RESUMO
Alignment-based RNA-seq quantification methods typically involve a time-consuming alignment process prior to estimating transcript abundances. In contrast, alignment-free RNA-seq quantification methods bypass this step, resulting in significant speed improvements. Existing alignment-free methods rely on the Expectation-Maximization (EM) algorithm for estimating transcript abundances. However, EM algorithms only guarantee locally optimal solutions, leaving room for further accuracy improvement by finding a globally optimal solution. In this study, we present TQSLE, the first alignment-free RNA-seq quantification method that provides a globally optimal solution for transcript abundances estimation. TQSLE adopts a two-step approach: first, it constructs a k-mer frequency matrix A for the reference transcriptome and a k-mer frequency vector b for the RNA-seq reads; then, it directly estimates transcript abundances by solving the linear equation ATAx = ATb. We evaluated the performance of TQSLE using simulated and real RNA-seq data sets and observed that, despite comparable speed to other alignment-free methods, TQSLE outperforms them in terms of accuracy. TQSLE is freely available at https://github.com/yhg926/TQSLE.
Assuntos
Algoritmos , Transcriptoma , RNA-Seq , Análise de Sequência de RNA/métodos , Software , Perfilação da Expressão Gênica/métodosRESUMO
SUMMARY: We propose RabbitKSSD, a high-speed genome distance estimation tool. Specifically, we leverage load-balanced task partitioning, fast I/O, efficient intermediate result accesses, and high-performance data structures to improve overall efficiency. Our performance evaluation demonstrates that RabbitKSSD achieves speedups ranging from 5.7× to 19.8× over Kssd for the time-consuming sketch generation and distance computation on commonly used workstations. In addition, it significantly outperforms Mash, BinDash, and Dashing2. Moreover, RabbitKSSD can efficiently perform all-vs-all distance computation for all RefSeq complete bacterial genomes (455 GB in FASTA format) in just 2 min on a 64-core workstation. AVAILABILITY AND IMPLEMENTATION: RabbitKSSD is available at https://github.com/RabbitBio/RabbitKSSD.
Assuntos
Genoma Bacteriano , Software , Evolução BiológicaRESUMO
Chronic kidney disease (CKD) is a complex gene-environmental disease affecting close to 10% of the US population. Genome-wide association studies (GWASs) have identified sequence variants, localized to non-coding genomic regions, associated with kidney function. Despite these robust observations, the mechanism by which variants lead to CKD remains a critical unanswered question. Expression quantitative trait loci (eQTL) analysis is a method to identify genetic variation associated with gene expression changes in specific tissue types. We hypothesized that an integrative analysis combining CKD GWAS and kidney eQTL results can identify candidate genes for CKD. We performed eQTL analysis by correlating genotype with RNA-seq-based gene expression levels in 96 human kidney samples. Applying stringent statistical criteria, we detected 1,886 genes whose expression differs with the sequence variants. Using direct overlap and Bayesian methods, we identified new potential target genes for CKD. With respect to one of the target genes, lysosomal beta A mannosidase (MANBA), we observed that genetic variants associated with MANBA expression in the kidney showed statistically significant colocalization with variants identified in CKD GWASs, indicating that MANBA is a potential target gene for CKD. The expression of MANBA was significantly lower in kidneys of subjects with risk alleles. Suppressing manba expression in zebrafish resulted in renal tubule defects and pericardial edema, phenotypes typically induced by kidney dysfunction. Our analysis shows that gene-expression changes driven by genetic variation in the kidney can highlight potential new target genes for CKD development.
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Regulação da Expressão Gênica , Variação Genética , Nefropatias/genética , Animais , Sequência de Bases , Técnicas de Silenciamento de Genes , Redes Reguladoras de Genes , Estudo de Associação Genômica Ampla , Humanos , Rim/metabolismo , Rim/patologia , Nefropatias/patologia , Desequilíbrio de Ligação/genética , Polimorfismo de Nucleotídeo Único/genética , Locos de Características Quantitativas/genética , Característica Quantitativa Herdável , Reprodutibilidade dos Testes , Peixe-Zebra/genética , beta-Manosidase/genéticaRESUMO
With the advent of high-throughput sequencing technologies, the rapid generation and accumulation of large amounts of sequencing data pose an insurmountable demand for efficient algorithms for constructing whole-genome phylogenies. The existing phylogenomic methods all use assembled sequences, which are often not available owing to the difficulty of assembling short-reads; this obstructs phylogenetic investigations on species without a reference genome. In this report, we present co-phylog, an assembly-free phylogenomic approach that creates a 'micro-alignment' at each 'object' in the sequence using the 'context' of the object and calculates pairwise distances before reconstructing the phylogenetic tree based on those distances. We explored the parameters' usages and the optimal working range of co-phylog, assessed co-phylog using the simulated next-generation sequencing (NGS) data and the real NGS raw data. We also compared co-phylog method with traditional alignment and alignment-free methods and illustrated the advantages and limitations of co-phylog method. In conclusion, we demonstrated that co-phylog is efficient algorithm and that it delivers high resolution and accurate phylogenies using whole-genome unassembled sequencing data, especially in the case of closely related organisms, thereby significantly alleviating the computational burden in the genomic era.
Assuntos
Algoritmos , Filogenia , Simulação por Computador , Enterobacteriaceae/classificação , Enterobacteriaceae/genética , Escherichia/classificação , Escherichia/genética , Gammaproteobacteria/classificação , Gammaproteobacteria/genética , Genoma Bacteriano , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Alinhamento de Sequência , Shigella/classificação , Shigella/genética , SoftwareRESUMO
Here, we develop k -mer substring space decomposition (Kssd), a sketching technique which is significantly faster and more accurate than current sketching methods. We show that it is the only method that can be used for large-scale dataset comparisons at population resolution on simulated and real data. Using Kssd, we prioritize references for all 1,019,179 bacteria whole genome sequencing (WGS) runs from NCBI Sequence Read Archive and find misidentification or contamination in 6164 of these. Additionally, we analyze WGS and exome runs of samples from the 1000 Genomes Project.
Assuntos
Biologia Computacional/métodos , Metagenômica/métodos , Software , Algoritmos , Bactérias/genética , Biologia Computacional/normas , Bases de Dados Genéticas , Genoma Bacteriano , Sequenciamento de Nucleotídeos em Larga Escala , Metagenômica/normas , Análise de Sequência de DNARESUMO
BACKGROUND: The availability of 12 fully sequenced Drosophila species genomes provides an excellent opportunity to explore the evolutionary mechanism, structure and function of gene families in Drosophila. Currently, several important resources, such as FlyBase, FlyMine and DroSpeGe, have been devoted to integrating genetic, genomic, and functional data of Drosophila into a well-organized form. However, all of these resources are gene-centric and lack the information of the gene families in Drosophila. DESCRIPTION: FlyPhy is a comprehensive phylogenomic analysis platform devoted to analyzing the genes and gene families in Drosophila. Genes were classified into families using a graph-based Markov Clustering algorithm and extensively annotated by a number of bioinformatic tools, such as basic sequence features, functional category, gene ontology terms, domain organization and sequence homolog to other databases. FlyPhy provides a simple and user-friendly web interface to allow users to browse and retrieve the information at multiple levels. An outstanding feature of the FlyPhy is that all the retrieved results can be added to a workset for further data manipulation. For the data stored in the workset, multiple sequence alignment, phylogenetic tree construction and visualization can be easily performed to investigate the sequence variation of each given family and to explore its evolutionary mechanism. CONCLUSION: With the above functionalities, FlyPhy will be a useful resource and convenient platform for the Drosophila research community. The FlyPhy is available at http://bioinformatics.zj.cn/fly/ .
Assuntos
Drosophila/genética , Genoma de Inseto/genética , Genômica/métodos , Filogenia , Software , Animais , Biologia Computacional , Bases de Dados Genéticas , Genes de Insetos , Interface Usuário-ComputadorRESUMO
Integrons are genetic platforms responsible for the dissemination of antimicrobial resistance genes among Gram-negative bacteria, primarily due to their association with transposable elements and conjugative plasmids. In this study, a cassette array containing four identical blaGES-5 genes embedded in a class 1 integron located on an IncP-1ß group plasmid from a clinical Pseudomonas aeruginosa strain was identified. Comparative genome analysis and conjugation assay showed that the plasmid pICP-4GES lacked the trbN, trbO and trbP genes but was conjugable. Antimicrobial susceptibility testing revealed that compared with single-copy blaGES-5 complementary strains, both the cloned and chromosome-targeted expression of four copies of blaGES-5 increased the minimum inhibitory concentration (MIC) by one to two dilutions for most of the selected antimicrobials. Quantitative real-time reverse transcription PCR (RT-qPCR) showed that the four consecutive cassettes increased blaGES-5 expression by approximately two-fold compared with the single-copy blaGES-5 strain, suggesting that the level of gene expression was not directly proportional to copy number. In addition, the gene cassette capture assay showed that the global blaGES-5 transfer frequency reached 5.38â¯×â¯10-4.
Assuntos
Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Integrons , Pseudomonas aeruginosa/genética , beta-Lactamases/genética , Antibacterianos/farmacologia , Sequência de Bases , Conjugação Genética , Elementos de DNA Transponíveis , Humanos , Testes de Sensibilidade Microbiana , Família Multigênica , Plasmídeos/química , Plasmídeos/metabolismo , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/isolamento & purificação , beta-Lactamases/metabolismoRESUMO
Similar to other CTX-M family enzymes, KLUC is a recently identified and emerging determinant of cefotaxime resistance that has been recovered from at least three Enterobacteriaceae species, including Kluyvera cryocrescens, Escherichia coli, and Enterobacter cloacae. Whether this extended-spectrum ß-lactamase (ESBL) has been disseminated among commonly isolated Enterobacteriaceae is worthy of further investigation. In this study, we screened 739 nosocomial Enterobacteriaceae isolates (240 Klebsiella pneumoniae and 499 E. coli strains) and found that one K. pneumoniae and four E. coli isolates harbored the blaKLUC gene. Three blaKLUC determinants isolated from E. coli were entirely identical to a blaKLUC-3 gene previously recovered in the same hospital. PFGE of four blaKLUC-harboring E. coli strains showed that prevalence of these determinants was most likely mediated by horizontal gene transfer but not clonal dissemination. However, the variant isolated from K. pneumoniae belonged to a novel member of the KLUC enzyme group. This newly identified enzyme (KLUC-5) has an amino acid substitution compared with previously identified KLUC-1 (G18S) and KLUC-3 (G240D). Antimicrobial susceptibility tests showed that KLUC-5 significantly reduced resistance activity to almost all the selected antimicrobials compared to previously identified KLUC-3. Site-directed mutagenesis showed that blaKLUC-5-D240G and blaKLUC-5-S18G significantly enhanced the MIC against its best substrate. Conjugation and S1-PFGE indicated that blaKLUC-5 was located on a transferable plasmid, which was further decoded by single-molecule, real-time sequencing. Comparative genome analysis showed that its backbone exhibited genetic homology to the IncA/C incompatibility group plasmids. A transposable element, ISEcp1, was detected 256-bp upstream of the blaKLUC-5 gene; this location was inconsistent with the previously identified blaKLUC-1 but congruent with the variants recovered from E. coli in the same hospital. These data provide evidence of the increasingly emerging KLUC group of ESBLs in China.
RESUMO
Background: Florfenicol is a derivative of chloramphenicol that is used only for the treatment of animal diseases. A key resistance gene for florfenicol, floR, can spread among bacteria of the same and different species or genera through horizontal gene transfer. To analyze the potential transmission of resistance genes between animal and human pathogens, we investigated floR in Klebsiella pneumoniae isolates from patient samples. floR in human pathogens may originate from animal pathogens and would reflect the risk to human health of using antimicrobial agents in animals. Methods: PCR was used to identify floR-positive strains. The floR genes were cloned, and the minimum inhibitory concentrations (MICs) were determined to assess the relative resistance levels of the genes and strains. Sequencing and comparative genomics methods were used to analyze floR gene-related sequence structure as well as the molecular mechanism of resistance dissemination. Results: Of the strains evaluated, 20.42% (67/328) were resistant to florfenicol, and 86.96% (20/23) of the floR-positive strains demonstrated high resistance to florfenicol with MICs ≥512 µg/mL. Conjugation experiments showed that transferrable plasmids carried the floR gene in three isolates. Sequencing analysis of a plasmid approximately 125 kb in size (pKP18-125) indicated that the floR gene was flanked by multiple copies of mobile genetic elements. Comparative genomics analysis of a 9-kb transposon-like fragment of pKP18-125 showed that an approximately 2-kb sequence encoding lysR-floR-virD2 was conserved in the majority (79.01%, 83/105) of floR sequences collected from NCBI nucleotide database. Interestingly, the most similar sequence was a 7-kb fragment of plasmid pEC012 from an Escherichia coli strain isolated from a chicken. Conclusions: Identified on a transferable plasmid in the human pathogen K. pneumoniae, the floR gene may be disseminated through horizontal gene transfer from animal pathogens. Studies on the molecular mechanism of resistance gene dissemination in different bacterial species of animal origin could provide useful information for preventing or controlling the spread of resistance between animal and human pathogens.
Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Genes Bacterianos , Infecções por Klebsiella/epidemiologia , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/genética , Tianfenicol/análogos & derivados , Antibacterianos/uso terapêutico , China/epidemiologia , Conjugação Genética , Eletroforese em Gel de Campo Pulsado , Genoma Bacteriano , Genômica/métodos , Humanos , Infecções por Klebsiella/tratamento farmacológico , Infecções por Klebsiella/transmissão , Klebsiella pneumoniae/classificação , Testes de Sensibilidade Microbiana , Filogenia , Análise de Sequência de DNA , Tianfenicol/farmacologia , Tianfenicol/uso terapêuticoRESUMO
Escherichia coli (E. coli) commonly reside in human intestine and most E. coli strains are harmless, but some serotypes cause serious food poisoning. This study identified and molecularly characterized blaSHV genes from 490 E. coli strains with multi-drug resistance in a hospital population. PCR and molecular cloning and southern blot were performed to assess functions and localizations of this resistant E. coli gene and the pulsed-field gel electrophoresis (PFGE) was utilized to demonstrate the clonal relatedness of the positive E. coli strains. The data showed that 4 of these 490 E. coli strains (4/499, 0.8%) carried blaSHV genes that included EC D2485 (blaSHV-5), EC D2487 (blaSHV-5), EC D2684 (blaSHV-11) and EC D2616 (blaSHV-195, a novel blaSHV). Analysis of blaSHV open-reading frame showed that blaSHV-5 had a high hydrolysis activity to the broad-spectrum penicillin (ampicillin or piperacillin), ceftazidime, ceftriaxone, cefotaxime and aztreonam. blaSHV-195 and blaSHV-11 had similar resistant characteristics with high hydrolysis activities to ampicillin and piperacillin, but low activities to cephalosporins. Moreover, the two blaSHV-5 genes were located on a transferable plasmid (23kb), whereas the other two blaSHV variants (blaSHV-11 and blaSHV-195) seemed to be located in the chromosomal material. Both EC D2485 and EC D2487 clones isolated in 2010 had the same DNA finger printing profile and they might be the siblings of clonal dissemination. The data from the current study suggest that the novel blaSHV and clonal dissemination may be developed, although blaSHV genes were infrequently identified in this hospital population. The results of the work demonstrate the necessity for molecular surveillance in tracking blaSHV-producing strains in large teaching hospital settings and emphasize the need for epidemiological monitoring.
Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Genes Bacterianos , beta-Lactamases/genética , China , Infecção Hospitalar/tratamento farmacológico , Infecção Hospitalar/microbiologia , Farmacorresistência Bacteriana Múltipla/genética , Escherichia coli/enzimologia , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/microbiologia , Genótipo , Hospitais de Ensino , Humanos , Filogenia , Fatores R/genéticaRESUMO
AmpG is a transmembrane protein with permease activity that transports meuropeptide from the periplasm to the cytoplasm, which is essential for the induction of the ampC encoding ß-lactamase. To obtain new insights into the relationship between AmpG structure and function, comparative genomics analysis, secondary and tertiary structure modeling, site-directed mutational analyses and genetic complementation experiments were performed in this study. AmpGs from different genera of bacteria (Escherichia coli, Vibrio cholerae and Acinetobacter baumannii) could complement AmpG function in Pseudomonas aeruginosa. The minimal inhibitory concentration (MIC) to ampicillin is 512 µg/ml for wild type strain PAO1, while it is 32 µg/ml for an ampG deletion mutant strain (PAO1ΔampG) with a corresponding decrease in the activity of the ampC-encoded ß-lactamase. Site-directed mutagenesis of conserved AmpG residues (G29, A129, Q131 and A197) resulted in a loss of function, resulting in a loss of resistance to ampicillin in PAO1ΔampG. The G29A, G29V, A129T, A129V, A129D, A197S and A197D mutants had lower resistance to ampicillin and significantly decreased activity of the AmpC ß-lactamase. The G29A, G29V, A129V, A197S and A197D mutants had decreased ampG mRNA transcript levels. The A129T and A129D mutants had normal ampG mRNA transcript levels, but the function of the protein was drastically reduced. Our experimental results demonstrate that the conserved amino acids played essential roles in maintaining the function of AmpG. Combined with the AmpG structural information, these critical amino acids can be targeted for the development of new anti-bacterial agents.
Assuntos
Proteínas de Bactérias/química , Proteínas de Membrana Transportadoras/química , Pseudomonas aeruginosa/enzimologia , Relação Quantitativa Estrutura-Atividade , Motivos de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Mutação de Sentido IncorretoRESUMO
We studied phycoerythrin (PE) in human SW480 tumor cells and the underlying molecular mechanisms of action. PE inhibited cell proliferation as evidenced by CCK-8 assay. The IC50 values of phycoerythrin were 48.2 and 27.4 µg/ml for 24 and 48 h of exposure, respectively. PE induced apoptosis and cell cycle arrest in SW480 cells as observed under electron microscopy and with flow cytometry. Apoptosis increased from 5.1 (controls) to 39.0% in 80.0 µg/ml PE-treated cells. Differences in protein expression were identified using proteomic techniques. Protein spots (1018±60 and 1010±60) were resolved in PE-treated and untreated group. Forty differential protein spots were analyzed with MALDI-TOF-MS, including GRP78 and NPM1. The expression as measured by qPCR and western blotting agreed with data from two-dimensional electrophoresis. GRP78, NPM1, MTHSP75, Ezrin and Annexin A2 were decreased and HSP60 was increased after PE treatment, indicating that PE may target multiple proteins to induce apoptosis.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ficoeritrina/farmacologia , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Chaperona BiP do Retículo Endoplasmático , Expressão Gênica/efeitos dos fármacos , Gracilaria/química , Humanos , NucleofosminaRESUMO
The aim of this study was to analyze the molecular epidemiologic characteristics of Acinetobacter baumannii. A total of 398 isolates were collected in 7 regions of South China from January to June of 2012. Drug sensitivity was tested toward 15 commonly used antibiotics; thus, 146 multi-drug-resistant strains (resistant to more than 7 drugs) were identified, representing 36.7% of all isolates. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were used for molecular subtyping. According to the PFGE results (with a cutoff of 70% similarity for the DNA electrophoretic bands), 146 strains were subdivided into 15 clusters, with cluster A being the largest (33.6%, distributed in all districts except Jiaxing). Cluster B was also widespread and included 14.4% of all strains. In addition, MLST results revealed 11 sequence types (ST), with ST208 being the most prevalent, followed by ST191 and ST729. Furthermore, 4 novel alleles and 6 novel STs were identified. Our results showed that multi-drug-resistant A. baumannii in South China shares the origin with other widespread strains in other countries. The nosocomial infections caused by A. baumannii have been severe in South China. Continuous monitoring and judicious antibiotic use are required.
Assuntos
Infecções por Acinetobacter/epidemiologia , Acinetobacter baumannii/genética , Infecção Hospitalar/epidemiologia , Farmacorresistência Bacteriana Múltipla/genética , Genótipo , Filogenia , Infecções por Acinetobacter/diagnóstico , Infecções por Acinetobacter/tratamento farmacológico , Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/classificação , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/isolamento & purificação , Alelos , Antibacterianos/farmacologia , Técnicas de Tipagem Bacteriana , China/epidemiologia , Infecção Hospitalar/diagnóstico , Infecção Hospitalar/tratamento farmacológico , Infecção Hospitalar/microbiologia , Eletroforese em Gel de Campo Pulsado , Humanos , Epidemiologia Molecular , Família Multigênica , Tipagem de Sequências MultilocusRESUMO
Phycocyanin (PC) from Spirulina platensis has inhibitory effects on tumor cell growth. In this research, the transcriptome study was designed to investigate the underlying molecular mechanisms of PC inhibition on human ovarian cancer cell SKOV-3 proliferation. The PC IC50 was 216.6µM and 163.8µM for 24h and 48h exposure, respectively, as determined by CCK-8 assay. The morphological changes of SKOV-3 cells after PC exposure were recorded using HE staining. Cells arrested in G2/M stages as determined by flow cytometry. The transcriptome analysis showed that 2031 genes (with > three-fold differences) were differentially expressed between the untreated and the PC-treated cells, including 1065 up-regulated and 966 down-regulated genes. Gene ontology and KEGG pathway analysis identified 18 classical pathways that were remarkably enriched, such as neurotrophin signaling pathway, VEGF signaling pathway and P53 signaling pathway. qPCR results further showed that PTPN12, S100A2, RPL26, and LAMA3 increased while HNRNPA1P10 decreased in PC-treated cells. Molecules and genes in those pathways may be potential targets to develop treatments for ovarian cancer.
Assuntos
Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Neoplasias Ovarianas/tratamento farmacológico , Ficocianina/farmacologia , Linhagem Celular Tumoral , Fatores Quimiotáticos/biossíntese , Feminino , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Laminina/biossíntese , Fatores de Crescimento Neural/metabolismo , Neoplasias Ovarianas/patologia , Proteína Tirosina Fosfatase não Receptora Tipo 12/biossíntese , Proteoglicanas/metabolismo , Proteínas Ribossômicas/biossíntese , Proteínas S100/biossíntese , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The present study was designed to determine the effects of phycocyanin (PC) on Human ovarian cancer SKOV-3 cells and the underlying molecular mechanisms of action. The inhibitory effects of PC on the cell proliferation were detected by MTT assay. The IC50 values of PC were 182.0µM and 133.6µM for 24h and 48h exposure, respectively. PC induced apoptosis in SKOV-3 cells was observed by electron microscopy and flow cytometry. The apoptosis rate was increased from 1.6% to 19.8% after PC exposure. The fluorescence intensity of ROS and the activities of Caspase-3, Caspase-8, and Caspase-9 were increased. Differentiated expression protein spots were selected and identified using proteomic techniques. There were 698±73 and 683±79 protein spots resolved in untreated and PC-treated cells, respectively. Forty five differential protein spots were analyzed by MALDI-TOF-MS, including mtSSB, PSME3, and nucleolin. The mRNA expression profiles determined by RT-PCR were consistent with that of the two-dimensional electrophoresis. The decreased proteins such as HSP60, nucleolin, PPase, peroxiredoxin-4 and the increased protein (mtSSB) were identified in SKOV-3 cells after PC treatment, indicating that the effects of PC on tumor cell apoptosis may be relate to multiple target proteins. And the mitochondrial pathway may be the main pathway for PC-induced apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Proteínas de Neoplasias/metabolismo , Ficocianina/farmacologia , Spirulina/química , Apoptose/genética , Caspases/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Eletroforese em Gel Bidimensional , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas de Neoplasias/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/ultraestrutura , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The homocysteine methyltransferase encoded by mmuM is widely distributed among microbial organisms. It is the key enzyme that catalyzes the last step in methionine biosynthesis and plays an important role in the metabolism process. It also enables the microbial organisms to tolerate high concentrations of selenium in the environment. In this research, 533 mmuM gene sequences covering 70 genera of the bacteria were selected from GenBank database. The distribution frequency of mmuM is different in the investigated genera of bacteria. The mapping results of 160 mmuM reference sequences showed that the mmuM genes were found in 7 species of pathogen genomes sequenced in this work. The polymerase chain reaction products of one mmuM genotype (NC_013951 as the reference) were sequenced and the sequencing results confirmed the mapping results. Furthermore, 144 representative sequences were chosen for phylogenetic analysis and some mmuM genes from totally different genera (such as the genes between Escherichia and Klebsiella and between Enterobacter and Kosakonia) shared closer phylogenetic relationship than those from the same genus. Comparative genomic analysis of the mmuM encoding regions on plasmids and bacterial chromosomes showed that pKF3-140 and pIP1206 plasmids shared a 21 kb homology region and a 4.9 kb fragment in this region was in fact originated from the Escherichia coli chromosome. These results further suggested that mmuM gene did go through the gene horizontal transfer among different species or genera of bacteria. High-throughput sequencing combined with comparative genomics analysis would explore distribution and dissemination of the mmuM gene among bacteria and its evolution at a molecular level.
Assuntos
Bactérias/enzimologia , Transferência Genética Horizontal/genética , Variação Genética/genética , Homocisteína S-Metiltransferase/genética , Filogenia , Sequência de Bases , Mapeamento Cromossômico , Análise por Conglomerados , Primers do DNA/genética , Sequenciamento de Nucleotídeos em Larga Escala , Dados de Sequência Molecular , RNA Ribossômico 16S/genética , Especificidade da EspécieRESUMO
In order to get insights into plasmid evolution and the dissemination of multidrug resistance, we performed extensive comparative genomics analyses of the Klebsiella pneumoniae plasmid pKF3-94 and some of its related plasmids. pKF3-94 is one of three plasmids isolated from the K. pneumoniae strain KF3. Of the 144 putative genes it harbors, 69 can be functionally assigned to be involved in transfer conjugation, transfer leading, antimicrobial resistance, transposon function, and plasmid replication. Comparison of plasmid replicon sequence types revealed that pKF3-94 carries two replicons that are distinct from those carried on the two sibling K. pneumonia plasmids pKF3-70 and pKF3-140, thereby allowing pKF3-94 to coexist with these latter plasmids in the same host cell. Comparative genomics analyses further showed that pKF3-94 is more similar to plasmids pK1HV and pC15-k, which were isolated from different K. pneumonia strains, than to pKF3-70 and pKF3-140. Interestingly, pK1HV contains a unique 49 kb region rich in mobile genetic elements and drug resistance genes, while pKF3-94 and pC15-k share a 15 kb homology region partitioned into a region rich in drug resistance genes and one containing a replicon. It is conceivable, therefore, that pK1HV and pC15-k have both arisen from a common pKF3-94-like plasmid. The comparisons lend further support for the role horizontal gene transfer plays in genome evolution and in the dissemination of genetic elements including drug resistance genes.
RESUMO
BACKGROUND: Klebsiella pneumoniae is a clinically significant species of bacterium which causes a variety of diseases. Clinical treatment of this bacterial infection is greatly hindered by the emergence of multidrug-resistant strains. The resistance is largely due to the acquisition of plasmids carrying drug-resistant as well as pathogenic genes, and its conjugal transfer facilitates the spread of resistant phenotypes. METHODOLOGY/PRINCIPAL FINDINGS: The 70,057 bp plasmid pKF3-70, commonly found in Klebsiella pneumoniae, is composed of five main functional modules, including regions involved in replication, partition, conjugation, transfer leading, and variable regions. This plasmid is more similar to several Escherichia coli plasmids than any previously reported K. pneumoniae plasmids and pKF3-70 like plasmids share a common and conserved backbone sequence. The replication system of the pKF3-70 is 100% identical to that of RepFII plasmid R100 from E. coli. A beta-lactamase gene ctx-m-14 with its surrounding insertion elements (ISEcp1, truncated IS903 and a 20 bp inverted repeat sequence) may compose an active transposon which is directly bordered by two putative target repeats "ATTAC." CONCLUSIONS/SIGNIFICANCE: The K. pneumoniae plasmid pKF3-70 carries an extended-spectrum beta-lactamase gene, ctx-m-14. The conjugative characteristic makes it a widespread plasmid among genetically relevant genera which poses significant threat to public health.