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1.
PLoS Biol ; 17(3): e2006716, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30856173

RESUMO

The interdependence of selective cues during development of regulatory T cells (Treg cells) in the thymus and their suppressive function remains incompletely understood. Here, we analyzed this interdependence by taking advantage of highly dynamic changes in expression of microRNA 181 family members miR-181a-1 and miR-181b-1 (miR-181a/b-1) during late T-cell development with very high levels of expression during thymocyte selection, followed by massive down-regulation in the periphery. Loss of miR-181a/b-1 resulted in inefficient de novo generation of Treg cells in the thymus but simultaneously permitted homeostatic expansion in the periphery in the absence of competition. Modulation of T-cell receptor (TCR) signal strength in vivo indicated that miR-181a/b-1 controlled Treg-cell formation via establishing adequate signaling thresholds. Unexpectedly, miR-181a/b-1-deficient Treg cells displayed elevated suppressive capacity in vivo, in line with elevated levels of cytotoxic T-lymphocyte-associated 4 (CTLA-4) protein, but not mRNA, in thymic and peripheral Treg cells. Therefore, we propose that intrathymic miR-181a/b-1 controls development of Treg cells and imposes a developmental legacy on their peripheral function.

2.
Eur J Immunol ; 2018 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-30281154

RESUMO

The interdependence of posttranscriptional gene regulation via miRNA and transcriptional regulatory networks in lymphocyte development is poorly understood. Here, we identified miR-191 as direct upstream modulator of a transcriptional module comprising the transcription factors Foxp1, E2A, and Egr1. Deletion as well as ectopic expression of miR-191 resulted in developmental arrest in B lineage cells, indicating that fine tuning of the combined expression levels of Foxp1, E2A, and Egr1, which in turn control somatic recombination and cytokine-driven expansion, constitutes a prerequisite for efficient B-cell development. In conclusion, we propose that miR-191 acts as a rheostat in B-cell development by fine tuning a key transcriptional program.

3.
World J Gastrointest Pathophysiol ; 8(2): 87-92, 2017 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-28573071

RESUMO

Chronic granulomatous disease (CGD) is a primary immune deficiency that is commonly diagnosed under the age of 5 years (95%) and is rarely seen in adulthood. CGD may manifest as inflammatory bowel disease (IBD) in childhood. Without proper diagnosis, these patients may be monitored for years as IBD; some may even be regarded as steroid-resistant ulcerative colitis (UC) and end up having a colectomy. In this case report, we described a patient who had been followed-up for years as UC and subsequently underwent colectomy, but was finally diagnosed in adulthood as primary immune deficiency.

4.
Nat Genet ; 49(5): 742-752, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28369036

RESUMO

We identify SMARCD2 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily D, member 2), also known as BAF60b (BRG1/Brahma-associated factor 60b), as a critical regulator of myeloid differentiation in humans, mice, and zebrafish. Studying patients from three unrelated pedigrees characterized by neutropenia, specific granule deficiency, myelodysplasia with excess of blast cells, and various developmental aberrations, we identified three homozygous loss-of-function mutations in SMARCD2. Using mice and zebrafish as model systems, we showed that SMARCD2 controls early steps in the differentiation of myeloid-erythroid progenitor cells. In vitro, SMARCD2 interacts with the transcription factor CEBPɛ and controls expression of neutrophil proteins stored in specific granules. Defective expression of SMARCD2 leads to transcriptional and chromatin changes in acute myeloid leukemia (AML) human promyelocytic cells. In summary, SMARCD2 is a key factor controlling myelopoiesis and is a potential tumor suppressor in leukemia.


Assuntos
Diferenciação Celular/genética , Redes Reguladoras de Genes , Neutrófilos/metabolismo , Fatores de Transcrição/genética , Animais , Animais Geneticamente Modificados , Sequência de Bases , Linhagem Celular Tumoral , Montagem e Desmontagem da Cromatina , Análise Mutacional de DNA , Saúde da Família , Feminino , Humanos , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Linhagem , Peixe-Zebra
6.
J Allergy Clin Immunol ; 140(4): 1112-1119, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28115216

RESUMO

BACKGROUND: Myb-like, SWIRM, and MPN domains 1 (MYSM1) is a transcriptional regulator mediating histone deubiquitination. Its role in human immunity and hematopoiesis is poorly understood. OBJECTIVES: We sought to investigate the clinical, cellular, and molecular features in 2 siblings presenting with progressive bone marrow failure (BMF), immunodeficiency, and developmental aberrations. METHODS: We performed genome-wide homozygosity mapping, whole-exome and Sanger sequencing, immunophenotyping studies, and analysis of genotoxic stress responses. p38 activation, reactive oxygen species levels, rate of apoptosis and clonogenic survival, and growth in immune and nonimmune cells were assessed. The outcome of allogeneic hematopoietic stem cell transplantation (HSCT) was monitored. RESULTS: We report 2 patients with progressive BMF associated with myelodysplastic features, immunodeficiency affecting B cells and neutrophil granulocytes, and complex developmental aberrations, including mild skeletal anomalies, neurocognitive developmental delay, and cataracts. Whole-exome sequencing revealed a homozygous premature stop codon mutation in the gene encoding MYSM1. MYSM1-deficient cells are characterized by increased sensitivity to genotoxic stress associated with sustained induction of phosphorylated p38 protein, increased reactive oxygen species production, and decreased survival following UV light-induced DNA damage. Both patients were successfully treated with allogeneic HSCT with sustained reconstitution of hematopoietic defects. CONCLUSIONS: Here we show that MYSM1 deficiency is associated with developmental aberrations, progressive BMF with myelodysplastic features, and increased susceptibility to genotoxic stress. HSCT represents a curative therapy for patients with MYSM1 deficiency.


Assuntos
Doenças da Medula Óssea/imunologia , Dano ao DNA/imunologia , Proteínas de Ligação a DNA/metabolismo , Deficiências do Desenvolvimento/imunologia , Hematopoese , Transplante de Células-Tronco Hematopoéticas , Síndromes de Imunodeficiência/imunologia , Fatores de Transcrição/metabolismo , Células Cultivadas , Consanguinidade , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Estudo de Associação Genômica Ampla , Genótipo , Histonas/metabolismo , Humanos , Linhagem , Deleção de Sequência/genética , Fatores de Transcrição/genética , Ubiquitinação , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
7.
Mol Biosyst ; 12(3): 994-1005, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26818782

RESUMO

Genome-Scale Metabolic Reconstructions (GSMRs), along with optimization-based methods, predominantly Flux Balance Analysis (FBA) and its derivatives, are widely applied for assessing and predicting the behavior of metabolic networks upon perturbation, thereby enabling identification of potential novel drug targets and biotechnologically relevant pathways. The abundance of alternate flux profiles has led to the evolution of methods to explore the complete solution space aiming to increase the accuracy of predictions. Herein we present a novel, generic algorithm to characterize the entire flux space of GSMR upon application of FBA, leading to the optimal value of the objective (the optimal flux space). Our method employs Modified Latin-Hypercube Sampling (LHS) to effectively border the optimal space, followed by Principal Component Analysis (PCA) to identify and explain the major sources of variability within it. The approach was validated with the elementary mode analysis of a smaller network of Saccharomyces cerevisiae and applied to the GSMR of Pseudomonas aeruginosa PAO1 (iMO1086). It is shown to surpass the commonly used Monte Carlo Sampling (MCS) in providing a more uniform coverage for a much larger network in less number of samples. Results show that although many fluxes are identified as variable upon fixing the objective value, majority of the variability can be reduced to several main patterns arising from a few alternative pathways. In iMO1086, initial variability of 211 reactions could almost entirely be explained by 7 alternative pathway groups. These findings imply that the possibilities to reroute greater portions of flux may be limited within metabolic networks of bacteria. Furthermore, the optimal flux space is subject to change with environmental conditions. Our method may be a useful device to validate the predictions made by FBA-based tools, by describing the optimal flux space associated with these predictions, thus to improve them.


Assuntos
Algoritmos , Genoma , Redes e Vias Metabólicas , Simulação por Computador , Análise Discriminante , Genoma Bacteriano , Genoma Fúngico , Análise de Componente Principal , Pseudomonas aeruginosa/genética , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
8.
BMC Genomics ; 16: 883, 2015 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-26519161

RESUMO

BACKGROUND: Pseudomonas aeruginosa is an environmentally ubiquitous Gram-negative bacterium and important opportunistic human pathogen, causing severe chronic respiratory infections in patients with underlying conditions such as cystic fibrosis (CF) or bronchiectasis. In order to identify mechanisms responsible for adaptation during bronchiectasis infections, a bronchiectasis isolate, PAHM4, was phenotypically and genotypically characterized. RESULTS: This strain displays phenotypes that have been associated with chronic respiratory infections in CF including alginate over-production, rough lipopolysaccharide, quorum-sensing deficiency, loss of motility, decreased protease secretion, and hypermutation. Hypermutation is a key adaptation of this bacterium during the course of chronic respiratory infections and analysis indicates that PAHM4 encodes a mutated mutS gene responsible for a ~1,000-fold increase in mutation rate compared to wild-type laboratory strain P. aeruginosa PAO1. Antibiotic resistance profiles and sequence data indicate that this strain acquired numerous mutations associated with increased resistance levels to ß-lactams, aminoglycosides, and fluoroquinolones when compared to PAO1. Sequencing of PAHM4 revealed a 6.38 Mbp genome, 5.9 % of which were unrecognized in previously reported P. aeruginosa genome sequences. Transcriptome analysis suggests a general down-regulation of virulence factors, while metabolism of amino acids and lipids is up-regulated when compared to PAO1 and metabolic modeling identified further potential differences between PAO1 and PAHM4. CONCLUSIONS: This work provides insights into the potential differential adaptation of this bacterium to the lung of patients with bronchiectasis compared to other clinical settings such as cystic fibrosis, findings that should aid the development of disease-appropriate treatment strategies for P. aeruginosa infections.


Assuntos
Bronquiectasia/microbiologia , Fibrose Cística/complicações , Genótipo , Fenótipo , Infecções por Pseudomonas/etiologia , Pseudomonas aeruginosa/fisiologia , Adaptação Biológica/genética , Alelos , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes , Doença Crônica , Biologia Computacional , Farmacorresistência Bacteriana , Perfilação da Expressão Gênica , Ordem dos Genes , Genoma Bacteriano , Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Mutação , Taxa de Mutação , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/isolamento & purificação , Pseudomonas aeruginosa/patogenicidade , Percepção de Quorum/genética , Metabolismo Secundário , Transcriptoma , Virulência/genética
9.
J Exp Med ; 212(10): 1589-601, 2015 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-26347471

RESUMO

Postnatal T cell development depends on continuous colonization of the thymus by BM-derived T lineage progenitors. Both quantitative parameters and the mechanisms of thymus seeding remain poorly understood. Here, we determined the number of dedicated thymus-seeding progenitor niches (TSPNs) capable of supporting productive T cell development, turnover rates of niche occupancy, and feedback mechanisms. To this end, we established multicongenic fate mapping combined with mathematical modeling to quantitate individual events of thymus colonization. We applied this method to study thymus colonization in CCR7(-/-)CCR9(-/-) (DKO) mice, whose TSPNs are largely unoccupied. We showed that ∼160-200 TSPNs are present in the adult thymus and, on average, 10 of these TSPNs were open for recolonization at steady state. Preconditioning of wild-type mice revealed a similar number of TSPNs, indicating that preconditioning can generate space efficiently for transplanted T cell progenitors. To identify potential cellular feedback loops restricting thymus colonization, we performed serial transfer experiments. These experiments indicated that thymus seeding was directly restricted by the duration of niche occupancy rather than long-range effects, thus challenging current paradigms of thymus colonization.


Assuntos
Linfócitos T/fisiologia , Timo/citologia , Animais , Linhagem da Célula , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores CCR/genética , Receptores CCR/metabolismo , Receptores CCR7/genética , Receptores CCR7/metabolismo , Receptores de Interleucina-17/genética , Células-Tronco/fisiologia , Linfócitos T/citologia , Timócitos/fisiologia , Timo/fisiologia , Timo/efeitos da radiação
10.
PLoS Pathog ; 11(2): e1004653, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25706310

RESUMO

The microbiome and the phage meta-genome within the human gut are influenced by antibiotic treatments. Identifying a novel mechanism, here we demonstrate that bacteria use the universal communication molecule AI-2 to induce virulence genes and transfer them via phage release. High concentrations (i.e. 100 µM) of AI-2 promote dispersal of bacteria from already established biofilms, and is associated with release of phages capable of infecting other bacteria. Enterococcus faecalis V583ΔABC harbours 7 prophages in its genome, and a mutant deficient in one of these prophages (i.e. prophage 5) showed a greatly reduced dispersal of biofilm. Infection of a probiotic E. faecalis strain without lytic prophages with prophage 5 resulted in increased biofilm formation and also in biofilm dispersal upon induction with AI-2. Infection of the probiotic E. faecalis strain with phage-containing supernatants released through AI-2 from E. faecalis V583ΔABC resulted in a strong increase in pathogenicity of this strain. The polylysogenic probiotic strain was also more virulent in a mouse sepsis model and a rat endocarditis model. Both AI-2 and ciprofloxacin lead to phage release, indicating that conditions in the gastrointestinal tract of hospitalized patients treated with antibiotics might lead to distribution of virulence genes to apathogenic enterococci and possibly also to other commensals or even to beneficial probiotic strains.


Assuntos
Biofilmes/crescimento & desenvolvimento , Endocardite Bacteriana/microbiologia , Enterococcus faecalis , Prófagos/fisiologia , Percepção de Quorum , Sepse/microbiologia , Fatores de Virulência/metabolismo , Liberação de Vírus/fisiologia , Animais , Biofilmes/efeitos dos fármacos , Células CACO-2 , Ciprofloxacino/farmacologia , Endocardite Bacteriana/patologia , Enterococcus faecalis/patogenicidade , Enterococcus faecalis/fisiologia , Enterococcus faecalis/virologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Ratos , Ratos Wistar , Sepse/patologia , Liberação de Vírus/efeitos dos fármacos
11.
Blood ; 125(3): 457-64, 2015 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-25411428

RESUMO

The origins of dendritic cells (DCs) and other myeloid cells in the thymus have remained controversial. In this study, we assessed developmental relationships between thymic dendritic cells and thymocytes, employing retrovirus-based cellular barcoding and reporter mice, as well as intrathymic transfers coupled with DC depletion. We demonstrated that a subset of early T-lineage progenitors expressed CX3CR1, a bona fide marker for DC progenitors. However, intrathymic transfers into nonmanipulated mice, as well as retroviral barcoding, indicated that thymic dendritic cells and thymocytes were largely of distinct developmental origin. In contrast, intrathymic transfers after in vivo depletion of DCs resulted in intrathymic development of non-T-lineage cells. In conclusion, our data support a model in which the adoption of T-lineage fate by noncommitted progenitors at steady state is enforced by signals from the thymic microenvironment unless niches promoting alternative lineage fates become available.


Assuntos
Células Dendríticas/imunologia , Células Mieloides/imunologia , Nicho de Células-Tronco/imunologia , Células-Tronco/imunologia , Linfócitos T/imunologia , Timo/imunologia , Animais , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Células Dendríticas/citologia , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos C57BL , Células Mieloides/citologia , Células-Tronco/citologia , Linfócitos T/citologia , Timo/citologia
12.
Nat Genet ; 46(9): 1021-7, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25129144

RESUMO

The analysis of individuals with severe congenital neutropenia (SCN) may shed light on the delicate balance of factors controlling the differentiation, maintenance and decay of neutrophils. We identify 9 distinct homozygous mutations in the JAGN1 gene encoding Jagunal homolog 1 in 14 individuals with SCN. JAGN1-mutant granulocytes are characterized by ultrastructural defects, a paucity of granules, aberrant N-glycosylation of multiple proteins and increased incidence of apoptosis. JAGN1 participates in the secretory pathway and is required for granulocyte colony-stimulating factor receptor-mediated signaling. JAGN1 emerges as a factor that is necessary in the differentiation and survival of neutrophils.


Assuntos
Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Células Mieloides/metabolismo , Neutropenia/congênito , Adolescente , Adulto , Apoptose/genética , Diferenciação Celular/genética , Sobrevivência Celular/genética , Criança , Pré-Escolar , Feminino , Glicosilação , Homeostase/genética , Humanos , Lactente , Recém-Nascido , Masculino , Proteínas de Membrana/metabolismo , Mutação , Neutropenia/genética , Neutropenia/metabolismo , Neutropenia/patologia , Neutrófilos/metabolismo , Receptores de Fator Estimulador de Colônias de Granulócitos/genética , Receptores de Fator Estimulador de Colônias de Granulócitos/metabolismo , Transdução de Sinais , Adulto Jovem
13.
Blood ; 123(24): 3811-7, 2014 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-24753537

RESUMO

Severe congenital neutropenia (SCN) is characterized by low numbers of peripheral neutrophil granulocytes and a predisposition to life-threatening bacterial infections. We describe a novel genetic SCN type in 2 unrelated families associated with recessively inherited loss-of-function mutations in CSF3R, encoding the granulocyte colony-stimulating factor (G-CSF) receptor. Family A, with 3 affected children, carried a homozygous missense mutation (NM_000760.3:c.922C>T, NP_000751.1:p.Arg308Cys), which resulted in perturbed N-glycosylation and aberrant localization to the cell surface. Family B, with 1 affected infant, carried compound heterozygous deletions provoking frameshifts and premature stop codons (NM_000760.3:c.948_963del, NP_000751.1:p.Gly316fsTer322 and NM_000760.3:c.1245del, NP_000751.1:p.Gly415fsTer432). Despite peripheral SCN, all patients had morphologic evidence of full myeloid cell maturation in bone marrow. None of the patients responded to treatment with recombinant human G-CSF. Our study highlights the genetic and morphologic SCN variability and provides evidence both for functional importance and redundancy of G-CSF receptor-mediated signaling in human granulopoiesis.


Assuntos
Mutação de Sentido Incorreto , Neutropenia/congênito , Receptores de Fator Estimulador de Colônias/genética , Sequência de Bases , Criança , Pré-Escolar , Feminino , Células HeLa , Homozigoto , Humanos , Lactente , Recém-Nascido , Masculino , Modelos Moleculares , Neutropenia/genética , Linhagem , Receptores de Fator Estimulador de Colônias/química
14.
PLoS One ; 9(2): e89941, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24587139

RESUMO

Pseudomonas aeruginosa is a highly versatile opportunistic pathogen capable of colonizing multiple ecological niches. This bacterium is responsible for a wide range of both acute and chronic infections in a variety of hosts. The success of this microorganism relies on its ability to adapt to environmental changes and re-program its regulatory and metabolic networks. The study of P. aeruginosa adaptation to temperature is crucial to understanding the pathogenesis upon infection of its mammalian host. We examined the effects of growth temperature on the transcriptome of the P. aeruginosa PAO1. Microarray analysis of PAO1 grown in Lysogeny broth at mid-exponential phase at 22°C and 37°C revealed that temperature changes are responsible for the differential transcriptional regulation of 6.4% of the genome. Major alterations were observed in bacterial metabolism, replication, and nutrient acquisition. Quorum-sensing and exoproteins secreted by type I, II, and III secretion systems, involved in the adaptation of P. aeruginosa to the mammalian host during infection, were up-regulated at 37°C compared to 22°C. Genes encoding arginine degradation enzymes were highly up-regulated at 22°C, together with the genes involved in the synthesis of pyoverdine. However, genes involved in pyochelin biosynthesis were up-regulated at 37°C. We observed that the changes in expression of P. aeruginosa siderophores correlated to an overall increase in Fe²âº extracellular concentration at 37°C and a peak in Fe³âº extracellular concentration at 22°C. This suggests a distinct change in iron acquisition strategies when the bacterium switches from the external environment to the host. Our work identifies global changes in bacterial metabolism and nutrient acquisition induced by growth at different temperatures. Overall, this study identifies factors that are regulated in genome-wide adaptation processes and discusses how this life-threatening pathogen responds to temperature.


Assuntos
Adaptação Fisiológica/fisiologia , Regulação Bacteriana da Expressão Gênica/fisiologia , Pseudomonas aeruginosa/metabolismo , Temperatura Ambiente , Transcriptoma/genética , Adaptação Fisiológica/genética , Regulação Bacteriana da Expressão Gênica/genética , Interações Hospedeiro-Patógeno , Análise em Microsséries , Pseudomonas aeruginosa/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
15.
J Clin Immunol ; 34(3): 331-9, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24519095

RESUMO

PURPOSE: Loss-of-function mutations in IL10 and IL10R cause very early onset inflammatory bowel disease (VEO-IBD). Here, we investigated the molecular pathomechanism of a novel intronic IL10RA mutation and describe a new therapeutic approach of T cell replete haploidentical hematopoietic stem cell transplantation (HSCT). METHODS: Clinical data were collected by chart review. Genotypes of IL10 and IL10R genes were determined by Sanger sequencing. Expression and function of mutated IL-10R1 were assessed by quantitative PCR, Western blot analysis, enzyme-linked immunosorbent assays, confocal microscopy, and flow cytometry. RESULTS: We identified a novel homozygous point mutation in intron 3 of the IL10RA (c.368-10C > G) in three related children with VEO-IBD. Bioinformatical analysis predicted an additional 3' splice site created by the mutation. Quantitative PCR analysis showed normal mRNA expression of mutated IL10RA. Sequencing of the patient's cDNA revealed an insertion of the last nine nucleotides of intron 3 as a result of aberrant splicing. Structure-based modeling suggested misfolding of mutated IL-10R1. Western blot analysis demonstrated a different N-linked glycosylation pattern of mutated protein. Immunofluorescence and FACS analysis revealed impaired expression of mutated IL-10R1 at the plasma membrane. In the absence of HLA-identical donors, T cell replete haploidentical HSCT was successfully performed in two patients. CONCLUSIONS: Our findings expand the spectrum of IL10R mutations in VEO-IBD and emphasize the need for genetic diagnosis of mutations in conserved non-coding sequences of candidate genes. Transplantation of haploidentical stem cells represents a curative therapy in IL-10R-deficient patients, but may be complicated by non-engraftment.


Assuntos
Transplante de Medula Óssea , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/terapia , Subunidade alfa de Receptor de Interleucina-10/metabolismo , Idade de Início , Processamento Alternativo , Sequência de Aminoácidos , Linhagem Celular , Membrana Celular/metabolismo , Criança , Pré-Escolar , Consanguinidade , Análise Mutacional de DNA , Feminino , Genótipo , Glicosilação , Transplante de Células-Tronco Hematopoéticas , Humanos , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/imunologia , Subunidade alfa de Receptor de Interleucina-10/química , Subunidade alfa de Receptor de Interleucina-10/genética , Íntrons , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Linhagem , Fenótipo , Conformação Proteica , Transporte Proteico , Alinhamento de Sequência , Transdução de Sinais , Linfócitos T/imunologia , Resultado do Tratamento
16.
BMC Biotechnol ; 13: 93, 2013 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-24168623

RESUMO

BACKGROUND: Genome scale metabolic reconstructions are developed to efficiently engineer biocatalysts and bioprocesses based on a rational approach. However, in most reconstructions, due to the lack of appropriate measurements, experimentally determined growth parameters are simply taken from literature including other organisms, which reduces the usefulness and suitability of these models. Pseudomonas putida KT2440 is an outstanding biocatalyst given its versatile metabolism, its ability to generate sufficient energy and turnover of NADH and NAD. To apply this strain optimally in industrial production, a previously developed genome-scale metabolic model (iJP815) was experimentally assessed and streamlined to enable accurate predictions of the outcome of metabolic engineering approaches. RESULTS: To substantially improve the accuracy of the genome scale model (iJP815), continuous bioreactor cultures on a mineral medium with glucose as a sole carbon source were carried out at different dilution rates, which covered pulling analysis of the macromolecular composition of the biomass. Besides, the maximum biomass yield (on substrate) of 0.397 gDCW · gglc-1, the maintenance coefficient of 0.037 gglc · gDCW-1 · h-1 and the maximum specific growth rate of 0.59 h-1 were determined. Only the DNA fraction increased with the specific growth rate. This resulted in reliable estimation for the Growth-Associated Maintenance (GAM) of 85 mmolATP · gDCW-1 and the Non Growth-Associated Maintenance (NGAM) of 3.96 mmolATP · gDCW-1 · h-1. Both values were found significantly different from previous assignment as a consequence of a lower yield and higher maintenance coefficient than originally assumed. Contrasting already published 13C flux measurements and the improved model allowed for constraining the solution space, by eliminating futile cycles. Furthermore, the model predictions were compared with transcriptomic data at overall good consistency, which helped to identify missing links. CONCLUSIONS: By careful interpretation of growth stoichiometry and kinetics when grown in the presence of glucose, this work reports on an accurate genome scale metabolic model of Pseudomonas putida, providing a solid basis for its use in designing superior strains for biocatalysis. By consideration of substrate specific variation in stoichiometry and kinetics, it can be extended to other substrates and new mutants.


Assuntos
Reatores Biológicos , Microbiologia Industrial , Pseudomonas putida/crescimento & desenvolvimento , Biocatálise , Biomassa , Carbono/metabolismo , Meios de Cultura/química , Glucose/metabolismo , Engenharia Metabólica , Modelos Moleculares , Transcriptoma
17.
PLoS One ; 8(9): e74838, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24069355

RESUMO

Here we describe a novel, spontaneous, 4035 basepairs long deletion in the DNA cross-link repair 1C (Dclre1c)-locus in C57BL/6-mice, which leads to loss of exons 10 and 11 of the gene encoding for Artemis, a protein involved into V(D) J-recombination of antigen receptors of T and B cells. While several spontaneous mutations of Artemis have been described to cause SCID in humans, in mice, only targeted deletions by knockout technology are known to cause the same phenotype so far. The deletion we observed causes a loss of Artemis function in the C57BL/6 strain and, consequently, the absence of T and B cells, in presence of normal numbers of NK cells and cells of the myeloid lineage. Thus, for the first time we present T(-)B(-)NK(+) severe combined immunodeficiency (SCID) phenotype after spontaneously occurring modification of Artemis gene in mice. Our mouse model may serve as a valuable tool to study mechanisms as well as potential therapies of SCID in humans.


Assuntos
Linfócitos B/metabolismo , Endonucleases/genética , Éxons , Proteínas Nucleares/genética , Deleção de Sequência , Imunodeficiência Combinada Severa/genética , Linfócitos T/metabolismo , Animais , Linfócitos B/imunologia , Sequência de Bases , Ordem dos Genes , Imunofenotipagem , Camundongos , Fenótipo , Polimorfismo de Nucleotídeo Único , Imunodeficiência Combinada Severa/imunologia , Linfócitos T/imunologia
18.
N Engl J Med ; 369(1): 54-65, 2013 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-23738510

RESUMO

BACKGROUND: Neutrophils are the predominant phagocytes that provide protection against bacterial and fungal infections. Genetically determined neutrophil disorders confer a predisposition to severe infections and reveal novel mechanisms that control vesicular trafficking, hematopoiesis, and innate immunity. METHODS: We clinically evaluated seven children from five families who had neutropenia, neutrophil dysfunction, bone marrow fibrosis, and nephromegaly. To identify the causative gene, we performed homozygosity mapping using single-nucleotide polymorphism arrays, whole-exome sequencing, immunoblotting, immunofluorescence, electron microscopy, a real-time quantitative polymerase-chain-reaction assay, immunohistochemistry, flow cytometry, fibroblast motility assays, measurements of apoptosis, and zebrafish models. Correction experiments were performed by transfecting mutant fibroblasts with the nonmutated gene. RESULTS: All seven affected children had homozygous mutations (Thr224Asn or Glu238Lys, depending on the child's ethnic origin) in VPS45, which encodes a protein that regulates membrane trafficking through the endosomal system. The level of VPS45 protein was reduced, as were the VPS45 binding partners rabenosyn-5 and syntaxin-16. The level of ß1 integrin was reduced on the surface of VPS45-deficient neutrophils and fibroblasts. VPS45-deficient fibroblasts were characterized by impaired motility and increased apoptosis. A zebrafish model of vps45 deficiency showed a marked paucity of myeloperoxidase-positive cells (i.e., neutrophils). Transfection of patient cells with nonmutated VPS45 corrected the migration defect and decreased apoptosis. CONCLUSIONS: Defective endosomal intracellular protein trafficking due to biallelic mutations in VPS45 underlies a new immunodeficiency syndrome involving impaired neutrophil function. (Funded by the National Human Genome Research Institute and others.).


Assuntos
Síndromes de Imunodeficiência/genética , Neutropenia/congênito , Proteínas de Transporte Vesicular/genética , Animais , Criança , Endossomos/metabolismo , Homozigoto , Humanos , Síndromes de Imunodeficiência/congênito , Síndromes de Imunodeficiência/imunologia , Mutação , Neutropenia/genética , Neutrófilos/fisiologia , Fenótipo , Transporte Proteico , Proteínas de Transporte Vesicular/metabolismo , Peixe-Zebra
19.
Mol Syst Biol ; 9: 653, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23549481

RESUMO

Mycoplasma pneumoniae, a threatening pathogen with a minimal genome, is a model organism for bacterial systems biology for which substantial experimental information is available. With the goal of understanding the complex interactions underlying its metabolism, we analyzed and characterized the metabolic network of M. pneumoniae in great detail, integrating data from different omics analyses under a range of conditions into a constraint-based model backbone. Iterating model predictions, hypothesis generation, experimental testing, and model refinement, we accurately curated the network and quantitatively explored the energy metabolism. In contrast to other bacteria, M. pneumoniae uses most of its energy for maintenance tasks instead of growth. We show that in highly linear networks the prediction of flux distributions for different growth times allows analysis of time-dependent changes, albeit using a static model. By performing an in silico knock-out study as well as analyzing flux distributions in single and double mutant phenotypes, we demonstrated that the model accurately represents the metabolism of M. pneumoniae. The experimentally validated model provides a solid basis for understanding its metabolic regulatory mechanisms.


Assuntos
Metabolismo Energético/genética , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Mycoplasma pneumoniae/genética , Mycoplasma pneumoniae/metabolismo , Simulação por Computador , Redes e Vias Metabólicas/genética , Modelos Biológicos , Mutação
20.
J Exp Med ; 210(3): 433-43, 2013 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-23440042

RESUMO

Primary immunodeficiencies (PIDs) represent exquisite models for studying mechanisms of human host defense. In this study, we report on two unrelated kindreds, with two patients each, who had cryptosporidial infections associated with chronic cholangitis and liver disease. Using exome and candidate gene sequencing, we identified two distinct homozygous loss-of-function mutations in the interleukin-21 receptor gene (IL21R; c.G602T, p.Arg201Leu and c.240_245delCTGCCA, p.C81_H82del). The IL-21R(Arg201Leu) mutation causes aberrant trafficking of the IL-21R to the plasma membrane, abrogates IL-21 ligand binding, and leads to defective phosphorylation of signal transducer and activator of transcription 1 (STAT1), STAT3, and STAT5. We observed impaired IL-21-induced proliferation and immunoglobulin class-switching in B cells, cytokine production in T cells, and NK cell cytotoxicity. Our study indicates that human IL-21R deficiency causes an immunodeficiency and highlights the need for early diagnosis and allogeneic hematopoietic stem cell transplantation in affected children.


Assuntos
Síndromes de Imunodeficiência/genética , Subunidade alfa de Receptor de Interleucina-21/genética , Mutação , Criança , Pré-Escolar , Feminino , Humanos , Síndromes de Imunodeficiência/etiologia , Síndromes de Imunodeficiência/imunologia , Subunidade alfa de Receptor de Interleucina-21/química , Subunidade alfa de Receptor de Interleucina-21/fisiologia , Células Matadoras Naturais/imunologia , Masculino , Fosforilação , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais
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