Isolation and identification of chlorate-reducing Hafnia sp. from milk.

Chlorate has become a concern in the food and beverage sector, related to chlorine sanitizers in industrial food production and water treatment. It is of particular concern to regulatory bodies due to the negative health effects of chlorate exposure. This study investigated the fate of chlorate in raw milk and isolated bacterial strains of interest responsible for chlorate breakdown. Unpasteurized milk was demonstrated to have a chlorate-reducing capacity, breaking down enriched chlorate to undetectable levels in 11 days. Further enrichment and isolation using conditions specific to chlorate-reducing bacteria successfully isolated three distinct strains of Hafnia paralvei. Chlorate-reducing bacteria were observed to grow in a chlorate-enriched medium with lactate as an electron donor. All isolated strains were demonstrated to reduce chlorate in liquid medium; however, the exact mechanism of chlorate degradation was not definitively identified in this study.

Panel sequencing links rare, likely damaging gene variants with distinct clinical phenotypes and outcomes in juvenile-onset SLE.

OBJECTIVES: Juvenile-onset systemic lupus erythematosus (jSLE) affects 15-20% of lupus patients. Clinical heterogeneity between racial groups, age groups and individual patients suggests variable pathophysiology. This study aimed to identify highly penetrant damaging mutations in genes associated with SLE/SLE-like disease in a large national cohort (UK JSLE Cohort Study) and compare demographic, clinical and laboratory features in patient sub-cohorts with 'genetic' SLE vs remaining SLE patients. METHODS: Based on a sequencing panel designed in 2018, target enrichment and next-generation sequencing were performed in 348 patients to identify damaging gene variants. Findings were integrated with demographic, clinical and treatment related datasets. RESULTS: Damaging gene variants were identified in ∼3.5% of jSLE patients. When compared with the remaining cohort, 'genetic' SLE affected younger children and more Black African/Caribbean patients. 'Genetic' SLE patients exhibited less organ involvement and damage, and neuropsychiatric involvement developed over time. Less aggressive first line treatment was chosen in 'genetic' SLE patients, but more second and third line agents were used. 'Genetic' SLE associated with anti-dsDNA antibody positivity at diagnosis and reduced ANA, anti-LA and anti-Sm antibody positivity at last visit. CONCLUSION: Approximately 3.5% of jSLE patients present damaging gene variants associated with younger age at onset, and distinct clinical features. As less commonly observed after treatment induction, in 'genetic' SLE, autoantibody positivity may be the result of tissue damage and explain reduced immune complex-mediated renal and haematological involvement. Routine sequencing could allow for patient stratification, risk assessment and target-directed treatment, thereby increasing efficacy and reducing toxicity.

Emerging principles for the allocation of academic work in universities.

Internationally, much has changed in the governance of universities since the adoption of corporate management approaches. A strong focus on efficiency, productivity and accountability arising from these approaches has been well documented in the literature. Reductions in government funding have caused universities to become more competitive and entrepreneurial. However, little is known about the impacts of these changes on the working lives of individual academics. This paper is part of an ongoing study exploring the lived experiences of 2526 Australian academics who responded to a national questionnaire. This paper builds on earlier work by holistically drawing together the earlier findings which separately analysed the teaching, research and administration/service aspects of their work. In examining the effectiveness of universities through the ability of their academics to undertake their roles, we found the voices of academics that need to be heard in the development and implementation of key policies, such as academic workload and performance, to preserve the essentially self-managed nature of their work. By combining the learning from the project through the literature review, the statistical analysis and themes from the open-ended questions, we developed a set of principles to underpin these policies in universities. These principles can guide universities to shift towards a more collaborative working relationship with academics, based on trust, and actively encourage them to play be more active in institutional decision-making, especially in relation to policies that directly affect their work. These results have implications for improving the productivity of academics and the institutions in which they work.

Life at the academic coalface: validation of a holistic academic workload estimation tool.

This paper reports on research exploring the academic workload and performance practices of Australian universities. This research has identified a suite of activities associated with teaching, research and service, each with an associated time value (allocation). This led to the development of the academic workload estimation tool (AWET). In 2020, to validate the findings, we contacted academics willing to participate further and conducted interviews. We used the AWET to estimate workload for each individual for the previous year and compared it to the workload allocated according to their institutional workload model. Discrepancies were then discussed to ascertain to what extent the AWET was able to capture their work. In general, the participants thought the AWET provided a more realistic estimate of their actual work and highlighted how much is underestimated or unaccounted for by the workload models used within their institutions. It also showed how academic performance policies, focussed primarily on research output, disadvantaged many individuals because they ignored or minimised many scholarly, teaching and service-related tasks inherent in the academic role. Overall, the findings showed the AWET was a useful tool to discuss academic work and assisted them to better capture the complexity and extent of what they did. We offer the AWET as a validated approach for academics to estimate their workload in a holistic and transparent manner. We suggest its implementation institution-wide, along with an aligned performance policy, will facilitate negotiation of reasonable performance expectations. This will rebuild trust in the processes and improve a university's effectiveness.

Hidden variation in polyploid wheat drives local adaptation.

Wheat has been domesticated into a large number of agricultural environments and has the ability to adapt to diverse environments. To understand this process, we survey genotype, repeat content, and DNA methylation across a bread wheat landrace collection representing global genetic diversity. We identify independent variation in methylation, genotype, and transposon copy number. We show that these, so far unexploited, sources of variation have had a significant impact on the wheat genome and that ancestral methylation states become preferentially "hard coded" as single nucleotide polymorphisms (SNPs) via 5-methylcytosine deamination. These mechanisms also drive local adaption, impacting important traits such as heading date and salt tolerance. Methylation and transposon diversity could therefore be used alongside SNP-based markers for breeding.

Assessment of the impact of mitochondrial genotype upon drug-induced mitochondrial dysfunction in platelets derived from healthy volunteers.

Mitochondrial DNA (mtDNA) is highly polymorphic and encodes 13 proteins which are critical to the production of ATP via oxidative phosphorylation. As mtDNA is maternally inherited and undergoes negligible recombination, acquired mutations have subdivided the human population into several discrete haplogroups. Mitochondrial haplogroup has been found to significantly alter mitochondrial function and impact susceptibility to adverse drug reactions. Despite these findings, there are currently limited models to assess the effect of mtDNA variation upon susceptibility to adverse drug reactions. Platelets offer a potential personalised model of this variation, as their anucleate nature offers a source of mtDNA without interference from the nuclear genome. This study, therefore, aimed to determine the effect of mtDNA variation upon mitochondrial function and drug-induced mitochondrial dysfunction in a platelet model. The mtDNA haplogroup of 383 healthy volunteers was determined using next-generation mtDNA sequencing (Illumina MiSeq). Subsequently, 30 of these volunteers from mitochondrial haplogroups H, J, T and U were recalled to donate fresh, whole blood from which platelets were isolated. Platelet mitochondrial function was tested at basal state and upon treatment with compounds associated with both mitochondrial dysfunction and adverse drug reactions, flutamide, 2-hydroxyflutamide and tolcapone (10-250 µM) using extracellular flux analysis. This study has demonstrated that freshly-isolated platelets are a practical, primary cell model, which is amenable to the study of drug-induced mitochondrial dysfunction. Specifically, platelets from donors of haplogroup J have been found to have increased susceptibility to the inhibition of complex I-driven respiration by 2-hydroxyflutamide. At a time when individual susceptibility to adverse drug reactions is not fully understood, this study provides evidence that inter-individual variation in mitochondrial genotype could be a factor in determining sensitivity to mitochondrial toxicants associated with costly adverse drug reactions.

A modified sequence capture approach allowing standard and methylation analyses of the same enriched genomic DNA sample.

BACKGROUND: Bread wheat has a large complex genome that makes whole genome resequencing costly. Therefore, genome complexity reduction techniques such as sequence capture make re-sequencing cost effective. With a high-quality draft wheat genome now available it is possible to design capture probe sets and to use them to accurately genotype and anchor SNPs to the genome. Furthermore, in addition to genetic variation, epigenetic variation provides a source of natural variation contributing to changes in gene expression and phenotype that can be profiled at the base pair level using sequence capture coupled with bisulphite treatment. Here, we present a new 12 Mbp wheat capture probe set, that allows both the profiling of genotype and methylation from the same DNA sample. Furthermore, we present a method, based on Agilent SureSelect Methyl-Seq, that will use a single capture assay as a starting point to allow both DNA sequencing and methyl-seq. RESULTS: Our method uses a single capture assay that is sequentially split and used for both DNA sequencing and methyl-seq. The resultant genotype and epi-type data is highly comparable in terms of coverage and SNP/methylation site identification to that generated from separate captures for DNA sequencing and methyl-seq. Furthermore, by defining SNP frequencies in a diverse landrace from the Watkins collection we highlight the importance of having genotype data to prevent false positive methylation calls. Finally, we present the design of a new 12 Mbp wheat capture and demonstrate its successful application to re-sequence wheat. CONCLUSIONS: We present a cost-effective method for performing both DNA sequencing and methyl-seq from a single capture reaction thus reducing reagent costs, sample preparation time and DNA requirements for these complementary analyses.

Investigating the Influence of Ribavirin on Human Respiratory Syncytial Virus RNA Synthesis by Using a High-Resolution Transcriptome Sequencing Approach.

UNLABELLED: Human respiratory syncytial virus (HRSV) is a major cause of serious respiratory tract infection. Treatment options include administration of ribavirin, a purine analog, although the mechanism of its anti-HRSV activity is unknown. We used transcriptome sequencing (RNA-seq) to investigate the genome mutation frequency and viral mRNA accumulation in HRSV-infected cells that were left untreated or treated with ribavirin. In the absence of ribavirin, HRSV-specific transcripts accounted for up to one-third of total RNA reads from the infected-cell RNA population. Ribavirin treatment resulted in a >90% reduction in abundance of viral mRNA reads, while at the same time no such reduction was detected for the abundance of cellular transcripts. The presented data reveal that ribavirin significantly increases the frequency of HRSV-specific RNA mutations, suggesting a direct influence on the fidelity of the HRSV polymerase. The presented data show that transitions and transversions occur during HRSV replication and that these changes occur in hot spots along the HRSV genome. Examination of nucleotide substitution rates in the viral genome indicated an increase in the frequency of transition but not transversion mutations in the presence of ribavirin. In addition, our data indicate that in the continuous cell types used and at the time points analyzed, the abundances of some HRSV mRNAs do not reflect the order in which the mRNAs are transcribed. IMPORTANCE: Human respiratory syncytial virus (HRSV) is a major pediatric pathogen. Ribavirin can be used in children who are extremely ill to reduce the amount of virus and to lower the burden of disease. Ribavirin is used as an experimental therapy with other viruses. The mechanism of action of ribavirin against HRSV is not well understood, although it is thought to increase the mutation rate of the viral polymerase during replication. To investigate this hypothesis, we used a high-resolution approach that allowed us to determine the genetic sequence of the virus to a great depth of coverage. We found that ribavirin did not cause a detectable change in the relative amounts of viral mRNA transcripts. However, we found that ribavirin treatment did indeed cause an increase in the number of mutations, which was associated with a decrease in virus production.

A clinical database to assess action levels and tolerances for the ongoing use of Mobius3D.

In radiation therapy, calculation of dose within the patient contains inherent uncertainties, inaccuracies, limitations, and the potential for random error. Thus, point dose-independent verification of such calculations is a well-established process, with published data to support the setting of both action levels and tolerances. Mobius3D takes this process one step further with a full independent calculation of patient dose and comparisons of clinical parameters such as mean target dose and voxel-by-voxel gamma analysis. There is currently no published data to directly inform tolerance levels for such parameters, and therefore this work presents a database of 1000 Mobius3D results to fill this gap. The data are tested for normality using a normal probability plot and found to fit this distribution for three sub groups of data; Eclipse, iPlan and the treatment site Lung. The mean (µ) and standard deviation (σ) of these sub groups is used to set action levels and tolerances at µ ± 2σ and µ ± 3σ, respectively. A global (3%, 3 mm) gamma tolerance is set at 88.5%. The mean target dose tolerance for Eclipse data is the narrowest at ± 3%, whilst iPlan and Lung have a range of -5.0 to 2.2% and -1.8 to 5.0%, respectively. With these limits in place, future results failing the action level or tolerance will fall within the worst 5% and 1% of historical results and an informed decision can be made regarding remedial action prior to treatment.

A comprehensive benchmarking study of protocols and sequencing platforms for 16S rRNA community profiling.

BACKGROUND: In the last 5 years, the rapid pace of innovations and improvements in sequencing technologies has completely changed the landscape of metagenomic and metagenetic experiments. Therefore, it is critical to benchmark the various methodologies for interrogating the composition of microbial communities, so that we can assess their strengths and limitations. The most common phylogenetic marker for microbial community diversity studies is the 16S ribosomal RNA gene and in the last 10 years the field has moved from sequencing a small number of amplicons and samples to more complex studies where thousands of samples and multiple different gene regions are interrogated. RESULTS: We assembled 2 synthetic communities with an even (EM) and uneven (UM) distribution of archaeal and bacterial strains and species, as metagenomic control material, to assess performance of different experimental strategies. The 2 synthetic communities were used in this study, to highlight the limitations and the advantages of the leading sequencing platforms: MiSeq (Illumina), The Pacific Biosciences RSII, 454 GS-FLX/+ (Roche), and IonTorrent (Life Technologies). We describe an extensive survey based on synthetic communities using 3 experimental designs (fusion primers, universal tailed tag, ligated adaptors) across the 9 hypervariable 16S rDNA regions. We demonstrate that library preparation methodology can affect data interpretation due to different error and chimera rates generated during the procedure. The observed community composition was always biased, to a degree that depended on the platform, sequenced region and primer choice. However, crucially, our analysis suggests that 16S rRNA sequencing is still quantitative, in that relative changes in abundance of taxa between samples can be recovered, despite these biases. CONCLUSION: We have assessed a range of experimental conditions across several next generation sequencing platforms using the most up-to-date configurations. We propose that the choice of sequencing platform and experimental design needs to be taken into consideration in the early stage of a project by running a small trial consisting of several hypervariable regions to quantify the discriminatory power of each region. We also suggest that the use of a synthetic community as a positive control would be beneficial to identify the potential biases and procedural drawbacks that may lead to data misinterpretation. The results of this study will serve as a guideline for making decisions on which experimental condition and sequencing platform to consider to achieve the best microbial profiling.

Genome-wide DNA methylation patterns in wild samples of two morphotypes of threespine stickleback (Gasterosteus aculeatus).

Epigenetic marks such as DNA methylation play important biological roles in gene expression regulation and cellular differentiation during development. To examine whether DNA methylation patterns are potentially associated with naturally occurring phenotypic differences, we examined genome-wide DNA methylation within Gasterosteus aculeatus, using reduced representation bisulfite sequencing. First, we identified highly methylated regions of the stickleback genome, finding such regions to be located predominantly within genes, and associated with genes functioning in metabolism and biosynthetic processes, cell adhesion, signaling pathways, and blood vessel development. Next, we identified putative differentially methylated regions (DMRs) of the genome between complete and low lateral plate morphs of G. aculeatus. We detected 77 DMRs that were mainly located in intergenic regions. Annotations of genes associated with these DMRs revealed potential functions in a number of known divergent adaptive phenotypes between G. aculeatus ecotypes, including cardiovascular development, growth, and neuromuscular development.

A simple procedure for directly obtaining haplotype sequences of diploid genomes.

BACKGROUND: Almost all genome sequencing projects neglect the fact that diploid organisms contain two genome copies and consequently what is published is a composite of the two. This means that the relationship between alternate alleles at two or more linked loci is lost. We have developed a simplified method of directly obtaining the haploid sequences of each genome copy from an individual organism. RESULTS: The diploid sequences of three groups of cattle samples were obtained using a simple sample preparation procedure requiring only a microscope and a haemocytometer. Samples were: 1) lymphocytes from a single Angus steer; 2) sperm cells from an Angus bull; 3) lymphocytes from East African Zebu (EAZ) cattle collected and processed in a field laboratory in Eastern Kenya. Haploid sequence from a fosmid library prepared from lymphocytes of an EAZ cow was used for comparison. Cells were serially diluted to a concentration of one cell per microlitre by counting with a haemocytometer at each dilution. One microlitre samples, each potentially containing a single cell, were lysed and divided into six aliquots (except for the sperm samples which were not divided into aliquots). Each aliquot was amplified with phi29 polymerase and sequenced. Contigs were obtained by mapping to the bovine UMD3.1 reference genome assembly and scaffolds were assembled by joining adjacent contigs that were within a threshold distance of each other. Scaffolds that appeared to contain artefacts of CNV or repeats were filtered out leaving scaffolds with an N50 length of 27-133 kb and a 88-98 % genome coverage. SNP haplotypes were assembled with the Single Individual Haplotyper program to generate an N50 size of 97-201 kb but only ~27-68 % genome coverage. This method can be used in any laboratory with no special equipment at only slightly higher costs than conventional diploid genome sequencing. A substantial body of software for analysis and workflow management was written and is available as supplementary data. CONCLUSIONS: We have developed a set of laboratory protocols and software tools that will enable any laboratory to obtain haplotype sequences at only modestly greater cost than traditional mixed diploid sequences.

Transcriptomic analysis of Shiga-toxigenic bacteriophage carriage reveals a profound regulatory effect on acid resistance in Escherichia coli.

Shiga-toxigenic bacteriophages are converting lambdoid phages that impart the ability to produce Shiga toxin to their hosts. Little is known about the function of most of the genes carried by these phages or the impact that lysogeny has on the Escherichia coli host. Here we use next-generation sequencing to compare the transcriptomes of E. coli strains infected with an Stx phage, before and after triggering of the bacterial SOS response that initiates the lytic cycle of the phage. We were able to discriminate between bacteriophage genes expressed in the lysogenic and lytic cycles, and we describe transcriptional changes that occur in the bacterial host as a consequence of Stx phage carriage. Having identified upregulation of the glutamic acid decarboxylase (GAD) operon, confirmed by reverse transcription-quantitative PCR (RT-qPCR), we used phenotypic assays to establish the ability of the Stx prophage to confer a greater acid resistance phenotype on the E. coli host. Known phage regulators were overexpressed in E. coli, and the acid resistance of the recombinant strains was tested. The phage-encoded transcriptional regulator CII was identified as the controller of the acid response in the lysogen. Infection of an E. coli O157 strain, from which integrated Stx prophages were previously removed, showed increased acid resistance following infection with a nontoxigenic phage, ϕ24B. In addition to demonstrating this link between Stx phage carriage and E. coli acid resistance, with its implications for survival postingestion, the data set provides a number of other potential insights into the impact of lambdoid phage carriage on the biology of E. coli.

Population genomics of the killer whale indicates ecotype evolution in sympatry involving both selection and drift.

The evolution of diversity in the marine ecosystem is poorly understood, given the relatively high potential for connectivity, especially for highly mobile species such as whales and dolphins. The killer whale (Orcinus orca) has a worldwide distribution, and individual social groups travel over a wide geographic range. Even so, regional populations have been shown to be genetically differentiated, including among different foraging specialists (ecotypes) in sympatry. Given the strong matrifocal social structure of this species together with strong resource specializations, understanding the process of differentiation will require an understanding of the relative importance of both genetic drift and local adaptation. Here we provide a high-resolution analysis based on nuclear single-nucleotide polymorphic markers and inference about differentiation at both neutral loci and those potentially under selection. We find that all population comparisons, within or among foraging ecotypes, show significant differentiation, including populations in parapatry and sympatry. Loci putatively under selection show a different pattern of structure compared to neutral loci and are associated with gene ontology terms reflecting physiologically relevant functions (e.g. related to digestion). The pattern of differentiation for one ecotype in the North Pacific suggests local adaptation and shows some fixed differences among sympatric ecotypes. We suggest that differential habitat use and resource specializations have promoted sufficient isolation to allow differential evolution at neutral and functional loci, but that the process is recent and dependent on both selection and drift.

Acute potentiating effect of depth jumps on sprint performance.

The purpose of this investigation was to determine whether the addition of 3 depth jumps to a dynamic warm-up (DYNDJ) protocol would significantly improve 20-m sprint performance when compared with a cardiovascular (C) warm-up protocol or a dynamic (DYN) stretching protocol alone. The first part of the study identified optimal drop height for all subjects using the maximum jump height method. The identified optimal drop heights were later used during the DYNDJ protocol. The second part compared the 3 warm-up protocols above to determine their effect on 20-m sprint performance. Twenty-nine subjects (age, 20.8 ± 4.4 years; weight, 82.6 ± 9.9 kg; height, 180.3 ± 6.2 cm) performed 3 protocols of a C protocol, a DYN protocol, and a DYNDJ protocol in a randomized order. A 20-m sprint was performed 1 minute after the completion of each of the 3 protocols. Results displayed significant differences between each of the 3 protocols. A significant improvement (p = 0.001) of 2.2% was obtained in sprint time between the C protocol (3.300 ± 0.105 seconds) and the DYN protocol (3.227 ± 0.116 seconds), a further significant improvement of 5.01% was attained between the C and the DYNDJ protocols (3.300 ± 0.10 vs. 3.132 ± 0.120 seconds; p = 0.001). In addition, a significant improvement (p = 0.001) of 2.93% was observed between the DYN protocol (3.227 ± 0.116 seconds) and the DYNDJ protocol (3.132 ± 0.116 seconds). The data from this study advocate the use of DYNDJ protocol as a means of significantly improving 20-m sprint performance 1 minute after the DYNDJ protocol.

GROND: a quality-checked and publicly available database of full-length 16S-ITS-23S rRNA operon sequences.

Sequence comparison of 16S rRNA PCR amplicons is an established approach to taxonomically identify bacterial isolates and profile complex microbial communities. One potential application of recent advances in long-read sequencing technologies is to sequence entire rRNA operons and capture significantly more phylogenetic information compared to sequencing of the 16S rRNA (or regions thereof) alone, with the potential to increase the proportion of amplicons that can be reliably classified to lower taxonomic ranks. Here we describe GROND (Genome-derived Ribosomal Operon Database), a publicly available database of quality-checked 16S-ITS-23S rRNA operons, accompanied by multiple taxonomic classifications. GROND will aid researchers in analysis of their data and act as a standardised database to allow comparison of results between studies.

The impact of live dietary microbes on health: A scoping review.

A systematic approach to collect, peruse, and summarize the available information relating to the potential benefits of consuming dietary microbes was pursued in this scoping review. This review focused on the research endpoints, experimental designs, and microbial exposure in experimental as well as observational research work. Using a structured- set of keywords, scientific databases were systematically searched to retrieve publications reporting outcomes pertaining to the use of dietary microbes in healthy, nonpatient populations. Searches were further tailored to focus on eight different health categories, namely, "antibiotic associated diarrhoea" (AAD), "gastrointestinal health" (GIH), "immunological health" (ImH), "cardiovascular health and metabolic syndrome" (CvHMS), "cancer prevention" (CanPr), "respiratory health" (ReH), "weight management" (WtMgt), and "urogenital health" (UrGH). Quality of evidence available in each publication was assessed using the Jadad scoring system. The search yielded 228 relevant publications describing 282 experimental cases comprising 62 research endpoints overall. A microbial dose of ≥ 2 × 10 9 $\ge 2\times 10^9$ CFU.day-1 was associated with non-negative reported outcomes. Older population groups with a median age of 39 years were associated with positive outcomes. More high-quality research is required investigating the role of dietary microbes in maintaining general health, particularly in the health categories of UrGH, WtMgt, and CanPr.

The gut microbiota posttranslationally modifies IgA1 in autoimmune glomerulonephritis.

Mechanisms underlying the disruption of self-tolerance in acquired autoimmunity remain unclear. Immunoglobulin A (IgA) nephropathy is an acquired autoimmune disease where deglycosylated IgA1 (IgA subclass 1) auto-antigens are recognized by IgG auto-antibodies, forming immune complexes that are deposited in the kidneys, leading to glomerulonephritis. In the intestinal microbiota of patients with IgA nephropathy, there was increased relative abundance of mucin-degrading bacteria, including Akkermansia muciniphila. IgA1 was deglycosylated by A. muciniphila both in vitro and in the intestinal lumen of mice. This generated neo-epitopes that were recognized by autoreactive IgG from the sera of patients with IgA nephropathy. Mice expressing human IgA1 and the human Fc α receptor I (α1KI-CD89tg) that underwent intestinal colonization by A. muciniphila developed an aggravated IgA nephropathy phenotype. After deglycosylation of IgA1 by A. muciniphila in the mouse gut lumen, IgA1 crossed the intestinal epithelium into the circulation by retrotranscytosis and became deposited in the glomeruli of mouse kidneys. Human α-defensins-a risk locus for IgA nephropathy-inhibited growth of A. muciniphila in vitro. A negative correlation observed between stool concentration of α-defensin 6 and quantity of A. muciniphila in the guts of control participants was lost in patients with IgA nephropathy. This study demonstrates that gut microbiota dysbiosis contributes to generation of auto-antigens in patients with IgA nephropathy and in a mouse model of this disease.

Mapping the available evidence on the impact of ingested live microbes on health: a scoping review protocol.

INTRODUCTION: It has been hypothesised that the regular consumption of safe, live microbes confers health-promoting attributes, including the prevention of disease. To address this hypothesis, we propose a scoping review approach that will systematically assess the large corpus of relevant literature that is now available on this research topic. This article outlines a protocol for a scoping review of published studies on interventions with live microbes in non-patient populations across eight health categories. The scoping review aims to catalogue types of interventions, measured outcomes, dosages, effectiveness, as well as current research gaps. METHODS AND ANALYSIS: The scoping review will follow the six-staged protocol as proposed by Arksey and O'Malley and will include the following stages: defining the research questions (stage 1); defining the eligibility criteria and finalising search strategy (stage 2); selection of studies based on the eligibility criteria (stage 3); development of a data extraction framework and charting of data (stage 4); aggregation of results and summarisation of findings (stage 5); and the optional consultation with stakeholders (stage 6), which will not be performed. ETHICS AND DISSEMINATION: Since the scoping review synthesises information from existing literature, no separate ethical approval is required. The findings of the scoping review will be communicated for publication to an open-access, peer-reviewed scientific journal, presented at relevant conferences, and disseminated at future workshops with all relevant data and documents being available online through the Open Science Framework (https://osf.io/kvhe7).

Genomic profiling of idiopathic peri-hilar cholangiocarcinoma reveals new targets and mutational pathways.

Peri-hilar cholangiocarcinoma (pCCA) is chemorefractory and limited genomic analyses have been undertaken in Western idiopathic disease. We undertook comprehensive genomic analyses of a U.K. idiopathic pCCA cohort to characterize its mutational profile and identify new targets. Whole exome and targeted DNA sequencing was performed on forty-two resected pCCA tumors and normal bile ducts, with Gene Set Enrichment Analysis (GSEA) using one-tailed testing to generate false discovery rates (FDR). 60% of patients harbored one cancer-associated mutation, with two mutations in 20%. High frequency somatic mutations in genes not typically associated with cholangiocarcinoma included mTOR, ABL1 and NOTCH1. We identified non-synonymous mutation (p.Glu38del) in MAP3K9 in ten tumors, associated with increased peri-vascular invasion (Fisher's exact, p < 0.018). Mutation-enriched pathways were primarily immunological, including innate Dectin-2 (FDR 0.001) and adaptive T-cell receptor pathways including PD-1 (FDR 0.007), CD4 phosphorylation (FDR 0.009) and ZAP70 translocation (FDR 0.009), with overlapping HLA genes. We observed cancer-associated mutations in over half of our patients. Many of these mutations are not typically associated with cholangiocarcinoma yet may increase eligibility for contemporary targeted trials. We also identified a targetable MAP3K9 mutation, in addition to oncogenic and immunological pathways hitherto not described in any cholangiocarcinoma subtype.