Gestational weight gain (GWG) is an important modifiable factor known to influence fetal outcomes including birth weight and adiposity. Unlike behaviors such as smoking and alcohol consumption, the effect of GWG throughout pregnancy on fetal development and other outcomes has not been extensively studied. The aim of this study was to investigate the relationship of GWG with endocrine factors such as adiponectin, leptin, and C-reactive protein which may be associated with inflammatory response, fetal growth, and adiposity later in life. Data were obtained from the Ulm Birth Cohort Study (UBCS) and the Ulm SPATZ Health Study, two methodologically similar birth cohort studies including newborns and their mothers recruited from 11/2000–11/2001 and 04/2012–05/2013. In the two included birth cohorts we consistently observed statistically significant positive associations between GWG beginning as early as the second trimester with fetal cord blood leptin and stronger association beginning as early as the first trimester with post-delivery maternal serum leptin. Total weight gain exceeding commonly accepted recommended guidelines was consistently associated with higher leptin levels in both cord blood and post-delivery maternal serum. These results suggest a potential pathomechanistic link between fetal environment and surrogate markers of long-term health. Mounting evidence suggests fetal environment and nutrition may play a role in the origin of a number of chronic diseases manifesting later in life3Though a number of confounding factors such as offspring gender, parity, and smoking are known to influence fetal growth57One potential mechanism has been identified in studies observing associations between cord blood levels of the hormones adiponectin and leptin and pre- and post-natal growth. These hormones are involved in signaling pathways associated with growth, metabolism, and the immune system thus providing a possible link between fetal growth and disease111213161719The primary aim of this analysis was to investigate associations between GWG throughout pregnancy and both fetal (cord blood) and maternal (serum collected at birth) levels of adiponectin and leptin. As we have previously identified potential associations between leptin and birth-related factors including duration of laborData were obtained from the Ulm Birth Cohort Study (UBCS) and the Ulm SPATZ Health Study, two methodologically similar birth cohort studies including newborns and their mothers recruited from the general population shortly after delivery in the University Medical Center Ulm, Southern Germany, respectively from 11/2000–11/2001 and 04/2012–05/2013. Details can be found elsewhere22Exclusion criteria were outpatient delivery, maternal age <18 years, transfer of the newborn or the mother to intensive care immediately after delivery, and/or insufficient knowledge of the German (UBCS and SPATZ), Turkish, or Russian (both UBCS only) language. At baseline, the UBCS and SPATZ cohorts respectively included 1090 newborns of 1066 mothers  and 1006 newborns of 970 mothers . For the purposes of this analysis, the study populations were restricted to singleton full-term  newborns. In order to ensure comparability among results, we also restricted to mother-infant pairs for whom results were obtained for all three biomarkers in both cord blood and post-delivery maternal serum. Ethical approval was obtained from and all study protocols were carried out in accordance with guidelines approved by the ethics board of Ulm University . Participation was voluntary and written informed consent was obtained in each case. Exposure, outcome, and confounder definitions as well as statistical methods were identical for both studies unless specifically stated otherwise.Maternal demographic and lifestyle data including age at delivery, education (<12 years or ≥12 years), parity (first birth or greater), smoking history (within the year prior to pregnancy) were collected using a self-administered questionnaire during the hospital stay following delivery. Clinical data related to the child’s delivery including gender, birthweight, delivery mode, and duration of labor were obtained from electronic hospital records. Clinical data related to the mother’s pregnancy, including gestational weight measurements, were obtained from routine paper documentation known in Germany as “Mutterpass”, which obstetricians are required to issue to their patients when pregnancy is clinically established and which are generally updated at each clinical visit during pregnancy.Pre-pregnancy weight and height were estimated based on self-reported data documented in “Mutterpass” during the mother’s first obstetric appointment at which pregnancy was clinically established. Trimester specific and total weight gain were calculated using obstetrician-documented weight measured nearest to the end of the first (day 85), second (day 190), and third (day closest to full-term delivery) trimesters. Start of pregnancy was calculated as 280 days before the obstetrician reported full-term delivery date estimated at approximately 12 weeks gestation using fetal ultrasound measurements. As weights were seldom recorded on the trimester end-date, only weights recorded on the closest day within 14 days of the target date were used in the analysisCord blood was collected in S-Monovette 7.5 ml serum-gel tubes  by midwives or obstetricians shortly after delivery, centrifuged, and stored in a refrigerator until further processed for long-term storage at −80 °C by trained study personnel. In SPATZ, average time until long-term storage was 2.4 days (sd = 0.8 days); in UBCS the process was similar, but the time was not recorded. Maternal blood was collected during routine blood collection following delivery (after 24 h for most mothers). Adiponectin, leptin , and high-sensitivity CRP  concentrations were measured by ELISA . Biomarker measurements reported as below detection limit (SPATZ: n = 1) were imputed to the lower detection limit. Hs-CRP levels above 200 μg/L have been associated with amniotic fluid infection, therefore subjects with cord blood hs-CRP measurements above 200 μg/L were excluded from the analysisFor UBCS, adiponectin and leptin were measured in 06-07/2005 in Heidelberg, Germany, whereas hs-CRP was measured in 08-10/2009 in a research lab at Medical Center Ulm from continuously frozen stored material. For SPATZ, all three markers were measured in the latter research lab in cord blood and post-delivery maternal serum in 06-08/2013 with the same equipment and by the same technician as in UBCS. Hs-CRP levels differed between both studies with the whole distribution shifted by approximately 10 μg/L towards higher levels in UBCSTo assess the impact of missing data and restrictions, characteristics (proportions or means) of each study sample were compared to 95% confidence intervals of the characteristics within the corresponding full cohort. Chi-square and Kruskall-Wallis tests were performed to identify significant differences  in characteristics across study cohorts. Biomarkers were log transformed to account for right skewed distributions (leptin and CRP) or for comparability (adiponectin). Biomarker measurements outside 3 standard deviations from the underlying mean were considered potential outliers and excluded in sensitivity analyses. Linear regression models were used to estimate geometric means ratios (GMR) for the effect of a one standard deviation difference from the underlying mean trimester specific and total GWG and of a 5 kg change in difference from IOM recommended weight gain separately with each biomarker . Maternal age, education, parity, smoking history, and pre-pregnancy BMI, as well as, the child’s gender, birthweight, mode of delivery, and duration of labor were tested as putative confounding factors. As crude effects were small, putative confounders producing a 1% change in GMR after single adjustment of models measuring the effect of total GWG on any cord blood biomarker were selected for inclusion in adjusted models. All statistical analyses were performed by using SAS 9.4 .Following restriction to singleton full-term newborns, complete GWG and valid biomarker data were available for 540 UBCS and 412 SPATZ subjects . Due to 2) cohort compared to that observed in UBCS (16% of subjects).Overall, GWG trajectories were comparable between cohorts with the exception of obese SPATZ mothers who appeared to exhibit a lower weight gain trajectory following the first trimester . By delird trimester in SPATZ . Following adjustment for maternal smoking history and duration of labor, associations were slightly attenuated but remained significant (p < 0.05) in UBCS and for the 3in SPATZ . Pattern in UBCS . No assoth week of pregnancy compared to those classified as having “low” or “normal total pregnancy weight gain.To further explore strong associations observed between difference from IOM-recommended weight gain and leptin, we plotted weight gain trajectories for subjects who were categorized as below (“low”), within , or above (“excessive”) specified recommended total weight gain ranges 25. In boIn our analyses of two birth cohorts recruited from the general population with identical sampling and measurement methodologies and a time lag of 11 years, we observed significant positive association between GWG beginning as early as the second trimester and cord blood leptin. GWG beginning as early as the first trimester was associated with higher levels of post-delivery maternal serum leptin measured shortly after birth. Total weight gain exceeding IOM recommendations was consistently associated with higher leptin levels in both cord and post-delivery maternal serum. Finally, first trimester GWG was indicative of IOM total weight gain category. These results suggest GWG beginning as early as the first trimester might influence expression of leptin in the fetus, the newborn and possibly later in life. Furthermore, lack of consistent association between GWG and hs-CRP suggests the association with leptin was not likely associated with the inflammatory response at birth.Though we identified two previous studies reporting similar findings with cord blood outcomes, we believe we are among the first to concurrently report on associations between total and trimester-specific GWG and adiponectin, leptin, and hs-CRP levels in both cord blood and post-delivery maternal serum. Our results support those of the Rhea group, who have previously identified similar positive associations between IOM-recommended GWG and cord blood leptin27rd trimester16Interestingly, we observed similar but stronger patterns of association between GWG and leptin levels measured in post-delivery maternal serum compared to those observed in cord blood. Maternal serum leptin increases beginning in early pregnancy before peaking in the 3in-vitro study has demonstrated the possibility of cross-placental transfer of circulating leptin in human choriocarcinoma BeWo cellsThough we observed correlation between fetal and maternal leptin levels in our cohorts , it is cMaternal leptin levels in pregnancy have also been associated with pregnancy-related health conditions including gestational diabetes and pre-eclampsiaThe key strength of our study was our ability to replicate results in two geographically and methodologically similar but demographically different cohorts. Despite the demographic differences, GWG, biomarker outcomes, and overall results were similar to one another. However, it should be noted that point estimates for both cord blood and post-delivery maternal serum leptin were consistently higher in the UBCS cohort. Though we were unable to identify a specific reason for this difference, factors associated with differences between studies in GWG trajectory amongst obese mothers may warrant further exploration.As with other studies of this nature, some key limitations must also be acknowledged. First, approximately 31% of eligible UBCS mothers and 43% of eligible SPATZ mothers were excluded from our analyses due primarily to restriction to mothers with gestational weight measurements reported within 14 days of the end of each trimester and delivery. In most cases, these subjects were missing first trimester weight measurements which may have been due to a number of factors, including late establishment of pregnancy. However, no significant differences in demographic or lifestyle factors were observed due to missing data. Furthermore, several sensitivity analyses in which we included all available data for each model, less stringent restriction, and/or wider acceptable date ranges for trimester-specific weight gain estimation produced similar point estimates to reported results. Second, biomarkers were only measured upon (cord blood) and after  delivery and post-delivery maternal serum collection time varied. As GWG was only marginally associated with hs-CRP in UBCS cord blood and not associated with hs-CRP in maternal serum, it is unlikely more proximal factors at birth, such as duration of labor, affected levels of the other biomarkers. Nevertheless, we were unable to specifically determine precisely when and how biomarkers may have changed over the course of pregnancy or the possibility of reverse causality. Lastly, although specific timing of post-delivery maternal serum collection was unavailable in UBCS, we observed a weak but significant correlation between duration from delivery to blood collection and maternal adiponectin and leptin respectively in SPATZ. Adjustment for time to collection did not affect SPATZ post-delivery maternal serum model results.Despite certain limitations, our results from two independent cohorts provide strong evidence supporting an association between GWG and fetal-maternal leptin. Although GWG is likely only one of a number of factors associated with leptin synthesis or expression, our results are in line with the idea that GWG may be an important exposure during early fetal development which may potentially influence long-term health in offspring. Therefore, further research may be warranted to prove and better understand causality of the association and the potential effects on fetal development, as well as the long-term consequences of hypo- and hyper-leptinemia during gestation.How to cite this article: Logan, C. A. et al. Gestational Weight Gain and Fetal-Maternal Adiponectin, Leptin, and CRP: results of two birth cohorts studies. Sci. Rep.7, 41847; doi: 10.1038/srep41847 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Stimulants, including amphetamines, are widely abused in the United States and worldwide. Amphetamine acts on the dopamine transporter to elevate and prolong extracellular dopamine levels in the nucleus accumbens that mediate the drug’s rewarding effects. Repeated exposure to amphetamine has profound effects on the plasticity of medium spiny neurons (MSNs) in the nucleus accumbens that are thought to contribute to long-lasting behavioral alterations underlying amphetamine abuse. MSNs make up the vast majority of the neurons in the nucleus accumbens, but their activity is regulated by several types of interneurons, including fast-spiking parvalbumin expressing (PV+) interneurons. Although PV+ interneurons make up only 1–2% of neurons in the nucleus accumbens, they provide powerful feed-forward inhibitory control of MSN firing. Although their effects on nucleus accumbens electrophysiology are well understood, the role of accumbens PV+ interneurons in behavior has been far less explored.Neuropsychopharmacology, Pvalb-T2A-Cre mice to selectively silence PV+ interneurons with tetanus toxin light-chain (TeLC) in the nucleus accumbens. Tetanus toxin light-chain cleaves synaptobrevin, eliminating vesicle release and effectively turning off PV+ interneuron signaling. Silencing these interneurons profoundly disrupted both locomotor sensitization and CPP for amphetamine.In this issue of One goal of the study by Furthermore, Inhibition of PV+ interneuron signaling was also associated with a large increase in Fos expression in the nucleus accumbens, confirming that PV+ neurons in the nucleus accumbens regulate the activity of MSNs. Increased Fos expression in the nucleus accumbens has previously been associated with locomotor sensitization to amphetamine . The authors declare no conflict of interest.


Task-specific rehabilitative training is commonly used for chronic stroke patients. Axonal remodeling is believed to be one mechanism underlying rehabilitation-induced functional recovery, and significant roles of the corticospinal pathway have previously been demonstrated. Brainstem-spinal pathways, as well as the corticospinal tract, have been suggested to contribute to skilled motor function and functional recovery after brain injury. However, whether axonal remodeling in the brainstem-spinal pathways is a critical component for rehabilitation-induced functional recovery is not known. In this study, rats were subjected to photothrombotic stroke in the caudal forelimb area of the primary motor cortex and received rehabilitative training with a skilled forelimb reaching task for 4 weeks. After completion of the rehabilitative training, the retrograde tracer Fast blue was injected into the contralesional lower cervical spinal cord. Fast blue-positive cells were counted in 32 brain areas located in the cerebral cortex, hypothalamus, midbrain, pons, and medulla oblongata. Rehabilitative training improved motor performance in the skilled forelimb reaching task but not in the cylinder test, ladder walk test, or staircase test, indicating that rehabilitative skilled forelimb training induced task-specific recovery. In the histological analysis, rehabilitative training significantly increased the number of Fast blue-positive neurons in the ipsilesional rostral forelimb area and secondary sensory cortex. However, rehabilitative training did not alter the number of Fast blue-positive neurons in any areas of the brainstem. These results indicate that rehabilitative skilled forelimb training enhances axonal remodeling selectively in the corticospinal pathway, which suggests a critical role of cortical plasticity, rather than brainstem plasticity, in task-specific recovery after subtotal motor cortex destruction. Stroke is a major cause of disability worldwide and rehabilitation is commonly used to treat chronic stroke patients. Disability of the upper extremities is a common impairment experienced by a large majority of stroke survivors. Many forms of rehabilitative therapy, especially task-specific training, have long been used to improve upper limb dexterity following stroke. In the recent guideline for stroke rehabilitation, task-specific training is still considered to be a more beneficial and reliable form of therapy than newly developed rehabilitative methods such as transcranial magnetic stimulation, transcranial direct current stimulation, robotic therapy, or virtual reality . FurtherAfter stroke, surviving neurons undergo morphological alterations such as axonal remodeling, dendritic arborization, and synapse formation to compensate for lost functions. Through these morphological changes, surviving neurons modify their innervations and reorganize remaining neural networks. The functional contribution of the newly formed connections has recently been proven using pathway-specific silencing techniques –5. BecauThe goal of the current study was to investigate the effect of rehabilitative training on axonal remodeling in the brainstem-spinal pathways during rehabilitation-induced functional recovery. We carried out rehabilitative training using a skilled forelimb reaching task (task-specific training) after subtotal destruction of the caudal forelimb area (CFA) in the primary motor cortex and analyzed the rehabilitation-induced alterations in the brainstem-spinal projections using a retrograde tracing method.ad libitum weight. Water was available ad libitum. All experimental procedures were in accordance with National Institutes of Health regulations and approved by the Animal Research Committee of Kawasaki Medical School.We used a total of 44 adult male Fisher 344 rats  for the experiments. We chose to conduct our experiments in Fisher 344 rats, because neural network remodeling and motor map reorganization induced by rehabilitative training have been well investigated in this strain –15. EachWe investigated the effects of rehabilitative skilled forelimb training (Rehab) on functional recovery and axonal remodeling in the descending spinal pathways after photothrombosis (PT) . Rats weThe skilled forelimb reaching task was carried out as previously described , with a The staircase test was used to assess non-trained skilled forelimb motor performance . The staThe ladder walk test was used to assess precise limb placing and stepping. Rats were tested for their ability to walk on a horizontal ladder with irregular spacing (1–3 cm) between rungs following a previously described procedure . AnimalsThe cylinder test was used to assess asymmetries in forelimb use for postural support during rearing . Rats wePhotothrombotic stroke was induced as described previously  in the CRats were anesthetized and maintained under a constant rate infusion of ketamine hydrochloride  via tail vein catheter using a syringe pump . After skin incision and muscle separation from the vertebrae, a laminectomy was performed at the cervical spinal level from C5 to C7. Rats were fixed to a stereotactic frame  by holding the dorsal processes at C4 and T1. The dura was then carefully opened and folded aside. Fast blue  was injected into the spinal cord using a 34G beveled needle  and a 10 μl syringe driven by an electric pump with a flow rate of 200 nl/min. Five injections of Fast blue (80 nl/injection) were made in the contralesional side of the cervical spinal cord  and transcardially perfused with 200 ml of 0.01 M phosphate-buffered saline, followed by 200 ml of 4% phosphate-buffered paraformaldehyde. After cryoprotection with 10%, 20%, and 30% sucrose, brains were embedded in Tissue-Tec  and rapidly frozen in a dry ice/ethanol bath. Brain and spinal cord tissues were cut into 20 μm thick coronal sections on a cryostat. Brain sections were prepared in 500 μm intervals from the rostral-most tip to the caudal end of medulla oblongata. The spinal cord sections were prepared at the C7 and C8 levels for injection validation. Frozen sections were mounted on hydrophilic glass  and coverslipped with Fluoromount .Images of the spinal cord sections at the C7 and C8 levels were taken using an all-in-one fluorescence microscope  and leakage of the tracer was carefully checked. Rats were excluded if leakage of the tracer was observed in the ipsilesional side of the spinal cord or in any part of the white matter. To confirm equivalent injections in the two groups, the diffusion of the tracer was examined by measuring the areas with increased background signals (intensity threshold 180–255) using imageJ software . The coordinates of the tracer diffusion centroid with the origin located at the center of the central canal were also measured.Images from 39 sections at 500 μm intervals were taken using an all-in-one fluorescence microscope . Images were tiled and stitched using automatic software . Images were analyzed with MetaXpress Imaging Analysis V2.0  to count the number of Fast blue-positive cells. The number of cells was multiplied by the ratio of section thickness and the number of intervals, allowing for an estimation of the number of cells in the whole brain.All data are reported as mean ± standard deviation (SD). Unpaired two-tailed t-tests were performed to compare the injection diffusion, the number of Fast blue-positive cells in each brain area and behavioral measurements in the final session of the skilled forelimb reaching test. We compared behavioral performance in multiple groups at different time points using two-way repeated measures analysis of variance (ANOVA). For post-hoc testing, Tukey’s multiple comparison tests were used to compare the differences between the different time points and Sidak’s multiple comparison tests were used to detect group differences at each time point. A statistically significant difference was defined as a P value less than 0.05. Nonsignificant tendencies were indicated as (not significant but p < 0.1). All statistical analyses were carried out using GraphPad Prism Version 7.0 .In this study, a total of 44 rats were used. Following the behavioral procedures, a total of 13 rats were excluded from the study because of a pre-infarct motor performance less than 30% (n = 11), or a post-infarct motor performance greater than 50% (n = 2). Thus, 31 rats were included in this study . For some rats, we could not obtain usable data for statistical analysis from the cylinder test , ladder walk test (no attempt to cross the ladder: n = 2), or staircase test . In the Fast blue tracer studies, nine rats were excluded because tracer leakages were observed in the ipsilesional spinal cord or the white matter.To investigate the effect of rehabilitative skilled forelimb training on functional recovery following stroke, motor performance was assessed using various motor tests. Cylinder tests and ladder walk tests were conducted for the evaluation of general motor performance. These tasks can be executed with little habituation  or without any training (cylinder test). The skilled forelimb test and staircase test were used to assess skilled motor performance. Performance of these tests requires a substantial training period (greater than 2 weeks).In the cylinder test  or C8 level . Similarly, the lateral and dorsal coordinates of the injection centroid were not significantly different between the groups at either the C7  or C8 level .Following a 4-week recovery period, all rats were injected with Fast blue in the contralesional lower cervical spinal cord (C7-8). The injection site was carefully checked and rats were excluded if leakage of the Fast blue injection was observed in any part of the white matter or on the contralateral side of the injection . Because each descending spinal pathway enters the gray matter of the spinal cord from different parts of the white matter, both the volume and location of the injection can affect the number of the Fast blue labeled cells in each brain area. Thus, we confirmed whether an equivalent volume of tracer was injected into the same area of the spinal cord for each group  forelimb function in the staircase test. Several studies have reported impaired ipsilesional motor performance in stroke patients , 26. TheSince each descending spinal pathway passes through a different part of the spinal white matter and has different terminal distribution patterns in the spinal grey matter , both thIn the analysis of the brainstem, we did not observe a significant difference in the number of Fast blue-positive cells in any areas. We analyzed 26 areas in the brainstem and did not find even a weak tendency towards change . These results indicate that rehabilitative skilled forelimb training promotes axonal remodeling in the corticospinal pathway but not in the brainstem-spinal pathways. Axonal remodeling in the brainstem-spinal pathways has been reported in spontaneous recovery after spinal cord injury  and stroIn the current study, rehabilitative training was carried out for 4 weeks, beginning on day 4 after stroke induction. A great deal of evidence from animal  and clinIt should be pointed out that we cannot exclude the functional contribution of the brainstem-spinal pathways. Studies using retrograde tracers cannot rule out axonal remodeling and synapse formation in the neurons that originally had neural projections to the injection site. Moreover, rehabilitative training may modify the function of the brainstem-spinal pathways by increasing the neural input from the cerebrobulbar pathways , 5. HoweIn conclusion, rehabilitative skilled forelimb training induces task-specific functional recovery and promotes axonal remodeling selectively in the corticospinal pathway. Our results suggest that cortical plasticity, rather than brainstem plasticity, plays a dominant role in task-specific recovery induced by rehabilitative training after subtotal destruction of the motor cortex.


There has been increasing interest and research into sarcopenia in community-dwelling older adults since the European Working Group on Sarcopenia in Older People (EWGSOP) agreed a consensus definition in 2010. Sarcopenia has been defined as loss of muscle mass with loss of muscle function , with measurements two Standard Deviations (SDs) below the mean of a young reference population. This definition does not necessitate longitudinal measurements, or the absence of acute illness and diagnosis can be made from single measurements. We hypothesise that hospitalisation, due to a combination of acute inflammatory burden and muscle disuse, leads to an acute decline in muscle mass and function and may lead to some individuals meeting criteria for sarcopenia, acutely, based on the EWGSOP definition. This may be partially recoverable or may lead to increased risk of developing sarcopenia long-term. We have denoted the term “acute sarcopenia” to refer to acute loss of muscle mass and function associated with hospitalisation. This review discusses some of the current available research in this context and also identifies some of the knowledge gaps and potential areas for future research. Sarcopenia refers to age-related loss of skeletal muscle mass and function; the term originates from Greek, meaning literally “loss of the flesh”. The European Working Group on Sarcopenia in Older People (EWGSOP) , InternaThe presence of sarcopenia has been demonstrated to relate more closely to adverse outcomes than reduced muscle mass alone . The EWGSarcopenia is distinct from but related to the diagnosis of frailty , 10. ThiThe EWGSOP refers to the principle that both primary and secondary forms of sarcopenia exist; primary sarcopenia has no other cause other than cumulative insults over time associated with ageing, whereas, secondary sarcopenia may be disease-related or nutrition-related . The AWGThe frequency of acute sarcopenia is currently unknown, but it is known that a higher proportion of individuals will meet criteria for sarcopenia at any one time in hospital compared to in the community. Acute sarcopenia is a significant problem and important area for future research. It is likely to be associated with increased financial cost secondary to increased length of stay, rehabilitation needs and increased social care needs , 17. It The prevalence of sarcopenia amongst older community-dwelling adults, using EWGSOP criteria, has been variably reported in 15 studies in economically developed countries as between 1-29%, in a recent systematic review . PrevaleThe precise mechanisms that lead to sarcopenia are only partially understood. However, it has been hypothesised to be related to an accumulation of multiple insults over time . ReducedAlthough individual differences exist and in particular a high level of physical activity in adulthood can prevent sarcopenia , a decliInflammatory cytokines such as Interleukin 6 (IL-6) and Tumour Necrosis Factor Alpha (TNF-α) have been proposed to contribute to catabolism in skeletal muscle. Inflammatory cytokines activate Nuclear Factor Kappa-light-chain-enhancer of activated B cells (NFκB) and Forkhead box O (FoxO) transcription factors in muscle resulting in proteolysis via induction of atrogenes . Raised Endocrine and inflammatory status interact to modulate muscle mass. Cortisol levels have been demonstrated to increase slightly with age, which in turn is associated with muscular weakness. A cohort study of adults aged 65 and older in the Netherlands demonstrated that increased salivary but not serum concentrations of cortisol were associated with reductions in grip strength . 11β-hydAdditional endocrine regulation occurs through growth hormone (GH) which induces insulin-like growth factor 1 (IGF-1) production in the liver. Both insulin and IGF-1 receptors are upstream of phosphatidylinositol 3 kinase/ protein kinase B (PI3K/AKT) pathway activation and can trigger protein synthesis. The PI3K/AKT pathway stimulates protein synthesis via activation of glycogen synthase kinase 3 (GSK-3) and mechanistic target of rapamycin (mTOR) pathways and prevents protein degradation via the FoxO pathway .Vitamin D has also been implicated in modulation of skeletal muscle mass. Muscle biopsies taken from patients with vitamin D deficiency have demonstrated atrophy of Type II muscle fibres . AdditioAn increased risk of hospitalisation amongst individuals with sarcopenia compared to those without sarcopenia has been reported in a longitudinal study in the USA . SimilarFew studies have assessed sarcopenia criteria during the acute phase of an illness. A prospective observational study involving 103 older adults admitted to geriatric medicine wards, who had evidence of or who were felt to be at risk of malnutrition, demonstrated a prevalence of sarcopenia of 21.4%. Notably, 22.3% of patients recruited to this study were unable to perform both the gait speed test and handgrip measurements due to acute illness. An association was seen between sarcopenia and increased mortality; further research is needed to assess the cause for this association .The presence of sarcopenia is associated with increased costs and mortality in general, vascular and liver transplant surgery specialties . ReducedLoss of lean leg mass during periods of bed rest in otherwise healthy adults has been extensively reported , 18, musIn rodents, skeletal muscle unloading has been most commonly modelled using hind limb immobilisation. The molecular mechanisms that have been implicated in the development of disuse muscle atrophy are the Atrogin-1/ Muscle atrophy F-Box (MaFbx)/ Muscle ring finger 1 (MuRF1) pathway, the IGF-1-AKT-mTOR pathway and the Myostatin pathway . MaFbx aImmobilisation studies in humans have shown that the mechanisms involved include induction of the atrogenes MuRF1, Foxo3 and atrogin 1, with just 8 days of immobilisation leading to a 51% increase in MuRF1 protein .Hypercortisolaemia has been demonstrated to exacerbate loss of muscle mass and muscle strength associated with bed rest. A controlled study involving timed hydrocortisone administered to healthy young men over a 28 day period of bed rest demonstrated greater loss of lean leg mass compared to a bed rest only model . CortisoAs described above the development of sarcopenia is positively related to oxidative stress  and inflIntensive Care Unit-Acquired Weakness (ICU-AW) is a recognised complication following admission to the Intensive Care Unit (ICU). ICU-AW is an all-encompassing term including critical illness myopathy (CIM), critical illness polyneuropathy (CIP) and critical illness neuromyopathy (CINM) . ProlongThe presences of sepsis or Systemic Inflammatory Response Syndrome (SIRS) criteria are known risk factors of ICU-AW. Local inflammation is found in ICU-AW, although specificity is unknown. Fourteen studies examining the presence of inflammatory cells within human muscle biopsies demonstrated a positive finding in 25% of cases (95% CI 18-34) . One stuICU-AW affects comparatively younger individuals than age-related sarcopenia, even though these patients may not have been exposed to multiple previous episodes of oxidative stress or inflammatory insults. However, age is a risk factor for ICU-AW, a USA study reported a mean age of 59.73 years in those with ICU-AW compared to 49.98 in those without . Frail oCurrent available research suggests that hospital admission is likely to have a negative impact upon muscle mass and muscle function . This leWe define acute sarcopenia as changes in muscle mass and muscle function within 28 days of a significant physiological stressor event, such as an acute illness, surgery, trauma or burns , 90, sufAs a specific example, sepsis is a life-threatening condition characterised by the presence of infection associated with organ dysfunction. Survivors of severe sepsis are at an increased risk of developing new cognitive impairment or functional disability long-term . Sepsis As described, chronic age-related sarcopenia is associated with progressive decline over time secondary to an accumulation of insults . We hypoIdentifying risk factors for acute sarcopenia may be important in preventing these long-term sequelae. Risk factors are likely to include, but not be confined to conditions that lead to endocrine dysregulation, increased inflammatory state and/or result in reductions in mobility; delirium , sleep dHowever, long-term data relating to outcomes following episodes of acute loss of muscle mass and function are currently lacking. There is some evidence that reduced signalling through anabolic pathways is of benefit with regards to reducing cancer risk and increasing longevity. Clearly, during acute illness upregulation of catabolic pathways helps to produce energy to overcome illness. Loss of muscle mass and function may be a trade-off of this. Further research is needed in the form of observational studies and subsequent translational studies to assess potential targets and long-term risk from treatments.During hospital admission bed rest should be minimalised where possible . This haThere is increasing evidence that early mobilisation and intensive physical activity regimes can help to improve outcomes. A recently published trial involving 92 non-delirious older medical inpatients demonstrated increased mobility at one month follow-up in those randomised to an inpatient mobility program compared to those who received usual care; no differences in ADL performance were reported . A previResistance exercise training in combination with moderate aerobic exercise have been shown to be beneficial in improving muscle mass and function in chronic sarcopenia . ResistaNutritional interventions have been trialled in chronic sarcopenia and there is increasing evidence that older adults have higher protein requirements than younger adults, with further increased requirements during acute illness . LeucineThere are clear health benefits for promotion of physical activity and nutritional optimisation. However, further research is needed to clarify appropriate dosages, duration, intensity and timing that provide economic as well as individual benefits.Neuromuscular Electrical Stimulation (NMES) involves the application of electrical current to stimulate muscular contraction and has been trialled in situations where mobilisation is not possible, such as in the ICU setting. A recent randomised controlled trial involving critically ill patients following cardiothoracic surgery demonstrated no effect of NMES applied bilaterally to quadriceps on muscle layer thickness, although patients regained muscle strength faster than the control group. The mean age in the NMES group was 63.3, compared to 69.7 in the control group . ConversNovel pharmacological agents for treatment of sarcopenia include calorie restriction mimetics (CRMs) and exercise mimetics (EMs); these are phytochemicals such as resveratrol with antioxidant and regenerative properties that partially mimic the molecular pathways leading to the favourable effects of calorie restriction and physical exercise . If provRecombinant growth hormone administration has previously been trialled with conflicting results. Growth hormone supplementation can increase skeletal muscle mass, but this is rarely accompanied by improved muscle strength and side-effects of this treatment are prevalent in older adults . TestostAcute sarcopenia secondary to hospital admission is hypothesised to be a related but distinct condition from chronic age-related primary sarcopenia. Acute illness or surgery lead to an acutely heightened inflammatory burden, which, coupled with reduced physical activity and muscle disuse, can lead to a reduction in muscle mass and function. The natural history of this condition needs further evaluation, including factors that affect rate and extent of development of this condition and long-term outcomes.It is unclear to what extent acute sarcopenia differs from chronic age-related sarcopenia in terms of biological and structural changes, physical phenotyping and long-term outcomes. Further research is needed on this subject, initially to demonstrate the association between hospitalisation, acute illness or surgery and sarcopenia and, subsequently, to examine long-term outcomes and the effect of early interventions including physical therapy, nutritional supplementation or using novel therapeutic targets. In-vivo physiology and muscle biopsy studies will be vital in assessing mechanisms driving acute sarcopenia.Acute sarcopenia should be considered as a separate entity to chronic age-related sarcopenia, much in the same way that acute kidney injury is considered a separate disease to chronic kidney disease. The same criteria may be met for the two conditions, but it is the timing and natural history of the conditions that differ. However, considering the analogy above, there are likely to be overlap between the two conditions, with chronic sarcopenia being a possible risk factor for acute sarcopenia and the development of acute sarcopenia potentially increasing the long-term risk of chronic sarcopenia.The presence of low muscle mass and function during acute illness is associated with increased health economic costs in terms of increased length of hospital stay, rehabilitation costs and the need for institutional care or social care on discharge. Preventing acute sarcopenia will have wider economic benefits as well as individual benefit to patients.Initial research should determine the feasibility of conducting research on this condition in the acute care setting. The aim is that observational studies should be conducted within the next five years to include evaluation of histological changes and biological mechanisms. Focus on specific postulated risk factors, such as delirium will guide future treatment. Interventional studies involving nutritional, physical activity and other interventions should continue concurrently with assessment of general health and economic benefits. Prevention and early treatment of acute sarcopenia may help to prevent older adults from meeting criteria for sarcopenia in the long-term.


Sample pooling enabled by dedicated indexes is a common strategy for cost-effective and robust high-throughput sequencing. Index misassignment leading to mutual contamination between pooled samples has however been described as a general problem of the latest Illumina sequencing instruments utilizing exclusion amplification. Using real-life data from multiple tumour sequencing projects, we demonstrate that index misassignment can induce artefactual variant calls closely resembling true, high-quality somatic variants. These artefactual calls potentially impact cancer applications utilizing low allelic frequencies, such as in clonal analysis of tumours. We discuss the available countermeasures with an emphasis on improved library indexing methods, and provide software that can assist in the identification of variants that may be consequences of index misassignment. As of now, next-generation sequencing technologies enable affordable assaying of variation in the entire tumour genome within days, but despite the availability of specialized software tools, somatic variant calling continues to pose challenges. Notably, the inherent complexity of tumours, as exemplified by aneuploidy, tumour heterogeneity, and sample impurity, often leads to important somatic variants only being detectable in low allelic fractions (AFs)7. AFs can be further affected due to technical reasons, such as the inability to accurately represent allelic ratios at genomic loci with low coverage8. As a consequence of the necessarily high required sensitivity, somatic variant calling is susceptible to random noise, systematic artefacts, and sample contamination10. When unnoticed, false positive variant calls can contribute to high costs incurred by follow-up analyses and experiments, and in the worst case support inadequate therapeutic strategies.Identification of somatic variants by next-generation sequencing has become an important technique in cancer research by pinpointing the genomic causes of tumour phenotypes. An increasing number of examples have further shown that genomic aberrations have prognostic value and can inform rational clinical deployment of targeted cancer drugs11, when material from multiple samples is being pooled together before sequencing, the sequencing technology itself can be a source of noticeable sample cross-contamination. Sample pooling  is a standard means of dividing the throughput of a sequencing instrument among multiple samples, relying on index sequences that uniquely barcode the material of each involved sample. Sample index misassignment, a phenomenon most evident on Illumina instruments utilizing exclusion amplification chemistry (ExAmp) and patterned flow cell technology , effectively leads to transfer of individual sequencing reads between samples included into a common pool11.As previously described in the context of RNA-seq applicationsOur analysis of high-coverage tumour sequencing data shows that index misassignment is a source of false positive somatic variant calls in a form of true variation obtained from co-multiplexed samples.http://cancergenomics.no) .We investigated tumour-normal sample pairs of three different tumour types: diffuse large B-cell lymphoma, follicular lymphoma, and sarcoma. All samples were collected from cancer patients in Norway, and deep exome sequencing  was carried out on local Illumina instruments: three HiSeq 2000/2500 instruments utilizing bridge amplification (generating 42 diffuse large B-cell lymphoma samples and 31 sarcoma samples) and a HiSeq 4000 employing ExAmp (generating 81 follicular lymphoma samples and 77 sarcoma samples). All samples were subject to standardized library preparation, sequencing, and bioinformatics analysis, as adopted by the Norwegian Cancer Genomics Consortium . For increased reliability, all tests and analyses were limited to SSNVs agreed upon by two independent variant callers (MuTect and Strelka)A schematic overview of all conducted experiments and their data dependencies is available on Fig. https://www.illumina.com/science/education/minimizing-index-hopping.html), but for the 16 libraries included in our testing, performing a gel-based library purification step in combination with bead purification did not provide improvements over bead purification alone . In accordance with previous reports by Illumina and Sinha et al., we concluded that ExAmp chemistry is the cause of sample contamination. The dependency on sample pooling indicated that co-multiplexed samples serve as the contaminants.During the analysis of hundreds of sequenced tumour-normal matched pairs, we noted that data generated on the ExAmp instrument showed significantly higher sample-wise contamination levels in comparison to data from bridge amplification instruments  . If control samples have been contaminated by true somatic variation, it might be necessary to adopt variant caller settings more permissive to somatic allele evidence in the matched normal material.For data that have already been generated, suitable variant post-processing should be chosen based on the type of contamination artefacts relevant for given project Table . CommonlWe believe our findings to be of relevance to other cancer sequencing projects that utilize the ExAmp chemistry, even though the impacts of index misassignment on somatic variant calling may vary depending on the combination of employed sequencing instruments, library preparation protocols, and bioinformatics analyses. In general, we expect the effects of index misassignment to dampen as the sequencing depth decreases and the allelic fraction threshold for accepted somatic variants increases. On the other hand, we note that besides single nucleotide variants, other types of somatic variation (e.g. insertions and deletions) are likely to be affected.Methods used for sample collection, DNA extraction, library preparation and sequencing, as well as full details of all bioinformatics analyses, are available in the methods section of Supplementary Information.18, Picard (http://broadinstitute.github.io/picard/) and GATK19 tools, before performing somatic calling with MuTect and Strelka . All analyses were performed using human reference genome build b37 with an added decoy contig and corresponding variation databases.All sequenced samples were pre-processed with BWA MEMThe pool complement of each individual tumour sample was formed by reads of all co-multiplexed samples originating from other individuals. For each identified somatic variant in a given tumour sample, all overlapping reads from the corresponding pool complement were extracted, and the pool complement allelic fraction  was calculated. Tumour samples without a matched control and/or tumour samples failing our quality metrics thresholds (Supplementary Information) were excluded from the analyses of potential contamination targets, but did serve as potential contamination sources in our PC-AF calculations.For evaluating the role of multiplexing in ExAmp-associated index misassignment, tumour-normal sample pairs from 8 selected individuals were sequenced (i) individually (one sample per dedicated flow cell lane), (ii) in a pool of all 8 tumour/normal samples and (iii) in a pool of all 8 tumour/normal samples after applied gel purification. For generating contamination estimates with Conpair, the separately sequenced normal sample of each individual was used as a control for the five other samples of given individual.Accuracy of Conpair contamination estimates was assessed with the help of artificially created exome-wide sample admixtures. Reads from multiple libraries were mixed to simulate contamination by 1, 2, 4 or 7 samples from as many different individuals, with the total contamination amounting to either ~2% or ~8% in each case, depending on the experiment. Conpair’s contamination estimates were compared to true contamination content, which was validated by library-specific depth of coverage calculations.NCGC’s cohort of normal samples consisted of blood samples of 789 different individuals living in Norway. The samples were processed according to “Best Practices for Germline SNP & Indel Discovery in Whole Genome and Exome Sequence” developed by the Broad Institute, utilizing allele-specific (rather than site-specific) variant calling.All statistical tests were performed in R (http:/www.r-project.org) as two-tailed non-parametric tests (either Wilcoxon rank sum test with continuity correction or Wilcoxon signed rank test) of the identity of two populations. All input test data are available as Supplementary Data. R session information, utilized R commands together with their outputs, as well as median and interquartile range values of all the tested distributions are available in the Supplementary Note. All distributions have been plotted as Supplementary Figures.All patients provided informed consent. The study was approved by the Regional Committee for Medical and Health Research Ethics of South-East Norway . All experiments were performed in accordance with relevant guidelines and regulations.All data that support the findings of this study and that do not compromise research participant privacy are available as Supplementary Data. All data that may compromise research participant privacy  are available for inspection upon request to the corresponding author [EH] on non-disclosure terms.https://github.com/danielvo/IBPC). R code for generating Figures Python code used for calculating PC-AF values is available at GitHub (Supplementary noteSupplementary Dataset Number 1Supplementary Dataset Number 2Supplementary Dataset Number 3Supplementary Dataset Number 4Supplementary Dataset Number 5Supplementary Dataset Number 6


It is an important question how human beings achieve efficient recognition of others’ facial expressions in cognitive neuroscience, and it has been identified that specific cortical regions show preferential activation to facial expressions in previous studies. However, the potential contributions of the connectivity patterns in the processing of facial expressions remained unclear. The present functional magnetic resonance imaging (fMRI) study explored whether facial expressions could be decoded from the functional connectivity (FC) patterns using multivariate pattern analysis combined with machine learning algorithms (fcMVPA). We employed a block design experiment and collected neural activities while participants viewed facial expressions of six basic emotions . Both static and dynamic expression stimuli were included in our study. A behavioral experiment after scanning confirmed the validity of the facial stimuli presented during the fMRI experiment with classification accuracies and emotional intensities. We obtained whole-brain FC patterns for each facial expression and found that both static and dynamic facial expressions could be successfully decoded from the FC patterns. Moreover, we identified the expression-discriminative networks for the static and dynamic facial expressions, which span beyond the conventional face-selective areas. Overall, these results reveal that large-scale FC patterns may also contain rich expression information to accurately decode facial expressions, suggesting a novel mechanism, which includes general interactions between distributed brain regions, and that contributes to the human facial expression recognition. Facial expression is an important medium for social communication as it conveys information about others’ emotion. Humans can quickly and effortlessly decode emotion expressions from faces and perceive them in a categorical manner. The mechanism under which enables human brain achieving the efficient recognition of facial expressions is intensively studied.The usual way in exploring facial expression perception is recoding the brain activity patterns while participants are presented with facial stimuli. Most of the past fMRI studies on facial expression perception employed univariate statistics to analyze expression stimuli induced increments of neural activity in specific brain areas. Due to the expected existence of interactions between different brain areas, the analyses of functional connectivity (FC) attracted more and more attention, which is measured as the temporal correlations in the fMRI activity between distinct brain areas . AnalysiMachine learning techniques make use of the multivariate nature of the fMRI data and are being increasingly applied to decode cognitive processes . PreviouThe present fMRI study explored the role of the FC patterns in the facial expression recognition. We hypothesized that expression information may also be represented in the FC patterns. To address this issue, we collected neural activities while participants viewed facial expressions of six basic emotions  in a block design experiment. Both static and dynamic expression stimuli were included in our experiment. After scanning, we conducted a behavioral experiment in accordance to previous study to assess the validity of the facial stimuli, in which we recorded the classification accuracy, the emotional intensity the participants perceived and the corresponding reaction times for each facial stimulus , 2015. AThe data used in this study were collected in our previous study . EighteeAll the participants were scanned using a 3.0-T Siemens scanner with an eight-channel head coil in Yantai Affiliated Hospital of Binzhou Medical University. Foam pads and earplugs were used to reduce the head motion and scanner noise. Functional images were obtained using a gradient echo-planar imaging (EPI) sequence . In addition, a three-dimensional magnetization-prepared rapid-acquisition gradient echo (3D MPRAGE) sequence  was used to acquire the T1-weighted anatomical images. The stimuli were displayed by high-resolution stereo 3D glasses within a VisualStim Digital MRI Compatible fMRI system .Figure 1A. We created the dynamic expression stimuli by cropping all videos to 1520 ms to retain the transition from a neutral expression to the expression apex, and the apex expression image was used as the static stimuli , which is a standard facial expression database containing both images and videos of basic emotions . Video c stimuli , 2015.Figure 1B shows the schematic representation of the employed paradigm. At the beginning of each run, there was a 10 s fixation cross, which was followed by a 24 s stimulus block (the same condition and expression) and then a 4 s button task. Successive stimulus blocks were separated by the presentation of a fixation cross for 10 s. In each stimulus block, 12 expression stimuli were presented (each for 1520 ms) with an interstimulus interval (ISI) of 480 ms. During the course of each stimulus block, participants were instructed to carefully watch the facial stimuli, and after the block, a screen appeared with six emotion categories and corresponding button indexes to instruct the participants to press a button to indicate the facial expression they had seen in the previous block  space, with a re-sampled voxel size of 3 mm × 3 mm × 3 mm. Finally, the functional data were spatially smoothed with a 4-mm full-width at half-maximum Gaussian kernel.Functional image preprocessing was conducted using SPM8 software2 .Estimation of the task-related whole-brain FC was carried out using the CONN toolbox2  in MATLAFigure 2 represents the framework overview of our fcMVPA classification. We performed a six-way expression classification separately for the static and dynamic facial expressions. Due to some evidence showed that the interpretation of the negative FCs remained controversial  q = 0.01. This procedure identified 1540 positive FCs for anger, 1792 positive FCs for disgust, 1466 positive FCs for fear, 1838 positive FCs for joy, 1799 positive FCs for sadness and 1726 positive FCs for surprise for the static expressions, while for the dynamic expressions, it correspondingly identified 1944, 1798, 1696, 1703, 1608, and 1822 positive FCs for each of the six basic expressions. Pooling the positive FCs together separately for static and dynamic conditions, we obtained a total of 3014 (for static) and 2986 (for dynamic) FCs that were significantly positive for at least one expression  was used to evaluate the performance (p < 0.05), which was only performed on the training data of each LOOCV fold to avoid peeking. The statistical significance of the decoding performance was evaluated with permutation test, in which the same cross-validation procedure was carried out for 1000 random shuffles of class labels  of the accuracies from all permutation tests exceeded the actual accuracy using correct labels, the results was thought to be significant.We employed multivariate pattern analysis and machine learning to examine whether facial expression information could be decoded from the FC patterns (fcMVPA). oversial , we alsopression . For claformance . For muls labels . The p-vFigure 3 shows the behavioral results. These results verified the validity of the facial stimuli used in our experiment as both static and dynamic facial expression stimuli could be successfully classified with high accuracies . A further comparison of the classification accuracies, intensity ratings and the corresponding reaction times between static and dynamic facial expressions, we found that participants showed higher classification accuracies for dynamic compared with static facial expressions . For the emotional intensity and the reaction times, there were no significant differences.Participants completed a behavioral experiment in which they classified the emotional categories and rated the perceived emotional intensities for each facial stimulus after scanning. Figures 5 show the results of group-level analysis of the FC patterns for each facial expression .We constructed the whole-brain FC patterns for each facial expression separately for the static and dynamic stimuli. For each participant, we got 12 FC matrices with each contained 6216 [(112 × 111)/2] connections between the pre-defined 112 brain notes. Second-level analysis was performed for each facial expression for the group comparisons of the differences in these ROI-to-ROI functional connections. t-test across participants with multiple comparisons (FDR q = 0.01) and by pooling the positive FCs together, we obtained 3014 (for static) and 2986 (for dynamic) FCs for the classification of static and dynamic facial expressions , we found that classification accuracies based on the FC patterns were significantly above the chance level for both static and dynamic facial expressions , indicating that expression information could be successfully decoded from the FC patterns.In this section, we explored whether facial expressions could be decoded from the FC patterns using fcMVPA. Since the interpretation of the negative FCs remained controversial , we focuressions . In the p < 0.05) composed the expression-discriminative networks. Figure 7 shows the expression-discriminative networks for the static and dynamic facial expressions, all of which were widely distributed in both hemispheres. We summarized the brain regions that were involved in both static and dynamic expression-discriminative networks in Table 1. We found conventional face-selective areas, including the insula, inferior frontal gyrus, superior temporal gyrus, lateral occipital cortex ; temporal fusiform cortex (fusiform gyrus) and amygdala, which were commonly studied in previous fMRI studies on facial expression perception (Furthermore, we identified the expression-discriminative networks for the static and dynamic facial expressions, defined as FCs that contributed significantly in discriminating between different expression categories. Connections that were selected over all iterations of LOOCV feature selection (consensus features, ANOVA rception . Moreoverception , was alsThe main purpose of this study was to explore whether the FC patterns effectively contributed to human facial expression recognition. To address this issue, we employed a block design experiment and conducted fcMVPA. We obtained the whole-brain FC patterns for each facial expression separately for static and dynamic stimuli and found that both static and dynamic facial expressions could be successfully decoded from the FC patterns. We also identified the expression-discriminative networks for the static and dynamic facial expressions, composed of FCs that significantly contributed to the classification between different facial expressions.Using multivariate connectivity pattern analysis and machine learning algorithm, we found the successful decoding of both static and dynamic facial expressions based on the FC patterns.Previous studies on facial expression recognition are dominated by identifying cortical regions showing preferential activation to facial expressions . AlthougNeuroscience studies on facial expressions have paid considerable attention to the face-selective areas which exhibited selectivity to facial stimuli based on traditional activation analyses. Previous fMRI studies have indicated that face-selective areas are involved in the processing of facial expressions . In our In addition, we found brain regions beyond these conventional face-selective areas participated in the expression-discriminative networks. The middle temporal gyrus, which was unanimously found sensitive to facial motion in the previous studies , was alsMoreover, it has been demonstrated a common neural substrate underlying the processing of static and dynamic facial expressions . Our resIn our present study, we employed comparable sample size as the previous fMRI studies on facial expression perception and MVPA-based analyses . Future In summary, we show that expression information can be successfully decoded from the FC patterns and the expression-discriminative networks include brain regions far beyond the conventional face-selective areas identified in previous studies. Our results highlighted the important role of the FC patterns in the facial expression decoding, providing new evidence that the large-scale FC patterns may also contain rich expression information and effectively contribute to the facial expression recognition. Our study extends the traditional research on facial expression recognition and may further the understanding of the potential mechanisms under which human brain achieve quick and accurate recognition of facial expressions.BL designed the study. YL, XL, and PW performed the experiments. YL analyzed the results and wrote the manuscript. YL and BL contributed to manuscript revision. All authors have approved the final manuscript.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.


The optimal design of pedagogical strategies for e-learning in graduate and postgraduate medical education remains to be determined. Video-based e-learning use is increasing, with initial research suggesting that taking short breaks while watching videos (independent of answering test questions) may improve learning by focusing attention on the content presented. Interspersed test questions may also improve knowledge acquisition and retention.To examine the effect of interspersed test questions and periodic breaks on immediate knowledge acquisition and retention at 6 months by pediatric residents engaged in video-based e-learning.First- and second-year pediatric residents were randomized to 1 of the following 3 groups: viewing the complete video uninterrupted (full video), viewing the video interrupted with unrelated logic puzzles (logic puzzles), or viewing the video interrupted with brief comprehension test questions (short answer questions). Residents answered pre- and post-tests before and after video viewing, followed by a retention test at 6 months. Primary outcome included comparison of the change in test scores between groups.F2,46=0.35, P=.71). Thirty-five residents completed retention testing with comparable degrees of knowledge retention in the full video and short answer test questions groups (P<.001), but no significant change in the logic puzzles group .A total of 49 residents completed the initial testing session. All 3 learning groups had comparable mean increases in immediate knowledge gain, but with no significant differences between groups (Improved knowledge gain was not demonstrated among residents answering interspersed questions or completing logic puzzles during interrupted online video viewing when compared with residents viewing uninterrupted video content. However, residents who either participated in uninterrupted video viewing or answered interspersed questions during interrupted video viewing demonstrated significant knowledge retention at 6 months. The recent introduction of e-learning initiatives in postgraduate medical education has been heralded as a disruptive change to efficiently scale knowledge and promote more effective learning. Although e-learning can deliver knowledge, research to inform the optimal learning design and teaching practices in this environment remains in its infancy -4. ReseaUsing interspersed test questions as an educational learning tool also allows for superior knowledge retention relative to passively restudying the same material when students are tested at extended intervals after the initial learning activity ,9. TestsDespite the increased use of e-learning platforms to educate postgraduate medical trainees, we were unable to identify any studies investigating the implementation of test-enhanced learning strategies in an e-learning platform for postgraduate medical education. Yet, many e-learning platforms and massive open online courses (MOOCs) utilize uninterrupted instructional videos as a means of learning, with or without pre- and post-tests for knowledge assessment -14.The purpose of this study was to evaluate the extent to which the use of interspersed test questions or taking periodic breaks while watching an online video would impact knowledge gain as compared with watching the same video without any breaks. As a proxy for knowledge gain, the primary study outcome was the difference between pre- and post-test scores between groups. Secondary outcomes included the difference between pre- and post-test scores within each group and the difference in retention test scores at 6 months compared with pretest scores between groups.We conducted a randomized, prospective, cohort study in 3 academic medical centers in Boston, Massachusetts between June 2014 and March 2016. Pediatric residents in their first or second year of postgraduate training were eligible to participate. Participation was completely voluntary, as this educational initiative was independent from educational obligations during their clinical pediatric intensive care unit (PICU) rotations. Residents were ineligible if they had previously completed a 4-week PICU rotation during residency because the study intervention assessed knowledge acquisition and retention of mechanical ventilation concepts that would have been encountered by residents on their first PICU rotation. Email invitations were sent to all eligible residents enrolled in the following pediatric residency programs: Boston Combined Pediatric Residency Program, Massachusetts General Hospital, and Tufts Floating Hospital for Children. Residents provided voluntary written consent for study inclusion. No residents who volunteered to participate actively refused participation at a subsequent time point during the study. Residents received a US $50 Amazon gift card and the chance to receive an iPad via random selection upon completion of 6-month retention testing. The Boston Children’s Hospital’s Institutional Review Board deemed this study exempt from informed consent, given no identifying data on study participants were collected. Affiliated institutions honored the exempt status. Initial testing was conducted at each of the 3 institutions with all sessions monitored by 1 of 2 study facilitators who were available to troubleshoot technical difficulties and monitor for dishonest behavior. Retention tests were administered via email. Residents were asked to abide by the honor code when completing the retention test.Pediatric residents were blindly randomized via concealed envelopes to 1 of the following 3 groups: full video, logic puzzles, or short answer questions . ResidenResidents completed all computer-based elements of this study via a single lesson plan created on the commercial e-learning platform Softchalk . All residents watched a peer-reviewed video about the basic principles of high frequency oscillatory ventilation (HFOV), assuming that pediatric residents would have limited baseline knowledge of this content as clinical exposure to ICUs is low early in residency training. In addition, this specific topic was chosen for educational use because although HFOV is an important mode of mechanical ventilation for advanced, refractory pediatric respiratory failure and requires basic conceptual understanding by residents, the use of this type of mechanical ventilation is an overall low-frequency event in most PICUs. The video was written and presented by a Harvard Professor who conducts research on HFOV, as part of an existing curriculum on OPENPediatrics, an open access, e-learning platform . This viThree content experts from the Division of Critical Care at Boston Children’s Hospital developed test questions and acceptable answers. All experts utilized the same content validity scoring system to evaluate questions. Questions scored as highly relevant  were included. All questions required free text answers. During the initial testing session, all residents completed a 10-question pretest prior to video watching and a 10-question posttest immediately afterwards. Six months after initial testing session completion, all residents were asked to complete a 10-question retention test. Different questions were included on pre-, post-, and retention tests, but all tested similar concepts. Three independent graders scored all test questions upon participant completion. Free text responses were scored on a binary scale . Scores were reported as percent correct, out of a possible 100% . No partial credit was given.F-tests to compare differences in the change in pre- and post-test scores among the 3 groups immediately and at 6-month follow-up  vs 39% [SD 16] vs P=.02).Pediatric residents were randomized to the following groups: full video (n=17), logic puzzles (n=16), and short answer questions (n=16). Mean initial pre- and post-test percent correct scores and 95% CIs for each group are reported in P=.80), all reporting caring for at least 3 patients receiving HFOV. No residents cared for more than 5 patients receiving HFOV (P=.67). Five residents reported repeated viewing of the HFOV video on OPENPediatrics prior to the 6-month posttest . Cross-reference of OPENPediatrics data verified that only 2 participants had actually watched the video in between the initial and 6-month retention testing. Mean retention test scores for each group are reported in Thirty-five residents 71%; 35/49) completed the 6-month retention test with similar numbers in each group . During this 6-month interval, 10 residents overall rotated through the PICU . Repeated measures mixed-model ANOVA demonstrated that the full video and short answer questions groups had a significant degree of knowledge retention at 6 months, although to a lesser degree than knowledge gained on initial mean posttest scores . The logic puzzle group did not demonstrate statistically significant knowledge retention at 6 months .One-way ANOVA revealed that all 3 groups had comparable degrees of mean knowledge retention between the initial pretest and retention test (P<.001), although to a lesser degree compared with the change in test scores between initial pre- and post-testing.This study did not demonstrate a significant difference related to overall initial knowledge gain when pediatric residents watched a video without structured breaks compared with those with interspersed breaks and completion of either logic puzzles or short answer questions. Despite this finding, we demonstrate that irrespective of group assignment, all groups had significantly higher mean percent correct test scores on initial posttests compared with pretests. This suggests that the online video itself is an effective teaching modality. Six-month retention analysis reveals a continued lack of statistical significance when comparing residents’ change in test scores between groups. However, there is an overall significant increase in the change in test scores between initial pretests and 6-month retention tests in the full video and short answer groups , but possibly due to time constraints related to clinical rotations or time away from residency during which they were unresponsive to email.We acknowledge several limitations to this study. First, the sample size reported here was designed to power to an 80% level, yet it is still possible that our sample size was too small to detect significant differences between groups. Second, the lack of a statistically significant difference in knowledge gain between groups may be related to several factors, including overall video duration, timing of when residents completed this study in the context of their clinical rotations, emotional state of residents during study completion , and their overall content interest. Third, this study may have some methodological insufficiency regarding the use of spaced learning for evaluation of knowledge retention, and we believe that this would be an interesting hypothesis to incorporate in a future study.The overall duration of the video used in this study was relatively short 23 min) such that this may have contributed to not finding a meaningful effect size difference between groups who were taking breaks while watching the video. Yet, data regarding the optimal video length for learner engagement are conflicting. Research in disciplines other than medicine suggests that shorter duration is generally better and that including breaks within longer videos helps reduce cognitive load. Data from TED talks suggests that the optimal video length is around 18 min, which is short enough to hold attention, yet long enough to succinctly communicate complex topics, both of which decreases cognitive overload by limiting the amount of time of active brain engagement, and forces the speaker to be clear and concise . Data fr min suchIn addition, inattentiveness and mind-wandering have been linked to poor knowledge gain, and these behaviors occur more frequently when students are experiencing an underlying negative emotional state, lack engagement, or experience stress related to learning . These aP=.67). If this were clinically significant, we would have expected to observe a greater increase in knowledge retention within all groups at 6 months’ follow-up. Similarly, numbers of residents rotating through the PICU between initial testing and 6-month follow-up were low and not statistically different between groups (P=.80). We continue to acknowledge the overall small sample size in interpretation of our 6-month follow-up analysis.Finally, several limitations must be considered when reviewing our secondary outcome of retention test score analysis. First, residents were not directly observed for dishonest behavior on retention test completion, which could potentially falsely elevate test scores. Second, we did not specifically control for “on-the-job” training. However, given the overall small exposure to patients ventilated by HFOV as self-reported by residents across all groups between the initial testing and the 6-month follow-up, we do not believe this has greatly impacted our findings as differences in exposure between groups lacked statistical significance , in which interspersed test questions and periodic breaks integrated into an online statistics lecture improved knowledge gain among undergraduate students. However, when our findings are viewed together with other previous studies , in whic,10,21, w