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1.
Br J Haematol ; 163(4): 496-500, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24032483

RESUMO

Recurrent gene mutations contribute to the pathogenesis of chronic lymphocytic leukaemia (CLL). We developed a next-generation sequencing (NGS) platform to determine the genetic profile, intratumoural heterogeneity, and clonal structure of two independent CLL cohorts. TP53, SF3B1, and NOTCH1 were most frequently mutated (16.3%, 16.9%, 10.7%). We found evidence for subclonal mutations in 67.5% of CLL cases with mutations of cancer consensus genes. We observed selection of subclones and found initial evidence for convergent mutations in CLL. Our data suggest that assessment of (sub)clonal structure may need to be integrated into analysis of the mutational profile in CLL.


Assuntos
Leucemia Linfocítica Crônica de Células B/genética , Mutação , Análise de Sequência de DNA/métodos , Estudos de Coortes , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Transdução de Sinais
2.
Leuk Lymphoma ; 54(8): 1831-5, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23621802

RESUMO

Sensitive identification of mutations in genes related to the pathogenesis of cancer is a prerequisite for risk-stratified therapies. Next-generation sequencing (NGS) in lymphoma has revealed genetic heterogeneity which makes clinical translation challenging. We established a 454-based targeted resequencing platform for robust high-throughput sequencing from limited material of patients with lymphoma. Hotspot mutations in the most frequently mutated cancer consensus genes were amplified in a two-step multiplex-polymerase chain reation (PCR) which was optimized for homogeneous coverage of all regions of interest. We show that targeted resequencing based on NGS technologies allows highly sensitive detection of mutations and assessment of clone size. The application of this or similar techniques will help the development of genotype-specific treatment approaches in lymphoma.


Assuntos
Transformação Celular Neoplásica/genética , Sequenciamento de Nucleotídeos em Larga Escala , Linfoma/genética , Oncogenes/genética , Substituição de Aminoácidos , Éxons , Frequência do Gene , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Linfoma/diagnóstico , Mutação
3.
Infect Immun ; 80(3): 1050-64, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22158741

RESUMO

The Yersinia pseudotuberculosis Ifp and InvC molecules are putative autotransporter proteins with a high homology to the invasin (InvA) protein. To characterize the function of these surface proteins, we expressed both factors in Escherichia coli K-12 and demonstrated the attachment of Ifp- and InvC-expressing bacteria to human-, mouse-, and pig-derived intestinal epithelial cells. Ifp also was found to mediate microcolony formation and internalization into polarized human enterocytes. The ifp and invC genes were not expressed under in vitro conditions but were found to be induced in the Peyer's patches of the mouse intestinal tract. In a murine coinfection model, the colonization of the Peyer's patches and the mesenteric lymph nodes of mice by the ifp-deficient strain was significantly reduced, and considerably fewer bacteria reached liver and spleen. The absence of InvC did not have a severe influence on bacterial colonization in the murine infection model, and it resulted in only a slightly reduced number of invC mutants in the Peyer's patches. The analysis of the host immune response demonstrated that the presence of Ifp and InvC reduced the recruitment of professional phagocytes, especially neutrophils, in the Peyer's patches. These findings support a role for the adhesins in modulating host-pathogen interactions that are important for immune defense.


Assuntos
Adesinas Bacterianas/metabolismo , Aderência Bacteriana , Células Epiteliais/microbiologia , Proteínas de Membrana Transportadoras/metabolismo , Fatores de Virulência/metabolismo , Yersinia pseudotuberculosis/patogenicidade , Adesinas Bacterianas/genética , Animais , Linhagem Celular , Modelos Animais de Doenças , Escherichia coli K12/genética , Escherichia coli K12/patogenicidade , Feminino , Expressão Gênica , Humanos , Intestinos/microbiologia , Linfonodos/microbiologia , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Endogâmicos BALB C , Nódulos Linfáticos Agregados/microbiologia , Virulência , Fatores de Virulência/genética , Infecções por Yersinia pseudotuberculosis/microbiologia
4.
PLoS Pathog ; 7(7): e1002117, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21750675

RESUMO

Many enteric pathogens are equipped with multiple cell adhesion factors which are important for host tissue colonization and virulence. Y. enterocolitica, a common food-borne pathogen with invasive properties, uses the surface proteins invasin and YadA for host cell binding and entry. In this study, we demonstrate unique cell adhesion and invasion properties of Y. enterocolitica serotype O:3 strains, the most frequent cause of human yersiniosis, and show that these differences are mainly attributable to variations affecting the function and expression of invasin in response to temperature. In contrast to other enteric Yersinia strains, invasin production in O:3 strains is constitutive and largely enhanced compared to other Y. enterocolitica serotypes, in which invA expression is temperature-regulated and significantly reduced at 37°C. Increase of invasin levels is caused by (i) an IS1667 insertion into the invA promoter region, which includes an additional promoter and RovA and H-NS binding sites, and (ii) a P98S substitution in the invA activator protein RovA rendering the regulator less susceptible to proteolysis. Both variations were shown to influence bacterial colonization in a murine infection model. Furthermore, we found that co-expression of YadA and down-regulation of the O-antigen at 37°C is required to allow efficient internalization by the InvA protein. We conclude that even small variations in the expression of virulence factors can provoke a major difference in the virulence properties of closely related pathogens which may confer better survival or a higher pathogenic potential in a certain host or host environment.


Assuntos
Aderência Bacteriana/fisiologia , Interações Hospedeiro-Patógeno , Yersiniose , Yersinia enterocolitica/fisiologia , Adaptação Fisiológica , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Regulação para Baixo , Feminino , Regulação Bacteriana da Expressão Gênica , Temperatura Alta , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Antígenos O/genética , Antígenos O/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , Yersinia enterocolitica/patogenicidade
5.
PLoS One ; 6(6): e20425, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21673990

RESUMO

A family of versatile promoter-probe plasmids for gene expression analysis was developed based on a modular expression plasmid system (pZ). The vectors contain different replicons with exchangeable antibiotic cassettes to allow compatibility and expression analysis on a low-, midi- and high-copy number basis. Suicide vector variants also permit chromosomal integration of the reporter fusion and stable vector derivatives can be used for in vivo or in situ expression studies under non-selective conditions. Transcriptional and translational fusions to the reporter genes gfp(mut3.1), amCyan, dsRed2, luxCDABE, phoA or lacZ can be constructed, and presence of identical multiple cloning sites in the vector system facilitates the interchange of promoters or reporter genes between the plasmids of the series. The promoter of the constitutively expressed gapA gene of Escherichia coli was included to obtain fluorescent and bioluminescent expression constructs. A combination of the plasmids allows simultaneous detection and gene expression analysis in individual bacteria, e.g. in bacterial communities or during mouse infections. To test our vector system, we analyzed and quantified expression of Yersinia pseudotuberculosis virulence genes under laboratory conditions, in association with cells and during the infection process.


Assuntos
Fusão Gênica Artificial , Bactérias/genética , Bactérias/patogenicidade , Infecções Bacterianas , Regulação Bacteriana da Expressão Gênica , Vetores Genéticos/genética , Proteínas Luminescentes/genética , Animais , Sequência de Bases , Linhagem Celular , Feminino , Camundongos , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , Yersinia pseudotuberculosis/genética , Yersinia pseudotuberculosis/patogenicidade
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