RESUMO
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.
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Conservação dos Recursos Naturais , Genoma , Marsupiais , Animais , Marsupiais/genética , Austrália , Polimorfismo de Nucleotídeo Único , Extinção BiológicaRESUMO
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.
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Microbiota , Probióticos , Cavalos , Masculino , Animais , Sêmen , Projetos Piloto , Prebióticos , RNA Ribossômico 16S/genética , Espermatozoides , Probióticos/farmacologiaRESUMO
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.
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Microbiota , Sêmen , Feminino , Masculino , Cavalos/genética , Humanos , Animais , RNA Ribossômico 16S/genética , FertilidadeRESUMO
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.
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Células Secretoras de Insulina , Insulina , Camundongos , Animais , Insulina/farmacologia , Apolipoproteína A-I/metabolismo , Células Secretoras de Insulina/metabolismo , Colesterol/metabolismo , Glucose/metabolismo , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/farmacologiaRESUMO
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.
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Insulina , Músculo Esquelético , Camundongos , Animais , Insulina/farmacologia , Insulina/metabolismo , Músculo Esquelético/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Mitocôndrias/metabolismoRESUMO
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.
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Leucemia Mieloide Aguda , Células-Tronco Neoplásicas , Fatores de Processamento de RNA , Hematopoese , Humanos , Leucemia Mieloide Aguda/metabolismo , Células-Tronco Neoplásicas/metabolismo , Proteômica , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismoRESUMO
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.
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Rodófitas , Alga Marinha , Regulação para Baixo/genética , Ecossistema , Imunidade , Alga Marinha/genéticaRESUMO
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.
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Células Endoteliais , Fatores de Transcrição , Dedos de Zinco , Proteína 1 de Resposta de Crescimento Precoce/genética , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Células Endoteliais/metabolismo , Regulação da Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Análise de Sequência de RNA , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
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.
Assuntos
Butanonas , Células Quimiorreceptoras/metabolismo , Suplementos Nutricionais , Regulação da Expressão Gênica , Infertilidade Masculina/metabolismo , Tephritidae/metabolismo , Animais , MasculinoRESUMO
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.
Assuntos
Esôfago de Barrett/etiologia , Esôfago de Barrett/metabolismo , Biomarcadores , Microambiente Celular , Suscetibilidade a Doenças , Esôfago/metabolismo , Adulto , Esôfago de Barrett/patologia , Microambiente Celular/genética , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Esôfago/microbiologia , Esôfago/patologia , Feminino , Refluxo Gastroesofágico/complicações , Refluxo Gastroesofágico/etiologia , Perfilação da Expressão Gênica , Infecções por Bactérias Gram-Negativas/complicações , Infecções por Bactérias Gram-Negativas/microbiologia , Interações Hospedeiro-Patógeno/genética , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Mastócitos/imunologia , Mastócitos/metabolismo , Metaplasia , Microbiota , Pessoa de Meia-Idade , Modelos Biológicos , RNA Ribossômico 16SRESUMO
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.
Assuntos
MicroRNAs , RNA Longo não Codificante , Humanos , MicroRNAs/genética , RNA Longo não Codificante/genética , RNA Mensageiro , RNA não Traduzido/genética , Transcriptoma/genéticaRESUMO
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.
Assuntos
Permeabilidade Capilar , Molécula 1 de Adesão de Célula Vascular , Inibidores da Angiogênese/farmacologia , Animais , Humanos , Camundongos , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Coelhos , Ratos , Molécula 1 de Adesão de Célula Vascular/metabolismo , Molécula 1 de Adesão de Célula Vascular/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
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.
Assuntos
Cárie Dentária/microbiologia , Variação Genética , Lacticaseibacillus rhamnosus/genética , Boca/microbiologia , Proteínas de Bactérias/genética , Evolução Molecular , Humanos , Lacticaseibacillus rhamnosus/classificação , Lacticaseibacillus rhamnosus/patogenicidade , Filogenia , Polissacarídeos Bacterianos/genética , Seleção Genética , Sistemas Toxina-Antitoxina/genéticaRESUMO
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.
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Fenômenos Biológicos , Leucemia Mieloide Aguda , Processamento Alternativo , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Mutação , Prognóstico , Splicing de RNA/genética , Fatores de Processamento de RNA/genéticaRESUMO
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.
Assuntos
Sequência Consenso/genética , Genômica/métodos , Alinhamento de Sequência/métodos , Análise de Sequência de DNA/métodos , Algoritmos , Genoma Bacteriano/genética , Sequenciamento de Nucleotídeos em Larga EscalaRESUMO
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/.
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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.
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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.