Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 26
Filtrar
1.
Nature ; 610(7933): 783-790, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36224385

RESUMO

Around birth, globin expression in human red blood cells (RBCs) shifts from γ-globin to ß-globin, which results in fetal haemoglobin (HbF, α2γ2) being gradually replaced by adult haemoglobin (HbA, α2ß2)1. This process has motivated the development of innovative approaches to treat sickle cell disease and ß-thalassaemia by increasing HbF levels in postnatal RBCs2. Here we provide therapeutically relevant insights into globin gene switching obtained through a CRISPR-Cas9 screen for ubiquitin-proteasome components that regulate HbF expression. In RBC precursors, depletion of the von Hippel-Lindau (VHL) E3 ubiquitin ligase stabilized its ubiquitination target, hypoxia-inducible factor 1α (HIF1α)3,4, to induce γ-globin gene transcription. Mechanistically, HIF1α-HIF1ß heterodimers bound cognate DNA elements in BGLT3, a long noncoding RNA gene located 2.7 kb downstream of the tandem γ-globin genes HBG1 and HBG2. This was followed by the recruitment of transcriptional activators, chromatin opening and increased long-range interactions between the γ-globin genes and their upstream enhancer. Similar induction of HbF occurred with hypoxia or with inhibition of prolyl hydroxylase domain enzymes that target HIF1α for ubiquitination by the VHL E3 ubiquitin ligase. Our findings link globin gene regulation with canonical hypoxia adaptation, provide a mechanism for HbF induction during stress erythropoiesis and suggest a new therapeutic approach for ß-haemoglobinopathies.


Assuntos
gama-Globinas , Humanos , Cromatina , Hemoglobina Fetal/biossíntese , Hemoglobina Fetal/genética , gama-Globinas/biossíntese , gama-Globinas/genética , Hipóxia/genética , Prolil Hidroxilases/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , RNA Longo não Codificante , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Eritropoese
2.
Blood ; 137(2): 155-167, 2021 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-33156908

RESUMO

The histone mark H3K27me3 and its reader/writer polycomb repressive complex 2 (PRC2) mediate widespread transcriptional repression in stem and progenitor cells. Mechanisms that regulate this activity are critical for hematopoietic development but are poorly understood. Here we show that the E3 ubiquitin ligase F-box only protein 11 (FBXO11) relieves PRC2-mediated repression during erythroid maturation by targeting its newly identified substrate bromo adjacent homology domain-containing 1 (BAHD1), an H3K27me3 reader that recruits transcriptional corepressors. Erythroblasts lacking FBXO11 are developmentally delayed, with reduced expression of maturation-associated genes, most of which harbor bivalent histone marks at their promoters. In FBXO11-/- erythroblasts, these gene promoters bind BAHD1 and fail to recruit the erythroid transcription factor GATA1. The BAHD1 complex interacts physically with PRC2, and depletion of either component restores FBXO11-deficient erythroid gene expression. Our studies identify BAHD1 as a novel effector of PRC2-mediated repression and reveal how a single E3 ubiquitin ligase eliminates PRC2 repression at many developmentally poised bivalent genes during erythropoiesis.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Eritropoese/fisiologia , Proteínas F-Box/metabolismo , Regulação da Expressão Gênica/fisiologia , Complexo Repressor Polycomb 2/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Linhagem Celular , Eritroblastos/metabolismo , Humanos , Proteólise
3.
Blood ; 133(23): 2518-2528, 2019 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-30971389

RESUMO

The microRNA (miRNA) locus miR-144/451 is abundantly expressed in erythrocyte precursors, facilitating their terminal maturation and protecting against oxidant stress. However, the full repertoire of erythroid miR-144/451 target messenger RNAs (mRNAs) and associated cellular pathways is unknown. In general, the numbers of mRNAs predicted to be targeted by an miRNA vary greatly from hundreds to thousands, and are dependent on experimental approaches. To comprehensively and accurately identify erythroid miR-144/451 target mRNAs, we compared gene knockout and wild-type fetal liver erythroblasts by RNA sequencing, quantitative proteomics, and RNA immunoprecipitation of Argonaute (Ago), a component of the RNA-induced silencing complex that binds miRNAs complexed to their target mRNAs. Argonaute bound ∼1400 erythroblast mRNAs in a miR-144/451-dependent manner, accounting for one-third of all Ago-bound mRNAs. However, only ∼100 mRNAs were stabilized after miR-144/451 loss. Thus, miR-144 and miR-451 deregulate <10% of mRNAs that they bind, a characteristic that likely applies generally to other miRNAs. Using stringent selection criteria, we identified 53 novel miR-144/451 target mRNAs. One of these, Cox10, facilitates the assembly of mitochondrial electron transport complex IV. Loss of miR-144/451 caused increased Cox10 mRNA and protein, accumulation of complex IV, and increased mitochondrial membrane potential with no change in mitochondrial mass. Thus, miR-144/451 represses mitochondrial respiration during erythropoiesis by inhibiting the production of Cox10.


Assuntos
Alquil e Aril Transferases/biossíntese , Eritropoese/genética , Regulação da Expressão Gênica/genética , Proteínas de Membrana/biossíntese , MicroRNAs/genética , Alquil e Aril Transferases/genética , Animais , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout
4.
Gene Ther ; 27(12): 545-556, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32341484

RESUMO

Autologous gene therapy using lentiviral vectors (LVs) holds promise for treating monogenetic blood diseases. However, clinical applications can be limited by suboptimal hematopoietic stem cell (HSC) transduction and insufficient quantities of available vector. We recently reported gene therapy for X-linked severe combined immunodeficiency using a protocol in which patient CD34+ cells were incubated with two successive transductions. Here we describe an improved protocol for LV delivery to CD34+ cells that simplifies product manipulation, reduces vector consumption, and achieves greater vector copy number (VCN) of repopulating HSCs in mouse xenotransplantation assays. Notable findings include the following: (1) the VCN of CD34+ cells measured shortly after transduction did not always correlate with the VCN of repopulating HSCs after xenotransplantation; (2) single-step transduction at higher CD34+ cell concentrations (2-4 × 106/ml) conserved LV without compromising HSC VCN; (3) poloxamer F108 (LentiBOOST) increased HSC VCN by two- to threefold (average from three donors); (4) although LentiBOOST + prostaglandin E2 combination further increased VCN in vitro, the VCN observed in vivo were similar to LentiBOOST alone; (5) cyclosporine H increased the HSC VCN to a similar or greater extent with LentiBOOST in vivo. Our findings delineate an improved protocol to increase the VCN of HSCs after CD34+ cell transduction with clinically relevant LVs.


Assuntos
Transplante de Células-Tronco Hematopoéticas , Lentivirus , Animais , Antígenos CD34 , Terapia Genética , Vetores Genéticos/genética , Células-Tronco Hematopoéticas , Humanos , Lentivirus/genética , Camundongos , Transdução Genética
5.
Stem Cells ; 36(6): 943-950, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29430853

RESUMO

Hematopoietic stem and progenitor cells (HSPCs) are necessary for life-long blood production and replenishment of the hematopoietic system during stress. We recently reported that nuclear factor I/X (Nfix) promotes HSPC survival post-transplant. Here, we report that ectopic expression of Nfix in primary mouse HSPCs extends their ex vivo culture from about 20 to 40 days. HSPCs overexpressing Nfix display hypersensitivity to supportive cytokines and reduced apoptosis when subjected to cytokine deprivation relative to controls. Ectopic Nfix resulted in elevated levels of c-Mpl transcripts and cell surface protein on primary murine HSPCs as well as increased phosphorylation of STAT5, which is known to be activated down-stream of c-MPL. Blocking c-MPL signaling by removal of thrombopoietin or addition of a c-MPL neutralizing antibody negated the antiapoptotic effect of Nfix overexpression on cultured HSPCs. Furthermore, NFIX was capable of binding to and transcriptionally activating a proximal c-Mpl promoter fragment. In sum, these data suggest that NFIX-mediated upregulation of c-Mpl transcription can protect primitive hematopoietic cells from stress ex vivo. Stem Cells 2018;36:943-950.


Assuntos
Células-Tronco Hematopoéticas/metabolismo , Fatores de Transcrição NFI/metabolismo , Receptores de Trombopoetina/metabolismo , Animais , Humanos , Camundongos , Transdução de Sinais
6.
Infect Immun ; 81(8): 2828-37, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23716605

RESUMO

Francisella tularensis, the bacterial cause of tularemia, infects the liver and replicates in hepatocytes in vivo and in vitro. However, the factors that govern adaptation of F. tularensis to the intrahepatocytic niche have not been identified. Using cDNA microarrays, we determined the transcriptional profile of the live vaccine strain (LVS) of F. tularensis grown in the FL83B murine hepatocytic cell line compared to that of F. tularensis cultured in broth. The fslC gene of the fsl operon was the most highly upregulated. Deletion of fslC eliminated the ability of the LVS to produce siderophore, which is involved in uptake of ferric iron, but it did not impair its growth in hepatocytes, A549 epithelial cells, or macrophages. Therefore, we sought an alternative means by which F. tularensis might obtain iron. Deletion of feoB, which encodes a putative ferrous iron transporter, retarded replication of the LVS in iron-restricted media, reduced its growth in hepatocytic and epithelial cells, and impaired its acquisition of iron. Survival of mice infected intradermally with a lethal dose of the LVS was slightly improved by deletion of fslC but was not altered by loss of feoB. However, the ΔfeoB mutant showed diminished ability to colonize the lungs, liver, and spleen of mice that received sublethal inocula. Thus, FeoB represents a previously unidentified mechanism for uptake of iron by F. tularensis. Moreover, failure to produce a mutant strain lacking both feoB and fslC suggests that FeoB and the proteins of the fsl operon are the only major means by which F. tularensis acquires iron.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Francisella tularensis/metabolismo , Ferro/metabolismo , Tularemia/metabolismo , Animais , Linhagem Celular , Modelos Animais de Doenças , Hepatócitos/microbiologia , Humanos , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Virulência/fisiologia
7.
Sci Adv ; 9(40): eadg9959, 2023 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-37801507

RESUMO

Lentiviral vector (LV)-based gene therapy holds promise for a broad range of diseases. Analyzing more than 280,000 vector integration sites (VISs) in 273 samples from 10 patients with X-linked severe combined immunodeficiency (SCID-X1), we discovered shared LV integrome signatures in 9 of 10 patients in relation to the genomics, epigenomics, and 3D structure of the human genome. VISs were enriched in the nuclear subcompartment A1 and integrated into super-enhancers close to nuclear pore complexes. These signatures were validated in T cells transduced with an LV encoding a CD19-specific chimeric antigen receptor. Intriguingly, the one patient whose VISs deviated from the identified integrome signatures had a distinct clinical course. Comparison of LV and gamma retrovirus integromes regarding their 3D genome signatures identified differences that might explain the lower risk of insertional mutagenesis in LV-based gene therapy. Our findings suggest that LV integrome signatures, shaped by common features such as genome organization, may affect the efficacy of LV-based cellular therapies.


Assuntos
Vetores Genéticos , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X , Humanos , Vetores Genéticos/genética , Terapia Genética , Retroviridae/genética , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/terapia , Linfócitos T
8.
J Bacteriol ; 194(3): 653-62, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22139502

RESUMO

Yersinia pestis is able to survive and replicate within murine macrophages. However, the mechanism by which Y. pestis promotes its intracellular survival is not well understood. To identify genes that are important for Y. pestis survival in macrophages, a library comprised of ∼31,500 Y. pestis KIM6+ transposon insertion mutants (input pool) was subjected to negative selection in primary murine macrophages. Genes underrepresented in the output pool of surviving bacteria were identified by transposon site hybridization to DNA oligonucleotide microarrays. The screen identified several genes known to be important for survival of Y. pestis in macrophages, including phoPQ and members of the PhoPQ regulon (e.g., pmrF). In addition, genes predicated to encode a glucose-1-phosphate uridylyltransferase (galU), a UDP-N-acetylglucosamine 2-epimerase (wecB) and a UDP-N-acetyl-d-mannosamine dehydrogenase (wecC) were identified in the screen. Viable-count assays demonstrated that a KIM6+ galU mutant and a KIM6+ wecBC mutant were defective for survival in murine macrophages. The galU mutant was studied further because of its strong phenotype. The KIM6+ galU mutant exhibited increased susceptibility to the antimicrobial peptides polymyxin B and cathelicidin-related antimicrobial peptide (CRAMP). Polyacrylamide gel electrophoresis demonstrated that the lipooligosaccharide (LOS) of the galU mutant migrated faster than the LOS of the parent KIM6+, suggesting the core was truncated. In addition, the analysis of LOS isolated from the galU mutant by mass spectrometry showed that aminoarabinose modification of lipid A is absent. Therefore, addition of aminoarabinose to lipid A and complete LOS core (galU), as well as enterobacterial common antigen (wecB and wecC), is important for survival of Y. pestis in macrophages.


Assuntos
Proteínas de Bactérias/metabolismo , Desidrogenases de Carboidrato/metabolismo , Elementos de DNA Transponíveis , Macrófagos/microbiologia , Peste/microbiologia , UTP-Glucose-1-Fosfato Uridililtransferase/metabolismo , Yersinia pestis/enzimologia , Animais , Proteínas de Bactérias/genética , Desidrogenases de Carboidrato/genética , Carboidratos Epimerases/genética , Carboidratos Epimerases/metabolismo , Linhagem Celular , Regulação Bacteriana da Expressão Gênica , Camundongos , Viabilidade Microbiana , Mutagênese Insercional , UTP-Glucose-1-Fosfato Uridililtransferase/genética , Yersinia pestis/genética , Yersinia pestis/crescimento & desenvolvimento
9.
Dis Model Mech ; 15(6)2022 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-35793591

RESUMO

We characterized the human ß-like globin transgenes in two mouse models of sickle cell disease (SCD) and tested a genome-editing strategy to induce red blood cell fetal hemoglobin (HbF; α2γ2). Berkeley SCD mice contain four to 22 randomly arranged, fragmented copies of three human transgenes (HBA1, HBG2-HBG1-HBD-HBBS and a mini-locus control region) integrated into a single site of mouse chromosome 1. Cas9 disruption of the BCL11A repressor binding motif in the γ-globin gene (HBG1 and HBG2; HBG) promoters of Berkeley mouse hematopoietic stem cells (HSCs) caused extensive death from multiple double-strand DNA breaks. Long-range sequencing of Townes SCD mice verified that the endogenous Hbb genes were replaced by single-copy segments of human HBG1 and HBBS including proximal but not some distal gene-regulatory elements. Townes mouse HSCs were viable after Cas9 disruption of the HBG1 BCL11A binding motif but failed to induce HbF to therapeutic levels, contrasting with human HSCs. Our findings provide practical information on the genomic structures of two common mouse SCD models, illustrate their limitations for analyzing therapies to induce HbF and confirm the importance of distal DNA elements in human globin regulation. This article has an associated First Person interview with the first author of the paper.


Assuntos
Anemia Falciforme , Hemoglobina Fetal , Anemia Falciforme/genética , Anemia Falciforme/terapia , Animais , Modelos Animais de Doenças , Hemoglobina Fetal/genética , Hemoglobina Fetal/metabolismo , Edição de Genes , Humanos , Camundongos , Fatores de Transcrição/genética , Transgenes , gama-Globinas/genética
10.
Mol Ther Methods Clin Dev ; 21: 693-701, 2021 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-34141824

RESUMO

Vector-mediated mutagenesis remains a major safety concern for many gene therapy clinical protocols. Indeed, lentiviral-based gene therapy treatments of hematologic disease can result in oligoclonal blood reconstitution in the transduced cell graft. Specifically, clonal expansion of hematopoietic stem cells (HSCs) highly expressing HMGA2, a chromatin architectural factor found in many human cancers, is reported in patients undergoing gene therapy for hematologic diseases, raising concerns about the safety of these integrations. Here, we show for the first time in vivo multilineage and multiclonal expansion of non-human primate HSCs expressing a 3' UTR-truncated version of HMGA2 without evidence of any hematologic malignancy >7 years post-transplantation, which is significantly longer than most non-human gene therapy pre-clinical studies. This expansion is accompanied by an increase in HSC survival, cell cycle activation of downstream progenitors, and changes in gene expression led by the upregulation of IGF2BP2, a mRNA binding regulator of survival and proliferation. Thus, we conclude that prolonged ectopic expression of HMGA2 in hematopoietic progenitors is not sufficient to drive hematologic malignancy and is not an acute safety concern in lentiviral-based gene therapy clinical protocols.

11.
Nat Genet ; 53(8): 1177-1186, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34341563

RESUMO

Hereditary persistence of fetal hemoglobin (HPFH) ameliorates ß-hemoglobinopathies by inhibiting the developmental switch from γ-globin (HBG1/HBG2) to ß-globin (HBB) gene expression. Some forms of HPFH are associated with γ-globin promoter variants that either disrupt binding motifs for transcriptional repressors or create new motifs for transcriptional activators. How these variants sustain γ-globin gene expression postnatally remains undefined. We mapped γ-globin promoter sequences functionally in erythroid cells harboring different HPFH variants. Those that disrupt a BCL11A repressor binding element induce γ-globin expression by facilitating the recruitment of nuclear transcription factor Y (NF-Y) to a nearby proximal CCAAT box and GATA1 to an upstream motif. The proximal CCAAT element becomes dispensable for HPFH variants that generate new binding motifs for activators NF-Y or KLF1, but GATA1 recruitment remains essential. Our findings define distinct mechanisms through which transcription factors and their cis-regulatory elements activate γ-globin expression in different forms of HPFH, some of which are being recreated by therapeutic genome editing.


Assuntos
Fator de Ligação a CCAAT/genética , Hemoglobina Fetal/genética , Fator de Transcrição GATA1/genética , gama-Globinas/genética , Animais , Sítios de Ligação , Células COS , Sistemas CRISPR-Cas , Linhagem Celular , Chlorocebus aethiops , Células Eritroides , Edição de Genes/métodos , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Regiões Promotoras Genéticas , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo
12.
Blood Adv ; 5(14): 2839-2851, 2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34283174

RESUMO

Individuals with monogenic disorders can experience variable phenotypes that are influenced by genetic variation. To investigate this in sickle cell disease (SCD), we performed whole-genome sequencing (WGS) of 722 individuals with hemoglobin HbSS or HbSß0-thalassemia from Baylor College of Medicine and from the St. Jude Children's Research Hospital Sickle Cell Clinical Research and Intervention Program (SCCRIP) longitudinal cohort study. We developed pipelines to identify genetic variants that modulate sickle hemoglobin polymerization in red blood cells and combined these with pain-associated variants to build a polygenic score (PGS) for acute vaso-occlusive pain (VOP). Overall, we interrogated the α-thalassemia deletion -α3.7 and 133 candidate single-nucleotide polymorphisms (SNPs) across 66 genes for associations with VOP in 327 SCCRIP participants followed longitudinally over 6 years. Twenty-one SNPs in 9 loci were associated with VOP, including 3 (BCL11A, MYB, and the ß-like globin gene cluster) that regulate erythrocyte fetal hemoglobin (HbF) levels and 6 (COMT, TBC1D1, KCNJ6, FAAH, NR3C1, and IL1A) that were associated previously with various pain syndromes. An unweighted PGS integrating all 21 SNPs was associated with the VOP event rate (estimate, 0.35; standard error, 0.04; P = 5.9 × 10-14) and VOP event occurrence (estimate, 0.42; standard error, 0.06; P = 4.1 × 10-13). These associations were stronger than those of any single locus. Our findings provide insights into the genetic modulation of VOP in children with SCD. More generally, we demonstrate the utility of WGS for investigating genetic contributions to the variable expression of SCD-associated morbidities.


Assuntos
Anemia Falciforme , Hemoglobina Fetal , Anemia Falciforme/complicações , Anemia Falciforme/genética , Criança , Hemoglobina Fetal/genética , Humanos , Estudos Longitudinais , Dor , Polimorfismo de Nucleotídeo Único
13.
Cancer Discov ; 11(5): 1082-1099, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33408242

RESUMO

Effective data sharing is key to accelerating research to improve diagnostic precision, treatment efficacy, and long-term survival in pediatric cancer and other childhood catastrophic diseases. We present St. Jude Cloud (https://www.stjude.cloud), a cloud-based data-sharing ecosystem for accessing, analyzing, and visualizing genomic data from >10,000 pediatric patients with cancer and long-term survivors, and >800 pediatric sickle cell patients. Harmonized genomic data totaling 1.25 petabytes are freely available, including 12,104 whole genomes, 7,697 whole exomes, and 2,202 transcriptomes. The resource is expanding rapidly, with regular data uploads from St. Jude's prospective clinical genomics programs. Three interconnected apps within the ecosystem-Genomics Platform, Pediatric Cancer Knowledgebase, and Visualization Community-enable simultaneously performing advanced data analysis in the cloud and enhancing the Pediatric Cancer knowledgebase. We demonstrate the value of the ecosystem through use cases that classify 135 pediatric cancer subtypes by gene expression profiling and map mutational signatures across 35 pediatric cancer subtypes. SIGNIFICANCE: To advance research and treatment of pediatric cancer, we developed St. Jude Cloud, a data-sharing ecosystem for accessing >1.2 petabytes of raw genomic data from >10,000 pediatric patients and survivors, innovative analysis workflows, integrative multiomics visualizations, and a knowledgebase of published data contributed by the global pediatric cancer community.This article is highlighted in the In This Issue feature, p. 995.


Assuntos
Anemia Falciforme/genética , Computação em Nuvem , Genômica , Disseminação de Informação , Neoplasias/genética , Criança , Ecossistema , Hospitais Pediátricos , Humanos
14.
BMC Bioinformatics ; 11: 33, 2010 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-20078885

RESUMO

BACKGROUND: With the rapid expansion of DNA sequencing databases, it is now feasible to identify relevant information from prior sequencing projects and completed genomes and apply it to de novo sequencing of new organisms. As an example, this paper demonstrates how such extra information can be used to improve de novo assemblies by augmenting the overlapping step. Finding all pairs of overlapping reads is a key task in many genome assemblers, and to this end, highly efficient algorithms have been developed to find alignments in large collections of sequences. It is well known that due to repeated sequences, many aligned pairs of reads nevertheless do not overlap. But no overlapping algorithm to date takes a rigorous approach to separating aligned but non-overlapping read pairs from true overlaps. RESULTS: We present an approach that extends the Minimus assembler by a data driven step to classify overlaps as true or false prior to contig construction. We trained several different classification models within the Weka framework using various statistics derived from overlaps of reads available from prior sequencing projects. These statistics included percent mismatch and k-mer frequencies within the overlaps as well as a comparative genomics score derived from mapping reads to multiple reference genomes. We show that in real whole-genome sequencing data from the E. coli and S. aureus genomes, by providing a curated set of overlaps to the contigging phase of the assembler, we nearly doubled the median contig length (N50) without sacrificing coverage of the genome or increasing the number of mis-assemblies. CONCLUSIONS: Machine learning methods that use comparative and non-comparative features to classify overlaps as true or false can be used to improve the quality of a sequence assembly.


Assuntos
Inteligência Artificial , Genômica/métodos , Análise de Sequência de DNA/métodos , Sequência de Bases , Bases de Dados de Ácidos Nucleicos , Genoma Bacteriano , Genoma Fúngico
15.
Infect Immun ; 78(9): 3700-15, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20566693

RESUMO

Yersinia pestis, the causative agent of plague, is a facultative intracellular pathogen. Previous studies have indicated that the ability of Y. pestis to survive inside macrophages may be critical during the early stages of plague pathogenesis. To gain insights into the biology of intracellular Y. pestis and its environment following phagocytosis, we determined the genome-wide transcriptional profile of Y. pestis KIM5 replicating inside J774.1 macrophage-like cells using DNA microarrays. At 1.5, 4, and 8 h postinfection, a total of 801, 464, and 416 Y. pestis genes were differentially regulated, respectively, compared to the level of gene expression of control bacteria grown in tissue culture medium. A number of stress-response genes, including those involved in detoxification of reactive oxygen species, as well as several metabolic genes involved in macromolecule synthesis, were significantly induced in intracellular Y. pestis, consistent with the presence of oxidative stress and nutrient starvation inside Yersinia-containing vacuoles. A putative stress-induced operon consisting of y2313, y2315, and y2316 (y2313-y2316), and a previously unidentified open reading frame, orfX, was studied further on the basis of its high level of intracellular expression. Mutant strains harboring either deletion, Deltay2313-y2316 or DeltaorfX, exhibited diverse phenotypes, including reduced effector secretion by the type III secretion system, increased intracellular replication, and filamentous morphology of the bacteria growing inside macrophages. The results suggest a possible role for these genes in regulating cell envelope characteristics in the intracellular environment.


Assuntos
Perfilação da Expressão Gênica , Macrófagos/microbiologia , Óperon/fisiologia , Estresse Fisiológico/genética , Yersinia pestis/genética , Animais , Proteínas de Bactérias/metabolismo , Divisão Celular , Linhagem Celular , Ciclo do Ácido Cítrico , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Virulência , Yersinia pestis/patogenicidade
16.
Infect Immun ; 77(6): 2251-61, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19289509

RESUMO

Yersinia pestis survives and replicates in phagosomes of murine macrophages. Previous studies demonstrated that Y. pestis-containing vacuoles (YCVs) acquire markers of late endosomes or lysosomes in naïve macrophages and that this bacterium can survive in macrophages activated with the cytokine gamma interferon. An autophagic process known as xenophagy, which destroys pathogens in acidic autophagolysosomes, can occur in naïve macrophages and is upregulated in activated macrophages. Studies were undertaken here to investigate the mechanism of Y. pestis survival in phagosomes of naïve and activated macrophages and to determine if the pathogen avoids or co-opts autophagy. Colocalization of the YCV with markers of autophagosomes or acidic lysosomes and the pH of the YCV were determined by microscopic imaging of infected macrophages. Some YCVs contained double membranes characteristic of autophagosomes, as determined by electron microscopy. Fluorescence microscopy showed that approximately 40% of YCVs colocalized with green fluorescent protein (GFP)-LC3, a marker of autophagic membranes, and that YCVs failed to acidify below pH 7 in naïve macrophages. Replication of Y. pestis in naïve macrophages caused accumulation of LC3-II, as determined by immunoblotting. While activation of infected macrophages increased LC3-II accumulation, it decreased the percentage of GFP-LC3-positive YCVs (approximately 30%). A viable count assay showed that Y. pestis survived equally well in macrophages proficient for autophagy and macrophages rendered deficient for this process by Cre-mediated deletion of ATG5, revealing that this pathogen does not require autophagy for intracellular replication. We conclude that although YCVs can acquire an autophagic membrane and accumulate LC3-II, the pathogen avoids xenophagy by preventing vacuole acidification.


Assuntos
Macrófagos/microbiologia , Fagossomos/química , Fagossomos/microbiologia , Yersinia pestis/imunologia , Yersinia pestis/fisiologia , Animais , Biomarcadores/análise , Contagem de Colônia Microbiana , Concentração de Íons de Hidrogênio , Camundongos , Camundongos Endogâmicos C57BL , Viabilidade Microbiana , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Fagossomos/ultraestrutura
18.
Leukemia ; 32(5): 1157-1167, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29479065

RESUMO

In vivo persistence of chimeric antigen receptor (CAR)-modified T cells correlates with therapeutic efficacy, yet CAR-specific factors that support persistence are not well resolved. Using a CD33-specific CAR in an acute myeloid leukemia (AML) model, we show how CAR expression alters T cell differentiation in a ligand independent manner. Ex vivo expanded CAR-T cells demonstrated decreased naïve and stem memory populations and increased effector subsets relative to vector-transduced control cells. This was associated with reduced in vivo persistence. Decreased persistence was not due to specificity or tumor presence, but to pre-transfer tonic signaling through the CAR CD3ζ ITAMs. We identified activation of the PI3K pathway in CD33 CAR-T cells as responsible. Treatment with a PI3K inhibitor modulated the differentiation program of CAR-T cells, preserved a less differentiated state without affecting T cell expansion, and improved in vivo persistence and reduced tumor burden. These results resolve mechanisms by which tonic signaling of CAR-T cells modulates their fate, and identifies a novel pharmacologic approach to enhance the durability of CAR-T cells for immunotherapy.


Assuntos
Imunoterapia Adotiva/métodos , Leucemia Mieloide Aguda/terapia , Fosfatidilinositol 3-Quinases/metabolismo , Receptores de Antígenos Quiméricos/uso terapêutico , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Ativação Linfocitária/efeitos dos fármacos , Inibidores de Fosfoinositídeo-3 Quinase , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/farmacologia , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/uso terapêutico , Linfócitos T , Carga Tumoral/efeitos dos fármacos
19.
J Comput Biol ; 17(11): 1549-60, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20973743

RESUMO

Genomic sequencing techniques introduce experimental errors into reads which can mislead sequence assembly efforts and complicate the diagnostic process. Here we present a method for detecting and removing sequencing errors from reads generated in genomic shotgun sequencing projects prior to sequence assembly. For each input read, the set of all length k substrings (k-mers) it contains are calculated. The read is evaluated based on the frequency with which each k-mer occurs in the complete data set (k-count). For each read, k-mers are clustered using the variable-bandwidth mean-shift algorithm. Based on the k-count of the cluster center, clusters are classified as error regions or non-error regions. For the 23 real and simulated data sets tested (454 and Solexa), our algorithm detected error regions that cover 99% of all errors. A heuristic algorithm is then applied to detect the location of errors in each putative error region. A read is corrected by removing the errors, thereby creating two or more smaller, error-free read fragments. After performing error removal, the error-rate for all data sets tested decreased (∼35-fold reduction, on average). EDAR has comparable accuracy to methods that correct rather than remove errors and when the error rate is greater than 3% for simulated data sets, it performs better. The performance of the Velvet assembler is generally better with error-removed data. However, for short reads, splitting at the location of errors can be problematic. Following error detection with error correction, rather than removal, may improve the assembly results.


Assuntos
Algoritmos , Biologia Computacional/métodos , Análise de Sequência de DNA/métodos , Genoma , Alinhamento de Sequência/métodos
20.
Vaccine ; 26(41): 5276-88, 2008 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-18692537

RESUMO

Vaccination of mice with Francisella tularensis live vaccine strain (LVS) mutants described so far have failed to induce protection in C57BL/6 mice against challenge with the virulent strain F. tularensis SchuS4. We have previously reported that a mutant of F. tularensis LVS deficient in iron superoxide dismutase (sodB(Ft)) is hypersensitive to oxidative stress and attenuated for virulence in mice. Herein, we evaluated the efficacy of this mutant as a vaccine candidate against respiratory tularemia caused by F. tularensis SchuS4. C57BL/6 mice were vaccinated intranasally (i.n.) with the sodB(Ft) mutant and challenged i.n. with lethal doses of F. tularensis SchuS4. The level of protection against SchuS4 challenge was higher in sodB(Ft) vaccinated group as compared to the LVS vaccinated mice. sodB(Ft) vaccinated mice following SchuS4 challenge exhibited significantly reduced bacterial burden in lungs, liver and spleen, regulated production of pro-inflammatory cytokines and less severe histopathological lesions compared to the LVS vaccinated mice. The sodB(Ft) vaccination induced a potent humoral immune response and protection against SchuS4 required both CD4 and CD8 T cells in the vaccinated mice. sodB(Ft) mutants revealed upregulated levels of chaperonine proteins DnaK, GroEL and Bfr that have been shown to be important for generation of a potent immune response against Francisella infection. Collectively, this study describes an improved live vaccine candidate against respiratory tularemia that has an attenuated virulence and enhanced protective efficacy than the LVS.


Assuntos
Vacinas Bacterianas/imunologia , Francisella tularensis/imunologia , Infecções Respiratórias/prevenção & controle , Tularemia/prevenção & controle , Animais , Anticorpos Antibacterianos/sangue , Proteínas de Bactérias/genética , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Citocinas/imunologia , Feminino , Francisella tularensis/genética , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infecções Respiratórias/imunologia , Infecções Respiratórias/microbiologia , Infecções Respiratórias/patologia , Superóxido Dismutase/genética , Análise de Sobrevida , Tularemia/imunologia , Tularemia/patologia , Regulação para Cima , Vacinas de Produtos Inativados/imunologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA