The first minutes after birth are critical to reducing neonatal mortality. Helping Babies Breathe (HBB) is a simulation-based neonatal resuscitation program for low resource settings. We studied the impact of initial HBB training followed by refresher training on the knowledge and skills of the birth attendants in facilities.We conducted HBB trainings in 71 facilities in the NICHD Global Network research sites , with a 6:1 ratio of facility trainees to Master Trainers (MT). Because of staff turnover, some birth attendants (BA) were trained as they joined the delivery room staff, after the initial training was completed . We compared pass rates for skills and knowledge pre- and post- initial HBB training and following refresher training among active BAs. An Objective Structured Clinical Examination (OSCE) B tested resuscitation skill retention by comparing post-initial training performance with pre-refresher training performance. We identified factors associated with loss of skills in pre-refresher training performance using multivariable logistic regression analysis. Daily bag and mask ventilation practice, equipment checks and supportive supervision were stressed as part of training.p < 0.001). Only 5% could ventilate a newborn mannequin correctly before initial training but 97% passed the post-initial ventilation training test (p < 0.0001) and 99% passed the OSCE B resuscitation evaluation. During pre-refresher training evaluation, a mean of 6.7 (SD 2.49) months after the initial training, 99% passed the knowledge test, but the successful completion rate fell to 81% for the OSCE B resuscitation skills test. Characteristics associated with deterioration of resuscitation skills were BAs from tertiary care facilities, no prior resuscitation training, and the timing of training .One hundred five MT (1.6 MT per facility) conducted initial and refresher HBB trainings for 835 BAs; 76% had no prior resuscitation training. Initial training improved knowledge and skills: the pass percentage for knowledge tests improved from 74 to 99%  contains supplementary material, which is available to authorized users. Millennium Development Goal 4 called for a two-thirds reduction in mortality of children under 5 years of age from 1990 rates by 2015 .The three leading causes of neonatal mortality worldwide are prematurity 36%), birth asphyxia (23%), and infections (23%) 6%, birth. An addiThe Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) Global Network for Women’s and Children’s Health Research , developed Helping Babies Breathe (HBB), a simulation-based curriculum to train facility BAs in resuscitation, in resource limited settings [The Neonatal Resuscitation Program (NRP) has been the standard of care for resuscitating newborns since 1987 . Subsequsettings –14.TM). HBB training materials use multiple approaches ; draw attention to critical decision points; and stress the need to initiate ventilation no later than the end of the first minute after birth [TM) that allows trainers to manipulate cardinal evaluation signs , and an Action Plan that uses these evaluation signs to guide decision-making and management of the newborn who may range from a healthy wailing newborn to one who needs extra attention before crying and breathing well, or one who needs BMV and advanced care.HBB focuses on the initial steps of resuscitation, including immediate drying of the baby, providing warmth and additional stimulation to breathe, followed by bag and mask ventilation (BMV) if needed, within the first 60 seconds after birth , based on delivery logs at each facility. The baseline characteristics of BAs were recorded at both initial and refresher training to help evaluate the initial skill level and experience, as well as skill retention at refresher training; these characteristics included age, type of health provider (physicians or nurses), health facility level , prior resuscitation training , the average monthly number of births attended and, at refresher training, the average monthly number of births attended in the prior six months, and the interval between initial and refresher training. The general and specialist doctors were categorized as physicians and the trained nurses, nurse midwives, auxiliary nurse midwives were categorized as nurses. Health facilities were classified as primary level if they did not perform Caesarean sections (C-sections), secondary level if C-section staff was available on call, and tertiary level when C-sections were available 24 h a day.Country-level training of facility MTs: As above, a group of three experienced central AAP MTs were paired with three experienced MTs at each of the participating Global Network sites to form a female–male, multi-disciplinary (physicians and nurses) country team of MTs to conduct a four-day training of the new facility MTs. The goals of this training of trainers were to rapidly scale up HBB training by training at least one MT per facility (vs. a training cascade), to preserve the integrity of the intervention and to minimize time and training costs. The training agenda was developed in collaboration with the AAP, based on lessons learned to date, including the need for trainers to do pre-training preparation by phone and in person and through post-training evaluation meetings. Each country MT trained a team of six new multidisciplinary facility MTs. The first two days were dedicated to hands-on learning of HBB. During the last two days, the new facility MTs taught small segments of HBB to their fellow team members and developed plans for rolling out training, as the central AAP MTs and country MTs monitored their teaching skills. A roving monitor observed whether the training groups were functioning well and provided feedback after consultations with the team members. Whenever needed, the new facility MT was provided an opportunity to improve his/her skills and extra training was provided until every trainee MT was proficient in resuscitation training. The unique features of these training sessions are summarized in Table Facility-level training of BAs: The study provided sufficient equipment for facility delivery rooms and resuscitation practice corners. The facility BAs were trained as HBB teams in three-day on-site facility courses by the newly-trained site MTs at their respective facilities between June and October 2012. This course included pre- and post-tests for all participants. Unlike the facility MT training, the primary focus of the BA courses was on training to optimally perform delivery room resuscitation. The BA trainees were taught to prepare for every birth; to focus on immediate, thorough, and vigorous drying of every newborn; to provide routine care  for babies that breathed spontaneously; to provide more vigorous stimulation and, if needed, suction of mouth and nose, for babies that did not breathe well after drying; and finally to initiate and establish early and effective ventilation within The Golden MinuteTM for all babies that did not breathe after the above initial steps. Rather than didactic sessions, skill training and open discussion were provided  to ensure that every BA could effectively resuscitate newborns. BAs were instructed to resuscitate every non-macerated birth, including those that might previously have been considered fresh stillbirths. High-quality HBB teaching materials and methods were used throughout the training. The HBB teaching materials included a neonatal simulator that felt and weighed like a newborn which could be manipulated by the MT to demonstrate the presence or absence of chest rise, cry and cord pulsations ; a Learner Workbook for each participant; a large Action Plan poster mounted near each group of six participants that pictured the color-coded paths for the three resuscitation outcomes; and a Facilitator Flipchart that guided teaching and discussions with pictures for the BA on the front and learning content for the MT on the back of each page. The teaching methods included demonstrations of each resuscitation step by the MTs, followed by individual and paired practice to encourage peer teaching and paired learning and standardized case scenarios and exercises for problem solving. This group training included a maximum ratio of six BAs per MT. Because all sites experienced staff turnover, newly-hired BAs were trained by the facility MTs in catch-up individual training or small group sessions, using the same materials and approach as in the initial training sessions.Refresher training: A half-day refresher training course for all “active” BAs was prospectively planned for approximately six months after the initial training course. If required to accommodate the staff schedules and clinical duties, multiple refresher training sessions were conducted in each participating health facility. The refresher training included a group review of the entire resuscitation algorithm and demonstration of critical skills through video clips developed by AAP, along with instructional narration by the MTs in local languages as required [required . The starequired . This waRapid Scale Up of HBB TrainingMonitoring ActivitiesMonitoring activities were introduced immediately after the initial facility-level training of BAs and carried out as a part of continuation of learning/training techniques best described as supportive supervision , 30. TheInformation from all the participating facilities was compiled by the site HBB coordinators. This included compliance with monitoring activities, details of each resuscitation event in each of the participating facilities, and the outcomes, using a standard format. Calls were conducted every two weeks between the central staff  and each site coordinator to discuss individually every baby that had required resuscitation in the 72 participating facilities, including confirming whether each live born baby (non-macerated) was immediately dried, whether the baby cried or breathed spontaneously after drying, required additional stimulation, whether the baby received BMV, and whether the baby was alive or dead (fresh stillbirth) at discharge from the delivery room  planning phase; (2) selection of facility staff to be trained as facility-level MTs and identification of all BAs in the participating facilities; (3) rapid scale up of HBB training done in three stages: (a) training of facility-level MTs (b) facility-level training of BAs, (c) refresher training of active BAs (defined as those who regularly attended deliveries); and (4) introduction of a multi-faceted monitoring program soon after the facility-level trainings.The knowledge evaluation of the BA trainees was done using the standard HBB Knowledge Check before and after (pre-post scores) initial and refresher training. The Knowledge Check was a 17-item written multiple choice questionnaire . The traTM [The skills evaluation was done with the neonatal simulator and included ability to perform BMV effectively (appropriate chest rise and ventilation rate) and successful completion of the Objective Structured Clinical Evaluation (OSCE) A and OSCE B. These evaluations were performed according to study protocol to minimize potential bias and inter-rater variability. The Facilitator Flip Chart provided detailed instructions for administering the three skill tests i.e. BMV, OSCE A and OSCE B. Every trainee was required to successfully complete the BMV Performance Evaluation (seven out of seven steps) before attempting the OSCE evaluations . OSCEs wTM . The traTM . TraineeThe study data were recorded on paper forms and then entered into a password protected electronic data base by study staff at each site. Data were securely transmitted on a regular basis to the central data center. Data entry software included consistency checks, with cross-form edits performed centrally at the data center and resolved locally. Each site performed local data review and edits.p < 0.05) in univariate models were retained as predictors in the final model. The variables in the final model were type of training , type of health facility , type of health provider , exposure to prior resuscitation training, number of births attended per month in the last six months , study site, and age of the BAs. The dependent variable was the probability of failing the pre-refresher OSCE B test after passing the post-initial OSCE B. Unadjusted odds ratios (ORs) and 95% confidence intervals (CIs) were estimated by fitting univariate models with each of the predictors. Adjusted ORs and 95% CIs were estimated by fitting a multi-variable logistic regression model with all of the predictors included.Chi-square tests of proportion were used to test differences in pass rates between the types of health providers. McNemar’s test was used for paired comparisons to test the proportion of BAs who passed the MCQ after initial training but failed it in the pre-refresher training. A logistic regression model was used to assess the association between BA characteristics and loss of skills. Those that were significant . Of the 291 physicians who participated in refresher training, 130 (45%) had received their initial training as catch-up training. All these physicians served in primary care facilities. Among nurse BAs, 99 (18%) had received their initial training as catch-up training. Of these nurse BAs, 98% were in secondary level facilities and 2% were in tertiary facilities.The 105 MTs trained 2227 BAs in the HBB curriculum during 236 workshops  or secondary level facilities . In contrast, many of the BAs in Nagpur and Belgaum were physicians; deliveries in Nagpur were predominantly in tertiary level facilities where C-sections were available 24 h a day.p < 0.0001). Physicians were significantly more likely than nurses to have a passing knowledge score before the initial HBB training , but, after the initial HBB training, all physicians and 98% nurses passed the post-training knowledge exam.Table p < 0.0001 pre- vs. post-initial training). OSCE A was assessed after initial and refresher trainings with 99.4 and 99.8% of BAs passing the test at both time points, respectively.Improvements in resuscitation skills after training were more impressive than the gains in knowledge, in part because BMV skills were so low initially. Prior to the initial HBB training, only 4.6% of BAs (3% of physicians and 5% of nurses) could ventilate a newborn mannequin effectively, as measured by the straightforward BMV skills test, but all physicians and 96% of nurses passed the test after initial training (p < 0.0001), but after refresher training 99% passed the OSCE B. Physicians were less likely than nurses to retain OSCE B skills pre-refresher training (p < 0.002). The interval from initial to refresher training for those who failed the pre-refresher OSCE B after passing it in post-initial assessment was not longer than for those that passed both .Table p = 0.002) failed pre-refresher training OSCE B. Among the seven characteristics in the regression model, facility type (p < 0.0001) and prior training (p < 0.01) were the two significant predictors of deterioration of OSCE B skills. BAs from tertiary facilities  and those with no prior resuscitation training  were most likely to fail the pre-refresher training OSCE B. However, those who received their initial training during catch-up rounds in individual or small-group training ; and the BAs from Nagpur site  were less likely to lose the skills over time.Table Our study is the first large multi-national pre-post study of the impact of initial HBB training, followed by refresher training, on resuscitation knowledge and skills over time, using a state-of-the-art curriculum, standardized training protocol and evaluation methods, and ongoing supportive supervision and monitoring activities. Our goal was to address the methodologic problems of previous research in resuscitation training .One of the most striking findings of our study of a large cohort of 835 active BAs trained and retrained in the HBB curriculum in 71 facilities in India and Kenya was that 76% of active BAs reported no prior training in neonatal resuscitation, despite efforts in India, Africa and other low and middle income countries to encourage facility deliveries and the relatively high BA delivery rates (47% of the BAs delivered between 11 and 40 babies per month and 19% delivered more than 40 babies per month.p < 0.0001) disappeared after HBB training when more than 98% of both doctors and nurses passed the knowledge tests and the proportion of both nurses and doctors who correctly ventilated a neonatal model increased from approximately 4% to 97%.The lack of resuscitation training was evidenced by a large initial knowledge-skills gap among the BAs: at the initial HBB training, 74% of BAs passed the pre-training knowledge assessment but only 5% were able to demonstrate effective ventilation of the neonatal mannequin. Our findings are consistent with Singhal et al., who also found a low concordance between knowledge and skills . A majorOur study provided sufficient ventilation equipment for facility delivery rooms, resuscitation practice corners, and adequate numbers of MTs to ensure ongoing mentoring and supportive supervision of BAs: daily ventilation practice, equipment checks, death audits, resuscitation debriefings, and announced and unannounced site visits at each facility, as well as unique biweekly site conference calls to review the management of each resuscitated newborn in detail and the outcomes of all births before discharge at each facility.p < 0.0001), suggesting that recent training may be associated with improved skill performance. The lack of information on the optimal interval for training was recently noted in the Part 7 2015 Neonatal Resuscitation Consensus document: no evidence-based, effective strategy for teaching, assessing, and maintaining resuscitation knowledge and skills has been identified and that the existing evidence is of low quality [However, the knowledge-skills gap that was evident before the initial HBB training recurred months later at refresher training despite ongoing mentoring and supportive supervision, when resuscitation skills, as measured by OSCE B, were less likely to be retained than knowledge (81% vs. 99%). At pre-refresher testing, BAs from tertiary facilities had nine times higher risk than those at lower-level facilities of failing the OSCE B, after having passed it at the end of initial training—a surrogate for loss of resuscitation skills. The higher skill-deterioration rate of BAs in tertiary facilities who care for high risk patients was unexpected. It may be due to a combination of factors, including less daily practice, whether due to busy schedules, timing of resident rotation, understaffing, increased workload or even a higher proportion of physicians in tertiary-level facilities, resulting in a less frequent BMV practice. During the biweekly reviews, the qualitative reports suggested that physicians were less compliant to daily BMV practice than the nurses, with assertions that they were ‘too busy” or “not in the delivery area.” Lack of previous resuscitation training prior to HBB training was associated with a 2.5 times greater risk of pre-refresher OSCE B failure. This may mean that all or a subset of BAs need repeated trainings to decrease the risk of failure. Those whose initial training was late (catch-up training) were less prone to skill deterioration; the mean time between initial and refresher trainings was also almost half compared to the initially trained BA cohort (3.8 ± 2.9 months vs. 7.8 ± 0.9 months,  quality . Hands-oOur large, rigorous pre-post cohort training study documented that facility BAs in 71 facilities in India and Kenya improved their knowledge and neonatal resuscitation skills after HBB training by MTs and supportive supervision during active monitoring and evaluation over the subsequent year. That 18% of BAs failed pre-refresher training skills tests suggests the need to identify such BAs earlier in order to provide the individualized training, supervision, and practice necessary to ensure that their resuscitation skills are durable over time or to reassign them to a less critical care area. The 18% failure rate should also prompt future researchers to compare different time intervals, structures or content of daily practice or different supervisory models to improve resuscitation skill retention. The risk factors for deterioration of resuscitation skills also suggest that resuscitation training should take place earlier and that administrators should ensure that staffing patterns in tertiary facilities with high delivery volume provide adequate time, motivation and accountability to maintain BA resuscitation skills.As the HBB program paves the way for its successor, Helping Babies Survive, which includes the HBB 2nd edition and care of a newborn after immediate delivery room resuscitation, it will provide opportunities to incorporate standardized data collection, quality improvement activities, and institutional support for registries .Given the low quality of evidence for simulation training, it is essential that future research addresses the knowledge gaps identified by the recent Wyllie et al. report , includi


Marine diatoms in tillites along the Transantarctic Mountains (TAMs) have been used to suggest a diminished East Antarctic Ice Sheet (EAIS) during Pliocene warm periods. Updated ice-sheet modelling shows significant Pliocene EAIS retreat, creating marine embayments into the Wilkes and Aurora basins that were conducive to high diatom productivity and rapid accumulation of diatomaceous sediments. Here we show that subsequent isostatic uplift exposed accumulated unconsolidated marine deposits to wind erosion. We report new atmospheric modelling utilizing Pliocene climate and derived Antarctic landscapes indicating that prevailing mid-altitude winds transported diatoms towards the TAMs, dominantly from extensive emerged coastal deposits of the Aurora Basin. This result unifies leading ideas from competing sides of a contentious debate about the origin of the diatoms in the TAMs and their link to EAIS history, supporting the view that parts of the EAIS are vulnerable to relatively modest warming, with possible implications for future sea-level rise. A long-standing debate regarding the Pliocene history of the East Antarctic Ice Sheet was spurred by the discovery of marine diatoms in the Transantarctic Mountains. Here the authors show that the diatoms were emplaced by wind following a retreat of the ice sheet into coastal basins and subsequent isostatic emergence. Once the occurrence of the diatoms in association with the high-altitude deposits was independently replicated2020The majority consensus that the key diatoms were emplaced by aeolian processes was widely interpreted as refutation of the ‘dynamic' EAIS hypothesis12131927144The ‘stabilist' view gained considerable support from an early generation ice-sheet model2331In contrast with earlier modelling efforts331783334363739Here we further evaluate aeolian processes and test the hypothesis that aeolian diatoms in the TAMs were the result of Pliocene EAIS retreat, rather than evidence against ice-sheet dynamic behaviour. We evaluate coastal isostatic response over a Pliocene Antarctic ice-sheet reconstruction derived from an ice-sheet model that incorporates ice-sheet grounding zone processes, including cliff failure and hydrofracturingSilt and sand-sized grains, including microfossils, are well known to be raised by saltation processes and then carried significant distances by wind. Diatom frustules and small clumps of diatomaceous sediment (microclasts) have an extremely high surface-area:mass ratio , making 42Once aloft, these particles can be carried great distances, as readily seen by the observed distribution of diatoms across the Antarctic ice-sheet surface. Pleistocene and modern long-distance transport and atmospheric precipitation of diatoms on the Antarctic ice sheet and the TAMs have been demonstrated through analysis of ice cores44444445The fact that marine diatoms typically dominate the diatom assemblages extracted from most Sirius tillite surfaces, despite a constant and ongoing rain of non-marine diatoms across these surfaces today, indicates that the accumulation of marine diatoms is not a recent phenomenon, but largely reflects a specific interval or intervals of significant aeolian input with distinct source beds of subaerially exposed Pliocene marine diatomaceous sediments. Diatoms are generally not lifted from ocean surfaceset al.20et al.The widespread distribution of aeolian marine diatoms in Sirius deposits  implies 2 and a warm austral summer orbitet al.373839A new version of an established Antarctic ice-sheet model PSUICE-3D)  transitions described in the ANDRILL-1B drillcore (defined as Motif 2)Following uplift, a subaerially exposed diatom-strewn landscape would have been highly susceptible to wind erosion, especially during summer with reduced snow cover , thus prThe entire process takes several thousand years following climate warming. With grounding line retreat and ice shelf collapse, a marine basin opens, with copious diatom production in open waters during summer with little sea ice. The outer basins shallow with the isostatic response following ice retreat, eventually leading to emergence of uplifted islands and coastal areas. Although the simulation in 533−1 in many locations adjacent to the freshly exposed sediments, most notably along the Aurora Basin coastline  over Antarcticaoastline . Wind spoastline  indicateIn contrast with surface winds, mid-tropospheric winds at 500 hPa, ∼5,000 m altitude, are generally cyclonic over the continental interior and show strongly favourable transport directions towards the TAMs and Sirius outcrop localities, from both Wilkes and Aurora basin marine sediment exposures , especiaet al.et al.Although ice-free bedrock patches also emerge around the islands of West Antarctica , both su39et al.Despite a paucity of exposed source beds today, there is widespread evidence for rare diatoms from coastal and Dry Valley sites being raised to the Polar Plateau, but these are dominantly of non-marine origin44445572 has now reached Pliocene levels and is expected to continue to rise. Scientific debates, such as the interpretation of Sirius Group diatom data, often turn on key details that if misinterpreted or interpreted through a rhetorical bias may obscure broader truths. The debate between ‘stabilists' and ‘dynamicists' focused on the configuration of the EAIS during the Pliocene, but none of the published studies presented data that was directly related to or indicative of EAIS geometry or thickness during the Pliocene. Furthermore, neither a ‘stable' nor ‘dynamic' ice sheet was adequately defined in context, and intermediate ice-sheet configurations, such as those suggested by new models, were not considered. The IODP Sites 1165 . Surface winds are from the lowest model level (σ=0.9950).Pliocene mean annual and summer (January) temperatures are calculated from the RCM . These hPlots for both the ice sheet and atmospheric model outputs were prepared using MATLAB software .Ice-sheet and RCM model outputs are available on request. Netcdf-format output file is available for the ice-sheet distribution in How to cite this article: Scherer, R. P. et al. Windblown Pliocene diatoms and East Antarctic ice sheet retreat. Nat. Commun.7, 12957 doi: 10.1038/ncomms12957 (2016).


Accumulating evidence supports the polygenic nature of most complex diseases, suggesting the involvement of many susceptibility genes with small effect sizes. Although hundreds of genes may underlie the genetic architecture of complex diseases, those involved in a given disease are probably not randomly distributed, but likely to be functionally related. Protein-protein interaction networks have been used to evaluate the functional relatedness of susceptibility genes. However, these networks do not account for tissue specificity, are limited to protein-coding genes, and are typically biased by incomplete biological knowledge. Here, we present Gene Link Inspector Through Tissue-specific coExpRession (GLITTER), a web-based application for assessing the functional relatedness of susceptibility genes, either coding or noncoding, according to tissue-specific gene expression profiles. GLITTER can also shed light on the specific tissues in which susceptibility genes might exert their functions. We further demonstrate examples of how GLITTER can evaluate the functional relatedness of susceptibility genes underlying schizophrenia and breast cancer, and provide clues about etiology. Accumulating evidence supports the polygenic nature of most complex diseases, suggesting the involvement of many susceptibility genes with effect sizes that are typically very small124http://han-lab.org/GLITTER.Complex diseases caused by susceptibility genes often show tissue-specific pathology. We reason that tissue-specific gene relationships may more closely reflect the functional relatedness of susceptibility genes than does a general PPI network. Here, we present the Gene Link Inspector through Tissue-specific coExpRession (GLITTER), a web-based application for assessing the functional relatedness of disease susceptibility genes, either coding or noncoding, according to tissue-specific gene expression profiles. GLITTER quantifies the functional relatedness of susceptibility genes by calculating the total number of connected gene pairs based on gene coexpression patterns. Its statistical significance is estimated by randomly sampled gene sets, while matching for gene numbers, gene sizes and GC content. GLITTER may also shed light on specific tissues in which susceptibility genes might exert their functions. A web-based application to implement GLITTER is freely available at An overview of GLITTER is provided in http://www.gtexportal.org/home/. We limited our analysis to 49 distinct tissues that each had sample size >20, including 13 brain region-specific tissues and 36 non-brain tissues. For each tissue, we considered genes expressed if they had a normalized RPKM value of ≥1 for at least 80% of the samples. Gene expression values were log-transformed (log2 [RPKM + 1]) for gene co-expression analysis.We downloaded RNA-Seq data (version 6), summarized to Gencode v19 gene-level reads per kilobase million mapped reads (RPKM) values, from the GTEx Portal website. Detailed information on tissue processing, experimental and bioinformatics procedures related to the RNA-Seq data is available at bicor function in the R-package WGCNAUtilizing the gene expression data from GTEx, we used gene coexpression patterns to evaluate the functional relatedness of susceptibility genes. GLITTER provides two options, Pearson correlation or biweight midcorrelation, for calculating gene expression correlations between two genes. Biweight midcorrelation is more robust against outliers than Pearson correlationGLITTER takes a list of user-defined genes as input, together with options for the type of correlation, correlation threshold, and the number of simulations for random gene sets. The gene names can either be official symbols or ENSEMBLE IDs. The two correlation-related options (correlation type and correlation threshold) determine the way to estimate the connections of genes in each tissue. Simulations are used for estimating the significance of the observed number of connected gene pairs among input genes. Random gene sets are generated from the human Gencode v19 gene annotation by default. The users can also choose to generate random gene sets from customized gene sets, such as those represented in a GWAS array. We note that the customized reference gene sets should contain as many genes as possible, so that the background distribution can be approximated by random gene sets. GLITTER returns two sets of results: (1) a summary text file with each line containing meta information for each tissue, including tissue name, total number of expressed genes, the number of input genes expressed, the number of connected gene pairs among input genes, and the p-value assessing the significance of the observed number of connected gene pairs; and (2) a series of PDF files for the histogram of the number of connected gene pairs from random gene sets for each tissue. The running time depends on the number of input genes and other selected parameters. For a set of 102 genes and 10,000 simulations, it takes about one hour. GLITTER sends an email notice to users once the job is done, alerting them that the results are ready to be downloaded from the website.corrected ≤ 0.05), suggesting their potentially important roles in the etiopathogenesis of the disease. These regions included the amygdala (p = 0.0005), cerebellar hemisphere (p = 0.0004), cortex (p = 0.0004), and hippocampus (p = 0.0006). However, we did not observe a similar pattern in non-brain tissues. These data strongly support the functional relatedness of schizophrenia genes in brain-related tissues, but not in other tissues.We applied GLITTER to evaluate the functional relatedness of 97 schizophrenia candidate genes implicated by the 108 independent schizophrenia risk loci identified in a recent genome-wide association study of schizophrenia from the Psychiatric Genomics Consortium (PGC)corrected = 0.07). There were only two other tissues with nominal levels of evidence for the non-random connectedness of breast cancer genes: kidney cortex (p = 0.03) and uterus (p = 0.04). To evaluate the generalizability of GLITTER, we also applied it to examine the functional relatedness of 49 breast cancer susceptibility genes identified through GWASFinally, as a comparison between GLITTER and an existing method of assessing the functional relatedness of gene sets, we used the Disease Association Protein-protein Link Evaluator (DAPPLE)In summary, we designed GLITTER, a user-friendly web-based tool, to examine the functional relatedness of susceptibility genes according to tissue-specific gene expression profiles. GLITTER employs the basic assumption that if two genes are highly co-expressed then there is a good chance they are functionally related. GLITTER first estimates the functional relatedness of susceptibility genes by counting the total number of connected gene pairs based on gene coexpression patterns. It then uses simulations to estimate the likelihood that the same or a greater number of connected gene pairs can be observed from random gene sets, while matching for basic features  found in the susceptibility gene list.The motivation underlying GLITTER is based on the fact that complex diseases often show tissue-specific pathology. We reason that gene relationships in the context of disease-relevant tissues may more closely reflect the functional relationships among susceptibility genes than those captured in a tissue-independent context through the general PPI network. Compared to PPI networks, gene co-expression networks are less biased by incomplete biological knowledge. Therefore, GLITTER has the advantage of comprehensiveness in defining all potential functionally related gene pairs. In addition, GLITTER can also shed light on specific tissues in which susceptibility genes might exert their functions and therefore enhance our understanding of disease etiology. We note that GLITTER is especially useful for evaluating susceptibility genes underlying psychiatric disorders, because it allows evaluation of gene connections in a number of brain regions, and thus provides opportunities to look into specific brain regions that may be related to underlying disease pathophysiology. Our case study illustrates how GLITTER can evaluate the functional relatedness of schizophrenia susceptibility genes and provide clues about etiopathogenesis. The case study of breast cancer genes further demonstrates the generalizability of GLITTER to other complex diseases.We note that GLITTER has also some limitations. For example, GLITTER uses a random sampling approach for estimating the statistical significance of connected gene pairs. Although random gene sets match for some basic features found in the input genes, such as gene numbers, gene sizes and GC content, the random sampling approach cannot fully account for all important features that determine the background distribution of the test statistic. In addition, while the gene co-expression network approach has the advantage of comprehensiveness, as it captures all potential functionally related gene pairs, it may also inevitably introduce a greater amount of noise compared to the PPI network approach. Therefore, we view GLITTER as by no means a replacement for current tools, such as DAPPPLE, but as a complementary tool for assessing the functional relationships among sets of potential disease genes.How to cite this article: Liu, X. et al. GLITTER: a web-based application for gene link inspection through tissue-specific coexpression. Sci. Rep.6, 33460; doi: 10.1038/srep33460 (2016).


The k-ecoli457 model contains 457 model reactions, 337 metabolites and 295 substrate-level regulatory interactions. Parameterization is carried out using a genetic algorithm by simultaneously imposing all available fluxomic data (about 30 measured fluxes per mutant). The Pearson correlation coefficient between experimental data and predicted product yields for 320 engineered strains spanning 24 product metabolites is 0.84. This is substantially higher than that using flux balance analysis, minimization of metabolic adjustment or maximization of product yield exhibiting systematic errors with correlation coefficients of, respectively, 0.18, 0.37 and 0.47 (k-ecoli457 is available for download at http://www.maranasgroup.com).Kinetic models of metabolism at a genome scale that faithfully recapitulate the effect of multiple genetic interventions would be transformative in our ability to reliably design novel overproducing microbial strains. Here, we introduce k Kinetic models of microbial metabolism have great potential to aid metabolic engineering efforts, but the challenge of parameterization has so far limited them to core metabolism. Here, the authors introduce a genome-scale metabolic model of E. coli metabolism that satisfies fluxomic data for a wild-type and 25 mutant strains in various growth conditions. Escherichia coli15Rapid and pervasive advances in genome editing457810E. coli metabolism by seamlessly integrating flux data for a wild-type and seven mutant strains under aerobic conditions with glucose as the carbon substrate. The model was accurate in recapitulating genetic perturbations as long as growth conditions remained aerobic and the list of deleted genes was in the neighbourhood of the ones used during model parameterization11Recent approaches first deconstruct complex kinetic expressions into their elementary stepsE. coli metabolism was parameterized by combining a machine-learning algorithm202122Km and 49 kcat values) extracted from BRENDAKm and kcat values, respectively, are within the experimentally reported ranges. This level of agreement of k-ecoli457 with experimental data exceeded the metrics reached by the core modelE. coli phenotypes under different growth conditions with a substantially higher accuracy than any other earlier modelling effort.The k-ecoli457 genome-scale kinetic model of E. coliiAF1260 model  upon imposing the flux data of the reference (wild-type) strain or blocked The k-ecoli457 model contains 457 reactions and 337 metabolites and includes all reactions from the largest previously published mechanistic kinetic model of Km and vmax) using the experimentally measured flux data for the nineteen mutant strains grown aerobically with glucose. This is achieved by identifying the best combination of the sampled kinetic parameters in the ensemble (see Km) were fixed and the levels of enzymes  were estimated under the other three growth conditions , separately, by solving the second step of the optimization procedure  strain was constructed see . A two-sdure see . In genedure see . This isdure see .pgi by the previously published core modelpfkA, ΔpfkB and ΔfbaB) that filled in information lost by removing the Δpgi data set. Unsurprisingly, model parameterization was not as robust for the two mutant strains under anaerobic conditions  as alluded by higher prediction deviations from experimental data upon removal of one of the two data sets . Because fluxes of the fermentative products  are significantly different between the two data sets, both data sets are needed as they cannot complement information upon the loss of one of the two. These discrepancies propagate to some extent in other parts of the network, however, model prediction is still adequate for the rest of reactions .We also performed leave-one-out and leave-two-out cross-validation analyses to assess the robustness of the estimated parameters in k-ecoli457 compared with that of the core model  of the predictions within the confidence ranges in the core model were predicted again within ranges by k-ecoli457. Notably, the computed Michaelis–Menten constants in k-ecoli457 for amino acid and pyruvate metabolism exhibited significantly higher agreement with experimental data compared with the values in the core modelKm and 14 kcat parameters. Comparisons revealed that for this set 36% of the estimated Km and 29% of the estimated kcat values are within the experimentally reported ranges. Overall, these analyses demonstrate the importance of integrating pathways distant to central metabolism into the model as they can affect the quality of parameterization through the pools of shared metabolites such as cofactors. This motivates the development of flux data sets in response to genetic perturbations distal from central metabolism pathways to improve overall model parameterization. It is also important to note that the large magnitude in the confidence ranges for both measured and computed kcat values detracts from the confidence for some of the estimated kcat values.To assess the accuracy of the estimated elementary parameters, we compared the corresponding Michaelis–Menten constants with experimental values from BRENDA20nges see . For thepgi, Δgnd, Δzwf, ΔrpiA, ΔrpiB and wild-type strain grown with acetate . The target products included 24 different chemicals under both aerobic (184 mutants) and anaerobic (136 mutants) conditions spanning biofuels , pharmaceuticals and nutraceuticals (artemisinin and naringenin), polymer precursors  and commodity and specialty chemicals  . A comparison also revealed that the product yield in 129 out of 320 designed strains are predicted within 20% of the experimental measurements by k-ecoli457, whereas FBA, MOMA and maximization of product yield predicted the product yield for only 16, 18 and 65 of the designed strains, respectively, with the same accuracy. In general, under aerobic conditions k-ecoli457 closely reproduces the yields for products that branch out from the PP pathway , the TCA cycle  and pyruvate metabolism . Both FBA and MOMA tend to underestimate their yields as more carbon is diverted towards biomass  were available for model parameterization implying insufficient training data as affirmed in the cross-validation analysis (see −1), while product yield data were often reported in higher dilution rates. These observations identify the limits of prediction and pinpoint needed flux measurements under different dilution rates, growth phases, alternate substrates and anaerobic conditions using both batchk-ecoli457 often underestimates the yield of acetate under aerobic conditions as well as ethanol and formate under anaerobic conditions see  due to iE. coli metabolism that approaches genome-scale coverage (457 reactions and 337 metabolites). Comparisons of k-ecoli457 with a previously constructed core modela posteriori correction may not be a fair representation of the proposed model and methodology. These limitations are likely to be ameliorated as expanded metabolomic  uptake rates to become an output of the kinetic model. In k-ecoli457 all mutant fluxes in the training data sets were scaled with the corresponding substrate uptake rate. Given substrate uptake rates data sets24Looking beyond substrate-level regulation, the increasing availability of data sets that provide genome-wide collections of interactions between mRNAs4E. coli metabolism composed of 457 reactions and 337 metabolites was constructed . Enzyme fraction is defined as the level of unbound enzyme e normalized by the total pool of the specific enzyme  in the iAF1260 model using FBA while imposing the experimental flux measurements (for 43 reactions)17=131,072 models, as no improvement in model predictions and convergence to the optimal solution was achieved for a larger ensemble size.The reference flux distribution was obtained by first maximizing biomass yield ldh) with glucose as the carbon substratezwf and Δgnd)E. coli biomass reactionModel parameterization was carried out using steady-state flux data for 25 mutant strains of Km and kcat) with multiple reported values in BRENDA162833575859iAF1260 model of E. coliConfidence intervals were provided only for the measured fluxes of the wild-type strain in the training data setsModel parameterization was performed in two stages: (a) estimation of the elementary kinetic parameters and isozyme activity using the experimentally measured flux data for the single knockout mutants growing under aerobic minimal glucose conditions ; and (b) estimation of enzyme levels under minimal glucose fermentative (anaerobic) conditions and alternate carbon substrates pyruvate and acetate .Km and vmax values in Michaelis–Menten description by permuting parameterizations for different reactions across models in the ensemble. The consequence of an enzyme knockout is captured by fixing the total pool of the normalized enzyme level pfkA, ΔpfkB, ΔfbaB, ΔgpmA, ΔgpmB, Δpgl, ΔrpiA, ΔrpiB, ΔtalA, ΔtalB, ΔtktA and ΔtktB under aerobic conditions with glucose as the carbon substrate) during step one , we next estimated vmax The objective function of the parameterization problem y is defined as the rate of reaction that produces the product per rate of uptake reaction (product mol per glucose mol).Product yield yexp and predicted ypre values.For comparisons, the majority of experimental studies reported only the measured product yield with no confidence range. Consequently, for each product yield the error was calculated based on relative deviation between the measured http://www.maranasgroup.com as a MAT-file (MathWorks Inc.).All parameterization problems were solved using a GA implementation. All calculations, including the ensemble construction and the GA problems, were implemented in MATLAB (MathWorks Inc.) and solved in parallel on three nodes of Intel Xeon E5-2670 v2 with 2.5 GHz (20 cores per node and 256 GB RAM) on the Penn State Lion-X clusters. The k-ecoli457 parameterization took ∼58,800 CPU-hour. Integrating k-ecoli457  for a given metabolic condition also takes up to a few minutes. The estimated elementary kinetic parameters as well as stoichiometry matrix of the model are available in http://www.maranasgroup.com as a MAT-file (MathWorks Inc.).The authors declare that the code supporting the findings of this study is available within the article's All data generated or analysed during this study are included in this published article and its How to cite this article: Khodayari, A. & Maranas, C. D. A genome-scale Escherichia coli kinetic metabolic model k-ecoli457 satisfying flux data for multiple mutant strains. Nat. Commun.7, 13806 doi: 10.1038/ncomms13806 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary methods, supplementary tables, supplementary figures, supplementary references.List of metabolites, reactions and regulatory interactionsEstimated kinetic parameters and predicted metabolic fluxes, metabolite concentrations and product yieldsMATLAB version of the model


Tissue plasminogen activator (tPA) thrombolysis remains the gold standard treatment for ischemic stroke. A time-constrained therapeutic window, with the drug to be given within 4.5 h after stroke onset, and lethal side effects associated with delayed treatment, most notably hemorrhagic transformation (HT), limit the clinical use of tPA. Co-administering tPA with other agents, including drug or non-drug interventions, has been proposed as a practical strategy to address the limitations of tPA. Here, we discuss the pharmacological and non-drug approaches that were examined to mitigate the complications—especially HT—associated with delayed tPA treatment. The pharmacological treatments include those that preserve the blood-brain barrier , enhance vascularization and protect the cerebrovasculature ), and exert their effects through other modes of action . The non-drug approaches include stem cell treatments and gas therapy with multi-pronged biological effects. Co-administering tPA with the abovementioned therapies showed promise in attenuating delayed tPA-induced side effects and stroke-induced neurological and behavioral deficits. Thus, adjunctive treatment approach is an innovative therapeutic modality that can address the limitations of tPA treatment and potentially expand the time window for ischemic stroke therapy. Stroke persists as one of the most prolific killers of Americans, and poses a considerable threat to millions of others worldwide . The theAn assortment of drugs ranging from those that augment neurogenesis  and otheDisruption of the blood-brain barrier (BBB), damage to microvessels, and the toxic and non-thrombolytic actions of tPA have been suggested as the mechanisms underlying delayed tPA-induced complications, especially HT ,11,12,13The HT following delayed tPA treatment can be curtailed by an intervention that could help preserve the integrity of the BBB. Of note, stabilizing the BBB after stroke has been suggested to enhance the overall efficacy of tPA reperfusion therapy . In lighThat glutathione and ascorbic acid (AA) levels decrease and free radical formation increases after ischemic brain injury (IBI) indicate the potential of AA supplementation to improve outcomes after IBI . AscorbiThe pleiotropic  effects of statins make them attractive co-treatments to reduce the complications of delayed tPA treatment, as well as to extend the therapeutic window of the drug . AtorvasBatimastat is a broad-spectrum MMP inhibitor . TreatmeThe efficacy of the protein kinase C (PKC) modulator bryostatin , given 2 h post MCAO to reduce delayed tPA -induced cerebral swelling, hemorrhage, and mortality at 24 h post MCAO in rats was investigated . NotablyCandesartan blocks the angiotensin II type 1 receptor and prevents injury due to ischemic stroke . Early tCilostazol is used for the treatment of intermittent claudication . CombinaDodecafluoropentane emulsion (DDFPe) is an oxygen-transporting perfluorocarbon given i.v. shown to provide neuroprotection in rabbits subjected to ischemic stroke . The effFasudil has been marketed in Japan for the treatment of cerebral vasospasms occurring after subarachnoid hemorrhage . It is aGranulocyte-colony stimulating factor (G-CSF) is an FDA-approved medical countermeasure to promote survival in patients exposed to myelosuppressive doses of radiation. Functionally, it is a cytokine which regulates the survival, proliferation, and differentiation of hematopoietic stem cells and hematopoietic progenitor cells . G-CSF (GM6001 attaches to the active sites of MMPs and prevents the conversion of pro-MMPs to active forms of matrix-degrading MMPs . GM6001 Imatinib is a platelet-derived growth factor α-receptor (PDGFR-α) inhibitor approved by the FDA for the treatment of chronic myelogenous leukemia and other cancers. The drug, given orally at a high dose of 200 mg/kg, at 1 h after ischemia via photothrombosis, was observed to reduce the extent of HT after delayed (5 h post stroke) treatment with tPA . It alsoIMM-H004 is a coumarin derivative which belongs to a class of organic heterocyclic compounds with numerous biological effects . In ratsClinically used for the treatment of acne vulgaris, minocycline  has been shown to reduce infarction and attenuate the brain hemorrhage observed 24 h after embolic stroke  in animaExpanding the time window for thrombolysis may not only be achieved through pharmacological means, but also through non-drug strategies . The mulMinocycline has been previously shown to reduce hemorrhage associated with delayed tPA treatment . IntracrOther types of stem cells have also been investigated and have shown promise in mitigating the complications associated with tPA treatment. Mesenchymal stem cells (MSCs) which have been shown to reduce stroke volume and behavioral deficits in stroke models , also r2) was found to attenuate the MMP-9 induction in the ischemic microvessels of tPA-treated rats  [NBO treatment affords neuroprotection when initiated early after ischemia onset . Previou h MCAO) . It also h MCAO) . Similar h MCAO) ,62 and i h MCAO) ,64 warraBrain imaging has been used to determine patient subgroups with increased risk for hemorrhage and poor clinical outcomes profile . This teWhile we have only included in this review the preclinical studies which specified delayed tPA treatment as >4.5 h post stroke onset, it is noteworthy that some other drugs also attenuated HT and other complications associated with tPA treatment initiated at <4.5 h after stroke  in animal models ,13,14. NPreclinical studies should also focus on interventions that exert neuroprotection in addition to attenuating HT. Notably, a recent meta-analysis involving 6756 participants in the nine clinical trials of intravenous alteplase versus controls showed that the increase in the occurrence of HT has been caused by a number of factors, including stroke severity . Thus, iBecause tPA is essential for reperfusion therapy, finding the right dosage and timing of initiating treatment in relation to tPA is important to enhance possible clinical application of the combined therapy. Specifically, it is very difficult to estimate the precise time of stroke onset to administer the drugs in combination with tPA in the clinics. The FDA standards require assurance that any intervention  that are given alongside tPA should not block the fibrinolytic activity of tPA . NeverthEnhancing tPA’s time window via combination therapy will not only significantly improve HT and other side effects associated with delayed tPA therapy, but will also result in enhancement of the risk–benefit ratio for thrombolytic therapy and increase the number of patients eligible for tPA therapy. An expanded treatment window will also allow the time window of neuroplasticity to remain open for a longer period resulting in better recovery and functional outcomes post-treatment. Another potential, significant clinical application of this strategy is the treatment of “wake-up strokes”, a case where patients awaken with stroke symptoms, which poses a significant challenge for acute stroke providers . CombiniAt the time of writing, efforts are still underway to discover other fibrinolytics or thrombolytic drugs with better reperfusion efficacy than tPA ,71,72. N


Salmonella typhimurium. We studied the antibiotic resistance pattern, phage type and virulence factors playing a role in the invasive nature of the pathogen since no such study from pericardial fluid was found in the literature.Among the infectious causes of pericarditis are various bacteria, viruses, fungal and parasitic infections. The course disease may progress to a chronic constrictive pattern especially with tubercular etiology. Non-typhoidal Salmonella has rarely been reported as a cause of pericarditis. We describe here a case from which the pericardial fluid from an old case of tubercular pericarditis sent for culture to microbiology laboratory grew a The isolate was sensitive only to cephalosporins and it was untypable. It showed amplification for five fimbrial operons, three colonization factors, and other genes (pef operon), gog B(Gifsy-1 encoded effector), sseI (Gifsy-2 encoded effector), sodC  & sopE (a guanine nucleotide exchange factors).The present case highlights the need for early detection of the exact causative agent and serovar in management, the likelihood of a different etiological agent other than the original to be kept in mind for timely management and the highly resistant pattern of non-typhoidal Salmonella (NTS) limiting the therapeutic options as in our case to only cephalosporins. The genes encoded from the NTS might be required for invasive cardiac manifestation in humans. Salmonellosis is an endemic disease in India. The most common clinical presentation is as typhoid fever. Nontyphoidal Salmonella (NTS) commonly cause self-limiting gastroenteritis, and bacteremia occurs in only five percent of patients, but severe extraintestinal focal infections including septic aortitis, meningitis, pneumonia, septic arthritis, osteomyelitis or cholangitis have been reported earlier . With thOver the years, there have been an increasing report of multidrug resistance in Salmonella and more so in NTS. The key to recovery is effective antibiotic usage.The Centre for Disease Control, Atlanta recognizes two species which are divided into seven subspecies: S.enterica (six subspecies) and S.bongori (one subspecies). Different methodologies for Salmonella enterica subspp enterica subtyping exist, serotyping being the most popular method. Other methods include antibody microarrays and phage typing. Molecular typing techniques which have been employed in various centers are pulse-field gel electrophoresis, deoxyribonucleic acid (DNA) base serotyping, multiplexed ligase chain reaction assays, polymerase chain reaction (PCR), subtyping based on whole -genome comparisons, multilocus sequence typing, single-nucleotide polymorphism (SNP) typing, multilocus variable number tandem repeat analysis and molecular typing with composite microarrays. Whole -cell matrix desorption ionization - a time of flight mass spectrometry (MALDI-TOF MS) is a direct bacterial profiling method based on detection of surrogate marker proteins. We have used a combination of methodologies available to us even though it is a single strain, and phage typing is not essential here as we do not expect many cases to be reported with such presentation and thus we studied the virulence factors and phage type to have a database for Salmonella Typhimurium.S .typhimurium is an intracellular pathogens, and different sets of specific virulence factors are responsible for its internalization, survival, replication and also act as effectors targeting various stages in the cellular processes of infection, invasion and intracellular infection. Salmonella can actively invade both phagocytic and nonphagocytic cells using a type III secretory system. Also, a group of effector proteins  induce rearrangements in the actin cytoskeleton resulting in internalization of the bacteria. Other factors like fimbriae, flagella may play a role.We report here a case of nontyphoidal Salmonella pericarditis in a patient with past history of tubercular constrictive pericarditis and we also looked for the probable virulence factors.A 46-year-old female who was a resident of Nigeria was admitted to our hospital with complaints of breathlessness on minimal exertion and swelling of her face and legs since last seven months. She was a known case of tubercular constrictive pericarditis diagnosed about 15 years back and treated with a full course of antitubercular therapy. She was on medical follow-up in her country and had minimal to no symptoms for several years. However, about seven months back she was hospitalized for abdominal pain of seven days duration. The hospital records of that time mentioned heart failure and advice for pericardiectomy and she was referred to our hospital for specialized care. On admission here, she appeared to be sick looking with an irregular pulse rate of 96/min, raised jugular venous pressure (JVP), bilateral pedal edema and muffled heart sounds. Routine blood tests were unremarkable. Sickle cell trait was negative. Anti- hepatitis C virus (HCV) antibodies were reactive. Ultrasound abdomen did not reveal any cirrhosis. The patient was human immunodeficiency virus (HIV) negative. Two- dimension echocardiogram (2-D ECHO) showed evidence of calcified constrictive pericarditis. Cardiac catheterization showed chamber pressures consistent with constriction. Coronary arteries were normal.The patient was taken up for pericardiectomy and during operation, the pericardium was found to be heavily calcified. While dissecting along the inferior surface, a small pocket of creamy white fluid was encountered. Excised pericardium was sent for histopathology and pericardial fluid to microbiology for aerobic culture and sensitivity, Ziehl Neelsen’s stain (ZN) and culture for mycobacteria.The patient was empirically put on amoxicillin plus clavulanate (amoxicillin 875 mg plus clavulanic acid 125 mg) twice a day & cefuroxime 500 mg twice a day till culture results were available. The aerobic culture put on Columbia blood agar and MacConkey agar showed pure growth of non-lactose fermenting colonies after 24 hours of incubation at 37o C. These were oxidase negative, motile, citrate positive, triple sugar iron (TSI) agar showed the alkaline/acid reaction with hydrogen sulfide (H2S) and gas production. Lysine decarboxylase was negative. The isolate was processed for identification of Vitek -2 Compact Systems and Vitek -MS . Serotyping was done using Salmonella antisera. The lactose non-fermenting isolate was identified as Salmonella group on Vitek -2 Compact Systems. The strain showed agglutination with Salmonella antisera polyvalent O, Salmonella O 4 antiserum and Salmonella antiserum H-i . On Vitek –MS (which is based on MALDI-TOF technology) it was identified as Salmonella group  and further as Salmonella enterica spp enterica by analyzing the mass data in Spectral Archive And Microbial Identification System  and processed with baseline correction, Gaussian smoothing and peak finding. The isolate was thus identified as Salmonella enterica subspp enterica serovar Typhimurium based on the above techniques.Antibiotic susceptibility testing was performed as per the Clinical and Laboratory Standards Institute (CLSI) guidelines by Kirby-Bauer method and also put in Vitek 2 compact  using antimicrobial susceptibility testing (AST-N280) card. The isolate was sensitive to ceftriaxone, cefixime and resistant to nalidixic acid, cotrimoxazole, chloramphenicol, ampicillin, and quinolones. The isolate was sent for phage typing to National Salmonella Phage Typing Centre, Lady Hardinge Medical College, New Delhi, keeping in view the high resistance pattern even though phage typing is done mostly for epidemiological investigation of outbreaks. The isolate was reported as ‘untypable’.To further understand the genetic profile of the invasive nontyphoidal serovar (i NTS) and know the virulence gene content of the isolate, polymerase chain reaction (PCR) was performed. Amplified PCR products were separated by agarose gel electrophoresis and stained with ethidium bromide to assess the presence of specific band size of each primer set Table  and gelsThe strain showed amplification for the five fimbrial operons , sseI, gogB, pef, sopE ), three colonization factors  and other genes- pef operon, gog B, sseI, sodC, sopE Figures  -3. No aHistopathology report of the excised pericardium showed chronic inflammation with fibrosis and calcification. No granulomatous inflammation was evident. ZN stain and GeneXpert assay were negative for Mycobacteria. Mycobacterial culture processed in mycobacteria growth indicator tube (MGIT 960)  showed no growth till six weeks of incubation.The patient was treated with ceftriaxone 2 gms intravenously twice a day for two weeks followed by Cefixime 400 mg once a day for four weeks. The patient has not shown any residual bloodstream infection on repeat blood cultures and no relapses till now.NTS pericarditis is uncommon. A literature review of 23 cases of NTS pericarditis between 1936- 1986 revealed four patients with pericarditis caused by Salmonella paratyphi. Paratyphoid fever with pericarditis was reported in an 11-year-old male in 2002 .In a study on infective pericarditis in children in Nigeria, 31% were due to Mycobacterium tuberculosis and three percent due to Salmonella typhi. Both causes can lead to a constrictiv pattern . The couMALDI-TOF MS is a direct bacterial profiling method based on detection of surrogate marker proteins and thus is quick in identification which can greatly help in early effective management.Comparative genomic hybridization using a Salmonella enterica microarray revealed a core of 3233 genes present in all of the iNTS strains, which include the Salmonella pathogenicity islands 1-5, 9, 13, 14; five fimbrial operons ; three colonization factors ; and the invasion gene, pagN. In the iNTS variable genome, Suez J, et al. identified in addition 16 novel genomic islets; various NTS virulence factors and six typhoid-associated virulence genes  [The virulence factors which might have played a role in invasion or causing pericarditis were found to be the five fimbrial operons , three colonization factors  and other genes- pef operon, gog B, sseI, sodC, sopE; although this cannot be conclusive based on one single case report.Pef operon encoded for plasmid-encoded fimbriae that mediate adhesion of Salmonella typhimurium to the small intestine of mice was present in our strain . SopE waOver the years the prevalence of multidrug resistance among Salmonella and resistance to clinically important antimicrobial agents such as fluoroquinolones has also emerged. Antimicrobial resistance of NTS to Trimethoprim-sulfamethoxazole, ampicillin and chloramphenicol is documented in Sub-Saharan Africa . AntibioA total of 283 Salmonella typhimurium strains isolated from 187 cases of human infections and 96 non-human sources from the state of Sao Paulo, Brazil were examined for antimicrobial susceptibility and the incidence of resistance was 38% and multiple resistance  was 15%. All 43 multidrug-resistant strains (MDR) and 13 susceptible ones were characterized by phage typing and PFGE. Three were untypable (UT) and one untypable phage type was resistance to ampicillin, nalidixic acid and Streptomycin [In a report from Lagos, Nigeria of Salmonella-associated community gastroenteritis out of nineteen Salmonella typhimurium including three sporadic isolates, the three sporadic strains was untypable . All theThe isolate in our case report was in addition resistant to nalidixic acid and cotrimoxazole limiting the use of these agents for the management. Third-generation cehalosporins have become the treatment of choice for pericarditis caused by ampicillin-resistant strains . The gooWe report a rare case of Salmonella Typhimurium pericarditis from India. The aim is to highlight the potential of these bacteria to cause pericarditis with an aggressive course and rapid deterioration, thickening, and constriction requiring surgery. We also emphasize the importance of sending a diagnostic tap of the pericardial effusion and thus the impact microbiology culture has in the early and right choice of antibiotics. The virulence factors namely five fimbrial operons , three colonization factors  and other genes- pef operon,gog B,sseI,sodC, sopE might have played a role in pericarditis; though further studies are needed to conclude their role.The likelihood of a different etiological agent other than the original one should be kept in mind every time when there is worsening of symptoms even in an old case of constrictive pericarditis. The previously damaged pericardium was susceptible to seeding during a transient bacteraemia. In our case, NTS worsened the pre-existing pericarditis and led to hasten the need for a pericardiectomy which otherwise might have been done at a later stage. This is to the best of our knowledge, the first case reported of NTS pericarditis in a previous case of tubercular pericarditis. We speculate that the healed tuberculous pericarditis might provide a nidus for Salmonella infection, though this may not be conclusive as this being the only known case.In addition, the highly resistant pattern of NTS limited the therapeutic options as in our case to cephalosporins is the only option.


BRAFV600E mutation in brainstem neurohistiocytosis, where no lesional tissue was readily obtainable, using a cell-free DNA approach.To identify a treatment-responsive BRAFV600E mutation was performed. Response to conventional treatment (corticosteroids and interferon) and targeted treatment with a BRAF inhibitor was assessed by clinical evaluation, gadolinium-enhanced MRI brain scan, and serial testing of urinary cell-free DNA for mutant alleles.Cell-free DNA was extracted from urine and allele-specific PCR for the BRAFV600E mutation could be readily identified in urinary cell-free DNA at an allele frequency of 4.2%. Treatment of Erdheim-Chester disease with corticosteroids and interferon was ineffective and associated with disease progression. Treatment with BRAF inhibitors was associated with clinical improvement and near-complete radiologic remission. Following 6 months of BRAF inhibitor therapy, no enhancing lesions could be detected in the brain and mutant alleles were cleared from the urine.BRAFV600E mutations allows rapid noninvasive identification of a highly treatment-responsive pathway, leading to clinical and radiologic remission of disease. Our case demonstrates that this assay may have a particular role in challenging neurohistiocytosis cases, where attempts at obtaining lesional tissue have failed or are not feasible.Analysis of urinary cell-free DNA using allele-specific PCR for This study provides Class IV evidence. This is a single observation study without controls. As such, it is difficult to diagnose and can mimic diseases such as multiple sclerosisBRAFV600E mutations.5 These findings present the possibility of making a molecular diagnosis and using targeted therapies, sometimes with dramatic benefit.6–8 However, identification of such mutations requires the extraction of DNA from lesional tissue. In patients with deep-seated brainstem disease, this may not be an option.4 Even when multisystem disease is present, bone biopsy is often highly fibrotic and may not be adequately enriched for somatic mutation or yield DNA of sufficient quality.3 A recent report has highlighted the potential utility of cell-free DNA to deliver an accurate molecular diagnosis in histiocytosis.9 Cell-free DNA is released by diseased cells into the bloodstream and filtered into the urine, where it can be purified and analyzed for the presence of mutant alleles.9Recent studies have shown that ECD is a proliferative disorder of the myeloid lineage, with a high frequency of somatic BRAFV600E mutation can be identified by a simple and rapid urine cell-free DNA test in a particularly challenging CNS ECD case, where lesional tissue could not be readily obtained. The identification of the mutation predicted a near-complete response of brain lesions to BRAF inhibition after the failure of conventional therapy.We demonstrate that a highly treatment-responsive A 62-year-old man presented with polydipsia and cranial diabetes insipidus, associated with an isolated pituitary stalk lesion . At pres6 options for obtaining biopsy material to look for BRAFV600E mutation were reviewed. Given the deep location of the brain lesions, the risk of brain biopsy was considered high. Two biopsies of bone revealed no lesional tissue (table e-1 at Neurology.org/nn). The lack of other involved organs presented little option for obtaining fresh lesional material in which to identify targetable kinase mutations. We therefore performed allele-specific BRAFV600E PCR on urine and peripheral blood.9In light of recent evidence suggesting a possible role for BRAF inhibition in the treatment of histiocytic disorders,BRAFV600E Taqman Detection Assay  diluted into EBV-transformed B cells was used as a positive control. The clinical response to BRAF inhibition was monitored through clinical assessment, serial gadolinium MRI scan of the brain, and detection of mutant alleles in the urine using the urine cell-free DNA assay described above.Cell-free DNA was extracted from urine and plasma using QIAamp DNA micro and circulating nucleic acid kits  and subjected to allele-specific PCR using a BRAFV600E alleles , which is used as first-line therapy for ECD. The patient reported progression of brainstem symptoms during this time and his radiologic appearances worsened  but developed moderately severe liver toxicity within 2 weeks of starting treatment and was changed to dabrafenib 150 mg bd. Dabrafenib, an alternative oral BRAF inhibitor, was tolerated well. Within a month of starting treatment with BRAF inhibitors, the patient experienced resolution of dysarthria and saccadic abnormalities. His use of nocturnal desmopressin spray halved. At 3 months of treatment, MRI scan of the brain showed >90% reduction in enhancing lesions with a concomitant 90% reduction in urinary mutant allele count (BRAFV600E allele count was undetectable in urine (After detection of le count . At 6 moin urine . ClinicaConfirmation of the molecular diagnosis was eventually obtained from a surgical tibial bone biopsy that was analyzed fresh (10 and other neuroinflammatory/infiltrative disease.11 Such presentations are associated with a poor response to standard therapy.4 The recent identification of targetable somatic BRAF mutations in these disorders highlights the potential benefit for patients with these challenging orphan diseases. However, if this potentially transformative therapeutic approach is to be exploited, lesional tissue is required, which we show here can present a major challenge to clinicians. Even when lesional tissue can be obtained from bone, identification of mutant DNA can fail since bone lesions can be highly fibrotic and cellular content low.9Histiocytic disorders presenting with isolated CNS disease are an important differential diagnosis of multiple sclerosisBRAFV600E mutation where multiple invasive approaches to obtaining lesional tissue had failed. The identification of this mutation identified a near-complete response to BRAF inhibition, in contrast to conventional therapy, which had been associated with relentless disease progression. This neuroradiologic response was observed across all brain MRI sequences used to assess disease activity, including T1 with contrast, T2, and fluid-attenuated inversion recovery, and was associated with clinical improvement.We show that a simple rapid noninvasive urine test can guide treatment with a highly effective targeted therapy for deep neurohistiocytosis, where standard therapy has failed. In this case, this test reliably identified a somatic 3Importantly, the mutated allele frequency detected in the urine correlated with clinical and radiologic responses, suggesting that this may provide a further noninvasive modality for monitoring response to therapy. This may be particularly important in ECD, where repeat bone biopsy to monitor disease activity may be challenging because of its invasive nature and lack of reliable involvement.BRAFV600E was at least as sensitive as fresh lesional DNA. Our case also shows that although treatment with vemurafenib can cause sufficiently serious liver toxicity to lead to discontinuation of the drug, alternative BRAF inhibitors remain an efficacious option in patients with a somatic BRAF mutation.In this case, urine cell-free DNA testing for Therefore, urine cell-free DNA presents an option to diagnose and monitor actionable somatic mutations in neurohistiocytic disorders, especially when lesional tissue is not readily available.


Doppler images of pulmonary vessels in pulmonary diseases associated with subpleural consolidations have been described. Color Doppler easily identifies such vessels within consolidations while spectral Doppler analysis allows the differentiation between pulmonary and bronchial arteries. Thus, Doppler helps in diagnosing the nature of consolidations. To our knowledge, Doppler analysis of pulmonary vessels within anesthesia-induced atelectasis has never been described before. The aim of this case series is to demonstrate the ability of lung ultrasound to detect the shunting of blood within atelectatic lung areas in anesthetized children.Three anesthetized and mechanically ventilated children were scanned in the supine position using a high-resolution linear probe of 6–12 MHz. Once subpleural consolidations were detected in the most dependent posterior lung regions, the probe was rotated such that its long axis followed the intercostal space. In this oblique position, color Doppler mapping was performed to detect blood flow within the consolidation. Thereafter, pulsed waved spectral Doppler was applied in the previously identified vessels during a short expiratory pause, which prevented interferences from respiratory motion. Different flow patterns were identified which corresponded to both, pulmonary and bronchial vessels. Finally, a lung recruitment maneuver was performed which leads to the complete resolution of the aforementioned consolidation thereby confirming the pathophysiological entity of anesthesia-induced atelectasis.Lung ultrasound is a non-invasive imaging tool that not only enables the diagnosis of anesthesia-induced atelectasis in pediatric patients but also analysis of shunting blood within this consolidation.The online version of this article (doi:10.1186/s13089-016-0055-7) contains supplementary material, which is available to authorized users. Anesthesia-induced atelectasis is a well-known entity observed in approximately 68–100% of pediatric patients undergoing general anesthesia . The colLung ultrasound (LUS) has demonstrated its high sensitivity and specificity for diagnosing the entity of anesthesia-induced atelectasis in mechanically ventilated patients , 12. LUSThe use of Doppler for the study of pulmonary vessels within consolidated lung areas has already been reported by several authors –16. YuanEven though it has been known that intra-pulmonary shunting is the main reason for the deterioration of gas exchange in anesthesia-induced atelectasis, the visualization of shunting blood usually requires technologies such as PET, SPECT or arteriography. As opposed to these complex diagnostic tools, the examination of pulmonary vessels within consolidated lung areas by Doppler is simple, non-invasive, non-ionizing and perfectly suitable to assess shunt at the bedside , 15. Fur2O, an inspiration/expiration (I:E) ratio of 1:1 and FIO2 of 0.5. Respiratory rate was adjusted to keep end-tidal CO2 between 35 and 40 mm Hg.Three pediatric patients aged two months, one year and four years of age undergoing general anesthesia for abdominal laparoscopic surgery were analyzed. A protective ventilation strategy was applied using volume-controlled ventilation with a tidal volume of 6 ml/kg ideal body weight, a positive end-expiratory pressure (PEEP) of 5 cmHLUS assessment was performed with the portable echograph MicroMaxx  using a linear probe of 6–12 MHz. Patients were examined in the supine position placing the probe longitudinally as recommended by expert consensus . Given tSubpleural consolidations were identified as atelectasis when they were associated with the following signs : absenceAtelectasis should be distinguished from other types of consolidation like pneumonia and pulmonary embolism. Pneumonia appears as a consolidation with irregular and somewhat blurred margins, commonly associated with dynamic tree-shaped air bronchograms and pleural effusion. The sonographic signs of pulmonary embolism consist of generally two or more small pleural based, hypoechoic consolidations with sharp margins and without central vascularization. Importantly, these two kinds of consolidations cannot be reverted by a lung recruitment maneuver.Once subpleural consolidations were detected in the most dependent lung areas, the probe was rotated to overcome the acoustic shadows of the ribs and an oblique intercostal view was used for further analysis Fig. a, b.Fig.Color Doppler ultrasound detected pulmonary vessels within consolidated lung areas –16. MostA high-impedance flow signal. This flow pattern originates from a pulmonary artery because as activation of the local hypoxic pulmonary vasoconstriction reflex results in high impedance .A low-impedance monophasic flow signal that corresponded to bronchial arteries .We observed two different arterial flow patterns within atelectatic areas Fig. :Fig. 3Sp2O of PEEP that was then increased in 5 cmH2O steps until airway opening pressure of 30 cmH20 was reached. At this point, LUS images confirmed the resolution of atelectasis, with the subsequent improvement in lung aeration. Thereafter, a step-wise decrease in PEEP allowed the detection of the minimum level that prevented the reappearance of atelectasis seen in the LUS images. The complete resolution of dependent lung atelectasis by the recruitment maneuver was confirmed 5 min after this maneuver by longitudinal and oblique LUS examination in three regions in each hemithorax, anterior, lateral y posterior; leading to the diagnosis of anesthesia-induced atelectasis [Finally, we performed a lung recruitment maneuver as previously described , 20. Thelectasis   clinical, which is related to the potential analysis of blood flow in vessels within lung consolidations. Thus, color and spectral Doppler confirm the existence of shunt while the Doppler flow pattern could provide a general idea about the vascular origin of those vessels . The spectral Doppler analysis of pulmonary vessels could be useful to assess the impact of hypoxic pulmonary vasoconstriction on arterial oxygenation or right ventricular afterload. The role of Doppler analysis of pulmonary and bronchial flow within atelectasis should be evaluated in new protocols.This finding has two important implications: (1) Therefore, many questions arise: are these regional changes in vascular flow representative of the blood flow pattern within the main pulmonary arteries? Do atelectasis have negative effects on right heart function? Can blood flow throughout the shunt pathways be manipulated by vasoactive drugs? Surely, future studies should be conducted to answer these and many other related questions and to determine the potential clinical value of the presented Doppler-based shunt analysis.Most published evidence was obtained in spontaneously breathing adults and is thus directly related neither to mechanically ventilated nor pediatric patients. Recently, Mongodi et al.  publisheLung ultrasound can easily detect the presence of anesthesia-induced atelectasis and Doppler shunting within them in pediatric patients. Beyond the effects on gas exchange, the clinical impact of this shunting on pulmonary circulation must be analyzed in future studies.