To assess the feasibility of a novel diagnostic algorithm targeting patients with risk factors for chronic liver disease in a community setting.Prospective cross-sectional study.Two primary care practices (adult patient population 10 479) in Nottingham, UK.Adult patients (aged 18 years or over) fulfilling one or more selected risk factors for developing chronic liver disease: (1) hazardous alcohol use, (2) type 2 diabetes or (3) persistently elevated alanine aminotransferase (ALT) liver function enzyme with negative serology.A serial biomarker algorithm, using a simple blood-based marker  and subsequently liver stiffness measurement using transient elastography (TE).Diagnosis of clinically significant liver disease (defined as liver stiffness ≥8 kPa); definitive diagnosis of liver cirrhosis.We identified 920 patients with the defined risk factors of whom 504 patients agreed to undergo investigation. A normal blood biomarker was found in 62 patients (12.3%) who required no further investigation. Subsequently, 378 patients agreed to undergo TE, of whom 98  had elevated liver stiffness. Importantly, 71/98 (72.4%) patients with elevated liver stiffness had normal liver enzymes and would be missed by traditional investigation algorithms. We identified 11 new patients with definite cirrhosis, representing a 140% increase in the number of diagnosed cases in this population.A non-invasive liver investigation algorithm based in a community setting is feasible to implement. Targeting risk factors using a non-invasive biomarker approach identified a substantial number of patients with previously undetected cirrhosis.The diagnostic algorithm utilised for this study can be found on clinicaltrials.gov (NCT02037867), and is part of a continuing longitudinal cohort study. The current study provides an alternative diagnostic algorithm to the usual flawed concept of investigating patients for liver disease on the basis of elevated liver function enzymes. We introduced a novel algorithm that targets established risk factors for chronic liver disease in a primary care setting. Patients were investigated utilising simple and cheap blood-based biomarkers followed by transient elastography .Patients with common lifestyle-related risk factors for chronic liver disease  were identified using a widely utilised prospective primary care database in the UK, from two general practices with an adult population of 10 479 patients. Our patient selection method, and subsequent investigation algorithm, would therefore be easy to adopt at other primary care sites in the UK.Further study is required in a number of areas to address limitations of the current study, including validation of the diagnostic algorithm in other regions of the UK, investigation using alternative simple fibrosis markers or transient elastography and health-economic analysis of earlier identification of chronic liver disease.Current strategies to identify liver disease in the general population rely on the use of liver function enzyme blood tests, which are non-specific markers of liver injury. In the UK, persistently elevated liver enzymes typically trigger referral to hospital-based gastroenterology and hepatology clinics, where liver biopsy is the reference standard investigation for staging of hepatic fibrosis. This is an inadequate strategy, as the absence of symptoms in early stages of liver disease, and poor sensitivity of liver function blood tests to detect hepatic fibrosis, often result in late diagnosis. A recent study using data from a large community database, the clinical practice research datalink, suggested that 50% of patients with cirrhosis are given their initial diagnosis of liver disease after the first hospitalisation with decompensation, which substantially impaired prognosis independent of stage of cirrhosis, versus patients who are diagnosed while ambulatory .These inefficient diagnostic algorithms have contributed to the fact that liver-related deaths have continued to rise in the UK,Despite the widespread recognition that significant liver disease exists in the context of normal LFTs, current management algorithms are still dependent on abnormal liver enzymes to initiate investigation. Our aim was to challenge this flawed concept by testing the feasibility of a novel diagnostic algorithm that directly targeted risk groups , rather than relying on LFTs. Importantly, we designed a pathway that integrated non-invasive diagnostic tests and liver specialists within a community setting. The pathway used simple tests followed by a validated test of liver fibrosis . We hypothesised this approach would detect a substantial number of hitherto undiagnosed cases of chronic liver disease.This was a prospective study with recruitment from two suburban general medical practices in Rushcliffe, Nottingham, UK (the least deprived borough in Nottingham), with a total patient population of 12 368. The study ran for 14 months from February 2012 to April 2013; local regulatory approval was obtained (10/HO405/8 and 13/EM/0123), and written informed consent was gained from patients. Both practices utilise the SystmOne general practice records system  for live recording of clinical, anthropometric and biochemical patient data. Data are stored as searchable numeric data or prospectively coded with ‘Read codes’ .Hazardous alcohol use—defined as >14 units per week ethanol consumption for women and >21 units per week ethanol consumption for men, or alcohol AUDIT questionnaire score ≥8, or presence of Read codes related to alcohol misuse.Type 2 diabetes—presence of Read codes related to type 2 diabetes (a full code list is available from the authors on request).Raised alanine aminotransferase (ALT)—as a comparator group, patients with persistently elevated serum ALT levels  without type 2 diabetes or hazardous alcohol use were assessed. In order to ensure only patients with alcohol-related liver disease or NAFLD were investigated, other causes of chronic liver disease were excluded by specific testing of chronic viral hepatitis B and C, autoimmune liver diseases, haemochromatosis and common genetic liver diseases, and no other relevant reason for transaminase elevation . Local laboratory cut-offs denoting elevated ALT are >35 U/L for women and >45 U/L for men.From the electronic general practice records, we identified adult patients (aged 18 years or older) with selected risk factors for lifestyle-related chronic liver disease at the start of the study period. Patients were eligible for study inclusion regardless of previous liver function blood test results, if identified with one or more of the following chronic liver disease risk factors:Identified patients were excluded from the study a priori if: (1) there was definitive evidence of hepatic fibrosis or cirrhosis from previous investigation, (2) there was a contraindication to TE  or (3) patients were unable to consent to investigation, or were housebound and could not attend the community practice.Patients identified with the defined risk factors were invited to the diagnostic algorithm see . Patient2, AST:ALT ratio ≥0.8 and type 2 diabetes, measured. A BARD score of less than 2 in hospital populations of NAFLD has a high negative predictive value to rule out significant hepatic fibrosis.Patients initially underwent a simple blood-based biomarker, with a high negative predictive value to accurately rule out hepatic fibrosis and therefore avoid the need for further tests. Hazardous alcohol users had the aspartate aminotransferase (AST):ALT ratio measured, a simple and cheap blood biomarker that has been shown to reflect disease severity in a number of chronic liver diseases, including alcoholic liver disease,2 underwent TE at the hospital using the XL probe (FS502 device), due to high rate of scan failures using the standard M probe above this BMI threshold.Patients with a high simple biomarker result underwent TE at the community practice. TE is a non-invasive imaging method that calculates liver stiffness via measurement of the propagation of an elastic shear wave through the liver substance. TE accurately predicts liver biopsy findings of fibrosis and cirrhosis in all major aetiologies of chronic liver disease.A TE threshold of 8.0 kPa or greater, which has been previously shown to accurately determine presence of hepatic fibrosis during community screening,2 test, or Fisher's exact test where appropriate.Statistical analysis was performed using SPSS V.19.0 (IBM). Categorical data are presented as numbers (percentage). Continuous data are presented as mean (SD) for parametric data and medians (range) for non-parametric data. Anthropometric and biochemical data were compared between patients with normal and elevated liver stiffness—continuous variables were compared using the two sample t test for parametric variables and Mann-Whitney test for non-parametric variables. Categorical variables were compared using χThe total patient population of the two participating general medical practices was 12 368, of which 10 479 patients were adults. In total, 5922 adult patients (56.5%) had alcohol consumption documented, with 6.3% of the total GP population (658 patients) meeting our definition for hazardous alcohol use. The adult prevalence of type 2 diabetes was 3.7% (390 patients). Both of these risk factors were found in 21 patients and thus 1027 patients were identified for the study. We excluded 107 patients and therefore 920 were invited to the study see . Of the Blood test uptake differed significantly between patients with type 2 diabetes and hazardous alcohol users . Normal blood-based biomarker results were seen in 62 patients (12.3%). In total, 378 patients underwent TE, of whom portable M-probe readings were performed in 361 patients (95.5%), while XL probe readings were performed in the remaining 17 patients. It was not possible to obtain a valid liver stiffness measurement in 12 patients (3.2%), and a further 24 patients (6.3%) had an unreliable measurement.Hazardous alcohol use was present in 174 patients (46.0%), type 2 diabetes in 211 patients (55.8%) and raised ALT in 54 patients . Overall, 57 patients (15.1%) had more than one chronic liver disease risk factor see . BaselinValid liver stiffness acquisition was possible in 366 patients (96.8%). A new diagnosis of clinically significant liver disease was made in 98 patients (26.8%) with valid TE measurement. This represents a substantial increase in diagnoses for these practices. A significantly greater percentage of patients with type 2 diabetes had elevated liver stiffness compared with hazardous alcohol users  were older, had a higher BMI and higher prevalence of the metabolic syndrome than those with normal liver stiffness (n=268). On laboratory testing, patients with elevated liver stiffness had lower platelet count, greater prevalence of thrombocytopaenia, higher serum ALT and lower AST:ALT ratio than patients with normal liver stiffness (see online supplementary table S1). The simple serum fibrosis scores for APRI and FIB4 were significantly higher in patients with elevated liver stiffness. However, these markers were only elevated in 2.0% and 10.2% of patients with elevated liver stiffness, respectively.Of 98 patients referred for community assessment by the hepatologists, liver biopsy was performed in 25 patients for whom there was diagnostic uncertainty on review of clinical data and TE results. Median biopsy length was 13 mm (IQR 7.5–18.0), and 12/25 patients had cores ≥15 mm. Hepatic fibrosis was confirmed in 20 patients undergoing liver biopsy; the remainder all had steatohepatitis (see online supplementary figure S1). Overall, 11 patients were newly diagnosed with liver cirrhosis during the study period based on clinical, radiological and/or histological assessment; 4 of these patients had additional evidence of portal hypertension (see online supplementary table S2). Prior to starting the study, only eight patients were known to have cirrhosis in this primary care population. The new observed cirrhosis prevalence of 19 patients after the study period therefore represents a 140% increase compared with before the study; usual care methods missed the majority of patients with very advanced liver disease.For local ALT cut-offs (>35 U/L for women and >45 U/L for men), 72.4% of patients with elevated liver stiffness, 60% with liver fibrosis on biopsy and 90.9% with liver cirrhosis had normal ALT levels, and would have been missed by standard diagnostic algorithms. Using conservative cut-offs (>19 U/L for women and >30 U/L for men) would still have missed 41.8% of patients with elevated liver stiffness and 18.2% with cirrhosis see . Simple This study uses a novel approach to detect clinically significant but asymptomatic chronic liver disease, based on: (A) directly targeting established risk factors and not simply using LFTs and (B) the integration of validated non-invasive tests of liver fibrosis and liver specialists within a community setting. The utilisation of a simple, low-cost algorithm detected a large number of previously undiscovered subclinical but significant chronic liver disease cases, including 98 patients with elevated liver stiffness , and the number of observed cases of liver cirrhosis in the primary care population of 10 479 adult patients from which our high-risk patients were drawn was increased by 140% compared with before the study. The study highlights the inadequacy of liver function enzymes as a stratification tool in primary care: 72% of patients with elevated liver stiffness, 60% with liver fibrosis on biopsy and 91% diagnosed with cirrhosis had normal ALT. In the absence of liver function abnormalities, or specific liver-related symptoms, their disease would have been missed by the standard diagnostic algorithms. We have demonstrated this diagnostic approach is feasible to implement and acceptable to patients, with more than 95% of investigations occurring in the community setting and non-attendance rates for TE appointments of less than 5%. This nurse-led TE service, which we originally established in the hospital setting,This study is the first to utilise blood biomarkers and TE to stratify patients at risk of liver disease in a community population of the UK. We have utilised a novel diagnostic algorithm, which is rapidly applicable in clinical practice, in a large community population of more than 10 000 adults. Targeting patients with well-defined risk factors for liver disease resulted in a high pretest probability of detecting hepatic fibrosis, with 26.8% of patients with TE results exceeding the threshold for clinically significant liver disease. Of 25 patients undergoing confirmatory liver biopsy, 80% had histological evidence of fibrosis. It is possible to argue that the remaining biopsies exposed patients to unnecessary risks of liver biopsy, although these five patients had histological evidence of steatohepatitis, suggesting a high risk of future progression to hepatic fibrosis or cirrhosis. This is a far greater diagnostic yield than traditional algorithms using elevated liver enzymes alone to stratify fibrosis risk,However, there are limitations to discuss from our approach. We investigated patients from specific medical practices within a distinct sociodemographic area of the UK. It is possible that both patient attendance and the detection of clinically significant liver disease may differ elsewhere, and further study of our algorithm in other regions is necessary. The pragmatic study design, both in terms of biomarker selection and investigation of selected risk factors for liver disease, means we also cannot formally assess the sensitivity of the algorithm, or the total fibrosis and cirrhosis prevalence, in this community population. Alcohol use was only screened in 57% of this community population, which is lower than the approximately 70% documentation of alcohol intake in primary care practices in the East Midlands region.et al32Previous studies of chronic liver disease stratification in the general population support our findings of an appreciable burden of previously undetected chronic liver disease. A study of presumed well army personnel and families in the USAet al,A recent study in the UK investigated 1118 patients with elevated liver enzymes in primary care; in those with suspected NAFLD, 7.6% were found to have significant disease based on an elevated NAFLD fibrosis score.The utilisation of a simple, low-cost and patient acceptable algorithm in the community resulted in the detection of a large number of cases of previously unidentified chronic liver disease. While the detection of these new cases is currently of unproven benefit, there are likely to be a number of advantages. Early identification of asymptomatic liver disease is likely to reduce future liver-related morbidity and mortality. Interventions for lifestyle-related disease risk factors form established treatments , can be performed in a community settingSecond, early identification of chronic liver disease is likely to be cost-effective compared with traditional investigation algorithms. The investigation of liver disease in specialist centres can be expensive for the healthcare system and inconvenient to the patient. Recent economic modelling from the UK has suggested that the substitution of elastography for liver biopsy in a hospital setting would save £520 per patient investigated.Lastly, there are no current medical treatments with widely proven efficacy to prevent fibrosis progression in either alcohol-related liver disease or NAFLD. Novel investigation algorithms, such as the one we have presented, identify a representative population of patients with clinically significant liver disease in whom appropriate therapies may be tested and developed. These, in conjunction with wider adoption of health-related lifestyle changes, may enable reduction of disease progression, symptomatic liver disease and liver disease-related deaths at a general population level.We have utilised a novel diagnostic approach in the general population by identifying patients with chronic liver disease risk factors and stratifying using non-invasive fibrosis biomarkers. This algorithm was feasible to implement and resulted in increased detection of asymptomatic but clinically significant liver disease compared to traditional liver enzyme-based algorithms, including a 140% increase in observed cirrhosis prevalence. Importantly, the majority of patients diagnosed with significant liver disease had normal liver function enzymes. Future studies are warranted to ensure the generalisability of our findings to other community populations, to identify the optimal biomarker algorithm for detecting clinically significant liver disease and, subsequently, the health-economic benefits of this approach compared with standard care.


The aim of the study was to investigate if (a) substituting total sedentary time or long sedentary bouts with standing or various types of physical activity and (b) substituting long sedentary bouts with brief sedentary bouts; is associated with obesity indicators using a cross sectional isotemporal substitution approach among blue-collar workers.brief (≤5 mins), moderate (>5 and ≤30 mins), and long (>30mins) bouts, were determined for the whole day and during work and non-work time separately. The obesity indicators, BMI (kg/m2), waist circumference (cm) and fat percentage were objectively measured. Isotemporal substitution modelling was utilized to determine the linear association with obesity indicators of replacing 30 min of total sedentary time or long sedentary bouts with standing, walking or MVPA and separately replacing 30 min of long sedentary bouts with brief sedentary bouts.A total of 692 workers from transportation, manufacturing and cleaning sectors wore an Actigraph GT3X+ accelerometer on the thigh for 1–4 working days. The sedentary (sit and lie), standing, walking, and moderate to vigorous physical activity (MVPA) time on working days was computed using validated Acti4 software. The total sedentary time and uninterrupted sedentary time spent in 2, %BF = 29.6 (9.5), waist circumference = 94.4 (13.0) cm] sat for 2.4 hours  across the day during work period and 5.5 hours  during non-work period. Most of the sedentary time was accrued in moderate bouts [work = 1.40 (SD = 1.09) hours] during work and in long bouts during non-work [2.7 (SD = 1.4) hours], while least in long sedentary bouts during work [work = 0.5 (SD = 0.9)] and in brief sedentary bouts [0.5 hours (SD = 0.3)] during non-work. Significant associations with all obesity indicators were found when 30 min of total sedentary time or long sedentary bouts were replaced with standing time (~1–2% lower) or MVPA (~4–9% lower) during whole day, work, and non-work periods. The exception was that a statistically significant association was not observed with any obesity indicator when replacing total sedentary time or long sedentary bouts with standing time during the work period. Significant beneficial associations were found when replacing the long sedentary bouts with brief sedentary bouts (~3–5% lower) during all domains.Workers [mean ; age = 45.1 (9.9) years, BMI = 27.5 (4.9) kg/mReplacing total sedentary time and long sedentary bouts, respectively, not only with MVPA but also standing time appears to be beneficially associated with obesity indicators among blue-collar workers. Additionally, replacing long sedentary bouts with brief sedentary bouts was also beneficially associated with obesity indicators. Studies using prospective design are needed to confirm the findings. The worldwide prevalence of obesity has almost doubled since 1980  with parsedentary time which has increased in western countries with many adults spending as much as 70% of their waking hours sitting  and body postures  across the day(s) with a sensitivity and specificity of more than 94% and 99%, respectively, during semi-standardized conditions . During th order Butterworth filter and split-up into 2 sec intervals with 50% overlap. Then, the occurrence of sedentary posture was defined as the posture in which the inclination of the thigh is above 45°. The daily reference measurements (i.e. standing in an upright position for 15sec on every measured day) were used to obtain the angle between the leg and accelerometer axis. The technical definitions of the identification of other activities using Acti4 program are reported in detail elsewhere  for BMI.Similar estimates to the primary analysis using isotemporal substitution were also observed when adjusting for time in bed (results not shown), except that the results of replacing 30 min of long sedentary bouts during work with MVPA became marginally non-significant  for fat percentage and replacing long sedentary bouts with standing became non-significant  for waist circumference during work.The most important finding of the present cross-sectional study is that among blue-collar workers beneficial associations with indicators of obesity were observed by theoretically replacing time spent on sedentary behavior with standing or higher intensity physical activity. Beneficial findings were also observed when time in long sedentary bouts was reallocated to short sedentary bouts.The amount of sedentary time in this study was similar to other studies among blue-collar workers , 39, 43.Workers who not only spent more time in long, but also less time in brief sedentary bouts had higher indicators of obesity and vice versa, even after adjusting for potential confounders and wear time (the single activity model) and other activities performed (the partition model). For example, spending 30 minutes more in long sedentary bouts during non-work time was associated with 0.98 cm (~1%) increase in waist circumference, while spending 30 minutes more in brief sedentary bouts during non-work time was associated with a decreased waist circumference by 2.94 cm (~3%) see , keepingDuring work, however, significant associations between total sedentary time or long sedentary bouts and obesity indicators were not observed, which is in contrast to some studies –58, but Our findings suggest that replacing 30 minutes of total sedentary time or long sedentary bouts with 30 minutes of MVPA during a day has a considerable beneficial meaningful association with obesity during all domains, which confirms the previously established beneficial role of MVPA in prevention and management of obesity , 63. HowReplacing prolonged sedentary time with brief sedentary bouts or standing may be more feasible than replacing it with MVPA among blue-collar workers who are already engaged in highly strenuous work tasks. We estimated that replacing ~45 minutes of long sedentary bouts with brief sedentary bouts or ~137 mins of long sedentary bouts with standing will have equal effect on obesity as replacing 30 min of long sedentary bouts with MVPA. In relative terms, ~45 min increase in brief sedentary bouts and ~137 min increase in standing corresponds to 69% or 37% increase in daily time spent in these activities, and may be more feasible among blue-collar workers than increasing 30 minutes in MVPA which corresponds to a 250% increase in their daily MVPA. Thus, the replacement of sedentary time with other activities such as standing and brief sedentary bouts may be relevant and practical in this cohort as long as the sufficient time is reallocated. Nevertheless, the observed results suggest that even a modest change such as replacing 30 minutes of long sedentary bouts with standing or brief sedentary bouts may reduce obesity among blue-collar workers. However, for these workers who may require sedentary time as ‘rest’ period between high strenuous working tasks, it might be appropriate to shift between job tasks to ensure reduction in prolonged, unbroken sedentary bouts instead of aiming to reduce total sedentary time. More research is needed to determine which activity-replacement, in practice, is most feasible for achieving sustainable favorable changes in obesity among blue-collar workers without affecting their work demands-recovery balance.Interestingly, we did not find any beneficial association with obesity indicators of replacing total sedentary time or long sedentary bouts with walking time. These findings are in contrast to a growing literature on the positive effect of light physical activities on health outcomes including obesity , 35. IndA major strength of our study is the study population of blue-collar workers, being homogeneous with respect to being employed in blue collar work, but offering a great variation in sedentary time and various types of physical activity. We also used the validated software, Acti4 to assess the exposure. It is capable of discriminating between different types of physical activity and body postures with excellent sensitivity and specificity . PreviouThe main limitation of the study is the cross-sectional study design, which does not allow for inferences about causal associations with obesity of replacing prolonged sedentary activities with brief sedentary bouts and various types of physical activity. Thus, future prospective studies are needed to assess the strength and direction of this association in different domains. Since our study included a sample of Danish blue-collar workers only, our results may not be generalized to the general population of blue-collar workers in Denmark, let alone in industrialized countries in general. Furthermore, the results may be affected by residual confounding from medication usage, prevalence of chronic diseases, working status, family history, and activity patterns during commuting to work. The adjustment for influence at work was only performed in the sensitivity analyses due to a large amount of missing values in the variable (~30%), although adjusting for this variable did not change the results of the primary analysis. Another limitation is that the use of traditional statistical analyses may not be suitable due to the constrained nature of the data used in this study . This reIn conclusion, total sedentary time and uninterrupted long sedentary bouts were positively associated with obesity indicators during whole day and non-work time while brief sedentary bouts were negatively associated with obesity indicators during all domains. Our findings indicate that beneficial health effects may be gained by replacing total sedentary time and uninterrupted long sedentary bouts not only with MVPA, but also with standing among blue-collar workers. Similar benefits may also be gained by replacing uninterrupted long sedentary bouts with brief sedentary bouts among these workers. However, these interpretations of the results need to be confirmed in future studies using a prospective study design.S1 DatasetThe data file ‘whole day’ contains data for investigating the association between sedentary patterns during whole day and obesity indicators (N = 692) and the data file ‘work and non-work’ contains data for investigating the association between sedentary patterns during work and non-work time domain and obesity indicators (N = 671).(XLSX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Functional brain signals are frequently decomposed into a relatively small set of large scale, distributed cortical networks that are associated with different cognitive functions. It is generally assumed that the connectivity of these networks is static in time and constant over the whole network, although there is increasing evidence that this view is too simplistic. This work proposes novel techniques to investigate the contribution of spontaneous BOLD events to the temporal dynamics of functional connectivity as assessed by ultra-high field functional magnetic resonance imaging (fMRI). The results show that: 1) spontaneous events in recognised brain networks contribute significantly to network connectivity estimates; 2) these spontaneous events do not necessarily involve whole networks or nodes, but clusters of voxels which act in concert, forming transiently synchronising sub-networks and 3) a task can significantly alter the number of localised spontaneous events that are detected within a single network. These findings support the notion that spontaneous events are the main driver of the large scale networks that are commonly detected by seed-based correlation and ICA. Furthermore, we found that large scale networks are manifestations of smaller, transiently synchronising sub-networks acting dynamically in concert, corresponding to spontaneous events, and which do not necessarily involve all voxels within the network nodes oscillating in unison. Functional magnetic resonance imaging (fMRI) signals are often decomposed into a few, large scale, distributed networks within which blood oxygenation level-dependent (BOLD) signals are highly temporally correlated . These nMost functional connectivity studies assume spatio-temporal stationarity in the coupling between network nodes. However some studies have begun to assess dynamic changes in functional connectivity using both fMRI –16 and ea priori). Paradigm free mapping (PFM) [In order to detect the BOLD response associated with spontaneous events, a model free analysis approach is required (i.e. one for which the timing of the events is not known ng (PFM)  is such ng (PFM) ,22,23. Ong (PFM) . HoweverUsing this methodology, we tested the hypotheses that: 1) spontaneous BOLD events are related to dynamic variations in network connectivity; 2) these events involve consistent, spatially-structured activity within and across nodes of distributed networks, but may not involve the whole network; 3) performance of a task can change the rate of detection of spontaneous events.The University of Nottingham ethics committee approved this research. Written informed consent was received from each subject in accordance with the ethics. Twelve subjects from our lab  took part in the study, and each performed two experiments: a ‘Motor’ and a ‘2-back’ experiment. Each subject was instructed to remain at rest with their eyes open for 0<t<300s, perform a task for 300<t<660s and rest again for 660<t<1020s. The motor task consisted of a continuous, unilateral right hand finger tapping, whilst a sequence of letters was presented for the two back task. This was repeated at 2s intervals and the subject was asked to respond with an index finger button press when the current letter matched the letter presented two previously (a 2-back match occurred every 10s on average with 36 +/- 1 targets in each experiment). Both tasks were effectively continuous and were aimed at modulating baseline brain activity between the three periods (rest/task/rest) of the paradigm, rather than generating event-like activations during the task.3, 30 slices, TE/TR: 25/2000 ms, SENSE: 3, bandwidth: 28 Hz). Cardiac and respiratory data were recorded using a vector cardiogram and respiratory belt. Data were initially realigned and slice timing corrected using SPM5 . Datasets were excluded if movement was greater than 1 voxel (none excluded). Subsequently, RETROICOR [A schematic diagram of the analysis workflow is shown in the supplementary information . T2*-weiIndividual subject data sets were analysed using the probabilistic spatial ICA algorithm implemented in FSL . Thirty Next, to identify spontaneous events Paradigm Free Mapping (PFM)  was applNodal ATSs were produced by summing the ATS over all voxels in each of the nodes of each network . Nodal EThe Voxelwise Event Rate was assessed by calculating the number of spontaneous events found in any voxel (of any node) in the network per minute, normalised by the total number of voxels within the network. This was done for each network and each paradigm period to identify changes in rates of local (voxelwise) spontaneous activity caused by the introduction of a task.th percentile to determine a significant correlation for that window length.To demonstrate how functional connectivity measures were affected by these detected events, for each network one Coordinated Network Event per subject was selected at random and the correlation map was computed for the 30s time window immediately following the onset of the event. The resulting correlation maps were transformed into MNI space using FLIRT  and wereTo study the effect of events detected by PFM on the correlation analysis, sliding window correlation analysis ,30,31 2 for eachFinally, temporal ICA (tICA) was used to detect regions showing consistent, temporally independent patterns of activity related to events. tICA is generally not applied to fMRI data due to the limited number of time points acquired in a time series, but tICA can be applied to the sparse output of PFM since the number of temporal independent signals in the data is substantially reduced relative to the raw data . Furthermore tICA was only applied within recognised functional networks to focus on a particular set of temporal components, whilst eliminating other confounding signals. For tICA analysis, data were masked using standard network masks for the MN, FPN and DMN ; standarAll statistical analyses were performed using 2-sided Wilcoxon signed rank tests since the data were not expected to be normally distributed given the variations in the numbers of events found between individuals.The ATS, averaged over all voxels within separate nodes of each network, can be seen in Sliding window seed correlation maps (30s window) computed at 40s time intervals from the pre-processed data are shown below each ATS in vice versa . The difference in average connectivity between the seed node and rest of the network for the period following a Coordinated Network Event is significantly higher than that during a null period (The subject-averaged correlation maps produced for 30 second windows following a Coordinated Network Event (for the three networks) demonstrate that full network structure can be defined from short periods of data Click here for additional data file.S2 FigComparison timecourses for the nodal ATS for (EPS)Click here for additional data file.S3 Fig(EPS)Click here for additional data file.S4 Fig(EPS)Click here for additional data file.S5 Fig(EPS)Click here for additional data file.S6 Fig(EPS)Click here for additional data file.S7 Fig(EPS)Click here for additional data file.S8 Fig(EPS)Click here for additional data file.S9 Fig(EPS)Click here for additional data file.S10 Fig(EPS)Click here for additional data file.S11 Fig(EPS)Click here for additional data file.S12 Fig(EPS)Click here for additional data file.S13 Fig(EPS)Click here for additional data file.S14 Fig(EPS)Click here for additional data file.S15 Fig(EPS)Click here for additional data file.


Markusse and colleagues recently investigated whether rheumatoid arthritis patient subgroups formed according to the presence of poor prognostic factors respond differently to initial monotherapy or combination therapy . Since bAs a general remark, the authors should be reminded that the EULAR recommendations primarily suggest combination of methotrexate with low-dose glucocorticoids because its efficacy is not surpassed by biologicals and it prevents overtreatment in 20 to 25 % of patients , 4; delasic) uses a different approach, namely C-reactive protein, erosion score and rheumatoid factor/anti-citrullinated protein antibody combination, to determine who had >50 % chance of rapid radiographic progression (≥5 Sharp–van der Heijde Score (SHS) units/year) [Markusse and colleagues propose the presence of three of four characteristics as the definition of PP . In contrast, the definition of PP established by Visser and colleagues in the same trial population (ts/year) , is muchThe odds of response to initial combination therapy versus initial monotherapy in the PP versus non-PP populations were much higher when using Visser and colleagues’ approach versus Markusse and colleagues’ approach . Separation of the Health Assessment Questionnaire score between PP and non-PP patients treated with initial combination therapy is only seen with Visser and colleagues’ definition. This highlights that definition of PP influences the effect of clinical outcomes.In accordance with the EULAR research agenda , we also


ATM locus for rs227060 , which remained significant after adjustment for multiple testing. In a subtype-specific analysis, associations were also observed for the ATM locus among both diffuse large B-cell lymphomas (DLBCL) and small lymphocytic lymphomas (SLL), however there was no association observed among follicular lymphomas (FL). In addition, our study provides suggestive evidence of an interaction between SNPs in MRE11A and NBS1 associated with NHL risk . Finally, an imputation analysis using the 1,000 Genomes Project data combined with a functional prediction analysis revealed the presence of biologically relevant variants that correlate with the observed association signals. While the findings generated here warrant independent validation, the results of our large study suggest that ATM may be a novel locus associated with the risk of multiple subtypes of NHL.Molecular and genetic evidence suggests that DNA repair pathways may contribute to lymphoma susceptibility. Several studies have examined the association of DNA repair genes with lymphoma risk, but the findings from these reports have been inconsistent. Here we provide the results of a focused analysis of genetic variation in DNA repair genes and their association with the risk of non-Hodgkin's lymphoma (NHL). With a population of 1,297 NHL cases and 1,946 controls, we have performed a two-stage case/control association analysis of 446 single nucleotide polymorphisms (SNPs) tagging the genetic variation in 81 DNA repair genes. We found the most significant association with NHL risk in the  The incidence of non-Hodgkin's lymphoma (NHL) in the U.S. has doubled over the past two decades. While the etiology of the disease remains largely unknown Associations between DNA repair genes and lymphoma risk have been reported previously All cases were ascertained through Memorial Sloan-Kettering Cancer Center (MSKCC) IRB-approved protocols, or a protocol approved by the IRB at the Dana Farber Cancer Institute (DFCI) or Hadassah-Hebrew University. These protocols required written informed consent either for identified use of specimens for research into the genetic basis of lymphoma, or research use of specimens permanently de-identified prior to genotyping. Controls were part of the New York Cancer Project (NYCP) and all subjects gave written consent for use of samples in genetic studies of any disease state.In total, the study involved 1,297 non-Hodgkin's lymphoma (NHL) cases from the combined resources at MSKCC, DFCI and Hadassah-Hebrew University, Israel as well as 1,946 controls collected from the NYCP, a study of 18,000 New York City residents originally designed to assess the role of environment and genetics in cancer risk, and described previously elsewhere 2) threshold <0.6 across selected gene regions (including 5 kb from 3′ and 5′ UTR), and minor allele frequency (MAF) >0.05. The tagging SNP selection has been performed using the CEU data  of HapMap Phase II, the most accurate resource available for this purpose at the time of the study design and still serves as the most validated reference for capturing the common genetic variation in European populations. In total, 531 tagging SNPs (tSNPs) were selected to tag the 87 DNA repair genes.The selection of candidate DNA repair genes was performed as summarized in For stage 1, the genotyping of 531 tSNPs from 87 selected DNA repair genes on 698 lymphoma cases and 1,041 controls was conducted using Sequenom MassARRAY iPLEX , multiplexed into a 16-plex design as per manufacturer's protocol and as described previously ggplot2 in R, and inflation factors (λ) were calculated based on 90% of least significant SNPs. Statistical analyses were performed using PLINK Single SNP associations with NHL risk were tested using a logistic regression model under a per-allele test, calculating odds ratios (OR) and 95% confidence intervals (95% CI), adjusted for age, gender and AJ status. The associations were analyzed for stage 1, stage 2, and the aggregate sample set including both stages. In the aggregate sample set we also used a per-allele logistic regression model to test fifteen SNP associations within the three most common NHL subtypes in our dataset: diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL) and small lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL). Quantile/quantile (Q/Q) plots were produced using Haplotypes were visualized by Haploview and haplotype associations were performed by logistic regression analysis adjusted for age, gender, and AJ status. We have also tested the homogeneity of the odds ratios between these three major subtypes by calculating the Breslow-Day statistics for each SNP.−5). This analysis was performed using PLINK MRE11A, NBS1), the multiplicative two-way gene-gene interactions were estimated using multiple logistic regression models. For each SNP pair, a logistic regression model was built to test case/control association based on the indicator variables  and the 2-SNP variable, for a total of 5 variables and an intercept. The 2-SNP variable was defined separately under three genetic models, based on the number of risk alleles in individual subjects.In order to explore possible epistatic interactions associated with NHL risk, logistic regressions were modeled by adding an interaction term between the genotypes of each SNP pair. First, pairwise comparisons were performed between each SNP from the list of fifteen associations with NHL (105 tests), followed by pairwise comparisons of fifteen SNPs with the additional 94 SNPs included in the aggregate analysis   = 3.3×10For the assessment of SNP-gene association by incorporating expression information (expressed quantitative trait loci – eQTL) we used Genevar The imputation of genotypes from 1,297 NHL cases and 1,946 controls was performed using IMPUTE2 The demographic and clinical composition of our sample population is summarized in In this study we applied a two-stage design: in stage 1 we performed the association analysis on 446 SNPs tagging 81 DNA repair genes in the population of 650 cases and 965 controls. There was no significant inflation in observed versus expected associations (λ = 1.07), indicating no detectable genotyping artifacts or population substructures impacting our findings .ATM locus: rs611646, rs419716 and rs227060, the latter showing the strongest effect . Other associations in stage 1 included tSNPs in MRE11A , GTF2H1 , and MSH2 .We first tested the association of DNA repair variants with NHL cases . The association analysis of NHL cases in stage 1 identified 28 SNPs associated with NHL risk (p<0.05). The strongest associations in stage 1 were found for 3 SNPs in the ATM with a more pronounced effect than in stage 1; rs227060 again shows the strongest association in stage 2 . Other SNPs that replicated in stage 2 include GTF2H1 , MSH2  and MRE11A . See The twenty-eight most significant SNPs from stage 1 and an additional 81 SNPs, as described in ATM locus: rs227060 and rs611646 . Importantly, the associations for both loci remained significant after Bonferroni correction, as detailed in adj  = 0.022; padj = 0.049, respectively, where padj is the Bonferroni corrected p-value for each SNP). The two SNPs show incomplete LD , suggesting the association signals observed for ATM SNPs are correlated. No other SNP associations in the aggregate analysis passed Bonferroni correction. To further explore the structure of associated loci, we also examined common haplotypes (MAF>0.05) for association with NHL risk. The strongest risk effect has been observed for a haplotype in ATM. Other loci have also shown specific haplotypes associated with NHL risk, however none were more significant than the associations from single SNP analyses , rs4952887 (MSH2), and rs702019 (POLQ), both AJ and non-AJ subsets contribute to the association signal. Therefore, all analyses in the study were also adjusted by AJ status.In the aggregate analysis of stage 1 and 2, fifteen SNPs showed associations with NHL risk . These ianalyses . As our analyses . Three Spulation . HoweverATM tSNPs in DLBCL (n = 412), with the strongest effect for rs611646 . Associations for rs611646 and rs227060 were also observed in SLL/CLL (n = 164). No ATM tSNPs were associated with risk in the FL subset (n = 301). In contrast, the strongest effects in FL was observed for CHEK1  and TDP1 , which were not associated with DLBCL or SLL/CLL. In SLL/CLL, the strongest association effect was found for rs4150606 tagging GTF2H1 ; this association was not seen in FL or DLBCL sub-analyses. The association of rs4150606 with SLL/CLL remains significant after Bonferroni correction for multiple testing (padj = 0.0016). The SNP/MAF plot between AJ and non-AJ , follicular lymphomas (FL), and small lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL). As shown in d non-AJ  identifid non-AJ .GTF2H1, p = 0.001) and rs565416 .We have also tested the association heterogeneity among the three major subtypes in our analysis of fifteen SNPs . The BreMRE11A with SNPs in NBS1 associated with NHL risk. Interestingly, both genes biologically interact in the MRN complex, a centerpiece of double strand break repair machinery, which prompted us to examine these interactions more closely using different genetic models. While all interactions involve rs1805812 in NBS1, for MRE11A there are four different variants which contribute to these associations . After examination of the four pairwise associations (rs1805812 in NBS1 with each of the four MRE11A SNPs) using three genetic models (Models 1–3 detailed in NBS1) x rs625245 (MRE11A) and rs1805812 (NBS1) x rs607974 (MRE11A). As shown in MRE11A, NBS1). The association observed for rs1805812 x rs625245 also shows the strongest effect under Model 3 , other imputed SNPs map within high-impact regulatory regions. In the ATM locus we found numerous putatively functional variants strongly correlated with associated tSNPs , T-cell lines (TCL), and fibroblast cell lines (FCL) derived from 75 individuals of European ancestry ATM SNPs associated with NHL risk in our data, the most significant eQTL effect was observed for rs227060 in LCL based on data from probe ILMN_1716231, showing that reduced expression correlates with the risk allele  or Nijmegen breakage syndrome  manifest with early onset lymphomas of various histological subtypes Extensive published data suggest that DNA repair pathways are associated with lymphoma susceptibility ATM locus, remaining significant after adjustment for multiple testing (Bonferroni). Although among FL patients there was no association observed, ATM SNPs did correlate with risk of DLBCL and SLL/CLL. While the FL subset was the second largest in the study (n = 301), many other subtypes were underrepresented (n<100) and hence it was not possible to assess risk among these smaller groups due to the limited power . It is also important to note that the associations of ATM SNPs are stronger among the pooled NHL analysis  compared to separate associations with DLBCL or SLL/CLL. This evidence suggests for the first time that ATM is a putatively novel candidate NHL susceptibility locus.In this study, we report the most significant association with risk of NHL for two SNPs in the ATM SNPs, which show the significant risk effect only in non-AJ population. While the frequency of the risk allele of both SNPs appears to be similar among AJ and non-AJ cases , the MAF in AJ and non-AJ controls differs by approximately 4% . While there is a possibility of underlying genetic substructure ATM locus in AJ as well as non AJ populations will be needed.In our study we have noted association differences in stratified analyses by AJ status . This reATM with lymphoid malignancies has been largely restricted to the somatic level; ATM somatic mutations were noted in particular lymphoma subtypes of DLBCL ATM (MAF<0.05) have previously been associated with CLL susceptibility 2<0.1). The study by Sipahimalani, et al. performed an extensive analysis exploring the association of six common genetic variants in ATM with lymphoma risk ATM SNP associated with NHL risk in our data, rs419716, is strongly correlated with one of the SNPs from that prior study, rs664982 (r2>0.9), no association was reported for rs664982 by Sipahimalani, et al. et al. provides another possible explanation. While our strongest signals appear to be driven by associations of ATM SNPs in DLBCL and SLL/CLL, the study population from Sipahimalani, et al. had a much higher proportion of FL, for which we did not observe an association in our study. DLBCL specific GWAS studies have been previously reported ATM was not among the reported associations in these prior studies, these reports focused only on the loci that passed the threshold of genome wide level of significance. For independent confirmation, a separate deeper analysis of this published data will be needed in the follow up study to validate the potential association effect of ATM with SLL/CLL and DLBCL in the large populations studied in these scans. Although the replication of our findings in NHL subtypes will be critical as part of the prior and upcoming GWAS studies from large consortia, it will also be very important to test the association effect of identified ATM variants in pooled NHL population, where we observed the most significant associations in our analysis. Such rationale is particularly relevant given the critical role of DNA repair pathways (with ATM as an important cell cycle checkpoint) in early development of progenitor B and T-cell lineages ATM is a critical checkpoint impacting both homologous recombination and non-homologous end joining ATM may affect the capacity of DSB repair in a way that would associate with the patterns of specific large-scale genomic alterations and rearrangements in a subset of cells in the tumor due to yet unknown genetic or microenvironment modifiers. Although in the context of this study it is highly speculative, given the associations with the ATM locus observed here, such a biological scenario is an attractive possibility and should be further investigated in detail on the somatic level of lymphomagenesis.Prior evidence linking XRCC1 and XRCC2BLMMGMT, a gene involved in DNA repair of alkylation damage XRCC1MRE11A and NBS1, which were associated with NHL risk in our study, were examined in a previous report on a small subset of NHL population MRE11A and NBS1 associated with lymphoma risk in our analysis.Numerous other studies have previously examined the germline variation in major DNA repair genes for their association with lymphoma risk MRE11A and NBS1, critical components of the MRN complex, associate with increased risk of NHL. Interestingly, these findings show that the carriers of at least one copy of the minor (protective) allele inherited in both MRE11A and NBS1 are at more than a 2-fold reduced risk of developing NHL compared to the single SNP effect of each locus separately. While these findings are novel they are also exploratory, given the reduced power of this analysis and the fact that none of these observations reached the significance threshold adjusted for multiple testing. Importantly however, these epistatic interactions were observed in both stage 1 and 2 separately (ATM or CHEK1/2) to the MRN interaction model found in our study would be of interest. This analysis, along with the independent validation of epistatic and single SNP associations identified here, will need to be performed in a large consortium as part of the analyses following up on recent reports One novelty of our study design compared to prior efforts is the exploratory assessment of potential epistatic effects in DNA repair networks contributing to lymphoma risk. Our data suggest that the interactions between several genetic variants in parately  suggestiATM were located within multiple functional regions, as annotated utilizing ENCODE data from lymphoblastoid cell lines. For example, rs228594, rs228599, and rs189037, which correlate with the associations observed in ATM, map within known transcription factor binding sites as well as chromatin marks, DNase I hypersensitivity clusters, and 5′ UTR, strongly supporting a possible impact on expression regulation.Our study provides a suggestive link between the associated SNPs and putatively functional variants to be pursued in subsequent molecular studies. By imputing our data from the public resources of 1KG we have identified several functional variants highly correlated with the SNPs associated with NHL risk in our study. Most notably, several of the variants in ATM region, and was detected in lymphoblastoid cell lines but not in fibroblast controls ATM correlating with the dosage of rs227060 risk allele. The reduction of ATM expression has been strongly linked with radiosensitivity and defective DNA damage-induced ATM-dependent signaling in various experimental studies, and was clearly shown to promote the tumor growth in lymphoma and other cancer models in-silico functional predictions as presented here can substantially improve subsequent fine mapping strategies of associated SNPs. At the same time this approach can reduce the need for a large scale re-sequencing by functional prioritizing the target variants for further molecular investigation.The eQTL association with our top SNP, rs227060, was the second most significant eQTL association within the ATM, other loci also showed association effects in pooled NHL or subtype-specific analyses. As power limitations of our study may also be a concern, subsequent validation of these findings should be performed in large consortia. These future studies, with the concomitant collection of epidemiologic data and clinical characteristics, will also allow for a more in-depth analysis of potential gene-environment interactions attributed to DNA repair pathways. At the same time, the utilization of data from completed and ongoing lymphoma GWAS will be critical for the assessment of other interacting molecular pathways that may define the complex genetic susceptibility to NHL. For example, the HLA locus has been consistently replicated as a low-penetrant allele in recent NHL GWAS and follow-up meta-analyses Besides Figure S1Strategy schema for the selection of candidate DNA repair genes in the study.(TIF)Click here for additional data file.Figure S210 vs. –log10 under different quality metric scores (info).Quantile/Quantile (Q/Q) plot comparing –log Based on imputation analysis of 109 genotyped SNPs using IMPUTE2. Q/Q analysis of imputed data was used to select quality score threshold with least inflation of significant SNPs, which we have set at 0.7.(TIF)Click here for additional data file.Figure S310 vs. −log10.Quantile/Quantile (Q/Q) plot of the association results from stage 1 SNPs genotyped in 650 NHL cases and 965 controls; −log The blue dashded line indicates the inflation factor based on 90% of the least significant SNPs (λ = 1.07).(TIF)Click here for additional data file.Figure S4LD structure generated by Haploview shown for ATM gene region. The triangle plot displays correlation between the three tagging SNPs genotyped in the study . The associations of individual SNPs are displayed as −log10 for each SNP from the main effect aggregate analysis.(TIF)Click here for additional data file.Figure S5Minor allele frequency plot for non-AJ vs. AJ samples. Data based on allele frequencies in the aggregate analysis (for 109 SNPs with genotype information in stage 1 and stage 2). Blue dots indicate those SNPs associated with NHL in the aggregate analysis. Only 15 SNPs (14%) showed a difference in MAF >0.05. Indicated by arrows are NHL associated SNPs which did show a MAF difference >0.05 between non-AJ and AJ samples.(TIF)Click here for additional data file.Figure S6Genotype vs. expression (eQTL) results for rs227060 and four expression probes across the ATM locus. Data generated from lymphoblastoid cell lines (GenCord-L), T-cell lines (GenCord-T) and fibroblastoid cell lines (GenCord-F) established from 5 individuals of European ancestry. The most significant association for rs227060 in GenCord-L has been observed for ILMN-1716231. Data were generated using Genevar.(TIF)Click here for additional data file.Table S1Summary of the demographic characteristics of the case/control study population.(XLSX)Click here for additional data file.Table S2List of DNA repair genes and tagging SNPs genotyped. Chromosome and base pair position are based on GRCh37/hg19 build. Allele frequencies calculated among our sample population.(XLSX)Click here for additional data file.Table S3Results of single SNP associations analysis of tSNPs in DNA repair genes with the risk of NHL observed in our study, including 109 SNPs genotyped in stage 1 and stage 2.(XLSX)Click here for additional data file.Table S4Full list of haplotype associations (p<0.05) for DNA repair genes associated with NHL in our study.(XLSX)Click here for additional data file.Table S5Stratified AJ and non-AJ results for SNPs associated with NHL in the main effect aggregate analysis.(XLSX)Click here for additional data file.Table S6Results from pairwise SNP-SNP interaction associations of tSNPs in DNA repair genes with NHL risk. A) Pairwise comparisons among the 15 tSNPs associated with NHL risk in the aggregate main effect analysis. B) Pairwise comparisons of 15 tSNPs with all additional SNPs genotyped in both stage 1 and stage 2 (n = 94). Associations between MRE11A and NBS1 are shaded in grey.(XLSX)Click here for additional data file.Table S7Pairwise interaction results between NBS1 (rs1805812) and MRE11A  and NHL risk under three different genetic models. Each model was tested for a different SNP x SNP combination between rs1805812 and 4 SNPs tagging MRE11A. Indicated are the different combinations of genotypes used for each respective genetic model.(XLSX)Click here for additional data file.Table S8Summary of tagging and imputed SNPs in DNA repair genes associated with NHL risk with high predicted functional impact. Functional information was annotated using ANNOVAR, and included data generated from the ENCODE project.(XLSX)Click here for additional data file.


Wolbachia induce a form of embryonic lethality called cytoplasmic incompatibility (CI). This property can be used to reduce the density of mosquito field populations through inundative releases of incompatible males in order to sterilize females . We have previously constructed the LR[wPip(Is)] line representing a good candidate for controlling field populations of the Culex quinquefasciatus mosquito in the islands of the south-western Indian Ocean. The main purpose of the present study was to fill the gap between laboratory experiments and field implementation, i.e. assessing mating competitiveness of these incompatible males under semi-field conditions. In a first set of experiments, we analyzed crossing relationships between LR[wPip(Is)] males and La Réunion field females collected as larvae in 19 distinct localities throughout the island. This investigation revealed total embryonic mortality, confirming the strong sterilizing capacity of LR[wPip(Is)] males. Subsequently, mating competitiveness of LR[wPip(Is)] males was assessed under semi-field conditions in the presence of field males and females from La Réunion. Confrontations were carried out in April and December using different ratios of LR[wPip(Is)] to field males. The results indicated that the LR[wPip(Is)] males successfully compete with field males in mating with field females, displaying even higher competitiveness than field males in April. Our results support the implementation of small-scale field tests in order to assess the feasibility of IIT against Cx. quinquefasciatus in the islands of southwestern Indian Ocean where this mosquito species is a proven competent vector for human pathogens.In mosquitoes, the maternally inherited bacterial  Wolbachia has been focusing increasing attention and is currently under development in several countries worldwide  line conferring total embryonic lethality in crosses between LR[wPip(Is)] males and field females sampled at a single site on each of the five investigated SWIO islands [wPip(Is)] males with La Réunion field males and demonstrated that a population crash could be reached in competition experiments involving 1:5 to 1:10 La Réunion:LR[wPip(Is)] males ratios  line in reciprocal crosses involving field mosquitoes sampled as larvae in 19 distinct localities on La Réunion Island. We then assessed incompatible males’ competitiveness under semi field conditions, an investigation that is required prior to open field release since these conditions are more closely related to mosquitoes’ natural habitat. Indeed, laboratory conditions are homogeneous in terms of temperature, relative humidity and light intensity; while field conditions are highly dynamic and diverse, which could affect LR[wPip(Is)] males competitiveness. Overall, this study provides data that can be used for operational implementation of IIT against Cx. quinquefasciatus in SWIO islands.In this study, we performed tests under semi-field conditions in order to assess the feasibility of IIT against er virus , and exher virus , 34. As er virus , 35, we Culex quinquefasciatus larvae and pupae were collected in breeding sites in 19 localities of La Réunion Island in March, April, May and June 2012; and in March, April, November and December 2013 (wPip(Is)] line, harboring the sterilizing wPip(Is) strain together with a nuclear background of La Réunion mosquitoes [ber 2013 . Mosquitber 2013  and mosqsquitoes  was usedad libitum with a mixture of rabbit and fish-food whilst adults were fed with 10% sucrose solution [w/v].Mosquitoes were maintained in the laboratory under conditions at 25 ± 2°C and 75 ± 2% relative humidity and a LD 12:12 h photoperiod. Larvae were reared at a density of approximately 500 larvae per tray (30×40 cm) containing 1 liter of water and were fed Cx. quinquefasciatus mosquito is not considered as an endangered or protected species. So, no specific permission was required to collect mosquito larvae and pupae in public areas.None of the samples used in this study were collected in protected areas and the wPip(Is)] line and field mosquitoes from 19 localities of La Réunion island (i.e. unembryonated) egg rafts were removed from the analyses.Reciprocal crosses were performed in the laboratory between the LR males were obtained from the mosquito line reared in standardized laboratory conditions since 2010 (i.e. during ~60 generations assuming 12 generations per year). Males and females were held in separate laboratory cages (30×30×30 cm) before being transported to the field cages where males were released before females. Only one-day-old virgin adult mosquitoes were used in the experiments. Two hundred field females were mixed with each of the four following ratios of field to LR[wPip(Is)] males: 1:0 , 1:1 , 1:5  and 0:1 . A total of 15 trials were performed , whilst LR[erformed . These iwPip(Is)] males was assessed by comparing the observed and expected frequencies of infertile egg rafts assuming random mating.Mosquitoes released in field cages were recaptured five days following releases. Surviving mosquitoes were collected using a mouth aspirator at the end of each confrontation. Males and females were then placed into separate laboratory cages (30×30×30 cm) and brought back to the laboratory. Males were immediately stored in 70% EtOH and surviving mosquitoes were next counted and genotyped (see below); while females were blood fed in the laboratory and sugar fed until oviposition. Egg rafts were then collected and analysed as described above. Mating competitiveness of LR[wPip(Is)] males. For males recaptured from cage confrontations, we measured the survival rate by counting surviving males when only one type of males was present in cages (i.e. in the 1:0 and 0:1 control ratios) and by genotyping Wolbachia infecting each male’s category to determine the ratio of field vs. LR[wPip(Is)] surviving males at the end of confrontations. Due to the high number of males released in the 1:5 ratio (N = 1200), this analysis was restricted to individuals from trials with the 1:1 ratio. The discrimination of Wolbachia strains infecting field males (carrying wPip-I group strains) and LR[wPip(Is)] males (infected with a wPip-IV group strain) was performed through the genotyping of the Wolbachia ank2 marker, an ankyrin domain encoding gene which allows distinguishing wPip-I and wPip-IV groups on the basis of the size of the PCR amplified fragments ] (N = 177) virgin males were introduced into separated laboratory cages (45×45×45 cm) and were transported in the semi-field setup. Males were fed with 10% sucrose solution and dead mosquitoes were checked twice a day. Survival data were fitted to the Cox proportional hazards models  or by cectively , 39). Es package ) and a r package .vs. LR[wPip(Is)]) with Kruskal-Wallis tests. All statistical analyses were performed using the R software  line and field Cx. quinquefasciatus specimens sampled in 19 localities in La Réunion Island (wPip(Is)] line over a wide range of natural populations. Full compatibility (HR >90%) was found in control crosses between La Réunion field males and females as well as in the cross between LR[wPip(Is)] males and females ] males and field females from the 19 localities were incompatible and displayed total embryonic mortality ] males towards field females sampled throughout La Réunion Island, this result also shows a temporal stability of CI intensity considering that the LR[wPip(Is)] line has been maintained in the laboratory for over four years.All crosses between LR[wPip(Is)] males, crosses between LR[wPip(Is)] females and field males from the 19 localities produced polymorphic phenotypes with both infertile (HR = 0%) and fertile egg rafts (HR >90%) depending on crosses (wPip(Is)] females were crossed with males from La Possession (#10), only infertile egg rafts were observed (N = 74); most egg rafts were infertile with males from St Denis (#11) [87% (N = 84/97)] whilst the frequency of infertile egg rafts was the lowest with males from Ste Rose (#17) [19% (N = 22/118)].In contrast to LR[ crosses . For inswPip(Is)] males and females with La Réunion field mosquitoes, two CI patterns were thus observed: bidirectional CI ] and field mosquitoes from a locality) and unidirectional CI (or UCI i.e. infertile egg rafts found in the cross between LR[wPip(Is)] males and field females, and fertile egg rafts in the reciprocal cross). BCI was the most frequent CI pattern, occurring in all examined localities, with a frequency depending on localities ] males were present with field females (0:1 ratio) (wPip(Is)] males (i.e. in 1:1 and 1:5 ratios) produced both fertile and infertile egg rafts. We never observed egg rafts with intermediate hatching rate, all egg rafts being either fully fertile or infertile.As expected, all egg rafts from cages with only field males and females (1:0 ratio) were fertile (HR >90%) and full sterility (HR = 0%) occurred when only LR[1 ratio) . ExperimwPip(Is)] males ratio (1:1 ratio) should produce 50% infertile egg rafts whilst 83.3% (5/6) of infertile egg rafts are expected in the 1:5 field to LR[wPip(Is)] males ratio. Observed frequencies of infertile egg rafts overall showed no significant deviation from theoretical expectations ; the mean frequency of infertile egg rafts produced by field females were 50% (N = 158 out of 313 egg rafts) and 80% (N = 148 out of 184 egg rafts) for the 1:1 and 1:5 ratios, respectively . In contrast to the results obtained in April, frequencies of infertile egg rafts observed in December were not significantly different from the expected frequency for three out of four trials. When the 1:5 ratio was implemented, no significant difference was noted between observed and expected frequencies of infertile egg rafts in April . However, in December, the observed frequencies of infertile egg rafts were either significantly higher  or significantly lower than expected ; however, significance did not resist the multiple Hommel’s sequential Bonferroni correction. A GLM model was then performed to test the effect of the experimental period (two-levels variable i.e. April and December) and ratio on the proportion of infertile egg rafts. Both variables had a significant effect (P < 0.0001).Under random mating and equal males’ mating competitiveness hypotheses, equilibrated field to LR[ectively . The anaectively . The frewPip(Is)] and field males. The overall survival of LR[wPip(Is)] males recaptured from cages at the end of confrontations was not significantly different from that of field males, when considering all replicates or only the 1:1 ratio replicates . Daily survival of field males (N = 220) and LR[wPip(Is)] males (N = 177) was also investigated by counting dead males placed in separated cages in the semi-field setup. The survival of field males was higher than that of LR[wPip(Is)] during the first 13 days of monitoring, whilst LR[wPip(Is)] males survival was the highest from day 13 to day 28 ] males under semi-field conditions. However, before performing tests in field cages, we first confirmed the sterilizing capacity of LR[wPip(Is)] males through extensive crossing experiments with field females sampled in 19 localities in La Réunion Island. Indeed, due to the previously reported rapid evolution of CI phenotypes in the Cx. pipiens complex  males and La Réunion field females showed that there was no alteration of sterilizing capacities of incompatible LR[wPip(Is)] males in spite of four years of laboratory maintenance. However, crosses between LR[wPip(Is)] females and field males revealed two phenotypes (compatibility and incompatibility) leading to both uni-and bidirectional CI phenotypes. This result complements previously reported data obtained with La Réunion mosquitoes sampled in a single locality St Denis (#11) in 2010  males, the success of an IIT vector control strategy will also depend on the ability of the released males to compete with indigenous wild males. So, intermediate tests under semi-field conditions are necessary to identify potential problems before proceeding to field implementation [wPip(Is)] males exhibit mating competitiveness that is indistinguishable from field collected Cx. quinquefasciatus males, confirming the results observed under laboratory conditions showing comparable competitiveness of LR[wPip(Is)] and La Réunion males [wPip(Is)] males as compared to field males was observed in April (corresponding to the end of the warm humid season); whilst in December (i.e. at beginning of warm humid season), LR[wPip(Is)] males displayed a mating competitiveness mostly comparable to that of wild males. Several factors including environmental conditions may affect mosquito quality and thus mating competitiveness of wild males in April. Larval rearing conditions such as density and food availability are known to modulate mosquito’s fitness [wPip(Is)] larvae were bred under low-crowding conditions and fed ad libitum, whilst larvae collected in natural breeding sites and used in the confrontations may have faced more challenging conditions. While LR[wPip(Is)] line was reared under controlled conditions of the insectary, crowding and food availability faced by wild mosquitoes surely vary along the year but were not controlled in our experimental setup.In addition to the sterilizing capacity of LR[entation . LR[wPipon males . An unex fitness –49. The Cx. pipiens mosquitoes infected by incompatible Wolbachia strains as previously reported [wPip(Is)] line and La Réunion field mosquitoes; unidirectional CI also occurs and must be taken into consideration. Altogether, our data set encourages the development of an effective sexing method strictly producing LR[wPip(Is)] males by any means ] females that would impair the success of such an attractive vector control strategy. Moreover, IIT as other sterile-male systems should be accompanied by regular molecular monitoring of field-caught mosquitoes in order to detect any impact of released mosquitoes on the evolution of natural populations. Although several studies have highlighted the potential of Wolbachia to inhibit pathogen replication [Wolbachia can also increase pathogen replication as observed with both artificial [wPip(Is)] females for pathogens of medical importance in the region such as lymphatic filarial and Rift Valley Fever virus are needed to fully investigate the impact of any accidental release of LR[wPip(Is)] females in the field.Finally, while CI and mating performances of incompatible males represent key parameters for implementing IIT, factors related to females such as mating choice of wild type females and accidental releases of females from the incompatible line can also affect the success of IIT. In this study, no evidence of mating choice was noted for field females, thus confirming random mating between reported , 50–52. nic, see –55) in olication –19; Wolbtificial  and natutificial  infectiowPip(Is)] males and evidenced a good competitiveness of LR[wPip(Is)] males compared to wild type males. In addition, crossing experiments confirm the coexistence of distinct CI phenotypes on several natural populations on the island, with limited but still not negligible expression of unidirectional CI. These data should be considered in future inundative releases as accidental releases of LR[wPip(Is)] females without a proper monitoring of Wolbachia dynamics in natural populations might compromise the success of IIT.Tests carried out in field cages in La Réunion Island confirm the stability of sterilizing performances of LR[S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.


Accumulating preclinical evidence suggests the use of amnion as a source of stem cells for investigations of basic science concepts related to developmental cell biology, but also for stem cells’ therapeutic applications in treating human disorders. We previously reported isolation of viable rat amniotic fluid-derived stem (AFS) cells. Subsequently, we recently reported the therapeutic benefits of intravenous transplantation of AFS cells in a rodent model of ischemic stroke. Parallel lines of investigations have provided safety and efficacy of stem cell therapy for treating stroke and other neurological disorders. This review article highlights the need for investigations of mechanisms underlying AFS cells’ therapeutic benefits and discusses lab-to-clinic translational gating items in an effort to optimize the clinical application of the cell transplantation for stroke. Stroke, the fourthleading cause of death and the leading cause of disability in the United States , 2 mM L-glutamine, 5 ng/ml basic fibroblast growth factor (FGF2) and incubated at 37°C with 5% humidified CO2. After 7 days, non-adherent cells are removed and the adherent cells are allowed to grow in the same medium, which is changed every 4 days. When the cell culture reaches confluency (about 20 days after the primary culture), cells are treated with 0.05% trypsin and 0.02% EDTA, then counted and replaced in 25 cm2 culture flasks. For routine AFS cell procedure, an approximate yield of about 22 million stem cells per amnion fluid aspirate is anticipated. The viability yield of AFS cells is about 70%, which would be an estimated 15 million stem cells per amnion fluid aspirate. An estimated range of several million AFS cells per milliliter of amnion fluid are therefore expected to be obtained.The following protocol allows the isolation of AFS cells from isolated amniotic fluid samples obtained from timed pregnant Sprague-Dawley rats at gestation age 16–18 weeks  need to be in place prior to transplantation of AFS cells as these are common cell release criteria in place for other donor stem cell types. Ultimately, the maintenance of therapeutic efficacy due to scarce data to date providing “efficacy” readout for cells prior to transplantation is a challenge. A simple assay of neurotransmitter release or growth factor secretion after thawing may need to be developed as an efficacy release criterion which should enhance the therapeutic outcome of AFS cell transplantation.Transplanted AFS cells-induced cell proliferation, in tandem with a decrease in neuronal loss, produced robust reduction in cerebral infarcts (Tajiri et al., CVB holds patents and has pending patents in stem cell biology and applications.


Oncorhynchus tshawytscha) ovarian fluid varied among females and then sought to examine the composition of this fluid. Ovarian fluid in chinook salmon was analyzed using 1D SDS PAGE and LC-MS/MS tryptic digest screened against Mascot and Sequest databases. We found marked differences in the number and concentrations of proteins in salmon ovarian fluid across different females. A total of 174 proteins were identified in ovarian fluid, 47 of which were represented by six or more peptides, belonging to one of six Gene Ontology pathways. The response to chemical stimulus and response to hypoxia pathways were best represented, accounting for 26 of the 174 proteins. The current data set provides a resource that furthers our understanding of those factors that influence successful egg production and fertilisation in salmonids and other species.The ovarian, or coelomic, fluid that is released with the egg mass of many fishes is increasingly found to play an important role in several biological processes crucial for reproductive success. These include maintenance of oocyte fertility and developmental competence, prolonging of sperm motility, and enhancing sperm swimming speed. Here we examined if and how the proteome of chinook salmon ( Ovarian fluid surrounds the eggs of many externally fertilising female fish and is released with the spawned eggs into fresh or salt water In salmonids, ovarian fluid  is derived from secretory epithelia in the ovaries and filtered blood plasma Oncorhynchus mykiss) ovulated eggs were stored in ovarian fluid for three days, they maintained fertility and developmental competence in contrast to those stored in trout artificial medium Sander vitreus) eggs Studies in a variety of species indicate that ovarian fluid is likely to have several important biological functions, most notably in reproduction Salvelinus alpinus), chinook salmon, rainbow trout, lake trout  and brown trout  is known to have an activating effect on sperm swimming speed, percent motility, and duration of sperm motility Salmo salar) and brown trout, potentially limiting the risks of genetic incompatibility through inter-specific hybridization Arguably, one of the most interesting aspects of ovarian fluid biology is its effect on sperm performance. Ovarian fluid in Arctic charr (Poecilia reticulata), ovarian fluid enhanced the sperm velocity of unrelated males in comparison to related males Emerging evidence suggests that ovarian fluid also affects fertilisation success in non-salmonid fishes; in the internally-fertilising guppy  and 2005 (n = 10) spawning seasons (April–May). Females were checked daily for ovulation (recognized by round and soft abdomens) and fish whose eggs had recently ovulated were killed with a single blow to the head and fish bled by severing the caudal vein. The fish were then cut open with a single cut down the abdomen and their eggs were expelled into a dry dish To examine general differences in protein composition we undertook 1D SDS-PAGE on ovarian fluids from 25 salmon . From this mixture, a 3 µl aliquot was run on SDS-PAGE and stained with homemade colloidal Coomassie Samples were re-solubilized in 5% [v/v] acetonitrile, 0.2% [v/v] formic acid in water and injected into an Ultimate 3000 nano-flow uHPLC-System  that was in-line coupled to the nanospray source of an LTQ-Orbitrap XL hybrid mass spectrometer . Peptides were separated on an in-house packed emitter-tip column  filled with C-18 material (5 µm bead size) to a length of 12 cm) and eluted by a 35 min. gradient starting at 5% [v/v] acetonitrile, 0.2% [v/v] formic acid and finishing at 80% [v/v] acetonitrile, 0.2% [v/v] formic acid in water at a flow rate of 500 nl/min.Full MS in a mass range between m/z 400–2000 was performed in the Orbitrap mass analyzer with a resolution of 60,000 at m/z 400 and an AGC target of 5e5 for each of the gel fractions. Preview mode for FTMS master scan was enabled to generate precursor mass lists. The strongest 5 signals were selected for CID (collision-induced dissociation)-MS/MS in the LTQ ion trap at a normalized collision energy of 35% using an AGC target of 2e4 and one microscan. Dynamic exclusion was enabled with 2 repeat counts during 30 s and an exclusion period of 180 s. Exclusion mass width was set to 0.01.www.matrixscience.com) and MS Amanda (http://ms.imp.ac.at/?goto=msamanda). The searches were set up for tryptic cleavages with a maximum of three missed cleavage sites and carboxyamidomethyl cysteine, oxidized methionine and deamidated asparagine were included as variable modifications. The precursor mass tolerance threshold was 10 ppm and the maximum fragment mass error 0.8 Da.Raw spectra were processed through the Proteome Discoverer software  using default settings. For protein identification, MS/MS spectra were first searched against a Teleostei subset of the NCBI nr amino acid sequence database  using three different search engines, SEQUEST (Thermo Scientific), Mascot (http://per-colator.com). Peptide hits were filtered for a FDR of q<0.01. In addition to the FDR filter, threshold score filters for each search engine were applied to eliminate very low scoring peptide hits that may have passed the Percolator FDR filter. The score thresholds were estimated by manual examination of very low scoring hits at a FDR of q<0.01. The following score thresholds for positive peptide identification were applied: Mascot peptide score of >20, MS Amanda score >100 and charge state (z)-dependent SEQUEST cross correlation scores (XCorr) of 2.25 (z = 2), 2.5 (z = 3), 3.0 (z = 4 or z = 5), 3.5 for all other charge states). All spectra that were not assigned to any sequence or did not pass the filters were searched again against all EST nucleotide sequences available for Salmonidae in the NCBI nr database  using the Mascot search engine. Search settings and filters were as described above. All searches were combined using the Proteome Discovery software. Only proteins/protein groups that were identified by two or more independent peptide hits and by at least two search engines (only for identifications from the Teleostei amino acid sequence database) were accepted as true positive hits. Identified proteins were assigned to biological processes (Gene Ontology annotation) using the GOanna search tool at the AgBase server (http://www.agbase.msstate.edu/cgi-bin/tools/GOanna.cgi).We have used different search programs and a strict statistic to filter out false discoveries. The Discovery Rate (FDR) was estimated using the Percolator algorithm  of the 25 individuals is presented in LC-MS/MS) 174 proteins were identified  contained six or more proteins , Drosophila, sea urchins and seastars Crinia georgiana, a similar pattern emerges, and sperm motility is affected by egg jelly leading to female and male×female interactive effects on fertilisation outcomes Clupea pallasii), in which two sperm-activating proteins were found Prior work from our team on chinook salmon found that concentrations of inorganic components in ovarian fluid vary and affect sperm function  and future work should include quantitative comparison of biological replicates of individual OF samples, to distinguish between ovarian fluid proteins and plasma contamination. While we only analysed a representative pool of 20 female's ovarian fluids, further work is needed to quantitatively compare the protein profiles of ovarian fluid isolated from different females.Several of the proteins we identified in chinook salmon were also found in a recent study on its close relative the rainbow trout response to chemical stimulus  ova Several other noteworthy proteins were found in the ovarian fluid of chinook salmon that are known to be important for reproductive functions, but could also be mediators of the effects previously observed between ovarian fluid and sperm in this system. For example, numerous forms of vitellogenin were identified in chinook salmon ovarian fluid, similar to that found in rainbow trout Dicentrachus labrax) and sea bream (Sparus aurata) Cathepsin D, another potential modulator of sperm performance, is an aspartic protease with diverse functions. Early work linked it to non-specific protein degradation in lysosomes While there is no direct evidence of cathepsin D influencing sperm function in fish, there is accumulating data that suggest cathepsin D may mediate aspects of sperm function in other systems. Cathepsin D is detected on the sperm surface in humans and undergoes modification to an active form during capacitation Mechanistically, we consider proteins involved in immune function and/or chemical signaling to be promising candidates for modulating sperm performance. Examples abound of molecular mechanisms that evolved in other biological systems to separate ‘self’ from ‘non-self’, yielding a glimpse of how ovarian fluid could signal ‘incompatibility’ (genetic similarity) to sperm – these examples tend to centre around the expression of (highly) polymorphic genes, which in some instances mediate their effects as secreted signaling molecules; thus, many flowering plants have mechanisms in place to prevent in-breeding through self-fertilisation. Three notably different mechanisms appear to have evolved and have been elegantly illustrated het) genes, and a difference between individuals among any of the 5–11 het loci, expressed on the plasma membrane can lead to fusion avoidance Filamentous fungi also evolved incompatibility genes, so-called heterokaryon  have long been recognized Finally, non-random sperm selection for like and dislike MHC class I and II genotypes, respectively, has been documented in chinook In conclusion, this study represents the first application of proteomics for the characterization of chinook salmon ovarian fluid and allowed the identification of 174 different proteins. Unfortunately, the genome of salmon is not yet fully sequenced, so there are many more proteins that remained unidentified; regardless, the current data set provides a resource that can contribute to furthering our understanding of factors that influence successful egg production and fertilisation in salmonids and other species. Analysis of the ovarian fluid proteomic profile in female chinook salmon may eventually reveal the identity of sperm-activating proteins that bring about differential effects on sperm swimming speed. Future work will focus on quantitative differences between females with known sperm motility patterns.Table S1Non-redundant list of significant protein identifications. Accession number (Accession) and description (protein group) from the Teleostei amino acid sequence database (Db: aa) or the est sequence database (Db: est). The number of different peptides (identified peptides) and the number of spectra acquired and identified per protein (Peptide spectrum match) as well as the average peak area of the three strongest peptide hits per protein (Area under the curve) can be used as a rough estimation of relative abundance. The total protein score is given for each search engine . Note the est database was only searched with Mascot and there is no area under the curve calculation available for this search. The molecular weight (MW) and isoelectric point (pI) of identified proteins have been calculated based on the amino acid sequence database entry. EST entries were not considered for MW and pI calculation.(XLSX)Click here for additional data file.Table S2Significant protein identifications for bands 1 and 2 excised from 1D SDS-PAGE gel (see ). Accession number (Accession) and description (protein group) from the Teleostei amino acid sequence database (Db: aa) or the est sequence database (Db: est).(XLSX)Click here for additional data file.Table S3List of significant protein identifications with gene ontology annotations based on the AgBase v2.00 database.(XLSX)Click here for additional data file.