The EuroSIDA observational cohort study was initiated in 1993 by Jens Lundgren (Copenhagen HIV Programme) and Andrew Phillips (University College London) as the successor of the AIDS in Europe Study, with the main objective of assessing the impact of antiretroviral drugs on outcomes for the general population of HIV-infected patients living in Europe. As of April 2014 EuroSIDA included 18,786 patients followed at 111 hospitals in 34 European countries plus Argentina and Israel. Since its inception, EuroSIDA has provided data published in 202 papers in peer-reviewed journals, among them 14 papers on hepatitis C virus (HCV) co-infection [As the HIV epidemic has evolved and the incidence of AIDS has decreased, the focus of EuroSIDA has shifted to addressing the research questions most pertinent to the clinical care of Europeans living with HIV on a long-term basis. With more than a quarter of all EuroSIDA patients positive for antibodies to HCV, and with growing concern about the burden of liver-related disease and death, hepatitis C research has come to play an increasingly important role in EuroSIDA in recent years.This article will describe key characteristics of the HIV/HCV co-infected population in EuroSIDA, and summarize the most important EuroSIDA co-infection studies.Patients eligible for enrolment in EuroSIDA are HIV-1 infected individuals aged 16 and older. To ensure that included patients are representative for patients followed at the participating clinics, EuroSIDA has, enrolled a new cohort of around 1200-2500 randomly selected patients every second year. EuroSIDA is the only cohort of HIV-infected individuals that include patients from all European geographical regions including the Russian Federation and other countries of the former Soviet Union  system .The central plasma repository, which was set up in 1997, receives plasma from most EuroSIDA patients at six-month intervals. In 2006, all patients with unknown hepatitis B and C status and stored plasma samples were identified and tested in a central reference laboratory for anti-HCV antibodies, HCV-RNA and genotype, as well as for relevant hepatitis B and D markers. This has established a cohort of almost 2,000 HCV antibody-positive patients who are well characterized with regards to HCV-RNA and genotype. The plasma repository has also been used to test all HCV antibody positive and hepatitis B surface antigen positive patients for plasma hyaluronic acid, a marker of liver fibrosis.Among the 18,786 HIV-positive individuals enrolled in EuroSIDA, 16,371 have been tested for HCV antibodies and 5,298 patients (32.4% (95% CI 31.6 - 33.1)) are known to be positive. Of those, 3,645 (68.8%) have been tested for HCV-RNA and 2,571 (70.5% (95% CI 69.1 – 72.0)) were positive for it.In 2008, the epidemiological and virological characteristics of 1,940 HCV antibody-positive EuroSIDA patients were published . These pTable Since HCV is more transmissible than HIV through blood contact, most PWID are already HCV-infected by the time they are diagnosed with HIV. Sexual transmission of HCV is less likely, but several outbreaks of acute hepatitis C (ACH) have been described in HIV-positive MSM ,17. ACH Persons who clear HCV-RNA either spontaneously or due to HCV treatment are at risk of becoming re-infected with HCV if they have continuous risk behavior. To investigate the occurrence of HCV re-infection in EuroSIDA patients, we measured HCV-RNA in the latest available follow-up plasma sample in all patients with prior spontaneous clearance included in an earlier study . Thirty-In the period from 2000 to 2013, around a quarter of all deaths among HCV treatment naïve co-infected patients could be attributed to their HCV infection. Other important causes of death among co-infected patients are AIDS (24%), cardiovascular disease (9%), infections (8%) and cancer (5%). The rate of liver-related death (LRD) peaked at age 35-45, and was two to four times lower in East and East Central compared with other EuroSIDA regions. Both lower CD4 cell count and elevated HIV-RNA were associated with increased risk of LRD. For example, the 5-year risk of LRD in patients with ≥F2 fibrosis (versus <F2) increased from 27-fold to 50-fold in patients with a CD4 cell count ≥200 cells/microliter and <200 cells/microliter, respectively .A EuroSIDA study from 2005, which included both anti-HCV positive and HBsAg positive patients, found an average annual decrease in the rate of LRD of 7% from 1994 to 2004. The decrease could be explained by increases in the CD4 cell count after starting cART. However, in patients with similar CD4 cell counts the rate of LRD increased by 13% per year of additional exposure to cART [In contrast to HBV infection, where there is evidence of an association between higher HBV-DNA levels and risk of liver-related death , studiesHistorically, liver biopsy has been considered the gold standard for diagnosis and monitoring of the progression of fibrosis in patients with chronic viral hepatitis and other liver diseases. However, the drawbacks of liver biopsy are its invasiveness, imprecision and cost. Non-invasive low-cost methods to evaluate liver fibrosis are therefore needed.EuroSIDA researchers assessed the reliability of plasma hyaluronic acid (HA) as a predictor of the risk of liver-related events  in all patients who were positive for either HCV antibodies or hepatitis B surface antigen (HBsAg) and had stored plasma samples available . Among 1Previous fibrosis marker research in hepatitis C has mainly been cross-sectional and has lacked relevant clinical end points. The results from EuroSIDA show that plasma HA may serve as a useful clinical tool for monitoring the progression of liver disease. Furthermore, HA has also proven to be an important research tool in EuroSIDA and other large studies, where a good estimate of liver disease severity is often lacking.per se. Since a large number of anti-HCV positive patients in EuroSIDA are well-characterized both in terms of HCV-RNA status and fibrosis, two studies have been conducted to disentangle the relative importance of these factors.HCV infection has been associated with an increased risk of chronic kidney disease (CKD) and other non-liver-related clinical events. However,2 (measured by the Cockcroft-Gault equation). Whereas there was no difference in incidence of CKD between the first two groups, patients with chronic HCV infection had a two-fold increased incidence of CKD . The incidence of HCV did not vary by HCV genotype. Different HCV-related nephropathies have been described, but this study was not able to characterize the type of CKD [In one study the incidence of CKD was compared across three groups of HIV-positive patients: those who were HCV antibody negative, those who had resolved HCV infection (anti-HCV positive and HCV-RNA negative) and those who were anti-HCV positive/HCV-RNA positive. CKD was defined as a confirmed (> 3 months apart) estimated glomerular filtration rate of ≤60 ml/minute per 1.73 m. A recent EuroSIDA study by Grint et al found that patients with chronic HCV infection had a 26% higher risk of antiretroviral drug discontinuation compared with patients with resolved HCV infection , but that the effect seems to be explained by more advanced liver fibrosis (as measured by elevated HA levels) in patient with chronic infection [Earlier EuroSIDA studies and other observational studies have found that anti-HCV positive persons more frequently discontinued antiretroviral drugs due to toxicity or physician choice compared with HCV negative patients ,6,23. AgUntil 2011, the standard of care for HIV/HCV co-infected patients in need of HCV treatment was weekly subcutaneous injections of pegylated interferon along with ribavirin taken orally twice per day for 48 weeks. This treatment eradicated the virus in about 30% to 70% of cases depending on the HCV genotype ,25. Due In EuroSIDA a quarter of co-infected patients received HCV treatment between 1998 and 2010. On average, the incidence of HCV treatment increased 27% per year between 1998 and 2007 before falling on average 12% per year between 2007 and 2010 figure . PatientThe EuroSIDA study follows one of the largest cohorts of HIV/HCV co-infected patients in the world, and is the only cohort to include patients from all geographical regions in Europe. The overall prevalence of HCV seropositivity among patients tested in EuroSIDA is 32.4% with the highest burden of co-infection seen in Eastern Europe where IDU is the most common mode of HIV transmission.The large number of patients, detailed virological and clinical characterization and long-term follow-up have enabled EuroSIDA researchers to describe important regional differences in treatment and the long-term clinical outcome of HIV/HCV co-infected patients in Europe, but also to disentangle the relative importance of viral replication, hepatic fibrosis and other risk factors for some extra-hepatic conditions, such as chronic kidney disease, seen more commonly in this complex patient population.Most co-infected patients in EuroSIDA are already HCV infected at the time of enrolment into EuroSIDA, but we have, in agreement with other cohort studies, documented a high risk of both primary HCV infection and HCV re-infection among PWID and MSM after enrolment in EuroSIDA. These findings underline importance of maintaining focus on preventive measures to reduce injecting drug use, sharing of contaminated needles and unprotected sex, but also that clinicians should maintain a high vigilance to identify patients with new HCV infection early and provide counseling to minimize the risk of onwards transmission and consider the need for HCV treatment.In the period from 2000 to 2013, around a quarter of all deaths among HCV treatment naïve co-infected patients could be attributed to their HCV infection, and despite the ageing of the population and hence longer time for liver fibrosis to develop, HCV-related mortality has decreased in the period. Reasons for this development, which could be due to more patients with advanced liver fibrosis accessing HCV treatment, better control of the HIV infection or competing risks of death, especially in Eastern Europe , are currently being explored.Striking regional differences in causes of death were observed, with AIDS and non-liver-related death dominating in Eastern Europe whereas rates of liver-related death still remained low compared with other European regions. The same study confirmed that lower CD4 cell counts, HIV viral replication and HBV co-infection are all risk factors for liver-related death. These findings highlight the importance of HIV treatment as part of an integrated strategy to decrease the burden of not only HIV-related death, but also HCV-related death among HIV/HCV co-infected patients.Until now, HCV treatment is likely to have improved the overall mortality of co-infected patients in EuroSIDA only minimally. We have shown that only a quarter of all co-infected patients in EuroSIDA have ever received interferon-based therapy, and among those who were treated only a third had evidence of significant liver fibrosis. On the other hand, 22% of all untreated patients have significant fibrosis. The reasons for the latter could be lack of access to or contraindications to therapy or fear of adverse effects of interferon and ribavirin.With the recent approval of potent and well-tolerated oral direct acting antivirals (DAAs) against HCV and the prospect of interferon-free treatment for all HCV genotypes, new HCV therapy could potentially eradicate the virus in nearly all treated patients and sharply reduce HCV related mortality . HoweverMonitoring of these important regional differences in uptake of DAA-based therapy across Europe, and to investigate the short- and long-term efficacy and adverse effects of these new drugs in a “real-life” co-infected population as compared with the generally healthier patients selected for clinical trials, will be an important future research priority for the EuroSIDA study.Before any study related activities are performed Local Ethical Committee approval of the study and procedure for obtaining informed consent from participants is obtained according to local and/or national regulations in all countries participating in the study as well as other national regulatory approvals as applicable. The senior investigator at each clinical site is responsible for obtaining and maintaining this/these approval(s) at all times during the conduct of the study.8 260694) programs. Current support also includes unrestricted grants by Gilead, Pfizer, BMS, Merck and Co. The participation of centers from Switzerland was supported by The Swiss National Science Foundation (Grant 108787).Primary support for EuroSIDA is provided by the European Commission BIOMED 1 (CT94–1637), BIOMED 2 (CT97–2713), the 5th Framework (QLK2–2000-00773) and the 6th Framework (LSHPCT- 2006-018632), and the 7th Framework (FP7/2007- 2013, EuroCoord nPYFU: person-years of follow-up; CI: confidence intervalThe authors declare that they have no competing interests.


Parus major), and the subsequent effects on offspring morphological traits. We manipulated perceived predation risk during incubation using taxidermic models of two predators: the short-tailed weasel posing a risk to incubating females and nestlings, and the sparrowhawk posing a risk to adults and offspring after fledging. To disentangle treatment effects induced during incubation from potential carry-over effects of parental behaviour after hatching, we cross-fostered whole broods from manipulated nests with broods from unmanipulated nests. Both predator treatments lead to a reduced on- and off-bout frequency, to a slower decline in on-bout temperature as incubation advanced and showed a negative effect on nestling body mass gain. At the current state of knowledge on predator-induced variation in incubation patterns alternative hypotheses are feasible, and the findings of this study will be useful for guiding future research.In birds, different types of predators may target adults or offspring differentially and at different times of the reproductive cycle. Hence they may also differentially influence incubation behaviour and thus embryonic development and offspring phenotype. This is poorly understood, and we therefore performed a study to assess the effects of the presence of either a nest predator or a predator targeting adults and offspring after fledging on female incubation behaviour in great tits ( Birds can assess predation risk and adjust behaviours adaptively , for exaThe effects of predation risk on incubation behaviour of birds are still poorly understood. To the best of our knowledge it has never been investigated whether the type of predator encountered may differentially alter incubation behaviour, depending on whether risk is directed at the parents and fledglings outside the nest, or to parents and their clutch/brood inside the nest. Parents may indeed be able to engage different mechanisms to cope with each enemy in a specific way . IncubatParus major) population breeding in artificial nest boxes to test whether female incubation behaviour is differentially affected by the presence of either a nest predator (the short-tailed weasel (Mustela erminea)) or of a predator targeting adults during incubation and offspring after fledging (the sparrowhawk (Accipiter nisus)). Predictions for the direction and magnitude of indirect effects of predation on prey are not straightforward, given that they are contingent on state-dependent life-history decisions [In this study, we used a wild great tit Parus majIncubation patterns may also affect offspring growth and condition. The few studies available report that sub-optimal incubation temperatures may lead to lower nestling body mass ,20 and sGreat tit incubation behaviour comprises incubation sessions where females warm the eggs, and off-bouts where females are out of the nests and eggs cool down. Females only develop a brood patch and incubate, while males may assist by providing incubating females with food. Incubation lasts 12–14 days and fledglings leave the nest 18–21 days after hatching . The stuTurdus philomelos) (C). Sparrowhawks are among the main predators of fledgling and adult great tits [F2,6 = 0.028, P = 0.972).We simulated increased predation risk in 6 plots , and 3 plots were assigned to a control treatment (see below). The remaining 11 plots were kept untreated and were used later as foster plots. We simulated the presence of predators near the nest boxes using stuffed models of two different predators: the sparrowhawk (Ps) as a post-fledging predator, and the short-tailed weasel (Pw) as a nest predator. As a control, we used stuffed models of the song thrush . We used the software combination of Raven (version 1.4) and Rhythm 1.0 [th incubation day until the day the data loggers were removed (day 10).On the third day of full incubation we weighed whole clutches from treated nests to the nearest 0.1 g. We measured clutch temperature and incubation rhythm by placing data loggers  at the bottom plate of the nest box, and the connected thermocouple in the bottom centre of the nest cup at level with its lining, with the eggs aggregated around it. Data loggers were programmed to record the temperature every 20 seconds during incubation with a resolution of 0.1°C. Specifically, they detected changes in temperature after initiation or termination of each new incubation session (incubation rhythm) ,32. Dataythm 1.0 ,34 to anWe excluded from all analyses nests where the female displaced the probe from its initial position or where the nest was deserted or predated (N = 8). Data for the whole incubation period were successfully recorded and subsequently analysed for 47 nests (N = 15 for adult predator (Ps); N = 18 for nest predator (Pw); N = 14 for control (C)).On the third day after hatching (day 3), whole broods of similar size (± 1 nestling) and similar hatching date (± 1 day) from nests exposed to predator and control treatments were swapped with broods from foster nests. Nestlings were individually marked by removing tuft feathers from their heads, backs and wings. 56 pairs of nests were successfully cross-fostered (N = 23 for adult predator (Ps); N = 17 for nest predator (Pw); N = 16 for control (C)).Before cross-fostering whole broods, nestlings of predator-exposed and control mothers were weighed to the nearest 0.1g. Body weight (to 0.1 g) and tarsus length (to 0.1 mm) were measured on day 8 and 15 post-hatch. On day 8 a small blood sample (Less than 5 μl) was collected from the nestlings’ meta-tarsal vein for molecular sexing using the primers 2917/3088 .χ2 = 0.768, P = 0.681), male incubation feeding was modelled using a zero-inflated Poisson model, with treatment included as a factor. Using a GLMM with Poisson error structure, we also analysed male incubation feeding restricting the analyses to the nests where the male actually fed the female to evaluate possible differences related to the treatment.Linear mixed effect models (LMM) with restricted maximum likelihood estimation (REML) were used to evaluate the effects of the parental treatment on incubation rhythm and behaviour as incubation advanced. We assessed treatment effects (1) on incubation constancy and on- and off-bout temperatures separately , (2) on average on- and off-bout duration and duration of the incubation period, as well as (3) on brood size and (4) on fledging age. Using a generalized linear mixed model  with PoNestling morphological traits (i.e. mass gain and tarsus growth) were analysed using a repeated measurements approach in two LMMs, using age as the repeated measure. Age was treated as a categorical variable, with three levels for the model of mass , and two for the model of tarsus (day 8 and day 15). To account for non-independence among siblings, we included nestling identity nested within nest of origin, and nested within plot as random factors. Our initial models included the two-way interaction between sex and treatment to account for potential differences among male and female nestlings, and brood size and hatching date as covariates. We also checked for correlations among nestling morphological traits and each incubation parameter. A GLMM with binomial errors was used to analyse fledging success (number of fledged nestlings over number of hatched nestlings), including brood size and hatching date as covariates.P > 0.05) were removed to improve interpretability of main effects [For all models, we calculated standardized effect sizes by z-transforming the independent continuous variables following Nakagawa and Cuthill ,38. Howe effects , while m effects  and the Given the elevated number of statistical tests performed, as may be typical for an exploratory study with this kind of complexity of the experimental design, the possibility of type I errors cannot be excluded. Hence, the results need to be interpreted with caution . All anaThis study was approved by the Ethical Committee of the Agricultural Office of the Canton of Bern, Switzerland (experimentation permit BE 19/13 to AB). Bird catching and ringing were performed with permission of the Federal Agency for the Environment of the Canton of Bern, Switzerland (ringing permit 2992). During the measurement, eggs were carefully stored in small cotton-padded boxes and kept warm. The entrance of the nest was blocked to prevent females from entering and finding an empty nest. The same procedure was used during the cross-fostering of the nestlings. Nestlings were marked by selectively removing combinations of tuft feathers, which are lost naturally once the head feathers grow. Less than 5 μL  of blooχ2 = 3.579, P = 0.167).Our treatment did not increase the probability of nest desertion . Also incubation constancy  was not significantly affected by the predator treatment , or by the interaction between treatment and day of incubation . However, incubation constancy increased as incubation advanced . None of the predator treatments, nor the interaction between treatment and day of incubation significantly influenced average off-bout duration , but duration of time the female spent off the nest significantly decreased as the incubation progressed . Average on-bout duration was also not significantly affected by the predator treatments or by the interaction between treatment and day of incubation , but there was a significant decrease in average duration as incubation advanced . On-bout temperature, i.e. average temperature of the on-bouts on each active day, was significantly affected by the interaction between day of incubation and predator treatment . No significant interaction  or main effect of the treatment  was detected on off-bout temperature.Predator treatment did not affect the total duration of incubation in the whole breeding period . However, when restricting the analyses to the nests in which males actually fed females at the nest, incubation feeding significantly increased in the Ps group compared to the control , while no significant difference was detected for the Pw group .Incubation feeding  was not significantly influenced by any of the predator treatments . Nestling growth, measured by mass gain and tarsus growth, showed no correlation (P>0.05) with any of the incubation parameters , and from day 8 to day 15 in the Pw group . We did not find any significant effect of the interaction between maternal predator treatments and nestling sex . No significant treatment-specific difference was found in the analyses of tarsus growth  and on fledging success .All the details related to these analyses are provided in rameters . In the Studies on incubation behaviour under increased predation risk found variation in female strategies ,44 when We predicted that on- and off-bouts, egg temperature and incubation constancy would change in response to the presence of either a nest predator or of a predator targeting adults during incubation and offspring after fledging. Among the measures related to incubation behaviour, only the number of on- and off-bouts and the on-bout temperature differed among predator treatments. Following our predictions, the number of on- and off-bouts under increased nest predation risk was lower compared to the control treatment, while a tendency only for a decreased number of on- and off-bouts was observed when predation risk was increased for adults outside the nest. This outcome is in line with previous studies where the frequency of movements close to the nest was reduced in the presence of a nest predator, possibly to minimize visual cues e.g. ,7,19,47),19,477,1http://www.meteosuisse.admin.ch)).A clearer understanding of female investment in current reproduction or on self-maintenance would require a link between frequency of movements to and from the nest (on- and off-bouts) and other incubation parameters . If females would prioritize investment in the current clutch, fewer off-bouts should be reflected in an increased time on the nest, achieved at the expenses of foraging. Consequently, resource-depleted females may in turn reduce steady-state incubation temperatures to achieve increased incubation constancy . AccordiAs increased predation risk (inside and outside the nest) affected some aspect of female incubation behaviour (i.e. off-bout frequency and on-bout temperature), we explored possible effects of the incubation rhythm on nestling growth.The decreased number of off-bouts in the presence of a nest predator and the slower decline of incubation temperatures in the presence of both types of predators may intuitively suggest enhanced development of offspring born from mothers exposed to predation risk since eggs were less exposed to frequent cooling and temperature fluctuations. Indeed, it has been demonstrated that variation in incubation investment with high temperature fluctuations may be directly harmful for developing young ,48. HoweIn conclusion, experimentally increased predation risk during incubation affected some aspects of female incubation behaviour (i.e. on- and off-bout frequency and on-bout temperature) as a function of the predator type. Even though the assessment of the direction and amplitude of female reproductive investment remains unclear and effect sizes seem to be rather small, both predator treatments induced modifications in incubation behavior, which appear to negatively affect the nestling condition. Predator-specific effects on incubation behaviour deserve further investigation, and the present study can be useful in guiding future work on predator-specific effects on incubation behaviour.S1 Table(DOCX)Click here for additional data file.


The CVV arepublic healthcare units, offering health care exclusively through the Brazilian PublicHealth Service (SUS), characterized as micro-regional average-complexity health carefacilities, which must work in an integrated manner with primary and tertiary care, seekingto ensure full attention to sexual and reproductive health and provide care to at-riskchildren, including preterm ones. In the municipality of Viçosa, this was a successful,integrated program that included actions developed in a reference hospital for women withhigh-risk pregnancies, including the structure to provide care to preterm infants andnutritional support with a milk bank. After hospital discharge, the preterm infant hasguaranteed care and follow-up at the CVV. The study by Freitas et al. shows the data from aretrospective cohort study of 103 preterm infants followed from 2010 to 2015 in this city.Breastfeeding rates were higher than that in full-term newborns in Brazil. In preterminfants younger than 37 weeks of gestation, the median duration of breastfeeding was 5.0months, with a 2.6-fold higher risk of breastfeeding interruption in infants younger than32 weeks. In preterm infants receiving supplemented human milk in the first visit afterdischarge, the risk of breastfeeding interruption was 3-fold higher, when compared withexclusive breastfeeding at the time. These good maternal breastfeeding indicators are theresults of the integration of tertiary and primary care in the municipality.-Breastfeeding promotion measures after hospital discharge, with adequate follow-up andcomprehensive care to preterm infants reduce early weaning.


In America, 60% of adults reported that they have driven a motor vehicle while feeling drowsy, and at least 15–20% of fatal car accidents are fatigue-related. This study translates previous laboratory-oriented neurophysiological research to design, develop, and test an On-line Closed-loop Lapse Detection and Mitigation (OCLDM) System featuring a mobile wireless dry-sensor EEG headgear and a cell-phone based real-time EEG processing platform. Eleven subjects participated in an event-related lane-keeping task, in which they were instructed to manipulate a randomly deviated, fixed-speed cruising car on a 4-lane highway. This was simulated in a 1st person view with an 8-screen and 8-projector immersive virtual-reality environment. When the subjects experienced lapses or failed to respond to events during the experiment, auditory warning was delivered to rectify the performance decrements. However, the arousing auditory signals were not always effective. The EEG spectra exhibited statistically significant differences between effective and ineffective arousing signals, suggesting that EEG spectra could be used as a countermeasure of the efficacy of arousing signals. In this on-line pilot study, the proposed OCLDM System was able to continuously detect EEG signatures of fatigue, deliver arousing warning to subjects suffering momentary cognitive lapses, and assess the efficacy of the warning in near real-time to rectify cognitive lapses. The on-line testing results of the OCLDM System validated the efficacy of the arousing signals in improving subjects' response times to the subsequent lane-departure events. This study may lead to a practical on-line lapse detection and mitigation system in real-world environments. Fatigue-related performance decrements such as lapses in attention and slowed reaction time could lead to catastrophic incidents in occupations ranging from ship navigators to airplane pilots, railroad engineers, truck and auto drivers, and nuclear plant monitors. Fatigue (or drowsiness) “concerns the inability or disinclination to continue an activity, generally because the activity has been going on for too long,” defined by European Transport Safety Council  Computer vision-based systems  translates neural activities into control signals to provide a direct communication pathway between the human brain and an external device  System that can continuously monitor EEG dynamics, predict fatigue-related lapses based on EEG signals, arouse the fatigued users by delivering arousing signals, and assess the efficacy of the arousing signal based on EEG spectra. This study hypothesized that (1) EEG spectral values would differ under different arousal states; (2) it is feasible to predict lapses based on the spectral changes in the spontaneous EEG; (3) arousing warning delivered to cognitively challenged subjects would mitigate cognitive lapse, and (4) the rectified performance would be accompanied by the changes in EEG power spectra. This study conducted an off-line experiment to explore the neurophysiological correlates of lapses, which tested the abovementioned hypotheses and guided the development of a truly OCLDM system. The system was then validated by an on-line driving experiment. Furthermore, to be practical for routine use in a car or workplace by freely moving individuals, the EEG-based lapse monitoring system must be non-invasive, non-intrusive, lightweight, battery-powered, and easily to put on and take off  with normal hearing and aged 20–28 years old participated in this study. All of them were free of neurological and psychological disorders. They were introduced how to manipulate the car and practiced ~10 min to get acquainted before the experiment started. None of them worked night shifts or traveled across multiple time zones in the previous 2 months. All participants were asked to read and sign the informed consent form before participating in the studies. After the experiments, subjects were asked to complete the questionnaire for assessing their cognitive states during the experiments.Experiments of this study were conducted in an 8-screen and 8-projector immersive virtual-reality (VR) environment that simulates the 1st person view scene of highway driving. This study adapted an event-related lane-departure driving paradigm originally proposed by Huang et al. ,b, 2009 Thirty-two channel EEG data were collected from participants by the NuAmp system . The electrodes were placed according to a modified international 10–20 system with a unipolar reference at the right earlobe. The EEG activities were recorded with 500 Hz sampling rate and 16-bit quantization level.Figure Figure The 32-channel EEG data were first down-sampled to 250 Hz, and a low-pass filter of 50 Hz and a high-pass filter of 0.5 Hz were applied. Channels or trials with severe artifacts (such as body movements or muscle activities) were manually removed . The remaining EEG data were segmented into several 115 s trials, each of them consisting of 15 s before and 100 s after the lane-deviation onsets. Independent Component Analysis  = 0.0004 for multiple comparisons). Note that the spectra shown here were calculated from the component activities prior to the lane-deviation onset. The nearly identical pre-lapse spectra of these three groups of non-responsive trials demonstrate the robustness of the broadband spectral augmentation preceding the behavioral lapses, suggesting the feasibility of using theta and alpha power from the lateral occipital areas to predict behavioral lapses in this sustained-attention driving task.Figure Next, this study explored temporal spectral dynamics preceding, during and following fatigue-related behavioral lapses and following arousing warning. Figure p < 0.01). The spectral difference between the trials with effective warning and without warning was significant from 7 to 18 s in alpha band and from 7 to 21 s in the theta band (p < 0.01). Furthermore, the spectral difference between the trials with effective and ineffective warning was significant from 7 to 16 s in both alpha and theta bands .Figure In sum, these results provided invaluable insights into the optimal electrode locations  and EEG features  for a practical OCLDM system detailed below. The EEG and behavioral data collected from this experiment were used to assess the EEG correlates of fatigue-related lapses and build a lapse prediction model for the second experiment.Our previous study  with normal hearing and aged 25–30 years old participated in the on-line closed-loop lane-departure driving experiments to evaluate the OCLDM System in a more naturalistic setting (in a regular office without any electromagnetic shielding). All of them were asked to read and sign the informed consent form before participating in the studies.Subjects seated in an armchair and the driving scene was displayed on a 27″ monitor, placed at ~60 cm in front of the subject.Subjects used a keyboard to control a vehicle cruising on a high way, i.e., a left key turns the simulated car to the left while a right key turns to the right.Four electrodes were placed over the lateral occipital area to collect EEG data non-invasively. The data were transmitted to a smartphone for processing via Bluetooth.When the averaged power spectra in alpha band met a certain criterion, arousing auditory warning (~65 dB 1750 Hz tone-burst) would be delivered in half of these lapse episodes through an ear set to the subjects. Note that, the subjects didn't know the warning before the experiments.The arousing tone-burst would be continuously delivered to the subjects until the averaged power spectra in the alpha band has dropped 3 dB from the lapse power.The entire experiment consisted of a 1 min training and a ~60 min driving periods. During the training session, subjects were asked to stay fully alert. The averaged alpha power collected in the BC session was used as an alert baseline to determine whether a subject is experiencing cognitive lapses in the driving task. The subject performed the lane-departure driving experiments following the protocol below:The cognitive lapses were detected when the subject's alpha-band power (Jung et al., The numbers of detected cognitive lapses varied across subjects. Table Figure Figure Many studies have shown that the brain dynamics correlated with behavioral lapses can be assessed from EEG data. Recent studies have also shown auditory signals can arouse drowsy subjects and affect EEG activities (Lin et al., The first experiment of this study showed that EEG power changes in either alpha or theta band can be used as an indicator for assessing the subjects' fatigue (cf. Figure In sum, this study demonstrated the feasibility of translating a laboratory-based passive BCI system to a neuroergonomic device that is capable of continuously monitoring and mitigating operator neurocognitive fatigue using a pervasive smartphone in real-world environments. The passive BCI technologies might also be applicable to other real-world cognitive-state monitoring, such as attention, distraction, comprehension, confusion, and emotion. We thus believe more real-world passive BCI implementations will emerge in the foreseeable future.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.


Following the Fukushima nuclear power plant disaster, assessment of internal radiation exposure was indispensable to predict radiation-related health threats to residents of neighboring areas. Although many evaluations of internal radiation in residents living north and west of the crippled Fukushima nuclear power plant are available, there is little information on residents living in areas south of the plant, which were similarly affected by radio-contamination from the disaster. To assess the internal radio-contamination in residents living in affected areas to the south of the plant or who were evacuated into Iwaki city, a whole body counter (WBC) screening program of internal radio-contamination was performed on visitors to the Jyoban hospital in Iwaki city, which experienced less contamination than southern areas adjacent to the nuclear plant. The study included 9,206 volunteer subjects, of whom 6,446 were schoolchildren aged 4–15 years. Measurements began one year after the incident and were carried out over the course of two years. Early in the screening period only two schoolchildren showed Cs-137 levels that were over the detection limit (250 Bq/body), although their Cs-134 levels were below the detection limit (220 Bq/body). Among the 2,760 adults tested, 35 (1.3%) had detectable internal radio-contamination, but only for Cs-137 (range: 250 Bq/body to 859 Bq/body), and not Cs-134. Of these 35 subjects, nearly all (34/35) showed elevated Cs-137 levels only during the first year of the screening. With the exception of potassium 40, no other radionuclides were detected during the screening period. The maximum annual effective dose calculated from the detected Cs-137 levels was 0.029 and 0.028 mSv/year for the schoolchildren and adults, respectively, which is far below the 1 mSv/year limit set by the government of Japan. Although the data for radiation exposure during the most critical first year after the incident are unavailable due to a lack of systemic measurements, the present results suggest that internal radio-contamination levels more than one year after the incident were minimal for residents living south of the crippled Fukushima nuclear plant, and that the annual additional effective doses derived from internal Cs contamination were negligible. Thus, internal radio-contamination of residents living in southern radio-contaminated areas appears to be generally well controlled. Following the Great East Japan Earthquake (GEJE) on March 11, 2011, various health threats have emerged at different levels and times ,4. Even Soon after the earthquake, local municipalities in the Fukushima prefecture began evaluating the internal radiation exposure of each resident by means of a whole body counter (WBC). In the local municipality Minamisoma, which is about 20 km north of the Fukushima nuclear plant, monitoring of internal radiation exposure to released radioactive Cesium (Cs) began 4 months (July 2011) after the nuclear plant incident . A six-mCompared to the systematic WBC monitoring in areas north of the Fukushima Daiichi nuclear power plant, there is less information on internal radio-contamination for residents living south of the plant , even thst, 2012 and December 31st, 2014 to assess internal radio-contamination among residents in the affected southern areas. Study subjects were among voluntary visitors to Jyoban Hospital in Iwaki city , which represented the total internal radiation exposure from radionuclides dispersed from the crippled nuclear power plant. Internal Cs contamination was measured as total body contamination. Prior to the WBC, study subjects changed into a hospital gown so that radio-contamination from their clothes was not detected.3 NaI scintillation detectors . Schoolchildren shorter than 130 cm stood on a 30-cm stool during the measurement. For a two-minute scan the Cs detection limits were 220 and 250 Bq/body for Cs-134 and Cs-137, respectively.The WBC device used for this study was a stereoscopic apparatus with two 3 x 5 x 16 inAnnual effective doses from internal Cs-137 contamination were calculated based on effective dose coefficients derived from the International Commission on Radiological Protection, Publication 67 , whereinThe present study was approved by the institutional review board of Jyoban Hospital, and written informed consent was obtained from all subjects enrolled in the study. For the children and minors, written informed consent was provided from the parents, relatives or caretakers.Using WBC, internal radiation exposure screening was performed for Jyoban Hospital visitors between April 2012 and December 2014. A total of 9,206 subjects, including 6,446 schoolchildren, were enrolled . The chiAmong the 6,446 children tested, only two boys (11 and 15 years old) showed internal radio-contamination with Cs . The detIn sharp contrast to the study schoolchildren, 1.26% of the adults in the study (35/2760) had detectable levels of internal Cs contamination . AlthougIn the present study, 37 of the 9,206 study subjects (0.4%) showed detectable levels of internal Cs contamination. With the exception of one examinee who was evaluated in November 2014, by the end of 2013 (within 3 years of the incident), no other examinees displayed internal Cs contamination using the WBC monitoring method. Among the 37 subjects who did show Cs radio-contamination, the levels were minimal. Combined with a recent report that assessed 2,839 residents living in Iwaki city who showed committed effective doses ranging from 0.01 to 0.06 mSv/year , the sitConsistent with the findings presented here, previous studies reported that internal Cs contamination could be detected for nearly one year after the incident, and after this period very few subjects had detectable contamination . We presRadionuclide inhalation could also be a source of the internal Cs contamination detected in this screening. However, given the half-life of Cs in the body (3 months) and air doses in the affected areas, and that WBC screening began one year after the incident, inhalation is an unlikely cause of internal radio-contamination. We therefore presume that most of the contamination in the study subjects occurred during the early post-incident period when residents continued to consume locally grown products.A notable limitation of the present study is that the WBC screening began one year after the incident, and no measurements were taken during the first year. As with other areas neighboring the affected nuclear plant, a mass of radionuclides was spread across southern areas, such that residents in those areas inhaled Cs intake and other highly radioactive compounds. In particular, immediately after the incident and up until the time when these residents were evacuated to areas such as Iwaki city, where contamination levels were far below the government-recommended limit of 1 mSv/year, these residents would have been exposed to high levels of contaminating radionuclides in the environment. In terms of health risk assessments, measurement of internal and external radiation exposure of residents living in areas south of the plant during the first year after the incident was critical. However, due to a lack of systemic measurement of exposure levels, especially during the first week after the incident, data for internal and external exposure of the residents are unavailable. As such, an accurate assessment of the health risks arising from radiation exposure is difficult, and the findings presented here can only partially describe the pattern of internal radio-contamination for residents living in areas to the south of the plant.A WBC screening program to determine internal exposure was initially performed with residents living north (Minamisoma city) and west of the crippled nuclear plant . ShortlyConsistent with the previous studies, the air dose in the affected areas likely does not reflect the internal radio-contamination assessed in the present study . More reTo avoid/reduce any chance of further internal Cs contamination, several strategies learned from previous incidents could be implemented. First, even if internal Cs contamination is detected, dietary interventions that promote consumption of inspected locally grown foods and commercially distributed food produced in unaffected areas appear to be effective in reducing contamination. For this purpose, continued WBC screening of affected residents and inspection of locally grown products is recommended. The importance of these efforts was verified by the long-term observations in areas affected by the Chernobyl incident . Second,On the other hand, excessive concerns about produce grown in affected areas may be detrimental to residents’ well-being, since such fears can discourage residents from engaging in outdoor activities, which may lead to decreased physical activity and an accompanying increase in the risk for non-communicable diseases . Even unInternal radio-contamination levels in residents living in affected areas south of the nuclear plant disaster assessed from one year after the incident were minimal and generally well controlled. Since the annual effective dose derived from internal Cs contamination was negligible, nearly all of the annual effective doses in residents of southern areas more than one year after the incident might be due to external radiation exposure.


The electronic Dietary Intake Assessment (e-DIA), a digital entry food record mobile phone app, was developed to measure energy and nutrient intake prospectively. This can be used in monitoring population intakes or intervention studies in young adults.The objective was to assess the relative validity of e-DIA as a dietary assessment tool for energy and nutrient intakes using the 24-hour dietary recall as a reference method.University students aged 19 to 24 years recorded their food and drink intake on the e-DIA for five days consecutively and completed 24-hour dietary recalls on three random days during this 5-day study period. Mean differences in energy, macro-, and micronutrient intakes were evaluated between the methods using paired t tests or Wilcoxon signed-rank tests, and correlation coefficients were calculated on unadjusted, energy-adjusted, and deattenuated values. Bland-Altman plots and cross-classification into quartiles were used to assess agreement between the two methods.Eighty participants completed the study . No significant differences were found between the two methods for mean intakes of energy or nutrients. Deattenuated correlation coefficients ranged from 0.55 to 0.79 (mean 0.68). Bland-Altman plots showed wide limits of agreement between the methods but without obvious bias. Cross-classification into same or adjacent quartiles ranged from 75% to 93% (mean 85%).The e-DIA shows potential as a dietary intake assessment tool at a group level with good ranking agreement for energy and all nutrients. The collection of accurate dietary consumption data is important in the field of nutritional epidemiology in order to establish true relationships between nutrition and health status. The food record (weighed or estimated portions) is a traditional method used to record amounts and types of foods and beverages consumed prospectively, thus limiting recall bias ,2. HowevWith 81% of Australians regularly using a mobile phone , the colA number of commercial mobile phone apps such as MyFitnessPal and Lose It provide a platform for users to digitally record foods and beverages consumed and have these records integrated with food composition databases to calculate nutrients . Only onStudents enrolled in a study aimed at assessing university students’ dietary intakes were invited to participate in this validation study. Recruitment methods for the larger study included email and poster advertisements on the university campus, which included a weblink to an online screening survey. Out of 313 students who completed the survey, 170 were eligible and 113 students were enrolled at an interview during which the study protocol was explained and written informed consent was obtained. From the enrolled students, 66 agreed to participate in the validation study and 57 completed both e-DIA and 24-hour dietary recalls from March to April 2014. To boost sample size, an additional 23 students were recruited in August 2014 by the same methods. This resulted in a final sample of 80 students . InclusiStudents downloaded the e-DIA app using an Android or iOS platform on their own mobile phone. To record intake, the user selects the meal occasion during which the food or beverage is consumed  which opens the Edit/Delete screen . On thisAt an initial clinic appointment on the university campus, anthropometric data were collected by the study investigators. Height was measured to the nearest 0.5 cm, weight to the nearest 0.1 kg (without heavy clothing or shoes), and waist circumference to the nearest 0.5 cm, according to the Anthropometry Procedures Manual from the National Health and Nutrition Examination Survey  .Participants were instructed to complete five consecutive days of food records including three weekdays and two weekend days using e-DIA. Participants practiced selecting and entering food items and weights, and written instructions were included on how to choose foods from the database, how to enter mixed recipes, and how to estimate portion sizes when eating away from home. Participants were asked to weigh foods using the scales supplied ; an instruction booklet was provided. If participants were unable to weigh the foods, they were instructed to estimate portion sizes using metric cups and spoons supplied. Starting days were staggered so that all days of the week were represented across the sample. Participants were sent a text message reminder prior to each collection day which encouraged them to maintain their usual diet.As a reference measure, three 24-hour dietary recalls were collected on three random days (including weekend days) during the five-day study period. Appropriate calling times were established at the convenience of the participants. The standard 24-hour dietary recall interview multi-pass script adapted from the Five-Step Multiple-Pass Method by the US Department of Agriculture  was usedAll entries were checked the following day by study investigators, and participants were contacted to clarify manually entered food items and obvious inconsistencies such as gross data entry errors and skipped meals.Data collected using the e-DIA mobile web app were stored in a cloud-based database, and records were linked to food items in the AUSNUT 2007. If the nutrient composition of manually entered food items was known, study investigators added the information to the database; if unknown, investigators coded to the closest match. Food intake data from the 24-hour dietary recalls were manually entered by trained study investigators into FoodWorks 7 Premium , a nutriEnergy and nutrient intakes from the 24-hour dietary recalls and e-DIA were examined for outliers and checked against the original 24-hour dietary recall for obvious errors in data entry. Errors made by the participant in the e-DIA were left unaltered, and no outliers were removed to provide a more accurate indication of the relative validity of the e-DIA method. Vitamin and mineral supplements were excluded from analysis.t tests  or Wilcoxon signed-rank test . Correlations between the two methods were measured using Pearson product-moment correlation coefficients (or Spearman rank correlation coefficients for skewed data) for unadjusted, energy-adjusted and deattenuated data. Energy-adjusted nutrients were obtained by applying the residual method [P value <.05 was considered statistically significant.Mean or median intakes of energy and nutrients from three days of 24-hour dietary recalls and five days of e-DIA were calculated and differences determined using paired l method . Cross-cl method  were usel method  and a P 2 (SD 3.8) with 63 participants (79%) in the healthy weight range (BMI 18.5-24.9), nine overweight (BMI 25.0-29.9), four obese (BMI>30.0), and three underweight (BMI<18.5). Mean waist circumference was 70 cm (SD 6.6) for females and 81 cm (SD 10.5) for males. One participant did not consent to disclosing her anthropometric data. The majority of participants lived at home with family (70%), with English being the most commonly spoken language at home (75%).A sample of 80 students  completed five days of e-DIA and three days of 24-hour dietary recalls . The maiMean and median intakes of energy and nutrients reported by 24-hour dietary recall and e-DIA are shown in P<.001). Correlations for unadjusted intakes were in the range 0.50 to 0.79 , energy-adjusted correlations were in the range 0.40 to 0.78 (mean 0.63), and deattenuated correlations were in the range 0.55 to 0.79 (mean 0.68). The highest correlations were found for protein and saturated fats while the lowest correlation was found for polyunsaturated fats. Deattenuated correlation coefficients were generally higher than unadjusted or energy-adjusted coefficients but differences were small.Quartile cross-classification of nutrients with the 24-hour dietary recalls and e-DIA placed 75% to 93% (mean 85%) of the participants into the same or adjacent quartile, with the highest ranking agreement for fiber and the lowest for iron. Cross-classification into extreme quartiles ranged from 0% to 9% (mean 1%) with monounsaturated fatty acids (MUFA), thiamine, and iron having the greatest proportion of extreme misclassification.Bland-Altman plots illustrating the agreement between the 24-hour dietary recalls and e-DIA for energy and selected nutrient intakes are shown in This study is the first to compare the energy and nutrient intakes using a mobile phone food diary app with 24-hour dietary recall as reference measure using an Australian food composition database. Mean intakes of energy and all nutrients were similar in both methods, with no consistently higher or lower values for either method. Correlation coefficients were moderate to strong ranging from 0.55 to 0.78. Cross-classification into quartiles revealed good agreement for energy and all nutrients. In addition Bland-Altman plots showed robust agreement between the e-DIA and 24-hour dietary recalls for energy and all nutrients, without bias and with most data points located within two standard deviations of the mean. The wide limits of agreement suggest that e-DIA is unsuitable to accurately estimate intake at an individual level. However, collectively the results suggest the potential of e-DIA as an assessment tool for dietary analysis at the population level.r=0.51-0.80, 7-day PDA vs 24-hour dietary recall r=0.72-0.85) [These findings are consistent with those of other researchers. Carter et al recently validated a mobile phone app  designed to support weight loss . Mean in72-0.85) ; and min72-0.85) ,22.Mobile phones are also being used for digital imaging to record food and beverage intake ,9,23-30.Although the use of 24-hour dietary recall was the preferred choice of reference method, it introduces several limitations to the study design. Reliance on memory is a well-documented limitation with participants likely to forget foods consumed the previous day, although the use of the multiple pass method and portion size aids are designed to minimize the impact of errors related to memory. As the 24-hour dietary recall was administered on days that the participants digitally recorded their food records into the e-DIA, there was potential for the recording process to have improved their recall of food and beverages. However, it should be noted that records were deleted from the app at midnight and recalls were conducted up to 22 hours after their deletion. As both methods relied on self-report, more objective measures of dietary intake such as biomarkers are needed to further validate the e-DIA.Compared with the 2011-2012 Australian Health Survey , energy The use of the e-DIA also has limitations, including the burden of recording foods prospectively for a prolonged period of time and trouble navigating within the e-DIA tool itself. When entering a food into the e-DIA, participants were presented with a long list of food options that was challenging to navigate. However, the presence of the favorites function relieves some of this burden by prioritizing the food options according to individual preferences. Commercial apps may have shorter lists but this is likely to result in less accurate food records and resulting nutrient intakes.One of the main strengths of the study is the ability of e-DIA to collect dietary intake data without alerting the participants to their ongoing caloric intake. The app is linked to the Australian national food composition database compiled by Food Standards Australia New Zealand which consists of over 4500 foods . This grThis validation study demonstrated good agreement between the e-DIA and 24-hour dietary recalls at a group level, and no evidence of bias for energy, macro-, and micronutrients was noted. With the growing popularity of mobile phones among young adults this method of collecting dietary intake is highly acceptable in this population group. Future studies should explore the validity of the e-DIA in larger, more representative samples and employ external biomarkers to reflect usual intakes. Studies assessing the e-DIA’s sensitivity to changes in dietary intake are also required. This would confirm its value as a tool to monitor dietary intake in intervention studies in public health and clinical trials.


Chromatin immunoprecipitation followed by sequencing (ChIP-seq) experiments revolutionized genome-wide profiling of transcription factors and histone modifications. Although maturing sequencing technologies allow these experiments to be carried out with short (36–50 bps), long (75–100 bps), single-end, or paired-end reads, the impact of these read parameters on the downstream data analysis are not well understood. In this paper, we evaluate the effects of different read parameters on genome sequence alignment, coverage of different classes of genomic features, peak identification, and allele-specific binding detection.in silico variations of these data as well as fully simulated data, revealed complex interplay between the sequencing parameters and analysis tools, and indicated clear advantages of paired-end designs in several aspects such as alignment accuracy, peak resolution, and most notably, allele-specific binding detection.We generated 101 bps paired-end ChIP-seq data for many transcription factors from human GM12878 and MCF7 cell lines. Systematic evaluations using Our work elucidates the effect of design on the downstream analysis and provides insights to investigators in deciding sequencing parameters in ChIP-seq experiments. We present the first systematic evaluation of the impact of ChIP-seq designs on allele-specific binding detection and highlights the power of pair-end designs in such studies.The online version of this article (doi:10.1186/s12859-016-0957-1) contains supplementary material, which is available to authorized users. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) is widely used for genome-wide profiling of histone modifications  and tranDrosophila melanogaster, they also investigated a limited comparison of PE and SE designs with the exact same read lengths in terms of the library complexity and coverage in repetitive regions using very high depth samples. The human genome has much more repetitive DNA and a greater genome size, and although the sequencing depths of human samples have also been increasing over time, they are still lagging far behind the Drosophila melanogaster study. Therefore, it remains largely unclear how the PE and SE designs and long and short reads influence the alignment rates and accuracy, coverage of various repetitive elements, sensitivity and specificity in peak calling and in allele-specific binding detection.Recent works on the design of ChIP-seq experiments did not adequately address the performance of PE and SE designs and long and short reads –15. Instin silico from these full data, and evaluated short (36 and 50 bps) and long (75 and 101 bps) PE and SE read designs for their impact on alignment, peak calling, and allele-specific binding (ASB) detection. We complemented these comparisons with evaluations on simulated data where the underlying truth was known and established advantages and disadvantages of different designs in terms of accuracy and power. Our study deepens the understanding on the impact of design in transcription factor ChIP-Seq experiments, and is likely to provide insights on other types of ChIP-Seq experiments.In this paper, we systematically and quantitatively investigated the impact of ChIP-seq read parameters on the alignment, peak identification, and allele-specific binding detection. We first generated PE ChIP-seq data for CTCF, BHLHE40 , and NONO from the human GM12878 cell line and MAFK from the human MCF7 cell line, as well as the control Input data from these two cell lines, with a read-length of 101 bps at typical depths (15–80 million reads per replicate). We generated data with other read parameters We generated ChIP-seq datasets for CTCF, NONO, and BHLHE40 DEC1) in GM12878 cells and MAFK in MCF7 cells as part of the phase 3 of the ENCODE project  in GM128We randomly sampled one end from each paired-end read to generate single-end reads. We used HOMER software  to trim 𝜗 -I 100 -X 700 -a –best –strata -m 1”, where 𝜗=0, 1, 2, 3 and “-I 100 -X 700" only applies to PE. Here, 𝜗 denotes the number of mismatches allowed in each read, and 100 and 700 the allowed range of the fragment lengths in PE alignments. These upper and lower bounds were chosen based on BWA estimates of the fragment length from the CTCF dataset to make this parameter comparable between the two aligners. We considered four different options for BWA: “BWA -q 0 -o 0 -n 0.04", “BWA -q 20 -o 0 -n 0.04", “BWA -q 20 -o 1 -n 0.04", and “BWA -q 20 -o 1 -n 8", where -q 0, -o 1 and -n 0.04 are default options of BWA. The option -q allows trimming the low quality base-pairs from their 3′ ends, option -o allows user-specified numbers of gaps in the alignment, and option -n controls the number of allowed mismatches. Specifically, when -n is an integer, it denotes the number of allowed mismatches. When -n is between 0 and 1, the number of allowed mismatches is set to the quantile of a Poisson distribution with mean 0.02 ×read-length (after trimming by option -q) with tail probability -n. We used “BWA -q 20 -o 1 -n 8" to generate a relaxed alignment set. After the BWA alignment of the PE designs, we considered keeping all aligned pairs with both ends uniquely mapping (Uni) and additionally kept those with one uniquely mapping end (UR). For SE designs, we kept all the reads that are uniquely mapped (Uni/UR).We initially compared the alignment results of both Bowtie -v mode  and BWA For simplicity, we introduced some abbreviations for the designs and the design-alignment combinations in Additional file 5 by default). Then the irreducible discovery rate (IDR)  as the true maternal probability in each 1000bps window around SNPs. We further assumed equal maternal probabilities in overlapping windows and maternal probability of 0.5 in regions outside of these windows. We sampled a based on the maternal probability of the sampled middle point position.Simulate the allele assignment z to the corresponding coordinate on the assigned allele za: We converted z to the corresponding coordinate on the assigned allele by the liftOver function in the Bioconductor package rtracklayer. For the positions on the reference sequence that cannot be mapped to the assigned allele, we slightly perturbed z iteratively until it was properly mapped.Convert za and L and extracted the true read sequences from both ends.Extract the read sequences at both ends for the sampled fragment from the assigned allele: We calculated the two end positions of the fragment based on Sample quality score: For each read, we sampled the quality score from the qualities of the CTCF data.Insert read errors based on the quality score: We calculated the base-wise error probability based on the sampled quality score, determined the read error locations based on the error probabilities, and inserted the read errors based on the error distribution provided in Additional file We simulated paired-end reads from the diploid sequences of chr19 in GM12878 cells using the following procedure. 6 reads and the Input sample contains 1.1 ×106 reads, so that the coverage of the simulated samples were similar to the coverage of chr19 in the real data.We performed simulation experiments for five times. Each experimental replicate contains one ChIP sample and one Input sample. The read-densities on the reference genome were from PE101v3 of pooled CTCF and Input samples of GM12878. The ChIP samples contain 2 ×10In our simulation, the reads were simulated from the diploid sequence. When assessing the accuracy in their alignment to the reference sequence, we treated a read as aligned correctly as long as the extended aligned read covered the true middle point position on the reference sequence. When assessing the accuracy of ASB detection, we defined the true ASB as the SNPs where the ratio between the maternal and the paternal alleles is larger than 1.5 .The first step of ChIP-seq data analysis is aligning reads to a reference genome, and the choice of alignment strategy impacts the downstream analysis. Thus we started with a detailed comparison of data alignments with different read parameters, and throughout this paper, we paid attention to matching designs and analysis protocols so that the read error rates, the numbers of reads, and the numbers of sequenced bases were comparable when necessary. For simplicity, we introduced some abbreviations for the designs and the design-alignment combinations in Additional file ′ end of long reads, a phenomenon commonly observed and well characterized for Illumina platforms [Alignment rates have a profound effect on the downstream analysis because higher alignment rates lead to more aligned reads at fixed sequencing depth and typically yield identification of more true signals with higher confidence. Our comparison in alignment rates revealed a complex interplay between the read parameters, alignment rules, and other biological factors  covered by at least five reads . One reFalse positives in alignment may cause false positives in detecting enrichment regions. We evaluated the alignment accuracy using both experimental data and fully simulated data, and found that both PE designs and longer reads improved alignment accuracy. We devised a strategy for evaluating alignment accuracy with the experimental data, even though the true origins of the reads were unknown. We used a relaxed alignment set, BWAq20o1n8 with the PE101 data as the “gold" standard, and labeled those reads that could not be aligned by this relaxed rule as false positives. Then we compared the false positive rates of the sequencing designs in pairs  and assessed the false positive rates in their alignment set differences . It also reports one selected alignment location for reads aligned to multiple locations (multi-reads). While the majority of our analysis is based on Bowtie -v mode, we investigated a comprehensive set of alignment strategies based on BWA and compared them to the Bowtie -v mode in this section. This investigation also led to many practical suggestions on trimming (option -q) and filtering multi-reads in BWA alignment. In Table 1relaxed peaks, a list of approximately 3×105 regions using peak caller SPP [optimal set of peaks is selected as the subset of the relaxed peaks with better peak score than the threshold needed for controlling the irreproducible discovery rate (IDR) [We next evaluated the impact of designs on the number of detected protein-DNA interactions, i.e., peaks. We adopted the robust ChIP-seq uniform processing pipeline  developeller SPP . Then, tte (IDR)  at a givWe found that PE designs led to larger numbers of optimal peaks than SE designs regardless of the IDR level  under different designs. We overlapped the top peaks of each design with satellite DNA, LINE, SINE, LTR elements, and SDRs , 3,181 of them underwent moderate rank change , 767 of them suffered from large changes in rank, and at most 48 of them were specific to PE36v1, and could not be recovered under SE36v1. In summary, we observed that (1) more than 80 % of the ranks effectively remained unchanged in any comparison; (2) the majority of the rank changes were moderate; and (3) only a small portion  of the peaks seemed specific to one design, with the exception of BHLHE40, where 10–15 % of the SE peaks were specific to the SE designs. We focused on the peaks in the last category (specific to one design), and further examined their ChIP and Input coverage. Surprisingly, we found that even for these peaks, the coverage around the summits were usually similar in different designs, and only a small percentage (≤50 % except for one case) exhibited a fold-change of at least 1.5 in any replicate of ChIP or Input samples . Such consistency indicated that all designs targeted the same overall signal pattern. In this section, we focused on the peak set differences, and investigated to what extent the differences could be attributed to the change in the ranking of the relaxed peaks and the alignment differences that led to significant changes in coverage. We first used SE36v1 as the baseline and investigated the changes in peak ranking and peak region coverage when switching to PE36v1 or doubling the read-length SE75v2). Additional file . AdditioAllele-specific binding (ASB) detection searches for differences in TF binding between the two alleles of the same individual at a given set of loci (SNPs). ASB studies provide insights in understanding genomic imprinting  and non-We compared the numbers of ASB loci detected by different designs at AlleleSeq default FDR level of 0.1 and observed an increasing trend with the number of sequenced bases Fig. a,b. At fp-values at 0.05 to compute the empirical FDR. We found that PE designs and longer reads had higher true positive rates regardless the range of the true maternal probability  and compared FIMO p-values of the motif matches in both alleles . In summary, we concluded that SE75v2 design was better than PE36v1 in ASB detection accuracy. This is largely because, for the ASB loci only identified under SE75v2 but not PE36v1, the overall improvement of the motif score in the winning allele over the losing allele was larger than those that were only identified under PE36v1  events as the peak regions where two or more TF bound in favor of the same allele. In contrast, we defined the peaks where binding by those factors favored different alleles as bi-allele-specific binding (BiASB). Partially due to the interaction between binding and open chromatin structure, we expected ASCB to be more common than BiASB. We then investigated ASB behaviors of CTCF and BHLHE40 in their co-binding regions. Similar to the last section, we adopted a more conservative criteria for ASB detection. We observed that the designs with similar amounts of sequenced bases yielded similar numbers of ASCB and BiASB, and the numbers of BiASB were much smaller than the numbers of ASCB as expected  to their SE counterparts, and the quality of PE peaks can be further improved if the peak caller is able to properly process PE information. On the other hand, read-length does not have much effect in peak calling. For ASB detection, both PE designs and long reads lead to more detected ASB with higher accuracy.′ end of reads is commonly seen [Our computational and data-driven experiments support PE designs for their higher alignment accuracy and higher coverage in repetitive elements especially for TFs where the antibody or other experimental conditions are far from ideal. We did not observe a clear advantage of read length of 101 bps. PE36 worked as good as other PE designs for peak calling and PE50 and PE75 worked best for ASB. One reviewer suggested that the observed design differences may depend on the particular error profiles of the data used. While this could be true, the typical error profiles from Illumina platforms have been well characterized and lower quality near the 3nly seen . Thus thWhile it is up to the individual investigator to decide whether the potential improvement in biological findings is worth the costs of the more expensive designs, this study provides a computational perspective and highlights the importance of using appropriate computational tools to maximize the power of the chosen design. For BWA alignments, we found that filtering improves the alignment accuracy, especially for SE and short reads, and trimming by option -q improves the coverage in aligning the long reads. However, despite the evidence presented in this paper and in the literature , we acknin silico data generated from ChIP-seq experimental data, instead of comparing publicly available ChIP-seq experiments using the same antibody and the cell line, but different designs. This is because our approach avoids the unnecessary variations in ChIP-seq experiments performed by different investigators using different facilities at different times. For example, ENCODE PE data are generally two to three years newer than their SE counterparts. In addition, generating in silico data via subsampling and trimming methods is common practice in the literature [We systematically investigated the design effects using terature , 12. Henterature , and 5-hterature .The impact of sequencing depth on the detection of enrichment regions and many other factors that affect alignment and peak calling in the context of ChIP-seq have been investigated by others , 12. OurAdditional file 1Supplementary materials. The file that contains all supplementary notes, figures and tables. (PDF 1330 kb)


The aim of the present study was to investigate the effectiveness of laparoscopic gallbladder-preserving surgery (L-GPS) for cholelithiasis and the feasibility and value of totally laparoscopic GPS (TL-GPS). A total of 517 patients underwent L-GPS, including 365 cases of laparoscopy-assisted GPS (LA-GPS), 143 cases of TL-GPS  and nine conversions to laparoscopic cholecystectomy. The surgeries were all performed by one medical team and the mean operating time was 72 min. All macroscopic calculi were removed through endoscopy. The number of calculi observed in the patients was between one and several dozen; diameters ranged between 0.1 and 2.5 cm. Only three cases of incisional infection were noted in the LA-GPS group and long-term follow-up showed a low recurrence rate of 1.2%. L-GPS is, therefore, an excellent approach to cure cholelithiasis and TL-GPS is a feasible and effective option that could avoid incisional complications. Cholelithiasis is a global disease. According to a previous study , China hConventional open gallbladder-preserving surgery may remove the calculus from the gallbladder but a long incision is necessary and the absence of a clear macroscopic view may lead to a high incidence of residual stone . LaparosA total of 517 consecutive patients , aged 21–67 years , who were diagnosed with cholelithiasis or cholelithiasis with polypus, underwent L-GPS in the Second Affiliated Hospital of Soochow University  between April 2009 and March 2014. A total of 143 patients underwent totally laparoscopic GPS (TL-GPS) and 365 patients received laparoscopy-assisted GPS (LA-GPS), with nine conversions to LC. One hundred and fifty-six patients had suffered from acute episodes of biliary colic at least once and 361 patients were asymptomatic or had non-specific upper abdominal discomfort. Sixty-eight patients had between one and three concurrent polypi. For all patients, a bland diet was demanded and a series of preoperative preparations, such as any necessary application of antibiotics or cholaneresis treatments, were adopted. Until clinical symptoms were apparently palliated, an ultrasonic examination was required for disease assessment. The present study was approved by the Ethics Committee of The Second Affiliated Hospital of Soochow University and all procedures were carried out in accordance with the Declaration of Helsinki. Informed, written consent was obtained from all patients (or patients’ families) prior to their inclusion in the study.Major equipment used included the mini-laparoscope-camera system , an inflexible cholecystoscope  and a soft choledochoscope . The supporting equipment (CHiAO) included a stone basket, biopsy forceps, electrocoagulation bar and a suction trunk.2 insufflation (12–14 mmHg). A trocar was then implanted and the mini-laparoscope was inserted to explore the gallbladder and extrahepatic bile duct.Patients underwent surgery under general anesthesia in the supine position, hands by the side. A 6-mm sub-umbilical incision was made, through which a pneumoperitoneum was created with simultaneous CO2O. Calculi that were ≥0.5 cm in diameter were removed using the basket . No bile duct injuries occurred intra-operatively and no bile leakage or intra-abdominal infection was observed postoperatively. Two patients were shown to have residual sedimental stone through ultrasonic examination. Three cases of incisional infection were noted in the patients undergoing LA-GPS. Typical types of calculi and the use of surgical equipment are shown in The follow-up time ranged from 3 to 54 months, during which time six recurrences were observed in two years postoperatively and four patients received LC. In the nine conversion cases, one subcostal incisional hernia was seen exhibiting a localized bulge with a diameter of 4.0 cm and two cases with a polypus in a borderline or malignant manner were followed up postoperatively without tumor recurrence at 24 and 36 months.Based on an in-depth knowledge of gallbladder function ,7, it isFor patients who are to be offered GPS, indication judgments are of vital importance prior to surgery as there is still no criterion of absolute indications. Satisfactory gallbladder function of patients with cholelithiasis is essential since the preservation of a nonfunctional gallbladder is unhelpful and should be considered as a contraindication. An ultrasonic examination and ‘fat meal test’  can assiWith regard to surgical approaches, LA-GPS and TL-GPS are both technically feasible. Even with extended exploration, no bile duct injury occurred during the surgeries in the present study. LA-GPS allows the gallbladder to be pulled out of the abdominal wall through a 2.0-cm subcostal incision ; howeverThese cases demonstrated the advantages of TL-GPS as it is not necessary to make a larger incision than the calculus size and the shorter contact period of spilt bile with the incision reduces the risk of incisional infection. Furthermore, in TL-GPS a titanium clamp can be used to temporarily obstruct the bile flow at the cystic duct to prevent calculus migration to the extrahepatic bile ducts and incarceration at the neck of the gallbladder or the bile duct opening. When complicated with extrahepatic bile duct calculus, the simultaneous laparoscopic common bile duct exploration can be performed, combined with fibercholedochoscope.Calculus residue is considered the primary cause of cholelithiasis recurrence ; thus, aIn conclusion, the adoption of GPS reflects the important application of minimally invasive endoscopic surgery in treating biliary diseases. GPS provides a complementary approach to cure cholelithiasis. L-GPS is effective and TL-GPS is feasible for cholelithiasis and is particularly favorable in the prevention of incisional complications.


MicroRNAs are the key post-transcriptional regulators of gene expression in development and stress responses. Thus, precisely quantifying the level of each particular microRNA is of utmost importance when studying the biology of any organism.http://www.combio.pl/mirex) provides a comprehensive platform for the exploration of microRNA expression data based on quantitative Real Time PCR and NGS sequencing experiments, covering various developmental stages, from wild-type to mutant plants. The portal includes mature and pri-miRNA expression levels detected in three plant species , and in A. thaliana miRNA biogenesis pathway mutants. In total, the database contains information about the expression of 461 miRNAs representing 268 families. The data can be explored through the use of advanced web tools, including (i) a graphical query builder system allowing a combination of any given species, developmental stages and tissues, (ii) a modular presentation of the results in the form of thematic windows, and (iii) a number of user-friendly utilities such as a community-building discussion system and extensive tutorial documentation . All data contained within the mirEX 2.0 database can be downloaded for use in further applications in a context-based way from the result windows or from a dedicated web page.The mirEX 2.0 web portal  contains supplementary material, which is available to authorized users. MicroRNAs are short, predominately 20–22 nucleotide small RNAs that function as versatile gene expression regulators in development and stress responses. Plant miRNAs are produced from primary transcripts (pri-miRNAs) that form a miRNA/miRNA*-containing stem-loop structure. They are processed by the DICER-LIKE 1 enzyme (DCL1)  proteinsGenes that encode transcripts that are processed to the same or similar mature microRNA species are grouped into families. In many cases it is only possible to observe the expression of all family members as a group rather than that of individual members by using Northern hybridization, RT-qPCR or sequencing approaches. However, individual members of a given microRNA family may be expressed in different developmental stages or in response to various biotic/abiotic stimuli –20. In aRecently, genome-wide analyses from several plant species have revealed that variation in miRNA expression and processing has an impact on micro RNA unique functionality , 22.At present, the amount of expression data produced by sequencing technologies in many cases overbalances the potential of data exploration tools. Therefore, it is of outmost importance to create user-friendly and powerful tools that will allow for new hypothesis testing and application of novel approaches for data exploration. Although a few databases include expression information of miRNAs –26, theiMIR gene expression at the level of transcript (pri-miRNA) and mature miRNA [Arabidopsis thaliana), monocots (Hordeum vulgare), and bryophytes (Pellia endiviifolia). Arabidopsis, the well-established reference plant model, is represented by wild-type plants as well as by several miRNA-biogenesis mutants. Barley represents one of the most agronomically important plants that can serve, in many aspects, as a model organism for other closely related grass species. P. endiviifolia belongs to liverworts, i.e., plants that are most probably the basal group of land invaders [We have developed mirEX – a portal dedicated to comparative data mining of microRNA expression information in plants, from the studies of wild-type and mutant species, covering various developmental stages and including data from RT-qPCR, northern blot and NGS experiments. The portal contains data, which present re miRNA . Currentinvaders . The mirThe mirEX 2.0 portal is dedicated to researchers working on specific microRNA functions and expression profiles of entire microRNA family members during a particular organ/developmental stage or on microRNA biogenesis and evolution.Pellia endiviifolia is represented by two types of plants: female and male thalli collected as environmental samples producing archegonia and antheridia, respectively, and female and male thalli grown in vitro without sex organs [The Arabidopsis developmental phases were classified according to Boyes et al.  and inclx organs .cbp20xcbp80) [dcl1-7) [hyl1-2) [serrate (se-1) (3). The selected mutants represent the key components of the microRNA biogenesis pathway in the plants [The mirEX 2.0 portal provides measurements of pri-miRNA expression levels in four arabidopsis mutants: the double mutant of the CBC subunits (0xcbp80) , dicer-l[hyl1-2) , and sere plants  and showe plants , 34. Sucdcl1-7 plants contain a point mutation in the DCL1 RNA-helicase domain that is manifested in small leaves with an altered shape, delayed bolting and floral transition. Additionally, the mutants show reduced pollen production and disturbed ovule development, causing female sterility [hyl1-2 plants are much shorter than the wild-type with characteristic narrow and hyponastic leaves. The roots show reduced gravitropic response and plagiotropic growth. The lack of the HYL1 protein leads to delayed flowering – the flowers are smaller and fertility is reduced, the siliques are short and twisted [hyl1 mutants show hypersensitivity to several phytohormones: abscisic acid (ABA), auxin and cytokinin [serrate mutant (se-1) are notched (serrated) and do not curl abaxially. The plants have fewer juvenile leaves and show delayed floral initiation and extended flowering time. The flowers of the se-1 mutant show extra sepals and petals [hyl1-2, se-1 shows hypersensitivity to ABA and amplified response to abiotic stress [se-1 mutation. The rosette leaves of the mutant plants have serrated margins and show retarded growth [The terility , 36. The twisted . Additioytokinin , as wellytokinin . The lead petals , 40. Simc stress . T-DNA id growth . Both prd growth –45 but dd growth .Arabidopsis thaliana miRNA sequences includes 299 pri-miRNAs representing 194 families. The expression data for microRNA primary transcripts from two-row spring barley Hordeum vulgare  as well as Northern blot hybridization experiments. The data set of ar Rolap , 48) andviifolia , 50 contviifolia .At1g07930), glyceraldehyde-3-phosphate dehydrogenase C subunit 1 , polyubiquitin 10  for arabidopsis [ACTIN1 for Pellia (GenBank AC: DQ100290) [For each developmental stage and organ studied we provide the expression data obtained by RT-qPCR representing pri-miRNA transcript levels. For the quantitative PCR experiments we include a number of reference genes: elongation factor 1-alpha  . The porThe content of the database is constantly being updated and the most recent number of records as well as documentation regarding employed methods and procedures can be found on the mirEX 2.0 web page. All data that have been used to create the mirEX 2.0 portal can be downloaded from the web page and from interactive windows, and can be used in subsequent data-mining applications.The database was implemented in mySQL 5.5  and the The mirEX 2.0 portal offers a modern and graphical interface that allows to query and explore all of the information contained in it. The data can be accessed by searching for a particular microRNA name or by browsing the database content. We employ a simple, two-step querying system despite the vast amount and variability of data contained in the mirEX 2.0 database. The first step in the data selection process is provided in the form of a graphical query builder Fig.  to supplSelecting a mutant ID Fig.  results The real potential of the mirEX 2.0 portal is based on its ability to combine different datasets into one display. There is no limit as to the number and type of selected plant species, developmental stages and microRNAs that can be combined in a single query. Depending on the complexity of the search, the interface determines the most user-friendly and informative way of presenting the results; for example, in addition to line and bar graph outputs, mirEX 2.0 offers a heat map-based display of multiple miRNA entries across complex (multi-tissue/multi-species) comparisons  a graphical presentation of the expression levels in all of the tested growth stages and tissues in the form of “Electronic Fluorescent Pictographs” , (ii) exThe portal is accessed through a clean, intuitive web design which provides the user with an optimal viewing experience of the website’s features by adapting the web page layout to the operated device . A major point of emphasis of the mirEX 2.0 design has been to develop individual pages that are intuitive, thus giving the user freedom to focus on the biological query being addressed.With this in mind, several user-centered solutions have been provided to enhance the general efficiency of the data mining experience. All results displayed in mirEX 2.0 are presented as separate, clearly labeled windows with the graphical elements providing contextual clues as well as mouse-over tooltips and short video tutorials with feature details. Users can adjust the amount of information that is present on the page to their needs, as any window can be minimized or expanded at any given moment. Depending on the data type, each of the windows may contain tools for data presentation, e.g., for sorting, filtering and changing the data source, thus allowing the user to generate customizable and integrated results. Other features that enhance the readability of the presented information include interactive charts as well as searchable and sortable table views.Other general functionalities incorporated in the portal include a status message prompter presented at every query-building step and results screens. This widget informs the user about the current status of the request; for example, the status window will contain information about selected species, stages and the number of microRNAs, or it will show the progress of data loading. Through the ‘Comment’ feature, users can comment on any resource present in the database and start discussions. We encourage feedback from the bioscience community in terms of verifying the information we provide as well as obtaining relevant data either from ongoing research or from previous research, both published and unpublished.We constantly continue to collect high-quality expression data for already incorporated plant species following new discoveries and annotations. In the future, we also plan to include data from other, not-well-established model organisms. The database schema as well as the user interface are now tuned to allow for the exploration of any number of combination of species/tissues/mutants and developmental stages. We also plan to incorporate microRNA expression profiles from plants exposed to various biotic and abiotic stresses. Work on drought and heat effects on the microtranscriptome in arabidopsis and barley is already under way. The currently established modular scheme of data presentation and wizard-like querying interface create an environment that guarantees further expansion toward the assimilation of new datasets and the development of novel visualization tools.Arabidopsis thaliana), economically (Hordeum vulgare), and evolutionarily (Pellia endiviifolia) attractive research models. The provided user-friendly tools allow to explore expression data in any combination of species, tissues and developmental stages, thus leading to the rapid discovery and hypothesis-building of underlying relations and regulatory mechanisms. The developed technology also allows for unlimited further expansion of the data content and provides an environment for the design of novel tools following the needs of the plant community involved in the exploration of microRNA biology.The mirEX 2.0 web-based portal is a one-stop solution for the exploration of plant microRNA expression data covering mutants and three plant species representing scientifically (Project name: mirEX 2.0http://www.combio.pl/mirexProject home pages: Operating system(s): Platform independent.License: Not required.Any restrictions to use by non-academics: None.


Massive cellular insults lead to necrosis. It appears, however, that cells have some choice in the mode of their demise depending on the severity of the insult. Hypoxic cores in solid tumors2 or perfusion-impaired brain tissue in stroke,3 for instance are typically necrotic, but possess an apoptotic shell.Cell death can take many shapes. Programmed ‘apoptotic' and uncontrolled ‘necrotic' cell death mark the extremes of the spectrum of possibilities.6 Lysosomes can rupture during ischemic or traumatic cell injury,7 giving rise to both apoptotic and necrotic outcomes.The same can be seen for lysosomal damage.8 dedicated to the degradation of both intracellular material, such as damaged or old organelles, and extracellular components such as matrix proteins. The release of the lysosomal proteases into the cytosol sentences the cell to death by autodigestion.Lysosomes are cellular organelles with a proteolytic function and possess an impressive set of protease enzymesCell Death Discovery article,9 we looked at the fate of the major regulators of apoptosis, the bcl-2 protein family, in order to understand how cell fate upon lysosomal rupture is directed toward necrosis or apoptosis. This protein family contains both pro- and anti-apoptotic members and has been shown to be part of the proteolytic spectrum of cathepsin lysosomal proteases.10 We reasoned that a regulated degradation of these proteins, by affecting the equilibrium of pro- and anti-apoptotic signaling, might offer a means for switching between cell death forms.In a recent To this end, we imaged the proteolytic degradation of the major bcl-2 family representatives bcl-xl, bid and bax upon induced lysosomal disruption in real time using förster resonance energy transfer (FRET) microscopy of single cells. We created proteolytic FRET sensors by sandwiching full-length bcl-2 proteins between cyan and yellow fluorescent proteins. A strong FRET signal is detected in the intact sensor, which is lost upon cleavage , left.The bcl-2 sensors show a common sigmoidal ‘delay-snap' cleavage behavior: after a delay of tens of minutes, cleavage occurs rapidly and is completed within minutes. Among the observed bcl-2 members, anti-apoptotic bcl-xl is degraded before pro-apoptotic bid and bax. Cells thus appear to accumulate pro-apoptotic reactants in the early stages of lysosomal rupture.11 Our work places the bid shunt very early in lysosomal cell death signaling and this is the first time this event is caught ‘on film'.The pro-apoptotic bid showed a conspicuous behavior. Before the overall sigmoidal degradation, a pronounced and very rapid cleavage was observed in the first minutes of lysosomal lysis. Fast proteolytic processing of bid is used in apoptosis and might serve the same role in lysosomal lysis. In apoptosis, the caspase 8 protease activates bid by its truncation, directly connecting the extrinsic and intrinsic apoptotic signaling pathways. This mechanism is called the ‘bid shunt' and processing of bid by cathepsin B has been suggested to operate similarly.In order to gain a better understanding of the regulation of bcl-2 protein levels by cathepsins and the involvement in programmatic steps in necrosis and apoptosis, we investigated the interplay of both systems in more detail.Given the relatively low amount of lysosomes that were disrupted in the early stages after lysosomal disruption, we selected the neutral-active cathepsin B as likely candidate for most of the early proteolytic actions. Hence, we repeated the experiments in the presence of a selective cathepsin B inhibitor and an inhibitor for the thiol-cathepsin class, to which cathepsin B belongs. Inhibition of the thiol-cathepsins confirmed their predominant role in the degradation of bcl-2 family proteins, as the proteolysis of all three bcl-2 constructs was inhibited. Selective cathepsin B inhibition helped uncover its role in steering cell death. We found that early bid truncation was abolished, confirming cathepsin B as the responsible protease. Furthermore, when we limited lysosomal protease action to the time window of the early bid truncation, we detected late-apoptotic caspase 3/7 activation, showing that cells had chosen an apoptotic exit.Unexpectedly and paradoxically, bax and also bid exhibited a new accelerated proteolytic behavior upon selective inhibition of cathepsin B. The delay phase before the onset of cleavage was completely abolished, in particular for bax. As a general inhibition of the thiol-cathepsin classes resulted in the complete inhibition of the proteolysis of these same sensors, we concluded that their accelerated degradation upon cathepsin B inhibition is most likely caused by another, unidentified, thiol-cathepsin. This proteolytic cascade prevents the degradation of apoptosis-supporting proteins, shifting the balance toward apotosis. The identity of this cathepsin requires further research.Cells thus appear to be able to pull an ‘emergency brake' upon lethal injury with damaging of lysosomes. Starting with cathepsin B, an early programmatic protease cascade is initiated that appears to avoid necrosis in favor of apoptosis. In this apoptotic exit effort, cathepsin B preferentially degrades anti-apoptotic bcl-xl, activates bid by its rapid and selective truncation, and degrades a thiol-cathepsin to avoid the removal of apoptotic bid and bax , right.