RESUMO
Ovarian cancer ascites is a native medium for cancer cells that allows investigation of their secretome in a natural environment. This medium is of interest as a promising source of potential biomarkers, and also as a medium for cell-cell communication. The aim of this study was to elucidate specific features of the malignant ascites metabolome and proteome. In order to omit components of the systemic response to ascites formation, we compared malignant ascites with cirrhosis ascites. Metabolome analysis revealed 41 components that differed significantly between malignant and cirrhosis ascites. Most of the identified cancer-specific metabolites are known to be important signaling molecules. Proteomic analysis identified 2096 and 1855 proteins in the ovarian cancer and cirrhosis ascites, respectively; 424 proteins were specific for the malignant ascites. Functional analysis of the proteome demonstrated that the major differences between cirrhosis and malignant ascites were observed for the cluster of spliceosomal proteins. Additionally, we demonstrate that several splicing RNAs were exclusively detected in malignant ascites, where they probably existed within protein complexes. This result was confirmed in vitro using an ovarian cancer cell line. Identification of spliceosomal proteins and RNAs in an extracellular medium is of particular interest; the finding suggests that they might play a role in the communication between cancer cells. In addition, malignant ascites contains a high number of exosomes that are known to play an important role in signal transduction. Thus our study reveals the specific features of malignant ascites that are associated with its function as a medium of intercellular communication.
Assuntos
Ascite/genética , Regulação Neoplásica da Expressão Gênica , Metaboloma/genética , Proteínas de Neoplasias/genética , Neoplasias Ovarianas/genética , Proteoma/genética , RNA Neoplásico/genética , Processamento Alternativo , Ascite/metabolismo , Ascite/patologia , Comunicação Celular , Linhagem Celular Tumoral , Exossomos/química , Exossomos/metabolismo , Feminino , Fibrose/genética , Fibrose/metabolismo , Fibrose/patologia , Humanos , Proteínas de Neoplasias/metabolismo , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Proteoma/metabolismo , RNA Neoplásico/metabolismo , Transdução de Sinais , Spliceossomos/química , Spliceossomos/metabolismo , Vesículas Transportadoras/química , Vesículas Transportadoras/metabolismoRESUMO
The avian bacterial pathogen Mycoplasma gallisepticum is a good model for systems studies due to small genome and simplicity of regulatory pathways. In this study, we used RNA-Seq and MS-based proteomics to accurately map coding sequences, transcription start sites (TSSs) and transcript 3'-ends (T3Es). We used obtained data to investigate roles of TSSs and T3Es in stress-induced transcriptional responses. We identified 1061 TSSs at a false discovery rate of 10% and showed that almost all transcription in M. gallisepticum is initiated from classic TATAAT promoters surrounded by A/T-rich sequences. Our analysis revealed the pronounced operon structure complexity: on average, each coding operon has one internal TSS and T3Es in addition to the primary ones. Our transcriptomic approach based on the intervals between the two nearest transcript ends allowed us to identify two classes of T3Es: strong, unregulated, hairpin-containing T3Es and weak, heat shock-regulated, hairpinless T3Es. Comparing gene expression levels under different conditions revealed widespread and divergent transcription regulation in M. gallisepticum. Modeling suggested that the core promoter structure plays an important role in gene expression regulation. We have shown that the heat stress activation of cryptic promoters combined with the hairpinless T3Es suppression leads to widespread, seemingly non-functional transcription.
Assuntos
Regulação Bacteriana da Expressão Gênica , Mycoplasma gallisepticum/genética , Transcrição Gênica , Proteínas de Bactérias/química , Perfilação da Expressão Gênica , Genoma Bacteriano , Temperatura Alta , Mycoplasma gallisepticum/metabolismo , Regiões Promotoras Genéticas , RNA Antissenso/biossíntese , RNA Bacteriano/química , RNA Bacteriano/metabolismo , Ribossomos/metabolismo , Estresse Fisiológico/genética , Sítio de Iniciação de Transcrição , Transformação BacterianaRESUMO
BACKGROUND: Protein degradation is a basic cell process that operates in general protein turnover or to produce bioactive peptides. However, very little is known about the qualitative and quantitative composition of a plant cell peptidome, the actual result of this degradation. In this study we comprehensively analyzed a plant cell peptidome and systematically analyzed the peptide generation process. RESULTS: We thoroughly analyzed native peptide pools of Physcomitrella patens moss in two developmental stages as well as in protoplasts. Peptidomic analysis was supplemented by transcriptional profiling and quantitative analysis of precursor proteins. In total, over 20,000 unique endogenous peptides, ranging in size from 5 to 78 amino acid residues, were identified. We showed that in both the protonema and protoplast states, plastid proteins served as the main source of peptides and that their major fraction formed outside of chloroplasts. However, in general, the composition of peptide pools was very different between these cell types. In gametophores, stress-related proteins, e.g., late embryogenesis abundant proteins, were among the most productive precursors. The Driselase-mediated protonema conversion to protoplasts led to a peptide generation "burst", with a several-fold increase in the number of components in the latter. Degradation of plastid proteins in protoplasts was accompanied by suppression of photosynthetic activity. CONCLUSION: We suggest that peptide pools in plant cells are not merely a product of waste protein degradation, but may serve as important functional components for plant metabolism. We assume that the peptide "burst" is a form of biotic stress response that might produce peptides with antimicrobial activity from originally functional proteins. Potential functions of peptides in different developmental stages are discussed.
Assuntos
Bryopsida/citologia , Bryopsida/metabolismo , Células Germinativas Vegetais/citologia , Células Germinativas Vegetais/metabolismo , Peptídeos/metabolismo , Células Vegetais/metabolismo , Protoplastos/citologia , Bryopsida/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Fotossíntese , Proteínas de Plantas/metabolismo , Proteoma/metabolismo , Protoplastos/metabolismo , Alinhamento de SequênciaRESUMO
Personalized nutrition is of increasing interest to individuals actively monitoring their health. The relations between the duration of diet intervention and the effects on gut microbiota have yet to be elucidated. Here we examined the associations of short-term dietary changes, long-term dietary habits and lifestyle with gut microbiota. Stool samples from 248 citizen-science volunteers were collected before and after a self-reported 2-week personalized diet intervention, then analyzed using 16S rRNA sequencing. Considerable correlations between long-term dietary habits and gut community structure were detected. A higher intake of vegetables and fruits was associated with increased levels of butyrate-producing Clostridiales and higher community richness. A paired comparison of the metagenomes before and after the 2-week intervention showed that even a brief, uncontrolled intervention produced profound changes in community structure: resulting in decreased levels of Bacteroidaceae, Porphyromonadaceae and Rikenellaceae families and decreased alpha-diversity coupled with an increase of Methanobrevibacter, Bifidobacterium, Clostridium and butyrate-producing Lachnospiraceae- as well as the prevalence of a permatype (a bootstrapping-based variation of enterotype) associated with a higher diversity of diet. The response of microbiota to the intervention was dependent on the initial microbiota state. These findings pave the way for the development of an individualized diet.
Assuntos
Dieta , Microbioma Gastrointestinal , Bacteroidetes/genética , Bacteroidetes/isolamento & purificação , Bifidobacterium/genética , Bifidobacterium/isolamento & purificação , Clostridium/genética , Clostridium/isolamento & purificação , Análise por Conglomerados , Fezes/química , Fezes/microbiologia , Humanos , Metagenoma , Methanobrevibacter/genética , Methanobrevibacter/isolamento & purificação , RNA Ribossômico 16S/genética , Tamanho da Amostra , Análise de Sequência de DNARESUMO
Bacteria of class Mollicutes (mycoplasmas) feature significant genome reduction which makes them good model organisms for systems biology studies. Previously we demonstrated, that drastic transcriptional response of mycoplasmas to stress results in a very limited response on the level of protein. In this study we used heat stress model of M. gallisepticum and ribosome profiling to elucidate the process of genetic information transfer under stress. We found that under heat stress ribosomes demonstrate selectivity towards mRNA binding. We identified that heat stress response may be divided into two groups on the basis of absolute transcript abundance and fold-change in the translatome. One represents a noise-like response and another is likely an adaptive one. The latter include ClpB chaperone, cell division cluster, homologs of immunoblocking proteins and short ORFs with unknown function. We found that previously identified read-through of terminators contributes to the upregulation of transcripts in the translatome as well. In addition we identified that ribosomes of M. gallisepticum undergo reorganization under the heat stress. The most notable event is decrease of the amount of associated HU protein. In conclusion, only changes of few adaptive transcripts significantly impact translatome, while widespread noise-like transcription plays insignificant role in translation during stress.
Assuntos
Adaptação Fisiológica/genética , Resposta ao Choque Térmico/genética , Mycoplasma gallisepticum/genética , Ribossomos/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Cromatografia Líquida de Alta Pressão , Perfilação da Expressão Gênica/métodos , Regulação Bacteriana da Expressão Gênica , Temperatura Alta , Mycoplasma gallisepticum/metabolismo , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Estresse Fisiológico , Espectrometria de Massas em TandemRESUMO
Numerous studies are devoted to the intestinal microbiota and intercellular communication maintaining homeostasis. In this regard, vesicles secreted by bacteria represent one of the most popular topics for research. For example, the outer membrane vesicles (OMVs) of Bacteroides fragilis play an important nutritional role with respect to other microorganisms and promote anti-inflammatory effects on immune cells. However, toxigenic B. fragilis (ETBF) contributes to bowel disease, even causing colon cancer. If nontoxigenic B. fragilis (NTBF) vesicles exert a beneficial effect on the intestine, it is likely that ETBF vesicles can be utilized for potential pathogenic implementation. To confirm this possibility, we performed comparative proteomic HPLC-MS/MS analysis of vesicles isolated from ETBF and NTBF. Furthermore, we performed, for the first time, HPLC-MS/MS and GS-MS comparative metabolomic analysis for the vesicles isolated from both strains with subsequent reconstruction of the vesicle metabolic pathways. We utilized fluxomic experiments to validate the reconstructed biochemical reaction activities and finally observed considerable difference in the vesicle proteome and metabolome profiles. Compared with NTBF OMVs, metabolic activity of ETBF OMVs provides their similarity to micro reactors that are likely to be used for long-term persistence and implementing pathogenic potential in the host.
Assuntos
Bacteroides fragilis/citologia , Metabolômica/métodos , Vesículas Secretórias/metabolismo , Bacteroides fragilis/patogenicidade , Cromatografia Líquida de Alta Pressão , Redes e Vias Metabólicas , Espectrometria de Massas em TandemRESUMO
MALINA is a web service for bioinformatic analysis of whole-genome metagenomic data obtained from human gut microbiota sequencing. As input data, it accepts metagenomic reads of various sequencing technologies, including long reads (such as Sanger and 454 sequencing) and next-generation (including SOLiD and Illumina). It is the first metagenomic web service that is capable of processing SOLiD color-space reads, to authors' knowledge. The web service allows phylogenetic and functional profiling of metagenomic samples using coverage depth resulting from the alignment of the reads to the catalogue of reference sequences which are built into the pipeline and contain prevalent microbial genomes and genes of human gut microbiota. The obtained metagenomic composition vectors are processed by the statistical analysis and visualization module containing methods for clustering, dimension reduction and group comparison. Additionally, the MALINA database includes vectors of bacterial and functional composition for human gut microbiota samples from a large number of existing studies allowing their comparative analysis together with user samples, namely datasets from Russian Metagenome project, MetaHIT and Human Microbiome Project (downloaded from http://hmpdacc.org). MALINA is made freely available on the web at http://malina.metagenome.ru. The website is implemented in JavaScript (using Ext JS), Microsoft .NET Framework, MS SQL, Python, with all major browsers supported.