.62--1.61) 1.09 (0.6--1.55) 0.619
------------------------------------------------------------------------------------------
######
Concentrations of plasma cytokines, leptin and IGF-I in patients with chronic bronchitis and emphysema on D15.
-------------------------------------------------------------------------------------------
**Markers**\ **Chronic bronchitis**\ **Emphysema**\ **p**
**(Median (25--75 percentile)** **(n = 29)** **(n = 23)**
--------------------------------- ------------------------- ------------------- -----------
**TNF-α (pg/ml)** 13.7 (10.5--19.8) 15.1 (10.5--20.1) 0.632
**IL-1β (pg/ml)** 3.9 (3.4--5.8) 3.7 (2--4.7) 0.273
**IL-6 (pg/ml)** 10.6 (7.4--14.1) 10.4 (5.8--16.2) 0.706
**IL-8 (pg/ml)** 17.9 (11.8--23.2) 19 (12.3--26.6) 0.612
**Leptin (ng/ml)** 15.6 (7.9--28.6) 15.7 (8.4--22.8) 0.847
**IGF-1 (ng/ml)** 112.7 (92.4--124.1) 84.7 (74.4--96.9) **0.000**
**Leptin/% FM** 0.45 (0.27--0.98) 0.56 (0.25--0.81) 0.768
-------------------------------------------------------------------------------------------
### Correlation analyses
A strong correlation between leptin and % FM was observed in controls (r = 0.780, p \< 0.001).
In order to elucidate the possible relationship of circulating cytokine levels with plasma leptin and IGF-I concentrations correlation analysis was performed on D1 and D15.
On D1 of the exacerbation, TNF-α demonstrated a significant positive correlation with leptin as well as with leptin adjusted to % FM (r = 0.620, p \< 0.001 and r = 0.650, p \< 0.001)(Figure [2](#F2){ref-type="fig"}). This correlation between TNF-α and leptin was true even when we studied patients with chronic bronchitis and emphysema separately (r = 0.719, p \< 0.001 and r = 0.505, p = 0.014 respectively). TNF-α was also positively correlated with leptin/% FM in both chronic bronchitis and emphysema (r = 0.774, p \< 0.001 and r = 0.499, p = 0.015 respectively). Moreover, the change in TNF-α levels (ΔTNF-α) was significantly related to the change in leptin levels (ΔLeptin) (r = 0.545, p \< 0.001).
{#F2}
On D15, no significant correlations could be revealed between any of the parameters.
IGF-I was not found to be related to any of the inflammatory cytokines neither on D1 nor on D15.
Discussion
==========
The present study was performed to examine circulating levels of leptin and IGF-I at the onset of COPD exacerbations (D1) and two weeks later (D15) and furthermore to investigate the relationship of plasma leptin and IGF-I concentrations with cytokine concentrations as a possible reflection of the enhanced inflammatory status observed during the COPD exacerbation.
We found that COPD exacerbations are characterized by increased levels of leptin and the proinflammatory cytokines TNF-α, IL-1β, IL-6 and IL-8 and decreased levels of IGF-I on D1. We also found that two weeks after the onset of the exacerbation (D15) cytokine levels did not significantly differ from those of healthy subjects, with the exception of IL-8. IL-8 levels, although significantly decreased in COPD patients between D1 and D15, they still remained higher on D15 compared to healthy subjects. Although circulating plasma leptin decreases and plasma IGF-I increases, they still remained different compared to healthy controls on D15. Leptin was positively related to TNF-α on D1 of the exacerbation and this correlation also applied to both subgroups of COPD patients, thus chronic bronchitis and emphysema. Finally, we found that IGF-I levels were significantly lower to emphysematous patients compared to patients with chronic bronchitis both on D1 and on D15. Leptin and cytokine levels did not differ between patients with chronic bronchitis and emphysema.
The elevated levels of TNF-α, IL-1β, IL-6 and IL-8 on admission to the hospital (D1), observed in our study are in keeping with numerous studies that have reported increased levels of circulating cytokines in the peripheral circulation of patients with COPD \[[@B3],[@B20],[@B21]\]. These abnormalities, albeit seen in clinically stable COPD patients, they are generally more pronounced during exacerbations of the disease \[[@B21]\]. The systemic inflammatory response is ameliorated 15 days after the onset of the exacerbation, as shown by the decrease in cytokine levels.
Circulating leptin levels were high on D1 of the exacerbation and remained elevated on D15 in COPD patients compared to healthy subjects. The elevated leptin concentrations during COPD exacerbation are in keeping with the results of other studies \[[@B7]\] and likely represented an up-regulation of leptin mRNA resulting in an enhanced leptin production that might have been induced by several factors. The positive correlation between leptin and TNF-α which was seen in our COPD patients on D1 of the exacerbation, supports an inflammatory-related disturbance in leptin metabolism in COPD. This correlation was true even when we studied separately the two subgroups of patients (chronic bronchitis -- emphysema). It has been shown that the administration of endotoxin or cytokines such as TNF-α and IL-1 can increase serum leptin levels both in hamsters and humans \[[@B8],[@B9]\]. A positive correlation between leptin and soluble TNF-α receptors (sTNF-R55) was also found during COPD exacerbations in the study by Creutzberg et al \[[@B7]\]. In a recent study by Calikoglu et al serum leptin and TNF-α levels were increased in COPD patients with exacerbation in comparison to COPD patients with stable disease and healthy controls \[[@B22]\]. Moreover, and in accordance with our findings, a strong correlation between TNF-α and leptin was observed only in COPD patients with exacerbation but not in stable COPD patients and healthy individuals \[[@B22]\]. Takabatake et al recently reported that serum leptin levels were significantly lower in COPD patients than healthy controls \[[@B23]\]. However, this study included patients with stable disease who were free of symptoms for at least three months. The relationship between the change in TNF-α levels (ΔTNF-α) and the change in leptin levels (ΔLeptin) observed in our study, further supports a TNF-α-related disturbance in leptin during COPD exacerbations.
A reason for the high leptin concentrations might have been the glucocorticosteroid treatment and that presents a limitation of our study. Reports concerning the effects of corticosteroids on leptin are contradictory. Two days of oral dexamethasone (1.5 mg/day) in healthy subjects resulted in significantly increased serum concentrations of leptin \[[@B24]\]. In another study, administration of dexamethasone for 4 days (2.5 mg/day) to healthy subjects also induced a significant increase in plasma leptin concentrations \[[@B25]\]. Glucocorticosteroids may have a stimulating effect on leptin via the induction of insulin resistance, as glucose and insulin are also able to induce leptin expression \[[@B26]\]. However, Tataranni et al demonstrated that acute intravenous administration of glucocorticosteroids or prolonged oral treatment did not affect serum leptin levels \[[@B27]\]. Our COPD patients had severe airflow obstruction and it was necessary to introduce steroid treatment to improve their clinical status. However, samples on D1 were obtained before the first corticosteroid administration and on D15 7 days after the last intravenous administration of prednisolone, according to the protocol used. Regarding the effects of adrenergic stimulation on leptin metabolism, no studies have been performed with specific β2-adrenergic stimulants such as salbutamol. Intravenous infusion of isoprenaline in young healthy volunteers resulted in a maximal suppression of plasma leptin of 20% of baseline values after 2 h, but in the recovery period of 1 h, leptin concentrations rapidly returned to normal \[[@B28]\]. In our study COPD patients received salbutamol by nebulizer and not intravenously.
Limited data is available regarding circulating levels of IGF-I in COPD. Growth hormone (GH) mediates its major metabolic effects predominantly through IGF-I \[[@B10]\]. The GH axis is suppressed in chronic diseases and this may partly explain the low IGF-I levels. However, increased concentrations of GH have been found in COPD patients, especially in those with hypoxaemia \[[@B29]\].
In our study, patients with COPD exacerbation had significantly lower IGF-I levels on D1 and also on D15 compared to healthy controls. Our finding of low IGF-I levels is compatible with other studies. Casaburi et al have also reported low levels of IGF-I in COPD patients \[[@B30]\]. Spruit et al found that IGF-I levels tended to be lower in patients with COPD exacerbation than in healthy subjects \[[@B12]\].
We are aware that the bioavailability and the effects of IGF-I are influenced by IGF-I binding proteins (IGFBP). Therefore, an increase in circulating levels of these proteins may decrease the levels of free IGF-I \[[@B31]\]. Cytokines increase IGFBP-1 and IGFBP-4 and this results in a decreased free IGF-I fraction. IL-6 performs its suppressive activity on IGF-I via increased production of its binding protein \[[@B32]\].
It is interesting to note that IGF-I levels were lower in patients with emphysema compared to those with chronic bronchitis, both at the onset of the exacerbation and 15 days later, whereas cytokine and leptin levels did not differ between the two subgroups. The decline in IGF-I levels may itself be deleterious. IGF-I mRNA levels were decreased in muscle biopsies from hospitalized patients due to an acute exacerbation of COPD \[[@B13]\]. Moreover, it has been demonstrated that pulmonary rehabilitation leads to an increase in muscle mRNA expression of IGF-I in COPD patients \[[@B33]\]. COPD patients often present with hypoxia during exacerbations. In animal studies, recombinant IGF-I has been shown to ameliorate the protein catabolism observed under hypoxic conditions and to promote anabolism \[[@B14]\]. The decreased levels of this anabolic factor, especially in emphysematous patients further enhance its possible role in metabolic derangements of COPD and mainly emphysema. The lower body weight, usually observed in patients with emphysema may be related to the lower IGF-I levels found in these patients, particularly during exacerbations. Low levels of anabolic hormones could act synergistically with the catabolic activity of cytokines and leptin and play a role in the weight loss and decreased muscular mass usually seen in COPD patients and mainly those with emphysema. This may also have important therapeutic implications, since an increase in anabolic factors in COPD patients, particularly during exacerbations may have a protective metabolic effect.
Conclusion
==========
During an acute exacerbation of COPD, elevated levels of the proinflammatory cytokines TNF-α, IL-1β, IL-6 and IL-8 and increased levels of leptin and decreased levels of IGF-I are observed. It seems that, although systemic inflammation, in terms of cytokines, is restored relatively quickly, the coexistent disturbance of leptin and IGF-I is preserved for a longer period of time.
Lower levels of the anabolic factor IGF-I are observed in emphysema compared to chronic bronchitis and this may be related to the more pronounced metabolic derangements observed in this subgroup. Further studies are needed to elucidate the role of increased leptin and decreased IGF-I levels during COPD exacerbations and their possible relationship with energy imbalance observed in these patients.
Competing interests
===================
The authors declare that they have no competing interests.
Authors\' contributions
=======================
PK and PB conceived of the study, and participated in its design and coordination and drafted the manuscript. AK, AH and EA carried out the immunoassays. SA contributed to the acquisition of the data and the statistical analysis. AR participated in its design and drafted the manuscript. All authors read and approved the final manuscript.
Pre-publication history
=======================
The pre-publication history for this paper can be accessed here:
<http://www.biomedcentral.com/1471-2466/9/11/prepub>
|
{
"pile_set_name": "PubMed Central"
}
|
{#sp1 .200}
|
{
"pile_set_name": "PubMed Central"
}
|
**Core tip:** Concomitant therapy is one of the most effective first line regimen for *Helicobacter pylori* eradication. The comparison with other regimens (sequential or triple) in a selected geographical region (China in this case) implies several issues. The low number of included studies, the lack of quality evaluation and the high heterogeneity may undermine the strength of a meta-analysis. Therefore, further studies are needed to prove which is the best first line eradication treatment in China, according to the geographical differences in antibiotic resistances.
TO THE EDITOR
=============
We read with interest the recent meta-analysis by Lin et al\[[@B1]\], who evaluated the effectiveness of concomitant regimen for *Helicobacter pylori* (*H. pylori*) in Chinese regions in a first line context. The choice of deal with this eradication treatment in a selected geographical area is absolutely appropriate, since, as we have already demonstrated\[[@B2]\], a single therapeutic approach is not fitting worldwide. Indeed, geographical variations in antibiotic resistance rates strongly influence the outcome, therefore the most proper regimen should be considered on the basis of the antibiotic resistance pattern in each area.
However, the results of this meta-analysis deserve special considerations. First, three studies compared 7-d concomitant versus 7-d conventional triple therapy, demonstrating that concomitant regimen is significantly superior to 7-d triple therapy (91.2% *vs* 77.9%, *P* \< 0.0001). However, it is a common belief that a triple therapy lasting 7 d should be definitively abandoned, due to the disappointingly low success rate. Currently, only the prolongation of triple therapy to 14 d can be advised to overcome this limit\[[@B3],[@B4]\]. Moreover, the outcome of a meta-analysis should be the comparison of two regimens that may have a similar effectiveness; the assessment of an old and outdated treatment versus a more recent and successful one could not bring novel and useful information. The search strategy of this systematic review did not retrieve trials evaluating concomitant versus 14-d triple therapy in China, but only this comparison could state whether concomitant is really more effective than triple therapy. Furthermore, the duration of 7 d for concomitant therapy is not considered optimal, since Guidelines recommend a 10-d regimen\[[@B3],[@B5]\]. Additionally, the comparison between 10-d concomitant and 10-d triple therapy showed a similar success, even if this analysis was based only on one study and this aspect could cause to judge the result questionable.
Another issue involves the comparison of concomitant versus sequential regimen. Herein, it has been demonstrated a similar eradication rate (86.9% and 86% respectively, *P* = 0.69). This finding is in agreement with other meta-analyses\[[@B6],[@B7]\] and previous experiences in Western countries\[[@B8],[@B9]\]. However, it is common opinion that sequential therapy is more prone to fail when metronidazole resistance occurs\[[@B10]\]. Indeed, Georgopoulos et al\[[@B11]\] showed that the success rate of sequential regimen decreased from 89.8% to 70%, while a reduction of only 2% occurred for concomitant regimen in the presence of metronidazole resistance. This observation does not seem to agree with the results of this meta-analysis, since China and Far East are considered as high metronidazole resistance areas\[[@B2]\]. Therefore, concomitant therapy would be expected to achieve a higher success rate than sequential therapy. Furthermore, in order to support this consideration, a recent trial in Korea showed eradication rates of 77.8% for concomitant and 70.6% for sequential regimens at intention-to-treat analysis\[[@B12]\]. This controversial aspect could be explained only by the analysis of metronidazole resistance in treated patients. On the other hand, Authors themselves observed that this issue was not investigated in included trials.
Finally, we believe that the lack of quality assessment and the high heterogeneity of included studies constitute a relevant limit to draw solid conclusions in this meta-analysis. Therefore, further studies and the knowledge of antibiotic resistance pattern will support with high quality evidence the assessment of the best regimen and its optimal duration\[[@B13]-[@B17]\].
Manuscript source: Unsolicited manuscript
Specialty type: Gastroenterology and hepatology
Country of origin: Italy
Peer-review report classification
Grade A (Excellent): A, A
Grade B (Very good): B, B
Grade C (Good): C
Grade D (Fair): 0
Grade E (Poor): 0
Conflict-of-interest statement: No potential conflicts of interest relevant to this article were reported.
Peer-review started: July 13, 2016
First decision: August 19, 2016
Article in press: September 12, 2016
P- Reviewer: Abadi ATB, Hori K, Lakatos PL, Tepes B, Zamani M S- Editor: Qi Y L- Editor: A E- Editor: Zhang FF
[^1]: Author contributions: Losurdo G, Di Leo A and Ierardi E conceived the article; Losurdo G and Ierardi E wrote the article; Giorgio F, Iannone A, Principi M and Barone M collected the data; all Authors read and approved the final version of the manuscript.
Correspondence to: Dr. Enzo Ierardi, Professor, Gastroenterology, Department of Emergency and Organ Transplantation, University of Bari, Piazza Giulio Cesare 11, 70124 Bari, Italy. <[email protected]>
Telephone: +39-80-5594034 Fax: +39-80-5593088
|
{
"pile_set_name": "PubMed Central"
}
|
Introduction
============
Ultrasound-based elastography is one of the most remarkable advances in the field of medical ultrasonography in recent decades, and it has greatly expanded the utility of ultrasonography in clinical practice. The biophysical information provided by ultrasonic elastography has led to its widespread use in the management of various diseases involving tissue stiffness, such as solid tumors, fibrosis associated with liver cirrhosis, and atheromas and calcifications associated with arteriosclerosis \[[@b1-usg-16026]\]. In 2015, the World Federation for Ultrasound in Medicine and Biology released guidelines and recommendations for the clinical use of ultrasound-based elastography, with a special focus on chronic liver disease (CLD) as an example of diffuse disease and on breast tumors as examples of focal lesions \[[@b2-usg-16026],[@b3-usg-16026]\]. Acoustic radiation force impulse (ARFI) imaging is an emerging technique with great promise in the field of ultrasonic elastography.
In ARFI imaging, an ultrasound probe creates a focused "push" pulse, which can induce tissue deformation in the region of interest (ROI). As a result, shear waves are generated and propagate away from the ROI. The same transducer is then used to monitor the propagation of the shear waves by radiofrequency-echo tracking, and ultimately displays their velocity (m/sec). Assuming that a given tissue displays simple physical behavior (i.e., linear, isotropic, and homogeneous), the speed of shear waves is related to the underlying stiffness of the material. Specifically, shear waves propagate faster through stiffer tissue.
ARFI imaging has many advantages over previously used strain elastography techniques. First, since the acoustic radiation force pulses automatically induce tissue displacement, ARFI imaging is not operator-dependent and can determine tissue elasticity quantitatively, whereas strain elastography can only determine qualitative and relative elasticity. Moreover, ARFI imaging does not rely on transducer compression, as strain elastography does, and it can therefore be applied to visualize deep organs, for which it is difficult to generate sufficient deformation via compression from the surface of the body.
Due to these remarkably promising characteristics, multiple studies have been carried out over the last decade to explore the application of ARFI imaging in kidney disease. With study populations ranging from healthy individuals to allograft patients, many promising results have been published, which have encouraged clinicians and researchers to explore the role of ARFI in chronic kidney disease (CKD). ARFI quantification as a method of estimating fibrosis has been validated in patients with CLD. As in CLD, fibrosis is the principal process underlying the progression of CKD, which displays a relatively uniform response involving glomerulosclerosis, tubular atrophy, interstitial fibrosis, and changes in the renal vasculature \[[@b4-usg-16026]\].
CKD is defined as abnormalities of kidney structure or function present for 3 months with implications for health. CKD comprises a group of pathologies, and is a progressive and irreversible syndrome that starts asymptomatically, continues through renal dysfunction, and ultimately leads to renal failure \[[@b5-usg-16026]\]. The incidence of CKD has increased significantly over recent decades, and its prevalence is estimated to be 8%-16% worldwide \[[@b6-usg-16026]\]. CKD has recently emerged as an increasingly significant public health issue. In particular, the risk of cardiovascular disease is notably greater in individuals with CKD. Diabetes and hypertension are the leading causes of CKD, although autoimmunity, atherosclerosis, infections, drugs and toxins, obstruction of the urinary tract, genetic defects, and other factors may initiate the disease \[[@b6-usg-16026]\].
To date, several studies on ARFI quantification in CKD have been published ([Table 1](#t1-usg-16026){ref-type="table"}) \[[@b7-usg-16026]-[@b18-usg-16026]\]. The aim of the present review was to identify points of agreement and disagreement on this topic and to outline possible avenues of future research.
Procedure of ARFI Quantification
================================
[Table 1](#t1-usg-16026){ref-type="table"} summarizes previously published studies on ARFI quantification in CKD patients. Variations in the procedure of ARFI quantification were present among the studies, but the following commonalities were nonetheless observed ([Fig. 1](#f1-usg-16026){ref-type="fig"}): (1) the kidney was displayed on the longitudinal plane to identify the intrarenal structures of the cortex, medulla, and sinus; (2) the sample volume (ROI) was located exclusively in the cortical region; (3) the sample line was radially oriented; (4) the applied transducer compression was minimized as much as possible; (5) patients were asked to hold their breath during the measurements; and (6) shear-wave velocity (SWV) measurements were repeated 5-10 times.
The above requirements had the goal of reducing the impact of renal anisotropy and other technical factors affecting the quality of the measurements, and thereby improving the reproducibility of SWV measurements. Unfortunately, these requirements appeared insufficient for achieving this goal. Two important factors should not be ignored in the standardization of ARFI quantification. First, although the sample volume was located exclusively in the cortical region, the ROI was allowed to encompass any part of the kidney (i.e., the upper, middle, or lower third). However, this may lead to violations of the requirement for the sample line to be radially oriented, particularly when sampling the renal cortex at the upper and lower parts of the kidney. Radial sampling has the goal of avoiding the effects of the anisotropic properties of the kidney, as non-radial orientations are susceptible to renal anisotropy, which may compromise the reproducibility of the measurements. Second, the position of the patient is an often-underestimated factor influencing the reproducibility of the measurements. In B-mode scans, patients must be turned with varying degrees of obliquity in order to obtain complete images of both kidneys. The literature on ARFI quantification indicates that multiple positions have been chosen, including the supine, prone, and lateral decubitus positions. The kidney is a deeply located retroperitoneal organ and is surrounded by anatomic structures with different depths from the renal capsule to the surface of the body. Therefore, different positions are associated with variations in scanning depth. Moreover, certain positions may require unrealistically large levels of compression in order to obtain clear images, especially in patients with a high body mass. Depth and compression have been proven to have a significant impact on SWV values \[[@b19-usg-16026],[@b20-usg-16026]\]. Therefore, further investigation is warranted in order to develop recommendations regarding the choice of patient position.
ARFI Quantification in Pediatric CKD
====================================
The etiological spectrum of CKD is different in children and adults. Some diseases are very important in pediatric nephrology, but are much less frequently seen in adult patients, such as vesicoureteral reflux (VUR). VUR is the second most common cause of pediatric CKD, accounting for nearly a quarter of pediatric CKD patients \[[@b21-usg-16026]\]. In children with VUR, the kidneys are exposed to the risk of a urinary tract infection, which may lead to renal parenchymal damage and ultimately to the development of hypertension and chronic renal failure \[[@b22-usg-16026]\].
The first study of ARFI quantification in VUR was presented by Bruno et al. \[[@b7-usg-16026]\]. Twenty-eight children (age range, 9 to 16 years) with high-grade VUR and 16 age-matched healthy controls were included in the study, and all underwent ARFI quantification and scintigraphy. They noted that the highest ARFI values were observed in kidneys with secondary VUR, followed by kidneys with primary reflux, unaffected kidneys contralateral to kidneys experiencing reflux, and normal kidneys. The authors hypothesized that the fibrotic damage in CKD induced by VUR gave rise to the high SWV values observed in this series. Their preliminary results illustrate the possibility of including elastography in renal diagnostic imaging protocols.
However, in a recent study including 88 children with VUR, contradictory findings were reported. Goya et al. \[[@b15-usg-16026]\] found that the SWV values observed in the renal cortex decreased with increasing VUR severity and scintigraphy-assessed renal damage. Significantly higher SWV values were found in non-damaged kidneys, whereas severely damaged kidneys and high-grade refluxing kidneys had the lowest SWV values. Significant negative correlations were found between SWV values and both the renal scarring score and the VUR grade. The authors did not explain the disagreement with the previous study. These two studies employed a similar research design and ARFI imaging procedures but obtained completely opposite findings. Further investigation remains necessary.
ARFI Quantification in Adult CKD
================================
Although CKD is a heterogeneous disease that consists of a group of various pathologies, the features common to CKD by definition include a progressive decrease in the glomerular filtration rate (GFR) and histological changes. Therefore, in studies of ARFI quantification in CKD, the primary concern should be to clarify the relationships of ARFI with renal dysfunction (CKD stage) and renal damage (pathologic alterations).
Three relevant studies have explored the correlations of SWV values with pathologic alterations but did not agree with each other. In a study of 163 adult patients, Hu et al. \[[@b10-usg-16026]\] reported that the mean SWV in kidneys with severe histologic impairment was significantly lower than in kidneys with mild histologic impairment, moderate histologic impairment, and controls. Additionally, SWV measurements showed a moderate negative correlation with pathologic parameters (r=-0.422 to -0.511). Cui et al. \[[@b12-usg-16026]\] reported that the mean SWV values found in the subcapsular parenchyma of kidneys with various percentages of renal fibrosis (\<25%, 25%-50%, \>50%) were higher than those of non-fibrotic kidneys, and that SWV values showed a positive linear correlation with fibrosis. In another CKD study that focused primarily on immunoglobulin A nephropathy (approximately 70% of subjects), Wang et al. \[[@b11-usg-16026]\] found that SWV values did not show any significant correlations with renal fibrosis.
The definitive reason underlying these discrepancies remains unknown. However, these three studies employed different pathologic scoring systems. Hu et al. \[[@b10-usg-16026]\] used a much more complicated scoring system than the other two studies, while Cui et al. \[[@b12-usg-16026]\] assessed pathologic alterations of the kidney using only the parameter of fibrosis percentage. This difference may have contributed to the discrepancy in their findings. In a renal transplant study, Grenier et al. \[[@b23-usg-16026]\] reported that renal cortical stiffness was not correlated with any single pathologic score or the level of interstitial fibrosis, although a significant correlation was observed between cortical stiffness and global histological deterioration. Furthermore, considering the complex pathophysiology of CKD, any correlation between cortical SWV values and renal histological alterations may be subject to various influencing factors. In an animal study, Warner et al. \[[@b24-usg-16026]\] found that in a 6-week chronic renal arterial stenosis model, cortical stiffness was not significantly different in the treatment group and in the control group, despite histologic evidence of renal tissue fibrosis. They concluded that hemodynamic variables modulate kidney stiffness and may mask the presence of fibrosis.
In contrast, despite failing to predict pathologic alterations, ARFI quantification has been shown to have a significant correlation with GFR and CKD stage in several studies. In 2013, Guo et al. \[[@b8-usg-16026]\] evaluated 64 adult CKD patients and reported lower SWV values in the renal parenchyma than were observed in 327 control subjects. In particular, a progressive reduction of SWV values was unexpectedly observed to correlate with increasing stages of CKD. Interestingly, this unexpected trend has also been reported in most subsequent studies \[[@b9-usg-16026],[@b10-usg-16026],[@b16-usg-16026],[@b17-usg-16026]\]. The only exception is the study carried out by Wang et al. \[[@b11-usg-16026]\], in which no relation was found between SWV values and GFR or CKD stage. In contrast to CLD, kidneys with more advanced CKD tend to present lower cortical SWV values than are observed in early-stage disease. Many authors have suggested that hemodynamic alterations of the kidney may affect renal parenchymal elasticity during the progression of CKD.
In ARFI quantification for CLD, SWV values increase in more advanced stages of the disease as progressive interstitial fibrosis affects tissue elasticity. However, the dominant factor affecting ARFI quantification in CKD has not been clearly elucidated. The kidney is an organ with rich perfusion, receiving 20% of cardiac output despite only constituting \<1% of body mass \[[@b25-usg-16026]\]. Its distension and stiffness may therefore be significantly affected by renal blood perfusion. Under normal conditions, in order to provide the large quantity of oxygen necessary to support massive levels of reabsorption, the renal tubules are surrounded by a dense vascular plexus in both the cortex and medulla \[[@b25-usg-16026]\]. As CKD progresses, glomerular sclerosis, tubular atrophy, and peritubular fibrosis gradually become aggravated. Accordingly, the blood flow in the peritubular vascular plexus decreases. Considering the remarkable extent of damage in the microcirculation in advanced CKD, it is conceivable that renal blood flow may significantly influence SWV values, perhaps even more than interstitial fibrosis.
Comparison of ARFI Quantification in CKD and CLD
================================================
Although ARFI quantification is a reliable indicator of the severity of liver fibrosis in CLD, ARFI measurements in CKD remain quite controversial, and previous studies have reported variable results. These conflicting findings may be, to some extent, due to anatomical and physiological differences between the liver and kidney.
Internal Architecture
---------------------
In contrast to the homogeneous echotexture of the liver, the kidney exhibits complex acoustic characteristics resulting from its heterogeneous histological structure, which consists of glomeruli, tubules, and stromal components. In addition, the regular arrangement of the internal tubular system gives rise to the anisotropic property of the kidney, whereas liver tissue is isotropic. In preclinical research, Gennisson et al. \[[@b26-usg-16026]\] found that cortical elasticity values were always higher when acquisitions were performed with the ultrasound main axis perpendicular to the main pyramid axis than when parallel, demonstrating the effect of renal anisotropy on renal stiffness.
Blood Supply
------------
As stated above, the kidney is a richly perfused organ with a high perfusion pressure. In contrast, despite possessing a dual-source blood supply, the liver is mainly perfused by the low-pressure portal vein. Interestingly, under normal conditions, SWV values in the renal cortex are approximately twice as high as those obtained in hepatic tissue \[[@b27-usg-16026]\], which might be partially attributable to their different perfusion patterns. Thus, as disease progresses, diminished blood perfusion may influence tissue stiffness more strongly in CKD than in CLD.
Age-Related Changes
-------------------
In studies of both children and adults, the liver did not show significant changes in tissue elasticity according to age \[[@b28-usg-16026],[@b29-usg-16026]\]. However, distinct changes in renal cortical stiffness with age have been reported in both children and adults. Lee et al. \[[@b29-usg-16026]\] found that cortical SWV values for the kidney increased with age in children under 5 years old. In contrast, Bota et al. \[[@b20-usg-16026]\] reported that renal SWV values decreased with age in adults (18-30 years, 2.94±0.60 m/sec; 31-50 years, 2.26±0.82 m/sec; 51-65 years, 2.48±0.8 m/sec; \>65 years, 1.82±0.63 m/sec). In a correlation analysis, age was negatively correlated with renal SWV values as assessed by ARFI quantification (r=-0.370).
Future Prospects
================
Considering the controversies regarding ARFI quantification in CKD, further studies are urgently necessary in order to clarify several important issues.
First, the mechanism underlying the impact of renal hemody-namics on tissue elasticity remains unknown. Asano et al. \[[@b9-usg-16026]\] reported that SWV values decreased concurrently with decreases in the estimated GFR. Meanwhile, low SWV values were obtained in patients with a high brachial-ankle pulse wave velocity. Since a high brachial-ankle pulse wave velocity represents the progression of arteriosclerosis in the large vessels, the authors concluded that the reduction of elasticity subsequent to the diminution of blood flow was likely to be the factor most strongly influencing the SWV in the kidneys.
Moreover, etiology-specific applications of ARFI quantification may be more valuable. For example, in a study of diabetic nephropathy, Goya et al. \[[@b16-usg-16026]\] found SWV values for the kidneys of 2.87 m/sec, 3.14 m/sec, 2.95 m/sec, 2.68 m/sec, and 2.55 m/sec in patients with stage 1, 2, 3, 4, and 5 diabetic nephropathy, respectively, in comparison with 2.35 m/sec for healthy controls. Using 2.43 m/sec as the threshold value for predicting diabetic nephropathy, they obtained diagnostic sensitivity and specificity values of 84.1% and 67.3%, respectively.
Finally, the natural course of renal SWV in the normal population is not fully understood. Age has been well established as a confounding factor for ARFI quantification in CKD, but it remains unclear to what extent age can mask the presence and progression of pathologic alterations in CKD patients. As an extreme example, Takata et al. \[[@b18-usg-16026]\] reported that no differences were identified between age-matched non-CKD controls and end-stage renal disease patients, reflecting the non-negligible impact of aging on kidney elasticity. Thus, age-adjustment of renal elasticity may be necessary in order to apply ARFI quantification to CKD.
Conclusion
==========
In summary, the quantification of tissue stiffness using ARFI is more complex for the kidney than the liver. It should be studied more both in animal models and in clinical patients, in order to better understand the pathophysiological mechanisms underlying renal elasticity. In addition, not all previous studies are comparable because they used different procedures \[[@b30-usg-16026]\]. Therefore, further research is warranted to improve this technique and to develop standardized guidelines.
No potential conflict of interest relevant to this article was reported.
{#f1-usg-16026}
######
Summary of previous studies on ARFI quantification in CKD
Study Patient information Renal disease ARFI quantification Conclusion
------------------------------------------- --------------------- -------------------------------------- --------------------- ---------------------------------------------------------------------------------------------------------- -------------------------------------------- ------------------------------------------ ----------------------------------- ------------------------------ ----- -------------------- -------------------------------------------------------- ----- ------------------- -------------------------------------------------------------------------------------------
Bruno et al. (2013) \[[@b7-usg-16026]\] 28 Children (12.1, 9-16 yr) 17:11 Vesicoureteral reflux (≥grade III) NA Scintigraphy Acuson S2000 with 4-MHz probe Supine Yes Longitudinal plane Cortex at upper, middle, lower third/radial 9 NA/NA SWV values increased in kidneys with vesicoureteral reflux
Guo et al. (2013) \[[@b8-usg-16026]\] 64 Adults (64.7, 23-89 yr) 37:27 CKD with unknown etiology I (11), II (11), III (20), IV (10), V (12) Serum markers Acuson S2000 with 1-4-MHz probe Prone NA Longitudinal plane Parenchyma (cortex and medulla) at middle third/NA 5 Positive/NA NA
Asano et al. (2014) \[[@b9-usg-16026]\] 319 Adults (62.0, 17-93 yr) 198:121 Glomerulonephritis (129), diabetic nephropathy (107), nephrosclerosis (83) I (29), II (59), III (99), IV (66), V (66) Biopsy, serum markers Acuson S2000 with 3.5-MHz probe Prone Yes Longitudinal plane Cortex at lower third/NA 5-6 Positive/NA NA
Hu et al. (2014) \[[@b10-usg-16026]\] 163 Adults (41.3, 18-79 yr) 91:72 Glomerulonephritis (143), diabetic nephropathy (13), interstitial disease (5), nephrosclerosis (2) NA Biopsy, serum markers Acuson S2000 with 1-4-MHz probe Lateral decubitus Yes Longitudinal plane Cortex at middle third/NA 10 Positive/negative NA
Wang et al. (2014) \[[@b11-usg-16026]\] 45 Adults (37.1, 18-72 yr) 23:22 Glomerulonephritis (36), nephrosclerosis (6), diabetic nephropathy (3) I (26), II (7), III (6), IV (6) Biopsy, serum markers Acuson S2000 with 1-4-MHz probe Lateral decubitus Yes Longitudinal plane Cortex at middle third/radial 15 No/No NA
Cui et al. (2014) \[[@b12-usg-16026]\] 76 Children and adults (40.4, 11-75 yr) 43:33 CKD with unknown etiology NA Biopsy Acuson S2000 with 2-5-MHz probe Prone Yes Longitudinal plane NA/radial 5 NA/positive NA
Sohn et al. (2014) \[[@b13-usg-16026]\] 30 Young children (4.5, 0-23 mo) 25:5 Hydronephrosis NA Scintigraphy, voiding cystourethrography Acuson S2000 with 4-9-MHz probe NA No Axial plane Parenchyma (cortex and medulla) at middle third/radial 3 NA/NA SWV values increased in kidneys with high-grade hydronephrosis
Yu et al. (2014) \[[@b14-usg-16026]\] 120 Adults (50, 32-68 yr) 66:54 Diabetic nephropathy NA Serum and urinary markers Acuson S2000 with 1-4-MHz probe NA Yes Longitudinal plane Cortex at middle third/radial 3 NA/NA SWV values increased in kidneys with diabetic nephropathy
Goya et al. (2015) \[[@b15-usg-16026]\] 88 Children (5.6, 1-17 yr) 46:42 Vesicoureteral reflux (grade I to V) NA Scintigraphy, voiding cystourethrography Acuson S2000 with 1-4.5-MHz probe Lateral decubitus Yes Longitudinal plane Cortex at upper, middle, lower third/NA 15 NA/NA SWV values decreased in kidneys with vesicoureteral reflux and DMSA-assessed renal damage
Goya et al. (2015) \[[@b16-usg-16026]\] 114 Adults (57, 23-89 yr) 67:47 Diabetic nephropathy I (42), II (28), III (13), IV (16), V (14) Serum and urinary markers Acuson S2000 with 1-4-MHz probe Supine and lateral decubitus Yes Longitudinal plane Cortex at upper, middle, lower third/NA 15 Positive/NA SWV values increased in kidneys with diabetic nephropathy
Bob et al. (2015) \[[@b17-usg-16026]\] 46 Adults 28:18 Diabetic nephropathy (16), nephrosclerosis (12), glomerulonephritis (9), pyelonephritis (3), unknown (6) NA Serum markers Acuson S2000 with 4-9-MHz probe Lateral decubitus Yes Longitudinal plane Parenchyma (cortex and medulla) at middle third/radial 5 Positive/NA NA
Takata et al. (2015) \[[@b18-usg-16026]\] 39 Adults (72, 38-86 yr) 25:14 End-stage renal disease NA Serum markers Acuson S2000 with 1-4-MHz probe NA Yes NA NA/radial 10 NA/NA SWV values were not associated with advanced renal impairment
ARFI, acoustic radiation force impulse; CKD, chronic kidney disease; ROI, region of interest; SWV, shear-wave velocity; GFR, glomerular filtration rate; NA, not available; DMSA, dimercaptosuccinic acid.
|
{
"pile_set_name": "PubMed Central"
}
|
Introduction
============
Lung cancer is an aggressive and heterogeneous malignancy, and one of the leading causes of cancer-associated mortalities in men throughout the world ([@b1-ol-0-0-4779]). Although lung cancers are treated by surgical, radiotherapeutic and chemotherapeutic approaches, the long-term survival rate is still not satisfactory ([@b1-ol-0-0-4779],[@b2-ol-0-0-4779]). Therefore, novel approaches/compounds are still required to increase the success of treatment.
The use of medicinal plants for the treatment of human diseases begins with the history of humanity, and represents the oldest and most widespread form of medication due to their healing properties ([@b3-ol-0-0-4779],[@b4-ol-0-0-4779]). Several types of plants are important as a source of effective anticancer agents, which indicates their therapeutic values ([@b5-ol-0-0-4779]). *Pelargonium* species are widely used as traditional remedies for several diseases in Southern Africa ([@b6-ol-0-0-4779]). A number of herbs belonging to the genus *Pelargonium* possess numerous properties that have medicinal importance. For example, 'Umckaloabo', ethanol extracts of the root of two species of *Pelargonium* (*P. sidoides* and *P. reniforme*), have been used in gastrointestinal, hepatic and respiratory diseases as a herbal remedy ([@b7-ol-0-0-4779]). *Pelargonium* species are rich in essential oils that are the source of their therapeutic value ([@b8-ol-0-0-4779],[@b9-ol-0-0-4779]). Among these, Geranium monoterpene oils exhibit anti-bacterial, antifungal antioxidant, insecticidal, anthelmintic and anticancer activity ([@b7-ol-0-0-4779]--[@b13-ol-0-0-4779]). There are also several *Pelargonium* species used in folk medicine that have been screened for their biological activities ([Table I](#tI-ol-0-0-4779){ref-type="table"}) ([@b8-ol-0-0-4779],[@b12-ol-0-0-4779],[@b13-ol-0-0-4779],[@b14-ol-0-0-4779]--[@b26-ol-0-0-4779]).
*Pelargonium quercetorum* Agnew (*P. quercetorum*) belongs to Geraniaceae family, and is known as Tolk in Gecitli, in the Hakkari province of Turkey ([@b27-ol-0-0-4779]). Kaval *et al* ([@b27-ol-0-0-4779]) reported that native people have used this plant to treat intestinal worms, and that study was the first report about the traditional use of *P. quercetorum*. However, to the best of our knowledge, there is no information regarding the cytotoxic activity of *P. quercetorum* against lung cancer cell lines in the literature.
In the present study, the anti-growth/cytotoxic effects of the methanol extract of *P. quercetorum* against non-small cell lung cancer cell lines (A549, PC3 and H1299) were investigated. The results demonstrated that *P. quercetorum* had cytotoxic activity in a dose-dependent manner, and resulted in an incomplete apoptosis, implying the requirement of further *in vivo* experiments for the elucidation of its cell death mechanism.
Materials and methods
=====================
### Plant material
The whole plant of *P. quercetorum* was collected from the Zap Valley at Sumbul Mountain in the Hakkari province of Turkey, located in the C10 square according to the Turkey\'s grid square system ([@b28-ol-0-0-4779]) in May 2006 by Mr. Mehmet Firat (Department of Biology, Yuzuncu Yil University, Van, Turkey). The specimen was identified using the standard text 'Flora of Turkey and the East Aegean lslands' ([@b29-ol-0-0-4779]). A voucher specimen (number MF.10111) was deposited in the Hacettepe University Herbarium (Ankara, Turkey).
### Extraction of P. quercetorum sample
The sample was air-dried at room temperature, cleaned of extraneous materials and then grounded into powder. A total of 15 g of the material (trunk and flower parts) was extracted by adding 150 ml of solvent methanol (Merck Millipore, Darmstadt, Germany) in a Soxhlet apparatus for 24 h, and the crude extract was concentrated in a rotary evaporator at 40°C. The residues were lyophilized and stored at −80°C until used.
### Determination of P. quercetorum chemical compounds
#### Direct thermal desorption (DTD)
To evaluate the volatile compounds present in *P. quercetorum*, DTD followed by analysis with comprehensive two-dimensional gas chromatography-time-of-flight/mass spectrometry (GCxGC-TOF/MS) was performed. The plant was directly loaded into the system. A GCxGC-TOF/MS system was used together with a dual stage commercial thermal desorption injector, which incorporated a thermal desorption unit (TDU) connected to a programmable-temperature vaporization injector \[Cooled Injection System (CIS)-4 Plus; Gerstel, Mülheim an der Ruhr, Germany\], using a heated transfer line. The injector was equipped with a MultiPurpose Sample (Gerstel). Empty glass thermodesorption tubes were conditioned at 400°C for 2 h prior to each use. Approximately 20--30 mg sample was placed into the thermodesorption tubes using tweezers to ensure no contamination of the sample. The initial desorption of the sample was conducted by heating the TDU from 40°C (initial time, 0.2 min) to 150°C at a rate of 120°C/min with a final hold time of 10 min under a helium flow of 1.5 ml/min in splitless mode. Volatile analytes released from this heating were cryo-focused at −40°C in the CIS, which was cooled with liquid nitrogen prior to injection. The CIS was then heated at a rate of 10°C/sec to a final temperature of 150°C. Analytes were transferred splitless to the GC column during the CIS temperature ramp.
#### Chromatographic analysis
The GCxGC-TOF/MS system consisted of a 6890 GC (Agilent Technologies, Inc., Santa Clara, CA, USA) and a Pegasus III TOF-MS system (LECO Corporation, Saint Joseph, MI, USA). The modulator between the first and second GC columns was based on a LECO liquid nitrogen two-stage cold jet system. Helium was used as a carrier gas at a constant flow of 1.0 ml/min. The first column was a non-polar BPX5 (30 mx0.32 mm i.d. ×0.25 µm film thickness), while the second column was a BPX50 (1.5 mx0.10 mm i.d. ×0.10 µm film thickness), both from SGE Analytical Science (Victoria, Australia). The combination of separations produced the overall two-dimensional chromatogram. Peak identification was performed using TOF/MS with electron ionization.
### Determination of cytotoxic activity
#### Cell culture and chemicals
Non-small cell lung cancer cell lines A549, H1299 (Dr Donner, Walther Oncology Center, Indiana University School of Medicine, Indianapolis, IN, USA) and PC3 (Dr Yokota, National Cancer Center Research Institute, Division of Genome Biology, Tokyo, Japan) were cultured in RPMI-1640 (Lonza Bioscience, Verviers, Belgium) medium supplemented with L-glutamine (Gibco^®^; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 10% fetal bovine serum (Lonza Bioscience), penicillin G (100 U/ml) and streptomycin (100 µg/ml) (HyClone; GE Healthcare Life Sciences, Logan, UT, USA) at 37°C in a humidified atmosphere containing 5% CO~2~. According to the American Type Culture Collection (Manassas, VA, USA), PC3 is often known as a prostate cancer cell line (CRL1435), but in the present study, it represents a non-small cell lung cancer cell line derived from the Japanese Collection Research Resources Bank (Osaka, Japan; JCRB, JCRB0077).
The lyophilized *P. quercetorum* extract (PQE) was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA) at a concentration of 100 mg/ml as a stock solution, aliquoted and stored at −80°C. PQE was used at different concentrations ranging from 3.13 to 100 µg/ml, and the dilutions were made in culture medium.
#### Adenosine triphosphate (ATP) assay
The ATP assay, a highly sensitive luciferin:luciferase-based assay, was performed to determine the level of cellular ATP as an indirect marker of the number of alive cells present in the sample ([@b30-ol-0-0-4779]). A549, PC3 and H1299 cells were seeded at a density of 1×104 cells/well in a 96-well plate in 200 µl medium. Cells were incubated either alone (as control) or in the presence of PQE for 48 h. The untreated/control cells received vehicle only (0.1% DMSO) without any drugs (which represented the maximum viability). Each experiment was conducted at least twice in triplicates. At the end of the treatment period (48 h), cell viability was determined by ATP assay with an ATP bioluminescent somatic cell assay kit (Sigma-Aldrich), according to the manufacturer\'s protocol with a slight modification, as explained previously ([@b31-ol-0-0-4779]). Morphological changes of cells were also observed under a phase-contrast microscope (CKX41; Olympus Corporation Tokyo, Japan).
### Determination of cell death mode
#### Annexin-V-fluorescein isothiocyanate (FITC) fluorescence imaging for apoptosis
The translocation of phosphatidylserine (PS) molecules from the inner to the outer side of the cell membrane is one of the earlier events of apoptosis ([@b32-ol-0-0-4779]). Annexin-V-FITC is able to bind to PS, thus allowing the apoptotic cells to become visible. Propidium iodide (PI) is normally used as a second dye to distinguish between early and late apoptosis as well as necrosis ([@b33-ol-0-0-4779]). In addition, a nucleus-staining fluorescent dye, Hoechst 33342 (200 µg/ml, 1:40; AppliChem GmbH, Darmstadt, Germany) was used in the present study to detect apoptosis on the basis of nuclear morphology. Hoechst 33342 dye stains all types of cells (alive and dead) ([@b34-ol-0-0-4779]). While early apoptotic cells are considered only Annexin-V-FITC-positive, late apoptotic cells (or secondary necrotic cells) are considered both Annexin-V-FITC- and PI-positive, with the presence of pyknotic nuclei and/or condensed chromatin ([@b33-ol-0-0-4779],[@b34-ol-0-0-4779]). Briefly, A549, PC3 and H1299 cells were seeded in a 96-well plate at a density of 1×104 cells/well, and treated for 12 and 24 h with PQE at a dose of 100 µg/ml. Upon treatment, cells were stained with Annexin-V-FITC and PI using the Annexin-V-FLUOS Staining kit (Roche Diagnostics GmbH, Mannheim, Germany), according to the manufacturer\'s protocol. The cells were then visualized under a fluorescence microscope.
#### M30 and M65 assays
Intact cytokeratin 18 (CK18, also known as M65) and caspase-cleaved CK18 (also known as M30) were measured using M30-Apoptosense^®^ and M65 EpiDeath^®^ enzyme-linked immunosorbent assay (ELISA) kits (Vivalavida AB, Nacka, Sweden). The M30-Apoptosense^®^ ELISA kit measures the levels of M30 produced during apoptosis, while the M65 EpiDeath^®^ ELISA kit measures the levels of both caspase-cleaved and intact CK18, which is released from cells undergoing necrosis ([@b35-ol-0-0-4779]). A total of 1×10^4^ A549, PC3 or H1299 cells were seeded per well in a 96-well plate in 200 µl culture medium in triplicates. After 24 h, cells were treated with PQE (100 µg/ml) for 48 h. At the end of the treatment period, cells were lysed with 10% NP-40 (Sigma-Aldrich) for 10 min on a shaker to perform the M30 assay, while the supernatants were collected for the M65 assay, according to the manufacturer\'s protocol. The absorbance was determined with an ELISA reader at 450 nm (FLASH Scan S12^®^; Analytik Jena AG, Jena, Germany).
#### Western blot analysis for further dissection of the apoptosis mechanism
A549, PC3 and H1299 cells were seeded in 25-cm^2^ flasks, and treated with PQE (100 µg/ml) for 24 h when the cells reached 70% confluency. Additionally, cisplatin (20 µM) for A549 and PC3 cells, and etoposide (5 µM) for H1299 cells, were used as positive controls for cleaved-poly (adenosine diphosphate-ribose) polymerase (PARP) ([@b36-ol-0-0-4779]--[@b38-ol-0-0-4779]). Cells were lysed in radioimmunoprecipitation assay lysis buffer (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), containing protease inhibitors. Equal amounts of protein (20 µg protein/lane) were subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a nitrocellulose membrane (Thermo Fisher Scientific, Inc.). Western blotting was performed using rabbit anti-β-actin and anti-PARP monoclonal antibodies at 1:1,000 dilution (catalog nos., 4970 and 9532, respectively; Cell Signaling Technology, Inc., Danvers, MA, USA) in 5% (w/v) bovine serum albumin (Amresco, LLC, Solon, OH, USA). Horseradish peroxidase (HRP)-linked anti-rabbit immunoglobulin G antibody (1:2,000 dilution; catalog no., 7074; Cell Signaling Technology, Inc.) was used to detect anti-β-actin and anti-PARP antibodies. Secondary antibody detection was performed according to the instructions of Phototope^®^-HRP Western Blot Detection System (Cell Signaling Technology, Inc.). Stripping was performed according to the manufacturer\'s reprobing protocol (Cell Signaling Technology, Inc.). Bound antibodies were visualized on a FUSION-FX7 imaging device (Vilber Lourmat, Marne-la-Vallée, France).
#### Statistical analysis
All statistical analysis were performed using SPSS 20.0 statistical software (IBM SPSS, Armonk, NY, USA). The significance was calculated using one-way analysis of variance. Significant differences in the M30 and M65 assays were determined using the Student\'s *t* test. P\<0.05 was considered to indicate a statistically significant difference. The results were expressed as the mean ± standard deviation.
Results
=======
### Chemical composition of P. quercetorum
The volatile compounds of *P. quercetorum* were analyzed by GCxGC-TOF/MS ([@b39-ol-0-0-4779]), and the qualitative and quantitative compositions were presented in [Table II](#tII-ol-0-0-4779){ref-type="table"}. A total of 23 compounds were identified in *P. quercetorum*, and the major components were tetracosane (40.92%), heneicosane (16.23%), 2-methyleicosane (12.37%), eicosane (5.15%), 2-pyrrolidinone (2.70%), octadecanol acetate (2.63%), 1-tetracosanol (2.61%), ylangene (1.36%) and 1-hexadecanol (1.06%). The rate of unknown compounds was 7.86%. All other components were present in \<1%.
### Anti-growth/cytotoxic effect of PQE by ATP assay
The cytotoxic effect of PQE on non-small cell lung cancer cell lines (A549, PC3 and H1299) were screened by ATP viability assay. Cells were treated with increasing doses of PQE (3.13--100 µg/ml) for 48 h. As shown in [Fig. 1A](#f1-ol-0-0-4779){ref-type="fig"}, PQE significantly reduced the cell viability levels in a dose-dependent manner (P\<0.05). PQE exhibited stronger anti-growth effect on PC3 cells at relatively lower doses, compared with A549 and H1299 cells. The cell death was clearly evident in all cell lines by phase-contrast microscopy ([Fig. 1B](#f1-ol-0-0-4779){ref-type="fig"}). The inhibitory concentration (IC)~50~ and IC~90~ values were calculated on the basis of the results of the ATP assay ([Table III](#tIII-ol-0-0-4779){ref-type="table"}).
### Fluorescence staining for confirmation of cell death mode
Annexin-V-FITC staining was performed in order to determine the presence of apoptosis and to distinguish early- from late-stage apoptosis. Hoechst dye 33342 was additionally used. Cells were treated with PQE (100 µg/ml) for 12 and 24 h. The images of 24-h treatment are shown in [Figs. 2](#f2-ol-0-0-4779){ref-type="fig"}--[4](#f4-ol-0-0-4779){ref-type="fig"}. The early apoptotic cells were positively stained for only Annexin-V-FITC (green), while the late apoptotic cells (also called secondary necrotic cells) were positively stained for PI (red). The presence of pyknotic nuclei (thin white arrows) on either early (yellow short arrows) or late (white short arrows) apoptotic cells was a well-known proof of apoptotic cell death ([Figs. 2](#f2-ol-0-0-4779){ref-type="fig"}--[4](#f4-ol-0-0-4779){ref-type="fig"}).
### Levels of total/intact and caspase-cleaved CK18 for cell death modes
Two different ELISAs were used, one for the estimation of total CK18 (M65, for primary or secondary necrosis) and the other one for the estimation of caspase-cleaved CK18 (M30, for apoptosis). [Fig. 5A](#f5-ol-0-0-4779){ref-type="fig"} indicates that the levels of M30 did not change in A549, PC3 or H1299 cells after 48-h PQE treatment, implying that the apoptosis resulted from PQE may not reach the fragmentation of the cytoskeleton. However, it is notable that M30 was substantially increased in A549 cells after treatment with paclitaxel, which is a positive control for caspase-cleaved CK18 (P=0.053) (data not shown).
With regard to M65 levels, they were observed to increase 6-fold in PC3 and H1299 cells, and \~4-fold in A549 cells, after treatment with PQE (100 µg/ml) for 48 h, compared with untreated control cells ([Fig. 5B](#f5-ol-0-0-4779){ref-type="fig"}). These increments may have resulted from secondary necrosis that normally occurs following apoptosis.
### Detection of PARP cleavage by western blotting
The cleavage of PARP was assayed by western blotting to further dissect the mechanism of apoptosis. Cisplatin and etoposide were used as positive controls for PARP-cleavage. It was observed that PARP was not cleaved in A549, PC3 or H1299 cell lines following the treatment with PQE (100 µg/ml) for 24 h ([Fig. 6](#f6-ol-0-0-4779){ref-type="fig"}). The lack of cleavage of PARP was considered as incomplete apoptosis, rather than typical apoptosis.
Discussion
==========
Lung cancer is the leading cause of cancer-associated mortalities worldwide due to its high incidence and mortality ([@b40-ol-0-0-4779]). Despite novel chemotherapy regimens, there is not sufficient success in its treatment. Therefore, new therapeutic approaches would be instrumental for better management of lung cancer patients. Since natural products are important in cancer therapy, the present study was conducted to evaluate the possible anti-growth/cytotoxic activity of PQE on lung cancer cell lines. Despite the fact that there are several studies on the anticancer activity of the *Pelargonium* genus ([@b13-ol-0-0-4779],[@b41-ol-0-0-4779]), the present study is the first one in the literature to demonstrate the cytotoxic activity of *P. quercetorum* on non-small cell lung cancer cell lines (A549, PC3 and H1299).
In the present study, *P. quercetorum* was observed to exert a significant anti-growth effect against A549, PC3 and H1299 lung cancer cell lines in a dose-dependent manner. According to the ATP assay results, the IC~50~ values were 62.1, 27.2 and 58.5 µg/ml for A549, PC3 and H1299 cell lines, respectively. In a previous study in which the crude acetone extracts of different *Pelargonium* species were used against transformed human kidney epithelium (Graham) cells, it was reported that the IC~50~ values of *P. sublignosum*, *P. citronellum, P. graveolens, P. betulinum*, *P. capitatum* and *P. tomentosum* were 11.9, 59.9, 83.3, 88.5, 101.5 and 195.1 µg/ml, respectively ([@b8-ol-0-0-4779]). This variability of IC~50~ values may be due to the use of different solvents for the extraction process or the different composition of the plant itself. In fact, biological activities of different species may vary by essential oil components.
A large number of *Pelargonium* species are aromatic and comprise geranium essential oil, which is one of the top 20 essential oils in the world ([@b9-ol-0-0-4779],[@b42-ol-0-0-4779]). It is known that geranium essential oils exhibit anticancer activity ([@b43-ol-0-0-4779],[@b44-ol-0-0-4779]). Therefore, in addition to certain unknown compounds, *P. quercetorum*-derived essential oils may be responsible for this cytotoxic effect. Despite the fact that these oils could not be analyzed in the present study, an extensive analysis of the chemical composition identified by GCxGC-TOF/MS was conducted ([Table II](#tII-ol-0-0-4779){ref-type="table"}). On the basis of this analysis, it was noticed that tetracosane, heneicosane and 2-methyleicosane were the most abundant components in *P. quercetorum*.
Furthermore, the present study investigated the mode of cell death resulted from PQE. Firstly, the morphology of the cells nuclei was evaluated for the presence of any apoptosis. The nuclei were observed to be pyknotic, implying that the mode of cell death was apoptosis, which was confirmed by Annexin-V positivity. To dissect further the mechanism of cell death/apoptosis, both the caspase-cleaved CK18 (M30) and intact CK18 (M65) levels were also measured. These assays perfectly discriminate two main cell death modes, apoptosis or necrosis ([@b35-ol-0-0-4779]). No increase in M30 levels was observed in these cell lines after treatment with PQE, despite the fact that the Annexin-V-FITC staining of the cell membrane and the morphological evaluation of the cell nuclei clearly implied an apoptotic cell death. For the lack of M30 increase, there could be two reasons, one being the lack of CK18 in the cells, and the other one being no fragmentation of CK18 actually occurring. The present authors recently reported that all the cell lines evaluated in the present study expressed CK18, being A549 cells the ones that exhibited the highest levels ([@b45-ol-0-0-4779]). This result implies that the apoptotic process may not include the fragmentation of CK18. That is why the type of cell death observed in the present study was described as incomplete apoptosis. Regarding the M65 levels, the explanation of increases in M65 levels is that the cells should be undergoing secondary necrosis following apoptosis ([@b46-ol-0-0-4779]).
In order to additionally investigate the mechanism of apoptosis, the levels of PARP, which are normally cleaved by active caspase-3 during apoptosis, were evaluated by the present study ([@b47-ol-0-0-4779],[@b48-ol-0-0-4779]). No significant alterations in the cleaved PARP levels were observed compared with untreated control cells by western blot analysis. Therefore, the present authors would like to refer to the resultant cell death in the present study as either incomplete apoptosis or apoptosis-like cell death. The latter is highly possible, since numerous different types of cell death have recently emerged ([@b49-ol-0-0-4779]). However, there are no data in the literature that could be used for comparison with results of the present study. Since both M30 production and PARP cleavage have been demonstrated to result from caspase-activation ([@b50-ol-0-0-4779]), it is reasonable to conclude that in the present study the cell death resulted from PQE may be caspase-independent. However, this should be the topic of future studies, as additional extensive experiments are required to elucidate this point.
To the best of our knowledge, the present is the first study to demonstrate the anti-growth/cytotoxic activity of *P. quercetorum i*n non-small cell lung cancer cells, warranting further evaluation *in vivo* for proof of concept.
The authors would like to thank the Research Fund of Uludag University (Bursa, Turkey) for funding the present study \[project number UAP (F)-2012/17\] and for providing the kits/chemicals required. The authors would also like to thank Dr Aysegul Cebi (Giresun University, Giresun, Turkey) for providing the plant; Dr Serap Celikler (Uludag University, Bursa, Turkey) for contributing to the extraction and lyophilization of the plant; and Dr Hakan Akca (Pamukkale University, Denizli, Turkey) for providing the cell lines used in the present study.
{#f1-ol-0-0-4779}
{#f2-ol-0-0-4779}
{#f3-ol-0-0-4779}
{#f4-ol-0-0-4779}
{#f5-ol-0-0-4779}
{#f6-ol-0-0-4779}
######
*Pelargonium* species used in folk medicine.
Genus: *Pelargonium* plant species Traditional uses (ref) Screened parts Previously screened activity (ref)
------------------------------------ -------------------------------------------------------------------------------------------------------------------------------------------------------------- ------------------------ -----------------------------------------------------------------------------------------
*P. reniforme* and *P. sidoides* Coughs, diarrhoea, hepatic disorders and tuberculosis ([@b14-ol-0-0-4779],[@b15-ol-0-0-4779]) (roots) Roots Antimycobacterial ([@b12-ol-0-0-4779],[@b16-ol-0-0-4779])
*P. graveolens* Antiasthmatic, antiallergic, antidiarrhoeic, diuretic, tonic, hemostatic, anti-hepatotoxic, stomachic and diabetic ([@b17-ol-0-0-4779]) (leaves and flowers) Aerial parts Antioxidant ([@b13-ol-0-0-4779]) and anticancer ([@b13-ol-0-0-4779],[@b18-ol-0-0-4779])
*P. endlicherianum* Anthelmintic ([@b19-ol-0-0-4779]) (roots and flowers) Aerial parts and roots Antioxidant ([@b20-ol-0-0-4779]) and antimicrobial ([@b21-ol-0-0-4779])
*P. radula* Mosquito repellent ([@b22-ol-0-0-4779]) (leaves and flowers) Leaves Antimicrobial ([@b23-ol-0-0-4779])
*P. betulinum* Coughs and other chest problems, wound healing and gastrointestinal-related problems ([@b24-ol-0-0-4779]) (leaves) Aerial parts Antibacterial and antioxidant ([@b8-ol-0-0-4779])
*P. cucullatum* Colic, diarrhoea and wounds ([@b24-ol-0-0-4779]) (leaves) Aerial parts Antibacterial and antioxidant ([@b8-ol-0-0-4779])
*P. glutinosum* Astringent ([@b25-ol-0-0-4779]) (all parts) Stems and leaves Antibacterial, antioxidant and cytotoxic ([@b8-ol-0-0-4779])
*P. citronellum* Culinary ([@b26-ol-0-0-4779]) (leaves) Stems and leaves Antibacterial and cytotoxic ([@b8-ol-0-0-4779])
######
Chemical composition of *Pelargonium quercetorum* extract.
Compound^[a](#tfn1-ol-0-0-4779){ref-type="table-fn"}^ ^1^tR^[b](#tfn2-ol-0-0-4779){ref-type="table-fn"}^ ^2^tR^[b](#tfn2-ol-0-0-4779){ref-type="table-fn"}^ \% Area^[c](#tfn3-ol-0-0-4779){ref-type="table-fn"}^
------------------------------------------------------- ---------------------------------------------------- ---------------------------------------------------- ------------------------------------------------------
Acetic acid 430 1.30 0.65
2,5-Dimethyl pyrazine 510 2.05 0.61
γ-Butyrolactone 545 2.68 0.47
Benzeneacetaldehyde 740 2.35 0.11
4-Hydroxy-2,5-dimethyl-3(2H)-furanone 760 2.27 0.15
2-Pyrrolidinone 805 3.17 2.70
5-Hydroxymethyldihydrofuran-2(3H)-one 1025 3.19 0.36
Hexahydrofarnesyl acetone 1980 1.54 0.73
Allyl octadecyl oxalate 2000 1.37 0.57
n-Tridecan-1-ol 2045 1.57 0.94
Ylangene 2075 1.70 1.36
Iso-palmitic methyl ester 2090 1.52 0.14
Nerolidol 2490 1.96 0.11
Farnesane 2810 1.37 0.68
Eicosane 2900 1.42 5.15
1-Tetracosanol 2920 1.56 2.61
3,7-Dimethylnonane 2965 1.48 0.70
1-Hexadecanol 2990 1.45 1.06
1-Octadecanol acetate 3005 1.53 2.63
Heneicosane 3075 1.56 16.23
2-Methyleicosane 3150 1.45 12.37
Tetracosane 3235 1.59 40.92
Oleic acid 3315 3.81 0.85
Unknown [--]{.ul} -- 7.86
As identified by LECO GCxGC-TOF/MS ChromaTOF^®^ version 2.3 software (LECO Corporation, Saint Joseph, MI, USA). The compounds were named according to the 2005 Automated Mass Spectral Deconvolution and Identification System (National Institute of Standards and Technology, US Department of Commerce) ([@b39-ol-0-0-4779]).
^1^tR and ^2^tR, retention times in the first and second dimension, respectively.
The percentage of each component was calculated as the peak area of the analyte divided by the peak area of the total ion chromatogram and multiplied by 100.
######
IC~50~ and IC~90~ values of *Pelargonium quercetorum* extract in non-small cell lung cancer cells lines.
Dose (µg/ml) A549 PC3 H1299
-------------- ------------ ------------ ------------
IC~50~ 62.13±1.90 27.18±3.21 58.53±2.96
IC~90~ 98.70±1.64 47.73±1.36 92.43±0.60
IC~50~, concentration inhibiting 50% of cell growth (viability). IC~90~, concentration inhibiting 90% of cell growth (viability). Values are shown as the mean ± standard deviation.
|
{
"pile_set_name": "PubMed Central"
}
|
INTRODUCTION
============
Middle East Respiratory Syndrome (MERS) is a severe respiratory infection caused by a novel beta coronavirus (MERS-CoV) \[[@b1-epih-40-e2018014]-[@b3-epih-40-e2018014]\]. The symptoms of MERS include fever, chills, cough, shortness of breath, gastrointestinal symptoms, expectoration, wheezing, chest pain, hemoptysis, sore throat, headache, myalgia, abdominal pain, vomiting, and diarrhea; it can also cause death in severe cases \[[@b3-epih-40-e2018014]-[@b6-epih-40-e2018014]\].
The causative pathogen of MERS is transmitted via 4 modes: animal-to-human, intra-familial, healthcare-associated, and travel-related \[[@b7-epih-40-e2018014],[@b8-epih-40-e2018014]\]. The 186 cases that occurred in South Korea (hereafter Korea) were predominantly caused by healthcare-associated transmission \[[@b7-epih-40-e2018014],[@b9-epih-40-e2018014]-[@b11-epih-40-e2018014]\], followed by intra-familial transmission.
According to data reported to the World Health Organization, the rates of asymptomatic or mild infection were 44 of 398 (28.60%) in Saudi Arabia, the United Arab Emirates, and the Islamic Republic of Iran between April and June 2014, and 32 of 113 (28.31%) in Saudi Arabia in June 2014 \[[@b12-epih-40-e2018014],[@b13-epih-40-e2018014]\]. However, Oboho et al. \[[@b14-epih-40-e2018014]\] reported that 78.79% (26 of 33) of initially reported asymptomatic patients had at least 1 symptom. In Korea, among the 186 confirmed cases, 3 asymptomatic cases were detected among healthcare workers via screening tests (1.61%) \[[@b15-epih-40-e2018014]\]. In serologic studies using indirect immunofluorescence tests for healthcare workers who were at MERS-affected hospitals, 2 of 457 (0.44%) had positive results \[[@b16-epih-40-e2018014]\]. However, no report has been published regarding the asymptomatic infection rate among non-healthcare workers in Korea. There is a considerable chance of human-to-human transmission, as well as direct infection via the dromedary camel \[[@b17-epih-40-e2018014]-[@b19-epih-40-e2018014]\]. Therefore, it is necessary to identify the rate of asymptomatic MERS infections in healthcare workers and non-healthcare workers.
MATERIALS AND METHODS
=====================
Selection and participation of individuals
------------------------------------------
This survey was conducted between August 2015 and February 2016 after the last MERS case diagnosed in July 5, 2015. Based on a database of quarantined individuals provided by the Department of Epidemiologic Investigation of the Korea Centers for Disease Control and Prevention (KCDC), individuals from 4 regions with major outbreaks---Seoul, Gyeonggi, Chungcheong, and Jeonbuk ---were selected. Individuals whose MERS status was diagnosed as positive using a polymerase chain reaction test were excluded from the analysis of this study. From the 14,831 quarantined individuals, 7,233 residents (48.8%) living in the 4 major MERS outbreak regions were selected. Of these individuals, calls requesting participation in this study were made to 3,291 individuals (45.5%) according to prioritization groupings. A total of 1,610 individuals (48.9%) ultimately participated in the study ([Figure 1](#f1-epih-40-e2018014){ref-type="fig"}). Those who refused to participate have been described in another study \[[@b20-epih-40-e2018014]\].
The study individuals were prioritized in groups according to the transmission intensity of the MERS case they were exposed to, as follows: contact with super-spreading events (5 or more individuals infected) \[[@b21-epih-40-e2018014]\], contact with spreaders who infected 1 to 4 individuals, and contact with non-spreaders. We selected study subjects according to this prioritization of groups, and the selection rates were 48.8, 16.4, and 15.8%, respectively. We also categorized the subjects according to their exposure intensity (i.e., status when they were exposed to the MERS case), as follows: inpatients or outpatients at a MERS-affected hospital, cohabiting family members or paid caregivers of the MERS case, visitors of the hospitalized MERS case, healthcare workers employed at a MERS-affected hospital, and colleague of the MERS case. We selected more subjects from the categories of family, patients, and visitors ([Table 1](#t1-epih-40-e2018014){ref-type="table"}).
Study performance
-----------------
Only individuals who consented to participate in the study through a telephone call and gave written consent at the time of study initiation underwent surveys at local public health centers. Each participant responded to a questionnaire on exposure and provided a blood sample. Following the first serologic test by an enzymelinked immunospecific assay (ELISA), positive or borderline cases were tested using an immunofluorescence assay (IFA) and a plaque reduction neutralization antibody test (PRNT).
Antibody tests
--------------
MERS-CoV antibody levels in all sera collected from contacts were measured using a recombinant S1 protein-coated human anti-MERS-CoV (immunoglobulin G \[IgG\]) ELISA kit (Euroimmun, Luebeck, Germany), which was used according to the manufacturer's protocol. The results of the tested samples were determined by calculating the ratio of the optical density (OD) value of the sample to the OD value of the calibrator. Ratios ≥ 1.1 were considered positive, ratios ≥ 0.8 to \< 1.1 were considered borderline, and ratios \< 0.8 were considered negative.
IFA slides were coated with MERS-CoV non-infected or infected Vero cells. Serum samples were diluted in phosphate-buffered saline (PBS) to 1:10, 1:100, 1:250, and 1:1,000, transferred to IFA slides, and incubated for 30 minutes at 37°C in a humidified chamber. The IFA slides were washed 3 times in PBS-T for 5 minutes each and incubated with fluorescein isothiocyanate-conjugated rabbit anti-human IgG (Abcam, Cambridge, MA, USA) diluted at 1:800 for 30 minutes at 37°C in a humidified chamber. After washing 3 times in PBS-T for 5 minutes, the slides were embedded with a mounting fluid, topped with a cover glass, and observed under a fluorescent microscope.
The neutralizing antibodies in the serum samples were measured by PRNT. The PRNT procedures were performed as follows. In brief, 1:10 diluted sera were heat-inactivated at 56°C for 30 minutes. Heat-inactivated sera were diluted serially 4-fold. After an equal volume of virus (MERS-CoV/KOR/KNIH/002_05_2015) was added to the volume of serum dilutions, these mixtures were incubated at 37°C for 2 hours. The mixtures were added to each well of the 24-well plate cultured with Vero cells. The plate was incubated at 37°C for 60 minutes, and 1 mL of 1.5% carboxymethylcellulose overlay medium was then added. After incubation for 3-4 days, cell staining was performed by crystal violet. The titer of neutralizing antibody by PRNT~50~ was calculated using the Kärber formula, as described previously \[[@b22-epih-40-e2018014]\]. A titer above 1:20 was interpreted as positive. All sera with a positive or borderline reaction in ELISA were tested by the IFA and PRNT assays for confirmation, and cases with positive results from any 2 assays were considered to be anti-MERS positive.
This study received approval from the bioethics committee of the KCDC (2015-08-EXP-03-P-A) and the institutional review board of the National Cancer Center (NCC 2016-0058). Informed consent was obtained from study participants or their parent or legal guardian for children under 14.
RESULTS
=======
Among the 3,291 selected individuals, the response rates of contacts with super-spreading events (5 or more individuals infected), contacts with spreaders who infected 1-4 individuals, and contacts with non-spreaders were 39.8% (552 of 1,388), 40.3% (139 of 345), and 59.0% (919 of 1,558), respectively. According to their status when they were exposed to the MERS case, the highest response rates were found for healthcare workers (99.4%), and family members (97.1%), followed by visitors (75.8%), colleagues (49.0%), and patients (36.8%) ([Table 1](#t1-epih-40-e2018014){ref-type="table"}).
The seropositive rate using ELISA was 1.05% (6 of 574) in patients, 0.33% (1 of 307) in healthcare workers, and 0.43% (7 of 1,610) overall. Among the 7 ELISA-positive individuals, 3 had contact with a super-spreading event (patient zero, case \#1) whoinfected 28 individuals \[[@b23-epih-40-e2018014]\], 1 had contact with a spreader (case \#118) who infected 2 individuals, and 3 had contact with a non-spreader (case \#89). Among the ELISA-positive individuals, only 1 was both IFA-positive and PRNT-positive. Therefore, the confirmed rates of asymptomatic MERS infection were 0.17% in patients and 0.060% (95% confidence interval, 0.002 to 0.346) overall ([Table 1](#t1-epih-40-e2018014){ref-type="table"}).
The confirmed asymptomatic case, patient zero, and 11 secondary MERS patients were hospitalized on the same floor of the hospital at the same time \[[@b23-epih-40-e2018014],[@b24-epih-40-e2018014]\]. The asymptomatic case was quarantined at home for 2 weeks after discharge. The case had underlying diseases that included hypertension, angina, and degenerative arthritis. The case reported no fever, cough, myalgia, or gastrointestinal symptoms during hospitalization or quarantine ([Table 2](#t2-epih-40-e2018014){ref-type="table"}).
DISCUSSION
==========
Limited information exists regarding MERS-CoV seroprevalence among populations other than confirmed MERS cases. Saudi Arabian data showed that the seroprevalence of MERS-CoV IgG among the general population was 0.15% \[[@b25-epih-40-e2018014]\], suggesting that a number of cases of asymptomatic or mild infections may be present in the general population.
Despite a high prevalence of 186 confirmed MERS cases during the outbreak of MERS in Korea, the rate of asymptomatic infection (1.60%) \[[@b15-epih-40-e2018014]\] was lower than expected. The rates of asymptomatic infection confirmed using IFA and PRNT in the present study were 0.06% (1 of 1,610) for all contacts and 0.17% (1 of 574) for patients. These results are markedly lower than the rates of 0.27% (2 of 737) among healthcare workers, and 0.44% (2 of 457) among healthcare workers at MERS-affected hospitals in Korea \[[@b16-epih-40-e2018014]\]. Moreover, the rate of asymptomatic or mild infection in Saudi Arabia, the United Arab Emirates, and the Islamic Republic of Iran was approximately 28.00% \[[@b12-epih-40-e2018014],[@b13-epih-40-e2018014],[@b18-epih-40-e2018014]\].
The confirmed asymptomatic case presented in this study was a patient at the same hospital as confirmed MERS cases and, unlike in previous studies, was neither an intra-familial infection nor a pediatric infection \[[@b26-epih-40-e2018014],[@b27-epih-40-e2018014]\]. The low rate of asymptomatic infection in Korea is attributable to the different transmission pathway of MERS infections compared to the Middle East. In Korea, most of the MERS cases were healthcare-associated infections, and none were from an animal. The low asymptomatic infection rate is also attributable to the extensive epidemiological investigation conducted in Korea, including close monitoring of contacts with MERS patients; this helped identify almost all MERS patients. This may also have been attributable to the promotion of proactive identification of patients via mass media and the establishment of communication networks by the government, leading to voluntary reports by people, active quarantine, and countermeasures to this public health crisis \[[@b10-epih-40-e2018014],[@b28-epih-40-e2018014],[@b29-epih-40-e2018014]\].
ELISA is appropriate as a screening tool, as it is 10-fold more sensitive than IFA. However, it may cross-react with seasonal human coronavirus antibodies, so a spike protein-specific IFA is required for confirmation. PRNT is a definitive test when ELISA and IFA have inconclusive results \[[@b25-epih-40-e2018014]\]. Only 10% of ELISA-positive results are positive on a neutralization assay \[[@b25-epih-40-e2018014]\]. In our study, 1 of 7 patients with borderline or positive ELISA results also had positive results in PRNT. Therefore, the results obtained in our study are accurate because ELISA, IFA, and PRNT were used.
A previous study predicted the pandemic potential of MERS-CoV to be ≤ 5%; however, this does not indicate that the risk has abated \[[@b30-epih-40-e2018014]\]. Prerequisites for reducing the risk include improved surveillance, active contact tracing, and the initiation of animal host searching \[[@b30-epih-40-e2018014],[@b31-epih-40-e2018014]\]. During the outbreak in Korea, MERS was classified as a notifiable infectious disease and was subjected to surveillance \[[@b32-epih-40-e2018014]\]. Since MERS is an imported disease in Korea, it is recommended that precautions be taken before travel and that the time of returning from travel and incubation period be considered.
This study showed a low seropositivity in the population of individuals quarantined due to contact with MERS cases. However, there is a possibility that the seropositivity rate was underestimated for the following reasons. Firstly, the participants of the present study were mostly non-healthcare workers and were relatively healthy. Thus, the risk of infection was low. Secondly, the overall participation rate was 48.9%, whereas it was 36.8% in the patient group at a higher risk of infection. Moreover, we only surveyed 10.9% of the quarantined individuals. Therefore, the actual rate of asymptomatic infections may be higher than reported in the present study. Lastly, the present study may have been conducted too late. In a previous study, MERS-CoV ELISA results indicated that the antibody response was highest after 3 weeks from symptom onset \[[@b33-epih-40-e2018014]\]. Although no reports have analyzed the duration of antibody presence in MERS patients regardless of symptoms, a recent study of severe acute respiratory syndrome and MERS reported that antibodies in some patients persisted for up to 2-3 years after infection \[[@b34-epih-40-e2018014],[@b35-epih-40-e2018014]\]. Blood sampling for serologic test in this study was performed on contacts between October and December 2015, while exposure to the confirmed case occurred between May and June 2015 (a gap of 5 months). Thus, a loss of the MERS-CoV antibody titer could have taken place despite actual asymptomatic infections; therefore, the actual rate of asymptomatic infection may be higher than the rate presented in this report.
In conclusion, among 1,610 contacts, only 1 non-healthcare worker who was a patient in a MERS-affected hospital had an asymptomatic MERS-CoV infection. To understand new emerging infectious diseases such as MERS, more intensive epidemiologic research is needed, including an analysis of asymptomatic infections.
The authors would like to thank Hee-Dong Jung and Jeong-Gu Nam (Korea Centers for Disease Control and Prevention) for laboratory assistance and technical support in production of the manuscript. Additionally, we would like to thank all the administrative and laboratory staff of the Korea Centers for Disease Control and Prevention who participated in the MERS-CoV outbreak control in Korea.
The authors have no conflicts of interest to declare for this study.
{#f1-epih-40-e2018014}
######
Study subjects and seropositive participants by characteristics of the MERS case they were exposed to, and status of subjects upon MERS exposure in the 2015 Korean outbreak
Characteristics of exposed MERS case Status of subjects when they were exposed to MERS^[1](#tfn1-epih-40-e2018014){ref-type="table-fn"}^ Selection or response rate (%)^[2](#tfn2-epih-40-e2018014){ref-type="table-fn"}^
--------------------------------------------- -------------------------------------- ----------------------------------------------------------------------------------------------------- ---------------------------------------------------------------------------------- ----------- ------- ------- ------------- ------- ------
Quarantined subjects Spreaders with 5 or more cases 1,141 71 417 558 3 657 2,847
Spreaders with 1 to 4 cases 1,067 64 364 198 35 375 2,103
Non-spreaders 3,489 321 1,613 1,260 453 2,745 9,881
Total 5,697 456 2,394 2,016 491 3,777 14,831
Selected subjects Spreaders with 5 or more cases 744 60 254 115 3 212 1,388 48.8
Spreaders with 1 to 4 cases 191 10 105 10 0 29 345 16.4
Non-spreaders 626 101 281 184 101 265 1,558 15.8
Total 1,561 171 640 309 104 506 3,291 22.2
Selection rate (%) 27.4 37.5 26.7 15.3 21.2 13.4 22.2
Study participants Spreaders with 5 or more cases 252 53 152 94 1 0 552 39.8
Spreaders with 1 to 4 cases 60 4 62 12 0 1 139 40.3
Non-spreaders 262 109 271 201 50 26 919 59.0
Total 574 166 485 307 51 27 1,610 48.9
Response rate (%) 36.8 97.1 75.8 99.4 49.0 5.3 48.9
Subjects with seropositive ELISA (IFA/PRNT) Spreaders with 5 or more cases 2 (1) 1 (0) 3 (1)
Spreaders with 1 to 4 cases 1 (0) 1 (0)
Non-spreaders 3 (0) 3 (0)
Total 6 (1) 1 (0) 7 (1)
Seropositive rate (%) 1.05 (0.17) 0.33 (--) 0.43 (0.06)
MERS, Middle East Respiratory Syndrome; ELISA, enzyme-linked immunospecific assay; IFA, immunofluorescence assay; PRNT, plaque reduction neutralization antibody test.
Inpatients or outpatients at a MERS-affected hospital, family of people living with or a paid caregiver of the MERS case, visitors of the hospitalized MERS case, healthcare workers employed at a MERS-affected hospital, and colleagues of co-workers of a MERS case.
Selection rates varied by characteristics of the MERS case subjects were exposed to (i.e. transmission intensity) and the subjects' status (i.e. exposure intensity).
######
Characteristics of MERS-seropositive subjects by ELISA, IFA, and PRNT in Korea, 2015
Subject ID Exposure date Sampling date Status at exposure Exposed MERS case ID^[1](#tfn3-epih-40-e2018014){ref-type="table-fn"}^ Underlying disease Symptoms after exposure ELISA IFA PRNT
------------ --------------- --------------- -------------------- ------------------------------------------------------------------------ -------------------------------- ------------------------- ------- ------------ ---------- ----------
1 May 15 Nov 1 Patient \#1 Angina None 0.996 Borderline Positive Positive
2 May 16 Nov 2 Patient \#1 None None 2.078 Positive Negative Negative
3 May 15 Nov 2 Healthcare worker \#1 None Fatigue 1.640 Positive Negative Negative
4 Jun 9 Oct 31 Patient \#118 Hypertension Blurred vision 1.116 Positive Negative Negative
5 Jun 4 Nov 8 Patient \#89 Hypertensive heart disease None 0.916 Borderline Negative Negative
6 Jun 5 Nov 9 Patient \#89 Lumbar spinal Fatigue stenosis 1.724 Positive Negative Negative
7 Jun 11 Nov 8 Patient \#89 None Fatigue and 0.985 Borderline Negative Negative
MERS, Middle East Respiratory Syndrome; ELISA, enzyme-linked immunospecific assay; IFA, immunofluorescence assay; PRNT, plaque reduction neutralization antibody test.
Patient zero (\#1) infected 28 cases (super-spreading event transmitting 5 or more cases), case \#118 infected 2 cases, and case \#89 did not transmit MERS to anyone during the 2015 Korean MERS outbreak.
[^1]: Song & Yang contributed equally to this work as joint first authors.
|
{
"pile_set_name": "PubMed Central"
}
|
INTRODUCTION {#rjy277s1}
============
Tracheal injuries can occur following esophageal resections in a variety of malignant and benign esophageal diseases. If injuries are diagnosed during the operation with a loss in the airway pressure during ventilation and visible or audible air leak, it must be repaired immediately with primary repair and reinforcement of the suture line with pericardial or intercostal muscle flaps. If not diagnosed early, the injury may lead to increased morbidity and mortality. Small injuries in the thoracic trachea, which are not diagnosed during operation, could cause a lung collapse, empyema and air leak through tracheostomy tube. The delayed diagnosis and failure of primary repairs are rarely reported in the medical literature. Therefore, the treatment has not been exactly defined. Treatment may include a wide spectrum of options, extending from conservative management to surgical intervention. Patients with fully expanded lungs and mild air-leak trough thoracostomy tubes (TT) and no evidence of empyema, or mediastinitiscan have to be managed conservatively. Yet, the presence of the more severe symptoms includes lung collapse, severe air-leak, respiratory complaints and empyema/mediastinitis, necessitating surgical intervention \[[@rjy277C1]--[@rjy277C3]\].
Here, we report a case of tracheal injury following thoracoscopic esophageal resection which was primarily repaired unsuccessfully with no flap reinforcement. The patient suffered right lung collapse and severe air leak thorough tracheostomy tube several days after surgery. We successfully repaired the injury with just a pericardial and intercostal muscle flaps since the primary repair was not feasible due to localized mediastinitis. To our knowledge, it is the first report of such a surgical intervention in repairing trachea injuries.
CASE REPORT {#rjy277s2}
===========
A 66-year-old male patient with esophageal cancer has undergone a thoracoscopic esophageal resection. He was a known case of stage IIb and with a history of neoadjuant chemoradiotherapy. During the thoracoscopic esophageal resection, the posterior membranous wall of the trachea was injured 1 cm above the carina. A cancer surgeon repaired the laceration primarily with a single interrupted suture with no flap reinforcement. He did not convert to thoracotomy. The patient suffered a right lung collapse and a severe air leak from TT on the fifth post-operative day. In consultation with a thoracic surgeon, video bronchoscopy was performed. The patient sustained tracheal injury in the range of 5 × 5 ml 1 cm above the carina, showing the failure of primary repair. Owing to the lung collapse and severe air leak, he was a definite candidate for reoperation.
Under general anesthesia with double-lumen endotracheal intubation, operative repair was done with a right postero-lateral thoracotomy through the fifth intercostal space. At first, the intercostal flap was prepared, whereupon the gastric flap was retracted to expose tracheal laceration. Tracheopleural fistula was seen on the posterior wall of trachea 1 cm above the carina (Fig. [1](#rjy277F1){ref-type="fig"}). Because of the severe inflammation, primary repair was not possible. Therefore, we had to repair the tear with flaps. First, the tear was covered with a rotated pericardial flap and was sutured into the laceration edge with interrupted Vicryl (4.0) (Fig. [2](#rjy277F2){ref-type="fig"}). Then the pericardial flap was reinforced with the intercostal muscle flap ([Video](#sup1){ref-type="supplementary-material"}). Care was taken not to puncture the endotracheal tube. Finally, the gastric flap was reverted and sutured to surrounding tissue. Two TT were inserted into the ipsilateral thoracic cavity both anteriorly and posteriorly.
{#rjy277F1}
{#rjy277F2}
He passed the post-operative period in the intensive care unit uneventfully. The air leak stopped on the fifth post-operative day. Video bronchoscopy on the sixth post-operative day showed near completion of the repair of the injury. The day after bronchoscopy the tubes were removed. The patient was discharged 24 h later and followed on out patient base.
DISCUSSION {#rjy277s3}
==========
Tracheal injuries occur in 1--10% of esophagectomies. Previous history of esophageal cancer, preoperative chemoradiation, extensive lymph node mediastinal dissection, peri-tumoral infection and closed proximity of esophageal tumor to carina are the most common predisposing factors \[[@rjy277C1]\]. Therefore, transhiatal esophagectomy for proximal tumors must be performed with caution after neoadjuant chemoradiotherapy \[[@rjy277C1]\]. When it is recognized during the surgery, tracheal injury must be repaired immediately. However, the management is challenging; the surgical approach depends upon the location of the injury.
In the case of proximal injury, cervical approach with or without partial sternotomy has to be done. Injuries at or above level of carina must be repaired with right postero-lateral thoracotomy after insertion of the double-lumen endotracheal tube. The primary repair of airway injury must be done with interrupted sutures and flap reinforcement. George *et al.*\[[@rjy277C1]\] reported optimal outcome even without flap. All the repairs were carried out during thoracotomy. In our patient, the cancer surgeon did not revert to thoracotomy and sutured the injury with no flap reinforcement. The primary repair without reinforcement and no conversion to thoracotomy might justify the failure of the management and inconsistency of the report by Baht *et al.* Uwatoko *et al.* reported tracheal injury during thoracoscopic esophagectomy in a patient with advanced cancer. They converted to minithoracotomy and repaired the injury with a primary sutured and reinforcement with gastric tube \[[@rjy277C4]\]. It seems once the injury is identified during thoracoscopy, minithoracotomy is the preferred approach to repair because the use of pediculated muscle flap is not possible with thoracoscopy. Pericardial flaps are not safe enough for the coverage of tracheal laceration due to poor vascularization and paradoxical movement especially under positive pressure ventilation \[[@rjy277C5]\]. Therefore, the reinforcement of pericardial flap with an intercostal muscle flap might increase the success of procedure.
In our technique, we have covered the defect with transposed pericardial flap and reinforced it with an intercostal muscle flap. There are successful reports of the primary repair with a pericardial reinforcement. Yet, the hypovascular nature of pericardium has been always a major concern \[[@rjy277C1]\]. Therefore, we buttressed the pericardial patch with intercostal muscle flap to avoid any failure. The ease of access and availability prompted us to use intercostal muscle flap. Lung expansion, and air leak stoppage on the fifth post-operative day, and bronchoscopy exam all demonstrated the success of this technique.
CONCLUSION {#rjy277s4}
==========
In conclusion, a tracheal injury during esophagectomy must be repaired immediately with a flap reinforcement. The preparation of the muscle flap is not feasible during thoracoscopic esophagectomy; it seems to be a reasonable option to convert to thoracotomy in case of tracheal injury. However, this issue must be addressed in further studies.
Supplementary Material
======================
######
Click here for additional data file.
CONFLICT OF INTEREST STATEMENT {#rjy277s6}
==============================
None declared.
|
{
"pile_set_name": "PubMed Central"
}
|
Dear Editor,
SARS-CoV-2 infection is associated with marked lymphopenia that correlates with morbidity and mortality \[[@CR1], [@CR2]\]. Here, we present the first report on serial immunophenotypic and functional changes in 13 consecutively recruited patients infected with SARS-CoV-2 virus during their first week of ICU stay (Supplementary Table 1) with 10 healthy donors used as controls.
Patients uniformly exhibited deep global and persisting T, NK and B cell lymphopenia from ICU admission (D0) to day 7 (D7) (Fig. [1](#Fig1){ref-type="fig"}a to d). On D0, median absolute lymphocyte count was dramatically reduced at 0.72 \[0.65--0.88\] G/L as were CD4 and CD8 T cell counts at 0.29 \[0.19--0.43\] and 0.08 \[0.05--0.1\] G/L (Fig. [1](#Fig1){ref-type="fig"}a, e, f), such CD4 T cell levels reflecting profound immunosuppression in HIV-infected patients. Few CD4 T cells transiently expressed CTLA-4 during the first 3 days (Fig. [1](#Fig1){ref-type="fig"}g) while expression of PD-1 was observed at D0 and increased until D7 (Fig. [1](#Fig1){ref-type="fig"}h). CD8 T cells significantly and persistently expressed PD-1 from D0 to D7 while CTLA-4 expression remained unchanged (Fig. [1](#Fig1){ref-type="fig"}i, j).Fig. 1Over time, ICU COVID-19 patients showed a profound and sustained lymphopenia correlated with increased percentages of CD4 and CD8 expressing exhaustion marks and increased frequency of immune suppressive cells. Box plot represent results for 10 healthy subjects (controls) and for the following time point and number of COVID-19 ICU patients. The number of patients is given below the horizontal axis of panel a. Boxes give the median with the first and the third quartile. Whiskers represent min to max. Lines with bracket and plain lines indicate a Mann--Whitney and ANOVA (Kruskall--Wallis test) comparison with controls or during the ICU stay respectively. Test *p* values are represented by \*, \*\* and \*\*\* for *p* ≤ 0.05, *p* ≤ 0.01 and *p* ≤ 0.001 respectively. Upper panel **a**, **b**, **c**, **d**, **e**, **f**: absolute lymphocytes count (ALC) (**a**), CD3 T-lymphocytes (**b**), Natural Killer (NK) cells (**c**), B lymphocytes (**d**), CD4 (**e**) and CD8 T cells (**f**). Light blue boxes represent controls and darker blue boxes represent patients. Middle panel **g**, **h**, **i**, **j**: percentages of CD4 (**g** and **h**) and CD8 (**i** and **j**) T cells expressing CTLA-4 (**g** and **i**) and PD-1 (**h** and **j**). Light green boxes represent controls and darker green boxes represent patients. Lower panel **k**, **l**, **m**, **n**, **o**: percentages of CD4 +/CD25 +/CD127low regulatory T cells (T-reg) \[[@CR3]\] (**k**) expressing CTLA-4 (**l**) and PD-1 (**m**) and CD14 monocyte counts (**n**) with quantification of the mHLA-DR at their surface membrane (**o**). Light orange boxes represent controls and darker orange boxes represent patients
Being heterogeneous at D0 (Fig. [1](#Fig1){ref-type="fig"}k), percentages of regulatory T cells (Tregs) increased during time. Few of them over-expressed CTLA-4 while PD-1 expression was strongly and stably increased until D7 (Fig. [1](#Fig1){ref-type="fig"}l, m). Total granulocytes were moderately increased with a transient egression of immature granulocytes in 4/10 patients at day 4--5 (Supplementary Figure 1). Monocyte counts increased during the first week. Nevertheless, HLA-DR expression was strongly down-regulated by a threefold factor at D0. Strikingly this decrease persisted unabated until D7, possibly impairing antigen presentation, and was associated with increased PD-L1 expression (Fig. [1](#Fig1){ref-type="fig"}n, o and Supplementary Figure 4d).
Being either an exhaustion or an activation marker, PD-1 is an inducer of CD8 T cell apoptosis when activated. Therefore, functional evaluation of T-lymphocytes was performed in three patients and controls for comparison. Meanwhile production of TNF-α and IL-2 was normal, CD4 T cell IFN-γ production was decreased (Supplementary Figure 2), indicating a CD4 exhaustion process. In contrast, CD8 T cells could be involved in anti-viral immune response since they produced higher levels of IFN-γ and TNF-α (Supplementary Figure 3). Consistently, percentages of effector CD4 T cells were decreased while those of effector memory and activated CD8 T cells were increased (Supplementary Figure 4a to 4c). Circulating levels of IL-6 and IL-8 were moderately but significantly and sustainly increased over time, reflecting the known SARS-CoV-2 related sub-acute inflammatory response of innate immune cells \[[@CR4]\] (Supplementary Figure 5).
Although our results warrant further confirmation in larger cohort, they strongly suggest a multifaceted devastating effect of the virus to cause depletion of virtually all classes of adaptive immune cells and to cause upregulation of potent T cell killing and immunosuppressive mechanisms in critically-ill COVID-19 patients. Since T cells are essential for definitive viral clearance, these results call into question therapies (e.g., anti-IL-6, corticosteroids, JAK inhibitors) that aim to block the ability of the patient to mount an effective immune response to the invading SARS-CoV-2. Knowing that almost all anti-inflammatory therapies have also chronically failed in sepsis, consideration to therapies that boost host immunity in selected severe ARDS ICU patients (e.g., IL-7, IFN-γ or checkpoint inhibitors) may be appropriate \[[@CR5], [@CR6]\].
Electronic supplementary material
=================================
{#Sec1}
Below is the link to the electronic supplementary material.Supplementary material 1 (PDF 229 kb)Supplementary material 2 (PDF 551 kb)
**Publisher\'s Note**
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Robin Jeannet, Thomas Daix and Remy Formento have contributed equally to this study.
The authors want to thank the hematology flow cytometry and the virology Laboratories from Limoges as well as the French Reference Laboratory for Herpesviruses, and Prs. Sylvie Rogez and Sophie Alain. The authors acknowledge that without the implication of the nurses from the Clinical Investigation Center this work would not have been possible. They also thank Prs. Philippe Vignon and Richard Hotchkiss for their discussion and proofreading of the letter.
TD, BF included patients. RJ, RF, JF analyzed the data. RJ, TD drafted the manuscript. JF, BF, and RF reviewed the manuscript. All authors read and approved the final version of the manuscript.
None.
None.
Biological collection Inserm CIC 1435: DC-2008-604.
All patients agreed on the use of anonymized information as per the French law on the General Data Protection Regulation (GDPR).
|
{
"pile_set_name": "PubMed Central"
}
|
Introduction {#sec1-1}
============
Cervical cancer is the third most common malignancy and the fourth leading cause of cancer-related death among women worldwide. Overall, the mortality incidence ratio is 52%, and cervical cancer caused 275,000 deaths in 2008 alone, about 88% of which occurred in developing countries (de Sanjose et al., 2007; Kroupis et al., 2007; Panotopoulou et al., 2007; Agorastos et al., 2014). Numerous epidemiological and molecular biology studies over the past two decades have shown that human papillomavirus (HPV) plays a central role in the etiology of cervical cancer (Munoz et al., 2003).
HPV is a small, double-stranded DNA virus that contains approximately 7,900 base pairs, and can specifically infect human skin and mucous membrane squamous epithelial cells, causing a variety of benign and malignant lesions (Clifford et al., 2005). More than 100 types of HPV have been identified, and 40 of these are associated with the anogenital area. HPV genotypes of the genital tract have been divided into low-risk and high-risk forms based on pathogenicity. Low-risk HPV types are responsible for 90% of genital warts, while the high-risk types are responsible for 95% of cervical cancers and cervical intraepithelial neoplasia (CIN)(Clifford et al., 2005; de Sanjose et al., 2010). HPV 16 and 18, specifically, have been identified as the cause of 70% of cervical cancers (HPV16 alone is responsible for 50% of cervical cancers) (Bosch et al., 2008).
High-risk HPV infection caused by CIN disorders is a long-term, reversible precancerous lesion, and early intervention can effectively prevent cervical cancer. Therefore, HPV detection through physical examination is an effective means to prevent cervical disease and is important in guiding clinical treatment and disease prognostication, as well as in tracking possible relapse after treatment (de Sanjose et al., 2010). Clarifying the type of HPV involved in infection further enhances such critical clinical decision-making and plan of action. The present study aimed to identify the prevalence of cervical HPV DNA in women undergoing routine physical examination.
Materials and Methods {#sec1-2}
=====================
Clinical data {#sec2-1}
-------------
During the time period between March and December 2013, data were collected from women volunteered for cervical cancer screening at Hua-Dong Sanatorium, a specialized facility for medical examination and health consulting services. The population was comprised of individuals spanning various enterprises and institutions in Shanghai, China. All participants reported experience in vaginal sexual intercourse and voluntarily joined the study free of charge. Informed written consent was obtained from all participants prior to examination. Exclusion criteria included: younger than 20 years of age, Chronic smoker, two or more sex partners, oral contraceptives use for more than 2 years, injection of HPV vaccine, hysterectomy, prior abnormal cytology, prior cervical cancer, pelvic radiotherapy and pregnancy. A total of 2,452 women were enrolled in the study. According to study objective, study participants, aged 20 to 80, were divided into 6 age-defined sub-groups including 20-29, 30-39, 40-49, 50-59, 60-69 and older than 70 years of age. The study was approved by the local ethics committee.
Methods {#sec2-2}
-------
In accordance with practice protocols, none of the participants had a history of vaginal drug delivery or flushing within 3 days prior to examination. A cervical smear was taken upon receipt of consent. To obtain the smear, the patient's cervix was exposed with a speculum. A gynecologist with more than 5 years of clinical experience collected samples of exfoliated cervical cells using a standard cytobrush at the transformation zone of the uterine cervix for HPV genotyping.
We performed the HPV Genotyping detection kit (Shenzhen Asia Energy Biotechnology Co, Ltd, in China)to simultaneously identify 23 HPV types, including HPV 6, 11, 16, 18, 31, 33, 35, 39, 42, 43, 45, 51-53, 56, 58, 59, 66, 68, 73, 81-83. According to the manufacturer's protocol, the kit uses in vitro amplification of PCR and DNA reverse dot blot hybridization combined with DNA chip technology. A positive and negative control was provided within the kit, and was used in the process of testing according to the directions. The process includes HPV DNA Extraction, PCR amplification, hybridization, washing membrane, developing color and results analyses. Specific as follows, HPV-DNA extraction: specimens are collected from cervical via HPV collecting kit (sufficiently elute the samples from the cervical brush), after a centrifuge of 13,000 r/min for 10 min, remove the supernatant and reserve the cell block in tube-bottom; then, add 50 ul of lysate for suspension and precipitate, water bath for 10 min at 100 °C, a centrifuge of 13,000 r/min for 10 min, reserve the supernatant; PCR amplification: add 5 μl of extracted sample in each reaction tube, with the total reaction volume of 25 μl, then add 1 drop mineral oil, a negative and positive control can be set in each test; PCR amplification follows the following condition: 50 °C for 15 min, 95 °C for 10min, 94 °C for 30 sec, 42 °C for 90 sec, 72 °C for 30 sec, 40 cycles, 72 °C for 5min; Hybridization: first, denature PCR amplification products to get single-stranded DNA; then, based on base-pair complementary, add the PCR amplification products in low density gene patches with total 23 gene different HPV subtype probe; then hybridize. Develop the color after the hybridization; Results analysis: HPV genotype hybridization membrane has 23 gene types as well as Biotin and internal control as quality control. HPV genotyping test result can be interpreted as negative when 2 quality control dots are positive while others are negative. If 2 quality control dots are positive, and there is 1 or more HPV genotype dots is positive, the HPV virus type can be determined by the positive point based on the distribution of HPV genotype profile on the membrane.
Any types of HPV detected were defined as HPV-positive, and all others were designated negative. HPV types were grouped into high-risk and low-risk types as follows: high-risk (16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 67, 68, 69, 70, 73, 82, and its subtype IS039) and low-risk (6, 11, 40, 42, 54, 55, 57, 61, 62, 64, 71, 72, 83, 84, and 89) (Munoz et al., 2003). A positive result for any one HPV type was defined as single HPV infection. HPV-positive samples with two or more identified HPV types were defined as multiple HPV infection.
Statistical analysis {#sec2-3}
--------------------
Statistical analyses were performed to characterize the prevalence of HPV subtype infection in the different aged groups as well as for the HPV-positive women. Prevalence was calculated by counting single and multiple infections. Chi-square tests were utilized for comparing differences in HPV-positive rates among different aged groups. All statistical analyses were performed with IBM SPSS Statistics 20 (IBM, Armonk, New York City, USA). All p values were two-sided with significance determined when p \< 0.05.
Results {#sec1-3}
=======
HPV DNA prevalence among different age groups
The mean age (Standard deviation, SD) of study participants was 47.7±9.8 years. The prevalence of HPV DNA in different age groups (20-29, 30-39, 40-49, 50-59, 60-69, ≥70) were 9.3%, 15.6%, 17.1%, 22.1%, 23.0% and 20.0%, respectively ([Table 1](#T1){ref-type="table"}). The prevalence of overall HPV DNA detection increased significantly along with increasing age ([Figure 1](#F1){ref-type="fig"}).
######
Incidence of HPV in Different Age Groups
Age group Cases HPV+ rate n (%) Proportion HPV women n (%) Proportion of all participants n (%)
----------- ------- ------------------ ---------------------------- --------------------------------------
20-29 54 5/54 (9.3) 5/463 (1.1) 5/2452 (0.2)
30-39 463 72/463 (15.6) 72/463 (15.6) 72/2452 (2.9)
40-49 861 147/861 (17.1) 147/463 (31.8) 147/2452 (6.0)
50-59 795 176/795 (22.1) 176/463 (38.0) 176/2452 (7.2)
60-69 239 55/239 (23.0) 55/463 (11.9) 55/2452 (2.2)
≥70 40 8/40 (20.0) 8/463 (1.7) 8/2452 (0.3)
Total 2452 463/2,452 (18.9) 100 463/2,452 (18.9)
{#F1}
Overall HPV DNA prevalence {#sec2-4}
--------------------------
Overall prevalence of any of the 23 HPV types within the study population was 18.9% (463/2452). HPV 52 was the most prevalent type of HPV observed in this study of high-risk HPV infection, followed by HPVs 58, 53, 16 and 68 in descending order of prevalence. HPV 52, 58, 53, 16, 51 were associated with 57.5% (257/447) of high-risk HPV-positive, including single and multiple infections. Among high-risk HPV-positive women, single infections accounted for 72.0% with the other 28.0% exhibiting multiple HPV infection. In this population, the prevalence of HPV 45 and 73 was low, and HPV 82 was not detected. HPV 81 was the most prevalent type of HPV found in cases of low-risk HPV infection, followed by HPV 43, 6, 42 and 11 in descending order of prevalence. HPV 81 and 43 were associated with 59.1% of low-risk HPV infections ([Table 2](#T2){ref-type="table"}).
######
Distribution of Detected HPV Infection
HPV type Single infection n Multiple infection n Overall infection n Overall Infection rate n/2,452(%)
----------- -------------------- ---------------------- --------------------- ----------------------------------- ------
Low-risk 6 16 15 31 1.3
11 6 5 11 0.4
42 17 11 28 1.1
43 24 9 33 1.3
81 36 32 68 2.8
High-risk 16 20 19 39 1.6
18 12 11 23 0.9
31 7 4 11 0.4
33 7 12 19 0.8
35 6 5 11 0.4
39 11 3 14 0.6
45 1 1 2 0.1
51 18 18 36 1.5
52 52 36 88 3.6
53 25 18 43 1.8
56 19 11 30 1.2
58 30 21 51 2.1
59 10 9 19 0.8
66 8 10 18 0.7
68 20 13 33 1.3
73 2 1 3 0.1
83 6 1 7 0.3
Total All types 353 265 618 24.3
Note, Single infection n, Number of Single infection cases; Multiple infection n, Number of Multiple infection cases; Overall infection n, Number of Overall infection cases; Overall Infection rate n/2,452 (%) , Proportion of Overall infection cases in all participants.
Single infection of HPV type {#sec2-5}
----------------------------
The single HPV DNA prevalence was 14.4% (353/2452), and prevalence of high-risk HPV types was found in 10.4% (254/2452) of the total study population. HPV 52 was found in 2.1% (52/2452) of the women tested and was the most prevalent HPV type. Other HPV types were HPV 58, 53, 16 and 51 in descending order of prevalence.
The prevalence of low-risk HPV types was found in 4.0% (99/2452) of the population. HPV 81 was the most prevalent of the low-risk types and was found in 1.5% (36/2452) of the women tested. Other HPV types were HPV 43, 42, 6 and 11 in descending order of prevalence.
Multiple infections of HPV types {#sec2-6}
--------------------------------
The multiple HPV DNA prevalence was 4.5% (110/2452). The prevalence of high-risk HPV types was observed in 106 participants. HPV 52 was the most prevalent type in this study, which was found in 36 individuals. Other HPV types identified were HPV 58, 16, 53 and 51 in descending order.
The prevalence of low-risk HPV types was observed in 60 cases. HPV 81 was found in 32 individuals, and was the most prevalent of the low-risk types. Other HPV types were HPV 6 and 42 in descending order of prevalence. Among multiple infection cases, one individual was infected with eight types of HPV (HPV6, 11, 16, 51, 52, 53, 58, 66), one was infected with five HPV types (HPV16, 18, 51, 85, 68), and 9 were infected with four HPV types (HPV31, 51, 52, 53).
Discussion {#sec1-4}
==========
In the present study, the HPV DNA prevalence was 18.9% among all women examined, including 14.4% in single and 4.5% in multiple HPV infection. Overall prevalence of HPV DNA detection increased significantly with increasing age. The characteristics of the participants were of a population that received a healthy check-up, which could represent infection rate in healthy individuals to some extent.
The study participants were 20 years and older, due to delegation of the 2012 consensus conference that proposed less intensive management for other young women with abnormal cytology, according to the American Society for Colposcopy and Cervical Pathology (ASCCP) Consensus Guidelines in 2012 (Massad et al., 2013).
The distribution of HPV types varies among different geographical regions and populations. The HPV DNA prevalence among women in the present study was higher than reported in previous studies in Europe. The worldwide crude HPV prevalence estimate among women with normal cytology was 10.0% (de Sanjose et al., 2007). In Greece, the HPV DNA prevalence was 5.8% (Agorastos et al., 2014). The overall prevalence of HPV in Nigeria was reported at 37% (Akarolo-Anthony et al., 2014). The worldwide HPV DNA prevalence in women without documented cervical abnormalities is approximately 11-12% with higher rates in sub-Saharan Africa (24%), Eastern Europe (21%) and Latin America (16%) (Forman et al., 2012).
In China, some studies have reported HPV prevalence and the distribution of the types of HPV in women in areas including Shanghai. According to one study, human papillomavirus was detected in 5,173 of 17,148 women (30.2%) with the highest prevalence occurring in the ≤20 age group (45.2%) (Xue et al., 2009). Another study reported that the rate of HPV-positive diagnosis was 12.6% in suburban areas of Shanghai, and the five top HPV types were HPV52, 16, 58, 18 and 33 (in descending order) (Zhang et al., 2013).
Our finding that HPV 52 was the most prevalent type is consistent with other Chinese studies(de Sanjose et al., 2007; Xue et al., 2009; Zhang et al., 2013; Agorastos et al., 2014) and similar to findings reported from South Africa and North India (Bhatla et al., 2008; Vinodhini et al., 2012). In contrast, HPV 16 was by far the most common high-risk HPV type in developed countries(de Sanjose et al., 2007; Kroupis et al., 2007; Panotopoulou et al., 2007; Agorastos et al., 2014). In Iranian, the HPV DNA prevalence types were HPV 16, 18, 6/11, 31, and 33, and HPV 16 was the most frequent type in all five different groups (Bhatla et al., 2008; Vinodhini et al., 2012).
At present, there are some different views about the association between HPV infection and age, some research reported that it showed "U" shape of age-specific prevalence of HPV infection occurring in developing countries or economic backward areas (Baseman and Koutsky, 2005). This study suggests that the prevalence of HPV DNA was significantly increased in more than 50 year-old female, with a high incidence of cervical cancer in China in the age of 45 to 60 years old. The observed increase in HPV prevalence in older age groups could be attributed to human estrogen and progesterone level changes affect the cervical epithelial metaplasia process and support the HPV replication; on the other hand, the declining of body's immune function leads up to decrease the ability of HPV clearance and increase the susceptibility to an HPV infection. Due to the gradual reduction of the sex in elderly women, the HPV DNA prevalence could reflect persistent or potential HPV infection. Therefore, it is important to carry out the detection of HPV infection in the elderly women in order to prevent the occurrence of cervical cancer and precancerous lesions.
The data provide valuable information for HPV based screening and prevention for women in China where robust screening and vaccination programs are not yet established. As the HPV types differ in the Chinese population from those in the Western countries, for optimal population coverage, in addition to HPV 16 and 18, next generation HPV prophylactic vaccines including HPV-52 and -58 may offer higher protection for Chinese women. The unique epidemiological feature of HPV52 and 58 in China should be considered in the design and evaluation of diagnostic assays of cervical screening intended for Chinese women.
Low-risk HPV causes genital warts, but its rate of detection is low, considering the harm is small, so the body is easy to exclude. This study found that HPV81 was the most common type of low-risk types, which was related to regional, ethnic differences, living environment, health habits and subjects. This study reflects the HPV carrying status among healthy people and conforms to the regional characteristics of HPV distribution. Further study is needed to address the prevalence profile of low-risk HPV types in both cytology-normal and cytology-abnormal populations.
In summary, this study covered a considerable proportion of the female population in Shanghai, the most populous city in China according to the latest census. Therefore, the study is somewhat representative of the wider general population.
As a next step, a meta-analysis of studies on genotype-specific prevalence of cervical HPV DNA in the Yangtze River Delta in China is planned and should expand our knowledge base on the overall prevalence and prevalence of specific types of HPV in China.
Statement conflict of Interest {#sec2-7}
------------------------------
The authors declare that they have no conflict of interest.
|
{
"pile_set_name": "PubMed Central"
}
|
Background {#Sec1}
==========
Fluorescent core/shell semiconductor quantum dots (QDs) are characterized by the excellent optical properties such as broader emitting range, higher photoluminescence (PL) quantum yields (QYs), and higher optical and chemical stability than traditional organic dyes. These advantages open up opportunities for revolutionary advances in fluorescent labels for biomedical diagnostics, molecular imaging, and photoelectric field \[[@CR1]--[@CR7]\]. According to the band alignment between core and shell materials, the core/shell QDs can be classified as type-I, reverse type-I, and type-II structures. Type-I QDs characterized by the "nested" band alignment structure, which can confine both electrons and holes to the core region to enhance radiative recombination and physically separate the surface of the optical active core from its surrounding medium, and thus improve the PL intensity and optical stability \[[@CR6]--[@CR9]\]. Despite these favorable properties, a small Stokes shift (only a dozen of nanometers), referred to as the difference between the absorption and PL spectra, generates a serious reabsorption, leading to an overall emission loss and limiting their application in quantitative determination \[[@CR10], [@CR11]\]. In contrast, type-II QDs with staggered bandgap alignment promote spatial separation of electron and hole into different regions of the core/shell structure. The subsequent band edge e--h recombination transition energy is smaller than the bandgap of either of the constituent material components, leading to a significant red-shifted emission, which is unavailable with either monocomponent material. The oscillator strength of the first exciton absorption feature of type-II QDs dramatically diminishes compared to that of core QDs \[[@CR12], [@CR13]\]. The largely redshifted emission and the flatted first exciton absorption peak both lower the overlap of the absorption and emission spectra, which suppresses the reabsorption, and benefits the biological quantitative detection. The typical type-II ZnSe/CdS QDs have tunable emission from bluish violet to red range and suppressed reabsorption \[[@CR13]\]. However, the electrons delocalized in CdS shell are vulnerable to trap from surface defects or surrounding medium, lead to low fluorescence quantum yield. A feasible solution is coating ZnSe/CdS QDs with ZnS outermost shell not only to passivate the surface for increasing the quantum yield and optical stability, but also to restrict the leak of toxic Cd element, reducing the biotoxicity. So far, the majority of researches have focused on type-I QDs, and only a few have been carried out on ZnSe/CdS/ZnS type-II/type-I QDs \[[@CR12]--[@CR15]\]. Moreover, all of the studies about the synthesis process of ZnSe/CdS/ZnS QDs used a two-step preparation by pre-purifying crude ZnSe core QDs and used toxic and expensive phosphines. In addition, none of them involved the application of ZnSe/CdS/ZnS QDs in biological detection.
Here, we report a phosphine-free one-pot method to synthesize high-quality red emission ZnSe/CdS/ZnS type-II/type-I core/shell QDs with the feature of reabsorption-suppression and the first using of the QDs to fabricate fluorescence-linked immunosorbent assay (FLISA). We used highly reactive and low toxic Se precursor (ODE-Se) and zinc oleate to synthesize high-quality ZnSe core QDs, and then achieved multishell growth without purification of core quantum dots. This shows great promise for large-scale synthesis of core/shell quantum dots. The quantum yield of as-prepared red emitting ZnSe/CdS/ZnS type-II/type-I QDs can reach as high as 82% with lower toxic cadmium content which is particularly important to reduce biotoxicity in biomedical field. Moreover, the QDs have large Stokes shift as well as flatted first absorption peak, which lead to low overlap of PL and absorption spectra and suppressed reabsorption effect.
C-reactive protein (CRP), as an acute phase protein from liver cells, has been regarded as an early indicator of infection and autoimmune disorders. Such diseases often commence at very low CRP levels. Therefore, the sensitive quantitative immunoassay analysis of CRP levels in biological samples is of critical importance for diagnosis and monitoring the evolution of diseases \[[@CR16]\]. Compared with traditional enzyme-linked immunosorbent assay (ELISA), FLISA is time-saving without enzymatic reaction and less sensitive to environmental conditions deriving from the optical quality of fluorescent QDs \[[@CR17]\]. Hence, FLISA has become a new research hotspot of quantitative immunoassay \[[@CR2], [@CR18]--[@CR21]\]. Here, we firstly demonstrated FLISA quantitative immunoassay using water-soluble ZnSe/CdS/ZnS type-II/type-I core/shell QDs as fluorescent probe. The limit of detection (LOD) for quantitative detection of CRP protein reached 0.85 ng/mL which was 15% more sensitive than that of the CdSe/ZnS type-I QDs based FLISA in control experiments. The high QYs, excellent optical stability, and low reabsorption effect may promote the application of ZnSe/CdS/ZnS type-II/type-I QDs in biomedicine and photoelectric field.
Methods {#Sec2}
=======
Chemicals {#Sec3}
---------
Cadmium oxide (CdO, 99.99%), zinc oxide (ZnO, 99.9%, powder), selenium (Se, 99.9%, powder), 1-octadecene (ODE, 90%), 1-octanethiol (OT, 98%), oleic acid (OA, 90%) Poly(maleic anhydride-alt-1-octadecene) (PMAO), and 2-(N-morpholino) ethanesulfonic acid (MES) were purchased from Aldrich. Paraffin oil (analytical grade), acetone (analytical grade), hexanes (analytical grade), and methanol (analytical grade) were obtained from Beijing Chemical Reagent Co., Ltd, China. NaOH, HCl, Na~2~CO~3~, NaHCO~3~, KH~2~PO~4~, Na~2~HPO~4~, H~3~BO~3~, Na~2~B~4~O~7~ · 10H~2~O, and Tween-20 were purchased from Shanghai Sangon Co., Ltd, China. Bovine serum albumin (BSA) and calf serum were purchased from Sigma. 1-ethyl-3-(3-(dimethylamino) propyl) carbodiimide (EDC), N-Hydroxysulfosuccinimide (sulfo-NHS) and the microplates were purchased from Thermo Fisher Scientific (USA). Mouse anti C-reaction protein monoclonal antibody and CRP antigen were obtained from Abcam (USA). All chemicals and solvents were used as received without further purification.
Stock Solution for the Se Precursor (0.1 M) {#Sec4}
-------------------------------------------
Se (6 mmol) and ODE (60 mL) were loaded in a 100 mL three-necked flask, and then heated to 220 °C for 180 min under nitrogen to obtain yellow clear solution.
Stock Solution for Zn Precursor (0.4 M) and Cd Precursor (0.2 M) {#Sec5}
----------------------------------------------------------------
ZnO (30 mmol), oleic acid (30 mL), and 45 mL ODE were loaded in a 100 mL three-necked flask and heated to 310 °C under nitrogen to obtain a clear solution. The resulting solution was allowed to cool down to 140 °C for injection. The preparation process for Cd precursor was same to Zn precursor except the concentration was adjusted to 0.2 M and the reaction temperature was set to 240 °C.
Typical Synthesis of ZnSe/CdS/ZnS Type-II/Type-I QDs {#Sec6}
----------------------------------------------------
As a typical synthetic procedure, 4 mL Se precursor and ODE (15 mL) were placed in a 100-mL round flask. The mixture was heated to 310 °C. At this temperature, 2 mL of Zn precursor was quickly injected into the reaction flask. Aliquots were extracted at different time intervals to monitor the evolution of PL position which coordinates with the particle size of QDs. When the core nanocrystals reached the desired dimension, the reaction temperature was reduced to 230 °C for CdS shell growth. Without any purification steps, as the mixture of Cd precursor and 1-octanethiol (the molar radio of OT and cation is 1:1.2) started to be added dropwise with a syringe pump at a rate of 3 mL/h, while the temperature was elevated to 310 °C. The same process was applied to shell growth of ZnS. Aliquots of QDs were taken during the reaction to analyse the development of ZnSe/CdS/ZnS core/shell QDs. The as-prepared core/shell QDs were purified by adding acetone, and then redispersed in chloroform.
Typical Synthesis of CdSe/ZnS Type-I QDs {#Sec7}
----------------------------------------
CdSe/ZnS QDs were synthesized as described in our previous reports \[[@CR7]\]. Subsequently, the process of phase transfer, QDs-antibody detection probes and the preparation of FLISA were same to them of ZnSe/CdS/ZnS QDs, which would be described below.
Phase Transfer of ZnSe/CdS/ZnS QDs for Bioapplication {#Sec8}
-----------------------------------------------------
Poly(maleic anhydride-alt-1-octadecene) (PMAO)---an amphiphilic oligomer whose hydrophobic ends interleave with the organic coating on QDs and hydrophilic end groups are free to interact with the surrounding buffer---has been used to transfer hydrophobic QDs into pure water. The ZnSe/CdS/ZnS QDs and PMAO were mixed and dissolved in chloroform with sonication (molar ratio of QD/PMAO was 1:7). After that, chloroform was then removed by rotary evaporation at 45 °C. Then, an equal volume of 0.1 M NaHCO~3~ aqueous solution (pH = 8.5) was added to dissolve the QDs-PMAO. PMAO encapsulated ZnSe/CdS/ZnS type-II/type-I QDs show no fluorescence loss and have high stability in aqueous solution under a wide range of pH environments.
Preparation of QDs-antibody Detection Probes {#Sec9}
--------------------------------------------
The procedure has been widely reported in previous literatures \[[@CR1]--[@CR3]\]. The QDs-PMAO were firstly conjugated with monoclonal CRP antibody through activation of these -COOH groups by EDC and sulfo-NHS. Next, a certain amount of monoclonal CRP antibodies were added into the QDs and dissolved in BS buffer and then were blocked by BSA. Finally, the product was washed by 5 mM BS buffer (pH = 8.0) under centrifugation. The QDs-mAb was stored in 50 μL BS buffer (5 mM, pH = 8.0).
Preparation of Antibody-Coated Fluorescence Microplate {#Sec10}
------------------------------------------------------
Primary antibody (the concentration of CRP monoclonal antibody was 1.8 mg/mL) was diluted with a carbonate-bicarbonate buffer (50 mM pH = 9.6, CB buffer) in each well of microplate. Subsequently, the microplate was covered with sealing film and incubated at 4 °C for 24 h. In order to remove extra coating antibody, the microplate was washed three times with a wash buffer (0.05% Tween-20 in 10 mM PBS, pH = 7.4). Then, excess binding sites were blocked with 0.5% (w/v) BSA in 10 mM PBS (pH = 7.4) for incubating overnight at 4 °C. This process ensured that all the available and remaining binding sides of the microplate wells were covered. The microplate was dried in a chamber with constant temperature and humidity for 24 h, then stored at 4 °C for future use.
Quantitative Detection of CRP by Fluorescence-Linked Immunoassay {#Sec11}
----------------------------------------------------------------
In each well of a 96-well microplate, contained coating antibody was added 100 μL of the standard antigen and diluted to a series of concentrations with the sample buffer. The plates were incubated at 37 °C for 30 min and then washed five times with a wash buffer. Next, 100 μL of the QDs-mAb probes were diluted with the probe buffer (10% calf serum (v/v) in 0.1 M PBS) into each well were incubated and washed same as the above-mentioned process.
Characterization {#Sec12}
----------------
Room temperature UV--vis absorption and PL spectra were measured with an Ocean Optics spectrophotometer (mode PC2000-ISA). PL quantum yields (QYs) were determined by comparison of the integrated fluorescence intensity of the QD samples in solution with that of standard of known QYs (Rhodamine 101 (R101) ethanol solution (0.01% HCl, QY = 100%) as the standard). Transmission electron microscopy (TEM) studies were performed using a JEOL JEM-2010 electron microscope operating at 200 kV. Phase determination of the products was carried out on an X-ray diffractometer (D8-ADVANCE) using Cu-Ka radiation (wavelength = 1.54 Å). The sizes of QDs and QD-antibody probe were recorded using dynamic light scattering (Nano-ZS 90, Malvern Instruments, UK).
Results and Discussion {#Sec13}
======================
The UV--vis absorption and PL spectra of shell growth process are shown in Fig. [1](#Fig1){ref-type="fig"}. The Stokes shift of ZnSe core is only 8 nm with the first absorption peak at 420 nm and emission peak at 428 nm, and emission full width at half maximum (FWHM) of 17 nm. However, when only one monolayer (ML) of CdS shell grew on ZnSe core, the Stokes shift significantly increased to 54 nm with the first absorption peak at 497 nm and emission peak at 551 nm, with emission full width at half maximum (FWHM) of 38 nm. Due to delocalization of the electron wavefunction, the PL emission of ZnSe/CdS QDs (629 nm) is redshifted with respect to ZnSe core QDs (428 nm), and the FWHM broadened to 52 nm with the deposition of CdS shell. The broadened PL FWHM originated from an enhanced Frölich-like exciton-phonon interaction \[[@CR22], [@CR23]\]. What is more, the oscillator strength of the first absorption peak rapidly weakened due to spatially indirect type-II transition from the valence band of the ZnSe to the conduction band of the CdS. The phenomenon is common in type-II QDs \[[@CR24]--[@CR26]\]. While the dramatic increase in absorption in blue spectral region (\<500 nm) was assigned to the band gap of bulk CdS material (2.42 eV). In consequence, the red emission, the flatted first exciton absorption peak and the robust absorption in short wavelength region (\<500 nm) of ZnSe/CdS type-II QDs resulted in large Stokes shift and suppressed reabsorption. With the sequential growth of ZnS shell, the PL was shifted to short wavelength and the FWHM was narrowed from 52 nm to 43 nm. This phenomenon was ascribed to the fact that the Zn-atoms diffuse into the Cd-rich regions to form gradient shell at high temperature, thus increasing the band-offset of the shell. The QYs could increase from 20 to 82% during the shell growth process of CdS and ZnS onto ZnSe cores.Fig. 1Evolution of the UV--vis absorption and PL spectra upon consecutive growth of ZnSe/CdS/ZnS core/shell QDs
It was noteworthy that the excess of Zn-OA precursor relative to Se precursor in the core solution was necessary to obtain high-quality and monodisperse ZnSe core QDs. As a result, ZnCdSeS alloy shell would be inevitably formed during the addition of Cd-OT shell precursor in the initial stage since the high temperature (\>200 °C) promoted the cation exchange and diffusion between Zn^2+^ and Cd^2+^, and the rich octanethiol in Cd-OT precursor could also react with the excess Zn-OA \[[@CR7], [@CR12], [@CR27], [@CR28]\]. The alloy shell can not only reduce the interfacial tension and defect to increase the QYs but also generate energy barrier for holes. The conduction band edge of ZnCdSeS alloy shell material was located between that of ZnSe and CdS, while the valence band edge was deeper than CdS. This formed a larger potential trough in the valence band edge as an additional blocking layer for the holes (Scheme [1](#Sch1){ref-type="fig"}) \[[@CR12]\]. This energy band structure can further reduce the overlap of electrons and holes to decrease the strength of the first exciton absorption peak and suppress the reabsorption.Scheme 1The schematic structure (*up*) and the band alignment (*bottom*) for ZnSe/CdS/ZnS type-II/type-I QDs based on the corresponding abrupt and alloyed interfaces, respectively
The information about core/shell band structure and the evolution of PL and absorbance during the shell growth can be further verified by the comparison of XRD, TEM, and HRTEM of core and core/shell nanocrystals. The powder XRD patterns of the ZnSe core, ZnSe/CdS, and ZnSe/CdS/ZnS core/shell (the left picture of Fig. [2](#Fig2){ref-type="fig"}) show that the diffraction peaks become sharp and shift to positions corresponding to bulk wurtzite (WZ) CdS or ZnS crystal structures. This result is consistent with the predicted values for the larger volume of CdS or ZnS shell compared to ZnSe core in the final core/shell QDs and testifies the growth of multishells. Moreover, a transformation from zinc blende (ZB) type ZnSe cores to WZ-type core/shell has happened with CdS and ZnS coating. This phenomenon has been reported in CdSe/CdS core/shell QD systems \[[@CR29], [@CR30]\]. TEM images of the core QDs and several core/shell QDs are shown in Fig. [2a](#Fig2){ref-type="fig"} − 2 F. All the TEM images show nearly monodisperse spherical QDs with gradually increscent average diameters from original ZnSe core (3.90 nm) to ZnSe/6CdS type-II QDs (7.98 nm) and ZnSe/6CdS/6ZnS type-II/type-I QDs (11.92 nm). As shown in the HRTEM image of ZnSe core QDs (inset of Fig. [2a](#Fig2){ref-type="fig"}), the lattice spacing of the (111) planes is 0.32 nm, and the QDs possess good crystallinity and monodispersity. With the growth of shells, the lattice parameter showed corresponding change (0.35 nm for CdS and 0.31 nm for ZnS) in accordance with the XRD data. The results have clearly suggested the controllable growth of CdS and subsequent ZnS shell materials.Fig. 2*Left*: XRD patterns of ZnSe/CdS/ZnS type-II/type-I QDs with different shell growth stages. The diffraction lines for zinc blende (ZB) ZnSe (bottom), WZ CdS (*middle*), and WZ ZnS (*top*) are indexed. *Right*: the corresponding TEM and HRTEM (*inset*, bar of 5 nm) images of the ZnSe core (**a**), ZnSe/CdS type-II QDs with 2 ML (**b**), 4 ML (**c**) and 6 ML (**d**) CdS shell, respectively, and ZnSe/CdS/ZnS type-II/type-I QDs with 3 ML (E) and 6 ML (F), respectively
Meanwhile, to confirm the evolution of composition during the growth of multishell, the energy dispersive x-ray spectroscopy (EDS) analysis has been taken for different stages of the core/shell growth as shown in Additional file [1](#MOESM1){ref-type="media"}: Table S1. The EDS data show that the corresponding changes of content of Cd, Se, Zn, and S are in accordance with the shell growth stage. But it is worth noting that Cd/(Zn + Cd) molar ratio in the resulting ZnSe/CdS QDs is higher than the Cd/(Zn + Cd) feed ratio due to the cation exchange between Zn^2+^ and Cd^2+^ during the process of coating CdS shell onto ZnSe core at above 200 °C. By comparison with the issued literature about typical type-I CdSe/CdS/ZnS QDs (Cd molar ratio \~ 40%) \[[@CR31]\], the type-II/type-I ZnSe/CdS/ZnS QDs contained far less Cd element (\~13%).
A visual comparison of hydrophobic QDs in chloroform and PMAO-capped QDs in water under sun light and UV light is shown in Additional file [1](#MOESM1){ref-type="media"}: Figure S1 (A). It appears that both QD solutions are undisturbed and no aggregation of nanoparticles. Both QDs emitted the same red light when they were illuminated with a hand-held UV lamp (365 nm). Additional file [1](#MOESM1){ref-type="media"}: Figure S1 (B) shows the UV−visible absorption and PL spectra of QDs before and after phase transfer. Compared with the hydrophobic QDs in chloroform, the PL spectrum of PMAO-capped QDs has negligible change, indicating no obvious change in the particle size and PL properties. Additional file [1](#MOESM1){ref-type="media"}: Figure S1 (C) and (D) present the TEM images of the QDs before and after phase transfer which further determines the morphology and state of PMAO-capped QDs. It appears that PMAO-capped QDs are well isolated and rarely observed as aggregates.
In order to confirm the formation of PMAO encapsulated QDs during phase transfer process, FTIR spectroscopy was used to characterize the functional groups on the surface of QDs (shown in Additional file [1](#MOESM1){ref-type="media"}: Figure S2). The decrease of peak at 1777 cm^−1^ (compared PMAO with QDs-PMAO) and the increase of peak at 1715 cm^−1^ (compared the three samples) were attributed to the decomposition of anhydride and the formation of -COOH. The FTIR results indicated PMAO amphiphilic polymer was successfully coated on the surface of the ZnSe/CdS/ZnS QDs.
The stability of as-prepared QDs is very important for the subsequent treatment. Figure [3a](#Fig3){ref-type="fig"} shows that the evolution of the relative PL stability of hydrophobic ZnSe/CdS/ZnS QDs upon purification steps. The PL intensity of ZnSe/CdS/ZnS core/shell QDs could maintain 85% upon many cycles of purification in hexanes. As shown in Fig. [3b](#Fig3){ref-type="fig"}, the colloidal stability of the QDs-PMAO in BS buffer (pH = 7.2) was estimated as a function of time at 25 °C. The PL intensity almost kept constant and the solution was clear even after 400 h. This indicates that the QDs-PMAO is stable in BS solution without any damage. Figure [3c](#Fig3){ref-type="fig"} shows the variation in PL intensity of QDs-PMAO which were immersed in acidic-to-basic pH (pH = 1-14, adjusted by HCl or NaOH) solution for 30 min. The PL intensity of hydrophilic QDs could retain over 85% except when the PH = 14. Figure [3d](#Fig3){ref-type="fig"} shows the effect of temperature parameter on the relative fluorescence intensity of QDs-PMAO. The fluorescence intensity gradually decreased with the temperature increase but still maintained 76% at 90 °C, while the PL peaks gradually shifted to longer wavelength due to the thermal expansion and electron--phonon coupling effect. All stability evaluation indicates that the ZnSe/CdS/ZnS type-II/type-I QDs and QDs-PMAO were very stable and thus suitable for biological applications.Fig. 3Stability test of hydrophobic QDs upon (**a**) repeated purification process steps; stability test of QDs-PMAO upon (**b**) BS buffer, (**c**) PH, and (**d**) temperature
CRP is an acute phase protein from liver cells, and the level of it is regarded as an early indicator of infection and autoimmune disorders. Here, the as-synthesized PMAO-capped ZnSe/CdS/ZnS QDs are coupled with CRP to demonstrate the possibility for application in quantitative immunoassay. Comparison diagram of the fluorescence spectra of aqueous ZnSe/CdS/ZnS QDs and the QDs-mAb are shown in Fig. [4a](#Fig4){ref-type="fig"}. Clearly, the PL peak shape of both samples are approximately identical except that the fluorescence intensity declines to 60% after coupling reaction due to inevitable sample loss during centrifuge separation process. It proves the excellent optical stability of ZnSe/CdS/ZnS type-II/type-I QDs even after the antibody protein coupling process.Fig. 4Fluorescence spectra (**a**) and dynamic light scattering (**b**) of the QDs-PMAO and QDs-mAb in buffer
To further investigate the effect of conjugation on the size of QDs, the aqueous QDs and QDs-mAb are characterized by dynamic light scattering (DLS). The DLS results (Fig. [4b](#Fig4){ref-type="fig"}) clearly show that both of the samples have narrow size distribution with good monodispersity and maintain a discrete form without aggregation, while the hydrodynamic size increases from 46 to 120 nm after coupling process. This demonstrates the success in conjugation with CRP antibodies.
Further, we used the ZnSe/CdS/ZnS type-II/type-I composite quantum dots instead of CdSe/ZnS type-I QDs as fluorescent probe to establish a FLISA for quantitative detection of CRP. The assemble process is shown in Additional file [1](#MOESM1){ref-type="media"}: Scheme S1. Figure [5a](#Fig5){ref-type="fig"} shows the relative fluorescence intensity of QDs fluorescence label for immunoassay in detection of various concentrations of CRP antigens (the standard CRP antigen is diluted to 0, 1, 5, 10, 50, 100, 200, 400 ng/mL). Obviously, the PL intensity gradually increases with the increase of concentration of CRP. Figure [5b](#Fig5){ref-type="fig"} shows that the correlation between the fluorescence intensity and the target CRP concentrations obey a quadratic regression curve equation of *y* = 44230 + 8121.1x-10.3x^2^ with a correlation coefficient of 0.9991, which is the closer to 1 the better. The working concentrations range from 0 to 400 ng/mL. The LOD is one of the key parameters for immunoassay for FLISA. By using the ZnSe/CdS/ZnS composite type-II/type-I QDs as fluorescent probe, the sensitivity of quantitative detection of CRP is 0.85 ng/mL, which is 15% more sensitive than that of FLISA based on CdSe/ZnS type-I QDs (1.00 ng/ml) (in Additional file [1](#MOESM1){ref-type="media"}: Figure S3).Fig. 5Photoluminescence spectra of FLISA for determination of different concentration of CRP antigen (**a**) and the standard curves (**b**)
Additionally, recovery experiments were used to evaluate the matrix effect of the FLISA with a series of known standard CRP antigens for analysis, and the final concentrations covered the low, medium, and high-risk levels. As shown in Table [1](#Tab1){ref-type="table"}, all of the recovery rates are within the range of 83.61-105.9%. These results indicate that the FLISA based on ZnSe/CdS/ZnS type-II/type-I QDs with reabsorption suppression property has high accuracy and is of great advantages in quantitative immunoassay detection.Table 1Recovery tests for CRP determinationSampleExperimental value (ng/mL)Theoretical value (ng/mL)Recovery (%)P1288.5330096.18P2148.7315099.15P374.997599.99P426.3225105.90P54.18583.61
Conclusions {#Sec14}
===========
We report a phosphine-free one-pot method to synthesize reabsorption-suppressed ZnSe/CdS/ZnS type-II/type-I core/shell QDs with large Stokes shift and flat first absorption peak. These characteristics reduce reabsorption and improve the level of fluorescence output. The as-synthesized QDs have high QY (82%) and high stability against various test conditions. Then, we first use ZnSe/CdS/ZnS QDs as fluorescence probe in FLISA for quantitative detection of CRP protein with high sensitivity (LOD of 0.85 ng/mL). It indicates that the reabsorption-suppressed ZnSe/CdS/ZnS type-II/type-I core/shell QDs have promising potential for application in biomedicine and photoelectric fields.
Additional file {#Sec15}
===============
Additional file 1:Visual, PL, and TEM comparison of QDs before and after phase transfer. FTIR characterizations of hydrophobic QDs, PMAO and water-soluble QDs-PMAO. The EDS data of QDs with different injection volume of shell precursor. The scheme of assemble process of FLISA for quantitative detection of CRP. The PL spectra and standard curve of FLISA based on CdSe/ZnS QDs. (DOCX 1332 kb)
BSA
: Bovine serum albumin
CRP
: C-reactive protein
DLS
: Dynamic light scattering
EDC
: 1-ethyl-3-(3-(dimethylamino) propyl) carbodiimide
EDS
: Energy dispersive x-ray spectroscopy
ELISA
: Enzyme-linked immunosorbent assay
FLISA
: Fluorescence-linked immunosorbent assay
FWHM
: Full width at half maximum
LOD
: Limit of detection
MES
: 2-(N-morpholino) ethanesulfonic acid
OT
: 1-octanethiol
PMAO
: Poly(maleic anhydride-alt-1-octadecene)
QDs
: Quantum dots
QYs
: Quantum yields
sulfo-NHS
: N-Hydroxysulfosuccinimide
**Electronic supplementary material**
The online version of this article (doi:10.1186/s11671-017-2135-4) contains supplementary material, which is available to authorized users.
The authors gratefully acknowledge the financial support from the research project of the National Natural Science Foundation of China (31570531, 61504040, and 61474037), Program for Science & Technology Innovation Talents in University of Henan Province (No. 14HASTIT009), and Funding of Jilin Provincial Education Department, China (grant numbers 2015--470).
Authors' Contributions {#FPar1}
======================
SW participated in the fabrication of ZnSe/CdS/ZnS quantum dots, measured and analyzed the data, and wrote the manuscript. JL and RW helped the water phase-transfer. YL prepared the FLISA and characterized it. HW, MX, HS, LSL, and XC supervised the study and revised the paper. All authors read and approved the final manuscript.
Competing Interests {#FPar2}
===================
The authors declare that they have no competing interests.
Publisher's Note {#FPar3}
================
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
|
{
"pile_set_name": "PubMed Central"
}
|
Introduction {#s1}
============
Behavioral Findings of Mirror Visual Feedback {#s1-1}
---------------------------------------------
Motor recovery after stroke depends on the intrinsic properties of the central nervous system to reorganize its structure, function and connections. Therefore, it is important that rehabilitation programs facilitate neural plasticity by including repetitive, intensive and task-relevant movement training (Takeuchi and Izumi, [@B53]). Mirror visual feedback (MVF) has been successfully applied to enhance motor performance without training not only in healthy adults (Nojima et al., [@B40]; Hoff et al., [@B25]; von Rein et al., [@B60]) but also in patients suffering from focal brain lesions (Altschuler et al., [@B2]; Yavuzer et al., [@B63]; Dohle et al., [@B14]; Michielsen et al., [@B37]; Thieme et al., [@B57]). For MVF-training, participants perform a task while observing the movements of their active limb in an orthogonally placed mirror providing MVF. Interestingly, MVF-induced performance improvements do not seem to be affected by hand dominance (Rjosk et al., [@B50]) which indicates that both hands can be equally well used for MVF-training.
However, it is known that to some extent, unilateral skill training results in performance gains of both the trained and untrained limb (Obayashi, [@B43]; Perez et al., [@B46]; Kwon et al., [@B31]). In the literature, this phenomenon called intermanual transfer, has been described for multiple motor tasks, like strength training (Carroll et al., [@B9]), sequential pinch force tasks (Camus et al., [@B7]) and reaching movements (Criscimagna-Hemminger et al., [@B10]). It seems to be influenced by several factors, like structural integrity of the corpus callosum or complexity of the task (Bonzano et al., [@B5]). Interestingly, neural mechanisms underlying intermanual transfer seem to be divergent from those mediating MVF-induced performance improvements (Nojima et al., [@B40], [@B41]) and further studies are needed to distinguish these mechanisms/phenomena.
Findings of Studies Combining Non-Invasive Brain Stimulation and Mirror Visual Feedback {#s1-2}
---------------------------------------------------------------------------------------
It has been shown that MVF is associated with functional alterations in primary motor cortex (M1) representing the resting hand as assessed with transcranial magnetic stimulation (TMS; Nojima et al., [@B40]; Kumru et al., [@B30]). Furthermore, studies applying facilitatory transcranial direct current stimulation (a-tDCS) over M1 representing the resting hand showed enhanced mirror illusion as well as superior MVF-induced performance improvements in healthy participants relative to sham stimulation (Hoff et al., [@B25]; Jax et al., [@B26]; von Rein et al., [@B60]).
Neural Correlates of Mirror Visual Feedback as Assessed with fMRI {#s1-3}
-----------------------------------------------------------------
Analysis of tasked-based functional magnetic resonance imaging (fMRI) providing MVF during a grasping task or finger-thumb opposition task showed that functional alterations are not limited to M1 but also involve other motor-related brain areas such as the premotor cortex (PMC) and the supplementary motor area as well as the primary somatosensory cortex (S1; Hamzei et al., [@B24]; Fritzsch et al., [@B17]). Moreover, also higher order areas of perceptual-motor coordination and visual attention seem to be involved in the processing of MVF such as the superior temporal gyrus and the secondary visual cortex (V2) ipsilateral to the moving hand as well as the bilateral anterior intraparietal sulcus (aIP; Matthys et al., [@B35]; Numata et al., [@B42]).
However, in these previous studies, brain activation was captured during MVF performance using task-based fMRI, thus one can only draw conclusions on instantaneous brain adaptations. Resting-state fMRI (rs-fMRI), however, provides unique insights in functional plasticity beyond instantaneous task-induced brain changes. Resting-state functional connectivity (rs-FC) represents neuronal activity that is not attributable to specific task-evoked fluctuations in blood-oxygen-level-dependent (BOLD) signal but represents signals that are intrinsically generated by the brain (Biswal et al., [@B4]; Fox and Raichle, [@B16]). These low-frequency changes are not random but highly organized, temporally correlated across distinct brain regions and of behavioral relevance (Buckner and Vincent, [@B6]; Guerra-Carrillo et al., [@B22]). Furthermore, it has been shown that these activity patterns reflect the subsequent processing and consolidation of information gained from earlier learning (Miall and Robertson, [@B36]; Peigneux et al., [@B45]). Albert et al. ([@B1]) demonstrated that after a visuomotor perturbation task, subsequent resting activity in task-specific networks was modulated. Interestingly, changes in rs-FC in this study reflected learning or adaptation processes rather than activity changes induced by movement execution as pure motor performance did not elicit any changes in rs-FC. Hence, we used rs-fMRI to describe learning-induced neuroplasticity (Buckner and Vincent, [@B6]; Guerra-Carrillo et al., [@B22]), without confound of pure motor performance, because participants perform no task during scanning (Albert et al., [@B1]; Ma et al., [@B34]; Vahdat et al., [@B59]). Furthermore, it has been shown that resting-state networks are consistent across experimental sessions and participants (Damoiseaux et al., [@B11]; Shehzad et al., [@B52]).
Thus, the present study aims at evaluating learning-related changes in functional network connectivity induced by MVF and aims at identifying brain regions mediating MVF-induced performance improvements of the untrained hand. We hypothesized that MVF (mirror group, MG) during right hand (RH) training results in superior performance improvement of the untrained left hand (LH) as compared to no MVF (control group, CG; Nojima et al., [@B40]; Hoff et al., [@B25]; von Rein et al., [@B60]). Furthermore, we hypothesized that MVF during training will result in additional rs-FC changes in MG as compared to CG. Based on previous findings, we expected significant alterations in sensorimotor as well as higher order visual areas (Matthys et al., [@B35]; Hamzei et al., [@B24]; Numata et al., [@B42]). Additionally, we investigated whether these rs-FC changes are associated with the individual behavioral gains of the untrained LH.
Materials and Methods {#s2}
=====================
Participants {#s2-1}
------------
Thirty-five healthy and task naïve participants (mean age: 26.91 ± 0.61 years; 16 females) took part in the study. All participants were right-handed as assessed with the Edinburgh Handedness Inventory (mean handedness score of 87.91 ± 2.79; Oldfield, [@B44]). The study was performed in accordance with the Declaration of Helsinki and was approved by the local ethics committee of the University of Leipzig. All participants gave their written informed consent. None of the participants was taking any centrally acting medication and none of the participants had a history of neurological illness. Highly skilled musicians and sportsmen were excluded from the study as well as participants who had contraindications for MRI measurements. Total hours of sports per week and hours of fine-motor training per week were assessed with a questionnaire. Seventeen participants were randomly assigned to the MG, the group that performed the ball-rotation task with MVF. The other 18 participants were assigned to the CG and performed the same task without MVF (see also Table [1](#T1){ref-type="table"} for group demographics). Before and after the experiment, all participants rated their levels of attention, fatigue and discomfort on a visual analog scale (VAS).
######
**Group demographics**.
Age (years) Gender (female/male) LQ Sports/week (hours) Fine-motor/week (hours)
----------------- -------------- ---------------------- -------------- --------------------- -------------------------
**MG** *n* = 17 26.53 ± 0.95 8/9 87.82 ± 3.92 2.71 ± 0.47 0.18 ± 0.18
**CG** *n* = 18 27.28 ± 0.75 8/10 88.00 ± 4.08 3.44 ± 0.75 0.33 ± 0.28
*Hours of sports per week and hours of fine-motor training per week were assessed with a questionnaire, handedness (Laterality Quotient, LQ) was assessed with the Edinburgh Handedness Scale (range: −100 (full left-handed) to +100 (full right-handed)). Statistical analysis revealed no significant differences in age, gender, LQ, sports/week or fine-motor training/week between groups. All values are depicted as mean ± standard error of the mean. MG, mirror group; CG, control group*.
Experimental Procedures {#s2-2}
-----------------------
### Study Design {#s2-2-1}
The study consisted of one experimental session per subject. Here, we first acquired a rs-fMRI (rs-fMRI_pre) and a T1-weighted anatomical image of each participant. Then, MG and CG performed a complex ball-rotation task outside of the scanner immediately followed by another rs-state fMRI (rs-fMRI_post) \~10 min after termination of the ball-rotation task. The only difference between groups was the condition used in the ball-rotation task: participants in MG received MVF during RH training, whereas participants in CG performed the same training without MVF (Figure [1](#F1){ref-type="fig"}). A between-group design was chosen to ensure that all subjects were task naïve before participating in the ball-rotation task. See below for a detailed description of the complex ball-rotation task.
{#F1}
### Ball-Rotation Task and MVF {#s2-2-2}
We adapted the ball-rotation task introduced by Nojima et al. ([@B40]). In short, participants were seated at a desk with their arms extended in front of them and their hands in a relaxed pronated position. To assess baseline performance, participants in both groups were asked to rotate two cork balls (diameter 30 mm; weight 10 g) for 1 min with their LH in a counterclockwise orientation as quickly as possible (LH_pre) while observing the performance of the LH. Subsequently, the training period was conducted: participants were instructed to rotate the balls with their RH but in a clockwise orientation as quickly as possible. Here, however, a wooden barrier placed over the RH prevented a direct view of the RH. Participants in MG were asked to observe the movements of their RH in a mirror (providing MVF) placed between their arms. The MVF-training was performed for 10 trials of 1 min each, separated by 30 s breaks. This led to a total of 15 min of MVF-training. During MVF-training, participants were instructed to concentrate on the mirror image and to relax their LH behind the mirror. Participants in CG performed the same training of the RH (10 trials of clockwise rotation, trial length 1 min with 30 s breaks), except that there was no mirror present. Participants in CG were instructed to observe their resting LH while a direct view of the RH during training phase was prevented by a wooden barrier placed over the RH. After this training phase, performance of the untrained LH was retested in both groups in the same manner as for LH_pre by rotating the balls with the LH in a counterclockwise direction for 1 min (LH_post). Motor performance was videotaped throughout the experiment. The number of ball-rotations/min was counted by an experimenter, who was blinded to the study procedures, and analyzed offline to assess motor dexterity.
### MRI Data Acquisition {#s2-2-3}
We used a Siemens Magnetom Tim Trio 3 T scanner equipped with a 32-channel head coil. Each experimental session consisted of two scans of echo-planar-imaging (EPI): one scan before the ball-rotation task (rs-fMRI_pre; \~7.6 min) and one scan after (rs-fMRI_post; \~7.6 min). Each scan was acquired with a total of 200 whole-brain volumes using the following parameters: acquisition matrix 64 × 64, 3 mm isotropic voxel, 1 mm gap between slices, 34 slices, TR = 2300 ms. For co-registration, we acquired T1-weighted anatomical images (MP2RAGE; \~5.12 min) before the ball-rotation task with the following parameters: voxel size = 1 mm × 1 mm × 1 mm, 176 sagittal slices, FOV = 256 × 240 mm. Participants were instructed to relax but to stay awake while keeping their eyes closed during image acquisition.
### MRI Data Analysis {#s2-2-4}
Analysis of the rs-fMRI data was performed with the FMRIB Software Library (FSL 5.0[^1^](#fn0001){ref-type="fn"}), utilizing the independent component analysis (ICA)-AROMA pipeline, the fastECM toolbox (Wink et al., [@B62]) and SPM12[^2^](#fn0002){ref-type="fn"} running in MATLAB version 8.6[^3^](#fn0003){ref-type="fn"}. First, the T1-weighted images were segmented using SPM12, and a skull-stripped version was created with fslmaths. Second, standard motion correction, spatial smoothing with a Gaussian kernel of 6 mm FWHM and linear registration of functional and T1-weighted images to each other and to the MNI space were performed. To further detect and remove motion-related artifacts ICA-AROMA (Pruim et al., [@B47],[@B48]) was used to perform an ICA on the functional data to identify and remove head motion related components by employing predefined temporal (high frequency content and maximum correlation) as well as spatial features (edge and cerebrospinal fluid fraction). Subsequently, additional nuisance correction was performed by regressing out signal from white matter and cerebrospinal fluid (physiological noise; Fox and Raichle, [@B16]) and by high pass filtering (0.01 Hz). Within the preprocessing, the functional data was also resampled at a resolution of 2 × 2 × 2 mm^3^ as this is the standard in most fMRI analyses.
For the analysis of functional connectivity, the eigenvector centrality maps (ECM) approach was used. Eigenvector centrality can quantify the relative importance, or centrality, of an individual node on a network as a whole. The centrality is high if a node is connected to many nodes that themselves are "central" (Lohmann et al., [@B33]). Hereby, the importance of points in brain networks can be measured and visualized (Lohmann et al., [@B33]). Centrality analyses are supposed to enable the interpretability of connectivity matrices used in graph analyses combined with the high spatial resolution of voxel-based methods (Wink et al., [@B62]). ECM, in particular, were used for our data analysis for its exploratory whole brain approach independently from predefined seed regions (Lohmann et al., [@B33]). Using the preprocessed functional data, an ECM analyses was performed for each subject and each scan time point (rs-fMRI_pre and rs-fMRI_post) separately using the fastECM toolbox (Wink et al., [@B62]). The fastECM algorithm was chosen for its shorter computation times and lower storage requirements for high-resolution fMRI data. Within the preprocessed functional images, the voxel-wise connectivities between all pairs of voxels were computed with the fastECM program within a study-specific gray matter mask. To create this study-specific mask, the individual gray matter masks derived from segmenting the T1-weighted images were added up. Then a threshold was applied to include only voxels that contained gray matter from all participants (total of 202519 voxel). Hence, a 3D voxel-wise ECM per subject and scan time point was created and used for further statistical analyses.
All statistical analyses were performed using SPM12. To assess the effect of MVF on the rs-FC single-subject ECM images were compared using a 2 × 2 flexible factorial design with the factors TIME (rs-fMRI_pre vs. rs-fMRI_post) and GROUP (MG vs. CG), and complemented with subsidiary *t*-tests. To analyze a potential relationship between changes in functional connectivity and behavioral improvements, a correlation analysis was performed with SPM12. First, ECM difference images (rs-fMRI_post minus rs-fMRI_pre) were created on the single subject level using the ImCalc-toolbox in SPM12. We then performed a correlation analysis per group correlating the difference images with the performance improvement of the untrained LH.
All stated findings are significant at *p* \< 0.05 with cluster-wise (*p* = 0.001) FWE correction for multiple comparisons.
Statistical Analyses {#s2-3}
--------------------
### Data Analyses: Ball-Rotation Task {#s2-3-1}
We used the Statistical Software Package for Social Sciences (IBM SPSS Version 22) for statistical analyses of the behavioral data. The number of ball-rotations/min both for the LH_pre and LH_post as well as for the RH (trials T1--T10) during the training period was used to assess motor performance. To test for differences in baseline performance, an independent samples *t*-test was used to compare the number of ball-rotations/min of the LH_pre between groups (MG vs. CG). Subsequently, a repeated-measures ANOVA (ANOVA-RM) with factor TRIAL (LH_pre vs. LH_post) and GROUP (MG vs. CG) was performed to assess the effect of MVF during the training period on performance improvements of the untrained LH. Here, the factor TRIAL was the independent variable or the within-subject factor with two levels (LH_pre vs. LH_post). The factor GROUP was the between-subjects factor (MG vs. CG). Supporting this, independent samples *t*-tests were conducted to compare the absolute and relative amount of performance improvement of the untrained LH after the training period across groups (MG vs. CG). Paired *t*-tests, comparing LH_pre vs. LH_post, were used to evaluate the performance improvement of the untrained hand within each group. Finally, the RH performance over the whole training period was evaluated using another ANOVA-RM with factor TRIAL (T1--T10) and GROUP (MG vs. CG) to test whether participants in both groups improved their RH during training period. Again, factor TRIAL was the within-subject factor (10 levels T1--T10) and factor GROUP was the between-subjects factor (MG vs. CG). Further within- and between-group comparisons were performed using *post hoc t-tests*. A Bonferroni corrected *p-value* of \<0.05 was considered to be significant. Greenhouse-Geisser correction was applied, if applicable. Behavioral data are presented as mean ± standard error (SE). The Eta-squared (*η*^2^) is reported for each ANOVA as a measure of the effect size. We considered an *η*^2^ of ≥0.02 as small, ≥0.13 medium and ≥0.26 large effect as proposed by Miles and Shevlin ([@B38]).
Results {#s3}
=======
Groups did not differ regarding age (*t*~(33)~ = −0.609, *p* = 0.547), gender (*t*~(33)~ = 0.151, *p* = 0.881), handedness (*t*~(33)~ = −0.031, *p* = 0.975), weekly hours of sports (*t*~(33)~ = −0.818; *p* = 0.419) or fine-motor training (*t*~(33)~ = −0.468; *p* = 0.643; see also Table [1](#T1){ref-type="table"}). Prior to the experiment, participants in both groups did not differ in their levels of attention (*t*~(33)~ = −0.336, *p* = 0.739), fatigue (*t*~(33)~ = −0.097, *p* = 0.924) or discomfort (*t*~(33)~ = 0.852, *p* = 0.400).
Behavioral Results: Ball-Rotation Task {#s3-1}
--------------------------------------
### Left Hand Performance {#s3-1-1}
Baseline performance of the LH did not differ between MG and CG (MG: 31.82 ± 2.94; CG: 35.33 ± 3.01 ball-rotations/min, *t*~(33)~ = −0.833; *p* = 0.411). Both groups showed significant performance improvements of LH after RH training (ANOVA-RM with factor TRIAL (LH_pre vs. LH_post) × GROUP (MG vs. CG): *F*~(1,\ 33)~ = 7.925; *p* = 0.008; *η*^2^ = 0.194) (Figure [2](#F2){ref-type="fig"}). However, LH performance improvements were more pronounced in MG as compared to CG. MG improved by 9.65 ± 1.33 ball-rotations/min (*t*~(16)~ = 7.233; *p* \< 0.001) (35.43 ± 5.63% \[*t*~(16)~ = 6.296; *p* \< 0.001\]). CG improved by 4.28 ± 1.36 ball-rotations/min (*t*~(17)~ = 3.146; *p* = 0.006) (16.29 ± 5.29% \[*t*~(17)~ = 3.077; *p* = 0.007\]). Subsequent analyses revealed significantly higher performance improvements of the untrained hand in MG compared to CG (absolute performance improvement: *t*~(33)~ = 2.815; *p* = 0.008; relative performance improvement: *t*~(33)~ = 2.480; *p* = 0.018; Figure [2](#F2){ref-type="fig"}).
{#F2}
### Right Hand Performance {#s3-1-2}
In both groups, performing the ball-rotation task during the training phase (trials T1--T10) resulted in significant performance gains of the RH. Participants in MG improved on average by 22.94 ± 3.02 ball-rotations/min (*t*~(16)~ = −7.608; *p* \< 0.001) and participants in CG by 10.17 ± 2.20 ball-rotations/min (*t*~(17)~ = −4.619; *p* \< 0.001). But there was a significant difference in the absolute amount (T10 − T1) of performance improvement of the trained RH between groups (ANOVA-RM with factor TRIAL (T1--T10) × GROUP (MG vs. CG): *F*~(3.796,125.278)~ = 4.666; *p* = 0.002; *η*^2^ = 0.124). *Post hoc* between-group comparisons showed a significantly higher amount of performance improvement in MG compared to CG (*t*~(33)~ = 3.450; *p* \< 0.002). However, there was no correlation between the amount of performance improvement of the untrained LH and performance improvements of the trained RH in either group (MG: *r* = 0.27; *p* = 0.299; CG: *r* = 0.213; *p* = 0.396).
For a description of behavioral data of participants in MG see also Rjosk et al. ([@B50]).
See also Table [2](#T2){ref-type="table"} for a complete breakdown of group data of the untrained LH and trained RH in the ball-rotation task.
######
**Group data of the untrained left hand (LH) pre and post training phase as well as of the trained right hand (RH) during training phase (trials T1--T10) in the ball-rotation task**.
LH_pre T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 LH_post
----------------- -------------- -------------- -------------- -------------- -------------- -------------- -------------- -------------- -------------- -------------- -------------- --------------
**MG** *n* = 17 31.82 ± 2.94 23.00 ± 4.43 27.94 ± 4.47 33.00 ± 4.37 36.41 ± 3.46 36.35 ± 3.62 39.24 ± 3.23 40.71 ± 3.37 43.29 ± 3.25 43.82 ± 3.15 45.94 ± 3.14 41.47 ± 3.17
**CG** *n* = 18 35.33 ± 3.01 35.83 ± 2.37 39.06 ± 2.86 39.22 ± 3.09 40.78 ± 2.73 43.67 ± 3.28 44.33 ± 3.06 44.50 ± 3.36 45.00 ± 3.45 47.67 ± 3.49 46.00 ± 3.35 39.61 ± 2.80
*Behavioral data for the untrained LH and trained RH (ball-rotations/min). Participants in the MG received MVF during training phase (T1--T10), while participants in the CG watched their resting untrained LH during training phase. Performing the ball-rotation task during training phase (T1--T10) resulted in significant performance gains of the untrained LH as well as trained RH in both groups, while there was a significant higher amount of performance improvement in both hands in MG. For details, see text. Data are depicted as mean ± standard error of the mean*.
Rs-fMRI Results: Centrality Changes {#s3-2}
-----------------------------------
### ECM {#s3-2-1}
The interaction of TIME × GROUP showed a significant increase in centrality in MG compared to CG in left V1, V2 ipsilateral to the untrained LH (Figure [3A](#F3){ref-type="fig"}). There were no regions that showed a significant decrease in centrality in MG compared to CG. Subsequently, a series of *t*-tests were conducted to further investigate changes in centrality within and between groups. No significant differences in baseline rs-FC were found between groups as assessed by an independent samples *t*-test. To assess changes in centrality due to MVF, a paired-sample *t*-test for MG was conducted, which showed a significant increase in centrality in left visual areas (V4) and a non-significant trend in left PMC (*p* = 0.082) as well as a significant centrality increase in bilateral primary sensorimotor cortices (SM1). It further indicated a significant decrease in centrality in left aIP in MG (Figure [3B](#F3){ref-type="fig"}). The corresponding *t*-test for CG showed a decrease in centrality in the right frontopolar cortex (FPC) only (Figure [3C](#F3){ref-type="fig"}). This pattern of results revealed by the post vs. pre comparison of MG was mirrored in the interaction contrast TIME × GROUP by supplementary but (for multiple comparisons) non-significant changes in rs-FC in right V2, in left PMC and in bilateral SM1.
{#F3}
### Correlation Analyses {#s3-2-2}
A correlation analyses of centrality changes in rs-fMRI and relative performance improvements of the untrained LH revealed a significant positive relationship in left SM1 for MG (*r* = 0.842; *p* \< 0.001; Figure [4](#F4){ref-type="fig"}). No such correlation could be observed for CG. In addition, there was no evidence for negative correlations between centrality changes and relative performance improvements within either group.
{#F4}
All results are summarized in Table [3](#T3){ref-type="table"}.
######
**MNI-coordinates of peak voxels of training-induced changes in eigenvector centrality (ECM)**.
Contrast Anatomical area MNI (*X Y Z*) *Z*-max No. of voxels
---------------------------------------- ----------------- --------------- --------- --------------- ------ -----
Interaction MG vs. CG *(TIME × GROUP)* left V1, V2 −16 −98 −12 4.11 169
MG *(rs-fMRI_pre \< rs-fMRI_post)* left V4 −24 −84 −16 4.24 440
left SM1 −34 −28 52 3.91 208
right SM1 46 −20 48 3.76 221
left PMC\* −6 −10 48 4.13 114
MG *(rs-fMRI_pre \> rs-fMRI_post)* left aIP −42 −52 50 3.9 170
CG *(rs-fMRI_pre \> rs-fMRI_post)* right FPC 50 48 14 4.03 185
MG *(correlation analysis)* left SM1 −66 −6 28 4.24 156
*Listed are all findings that are significant at *p* \< 0.05 in the corresponding contrast with cluster-wise FWE correction for multiple comparisons. \*Denotes a non-significant trend only (*p* = 0.082). Please note that we found a positive correlation only in MG between left SM1 and performance improvements of the LH, for details see "Results" Section and Figure [4](#F4){ref-type="fig"}. MG, mirror group; CG, control group; V1--V4, visual cortices; SM1, primary sensorimotor cortices; PMC, premotor cortex; aIP, anterior intraparietal sulcus; FPC, frontopolar cortex*.
Discussion {#s4}
==========
The aim of the present study was to investigate learning-related rs-FC changes associated with MVF-induced behavioral changes in the untrained LH. In line with our hypotheses, we observed superior performance improvements in MG compared to CG. The ECM analyses revealed no differences in baseline rs-FC between groups but indicated a significant increase of centrality in left visual areas (V1, V2) in MG due to MVF-training compared to CG. Furthermore, subsidiary within group comparisons showed further functional alterations in left V4, bilateral SM1 and left aIP in MG, only.
While MG and CG both showed significant improvements in LH performance, the effect was more pronounced in MG. This finding seems to be in contrast with a control experiment performed by Nojima et al. ([@B40]) showing no performance improvements of the untrained LH when motor training with the RH was performed without MVF in the same complex ball-rotation task. However, Reissig et al. ([@B49]) could also not replicate the findings by Nojima et al. ([@B40]). One potential explanation for our divergent results might be that participants in our study performed the task with the LH twice as long as participants in the study of Nojima et al. ([@B40]) (1 min of ball-rotation instead of 30 s) giving our participants in CG more time to familiarize with the task. Furthermore, participants receiving MVF during motor training improved their dexterity above this simple familiarization effect. As we hypothesized, participants in MG showed significantly stronger behavioral gains of the untrained hand compared to CG indicating the superior effect of MVF.
Considering intermanual transfer, one might argue that MVF is not solely the driving mechanism behind the performance improvements of the untrained hand. However, the underlying neural mechanisms of these phenomena seem to be divergent. Intermanual transfer seems to be mediated via alterations in intracortical and interhemispheric inhibition (IHI) between homologous M1s (Perez et al., [@B46]; Camus et al., [@B7]), whereas Nojima et al. ([@B40]) did not detect any alterations in IHI due to MVF. Furthermore, MVF-induced performance improvements were even shown in callosotomized patients (Nojima et al., [@B41]).
Contrary to our hypotheses, we did not find significant centrality changes between groups in higher order visual areas, but in left V1 and V2. However, within group comparisons revealed a significant increase in centrality in left V4 only in MG. This increase in rs-FC that we observed due to MVF-training in left visual areas is in line with previous findings of Lewisa et al. ([@B32]), who demonstrated that visual perceptual learning can modify the resting activity between the trained visual cortex and areas involved by the task. Furthermore, regarding the underlying mechanisms for MVF in motor rehabilitation, Altschuler et al. ([@B2]) proposed that the visual image might recruit the PMC and hereby connect the visual input to the motor system. V1, V2 and V4 are embedded into the dorsal visual stream and hereby connected to the posterior parietal cortex (PPC; Goodale and Milner, [@B20]). The PPC is involved in motor control and action planning and is specialized in visuomotor transformations required for visually guided movements (Fogassi and Luppino, [@B15]). The ability to coordinate and integrate somatosensory and visual information with motor signals is represented in areas of the inferior parietal lobule (IPL; Jeannerod and Jacob, [@B27]). Furthermore, the left IPL is thought to be preferentially involved in performing more complex actions as well as in storing these complex representations (Glover, [@B19]). Based on this, our reported activation in the left visual areas only in MG may indicate an influence of MVF on the left PPC and IPL, and thus may suggest a potential influence of MVF on storage of representations after visuomotor learning (Goodale and Milner, [@B20]; Glover, [@B19]; Fogassi and Luppino, [@B15]; Jeannerod and Jacob, [@B27]). However, results on hemispheric specific involvement of visual areas are heterogeneous as Matthys et al. ([@B35]) found the right superior occipital gyrus (located in V2) to be associated with MVF in a fingertapping task with the RH.
Importantly, we showed a positive correlation of performance improvement of the untrained LH and centrality changes in the left SM1. This might indicate that S1 representing the trained hand plays an important role in mediating the MVF-induced effects. Interestingly, when considering the anatomical somatotopy of SM1, the cluster of the correlation analysis is not located in the hand area but in the ventral part of SM1. However, it is discussable whether the MRI resolution used in the current study is high enough to disentangle sub regions of the sensorimotor system and whether features of brain structure are an appropriate tool to assign functional properties (Wang et al., [@B61]).
By taking a closer look at the underlying modifications in rs-FC within each group via paired *t*-tests, we found a significant increase in centrality in MG in bilateral SM1 and left V4 and a non-significant trend towards an increase in left PMC ipsilateral to the untrained LH. Furthermore, we observed a decrease in centrality in the left aIP in MG within these analyses.
The observed rs-FC changes in MG in bilateral SM1 and the trend towards an increase in centrality in left PMC are in line with previous TMS- and task-based fMRI-studies reporting similar regions to be associated with MVF. Hamzei et al. ([@B24]) showed MVF-specific activation changes within the bilateral PMC as well as in M1 representing the trained hand during action observation and imitation in a grasping task using task-based fMRI. Garry et al. ([@B18]) observed enhanced excitability of the M1 representing the resting hand during MVF, and Kumru et al. ([@B30]) found a cortical disinhibition in the ipsilateral M1 using TMS. However, Fritzsch et al. ([@B17]) observed activity changes in S1 and argued that not M1 but S1 representing the untrained hand is directly modulated by a mirror task. In line with this, Moseley and Wiech ([@B39]) showed an improvement of tactile discrimination in patients with complex regional pain syndrome already after one intervention with MVF, whereas improvement of motor function in patients with a hemiparesis after stroke requires repetitive training (Thieme et al., [@B57]). Taken these results together, we observed an increase in centrality in bilateral SM1 and a non-significant trend in the left PMC representing the trained RH.
However, it is discussable, whether these bilateral modulations in SM1 are solely due to MVF or due to bilateral sensorimotor training *per se*. Tamè et al. ([@B54]) proposes, that S1, together with the secondary somatosensory cortex, processes tactile information from both sides of the body, especially during demanding bilateral tasks.
Considering the properties of the aIP and its connection to the ipsilateral ventral PMC enabling visually guided, object-directed hand actions (Fogassi and Luppino, [@B15]), our observed connectivity changes in the aIP, PMC and SM1 fit into the model of gating MVF-induced activation from the aIP into the motor system as proposed by Numata et al. ([@B42]). They found a significant activation in the bilateral aIP in a finger-thumb opposition task-based fMRI only when MVF was provided. Contrary, we found a decrease in connectivity in left aIP. However, an anti-correlation of spontaneous BOLD activity between parts of a network (here aIP, visual and motor areas) was explained to be an efficient computational state to promote task recruitment and flexibility and may prevent the network elements from interfering with each other (Guidotti et al., [@B23]). Thus, our observed decrease in centrality in left aIP in MG might indicate a rise in efficiency of connectivity between involved network elements.
However, the impact of our findings within MG is limited due to the fact that functional alterations in left V4, bilateral SM1, left PMC and left aIP did not reach significance in the interaction contrast between groups.
When taking a closer look at modifications in rs-FC within CG, paired *t*-test analyses revealed only a significant decrease in centrality in the right FPC. This area is cyto-architectonically defined as the lateral part of Brodmann's area 10 and as a component of the frontal lobe involved in decision-making (Koechlin and Summerfield, [@B29]). However, according to Koechlin and Hyafil ([@B28]), the main function of FPC is "cognitive branching" meaning the ability to switch between independent tasks and to temporarily suspend a task while another is being performed. Changes in centrality in FPC may indicate that participants in CG experienced the performance of the ball-rotation task with the LH and RH as separate and independent task units. Contrarily, receiving MVF during performance of the RH training may have induced the illusion of pure LH training since participants in MG did not show changes in connectivity in FPC. However, changes in centrality in FPC also did not reach significance in the interaction contrast.
Limitations of the Study {#s4-1}
------------------------
Since participants in our study performed the task only once and did not receive multiple MRI scans after the ball-rotation task, we cannot make inferences about the exact time course of the observed rs-FC changes. However, there is evidence that plasticity changes after training depend on the length of the training (Dayan and Cohen, [@B12]). Immediate changes, as assessed in the present study, might differ from later and long term changes (Taubert et al., [@B55], [@B56]; Ma et al., [@B34]; Gryga et al., [@B21]). The fact that we showed a correlation between SM1 rs-FC changes and gains in performance of the LH does not explicitly highlight a causal relation between functional plasticity and training effects. Hence, future studies should use non-invasive brain stimulation to selectively target these brain regions (e.g., by down-regulating its activity) and thereby investigate this causal relationship in more detail.
Conclusions and Clinical Implications {#s4-2}
-------------------------------------
To our knowledge, the present study is the first to investigate the effect of MVF-induced performance improvements by means of rs-fMRI. In support of our hypotheses, we observed superior performance improvements in MG compared to CG. Additionally, we identified learning-related changes induced by MVF in left visual areas. Further functional alterations in left V4, left PMC and left aIP ipsilateral to the untrained LH as well as in bilateral SM1 were revealed by within group comparisons in MG, only. These findings might indicate that the effects of MVF-training are likely the result of interactions between perceptual and motor cortical regions.
Our results suggest that the hemisphere ipsilateral to the untrained hand (which would correspond to the unaffected hemisphere in patients) plays an important role in the underlying mechanism of MVF, since we found a positive correlation between centrality changes in left SM1 and performance improvements of the untrained LH. In line with this, Deconinck et al. ([@B13]) hypothesized that MVF-induced recruitment of ipsilateral motor pathways might attribute to the behavioral gains due to MVF. Interestingly, activity in ipsilateral pathways has been discussed in the context of stroke to be beneficial for motor recovery (Benecke et al., [@B3]; Carr et al., [@B8]; Schwerin et al., [@B51]).
Since we showed that MVF is capable of inducing learning-related neuroplastic modifications indicated by changes in rs-FC, our results might be of clinical relevance. Our findings support the application of MVF in neurorehabilitation to facilitate neural plasticity in patients suffering from unimanual motor impairments and have identified additional target regions for non-invasive brain stimulation techniques.
Author Contributions {#s5}
====================
VR, PR and AV designed the study. VR performed the experiment. PR, VR, CJS and JL analyzed the data. VR, EK, MH, BS, JL, CJS and PR interpreted results of the experiment; edited and revised the manuscript. PR and VR drafted the manuscript. All authors approved the final version of the manuscript.
Conflict of Interest Statement {#s6}
==============================
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
EK was funded by the Fazit Stiftung, Frankfurt/Main, Germany.
^1^<http://www.fmrib.ox.ac.uk/fsl>
^2^<http://www.fil.ion.ucl.ac.uk/spm/software/spm12>
^3^<http://mathworks.com>
aIP
: anterior intraparietal sulcus
CG
: control group
ECM
: eigenvector centrality maps
fMRI
: functional magnetic resonance imaging
FPC
: frontopolar cortex
IHI
: interhemispheric inhibition
IPL
: inferior parietal lobule
LH
: left hand
M1
: primary motor cortex
MG
: mirror group
MVF
: mirror visual feedback
PMC
: premotor cortex
PPC
: posterior parietal cortex
RH
: right hand
Rs-FC
: resting-state functional connectivity
Rs-fMRI
: resting-state functional magnetic resonance imaging
S1
: primary somatosensory cortex
tDCS
: transcranial direct current stimulation
TMS
: transcranial magnetic stimulation
V1--4
: visual cortices.
[^1]: Edited by: Stephane Perrey, University of Montpellier, France
[^2]: Reviewed by: Tamer Demiralp, Istanbul University, Turkey; Luigi Tamè, Birkbeck, University of London, UK
|
{
"pile_set_name": "PubMed Central"
}
|
Dear Editor,
The self-renewing intestinal epithelium is functionally organized into proliferating crypts and differentiating villus^[@CR1]^. At the bottom of the crypts, Lgr5^+^ intestinal stem cells (ISCs) reside between terminally differentiated Paneth cells and actively drive the self-renewal of intestinal epithelium^[@CR1],\ [@CR2]^. The balance between proliferation and differentiation of Lgr5^+^ ISCs is critical for the intestinal homeostatic maintenance^[@CR1]^. The establishment of the three-dimensional (3D) self-renewing organoid culture system in Matrigel supplemented with the growth factors EGF, Noggin and R-spondin (ENR) provides an ideal system to dissect how stem cells integrate multiple signals to maintain stemness and undergo malignant transformation^[@CR3]^. However, the 3D geometry nature of organoids prevents visual tracing of stem cells and challenges the dissection of interaction between stem cells and niche cells with microscopy. Much effort has been made to establish two-dimensional (2D) culture of intestinal epithelial cells, but whether these systems are suitable for adult Lgr5^+^ stem cell study remains elusive^[@CR4]--[@CR6]^. Thus, it would be of great value to establish *a bona fide* 2D monolayer culture system to study Lgr5^+^ ISCs and facilitate drug screening. Also, it is interesting to determine whether Lgr5^+^ cells are able to maintain stem cell activity on monolayer without the curvature structure.
To establish a 2D monolayer culture system, we first explored whether Lgr5^+^ ISCs could survive and maintain stemness on a matrix-coated plate. Isolated intestinal crypts from *Lgr5-GFP-IRES-CreERT2* mice were re-suspended in the ENR-containing organoid culture medium and seeded on collagen I- or Matrigel-coated plates with or without the non-muscle myosin IIA inhibitor blebbistatin as our previous work showed that blebbistatin could significantly improve the survival of Lgr5^+^ ISCs and the growth of organoids^[@CR7]^. Although a few cells survived in the absence of blebbistatin, the addition of blebbistatin greatly enhanced the attachment and growth of intestinal epithelial cells on Matrigel-coated plate, much better than Y-27632 (Supplementary Fig. [S1A](#MOESM1){ref-type="media"}), and supported the survival and growth of Lgr5^+^ ISCs (Fig. [1a](#Fig1){ref-type="fig"}). However, no Lgr5^+^ ISCs were observed in the collagen I-coated plates even in the presence of blebbistatin (data not shown). To examine whether uniform thickness would improve the growth of Lgr5^+^ ISCs in 2D culture, we developed a novel method to generate a thin layer of Matrigel with uniform thickness on glass sheets based on our previous work^[@CR8]^. Briefly, a single layer of Matrigel was formed between a pre-treated coverslip and a hydrophobic glass slide (see [Supplementary Methods](#MOESM1){ref-type="media"}), and the thickness can be easily controlled by Matrigel volume and coverslip dimension. We found that the 10 μm-thick Matrigel best supported the growth of Lgr5^+^ ISC monolayer, whereas thicker Matrigel layer (50 μm) allowed the formation of an organoid-like 3D structure (Supplementary Fig. [S1B](#MOESM1){ref-type="media"}). Thinner Matrigel layers such as 5 μm could still support the growth of Lgr5^+^ ISCs, but the efficiency was lower, similar to the Matrigel-coated culture. The bright-field microscopy revealed that the monolayer exhibited heterogeneity in both cell morphology and densities (Fig. [1a](#Fig1){ref-type="fig"}). Highly dense compartments with small sized cells were enriched with Lgr5^+^ ISCs, which were surrounded by large differentiated cells. We confirmed the epithelium origin by staining of E-cadherin and ZO-1 and determined the monolayer nature by Z-stack modeling (Fig. [1b, c](#Fig1){ref-type="fig"}; Supplementary Fig. [S2](#MOESM1){ref-type="media"} and [Movie](#MOESM3){ref-type="media"}).Fig. 1Establishment of a self-renewing 2D monolayer culture of Lgr5^+^ ISCs.**a** Representative bright-field and Lgr5-EGFP fluorescence images of small intestinal epithelial cells cultured in 2D system with blebbistatin, EGF, Noggin, and R-spondin1 (BENR) for 2 days, followed by culturing in BLRC for 4 days. **b** E-cadherin staining of epithelium and GFP of Lgr5^+^ stem cells in 2D-cultured monolayers. **c** Confocal images of monolayers stained for EpCam (green) and DAPI (blue). Right and bottom panels suggest the horizontal (H) and vertical (V) projections. **d** Confocal images staining with EphB2 (red) showed the location of crypt, stem cells and Paneth cells. Asterisks mark the Paneth cells. **e** Confocal images stained for proliferation (Ki67^+^), Enterocytes (Vil^+^), Paneth cells (Lyz^+^), Goblet cells (Muc2^+^), enteroendocrine cells (Chga^+^), and apoptosis (TUNEL^+^). **f**, **g** Representative FACS analysis (**f**) and gene expression (**g**) in 2D or 3D system. The data were analyzed by Student's *t*-test and shown as mean ± SD. \**P* \< 0.05, \*\**P* \< 0.01. **h** Muc2 staining of Goblet cells from cells initially cultured in 2D system (7 d) and then transferred to ENR or ENR plus ID (5 d). I: IWP-2. D: DAPT. Scale bars, 50 μm
To better sustain Lgr5^+^ ISCs on the 2D monolayer, we modified the culture medium with small molecules. EGF is dispensable in this system, as removal of EGF only led to a slight decrease of Lgr5^+^ ISCs (Supplementary Fig. [S3](#MOESM1){ref-type="media"}). Noggin could be replaced by the BMP type I receptor inhibitor LDN-193189 (LDN) after first two days, and the combination of LDN and the GSK-3 inhibitor CHIR-99021 greatly increased the proportion of the Lgr5^+^ ISCs (Supplementary Fig. [S3](#MOESM1){ref-type="media"}). The BLRC (blebbistatin, LDN, R-spondin, and CHIR) culture medium supported a robust monolayer culture of Lgr5^+^ ISCs without affecting differentiated lineage cells (Fig. [1d--f](#Fig1){ref-type="fig"}; Supplementary Fig. [S3](#MOESM1){ref-type="media"}). With the modified 2D system, we tested the culture efficiency from different portions of small intestine, and found that the survival rate was highest in the duodenum (Supplementary Fig. [S4A](#MOESM1){ref-type="media"}). Furthermore, the monolayer culture could be applied to APC KO crypts (Supplementary Fig. [S4B](#MOESM1){ref-type="media"}).
Next, we assessed whether the cells cultured with this 2D system resembled the in vivo intestinal epithelium. We have noticed that in our 2D culture, Lgr5^+^ ISCs were intermixed with visible large Paneth cells, reminiscent of the in vivo crypt base (Fig. [1d](#Fig1){ref-type="fig"}). Proliferation marker Ki67 staining indicated that the cells in the stem cell zone marked by Lgr5 were actively proliferating. Other cell lineage markers were expressed in the non-stem cell compartment: villin (enterocytes), mucin 2 (goblet cells), and chromogranin A (enteroendocrine cells) (Fig. [1e, g](#Fig1){ref-type="fig"}), indicating that normal differentiation occurs in the monolayer culture. Moreover, the cells at the border underwent apoptosis (Fig. [1e](#Fig1){ref-type="fig"}), resembling the in vivo villus tip. Tracing of the progeny of Lgr5^+^ cells from *Lgr5-CreER; loxp-Stop-tdTomato* mice in the 2D culture revealed that Lgr5^+^ ISCs sustained the self-renewal of this monolayer (data not shown).
We next assessed whether this 2D system could resemble the previously established in vitro 3D organoid culture system. The isolated crypts were cultured either in the 3D Matrigel using the ENR-containing medium or on the 2D Matrigel layer with the BLRC-containing medium for 5 days. FACS analysis revealed that the proportion of Lgr5^+^ ISCs in the 2D monolayer culture was significantly higher than the one in the 3D organoid culture (22.2% vs. 10.3%) (Fig. [1f](#Fig1){ref-type="fig"}). The Lgr5^+^ ISCs in monolayer exhibited stem cell properties as they efficiently formed 3D organoids in Matrigel (Supplementary Fig. [S5A](#MOESM1){ref-type="media"} and [S5B](#MOESM1){ref-type="media"}) and possessed a similar gene expression profile to Lgr5^+^ ISCs from organoids except for ID1 and ID2 (Fig. [1g](#Fig1){ref-type="fig"}). To show that the Lgr5^+^ ISCs could be directed to differentiate into specific cell lineage, we changed the BLRC medium to ENR medium, which allows differentiation into mature cell types^[@CR9]^. Indeed, the combination of the Wnt inhibitor IWP-2 (2 μM) and the Notch inhibitor DAPT (10 μM) efficiently induced goblet cell differentiation (Fig. [1h;](#Fig1){ref-type="fig"} Supplementary Fig. [S5C](#MOESM1){ref-type="media"}, [D](#MOESM1){ref-type="media"}).
In summary, we successfully established a 2D culture system that could support self-sustaining of Lgr5^+^ intestinal stem cells in monolayer. Importantly, our approach has a number of advantages over the existing in vitro organoid culture system. The 2D culture system provides an intuitionistic method for observation of the dynamics of Lgr5^+^ stem cells and facilitates cell imaging studies. In contrast to the inaccessibility of the apical domain of epithelium cells in organoids, exposure of the apical domain in the monolayer culture also provides a good system for the microbe--epithelial cell interaction study and drug screening.
Note: During the preparation of our manuscript, Thorne and their colleagues reported a similar 2D culture system of intestinal epithelium^[@CR10]^. Different from their system, we found that the addition of blebbistatin and Matrigel of uniform thickness could better sustain Lgr5^+^ ISCs.
Electronic supplementary material
=================================
{#Sec2}
Supplementary methods and figures video legend 3D reconstitution video
These authors contributed equally: Yuan Liu, Zhen Qi.
**Electronic supplementary material**
**Supplementary Information** accompanies the paper at 10.1038/s41421-018-0036-z.
This work was supported by grants from the National Natural Science Foundation of China (31330049) and the National Key Research and Development Program of China (2017YFA0103601) to Y.-G.C.
Y.L., Z.Q., and Y.-G.C. conceived the experiments. Y.L., Z.Q., and X.L. performed the experiments and analyzed the data. Y.L., Z.Q., Y.D., and Y.-G.C. analyzed the data. Y.L., Z.Q., and Y.-G.C. wrote the manuscript.
Conflict of interest {#FPar1}
====================
The authors declare that they have no conflict of interest.
|
{
"pile_set_name": "PubMed Central"
}
|
Introduction {#Sec1}
============
The brown marmorated stink bug (BMSB), *Halyomorpha halys*, is a highly polyphagous insect that feeds on more than 120 plants including field crops, trees, vegetables, and ornamentals^[@CR1]^. Native to eastern Asia, the BMSB was first discovered in Pennsylvania, then rapidly spread in the Mid-Atlantic region of the USA; damaging crops and distressing homeowners due to their overwintering behavior^[@CR2]^. In a recent special issue of Journal of Pest Science, three review articles^[@CR3]--[@CR5]^ summarized chemical ecology and chemical and biological control methods of this pest. In addition, 23 original papers have reported on the latest research on biology and management of BMSB^[@CR6]^.
After the discovery of RNA interference (RNAi) in the nematode, *Caenorhabditis elegans* and demonstration of its functioning in insects, several groups started using this technology in basic research as well as for the development of methods to control insect pests and disease vectors^[@CR7]--[@CR10]^. Double-stranded RNA (dsRNA) is delivered to insects in a variety of ways, including injection, feeding or through transgenic plants or microorganisms such as bacteria^[@CR7],[@CR10]--[@CR12]^. RNAi efficiency varies among insects depending on the species, delivery method and genes targeted. For example, RNAi works efficiently and is systemic in most of the coleopteran insects tested^[@CR7],[@CR10],[@CR13]^. However, RNAi efficiency is quite variable in most other insects, including those belonging to Lepidoptera, Hemiptera, and Diptera^[@CR14],[@CR15]^. Differences in degradation of dsRNA by dsRNases, transport into and within cells, processing of dsRNA and differences in expression and structures of proteins involved in RNAi are among the major contributors to differential RNAi efficiency among insects^[@CR15],[@CR16]^. Comparison of dsRNA transport and processing between lepidopteran and coleopteran cells and tissues demonstrated that dsRNAs are taken up by coleopteran cells and are processed into siRNAs, resulting in silencing of target genes. Conversely, lepidopteran cells take up dsRNAs, but they are accumulated in acidic bodies and hence not processed into siRNAs. Thus, the target genes are not silenced efficiently^[@CR15]^. Recent studies by Yoon *et al*.^[@CR17]^ identified the acidic bodies where dsRNAs are accumulated as early and late endosomes in the lepidopteran insect, *Spodoptera frugiperda*; suggesting that endosomal entrapment is one of the major contributors to RNAi inefficiency.
The efficiency of RNAi is also variable among hemipteran insects tested so far. RNAi appears to work well in heteropteran insects but does not perform as effectively in homopteran insects such as aphids and whiteflies. However, some published reports showed successful silencing of target genes by delivering dsRNA to homopteran insects by injection, feeding or through transgenic plants^[@CR18]--[@CR20]^. In BMSB, Bansal *et al*.^[@CR21]^ injected adult BMSB with dsRNA targeting the catalase gene and observed knockdown. In addition, knockdown of genes coding for Juvenile hormone acid O-methyltransferase (JHAMT) and vitellogenin (Vg) in BMSB nymphs after orally delivering dsRNA targeting these genes through green beans was also reported recently^[@CR22]^. However, in these studies, a significant mortality of BMSB after knockdown of these genes was not observed.
To identify target genes that could be used in the development of RNAi-based control methods for BMSB, we identified homologs of 13 genes that worked well as RNAi targets in other insects and screened them by injecting dsRNA targeting these genes in adult BMSB. Five out of 13 dsRNAs tested caused significant corrected percent mortality. Feeding dsRNA targeting three of these genes (*IAP, SNF7* and *PPI*) to BMSB nymphs caused more than 70% mortality. The data included in this study demonstrate that feeding dsRNA causes knockdown of target genes and mortality in BMSB.
Materials and Methods {#Sec2}
=====================
Rearing of BMSB and Tobacco budworm {#Sec3}
-----------------------------------
BMSB were collected from South and North Farms of the University of Kentucky between July and September 2017. Insects were reared in a greenhouse following the published methods (see Medal *et al*.)^[@CR23]^. BugDorm-2120 insect rearing cages (61 × 61 × 61 cm) were kept in a greenhouse and maintained at a photoperiod of 16:8 h L:D, 26 °C ± 2, and 50--55% RH. Ornamental plants (*Peperomia obtusifolia Variegata*) were provided for shelter and resting, green bean and sweet corn plants were provided for oviposition, and organic green beans and peanuts were offered to the insects as dietary supplements. The tobacco budworm (TBW) *Heliothis virescens* was reared on an artificial diet as described previously^[@CR24]^.
Isolation of total RNA and cDNA synthesis {#Sec4}
-----------------------------------------
Total RNA was isolated from BMSB adults using TRI Reagent® RT (Molecular Research Center Inc., Cincinnati, OH). The DNase1 was used to remove the contaminating DNA from the total RNA (Ambion Inc., Austin, TX). The purified RNA was stored at −80 °C until further use. The integrity and quality of total RNA were analyzed on 1.2% agarose gels and quantified in Nanodrop 2000 Spectrophotometer (Thermo scientific). Two micrograms of total RNA for each sample was used for cDNA synthesis using M-MLV Reverse Transcriptase (Invitrogen).
Gene amplification, purification and dsRNA synthesis {#Sec5}
----------------------------------------------------
The cDNA was used as a template to amplify fragments of target genes using gene-specific primers (Table [S1](#MOESM1){ref-type="media"}). PCR reaction contained 1 μl of cDNA template, 1 μl of 10 μM each primer, 25 μl of Taq 2× Master Mix (NEB, USA) in a total volume of 50 μl. PCR conditions used are as follows: 5 min at 95 °C for initial denaturation followed by 30 sec at 95 °C, 30 sec at 55 °C and 45 sec at 68 °C for 35 cycles and final extension 10 min at 68 °C. The amplified products were analyzed on 1% agarose gels and purified using PCR purification kit (Qiagen). The purified PCR products were quantified using a Nanodrop 2000 Spectrophotometer (Thermo Scientific) and stored at −20 °C until further use. Double-stranded RNA (dsRNA) targeting gene coding for green fluorescence protein (GFP, control) and 13 target genes were synthesized by using MEGAscript® T7 RNAi kit (Ambion, USA) following manufacturer's instructions. The integrity of the dsRNA was analyzed on 1% agarose gels, and the concentration was determined by Nanodrop 2000 Spectrophotometer (Thermo scientific).
Collection of hemolymph from BMSB and Tobacco budworm {#Sec6}
-----------------------------------------------------
Hemolymph was collected from BMSB and last instar larvae of Tobacco budworm. The larvae and adults were placed on ice, the forelegs of larvae and thoracic region of adults were pierced with a needle and hemolymph was collected into 1.5 ml tubes containing Phenylthiourea (Sigma-Aldrich). The hemocytes and other cell debris were removed from hemolymph by centrifugation and supernatant were used for dsRNA stability assay.
Collection of watery saliva from BMSB {#Sec7}
-------------------------------------
Watery saliva was collected from BMSB adults as described Peiffer and Felton^[@CR25]^. BMSB adults were chilled on ice for five minutes then placed ventral side up and observed under a microscope. BMSB secreted saliva from the tip of the beak when they returned to room temperature. This saliva was collected into a 1.5 ml tube placed on ice by using 10 µl pipet tip containing 3 µl of 1× PBS buffer.
^32^P- labelling of dsGFP {#Sec8}
-------------------------
The ^32^P-labelled dsGFP was prepared using MEGA script® T7 RNAi kit (Ambion, USA) as described recently^[@CR15]^.
dsRNA stability assay {#Sec9}
---------------------
Total protein in the hemolymph and watery saliva was estimated by Bradford method^[@CR26]^. Different dilutions of hemolymph (1.2, 0.6, 0.3 and 0.15 mg/ml), and saliva (4.8, 2.4, and 1.2 mg/ml) were prepared, and 250 ng of dsRNA was added to the hemolymph and saliva. After an hour of incubation at room temperature (RT), samples were mixed with 2 μl of 6× loading dye, loaded and analyzed on 1% agarose gel and photographed by using AlphaImager Gel Imaging System (Alpha Innotech, San Leandro, CA). The hemolymph samples were also incubated 1hr with 8000 CPM ^32^P-UTP labelled dsGFP and run on 16% polyacrylamide-8M urea gel using 1X TBE buffer. Gels were washed and fixed with a solution containing 0.5X TBE buffer, 10% ethanol, and 10% methanol. The gels were dried using the gel drier. Gels were exposed to Phosphor-Imager screen and scanned using Typhoon FLA 9500 Laser Scanner (GE Healthcare Life Sciences).
Injection of dsRNA into BMSB adults {#Sec10}
-----------------------------------
One microgram of dsRNA in 5 μl of distilled water was injected into the thoracic region of adults using insulin syringe (1/2 cc U-100 insulin syringe). Each replicate used 10 adults, and 3--4 replicates were used for each treatment, and the experiments were repeated twice. Adults were collected at three days after injection for determining knockdown efficiency, and mortality was recorded at seven days post-injection.
Feeding of dsRNA {#Sec11}
----------------
Certified organic green beans (*Phaseolus vulgaris* L.) were used for feeding dsRNA as described previously^[@CR22]^. Lean green beans were selected to ensure their fit in the 2 ml Eppendorf tubes. The beans were washed three times with ddH2O and trimmed from the calyx end to a total length of 5 cm. The beans were immersed in a capless 2 ml microcentrifuge tube containing 300 μl nuclease free water and 20 μg of dsRNA (added daily for 3 days). The tubes were sealed with parafilm to prevent evaporation of the solution and to prohibit insects from entering the solution. Beans were immersed in water containing dsRNA 3 hr prior to feeding. The tubes were then placed in a solid platform to keep them upright and enclosed within magenta jars (Sigma-Aldrich). Second instar BMSB nymphs were starved overnight prior to feeding. 15 nymphs were released per magenta jar containing one green bean in a microcentrifuge tube containing dsRNA (20 µg daily for three days) solution. Samples were collected for RT-qPCR study on the 4^th^ day after initiation of feeding and mortality was recorded on the 7^th^ day.
Selection of candidate reference gene, RT-qPCR primers and analysis of amplification efficiency {#Sec12}
-----------------------------------------------------------------------------------------------
The candidate reference genes were selected based on their previous reports in other insects. These include 1*8S ribosomal RNA* (XM_014421522.1), *Elongation factor 1 alpha* (XM_014438029.1), *Actin* (XM_014431329.1), *Ubiquitin* (XM_014429239.1), *60S ribosomal protein* (XM_014430141.1) and *Beta Tubulin* (XM_014438117.1). For each candidate reference gene, BLASTN and BLASTX were carried out in NCBI (<http://www.ncbi.nlm.nih.gov/>) and CDS region was identified using ExPASy translate tool (<http://web.expasy.org/translate/>). The IDT PrimerQuest software (<http://eu.idtdna.comwebcite>) was used for designing primers. The primer sets were checked for amplification specificity and annealing temperature. The specificity of the PCR amplified product was characterized by electrophoresis. Primer sets that amplified a single-specific product were chosen for RT-qPCR amplification efficiency test (Fig. [S1](#MOESM1){ref-type="media"}).
Reverse-transcriptase quantitative real-time PCR (RT-qPCR) {#Sec13}
----------------------------------------------------------
RT-qPCR experiments used StepOnePlus™ Real-Time PCR System (Applied Biosystems, USA). A 10 μl reaction volume \[containing, 2 μl of diluted cDNA (1:2), 5 μl of iTaq™ universal SYBR® Green Supermix (Bio-Rad), 0.2 μl of each primer\] was used. The RT-qPCR was performed under the following conditions: an initial denaturation step for 20 sec at 95 °C, followed by 40 cycles of amplification with 5 sec of denaturation at 95 °C, 30 sec of annealing and extension at 55 °C. The melt curve was obtained by heating the amplicon from 60 to 95 °C. A non-template control (NTC) was also included in each run for each gene. The *18S rRNA gene* was used as a housekeeping gene to normalize the RT-qPCR data.
Statistical analysis {#Sec14}
--------------------
The stability levels of the six candidate reference genes from BMSB were determined using four statistical algorithms, geNorm^[@CR27]^, NormFinder^[@CR28]^, BestKeeper^[@CR29]^ and RefFinder^[@CR30]^. The corrected percent mortality was calculated based on Schneider-Orelli's formula^[@CR31]^. Double delta Ct (^ΔΔ^Ct) method was used for RT-qPCR data analysis^[@CR32]^. A one-tailed t-test was used to compare the mean of a single variable.
Results and Discussion {#Sec15}
======================
Comparison of dsRNase activity {#Sec16}
------------------------------
For the systemic spread of fed dsRNAs from the midgut to other tissues, the dsRNAs have to travel through hemolymph where they may encounter dsRNases. Our previous studies showed that the activity of dsRNases is higher in the hemolymph of *Heliothis virescence* (Lepidoptera) where RNAi does not work well, as opposed to their activity in the hemolymph of *Leptinotarsa decemlineata* (Coleoptera) where RNAi works well^[@CR15]^. To determine if the dsRNase activity in the hemolymph of BMSB could be detrimental to RNAi in this insect, we compared dsRNase activity in the hemolymph of BMSB and TBW (Lepidoptera). About 250 ng of dsGFP was incubated with serial dilutions of hemolymph and watery saliva (diluted with 1xPBS in a total volume of 5 μl based on total protein concentration) for an hour at room temperature. After incubation, the dsRNA samples were analyzed by 1% agarose gel or 16% polyacrylamide 8 M urea gels. The hemolymph collected from TBW degraded dsRNA at a concentration (0.3 mg/ml) or higher (Fig. [1A](#Fig1){ref-type="fig"}). The more sensitive method using ^32^P-labeled dsRNA also showed similar results (Fig. [1B](#Fig1){ref-type="fig"}). However, the hemolymph and saliva collected from BMSB did not completely degrade dsRNA even at 1.2 mg/ml concentration (Fig. [1A--C](#Fig1){ref-type="fig"}). These data suggest that the dsRNase activity in the BMSB is lower than that of TBW; thus, RNAi efficiency may be higher in BMSB when compared to that in TBW. The previous report suggested that higher levels of dsRNases are one of the major factors contributing to inefficient RNAi in pea aphid^[@CR16]^. Recently, Bansal *et al*.^[@CR21]^ demonstrated silencing of catalase gene by injection of dsRNA in BMSB. In another study, Ghosh *et al*.^[@CR22]^ developed a green bean-based feeding method and showed effective silencing of target genes coding for JHAMT and Vg by feeding dsRNA. ^32^P labelled dsRNA injected into BMSB adults is processed into siRNA^[@CR33]^, suggesting that these insects possess machinery to take up dsRNA and process to siRNA. The results from the previous reports and our finding of lower levels of dsRNase activity in the hemolymph and saliva of BMSB suggest that the RNAi could work well in BMSB and may be a viable option for controlling this invasive pest.Figure 1Analysis of dsRNA stability in Tobacco budworm and BMSB hemolymph. Agarose gel electrophoresis analysis of dsRNA degradation products exposed to Tobacco budworm (TBW), *Heliothis virescens* (Lepidoptera) and BMSB, *Halyomorpha halys* (Hemiptera) hemolymph and salivary gland secretions. (**A**) About 250 ng of dsGFP was exposed to various concentrations (1.2--0.15 mg/ml) of hemolymph from TBW or BMSB for an hour at room temperature, and the products were resolved on 1% agarose gels, and the gels were stained with ethidium bromide. L, 1 kb plus DNA ladder; dsR, 270 ng dsGFP alone and H, hemolymph of TBW or BMSB. (**B**) About 8000 CPM of ^32^P-labeled dsGFP was exposed to various concentrations of hemolymph (1.2--0.15 mg/ml) for an hour at room temperature, and the products were resolved on 16% polyacrylamide 8 M urea gels. The gels were dried and exposed to a PhosphorImager screen, and the image was scanned using a PhosphorImager. dsR, labeled dsGFP. (**C**). The BMSB saliva was collected as described in the Materials and Methods section. Protein concentration was determined and various concentrations (4.8--1.2 mg/ml) of saliva added to 250 ng dsGFP and incubated at room temperature for an hour. The products were resolved on 1% agarose gel, and the gel were stained with ethidium bromide. L, 1 kb plus DNA ladder and dsR, 270 ng dsGFP alone.
Selection of candidate reference genes {#Sec17}
--------------------------------------
To determine the knockdown efficiency of target gene after dsRNA administration, the mRNA or protein levels of target genes need to be quantified. Since antibodies are not available for most of the target genes being tested, quantification of mRNA levels is the most commonly used method. To quantify mRNA levels by RT-qPCR, a reliable reference gene is a prerequisite. Although some reference genes have been identified in BMSB, in the preliminary studies, we did not find them stable across the dsRNA treatments in our experiments. Therefore, we conducted experiments to identify reference genes.
To identify suitable reference genes, six genes (*Ubiquitin*, *Elongation factor*, *60S RP*, *Actin*, *18S rRNA* and *β-Tubulin*) were selected based on previous reports in other insects. Information on the selected reference genes is shown in Table [S2](#MOESM1){ref-type="media"}. Melt curve analysis was performed to confirm the specific amplification of each reference gene and a single peak with no visible primer-dimer formation and genomic DNA contamination was observed and no signals were detected in the nontemplate controls (NTC) (Fig. [S1](#MOESM1){ref-type="media"}). The candidate reference genes, primer sequences, and amplicon sizes are shown in Table [S3](#MOESM1){ref-type="media"}.
Expression levels of six candidate reference genes were measured using RNA isolated from dsRNA injected or fed BMSB adults and nymphs. The Ct values for these genes varied from 15--40 (Fig. [2](#Fig2){ref-type="fig"}). *18S* rRNA, Ubiquitin, actin and *60S* RP showed lower variation in their Ct values when compared to those of the other two genes tested (Fig. [2](#Fig2){ref-type="fig"}). The geNorm algorithm^[@CR27]^ was used to calculate the average expression stability value (*M*-value), using Ct values of each gene among the dsRNA treatments. The genes with the lowest *M*-value were considered as the most stable. The geNorm analysis identified *18S rRNA* and *60S RP* as the most stable genes (Table [1](#Tab1){ref-type="table"}) across the dsRNA treatments. The stability of the six housekeeping genes was further analyzed using the NormFinder algorithm^[@CR28]^. The NormFinder analysis of the datasets estimated the stability of genes based on intra- and intergroup variation. The genes with fewer stability values were considered to be the most stable. This program also identified *18S rRNA* and *60S RP* as the most stable genes across the dsRNA treatments (Table [1](#Tab1){ref-type="table"}).Figure 2Identification of stable reference genes. Variability of the Ct values for six reference genes in all samples (Injection and feeding dsRNA) of BMSB nymphs and adults tested. Total RNA was isolated after injection/feeding dsRNA. The RNA was converted to cDNA, and the cDNA and gene-specific primers were used in RT-qPCR to determine Ct values. Mean ± SD of Ct values are shown.Table 1Ranking of the candidate housekeeping genes according to their stability value by geNorm, NormFinder, and BestKeeper analysis. M, the gene expression stability measure; SD, standard deviation value; SV, stability value; GM, Geomean value and R, Ranking.Gene NamegeNormNormFinderBestKeeperΔCTComprehensiveMRSVRSDRSDRGMRAdult dsRNA injected samples18s rRNA0.73310.3310.5213.91111Ubiquitin0.82120.38430.9944.0533.46460S RP0.73310.3720.6434.0122.062Actin0.94830.3310.6124.1242.833β-Tubulin3.90148.6548.0169.3355.235EF1α5.87759.24856.1559.8365.736Nymph dsRNA fed samples18s rRNA0.35610.1810.5121.5111.191Ubiquitin0.35610.4720.4311.5721.41260S RP0.65620.4830.5231.65333Actin2.10551.1262.5263.57666β-Tubulin1.09731.9651.3652.354.735EF1α1.37243.4441.1442.0344.234Combined adult injected and nymph fed samples18s rRNA0.89310.44610.8913.7511.191Ubiquitin1.15221.42522.0744.0633.22360S RP0.89310.44611.3323.7921.412Actin1.82331.9631.3734.4743.724β-Tubulin5.35757.69254.6168.27666EF1α3.90347.12945.9257.81555
The descriptive statistics of all six housekeeping genes used in the study were computed by the BestKeeper algorithm^[@CR29]^. *18S rRNA* showed standard deviation (SD) values less than 1 an indicator of the consistent and stable performance. The coefficient of variation (*CV*) of housekeeping genes ranged from 0.89% for *18S rRNA* to 4.61% for *β-Tubulin* (Table [1](#Tab1){ref-type="table"}).
RT-qPCR is a powerful technique to study the gene expression due to its high sensitivity, accuracy, specificity, and reproducibility. During RNA isolation, cDNA conversion and assembling reactions variability could be introduced. This could be countered using appropriate reference genes. Also, use of multiple reference genes is important because using a single reference gene may not be sufficient to control variability across all treatments^[@CR34]^. In the present study, six reference genes were selected and validated in adult and nymph stages treated with different dsRNAs by injection and feeding and the data were analyzed by four statistical algorithms: geNorm, NormFinder, BestKeeper, and RefFinder. The results showed that *18S rRNA*, *60S RP* and *Ubiquitin* are the most stable reference genes in nymphs (Supp. Figs [S3](#MOESM1){ref-type="media"}B and [S4B](#MOESM1){ref-type="media"}; Table [S5](#MOESM1){ref-type="media"}); *18S rRNA* and *60S RP* in adults, (Supp. Figs [S3](#MOESM1){ref-type="media"}A and [S4A](#MOESM1){ref-type="media"}; Table [S4](#MOESM1){ref-type="media"}) and when both nymphs and adults were compared, the *18S rRNA* and *60S RP* are identified as the most stable genes (Table [1](#Tab1){ref-type="table"}). In a recent study, ten housekeeping genes were evaluated for their stability across various treatments in BMSB and showed that *ARP8* and *Ubiquitin E4A* as the most stable genes across tissues and developmental stages treatments tested. *Ubiquitin* is the common gene identified in both these studies and may be the most stable genes to target across developmental stages and treatments. These studies also confirm the previous finding that the same housekeeping gene may not work well for all stages and treatments. Therefore one needs to identify one or more reference genes for the specific treatments compared by RT-qPCR.
Screening of target genes and feeding RNAi {#Sec18}
------------------------------------------
To identify target genes that could be used for RNAi-mediated control of BMSB, we screened 13 genes that are known to cause mortality in other insects after their knockdown by RNAi. 1.0 μg of dsRNA targeting each of the selected genes or gene coding for GFP as a control were injected into each BMSB adult. At seven days after injection, the mortality was recorded. Five out of the 13 dsRNAs tested caused more than 70% mortality by seven days after injection (Fig. [3](#Fig3){ref-type="fig"}). Quantification of mRNA levels of three of these genes (IAP, PP1 and ATPase) using RT-qPCR showed 40--75% knockdown in the expression of these target genes in insects injected with dsRNA targeting each of these genes (Fig. [4](#Fig4){ref-type="fig"}). Considerable differences in the efficacy of the 13 dsRNAs in causing mortality of BMSB was observed. This may be due to the differences in function of these target genes. Also, differences in knockdown efficiency of the target genes by the dsRNAs used could also account for some of the differences in the efficacy of dsRNAs tested.Figure 3Screening of RNAi target genes in BMSB adults by injection of dsRNA. One microgram of dsRNA targeting each of the 13 select genes, ATPase (Putative ATPase N2B, Acc. no: XM_014420983.1); PP1 (Serine/threonine-protein phosphatase PP1-beta catalytic subunit, Acc. no: XM_014431150.1); GPCR (G protein-coupled receptor 161-like, Acc. no. XM_014438952.1); IAP (Baculoviral IAP repeat-containing protein 7-B-like, Acc. no: XM_014435389.1); SNF7 (Charged multivesicular body protein 4b, Acc. no: XM_014427464.1); SRP (Signal recognition particle 54 kDa protein, Acc. no: XM_014419857.1); Actin (Actin-5C, Acc. no: XM_014433214.1); NSF (Alpha-soluble NSF attachment protein, Acc. no: XM_014434606.1); Kanesin (Uncharacterized Kanesin, Acc. no: XM_014426093.1.); Dynamin (Dynamin, Acc. no: XM_014433358.1); *26*S (*26*S protease regulatory subunit 6B, Acc. no: XM_014421516.1); ROP (Protein ROP, Acc. no. XM_014418690.1); HSP70 (Heat shock 70 kDa protein cognate 3, Acc. no: XM_014425902.1) was injected into BMSB adults. The mortality was recorded on the 7th day after injection. dsRNA targeting gene coding for GFP was used as a control.Figure 4Knockdown efficiency determined by RT-qPCR in dsRNA injected BMSB. One microgram of dsRNA targeting IAP, ATPase, PPI or GFP (control) was injected in BMSB adults. Total RNA was isolated on the 3rd day after injection of dsRNA. The RNA was converted to cDNA, and the cDNA and gene-specific primers were used to quantify mRNA levels of IAP, ATPase, and PPI using RT-qPCR. The *18*S rRNA gene was used to normalize expression. The mean of relative mRNA levels and SE (n = 3--5) in dsGFP and dsIAP (**A**), dsATPase (**B**) or dsPP1 (**C**) injected insects are shown. A one-tailed t-test was used to compare the means of a single variable.
BMSB frequently feed on fruit crops and beans in agricultural systems using their needle-like stylets by alternate salivation and ingestion^[@CR25]^. BMSB are highly attracted to green beans and are major pests of many bean varieties. Green beans (*Phaseolus vulgaris* L.,) were selected for delivery of dsRNA. Slender green beans were trimmed from the calyx, inverted and immersed into dsRNA solution (0.066 μg/μl) in a 2 ml microcentrifuge tube. Each bean was placed in a magenta vessel, and 15 nymphs were released per vessel (Fig. [S2](#MOESM1){ref-type="media"}). Second instar nymphs were starved overnight prior to exposure to dsRNA. Five dsRNAs targeting *IAP, ATPase, SNF7, GPCR*, and *PPI* were tested in feeding RNAi bioassay. Three of the five dsRNAs tested caused more than 70% corrected mortality (Fig. [5](#Fig5){ref-type="fig"}). RT-qPCR analysis showed 60--20% knockdown in the expression of five of these genes in BMSB nymphs fed on dsRNA targeting each of these genes (Fig. [6](#Fig6){ref-type="fig"}). These data suggest that feeding dsRNA causes significant knockdown of target gene and mortality in BMSB.Figure 5Feeding dsRNA causes mortality in BMSB. Certified organic beans were inserted in 2 ml tubes containing 300 µl of nuclease-free water and 20 µg of dsRNA (20 µg of dsRNA in water was added to the tube on the second and third day). Fifteen 2^nd^ instar nymphs were fed on each bean for seven days. The mortality was recorded on the 7^th^ day, and the corrected percent mortality was calculated using Schneider-Orelli's formula. Mean + S.E (n = 3) are shown. The asterisk reflects significant differences in mortality rate (ANOVA, Student-Newman-Keuls Method, P \< 0.05).Figure 6Knockdown efficiency determined by RT-qPCR in dsRNA fed BMSB. Twenty micrograms of dsRNA targeting IAP, SNF7, PPI, ATPase, GPCR or GFP (control) was fed to BMSB nymphs on each day for three days. On the fourth day, the nymphs were fed on beans. Total RNA was isolated on the 4th day after initiation of feeding dsRNA. The RNA was converted to cDNA, and the cDNA and gene-specific primers were used to quantify mRNA levels of IAP, SNF7, ATPase, PPI and GPCR using RT-qPCR. The *18S* rRNA mRNA levels were used to normalize expression. The mean of relative mRNA levels and SE (n = 3--4) in dsGFP and dsIAP (**A**), dsSNF7 (**B**), dsPPI (**C**), dsATPase (**D**) or dsGPCR (**E**) fed nymphs are shown. A one-tailed t-test was used to compare the mean of a single variable.
In this present study, oral delivery of dsRNA through green beans caused mortality in BMSB, confirming the effectiveness of RNAi in BMSB and demonstrating that feeding dsRNA could induce RNAi and mortality in this insect. Both injection and feeding of dsIAP caused mortality, and gene knockdown data suggests that the *IAP* gene may be one of the best target genes to control the BMSB using RNAi. The *IAP* gene from *Bombyx mori* was identified and shown to function as a caspase inhibitor to block apoptosis^[@CR35]^. The functioning of RNAi in the tarnished plant bug, *Lygus lineolaris* was demonstrated using the *IAP* gene as the target by delivering dsIAP to the nymphs and adults through microinjection^[@CR36]^. The dsIAP treated insects showed a significant reduction in the lifespan when compared with those injected with control dsRNA. The I*AP1* gene was identified in *Aedes aegypti* and showed that the gene product inhibits both initiator and effector caspases^[@CR37]^. In Aag-2 cell, five genes coding for *IAPs* (1, 2, 5, 6 and 9) were identified. Treating these cells with dsRNA targeting these genes caused a significant reduction in the mRNA levels of target genes but only dsIAP1 induced apoptosis phenotype^[@CR38]^. In Lepd-SL1 cell line, a gene coding *IAP1* was identified and exposing these cells to dsIAP1 induced apoptosis^[@CR39]^. These investigators used *IAP1* to develop an assay to identify genes critical of RNAi pathway in these cells. Exposing Ledp-SL1 cells to dsRNA targeting RNAi pathway genes followed by dsIAP1 showed that five genes (*Argonaute-1, Argonaute-2a, Argonaute-2b, Aubergine and V-ATPase 16* *kDa subunit 1* and *Vha16*) are essential for successful RNAi in these cells. Recently, Rodrigues *et al*.^[@CR40],[@CR41]^ targeted *IAP1* gene in two invasive forest pests, *Agrilus planipennis* and *Anoplophora glabripennis* and showed significant knockdown of *IAP1* gene after injection of dsIAP1.
The previous two reports and the data included in this paper showed that RNAi works in BMSB and feeding dsRNA could be a viable control option for this pest. The next major challenge is the delivery of dsRNA: What is the best way to get dsRNA to BMSB in the field?, Expression in crop plants, delivery through green beans drenched with dsRNA and placed as bait stations in the field, dsRNA spray on the foliage and dsRNA applied to the soil are just a few of the possible routes of application that need to be evaluated.
Electronic supplementary material
=================================
{#Sec19}
Supplementary Information
**Electronic supplementary material**
**Supplementary information** accompanies this paper at 10.1038/s41598-018-22035-z.
**Publisher\'s note:** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
This is publication number 18-08-027 from the Kentucky Agricultural Experimental Station and is published with the approval of the director. This work was supported by the USDA HATCH under 2351177000. We thank Dr. Ric Bessin for help with collection and rearing of insects.
K.M. and S.R.P. conceived the experiments; K.M. and J.H. conducted the experiments; K.M., J.H. and R.P. analyzed the results and prepared the manuscript. All authors approved the manuscript.
Competing Interests {#FPar1}
===================
The authors declare no competing interests.
|
{
"pile_set_name": "PubMed Central"
}
|
INTRODUCTION {#sec1-1}
============
Obesity is epidemic in the world\[[@ref1]\] and its prevalence has increased significantly.\[[@ref2]\] Obesity, especially central obesity is associated with excess deaths in the population.\[[@ref3]\] The prevalence of central obesity in Iran is 53.6%.\[[@ref4]\] Previous studies provided strong evidence that the metabolic problems of central obesity such as insulin resistance, hypertension, hypertriglyceridemia, low high density lipoprotein cholesterol (HDL-C) and steatosis are marked in South Asians including Iranians at lower amounts of total body fat compared to whites.\[[@ref5]\] These differences can be interpreted by high amount of central adipose tissue in South Asians.\[[@ref5]\] Healthy foods are protective factor for metabolic syndrome.\[[@ref6]\] Legumes are one of the healthy and inexpensive foods. They are high in phytochemicals, fibre, protein, minerals and vitamins. Most of the researches that have investigated the effect of legume consumption on metabolic features studied soybeans rather than non-soybean legumes. The effects of soy bean on metabolic features are well-known.\[[@ref7]\] In Iran low amounts of soy beans are consumed, while non-soy legume such as white, red and wax beans, chickpeas, cowpea, lentils and split peas are conventional foods. Anderson and Major in 2002 performed a meta-analysis on secondary outcomes of eleven clinical trials and showed consumption of non-soy legumes was associated with increasing of HDL-C and decreasing of triglyceride (TG) and weight.\[[@ref8]\] After Anderson and Major meta-analysis several randomized controlled trials (RCTs) were studied the effects of legumes on metabolic features. Zhang *et al*. tested the effects of legume on biomarkers of insulin resistance among males in isocaloric and hypocaloric diets. Despite isocaloric diet, in hypocaloric period of intervention, mean body weight, body mass index (BMI), Levels of serum TG, C-peptide and fasting plasma glucose, insulin and C-reactive protein were significantly reduced.\[[@ref9]\] In Hermsdorff study, systolic blood pressure (SBP) was improved only with the legume-based hypocaloric diet compared to calorie-restricted legume-free diet.\[[@ref10]\] Inconsistent with Zhang *et al*. study, Crujeiras *et al*. and Hartman *et al*. showed baseline and endpoint values of insulin, C-peptide and glucose were not statistically different after following hypocaloric and isocaloric diets with or without legumes.\[[@ref11][@ref12]\] Even Hartman *et al*. showed high-legume diet increased fasting blood sugar (FBS) compared to legume-less diet.\[[@ref12]\] Due to paradoxical results this study was planned. The present research takes advantages of higher consumption of non-soy legume among participants at pre-study period in comparison with other similar researches.\[[@ref13][@ref14][@ref15][@ref16]\] In Iran, the eating of non-soy legume is more common than western countries. The mean consumption of non-soy legume among Iranians is nearly 3 servings/week compared to 2 servings/week in US and Europe.\[[@ref13][@ref14][@ref15][@ref16]\] The average intake of non-soy legume in subjects of current study compared with previous trials was approximately triple.\[[@ref10][@ref12]\] To the best of our knowledge, this is the first study that investigates the role of high-legume hypocaloric diet on metabolic features exclusively among women.
METHODS {#sec1-2}
=======
Study design and participants {#sec2-1}
-----------------------------
The study was approved by the Ethics Committee of Tabriz University of Medical Sciences (Tabriz, Iran) and registered at www.irct.ir (irct ID: irct138712101720N1). Written informed consent was achieved from all selected participants.
The sample size for each intervention group was calculated regarding to the studies conducted on women with central obesity.\[[@ref17][@ref18]\] With a 1 -- α=95% and 1 -- β=95%, the maximum sample size was obtained from waist circumference (WC) marker via the formula:
*n* = *a*σ^2^ ϕ^2^ / ∑*t*~i^2^~ = 16.49 16
in which *a* = 4, σ^2^ = 59.9, ϕ^2^ (the indicator curve) =2.5 and ∑t~i~^2^ = 36.46.
Finally, samples for each group were calculated to be 16 participants.
The study was a RCT with a 2 week pre-trial period and a 6 week trial period. After advertising in local newspapers, 257 pre-menopausal women were eligible to enter the study.
Inclusion criteria were: Pre-menopausal women aged 20-50 years, WC \> 88 cm, no involvement in weight-loss programs and maintenance of a stable weight during the previous 6 months (±2 kg).
Exclusion criteria were: Treatment with insulin or oral hypoglycemic agents, anti-hypertensive drugs or anti-lipemic drugs; any secondary cause of hypertension or hyperglycemia; consumption of mineral or vitamin supplements or antacids containing calcium or magnesium; untreated hypothyroidism; psychiatric disorders; cancer; systemic, hepatic, renal, pulmonary, or cardiovascular disease; infectious or inflammatory disease; alcoholism; smoking; and legume intolerance. [Figure 1](#F1){ref-type="fig"} shows the flowchart of the participants of the study.
{#F1}
Diets {#sec2-2}
-----
The energetic needs were calculated individually by the formula from the Food and Nutrition Board of the Institute of Medicine.\[[@ref19]\] The subjects consumed an isocaloric diet for 2 weeks in the run-in period. In the intervention period, intervention group ate hypocaloric diet enriched in legumes (HDEL) (which included 1 cup/day of cooked non-soy legumes including white, red and wax beans, chickpeas, cowpea, lentils and split peas instead of meat) and control group ate hypocaloric diet without legumes (HDWL). Participants in HDWL group increased consumption of animal proteins (meat, poultry, fish, egg or cheese) as much as 2 servings/day (60 g) instead of legumes and reduced consumption of fats as much as 2 servings/day to compensate increased intake of animal fats. The amount of cereals in daily diet of both intervention groups was equivalent but participants in HDEL group were prescribed to consume 2 servings of cereals as legumes. The macronutrient content of both diets was 55% carbohydrate, 30% fat and 15% protein. In the intervention period, all of subjects in both groups were prescribed a hypocaloric diet (500-kcal less than their isocaloric needs). Diets were given individually. Participants were being visited every week for 20-30 min. The nutritionist explained the advantages of diets for the participants and trained participants how to write "food diaries." Each participant had to write her 3-day physical-activity and diet records before the run-in period as well as before, in the middle and at the end of the intervention period. Participant compliance was evaluated by weekly visits and evaluating the 3-day food diaries. Subjects who did not complete ≥80% of the planned diets for 2 consecutive weeks were excluded from the study (*n* = 6).
Study procedures {#sec2-3}
----------------
We planned a run-in period to getting detailed information about the study population and to standardize macronutrient consumption. The true isocaloric needs of some of the participants were different from the amount calculated in the formula from the Food and Nutrition Board at the Institute of Medicine. Among individuals eligible to enter the study, only those who maintained their weight at the end of the run-in period were chosen. After the run-in period on an isocaloric diet for 2 weeks, subjects were randomly allocated to two intervention groups for 6 weeks: (1) HDEL and (2) HDWL. For allocation of the participants, a computer-generated list of random numbers was used.
We repeated random allocation several times to obtain most homogenous groups. The dependent variables were measured before, in the middle and at the end of the intervention. Subjects were asked not to vary their common physical activities during the study.
Measurements {#sec2-4}
------------
All measurements were carried out by the unchanged investigator and the unchanged tool in the first and follow-up evaluations. WC was measured (to the nearest 0.1 cm) at the narrowest point without pressure to the body surface by the light clothing using a tape measure.
After a 12-h fast, blood samples were taken. Samples were centrifuged at 500 ×g for 10 min at 4°C and the serum separated. All parameters except insulin were measured on the day of blood collection. Serum was frozen at --80°C until it was analyzed for insulin.
Levels of fasting blood glucose (FBG), HDL-C and TG were measured enzymatically (ParsAzmoun, Tehran, Iran). Plasma levels of insulin were measured by a human insulin enzyme-linked immunosorbent assay test kit\[[@ref20]\] (Diaplus, San Francisco, CA, USA) according to manufacturer instructions. Insulin resistance was calculated on the basis of the homeostasis model assessment of insulin resistance (HOMA-IR).\[[@ref21]\] Both alanine aminotransferase (ALT) and aspartate aminotransferases (AST) were measured by International Federation of Clinical Chemistry method without adding prydoxal phosphate (Pars Azmoon kit, Tehran, Iran).
Inter- and intra-assay coefficients of variation were 1.19 and 1.28% for FBG, 1.8 and 0.73% for HDL, 1.04 and 1.47% for TG, 8 and 8% for insulin, 3.08 and 6.22% for ALT and 4.40 and 3.25% for AST, respectively.
Confounding factors was obtained by questionnaires. According to this information "Chronic dieters" were distributed among the groups. Participants were classified into three levels of education (did not obtain a high-school diploma, obtained a high-school diploma and university graduates); income (no income, \< US\$350/month and \> US\$350/month); family income (\< US\$350/month, US\$350-700/month and \> US\$700/month); and overweight subjects and the metabolic syndrome in the family (any relative, first-degree relative and second-degree relative). Overweight was defined as (BMI) \>25 kg/m^2^. Metabolic syndrome was defined according to criteria set by the Adult Treatment Panel III.\[[@ref22]\]
Statistical analysis {#sec2-5}
--------------------
Two ways were applied for statistical analyses. In the first way, we used multifactor model of nested multivariate analysis of variance (MANOVA) repeated measurements by Minitab Package (v13) as followed:
Variation of dependent variables = Intra-individual variation + variation because of hypocaloric diet or time + variation because of legumes or diet (time) + variation because of legumes \* time + error.
In this method, we also controlled the effect of WC:
Variation of dependent variables = Intra-individual variation + variation because of hypocaloric diet or time + variation because of legumes or diet (time) + variation because of legumes \* time + error + (B~1~ \* WC).
In the model described above, "Error" represents the random changes during the study. "B~1~" is regression co-efficient. "B~1~ \* WC" represents the effect of WC on dependent variables. In this model, the concurrency of analyses instead of multiple comparisons minimized the probability of false-positive results.
In second way, we used a paired *t*-test or its non-parametric equivalent (Wilcoxon test) for comparing the amount of variables in different times within groups. Furthermore, we used an independent *t*-test or the Mann--Whitney U-test for comparing the percentage changes in variables during different times (T3--T1, T2--T1 and T3--T2) in the HDWL group with a change in the HDEL group. Histograms were used to recognize normal distributions. These analyzes were conducted using SPSS 13.0 (SPSS, Chicago, IL, USA).
We used Chi-square test and independent *t*-test to find significant differences in baseline values among two intervention groups. For appropriate variables, we merged subclasses of variables and then used the Chi-square test. Two-tailed *P* \< 0.05 was considered to be significant. All values expressed as means ± standard error.
RESULTS {#sec1-3}
=======
The general characteristics of the groups are shown in [Table 1](#T1){ref-type="table"}. Food intake of the groups, calorie intake and calories expended in activities before the run-in period are shown in [Table 2](#T2){ref-type="table"}. The mean consumption of fruit and milk in both groups was low. There were no differences in food intake between the groups before the run-in period.
######
Baseline characteristics of the groups
######
Intake of food, calorie intake and calories expended in activity before the run-in period
The effect of interventions on metabolic features using multifactor model of nested MANOVA repeated measurements are outlined in [Table 3](#T3){ref-type="table"}. There were no significant differences among basal (before intervention) measurements in two groups \[not shown in [Table 3](#T3){ref-type="table"}\].
######
Effect of interventions on metabolic features by nested MANOVA repeated measurements of multi-factor model
After 6 weeks of intervention the following results were obtained by repeated measurements of MANOVA \[[Table 3](#T3){ref-type="table"}\]: (1) HDEL and HDWL significantly reduced the WC (*P* = 0.001). (2) HDEL significantly reduced the SBP (*P* = 0.001). This significant effect maintained after adjusting for weight and/or waist. (3) There was not shown any significant effects on diastolic blood pressure (DBP), FBS, TG, HDL-C, Insulin, HOMA-IR, AST and ALT in this model.
With Wilcoxon or paired *t*-test, the following results were obtained (paired *t*-test was used only about HDL-C) \[[Table 4](#T4){ref-type="table"}\]: (1) HDEL and HDWL reduced WC in 6 weeks (4.6%, *P* = 0.000; 5.9%, *P* = 0.000, respectively); (2) HDEL decreased SBP after 3 and 6 weeks (4%, *P* = 0.06; 8%, *P* = 0.009); (3) In HDWL group percent of increase in SBP, DBP, FBS and TG between 3^rd^ and 6^th^ weeks was significant (6.2%, *P* = 0.005; 5%, *P* = 0.017; 3%, *P* = 0.03; 22%, *P* = 0.01); (4) In HDEL group percent of decrease in TG between 3^rd^ and 6^th^ weeks and 1^st^ and 6^th^ weeks was significant (9%, *P* = 0.009; 12%, *P* = 0.05); (5) Both HDEL and HDWL significantly increased fasting concentration of insulin after 3 weeks (HDEL: 31%, *P* = 0.039; HDWL: 39%, *P* = 0.03), but their significant effects disappeared after 6 weeks.; (6) Both HDEL and HDWL significantly increased HOMA-IR in the 1^st^ 3 weeks (HDEL: 35%, *P* = 0.002; HDWL: 38%, *P* = 0.049) but HDEL returned it to basal levels in the subsequent 3 weeks (29%, *P* = 0.031); (7) In HDEL group percent of decrease in AST and ALT between 3^rd^ and 6^th^ weeks was significant (AST: 30%, *P* = 0.000; ALT: 46%, *P* = 0.038).
######
Effect of interventions on metabolic features with in groups
With an independent *t*-test or Mann--Whitney U-test we obtained the following results.(Mann--Whitney U-test was used for comparing the percentage changes in WC and AST during T3 and T1, the percentage changes in SBP during T2 and T1 and the percentage changes in TG and AST during T3 and T2. Independent *t*-test was used in the rest of variables): (1) Both HDEL and HDWL increased HOMA-IR in the 1^st^ 3 weeks of intervention. Percent of HOMA-IR change in the 1^st^ 3 weeks of intervention in HDWL group was marginally (*P* = 0.072) less than HDEL group. (2) HDEL increased HDL-C and HDWL decreased it after 3 weeks, the HDEL group had a marginally increased HDL-C compared with that in the HDWL group (*P* = 0.058). (3) HDEL decreased TG and HDWL increased it after 6 weeks, the HDEL group had a significantly decreased TG compared with that in the HDWL group (*P* = 0.021). This difference was made in second half of the study. (4) HDEL decreased SBP and HDWL increased it after 6 weeks, the HDEL group had a significantly decreased SBP compared with that in the HDWL group (*P* = 0.003). This difference was made in second half of the study.
DISCUSSION {#sec1-4}
==========
This clinical trial explored the effects of high-legume hypocaloric diet on metabolic features among a population of women with central obesity. Men and women have different responses to some exposures on cardiometabolic risk factors due to their physiological differences in sex hormones. Previous RCTs were conducted on men or men/women participants. This study was the first exclusively female study of this type. In this study legumes significantly reduced the SBP. This finding was shown in Hermsdorff and Papanikolaou study, too.\[[@ref10][@ref23]\] Legumes are commonly rich in fiber, calcium, potassium and magnesium and low in sodium.\[[@ref24]\] Meta-analysis showed that increasing fiber consumption as much as approximately 17 g/day will decrease SBP by 1.15 mmHg and DBP by 1.65 mmHg\[[@ref25]\] The mechanisms contributed to hypotensive effects of high-fiber foods are uncertain and several factors may be involved.\[[@ref26]\] In epidemiologic studies, High consumption of calcium, potassium and magnesium and low consumption of sodium have been associated with reduced metabolic risks.\[[@ref27][@ref28]\] The Dietary Approaches to Stop Hypertension (DASH) clinical trial was a milestone study in treatment and prevention of hypertension.\[[@ref29]\] The diet was rich in legumes, vegetables, fruits, vegetables and whole grains. The DASH diet significantly reduced blood pressure.\[[@ref29]\] The follow-up clinical trial studied the effects of sodium intake as part of the DASH diet and showed a low sodium intake as part of the DASH diet decreased SBP by 8.9 mmHg and DBP by 4.5 mmHg.\[[@ref30]\] These studies suggest that high consumption of legumes may have a beneficial effect on blood pressure.
In this study legumes had beneficial effects on TG compared to legume-less diet in consistent with Anderson and Major meta-analysis and Zhang *et al*. study.\[[@ref8][@ref9]\] Probably legumes decreased TG due to high fiber and specific protein content. Sandström *et al*. indicated pea fiber reduced fasting and postprandial serum TG concentrations in healthy people.\[[@ref31]\] Lasekan *et al*. showed pea proteins significantly decreased blood TG in rats.\[[@ref32]\] In Boualga *et al*. study proteins of lentil and chickpea reduced TG more than casein in growing rats.\[[@ref33]\]
In HDEL group percent of decrease in AST and ALT between 3^rd^ and 6^th^ weeks was significant. No effect of legumes in the 1^st^ 3 weeks of the study and its beneficial effects in subsequent 3 weeks represent probability of beneficial effects of legumes on hepatic function in long period. Recent studies have indicated that liver enzymes are correlated with insulin resistance and cardiovascular diseases.\[[@ref34]\] Due to blood liver enzymes levels as a new component of metabolic syndrome and their association with insulin resistance, our study provides new evidence for the benefits of consuming a specific food, like legumes, for women with central obesity.
In consistent with most of previous studies in healthy or obese participants, legumes had not beneficial effects on FBS, insulin and HOMA-IR.\[[@ref10][@ref35][@ref36][@ref37][@ref38][@ref39][@ref40]\] However, studies on diabetic or insulin resistant participants showed beneficial effects of legumes on insulin resistance parameters.\[[@ref9][@ref41]\] Another reason contributed to beneficial effects of legumes on insulin resistant parameters in Zhang *et al*. study can be the high amount of legumes in their hypocaloric diet.\[[@ref9]\] The amount of legumes in legumes enriched hypocaloric diet of Zhang *et al*. study (3.8 servings/day) was higher than all of the previous RCTs. Furthermore, we showed in HDWL group percent of increase in FBS between 3^rd^ and 6^th^ weeks were significant and after enhancement of HOMA-IR in 1^st^ 3 weeks in both groups only HDEL returned it to basal levels in the subsequent 3 weeks. These results represent probability of beneficial effects of legumes on insulin resistance in long period.
HDEL and HDWL significantly reduced the WC and legumes had no advantage in 3 and 6 weeks.
In HDWL group percent of increase in SBP, DBP, FBS and TG between 3^rd^ and 6^th^ weeks was significant. These findings confirmed beneficial effects of legumes on metabolic features and showed that omitting of legumes from diet may have harmful effects on metabolic features and increase the cardiovascular risk. In HDWL group participants stopped their usual intake of legumes and replaced it and some of diet liquid fats with animal proteins and fats. Probably the effect of this change on increasing TG is more than lowering effect of hipocaloric diet.
In our study, legumes marginally increased HDL-C compared to legume-less diet only in 1^st^ 3 weeks of the intervention. Hirshberg *et al*. exhibited a small positive correlation between pulses intake and HDL-C.\[[@ref42]\] Short-term effect of legumes on HDL-C levels can be contributed to their specific proteins. Lasekan *et al*. represented in rats that pea proteins significantly increased HDL-in 4 weeks.\[[@ref32]\] In consistent with Zahradka study,\[[@ref36]\] HDEL had no advantage in increasing HDL-C compared to HDWL in 6 weeks but Abet *et al*. showed legumes reduced HDL-C.\[[@ref35]\] Probably inconsistent result of Abet *et al*. study was created because of different amount of fat in their interventional diet.
In this study, the mean consumption of legume in pre-study period was 2.94 servings/week compared to 1serving/week in Hermsdorff study\[[@ref10]\] and 1.3 servings/week in Zhang *et al*. and Hartman *et al*. study.\[[@ref9][@ref12]\] In fact, the pre-study consumption of legume in our study was almost 3 times more than pre-study legume consumption in previous RCTs. In Crujerias and Hermsdorff studies the legumes consumption even after intervention reached to the pre-study level of the current study.\[[@ref10][@ref11]\] The beneficial effects of different doses of legumes such as 4 serving/week in Hermsdroff study,\[[@ref10]\] 2 servings/d in current study, 3 servings/d in Hartman *et al*. and Zhang *et al*. studies,\[[@ref9][@ref12]\] on BP, TG, HDL-C and liver enzymes and more beneficial effects of 3.8 servings/d in Zhang *et al*. study\[[@ref9]\] not only on motioned metabolic features but also on C-peptide and fasting plasma glucose and insulin indicates probably there are liner relationship between the legumes consumption and SBP and fasting blood TG, HDL-C, liver enzymes, glucose, insulin and C-peptide. Legumes beneficial effects on these parameters did not reach to a plateau.
To the best of our knowledge, this is the first research studied a high-legume hypocaloric diet exclusively in women. The advantage of current research was the particular population of our research which their mean usual intake of non-soy legumes was nearly threefold of usual intakes in preceding RCTs.\[[@ref10][@ref12]\] This study offered an opportunity to discover the effects of high-legume diet on metabolic features in subjects with high basal intake of legumes. The present study had two limitations: First, The subjects' explanations for leaving the research were not assessed in the current study. Second, Intervention diets had inevitable differences in animal protein and fat content in addition to legumes content and some of observed results could be related to this diversity.
CONCLUSIONS {#sec1-5}
===========
HDEL significantly reduced the SBP and TG. Both HDEL and HDWL significantly increased fasting concentration of insulin and HOMA-IR after 3 weeks, but their significant effects on insulin disappeared after 6 weeks and HDEL returned HOMA-IR to baseline levels in the subsequent 3 weeks. In HDEL group percent of decrease in AST and ALT between 3^rd^ and 6^th^ weeks was significant. In HDWL group percent of increase in SBP, DBP, FBS and TG between 3^rd^ and 6^th^ weeks was significant. The study indicated beneficial effects of hypocaloric diets on central obesity and legumes on blood pressure, metabolic features and hepatic function. Long-term studies for approving these results are necessary.
The authors would like to thank Tabriz University of medical sciences, Nutrition Research Center and Liver and Gastrointestinal Disease Research Center for their support and also thank the participants of this study for their enthusiastic support.
**Source of Support:** This work was supported by Tabriz University of medical sciences (Grant no. 5.4.8491), Nutrition Research Center (Grant no. 5.71.2419) and Liver and Gastrointestinal Disease Research Center (Grant no. GT-660)
**Conflict of Interest:** None declared.
|
{
"pile_set_name": "PubMed Central"
}
|
Malignant gliomas are a highly heterogeneous group of brain tumours that exhibit diverse invasiveness, aggressiveness and treatment responsiveness. Diffuse lower-grade gliomas (LGGs, World Health Organization (WHO) grades II and III) demonstrate highly variable clinical behaviour ranging from indolent to rapidly progressive^[@R1]^. LGGs often recur as glioblastomas (GBMs, WHO grade IV glioma), which are inarguably the most advanced and lethal adult brain tumour, with poor treatment responsiveness and a high recurrence rate contributing to poor patient outcome.
Mutations in the metabolic enzyme isocitrate dehydrogenase (IDH) characterize the majority of LGGs and a small percentage of GBMs, and define subtypes that associate with better responses to radiation treatment and an improved prognosis compared with gliomas with wild-type (WT) IDH^[@R1]--[@R4]^. Oncogenic IDH mutations divert metabolic flux resulting in high levels of (*R*)-2-hydroxyglutarate, an onco-metabolite implicated in hypoxia-inducible factor 1 alpha (HIF1α) stability, epigenetic modifications and extracellular matrix (ECM) remodelling^[@R5]^. The relationship between IDH mutations and downstream HIF1α signalling remains unclear with conflicting reports suggesting elevated HIF1α protein in IDH-mutant tumours^[@R6]^ or, conversely, blunted hypoxia sensing through HIFα mediated by elevated activity of prolyl 4-hydroxylases^[@R7]^. As tumours are characterized by altered tissue-level and cellular mechanics, including ECM remodelling and stiffening, and a stiffened ECM can compromise blood vessel integrity to induce hypoxia and activate HIF1α (refs [@R8]--[@R10]), we investigated the interplay between IDH1 mutation status, ECM mechanics, HIF1α signalling and GBM aggression.
Results {#S1}
=======
ECM stiffness associates with IDH1 mutations in primary tumours {#S2}
---------------------------------------------------------------
To determine whether glioma aggression associates with ECM stiffness, we utilized atomic force microscopy (AFM) to quantify the stiffness^[@R11]^ of the ECM in a cohort of fresh-frozen human brain biopsies representing non-tumour gliosis, *de novo* (primary) LGGs (WHO grades II and III) and *de novo* primary GBMs (WHO grade IV). We determined that gliotic tissue had the lowest ECM stiffness (Young\'s modulus, *E*;10--180 Pa) while LGGs (50--1,400 Pa) and GBMs (70--13,500 Pa) were progressively stiffer ([Fig. 1a](#F1){ref-type="fig"}). Consistent with the elevated ECM stiffness, mechanosignalling increased progressively from gliotic tissue to the stiffer GBMs, as indicated by higher phosphorylated focal adhesion kinase (pFAK residue Tyr397) and myosin light chain 2 (pMLC2 residue Ser19) ([Fig. 1b](#F1){ref-type="fig"}). NanoString nCounter gene expression analysis^[@R12]^ of an outcome-predictive gene cluster^[@R13]^ (aggressiveness binned on a scale of 1--10 with 1 indicating the most aggressive) of human GBM tissue biopsies indicated a significant correlation between the proportion of highly stiff areas within a GBM tissue (*E* \>1,400 Pa) and worst patient prognosis score ([Fig. 1c](#F1){ref-type="fig"}, red). By contrast, those tissues that contained a high proportion of soft ECM regions (*E* \< 200 Pa) had the best patient prognosis score^[@R13]^ ([Fig. 1c](#F1){ref-type="fig"}, blue).
Interestingly, despite a strong overall correlation between ECM stiffness and glioma grade, relatively stiff and compliant subsets of patient tumours were evident in both the LGG and GBM samples. To explore this finding, we assessed the IDH1 mutational status in our cohort of patient samples. Mutations in IDH genes characterize the majority of LGGs and a small percentage of GBMs^[@R1]--[@R4]^. For both the LGG and GBM samples, we noted that the stiffness of the ECM in the R132H IDH1 tumours was significantly less than the ECM measured in the WT IDH1 tumours ([Fig. 1d,e](#F1){ref-type="fig"}). To this end, the R132H IDH1 GBM samples exhibited mechanical characteristics more concordant with the LGG samples ([Fig.1d,e](#F1){ref-type="fig"}). R132H IDH1 GBM samples also had lower pFAK and pMLC2 levels, reflecting reduced mechanosignalling ([Fig. 1f](#F1){ref-type="fig"}). These data demonstrate that ECM stiffness correlates with glioma aggression and that IDH1 mutational status associates with a softer ECM, regardless of histological grade.
TNC modifies ECM stiffness and mechanosignalling {#S3}
------------------------------------------------
To examine the relationship between IDH mutation and ECM stiffness, we used a candidate approach to profile major ECM constituents in GBM, hyaluronic acid (HA), and HA-interacting molecules^[@R14],[@R15]^. Immunohistochemistry revealed that the elevated ECM stiffness in the patients with poorer prognosis was accompanied by a substantial increase in HA expression as well as increased levels of TNC, and indicated that these levels increased progressively from gliosis to LGGs and to the GBMs ([Fig. 2a](#F2){ref-type="fig"} and [Supplementary Fig. 2a,b](#SD1){ref-type="supplementary-material"})^[@R16]^. Importantly, ECM stiffness did not correlate with levels or distribution of type I collagen, vasculature or cellularity ([Supplementary Fig. 1a--g](#SD1){ref-type="supplementary-material"}). Indeed, a survey of the ECM status of the softer ECMs in the IDH1-mutant human GBM biopsies revealed a reduction in the levels of TNC ([Fig. 2b](#F2){ref-type="fig"}). Further, bioinformatics analysis of publicly available messenger RNA expression data^[@R1]^ revealed a significant correlation between IDH1 mutational status and TNC expression in LGG tumours, with stratification of LGG patients by TNC upregulation revealing reduced patient survival ([Fig. 2c,d](#F2){ref-type="fig"}).
Similarly, IDH1 mutational status correlated significantly with TNC mRNA expression in GBM^[@R17],[@R18]^ tumours ([Fig. 2e](#F2){ref-type="fig"}). NanoString nCounter gene expression analysis^[@R12]^ of TNC expression (where lower scores correspond to lower expression) in GBM samples yielded a significant correlation between TNC and the tumour aggressiveness score derived via the outcome-predictive gene expression signature (where lower scores correlated with reduced overall survival, [Fig. 2f](#F2){ref-type="fig"}).
Consistent with a relationship between IDH1 mutational status, ECM stiffness and glioma aggression, ectopic expression of the R132H IDH1 mutation significantly improved the survival of nude mice bearing xenografts derived from either primary GBM cells or the U87 cell line ([Fig. 2g](#F2){ref-type="fig"} and [Supplementary Fig. 2c,d](#SD1){ref-type="supplementary-material"}). The resultant tumours were also softer and exhibited reduced mechanosignalling, as revealed by decreased pMLC2 and pFAK ([Fig. 2h](#F2){ref-type="fig"} and [Supplementary Fig. 2e,f](#SD1){ref-type="supplementary-material"}). In both the primary and U87 xenograft models, we noted that the softer IDH1-mutant-expressing GBM tumours had low to negligible TNC expression ([Fig. 2i](#F2){ref-type="fig"} and [Supplementary Fig. 2g](#SD1){ref-type="supplementary-material"}).
To experimentally test the role of TNC in ECM stiffness and glioma aggression, we knocked down TNC in primary GBM cells (WT IDH1, [Supplementary Fig. 2h,i](#SD1){ref-type="supplementary-material"}) and assessed the resulting effects on ECM stiffness and tumour aggression. Nude mice injected orthotopically with GBM cells with short hairpin RNA (shRNA)-reduced TNC not only survived longer ([Fig. 2j](#F2){ref-type="fig"}), but the tumours were softer and had lower mechanosignalling, as revealed by reduced pFAK and pMLC2 ([Fig. 2k,l](#F2){ref-type="fig"}). These findings demonstrate that TNC can impact glioma aggression by increasing ECM stiffness and suggest a molecular role for IDH1 mutational status in perturbing this relationship.
HIF1α directly regulates TNC expression {#S4}
---------------------------------------
Interestingly, we noted that compliant ECM regions in xenograft tumours with TNC knocked down ([Fig. 2j--l](#F2){ref-type="fig"}) had reduced HIF1α expression^[@R19]^ ([Supplementary Fig. 3a,b](#SD1){ref-type="supplementary-material"}). Previous work implicated hypoxia and HIF1α signalling in TNC expression^[@R20],[@R21]^. A bioinformatics analysis revealed a strong correlation between TNC mRNA upregulation and expression of hypoxia-induced genes in both WT and R132H IDH1 patient GBMs ([Fig. 3a](#F3){ref-type="fig"}), and expression of carbonic anhydrase 9 (CA9), a HIF1α-target gene, was reduced in R132H tumours ([Fig. 3b](#F3){ref-type="fig"}). Accordingly, we explored the relationship between GBM aggression and HIF1α-induced TNC expression.
TNC protein expression increased in (WT IDH1) human GBM cells cultured *in vitro* under hypoxia ([Fig. 3c](#F3){ref-type="fig"} and [Supplementary Fig. 3d,e](#SD1){ref-type="supplementary-material"}). shRNA-mediated knockdown of HIF1α reduced hypoxia-dependent induction of TNC ([SupplementaryFig.3c--f](#SD1){ref-type="supplementary-material"}). To investigate the role of reduced HIF1α expression *in vivo*, we orthotopically injected the WT IDH1 primary GBM cells with shRNA-reduced HIF1α. Consistent with a direct link between HIF1α and TNC expression, reducing HIF1α expression increased survival and reduced mechanosignalling ([Fig. 3d,e](#F3){ref-type="fig"}). Importantly, reducing HIF1α expression significantly correlated with reduced TNC mRNA and protein expression levels ([Fig. 3f,g](#F3){ref-type="fig"}).
To determine whether HIF1α directly regulates TNC expression, we identified putative hypoxia-response elements in the promoter of TNC (provided in the Methods). We performed chromatin immunoprecipitation assays using HIF1α as the bait in WT IDH1 human GBM cells and found that HIF1α binds directly to the TNC promoter as indicated by co-precipitation of HIF1α with the TNC promoter ([Fig. 3h](#F3){ref-type="fig"}). As expected, this interaction was largely dependent on HIF1α stabilization under reduced oxygen tension (1% O~2~, hypoxia, [Fig. 3h](#F3){ref-type="fig"}). These data suggest that tissue mechanics and HIF1α could modify glioma aggression via a positive feedback loop.
R132H IDH1 primary GBMs cannot tune HIF1α {#S5}
-----------------------------------------
The presence of the IDH1 mutation could enhance GBM patient survival by increasing the production of the onco-metabolite (*R*)-2-hydroxyglutarate, which can induce genome-wide methylation alterations and alter cellular redox state to promote cellular transformation^[@R5],[@R22]^. IDH1 could also influence tumour phenotype by regulating HIF1α (refs [@R6],[@R7],[@R23],[@R24]). Analysis of WT and R132H IDH1 GBM xenografts revealed that R132H IDH1 tumours had low to barely detectable nuclear HIF1α (and TNC) despite evident necrosis (asterisks) and hypoxia ([Fig. 4a](#F4){ref-type="fig"} and [Supplementary Fig. 3g](#SD1){ref-type="supplementary-material"}).
Immunoblot analysis confirmed that the IDH1-mutant GBMs had significantly reduced total tissue HIF1α protein ([Fig. 4b](#F4){ref-type="fig"}) suggesting a profound dysregulation of the hypoxic response in xenograft tumours expressing the R132H IDH1 mutation.
Experimentally, we observed a lack of upregulation of either HIF1α or TNC in R132H IDH1 GBM cells cultured *in vitro* under hypoxia, a finding that was recapitulated with experimental models endogenously expressing R132H IDH1 ([Fig. 4c](#F4){ref-type="fig"} and [Supplementary Fig. 4a--c](#SD1){ref-type="supplementary-material"}). When we ectopically expressed a constitutively active HIF1α (CA-HIF)^[@R25]^ in the R132H IDH1 cells, we found a robust upregulation of TNC expression both under normoxia and hypoxia ([Fig. 4c](#F4){ref-type="fig"} and [Supplementary Fig. 4c](#SD1){ref-type="supplementary-material"}). Importantly, survival was decreased in mice bearing orthotopic R132H IDH1 GBM tumours expressing the constitutively active HIF1α (CA-HIF) compared with vector-only tumours ([Fig. 4d](#F4){ref-type="fig"}). Consistently, both mechanosignalling and TNC were elevated in constitutively active HIF1α tumours ([Fig. 4e, f](#F4){ref-type="fig"}). These data suggest that the R132H IDH1 mutation blunts hypoxia sensing to reduce HIF1α-dependent TNC expression, and provide a potential explanation for why patients bearing tumours with mutations in IDH experience improved survival.
Mechanosignalling promotes R132H IDH1 tumour aggression {#S6}
-------------------------------------------------------
While IDH mutations may confer prolonged survival in glioma patients suffering from gliomas, IDH-mutation-bearing gliomas still frequently recur despite surgical resection and treatment^[@R26],[@R27]^. Standard of care for GBM patients involves surgical resection followed by high-dose radiation treatment and chemotherapy. These treatment modalities can cause dramatic changes in the post-treatment tumour microenvironment, including the deposition and remodelling of ECM molecules, which we and others have shown to be associated with stromal stiffening^[@R28]^. Accordingly, we asked whether the post-treatment (secondary) IDH1-mutant GBMs were stiffer than the patient-matched primary LGG or GBM tumours. AFM analysis of secondary IDH1-mutant patient GBMs revealed highly stiff ECMs that were significantly stiffer than those measured in primary IDH1-mutant GBMs and were as stiff, if not stiffer, than those measured in primary WT IDH1 GBMs ([Fig. 5a](#F5){ref-type="fig"} left compared with [Fig. 1e](#F1){ref-type="fig"}). Indeed, mechanical analysis of patient-matched IDH1-mutant GBM pairs, at initial diagnosis (primary tumour) and at recurrence (secondary tumour excised after treatment with gamma radiation and temozolomide chemotherapy), revealed a remarkable increase in the stiffness of the associated ECMs in the secondary IDH1-mutant GBMs ([Fig. 5a](#F5){ref-type="fig"}, right). Further, we noted a strong increase in TNC expression in secondary tumours for both LGG and GBM patient cohorts ([Fig. 5b](#F5){ref-type="fig"}).
As such, we explored whether we could increase the aggression of R132H IDH1 GBM cells by enhancing mechanosignalling through plating the cells on a stiff substrate. We plated the R132H IDH1 GBM cells on two-dimensional polyacrylamide (PA) gels with a calibrated stiffness ranging from very soft (140 Pa, representing normal brain^[@R29],[@R30]^) to stiff (6,000 Pa, representing the upper range of elastic moduli in GBM samples, as presented in [Fig. 1d](#F1){ref-type="fig"}). We compared their characteristic cell spreading behaviour and found that R132H IDH1 cells spread better on stiff substrates ([Fig. 5c](#F5){ref-type="fig"}). When R132H IDH1 cells were subjected to hypoxia, they showed a robust upregulation of HIF1A, TNC and Glut1 expression ([Fig. 5d](#F5){ref-type="fig"}). By contrast, when plated on a soft substrate analogous to the soft ECMs we measured *in vivo*, they showed a blunted hypoxia sensing response ([Fig. 5d](#F5){ref-type="fig"}). These findings indicate that a stiffened ECM overrides the blunted hypoxia sensing conferred by expression of the mutant R132H IDH1.
To directly assess whether enhancing mechanosignalling could override the blunted hypoxia sensing conferred by the R132H IDH1 gene, we ectopically expressed an auto-clustering β1 integrin mutant (V737N) in the R132H IDH1 cells to recapitulate downstream stiffness-dependent mechanosignalling through elevation of FAK signalling^[@R29]^. We found that R132H IDH1 GBM cells expressing the auto-clustering V737N mutated β1 integrin spread similarly on the soft and stiff substrates ([Fig. 5c](#F5){ref-type="fig"}). Consistent with cell spreading, V737N R132H IDH1 GBM cells responded to hypoxia largely independently of the underlying substrate stiffness with robust increases in HIF1α, TNC and Glut1 expression levels on both soft and stiff substrates ([Fig. 5d](#F5){ref-type="fig"}). These data indicate that ECM stiffness can induce HIF1α and suggest that the relationship between IDH1 mutation and hypoxia sensing through HIF1α may be largely dependent on the stiffness of the ECM micro environment of the tissue.
We performed xenograft injections of R132H IDH1 cells expressing either WT or V737N β1 integrin to test whether enhancing cellular mechanosignalling in mutant GBMs would increase tumour aggression and, if so, whether this increase would be associated with elevated HIF1α and TNC expression. Survival was decreased in mice bearing the V737N β1 tumours compared with WT β1 tumours ([Fig. 5e](#F5){ref-type="fig"}). Tumours derived from R132H IDH1 GBM cells expressing the V737N integrin demonstrated increased mechanosignalling ([Fig. 5f](#F5){ref-type="fig"}), and expressed elevated levels of both HIF1α and TNC ([Fig. 5g](#F5){ref-type="fig"}). In contrast, R132H IDH1 GBM tumours expressing WT β1 integrin had low to negligible HIF1α and TNC protein expression, despite pronounced hypoxia ([Fig. 5g](#F5){ref-type="fig"}). Interestingly, V737N tumours were substantially stiffer than control tumours ([Fig. 5h](#F5){ref-type="fig"}), suggesting a positive feedback whereby elevated mechanosignalling increases TNC expression to increase ECM stiffness. These findings further link GBM aggression to tissue mechanics, and suggest that elevated mechanosignalling can bypass the protective activity imparted by the R132H IDH1 to promote tumour aggression.
miR-203 targets HIF1α and TNC {#S7}
-----------------------------
Next, we explored the putative positive feedback whereby elevated mechanosignalling increases TNC expression to increase ECM stiffness. We previously found that ECM stiffness regulates microRNA expression to alter tumour progression, including miR-203, a microRNA implicated in GBM aggression, recurrence, and treatment responsiveness^[@R9],[@R31]--[@R34]^. We found that R132H IDH1 cells expressed fivefold higher levels of miR-203 on soft PA gels compared with stiff gels ([Fig. 6a](#F6){ref-type="fig"}). Further, we identified several putative consensus sites in the 3′ UTRs of both HIF1α and TNC mRNA ([Supplementary Fig. 5a](#SD1){ref-type="supplementary-material"}). Consistent with the hypothesis that tissue mechanics may induce HIF1α and TNC in GBMs expressing the mutant IDH1 by reducing levels of miR-203, reporter assays using the wild-type and mutated 3′UTR consensus sites confirmed that miR-203 interacts with and inhibits HIF1α transcription at three out of three predicted sites and TNC at one of two predicted sites ([Fig. 6b](#F6){ref-type="fig"}). Experimentally, we tested whether antagomiR-mediated knockdown^[@R35]^ of miR-203 could elevate HIF1α and TNC levels in R132H IDH1 GBM cells grown in culture (on a soft ECM where miR-203 levels are elevated) and *in vivo* ([Supplementary Fig. 5b](#SD1){ref-type="supplementary-material"}). Consistent with a mechanistic interaction, we observed elevated HIF1α and TNC levels in R132H IDH1 GBM cells with reduced miR-203 (ant-203) compared with vector controls ([Supplementary Fig. 5c,d](#SD1){ref-type="supplementary-material"}), with a significant correlation between their protein expression ([Fig. 6c](#F6){ref-type="fig"}). Importantly, orthotopically injected R132H IDH1 tumours with reduced miR-203 exhibited decreased survival ([Fig. 6d](#F6){ref-type="fig"}), increased TNC expression ([Fig. 6e](#F6){ref-type="fig"}), and elevated ECM stiffness ([Fig. 6f](#F6){ref-type="fig"}).
Next, we assessed the expression of miR-203 in our patient-matched primary and secondary tumours, using *in situ* and quantitative PCR, and found that the stiffer secondary tumours exhibited reduced miR-203 expression compared with their patient-matched primary tumour counterparts for patients who originally presented with either LGG or GBM tumours ([Figs 5a](#F5){ref-type="fig"} and [6g](#F6){ref-type="fig"}). Taken together, these data suggest that tumour therapy may contribute to the development of more aggressive IDH1-mutant GBMs by increasing ECM stiffness and reducing miR-203 expression ([Fig. 6h](#F6){ref-type="fig"}).
Discussion {#S8}
==========
Here, we establish a clinically relevant role for ECM mechanics in glioma aggression. We link HIF1α-dependent hypoxia sensing and TNC expression with an aggressive tumour phenotype, and demonstrate that ECM stiffness directly represses miR-203 expression to activate HIF1α-dependent TNC deposition via a positive feedback loop. The relationship between IDH1 mutations and HIF1α is controversial^[@R6],[@R7],[@R24],[@R36],[@R37]^. Our studies highlight the cellular plasticity and sensitivity of R132H IDH1 GBM cells to ECM stiffness and oxygen tension. In all likelihood, mutations in IDH1 will exert a myriad of context-dependent effects to varying consequence depending on the origin of the tumour, the stage of the disease, the oxygen tension within the tumour and the duration of the disease. Gain-of-function IDH mutations often induce a CpG island methylator phenotype (G-CIMP)^[@R20],[@R38]^ that is associated with improved outcome^[@R38]^. IDH mutations are most prevalent in LGG, which, as a result of being less proliferative and diffusely infiltrative, may be less hypoxic than higher-grade GBMs^[@R1],[@R39],[@R40]^. Therefore, LGGs frequently have a relatively long disease course where the long-term effect of IDH mutations (global DNA hypermethylation) is likely to dominate LGG pathobiology. On the contrary, for the rapidly progressing GBMs, the acute effects of IDH1 mutation may profoundly dictate GBM cell behaviour. Our work presents an example of one mechanism, outside of the sustained effects imparted by hypermethylation, whereby expression of the mutant IDH1 can compromise HIF1α induction in response to hypoxia to modify the ECM and alter tissue mechanics and tumour aggression. Our findings suggest that ECM mechanics provides putative promising targets for therapeutic interventions aimed at disabling biomechanically driven pathways that intersect with glioma aggression to enhance treatment efficacy, delay disease progression, and improve patient survival.
Methods {#S10}
=======
Human samples {#S11}
-------------
Human tissue samples, lacking any patient-identifying information, were either collected under approved study (protocol number 11-07588) and in accordance with the University of California, San Francisco Committee on Human Research policy, or obtained from the University of California, San Francisco Brain Tissue Bank. All human tissue samples were collected in compliance with informed consent policy. Being fully aware of the histopathological heterogeneity of glioblastoma 'multiforme', we performed a meticulous region-to-region analysis correlating the mechanical properties of the patient gliosis and tumour samples underlying [Fig. 1](#F1){ref-type="fig"}. We worked with a pathologist to identify specific regions within the patient samples to correlate ECM stiffness with mechanosignalling in adjacent sections. In H&E-stained serial sections, distinct regions (varying from approximately 1--3 mm^2^ in area) of the patient tumours were pathologist identified, with care taken to avoid vasculature and adjacent non-malignant tissue. Stiffness (as measured by AFM), immunostaining and *in situ* hybridization analyses were performed in region-matched adjacent serial sections.
Mouse studies {#S12}
-------------
All mice were maintained in accordance with University of California Institutional Animal Care and Use Committee (IACUC) guidelines under protocol number AN109372-01. For all mouse xenograft studies, an hour before intracranial injection, cells were washed once with PBS solution, collected by trypsinization, counted, and re-suspended in PBS at 100,000 cells per microlitre. For intracranial injections, 5--6-week-old NCR nude female mice were anaesthetized with 2% isoflurane, injected subcutaneously with buprenorphine (0.03 mgkg^−1^), and slowly injected with a 3 μl tumour cell suspension (300,000 cells per 3 μl injection) into the striatum, as described previously^[@R41]^. For TNC shRNA studies, TNC knockdown was induced by addition of 20 mM IPTG in the drinking water of mice for the duration of the entire experiment. Mice were euthanized when exhibiting 15% weight loss. For hypoxia experiments, mice were injected via intraperitoneal injection with 60 mg kg^−1^ hypoxyprobe 70 min prior to euthanization.
Immunostaining and immunoblotting {#S13}
---------------------------------
Immunofluorescence and immuno blotting analysis were performed as described previously^[@R29],[@R30]^. For all immunoblotting, cells were lysed in RIPA or Laemmli buffer^[@R42]^. Antibodies used for immunofluorescence and immunoblotting analysis are listed below.
Antibodies and reagents {#S14}
-----------------------
Antibodies were as follows (used at 1μg ml^−1^ for western blotting and chromatin immunoprecipitation (ChIP) and at indicated concentrations for immunofluorescence studies): tenascin C (Abcam ab108930; 1:1,000), pY397-FAK (Invitrogen 44625; 1:200), total FAK (BD Biosciences 610088), pMLC2 (Cell Signaling 3671; 1:200), total MLC2 (Abcam ab92721, clone EPR3741; 1:200), p-MyPT1 (Millipore ABS45; 1:200), hyaluronic acid binding protein (Calbiochem, 385911; 1:500), aggrecan (Abcam ab3778; 1:500), versican (Abcam ab19345; 1:500), collagen 1 (Abcam ab34710; 1:1,000), propidium iodide (AcrosOrganics 440300250; 1 μg ml^−1^), β-actin (Sigma-Aldrich a5441), HIF1α (Abcam ab-1, for immunofluorescence; 1:200; Abcam ab1, for ChIP; Novus 100-449, for western blotting), hypoxyprobe (Hypoxyprobe, HP1-100 Kit; per manual), CD31 (BD 550389; 1:500), laminin (Abcam ab11575; 1:500), RNA polymerase II (Millipore 05-623B), rabbit IgG isotype control (Cell Signaling 2729; 1:500), Alexa Fluor-conjugated goat secondary anti-mouse IgG and anti-rabbit IgG antibodies (Invitrogen A11012 and A11005; 1:500) and HRP-conjugated rabbit secondary antibody (GE Healthcare Life Sciences NA934VS; 1:5,000).
Cell culture conditions {#S15}
-----------------------
All cells were maintained at 37 °C and 5% CO~2~. Primary human GBM cells (GBM43) have been previously described^[@R43]^, and were cultured in Neurobasal-A media (Invitrogen) supplemented with B27 Supplement (Invitrogen), N2 Supplement (Invitrogen), 20ngml^\_1^ epidermal growth factor (Peprotech), 20ngml^−1^ fibroblast growth factor (Peprotech), and 100 units ml^−1^ penicillin/streptomycin. U87 cells (obtained from and authenticated by ATCC) were grown in DMEM supplemented with 10% fetal bovine serum (Hyclone), 2mM L-glutamine, and 100 units ml^−1^ penicillin/streptomycin. TS603 cells (harbouring endogenous R132H IDH1) and TS667 cells (expressing WT IDH1) have been previously described^[@R44]^, and were grown in Neurobasal-A media (Invitrogen) supplemented with B27 Supplement (Invitrogen), N2 Supplement (Invitrogen), 20ngml^−1^ epidermal growth factor (Peprotech), 20ngml^−1^ fibroblast growth factor (Peprotech), 20ngml^−1^ platelet-derived growth factor AA (Peprotech), and 100 units ml^−1^ penicillin/streptomycin. BT142 cells harbouring endogenous R132H were obtained from and authenticated by ATCC, and were grown in Neurobasal-A media (Invitrogen) supplemented with B27 Supplement (Invitrogen), N2 Supplement (Invitrogen), 20ngml^−1^ epidermal growth factor (Peprotech), 20 ng ml^−1^ fibroblast growth factor (Peprotech), 20 ng ml^−1^ platelet-derived growth factor AA (Peprotech), and 100 units ml^−1^ penicillin/streptomycin. All primary cells and cell lines were authenticated by ATCC (where applicable), or by analysis of morphological and phenotypic characteristics as well as gene and protein expression.
No cell lines used in this study were found in the database of commonly misidentified cell lines that is maintained by ICLAC and NCBI Biosample. All primary cells and cell lines were tested for mycoplasma contamination using a commercially available kit (PCR-Mycoplasma Test Kit I/C (Promokine PK-CA91-1024), according to the manufacturer\'s instructions, at the onset of the work (tested negative) and have never exhibited contamination symptoms after initial testing. Derivative cells were transduced *in vitro* with a lentiviral vector-encoding firefly Luciferase and either eGFP (U87 lines) or mCherry (primary GBM lines) fluorescent proteins for combined bioluminescent imaging and flow sorting. For stable shRNA cell lines harbouring isopropyl β-[d]{.smallcaps}-1-thiogalactopyranoside (IPTG)-inducible transgenes, expression was induced with 100 μM IPTG five days before experimentation. For polyacrylamide hydrogel studies, cells were plated on fibronectin-conjugated gels for either 8 h (protein analysis) or 24 h (mRNA analysis) prior to harvesting cell lysates.
Quantitative real-time PCR and *in situ* hybridization {#S16}
------------------------------------------------------
Total cellular RNA was isolated using the mirVana miRNA isolation kit (Life Technologies AM1560), according to the manufacturer\'s instructions. Reverse transcription was performed from 2 μg of total RNA using random primers (Amersham Biosciences). PCR reactions were performed in triplicate with LightCycler Fast Start DNA Master SYBR Green Mix (Roche) using a Realplex^[@R29],[@R30]^ epGradient S Mastercycler (Eppendorf) and the relative amount of cDNA was calculated by the comparative Ct method using the RPS20 ribosomal protein as a control. Primer sequences are: human TNC forward primer 5′-TCTCAGGGTCATTCACCACA-3′, human TNC reverse primer 5′-CA CCGTGCGTGTAATTTCTG-3′, human HIF1α forward primer 5′-CAGTCGACA CAGCCTGGATA-3′, human HIF1α reverse primer 5′-TGTCCTGTGGTGACTT GTCC-3′, SLC2A1 forward primer 5′-GGCATCAACGCTGTCTTCTA-3′, SLC2A1 R 5′-CAGCGAGGCCTATGAGGTG-3′, RPS20 forward primer 5′-AGGACCAGT TCGAATGCCTA-3′, and RPS20 reverse primer 5′-GATTCGATCAACTCAACTC CTG-3′.
For miRNA qPCR analysis, reverse transcription of specific miRNAs (from 10 ng of total RNA) was carried out using the real-time loop primers for each type of miRNA and the TaqMan miRNA RT Kit from Applied Biosystems (Life Technologies 4366596), according to the instructions. cDNA obtained from this step was used to carry out real-time quantitative TaqMan PCR for miR-203 (Life Technologies 4427975, Assay 000507) using the real-time primers provided, according to the instructions. Ct values were converted to fold expression changes (2 ---ΔΔCt values) following normalization to U6 small nuclear RNA (Life Technologies 4427975, Assay 001973) or SNORD48 (Life Technologies 4427975, Assay 001006).
miRNA *in situ* hybridization was performed as previously described using formalin-fixed, paraffin-embedded tissue sections (5 μm thickness)^[@R45]^. 5′-DIG labelled probes targeting miR-203 (Exiqon 35029-01), U6 small nuclear RNA (positive control, Exiqon 99002-01) or Scramble-miR (negative control, Exiqon 99004-01) were diluted to 40 nM in hybridization buffer. RNA was hybridized overnight at 58 °C for all probes.
Chromatin immunoprecipitation (ChIP) studies {#S17}
--------------------------------------------
Samples were cultured under either normoxia or hypoxia (1% oxygen) and ChIP was performed using the ChIP-IT Express kit (Active Motif 5300), according to the instructions. Primer pairs are: intronic TNC set 1 (90 bp) forward primer 5′-AAAGCAACCGTAGGATGACC-3 and reverse primer 5′-ATCCCCACAGTTATTATTGTT-3′, intronic TNC set 2 (149 bp) forward primer 5′-GGCTGTGATTTCTACACAAATGG-3′ and reverse primer 5′-AGGGAATACACCTGGGAAGTG-3′, intronic TNC set 3 (152 bp) forward primer 5′-CTGGTCCCATTTCCAGCTT-3′ and reverse primer 5′-CGA CCCCGAGTAGCTGTTAG-3′, exonic TNC set 4 (142 bp) forward primer 5′-ACA GCCTGCCTACTGTCACC-3′ and reverse primer 5′-GGAAGAAGTACCTGGAG TGTGG-3′, intronic TNC negative control (149 bp) forward primer 5′-GTGAAAT TCAAAATTAAGTTCAACAA-3′ and reverse primer 5′-CAAGTCGCATCCACT CTTGA-3′, and GAPDH positive control (149 bp) forward primer 5′-TACTAGCG GTTTTACGGGCG-3′ and reverse primer 5′-AGAGCGAAGCGGGAGGCT-3′.
Atomic force microscopy measurements {#S18}
------------------------------------
Atomic force microscopy (AFM) and analysis were performed using an MFP3D-BIO inverted optical atomic force microscope mounted on a Nikon TE2000-U inverted fluorescent microscope (Asylum Research) as described previously ([Supplementary Fig. 1a](#SD1){ref-type="supplementary-material"})^[@R11]^. Briefly, for pathologist-identified, regionally matched correlations between ECM stiffness and mechanosignalling, patient tissues were immunostained with multiple specific, patient-representative regions (4--10 regions per tumour) identified (with the *x*-and *y*-coordinates noted and images acquired) for further AFM testing in serial sections ([Supplementary Fig. 6c](#SD1){ref-type="supplementary-material"} left). After patient tumour sections were secured for mechanical testing, the identified testing region was located via *x*- and *y*-coordinates using the optical microscope ([Supplementary Fig. 6c](#SD1){ref-type="supplementary-material"} right). Individual patient data were combined and graphed to depict correlations and the patient variability within the cohort(s) ([Supplementary Fig. 6d,e](#SD1){ref-type="supplementary-material"}).
Nanostring nCounter gene expression signature assessment {#S19}
--------------------------------------------------------
NanoString nCounter gene expression analysis^[@R12]^ of human GBM tissue biopsies was used to assess the 9-gene expression signature used as a surrogate for tumour aggressiveness^[@R13]^. Derived from a 38-gene predictor (validated as a significant predictor of overall survival), the top-ranked 9 genes were selected on the basis of the significance of survival association (fold change level) and technical considerations (including abundance and consistency of amplification of the target gene in FFPE samples)^[@R13]^. Aggressiveness reflects the overall survival prediction based on the meta-score calculated with the 9-gene signature^[@R13]^.
Lentivirus-mediated ectopic expression or knockdown {#S20}
---------------------------------------------------
For ectopic expression or knockdown studies, lentiviral vectors (pLV lacI NeoR; gift from J. Lakins, UCSF, USA) were custom built to facilitate cloning of shRNA under inducible IPTG control. Briefly, oligonucleotides corresponding to validated shRNA from The RNAi Consortium (TRC) were designed and annealed to yield duplexes with 5′ and 3′ overhangs complementary to AgeI and EcoRI sites of the modified U6 promoter containing three lacO binding sites for the bacterial repressor protein lacI (Sigma). pLV lacI_NeoR also provides for constitutive nuclear expression via the human EEF1a promoter of lacI tagged at the N terminus with three tandem repeats of the 9E10 myc epitope and at the C terminus with a nuclear localization sequence from SV40 large T antigen. The latter is co-expressed with the neomycin phosphotransferase gene from a bicistronic mRNA via an internal ribosome entry site (IRES) to provide selection of transduced cells with G418. Utilized TNC shRNA was TRCN0000230788.
Preparation of polyacrylamide gels substrates {#S21}
---------------------------------------------
Glass and silicon substrates were prepared by glutaraldehyde activation followed by conjugation with 10μg ml^−1^ (glass) or 20 μg ml^−1^ (silicon) fibronectin as described previously^[@R29]^. Polyacrylamide hydrogel substrates (soft: 2.5% acrylamide, 0.03% Bis-acrylamide; stiff: 10% acrylamide, 0.5% Bis-acrylamide) were prepared as previously described with one modification: functionalization with succinimidyl ester was with 0.01% N6, 0.01% Bis-acrylamide, 0.025% Irgacure 2959, and 0.002% di(trimethylolpropane) tetraacrylate (Sigma)^[@R46]^. Following functionalization with succinimidyl ester, hydrogels were conjugated overnight with 20 μg ml^−1^ fibronectin at 4 °C and rinsed twice with PBS and DMEM before cell plating.
Immunofluorescence and imaging {#S22}
------------------------------
Samples were fixed and labelled as previously described^[@R29]^, with multiple independent fields imaged on a Zeiss LSM 510 microscope system with either a 20 × Apo NA0.75 air or 40 × ApoLWDNA 1.15 water objective and 488 nm argon, 543 nm HeNe, and 633 nm HeNe excitation lines.
Clinical data analysis {#S23}
----------------------
Previously published and reanalysed microarray data were obtained from the TCGA Research Network (<http://cancergenome.nih.gov>) and downloaded from the CBio Portal for Cancer Genomics (<http://www.cbioportal.org/public-portal/index.do>) under the Low Grade Glioma^[@R1]^ and the Glioblastoma Multiforme^[@R17]--[@R19]^ data sets, with mean normalized expression scores (*z* scores) for genes of interest determined. Statistical significance of differences was determined using a two-tailed Mann--Whitney test, with *P* \< 0.05 considered to be significant.
Genes used in the hypoxia gene signature include: *ANXA2*, *BTG1*, *CA9*, *EDN1*, *EGR1*, *HMOX1*, *IER3*, *LDHA*, *LGAL3*, *LOXL2*, *PDK1*, *SLC2A1*, *TFRC*, *VDAC1* and *VEGFA*.
Statistics and reproducibility {#S24}
------------------------------
All quantitative results were assessed by unpaired Student\'s *t*-test after confirming that the data met appropriate assumptions (normality, homogeneous variance, and independent sampling), non-parametric Wilcox/Mann--Whitney exact test (using the normal approximation for the U score), or the Kolmogorov--Smirnov distribution test (α = 0.05), all two-tailed. Unless otherwise indicated, all data were plotted with standard deviation error bars, and variances between groups being statistically compared are similar. For AFM mechanical testing of clinical specimens, multiple regions were assessed (10 regions per patient), and regions were pooled per patient to establish a mean Young\'s modulus per patient; statistical analysis was performed on patient means (unless otherwise noted). Animal cohort and *in vitro* sample sizes were chosen to provide 85% power to detect an effect size of 2.5 with a two-sided error of less than or equal to 5%. All results were reproduced at least twice in the laboratory and the n-value denotes the sample size used in statistical testing. Graphpad/Prism software was used to conduct the statistical analysis of the data. *P* values less than or equal to 0.05 were considered to be significant. For animal studies, animals were randomly distributed among the different conditions by the investigator since the animals did not exhibit any size or appearance differences at the onset of the experiments. No animals were excluded. For mouse and clinical studies, mechanical testing was performed blinded and immunostaining intensity of tissue sections was scored blinded.
Data availability {#S25}
-----------------
Previously published microarray data^[@R1],[@R17],[@R18]^ that were reanalysed here are available from the TCGA Research Network (<http://cancergenome.nih.gov>) via download from the CBio Portal for Cancer Genomics (<http://www.cbioportal.org/public-portal/index.do>) under the Low Grade Glioma^[@R1]^ and the Glioblastoma Multiforme^[@R17]--[@R19]^ data sets. All other relevant data that support the findings and conclusions of this study are available from the corresponding author on request.
Supplementary Material {#S26}
======================
######
**Supplementary Figure 1** Cellularity, fibrillar collagen and vascularity are independent of ECM stiffness. a, Scatter plots of ECM stiffness measured by AFM in a human patient sample analyzed immediately post resection from the operating room (Fresh), post snap freezing and analyzed either within 30 minutes of being thawed in a cocktail of protein inhibitors (Frozen) or treated with either PBS (PBS) or hyaluronidase (HAse) for 60 minutes (6 samples/group were tested for all groups with the exception of the "HAse 60 mins" group where 5 samples were assessed). All samples were pooled per group with 5-6 regions tested per sample (pooled group means indicated with red lines). Group means are indicated in red (6 samples/ group were tested for all groups with the exception of the "HAse 60 mins" group where 5 samples were assessed). b, Immunohistochemistry images of patient tissue immunostained for neuronal (SMI31) or astrocytic (GFAP) processes indicating a switch in the type but not density of the cellular processes in normal versus GBM brains. Scale Bar 70μm. c, Correlation between measured ECM stiffness and cell number in the analyzed area revealing no relationship between the two variables (linear and exponential regressions, n=100 areas (10 patient biopsies with 10 areas/patient), insert: representative image of the AFM method for tissue analysis). Scale Bar 150μm. d, Second harmonic generation (SHG) image of fibrillary collagen (purple) and CD31-immunostained vasculature (red) in human GBMs (insert: composite image of SHG and CD31 in a normal human brain). Scale Bar 70μm. e, Immunofluorescence images of astrocytic processes (GFAP, red) and vasculature (Collagen 1, green) in human gliotic (top) and GBM (bottom) biopsies. Scale Bar 50μm. f, Correlation between measured ECM stiffness and percent vascularity of the tissue (percent area positive for FVIII stain) revealing no relationship between the two variables (linear and exponentia regression, n=10 patients). g, Distribution of ECM stiffness measured by AFM in two (labeled as mGBM model 1 and mGBM model 2) mouse xenograft models of GBM (n=4 mice/group), normal mouse brain (n=3 mice) as well as mGBM model 2 treated with an antiangiogenic agent, avastin (labeled as mGBM model 2 + B20, n=5 mice) revealing a slight but nonsignificant reduction in ECM stiffness upon B20-mediated normalization of GBM vasculature in mouse models of GBM. All samples were pooled per group with 5 regions tested per sample (pooled group means indicated with yellow lines).
**Supplementary Figure 2** R132H IDH1 expression modifies TNC expression and ECM stiffness. a, Immunofluorescence images and quantifications of WT and R132H IDH1 patient tissues immunostained for DAPI(blue), aggrecan (green), and versican (red) (mean+/- s.e.m., n=7 mice/group, ns by unpaired 2-sided *t*-test). b, Immunofluorescence images of patient tissues immunostained for the lecticans, aggrecan (green) and versican (red). c, Immunoblot analysis confirming the R132H and WT IDH1 status in U87 GBM line. d, Kaplan-Meier graph showing survival of xenograft mice injected orthotopically with either U87 WT IDH1 (blue, n=7 mice) or U87 R132H IDH1 (red, n=7 mice) human GBM cells. e, Histogram showing the distribution of ECM stiffness measured by AFM in xenograft tumors derived from U87 WT (blue) or R132H mutant (red) IDH1 cells (n=5 mice/group WT IDH1, n=4 mice/group R132H IDH1, two-sided Kolmogorov-Smirnov test P=9.7e-4). f, Immunofluorescence mages and quantification of U87 IDH1 WT and R132H mutant xenograft tumors immunostained for pMLC2 (green, top) and pFAK397 (green, bottom) with propidium iodide (PI, red) (mean+/-s.e.m., n=7 mice/group, unpaired 2-sided *t*-test P=0.0007). g, Immunofluorescence images and quantification of TNC (green) with PI (red) in xenograft tumors derived from either WT or R132H IDH1 human primary cells (mean+/-s.e.m., n=7 mice/group, unpaired 2-sided *t*-test P=0.0087). h, Immunoblot analysis confirming lentiviral shRNA construct targeting of TNC (top). immunofluorecence images of cellular morphology (F-actin in green and DAPI in blue) of scramble shRNA and TNC shRNA cells (bottom). Scale Bar 10μm. i, TNC knockdown in mouse xenograft tumors by mRNA (top) and immunofluorescence (bottom) in scramble and TNC shRNA constructs (mean+/-s.d., n= 5 mice/group, unpaired 2-sided *t*-test P=0.014). Scale Bars 50μm, unless otherwise indicated.
**Supplementary Figure 3** HIF1α and miR-203 regulate TNC expression. a, Distribution of ECM stiffness measured by AFM in xenograft tumors arising from primary R132H IDH1 cells in either HIF1α-positive or -negative regions surrounding areas of necrosis (n=4 mice, two-sided Kolmogorov-Smirnov test P=3.7e-2). All samples were pooled per group with 5 regions tested per sample (pooled group means indicated with yellow lines). b, Immunofluorescence images of xenograft tumors derived from cells expressing a control scramble shRNA or an shRNA for TNC shRNA immunostained for HIF1α with PI (red). c, mRNA expression of HIF1α (left) and TNC(right) in WT IDH1 cells expressing either scramble shRNA or shRNA targeting HIF1α, represented as % of shCNL (mean+/-s.e.m., n=6/group, unpaired 2-sided test P=0.001). d, Quantification of HIF1α (left) and TNC(right) protein expression in IDH1 WT cells with and without hypoxia, as indicated (mean+/-s.e.m., n=6/group, one-way ANOVA with Tukey\'s multiple comparisons test P=0.0013). e, Representative immunoblot of WT and R132H IDH1 primary GBM cells plated on laminin-coated tissue culture plates under normoxic and hypoxic (1% oxygen) conditions probed for TNC and HIF1α. f, Immunoblot of WT IDH1 primary human GBM cells, expressing either scramble control shRNA or shRNA targeting HIF1α, plated on lamin-coated tissue culture plates under normoxic or hypoxic (1% oxygen) conditions, as indicated, and probed for TNC induction. g, immunofluorescence images of xenograft tumors derived from either WT or R132H IDH1 primary human GBM cells immunostained for HIF1α (green) with PI (red); asterisks indicate areas of necrosis. Scale Bars 50μm.
**Supplementary Figure 4** Endogenous and ectopic R132H IDH1 1° GBMs cannot tune HIF1α. a, Immunoblots and quantification of primary human glioma cells harboring an endogenous R132H IDH1 mutation (top: TS603, bottom: BT142) plated on soft (140Pa) or stiff (\>6kPa) substrates under normoxic and hypoxic (1% oxygen) conditions and probed for HIF1α and TNC protein expressions, as well as actin (graph shows means of 2 biological replicate samples per group) b, Immunoblots and quantification of WT IDH1 primary human glioma cells (TS667) plated on soft (140Pa) or stiff (\>6kPa) substrates under normoxic and hypoxic (1% oxygen) conditions and probed for HIF1α and TNC protein expressions, as well as actin (graph shows means of 2 biological replicate samples per group,) c, Immunoblot of R132H IDH1 primary human GBM cells expressing either vector control or constitutively active HIF1α (CA- HIF1α) plated on soft (140Pa) gels under normoxic and hypoxic (1% oxygen) conditions, as indicated, and probed for HIF1α protein expression, as well as HIF1α -driven protein expressions of TNC and LOXL2.
**Supplementary Figure 5** miR-203 targets the HIF1α and TNC 3′ UTRs. a, Diagram of miR-203 targeting seed sequences located in 3′-untranslated regions (3′ UTRs) of human HIF1α and TNC mRNAs. b, Quantification of miR-203 expression after RNA immunoprecipitation (RIP) using AGO2 as a bait validates that antagomiR-mediated targeting of miR-203 reduces miR-203 loading into the RISC (graph shows mean of 2 IP lysates per group). c, Immunoblots for TNC, HIF1α, LOXL2 (as HIF1α target gene) and actin from primary human GBM cells expressing R132H IDH1 (+/-an antagomiR control or an antagomiR targeting miR-203, +/- 1% hypoxia). d, Quantification of HIF1α and TNC protein expressions in primary human GBM cells expressing R132H IDH1 (+/-an antagomiR control or an antagomiR targeting miR-203, +/- 1% hypoxia) (means+/-s.e.m., n=3 lysates/group, One-way ANOVA with Tukey\'s multiple comparisons test P=0.0011).
**Supplementary Figure 6** Regional assessment of ECM stiffness and mechanosignaling. a, Maps of ECM stiffness are obtained using an AFM tip where a 5μm diameter bead is attached to the end of a cantilever. The AFM tip was brought down to indent the sample, during which the motion of the z-piezo and the applied force were recorded. The ECM stiffness was computed from the displacement of the z-piezo minus the bending of the cantilever. The number of ECM stiffness measurements made was operator-determined by the x- and y-step sizes input for a 90μm by 90μm grid. The example shown demonstrates how step sizes of 14 (in the x-and y-directions) result in 196 independent mechanical measurements. b, For regionally-matched correlations between ECM stiffness and mechanosignaling, patient (and xenograft) tissues were immunostained for TNC (green) and pFAK (red). Multiple specific, patient-representative regions (4-10 regions per tumor) were identified (with the x- and y-coordinates noted and images acquired) for further AFM testing in a serial section. For subsequent AFM testing, after patient (and xenograft) tumor sections were secured for testing, the identified testing region was located via x- and y-coordinates using the optical microscope. c, Left: Immunohistochemistry mages of patient tumors immunostained for TNC (green) and pFAK (red) with DAPI (blue) with regions for AFM analysis in serial sections denoted by white boxes. Graphs illustrate individual patient correlations between TNC and pFAK. Right: AFM maps of patient tumors. Graphs illustrate individual patient correlations between elastic modulus (stiffness) and pFAK. d, The data underlying (c left) was combined into graphs depicting regiona matching of ECM stiffness and pFAK (left) and TNC and pFAK (right) to illustrate not only the correlations between the variables, but also the variability between patients within the cohort (n=3 patient samples/group, One-way ANOVA with Tukey\'s comparison P=0.0011). e, The data underlying (c right) was combined into a graph depicting both the patient-specific trends in elastic modulus (stiffness) and the patient variability within the cohort (n=10 patients, 4 regions/patient sample, linear regression analysis yielding p\<0.0001 for both plots with R2=0.4812 for pFAK:stiffness data and R2=0.5850 for pFAK:TNC data). Scale Bars 200μm (left panels) and 20μm (right panels), as indicated.
**Supplementary Figure 7** Full Scans of Key Blots
We thank K. Lu for the B20 mouse experiment with input from G. Bergers; C. Cowdrey and A. Shai for tissue collection support; and I. Acerbi for IRB paperwork. Animal handling and tissue preparation was supported by L. Korets and N. Korets. This work was supported by US National Institutes of Health NCI R01 grants CA138818-01A1, CA192914-01, CA174929-01, and 2R01CA085482-11A1 (VM.W.), U54 grants CA163155-01 and CA143836-01 (VM.W.), US National Institutes of Health NCI F31CA180422-01A1 (Y.A.M.), NSF GRFP 1144247 (Y.A.M.), US DOD BCRP grant W81XWH-07-1-0538 (J.K.M.), NINDS R01 NS081117 (J.J.P.), NINDS R21 NS088114 and NBTS (A.I.P.).
Note: [Supplementary Information](#SD1){ref-type="supplementary-material"} is available in the online version of the paper
**Author Contributions:** V.M.W., Y.A.M. and J.K.M. conceived the project. T.R.M., K.L., J.J.P. and Y.A.M. were involved in patient recruitment and human tissue collection. J.J.P. performed human patient sample selection and performed NanoString nCounter gene expression analysis with G.F.R. Y.A.M. and J.M.B. performed mechanical testing. Y.A.M. and J.K.M. performed immunohistological analyses, imaging analyses, and quantification. J.M.B. conducted animal injections. J.M.B., Y.A.M. and J.K.M. monitored mouse colonies, and processed and analysed mouse and human tissues. J.K.M. and Y.A.M. performed all *in vitro* studies. J.N.L. and Y.A.M. designed and built all shRNA, microRNA and IDH1 expression constructs. M.W.P performed the bioinformatics analysis. A.I.P. assisted Y.A.M. with primary GBM culture and manipulations. E.C.H. and YK. provided the TS603 and TS667 glioma cells. V.M.W., J.K.M. and Y.A.M. wrote the manuscript with input from all authors.
**Competing Financial Interests:** The authors declare no competing financial interests.
{#F1}
{ref-type="fig"} correlating TNC expression with raw NanoString nCounter aggressiveness scores (higher score indicating higher aggressiveness, *n* = 10 patient samples, linear regression where *R*^2^ = 0.8245). **(g)** Survival of xenograft mice injected with either WT IDH1 (red, *n* = 8 mice) or R132H IDH1 (orange, *n* = 8 mice) human GBM cells (log-rank (Mantel--Cox) test, *P* = 0.04). **(h)** Distribution of ECM stiffness in xenograft tumours from human GBM cells infected with either WT or R132H IDH1 cells (*n* = 4 mice per group, *n* was used to derive statistics, histogram encompasses all measurements across the five regions of four mice, two-sided Kolmogorov-Smirnov test, *P*=1.3×10^−3^). **(i)** Immunofluorescence images and quantification for TNC with propidium iodide (PI) in xenograft tumours derived from either WT or R132H IDH1 human GBM cells (mean ± s.e.m., *n* = 4 WT mice, *n* = 5 R132H IDH1 mice, two-sided unpaired *t*-test, \*\**P* = 0.00023). Dashed lines indicate tumour edges. **(j)** Survival of xenograft mice injected with WT IDH1 human GBM cells expressing either a control (CNL) scramble shRNA (red) or an shRNA targeting TNC (grey) (*n* = 8 mice per group, log-rank (Mantel--Cox) test, *P* = 0.03). **(k)** Distribution of ECM stiffness in xenograft tumours derived from WT IDH1 primary human GBM cells expressing either a control scramble shRNA (red) or shRNA targeting TNC (grey) (*n* = 6 mice per group, *n* was used to derive statistics, histogram encompasses all the measurements across all regions of all six mice, two-sided Kolmogorov-Smirnov test, *P*= 1.73 × 10^−2^). **(l)** Immunofluorescence images and quantification for pFAK397 (\*\*\**P* = 0.0008) and pMLC2 (\**P* = 0.009) with DAPI in xenograft tumours expressing control scramble shRNA or shRNA targeting TNC (mean ± s.e.m., *n* = 4 mice per group, two-sided unpaired *t*-test). Scale bar, 50 μm.](nihms852654f2){#F2}
{ref-type="supplementary-material"}.](nihms852654f3){#F3}
{ref-type="supplementary-material"}.](nihms852654f4){#F4}
{ref-type="supplementary-material"}.](nihms852654f5){#F5}
{ref-type="fig"}. (e) Immunofluorescence images and quantification for TNC (red) in xenograft tumours derived from primary human GBM cells expressing either CNL or ant-203 vectors (mean ± s.d., *n* = 4 mice per group, two-sided unpaired *t*-test, \**P* = 0.0491). (f) Histogram showing the distribution of ECM stiffness in xenograft tumours derived from primary R132H IDH1 human GBM cells expressing either a CNL or ant-203 constructs (n = 5 mice per group, *n* was used to derive statistics, two-sided Kolmogorov-Smirnov test, \*\*P\<0.009). (g) Bright-field images (left) of miR-203 *in situ* hybridization in patient-matched primary and recurrent secondary R132H IDH1 patient glioma tumours. Top: patient-matched primary LGG and secondary GBM. Bottom: patient-matched primary GBM and secondary GBM, with corresponding U6-positive control and scramble negative control. Insets show staining with DAPI. Right: qPCR quantification of miR-203 expression in the patient samples shown on the left; miR-203 expression was normalized by SNORD48 small nucleolar RNA expression (mean ± s.d., two-sided paired *t*-test, for LGG to GBM, *n* = 5 patients and \*\*\**P* = 0.0034; for GBM to GBM, *n* = 3 patients and \*\*\**P* = 0.0006). (h) Graphic depicting a model of ECM stiffness-driven repression of miR-203 expression to activate HIF1α/TNC. Scale bars, 50 μm.](nihms852654f6){#F6}
[^1]: These authors contributed equally to this work.
|
{
"pile_set_name": "PubMed Central"
}
|
Background
==========
The benefits of breastfeeding for infants, mothers and communities are widely acknowledged \[[@B1]\]. Breastfeeding can decrease the incidence and severity of infectious diseases and thereby the risk for post neonatal infant death \[[@B1]\]. Exclusive breastfeeding has been found to prevent obesity \[[@B1]\]. Breastfeeding has also been found to be associated with better cognitive development \[[@B1]\] as well as reduced occurrence of postpartum bleedings, breast cancer and ovarian cancer of mothers \[[@B2]\]. In addition, benefits of breastfeeding on the economic situation of the family and the environment have been reported \[[@B1]\]. The World Health Organization (WHO) recommends exclusive breastfeeding for the first 6 months of life, with continued breastfeeding through the second year of life \[[@B3]\]. However, these recommendations are not followed everywhere: in 2007, a national survey in USA reported that 26% of all women, with children aged from 0 to 5 years, did not give any breastfeeding at all \[[@B2]\]. In a study of five Asian countries 2002 to 2005, the percentages of exclusive breastfeeding of infants younger than 6 months were reported as 30.7% in Timor-Leste, 33.7% in Philippine, 38.9% in Indonesia and 60.1% in Cambodia. The fifth country, Vietnam, reported only 15.5% \[[@B3]\].
In Vietnam breastfeeding has been promoted by health authorities since the early 1980s. The government then adopted a policy for exclusive breastfeeding to at least six months of age as part of the national strategy and advocated initiation of breastfeeding during the first hour after birth \[[@B4]\].
In practice the recommendations have not been followed \[[@B3]\]. A study from Ho Chi Minh city in 2000 reported that only 47.5% of all newborns were given breast milk as the first feed \[[@B5]\]. In a rural area in 2002, initiation of breastfeeding during the first hour of life was reported for 73.6% of all newborn and 83.6% were exclusively breastfed at one week of age \[[@B6]\]. However, exclusive breastfeeding dropped to zero by 6 months \[[@B7]\]. In another study 77% and 22% of children continued any breastfeeding at 12 months and 24 months of age respectively \[[@B8]\].
In Vietnam, it is generally poorly understood by health professionals what factors are associated with breastfeeding practices \[[@B9]\]. Cultural and traditional beliefs, socioeconomic situation \[[@B10]\], mother's work outside home \[[@B11]\], mother's educational level, father's occupation \[[@B7]\], mother's lack of knowledge and misinformation \[[@B12]\], the delivery method and delivery location as well as health problems of the mother \[[@B6]\] have been proposed to influence the breast feeding practices. Another potential factor of influence is the maternity leave regulation. In Vietnam the maternity leave is at present 4 months \[[@B13]\].
Rural--urban differences in breastfeeding initiation, exclusive breastfeeding, and duration of breastfeeding have been reported from some countries \[[@B2],[@B14]-[@B17]\]. The prevalence of breastfeeding was higher in an urban setting as compared to a rural in USA and Tanzania \[[@B2],[@B17]\]. The opposite was found in China \[[@B14]\].
Longitudinal studies like the present that compare breastfeeding practices between urban and rural areas have not been conducted in Vietnam. A hypothesis is that there are different breastfeeding practices of infants in rural and urban areas in Vietnam, possibly due to differences in economic resources and the educational level of mothers. The aim of this study is therefore to quantitatively describe breastfeeding practices during the first year of life in one rural and one urban area of Vietnam using information from repeated interviews with mothers and to study associations between child, mother and household factors and these breastfeeding practices.
Methods
=======
Study sites
-----------
The study was conducted in two Health and Demographic Surveillance Sites (HDSS), one urban and one rural, located within the municipality of Hanoi, the capital city of Vietnam. Dong Da is a central, old district of urban Hanoi. The total population is about 352,000 persons living in 21 communes with a total area of around 10 km^2^. The socio-economic characteristics are relatively typical for urban Vietnamese areas in big cities with an estimated income per capita above 1,000 USD per year. Three communes with 37,000 persons in 10,500 households strategically selected to have different economic levels, were defined as the DodaLab HDSS \[[@B18]\].
Ba Vi is a rural district within the municipality of Hanoi with a population of 250,000 persons. Farming is the main occupation with an income per capita of about 300 USD per year. A sample called FilaBavi HDSS, of 11,089 households with 51,024 persons has been followed since 1999 \[[@B19]\].
A cross-sectional baseline study was undertaken in DodaLab from the end of 2007 and into the beginning of 2008 to obtain information about persons and households, mainly on demographic characteristics, education and occupation as well as the social and economic household situation. Corresponding surveys are conducted every second year in FilaBavi, the two latest in 2007 and 2009. Information from the 2007 surveys in both sites has been used for this study. At both sites, all households are routinely visited every three months to record vital events, births, deaths and migrations. Every year, about 500 children are born in DodaLab and about 900 are born yearly in FilaBavi. The quarterly surveys have been used to identify pregnant women for the present study but the subsequent follow up of pregnant women and their children have then been conducted separately.
Study design and subjects
-------------------------
All mothers of infants born from 1^st^ March, 2008 to 30^th^ June 2010, were invited to participate in the study. All had taken part in a previous study of antenatal and delivery care \[[@B18]\]. The infants included were then followed from birth to one year of age with respect to breastfeeding. Information about breastfeeding and exclusive breastfeeding was collected in monthly interviews. Totally 2,690 babies were enrolled for the study. During the first months of the data collection, some observations in FilaBavi were missed due to mistakes during the field work. For 57 infants no breastfeeding data was received resulting in 2,633 remaining for the study (1,145 in DodaLab, 633 boys and 512 girls and 1,488 in FilaBavi, 823 boys and 665 girls). The total numbers of breastfeeding interviews were 10,189 in DodaLab and 17,008 in FilaBavi.
Data collection
---------------
Information on the infant feeding was obtained through interviews with the mothers using a structured questionnaire constructed by the investigators with references to Scott JA \[[@B20]\] and Dhandapany G et al. \[[@B21]\]. The data collection was approved by the Research Committee of Hanoi Medical University. The questionnaire was pretested for cultural sensitivity before the actual data collection. The mothers were interviewed each month from delivery to child age of twelve months. At each interview the mothers were asked if the baby had received only breastfeeding or breastfeeding together with additional food or no breastfeeding at all during the period since the previous interview. Data was collected by 106 HDSS interviewers (46 in FilaBavi and 60 in DodaLab), who were trained on household interview skills and the content of the questionnaire. The interviewers were trained to carefully ask about the feeding details of the infants during the period since the last interview, or birth in the first interview. As a quality control 3% of the randomly selected forms for re-interview by field supervisors. One hundred percent of the forms were rechecked by field supervisors and data clerks before entering into computers.
WHO breastfeeding definitions
-----------------------------
To describe breastfeeding, the World Health Organisation provides the following definitions \[[@B22]\]:
Exclusive breastfeeding: The infant receives breast milk, from the breast of the mother or a wet nurse or expressed, with the only additional oral intake of oral rehydration solutions (ORS) or medication including vitamins or minerals.
*Any breastfeeding: The infant receives breast milk, from the breast of the mother or a wet nurse or expressed, with or without additional oral foods. This category includes the WHO definitions of exclusive breastfeeding as well as non-exclusive breastfeeding, that is predominant breastfeeding and complementary feeding according to the WHO definitions*.
Given the way data has been obtained for this article we are not able to discuss exclusive breastfeeding in a strict sense. Even if a mother by the first interview report that the child has not been given anything but breast milk we can't be sure that prelacteal feeds have not been given. Early single meals of formula given by the maternity ward staff cannot be fully excluded. The importance of such deviations from exclusive breastfeeding is different in different situations. e.g. in a study of exclusive breastfeeding and allergy development it can be extremely important to know about prelacteal feeds. For studies of child growth the importance could be considered minute.
For the aim of this paper we wanted to study three components. The first is the frequency of early onset of breastfeeding defined as within one hour after delivery. The second is the duration for which mothers report that the infant is only given breast milk (Exclusive breastfeeding). The third is the duration for which the infant is reported to be given breast milk with or without additional food (Any breastfeeding) If a visit with report of exclusive breastfeeding was followed by a visit reporting any supplementary food or no breastfeeding, but exclusive breastfeeding was reported at later visits, the end time of exclusive breastfeeding was defined by the first interruption.
Statistical analysis
--------------------
### Outcomes
As described above, two time variables measuring breastfeeding time were defined, (i) the number of days from birth to the maximum time with exclusive breastfeeding reported and (ii) the number of days from birth to the maximum time with any breastfeeding reported. A dichotomous outcome variable "early commencement of breastfeeding" was defined as initiation of breastfeeding in the first hour after delivery.
### Explanatory variables
At the child level we included sex of the child, birth weight, delivery in hospital and in particular use of Caesarean section as explanatory variables.
For mothers, the following variables were used: age, education, occupation and number of antenatal care visits. Parity was not included as it is strongly correlated with age creating risk for collinearity problems in the estimation. Also, parity is less informative since it is commonly only zero (nullipara) or one due to the two-child policy. Education and occupation are also correlated but for breastfeeding we should expect occupation to have some importance *per se,* predominantly farming in the rural area and office employment in the urban.
The durations of exclusive breastfeeding or any breastfeeding were described using Kaplan Meier curves and analysed by means of standard survival analysis technique including stratification, regression and Cox regression models. In the analysis of exclusive breastfeeding, infants never reported to have been exclusively breastfed were not included and infants never reported to have been breastfed were not included in the analysis of any breastfeeding.
Two variables were used for stratification throughout the analysis: context, i.e. living in urban or rural area, and sex of the child. We have previously found that these variables define natural strata with respect to birth weight and growth during the first year of life \[[@B23]\]. Cox regressions of breastfeeding duration were conducted separately within each stratum. In addition specific studies of medians of exclusive breastfeeding were done for selected variables. Associations between the outcome variable "early initiation" and the explanatory variables were investigated using multiple logistic regressions. Statements of statistical significance have p\<0.05 as the default level.
Ethical consideration
---------------------
Approval for the project was given by the Scientific and Ethical Committee of Hanoi Medical University, the Hanoi Health Bureau and the Dong Da district authorities. The proposal was also approved and given permission by Ministry of Health after the baseline survey.
The participants were informed about the purpose of the studies and their right to decline participation or to withdraw at any time and without any conditions. Consent was sought from all eligible mothers and those who consented were included in the study. Data were analysed and results were presented anonymously. All results were duly disseminated to communities and authorities.
Results
=======
Early initiation of breastfeeding during the first hour was significantly more common in urban as compared to rural newborns (Table [1](#T1){ref-type="table"}). The differences between sites as well as between the sexes were statistically significant (p\< 0.01). A logistic regression using all listed independent variables showed that high birth weight significantly increased the probability for early initiation (p\<0.001). The use of Caesarean section on the other hand delayed initiation. The odds ratio for early initiation was 0.12 with p\<0.001 comparing children delivered with Caesarean section to other. Increasing number of household assets showed a tendency towards increasing probability of early initiation though not statistically significant (p=0.068).
######
Initiation of breastfeeding by site and child sex
**Urban boys** **Urban girls** **Rural boys** **Rural girls**
----------------------------------------------------------------------- ---------------- ----------------- ---------------- -----------------
Total number of children 633 512 823 665
Number of children with breastfeeding initiated during first hour 254 (40%) 251 (49%) 291 (35%) 270 (40%)
Number of children with breastfeeding initiated during first 24 hours 272 (43%) 351 (68.5%) 414 (50.3%) 328 (49.3%)
Exclusive breastfeeding in the first months was significantly more common in the rural area than in the urban (Figure [1](#F1){ref-type="fig"}, Table [2](#T2){ref-type="table"}). The differences at age three months were 12 percent units, both for boys and girls. A reverse relation was observed at a higher age, however based on observations from fewer children and therefore more likely to be a matter of chance. Also in a corresponding figure based only on children with 9--12 visits this difference became weaker and not statistically significant. At all ages and at both sites girls were more frequently exclusively breastfed but the differences were not statistically significant. The differences in medians of the duration of exclusive breastfeeding between areas were statistically significant (p \< 0.001) as well as sexes (p = 0.016) (Table [2](#T2){ref-type="table"}).
{#F1}
######
Exclusive breastfeeding at selected ages and duration of exclusive breastfeeding by site and child sex
**Urban boys** **Urban girls** **Rural boys** **Rural girls**
------------------------------------------------------------------------------------ ---------------- ----------------- ---------------- -----------------
Exclusive breastfeeding at 1 month (%) 95% confidence interval 81 (78;84) 86 (83;89) 91 (88;94) 91 (89;93)
Exclusive breastfeeding at 3 month (%) 95% confidence interval 46 (42;50) 53 (49;58) 58 (53;60) 65 (60;68)
Exclusive breastfeeding at 6 months (%) 95% confidence interval 8 (4;12) 11 (7;15) 4 (0;9) 4 (0;9)
Medians of duration of exclusive breastfeeding with 95% confidence interval (days) 81 (69;89) 91 (84;95) 97 (94;102) 102 (98;105)
Most infants, in both areas, were given any breastfeeding for the first 6--9 month. Later, breastfeeding declined, most rapidly in the urban area (Figure [2](#F2){ref-type="fig"}).
{#F2}
In the urban area a trend to shorter exclusive breastfeeding for mothers with longer education was seen. The median durations were 108 days for mothers with secondary school, 88 days for high school and 82 days for higher education. This trend was however, not statistically significant. In the rural area the trend was opposite with longer breastfeeding in mothers with longer education; 98, 98 and 108 days, respectively. Here the difference was statistically significant.
Mothers reporting farming as occupation in the rural area and being employed in offices and business in the urban area did not differ from others with respect to exclusive breastfeeding duration medians. Likewise no statistically significant associations or systematic patterns were found between exclusive breastfeeding duration and mother's age, household economy indicators or household size.
The median exclusive breastfeeding duration for those attending less than three antenatal care visits was 105 days, whereas the duration for those attending three visits or more was significantly shorter, 92 days (p\<0.05). The majority of urban infants were born in hospitals. Those infants had shorter duration of exclusive breastfeeding than the urban infants born outside the hospital (medians are 85 days and 129 days). In the rural area, the opposite trend was found (medians of 113 and 98 days). Both differences were however, non-significant. For children delivered by Caesarean section the median duration was 78 days. The difference was statistically significant from the 98 days for normal deliveries with the same tendency for both sites.
The results of the Cox regressions of exclusive breastfeeding duration on all independent variables within the four strata formed by child sex and area type largely confirmed the association between duration of exclusive breastfeeding and use of Caesarean section. The hazard ratio (HR) for ending exclusive breastfeeding was estimated to 1.25 in the urban and 1.07 in the rural area. High household income turned out to be significantly associated with a HR above one in three out of the four strata. For urban boys, the mother's age and education were significantly associated with the risk of ending exclusive breastfeeding. Older mothers showed lower risk whereas better educated women exhibited increased risk compared to the overall risk.
The results for median duration of exclusive breastfeeding and the results of Cox regression mainly coincide. The approaches are based on different assumptions, different statistical tests are used and statistical significance (p-values) will differ. Strict application of conventional rules for significance might be considered as creating conflicting results but the discrepancies are mainly indications of a rather complex situation.
Looking at infants given any breastfeeding, the frequency at 12 months of age is high, more than two out of three, in all groups (Table [3](#T3){ref-type="table"}). The survival curves for any breastfeeding differ significantly between the urban and the rural areas (p\<0.001) but not between sexes (p=0.44) (Figure [2](#F2){ref-type="fig"}).
######
Any breastfeeding at 6 and 12 months of age
**Urban boys** **Urban girls** **Rural boys** **Rural girls**
---------------------------------------------------------------- ---------------- ----------------- ---------------- -----------------
Total number of children 633 512 823 665
Number and percentage of children who report any breastfeeding 623 (98.5%) 507 (99%) 820 (99.5%) 665 (99.5)
Any breastfeeding at 6 month (%) 95% confidence interval 85 (81;87) 87 (84;90) 94 (92;96) 96 (94;98)
Any breastfeeding at 12 month (%) 95% confidence interval 66 (61;71) 70 (64;75) 89 (86;91) 90 (88;92)
Discussion
==========
A key finding of this study was that early initiation of breastfeeding was more common in the urban area than in the rural. A similar finding has been reported from Tanzania where the percentage of initiation of breastfeeding during the first hour after birth was estimated to be 82% in an urban area and 52% in a rural. Only 10% of the urban mothers discarded colostrum compared to 43% of the rural mothers \[[@B16]\]. Delayed initiation of breastfeeding and discarding of colostrum has also been found to be very common in a rural area in Bangladesh. Only 12 % the mothers used colostrum for the first feeding of their newborns and a relationship was found between mother knowledge and the practice of giving colostrum \[[@B24]\]. In Vietnam, comparisons between breastfeeding patterns in rural and urban areas have not previously been published. A study of Vietnamese women in Australia suggested that the proportion of early initiation of breastfeeding was low due to mothers having negative views on colostrum. Only 25.7% thought that colostrum was healthier for babies than formula, 64.9% said that it was equally healthy and 40% gave their babies formula milk in the hospital \[[@B25]\]. In Vietnam, the level of education of mothers in urban areas is generally higher than in rural areas. This might indicate that there is a difference in knowledge about colostrum and the value of early breastfeeding. Vietnam began to implement the policy of "Baby-Friendly Hospitals' in 1995, which is strongly supportive of breastfeeding \[[@B5]\]. In our study, the proportion of urban mothers who delivered in hospitals was higher than in the rural area \[[@B26]\], which could have contributed to a higher level of early breastfeeding in urban areas. Birth weight, number of household assets and Caesarean section were associated with the pattern of early initiation of breastfeeding in the present study. We also knew that mean birth weight was higher in the urban area than in the rural area \[[@B23]\], Caesarean section was more common \[[@B26]\] and household income was higher \[[@B23]\]. The factors associated with initiation of breastfeeding thus differ between the urban and rural areas. This may partly explain the differences we found in the early initiation of breastfeeding.
The percentages of initiation of breastfeeding during the first hour after birth in the present study were much lower in both areas than the 73.6% found in a rural area of Vietnam in 2002 \[[@B6]\] and the 61.7% reported in a cross-sectional study of National Institute of Nutrition in 2010 \[[@B8]\]. This may indicate that the level of early initiation of breastfeeding does not improve.
An important finding of this study is that exclusive breastfeeding during the first three months was more common in the rural area than in the urban one. This is in line with results from China where exclusive breastfeeding was more common in a rural area (61%) than in an urban (38%) \[[@B14]\]. The opposite was reported from Tanzania where the mean duration of exclusive breastfeeding was longer in the urban setting (23 days) than in the rural (9 days) \[[@B16]\].
Some factors can be suggested to be associated with the differences of exclusive breastfeeding between the two sites. The use of Caesarean section as delivery method has been seen to increase the risk for not breastfeeding in Vietnam and China \[[@B6],[@B15]\]. After surgery, the babies are often taken away from the mother. Mothers can also be worried about side effects of medicines like postoperative antibiotics which may pass to their babies through the breast milk \[[@B15]\]. In the present study the median duration of exclusive breastfeeding of children delivered using Caesarean section was significantly shorter than in other groups in both settings. The percentage of women having Caesarian section was substantially higher in the urban area (38.9%) than in rural area (12.2%) \[[@B26]\]. Caesarian section could be one of the factors behind the differences found in exclusive breastfeeding between the two sites in our study.
Another factor may be marketing of formula milk which has been shown to affect the breastfeeding behaviors of mothers \[[@B7],[@B10]\]. Mothers are given the impression that formula milk is as good as, or better than, breast milk \[[@B10]\]. Marketing of formula milk may be more aggressive in urban areas than in rural areas in Vietnam. Economic conditions are also better in the urban area. Financial constraints may to a higher extent prevent women from buying formula milk in the rural areas \[[@B7]\].
The associations between household economic condition and duration of exclusive breastfeeding found in this study might partly explain the differences of breastfeeding practices of infants between the two settings.
Another factor behind the differences of breastfeeding practices between the two sites may be education. An earlier study in rural area of Vietnam showed that non-exclusive breastfeeding women had less education than exclusively breastfeeding women \[[@B7]\]. In the present study, the median duration of exclusive breastfeeding for the group of the highest level of education in the urban area was shortest. The Cox regression analysis pointed to a significant influence in the urban boy stratum. In the rural area, the result was the opposite: the median duration of exclusive breastfeeding for the group with the highest level of education was the longest. A problem for the interpretation of these results is that the statistical significance is very much dependent on numbers of observations. For education, the distributions are radically different between the urban and rural areas. Two reasonable hypotheses could be that the higher educated women in the urban area, who are the majority, tend to give exclusive breastfeeding shorter than others, and that the rather few women with higher education in the rural area for unclear reasons, breastfeed exclusively longer than rural women with low education.
The duration of exclusive breastfeeding by mothers with three antenatal care visits or more was shorter than the duration of exclusive breastfeeding of mothers with less than three visits. The result may indicate that many antenatal visits did not necessarily give mothers more information about breast feeding. A study in Vietnam found that lack of information and misunderstanding of mothers about breastfeeding were barriers to exclusive breastfeeding \[[@B12]\].
The results of this article show that exclusive breastfeeding decreased rapidly with increasing child age and was uncommon at six months of age at both sites. Similar results have been seen in China and in other studies in Vietnam. The percentage of children with exclusive breastfeeding dropped from 83.6% at the age of one week to zero at week 24 in a previous study in rural Vietnam \[[@B7]\]. The percentages of infants less than 6 months of age, who were exclusively breastfed, was 19.6% in Vietnam in 2010 \[[@B8]\]. In China, exclusive breastfeeding at six months of age dropped to 0.2% in an urban area and to 7.2% in a rural area \[[@B14]\]. The most important reason was that the mothers had to return to work \[[@B7],[@B11],[@B27]\]. Vietnamese mothers have a legal right to four months maternity leave \[[@B13]\], which is insufficient in relation to the WHO recommendation about six months of exclusive breastfeeding. Mothers commonly see formular milk as the best choice for the child when they return to work \[[@B10]\]. Another reason for giving complementary food was discussed in China, suggesting there is a belief that it will improve weight gain and lead to healthier babies \[[@B14]\]. A Chinese tradition is also that friends and relatives come to visit the mother and child after delivery. The most popular gift is infant milk formula. This is one reason for the extensive use of early formula milk in China \[[@B15]\].
The last major finding of this study is that most infants in both areas are reported to be given any breastfeeding for the first 12 months. Towards the end of the first year, breastfeeding is most rapidly declining in the urban area. Similar results were seen in Tanzania. Mean duration of breastfeeding was 27 ± 5 months in a rural compared to 24 ± 5 months in an urban area \[[@B16]\]. The opposite results were found in the USA: the prevalence of breastfeeding was significantly lower in a rural area than in an urban area \[[@B2]\]. Results from National Institute of Nutrition in Vietnam showed that 77% of infants in 2009--2010 continued breastfeeding at 12 months of age and that 22% continued breastfeeding at 24 months of age \[[@B8]\]. In Tanzania, reasons to stop breastfeeding early were that the child was considered old enough, mothers started a new pregnancy, the child refused the breastfeeding, infant or maternal illness or that mothers did not want to continue breastfeeding \[[@B16]\]. In Vietnam, some common reasons to stop breastfeeding have been found to be perceived insufficient breast milk supply, mothers return to work, that infants stopped taking breast milk and the mother's health condition \[[@B5]\]. Even though the educational level of mothers was higher in the urban area, we found that any breastfeeding was lower in the urban compared to the rural area and also lower than data from National Institute of Nutrition in Vietnam \[[@B8]\]. One important reason behind that finding could be that urban mothers returned to work early.
Boys are considered more important than girls in Vietnam \[[@B28]\], but the present study found that boys were rather given less breastfeeding than girls in both sites at all ages. A reason may be that formula feeding is considered better than breastfeeding and hence preferred for the boys.
One limitation of the present study is the restriction to one year of follow-up. A second limitation of the study is that it is based on interview data from the mothers. Even if the interviewers were carefully trained to ask for data e.g. on additional foods from the whole period after birth or last interview, respectively, recall bias could not be excluded.
Moreover, it could not be excluded that hospital staff may have given early formula meals without the knowledge of the mother, particularly to babies born with Caesarean section. However, the present study shares these methodological problems with most other published breast feeding studies. Consequently, results from this study allows for comparison to data from other studies.
In conclusion, exclusive breastfeeding is still low in rural and more common in urban Vietnam. Intervention programs to promote breastfeeding are necessary particularly regarding early initiation and prolonged breastfeeding. To improve the latter a prolonged period of maternity leave to at least until the infant is six month old would be desirable.
Conclusion
==========
Initiation of breastfeeding during the first hour after birth was more common in the urban area than in the rural. Exclusive breastfeeding during the first months of life and breastfeeding in general was more frequent in the rural area than in the urban. Exclusive breastfeeding at six months of age of infants was uncommon in both areas.
The results suggest that the different breastfeeding practices between the two sites are not only due to the geographic location as such but rather to conditions associated with living in urban or rural settings.
To reduce the impact of such factors we propose that intervention programs with the aim of increasing the mother's knowledge about breastfeeding, particularly rural mothers, should be implemented. A minimum of six months maternity leave would probably be an important measure to prolong the duration of exclusive breastfeeding. Limitation of advertisement and marketing of formula milk should be imposed.
Competing interests
===================
The authors declare that our findings have not been influenced by our personal or financial relationships with other persons or other organizations.
Authors' contributions
======================
HNT led and supervised the fieldwork and data management. She also drafted and completed this paper. BE assisted in the research design as well as in the statistical analyses, interpretation of results and revising the manuscript. HA assisted particularly with paediatric expertise and was together with MP, GB, TTK, CNTK and NTL involved in the design of the study, supervised the study and revised the manuscript. All authors have read and approved the final manuscript.
Pre-publication history
=======================
The pre-publication history for this paper can be accessed here:
<http://www.biomedcentral.com/1471-2458/12/964/prepub>
Acknowledgements
================
The authors would like to thank all field workers, mothers of infants and infants at the two Health and Demographic Surveillances sites: FilaBavi and DodaLab for their contribution to data collection. The study was supported by grants from Sida (Swedish International Development Cooperation Agency), Swedish Research Council and the Nordic School of Public Health, Sweden.
|
{
"pile_set_name": "PubMed Central"
}
|
Background {#Sec1}
==========
Historically focussed on cancer care, there is a growing expectation that palliative care services should provide care to patients with any life limiting condition \[[@CR1]\]. This, along with an ageing population with a growing burden of comorbidities has led to increasing strain on palliative care services. Several studies have shown the benefit of palliative care to patient quality of life \[[@CR2], [@CR3]\]. Despite this, work completed by Marie Curie highlights the inequalities in service provision for palliative care patients, with particular reference to patients with non-cancer diagnoses and out of hours care \[[@CR1]\]. The provision of palliative care across rural communities should also be highlighted as a challenge for the specialist palliative care service \[[@CR4]\].
Supporting patients with palliative care needs to access services and avoid hospital admission requires increasing support by community general and specialist palliative care services \[[@CR5]\]. Several studies in the UK have indicated that the majority of patients wish to die at home \[[@CR2]\], and a systematic review by Cochrane demonstrated that home-based end of life care increased the likelihood of dying at home \[[@CR6]\].
Telehealth is a new and growing industry which comprises healthcare services, information technology and mobile technology. The World Health Organisation (WHO) state "Telehealth involves the use of telecommunications and virtual technology to deliver health care outside of traditional health-care facilities" \[[@CR7]\]. The Health Resources and Service Administration expand on this description in their definition to include education; "the use of electronic information and telecommunications technologies to support and promote long-distance clinical health care, patient and professional health-related education, public health and health administration" \[[@CR8]\].
The use of telehealth in a variety of chronic health conditions, such as heart failure and chronic obstructive pulmonary disease (COPD), has been studied \[[@CR9], [@CR10]\] and there is a growing body of research into its application in palliative medicine. Although there have been some large-scale notable uses of telehealth; for example, the use of electronic patient record systems in the UK \[[@CR11]\], the importance of telehealth in providing quality healthcare has only recently become more widely accepted. Used to its full potential, telehealth technology could be particularly vital in improving access to healthcare over geographical distance and outside of normal working hours \[[@CR12]\]. Telehealth has also been postulated as a solution to reduce acute hospital admissions which currently account for around 65% of hospital bed days in England \[[@CR13]\].
The potential for telehealth to be utilised in the provision of palliative care services has already been identified in national publications. NHS Scotland identified 'providing telehealth and telecare support' as one of their actions as part of the 'Living and Dying Well' action plan \[[@CR14]\] and the National Palliative Care and End of Life Care Partnership also identified the use of technology in their 'ambitions' for 2015--2020 \[[@CR15]\]. In 2017, the UK government published its Digital Service Strategy which outline the ambitions to grow digital services across a variety of sectors \[[@CR16]\]. This includes a service manual which explains how teams can build a good digital service. As a part of this work, the government also developed a Digital Service Standard \[[@CR17]\]. This standard is a list of criteria to help create and run good digital services. The criteria include detailed information on why each element is important and what it means when designing and delivering a service. Some of the criteria include understanding user needs, making a service simple to use and protects user data. The digital service standard will be discussed further in the results section.
A review undertaken in the use of telehealth in palliative care in the UK was published in 2010 \[[@CR18]\]. This paper examined the use of telehealth in palliative care settings in the UK between 1999 and 2009. The paper showed that telehealth was becoming increasingly accepted as useable by patients and healthcare professionals in this field; however there was an identified lack of clear evidence-based research to support the use of telehealth in palliative care in the UK \[[@CR18]\].
Telehealth may provide a solution to meeting the growing demands of palliative care services across geographical regions with limited resources. Ready access to general and specialist palliative care services for patients with a variety of life-limiting conditions may prove beneficial in reducing the need for emergency services. This review will examine the current status of telehealth in palliative care in the UK and any evidence to suggest an effect on access to emergency or unscheduled care. In light of the recent UK government publications on digital services, the review will also examine to what extent current initiatives meet these standards. The issues described are not unique to the UK- studies in Australia \[[@CR19], [@CR20]\], the USA \[[@CR21]\] and Europe \[[@CR22]\] demonstrate a global interest in the potential of telehealth for meeting the needs of the palliative care population. Although this review focusses on UK studies, the results are undoubtedly transferable to an international audience.
Methods {#Sec2}
=======
The protocol for this systematic review was registered with PROSPERO and can be found at <http://www.crd.york.ac.uk/PROSPERO/display_record.php?ID=CRD42017080038>.
The aim of this systematic review is to describe and evaluate the current use of telehealth technology in the provision of generalist and specialist palliative care in the UK. The authors examined the UK specifically in line with the 2010 review and also due to the unique way healthcare services- specifically, palliative care- are structured and funded via the National Health Service (NHS), meaning direct comparison with other countries would not be possible. The NHS is a government funded health and medical service which is free at the point of access to all UK residents \[[@CR23]\]. Although a proportion of palliative care services are commissioned and funded by the NHS, much of palliative and end of life care is provided by hospices based in the voluntary sector \[[@CR24]\]. To this end, the systematic review will answer the following question: What are the current published uses of telehealth in palliative care in the UK?
In addition to the primary objective of describing telehealth use in palliative care, the review will also address the evaluation of these services. The secondary questions to be answered are: 2.If telehealth is being used for patients and/or carers, does this meet the criteria of a digital service as described by UK government?3.Is there any evidence that compared with standard care, telehealth initiatives reduce the need for access to emergency/acute services for the palliative care population in the UK?
The authors used similar methods to those in the 2010 review paper in terms of selected electronic databases and grey literature searches. The search terms used for this review included those in the 2010 review however additional terms were added (adapted from previous Cochrane reviews \[[@CR25], [@CR26]\]) to maximise identification of suitable literature. Additionally, the authors opted not to include 'United Kingdom' and other synonyms in the search terms as it was felt this may result in missing relevant literature which referenced specific UK locations such as towns or regions.
Thematic synthesis was used to examine the results of the review \[[@CR27]\].
Inclusion criteria {#Sec3}
------------------
Due to the descriptive nature of literature available, and initial scoping indicated the amount of literature available on this topic was not expected to be substantial, all study types including case series were included for review, with the exception of anecdotal opinion pieces and editorials. Research published on or after 1st January 2010 was included in this systematic review to allow comparison to the 2010 review article. Due to the nature of the review examining only studies from the UK, only articles reported in the English language were included.
Of interest were studies which described the use of any telehealth initiative in the delivery of palliative care in the UK. We included studies which described the use of telehealth to facilitate multi-disciplinary working or for the purpose of staff education and support, as well as to provide service to patients with palliative needs. Paediatric studies were included.
Studies describing the use of any mode of telehealth intervention were included, such as remote patient monitoring, digital support via telephone or video for patients or carers, remote support and advice for healthcare professionals in managing the stated population, or facilitation of the education and networking of healthcare professionals delivering palliative care.
Outcomes {#Sec4}
--------
The primary outcome of the review was to describe telehealth use in palliative care in the UK. The secondary outcome was to judge whether the telehealth initiative described met the digital service standard. The UK government published its 'Digital Service Standard' in 2016 which is a set of criteria to help create and run good digital services \[[@CR17]\]. The authors adapted these criteria and judged the telehealth interventions in the included studies against this standard. Additional outcomes included any reduction in the requirement of acute or emergency services when compared with standard care, cost effectiveness of telehealth interventions and participant perception of the technology.
Search methods for identification of studies {#Sec5}
--------------------------------------------
We identified studies from a search of five databases conducted November 2017: EMBASEMEDLINECINAHLPsychinfoCochrane central register of controlled trials
Search terms were adapted from existing strategies used in Cochrane systematic reviews \[[@CR25], [@CR26]\] and developed with input from our academic liaison librarian team (Table [1](#Tab1){ref-type="table"}). Search terms were tailored to the five electronic databases accordingly. Table 1Terms used in search strategy1. Identification of palliative carePalliative care OR Terminal care OR Terminally ill OR advanced/end stage/terminal disease/illness/cancer OR last year of life OR end of life OR macmillan/marie curie nurse2. TelehealthTelemedicine OR Telecommunications OR Telecare OR telemonitor OR teleconsult OR teleconference OR telephone OR telehealth OR remote consult OR ehealth OR mobile health
A search of the grey literature with search terms modified from the above was also conducted using Google search engine. Additionally, we handsearched the conference literature from the European Association for Palliative Care, EThOS doctoral theses and reference lists from included papers. Where suitable abstracts were identified from conference or thesis abstracts, authors were contacted for full published papers and one expert in the field was approached for discussion. If full papers had not been published, these were excluded.
Data collection and analysis {#Sec6}
----------------------------
Results of the literature search were uploaded on to Covidence, an online tool to support literature screening. Two authors (SH and HJ) screened the titles and abstracts for relevance, to judge eligibility and remove duplicates. Full text of all potentially relevant studies was assessed by SH and HJ. Disagreements were resolved by consensus or by consulting a 3rd reviewer (AG or NP).
Data from each study were entered on a data extraction form. Specifically, data on the study population including sample size and diagnosis, study intervention, study design including data collection and analysis methods, and results or recommendations were extracted. The form was piloted by the two reviewers to ensure usability and consistency. All studies were extracted by SH with HJ independently completing data extraction on 80% of the studies included. Following extraction of baseline data (study type, number and nature of participants), thematic synthesis was conducted from the included studies following the methods of Thomas and Harden \[[@CR27]\]. Following familiarisation with the study by repeat reading, any qualitative data was initially coded by hand and connections and overlaps in the data brought together in to descriptive themes. The themes were then reviewed in line with the review objectives to ensure the themes were able to capture crucial aspects of the data and address the review questions.
Critical appraisal {#Sec7}
------------------
Where possible, critical appraisal of the methodology of the paper was conducted using criteria adapted from Wallace et al's 2004 paper on meeting the challenge of developing systematic reviewing in social policy \[[@CR28]\]. A critical appraisal tool was not used in the 2010 review. This appraisal methodology allows for qualitative research evidence, which was felt to be important for this systematic review. The purpose of this appraisal was to provide an overview of the quality of the papers- studies were not excluded purely on the basis of the critical appraisal. Wallace et al's original critical appraisal criteria can be found in Additional file [1](#MOESM1){ref-type="media"}.
Results {#Sec8}
=======
A total of 30 articles met the inclusion criteria and were included in the synthesis. The search results are shown as a PRISMA flowchart in Fig. [1](#Fig1){ref-type="fig"}. Fig. 1PRISMA flowchart of results
A table of the included studies \[[@CR22]--[@CR49]\] can be found below \[[@CR29], [@CR30]\] (Table [2](#Tab2){ref-type="table"}). Studies are grouped according to the predominant outcomes; quantitative, qualitative, protocols and mixed methods. Table 2Summary table of included studiesAuthor (year)Research questionParticipantsTelehealth initiativeSettingKey findingsOverview of qualityQuantitative Chatwin (2016)\[[@CR22]\]Randomised crossover trialEffect of home telemonitoring on interaction with acute services and quality of life and self-efficacy68 patients with COPD or non-COPD respiratory failurePhilips Motiva home telemonitoring system with heart rate monitor, pulse oximeter, blood pressure monitor and weighing scalesPatient's homeAdmission rates and home visits increased in the telemonitoring armTime to first exacerbation did not differ between groups.Self-efficacy fell in the telemonitored group.**Score = 18** Dey (2016)\[[@CR25]\]Prospective interventional pilot studyAcceptability of technology to monitor symptoms in advanced peritoneal dialysis patients22 adults with end stage renal failure on peritoneal dialysisSpecialised software on tablets for inputting symptom data and blood pressure measurementPatient's homeClaimed 36 admissions avoided, support to manage at home provided on 154 instances.91% retention rate in study excluding medical reasons.No significant change in QOL scores. QUEST scores high indicating satisfaction with the technology.Score = 12 Dierckx (2015)\[[@CR26]\]Retrospective observational analysisDoes home telemonitoring reduce heart failure mortality?333 patients with heart failure referred- 278 agreed to telemonitoringMotiva home telemonitoring system for symptom data and clinical measurements. Can also transmit educational videos.Patient's homeTelemonitoring was associated with reduced all-cause mortality and overall improved survival. The number of admissions and time to first hospitalization was the same in each group. Patients who refused telemonitoring were generally older.Score = 10 Hall (2013)\[[@CR29]\]Service evaluationAn evaluation of the 7 day specialist palliative care serviceN/ATelephone advice service within an acute hospitalHospital99 calls with professionals and 37 with carers in a one-year period. Consultant contacted on average once per day at the weekend.N/A Hamad (2015)\[[@CR31]\]Prospective observationalDescription of the use of telehealth data in a COPD MDT95 patientsTelehealth platform via smartphone or tablet to provide symptom status and oxygen saturationsHospital18 of 95 patients had no recommendations from the MDT. There were 141 recommendations generated for the remainder eg. referral to palliative care, smoking cessation.N/A Lewis (2010)\[[@CR32]\]Randomised controlled trialDoes home telemonitoring reduce healthcare use in COPD?40 patients with COPDHealthHub system via Freephone line to monitor symptoms and pulse oximetry.Patient's homeReduced contact with primary care in intervention group- not statistically significant but may be clinically important. No difference in ED attendance or hospital admissions.Patients uploaded median 97% data and no difficulties using technology.**Score = 17** Plummer (2011) \[[@CR33]\]Service evaluationAnalysis of the specialist palliative care advice line70 patients and carers24/7 telephone advice line for patients, carers or professionalsHospiceMost calls weekday after 5 pm and from patients or carers. Primary reason for call was symptom management. 65% callers remained at home following call.Score = 8 Purdy (2015)\[[@CR34]\]Retrospective observational studyImpact of the delivering choice programme on place of death and hospital usageAnalysis of 3594 patientsElectronic end of life register, out of hours advice line (plus two non-telehealth components)Hospice and community21--24% accessed some element of the programme. Patients using programme more likely to have cancer diagnosis. Care coordination centres most effective intervention. OOH advice line associated with reduced ED attendance in last week of life only. Patients using centres or entered on end of life register were less likely to die in hospital. Hospital admissions were lower in both counties for patients using the programme in 30 days prior to death.**Score = 18** Warren (2011)\[[@CR35]\]Prospective observational studyReview of telephone support for patients with advanced breast cancer229 calls related to patients with metastatic breast cancerTelephone advice lineHospitalLargest contact group was patients followed by professionals. Total time spent on calls was 63 h (30% of CNS working time). 1281 interventions generated from the calls.Score = 12 Wye (2016)\[[@CR36]\]Service evaluationAnalysis of electronic palliative care coordinating systems (EPaCCS)101 health care professionalsElectronic palliative care recordCommunityEPaCCS used in small proportion of patients (9--13%).Where EPaCCS used, seems to correlate with a home death.**Score = 17**Qualitative Carlebach (2010)\[[@CR21]\]Qualitative interviewsWhat are the experiences and opinions of users of telephone support service?Palliative care diagnosis not specified.6 patients, 8 carers, 13 health professionalsHospice 24 h advice lineHospiceRelatives and carers were group most likely to use service, followed by district nurses. The service was valued by users.Score = 8 Duxbury (2015)\[[@CR27]\]Qualitative interviewsWhat are the barriers and enablers to adoption of Coordinate My Care?8 professionalsAn online tool completed by health professionals for palliative patientsCommunityUseful and relevant however process of completing 'laborious' and issues with connectivity- lack of remote access and some providers not connected.N/A Faull (2016)\[[@CR28]\]Description of initiativeDescription of online learning tool.For professionalse-ELCA online education tool for palliative care professionalsN/ANone- description only.N/A Hall (2012)\[[@CR29]\]Qualitative interviewsThe opinion of electronic palliative care summary record in Scotland16 professionals, 6 patients/carersElectronic palliative care system to allow recording of patient data and sharing with out of hours servicesCommunityUseful and feasible innovation. Felt to be more specific to cancer patients. Felt that not enough emergency providers know of its existence. Reassuring for patients and carers.Score = 11 Hobson (2018)\[[@CR37]\]Qualitative interviewsIdentify how technology may improve the service for motor neurone disease3 patients, six carers and 1 motor neurone disease specialist nurseTablet computer app which patients use to input health and wellbeing dataHospice/ CommunityTelehealth was acceptable to patients and carers. They wanted more information to help self-manage. The touch screen layout will be redesigned following observation of users.Score = 15 Johnston (2011)\[[@CR38]\]Qualitative interviewsEvaluating the use of telehealth in palliative care across Scotland22 patients and carers, 8 healthcare professionalsVariety of telehealth interventions discussedHospice/ CommunityPatients generally unaware of the term 'telehealth' but aware of the existence of technologies.Stakeholder telehealth activity consisted of videoconferencing for MDT, networking and education.Patients/carers aware of telephone advice lines, internet forums and personal safety alarms and found these useful.Felt to be used more in remote locations. Felt should supplement rather than replace existing support. Barriers to use were broadband coverage, funding and lack of awareness.Score = 14 Leadbeater (2014)\[[@CR39]\]Qualitative interviewsTo review the organisation of community palliative care teams in rural England6 specialist palliative care nursesVariety of telehealth interventions discussedCommunityTwo teams used videoconferencing for MDT meetings.40--75% patient contact via telephone. 3 teams had laptops and 2 had remote access to patient records.N/A Middleton-green (2016)\[[@CR40]\]Qualitative interviewsEvaluation of palliative care telephone advice line8 patients and 6 carersTelephone advice line or video call from iPad with hub staffed by nursing professionalsHospice5106 telephone calls received related to 1813 patients.Service found to be beneficial for emotional support and practical advice. Reports from patients of how advice prevented avoidable admission or expedited appropriate admission. Reported 98.5% of calls resulted in patients remaining in place of residenceScore = 10 Nwosu (2012)\[[@CR41]\]DescriptionDescribe smartphone applications for palliative medicineN/ASmartphone applicationsN/ASix applications identified- 2 'blog' style, 2 with guidance to facilitate learning or practice and 2 apps to facilitate opioid prescribing.N/A Rafter (2016)\[[@CR42]\]Description of serviceDescription of e-health in managing pressure ulcers in palliative care2 case examplese-Health system for staff to upload information/picture of ulcers.HospiceAll patients received care pathways within 24 h of referral. Staff gave positive feedback- easy to use and increased job satisfaction.N/A Wye (2014)\[[@CR43]\]Realist evaluation'Real life' evaluation of what facilitates home deaths and reduces hospital admissions for palliative patients43 family carers and 105 professionalsElectronic end of life register, out of hours advice line (plus two non-telehealth components)Hospice/ CommunityHaving skilled, experienced professionals with sufficient and dedicated time was important to the success of the project.Overall there was high carer satisfaction, low hospital utilization and more deaths in the community among project users. Patchy uptake of the service.**Score = 17**Protocols Aiyegbusi (2017)\[[@CR20]\]Study protocolDoes the use of patient-reported outcome measures promote care and safety in managing advanced CKD?Stage 4 or 5 chronic kidney disease.Electronic questionnaire on smartphone/ tablet/laptop/ computer of symptomsPatient's homeN/AN/A Choyce (2017)\[[@CR23]\]Study protocolEffect of home telemonitoring on clinical parameters and quality of lifeCystic Fibrosis patients with admission for IV antibiotics in last 24 monthsMobile phone for symptom reporting and Bluetooth spirometerPatient's homeN/AN/A Hudson (2016)\[[@CR44]\]Study protocolFeasibility of online cognitive behavioural therapy interventionEnd stage renal failure on dialysis with depression or anxietyOnline cognitive behavioural therapy using an iPad accompanied by telephone support.Patient's homeN/AN/AMixed Cox (2011)\[[@CR24]\]Intended mixed-methods. Qualitative interviews with cliniciansTo assess the acceptability of technology to monitor symptoms following palliative radiotherapy for lung cancerPatients with lung cancer receiving radiotherapy- none successfully recruitedCareHub device to monitor symptoms reported by patientsPatient's home21 patients identified, consent from clinician withheld for 20. 1 other patient declined to participate.9 of 13 clinicians felt e-technology inappropriate in this group due to age. Themes of gatekeeping due to concern of burden of research on this population. Concerns their clinical judgement replaced by technology.Score = 9 Hattink (2015)\[[@CR45]\]Randomised controlled trialDoes an e-learning course increase empathy and understanding in dementia caregivers?57 caregivers of people with advanced dementiaWeb-based training portal on different aspects of dementia careCarer's home30/57 UK participants did not complete the course. Modules rated useful and user-friendly. Empathy, perspective and coping with stress improved in the intervention group (though UK/Dutch results pooled).**Score = 16** Hudson (2017)\[[@CR46]\]Randomised controlled trialFeasibility of online cognitive behavioural therapy (CBT) intervention25 patients- 18 intervention, 7 controlOnline cognitive behavioural therapy using an iPad accompanied by telephone support.Patient's home410 patients approached and 25 agreed to participate with 23 completing follow up. Adherence with online CBT higher in control arm. Numbers not large enough to show statistical difference for patient reported outcomes. Calculated QALY gain for supported arm £82,283 though wide confidence intervals.**Score = 17** Lisk (2012)\[[@CR47]\]Mixed methodsDoes geriatrician input for nursing home patients reduce emergency admissions?Audit of 1954 nursing home residents with 3 nursing homes involved in pilotTelephone advice line to speak to geriatrician and e-mail alert to geriatrician when patient admittedHospital and communityReduction of bed days from 90 to 33 in initial pilot with calculated cost saving of £2630. In second phase of study calculated reduction of 250 bed days over 4 months with potential cost savings of £74,383. Service was well received by GPs.N/A Milton (2012)\[[@CR48]\]Service evaluationDescribe the 7 day community specialist palliative care service20 patients/carers6 professionalsProactive and reactive telephone support run by community specialist nursesCommunity36% of contact from the service was unplanned. There were 132 telephone contacts in a 6 month period.Viewed positively by all staff in the focus group and valued by patients.Score = 9 White (2016)\[[@CR49]\] Prospective longitudinal cohort studyReview of 'ECHO' education project for hospice nursing staff34 community hospice nursesWeekly educational session facilitated by videoconferencingHospice28/34 completed pre and post intervention evaluation. Mean knowledge score improved by 11.3% (*p* = 0.0005) and all domains of self-efficacy improved.Project received positively by participants.**Score = 18**Studies with overview of quality scores highlighted in bold in the table met all of the nine quality criteria completely or to some extent
Overview of studies {#Sec9}
-------------------
A wide variety of study designs were used, with the most common being qualitative methods used in seven of the papers \[[@CR32], [@CR35], [@CR36], [@CR38], [@CR41], [@CR42], [@CR50]\]. Four of the papers were service evaluations \[[@CR30], [@CR47], [@CR51], [@CR52]\] and there were three randomised controlled trials \[[@CR34], [@CR48], [@CR49]\]. Three of the papers were protocols \[[@CR29], [@CR33], [@CR45]\] and three of the papers simply gave a description of the intervention without any identifiable study design \[[@CR39], [@CR53], [@CR54]\] which served to address objective 1 of the study (description of current telehealth use). Other study designs included randomised crossover trial \[[@CR31]\] mixed methods \[[@CR37], [@CR43]\] realist evaluation \[[@CR55]\], prospective interventional \[[@CR44]\], prospective longitudinal cohort \[[@CR56]\], prospective observational \[[@CR40], [@CR57]\] and retrospective observational \[[@CR46], [@CR58]\].
The majority of included studies had relatively small sample sizes. Where qualitative or service evaluation type studies were conducted that involved interviews with participants, the majority had sample sizes less than 30 which may be expected given the stated methodology. The exception to this is in the two studies conducted by Wye et al. \[[@CR30], [@CR55]\] where 148 and 101 participants were interviewed respectively. Similarly, in the studies which examined an intervention, sample sizes were low (range 22--68) and the median number of participants was 40.
Fourteen of the included studies had a mix of participants (patients, carers and professionals) \[[@CR30], [@CR32], [@CR36], [@CR37], [@CR40]--[@CR43], [@CR47], [@CR50]--[@CR52], [@CR55], [@CR57]\], nine were patient centred \[[@CR29], [@CR31], [@CR33], [@CR34], [@CR44]--[@CR46], [@CR49], [@CR58]\]and five were studies with telehealth interventions aimed at professionals \[[@CR35], [@CR38], [@CR39], [@CR54], [@CR56]\]. Only one study by Hattink et al. \[[@CR48]\] described a telehealth intervention specifically for carers; an online education tool for carer-givers of those with advanced dementia. One study was a description of palliative care mobile phone applications and did not have any participants \[[@CR53]\].
Where studies included patients, the majority did not specify a particular diagnosis \[[@CR32], [@CR34], [@CR36], [@CR42], [@CR43], [@CR47], [@CR50]--[@CR52], [@CR54], [@CR55], [@CR58]\]. Specific diagnoses included end stage renal disease, COPD, cystic fibrosis, heart failure, dementia, motor neurone disease and specific cancer sites (lung and breast).
Overview of quality {#Sec10}
-------------------
Using guidance provided by Wallace et al's 2004 paper \[[@CR28]\], the authors appraised the methodology of 19 of the 30 papers. For the 11 which were not able to be assessed, this was due to the paper being descriptive in nature with insufficient detail on study design.
The 19 papers suitable for appraisal were reviewed against a set of nine criteria developed by the authors which had been adapted from Wallace et al. \[[@CR28]\] Two authors independently scored eligible papers against the nine criteria. Authors judged whether papers met the criteria fully (scoring 2), to some extent (scoring 1), not at all or unable to say from the information in the paper (scoring 0). The maximum score was therefore 18; scores for suitable papers are included in the table below.
Eight of the 19 papers met all of the nine criteria completely or to some extent \[[@CR30], [@CR31], [@CR34], [@CR48], [@CR49], [@CR55], [@CR56], [@CR58]\]. These papers scores are highlighted in bold in the table. The majority of papers which did not meet the nine criteria did so because of insufficient sample to explore the subject, or insufficient description of data collection methods.
Types of telehealth interventions {#Sec11}
---------------------------------
The first objective of this review was to describe the current uses of telehealth in palliative care in the UK which will be discussed here.
There was a variety of telehealth interventions described in the included studies. The majority of interventions described a form of home telemonitoring (using telephone or computer software to record clinical symptoms or signs from the patient's home) \[[@CR29], [@CR31], [@CR37], [@CR40], [@CR41], [@CR44]--[@CR46], [@CR49]\]. Home telemonitoring was used in a variety of conditions; respiratory disease, heart failure, motor neurone disease, cystic fibrosis and end stage renal failure. This required patients to input data using their telephone landline, their television or using computer hardware and software provided to the patient for this purpose. All home telemonitoring studies required patients to input specific data regarding symptoms specific to their illness, such as breathlessness in respiratory disease, and some studies also required patients to provide physical parameters. For example, pulse oximeter measurements in respiratory disease studies \[[@CR31], [@CR49]\] and weight and blood pressure measurements in heart failure and renal failure studies \[[@CR44], [@CR46]\]. The majority of these studies included some form of telephone support either in response to patient data triggering an alert or as an additional support for patients at home.
Several papers described a telephone advice line as the telehealth intervention \[[@CR36], [@CR47], [@CR50]--[@CR52], [@CR57]\]. These were a mix of 'reactive' and 'proactive' telephone services and tended to be for the palliative population generally rather than a specific diagnosis. The majority of these papers were descriptive of the service and used qualitative measures to obtain feedback from users on the service.
Three studies described the use of electronic patient records as a telehealth intervention \[[@CR30], [@CR32], [@CR38]\]. Five studies described the use of online or videoconferencing platforms to facilitate education; these were either to support patients and carers \[[@CR33], [@CR34], [@CR48]\] or to provide education and networking opportunities for professionals \[[@CR39], [@CR56]\].
A number of studies had a mix of interventions- from studies evaluating participant opinion on a number of interventions \[[@CR35], [@CR42]\], to studies which had a combination of elements to their intervention (a combination of telephone advice line, electronic patient record and non-telehealth interventions such as 'end of life care facilitators') \[[@CR43], [@CR55], [@CR58]\].
Synthesis of findings {#Sec12}
---------------------
Results from included studies have been grouped in to quantitative and qualitative outcomes. Quantitative results included descriptive data on the number and demographic information of users of a telemedicine service. Studies which examined specific outcomes such as number of acute hospital admission, length of admission, primary care contacts and number of contacts needed from a telemedicine provider tended to be associated with studies which described use of a home telemonitoring system. Some quantitative data was specific to the condition monitored, for example spirometry values in cystic fibrosis patients. The study by Lisk et al. which described a multi-modal intervention of telephone advice line, multi-disciplinary team meetings and e-mail alerts on hospital admission for nursing home patients discussed cost reduction as a result of the intervention \[[@CR43]\]. The study calculated a saving of £2630 as a result of their intervention and predicted a £74,383 cost reduction for the next, larger-scale stage of their study. These calculations were reached by comparing the number of inpatient bed-days during the intervention period with the same period from the previous year and hence are estimations. It is not possible to ascertain which element of their intervention resulted in this outcome.
Qualitative results included participant and healthcare provider opinions on either a specific service or telemedicine in general. The results from these studies were generally positive and indicated an openness to telemedicine in palliative care \[[@CR32], [@CR36], [@CR38], [@CR41], [@CR42]\].Specifically, healthcare professionals reported telemedicine interventions to be 'useful' and 'relevant' whilst patients reported telehealth as an acceptable provision of care \[[@CR32], [@CR38], [@CR41]\].However, the study by Johnston et al. \[[@CR42]\] also highlighted some of the potential barriers to telehealth following interviews with patients, carers and healthcare professionals, including a need to improve infrastructure to support telehealth, perceived potential for patient difficulty in managing the technology and a lack of funding and broadband coverage. The paper by Cox et al. \[[@CR37]\] aimed to introduce a home telemonitoring system for patients receiving palliative radiotherapy in lung cancer. Unfortunately, the study was unable to take place due to refusal of consent to approach patients by their clinicians. On interviewing clinicians regarding this there was clear evidence of gatekeeping preventing participation in the research; many clinicians felt technology was inappropriate in this specific population due to age, burden of illness and rapidity of deterioration.
A number of the studies did not outline specific outcomes and were purely descriptive of the telemedicine service or intervention.
Telehealth and the digital service standard {#Sec13}
-------------------------------------------
The second objective of this review was to assess whether telehealth initiatives met the standard of a UK digital service set out by the UK government; this will be discussed here.
Ten papers from the review were suitable for review of the telehealth intervention using criteria adapted from the UK government's digital service standard \[[@CR17]\]. The remaining 20 studies did not contain sufficient detail of the service to complete this review, or detailed a multi-faceted intervention where telehealth was only a component.
Two authors (SH and HJ) independently scored eligible papers against eight criteria using the same system as above (criteria met fully, to some extent, not at all or unable to say). Of these 10 papers, only one paper met all 8 criteria completely or to some extent (Table [3](#Tab3){ref-type="table"}) \[[@CR44]\]. Table 3Performance of studies suitable for telehealth intervention appraisal, where white is 'meets all criteria', /\\ pattern 'to some extent', black is 'not at all' and grey is 'unable to say'
It is clear from the table above that the majority of papers did not detail any information on security and privacy, and making a plan for being offline. Identification of performance indicators and collecting performance data was also not detailed or only partially detailed by many of the included papers.
Access to emergency care {#Sec14}
------------------------
The third objective of this review was to examine for any evidence of reduction in access to emergency care as a result of the telehealth initiative; this will be discussed here.
Seven of the studies made specific reference to reduction in access to emergency or acute care services \[[@CR31], [@CR36], [@CR44], [@CR46], [@CR49], [@CR52], [@CR58]\].
Two of the studies which examined the use of telephone advice lines reported a reduction in admission. In the Middleton-Green et al. study \[[@CR36]\], the authors state that '98.5% of calls resulted in patients remaining in their place of residence' and the service evaluation of the telephone advice line carried out by Plummer et al. \[[@CR52]\] discusses that 70% of callers were not admitted to hospital- possibly as a result of advice given by the call handler.
The studies by Wye et al. \[[@CR55]\] and Purdy et al. \[[@CR58]\] describe the 'Delivering Choice Programme' (DCP). In their study, Purdy et al. describe how participants receiving DCP were less likely to die in hospital, less likely to be admitted to hospital in the 30 days prior to death and less likely to visit the emergency department.
Four studies of home telemonitoring discussed access to emergency care. Dey et al. \[[@CR44]\] found that 36 admissions were avoided using their home telemonitoring system in renal failure patients. Dierckx et al. \[[@CR46]\] undertook a retrospective observational analysis of telemonitoring in heart failure patients and described that telemonitoring seemed to be associated with a reduction in all-cause mortality, however the number of admissions due to heart failure and the time to first hospitalisation was similar between the two groups (receiving telemonitoring and not). The randomised crossover trial by Chatwin et al. \[[@CR31]\] examining telemonitoring in respiratory failure resulted in increased respiratory admissions and home visits in the telemonitoring group. Interestingly, the randomised controlled trial of telemonitoring in COPD patients by Lewis et al. \[[@CR49]\] showed no difference in hospital admissions, days in hospital or emergency department attendances, but a reduction of contact with primary care though this was not statistically significant as the study was underpowered.
Discussion {#Sec15}
==========
Similar to the review published by Kidd et al. in 2010 \[[@CR18]\], this paper provides a useful overview and description of how telehealth initiatives are being used in palliative care in the UK. It is notable that despite a growth in the use of technology, the numbers of papers eligible for inclusion have not increased as expected. During the search, the authors noted a lack of conversion of abstracts to full publication; 12 interesting and potentially eligible abstracts were identified during the grey literature search which had not been converted to full publication. In keeping with these observations, Hanchanale et al. report that just over half of palliative care conference abstracts subsequently go on to full publication \[[@CR59]\]. Although a Cochrane review in 2007 demonstrated a similar publication rate across all specialties \[[@CR60]\], the article by Walshe in 2017 highlights the trend for observational rather than interventional research and a low publication rate of trials in palliative care \[[@CR61]\]. This may account for the relatively low numbers of studies.
Despite this, we have found a number of papers which describe the varying uses of telehealth. Although telephone advice lines and the use of telehealth in providing patient or professional education continue to feature in this review, there was a notable increase in the number of home telemonitoring initiatives implemented compared to the 2010 review. This may be due to the improvement in these technologies. It was interesting to note that all of the home telemonitoring studies were undertaken in participants with specific diagnoses (for example, heart failure) rather than a general palliative care population. This may be appropriate given different diagnoses may result in different symptoms but may also be a barrier when thinking about the number and variety of telehealth applications needed to meet the demands of the palliative care population as a whole.
Where telehealth is described, the detail included in the paper was often insufficient for the authors to judge the initiative against the digital service standard. The majority of the papers which could be judged against this standard did not meet the criteria. This may reflect how recent this digital service standard was published (some papers included were published prior to the standard) but may also corroborate with the overall lack of robust study design noted across the review. Given this standard is now widely available, it may be that future telehealth initiatives align with the criteria more closely. It is worth noting that the criteria were adapted by the authors. For example, the requirement to 'test with the minister' was felt to be inappropriate for this review. The digital service standard was updated in July 2019 after completion of this review and the new criteria seem to reflect some of the challenges identified, including removal of the aforementioned point \[[@CR62]\].
Kidd et al. \[[@CR18]\] comment that telehealth is reported to advantage patient care by improving the patient and carer experience, however there is little known about the clinical benefits and feasibility of providing telehealth services. This review includes papers which comment on clinical benefits and reduced need for emergency care but there are limitations to these findings. Purdy et al. find a reduction in hospital admission, emergency department attendances and deaths in hospital, however their intervention was multi-faceted and they acknowledge that their 'coordination centres' which organise care and equipment for patients seemed to provide the most benefit, rather than the telehealth aspects. Although Dey et al. \[[@CR44]\] report that admissions were avoided, the sample size for the study was small and it is unclear how the authors have reached this conclusion. Dierckx et al. \[[@CR46]\] report a reduction in all-cause mortality, however this was a retrospective observational study where patients were offered telemonitoring rather than allocated. If patients opting to engage with telemonitoring are in general more engaged with healthcare, this may account for some of their findings. The study design in the paper by Middleton-Green \[[@CR40]\] was insufficient to demonstrate that patients remaining in their usual place of residence was as a direct result of their telehealth initiative.
Although there is an increase in the use of home telemonitoring, and a growing appreciation for the use of telehealth in palliative care (as evidenced by the qualitative nature of some of these studies) there remains a lack of evaluation of telehealth interventions. Where evaluation was undertaken, it was difficult to attribute the results to telehealth as many studies implemented a multi-faceted intervention (for example, telephone advice line with a face-to-face support element). Most of the literature continues to be purely descriptive and without robust study design. Without this, it is not possible to clearly demonstrate a clinical benefit of telehealth in palliative care in this review.
Limitations {#Sec16}
-----------
There are several limitations to this review. Although article screening and data extraction was conducted by two reviewers, the synthesis was conducted by only one reviewer which limits the objectiveness and introduces opportunity for error. The studies were not homogenous in nature, which also makes synthesis of the findings difficult. The variety of key terms used in the literature for both palliative care and telehealth made searching for articles challenging and although the search was thorough, omission of relevant articles cannot be ruled out.
It is worth noting that although the criteria used for review of study quality were adapted from existing literature, they were developed by the authors and assessed by the authors, creating scope for bias. Rather than being used as a specific and rigorous assessment of each paper, it served to emphasise the lack of clear study design or method used in the majority of the included papers, resulting in many of these studies being very difficult to reproduce. It is also worth noting that the failure of some papers to meet the nine criteria may actually reflect the written report of the study, rather than the rigor of the method. Similarly, the criteria used by the authors to judge telehealth initiatives against the digital service standard are subject to similar levels of bias for the reasons detailed above.
Strengths {#Sec17}
---------
Despite these limitations, the included studies and synthesis have been able to address the three review questions. The literature search was conducted rigorously and is replicable. Inclusion of all applicable studies in the review allowed for a broad overview of this topic. Screening of papers, data extraction and assessment of quality were undertaken by two reviewers to attempt to minimize bias. Interpretation and synthesis of themes was discussed amongst all authors. The results reinforce some of the findings from the 2010 review used as a starting point for this review and go further to examine some new areas relevant for future work, such as the comparison against the digital service standard.
Impact on policy and practice {#Sec18}
-----------------------------
Although confirming that telehealth initiatives continue to be implemented in palliative care, this review highlights the need for further studies on the use of telehealth in palliative care. Important questions regarding the acceptability and effectiveness of telehealth in this setting remain unanswered.
It was also noted by the authors that although some included studies examined the relationship between telehealth and access to emergency care, none of the studies specifically examined the effect on out of hours service provision. The palliative care and end of life priority setting partnership, identified the provision of palliative care outside of normal working hours as it's number one priority \[[@CR63]\]. This, coupled with the service delivery guideline for adults in the last year of life currently in progress by NICE \[[@CR64]\] indicate that planning of specialist palliative care service provision is of great importance. Hence, the authors suggest that future work examining the use of telehealth in meeting the demands of an out of hours specialist palliative care service could have significant impact on future clinical practice.
Conclusions {#Sec19}
===========
This review demonstrates that a variety of UK palliative care telehealth initiatives continue to be described in the published literature. Since the 2010 review there particularly appears to have been an increase in the number of home telemonitoring interventions, perhaps because of an improvement in this technology. However, where sufficient detail of the telehealth initiative allowed review against a standard, the majority of interventions did not meet the requirements of a UK digital service. Despite the description of telehealth development and implementation, there remains a lack of robust study design and evaluation of these interventions meaning that clear conclusions around the benefit of telehealth in palliative care cannot be drawn; there is insufficient high quality evidence to comment on any influence on access to emergency or unscheduled care. Further work to evaluate the use of telehealth in palliative care, and to specifically examine its use in out of hours specialist palliative care provision is recommended.
Supplementary information
=========================
{#Sec20}
**Additional file 1.** Critical appraisal criteria from Wallace et al. (2004) \[[@CR19]\].
CBT
: Cognitive behavioural therapy
CNS
: Clinical Nurse Specialist
COPD
: Chronic Obstructive Pulmonary Disease
DCP
: Delivering Choices Programme
ED
: Emergency department
EPaCCS
: Electronic Palliative Care Coordination System
MDT
: Multi-disciplinary team
NHS
: National Health Service
NICE
: The National Institute for Health and Care Excellence
OOH
: Out of hours
QALY
: Quality adjusted life years
QOL
: Quality of life
UK
: United Kingdom
USA
: United States of America
WHO
: World Health Organization
**Publisher's Note**
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary information
=========================
**Supplementary information** accompanies this paper at 10.1186/s12904-019-0495-5.
Not applicable.
SH designed the review, collected and analysed the data and drafted the manuscript. AG contributed to the design of the review and critical revision of the manuscript. HJ contributed to data collection as second review. NP provided critical revision of the manuscript. All authors read and approved the final manuscript.
Dr. Sophie Hancock completed this work as part of a NIHR funded academic clinical fellowship. The funding body did not contribute to the design of the study, collection, analysis and interpretation of data or writing the manuscript.
No primary data collected in this study. Detailed search strategy and further information on included studies available on request from Dr. Sophie Hancock.
Not applicable.
Not applicable.
Professor Nancy Preston is a member of the editorial board for BMC Palliative Care.
|
{
"pile_set_name": "PubMed Central"
}
|
Introduction
============
Gastric cancer is the fifth most common malignant tumor in the world and the third most common cause of malignant tumor-related death [@B1]. Though the overall survival (OS) of gastric cancer patients has improved with the development of standardized D2 lymphadenectomy [@B2] and subsequent adjuvant chemotherapy in recent years [@B3], [@B4], the long-term survival rate is still unsatisfactory. Moreover, the majority of gastric cancer patients in China were diagnosed as advanced gastric cancer (AGC) at an early visit. The overall 5-year OS for AGC patients is below 20%, and it is approximately 30% for those who undergo surgery. Even after curative resection, only 30-50% of patients are still alive after 5 years [@B5]. Among AGC T4 gastric cancer patients, which includes tumor perforating serosa (T4a) and invasion of the adjacent structure (T4b), patients often suffered from recurrence and had dismal overall survival, even after R0 resection. Several studies have reported potential prognostic factors affecting the survival of T4 gastric cancer patients after curative resection, including lymph node metastasis, venous invasion, peritoneal washing cytology and tumor diameter[@B6]-[@B8]. However, the sample sizes of previous studies were small.
Recurrence is the most important factor affecting the survival of patients with GC. After curative resection for GC, recurrence patterns include local-regional recurrence, distant lymph node metastasis, hematogenous metastasis, and peritoneum implantation. According to soil and seed theory, T4 GC patients are prone to suffering peritoneal seeding. However, there are few studies exploring the recurrence patterns for T4 GC after curative resection.
In the present study, we aimed to clarify the prognostic factors and recurrence patterns for T4 GC after curative resection, which would help clinicians perform the appropriate treatment in order to improve survival.
Methods and patients
====================
Between January 2004 and December 2014, a total of 249 gastric cancer patients were treated with curative resection (R0 resection) and diagnosed as pathological T4 stage in Sun Yat-sen University Cancer Center. Standardized D2 lymphadenectomy or expanded resections for gastric cancer (including D2 + lymphadenectomy, or D2 lymphadenectomy plus the resection of other adjacent organs) were performed in these patients with curative resection. Tumors were staged according to the seventh edition of the American Joint Committee on Cancer (AJCC) tumor-node-metastasis (TNM) staging system [@B9]. Patients were excluded if they received neoadjuvant treatment, were diagnosed as late-stage gastric cancer or underwent R1/2 (microscopically/macroscopically residual tumor) resection. According to NCCN Guidelines [@B10], our clinicians recommended that all the included patients received adjuvant chemotherapy. However, in clinical practice, whether or not they received adjuvant chemotherapy was determined by the will of the patients.
All patients were followed up every 3 months in the first 2 years, 6 months in the 3-5 years, and 12 months annually thereafter, the median follow-up time was 25.87 months (range, 0.3-137).
The following clinicopathological data were collected prospectively through a constructed database: gender, age, tumor size, weight loss, tumor location, type of gastrectomy, extent of lymphadenectomy, intraoperative blood loss, postoperative complications, histological type, harvested lymph nodes, N stage, Lauren type, lymphatic invasion, venous invasion, perineural invasion and adjuvant chemotherapy. OS time was calculated from the time of surgery to death or the last follow-up date. Recurrence patterns were diagnosed based on imaging examination, gastroscopy with biopsy, and/or cytological examination of ascites, or intraoperative findings in patients who underwent reoperation.
Statistical analysis
--------------------
Statistical analysis was performed using SPSS 17.0 for Windows (SPSS Inc, Chicago, IL, USA). Categorical variables, presented as rate, were compared by the χ2 (Chi-square) test. Continuous data, expressed as the mean ± standard deviation (SD), were compared by Student\'s t-test. Survival outcomes were analyzed using the Kaplan-Meier method and compared using the log-rank test. Factors that were considered as significant in the univariate analysis (P \< 0.05) were included in the multivariate analysis by Cox regression. HR with 95% confidence intervals (CIs) were presented. P \< 0.05 was considered statistically significant.
Results
=======
Patient characteristics
-----------------------
The clinicopathologic characteristics of 249 patients were summarized in Table [1](#T1){ref-type="table"}. There were 221 T4a and 28 T4b GC patients receiving curative resection, comprising 165 males and 84 females, with a mean age of 56.10 years (range, 21-82 years). Some 221 patients underwent standard D2 lymphadenectomy, while 28 patients had expanded resection. The postoperative morbidity rate was 6.0%. A total of 184 patients (73.9%) received adjuvant chemotherapy after surgery. As shown in Table [2](#T2){ref-type="table"}, the main organs invaded with T4b were the pancreas and spleen.
Survival
--------
The median follow-up duration was 25.87 months (range, 0.3-137.0 months). The OS rate of 249 patients was 92.5% at 1 year, 65.3% at 3 years and 46.8% at 5 years (Fig [1](#F1){ref-type="fig"}).
Our results showed that the MST for T4a gastric cancer was longer than that for T4b gastric cancer \[59.47 (95% CI: 32.64-86.30) months versus 25.90 (95% CI: 12.32-39.48) months\] (Fig. [2](#F2){ref-type="fig"}A). The MST difference was significant (P = 0.034). In the univariate analysis, age (P = 0.007) (Fig. [2](#F2){ref-type="fig"}B), weight loss (P = 0.013), tumor location (P\< 0.001) (Fig. [2](#F2){ref-type="fig"}C), extent of lymphadenectomy (P = 0.034), intraoperative blood loss (P \< 0.001) (Fig. [2](#F2){ref-type="fig"}D), postoperative complications (P = 0.038) and N stage (P = 0.012) were significant predictors of OS (Table [3](#T3){ref-type="table"}). Multivariate analysis revealed that age (P = 0.006), tumor location (P \< 0.001) and intraoperative blood loss (P = 0.010) remained as independent prognostic factors for long-term survival (Table [3](#T3){ref-type="table"}).
Pattern of recurrence and risk factors of recurrence
----------------------------------------------------
After a median follow up of 25.87 months, a total of 109 (43.8%) patients experienced recurrence. The recurrence rates for T4a and T4b patients were 92/221 (41.6%) and 17/28 (60.7%), respectively. Of these, the recurrence sites were obtained in a total of 90 patients, with a median of 15.90 (range: 1-71.1) months relapsing after curative surgery. The recurrence patterns were shown in Table [4](#T4){ref-type="table"}. Among those 90 patients, peritoneal metastasis (62.2%) was the most frequent recurrence site, followed by hematogenous metastasis (26.7%), distant lymph node metastasis (24.4%) and locoregional metastasis (15.6%). The peritoneal metastasis was the most common recurrence pattern, accounting for 88.3% (10/12) recurrence in T4b, and 59.0% (46/78) in T4a. The multivariate analysis showed that the N stage and intraoperative blood loss were the independent risk factors for recurrence (Table [5](#T5){ref-type="table"}).
Discussion
==========
Even with the development of standardized D2 lymphadenectomy [@B2] and subsequent adjuvant chemotherapy [@B4], [@B11] for gastric cancer, the survival outcomes are still unsatisfactory, causing a third of cancer-related deaths worldwide [@B1]. Patients often suffer from recurrence after curative resection in the first 5 years, especially T4 patients. Better understanding of the prognostic factors and recurrence patterns for T4 gastric cancer will help clinicians to perform more intensive treatment strategies for high-risk patients. In the present study, our results demonstrated that age, tumor location and intraoperative blood loss were the prognostications of OS for T4 gastric cancer after curative resection. Moreover, peritoneal metastasis was the most common pattern of recurrence.
Several studies [@B6]-[@B8], [@B12], [@B13] had reported the prognostic factors for T4 gastric cancer. Fukuda et al [@B6] reported that number of metastatic lymph nodes, venous invasion and peritoneal washing cytology influenced the prognosis of T4 gastric cancer after potentially curative resection. Li et al [@B7] found that lymph node involvement was the only prognostic factor of OS. However, the sample sizes of previous studies were small, and they included patients with noncurative resection, such as R1/2 resection. While previous studies did not report the survival discrepancy regarding age, our results indicated that elderly patients (\>60 years) had worse survival outcomes. The reasons for this might be considered as follows. First, disorder of immune function in elderly GC patients might relatively weaken antitumor capacity and lead to poor prognosis [@B14]. Second, compared to young patients, elderly patients cannot tolerate extensive lymphadenectomy and the toxicities of chemotherapy. Therefore, elderly patients get inadequate treatment, which affects survival.
The impact of intraoperative blood loss on long-term outcomes had been reported in various types of malignancy [@B15]-[@B18]. Dhar et al [@B19] reported that more than 500 ml blood loss during surgery was an independent predictor of survival in gastric cancer patients with transmural depth invasion following a curative resection. Liang et al [@B20] also found intraoperative blood loss was an independent prognostic factor for gastric cancer after curative resection. In line with previous studies, our results showed that more intraoperative blood loss (\>110 ml) during surgery was an independent prognosis factor in T4 gastric cancer after curative resection. The exact mechanism of how blood loss affects the survival of patients remained unclear. It was considered that excessive intraoperative blood loss reduces the body\'s immunity, thus affecting the patients\' ability to fight cancerous cells [@B19]. Therefore, our results implied that operating carefully to reduce intraoperative blood loss might improve the survival of T4 gastric cancer after curative resection.
In spite of improvements in surgical technique and adjuvant chemotherapy, advanced gastric cancer patients always suffered recurrence after curative resection. The majority of recurrences happened within 2 years postsurgery [@B21]-[@B25]. Previous studies had reported the recurrence patterns of gastric cancer [@B25]-[@B28], especially for patients with tumor invading serosa (T4 stage). However, the recurrence patterns of T4 gastric cancer with curative resection have not been reported. In the present study, 109 of 249 (43.8%) patients experienced relapse. Among them, 90 patients had specifically documented recurrence sites, and peritoneal metastasis was the most frequent recurrence pattern (n = 56), accounting for 62.2% of all relapsed patients. For T4b patients, the peritoneal metastasis ratio reached 88.3%. According to the seed and soil theory, when gastric cancer invades the serosa (T4), the cancer cells have a high possibility of falling into the peritoneal cavity where a suitable environment is provided for the cancer cells to grow. Kondo et al. reported that free cancerous cells could be found in 40% of stage II or III gastric cancers after peritoneal washings [@B29], suggesting that microscopic metastasis still remained after curative resection. Another study also reported that for advanced gastric cancer with serosal involvement, peritoneal metastasis usually occurred, with 15%-50% of patients having peritoneal metastasis at the time of surgery [@B30].
Peritoneal metastasis has been considered the terminal stage of gastric cancer, with a median overall survival of less than 6 months [@B31], accounting for almost 60% of all causes of GC death [@B32]. Therefore, understanding how to prevent and treat peritoneal metastasis is clinically important. In 1988, Fujimoto et al reported that hyperthermic intraperitoneal chemotherapy (HIPEC) was effective for gastric cancer patients with peritoneal seeding. A large number of studies also reported similar results [@B33]-[@B37]. Moreover, several prospective randomized controlled trials [@B38]-[@B41] also reported that curative resection with HIPEC was a useful treatment to prevent occurrence of peritoneal metastasis in advanced gastric cancer. Theoretically, HIPEC after curative resection could help eliminate free cancer cells and micro metastases in the abdominal cavity for T4 gastric cancer, which might prolong survival of patients. Nevertheless, this remained to be confirmed through well-designed randomized controlled trials.
There were several limitations in the present study. First, the present study was a retrospective, single-center research project. Second, the time span of this study was long (10 years). Therefore, the results of this study should be interpreted cautiously. A large-scale, validation study is required to confirm the results.
Conclusions
===========
For T4 gastric cancer patients after curative resection, older age, entire gastric cancer and more intraoperative blood loss were associated with poor OS. The recurrence rate after curative resection for T4 was high, and the most common recurrence pattern was peritoneal metastasis.
{#F1}
{#F2}
######
Clinicopathologic characteristics for 249 patients with T4 gastric cancer after curative resection.
Variables n (%)
--------------------------------- ------------
Gender
Male 165 (66.3)
Female 84 (33.7)
Age (y)
≤60 156 (62.7)
\>60 93 (37.3)
Tumor diameter (cm)
≤6.5 176 (70.7)
\>6.5 73 (29.3)
Weight loss (kg)
≤5 197 (79.1)
\>5 52 (20.9)
Location of tumor
Upper 1/3 93 (37.3)
Middle 1/3 38 (15.3)
Lower 1/3 97 (39.0)
Entire 21 (8.4)
Type of gastrectomy
Distal 103 (41.4)
Proximal 47 (18.9)
Total 99 (39.7)
Lymphadenectomy
D2 221 (88.8)
Expanded D2 28 (11.2)
Intraoperative blood loss (ml)
≤110 142 (57.0)
\>110 107 (43.0)
Postoperative complications
Absence 234 (94.0)
Presence 15 (6.0)
Histological type
Moderate or well differentiated 26 (10.4)
Poorly differentiated 223 (89.6)
Number of harvested lymph nodes
\<15 21 (8.4)
≥15 228 (91.6)
N stage
N0-N1 77 (30.9)
N2-N3 172 (69.1)
Lauren classification
Intestinal 99 (39.8)
Diffuse 25 (10.0)
Mixed 38 (15.2)
Lymphatic invasion
Absence 138 (55.4)
Presence 111 (44.6)
Venous invasion
Absence 151 (60.6)
Presence 98 (39.4)
Perineural invasion
Absence 113 (45.4)
Presence 136 (54.6)
Postoperative chemotherapy
No 65 (26.1)
Yes 184 (73.9)
######
Organs Invaded by T4b Gastric Cancer.
Invaded organs n \%
------------------------------- ---- ------
Pancreas 15 53.6
Spleen 13 46.4
Liver 4 14.3
Transverse colon or mesocolon 3 10.7
Adrenal gland 2 7.2
Gallbladder 1 3.6
Diaphragm 1 3.6
No. of invaded organs
One 20 71.4
Two 6 21.4
Four 2 7.2
######
Univariate analysis of risk factors and Multivariable Cox regression analysis of risk factors for patients with T4 gastric cancer.
----------------------------------------------------------------------------------------------------------------
Variables Univariate analysis Multivariate analysis
--------------------------------- ---------------------- ------- ----------------------- -----------------------
Gender 0.555
Male 1
Female 1.144 (0.732\~1.787)
Age (y) 1 0.007 0.006
≤60 1.769 (1.160\~2.697) 1
\>60 1.859 (1.195\~2.892)
Tumor diameter (cm) 0.026 0.561
≤6.5 1 1
\>6.5 1.622 (1.055\~2.494) 0.844 (0.477\~1.494)
Weight loss (kg) 0.013 0.067
≤5 1 1
\>5 1.785 (1.120\~2.844) 1.593 (0.968\~2.623)
Location of tumor 0.000 0.000
Upper 1/3 1 1
Middle 1/3 1.016 (0.526\~1.963) 1.252 (0.626\~2.503)
Lower 1/3 0.966 (0.585\~1.593) 0.899 (0.537\~1.506)
Entire 4.854 (2.463\~9.567) 5.640 (2.463\~12.915)
Type of gastrectomy 0.204
Proximal 1
Distal 1.195 (0.682\~2.094)
Total 1.535 (0.954\~2.468)
Lymphadenectomy 0.034 0.819
D2 1 1
Expanded D2 1.801 (1.037\~3.125) 1.072 (0.588\~1.955)
Intraoperative blood loss (ml) 0.000 0.010
≤110 1 1
\>110 2.148 (1.392\~3.316) 1.851 (1.162\~2.950)
Postoperative complications 0.038 0.223
Absence 1 1
Presence 2.130 (1.027\~4.420) 1.606 (0.750\~3.439)
Histological type 0.913
Moderate or well\ 1
differentiated
Poorly differentiated 1.037 (0.536\~2.007)
Number of harvested lymph nodes 0.156
≤15 1
\>15 0.663 (0.374\~1.175)
N stage 0.012 0.113
N0-N1 1 1
N2-N3 1.874 (1.137\~3.089) 1.520 (0.906\~2.551)
Lauren classification 0.849
Intestinal 1
Diffuse 0.896 (0.370\~2.173)
Mixed 0.790 (0.344\~1.816)
Lymphatic invasion 0.082
Absence 1
Presence 1.458 (0.951\~2.235)
Venous invasion 0.923
Absence 1
Presence 1.023 (0.644\~1.625)
Perineural invasion 0.549
Absence 1
Presence 0.871 (0.555\~1.368)
Postoperative chemotherapy 0.272
No 1
Yes 0.760 (0.465\~1.242)
T4 stage 0.034
T4a 1
T4b 1.801 (1.037\~3.125)
----------------------------------------------------------------------------------------------------------------
######
Sites of recurrence in 90 patients.
Recurrence site Total group (%) T4a T4b P value
----------------------- ----------------- ----- ----- ---------
peritoneum implanting 56 (62.2) 46 10 0.105
Hematogenous 24 (26.7) 21 3 0.888
distant lymph node 22 (24.4) 20 2 0.501
Locoregional 14 (15.6) 14 0 0.110
No. of sites
One 67 (74.4)
Two 19 (21.1)
Three 4 (4.5)
######
Relationship between the recurrence and clinicopathologic characteristics by univariate and multivariable analysis in T4 gastric cancer patients after curative resection.
Variables Univariate analysis χ^2^test Multivariate analysis
--------------------------------- --------------------- --------- ---------- ---------------------- -----------------------
No. of patients (n=140) (n=109)
Gender 0.835
Male 48 36
Female 92 73
Age (y) 0.385
≤60 91 65
\>60 49 44
Tumor diameter (cm) 0.090
≤6.5 105 71
\>6.5 35 38
Weight loss (kg) 0.050
≤5 117 80
\>5 23 29
Location of tumor 0.115
Upper 1/3 58 35
Middle 1/3 21 17
Lower 1/3 54 43
Entire 7 14
Type of gastrectomy 0.291
Proximal 58 45
Distal 22 25
Total 60 39
Lymphadenectomy 0.055
D2 129 92
Expanded D2 11 17
Intraoperative blood loss (ml) 0.004 0.041
≤110 91 51 1
\>110 49 58 1.778 (1.024\~3.088)
Postoperative complications 0.191
Absence 134 100
Presence 6 9
Histological type 0.796
Moderate or well differentiated 14 12
Poorly differentiated 126 97
Number of harvested lymph nodes 0.359
≤15 14 15
\>15 126 94
N stage 0.016 0.019
N0-N1 52 25 1
N2-N3 88 84 1.992 (1.119\~3.545)
Lauren classification 0.308
Intestinal 59 40
Diffuse 16 9
Mixed 28 10
Lymphatic invasion 0.879
Absence 77 61
Presence 63 48
Venous invasion 0.308
Absence 81 70
Presence 59 39
Perineural invasion 0.029 0.139
Absence 55 58 1
Presence 85 51 0.659 (0.379\~1.145)
Postoperative chemotherapy 0.315
No 40 25
Yes 100 84
T4 stage 0.055
T4a 128 92
T4b 11 17
[^1]: ^\#^ These authors contributed equally to this study.
[^2]: ^\*^ These authors also contributed equally to this study.
[^3]: Competing Interests: The authors have declared that no competing interest exists.
|
{
"pile_set_name": "PubMed Central"
}
|
Background {#Sec1}
==========
Intervertebral disc (IVD) degeneration appears to be the foremost cause of the back pain \[[@CR1]\], which causes a tremendous economic burden to society \[[@CR2]\]. Nucleus pulposus (NP) cells presenting in the intervertebral disc maintain the extracellular matrix \[[@CR3]\]. The decrease in NP cell viability lead to the disturbing disc homeostasis, characterized by loss of extracellular matrix. Exogenous mesenchymal stem cells (MSCs) have shown an ability to regenerate disc cells and maintain the normal structure of degenerated IVD \[[@CR4]\]. Recently, endogenous MSCs stimulation and recruitment is indicated to be an essential way to repair IVD degeneration \[[@CR5]\]. Previous studies have confirmed that human degenerated and normal IVD contained human nucleus pulposus-derived mesenchymal stem cells (hNP-MSCs) \[[@CR6]--[@CR9]\] and hNP-MSCs fulfilled morphological, immunophenotypic, and differentiation definition criteria described by the International Society of Cell Therapy for mesenchymal stromal cells. These cells could retard the process of IVD degeneration \[[@CR10]\]. The aim of endogenous hNP-MSCs therapy is to make hNP-MSCs differentiate into NP-like cells and promote disc cells maintaining metabolic balance and biomechanical functions of IVD. However, it is hard to maintain the number of the active hNP-MSCs under the adverse microenvironment in the degenerated IVD and leads to repair failure of endogenous hNP-MSCs \[[@CR11]\].
The IVD obtains all essential nutrients through the cartilage endplate \[[@CR12]\]. Some studies found that there were steep gradients in the concentration of nutrients across the IVD with oxygen level ranging from 1 to 5% \[[@CR13]\]. With the processing of aging and degeneration, the supply of nutrients such as oxygen, glucose, and serum reduces significantly and even disappears, making the microenvironment more hypoxic and nutrition deficiency, which leads to metabolic disturbance and decrease of nucleus pulposus cells and nucleus pulposus progenitor cells \[[@CR14]\]. Previous studies have investigated the single effect of hypoxia \[[@CR15]\] and nutrition deprivation \[[@CR16]\] presenting in the IVD on the biological activities of IVD cells, but microenvironment in the degenerated IVD is complex, consisting of hypoxia and nutrition deficiency. There have been no study that investigates whether hypoxia and nutrition deficiency are able to change the activities of the hNP-MSCs. Thus, in our present study, we investigated the effect of hypoxia (1% O~2~) and nutrition deficiency (no glucose and no fetal serum), mimicking the microenvironment in the degenerated IVD on the hNP-MSCs as in the previous study \[[@CR17]\]. We hypothesized that microenvironment of hypoxia and nutrition deficiency may inhibit the biological activities of hNP-MSCs.
The phosphatidylinositol 3-kinase (PI3K)/Akt pathway plays a critical role in cell proliferation, apoptosis, and differentiation during physiological and pathological conditions \[[@CR18], [@CR19]\]. PI3K is a unique family of intracellular lipid kinases and Akt is a serine/threonine kinase. Once activated by different agents, PI3Ks change phosphatidylinositol 4,5-biphosphate (PIP2) into phosphatidylinositol 3,4,5-triphosphate (PIP3, \[[@CR20]\]) and further activate Akt. Activated Akt modulates biological processes, including cell proliferation and apoptosis through interaction with downstream proteins. More importantly, PI3K/Akt signaling activation has shown protective effect on IVD degeneration. Activation of PI3K/Akt signaling increases SOX9 expression and activity, which induces expression of aggrecan in NP cells \[[@CR21]\]. Insulin-like growth factor-1(IGF-1) promotes IVD cells proliferation by activating this pathway \[[@CR22]\]. Many adverse factors contribute to IVD degeneration, such as adverse microenvironment, cytokines. Previous studies indicated that activation of PI3K/AKT signaling attenuated NP cells apoptosis induced by high-magnitude compression \[[@CR23]\] or hyperosmotic conditions \[[@CR24]\]. In addition, PI3K/AKT could protect NP cells against apoptosis induced by some cytokines, such as IL-1β \[[@CR25]\] and TNF-ɑ \[[@CR26]\]. Thus, we supposed that whether PI3K/Akt also played a protective effect on hNP-MSCs under hypoxia and nutrition deficiency.
In our present study, we speculated that hypoxia and nutrition deficiency could inhibit biological behavior of hNP-MSCs and PI3K/Akt pathway could protect hNP-MSCs from hypoxia and nutrition deficiency. We isolated cells from human degenerated lumbar disc and identified whether this type of cell fulfilled the definition criteria of MSCs established by an expert panel from the International Society for cellular Therapy (ISCT). Afterwards, we investigated the effects of hypoxia and nutrition deficiency on the biological behavior of hNP-MSCs in vitro, and studied the role of PI3K/Akt signaling in the process.
Materials and methods {#Sec2}
=====================
Cell isolation and culture {#Sec3}
--------------------------
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. This research has been approved by the IRB of the authors' affiliated institutions. Informed consent was obtained from all individual participants included in the study.
Our present study collected three disc samples from patients who underwent spine surgery for degenerative spine diseases. All data about the source of the disc material were showed in Table [1](#Tab1){ref-type="table"}. Briefly, gel-like NP tissues taken from human degenerated IVD were carefully separated from the AF using a microscope under aseptic conditions. Thereafter, NP tissue was minced into small pieces before enzymatic digestion with 0.02 mg/ml collagenase type II (Gibco, USA) at 37 °C overnight. The suspension was centrifuged at 1500 rpm for 5 min and the supernatant was discarded. The remaining tissue and cells was cultured in standard MSC culture medium, consisting of Dulbecco's modified Eagle's medium-low glucose (HyClone, USA), 10% fetal calf serum (Gibco, USA), and 1% penicillin/streptomycin (Gibco, USA) at 37 °C in 5% CO~2~ for 24 h. Suspended cells and tissue were abandoned, and adherent cells were cultured in standard MSC expansion medium with complete replacement of the medium every 3 days. When adherent cells reached 80% to 90% confluence, they were subcultured at 1:3. Cells at passage 2 were used for subsequent experiments. Table 1Patients characteristicsAge (year)GenderModified Pfirrmann gradingLevelCase131FemaleIIIL4--5Case244FemaleVIL5--S1Case366MaleIVL5--S1
Immunophenotype of hNP-MSCs {#Sec4}
---------------------------
Cells were rinsed and suspended in PBS (Sigma, USA) at 1 × 10^5^ cells/ml. Further, 100 μl cell suspension solution was aliquoted per tube and was respectively incubated with a solution of CD34, CD73, CD45, CD90, CD105, and HLA-DR (eBioscience, USA) at room temperature for 30 min. Isotype control was used in each case. Cells were rinsed in PBS and suspended in 500 μl of PBS prior flow cytometry analyses.
Multilineage differentiation {#Sec5}
----------------------------
Mesenchymal Stem Cell Differentiation Medium (Cyagen Biosciences, Guangzhou, China) were purchased for differentiation assays according to the manufacturer's instructions, which includes osteogenic differentiation, adipogenic differentiation, and chondrogenic differentiation. The cells were planted at 2 × 10^4^ cells/cm^2^ for osteogenic differentiation, 2 × 10^4^ cells/cm^2^ for adipogenic differentiation, and 5 × 10^5^ cells/ml for chondrogenic differentiation, respectively. The cells were then respectively stained with Alizarin Red, Oil Red O, and Alcian Blue. The entire stained areas were visualized under inverted microscopy.
Preparation of four experimental groups {#Sec6}
---------------------------------------
Cells, isolated from all three disc samples, were cultured and passaged. For each experiment, cells from three disc samples were selected for each assay. These cells were divided into four groups, including the control group (group1), the hypoxia and nutrition deficiency group (group2), the LY294002 group (group3), and IGF-1 group (group4). The cells in the control group were cultured under normoxia with standard MSC expansion medium (21% O~2~, 10% fetal calf serum, 5 mM glucose). The cells in the hypoxia and nutrition deficiency group were cultured with hypoxia and nutrition deficiency (1% O~2~, no fetal calf serum, no glucose). LY294002, a specific PI3K inhibitor, with a concentration of 30--50 μM, was shown to specifically inhibit PI3K activity without inhibiting other lipid and protein kinases \[[@CR27]\]. The cells in the LY294002 group were added into 30 μmol/L LY294002 for 1 h before cultured under hypoxia and nutrition deficiency conditions. The cells in the IGF1 group were added into 200 μg/L IGF1, a PI3K activator, for 1 h before cultured under hypoxia and nutrition deficiency conditions.
Apoptosis assay {#Sec7}
---------------
hNP-MSCs were planted at 1 × 10^5^ cells/ml in 6-well plates, cultured under normal condition for 24 h, and then divided into above four groups and cultured for 24 h. Then harvested cells were incubated with 5 μl Annexin V-FITC and 5 μl PI (KeyGEN BioTECH, China) for 5 min at 37 °C in the dark and analyzed by Flow cytometry (BD, USA).
CCK-8 assay {#Sec8}
-----------
The viability of hNP-MSCs cultured in different conditions was evaluated by Cell-Counting Kit-8 (CCK-8). hNP-MSCs were planted at 5 × 10^4^ cells/ml in 96-well plates. These cells were cultured under normal condition for 1 day and then divided into above four groups and cultured for 1 day. Finally, the cells were incubated with 10 μl CCK-8 reagent (DOJINDO, Japan) for 4 h at 37 °C in the dark. Isotype group had no cells. The absorbance of different groups was measured at 450 nm using a Spectra MAX microplate reader.
Caspase3 activity {#Sec9}
-----------------
hNP-MSCs were planted at 1 × 10^5^cells/ml in 6-well plates, cultured under normal condition for 24 h, and then divided into above four groups and cultured for 24 h. The caspase tetrapeptide substrate Ac-DEVD-pNA (Beyotime, China) was used to detect caspase 3 activity. Cells were washed with PBS and lysed with 100 μl of lysis buffer (Beyotime, China) on ice for 15 min. Then 10 μl of lysate was added to mixture solutions, consisting of 80 μl of reaction buffer and 10 μl of Ac-DEVD-pNA and incubated for 1 h at 37 °C. A microplate spectrophotometer was used to measure the optical density of free pNA.
Gene expression of PI3K, Akt, aggrecan, collagen I, collagen II, stem cell-related genes (Oct4, Nanog, Jagged, and Notch1) {#Sec10}
--------------------------------------------------------------------------------------------------------------------------
hNP-MSCs were planted at 1 × 10^5^ cells/ml in 6-well plates, cultured under normal condition for 24 h, and then divided into above four groups and cultured for 24 h. Total RNA was extracted from harvested cells using TRIReagent (Ambion, USA) according to the product instructions. Then, total RNA was reverse transcribed into the cDNA using a reverse-transcribed reagent (Takara, Japan). GAPDH was used as the reference gene. The obtained cDNA used in quantitative real-time PCR was analyzed using A SYBR Premix Ex Taq PCR kit (Takara, Japan) and LightCycler (Roche, Switzerland). Primers sequences of all genes were synthesized according to the software Premier 5.0, as shown in Table [1](#Tab1){ref-type="table"}.
Statistical analysis {#Sec11}
====================
All data were presented as means ±standard deviation (SD) of independent experiments (*n* = 3). All measurements were performed in triplicate. The SPSS version 17.0 software (IBM, USA) were used to analyze data. ANOVA with a post-hoc test were performed to analyze the results. *P* values \< 0.05 were considered statistically significant (Table [2](#Tab2){ref-type="table"}). Table 2Primers used in qRT-PCRGenesSense primerAntisense primerOct4GTGAGAGGCAACCTGGAGAAGAACCACACTCGGACCACATjaggedCGAGGACTATGAGGGCAAGACTTCAGGTGTGTCGTTGGAANotch1GCCAGAGTGGACAGGTCAGTACACACACGCAGTTGTAGCCNanogAGGCAAACAACCCACTTCTGTCTGCTGGAGGCTGAGGTATCollagen ICCTGGAAAGAATGGAGATGATGATCCAAACCACTGAAACCTCTGCollagen IIGGTAAGTGGGGCAAGACTGTTATGTTGTTTCTGGGTTCAGGTTTAggrecanGTCAGATACCCCATCCACACTCCATAAAAGACCTCACCCTCCATPI3KACCAGCACTGCCTCCTAAACTCTTCATCATCTTCCACCAGTGAktACTCTTTCCAGACCCACGACCCAAAGAAGCGATGCTGCATG
Results {#Sec12}
=======
Isolation and characterization of hNP-MSCs {#Sec13}
------------------------------------------
Primary cells were observed after 3--5 days of initial cell culture and presented short spindle-shape (Fig. [1](#Fig1){ref-type="fig"}a). The cells grew significantly faster when cultures were passaged and grew in spiral formation and also consistently presented the characteristic spindle-shape (Fig. [1](#Fig1){ref-type="fig"}b). These cells isolated from degenerated IVD were highly positive for CD73, CD90, and CD105, and were negative for CD34, CD45, and HLA-DR (Fig. [1](#Fig1){ref-type="fig"}c, d). Alizarin Red staining showed that cells formed mineralized nodules. Oil Red O staining revealed that cells produced intracellular lipid vacuoles. Alcian Blue staining indicated that cells exhibited sulfated proteoglycan (Fig. [1](#Fig1){ref-type="fig"}e). These results suggested that these cells fulfilled the definition criteria of MSCs \[[@CR28]\] and hNP-MSCs were successfully obtained from human degenerated NP. Fig. 1Primary hNP-MSCs present short spindle-shape (**a**). P2 hNP-MSCs presented the characteristic spindle-shape and grew in spiral formation (**b**). These cells were highly positive for CD73, CD90, CD105, and negative for CD34, CD45, HLA-DR (**c, d**). hNP-MSCs possessed osteogenic, adipogenic, and chondrogenic differentiation (**e**)
Hypoxia and nutrition deficiency increased the gene expression of PI3K and Akt {#Sec14}
------------------------------------------------------------------------------
The relative gene expression of PI3K and Akt in group 2 was notably higher than that in group 1 (*P* \< 0.05), which indicated that PI3K/Akt pathway could be involved in the process under hypoxia and nutrition deficiency (Fig. [2](#Fig2){ref-type="fig"}). Fig. 2The relative gene expression of PI3K and Akt in normal condition, hypoxia and nutrition deficiency condition evaluated by qRT-PCR. \**p* \< 0.05 indicated significant difference between groups
Hypoxia and nutrition deficiency inhibited the proliferation of hNP-MSCs and inhibiting PI3K by LY294002 could aggravate this inhibiting effect while activating of PI3K by IGF-1 could improve the biological activity {#Sec15}
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
The cell proliferation of group 2 was notably lower than that of group 1 (*P* \< 0.05), which indicated that hypoxia and nutrition deficiency could inhibit the proliferation of hNP-MSCs. Meanwhile, after blocking PI3K by LY294002, the proliferation in group 3 was significantly lower than that in group 2 (*P* \< 0.05); however, after activating of PI3K by IGF1, the proliferation in group 4 was obviously higher than that group 2 (*P* \< 0.05) (Fig. [3](#Fig3){ref-type="fig"}). Fig. 3The viability of hNP-MSCs cultured in normal condition (group 1), hypoxia and nutrition deficiency condition (group 2), the LY294002 condition (group 3), and the IGF-1 condition (group 4) evaluated by cell-counting kit-8 (CCK-8). \**p* \< 0.05 indicated significant difference between groups
Hypoxia and nutrition deficiency induced apoptosis of hNP-MSCs and inhibiting PI3K by LY294002 could aggravate this effect while activating of PI3K by IGF-1 could attenuate the apoptosis {#Sec16}
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
The cell apoptosis of group 2 was significantly higher than that of group 1 (*P* \< 0.05), which indicated that hypoxia and nutrition deficiency could induce the apoptosis of hNP-MSCs. Meanwhile, after blocking PI3K by LY294002, the apoptosis of group 3 was significantly higher than that of group 2 (*P* \< 0.05); however, after activating of PI3K by IGF1, the apoptosis of group 4 was obviously lower than that of group 2 (*P* \< 0.05) (Fig. [4](#Fig4){ref-type="fig"}). Fig. 4The apoptosis of hNP-MSCs cultured in normal condition (group 1), hypoxia and nutrition deficiency condition (group 2), the LY294002 condition (group 3), and the IGF-1 condition (group 4) evaluated by flow cytometry. \**p* \< 0.05 indicated significant difference between groups
Hypoxia and nutrition deficiency increased caspase 3 activity and inhibiting PI3K by LY294002 could aggravate this effect while activating of PI3K by IGF-1could inhibit the caspase3 activity {#Sec17}
----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
The optical density value of group 2 was significantly higher than that of the group 1 (*P* \< 0.05), which indicated that hypoxia and nutrition deficiency could increase caspase3 activity. Meanwhile, after blocking PI3K by LY294002, the value of group 3 was significantly higher than that of group 2 (*P* \< 0.05); however, after activating of PI3K by IGF1, the value of group 4 was obviously lower than that of group 2 (*P* \< 0.05) (Fig. [5](#Fig5){ref-type="fig"}). Fig. 5The caspase 3 activity of hNP-MSCs cultured in normal condition (group 1), hypoxia and nutrition deficiency condition (group 2), the LY294002 condition (group 3), and the IGF-1 condition (group 4) evaluated by Ac-DEVD-pNA. \**p* \< 0.05 indicated significant difference between groups
Hypoxia and nutrition deficiency downregulated the expression of functional genes and stem cell-related genes and blocking PI3K by LY294002 could aggravate this effect while activating of PI3K by IGF-1 could upregulate the expression levels {#Sec18}
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
The relative expression of functional genes and stem cell-related genes in group 2 was significantly lower than that of group 1 (*P* \< 0.05), which indicated that hypoxia and nutrition deficiency could inhibit the gene expression of functional genes and stem cell-related genes in hNP-MSCs. Meanwhile, after blocking PI3K by LY294002, the relative expression level of group 3 was significantly lower than that of group 2 (*P* \< 0.05); however, after activating of PI3K by IGF1, the relative expression level of group 4 was obviously higher than that of group 2 (*P* \< 0.05) (Fig. [6](#Fig6){ref-type="fig"}). Fig. 6The relative gene expression of PI3K, Akt, aggrecan, collagen I, and collagen II in hNP-MSCs cultured in normal condition (group 1), hypoxia and nutrition deficiency condition (group 2), LY294002 condition (group 3), and IGF-1 condition (group 4) evaluated by qRT-PCR. \**p* \< 0.05 indicated significant difference between groups
Discussion {#Sec19}
==========
The nucleus pulposus is the largest avascular tissue in the human body and nutrients diffuse across the cartilage endplate to reach disc cells and disc cells thus consume glucose and oxygen to generate energy to maintain cellular metabolism \[[@CR29]\]. The microenvironment becomes hypoxic and nutrition deficient with the processing of degeneration. Hypoxia and nutrition deficiency have been respectively shown to inhibit viability and proliferation of nucleus pulposus cells and NP stem cells. But whether hypoxia and nutrition deficiency synergy have similar effects on hNP-MSCs has, until now, remained unknown. This is the first study to investigate whether hypoxia and nutrition deficiency, mimicking the complex conditions in the degenerated disc, could result in detrimental effects on hNP-MSCs.
In our present study, we cultured the hNP-MSCs in the hypoxia and nutrition deficiency condition in vitro, which mimicked the disc degenerative microenvironment. Trial results showed that the hypoxia and nutrition deficiency inhibited proliferation of hNP-MSCs; what is more, the detrimental microenvironment increased the apoptosis rate and enhanced the caspase 3 activity. Our data suggested that hypoxia and nutrition deficiency could induce apoptosis and inhibit the proliferation of hNP-MSCs. Aggrecan, collagen II, and collagen I were used as representative of the IVD matrix. PCR results showed that hNP-MSCs cultured in hypoxia and nutrition deficiency had decreased expression of aggrecan, collagen type II and I, indicating that hypoxia and nutrition deficiency could lead to metabolic disturbance in hNP-MSCs. hNP-MSCs expressed stem cell-related genes (Oct4, Nanog, Jagged, and Notch1) \[[@CR30]\] and hypoxia and nutrition deficiency downregulated the expression levels of these genes. Previous study found that hypoxia inhibited the viability and proliferation of NP-MSCs, but promoted the chondrogenic differentiation of NPMSCs \[[@CR15]\]. These results were a little different with our study. The microenvironment in the IVD is complex and presents hypoxia, nutrition deficiency, and so on. Thus, biological behavior of cells in the hypoxia and nutrition deficiency conditions may differ with that in the hypoxia conditions. Hypoxia and nutrition deficiency could not only inhibit proliferation and matrix synthesis but also promote the apoptosis of hNP-MSCs, which may explain why these hNP-MSCs could not maintain the stability of IVD.
Dysregulation of various signaling pathways has been shown to be involved in the disc degeneration \[[@CR31]\]. PI3K/Akt signaling plays a critical role in cell proliferation, apoptosis, and differentiation during physiological and pathological conditions \[[@CR18], [@CR19]\]. PI3K/Akt pathway has been shown to protect nerve cells from cerebral infarction \[[@CR32]\] and protect cardiomyocytes from hypoxia-induced cell injury \[[@CR33]\]. More importantly, targeting this pathway has been shown to execute protective role in IVDD \[[@CR34]\]. Our study found that hypoxia and nutrition deficiency could upregulate the expression of PI3K/Akt, which indicated that PI3K/Akt pathway could be involved in the process under hypoxia and nutrition deficiency condition.
LY294002 is regarded as a specific inhibitor of PI3K \[[@CR27]\] and have been shown to reverse the protective of PI3K/Akt pathway \[[@CR35]\]. Thus, in the study, we further assessed the effect of blocking PI3K/Akt pathway by LY294002 on the biological activities of hNP-MSCs. Our study observed that treatment of hNP-MSCs with LY294002 under hypoxia and nutrition deficiency aggravated the decrease in cell proliferation and matrix gene expression. Blocking PI3K/Akt pathway by LY294002 could also increase cell apoptosis. Additionally, treatment of hNP-MSCs with LY294002 aggravated the decrease in the expression of stem cell-related genes.
Insulin-like growth factor 1(IGF-1) has a significant protective effects against IVD degeneration \[[@CR36]\]. Some studies found that IGF-1 dramatically increased collagen and aggrecan content in NP cells by activating the PI3K/Akt pathway \[[@CR37]\]. Meanwhile, IGF-1 can promote cell proliferation in human IVD cells through activation of this pathway \[[@CR22]\]. Thus, in our study, we further assessed the effect of activation of PI3K/Akt pathway by IGF-1 on the biological activities of hNP-MSCs. Our study observed that addition of IGF-1 to hNP-MSCs under hypoxia and nutrition deficiency could meliorate the decrease in cell proliferation and matrix gene expression, and also inhibit cell apoptosis. Additionally, treatment of hNP-MSCs with IGF-1 improves the decrease in the expression of stem cell-related genes.
All above results showed that inhibition of PI3K/Akt pathway could aggravate the harmful effect induced by hypoxia and nutrition deficiency condition while activation of PI3K/Akt pathway could meliorate the inhibiting effect, which demonstrated that PI3K/Akt signal pathway has protective effects on hNP-MSCs against hypoxia and nutrition deficiency. However, the downstream signaling pathway of PI3K/Akt still remains unclear. mTOR, a key protein of downstream signal pathway of PI3K/Akt \[[@CR38]\], acts as major signal of cell growth and is involved in proliferation, protein translation, and the anti-apoptosis process \[[@CR24], [@CR39]\]. Previous studies suggested that PI3K/Akt/mTOR signaling pathways was involved in protection against IL-1β \[[@CR25]\] or serum deprivation-induced IVD cells apoptosis \[[@CR40]\] and activation of mTOR suppressed activated caspase3. In our present study, PI3K/Akt was involved in the hypoxia and nutrition deficiency conditions and the results showed that activation of PI3K/Akt decreased caspase 3 activity, so we could make an assumption that PI3K/Akt/mTOR/caspase 3 signaling pathway may be involved in this process and needs further work to demonstrate it.
There are some limitations in our study. Firstly, the effect of hypoxia and nutrition deficiency on hNP-MSCs was performed in vitro and in 2D culture medium, so further studies need to be carried out in vivo and 3D culture system. Secondly, our study mainly investigated the relative expression of genes to indirectly reflect the changes appeared in hypoxia and nutrition deficiency microenvironment, lacking of protein evidence to directly reflect the changes. Finally, the precise mechanism of PI3K/Akt pathway in regulating hNP-MSCs in response to hypoxia and nutrition deficiency remains unknown.
Conclusions {#Sec20}
===========
PI3K/Akt signal pathway may have protective effects on human nucleus pulposus mesenchymal stem cells against hypoxia and nutrition deficiency. We could protect endogenous stem cells from nutrient deficiency-induced death by activating the pathway and provide seeds to repair IVD.
hNP-MSCs
: Human nucleus pulposus mesenchymal stem cells
IGF-1
: Insulin-like growth factor-1
ISCT
: International Society for cellular Therapy
IVD
: Intervertebral disc
MSCs
: Mesenchymal stem cells
NP
: Nucleus pulposus
**Publisher's Note**
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Da Sheng Tian and Jianjun Liu contributed equally to this work.
Not applicable.
TDS and LJJ contributed to study conception and design, and were major contributors in data collection and paper writing. CL performed data acquisition and interpretation. ZB contributed data analysis. JJH assisted in drafting paper and revised it. All authors read and approved the final manuscript.
The work was supported by grants from the National Nature Science Foundation of China (NO.61872407).
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
This research has been approved by the IRB of the authors' affiliated institutions. Informed consent was obtained from all individual participants included in the study.
Not applicable.
The authors declare that they have no competing interests.
|
{
"pile_set_name": "PubMed Central"
}
|
Besides the numerous memory T-cell subsets that arise following antigenic challenge, it is now clear that memory phenotype (MP) CD8 T cells can be found in all mice regardless of prior pathogen exposure. Many of these MP subsets, such as CD8 intraepithelial lymphocytes or innate CD8s, have a well-described development that depends on thymic signalling[@b1][@b2][@b3]. Much less is known about the development of another MP subset, CD44hi/CD122hi/CD49dlo CD8 cells, which is specific for nominal antigen but present in antigen-inexperienced mice. While we and our collaborators coined the term 'virtual memory\' (VM) to described this cellular subset, the presence of MP cells in the unprimed host had been long been known[@b4], but were largely assumed to represent cells that had undergone antigen-mediated expansion to microbiome- or food-associated antigens. As a result, the repertoire of these MP cells was not expected to possess any cells specific to nominal/novel antigens except as a result of cross-reactivity to related antigens. In our original description of VM cells, we demonstrated their development depended on homeostatic, not antigenic, cues in the environment, and that within their ranks were included T cells specific to nominal antigens[@b5]. Since then, we and others have shown that VM cells arise in the periphery[@b6] in a PLZF/IL-4/NKT cell-independent, but interleukin (IL)-15-dependent, manner[@b7], once developed they can respond vigorously to cytokines such as IL-4 (ref. [@b8]) and type I interferon (IFN)[@b9], and that they accumulate in the aged host[@b10]. As with memory cells in general, VM cells make IFNγ in response to stimulation with IL-12 and IL-18 (ref. [@b5]), and, similar to homeostatic proliferation (HP) memory T cells derived from a lymphopenic environment, are efficient in mediating a protective response against a cognate antigen-expressing pathogen[@b7][@b11]. Considering that VM cells make up 15--25% of the unprimed CD8 pool (in unmanipulated B6 mice), functional benefits commensurate with their prevalence in the repertoire have yet to be clarified.
The identification of VM cells contributes to the growing recognition that, much similar to the antigen-experienced repertoire of memory T cells, the antigen-inexperienced repertoire displays substantial heterogeneity. More recent evidence shows that the naive (CD44lo) CD8 pool in the periphery has different functionality dependent upon selection signals received in the thymus. Indeed, data have shown that T cells emerging from the thymus with higher affinity for self-antigens (expressing high levels of CD5 \[CD5hi\]) display a distinct advantage in becoming engaged in both homeostatic and antigen-mediated response when compared with their CD5lo counterparts[@b12][@b13]. Recent data examining the gene expression profile of CD5hi and CD5lo naive T cells suggests that CD5hi cells are transcriptionally poised to engage both proliferative and effector functions far more rapidly than CD5lo cells of the same specificity[@b14]. While these studies are informative as to the naive T-cell response to antigen in an inflammatory setting, the cues by which a naive phenotype T cell within the periphery integrates tonic and cytokine signals in a non-lymphopenic environment to become a member of the VM pool are still poorly defined. Furthermore, VM cells have thus far only been studied in mice, although putative human analogues have been suggested[@b15][@b16].
In the present work, we now provide conclusive evidence that VM cell development is a natural consequence of the heterogeneity of the naive CD8 T-cell pool. We show that VM cells are not only derived from cells with increased affinity for self-antigens but they also have higher affinity for their cognate antigens than naive phenotype T cells of the same specificity. As has been described for naive T cells during an antigen-specific response, naive CD8 T cells of the highest self-affinity (as measured by CD5) are the most likely ones to undergo IL-15-dependent HP that leads to VM development in the lymphoreplete host. RNA sequencing (RNAseq) comparing naive and VM subsets confirms recently published data on naive T cells[@b14] and further connects VM cell development to the CD5hi naive repertoire. Differences between CD5hi naive, VM and antigen-experienced memory cells in their expression of NKG2D and GzmB further predicted a role for VM cells in mediating bystander immune protection[@b17]. Surprisingly, in the absence of IL-15, VM expression of granzyme B and NKG2D was nearly ablated, as was their capacity to mediate bystander protection. These data give a new functional role for VM cells during exposure to pathogens independent of antigen specificity as well as a new role for IL-15 during the primary response. Finally, we show that a previously identified VM-like subset in humans traffics extensively to the liver, accumulates with age and collectively displays the phenotypic and functional traits identified in the mouse.
Results
=======
CD5 expression correlates with increase VM development
------------------------------------------------------
VM cells are CD8+CD44hi,CD49dlo, allowing their reliable separation from naive (CD44lo) and antigen-experienced memory cells (CD44hi,CD49dhi)[@b5] ([Fig. 1a](#f1){ref-type="fig"}). Other than the fact that these cells arise in the periphery and are dependent on IL-15, little else was known about VM development. A non-stochastic mechanism was initially suggested by the difference in CD5 expression between VM and naive cells. Expression of CD5 reflects a T cell\'s affinity for the self-ligands on which it was selected in the thymus[@b18]. Numerous labs have reported that naive T cells expressing higher CD5 show increased proliferation in response to lymphopenia[@b13][@b19] or antigenic stimulation[@b12][@b14] relative to their CD5lo counterparts. Analysis of bulk naive and VM CD8+ T cells within an unmanipulated B6 host for CD5 expression levels revealed a statistically significant increased expression of CD5 on all VM cells relative to naive ([Fig. 1b](#f1){ref-type="fig"}). Further, CD5^−/−^ hosts showed an increase in VM cells relative to wild-type (WT; [Supplementary Fig. 1](#S1){ref-type="supplementary-material"}), allowing us to rule out any role for direct CD5 signalling in VM cell formation. Considering T cells developing in the CD5^−/−^ host experience stronger T cell receptor (TCR) stimulation during selection[@b20], these data in conjunction with [Fig. 1b](#f1){ref-type="fig"} are consistent with the conclusion that the VM pool was likely derived from cells that had been selected with high affinity/avidity on self-antigens in the thymus.
We previously showed that VM cells are present even in TCR transgenic crossed onto Rag-deficient hosts (Rag^−/−^; ref. [@b5]), a result that supported the conclusion that VM cells develop independently of their encounter with cognate antigens. In comparing VM cells from WT B6 mice with T cells from either the gBT TCR transgenic (specific for a peptide from herpes simplex virus (HSV) glycoprotein B) or the F5 TCR transgenic (specific for a peptide from influenza nucleoprotein (NP)), each crossed to the Rag^−/−^ (gBTxRag, F5xRag), we noted a direct correlation between CD5 expression and the frequency of VM cells. T cells from the gBT and F5 TCR transgenics have higher and lower CD5 expression levels, respectively, as compared with the polyclonal repertoire in WT B6 ([Fig. 1c](#f1){ref-type="fig"})[@b19]. When plotted against the percentage of T cells in these hosts that are of VM phenotype, we obtained a near straight-line relationship ([Fig. 1d](#f1){ref-type="fig"}). This was further evidence that the affinity of a T cell for selecting ligands in the thymus (as gauged by CD5 expression) may be a predictor of a cell\'s propensity to convert to a VM phenotype.
VM derive from naive cells with high self-affinity
--------------------------------------------------
It was conceivable that VMs had increased CD5 levels as a result of cellular activation, as some data have suggested[@b20], giving the appearance of having increased affinity for self-antigen. To establish a causal relationship between CD5 expression and propensity for VM conversion, we isolated CD5hi and CD5loCD44lo polyclonal CD8+ T cells from unmanipulated B6 WT mice and adoptively transferred them into separate congenic, lymphoreplete WT mice. After 3 weeks, the donor cells were isolated and phenotyped. As anticipated, a significantly greater percentage of the transferred CD44loCD5hi cells had acquired a VM phenotype (CD44hi, CD49dlo) as compared with the transferred CD5lo cells ([Fig. 2a,b](#f2){ref-type="fig"}), in agreement with recent data from Jameson and colleagues[@b14]. The few VM cells derived from the CD5lo transfers maintained a lower expression of CD5 compared with CD5hi-derived VM converts ([Supplementary Fig. 2](#S1){ref-type="supplementary-material"}). Although we cannot discount the possibility of minor fluctuations in CD5 levels, these data indicate that CD5 expression is not markedly upregulated during the process of VM conversion. Regardless of minor changes in CD5 expression, these data are consistent with a causal relationship between the CD5 expression profile of a naive CD8+ T cell and its propensity to convert into VM cell within the periphery.
CD5hi naive T cells have also shown an increased response to antigenic challenge[@b12][@b14][@b21]. The preferential responsiveness of CD5hi over CD5lo T cells can be observed even between T cells with identical TCRs, effectively separating the responsiveness of the T cell from its affinity for cognate antigen[@b12][@b14]. Using T cells from the gBTxRag host, we examined whether the conversion of CD5hi T cells to VM could similarly occur irrespective of TCR affinity. Similar to the results with bulk naive T cells, transferred CD44loCD5hi gBT cells were significantly more likely to convert to VM phenotype compared with transferred CD44loCD5lo gBT cells ([Fig. 2c](#f2){ref-type="fig"}).
Previous studies have looked at the relationship between CD5 levels and affinity for foreign antigen, but none included VM cells[@b12][@b14][@b21]. As such, we used a tetramer staining and magnetic enrichment method[@b5] to compare naive and VM cells\' affinity for the vaccinia virus epitope, B8R. Using flow analysis software, we divided the tetramer mean fluorescent intensity (MFI) for each event against its own CD3 MFI. This method corrects for any reduced tetramer staining that is a result of reduced TCR levels and produces a quantification of relative affinity of the TCR for antigen[@b22]. VM cells demonstrated a significantly higher apparent affinity for cognate antigen as compared with naive phenotype T cells of the same specificity ([Fig. 2d](#f2){ref-type="fig"}). This was not an artefact of B8R-specific T cells, as this pattern held true for T cells specific for Ova, HSVgB and LCMV GP33 ([Fig. 2e](#f2){ref-type="fig"}).
These data supported a possible connection between a T cell\'s affinity for self-ligands and its affinity for cognate antigens. However, naive and memory T cells are known to interact with multimeric peptide:major histocompatibility complex (MHC) with very different avidities due to changes in cell membrane composition and not TCR affinity[@b23]. Again, analysing T cells from the TCR transgenics, we observed that VM cells with the same TCR as their naive counterparts maintained an increased tet:CD3 ratio ([Fig. 2f](#f2){ref-type="fig"}), consistent with their having an overall higher avidity for peptide:MHC. Although it is still conceivable that VM cells in the polyclonal repertoire also have an increased affinity for cognate antigens, this parameter is obscured by an increase in avidity endowed by their conversion to MP. Overall, these data continue to indicate that the VM pool is not derived stochastically from the entire naive T-cell repertoire. Rather, it is derived from the subset of naive T cells that were selected on higher affinity self-ligands, ultimately producing MP cells with increased binding to multimeric cognate peptide:MHC as well.
IL-15 availability dictates VM cell conversion
----------------------------------------------
Previous reports indicated that CD5 expression also predicts the responsiveness of CD8+ T cells to undergo HP in response to IL-2/15 under non-lymphopenic conditions[@b19]. This fit well with our data, as we and others have already established that VM cells form in the periphery as a result of IL-15-dependent HP[@b7][@b11] ([Fig. 3a](#f3){ref-type="fig"}). Combined, these data suggest that the size of the VM pool is dictated both by the affinity of the T-cell repertoire as well as the availability of IL-15. To test this prediction, we injected the IL-15^−/−^ host with IL-15/Rα complexes[@b24] (IL-15c). As shown previously, an IL-15^−/−^ host has no VM cells ([Fig. 3a](#f3){ref-type="fig"}). Four days post-IL-15c injection, there is a dose-dependent increase in VM cells ([Fig. 3b](#f3){ref-type="fig"}). There was also an inverse relationship between the CD5 geometric mean fluorescence intensity (gMFI) of the VM cells generated and the dose of IL-15c injected ([Fig. 3c](#f3){ref-type="fig"}). This was not because IL-15 stimulation caused reduced CD5 expression, as OT1 VM cells transferred into a naive recipient maintained the same levels of CD5 with or without subsequent IL-15c injection ([Supplementary Fig. 3](#S1){ref-type="supplementary-material"}). Thus, as more IL-15 becomes available, T cells with lower affinity for self-antigens convert to VM phenotype, such that the gMFI of CD5 on the VM cells becomes indistinguishable from that of the naive phenotype cells ([Fig. 3c](#f3){ref-type="fig"}). Collectively, these data ([Figs 1](#f1){ref-type="fig"}, [2](#f2){ref-type="fig"}, [3](#f3){ref-type="fig"}) indicate that the pool of VM phenotype cells arises as a result of naive T cells with the high affinity for self-antigens responding to limiting amounts of IL-15 trans-presented in the periphery.
RNA profile shows homeostatic and inflammatory sensitivity
----------------------------------------------------------
The transcription factor Eomes and its induction of the IL-2/15Rβ CD122 are essential for the CD8+ T-cell response to IL-15 (ref. [@b25]). These gene products are also elevated in VM cells relative to the total naive pool[@b7][@b11]. We observed a substantial difference between CD44loCD5lo and CD44loCD5hi T cells in their expression of these proteins, with essentially all Eomes and CD122 expression contained within the CD5hi\'s ([Fig. 4a,b](#f4){ref-type="fig"}). To better examine gene expression, we performed RNAseq by comparing the total RNA expression in CD5loCD44lo, CD5hiCD44lo and VM cells ([Supplementary Data 1](#S1){ref-type="supplementary-material"}). By comparing the CD5lo and CD5hi naive T-cell profiles, we found ∼600 genes that were differentially expressed ([Supplementary Data 2](#S1){ref-type="supplementary-material"}); a gene set that was in good agreement with recently published RNAseq data from Fulton *et al*.[@b14] Kyoto encyclopedia of genes and genomes (KEGG) analysis revealed 'cytokine/cytokine receptor interaction\' as the dominant cellular pathway associated with these genetic differences ([Table 1](#t1){ref-type="table"}). Gene ontology (GO) analysis identified 10 cellular functions as significantly increased ([Table 2](#t2){ref-type="table"}); the majority of which related to the regulation of life and death pathways. Curiously, other than the chemokine receptors *Cxcr3* and *Ifngr1*, genes that might predict increased responsiveness to the inflammatory milieu (for example, *Cxcr5*, *Il18rap*, *Il12rb1*, *Il18r1*, *Stat4* and *Ccr2*) only showed substantial changes in expression (\>15 transcripts per million) after differentiation to VM cells ([Table 1](#t1){ref-type="table"}). In contrast, the greatest increases were seen in cytokine genes associated with homeostatic cytokine responsiveness (*Il2rb*, *Eomes* and *Il4r*).
In comparing gene expression between CD5hi T cells and VM cells, substantially more genes (1909; [Supplementary Data 3](#S1){ref-type="supplementary-material"}) and gene ontology pathways ([Table 2](#t2){ref-type="table"}) showed differential expression, although 'cytokine signalling\' continued to be the major associated KEGG pathway ([Table 1](#t1){ref-type="table"}). In addition to even greater increases in the genes necessary for response to homeostatic cytokines IL-2/7/15 (*Il2rb*, *Il4r*, *Il7r*, *Eomes* and *Tbx21*), we also observed robust increases in genes involved in producing and/or sensing the pro-inflammatory milieu (*Ccl3/4/5*, *Il18r1*, *Il18rap*, *Il12r1*, *Stat4* and tumor necrosis factor *Tnf*), chemotaxis (*Ccr2/5*, *Cxcr3/5* and *Xcl1*), sensing costimulatory ligands (*Tnfsf14* and *Tnfrsf9*) and effector molecules associated with target recognition (*Nkg2d*) and cell killing (*Grzb*, *Ifng* and *Fasl*) ([Table 1](#t1){ref-type="table"}). These molecules indicate that the conversion of CD5hi naive T cells into VM cells results in their increased sensitivity to both homeostatic and inflammatory cues, consistent with the functional attributes of VM cells previously described[@b5][@b7][@b11].
VM cells effect bystander protection
------------------------------------
Besides showing strong upregulation of cytokine and chemokine signalling networks, the RNAseq data also showed that VM cells potently express molecules (IL-12R, IL-18R, IFNγ, GrzB and NKG2D) known to enhance innate-like effector functions, which have been connected to so-called 'bystander protection\' of memory CD8+ T cells[@b17]. Increased expression of IL-12R and IL-18R facilitate the VM cell production of IFNγ even in the absence of TCR stimulation[@b5], and GrzB and NKG2D expression can mediate antigen-independent killing of infected target cells by antigen-experienced memory T cells[@b17]. We therefore speculated that VM cells may be involved in bystander killing.
Given previously published work on listeria monocytogenes (LM) infection as a model for CD8 bystander killing[@b17], we used an LM-ova protection assay with the adoptive transfer of VM cells isolated from antigen-specific (OT1) or antigen-irrelevant (gBT-1) donors ([Fig. 5a](#f5){ref-type="fig"}). VM cells typically comprise anywhere from 15 to 25% of CD8+ T cells in an unmanipulated B6 host, at least some of which are specific for nominal antigens such as ovalbumin[@b5], the presence of which confounds attempts to measure a non-antigen-specific challenge. We therefore utilized two different hosts as VM transfer recipients (3KxRag and IL-15^−/−^), both chosen because of their lack of endogenous VM cells. First, we utilized a Rag^−/−^ crossed to the 3K CD4+ TCR transgenic host (3KxRag)[@b26]. This host has no VM cells because it lacks all CD8+ T cells and there is no VM equivalent in the CD4+ T-cell compartment[@b5][@b7]. Importantly, however, the presence of the irrelevant CD4+ T cells prevents canonical lymphopenia-induced T-cell expansion of any transferred T cells. VM cells were sorted from either OT1 or gBT-1 transgenic hosts, transferred into Rag3K mice, and 24 h later the recipients were challenged with LM-ova. Four days after challenge, the mice were killed and splenic bacterial counts were determined. As previously published[@b7][@b11], OTI cells protected against LM-ova challenge, mediating a substantial reduction in splenic bacterial CFUs. gBT cells also protected against LM-ova challenge to a magnitude that was, surprisingly, indistinguishable from that mediated by OTI cells ([Fig. 5b](#f5){ref-type="fig"}). Thus, VM cells are capable of mediating potent immunological protection against bacterial challenge even in the absence of their cognate antigen.
IL-15 is required for VM bystander protection
---------------------------------------------
In addition to the 3KxRag recipient above, we performed similar bystander protection experiments using IL-15^−/−^ mice as recipients because these hosts also lack all VM cells ([Fig. 3](#f3){ref-type="fig"}). In contrast to the results in the 3KxRag hosts, only the antigen-specific VM cell transfers (OTI) showed any protective effect in this system ([Fig. 5c](#f5){ref-type="fig"}). Although the IL-15^−/−^ host is also devoid of both natural killer (NK) and natural killer T (NKT) cells, previously published data showed that protection against LM challenge does not require either of these cell types[@b27][@b28][@b29]. The lack of bystander protection in the IL-15^−/−^ therefore indicated that IL-15 was necessary for VM cells to mediate this function. To examine the effect of IL-15 on T-cell effector functions under conditions of bystander killing/protection, we performed adoptive transfer of gBT VM cells into WT and IL-15^−/−^ hosts and challenged with LM-ova as before. On day 4 post infection, we performed an *in vivo* intracellular cytokine stain (ICCS) on transferred cells, looking at molecules crucial to bystander killing[@b5][@b17]. Most strikingly, gBT VM cells in the IL-15^−/−^ host showed substantially reduced GrzB, NKG2D and IFNγ expression ([Fig. 5d](#f5){ref-type="fig"}). These differences in GrzB and IFNγ were also observed in VM cells after overnight incubation with WT or IL-15^−/−^ splenocytes ([Fig. 5e](#f5){ref-type="fig"}). Reduced IFNγ and GrzB expression have previously been reported during the primary T-cell response in IL-15^−/−^ mice[@b30][@b31]. Our data now reveal that IL-15 is also necessary for maximal expression of these effector molecules in the presence or absence of inflammation. We conclude from these data that VM cells are able to mediate robust, non-cognate-antigen bystander protection against bacterial challenge. However, maintaining expression of the effector molecules necessary for this function is dependent on the continued presence of IL-15.
Trafficking of VM cells
-----------------------
The unique profile of chemokine receptor and effector molecule expression, ([Table 1](#t1){ref-type="table"}) and adhesion marker ([Table 3](#t3){ref-type="table"}) expression in VM cells revealed by the RNAseq data suggested unique tissue trafficking preferences of VM cells as compared with naive. We therefore determined the tissue tropism of VM cells, first focusing on the steady-state distribution of CD8 T cells in unmanipulated, adult (\>5 weeks) mice. Using intravenous injection of a labelled anti-CD8b antibody injection to discriminate against vascular and tissue-resident T cells ([Fig. 6a](#f6){ref-type="fig"})[@b32], we isolated various tissues and determined relative percentages of naive, VM and antigen-experienced memory cells by flow cytometry. Perhaps, most striking is the significant proportion of the liver-resident T cells that are VM cells, being generally upwards of 60--70%. Previous studies have described the presence of a considerable pool of memory T cells in the liver[@b33], largely assumed to be antigen experienced. Our data presented here indicate that these liver-associated memory cells are overwhelmingly VM cells. In contrast to the liver, the small intestine has essentially no VM cells ([Fig. 6b](#f6){ref-type="fig"}), consistent with their lack of CCR9 and α4β7 expression ([Table 3](#t3){ref-type="table"}). Adoptive transfer of VM cells showed a similar pattern of tissue tropism, suggesting that VM cells migrate to these sites as opposed to developing *in situ* ([Fig. 6c](#f6){ref-type="fig"}).
Tissue-specific protection of VM cells
--------------------------------------
These migratory preferences of VM cells were highly consistent with their protective capacity against LM, a pathogen that replicates primarily in the liver and spleen after a systemic challenge[@b6][@b7]. To assess whether VM cells were also highly protective against an infectious challenge initiated outside of their tissue trafficking patterns, we utilized a strain of LM-ova expressing modified internalin-A capable of intestinal epithelial cell invasion in the mouse[@b34][@b35]. Mice were orally challenged with the recombinant LM 1 day after adoptive transfer of naive or VM OTI T cells. Four days after challenge, we measured bacterial counts in both the small intestine and in the spleen ([Fig. 6d](#f6){ref-type="fig"}). The bacterial load in the small intestine was the same after either naive or VM T-cell transfers, indicating that, at least for the gut, VM cells did not display any increased protective capacity relative to naive T cells ([Fig. 6d](#f6){ref-type="fig"}). Bacterial loads in the spleen, however, recapitulated our previous protection assays, showing increased protection in mice transferred with VM cells over that of naive ([Fig. 6d](#f6){ref-type="fig"}). Thus, while VM cells did not provide increased gut-specific protection, they still mediated potent systemic protection against an infection instigated via a gut-specific route.
Phenotypic similarities in putative human VM cells
--------------------------------------------------
Thus far, VM cells have been documented only in mice, and it was not clear whether humans produced an equivalent subset. Establishing human equivalents of mouse cellular subsets is often complicated by the disparity in surface marker expression between the two species. Given its centrality in governing mouse VM development, we initially focused on IL-15 responsiveness as a possible identifier of human VM cells. In contrast to CD5 expression in the mouse, human T-cell expression of CD5 is progressively decreased during the process of memory T-cell differentiation. Further, the T cells with the lowest CD5 expression have the highest response to IL-15, express the highest levels of CD122 and are CD45RA^+^/CD27- (refs [@b36], [@b37]; [Fig. 7a](#f7){ref-type="fig"}). Although these markers place the putative VM cells within a subset previously considered terminally differentiated (terminal effector memory RA+), they bear considerable similarity to mouse VM cells in their higher expression of CD122, responsiveness to IL-15 and expression of both memory and naive phenotypic markers. Furthermore, when analysing Nur77 and Eomes expression in CD45RA+, RO- CD8+ T cells, there was a clear increase in both molecules as CD27 levels drop ([Fig. 7a](#f7){ref-type="fig"}). The Nur77 profile suggests a higher level of basal TCR interactions (higher self-affinity), whereas the Eomes expression suggests a history of exposure to IL-15.
A cell type with a very similar phenotype (CD45RA+CD122hiEomes+) was previously observed in fetal spleen and thymus, although the population was markedly reduced in cord blood and postnatal thymus[@b38]. Recently, the Eomes+ cells within the CD45RA+ CD8+ T cells were shown to be better identified by co-staining with pan-KIR and NKG2A antibodies[@b15]. The functional capacity of these CD45RA+Eomes+KIR+NKG2A+ T cells was essentially identical to mouse VM cells: coexpressing Tbet and producing IFNγ in response to IL-12/18 stimulation. Using these markers, we confirmed the observations ([Fig. 7b,c](#f7){ref-type="fig"}) that this subset represents a sizable fraction of total circulating adult (∼5%) and cord blood (∼1%) CD45RA+ T cells.
The presence of these cells in cord blood indicated that it was not necessary for them to be derived from antigen-experienced memory T cells, consistent with their designation as potential VM cells. Besides preferentially trafficking to the liver in mice ([Fig. 5](#f5){ref-type="fig"}), VM cells also accumulate with age[@b39]. We reasoned that if CD45RA+ KIR+NKG2A+ T cells were VM, then they might display similar trafficking patterns and age distributions in humans. We therefore compared the frequency of CD45RA+ KIR+NKG2A+Eomes+ T cells from normal human liver samples with that observed in the blood. In addition, we examined the frequency of these cells in non-diseased human spleen samples from patients ranging between 30 and 70 years old. As in the mouse, we found the frequency of these cells markedly elevated in the liver ([Fig. 7d](#f7){ref-type="fig"}). Furthermore, we saw a reasonable correlation between age and frequency of KIR+NKG2A+Eomes+ T cells, with older patients achieving frequencies of 20--30% of CD45RA+ T cells in the spleen ([Fig. 7e](#f7){ref-type="fig"}). Collectively, these data suggest that CD45RA+ KIR+NKG2A+Eomes+ T cells may represent the human equivalent of VM cells.
Discussion
==========
We present above three major findings regarding the development and functional properties of VM cells. First, we show that the development of VM cells is not a stochastic process, but rather proceeds as a result of T cells with high affinity for self-antigens responding to IL-15 in the periphery. The end result is an antigen-inexperienced memory T-cell repertoire skewed towards having a higher affinity for self-antigens and higher avidity for foreign antigens. The fact that all of these events proceed in the lymphoreplete host indicates that HP occurs in the WT environment in the absence of overt lymphopenia. Second, besides having higher affinity for self-antigens, VM cells also express adhesion, chemotactic and effector molecules that facilitate an 'innate-like\' response to inflammatory cues. The combination of these attributes make VM cells ideally suited for mediating both antigen-specific and bystander protective immunity even against infectious challenges that have never previously been encountered. Third, the collective properties of IL-15 sensitivity, apparent affinity for self-antigen, sensitivity to the inflammatory milieu and immediate effector functions are maintained in a unique population of cells that most likely represent the human VM equivalent. These human VM cells additionally reflect their mouse VM counterparts in their preferential trafficking to the liver and their increasing presence in the repertoire during ageing.
The predisposition of CD5hi naive T cells to proliferate in response to antigen or a lymphopenic environment over their CD5lo counterparts is well established[@b12][@b13][@b14]. It now seems clear that the central determinant favouring the response of CD5hi T cells over CD5lo T cells is not dictated by the TCR specificity or its affinity for cognate antigen. Recent experiments from the Jameson and Allen labs convincingly showed that T-cell responsiveness during antigen challenge was stratified by CD5 expression, even when using a fixed TCR[@b12][@b14]. Jameson and colleagues proposed that the sensitivity of CD5hi T cells to inflammatory cues facilitated their CD5-stratified response[@b14]. Our data presented here are in general agreement, but further suggest that sensitivity to IL-15 may be central to this increased responsiveness. This sensitivity may be related to more than just expression of receptor components. For example, Surh and colleagues suggested that an increased sensitivity to IL-2/15 was due to the incorporation of their receptors into lipid raft microdomains[@b40]. That said, as we have shown here ([Fig. 4a--c](#f4){ref-type="fig"}), there are indeed changes in the expression of gene products necessary for transmission of cytokine signals (*Il2rb*, *Eomes* and *Il4r*), which progressively increase along the CD5lo-memory differentiation continuum ([Table 1](#t1){ref-type="table"}). However, this is not true for the receptors for IL-7, IL-12 or the common gamma receptor, on which signalling through IL-2, -4, -7 and -15 depends. Thus, the increased efficiency of cytokine signalling in naive CD5hi and VM cells is likely influenced by both progressive expression of signalling components and a subcellular organization that is optimized for signal transduction.
These data ultimately support a model in which selection in the thymus on high-affinity ligands enacts a cellular programme that augments responsiveness to cytokines. For CD8+ T cells, a major outcome of the increased sensitivity to IL-15 is the conversion of a subset of CD5hi T cells into VM. Undergoing VM conversion results in the induction of a host of genes critical for sensing and producing inflammation, thereby facilitating the VM cell response against infectious challenges regardless of antigen specificity. This model lends increasing support for the importance of so-called 'inflammatory\' IL-15, produced during the earliest stages of bacterial or viral infection[@b41], as being central for mediating maximal function of both naive CD5hi (ref. [@b14]) and memory CD8+ T cells[@b42]. Thus, thymic selection not only produces T cells capable of response to foreign antigens but it also produces cells optimized for diversifying the peripheral T-cell repertoire (via cytokine responsiveness) regardless of antigen encounter.
Taken more broadly, our data support a new mechanistic understanding of the progressive differentiation from naive to memory T cells. Bevan and colleagues showed that, contradictory to conventional thought, memory T cells are actually less sensitive to antigen, displaying weaker TCR signalling than naive T cells at the same concentration of antigen[@b43]. However, memory cells are more sensitive to cytokine-mediated stimuli, engaging both proliferative and effector responses at far lower cytokine concentrations than their naive counterparts. Indeed, the efficiency of the memory cell response to homeostatic cytokines is such that they no longer require 'tonic\' stimulation via their TCR and are able to survive in MHC-deficient hosts[@b44]. This suggests that at each increasing step in the differentiation process (CD5lo/CD5hi/effector/memory/VM), a CD8+ T cell has increasingly wired its response to be based less on TCR-derived signals and more on signals derived from environmental/cytokine cues. The data we present here demonstrate this principle at each progressive stage of T-cell differentiation with CD5lo cells being relatively quiescent, CD5hi cells being responsive for VM conversion and VM cells being fully recruited into a protective response, all dependent on IL-15, and independent of overt TCR-mediated signals.
It has become increasingly clear that memory T cells possess substantial innate-like effector functions that are capable of mediating potent (in some instances nearly sterilizing) antigen non-specific protective immunity[@b17][@b45][@b46][@b47][@b48][@b49] As a result, memory T cells can influence not just the response to secondary antigen encounters, but to any response accompanied by inflammatory or homeostatic cytokine induction. As we show here ([Fig. 5](#f5){ref-type="fig"}), mice devoid of memory CD8+ T cells (3KxRag) are far less capable of managing their bacterial burden than mice with a population of antigen non-specific memory CD8+ T cells. Our data on VM cells perfectly recapitulate the bystander protective function observed for antigen-experienced memory T cells[@b17], and further add a necessary role for IL-15 in this process. While the combined action of IL-12/18/15 was known to induce CD8+ T-cell GrzB and NKG2D expression[@b17][@b48], our data reveal that IL-15 is required to sustain the expression of these molecules *in vivo* during a bacterial challenge ([Fig. 5](#f5){ref-type="fig"}).
Finally, we provide here convincing evidence for a population of T cells that likely represent a human equivalent of VM cells. While it was previously suggested that this subset represented human 'innate CD8\' T cells, the phenotypic and functional similarities between innate CD8 and VM cells (at least in the mouse) allow either conclusion for this population. Despite their phenotypic similarities, each cell is the culmination of very different differentiation processes; innate CD8s depend on PLZF, NKT cells and IL-4 in the thymus, and VM cells depend on IL-15 responsiveness in the periphery[@b2][@b6][@b7]. Although their development is well established, a location and function for innate CD8 cells outside of the thymus has been difficult to clarify, and little is known of what becomes of these cells within a lymphoreplete, WT host. In contrast, VM cells (i) represent the dominant subset of MP cells within the antigen-inexperienced B6 host[@b7]; (ii) are present even in the complete absence of all the factors (PLZF, IL-4 and NKT cells) required to produce innate CD8s (refs [@b6], [@b7]); (iii) do not require any prior antigen exposure evidenced by their presence in germ-free and TCRxRag^−/−^ hosts[@b5][@b7]; (iv) traffic extensively to the liver ([Fig. 6](#f6){ref-type="fig"}); (v) display a phenotype consistent with high affinity for self/foreign antigens ([Figs 1](#f1){ref-type="fig"} and [2](#f2){ref-type="fig"}); (vi) respond to IL-15 by maintaining expression of effector molecules that mediate bystander protective function ([Fig. 5](#f5){ref-type="fig"}); and (vii) increase substantially in their frequency over the course of ageing[@b39]. While admittedly lacking in definitive proof, the putative human VM equivalent shares many of these qualities in that they are found in cord blood (no/limited prior antigen exposure), demonstrate a phenotype consistent with an higher affinity for self ([Fig. 7](#f7){ref-type="fig"}) and responsiveness to IL-15 (ref. [@b37]), express effector molecules important for bystander protection[@b15], and are found in high frequency in the liver and increasing frequency during ageing ([Fig. 7](#f7){ref-type="fig"}). While the formation of these VM cells in human, as well as those in the mouse, do not appear to require antigenic experience, others have shown that a VM phenotype may be acquired from antigen-experienced memory cells following incubation with IL-2/7/15 (ref. [@b36]). Thus, it remains to be determined to what degree the peripheral pool of VM cells, in mouse or human, can be derived from antigen-experienced memory T cells or naive precursors. In addition, more work is needed to establish actual innate protective capacity of these cells in the human in the absence of antigenic stimulation.
Given the collective data discussed above, it seems reasonable to conclude that the host is generally pre-disposed towards favouring the formation of memory T cells, regardless of specificity. This is, perhaps, the context in which to best view the developmental and functional role of VM cells: by promoting the differentiation of the T cells with highest affinity to self-antigens into VM cells, the host acquires a population of effector cells that are preferentially and rapidly responsive to cytokines but less so to TCR stimulation, ultimately enhancing protective immunity while, possibly, reducing the likelihood of autoimmunity. The need for this dual benefit is most logically centred on the elderly where the decline in lymphocyte development means that the protection of the host to novel antigenic challenges must be increasingly dependent on the functional response of memory cells, with or without antigen specificity. The data we present here on the development, gene expression profile, trafficking and function of VM cells (in mouse and human) draw attention to this populous and versatile T-cell subset as a likely mediator of host protection to a spectrum of immunological challenges.
Methods
=======
Mice and reagents
-----------------
Female C57/Bl6 mice, 6--12 weeks old, were purchased from the National Cancer Institute. IL-15^−/−^, RAG^−/−^3k TCR tg, RAG^−/−^ gBT TCR tg, RAG^−/−^ F5 TCR tg and OTI TCR tg mice (all on the C57/Bl6 background) were bred in either the Biological Resource Center at the National Jewish Center for Health or the vivarium at the University of Colorado Anschutz Medical Center. CD5KO mice were a gift from the Raman lab at the University of Alabama. All mice used for experiments were between 8 weeks and 4 months old, and of either sex. Peptides were purchased from Peprotech and R&D Systems. Fluorochrome-conjugated antibodies against CD3, CD4, CD5, CD8a, CD8b, CD44 and CD45.1 (1:400); CD49d, CD122 and NKG2D (1:200); IFNg and Gzmb (1:50) were purchased from Biolegend or eBioscience. MHC class I tetramers against B8R \[TSYKFESV\], LCMV \[KAVYNFATC\], OVA \[SIINFEKL\] and HSV \[SSIEFARL\] (1:200) were either purchased from Beckman Coulter or produced as previously described[@b50][@b51]. All strains of Listeria monocytogenes used are of the 10403s parental strain. The recombinant Listeria monocytogenes 10403s expressing OVA (Lm-OVA) was a gift from the Bevan lab at the University of Washington. The recombinant Listeria monocytogenes 10403s internalin-A mutant strain was a gift from the LeFrancois lab at the University of Connecticut. All mouse protocols were approved by the Institutional Animal Care and Use Committees at either National Jewish Health or the University of Colorado.
Tissue perfusion and processing
-------------------------------
For distinguishing between tissue and vascular CD8s, anti-CD8b (0.6 μg) was mixed with 50 μl heparin and 150 μl Hanks\' balanced salt solution (HBSS) and injected intravenously Ten minutes later, mice were killed and perfused with PBS through the vena cava until the liver and lungs appeared completely perfused (enlarged and whitish). All tissues were then processed as previously described[@b51][@b52].
Cell sorting and transfers
--------------------------
Mouse spleens were dissociated as previously described[@b51]. Splenocytes were then negatively selected for CD8s using the StemCell CD8 selection kit (cat. no. 19753A). The negative fraction was then stained with CD5 (PE), CD44 (PerCP-Cy5.5) and CD49d (AlexaFluor 647). Cells were sorted on a BD FACSAria into 'CD5lo\' (CD44lo, lowest 20% expressers of CD5), 'CD5hi\' (CD44lo, highest 20% expressers of CD5) and 'VM\' (CD44hi, CD49dlo) populations. Cells were then resuspended in PBS and injected via tail vein.
Tetramer/CD3 stain and calculation
----------------------------------
Mouse splenocytes were stained for tetramer and pulled down as described previously[@b7]. Resulting fractions were then stained for various surface molecules and run on a Dako CyAn flow cytometer. Data were analysed on FlowJo 7.6.5. Tetramer:CD3 ratios were obtained by creating the ratio equation under 'derived parameters\', applying the ratio to the appropriate population and subsequently measuring the population for the gMFI of the derived Tet:CD3 ratio.
Generation of IL-15 complexes
-----------------------------
Resuspended IL-15 and IL-15Ra were incubated at a ratio of 2:9 (for example, for 1 μg IL-15c, 0.18 μg IL-15 and 0.82 μg IL-15Ra) for 30 min at 37°. Resulting complexes were then resuspended in PBS and injected via tail vein. Four days later, mice were killed and splenocytes were analysed for percentage of VM cells.
LM protection assay
-------------------
Splenocytes were sorted into the appropriate population (as described above), and 2.0 × 10^5^ VM cells were injected into recipient mice via tail vein injection. Twenty-four hours later, mice received an injection of Listeria monocytogenes via tail vein (1.0 × 10^5^ colony forming units (CFU) per mouse for WT and 2.5 × 10^3^ CFU per mouse for IL-15^−/−^). Four days later, mice were killed and splenic CFUs were determined as described previously[@b50].
Oral Lm infection
-----------------
Splenocytes were sorted into the appropriate population (as described above), and 2.0 × 10^5^ VM cells were injected into recipient mice via tail vein injection. The next day, mice were deprived of food and water for 4 h. They were then isolated into separate cages and given a piece of bread containing 2 × 10^10^ CFU internalin-A mutant Listeria monocytogenes. Mice remained isolated until entire piece of bread was consumed, and subsequently returned to normal housing. Four days later, splenic CFUs were determined as described previously[@b50]. Small intestines were removed from the mice that were killed, bisected and scraped to clean away mucous. The intestines were then homogenized and CFUs were determined in the same way as the spleen.
*In vivo* ICCS
--------------
Mice were injected with 250 μg of Brefeldin A/mouse (in 500 μl of PBS) 1.5 h before killing. Spleens were then processed and stained as previously described, with the caveat that all steps were completed with buffers containing Brefeldin A.
*In vitro* cytokine stimulation
-------------------------------
WT CD45.1 splenocytes were negatively selected for CD8 T cells using the StemCell enrichment kit (see above). CD45.1 T cells (1 × 10^6^) were then incubated with 1 × 10^7^ splenocytes from either WT CD45.2 or IL-15^−/−^ (CD45.2) mice in 50 U ml^−1^ IL-2±50 ng ml^−1^ of both IL-12/IL-18. Four hours before collection for staining, Brefeldin A was added to the cultures. All CD45.1+ VM cells were analysed by FACS for IFNg and GrzB.
RNA isolation
-------------
WT C57/Bl6 mouse splenocytes were negatively selected for CD8s using the StemCell CD8 selection kit (cat. no. 19753A). The negative fraction was stained with CD5 (PE), CD44 (PerCP-Cy5.5), CD49d (AlexaFluor 647) and CD8 (APC). Cells were sorted on a BD FACSAria through a CD8+ gate, then sorted into 'CD5lo\' (CD44lo, lowest 20% expressers of CD5), 'CD5hi\' (CD44lo, highest 20% expressers of CD5) and 'VM\' (CD44hi, CD49dlo). Resulting populations were processed for total mRNA using a Qiagen RNeasy plus kit (cat. no. 74134).
Next-generation sequencing of the transcriptome (RNAseq)
--------------------------------------------------------
The isolated total RNA from the splenocytes was processed for next-generation sequencing library construction as developed in the NJH Genomics Facility for analysis with a Life Technologies (Carlsbad, CA, USA) Ion Proton next-generation sequencing platform. A modified Kapa Biosystems (Wilmington, MA, USA) KAPA Stranded mRNA-Seq kit for whole-transcriptome libraries was used to primarily target all poly-A RNA. Briefly, library construction proceeded from isolation of total RNA species, followed by mRNA (poly-A) isolation, first and second strand cDNA synthesis, Life Technologies Ion adaptor ligation, amplification and bead templating. Once validated, the libraries were sequenced as barcoded-pooled samples on a P1 Ion Proton chip. Sequence reads of 30 or more nucleotides were mapped to the mm10 (GRCm38) assembly of the mouse genome with the STAR RNA-Seq aligner version 2.4.1d (ref. [@b53]) using gene annotation data from Ensembl version 78 (ref. [@b54]). For each gene in the Ensembl 71 annotation, reads were counted using the HTSeq software version 0.6.0 (ref. [@b55]). Statistical comparisons of the gene expression between the cell types were performed with the DESeq2 package version 1.8.1 (ref. [@b56]) and for the R statistical software version 3.2.0 (ref. [@b57]). All sequencing data are stored in the GEO repository and is accessible under the accession number [GSE78247](GSE78247).
Human reagents
--------------
Fluorochrome-conjugated antibodies against CD3, CD4, CD5, CD8a, CD27, CD45RA, CD45RO, CD122, Nur77, Eomes, NKG2A, NKG2D, KIR2D and KIR3DL1 were purchased from eBioscience, Biolegend or Miltenyi Biotec.
Human cell sources and methods
------------------------------
Human peripheral blood mononuclear cells (PBMCs) were obtained from apheresis LRS chambers that were left over after blood donation. Adult PBMCs were obtained from nine patients, six male and three female, ranging in age from 29 to72 (mean 50) years. Samples were washed with PBS several times and aliquoted before staining. Cord blood samples were obtained from the University of Colorado Cord Blood bank. Samples were diluted 1:1 and run on a Ficoll gradient to isolate the mononuclear layer, which was then isolated and washed. Human spleen and liver samples were obtained from the surgical banks of Drs McCarter and Rosen, respectively. Spleen samples were obtained from surgeries unrelated to haematological malignancies (*n*=7 benign pancreatic mass and *n*=2 non-functioning neuroendocrine tumour), and were determined by a pathologist to be histologically 'normal\'. Healthy liver biopsies were obtained from five normal patients, four males and one female, age ranging from 23 to 56 (mean 40) years. Samples (stored in single-cell suspensions) were thawed, plated, allowed to equilibrate over a few hours in media, washed and stained. All human samples were de-identified by the collecting body before our analysis.
Additional information
======================
**How to cite this article:** White, J. T. *et al*. Virtual memory T cells develop and mediate bystander protective immunity in an IL-15-dependent manner. *Nat. Commun.* 7:11291 doi: 10.1038/ncomms11291 (2016).
Supplementary Material {#S1}
======================
###### Supplementary Figures
Supplementary Figures 1-3
###### Supplementary Data 1
A master list of all genes identified by RNAseq of sorted, polyclonal CD5lo, CD5hi and VM cells from unmaniupulated B6 mice.
###### Supplementary Data 2
A comparison of gene expression (RNAseq) between CD5lo and CD5hi naive CD8+ T cells from unmaniupulated B6 mice.
###### Supplementary Data 3
A comparison of gene expression (RNAseq) between naive CD5hi and VM CD8+ T cells from unmaniupulated B6 mice.
We thank G. Hedlund for assisting with FACSAria sorting and K. Walton for creating libraries and running RNAseq.
**Author contributions** J.T.W. and R.M.K. designed experiments and co-wrote the manuscript. J.T.W. performed all experiments and T.D. mapped the RNAseq reads and ran DESeq2 on the expression data. The remaining authors provided technical and material support.
![VM cells resemble cells that have undergone HP in both integrin expression and affinity.\
(**a**) Flow cytometry on splenocytes isolated from unmanipulated WT mice. Gating on CD44hi/CD122hi CD8 T cells (left, percentage of CD8 above gate). Gated population visualized by CD44/CD49d (right, percentage of VM cells \[CD44hi, CD49dlo\] above population). (**b**) Naive (CD44lo) and VM (CD44hi, CD49dlo) CD8 T cells from the same mouse stained for CD5. Representative histogram overlay of the two populations (left), gMFI comparison (right; \*\**P*\<0.01, paired *t*-test, representative of two experiments of *n*=4 per group and *n*=3 per group). (**c**,**d**) Naive (CD44lo) CD8 T-cell populations from polyclonal and WT mice were stained for CD5. (**c**) Representative flow histogram overlay of naive CD5 distributions for WT (blue), F5xRag^−/−^ (red) and gBTxRag^−/−^ (orange) (one mouse per histogram; representative of three experiments). (**d**) gMFI of naive populations graphed against per cent VM cells found in each mouse (linear regression, *r*^2^: 0.97, *P*\<0.0001; representative of two experiments with at least *n*=2 per group).](ncomms11291-f1){#f1}
![CD5 expression is a predictor of a naive cell\'s propensity to become VM.\
(**a**) Naive (CD44lo), CD5hi (the 20% highest CD5 expressers) or CD5lo (the 20% lowest CD5 expressers) CD8 T cells were sorted out of unmanipulated WT mice, and 4.0 × 10^5^ of each population were adoptively transferred into congenic WT recipients, which were left undisturbed for 3 weeks (left). Recipients were then killed and splenocytes were analysed for transferred cells changing phenotype (right, per cent VM in quadrant). (**b**,**c**) Comparisons between WT mice receiving WT (**b**) or gBTxRag^−/−^ (**c**) transferred cells (\**P*\<0.05, \*\*\**P*\<0.001, *t*-test; *n*=3 per group). (**d**,**e**) Tetramer staining and pulldown of CD8s specific for foreign cognate antigen in WT mice. Representative contour flow plot of CD44/tetramer staining for B8R tetramer (left), overlay of pseudocolour dot plots for CD3 and tetramer staining in naive (blue) and VM (red) CD8 T cells. Comparison within mice of tetramer:CD3 gMFI staining on individual tetramer+ (Tet+) cells (**d**, B8R \[TSYKFESV\]; **e**, HSV \[SSIEFARL\], OVA \[SIINFEKL\], LCMV \[KAVYNFATC\]; \**P*\<0.05, \*\**P*\<0.01, paired *t*-test, representative of 2--4 experiments with *n*=3 per group). (**f**) PBMCs from gBT-1 and OTI TCR transgenic mice were stained with the HSV and OVA MHCI tetramers, respectively, as described above. VM and naive populations were compared within mice for Tet:CD3 ratios (\*\**P*\<0.01, \*\*\**P*\<0.001, paired *t*-test; *n*=5 per group, error bars represent s.e.m.).](ncomms11291-f2){#f2}
{#f3}
{#f4}
{#f5}
{#f6}
{#f7}
###### RNAseq data for cytokine-cytokine receptor KEGG pathway and relevant genes across VM and naive CD5lo/hi.
------------------------------
{#i2}
------------------------------
RNAseq, RNA sequencing; TPM, transcripts per million; VM, virtual memory.
Darker shading indicates increased absolute gene expression.
###### GO annotations associated with genes upregulated in both CD5 compared with CD5lo and VM compared with CD5hi.
**Gene ontology annotation: CD5lo\<CD5hi** **Bayes factor**
------------------------------------------------------------------------- ------------------
GO:0050877 \[4\]: neurophysiological process 20.87
GO:0009581 \[5\]: detection of external stimulus 18.05
GO:0007600 \[5\]: sensory perception 17.85
GO:0009605 \[4\]: response to external stimulus 11.08
GO:0042981 \[6\]: regulation of apoptosis 6.76
GO:0043067 \[5\]: regulation of programmed cell death 6.59
GO:0007049 \[5\]: cell cycle 6.3
GO:0008283 \[4\]: cell proliferation 6.25
GO:0006915 \[6\]: apoptosis 6.25
GO:0012501 \[5\]: programmed cell death 6.02
**GO annotation: CD5hi\<VM** **Bayes factor**
GO:0050877 \[4\]: neurophysiological process 47.67
GO:0009581 \[5\]: detection of external stimulus 43.54
GO:0007600 \[5\]: sensory perception 43.07
GO:0007186 \[6\]: G-protein-coupled receptor protein signalling pathway 32.8
GO:0009605 \[4\]: response to external stimulus 24.63
GO:0007242 \[5\]: intracellular signalling cascade 13.97
GO:0008219 \[4\]: cell death 11.57
GO:0016265 \[3\]: death 11.07
GO:0006793 \[5\]: phosphorus metabolism 10.35
GO:0006796 \[6\]: phosphate metabolism 10.35
GO:0051244 \[4\]: regulation of cellular physiological process 10.17
GO:0043170 \[4\]: macromolecule metabolism 9.35
GO:0006468 \[8\]: protein amino-acid phosphorylation 9.12
GO:0044260 \[5\]: cellular macromolecule metabolism 8.84
GO:0006464 \[7\]: protein modification 8.67
GO:0016310 \[7\]: phosphorylation 8.23
GO:0006915 \[6\]: apoptosis 8.08
GO:0012501 \[5\]: programmed cell death 7.68
GO:0050874 \[3\]: organismal physiological process 7.29
GO:0007166 \[5\]: cell surface receptor linked signal transduction 7.28
GO:0050794 \[3\]: regulation of cellular process 6.58
GO:0044267 \[6\]: cellular protein metabolism 6.32
GO:0042981 \[6\]: regulation of apoptosis 6.09
GO, gene ontology; VM, visual memory.
###### List of integrins and adhesion molecules showing significant differences between the VM and naive pools.
------------------------------
{#i3}
------------------------------
TPM, transcripts per million; VM, virtual memory.
Red values indicate increasing gene expression.
|
{
"pile_set_name": "PubMed Central"
}
|
Background
==========
In a previous descriptive study of international variation in the incidence of adult primary brain cancer, we found that: 1) White populations had the highest incidence rates, North American Blacks had intermediate rates, and populations of eastern and southeastern Asian origin had the lowest rates; 2) incidence rates increased less steeply with age in the latter populations; 3) the male/female incidence rate ratio was between 1.4 and 1.5 among populations throughout the world; and 4) the male/female rate ratio was higher for peri- and post-menopausal ages than for pre-menopausal ages \[[@B1]\]. The lower incidence rate among non-Whites and the male excess had previously been noted by other investigators \[[@B2]-[@B6]\].
Although we examined brain cancer as a whole (due to limitations of the data we were unable to examine variation by morphologic type), our results were most likely driven by variation in the incidence of glioma, the predominant morphologic type of adult primary malignant brain tumors \[[@B2],[@B6]\]. However, because gliomas themselves are heterogeneous, we now hypothesize that race/ethnic group, sex, and/or age variation in adult glioma incidence differs by morphologic subtype. To test these hypotheses, we utilized data from the population-based cancer registries of the National Cancer Institute\'s Surveillance, Epidemiology, and End Results (SEER) Program. We also tested the hypothesis that calendar period trends in adult glioma incidence varied by subtype.
The three main categories of adult glioma according to traditional pathological classification are astrocytoma, oligodendroglioma, and mixed oligoastrocytoma \[[@B7]-[@B9]\]. Each of these categories is further sub-classified by grade (grade II, grade III, or grade IV, the latter grade reserved for glioblastoma \[GBM\], which is grade IV astrocytoma) \[[@B8],[@B9]\]. Secondary GBM develops through progression from a lower grade astrocytoma, whereas primary GBM appears to arise *de novo*, with no evidence of a lower-grade precursor \[[@B9]\].
There is considerable variability in the diagnosis of the traditional glioma categories and grades among pathologists, across geographic regions, and over time \[[@B10]\]. Nevertheless, epidemiologists and neuropathologists have reached a consensus that a pathology report of GBM is likely to be valid (although a small proportion of GBMs may be incorrectly classified as grade III \[anaplastic\] astrocytomas), whereas valid classification of non-GBM subtypes requires centralized pathologic review \[[@B10]\]. Thus, adult gliomas can validly be categorized broadly as GBM versus non-GBM based on pathology report diagnoses (which are utilized by SEER cancer registries), but non-GBM cannot be sub-divided further without compromising validity. Primary GBM, which constitute 90-95% of GBM \[[@B9]\], differ from non-GBM with respect to DNA copy number \[[@B11]\], gene expression \[[@B11]\], DNA methylation \[[@B12]-[@B14]\], and isocitrate dehydrogenase (IDH) 1 or 2 gene mutation status \[[@B11]-[@B16]\], providing a molecular rationale for the two broad glioma subtypes of GBM and non-GBM.
Methods
=======
Study population
----------------
We utilized publicly-available data from the SEER population-based cancer registries (SEER Research Data \[1973-2007\], released April 2010, based on the November 2009 submission) and associated SEER U.S. population data (SEER Program Populations \[1969-2007\] <http://www.seer.cancer.gov/popdata>, released November 2009) \[[@B17]\]. We included in our analyses adult glioma cases diagnosed between 1992 and 2007. Cases diagnosed between 1992 and 1999 were from the SEER 13 Registries Database (Alaska Native, Atlanta, Connecticut, Detroit, Hawaii, Iowa, Los Angeles, New Mexico, Rural Georgia, San Francisco-Oakland, San Jose-Monterey, Seattle-Puget Sound, and Utah). Cases diagnosed between 2000 and 2007 were from the SEER 17 Registries Database (SEER 13 plus Greater California, Kentucky, Louisiana, and New Jersey). The SEER 13 registries cover about 14% and the SEER 17 registries cover about 26% of the total U.S. population.
We restricted our analyses to adult glioma because different morphologic types of glioma predominate in adults compared to children \[[@B2],[@B5],[@B6]\]. We defined glioma as a malignant brain neoplasm (International Classification of Diseases for Oncology, third edition \[ICD-O-3\] topography codes C710-C719 and behavior code 3) with an ICD-O-3 morphology code of 9380 (glioma, malignant; glioma, not otherwise specified \[NOS\]), 9382 (mixed glioma), 9400 (astrocytoma, NOS), 9401 (astrocytoma, anaplastic), 9410 (protoplasmic astrocytoma), 9411 (gemistocytic astrocytoma), 9420 (fibrillary astrocytoma), 9440 (glioblastoma, NOS), 9441 (giant cell glioblastoma), 9442 (gliosarcoma), 9450 (oligodendroglioma, NOS), 9451 (oligodendroglioma, anaplastic), or 9460 (oligodendroblastoma) \[[@B18]\]. We defined GBM as ICD-O-3 morphology codes 9440, 9441, and 9442. We defined non-GBM as all other codes except 9380 (glioma, malignant; glioma, NOS).
For valid classification of adult glioma as GBM versus non-GBM, we made two necessary exclusions. First, we excluded cases with the non-specific code 9380 because such cases could not be classified as GBM versus non-GBM. Second, we excluded cases that were not microscopically confirmed because valid distinction between GBM and non-GBM requires microscopic examination of tumor tissue and cannot be accomplished by imaging alone \[[@B19]-[@B21]\].
Given these exclusions, we sought to minimize selection bias due to differences in the proportion of cases excluded according to age, calendar period, sex, or race/ethnic group. First, we required that the proportion of cases that were both microscopically confirmed and had a specific morphology code (a code other than 9380) be greater than 90% in each included 5-year age group, resulting in restriction of the age range to 30-69 years (Table [1](#T1){ref-type="table"}). Overall, in this age range 92.3% of cases were both microscopically confirmed and had a specific morphology code. Then, we examined this proportion within this age range according to period, sex, and race/ethnic group and found that the proportion did not vary meaningfully by these characteristics, although it was slightly lower among Blacks (89.8%) and Asians/Pacific Islanders (89.9%) than among other race/ethnic groups (Table [1](#T1){ref-type="table"}). Overall, in the age range 30-69 years, 4.6% of cases were excluded due to having morphology code 9380 and 3.1% were excluded due to lack of microscopic confirmation (3.8% of GBM cases and 2.4% of non-GBM cases). We included a total of 15,088 GBM cases and 9,252 non-GBM cases in our analyses.
######
Number and percent of glioma cases that were both microscopically confirmed and had specific ICD-O-3 morphology codes (not 9380), by demographic characteristic, SEER, 1992-2007
Characteristic Glioma Cases
----------------------------------------- -------------- ------------ -----------
**Age (years)**
20-24 812 699 86.1%
25-29 1,308 1,171 89.5%
**30-34** **1,709** **1,553** **90.9%**
**35-39** **2,194** **2,038** **92.9%**
**40-44** **2,730** **2,522** **92.4%**
**45-49** **3,224** **3,003** **93.1%**
**50-54** **3,879** **3,607** **93.0%**
**55-59** **4,133** **3,835** **92.8%**
**60-64** **4,253** **3,917** **92.1%**
**65-69** **4,252** **3,865** **90.9%**
70-74 4,530 3,948 87.2%
75-79 4,081 3,353 82.2%
80-84 2,579 1,793 69.5%
85+ 1,448 663 45.8%
**Total, age 30-69 years** **26,374** **24,340** **92.3%**
**Period (age 30-69 years)**
1992-1995 4,121 3,813 92.5%
1996-1999 4,345 4,006 92.2%
2000-2003 8,606 7,916 92.0%
2004-2007 9,302 8,605 92.5%
**Sex (age 30-69 years)**
Male 15,607 14,414 92.4%
Female 10,767 9,926 92.2%
**Race/ethnic group (age 30-69 years)**
Non-Hispanic White 20,860 19,354 92.8%
Hispanic White 2,735 2,487 90.9%
Black 1,444 1,297 89.8%
Asian/Pacific Islander 1,222 1,098 89.9%
American Indian/Alaskan Native
Native 113 104 92.0%
^a^GBM (codes 9440, 9441, 9442), non-GBM (codes 9382, 9400, 9401, 9410, 9411, 9420, 9450, 9451, 9460), and non-specific glioma, malignant (9380)
Data analysis and statistical methods
-------------------------------------
We downloaded sex-, age-, race-, Hispanic-ethnicity-, and calendar-year-specific numbers of cases and person-years at risk from the SEER website. We classified age into eight five-year age groups ranging from 30-34 to 65-69. We divided calendar-years into four four-year periods (1992-1995, 1996-1999, 2000-2003, and 2004-2007). Our race/ethnic group categories were non-Hispanic White, Hispanic White, Black, Asian/Pacific Islander, and American Indian/Alaskan Native.
For each of our two glioma subtypes, we calculated sex-and age-specific incidence rates (for each race/ethnicity group) by dividing the number of cases by the number of person-years at risk. We calculated sex-specific, age-standardized incidence rates (ASRs) and 95% confidence intervals (CIs) (for each race/ethnicity group) by direct standardization of the age-specific incidence rates to the 2000 U.S. Standard Population (Census P25-1130). We used Poisson regression to calculate adjusted rate ratios (RRs) and 95% CIs. We modeled age as a categorical variable or as the natural logarithm of age (log~e~(age)), where age was defined as the midpoint of the five-year age group. We modeled calendar period as a categorical or interval variable, the latter being used to calculate the p-value for period trend. Results for models with and without adjustment for cancer registry did not meaningfully differ; we only present the unadjusted results. To determine a p-value for heterogeneity, we entered the appropriate cross-product term into the Poisson model and conducted a likelihood ratio test for its addition, with the appropriate degrees of freedom. To further explore the relationship between glioma subtype incidence and age, we calculated adjusted RRs for 5-year age groups using Poisson regression and used the calculated RRs to perform weighted linear least squares regression of log~10~(RR) versus log~10~(age). We also performed weighted linear least squares regression of log~10~(age-specific incidence rate) versus log~10~(age). We performed Poisson regressions using Proc Genmod of SAS version 9.1; statistical tests were two-sided with α = 0.05.
Results
=======
Race/ethnic group
-----------------
In Table [2](#T2){ref-type="table"}, we tested the hypothesis that race/ethnic group variation in incidence differs according to glioma subtype. The reference group for the RRs was non-Hispanic Whites. Except for American Indians/Alaskan Natives, 95% CIs for ASRs and RRs were relatively tight due to large numbers of cases. For each subtype, ASRs and RRs varied by race/ethnic group similarly for males and females. For GBM, rates varied about three-to-four-fold, with highest rates among non-Hispanic Whites, followed by Hispanic Whites, Blacks, Asian/Pacific Islanders, and American Indians/Alaskan Natives. As with GBM, rates for non-GBM were highest among non-Hispanic Whites, followed by Hispanic Whites, with rates among Blacks, Asian/Pacific Islanders, and American Indians/Alaskan Natives each about 40% of the rates among non-Hispanic Whites. The main differences in race/ethnic variation between GBM and non-GBM were that Blacks had lower RRs, and Asian/Pacific Islanders and American Indians/Alaskan Natives had higher RRs, for non-GBM than for GBM. Overall, race/ethnic group variation was less for non-GBM than for GBM. The difference in race/ethnic variation between GBM and non-GBM was highly significant among both males and females (p-value for heterogeneity \< 0.0001 in each sex).
######
Sex- specific, age-standardized incidence rates, age- and period-adjusted rate ratios, and age- and period-adjusted male/female rate ratios, by race/ethnic group and glioma subtype, age 30 to 69 years, SEER, 1992-2007
Glioma
------------- -------------------------------- ------- ------------------- ------------------- ------- ------------------- ------------------- ---------------------
**GBM**
Non-Hispanic White 7,458 5.20 (5.06, 5.32) 1.00 (reference) 4,745 3.16 (3.07, 3.25) 1.00 (reference) 1.64 (1.58, 1.70)
Hispanic White 769 3.08 (2.86, 3.31) 0.58 (0.54, 0.62) 625 2.34 (2.16, 2.53) 0.74 (0.68, 0.80) 1.31 (1.18, 1.45)
Black 470 2.51 (2.28, 2.74) 0.48 (0.44, 0.53) 352 1.56 (1.40, 1.73) 0.49 (0.44, 0.55) 1.61 (1.40, 1.85)
Asian/Pacific Islander 372 1.94 (1.75, 2.14) 0.38 (0.34, 0.42) 245 1.12 (0.98, 1.26) 0.35 (0.31, 0.40) 1.74 (1.48, 2.05)
American Indian/Alaskan Native 29 1.26 (0.79, 1.73) 0.25 (0.17, 0.36) 23 0.95 (0.56, 1.35) 0.30 (0.20, 0.45) 1.36 (0.78, 2.34)
Total 9,098 5,990 1.61^e^(1.56, 1.66)
**Non-GBM**
Non-Hispanic White 4,175 3.01 (2.91, 3.10) 1.00 (reference) 2,976 2.12 (2.04, 2.19) 1.00 (reference) 1.42 (1.36, 1.49)
Hispanic White 577 1.85 (1.69, 2.00) 0.59 (0.54, 0.65) 516 1.64 (1.49, 1.78) 0.76 (0.70, 0.84) 1.10 (0.98, 1.24)
Black 269 1.30 (1.14, 1.45) 0.43 (0.38, 0.49) 206 0.85 (0.74, 0.97) 0.40 (0.35, 0.46) 1.52 (1.26, 1.82)
Asian/Pacific Islander 264 1.29 (1.13, 1.45) 0.43 (0.38, 0.49) 217 0.95 (0.82, 1.08) 0.45 (0.39, 0.52) 1.35 (1.13, 1.62)
American Indian/Alaskan Native 31 1.24 (0.79, 1.69) 0.40 (0.28, 0.57) 21 0.77 (0.44, 1.11) 0.37 (0.24, 0.57) 1.54 (0.88, 2.67)
Total 5,316 3,936 1.38^e^(1.32, 1.44)
^a^Age-standardized rate (per 100,000 person-years; 2000 U.S. Standard Population (Census P25-1130))
^b^Confidence interval
^c^Sex-specific, race/ethnic group rate ratio, adjusted for age and period by Poisson regression
^d^Race/ethnic group-specific male/female rate ratio, adjusted for age and period by Poisson regression
^e^Male/female rate ratio, adjusted for race/ethnic group, age, and period by Poisson regression
Sex
---
In Table [2](#T2){ref-type="table"}, we also tested the hypothesis that sex variation in incidence differs according to glioma subtype. For each race/ethnic group and glioma subtype, males had a higher incidence rate than females. For each race/ethnic group, the male/female RR was higher for GBM than for non-GBM, except for American Indians/Alaska Natives (for whom the 95% confidence intervals were wide). The overall male/female RR, derived from a Poisson model with sex as the independent variable and age, period, and race/ethnic group as covariates, was 1.61 (95% CI = 1.56, 1.66) for GBM versus 1.38 (95% CI = 1.32, 1.44) for non-GBM. This difference was highly significant (p-value for heterogeneity \< 0.0001).
The p-value for heterogeneity for the variation in male/female RR by race/ethnic group was 0.0013 for GBM and 0.0011 for non-GBM, indicating that the male/female RR varied by race/ethnic group for each glioma subtype. However, when we excluded Hispanic Whites, among whom we observed the lowest male/female RR for both GBM and non-GBM, the p-value for heterogeneity was no longer significant (p = 0.76 for GBM and p = 0.85 for non-GBM).
In Table [3](#T3){ref-type="table"} we tested the hypothesis that the male/female RR is higher for peri- and post-menopausal ages than for pre-menopausal ages. The male/female RR did not significantly vary among these menopausal age groups (p-value for heterogeneity = 0.38 for GBM and 0.087 for non-GBM). Because we observed in our previous study that the male/female RR was higher for peri- and post-menopausal ages than for pre-menopausal ages for adult brain cancer as a whole \[[@B1]\], most of which is glioma, in the current study we also examined GBM and non-GBM combined (data not shown) and did observe a borderline-significantly higher male/female RR for peri- and post-menopausal ages than for pre-menopausal ages (p-value for heterogeneity = 0.047).
######
Age-, period-, and race/ethnic group-adjusted male/female rate ratios by menopausal age group and glioma subtype, age 30 to 69 years, SEER, 1992-2007
GBM Non-GBM
---------------------------------- ------- ------------------- ------- -------------------
30-44 (pre-menopausal) 2,056 1.70 (1.56, 1.86) 4,057 1.33 (1.25, 1.41)
45-54 (peri-menopausal) 4,135 1.61 (1.51, 1.71) 2,475 1.36 (1.25, 1.47)
55-69 (post-menopausal) 8,897 1.59 (1.52, 1.66) 2,720 1.48 (1.37, 1.60)
p-value for heterogeneity**^c^** 0.38 0.087
^a^Adjusted for age (5-year age categories), period, and race/ethnic group by Poisson regression
^b^Confidence interval
^c^Calculated from the likelihood ratio test for the addition of the cross-product term for sex by menopausal age group to the Poisson model
Age
---
In Poisson models including age (categorical variable with 5-year age groups), sex, and a cross-product term between age and sex, adjusted for period and race/ethnic group, the p-value for the cross-product term was 0.37 for GBM and 0.12 for non-GBM, indicating homogeneous age effects in males and females for GBM and non-GBM, respectively. However, the difference in age effect between GBM and non-GBM was highly significant (p-value for heterogeneity \< 0.0001).
To further explore the hypothesis that age variation in incidence differs according to glioma subtype, for each subtype we calculated adjusted RRs for age by 5-year age group using Poisson regression, adjusting for period, race/ethnic group, and sex. We then used these adjusted RRs in weighted least squares regressions of log~10~(RR) versus log~10~(age) and found reasonable fits, with the GBM incidence rate increasing proportionally with approximately the 4^th^power of age (slope = 4.17; 95% CI: 3.80, 4.54; adjusted-R^2^= 0.99), and the non-GBM incidence rate increasing proportionally with approximately the square root of age (slope = 0.48; 95% CI = 0.25, 0.72; adjusted-R^2^= 0.78), showing the marked difference in age effect between GBM and non-GBM. We then calculated unadjusted RRs by 5-year age group and subtype using Poisson regression and used the unadjusted RRs in weighted least squares regression of log~10~(RR) versus log~10~(age). We found slopes and adjusted-R^2^s similar to those found when using adjusted RRs, showing that it would be valid to plot log~10~(age-specific incidence rate) versus log~10~(age) without adjustment, which we show in Figure [1](#F1){ref-type="fig"}.
{#F1}
Finally, we modeled age as log~e~(age) in Poisson models (Table [4](#T4){ref-type="table"}) and found the period-, sex-, and race/ethnic-group-adjusted regression coefficients (intersections of \"Total\" row and \"Total\" column) to be identical to the corresponding slopes observed in the weighted least squares regression models that used the adjusted RRs for 5-year age groups. The p-value for heterogeneity for the sex difference in the log~e~(age) effect was 0.33 for GBM and 0.18 for non-GBM, confirming the sex homogeneity; the p-value for heterogeneity for the difference in log~e~(age) effect between GBM and non-GBM was highly significant (p \< 0.0001).
######
Race/ethnic group-specific, period-adjusted Poisson regression coefficients (slopes) for the natural logarithm of age (log~e~(age)) by glioma subtype, age 30 to 69 years, SEER, 1992-2007
------------- -------------------------------- ------------------------------------- ------------------------------------- -------------------------------------
**Glioma** **Male^a^** **Female^a^** **Total^b^**
**Subtype** **Race/ethnic group** **Slope (log~e~(age)) (95% CI^c^)** **Slope (log~e~(age)) (95% CI^c^)** **Slope (log~e~(age)) (95% CI^c^)**
**GBM**
Total**^d^** 4.12 (4.01, 4.23) 4.24 (4.10, 4.38) 4.17 (4.08, 4.26)
Non-Hispanic White 4.09 (3.96, 4.21) 4.23 (4.07, 4.39) 4.14 (4.05, 4.24)
Hispanic White 4.51 (4.17, 4.86) 4.39 (3.99, 4.78) 4.46 (4.20, 4.72)
Black 4.29 (3.81, 4.76) 4.38 (3.83, 4.94) 4.33 (3.97, 4.69)
Asian/Pacific Islander 3.70 (3.19, 4.21) 3.83 (3.19, 4.47) 3.75 (3.35, 4.15)
American Indian/Alaskan Native 3.03 (1.28, 4.77) 4.39 (2.21, 6.56) 3.59 (2.23, 4.94)
p-value for heterogeneity^e^ 0.044 0.68 0.021
**Non-GBM**
Total**^d^** 0.52 (0.40, 0.64) 0.43 (0.29, 0.56) 0.48 (0.39, 0.57)
Non-Hispanic White 0.43 (0.29, 0.56) 0.38 (0.22, 0.54) 0.41 (0.30, 0.51)
Hispanic White 1.13 (0.77, 1.49) 0.76 (0.38, 1.14) 0.95 (0.69, 1.21)
Black 0.88 (0.34, 1.41) 0.68 (0.07, 1.29) 0.79 (0.39, 1.19)
Asian/Pacific Islander 0.38 (-0.15, 0.91) 0.08 (-0.51, 0.66) 0.24 (-0.15, 0.63)
American Indian/Alaskan Native 0.46 (-1.13, 2.05) 0.01 (-2.29, 1.61) 0.14 (-1.09, 1.38)
p-value for heterogeneity^e^ 0.0053 0.22 0.0005
------------- -------------------------------- ------------------------------------- ------------------------------------- -------------------------------------
^a^Log~e~(age) coefficient adjusted for period by Poisson regression
^b^Log~e~(age) coefficient adjusted for period and sex by Poisson regression
^c^Confidence interval
^d^Log~e~(age) coefficient additionally adjusted for race/ethnic group.
**^e^**Calculated from the likelihood ratio test for the addition of the cross-product term for race/ethnic group by log~e~(age) to the Poisson model
In Table [4](#T4){ref-type="table"}, we also tested the hypothesis that the log~e~(age) effect differs according to race/ethnic group for each subtype. For GBM, the slope for log~e~(age) for each race/ethnic group was approximately 4. However, there was some evidence for heterogeneity (p-value for heterogeneity = 0.021), with Asians/Pacific Islanders and American Indians/Alaskan Natives having smaller slopes than the other race/ethnic groups. For non-GBM, the slopes by race/ethnic group ranged form 0.14 to 0.95, with a p-value for heterogeneity of 0.0005, with the smallest slopes again observed among Asians/Pacific Islanders and American Indians/Alaskan Natives.
Calendar period
---------------
In Table [5](#T5){ref-type="table"} we tested the hypothesis that calendar period variation in incidence differed according to glioma subtype. For GBM, we observed a significant trend of increasing RR with more recent period; however, the magnitude of the increase in incidence rate was not large (6% increase between 1992-1995 and 2004-2007). For non-GBM, we observed a significant trend of decreasing RR with more recent period, with a 16% decrease in incidence rate between 1992-1995 and 2004-2007. The difference in period effect between GBM and non-GBM was highly significant (p-value for heterogeneity \< 0.0001). Overall, for GBM and non-GBM combined, we observed a slight trend of decreasing incidence rate with more recent period (3% decrease between 1992-1995 and 2004-2007).
######
Age-, sex-, and race/ethnic group-adjusted rate ratios for period by glioma subtype, age 30 to 69 years, SEER, 1992-2007
GBM Non-GBM Total^a^
------------------- ------- ------------------- ---------- ------------------- ------- -------------------
1992-1995 2,181 1.00 (reference) 1,632 1.00 (reference) 3,813 1.00 (reference)
1996-1999 2,351 1.03 (0.97, 1.09) 1,655 0.97 (0.91, 1.04) 4,006 1.00 (0.96, 1.05)
2000-2003 4,930 1.02 (0.97, 1.07) 2,986 0.87 (0.82, 0.92) 7,916 0.95 (0.92, 0.99)
2004-2007 5,626 1.06 (1.01, 1.12) 2,979 0.84 (0.79, 0.89) 8,605 0.97 (0.93, 1.01)
p-value, trend^d^ 0.012 \< 0.0001 0.037
^a^GBM and non-GBM combined
^b^Adjusted for age, sex, and race/ethnic group by Poisson regression
^c^Confidence interval
^d^Calculated from the likelihood ratio test for the addition of period (as an interval variable) to the model
Discussion
==========
Although we observed statistically significant differences between GBM and non-GBM in the variation of incidence rates according to age, race/ethnic group, and sex (we will discuss period separately), only the difference in age effect, which has been observed by others \[[@B2],[@B5],[@B22]\], was sizeable, with the incidence rate of GBM rising steeply with age and the incidence rate of non-GBM rising comparatively slightly with age. In spite of the statistically significant differences, the commonalities between GBM and non-GBM with respect to race/ethnic group variation and sex were more notable than the somewhat subtle differences.
Age
---
The GBM incidence rate increased in proportion to approximately the 4^th^power of age, similar to what was found in an analysis of SEER data among Whites in 1973-1982 \[[@B22]\]. Because secondary GBM cases are known to have a younger age distribution than primary GBM cases \[[@B9]\], the increase in primary GBM incidence with age (after removing secondary GBM) must be somewhat steeper, rising toward the 5^th^power, which would make the age-incidence curve for primary GBM similar to that of many carcinomas, which typically increase in incidence approximately in proportion to between the 4^th^and 6^th^power of age \[[@B23]\]. On the other hand, the non-GBM incidence rate increased in proportion to approximately the square root of age, an age-incidence pattern that is quite unusual \[[@B23]\]. The previous SEER analysis among Whites in 1973-1982 found slopes of 1.7 for astrocytomas and 1.0 for oligodendrogliomas \[[@B22]\].
The pronounced difference in age-incidence curves between GBM (mainly primary GBM) and non-GBM suggests a fundamental difference in the genesis of these glioma subtypes, as has been suggested previously \[[@B22]\]. Mathematical models of carcinogenesis suggest that development of cancer types with steep log(incidence)-log(age) slopes, such as primary GBM, involve a large number of stable genetic or epigenetic changes (slope plus one; thus 5-6 changes for primary GBM) \[[@B24],[@B25]\] or rapid expansion of a premalignant clone \[[@B26]\], whereas development of cancer types with shallow log(incidence)-log(age) slopes, such as non-GBM, involve a small number of stable genetic or epigenetic changes (as few as one or two) or slow expansion of a premalignant clone.
Indeed, GBM exhibits greater molecular complexity than non-GBM, with the implication that the path to GBM involves a greater number of molecular alterations \[[@B7]\]. Furthermore, the steeper age-incidence curve for GBM may indicate a greater role for age-related processes such as immunosenescence \[[@B27]\] or reduced efficiency of DNA repair mechanisms \[[@B28]\]. Given that among malignancies (brain or otherwise), non-GBM (as well as secondary GBM) are unique in exhibiting a high frequency (as high as 80%) of IDH mutations \[[@B15],[@B16],[@B29],[@B30]\], which appear to be early events in non-GBM development \[[@B16]\], one can speculate that these mutations drive an atypical path to malignancy.
For both GBM and non-GBM, the log(age) slope was similar for males and females. However, within the context of the large difference in age-incidence curves between GBM and non-GBM, we observed subtle, but statistically significant, variation in age-incidence curves among race/ethnic groups. For both subtypes, Asians/Pacific Islanders and American Indians/Alaskan Natives had the smallest slopes. We observed a similar result for Asians/Pacific Islanders in our previous work on brain cancer as a whole \[[@B1]\].
Race/ethnic group
-----------------
For race/ethnic group variation, we observed an important commonality between GBM and non-GBM. For each subtype, compared to non-Hispanic Whites, the incidence rate among Blacks, Asian/Pacific Islanders, and American Indians/Alaskan Natives was substantially lower (one-fourth to one-half for GBM; about two-fifths for non-GBM). However, secondary to this primary effect, race/ethnic group variation in incidence was less for non-GBM than for GBM, a difference that was highly statistically significant but only moderate in magnitude.
There is evidence for race/ethnic group differences in genetic pathways to glioma \[[@B31]-[@B33]\]. Furthermore, genome-wide association studies have identified several genetic susceptibility regions for glioma \[[@B34],[@B35]\]. Given the genotype variability across race/ethnic groups \[[@B36]\], it is possible that variation in the frequency of susceptibility alleles across race/ethnic groups explains at least some of the race/ethnic group variation in glioma incidence, including the race/ethnic group heterogeneity in the relationship between glioma incidence and age. The commonality between GBM and non-GBM in race/ethnic group variation suggests that at least some of the susceptibility loci that may help explain race/ethnic group variation in glioma incidence would be the same for GBM and non-GBM, although some susceptibility loci appear to show specificity with respect to glioma subtype \[[@B37]-[@B39]\].
Sex
---
As with race/ethnic group variation, we observed an important commonality between GBM and non-GBM for sex. For each subtype, the incidence rate was higher for males than for females; this male excess of glioma is well known \[[@B2],[@B3],[@B5],[@B6]\]. However, we did find the male/female RR to be somewhat higher for GBM (1.6) than for non-GBM (1.4), a result that was highly statistically significant. We previously suggested that the male/female difference in brain cancer incidence is biologically based \[[@B1]\], and that an explanation should be sought in genetic differences between males and females, sex hormones, and/or female reproductive factors \[[@B40]\]. Now we would add that any explanation should take into account the difference in the male/female RR between glioma subtypes.
For GBM and non-GBM, respectively, we observed similar male/female RRs among race/ethnic groups, with the exception of Hispanic Whites, among whom the male/female RR was anomalously low. Further work is required to determine whether the latter result has a biological basis or stems from an unidentified bias. It is noteworthy that the quality of the SEER Hispanic ethnicity variable was found to be moderate to substantial, but not excellent \[[@B41]\].
In our previous work, we found that for adult brain cancer as a whole (most of which are glioma), the male/female RR was higher in the peri- and post-menopausal age range than in the pre-menopausal age range \[[@B1]\]. However, in the current study, we found no evidence for an association between male/female RR and age for GBM and only suggestive evidence (p = 0.09) for a higher RR in the post-menopausal age range for non-GBM. We also found that the result for all glioma appeared to have been confounded by glioma subtype, with non-GBM both having a lower male/female RR and occurring at younger ages compared to GBM, which would explain the result from our previous work.
Calendar period
---------------
We observed trends of increasing GBM incidence rate and decreasing non-GBM incidence rate with more recent period, with an overall trend for GBM and non-GBM combined of slightly decreasing rate. Although the latter trend was statistically significant (p = 0.037), we do not conclude that the overall incidence of glioma decreased over the 16 year period of observation because the small 3% decrease between 1992-1995 and 2004-2007 could easily be explained by an unrecognized bias. Furthermore, given the well-known inter-observer variability in glioma diagnosis \[[@B10],[@B42]-[@B45]\], the increasing trend of GBM incidence coupled with the decreasing trend of non-GBM incidence may at least partly be due to a secular trend in diagnostic fashion, as opposed to real changes in incidence of these two subtypes.
Limitations
-----------
Due to the known inter-observer variability in the detailed morphologic classification of non-GBM in particular \[[@B10]\], we did not attempt to sub-classify these tumors into the traditional categories of astrocytoma, oligodendroglioma, and mixed oligoastrocytoma or by grade. Furthermore, we were unable to distinguish primary from secondary GBM and were unable to take into account known molecular heterogeneity within the two broad categories of GBM \[[@B46]\] and non-GBM \[[@B47]\]. Another limitation was possible selection bias due to differential diagnosis of microscopically confirmed glioma that had a specific morphology code (not the nonspecific code 9380), according to demographic factors. However, our restriction to ages 30-69 years, an age range with a high proportion of cases both microscopically confirmed and with a specific morphology code, tended to minimize the potential for any such bias appreciably distorting the results. This restriction was at the expense of generalizability to a broader age range.
Conclusions
===========
We found statistically significant differences between GBM and non-GBM in the variation of incidence rates according to age, race/ethnic group, sex, and period. The difference in age effect, which was substantial, suggests a fundamental difference in the genesis of primary GBM (the driver of GBM incidence) versus non-GBM. However, the commonalities between GBM and non-GBM with respect to race/ethnic group and sex variation were more notable than the somewhat subtle, albeit statistically significant, differences. For each subtype, the incidence rate among Blacks, Asian/Pacific Islanders, and American Indians/Alaskan Natives was substantially lower (one-fourth to one-half for GBM; about two-fifths for non-GBM) than the rate among non-Hispanic Whites, and the rate among females was about two-thirds the rate among males. These similarities suggest that within the context of a fundamental difference in the genesis of these subtypes, some aspects of the complex process of gliomagenesis are shared by these subtypes as well. The increasing calendar time trend of GBM incidence coupled with the decreasing calendar time trend of non-GBM incidence may at least partly be due to a secular trend in diagnostic fashion, as opposed to real changes in incidence of these two subtypes.
Competing interests
===================
The authors declare that they have no competing interests.
Authors\' contributions
=======================
RD conceived of the study, participated in the statistical analysis, and drafted the manuscript. ASD conducted the statistical analysis and provided important input into the manuscript draft. Both authors designed the study, participated in interpretation of data, and read and approved the final manuscript.
Pre-publication history
=======================
The pre-publication history for this paper can be accessed here:
<http://www.biomedcentral.com/1471-2407/11/325/prepub>
Acknowledgements
================
We are grateful to the SEER Program for making its data publicly available.
|
{
"pile_set_name": "PubMed Central"
}
|
Many stillbirths result from pregnancy complications, whose root cause is abnormal development and function of the placenta \[[@B1]\]. In order to prevent stillbirths, we need to have a better understanding of how the human placenta develops, both in normal and abnormal pregnancies. This lack of understanding of the human placenta has recently been acknowledged, and "The Human Placenta Project" launched, by the National Institute of Child Health and Human Development (NICHD) \[[@B2]\]. In fact, the human placenta is difficult to study because of the lack of both "in vivo" animal models and placental cell lines able to be cultured "in vitro" in a tissue culture dish. Specifically, mice and rats have placentas which differ from the human both in structure and at the molecular level \[[@B3]\]; in addition, the human placental cell lines behave differently in culture, compared to the placental cells as they exist "in vivo" in the pregnant patient \[[@B4]\]. Over the last 5 years, our laboratory has set out to use human pluripotent stem cells (hPSCs) to model placental development in a dish \[[@B5]\]. "Pluripotent" stem cells have the ability to differentiate, or turn into, any cell type in the body, including the placental cell type, "trophoblast"\[[@B6],[@B7]\]. While initially hPSCs had to be derived from human embryos, in 2007, Yamanaka et al. developed a method for generating such cells from any proliferative cell type \[[@B8]\]. hPSCs have now been derived from numerous cell types, including amnion cells of the placenta \[[@B9]\].
We have developed a method for step-wise differentiation of such hPSCs, first into trophoblast precursor cells and then into terminally differentiated, functional trophoblast, including multinucleated syncytiotrophoblast (STB) and invasive extravillous trophoblast (EVT). These two cell types are the functional units of the placenta: STB carry out nutrient and gas exchange, while the EVT invade the maternal uterus and establish blood flow to the feto-placental unit. Our differentiation method is both reproducible and highly efficient, with \>95% of cells becoming trophoblast in the culture dish, based both on expression of specific genes and on functional assays such as secretion of the pregnancy hormone, hCG. We recently applied this method to hPSCs carrying a chromosomal aneuploidy, Trisomy 21 (T21). It is known that trophoblast isolated from T21 placentas have a defect in differentiation into multinucleated, hCG-secreting STB \[[@B10]\]. We asked whether this defect could be reproduced in culture when differentiating T21 hPSCs into trophoblast. We observed that T21 hPSCs indeed show delayed differentiation into functional STB, secreting significantly less hCG into the media compared to trophoblast derived from hPSCs with a normal karyotype. These results confirm the utility of hPSCs in modeling human placenta, both during normal development and in disease. We are currently collecting and banking amnion epithelial cells from placentas of patients with pregnancy complications, focusing on early-onset severe preeclampsia, which is highly associated with both maternal and neonatal morbidity and mortality. We believe that, once reprogrammed into hPSCs, these cells hold great promise, both in advancing our understanding of the mechanisms of placental dysfunction, and also in providing a platform for drug screening to reverse the disease phenotype.
|
{
"pile_set_name": "PubMed Central"
}
|
INTRODUCTION {#sec1-1}
============
The orofacial region is very vascular and significant blood loss can occur and a subsequent need for blood transfusion is often encountered. The major sources of bleeding in maxillofacial surgery including Le Fort I and sagittal split osteotomies are the descending palatine and inferior alveolar arteries respectively. Although bleeding from these arteries is usually controllable, there may be significant blood loss in long time maxillofacial surgeries.
Several approaches have been used to reduce intraoperative blood loss, including: Hypotensive anesthesia which can reduces perfusion of vital organs especially in patients with altered baseline auto regulatory mechanisms (hypertension) or those likely to be particularly vulnerable (eg, diabetes, coronary artery disease, stroke, and chronic renal failure).\[[@ref1][@ref2]\]
The alternate approaches are administration of antifibrinolytic agents such as aprotinin, aminocaproic acid and tranexamic acid perioperatively to stabilize the multiple micro clots that form within the surgical wound.\[[@ref3][@ref4]\]
Tranexamic acid is a synthetic derivative of the amino acid lysine that exerts its antifibrinolytic effect through the reversible blockade of lysine binding sites on plasminogen molecules.
Lysine exerts its antifibrinolytic effect by competitively inhibiting the activation of plasminogen thereby reducing the conversion of plasminogen to plasmin. It can also directly inhibit plasmin activity.
Adverse effects of tranexamic acid including nausea, diarrhea and occasional orthostatic events, are uncommon. Isolated cases of thromboembolism after the use of tranexamic acid have been reported; however, these observations have not been confirmed by the results of controlled clinical studies.\[[@ref5]\]
Tranexamic acid has been used in neuro, orthopedic, cardiac, spine and maxillofacial surgeries and has reduced the amount of blood loss and subsequent need for blood transfusion.\[[@ref6]--[@ref11]\]
The most common application of topical tranexamic acid in oral and maxillofacial surgery is in patients with congenital or acquired coagulation disorders.\[[@ref12][@ref13]\]
Despite of mentioned studies, in a study by Kaewpradub *et al*. on 40 patients, tranexamic acid in an irrigant fluid did not significantly decrease intraoperative blood loss compared with the placebo during orthognathic surgery.\[[@ref13]\]
The aim of this study is to evaluate the efficacy of preoperative IV tranexamic acid on intraoperative blood loss during bimaxillary surgeries.
METHODS {#sec1-2}
=======
This randomized double blind clinical trial was performed in Dr. Shariati Hospital of Tehran University of Medical Sciences from August 2010 to January 2011. The study protocol conformed to the ethical guidelines of the 1989 Declaration of Helsinki.
Ethics {#sec2-1}
------
Ethical approval for this study was provided by the Ethical Committee of Tehran University of Medical Sciences, Tehran, Islamic Republic of IRAN, protocol number 220, on April 20, 2010.
All American Society of Anesthesiologists (ASA) Class I patients between 18 and 40 years of age scheduled for bimaxillary osteotomy at Dr. Shariati Hospital between August 2010 to January 2011 were consecutively recruited to the study after written informed consent. Exclusion criteria were patients with uncontrolled systemic diseases, anticoagulant consumption, simultaneous temporomandibular joint (TMJ) surgery or rhinoplasty, concomitant craniofacial surgery, bone disease (eg, fibrous dysplasia), or massive autogenous graft. Randomization was by means of computer-generated codes. Sealed envelopes containing the information of the randomization allocation were prepared and kept by the staff not involved in the study. The specific envelope was transferred to a specific member of the anesthesia team not involved in the study before the induction of anesthesia. The study drug was prepared by this member and was transferred to the anesthetist for administration after induction of anesthesia. The envelope was sealed again and kept in the patient\'s folder until the end of the study period. All members of the surgical team, nursing staff, and the anesthetist were unaware of the allocation. Subject enrollment and allocation is summarized in a CONSORT flow diagram \[[Figure 1](#F1){ref-type="fig"}\].
{#F1}
IV infusion of normal saline 4 ml/kg/h was started 1 h before patients, were transferred to the operating room. Standard monitoring including electrocardiogram, arterial blood pressure, arterial oxygen saturation, core temperature, and end-tidal carbon dioxide were used throughout the operation.
Patients were premedicated with 1 mg of modazolam and 3 μg/kg of fentanyl intravenously.
Anesthesia was induced with 5 mg/kg of thiopental sodium intravenously. Patients were nasotracheally intubated after IV administration of 0.5 mg/kg atracurium and 1 mg/kg lidocine. Anesthesia was maintained with 1%--2% isoflurane and 50% nitrous oxide in oxygen. Arterial blood pressure was monitored continuously via radial catheter and was kept 20% below the patient\'s mean arterial pressure (mild hypotension) by changing isoflurane concentration and infusion of 0.15-0.5 μg/kg/h remifentanil. Intraoperative fluid therapy was obtained with 0.9% saline or lactated Ringer\'s solution 4ml/kg/h and blood loss was replaced with three fold saline in order to maintain the urine output more than 0.5 ml/kg/h. When 15% of the estimated blood volume (EBV) was lost, hydroxyethyl starch (Voluven; Fresenius Kabi Pharmaceutical, Bad Homburg, Germany) was given on a matching basis (milliliter for milliliter) or according to the vital signs (pulse and blood pressure) and urine output of more than 0.5 ml/kg/h.
The surgeon injected 20 mL saline containing 1/200,000 epinephrine into the surgical field a few minutes before the incisions. Surgeons and the technique of surgery were all the same for patients and consisted of Le Fort I osteotomy of the maxilla and bilateral sagital split osteotomy of the mandible followed by internal fixation.
During the surgery, the amount of blood loss was meticulously measured as follows:
The amount of irrigation solution used during the surgical procedure was detracted from the total amount of fluid accumulated in portable suction deviceThe average weight of standard 20-cm gauze was detracted from the mean weight of completely blood-soaked gauze and the total amount of 7 mL blood was considered for each blood-soaked gauzeThe sum of blood accumulated in suction device and surgical gauze was measured and considered as the estimated blood loss (EBL).
In patients undergoing concomitant genioplasty, the total amount of blood loss during this procedure was recorded and detracted from the total blood loss.
Hemoglobin (Hb) and hematocrit (Hct) concentrations were checked at 1^th^ and 6^th^ hours after operation to control the patient\'s status and verify the need for blood transfusion. Any complications related to tranexamic acid injection, blood transfusion rate, length of hospital stay, and surgery time were also recorded.
Statiatical analysis {#sec2-2}
--------------------
According to the previous studies, the blood loss of bimaxillary osteotomy was within a range of 600 mL to 2,000 mL, with a mean of 1,000 mL and a standard deviation (SD) of 350 mL, and the use of tranexamic acid was associated with a reduction of intraoperative blood loss by 30%.\[[@ref11]\]
A power analysis using these assumptions showed that 16 patients per group was sufficient to detect a 30% difference in the intraoperative blood loss, assuming a power of 85% and a significance level of 5%. Normality of distribution was tested by Kolmogorov Smirnov test. Data were analyzed by SPSS version 11.5 (SPSS, Chicago, IL, USA). Independent sample *t*-test and Chi-squared tests were used for comparing demographic data. Repeated measures of analysis of variance (ANOVA) (within subject and between subject) was used for comparing mean Hb and Hct concentration.
RESULTS {#sec1-3}
=======
Demographic data, duration of surgery and hospital stay time were not statistically different between the study groups \[[Table 1](#T1){ref-type="table"}\].
######
Comparing demographic data, duration of surgery and hospital stay time between the study groups
Twenty-eight patients (87.5%) underwent bimaxillary orthognathic surgery and the same procedure plus genioplasty was performed on the remaining 4 patients \[[Table 2](#T2){ref-type="table"}\].
######
Comparing surgical treatment plan between the study and control groups
Intraoperative blood loss in the tranexamic group and control group were 585.9ml and 790ml respectively (*P*=0.008) \[[Figure 2](#F2){ref-type="fig"}\].
{#F2}
In the txa group, preoperative mean Hb concentration was 14.11 g/dl which was decreased to 11.56 g/dl at 1^th^ and 11.8 g/dl at 6^th^ hour after operation respectively. In the control group, the preoperative mean Hb concentration was 13.78 g/dl which was reduced to 10.68 g/dl at 1^st^ and 10.84 g/dl at 6^th^ hour after operation respectively (repeated measures ANOVA, within subject, *P*\>0.05) \[[Figure 3](#F3){ref-type="fig"}\].
{#F3}
In the txa group, preoperative mean Hct concentration was 41.46%, which was decreased to 34.03% at the 1^th^ and 34.93% at the 6^th^ hour after operation respectively. In the control group, the preoperative mean Hct concentration was 40.67% which was reduced to 32.06% at first and 33% at sixth hour after operation respectively (repeated measures ANOVA, within subject, *P*\>0.05 \[[Figure 4](#F4){ref-type="fig"}\].
{#F4}
There was a statistical difference in the mean Hb concentration at 6^th^ hour after operation between the study groups (repeated measures ANOVA, between subject, *P*=0.02) \[[Figure 3](#F3){ref-type="fig"}\].
There was no significant difference in postoperative Hct concentration between the study groups (repeated measures ANOVA, between subjects, *P*\>0.05).
Only 1 patient in the control group received packed red blood cell. Although there was a statistical difference in the blood loss between both the groups but there was no difference in the blood transfusion rate because the Hb concentrations were higher than 10 g/dl. Hospital stay time and duration of surgery were not statistically different between the study groups. No complications related to tranexamic acid injection were recorded.
DISCUSSION {#sec1-4}
==========
Tranexamic acid has been used to reduce blood loss and the subsequent need for transfusion in neuro, orthopedic, spinal, and cardiac surgery.\[[@ref6]--[@ref10]\]
Orthognathic surgery can be associated with significant bleeding and strategies have been used to reduce the amount of bleeding during this type of surgeries including: Antifibrinolytic agents, moist pressure dressings, diluted hydrogen peroxide, microfibrillar collagen, topical solutions containing thrombin, patient\'s positioning (reverse Trendelenburg), vasoconstrictors, and hypotensive anesthesia.\[[@ref11]\]
In a study by Choi *et al*. the effect of tranexamic acid on blood loss during orthognathic surgery was evaluated. They found that the total blood loss and intraoperative bleeding was reduced significantly in the txa group compared with control group.\[[@ref11]\]
We did not calculate postoperative oozing(concealed blood loss). It is not easy to measure the amount of oozing precisely because:
Application of suction systems, such as hemovac In oral and maxillofacial surgeries, as a result of confined surgical field and intraoral incisions, is not practicalThe variation of salivary secretion in different patients may interfere with reliable measurement of intraoral oozingA significant proportion of oozing after orthognathic surgeries is post pharyngeal which can be easily swallowed by patients and is concealed.
In another study by Kaewpradub *et al*.,\[[@ref13]\] 0.05% tranexamic acid in normal saline solution or normal saline as placebo was used as an irrigant fluid during orthognatic surgery on 40 patients and intraoperative blood loss was evaluated. They found no difference in blood loss during bimaxillary surgery in the txa group compared with the control group.
This finding was different from our study because they used 0.05% tranexamic acid in normal saline for tissue irrigation. This concentration was approximately equal to 5 fold of that in which 15 to 25 mg/kg tranexamic acid was administered intravenously.
Application of tranexamic acid has reduced the amount of blood loss during surgeries that have been conducted in other fields; these findings were also correlated to our study.\[[@ref6]--[@ref11]\]
There is no strong evidence for risk of thromboembolism associated with the use of tranexamic acid. However, it should be used with caution in patients with risk for thrombosis (eg, history of a thromboembolic event or a family history of thromboembolic disease). Because tranexamic acid is excreted in the urine, dose reduction may also be required in patients with renal insufficiency.
In conclusion, this study showed that the preoperative IV administration of tranexamic acid significantly reduced intra operative bleeding in bimaxillary surgery.
**Source of Support:** Nil
**Conflict of Interest:** None declared.
|
{
"pile_set_name": "PubMed Central"
}
|
Søgaard M, Nielsen PB, Skjøth F, Kjældgaard JN, Larsen TB. Risk of recurrence and bleeding in patients with cancer‐associated venous thromboembolism treated with rivaroxaban: A nationwide cohort study. Cancer Med. 2019;8:1044--1053. 10.1002/cam4.1997
**Funding information**
This study was supported by Bayer AG, Berlin, Germany. The sponsor had no role in study design; in the collection, analysis and interpretation of data; in the writing of the report; or in the decision to submit the article for publication.
1. INTRODUCTION {#cam41997-sec-0005}
===============
Venous thromboembolism (VTE) is a frequent complication in patients with cancer.[1](#cam41997-bib-0001){ref-type="ref"} Oral anticoagulant (OAC) treatment is vital but also challenging because cancer can be associated with a hypercoagulable state, multi medication, invasive procedures, and increased risk of bleeding. As a consequence, both recurrent VTE and bleeding during treatment are more prevalent than in patients without cancer.[2](#cam41997-bib-0002){ref-type="ref"} Current clinical practice guidelines recommend treatment with low molecular weight heparin (LMWH) over vitamin K antagonists (VKA) or non‐VKA OAC (NOAC); this recommendation is well‐demonstrated with strong evidence for at least a 3‐month treatment duration (Grade 1A).[3](#cam41997-bib-0003){ref-type="ref"}, [4](#cam41997-bib-0004){ref-type="ref"}, [5](#cam41997-bib-0005){ref-type="ref"}
For treatment of VTE in patients without cancer, the American College of Chest Physicians Evidence‐Based Clinical Practice Guidelines for antithrombotic therapy for VTE disease recently recommended NOACs including rivaroxaban as long‐term anticoagulant treatment (3 months or longer).[5](#cam41997-bib-0005){ref-type="ref"} The regulatory approval of the factor Xa inhibitor rivaroxaban for VTE was based on the EINSTEIN‐DVT and the EINSTEIN‐PE phase 3 noninferiority trials.[6](#cam41997-bib-0006){ref-type="ref"}, [7](#cam41997-bib-0007){ref-type="ref"} A pooled post‐hoc analysis of these trials indicated that compared with VKA, rivaroxaban was effective at reducing recurrent VTE and could be used for cancer patients without compromising safety.[8](#cam41997-bib-0008){ref-type="ref"} Recently, the results of the first two head‐to head comparisons between edoxaban and rivaroxaban, respectively, and LMWH also indicated that these regimens were comparable in terms of safety and effectiveness.[9](#cam41997-bib-0009){ref-type="ref"}, [10](#cam41997-bib-0010){ref-type="ref"}
Rivaroxaban could be an attractive alternative to LMWH due to the once daily, oral dosing, which obviate the need for daily parenteral injections combined with short half‐life and lower price. Due to insufficient data examining outcomes in rivaroxaban and LMWH in cancer patients, we examined the safety and effectiveness of rivaroxaban in a nationwide cohort of patients with active cancer and VTE, and compared our findings with previous published studies reporting on treatment exposure and associated VTE recurrence in cancer patients.
2. MATERIAL AND METHODS {#cam41997-sec-0006}
=======================
2.1. Setting and data sources {#cam41997-sec-0007}
-----------------------------
The source population for this cohort study comprised the entire population of Denmark encompassing 5.6 million inhabitants. Denmark has a tax‐funded universal health care system, with equal access to hospitals and primary care for all residents, and partial reimbursement of the costs of most prescribed medications, including OAC treatments. The health care system records most contacts with the health system, including births, deaths, hospital visits, and prescription claims.[11](#cam41997-bib-0011){ref-type="ref"} As a result, data on diagnoses and prescription claims are compiled in longitudinal national registries allowing a true nationwide population‐based study with no loss to follow up. This study was based on linkage of three nationwide registries; (a) the National Patient Register,[12](#cam41997-bib-0012){ref-type="ref"} including information from all inpatient stays and outpatient visits at Danish hospitals; (b) the National Prescription Register,[13](#cam41997-bib-0013){ref-type="ref"} which hold data on all prescription purchases by Danish residents since 1995; and (c) the Danish Person Registry,[14](#cam41997-bib-0014){ref-type="ref"} which contain data on sex, date of birth, vital, and emigration status. These registries were linked using the unique 10‐digit personal registration number assigned to each Danish citizen at birth and to residents upon immigration.[14](#cam41997-bib-0014){ref-type="ref"}
2.2. Ethical considerations {#cam41997-sec-0050}
---------------------------
The study was approved by the Danish Data Protection Agency (ref. 2015‐57‐0001). Ethical approval is not required for anonymous register‐based studies in Denmark. The data was provided by the Danish Health Data Authority.
2.3. Study population {#cam41997-sec-0008}
---------------------
We used the National Patient Register to identify all adult patients aged 18 years and older with active cancer and an inpatient or outpatient primary or secondary discharge diagnosis of VTE. As emergency room VTE diagnoses have low validity (positive predictive value of 31%), we did not consider these diagnoses.[15](#cam41997-bib-0015){ref-type="ref"} To ensure sufficient clinical record history for treatment and diagnoses, we excluded patients who had not been residents of Denmark for at least 1 year before the VTE diagnosis. Active cancer was defined as a diagnosis of cancer other than basal‐cell carcinoma of the skin, within 6 months of the VTE event, any treatment for cancer (see Table for codes) within the 6 months, or recurrent or metastatic cancer as described previously.[8](#cam41997-bib-0008){ref-type="ref"}, [16](#cam41997-bib-0016){ref-type="ref"} We classified cancers according to cancer site (breast, gastrointestinal, lung including pleura, genitourinary, gynecological, hematological, metastatic cancer, and other cancers) and cancer stage according to the TNM (Tumor, Node, Metastasis) classification categorized as localized, regional spread, distant spread, unstaged, and stage not recorded, (Table ).
Since we focused on outcomes under rivaroxaban exposure, patients were eligible for study inclusion; if they claimed any prescription for rivaroxaban within 30 days of discharge from the index VTE in the period from January 1, 2012 (rivaroxaban was introduced to the Danish market for recurrent VTE prophylaxis December 9, 2011) to December 31, 2017 (end of inclusion). We excluded patients with concomitant prescriptions for LMWH, warfarin, dabigatran, or apixaban within 30 days of the index VTE.
2.4. Study outcomes and comorbidity {#cam41997-sec-0009}
-----------------------------------
We derived all primary outcomes from discharge diagnoses recorded in the National Patient Register (excluding emergency room diagnoses). The primary effectiveness outcome was the first recorded episode of recurrent VTE during follow up. Recurrent VTE was defined by a primary inpatient or outpatient diagnosis combined with an objectively confirmed VTE diagnosis using imaging in order to rule out repeated coding of the index event. According to a recent validation study, this ensures a positive predictive value of 82%.[17](#cam41997-bib-0017){ref-type="ref"} In addition, follow‐up began 10 days after the date of diagnosis of the index VTE to avoid repeated coding of the index event. The primary safety outcome was major bleeding events recorded as intracranial, gastrointestinal, and major bleeding in various anatomical positions and reported in total as "any bleeding." Bleeding events were required to be primary inpatient diagnoses to increase the validity of the coded diagnosis. All‐cause death was included as a secondary outcome.
We ascertained comorbidities at baseline according to medication claims within the year before the index VTE event and/or history of primary or secondary hospital discharge diagnoses (excluding emergency room diagnoses) since 1994 (introduction of ICD‐10 in Denmark). Comorbidity information included cardiovascular and metabolic diseases, lifestyle‐related diseases, and indicators for surgery (Table ). We further combined covariate information into the Charlson Comorbidity Index[18](#cam41997-bib-0018){ref-type="ref"} (Table ).
2.5. Statistical analysis {#cam41997-sec-0010}
-------------------------
Baseline characteristics at the time of index VTE diagnosis were described using means and standard deviations for continuous measures and percentages for categorical measures. Patients were followed until the outcome of interest, emigration, death, or end of study period (March 23, 2018), whichever came first. We used time‐to‐event analysis to analyze the risk of study outcomes at 6‐month follow‐up.
To enable comparison with other studies, we first calculated crude incidence rates as the number of events divided by person‐time. Then, we used the pseudovalue approach to estimate the cumulative incidence within 6‐month follow‐up, assuming death as competing risk.[19](#cam41997-bib-0019){ref-type="ref"} Allowing for a thorough evaluation, the main analysis was supplemented by analyses stratified by cancer site and cancer stage, and anticancer treatments within 30 days before the index VTE.
The analyses were performed using Stata/MP, version 15 (StataCorp) and R version 3.3.3 (The R Foundation).
2.6. Literature search methods {#cam41997-sec-0011}
------------------------------
We searched PubMed Medline using Medical Subject Headings (MeSH) and free text to identify and summarize previous observational studies on the outcomes of rivaroxaban treatment in patients with active cancer and VTE. Development of the search strategy described in detail in Table was assisted by a trained medical librarian. All searches were performed at the end of December 2018. The first author reviewed the titles and abstracts and removed articles not relevant according to the population, intervention (exposure), comparison, and/or outcome (PICO criteria). The information summarized by our review was collected from the remaining articles and prior publications cited by these articles.
3. RESULTS {#cam41997-sec-0012}
==========
3.1. Nationwide cohort study in Denmark {#cam41997-sec-0013}
---------------------------------------
From January 1, 2012 to December 31, 2017, we identified 8901 patients aged 18 years or older with active cancer and VTE. Of these, 914 redeemed a prescription for rivaroxaban at any time following the index VTE; 744 initiated rivaroxaban within the first year and 476 within 30 days after the index VTE diagnosis. The final study population comprised these 476 patients with mean age 71.5 years (42% females, 57% with pulmonary embolism) (Figure [1](#cam41997-fig-0001){ref-type="fig"}, Figure ). Only 10 patients were diagnosed in 2012; the majority of patients was diagnosed in the latter part of the study period (Table [1](#cam41997-tbl-0001){ref-type="table"}). Median time from last cancer diagnosis to prescription redemption of rivaroxaban was 31 days (interquartile range, IQR, 12‐73 days); median time from the date of last anticancer treatment to prescription redemption was 67 days (IQR 28‐184 days) (Table [1](#cam41997-tbl-0001){ref-type="table"}). Forty‐nine patients (10.3%) had a previous VTE before cancer diagnosis and 8.2% were prior users of OACs. The most frequent cancer type was gastrointestinal cancer (26.1%) followed by genitourinary (23.3%) and hematological cancer (12.6%) (Table [1](#cam41997-tbl-0001){ref-type="table"}). Among the 406 patients with solid tumors, except for brain cancer, 7.1% had distant metastases. Cancer stage was unknown in 54.9% patients and not registered in 6.9%. Approximately one‐fifth (21.6%) had received anticancer treatment within 30 days before, in particular chemotherapy (15.5%) (Table [1](#cam41997-tbl-0001){ref-type="table"}).
{#cam41997-fig-0001}
######
Patient characteristics
Characteristic \% (n)
------------------------------------------------------------------------------------------ -------------------
N 476
Study year
2012 2.1 (10)
2013 13.0 (62)
2014 15.8 (75)
2015 19.7 (94)
2016 25.4 (121)
2017 23.9 (114)
Female 42.0 (200)
Mean age (SD) 71.5 (10.8)
Cancer‐related characteristics
Median time from last cancer diagnosis to first rivaroxaban prescription, days (IQR) 31.0 (12.0‐73.0)
Median time from last anticancer treatment to first rivaroxaban prescription, days (IQR) 66.5 (28.0‐184.0)
Any anticancer treatment with 30 d before VTE 21.6 (103)
Radiotherapy 5.9 (28)
Chemotherapy 15.5 (74)
Immune modulating, hormone or biological treatment 7.1 (34)
Cancer site
Breast cancer 9.7 (46)
Gastrointestinal cancer 26.1 (124)
Lung cancer 10.1 (48)
Genitourinary cancer 23.3 (111)
Gynecological cancer 6.1 (29)
Hematological cancer 12.6 (60)
Metastatic cancer 2.1 (10)
Other cancer sites 10.1 (48)
Cancer stage for solid tumors except brain cancer[a](#cam41997-note-0003){ref-type="fn"}
Localized 16.3 (66)
Regional 14.8 (60)
Distant 7.1 (29)
Unstaged 54.9 (223)
Stage not recorded 6.9 (28)
Medical history
Prior VTE 10.3 (49)
Atrial fibrillation 8.4 (40)
Major surgery within 3 mo 36.8 (175)
Renal dysfunction 5.7 (27)
Alcohol‐related disease 5.3 (25)
Pneumonia within 3 mo 13.0 (62)
Vascular disease 11.1 (53)
Diabetes 12.8 (61)
Prior bleeding 14.3 (68)
Heart failure 10.1 (48)
Prior stroke 10.5 (50)
Chronic pulmonary disease 18.7 (89)
Myocardial infarction 4.6 (22)
Mean Charlson comorbidity index score (SD) 2.6 (2.4)
Concomitant drugs
Prior oral anticoagulants 8.2 (39)
Systemic corticosteroids 18.7 (89)
Clopidogrel 5.9 (28)
Aspirin 22.1 (105)
Renin‐angiotensin inhibitor 35.7 (170)
NSAID 27.5 (131)
Statins 29.8 (142)
Loop diuretics 17.0 (81)
Non‐loop diuretics 28.6 (136)
Calcium channel blocker 18.9 (90)
SD; standard deviation, IQR, interquartile range; NSAID; nonsteroidal anti‐inflammatory drugs.
406 patients had solid tumors except brain cancer
John Wiley & Sons, Ltd
Figure [2](#cam41997-fig-0002){ref-type="fig"} displays cumulative incidence curves for recurrent VTE and major bleeding. As appears, most events occurred shortly after the index event. During 6 months follow‐up, 28 recurrent VTE events occurred (absolute risk 6.1%, rate of 15.1 events per 100 person‐years) with the highest absolute risk in patients with genitourinary cancer (8.1%), gastrointestinal cancer (7.3%), breast cancer (6.5%), and regional cancer stage (8.2%). Nine major bleed were diagnosed (absolute risk 1.9%, rate of 4.7 events per 100 person‐years), in particular, in genitourinary cancer (4.5%), lung cancer (4.2%), and patients with missing cancer stage (3.6%). Restriction to patients who received any anticancer treatment had little impact on the estimates (8.8% had a recurrent VTE, 1.0% experienced major bleeding). Among patients with chemotherapy treatment corresponding estimates were 6.8% and 1.4%, respectively.
{#cam41997-fig-0002}
Eighty deaths (17.8%) were occurred during 6 months follow‐up, equivalent to a rate of 40.6 events per 100 person‐years.
3.2. Literature review of previous observational studies {#cam41997-sec-0014}
--------------------------------------------------------
Our initial search yielded 151 studies, of which 24 full‐text articles were retrieved and assessed for eligibility. After further exclusion of 11 studies, our review included 13 cohort studies (Figure [3](#cam41997-fig-0003){ref-type="fig"}). Study populations ranged from 41 cancer patients to 949, with 5 of the 12 studies including less than 100 patients. Two studies were restricted to patients with catheter‐related thrombosis and one study included only women with gynecological cancer. Duration of follow‐up varied across studies from 3 months to a mean follow‐up of 1.36 years. A detailed description of the studies is provided in Table .
{#cam41997-fig-0003}
Table [2](#cam41997-tbl-0002){ref-type="table"} presents the absolute risk of VTE recurrence and major bleeding for our study compared with estimates retrieved by our review. The risk of recurrent VTE ranged from 0% to 13.2% with the most extreme estimates observed in studies including very few patients or not accounting competing risk of death leading to overestimation of event rates[20](#cam41997-bib-0020){ref-type="ref"}, [21](#cam41997-bib-0021){ref-type="ref"}; in the majority of studies recurrence risk was approximately 3%‐5%. The risk of major bleeding ranged from 0% to 17%, again with the most extreme estimates obtained in studies with very few patients, for example the estimate of a 17% risk of major bleeding originated from studies with 18 and 48 patients, respectively.[22](#cam41997-bib-0022){ref-type="ref"}, [23](#cam41997-bib-0023){ref-type="ref"} Six studies provided estimates of clinically relevant nonmajor bleeding, which ranged from 1.2% to 12.2% (5 events) (Table [2](#cam41997-tbl-0002){ref-type="table"}; Table ). In addition to our study, eight studies provided data on all‐cause mortality which varied widely from 0.8% to 31.4% (Table [2](#cam41997-tbl-0002){ref-type="table"}).
######
Event risk from observational studies on patients with active cancer and venous thromboembolism treated with rivaroxaban
Author, year Follow‐up Cancer patients with rivaroxaban, N Recurrent VTE % (n) Major bleeding % (n) Clinically relevant nonmajor bleeding % (n) All‐cause mortality % (n)
--------------------------------------------------------------------- ----------------------------- ------------------------------------- --------------------- ---------------------- --------------------------------------------- ----------------------------------------------------
Søgaard et al, 2019 (present study) 6 mo 476 6.1% (28) 1.9% (9) NA 17.8% (80)
Studies with 3 mo follow‐up
Simmons et al,[31](#cam41997-bib-0031){ref-type="ref"} 2018 3 mo 98 1.0% (1) 5.1% (5) 6.6% (6) 4.1% (4)
Davies et al,[32](#cam41997-bib-0032){ref-type="ref"} 2017 3 mo 70 1.4% (1) 10% (7) 5.7% (4) 1.4% (1)
Laube et al,[33](#cam41997-bib-0033){ref-type="ref"} 2017 3 mo 83 3.6% (3) 2.4% (2) NA 7.2% (6)
Nicklaus et al,[22](#cam41997-bib-0022){ref-type="ref"} 2017 3 mo 45 8.9% (NA) 17% (NA) NA NA
Studies with 6 mo follow‐up
Yhim et al,[34](#cam41997-bib-0034){ref-type="ref"} 2018 6 mo 124 5.9% (7) 5.3% (6) 10.2% (11) 24.0% (28)
Kohn et al,[35](#cam41997-bib-0035){ref-type="ref"} 2018 6 mo 949 4.0% (37) 2.7% (22) NA 11.3% (105)[a](#cam41997-note-0005){ref-type="fn"}
Streiff et al,[20](#cam41997-bib-0020){ref-type="ref"} 2018 6 mo 707 13.2% (119) 6.0% (63) NA NA
Chaudhury et al,[36](#cam41997-bib-0036){ref-type="ref"} 2017 6 mo 107 4.9% (3) 2.8% (3) NA NA
Mantha et al,[37](#cam41997-bib-0037){ref-type="ref"} 2017 6 mo 200 4.4% (8) 2.2% (4) NA 17.6% (31)
Signorelli and Gandhi,[23](#cam41997-bib-0023){ref-type="ref"} 2019 6 mo 18 0 17% (2) NA NA
Studies with follow‐up\>6 mo
Bott‐Kitslaar et al,[38](#cam41997-bib-0038){ref-type="ref"} 2016 Mean follow‐up 1.36 ±0.5 y 118 3.4% (4) 2.5% (3) 3.4% (4) 31.4% (37)
Studies with unclear duration of follow‐up
Wells et al,[39](#cam41997-bib-0039){ref-type="ref"} 2016 NA 237 3.8% (9) 1.3% (3) 1.2% (1) 0.8% (2)
Xavier et al,[40](#cam41997-bib-0040){ref-type="ref"} 2017 NA 41 12.2% (5) None 12.2% (5) NA
NA, not available
Defined by in‐hospital deaths or hospice claims.
John Wiley & Sons, Ltd
4. DISCUSSION {#cam41997-sec-0015}
=============
The findings of this nationwide cohort study of patients with active cancer and VTE indicate that use of rivaroxaban were safe and effective. The VTE recurrence rate of 6.1% and 1.9% major bleeds at 6 months follow‐up are in line with the estimates demonstrated by our review of previous cohort studies. Our findings also reveal that rivaroxaban was used infrequently in Danish cancer patients and presumably mostly for selected cancer patients. However, rivaroxaban was increasingly used during the study period.
4.1. Comparison with other studies {#cam41997-sec-0016}
----------------------------------
In 2014, rivaroxaban was prescribed in 20% of US patients with cancer despite lack of evidence and guideline recommendations to support this treatment.[24](#cam41997-bib-0024){ref-type="ref"} Sub‐group analyses of patients with cancer from large pivotal phase 3 trials have suggested that NOACs are noninferior to LMWH.[8](#cam41997-bib-0008){ref-type="ref"}, [25](#cam41997-bib-0025){ref-type="ref"} Yet, none of these trials were dedicated to cancer patients and less than 5% of the original trial populations had cancer. Considering that cancer patients typically represent 20% of all patients with VTE in the community, the included trial patients likely represent a highly selected subset of cancer patients encountered in routine clinical care. Nonetheless, based on available post‐hoc analyses of these RCTs and meta‐analyses, the updated International Initiative on Thrombosis and Cancer (ITAC‐CME) consensus statement recommendations from 2016 are that NOACs can be considered for VTE treatment of patients with stable cancer not receiving systemic anticancer therapy, and in cases where VKA is an acceptable, but not available, treatment choice.[3](#cam41997-bib-0003){ref-type="ref"}
In 2017, results of the first two head‐to head comparisons between LMWH and NOAC for the initial and long‐term treatment of VTE in patients with cancer were presented.[9](#cam41997-bib-0009){ref-type="ref"}, [10](#cam41997-bib-0010){ref-type="ref"} The Hokusai VTE cancer trial randomized 1050 cancer patients with acute VTE to edoxaban or LMWH.[9](#cam41997-bib-0009){ref-type="ref"} Over 12 months of therapy, recurrent VTE developed in 7.9% in the edoxaban group and in 11.3% in the LMWH group; major bleeding complications occurred in 6.9% vs 4.0%. The excess bleeding events in the edoxaban group were mainly driven by upper gastrointestinal bleeds in patients with gastrointestinal cancer. The 2‐phase pilot trial SELECT‐D, which randomized 406 cancer patients to rivaroxaban and LMWH reported a 6 months recurrence rate of 4% in the rivaroxaban group and 11% in LMWH group; rates of major bleeding were 4% vs 3%, and there were substantially more clinically relevant nonmajor bleeds with rivaroxaban than LMWH (13% vs 2%).[10](#cam41997-bib-0010){ref-type="ref"} Several other trials are ongoing.[3](#cam41997-bib-0003){ref-type="ref"}
Limited data are available on the selection and uses of rivaroxaban in clinical practice outside clinical trials, and it is debatable whether results from carefully selected trial populations can be extrapolated to routine clinical care. Different factors, patient‐related or based on a clinical decision, can affect the preferred treatment. The present study therefore adds to the existing knowledge. The risk of recurrent VTE in the present study is in line with the estimates of previous trials whereas the risk of bleeding was substantially lower and no excess bleeding events in gastrointestinal cancers were observed. This indicates that in selected cancer patients with VTE encountered in routine care, rivaroxaban may be a safe and effective alternative to LMWH. Taken together with the findings of our review, cumulative evidence suggests that rivaroxaban could be a favorable alternative to LMWH in selected cancer patients. However, the output and insights from the ongoing RCTs should confirm our observations before strong treatment recommendations can be presented.
Cancer patients with VTE is a heterogeneous and complex group of patients. Nonetheless, current practice recommendations apply to all cancer patients, irrespective of cancer site, spread, and anticancer treatments. It is very likely that the biological and clinical heterogeneity influence thrombogenicity and response to treatment. Treatment with LMWH can be burdensome for the patients because the treatment requires daily subcutaneous injections. The need for once or twice daily injection with LMWH might be acceptable for cancer patients but oral alternatives would undoubtedly increase quality of life and prevent post‐injection subcutaneous fibrosis.[26](#cam41997-bib-0026){ref-type="ref"} In a recent cohort study the cumulative 6‐month incidence of LMWH discontinuation among patients with cancer‐associated VTE was approximately 20%.[27](#cam41997-bib-0027){ref-type="ref"} The most common reason for discontinuation was pain at the injection site, followed by injection site hematoma and allergic reactions.[27](#cam41997-bib-0027){ref-type="ref"} Rivaroxaban is administered orally once daily and the short half‐life (5‐9 hours)[28](#cam41997-bib-0028){ref-type="ref"} facilitate temporary interruptions for procedures or periods of thrombocytopenia. However, despite practical advantages of rivaroxaban, the use may be limited by the potential increased risk of gastrointestinal bleeding especially in gastrointestinal cancers. An antidote to immediately reverse the action in case of bleeding has been tested in a phase 3 trial but is not currently on the market.[29](#cam41997-bib-0029){ref-type="ref"} Furthermore, there is a risk of drug interactions with anticancer treatments, especially those that interacts with P‐glycoprotein and cytochrome P450 3A4.[3](#cam41997-bib-0003){ref-type="ref"} Whether these interactions are clinically important remain unclear. In this study, event rates were not notably different among patients with recent chemotherapy or other anticancer treatments. It is likely that more profound knowledge and understanding of various anticoagulant options would allow clinicians to individualize management according to primary tumor site, anticancer treatment, concomitant medications, and interventions and thereby enhance patient outcomes and quality of care.
4.2. Strengths and limitations {#cam41997-sec-0017}
------------------------------
Strengths of this study include data from a nationwide real‐world population with access to free health care, which is not subject to the selection biases that could affect the previous observational studies that included only subsets of cancer patients. All data were collected prospectively within the nationwide registries, which have full coverage for hospital admissions, outpatient care visits, and filled prescriptions with no patients lost for follow‐up.
This study also has limitations. The lack of a comparison group limits the interpretation of our results. We had no data on in‐hospital treatments; therefore, we were unable to identify a cohort of LMWH users since in Denmark, this treatment in cancer patients is generally administered by the hospitals and not recorded in the prescription database. Using VKA users as comparisons are problematic because this treatment is not the guideline recommended treatment and has been demonstrated as less effective than LMWH in cancer patients despite maintenance of INR within therapeutic range.[30](#cam41997-bib-0030){ref-type="ref"} Another limitation is our reliance on prescription purchase as proxy for medication usage, since patients might not take their anticoagulant drug.
5. CONCLUSION {#cam41997-sec-0018}
=============
In conclusion, this nationwide cohort study revealed that rivaroxaban was rarely but increasingly used to treat VTE in patients with active cancer in routine clinical practice in Denmark. In patients selected to this treatment, rivaroxaban appeared safe and effective with rates comparable to what has been reported in randomized trials and in minor observational studies based on selected patient populations. Additional studies are needed to delineate what types of cancer patients that might benefit from NOAC treatment.
CONFLICTS OF INTERESTS {#cam41997-sec-0019}
======================
Associate Professor Larsen has served as an investigator for Janssen Scientific Affairs, LLC and Boehringer Ingelheim, and has been on the speaker bureaus for Bayer, BMS/Pfizer, Roche Diagnostics, Boehringer Ingelheim and Takeda Pharma. Flemming Skjøth has been consultant for Bayer. Peter Brønnum Nielsen has received speaking fees from Boehringer Ingelheim, consulting fees from Bayer; and grant support from BMS/Pfizer. Other authors -- none declared.
|
{
"pile_set_name": "PubMed Central"
}
|
Introducción {#sec0005}
============
La gripe es una enfermedad altamente contagiosa que ocasiona una gran carga de morbimortalidad en todo el mundo, principalmente en personas mayores de 65 años o con alto riesgo de complicaciones[@bib0125]. Se estima que afecta al 5-15% de la población mundial cada año, y en torno a 38.500 muertes anuales en Europa son atribuidas a la gripe[@bib0130]. En España se registró una tasa media de 3,37 y 1,61 defunciones por 100.000 habitantes para las temporadas en las que circuló el virus A(H3N2) y A(H1N1), respectivamente[@bib0135].
Los trabajadores sanitarios están en riesgo de contraer la gripe al estar más expuestos en su trabajo, y pueden actuar como vectores de transmisión nosocomial. En este sentido, el personal de atención primaria juega un papel primordial, ya que está expuesto a la inmensa mayoría de los pacientes con gripe y juega un papel fundamental en la vacunación de los pacientes[@bib0140]. Para generar inmunidad de grupo e interrumpir la transmisión de la gripe en los centros sanitarios sería necesaria una cobertura de vacunación del 80% en los sanitarios[@bib0145]; sin embargo, el porcentaje más alto registrado en los países europeos es del 26,3%, mientras que en España esta cobertura se ha estimado en el 25,4%[@bib0150].
Aunque existe evidencia científica suficiente para recomendar la vacunación antigripal en el personal sanitario, en este trabajo se ha querido ahondar en el triple argumento de autoprotección (principal motivo para vacunarse en los sanitarios[@bib0155]), ética (en atención a los pacientes más vulnerables, la vacunación de los profesionales genera inmunidad de grupo[@bib0145]) y ejemplaridad (aumenta la confianza en la población general[@bib0160]) propuesto por las principales sociedades científicas en aras de aumentar las coberturas vacunales[@bib0165]. El objetivo de este trabajo es estimar el efecto de la vacunación antigripal en los trabajadores de centros asistenciales de atención primaria y en la población atendida durante la campaña 2015-2016.
Métodos {#sec0010}
=======
Diseño del estudio {#sec0015}
------------------
Se realizó un estudio observacional transversal en el que se utilizaron dos bases de datos, una de trabajadores de atención primaria de Gran Canaria (PeopleNet) y otra de la población grancanaria (Drago-AP).
Sujetos de estudio y variables {#sec0020}
------------------------------
Se incluyó, por un lado, a los profesionales (sanitarios y no sanitarios) que tuvieran un contrato de forma continuada en el mismo puesto de trabajo en un centro asistencial de la Gerencia de Atención Primaria entre el 15 de octubre de 2015 y el 31 de marzo de 2016. Se excluyó a los que no cumplían este criterio, así como a aquellos con un proceso de incapacidad temporal (IT) previo al inicio del estudio, o con \> 30 días de IT entre el 1 de octubre y el 15 de noviembre de 2015, por la menor probabilidad de vacunarse que es mayor en ese periodo. De cada uno de ellos se obtuvo: número de identificación, código de identificación de área sanitaria (CIAS), edad, sexo, días en el puesto de trabajo, categoría profesional, zona básica de salud, antigüedad, vacunación antigripal, gripe declarada (EDO) y procesos de IT. Por otro lado, se contó con los usuarios del Área de Salud de Gran Canaria con tarjeta sanitaria y sus características: número de identificación, CIAS, edad, sexo, vacunación antigripal, grupo de riesgo ([fig. 1](#fig0010){ref-type="fig"}), gripe declarada y recordatorio de vacunación. Esta última consistía en la aparición de un mensaje cuando se abría la historia clínica de un paciente susceptible. Las variables resultado fueron: coberturas vacunales, gripes declaradas al Sistema de Vigilancia Epidemiológica y días de IT por enfermedad en 2016 en sanitarios; coberturas vacunales y gripes declaradas según el estado vacunal del sanitario en población general.Figura 1Coberturas vacunales por grupo de riesgo en la población general. Gran Canaria, 2015-2016.^a^ Excepto en AAS: p = 0,001 en hombres y p = 1,0 en mujeres, y en enfermedad hematológica: p = 0,240 en hombres y p = 0,017 en mujeres. ≥ 40: IMC ≥ 40 kg/m2; ≥ 65: ≥ 65 años; AAS: \< 18 años tratados con ácido acetilsalicílico; AD: atención domiciliaria; CD: cuidados domiciliarios; COG: enfermedad cognitiva; DM: enfermedad metabólica; EMB: embarazo; EVA: enfermedad cardiovascular crónica; HEM: enfermedad hematológica; HEP: enfermedad hepática crónica; INM: enfermedad inmunológica; NRL: enfermedad neurológica crónica; NRM: enfermedad neuromuscular; ONC: enfermedad oncológica; REN: enfermedad renal; RESP: enfermedad pulmonar crónica.
Análisis de los datos {#sec0025}
---------------------
Se hizo un cruce de bases de datos para poder asignar la variable «fecha de vacunación» y así poder cuantificar la cobertura en los profesionales. Al no disponer del diagnóstico de los procesos de IT, la variable de gripe en profesionales se generó a partir de la notificación automática al Sistema de Vigilancia Epidemiológica. Además, teniendo en cuenta que generalmente los síntomas de la gripe mejoran dentro de una semana[@bib0170], se dicotomizó la variable de procesos según su duración: 1-7 días y \> 7 días. Se excluyeron los días de baja por accidentes laborales y las ausencias sin IT. Adicionalmente, para valorar el efecto que tenía la vacunación de los profesionales en la población asignada se consideró la Unidad de Atención Familiar/Pediátrica (UAF/UAP), compuesta por un médico y un enfermero, y se estudió en conjunto e individualmente.
En el análisis bivariado se empleó el test ji-cuadrado para las variables categóricas, mientras que las variables cuantitativas se compararon con el test t de Student. La magnitud de asociación entre la vacunación antigripal y la morbilidad en profesionales y población general fue estimada separadamente para hombres y mujeres mediante odds ratio ajustada con su intervalo de confianza al 95% obtenida a partir de modelos de regresión logística. Los análisis estadísticos se realizaron con el paquete estadístico SPSS versión 20 (SPSS INC., Chicago, IL, EE.UU.).
Aspectos éticos {#sec0030}
---------------
El estudio se realizó en conformidad con los principios de la Declaración de Helsinki de la Asociación Médica Mundial[@bib0175]. El tratamiento de los datos de carácter personal requeridos en este estudio se rigió por la Ley Orgánica de Protección de Datos de Carácter Personal 15/1999. Al tratarse de un estudio observacional no se realizó intervención sobre los sujetos de estudio que pudiera suponer un riesgo para su salud. La información respecto de ellos fue tratada de modo confidencial y las bases de datos estaban debidamente anonimizadas. Además, el estudio fue evaluado y autorizado por la Comisión de Investigación de la Gerencia de Atención Primaria de Gran Canaria el 11 de abril de 2016 (número de registro: 193807; SCS: 43062) y por el Comité de Ética de la Investigación con medicamentos del Complejo Hospitalario Universitario Insular de Gran Canaria el 28 de abril de 2016 (Id: CEIm-CHUIMI-2016/852).**Esquema general del estudio**. Estudio transversal de los trabajadores de atención primaria del Área de Salud de Gran Canaria y la población grancanaria con tarjeta sanitaria.
Resultados {#sec0035}
==========
Se estudió a 1.868 profesionales (33,5% hombres y 66,5% mujeres) y 795.605 personas de la población general (49,4% hombres y 50,6% mujeres). La edad media de los profesionales que se vacunaron fue de 52,76 años en hombres y 49,68 años en mujeres, y en la población general, 65,47 y 65,68 años, respectivamente.
En la [tabla 1](#tbl0005){ref-type="table"} se presentan las características descriptivas de los sujetos de estudio según el sexo, el estado de vacunación y el proceso gripal. En los profesionales de atención primaria la cobertura de vacunación fue del 25,4%, más alta en las mujeres sanitarias (26,7%), en los profesionales masculinos no pertenecientes a urgencias (31,0%) y, por categoría profesional, en las mujeres facultativas (34,3%) y en los y las pediatras (42,3 y 48,1%). Por UAF/UAP, en el 49,2% ninguno de los profesionales estaba vacunado, mientras que en el 10,4% tanto el médico como el enfermero estaban vacunados. El número de gripes declaradas en las mujeres trabajadoras de los equipos de urgencias fue 3,2 veces el de las mujeres trabajadoras de los equipos que no eran de urgencias (IC 95%: 1,5-6,7) (datos no mostrados). No hubo diferencias en los días de IT según las coberturas de vacunación. En la población general, la cobertura vacunal fue del 7%. Las mujeres y los hombres con factores de riesgo se vacunaron 15,9 veces más (IC 95%: 15,3-16,4) y 10,9 (IC 95%: 10,6-11,3), respectivamente, que los que no los tenían; en las personas que se vacunaron se redujo el riesgo de tener gripe en un 25% (0,7; IC 95%: 0,7-0,8 en hombres, y 0,76; IC 95%: 0,7-0,8 en mujeres) con respecto a la población no vacunada (datos no mostrados).Tabla 1Características de los profesionales de atención primaria y de la población general. Gran Canaria, 2015-2016Tabla 1Profesionales sanitariosSexoVacunaciónGripeTotalHombresMujeresTotalHombresMujeresTotalHombresMujeres*Edad en años, media (DE)*50,3 (8,1)[\*](#tblfn0005){ref-type="table-fn"}52,1 (7,7)49,4 (8,1)65,6 (17,7)65,5 (17,3)65,7 (18,0)36,6 (20,2)34,8 (20,2)38,2 (20,1)
*Personal* Sanitario1.476 (79,0%)[\*](#tblfn0005){ref-type="table-fn"}491 (78,4%)985 (79,3%)403 (27,3%)[\*](#tblfn0005){ref-type="table-fn"}140 (28,5%)263 (26,7%)[\*](#tblfn0005){ref-type="table-fn"}50 (3,4%)15 (3,1%)35 (3,6%) No sanitario392 (21,0%)135 (21,6%)257 (20,7%)72 (18,4%)37 (27,4%)35 (13,6%)13 (3,3%)4 (3,0%)9 (3,5%)
*Equipo* Urgencias228 (12,2%)[\*](#tblfn0005){ref-type="table-fn"}117 (18,7%)111 (8,9%)38 (16,7%)[\*](#tblfn0005){ref-type="table-fn"}19 (16,2%)[\*](#tblfn0005){ref-type="table-fn"}19 (17,1%)11 (4,8%)1 (0,9%)10 (9,0%)[\*](#tblfn0005){ref-type="table-fn"} No urgencias1.640 (87,8%)509 (81,3%)1.131 (91,1%)437 (26,6%)158 (31,0%)279 (24,7%)52 (3,2%)18 (3,5%)34 (3,0%)
*Categoría profesional* Enfermero812 (43,5%)[\*](#tblfn0005){ref-type="table-fn"}191 (30,5%)621 (50,0%)203 (25%)[\*](#tblfn0005){ref-type="table-fn"}65 (34,0%)138 (22,2%)[\*](#tblfn0005){ref-type="table-fn"}24 (3,0%)5 (2,6%)19 (3,1%) Facultativo664 (35,5%)300 (47,9%)364 (29,3%)200 (30,1%)75 (25,0%)125 (34,3%)26 (3,9%)10 (3,3%)16 (4,4%) Pediatra107 (5,7%)[\*](#tblfn0005){ref-type="table-fn"}26 (4,2%)81 (6,5%)50 (46,7%)[\*](#tblfn0005){ref-type="table-fn"}11 (42,3%)[\*](#tblfn0005){ref-type="table-fn"}39 (48,1%)[\*](#tblfn0005){ref-type="table-fn"}7 (6,5%)3 (11,5%)4 (4,9%) Otros392 (21,0%)135 (21,6%)257 (20,7%)72 (18,4%)37 (27,4%)35 (13,6%)13 (3,3%)4 (3,0%)9 (3,5%)
*Días de IT* 0 o \> 7 días1.792 (95,9%)597 (95,4%)1.195 (96,2%)457 (25,5%)171 (28,6%)286 (23,9%)52 (2,9%)[\*](#tblfn0005){ref-type="table-fn"}15 (2,5%)[\*](#tblfn0005){ref-type="table-fn"}37 (3,1%)[\*](#tblfn0005){ref-type="table-fn"} 1-7 días76 (4,1%)29 (4,6%)47 (3,8%)18 (23,7%)6 (20,7%)12 (25,5%)11 (14,5%)4 (13,8%)7 (14,9%)Población general*Edad en años, media (DE)*41,1 (21,5)[\*](#tblfn0005){ref-type="table-fn"}40,3 (21,1)42,0 (21,9)50,8 (8,1)52,7 (7,4)49,7 (8,4)49,7 (8,4)49,9 (8,8)49,7 (8,3)
*Grupo de riesgo* No520.734 (65,5%)[\*](#tblfn0005){ref-type="table-fn"}268.617 (68,4%)252.117 (62,6%)8.173 (1,6%)[\*](#tblfn0005){ref-type="table-fn"}3.743 (1,4%)[\*](#tblfn0005){ref-type="table-fn"}4.430 (1,8%)[\*](#tblfn0005){ref-type="table-fn"}10.728 (2,1%)5.172 (1,9%)5.556 (2,2%) Sí274.871 (34,5%)124.108 (31,6%)150.763 (37,4%)47.423 (17,3%)22.742 (18,3%)24.681 (16,4%)5.598 (2,0%)2.301 (1,9%)3.297 (2,2%)[^1]
El grupo de riesgo poblacional que registró una mayor cobertura vacunal fue el de los pacientes en atención domiciliaria (41,1% en hombres y 39,3% en mujeres); la menor cobertura se encontró en embarazadas (5,3%). Las diferencias fueron estadísticamente significativas por sexo en la mayoría de los grupos, siendo los hombres los que presentaron coberturas más elevadas en la mayoría de los grupos de riesgo ([fig. 1](#fig0010){ref-type="fig"}).
En la [tabla 2](#tbl0010){ref-type="table"} se muestra la asociación entre estado vacunal y gripe en profesionales y en población general; además, su asociación con los días de IT en profesionales. Los profesionales sanitarios que no se vacunaron tuvieron un riesgo de tener gripe 1,7 veces superior al de los profesionales que se vacunaron, aunque la diferencia no fue estadísticamente significativa. La población general mostró mayor riesgo de tener gripe cuando no se había vacunado (OR: 1,2; IC 95%: 1,1-1,4), y esta asociación se mantuvo y fue mayor en la población femenina tras ajustar por los posibles factores de confusión (OR: 1,3; IC 95%: 1,1-1,5).Tabla 2Efecto de la no vacunación antigripal en la morbilidad de los profesionales y en la morbilidad de la población general. Gran Canaria, 2015-2016Tabla 2ProfesionalesPoblaciónnOR (IC 95%)[a](#tblfn0010){ref-type="table-fn"}OR (IC 95%)[b](#tblfn0015){ref-type="table-fn"}nOR (IC 95%)[a](#tblfn0010){ref-type="table-fn"}OR (IC 95%)[c](#tblfn0020){ref-type="table-fn"}**Total** *Gripe* No1.805779.279 Sí631, 6 (0,8-3,2)1,7 (0,9-3,3)16.3261,3 (1,2-1,4)1,2 (1,1-1,4) *Días de IT* 0 o \> 7 días1.792− 1-7 días761,1 (0,6-1,9)1,0 (0,6-1,7)−−−
**Hombres** *Gripe* No607385.252 Sí191,1 (0,4-3,1)1,2 (0,4-3,3)7.4731,4 (1,2-1,5)1,0 (0,8-1,2) *Días de IT* 0 o \> 7 días597− 1-7 días291,5 (0,6-3,8)1,3 (0,5-3,3)−−−
**Mujeres** *Gripe* No1.198394.027 Sí442,0 (0,9-4,9)2,2 (0,9-5,4)8.8531,3 (1,2-1,5)1,3 (1,1-1,5) *Días de IT* 0 o \> 7 días1.195− 1-7 días470,9 (0,5-1,8)0,9 (0,4-1,7)−−−[^2][^3][^4]
La cobertura de vacunación en la población general aumentó significativamente a medida que aumentaba el número de miembros vacunados de la UAF/UAP que la atendían (OR: 1,3; IC 95%: 1,3-1,4 en hombres, y OR: 1,3; IC 95%: 1,2-1,3 en mujeres, cuando médico y enfermero estaban vacunados). Por su parte, las gripes declaradas en la población disminuyeron significativamente en ambos sexos cuando el enfermero que la asistía estaba vacunado (OR: 0,9; IC 95%: 0,9-0,9) ([tabla 3](#tbl0015){ref-type="table"}).Tabla 3Efecto de la vacunación antigripal de la UAF en la vacunación y en la morbilidad de la población general. Gran Canaria, 2015-2016Tabla 3HombresMujeresnVacunaciónGripenVacunaciónGripeOR (IC 95%)[a](#tblfn0025){ref-type="table-fn"}OR (IC 95%)[b](#tblfn0030){ref-type="table-fn"}OR (IC 95%)[a](#tblfn0025){ref-type="table-fn"}OR (IC 95%)[b](#tblfn0030){ref-type="table-fn"}OR (IC 95%)[a](#tblfn0025){ref-type="table-fn"}OR (IC 95%)[b](#tblfn0030){ref-type="table-fn"}OR (IC 95%)[a](#tblfn0025){ref-type="table-fn"}OR (IC 95%)[b](#tblfn0030){ref-type="table-fn"}*UAF vacunada* No204.639201.386 Médico o enfermero156.8441,1 (1,1-1,2)1,2 (1,2-1,2)1,0 (0,9-1,0)1,0 (0,9-1,0)151.1511,1 (1,1-1,1)1,1 (1,1-1,2)1,0 (0,9-1,0)1,0 (0,9-1,0) Médico y enfermero41.3971,3 (1,2-1,3)1,3 (1,3-1,4)1,0 (0,9-1,0)0,9 (0,9-1,0)40.1881,2 (1,2-1,3)1,3 (1,2-1,3)0,9 (0,9-1,0)0,9 (0,9-1,0)
*Médico vacunado* No283.206277.839 Sí119.6741,1 (1,1-1,1)1,2 (1,2-1,2)1,1 (1,0-1,1)1,0 (1,0-1,1)114.8861,1 (1,1-1,1)1,2 (1,1-1,2)1,0 (1,0-1,1)1,0 (1,0-1,1)
*Enfermero vacunado* No282.916276.084 Sí119.9641,2 (1,1-1,2)1,2 (1,1-1,2)0,9 (0,9-0,9)0,9 (0,9-0,9)116.6411,1 (1,1-1,1)1,1 (1,1-1,2)0,9 (0,9-0,9)0,9 (0,9-0,9)[^5][^6][^7]
Discusión {#sec0040}
=========
Las coberturas de vacunación antigripal en los trabajadores de atención primaria obtenidas en el presente estudio se situaron en torno a las registradas en otros países europeos[@bib0150], y tuvieron un efecto en la población que atendían en términos de cobertura vacunal y probablemente en número de gripes declaradas. No se encontró asociación entre vacunación y días de IT en profesionales sanitarios, pero sí un posible efecto protector en la población general que había recibido la vacuna.
Las coberturas encontradas en sanitarios, próximas del objetivo planteado por el Ministerio de Sanidad, Servicios Sociales e Igualdad[@bib0180], aún distan del propuesto por los *Centers for Disease Control and Prevention*[@bib0145]. Otros estudios que revelan coberturas del 50-70%[@bib0185], [@bib0190] han podido sobreestimar sus resultados al utilizar encuestas autocumplimentadas, con una probable mayor predisposición a responder favorablemente[@bib0195]. La inclusión de las coberturas vacunales en incentivos[@bib0185], la vacunación de directivos o establecer un *feedback* han mostrado ser estrategias efectivas[@bib0200], [@bib0205], pero no fueron consideradas en este estudio. La mayor cobertura en población general se detectó en pacientes en atención domiciliaria; sin embargo, no dispusimos de datos con los que contrastar estos resultados. En cambio, en ≥ 65 años, el tercer grupo más vacunado, Blank et al.[@bib0150] encontraron cifras muy dispares en un estudio realizado en once países europeos, desde el 70,2% en el Reino Unido hasta el 13,9% en Polonia. En el presente estudio, esta cifra se situó en el 29,4%, valor inferior al registrado a nivel nacional (56,1% en 2015/2016) y muy por debajo de la meta propuesta por el Ministerio (65%)[@bib0180]. En cuanto a la menor cobertura hallada en embarazadas, resulta difícilmente comparable por la variabilidad en el registro y la semana en que se indica la vacunación; solo siete países europeos informan de sus coberturas[@bib0210].
En cuanto a la asociación entre vacunación antigripal y absentismo laboral en sanitarios, se ha encontrado un efecto protector con respecto a bajas por afecciones de tipo gripal o respiratorias[@bib0215]; sin embargo, otros hallazgos no defienden con suficiente evidencia una reducción de días por procesos respiratorios febriles[@bib0220]. Esto último coincide con los resultados del presente estudio, que podrían deberse, además de a las bajas coberturas registradas en los profesionales, a la baja actividad gripal que se registró en España en la temporada 2015-2016[@bib0225]. Asimismo, la magnitud de la asociación encontrada en la población general pudo verse también afectada por esa reducción en el número de gripes declaradas en la misma temporada.
Aunque no se ha medido el efecto de la vacunación de los profesionales en mortalidad poblacional como otros autores[@bib0230], sí se ha analizado su asociación con la cobertura y la morbilidad de la población atendida. Godoy et al.[@bib0190] concluyeron que las coberturas poblacionales son en parte consecuencia de la actitud de los médicos, siendo mayores en población ≥ 65 años cuando estos están vacunados. Por su parte, en un estudio realizado en Francia calcularon que una cobertura vacunal ≥ 35% en sanitarios protegería a los pacientes de la gripe adquirida en el hospital con una odds ratio de 0,07 (IC 95%: 0,005-0,98)[@bib0235]. El nivel de protección encontrado en nuestro trabajo, en el ámbito de atención primaria, se evidenció cuando el profesional vacunado era enfermero, con una cobertura vacunal del 25%. Aunque el efecto de la vacunación de los profesionales en la población general hallado en el estudio sea de pequeña magnitud, asociada probablemente a la baja actividad gripal que caracterizó a la temporada 2015-2016, no por ello se debe presuponer que su impacto sea reducido.
En nuestra opinión, una de las fortalezas del presente estudio es la potencia al incluir al conjunto de la población diana. El análisis de la información extraída directamente de las bases de datos originales ha permitido presentar unos resultados que reflejan el verdadero proceder de los profesionales y los efectos de la vacuna, en comparación con los cuestionarios autocumplimentados. Otro de los puntos fuertes es haber podido estudiar y comparar el comportamiento de la población en función de la conducta del médico y enfermero que la atendía en el mismo periodo de tiempo. Además, debido a la importante variabilidad climática que existe en la isla[@bib0240], y que las tasas de gripe llegan a ser superiores a las registradas en otras regiones del país[@bib0225], nuestros resultados son comparables a los de otros estudios de ámbito nacional. Sin embargo, también podemos detectar algunas limitaciones. Obviamente, la principal obedece al diseño transversal del estudio, que no permite establecer una relación causal entre la vacunación antigripal y el riesgo de tener gripe. Se utilizaron características de la población como variables de ajuste; no obstante, otras variables, como lugar de residencia, fármacos, nivel educativo, visita al centro de salud o negativa a la vacunación, podrían explicar parte de las asociaciones encontradas. Asimismo, no se excluyeron aquellos con contraindicaciones a la vacunación, como se ha hecho en otros estudios[@bib0190]. Otra limitación es haber considerado los procesos de 1 a 7 días como una aproximación a los procesos gripales; aunque estos procesos se resuelven mayoritariamente en unos días, otras afecciones, tales como procesos víricos no gripales, gastroenteritis, etc., pueden tener una duración similar y coexistir con estados gripales, lo que hace muy cuestionable la validez de esta variable. Además, aunque no se ha realizado confirmación microbiológica del virus, hasta el 23% de los sanitarios puede tener una serología positiva durante la temporada de la gripe, presentando una sintomatología leve o de manera subclínica[@bib0220]. Por último, otras infecciones del aparato respiratorio han podido ser declaradas como gripes, y viceversa.
En conclusión, los resultados de este estudio apoyan un posible efecto protector de la vacunación antigripal en la población general, así como la influencia que tienen los trabajadores de atención primaria en la conducta de los pacientes frente a la vacuna. Sin embargo, las coberturas en los sanitarios no alcanzan las mínimas establecidas para intentar garantizar la inmunidad de grupo. Sería conveniente ahondar en los motivos por los cuales este colectivo se muestra reticente a vacunarse. Conocimientos y creencias acerca de la efectividad de la vacuna, así como experiencias con vacunaciones previas, podrían ser áreas donde implementar medidas que propicien una actitud más favorable frente a la vacunación contra la gripe.Lo conocido sobre el tema•La gripe es una enfermedad contagiosa y con una gran carga de morbimortalidad en todo el mundo, principalmente en personas mayores de 65 años o con alto riesgo de complicaciones.•El personal sanitario, especialmente de atención primaria, está en riesgo de contraer la gripe al estar más expuesto en su trabajo, pudiendo actuar además como vector de su transmisión nosocomial.•Los estudios que analizan la repercusión que tiene la actitud de los profesionales sanitarios frente a la vacunación antigripal en la población que atienden son escasos y la mayoría se basan en resultados obtenidos mediante encuestas.Qué aporta este estudio•El uso de bases de datos originales permite mostrar resultados más fidedignos que los obtenidos mediante cuestionarios autocumplimentados.•La vacunación antigripal en los trabajadores de atención primaria tiene un impacto en la población que atienden en términos de cobertura vacunal y probablemente en incidencia de gripe.•Sería conveniente explorar los motivos por los que el personal sanitario y la población general no se vacunan para poder diseñar estrategias que mejoren la actitud de ambos colectivos frente a la vacunación antigripal.
Conflicto de intereses {#sec0055}
======================
Los autores declaran no tener ningún conflicto de intereses.
[^1]: p \< 0,05.
[^2]: Odds ratio bruta.
[^3]: Odds ratio ajustada por edad, personal, equipo, categoría profesional y días de IT en el caso de la gripe, y odds ratio ajustada por edad, personal, equipo, categoría profesional y gripe en el caso de los días de IT.
[^4]: Odds ratio ajustada por edad, grupo de riesgo (65 años o más, embarazo, IMC ≥ 40 kg/m^2^, cuidador, enfermedad cardiovascular, \< 18 años tratados con ácido acetilsalicílico, atención domiciliaria, enfermedad respiratoria, enfermedad metabólica, enfermedad renal, enfermedad hepática, enfermedad neuromuscular, enfermedad hematológica, enfermedad inmunológica, enfermedad oncológica, enfermedad neurológica, enfermedad cognitiva, enfermedad auditiva) y mensaje recordatorio de vacunación.
[^5]: UAF: unidad de atención familiar.
[^6]: Odds ratio bruta.
[^7]: Odds ratio ajustada por edad, grupo de riesgo (65 años o más, embarazo, IMC ≥ 40 kg/m^2^, cuidador, enfermedad cardiovascular, \< 18 años tratados con ácido acetilsalicílico, atención domiciliaria, enfermedad respiratoria, enfermedad metabólica, enfermedad renal, enfermedad hepática, enfermedad neuromuscular, enfermedad hematológica, enfermedad inmunológica, enfermedad oncológica, enfermedad neurológica y enfermedad cognitiva) y mensaje recordatorio de vacunación.
|
{
"pile_set_name": "PubMed Central"
}
|
Bacteria exposed to antibiotics mount complex stress responses that promote survival^[@R9]--[@R14]^, and accumulating evidence suggests that inhibiting such responses potentiates antimicrobial activity in drug-sensitive, tolerant and resistant organisms^[@R2],[@R3],[@R5],[@R8],[@R15]--[@R18]^. In both prokaryotes and eukaryotes, genetic pathways underlying responses to environmental insults have been widely studied and involve some of the most phylogenetically conserved proteins known^[@R19]^. In eukaryotes, stress can also elicit epigenetic modification of histones and DNA that support long-lasting downstream responses^[@R20]--[@R23]^. The role of prokaryotic epigenomes in stress, however, is much less clear.
Bacteria lack histones, but harbor a diverse group of enzymes able to insert epigenetic modifications in the form of sequence-specific methylation of DNA bases^[@R24]^. Prokaryotic DNA methyltransferases (MTases) function either alone or as part of restriction-modification systems, participating in various cellular processes including anti-viral defense, cell cycle regulation, DNA replication and repair, and transcriptional modulation^[@R24]--[@R26]^. While several methylation-dependent epigenetic switches have been described^[@R27]--[@R32]^, genome-wide methylation patterns and kinetics have, until recently, been difficult or impossible to study in a high-throughput manner^[@R33]--[@R36]^. In this study, we use genetic and genomic tools to explore the function and behavior of the bacterial methylome during antibiotic stress.
To assess the role of DNA methylation in antibiotic stress survival, we first tested the ability of *E. coli* lacking different MTases to withstand sub-lethal doses of β-lactam antibiotics. Laboratory *E. coli* K12 possesses four functional MTases that methylate adenines or cytosines within distinct target sequences^[@R24],[@R36]--[@R40]^ ([Fig. 1a](#F1){ref-type="fig"}). Survival of sub-inhibitory ampicillin exposure by log-phase *E. coli* was unaffected in mutants lacking HsdM, YhdJ or Dcm MTases. However, bacteria deficient in DNA adenine methyltransferase (Dam) were highly susceptible to this low drug dose ([Fig. 1b](#F1){ref-type="fig"} and [Sup. Fig. 1a,b](#SD2){ref-type="supplementary-material"}). Increased ampicillin susceptibility in *dam*-deficient *E. coli* was also reflected in a reduced minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) ([Sup. Fig. 1c](#SD2){ref-type="supplementary-material"}). Complementation with a plasmid expressing *dam*, but not *gfp*, restored wild-type survival levels in Δ*dam E. coli* ([Fig. 1c](#F1){ref-type="fig"}, [Sup. Fig. 2a, b](#SD2){ref-type="supplementary-material"}). Because Dam might also behave as a transcriptional repressor independently of its DNA methyltransferase function^[@R37]^, we tested the ability of plasmids expressing previously characterized methylation-incompetent Dam variants^[@R38]^ ([Sup. Fig. 2a](#SD2){ref-type="supplementary-material"}) to rescue ampicillin hypersensitivity in Δ*dam E. coli*. Consistent with a role for GATC methylation, mutant Dam expression minimally altered the ampicillin hypersensitivity of Δ*dam E. coli*, if at all ([Sup. Fig. 2b, c](#SD2){ref-type="supplementary-material"}). Finally, we sought to determine whether Δ*dam E. coli* hypersensitivity extended to drugs other than ampicillin. Sub-inhibitory treatment with aztreonam, meropenem and cephalexin, other β-lactams commonly used in the clinic, was also significantly potentiated in the absence of *dam* ([Fig. 1d](#F1){ref-type="fig"}). Together, these results suggest that Dam-dependent methylation is important for bacterial survival during β-lactam stress.
Dam methylates GATC sites throughout the genome of organisms belonging to multiple orders of γ-proteobacteria, including the clinically relevant genera *Escherichia, Salmonella*, *Yersinia* and *Vibrio*^[@R24]^. To explore the behavior of the Dam methylome in the context of antibiotic pressure, we extracted genomic DNA from *E. coli* growing in the presence or absence of ampicillin stress, and analyzed genome-wide GATC methylation over time using single molecule real-time (SMRT) sequencing. With SMRT technology, epigenetic modifications on template DNA strands are inferred through the unique kinetic signature they engender during sequencing^[@R35],[@R39]^, and the fraction of DNA molecules methylated ('frac') at each GATC site is estimated ([Fig. 2a](#F2){ref-type="fig"}). In all samples, consistent with Dam's processive kinetics^[@R40]^, the majority of GATC sites were detected as methylated in a high fraction of DNA molecules sequenced (0.97±0.05 on average) ([Fig. 2b](#F2){ref-type="fig"}, [Sup. Data Set 1](#SD1){ref-type="supplementary-material"}). Notably, during the log-to-stationary phase transition, we identified 19 GATC sites that appeared transiently or stably non-methylated, or hemimethylated ([Fig. 2c, d](#F2){ref-type="fig"}; [Sup. Table 1, 2](#SD1){ref-type="supplementary-material"}; [Sup. Data Set 1](#SD1){ref-type="supplementary-material"}). Transiently non-methylated sites typically became steadily more or less methylated over time, following clear temporal patterns ([Fig. 2c, d](#F2){ref-type="fig"}). Because prokaryotes lack demethylases, non-methylated GATC sequences exist mainly where DNA-binding proteins sterically hinder Dam activity immediately following DNA replication^[@R24]^. Consistent with this notion, 18 of these 19 sites fell within intergenic regions, mostly overlapping with or closely neighboring footprints of transcription factors ([Sup. Table 1](#SD1){ref-type="supplementary-material"}). To our knowledge, only five of these sites have been previously reported as protected^[@R24],[@R41]--[@R44]^.
Remarkably, the GATC methylome and its kinetics were largely unaltered by ampicillin stress. The genome-wide distribution of frac values was similar in treated and untreated cells over time, indicating that global methylation levels were not increased or decreased by drug exposure ([Fig. 2b](#F2){ref-type="fig"}). Furthermore, methylation at the vast majority of GATC sites, including those displaying dynamic methylation patterns, remained unchanged by treatment ([Fig. 2d](#F2){ref-type="fig"}, [Sup. Table 2](#SD1){ref-type="supplementary-material"}, [Sup. Data Set 1](#SD1){ref-type="supplementary-material"}). Comparison of treated versus untreated samples at each timepoint revealed only one GATC site (site 19) displaying statistically significant differential methylation, which occurred at a single timepoint and only on one strand ([Sup. Table 2](#SD1){ref-type="supplementary-material"}, [Sup Fig. 3](#SD2){ref-type="supplementary-material"}). This event's biological consequence is unclear, however, as expression of the surrounding gene (*gdhA*) was unperturbed by ampicillin treatment (data not shown). Thus, ampicillin stress does not majorly alter the *E. coli* Dam methylome.
Given the remarkable stability of adenine methylation during antibiotic exposure and the contrasting drug-sensitive phenotype of *dam*-deletion mutants, we reasoned that the GATC methylome must provide structural rather than regulatory support for bacterial survival during antibiotic stress. Widespread genomic Dam methylation enables cellular processes requiring discrimination between the fully methylated parental DNA strand, and newly synthesized DNA whose GATC sites are not yet modified^[@R45]^. Specifically, transient hemimethylation at replication forks orients the methyl-dependent mismatch repair (MMR) system, guiding replacement of mismatched bases to nascent DNA strands only^[@R46]^. Importantly, without GATC methylation, the methyl-dependent endonuclease MutH can introduce double-strand breaks (DSB) near mismatches targeted for repair^[@R47]--[@R51]^. Mismatches are rare in log-phase *E. coli*^[@R52]^ (\<1 per replication cycle), but under conditions of stress, their frequency can increase due in part to induction of the error-prone polymerase IV (PolIV, encoded by *dinB*)^[@R53]--[@R55]^. We thus hypothesized that potentiation of β-lactam killing in the absence of Dam was a result of drug-induced mutagenesis fueling a genotoxic MMR pathway.
To test this, we assessed the effect of deleting *dinB*, *mutH* or the mismatch-binding component of the MMR complex, *mutS*, on antibiotic hypersensitivity in Δ*dam* bacteria. Strikingly, without the mutagenic polymerase PolIV, Δ*dam E. coli* survival of ampicillin stress returned to wild-type levels ([Fig 3a](#F3){ref-type="fig"}; [Sup. Fig. 4a](#SD2){ref-type="supplementary-material"}). Similarly, removal of *mutS* or *mutH* on the Δ*dam* background also abrogated ampicillin hypersensitivity ([Fig. 3b](#F3){ref-type="fig"}, [Sup. Fig. 4b](#SD2){ref-type="supplementary-material"}). In Δ*mutH* Δ*dam* bacteria, optical density (OD) was somewhat diminished in ampicillin ([Sup. Fig. 4b](#SD2){ref-type="supplementary-material"}), but this did not reflect decreased viability during treatment ([Fig. 3b](#F3){ref-type="fig"}). Further consistent with our hypothesis, we found that Δ*dam*, but not Δ*dinB* Δ*dam* or Δ*mutH* Δ*dam* bacteria, developed significantly more DNA damage than wild-type cells during ampicillin treatment, as assessed by terminal deoxynucleotidyl transferase nick end-labeling (TUNEL)^[@R56]^ ([Fig. 3c, d](#F3){ref-type="fig"}). Thus, without the GATC methylome, β-lactam-elicited PolIV introduces mismatches that are converted into lethal DNA strand breaks by a deleterious MMR system ([Fig. 3e](#F3){ref-type="fig"}).
The finding that genomic GATC methylation supports β-lactam stress survival in *E. coli* evokes the possibility of targeting Dam to therapeutically potentiate antibiotic drug activity. Dam is an attractive target as it lacks mammalian homologs but is conserved in several enteric pathogens^[@R57]--[@R59]^. Furthermore, because muliple drugs can induce mutagenic responses in bacteria^[@R9],[@R12],[@R55],[@R60]--[@R62]^, treatment with antibiotics other than β-lactams should also be potentiated in the absence of GATC methylation. Indeed, survival of *dam*-deficient *E. coli* in the presence of sub-inhibitory doses of the quinolones norfloxacin, ofloxacin and ciprofloxacin was severely compromised compared to wild-type bacteria ([Fig. 4a](#F4){ref-type="fig"}). As seen with ampicillin, hypersensitivity to ofloxacin could be abrogated by deleting *dinB*, *mutH* or *mutS,* in Δ*dam E. coli* ([Sup. Fig. 5a, b](#SD2){ref-type="supplementary-material"}). Consequently, drug potentiation in the absence of GATC methylation occurs via a similar mechanism across different antibiotic classes, and may be broadly exploitable.
Next, we sought to determine whether virulent clinical isolates could also be sensitized to treatment by the removal of Dam. As in *E. coli* K12, *dam* deletion in uropathogenic *E. coli* (UPEC) UTI89^[@R63]^ significantly increased sensitivity to ciprofloxacin ([Fig. 4b](#F4){ref-type="fig"}). Ciprofloxacin is a valuable drug for UPEC treatment, but its use is increasingly restricted by the spread of quinolone resistance^[@R64]^. To assess whether targeting Dam might allow re-sensitization of resistant strains, we deleted *dam* in a highly ciprofloxacin resistant (Cipro^R^) clinical UPEC isolate bearing multiple common quinolone resistance-conferring mutations ([Sup. Table 3](#SD1){ref-type="supplementary-material"}). Remarkably, though *dam* deletion did not restore full sensitivity to this isolate, the ciprofloxacin MIC for Cipro^R^ UPEC was reduced by over half, and its MBC~90~ by 4.6 fold ([Fig. 4c](#F4){ref-type="fig"}). Thus, removing GATC methylation can potentiate antibiotic lethality in both drug-sensitive and drug-resistant pathogenic organisms.
Together, our results define an important structural role for the bacterial epigenome in antibiotic stress survival. Characterization of the adenine methylome revealed highly stable global GATC methylation levels during log-to-stationary phase transition and sub-inhibitory β-lactam stress, and while we identified several previously uncharacterized GATC sites with variable methylation over time, antibiotic stress did not significantly alter these patterns.
Despite the remarkable stability of the GATC methylome, *E. coli* lacking Dam are hypersensitive to antibiotic stress. Deletion of *E. coli dcm* or *Neisseria meningitides* Mod11A (an adenine Mtase) was also reported to alter bacterial sensitivity to toxic compounds, but increased resistance, not hypersensitivity, was observed, and attributed to altered gene expression^[@R36],[@R69]^. While we cannot exclude additional involvement of transcriptional dysregulation, our data suggest that the GATC methylome represents an important backbone structure enabling DNA repair processes to function in the context of β-lactam and quinlone stress. Specifically, GATC methylation likely supports antibiotic-elicited mutagenesis dependent on PolIV, an error-prone polymerase induced transcriptionally or post-translationally in the presence of several antibiotics^[@R53]--[@R55]^. In the absence of GATC methylation, MMR machinery can convert post-replicative mismatches to DSBs^[@R47]^, which accumulate to toxic levels in mutagenizing drug-exposed *dam-*deficient bacteria. In Δ*dam E. coli,* the DNA damage response program (SOS) is constitutively sub-induced^[@R65]^. *dinB* is within the SOS regulon^[@R66],[@R67]^, thus Δ*dam E. coli* may be primed for rapid PolIV synthesis, enhancing their sensitivity. In addition, DNA breaks caused by MMR in mutating, drug-exposed Δ*dam* bacteria likely promote SOS pathway induction further, leading to more PolIV activity^[@R68]^. Consequently, during antibiotic stress, a toxic feedback loop may establish itself ([Fig. 3e](#F3){ref-type="fig"}). This model is consistent with earlier observations that DNA-damaging agents cause MMR-dependent genotoxicity in Δ*dam* bacteria^[@R49],[@R50],[@R69]--[@R71]^; however, our data further suggest that any initial DNA damage caused by antibiotics directly is not sufficient kill Δ*dam* bacteria, as the error-prone PolIV is required for hypersensitivity ([Fig. 3a](#F3){ref-type="fig"} and [Sup. Fig. 5a](#SD2){ref-type="supplementary-material"}). Measuring mutagenesis rates in Δ*dam* bacteria is challenging (due to MMR toxicity to mutating cells) and we cannot completely exclude a requirement for PolIV in introduction of initial DNA damage. This seems unlikely, however, given that similar levels of damage were recorded in wild-type and Δ*dinB* bacteria during drug treatment ([Fig. 3d](#F3){ref-type="fig"}). Thus, our data support a model in which antibiotic stress becomes lethal as mutagenic PolIV activity fuels a genotoxic MMR response in the absence of GATC methylation.
Our findings raise the possibility of targeting Dam to enhance the therapeutic activity of existing drugs. Several classes of antibiotics induce mutagenesis at sub-inhibitory concentrations^[@R9],[@R12],[@R55],[@R60]--[@R62]^, and may thus be subject to potentiation by this mechanism. While enhancement of drug activity could be harnessed to lower effective therapeutic doses in drug-sensitive infections, it may also allow re-sensitization of resistant organisms. Indeed, our data suggest that targeting Dam methylation can partially reverse ciprofloxacin resistance in UPEC. More broadly, this observation suggests that mutagenic stress responses can occur and be therapeutically exploited in highly drug-resistant pathogenic organisms. In addition to drug potentiation, inhibiting Dam has been proposed as a strategy to weaken bacterial pathogenicity in vivo^[@R25],[@R72]--[@R74]^, as GATC methylation controls virulence gene expression in some organisms. While elevated rates of mutagenesis and induction of certain prophages^[@R75]^ in the absence of Dam could complicate a Dam inhibitor-based monotherapy, these drawbacks may be mitigated in the context of combination treatment. In summary, our results suggest that targeting bacterial epigenomic structures that support mutagenic stress responses may be a viable strategy to enhancing antibiotic activity.
Online methods {#S1}
==============
Bacterial strains and plasmids {#S2}
------------------------------
Laboratory bacterial strains used are derived from *E. coli* K12 (BW25113 obtained from the Coli Genetic Stock Center or MG1655 obtained from ATCC). The uropathogenic *E. coli* strain UTI89 was kindly provided to us from Matt Conover and Scott Hultgren. The ciprofloxacin-resistant uropathogenic *E. coli* isolate (UPEC Cipro^R^) was collected from the Brigham and Women's Hospital Specimen bank ([Sup. Table 3](#SD1){ref-type="supplementary-material"}). Deletion mutants on the BW25113 background were derived from the KEIO collection following Kan^R^ cassette removal. Deletion mutants on the MG1655 background were constructed by allelic transduction from KEIO collection strains using classical P1 phage transduction, followed by Kan^R^ cassette excision. The *dam* null phenotype was confirmed by PCR alone or with electrophoresis of genomic DNA digested with DpnII, which cleaves unmethylated GATC sites only. For construction of the Δ*dam* UTI89 and Δ*dam* UPEC Cipo^R^ strains, the parent strain bearing a KM208 plasmid-based Red-recombinase system was electroporated with a PCR amplicon encoding the Δ*dam*::Kan^R^ allele. Recovered cells were selected for kanamycin-resistant homologous recombinants. The plasmid was cured and the Kan^R^ cassette was removed. The genotype of each deletion strain was verified by colony PCR. The plasmid used in the dam complementation studies, namely pZS\*31 ([Fig. 1c](#F1){ref-type="fig"}, [Sup. Fig. 2](#SD2){ref-type="supplementary-material"}), was obtained from Expressys and belongs to the pZ vector family. pZS\*31 has a pSC101\* origin of replication (which yields a low copy number of 3--5 plasmids per cell) and a chloramphenicol-resistance marker. Genes encoding either Dam (with 500bp upstream flanking region) or GFP were inserted into the multiple cloning site. For complementation experiments using mutated versions of *dam*, the plasmid containing the *dam* insert was engineered using either Gibson cloning or site-directed mutagenesis (NEB, Q5 site-directed mutagenesis kit). Quinolone resistance-conferring mutations in the Cipo^R^ UPEC clinical isolate were identified though whole-genome Illumina sequencing of genomic DNA (PureLinK Pro-96 Genomic Purification Kit; Life Technologies). Libraries were prepared as previously described^[@R76]^. Raw sequencing reads were processed by trimming adapter sequences and discarding reads shorter then 28bp. Processed reads were aligned to the *E. coli* MG1655 genome using breseq^[@R77]^. The genome alignments were searched for known quinolone resistance-conferring mutations in *acrA*, *acrR*, *beaS*, *cpxA*, *cpxB*, *envZ*, *gyrA*, *gyrB*, *marA*, *marR*, *mdtA*, *mdtB*, *mdtC*, *ompC*, *ompF*, *ompR*, *parC*, *parE*, *soxR*, *soxS* and *tolC* genes and their regulatory regions.
Bacterial kill curves, MBC and MIC determination {#S3}
------------------------------------------------
For timecourse kill curves and MBC assays, stationary-phase bacterial cultures were diluted at 1:1,000 in 25mLs of LB medium in 250mL baffled flasks. Cultures were grown at 37°C and 200rpm until they reached an OD of \~0.3. Cultures were transferred to 24-well plates at 500 μl per well, or to 96-well plates at a final volume of 150ul per well, and either left untreated or treated with the indicated drugs at specified doses. Plates were sealed using breathable membranes (BreatheEasy, Cat \#: BEM-1) and incubated at 37°C and 900rpm for the remainder of the experiment. CFUs were enumerated at desired time points (4 hours for MBC determination) by spot plating 5 μl of ten-fold serially diluted culture onto LB agar and counting colonies after overnight growth at 37°C. Percent survival at each timepoint was calculated in relation to the CFU immediately before treatment (0h). For MIC determination, antibiotics were serially diluted in a 96-well plate and mixed with stationary-phase bacterial cultures diluted at 1:10,000 in a final volume of 150 μl LB per well. OD was measured from plates after 24hrs of growth at 900rpm and 37C.
Genomic DNA extraction and PacBio sequencing {#S4}
--------------------------------------------
Genomic DNA (gDNA) was extracted from *E. coli* K12 MG1655 LB cultures grown in the presence or absence of ampicillin using the GenElute Bacterial Genomic DNA Extraction Kit (Sigma). To assess genomic methylation status, gDNA extracted from stationary-phase cultures was quantified, digested using DpnII (NEB) and run on an 0.8% agarose gel containing ethidium bromide. For methylome analyses, samples were sent to UMass Medical School Deep Sequencing Core, where methylome data were obtained by PacBio Core Enterprise instrument SMRT. SMRTbell™ DNA template libraries for SMRT sequencing were prepared according to the instructions described in the 'Procedure & Checklist for 10 kb Template Preparation and Sequencing' document (Pacific Biosciences). Briefly, genomic DNA samples were first sheared to a target shear size of 10kb using g-Tube devices (Covaris, Inc.), treated with DNA damage repair mix, end-repaired and ligated to hairpin adapters. The SMRTbell libraries were prepared using the DNA Template Prep Kit 2.0 (3--10kb) fro Pacific Biosciences. Incompletely formed SMRTbell templates were digested using Exonuclease III (New England Biolabs) and Exonuclease VII (Affymetrix). The prepared SMRTbell libraries were sequenced using a 120-min movie acquisition time and P4 polyerase-C2 DNA sequencing reagent kits following standard instructions for a PacBio RS II instrument (Pacific Biosciences). Each *E. coli* sample was sequenced on four or more SMRT cells yielding a total of approximately 200-fold double-stranded coverage of the bacterial genome, and two or three biological replicates were sequenced for each antibiotic treatment condition ([Sup. Data Set 2](#SD1){ref-type="supplementary-material"}). Sequencing coverage was comparable between methylated and non-methylated sites ([Sup. Table 1](#SD1){ref-type="supplementary-material"}, [Sup. Data Set 2](#SD1){ref-type="supplementary-material"}), ruling out coverage loss as an explanation for the absence of methylation.
Bioinformatics analyses of SMRT sequencing data {#S5}
-----------------------------------------------
Genome-wide detection of base modification and the affected motifs was performed using the standard (default) settings in the 'RS_Modification_and_Motif_Analysis.1' protocol included in SMRT Analysis version 2.3.0 Patch 5. The FASTA reference genome sequence (E. coli K12 MG1655, NCBI NC_000913.2) used for the base modification detection analyses was obtained from Pacific Biosciences. For motif identification, the base modification Quality Value (QV) threshold setting was left at the default value of 30. Interpulse duration (IPD) values were measured for all nucleotide positions in the genome and compared with expected durations in an *in silico* kinetic model of the polymerase for significant associations. 'Frac' values were calculated in SMRT Analysis using a standard mixture model analysis of the pooled kinetic data for a given sample. The frac output value provides information about the fraction of individual molecules displaying a methylation signal at each identified motif site within the genome ([Sup. Data Set 1](#SD1){ref-type="supplementary-material"}). Methylation frac values were derived from IPD data within the SMRT pipeline using the single site mixture model^[@R39]^. The value 0 was substituted for frac values that were below detection limits. The values from two or three experimental replicates were compared by Student's T-test and FDR adjusted *p*-values were obtained by the method of Benjamini and Hochberg ([Sup. Data Set 1](#SD1){ref-type="supplementary-material"}). Circular graphs were generated using the Circos software package
Flow cytometric assessment of DNA damage {#S6}
----------------------------------------
*E. coli* log-phase cultures were transferred to a 96-well plate (200 μl/well) and treated with ampicillin (2.5 μg/mL) or hydrogen peroxide (100mM) for 30 minutes to 2 hours at 37°C and 900rpm. Bacteria were pelleted by centrifugation at 3,000× g for 5 minutes. The supernatant was discarded. Cell pellets were resuspended vigorously in 200 μl of cold 4% paraformaldehyde/PBS and incubated at room temperature for 30 minutes to allow fixation. Bacteria were centrifuged again, then resuspended in 200 μl of cold permeabilization buffer (0.1%TritonX-100 in 0.1% sodium citrate). After 2 minutes at room temperature, bacteria were centrifuged and washed in PBS. After pelleting the cells and discarding the supernatant, cells were resuspended in 50 μl of TUNEL labeling mix (dUTP-FITC and TdT enzyme) or 50 μl TUNEL labeling reagent (dUTP-FITC) according to manufacturer's instructions (Roche; *in situ* cell death detection kit, fluorescein). Bacteria were stained for 1h at 37°C. Cells were then washed twice with PBS, resuspended in 1 μg/mL PI/PBS and analyzed by flow cytometry (BD LSR Fortessa). PI negative cells, which lack genomic material, were excluded from the analysis. Gating was determined using single color and unstained controls as references. For [Fig. 3d](#F3){ref-type="fig"} and statistical analysis, background staining with labeling reagent only was subtracted for each sample to account for treatment dependent shifts in auto-fluorescence or stain retention.
Statistical analyses {#S7}
--------------------
Statistical analysis performed on log10-transformed data (for survival experiments) or on untransformed data (for TUNEL assay) using a two-way ANOVA followed by a post-hoc t-test using Sidak's multiple comparison test correction. In all cases, p-values indicated are multiplicity adjusted.
Supplementary Material {#S8}
======================
We thank Tom Ferrante, Ewen Cameron, Kyle Allison, Jonathan Winkler, Jeff Way, Charley Gruber, Prerna Bhargava, Michio Painter, Nawang Sherpa, Tami Lieberman, Laura Certain and Yoshikazu Furuta for critical feedback and technical support over the course of this study. We also thank Matt Conover and Scott Hultgren for generously providing the UTI89 strain, as well as related reagents and protocols. Additionally, we are grateful for SMRT sequencing support from Maria Zapp and Ellie Kittler at the UMassMed Sequencing core, and from Sonny Mark, Khai Luong, Michael Weiand and Onureena Banerjee at Pacific Biosciences. This work was supported by funding from the Defense Threat Reduction Agency grant HDTRA1-15-1-0051, the National Institutes of Health grant 1U54GM114838-01, the Mayo Clinic Center for Individualized Medicine and Donors Cure Foundation, the Howard Hughes Medical Institute, the Banting Postdoctoral Fellowship from the Canadian Institutes of Health and Research, and the Wyss Institute for Biologically Inspired Engineering, Harvard University.
**Competing financial interests**
The authors have no competing financial interests to declare.
**Author contributions**
N. R. C. conceived of project, designed and performed experiments and wrote the manuscript; C. A. R. performed bioinformatics analyses. S. J. performed experiments; R. S. S. performed experiments and bioinformatics analyses; A. G. contributed intellectually to the project and helped design experiments; P. B. contributed intellectually to the project; H. L. performed bioinformatics analyses and provided mentorship; J. J. C. oversaw the project and provided mentorship.
{#F1}
{#F2}
{#F3}
{#F4}
[^1]: These authors have contributed equally to this study
|
{
"pile_set_name": "PubMed Central"
}
|
Background {#Sec1}
==========
MicroRNAs (miRNAs) are short non-coding RNAs that induce silencing and destabilization of messenger RNAs (mRNAs) by binding to specific target sites \[[@CR1], [@CR2]\]. Several studies have recently investigated miRNA and mRNA co-expression to identify post-transcriptional regulations involved in proliferative and degenerative diseases \[[@CR3]--[@CR8]\], based on the fact that the up/down-regulation of a miRNA causes the inverse down/up-regulation of its target mRNAs, and this would result in a negative correlation between miRNA and mRNA expressions. On the other hand, most studies have so far shown the co-existence of negative and positive miRNA-mRNA correlations, which are consistent with the presence of a more complex network that involves not only inhibition of miRNA targets (resulting in negative miRNA-mRNA correlations) but also feed-forward regulation activated by common transcription factors \[[@CR9]--[@CR11]\], resulting in positive miRNA-mRNA, miRNA-miRNA and mRNA-mRNA correlations. Moreover, there is increasing evidence for the existence of miRNA-miRNA \[[@CR12]--[@CR15]\] and mRNA-mRNA \[[@CR16], [@CR17]\] direct interactions.
A long recognized problem in correlation studies is the presence of covariates \[[@CR18]\], which result in spurious correlations that confound the true correlations. In gene expression studies of diseases, critical covariates are represented by the different degree or extension of the histopathological lesions of samples. This is particularly relevant to human tissues, whose histological conditions are not as homogeneous as in experimental laboratory models.
Another problem concerns the visualization of data. Genome-wide studies are able to assess the expression of more than 35,000 well-characterized human genes and more than 1000 mature miRNAs. Even restricting the study to a small subset of genes differentially expressed in specific diseases or experimental conditions, the number of potential correlations is very high, and needs robust multivariate methods to be conveniently summarized by a small set of significant data \[[@CR19]\].
These issues were addressed in this study, which was aimed at investigating the joint expression of miRNAs and mRNAs in pathologic livers obtained from patients with HBV-associated acute liver failure (ALF), a dramatic disease characterized by hepatocellular necrosis. Our previous studies in HBV-associated ALF have shown a prominent expression of B cell-related genes as well as of genes involved in liver regeneration and fibrogenesis \[[@CR20], [@CR21]\].
The study involved various steps. First, miRNAs and mRNAs differentially expressed in ALF were merged into a single gene-by-sample matrix. Then, partial nonparametric correlations between each gene pair (including all miRNA-mRNA, miRNA-miRNA and mRNA-mRNA combinations) were calculated to remove the effect of necrosis. Nonparametric correlations were transformed into nonmetric distances, and multidimensional scaling (MDS) was then applied to transform distances into spatial coordinates. MDS provided a comprehensive framework for thematic maps showing different features of the miRNA-mRNA, miRNA-miRNA and mRNA-mRNA interrelationships.
Methods {#Sec2}
=======
Patients and liver specimens {#Sec3}
----------------------------
Thirteen liver specimens were obtained at the time of liver transplantation from 4 patients with HBV-associated ALF. The demographic, clinical, biochemical, virological and histopathological data have been previously reported \[[@CR20], [@CR21]\]. The control group comprised 10 liver donors and 7 subjects who underwent hepatic resection for liver angioma. Liver specimens were received under code to protect the identity of the subjects. Written informed consent was obtained from each patient or the next of kin. The study received approval by the NIH Office of Human Subjects Research, granted on the condition that all samples were made anonymous.
RNA extraction and microarray analysis {#Sec4}
--------------------------------------
miRNA analysis was performed using Affymetrix GeneChip miRNA 2.0 arrays (Affymetrix, Santa Clara, CA), which contain 1105 pre-miRNA (mir-), and 1105 mature miRNA (miR-) probe sets, whose nomenclature refers to miRBASE release 15 \[[@CR22]\]. However, miRNAs removed from next miRBase releases were also excluded from the study. Total RNA was extracted from frozen liver specimens using the miRNeasy Mini Kit (Qiagen, Valencia, CA). 500 ng of total RNA, including microRNA, was poly(A)-tailed and then directly ligated to a fluorescent dendrimer (a branched single- and double-stranded DNA molecule conjugated to biotin) using the FlashTag Biotin HSR RNA Labeling Kit (Affymetrix). An ELOSA was performed prior to hybridization and analysis of the arrays in order to verify that all miRNAs were correctly labeled with the biotin molecule at the 3′ end. mRNA analysis was performed using Affymetrix Human U133 Plus 2 arrays, which contain 54,675 probe sets representing approximately 38,000 known human genes. Total RNA was extracted from frozen liver specimens using Trizol (Invitrogen, Life Technologies, Carlsbad, CA). Total liver RNA (50 ng) was subjected to two successive rounds of amplification. RNA quality and integrity were assessed with the RNA 6000 Nano Assay on the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Standard Affymetrix protocols were used for hybridization, staining, washing, scanning and quality control of the arrays \[[@CR23]\].
Statistical analysis {#Sec5}
--------------------
Raw microarray data (cel files) were imported into BRB-ArrayTools \[[@CR24]\] and probe set summaries were computed using the RMA algorithm. Multiple transcripts of known gene were averaged, whereas transcripts of unknown genes were discarded. Signed fold changes were calculated as the ratio between the geometric means of ALF and normal livers. A multivariate permutation F-test \[[@CR25]\] with a maximum false discovery rate of 1 % with 80 % confidence level identified 109 miRNAs and 3239 mRNAs differentially expressed in ALF. To ensure a more robust analysis, the number of mRNAs was then reduced to 531 by selecting only mRNAs with absolute fold changes \>5. The list and fold changes of miRNAs and mRNAs are shown in the Additional file [1](#MOESM1){ref-type="media"}: Tables S1-S2. Log~2~-transformed miRNAs and mRNAs expressions of ALF and control livers were merged into two separate genes × samples matrices, a 642 × 13 matrix for ALF livers, and a 642 × 17 matrix for control livers. Pairwise nonparametric partial Kendall correlations were calculated for the matrix of ALF livers, setting the degree of liver necrosis as covariate, whereas simple Kendall correlations were calculated for the matrix of normal livers, which were not affected by necrosis. The choice of a nonparametric correlation, rather than Pearson's correlation, was forced by the fact that the expression levels of miRNAs and mRNAs, particularly in ALF samples, were not normally distributed (data not shown). Correlations were then transformed into nonmetric distances using the formula: (−1) × Kendall tau, rather than 1 - Kendall tau, to maintain the data zero-centered. The distance matrices were finally processed by MDS to obtain a dimensionally reduced map of gene coordinates. The MDS method was preferred to analogous methods (i.e., Principal Coordinates) as it allows data to be preliminarily processed by partial nonparametric correlations. MDS was computed using the singular value decomposition (SVD) method \[[@CR19]\], which ensures a matrix factorization numerically accurate even in the presence of a high degree of multicollinearity (i.e., multiple correlation). Multivariate analyses and graphics were made using the following R functions available from the CRAN repository \[[@CR26]\]: pcor.test {ppcor}; svd {base}; sammon, isoMDS {MASS}; ordiellipse {vegan}; ellipse3d, plot3d {rgl}; lm, density {stats}; ppp {spatstat}. An example of R code of MDS achieved using SVD is shown in \[[@CR27]\]. MiRNA seed sequences and chromosomal loci were obtained from Affymetrix annotations to the GeneChip miRNA 2.0 \[[@CR23]\], and verified in miRBase \[[@CR22]\]. Only the chromosomal locus of miR-199a-3p was found in miRBase, but not in Affymetrix annotations. The identification of target mRNAs was obtained from the microRNA.org database \[[@CR28]\], selecting miRNA-mRNA pairs with conserved miRNAs and a good (\<= −0.1) mirSVR score.
Results and discussion {#Sec6}
======================
The effect of the tissue condition on spurious correlations {#Sec7}
-----------------------------------------------------------
In ALF livers, nonparametric correlations between each gene pair (including all miRNA-mRNA, miRNA-miRNA and mRNA-mRNA combinations) showed a prominent bimodal distribution due to a large number of highly negative and positive correlations, contrasting with the more regular, unimodal distribution of correlations of control livers (Fig. [1](#Fig1){ref-type="fig"}). This was an evident effect of the disease, as necrosis involves a loss of hepatocytes and an increase of infiltrating cells. Thus, pairs of genes prevalently expressed by hepatocytes (both ↓↓) or infiltrating cells (both ↑↑) result in spurious positive correlations, whereas pairs of genes expressed by hepatocytes and infiltrating cells (one ↓ and one ↑, respectively) result in spurious negative correlations. The impact of histopathological changes on the apparent gene expression was also indicated by the fact that about one third of genes differentially expressed in ALF livers correlated with the level of necrosis with very high correlation coefficients (\|R\| \> 0.9). This means that more than 81 % (R squared) of the overall variability of gene expression was due to the changes in the histological composition of ALF livers. A representative sample of genes positively and negatively correlated with necrosis with \|R\| \> 0.9 is shown in Fig. [2](#Fig2){ref-type="fig"}. We also calculated the regression between the level of necrosis (independent variable) and the mRNA concentration (dependent variables), in order to estimate the gene expression expected for zero necrosis (intercept). This involved a certain statistical licence, because zero necrosis was out of the range of data inputted in the model (some amount of necrosis is invariably present in all ALF livers).Fig. 1Frequency distribution comprehensive of all pairwise miRNA-mRNA, miRNA-miRNA and mRNA-mRNA Kendall correlations. **a** ALF livers. **b** normal livers. **c** ALF livers after partial correlations calculated for the level of necrosisFig. 2Correlation between gene expression and level of necrosis in ALF livers. The plots show a selection of representative mRNAs positively (*left*) or negatively (*right*) correlated with the degree of necrosis in ALF livers with \|R\| \> 0.9. The level of hepatic necrosis is on the X axis; the gene expression is on the Y axis. Presumably, mRNAs positively correlated with necrosis are produced by non-hepatocyte cells, whereas those negatively correlated with necrosis are produced by hepatocytes. Gene expressions were standardized to fit the same scale range. Multiple dots of the same gene (*color*) for each level of necrosis represent data of multiple samples
Using the same subset of genes as in Fig. [2](#Fig2){ref-type="fig"}, the mRNA levels of control and ALF livers, and those expected for ALF livers with zero necrosis, are shown in Fig. [3a-c](#Fig3){ref-type="fig"}. The correlation between the original mRNA levels of control and ALF livers was very low (*R* = 0.34, Fig. [3d](#Fig3){ref-type="fig"}). However, using the mRNA levels of ALF livers calculated for zero necrosis, the correlation became very high (*R* = 0.99, Fig. [3e](#Fig3){ref-type="fig"}). This finding confirmed not only that gene expressions were strongly biased by the level of necrosis, but also showed that the impact of necrosis could be effectively removed. It is also noteworthy that the analysis included both hepatocyte genes and non-hepatocyte genes (i.e., genes negatively and positively correlated with necrosis, respectively). This suggests that the opposite effects of the loss of hepatocytes and the increase of infiltrate were equally removed, thus making hepatocyte and non-hepatocyte gene expressions balanced and comparable to those of control normal livers. These preliminary findings prompted us to estimate the partial correlations for necrosis in order to investigate the genuine relationships among miRNA and mRNA gene expressions of ALF livers.Fig. 3Gene expression corrected for necrosis. Data refer to the same mRNAs shown in Fig. [2](#Fig2){ref-type="fig"}. **a** control livers. **b** ALF livers. **c** ALF livers data expected for zero necrosis, obtained as the intercept of the regression between necrosis (*X variable*) and gene expression (*Y variable*). **d** correlation between control livers and original ALF livers data. **e** correlation between control livers and ALF livers data expected for zero necrosis
Partial correlations and multidimensional scaling {#Sec8}
-------------------------------------------------
Partial nonparametric (Kendall) correlations of ALF livers showed a unimodal distribution, similar, although somewhat flatter, to that of control livers (Fig. [1c](#Fig1){ref-type="fig"}). Partial correlations were transformed into distances and then processed by MDS using the singular value decomposition method. MDS provided a general framework for thematic maps showing different features of miRNA-mRNA, miRNA-miRNA and mRNA-mRNA interrelationships (Figs. [4](#Fig4){ref-type="fig"}, [5](#Fig5){ref-type="fig"}, [6](#Fig6){ref-type="fig"}, [7](#Fig7){ref-type="fig"} and [8](#Fig8){ref-type="fig"} for ALF livers; Figs. [9](#Fig9){ref-type="fig"}, [10](#Fig10){ref-type="fig"}, [11](#Fig11){ref-type="fig"}, [12](#Fig12){ref-type="fig"} and [13](#Fig13){ref-type="fig"} for control livers). Though all 531 mRNAs differentially expressed in ALF livers were included in the analyses, for reasons of clarity only the symbols of a subset of 87 mRNAs, attributable to 9 well-defined functional groups (CYP450, transcription factors, complement, proliferation, HLA class II, monocytes/macrophages, T cells, T-NK cells and B cells, whose genes are listed in Table [1](#Tab1){ref-type="table"}) are shown in MDS maps. The remaining mRNAs are graphically represented by points.Fig. 4MDS mapping of mRNAs differentially expressed in ALF livers. MDS was applied to a 640 × 640 square matrix including the Kendall correlations among 109 miRNAs and 531 mRNAs differentially expressed in ALF livers. This figure shows only a subset of 87 mRNAs functionally related to CYP450, transcription factors, complement, HLA class II, monocytes/macrophages, T cells, T-NK cells and B cells. These nine functional classes are delimited by dispersion ellipses with a confidence of 1.6 standard deviations. A 360° rotation of 3D ellipses is shown in the Additional file [2](#MOESM2){ref-type="media"}: Movie 1. The nine ellipses are also shown in the next Figs. [5](#Fig5){ref-type="fig"}, [6](#Fig6){ref-type="fig"}, [7](#Fig7){ref-type="fig"} and [8](#Fig8){ref-type="fig"} for reference. The numbers in parentheses, on the right of gene symbols, are the fold changes of original data, not corrected for necrosis. A prominent segregation of leukocyte-related mRNAs from hepatocyte-related mRNAs is apparentFig. 5MDS density plot of mRNAs differentially expressed in ALF livers. The density plot was calculated for the MDS map of 531 mRNAs differentially expressed in ALF livers, including the 87 mRNAs of Fig. [3](#Fig3){ref-type="fig"} (shown by large dots, symbols, original fold changes and dispersion ellipses) and the remaining 444 mRNAs (shown by small dots)Fig. 6MDS density plot of miRNAs differentially expressed in ALF livers. The numbers in parentheses, on the right of miRNA symbols, are the fold changes of original data, not corrected for necrosis. The 17 green-outlined points are the miRNAs whose median correlation with mRNAs decreased more than 0.15 Kendall tau in ALF livers, whereas the red-outlined point is the only miRNA whose median correlation increased more than 0.15 Kendall tau (Additional file [4](#MOESM4){ref-type="media"}: Figure S1). The dispersion ellipses of functional mRNA clusters are shown for reference. The inset shows the complementarity of miRNA (cyan) and mRNA (yellow) MDS density plotsFig. 7MDS mapping of the network of miRNAs and target mRNAs differentially expressed in ALF livers. For clarity, only the 87 mRNAs of the nine functional groups are shown. Target mRNAs were obtained from the microRNA.org database, selecting miRNA-mRNA pairs with conserved miRNAs and a good (\<= −0.1) mirSVR score. The dispersion ellipses of functional mRNA clusters are shown for referenceFig. 8MDS mapping of miRNAs with the same seed sequence, found among miRNAs differentially expressed in ALF livers. MiRNAs with the same seed sequence are encircled by small ellipses. Small blue and red ellipses indicate down-regulated and up-regulated miRNAs, respectively. The dispersion ellipses of functional mRNA clusters are also shown for reference. The complete sequence of these miRNAs is shown in the Additional file [5](#MOESM5){ref-type="media"}: Table S3Fig. 9MDS mapping of mRNAs expressed in control livers. This figure is to be compared with Fig. [4](#Fig4){ref-type="fig"}. A 360° rotation of 3D ellipses is shown in the Additional file [3](#MOESM3){ref-type="media"}: Movie 2. The nine ellipses are also shown in next Figs. [10](#Fig10){ref-type="fig"}, [11](#Fig11){ref-type="fig"}, [12](#Fig12){ref-type="fig"} and [13](#Fig13){ref-type="fig"} for reference. The numbers in parentheses, on the right of gene symbols, are the fold changesFig. 10MDS density plot of mRNAs expressed in control livers. This figure is to be compared with Fig. [5](#Fig5){ref-type="fig"}Fig. 11MDS density plot of miRNAs expressed in control livers. This figure is to be compared with Fig. [6](#Fig6){ref-type="fig"}Fig. 12MDS mapping of the network of miRNAs and target mRNAs expressed in control livers. This figure is to be compared with Fig. [7](#Fig7){ref-type="fig"}Fig. 13MDS mapping of miRNAs with the same seed sequence expressed in control livers. This figure is to be compared with Fig. [8](#Fig8){ref-type="fig"}Table 1Functional classes of genesCYP family CYP8B1 (-19), CYP4F3 (-28), CYP4F2 (-18), CYP4A11 (-7), CYP3A7 (-6), CYP3A5 (-5), CYP3A4 (-7), CYP39A1 (-7), CYP2J2 (-6), CYP2E1 (-14), CYP2D6 (-8), CYP2C9 (-8), CYP2C8 (-12), CYP2C19 (-7), CYP2C18 (-7), CYP2B6 (-10), CYP26A1 (-5), CYP1A2 (-14), CYP1A1 (-7)Transcription factors NR1I3 (-7), NR1I2 (-6), HNF4A (-5), FOXA3 (-7)HLA class II HLA-DRA (7), HLA-DQB1 (7), HLA-DQA1 (14), HLA-DPB1 (9), HLA-DPA1 (6), HLA-DOA (8), HLA-DMB (6), HLA-DMA (10), CD74 (6)B cells TNFRSF17 (14), SEL1L3 (6), POU2AF1 (32), MZB1 (18), IGLL3P (10), IGLJ3 (6), IGKV4-1 (7), IGKV1-5 (12), IGKC (13), IGHM (12), IGHD (5), FCRL5 (11)Monocytes/macrophages CD86 (5), CD163 (5), C1QC (6), C1QB (13), C1QA (10)Cell proliferation ZWINT (5), TOP2A (7), RRM2 (7), PRR11 (7), PRC1 (5), NDC80 (5), DLGAP5 (6), CDC20 (6), CCNB1 (5), BUB1B (5), ANLN (5), AKR1B10 (18)T cells VTCN1 (6), VSIG4 (10), TRAC (5), LAX1 (5), CD8A (7), CD3D (5), CD2 (8)T-NK cells SLAMF7 (9), RASGRP1 (7), PRDM1 (6), NKG7 (6), KLRK1 (6), GZMK (5), GZMH (7), GZMB (13), GZMA (12), GNLY (6)Complement^a^ C9 (-118), C8G (-6), C8B (-36), C8A (-39), C6 (-10), C5 (-15), C4BPB (-20), C4BPA (-18), C3P1 (-19), C1S (-6)In parentheses are the fold changes of original data, not corrected for necrosis^a^Complement components C1QC, C1QB and C1QA were attributed to monocytes/macrophages as these proteins are mostly produced by these cells
miRNA and mRNA co-expression maps {#Sec9}
---------------------------------
ALF livers showed a clear segregation of leukocyte-related mRNAs (HLA class II, monocytes/macrophages, T cells, T-NK cells and B cells) from hepatocyte-related mRNAs (CYP450, transcription factors, complement) (Fig. [4](#Fig4){ref-type="fig"}), at variance with normal liver mRNAs which were densely interconnected (Fig. [9](#Fig9){ref-type="fig"}). A 360° rotation of the MDS maps of these mRNAs in ALF and control livers is shown in Additional files [2](#MOESM2){ref-type="media"} and [3](#MOESM3){ref-type="media"}: Movies 1--2. A clustered arrangement of the five groups of leukocyte-related mRNAs was also evident in ALF livers, in spite of the wider overall spreading. A major overlap was found between T and T-NK cell mRNAs, consistent with the strong functional interrelation of T and NK cells. Interestingly, MDS enabled the identification of a unique relationship between T and T-NK cell mRNAs and B cell mRNAs in correspondence of regulatory genes, but not of Ig genes, which encode the terminal effectors of humoral immunity. In addition, B cell mRNAs were located in a region characterized by the lowest mRNA density (Fig. [5](#Fig5){ref-type="fig"}). This suggests that B cell mRNAs were those which deviated more markedly from the configuration of normal livers. Paradoxically, the region with the lowest density of mRNAs was also the one with the highest density of miRNAs, revealing a sort of complementarity of miRNA and mRNA maps on a large scale (Fig. [6](#Fig6){ref-type="fig"}, inset). A similar complementarity was also seen in normal livers (Fig. [11](#Fig11){ref-type="fig"}). Other nonmetric MDS methods such as Kruskal's MDS and Sammon's mapping \[[@CR29], [@CR30]\] produced MDS maps substantially similar to those obtained using Kendall correlation. On the other hand, less comparable patterns were obtained using MDS based on metric (i.e., Euclidean) distances.
Numerical comparison of miRNA-mRNA relationships in ALF and control livers {#Sec10}
--------------------------------------------------------------------------
To compare numerically the miRNA-mRNA relationships in ALF and control livers, for each miRNA we computed the median correlation between that miRNA and all mRNAs (Additional file [4](#MOESM4){ref-type="media"}: Figure S1). Using an arbitrary threshold of ± 0.15 Kendall tau, a decreased correlation was found in 17 miRNAs (miR-143-star, 625, 542-5p, 30c-1-star, 18a-star, 200a, 629, 150, 125b-2-star, 30e, 155, 154, 192-star, 30a, 15a, 487a, 148a), all located within or very close to the B and T cell mRNAs in the MDS map of ALF livers (shown as green-outlined points in Fig. [6](#Fig6){ref-type="fig"}). Conversely, an increased correlation was found only in a single miRNA (miR-665), located on the opposite side of the MDS map (shown as a red-outlined point in Fig. [6](#Fig6){ref-type="fig"}). The discrepancy between these two findings is in agreement with the inhibitory effect of miRNAs.
MDS location of miRNA-mRNA target pairs {#Sec11}
---------------------------------------
We also mapped miRNA-mRNA target pairs (Figs. [7](#Fig7){ref-type="fig"} and [12](#Fig12){ref-type="fig"}). In general, miRNAs and target mRNAs were located far apart from each other. In view of the fact that the distance in the MDS plot accounts for a negative correlation, this finding appeared to be suggestive of the inhibitory relationship between miRNA-mRNA target pairs. To test this hypothesis, we simulated an alternative MDS plot by inverting the sign of miRNA-mRNA correlations. Surprisingly, the average distance between miRNAs and their target mRNAs was unchanged. This means that the distance between single miRNA-mRNA pairs does not reflect only the negative regulation of miRNA targets, but also positive feed-forward co-expressions mediated by transcription factors \[[@CR9]--[@CR11]\]. This hypothesis is consistent with the symmetric distribution of positive and negative miRNA-mRNA correlations observed in this and previous studies \[[@CR3]--[@CR7], [@CR31]--[@CR33]\]. On the other hand, it must be also considered that the 2D MDS map does not exhaust the whole multidimensional structure of data. We therefore performed a multiple regression between the mirSVR score, a conventional estimate of mRNA down-regulation calculated for each miRNA-mRNA pair \[[@CR28]\], and the distance between the same miRNA-mRNA pair in each of the first 50 MDS dimensions. This analysis showed a statistically significant relationship (*p* = 0.032), although predictively poor (multiple R-squared = 0.131), between mirSVR scores and MDS distances. By contrast, the same test performed on control livers was not statistically significant. This may be attributed to the fact that the miRNAs and mRNAs under investigation were those differentially expressed in ALF livers.
MDS location of miRNAs with the same seed sequence {#Sec12}
--------------------------------------------------
Finally, we also mapped 8 groups of miRNAs which showed the same seed sequence (Figs. [8](#Fig8){ref-type="fig"} and [13](#Fig13){ref-type="fig"}). The complete sequence and chromosomal origin of these miRNAs is reported in the Additional file [5](#MOESM5){ref-type="media"}: Table S3. Some miRNAs were similar through their entire sequence, differing by just one or two bases. Two miRNAs in particular (199a-3p and 199b-3p) were perfectly identical despite the different chromosomal origin; thus that their distance, however small, could be ascribed to purely technical factors. On the other hand, other miRNAs (i.e., miR-221 and 222; miR-30b and 30a/30e) showed several differences in the base sequence. Interestingly, in ALF livers these miRNAs were located in the B-cell region.
Knowledge-based and knowledge-independent methods {#Sec13}
-------------------------------------------------
The functional characterization of a set of differentially expressed genes is generally based on enrichment analysis \[[@CR34]\] using Gene Ontology \[[@CR35]\] and/or other gene annotations. A major limit of these methods is that they depend on the knowledge publicly available at the time of the investigation. This is particularly relevant, for example, for miRNAs whose list is continuously growing (known mature human miRNAs are at present 2588, but they were only 313 about ten years ago), and the number of miRNA targets experimentally validated is only a minimal fraction (less than 2 %) of those numerically predicted. Alternatively, gene expressions can be investigated using knowledge-independent methods. One of the most used methods is hierarchical clustering, often associated with heat maps. On the other hand, hierarchical clustering methods are strongly sensitive to the linkage method adopted and, in addition, their standard output, the tree diagram, is not adequate to represent a relational network. A more suitable but less used method is MDS, which allows a number of useful options such as the preliminary control of covariates and the adoption of nonmetric distances, less sensitive to nonlinear relationships.
Hidden covariates and confounding factors {#Sec14}
-----------------------------------------
The problem of covariate interference is of great importance in correlations studies, in particular when large ('omics') data sets are investigated from tissues affected by pathological alterations, including, but not limited to, tumors, inflammation, necrosis and fibrosis, present with different severity in different samples, and whenever gene expression profiles are associated to phenotypic traits \[[@CR36]\]. In this study, it was relatively easy to recognize necrosis as a confounding covariate, as samples where histologically characterized. We have shown that removing the effect of necrosis (re)establishes a linear relationship between gene expressions of control and ALF livers, including both hepatocyte and non-hepatocyte-related genes (Figs. [2](#Fig2){ref-type="fig"} and [3](#Fig3){ref-type="fig"}). However, it is very likely that other latent covariates may exist, possibly associated with the origin of ALF, rather than with its final outcome resulting in necrosis. But the biological factors and genetic predisposition involved in ALF pathogenesis are still largely unknown. A drastic alternative would be that of calculating partial correlations 'within' genes (i.e., each gene versus all others, as in some prediction-oriented methods), but this would also remove the genuine interactions at the molecular level, representing gene co-regulations.
Emerging evidences for a complex regulatory network {#Sec15}
---------------------------------------------------
mRNA destabilization induced by miRNAs has been successfully demonstrated in strictly controlled experimental conditions by hyper-expressing or silencing a single or a few miRNAs at a time \[[@CR37], [@CR38]\], but this is less achievable in observational studies, due to the simultaneous presence of a high number of genes differentially expressed. On the other hand, the balanced number of positive and negative miRNA-mRNA correlations observed in this and previous studies \[[@CR3]--[@CR7], [@CR31]--[@CR33]\] is consistent with the presence of a complex network involving not only the inhibitory regulation of miRNA-targeted mRNAs, but also feed-forward regulations of both miRNAs and mRNAs, activated by common transcription factors \[[@CR9]--[@CR11]\], as well as miRNA-miRNA \[[@CR12]--[@CR15]\] and mRNA-mRNA \[[@CR16], [@CR17]\] direct interactions.
Conclusions {#Sec16}
===========
The symmetric distribution of positive and negative correlations between miRNA and mRNA expression suggests that miRNAs are involved in a complex bidirectional molecular network including, but not limited to, the inhibitory regulation of miRNA targets. Different features of this network can be represented as thematic maps within the framework of a MDS analysis applied to the whole set of pairwise correlations. MDS made it possible to visualize: (a) a prominent displacement of miRNAs and mRNAs in ALF livers, indicative of gene expression dysregulation; (b) a clustering of mRNAs consistent with their functional annotation; (c) a tendency of miRNAs and mRNAs to populate distinct regions of MDS; (d) a map of miRNA-mRNA target pairs.
Availability of supporting data {#Sec17}
===============================
MicroRNA and mRNA microarray data sets supporting the results of this article are available in Gene Expression Omnibus at <http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE62037>.
Additional files {#Sec18}
================
Additional file 1: Table S1.List of 109 miRNAs differentially expressed in patients with HBV-associated acute liver failure (ALF). **Table S2.** List of 531 mRNAs differentially expressed in patients with HBV-associated acute liver failure (ALF). (XLSX 37 kb)Additional file 2: Movie 1.360° rotation of 3D MDS dispersion ellipses of mRNA functional clusters of ALF livers. Labels are shown in the two last frames (38--39) at the end of the rotation cycle. If the media player runs continuously, it should be stopped manually to visualize the labels. The original frame size is 731x712. The player window should be properly resized to optimize the image quality. (SWF 1150 kb)Additional file 3: Movie 2.360° rotation of 3D MDS dispersion ellipses of mRNA functional clusters of control livers. Labels are shown in the two last frames (38--39) at the end of the rotation cycle. If the media player runs continuously, it should be stopped manually to visualize the labels. The original frame size is 731x712. The player window should be properly resized to optimize the image quality. (SWF 1101 kb)Additional file 4: Figure S1.Median Kendall correlations between each miRNA and all mRNAs, in normal and ALF livers. The vertical distance between each data point and the diagonal (equality line) indicates the correlation change. An arbitrary threshold of ±0.15 Kendall tau is traced by the dashed lines parallel to the main diagonal. Based on this threshold, seventeen miRNAs (green symbols) show a decreased correlation in ALF livers, while only one miRNA (red symbol) shows an increased correlation. (PDF 258 kb)Additional file 5: Table S3.MiRNAs with the same seed sequence, found among the 109 miRNAs differentially expressed in patients with HBV-associated acute liver failure (ALF). (XLSX 12 kb)
**Competing interests**
The authors declare that they have no competing interests.
**Authors' contributions**
GD conceived the study, performed the statistical analysis and wrote the manuscript. FZ contributed to the design of the study and provided liver biopsy specimens. AT carried out RNA extractions and microarrays. PF participated in the design and coordination of the study and helped to write the manuscript. All authors read and approved the final manuscript.
Funding {#FPar1}
=======
This work was supported by the Intramural Research Program of the National Institutes of Health, National Institute of Allergy and Infectious Diseases; and Regione Autonoma della Sardegna \[grant number CRP-60086\] to GD.
|
{
"pile_set_name": "PubMed Central"
}
|
I[NTRODUCTION]{.smallcaps} {#sec1-1}
==========================
Pain is a prevalent symptom of cancer, affecting approximately 75% of people with advanced cancer\[[@ref1]\] and almost all patients with end-stage disease.\[[@ref2]\] The World Health Organization pain ladder is the cornerstone of pain management, with opioids playing a major role in the treatment of cancer pain. Morphine by mouth, in immediate-release or extended formulations, is the strong opioid of choice for cancer pain globally.
Morphine is a phenanthrene opioid receptor agonist. Bioavailability of oral morphine is 20%--40%. Its main effect is binding to and activating μ-opioid receptor in central nervous system. Activation of μ-opioid receptor is associated with analgesia, sedation, euphoria, physical dependency, and respiratory depression. It also has a weak kappa agonist activity. It is mainly metabolized to morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G). While M6G has more potent analgesic activity than morphine, M3G might be responsible for side effects, especially hyperalgesia/allodynia and myoclonus seen after high-dose morphine administration. Both M3G and M6G accumulate in patients with renal failure. Hence, its use is restricted by renal failure which increases the adverse events (AEs) such as nausea, vertigo, constipation, and respiratory depression culminating in poor analgesic tolerance. Further, the limitation of morphine use is imposed by cultural barriers, unavailability, and political ideology.
Buprenorphine is a newly developed semisynthetic opioid derivative of thebaine having morphine-like pure μ-agonistic and antagonistic activities at kappa opioid receptor.\[[@ref3]\] High lipid and water solubility, low molecular weight of 467 g/mol, and low melting point highlight the unique physicochemical properties of buprenorphine, making it a suitable candidate for transdermal (TD) administration.\[[@ref4]\] It has been tested widely and extensively in postoperative and chronic pain\[[@ref5][@ref6][@ref7][@ref8]\] and pain in myocardial infarction.\[[@ref9]\] Use of buprenorphine in the sublingual or TD patch has been evaluated in cancer pain management, but data on comparative studies with oral morphine are limited. As oral morphine use is often restricted due to AEs, alternative opioids such as buprenorphine TD patch remain a good choice to circumvent the toxicities of severe nausea, vomiting, and constipation. Different TD strengths are available with 10 μg/h and 20 μg/h being available from our hospital pharmacy. The limited comparative studies had prompted us to design this study to compare the efficacy and adverse effects of buprenorphine TD (20 μg/h, extended 7-day formulation) to that of oral morphine (immediate-release formulation) in the pain management of cancer patient.
Aims and objectives {#sec2-1}
-------------------
A randomized open-labeled prospective study was done at a palliative cancer pain clinic in a tertiary care medical college with the following objectives:
To compare the efficacy of TD buprenorphine with oral morphineTo compare the AEs of TD buprenorphine with oral morphine.
M[ATERIALS AND]{.smallcaps} M[ETHODS]{.smallcaps} {#sec1-2}
=================================================
Patients with solid tumor malignancies, suffering from moderate-to-severe chronic pain (visual analog scale \[VAS\] \>40) attending our palliative care clinic, were included in our study according to the inclusion and exclusion criteria. Patients in the age group of 18--70 years, with duration of pain \>3 months, were included in the study, provided that they had not received any opioid group of drugs over the past 3 months. In this regard, hospital records and physicians\' prescriptions were scrutinized. Only analgesics allowed before the study entry were paracetamol, ibuprofen, and diclofenac sodium along with adjuvants such as dexamethasone, pregabalin, and benzodiazepines. Patients were needed to understand VAS and respond appropriately. Minimum life expectancy of \>2 months along with no signs of neurological deficits was another inclusion criterion for the study. While all patients recruited were entitled to give consent, patients\' caregivers were also counseled regarding potential toxicities arising from drugs\' administration and their remedies. Patients recruited in this study were randomized into two arms using computer-based randomization with 1:1 ratio.
The patients in study arm A (experimental arm) received TD buprenorphine (20 μg/h, over a period of 7 days); replaced every 7 days. Patients in Arm B (control arm) received oral morphine (immediate-release formulation) 10 mg/tab in 4 hourly divided dose, with dose modifications allowed every 7 days.
Additional drugs allowed were paracetamol (1 g every 6 h, maximum of 4 g daily dose in individuals with normal liver function tests), diclofenac sodium (50 mg QDS), lorazepam (1 mg), and short-course dexamethasone (8 mg BDPC for 3--4 days) and other adjuvants such as pregabalin or amitriptyline as per requirements. Both the drugs were available through hospital pharmacy with appropriate permission from the government health and narcotic departments.
The patients were assessed by the VAS weekly for at least four observations. For Arm A patients, if subsequent VAS scores were \>40, then the dose of TD buprenorphine was increased by 20 μg/h every week. For Arm B patients, oral morphine was increased by 30 mg/day if VAS scores were \>40.
The occurrence of AEs was measured in both the arms using Common Terminology Criteria for Adverse Events v4.1. Statistical analysis was done using Statistical analysis was done using R software v3.5.0, (Vinnea, Austria).
R[ESULTS]{.smallcaps} {#sec1-3}
=====================
The study was conducted between August 2017 and January 2018. A total of 63 patients fulfilling our selection criteria were analyzed. Baseline parameters were comparable \[[Table 1](#T1){ref-type="table"}\]. The most common sites of primary cancer were the breast in females and the head and neck in males in both the arms. Baseline VAS scores were comparable in both the arms. The initial VAS scores of Arm A and Arm B were 81.25 and 82.26, respectively \[[Table 2](#T2){ref-type="table"}\]. At the end of the 1^st^ week, 11 Arm A patients were relieved from pain (VAS ≤40 mm) and the rest needed an increase in the dose of TD buprenorphine by 20 μg/h. Another 17 patients of Arm A became pain free at the end of the 2^nd^ week with a total dose of 40 μg/h. Only four patients needed 60 μg/h dose to get relief from the pain.
######
Baseline profile comparison between the two study arms
######
Changes in visual analog scale scores over the study period
In Arm B, two patients were relieved by 1 week with a dose of 30 mg/day of oral morphine. Eleven patients were relieved by 60 mg/day at the end of the 2^nd^ week and were stable with this dose. Twelve patients were relieved with 90 mg/day at the end of the 3^rd^ week and were stabilized with that dose. Six patients were relieved with 120 mg/day dose at the end of the 4^th^ week and were stabilized with that dose of oral morphine.
Common side effects in both the arms were constipation and nausea. Both nausea and constipation were statistically higher in Arm B compared to that of Arm A. However, the toxicities were manageable, and no patient was discontinued from the study due to toxicity \[[Table 3](#T3){ref-type="table"}\].
######
Adverse events on both arms
Our study was not designed to measure changes in quality-of-life parameters due to narcotic administration, which is one of the drawbacks of our study.
D[ISCUSSION]{.smallcaps} {#sec1-4}
========================
The benefits of sublingual and TD buprenorphine in the treatment of chronic cancer pain were demonstrated in a number of randomized trials as well as cohort studies.\[[@ref5][@ref6][@ref7][@ref8]\] The TD formulation offered several advantages such as noninvasive and controlled release and stable plasma concentration of buprenorphine. In the year 2001, the buprenorphine TD delivery system was introduced to deliver the drug at 35, 52.5, and 70 μg/h for the treatment of chronic cancer and noncancer pain. Later on, 10 μg/h and 20 μg/h "7-day extended release" formulations were commercially available. There are conflicting reports on "daily morphine equivalent dose" of TD buprenorphine dose, with some studies mentioning TD buprenorphine to be 70--115 times more potent than PO morphine.\[[@ref10][@ref11][@ref12][@ref13]\]
Poulain *et al*. in a randomized multicentric, double-blinded, placebo-controlled trial showed efficacy and safety of TD buprenorphine (70 μg/h) in 289 patients with severe cancer pain. Patients were randomized to receive either buprenorphine or placebo patches during the 14-day double-blind phase. The superior efficacy of buprenorphine during the double-blind phase was statistically significant despite the high placebo effect of the patch. This was also confirmed by secondary end-points such as pain intensity and consumption of rescue medicine. AEs were comparable in both arms.\[[@ref14]\]
Efficacy of TD and sublingual buprenorphine has been proven in numerous trials for cancer and noncancer pain.\[[@ref15][@ref16][@ref17][@ref18]\] In a multicentric Polish trial, TD buprenorphine was evaluated for efficacy and AEs in patients with moderate-to-severe cancer pain and in patients with severe, nonmalignant pain in the course of other diseases, after failure with nonopioid analgesics. The common dosage was TD buprenorphine (Transtec^®^ 35, 52.5, and 70 μg/h).
Cancer patients accounted for 81% of the 4030 patients. Mean pain intensity on VAS (0--100 mm) gradually decreased from a mean value of 62.5 mm at the baseline visit to the value of 16.5 mm at the final study assessment. Although no specific mention was made on benefits of cancer patients, this postmarketing study definitively confirmed high efficacy and good tolerability of buprenorphine.\[[@ref19]\]
In our study, we also found similar outcome in both the arms, the dose stabilized within 4 weeks with 1 week earlier in Arm A. The global score of quality of life also increased in both arms, but more in Arm A. The toxicities were mainly nausea, vomiting, and constipation in both arms and statistically significantly lower in Arm A.
Pace *et al*. compared TD buprenophine with oral morphine in which it was showed that patients receiving buprenorphine had significant physical pain relief as well as improvement in mental health and lower interference with sleep than those in morphine group. The toxicities (nausea, vertigo, and constipation) were significantly lower in TD buprenorphine group.\[[@ref2]\]
In one of the studies, 42 (21% with cancer) patients receiving high-dose morphine (\>120 mg/day) were switched to TD buprenorphine because of inadequate analgesia and severe adverse effects. Improved overall satisfaction and quality of sleep (good/very good increased from 14% to 74%, *P* \< 0.005) highlighted pain relief which increased from 5% to 76% (*P* \< 0.001). Only 5% of patients reported insufficient relief.\[[@ref20]\]
C[ONCLUSION]{.smallcaps} {#sec1-5}
========================
In comparative studies, buprenorphine was found efficacious against tramadol, fentanyl, and even oral morphine with a better safety profile. The patients\' acceptance was also better with TD formulation. Our trial might be considered as a pilot study being first of its kind in India paving the way for promising future for cancer pain management where morphine administration still poses a problem for palliative care specialists.
Financial support and sponsorship {#sec2-2}
---------------------------------
Nil.
Conflicts of interest {#sec2-3}
---------------------
There are no conflicts of interest.
|
{
"pile_set_name": "PubMed Central"
}
|
Background {#Sec1}
==========
Heart failure (HF) and atrial fibrillation (AF) each affect 1--2 % of the general population and the prevalence of both increases with age \[[@CR1], [@CR2]\]. AF is the most common sustained arrhythmia and occurs in 15 to 30 % of patients with HF \[[@CR3], [@CR4]\]. Since AF is a marker of increased cardiovascular mortality and morbidity in patients with HF \[[@CR5], [@CR6]\], it is important to investigate the predictors of AF occurrence in these patients. The development of AF in HF appears to be a multifactorial process, involving structural and neurohormonal processes and electrical remodeling \[[@CR7]\]. The heart's vulnerability to changes in loading affects the prognosis of HF and the development of AF. As this vulnerability is affected by ventricular stiffness and ventriculo-arterial interaction \[[@CR8], [@CR9]\], it is reasonable to hypothesize that those factors are closely associated with the development of AF. However, the relationship has still not been determined in HF patients. Therefore, the aim of this study was to investigate indexes of ventricular stiffness and ventriculo-arterial interaction as predictors for the development of AF in patients with HF.
Methods {#Sec2}
=======
Study populations {#Sec3}
-----------------
We retrospectively investigated 349 consecutive patients (238 men; age 62 ± 15 years) with stable HF with normal sinus rhythm who had undergone echocardiography between January 2007 and December 2008 in our institution. Reduced left ventricular (LV) contractility was defined as either a reduced ejection fraction (EF) (EF \< 40 %) or a borderline EF (40 % ≤ EF \< 50 %), based on two-dimensional echocardiography \[[@CR10]\]. Exclusion criteria were (1) muscular dystrophy, (2) more than moderate valvular disease, (3) previously documented AF or atrial flutter on the electrocardiogram (ECG) or Holter monitoring, (4) significant lung disease and (5) a New York Heart Association functional classification III or IV.
We reviewed medical history, physical examinations, laboratory tests, medication records, chest radiographs, 12-lead ECG, and echocardiography findings in all patients. We followed the study population for the development of AF via a review of their medical records. New-onset AF was defined as the first episode of paroxysmal or persistent AF, as documented by resting ECG or 24-h Holter monitoring, during the follow-up period. When the patient presented symptoms such as palpitations or dizziness, 24-h Holter monitoring was performed. Paroxysmal AF was defined as AF that terminated spontaneously within seven days, and persistent AF as continuous AF that was sustained beyond seven days \[[@CR5]\]. Study protocols were approved by our institutional review board, and informed consent from patients was waived due to retrospective nature of the study.
Conventional echocardiography {#Sec4}
-----------------------------
Two-dimensional, pulsed-Doppler, and tissue-Doppler echocardiographic examinations were performed using an appropriate ultrasound device (Sonos 7500, Philips, or Vivid 7, GE healthcare). Standard 2-dimensional measurements---LV diastolic and systolic dimension, ventricular septum and posterior wall thicknesses, left atrial (LA) volume, and LV outflow tract diameter---were obtained, based on the recommendations of the American Society of Echocardiography \[[@CR6]\]. LVEF was calculated from minimal (LV end-systole just before mitral valve opening) and maximal (LV end-diastole at the start of the QRS) LV volumes, which were obtained using a modified Quinones method \[[@CR5]\]. LA volume was determined by the prolate ellipsoid formula and was indexed to the body surface area \[[@CR6]\]. Peak velocities of early diastolic filling (E) and late diastolic filling (A) were measured by the pulsed-wave Doppler method at the level of the mitral valve leaflet tips. Peak early diastolic velocity (e') and late diastolic velocity (a') were measured by tissue Doppler imaging from the septal mitral annulus. The E/A and E/e' ratios were calculated to estimate the LV filling pressure \[[@CR7]\]. Echocardiographic data were analyzed by two cardiologists who were blinded to the patients' other information.
Echo-Doppler--derived parameters of ventricular stiffness and ventriculo-arterial interaction {#Sec5}
---------------------------------------------------------------------------------------------
For the evaluation of the vulnerability of the LV, we measured ventricular stiffness and end-systolic ventriculo-arterial interaction using echo-Doppler \[[@CR8], [@CR9]\]. Ventricular diastolic elastance (Ed) was calculated as E/e' divided by stroke volume \[[@CR11], [@CR12]\]. Ventricular end-systolic elastance (Ees) was required to describe the contractility of the LV and was calculated based on pulsed-wave Doppler echocardiography of trans-LV outflow tract (LVOT) as peak velocity divided by acceleration time \[[@CR13]\]. Peak velocity of LVOT was measured at the point of maximal velocity and acceleration time was measured as the time from the onset to the peak velocity. Effective arterial elastance (Ea) provides an integrative measure of arterial load and was defined as the ratio of end-systolic pressure to stroke volume where end-systolic pressure was calculated as (2 × systolic blood pressure + diastolic blood pressure)/3 \[[@CR11], [@CR12]\]. To evaluate the interaction of the ventricular and arterial systems, the ventricular--vascular coupling index (VVI) was calculated as the ratio Ea/Ees \[[@CR14], [@CR15]\].
Statistical analysis {#Sec6}
--------------------
Baseline characteristics were summarized for all participants using standard descriptive statistics. Continuous variables are presented as mean ± standard deviation and categorical variables as number and percentage. Baseline characteristics of groups were compared using independent t-tests for continuous variables and chi-square tests for categorical variables. Variables without normal distribution were presented as median (inter-quartile range) and Mann-Whitney *U* test were used for their comparisons.
Linear regression analyses were performed to determine the association between echo-derived parameters and the HF severity, estimated by EF and levels of log-transformed brain natriuretic peptide (BNP). To achieve a constant variance, BNP values were transformed logarithmically. Cox proportional hazard models were used to determine the association between Ed, Ees, Ea, and VVI, and the development of new-onset AF. Adjustment variables were selected based on the differences between two groups (AF vs. sinus rhythm) and clinical rationale; they included age, LA volume index(LAVI), and underlying disease. We also used receiver operating characteristic curves to derive cutoff values for predictors. Time-to-event data are reported and displayed using the Gehan's generalized Wilcoxon method, with comparisons between groups performed using the log-rank testDifferences were considered statistically significant if the *p*-value was \<0.05 when using two-sided tests. All statistical analyses were performed using SPSS 18.0 (SPSS Inc., Chicago, Illinois).
Results {#Sec7}
=======
Patient characteristics and echocardiographic parameters are presented in Table [1](#Tab1){ref-type="table"}. New-onset AF developed in 57 of the 349 patients studied (16.3 %). The mean age of the study population was 62 ± 15 years and 238 (68 %) were male. At the beginning of the study, all patients were in normal sinus rhythm. The median follow-up period was 30.3 months. Anticoagulation medication was administered to all patients who developed AF and had appropriate indications.Table 1Intergroup comparison of clinical characteristics and echocardiographic parametersCharacteristicSinus rhythmNew-onset AF*p*-value(*n* = 292)(*n* = 57)Age (years)61.2 ± 15.767.7 ± 12.8\*0.003Male, n (%)203 (69.5)35 (61.4)0.229Body surface area (m^2^)1.71 ± 0.231.68 ± 0.190.320SBP (mmHg)122.4 ± 23.3115.4 ± 25.20.094DBP (mmHg)74.1 ± 16.068.1 ± 14.7\*0.032Hypertension, n (%)94 (32.2)28 (49.1)\*0.014Diabetes mellitus, n (%)52 (17.8)19 (33.3)\*0.008Current smoker, n (%)57 (19.5)12 (21.1)0.791Ischemic CMP, n (%)164 (56.2)32 (56.1)0.997Chronic kidney disease, n (%)29 (12.0)10 (18.5)0.264BUN (mg/dL)21.7 ± 14.924.4 ± 21.50.259Creatinine (mg/dL)1.4 ± 1.52.0 ± 2.30.095BNP (pg/ml)Median: 216.0 (81.8--749)Median: 566.0(180.5-1302.5)\*0.011Medications Antiplatelet agent, n (%)218 (74.7)46 (80.7)0.400 RAS blockade, n (%)243 (83.2)49 (86.0)0.608 Diuretics, n (%)131 (44.9)42 (73.7)\* \< 0.001 Beta blockade, n (%)191 (65.4)31 (54.4)0.114 Calcium channel blockade, n (%)88 (30.1)21 (36.8)0.318 Statin, n (%)152 (52.1)26 (45.6)0.374Echocardiographic parameters Ejection fraction (%)36.9 ± 9.435.3 ± 10.30.249 LVEDD (mm)52.4 ± 7.152.4 ± 7.90.964 LVESD (mm)42.0 ± 8.142.3 ± 8.70.814 LA-AP (mm)37.8 ± 7.440.3 ± 7.5\*0.023 LA-ML (mm)43.9 ± 7.547.8 ± 8.7\*0.001 LA-SI (mm)52.8 ± 8.757.5 ± 9.7\* \< 0.001 LA volume index (mm^3^/m^2^)28.4 ± 12.736.1 ± 14.8\* \< 0.001 LV mass index (g/m^2^)73.8 ± 27.674.8 ± 18.90.738 E velocity (m/s)0.65 ± 0.230.79 ± 0.30\* \< 0.001 A velocity (m/s)0.72 ± 0.220.71 ± 0.250.706 E/A1.02 ± 0.631.22 ± 0.75\*0.036 E/DT (cm/s)0.39 ± 0.270.55 ± 0.37\*0.003 e' (m/s)0.05 ± 0.020.04 ± 0.020.278 a' (m/s)0.08 ± 0.020.07 ± 0.03\*0.001 S' (m/s)0.05 ± 0.020.05 ± 0.020.223 E/e'15.2 ± 6.819.1 ± 7.2\* \< 0.001 Ed (1/ml)0.36 ± 0.220.47 ± 0.27\*0.009 Ees (m/s)1.02 ± 0.241.11 ± 0.370.195 Ea (mmHg/ml)2.67 ± 0.962.75 ± 1.430.717 VVI25.5 ± 9.132.1 ± 16.80.115*Abbreviations*: *AF* atrial fibrillation, *CMP* cardiomyopathy, *BUN* blood urea nitrogen, *RAS* renin-angiotensin system, *LVEDD* left ventricular end-diastolic dimension; *LVESD*, left ventricular end-systolic dimension; *LA* left atrial; *AP* anteroposterior; *ML* mediolateral; *SI* Superoinferior; *DT* Deceleration time; *Ed* left ventricular (LV) end-diastolic elastance; *Ees*, LV end-systolic elastance; *Ea*, effective arterial elastance; *VVI*, ventricular-vascular coupling index\* *p* \< 0.05
The new-onset AF group was older (67.7 ± 12.8 vs. 61.2 ± 15.7, *p* = 0.003) and had a higher prevalence of hypertension (49.1 % vs. 32.2 %, *p* = 0.014) and diabetes (33.3 % vs. 17.8 %, *p* = 0.008). The rate of ischemic cardiomyopathy did not differ between the two groups. The new-onset AF group used more diuretics (73.7 % vs. 44.9 %, *p* \< 0.001) than the sinus rhythm group (Table [1](#Tab1){ref-type="table"}). This finding suggests that patients with AF may have more symptoms of heart failure and more volume overloading conditions. The median BNP level was higher (216.0 vs. 566.0, *p* = 0.011) in patients with AF.
There was no difference in LV end-diastolic diameter (52.4 ± 7.1 vs. 52.4 ± 7.9 mm, *p* = 0.964), LV systolic function (EF: 36.9 ± 9.4 % vs. 35.3 ± 10.3 %, *p* = 0.249), LV mass index (73.8 ± 27.6 vs. 74.8 ± 18.9 g/m^2^, *p* = 0.738), A (0.72 ± 0.22 vs. 0.71 ± 0.25 m/s, *p* = 0.706), or e' (0.71 ± 0.25 vs. 0.04 ± 0.02 m/s, *p* = 0.278). However, the new-onset AF group had a larger LA volume index (36.1 ± 14.8 vs. 28.4 ± 12.7 mL/m^2^, *p* \< 0.001), a higher E (0.79 ± 0.30 vs. 0.65 ± 0.23 m/s, *p* \< 0.001), and a higher E/e' ratio (19.1 ± 7.2 vs. 15.2 ± 6.8, *p* \< 0.001). A' was significantly lower in the new-onset AF group (0.07 ± 0.03 vs. 0.08 ± 0.02 m/s, *p \<* 0.001) (Table [1](#Tab1){ref-type="table"}). Among the echo-derived Doppler indexes, Ed was significantly higher (0.47 ± 0.27 vs. 0.36 ± 0.22 /mL, *p* = 0.009) in the new-onset AF group. Ees, Ea, and VVI did not differ significantly between the two groups.
In linear regression, EF measured by echocardiography was significantly correlated with Ed (β: -0.496, *p* \< 0.001) and Ea (β: -0.314, *p* \< 0.001). In addition, BNP level was significantly correlated with Ed (β: 0.609, *p* \< 0.001), Ees (β: 0.218, *p* =0.020) and Ea (β: 0.212, *p* =0.031) (Table [2](#Tab2){ref-type="table"}). These findings suggest that there are significant relationships between HF severity, estimated by EF and BNP level, and echo-derived Doppler parameters that reflect ventricular stiffness (Ed) and arterial load (Ea).Table 2Association of HF severity with ventricular stiffness and ventriculoarterial interaction index by univariate linear regressionEjection fractionBNP(Log-transformed)ß*p*-valueß*p*-valueEd (1/ml)−0.496\* \< 0.0010.609^\*^ \<0.001Ees (m/s)0.0010.9910.218^\*^ 0.020Ea (mmHg/ml)−0.314\* \< 0.0010.212^\*^ 0.031VVI−0.1900.0640.0400.735*Ed* left ventricular (LV) diastolic elastance; *Ees* LV end-systolic elastance; *Ea* effective arterial elastance; *VVI* ventricular-vascular coupling index
Over a median follow up of 30.3 months, new-onset AF developed in 57 patients (16.3 %). Lower EF was significantly associated with new-onset AF (hazard ratio \[HR\] 0.97, *p* = 0.025). After adjustment for age, hypertension, diabetes mellitus, use of diuretics and LAVI, EF was not correlated with AF (HR 0.98, *p* = 0.250). Ed was also significantly associated with new-onset AF, with a greater than 10-fold risk in an unadjusted model (HR 10.40, *p* \< 0.001). This association remained significant after adjustment for age, hypertension, diabetes mellitus, use of diuretics and LAVI, (HR 5.49, *p* = 0.018). Ea and VVI were associated with new-onset AF after adjustment for age, hypertension, diabetes mellitus, use of diuretics and LAVI (HR 1.66 and 1.06, *p* = 0.026 and 0.001, respectively) (Table [3](#Tab3){ref-type="table"}). For subgroup analysis, we devided our study population into two groups: (1) heart failure with reduced EF (HFrEF, EF \< 40 %) and (2) heart failure with preserved EF (HFpEF, EF ≥ 40 %). In patients with HFrEF, Ea and VVI were associated with new-onset AF after adjustment for age, hypertension, diabetes mellitus, use of diuretics and LAVI (HR 1.73 and 1.04, *p* = 0.045 and 0.035, respectively) (Additional file [1](#MOESM1){ref-type="media"}). Whereas, there was no independent parameter of LV stiffness that associated with new-onset AF in patients with HFpEF.Table 3Association between echocardiographic parameters and new-onset atrial fibrillationUnadjustedAdjustedHazard ratio*p*-valueHazard ratio*p*-valueEjection fraction (%)0.97\*0.0250.980.250Ed (1/ml)10.40\* \< 0.0015.49\*0.018Ea (mmHg/ml)1.700.0111.66\*0.027Ees (m/s)1.580.4970.950.940VVI1.040.0041.06\*0.001Adjusted for age, hypertension, DM, use of diuretics and left atrial volume index*Ed* left ventricular (LV) diastolic elastance; *Ea* effective arterial elastance; *Ees* LV end-systolic elastance; *VVI* ventricular-vascular coupling index\**p* \< 0.05
Further evaluation was performed to determine the power of Ed in predicting AF occurrence. The optimal cutoff value to predict AF was an Ed value of 0.33, with corresponding sensitivity and specificity values of 71 and 58 %, respectively (receiver operating characteristic curve: area under curve \[AUC\] = 0.646, *p* \< 0.001) (Fig. [1](#Fig1){ref-type="fig"}). Figure [2](#Fig2){ref-type="fig"} shows the cumulative survival curves for AF occurrence. The cumulative survival rate free of AF occurrence was significantly higher in patients with higher Ed (≥0.33) than in patients with lower Ed (\<0.33) (*p* \< 0.001).Fig. 1Receiver operating characteristic (ROC) curve for ventricular diastolic elastance (Ed) Fig. 2Cumulative AF occurrence free rate in relation to Ed (*p* \< 0.001)
Discussion {#Sec8}
==========
The principle findings of this study are as follows: (1) Ventricular stiffness and ventriculo-arterial interaction could be measured by echo Doppler, and (2) Ed, Ea and VVI were closely correlated with new-onset AF in patients with HF.
The variable effect of AF on patients with HF {#Sec9}
---------------------------------------------
AF is the most common arrhythmia \[[@CR4], [@CR9]\] and further aggravates symptoms in patients with HF \[[@CR9], [@CR16]\]. AF occurs in 10--20 % of mild to moderate cases of HF and in up to 50 % of patients with more advanced HF. The prevalence of AF generally depends on the severity of the underlying HF \[[@CR17], [@CR18]\]. In this study, only ambulatory patients with stable HF were enrolled and new-onset AF developed in 16.3 % of patients.
The new-onset AF group was receiving diuretics at a statistically significantly higher rate (73.7 % vs. 44.9 %, *p* \< 0.001). This finding suggests that these patients were clinically identified as having volume overload or elevated LV filling pressure and were being treated as such. Recently, Cambell et al. showed that renal impairment was strong predictor of AF in patients with HF and should be regularly screened. In our study, prevalence of chronic kidney disease was not different between AF group and sinus rhythm group.
While LV systolic function was similar between the two groups, patients with new-onset AF had significantly higher LAVI, E/e', and Ed. These findings suggest that atrial dilation and reduced LV relaxation are related to the development of AF and could be explained by diastolic dysfunction.
The correlation between AF and diastolic dysfunction {#Sec10}
----------------------------------------------------
Diastolic dysfunction, like systolic dysfunction, is known to be associated with the development of AF \[[@CR11], [@CR13]\]. The presence of diastolic dysfunction is also a strong predictor of AF occurrence and is closely associated with a poor prognosis in patients with HF \[[@CR19]\]. Diastolic dysfunction leads to elevated LV filling pressures and atrial remodeling. The relationship between atrial remodeling and AF has been investigated in many studies. Diastolic dysfunction with an increase in LV filling pressures causes atrial remodeling and is associated with a more than 5-fold increased risk of AF occurrence compared with normal diastolic function \[[@CR20]\].
The assessment of diastolic dysfunction by echocardiography {#Sec11}
-----------------------------------------------------------
LV diastolic filling pressure can be measured by Doppler echocardiography. Mitral inflow velocity (E) correlates well with LV filling pressure in heart failure; however, myocardial relaxation and filling pressure could affect the mitral E velocity. The mitral e' velocity reflects relaxation of the myocardium and the E/e' ratio correlates well with LV filling pressure or pulmonary capillary wedge pressure \[[@CR21]--[@CR23]\]. In this study, the AF group had significantly higher E/e' values compared with patients in normal sinus rhythm.
In HF patients, increased ventricular stiffness induces diastolic dysfunction, thereby causing an elevation of LV filling pressure and increasing vulnerability to an acute loading change \[[@CR14]\]. E/e' is significantly correlated with LV filling pressure \[[@CR22]\], while stroke volume can be used as an indicator of LV filling volume. Thus, combining these parameters, dividing E/e' by stroke volume, represents the ratio of LV filling pressure to filling volume and can be used as the Ed \[[@CR11]\]. We used Ed for the evaluation of ventricular stiffness in this study. We found that Ed was significantly correlated with EF and BNP, which represent HF severity. Furthermore, a higher Ed was independently associated with AF, after adjustment for age, hypertension, diabetes mellitus, use of diuretics and LAVI. In addition, using receiver operating characteristic analysis, we were able to determine a cutoff value for LV filling pressure/volume, as represented by the Ed value. According to our results, the optimal cutoff value of Ed for the prediction of new-onset AF was 0.33, with a sensitivity of 71 % and a specificity of 58 %.
Ventriculo-arterial interaction {#Sec12}
-------------------------------
Increased afterload is also important in the pathophysiology of HF \[[@CR24]\]. The Ea was used as the total arterial afterload and can be measured noninvasively \[[@CR11], [@CR12]\]. In addition, the Ees is related to LV contractility, independently of loading conditions \[[@CR25]\]. The combination of these parameters, called VVI, is the ratio Ea/Ees and is used as an index of ventriculo-arterial coupling, as previously described \[[@CR14]\]. In our study, we found that Ea was closely related to HF severity. Interestingly, our study showed that Ea and VVI, as well as Ed, were predictors of new-onset AF in patients with HF, whereas Ees was not. These results suggest that ventricular stiffness and ventriculo-arterial coupling are important predictors of AF occurrence in HF patients. In addition, ventricular stiffness and ventriculo-arterial coupling are more relevant than LV contractility alone.
Limitations {#Sec13}
-----------
The present study had some limitations. First, since it was a single-center study with a retrospective design, only limited data analysis was possible and follow-up durations were arbitrary. Second, the occurrence of new-onset AF might have been underestimated. Capturing AF is notoriously difficult, unless careful transtelephonic monitoring or regular Holter recordings can be performed. We know that relying on symptomatic presentation will underestimate the occurrence of AF, given that much of AF is asymptomatic. There is no way of knowing whether patients in the "sinus rhythm" group actually had paroxysmal AF or not. Finally, the male predominance in the study patients was a potential limitation, in that it did not reflect the general clinical HF population; however, this is a common failing shared with many other contemporary HF studies.
Conclusions {#Sec14}
===========
Echo-Doppler--derived indexes of ventricular stiffness and ventriculo-arterial coupling are associated with HF severity and are important predictors of new-onset AF in patients with HF. Therefore, HF patients with increased ventricular stiffness may need close observation for the development of AF, using techniques such as frequent Holter monitoring.
Additional file {#Sec15}
===============
Additional file 1:**Association between echocardiographic parameters and** **new-onset** **atrial fibrillation in patients with heart failure with reduced ejection fraction.** (DOCX 16 kb)
**Electronic supplementary material**
The online version of this article (doi:10.1186/s12947-016-0050-y) contains supplementary material, which is available to authorized users.
**Competing interests**
The authors declare that they have no competing interests.
**Authors\' contribution**
Drs J-YK, JHY and HC designed study, Drs M-HK and JHY collected, analyzed and interpreted data. Dr JHY wrote article. Drs JHY, E-YC, P-KM, BKL, YWY, B-KH, S-JR and HMK collected, analyzed and interpreted clinical and echocardiographic data. This paper is not under consideration elsewhere; none of the paper\'s contents have been previously published, and all authors have read and approved the manuscript.
We would like to thank the *Medical Research Supporting Section* of *Yonsei University College of Medicine* for providing excellent assistance with the statistical analysis.
Funding {#FPar1}
=======
This research received no grant from any funding agency in the public, commercial, or not-for-profit sectors.
|
{
"pile_set_name": "PubMed Central"
}
|
Chagas disease was initially described as an endemic health problem in a few countries in South America -- mainly Argentina and Brazil -- and one of the consequences of the disease, heart damage, made it an interesting issue for healthcare professionals, from epidemiologists to cardiologists.\[[@r1]\] Chagas cardiomyopathy (ChCM) is now recognised as a cardiovascular disorder, diagnosed and treated not only in the original region, but also in Europe, North America and even in Asia (*[Figure 1](#fig1){ref-type="fig"}*). This is a result of increased migration around the world.\[[@r2]--[@r4]\] It is estimated that Chagas disease affects 6--7 million people in Latin America and more than 300,000 people in the US.\[[@r5],[@r6]\] The natural history of the disease shows that after two to three decades up to 30% of infected individuals exhibit evidence of chronic cardiomyopathy and a proportion of these develop heart failure (HF) with reduced ejection fraction (HFrEF).\[[@r7]\] Despite the high prevalence of Chagas disease, little is known about morbidity and mortality in patients with HFrEF caused by Chagas disease, compared with other aetiologies, especially in the modern era of HF therapies.\[[@r8],[@r9]\] Future trials should consider recruiting larger numbers of patients with ChCM to allow adequately powered subgroup analysis. We still treat patients with HF caused by ChCM empirically with therapies recommended by guidelines and this is another reason to specifically promote more research in ChCM.\[[@r10]\] This article will analyse and discuss Chagas disease and HF, from epidemiology through to the latest treatment and prevention strategies.
The Expanding Epidemiology
==========================
Chagas disease is an important public health problem, with a frequency 140 times higher than HIV. Initiatives in the Americas have helped to achieve significant reductions in the number of acute cases of Chagas disease and the presence of domiciliary triatomine vectors in endemic areas. The estimated number of people infected with *Trypanosoma cruzi* worldwide dropped from 30 million in 1990 to 8--10 million in 2010, the annual incidence of infection decreased from 700,000 to 28,000 new cases over the same period and the burden of disease decreased from 2.8 million disability-adjusted life-years lost to \<0.5 million years between 1990 and 2006.\[[@r11]\] Approximately 108 million people are exposed to Chagas disease in 21 South American countries, with 8 million infected and 41,200 new cases per year. About 20--40% of infected people will develop cardiac damage.\[[@r12]--[@r14]\] The prevalence of Chagas disease varies between countries, from 1.3% in Brazil to 20.0% in Bolivia. In the US, estimates claim that over 0.5 million immigrants are infected with *T cruzi*. The number of houses infested by triatomine vectors has been reduced as a result of campaigns and the work of teams throughout South America. Vectorial transmission has been stopped in Uruguay, Chile and Brazil, and in large areas of Argentina and Venezuela, achieving an average reduction in prevalence and incidence of 70%.
Chagas disease is an important cause of dilated cardiomyopathy in Latin America, where the disease is endemic.\[[@r15],[@r16]\] Chagas heart disease (ChHD) commonly presents with symptomatic ventricular arrhythmias, symptomatic bradyarrhythmias, sudden death, HF, embolic events, chest pain and high susceptibility to proarrhythmia.\[[@r15]\] The clinical picture mimics that of coronary artery disease and idiopathic dilated cardiomyopathy. The prognosis is poor for patients with malignant ventricular arrhythmias, HF, left ventricular aneurysm or global systolic dysfunction.\[[@r16]\] Moreover, in an Argentinian survey, HF was the most frequent finding, leading clinicians to suspect Chagas disease.\[[@r17]\]
HF may occur in approximately 10% of subjects who have Chagas disease with cardiac involvement.\[[@r18],[@r19]\] A meta-analysis of 143 studies of HF from Latin America reported the incidence of HF in Chagas disease to be 137 per 100,000 people per year and annual mortality to be 1.12--7.18 per 100,000 people per year. Higher in-hospital mortality was also found in patients with Chagas disease, compared with a non-Chagas disease aetiology, representing 36% of the aetiology of HF.\[[@r20]\] The estimated prevalence of Chagas disease within HF populations in Argentina is about 15%.\[[@r21]\] However, data from different surveys reveal a Chagas disease prevalence of 4.0--6.0% in outpatient registries and 1.3--8.4% in decompensated HF settings in Argentina, and of 0.6--20.0% in Latin American registries.\[[@r22]--[@r24]\] This discrepancy may be attributable to actual lower prevalence, underrepresentation of rural inhabitants in the surveys, low use of screening tests or because some cases of Chagas disease might represent a comorbidity. The Grupo de Estudio de la Sobrevida en la Insuficiencia Cardíaca en Argentina (GESICA) Registry has shown a prevalence of true ChCM of 6.4%.\[[@r25]\] Patients with Chagas disease had a different clinical profile from, and were treated differently to, those without Chagas disease. Patients with ChCM were admitted more frequently for decompensated HF than other patients.\[[@r26]\] It has been reported that long-term outcomes in patients with chronic systolic HF secondary to ChCM are poorer than those in patients with idiopathic and ischaemic aetiologies.\[[@r27],[@r28]\]
Acute decompensated HF (ADHF) is a common clinical condition in ChCM. A study has shown that patients with ChCM had the highest proportion of hospital admissions for cardiogenic shock and arrhythmia, with lower systolic blood pressure and a higher proportion of right ventricular HF than other aetiologies.\[[@r29]\] Outcomes were also influenced by aetiology, and ChCM had the lowest proportion of hospital discharge and the highest proportion of cardiac transplant when compared with other aetiologies, mainly in Latin American countries. So, the poorer prognosis of ChCM in comparison with other common causes of cardiomyopathy is also applicable to vulnerable ADHF, related to the severity of presentation and other issues, including socioeconomic factors.
Typically, the clinical profile of ChCM includes younger people, more often women, with lower prevalence of hypertension, diabetes and renal impairment, compared with non-ChCM patients. In contrast, health-related quality of life was worse and the prevalence of stroke and pacemaker implantation were higher in ChCM compared with non-ChCM aetiologies. Despite these differences, the rates of cardiovascular death, HF hospitalisation and all-cause mortality were higher in patients with Chagas disease than other non-ischaemic and ischaemic patients.\[[@r9]\]
{#fig1}
Main Pathophysiologic Pathways
==============================
Chagas disease may be diagnosed in the infected patient in either acute or chronic clinical presentation. In some cases, the acute phase is light and can go undiagnosed by the patient and the medical team.
Acute Chagas disease is an immunological reaction typically characterised by diffuse lymphadenopathy, hepatomegaly and splenomegaly. In this period the myocardium and the gastrointestinal system are severely infected by the parasite and show important tissue inflammation.\[[@r30]\] Sometimes, acute myocarditis may be diagnosed with signs of cardiac dilatation and pericardial effusion. The most severe presentations may produce pancarditis and even vasculitis. In these cases, development of acute HF is frequent and the prognosis is usually poor because multiorgan involvement may occur.\[[@r31],[@r32]\]
Development of HF linked to Chagas disease most commonly presents chronically. The typical dilated cardiomyopathy observed in most patients with chronic HF related to Chagas disease consists of chronic myocarditis producing fibrosis with particular invasion of His bundle branches, causing different types and grades of cardiac block and progressive dysfunction.\[[@r33]\] The exact mechanism whereby parasitism causes tissue damage in the chronic phase is not clear and could be related to chronic immune reactions. The severity and extension of the inflammation and fibrosis depend on many factors, mainly the aggressiveness of the parasite, the immunologic reaction of the patient and the concomitant cardiovascular risk factors.\[[@r34]--[@r36]\] Some patients with ChHD may also have digestive disturbances, often dysfunctions in the oesophagus and bowel as a result of inmunoinflammatory reactions in these organs, which ends in megaoesophagus, megacolon and/or megarectum.\[[@r37]\]
An Update on Diagnosis
======================
In the acute phase of Chagas disease, the diagnosis might be suspected, taking account of the different transmission forms and patient history. Most patients are oligosymptomatic with non-specific symptoms of weakness, fever and malaise. Patients could present with the pathognomonic chagoma or unilateral eyelid swelling, which is often treated as viral conjunctivitis. In some cases -- mainly in immunocompromised patients or in those who were infected with *T cruzi* through oral transmission -- fulminant disease is present with acute myocarditis, pericardial effusion or meningoencephalitis.\[[@r38]--[@r40]\]
After the acute phase of infection, most patients develop the chronic form. This is defined by positive serology and slow progression or even absence, during a non-specific period of time, of physical signs or symptoms of cardiac electrogenic conduction abnormalities, myocardial contractile dysfunction, arrhythmias thromboembolism, colon, rectum or oesophagus abnormalities.\[[@r41]--[@r46]\] ChCM is the most important clinical manifestation of Chagas disease, resulting in the majority of Chagas disease morbidity and mortality.\[[@r39],[@r41],[@r47],[@r48]\]
ECG and Holter monitor
----------------------
The ECG and Holter monitor may show tachycardia (out of proportion to fever), different degrees of atrioventricular (AV) block, QT prolongation, low voltage and repolarisation abnormalities in the acute phase of the disease.
Later in the disease progression, the ECG plays an important role in diagnosis and prognosis of ChCM. It could be normal, with slight abnormalities, premature ventricular beats, ventricular tachycardia, AF or flutter, complete AV block, anterior and inferior fibrosis, complete right bundle branch block alone or in combination with left anterior fascicular block, with or without different degrees of AV block.\[[@r49]\]
Radiology
---------
X-ray is useful in assessment and follow-up of Chagas disease. Enlargement of all four cardiac chambers with or without signs of pulmonary congestion suggests ChCM.\[[@r41]\] X-ray is also useful in the diagnosis of megacolon and megaoesophagus, which can be corroborated with endoscopy.
Parasitaemia
------------
In the acute phase of Chagas disease, parasitaemia can be observed with a microscopic blood examination.\[[@r49]\] Microhaematocrit is a widely used method of identifying congenital infection. The trypomastigotes present in the blood can be seen during the first 8--12 weeks and, after that, parasitaemia falls below detectable levels.\[[@r50]\]
Serology and C-reactive Protein
-------------------------------
Indirect immunofluorescence, haemagglutination, and enzyme-linked immunosorbent assays are commonly used in screening for Chagas disease.\[[@r51]--[@r53]\] The WHO recommends diagnosing Chagas disease with two conventional laboratory tests and doing a third test in cases of discordance. C-reactive protein testing is the most sensitive test in acute infection or reactivation in patients with Chagas disease who are organ recipients or are otherwise immunocompromised.\[[@r54]--[@r56]\]
Echocardiography
----------------
Echocardiography may find segmental wall motion abnormalities, including akinesia, hypokinesia or dyskinesia with preserved septal contraction, left ventricular aneurysm (most common in apex), left ventricular diastolic dysfunction, dilated cardiomyopathy involving both ventricles, and mural thrombus, mitral and tricuspid regurgitation.
Assessment of myocardial strain through speckle tracking could help to detect myocardial damage in early periods of the disease, and some authors describe decreased global radial strain during the indeterminate stage, even in patients with a normal ECG and common echocardiogram.\[[@r57]--[@r59]\] Left ventricular global dysfunction with low ejection fraction is one of the most important predictors of death in ChCM.\[[@r57],[@r59]--[@r61]\]
Multigated Radionuclide Ventriculography
----------------------------------------
Multigated radionuclide ventriculography can be particularly helpful for the assessment of biventricular systolic function in patients with poor echocardiographic windows and a contraindication to cardiac MRI. It is an excellent method for providing LVF information in patients with Chagas disease.\[[@r62],[@r63]\] It can also provide us with quantification of right ventricular function -- although this is less precise -- and with echocardiographic analysis can be used to qualitatively assess ventricular dyssynchrony.\[[@r64]--[@r66]\] These data could all be improved with myocardial perfusion assessment.\[[@r67]\]
PET
---
PET is not included as a routine investigation in Chagas disease but perfusion defects and areas of fibrosis can be detected with PET in early stages and they correlate with ventricular wall motility abnormalities in the absence of coronary artery lesions.\[[@r68],[@r69]\]
The presence of chest pain -- mostly non-typical angina pectoris -- in patients with Chagas disease could be attributable to microvascular perfusion abnormalities.\[[@r69],[@r70]\]
Cardiac MRI
-----------
Cardiac MRI with or without gadolinium is useful to obtain more precise information about left ventricular ejection fraction and right ventricular ejection fraction, thrombus and fibrosis, giving us a good diagnostic and prognostic base.\[[@r17],[@r71]--[@r74]\]
Cardiac Catheterisation
-----------------------
Although most patients with Chagas disease have normal epicardial coronary arteries, cardiac catheterisation is necessary to rule out epicardial coronary artery disease, which could coexist with Chagas disease, even in patients without angina pectoris or with non-characteristic chest pain.
Ventriculography allows the detection small aneurysms, which would not be detected with echocardiography, and left ventricular wall motion abnormalities. Right and left cardiac catheterisation is also indicated in patients with advanced HF to assess and more effectively tailor therapy and determine the feasibility of cardiac transplantation or the implant of a ventricular assist device.
In addition, continuous ambulatory pulmonary pressure monitoring could reduce decompensation episodes and reduce the number of hospitalisations in patients with HF.\[[@r75]--[@r77]\]
Drug Heart Failure Management: Similar or Different?
====================================================
This update is specifically focused on the management of HF and not on the parasite-related therapeutic issues, nor on the other organs damaged by Chagas disease.
Treatment of cardiac dysfunction should be similar in populations with and without Chagas disease, as the haemodynamics and pathophysiology are similar. Treatment approaches have been based on evidence from other forms of HF and most clinical trials confirming a survival advantage did not include Chagas disease. So, information about treatment in patients with Chagas disease and HF derives from non-randomised studies or clinical trials of HF that included only small proportion of ChCM.\[[@r78]\] Consequently, there are few therapies with strong recommendations and most of therapies are based on evidence from small trials and expert opinions.
###### Recommendations of Argentinean Consensus of Chagas Disease
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Drug Clinical Scenario Recommendation Level of evidence
--------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ ---------------- -------------------
ACE inhibitors EF \<40%, FC I--IV, stages B, C and D I B
Angiotensin receptor blockers EF \<40%, FC I--IV, stages B, C and D, ACE inhibitor intolerantEF \<40% and symptomatic HF, optimal treatment with ACE inhibitor and beta-blocker I\ B\
IIb B
Beta-blockers EF \<40%, FC II--IV, stages B, C and DContraindications: bronchospasm, AV block, sinus sick syndrome, symptomatic bradychardia (\<50) IIa\ B\
III B
Mineralocorticoid receptor antagonist EF \<35% and FC III--IVModerated HF (FC II, stage C) I\ B\
IIa C
Digoxin AF with high ventricular rateAF with moderated ventricular rate, and FC III--IVSinus rhythm, EF \<40%, FC III--IV, optimal treatmentContraindications: AF with low ventricular rate, bronchospasm, AV block, sinus sick syndrome, and severe conduction system abnormalities I\ C\
IIa\ C\
IIb\ B\
III B
Diuretics HF and clinical congestion; FC II--IV, stages C and D I B
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
ACE = angiotensin-converting enzyme; AV = atrioventricular; EF = ejection fraction; FC = functional class; HF = heart failure.
While specific pharmacological treatment of HF is common to other aetiologies, there are some significant features in the context of HF as a result of ChCM that should be considered. *[Table 1](#tab1){ref-type="table"}* shows the main recommendations from the Argentinian consensus document on Chagas disease.\[[@r79]\]
Routinely, beta-blockers, angiotensin-converting enzyme inhibitors (ACEI) or angiotensin receptor blockers and mineralocorticoid receptor antagonists (MRA) represent the ideal combination in subjects with reduced ejection fraction, in the absence of any contraindications. In addition, digoxin, diuretics and anticoagulation are used as needed.\[[@r80],[@r81]\] Small trials in patients with cardiomyopathy have shown that these treatments can improve functional class, lower BNP and have a beneficial effect on neurohormones with a reduction in heart rate and decreased incidence of ventricular arrhythmias.\[[@r3],[@r82]\] Evidence for the role of angiotensin receptor neprilysin inhibitors (ARNI) is lacking and only 7.6% of 2,552 Latin American patients with HFrEF randomised in the Prospective Comparison of ARNI with ACEI to Determine Impact on Global Mortality and Morbidity in Heart Failure (PARADIGM-HF) trial and Aliskiren Trial of Minimizing OutcomeS in Patients With HEart Failure (ATMOSPHERE) had ChCM.\[[@r83]\] Compared with other aetiologies, patients with ChCM less frequently received beta-blockers and digitalis, but a higher proportion were treated with amiodarone, MRA and anticoagulants, with no differences in the use of ACEI.
Although several clinical trials have demonstrated the utility of adrenergic blockers in dilated cardiomyopathy different from Chagas disease in terms of reducing mortality and the number of hospitalisations, in ChCM, the presence of significant bradycardia and autonomic nervous system disorders with central and peripheral dysautonomia lead to greater precautions for routine use. The most used drug in this condition is carvedilol. Three trials evaluating carvedilol in ChCM were identified, with a total of 108 participants.\[[@r84]\] A lower proportion of all-cause mortality was found in the carvedilol groups compared with the placebo groups (RR 0.69; 95% CI \[0.12--3.88\]), with no difference in hospital readmissions. However, the authors found that the available evidence was low quality and there were no conclusive data to support or reject the use of carvedilol.
Diuretics should be used at the lowest possible dosage to obtain a negative balance, thus avoiding electrolyte and metabolic disorders caused by high dosages. Furosemide has the strongest diuretic effect and electrolytes should be checked frequently because excessive depletion can generate malignant arrhythmia.
Patients with severe HF as a result of heart disease not related to Chagas disease have shown improvement in quality of life and fewer hospitalisations when treated with digoxin, compared with placebo. However, ChCM has been associated with changes in automaticity and conduction related to malignant ventricular arrhythmia, and dysautonomia favours the appearance of bradycardic rhythms. Consequently, as digoxin can aggravate these disorders of rhythm and conduction, it has a restricted use in Chagas disease. Low-dose amiodarone has been associated with reduced HF mortality and sudden death in an Argentinian study that included patients with ChCM. A such, amiodarone can be safely used in the presence of arrhythmias. Anticoagulation in ChCM shares its common indication with other aetiologies, including permanent or paroxysmal episodes of AF, a previous thromboembolic event, the presence of a cardiac thrombus and apical aneurysms. A subanalysis of the Systolic Heart failure treatment with the If inhibitor ivabradine Trial (SHIFT) reported that ivabradine was effective in reducing heart rate in ChCM and improving functional class, suggesting that ivabradine may have a favourable benefit-risk profile in this population.\[[@r85]\] Specific drugs to eliminate the parasite or its reactions and non-pharmacological or invasive therapies are explained in the following section.
Specific Antitrypanosomal Therapy
=================================
Treatment for Chagas disease is mainly non-invasive in the early stages, when nifurtimox or benznidazole are used. More invasive procedures could be applied in advanced stages of the pathology, for example, where there is pericardial effusion, cardiac tamponade, arrhythmia and HF.
Benznidazole is a nitroimidazole with antiparasitic effects. The side-effects include rash, numbness, fever, muscle pain, anorexia and weight loss, nausea, vomiting and insomnia, plus a rare but important symptom, bone marrow suppression, which can lead to low blood cell levels.
Nifurtimox forms a nitro-anion radical metabolite that reacts with parasite nucleic acids causing significant DNA breakdown. It is used as a second specific treatment option in early Chagas disease because it has more serious side-effects than benznidazole, including anorexia, weight loss, nausea, vomiting, headache, dizziness, amnesia, rash, depression, anxiety, confusion, fever, sore throat, chills, seizures, impotence, tremors, muscle weakness and numbness.
Invasive Treatment of Gastrointestinal Chagas Disease
=====================================================
Invasive procedures to treat megacolon and megaoesophagus may be necessary. Megacolon can be treated with the Duhamel procedure or a modified version of the procedure, introduced by Haddad.\[[@r86]--[@r88]\] Laparoscopic procedures could also be applied. Megaoesophagus can be treated with wide oesophago-cardiomyectomy on the anterior oesophagogastric junction, combined with an antireflux valvuloplasty procedure or oesophagogastroplasty through the oesophageal bed.\[[@r89]\] These procedures could be extremely useful when advanced stages of HF are present and it is necessary to offer more invasive procedures.
Invasive Treatment of Cardiovascular Chagas Disease
===================================================
Cardiac manifestations of Chagas disease may also need invasive surgical procedures to preserve the patient's life or improve their functional class. Clinical management of patients with chronic Chagas disease requires proper clinical risk stratification and the identification of patients at high risk of sudden cardiac death (SCD). Recognising high-risk patients who require specific therapies -- especially invasive procedures such as the implantation of pacemakers, automatic ICDs, ablative procedures and even cardiac resynchronisation therapy (CRT) -- is a major challenge in clinical practice.\[[@r90]\]
Pacemakers
----------
Electrophysiological abnormalities of sinus node, AV node and His-Purkinje conduction can be detected in about one-third of patients with Chagas disease and pacemakers have shown utility in patients with this manifestation, but in some places this is still an unmet need.\[[@r91]--[@r93]\] AV block and symptomatic sinus sick syndrome are the main indications for pacemaker implantation in these patients, preferably electrode implantation in the mid-septal of the right ventricle to the apical site.\[[@r94]\]
Automatic ICDs
--------------
Malignant ventricular arrhythmia and SCD are more frequent in patients with Chagas disease.\[[@r91],[@r95],[@r96]\] Automatic ICDs may be used in the treatment of severe arrhythmias with high risk of SCD.\[[@r97]\] Primary or secondary prevention should be a routine indication for patients with Chagas disease with malignant arrhythmias.\[[@r98]\] The combination of ablation procedures, amiodarone and/or beta-blockers might be considered in special cases to reduce the number of automatic ICD therapies, as in other types of cardiomyopathy.\[[@r99]\]
Ablation Therapy
----------------
Ablation ventricular tachycardia therapy should be considered after oral medication failure.\[[@r100]\] CRT could be used in the treatment of patients with severe HF and left bundle branch block but there is scarce evidence to support resynchronisation therapy for patients with ChCM and right bundle branch block or its combination with left anterior fascicular block, where experience is not conclusive.\[[@r101],[@r102]\]
Ambulatory Pulmonary Pressure Monitoring
----------------------------------------
Other less invasive procedures such as ambulatory pulmonary pressure monitoring, which are useful in patients with HF, need to be tested in patients with Chagas disease HF and could probably prevent recurrent episodes of decompensation.\[[@r75],[@r77]\]
Heart Transplant
----------------
Heart transplantation and left ventricular assist devices (LVAD) have an important place in the treatment of irreversible HF in patients with Chagas disease.
Chagas disease was initially considered a contraindication for transplant because of the possible reactivation after transplantation and immunosuppression, but advances in immunosuppression programmes since the 1990s mean that there is now a similar survival and quality of life in HF patients with Chagas cardiomyopathy.\[[@r103]\] Selection criteria are similar to those for general heart transplant, including pulmonary artery pressures that could be elevated in some cases as a result of chronic left ventricular failure or undiagnosed pulmonary microembolism. However, the clinician also needs to consider the presence of megaoesophagus or megacolon, which could constitute a contraindication because of the possibility of complications (perforation) with the use of antiproliferative therapy such as mycophenolic acid derivatives or mammalian target of rapamycin inhibitors. The administration of prophylactic antitrypanosomal therapy is not recommended because of the higher risk of malignant neoplasms after transplant in patients who received reactivation prophylactic antitrypanosomal therapy.\[[@r104]\] The lowest immunosuppression regimen, high suspicion of possible reactivation and early detection and introduction of medical treatment with benznidazole offer a secure treatment of Chagas disease reactivation after transplant. Lifelong *T cruzi* monitoring is required, mainly during increased immunosuppression therapy for transplant organ rejection.\[[@r105],[@r106]\]
Circulatory Assist Devices
--------------------------
Different devices for cardiac assist could be used in patients with end-stage HF as a result of Chagas disease. These could be applied as a bridge to transplant, a bridge to recovery during the acute period or as a bridge to decision, or even as destination therapy depending on the general state of the patient and meticulous clinical evaluation of the possibility of survival and better quality of life.\[[@r107]--[@r110]\]
As in the transplant population, previous evaluation of the pulmonary pressures plus poor right ventricular function could be a contraindication for LVAD alone.
Global Action on Chagas Disease
===============================
ChHD is a preventable non-communicable disease (NCD) that mainly affects the poorest and most vulnerable populations of South America. Driven by poverty, poor access to health services and other health system weaknesses, the majority of people with this condition live in low- and middle-income countries.
The need for concerted global action to control NCDs is a high priority on the global health agenda. This is evident in the UN political declarations on the prevention and control of NCDs -- the 25x25 target aims for a 25% reduction in premature mortality from NCDs by 2025 -- the WHO Global NCD Action Plan, and the UN Sustainable Development Goals.\[[@r111],[@r112]\]
Cardiovascular disease (CVD) is the leading cause of premature mortality worldwide and with more than 80% of deaths occurring in low- and middle-income countries, leading the World Heart Federation (WHF) to launch its Roadmap Initiative in 2014 to guide and support those seeking to improve CVD control.
The WHF Roadmaps are global implementation strategies designed to help governments, employers, non-governmental organisations, health activists, academic and research institutions, healthcare providers and people affected by CVD, take action to better prevent and control CVD.\[[@r113],[@r114]\] The Roadmaps synthesise existing evidence on the efficacy, feasibility and cost-effectiveness of various strategies. They also identify potential barriers (roadblocks) to implementation and propose solutions to bypass them. The WHF and Inter-American Society of Cardiology Roadmap for reducing morbidity and mortality through improved prevention and control of ChHD complements existing Roadmaps on tobacco control, hypertension, secondary prevention of CVD, rheumatic heart disease, cholesterol, AF, diabetes and HF. The ChHD Roadmap is a resource to raise the profile of ChHD and provides a framework to guide and support the strengthening of national, regional and global ChHD control efforts. The process also requires a range of local expertise, including knowledge of medicine, cardiology, cultural and social contexts, prevention, health promotion, health systems, economics and government priorities.
Conclusion
==========
Chagas disease -- originally a South American endemic health problem -- is expanding worldwide as a result of people migrating. The pathology of Chagas disease is based in an inmunoinflammatory reaction producing fibrosis and remodelling, mainly in the myocardium. In many cases these mechanisms result in a dilated cardiomyopathy with HF and reduced ejection fraction, frequent cardiac arrhythmias and different types of heart block. The diagnosis and treatment of HF as a result of Chagas disease include the usual steps for other aetiologies, plus the need for laboratory techniques for parasite-related issues. International scientific organisations are concerned about this health problem and about delivering recommendations for prevention and early diagnosis.
[^1]: **Disclosure**: FM, EP and ASL have no conflicts of interest to declare. SVP is a member of advisory groups for Novartis, Ferrer, Abbott and Servier, and has received conference fees from these companies.
|
{
"pile_set_name": "PubMed Central"
}
|
Introduction {#S5}
============
Ventilator-associated pneumonia (VAP) is a complication of mechanical ventilation support in critically ill children and is the second most common hospital acquired infection among patients in the pediatric intensive care unit (PICU) \[[@R1]\]. VAP is associated with a substantial increase in resource utility, length of stay, and morbidity \[[@R2]\], yet limited understanding of the microbial factors associated with VAP pathogenesis has precluded development of effective prevention strategies.
It has been postulated that the presence of an endotracheal tube (ETT) contributes to the development of VAP via colonization and formation of biofilms, providing a conduit for potential pathogens to the lower respiratory tract. Multiple studies have indicated that ETTs are quickly colonized with microorganisms and lower airways are exposed to these organisms, increasing risk of VAP or other systemic infection \[[@R3]\]. Biofilms are also relatively protected from the host immune defense and from systemically administered antibiotics \[[@R4],[@R5],[@R6]\]. Further understanding of the composition of microbial communities in ETT biofilms and the timing of colonization could provide insight into mechanisms leading to VAP and creation of preventive interventions.
Tracheal aspirates are often utilized for the diagnosis of VAP in lieu of gold standard methods, such as culture of lower airway samples or lung biopsy, because of the invasiveness of these techniques \[[@R7],[@R8],[@R9]\]. Unfortunately, a recent study by Willison et al. demonstrated that, while tracheal aspirates in the pediatric population are fairly sensitive, they lack specificity and poorly distinguish between infection and colonization, even when stringent requirements for number of colony forming units and polymorphonulcear leukocytes are used to define infection \[[@R10]\]. Furthermore, molecular methods of bacterial identification have demonstrated enhanced detection of pathogenic bacteria compared to traditional culture of bronchoalveolar lavage samples in cystic fibrosis patients \[[@R11]\]. Similar results have been found when using 16S ribosomal RNA (16S rRNA) to analyze central venous catheters \[[@R12]\]. These studies, and others, have called into question the accuracy of traditional methods to identify the most abundant or, potentially, pathogenic bacteria when compared to molecular diagnostics \[[@R12],[@R13],[@R14]\].
There has been limited application of 16S rRNA sequencing to detect bacteria in ETT biofilms and tracheal aspirates. Existing studies, performed in adults, suggest that molecular diagnostics can characterize a larger proportion of the microbial community and provide additional data to better determine whether organisms are more likely to represent infection or colonization compared to traditional approaches. These techniques may also provide insight into the timing of colonization of the lower airways as well as the transition from colonization to infection \[[@R9],[@R12],[@R15],[@R16]\].
The goal of this study was to examine the bacterial composition of ETT biofilms and tracheal aspirates of mechanically ventilated children on the day of extubation to determine whether pathogenic bacteria are disproportionally represented in the ETT biofilm and how the biofilm composition compares to the bacterial communities in the lower airways as characterized by the tracheal aspirate. We hypothesized that the bacterial composition of ETT biofilms and tracheal aspirates are likely similar and will also contain high levels of opportunistic pathogens implicated in lower airway infections.
Materials and methods {#S6}
=====================
Data collection {#S7}
---------------
The data and specimens for this analysis were obtained from a prospective study conducted in the Children's Hospital Colorado PICU between April 2010 and April 2011. The Colorado Multiple Institutional Review Board approved the protocol, and parents or guardian provided informed consent for patients. Children between the age of 4 weeks and 18 years of age who required mechanical ventilator support via ETT for at least 72 hours were eligible for this study. Exclusion criteria included gestational age less than 37 weeks at birth for children less than one year at the time enrollment, indwelling tracheostomy or tracheostomy expected to be placed within seven days of PICU admission, an ETT present without mechanical ventilator support, and contraindication to deep tracheal suctioning.
All patients were subject to the VAP Event Bundle instituted at Children's Hospital Colorado which includes regular oral care, in-line suctioning, and other infection prevention measures defined in ventilator care guidelines.
Tracheal aspirate specimens from eligible subjects were collected with the first routine suctioning of the ETT occurring on the planned day of extubation via in-line suction and sterile specimen trap. Depth of suctioning was standardized by protocol as 1 cm below the ETT. For samples with low volume, up to 1 mL of sterile saline was used to facilitate collection. Specimens were aseptically transferred from mucous traps to 2 mL cryovials, flash frozen in liquid nitrogen, and stored at −80° C.
ETTs were collected upon extubation of patients, regardless of when extubation occurred. Each ETT was placed in a sterile specimen bag and immediately frozen at −80° C until processed. The proximal 5 cm (defined as the end protruding from the mouth) and the distal 5 cm (defined as the end residing in the trachea) were excised. Two separate standard culture swabs were used to sample the proximal lumen and distal lumen. The ends of the swabs were placed in 400 microliters of QIAGEN Buffer G2 (Germantown, Maryland). The swabs and buffer were then heated to 37° C for 30 minutes and vortexed to help release the biofilm. Afterwards, 200 microliters of the fluid were then used for DNA extraction using QIAGEN EZ1 Advanced DNA Bacterial DNA purification system per the manufacturer's instructions \[[@R17]\]. The purified DNA was used to determine bacterial load by quantitative PCR (qPCR) \[[@R18]\].
High-throughput DNA sequencing for microbiome analysis {#S8}
------------------------------------------------------
### 16S Amplicon Library Construction {#S9}
Bacterial profiles were determined by broad-range amplification and sequence analysis of 16S rRNA genes as previously described \[[@R19],[@R20]\].
### Analysis of Illumina Paired-end Reads {#S10}
Illumina MiSeq paired-end reads were aligned to human reference genome Hg19 with bowtie2 and matching sequences were discarded \[[@R21],[@R22]\]. The remaining non-human paired-end sequences were sorted by sample via barcodes in the paired reads with a python script \[[@R20]\]. Sorted paired-end sequence data were deposited in the NCBI Sequence Read Archive under accession number SRP063527. The sorted paired reads were assembled using phrap, and pairs that did not assemble were discarded \[[@R23],[@R24]\]. Assembled sequence ends were trimmed over a moving window of five nucleotides until average quality met or exceeded 20. Trimmed sequences with more than one ambiguity or shorter than 200 nucleotides were discarded. Potential chimeras identified with Uchime (usearch6.0.203_i86linux32) using the Schloss Silva reference sequences were removed from subsequent analyses \[[@R25],[@R26]\]. Assembled sequences were aligned and classified with SINA (1.2.11) using the 418,497 bacterial sequences in Silva 115NR99 as reference configured to yield the Silva taxonomy \[[@R27],[@R28]\]. Operational taxonomic units were produced by clustering sequences with identical taxonomic assignments. This process generated 6,270,141 sequences for 93 samples (average sequence length: 314 nucleotides; average sample size: 67,421 sequences/sample; minimum sample size: 7,059; maximum samples size: 182,870). The median Goods coverage score was = 99.6% at the rarefaction point of 7,059. The software package Explicet (v2.10.5, [www.explicet.org](www.explicet.org)) was used for display and statistical analysis \[[@R29],[@R30]\].
Statistical analysis {#S11}
--------------------
Sequence counts were analyzed using two-part statistics, calculated as the sum of two Chi squared statistics, the McNemar's test for paired proportions, and the Wilcoxon signed rank sum test as described elsewhere \[[@R30]\]. Morisita Horn (MH) indices were calculated to determine similarity between sample sites. Values range from 0 to 1, with 1 representing complete similarity in the proportion and identity of taxa, and 0 representing no similarity. Wilcoxon signed rank sum tests were performed to analyze differences between each comparison group for both MH and bacterial load. Further bacterial load analysis was done using random coefficients model with random intercept. Additional analyses for sequence (relative abundance of specific taxa), MH, and qPCR were performed using SAS version 9.4, SAS Institute, Cary, NC. Sequence and MH data were calculated in Explicet \[[@R31]\].
Results {#S12}
=======
Fifty-seven ETTs were collected. Five ETTs were excluded because the culture swab was unable to pass through the lumen of the ETT (ETT diameter \<3.5 mm). Fifty-two ETTs were subject to qPCR amplification. Fifteen ETTs did not have more bacterial DNA than reagent blanks and were eliminated from analysis leaving a total of 37 ETTs for 16S rRNA analysis. Four subjects only had one sample site (proximal or distal ETT or tracheal aspirate) with sufficient biomass to undergo sequencing, leaving 33 patients for comparative analysis. There were 23 paired tracheal aspirate and proximal ETT samples, 30 paired tracheal aspirate and distal ETT samples, and 25 paired distal and proximal ETT samples.
The 33 patients ranged in age from 2 months to 17 years of age. Patients were intubated between 3 days to 22 days with an average duration of intubation of 8.8 days (SD ± 5.0 days). Male patients comprised 58% of the study population. The most common diagnoses were lower respiratory tract infections, seizures, and sepsis. Eight patients received antibiotics 24 hours prior to extubation ([Table 1](#T1){ref-type="table"}).
Distribution of bacteria identified {#S13}
-----------------------------------
Bacterial communities from the proximal and distal ETT and tracheal aspirates were determined by 16S rRNA analysis. *Prevotella* was the most common genus identified in all sites comprising 36.8% of the overall sequences. *Streptococcus* and *Staphylococcus* were the next most common bacterial genera at 21.6% and 10.2%, respectively ([Table 2](#T2){ref-type="table"}). All sites contained *Prevotella* and *Streptococcus. Staphylococcus* was absent from two distal biofilms and two tracheal aspirates although not from the same patients. *Staphylococcus, Stenotrophomonas,* and *Veillonella* had a higher abundance in the proximal ETT biofilm compared to the distal ETT biofilm (p\<0.01; [Table 3](#T3){ref-type="table"}). There was a significant difference in the proportion of *Haemophilus* in the tracheal aspirate compared to the proximal ETT (1.51% vs. \<1%, p=0.02). *Burkholderia* was more abundant in tracheal aspirate samples (7.35%) versus proximal and distal ETT biofilm samples (1.85% and 1.95%, respectively). However, a significant difference was only observed between the distal ETT and tracheal aspirate (p=0.02, [Table 3](#T3){ref-type="table"}).
Bacterial load {#S14}
--------------
qPCR determined bacterial load at each sample site. There was no significant difference between bacterial load of the proximal ETT biofilm and tracheal aspirate. As a group, the sampled tracheal aspirates had a significantly higher bacterial load compared to the distal ETT biofilms (p\<0.04). The proximal end of the ETT had greater bacterial load compared the distal end (p\<0.01). Random coefficients model with random intercept was used to determine that bacterial load was not significantly associated with length of intubation at any of the sampled sites. There was not a significant interaction between duration of intubation and the comparison of bacterial load across the sampled sites (p=0.35).
Comparison of diversity {#S15}
-----------------------
The median index for MH comparison was 0.94 between tracheal aspirates with proximal ETT biofilms, 0.94 between tracheal aspirates and distal ETT biofilms, and 0.87 between the distal ETT biofilms and proximal ETT biofilms ([Figure 1](#F1){ref-type="fig"}). While a majority of patients had relative similarity between the sample sites, there was a subset of patients (n=9, 27.3%) with MH index scores less than 0.7, indicating less similarity. These patients were not consistent in which sampling site was responsible for the dissimilarity. There were also various predominant genera in these dissimilar communities that were not consistent between patients ([Figure 2](#F2){ref-type="fig"}). We examined the clinical and demographic data for the subset of patients with a MH index score less than 0.7 in attempt to determine factors that contribute to divergent bacterial populations. There was no significant difference in type of admission diagnosis, age, length of intubation, or total bacterial load between these 9 patients and the remaining cohort. Of the nine patients with MH \<0.7, only three patients were on antibiotics 24 hours prior to extubation.
Discussion {#S16}
==========
We examined the bacterial composition of ETT biofilms and tracheal aspirates collected on the day of extubation from mechanically ventilated children to determine the relationship between biofilms and lower airway colonization. We found that molecular analysis techniques reveal a wide variety of microbial taxa, well beyond that of standard culture techniques, and may have implications for future strategies to prevent ventilator-associate infections.
This study compared the microbiology of lower airway secretions to that of the ETT biofilm and provides new insight into the complex composition of ETT biofilms and airway colonization which could impact the risk of ventilator-associated infections. 16S rRNA sequencing demonstrated that *Prevotella* was the most prevalent genus in this pediatric population. *Prevotella* is a known constituent of the oropharyngeal microbiota and likely represents a common inoculum within the ETT through contamination with oral secretions either during the process of intubation or via aspiration of oral secretions after intubation has occurred. *Streptococcus* and *Staphylococcus,* other common constituents of the oropharyngeal microbiota, were also prominent in the ETT biofilms and tracheal aspirates. Yet, all three sample sites contain taxa with species that have pathogenic potential \[[@R32]\]. The predominance of *Staphylococcus* in the proximal section of the ETT was an interesting finding and may be a product of external manipulation of the ETT and subsequent introduction of bacteria to the endotracheal circuit. In addition, the proximal aspect of the ETT is least likely to be affected by immune response or antibiotics \[[@R33]\].
*Burkholderia, Neisseria*, *Moraxella,* and *Haemophilus* sequences are potential pathogens that were identified in proximal and distal biofilms as well as tracheal aspirates. While not all of these genera demonstrated a statistically significant difference between sample sites, they were represented in a higher abundance in the distal ETT biofilms and tracheal aspirates. This is not unexpected as these genera are often thought to be potential infectious pathogens in the lower airways \[[@R9]\]. While many investigations of VAP discuss the risk of oral microbiota being introduced to the lower airways through tracheal intubation, it is also evident that the microbiota of the lower airways impact the distal biofilms as well \[[@R9],[@R16]\]. The median MH value on site comparison ranged from 0.87 to 0.94 indicating that the bacterial communities of the tracheal aspirate and distal and proximal biofilms were consistent with each other. There were, however, nine patients with MH values less than 0.7 for one or more comparison between sampling sites. Subsequent analyses did not provide an explanation for the divergence observed in this subset of patients.
Patients with MH index less than 0.7 were neither more likely to be on antibiotics prior to extubation, nor were they noted to have a greater bacterial load at sampled sites than the rest of the cohort. While possible contamination could explain some of the differences observed, that fact that differences were seen within the ETT itself suggests there may be alternative explanations as well. These patients are interesting outliers and further investigation is required to identify factors that contribute to this diversity and its clinical impact on patients.
It has been suggested that strategies to reduce ETT biofilm accumulation may decrease the risk of VAP \[[@R3]\]. However, total bacterial load was found to be significantly higher in the tracheal aspirate compared to the distal ETT. This finding may suggest that the ETT biofilm is a reservoir for infection whereas bacterial replication and biomass is more robust in the airways where nutritive resources are more abundant. Furthermore, there was increased bacterial load at the proximal biofilm when compared to the distal biofilm among patients. This is in contrast to previous data that has reported higher bacterial load in the distal ETT biofilms \[[@R34],[@R35],[@R36]\]. This discrepancy may represent the effect of exposure to the external environment on each section of the ETT or the fact that the proximal ETT is protected from delivery of antibiotics or host immune responses. The duration of intubation did not appear to affect bacterial load at any of the three sample sites, which is consistent with at least one other study \[[@R15]\]. Given that biofilms and tracheal aspirate samples were obtained on the day of extubation, it is difficult to determine the effect of time on bacterial load for each individual patient. However, increased length of intubation as an independent variable does not appear to result in increased bacterial load when assessed in subjects intubated greater than 72 hours. It should be noted that ETTs analyzed just 72 hours after intubation had high levels of bacteria present, demonstrating how quickly bacterial biofilms are formed.
Our data showed that *Streptococcus* was the second most prominent genus in this patient population. *Streptococcus* has been described as forming biofilms especially in conjunction with other bacteria including *Actinomyces* species and *Veillonella* species \[[@R37],[@R38]\]. *Veillonella* species were found in both proximal and distal biofilms whereas *Actinomyces* species were not prominent in our study. Previous studies have isolated *Enterobacteriaceae* species within ETT biofilms though these species were not highly represented in this study \[[@R39],[@R40]\]. The internal lumen of the ETT was sampled in this study which is in contrast to similar studies which have sampled the external and internal aspects of the ETT \[[@R15],[@R16]\]. The internal lumen was selected as it was thought to be least impacted by the host immune response and systemic antimicrobial agents and also less likely to be affected by communication with the external environment with exposure to the bidirectional movement of ventilated gas.
This study does have several limitations. While this study is relatively large compared to similar published work performed utilizing 16S rRNA sequencing of ETT, the study size is still insufficient to make generalized conclusions within this patient population. Fifteen of the ETT did not have more bacterial DNA compared to reagent blanks after amplification with qPCR. Since neither the proximal nor distal portions amplified, the assumption was made that there was not significant biomass in the ETT biofilms. Given that a majority of samples did produce sufficient biomass, it is unlikely that sampling technique contributed to the lack of amplification. It may have been possible to increase yield of sampling using flocked swabs instead of standard culture swabs. Another limitation of the methodology employed for bacterial molecular identification is the inability to accurately identify organisms at the species level. Thus, further investigations are required to determine whether genera like *Staphylococcus* and *Streptococcus* are comprised of mostly commensal species versus potentially pathogenic ones. In addition, the 16S rRNA methodology does not detect fungi or viruses which may impact bacterial load and composition as well as host immune responses. Furthermore, there are multiple patient variables that could theoretically impact the microbiome of the subjects including the total use of antibiotics, nasal versus oral placement of ETT, or presence of a cuff. An exhaustive assessment of these variables was beyond the scope of this initial study, although several of the most impactful interventions such as antibiotics within 24 hours of extubation were evaluated. While data such as infection status on admission and antibiotic exposure were analyzed in this study, other factors such as suctioning frequency and consistency of technique were not examined, although each patient was managed per a standardized VAP bundle. Each of these elements could introduce variability in the quantitative assessment of bacterial communities, and a more exhaustive analysis of these factors may be warranted. Investigations evaluating the entire spectrum of microbiota and host responses are likely to produce further insights into lower airway colonization and the risks for transition from colonization to infection in mechanically ventilated children.
Conclusions {#S17}
===========
We found that there is a broad microbiota in tracheal aspirates and ETT biofilms from intubated pediatric patients. Oropharyngeal bacteria, which are a main source of inoculation of the airways, were most highly represented. However, there were significant numbers of potentially pathogenic genera present in variable abundances demonstrating the complex interaction between the upper and lower airways and the ETT. While a majority of sample sites showed similar microbial communities within patients there were differences between sample sites in nine patients that was not related to age, admission diagnosis, total bacterial load, or antibiotic administration 24 hours prior to extubation. Further investigations are warranted to determine the cause and clinical impact of such differences.
**Sources of funding:** Funding provided by an institutional CCTSI Grant, NIH/NCATS Colorado CTSA Grant UL1 TR001082 and NIH NHLBI R01 HL 124103.
**Competing interests:** The authors have declared that no competing interests exist.
**Ethical approval**
The Colorado Multiple Institutional Review Board approved the protocol, and parents or guardian provided informed consent for patients. COMIRB Protocol 09-1094. Initial approval 04-Dec-2009.
**Author Contribution**
M.K. Leroue, P.M. Mourani, and J.K. Harris contributed to conception and design of study.
M.K. Leroue, J.K. Harris, M.J. Stevens, Y.L. Sierra, and P.M. Mourani contributed to the acquisition of data.
M.K. Leroue, J.K. Harris, K.M. Burgess, J.I. Miller, M.K. Sontag, B.D. Wagner, and P.M. Mourani were involved in analysis and interpretation of data.
M.K. Leroue, J.K. Harris, P.M. Mourani, K.M. Burgess, M.K. Sontag, B.D. Wagner, Y.L. Sierra were involved in drafting the manuscript and revising the manuscript critically for important intellectual content.
M.K. Leroue, J.K. Harris, K.M. Burgess, M.J. Stevens, J.I. Miller, M.K. Sontag, Y.L. Sierra, B.D. Wagner, and P.M. Mourani approved the version of the manuscript published.
Guarantor: Peter M. Mourani, MD
{ref-type="fig"}. Asp: aspirate; Dis: distal; Prox: proximal.](nihms919553f1){#F1}
{#F2}
######
Patient characteristics (n=33)
Range Mean (SD)
--------------------------------------------------- -------------------- ---------------
Age (years) 0.2--17 years 6.1 (±5.1)
Height (cm) 55--167 cm 109.5 (±30.1)
Weight (kg) 4.1--100 kg 24.8 (±20.0)
Days intubated 3.0--22.0 days 8.8 (±5.0)
PICU length of stay 4.0--41.0 days 13.6 (±8.6)
Hospital length of stay 5.9--143.9 days 30.6 (±26.9)
Number of patients Percent
Gender
Male 19 57.6
Admission category
Medical 24 72.7
Surgical 1 3.0
Trauma 8 24.3
Admission medical diagnosis
Lower respiratory tract infection 4 16.7
Seizures 6 25.0
Sepsis 3 12.5
Non-infectious airway obstruction 3 12.5
Other 8 33.3
Received antibiotics 24 hours prior to extubation 8 24.2
######
Mean relative abundance of bacteria in sampled sites
Bacteria Overall Proximal Distal Tracheal Aspirate
----------------------- --------- ---------- -------- -------------------
*Prevotella* 36.78% 41.09% 35.54% 35.22%
*Streptococcus* 21.55% 16.64% 24.57% 21.69%
*Staphylococcus* 10.21% 16.77% 7.60% 8.59%
*Burkholderia* 3.94% 1.85% 1.95% 7.35%
*Moraxella* 3.92% \<1% 6.33% 3.78%
*Porphyromonas* 2.79% 1.51% 2.41% 4.01%
*Neisseria* 2.70% 1.46% 3.15% 3.04%
*Bacilli* 1.99% 2.45% 3% \<1%
*Veillonella* 1.80% 2.51% 1.81% \<1%
*Stenotrophomonas* 1.34% \<1% \<1% 2.38%
*Gammaproteobacteria* \<1% \<1% \<1% 1.82%
*Haemophilus* \<1% \<1% 1.41% 1.51%
*Lacobacillales* \<1% 1.48% \<1% \<1%
*Carnobacteriaceae* \<1% 1.45% \<1% \<1%
Other 12.98% 12.79% 12.25% 10.61%
######
Comparison of bacterial genera across sampled sites
Bacteria Distal vs Proximal Aspirate vs Proximal Aspirate vs Distal
------------------ -------------------- ---------------------- ------------------------------------------ ---------- -------- ---------------------------------------- ----------- -------- ----------------------------------------
Acinetobacter 7 (0.28) 0.002 0.39 9 (0.39) −0.006 0.11 11 (0.35) 0.001 0.45
Burkholderia 8 (0.32) −0.001 0.81 9 (0.39) −0.005 0.07 10 (0.32) −0.005 *0.02*[\*](#TFN1){ref-type="table-fn"}
Haemophilus 6 (0.24) 0.006 0.37 5 (0.22) −0.029 *0.02*[\*](#TFN1){ref-type="table-fn"} 6 (0.19) −0.002 0.91
Moraxella 10 (0.40) −0.002 0.29 7 (0.30) −0.028 0.06 15 (0.48) −0.010 0.58
Neisseria 0 (0.00) 0.011 0.29 3 (0.13) 0.033 0.08 3 (0.10) 0.001 0.47
Porphyromonas 4 (0.16) 0.002 0.32 1 (0.04) 0.076 0.97 6 (0.19) −0.008 0.26
Prevotella 0 (0.00) −0.063 0.98 0 (0.00) 1.929 0.54 0 (0.00) 0.221 0.55
Staphylococcus 0 (0.00) 1.854 *\<0.01*[\*](#TFN1){ref-type="table-fn"} 0 (0.00) 0.052 0.99 0 (0.00) −0.040 0.16
Stenotrophomonas 11 (0.44) 0.007 *\<0.01*[\*](#TFN1){ref-type="table-fn"} 9 (0.39) 0.004 0.53 13 (0.42) −0.001 0.08
Streptococcus 0 (0.00) −0.003 0.74 0 (0.00) 1.463 0.45 0 (0.00) 0.016 0.44
Veillonella 0 (0.00) 0.566 *\<0.01*[\*](#TFN1){ref-type="table-fn"} 0 (0.00) 0.530 0.08 2 (0.06) 0.009 0.78
p\<0.05 Negative values represent a higher relative abundance observed in the Distal site compared to the Proximal siteNegative values represent a higher relative abundance observed in the Aspirate site compared to the Proximal siteNegative values represent a higher relative abundance observed in the Aspirate site compared to the Distal site
|
{
"pile_set_name": "PubMed Central"
}
|
In the last two decades, there has been a significant increase in the incidental diagnosis of small renal masses by various diagnostic imaging modalities, as well as in the variety of treatments available for such masses. That has led to notable changes in the management of kidney cancer, which has evolved from open surgery to minimally invasive, nephron-sparing procedures. Strategies for the localized treatment of suspicious renal masses include radical nephrectomy, partial nephrectomy, thermal ablation, and active surveillance. The American Urological Association consensus guidelines recently included percutaneous ablation as an acceptable treatment option for high-risk surgical patients^([@r1])^. The ideal renal tumor for percutaneous thermal ablation is one that measures ≤ 3 cm and is in a location favorable to preserving the largest possible amount of normal renal tissue^([@r2])^.
In patients diagnosed with a renal mass and submitted to any of the various treatments, the preservation of renal function has become an important consideration. A decrease in renal function is associated with an increased risk of cardiovascular events, hospitalization, and mortality^([@r3],[@r4])^. It is well established that radical nephrectomy has a significantly greater impact on renal function than does partial nephrectomy^([@r5],[@r6])^, and that patients undergoing radical nephrectomy are at a higher risk of developing chronic kidney disease^([@r7])^. However, in studies comparing renal mass ablation and partial nephrectomy, both of which are nephron-sparing approaches, the differences in renal functional changes between the two treatment strategies have not been fully established. Therefore, although ablative techniques can offer advantages such as shorter recovery times and fewer complications, the decline in renal function is still the object of study in relation to the choice of treatments. Given the complexities associated with partial nephrectomy, percutaneous cryoablation can be considered an excellent alternative treatment in selected patients.
In a systematic review and meta-analysis carried out by Patel et al.^([@r8])^, renal function was assessed after surgical excision, ablation, and active surveillance of localized renal tumors. The authors identified 58 relevant articles and concluded that the implications for renal function varied among management strategies for localized renal masses, postoperative renal function being worse for patients undergoing radical nephrectomy than for those treated with other strategies and the results obtained with thermal ablation being similar to those achieved with partial nephrectomy. They called attention to the need for further studies to quantify changes in renal function associated with active surveillance and nephron-sparing approaches in patients with pre-existing chronic kidney disease.
The current issue of **Radiologia Brasileira** features an interesting article by Staziaki et al.^([@r9])^ entitled "Early trends and predictors of renal function following computed tomography-guided percutaneous cryoablation of a renal mass in patients with and without prior renal impairment". In that retrospective study, 39 patients underwent renal mass cryoablation. Despite its limitations (well detailed by the authors), such as the small number of patients evaluated, the retrospective nature of the study, the relatively short follow-up period, and the lack of a control group, their study showed that the trend in renal function after cryoablation did not differ between the patients with previous renal impairment and those without. In addition, the authors found that there were no post-cryoablation changes in the stage of renal disease in either group. They also observed that the glomerular filtration rate at baseline was predictive of the mean glomerular filtration rate in the early post-cryoablation period.
Given the ongoing technological advances in the area of diagnostic imaging and in medical knowledge, we must seek to care for our patients through the use of methods that are increasingly less invasive, in order to obtain the best possible result and with the fewest possible complications. In view of that, we believe that the work conducted by Staziaki et al.^([@r9])^ makes a noteworthy contribution.
|
{
"pile_set_name": "PubMed Central"
}
|
1. Rims of atrial septal defect (ASD) on the left hand {#sec0010}
======================================================
As illustrated in [Fig. 1](#fig0005){ref-type="fig"}, the left hand with its thumb and index finger stretched out represents the atrial septum ([Fig. 1](#fig0005){ref-type="fig"}a). The defect with the rims is illustrated in [Fig. 1](#fig0005){ref-type="fig"}b shows anteriorly the aorta and the aortic (anterior) rim and posteriorly the posterior rim. The posterosuperior and posteroinferior margins are the SVC and IVC rims respectively. Free atrial and mitral (AV valve) rims are of the nature anterosuperior and anteroinferior respectively.
2. Rims of ASD on trans-esophageal echo (TEE) {#sec0015}
=============================================
As illustrated in [Fig. 2](#fig0010){ref-type="fig"}, the blue line represents the long axis of the TEE probe and red line the axis of the visualized plane, that is the TEE visual angle. On TEE, the free atrial and the mitral rims are seen in 0° both red and blue line merged ([Fig. 2](#fig0010){ref-type="fig"}b). The hand is showing both the rims ([Fig. 2](#fig0010){ref-type="fig"}b) and the TEE image ([Fig. 2](#fig0010){ref-type="fig"}c).
As illustrated in [Fig. 3](#fig0015){ref-type="fig"}, at 45°, angle as shown by angulation between red and blue lines, the aortic and posterior rims are visualized ([Fig. 3](#fig0015){ref-type="fig"}a). The aortic and posterior rims at 45° are profiled in the hand image ([Fig. 3](#fig0015){ref-type="fig"}b) and TEE image ([Fig. 3](#fig0015){ref-type="fig"}c).
As illustrated in [Fig. 4](#fig0020){ref-type="fig"}, at 120° angle, as shown by angulation between the red and blue lines, the SVC and IVC rims are visualized, with slight rotation ([Fig. 4](#fig0020){ref-type="fig"}a). The SVC and the IVC rims are profiled in hand image at 120° plane ([Fig. 4](#fig0020){ref-type="fig"}b) and the TEE image ([Fig. 4](#fig0020){ref-type="fig"}c).
Conflicts of interest {#sec0020}
=====================
The author has none to declare.
The author acknowledges Dr Vaidehi Dande.
{#fig0005}
{#fig0010}
{#fig0015}
{#fig0020}
|
{
"pile_set_name": "PubMed Central"
}
|
1. Introduction {#sec1}
===============
Soybeans contain a wide range of isoflavones, such as daidzein, glycitein, genistein, and so forth. Soy isoflavones are naturally occurring polyphenol compounds and structurally similar to estradiol. Soybeans contain 3 mg g^--1^ (dry weight) of isoflavones.^[@ref1]^ Meanwhile, isoflavones are similar to the antioxidant flavonoids that are found in other vegetables, plants, and flowers. Among all the soy isoflavones, genistein and daidzein are the major portions of isoflavones in soybeans. Daidzein and genistein from soybeans are a source of phytoestrogens for humans. On the other hand, most of the natural estrogenic substances show weak activities. As a result, they have been widely investigated for their important health-enhancing properties such as prevention of sex hormone-dependent cancer, improvement of bone health, and so forth.^[@ref2]^ For instance, genistein has many health benefits as an antioxidant, inhibitor to regulate cell divisions and cells survival, antiangiogenetic agent, and so forth.^[@ref3],[@ref4]^ Their estrogenic activities have been demonstrated to bind to estrogen receptors from different animals, such as mice, rats, sheep, and so forth.^[@ref5]^
Meanwhile, glycitein (C~16~H~12~O~5~, 7,4′-dihydroxy-6-methoxyisoflavone) is about 5--10% of the total soy isoflavones.^[@ref6]^ Thus, it is essential to assess the chemical and physical activity of glycitein. One of the studies showed that glycitein is much weaker in estrogenic activity than other soy isoflavones.^[@ref1]^ In contrast, glycitein actually has a stronger estrogenic response on an equal amount basis in the mice uterine enlargement assay.^[@ref7]^ However, there are a variety of impurities in soy which can influence the quality of the isoflavones during production. Therefore, removal of the impurities below the acceptable level is required. One of the commonly employed separation method is extraction, and isoflavones are normally extracted from foods with methanol, ethanol, acetonitrile, and so forth.^[@ref8]−[@ref10]^ The extraction can be carried out either at room temperature or above.^[@ref11]^ Meanwhile, hydrogen bonding interactions between solute and polar organic solvents play a crucial role in the extraction reaction.^[@ref12]^ However, few studies have been focused on the extraction mechanism. Density functional theory (DFT) calculations are one of the effective methods to obtain the conformation, electronic structures, and inter/intramolecular interactions of isoflavones, such as daidzein, genistein, and so forth.^[@ref13],[@ref14]^ On the other hand, the intermolecular interaction in solvents, namely, hydrogen bonding interaction, has a great influence on the extraction process.^[@ref15]^ The main aim of this study is to calculate the hydrogen bonding interaction between glycitein and methanol (MeOH), ethanol (EtOH), or acetone in the extraction reaction from a theoretical point of view.
2. Results and Discussion {#sec2}
=========================
2.1. Conformational Analysis {#sec2.1}
----------------------------
The framework and atom numbering of glycitein are presented in [Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"}. There are two phenyl rings (**I** and **III**) and one heterocyclic ring (**II**) in glycitein. They contain several functional groups: −OCH~3~, −OH, and −C=O. The optimization of the glycitein monomer at the B3LYP-D3 DFT functional level converged to eight different conformers when one rotates the −O~a~CH~3~, −O~b~H~b~, or −O~e~H~e~ functional groups ([Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}). Then, it can be noticed that all the eight conformers have nonplanar structures, and they can be divided into two groups: O~b~--H~b~···O~a~ intramolecular hydrogen bonded structures ([Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}, A, B) and non-hydrogen-bonded structures ([Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}, C--H). Meanwhile, there is a torsion angle between the **II** and **III** rings as seen in [Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"}. In a previous study, the conformational absolute minimum of glycitein by rotating the **II** and **III** rings was found with a torsion angle of 40° (B3LYP/6-311G(d,p)).^[@ref16]^ In this study, the torsion angles were calculated to be 40.9°--41.9° for all eight conformers. As a result, the coplanarity between the **II** and **III** rings is lost, whereas the coplanarity remains between the **I** and **II** rings. In the most stable conformers \[glycitein (A) and glycitein (B), [Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}\], the hydroxyl group O~b~--H~b~ interacts with the O~a~ carbonyl atom to form an intramolecular hydrogen bond. This makes the glycitein (A) and glycitein (B) conformers at least ∼19 kJ mol^--1^ more stable than the non-hydrogen-bonded conformers. Meanwhile, the geometric difference between glycitein (A) and glycitein (B) is the orientation of the −O~e~H~e~ group. Glycitein (A) is only slightly about 0.8 kJ mol^--1^ more stable than glycitein (B). In contrast, the rotational barrier between the two conformers is much higher about 14.3 kJ mol^--1^. However, different orientations of the −O~e~H~e~ group are unlikely to affect the relative stability of the molecular interaction between glycitein and various solvents in this work, and the strongest interaction is the one between carbonyl oxygen and solvents. Meanwhile, the EtOH monomer has two conformers: a *trans*-conformer and a *gauche*-conformer.^[@ref17]^ The *gauche*-conformer is about 0.3 kJ mol^--1^ \[B3LYP-D3/cc-pVTZ, corrected with zero-point vibrational energy (ZPVE)\] more stable than the *trans*-conformer. Thus, only the most stable glycitein (A) and the *gauche*-EtOH conformer will be used to study the molecular interaction in this study. The notations "glycitein" and "EtOH" in the following text will refer to glycitein (A) and *gauche*-EtOH, respectively.
{#fig1}
{#fig2}
Glycitein interacts with solvents (such as MeOH, EtOH, and acetone) during extraction as either a hydrogen bond acceptor or a hydrogen bond donor. When it acts as a hydrogen bond acceptor, there are five different docking sites for the hydrogen atom of MeOH, that is, O~a~, O~b~, O~c~, O~d~, and O~e~ ([Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"}). The labels of hydrogen, carbon, and oxygen atoms are in white, gray, and red colors, respectively. The O~b~ atom together with the O~e~ atom belongs to the hydroxyl groups of the glycitein molecule. The O~b~ and O~d~ atoms, on the other hand, belong to an ether group while O~c~ is derived from a carbonyl group. As a hydrogen bond donor, the two hydroxyl groups, −O~b~H~b~, and −O~e~H~e~, can donate their hydrogen atoms to a hydrogen bond acceptor, such as acetone. The most stable structures of the MeOH--glycitein complexes at the B3LYP-D3/cc-pVTZ level are presented in [Figure [3](#fig3){ref-type="fig"}](#fig3){ref-type="fig"}. The MeOH molecule acts as a hydrogen bond donor and acceptor in MeOH--glycitein (A). MeOH is the hydrogen bond donor approaching to glycitein in MeOH--glycitein (B--E), while MeOH is the hydrogen bond acceptor in MeOH--glycitein (F). The binding energy (BE) is one of the most effective indicators to reveal the relative stability of a structure. BE of a stable interacted complex is often negative. The lower BE means a stronger molecular interaction. Meanwhile, ZPVEs are quite large about 4.6--6.6 kJ mol^--1^ for the studied systems, and basis set superposition errors (BSSEs) vary from 6.8 to 9.9 kJ mol^--1^ (Table S1, [Supporting Information](http://pubs.acs.org/doi/suppl/10.1021/acsomega.9b02464/suppl_file/ao9b02464_si_001.pdf)). The binding energies in [Figure [3](#fig3){ref-type="fig"}](#fig3){ref-type="fig"} were calculated at ambient conditions (298.15 K and 1 atm) at the B3LYP-D3/cc-pVTZ level and corrected with ZPVE and BSSE. Based on the binding energies, the strength as a hydrogen bond acceptor can be sorted as O~c~ \> O~b~ \> O~e~ \> O~a~ \> O~d~. This indicates that the carbonyl oxygen is the best hydrogen bond acceptor, and this is in line with previous studies: (i) the O--H···O=C (carbonyl oxygen) hydrogen bonding interactions are about 9.6--11.0 kJ mol^--1^ (B3LYP/6-31+G(d)) more favorable than the O--H···O (ester oxygen) hydrogen bonding interaction in the MeOH−α-hydroxyester systems, where α-hydroxyester is methyl glycolate, methyl lactate, or methyl α-hydroxyisobutyrate;^[@ref18]^ (ii) the O--H···O=C (carbonyl oxygen) hydrogen bonding interaction is about 9.7 kJ mol^--1^ also more stable than the corresponding O--H···O (ester oxygen) hydrogen bonding interaction in the MeOH--methyl lactate system.^[@ref19]^ All these imply that the most favorite docking site for the incoming MeOH is the carbonyl group oxygen O~c~.
{#fig3}
Meanwhile, the strength as a hydrogen bond donor is sorted as O~e~H~e~ \> O~a~H~a~. Thus, for the EtOH--glycitein and acetone--glycitein systems, only the most stable structures formed between EtOH/acetone and the O~c~ or O~e~H~e~ group of glycitein were studied. The most stable structures of the EtOH--glycitein and acetone--glycitein complexes at the B3LYP-D3/cc-pVTZ level are presented in [Figure [4](#fig4){ref-type="fig"}](#fig4){ref-type="fig"}. This demonstrates that the driving force for extracting glycitein is the hydrogen bonding interaction between the solvent (a hydrogen bond donor, such as MeOH and EtOH) and carbonyl oxygen O~c~ of glycitein, or between the solvent (a hydrogen bond acceptor, such as acetone) and the hydroxyl group O~e~H~e~ of glycitein.
{#fig4}
2.2. Solvent Effects and Their Influence on the Hydrogen Bond {#sec2.2}
-------------------------------------------------------------
In order to study the effects of solvents on the electronic energies, geometrical parameters, and IR frequencies of the glycitein-containing complexes, the most stable conformers of the MeOH--glycitein, EtOH--glycitein, and acetone--glycitein structures were fully optimized within the polarizable continuum model (PCM) at the B3LYP-D3/cc-pVTZ level of theory. The calculated geometrical parameters and interaction energies of the most stable conformers of MeOH--glycitein, EtOH--glycitein, and acetone--glycitein in different solvents are present in [Table [1](#tbl1){ref-type="other"}](#tbl1){ref-type="other"}. The relative permittivity (ε) values of acetone, ethanol, and methanol are 20.493, 24.852, and 32.613, respectively.^[@ref20]^ The structure is stabilized in the solvent, and it is due to immersion in the solvent. The stabilized energy (Δ*E*~S~) can be calculated as followswhere *E*~(solvent)~ is the electronic energy in the solvent, and *E*~(gas)~ is the electronic energy in the gas phase. Meanwhile, the major components of the hydrogen bonding interaction energy are electrostatic and charge-transfer; thus, the effect of the solvent polarity on the hydrogen bond is expected.^[@ref21]^ In this study, the polarity of the solvent plays an important role in extraction, and this is due to the hydroxyl groups of glycitein. MeOH--glycitein is stabilized by 38.2 kJ mol^--1^ (in the MeOH solvent), 37.5 kJ mol^--1^ (in the EtOH solvent), and 36.8 kJ mol^--1^ (in the acetone solvent) as compared with the electronic energy in the gas phase, whereas the EtOH--glycitein complex is stabilized by 37.4--38.8 kJ mol^--1^ in the three solvents. In contrast, the acetone--glycitein complex is favored by 44.2--45.7 kJ mol^--1^ in the three solvents. It should be mentioned that the dipole moments of the three complexes were found to be 1.86, 2.87, and 5.96 D for MeOH--glycitein, EtOH--glycitein, and acetone--glycitein, respectively, at the B3LYP-D3/cc-pVTZ level of theory (gas phase). To summarize, the PCM model indicates that acetone--glycitein is stabilized more than EtOH--glycitein and MeOH--glycitein. This is due to the higher dipole moment of the former when the solvent effects of MeOH, EtOH, and acetone solvents were taken into account. For the glycitein-containing complexes, BEs were obtained at −39.1 to −36.2 kJ mol^--1^ in the gas phase. This is much more stable than some MeOH-containing hydrogen bonded systems (gas phase), where MeOH--dimethylamine (DMA), MeOH--trimethylamine (TMA), and MeOH--dimethylether (DME) were obtained to be about −21.2 to −19.7 kJ mol^--1^ (B3LYP/aug-cc-pVTZ).^[@ref22],[@ref23]^ Meanwhile, BEs were calculated to be −34.6 to −32.7 kJ mol^--1^ in the three solvents. This means that the monomers in the three solvents bind to each other slightly less favored than the monomers in the gas phase bind to each other.
###### Selected Parameters of the Most Stable Glycitein-Containing Complexes Calculated by Applying the B3LYP-D3 Method Using the cc-pVTZ Basis Set[a](#t1fn1){ref-type="table-fn"}
conformer solvent BE Δ*ṽ*[a](#t1fn1){ref-type="table-fn"} Δ*r*~(OH)~ Δ*E*(H) ρ(BCP) ∇^2^ρ(BCP) dipole moment
-------------------- --------- -------- -------------------------------------- ------------ --------- -------- ------------ ---------------
MeOH--glycitein gas --37.2 255 0.014 0.041 0.024 0.145 1.86
acetone --25.2 294 0.015 0.050 0.027 0.166 2.80
MeOH --25.1 312 0.015 0.050 0.027 0.166 2.89
EtOH --24.9 311 0.015 0.050 0.027 0.166 2.84
EtOH--glycitein gas --39.1 227 0.012 0.045 0.024 0.137 2.87
acetone --27.8 282 0.013 0.047 0.026 0.153 3.40
MeOH --27.7 284 0.013 0.047 0.026 0.153 3.49
EtOH --27.6 283 0.013 0.047 0.026 0.153 3.44
acetone--glycitein gas --36.2 298 0.015 0.051 0.025 0.143 5.96
acetone --28.3 424 0.020 0.061 0.028 0.177 5.87
MeOH --28.2 426 0.020 0.061 0.028 0.177 5.88
EtOH --28.1 428 0.020 0.061 0.028 0.177 5.87
BEs are given in kJ mol^--1^. Δ*ṽ* = *ṽ*~monomer~ -- *ṽ*~dimer~ (in cm^--1^). Δ*r*~(OH)~ = *r*~dimer~ -- *r*~monomer~ (in Å), is the change in the OH bond length upon complexation. QTAIM parameters \[Δ*E*(H), ρ(BCP), ∇^2^ρ(BCP)\] are given in au. Dipole moment is given in debye.
The OH-stretching vibrational frequencies of MeOH, EtOH, and glycitein monomers are red-shifted (Δ*ṽ* = *ṽ*~monomer~ -- *ṽ*~dimer~) by about 11--20 cm^--1^ in the three solvents when compared with the values in the gas phase. The corresponding OH bond lengths are increased by 0.001--0.002 Å in the three solvents. Meanwhile, the C=O stretching vibrational frequencies of acetone and glycitein are also red-shifted by 36--56 cm^--1^ in the solvent, and the C=O bond distances are elongated by 0.006--0.007 Å in the three solvents as well. On the other hand, there is a change in the hydrogen bond length during complexation. Thus, it leads to a red shift between the free and the hydrogen-bonded vibrational transitions. The quantity of red shift is commonly used to justify the relative strength of the hydrogen bonding interaction.^[@ref24],[@ref25]^ The B3LYP-D3/cc-pVTZ-calculated red shifts of the OH-stretching fundamental transition upon complexation and the corresponding changes of the OH bond are listed in [Table [1](#tbl1){ref-type="other"}](#tbl1){ref-type="other"}. The red shifts of the OH-stretching vibrational frequencies are 255--298 cm^--1^ in the gas phase as compared with the monomer (gas phase). The corresponding red shifts are significantly increased to 282--428 cm^--1^ in the three solvents. This is because the strength of the hydrogen bond dramatically increases in the three solvents. Upon complexation, the changes of the OH bond length of the glycitein-containing complexes vary from 0.012 to 0.020 Å. These are similar with the previous studies in the O--H···O hydrogen bonded system. In the study of the MeOH--DMA and MeOH--TMA complexes (gas phase), the OH bond lengths in the O--H···O hydrogen bonds were calculated to be elongated by 0.016--0.018 Å during complexation.^[@ref22]^ However, the NH bond distances in the N--H···N hydrogen bonds (gas phase) were only increased by 0.003--0.004 Å (QCISD/aug-cc-pVTZ) in DMA--DMA and 0.005 Å \[CCSD(T)-F12a/VDZ-F12\] in DMA--TMA upon hydrogen bond formation.^[@ref25],[@ref26]^ Consequently, comparable red shifts were also found in MeOH-containing complexes (gas phase): MeOH--TMA (333 cm^--1^, local mode model), MeOH--DME (234 cm^--1^, B3LYP-D3/aug-cc-pVTZ), and MeOH--DMA (301 cm^--1^, local mode model).^[@ref22],[@ref23]^
The most stable conformers of glycitein and MeOH--glycitein in both the gas phase and the MeOH solvent were used to simulate the ultraviolet--visible (UV--vis) absorption spectra. The first 200 singlet → singlet spin-allowed excited states were calculated, and the max absorption wavelengths λ~max~, the electronic excitation energies, and the oscillator strengths *f* were obtained using time-dependent (TD)-DFT at the B3LYP-D3/cc-pVTZ level. The simulated 200--350 nm UV--vis spectra are displayed in [Figure [5](#fig5){ref-type="fig"}](#fig5){ref-type="fig"}. The simulated spectra were formed because of the electronic transitions from the highest occupied molecular orbitals (HOMOs) to the lowest unoccupied molecular orbitals (LUMOs). The five important frontier molecular orbitals of glycitein (in the MeOH solvent): HOMO -- 2, HOMO -- 1, HOMO, LUMO, and LUMO + 1, are illustrated in [Figure [6](#fig6){ref-type="fig"}](#fig6){ref-type="fig"}. It is clear that the five frontier molecular orbitals are the π and π\* molecular orbitals locating at the **I**, **II**, and **III** rings of glycitein (in the MeOH solvent). One can notice that it is the π → π\* transitions taking place in the UV--vis region with high extinction coefficients. The max absorption wavelengths λ~max~ of glycitein were calculated to be 271.63 nm (gas phase) and 270.49 nm (in the MeOH solvent). The max absorption wavelengths λ~max~ of MeOH--glycitein were calculated to be 271.62 nm (gas phase) and 273.54 nm (in the MeOH solvent). Moreover, the experimental UV--vis spectral data for glycitein were λ~max~ 257 nm (in the MeOH solvent).^[@ref1]^ Our calculated λ~max~ is slightly larger than the experimental value, and this may be due to the DFT method used which overestimates the UV--vis spectra. Moreover, our calculations show that the max absorption is mainly formed by three excitations: +0.66(HOMO → LUMO + 1), +0.14(HOMO -- 1 → LUMO + 1), and 0.15(HOMO -- 1 → LUMO). The interacting site in MeOH--glycitein is the carbonyl group of glycitein. The interaction only has a slight effect on the π and π\* molecular orbitals located at the **I**, **II**, and **III** rings of glycitein. Thus, the max absorption wavelength is only marginally influenced by the hydrogen bonding interaction.
{#fig5}
{#fig6}
2.3. Nature of Hydrogen Bond: Quantum Theory of Atoms in Molecules Analysis {#sec2.3}
---------------------------------------------------------------------------
The topological quantum theory of atoms in molecules (QTAIM) was unitized to analyze the chemical bond between two neighboring atoms. Then, the parameters to describe the nature of a chemical bond, such as, bond critical points (BCPs), ring critical points (RCPs), cage critical points (CCPs), electron density ρ(*r*) and Laplacian ∇^2^ρ(*r*) at BCPs, and atomic charge Δ*q*(H) and atomic energy \[Δ*E*(H)\] of the hydrogen bond donor atom, were calculated with the AIM2000 program package. The QTAIM topological plots of the most stable MeOH--glycitein, EtOH--glycitein, and acetone--glycitein conformers with BCPs, RCPs, and electron density paths are displayed in [Figure [7](#fig7){ref-type="fig"}](#fig7){ref-type="fig"}. The corresponding parameters in the gas phase and the solvents are listed in [Table [1](#tbl1){ref-type="other"}](#tbl1){ref-type="other"}. Moreover, the strength of a hydrogen bond can be classified as follows: (a) weak hydrogen bond: Δ*E*(H) \< 0.019 au, ∇^2^ρ(*r*) \> 0; (b) medium hydrogen bond: 0.019 au \< Δ*E*(H) \< 0.038 au, ∇^2^ρ(*r*) \> 0; and (c) strong hydrogen bond: Δ*E*(H) \> 0.038 au, ∇^2^ρ(*r*) \< 0.^[@ref27]^
{#fig7}
As seen in [Table [1](#tbl1){ref-type="other"}](#tbl1){ref-type="other"}, the Laplacian of total electronic densities, ∇^2^ρ(*r*), at BCPs are all positive, 0.137--0.177 au. This indicates that the electronic charges are depleted in the interatomic path. Thus, the hydrogen bonding interactions are classified as closed-shell molecular interactions. According to the abovementioned classification, the O--H···O~c~ and O~e~--H~e~···O hydrogen bonds are strong hydrogen bonds \[Δ*E*(H) = 0.041--0.061 au\] in both the solvents and the gas phase. Moreover, the strengths of the studied hydrogen bonds increase in solvents. However, the dielectric constants of the three solvents are very close to each other, so the increase in the strength of the hydrogen bond seems very close in the three solvents as well.
For a hydrogen bond, the electron density ρ(BCP) and the Laplacian of charge density ∇^2^ρ(BCP) at BCPs should be in the range 0.002--0.040 au and 0.014--0.139 au, respectively.^[@ref28],[@ref29]^ For the studied systems in [Table [1](#tbl1){ref-type="other"}](#tbl1){ref-type="other"}, ρ(BCP) and ∇^2^ρ(BCP) were calculated to be in the ranges 0.024--0.028 and 0.137--0.177 au, respectively. However, the ∇^2^ρ(BCP) values are higher than the upper range of the Laplacian criteria for a hydrogen bond.^[@ref28],[@ref29]^ The ∇^2^ρ(BCP) values for the interactions between benzoic acid/*cis*-pinonic acid--sulfuric acid were also calculated to be exceeding the upper range of hydrogen bond criteria.^[@ref30]^ This is due to the formation of strong hydrogen bonds. On the other hand, there is also a charge transfer (CT) when a hydrogen-bonded complex is formed, leading to a decreased charge on the hydrogen atom.^[@ref31]^ The quantity of CT reveals a part of the stabilization energy of the hydrogen bonded system, and it determines the electron delocalization interaction between the two interacted systems.^[@ref32]^ This means that the more electron transfer it does, the more stable the system is. In this study, there are about 0.09--0.11 electrons from the hydrogen bond acceptor to the donor. The results of Δ*q*(H) demonstrate that the hydrogen bond strengths in the gas phase are greater than the ones in the solvent.
3. Concluding Remark {#sec3}
====================
In the present study, DFT has been used to investigate the naturally occurring isoflavonoid compound, glycitein. The hydrogen bonds were analyzed using the B3LYP-D3 level of theory at the cc-pVTZ basis set. The effects of various solvents (methanol, ethanol, and acetone) on the hydrogen bond between glycitein and various solvents were investigated. This study aimed to determine the electronic energies, geometric parameters, solvent effects, and so forth of the molecules in question. The results obtained from DFT and the topological parameters suggest that the most stable clusters are found to be stabilized by hydrogen bonds formed with the hydroxyl group of glycitein and the carbonyl group of acetone or with the carbonyl group of glycitein and MeOH/EtOH. The UV--vis adsorption spectra were calculated with TD-DFT to investigate the electronic properties. The analysis of the solvent effect demonstrated that the polar solvent stabilizes the complexes. The changes of the OH bond lengths are larger in the three solvents because of the polar environments.
4. Methodology {#sec4}
==============
All the computations have been performed using the Gaussian 09 Revision E.01 software package^[@ref20]^ Because of the excellent computational time and electronic properties (such as electronic structures, vibrational frequencies, and so forth), the DFT method has been used. The B3LYP-D3 approach was carried out. This approach is a hybrid functional of the DFT method, and it contains the Becke's three-parameter nonlocal exchange functional with the correlation functional of the Lee--Yang--Parr (B3LYP) method and the Grimme's D3 dispersion correction.^[@ref33]^ Previous studies on small hydrogen-bonded molecular clusters were found to offer very accurate electronic energetics, vibrational frequencies, structural information, and so forth.^[@ref24],[@ref34]−[@ref38]^ Moreover, the Dunning's correlation consistent triple-zeta basis set (cc-pVTZ) was used throughout the computational process. Vibrational frequencies of the optimized structures were computed at the same level of theory to confirm the nature of stationary points. The corresponding ZPVE correction and thermodynamic corrections were added to electronic energies. Meanwhile, BSSE was added to BEs by using the typical counterpoise method.^[@ref39]^ For UV--vis calculations, the electronic maximum absorption wavelengths λ~max~ of glycitein and MeOH--glycitein in the MeOH solvent were computed using the TD-DFT method at the B3LYP-D3/cc-pVTZ level.
Several different solutions (methanol, ethanol, and acetone) to investigate the influence of the solvent on hydrogen bonds were used, and the corresponding effects were compared with those in the gas phase. The solvation effects were calculated considering the cavity of series of spheres by the aid of the means of the self-consistent reaction field method with the integral equation formalism variant model.^[@ref40]^ The analysis of the electronic charge density (ρ), its Laplacian ∇^2^ρ at BCPs, changes in atomic charge Δ*q*(H), and changes of atomic energy Δ*E*(H) at the H atom was performed by making use of the theory of molecular structure to investigate the nature of hydrogen bonds. The topological QTAIM was performed using the "output = WFN" option for the AIM keyword as implemented in Gaussian 09 Revision E.01 and AIM2000 software.
The Supporting Information is available free of charge on the [ACS Publications website](http://pubs.acs.org) at DOI: [10.1021/acsomega.9b02464](http://pubs.acs.org/doi/abs/10.1021/acsomega.9b02464).Calculated BE, ZPVE, BSSE, enthalpy of formation (Δ*H*~289K~^θ^), and Gibbs free energy of formation (Δ*G*~289K~^θ^) of MeOH--glycitein at the B3LYP-D3/cc-pVTZ level ([PDF](http://pubs.acs.org/doi/suppl/10.1021/acsomega.9b02464/suppl_file/ao9b02464_si_001.pdf))
Supplementary Material
======================
######
ao9b02464_si_001.pdf
The authors declare no competing financial interest.
This research is funded by the Province Key Laboratory of Cereal Resource Transformation and Utilization, Henan University of Technology under the grant number: PL2017003. We also thank Shandong University for providing Gaussian 09 Revision E.01 software package and high-performance computation.
|
{
"pile_set_name": "PubMed Central"
}
|
Background {#Sec1}
==========
Hepatocellular carcinoma (HCC) is one of the most aggressive malignancies worldwide, being recognized as the third leading cause of cancer-related deaths \[[@CR1], [@CR2]\]. HCC is the fifth most common malignancy in men and the seventh among women \[[@CR3]\]. The incidence of HCC depends on geography, and most of the burden is in developing countries, occurred with hepatitis. The situation is more severe in China, with poorer 5-year survival \[[@CR4]\]. The tumorigenesis of HCC is a multistage process including noncoding and protein-coding genes. MiRNAs are found to be deregulated in most malignancies, affecting carcinogenesis, progression, metastasis and tumor recurrence. In HCC, it has been reported that aberrant expression of let-7 miRNAs contributed to the development and progression of HCC \[[@CR5], [@CR6]\].
Research has indicated that some miRNAs may function as oncogenes when up-regulated in HCC; to the contrary, the down-regulated miRNAs suggested them as tumor suppressors \[[@CR7], [@CR8]\]. Functioning as tumor suppressors, let-7 miRNAs were found to repress Ras, Bcl-xl, MAPK, c-Myc, cyclin D1 and other oncogenes in HCC \[[@CR8], [@CR9]\]. Wnt1 stimulates the Wnt/β-catenin/TCF pathway, leading to different cell fates, and then regulating the transcription of many downstream genes which contain the TCF/LEF1 motif, affecting biological functions and the maintenance of self-renewal of cancer stem cells (CSCs) \[[@CR10]--[@CR12]\]. However, there is no research focused on the relationship between let-7 and the Wnt signaling pathway.
Tumors consist of cells with heterogeneity, with different characteristics, and the CSCs are demonstrated to be steady and stable through clinical chemo-radiotherapy, due to the slow cell cycles and low proliferative ability, contributing to tumor relapse and occurrence of resistance \[[@CR8], [@CR13]\]. Therefore, treatments targeting these silent CSCs will show great potential in eliminating the tumor group entirely, helping to overcome resistance to therapy and recurrence of the tumor. However, the underlying mechanism by which let-7 works to inhibit CSCs in HCC remains largely unknown.
Methods {#Sec2}
=======
Cell culture, transfection and infection {#Sec3}
----------------------------------------
MHCC97-H and HCCLM3 human HCC cells were purchased from ATCC and maintained at the central laboratory of the Qilu Hospital of Shandong University. The cells are cultured in DMEM medium (Invitrogen, USA), containing 10 % fetal bovine serum (FBS), 1 % penicillin and 1 % streptomycin (Invitrogen, USA). The spheres (hepatospheres) were cultured in DMEM/Ham's F-12 medium compounded with 10 ng/ml epidermal growth factor, 10 ng/ml human basic fibroblast growth factor, 4 μg/ml insulin, 1 % penicillin and 1 % streptomycin (Invitrogen, USA). Oligonucleotides encoding mature let-7a/b/c/d/e/f/g/i miRNAs and miRNA-LSC1 were synthesized by Invitrogen and cloned into the lentiviral vector lentilox3.7 (pLL3.7). SiRNAs targeting Wnt1were synthesized and purchased from GenePharma Inc. (Shanghai, China). Transfections were performed using Lipofectamine 2000 (Invitrogen, USA), and the siRNAs were added the second days after cells were plated. Transiently transfected HCC cells were harvested 48 h post-transfection.
Reverse transcription PCR and real-time PCR {#Sec4}
-------------------------------------------
Total RNA was isolated from fresh clinical specimen using TRIzol® Reagent (Invitrogen, USA) following mechanical tissue homogenization, or from or cultured cells using Trizol reagent after treatment with 5 μg/ml puromycin for 48 h. Approximately 1 μg of RNA was reverse-transcribed to single strand cDNA using PrimeScript RT Master Mix (Takara Biotechnology, China). The real-time PCR was conducted in a total volume of 25 μl as previously reported \[[@CR7], [@CR14]\]. The cycle threshold (Ct) was automatically calculated by iQ™-5 Optical Module software (Bio-rad, USA). The relative expression of let-7 family members was normalized to U6 expression, and then calculated using the formula 2^−ΔΔCt^ method, versus the scramble control.
Sphere formation assays {#Sec5}
-----------------------
The spheres formation assay is often used to enrich stem-like cells, being are used to isolate and expand population enriched in CSCs \[[@CR15]--[@CR18]\]. Cells of different groups were seeded in ultra-low adherent-conditioned plates (Corning, USA) to test their ability of forming primary spheres, in the presence of puromycin. In total, 12,000 cells were seeded per 6 cm plate. On day 9, cell sphere number of spheres was counted using an inverted microscope, a sphere was defined as contaning more than 10 cells. The sphere-forming efficiency was calculated as the percentage of counted spheres versus seeded cells \[[@CR19]\]. Spheres of different groups were disaggregated, and 12,000 cells were then re-suspended per 6 cm plate to test their self-renewal ability.
Immunofluorescence and IHC {#Sec6}
--------------------------
Cells were seeded on glass chamber slides for 24 h, and then fixed by 10 % formalin for 15 min. Antigens were blocked with 2 % normal goat serum (ab7481, Abcam, USA), cells werethen incubated with the β-catenin antibody for 1 h in PBS, and then incubated with Alexa Fluor® 488 (Life technologies, USA) for 30 min, washed in PBS, incubated for 10 min with 2 μg/ml of Hoechst 33342 (Life technologies, USA), and washed with PBS again. For IHC, the paraffin tissues were rehydrated in an alcohol gradient and then were rinsed in deionized water. Endogenous peroxidase activity was blocked using a 0.03 % hydrogen peroxide solution. 1:200 IHC-TekTM Antibody Diluent (IW-1000, Ellicott City, MD, USA) was used to reduce background staining. The section was then incubated with 1 μg/ml ab16051 for 1 h at room temperature and detected using an HRP, DAB was used as the chromogen.
Flow cytometry analysis {#Sec7}
-----------------------
The propidium iodide (PI) and Annexin V-FITC (Green) kits were obtained from BD Biosciences (San Jose, CA). Cells of different groups were collected and trypsinized into a single cell, and then washed twice with PBS. All cells were resuspended in 500 μl binding buffer respectively, and 5 μl of Annexin V-FITC and 10 μl of PI were added. The mixture was gently vortexed and then incubated for 15 min at room temperature in the dark. The cells were analyzed by FACSAria cytometry within 1 h of incubation using BD FACSuite Software. For cell cycle analysis, 5 × 10^5^ cells were collected and washed twice with PBS. Cells were fixed with ice-cold 70 % ethanol at 4 °C for 24 h. Before detection, the fixed cells were stained with PI for 30 min at 37 °C, followed by FACSAria cytometry. All tests were performed in triplicate. Cell cycle analysis of DNA content was performed using MultiCycle software. Instrument Setup instructions were followed as presenteding online documentation: <https://www.bdbiosciences.com/documents/BD_FACSVerse_Apoptosis_Detection_AppNote.pdf>.
Luciferase assay {#Sec8}
----------------
The TCF/lymphoid enhancer factor luciferase reporter (TOP) and its negative control (FOP) plasmids were obtained from Upstate Biotechnology (NY, USA). Cells were plated at 30--40 % confluence in 24-well plates 16 h prior to transfection by using FuGENE 6 (Roche, USA) \[[@CR20]\].
Let-7 and Wnt1 expression in clinical tissues {#Sec9}
---------------------------------------------
To explore the role of let-7a in HCC, we first collected specimens from 58 patients, who underwent surgery at the Department of General Surgery, the Second Hospital of Jilin University from June 2008 to November 2013. Of the samples collected, 20 slides were prepared for IHC using a Wnt 1 (1:200; sc-6280, Santa Cruz, USA). All of the patients were diagnosed and confirmed by pathological examination. No preoperative chemotherapy or radiotherapy was performed in any of these patients. Specimens were stored in liquid nitrogen. This study was conducted under the supervision of the Ethical Board of Jilin University.
Statistical analysis {#Sec10}
--------------------
All data were obtained from at least three independent experiments, are expressed as mean ± SD and were analyzed by Student's *T*-test and *χ* ^2^ test using SPSS for Windows version 16.0 (IBM, USA) and Excel 2010 (Microsoft, USA).
Results {#Sec11}
=======
The function of let-7 miRNAs in HCC cells {#Sec12}
-----------------------------------------
RFP based let-7a/b/c/d/e/f/g/i lentiviral vectors were successfully infected into HCC cells, asshown in Fig. [1](#Fig1){ref-type="fig"}. Inhibition of cell proliferation was detected using MTT assay at 48 h. The proliferation of let-7a-overexpressing HCC cells was suppressed most effectively compared to scramble and empty vector groups, as determined by the Student's *T*-test (*p* \< 0.01) and ANOVA (*p* \< 0.01) analysis with Bonferroni correction (Fig. [2a](#Fig2){ref-type="fig"}).Fig. 1The construction of let-7 miRNAs overexpressing HCC cells. **a**. The expression levels of let-7 miRNAs in HCC cells were detected after lentivirals infection, and results showed that we successfully constructed let-7 overexpressing HCC cells. The lentivirals vectors infected cells were *red* when observed under Inversed Fluorescent Microscope Fig. 2The inhibitory effects let-7 on HCC cells. **a**. After let-7 miRNAs were successfully overexpressing in HCC cells, the effects of let-7 on cell proliferation were detected by MTT assay. Let-7a was demonstrated to exert the strongest repression on cell proliferation, defined by student *t* test and Two-way ANOVA, \* *p* \< 0.01. Let-7a induced more cell apoptosis in MHCC97-H and HCCLM3 cells (**b**); let-7a also increased the portion of cells staying in G0-G1 stage and decreased the cells in S phase (**c**), compared to Scramble group. \* *p* \< 0.01. The representative images of the FACS derived apoptosis ratios (**d**) and FACS derived cell cycle analysis (**e**)
Overexpressing let-7a induced apoptosis and cell cycle arrest of HCC cells {#Sec13}
--------------------------------------------------------------------------
To examine whether the enforced let-7a could induce cell apoptosis and cell cycle arrest, both MHCC97-H and HCCLM3 cell lines were subjected to Flow cytometry analysis. We found that both of these HCC cell lines exhibited higher apoptosis ratios (*T*-test, *p* \< 0.01, Fig. [2b](#Fig2){ref-type="fig"}). Let-7a also induced cell cycles arrest at G1 (*T*-test, *p* \< 0.01, Fig. [2c](#Fig2){ref-type="fig"}). Representative images of apoptosis and cell cycle are shown in Fig. [2d-e](#Fig2){ref-type="fig"}.
Let-7a suppressed the sphere formation efficiency of HCC cells {#Sec14}
--------------------------------------------------------------
Sphere formation efficiency of MHCC97-H-let-7a and HCCLM3-let-7a cells was significantly lower than that of Scramble groups (*T*-test, *p* \< 0.01, Fig. [3a](#Fig3){ref-type="fig"}). Disaggregation of primary hepatospheres and secondary plating of suspending cells led to the formation of hepatospheres again, and the sphere forming efficiency of MHCC97-H-let-7a and HCCLM3-let-7a secondary spheres was lower than that of Scramble groups (*T*-test, *p* \< 0.01,Fig. [3b](#Fig3){ref-type="fig"}), as were shown in Fig. [3c](#Fig3){ref-type="fig"}. To further confirm the effects of let-7a1 on HCC stem cells, continuous sphere culture assay was applied, and up to six generations were cultured and detected. Let-7a1 showed significant impacts on HCC stem cells, and the inhibitive influence could be accumulated (Fig. [3d](#Fig3){ref-type="fig"}).Fig. 3Let-7a1 inhibits the capacity of self-renewal of HCC stem cells. The HCC cells were seeded in ultra-low attachment plates to form the 1st generation of spheres. Then the cell form spheres were reseeded to acquire the 2nd generation of spheres. The sphere forming efficiency of the first generation (**a**) and the second generation (**b**) of HCC stem-like cells infected with let-7a1 and Scramble vector, \* *p* \< 0.01, with representative images shown in (**c**). **d** Let-7a1 inhibits the self-renewal of HCC stem cells group in continuously cultured spheres, showing much stronger inhibition than that of control group in total six generations
Overexpressing let-7a suppressed the EMT factors of HCC cells and Wnt signaling pathway of HCC stem-like cells {#Sec15}
--------------------------------------------------------------------------------------------------------------
We first detected markers related to apoptosis and cell cycle (Fig. [4a](#Fig4){ref-type="fig"}). To explore the possible mechanisms by which let-7a represses sphere number, we hypothesized that increased let-7a in HCC cells and HCC stem cells inhibited malignant cellular behaviors through down-regulating N-cadherin and Snail, that has been typical pathological markers for epithelial trait was up-regulated; meanwhile, was involved in the process of epithelial-mesenchymal transition (EMT) (Fig. [4b](#Fig4){ref-type="fig"}, Left). In HCC stem-like cells, key molecules of the Wnt signaling pathway universally decreased due to overexpressing let-7a, indicating that let-7a inhibited the Wnt1/Frizzled/β-catenin pathway in a population enriched with HCC stem cells (Fig. [4b](#Fig4){ref-type="fig"}, right). Further, using the luc-reporter assay and immunofluorescence staining, we found that let-7a inhibited Wnt signaling, which was achieved by decreasing TCF-4 promoter activity (*T*-test, *p* \< 0.01, Fig. [4c](#Fig4){ref-type="fig"}) and β-catenin expression (Fig. [4d](#Fig4){ref-type="fig"}).Fig. 4The suppressions of EMT factors of HCC cells and Wnt activation of HCC stem cells were related to let-7a functions. **a**. Gene expression levels of let-7 targeted genes, which were implicated in cell apoptosis and cell cycle regulations. Let-7a decreased HMGA2, Bcl-xl, MAPK, cyclin d1, and increased Bcl-2 in both cell lines. **b**. Genes related to EMT were detected, and results showed that let-7 inhibited the EMT of HCC cells through regulating E-cadherin, N-cadherin and Snail, of which, E-cadherin was up-regulated, whereas, the mesenchymal biomarker N-cadherin and Snail were decreased. **c**. Let-7a1 inhibited the TCF-4 promoter activity of HCC cells, and inhibited Wnt1/Frizzled/β-catenin signaling of HCC stem cells, which was demonstrated to promote the self-renewal ability of cancer stem cells. **d**. The results of immunofluorescence showed that β-catenin was decreased due to let-7 overexpression in HCC stem cells
Decreased Wnt1 is required for let-7a-induced renewal inhibition of hepatospheres {#Sec16}
---------------------------------------------------------------------------------
We used the RNA interference assay to suppress Wnt1 activity in HCC cells by three independent siRNA. As with let-7a1 overexpression, knockdown of Wnt1 significantly inhibited the self-renewal ability of stem-like cells, and inhibited Wnt signaling pathway factors (Fig. [5a](#Fig5){ref-type="fig"}). In the presence of Wnt1 siRNA, let-7a1 did not show significant inhibition on sphere formation ability in continuous stem cell culture compared to the scramble control (Fig. [5b](#Fig5){ref-type="fig"}). The knockdown of Wnt1 decreased TCF-4 activity significantly, abolishing let-7a functions (Fig. [5c](#Fig5){ref-type="fig"}). To identify whether Wnt1 is critical for let-7a-repressed self-renewal ability of HCC stem cells, MHCC97-H and HCCLM3 cells were cultured in ultralow attachment plates with recombinant Wnt1 protein (50 ng/ml) for 8 days. The addition of recombinant Wnt1 protein increased the sphere formation efficiency of both let-7a1 and scramble controls, reversing the suppressive roles of let-7a (Fig. [5d](#Fig5){ref-type="fig"}), and also increased TCF-4 activity (Fig. [5c](#Fig5){ref-type="fig"}).Fig. 5Let-7a inhibits the HCC spheres through Wnt1. **a**. The knockdown of Wnt1 (left) decreased the sphere forming efficiency of HCC stem cells, and abolished the functions of let-7a on self-renewal ability (right), which functioned through regulations of Wnt1/Frizzled/β-catenin pathway (left). **b**. In continuous culture of HCC spheres, the Wnt1 siRNA didn't affect the self-renewal ability of let-7a1 overexpressing HCC stem-like cells, compared to Control group (VEH). **c**. The knockdown of Wnt1 decreased TCF-4 activity, abolished let-7a effects; similarly, the addition of Wnt1 reversed let-7a effects, increasing TCF-4 activity, and no significant differences between let-7a and Scramble group were detected. **d**. The addition of Wnt1 reversed the suppressive effects of let-7a on MFE
Let-7a sensitized HCC stem-like cells to cis-platinum-induced self-renewal inhibition {#Sec17}
-------------------------------------------------------------------------------------
Cis-platinum is one of the most commonly used anticancer drugs in clinical treatment; however its role in the regulation of CSCs is not well understood. We found that cis-platinum reduced spheres number (Fig. [6a-b](#Fig6){ref-type="fig"}) through the inhibition of Wnt1 expression (Fig. [6c](#Fig6){ref-type="fig"}); the effects were reversed by recombinant Wnt1 protein (Fig. [6a](#Fig6){ref-type="fig"}). The combined use of cis-platinum and let-7a significantly decreased the sphere number, inhibiting the self-renewal ability of HCC stem-like cells through concurrent effects on Wnt signaling.Fig. 6Let-7a sensitized HCC stem-like cells to cis-platinum induced self-renewal inhibition. **a**. Cis-platinum reduced sphere number through inhibition of Wnt1 expression. **b**. The combined use of cis-platinum and let-7a significantly decreased the sphere number, inhibiting the self-renewal ability of HCC stem-like cells through concurrent effects on Wnt signaling. **c**. Both let-7a and cis-platinum inhibited Wnt1 explression level, and exerted sygnergic inhibition on Wnt1 activity
The loss of let-7a was related to the occurrence and progress of HCC {#Sec18}
--------------------------------------------------------------------
IHC studies show that the level of β-catenin was much higher in tumors with later clinical stages (Fig. [7a-b](#Fig7){ref-type="fig"}). In clinical HCC samples, let-7a expression was much lower in tumor tissues than adjacent normal tissue (Fig. [7c](#Fig7){ref-type="fig"}), indicating an inverse relationship between let-7a expression and HCC occurrence. Likewise, let-7a expression level was inversely correlated with Wnt1 mRNA in HCC tissues (Fig. [7d](#Fig7){ref-type="fig"}).Fig. 7Let-7a was inversely related with Wnt1 and indicated better clinical prognosis. **a**. Representative images of immunohistochemical staining for β-catenin in tissues of different clinical stages. **b**. **d**. Quantification of relative immunostaining intensity of β-catenin, StagingIhad the least expression of β-catenin. **c**. Quantification of let-7a in HCC tissues and adjacent normal tissues, the relative quantity was calculated by 2^-ΔΔCt^, with U6 acting as the internal reference. The statistical analysis was performed using *T* test. **d**. Wnt1 mRNA expression in tissues from 20 patients were tested, and there is inverse correlationship between let-7a miRNA and Wnt1 mRNA was found, Pearson = −0.722, *p* \< 0.01
Discussion {#Sec19}
==========
Let-7 is a family consisting of 13 members located on nine different chromosomes whose expression is usually lost, reduced, or deregulated in most human malignancies \[[@CR21]\]. Growing evidence suggests that the restoration of let-7 expression effectively repressed cell proliferation, invasion, metastasis, and resistance to therapy. The findings of let-7 repression on CSC self-renewal indicated that let-7 restoration may be a useful therapeutic option in HCC and stem-like cells, which was more crucial for curing the cancer \[[@CR8], [@CR22]--[@CR24]\]. Recent studies found that cholesterol-conjugated let-7a inhibited cell proliferation, growth, and metastasis, and mainly functioned in the cytoplasm through directly reaching HCC orthotropic tumors \[[@CR25]\]. What's more, the therapeutic trial of let-7 mimics showed suppressed effects on tumor growth in pre-clinical studies \[[@CR4]\]. Especially, nanoparticle-based let-7 replacement therapy had been successfully applied in vivo, together with other delivery methods, including lentivirus-mediated pre--let-7 s, adenovirus-mediated hairpin sequences of mature let-7, cationic liposome--mediated pre--let-7, and electroporation of synthetic let-7 \[[@CR8], [@CR26]\].
In this study, we show that overexpressing let-7a exerted inhibitory effects on HCC, consistent with previously published results for other malignancies \[[@CR27], [@CR28]\]. EMT inducers, including Snail, Slug, Twist1, ZEB1 and ZEB2, suppress the expression of adherence proteins to induce cellular malignancies. EMT is a major mechanism for cancer generation, metastasis and progression \[[@CR8]\], which ultimately promote the growth of tumor bulk and cell proliferation, and during the EMT process, CSCs are generated \[[@CR29]\]. We found that increased let-7a could inhibit sphere formation efficiency through alleviating EMT via down-regulating N-cadherin and Snail in HCC cells. In HCC stem-like cells, overexpressing let-7a inhibited the Wnt1/Frizzled/β-catenin signaling pathway, which was involved in maintaining the self-renewal ability of stem cells. We further identified that repressed Wnt1/Frizzled/β-catenin signaling in a CSC-enriched population was attributed to enforced let-7 and let-7 enhanced cis-platinum functions, helping to inhibit the self-renewal of stem-like cells. Our results suggest that overexpression of let-7a could be used as a therapeutic agent and prognostic indicator in the management of HCC against Wnt activation, and help to understand the mechanisms through which let-7 regulated HCC stem cells.
Let-7 functions are detailed explored in many kinds of tumors, and let-7 acted through post-transcriptional regulations of the targeted genes \[[@CR30]\]. However, the roles of let-7 in HCC stem-like cells are less involved. For the first time, we identified the let-7 controlled Wnt signaling activity, which was accused for maintaining of cell pluripotency. Wnt/β-catenin transactivation of let-7 in breast cancer further suggested the regulatory roles of let-7 in stem cells' regulations \[[@CR31]\]. Overall, our results suggest that overexpression of let-7a could be used as a therapeutic agent and prognostic indicator in the management of HCC via repression of Wnt signaling activation in stem cells, and to help understand the mechanisms through which let-7 regulates HCC stem cells.
We appreciate the great help of the central laboratory and the center of translational medicine of the Qilu Hospital of Shandong University. We also appreciated very much for the help of Shou-Ching Tang (Georgia Regents University Cancer Center, Augusta, GA 30912 USA, Tianjin Medical University Cancer Institute and Hospital, Tianjin, China), who worked hard at language polishing, editing and consent resembling, which did not match the criteria for authorship of BMC Cancer.
Funding {#FPar1}
=======
This study was supported by the National Natural Science Foundation of China (81100323), the Fundamental Research Funds of Shan Dong University (Qilu Hospital Research Project, 2014QLKY18), and the Project Fund of Techpool (UF201330).
Availability of data and materials {#FPar2}
==================================
All data of this paper belonged to the center of translational medicine, the Qilu Hospital of Shandong University, and could not be shared as public data. The data could be shared personally after negotiation with signed document. All the data will be applied for answering and finishing the National Grant. If anyone have trouble in getting certain materials listed in this study, the authors could be contacted via email listed below. Supportive methods were involved in "Supplemental data".
Authors' contributions {#FPar3}
======================
BJ: Conception and design, Financial support, Administrative support, Provision of study material or patients, Coordination of experiments, Manuscript writing, Statistical analysis. WW: Immunohistochemistry staining, Sphere-formation assays, Collection and assembly of data. XM: Conception and design, Manuscript writing. GD: Quantitative Real-time PCR, Immunohistochemistry staining, Luciferase assay, Transduction of lentiviral vectors. JL: Cell culture, Western blot, Sphere-formation assays. SZ: Cell culture, Western blot, Luciferase assay, Sphere-formation assays. BZ: Provision of study material or patients, Manuscript writing. ZF: Western blot, Collection and assembly of data. All scholars contributing to this project are all listed as co-authors, and all co-authors are agreed of publishing this paper generated from our research. All authors read and approved the final manuscript.
Competing interests {#FPar4}
===================
The authors declare that they have no competing interest.
Consent for publication {#FPar5}
=======================
Not applicable.
Ethics approval and consent to participate {#FPar6}
==========================================
Written informed consent was obtained according to the guidelines approved by the Institutional Review Board. This study is approved by the Ethics Committee of the Qilu Hospital of Shandong University, and consent to publish has been obtained from all participants. All patients whose cancer tissues were collected and used agreed to participate in this study, and informed consents together with the consents for publication were obtained and kept in the Department of Ethics Committee. No individual patient data was acquired in this study, and the private information of individual patient are not and will not be displayed.
|
{
"pile_set_name": "PubMed Central"
}
|
The collection of papers included in this Research Topic represents the outcome of one of the activities of the COST Action FA1301---"A network for improvement of cephalopod welfare and husbandry in research, aquaculture, and fisheries" (Cephs*In*Action)---that operated for 4 years from 2013 to 2017. The idea of a "Cephs*In*Action" Research Topic entitled "Towards Future Challenges for Cephalopod Science" emerged at one of the last meetings of the COST Action FA1301: Cephs*In*Action and CIAC Meeting "Cephalopod Science from Biology to Welfare[^1^](#fn0001){ref-type="fn"}" (Hellenic Centre for Marine Research, Heraklion, Crete, Greece, 28--31 March 2017), and from some editorial initiatives discussed at that time. This Research Topic (RT) is just one example of several RTs, and indeed other separate papers, dedicated to cephalopod molluscs (Hanke and Osorio, [@B7]; Ponte et al., [@B12])[^2^](#fn0002){ref-type="fn"} that have been hosted by Frontiers over the last few years.
This highlighting of cephalopod science is important as it has much to offer not only the life sciences community, but also more broadly the public perception of science and its understanding and relationship with scientific endeavor. To make this contribution, there are logistical challenges facing the cephalopod research community that need to be overcome. Importantly, given that cephalopod science is a relatively small, globally distributed research community, there is a need for rapid and effective mechanisms for exchange of knowledge and resources, encompassing everything from the sharing of laboratory protocols, videos, tissues and samples, and data-sets to innovation in public engagement. This also presents strategic challenges in convincing globally distributed policy makers and funders of the relevance of cephalopods in scientific advances. There are regulatory aspects too, as cephalopods are the only invertebrates whose use is regulated in Europe in a research context (e.g., Fiorito et al., [@B4], [@B5]; Di Cristina et al., [@B3]), which increases the need for an integrated oversight and direction in terms of ethics and animal welfare (Ponte et al., [@B13]). This links to the important recognition that cephalopods are not "simple" laboratory animals and that we need to understand their physiology and behavior by intersecting studies in their natural environment with those in standardized settings such as the lab-bench. Only by a better understanding of the normal range of behaviors of distinct species of cephalopods can their welfare be improved. There is the need for better phylogenetic resolution and for more accurate field data to facilitate this.
This Research Topic also aligns with the interests of the cephalopod community in stimulating public interest in cephalopods and their artistic interpretation. This extends to a broader audience that could include chefs and gourmets[^3^](#fn0003){ref-type="fn"}, fishers and scientists aiming to develop sustainable food resources. School children\'s natural fascination with cephalopods (e.g., Sperduti et al., [@B15]) can excite their interest in scientific discovery and encourage them to engage in conversations about the scientific process and what it means. The importance of such conversations cannot be underestimated in a world in which the public needs to be scientifically literate, as they must be equipped to make important socio-economical and political decisions facing the current condition of the world, whilst being confronted with 'fake news\', 'alternative truths\', and when expert opinion is often derided. There is great potential for innovative schemes for public engagement that springs from the natural wonderment the cephalopods incite.
The last five years have been extremely challenging, but also very innovative for cephalopod science and have continued the outstanding tradition of biological contribution with cephalopod molluscs as key players (e.g., Keynes, [@B9]; De Sio, [@B2]; Albertin et al., [@B1]; Garrett and Rosenthal, [@B6]; Huffard, [@B8]; Liscovitch-Brauer et al., [@B10]; Marini et al., [@B11]; Sanchez et al., [@B14]).
This Research Topic includes 13 papers from about 40 authors representing ten different countries, thus overlapping with the original parties that contributed to the COST FA1301. Three papers present original data and 10 others are reviews and perspective articles on various topics, as examples of the interest that humans have for these fascinating marine molluscs. This Research Topic offers a journey that spans cephalopod gastronomy ([Mouritsen and Styrbæk](https://doi.org/10.3389/fcomm.2018.00038)) offering a glance at the interest among chefs and gastroscientists to explore these organisms as a counterpoint to other seafood, a look at new protein sources to replace meat from land-animal production, and a test of texture and flavor properties of cuttlefish, squid and octopus and how these provide the ground for a variety of culinary transformations. "CephsInAction: Towards Future Challenges for Cephalopod Science" also offers an "OctopusEye,": a "refracted spectatorship" perspective and conceptual analysis of the film "The Love Life of the Octopus (Les Amours de la pieuvre) (1965)" ([Hayward](https://doi.org/10.3389/fcomm.2018.00050)). But cephalopods are also at the boundaries between "science, art and engineering" ([Nakajima et al.](https://doi.org/10.3389/fcomm.2018.00020)). They are among the most enthusiastically visited animals in public aquaria, providing a way of communicating science and conservation ([Marchio](https://doi.org/10.3389/fcomm.2018.00017)), and offer various "Critical Challenges Ahead" ([O\'Brien et al.](https://doi.org/10.3389/fphys.2018.00700)) as envisioned by three junior "researchers who have recently embarked on careers in cephalopod biology" and that provide their suggestions on a variety of topics spanning from genetics, to welfare, behavior, cognition, and neurobiology.
This volume includes studies on the effects of maternal and embryonic stress on the behavior of offspring (*Sepia officinalis*, [O\'Brien et al.](https://doi.org/10.3389/fphys.2017.00981)), presenting evidence for age-related differences in defensive behaviors in the sepiolid *Euprymna* ([Seehafer et al.](https://doi.org/10.3389/fphys.2018.00299)), or the development of swimming abilities of paralarvae of *Doryteuthis opalescens* ([Vidal et al.](https://doi.org/10.3389/fphys.2018.00954)). A preliminary analysis of the expression of protocadherins in *Octopus vulgaris* is also included providing the ground for future analysis of the way these genes may drive neural wiring during development and in the cases of biological and neural plasticity in the adult ([Styfhals et al.](https://doi.org/10.3389/fphys.2018.01905)).
Finally, several reviews are included in this Research Topic, (i) examining parasites that cephalopod host, (ii) raising the possibility of the existence of stem cells in cephalopod brains, (iii) considering possible cases of functional and convergent evolution of neural-systems, when compared with vertebrates, and (iv) overviewing the extraordinary and historically well-studied biological cases of tissue and neural regeneration ([Deryckere and Seuntjens](https://doi.org/10.3389/fphys.2018.01160); [Imperadore and Fiorito](https://doi.org/10.3389/fphys.2018.00593); [Roumbedakis et al.](https://doi.org/10.3389/fphys.2018.01573); [Shigeno et al.](https://doi.org/10.3389/fphys.2018.00952)).
In sum, all contributions reflect a broad, interdisciplinary active and vital scientific community.
Author Contributions {#s1}
====================
All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.
Conflict of Interest Statement
------------------------------
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
This editorial and the e-book are based upon collaboration under the COST Action FA1301 Cephs*In*Action that also supported the meeting and partially funded the open access costs of the papers included in this RT. The Association for Cephalopod Research CephRes supported this work. We are deeply thankful to the CretAquarium (Heraklion) for help for the organization of the meeting, and in particular to Dr. Panagiotis Grigoriou and Dr. Aspasia Sterioti. We are also grateful to Dr. C. Balestrieri (CephRes) for her scientific and managerial assistance. The studies included herein are considered a contribution to the COST (European COoperation on Science and Technology) Action FA1301.
^1^<http://www.cephsinaction.org/activities/meetings/year2016/cephsinactioncretaquarium/>
^2^see also: <https://www.frontiersin.org/research-topics/10233/vision-in-cephalopods-part-ii>; <https://www.frontiersin.org/research-topics/9997/cephalopod-research-across-scales---molecules-to-ecosystems>
^3^See for example the Atlantic Area Interreg Project "Cephs and Chefs" <https://www.cephsandchefs.com/>
[^1]: Edited and reviewed by: Sylvia Anton, Institut National de la Recherche Agronomique (INRA), France
[^2]: This article was submitted to Invertebrate Physiology, a section of the journal Frontiers in Physiology
|
{
"pile_set_name": "PubMed Central"
}
|
Plasmons, the collective excitations of conduction electrons supported by metallic nanostructures, enable the confinement of electromagnetic radiation on a subwavelength nanometer scale. This phenomenon holds great potential for a vast range of applications in optics, including metamaterials design,^[@ref1]−[@ref3]^ biosensing,^[@ref4],[@ref5]^ therapeutics,^[@ref6],[@ref7]^ solar energy harvesting,^[@ref8]^ and photocatalysis.^[@ref9]^ Alongside the continued progress in the synthesis of noble metal nanoparticles with new exotic shapes and increased monodispersity, the manufacture of bimetallic nanostructures represents an alternative strategy to enrich the library of plasmonic structures at our disposal.^[@ref10]^ Epitaxial seeded growth is one of the most widely used approaches for the preparation of such structures, where one or more metal precursors are reduced or coreduced on the surface of a previously prepared core of a different metal.^[@ref11]^ The geometry of the obtained bimetallic system mainly depends on the lattice matching of the metal species involved,^[@ref12],[@ref13]^ on the seed morphology,^[@ref14]^ and on the growth mode on different crystallographic facets, which can be influenced by facet-specific capping agents.^[@ref15]^ The particular case of Au\@Ag bimetallic nanoparticles has attracted much attention from the scientific community because of their complementary properties: though gold nanoparticles can be easily modulated in shape and size,^[@ref16]−[@ref19]^ the lower optical losses of silver render a better plasmonic performance, for instance, in the amplification of weak optical processes such as Raman scattering,^[@ref20]^ fluorescence,^[@ref21]^ and IR spectroscopy.^[@ref22]^ Among many other examples, Seo et al. reported the preparation of silver--gold--silver pentatwinned nanorods through epitaxial growth of silver on a pentatwinned gold nanorod core in an ethylene glycol solution of polyvinylpyrrolidone.^[@ref23]^ Recently, Li et al. performed the same reaction in water,^[@ref24]^ which was further studied by Gómez-Graña et al. to elucidate the growth mechanism behind the formation of the silver shell using high-resolution electron microscopy in combination with density functional theory (DFT) calculations.^[@ref19]^ It is important to underline that the production of high aspect ratio nanowires relies on the selective deposition of Ag on {111} tip facets, whereas a similar overgrowth reaction with gold would lead to a decrease in aspect ratio, as previously reported.^[@ref25]^ Such elongated structures can be regarded as the plasmonic analogues of radiofrequency antennas, but with the resonance shifted into the visible-near IR (Vis-NIR) range of the electromagnetic spectrum.^[@ref26]^ Like their radiofrequency counterparts, optical antennas are characterized by several multipolar plasmon oscillations, which can be separated into bright and dark modes, depending on their ability to couple efficiently (bright) or not (dark) to incident/scattered far-field radiation.^[@ref27],[@ref28]^ Although the former can be exploited in the development of signal processing devices^[@ref29]^ and Raman/IR/fluorescence-based sensors, the latter is useful for enhanced absorption spectroscopy and photothermia.^[@ref30]^
In order to efficiently engineer the near-field electromagnetic confinement and implement the application of optical antennas, it is useful to have a detailed understanding of the relationship between the antenna structure and the spatial/spectral distributions of the different plasmon modes.^[@ref31],[@ref32]^ In this respect, the near-field properties of pure gold and silver nanorods have been investigated by various research groups, both theoretically^[@ref28],[@ref33]^ and experimentally, using different imaging techniques that exploit either resonant optical illumination (e.g., dark-field spectroscopy,^[@ref34],[@ref35]^ apertureless scanning near-field optical microscopy^[@ref36]−[@ref38]^ (aSNOM), and photoemission electron microscopy^[@ref39],[@ref40]^) or fast electrons (cathodoluminescence^[@ref41]^ and electron energy-loss spectroscopy^[@ref27],[@ref42]−[@ref44]^ (EELS)). In contrast, detailed studies of the plasmon near-field behavior of Au\@Ag bimetallic nanostructures have not been reported. Rodríguez-González et al. studied the effect of a silver shell on the plasmonic behavior of gold nanodumbbell cores, describing a complex plasmonic scenario where the transversal mode of the core--shell system cannot be directly related to an equivalent silver nanorod.^[@ref45]^ To the best of our knowledge, the only available study of the plasmonic properties of silver--gold--silver nanorods is the work by Ahn et al. using dark-field spectroscopy, which indicated no influence of the gold core.^[@ref35]^
The most important prerequisites for a precise engineering of the plasmonic properties of noble metal nanoparticles are monodispersity and size tunability. As maintaining a narrow size distribution becomes more difficult when anisotropy is increased, we developed, as we report here, the concept of *controlled living nanowire growth* for the production of monodisperse silver--gold--silver nanowires (AgAuAg NWs), by analogy with controlled living polymerization reactions.^[@ref46]^ Controlling the addition of silver precursors by means of a syringe pump device, we managed to achieve a linear growth rate of silver on the gold cores. This procedure allowed us to prevent the nucleation of silver nanoparticles during nanowire growth, to significantly improve the monodispersity of the product, and to accurately predict the final dimensions of the bimetallic system. As a result, AgAuAg NW colloids were obtained which display up to nine well-defined plasmon resonance peaks spreading over the entire Vis-NIR wavelength range, with a tight control on the total NW length up to several microns, corresponding to aspect ratios above 100. Direct electron-microscopy-based visualization of the plasmon near-field spatial distributions provided essential information toward eventual optimization and rational application of this bimetallic system. We thus present here a complete optical-extinction and EELS analysis of plasmon modes supported by AgAuAg NWs, which are compared to boundary-element method (BEM) electromagnetic simulations,^[@ref47],[@ref48]^ allowing us to assess the influence of the central gold nanorod. We show that the presence of the gold core influences the high-energy spectral range, whereas it becomes progressively irrelevant in the Vis-NIR region, where the spectroscopic behavior resembles that of a monometallic silver nanowire.
To push the quality of Ag NW synthesis beyond the existing limits, any side reactions and secondary nucleation should be suppressed completely. Thus, the aims in NW synthesis are similar to those in polymer synthesis, where a small polydispersity index is desired. Consequently, we can apply the well-known concept of *controlled living polymerization reactions (CLPR)* to the one-dimensional NW growth reaction. The criteria for CLPR as defined by IUPAC standards involve the complete suppression of termination and side reactions and a constant number of actively growing chains throughout the polymerization.^[@ref46]^ Furthermore, the initiation reaction should be much faster than the propagation reaction. As a consequence, the degree of polymerization---and thus the resulting chain length---is determined only by the ratio of monomer to initiator concentrations *P*~n~ = \[*M*~0~\]/\[*I*~0~\]. In the following, we introduce the concept of *controlled living nanowire growth*, which allows us to synthesize bimetallic AgAuAg NWs with remarkably narrow size distributions and nanometer precision in length. As previously described by various groups, epitaxial silver overgrowth of pentatwinned gold nanorods (PT Au NRs) in the presence of surfactants with chloride counterions leads to one-dimensional growth of bimetallic AgAuAg rods/wires.^[@ref19],[@ref24],[@ref35]^ This can be explained by the adsorption of chloride onto the lateral {100} facets resulting in less favored silver reduction. Consequently, silver is only reduced and deposited epitaxially on the {111} facets at the NR tips, so the PT Au NR seeds can act as bifunctional initiators bearing two initiation sites. After deposition of the very first silver monolayer the initiator becomes an active species that is subsequently overgrown in one-dimensional fashion by continuous reduction of silver ions at the metal surface during NW growth. We identify the following requirements to obtain a living controlled nanocrystal growth mechanism: (i) the initiation of crystal growth takes place simultaneously and much faster than the continuous reduction of Ag^+^ ions at the metal surface of the active species; (ii) all particles have to persist as active growing species for selective deposition of Ag atoms throughout the complete experiment; and (iii) the reduction and deposition of Ag^+^ must be quantitative, so that the growth of the particles is proportional to the amount of added silver precursor and a precise control over the final NW dimensions can be achieved.(i)The first condition is met by using PT Au NRs with a narrow size distribution as seeds, uncoupling completely nucleation and elongation. Furthermore, the ex-situ synthesis of the PT initiator guarantees a homogeneous and simultaneous initiation at both ends upon addition of silver precursor. Consequently, the preparation of high quality PT Au NR seeds is of primary importance toward a precise growth of monodisperse AgAuAg NWs. PT Au NRs with average length of 210 ± 10 nm and width of 34 ± 1 nm were prepared as previously described by Pérez-Juste et al., with minor modifications (see Experimental Section).^[@ref49]^ The purified PT Au NRs show a shape yield around 99%, (Figure S1A--C, [Supporting Information](#notes-1){ref-type="notes"}) and can be used as seeds after redispersion in 10 mM benzyldimethylhexadecylammonium chloride (BDAC). As evidenced by UV--vis-NIR spectroscopy (Figure S1F, [Supporting Information](#notes-1){ref-type="notes"}), the characteristic transversal and longitudinal dipolar, as well as longitudinal quadrupolar plasmon modes can be identified for the pure PT Au NR dispersion. Further inspection of the high-energy region reveals the presence of an octupolar mode as a shoulder below 600 nm (inset Figure S1F, [Supporting Information](#notes-1){ref-type="notes"}). The narrow dipolar plasmon band, with a quality factor of 6.45 (Figure S2, [Supporting Information](#notes-1){ref-type="notes"}), and the high intensity ratio between the dipolar and the transversal modes confirm the narrow size distribution of the PT Au NR seeds.(ii)The second requirement can be fulfilled through careful adjustment of a low silver reduction rate to facilitate anisotropic growth.^[@ref10],[@ref50]^ Therefore, the silver overgrowth reaction conditions were set according to the following three conditions: (1) slow silver reduction at slightly acidic conditions and elevated temperature;^[@ref19]^ (2) BDAC as surfactant, which drastically reduces the reduction rate as compared to nonaromatic surfactants;^[@ref51]^ and most importantly, (3) continuous addition of silver nitrate and ascorbic acid by means of a microfluidic pump setup from separated reservoirs. The continuous and slow addition prevents the accumulation of unreacted Ag^+^ within the growth solution, which might lead to secondary nucleation and nonspecific silver deposition.UV--vis-NIR extinction spectra after various reaction times during NW growth were correlated with TEM images. All spectra were collected after transferring the NWs into heavy water so as to expand the detection wavelength window up to 2500 nm, avoiding the strong absorbance of water around 1350 nm (see experimental section). We observed an initial blue shift of about 20 nm, along with a slight reduction of the overall aspect ratio when a thin silver layer (approximately 2 nm) grows on all facets (sides and tips). However, after coverage of the PT Au NR cores with this initial thin layer, silver deposits on the nanowire tips only. This results in a significant and gradual redshift of all plasmon modes, whereas multipolar, higher energy plasmon modes emerge as silver deposition continues. Optical extinction spectra of the resulting length-controlled AgAuAg NWs are shown in [Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"}A, top, along with representative TEM images for selected sizes ([Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"}D--G), illustrating the striking monodispersity typically achieved by the growth method described above (see Table S1 and Figure S3, [Supporting Information](#notes-1){ref-type="notes"}). In particular, the plasmon spectral features are narrow and mainly limited by intrinsic absorption and radiative losses, rather than by particle size dispersion (see below). The corresponding BEM calculated extinction spectra ([Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"}A, bottom solid lines, see Methods) show an excellent agreement with the experimental spectra, as well as a gradually increasing similarity with spectra calculated for pure Ag NWs with the same overall dimensions ([Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"}A, bottom dashed lines).The quality factor (Q-factor) is the characteristic figure of merit of any resonator, as it indicates how many oscillations are undergone by a particular oscillator (see [Supporting Information](#notes-1){ref-type="notes"} for the calculation details). During silver elongation, the Q-factor of the dipolar mode decays exponentially (Figure S2, [Supporting Information](#notes-1){ref-type="notes"}), which can be explained considering that radiative coupling to the far-field increases with the length of the antenna, together with the relative dissipation, causing the peaks to broaden.^[@ref52]^ When comparing the experimental Q-factors with the ones obtained from the BEM simulated spectra of [Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"}A, the calculated ones are found to be only 25% higher compared to the experimental ones. This clearly emphasizes the low polydispersity of the samples. Interestingly, the decay slope is similar in both experiment and theory, suggesting again that there is no significant increase in the polydispersity of AgAuAg NWs during silver growth. Overall, the resulting polydispersity of the product is remarkably narrow and clearly emphasized by the narrowness of the plasmon bands in [Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"}A. In order to achieve a more meaningful comparison between the widths of experimental and calculated spectra, we used TEM analysis to predict the contribution of the polydispersity to the FWHM, which is missing in the calculation. In fact, the FWHM of the experimental peaks is constituted by a Lorentzian component, intrinsically related to the nanoparticle properties, and a Gaussian component, related to the size distribution in the colloidal solution. The latter is what we normally evaluate using different techniques like transmission electron microscopy (TEM), dynamic light scattering (DLS), or small-angle X-ray scattering (SAXS). The finite size distribution within each sample accounts for a moderate increase in FWHM of the observed plasmon features (see Figure S4, [Supporting Information](#notes-1){ref-type="notes"}). A more precise assessment is provided by comparing the wavelength width of the measured (Δ*λ*~exp~) and theoretical (Δ*λ*~th~) spectra, assuming that the excess in the former one originates from the finite size distribution of NW sizes for each given sample (Δ*L*). We then have Δ*λ*~exp~ ∼ Δ*λ*~th~+*m*·Δ*L*, where *m* is the slope of the plot of plasmon wavelength peak vs NW length, extracted from [Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"}B. In [Table [1](#tbl1){ref-type="other"}](#tbl1){ref-type="other"} the values of Δ*L* extracted from this analysis of the optical spectra (Δ*L*~OPT~*)* are compared with the size distributions obtained from TEM analysis (Δ*L*~TEM~). The results show an acceptable agreement, considering that the number of NWs measured by TEM for each sample is much smaller than those contributing to UV--vis-NIR spectra.(iii)The last requirement to achieve a living polymerization reaction (linear growth) can be met by selecting an addition rate that is slower than the reaction rate. In this way, a linear zero-order kinetic path is enforced and the resonance wavelengths of the longitudinal modes, as well as the corresponding aspect ratios evaluated from TEM measurements (see Table S1, [Supporting Information](#notes-1){ref-type="notes"}), turn out to scale linearly with time. Furthermore, as the conversion rate for silver is close to 100%, the linear shifts are directly proportional to the amount of added Ag^+^. This proportionality is referred to in what follows as the *degree of silver elongation, AgEn*, defined as the molar ratio of added silver salt to gold seeds (i.e., \[Ag^+^\]/\[Au^0^\]), in analogy to the concept of *degree of polymerization*. We plotted in [Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"}B the resonance wavelengths for all the longitudinal plasmons that were recorded within our measurement spectral window (ranging from the dipolar mode up to the fifth order mode) *vs AgEn*. Note that we disregarded some high-energy spectral features that cannot be clearly resolved apart from the transverse mode. This plot clearly reveals a linear slope for all modes, up to aspect ratios above 20 and *AgEn* value of almost 6. In other words, this plot indicates that the reaction proceeds according to zero order kinetics and that no termination or passivation occurs. The corresponding regression fits (solid lines) yield a Pearson *R*^2^ above 0.999 for all the modes and for the aspect ratio. As a control experiment, we increased the rate of Ag^+^ addition by a factor of 1.25. The resulting plasmon shifts were plotted in [Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"}C (red dots), showing that the faster addition rate leads to nonlinear shifts. Because the reaction does not comply in the case of a higher addition rate, the apparent *AgEn* increases significantly faster than the plasmon band shift, which represents the actual growth rate. Furthermore, under these conditions, excess silver nitrate accumulates in solution, leading to secondary nucleation, formation of AgCl nuclei, nonspecific deposition of silver on the growing particle surface, and uncontrolled increase of the reaction rate. All these effects reduce the amount of available silver ions for deposition on the NW tips and consequently compromise the linear growth and impair an accurate prediction of the final length of the produced AgAuAg NWs. Interestingly, spherical particles with diameters around 500 nm were observed upon TEM inspection of the resulting colloid, which are likely due to crystallization of AgCl present in solution upon drying of the dispersion on the TEM grid (Figure S5, [Supporting Information](#notes-1){ref-type="notes"}). We also found that the thickness of the NWs was significantly larger than expected, in contrast to living reaction conditions (see Table S2, [Supporting Information](#notes-1){ref-type="notes"}). An increase of the reaction temperature also leads to faster reaction rates and uncontrolled growth, so we conclude that the living reaction conditions are highly sensitive to changes in reaction kinetics, either induced by increased temperature or faster precursor addition. The generality of this method was confirmed by carrying out different experiments under *controlled living nanowire growth* conditions but starting with different PT Au NR core dimensions, which also led to linear growth and reproducible evolution of the plasmonic features ([Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"}C). We used the silver to gold volume ratio of a single bimetallic nanowire as the *AgEn* parameter for BEM simulations ([Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"}A, bottom), so as to estimate the theoretical linear behavior (dashed line in [Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"}C). The observed deviation can be explained taking into account the presence of a small amount of Au byproducts (different particle shapes), on which silver is also reduced, as well as some uncertainty in the initial Au nanorod concentration, due to the purification step (\[Au^0^\] was estimated using the absorbance at 400 nm, as detailed in the experimental section). The obtained agreement is a strong confirmation that all added silver ions were reduced and selectively deposited on the AgAuAg NW tips. It is worth noting that we have not found any limitation in the final length of the NWs, our longest experiment resulting in 3.4 μm NWs (corresponding to a *AgEn* of 40.32) (Figure S6, [Supporting Information](#notes-1){ref-type="notes"}).
###### Total Length Distribution for AgAuAg NWs with Increasing Elongation Degrees *AgEn*, Evaluated by TEM Analysis, Δ*L*~TEM~, and Comparing Experimental and Calculated Optical Spectra, Δ*L*~OPT~
AgEn Δ*L*~TEM~ (nm) Δ*L*~OPT~ (nm)
------ ---------------- ----------------
0.24 20.3 17.6
0.36 19.5 18.7
0.48 19.8 24.3
0.72 21.9 27.5
0.84 29.2 29.7
1.20 36.3 29.7
1.92 39.8 34.2
2.40 42.9 39.1
{#fig1}
We selected two samples with significantly different NW lengths to discuss the influence of the PT Au NR core on the optical properties of the bimetallic particles. We analyzed the optical extinction spectra from NWs in heavy water solution ([Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}) and carried out a detailed EELS analysis ([Figures [3](#fig3){ref-type="fig"}](#fig3){ref-type="fig"},[4](#fig4){ref-type="fig"}). Both experimental methods were supported by BEM calculations. The first sample had *AgEn* = 0.5, corresponding to a total aspect ratio of 6.7, and 25 nm Ag extending beyond each tip of the PT Au NR core. We compare in [Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}A the Vis-NIR spectrum (black solid curve) with the calculated extinction spectrum of a NW with the average dimensions, either containing the PT Au NR core (red-dotted curve) or being made of pure Ag (blue dash-dotted curve). The simulations reveal in this case a clear influence of the PT Au NR core on all the plasmon modes, particularly in the short wavelength (high energy) region. The second sample corresponds to *AgEn* = 5.3, aspect ratio 25, and 360 nm of Ag from each tip. The 3D reconstructed volume of the two samples obtained by the Total Variation Minimization (TVM)^[@ref53]^ technique, applied to a tilt series of HAADF-STEM images ([Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}B and [2](#fig2){ref-type="fig"}D) clearly shows that the PT Au NR core is located in the center, with 2 nm of silver on the lateral facets (also considered for the simulations). The pentagonal cross section of the particle--characteristic for PT NRs--is preserved, as evidenced by the cross section in [Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}B and [2](#fig2){ref-type="fig"}D. Remarkably, the low polydispersity of the synthesized AgAuAg NWs allowed us to detect nine LSPR bands in solution ([Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}C), and we find again a good agreement with the BEM simulated extinction spectrum. In this case, however, the differences between simulations with and without the gold core are significantly smaller, and mainly observed in the high-energy region. It should be noted that the permittivities of silver and gold are well described through a common Drude expression ε(ω) = ε~b~ -- ω~p~^2^/ω(ω + iγ) in the IR region, where the so-called classical plasma energy is given by ℏω~p~ ∼ 9 eV,^[@ref54]^ as determined by the density of *s* conduction electrons (i.e., the same in both metals, because each atom contributes with one *s* electron and their atomic densities are nearly identical). The difference between these two metals lies in the level of losses (ℏγ = 25 meV for silver and 71 meV for gold) and in the background screening produced by *d*-band polarization (ε~b~ = 4 in silver and 9.5 in gold). Consequently, in the infrared spectral region (i.e., λ \> ∼1000 nm), the second term dominates in the Drude expression, the precise value of ε~b~ becomes irrelevant, and both silver and gold behave in a similar fashion, except that the latter produces more inelastic optical losses (through a larger damping rate γ). Besides these intrinsic material properties, which lead to similar optical behavior in the NIR, there is a geometrical effect associated with the NW: for a given mode order (e.g., the lowest-order dipolar mode), the wavelength shifts deeper into the IR with increasing NW length, and eventually the mode size becomes comparable to λ/2, giving rise to a relatively larger contribution of radiative damping, which again makes the two metals look more similar. These considerations explain why the AgAuAg bimetallic NWs have similar properties to those of pure Ag wires with the same outer geometry, particularly for infrared modes and long NWs ([Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"}A).
{#fig2}
To confirm these observations, we carried out a detailed near-field study by EELS, also supported by BEM calculations. Plasmon mapping confirmed the symmetry and standing-wave nature of the modes under study. We summarize in [Figures [3](#fig3){ref-type="fig"}](#fig3){ref-type="fig"} and S8 ([Supporting Information](#notes-1){ref-type="notes"}) the results for the short NWs (*AgEn* = 0.5): all of the extinction bands observed in the far-field Vis-NIR spectrum are also found in EELS ([Figure [3](#fig3){ref-type="fig"}](#fig3){ref-type="fig"}A). The spatial distribution of the plasmons clearly indicates the even or odd nature of each mode: modes (1--3) are the dipolar, quadrupolar, and octupolar longitudinal modes, respectively, whereas modes (4--5) are the accumulation of several modes (see below). Incidentally, only odd modes can be excited using light incident with its electric field parallel to the NW, in contrast to EELS in which even modes are also excited due to the multipolar character of the electron exciting field. This conclusion can be actually extended to NWs of arbitrary orientation relative to the externally applied field when they are sufficiently small as to neglect retardation effects (e.g., an exp(i2π*r*/λ) dependence on position *r* and light wavelength λ). However, we are dealing here with long NWs, with lengths that are comparable to λ, so there is strong retardation and this is the reason why our optical spectra reveal modes with both odd and even symmetry.^[@ref30],[@ref37],[@ref41],[@ref55]^ The EELS experimental maps were compared to numerically computed maps using BEM. Simulations were carried out both in the presence (middle panel) and in the absence (bottom panel) of the PT Au NR core. Although the agreement between theoretical and experimental plasmon energies in the optical measurements is excellent (see above), the EELS modes are noticeably red-shifted with respect to theory, presumably because of the effect of the substrate, which is not accounted for in our simulations. However, the features in the measured and calculated EELS spectra are in good mutual agreement (see Figure S7, [Supporting Information](#notes-1){ref-type="notes"}), and we argue that the spatial distribution of the plasmon excitation should not be too sensitive to the observed shift. This intuition is corroborated when comparing modes of the same symmetry, which yield very similar spatial distributions in theory and experiment. It is important to keep in mind that the highest-order modes observed in EELS (modes 4 and 5 in [Figure [3](#fig3){ref-type="fig"}](#fig3){ref-type="fig"}) are in fact the accumulation of several modes, which are integrated over the finite energy range covered within the energy-filtered images. Note that we are instead showing monochromatic maps in the calculations, so that these high-order maps are understandably different from those experimentally acquired. Additionally, the number of plasmon modes (an infinite, discrete set) has an accumulation point toward the electrostatic planar surface plasmon (signaled by ε~1~ = −**1** in vacuum), as they undergo fast oscillations, so that the surface is locally seen as flat. This explains the rather uniform distribution of the observed map at that energy (3.7 eV for the silver-vacuum interface). The theoretical calculations also allow us to explore the role of the PT Au NR core in the plasmonic response, which essentially produces a redshift of the plasmon energies, along with additional broadening in the EELS spectra (see Figure S7, [Supporting Information](#notes-1){ref-type="notes"}). It is thus not surprising that the silver NW has better-defined higher-order modes (second maps from the left in [Figure [3](#fig3){ref-type="fig"}](#fig3){ref-type="fig"}B,C).
{ref-type="fig"}](#fig2){ref-type="fig"}A. (B) BEM simulations of EELS maps associated with the plasmon modes shown in A, for a NW containing the PT Au NR core. (C) Simulations for a pure Ag NW with the same dimensions. The simulated maps are normalized to the maximum intensity in each case. The silver (gold) surface is indicated with solid (broken) white lines in the calculated maps.](nl-2015-018338_0006){#fig3}
For a long NW ([Figure [4](#fig4){ref-type="fig"}](#fig4){ref-type="fig"} and S9, [Supporting Information](#notes-1){ref-type="notes"}, *AgEn* = 5.3), EELS characterization did allow us to identify the dipolar longitudinal mode (1), as well as six additional multipolar modes (2---7 corresponding to increasing order, that is, number of sign changes in the associated induced charge along the wire), whereas the last two peaks (8--9) correspond to an accumulation of several modes, as discussed above for short NWs. In this case, the effect of the PT Au NR core is qualitatively similar, but nearly marginal for low energy modes. In particular, the lowest-order dipolar plasmon involves high plasmon strength (larger enhancement of the near field) near the NW tips, and consequently the near field plot looks nearly identical for the calculated AgAuAg ([Figure [4](#fig4){ref-type="fig"}](#fig4){ref-type="fig"}B) and the pure-Ag NW ([Figure [4](#fig4){ref-type="fig"}](#fig4){ref-type="fig"}C), also in excellent agreement with the experimental EELS maps ([Figure [4](#fig4){ref-type="fig"}](#fig4){ref-type="fig"}A). Because the NIR response of gold and silver is similar, as they both behave as Drude metals, and interband transitions contribute marginally in this spectral region, the position of the low-energy, low-order plasmons should not change significantly between pure Au and pure Ag NWs; nonetheless we expect to observe additional broadening in the case of gold, because it shows higher intrinsic damping losses compared to silver (71 vs 21 meV). In Figure S10 ([Supporting Information](#notes-1){ref-type="notes"}), we show calculated spectra for the corresponding pure Au NWs. In conjunction with a higher broadening, we observed a reduction in the plasmon extinction cross section. Therefore, the PT Au NR core can only be identified in the higher-order modes, which involve a large intensity in the central NW region, whereas their high energies involve very different and relatively moderate values of the dielectric functions of the two metals. Interestingly, the second-order mode, which also has a large weight in the center of the NW, appears at a sufficiently low energy as to feel a similar response from both gold and silver, as we argued above, and consequently, the calculated energies of this mode in the long NWs are similar to those obtained without the PT Au NR core. For such low order modes, the NW acts as a whole plasmonic entity. In contrast, the vacuum/silver interface plasmon is both experimentally and theoretically found to be localized on the silver parts only. For this mode, corresponding to high wave vectors (short wavelengths), the plasmons feel differently each metal, and consequently the gold and silver parts act as two separated plasmonic entities in this regime.
{ref-type="fig"}](#fig2){ref-type="fig"}A. (B) BEM simulations of EELS maps associated with the plasmon modes shown in (A), for a NW containing the PT Au NR core. (C) Simulations for an Ag NW with the same dimensions. The simulated maps are normalized to the maximum intensity in each case. The silver (gold) surface is indicated with solid (broken) white lines in the calculated maps.](nl-2015-018338_0001){#fig4}
In conclusion, the synthesis of AgAuAg bimetallic nanowires has been largely improved by enforcing living growth conditions to the deposition of silver on the tips of pentatwinned Au nanorods, which allowed us to achieve high quality colloidal dispersions with remarkably low polydispersity. As a result, the plasmon bands observed in colloidal NW dispersions are much narrower than those reported for other types of both lithographic and colloidal nanoparticles within the same IR spectral range (see [Figure [5](#fig5){ref-type="fig"}](#fig5){ref-type="fig"}). The measured quality factor (i.e., the ratio of plasmon energy to spectral energy FWHM, which is equal to 1/(2π) times the number of plasmon oscillations before the near-field intensity is attenuated by a factor 1/e) is in fact reasonably close to the calculated one for NWs of well defined size, and as we discuss above, the difference between these two is well explained in terms of the experimental finite size distribution, thus emphasizing our understanding of the optical response of the NWs in terms of the local permittivities of gold and silver. The new protocol provides the opportunity to fine-tune the NWs length up to the micrometer scale (AR \> 100), so that the obtained optical antennas displayed a large number of plasmonic modes that spread over the entire UV--vis-IR region (up to nine detectable modes directly in the colloidal suspension). The role of the gold cores on the optical response was carefully investigated by EELS, and we demonstrated that it does not have a large effect on plasmon propagation along the NW surface, whereas its influence is limited to higher energy modes. The procedure opens up new possibilities for the exploitation of plasmon resonances in the near and mid IR regions.
![Comparison of the quality factor of vis-NIR plasmons reported in the literature for different nanoparticles preparation: lithography (black opened circles) and wet-chemistry (green opened triangles). These are compared with our results from AgAuAg NWs experiments (pink diamonds) and calculations (blue triangles).^[@ref63]−[@ref75]^](nl-2015-018338_0008){#fig5}
Materials {#sec1.1}
=========
Benzyldimethylhexadecylammonium chloride, hexadecyltrimethylammonium bromide (CTAB, ≥96.0%), hexadecyltrimethylammonium chloride (CTAC, 25 wt % in water), hydrogen tetrachloroaurate trihydrate (HAuCl~4~·3H~2~O, ≥99.9%), silver nitrate (AgNO~3~, ≥99.9%), [l]{.smallcaps}-ascorbic acid (AA, ≥99%), deuterium oxide (D~2~O, 99.9 atom % D), sodium borohydride (NaBH~4~, 99%), and trisodium citrate (≥98%) were purchased from Aldrich. All chemicals were used as received. Milli-Q water (resistivity 18.2 MΩ·cm at 25 °C) was used in all experiments. All glassware was washed with aqua regia, rinsed with water, sonicated three times for 3 min with Milli-Q water, and dried before use.
PT Au NR Synthesis {#sec1.2}
==================
The pentatwinned gold nanorod synthesis was adopted from the protocol of Pérez-Juste et al. with some minor modifications.^[@ref49]^ These changes mainly refer to an increase of NaBH~4~ concentration during citrate-capped seed growth to improve reproducibility and increase of AA concentration during AuNR growth to improve the final yield.
3.5 nm Citrate-Capped Au Seeds {#sec1.3}
==============================
Briefly, 20 mL of a 0.125 mM HAuCl~4~, 0.25 mM trisodium citrate aqueous solution was prepared and stirred for 10 min at room temperature. Next, 600 μL of a freshly prepared 0.1 M NaBH~4~ solution was added quickly under vigorous stirring. After 2 min, the stirring rate was reduced and the seeds were aged 40 min under slow stirring at room temperature. To ensure for complete removal of excessive NaBH~4~, the solution was stirred at 40---45 °C for another 15 min.
5.5 nm CTAB-Capped Au Seeds {#sec1.4}
===========================
A 5 mL growth solution was prepared consisting of 40 mM CTAB and 0.125 mM HAuCl~4~. The Au(III) was reduced to Au(I) with 12.5 μL of a 0.1 M AA solution (f.c. 0.25 mM), indicated by a fast color change from yellow-orange to transparent. Then, 835 μL of the citrate-capped Au seeds was added quickly and the solution was mixed by hand-shaking thoroughly. The 5.5 nm CTAB-capped Au seeds were aged for 3 h at 23 °C prior final overgrowth.
PT Au NR Synthesis {#sec1.5}
==================
A 500 mL growth solution was prepared containing 8 mM CTAB and 0.125 mM HAuCl~4~. The solution was thermostated at 20 °C. Next 1560 μL of a 0.1 M AA (f.c. 0.313 mM) was added and gently stirred leading to a clearance of the solution. Finally, 750 μL of 5.5 nm CTAB-capped Au seeds was added quickly and mixed thoroughly. The solution was thermostated at 20 °C overnight.
PT Au NR Purification {#sec1.6}
=====================
Purification was carried out as described by Scarabelli et al.^[@ref56]^ For PT Au NRs of length and width ca. 200 and 30 nm, respectively, the final surfactant concentration was set to 0.1 M through the addition of 67.5 mL of a 25 wt % CTAC solution, leading to flocculation and sedimentation overnight (see [Supporting Information](#notes-1){ref-type="notes"} for more details about the calculation). The supernatant was discarded and the sediment containing the NRs was redispersed in a 10 mM BDAC solution to obtain a final Au(0) concentration of 0.25 mM (measured using the absorbance at 400 nm).^[@ref57],[@ref58]^
AgAuAg NWs Synthesis {#sec1.7}
====================
A total of 20 mL of the purified PT Au NR solution containing 10 mM BDAC and 0.25 mM Au(0) was heated to 60 °C. AgNO~3~ (0.004 M in water) and AA (0.016 M in 20 mM BDAC, in order to keep the BDAC concentration constant) were added continuously in separate syringes by a syringe pump with a rate of 0.24 mol of Ag(I) per mol of Au(0) per hour (effective rate starting at 300 μL/h) under slow stirring at 60 °C. Samples of 1 mL were taken after defined time frames and the effective rate adjusted to maintain the rate of 0.24 mol of Ag(I) per mol of Au(0) per hour during the whole experiment (see Table S3, [Supporting Information](#notes-1){ref-type="notes"} for the effective rates). For UV--vis-NIR measurements beyond the water limit at around 1350 nm, the path length was reduced using a 1 mm cuvette and water was exchanged by deuterated water to reduce vibrational modes and consequently reduce overall extinction. The samples were washed three times by centrifugation (1000---3000 rpm) and redispersing in deuterated water (concentrating the sample down to 400 μL) to allow UV--vis-NIR measurements in the range of 300---2500 nm in a 1 mm pathway cuvette to be performed.
Spectroscopic and TEM Characterization {#sec1.8}
======================================
Transmission electron microscopy (TEM) images were collected with a JEOL JEM1400PLUS instrument operating at 120 kV with carbon-coated 400 square mesh copper grids. All samples were centrifuged twice before blotting on the grid to reduce surfactant concentration. Optical extinction spectra were recorded using an Agilent Cary 5000 UV--vis-NIR spectrophotometer in deuterated water. All the presented UV--vis-NIR spectra were multiplied by the respective dilution factors to facilitate comparison of the data. HAADF-STEM images and electron tomography series were acquired using an aberration corrected cubed FEI Titan 60---300 electron microscope operated at 200 kV. For the reconstruction of the series, the total variation minimization technique (TVM) was used. EELS plasmon maps, were acquired using a monochromated double aberration corrected cubed FEI Titan 50---80 electron microscope operated at 300 kV and yielding an energy resolution of 0.12 eV. For the analysis of the two EELS data sets, first a Richardson--Lucy deconvolution of the ZLP from the data was performed and then the ZLP was fitted and subtracted from the result, by using Hyperspy.^[@ref59]^ Because the points of the map passing over the rod still present a large background due to multiple phonon scattering and are also noisier due to the lower counts, reducing the signal quality from those areas, a mask was used in the place of the NW.^[@ref60]^
Electromagnetic and EELS Simulations {#sec1.9}
====================================
Optical extinction spectra, electric near-field intensity maps, and EELS intensities are calculated by solving the Maxwell equations in the presence of either an external light plane wave or an electron point charge moving with constant velocity (*v* = 0.78*c*, corresponding to an acceleration voltage of 300 kV), respectively. The extinction calculations are averaged over NW orientations and light polarizations. For EELS, the electron is incident along a direction normal to the long wire axis. For simplicity, the wires are simulated as axially symmetric rods of the same volume as the actual PT NWs, using the boundary-element method and exploiting the axial symmetry of the particles, as described elsewhere.^[@ref48]^ More precisely, the outer gold and silver NW interfaces are modeled as circular cylinders with hemispherical caps. The radius of the silver wire is considered to be 2 nm larger than that of the gold core. The latter has a length (diameter) of 210 nm (34 nm) in the simulations of [Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"} and 180 nm (32 nm) in [Figures [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}--[4](#fig4){ref-type="fig"}. Gold and silver are represented by their tabulated frequency-dependent complex dielectric functions.^[@ref61]^ The NWs are considered to be in vacuum (ε = 1) for EELS and in deuterated water (ε = 1 -- 0.30637 λ^2^/(λ^2^ + 47.26686) + 0.74659λ^2^/(λ^2^ -- 0.00893), where λ is the free-space light wavelength in microns under optical illumination^[@ref62]^) for optical extinction.
Further experimental details, optical data, theoretical derivations and detailed interpretations are provided in Supporting Information. The Supporting Information is available free of charge on the [ACS Publications website](http://pubs.acs.org) at DOI: [10.1021/acs.nanolett.5b01833](http://pubs.acs.org/doi/abs/10.1021/acs.nanolett.5b01833).
Supplementary Material
======================
######
nl5b01833_si_001.pdf
^○^ Martin Mayer and Leonardo Scarabelli contributed equally.
The authors declare no competing financial interest.
L.M.L.-M. acknowledges funding from the European Research Council Advanced Grant PLASMAQUO (No. 267867) and from the Spanish MINECO (grant MAT2013-46101-R). S.B. acknowledges funding from ERC Starting Grant COLOURATOMS (335078). The research leading to these results has received funding from the European Union Seventh Framework Programme under Grant Agreements 312483 (ESTEEM2) and 262348 (ESMI). M.M., M.T., and A.F. acknowledge funding from the European Research Council starting grant METAMECH (No 306686). M.T. was supported by the Elite Network Bavaria in the frame of the Elite Study Program "Macromolecular Science" and funded via a grant for Ph.D. candidates according to Bavarian elite promotion law (BayEFG). F.J.G.deA. acknowledges funding from the Spanish MINECO (grant MAT2014-59096-P).
|
{
"pile_set_name": "PubMed Central"
}
|
1.. Introduction {#s1}
================
Cassava, *Manihot esculenta* Crantz, is a tropical crop that is important for food security and income generation for many poor farmers in several Asian and African countries. More than 240 million tons of cassava are produced per year, and cassava serves as the primary food source for \>750 million people. Cassava is one of the most efficient producers of carbohydrates and energy among all the food crops.^[@DSS016C1]^ Cassava is known for its adaptability to different soils and environmental conditions, particularly for its tolerance to drought. Cassava can withstand short and longest period of drought of around 4--6 months.^[@DSS016C2]^ The drought stress response in cassava is followed by dehydration avoidance through deep root system, closure of stomata in dry air, and shedding of older leaves in which these features are effective for survival under drought conditions. Upon recovery from water stress, cassava rapidly regenerates new leaves and leaf area index becomes higher compared with non-stressed plants.^[@DSS016C3]^
Partial sequencing of cDNA clones has been used as an effective method for gene discovery and in the last decade, the development of several EST collections has led to functional genomics studies in several plant species.^[@DSS016C4],[@DSS016C5]^ Large-scale cassava EST sequencing projects have been performed in various cassava research groups.^[@DSS016C2],[@DSS016C6],[@DSS016C7]^ Sakurai *et al*. constructed a full-length cDNA enriched library from cassava leaves and roots subjected to drought, heat, and acidic stress treatments, as well as from roots subjected to post-harvest physiological deterioration (PPD), a major obstacle for cassava commercialization.^[@DSS016C8]^ The cassava genome sequence is now publicly available and the initial assembly spans 419.5 Mb, covering 54% of the estimated cassava genome size (770 Mb).^[@DSS016C9]^ At present, 30 666 protein-coding genes have been predicted from the genome sequence and 3485 alternative splice forms have been supported by the ESTs.^[@DSS016C9]^
Microarray technology has demonstrated the power of high-throughput approaches to unravel key biological processes and identify useful candidate genes and promoters for genetic engineering.^[@DSS016C10]--[@DSS016C12]^ In cassava, a cDNA microarray containing ∼5700 unique cassava cDNA sequences has been prepared and used for studying expression profiles in response to *Xanthomonas axonopodis* pv. *manihotis* infection and during the PPD response.^[@DSS016C13],[@DSS016C14]^ Oligo-DNA microarrays are gradually gaining importance due to the number of genes contained on each microarray, easier management of the system, and a greater dynamic range in the evaluation of expression levels.^[@DSS016C15],[@DSS016C16]^ Recently, Yang *et al*.^[@DSS016C17]^ prepared an Agilent 60-mer oligo microarray representing ∼20 000 genes and applied this microarray to study the expression profile of cassava genes during the tuberization process. Although previous studies using cassava microarrays have provided valuable transcriptome information towards cassava molecular breeding, no reports on the cassava microarray studies under drought stress have been reported and only one genotype was used in the previous microarray studies.
More than 6500 cassava germplasm accessions with varying phenotypes for biomass, abiotic stress tolerance, and resistance to harmful pathogens are available from the Genetic Resources Program of CIAT (<http://isa.ciat.cgiar.org/urg/>).^[@DSS016C18]^ Therefore, it is necessary to develop a cassava microarray that can be applied to various cassava genotypes that is essential for identifying useful genes for genetic engineering and advancing molecular breeding in cassava.
In this study, we used three useful cassava genotypes: MTAI16 (also called KU50), MECU72, and MPER417-003. The detailed information for the three genotypes is shown in Section 2. Here we apply our newly developed cassava oligomicroarray to study the expression profile under drought stress and report that our cassava oligomicroarray can be used for analysing the cassava transcriptome in various cassava genotypes.
2.. Materials and methods {#s2}
=========================
2.1.. Plant materials and drought stress treatment {#s2a}
--------------------------------------------------
The three cassava genotypes for the microarray analysis were used: (i) MTAI16 (also known as KU50), which is one of the most important cassava genotype (*M. esculenta* Crantz), especially in Southeast Asia (including Thailand). It was developed through cross-breeding between Rayong 1 and Rayong 90 by breeders of the Kasetsart University, Department of Agriculture, Ministry of Agriculture and CIAT, and released officially in 1993 (<http://www.tapiocathai.org/English/K2_e.html>). It has high root yield, high starch content in root tubers, and vigorous plant growth with wide adaptability to unfavourable environmental conditions^[@DSS016C19],[@DSS016C20]^. (ii) MECU72, a naturally occurring cassava genotype (*M. esculenta* Crantz) with whitefly (*Aleurotrachelus socialis*) resistance and was isolated in Ecuador and (iii) MPER417-003, a wild landrace of *M. esculenta* subsp. *peruviana*, which shows resistance to whitefly and mealybug (*Phenococcus herreni*). MTAI16 and MECU72 might be closely related, because they are classified as the same species. Previous polymorphism studies using AFLP and RAPD markers reported that the mean genetic similarity between *M. esculenta* and *M. peruviana* was 0.59, suggesting that *M. esculenta* and *M. peruviana* are closely related when compared with between *M. esculenta* and other wild species.^[@DSS016C21],[@DSS016C22]^ Previously, the impact of water stress on yield and quality of cassava starch was studied in six varieties and this study indicated that MTAI16 recovered quickly from water stress and also had high starch content under recovery after drought stress compared with other varieties studied.^[@DSS016C19]^ No studies on drought stress tolerance in MECU72 and MPER417-003 have been reported.
The preparation of *in vitro* cassava plantlets was performed as follows: after plantlets were sterilized with 1% sodium hypochlorite solution, the plantlet was transferred to a glass pot with Murashige and Skoog (MS) media (pH5.8) containing 20 g/l sucrose, 4.4 g/l MS salts containing vitamins (Duchefa), 2 µM CuSO~4~ (Wako), and 3.0 g/l gelrite (Wako). After cutting ∼2--3 cm from the shoot top of the cassava plantlets, three shoot cuttings were transferred to a glass pot with the MS media and grown under 16-h illumination of 40--80 μmol photons m^−2^ s^−1^ at 30°C. The roots were formed from the sections of the cutting shoots and the plantlets were grown until ∼5 cm high during 1 month and then used as experimental materials. The phenotypes of these genotypes under non-treated conditions were very similar (Fig. [1](#DSS016F1){ref-type="fig"}). For the untreated control samples, the shoots were harvested and frozen in liquid nitrogen. The plantlets were also subjected to drought treatment by transferring the plantlets from the glass pot onto a plastic plate and maintaining them for 1 h under 40--80 μmol photons m^−2^ s^−1^ at 30°C in 50% relative humidity. After the drought stress treatment, all of the leaves from the three cassava genotypes wilted (Fig. [1](#DSS016F1){ref-type="fig"}). After removing roots from the plantlets, the shoots were immediately frozen in liquid nitrogen and stored at −80°C until the RNA preparation. Three independent biological replicative experiments were performed for each treatment and each genotype as follows: (i) MTAI16 (untreated control), (ii) MTAI16 (1 h drought treatment), (iii) MECU72 (untreated control), (iv) MECU72 (1 h drought treatment), (v) MPER417-003 (untreated control), and (vi) MPER417-003 (1 h drought treatment). Figure 1.Phenotype of cassava genotypes after 1-h drought-stress treatment.
2.2.. RNA extraction {#s2b}
--------------------
Total RNA was extracted from six shoots (∼800 mg fresh weight (FW)) per experiment using an RNeasy Plant Mini Kit (QIAGEN) according to the manufacturer\'s instructions (QIAGEN). The total RNA extracts were treated with RNase-free DNase I (QIAGEN) to completely remove genomic DNA. The total RNA quality was evaluated with electrophoresis using the Bioanalyzer system (Agilent). The extracted total RNA was stored at −80°C until further use.
2.3.. Oligomicroarray design {#s2c}
----------------------------
Figure [2](#DSS016F2){ref-type="fig"} shows an overview of the development of our cassava oligomicroarray. We retrieved 76 566 ESTs and 86 cDNA (from high-throughput cDNA category) sequences from GenBank in February 2009 as starting sequences for the microarray probe design. The sequences used are derived from various cassava genotypes, such as MTAI16, CAS36.01, CAS36.04, CM21772, CM523-7, MCol22, IAC 12.829, MBra685, MCol1522, MNga2, MPer183, Mirassol, SG107-35, Sauti, Gomani, Mbundumali, TME 1, MkondeziTMS30572, and CM2177-2. Some sequences deposited in GenBank were contaminated by non-native sequences derived from cloning vectors, bacterial hosts, and other sources. In addition, abundant repetitive elements in a sequence set decreased our ability to accurately assemble the sequence. Therefore, contamination and repetitive elements in the retrieved sequences were masked via the cross match program in the Phred/Phrap package^[@DSS016C23]^ by using the −minmatch 10 −minscore 20 parameters and comparison with the NCBI UniVec and MSU Plant Repeat Database, respectively. Similarly, polyA and polyT sequences were also masked. We then omitted sequences that are shorter than 100 bp from the design process. In order to eliminate the sequence redundancy, the remaining sequences were assembled with the CAP3 program^[@DSS016C24]^ by using the -p 95 parameter. This condition is tighter than one for redundancy omission alone. All the public ESTs which were used in our assembly were not deposited as the sense strand and the contigous sequences in our assembly included antisense sequences. Therefore, we evaluated the contiguous sequences using the public protein sequence sets. The sense strands of the assembled sequences were evaluated using the BLASTX program^[@DSS016C25]^ against the three databases, NCBI RefSeq plant (released January 20, 2009),^[@DSS016C26]^ UniProt TrEMBL plant (released 3 March 2009),^[@DSS016C27]^ and the predicted protein sequences from poplar (JGI Poptr1_1) and castor bean genome sequences (GenBank accession numbers EQ973772--EQ999533). We identified the sequence that aligned with the three protein sequence sets with the same translation frame as the appropriate sequence for the microarray probe design. Figure 2.Design of the cassava oligo-DNA microarray.
Finally, the sequences filtered through the sense direction evaluation were submitted to the eArray application (<https://earray.chem.agilent.com/earray/>) of Agilent Technologies (Santa Clara, CA, USA) to design the 60-mer oligomicroarray probes using 'Best probe methodology' and 'Design with 3′ bias' options in order to prevent the occurrence of cross-hybridization. Note that only 2.6 and 0.6% had \>80 and 90%, respectively, similarity with other probes on the array ([Supplementary Table S1](http://dnaresearch.oxfordjournals.org/lookup/suppl/doi:10.1093/dnares/dss016/-/DC1)). About 60% of the probes had \>1-bp overlap with the structural portion of genes. Sixty probes are added as non-plant or negative/positive control sequences on the array. The information is available at the Gene Expression Omnibus (GEO), accession number GPL14139.
2.4.. Microarray hybridization {#s2d}
------------------------------
Cyanine-3 (Cy3)-labeled cRNA was prepared from 0.5 μg of total RNA using the Quick Amp Labeling kit (Agilent Technologies) according to the manufacturer\'s instructions (<http://www.chem.agilent.com/Library/usermanuals/Public/G4140-90040_GeneExpression_One-color_v6.5.pdf>), followed by RNeasy column purification (Qiagen). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer. The Cy3-labelled cRNA (1.65 μg) was fragmented at 60°C for 30 min according to the manufacturer\'s instructions. Agilent hybridization buffer was then added to the fragmentation mixture and hybridized to the Agilent microarray (GPL14139) for 17 h at 65°C in a rotating Agilent hybridization oven. After hybridization, the microarrays were washed using the optimized protocol (<http://www.chem.agilent.com/Library/usermanuals/Public/G4140-90040_GeneExpression_One-color_v6.5.pdf>) recommended by Agilent technologies. Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 4 × 44 K array slides. Signal intensities were detected from the obtained digital images using Feature Extraction software (Ver. 9.1; Agilent Technologies). Three independent biological replicative experiments were performed for each treatment and for each genotype.
2.5.. Microarray data analysis {#s2e}
------------------------------
Total 18 expression data obtained by microarray analysis were exported to GeneSpring GX (Agilent Technologies) and per chip normalization to the quantile expression level and per gene normalization to the median expression intensity were performed in all samples. Data were transformed into the log 2 ratio for display and analysis. The following calculations were performed according to the manufacturer\'s instructions of GeneSpring GX. Briefly, the microarray data were filtered to remove control probe sets and those probe sets with an intensity value close to background levels. The remaining genes were filtered based on the deviation of the intensity values within a condition. The remaining genes were placed together in one list. The genes selected in this way were further filtered to remove those probe sets whose expression change under all experimental conditions was below a threshold, based on median normalized intensity values, which was considered to be the no-change threshold. The resulting working gene list of transcripts for each experiment was used for the statistical analysis. To test for differential expression, an analysis of variance test was carried out between non-treated and drought-treated samples. The changes in gene expression were statistically analysed by the unpaired *t*-test (threshold was set at *P*\< 0.01) for two groups (non-treated and drought-treated samples). The false discovery rate (*q*-value) was calculated for each *P* value according to the method of Benjamini and Hochberg (1995).^[@DSS016C28]^ The information from the cassava oligomicroarray is available at the GEO of NCBI. The accession numbers are Platform, GPL14139; Series, GSE31749; Samples, GSM787966--GSM787983.
When the total 21 522 sequences on the array were searched by BLASTX program against 35 176 *Arabidopsis* protein sequence data sets in the nuclear genome. (TAIR10, <ftp://ftp.arabidopsis.org/home/tair/Genes/TAIR10_genome_release/TAIR10_blastsets/TAIR10_pep_20101214>), 20 436 sequences had hits with *Arabidopsis* proteins at *E* value ≤ 1e^−5^. The top hits were used for gene annotations, and the corresponding AGI code was used for the functional classification using the gene ontology (GO) of TAIR10 (<http://www.arabidopsis.org/tools/bulk/go/index.jsp>). The percentage of the up-regulated, down-regulated, overrepresented and underrepresented genes was calculated as follows: percentage (%) = (number of the genes classified into the GO term)/(total number of the genes used for the classification).
2.6.. Quantitative real-time RT-PCR (qPCR) analysis {#s2f}
---------------------------------------------------
First-strand cDNA synthesis was performed with a SuperScript^®^ VILO™ cDNA synthesis kit (Invitrogen) using random hexamer primers. After denaturing the total RNA (2.0 µg) at 70°C for 5 min, reverse transcription was performed for 1 h at 42°C with total RNA, 4 µl of SuperScript VILO Reaction Mix, and 2 µl of SuperScript Enzyme Mix in a 20 µl total volume. The reaction was stopped by heating for 5 min at 85°C. The first-strand cDNA preparations were stored at −30°C until use. The basic procedure of qPCR was carried out on an ABI PRISM 7000 (Applied Biosystems) using a cDNA mixture corresponding to 1.5 ng of total RNA, 10 µl of Fast SYBR^®^ green master mix (Applied Biosystems), 0.5 µM of both forward and reverse primers in a 20 µl total volume. The gene-specific primers used for qPCR are listed in [Supplementary Table S2](http://dnaresearch.oxfordjournals.org/lookup/suppl/doi:10.1093/dnares/dss016/-/DC1). The sequences of all primer sets were designed using the Primer 3 program (<http://primer3.sourceforge.net/>). The PCR cycling conditions were as follows: 95°C for 20 s, for initial denaturation followed by 45 cycles of 95°C for 5 s and 60°C for 30 s. The specificity of the PCR amplification was evaluated with a melting curve analysis (from 55°C to 95°C) of the band pattern of the amplification product after the final cycle of the PCR. Each plate also incorporated a no-template control. We employed probes specific for the ubiquitin-conjugating enzyme 4 (UBC4) gene from cassava as references. The qPCR data was analysed with the ΔCT method using a reference gene. For each sample, the mRNA levels of target genes were normalized to that of the UBC4 mRNA.
3.. Results and discussion {#s3}
==========================
3.1.. Cassava oligomicroarray design {#s3a}
------------------------------------
For the development of the cassava oligomicroarray, we designed 60-mer oligonucleotide probes based on 76 652 cassava cDNA sequences (Fig. [2](#DSS016F2){ref-type="fig"}; 76 566 ESTs and 86 high-throughput cDNA sequences retrieved from GenBank in February 2009). We omitted non-native sequences derived from cloning vectors, bacterial host sequences, and sequences shorter than 100 bp. As a result of this cleaning process, the number of sequences was reduced to 76 568. In order to eliminate the sequence redundancy, the remaining sequences were assembled using the CAP3 program, which resulted in the identification of 29 636 non-redundant sequences (11 422 contigs and 18 214 singlets). We then performed a BLASTX search with the target sequences against NCBI RefSeq plant, UniProt TrEMBL plant, and the predicted protein sequences from poplar and castor bean genome sequences to evaluate the transcriptional direction, because some public ESTs were not deposited as sense strand sequences. This analysis identified 25 708 potential target sequences (average size: 853 bp) for the 60-mer probe design. To obtain more individual sequences among cassava varieties, we assembled the cassava expressed-sequences using the option p 95. We then applied the selected probes to the design via Agilent e-array (<https://earray.chem.agilent.com/earray/>) using 'Best probe methodology' and 'Design with 3′ bias' options to prevent cross-hybridization (Fig. [2](#DSS016F2){ref-type="fig"}). Finally, 21 522 unique microarray probes were selected via the Agilent\'s eArray application. About 52% (17 753) of the total predicted cassava CDS (34 151) are supported by 21 522 probes. Please note that when we designed the cassava microarray in 2009, 18 591 cassava CDS have been supported by the ESTs and this array has the probes corresponding to 89% (16 619) of them.
When the total 21 522 sequences were searched by BLASTX program against 35 176 *Arabidopsis* protein sequences present in the nuclear genome (TAIR10, <ftp://ftp.arabidopsis.org/home/tair/Genes/TAIR10_genome_release/TAIR10_blastsets/TAIR10_pep_20101214>), 20 436 sequences had hits with *Arabidopsis* proteins. Among them, 16 875 sequences had hits with 9748 *Arabidopsis* proteins at *E* value ≤1e^−5^. The top hits were used for gene annotation and analyses of the GO (please see below).^[@DSS016C29]^ Note that the percentage of the probes on the array having \>80 or 90% similarity with other probes is 2.6 or 0.6, respectively ([Supplementary Table S1](http://dnaresearch.oxfordjournals.org/lookup/suppl/doi:10.1093/dnares/dss016/-/DC1)). We then adopted the Agilent 44k oligomicroarray platform and the selected probes were duplicated in the randomized layout on the array. We have also added the 60 probes of non-plant or negative/positive control sequences on the array.
Similar type of 60-mer Agilent cassava oligomicroarray has been also developed^[@DSS016C17]^ and applied to study the expression profiles during storage root formation in cassava. The previous studies have applied it to only one cultivar, TMS60444 and have not demonstrated that the probes on the array could hybridize with the cRNAs prepared from various cassava genotypes. Our array differs from the previous array in the following ways: (i) slightly more ESTs were used to design the array (76 566 vs. 71 520), (ii) more genes are represented on our arrays (21 522 vs. 20 840), and (iii) care was taken to examine the transcript direction on our array.
3.2.. Cassava oligomicroarray can be used for transcriptome analyses in various cassava genotypes {#s3b}
-------------------------------------------------------------------------------------------------
We applied the 22-k cassava oligomicroarray to study the expression profiles of three cassava genotypes, MTAI16, MECU72, and MPER417-003 under drought stress (Fig. [1](#DSS016F1){ref-type="fig"}) as described in Section 2. After filtering (see Section 2), the genes with the following characteristics were selected: (i) the signal intensity is higher than the local background plus 2.6 times of the standard deviation; (ii) the signal intensity is not saturated; and (iii) the spot is uniform. We adopted the Agilent standard protocol for the oligo-microarray kit in order to keep the objectivity of the statistics (<http://www.genomics.agilent.com/files/Manual/G4460-90026_FE_Reference.pdf>). The total numbers of valid gene probes that were identified as 'expressed' with the above characteristics were 16 888, 17 772, and 17 037 from MTAI16, MECU72, and MPER417-003, respectively (Table [1](#DSS016TB1){ref-type="table"}). These results showed that 77--83% from a total of 21 522 genes were detectable by hybridization to the cRNA from the tissues. Table 1.Number of expressed genes and data reproducibility among experiments using three cassava genotypesCassava genotypesNumber of expressed genes^a^Correlation coefficientMTAI16MECU72MPER417-003MTAI16168881.000------MECU72177720.959 ± 0.0141.000---MPER417-003170370.967 ± 0.0050.879 ± 0.0141.000[^2]
To show the feasibility of our oligomicroarray for cassava genotypes with heterologous chromosomes, the signal intensities of the above-selected probes were plotted, and the correlation coefficients (*R*^2^) among three cassava genotypes under a non-treated condition were evaluated (Table [1](#DSS016TB1){ref-type="table"}). The *R*^2^ values were 0.959, 0.967, and 0.879 between MTAI16 and MECU72, MTAI16 and MPER417-003, and MECU72 and MPER417-003, respectively. It is worth noting that the *R*^2^ value between MECU72 and MPER417-003 was slightly lower than that of other combinations. This might be due to the following reasons: (i) the MTAI16 ESTs have been used for the microarray design, but the ESTs from MECU72 and MPER417-003 have not been used and (ii) the chromosomes of MECU72 and MPER417-003 might be more heterologous compared with MTAI16. To examine the reproducibility of the experiments, we calculated the coefficient of variation (CV) value for the signal intensities in the three replicative microarray experiments. In this study, we used four types of classification based on the CV value, that is, \<0.25, between 0.25 and 0.50, between 0.50 and 0.75, and ≥0.75. More than 90% of all signals have CV values of \<0.50 in both the control and drought-treated conditions (Fig. [3](#DSS016F3){ref-type="fig"}). These results indicate that there are few significant differences in the signal intensities among the three genotypes and that our oligomicroarray can be applied to multiple cassava genotypes. Figure 3.Variation in signal intensities in microarray experiments using three cassava genotypes. Black and white bars indicate non-treated and drought-treated samples, respectively. CVs were calculated for signal intensities in the three independent hybridizations.
Genes whose expression levels changed \>2-fold in response to drought treatment were selected using statistical methods (see Section 2). The following three criteria were used for the gene selection: (i) the signal intensity changed \>2-fold between the non-treated and drought-treated samples; (ii) The *P* value from the *t*-test for two groups (non-treated and drought-treated samples) was \<0.01 and (iii) FDR (*q*-value) according to the method of Benjamini and Hochberg was \<0.1.^[@DSS016C28]^ The number of drought stress up-regulated genes was 1078, 305, and 671 in MTAI16, MECU72, and MPER417-003, respectively (Fig. [4](#DSS016F4){ref-type="fig"}) and the number of drought stress down-regulated genes was 597, 419, and 238 in MTAI16, MECU72, and MPER417-003, respectively (Fig. [4](#DSS016F4){ref-type="fig"}). These results suggest that MTAI16 has the specific system for adaptability under various stress conditions, such as drought stress. Previous studies reported that MTAI16 recovered quickly from stress and also had high starch content under drought recovery compared with other varieties studied.^[@DSS016C19]^ Sequencing analysis of the full-length cDNA clones from MTAI16 subjected to various stress treatments, such as drought, identified many putative gene duplications that might have played a role in cassava stress responses.^[@DSS016C8]^ A total of 168 genes (Fig. [4](#DSS016F4){ref-type="fig"}; [Supplementary Table S3](http://dnaresearch.oxfordjournals.org/lookup/suppl/doi:10.1093/dnares/dss016/-/DC1)) and 69 genes (Fig. [4](#DSS016F4){ref-type="fig"}; [Supplementary Table S4](http://dnaresearch.oxfordjournals.org/lookup/suppl/doi:10.1093/dnares/dss016/-/DC1)) were up-regulated and down-regulated, respectively, by drought stress in all three genotypes. Figure 4.Venn diagram analysis of the genes up-regulated (A) or down-regulated (B) by drought stress treatment in the three cassava genotypes. In each genotype, the drought stress-up-regulated genes were identified as follows: (i) ratio (drought treatment/no treatment) ≥2, (2) *t*-test, *P*-value \< 0.01, and (3) FDR (*q*-value) \<0.1 according to the method of Benjamini and Hochberg (1995). The drought stress-down-regulated genes were identified as follows: (i) ratio (drought treatment/no treatment) ≤0.5, (ii) *t*-test, *P*-value \< 0.01, and (iii) FDR (*q*-value) \<0.1 according to the method of Benjamini and Hochberg (1995).
3.3.. Cassava has similar mechanisms for drought stress response and tolerance as other plants, such as arabidopsis {#s3c}
-------------------------------------------------------------------------------------------------------------------
Drought can be a major environmental constraint affecting the growth and physiology of many plant species. As a result, many plants have developed strategies to defend against damage caused by drought stress. In this study, we identified 168 genes that were up-regulated by drought stress in three cassava genotypes. Among them, there were many homologs of drought-inducible genes that were identified in previous studies of other plant species, such as *Arabidopsis* and rice ([Supplementary Table S3](http://dnaresearch.oxfordjournals.org/lookup/suppl/doi:10.1093/dnares/dss016/-/DC1)).^[@DSS016C30]--[@DSS016C32]^
Functional category classification using the GO of the *Arabidopsis* Information Resource (TAIR10; <http://www.arabidopsis.org/tools/bulk/go/index.jsp>) for three components ('biological process', 'molecular function' and 'cellular component') was performed on the 168 drought stress up-regulated or 69 down-regulated genes (Fig. [5](#DSS016F5){ref-type="fig"}). Many drought stress up-regulated genes were classified into the GO terms 'other cellular processes', 'other metabolic processes', 'unknown biological processes', 'response to abiotic or biotic stimulus' and 'response to stress' for biological process. Figure 5.Percentage of GO terms for (A) biological process, (B) molecular function, and (C) cellular component of the genes up-regulated (168 genes in [Supplementary Table S3](http://dnaresearch.oxfordjournals.org/lookup/suppl/doi:10.1093/dnares/dss016/-/DC1); black bar) and down-regulated (69 genes in [Supplementary Table S4](http://dnaresearch.oxfordjournals.org/lookup/suppl/doi:10.1093/dnares/dss016/-/DC1); white bar) by drought stress treatment in the 3 genotypes (MTAI16, MECU72, and MPER417-003).
The drought stress up-regulated genes classified in a GO term 'response to stress' ([Supplementary Table S5](http://dnaresearch.oxfordjournals.org/lookup/suppl/doi:10.1093/dnares/dss016/-/DC1)) include the homolog of a key gene in the biosynthetic pathway for abscisic acid (ABA), AtNCED3 (AT3G14440),^[@DSS016C33]^ which encodes a member of the 9-cis-epoxycarotenoid dioxygenases; an NAC transcription factor homolog, RD26 (AT4G27410)^[@DSS016C34]^ that is involved in the ABA-dependent drought stress signaling pathway; and a homolog of a drought-inducible galactinol synthase, AtGolS1 (AT2G47180),^[@DSS016C35]^ which encodes the key step in the biosynthesis of raffinose family oligosaccharides, osmolytes that play a role in drought stress tolerance. Among the drought-inducible genes identified, we also found homologs of several jasmonate ZIM-domain (JAZ) proteins^[@DSS016C36]^ that function in jasmonate pathway responses, such as wounding. This is most likely the result of cross-talk between ABA and JA signaling that has been observed in previous studies.^[@DSS016C37]^ In cassava, the interaction between ABA and JA might function in protecting the plants from the water loss that occurs under drought stress conditions. This might also be due to the fact that the cassava plants used for this microarray analysis were subjected to a wounding stress when the plantlets were removed from the gelrite medium during the drought stress treatment and when the shoots were cut from the plantlets prior to storage at −80°C.
Many drought stress down-regulated genes were classified into the GO terms 'other intracellular components', 'other cytoplasmic components', 'chloroplast', 'other membranes' and 'plastid' for cellular component (Fig. [5](#DSS016F5){ref-type="fig"}C). The drought stress down-regulated genes classified into the GO terms 'chloroplast' or 'plastid' include the homologs of a light-harvesting chlorophyll *a*/*b*-binding protein, LHCA4 (AT3G47470) and a chloroplast triose phosphate/3-phosphoglycerate translocator, APE2 (AT5G46110) genes ([Supplementary Table S4](http://dnaresearch.oxfordjournals.org/lookup/suppl/doi:10.1093/dnares/dss016/-/DC1)). These results are consistent with previous reports that drought stress inhibits photosynthesis.^[@DSS016C38]^ Repression of photosynthesis under drought stress also occurs in cassava in the same way as other plants and might help the plants to survive under the stress.
Hierarchical clustering analysis of the 168 drought stress up-regulated and 69 down-regulated genes revealed similar expression pattern of the genes in both non-treated and drought stress-treated conditions among the three genotypes (Fig. [6](#DSS016F6){ref-type="fig"}). The venn diagram analysis also identified many genes up-regulated or down-regulated specifically in each genotype by drought stress treatment (Fig. [4](#DSS016F4){ref-type="fig"}; [Supplementary Tables S6--S17](http://dnaresearch.oxfordjournals.org/lookup/suppl/doi:10.1093/dnares/dss016/-/DC1)). These genes include genes with unknown function and ones with various functional categories, such as transcription factors, protein kinases, stress response-related ones and metabolism-related ones. The GO term analyses for biological process showed that many genes up-regulated specifically in each genotype by drought stress were classified into the GO terms 'other cellular processes' and 'other metabolic processes'. We could not find big differences on the pattern of GO term among the differentially regulated gene groups in each genotype ([Supplementary Figs S1--S3](http://dnaresearch.oxfordjournals.org/lookup/suppl/doi:10.1093/dnares/dss016/-/DC1)). Figure 6.Hierarchical clustering analysis of the genes up-regulated (168 genes) and down-regulated (69 genes) by drought stress treatment in the three cassava genotypes. The signal intensity values for each sample were transformed to log~2~ values and subjected to hierarchical clustering using standard correlation. The genes with higher and lower signal intensity values are shown in red and blue, respectively. The genes with the signal intensity value of a median level are shown in yellow.
Many differentially expressed genes under the same conditions (no treatment and drought stress treatment) were identified between the genotypes ([Supplementary Tables S18--S30](http://dnaresearch.oxfordjournals.org/lookup/suppl/doi:10.1093/dnares/dss016/-/DC1)) and the GO term analyses of the genes were performed ([Supplementary Figs S4--S6](http://dnaresearch.oxfordjournals.org/lookup/suppl/doi:10.1093/dnares/dss016/-/DC1)). The number of the differentially expressed genes was larger in MPER417-003/MECU72 compared with that in MTAI16/MECU72 and MTAI16/MPER417-003. The differentially expressed genes between the genotypes include the genes with unknown function and the genes with various functional categories, such as transcription factors, protein kinases, transporters and metabolism-related ones.
3.4.. Validation of the microarray data by qPCR {#s3d}
-----------------------------------------------
To evaluate the expression profiles obtained by microarray analysis, we also performed qPCR analysis. Nine genes were randomly selected from the genes shown in [Supplementary Tables S1 and S3](http://dnaresearch.oxfordjournals.org/lookup/suppl/doi:10.1093/dnares/dss016/-/DC1). The cassava homolog of the ubiquitin UBC4 gene ([Supplementary Table S2](http://dnaresearch.oxfordjournals.org/lookup/suppl/doi:10.1093/dnares/dss016/-/DC1)) was used as a control because its expression level was constant between no treatment and the 1-h drought treatment. The results of the qPCR analysis were consistent with those of the microarray analysis (Fig. [7](#DSS016F7){ref-type="fig"}). The *R*^2^ between the two experiments for the 27 total plots was 0.904. These results show that the cassava microarray provides reliable data and can be used for transcriptome analyses in various cassava genotypes with heterologous chromosomes. Figure 7.Confirmation of microarray data by qPCR analysis. A scatter plot between the log~2~-transformed ratio (drought treatment/no treatment) measured by qPCR analysis (*X* axis) and those (drought treatment/no treatment) obtained by the microarray analysis (*Y* axis). White circles, black circles and white squares indicate the data from MTAI16, MECU72 and MPER417-003, respectively. Correlation coefficient (*R*^2^) was 0.904.
Supplementary Data {#s4}
==================
[Supplementary Data are available at www.dnaresearch.oxfordjournals.org](http://dnaresearch.oxfordjournals.org/lookup/suppl/doi:10.1093/dnares/dss016/-/DC1).
Funding {#s5}
=======
This work was supported by Strategic Funds for the Promotion of Science and Technology of the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan and the Grant-in-Aid for Scientific Research (Young Scientists (B) 21710205) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and the core fund of CIAT.
Supplementary Material
======================
###### Supplementary Data
The authors would like to thank Dr Sarah Ayling and Ms Angela Fernando for reading the manuscript and for providing helpful comments.
[^1]: Edited by Satoshi Tabata
[^2]: ^a^Total number of the gene probes with the following characteristics in six experiments (both non-treated and drought-treated samples): (i) the signal intensity was higher than the local background plus 2.6 times of the standard deviation; (ii) the signal intensity was not saturated, and (iii) the spot is uniform.
|
{
"pile_set_name": "PubMed Central"
}
|
Background {#Sec1}
==========
A vermiform appendix located within an inguinal hernia sac is termed Amyand's hernia (AH); Claudius Amyand reported a case of a perforated appendix in an inguinal hernia sac in 1735 \[[@CR1]\]. The incidence of an appendix within an inguinal hernia is seen in 0.1% of all inguinal hernias, and the diagnosis is usually made intraoperatively \[[@CR2]\]. Therefore, most of these cases are managed during surgery. We present a case of a preoperatively diagnosed Amyand's hernia (AH) in a man who underwent treatment by simultaneous laparoscopic totally extraperitoneal (TEP) repair and laparoscopic appendectomy.
Case presentation {#Sec2}
=================
A 76-year-old Japanese man was referred to our department with a several-week history of right inguinal pain and discomfort in his right femur that worsened with movement. Laboratory tests showed a normal white blood cell count and C-reactive protein level. Ultrasound and computed tomography examinations indicated a vermiform appendix in an inguinal hernia sac, with no remarkable findings of inflammation in the appendix (Fig. [1](#Fig1){ref-type="fig"}a, b). He was clinically diagnosed as having an AH without appendicitis. Reduction of the hernia was attempted under ultrasound but was unsuccessful. Thus, we planned combined TEP with mesh repair and laparoscopic appendectomy after laparoscopic reduction.Fig. 1Preoperative imaging. **a** Ultrasonography showing non-inflamed appendix (arrow) inside the right inguinal canal. **b** Axial computed tomography (CT) scan showing non-inflamed appendix (arrow) in right inguinal hernia canal
He was placed in a supine position and underwent general anesthesia by tracheal intubation. A laparoscopic transabdominal approach was initially performed after establishment of pneumoperitoneum. A 5-mm direct umbilical trocar and a needle forceps (Endo Relief™; Hirata Precisions, Chiba, Japan) were introduced into the upper right abdominal quadrant to inspect the hernia canal for the absence of appendicitis and reduce the appendix laparoscopically (Fig. [2](#Fig2){ref-type="fig"}). This inspection revealed a 3 × 2 cm right external inguinal hernia defect with the appendix; no other intra-abdominal pathology was identified. The vermiform appendix was pulled out and placed in the abdominal cavity without tearing the appendix (Fig. [2](#Fig2){ref-type="fig"}). Next, the hernia sac was reduced into the abdomen via the laparoscopic TEP approach. Our patient was placed in the 30° Trendelenburg position. The rectus muscle was lateralized and a Covidien Balloon Dissector (Medtronic, Minneapolis, MN, USA) was inserted preperitoneally from the umbilical incision of the skin to the symphysis pubis. The balloon was insufflated to open the extraperitoneal area. Additional trocars were introduced as follows: a 12-mm trocar in the initial umbilical incision of the skin and anterior right fascia of the rectus, a 5-mm trocar at the symphysis pubis in the midline, and a 5-mm midline trocar between the symphysis pubis trocar and the umbilical trocar. To cover the myopectineal orifice, Hesselbach's area, and the femoral canal orifice, a 7.9- × 13.4-cm mesh (3DMax™ mesh; Bard, Murray Hill, NJ, USA) was fixed to Cooper's ligament and the rectus muscle with an absorbable fixation device (AbsorbaTack™; Medtronic). Finally, we removed the trocars and newly inserted two 5-mm trocars at the umbilical region for the intraperitoneal operation with the initial use of needle forceps. The appendectomy was completed via a laparoscopic approach, and the appendix was removed in a sterile bag via the umbilical region. The total estimated blood loss was 5 mL, and the total operation time was 111 minutes. Our patient was started on intravenously administered cefmetazole at 2.0 g intraoperatively. A histopathological examination confirmed chronic appendicitis with fibrosis and inflammatory cells. Postoperatively, he was discharged and had an uneventful recovery. He was followed up at 6 months postoperatively. He had no recurrence of the hernia, and the wound had healed without inflammatory signs.Fig. 2Intraoperative findings. **a** Appendix located within an external inguinal hernia canal. **b** Normal appearance of the appendix having successfully reduced from the inguinal canal, no adhesions between the vermiform appendix and surrounding hernia sac
Discussion and conclusions {#Sec3}
==========================
AH is a rare condition, and the diagnosis is usually made incidentally during surgery. With the widespread use of helical computed tomography in current practice, however, several authors have recently reported the ability to diagnose AH by preoperative imaging \[[@CR3]--[@CR5]\]. Surgical treatment of AH requires both appendectomy and hernia repair. The treatment algorithm for AH (Table [1](#Tab1){ref-type="table"}) is generally accepted and recommends different management strategies depending on the severity of the condition of the appendix \[[@CR6]\]. AH of type 3--4 is considered to be complicated by appendicitis and requires surgical treatment with avoidance of mesh. However, the efficacy of combining appendectomy and inguinal hernia repair with or without mesh for other types of AH (type 1--2) remains unclear. Some reports have described appendectomy for inflamed appendices (type 2) combined with mesh inguinal hernia repair \[[@CR7]--[@CR12]\]. Therefore, some authors consider that tension-free inguinal hernia repair with mesh and appendectomy is acceptable for both non-inflamed and inflamed appendices \[[@CR3], [@CR8], [@CR10], [@CR12]\]. In addition, Kose *et al*. \[[@CR13]\] proposed using the presence of fibrous connections between the vermiform appendix and the surrounding hernia sac as an indicator for performing appendectomy with mesh inguinal hernia repair. Regarding the treatment of AH, several authors have suggested that laparoscopy can be a safe method for reduction of the appendix without contamination of the inguinal canal and allows the physician to rule out other pathologies \[[@CR12], [@CR14]\]. Mullinax *et al.* \[[@CR14]\] published a report of a type 2 AH treated by laparoscopic hernia repair and appendectomy. Only a single report of endoscopic total extraperitoneal management of an intraoperatively diagnosed AH (type 2) has been published \[[@CR15]\].Table 1Classification systems for Amyand's hernia \[[@CR6]\]TypesSalient featuresSurgical managementType 1Normal appendixReduction or appendectomy(depending on age), mesh hemioplastyType 2Acute appendicitis localized in the sacAppendectomy through hernia, endogenous repairType 3Acute appendicitis, peritonitisAppendectomy through laparotomy, endogenous repairType 4Acute appendicitis, other abdominal pathologyAppendectomy, diagnostic workup and other procedures as appropriate
We performed preperitoneal mesh placement and total laparoscopic appendectomy after reducing the appendix by an intraperitoneal approach to treat a preoperatively diagnosed AH. This process was introduced to allow inspection of the hernia canal and confirm the absence of a perforated appendix or peritonitis, as well as observe the degree of fibrous connections between the vermiform appendix and the surrounding hernia sac, which helped to avoid tearing the appendix. The main reasons for selecting TEP repair are that the procedure is not influenced by intra-abdominal conditions and avoids entering the peritoneal cavity, thus protecting the mesh from bacterial contamination.
In conclusion, a laparoscopic mesh inguinal hernia repair combined with laparoscopic appendectomy can be performed for the surgical treatment of AH type 1 and select cases of AH type 2. It may be regarded as a safe technique with minimal morbidity to the patient. In particular, TEP repair of an inguinal hernia with mesh after laparoscopic hernia reduction may help to avoid mesh contamination in patients with an AH.
AH
: Amyand's hernia
TEP
: Totally extraperitoneal
**Publisher's Note**
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
We thank Angela Morben, DVM, ELS, from Edanz Group (www.[Edanzediting.com/ac](http://edanzediting.com/ac)), for editing a draft of this manuscript.
All authors declare that the paper is being submitted for consideration for publication in *Journal of Medical Case Reports*, that the content has not been published or submitted for publication elsewhere. All authors read and approved the final manuscript.
Authors' information {#FPar1}
====================
I am Daisuke Muroya of Munakata Suikokai Hospital in Japan. I am in charge of the Department of surgery. I worked as a surgeon in Kurume University Hospital and associated hospital for 10 years.
Qualification: PhD in medicine, Kurume University, Fukuoka (2017)
No funding.
Not applicable.
Not applicable.
Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.
The authors declare that they have no competing interests.
|
{
"pile_set_name": "PubMed Central"
}
|
Introduction
============
Lactic acid bacteria in the gastrointestinal tract, such as lactobacilli and bifidobacteria, play an important role in the health of the host ([@b18]). In particular, these bacteria contribute to the modulation of immune responses ([@b31]; [@b500]). Consumption of probiotic foods, mainly yogurt, has been reported to ameliorate abnormal immune functions, including allergy ([@b24]; [@b6]). Recent studies have found that not only viable cells of lactic acid bacteria but also heat-killed cells exhibit immunomodulatory effects ([@b14]; [@b13]). [@b5]) and [@b23]) also reported that heat-killed lactic acid bacteria skewed the immune response toward T-helper 1 (Th1) polarization. Cell surface components in lactic acid bacteria, such as peptidoglycan, lipoproteins, and diacylated lipopeptide, act as ligands for immune cells ([@b27]; [@b17]). Nitric oxide (NO) is involved in a variety of important physiological processes such as vasodilatation, neurotransmission, and host defense against invading pathogens ([@b3]). However, an excessive amount of NO is detrimental, resulting in rheumatoid arthritis, gastritis, bowel inflammation, multiple sclerosis, and bronchitis ([@b1]; [@b9]). In macrophages, NO is synthesized by the inducible isoform of nitric oxide synthase (iNOS), which catalyzes the conversion of [l]{.smallcaps}-arginine to [l]{.smallcaps}-citrulline and NO, in response to various stimuli such as lipopolysaccharide (LPS), interferon (IFN), tumor necrosis factor-α (TNF-α), and interleukin (IL)-1β ([@b16]). Peptidoglycan, lipoproteins, and diacylated lipopeptide also bind to Toll-like receptor (TLR) 2 and TLR1 or 6 heterodimers on the macrophage cell surface and then stimulate NO production via the canonical NF-κB pathway ([@b15]; [@b30]). Thus, cell surface components in lactic acid bacteria are thought to be immunomodulators.
In 1988, we isolated a lactic acid bacterium from tempeh, a traditional side-dish fermented food, which is widely consumed in Southeast Asia ([@b19], [@b20]). We further examined the biochemical properties of the isolated lactic acid bacterium and identified it as *Enterococcus faecalis* TH10 ([@b19], [@b20]). *Enterococcus faecalis is* generally present in the intestinal tract of humans and animals. This Gram-positive, nonmotile organism shows homolactic fermentation, among other characteristics ([@b21]). Hitherto, we found an anti-*Escherichia coli* O-157 component produced by *E. faecalis* TH10 ([@b22]). In this study, we showed the immunomodulatory effect of heat-killed *E. faecalis* TH10 (hk-TH10) and its signal transduction on murine macrophage RAW 264 cells.
Materials and Methods
=====================
Preparation of heat-killed *Enterococcus faecalis* TH10
-------------------------------------------------------
*Enterococcus faecalis* TH10 was cultured in glucose--yeast--peptone medium at 37°C for 24 h. After incubation, the TH10 cells were harvested by centrifugation at 3000 × *g* for 20 min. The cells were washed twice with sterilized phosphate-buffered saline (0.85% NaCl, 2.86 mmol/L KCl, 10 mmol/L Na~2~HPO~4~, and 1.76 mmol/L KH~2~PO~4~ at pH 7.7) and then heat-killed at 121°C for 15 min by autoclaving.
Reagents
--------
Antibodies against p44/42 mitogen-activated protein kinases (MAPKs) or extracellular signal-regulated kinase (ERK), phospho-p44/42 MAPK (Thr202/Tyr204; p-ERK), p38 MAPK, phospho-p38 MAPK (Thr180/Tyr182; p-p38), stress-activated protein kinase (SAPK)/c-Jun NH~2~-terminal protein kinase JNK (JNK), phospho-SAPK/JNK (Thr183/Tyr185; p-JNK), iNOS, COX2, Stat1, phospho-Stat1, IκBα, and NF-κB (p65) were obtained from Cell Signaling Technology (Beverly, MA, USA). Antibody against β-actin was purchased from Sigma-Aldrich (St. Louis, MO, USA). The TLR4 peptide inhibitor set VIPER was purchased from IMGENEX (San Diego, CA, USA). The NF-κB inhibitor ammonium pyrrolidine dithiocarbamate (APDC) was purchased from Merck Millipore (Darmstadt, Germany).
Cell culture and treatment
--------------------------
Murine macrophage RAW264 cells were purchased from RIKEN Bio Resource Center (Tsukuba, Japan). Cells were cultured in Dulbecco\'s Modified Eagle\'s Medium containing 10% heat-inactivated fetal bovine serum (Invitrogen, Carlsbad, CA, USA), 100 U/mL of penicillin, and 100 μg/mL streptomycin in a humidified atmosphere of 5% CO~2~ at 37°C. RAW 264 cells (2 × 10^5^ cells/well) were seeded into a 24-well multiplate and cultured for 12 h. Hk-TH10 (5--100 μg/mL) or LPS (final concentration; 200 ng/mL; Sigma-Aldrich) was then added to the cells, and culturing was continued for 24 h.
Small-interfering RNA experiments
---------------------------------
Before transfection, RAW264 cells (2 × 10^5^ cells/well) were seeded into a 24-well multiplate and cultured for 12 h. Four small-interfering RNA duplexes (siRNAs) specific for mouse *TLR1*, *TLR2*, and *TLR6* genes and a nonsilencing control siRNA without any homology to known mouse genes were synthesized by Sigma-Aldrich. RAW264 cells were transfected with each of the siRNAs by using the FuGENE® HD Transcription reagent (Promega, Madison, WI, USA). After an additional incubation of 48 h at 37°C, the downregulation levels of *TLR1, TLR2*, and *TLR6* were assessed by Western blot analyses using anti-TLR1, -TLR2, and -TLR6 antibodies. After siRNA transfection, the RAW 264 cells were stimulated with hk-TH10 (25 μg/mL) and cultured for 24 h.
Measurement of NO production
----------------------------
After stimulation with LPS or hk-TH10 for 24 h, cell culture media were collected and then centrifuged at 4°C for 5 min. A Griess reagent kit (Promega) was used to measure the amount of nitrite, a stable metabolite of NO, in the supernatants. Briefly, 50 μL of each culture medium was added to 96-well multiplate in triplicate, and the same volume of sulfanilamide solution was dispensed. After incubation at room temperature for 10 min, dispensed 50 μL of *N*-1-naphthylethylenediamine dihydrochloride solution was dispensed to all wells. After incubation at room temperature for 10 min, the absorbance was measured at 540 nm using a colorimetric microplate reader (Corona Grating Microplate Reader SH-9000; Corona Electric Co., Ltd., Hitachinaka, Japan).
Quantitative real-time PCR
--------------------------
Total RNA was extracted from cells using TRIzol reagent (Invitrogen) followed by DNase I treatment. cDNA was synthesized from 0.25 μg of total RNA using a PrimeScript reagent kit (TaKaRa Bio, Ohtsu, Japan). cDNA was subjected to quantitative real-time PCR using a StepOne Real-Time PCR system (Applied Biosystems). Primers for *iNOS, COX-2,* and the gene-encoding *glyceraldehyde-3-phosphate dehydrogenase* (*GAPDH*) were purchased from TaKaRa Bio. The expression level of each gene was determined using the comparative *C*~t~ method and normalized to that of *GAPDH*, an internal control. The PCR reaction consisted of 45 cycles (95°C for 10 s and 60°C for 40 s) after an initial denaturation step (95°C for 10 min).
Western blotting
----------------
Whole-cell extracts were prepared by lysis of cells in RIPA buffer (10 mmol/L Tris--HCl (pH 7.5), 150 mmol/L NaCl, 1 mmol/L ethylenediamine-*N*,*N*,*N*′,*N*′-tetraacetic acid, 1% NP-40, 0.1% sodium deoxycholate, and 0.1% sodium dodecyl sulfate \[SDS\]) containing complete protease and phosphatase inhibitor cocktails (Roche, Penzberg, Germany). Samples were subjected to SDS-polyacrylamide gel electrophoresis and electroblotted onto PVDF membranes. The membranes were incubated with a primary antibody, followed by incubation with a horseradish peroxidase-conjugated secondary antibody. Immunolabeled proteins were detected using an ECL chemiluminescence kit (GE Healthcare, Piscataway, NJ, USA) and a LAS-4000 lumino-image analyzer (Fuji Film, Tokyo, Japan).
Statistical analysis
--------------------
All data were analyzed using Student\'s *t*-test or one-way ANOVA followed by Fisher\'s multiple range test.
Results
=======
Heat-killed *Enterococcus faecalis* TH10 stimulates NO production in RAW 264 cells
----------------------------------------------------------------------------------
The concentration of nitrite was measured as an index of NO production. As shown in [Figure 1](#fig01){ref-type="fig"}, NO was markedly stimulated by hk-TH10. The NO production level gradually increased as the concentration of hk-TH10 increased to 25 μg/mL, but the NO production levels did not change at concentrations above 25 μg/mL. The NO production level at 25 μg/mL hk-TH10 was approximately 50% of NO production level at 200 ng/mL LPS.
{#fig01}
To gain more information regarding the mechanisms by which hk-TH10 stimulates NO production, we examined the mRNA and protein expression levels of iNOS and COX-2 by using quantitative real-time PCR and Western blot analyses, respectively. Both *iNOS* and *COX-2* mRNA levels were upregulated by hk-TH10 treatment ([Fig. 2](#fig02){ref-type="fig"}A), thereby upregulating both iNOS and COX-2 protein levels ([Fig. 2](#fig02){ref-type="fig"}B). The mRNA and protein expression levels of iNOS and COX-2 were upregulated in a dose-dependent manner.
{#fig02}
Heat-killed *Enterococcus faecalis* TH10-stimulated NO production is not mediated through the TLR4 pathway
----------------------------------------------------------------------------------------------------------
To understand the mechanisms underlying hk-TH10-stimulated NO production, we first examined whether hk-TH10-stimulated NO production was mediated through the TLR4 pathway by using a TLR4 inhibitor. LPS-stimulated NO production was significantly suppressed by TLR4 inhibitor (VIPER (VP, amino acid sequence of the viral inhibitory peptide of TLR4 : KYSFKLILAEYRRRRRRRRR, molecular weight: 2780.3)) treatment ([Fig. 3](#fig03){ref-type="fig"}A). It is well known that LPS binds to the cell surface receptor CD14, which triggers activation of TLR4 and downstream signaling molecules such as IκBα and MAPKs including JNK, p38 MAPK, and ERK, culminating in the expression of pro-inflammatory genes such as *iNOS*, *COX-2*, *TNF-α,* and *IFN-β* ([@b15]; [@b30]). In contrast, the hk-TH10-stimulated NO production was not changed by TLR4 inhibitor. The CP (inert control peptide for VP amino acid sequence of the control peptide: RNTISGNIYSARRRRRRRRR, molecular weight: 2601) treatment did not suppress the LPS-stimulated or hk-TH10-stimulated NO production. From these results, we can conclude that hk-TH10-stimulated NO production is not mediated through the TLR4 pathway.
{#fig03}
Heat-killed *Enterococcus faecalis* TH10-stimulated NO production is mediated through the TLR2-TLR1/6 pathway
-------------------------------------------------------------------------------------------------------------
It is well known that peptidoglycan, lipoproteins, and diacylated lipopeptide, which are surface components of lactic acid bacteria, bind to TLR2 and TLR1/6 on the macrophage cell surface and then produce NO via the canonical NF-κB pathway. In order to investigate whether hk-TH10-stimulated NO production is mediated through the TLR2-TLR1/6 pathway, we examined whether knockdown of endogenous TLR1, TLR2, and TLR6 by each siRNA affected hk-TH10-stimulated NO production. These TLR1-, TLR2-, and TLR6-siRNA treatments significantly suppressed hk-TH10-stimulated NO production. In particular, the level of NO production in TLR2-siRNA-treated RAW264 cells was lower than that in TLR1- or TLR6-siRNA-treated RAW264 cells ([Fig. 3](#fig03){ref-type="fig"}B). Conversely, there was no effect on hk-TH10-stimulated NO production in the nonsilencing control siRNA-treated RAW264 cells. Next, we further examined whether the NF-κB inhibitor APDC (25--100 μmol/L) could affect hk-TH10-stimulated NO production. The NF-κB inhibitor treatment greatly suppressed NO production ([Fig. 3](#fig03){ref-type="fig"}C).
TLR2-TLR1/6 signaling enhances the phosphorylation of MAPKs and IκBα, and thereby activates transcription factors such as AP-1, ELK1, and NF-κB. These transcription factors are activated or upregulated by peptidoglycan, lipoproteins, and diacylated lipopeptide, which bind to the iNOS promoter and enhance NO production. Hk-TH10 stimulation enhanced phosphorylation of MAPKs including p38 ([Fig. 4](#fig04){ref-type="fig"}, left panel). Phosphorylation of IκBα proteins leads to its degradation and the translocation of NFκB into the nucleus. As shown in [Figure 4](#fig04){ref-type="fig"} (right panel), hk-TH10 stimulation facilitated the phosphorylation of IκBα. Taken together, these results suggest that hk-TH10-stimulated NO production is mediated through the TLR2-TLR1/6 pathway. The activation levels of each molecule were lower than those in LPS-stimulated RAW264 cells.
{#fig04}
Discussion
==========
In this study, we showed that the immunomodulatory effects of hk-TH10 are mediated via the TLR2-TLR1/6 pathway ([Fig. 5](#fig05){ref-type="fig"}). Ten TLRs have been identified in humans and 12 in mice ([@b26]; [@b12]; [@b32]). TLR1, 2, 4, 5, 6, and 11 recognize microbial membrane components, whereas TLR3, 7, 8, and 9 recognize nucleic acid of bacteria and virus ([@b26]; [@b12]; [@b32]). Cell wall components of lactobacilli can bind to TLR2 in combination with TLR6 ([@b17]; [@b30]). The diacyl lipid chains of diacylated lipoprotein bind to a hydrophobic pocket in the extracellular domain of TLR2 ([@b11]; [@b10]). In addition, the third lipid chain of triacylated lipoprotein binds to the hydrophobic pocket in TLR1 ([@b11]; [@b10]). Because of these interactions, MyD88/TRAF6/TAK1 signaling is activated, leading to the activation of MAPKs and the canonical NF-κB pathway. In this study, NO production levels in RAW264 cells were significantly suppressed by TLR1-, TLR2-, or TLR6-siRNA treatments and by NF-κB inhibitor APDC treatment. These results suggest that NO production stimulated by hk-TH10 is mediated through the TLR2-TLR1/6 pathway. Moreover, the degree of immunomodulation in hk-TH10-stimulated RAW264 cells was distinctly lower than the degree of that caused by LPS stimulation. Recent studies have shown that the production levels of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 differ among lactic acid bacteria strains or between viable and heat-killed cells of lactic acid bacterium ([@b25]; [@b7]). TLR and cytokine expression levels after stimulation with heat-killed Gram-positive and Gram-negative bacteria are different, as is their dose-dependent mechanism of action ([@b2]). From these reports, we thought that the amounts and thermostability of active components in the cells could contribute to differences in macrophage activation. We also thought that this modest activation in hk-TH10-stimulated RAW264 cells might be good for the health of the host because an excessive amount of NO induces inflammatory diseases.
{#fig05}
In conclusion, hk-TH10 stimulates NO in RAW 264 cells through the activation of the TLR2-TLR1/6 pathway. NO plays an important role in NK cells, mast cells, and neutrophil cells in the production of several cytokines ([@b28]; [@b29]). These cytokines initiate immune responses such as bacterial cell killing and T or B cell proliferation ([@b4]; [@b8]). Thus, hk-TH10 may be useful in foods for the facilitation of host immunomodulation.
Conflict of Interest
====================
None declared.
[^1]: **Funding Information** No funding information provided.
|
{
"pile_set_name": "PubMed Central"
}
|
Introduction {#s1}
============
In typical studies of propagative phenomena in single cells and tissue, the experimenter deliberately induces pulses in an otherwise quiescent, resting system. During most physiological processes, however, pulses are generated in a recurring manner because of, for instance, a persisting stimulus or a system-inherent pacemaker mechanism that generates auto-excitations. It is of fundamental interest to understand the mechanisms that underlie these processes.
The present work is concerned with the effect of environmental temperature on blood vessel pulsations in the poikilotherm *Lumbriculus variegatus*. *Lumbriculus* is a small annelid with a body that is widely transparent and thus well suited for observations of physiological processes within. The dorsal blood vessel of the animal is a contractile, muscular tube, which phyllogenetically represents a predecessor of the hearts of higher organisms ([Fig. 1](#pone-0066773-g001){ref-type="fig"}). From an experimentalist's point of view, blackworms are an excellent model system to study basic principles of rhythmical excitation processes [@pone.0066773-Lesiuk1]. The propagation velocity of vessel contractions as well as the pulse frequency in the dorsal vessel can be extracted readily by light microscopy. Studies of the effect of temperature on these parameters are of twofold interest. First, they can provide insight into how bodily functions of poikilotherms are affected by as well as adapted to *e.g.* diurnal or annual variations of environmental temperature. Second, temperature represents a thermodynamic state variable that can be controlled comparatively well in experiments with whole animals. Thus, such studies can facilitate an understanding of excitable cells and tissues within the theoretical framework of thermodynamics.
{#pone-0066773-g001}
It is the particular aim of the present work to elaborate on the latter. To establish a solid experimental basis for further discussions, the propagation velocity as well as frequency of blood vessel pulsations in *Lumbriculus* will be determined as functions of temperature. These data will be interpreted in the framework of a thermodynamic theory. It has been suggested in the past [@pone.0066773-Wilke1], [@pone.0066773-Kaufmann1] and more recently [@pone.0066773-Andersen1], [@pone.0066773-Griesbauer1] that excitation processes in biological systems could be based on acoustic waves. Strong support for such a view is lent by the macroscopic thermal [@pone.0066773-Ritchie1]--[@pone.0066773-Tasaki2], mechanical [@pone.0066773-Sandlin1]--[@pone.0066773-Castro1], optical [@pone.0066773-Cohen1]--[@pone.0066773-FernandesDeLima1] *and* electrical phenomenology [@pone.0066773-FernandesDeLima1], [@pone.0066773-Hodgkin1] of action potentials in single cells as well as spreading depression waves in nervous tissue. Thermodynamically, the assumption of propagating acoustic waves leads to testable predictions about the temperature-dependence of the macroscopic mechanical material properties of the excitable medium and its inherent relaxation. It will be shown that a thermodynamic relation between compressibility (κ) and relaxation time (τ) can be used to predict the temperature-dependent pulse frequency of blackworms correctly. Finally, the same concept will be applied to interpret data from the literature on propagating (chemical) waves in gels [@pone.0066773-Miyakawa1] and action potentials in human nerves [@pone.0066773-Lowitzsch1]. The general agreement between theory and experiment across living and non-living systems underlines the consistency and integrative nature of the presented thermodynamic approach.
Materials and Methods {#s2}
=====================
1.1 *Lumbriculus variegatus* {#s2a}
----------------------------
*Lumbriculus variegatus* was obtained from Carolina Biological (Burlington, NC, USA). The worms were cultivated in spring water (Poland Spring; Poland, ME, USA) at 9--11°C for at least four weeks prior to experiments. Studies with *Lumbriculus variegatus* do not require approval by the institutional animal care committee.
1.2 Setup to Study Blood Vessel Pulsations {#s2b}
------------------------------------------
After 20 minutes of pre-incubation in spring water buffered to pH 7.0 with 5 mM HEPES/NaOH, a worm was gently aspirated into a glass capillary (20 µL; Drummond Scientific Company, Broomall, PA, USA). This step had to be done with particular care in order to avoid injury to the animal. The capillary was subsequently trimmed with a glass cutter to the worm's body length and capped with rubber plugs. The worm-filled capillary was immersed in a petri dish whose temperature was controlled by a subjacent Peltier element ([Fig. 1B](#pone-0066773-g001){ref-type="fig"}). The dorsal blood vessel and pulsations therein are clearly observable by light microscopy ([Fig. 1C](#pone-0066773-g001){ref-type="fig"}). After every change of temperature a worm was left to equilibrate for 2--3 minutes. This period was typically sufficient for the pulse frequency to stabilize. Vessel pulsations at the tail end of the worm are generally more frequent and irregular than in the mid-body and head regions [@pone.0066773-Lesiuk1]. In order to ensure comparability, segments in the mid-body region of the worm were studied. The number of pulse waves travelling through an arbitrarily chosen segment was counted for one minute. This procedure was repeated six times at each temperature level and from the collected data an average pulse frequency was obtained. Generally, 3--4 temperature levels were studied per worm within about 45 minutes. Worms were not reused in later experiments.
The pulse propagation velocity was obtained as follows: The projected length of the vessel within the optical field of view was determined by manual delineation of the vessel edge in ImageJ (NIH, Bethesday, MA, USA). The typical projected vessel length was ∼3.3 mm. The propagation velocity was calculated by dividing this length by the time that it took for a pulse to cross it.
Results and Discussion {#s3}
======================
2.1 Effect of Temperature on Blood Vessel Pulsation {#s3a}
---------------------------------------------------
### Pulse propagation velocity {#s3a1}
When blackworms were exposed to different environmental temperatures, a characteristic variation of the propagation velocity *c* of blood vessel pulsations in the dorsal vessel was observed ([Fig. 2](#pone-0066773-g002){ref-type="fig"}). It was found that *c* varies linearily with temperature in the range between 0°C and 30°C. At 9.2±0.3°C the average pulse wave velocity was 0.19±0.05 mm s^−1^. Since the worms had been adapted to an environmental temperature of 9--11°C for four weeks prior to the measurements, this value will be denoted as the "basal velocity". It is important to bear in mind that the pulse velocities discussed herein were calculated based on the projected length of the blood vessel as extracted by microscopy. The "real" distance that a pulse covers in the excitable medium could certainly be much larger. For example, folds at the cell (plasma membrane) and tissue level (blood vessel surface) might lead to a considerable difference between actual path length and projected vessel length. However, unless the ratio between projected length and actual path length varies strongly with temperature, it is to be expected that the temperature-dependence in [Fig. 2](#pone-0066773-g002){ref-type="fig"} will be conserved and that the real velocities are simply offset by a constant.
{#pone-0066773-g002}
At the lowest temperature tested (∼0°C), *c* was reduced to about half of the basal velocity while at 20°C and 30°C an about 2 and 2.8 fold higher pulse velocity was observed respectively. Above 30°C, blood vessel pulsations became irregular and when the temperature was increased further they ceased altogether as will be discussed below. The relatively higher standard deviation of the velocity data at temperatures ≥20° ([Fig. 2](#pone-0066773-g002){ref-type="fig"}) is probably a measurement artifact. A blackworm in a glass capillary remains rather motionless in the temperature range between 0°C and 20°C. However, at higher temperatures motility of the worms - in the form of repeated body reversal, stretching as well as back-and-forth movement - clearly increases. These translocations of the worm lead to irregular changes of the projected vessel length in the field of view and thereby could result in a higher variability of the velocity data. The black envelopes in the temperature range between the last data points and the heat block temperatures ([Fig. 2](#pone-0066773-g002){ref-type="fig"} and [Fig. 3](#pone-0066773-g003){ref-type="fig"}) are interpolations. Blood vessel pulsations are highly irregular at these temperatures and thus it was hard to obtain meaningful data.
{#pone-0066773-g003}
The velocity-temperature relationship for vessel pulsations in *Lumbriculus* ([Fig. 2](#pone-0066773-g002){ref-type="fig"}) follows a pattern that seems to be conserved for many types of pulses in excitable systems: a linear increase of *c* with increasing temperature which is ultimately interrupted by a heat block. Qualitatively identical results have, for instance, been reported for action potentials in single cells such as squid giant axons [@pone.0066773-Hodgkin2], [@pone.0066773-Chapman1], nerve fibers from frog [@pone.0066773-Hutchinson1]--[@pone.0066773-Tasaki4] as well as cat [@pone.0066773-Engelhardt1], [@pone.0066773-Franz1] and humans [@pone.0066773-Ludin1]. Moreover, similar observations have been made for excitation phenomena on the tissue level. The temperature-velocity relationship of Ca^2+^-waves as well as that of spreading depression waves in brain cortex are well studied examples [@pone.0066773-MartinsFerreira1], [@pone.0066773-Jaffe1]. It also has to be underlined that the discussed phenomenology is not a peculiarity of animal or human organisms. Equivalent results have indeed been obtained for action potential propagation in *Chara* and *Nitella* plant cells by our lab (unpublished data) and by other groups [@pone.0066773-Beilby1], [@pone.0066773-Blatt1]. Finally, even pulses in non-living excitable media such as gel-based Belousov-Zhabotinsky reaction systems are characterized by a very similar temperature-velocity profile [@pone.0066773-Miyakawa1]. This remarkable conservation of the temperature dependence of the pulse propagation velocity has to be the consequence of a well-conserved physical mechanism.
### Pulse frequency {#s3a2}
It is fundamentally interesting to study if the pulse velocity and frequency of periodic excitations in cells and tissues (*e.g.* heart, intestine, etc.) are related. Thus, in addition to the temperature dependence of *c*, the temperature-frequency dependence of blood vessel pulsations was determined. As illustrated in [Fig. 3](#pone-0066773-g003){ref-type="fig"}, the pulse frequency follows a similar qualitative behavior, but with a steeper rise. The deviation from linearity becomes particularly evident between 30--35°C where up to 6-fold higher frequencies as compared to the basal rate (4.4±0.8 beats min^−1^ at 9.3±0.7°C) were observed.
### Heat-block of blood vessel pulsations {#s3a3}
Blood vessel pulsations in blackworms cease altogether if the worm is heated above a critical temperature. The absolute temperature at which such a heat block occurs varies between species and is moreover shifted by adaptation to environmental factors like *e.g.* temperature in poikilotherms [@pone.0066773-Roots1]. In *Lumbriculus*, cardiac arrest typically sets in at 37.2±2.7°C (lowest: 33°C, highest: 43°C). However, unless this temperature or an even higher one is sustained for several minutes, the worm can be brought back to life by rapid cooling to ∼5°C below the individual critical temperature. Upon subsequent expulsion of the animal from the glass capillary, partial deformations of the body segments were observed in several cases. The severity of such deformations seemed to increase with prolonged exposure of the worm to high temperature. Despite these irreversible effects it is remarkable that in most cases blood vessel pulsations were -- with slight hysteresis -- restored to the typical frequencies by rapid cooling ([Fig. 4](#pone-0066773-g004){ref-type="fig"}). This underlines the reversible component of the heat block phenomenon which has also been observed in other biological systems [@pone.0066773-Hodgkin2], [@pone.0066773-Chapman1].
{#pone-0066773-g004}
2.2 Thermodynamic Concept for Pulse Propagation and Predictions {#s3b}
---------------------------------------------------------------
The temperature dependence of the propagation velocity of pulses in nerve and muscle is typically ascribed to temperature sensitivities of molecular components of the excitable membrane (*i.e.* ion channels). However, such molecular interpretations are inherently difficult to test since ion channels presently can not be studied in absence of a membrane which -- by itself -- has a temperature sensitivity. Furthermore, the similarities between the temperature-velocity relationships for pulses in living [@pone.0066773-Chapman1], [@pone.0066773-Engelhardt1], [@pone.0066773-Ludin1], [@pone.0066773-Jaffe1], [@pone.0066773-Blatt1] and non-living systems [@pone.0066773-Miyakawa1], indicate that -- after all -- a more general theory will be necessary to explain the underlying mechanisms.
### A phenomenological thermodynamic approach {#s3b1}
It has been suggested that pulses in biological systems are thermodynamic processes that resemble acoustic waves [@pone.0066773-Wilke1]--[@pone.0066773-Griesbauer1]. This view is strongly supported by the macroscopic thermal [@pone.0066773-Ritchie1]--[@pone.0066773-Tasaki2], mechanical [@pone.0066773-Sandlin1]--[@pone.0066773-Castro1], optical [@pone.0066773-Cohen1]--[@pone.0066773-FernandesDeLima1] *and* electrical phenomenology [@pone.0066773-FernandesDeLima1], [@pone.0066773-Hodgkin1] of action potentials as well as spreading depression waves. In fact, it has been shown very recently that the propagation velocity *c* of pulses in lipid monolayers -- the simplest model of a cell membrane -- can be described by an expression similar to the well-established expression for linear soundwith κ*~S~* as the isentropic mechanical compressibility and ρ as the density of the medium [@pone.0066773-Griesbauer1], [@pone.0066773-Griesbauer2]. The compressibility of a 2-dimensional medium (*e.g.* an excitable cell membrane) is a macroscopic material property defined as with *A* as the surface area and π as the lateral pressure. It represents the mechanical susceptibility or "softness" of a system. κ, as well as all other macroscopic material properties such as the heat capacity *c~P~*, electrical capacitance *C~T~*, thermal expansion coefficient α*~T~*, etc., depend on the thermodynamic state of the system and thus can be obtained from state diagrams. Any state change, for example by heating/cooling, stretching/compression, exposure to high pH levels or ion concentrations, etc. will lead to the realization of a new set of state diagrams or material properties. It is clear from Eq. (1) that state change-induced variations of κ should be reflected in the velocity of pulses in the system.
To test if an acoustic theory and Eq. (1) adequately describe the velocity of pulses in nerve and muscle would require measurements of the state-dependent κ of the excitable medium (*e.g.* temperature dependence of κ; such experiments are presently underway in our lab). It is equally interesting, however, to use the experimentally observed temperature-velocity relationship for blood vessel pulsations in blackworms ([Fig. 2](#pone-0066773-g002){ref-type="fig"}) in order to make predictions about κ of the excitable medium. In fact, to determine a system's compressibility from the velocity of a sound wave in it is a typical approach in material science. Essentially, low propagation velocities are expected to correspond to relatively higher compressibilities ("softer" system) and vice versa. From the data in [Fig. 2](#pone-0066773-g002){ref-type="fig"} it is consequently predicted that the excitable medium in the dorsal blood vessel of the worm "hardens" with heating and that its material properties change profoundly in the vicinity of the heat block temperature [Fig. 5A](#pone-0066773-g005){ref-type="fig"} (middle, inset). The latter interpretation is supported by observations of discontinuous thermodynamic transitions in excitable gel rods [@pone.0066773-Miyakawa1], squid giant axons [@pone.0066773-Inoue1] and protoplasmic droplets of excitable cells [@pone.0066773-Ueda1] close to the typical heat block temperature.
![Thermodynamic approach: Experiments, predictions and comparisons.\
Based on experimentally obtained temperature-pulse velocity profiles *c* (left column), temperature-dependent compressibilities κ and relaxation times τ (middle column) were calculated for blood vessel pulsations in worms (A), action potentials in human nerves (B; data taken from Fig. 1 in [@pone.0066773-Lowitzsch1]) and waves in gel rods (C; data taken from Fig. 3 and 4 in [@pone.0066773-Miyakawa1]). Comparison of predicted relaxation times τ and frequencies ν with their experimentally obtained counterparts (right column). Experimental data are always plotted as open triangles connected by broken lines while predictions are plotted as filled circles connected by solid lines.](pone.0066773.g005){#pone-0066773-g005}
### Prediction of Relaxation Times {#s3b2}
In a further step, the mechanical compressibilities estimated from the velocity-temperature relationship can be used to tentatively predict the timescales for relaxation processes in the system. By combining Einstein's approach to thermodynamics (see [Text S1](#pone.0066773.s001){ref-type="supplementary-material"}) with an Onsager-type Ansatz, one can show that thermodynamic susceptibilities of a system (*e.g.* heat capacity, compressibility, etc.) are related to relaxation times τ. For lipid bilayer [@pone.0066773-Grabitz1] and monolayer (see [Text S1](#pone.0066773.s001){ref-type="supplementary-material"}), where thermodynamic observables (area *A*, enthalpy *H*, charge *Q*, etc.) are coupled linearly, the expression becomes particularly simple:where *T* is temperature, *A* the system's surface area, *L* a phenomenological parameter and κ~T~ the isothermal compressibility. A similar relation between τ and the heat capacity *c~P~* has been derived [@pone.0066773-Grabitz1]. If one assumes that the surface area as well as *L* of the excitable system does not change appreciably with temperature, relaxation times of the system can be predicted from temperature-dependent compressibilities. τ is expected to increase with increasing compressibility, which essentially means that softer systems relax slower. By combining Eq. (1) and Eq. (2) one also finds that or expressed in terms of frequency ν ():
Hence, propagation velocities are related to relaxation times and frequencies.
The thermodynamic relaxation times should for instance correlate to refractory periods after action potentials or spreading depression waves. In the case of systems with recurring auto-excitations (*e.g.* hearts) the reciprocal of the relaxation time is expected to represent the limiting (*i.e.* highest) frequency at which pulses can be generated.
### Comparison with Experimental Data {#s3b3}
The predictions of the presented thermodynamic concept can now be compared with experimental data. The temperature-velocity profile of blackworm blood vessel pulsations ([Fig. 5A](#pone-0066773-g005){ref-type="fig"}, left) served as a basis to calculate compressibilities and relaxation times ([Fig. 5A](#pone-0066773-g005){ref-type="fig"}, middle). The reciprocals of the relaxation times represent frequencies ν, which indeed agree well with the temperature-dependent pulse rates measured in blackworms ([Fig. 5A](#pone-0066773-g005){ref-type="fig"}, right). At least two interpretations are conceivable. First, two decoupled mechanisms determine the propagation velocity and pulse frequency in the dorsal vessel of *Lumbriculus*. The excitable medium in the blackworm's blood vessel could be capable of transmitting pulses at higher frequencies than those observed experimentally. If this were the case, the temperature-frequency dependence in [Fig. 3](#pone-0066773-g003){ref-type="fig"} would probably reflect the temperature dependence of an independent pacemaker mechanism. Second, the macroscopic thermodynamic properties of the excitable medium determine the propagation velocity *and* control the typical pulse frequency in the system. While this latter interpretation seems appealing by virtue of its integrative treatment of the system and a minimum of assumptions about a pacemaker mechanism, further experiments will have to test its validity.
To illustrate that the presented framework is by no means limited to a specific type of pulse or system, data on action potentials in human nerves [@pone.0066773-Lowitzsch1] and on Belousov-Zhabotinsky reaction waves in gel rods [@pone.0066773-Miyakawa1] were extracted from the literature. The experimentally obtained temperature-pulse velocity profiles ([Fig. 5](#pone-0066773-g005){ref-type="fig"} B and C, left) of these two systems were used to predict compressibilities and relaxation times ([Fig. 5](#pone-0066773-g005){ref-type="fig"} B and C, middle). In the case of nerve action potentials, the calculated relaxation times correspond well with experimental data on refractory periods ([Fig. 5B](#pone-0066773-g005){ref-type="fig"}, right). Similarly, the temperature-dependent frequencies predicted for chemical waves in gels are in quite good agreement with the experimentally found ones ([Fig. 5C](#pone-0066773-g005){ref-type="fig"}, right). This overall consistency further underlines the general applicability and predictive power of the presented thermodynamic approach.
### Comments and Outlook {#s3b4}
A short comment concerning the classical interpretation of refractory periods after excitation waves in tissue should be made. Typically, it is assumed that the duration of the refractory period, for example ensuing a spreading depression wave, is determined by the timescales required by metabolic processes to re-establish ion gradients [@pone.0066773-Weimer1]. It should be emphasized that for the derivation of the relaxation times ([Fig. 5](#pone-0066773-g005){ref-type="fig"}) no assumptions about metabolic reactions or equilibration processes outside of the excitable medium had to be made. Thus, we believe that it would be worthwhile to scrutinize if it is indeed necessary to invoke metabolic reactions or other mechanisms to explain refractory periods in biological systems.
Finally, it is important to point out key questions that have been left untouched herein. *(i)* Measurements of the temperature-dependent compressibility of excitable media will allow to directly challenge the presented concept (such experiments are currently underway in our lab). *(ii)* While the temperature of the preparation (worm, nerve, etc.) is rather easily "clamped" in experiments, the other thermodynamic state variables of the cell or tissue are free to vary (*e.g.* lateral pressure of the cell membrane, pH close to the cell membrane, etc.). This has to be kept in mind when interpreting phenomena in the system. *(iii)* It has to be emphasized that the expression for linear sound (Eq. (1)) can only serve as a reasonable first order approximation. Indeed, we do not believe that the observed pulses are linear phenomena over the entire temperature range. The square root dependence between *c* and κ, however, is a fairly good approximation as longs as the variations of κ with density are rather moderate compared to κ [@pone.0066773-Hamilton1]. *(iiii)* Dispersion and collision phenomena, which are readily observed in nerves [@pone.0066773-Tasaki5], nervous tissue [@pone.0066773-FernandesDeLima1] and non-living excitable media [@pone.0066773-Wood1], cannot be described in the linear regime. The characteristic annihilation of pulses is certainly a main challenge since neither a linear sound nor a soliton model [@pone.0066773-Heimburg1] is capable of explaining it. However, the existence of a transition (state-change) during pulse propagation as proposed in the soliton model appears to open new doors to tackle this problem [@pone.0066773-Kaufmann1].
Conclusions {#s3f}
-----------
Experimental data on the effect of temperature on blood vessel contraction velocity and frequency in *Lumbriculus variegatus* were presented. These results were interpreted in the framework of a thermodynamic theory. Testable predictions about the temperature dependence of the excitable medium's mechanical material properties were made. Based on these predictions, temperature-dependent relaxation timescales were derived which correlate with experimentally obtained data from blackworms, nerves and gels.
When trying to find a physical explanation for phenomena of such remarkable generality, one has to carefully consider the assumptions made. In this spirit, we believe that the universal character of thermodynamics combined with as few specific assumptions as possible is the most promising approach to provide a robust and testable explanation.
Supporting Information {#s4}
======================
######
Einstein's approach to thermodynamics -- Derivation of Eq. (2).
(PDF)
######
Click here for additional data file.
We thank K. Kaufmann for reviving interest in the basic mechanisms underlying pulse propagation in biological systems. Moreover, we are thankful for his stimulating lectures and the ensuing discussions on the thermodynamics of soft interfaces.
[^1]: **Competing Interests:**The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: CF MFS. Performed the experiments: CF. Analyzed the data: CF MFS. Contributed reagents/materials/analysis tools: CF MFS. Wrote the paper: CF MFS.
|
{
"pile_set_name": "PubMed Central"
}
|
Introduction {#s1}
============
Redox signalling is an essential component of cellular energy homeostasis and responses to the environment in animals and plants, redox-sensitive proteins functioning as sensors that trigger repair mechanisms and regulate cell division, growth and defence processes. Despite a growing acceptance in the animal and plant literature that ROS accumulation and programmed cell death are not the enemy but rather hallmarks of survival \[[@BCJ-2016-0814CC1],[@BCJ-2016-0814CC2]\], old paradigms die hard. The concept that ROS mediate their principal effects by causing indiscriminate irreversible inactivation of proteins and/or loss of function of other cellular components (i.e. damage) became strongly anchored within the literature with the advent of initiatives to confer general stress tolerance on plants by overexpression of antioxidative enzymes. This notion remains surprisingly persistent to this day, probably because it is extremely simple. According to the 'damage' paradigm, overproduction of ROS in conditions such as excess light availability induces a general loss of cellular functions through processes such as photoinhibition, lipid peroxidation and protein oxidation, the accumulation of damage leading ultimately to death. Not before time, this simple paradigm is finally being laid to rest. Evidence that is often cited in apparent support of the 'reduction good/oxidation bad' paradigm is that oxidation leads to loss of enzyme activity. However, such effects have often only been demonstrated *in vitro*, sometimes using oxidant concentrations that are not biologically relevant. Furthermore, the literature contains physiologically relevant counterexamples, such as oxidative activation of chloroplast glucose-6-phosphate dehydrogenase \[[@BCJ-2016-0814CC3],[@BCJ-2016-0814CC4]\] and protein kinase signalling cascades \[[@BCJ-2016-0814CC5]\]. Crucially, the biological relevance of oxidative changes must be understood within the context of cellular functions: loss of activity of a given protein may activate a function at the cellular level.
ROS at the heart of intracellular and cell-to-cell signalling {#s2}
=============================================================
ROS play numerous important roles in plant development and environmental responses. ROS functions in plants are tightly intertwined with signalling pathways through phytohormones. It has long been apparent from studies of plant responses to the gaseous pollutant ozone that ROS interact with stress hormones such as ethylene, salicylic acid and jasmonic acid \[[@BCJ-2016-0814CC6]\]. More recently, it has been established that ROS and related molecules such as thiols interact with auxins, gibberellins and cytokinins to control plant growth and development \[[@BCJ-2016-0814CC7]--[@BCJ-2016-0814CC9]\]. ROS-related redox processes probably intervene at multiple levels of signalling for any given phytohormone. For example, thiol-dependent steps are probably involved in both synthesis and signalling of salicylic acid \[[@BCJ-2016-0814CC10]\]. In this regard, ROS can act either as a life or a death signal, dependent on the molecular and cellular context in which ROS accumulate. The outcomes of such signalling depend on many parameters, principally the chemical nature of the ROS form produced (i.e. superoxide, hydrogen peroxide or singlet oxygen) and the nature of the interacting partner (protein thiol, metabolite, lipid or DNA molecule), as well as cell identity, but all types of oxidative modification (reversible or irreversible) can be viewed as part of the redox signalling matrix because they induce regulatory, repair or death responses. Together with cellular oxygen tension, ROS control many crucial aspects of animal and plant biology, not least cell proliferation, stem cell homeostasis and differentiation lineage commitment \[[@BCJ-2016-0814CC11]--[@BCJ-2016-0814CC14]\].
Publications continue to appear that are based on the premise that low doses of ROS play beneficial roles in signalling, while higher doses have detrimental effects. Cell death is often cited as an example of the latter, supposedly undesirable effects, even though this process is central to renewal, immunity and defence responses. In multicellular organisms, the elimination of certain cells is a beneficial process, whether it is in control of uncontained cell division in mammalian cells or in the hypersensitive response in the case of plants resisting pathogen attack. Even at the local (cellular) level, there is abundant evidence that cell death in plants does not only occur generally through damage that overwhelms the cell\'s defences, but rather through genetically programmed pathways, with signalled processes that are controlled by specific genes and that may involve the programmed withdrawal of antioxidative systems. The importance of specific 'executor' genes was first reported in plants accumulating singlet oxygen \[[@BCJ-2016-0814CC15]\]. Genetic control over ROS-induced cell death is equally apparent from studies of the effects of H~2~O~2~ in catalase-deficient plants, using both reverse and forward genetics \[[@BCJ-2016-0814CC16]--[@BCJ-2016-0814CC18]\].
ROS and photosynthesis {#s3}
======================
Chloroplasts were one of the very first sources of superoxide and H~2~O~2~ to be described in plants \[[@BCJ-2016-0814CC19],[@BCJ-2016-0814CC20]\]. Electron flow to oxygen rather than NADP^+^ relieves reductive pressure within the electron transport chain and balances ATP:NADPH ratios by allowing proton pumping without net reductant generation ([Figure 1](#BCJ-2016-0814CF1){ref-type="fig"}). Moreover, like ROS formed at other subcellular locations, thylakoid-generated ROS may play crucial roles as signal transducers ([Figure 1](#BCJ-2016-0814CF1){ref-type="fig"}). ROS generated during light capture and electron transport are situated at the interface between the environment and the molecular machinery of photosynthesis, thereby providing the cell with crucial information on current status \[[@BCJ-2016-0814CC21],[@BCJ-2016-0814CC22]\]. Figure 1.Matching supply and demand in photosynthesis.Light energy drives otherwise thermodynamically unfavourable electron transfer at PSI and PSII to enable the reduction in ferredoxin (*F*~d~) and NADP^+^ in the stroma. Electron transfer is accompanied by the release of protons into the intrathylakoid space during water-splitting at PSII and plastoquinol oxidation at the cytochrome b~6~f complex (CBF). The protons are used by the coupling factor to produce ATP which, together with the NADPH generated from electron transport, drives metabolism in the stroma. If the proton concentration inside the thylakoid reaches a certain value, non-photochemical quenching (NPQ) mechanisms are activated to enable energy dissipation as heat. Oxygen oils the wheels of the whole process: the continuous production of ROS at various sites in the electron transport chain serves numerous functions, including contributing to the proton gradient required for ATP generation, redox poising (adjustments of the ratios of reduced to oxidised forms of electron transfer components) and providing information on current status through signalling pathways.
As well as superoxide and H~2~O~2~ production by the photosynthetic electron transport chain, energy transfer within the photosystems leads to generation of singlet oxygen, a ROS that is formed by excitation energy transfer from triplet chlorophyll to O~2~ \[[@BCJ-2016-0814CC23],[@BCJ-2016-0814CC24]\]. The high reactivity of membrane proteins and lipids to singlet oxygen makes these prime targets for signalling from photosystem II (PSII) to the nucleus. While oxidative modification of PSII structural and repair proteins and lipids is unavoidable \[[@BCJ-2016-0814CC25],[@BCJ-2016-0814CC26]\], considerable uncertainty remains concerning the extent to which such processes impair PSII function within a physiological context.
Photoinhibition and regulation of PSII {#s4}
======================================
The management of light interception and energy conversion is one of the most fundamental concepts in understanding the regulation of photosynthesis. The chloroplast is faced with the problem of balancing light harvesting with the generation of ATP and NADPH, in appropriate ratios, at rates that match the demands of metabolism \[[@BCJ-2016-0814CC27]\]. The fast turnover of these pools in the light leaves little room for imbalances in rates of energy production and consumption, explaining the evolution of a plethora of stabilising mechanisms that come into play, as required, to ensure smooth running of the system over the wide range of irradiances that occur in the natural and field environments. Such mechanisms include pH-triggered non-photochemical chlorophyll fluorescence quenching (NPQ) at PSII and the direct transfer of energy and electrons to oxygen leading to the production of reactive oxygen species (ROS) such as singlet oxygen, superoxide and H~2~O~2~ ([Figure 1](#BCJ-2016-0814CF1){ref-type="fig"}), all of which play important roles in the regulation of photosynthesis \[[@BCJ-2016-0814CC27]\].
An extensive body of literature has accumulated over the last 30 years on phenomena that are included under the term 'photoinhibition'. Considerable confusion persists because this term includes both photodamage and down-regulation of PSII function. Even though increasing evidence suggests that photodamage is not generally the dominant component, photoinhibition tends still to be equated with 'damage'. For this reason, in the following critique, photoinhibition is used to denote this long-standing notion of 'photodamage'.
Excess sunlight saturates PSII, causing build-up of excess excitation energy in its antenna. This unused energy is potentially dangerous because it can lead to the inactivation of the reaction centres (RCIIs), resulting in a sustained decrease in the quantum efficiency of PSII and the subsequent electron transport rate, a phenomenon termed photoinhibition \[[@BCJ-2016-0814CC28]--[@BCJ-2016-0814CC30]\]. Indeed, the photosynthetic pigments of oxygen-evolving PSII should be potentially vulnerable to photoinhibition since the RCII possesses a very strong oxidative potential of ∼1.17 V that is required to oxidise water. Under conditions where electron donation to P680 is less efficient than its photo-oxidation, an increase in the P680^+^ lifetime will occur. This powerful oxidant may oxidise the nearest pigments and amino acids, causing their degradation and a subsequent degradation of the key RCII D1 protein \[[@BCJ-2016-0814CC29]\]. In other circumstances, when the acceptor side is less efficient, a radical pair will be formed. The recombination of this pair will lead to the formation of a P680 triplet state that was proposed to interact with atmospheric triplet oxygen, causing formation of highly reactive singlet oxygen, which in turn can lead to the degradation of the key RCII component, D1 protein \[[@BCJ-2016-0814CC30]--[@BCJ-2016-0814CC33]\]. Hence, initially photoinhibition was thought to lead to a decreased number of active RCIIs.
Mechanisms to deal with high light exposure are required to minimise the build-up of potentially photodamaging excess energy in PSII. An imbalance between ATP generation and utilisation, caused, for example, by a failure of metabolism to keep pace with the thylakoid reactions, will cause protons to rapidly accumulate in the intrathylakoid space ([Figure 1](#BCJ-2016-0814CF1){ref-type="fig"}). This leads to decreased PSII light-harvesting efficiency through a process called NPQ that protects RCII from the damage via prompt dissipation of excess energy as heat \[[@BCJ-2016-0814CC34]\]. Apart from being triggered by the proton gradient (ΔpH), NPQ is strongly enhanced by the xanthophyll cycle activity that leads to conversion of violaxanthin into zeaxanthin. This reaction is also dependent on the ΔpH, albeit on a somewhat slower timescale than onset of either ΔpH or, in many cases, the pH-dependent NPQ. Importantly, PSII quantum efficiency can be decreased by both photodamage to RCII and NPQ (see below). Until recently, the only criterion that was used to separate photodamage from the protective reduction in the PSII yield (otherwise called *down-regulation*) was the timescale of the recovery of these processes in the dark. Indeed, while the repair from the damage to RCIIs takes hours, the down-regulation via NPQ was believed to take only minutes to recover \[[@BCJ-2016-0814CC33]\]. The evidence for the former was taken from biochemical data (D1 protein repair) \[[@BCJ-2016-0814CC34]\], whereas the evidence for the latter was taken from the so-called pulse amplitude-modulated chlorophyll fluorescence analysis, the interpretation of which remains a source of controversy \[[@BCJ-2016-0814CC35]\].
Interpreting chlorophyll fluorescence {#s5}
=====================================
Chlorophyll fluorescence has been used for several decades for prompt and non-destructive assessment of PSII efficiency in a variety of photosynthetic organisms \[[@BCJ-2016-0814CC34],[@BCJ-2016-0814CC36]\]. A typical fluorescence-quenching experiment is depicted in [Figure 2](#BCJ-2016-0814CF2){ref-type="fig"}. The active (open) PSII RCIIs are efficient quenchers of antenna chlorophyll fluorescence (*F*~o~) excited by a very weak light (*measuring light*). Application of a saturating light pulse (10 000 µmol m^−2^ s^−1^) for 1 s closes all RCIIs, bringing the fluorescence to the *F*~m~ level. Now, the quantum efficiency of PSII can be expressed as Φ~PSII~ = (*F*~v~)/*F*~m~, where *F*~v~ = *F*~m~ − *F*~o~. Application of continuous illumination for 5 min causes a gradual decline not only in *F*~s~, a steady-state fluorescence level, but also in *F*~m~, which becomes *F*′~m~. The decline in *F*~m~ is called non-photochemical quenching (NPQ) and is often expressed as NPQ = (*F*~m~ − *F*′~m~)/*F*′~m~. NPQ decreases the yield of PSII under continuous illumination that can now be expressed as$$\Phi_{PSII} = {\frac{F_{v}^{\prime}}{F_{m}^{\prime}} = qP \times {\frac{(F_{v}/F_{m})}{\lbrack 1 + (1 - F_{v}/F_{m}) \times {NPQ}\rbrack},}}$$where *qP* is photochemical quenching \[*qP* = (*F*′~m~ − *F*~s~)/(*F*′~m~ − *F*′~o~)\]. Hence, the yield of PSII is a function of both NPQ and *qP*. In the dark, following moderate levels of illumination, *qP* = 1 and NPQ recovers gradually but not completely ([Figure 2](#BCJ-2016-0814CF2){ref-type="fig"}). The slowly reversible NPQ component is called *qI* \[equal to (*F*~m~ − *F*~m~″)/*F*~m~″\] and was first proposed to reflect the photodamage to RCII \[[@BCJ-2016-0814CC36],[@BCJ-2016-0814CC37]\] diminishing the PSII yield in the dark as can be seen from the formula (1). However, later it was discovered, mainly by the groups of Adams and Demming-Adams, that a large part of *qI* does not reflect the damage to RCIIs but relates to the synthesis of zeaxanthin, and promotion of the slowly reversible NPQ components \[[@BCJ-2016-0814CC38],[@BCJ-2016-0814CC39]\] at high light and low temperature conditions \[[@BCJ-2016-0814CC40]--[@BCJ-2016-0814CC43]\]. Hence, the PSII yield in the classical fluorescence measurements was established to be dependent on both the RCII photodamage and the sustained NPQ. To quantify the true effect of photodamage upon Φ~PSII,~ a new method has been developed \[[@BCJ-2016-0814CC44],[@BCJ-2016-0814CC45]\]. It uses a gradually increasing actinic light illumination and the periodic measurements of Φ~PSII~ \[(*F*′~m~ − *F*~s~)/*F*′~m~\] and compares them with the analytically derived Φ~PSII~ using the formula (1). The measured and calculated values of Φ~PSII~ match each other very well at somewhat low actinic light intensities, but gradually fall apart at somewhat higher light. This disparity can be easily corrected if the *qP* values in the dark were \<1. Indeed, these values have been obtained using the comparison between measured and calculated values of *F*′~o~ \[[@BCJ-2016-0814CC46]\]. Therefore, the *qP* in the dark (*qP*~d~) has been proposed as a true indicator of photoinhibition reflecting the percentage of the inactivated RCIIs \[[@BCJ-2016-0814CC46]\]. The use of this parameter demonstrated that a prolonged exposure to the saturating light intensities (1500--2000 µmol m^−2^ s^−1^) only caused inhibition to at most 25% of PSII RCIIs of established mature plants \[[@BCJ-2016-0814CC45],[@BCJ-2016-0814CC47]\]. Hence, photoinhibition may not be a common phenomenon in nature. If the D1 repair process is ongoing and recovery without damage is facilitated by the protective component of NPQ, the loss of the PSII yield due to photoinhibition will greatly decrease \[[@BCJ-2016-0814CC45],[@BCJ-2016-0814CC47]\]. Therefore, the sustained decline in *F*~v~/*F*~m~ should be interpreted as predominantly related to PSII down-regulation via sustained components of protective NPQ and not as damage resulting from photoinhibition, and the *qP*~d~ parameter should be used to quantify this change \[[@BCJ-2016-0814CC44],[@BCJ-2016-0814CC45],[@BCJ-2016-0814CC47],[@BCJ-2016-0814CC48]\]. Figure 2.A typical pulse amplitude-modulated chlorophyll fluorescence induction measurement.The modulated low intensity measuring light of ∼1 µmol m^−2^ s^−1^ is used to excite chlorophylls of the PSII antenna fluorescence (*F*~o~ level). In these conditions, fluorescence is highly quenched by working RCs (RCIIs). Application of a saturating light (10 000 µmol m^−2^ s^−1^) for 1 s causes the closure of all reaction centres for the measuring light so that they stop quenching of the antenna fluorescence and the level rises to *F*~m~. After ∼1 min, a continuous illumination is applied (actinic light) of an intensity of ∼800 µmol m^−2^ s^−1^. This causes gradual quenching of *F*~m~ to the *F*′~m~ level. This quenching is triggered by the proton gradient and called NPQ. After ∼5 min, the actinic light is turned off and NPQ begins to recover. The recovery that is not complete within 5 min of darkness is termed *qI*.
Conclusions and perspectives {#s6}
============================
The Manichean notion that sets evil ROS on one side and benevolent antioxidants on the other is impossible to defend. Different oxidants may antagonise each other, and antioxidants such as glutathione may play an integral part not only in controlling ROS but also in transmitting oxidative signals \[[@BCJ-2016-0814CC10],[@BCJ-2016-0814CC49]\]. Enzymes that play important antioxidative roles can also promote ROS production or ROS-dependent processes \[[@BCJ-2016-0814CC50],[@BCJ-2016-0814CC51]\]. While plant cell functions operate at rather negative redox potentials in the soluble phase \[[@BCJ-2016-0814CC21],[@BCJ-2016-0814CC22]\], the paradigm that oxidation is bad, while reduction is good, is too simplistic given the complexity of redox interactions \[[@BCJ-2016-0814CC52]\]. This is particularly true in photosynthesis, which is driven by large redox and energy gradients, and which is the major source of ROS in plant cells. ROS production, signalling and removal associated with photosynthesis provide flexibility and control in the management of high light stress. Grasping the implications of this paradigm shift is key to addressing global issues such as food security and the production of crops in a sustainable manner for a growing world population. This challenge has led to an upsurge of interest in improving photosynthesis through the manipulation of processes that alter the light use efficiency of photosynthesis \[[@BCJ-2016-0814CC48]\]. Current initiatives such as the introduction of characteristics of C~4~ photosynthesis into important C~3~ species such as rice and, more generally, improving the capture of light energy and its conversion into biomass \[[@BCJ-2016-0814CC45],[@BCJ-2016-0814CC53]\] might benefit from an enlightened appreciation of the beneficial roles of ROS as a central integrator of functions at the cellular and whole-plant level.
PSII
: photosystem II
RCIIs
: reaction centres
ROS
: reactive oxygen species.
Funding {#s8}
=======
A.V.R. thanks the Royal Society for a Wolfson Research Merit Award. C.H.F. thanks Biotechnology and Biological Sciences Research Council (U.K.) for financial support \[Grant BB/M009130/1\].
Competing Interests {#s9}
===================
The Authors declare that there are no competing interests associated with the manuscript.
|
{
"pile_set_name": "PubMed Central"
}
|
Introduction {#sec1-1}
============
Emergency medical technician (EMT) or ambulance technician are terms used in some countries to denote a healthcare provider of emergency medical services. Structured curricula in other disciplines, such as internal medicine, have been shown to improve resident knowledge base in that particular discipline.\[[@ref1][@ref2]\] We wanted to evaluate whether structured educational curricula in airway management can improve EMTs performance. The purpose of the current study was to investigate whether a certain set of skills acquired recently from one-training module, if re-employed and reinforced during the course of the next training module, could result in improvement of the competence of students in the former set of skills. We hypothesized that EMTs competence in airway management, gained after a 2-week airway training module, would improve further after immediate subsequent deployment of these airway skills in a 2-week cardiopulmonary resuscitation (CPR) training module in a sequential, structured educational curriculum. To test our hypothesis, 76 EMTs were assigned to a 2-week airway module with a structured curriculum followed by a 2-week CPR module and were tested for their airway management skills before and after the completion of the CPR module. The structured curriculum was developed following a review of the literature and common airway maneuvers and devices utilized by EMTs for basic level training were included. Since some patients receive cervical spinal immobilization, this was included in the curriculum. EMTs completed a postmodule questionnaire evaluating their perceptions of the degree to which goals for knowledge change were achieved and their preferences for the various teaching methods that were utilized.
Materials and Methods {#sec1-2}
=====================
The study protocol was reviewed and approved by the Institutional Review Board. Subjects included 76 EMTs of our Institution. Those EMTs eligible and those who agreed to participate in the study were enrolled after signing an informed consent. There were no EMTs who did not agree to participate in the study.
The EMTs underwent a 2-week basic airway training module, followed by preCPR module test and then went on to complete the CPR module where the skills learnt in the airway management module were employed. They were then were subjected to the postCPR module test. Both the airway and CPR training modules consisted of a series of lectures given by the faculty and senior residents of the Department of Anesthesiology as well as individual skill demonstration on the mannequins, under supervision by the faculty. A total of 36 h of training was incorporated into 2 weeks for each of the two modules. Both the preCPR and postCPR tests involved checking the skill levels in airway management of the EMTs. The tests consisted of identical patterns of a viva on the theoretical aspects of each domain, followed by a practical demonstration of skills by each EMT on a mannequin. A flow-chart showing the sequence of the training program for the 76 EMTs is shown in [Figure 1](#F1){ref-type="fig"}. Both the preCPR and the postCPR module tests consisted of five performance domains. The marks were entered onto a computer spreadsheet for data analysis. The five domains for airway assessment testing were: Positive pressure ventilation with a bag valve mask, oropharyngeal airway insertion, nasopharyngeal airway insertion, supplemental oxygen administration, and cervical spinal immobilization. The maximum score was 25 points, and a minimum score was 0. All domains were tested on the Laerdal Airway Management Trainer. The supplemental oxygen administration test consisted of five different oxygen administration devices.
{#F1}
The residents were not given any feedback on test performance to protect the confidentiality of the examination content.
The airway module consisted of a structured curriculum incorporated in 2 weeks training in both the theoretical aspects and practical demonstration of use of airway devices on the Airway Management Trainer. The CPR module consisted of theoretical aspects of basic life support and advanced cardiac life support, use of defibrillation and use of the automated external defibrillator, as well as actual performance of CPR on the SimMan (Laerdal). During this training, the airway skills picked up during the airway module were reinforced. Each session of airway/CPR lasted around 1 h, and each EMT was asked individually to demonstrate prowess on the mannequin.
A postrotation questionnaire was completed by the EMTs for curriculum evaluation. The questionnaire was a feedback of the entire training experience that the EMTs underwent. This survey was identified by examination number, not name. The residents were asked to rank on a scale of 1-5 (1 = did not address the purpose, 2 = achieved the purpose 30%, 3 = clearly achieved the purpose, 4 = exceeded the achievement of the purpose \>50%, 5 = significantly achieved the purpose consistently) the following topics and purposes of the curriculum:
Syllabus purpose: To add to your knowledge base in the evaluation and optimization of patients with airway compromisePositive pressure ventilation with a bag valve mask: To improve skills of bag valve mask ventilationOropharyngeal airway: To improve skills in inserting itNasopharyngeal airway: To consider the possibility of insertion and improve skills in its useSupplemental oxygen administration: To improve methods of oxygen administration with a variety of devicesImmobilizing the cervical spine: To make you consider the possibility of cervical spine injury in trauma patients.
The data was consolidated in a computerized table by an independent party and analyzed. The EMTs also made qualitative comments on each of the above points.
Statistical analysis {#sec2-1}
--------------------
For purposes of the study, a convenient sample of 76 EMTs who belonged to three successive batches of the EMT training program, coming for airway and CPR training modules were enrolled. Subsequent *posthoc* power analysis was done based on the assumption that a 30% increase in the mean scores of the postCPR module over the preCPR module, represents a significant improvement in skills. Assuming α = 0.05, such calculation indicated the power of the study to be 86.4%.
Statistical analysis was performed using SPSS Statistical Software Version 19 (IBM corp., Armonk, New York, USA). Results were considered significant at *P* \< 0.05. Repeated measures ANOVA analysis was used to test the difference between preCPR module and postCPR module mean test scores. Normality assumption was checked for the total scores and the scores in each domain.
For the postrotation questionnaire, a two-sided Wilcoxon rank sum test was used.
Results {#sec1-3}
=======
Performance {#sec2-2}
-----------
Emergency medical technicians' performance level in basic airway management increased after a sequential, structured curriculum in which the airway module of 2 weeks was followed by 2-week CPR module, except for bag valve mask ventilation. We compared the preCPR module test mean scores for the entire test and in each domain with the postCPR module test mean scores. The postCPR test mean total score and the postCPR test mean scores of oropharyngeal airway insertion, nasopharyngeal airway insertion, supplemental oxygen administration and cervical spinal immobilization were significantly more than those of the preCPR (but postairway module) test mean scores \[[Table 1](#T1){ref-type="table"}, *P* \< 0.05\]. The bag valve mask ventilation postCPR test mean scores, however, were not significantly higher than the preCPR test mean scores \[[Table 1](#T1){ref-type="table"}, *P* \> 0.05\].
######
Comparison of preCPR module test and postCPR module test mean scores\*
Curriculum evaluation {#sec2-3}
---------------------
Nasopharyngeal airway insertion was the intervention with the highest mean score of 4.3, that is, exceeded the achievement of the purpose \>50% \[[Table 2](#T2){ref-type="table"}\]. Nearly 60% of the responses were proportioned as the highest score of 5 for nasopharyngeal airway insertion \[[Table 2](#T2){ref-type="table"}\]. Furthermore, this component of the evaluation received the highest percentages of the residents' responses as a score of 5 \[[Table 2](#T2){ref-type="table"}\]. Supplemental oxygen administration received lower scores on the EMTs' evaluations. The residents' evaluation of a structured curriculum indicated that the syllabus was a good reference, but it contained too much material in the airway section to cover in just 2 weeks. Per the EMTs' comments, doing the CPR curriculum following the airway module, enhanced their confidence in the airway management of patients ("I feel more confident about the types of airway management decisions after doing both the airway and CPR modules than after doing only the airway module").
######
Postmodule questionnaire scoring response summaries of syllabus, bag valve mask ventilation, oropharyngeal airway, nasopharyngeal airway, oxygen administration, and cervical spine immobilization\*
Discussion {#sec1-4}
==========
This study shows that a sequential, structured educational curriculum in CPR training, immediately following an airway module, increased EMTs' performance in airway management except for the bag valve mask ventilation domain. Furthermore, the EMTs' evaluation of this curriculum indicated that it was useful to have the CPR module immediately following the airway module to improve their performance, and that enhanced their confidence in their airway management skills. The EMTs' performed best on the nasopharyngeal airway insertion domain.
The EMTs' performance in the bag valve mask domain did not improve even after the CPR module. Factors that can explain this result include the more difficult nature of this domain as compared to others, learning style, the content of the educational experience, the frequency of the instruction and the EMTs' effort.
The EMTs' evaluation of the curriculum indicated that the syllabus was a good reference, but it contained too much material to cover in the airway module within just 2 weeks. Potentially, the syllabus could be given to the resident several weeks prior to the airway training module, or the content of the syllabus could be stream-lined, an approach we need to take.
We believe that the beneficial effects of a structured educational curriculum are found not only in the specific content of the curriculum but also in the way the curriculum is structured to adapt to the diverse learning styles of the residents. It has been shown that individuals have different learning styles. Some prefer to learn by hearing the information and some prefer to visualize the information. Using different teaching methods helps overcome individual preferences for learning, helps maintain the students' interest, and helps reinforce the information.\[[@ref3]\] This reinforcement of learning can enhance the depth of learning, promote the retention of information, and enhance the ability to apply what has been learned.\[[@ref3]\] Our sequential, structured curriculum included a hands-on airway management module, which was followed by a CPR module, where the skills learnt during the airway module were well-utilized leading to better performances in four out of the five domains that we tested for.
The training on the use of the nasopharyngeal airway was well received by the EMTs; however, the domain related to administration of supplemental oxygen received lower scores on the EMTs' evaluations secondary to the problems of adequately explaining the intricacies of oxygen delivery methods within a very limited time frame. Further thought should go into streamlining the curriculum in the future.
The EMTs expressed that doing the CPR module immediately after going through the airway module contributed significantly to improving their airway management skills. The postCPR module test, mean scores of only 14.3 can be significantly improved upon. Furthermore, the interest of available clinical anesthesiologists and staff in EMT training modules may not be present, unless full-time personnel are spared for these training programs. In the United States, to maintain the National Registry of EMTs certification, basic level EMTs must obtain at least 48 h of additional education and either complete a 24 h refresher course or complete an additional 24 h of Continuing Education that would cover, on an hour by hour basis, the same topics as the refresher course would. At our Institute, EMT training for airway and CPR is provided by clinical Anesthesiologists, who take time off from their clinical duties to run the module.
Conclusions {#sec1-5}
===========
In summary, this study shows that a sequential, structured educational curriculum in airway management followed immediately by a CPR training module, enhanced EMTs' airway skills, except in bag valve mask ventilation. This improvement in airway management skills can contribute to the optimization of a sick patient\'s medical status prior to hospital arrival and ultimately lead to achieving all of the benefits that derive from a robust trauma care system, such as prompt response to emergency calls, performance of certain out-of-hospital medical procedures and safe transport of patients to hospital in accordance with protocols and guidelines established by physician medical directors.
We feel that this kind of structured educational curriculum in EMT training is useful and should be adaptable for other programs. One of the limitations of our study is that there will be a need to use validated outcome and assessment measures in the future. Another limitation is the small number of participants, which limits the ability to generalize results. However, because of the great impact of airway management on the optimization of a patient\'s medical status and the subsequent impact on patient care, we feel that EMT training programs can benefit from such structured educational curriculum in the Emergency Medical Department.
**Source of Support:** Nil
**Conflict of Interest:** None declared.
|
{
"pile_set_name": "PubMed Central"
}
|
Introduction
============
As a result of the needs of a globalized society, immersed in Information and Communication Technologies (ICT) and going through a continuous process of scientific and technological modernization, teaching in health and nursing has undergone transformations, adapting competencies, critical thinking and decision making skills[@B1] ^-^ [@B3].
To satisfy these needs, the professional education underwent restructuring, which has slowly provoked the evolution of knowledge and complex thinking, aiming to prepare more critical and reflexive professionals, capable of acting in a wide range of situations. In that context, the teaching institutions have reconsidered the educational practices and employed innovative strategies, with a view to stimulating competent professionals, which has highlighted the use of clinical simulation as a necessary and valued tool in the teaching-learning process[@B1] ^-^ [@B4].
The act of teaching through clinical simulation has frequently been part of the undergraduate curriculum, and also of health professionals' training. Nevertheless, as a result of the advances in the structuring of the strategy and the increased capacity to gain competencies, critical reasoning, decision making and teamwork and to strengthen the professionals' self-confidence, it has been increasingly valued and enhanced as a teaching strategy[@B5] ^-^ [@B8].
In simulated clinical practice, several resources can be used, ranging from dramatization to the use of inanimate anatomic pieces and/or advanced simulators, which incorporate high computer and robotic technology and lead to many interaction possibilities, with great variation in the costs involved. In the construction of the simulated scenarios, physical and material resources are employed that approach the actual activities of clinical practice involving patients with a high degree of realism. The resources are defined according to the learning objectives and are classified according to their technological potentials[@B6] ^,^ [@B9].
Among the resources applied in this study, the dramatization technique will be highlighted. Dramatization can be defined as a theatre representation, determined based on a focus or theme. This resource grants meanings and permits the contents taught to be experienced in a context similar to those experienced in the actual practice[@B10]. Dramatization allows the student to integrate theory and practice, it is flexible and adjustable to different contexts, permits experiencing different perspectives and viewpoints and offers the student the opportunity to explore the individual vulnerability in a safe environment[@B11].
In dramatization, the techniques explored can be role play and the use of simulated patients, mixed models and standardized patients.
Role play is the situation in which the learner, facilitator and/or instructor play different roles in the simulated scenario as if they were taking part in a clinical case, for the purpose of teaching and training[@B10]. This strategy grants learning opportunities, involving both the student's affective and cognitive process, as they permit experiencing feelings, such as the experience of the patient's and other professionals' roles[@B12].
Educators and clinical simulation researchers frequently use the expressions "simulated patient" and "standardized patient" interchangeably or as synonyms in the literature, although differences exist between them. Simulated patients are trained individuals and/or actors who play a role, exhibiting a story within the simulation for the purpose of teaching or assessment[@B13].
The term standardized patient can be defined as: a member of the community (child, adolescent, adult, elderly) who agreed to play the role of a patient for a learning activity, through a legal contract with the teaching institution. The standardized patients do not play a role to perform the characteristics of another person or patient, but they answer any inquiry about the medical and social history based on their own lives[@B13]. This resource has served as a concrete possibility to provide clinical skills teaching and training, in function of its potential to comply with conditions closer to the ideal, guaranteeing the reliability of human interaction with communication and empathy[@B10]. For ethical and legal reasons, this technique has not been much used in Brazil[@B14].
The mixed models enable the learner to develop technical and behavioral skills. They combine the simulated patient with a low-fidelity simulator to develop a specific activity in a scenario, such as an arm coupled to a student in a blood collection scenario for example[@B14] ^-^ [@B15].
Due to its reasonable cost and great application possibility, the use of simulated practices with dramatization resources can turn into an excellent ally for the qualification of professionals with critical and reflexive thinking, who are capable of reaching clinical judgments and making decisions. Nevertheless, to better use the technique, its use should be based on scientific evidences that demonstrate the positive or negative results of this teaching and learning strategy.
In that context, to better understand and employ the available resources related to the theme, the objective in this study was to identify, in the literature, the gains the health students and professionals perceived in the use of clinical simulation with dramatization resources.
Method
======
An integrative review was undertaken, using the method of the Joanna Briggs Institute (JBI), which is focused on the feasibility, adequacy, significance and efficacy of the health interventions. This method can be used to map the main concepts that sustain a research area, as well as to clarify the operational definitions and/or conceptual limits of a topic[@B16].
To construct the research question, the PICO strategy was used in the quantitative articles: P - Students and professionals; I - Clinical simulation using dramatization; O - Perceived gains from clinical simulation using dramatization; and PICo in the qualitative articles PICo: P - Students and professionals; I - Clinical simulation and dramatization; Co - Perceived gains from clinical simulation using dramatization[@B17].
This strategy permitted formulating the following guiding question: *What are the gains the health students and professionals perceive from the use of clinical simulation with dramatization resources?*
Thus, after establishing the question, an initial search was undertaken in the portal PubMed (Public Medline) and in the database CINAHL (Cumulative Index to Nursing and Allied Health Literature), in order to identify the main descriptors and key words used in the studies that discussed the theme of interest in this review.
To answer the research question, the controlled and non-controlled descriptors were selected, related to each of the components of the PICO and PICo strategy, used according to the Health Sciences Descriptors (DEsCS) and Medical Subject Headings (MeSH).
The research was developed between June and December 2015 without any restrictions in terms of time, presentation or publication type, using the following controlled descriptors: Students; Role Playing; Patient Simulation; Education; Perception; and the non-controlled descriptors: Professional; Patients Standardized; Standardized Patient; Dramatization; Clinical Simulation; Experience. In between the descriptors, the following Boolean operators were considered: *Students* AND *Professional* AND *Role Playing* OR *Patient Simulation* OR *Patients Standardized* OR *Standardized Patient* OR *Dramatization* OR *Clinical Simulation* AND *Education* OR *Perception* OR *Experience.*
Inclusion and exclusion criteria were established for the research, considering a number of study types: 1) studies involving health students and professionals; 2) studies that discussed the theme simulation with dramatization, that is, role play, standardized patients, patient simulation, mixed patient; 3) studies with a quantitative and/or qualitative focus, which answered the question established, independently of the knowledge area they were linked to and 4) studies published in Portuguese, English and Spanish. Publications of opinions, consensus statements, retractions, editorials and experience reports were excluded.
To identify the studies, the following electronic databases were used: Latin American and Caribbean Health Sciences Literature (LILACS), Web of Science, National Library of Medicine (PubMed), Cumulative Index to Nursing and Allied Health Literature (CINAHL), The Cochrane Library, Scopus, Scientific Electronic Library Online (SciELO).
In total, 6,826 studies were found, which were moved to Web ENDNOTE. Of these, 1,414 were excluded because the studies had been published in more than one database, resulting in 5,412 studies. After reading the titles and abstracts of the 5,412 research articles, 5,103 were excluded because they did not answer the research question and 309 were selected to read the full article. Among the 309 studies analyzed, 53 were included in the research because they answered the question and because they complied with the inclusion criteria established.
Next, the research data were analyzed with the help of a tool the researchers had constructed, in accordance with the JBI instructions[@B16], including: study title, authorship, journal, year of publication, place of study (country), research objective(s), methodological details, sample details, main outcomes and conclusions found. In the critical analysis of the selected articles, the research design was analyzed[@B18].
Results
=======
Among the 53 (100%) studies in the sample, the majority had been published in English. The studies had been mostly developed on the American (n=27, 50.94%), Asian (n=9, 17.0%), Oceania (n=9, 17.0%) and European continents (n=8, 15.1%).
When the type of dramatization the studies employed was analyzed, it was verified that 28 (52.9%) used a simulated patient; 18 (34.0%) role play; 4 (7.5%) dramatization with standardized patient; 2 (3.7%) simulated patient plus role play and 1 (1.9%) mixed patient (simulated patient plus pelvis).
As demonstrated in [Figure 1](#f1){ref-type="fig"}, as regards the method used, among the studies analyzed, 23 were descriptive (43.4%), 13 experimental (24.5%), 8 quasi-experimental (15.1%), 4 qualitative (7.5%), 2 mixed (3.8%), 1 cohort (1.9%), 1 multiple case study (1.9%) and 1 (1.9%) meta-analysis. The year of publication, type and number of participants have been described in [Figure 1](#f1){ref-type="fig"}.
Figure 1Method, year of publication, type and number of participants, 2016
Discussion
==========
Simulation has turned into a fundamental tool for the education and recycling of health professionals. It permits modeling clinical events in a safe environment, resulting in learning gains due to the possibility for the student to develop competencies, critical reasoning, decision making, teamwork and, mainly, to contribute to the strengthening of self-confidence[@B5] ^-^ [@B8].
Simulation with dramatization resources has been used as a teaching strategy through clinical simulation, in the education of future professionals as well as in the training of active ones. When applied as such, it is able to offer the students the possibility to train skills and even competencies at a reasonable cost, in a safe environment, through the creation of scenarios with a wide range of complexities. In addition, it realistically reproduces an encounter with the (simulated) patient, which can strongly contribute to the learning objectives outlined[@B71]. It also offers the possibility of feedback by the simulated patient, which contributes and enriches the teaching-learning process[@B55].
This study was aimed at identifying the gains health students and professionals perceived in clinical simulation using dramatization resources. Although the grey literature was not included, which can be considered a limiting factor, a large number of studies could be identified, observing that simulation with dramatization resources has been used expressive and effectively in the teaching and training process of health professionals in a wide range of scientific areas, also aiming to develop interprofessional competencies ([Figure 1](#f1){ref-type="fig"} and [Figure 2](#f2){ref-type="fig"}).
Figure 2Perceived gains by students and professionals using dramatization resources and frequency, 2016\*More than one perceived gain per article.
The dramatization strategy used needs to support the learning objectives of the activity. Different dramatization strategies were employed in the studies assessed; among these, the use of the simulated patient and role play stood out.
The simulated patient participates actively in the activity and, in the debriefing process, permits interactivity in the learner's reflection. In addition, the patient needs to be engaged in the assessment of the activity. The use of the role play strategy allows the learner to empathetically experience the role of the patient, relative and/or of another professional, in an active, involving and dynamic manner, supporting the construction process of clinical competencies and effective communication. Clinical competence is a fundamental quality for professionals who are apt and capable of delivering high-quality care. The use of simulation can be considered an admirable tool for the students to find their action sphere, autonomy, adaptation and flexibility in the course of their development, in different realities[@B72].
Among the gains identified in the studies analyzed, the enhancement of knowledge, development of empathy, of communication skills, satisfaction with the teaching-learning process, self-confidence, realism, reduction of the anxiety level, comfort, motivation to learn, capacity to reflect and think critically and teamwork skills were observed.
Communication was the gain that stood out in the studies analyzed. Health educators have been increasingly concerned with the inclusion of teaching-learning strategies for the development of communication skills, as effective communication is an essential clinical competency for the practice of health professions. It can be taught and qualified effectively by means of dramatization in simulated practices[@B20] ^-^ [@B21] ^,^ [@B25] ^-^ [@B27] ^,^ [@B29] ^,^ [@B31]. In the selected sample, the following dramatization strategies were widely used to develop communication: role play and simulated patient, mainly in situations that were difficult for the professionals to cope with, such as ethical dilemmas, communication of bad news, conflicts in the interprofessional team, among others[@B22] ^,^ [@B25] ^,^ [@B27] ^,^ [@B31] ^,^ [@B65] ^-^ [@B66].
Satisfaction with the clinical simulation method has been increasingly valued at health institutions and is related to the motivation process for learning[@B30]. It is an indicator of best practices in the teaching-learning process and of good work conditions for the educators. It can be influenced by the desire and experience of the teaching staff. In the studies analyzed, the use of simulated patients and the realism of the strategy were the main indicators of this perceived gain[@B45] ^,^ [@B73].
The realism benefits the activity and makes it successful, as it makes the participants consider the strategy as legitimate and authentic[@B32] ^,^ [@B38] ^,^ [@B58] ^,^ [@B63] ^,^ [@B65] ^,^ [@B74]. During the simulation, the realism can be translated by the fidelity of the simulated experience in approaching the actual environment. High-fidelity simulation approaches the practice with patients as closely as possible[@B75]. In the sample, the studies analyzed demonstrated that the learners perceived the use of the simulated patient as very close to the real patients. In addition, the following also contributed to the realism: the extent to which the environment approaches the facilities in practice, as well as the educators' knowledge and preparation to trigger the emotions[@B19] ^,^ [@B58] ^,^ [@B63] ^,^ [@B65] ^,^ [@B76]. An environment close to the reality provokes the same psychological reactions in the individuals as they would have in practice, which makes the learners develop critical thinking and the decision-making skills required in an actual clinical scenario[@B5] ^,^ [@B77] ^-^ [@B78].
What the teaching-learning process, knowledge and critical thinking are concerned, simulation with dramatization showed to be an innovative and diversified teaching-learning tool, which promotes the students' opportunities to reflect on the practice[@B37], strengthen the background knowledge[@B22] ^,^ [@B35] ^,^ [@B41] ^,^ [@B50] ^,^ [@B54] ^,^ [@B56], understand the strong and weak points of their learning[@B60], develop critical thinking[@B37] ^,^ [@B62] and the opportunity to use previously acquired knowledge and skills[@B62] and, therefore, enhances the awareness on the students' actual capacities. In the studies observed, role play showed to be an interesting tool in the teaching-learning process[@B25], in view of the learners' level of acceptance[@B32], as it makes the theoretical and practical knowledge significant, integrates and transforms it at the individual and collective levels[@B21] ^,^ [@B28]. It is also important to highlight that the simulated practices permit measuring and assessing the results obtained through instruments and/or video recordings for future clarifications[@B58].
The studies also demonstrated that the simulations made the learners more trusting, minimizing the fear to undertake the procedures with the patients[@B20] ^,^ [@B26] ^,^ [@B30] ~**,**~ mainly in the physical examination and communication processes[@B33] ^,^ [@B36] ^,^ [@B41] ^,^ [@B49] ^,^ [@B54]. Self-confidence also leads to the reduction of the anxiety level[@B26] ^,^ [@B44] ^,^ [@B47] ^,^ [@B63] and increased comfort[@B44] ^,^ [@B47].
Anxiety is a natural reaction, produced in response to certain situations in which the person needs adaptive resources. When confronted with critical activities for which they do not feel prepared, the learners report anxiety, tension, mainly when the care targets children and patients in severe and/or terminal conditions[@B79]. The stress and anxiety can negatively contribute and interfere in the teaching-learning process. The two main sources of anxiety in clinical practice are lack of knowledge and lack of skills[@B79].
In the gains the learners perceived, the development of empathy could be observed, which involves the feeling of sensitization for the changes the other person feels and reflects moment by moment[@B80]. Empathy was a gain perceived in some studies analyzed[@B29] ^,^ [@B59] ^,^ [@B66] ^-^ [@B67] ^,^ [@B69] and measured during the role play strategy[@B69].
It is important to highlight that, in technical competency development, the dramatization comes with some limitations, as not all procedures can be executed on the simulated patients. To solve that difficulty, sometimes, mixed patients are used, like when a pelvis is attached to the simulated patient during urinary catheterization. In the sample of this review, it could be identified that simulation with dramatization was used in anamnesis [@B46], physical examination[@B19] ^,^ [@B38], pelvic examination[@B23] ^,^ [@B33] ^,^ [@B47] ^,^ [@B49] and postoperative pain assessment skills[@B36]. It was also observed that dramatization was used to develop critical thinking in punctual[@B30] ^,^ [@B35] ^,^ [@B62] ^,^ [@B68] ^)^ studies, perhaps due to the fact that the physiological outcomes cannot be controlled in simulated patients.
Conclusion
==========
The large number of studies found in this research demonstrates that simulation with dramatization is a tool in the teaching-learning process, largely used in the education and qualification of health professionals.
In this process, in a wide range of health areas and also involving different professionals, different gains are obtained, among which satisfaction, self-confidence, knowledge, empathy, realism, reduced anxiety, comfort, communication, motivation, capacity to reflect and think critically and teamwork stand out. The evidences demonstrate the great possibility to use dramatization in the clinical simulation context.
|
{
"pile_set_name": "PubMed Central"
}
|
1. Introduction {#sec1}
===============
Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is an autoimmune disease that is characterized by the presence of neuropsychiatric symptoms. The standard for diagnosis is the detection of anti-NMDAR GluN1 subunit Immunoglobulin G (IgG) antibodies in the cerebrospinal fluid (CSF) \[[@B1]\]. As reviewed in \[[@B1]\], anti-NMDAR IgG antibodies present in the CSF bind to the extracellular N-terminal domain of the NMDAR NR1 subunit, embedded within postsynaptic membranes of central nervous system (CNS) neurons. This leads to the cross-linking and internalization of NMDARs, which reduces the number of NMDARs and NMDAR clusters on the surface of neurons, leading to dysfunction and disease. Anti-NMDAR encephalitis is most commonly diagnosed in children and young people and is often associated with tumors, such as ovarian teratomas, which more frequently occur in women over 18 years of age. In addition, viral infections such as herpes simplex virus can also induce anti-NMDAR encephalitis. Typical clinical manifestations include psychiatric and behavioral abnormalities, seizures, recent memory impairments, involuntary movements, speech disorders, conscious disturbances, and autonomic dysfunction. Currently, symptomatic treatment and immunotherapy (intravenous methylprednisolone (IVMP), intravenous immunoglobulin (IVIG), or plasma exchange (PE), etc.) are available for the treatment of anti-NMDAR encephalitis.
The disease was formally proposed by Dalmau and others in 2008 \[[@B1]\]. Its incidence and overall prognosis are currently unknown, although a Dutch study estimated the incidence to be 2-3 cases per 1 million people \[[@B2]\]. As clinicians gradually understand the disease, an increasing number of patients are diagnosed \[[@B3]\]. The British California Encephalitis Study showed that the incidence of anti-NMDAR encephalitis has exceeded that of any single virus-induced encephalitis. It further showed that anti-NMDAR encephalitis is the most common antineuronal antibody-mediated encephalitis \[[@B3]\]. In the current largest anti-NMDAR encephalitis cohort study published in 2013 \[[@B4]\], it was shown that after 24 months of follow-up, 78% of patients achieved a good prognosis, 13% of patients relapsed, and 5% of patients died. Since then, no other similar large-scale study on anti-NMDAR encephalitis has been conducted. However, in 2017, de Montmollin \[[@B5]\] and others analyzed 77 anti-NMDAR encephalitis patients admitted to the ICU and showed that 77% had a positive prognosis after 2 years of follow-up, while 4% died. Many factors can affect a patient\'s prognosis. However, both primary research and relevant reviews focusing on the prediction of anti-NMDAR encephalitis are scarce. In this review, we summarize the current progress on understanding and predicting the prognosis for anti-NMDAR encephalitis given its pathogenesis, clinical manifestations, auxiliary inspection, and treatments.
2. Pathogenesis {#sec2}
===============
Autoimmune encephalitis (AE) is an inflammatory disease of the CNS which is caused by an abnormal immune response against the body\'s own neuronal components. It can be divided into paraneoplastic AE and nonparaneoplastic AE, based on whether a tumor is present or not. Paraneoplastic AE can further be divided into intracellular antigen-antibody-associated encephalitis and cell surface antigen-antibody-associated encephalitis based on the location of the specific autoimmunogenic antigens. Intracellular antigen antibody encephalitis includes anti-Hu, anti-Ma2, and anti-GAD antibody encephalitis. Cell surface antigen antibody encephalitides are more common \[[@B6]\] and include anti-NMDAR, anti-leucine-rich glioma inactivating protein 1 (LGI1) antibody-related, and anti-*γ*-aminobutyric acid type B receptor (GABABR) antibody-related brain encephalitis, with the first having the highest incidence.
Anti-NMDAR encephalitis is an autoimmune neuroinflammatory disease mediated by antibodies against the NMDAR GluN1 subunit. In an endogenous rodent model of anti-NMDAR, Linnoila et al. showed that 67% of mice developed serum anti-NMDAR antibodies after infection with herpes simplex virus 1 \[[@B7]\]. In addition to viral infections, tumors and other unknown causes could also lead to autoimmune response against exposed neuronal antigens. In the case of anti-NMDAR encephalitis, exposed GluN1 subunit antigens become the target of autoantibodies against NMDAR \[[@B8]\]. In this process, the immune system first produces memory B cells, which could subsequently be recruited to the brain through the activity of various cytokines, chemokines, and inflammatory mediators. In the brain, memory B cells can be stimulated by antigens and can mature, proliferate, and differentiate into plasma cells, in order to produce antibodies \[[@B8]--[@B11]\].
The NMDAR is widely present in the CNS, especially in the frontal cortex, hippocampus, and thalamus. It participates in synaptic transmission and signal transmission between neurons and is related to advanced neuronal functions, including memory, cognition, and behavior. Neurons and animals exposed to patient anti-NMDAR antibodies *in vitro* and *in vivo*, respectively, confirmed that the specific binding of CSF anti-NMDAR antibodies to their cognate receptor does not lead to the death of nerve cells, but rather to the reversible reduction and functional decline of NMDAR \[[@B12], [@B13]\]. A series of abnormal psychiatric and behavioral movement disorders and other clinical symptoms became evident after the antibody-mediated reduction of NMDAR. Unlike T-cell-mediated anti-Hu antibody encephalitis and other types of intracellular neuronal encephalitis, neuronal death is not observed in anti-NMDAR encephalitis \[[@B14]\]. As such, anti-NMDAR encephalitis has a relatively good prognosis after treatment \[[@B1], [@B15], [@B16]\].
3. Clinical Manifestations {#sec3}
==========================
3.1. Psychiatric Symptoms {#sec3.1}
-------------------------
Psychiatric symptoms are the earliest, most common, and in some cases, the only clinical manifestations of anti-NMDAR encephalitis \[[@B17], [@B18]\]. They are more common in young and female patients \[[@B19], [@B20]\]. Common psychiatric symptoms include visual and auditory hallucinations, schizophrenia-like symptoms, anxiety and depression, mania, and abnormal behavior. Because the identification of psychiatric symptoms is difficult, the median admission time for patients presenting mainly with psychiatric symptoms is significantly longer than that of those with neurological dysfunctions, such as epilepsy (14 vs. 2 days, respectively), where neurological dysfunctions are the first manifestations \[[@B21]\]. Consequently, patients presenting mainly with psychiatric symptoms experience a significant delay in the administration of immunotherapy. In turn, this may affect their recovery, especially in terms of psychiatric symptoms and cognitive impairment. Previous studies have mostly focused on positive symptoms, such as delusions and mania. However, Gibson and others have shown that patients with anti-NMDAR encephalitis have obvious negative symptoms, cognitive impairments, and psychiatric disorders that do not match the severity of the symptoms \[[@B19]\]. The clinical manifestations of the negative symptoms can also be explained by the mechanism of antibody-mediated NMDAR downregulation. Indeed, in anti-NMDAR encephalitis, animal models which mimic the clinical course of the disease are characterized by memory loss and lack of pleasure \[[@B12]\]. Cainelli et al. conducted a long-term follow-up study of children diagnosed with anti-NMDAR encephalitis and found that about half of them had long-term attention disorders and executive dysfunction, while some of them had long-term mental behavior abnormalities \[[@B22]\]. Positive symptoms can be almost completely ameliorated after active treatment. By contrast, negative symptoms, including cognitive and memory impairments and thinking disorders, can persist in patients over the long-term and even be permanent, leading to a poor prognosis \[[@B23], [@B24]\]. In children, since the nervous system is not fully developed at the time of onset, the impact of the disease may be more pronounced \[[@B23]\]. Ameliorating cognitive impairments is of great significance for patients\' long-term rehabilitation and for improving their quality of life. However, research on cognitive impairment is still limited, and further large-scale and prospective studies are needed.
3.2. Seizure {#sec3.2}
------------
Seizures are also a core clinical manifestation of anti-NMDAR encephalitis, occurring in about 60-70% of patients \[[@B4], [@B25], [@B26]\] and being more common in children and young patients. Before puberty, epilepsy is more common in females than in males. After puberty, the situation is reversed, possibly due to changes in sex hormone levels \[[@B4], [@B25], [@B27], [@B28]\]. Seizure symptoms are diverse and can include typical generalized tonic-clonic seizures, focal seizures (with or without loss of consciousness), a persistent state of epilepsy, and a persistent state of refractory epilepsy. Some patients have 2 or more types of seizures \[[@B20], [@B26]\]. Epilepsy in patients with anti-NMDAR encephalitis is mediated by an autoimmune mechanism, and most patients have significantly reduced seizures after their encephalitis has been treated. For example, de Bruijn et al. assessed whether seizures could be ameliorated in patients with anti-NMDAR encephalitis who experienced seizures throughout the courses of their disease \[[@B29]\]. Most patients saw an improvement as a result of immunotherapy alone. For some, the addition of antiepileptic drugs after the immunotherapy was necessary to eliminate seizures \[[@B29]\]. In another study where epilepsy occurred in 81% of 109 patients, a single-agent antiepileptic response eliminated seizures for up to 2-weeks after the administrations. Moreover, all patients had no apparent seizures after 2 years of follow-up \[[@B28]\]. de Bruijn et al. suggested that epilepsy is a clinical symptom of anti-NMDAR encephalitis in a particular stage and can be gradually eliminated with the use of immunotherapy combined with the use of antiepileptic drugs \[[@B29]\]. This view is in line with the results of Taraschenko et al. \[[@B14]\]. Their group has recently shown a significant increase in seizures in mice exposed to anti-NMDAR antibodies, hinting that autoantibodies *per se* have the potential to induce seizures. Therefore, some believe that epilepsy or epilepsy status does not affect the prognosis of patients \[[@B30]\]. Although patients with anti-NMDAR encephalitis have epileptic symptoms, they should not be immediately diagnosed with epilepsy. However, if they are diagnosed with epilepsy or need to take antiepileptic drugs for prolonged periods, patients should be followed up for at least 1 year. It is further recommended that antiepileptic treatments are phased out during the recovery period \[[@B26], [@B28]\].
3.3. Movement Disorders {#sec3.3}
-----------------------
Abnormal movements are another common manifestation of anti-NMDAR encephalitis and mainly present as facial dyskinesia or involuntary movements of the limbs. In addition, patients can present with complex clinical manifestations, such as myotonia, dystonia, bradykinesia, and eyelid spasms. About 75% of adults and 95% of children develop movement disorders \[[@B1], [@B4], [@B31]\]. Some scholars believe that abnormal psychiatric and behavior symptoms and seizures are early clinical manifestations after the onset of anti-NMDAR encephalitis, which may last, in average, 10 to 20 days \[[@B26], [@B32]\]. The presence of movement disorders indicates that the disease has progressed to a more advanced stage. To explain this theory, it is postulated that early in the disease, serum antibodies act on the gray matter of the cerebral cortex to produce a series of clinical symptoms. As the disease progresses, intrathecally synthesized antibodies are produced and are able to act on the subcortical structure to produce additional clinical symptoms. Studies have shown the interval between different stages to be about 18.6 days \[[@B32], [@B33]\]. As a result of the facial dyskinesia, some patients may suffer from secondary local organ damage, such as to the oral cavity and tongue. Symptomatic treatment can improve the symptoms of patients. Nevertheless, the focus of treatment should still be immunotherapy. After active treatment, the symptoms are mostly ameliorated and there are no obvious sequelae. No studies have confirmed the association between movement disorders and poor prognosis.
3.4. Autonomic Dysfunction {#sec3.4}
--------------------------
Common autonomic dysfunctions caused by anti-NMDAR encephalitis include tachycardia, bradycardia, arrhythmia, cardiac arrest, diarrhea, central hypoventilation, and excessive ventilation \[[@B34], [@B35]\]. Autonomic dysfunctions can directly affect the patient\'s prognosis and long-term recovery. Symptoms such as arrhythmia, cardiac arrest, and central hypoventilation may be the main reasons for ICU support or death. Some patients with cardiac arrest may need a pacemaker. Heartbeat activity is regulated by cardiac sympathetic and parasympathetic nerves. Animal studies have shown that multiple regions of the medial cerebrum including the island lobe, cingulate gyrus, and amygdala are involved in the regulation of cardiac sympathetic and parasympathetic nerves. Consequently, intracranial lesions may affect the neuromodulation of the heart, leading to arrhythmia and cardiac arrest \[[@B35]\]. Simultaneously, anti-NMDAR encephalitis-related epileptic activity may induce synchronous firing of the autonomic nerves of the heart, leading to lethal arrhythmias \[[@B36], [@B37]\]. In a study with anti-NMDAR encephalitis patients, Wang et al. \[[@B20]\] showed that 28% of patients had hypoventilation, 26% required mechanical ventilation, and 20% had a tracheotomy and required ICU support. Moreover, studies have shown that ICU treatment can be used an independent risk factor for death \[[@B38]\]. Schubert \[[@B34]\] and others have pointed out that the presence of autonomic nervous dysfunction during hospitalization and the use of mechanical ventilation were significantly associated with poor neurological function at discharge and may be associated with poor long-term prognosis \[[@B39]\]. Therefore, the emergence of autonomic nerve dysfunction during the course of disease can lead to other serious complications and affect the prognosis of patients. The relationship between autonomic nerve dysfunction and poor prognosis requires further study.
The clinical manifestations of anti-NMDAR encephalitis are complex. Symptoms such as epilepsy and movement disorders can gradually disappear with treatment and have little correlation with prognosis. However, patients may have symptoms, such as cognitive impairment, which have a slow recovery process and may persist long-term, thereby impacting the patients\' quality of life. Autonomic dysfunction is strongly related to prognosis, which may lead to serious complications and poor prognosis, which requires active intervention.
4. Auxiliary Inspection {#sec4}
=======================
4.1. EEG {#sec4.1}
--------
In the diagnosis, treatment, and evaluation of anti-NMDAR encephalitis, EEG plays an important role due to its convenience, speed, and noninvasiveness. Seizures and movement disorders in anti-NMDAR encephalitis are easily confused \[[@B40]\], and EEG has been posited as a necessary auxiliary test for the differential diagnosis \[[@B41]\]. Studies have shown that in the most severe cases of anti-NMDAR encephalitis, EEG abnormalities are more helpful for diagnosing the disease than magnetic resonance imaging (MRI) (98.4% vs. 46.8%) \[[@B42]\]. In addition to seizures, common EEG abnormalities in anti-NMDAR encephalitis include nonspecific diffuse slow waves \[[@B43]\] and specific delta brushes (extreme delta brush (EDB)), among others \[[@B41]\]. Since EDB is more common in comatose patients and other severe cases, it is currently considered to indicate a poor prognosis \[[@B21], [@B44], [@B45]\]. However, some studies suggest that EDB is of limited significance in predicting patient prognosis; so with respect to anti-NMDAR encephalitis, more research is needed to understand the relationship between EDB and prognosis \[[@B42]\]. Besides, Zhang and others have shown that patients with a normal EEG background, epileptic discharge, polymorphic delta rhythm, and diffuse beta activity of EEG have a good long-term prognosis \[[@B42]\].
4.2. Imaging {#sec4.2}
------------
Imaging findings in anti-NMDAR encephalitis are complex, show poor specificity, and have limited prognostic value. Common imaging tools used for the diagnosis of anti-NMDAR encephalitis include MRI and FDG-PET. The incidence of abnormal MRI findings in cases of anti-NMDAR varies from 11% to 83% \[[@B18], [@B46]\]. Indeed, a systematic review of 56 clinical studies showed that less than 50% of patients had abnormal MRI findings \[[@B47]\], with most abnormalities coming from T2-weighted scans as well as a high FLAIR signal. Abnormal findings are most commonly found in the temporal lobe, in addition to the cerebral cortex and subcortical white matter regions. The frontal lobe, hippocampus, and cerebellum can also be affected. However, Gabilondo et al. \[[@B48]\] suggested that abnormal MRI manifestations were not associated with anti-NMDAR encephalitis recurrence. In addition to abnormal MRI findings, some patients can show reversible diffuse brain atrophy (DCA) and even brain atrophy, including progressive cerebellar atrophy. Studies have shown that DCA patients require more mechanical ventilation and have longer hospital stays \[[@B49]\]. However, in a clinical study with a median follow-up of 68 months, 33% of patients with DCA and some brain atrophy patients showed strong recoveries. Nevertheless, progressive cerebellar atrophy was irreversible, and in this study, cerebellar atrophy was strongly associated with a poor prognosis \[[@B49]\]. By using multimodal MRI, Finke et al. found that the hippocampal connectivity and fractional anisotropy in the white matter of anti-NMDAR encephalitis patients were reduced, mainly the cingulate gyrus. Importantly, these changes were not detected when using conventional MRI \[[@B50]\]. The abovementioned pathological changes may be related to the patient\'s cognitive impairment and severity of the disease, which may affect the prognosis.
Compared with lower-sensitivity MRI, FDG-PET is extremely sensitive for detecting the most severe stages of anti-NMDAR encephalitis \[[@B47], [@B51]\]. Typically, anti-NMDAR encephalitis is characterized by hyperfrontal and temporal lobe metabolism and a decrease in parietal and occipital lobe metabolism. The recovery period is mainly characterized by diffuse cortical metabolism \[[@B52]\]. The metabolic performance, measured by PET-computed tomography (CT), is almost normal in patients with negative antibody recovery and no obvious clinical symptoms. However, it may appear abnormal again as the patient relapses. Thus, PET-CT can be used to assist in the diagnosis of anti-NMDAR encephalitis, helping to determine the degree of disease progression based on the specific metabolic pattern of disease changes. However, further research is needed to guide long-term prognosis assessments.
4.3. Laboratory Tests {#sec4.3}
---------------------
At present, the detection of IgG antibodies against the NMDAR GluN1 subunit in the CSF is the main diagnostic method for anti-NMDAR encephalitis. Studies have shown that the CSF antibody titer is maximal when the disease is most severe. If the disease is controlled, the CSF antibody titer gradually decreases with time, although it may remain positive for a long time \[[@B53]\]. Gresa-Arribas and others have shown that patients with poor outcomes had higher levels of CSF and serum anti-NMDAR antibodies than patients with positive outcomes \[[@B53]\]. Likewise, the concentration of CSF antibodies increased when encephalitis recurred. This conclusion is also supported by the findings of Schneider et al. \[[@B45]\], given that intrathecally synthesized antibodies can be retained for many years after the clinical symptoms have disappeared. Therefore, even though antibody titers are related to the onset of the disease, the improvement of symptoms is not associated with a decline in antibody titers \[[@B5], [@B30]\]. Consequently, Dalmau and others believe that clinical evaluation, rather than antibody levels, should be the main decision-making tool to guide treatment \[[@B26]\]. The relationship between CSF antibody titer, prognosis, and relapse is not clear and warrants further research.
5. Treatment {#sec5}
============
5.1. Immunotherapy {#sec5.1}
------------------
There is no uniform standard immunotherapy for anti-NMDAR encephalitis. At present, immunomodulatory therapy is mainly used. Immunomodulatory drugs can be divided into first-line and second-line. First-line immunotherapy drugs include intravenous methylprednisolone (IVMP), intravenous immunoglobulin (IVIG), or PE. High-dose methylprednisolone therapy can regulate T lymphocyte function and reduce inflammatory responses \[[@B54]\]; it is currently the most commonly used immunotherapy and is given as an intravenous infusion of 30 mg/(Kg·d), which is gradually reduced during the course of the treatment \[[@B55]\]. IVIG can inhibit humoral and cellular immunity and regulate immune responses through a variety of mechanisms \[[@B56]\]. Commonly, IVIG is given at a dosage of 0.4 g/Kg for 5 days, and it can be reused later according to the condition of the disease \[[@B57]\]. PE can reduce the CSF anti-NMDAR antibody titer by removing these antibodies from the blood, thereby ameliorating the disease \[[@B57]\]. In the course of immunotherapy, IVMP, IVIG, and PE therapy can be used in combination, depending on the patient\'s condition. However, currently, the order of drug administration and choice of protocols are controversial \[[@B57], [@B58]\]. Second-line immunotherapy drugs include rituximab, cyclophosphamide, azathioprine, and mycophenolate mofetil \[[@B26]\]. Studies have shown that second-line immunotherapies can be started in patients who have failed to respond to first-line immunotherapies. In those cases, second-line immunotherapies have improved the prognosis of patients when compared to those who did not have any further form of immunotherapy \[[@B4]\]. Moreover, patients who were started on second-line immunotherapy without receiving any first-line immunotherapy drug had a better prognosis than untreated patients \[[@B4]\]. For patients who did not improve after first-line and second-line treatments, clinical trials are currently testing the effectiveness of intrathecal injections of methotrexate and glucocorticoids as an attempt to block the intrathecal synthesis of anti-NMDAR antibodies \[[@B59]\]. So far, this treatment showed promise by improving the symptoms of patients and reducing CSF antibody titers.
Current research suggests that early initiation of immunotherapy can significantly improve the prognosis of patients and reduce the recurrence rate \[[@B4], [@B48]\]. Studies on second-line treatment have shown that rituximab can significantly improve the prognosis of patients \[[@B60]\]. By contrast, some research has indicated that starting immunotherapy as soon as possible does not reduce mortality of anti-NMDAR encephalitis patients \[[@B38]\]. However, some researchers have proposed that third-line treatments should be used if second-line treatments fail. Bortezomib and tocilizumab are examples of third-line treatments; however, the effectiveness of bortezomib remains controversial \[[@B26]\]. Those who continued to be treated with tocilizumab after initial treatment failure may have shown a better prognosis at follow-up after 24 months \[[@B61]\]. Although most current studies on the prognosis of anti-NDMAR encephalitis demonstrate immunotherapy to be efficacious and able to gradually restore patients to baseline levels, current research has gradually progressed toward addressing patients who have not fully recovered \[[@B62]\]. More research is needed to understand the sequelae following immunotherapy in anti-NDMAR encephalitis patients.
5.2. Tumor Treatment {#sec5.2}
--------------------
Anti-NMDAR encephalitis is often associated with tumors. Patients who present with tumors also need to undergo removal of the tumor. The occurrence of tumors depends on age, sex, and ethnicity. Female patients mostly develop ovarian tumors, and patients over 12 years of age have a 52% chance of developing a teratoma \[[@B4]\]. The younger the patient, the less likely they are to have tumors \[[@B63]\]. Most tumors in men are germ cell tumors, and only 5% of male anti-NMDAR encephalitis patients over 18 years of age have underlying tumors \[[@B63]\]. In addition, liver neuroendocrine tumors, uterine neuroendocrine tumors, small cell lung cancer, and cancers of unknown origin have recently been associated to anti-NDMAR encephalitis \[[@B62]\]. In these cases, tumor resection in conjunction with simultaneous immunotherapy can significantly accelerate the amelioration of the disease and reduce the need for second-line treatments \[[@B63]\]. In a 2011 study by Dalmau et al., 80% of tumor patients showed a substantial improvement after a combination of tumor resection and first-line immunotherapy. By contrast, only 48% of tumor-free patients showed similar improvements after first-line immunotherapy. Some studies suggest that patients without tumors show more significant cognitive deficits. However, in assessing the long-term prognosis, follow-up results show that patients with and without tumors are statistically indistinguishable and both will eventually show a substantial improvement in symptoms \[[@B63]\]. However, patients without tumors who do not receive (or are delayed in receiving) immunotherapy may have a poor prognosis \[[@B31]\]. A 2013 study by Titulaer and others also demonstrated that there was no obvious correlation between the presence of tumors and long-term prognosis \[[@B4]\]. Moreover, they found that patients who had their tumors resected had a lower recurrence rate for anti-NMDAR encephalitis during follow-up. Similarly, in a cohort study of severe anti-NMDAR encephalitis patients who required ICU treatment, the presence of a tumor did not affect patient prognosis \[[@B5]\]. Indeed, a systematic analysis by Broadley et al. \[[@B30]\] in 2019 supports that anti-NMDAR encephalitis patients with an underlying tumor may have shorter recovery times and lower recurrence rates.
5.3. ICU Admission {#sec5.3}
------------------
A subset of anti-NMDAR encephalitis patients can display severe clinical symptoms with complications such as cognitive impairment, unconsciousness, status epilepticus, hypoventilation, and cardiac arrest. In this aspect, the ICU can perform a number of supportive treatments to relieve the condition. While there is a necessity for ICU treatments, the long-term prognosis for patients admitted to the ICU remains inconclusive. For example, in a 2013 clinical study involving 577 patients with anti-NMDAR encephalitis, Titulaer et al. showed that in univariate and multivariate analyses, not requiring ICU support in 4 weeks after admission was significantly associated with favorable patient outcomes \[[@B4]\]. de Montmollin et al. showed that, across 77 anti-NMDAR encephalitis patients from 52 ICU treatment centers, even if ICU treatment was required, the prognosis remained satisfactory \[[@B5]\]. They further affirmed that the early initiation of immunotherapy was beneficial and is an independent factor of a good prognosis \[[@B5]\]. However, when analyzing the cause of death of 96 patients with anti-NMDAR encephalitis, Chi and Zhou showed that the mortality rate was as high as 11.46%, and significantly associated with continued ICU treatment \[[@B38]\]. Broadley et al. conducted a systematic review and confirmed that on-admission ICU support treatment was necessary and could impact the long-term prognosis of anti-NMDAR encephalitis patients \[[@B30]\]. Therefore, patients who require ICU support treatment to address severe manifestations of anti-NMDAR encephalitis have a significantly higher risk for a poor prognosis and death. Moreover, the overall prognosis of the disease is significantly related to a timely administration of immunotherapy. Importantly, patients who have needed ICU support can still achieve a good prognosis. Thus, in the ICU, a multidisciplinary and comprehensive treatment strategy based on the patient\'s condition can significantly improve the patient\'s prognosis \[[@B45]\].
6. Conclusions {#sec6}
==============
The clinical manifestations of anti-NMDAR encephalitis are complicated. Patients tend to exhibit severe symptoms, and recovery is slow. At present, research on the prognosis of anti-NMDAR encephalitis remains limited. We summarized the relevant research on the anti-NMDAR encephalitis, with a focus on factors affecting prognosis, such as disease pathogenesis, clinical manifestations, auxiliary examination, and treatments. Autonomic dysfunction and related complications may affect long-term prognosis, with cognitive impairment being the main dysfunction encountered during the long-term recovery of patients with anti-NMDAR encephalitis. When the abovementioned clinical manifestations appear, they should be actively treated as soon as possible in order to improve the chances for a positive outcome. Auxiliary inspection has gradually shown a capacity to predict prognosis. The value of EEG for predicting the long-term prognosis of patients is gradually highlighting its value, but the relationship between CSF antibodies, imaging, and prognosis needs further exploration. Immunotherapy started at an early stage is still the best treatment plan to obtain a good prognosis, but for some patients with poor curative effects, more and more effective treatment methods need to be explored to shorten the clinical course of patients, reduce disease complications and sequelae, and obtain a better prognosis. At present, there are still a limited number of studies focusing on the prognosis of anti-NMDAR encephalitis. Our review showcases future research directions. Clinical manifestations, auxiliary examinations, treatment options, and complications may all become prognostic indicators for patients, and we look forward to further prospective research in the future that is aimed at overcoming these difficulties.
The author would like to thank Professor Xiao for her invaluable clinical advice and assistance with medical ethics. This study was supported by the National Natural Science Foundation of China (grants number 81071040 and 81471320).
Conflicts of Interest
=====================
The authors declare that there is no conflict of interest regarding the publication of this paper.
[^1]: Academic Editor: Bence Racz
|
{
"pile_set_name": "PubMed Central"
}
|
All relevant data are within the manuscript.
Introduction {#sec001}
============
Mycetoma is a neglected tropical disease frequently affecting the poorest of the poor in remote, poor communities \[[@pntd.0007019.ref001]\]. It is endemic in many tropical and subtropical countries around the world including Sudan \[[@pntd.0007019.ref002], [@pntd.0007019.ref003]\]. The geographical distribution of mycetoma depends on a range of environmental factors such as rainfall, humidity and temperature \[[@pntd.0007019.ref004], [@pntd.0007019.ref005]\]. Mycetoma has a higher prevalence in rural areas with poor domestic hygiene \[[@pntd.0007019.ref006], [@pntd.0007019.ref007]\]. The disease is caused by more than 50 microorganisms of fungal or bacterial origin, and hence it is classified as eumycetoma and actinomycetoma respectively \[[@pntd.0007019.ref008], [@pntd.0007019.ref009]\]. Mycetoma is believed to occur as a result of traumatic implantation of the causative organism into the subcutaneous tissue via minor trauma or injury \[[@pntd.0007019.ref010], [@pntd.0007019.ref011]\]. It then spreads to involve the skin, deep tissues and bone leading to massive destruction, deformities and disabilities \[[@pntd.0007019.ref012], [@pntd.0007019.ref013]\]. If untreated it can have a major impact on the affected patients, communities and the health system in endemic countries \[[@pntd.0007019.ref014], [@pntd.0007019.ref015]\].
The disease is characterised by the triad of subcutaneous mass, multiple sinuses and discharge containing grains \[[@pntd.0007019.ref016], [@pntd.0007019.ref017]\]. It is most frequently seen in the foot and hand, which account for 86% of reported cases, though no site is exempt \[[@pntd.0007019.ref018], [@pntd.0007019.ref019]\]. Mycetoma is most prevalent amongst children and young adults, with patients frequently presenting with advanced disease. Delayed presentation may result from patients' low socio-economic status and health education levels, and the paucity of health services in distant, isolated endemic areas. Overall, these factors lead to a devastating socio-economic impact \[[@pntd.0007019.ref020], [@pntd.0007019.ref021], [@pntd.0007019.ref022], [@pntd.0007019.ref023], [@pntd.0007019.ref024]\]. Currently, the available treatments (combination antimicrobial therapy for actinomycetoma, or antifungal therapy for eumycetoma) have proved ineffective and expensive, with a range of side effects and high recurrence rate \[[@pntd.0007019.ref025], [@pntd.0007019.ref026]\]. Late presentations often necessitate amputation or destructive surgical excisions \[[@pntd.0007019.ref027], [@pntd.0007019.ref028], [@pntd.0007019.ref029]\]. Patients require long-term follow-up to monitor recovery and recurrence, and unfortunately these factors as well as travel and opportunity cost of attending clinic lead to a high loss to follow up.
The disabling consequences of mycetoma are poorly understood. A medical literature search revealed no report addressing the present study objectives, and hence this study was designed to determine the disabling impact of mycetoma upon patients. We aim to identify key areas for future study with the ultimate goal of building evidence-based intervention and appropriate health care for those affected.
The mycetoma-related disability burden is hypothesised to be significant for several reasons. Firstly, the disease leads to structural impairment of the limbs, the most commonly affected parts, and disability arises from the direct loss of function. Secondly, current treatment options are limited and suboptimal, with disease potentially lasting for decades, resulting in increasing impairments over time. Finally, support and adaptation for individuals with any disabling consequences of mycetoma are limited in Sudan, as indeed they are for many other disabling neglected conditions around the world.
As far as the authors know, this is the first-ever study of the disabling consequences of mycetoma.
Material & methods {#sec002}
==================
Study design {#sec003}
------------
The study was designed using the International Classification of Functioning, Disability and Health (ICF). The ICF is a standardized framework and classification for the description of health and health related states \[[@pntd.0007019.ref030]\]. It conceptualises disability as an interaction between the patient health condition and his/her environment. Functions relate to body functioning (emotional state, pain, physical movement etc.), activities (driving, walking, etc.), and participation in society (engaging with religious ceremonies, etc.). Disability in this framework is a term for impairments or restrictions in functioning \[[@pntd.0007019.ref031]\].
Study design, questions and qualifiers were taken directly from the ICF. Questions were selected based on their relevance to mycetoma and were purposefully broad, encompassing a range of possible impacts. All the possible questions in the ICF were reviewed; six questions from Body Functioning and 27 from Activities and Participation, thus 33 variables were selected. Answers to the questions were not 'yes' or 'no'. Rather, grades of severity of the impairment or difficulty with a scale of mild, moderate, severe, or complete were used to further delineate the impact of the disease on individuals. These qualifiers for each grade were taken from the ICF ([Table 1](#pntd.0007019.t001){ref-type="table"}).
10.1371/journal.pntd.0007019.t001
###### Description of qualifiers.
{#pntd.0007019.t001g}
Qualifier Description Score Given
----------------------------------- -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- -------------
No impairment or difficulty A person has no problem 0
Mild impairment or difficulty A problem that is present less than 25% of the time, with an intensity which a person can tolerate and which happens occasionally over the last 30 days 1
Moderate impairment or difficulty A problem that is present less than 50% of the time, with an intensity which is interfering in a person's day to day life and which happens occasionally over the last 30 days 2
Severe impairment or difficulty A problem that is present more than 50% of the time, with an intensity which is partially disrupting the person's day to day life and which happens frequently over the last 30 days 3
Complete impairment or difficulty A problem that is present more than 95% of the time, with an intensity which is totally disrupting to the person's day to day life and which happens every day over the last 30 days 4
Data collection {#sec004}
---------------
The data was collected by four recently graduated doctors. Prior to the data collection, they had a four-day training course on data collection and interview skills provided by the first author. The training also included role-play, real-life interview assessment and an examination at the end of the course, following which their first two patients' interviews were additionally observed for performance quality. They each passed the different parts of the training course.
The interviews were conducted each Monday at the Mycetoma Clinic of the Mycetoma Research Centre (MRC), University of Khartoum, between May 2016 and January 2017. To prevent selection bias, patients with confirmed mycetoma were given a number on arrival. Every third patient was chosen to be part of the study. Patients attended the clinic for their regular appointment and were interviewed either before or after their review. Patients who attended clinic Tuesday-Friday were not interviewed. We were able to survey 300 patients within the defined time scale of the project. Patients were given the option to decline and the interview was conducted in privacy. Ethical clearance was obtained from the Mycetoma Research Centre IRB and every patient gave verbal informed consent.
Data analysis {#sec005}
-------------
A scoring system for the various mycetoma-related disability variables studied was created. The variables included impairment in bodily functions or difficulty in activities and participation in society. A mild impairment or difficulty was given a value of one; that of moderate was given a value of two; severe was given a value of three, and complete inability to perform a given task or participate in activity was given a value of four. These values were added up over the 33 variables into a cumulative disability score that was used during data analysis. The statistical software used was Statistical Package for the Social Sciences and a P value of \<0.05 denoted statistical significance. Correlations were determined using a Pearson co-efficient bivariate two-tailed test.
Results {#sec006}
=======
Demographic {#sec007}
-----------
Between May 2016 and January 2017, 300 patients were interviewed; of these 228 (76%) were males and 72 (24%) were females with a gender ratio of 3:1. The mean age of the studied participants was 32.1 years with a sample standard deviation of 14.0 years; the youngest was 5 years old and the oldest was 74 years old; 39% of them were between the ages of 5--25 years, 49% between 26--50 years, and 12% were aged 51 to 74 years. The participants were most commonly students (16%), farmers (13%), and housewives (11.3%). In 36% of participants, the occupation was unspecified.
The mycetoma mean duration was 7.8 years with a sample standard deviation of 6.6 years and a range of three months to 34 years. 50% of participants had had mycetoma for 0--5 years, 23% for 5--10 years, and 27% for more than 10 years. Most individuals (270, 90%) reported a mycetoma lesion in the lower limb (hip through to toes), 23 patients (8%) had a mycetoma lesion in the upper limb, six patients (2%) reported the lesion in the head and neck, and one participant reported mycetoma in multiple sites. Most patients (203, 68%) had had local surgical excision (most frequently at rural hospital sites), of these 140 (69%) had had recurrence. Seven patients had undergone amputation, of whom two had had recurrence.
Ethics statement {#sec008}
----------------
Ethical clearance was obtained from the Mycetoma Research Centre IRB and every patient gave verbal informed consent. Written consent was decided to be not necessary as the patients' identity was not disclosed in this article. The patients' verbal informed consent acceptance was documented in each questionnaire during the interview.
Cumulative disability scores {#sec009}
----------------------------
The study showed that 49 (16.3%) of all participants had no difficulty or impairment in any of the 33 studied variables ([Table 2](#pntd.0007019.t002){ref-type="table"}). These patients were slightly younger, with a mean age of 29 years and an age range of 7 to 65; and they had had mycetoma for a shorter duration than the average (mean 6.9 years, range 0.5--20 years). Their most common occupations were students (22%) or housewives (16%). All other patients (251, 83.7%) experienced disability ranging from mild to severe. The mean cumulative disability score for all degrees of disability among the studied patients was 10.7 with a range of 0 to 68. Most of the patients (133, 44.3%) had a score between 1 and 10. 62 patients (20.7%) had a score of 10--20, 31 patients (10.3%) had a score of 20--30, and 25 patients (8.3%) had a score of more than 30. The study showed that 181 patients (60.3%) had moderate impairment or difficulty in at least one domain variable. The scores were highest among farmers, those not in income-generating work, elderly patients and those with a longer disease duration.
10.1371/journal.pntd.0007019.t002
###### Overall assessment of mycetoma disability using cumulative disability scoring.
{#pntd.0007019.t002g}
Disability Scoring No. \%
-------------------- -------------------------- ------- ------
All patients No difficulty/Impairment 49 16.3
Score of 1--10 133 44.3
Score of 10--20 62 20.7
Score of 20--30 31 10.3
Above 30 25 8.3
Total 300 100.0
Results by variable {#sec010}
-------------------
In this study, 103 patients (34.3%) had local pain of various degrees at the mycetoma site ([Table 3](#pntd.0007019.t003){ref-type="table"}). Mild pain was confirmed by 70 patients (23.3%), 23 patients (8%) had moderate pain, and 10 (3%) had severe pain. Worsening of pain was statistically significantly correlated with advanced age; (*p*\<0.01). No results are available for analgesic control in this population ([Fig 1](#pntd.0007019.g001){ref-type="fig"}).
{#pntd.0007019.g001}
10.1371/journal.pntd.0007019.t003
###### Impairments in body function.
{#pntd.0007019.t003g}
Degree of Impairment Energy and Drive Sleep Emotional Body Image Pain Sensation Weight Maintenance
-------------------------------- ------------------ ------- ----------- ------------ ---------------- --------------------
None 220 269 224 216 197 236
Mild 52 18 47 37 70 46
Moderate 26 11 28 42 23 17
Severe 01 02 01 03 10 01
Complete 00 00 00 00 00 00
Missing records 01 00 00 02 00 00
Total no. with some impairment 79 31 76 82 103 64
\% with impairment 26.4 10.3 25.3 27.5 34.3 21.3
In this study, 119 patients (39.7%) reported walking difficulty, ([Table 4](#pntd.0007019.t004){ref-type="table"}): 54 patients (18%) had mild difficulty, 36 patients (12%) had moderate difficulty, 23 patients (8%) had severe difficulty, and six patients (2%) were completely unable to walk. Worsening of walking ability was statistically significantly associated with advanced age (*p*\<0.01), longer disease duration (*p*\<0.01), and pain at the lesion site (*p*\<0.01).
10.1371/journal.pntd.0007019.t004
###### Difficulties in mobility.
{#pntd.0007019.t004g}
Capacity Hand & Arm Use Walking Transportation Driving
--------------------------- ---------------- --------- ---------------- ---------
No difficulty 273 181 214 93
Mild Difficulty 19 54 47 09
Moderate Difficulty 05 36 19 01
Severe Difficulty 01 23 09 04
Complete Inability 01 06 04 14
Missing records 01 00 00 00
Not applicable 00 00 07 179
Total no. With difficulty 26 119 79 28
\% with impairment 8.7 40.0 26.3 23.1
In self-care, the activities that predominantly require upper limb mobility are least affected by mycetoma and score lowest (eating, drinking, etc.; [Table 5](#pntd.0007019.t005){ref-type="table"}). Impaired ability to self-dress was reported by 112 patients (38%): mild impairment in 86 patients (29%), moderate in 22 patients (7%), severe in three patients (1%) and complete in one patient (0.3%). Impairment inability to dress oneself was statistically significantly correlated to the presence of pain at the mycetoma site (*p*\<0.01); but not with advanced patient age or longer duration of the mycetoma. Proportionally, most impairment in self-care is marked as mild (dressing 76.8%, washing 71.2%, toileting 74.6%; [Fig 2](#pntd.0007019.g002){ref-type="fig"}).
{#pntd.0007019.g002}
10.1371/journal.pntd.0007019.t005
###### Difficulties in self care.
{#pntd.0007019.t005g}
Capacity Washing Toileting Dressing Eating Drinking
--------------------------- --------- ----------- ---------- -------- ----------
No difficulty 225 242 187 291 292
Mild Difficulty 52 41 86 06 04
Moderate Difficulty 16 8 22 02 00
Severe Difficulty 02 02 03 00 00
Complete Inability 03 04 01 00 00
Missing 00 02 00 00 01
Not applicable 02 01 01 01 03
Total no. with difficulty 73 55 112 08 04
\% with impairment 25.5 18.5 40.8 2.7 1.4
The study showed that 164 patients (55%) had difficulty in going about their daily life, ([Table 6](#pntd.0007019.t006){ref-type="table"}). A mild difficulty was reported by 63 patients (23%), 28 (9%) had moderate difficulty, 22 (7%) had severe difficulty, and 51 (17%) were completely unable to go about at least one aspect of their daily life because of the mycetoma. Many patients marked these sections as not applicable to them which was largely to do with male/female gender roles in domestic life. Generally speaking, in Sudan males are less likely to be involved in domestic life activities than females.
10.1371/journal.pntd.0007019.t006
###### Difficulties in domestic life activities.
{#pntd.0007019.t006g}
Capacity Shopping Gathering Daily Necessities Preparing Meals Doing Housework
--------------------------- ---------- ----------------------------- ----------------- -----------------
No difficulty 175 132 87 119
Mild Difficulty 23 12 10 18
Moderate Difficulty 07 05 04 12
Severe Difficulty 04 09 05 04
Complete Inability 15 20 07 09
Missing 00 01 01 01
Not applicable 76 121 186 137
Total no. with difficulty 49 46 26 43
\% with impairment 21.9 25.8 23.0 26.5
Regarding interpersonal relationships, 57 (19%) patients reported difficulty, ([Table 7](#pntd.0007019.t007){ref-type="table"}). Mild difficulty was reported by 33 patients (11%), 15 patients (5%) reported moderate difficulty, six patients (2%) reported severe difficulty and three patients (1%) reported a complete inability to relate in the area of interpersonal interaction.
10.1371/journal.pntd.0007019.t007
###### Difficulties in interpersonal interactions and relationships.
{#pntd.0007019.t007g}
Capacity Informal Social Relationships Family Relationships Intimate Relationships
-------------------------------- ------------------------------- ---------------------- ------------------------
No difficulty 267 283 140
Mild Difficulty 15 13 5
Moderate Difficulty 10 2 3
Severe Difficulty 4 1 1
Complete Inability 3 0 0
Missing record 0 0 0
Not applicable 1 1 151
Total no. with some difficulty 32 16 9
\% with impairment 10.7 5.4 6.0
Of the 300 interviewed patients, 37 were of school age ([Table 8](#pntd.0007019.t008){ref-type="table"}). Of these, 21 reported no difficulty at attending school while 16 reported present or previous difficulty. Twelve patients reported that mycetoma rendered them completely unable to continue their school education. Eighteen patients had the potential to gain higher education of whom seven experienced difficulty due to their mycetoma: four experienced mild difficulty, one experienced moderate difficulty, one experienced severe difficulty, and one was completely unable to continue their higher education because of their mycetoma. While the survey format did not allow us to collect information from all who reported that mycetoma had made continuing in school difficult or impossible, anecdotal comments to the data collectors identified mobility impairments that made walking to school difficult or impossible as the key barrier to continuing school attendance.
10.1371/journal.pntd.0007019.t008
###### Difficulties in attending education.
{#pntd.0007019.t008g}
Capacity School Education Higher Education
-------------------------------- ------------------ ------------------
No difficulty 21 11
Mild Difficulty 01 04
Moderate Difficulty 02 01
Severe Difficulty 01 01
Complete Inability 12 01
Missing 00 00
Not applicable 263 282
Total no. with some difficulty 16 07
\% with impairment 44.4 38.9
Using the ICF definition of 'economic status' as "having economic resources from any source enough to ensure economic security in the present and the future" in the study, 126 patients (46.7%) reported a reduction in their ability to economically sustain themselves because of their mycetoma ([Table 9](#pntd.0007019.t009){ref-type="table"}). Mild difficulty was reported by 47 patients (17%) in their ability to be economically self-sufficient because of their mycetoma, 36 patients (13%) reported moderate difficulty, 19 patients (7%) reported severe difficulty, and 24 patients (9%) were completely economically non-self-sufficient because of their mycetoma.
10.1371/journal.pntd.0007019.t009
###### Difficulties in economic status.
{#pntd.0007019.t009g}
Capacity Remunerative Employment Economic Self Sufficiency
-------------------------------- ------------------------- ---------------------------
No difficulty 104 143
Mild Difficulty 24 47
Moderate Difficulty 10 36
Severe Difficulty 06 19
Complete Inability 29 24
Missing 00 01
Not applicable 127 30
Total no. with some difficulty 69 126
No. with impairment % 39.9 46.7
Specifically, 69 patients (40%) out of a relevant 173 patients found difficulty in their remunerative employment: 24 patients (14%) experienced mild difficulty, 10 patients (6%) moderate, six patients (4%) severe and 29 patients (17%) were completely unable to work for remunerative gain because of their mycetoma. There were 127 non-applicable individuals, who were not in work previously.
Impairment in remunerative employment was significantly correlated with the presence of pain (*p*\<0.05), impairment in walking (*p*\<0.01) and impairment in economic self-sufficiency (*p*\<0.01). Impairment in economic self-sufficiency was also significantly correlated with the presence of pain (*p*\<0.01) and impairment in walking (*p*\<0.01) but also advanced age (*p*\<0.01). Neither was correlated with duration of the mycetoma.
Of the 296 patients who reported that participation in a range of community activities were important to them, 63 (21.3%) experienced difficulty in participation because of the mycetoma, with 20 patients (7%) reporting mild, 22 patients (8%) moderate, and six patients (2%) severe limitations ([Table 10](#pntd.0007019.t010){ref-type="table"}). An additional 15 patients (5%) reported that they were completely unable to participate in community events or ceremonies. Of the 216 patients to whom recreation or leisure was important, 21 patients (9.7%) experienced difficulty in participation. Of the 107 patients to whom sports were relevant, 68 patients (63.6%) experienced difficulty, of whom four patients (4%) experienced mild difficulty, six patients (6%) moderate difficulty, seven patients (7%) severe, and 51 (48%) reported being completely unable to participate in sports because of their mycetoma. Of this study population, 54 patients out of 290 (19%) experienced difficulty in socialising, whilst 71 patients out of 295 (24.1%) experienced difficulty in participating in religious activities ([Fig 3](#pntd.0007019.g003){ref-type="fig"}).
{#pntd.0007019.g003}
10.1371/journal.pntd.0007019.t010
###### Impairments in community, social and civic life.
{#pntd.0007019.t010g}
Capacity Ceremonies Recreation & Leisure Sports Socialising Religion
-------------------------------- ------------ ---------------------- -------- ------------- ----------
No difficulty 233 195 39 236 224
Mild Difficulty 20 09 04 26 39
Moderate Difficulty 22 03 06 10 24
Severe Difficulty 06 02 07 8 3
Complete Inability 15 07 51 10 05
Missing 00 0 0 0 00
Not applicable 04 84 193 10 05
Total no. with some difficulty 63 21 68 54 71
No. with impairment % 21.3 9.7 63.6 18.6 24.1
Discussion {#sec011}
==========
There is a general paucity of literature investigating the nexus of neglected tropical diseases and disability, though existing work supports our main findings. A study addressing leprosy-related disability highlights the disease's economic impact and association with extreme poverty. It also shows that people with a leprosy-related disability achieved fewer individual and family related objectives without interventions recognising their disability \[[@pntd.0007019.ref032]\]. Work in malaria and tuberculosis also shows the importance of qualitative approaches in elucidating the nature of the disease-related disability, its interaction with poverty and related barriers to care \[[@pntd.0007019.ref033], [@pntd.0007019.ref034]\].
Our study is the first which measures and characterises mycetoma-related disability. It also clearly links mycetoma-related disability with a negative impact on economic livelihood. Overall, 83.7% of the studied patients report disability in one or more of the 33 ICF domains examined, with 60.3% reporting at least moderate disability in one or more domain. Further analysis reveals areas of differential impact along the lines of gender and age, alongside specific vulnerabilities common to all those with mycetoma, including pain, impaired mobility and stigma.
Four times as many males are diagnosed with this disease as females, suggesting variations either in incidence patterns or in societal responses, and hinting at gender-based vulnerabilities requiring further research. There may be true gender variation in disease incidence, relating to increased likelihood of traumatic injury allowing pathogenic inoculation, or to a hormonal influence on disease progression in males \[[@pntd.0007019.ref035]\]. An alternative explanation is that women may be less likely to seek or receive medical care due to stigma, financial or 'gender-role' considerations, leading to reporting bias. This alternative is supported by a village level survey which showed that gender and age distributions were more evenly distributed than those measured at the tertiary level \[[@pntd.0007019.ref015]\]. Indeed in endemic areas, females may in fact have similar risks of exposure as men, due to extensive outdoor activities. Either explanation hints at a particular vulnerability rooted in gender.
There are two striking age-related impacts. Firstly, older adults bear a greater burden of disability overall than younger patients or students, suggesting that symptoms may correlate with both disease duration, and therefore disease progression, and with physical activity and pain. Secondly, there is a strong effect on educational attendance in the school-age population where 44.4% of students with mycetoma experience difficulty in attending primary school, and 38.9% struggle to attend higher education. Our study did not formally assess this effect further, but physical inability to walk to school was mentioned by several interviewees as an underlying cause. For school-age children with little or no access to transport to school facilities, this would represent a strong barrier to accessing education. Illness and disease have also been shown to negatively impact a child's learning and school time in less developed settings \[[@pntd.0007019.ref036]\]. The combined effects of decreased access and poorer quality of learning when in lessons are likely to result in unfulfilled potential and loss of future opportunities.
Three further mycetoma-related effects are worth discussing for their creation of particular vulnerabilities common to all individuals with the disease. These include pain, limited mobility and stigma. Mycetoma is typically characterised as a painless disease, although there are reports of local pain at the mycetoma site and that is frequently due to secondary bacterial infection \[[@pntd.0007019.ref037], [@pntd.0007019.ref038], [@pntd.0007019.ref039]\]. However, in this study over a third of respondents (103 patients, 34.3%) reported local pain, higher than has been previously reported \[[@pntd.0007019.ref002], [@pntd.0007019.ref004], [@pntd.0007019.ref020]\]. Whilst most cases were mild, infrequent and tolerable, this challenges the idea that mycetoma is generally painless. It is likely to result in vulnerabilities correlating with poorer development and achievement outcomes including school attendance, academic performance and the ability to perform physical tasks, both domestic and professional.
Walking itself receives high impact scores with 119 patients (40.0%) complaining of restriction in their ability to walk without assistance. The assessment questions point towards impairment being produced by the mycetoma itself, rather than other co-morbidities or age-associated frailties. In general, co-morbidities are rare in mycetoma as most of the patients by number are young adults and children (though notably those with the most disabling consequences are the elder patients). The associations between a sedentary lifestyle and non-communicable diseases are well-known however and those with mycetoma-related physical restriction are likely to be at increased risk for them.
A substantial number of respondents reported reduced participation in education, community, religious, social and civic life. This could be partly a result of stigma and social isolation, either because of specific beliefs about the disease e.g. contagion, linkage of physical and spiritual malady, or due to broader prejudice against individuals with a disability.
However, less than 20% of respondents reported difficulties in socialising (18.6%), with just 7% reporting difficulties in interpersonal relationships overall. Furthermore, relatively few patients had impairments in such bodily functions as energy and drive (26%), emotional function (25%) or body image impairment (27.5%). These findings might be explained by the localised, slowly progressive nature of the disease, together with the rarity of cosmetically 'significant' areas, such as the face, being affected.
It is also interesting to note that few patients had impairment in informal social- (11%), family- (5%) or intimate- (6%) relationships. This may be a finding particular to the study setting, partly related to the strong concept of the extended families in the Sudan, where patients receive support and care from many family members even if they are not close relatives.
Overall these findings suggest that in the context of Sudan reduced participation in aspects of public life may be influenced as much by physical issues as by social stigma. Further work is needed to explore this complex area.
The most common limitation, by a number of people affected, was in economic self-sufficiency (126 affected, 46.8% of applicable patients). Self-sufficiency here was defined as having enough economic resources from any source to ensure economic security in the present and the future \[[@pntd.0007019.ref030]\]. A component of this is likely to be disease-related inability to work. Indeed 40% of those who would otherwise be in work were partially or completely unable to engage in remunerative employment because of their mycetoma. Our results do not explore the exact nature of this loss in economic self-sufficiency, however we know that even where treatment is available free of charge, such as that provided at the MRC, indirect healthcare costs (e.g. cost of transport, loss of paid work) are consistently a significant burden on household economies \[[@pntd.0007019.ref040]\]. An emerging body of research evidence also shows that individuals living with a disability, and households with disabled members, face increased costs compared to those without, e.g. for medical care, transportation, loss of paid work both by the affected individual and by those who remain at home to provide care \[[@pntd.0007019.ref041],[@pntd.0007019.ref042]\].
In this study, there were four main limitations. Firstly, data collection was based on the ICF Model, rather than the Washington Group (WG) Questions on Disability Statistics \[[@pntd.0007019.ref043]\]. This was because the project was initiated and data was collected prior to awareness of the WG methodology by field research staff. There is some overlap, since the WG questions are based upon the ICF Model, however the WG methodology captures dimensions of individual functioning more precisely and should be the basis of future work. Nevertheless, we believe our data remains valid, relevant and yields new insight into mycetoma-related disability. Secondly, data collectors were recent medical graduates without backgrounds in research or disability. Therefore extensive training and support was given to minimise the risk of decreased accuracy or reliability of results. Third, our data is predominantly quantitative, save for anecdotes reported to data collectors. A qualitative component in further research could add further valuable insight into how individuals and societies are affected. And finally, although this was not the purpose of the study, we did not record whether patients had actinomycetoma or eumycetoma and are unable to perform a subgroup analysis of these groups to look for differences in disability.
In conclusion, this is the first study to outline the burden of disability caused by mycetoma. It shows clear evidence of mycetoma-related impact on individuals\' body function, mobility, and ability to self-care, with significant ramifications for domestic life, interpersonal relationships, educational attainment, economic status, and civic engagement. There is sufficient evidence to support specific interventions aiming to mitigate and adapt to the disabling consequences of mycetoma. However, better detection and disease education are also important for preventing infection and the development of mycetoma-related disability.
Clinically, effective pain management should be routinely integrated into mycetoma services, particularly for those groups identified as higher risk such as farmers and older adults. Different analgesic agents can be evaluated for efficacy against mycetoma-related pain. More definitive treatment should include the elimination of the secondary bacterial infection by appropriate anti-pathogenic agents and repeated lesion debridement. Further addition of physiotherapy services could begin to address limitations of mobility and function. Simple walking aids could be a starting point followed by in-house physiotherapy services. They could also provide a starting point for reducing risks associated with sedentary lifestyles.
In education, services could include assessment of a student's mycetoma-related disability using a pragmatic screening tool based on our results. This would inform adaptations in educational provision, including greater local awareness and consideration of learning aids or adaptations that allow distance learning, participation in physical and sporting activities, in modifying transport to and from school as well as mitigating stigma in educational settings.
This study shows evidence of impaired revenue generation at an individual level and a negative impact on economic self-sufficiency and household income. While this establishes that diseases such as mycetoma have significant development and public health implications, further research is needed to quantify its financial effects at the individual, household and societal levels. This includes effects on revenue generation, career opportunities and health-related costs, including the reallocation of disposable assets.
Above all, our data emphasise that the multi-faceted, long-term disabling consequences of NTDs, such as mycetoma, must not be overlooked. Their effects are likely to be felt at the level of individuals, families and communities, across multiple social and economic dimensions. Importantly, such effects are likely to be modifiable and therefore must be considered and addressed in future policy and programming.
[^1]: The authors have declared that no competing interests exist.
|
{
"pile_set_name": "PubMed Central"
}
|
INTRODUCTION {#s1}
============
Angiomyolipoma (AML) are benign tumours composed of varying amounts of thick-walled dysplastic blood vessels, smooth muscle and mature adipose tissue derived from epithelioid cells. They are the commonest benign tumours found in the kidney and can be associated with tuberous sclerosis complex, although the majority occur sporadically ([@R1],[@R2]). Primary AML of the pancreas ([@R3]) and liver ([@R4]) have been described before, however there are no cases of multifocal AML involving the liver and pancreas reported in the literature.
CASE REPORT {#s2}
===========
We report the case of a 61 year old man, previously treated for follicular lymphoma in the left groin with radiotherapy, presentING to our hepatobiliary department with painless obstructive jaundice ALP 592 iu/L (40-130), ALT 86 iu/L (2-53) and Bil 180 (3-17). His CA19.9 was 156 u/l and there was no clinical evidence of tuberous sclerosis. Cross-sectional imaging in the form of a CT and MRI scan reported multiple suspicious lesions in the liver (Fig. 1) and a small lesion in the uncinate process of the pancreas ([Fig. 2](#fig2){ref-type="fig"}) causing intrahepatic and common bile duct dilatation. The radiological diagnosis at this point was of a metastatic (and hence inoperable) pancreatic cancer. He underwent an ERCP and insertion of a CBD stent to relieve his jaundice and an attempted percutaneous biopsy of his pancreatic mass yielded inadequate cells. He then underwent a laparoscopy and attempted biopsy of his pancreatic and/or liver lesions. The pancreatic mass was difficult to visualise on intraoperative ultrasound however one of the liver lesions was biopsied and this was reported as an angiomyolipoma.
After re-discussion at our multi-disciplinary meeting it was felt that on balance the liver lesions were an incidental finding and in view of his age, presentation and imaging he most likely had a small pancreatic carcinoma. He underwent a pancreaticoduodenectomy and made an uneventful recovery. Histological analysis of the specimen revealed a small completely excised angiomyolipoma. Several months after his operation, he was admitted with vague abdominal pain, which resolved spontaneously. However, a CT scan performed during his admission revealed several intra-hepatic lesions of varying sizes and a growth of the original segment VI lesion. A percutaneous biopsy of one of the new lesions once again confirmed AML.
{#fig2}
DISCUSSION {#s3}
==========
This is an unusual case for several reasons. Firstly, pancreatic AML is extremely rare, with only one case reported in the literature ([@R3]). Secondly, we believe this is the only reported case of multifocal AML affecting the pancreas and liver. There are two cases in the literature describing multi-focal AML, one involving the lung and liver ([@R2]) and the other involving the pancreas and right kidney ([@R5]). Thirdly, multiple hepatic AMLs not associated with tuberous sclerosis are rare and only a handful of cases have been reported in the past ([@R6]).
Angiomyolipomas are considered to be benign in nature although there have been a few reported cases of excised renal ([@R7]) and hepatic ([@R8]) AMLs, uncharacteristically displaying malignant potential as evidenced by their immunoreactivity to *p53* on pathological analysis (which was positive in this specimen). There still remains a scarcity of data describing their natural progression and behaviour and this often makes surgical management of these lesions difficult. In this case, the pancreatic lesion was small and the consensus opinion was to treat this as a carcinoma, a reasonable assumption considering the clinical presentation and imaging results in a relatively fit 61 year old.
The next dilemma facing our team is how to deal with his enlarging hepatic AMLs. The clinical differential of hepatic AML includes lipoma, haemangioma, focal nodular hyperplasia hepatocellular carcinoma and hepatic adenoma. Although change of size in renal AMLs is a well described phenomenon ([@R9]), it is a relatively rare occurrence in the case of hepatic AMLs ([@R10]). We have decided to follow-up this gentleman with serial 6-monthly MRI scans; however this is based on anecdotal rather than robust clinical evidence.
In hindsight, although the patient underwent major surgery for a benign tumour, it is difficult to see how this could have been prevented by any further pre-operative investigations. Endoscopic ultrasound with biopsy of the lesion may have been of value in obtaining a preoperative tissue diagnosis of the primary pancreatic lesions. However, for a fit patient with a potentially resectable pancreatic tumour (on cross-sectional imaging) we believe it to be best practice to proceed to resection in the absence of definitive preoperative or intraoperative confirmation of malignancy. This remains a consensus view followed by many other pancreatic units worldwide.
We have described a fascinating and rare case of multifocal angiomyolipoma, however there are still questions regarding the ideal management of patients with these lesions, particularly those growing in size in the liver. Reporting of such cases is essential to further delineate the natural history of this pathology.
|
{
"pile_set_name": "PubMed Central"
}
|
Introduction
============
Mineral Trioxide Aggregate (MTA) is a calcium silicate-based biomaterial and is widely used in endodontics due to its favorable properties, including sealing ability \[[@B1]\], setting capacity in the presence of blood/moisture \[[@B2]\] bio-compatibility \[[@B3], [@B4]\], and the ability to induce regeneration in the pulp and periapical tissues \[[@B5]\]. Its applications in endodontics include as a pulp covering agent, as an apical plug/root-end fillings and as a perforation repair biomaterial. However, MTA has a long setting time and its handling is difficult \[[@B6]\]. Some studies have shown its poor antimicrobial properties against resistant bacteria such as *Enterococcus faecalis* \[[@B7]\].
Several studies have evaluated and suggested different strategies to improve the properties of MTA. One strategy is to incorporate ingredients into MTA to decrease its setting time. Disodium hydrogen phosphate is a buffering agent which has an effect on decreasing the setting time of MTA \[[@B8], [@B9]\].
Due to the antibacterial properties of silver nanoparticles, they have been incorporated into MTA \[[@B10]\]. These particles have a high surface-to-volume ratio and when added in small amounts interfere with peptidoglycans of bacterial cell wall and increase their permeability. In addition, they react with the sulfhydryl groups of bacterial proteins and prevent DNA replication, resulting in bacterial death \[[@B11]\].
When ingredients are incorporated into a biomaterial to improve its properties, important factors that should be taken into account are the cytotoxicity and genotoxicity of the final product \[[@B12]\]. Studies on the cytotoxicity of MTA after incorporation of disodium hydrogen phosphate have shown its biocompatibility \[[@B13]-[@B15]\]. In addition, incorporation of silver nanoparticles at 1 wt% and at 23-47 ppm concentrations has resulted in tissue reactions similar to those mounted to MTA alone; *i.e.* the resultant material has been shown to be biocompatible \[10, 16\]. Also, due to the results reported by Li *et al.*\[17\], the Ames was negative regarding the genotoxicity of silver nanoparticles and suggested that this result can be due to the inability of these nanoparticles to penetrate the bacterial cell wall.
No study is available on the genotoxicity of MTA after incorporation of disodium hydrogen phosphate and silver nanoparticles. Genotoxicity is defined as the induction of gene damage, gene mutation and chromosomal breakage. Of all the tests that evaluate genotoxicity, those that evaluate gene mutations are very reliable. The *Salmonella typhimurium*assay or Ames test is a bacterial test widely used to determine the potency of substances that can produce genetic damage and gene mutations \[[@B18], [@B19]\]. The Ames test utilizes *Salmonella*strains with preexisting mutations that are unable to synthesize the required amino acid, histidine, and therefore cannot grow and form colonies in the medium devoid of histidine. New mutations at the site of these preexisting mutations can restore the genes' function and allow the cells to synthesize histidine. These newly mutated cells can grow in the absence of histidine and form colonies. The *Salmonella*mutagenicity test was specifically designed to detect chemically induced mutagenesis \[[@B20], [@B21]\].
The aim of the present *in vitro*study was to evaluate the genotoxicity of MTA, MTA/disodium hydrogen phosphate and MTA/silver nanoparticles using Ames test.
Materials and Methods
=====================
In this study, TA100 strain of *Salmonella typhimurium*was used in order to investigate mutagenicity of MTA (Angelus Dental Industry Products, Londrina, Brazil), MTA/silver nanoparticles (produced in the laboratory)\[[@B21]\], and MTA/disodium hydrogen phosphate (Merck, Darmstad, Germany) with and without S9 mix fraction.
MTA, MTA/silver nanoparticles (average nanoparticle size of 20 nm) and MTA/disodium hydrogen phosphate were prepared at 0.1, 0.01, 0.001 and 0.0001 concentrations \[[@B21]\].
The *Salmonella typhimurium*bacterial strain used in the Ames test carried a mutant gene preventing it from synthesizing the essential amino acid histidine from the ingredients found in the standard bacterial culture medium. In this study, negative and positive control groups were included to assess the accuracy of the tests. The negative control group consisted of 1% dimethyl sulfoxide (DMSO) and distilled water. The positive control group, used to compare the results, consisted of sodium azide (0.5 µg/plate) and 2-aminoanthracene (2.5 µg/plate) as strong mutagenic agents in the absence of metabolic and metabolic activation systems. The TA100 strain of *Salmonella typhimurium* was cultured overnight in nutrient broth. Different concentrations of the test materials were prepared in a histidine-free medium. Glucose minimal (GM) agar plate consisted of distilled water, agar, Vogel-Bonner (VB) salt solution and glucose solution (10% v/v). VB salt medium consisted of warm distilled water, magnesium sulfate, citric acid monohydrate, potassium phosphate dibasic anhydrous, and sodium ammonium phosphate. Before the incubation, each test material and *Salmonella typhimurium*(50 µl) were incorporated into 2 mL of top agar containing distilled water, agar, sodium chloride and histidine/biotin solution (0.5 mM). The mixture was poured on GM agar plates. After solidification of the superficial agar, the plates were incubated at 37^°^C for 48 to 72 h. After proliferation of bacteria on the glucose-minimal agar plate, the histidine-revertant clones were counted using a colony counter. Rat liver enzymes (S9) were used as a metabolic activator. The microsomal enzyme system was induced by phenobarbital in rats and the rat liver was homogenized after 7 days in order to isolate the enzyme. A suspension of *Salmonella typhimurium* with a mixture of rat liver enzymes (S9 mix) and different cofactors, such as glucose-6 phosphate and NADP, were added to the superficial agar. Subsequently, the plates were incubated for 48 to 72 h in 37^°^C without shaking. Then the number of colonies was counted per plate. The ratio of the number of histidine-revertant colonies to spontaneous revertants of the negative control colonies was obtained; if the ratio was ≥2, the experimental material was considered as a mutagenic agent.
Results
=======
[Table 1](#T1){ref-type="table"} presents the number of initial and secondary colonies and their ratios (S9+/S9-). In all experimental groups and at all the concentrations these ratios were \<2; therefore, there was no genotoxicity and there was no need for statistical analysis. The findings of positive and negative controls were as expected in the present study.
Discussion
==========
In this *in vitro* study, the genotoxicity of different concentrations of MTA/silver nanoparticles and MTA/disodium hydrogen phosphate was evaluated, using Ames test. The results showed the safety of these materials in relation to genotoxicity. MTA is a silicate-based cement with numerous therapeutic applications in endodontics. Efforts have always been made to achieve favorable clinical outcomes by improving the physicochemical properties of MTA \[[@B22], [@B23]\]. Some studies have been undertaken in an attempt to decrease its setting time and increase its antimicrobial activity, which are prerequisites for achieving favorable treatment outcomes. In this context, some materials have been incorporated into the MTA powder.
One of the materials incorporated into the MTA composition was disodium hydrogen phosphate. Based on the results of previous studies, incorporation of this material into MTA results in a decrease in setting time, in an increase in tensile strength and in an increase in push-out bond strength; however, the setting pH is not affected \[[@B8], [@B9], [@B13], [@B24]\]. Studies on the cytotoxicity of this composition have reported it to be biocompatible \[[@B14], [@B15], [@B25]\]. Previous studies have used 2.5 and 15 wt% of disodium hydrogen phosphate \[[@B8], [@B26]\], which is different from the present study (100-200 ppm and %1 weight) and it has been recommended that future studies on the genotoxicity of MTA be carried out by incorporating these weight percentages of disodium hydrogen phosphate.
Silver ions have drawn attention in the medical field as significant antimicrobial agents. Silver nanoparticles exhibit less toxicity compared to silver ions due to their small size and high surface-to-volume ratio at a similar concentration, with antimicrobial activity comparable to that of silver ions \[[@B10], [@B16]\]. Studies have shown that incorporation of silver nanoparticles
(100-200 ppm and %1 weight) into MTA results in an increase in their antibacterial activity against *E. faecalis* and *C. albicans* \[[@B11], [@B27]\], which are microorganisms resistant to antimicrobial agents and are found in the majority of cases of endodontic failures \[[@B28]-[@B30]\]. Silver is a toxic agent and its toxicity is concentration-dependent \[[@B23]\]. Previous studies have shown that concentrations of 23-47 ppm are safe. In addition, incorporation of silver nanoparticles into MTA at 1 wt% did not result in a significant difference in the inflammatory reactions induced in rat connective tissue compared to pure MTA \[[@B15]\]. In the present study, incorporation of silver nanoparticles at weight percentages mentioned above did not result in genotoxicity.
######
Mean revertant colony counts after exposure to four concentrations of test materials with and without S9 mix fraction
**Experimental materials** **Colony counts of TA100** **Ratio of TA 100**
------------------------------ ---------------------------- --------------------- ------ ------ -----
**MTA** **0.1** 250 335 0.8 0.7
**0.01** 58 11 0.1 0.6
**0.001** 85 200 0.2 0.5
**0.0001** 60 250 0.1 1.45
**MTA/NaHPO** ~4~ **0.1** 53 0 0.18 0.7
**0.01** 83 200 0.2 0.6
**0.001** 350 650 1 0.5
**0.0001** 395 200 1.27 1.45
**MTA/Silver nanoparticles** **0.1** 400 405 1.3 0.8
**0.01** 3 43 0.2 0.07
**0.001** 80 660 0.2 1.8
**0.0001** NA NA NA NA
In many studies, the biocompatibility of silver nanoparticles and disodium hydrogen phosphate was evaluated by placing their implants in the subcutaneous tissues of rats or in exposure with cells in the cultures media; the histological cross-sections showed the safety of these materials \[[@B12]-[@B15], [@B25]\]. However, the genotoxicity and mutation induction capacity of these materials have not been evaluated to date. Ames test was used in the present study to evaluate the genotoxicity of these materials. In the design of this test, changes are induced in the genome area of *Salmonella* responsible for production of histidine. Therefore, these mutagenic strains cannot proliferate in an environment without histidine; however, if a material has mutagenic activity, it can result in the repair of the iatrogenic defect in the bacterial genome and the bacteria will be able to synthesize histidine again and will be able to proliferate in the culture medium. If the ratio of these new colonies to the initial colonies is ≥2, the material is considered to be mutagenic \[20, 21\]. In the present study, this test was used and none of the materials was shown to be mutagenic.
Conclusion
==========
Based on the results of the present *in vitro* study, MTA/silver nanoparticles and MTA/disodium hydrogen phosphate had no genotoxicity at the concentrations used.
The authors would like to thank the biotechnology research center of Tabriz University of Medical Sciences for their supports.
Conflict of Interest:
=====================
'None declared'.
|
{
"pile_set_name": "PubMed Central"
}
|
{#sp1 .58}
{#sp2 .59}
{#sp3 .60}
{#sp4 .61}
{#sp5 .62}
{#sp6 .63}
{#sp7 .64}
{#sp8 .65}
{#sp9 .66}
{#sp10 .67}
{#sp11 .68}
{#sp12 .69}
{#sp13 .70}
{#sp14 .71}
{#sp15 .72}
{#sp16 .73}
{#sp17 .74}
{#sp18 .75}
{#sp19 .76}
{#sp20 .77}
{#sp21 .78}
{#sp22 .79}
{#sp23 .80}
{#sp24 .81}
{#sp25 .82}
{#sp26 .83}
{#sp27 .84}
{#sp28 .85}
{#sp29 .86}
|
{
"pile_set_name": "PubMed Central"
}
|
INTRODUCTION {#rjz132s2}
============
Duplication of the appendix is a rare congenital anomaly with an incidence rate ranging from 0.004 to 0.0009 \[[@rjz132C1]\]. Due to the rarity of these anomalies, correct identification is important in order to avoid any harmful medico-legal consequences associated with missing a second appendix during surgery.
CASE REPORT {#rjz132s3}
===========
A 21-year-old female patient presented to the emergency department complaining of abdominal pain that started 12 hours ago. The pain started gradually as a stabbing pain around the umbilicus with eventual shift towards the right iliac fossa. She had loss of appetite and persistent nausea that was occasionally accompanied by vomiting. She denied any change in bowel habit or history of similar pain in the past. She had regular periods with no gynecological complaint. The patient was otherwise healthy with no previous past medical or surgical history.
On physical examination, the patient looked ill and tired. She had tachycardia but was afebrile. Examination of the abdomen revealed sever localized tenderness at the right iliac fossa, no guarding or rigidity. Blood tests were within normal values.
Abdominal ultrasonography was performed and revealed a distended appendix measuring 10 mm with appendicolith seen at the base and increased echogenicity surrounding fatty planes. A diagnosis of acute appendicitis was made based on the Ultrasonography findings and the patient was planned to undergo laparoscopic appendectomy. Initial diagnostic laparoscopy showed a small amount of free fluid in the abdomen and a swollen and slightly erythematous partially duplicated 'bifid' appendix Fig. [1](#rjz132F1){ref-type="fig"}. A traditional laparoscopic appendectomy was performed by dissecting the mesoappendix and ligating the base of the appendix. The appendix was then exteriorized and sent for histopathology which showed acute inflammation in the two lumens that were lined by appendiceal mucosa and two layers of musculature due to fecalith impaction which is consistent with true duplication of the appendix Fig. [2](#rjz132F2){ref-type="fig"}.
{#rjz132F1}
{#rjz132F2}
The post-operative course was uneventful and the patient had a full recovery without complications and was discharged on the second postoperative day.
DISCUSSION {#rjz132s4}
==========
Anatomical anomalies of the vermiform appendix are often asymptomatic and their discovery is done intraoperatively the majority of the time. However correctly identifying these anomalies in the setting of acute appendicitis is very important due to the life threatening consequences associated with the failure of treatment and they can often represents a manifestation of more complex developmental intestinal, genitourinary or vertebral abnormalities specially in children.
To better understand these anomalies, we should study the embryology of the normal appendix. During the fifth week of gestation, the appendix originates from a bud at the junction of the small and large bowel and undergoes rapid growth into a pouch. It is only after the 20th week of gestation that the proximal end of this pouch starts growing differentially to give rise to the true cecum that continues to develop into infancy \[[@rjz132C2]\]. Due to the common origin of the appendix and the cecum, true appendiceal duplication is only confirmed when histopathology shows that both specimens are lined with appendiceal mucosa and coated with two layers of musculature which was present in our case \[[@rjz132C3]\]. Although normal embryogenesis of appendix is known, the pathogenesis of its duplication is unclear.
The first to publish a clinical report describing appendiceal duplication was Picoli in 1892. However, with more than one hundred cases reported in the literature to date, multiple classifications have been proposed throughout the years to classify these anomalies and provide the proper management for each variant. Nowadays, the most widely accepted classification is the 'Cave--Wallbridge' classification which was first proposed by Cave in 1936 and later modified by C. Waugh and Wallbridge in 1963 \[[@rjz132C4]\]. It was later modified again by Biermann in 1993 \[[@rjz132C5]\].
In the 'Cave--Wallbridge' classification, anatomical variation of the appendix were grouped into three major categories based on the appendicular localization, namely category A, B and C, with category B and C being further divided into more subgroups.
The first category in the 'Cave--Wallbridge' classification, Category A, describes the presence of a single cecum that gives rise to a completely or partially duplicated appendix with a common base.
However, when two completely separated appendices are present, they are either classified as a type B1 'avian type' when both appendices are located symmetrically on either sides of the ileocecal valve or a type B2 'tenia coli type' when one appendix arises from the cecum normally while the other arises from the anterior tinea coli muscles at a variable distance from the first.
The last, and also the rarest, variant is the type C category which corresponds to the presence of two separate cecums each with its own appendix along with other more rare anomalies such as horseshoe and triple appendices.
In 2017, Nageswaran *et al.* published a comprehensive review of more than 140 cases of appendiceal anomalies published in the English and non-English literature \[[@rjz132C6]\]. According to Nageswaran *et al.*, type B2 was by far the most common with 73(59%) cases Table [1](#rjz132TB1){ref-type="table"}. Table 1The modified Cave--Wallbridge classification and the frequency of each type of appendiceal duplication.Category of anatomical variationFrequency *n*(%)\*CW Type22 (18)CW Type B73 (59) Type B18 (6) Type B246 (37) Type B unable to classify further19 (15)CW Type C10 (8)Horseshoe6 (5)Triple2 (2)A Miscellaneous configuration not fitting any category6 (5)[^1]
Since the Nageswaran *et al.* review, only four cases of appendiceal anomalies have been reported in the literature other than our case.
The first of these was published by Triki *et al.* \[[@rjz132C7]\] in 2017, when they reported on a case of a 35-year-old female who presented with right iliac fossa pain. Preoperative workup leads to a diagnosis of complicated acute appendicitis. During surgery, a localized peritonitis in relation to two phlegmonous appendages linked by a common base was identified. Histological examination confirmed the duplicated and phlegmonous appearance of the appendix consistent with a Cave--Wallbridge category A.
Ayoub *et al*. \[[@rjz132C8]\] also published an interesting case of a 30-year-old female who underwent open appendectomy for acute appendicitis. During surgery, two appendices on either side of the ileocecal valve were identified with acute appendicitis in only one of them which is consistent with a Cave--Wallbridge category B1 duplication. Both appendices were resected to avoid any future diagnostic confusion.
Lieu *et al*. \[[@rjz132C9]\] was the last to report a case of duplicated appendix in a 22-year-old man who was found to have a horseshoe appendix with the two bases forming a circle, each communicating with the cecum.
CONCLUSION {#rjz132s5}
==========
Although appendiceal duplication is rare, it should not be ignored by the surgeon as it may lead to serious ethical and legal issues if not recognized. Surgeons should always be aware of this possibility even in a patient with a history of a previous appendectomy. To the best of our knowledge, this case is the first to be reported in Jordan.
CONFLICT OF INTEREST STATEMENT {#rjz132s6}
==============================
None declared.
FUNDING {#rjz132s7}
=======
The authors received no financial support for the research and/or authorship of this article.
[^1]: As reported by Nageswaran *et al*.
|
{
"pile_set_name": "PubMed Central"
}
|
Background {#sec1}
==========
Hepatitis A virus (HAV) is primarily spread via the faecal-oral route, either by ingestion of contaminated food and water or direct contact with an infected person. The disease is characterised by fever, malaise, anorexia, nausea, abdominal pain and jaundice though some persons may have mild or no symptoms. Asymptomatic infection is particularly common in children under 5 years of age \[[@ref1]\].
The incidence of HAV infection has markedly declined in Australia since the 1990s when the notification rate of HAV was consistently above 10 cases per 100 000 population, but since 2010 has averaged less than one case per 100 000 population \[[@ref2]\]. Currently the most common source of HAV acquisition in Australia is in people who have travelled to high prevalence countries and among men who have sex with men (MSM). In Australia the HAV vaccine is recommended for people who are at higher risk for infection and since 2005 has been offered to indigenous children ⩽2 years in four Australian states \[[@ref3]\].
Widespread foodborne hepatitis A outbreaks have occurred in Australia, the largest being in 1997 associated with contaminated locally produced oysters \[[@ref4]\], followed by an outbreak in 2009 associated with imported sun-dried tomatoes \[[@ref5]\]. More recently in 2015 and 2017 imported frozen berries were found to be the source of infection for two multi-state outbreaks in Australia \[[@ref6], [@ref7]\]. In addition, there has been a rise in locally acquired HAV among MSM in several Australian states and territories since 2017 related to the ongoing outbreak of HAV genotype 1A among this high risk group in Europe \[[@ref8], [@ref9]\].
In March 2018 a multi-state investigation was launched following the identification of locally acquired HAV cases in multiple Australian states. These cases had not travelled outside of Australia and reported no other high risk behaviour for HAV. Routine genetic sequencing of the virus identified a unique strain of HAV genotype IB that had not been seen in Australia previously. Initial case interviews using hypothesis generating questionnaires revealed high levels of consumption of frozen pre-packed fruits, with 100% of early cases having consumed the same imported frozen pomegranate aril product. This paper describes the investigation into this outbreak and the evidence confirming the source.
Methods {#sec2}
=======
Case finding {#sec2-1}
------------
Hepatitis A infection is a nationally notifiable disease under legislation in Australia and laboratories and medical practitioners must report confirmed cases to public health authorities. All cases are interviewed by trained public health officers using a national standardised questionnaire.
In Australia, a confirmed case of HAV is defined as a person with either laboratory definitive evidence (detection of HAV by nucleic acid testing) or laboratory suggestive evidence (detection of HAV-specific immunoglobulin M in the absence of recent vaccination) and clinical evidence or laboratory suggestive evidence and epidemiological evidence \[[@ref10]\].
Outbreak case definition {#sec2-2}
------------------------
For the outbreak investigation, cases were classified as confirmed or probable.
A confirmed case was defined as a person who met the national hepatitis A surveillance case definition, genotyped as HAV IB with the characteristic outbreak genetic sequence, and with date of symptom onset (or date of testing if onset date not available) from 1 January 2018 and who must have spent some of their exposure period (15--50 days prior to onset of illness) in Australia.
A probable case was defined as a person who met the national hepatitis A surveillance case definition, with genotype IB but genetic sequence not yet compared (or met the probable national hepatitis A surveillance case definition \[[@ref10]\] and had an epidemiological link to a confirmed outbreak case), and onset from 1 January 2018, and who must have spent some of their acquisition period (15--50 days prior to onset of illness) in Australia.
Epidemiological investigation {#sec2-3}
-----------------------------
A national standardised questionnaire is used for all HAV cases in Australia that focuses on food consumption and high risk activity for the acquisition of HAV. Following initial interviews this questionnaire was enhanced for further notified cases to include more questions on salad items and frozen fruit consumption. Initial cases were re-interviewed.
In addition, a prospective, frequency matched case--control study was conducted, in order to generate additional analytic evidence to support a causal association between consumption of the suspected source product and the outbreak of locally acquired HAV. This study was conducted under the Public Health Act for each jurisdiction so ethical approval was not required.
The confirmed case definition for the case--control study was consistent with the outbreak investigation case definition, except that cases were excluded if they were non-English speaking or unable to provide coherent answers to interview questions, unable to provide date of onset of illness or jaundice, or had close contact with a person known or suspected to have hepatitis A during the case\'s exposure period. One case was excluded due to not being able to provide a date of onset and two cases were excluded that were secondary cases. Cases notified between 13 April 2018 and 8 June 2018 were enrolled and interviewed using a standardised questionnaire containing questions on eligibility, consent and demographics and structured questions on a limited range of shellfish, dried fruits and fresh and frozen fruits. A probable case definition was also defined for the study, but was not required as all enrolled cases were confirmed.
Controls were recruited from jurisdictional notifiable disease databases, being either previously notified cases of salmonellosis, campylobacteriosis or cryptosporidiosis where necessary. It was attempted to frequency match controls to cases by age group (0--14, 15--39, ⩾40 years), in a 2:1 ratio. During this period, unrelated but concurrent disease outbreaks in multiple jurisdictions constrained public health resource capacity to continually recruit eligible controls; therefore a consensus decision was reached to close the case--control study once sufficient study power had been reached to demonstrate a statistically significant association between consumption of the implicated food source and HAV infection. To minimise selection bias, controls were recruited from the same or a neighbouring local government area as cases in order to account for potential variability in retail supply distribution of the suspected food source, and hence potential for exposure. To ensure the control was likely to be well and eating normally during the exposure period of the corresponding hepatitis A case, the specimen collection date for controls was required to be within the 2 weeks prior to the onset date of the corresponding hepatitis A case. Controls were questioned on potential exposures during the 5 week period prior to their onset of illness. Control exclusion criteria included: past infection with HAV; previous vaccination for HAV or receiving normal human immunoglobulin in the 2 months before date of diarrhoea onset; being an enteric pathogen case included in a separate outbreak investigation; not being contactable by telephone; non-English speaking or unable to provide coherent answers to interview questions; having travelled overseas in the 2 months prior to their date of diarrhoea onset; having lived in a country with high HAV endemicity for at least 1 year of the first 5 years of life; or having close contact with a known or suspected HAV case in the 2 months prior to their date of diarrhoea onset. All participants gave verbal consent to participate and were interviewed by telephone.
Questionnaire responses were entered into a centralised database, then exported and analysed using Stata™, version 13 \[[@ref11]\]. Case and control demographic details were compared using Pearson *χ*^2^ and Wilcoxon rank sum test for gender and age respectively. Unconditional univariable analysis was used to calculate odds ratios (OR) and 95% confidence intervals (CIs) for the association between illness and foods consumed using Fisher\'s exact test, with *P* values \<0.05 considered statistically significant. Where there was zero occurrence of an outcome for exposure to a variable in cases or controls, exact logistic regression was used to generate ORs and *P* values. Multivariable analysis employed stepwise logistic regression, with hepatitis A illness as the dependent variable, and food exposures with a *P* value \<0.1 on univariable analysis included as independent variables, and excluded from the multivariable models in a reverse step-wise fashion. To account for frequency matching by age group, age was included in all multivariable models.
Laboratory investigation {#sec2-4}
------------------------
Serum samples for serologically diagnosed cases of HAV are routinely sent for further laboratory analysis. Samples were tested for HAV RNA by reverse transcription-polymerase chain reaction (RT-PCR) and if positive, HAV was sequenced employing the HAV network (HAVNET) protocol \[[@ref12]\]. This procedure uses a nested RT-PCR to amplify a fragment of approximately 500 nucleotides spanning the HAV VP1/2A junction region which can be directly sequenced. After being analysed, the sequence data were uploaded to the National Centre for Biotechnology Information (NCBI) BLAST (Basic Local Alignment Search Tool) to determine the HAV genotype. Phylogenetic analysis compares isolate sequences against other sequences in the global HAVNET database.
Food investigation {#sec2-5}
------------------
Opened and unopened packets of the implicated product belonging to the outbreak cases were collected for HAV testing as were unopened packets collected from retail stores and the supplier. The same product from a subsequent import batch that had not been repackaged or put out to sale was also sampled at the implicated factory. Other leftover frozen fruit products from cases freezers were also sampled opportunistically when they were volunteered by cases. Food samples were tested for HAV at the National Measurement Institute.
The testing method was based on ISO/TS Specification 15216-1, with Viral RNA Extraction by a Zymo Research ZR Viral RNA kit and HAV RNA sequence detection by a Genesig HAV Real-Time PCR kit. The sequence selected is proprietary but HAV specific. Controls included: positives (HAV sequence and internal spikes), negatives (template and extraction blanks) and replicates to monitor extraction and assay performance.
Controls included: positives (extraction blank spike, matrix spike, standard curve); negatives (extraction blank, non-template controls); internal control for all samples to ensure reverse transcription for the reverse transcription real time quantities PCR assay. Any food samples found positive for HAV were then forwarded to the laboratory that processed the human clinical samples to attempt to recover the HAV genotype and sequence by the method mentioned above.
The supply chain of the implicated product was traced back as far as possible by the Australian Federal Department of Agriculture. Local health authorities inspected the Australian food processing facility and assessed their operating procedures.
Results {#sec3}
=======
Description of the outbreak {#sec3-1}
---------------------------
Thirty confirmed outbreak cases of HAV infection, genotype IB with identical sequences were identified during this investigation. Outbreak cases were reported from all but one Australian jurisdiction including New South Wales (15 cases), Victoria (six cases), Western Australia (three cases), Northern Territory (two cases), South Australia (two cases), Queensland (one case) and Australian Capital Territory (one case). Cases included eighteen females and twelve males, age range 4--74 years (median 30.5 years), with 25 of the 30 cases (83%) hospitalised for their illness. One death was reported, but the cause of death is yet to be determined. Three cases were secondary infections, epidemiologically linked to an earlier confirmed case and thought to have been acquired by sexual contact. The outbreak occurred over a 123 day period, onsets ranged from 31 January 2018 to 18 June 2018 ([Fig. 1](#fig01){ref-type="fig"}). Fig. 1.Weekly epidemic curve of confirmed outbreak cases by onset date (*n* = 30).
Food consumption investigation {#sec3-2}
------------------------------
[Table 1](#tab01){ref-type="table"} describes the risk factors of cases. Food consumption was available for 26 primary cases, excluding the case that died. A high proportion of cases (23/26, 88%) reported purchasing groceries from one specific supermarket chain (supermarket A). Twenty-two of 26 (85%) primary cases reported consuming at least one type of frozen fruit. The median number of different frozen fruit products consumed per case was three (range 1--8). Consumption of frozen pomegranate arils was the most frequently reported food (18/26, 69%). Of these, 18/18 (100%) reported purchasing frozen pomegranates arils at supermarket A. This supermarket was the exclusive stockist of the product. Of the remaining eight cases: one case consumed pomegranate arils in a salad prepared at a cafe, which on investigation was revealed to be the implicated frozen pomegranate aril product. One case consumed salad containing pomegranate arils from several cafes but follow-up was not possible to find out what brand of arils were used in the salads. The final six cases could not recall eating pomegranate arils. Additionally the case that died had an unopened packet of the implicated product in her freezer. Table 1.Risk factors among outbreak casesRisk factorNumber exposedPer cent exposed confirmed casesAll outbreak cases (*n* = 30)Contact with HAV case in previous 15--50 days3/3010Travel outside Australia1/303Primary cases excluding the deceased case (*n* = 26)Food consumption Groceries from supermarket A23/2688 Groceries from supermarket B15/2658 Fresh berries -- strawberry12/2646 Fresh berries -- blueberry12/2646 Bagged lettuce -- mixed leaf15/2658 Dried fruit -- any13/2650 Shellfish -- any5/2619 Tomatoes sun/semi-dried4/2615 Frozen mixed berries8/2631 Frozen raspberries11/2642 Frozen strawberries10/2638 Frozen blueberries11/2642 Frozen blackberries3/2612 Frozen cherries5/2619 Frozen mango8/2631 Frozen smoothie mix8/2631 Frozen coconut2/268 Frozen pineapple5/2619 Frozen banana3/2612 Frozen acai puree3/2612** Frozen pomegranate arils18/2669**Cases who purchased frozen pomegranate arils (*n* = 18)** Frozen pomegranate arils purchased at supermarket A18/18100**
[Figure 2](#fig02){ref-type="fig"} shows the recall of any pomegranate aril consumption by primary cases over time. All cases (10/10) in the first half of the outbreak reported consumption of pomegranate arils whereas only 10/16 (62.5%) could recall eating any pomegranate arils in the last half of the outbreak. Fig. 2.Weekly epidemic curve of primary outbreak cases with food exposure information by onset date and pomegranate aril consumption (*n* = 26).
Case--control study {#sec3-3}
-------------------
Thirteen cases (*n* = 13) and 21 controls (*n* = 21) were enrolled into the study, which ran for eight weeks, from 13 April 2018 to 8 June 2018.
There was no statistically significant difference in gender (*P* = 0.36); or age (*P* = 0.24) between cases and controls.
The results of the univariable analysis found that consumption of 'frozen pomegranate arils' had the strongest association and was significantly associated with illness (OR 45.0, 95% CI 3.8--2065.4, *P* \< 0.001); as was consumption of 'frozen strawberries' (OR 20.6, 95% CI 2.6--∞, *P* = 0.001) and 'frozen raspberries' (OR 17.1, 95% CI 1.5--826.4, *P* = 0.007). No other items consumed were significantly associated with illness ([Table 2](#tab02){ref-type="table"}). Table 2.Univariate analysis of selected food exposuresFood item consumedCasesControlsCrude OR95% CI*P* value*n*%*n*%ShellfishOysters[^a^](#tfn2_1){ref-type="table-fn"}0029.520.70.0--8.70.513Mussels17.714.81.70.02--137.31Other shellfish215.4314.30.80.07--10.61Frozen fruitsFrozen mixed berries430.8523.81.40.2--8.60.704Frozen strawberries[^a^](#tfn2_1){ref-type="table-fn"}646.20020.62.6--∞0.001[\*](#tfn2_2){ref-type="table-fn"}Frozen blueberries646.2314.35.10.8--38.90.057Frozen raspberries646.214.817.11.5--826.40.007[\*](#tfn2_2){ref-type="table-fn"}Frozen pitted cherries215.414.83.60.2--225.00.544Frozen mango430.8314.33.00.4--24.70.377Frozen pineapple chunks430.829.54.20.5--52.50.173Frozen banana chunks17.6929.520.80.01--16.91Frozen acai puree[^a^](#tfn2_1){ref-type="table-fn"}17.69001.60.9--∞0.382Frozen pomegranate arils969.214.845.03.8--2065.4\<0.001[\*](#tfn2_2){ref-type="table-fn"}Other frozen fruit[^a^](#tfn2_1){ref-type="table-fn"}215.4004.50.92--∞0.0478[\*](#tfn2_2){ref-type="table-fn"}Other food itemsOther pomegranates[^a^](#tfn2_1){ref-type="table-fn"}00010.80.5--0.90.015Semi-dried tomatoes323.129.52.70.3--36.40.364[^1][^2]
In the multivariable analysis models the only variables that remained in the final model were 'frozen pomegranate arils' and 'age' (OR 43.4, 95% CI 4.2--448.8, *P* = 0.002).
Environmental investigation, product trace back and control measures {#sec3-4}
--------------------------------------------------------------------
The implicated product was 180 g bags of frozen pomegranate arils. This product was stocked exclusively by supermarket A and distributed to all states and territories. Food Standards Australia and New Zealand (FSANZ) co-ordinated a national consumer level recall of the product on 7 April 2018. The product originated from a manufacturer in Egypt, repacked in Australia (transferred into branded packaging) and placed on the Australian market from October 2017 until the recall on 7 April 2018. All but one primary case in this outbreak had illness within one incubation period of the product recall. The one primary case that had an onset of 55 days after the recall was unaware of the recall and reported consumption of the product up until their onset date.
The investigation of the Australian food processor that repackaged the product in Australia found they were operating with all appropriate hygiene and food safety control processes, concluding that the local food processor had no process deficits to suggest it was the cause of the contamination, and that contamination most likely occurred before the product arrived in Australia.
Thirteen different packets of the implicated product were sampled by health authorities as were other frozen fruit packets surrendered by cases, including frozen banana \[[@ref2]\], strawberries \[[@ref1]\], coconut \[[@ref1]\] and acai puree \[[@ref1]\]. HAV was only detected in one opened packet of the pomegranate product and one unopened packet of a frozen banana product collected from the same case\'s house. An investigation suggested that the positive banana result was likely due to cross-contamination of specimens in the laboratory. Further testing of unopened banana chunks packets from the factory did not detect any HAV.
Testing of the subsequent imported batch of the implicated product that had not been repackaged or released to market was conducted by the Australian wholesaler and one of the 10 pomegranate arils sub samples from this testing was detected with HAV.
All the PCR positive food samples that were referred for further testing did not have HAV detected at the referring laboratory and so were unable to be genotyped or sequenced. This was likely due to the difference in test methods and test sensitivities used between laboratories.
Documentation provided by the Australian importing company suggested that the Egyptian manufacturer of the suspected source product was the same manufacturer linked to a 2012 outbreak of HAV genotype IB associated with imported frozen pomegranate arils in Canada \[[@ref13]\]. In response to this outbreak the Australian Department of Agriculture and Water Resources commenced a 100% inspection and testing schedule of future consignments from the Egyptian manufacturer of the pomegranate arils linked to outbreak.
Discussion {#sec4}
==========
Australia experienced a nationwide outbreak of HAV in the first half on 2018 affecting 30 people (27 primary cases and three secondary cases). Analysis of the HAV sequence of the 30 cases identified all cases were HAV genotype 1B with an identical genetic sequence that was unique on the global HAVNET database. Based on food consumption history, imported frozen pomegranate arils were identified to be likely source of the infection with 19 of the 26 primary cases that could be interviewed reporting consumption of a specific product imported from Egypt. Additional evidence was obtained from the case--control study that demonstrated that the implicated frozen pomegranate aril product had the strongest association with illness. The detection of HAV RNA in food samples provided further support for frozen pomegranate arils being the source of the outbreak.
An enhanced national surveillance of hepatitis A project began in Australia in July 2017 prior to the detection of this outbreak. This project aims to gain improved understanding of the risk factors and molecular epidemiology of HAV in Australia, and to detect clusters of locally acquired hepatitis A to enable rapid public health action. The project was proposed after the national foodborne HAV outbreak associated with the consumption of frozen mixed berries in 2015 \[[@ref6]\], where HAV sequencing assisted in identifying outbreak cases. A similar surveillance project in the Netherlands allowed health authorities to identify food-associated infections that would not have been detected otherwise, this included cases that could be linked to international outbreaks. Additionally, these potentially foodborne clusters were able to be identified without an increase in cases \[[@ref14]\]. Prior to 2017, locally acquired cases in Australia were very rare, so an increase in locally acquired cases such as in 2009 with the sundried-tomato outbreak \[[@ref5]\] and 2015 in the frozen mixed berries outbreak \[[@ref6]\] stood out as unusual and prompted public health investigation. In 2017--2018, several states in Australia saw an increase of locally acquired HAV (genotype 1A) due to person-to-person spread, among cases with either male to male sex or injecting drug use as a risk factor \[[@ref8], [@ref9]\]. Fifty-three per cent of HAV cases in NSW in 2017 were locally acquired, a 250% increase on the proportion locally acquired in the previous 5 years \[[@ref7]\]. Considering the small number of cases in this outbreak spread over many months, against a background of increased locally acquired cases, without genetic sequencing information this outbreak may not have been detected in such a timely manner.
A suspect food vehicle for this outbreak was detected 70 days after the onset of the first case, after nine cases had been reported, five of which were confirmed as having the HAV 1B genetic sequence. At this stage of the investigation all nine cases had reported eating the same nationally distributed frozen pomegranate aril product that was imported from Egypt, where HAV genotype 1B is reported to be common \[[@ref15]\]. No other food had a similar high frequency of consumption, the biological plausibility and a geographic distribution that matched the cases in this outbreak. This was deemed sufficient evidence to implicate the food product and trigger a recall. A case--control study was nevertheless undertaken to provide additional analytic evidence to support the association between consumption of the product and illness. This was considered to be a prudent step because obtaining confirmatory microbiological evidence of HAV in suspect foodstuff is difficult due to the variable distribution of the virus on the food and owing to the level of the virus in the food being below the level that can be detected; this has been the case during previous HAV outbreak investigations in Australia and overseas \[[@ref5], [@ref16]\].
It was interesting to note that all 10 (100%) of the cases that fell sick prior to the recall could recollect eating any pomegranate arils whereas only 10 of 16 (63%) could recollect eating any pomegranate arils after the recall. Typically a nationwide recall and the media surrounding such a recall would bias recollection towards the implicated product, but in this case that did not happen. Given the certainty that the latter cases had the same source of infection based on the 100% homology of this sequence type in the outbreak cases, it may be that later cases who reported not eating pomegranate arils either did so as an ingredient in a food dish and didn\'t realise it or were in fact secondary cases to unidentified primary cases. Spill-over of HAV from food-exposed people to the general population is an important consideration in such investigations. Much of the public health intervention in individual HAV cases is follow-up of contacts for vaccination, but undiagnosed cases represent an additional risk for spill-over. A recent investigation of spill-over from a MSM outbreak in the Netherlands \[[@ref17]\] found that for every two HAV cases with MSM risk factors notified with the MSM strain, there was one notified who did not have MSM risk factors. If this occurs during a foodborne outbreak it would mean that as time goes on the epidemiological investigation becomes more difficult as the strength between the relationship of consumption and disease weakens. This highlights the importance of being able to identify related cases early, when their exposure risks are more likely to be limited to the source food.
Even without spill-over effects, identification of a specific food vehicle can be difficult in outbreaks due to poor recall by cases, especially considering the long incubation period of HAV. Although imported, minimally processed foods that may be consumed without cooking are often the focus of HAV foodborne investigations, investigators cannot discount an infected worker potentially contaminating a local ready-to-eat product or ingredient. Initially bagged lettuce and fresh local blueberries both had high consumption rates in this investigation, but the feasibility of these locally farmed products creating the temporal and spatial pattern of infection was investigated and found to be untenable. Many different frozen fruit items were mentioned by a number of cases which led to the revision of the national questionnaire that included all imported frozen fruits that were currently on sale at the implicated supermarket. It was not until cases were specifically asked about these individual products that many recalled consuming them. An additional difficulty was then found as cases more often than not reported consuming numerous frozen fruit products. This could present a problem for identification of the source in the analytical analysis and may also raise concerns about cross contamination at the packing plant. In this outbreak the presence of the genotype 1B helped direct lines of investigation of the source as this genotype had been associated with HAV outbreaks with foods imported from Egypt \[[@ref13]\] and Turkey \[[@ref5], [@ref16]\] and at the time of the investigation the only frozen fruits on shelves at supermarket A imported from that region were frozen pomegranate arils from Egypt and frozen cherries from Turkey.
HAV was detected in three food samples. One pomegranate aril sample that had not been repackaged or left the factory, one pomegranate aril sample and one banana sample which were collected from a case\'s house. The genotype was not able to be determined from these food samples, which is often the case in HAV outbreaks \[[@ref13], [@ref16]\]. However, the combination of other supporting descriptive epidemiological evidence, analytical epidemiological evidence and spatio-temporal distribution of the product indicates that the frozen pomegranate aril product was the source of the outbreak without this genotyping information.
Imported frozen pomegranate arils have been associated with HAV outbreaks previously, in Canada from an imported Egyptian product in 2012 \[[@ref13]\] and in the USA from an imported Turkish product in 2013 \[[@ref16]\]. Although it was discovered that the same Egyptian manufacturer likely provided the pomegranate arils for this current Australian outbreak and the 2012 Canadian outbreak, a comparison of the HAV genotype IB sequences from the two outbreaks showed seven nucleotide differences from an overlapping 363 nucleotides (97.5% homology) of the HAV VP1-2A region, indicating that they were unlikely to be genetically related. As no farm or processing facility trace back in Egypt was possible, it is not feasible to identify how the product became contaminated before entering Australia. However, evidence that pomegranate arils from this region have been previously associated with HAV contamination and that HAV is endemic in this area \[[@ref15], [@ref18]\] supports the likelihood of some pre-import processes being the source of the contamination. It is therefore reasonable to infer that minimally processed, ready-to-eat foods such as frozen fruit products that come from this HAV endemic area are susceptible to HAV contamination and may be of greater risk to susceptible consumers.
In a population like Australia with low background immunity there remains potential for further foodborne outbreaks of hepatitis A infection. A large number of cases in this outbreak were hospitalised (83%) and one person died, demonstrating that relatively small outbreaks such as this have the potential for serious morbidity, mortality and health system costs. Growing international food trade and the popularity of lower cost convenience foods means that imported frozen fruits will remain a commonly consumed product.
The onus for preventing unsafe food from coming into Australia is placed on the importer. It is an offence to import food into Australia if the importer knows, or ought reasonably to have known, that it poses a risk to human health. Importers must ensure that supply chains for foods have effective control strategies in the form of good agricultural practices and good hygienic practices. After the 2015 frozen berry outbreak, the Australian Department of Agriculture and Water Resources implemented a programme of inspection and testing on imported berries. However, this only includes tests for *Escherichia coli* as an indicator of overall process hygiene and only to fresh and frozen berries. It is not reasonable to test for HAV at the border due to the difficulties in detecting HAV in food described above. Importantly, although being of equal risk to frozen imported berries owing to the minimal processing of the product prior to consumption, other imported frozen fruit products including pomegranate arils, are not included in this screening process.
There is an urgent need for food safety recommendations in Australia to be re-assessed in relation to non-berry frozen fruit products. In the absence of control of HAV in imported food, consumers must be made more aware of the risks involved in consuming imported minimally processed ready-to-eat foods, such as imported frozen pomegranate arils and other frozen fruit products. They also need to be made more aware of the steps required to prevent disease, such as being vaccinated for HAV and the cooking steps required to eliminate pathogens such as HAV in food.
Conclusions {#sec5}
===========
Routine genetic sequencing of HAV is a necessary tool to detect and investigate outbreaks in Australia where person-to-person locally acquired infections are re-emerging in certain populations. Imported frozen fruits have now been associated with three recent outbreaks of HAV in Australia, and given the difficulty in detecting HAV in food and the growing popularity of such convenient health foods, consumers need to be informed of the risks of these products if they are consuming them without further cooking. A re-assessment of the risk of these types of imported foods to a susceptible population is strongly recommended.
The authors acknowledge the contribution of surveillance officers in each of the states and territories of Australia; OzFoodnet which is funded by the Australian Government Department of Health and the multijurisdictional outbreak investigation team including the Food Safety Unit from the Victorian Department of Health and the Victorian Infectious Diseases Reference Laboratory that conducted the genetic sequencing of HAV isolates the Food Safety and the National Measurement Institute that conducted the food testing.
Conflict of interest {#nts3}
====================
None.
N. Franklin [0000-0002-1529-9979](0000-0002-1529-9979).
This research received no specific grant from any funding agency, commercial or not-for-profit sectors.
[^1]: Exact logistic regression.
[^2]: Statistically significant at *α* = 0.05.
|
{
"pile_set_name": "PubMed Central"
}
|
Introduction {#Sec1}
============
The purpose of the Materials Genome Initiative (MGI) is to accelerate the discovery of novel materials by means of modern computational techniques and data mining methods^[@CR1]--[@CR3]^. Successful examples reported so far include solar water splitters, solar photovoltaics, topological insulators, scintillators, clean energy materials, piezoelectrics, thermoelectrics, catalysts, hydrogen storage materials, and Li-ion batteries^[@CR2],\ [@CR3]^. With the emergence of MGI, an exciting trend has recently appeared, utilizing collaborative informatics technology, e.g. data collection and data-mining, to extract meaningful information and patterns from massive data for the fast design and development of novel materials^[@CR4]^. The core task is to efficiently leverage various open-source data with state-of-the-art data-mining algorithms. In the fields of physical metallurgy, vast new data have been recently collected and reported for the mixing properties of binary alloy systems. This provides a unique opportunity to construct a visual miscibility map for binary alloy systems by mining these large numbers of data sources.
The miscibility of a binary alloy system, which indicates the tendency of two elements to form either a homogeneous solid solution or an intermetallic compound, plays an important role in the development of high-performance metallic materials such as diffusion barrier materials, bulk metallic glasses, damage-resistant nanomaterials, microelectronics, or superalloys^[@CR5],\ [@CR6]^. From a thermodynamical point of view, an immiscible system corresponds to a positive (i.e., endothermic) formation enthalpy^[@CR7]--[@CR9]^ (e.g., Cu-Nb systems^[@CR10],\ [@CR11]^). At the other extreme, a binary alloy system may readily form intermetallic compounds, or a homogeneous or clustered solid solution, typically with a solubility of about 10%^[@CR5],\ [@CR12]--[@CR14]^. For such miscible cases, the alloying process is exothermic, with a negative formation enthalpy (e.g., Cu-Ti system)^[@CR15]^. Several heuristic rules have been proposed to qualitatively predict the alloying behavior or reactivity of two metals, of which the most important ones are: the electronegativity rule^[@CR16]^, the Hume-Rothery rule^[@CR17]^ and the Darken and Gurry maps^[@CR18],\ [@CR19]^. A very successful semi-quantitative theory has been proposed by Miedema *et al*.^[@CR12]^ in the late 1970's to distinguish the negative (miscible) and positive (immiscible) systems. This model makes it possible to predict the formation enthalpies of binary alloy systems in a semi-quantitative manner^[@CR20]^. However, the model is sensitive to the choice of its parameters, which are determined empirically. The most critical problems with Miedema's model are: a) it needs more reliable experimental data to validate, improve and extend it^[@CR12],\ [@CR13],\ [@CR21]--[@CR23]^; b) as will be shown later, a large scatter of the results occurs when the formation enthalpy of the system is close to zero, i.e., for weakly alloying systems; and c) several outliers need be sorted out before it can be reliably used to guide the development of novel alloys.
The validation of such empirical and semi-quantitative rules depends generally on the availability of reliable and sufficient thermodynamic data. The recent progress in the development of the experimental techniques, thermodynamic analysis and modern first-principles methods provides an opportunity to improve the database of alloying behavior. For example, Kleppa *et al*.^[@CR24]--[@CR35]^ published experimental values of formation enthalpies with high accuracy, leading to updates in the Landolt-Boernstein database^[@CR7]^, the handbook of binary alloy phase diagrams compiled by Okamoto^[@CR9]^, and the alloy phase diagram database organized by ASM community^[@CR8]^. In addition, modern high-throughput first-principles (HTFP) calculations^[@CR2],\ [@CR36]--[@CR40]^ provide a unique way to check and compare the database from different sources. All of these data sources provide not only a pathway for a global data mining of alloying behavior of binary alloy systems, but an opportunity to validate the widely used Miedema theory and compare with HTFP method^[@CR21]--[@CR23]^.
In this article we first present a global collection and survey of the alloying behavior of binary systems based on the most recent phase diagrams, thermodynamic data and HTFP calculations. Second, we propose a data mining scheme to classify miscible and immiscible systems based on a general consideration of thermodynamic rules. Note that such data mining is not a trivial task; one may obtain a misleading conclusion if the data interpretation is not accompanied by a proper statistical analysis. It should be also noted that the requirement of massive data provides a foundation of the reliability of the present scheme for data mining, which however limits the present method to be used when sufficient data are unavailable for statistical analysis. Third, a series of two-dimensional miscibility maps are constructed based on several physically relevant descriptors to distinguish the immiscible systems from miscible ones by pattern recognition. Fourth, the neural network algorithm and elemental similarity are used to predict the alloying behavior of unknown systems, thus providing a complete miscibility map. Finally, a thorough validation of the classical Miedema theory and a comprehensive comparison with HTFP method is performed by statistical analysis.
Results {#Sec2}
=======
Classification of miscible and immiscible alloy systems {#Sec3}
-------------------------------------------------------
In all, 44 metallic elements comprise the 27 transition metals (TM), including Zn, Cd and Hg, and 17 lanthanide (rare earth (RE)) metals, including Sc and Y. Because of the scarcity of experimental and HTFP data for binary alloy systems consisting of binary lanthanide metals, i.e. RE-RE, we shall not discuss this group except for three binary systems of Sc-Y, Sc-La and La-Y. Consequently, we investigate a total of $\documentclass[12pt]{minimal}
\usepackage{amsmath}
\usepackage{wasysym}
\usepackage{amsfonts}
\usepackage{amssymb}
\usepackage{amsbsy}
\usepackage{mathrsfs}
\usepackage{upgreek}
\setlength{\oddsidemargin}{-69pt}
\begin{document}$${C}_{27}^{2}$$\end{document}$ = 351 + 459 + 3 = 813 binary alloy systems in the present study. The classification of different solid solutions (with positive and negative formation enthalpy) is not an easy task since there is no direct unified experimental data to distinguish them. However, the formation enthalpy provides an energetic criterion for this purpose. After a global survey, we found 35 binary alloy systems where a disagreement appeared between phase diagram analysis and HTFP calculations. These are listed in Table [1](#Tab1){ref-type="table"} as "outliers". As regards this group, our final choice of miscibility is based on an evaluation of the available experimental information, the similarity of chemical elements, and a global justification by comparison of different data sources. Remarks are added in the last column: SSL: low solid solubility; SSH: high solid solubility; COM: formation of compounds; COM1: only one compound reported; IMM: immiscibility gap exists.Table 1The binary alloy systems disagreed between phase diagram information and high throughput first principles (HTFP) calculations.Alloy systemsAlloying behaviorRemarks^\*^HTFPPhase diagramPresent choiceAg-Hg+−−COMAu-Cr+−+COM1Au-Hg+−−COMCd-Cu+−−COMCd-Nb+−+COMCo-Cr+−−COMCo-Ir+−−SSHCo-Os+−−SSHCo-Rh+−−SSHCo-Ru+−−SSHCr-Fe+−−COM1Cr-Mn+−−COMCr-Re+−−SSHCr-Ru+−−SSHCr-Tc+−−COMCr-Zn+−−COMCu-Hg+−−COM1Cu-Nb−++IMMFe-Os+−+SSHFe-Ru+−+SSHHf-V+−−COMHg-Mn+−−COMHg-Ni+−−COMHg-Zn+−−COMMn-W−+−IMMMn-Y+−−COMMo-Sc−++SSLMo-Tc+−−COMNi-Re−+−SSLOs-Pt+−+SSHRe-W+−−COMSc-Y+−+SSHTc-W+−−COMTi-Zr+−+SSHV-Zr+−−COM1The present choice is based on the estimation on the reliability of available information of phase diagram and thermodynamic data and elemental similarity. Plus symbol represents the immiscible systems, and minus symbol indicates the miscible or compound-formation systems.\*Note: SSL: low solid solubility; SSH: high solid solubility; COM: formation of multiple compounds; COM1: only one compound reported; IMM: immiscibility gap.
Special binary systems deserve particular attention. For the Ag-Hg, Au-Hg, Co-Cr, Cr-Mn, Cr-Tc, Cr-Zn, Hg-Mn, Hg-Ni, Hg-Zn, Mn-Y, Mo-Tc, Re-W, and Tc-W systems, the formation of stable intermetallic compounds have been reported in the published phase diagrams. Thus, we believe that these systems should be identified as miscible systems with a negative formation enthalpy, although the HTFP calculations provide opposite results. For Ag-Mn, Au-Cr, Fe-Os, Fe-Ru, Mn-W, Os-Pt, and Ta-Ti, the HTFP calculations are accepted because there is no information on phase diagrams or thermodynamic data available. For other binary alloy systems listed in Table [1](#Tab1){ref-type="table"}, our judgment is based on the elemental similarity, as will be discussed later.
Selection of physical descriptors {#Sec4}
---------------------------------
In order to enable pattern recognition, several physical descriptors, i.e. semi-empirical parameters and scales, are considered for a systematic description (or prediction) of the alloying behavior of the different metals. The physical descriptors are based on the similarity of atomic, physical and chemical properties of the elements, such as the atomic number (AN), Pauling electronegativity (PEN)^[@CR16],\ [@CR41],\ [@CR42]^, Teatum metallic radii of elements with coordinate number CN12 (Rij)^[@CR43]--[@CR45]^, Martynov-Batsanov electronegativity (MBEN)^[@CR35],\ [@CR46]^, Zunger's pseudopotential radii sum (Rsp)^[@CR35],\ [@CR47]^, Miedema's work function (Phi)^[@CR5],\ [@CR14]^, Miedema's electron density at the Wigner-Seitz cell boundary (Nws)^[@CR5],\ [@CR14]^, Miedema's molar volume (Vm)^[@CR5],\ [@CR14]^, and Mendeleev number or Pettifor chemical scale (Pcs)^[@CR48]^. The choice of these parameters or scales is based on the success of these parameters in the description of different alloying behaviors.
The first choice is the atomic number, which uniquely identifies a chemical element. The second criterion is based on the consideration of the diagrams developed by Darken and Gurry for solid solution predictions^[@CR19]^. The two coordinates represent the Pauling electronegativity and the atomic size of the elements. Electronegativity is a chemical property that describes the tendency of an atom or a functional group to attract electrons (or electron density). Pauling originally proposed the concept of electronegativity as an explanation of the fact that the covalent bond between two different atoms (A-B) is stronger than that would be expected by taking the average of the strengths of the A-A and B-B bonds. Allred^[@CR16],\ [@CR41],\ [@CR42]^ updated Pauling's original values by taking into account the available thermodynamic data. These "revised Pauling" values of the electronegativity are most often used.
The atomic radius of a chemical element is usually the mean or typical distance from the nucleus to the boundary of the surrounding cloud of electrons. Since the boundary is not well-defined, there are various non-equivalent definitions of atomic radii. Four widely used definitions are the Van der Waals radius, ionic radius, covalent radius and metallic radius. Among these definitions, the metallic radius proposed by Teatum^[@CR43]--[@CR45]^ is probably the most useful and widely accepted for metallic alloys, and will be adopted in the present work.
The third group of parameters come from the work by Villars, who analyzed the behavior of alloy systems on the properties of the component elements^[@CR35]^. He used a systematic elimination procedure to find atomic properties which could be used to distinguish the crystal structures of intermetallic compounds. 182 sets of tabulated physical properties and calculated atomic properties were considered. The best separation was obtained using three dimensional maps with the following variables as coordinates: (1) the mean number of valence electrons of the two elements, (2) the electronegativity difference proposed by Martynov-Batsanov in 1980^[@CR35],\ [@CR46]^, and (3) the difference of Zunger's pseudopotential radii sum^[@CR35],\ [@CR47]^. In view of the success of Villars' structure map, the two critical parameters of Martynov-Batsanov electronegativity and Zunger's pseudopotential radii sum are adopted in the present study.
The fourth group of parameters is derived from the famous Miedema's model because of its success in the description of the energy effects during alloying. Positive (immiscible) and negative (miscible) systems can be separated by two parameters of the constituent chemical elements: the chemical potential for electronic charge (electronegativity), Phi and the electron density at the boundary of the Wigner-Seitz atomic cell, Nws. Miedema's model became very popular due to the scarcity of experimental data, and the model provided a semi-quantitative evaluation. By assigning the two parameters as two coordinates it was possible to build a map in which a clear separation was observed between all binary alloys with positive heats of formation and those with negative values^[@CR5],\ [@CR12]--[@CR14]^. Additional justification will be provided later, in section 3.5. A quantum-mechanical interpretation of Miedema's parameters has been proposed by Chelikowsky and Phillips^[@CR49]^.
The last criterion is based on Pettifor's work on the chemical scale (or Mendleev number, M)^[@CR48]^. This has been set up by ordering the elements along a single axis so that the Mendeleev-type features of the periodic table are preserved. The Mendeleev numbers start with the least electronegative elements and end with the most electronegative ones. By using them, an excellent separation of similar structures is achieved for numerous A~m~B~n~ phases with a given stoichiometry within M~A~-M~B~ maps. Although the chemical scale is entirely phenomenological and has no a priori significance other than that it orders the elements relative to each other, Pettifor has shown that the corresponding two-dimensional structure maps achieve excellent structural separation. It will be shown in the following sections that the progressive order of chemical scale provides an excellent pattern clustering in the miscibility map of binary alloy systems.
Table [S1](#MOESM1){ref-type="media"} (in the Supplemental Materials) lists the adopted values of Pauling electronegativity (Pen)^[@CR16],\ [@CR41],\ [@CR42]^, Teatum metallic radii of elements with coordinate number CN12 (Rij)^[@CR43]--[@CR45]^, Martynov-Batsanov electronegativity (Men)^[@CR35],\ [@CR46]^, Zunger's pseudopotential radii sum (Rsp)^[@CR35],\ [@CR47]^, Miedema's work function (Phi)^[@CR5],\ [@CR14]^, Miedema's electron density at the Wigner-Seitz cell boundary (Nws)^[@CR5],\ [@CR14]^, Miedema's molar volume (Vm)^[@CR5],\ [@CR14]^, the Mendeleev number or the Pettifor chemical scale (Pcs)^[@CR48]^, and atomic number (AN) for ordering the 44 elements (27 transition metals and 17 Lanthanides). We refer the reader to the original publications on these parameters for the detailed physical meaning and their derivations.
The miscibility map sorted by different descriptors {#Sec5}
---------------------------------------------------
We use the descriptors ("parameters") defined in the previous section to analyze the pattern as it appears in the two-dimensional miscibility maps. Figure [S1](#MOESM1){ref-type="media"} (in the Supplemental Materials) shows the miscibility map ordered by Atomic number, Pauling electronegativity, Teatum metallic atomic radii, Martynov-Batsanov electronegativity, and Zunger's pseudo radii sum, respectively. These physical descriptors belong to the first three groups of variables identified in the preceding section. It can be seen that the atomic number, Pauling electronegativity, Teatum metallic atomic radii, Martynov-Batsanov electronegativity, and Zunger's pseudo radii sum can distinguish ("cluster") the immiscible systems distinctly. This indicates that these parameters provide elemental similarity and the underlying physics. However, the clustering patterns ordered by Pauling electronegativity, Teatum metallic atomic radii, Martynov-Batsanov electronegativity, and Zunger's pseudo radii, do not provide any better feature identification than that by atomic number. Interestingly, the descriptors belonging to the fourth and fifth group of variables are found to divide the immiscible systems within clusters much better than those in the former three criteria. Figure [S2](#MOESM1){ref-type="media"} (in the Supplemental Materials) shows the clustering feature of immiscible alloy systems ordered by Miedema's electronegativity, Miedema's electron density, Miedema's molar volume, and especially by Mendeleev number or the Pettifor chemical scale. Although Miedema's two parameters were shown to be a great success for predicting the formation enthalpies of intermetallic compounds, no single one provides a better ordering sequence than those according to criteria 1, 2 and 3. We further see that the Pettifor chemical scale shows the best separation between the miscible and immiscible systems and the distinct clustering of immiscible systems.
After an in-depth analysis of the pattern feature ordered by Mendeleev number (Pettifor chemical scale), we found that some outliers appear to break the continuous clustering feature for the lanthanide-based systems (see the two green points circled by blue squares in Fig. [1a](#Fig1){ref-type="fig"}). In order to avoid this breaking of continuity of the distribution of immiscible systems, we slightly modify the order of rare earth/lanthanide elements. Figure [1b](#Fig1){ref-type="fig"} shows the modified miscibility map ordered by the modified Mendeleev number or the Pettifor chemical scale with the new elemental ordering as: Yb \< Eu \< Lu \< Tm \< Er \< Ho \< Dy \< Tb \< Gd \< Sm \< Pm \< Nd \< Pr \< Ce \< La \< Y \< Sc \< Zr \< Hf \< Ti \< Nb \< Ta \< V \< Mo \< W \< Cr \< Tc \< Re \< Mn \< Fe \< Os \< Ru \< Co \< Ir \< Rh \< Ni \< Pt \< Pd \< Au \< Ag \< Cu \< Hg \< Cd \< Zn. It is seen from Fig. [1b](#Fig1){ref-type="fig"} that the new elemental sequence clearly divides the immiscible systems into two major clustered regions. Such clustering can be understood via similarity and dissimilarity of the elements in physics and chemistry.Figure 1The incomplete miscibility maps of binary alloy systems ordered by (**a**) the original Pettifor chemical scale and (**b)** the slightly modified ordering the RE elements. The red and green symbols indicate immiscible and miscible systems, respectively. The white symbols represent the systems with the unavailable experimental information and are not considered in the present study, while the blue ones indicate the boundary of alloys consisting of dissimilar elements.
Prediction of unknowns via artificial neural network algorithm {#Sec6}
--------------------------------------------------------------
We now apply the incomplete miscibility maps (Fig. [1b](#Fig1){ref-type="fig"}) and the radial basis function artificial neural network (RBF-ANN) algorithm as described in Methods section to predict the unknown classification of binary alloy systems in order to obtain a complete miscibility map. Figure [2a](#Fig2){ref-type="fig"} shows the filled miscibility map of binary alloy systems ordered by the slightly modified Mendeleev number or the Pettifor chemical scale based on the RBF-ANN analysis. The three combinations are binary transition metal alloys (TM + TM), a combination of transition metals and rare earth metals (TM + RE), and a binary combinations of rare earth metals (RE + RE). Two immiscible regions are located at TM + RE, and TM-TM combinations as seen in Fig. [2a](#Fig2){ref-type="fig"}.Figure 2The complete miscibility map of binary alloy systems ordered by the slightly modified Pettifor chemical scale based on (**a**) the RBF-ANN analysis, (**b**) the RBF-ANN analysis plus elemental similarity between Re and Tc.
It is seen that the prediction is mostly in agreement with the empirical prediction based on simple elemental similarity, with a few outliers for Tc-based systems. The agreement between the elemental similarity and the RBF-ANN analysis can be summarized as follows:For Pm-TM binary alloy systems: based on the similarity between Pm, Sm and Nd, one may see that the transition metals Zr \< Hf \< Ti \< Nb \< Ta \< V \< Mo \< W \< Cr are immiscible with Pm, whereas Pm is miscible with Tc \< Re \< Mn \< Fe \< Os \< Ru \< Co \< Ir \< Rh \< Ni \< Pt \< Pd \< Au \< Ag \< Cu \< Hg \< Cd \< Zn.For binary alloy systems between elements consisting of Zr \< Hf \< Ti \< Nb \< Ta \< V \< Mo \< W \< Cr and Yb \< Eu \< Lu \< Tm \< Er \< Ho \< Dy \< Tb \< Gd \< Sm \< Pm \< Nd \< Pr \< Ce, the immiscibility is readily chosen for these classes of binary alloy systems based on the elemental similarity.For Eu-(Co, Ru, Os) binary alloy systems: based on the elemental similarity between Rh(Ir) and Ru(Os), a miscible alloying behavior is an appropriate choice for the three Eu-(Co, Ru, Os) binary alloy systems.
Because the binary Tc-based alloy systems are located at the boundary of miscible and immiscible systems, one needs to further justify the miscibility of the related outliers based on the elemental similarity. Although our RBF-ANN analysis suggests that Tc-Eu, Tc-Yb, Tc-Pm and Tc-Nd systems are mostly immiscible, the elemental similarity between Tc and Re give an opposite conclusion, i.e. these systems should be miscible (negative formation enthalpy). Based on the similarity of valence electrons between Re and Tc, we would classify the four systems as miscible ones, and accordingly, Fig. [2b](#Fig2){ref-type="fig"} gives the complete miscibility map. Table [S2](#MOESM1){ref-type="media"} (in the Supplemental Materials) lists all of the 262 immiscible binary alloy systems identified from the 813 candidates. Figure [3](#Fig3){ref-type="fig"} shows the numbers of the immiscible binary alloys formed from different transition or rare earth metals M. It is seen that Cr, Nb, Ta, W, Mo -based binaries have the largest number of immiscible elements.Figure 3The numbers of immiscible binary alloy systems of different M-based binary systems (M is transition metals or rare earth metals). Significant feature is found that Cr, Nb, Ta, W, Mo -based systems possess the most number of immiscibility.
Comparison with Miedema's theory and high throughput first principles methods {#Sec7}
-----------------------------------------------------------------------------
Miedema's theory is generally regarded as the most successful empirical model to describe the energy effects during alloying. The electronegativity difference and electronic density discontinuity, used as parameters, can be regarded as a 2-dimensional (2D) basis for alloy behavior since it can separate the positive and negative systems with a reasonable precision. As shown in Fig. [S2](#MOESM1){ref-type="media"} (in the Supplemental Materials), however, the single parameter of Miedema's model cannot separate unambiguously the immiscible systems into large clusters. However, assigning two coordinates to each transition element it was possible to separate all binary alloys with positive heats of formation from those with negative ones. The existence of intermetallic compounds in the binary phase diagram indicates a negative formation enthalpy. The same applies to the mutual solute solubility. A negative formation enthalpy is expected if one or more intermetallic compounds occur in the phase diagram, or if there is an appreciable solid solubility between the given elements. If neither condition is satisfied, a positive formation enthalpy is expected. Figure [4a](#Fig4){ref-type="fig"} shows the miscibility map of binary alloy systems ordered by the slightly modified Mendeleev number or the Pettifor chemical scale based on Miedema's theory. Compared with Fig. [2b](#Fig2){ref-type="fig"} (or Fig. [S3](#MOESM1){ref-type="media"} in the Supplemental Materials), it is clear that the majority of the clustering region matches to each other with only minor disagreements, validating the success of Miedema's theory. We recall the equation of Г parameter for formation enthalpy in the form^[@CR5],\ [@CR14]^:$$\documentclass[12pt]{minimal}
\usepackage{amsmath}
\usepackage{wasysym}
\usepackage{amsfonts}
\usepackage{amssymb}
\usepackage{amsbsy}
\usepackage{mathrsfs}
\usepackage{upgreek}
\setlength{\oddsidemargin}{-69pt}
\begin{document}$${\rm{\Gamma }}=\frac{1}{{({{\rm{n}}}_{{\rm{ws}}}^{-1/3})}_{{\rm{av}}}}\{-{\rm{P}}{({{\rm{\Delta }}{\rm{\Phi }}}^{\ast })}^{2}+{\rm{Q}}{({{\rm{\Delta }}n}_{{\rm{ws}}}^{1/3})}^{2}\}$$\end{document}$$ Figure 4The miscibility map of binary alloy systems ordered by the slightly modified Pettifor chemical scale based on (**a**) Miedema's theory, and (**b**) HTFP calculations. Three different regions are divided as: TM+TM indicates binary transition metal alloy systems, TM+RE for the combinations of transition metals and rare earth metals, RE+RE for the combinations of binary rare earth metals.
Accordingly, the value of $\documentclass[12pt]{minimal}
\usepackage{amsmath}
\usepackage{wasysym}
\usepackage{amsfonts}
\usepackage{amssymb}
\usepackage{amsbsy}
\usepackage{mathrsfs}
\usepackage{upgreek}
\setlength{\oddsidemargin}{-69pt}
\begin{document}$${\rm{\Gamma }}=0$$\end{document}$ should separate the positive (immiscible) and negative (miscible) systems. A zero value of Г means that$$\documentclass[12pt]{minimal}
\usepackage{amsmath}
\usepackage{wasysym}
\usepackage{amsfonts}
\usepackage{amssymb}
\usepackage{amsbsy}
\usepackage{mathrsfs}
\usepackage{upgreek}
\setlength{\oddsidemargin}{-69pt}
\begin{document}$$-{\rm{P}}{({{\rm{\Delta }}{\rm{\Phi }}}^{\ast })}^{2}+{\rm{Q}}{({{\rm{\Delta }}n}_{{\rm{ws}}}^{1/3})}^{2}=0$$\end{document}$$
Thus, a straight line in a plot between $\documentclass[12pt]{minimal}
\usepackage{amsmath}
\usepackage{wasysym}
\usepackage{amsfonts}
\usepackage{amssymb}
\usepackage{amsbsy}
\usepackage{mathrsfs}
\usepackage{upgreek}
\setlength{\oddsidemargin}{-69pt}
\begin{document}$${{\rm{\Delta }}{\rm{\Phi }}}^{\ast }$$\end{document}$ versus $\documentclass[12pt]{minimal}
\usepackage{amsmath}
\usepackage{wasysym}
\usepackage{amsfonts}
\usepackage{amssymb}
\usepackage{amsbsy}
\usepackage{mathrsfs}
\usepackage{upgreek}
\setlength{\oddsidemargin}{-69pt}
\begin{document}$${{\rm{\Delta }}n}_{{\rm{ws}}}^{1/3}$$\end{document}$ should separate regions with positive and negative formation enthalpy.
Figure [5](#Fig5){ref-type="fig"} shows such plot for a large number of TM-TM and TM-RE alloys. Indeed the straight line y = x/9.4 separates two regions: one for binary alloy systems with negative formation enthalpy (green minus symbols) and one for systems with positive formation enthalpy (red plus symbols). The straight line gives the empirical value of the ratio Q/P = 9.4. As seen in Fig. [5a](#Fig5){ref-type="fig"}, Miedema's model provides a clear distinction between positive and negative systems when their formation enthalpy is sufficiently large. The agreement between the prediction and our data mining results is beyond 94%, which corresponds to large values of $\documentclass[12pt]{minimal}
\usepackage{amsmath}
\usepackage{wasysym}
\usepackage{amsfonts}
\usepackage{amssymb}
\usepackage{amsbsy}
\usepackage{mathrsfs}
\usepackage{upgreek}
\setlength{\oddsidemargin}{-69pt}
\begin{document}$${{\rm{\Delta }}{\rm{\Phi }}}^{\ast }$$\end{document}$ or $\documentclass[12pt]{minimal}
\usepackage{amsmath}
\usepackage{wasysym}
\usepackage{amsfonts}
\usepackage{amssymb}
\usepackage{amsbsy}
\usepackage{mathrsfs}
\usepackage{upgreek}
\setlength{\oddsidemargin}{-69pt}
\begin{document}$${{\rm{\Delta }}n}_{{\rm{ws}}}^{1/3}$$\end{document}$. However, when we zoom in the region with small values of $\documentclass[12pt]{minimal}
\usepackage{amsmath}
\usepackage{wasysym}
\usepackage{amsfonts}
\usepackage{amssymb}
\usepackage{amsbsy}
\usepackage{mathrsfs}
\usepackage{upgreek}
\setlength{\oddsidemargin}{-69pt}
\begin{document}$${({{\rm{\Delta }}{\rm{\Phi }}}^{\ast })}^{2}$$\end{document}$ from 0 to 0.7 and $\documentclass[12pt]{minimal}
\usepackage{amsmath}
\usepackage{wasysym}
\usepackage{amsfonts}
\usepackage{amssymb}
\usepackage{amsbsy}
\usepackage{mathrsfs}
\usepackage{upgreek}
\setlength{\oddsidemargin}{-69pt}
\begin{document}$$({{\rm{\Delta }}n}_{{\rm{w}}{\rm{s}}}^{1/3}{)}^{2}$$\end{document}$ from 0 to 0.05, as seen in Fig. [5b](#Fig5){ref-type="fig"}, the separation between the "positive" and "negative" systems is not unambiguous. This illustrates the aforementioned weakness of Miedema's model when it is used for binary alloy systems with formation enthalpies close to zero. A further demonstration of the correlation between the miscibility and formation enthalpies by Miedema's theory is shown in Fig. [6](#Fig6){ref-type="fig"}. It shows clearly that the distributions of highly positive (\>10 kJ/mol) and negative (\<−10kJ/mol) binary alloy systems calculated by Miedema's model correspond to the regions of highly miscible and immiscible systems classified in the present studies (see Fig. [2b](#Fig2){ref-type="fig"}).Figure 5(**a**) The separation map of miscible and immiscible systems coordinated by the two Miedema's parameters as two-dimensional axes. Plus symbols '+' represents the immiscible systems, while minus symbols '−' stands for the miscible systems with negative formation enthalpy, i.e. no intermetallic compounds exist or solubilities are generally smaller 10%. (**b**) The zoomed-in region with $\documentclass[12pt]{minimal}
\usepackage{amsmath}
\usepackage{wasysym}
\usepackage{amsfonts}
\usepackage{amssymb}
\usepackage{amsbsy}
\usepackage{mathrsfs}
\usepackage{upgreek}
\setlength{\oddsidemargin}{-69pt}
\begin{document}$${({{\rm{\Delta }}{\rm{\Phi }}}^{\ast })}^{2}$$\end{document}$ from 0 to 0.7 and $\documentclass[12pt]{minimal}
\usepackage{amsmath}
\usepackage{wasysym}
\usepackage{amsfonts}
\usepackage{amssymb}
\usepackage{amsbsy}
\usepackage{mathrsfs}
\usepackage{upgreek}
\setlength{\oddsidemargin}{-69pt}
\begin{document}$$({{\rm{\Delta }}n}_{{\rm{w}}{\rm{s}}}^{1/3}{)}^{2}$$\end{document}$ from 0 to 0.05. Figure 6The distribution map of formation enthalpies of binary alloy systems with 1:1 stoichiometry calculated by Miedema's model to indicate the distribution regions of highly miscible and immiscible systems. Red and green colored circles correspond to the positive formation enthalpy larger than 10kJ/mol and negative ones lower than −10kJ/mol respectively. The white symbols represent the systems with the unavailable experimental information and are not considered in the present study. The elemental sequence is ordered by the slightly modified Pettifor chemical scale.
Table [2](#Tab2){ref-type="table"} lists the 46 immiscible binary systems that exhibit disagreement between the results of our data mining-generated map and Miedema's theory. It should be noted that our choice is based on the estimation on the reliability and validity of the available information and similarity of different elements. The inconsistency between our choice and Miedema's theory is about 5%. In view of the uncertainty of each method, these systems warrant further careful study, both experimentally and theoretically.Table 2The binary alloy systems whose miscibility shows disagreement between present data mining and Miedema's theory.SystemsAlloying behaviorSystemsAlloying behaviorPresentMiedema(kJ/mol)PresentMiedema(kJ/mol)Au-Cr+−0.2Ir-Rh−+0.8Au-Os−+21.8Mn-Mo−+6.0Cd-Co+−1.8Mn-W−+7.6Cd-Ir+−12.6Mn-Yb+−4.2Cd-Mn+−8.1Mo-V−+0.0Ce-Fe−+2.5Mo-Zn−+6.3Ce-Re+−0.1Nb-Ta−+0.0Co-Os−+0.1Ni-Re−+2.8Co-Re−+2.5Ni-Tc−+0.7Cr-Mn−+2.6Os-Pt+−0.6Cu-Hg−+0.4Os-Rh−+2.5Cu-Ni−+4.3Os-Ru−+0.1Eu-Fe+−1.0Os-Tc−+0.2Eu-Mn+−1.6Os-Zn+−13.7Fe-Nd−+0.7Pd-Pt−+2.4Fe-Os+−4.9Pd-Re−+7.9Fe-Pr−+0.7Pr-Re+−2.5Fe-Ru+−5.6Re-Rh−+1.2Hf-Sc−+6.2Re-Tc−+0.2Hf-Ti−+0.2Rh-Ru−+1.5Hg-Ni−+1.1Sc-Zr−+4.9Hg-Zn−+1.2Ti-Zr+−0.3Tc-Ce+−25.9Tc-Pr+−28.2The present choice is based on the estimation on the reliability of the available information and elemental similarity. Plus symbol represents the immiscible systems, and minus symbol indicates the miscible or compound-formation systems.
For comparison, we have also constructed the miscibility map based on the HTFP data shown in Fig. [4b](#Fig4){ref-type="fig"}. This choice of the miscibility properties is based on the results of HTFP data by Curtarolo *et al*.^[@CR25],\ [@CR32]--[@CR34],\ [@CR36]--[@CR40]^. One can see that there is some disagreement (\~10%) with the results in Figs [2](#Fig2){ref-type="fig"}b and [4b](#Fig4){ref-type="fig"}. This implies that much more future work based on HTFP calculations is needed, and our present work shows the direction. For instance, further HTFP calculations based on a larger structure prototype library, e.g. ICSD or PCD, may help to validate the present results, although it is much computationally expensive for the TM-RE and RE-RE systems.
Discussion {#Sec8}
==========
The data mining presented in this paper (and in the prior cited studies^4^) provides a unique approach to discover novel materials, their properties and the supporting physical mechanisms. Our present study demonstrates a simple and illustrative example of obtaining the miscibility map of binary alloy systems from a large amount of raw data sources. It also provides a general pathway for one-, two- and three-dimensional spaces. The basic idea of data mining is not limited to the binary alloys, and therefore one may extend it for multicomponent alloy systems. To be noted that Miedema's theory is also applicable for ternary, quaternary and multicomponent systems via geometrical methods^[@CR20]^, while the HTFP mothed can also be readily applied for the ternary and even higher-order alloys when their structures are known or can be predicted. The success of Miedema's model and the HTFP method is clearly revealed, and some outliers are identified for further experimental and theoretical investigation. Because of the scarcity of reliable experimental and first-principles data for binary systems consisting of dissimilar lanthanides, it is of particular urgency to perform HTFP calculations on this group of systems.
Methods {#Sec9}
=======
Data collection and classification rule {#Sec10}
---------------------------------------
A total of 946 combinations exist for binary alloy phase diagrams consisting of transition and lanthanide metals, of which about 800 are available in the Landolt-Boernstein Database^[@CR7]^, in the "Desk handbook: Phase diagrams for binary alloys" compiled by Okamoto^[@CR9]^, and in the ASM alloy phase diagrams database organized by the ASM community^[@CR8]^. In addition, the experimental standard formation enthalpies determined by Kleppa *et al*.^[@CR24]--[@CR35]^ provide a further validation of the compound-formation systems. The recent computational binary alloy project, hosted in the *aflowlib.org* consortium repository^[@CR40]^, contains the formation enthalpies for hundreds of thousands of intermetallics comprising all of the transition metal systems. However, this repository does not include any alloying data between transition and lanthanide metals, and therefore a throughout data mining is much demanded for further design of novel alloys.
The binary solid phases consisting of elements A and B, may be classified into three groups: mechanical mixtures, solid solutions, and ordered intermetallic compounds. The mechanical mixture corresponds to binary systems with a positive formation enthalpy, while the intermetallic compound is favored for a negative formation enthalpy. The solid solution has three subtypes: clustered solid solution, disordered solid solution, or ordered solid solution. For the former two cases, the different kinds of atoms are energetically preferable to mix, while for the last case the different kinds of atoms prefer to segregate into different clusters. In comparison to the formation of intermetallic compounds with a negative formation enthalpy, the first two cases may be classified as poorly miscible systems, while the latter case can be classified as poorly immiscible systems since the formation enthalpy is close to zero^[@CR20]^.
With this classification rule of different solid phases and the five alloy databases at hand, a data mining scheme is proposed to separate the immiscible systems from those miscible ones in which the homogeneous disordered solid solution and intermetallic compounds may form. Figure [7a](#Fig7){ref-type="fig"} shows the proposed flowchart of the classification scheme that we shall use. Note that the miscible systems are those in which one or more intermetallic compounds, or one or more ordered solid phases, or a disordered solid solution exist with negative formation enthalpy. The immiscible systems correspond to those with positive formation enthalpy, in which case either a mechanical mixture or clustered solid solution may form at low temperature.Figure 7(**a**) A classification flowchart used in data mining to distinguish the miscible and immiscible systems based on the binary alloy phase diagrams, thermodynamic data and high-throughput first principles (HTFP) calculations. (**b**) A schematic diagram of radial basis function artificial neural network (RBF-ANN) for the prediction of the unknown systems.
Artificial neural network algorithm for prediction {#Sec11}
--------------------------------------------------
We use a radial basis function artificial neural network (RBF-ANN) algorithm to predict the immiscibility of an unknown system. The RBF-ANN has a three-layer structure: an input layer, a hidden layer and a linear output layer, as shown in Fig. [7b](#Fig7){ref-type="fig"}. In this work, the input and output of RBF-ANN are the map coordinates e.g. the "X- and Y-coordinate" in Fig. [7b](#Fig7){ref-type="fig"} and the miscible/immiscible classification, respectively. The input layer only transfers input data to the hidden layer. The hidden layer uses Gaussian functions as the radial basis function *Ф*. It can produce a localized response to the input and introduce non-linearity into the network^[@CR50]^.$$\documentclass[12pt]{minimal}
\usepackage{amsmath}
\usepackage{wasysym}
\usepackage{amsfonts}
\usepackage{amssymb}
\usepackage{amsbsy}
\usepackage{mathrsfs}
\usepackage{upgreek}
\setlength{\oddsidemargin}{-69pt}
\begin{document}$${{\rm{\Phi }}}_{i}(X)=\exp [-\frac{{\Vert X-{c}_{i}\Vert }^{2}}{2{\sigma }_{i}^{2}}],\,i=1,\,2,\,\ldots ,\,{\rm{n}}$$\end{document}$$Where X is the input vector, c~i~ is the centroid of the basis function of the i^th^ hidden layer node, and σ~i~ is the variance of basis function.
Before the RBF-ANN is trained, all data are scaled to fit within the interval \[−1,1\]. During the training processes, the center vectors c~i~ and variances σ~i~ of basis functions in the hidden layer are determined using k-means clustering^[@CR51]^. First the k samples are randomly chosen as the initial values of the center vectors c~i~, and the other samples are classified to form k subclasses according to the Euclidean distance between samples and center vectors. The new center vectors (i.e. the mean values of subclass data) are continually calculated by an iterative technique until they do not change. After the center vectors are determined, the variances σ~i~ can be obtained by the maximum distance among the center vectors. Subsequently, the weights w~i~ between the hidden and output layers (see Fig. [7b](#Fig7){ref-type="fig"}) are modified by the least mean square algorithm. The training of the network is completed after the number of neurons in the hidden layer reaches the sample dataset size (n = 770). The mean square errors of training are 4.7 × 10^−2^. We then use this network topological structure to predict the classification of unknown alloy systems.
Electronic supplementary material
=================================
{#Sec12}
Supplementary Information
**Electronic supplementary material**
**Supplementary information** accompanies this paper at doi:10.1038/s41598-017-09704-1
**Publisher\'s note:** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
This work is supported by the National Natural Science Foundation of China (NFSC) with Nos. 51471018 and 51672015, National Key Research and Development Program of China (Nos. 2016YFC1102500 and 2017YFB0702100), National Thousand Young Talents Program of China and Fundamental Research Funds for the Central Universities. D. L. acknowledges support by The Ministry of Education, Youth and Sports from the Large Infrastructures for Research, Experimental Development and Innovations project "IT4 Innovations National Supercomputing Center -- LM2015070" and the project No. 17-23964 S of the Grant Agency of the Czech Republic. We would also like to thank Prof. Dr. S. Veprek for constructive comments and suggestions to the manuscript, and Prof. S. Curtarolo for his comments and providing online HTFP data.
R.F.Z., X.F.K., S.H.Z. and S.H.S. performed the data collection, data mining, data verification and thermodynamic calculations. H.T.W. and S.S. performed the data analysis by radial basis function artificial neural network (RBF-ANN) algorithm and other approaches. D.L., K.R. and T.C.G. was involved in data analysis, verification and paper organization. All authors contributed the idea and participated in the scientific discussions, manuscript comments and corrections.
Competing Interests {#FPar1}
===================
The authors declare that they have no competing interests.
|
{
"pile_set_name": "PubMed Central"
}
|
Background {#Sec1}
==========
Spiritual care is an important part of holistic palliative care \[[@CR1]\]. In the Dutch guideline on 'Spirituality and meaning in the last phase of life', spirituality is defined as "the dynamic dimension of human life that relates to the way persons experience, express and/or seek meaning, purpose and transcendence, and the way they connect to the moment, to self, to others, to nature, to the significant and/or the sacred" \[[@CR2]\]. Spiritual care covers a multidimensional field of \[[@CR1]\] existential issues, e.g. identity, meaning, suffering and death \[[@CR2]\]; considerations and values, e.g. important issues and values for oneself, and \[[@CR3]\] religious considerations and sources, e.g. faith, beliefs and practices \[[@CR2], [@CR3]\]. Receiving spiritual care as part of holistic spiritual care is associated with positive effects on patients' quality of life and well-being. Patients and their relatives are open for discussing spiritual issues with their healthcare provider and appreciate these conversations. On the other side, a lack of spiritual care by healthcare professionals is associated with, amongst others, a poor quality of life \[[@CR4]--[@CR13]\].
Insufficient attention for spiritual issues has been identified as one of the barriers in providing holistic palliative care in the Netherlands \[[@CR14], [@CR15]\]. Palliative care is not a medical specialty in the Netherlands and it is preferably provided in the primary care setting, where it primarily falls under the responsibility of general practitioners (GPs) in close collaboration with district nurses. In respect of palliative care, Dutch GPs are encouraged to work together with district nurses and other healthcare providers in local PaTz-groups (palliative home care groups) \[[@CR16]\]. PaTz-groups meet at least six times per year under supervision of a palliative care consultant \[[@CR17]\]. The goal of these meetings is to identify patients facing a life-threatening illness, e.g. by using the surprise question, and to discuss current and future care needs of these patients, and to arrange and plan care accordingly.
Although spiritual issues are considered relevant by Dutch GPs \[[@CR18]\], they often struggle to provide adequate spiritual care, due to lack of time or attention for spiritual issues, or insufficient expertise and training \[[@CR19]\]. When a GP finds him- or herself unable to provide adequate spiritual care, for example in a case with complex spiritual needs or when crisis intervention is needed, they can theoretically refer their patient to a professional spiritual caregiver, a healthcare provider with specific expertise in the assessment of spiritual needs and the delivery of spiritual care \[[@CR2], [@CR20]\]. In reality, only half of the Dutch GPs occasionally involve a spiritual caregiver. When asked what reasons they have to not refer patients to spiritual caregivers, GPs mention that it is 'not needed', 'not my job', or that they 'do not know where to find them' \[[@CR21]\]. In addition, they experience barriers in finding and engaging spiritual caregivers \[[@CR18]\], and cooperation between spiritual caregivers and other healthcare providers has been reported to be poor \[[@CR19], [@CR22]\]. Thus, in practice, although providing spiritual care as part of holistic palliative care in cooperation with spiritual caregivers is considered to be very relevant and essential, it is often neglected and problematic, a phenomenon which has also been reported in international literature \[[@CR23]--[@CR26]\].
There is anecdotal evidence with regard to involving spiritual caregivers in palliative care in order to strengthen the spiritual domain of palliative care. In Scotland, a Chaplain Community service aimed at seriously ill patients, known as listening consultations, was perceived by patients, GPs and spiritual caregivers to be very beneficial. Activities of spiritual caregivers included therapeutic listening; being present; recognition of fear, loss and sadness; building trustful relationships in which difficult topics could be discussed; and helping patients to find hope, resilience or inner strength in times of illness, loss and death \[[@CR27]\]. Knowing each other and each other's activities well proved to be pivotal for the cooperation between spiritual caregivers and other healthcare providers \[[@CR27]\].
In the Netherlands, the above-mentioned PaTz-groups may provide an opportunity for fruitful cooperation between healthcare providers and spiritual caregivers, potentially resulting in improved spiritual care in the primary care setting. With the Chaplain Community project serving as an example, the PaTz-foundation launched a pilot in which a listening consultation service by spiritual caregivers was connected to GPs and nurses in PaTz-groups in an effort to strengthen the spiritual domain in Dutch primary palliative care.
In this explorative study, we aimed to evaluate and describe the listening consultation service with regard to its feasibility and perceived added value for healthcare providers and patients. Our research questions were:
1\. How did the process of implementation of a listening consultation service within PaTz-groups go, and what are barriers and facilitators for implementation?
2\. What is the perceived added value of a listening consultation service for healthcare providers and for patients?
Methods {#Sec2}
=======
Design {#Sec3}
------
For a period of ten months, the listening consultation services ran in three PaTz-groups. Qualitative data for the evaluation of the listening consultation service was collected through questionnaires as well as individual and group interviews, supplemented with quantitative data on personal and consultation characteristics. A description of the intervention is shown in Fig. [1](#Fig1){ref-type="fig"}. The consolidated criteria guidelines for reporting qualitative studies (COREQ) were followed for reporting on qualitative data \[[@CR17]\]. Fig. 1Overview of the intervention: Listening consultation services
Recruitment for the intervention {#Sec4}
--------------------------------
PaTz-groups were recruited via the PaTz-foundation and via the Palliative Care Consortium in Noord-Holland and Flevoland. Chairmen of four interested PaTz-groups were provided with information during a meeting with the researcher and after consenting to participate in the pilot, the researcher visited a meeting of each PaTz-group in which the spiritual caregiver was introduced. In these meetings, the researcher provided all PaTz-group members with flyers which could be handed out to patients. Finally, three interested PaTz-groups participated in the pilot.
Spiritual caregivers were included in collaboration with the 'Center for Life Questions' (in Dutch: Centrum voor Levensvragen), who selected interested spiritual caregivers based on their availability and their fit to the patient population of involved general practices. In total, seven spiritual caregivers were recruited (two per PaTz-group, with one backup and one who was recruited after drop-out of another spiritual caregiver). Four of them had a humanistic denomination, one an Islamic denomination, one Christian and one Buddhist. All spiritual caregivers were affiliated with the Dutch Association of Spiritual Caregivers (VGVZ), which is the national professional association of spiritual caregivers with approximately 1000 members who are currently employed in a care facility or in primary care. This association uses a professional standard for providing spiritual care. All spiritual caregivers were registered in the Quality Register of Spiritual Caregivers (SKVG), an association that manages the professional register of spiritual carers. The purpose of this register is to guarantee the professional level of the profession and to promote social recognition. All spiritual caregivers had to be available during planned PaTz-meetings.
During the study, patients and relatives with spiritual care needs were asked by the participating GPs whether they were interested in receiving the listening consultation service, or informed by a flyer in the waiting room. If so, they were provided with information, and were asked for permission to be approached by the spiritual caregiver attached to the GP's practice. The spiritual caregiver then contacted the patient for a first introduction and schedule a meeting. Patients and relatives who received the listening consultation services could also involve their relatives. Consultation with patients or relatives were free of charge. For time spend on consultations and attendance at PaTz-groups, spiritual caregivers were reimbursed from the project budget.
Data collection {#Sec5}
---------------
Data was collected from December 2018 until September 2019. Several qualitative and quantitative data collection methods were used to collect data on several aspects of the implementation process, the intervention and the perceived added value. Questionnaires with structured and open questions on characteristics and content of the consultation were filled out by spiritual caregivers after each consultation, which resulted in an overview of characteristics of users of consultations, and content of consultations. Additionally, at the end of the pilot study, questionnaires with structured and open questions on experiences with and perceived added value of spiritual care were filled out by referrers for all patients and relatives who used the listening consultation service. For the qualitative part, all participants were recruited by opportunity sampling and informed by an information letter. Semi-structured in-depth interviews were held with spiritual caregivers (*n* = 5) at the end of the pilot study or when they were not longer involved in the pilot study. In addition, semi-structured in-depth interviews were held with patients and relatives (*n =* 5). They were recruited by the spiritual caregiver, who provided an information letter and asked if the patient was willing to participate in an interview on experiences with the consultation service. If the patient or relative was interested, the spiritual caregiver provided contact details to the researcher, who contacted and informed the patient by phone. With their consent, an appointment for the interview was made. Finally, short group interviews were held with PaTz-groups at the end of the pilot study (*n =* 3, with 17 GPs and 10 nurses). All interviews were performed by one female researcher in palliative care (HK) who was trained in qualitative research, and were conducted at the participants' location of choice. Duration of interviews was between 20 and 60 min for patients, between 40 and 70 min for spiritual caregivers and between 20 and 45 min for PaTz-groups. No participants refused to participate. All interviews were guided by semi-structured topic lists (Additional file [1](#MOESM1){ref-type="media"}), and audio recorded and transcribed verbatim. A weekly diary was used to monitor the implementation process in this pilot. The characteristics of all participants in the interviews are presented in Table [1](#Tab1){ref-type="table"}. Table 1Characteristics of participants involved in data-collectionType of participant(s)Type of data-collection methodSexAge range (years)^**c**^ / denomination^**b**^Relative of deceased family member, patient of GPIndividual interviewF50--55NoneRelative of deceased family member, patient of GPIndividual interviewF55--60NoneRelative of deceased family member, patient of GPIndividual interviewF20--25NoneRelative of (deceased)^c^family member, patient of GPIndividual interviewM75--80Christian, otherRelative of deceased family member, patient of GPIndividual interviewF60--65BuddhistSpiritual caregiverIndividual interviewF60--65HumanisticSpiritual caregiverIndividual interviewM35--40IslamicSpiritual caregiverIndividual interviewF60--65HumanisticSpiritual caregiverIndividual interviewF55--60BuddhistSpiritual caregiverIndividual interviewF30--35Humanistic7 GPs, 5 district nurses, 1 palliative care consultantGroup interview4 M9FN/AN/A5 GPs, 5 district nurses, 1 nurse specialized in palliative care, palliative care consultantGroup interview2 M10FN/AN/A3 GPs, 4 district nurses, palliative care consultantGroup interview8FN/AN/A^a^In case of patients / relatives who had one or more consultations with the spiritual caregivers^b^In case of spiritual caregivers^c^ Family member deceased during the pilot period
Data analysis {#Sec6}
-------------
Various methods of analysis were used to involve several perspectives in the analysis. After answers to open questions were categorized by one researcher (IK) and checked by a second (HK), descriptive analyses took place for quantitative data using SPSS 26.0. Qualitative data of the semi-structured interviews with spiritual caregivers, patients and relatives, and health care professionals in PaTz-groups, were analysed following the principles of thematic analysis \[[@CR28]\]. After rereading transcripts, one researcher (HK) derived codes inductively from the data using Atlas.ti 8. The codes were checked by a second researcher (IK). Interpretation of the codes and themes was discussed regularly between HK, IK and BO to ensure peer debriefing and to function as a mechanism against interpretation of one single researcher. Negative case analysis was also included to ensure that results were based on both positive and negative attitudes towards aspects of the intervention or the intervention as a whole. Finally, codes were grouped into themes and all themes were discussed in the research team, until no new themes occurred (HK, IK, BO).
Ethics {#Sec7}
------
Patients, spiritual caregivers and healthcare providers who participated in the interviews gave written informed consent prior to the interview. Patients received a gift voucher for their participation. To ensure anonymity of participants, any personal identifying information was removed from the transcripts. Access to the data was limited to three researchers (HK, IK, BO).
Results {#Sec8}
=======
First, characteristics of the implementation process of listening consultation services including involvement of a spiritual caregiver to PaTz-groups and experienced facilitators and barriers are described. Second, the perceived added value and experiences regarding the listening consultation services and involvement of spiritual caregivers to PaTz-groups, are provided.
Implementation of the intervention {#Sec9}
----------------------------------
### Process of involvement of spiritual caregivers in PaTz-groups {#Sec10}
Spiritual caregivers attended eleven of fourteen PaTz-meetings during the pilot period. They were involved in patient discussions, asked questions concerning spiritual issues to the present GPs and nurses, and answered their questions. The time investment and effort required from spiritual caregivers turned out to be more than expected, due to the time-intensive start-up of referrals. The combination of collaboration between GPs, nurses and spiritual caregivers in PaTz-groups, and possibilities of referring to a spiritual caregivers hardly led to referrals. For GPs and nurses, recognition of spiritual issues appeared to be a barrier in this. Therefore, in all PaTz-groups a spiritual caregiver provided at least one training in recognizing and discussing spiritual issues in patients, as well as referring to spiritual caregivers. Further, activities such as (preparation for) training and meetings with other spiritual caregivers and the researcher needed time and effort in the beginning phase of the pilot.
### Process of referrals {#Sec11}
The consultation process started slowly, but consultations became more frequent during the pilot period. Consultations had been offered to both patients and relatives, but, we found that mainly relatives made use of the possibility of consultations. Referral took place not only by GPs, but also by relatives who used or had used consultations. After the training given by a spiritual caregiver, the numbers of referrals increased over time.
### Characteristics of consultations {#Sec12}
Spiritual caregivers held 46 consultations with 19 individuals (patients with a life-threatening illness and their relatives) with an mean age of 73. The majority was female (15/19), had no specific religious beliefs (8/19) or a Christian belief (4/19), were of Dutch descent (18/19) and were referred by their GP (13/19) or a family member (6/19). Nurses did not refer at all. They did not indicate a clear reason for this, except that they thought the patient had sufficient resources for spiritual care. The listening consultation service was used mainly by relatives (13/19) of terminal or deceased patients. Although the initial idea was to organise walk-in consultation hours in general practice, this turned out not to be feasible; home visits were the preferred alternative. Mainly one-to-one consultations were held (33/46), but group consultations with several relatives (7/46) or relatives and patients (6/46) were also held in varying compositions. Relatives used more consecutive consultations (M = 3.6) than patients (M = 1.8). Most care requests contained existential (32/46) or relational (24/46) components. The most often discussed topics were loss, grief and identity. Table [2](#Tab2){ref-type="table"} provides an overview of characteristics of participants who had consultations with the spiritual caregivers. Table 2Characteristics of participants who had consultations with spiritual caregiversn/NMean (range)Total unique users Patients6/19 Relatives13/19Sex Female15/19 Male4/19Conviction None8/19 Christian4/19 Other3/19 Unknown4/19Nationality Dutch18/19 Surinamese1/19Referred by GP13/19 Relative (already involved)6/19 Total unique consultations46 First consultation14/46 Follow-up consultation32/46 One-to-one consultations33/46 Patients at the end of life5/33 Relatives28/33 Group consultations13/46 Patient and relative6/13 Multiple relatives7/13 Consultations per user3.1 (1--10) Per patient1.8 (1--3) Per relative3.6 (1--10) Lengths of consultations (minutes)69 (15--150) One-to-one consultations (minutes)62 (15--100) Group consults (minutes)89 (45--150)Care request^a^ Existential32/46 Relational24/46 Psychological12/46 Religious3/46Discussed topics^b^ Grief33/46 Loss30/46 Identity24/46 Death / passing away22/46 Support21/46 Meaning19/46 Fear18/46 Finding strength18/46 Hope11/46 Other / diverse33/46^a^ Participants could have more than one care request^b^ Spiritual caregivers could report more than one discussed topic
A practical example of a consultation of spiritual caregivers by a patient is provided in Fig. [2](#Fig2){ref-type="fig"}. Fig. 2Practical example of a consultation of a spiritual caregiver by a patient
Facilitators and barriers for the listening consultation service {#Sec13}
----------------------------------------------------------------
### Facilitators for implementing spiritual care into palliative care in primary care {#Sec14}
First, frequent contact between spiritual caregivers and healthcare providers, in terms of spiritual caregivers attending PaTz-groups regularly and providing feedback about consultations to the referrer, proved to be a facilitator for implementation. This frequent contact resulted in health care providers more often thinking of spiritual issues and also referring patients more easily. Second, customization and flexibility in setting up listening consultation services and involvement of spiritual caregivers proved to be encouraging, e.g. consultations by home visits or a focus on relatives as a target group. This resulted in a service that was feasible and useful for those involved. Third, a project manager proved to be valuable when integration of spiritual care into already existing PaTz-groups took more time and effort than expected. Fourth, freedom of spiritual caregivers, e.g. consultations on appointment instead of a walk-in consultation hour or an unlimited number of consultations, was found to be a facilitator, as were the enthusiastic PaTz chairmen. Lastly, training given by spiritual caregivers on recognizing spiritual issues and discussing them with patients, was found to facilitate the integration of spiritual issues in primary palliative care.
### Barriers for implementing listening consultation services {#Sec15}
Some factors impeded the implementation and functioning of the listening consultation service. Spiritual caregivers mentioned that they struggled to get into contact with PaTz-groups and that the PaTz-groups did not meet as regularly as expected. The other healthcare providers mentioned the limited availability and occasional last-minute cancellations of spiritual caregivers as barriers for cooperation. Also, the terms 'spirituality' and 'spiritual care' turned out to be barriers, as these were often associated with religion or were difficult to concretize. Furthermore, some GPs had privacy concerns when referring a patient to a spiritual caregiver. Finally, the relatively short pilot period turned out to be a barrier, as spiritual caregivers and healthcare professionals needed more time to get to know and find each other, and, addressing and recognizing spiritual issues by GPs increased over time.
Perceived added value of the listening consultation services {#Sec16}
------------------------------------------------------------
Experiences of healthcare professionals, patients and patients' relatives and spiritual caregivers, are illustrated by quotes in Table [3](#Tab3){ref-type="table"}. Table 3Quotes on the perceived added value of the listening consultation serviceThemeQuoteFor HCP's*"R1: The fact that she \[spiritual caregiver\] is involved in PaTz-groups, means that you think of it as well, you see someone, and then you think of consultations, and of a different perspective, or of some extra possibilities. R2: R2: Because here \[PaTz-group meeting\] you sometimes can get stuck in the medical issues. Or when you have issues with providing care to a patient or whatever, and then you can say: maybe it's an idea that ...*" *(GPs in PaTz-group)*For patients /relatives*"Well, it just seems a bit like a soft way to talk about loss, without having a label or something, I experienced that as very pleasant. Because sometimes I have discussed these issues with my friends or sometimes family when I felt sad at certain moments. Then they say that you should just take a pill. Sometimes I have received that advice. Or that I had to engage a psychologists. But I don't think I want that at all. That's not the point at all. Then you feel somewhat misunderstood. And then I'd rather talk to an expert about it." (Patients' relative, 50y)*For spiritual caregivers*"Maintaining the part of communication with each other, that has had quite a lot of attention in such a start-up phase. (...*) *And then, I just think, that is worth the investment you know, if you can find each other well at the moments that matter. And if patients sometimes appreciate it if you give something back to the GP, yes, then you just work together on good, holistic patient care. So sometimes the investment is that it costs you extra time, but I think it is definitely worth it over time.." (Spiritual caregiver involved in pilot)*
### Experiences of healthcare professionals participating in PaTz-groups {#Sec17}
Enthusiasm for and perceived value of the listening consultation services varied per healthcare professional. Most GPs and district nurses who participated in the PaTz-groups felt the listening consultation services' added value. Firstly, the additional expertise of spiritual caregiver provided them with a broader perspective on the patient or relative(s) which was often focused on (psycho) social and spiritual wellbeing of the patient. Secondly, healthcare professionals felt that the listening consultation services facilitated identification and discussion of spiritual issues with the patient, although addressing spiritual issues in daily practice remained difficult. Also, healthcare professionals mentioned that spiritual caregivers sometimes spoke a "different language". A small amount of GPs and district nurses did not experience added value of the listening consultation services, most often due to available alternatives, such as a centre for relief and support for people with cancer, a nurse specialist in mental health (POH-GGZ), or because of their own perceived capacities in the field of spiritual care.
### Experiences of patients and relatives using the listening consultation services {#Sec18}
Interviewed relatives who had one or more consultations with spiritual caregivers, perceived the conversations as very valuable, especially in the recognition of normal feelings in times of loss and the recognition of grief as a normal aspect of life. Also, they experienced added value when they were mourning, and they appreciated this low-threshold and free initiative of listening consultation services. In particular, the role of the spiritual caregiver as a "humane person" who provides professional support, was well-appreciated. In addition, according to PaTz-group members, their patients perceived the consultations as valuable in a similar way as relatives did.
### Experiences of spiritual caregivers who were involved in consultations and PaTz-groups {#Sec19}
Spiritual caregivers felt that the listening consultation service added to holistic palliative care and the possibility to integrate spiritual care as a professional discipline into palliative care. They felt that the nature of care requests and discussed themes with patients fitted their mission as a spiritual caregiver: using listening as an essential and important instrument, providing meaning to illness and life stories, and connecting relatives and patients to God, religion or the ultimate, and providing support and rituals. At the same time, spiritual caregivers experienced that they needed time to become familiar with the PaTz-group members and their professions, and that integrating spiritual care into palliative care took more time and effort than expected. As a positive side effect of this intervention, some spiritual caregivers mentioned increased knowledge of possibilities for spiritual care among healthcare professionals, as well as increased reach of patients in palliative care.
Discussion {#Sec20}
==========
Summary of results {#Sec21}
------------------
This study evaluated and described a pilot of a listening consultation service by spiritual caregivers in PaTz-groups. It showed that, although time-intensive and difficult at start, the intervention could be feasible and has added value. Consultations were on existential and relational issues, mainly on loss, grief and identity were main themes. After a period of gaining momentum, the listening consultation services resulted in more attention for spiritual issues of patients and relatives in particular, who both highly appreciated this. Healthcare providers in PaTz-groups, particularly GPs, were more aware of (addressing) spiritual issues that could be relevant for their patients. They also experienced added value in the complementary expertise of spiritual caregivers. Still, the enthusiasm among GPs varied and nurses did not refer patients to spiritual caregivers at all. Involving spiritual caregivers in PaTz-groups seemed to be a good method to improve spiritual care in the primary care setting and cooperation between healthcare professionals and spiritual caregivers.
Scarce evidence on integrating spiritual care in palliative care {#Sec22}
----------------------------------------------------------------
Previous research has shown that healthcare professionals consider spiritual issues in palliative care to be relevant \[[@CR29], [@CR30]\], and that attention for spiritual issues positively affects the patients' relationship with their care provider, reduces discomfort, and increases quality of life \[[@CR29], [@CR31]\]. This is, to our knowledge, the first intervention in which spiritual caregiver consultations of patients with a life-threatening illness and relatives, are combined with training of healthcare professionals in spiritual issues as well as enhanced collaboration between primary healthcare providers and spiritual caregivers. This study provides useful insights into the integration of spiritual care in primary palliative care, and into the perceived added value of listening consultation services from the perspectives of patients and relatives, and all healthcare professionals that are involved.
Training as core element of integrating spiritual care into palliative care {#Sec23}
---------------------------------------------------------------------------
Several European studies have shown that spiritual caregivers can play an important role in the training of other healthcare providers to discuss spiritual issues in palliative care \[[@CR29]\]. Training turned out to be an essential part of this intervention, especially in the ability of healthcare providers to recognize and address spiritual issues in patients. Despite the relatively small set-up of this pilot study, our results indicate that training combined with close collaboration and regular meetings between GPs, nurses and spiritual caregivers gradually resulted in more awareness of and skills in spiritual care. Our results with regard to training and education are in line with other studies, that showed some positive effects of training hospice staff and hospice volunteers \[[@CR32]--[@CR34]\].
Comparison to the Scottish chaplain community listening services {#Sec24}
----------------------------------------------------------------
Similar to the Scottish Chaplain Community Listening service \[[@CR27]\], our pilot study showed unfamiliarity of healthcare professionals with spiritual issues. Healthcare providers found it difficult to recognize and address spiritual issues of patients, and often associated spiritual issues with religious care, like in the Scottish example. Also similar to the Scottish example was that this intervention seemed a good alternative for psychotherapy or counselling when patients experienced mourning and feelings of sadness due to (nearing) loss of a loved one. Both patients in the Scottish and Dutch situation appreciated this type of spiritual care particularly because of the non-judgemental and non-stigmatizing approach. This could be helpful for patients who do not want to be referred to other specialists or care providers. Lastly, similar to the Scottish situation, Dutch patients valued "safe space" by visits and availability of time of spiritual caregivers, as well as an unlimited number of consultations and no obligations, as positive. In addition, our study showed the benefits of a combined approach that consists of a spiritual caregiver using existing infrastructure in palliative care such as PaTz, and consultations with patients by the same spiritual caregiver. This combined approach enhanced close collaboration between healthcare providers and spiritual caregivers as well as competency and skills of especially GPs in providing holistic palliative care.
Financial considerations {#Sec25}
------------------------
In this pilot, the time spiritual caregivers spent attending PaTz-meetings, training healthcare providers and in consultations with patients and relatives was funded from the projects budget, removing a potential hurdle for healthcare providers and patients to make use of the listening consultation service. Without funding, the accessibility of these services may be limited as people may not be willing or able to pay for these consultations. In 2018, a governmental budget for consultations with spiritual caregivers, training and activities to increase awareness of the (possibilities of) spiritual caregivers was introduced for a period of 2 years \[[@CR20]\]. Since 2019, this budget has been managed by local networks of palliative care \[[@CR35]\]. As a result, financing this care does not have to be a barrier to the deployment of spiritual care in the Netherlands. In other countries, if there is no financial arrangement for spiritual care, this could be a barrier.
Strengths and limitations {#Sec26}
-------------------------
A strength of our study is that we were able to adapt the intervention to the needs of the participants, adding training for healthcare providers and substituting the walk-in consultation hours for home visits. By these changes, this study offers insight into facilitators and barriers to the provision of holistic palliative care, resulting in a list of practical recommendations before starting an intervention focused on the integration of spiritual care in palliative care (Additional file [2](#MOESM2){ref-type="media"}).
No baseline data was collected, which could be seen as a limitation as stating "added value" could also be of other reasons. However, since the aim of this study was to describe the process and perceived added value in a mainly qualitative and descriptive way, we explored a need and feasibility for this intervention and how to design this intervention. We believe that baseline data collection would be a good second step in the further development of this intervention. Also, the small scale of the pilot impacts the generalizability of the results. Although the listening consultation services seemed beneficial in the three participating PaTz-groups, this may be different in other PaTz-groups that may be less receptive to spiritual care. In addition, due to the small-scaled piloted intervention and explorative nature of the data, we did not collect large-scaled data on which we could establish significant relationships and influences, e.g. on perspectives on spirituality versus age and gender. More research is needed to understand large-scale provision of spiritual care in primary palliative care, also in a wider context such as other countries. Further, the district nurses in this study did not refer to spiritual caregivers, but we found no clear explanation for this. It may be related to their perspective on spirituality, or on their own resources on providing spiritual care or referring to spiritual caregivers in their own network. However, we did not conduct additional interviews due to lack of budget and time. We recommend further investigation of the role, perspectives and resources of (district) nurses regarding spirituality in palliative care in future research. Also, this study used mainly qualitative or descriptive quantitative methods in order to monitor implementation and to evaluate perceived added value. A more rigid, large scale method such as an RCT would be recommendable for the effect of this intervention over time. Further, while we know that patients and relatives valued the consultations, we do not know why patients and patients' relatives with spiritual issues who were offered but did not use the consultations, did not participate. Finally, this study does not provide any insights into the relatively high use of consultations by relatives instead of patients. We recommend these issues for future research.
Conclusion {#Sec27}
==========
If sufficient effort and time is given to implementation, the listening consultation service can be a good method for PaTz-groups to cooperate with spiritual caregivers, to receive training in spiritual care skills and to refer patients and relatives with spiritual needs to spiritual caregivers. Listening consultation services could also serve as a good method for integrating spiritual care in primary palliative care other contexts, such as multidisciplinary meetings between healthcare professionals in other countries or contexts.
Supplementary information
=========================
{#Sec28}
######
**Additional file 1.** Topic lists of semi-structured individual interviews with spiritual caregivers
######
**Additional file 2.** Practical recommendations for interventions concerning integration of spiritual care in palliative care
PaTz-group
: Palliative home care groups
WHO
: World Health Organization
EAPC
: European Association for Palliative Care
GP
: General practitioner
PaTz
: Palliatieve ThuisZorg (Palliative Care at Home)
COREQ
: Consolidated criteria guidelines for reporting qualitative studies
VGVZ
: Dutch Association of Spiritual Caregivers
SKVG
: Quality Register of Spiritual Caregivers
POH-GGZ
: Nurse specialist in mental health
RCT
: Randomized Controlled Trial
**Publisher's Note**
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary information
=========================
**Supplementary information** accompanies this paper at 10.1186/s12904-020-00595-0.
Not applicable.
BO and BS designed the study and acquired funding. HK conducted the pilot study and collected data. HK and IK analysed and interpreted the data, which was discussed with BO. HK and IK drafted the first version of the manuscript, which was critically revised by BO, BS and EJ in several rounds of feedback. All authors have approved the submitted version and have agreed to be accountable for their contributions as well as for accuracy and integrity for any part of the work.
Funding for this study was provided by the Pieter van Foreest Foundation, who had no role in design, data collection, analysis, interpretation of data and writing of the manuscript.
The datasets generated and/or analysed during the current pilot study are not publicly available due to the small scale of this pilot study and the easily traceable nature of the data, but are available from the corresponding author on reasonable request.
On November 30, 2018, the Ethics Review Committee of VU University Medical Centre provided a waiver as ethical approval was not needed under Dutch law (METC VUmc 2018.540). All interviewed participants gave written informed consent prior to the interview.
All individual person's data were anonymized in this publication. Consent for anonymized use in publication was included in the informed consent form.
The authors declare that they have no competing interests.
|
{
"pile_set_name": "PubMed Central"
}
|
Due to a typesetting error, the headings in Tables [1](#pone.0152634.t001){ref-type="table"}--[9](#pone.0152634.t009){ref-type="table"} appear incorrectly. The publisher apologizes for the errors. Please see the corrected tables here.
10.1371/journal.pone.0152634.t001
###### Different datasets of occurrences.
{#pone.0152634.t001g}
Taxon or Period Taphonomic Category or Epoch Dataset I Dataset II Dataset III Dataset IV
---------------------- ------------------------------ ----------- ------------ ------------- ------------ ---- ---- --- ----
**Abeli. (72)** **1** 1 26 1 12 1 24 1 11
**2** 2 37 2 13 1 29 1 8
**Not specified** 0 0 0 0 0 0 0 0
**Carch. (66)** **1** 0 15 0 11 0 15 0 10
**2** 9 41 6 20 5 27 5 13
**Not specified** 1 0 1 0 0 0 0 0
**Spino. (82)** **1** 6 12 3 10 5 10 3 8
**2** 8 52 7 20 4 32 4 13
**Not specified** 2 1 1 1 0 0 0 0
**Cretaceous (194)** **Early** 14 77 9 34 10 54 8 27
**Late** 8 88 4 36 3 74 3 28
C and T refer to the costal and terrestrial paleoenvironmental categories, respectively, while taxa are indicated by Abeli. (Abelisauridae), Carch. (Carcharodontosauridae), and Spino. (Spinosauridae). On the other hand, taphonomic categories are indicated by their respective numbers except for those occurrences lacking data about the nature of their fossil record, whose taphonomic categories were considered as not specified. Within brackets is the total number of occurrences of each taxon or period.
10.1371/journal.pone.0152634.t002
###### Results of Chi-square Test 1 as presented by software R.
{#pone.0152634.t002g}
Test 1 Dataset I Dataset II Dataset III Dataset IV
------------- ----------- ------------ ------------- ------------ --------- -------- ----------- --------
**Abeli.** \- n/a n/a n/a n/a n/a n/a n/a
**Carch.** \+ n/a n/a n/a n/a n/a n/a n/a
**Spino.** \+ n/a n/a n/a n/a n/a n/a n/a
**χ^2^** 8.7586 0.7283 4.5714 0.8276 4.625 1.7664 2.7143 0.381
**p-value** 0.01253\* 0.6948 0.1017 0.6611 0.09901 0.4135 0.2894^‡^ 0.8266
C and T refer to the coastal and terrestrial paleoenvironmental categories, respectively, while taxa are indicated by Abeli. (Abelisauridae), Carch. (Carcharodontosauridae), and Spino. (Spinosauridae). Positive, negative or lack of any association are signalized by +, -, and n/a, respectively. For the residual analysis values that indicate the type of association see S1 File.
10.1371/journal.pone.0152634.t003
###### Results of Chi-square Test 2 as presented by software R.
{#pone.0152634.t003g}
Test 2 Dataset I Dataset II Dataset III Dataset IV
-------------- ---------------------------------------------- ------------ ----------------------------------------------- ---------------------------------------------- ----- ----- ----- -----
**Abeli.** \- \+ n/a n/a n/a n/a n/a n/a
**Carch.** \+ \- n/a n/a n/a n/a n/a n/a
**Spino.** \+ \- n/a n/a n/a n/a n/a n/a
**χ**^**2**^ 7.3431 2.6081 5.5498 1.9352
**p-value** 0.02544[\*](#t003fn002){ref-type="table-fn"} 0.2714 0.07246[^‡^](#t003fn003){ref-type="table-fn"} 0.4308[^‡^](#t003fn003){ref-type="table-fn"}
C and T refer to the coastal and terrestrial paleoenvironmental categories, respectively, while taxa are indicated by Abeli. (Abelisauridae), Carch. (Carcharodontosauridae), and Spino. (Spinosauridae). Positive, negative or lack of any association are signalized by +, -, and n/a, respectively. For the residual analysis values that indicate the type of association see **S1 File**.
\* Significant p-value.
^‡^ p-value obtained in Chi-square test using the Monte Carlo analysis.
10.1371/journal.pone.0152634.t004
###### Results of Chi-square Test 3 as presented by software R.
{#pone.0152634.t004g}
Test 3 Dataset I Dataset II Dataset III Dataset IV
-------------- ----------------------------------------------------------------------------------- ----------------------------------------------- ------------------------------------------------------------------------------------ ------------ ----------------------------------------------- ----------------------------------------------- ---------------------------------------------- --------
**Abeli. 1** \- \- \- n/a n/a \+ n/a n/a
**Abeli. 2** \- \+ \- n/a n/a \+ n/a n/a
**Carch. 1** \- \- \- n/a n/a \- n/a n/a
**Carch. 2** \+ \+ \+ n/a n/a \+ n/a n/a
**Spino. 1** \+ \- \- n/a n/a \- n/a n/a
**Spino. 2** \+ \+ \+ n/a n/a \+ n/a n/a
**χ^2^** 16.9231 39.918 12.2632 7.0698 9.5 16.0657 8.2857 2.4286
**p-value** 0.0065[\*](#t004fn002){ref-type="table-fn"}[^‡^](#t004fn003){ref-type="table-fn"} 1.55E-07[\*](#t004fn002){ref-type="table-fn"} 0.03698[\*](#t004fn002){ref-type="table-fn"}[^‡^](#t004fn003){ref-type="table-fn"} 0.2155 0.09845[^‡^](#t004fn003){ref-type="table-fn"} 0.006659[\*](#t004fn002){ref-type="table-fn"} 0.1554[^‡^](#t004fn003){ref-type="table-fn"} 0.7872
C and T refer to the coastal and terrestrial paleoenvironmental categories, respectively, while taxa are indicated by Abeli. (Abelisauridae), Carch. (Carcharodontosauridae), and Spino. (Spinosauridae). Numbers after the taxa represent the taphonomic categories. Positive, negative or lack of any association are signalized by +, -, and n/a, respectively. For the residual analysis values that indicate the type of association see **S1 File**.
\* Significant p-value.
^‡^ p-value obtained in Chi-square test using the Monte Carlo analysis.
10.1371/journal.pone.0152634.t005
###### Results of Chi-square Test 4 as presented by software R.
{#pone.0152634.t005g}
Test 4 Dataset I Dataset II Dataset III Dataset IV
-------------- ------------------------------------------------------------------------------------ ---------------------------------------------- ------------------------------------------------------------------------------------ ---------------------------------------------- ---- ---- ----- -----
**Abeli. 1** \- \+ n/a n/a \- \+ n/a n/a
**Abeli. 2** \- \+ n/a n/a \- \+ n/a n/a
**Carch. 1** \- \+ n/a n/a \- \+ n/a n/a
**Carch. 2** \+ \- n/a n/a \+ \- n/a n/a
**Spino. 1** \+ \- n/a n/a \+ \- n/a n/a
**Spino. 2** \+ \- n/a n/a \+ \- n/a n/a
**χ**^**2**^ 14.6137 5.3791 13.8029 5.3592
**p-value** 0.01249[\*](#t005fn002){ref-type="table-fn"}[^‡^](#t005fn003){ref-type="table-fn"} 0.3923[^‡^](#t005fn003){ref-type="table-fn"} 0.01549[\*](#t005fn002){ref-type="table-fn"}[^‡^](#t005fn003){ref-type="table-fn"} 0.3758[^‡^](#t005fn003){ref-type="table-fn"}
C and T refer to the coastal and terrestrial paleoenvironmental categories, respectively, while taxa are indicated by Abeli. (Abelisauridae), Carch. (Carcharodontosauridae), and Spino. (Spinosauridae). Numbers after the taxa represent the taphonomic categories. Positive, negative or lack of any association are signalized by +, -, and n/a, respectively. For the residual analysis values that indicate the type of association see **S1 File**.
\* Significant p-value.
^‡^ p-value obtained in Chi-square test using the Monte Carlo analysis.
10.1371/journal.pone.0152634.t006
###### Results of Chi-square Test 5 as presented by software R.
{#pone.0152634.t006g}
Test 5 Dataset I Dataset II Dataset III Dataset IV
--------------- ----------------------------------------- ----------------------------------------- ----------------------------------------- ----------------------------------------- ----- ----- ----- -----
**Abeli. 1** n/a n/a n/a n/a n/a n/a n/a n/a
**Abeli. 2** n/a n/a n/a n/a n/a n/a n/a n/a
**χ^2^** 0.0746 0.2317 0.0173 0.0461
**p-value** 1[^‡^](#t006fn002){ref-type="table-fn"} 1[^‡^](#t006fn002){ref-type="table-fn"} 1[^‡^](#t006fn002){ref-type="table-fn"} 1[^‡^](#t006fn002){ref-type="table-fn"}
**Fischer's** 0.71509 0.55326 1.20416 0.73856
**p-value** 0.8003 0.8611 0.7071 0.8286
C and T refer to the coastal and terrestrial paleoenvironmental categories, respectively. Numbers after Abeli. (Abelisauridae) represent the taphonomic categories. Positive, negative or lack of any association are signalized by +, -, and n/a, respectively. For the residual analysis values that indicate the type of association see **S1 File**.
^‡^ p-value obtained in Chi-square test using the Monte Carlo analysis.
10.1371/journal.pone.0152634.t007
###### Results of Chi-square Test 6 as presented by software R.
{#pone.0152634.t007g}
Test 6 Dataset I Dataset II Dataset III Dataset IV
--------------- ---------------------------------------------- ---------------------------------------------- ---------------------------------------------- ---------------------------------------------- ----- ----- ----- -----
**Carch. 1** n/a n/a n/a n/a n/a n/a n/a n/a
**Carch. 2** n/a n/a n/a n/a n/a n/a n/a n/a
**χ^2^** 3.1339 3.0298 2.6228 3.3816
**p-value** 0.1054[^‡^](#t007fn002){ref-type="table-fn"} 0.1669[^‡^](#t007fn002){ref-type="table-fn"} 0.1599[^‡^](#t007fn002){ref-type="table-fn"} 0.1329[^‡^](#t007fn002){ref-type="table-fn"}
**Fischer's** 0 0 0 0
**p-value** 0.1033 0.1505 0.1617 0.1282
C and T refer to the coastal and terrestrial paleoenvironmental categories, respectively. Numbers after Carch. (Carcharodontosauridae) represent the taphonomic categories. Positive, negative or lack of any association are signalized by +, -, and n/a, respectively. For the residual analysis values that indicate the type of association see **S1 File**.
^‡^ p-value obtained in Chi-square test using the Monte Carlo analysis.
10.1371/journal.pone.0152634.t008
###### Results of Chi-square Test 7 as presented by software R.
{#pone.0152634.t008g}
Test 7 Dataset I Dataset II Dataset III Dataset IV
--------------- ----------------------------------------------- ---------------------------------------------- ----------------------------------------------- ----------------------------------------- ----- ----- ----- -----
**Spino. 1** n/a n/a n/a n/a n/a n/a n/a n/a
**Spino. 2** n/a n/a n/a n/a n/a n/a n/a n/a
**χ^2^** 3.7607 0.038 3.5979 0.0499
**p-value** 0.08196[^‡^](#t008fn002){ref-type="table-fn"} 0.8455[^‡^](#t008fn002){ref-type="table-fn"} 0.09645[^‡^](#t008fn002){ref-type="table-fn"} 1[^‡^](#t008fn002){ref-type="table-fn"}
**Fischer's** 3.19102 0.86042 3.8733 1.21006
**p-value** 0.07777 0.5857 0.1022 0.7505
C and T refer to the coastal and terrestrial paleoenvironmental categories, respectively. Numbers after Spino. (Spinosauridae) represent the taphonomic categories. Positive, negative or lack of any association are signalized by +, -, and n/a, respectively. For the residual analysis values that indicate the type of association see **S1 File**.
^‡^ p-value obtained in Chi-square test using the Monte Carlo analysis.
10.1371/journal.pone.0152634.t009
###### Results of Chi-square Test 8 as presented by software R.
{#pone.0152634.t009g}
Test 8 Dataset I Dataset II Dataset III Dataset IV
---------------------- ----------- ---------------------------------------------- ------------- ------------ ----- ----- ----- -----
**Early Cretaceous** n/a n/a n/a n/a n/a n/a n/a n/a
**Late Cretaceous** n/a n/a n/a n/a n/a n/a n/a n/a
**χ^2^** 2.2376 5.7445 1.8742 2.056
**p-value** 0.1347 0.01654[\*](#t009fn002){ref-type="table-fn"} 0.171 0.1516
**Fischer's** 1.99268 4.52091 2.35822 2.72479
**p-value** 0.1739 0.02031[\*](#t009fn002){ref-type="table-fn"} 0.231 0.1955
C and T refer to the coastal and terrestrial paleoenvironmental categories, respectively. Positive, negative or lack of any association are signalized by +, -, and n/a, respectively. For the residual analysis values that indicate the type of association see **S1 File**.
\* Significant p-value.
|
{
"pile_set_name": "PubMed Central"
}
|
Subsets and Splits
No community queries yet
The top public SQL queries from the community will appear here once available.