ABSTRACT
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.
Subject(s)
Leukemia, Myeloid, Acute , Neoplastic Stem Cells , RNA Splicing Factors , Hematopoiesis , Humans , Leukemia, Myeloid, Acute/metabolism , Neoplastic Stem Cells/metabolism , Proteomics , RNA Splicing Factors/genetics , RNA Splicing Factors/metabolismABSTRACT
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.
Subject(s)
Butanones , Chemoreceptor Cells/metabolism , Dietary Supplements , Gene Expression Regulation , Infertility, Male/metabolism , Tephritidae/metabolism , Animals , MaleABSTRACT
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.
Subject(s)
Barrett Esophagus/etiology , Barrett Esophagus/metabolism , Biomarkers , Cellular Microenvironment , Disease Susceptibility , Esophagus/metabolism , Adult , Barrett Esophagus/pathology , Cellular Microenvironment/genetics , Epithelial Cells/metabolism , Epithelial Cells/pathology , Esophagus/microbiology , Esophagus/pathology , Female , Gastroesophageal Reflux/complications , Gastroesophageal Reflux/etiology , Gene Expression Profiling , Gram-Negative Bacterial Infections/complications , Gram-Negative Bacterial Infections/microbiology , Host-Pathogen Interactions/genetics , Humans , Macrophages/immunology , Macrophages/metabolism , Male , Mast Cells/immunology , Mast Cells/metabolism , Metaplasia , Microbiota , Middle Aged , Models, Biological , RNA, Ribosomal, 16SABSTRACT
Existing compendia of non-coding RNA (ncRNA) are incomplete, in part because they are derived almost exclusively from small and polyadenylated RNAs. Here we present a more comprehensive atlas of the human transcriptome, which includes small and polyA RNA as well as total RNA from 300 human tissues and cell lines. We report thousands of previously uncharacterized RNAs, increasing the number of documented ncRNAs by approximately 8%. To infer functional regulation by known and newly characterized ncRNAs, we exploited pre-mRNA abundance estimates from total RNA sequencing, revealing 316 microRNAs and 3,310 long non-coding RNAs with multiple lines of evidence for roles in regulating protein-coding genes and pathways. Our study both refines and expands the current catalog of human ncRNAs and their regulatory interactions. All data, analyses and results are available for download and interrogation in the R2 web portal, serving as a basis for future exploration of RNA biology and function.
Subject(s)
MicroRNAs , RNA, Long Noncoding , Humans , MicroRNAs/genetics , RNA, Long Noncoding/genetics , RNA, Messenger , RNA, Untranslated/genetics , Transcriptome/geneticsABSTRACT
Vascular permeability and angiogenesis underpin neovascular age-related macular degeneration and diabetic retinopathy. While anti-VEGF therapies are widely used clinically, many patients do not respond optimally, or at all, and small-molecule therapies are lacking. Here, we identified a dibenzoxazepinone BT2 that inhibits endothelial cell proliferation, migration, wound repair in vitro, network formation, and angiogenesis in mice bearing Matrigel plugs. BT2 interacts with MEK1 and inhibits ERK phosphorylation and the expression of FosB/ΔFosB, VCAM-1, and many genes involved in proliferation, migration, angiogenesis, and inflammation. BT2 reduced retinal vascular leakage following rat choroidal laser trauma and rabbit intravitreal VEGF-A165 administration. BT2 suppressed retinal CD31, pERK, VCAM-1, and VEGF-A165 expression. BT2 reduced retinal leakage in rats at least as effectively as aflibercept, a first-line therapy for nAMD/DR. BT2 withstands boiling or autoclaving and several months' storage at 22°C. BT2 is a new small-molecule inhibitor of vascular permeability and angiogenesis.
Subject(s)
Capillary Permeability , Vascular Cell Adhesion Molecule-1 , Angiogenesis Inhibitors/pharmacology , Animals , Humans , Mice , Neovascularization, Pathologic/drug therapy , Neovascularization, Pathologic/genetics , Proto-Oncogene Proteins c-fos/metabolism , Rabbits , Rats , Vascular Cell Adhesion Molecule-1/metabolism , Vascular Cell Adhesion Molecule-1/pharmacology , Vascular Endothelial Growth Factor A/metabolismABSTRACT
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.
Subject(s)
Dental Caries/microbiology , Genetic Variation , Lacticaseibacillus rhamnosus/genetics , Mouth/microbiology , Bacterial Proteins/genetics , Evolution, Molecular , Humans , Lacticaseibacillus rhamnosus/classification , Lacticaseibacillus rhamnosus/pathogenicity , Phylogeny , Polysaccharides, Bacterial/genetics , Selection, Genetic , Toxin-Antitoxin Systems/geneticsABSTRACT
PURPOSE: RNA splicing is a fundamental biological process that generates protein diversity from a finite set of genes. Recurrent somatic mutations of splicing factor genes are common in some hematologic cancers but are relatively uncommon in acute myeloid leukemia (AML, < 20% of patients). We examined whether RNA splicing differences exist in AML, even in the absence of splicing factor mutations. EXPERIMENTAL DESIGN: We developed a bioinformatics pipeline to study alternative RNA splicing in RNA-sequencing data from large cohorts of patients with AML. RESULTS: We have identified recurrent differential alternative splicing between patients with poor and good prognosis. These splicing events occurred even in patients without any discernible splicing factor mutations. Alternative splicing recurrently occurred in genes with specific molecular functions, primarily related to protein translation. Developing tools to predict the functional impact of alternative splicing on the translated protein, we discovered that approximately 45% of the splicing events directly affected highly conserved protein domains. Several splicing factors were themselves misspliced and the splicing of their target transcripts were altered. Studying differential gene expression in the same patients, we identified that alternative splicing of protein translation genes in ELNAdv patients resulted in the induction of an integrated stress response and upregulation of inflammation-related genes. Finally, using machine learning techniques, we identified a splicing signature of four genes which refine the accuracy of existing risk prognosis schemes and validated it in a completely independent cohort. CONCLUSIONS: Our discoveries therefore identify aberrant alternative splicing as a molecular feature of adverse AML with clinical relevance.See related commentary by Bowman, p. 3503.
Subject(s)
Biological Phenomena , Leukemia, Myeloid, Acute , Alternative Splicing , Humans , Leukemia, Myeloid, Acute/diagnosis , Leukemia, Myeloid, Acute/genetics , Mutation , Prognosis , RNA Splicing/genetics , RNA Splicing Factors/geneticsABSTRACT
The de-novo genome assembly is a challenging computational problem for which several pipelines have been developed. The advent of long-read sequencing technology has resulted in a new set of algorithmic approaches for the assembly process. In this work, we identify that one of these new and fast long-read assembly techniques (using Minimap2 and Miniasm) can be modified for the short-read assembly process. This possibility motivated us to customize a long-read assembly approach for applications in a short-read assembly scenario. Here, we compare and contrast our proposed de-novo assembly pipeline (MiniSR) with three other recently developed programs for the assembly of bacterial and small eukaryotic genomes. We have documented two trade-offs: one between speed and accuracy and the other between contiguity and base-calling errors. Our proposed assembly pipeline shows a good balance in these trade-offs. The resulting pipeline is 6 and 2.2 times faster than the short-read assemblers Spades and SGA, respectively. MiniSR generates assemblies of superior N50 and NGA50 to SGA, although assemblies are less complete and accurate than those from Spades. A third tool, SOAPdenovo2, is as fast as our proposed pipeline but had poorer assembly quality.
Subject(s)
Consensus Sequence/genetics , Genomics/methods , Sequence Alignment/methods , Sequence Analysis, DNA/methods , Algorithms , Genome, Bacterial/genetics , High-Throughput Nucleotide SequencingABSTRACT
Bacteria capable of dechlorinating the toxic environmental contaminant dichloromethane (DCM, CH2Cl2) are of great interest for potential bioremediation applications. A novel, strictly anaerobic, DCM-fermenting bacterium, "DCMF", was enriched from organochlorine-contaminated groundwater near Botany Bay, Australia. The enrichment culture was maintained in minimal, mineral salt medium amended with dichloromethane as the sole energy source. PacBio whole genome SMRTTM sequencing of DCMF allowed de novo, gap-free assembly despite the presence of cohabiting organisms in the culture. Illumina sequencing reads were utilised to correct minor indels. The single, circularised 6.44 Mb chromosome was annotated with the IMG pipeline and contains 5,773 predicted protein-coding genes. Based on 16S rRNA gene and predicted proteome phylogeny, the organism appears to be a novel member of the Peptococcaceae family. The DCMF genome is large in comparison to known DCM-fermenting bacteria. It includes an abundance of methyltransferases, which may provide clues to the basis of its DCM metabolism, as well as potential to metabolise additional methylated substrates such as quaternary amines. Full annotation has been provided in a custom genome browser and search tool, in addition to multiple sequence alignments and phylogenetic trees for every predicted protein, http://www.slimsuite.unsw.edu.au/research/dcmf/.
ABSTRACT
Ocean acidification (OA) can be detrimental to calcifying marine organisms, with stunting of invertebrate larval development one of the most consistent responses. Effects are usually measured by short-term, within-generation exposure, an approach that does not consider the potential for adaptation. We examined the genetic response to OA of larvae of the tropical sea urchin Echinometra sp. C. raised on coral reefs that were either influenced by CO2 vents (pH ~ 7.9, future OA condition) or nonvent control reefs (pH 8.2). We assembled a high quality de novo transcriptome of Echinometra embryos (8 hr) and pluteus larvae (48 hr) and identified 68,056 SNPs. We tested for outlier SNPs and functional enrichment in embryos and larvae raised from adults from the control or vent sites. Generally, highest F ST values in embryos were observed between sites (intrinsic adaptation, most representative of the gene pool in the spawned populations). This comparison also had the highest number of outlier loci (40). In the other comparisons, classical adaptation (comparing larvae with adults from the control transplanted to either the control or vent conditions) and reverse adaptation (larvae from the vent site returned to the vent or explanted at the control), we only observed modest numbers of outlier SNPs (6-19) and only enrichment in two functional pathways. Most of the outliers detected were silent substitutions without adaptive potential. We conclude that there is little evidence of realized adaptation potential during early development, while some potential (albeit relatively low) exists in the intrinsic gene pool after more than one generation of exposure.
ABSTRACT
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.
Subject(s)
Bacteria/metabolism , Colitis, Ulcerative/therapy , Gastrointestinal Microbiome , Bacteria/classification , Bacteria/genetics , Bacteria/growth & development , Biomarkers/metabolism , Colitis, Ulcerative/diagnosis , Colitis, Ulcerative/microbiology , Double-Blind Method , Fecal Microbiota Transplantation/adverse effects , Feces/microbiology , Humans , Metabolomics , New South Wales , Remission Induction , Ribotyping , Time Factors , Treatment OutcomeABSTRACT
BACKGROUND: The esophageal microbiome has been proposed to be involved in a range of diseases including the esophageal adenocarcinoma cascade; however, little is currently known about its function and relationship to the host. Here, the esophageal microbiomes of 106 prospectively recruited patients were assessed using 16S rRNA and 18S rRNA amplicon sequencing as well as shotgun sequencing, and associations with age, gender, proton pump inhibitor use, host genetics, and disease were tested. RESULTS: The esophageal microbiome was found to cluster into functionally distinct community types (esotypes) defined by the relative abundances of Streptococcus and Prevotella. While age was found to be a significant factor driving microbiome composition, bacterial signatures and functions such as enrichment with Gram-negative oral-associated bacteria and microbial lactic acid production were associated with the early stages of the esophageal adenocarcinoma cascade. Non-bacterial microbes such as archaea, Candida spp., and bacteriophages were also identified in low abundance in the esophageal microbiome. Specific host SNPs in NOTCH2, STEAP2-AS1, and NREP were associated with the composition of the esophageal microbiome in our cohort. CONCLUSIONS: This study provides the most comprehensive assessment of the esophageal microbiome to date and identifies novel signatures and host markers that can be investigated further in the context of esophageal adenocarcinoma development.
Subject(s)
Adenocarcinoma/etiology , Bacteria/classification , Bacteriophages/classification , Esophageal Neoplasms/etiology , Esophagus/microbiology , Lactic Acid/metabolism , Metagenomics/methods , Polymorphism, Single Nucleotide , Adenocarcinoma/microbiology , Age Factors , Aged , Bacteria/genetics , Bacteria/isolation & purification , Bacteria/metabolism , Bacteriophages/genetics , Bacteriophages/isolation & purification , DNA, Bacterial/genetics , DNA, Ribosomal/genetics , Esophageal Neoplasms/microbiology , Female , High-Throughput Nucleotide Sequencing , Humans , Male , Middle Aged , Nerve Tissue Proteins/genetics , Oncogene Proteins/genetics , Phylogeny , Prevotella/classification , Prevotella/genetics , Prevotella/isolation & purification , Prospective Studies , RNA, Ribosomal, 16S/genetics , RNA, Ribosomal, 18S/genetics , Receptor, Notch2/genetics , Sequence Analysis, DNA , Streptococcus/classification , Streptococcus/genetics , Streptococcus/isolation & purificationABSTRACT
Myelodysplastic syndromes and chronic myelomonocytic leukemia are blood disorders characterized by ineffective hematopoiesis and progressive marrow failure that can transform into acute leukemia. The DNA methyltransferase inhibitor 5-azacytidine (AZA) is the most effective pharmacological option, but only â¼50% of patients respond. A response only manifests after many months of treatment and is transient. The reasons underlying AZA resistance are unknown, and few alternatives exist for non-responders. Here, we show that AZA responders have more hematopoietic progenitor cells (HPCs) in the cell cycle. Non-responder HPC quiescence is mediated by integrin α5 (ITGA5) signaling and their hematopoietic potential improved by combining AZA with an ITGA5 inhibitor. AZA response is associated with the induction of an inflammatory response in HPCs in vivo. By molecular bar coding and tracking individual clones, we found that, although AZA alters the sub-clonal contribution to different lineages, founder clones are not eliminated and continue to drive hematopoiesis even in complete responders.
Subject(s)
Azacitidine/administration & dosage , Drug Resistance , Genomics , Myelodysplastic Syndromes , Aged , Aged, 80 and over , Drug Resistance/drug effects , Drug Resistance/genetics , Female , Humans , Integrin alpha Chains/genetics , Integrin alpha Chains/metabolism , Middle Aged , Myelodysplastic Syndromes/drug therapy , Myelodysplastic Syndromes/genetics , Myelodysplastic Syndromes/metabolismABSTRACT
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.
Subject(s)
Endoribonucleases/metabolism , Escherichia coli/chemistry , Escherichia coli/enzymology , Gene Expression Regulation, Bacterial , RNA, Messenger/analysis , RNA, Small Untranslated/analysis , Escherichia coli/genetics , Protein Binding , RNA, Messenger/genetics , RNA, Messenger/isolation & purification , RNA, Small Untranslated/genetics , RNA, Small Untranslated/isolation & purification , Sequence Analysis, DNAABSTRACT
The epithelial response to the opportunistic pathogen Campylobacter concisus is poorly characterised. Here, we assessed the intestinal epithelial responses to two C. concisus strains with different virulence characteristics in Caco-2 cells using RNAseq, and validated a subset of the response using qPCR arrays. C. concisus strains induced distinct response patterns from intestinal epithelial cells, with the toxigenic strain inducing a significantly more amplified response. A range of cellular functions were significantly regulated in a strain-specific manner, including epithelial-to-mesenchymal transition (NOTCH and Hedgehog), cytoskeletal remodeling, tight junctions, inflammatory responses and autophagy. Pattern recognition receptors were regulated, including TLR3 and IFI16, suggesting that nucleic acid sensing was important for epithelial recognition of C. concisus. C. concisus zonula occludens toxin (ZOT) was expressed and purified, and the epithelial response to the toxin was analysed using RNAseq. ZOT upregulated PAR2 expression, as well as processes related to tight junctions and cytoskeletal remodeling. C. concisus ZOT also induced upregulation of TLR3, pro-inflammatory cytokines IL6, IL8 and chemokine CXCL16, as well as the executioner caspase CASP7. Here, we characterise distinct global epithelial responses to C. concisus strains, and the virulence factor ZOT, and provide novel information on mechanisms by which this bacterium may affect the host.
ABSTRACT
BACKGROUND: Pancreas, a relatively inaccessible organ, poses diagnostic difficulties with overlapping presentation among benign and malignant tumors. In the present study, pancreatic aspirates obtained by computed tomography (CT) guided procedures were used for cytodiagnosis. Our study aims at correlating clinical, cytological, biochemical, and histopathological results in obtaining a final diagnosis. METHODOLOGY: A retrospective study of 2 years was done which included 32 cases of pancreatic tumors at a tertiary care center. Patient data were retrieved and analyzed. RESULTS: Twenty-seven of the 32 (84.37%) cases were malignant tumors. Age distribution in malignant tumors was predominantly seen in the fourth to eighth decade, whereas in benign, it ranged in the second to third decade. Thirteen out of the 32 (40.62%) cases reported were females, with male:female ratio of 1.46:1. The most common presenting symptom was abdominal pain followed by jaundice and vomiting. Three of the 32 cases had visceral metastasis at the time of diagnosis. CT-guided aspirates in most cases yielded diagnostic material. Cytological and histopathological results concurred except for three cases. Cancer Antigen 19-9 was worked up for 14 of 27 malignant cases, 11 of which showed grossly elevated values (700-7000), and three cases showed mildly elevated values (100-300). Three of the four benign cases worked up for CA 19-9 showed normal values. CONCLUSIONS: Among the mass forming lesions in pancreas, malignancy was more common compared to benign tumors. A multidisciplinary approach in the assessment and diagnosis of pancreatic tumors yields accurate results in spite of the limitations faced in obtaining adequate samples by needle aspirates.
Subject(s)
Carcinoma, Ductal , Pancreatic Neoplasms , Adult , Aged , Carcinoma, Ductal/pathology , Female , Humans , Male , Middle Aged , Pancreatic Neoplasms/pathology , Retrospective Studies , Tertiary Care CentersABSTRACT
The B2 phylogenetic group of Escherichia coli contains important pathogens such as extraintestinal pathogenic, adherent-invasive, and uropathogenic strains. In this study, we used comparative genomics and statistical methods to identify genetic variations that define a subset of pathogenic strains belonging to the B2 phylogenetic group. An initial proof of concept analysis indicated that five of the 62 E. coli strains available in the Kyoto Encyclopedia of Genes and Genomes database showed close association with B2 adherent-invasive E. coli, forming a subgroup within the B2 phylogenetic group. The tool, kSNP which uses a k-mer approach, and the statistical phenotype prediction tool PPFS2 were then employed to identify 29 high-resolution SNPs, which reaffirmed this grouping. PPFS2 analysis also provided indications that the clustering of this subgroup was highly consistent, and thus, could have a strong phenotypic basis rather than being only evolutionary. Protein homology analyses identified three proteins to be conserved across this subgrouping, two CRISPR-Cas proteins and a hypothetical protein. Functional analyses of these genetic and protein variations may provide insights into the phenotype of these strains.
Subject(s)
Escherichia coli/classification , Escherichia coli/genetics , Genetic Markers , Genotype , Polymorphism, Single Nucleotide , Biostatistics , Cluster Analysis , Computational Biology , Conserved Sequence , Phenotype , Phylogeny , Sequence Homology, Amino AcidABSTRACT
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.
Subject(s)
Campylobacter Infections/immunology , Campylobacter/immunology , Gastrointestinal Diseases/immunology , Macrophages/immunology , Receptors, Pattern Recognition/immunology , Base Sequence , Cell Line, Tumor , Cyclic AMP Response Element-Binding Protein/metabolism , Gastrointestinal Diseases/microbiology , Gene Expression Profiling , Humans , Immunity, Innate , Inflammation/immunology , Interferon Regulatory Factors/metabolism , Macrophages/microbiology , Mass Spectrometry , MicroRNAs/genetics , Microscopy, Confocal , NF-kappa B/metabolism , Nuclear Proteins/biosynthesis , Phosphoproteins/biosynthesis , Protein Interaction Maps , Proteomics , RNA, Long Noncoding/genetics , Receptors, Pattern Recognition/genetics , STAT Transcription Factors/metabolism , Sequence Analysis, RNAABSTRACT
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.