High-throughput sequencing technologies could improve diagnosis and classification of TBI subgroups. Because recent studies showed that circulating microRNAs (miRNAs) may serve as noninvasive markers of TBI, we performed miRNA-seq to study TBI-induced changes in rat hippocampal miRNAs up to one year post-injury. We used miRNA PCR arrays to interrogate differences in serum miRNAs using two rat models of TBI . The translational potential of our results was evaluated by miRNA-seq analysis of human control and TBI (acute and chronic) serum samples. Bioinformatic analyses were performed using Ingenuity Pathway Analysis, miRDB, and Qlucore Omics Explorer. Rat miRNA profiles identified TBI across all acute and chronic intervals. Rat CCI and FPI displayed distinct serum miRNA profiles. Human miRNA profiles identified TBI across all acute and chronic time points and, at 24 hours, discriminated between focal and diffuse injuries. In both species, predicted gene targets of differentially expressed miRNAs are involved in neuroplasticity, immune function and neurorestoration. Chronically dysregulated miRNAs  are linked to psychiatric and neurodegenerative disorders. These data suggest that circulating miRNAs in biofluids can be used as “molecular fingerprints” to identify acute, chronic, focal or diffuse TBI and potentially, presence of neurodegenerative sequelae. One potential approach is a “liquid biopsy,” a blood test such as that recently reported to permit earlier detection of eight common types of cancer2. Because small, non-coding microRNAs (miRNAs) are stable in circulating biofluids and have been identified as potential, non-invasive disease-specific biomarkers3, we hypothesized that circulating miRNAs may identify the occurrence of TBIs in acute and long-term survivors and that distinct miRNA profiles could potentially discriminate among subgroups of TBI patients.Among the global health challenges posed by traumatic brain injuries (TBI)4. In the brain, altered miRNA expression is linked to neurodegenerative disorders5. In previous rat TBI studies, we reported that 24 h after experimental TBI, injury-induced miRNAs suppressed expression of pro-survival genes in dying hippocampal neurons6. These miRNA target genes, according to the Online Mendelian Inheritance in Man (OMIM) database, lead to human disease when dysregulated, mutated or deleted7. To test our hypothesis that persistent, long-term dysregulation of hippocampal miRNAs could partly explain chronic neurodegeneration after TBI, we designed a long-term survey of miRNA expression in rat hippocampus after TBI using unbiased next-generation sequencing (NGS).MiRNAs post-transcriptionally regulate gene expression by translationally repressing mRNA targets; they regulate most cell signaling pathways involved in development, cell function and disease8. Since our objective was to evaluate the translational potential of rat miRNA-seq results, we sequenced a limited set of archived human TBI serum samples representing multiple acute and chronic post-injury intervals and age-matched control serum. In these exploratory studies with rat and human tissues, we tested three hypotheses, 1) miRNA-seq profiles would identify TBI at all acute and chronic post-injury intervals, 2) miRNA-seq profiles would discriminate between focal and diffuse TBI and 3) serum miRNA-seq would reveal potential miRNA markers of neuropsychiatric and neurodegenerative sequelae.Advances in NGS technologies have great potential to improve precision medicine and clinical practiceThe miRNA-seq and data analyses workflow is shown in Fig. In the rat studies, for each post-injury interval, we sequenced four TBI and four sham-control hippocampi, thus 56 rats were used for miRNA-seq analysis. In the human studies, we sequenced 51 serum samples . Demographic characteristics are shown in Table et al.9. Serum from rats subjected to FPI and CCI was used for miRNA isolation and PCR array analysis as described in Supplementary Methods.Total RNA from rat hippocampal tissue was extracted and purified using Ribopure (Ambion) as in Boone and Weisz 10, and informed consent was obtained from all subjects or their legally authorized representatives for the samples remaining after analyses performed for the trial were completed to be banked and used in future studies. The de-identified samples were stored and distributed under IRB protocol H-13606 at Baylor College of Medicine . Use of these samples in the present study was deemed as exempt from review as a human subjects protocol by the IRB at the University of Texas Medical Branch at Galveston, as the materials were from an established tissue/sample bank, had been de-identified, etc. All procedures performed as part of this study were performed in accordance with institutional, state, and U.S. federal guidelines and regulations.Archived human TBI serum was used for this pilot study. The samples were initially collected as part of a clinical trial of erythropoietinThe samples that were selected were from six patients who had an injury that was predominantly a diffuse injury pattern with either a normal CT scan or scattered shear injuries, and 15 patients that had a focal contusion. Detailed miRNA isolation procedures are provided in Supplementary Methods.Small RNA libraries were made using the NEBNext small RNA Multiplex kit . The library preparation method included a final size fractionation to enrich for inserts of ~22 bases. After purification by polyacrylamide gel-electrophoresis, the sample libraries were pooled and sequenced by the UTMB Next Generation Sequencing Core on an Illumina NextSeq550 (single end 75 base) using TruSeq SBS kit v3 (Illumina) and protocols defined by the manufacturer. The miRDeep2 software package, version 2.0.0.8, was used to trim adapter sequences from the reads and quantify miRNA read counts using the miRBase database, release 22. On average, about 13 million reads were obtained per sample, approximately 80% of the reads passed trimming and filtering and approximately 1.3 million reads per sample mapped to miRNAs. The rat and human miRNA-seq data has been deposited in the National Center for Biotechnology Information’s Gene Expression Omnibus (GEO Accession numbers GSE131695 and GSE131704).To infer the regulatory actions of miRNA from sequencing data and improve our confidence that these miRNAs are representative of brain pathology, we performed an iterative process for miRNA target prediction and pathway analysis using a miRNA target prediction online suite, miRDB and Ingenuity Pathway Analysis software; details provided in Supplementary methods.Differential expression analysis of the miRNA-seq counts was done using two R packages: Empirical Analysis of Digital Gene Expression Data in R (EdgeR) and Differential gene expression analysis based on the negative binomial distribution (DESeq2). Both use the negative binomial distribution to assess differences in miRNA expression using count data and a general linear model (glm) analysis framework. The glm procedure was used to estimate two exact tests: the quasi-likelihood (QL) F-test  and the likelihood ratio test (LR test). The top tags from each were read out and compared. Additional details are provided in Supplementary Methods.11 which is derived from calculation of the covariance among the original variables in the data. In essence, PCA extracts essential information from high-dimensional data by finding the directions of maximum variance  and then captures or projects the variance in these many variables into a smaller, easier-to-analyze set of variables. The interactive, PCA approach facilitates heuristic data analysis and enables visualization of the miRNA data in a three-dimensional space after filtering out variables with low overall variance and statistical testing. Because PCA is used both for exploratory data analysis and as a machine learning tool for predictive models, this unsupervised statistical technique is suitable for finding patterns in high-dimensional data such as miRNA-seq.Qlucore Omics Explorer  is an intuitive data analysis and data mining software tool that combines speed and advanced analytics for interactive exploration and instant visualization of multivariate data; QOE is built on state-of-the-art mathematical and statistical methods . The dynamic visualization tool in QOE is principal component analysis (PCA), an unsupervised (agnostic to variable and sample annotations), descriptive and data reduction techniqueThis study was supported by the Moody Project for Translational Traumatic Brain Injury Research. The funders had no role in the study design, data curation, data analysis, data interpretation, writing of the report or decision to submit for publication.9.We examined FPI-induced miRNA changes in rat hippocampus Fig.  at sevenTo test our hypothesis that circulating miRNAs may reflect TBI-induced changes induced by focal or diffuse injuries, we performed serum miRNA PCR array analysis of rats subjected to CCI  or FPI . CCI and FPI produced distinct miRNA expression profiles Fig. .We performed and analyzed miRNA-seq data Fig.  from arc12 in diffuse injuries , we found that serum miRNA profiles clearly discriminate between focal and diffuse TBIs Fig. . Each inies Fig. . Howeveries Fig.  and S6 iIn silico analysis showed that four miRNAs  and age-matched controls Fig. . PrincipNAs Fig. , differeNAs Fig. .Figure 5At the miRNA level, the rat and human results were not concordant. However, examination of commonly altered pathways populated by genes regulated by upregulated and downregulated miRNAs in rat and human TBI Fig.  indicate13, bioinformatic and in silico analyses resulted in three key findings that may inform the potential of blood-based tests for TBI.This proof-of-concept study began with the goal of deciphering the underlying molecular progression of chronic TBI in the rat hippocampus using miRNA sequencing. With several insights gained from these data and from analyses of serum miRNA profiling data generated in two rat TBI models, we conducted a pilot study of human TBI using a limited number of archived acute and chronic TBI serum samples. From our survey of existing human TBI miRNA studies , we surmised that the key results of our pilot study have not been previously reported. Given the current interest in blood-based tests for human diseases14. Moreover, a miRNA-based blood test would also serve as additional confirmation of chronic TBI obtained via neuroimaging15. This study also indicates that, using miRNA-seq profiling of peripheral biofluids, we would be able to study the continuum and progression of TBI at the molecular genetic level to identify therapeutic windows throughout the lifetime of TBI survivors.First, despite the relatively poor concordance of rat and human miRNA data, in both species, miRNA profiles clearly distinguish all cases of brain injury from uninjured, age-matched controls across all acute and chronic post-injury intervals. Notably, the unequivocal identification of human TBI survivors up to 32 years post-injury is not only clinically relevant but, at the molecular level, demonstrates that TBI is indeed a chronic disease process, not just a single event16, we show here the first evidence that blood miRNA profiles could reflect the heterogeneity and injury-specific pathogenic changes induced in the brain by a diffuse or focal TBI which, at present, are only conclusively identifiable by sophisticated neuroimaging15. Given that genetic factors affect the heterogeneous presentation of TBI and functional outcome after TBI17, it is notable that discriminating focal from diffuse injuries by principal component analysis is congruent with the diagnosis by CT imaging. In future, we may be able to define minimally invasive circulating miRNA profiles that correspond to other specific subtypes of TBI such as hemorrhagic, penetrating and ischemic injuries.Second, in both rats and humans, focal and diffuse brain injuries result in distinct serum miRNA profiles. Although a previous study showed differences in focal and diffuse injuries in living biopsies of human brain tissuein silico analyses indicated that at longer times (two – 32 years) after TBI, differentially expressed miRNAs regulate critical CNS functions that are linked to well-known neuropsychiatric and neurodegenerative sequelae of TBI18. Given recent studies demonstrating that common neurobiological substrates (i.e. shared molecular genetic mechanisms) underlie different brain disorders19, these data suggest that some TBI dysregulated miRNAs may be causally implicated in the increased risk of, or may potentially serve as surrogate markers of, these brain disorders. For instance, changes in miR-199 and miR-34 have been linked to status epilepticus20. And chronically upregulated miR-1908-5p is predicted to regulate multiple genes involved in glutamatergic synaptic function21. Notably, miR-204-5p which was downregulated in chronic TBI was also reported downregulated in frontotemporal dementia22, a finding that suggests that this miRNA might serve as a useful biomarker of early stage, subclinical dementia in neurodegenerative disorders. TBI-dysregulated miRNAs also regulate multiple genes involved in neurodevelopmental functions23; Schork et al., showed in their study of shared risk across psychiatric disorders, that all share dysregulated expression of common gene variants that regulate neural development24. In fact, two isoforms of let-7, which are critically involved in brain development25, can identify chronic TBI patients two to 32 years post-injury. That these miRNAs are still dysregulated at 32 years after injury is evidence of long-lasting changes after a TBI, suggesting that psychological and cognitive impairment can persist for long times after TBI. Therefore, treatment of chronic TBI survivors is worthwhile.Third, 26. Bioinformatic analyses indicated that these AD-associated genes are regulated by multiple TBI-associated miRNAs, notably miR-9 and miR-125, which are downregulated in chronic TBI; this suggests that these AD-associated genes are upregulated. Given evidence that TBI accelerates brain aging27, we found that at 24–96 h post-injury and as long as 32 years post-injury, miRNAs implicated in brain aging28 are still found to be dysregulated. One positive finding is that the persistent upregulation of some miRNAs, such as miR-451, in chronic TBI is a sign of a restorative response; for example, increased levels of miR-451 via diet has been shown to protect cells from oxidative stress29.A recent study of epigenetic dysregulation in a spectrum of Alzheimer’s Disease (AD) patients showed hypomethylation of AD-associated enhancer genes; hypomethylation of enhancers in prefrontal cortex neurons results in aberrant activation of cell cycle and upregulation of AD-associated genes such as BACE16 and suggests both positive and negative consequences of dysregulated miRNAs. Some fish, amphibians and reptiles have regenerative properties which are not found in humans30. A potential explanation is that in humans, injury-induced miRNAs such as miR-155 decreased expression of antioxidant genes such as catalase and superoxide dismutase31, thus inhibiting regenerative signaling32. Moreover, as both miR-155 and miR-223 have functional roles in mitochondria and cytoplasm33, their dysregulation for several months after TBI suggests persistent mitochondrial dysfunction. On the other hand, upregulation of miR-223 may reflect a protective, regenerative response. MiR-223 inhibits inflammasome activation through caspase-1 and IL-1β34. By targeting glutamate receptors, GluR2 and NR2B, miR-223 also inhibits calcium influx and protects neurons from excitotoxic damage35. We also inferred that upregulation of miR-21 may contribute to neurodegenerative signaling given its role in proliferation and differentiation of neural stem cells via regulation of AKT and GSK-3β36.The rat hippocampal miRNA-seq data are largely congruent with the long-lasting immune dysregulation that we found in our recent study of chronic TBI-induced gene expression37. However, we show that, despite the differences in individual miRNAs, there are many commonly affected gene targets in cellular pathways collectively affected by TBI-dysregulated miRNAs in both rat and human tissues. Thus, in lieu of focusing on individual genes or miRNAs, it may be more relevant to study the commonly modulated CNS pathways affected by TBI in both species.Recent studies show that the difficulties in translating rodent pathophysiological processes to human is because TBI-induced pathological changes occur on different timescales and there is a lack of a universal “conversion” rate6, we showed in dying hippocampal neurons, miR-15b suppressed the expression of brain-derived neurotrophic factor (BDNF), a neurotrophic factor that is essential for brain survival and cognitive function38. Examination of the 3′UTR of the human BDNF gene shows multiple binding sites for human miRNAs chronically altered after injury, suggesting that dysregulated BDNF levels are involved in decline of cognitive functions after TBI. Given evidence of disrupted brain connectivity networks in brain diseases such as depression and Alzheimer’s39, our bioinformatic analysis indicated that miRNAs altered by acute and chronic TBI collectively regulate myriad gene networks involved in brain connectivity41.In previous studiesWe present this analysis with some caution since, in addition to the limited sample sizes in this exploratory pilot study, it is possible that some of the miRNA changes may not be specific to brain injury given that some of the TBI patients had some elements of polytrauma. Moreover, the samples were selected for simple diffuse injury and focal injury to help test our initial hypothesis. It is likely that a more general TBI population would have combinations of these injuries. We also note that, other than post-injury interval and age of survivors, we have no information about the nature of the injuries in the chronic TBI group. Finally, particularly for human studies, we need to point out that the interpretation of the functional relevance of circulating miRNA changes is based on their predicted effects on gene targets identified by miRNA target prediction algorithms. This informs and provides a cautionary note to our understanding of the potential functional roles played by any differentially expressed miRNAs in rat and human TBI.42. In our study, PCA of transcriptome-wide miRNA expression profiles, including those with small but significant effect sizes, facilitated the clear identification of TBIs and TBI subgroups despite the variability among the patients. This type of analysis may prove to be valuable for identifying mild TBIs that are often missed at initial assessments but have been found to result in chronic cognitive impairment43. Our study also indicates that TBI leaves a long-lasting “miRNA fingerprint” in circulating biofluids which may serve to identify injured individuals across all post-injury intervals as well as portend risk of later neurodegenerative disorders.The heterogeneity of the TBI population actually points to a reason why PCA of miRNA profiles may help identify TBI better than individual biomarkers. It is instructive to recognize that complex traits like intelligence, height and life expectancy are polygenic traits, i.e., there is no single gene that determines these characteristics; rather, the phenotype is due to huge numbers of common gene variants with tiny effect sizes44. Single outcome measures have been deemed insufficient to assess individuals after a TBI, thus multiple assessments are called for1. Since the idea that predictive analytics  can improve medical care is increasingly acknowledged45, miRNA-sequencing of biofluids, which is becoming more cost-effective to perform, would be an ideal adjunct to current imaging modalities and neuropsychological tests46 that may aid in a precision-health approach towards better stratification of patients into subgroups as well as other brain disorders8.To improve care for TBI, it is necessary to address the need for better characterization and mechanistic understanding of the life-long consequences of TBI, especially the cognitive and psychiatric sequelaeSupplementary Materials.


Objective: Cardiac-implantable electronic device (CIED) infections are associated with significant morbidity and mortality. In this review, we describe the risk factors and pathogenesis of CIED infections and review the rationale and the evidence for the use of antibiotic-eluting envelopes (ABEs) in patients at increased risk for CIED infections.Findings: The majority of CIED infections are caused by staphylococci that involve generator pocket and occur due to contamination of the device or the pocket tissues at the time of implantation. Clinical trials have shown that extending the duration of post-operative systemic antibacterial therapy is not beneficial in reducing CIED infection rate. However, ABEs that reduce device migration after implantation and provide sustained local delivery of prophylactic antibiotics at the pocket site, may provide benefit in reducing infection. Currently, there are two types of commercially available CIED envelope devices in the United States. The first ABE device  is composed of a synthetic absorbable mesh envelope that elutes minocycline and rifampin and has been shown to reduce CIED pocket infections in a large multi-center randomized clinical trial. The second ABE device  is composed of decellularized extracellular matrix (ECM) and was originally designed to stabilize the device within the pocket, limiting risk for migration or erosion, and providing a substrate for tissue ingrowth in a preclinical study. This device has shown promising results in a preclinical study with local delivery of gentamicin. Compared with artificial materials, such as synthetic surgical mesh, biologic ECM has been shown to foster greater tissue integration and vascular ingrowth, a reduced inflammatory response, and more rapid clearance of bacteria.Conclusions and Relevance: ABE devices provide sustained local delivery of antibiotics at the generator pocket site and appear beneficial in reducing CIED pocket infections. Given the continued increase in the use of CIED therapy and resultant infectious complications, innovative approaches to infection prevention are critical. Infections are a devasting complication of cardiac-implantable electronic devices (CIED) therapy and are associated with increased morbidity, mortality, and cost. Moreover, reported rates of CIED infection are rising faster even when accounting for the continuous growth in CIED implantation . This miCIED infections: Incidence, costs, and risk factorsIn the absence of comprehensive and mandatory registries, the estimates of the incidence of CIED infections in North America mostly rely on observational studies and vary based on study type, CIED type, and follow-up duration. A recent analysis of a claims database from 2016 reported a cumulative incidence of CIED infection within one-year post-implant of 1.18% for initial implantation and 2.37% for device replacement . Other sRisk factors for CIED infection include both patient and procedural factors and are summarized in Table Microbiology and pathogenesis of CIED-related infectionsStaphylococcus epidermidis) and S. aureus account for >70% of all CIED infections  differs based on the study and geographic location of the study population and must be considered when managing CIED infections [Coagulase-negative Staphylococci  proteins produced by the host that coat the device surface in the pocket; this mechanism is particularly relevant to new device technologies, such as CIED envelopes. As additional bacteria attach to the bacteria already adhered to the device surface, a biofilm of bacterial layers and intercellular adhesins develops, which makes the bacteria more resistant to antibiotics and immune defenses, specifically by inhibiting penetration to the local area .Prevention of CIED-related InfectionsStandard Prophylaxis (Excluding Antibiotic-Eluting Device Envelopes)Prophylaxis is the preferred strategy to reduce morbidity and mortality associated with CIED infection . EvidencS. aureus (MRSA), and irrigation of the surgical pocket with antibiotics. It is worth noting that evidence does not definitively support their efficacy for the prevention of CIED infections [Skin antisepsis is another standard preoperative technique for reducing CIED infection risk . Some evfections . Similarfections . Howeverfections .Antibiotic-Eluting CIED EnvelopesAntimicrobial-impregnated CIED envelopes have been developed to hold devices securely in place to provide a stable environment and to reduce infection risk . These dTwo CIED envelope devices are currently available for use in the US. Both are absorbable. One is made from absorbable multifilament block copolymer comprised of glycolide, caprolactone, and trimethylene carbonate mesh that is coated with an absorbable polyacrylate polymer containing minocycline and rifampin. The other envelope is made from decellularized, non-crosslinked ECM, which is derived from porcine intestinal submucosa; this device is hydrated in a gentamicin solution prior to implantation Figure . These eSynthetic Mesh EnvelopeThe CIED envelope made of synthetic mesh  elutes minocycline and rifampin for a minimum of seven days. An earlier version of the device (AEGIS) consisted of non-absorbable polypropylene but was associated with significant pocket fibrosis, making removal of a generator and/or leads difficult. A newer version was developed that is fully absorbed within approximately nine weeks after implantation. Initial retrospective studies suggested potential benefits to this device, and these findings were further supported by the prospective CITADEL and CENTURION studies -22. CombImportantly, the efficacy of the synthetic envelope was recently reported in a prospective multicenter randomized clinical trial (WRAP-IT 2019) . This trAlthough the results of this well-designed study (WRAP-IT) achieved statistical significance , the findings were not as robust as anticipated due to lower than anticipated overall infection rate. Moreover, the number-needed-to-treat (NNT) for this study to prevent one CIED infection was 200 patients, an elevated number compared to other preventive therapies. Therefore, further refinements are needed to optimize the clinical benefits and costs of these novel envelopes.ECM EnvelopeAnother CIED envelope that uses different materials and antibiotics is currently available. The CanGaroo-G™ envelope  is made with a biologic material that utilizes ECM, rather than synthetic mesh, to create a bioscaffold for tissue integration and neovascularization Figure . This ECSynthetic vs. biologic materials The inflammatory response to synthetic and cross-linked materials is characterized by a predominance of M1 phenotype, which facilitates the formation of scar tissue and encapsulation of the material [Compared with artificial materials, such as synthetic surgical mesh, biologic ECM has been shown to foster greater tissue integration and vascular ingrowth, a reduced inflammatory response, and more rapid clearance of bacteria -33. Numematerial ,37. Basematerial .Envelope-delivered antibioticsStudies of multiple surgical procedures have reported reduced infection rates with the local application of antibiotics. Based on this evidence, the microbiology of CIED infections, and bactericidal data, current CIED envelope devices utilize either rifampin/minocycline or gentamicin for infection prophylaxis.Rifampin and MinocyclineS. aureus and coagulase-negative Staphylococci isolated from CIED infections is ~0.5 to 2 mg/L [S. epidermidis biofilms, especially in combination with certain other agents, whereas many other tested antibiotics were ineffective [Rifampin inhibits bacterial DNA-dependent RNA synthesis, whereas minocycline inhibits bacterial protein synthesis. Both agents are active against Staphylococcus spp. . The repo 2 mg/L . In vitrffective . The addffective . The comffective .Escherichia coli, and other bacterial species [The combination of rifampin/minocycline has been used extensively in antibiotic-impregnated catheters. Studies report significant reductions in the risk for infection with rifampin/minocycline-impregnated catheters compared to standard catheters in neurosurgical procedures , chemoth species . The afo species .GentamicinGentamicin is an aminoglycoside that inhibits bacterial protein synthesis and has a broad-spectrum bactericidal activity that includes Staphylococcus spp. and aerobic Gram-negative organisms. Susceptibility studies demonstrate >3-log reductions in bacterial colonization , both in vitro and in vivo including the bacteria most commonly isolated from CIED infections. The minimum inhibitory concentration (MIC) for gentamicin for common CIED pathogens  has been reported to be ~0.5 to 4 mg/L ,47.S. aureus inoculates across a sixfold range of gentamicin concentrations [Gentamicin has demonstrated efficacy in vitro against Staphylococcal biofilms; in one study, only daptomycin had similar efficacy among single-agent antibiotics . Similartrations .Some clinical data and animal studies also suggest the antimicrobial efficacy of gentamicin for the prevention of CIED-related infections . Local uThe main limitation of the systemic use of gentamicin is its association with risk for nephrotoxicity and ototoxicity. Consequently, systemic administration of aminoglycosides has been used sparingly for several decades, thereby limiting the development of resistance to this class of antibiotics. Conversely, as described above, local administration of gentamicin has demonstrated efficacy in multiple surgical procedures, without the risks of systemic exposure -17,23,50S. epidermidis was present, presumably due to the high local concentrations of gentamicin [The effects of gentamicin are concentration-dependent, requiring a high concentration in the surgical site to produce high bactericidal activity, even against organisms with low susceptibility or resistance to this agent. When administered into the surgical site, gentamicin concentrations in local tissues can exceed 300 mg/L, whereas systemic exposure is limited to ~1-2 mg/L . In one ntamicin . TherefoCurrently, the efficacy of gentamicin delivering ECM envelope has been reported in preclinical animal infection models and clinical case report ,27. A prMinimizing risks of adverse outcomes associated with CIED implantation, especially infection, is of paramount importance to prevent serious complications that warrant device removal. New CIED envelope technologies demonstrate promise to reduce generator pocket infection. Preclinical and clinical data support the efficacy of local antibiotic delivery through synthetic or ECM-based CIED envelopes eluting rifampin/minocycline and gentamycin, respectively. Moreover, the ECM envelope typically stimulates tissue remodeling and angiogenesis, thereby minimizing inflammation and promoting bacterial clearance. Large, prospective studies are needed to help refining indications and judicious use of the ECM envelope to prevent CIED infection.


APP gene is present on chromosome 21, but other genes are likely to also have a role in AD in DS (AD-DS). Our aim is to study the effect of triplication of chromosome 21 genes other than APP on APP processing. We use Mouse Embryonic Fibroblasts (MEFs) derived from a DS mouse model (Dp1Tyb) that carries a segmental duplication of 23Mb of mouse chromosome 16, homologous to chromosome 21. We will focus on the expression and half-life of APP-derived fragments and on alteration in the structure of endosomes, a main site of APP processing. The ultimate aim of our research is to identify the genes responsible for altered APP processing in AD-DS. To do this we plan to use mouse models that have segmental duplications of progressively smaller regions of the mouse chromosomes homologous of human chromosome 21.Trisomy of human chromosome 21, which causes Down syndrome (DS), is the most common genetic cause of dementia. By the age of 40, virtually all people with DS have amyloid plaques and neurofibrillary tangles, the main pathological hallmarks of Alzheimer disease (AD). The main component of amyloid plaques, amyloid beta, is produced through sequential cleavage of Amyloid Precursor Protein (APP). The  Cornelia de Lange Syndrome (CdLS) is a developmental disorder caused by mutations that compromise the function of cohesin, a major regulator of 3-D genome organisation. A universal yet unexplained feature of CdLS is cognitive impairment. In particular, it is not known whether neuronal dysfunction arises primarily from developmental defects in neuronal progenitors, or from an ongoing requirement for cohesin in post-mitotic neurons. To address these questions, we investigated the impact on neuronal gene expression of depleting cohesin at distinct points in development - in the germline, in immature post-mitotic neurons, or in mature neurons. Unexpectedly, neuronal genes with key roles in synaptic transmission, connectivity, neuronal development and signaling were selectively downregulated, regardless of whether cohesin function was compromised throughout development, during the differentiation of immature post-mitotic neurons, or in mature neurons for just 24 hours. Our findings establish that cohesin is required to sustain the expression of functionally important neuronal genes, and provide a tractable approach to neuronal dysfunction in CdLS. Embryonic epithelia generate mechanical forces which sculpt the embryo. During neural tube closure, actomyosin-dependant neuroepithelial apical constriction and interkinetic nuclear migration (IKNM) facilitate apposition of the posterior neuropore (PNP) neural folds. Both apical constriction and IKNM are force generating mechanisms which co-regulate epithelial apical dimensions, but how they co-exist in the neuroepithelium is unknown.To investigate this, E9 CD1 mouse embryos were cultured in the Rho-associated kinase (ROCK) inhibitor Y-27632. Progression past S-phase was blocked with hydroxyurea. Whole-mount confocal immunofluroscopy was used to visualise cell cycle markers  and cell outlines (Scrib).ROCK inhibition widened the PNP and increased neuroepithelial apical dimensions without substantially changing mitotic index. In vehicle-treated embryos, pRB+ cells predominantly had small apical areas. ROCK inhibition (2hrs) significantly increased their apical dimensions. pHH3+ cells’ apical dimensions ranged from the smallest to the largest observed; surprisingly, this was unchanged by ROCK inhibition (2hrs). This suggests neuroepithelial cells adopt small apical dimensions independently of ROCK during M-phase, but require ROCK to maintain apical constriction when entering G1. Trapping neuroepithelial cells in S-phase for 8hrs narrowed the PNP and prevented its widening following ROCK inhibition. Thus, ROCK-dependent apical constriction is temporally coordinated with IKNM during PNP closure. CTNNB1 during development can drive this tumour-formation. SOX2 + ve pituitary stem cells targeted with oncogenic beta- catenin form senescent cell clusters that can induce tumourigenesis non-cell-autonomously, through a senescent-associated-secretory-phenotype (SASP), comprised of cytokines, chemokines, growth factors, etc. To elucidate the role of senescence in ACP I am generating a mouse model (FDR/+p21) allowing detection and ablation of senescent cells in-vivo. Selective ablation of cluster cells at time-points during tumour formation will determine whether the cells are required for tumour initiation, or solely in tumour maintenance and growth. Additionally a STOP-mBRF1/+Rosa2 mouse will attenuate activities of SASP genetically, to determine the crucial secretory drivers of tumourigenesis, as well as to determine the developmental roles of cytokines and chemokines in the pituitary. This will provide insight into senescence in ACP pathogenesis and pituitary development.ACP is the most common non-neuroepithelial developmental brain-tumour. Mouse models generated in our group have demonstrated that mutations in  in vivo correction of a mutation which results in phenotypic rescue, previously only described in autosomal recessive skin diseases with secondary somatic DNA mutations. We describe here epigenetic revertant mosaicism in three patients with congenital melanocytic naevi (CMN), a condition caused by in utero somatic heterozygous mutations in critical signaling pathway gene NRAS. Patients presented with new islands of normal skin within naevi, and biopsies were obtained from both areas. Unexpectedly, the known DNA mutation was present in all areas, as were equal numbers of naevus cells, however the expression within the phenotypically normal skin was strikingly monoallelic, and restricted to the normal allele. This was confirmed using an optimized and highly specific gene expression assay for the mutant alleles. Investigation of the underlying mechanism has excluded secondary promoter mutations in NRAS, and allele-specific differences in CpG methylation. This phenomenon of natural genetic therapy in vivo is proof of concept that therapeutic allele-specific targeting of gene expression would be sufficient to correct the clinical phenotype.Revertant mosaicism is the process of  MRC Harwell is involved in many national and international projects using mouse models to study the relationship between genes and disease. We have many resources available to the biomedical community which we are keen to share.Archiving - We have a frozen embryo and sperm archive where we can cryopreserve your mouse strains for free. These can then be sent anywhere in the world.Genome Editing Mice for Medicine - Our Genome Editing Mice for Medicine programme is an MRC programme aimed at generating scientifically important, novel mouse lines taking advantage of advances in genome editing. UK scientists are invited to nominate genetically altered mouse lines to be made to advance their own research and be of widespread use in biomedical science.Training - We have a range of different training courses for laboratory and animal science. These courses not only include technical courses in transgenesis, pathology and cryopreservation but also taught courses in mouse genetics and genome editing.IMPC - We are a member of the International Mouse Phenotyping Consortium (IMPC) a global programme looking to find the function of every protein-coding gene in the mouse genome, with the data freely accessible online. If you are interested in a particular gene knockout, you can nominate it through us. Defects in ear canal development can cause severe hearing loss, however very little is known about how the canal initiates, extends and opens. Here we have studied mammalian ear canal development and show that the canal undergoes a complex system of closure and reopening as it forms. The more superficial part of the canal formed from an open primary canal, initiating at the junction between the first and second arch, which later collapsed and then reopened. In contrast, the deeper part of the canal formed from a solid meatal plate that extended from the primary canal into the first arch and later opened. As the ear canal developed, the different parts displayed distinct patterns of proliferation and keratin expression, with collapse of the primary canal linked to loss of periderm. Final opening of the canal was triggered by terminal differentiation of the epithelium. Interestingly, the meatal plate opened asymmetrically, associated with differential proliferation, to create the thin outer surface of the ear-drum. Understanding these complex processes involved in canal development can shed light on the underlying causes of canal atresia. Autosomal dominant North Carolina Macular Dystrophy (NCMD) is believed to represent a failure of macular development with consequent central vision loss. Prior genetic linkage pinpointed the disease locus to chromosome 6q16 and 5p15. The aim was to identify causative variants and further the understanding of the disease mechanism.IRX1 andADAMTS16 (chr5 in hg19 and chr13 in mm9) recurrently identified in NCMD families by whole genome sequencing. Gene expression studies were performed on developing fetal eye and limb-bud. Chromosome conformation capture (c-HI-C) and chromatin immunoprecipitation performed on the same tissues, mapped chromatin architectural folding and defined putative regulatory elements.CRISPR technology was used to reproduce a 50 kb non-coding tandem duplication between IRX1 and IRX2 was increased in the limb-bud tissue at E12·5 in the mouse model versus control. Subsequently these genes are downregulated at E14·5, suggesting temporal dysregulation of the IRXA cluster. c-Hi-C at the 5p locus showed unique chromatin interactions in developing wild-type eye versus limb-bud, suggesting distinct tissue-specific regulation.Expression of Chromatin organization findings highlight the relevance of tissue specific DNA contacts in development and disease. Further characterization of mouse model and exploration of the nine unsolved cases may uncover additional molecular targets for NCMD. Juene syndrome is a rare autosomal recessive ciliopathy affecting about 1 in 100,000 – characterized by shortening of long bones, constriction of the thorax and polydactyly. Here we characterize a Tctex1d2 gene trap mouse model creating successful knock-down of gene expression particularly in the testes and bone. Tctex1d2 is a light chain of the ciliary intra-flagellar transport (IFT) dynein-2 motor complex, essential for motor stability and trafficking of protein cargos required for ciliogenesis. Although no gross skeletal abnormalities were evident, micro-CT scans revealed mild elevation in long bone (tibia) mass confirming a skeletal phenotype. Furthermore, a significant reduction in sperm number and motility was observed in mutant epididymis with several spermatogenesis defects including amorphous heads, short/bent flagella and swollen tail tips. During spermatid differentiation, protein transport is based on IFT and intra-manchette transport. In the mutants, Tctex1d2 protein was seen to be absent in the flagellum of developing spermatids and mature sperms. Hence, to determine the stage of spermatogenesis at which Tctex1d2 is required for cellular cargo trafficking selective IFT components were studied. We found markedly increased staining along the flagella of IFT components in the mutants, suggesting that transport of IFT proteins is disrupted. We therefore conclude that Tctex1d2 is an integral component of the sperm retrograde IFT machinery. The adrenal gland is an endocrine organ responsible for the stress response and is involved in the regulation of the immune system and metabolism. The adrenal gland is composed of an outer cortex and an inner medulla, which have distinct functions. It is a dynamic organ, capable of responding to the variable physiological demand requiring production of steroids and catecholamines. While stem and progenitor cell populations in the adrenal cortex have been identified and characterised, investigation of the adrenal medulla cell hierarchy is still ongoing.The adrenal medulla derives from the neural crest, from which a common progenitor generates both sympathetic neurons and neuroendocrine chromaffin cells. SOX2 is a marker of multiple stem cell/progenitor lineages and is found to be upregulated in many tumours including pheochromocytomas and paragangliomas, tumours of the adrenal medulla.Here we identify and characterise SOX2-positive cells in the murine adrenal medulla during homeostasis. SOX2-positive cells do not colocalise with other known progenitor markers in the medulla. Using genetic lineage tracing in the juvenile postnatal animal, we identify SOX2-positive cells to be an expanding population which gives rise to chromaffin cells. Taken together our data point towards a new candidate progenitor cell population of the adrenal medulla. agouti. These results reveal that early developmental landmarks can shape periodic patterns upstream of late local dynamics, and thus constrain their evolution.Spots, stripes… These periodic patterns often seen in animals have been largely viewed as self-organizing. Do they also depend on preexisting positional information? In juvenile galliform birds we show that signaling from the somite sets the position of stripes in the plumage, while their width is controlled by the expression of  Axial defects (Axd) model, a Grhl2 overexpressing model, has spinal defects. Grhl2 is expressed in the non-neural ectoderm or surface ectoderm, a monolayer of cells covering and wrapping around the neural folds that ultimately will give rise to the epidermis. We investigated the molecular and cellular mechanisms by which either excess or insufficient Grhl2 expression can cause NTDs. We performed RNA-seq experiments on both knockout and overexpressing models (Axd) and identified multiple pathways regulated inversely in both models. Genes encoding proteins of the junctional apical complex were highly regulated in both datasets. We further validated a number of adherens junction molecules (such as E-cadherin) and tight junction molecules (a number of claudins) to be regulated by Grhl2 in the caudal neural tube. Consistent with the fact that these molecules are pivotal for cell-cell adhesion and connected to the cytoskeleton, we also identified abnormalities in F-actin and p-MLCII organization. In addition, absence of Grhl2 altered the nature of the surface ectoderm cells which acquired a more neuroepithelial phenotype. Finally, functional experiments showed that the surface ectoderm cells in the presence of elevated or diminished Grhl2 expression, were under abnormal tension. In conclusion, Grhl2 defines the epithelial nature of the surface ectoderm monolayer, making it a determining factor for neural tube closure.Grainyhead-like proteins (Grhls) are evolutionary conserved transcription factors essential for neural tube closure. Grhl2 knockout models display both cranial and spinal neural tube defects (NTDs), whereas the  ERT; Rosa26RmTmG and Axin2-CreERT; Rosa26Tomato mice were induced with tamoxifen 2 days prior to osteoctomy, whereas Wnt1-Cre; Rosa26Tomato was constitutively active. We found that, within the first week post-surgery, not only a larger number of labelled stem cells reside on the inter-frontal suture when compared to the sagittal, but a higher proportion of them infiltrate the frontal wound in comparison to the parietal. Moreover we provide insights on the neural crest domain at the sagittal suture, which suggest that the position in relation to the surrounding sutures might be a determinant on the efficiency of parietal wound healing.The craniofacial skeleton is formed from the neural crest and mesodermal lineages, both of which contribute mesenchymal precursors during formation of frontal and parietal bones respectively. Increased healing capacity in frontal bones has been previously described. In culture, frontal bone cells are more osteogenic than parietals, as seen by formation of more mineralized nodules. Although differences in molecular signalling and osteogenic potential have been well studied, the recruitment of suture mesenchymal cells and specific stem cell subsets has not yet been investigated. Here we combine lineage tracing techniques in a calvarial wound model to assess the infiltration of previously reported stem cell populations, as well as the availability and recruitment of neural crest derivatives. 2 mm defects were performed on frontal and parietal bones of 6 weeks old mice. Gli1-Cre The dynamic nature of bone remodelling enables skeletal elements to adapt to their environment, thus playing a crucial role in normal development. Here, we investigated the interaction between abnormal bone remodelling (c-Fos knockout mice) and postnatal skull growth by utilising 3D morphometrics to characterise phenotypic change and link such changes to cellular involvement. MicroCT scans indicated a delayed development of c-Fos mutants compared with WT littermates, illustrated by their domed skull phenotype which lacked tooth eruption and displayed altered morphology of several bones: parietal, nasal and mandible. The two principle components (PCs) contributing to overall shape variation were skull height (37%) and width (31%) with the c-Fos mutants occupying the heightened and wider region of morphospace. Interestingly, this phenotype of the mutants increasingly diverged from WT littermates across postnatal ontogeny. Unexpectedly, microCT reconstructions indicated premature suture fusion (craniosynostosis) in c-Fos mutants. In WT mice at early stages of postnatal development (P4), TRAP-positive osteoclasts lined the bone margins along the patent coronal and sagittal sutures. At later stages of development (P20), osteoclast activity had decreased and was associated with increased bone deposition at the suture locale. Our results suggest an unappreciated role for osteoclasts in maintain suture patency across ontogeny.


Many studies exclude loci that exhibit linkage disequilibrium (LD); however, high LD can signal reduced recombination around genomic features such as chromosome inversions or sex-determining regions. Chromosome inversions and sex-determining regions are often involved in adaptation, allowing for the inheritance of co-adapted gene complexes and for the resolution of sexually antagonistic selection through sex-specific partitioning of genetic variants. Genomic features such as these can escape detection when loci with LD are removed; in addition, failing to account for these features can introduce bias to analyses. We examined patterns of LD using network analysis to identify an overlapping chromosome inversion and sex-determining region in chum salmon. The signal of the inversion was strong enough to show up as false population substructure when the entire dataset was analyzed, while the effect of the sex-determining region on population structure was only obvious after restricting analysis to the sex chromosome. Understanding the extent and geographic distribution of inversions is now a critically important part of genetic analyses of natural populations. Our results highlight the importance of analyzing and understanding patterns of LD in genomic dataset and the perils of excluding or ignoring loci exhibiting LD. Blindly excluding loci in LD would have prevented detection of the sex-determining region and chromosome inversion while failing to understand the genomic features leading to high-LD could have resulted in false interpretations of population structure. Eda gene that influences armor plating in stickleback   e.g.,  is increChromosome inversions have been associated with widely divergent adaptive variation ranging from social behavior  from western Alaska. Chum salmon exhibit an anadromous life history where individuals emerge in freshwater and then migrate to the ocean to mature before returning to freshwater streams to spawn. Populations often exhibit life-history variation in the timing of return migration. We used patterns of LD combined with network analysis to discover a chromosomal inversion in chum salmon that contains the sex-determining region. The inversion exhibits spatial variation in frequency throughout western Alaska and includes putatively adaptive genes associated with life-history variation in other salmonids.Here, we describe the detection and genetic architecture of multiple genomic features in chum salmon  using RAD sequencing . These cSbfI enzyme using a modification of the improved RADseq library preparation protocol developed in STACKS v1.47  using bowtie2 v2.2.1  to identify groups of linked SNPs, hereafter referred to as ‘sets’. This was done to determine if multiple patterns of LD were present within a single genomic region. For network analysis, SNP pairs with an r2 < 0.3 were removed. SNPs that were in LD with fewer than 3 other SNPs were also excluded to reduce the number of SNPs that were linked only by close physical proximity. Following network analysis, chromosomal positions of SNPs in LD sets were examined to determine if they could be attributed to genomic structures. Finally, patterns of variation for SNPs within LD sets were visualized using PCA.Genomic features were identified by examining LD between markers. Pairwise LD was estimated between SNPs using the r-squared method in Two putative, co-occurring genomic features were identified from the RADseq data (see results): a chromosome inversion and a sex-determining region.bowtie2; custom perl scripts were used to obtain flanking sequence for primer design. Primers were designed using BatchPrimer3 (GT-seq assays were developed for 39 loci diagnostic for the putative inversion and 21 loci diagnostic for the putative sex-determining region. Multiple loci for each genomic feature were included to provide a control for genotyping error and because some loci would be routinely lost during panel optimization (described below). Filters were applied prior to and during primer design to remove loci that were likely to amplify off-target sequence with GT-seq following the methods of GTscore . All GT-seq loci were used for evaluating sample quality even though loci developed for the separate project are not included in this study. Samples were evaluated for quality based on a minimum 90% genotype rate and visualization of allele scatter plots.An expanded sample of individuals was genotyped using the GT-seq panel to better characterize genomic structure across the region. This included additional individuals from the ascertainment collections as well as additional collections from Norton Sound, and the Yukon, Kuskokwim, and Nushagak rivers . Populathttps://doi.org/10.25387/g3.11903409.Raw RADseq is available in NCBI BioProject PRJNA611968. Raw GT-seq data are available in NCBI BioProject PRJNA609360.The authors affirm that all data necessary for confirming the conclusions of this article are represented fully within the article and its tables and figures. Supplemental material available at figshare: STACKS initially outputted 222,668 SNPs within 94,002 RAD tags. A total of 30,006 SNPs within 22,693 RAD tags were retained after applying all filters (Table S1). Of these loci, 13,015 SNPs within 10,821 RAD tags were aligned to the rainbow trout genome. After all filtering steps, 267 individuals were retained for SNP discovery.O. mykiss chromosome 28. Network analysis identified three distinct sets of loci that exhibited high LD  were plotted by SNP set and position to better visualize genotype patterns ; this plLoci in set 2 exhibited a more complex pattern that is consistent with a sex-determining region. Putative males in the top half of GTscore are in Table S3. A total of 43 individuals were removed for failing quality control: 19 samples had <90% genotype rate and an additional eight had broad allelic scatter due to reduced read depth , five individuals showed elevated heterozygosity and indistinct allelic clusters suggesting contamination , one individual showed four clusters consistent with triploidy , two individuals showed five clusters consistent with tetraploidy , and one individual had no heterozygous genotypes and was likely a different species of salmon .Development of the GT-seq panel involved filtering steps during primer design and panel optimization. After evaluation and optimization, the final GT-seq panel contained 22 SNPs diagnostic for a putative inversion and 18 SNPs diagnostic for a putative sex-determining region. Primer sequences for final loci are located in Table S2 and bioinformatic probes used for genotyping in Inversion type and sex were assigned by clustering samples with PCA ; this PCSimilar to the RADseq data alone, within the full dataset the inversion was found in every collection but showed regional variation in frequency. Inversion frequency ranged from 2 to 22% and was most common in the Lower Yukon collections followed by the Kuskokwim River collections . OutsideAnopheles sp.) (vs. residency in rainbow trout (Oncorhynchus mykiss)  (Genomic structures including inversions and sex-determining regions provide important contributions to adaptive variation and population structure (uttatus) . InhibitPopulation substructure shaped by the inversion and sex-determining region was only determined through a combination of LD and network analysis. Both the inversion and sex-determining region exhibited LD spanning the same genomic region, which would be difficult to ascertain manually. Network analysis and community detection was a simple method to automate detection of different groups of linked markers that contributed to this overall pattern of LD. Previous work has shown that a combination of LD and network analyses facilitates detection of genomic features even in the absence of reference genome (INV, XINVXINV, XY, XINVY. This agrees with the observed pattern of 5 sample clusters on the individual PCA (INV. Levels of LD for sex-associated loci varied depending on the X-chromosome type, suggesting different levels of divergence between the Y-chromosome and the ancestral and inverted X-chromosomes.The extended LD combined with the specific genotype patterns observed for high-LD loci suggests a genomic inversion that overlaps with the sex-determining region in chum salmon. The genotype patterns also suggest that the inversion arose on the X chromosome. An inversion arising on the X chromosome should result in 5 different chromosomal combinations: XX, XXdual PCA . If the Salmonids exhibit sex-specific recombination patterns where females recombine across the full length of chromosomes while recombination in males is restricted to telomeric regions (vs. resident life history in rainbow trout (Greb1L gene. This gene has been associated with variation in the timing of migration return in both Chinook salmon and rainbow trout (An inversion on the X chromosome containing the sex-determining region has interesting evolutionary implications. Females can exhibit all three genotypes of the inversion, while males can be homozygous for the ancestral form or heterozygous for the inversion but never homozygous for the inversion. Inversions can lead to fixation of genetic variants on alternate chromosomes and facilitate adaptive divergence (reviewed in i.e., Chum salmon exhibit two ecotypes that vary in season of migration into freshwater: summer migrants and fall migrants. In the Yukon River, summer chum salmon enter the river between early June and early July and spawn primarily in tributaries in the lower 700 km of the river. Fall chum salmon, the less-abundant ecotype, enter in mid-July to late August and, unlike the majority the chum salmon populations throughout the range, undertake long migrations with populations extending to the headwaters of Yukon River over 2800 km upriver (sdY (sdY has been identified on three different chromosomes (sdY gene in Atlantic salmon. Interestingly, the homeolog of LG15 is orthologous to the sex chromosome in coho salmon, lake whitefish, and sockeye salmon (sdY exon loss (sdY (Sex in salmon is genetically controlled  and is goss (sdY . DespiteExamining genome-wide patterns of LD is an important tool for evolutionary analysis. Many studies explicitly exclude loci with LD; these may be missing important patterns of genomic variation. We identified a co-locating inversion and sex-determining region in chum salmon by performing network analysis on patterns of LD. The signal of these features was strong enough to drive PCA of the full dataset, resulting in false population structure. Attempting to identify and account for genomic features should be standard practice in genome-scale datasets.


Abnormal metabolic pathways have been considered as one of the hallmarks of cancer. While numerous metabolic pathways have been studied in various cancers, the direct link between metabolic pathway gene expression and cancer prognosis has not been established.Using two recently developed bioinformatics analysis methods, we evaluated the prognosis potential of metabolic pathway expression and tumor-vs-normal dysregulations for up to 29 metabolic pathways in 33 cancer types. Results show that increased metabolic gene expression within tumors corresponds to poor cancer prognosis. Meta differential co-expression analysis identified four metabolic pathways with significant global co-expression network disturbance between tumor and normal samples. Differential expression analysis of metabolic pathways also demonstrated strong gene expression disturbance between paired tumor and normal samples.Taken together, these results strongly suggested that metabolic pathway gene expressions are disturbed after tumorigenesis. Within tumors, many metabolic pathways are upregulated for tumor cells to activate corresponding metabolisms to sustain the required energy for cell division.The online version contains supplementary material available at 10.1186/s12864-020-07251-0. Gene expression and metabolism are two essential biological processes critical to all living organisms. Gene expression is the fundamental information flow that transforms the heritable genetic information of individual genes to cellular functioning entities, including constituent proteins and catalytic enzymes. Metabolism refers to the system of all chemical reactions that are synergistically inter-connected to fulfill all respects of viability necessities. Abnormal metabolism has been added to the original six hallmarks of cancer in 2011 . Most reA metabolic pathway consists of a cascade of enzyme-catalyzed chemical reactions occurring in a cell that are orchestrated to fulfill one relatively independent cellular function. In a metabolic pathway, the product of one upstream reaction acts as the substrate for the successive reaction, and all those substrates/products are termed metabolites in general. Many of the metabolites in these pathways have significant implications in cancer. For example, folate exerts its effect on cancer through nutrient-gene interaction as to genes within the folate metabolic pathway. Deficiency of folate has been linked to increased risk of cancers , and a cMany metabolic pathways and corresponding metabolites have been thoroughly studied in cancers. These studies mostly focused on the cancer risk associated with nutrient supplement intake in the case-control type of epidemiology studies, or functional mechanism analysis of the regulatory effects between metabolism and gene expression. Previous pan-cancer metabolic studies –16 have PID [Twenty-nine major metabolic pathways curated by three sources: PID , PANTHERPID , and INOPID  were extPID . Pathwayxi denotes the standardized log-transformed expression value of the ith gene  and the βi denotes the score test coefficient for the same gene from a univariate Cox-proportional hazards regression. The normalization factor m captures the median of the linear combination term  data were downloaded from Genomic Data Commons for this study. Overall, processed RNA-seq data of 11,069 samples from 10,274 subjects of 33 cancer types were downloaded. Out of the 11,096 samples, 795 were adjacent normal tissues, and the rest were tumor tissues. The 33 cancer types alongside their abbreviations and sample sizes are listed in Supplementary Table Sing RNAs .p-value but is devoid of effect size and corresponding standard error. We adapted the GSNCA method into a meta analysis approach where multiple independent datasets can be used to determine the overall significance of co-expression changes between two conditions. To achieve this, we adapted a bootstrap approach to estimate the confidence interval. The effect size of an individual dataset can be estimated using the following formula: p-value. The meta analysis and forest plots were conducted using the generalized linear mixed effect model from R package Metafor [p-value < 0.05 is considered significant. The meta co-expression analysis has been built into a web application and can be accessed freely at http://www.innovebioinfo.com/Gene_Expression_Analysis/Meta_GSCA/MetaGSCA1.php.Our second analysis of metabolic pathways focused on the expression difference between tumor and normal tissues. Two types of differences were interrogated: differential co-expression and differential expression. Differential co-expression analysis was assisted by Gene Sets Net Correlations Analysis (GSNCA) , which i Metafor . In paraComprehensive survival analyses were conducted to evaluate the overall prognosis conferred by metabolic pathway expressions in cancers. Two types of survival data were obtained from publication by Liu et al. : overallp-value with R package heatmap3 [Cluster analysis was conducted on the adjusted heatmap3  using EuFurther exploring OS and DSS analysis results reveal that all significant survival results have the same direction. That is, increased overall gene expression level of a metabolic pathway always corresponds to worse survival outcome. Using MP5 (folate metabolism), an important cancer related metabolic pathway as an example, we plotted the Kaplan-Meier curves for cancer types KIRC, LGG and UVM Fig. c in bothn > 9). Meta differential co-expression analysis was conducted for each metabolic pathway, with 17 cancer types regarded as multiple datasets. Of the 29 metabolic pathways tested, four showed significant co-expression difference. They are folate metabolism, glutamic acid and glutamine metabolism, glycine and serine metabolism, and purine nucleotide metabolism. Folate is an important member of vitamin B complex and it is essential for cell division and homeostasis. Folate metabolism showed significant co-expression disturbance in 11 of the 17 cancer types, thus resulting in a meta p-value of 0.045  and lung squamous cell carcinoma (LUSC) formed a unique cluster branch  cancer-pathway combinations, 328 (66%) showed significantly different expression post multiple test adjustment, signaling significant expression alteration between tumor and normal samples for the metabolic pathways tested. Of these 328 dysregulated cancer-pathway combinations, 126 had higher expression in tumor samples and 202 had higher expression in normal samples Fig. . Some meTumor cells reprogram metabolism to sustain cell proliferation. This is designated as the Warburg effect, which refers to the fact that tumor cells prefer metabolism via glycolysis rather than the efficient oxidative phosphorylation pathway. A myriad of cancer metabolism studies has proved that cancer cells require alternative activation of metabolic pathways to obtain the necessary energy source for cell growth. The direct relation between metabolic pathway expression and cancer prognosis has not been established. Since a metabolic pathway includes multiple genes, survival analyses evaluating a pathway as a single entity has been difficult. Using a newly developed method AESA, we constructed a composite gene expression score to represent the expression level of a single pathway and thus managed to conduct OS and DSS analyses based on these scores. Overall, 166 and 170 cancer-pathway combinations were found to be significantly associated with survival for OS and DSS respectively. All significant results show that higher expression of metabolic pathways resulted in worse survival outcomes. These results strongly suggest that upregulation of metabolic pathway genes reinforced/activated select metabolic pathways, which translated to poor prognosis. Not to confuse with our analysis between tumor and normal, the survival analysis was conducted entirely using tumor samples. As we have shown, the gene expression of metabolic pathways was not always higher in tumor than normal. Additionally, the survival analysis results also show that cancer types have varied survival sensitivity toward metabolic pathway expression change. LGG and KIRC had strong survival associations with all tested metabolic pathways, while some cancer types had no survival association at all.Our second analysis dealt with the co-expression disturbance of metabolic pathways between tumor and normal samples. This analysis identified four metabolic pathways that showed repetitive co-expression disturbance across multiple cancers. They are folate metabolism, glutamic acid and glutamine metabolism, glycine and serine metabolism, and purine nucleotide metabolism. Folate, also known as vitamin B9, is essential for DNA and RNA synthesis and maintenance of methylation reaction in cells. Folate metabolism is pivotal for cell replication and survival. Interruption of folate metabolism pathway may produce substantial toxic effect on cell division process, the key to tumorigenesis. Inhibition of folate metabolism pathway has been used in cancer treatment . It has Our last analysis is the differential gene expression analysis of each metabolic pathway between paired tumor and normal samples. It is important to distinguish this analysis from the aforementioned survival analysis, which was based on only tumor gene expression data. In the survival analysis, we observed universal poor prognosis for higher expression of metabolic pathways. However, in the differential expression analysis, we observed more down regulation of metabolic pathway expression in tumor compared to normal. Most metabolites are nutrients for cellular functions. Healthy metabolisms are essential for normal cell growth. Deficiency in certain metabolites can increase cancer risk. For example, chronicle insufficient intake of folate may increase risk of many cancer types . HoweverOur genomic data source TCGA has several limitations. The OS data in TCGA was subjected to immortal time bias , the biaOur study discovered several important findings. The most novel finding is that higher metabolic pathway expression corresponds to worse survival. The differential co-expression and differential expression analyses demonstrated the disruption of metabolic pathway gene expression between tumor and normal tissues. In summary, these results show that heterogeneous tumor types bear varied sensitivity to metabolic pathway expression changes.Additional file 1.Additional file 2.Additional file 3.Additional file 4.Additional file 5.


A growing number of studies suggest that cancer-associated fibroblasts (CAFs) modulate both myeloid and lymphoid cells through secretion of molecules  and production of the extracellular matrix (ECM), i.e., physical function. Even though targeting functions CAFs is a relevant strategy, published clinical trials solely aimed at targeting the stroma showed disappointing results, despite being based on solid preclinical evidence. Our review dissects the interactions between CAFs and immune cells and explains how a deeper understanding of CAF subpopulations is the cornerstone to propose relevant therapies that will ultimately improve survival of patients with cancer.Cancer-associated fibroblasts (CAFs) are prominent cells within the tumor microenvironment, by communicating with other cells within the tumor and by secreting the extracellular matrix components. The discovery of the immunogenic role of CAFs has made their study particularly attractive due to the potential applications in the field of cancer immunotherapy. Indeed, CAFs are highly involved in tumor immune evasion by physically impeding the immune system and interacting with both myeloid and lymphoid cells. However, CAFs do not represent a single cell entity but are divided into several subtypes with different functions that may be antagonistic. Considering that CAFs are orchestrators of the tumor microenvironment and modulate immune cells, targeting their functions may be a promising strategy. In this review, we provide an overview of (i) the mechanisms involved in immune regulation by CAFs and (ii) the therapeutic applications of CAFs modulation to improve the antitumor immune response and the efficacy of immunotherapy. Control and elimination of tumor cells by the immune system  is a defense system that plays a major role in the prevention of cancers . HoweverThe tumor microenvironment (TME) is actively involved in immune evasion leading to cancer progression and metastasis . This coUpon tissue injury, such as in wound healing or in cancer, they differentiate into myofibroblasts  to repair damaged tissue by secreting the ECM and by interacting with immune cells . These fOnce activated, CAFs produce several molecules to interact with immune cells, such as growth factors and cytokines . ThroughTAMs comprise different subpopulations with distinct functionalities and derive from circulating monocytes or resident tissue macrophages. CAFs enrich the TME with TAMs by promoting monocytes precursors recruitment. In 2012, Ren, et al. showed that mesenchymal stromal cells from lymphoma, one of the precursors for CAFs, led to the intratumoral accumulation of CD11b+Ly6C+circulating monocytes by secreting chemokine-ligand 2 (CCL2) . This chMDSCs are immature myeloid cells that are only present in pathological conditions associated with chronic inflammation. In the case of cancer, they exert an immunosuppressive function by inhibiting T cell functions and promoting carcinogenesis . Like TAThe CTL antitumoral response requires the recognition of tumor epitopes presented by antigen presenting cells (APCs). APCs ingest tumor antigens and process them for presentation to T cells. Further, the activation of naive CD8+ T cells, leading to their proliferation and differentiation, also requires a costimulation signal in addition to antigen recognition. Once activated, tumor antigen-specific T cells are able to recognize and destroy pathologic cells presenting the same epitope. Tumor cells express major histocompatibility complex (MHC) class I molecules but often do not express costimulatory molecules. DCs can recognize antigens from a cell  and activate another cell ; this is called cross-priming . DC moduTANs are associated with poor prognosis in several cancer types, including melanoma, renal, pancreatic, colorectal and gastric cancers . Like TAMast cells are tissue-resident quiescent cells acting as sentinels, releasing cytokines and chemokines when activated. They play an immunosuppressive role in the TME by secreting free adenosine and IL-13 that, respectively, inhibit CTL and activate M2 polarization, as well as promoting MDSCs and regulatory T cells (Tregs). Ellem, et al.  describeThe main immune mechanism of tumor eradication is driven by CTLs specific for tumor antigens. Indeed, a strong intra-tumoral infiltration by CD3+/CD8+ CTLs, and CD45RO+ memory T lymphocytes is correlated with longer OS in most cancers . SeveralAs mentioned above, an enrichment in cytotoxic and memory T-lymphocytes is associated with a favorable prognosis. This infiltration is associated with a Th1-type immune orientation of CD4+ T lymphocytes involving T-bet, STAT-1 and IRF1 transcription factors leading to the local production of IL-12 and interferon γ. In order to counteract this antitumor immune phenotype, CAFs promote a Th2 orientation, mutually exclusive of Th1, leading to the secretion of the immunosuppressive IL-10 cytokine. This Th2 phenotype can be induced either by CAF-secreting Chi3L1, or by orientating DCs to secrete Th2 chemokines, such as IL-13, in breast  and pancTregs are CD4+ T cells whose function is to inhibit or attenuate immune responses. Most of Tregs strongly express CD25  and the transcription factor FoxP3. The prognostic value associated with a high Treg density within the tumor is controversial . A negatNK cells, or type I innate lymphoid cells, are lymphocytes belonging to innate immunity and do not express antigen receptors clonally synthesized by B and T lymphocytes. NK cells exert an antitumor activity by secreting pro-Th1 cytokines or by directly killing tumoral cells through the release of cytotoxic granules containing perforins and granzymes . PreviouCAFs secrete excess deposits of collagenous and noncollagenous ECM along with degradation enzymes that promote metastasis and cancer progression . BesidesFinally, CAF-induced fibrosis generates a high interstitial pressure leading to a poorly vascularized, hypoxic TME, which constrains access to cell nutrients and, thus, cellular phenotype and metabolism. Moreover, a positive-feedback loop exists since fibrosis is enhanced in hypoxic regions . Indeed,The literature predominantly supports the tumor-promoting role of CAFs, but there is also evidence that some CAF subsets restrain tumor activity. This is particularly important because therapeutic ablation of antitumoral CAFs can be deleterious. Indeed, nonspecific depletion of α-SMA+ myofibroblasts and SHH-dependent CAFs in pancreatic cancer generated more aggressive tumors  worsening patient survival ,94,95. CIdentifying tumor-restraining CAFs is not an easy task. First, studies can be conflicting regarding the same marker. As an example, podoplanin-expressing CAFs were shown to be associated with good prognoses in breast  and coloTo date, the exact phenotypes of good tumor-restraining CAFs remains unknown. Some studies suggested that normal quiescent fibroblasts resistant to activation may suppress tumorigenesis and metastasis ,110,111.It is now acknowledged that increasing tumor immunogenicity is relevant to induce cancer regression and prolonged survival in cancer patients . ConsideThe first strategy is to reduce ECM production. The clinical use of all-trans retinoic acid (ATRA) is well established in acute promyelocytic leukemia for its differentiating effect. ATRA has also been associated with ECM suppression and pancreatic stellate cells (pancreatic resident fibroblasts) inhibition  . ATRA isFinally, the hedgehog signaling pathway involving the Sonic Hedgehog (SHH) proteins promoted desmoplasia  and stimThe third therapeutic approach is to target the CAF-secreted molecules that promote the immunosuppressive TME, such as CCL2, CXCL12 and IDO. As mentioned above, the CCR2-CCL2 axis is involved in the recruitment of M2 TAM, and its blockade consequently reduces TAM infiltration . HoweverOverall, combining selective stromal modulation with ICI is being actively explored in early phase trials  since thCAFs are highly involved in tumor immune evasion by physically and chemically impeding the immune system and interacting with both myeloid and lymphoid cells. However, CAFs do not represent a single cell entity but are divided into several subtypes with different functions that may be antagonistic. Targeting CAFs functions is a promising strategy currently being explored in early phase trials. However, published clinical trials solely aimed at targeting the stroma showed disappointing results, despite being based on solid preclinical evidence. This shows that a deeper understanding of CAF subpopulations and heterogeneity in the context of immune evasion is the cornerstone to propose relevant therapies that will ultimately improve survival of patients with cancers . To achi


Hepatitis B virus (HBV) infection affects ~350 million people and poses a major public health problem worldwide. HBV is a major cause of cirrhosis and hepatocellular carcinoma. Fewer than 5% of HBV-infected adults (but up to 90% of HBV-infected infants and children) develop chronic HBV infection as indicated by continued, detectable expression of hepatitis B surface antigen (HBsAg) for at least 6 months after the initial infection. Increasing evidence indicates that HBV interacts with innate immunity signaling pathways of hepatocytes to suppress innate immunity. However, it is still not clear how HBV avoids monitoring by the innate immunity of hepatocytes and whether the innate immunity of hepatocytes can be effective against HBV if re-triggered. Moreover, a deep understanding of virus–host interactions is important in developing new therapeutic strategies for the treatment of HBV infection. In this review, we summarize the current knowledge regarding how HBV represses innate immune recognition, as well as recent progress with respect to in vitro models for studying HBV infection and innate immunity. Chronic infection with hepatitis B virus (HBV) is the main cause of liver cirrhosis and hepatocellular carcinoma (HCC) worldwide. Although there is a prophylactic vaccine to prevent HBV infection, HBV still infects ~350 million people and contributes toward viral hepatitis-associated morbidity and mortality. The HBV genome consists of a partially double-stranded DNA molecule approximately 3.2 kb long that replicates via an RNA intermediate ,2,3. BasIt is widely accepted that the adaptive immune responses play vital roles in the clearance of HBV infection. Early innate immune response is essential for the following induction of adaptive immunity. Restoring or boosting innate immunity linked to adaptive immunity may help eradicate chronic HBV infection. However, the role of innate immunity during HBV infection appears not to be well understood, which can be attributed to the fact that the recruitment of patients in the very early, asymptomatic phase of HBV infection is very difficult ,6. HBV bIn this review article, we summarize the recent knowledge and debate regarding HBV and innate immunity signaling pathways in hepatocytes, the different HBV proteins that target innate immunity, and different advanced in vitro cell models for the study of virus–host interactions. Collation of such information will provide new insights into novel therapeutic treatments for chronic hepatitis B infection (CHB).HBV is a member of the hepadnavirus family. The genetic organization HBV is shown in After entry into hepatocytes, the HBV genome is delivered into the nucleus where it is repaired to form covalently closed circular DNA (cccDNA) that serves as a template for the pregenomic RNA (pgRNA) and subgenomic RNAs for translation to HBV proteins , HBsAg, hepatitis B X protein (HBx), the core protein, and the precore protein ). The prGlobally, chronic hepatitis B infection affects around 350 million people worldwide , resulting in 600,000 deaths annually from cirrhosis, liver failure, and hepatocellular carcinoma . The numInnate immunity is the first line of defense against microbial pathogens, including viruses. Viral infection triggers the induction of type-I interferons  and other proinflammatory cytokines through two distinct signaling pathways .One of these pathways utilizes a subfamily of Toll-like receptors  to detect HBV nucleic acids in the endosome after the endocytosis of viral particles. These TLRs are localized in the endosomal membranes of specialized cell types, such as plasmacytoid dendritic cells (pDCs) , and theThe other HBV/innate immunity signaling pathway utilizes the retinoic acid inducible gene I (RIG-I) to detect HBV double-stranded RNA (dsRNA) in the cytosol . RIG-I bThe production of pro-inflammatory cytokines and IFNs and the activation of natural killer (NK) cells is frequently observed during the early phase of viral infections. Previously, HBV was considered as a “stealth virus” that could establish persistent infection in hepatocytes by evading the host innate immune system . Using aOn the other hand, in another study Mutz et al. elucidated the interactions between HBV and innate immunity to better understand the mechanisms of immune activation in HBV infection and reported that HBV does not trigger the IFN response of hepatocytes or interfere with the innate immune-sensing functions of hepatocytes, and nor does it inhibit the IFN-stimulated pathways of hepatocytes based on absence of interferon and cytokine production . On the In conclusion, these studies indicate that HBV can target the TLR system and thus attenuate the anti-HBV responses of the innate immune system, which implies that HBV does not affect at least some aspects of the innate immunity system. HBV, particularly HBx protein, was reported to disrupt RIG-I-mediated IFN-β induction by downregulating MAVS ,41. HBx TLR expression on Kupffer cells, peripheral monocytes, and hepatocytes is reduced in HBeAg-positive CHB compared with patients with HBeAg-negative CHB . HoweverHBV core protein interfered with IFN-β expression through binding to the IFN-β promoter as a transacting silencer in murine fibroblasts . SubsequPrevious reports have also shown that HBx promotes MAVS ubiquitination to trigger its proteasome-mediated degradation through the Lys136 site, and that MAVS K136R elicits a higher level of IFN-β activation compared with wild type MAVS, suggesting that the MAVS Lys136 site could be the ubiquitination site targeted by the ubiquitin E3 ligase RNF125 , enablinThree observations are worthy of mention to discuss the impact of HBV Pol on IFN signaling. First, previous data indicated the inhibitory effect of HBV Pol on IRF signaling and to a lesser extent on NF-κB signaling, which also contributes to IFN-β production. Second, abundant detection of HBV Pol in the nonencapsidated state indicates that HBV Pol could contribute to viral pathogenesis or immune evasion. Lastly, HBV Pol has been previously identified as one of the viral proteins that confers resistance to IFN treatment . PreviouIn the current review, we set out to elucidate the interactions between HBV and innate immunity to better understand the mechanisms of immune activation in HBV infection. Previously, HBV was considered a “stealth” virus that did not interfere with the innate immune-sensing functions of hepatocytes. However, data obtained from recent studies suggest that circulating innate immune cells, as well as liver cell populations, can sense and respond to HBV infection, which enables the innate immune system to detect and restrict the invading virus. In addition, it is worth noting that HBV induced IFN responses in hepatocytes are relatively weak as compared with other viral infections, which is consistent with the observations from the studies obtained in chimpanzee  and mousIn contrast, several other studies have reported that HBV replication can inhibit those aforementioned PRR functions. One study showed that HBeAg-positive chronic hepatitis B patients have downregulated TLR2 expression on hepatocytes . One stuWe believe that contradictory reports are mainly related to three aspects. The first is the infection stage. It is important to find evidence of innate immune responses in the early phase of acute hepatitis B infection even though no innate immune responses are observed in patients with chronic HBV. The second aspect is related to the experimental models. It is critical how similar the innate immunity pathways are in these models and primary human hepatocytes (PHH). The third aspect relates to the HBV genotypes. There are differences in innate immunity response among different HBV genotypes.To date, HBV research has been hampered by a distinct lack of robust infectious model systems that both support productive HBV infection and accurately mimic virus–host interactions. Hepatoma-derived cell lines support HBV replication and particle assembly following the transfection of cloned viral genomes ,63. AfteHowever, there is thus an urgent need for a novel in vitro HBV infection model. Recently, human hepatocyte-like cells differentiated from hESCs and hiPSCs have gained much attention not only due to their promise for regenerative medicine, but also due to their potential for modeling drug metabolism and pathogen infection in vitro ,72,73,74The innate immune system is the first line of host defense against HBV infection and plays an important role in anti-HBV. HBV only triggers little innate response in host cells after infection and has evolved escape strategies from the innate immune system. HBV proteins, including HBx, HBV polymerase, HBs, HBc, and HBe, block TLRs, the Jak-Stat pathway (type I/III IFN response), TBK1/IKKε (the effector kinase of the IRF-3/NF-κB pathway), and cytokines.Current evidence obtained from experiments has highlighted the importance of innate immunity in the early control of HBV spread. However, how HBV succeeds to avoid innate immune recognition has still not been fully clarified. The recent development of varied novel models, such as HepaRG cells, stem-cell derived hepatocytes, and liver organoids, may greatly contribute to clarifying the interaction between HBV and host innate immunity. Given the central role that innate immunity plays in antiviral responses, the enhancement of HBV-induced innate immunity may provide fundamental insights into the therapy of HBV. We believe that a better understanding of HBV–host interactions will be achieved by using these novel models, eventually leading to effective treatment strategies for hepatitis B.


The fecal viral load was positively associated with disease severity and negatively associated with serum anti-rotavirus immunoglobin A. High and persistent rotavirus shedding among vaccinated children with breakthrough disease may contribute to ongoing transmission in this setting.Quantifying rotavirus shedding among vaccinated individuals will aid understanding of vaccine indirect effects. Serial stool samples were collected from 196 children who presented with rotavirus gastroenteritis to health facilities in Blantyre, Malawi, and were tested for rotavirus using a VP6 semi-quantitative, real-time polymerase chain reaction. The median duration of fecal shedding was 28 days . The median copy numbers for peak shedding were 1.99 × 10 Serial stool samples from Malawian children with rotavirus gastroenteritis tested for rotavirus using polymerase chain reaction had high levels of viral shedding despite vaccination. High and persistent rotavirus shedding among vaccinated children with breakthrough disease may contribute to ongoing transmission. Rotavirus vaccine has been introduced in over 90 countries worldwide, including 45 low-income or GAVI Alliance-eligible countries . HoweverThe dynamics of fecal shedding of rotavirus in unvaccinated populations has been examined in children with both asymptomatic rotavirus infection and with clinical rotavirus disease using electron microscopy, enzyme-immune assay, and polymerase chain reaction (PCR) . Fecal sThere is a lack of shedding data from vaccinated infants in any setting, including infants from sub-Saharan Africa, in whom differences in intestinal integrity, nutritional state, comorbidities, and immune responses may lead to variations in shedding, with concomitant impacts on transmission. We aimed to describe patterns of wild-type rotavirus shedding over time and identify the factors associated with fecal viral load in children age-eligible for vaccination with symptomatic rotavirus disease in Malawi, an LIC in southern Africa. The live oral monovalent rotavirus vaccine was introduced into Malawi’s national immunization program on 29 October 2012, with doses at 6 and 10 weeks of age.Children age-eligible for vaccination (born on or after 12 September 2012 and >6 weeks old) presenting with acute gastroenteritis to Queen Elizabeth Central Hospital and to 3 government health centers in Blantyre, Malawi, between 16 February 2015 and 11 November 2016 were tested for rotavirus using immunochromatographic tests on a fecal sample. Following informed consent, rotavirus-positive children had demographic data and 2 mls of serum collected at recruitment to measure anti-rotavirus immunoglobin (Ig) A titres; a second bulk stool sample was obtained 48 hours after presentation (primary cohort). A subset of children had more intensive stool sampling carried out for 28 days (intensive cohort); samples were collected daily from the time of presentation for the first 7 days after symptom onset, twice weekly from 7 until 14 days, and weekly from day 14 until day 28.Disease severity was defined using the 20-point Vesikari score . Severe acute malnutrition was defined by either a weight-for-height Z-score (WHZ) less than -3 standard deviations, a mid–upper arm circumference <115 mm, or nutritional edema . WeightsStool samples were tested for wild-type rotavirus using a real-time semi-quantitative reverse transcription PCR . An addiSerum anti-rotavirus IgA titres were measured using a semi-quantitative sandwich enzyme-linked immunosorbent assay . ResultsP value of ≤.1 on a univariate analysis were selected for evaluation in the multivariable model. Nested models were compared using F tests. Variables which improved the model fit (P ≤ .05) were retained in the final model.The viral load data did not follow a normal distribution, so were log-transformed for analysis. Risk factors for fecal viral shedding density were investigated using multivariable linear regression, where the outcome variable was the peak log-viral load  in children from the primary cohort. Variables achieving a Wald test Data from the intensive cohort were used to evaluate changes in viral load over time using linear mixed models with a random intercept to account for within-child clustering; for this analysis, all data were included, regardless of viral load. Polynomial terms (quadratic and cubic) were included to account for the non-linear relationship between fecal viral load and time.A time-to-event analysis was used to estimate the duration of viral shedding in children from the intensive cohort. The event of interest was defined as the cessation of shedding and the start time for analysis was the onset of symptoms. Cessation of shedding was defined as the last time point from which rotavirus could be detected until censoring. Thus, an individual with no detectable rotavirus at a given analytical time point but who was shedding rotavirus in subsequent samples was classified as having ongoing shedding at the time of analysis. The follow-up was limited to 28 days.We recruited 196 index children, from whom 374 fecal samples were collected . The 2 unvaccinated children had peak viral loads comparable to the vaccinated children . In a multivariable analysis, a positive association between peak shedding density and Vesikari score and a negative association between peak shedding density and IgA titres were identified  protection appears limited despite high vaccine coverage . HoweverIncreasing anti-rotavirus IgA titres at presentation were negatively associated with fecal viral load, independent of disease severity. Serum anti-rotavirus IgA titres are known to correlate with intestinal IgA titres , and it We found that rotavirus viral shedding density is negatively associated with WHZ, implying that increasingly malnourished children shed more of the virus. Careful interpretation is required, as the standard deviations for our anthropometric measurements are outside the World Health Organization range for data quality assessment purposes . NeverthChildren in Malawi shed large quantities of rotavirus for an extended period following an episode of moderate-to-severe diarrhea, despite prior vaccination. Persistently high fecal virus shedding may contribute to the high prevalence of asymptomatic infections in young children in the community and to ongoing rotavirus transmission. While reduced disease severity in Malawian children was associated with lower viral shedding density, the potential impact of this on population-level rotavirus transmission remains to be determined.Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at The jiz612_suppl_TableS1Click here for additional data file.jiz612_suppl_TableS2Click here for additional data file.jiz612_suppl_TableS3Click here for additional data file.jiz612_suppl_TableS4Click here for additional data file.