RESUMO
BACKGROUND: Alzheimer's disease (AD) is a chronic neurodegenerative disease with pathological hallmarks including the formation of extracellular aggregates of amyloid-beta (Aß) known as plaques and intracellular tau tangles. Coincident with the formation of Aß plaques is recruitment and activation of glial cells to the plaque forming a plaque niche. In addition to histological data showing the formation of the niche, AD genetic studies have added to the growing appreciation of how dysfunctional glia pathways drive neuropathology, with emphasis on microglia pathways. Genomic approaches enable comparisons of human disease profiles between different mouse models informing on their utility to evaluate secondary changes to triggers such as Aß deposition. METHODS: In this study, we utilized two animal models of AD to examine and characterize the AD-associated pathology: the Tg2576 Swedish APP (KM670/671NL) and TgCRND8 Swedish plus Indiana APP (KM670/671NL + V717F) lines. We used laser capture microscopy (LCM) to isolate samples surrounding Thio-S positive plaques from distal non-plaque tissue. These samples were then analyzed using RNA sequencing. RESULTS: We determined age-associated transcriptomic differences between two similar yet distinct APP transgenic mouse models, known to differ in proportional amyloidogenic species and plaque deposition rates. In Tg2576, human AD gene signatures were not observed despite profiling mice out to 15 months of age. TgCRND8 mice however showed progressive and robust induction of lysomal, neuroimmune, and ITIM/ITAM-associated gene signatures overlapping with prior human AD brain transcriptomic studies. Notably, RNAseq analyses highlighted the vast majority of transcriptional changes observed in aging TgCRND8 cortical brain homogenates were in fact specifically enriched within the plaque niche samples. Data uncovered plaque-associated enrichment of microglia-related genes such as ITIM/ITAM-associated genes and pathway markers of phagocytosis. CONCLUSION: This work may help guide improved translational value of APP mouse models of AD, particularly for strategies aimed at targeting neuroimmune and neurodegenerative pathways, by demonstrating that TgCRND8 more closely recapitulates specific human AD-associated transcriptional responses.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Córtex Cerebral/metabolismo , Citocinas/metabolismo , Regulação da Expressão Gênica/genética , Fatores Etários , Doença de Alzheimer/genética , Doença de Alzheimer/imunologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/genética , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Córtex Cerebral/patologia , Correlação de Dados , Modelos Animais de Doenças , Humanos , Microdissecção e Captura a Laser , Camundongos , Camundongos Transgênicos , Proteínas dos Microfilamentos/metabolismo , Mutação/genética , Placa Amiloide/patologia , RNA Mensageiro/metabolismo , TranscriptomaRESUMO
Reversible janus associated kinase (JAK) inhibitors such as tofacitinib and decernotinib block cytokine signaling and are efficacious in treating autoimmune diseases. However, therapeutic doses are limited due to inhibition of other JAK/signal transducer and activator of transcription pathways associated with hematopoiesis, lipid biogenesis, infection, and immune responses. A selective JAK3 inhibitor may have a better therapeutic index; however, until recently, no compounds have been described that maintain JAK3 selectivity in cells, as well as against the kinome, with good physicochemical properties to test the JAK3 hypothesis in vivo. To quantify the biochemical basis for JAK isozyme selectivity, we determined that the apparent Km value for each JAK isozyme ranged from 31.8 to 2.9 µM for JAK1 and JAK3, respectively. To confirm compound activity in cells, we developed a novel enzyme complementation assay that read activity of single JAK isozymes in a cellular context. Reversible JAK3 inhibitors cannot achieve sufficient selectivity against other isozymes in the cellular context due to inherent differences in enzyme ATP Km values. Therefore, we developed irreversible JAK3 compounds that are potent and highly selective in vitro in cells and against the kinome. Compound 2, a potent inhibitor of JAK3 (0.15 nM) was 4300-fold selective for JAK3 over JAK1 in enzyme assays, 67-fold [interleukin (IL)-2 versus IL-6] or 140-fold [IL-2 versus erythropoietin or granulocyte-macrophage colony-stimulating factor (GMCSF)] selective in cellular reporter assays and >35-fold selective in human peripheral blood mononuclear cell assays (IL-7 versus IL-6 or GMCSF). In vivo, selective JAK3 inhibition was sufficient to block the development of inflammation in a rat model of rheumatoid arthritis, while sparing hematopoiesis.
Assuntos
Doenças Autoimunes , Janus Quinase 1 , Janus Quinase 3 , Piperidinas/farmacologia , Pirimidinas/farmacologia , Pirróis/farmacologia , Animais , Artrite Experimental/tratamento farmacológico , Doenças Autoimunes/tratamento farmacológico , Doenças Autoimunes/metabolismo , Relação Dose-Resposta a Droga , Monitoramento de Medicamentos/métodos , Humanos , Isoenzimas , Janus Quinase 1/antagonistas & inibidores , Janus Quinase 1/química , Janus Quinase 1/metabolismo , Janus Quinase 3/antagonistas & inibidores , Janus Quinase 3/química , Janus Quinase 3/metabolismo , Monitorização Imunológica/métodos , Inibidores de Proteínas Quinases/farmacologia , RatosRESUMO
Psychrobacter arcticus strain 273-4, which grows at temperatures as low as -10 degrees C, is the first cold-adapted bacterium from a terrestrial environment whose genome was sequenced. Analysis of the 2.65-Mb genome suggested that some of the strategies employed by P. arcticus 273-4 for survival under cold and stress conditions are changes in membrane composition, synthesis of cold shock proteins, and the use of acetate as an energy source. Comparative genome analysis indicated that in a significant portion of the P. arcticus proteome there is reduced use of the acidic amino acids and proline and arginine, which is consistent with increased protein flexibility at low temperatures. Differential amino acid usage occurred in all gene categories, but it was more common in gene categories essential for cell growth and reproduction, suggesting that P. arcticus evolved to grow at low temperatures. Amino acid adaptations and the gene content likely evolved in response to the long-term freezing temperatures (-10 degrees C to -12 degrees C) of the Kolyma (Siberia) permafrost soil from which this strain was isolated. Intracellular water likely does not freeze at these in situ temperatures, which allows P. arcticus to live at subzero temperatures.
Assuntos
DNA Bacteriano/química , DNA Bacteriano/genética , Genoma Bacteriano , Psychrobacter/genética , Temperatura Baixa , Congelamento , Dados de Sequência Molecular , Psychrobacter/isolamento & purificação , Psychrobacter/fisiologia , Análise de Sequência de DNA , Sibéria , Microbiologia do SoloRESUMO
Although the potential value of RNA as a target for new small molecule therapeutics is becoming increasingly credible, the physicochemical properties required for small molecules to selectively bind to RNA remain relatively unexplored. To investigate the druggability of RNAs with small molecules, we have employed affinity mass spectrometry, using the Automated Ligand Identification System (ALIS), to screen 42 RNAs from a variety of RNA classes, each against an array of chemically diverse drug-like small molecules (~50,000 compounds) and functionally annotated tool compounds (~5100 compounds). The set of RNA-small molecule interactions that was generated was compared with that for protein-small molecule interactions, and naïve Bayesian models were constructed to determine the types of specific chemical properties that bias small molecules toward binding to RNA. This set of RNA-selective chemical features was then used to build an RNA-focused set of ~3800 small molecules that demonstrated increased propensity toward binding the RNA target set. In addition, the data provide an overview of the specific physicochemical properties that help to enable binding to potential RNA targets. This work has increased the understanding of the chemical properties that are involved in small molecule binding to RNA, and the methodology used here is generally applicable to RNA-focused drug discovery efforts.
Assuntos
Descoberta de Drogas , Terapia de Alvo Molecular , RNA/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Humanos , Ligantes , Espectrometria de Massas , Preparações Farmacêuticas , RNA/genética , Bibliotecas de Moléculas Pequenas/químicaRESUMO
We present a tool to measure gene and protein expression levels in single cells with DNA-labeled antibodies and droplet microfluidics. Using the RNA expression and protein sequencing assay (REAP-seq), we quantified proteins with 82 barcoded antibodies and >20,000 genes in a single workflow. We used REAP-seq to assess the costimulatory effects of a CD27 agonist on human CD8+ lymphocytes and to identify and characterize an unknown cell type.
Assuntos
Proteínas/metabolismo , Análise de Célula Única/métodos , Linfócitos T CD4-Positivos/metabolismo , Humanos , Ativação Linfocitária/imunologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de Proteína , Análise de Sequência de RNARESUMO
The discovery of a potent selective low dose Janus kinase 1 (JAK1) inhibitor suitable for clinical evaluation is described. As part of an overall goal to minimize dose, we pursued a medicinal chemistry strategy focused on optimization of key parameters that influence dose size, including lowering human Clint and increasing intrinsic potency, bioavailability, and solubility. To impact these multiple parameters simultaneously, we used lipophilic ligand efficiency as a key metric to track changes in the physicochemical properties of our analogs, which led to improvements in overall compound quality. In parallel, structural information guided advancements in JAK1 selectivity by informing on new vector space, which enabled the discovery of a unique key amino acid difference between JAK1 (Glu966) and JAK2 (Asp939). This difference was exploited to consistently produce analogs with the best balance of JAK1 selectivity, efficacy, and projected human dose, ultimately culminating in the discovery of compound 28.
Assuntos
Janus Quinase 1/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/química , Pirazóis/farmacologia , Animais , Cães , Descoberta de Drogas , Halogenação , Humanos , Janus Quinase 1/química , Janus Quinase 1/metabolismo , Simulação de Acoplamento Molecular , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacocinética , Pirazóis/administração & dosagem , Pirazóis/farmacocinética , Ratos , Relação Estrutura-AtividadeRESUMO
Genotyping of hepatitis C virus (HCV) plays an important role in the treatment of HCV. As new genotype-specific treatment options become available, it has become increasingly important to have accurate HCV genotype and subtype information to ensure that the most appropriate treatment regimen is selected. Most current genotyping methods are unable to detect mixed genotypes from two or more HCV infections. Next generation sequencing (NGS) allows for rapid and low cost mass sequencing of viral genomes and provides an opportunity to probe the viral population from a single host. In this paper, the possibility of using short NGS reads for direct HCV genotyping without genome assembly was evaluated. We surveyed the publicly-available genetic content of three HCV drug target regions (NS3, NS5A, NS5B) in terms of whether these genes contained genotype-specific regions that could predict genotype. Six genotypes and 38 subtypes were included in this study. An automated phylogenetic analysis based HCV genotyping method was implemented and used to assess different HCV target gene regions. Candidate regions of 250-bp each were found for all three genes that have enough genetic information to predict HCV genotypes/subtypes. Validation using public datasets shows 100% genotyping accuracy. To test whether these 250-bp regions were sufficient to identify mixed genotypes, we developed a random primer-based method to sequence HCV plasma samples containing mixtures of two HCV genotypes in different ratios. We were able to determine the genotypes without ambiguity and to quantify the ratio of the abundances of the mixed genotypes in the samples. These data provide a proof-of-concept that this random primed, NGS-based short-read genotyping approach does not need prior information about the viral population and is capable of detecting mixed viral infection.
Assuntos
Hepacivirus/genética , Hepatite C/diagnóstico , Genes Virais , Genótipo , Hepatite C/sangue , Hepatite C/virologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Técnicas de Diagnóstico Molecular , Tipagem Molecular , Filogenia , Análise de Sequência de DNARESUMO
OBJECTIVES: Human airway epithelial cells are the principal target of human rhinovirus (HRV), a common cold pathogen that triggers the majority of asthma exacerbations. The objectives of this study were 1) to evaluate an in vitro air liquid interface cultured human airway epithelial cell model for HRV infection, and 2) to identify gene expression patterns associated with asthma intrinsically and/or after HRV infection using this model. METHODS: Air-liquid interface (ALI) human airway epithelial cell cultures were prepared from 6 asthmatic and 6 non-asthmatic donors. The effects of rhinovirus RV-A16 on ALI cultures were compared. Genome-wide gene expression changes in ALI cultures following HRV infection at 24 hours post exposure were further analyzed using RNA-seq technology. Cellular gene expression and cytokine/chemokine secretion were further evaluated by qPCR and a Luminex-based protein assay, respectively. MAIN RESULTS: ALI cultures were readily infected by HRV. RNA-seq analysis of HRV infected ALI cultures identified sets of genes associated with asthma specific viral responses. These genes are related to inflammatory pathways, epithelial structure and remodeling and cilium assembly and function, including those described previously (e.g. CCL5, CXCL10 and CX3CL1, MUC5AC, CDHR3), and novel ones that were identified for the first time in this study (e.g. CCRL1). CONCLUSIONS: ALI-cultured human airway epithelial cells challenged with HRV are a useful translational model for the study of HRV-induced responses in airway epithelial cells, given that gene expression profile using this model largely recapitulates some important patterns of gene responses in patients during clinical HRV infection. Furthermore, our data emphasize that both abnormal airway epithelial structure and inflammatory signaling are two important asthma signatures, which can be further exacerbated by HRV infection.
Assuntos
Asma/genética , Asma/virologia , Diferenciação Celular/genética , Células Epiteliais/virologia , Infecções por Picornaviridae/genética , Sistema Respiratório/virologia , Adolescente , Adulto , Células Cultivadas , Quimiocinas/genética , Criança , Feminino , Expressão Gênica/genética , Humanos , Inflamação/genética , Inflamação/virologia , Masculino , Pessoa de Meia-Idade , Infecções por Picornaviridae/virologia , Rhinovirus , Transdução de Sinais/genéticaRESUMO
Alternaria alternata is a fungal allergen linked to the development of severe asthma in humans. In view of the clinical relationship between A. alternata and asthma, we sought to investigate the allergic activity of this antigen after direct application to the lungs of Brown Norway rats. Here we demonstrate that a single intratracheal instillation of A. alternata induces dose and time dependent eosinophil influx, edema and Type 2 helper cell cytokine production in the lungs of BN rats. We established the temporal profile of eosinophilic infiltration and cytokine production, such as Interleukin-5 and Interleukin-13, following A. alternata challenge. These responses were comparable to Ovalbumin induced models of asthma and resulted in peak inflammatory responses 48h following a single challenge, eliminating the need for multiple sensitizations and challenges. The initial perivascular and peribronchiolar inflammation preceded alveolar inflammation, progressing to a more sub-acute inflammatory response with notable epithelial cell hypertrophy. To limit the effects of an A. alternata inflammatory response, MK-7246 was utilized as it is an antagonist for Chemoattractant Receptor-homologous molecule expressed in Th2 cells. In a dose-dependent manner, MK-7246 decreased eosinophil influx and Th2 cytokine production following the A. alternata challenge. Furthermore, therapeutic administration of corticosteroids resulted in a dose-dependent decrease in eosinophil influx and Th2 cytokine production. Reproducible asthma-related outcomes and amenability to pharmacological intervention by mechanisms relevant to asthma demonstrate that an A. alternata induced pulmonary inflammation in BN rats is a valuable preclinical pharmacodynamic in vivo model for evaluating the pharmacological inhibitors of allergic pulmonary inflammation.
Assuntos
Alternaria/efeitos dos fármacos , Anti-Inflamatórios/farmacologia , Carbolinas/farmacologia , Pneumonia/tratamento farmacológico , Receptores de Formil Peptídeo/metabolismo , Células Th2/efeitos dos fármacos , Alérgenos/imunologia , Alternaria/imunologia , Animais , Asma/tratamento farmacológico , Asma/imunologia , Asma/metabolismo , Citocinas/imunologia , Citocinas/metabolismo , Eosinófilos/efeitos dos fármacos , Eosinófilos/imunologia , Eosinófilos/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Interleucina-13/imunologia , Interleucina-13/metabolismo , Interleucina-5/imunologia , Interleucina-5/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/metabolismo , Masculino , Ovalbumina/imunologia , Ovalbumina/farmacologia , Pneumonia/imunologia , Pneumonia/metabolismo , Ratos , Ratos Endogâmicos BN , Receptores de Formil Peptídeo/imunologia , Células Th2/imunologiaRESUMO
Accumulating evidence indicates protective actions of mineralocorticoid antagonists (MR antagonists) on cardiovascular pathology, which includes blunting vascular inflammation and myocardial fibrosis. We examined the anti-inflammatory and anti-fibrotic potential of MR antagonists in rodent respiratory models. In an ovalbumin allergic and challenged Brown Norway rat model, the total cell count in nasal lavage was 29,348 ± 5451, which was blocked by spironolactone (0.3-60 mg/kg, p.o.) and eplerenone (0.3-30 mg/kg, p.o.). We also found that MR antagonists attenuated pulmonary inflammation in the Brown Norway rat. A series of experiments were conducted to determine the actions of MR blockade in acute/chronic lung injury models. (1) Ex vivo lung slice rat experiments found that eplerenone (0.01 and 10 µM) and spironolactone (10 µM) diminished lung hydroxyproline concentrations by 55 ± 5, 122 ± 9, and 83 ± 8%. (2) In in vivo studies, MR antagonists attenuated the increases in bronchioalveolar lavage (BAL) neutrophils and macrophages caused by lung bleomycin exposure. In separate studies, bleomycin (4.0 U/kg, i.t.) increased lung levels of hydroxyproline by approximately 155%, which was blocked by spironolactone (10-60 mg/kg, p.o.). In a rat Lipopolysaccharide (LPS) model, spironolactone inhibited acute increases in BAL cytokines with moderate effects on neutrophils. Finally, we found that chronic LPS exposure significantly increased end expiratory lung and decreased lung elastance in the mouse. These functional effects of chronic LPS were improved by MR antagonists. Our results demonstrate that MR antagonists have significant pharmacological actions in the respiratory system.
Assuntos
Bleomicina/efeitos adversos , Antagonistas de Receptores de Mineralocorticoides/farmacologia , Pneumonia/tratamento farmacológico , Receptores de Mineralocorticoides/metabolismo , Animais , Modelos Animais de Doenças , Elasticidade/efeitos dos fármacos , Fibrose , Hidroxiprolina/metabolismo , Hipersensibilidade/tratamento farmacológico , Hipersensibilidade/metabolismo , Hipersensibilidade/patologia , Hipersensibilidade/fisiopatologia , Lipopolissacarídeos/efeitos adversos , Pulmão/efeitos dos fármacos , Pulmão/patologia , Pulmão/fisiopatologia , Masculino , Camundongos , Antagonistas de Receptores de Mineralocorticoides/uso terapêutico , Pneumonia/metabolismo , Pneumonia/patologia , Pneumonia/fisiopatologia , Ventilação Pulmonar/efeitos dos fármacos , RatosRESUMO
High-throughput sequencing studies during the last decade have uncovered that bacterial genomes are very diverse and dynamic, resulting primarily from the frequent and promiscuous horizontal gene exchange that characterizes the bacterial domain of life. However, a robust understanding of the rates of genetic exchange for most bacterial species under natural conditions and the influence of the ecological settings on the rates remain elusive, severely limiting our view of the microbial world. Here, we analyzed the complete genomic sequences and expressed transcriptomes of several Shewanella baltica isolates recovered from different depths in the Baltic Sea and found that isolates from more similar depths had exchanged a larger fraction of their core and auxiliary genome, up to 20% of the total, compared with isolates from more different depths. The exchanged genes seem to be ecologically important and contribute to the successful adaptation of the isolates to the unique physicochemical conditions of the depth. Importantly, the latter genes were exchanged in very recent past, presumably as an effect of isolate's seasonal migration across the water column, and reflected sexual speciation within the same depth. Therefore, our findings reveal that genetic exchange in response to environmental settings may be surprisingly rapid, which has important broader impacts for understanding bacterial speciation and evolution and for modeling bacterial responses to human-induced environmental impacts.
Assuntos
Transferência Genética Horizontal/genética , Shewanella/genética , Adaptação Biológica/genética , Evolução Biológica , Especiação Genética , Genoma Bacteriano/genética , Humanos , Oceanos e Mares , Análise de Sequência com Séries de Oligonucleotídeos , Shewanella/classificaçãoRESUMO
DNA-DNA hybridization (DDH) values have been used by bacterial taxonomists since the 1960s to determine relatedness between strains and are still the most important criterion in the delineation of bacterial species. Since the extent of hybridization between a pair of strains is ultimately governed by their respective genomic sequences, we examined the quantitative relationship between DDH values and genome sequence-derived parameters, such as the average nucleotide identity (ANI) of common genes and the percentage of conserved DNA. A total of 124 DDH values were determined for 28 strains for which genome sequences were available. The strains belong to six important and diverse groups of bacteria for which the intra-group 16S rRNA gene sequence identity was greater than 94 %. The results revealed a close relationship between DDH values and ANI and between DNA-DNA hybridization and the percentage of conserved DNA for each pair of strains. The recommended cut-off point of 70 % DDH for species delineation corresponded to 95 % ANI and 69 % conserved DNA. When the analysis was restricted to the protein-coding portion of the genome, 70 % DDH corresponded to 85 % conserved genes for a pair of strains. These results reveal extensive gene diversity within the current concept of "species". Examination of reciprocal values indicated that the level of experimental error associated with the DDH method is too high to reveal the subtle differences in genome size among the strains sampled. It is concluded that ANI can accurately replace DDH values for strains for which genome sequences are available.
Assuntos
Técnicas de Tipagem Bacteriana , Genoma Bacteriano/genética , Hibridização de Ácido Nucleico/métodos , Análise de Sequência de DNA , DNA Bacteriano/análise , DNA Bacteriano/genética , Bactérias Gram-Negativas/classificação , Bactérias Gram-Negativas/genética , Bactérias Gram-Positivas/classificação , Bactérias Gram-Positivas/genéticaRESUMO
Anaerobic cultures of Shewanella oneidensis MR-1 grown with nitrate as the sole electron acceptor exhibited sequential reduction of nitrate to nitrite and then to ammonium. Little dinitrogen and nitrous oxide were detected, and no growth occurred on nitrous oxide. A mutant with the napA gene encoding periplasmic nitrate reductase deleted could not respire or assimilate nitrate and did not express nitrate reductase activity, confirming that the NapA enzyme is the sole nitrate reductase. Hence, S. oneidensis MR-1 conducts respiratory nitrate ammonification, also termed dissimilatory nitrate reduction to ammonium, but not respiratory denitrification.
Assuntos
Amônia/metabolismo , Nitratos/metabolismo , Nitritos/metabolismo , Shewanella/metabolismo , Anaerobiose , Genoma Bacteriano , Mutação , Nitrato Redutase/genética , Nitrato Redutase/metabolismo , Oxirredução , Shewanella/genética , Fatores de TempoRESUMO
Dangling ends and surface-proximal tails of gene targets influence probe-target duplex formation and affect the signal intensity of probes on diagnostic microarrays. This phenomenon was evaluated using an oligonucleotide microarray containing 18-mer probes corresponding to the 16S rRNA genes of 10 waterborne pathogens and a number of synthetic and PCR-amplified gene targets. Signal intensities for Klenow/random primer-labeled 16S rRNA gene targets were dissimilar from those for 45-mer synthetic targets for nearly 73% of the probes tested. Klenow/random primer-labeled targets resulted in an interaction with a complex mixture of 16S rRNA genes (used as the background) 3.7 times higher than the interaction of 45-mer targets with the same mixture. A 7-base-long dangling end sequence with perfect homology to another single-stranded background DNA sequence was sufficient to produce a cross-hybridization signal that was as strong as the signal obtained by the probe-target duplex itself. Gibbs free energy between the target and a well-defined background was found to be a better indicator of hybridization signal intensity than the sequence or length of the dangling end alone. The dangling end (Gibbs free energy of -7.6 kcal/mol) was found to be significantly more prone to target-background interaction than the surface-proximal tail (Gibbs free energy of -64.5 kcal/mol). This study underlines the need for careful target preparation and evaluation of signal intensities for diagnostic arrays using 16S rRNA and other gene targets due to the potential for target interaction with a complex background.
Assuntos
Bactérias/genética , DNA Bacteriano/química , Genes de RNAr , Conformação de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos/métodos , RNA Ribossômico 16S/genética , Bactérias/química , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sequência de Bases , Humanos , Dados de Sequência Molecular , Sondas de Oligonucleotídeos/química , Reação em Cadeia da Polimerase , TermodinâmicaRESUMO
The gamma-proteobacterium Shewanella oneidensis strain MR-1 is a metabolically versatile organism that can reduce a wide range of organic compounds, metal ions, and radionuclides. Similar to most other sequenced organisms, approximately 40% of the predicted ORFs in the S. oneidensis genome were annotated as uncharacterized "hypothetical" genes. We implemented an integrative approach by using experimental and computational analyses to provide more detailed insight into gene function. Global expression profiles were determined for cells after UV irradiation and under aerobic and suboxic growth conditions. Transcriptomic and proteomic analyses confidently identified 538 hypothetical genes as expressed in S. oneidensis cells both as mRNAs and proteins (33% of all predicted hypothetical proteins). Publicly available analysis tools and databases and the expression data were applied to improve the annotation of these genes. The annotation results were scored by using a seven-category schema that ranked both confidence and precision of the functional assignment. We were able to identify homologs for nearly all of these hypothetical proteins (97%), but could confidently assign exact biochemical functions for only 16 proteins (category 1; 3%). Altogether, computational and experimental evidence provided functional assignments or insights for 240 more genes (categories 2-5; 45%). These functional annotations advance our understanding of genes involved in vital cellular processes, including energy conversion, ion transport, secondary metabolism, and signal transduction. We propose that this integrative approach offers a valuable means to undertake the enormous challenge of characterizing the rapidly growing number of hypothetical proteins with each newly sequenced genome.
Assuntos
Perfilação da Expressão Gênica , Shewanella/genética , Regulação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , Fases de Leitura Aberta , Proteoma/análise , Shewanella/metabolismo , Shewanella/efeitos da radiaçãoRESUMO
We report the phylogenetic and physiological characterization of a mesophilic and halophilic member of the filamentous anoxygenic phototrophic (FAP) bacteria, provisionally named ' Candidatus Chorothrix halophila' gen. nov. sp. nov., that has been maintained in a highly enriched culture in our laboratory for over a decade. Phylogenetic analysis of small-subunit RNA-encoding sequences places ' Candidatus Chlorothrix halophila' in a clade that includes cultivated members of the genera Chloroflexus and Oscillochloris. Physiological studies demonstrated sulfide-dependent photosynthetic uptake of (14)C-labeled bicarbonate. Enzymatic assays for the activity of propionyl-coenzyme A synthase indicated that ' Candidatus Chlorothrix halophila' does not use the 3-hydroxypropionate cycle of Chloroflexus aurantiacus OK-70-fl for autotrophic carbon assimilation. New concepts regarding the taxonomy and phylogeny of FAP bacteria have emerged from this work.