ApoA-I Protects Pancreatic ß-Cells From Cholesterol-Induced Mitochondrial Damage and Restores Their Ability to Secrete Insulin.

BACKGROUND: High cholesterol levels in pancreatic ß-cells cause oxidative stress and decrease insulin secretion. ß-cells can internalize apo (apolipoprotein) A-I, which increases insulin secretion. This study asks whether internalization of apoA-I improves ß-cell insulin secretion by reducing oxidative stress. METHODS: Ins-1E cells were cholesterol-loaded by incubation with cholesterol-methyl-ß-cyclodextrin. Insulin secretion in the presence of 2.8 or 25 mmol/L glucose was quantified by radioimmunoassay. Internalization of fluorescently labeled apoA-I by ß-cells was monitored by flow cytometry. The effects of apoA-I internalization on ß-cell gene expression were evaluated by RNA sequencing. ApoA-I-binding partners on the ß-cell surface were identified by mass spectrometry. Mitochondrial oxidative stress was quantified in ß-cells and isolated islets with MitoSOX and confocal microscopy. RESULTS: An F1-ATPase ß-subunit on the ß-cell surface was identified as the main apoA-I-binding partner. ß-cell internalization of apoA-I was time-, concentration-, temperature-, cholesterol-, and F1-ATPase ß-subunit-dependent. ß-cells with internalized apoA-I (apoA-I+ cells) had higher cholesterol and cell surface F1-ATPase ß-subunit levels than ß-cells without internalized apoA-I (apoA-I- cells). The internalized apoA-I colocalized with mitochondria and was associated with reduced oxidative stress and increased insulin secretion. The IF1 (ATPase inhibitory factor 1) attenuated apoA-I internalization and increased oxidative stress in Ins-1E ß-cells and isolated mouse islets. Differentially expressed genes in apoA-I+ and apoA-I- Ins-1E cells were related to protein synthesis, the unfolded protein response, insulin secretion, and mitochondrial function. CONCLUSIONS: These results establish that ß-cells are functionally heterogeneous, and apoA-I restores insulin secretion in ß-cells with elevated cholesterol levels by improving mitochondrial redox balance.

Pathogen exposure leads to a transcriptional downregulation of core cellular functions that may dampen the immune response in a macroalga.

Diseases in marine eukaryotic organisms caused by opportunistic pathogens represent a serious threat to our oceans with potential downstream consequences for ecosystem functioning. Disease outbreaks affecting macroalgae are of particular concern due to their critical role as habitat-forming organisms. However, there is limited understanding of the molecular strategies used by macroalgae to respond to opportunistic pathogens. In this study, we used mRNA-sequencing analysis to investigate the early antipathogen response of the model macroalga Delisea pulchra (Rhodophyta) under the environmental conditions that are known to promote the onset of disease. Using de novo assembly methods, 27,586 unique transcripts belonging to D. pulchra were identified that were mostly affiliated with stress response and signal transduction processes. Differential gene expression analysis between a treatment with the known opportunistic pathogen, Aquimarina sp. AD1 (Bacteroidota), and a closely related benign strain (Aquimarina sp. AD10) revealed a downregulation of genes coding for predicted protein metabolism, stress response, energy generation and photosynthesis functions. The rapid repression of genes coding for core cellular processes is likely to interfere with the macroalgal antipathogen response, later leading to infection, tissue damage and bleaching symptoms. Overall, this study provides valuable insight into the genetic features of D. pulchra, highlighting potential antipathogen response mechanisms of macroalgae and contributing to an improved understanding of host-pathogen interactions in a changing environment.

Small RNA interactome of pathogenic E. coli revealed through crosslinking of RNase E.

RNA sequencing studies have identified hundreds of non-coding RNAs in bacteria, including regulatory small RNA (sRNA). However, our understanding of sRNA function has lagged behind their identification due to a lack of tools for the high-throughput analysis of RNA-RNA interactions in bacteria. Here we demonstrate that in vivo sRNA-mRNA duplexes can be recovered using UV-crosslinking, ligation and sequencing of hybrids (CLASH). Many sRNAs recruit the endoribonuclease, RNase E, to facilitate processing of mRNAs. We were able to recover base-paired sRNA-mRNA duplexes in association with RNase E, allowing proximity-dependent ligation and sequencing of cognate sRNA-mRNA pairs as chimeric reads. We verified that this approach captures bona fide sRNA-mRNA interactions. Clustering analyses identified novel sRNA seed regions and sets of potentially co-regulated target mRNAs. We identified multiple mRNA targets for the pathotype-specific sRNA Esr41, which was shown to regulate colicin sensitivity and iron transport in E. coli Numerous sRNA interactions were also identified with non-coding RNAs, including sRNAs and tRNAs, demonstrating the high complexity of the sRNA interactome.

Intra-species variation within Lactobacillus rhamnosus correlates to beneficial or harmful outcomes: lessons from the oral cavity.

BACKGROUND: The origin of most of the Lactobacillus rhamnosus genome sequences lodged in NCBI can be traced to food and faecal isolates followed by blood and tissue sites but with minimal representation from oral and vaginal isolates. However, on the L. rhamnosus phylogenetic tree no apparent clade is linked to the origin of isolation or to the relevant clinical source, except for a distinct clade exclusively shared by L. rhamnosus isolates from early stages of dental pulp infection (LRHMDP2 and LRHMDP3) and from bronchoalveolar lavage (699_LRHA and 708_LRHA) from a critical care patient. These L. rhamnosus strains, LRHMDP2, LRHMDP3, 699_LRHA and 708_LRHA isolated from different continents, display closest genome neighbour gapped identity of 99.95%. The aim of this study was to define a potentially unique complement of genes of clinical relevance shared between these L. rhamnosus clinical isolates in comparison to probiotic L. rhamnosus strains. RESULTS: In this analysis we used orthologous protein identification tools such as ProteinOrtho followed by tblastn alignments to identify a novel tyrosine protein phosphatase (wzb)-tyrosine-protein kinase modulator EpsC (wzd)- synteny exopolysaccharide (EPS) cluster. This EPS cluster was specifically conserved in a clade of 5 clinical isolates containing the four L. rhamnosus clinical isolates noted above and Lactobacillus spp. HMSC077C11, a clinical isolate from a neck abscess. The EPS cluster was shared with only two other strains, L. rhamnosus BPL5 and BPL15, which formed a distant clade on the L. rhamnosus phylogenetic tree, with a closest genome neighbour gapped identity of 97.51% with L. rhamnosus LRHMDP2 and LRHMDP3. Exclusivity of this EPS cluster (from those identified before) was defined by five EPS genes, which were specifically conserved between the clade of 5 clinical isolates and L. rhamnosus BPL5 and BPL15 when compared to the remaining L. rhamnosus strains. Comparative genome analysis between the clade of 5 clinical isolates and L. rhamnosus BPL5 and BPL15 showed a set of 58 potentially unique genes characteristic of the clade of 5. CONCLUSION: The potentially unique functional protein orthologs associated with the clade of 5 clinical isolates may provide understanding of fitness under selective pressure.

Specific Bacteria and Metabolites Associated With Response to Fecal Microbiota Transplantation in Patients With Ulcerative Colitis.

BACKGROUND & AIMS: Fecal microbiota transplantation (FMT) can induce remission in patients with ulcerative colitis (UC). In a randomized controlled trial of FMT in patients with active UC, we aimed to identify bacterial taxonomic and functional factors associated with response to therapy. METHODS: We performed a double-blind trial of 81 patients with active UC randomly assigned to groups that received an initial colonoscopic infusion and then intensive multidonor FMT or placebo enemas, 5 d/wk for 8 weeks. Patients in the FMT group received blended homogenized stool from 3-7 unrelated donors. Patients in the placebo group were eligible to receive open-label FMT after the double-blind study period. We collected 314 fecal samples from the patients at screening, every 4 weeks during treatment, and 8 weeks after the blinded or open-label FMT therapy. We also collected 160 large-bowel biopsy samples from the patients at study entry, at completion of 8 weeks of blinded therapy, and at the end of open-label FMT, if applicable. We analyzed 105 fecal samples from the 14 individual donors (n = 55), who in turn contributed to 21 multidonor batches (n = 50). Bacteria in colonic and fecal samples were analyzed by both 16S ribosomal RNA gene and transcript amplicon sequencing; 285 fecal samples were analyzed by shotgun metagenomics, and 60 fecal samples were analyzed for metabolome features. RESULTS: FMT increased microbial diversity and altered composition, based on analyses of colon and fecal samples collected before vs after FMT. Diversity was greater in fecal and colon samples collected before and after FMT treatment from patients who achieved remission compared with patients who did not. Patients in remission after FMT had enrichment of Eubacterium hallii and Roseburia inulivorans compared with patients who did not achieve remission after FMT and had increased levels of short-chain fatty acid biosynthesis and secondary bile acids. Patients who did not achieve remission had enrichment of Fusobacterium gonidiaformans, Sutterella wadsworthensis, and Escherichia species and increased levels of heme and lipopolysaccharide biosynthesis. Bacteroides in donor stool were associated with remission in patients receiving FMT, and Streptococcus species in donor stool was associated with no response to FMT. CONCLUSIONS: In an analysis of fecal and colonic mucosa samples from patients receiving FMT for active UC and stool samples from donors, we associated specific bacteria and metabolic pathways with induction of remission. These findings may be of value in the design of microbe-based therapies for UC. ClinicalTrials.gov, Number NCT01896635.

Transcriptional response of Nautella italica R11 towards its macroalgal host uncovers new mechanisms of host-pathogen interaction.

Macroalgae (seaweeds) are essential for the functioning of temperate marine ecosystems, but there is increasing evidence to suggest that their survival is under threat from anthropogenic stressors and disease. Nautella italica R11 is recognized as an aetiological agent of bleaching disease in the red alga, Delisea pulchra. Yet, there is a lack of knowledge surrounding the molecular mechanisms involved in this model host-pathogen interaction. Here we report that mutations in the gene encoding for a LuxR-type quorum sensing transcriptional regulator, RaiR, render N. italica R11 avirulent, suggesting this gene is important for regulating the expression of virulence phenotypes. Using an RNA sequencing approach, we observed a strong transcriptional response of N. italica R11 towards the presence of D. pulchra. In particular, genes involved in oxidative stress resistance, carbohydrate and central metabolism were upregulated in the presence of the host, suggesting a role for these functions in the opportunistic pathogenicity of N. italica R11. Furthermore, we show that RaiR regulates a subset of genes in N. italica R11, including those involved in metabolism and the expression of phage-related proteins. The outcome of this research reveals new functions important for virulence of N. italica R11 and contributes to our greater understanding of the complex factors mitigating microbial diseases in macroalgae.

Transcriptomic and proteomic analyses reveal key innate immune signatures in the host response to the gastrointestinal pathogen Campylobacter concisus.

Pathogenic species within the genus Campylobacter are responsible for a considerable burden on global health. Campylobacter concisus is an emergent pathogen that plays a role in acute and chronic gastrointestinal disease. Despite ongoing research on Campylobacter virulence mechanisms, little is known regarding the immunological profile of the host response to Campylobacter infection. In this study, we describe a comprehensive global profile of innate immune responses to C. concisus infection in differentiated THP-1 macrophages infected with an adherent and invasive strain of C. concisus. Using RNA sequencing (RNA-seq), quantitative PCR (qPCR), mass spectrometry, and confocal microscopy, we observed differential expression of pattern recognition receptors and robust upregulation of DNA- and RNA-sensing molecules. In particular, we observed IFI16 inflammasome assembly in C. concisus-infected macrophages. Global profiling of the transcriptome revealed the significant regulation of a total of 8,343 transcripts upon infection with C. concisus, which included the activation of key inflammatory pathways involving CREB1, NF-κB, STAT, and interferon regulatory factor signaling. Thirteen microRNAs and 333 noncoding RNAs were significantly regulated upon infection, including MIR221, which has been associated with colorectal carcinogenesis. This study represents a major advance in our understanding of host recognition and innate immune responses to infection by C. concisus.

Next-generation sequencing: a challenge to meet the increasing demand for training workshops in Australia.

The widespread adoption of high-throughput next-generation sequencing (NGS) technology among the Australian life science research community is highlighting an urgent need to up-skill biologists in tools required for handling and analysing their NGS data. There is currently a shortage of cutting-edge bioinformatics training courses in Australia as a consequence of a scarcity of skilled trainers with time and funding to develop and deliver training courses. To address this, a consortium of Australian research organizations, including Bioplatforms Australia, the Commonwealth Scientific and Industrial Research Organisation and the Australian Bioinformatics Network, have been collaborating with EMBL-EBI training team. A group of Australian bioinformaticians attended the train-the-trainer workshop to improve training skills in developing and delivering bioinformatics workshop curriculum. A 2-day NGS workshop was jointly developed to provide hands-on knowledge and understanding of typical NGS data analysis workflows. The road show-style workshop was successfully delivered at five geographically distant venues in Australia using the newly established Australian NeCTAR Research Cloud. We highlight the challenges we had to overcome at different stages from design to delivery, including the establishment of an Australian bioinformatics training network and the computing infrastructure and resource development. A virtual machine image, workshop materials and scripts for configuring a machine with workshop contents have all been made available under a Creative Commons Attribution 3.0 Unported License. This means participants continue to have convenient access to an environment they had become familiar and bioinformatics trainers are able to access and reuse these resources.

Analysis of the human protein interactome and comparison with yeast, worm and fly interaction datasets.

We present the first analysis of the human proteome with regard to interactions between proteins. We also compare the human interactome with the available interaction datasets from yeast (Saccharomyces cerevisiae), worm (Caenorhabditis elegans) and fly (Drosophila melanogaster). Of >70,000 binary interactions, only 42 were common to human, worm and fly, and only 16 were common to all four datasets. An additional 36 interactions were common to fly and worm but were not observed in humans, although a coimmunoprecipitation assay showed that 9 of the interactions do occur in humans. A re-examination of the connectivity of essential genes in yeast and humans indicated that the available data do not support the presumption that the number of interaction partners can accurately predict whether a gene is essential. Finally, we found that proteins encoded by genes mutated in inherited genetic disorders are likely to interact with proteins known to cause similar disorders, suggesting the existence of disease subnetworks. The human interaction map constructed from our analysis should facilitate an integrative systems biology approach to elucidating the cellular networks that contribute to health and disease states.

Tools to covisualize and coanalyze proteomic data with genomes and transcriptomes: validation of genes and alternative mRNA splicing.

Direct links between proteomic and genomic/transcriptomic data are not frequently made, partly because of lack of appropriate bioinformatics tools. To help address this, we have developed the PG Nexus pipeline. The PG Nexus allows users to covisualize peptides in the context of genomes or genomic contigs, along with RNA-seq reads. This is done in the Integrated Genome Viewer (IGV). A Results Analyzer reports the precise base position where LC-MS/MS-derived peptides cover genes or gene isoforms, on the chromosomes or contigs where this occurs. In prokaryotes, the PG Nexus pipeline facilitates the validation of genes, where annotation or gene prediction is available, or the discovery of genes using a "virtual protein"-based unbiased approach. We illustrate this with a comprehensive proteogenomics analysis of two strains of Campylobacter concisus . For higher eukaryotes, the PG Nexus facilitates gene validation and supports the identification of mRNA splice junction boundaries and splice variants that are protein-coding. This is illustrated with an analysis of splice junctions covered by human phosphopeptides, and other examples of relevance to the Chromosome-Centric Human Proteome Project. The PG Nexus is open-source and available from https://github.com/IntersectAustralia/ap11_Samifier. It has been integrated into Galaxy and made available in the Galaxy tool shed.

The draft genome of the pest tephritid fruit fly Bactrocera tryoni: resources for the genomic analysis of hybridising species.

BACKGROUND: The tephritid fruit flies include a number of economically important pests of horticulture, with a large accumulated body of research on their biology and control. Amongst the Tephritidae, the genus Bactrocera, containing over 400 species, presents various species groups of potential utility for genetic studies of speciation, behaviour or pest control. In Australia, there exists a triad of closely-related, sympatric Bactrocera species which do not mate in the wild but which, despite distinct morphologies and behaviours, can be force-mated in the laboratory to produce fertile hybrid offspring. To exploit the opportunities offered by genomics, such as the efficient identification of genetic loci central to pest behaviour and to the earliest stages of speciation, investigators require genomic resources for future investigations. RESULTS: We produced a draft de novo genome assembly of Australia's major tephritid pest species, Bactrocera tryoni. The male genome (650-700 Mbp) includes approximately 150 Mb of interspersed repetitive DNA sequences and 60 Mb of satellite DNA. Assessment using conserved core eukaryotic sequences indicated 98% completeness. Over 16,000 MAKER-derived gene models showed a large degree of overlap with other Dipteran reference genomes. The sequence of the ribosomal RNA transcribed unit was also determined. Unscaffolded assemblies of B. neohumeralis and B. jarvisi were then produced; comparison with B. tryoni showed that the species are more closely related than any Drosophila species pair. The similarity of the genomes was exploited to identify 4924 potentially diagnostic indels between the species, all of which occur in non-coding regions. CONCLUSIONS: This first draft B. tryoni genome resembles other dipteran genomes in terms of size and putative coding sequences. For all three species included in this study, we have identified a comprehensive set of non-redundant repetitive sequences, including the ribosomal RNA unit, and have quantified the major satellite DNA families. These genetic resources will facilitate the further investigations of genetic mechanisms responsible for the behavioural and morphological differences between these three species and other tephritids. We have also shown how whole genome sequence data can be used to generate simple diagnostic tests between very closely-related species where only one of the species is scaffolded.

The semen microbiome of miniature pony stallions.

CONTEXT: Little is known about the microbial composition of stallion semen. AIMS: To describe the microbiota detected in equine semen of healthy miniature pony stallions. METHODS: Semen specimens were collected using a Missouri artificial vagina at a single time point. PacBio (Pacific Biosciences) genomic DNA sequencing of the 16S rRNA gene was performed on these specimens, following which next-generation microbiome bioinformatics platform QIIME2 was used to process fastq files and analyse the amplicon data. The data were categorised into genus, family, class, order and phylum. KEY RESULTS: Firmicutes and Bacteroidetes phyla predominated (76%), followed by Proteobacteria (15%). Bacteroidales, Clostridiales and Cardiobacteriales predominated the microbial rank of order (86%). Class was mainly composed of Bacteroidia, Clostridia and Gammaproteobacteria (87%), while family was mainly composed of Porphyromonadaceae , Family_XI and Cardiobacteriaceae (62%). At the level of genus, 80% of the abundance was composed of seven genera, namely Porphyromonas, Suttonella, Peptoniphilus, Fastidiosipila, Ezakiella, Petrimonas and an unknown taxon. CONCLUSIONS: The findings indicate that specific microbiota may be characteristic of healthy miniature pony stallions' semen with some inter-individual variations observed. IMPLICATIONS: Larger equine studies involving fertile and infertile subjects could be informed by this study and could explore the relationship of the semen microbiome to male fertility.

Effect of probiotics and prebiotics on the composition of the equine fecal and seminal microbiomes and sperm quality: A pilot study.

Probiotic and prebiotic effects on equine semen and gastrointestinal microbiome composition and sperm quality are unknown. This study aimed to evaluate the effects of pre-, pro- or synbiotic supplementation on fecal and semen microbiome composition and sperm quality parameters of stallions. This Latin square crossover trial involved four miniature pony stallions receiving control diet only, or addition of a pro-, pre- or synbiotic formulation. Full-length 16S rRNA gene amplicon sequencing was used to measure diversity of semen and fecal microbiomes. Total sperm count, total motility, progressive motility, DNA integrity, lipid peroxidation and mitochondrial oxidative stress, biomarkers of sperm quality, were measured after each intervention. A general linear model was employed to analyse and compare microbiome diversity measures and sperm quality data across four time points. Shannon's diversity index (alpha-diversity), and evenness of semen and gastrointestinal microbiomes were significantly different (p<0.001). A trend was observed for prebiotic effects on the diversity indices of the GI microbiome (p= 0.07). No effects of treatments were observed on either semen microbiome or sperm quality. Pre-, pro- and synbiotic supplements showed no negative effect on sperm quality parameters observed. This proof of concept provides preliminary data to inform future studies exploring the relationship between microbiomes and fertility.

Effect of insulin insufficiency on ultrastructure and function in skeletal muscle.

BACKGROUND: Decreased insulin availability and high blood glucose levels, the hallmark features of poorly controlled diabetes, drive disease progression and are associated with decreased skeletal muscle mass. We have shown that mice with ß-cell dysfunction and normal insulin sensitivity have decreased skeletal muscle mass. This project asks how insulin deficiency impacts on the structure and function of the remaining skeletal muscle in these animals. METHODS: Skeletal muscle function was determined by measuring exercise capacity and specific muscle strength prior to and after insulin supplementation for 28 days in 12-week-old mice with conditional ß-cell deletion of the ATP binding cassette transporters ABCA1 and ABCG1 (ß-DKO mice). Abca1 and Abcg1 floxed (fl/fl) mice were used as controls. RNAseq was used to quantify changes in transcripts in soleus and extensor digitorum longus muscles. Skeletal muscle and mitochondrial morphology were assessed by transmission electron microscopy. Myofibrillar Ca2+ sensitivity and maximum isometric single muscle fibre force were assessed using MyoRobot biomechatronics technology. RESULTS: RNA transcripts were significantly altered in ß-DKO mice compared with fl/fl controls (32 in extensor digitorum longus and 412 in soleus). Exercise capacity and muscle strength were significantly decreased in ß-DKO mice compared with fl/fl controls (P = 0.012), and a loss of structural integrity was also observed in skeletal muscle from the ß-DKO mice. Supplementation of ß-DKO mice with insulin restored muscle integrity, strength and expression of 13 and 16 of the dysregulated transcripts in and extensor digitorum longus and soleus muscles, respectively. CONCLUSIONS: Insulin insufficiency due to ß-cell dysfunction perturbs the structure and function of skeletal muscle. These adverse effects are rectified by insulin supplementation.

Extant and extinct bilby genomes combined with Indigenous knowledge improve conservation of a unique Australian marsupial.

Ninu (greater bilby, Macrotis lagotis) are desert-dwelling, culturally and ecologically important marsupials. In collaboration with Indigenous rangers and conservation managers, we generated the Ninu chromosome-level genome assembly (3.66 Gbp) and genome sequences for the extinct Yallara (lesser bilby, Macrotis leucura). We developed and tested a scat single-nucleotide polymorphism panel to inform current and future conservation actions, undertake ecological assessments and improve our understanding of Ninu genetic diversity in managed and wild populations. We also assessed the beneficial impact of translocations in the metapopulation (N = 363 Ninu). Resequenced genomes (temperate Ninu, 6; semi-arid Ninu, 6; and Yallara, 4) revealed two major population crashes during global cooling events for both species and differences in Ninu genes involved in anatomical and metabolic pathways. Despite their 45-year captive history, Ninu have fewer long runs of homozygosity than other larger mammals, which may be attributable to their boom-bust life history. Here we investigated the unique Ninu biology using 12 tissue transcriptomes revealing expression of all 115 conserved eutherian chorioallantoic placentation genes in the uterus, an XY1Y2 sex chromosome system and olfactory receptor gene expansions. Together, we demonstrate the holistic value of genomics in improving key conservation actions, understanding unique biological traits and developing tools for Indigenous rangers to monitor remote wild populations.

Comparative genomics of Campylobacter concisus isolates reveals genetic diversity and provides insights into disease association.

BACKGROUND: In spite of its association with gastroenteritis and inflammatory bowel diseases, the isolation of Campylobacter concisus from both diseased and healthy individuals has led to controversy regarding its role as an intestinal pathogen. One proposed reason for this is the presence of high genetic diversity among the genomes of C. concisus strains. RESULTS: In this study the genomes of six C. concisus strains were sequenced, assembled and annotated including two strains isolated from Crohn's disease patients (UNSW2 and UNSW3), three from gastroenteritis patients (UNSW1, UNSWCS and ATCC 51562) and one from a healthy individual (ATCC 51561). The genomes of C. concisus BAA-1457 and UNSWCD, available from NCBI, were included in subsequent comparative genomic analyses. The Pan and Core genomes for the sequenced C. concisus strains consisted of 3254 and 1556 protein coding genes, respectively. CONCLUSION: Genes were identified with specific conservation in C. concisus strains grouped by phenotypes such as invasiveness, adherence, motility and diseased states. Phylogenetic trees based on ribosomal RNA sequences and concatenated host-related pathways for the eight C. concisus strains were generated using the neighbor-joining method, of which the 16S rRNA gene and peptidoglycan biosynthesis grouped the C. concisus strains according to their pathogenic phenotypes. Furthermore, 25 non-synonymous amino acid changes with 14 affecting functional domains, were identified within proteins of conserved host-related pathways, which had possible associations with the pathogenic potential of C. concisus strains. Finally, the genomes of the eight C. concisus strains were compared to the nine available genomes of the well-established pathogen Campylobacter jejuni, which identified several important differences in the respiration pathways of these two species. Our findings indicate that C. concisus strains are genetically diverse, and suggest the genomes of this bacterium contain respiration pathways and modifications in the peptidoglycan layer that may play an important role in its virulence.

RNA sequencing identifies genes reliant upon Ser26 in the zinc finger transcription factor, early growth response-1.

Early growth response-1 (Egr-1) is an inducible master regulatory transcription factor that orchestrates gene expression in vascular endothelial cells. We recently determined that Ser26 in Egr-1 undergoes phosphorylation and plays a critical functional role in a range of pro-angiogenic processes. To better understand the effect of Ser26 on Egr-1-dependent gene expression, in this study, we performed RNA-seq and bioinformatics analysis with human microvascular endothelial cells bearing a germline mutation (M) in Ser26 to Ala (M26 cells) exposed to the mitogen and chemoattractant fibroblast growth factor-2 (FGF2) as compared with wildtype (WT) cells. In WT cells, FGF2 increased the expression of numerous growth factors and hormones, cytokines, signaling molecules and transcriptional regulators. Comparison of FGF2-inducible WT and M26 cells enabled identification of differentially expressed genes, including genes reliant or not reliant upon Ser26. For example, Ser26 in Egr-1 was required for FGF2 inducible LIF expression but not for FGF2 inducible IL11. Ser26 was also required for FGF2 inducible NKX2-8 and RIPK2 expression but not for FGF2 inducible CREB5 or ALPK2 expression. Conversely, FGF2 inhibited genes such as TIE1, GPR146 and EPHB3, and Ser26 was required for FGF2's effect on TIE1 and GPR146 but not for EPHB3. Enrichment analysis also identified a range of gene ontologies upregulated and downregulated by FGF2. These findings demonstrate the importance of Ser26 in Egr-1 in programs of endothelial gene expression modulated by FGF2.

The splicing factor RBM17 drives leukemic stem cell maintenance by evading nonsense-mediated decay of pro-leukemic factors.

Chemo-resistance in acute myeloid leukemia (AML) patients is driven by leukemic stem cells (LSCs) resulting in high rates of relapse and low overall survival. Here, we demonstrate that upregulation of the splicing factor, RBM17 preferentially marks and sustains LSCs and directly correlates with shorten patient survival. RBM17 knockdown in primary AML cells leads to myeloid differentiation and impaired colony formation and in vivo engraftment. Integrative multi-omics analyses show that RBM17 repression leads to inclusion of poison exons and production of nonsense-mediated decay (NMD)-sensitive transcripts for pro-leukemic factors and the translation initiation factor, EIF4A2. We show that EIF4A2 is enriched in LSCs and its inhibition impairs primary AML progenitor activity. Proteomic analysis of EIF4A2-depleted AML cells shows recapitulation of the RBM17 knockdown biological effects, including pronounced suppression of proteins involved in ribosome biogenesis. Overall, these results provide a rationale to target RBM17 and/or its downstream NMD-sensitive splicing substrates for AML treatment.

Raspberry ketone diet supplement reduces attraction of sterile male Queensland fruit fly to cuelure by altering expression of chemoreceptor genes.

Sterile male Queensland fruit fly, Bactrocera tryoni (Froggatt), fed as immature adults on the plant compound raspberry ketone (RK), show a reduced attraction to cuelure, a synthetic analogue of RK used as an attractant in Male Annihilation Technique. We hypothesized the reduced attraction of RK-fed adult males to cuelure may be a consequence of altered expression of chemoreception genes. A Y-tube olfactometer assay with RK-fed and RK-unfed sterile B. tryoni males tested the subsequent behavioural response to cuelure. Behavioral assays confirmed a significant decrease in attraction of RK-fed sterile males to cuelure. RK-fed, non-responders (to cue-lure) and RK-unfed, responders (to cue-lure) males were sampled and gene expression compared by de novo RNA-seq analysis. A total of 269 genes in fly heads were differentially expressed between replicated groups of RK-fed, cuelure non-responders and RK-unfed, cuelure responders. Among them, 218 genes including 4 chemoreceptor genes were up regulated and 51 genes were down regulated in RK-fed, cuelure non-responders. De novo assembly generated many genes with unknown functions and no significant BLAST hits to homologues in other species. The enriched and suppressed genes reported here, shed light on the transcriptional changes that affect the dynamics of insect responses to chemical stimuli.

Multi-omics of the esophageal microenvironment identifies signatures associated with progression of Barrett's esophagus.

BACKGROUND: The enrichment of Gram-negative bacteria of oral origin in the esophageal microbiome has been associated with the development of metaplasia. However, to date, no study has comprehensively assessed the relationships between the esophageal microbiome and the host. METHODS: Here, we examine the esophageal microenvironment in gastro-esophageal reflux disease and metaplasia using multi-omics strategies targeting the microbiome and host transcriptome, followed by targeted culture, comparative genomics, and host-microbial interaction studies of bacterial signatures of interest. RESULTS: Profiling of the host transcriptome from esophageal mucosal biopsies revealed profound changes during metaplasia. Importantly, five biomarkers showed consistent longitudinal changes with disease progression from reflux disease to metaplasia. We showed for the first time that the esophageal microbiome is distinct from the salivary microbiome and the enrichment of Campylobacter species as a consistent signature in disease across two independent cohorts. Shape fitting and matrix correlation identified associations between the microbiome and host transcriptome profiles, with a novel co-exclusion relationship found between Campylobacter and napsin B aspartic peptidase. Targeted culture of Campylobacter species from the same cohort revealed a subset of isolates to have a higher capacity to survive within primary human macrophages. Comparative genomic analyses showed these isolates could be differentiated by specific genomic features, one of which was validated to be associated with intracellular fitness. Screening for these Campylobacter strain-specific signatures in shotgun metagenomics data from another cohort showed an increase in prevalence with disease progression. Comparative transcriptomic analyses of primary esophageal epithelial cells exposed to the Campylobacter isolates revealed expression changes within those infected with strains with high intracellular fitness that could explain the increased likelihood of disease progression. CONCLUSIONS: We provide a comprehensive assessment of the esophageal microenvironment, identifying bacterial strain-specific signatures with high relevance to progression of metaplasia.