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1.
Bioessays ; 42(9): e2000047, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32643177

RESUMO

Large deletions and genomic re-arrangements are increasingly recognized as common products of double-strand break repair at Clustered Regularly Interspaced, Short Palindromic Repeats - CRISPR associated protein 9 (CRISPR/Cas9) on-target sites. Together with well-known off-target editing products from Cas9 target misrecognition, these are important limitations, that need to be addressed. Rigorous assessment of Cas9-editing is necessary to ensure validity of observed phenotypes in Cas9-edited cell-lines and model organisms. Here the mechanisms of Cas9 specificity, and strategies to assess and mitigate unwanted effects of Cas9 editing are reviewed; covering guide-RNA design, RNA modifications, Cas9 modifications, control of Cas9 activity; computational prediction for off-targets, and experimental methods for detecting Cas9 cleavage. Although recognition of the prevalence of on- and off-target effects of Cas9 editing has increased in recent years, broader uptake across the gene editing community will be important in determining the specificity of Cas9 across diverse applications and organisms.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Sistemas CRISPR-Cas/genética , Genoma , Genômica , Humanos , RNA Guia de Cinetoplastídeos/genética
2.
Proc Natl Acad Sci U S A ; 116(40): 20135-20140, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31527262

RESUMO

Staphylococcus aureus small-colony variants (SCVs) are associated with unusually chronic and persistent infections despite active antibiotic treatment. The molecular basis for this clinically important phenomenon is poorly understood, hampered by the instability of the SCV phenotype. Here we investigated the genetic basis for an unstable S. aureus SCV that arose spontaneously while studying rifampicin resistance. This SCV showed no nucleotide differences across its genome compared with a normal-colony variant (NCV) revertant, yet the SCV presented the hallmarks of S. aureus linked to persistent infection: down-regulation of virulence genes and reduced hemolysis and neutrophil chemotaxis, while exhibiting increased survival in blood and ability to invade host cells. Further genome analysis revealed chromosome structural variation uniquely associated with the SCV. These variations included an asymmetric inversion across half of the S. aureus chromosome via recombination between type I restriction modification system (T1RMS) genes, and the activation of a conserved prophage harboring the immune evasion cluster (IEC). Phenotypic reversion to the wild-type-like NCV state correlated with reversal of the chromosomal inversion (CI) and with prophage stabilization. Further analysis of 29 complete S. aureus genomes showed strong signatures of recombination between hsdMS genes, suggesting that analogous CI has repeatedly occurred during S. aureus evolution. Using qPCR and long-read amplicon deep sequencing, we detected subpopulations with T1RMS rearrangements causing CIs and prophage activation across major S. aureus lineages. Here, we have discovered a previously unrecognized and widespread mechanism of reversible genomic instability in S. aureus associated with SCV generation and persistent infections.


Assuntos
Instabilidade Cromossômica , Cromossomos Bacterianos , Fenótipo , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genética , Translocação Genética , Inversão Cromossômica , Ordem dos Genes , Genoma Bacteriano , Hemólise , Humanos , Fagos de Staphylococcus/fisiologia , Staphylococcus aureus/virologia
3.
Eur J Immunol ; 49(5): 770-781, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30729501

RESUMO

Mutation of Dedicator of cytokinesis 8 (DOCK8) has previously been reported to provide resistance to the Th17 cell dependent EAE in mice. Contrary to expectation, we observed an elevation of Th17 cells in two different DOCK8 mutant mouse strains in the steady state. This was specific for Th17 cells with no change in Th1 or Th2 cell populations. In vitro Th cell differentiation assays revealed that the elevated Th17 cell population was not due to a T cell intrinsic differentiation bias. Challenging these mutant mice in the EAE model, we confirmed a resistance to this autoimmune disease with Th17 cells remaining elevated systemically while cellular infiltration in the CNS was reduced. Infiltrating T cells lost the bias toward Th17 cells indicating a relative reduction of Th17 cells in the CNS and a Th17 cell specific migration disadvantage. Adoptive transfers of Th1 and Th17 cells in EAE-affected mice further supported the Th17 cell-specific migration defect, however, DOCK8-deficient Th17 cells expressed normal Th17 cell-specific CCR6 levels and migrated toward chemokine gradients in transwell assays. This study shows that resistance to EAE in DOCK8 mutant mice is achieved despite a systemic Th17 bias.


Assuntos
Suscetibilidade a Doenças , Encefalomielite Autoimune Experimental/etiologia , Fatores de Troca do Nucleotídeo Guanina/genética , Contagem de Linfócitos , Mutação , Células Th17/imunologia , Células Th17/metabolismo , Animais , Biomarcadores , Quimiotaxia de Leucócito/genética , Quimiotaxia de Leucócito/imunologia , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/patologia , Expressão Gênica , Predisposição Genética para Doença , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo
4.
PLoS One ; 17(11): e0277019, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36318583

RESUMO

Acinetobacter baumannii is an emerging nosocomial, opportunistic pathogen with growing clinical significance globally. A. baumannii has an exceptional ability to rapidly develop drug resistance. It is frequently responsible for ventilator-associated pneumonia in clinical settings and inflammation resulting in severe sepsis. The inflammatory response is mediated by host pattern-recognition receptors and the inflammasomes. Inflammasome activation triggers inflammatory responses, including the secretion of the pro-inflammatory cytokines IL-1ß and IL-18, the recruitment of innate immune effectors against A. baumannii infection, and the induction programmed cell death by pyroptosis. An important knowledge gap is how variation among clinical isolates affects the host's innate response and activation of the inflammasome during A. baumannii infection. In this study, we compared nine A. baumannii strains, including clinical locally-acquired isolates, in their ability to induce activation of the inflammasome and programmed cell death in primary macrophages, epithelial lung cell line and mice. We found a variation in survival outcomes of mice and bacterial dissemination in organs among three commercially available A. baumannii strains, likely due to the differences in virulence between strains. Interestingly, we found variability among A. baumannii strains in activation of the NLRP3 inflammasome, non-canonical Caspase-11 pathway, plasmatic secretion of the pro-inflammatory cytokine IL-1ß and programmed cell death. Our study highlights the importance of utilising multiple bacterial strains and clinical isolates with different virulence to investigate the innate immune response to A. baumannii infection.


Assuntos
Acinetobacter baumannii , Inflamassomos , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Interleucina-1beta/metabolismo , Caspases/metabolismo , Macrófagos/metabolismo
5.
Cells ; 10(10)2021 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-34685766

RESUMO

The morbidity and mortality caused by the globally prevalent human respiratory pathogen respiratory syncytial virus (RSV) approaches that world-wide of influenza. We previously demonstrated that the RSV matrix (M) protein shuttles, in signal-dependent fashion, between host cell nucleus and cytoplasm, and that this trafficking is central to RSV replication and assembly. Here we analyze in detail the nuclear role of M for the first time using a range of novel approaches, including quantitative analysis of de novo cell transcription in situ in the presence or absence of RSV infection or M ectopic expression, as well as in situ DNA binding. We show that M, dependent on amino acids 110-183, inhibits host cell transcription in RSV-infected cells as well as cells transfected to express M, with a clear correlation between nuclear levels of M and the degree of transcriptional inhibition. Analysis of bacterially expressed M protein and derivatives thereof mutated in key residues within M's RNA binding domain indicates that M can bind to DNA as well as RNA in a cell-free system. Parallel results for point-mutated M derivatives implicate arginine 170 and lysine 172, in contrast to other basic residues such as lysine 121 and 130, as critically important residues for inhibition of transcription and DNA binding both in situ and in vitro. Importantly, recombinant RSV carrying arginine 170/lysine 172 mutations shows attenuated infectivity in cultured cells and in an animal model, concomitant with altered inflammatory responses. These findings define an RSV M-chromatin interface critical for host transcriptional inhibition in infection, with important implications for anti-RSV therapeutic development.


Assuntos
Cromatina/metabolismo , Infecções por Vírus Respiratório Sincicial/genética , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/fisiologia , Transcrição Gênica , Proteínas da Matriz Viral/metabolismo , Animais , Arginina/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Chlorocebus aethiops , DNA Viral/metabolismo , Modelos Animais de Doenças , Humanos , Lisina/metabolismo , Camundongos Endogâmicos BALB C , Modelos Biológicos , Proteínas Mutantes/metabolismo , Mutação/genética , Ligação Proteica , Domínios Proteicos , RNA Viral/metabolismo , Células Vero , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genética , Viremia/virologia
6.
J Med Chem ; 64(8): 4478-4497, 2021 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-33792339

RESUMO

Malaria-causing Plasmodium parasites are developing resistance to antimalarial drugs, providing the impetus for new antiplasmodials. Although pantothenamides show potent antiplasmodial activity, hydrolysis by pantetheinases/vanins present in blood rapidly inactivates them. We herein report the facile synthesis and biological activity of a small library of pantothenamide analogues in which the labile amide group is replaced with a heteroaromatic ring. Several of these analogues display nanomolar antiplasmodial activity against Plasmodium falciparum and/or Plasmodium knowlesi, and are stable in the presence of pantetheinase. Both a known triazole and a novel isoxazole derivative were further characterized and found to possess high selectivity indices, medium or high Caco-2 permeability, and medium or low microsomal clearance in vitro. Although they fail to suppress Plasmodium berghei proliferation in vivo, the pharmacokinetic and contact time data presented provide a benchmark for the compound profile likely required to achieve antiplasmodial activity in mice and should facilitate lead optimization.


Assuntos
Antimaláricos/química , Isoxazóis/química , Ácido Pantotênico/análogos & derivados , Tiadiazóis/química , Triazóis/química , Animais , Antimaláricos/metabolismo , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Células CACO-2 , Proliferação de Células/efeitos dos fármacos , Estabilidade de Medicamentos , Eritrócitos/citologia , Eritrócitos/parasitologia , Feminino , Meia-Vida , Humanos , Malária Falciparum/tratamento farmacológico , Camundongos , Camundongos Endogâmicos BALB C , Ácido Pantotênico/química , Ácido Pantotênico/metabolismo , Ácido Pantotênico/farmacologia , Ácido Pantotênico/uso terapêutico , Plasmodium falciparum/efeitos dos fármacos , Plasmodium knowlesi/efeitos dos fármacos , Relação Estrutura-Atividade
7.
Sci Rep ; 10(1): 2303, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-32024943

RESUMO

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

8.
Artigo em Inglês | MEDLINE | ID: mdl-33014890

RESUMO

An important component in host resistance to malaria infection are inherited mutations that give rise to abnormalities and deficiencies in erythrocyte proteins and enzymes. Understanding how such mutations confer protection against the disease may be useful for developing new treatment strategies. A mouse ENU-induced mutagenesis screen for novel malaria resistance-conferring mutations identified a novel non-sense mutation in the gene encoding porphobilinogen deaminase (PBGD) in mice, denoted here as PbgdMRI58155. Heterozygote PbgdMRI58155 mice exhibited ~50% reduction in cellular PBGD activity in both mature erythrocytes and reticulocytes, although enzyme activity was ~10 times higher in reticulocytes than erythrocytes. When challenged with blood-stage P. chabaudi, which preferentially infects erythrocytes, heterozygote mice showed a modest but significant resistance to infection, including reduced parasite growth. A series of assays conducted to investigate the mechanism of resistance indicated that mutant erythrocyte invasion by P. chabaudi was normal, but that following intraerythrocytic establishment a significantly greater proportions of parasites died and therefore, affected their ability to propagate. The Plasmodium resistance phenotype was not recapitulated in Pbgd-deficient mice infected with P. berghei, which prefers reticulocytes, or when P. falciparum was cultured in erythrocytes from patients with acute intermittent porphyria (AIP), which had modest (20-50%) reduced levels of PBGD. Furthermore, the growth of Pbgd-null P. falciparum and Pbgd-null P. berghei parasites, which grew at the same rate as their wild-type counterparts in normal cells, were not affected by the PBGD-deficient background of the AIP erythrocytes or Pbgd-deficient mice. Our results confirm the dispensability of parasite PBGD for P. berghei infection and intraerythrocytic growth of P. falciparum, but for the first time identify a requirement for host erythrocyte PBGD by P. chabaudi during in vivo blood stage infection.


Assuntos
Malária , Plasmodium chabaudi , Porfiria Aguda Intermitente , Animais , Eritrócitos , Humanos , Camundongos , Plasmodium berghei/genética , Plasmodium falciparum
9.
Sci Rep ; 9(1): 6356, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015511

RESUMO

Plasmodium falciparum malaria causes half a million deaths per year, with up to 9% of this mortality caused by cerebral malaria (CM). One of the major processes contributing to the development of CM is an excess of host inflammatory cytokines. Recently K+ signaling has emerged as an important mediator of the inflammatory response to infection; we therefore investigated whether mice carrying an ENU induced activation of the electroneutral K+ channel KCC1 had an altered response to Plasmodium berghei. Here we show that Kcc1M935K/M935K mice are protected from the development of experimental cerebral malaria, and that this protection is associated with an increased CD4+ and TNFa response. This is the first description of a K+ channel affecting the development of experimental cerebral malaria.


Assuntos
Ativação do Canal Iônico , Malária Cerebral/metabolismo , Malária Cerebral/prevenção & controle , Membro 4 da Família 12 de Carreador de Soluto/metabolismo , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Citocinas/metabolismo , Resistência à Doença , Feminino , Mediadores da Inflamação/metabolismo , Malária Cerebral/imunologia , Malária Cerebral/parasitologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mutação/genética , Plasmodium berghei/fisiologia , Membro 4 da Família 12 de Carreador de Soluto/genética
10.
Pathog Dis ; 76(9)2018 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-30657912

RESUMO

Acinetobacter baumannii is an emerging nosocomial, opportunistic pathogen with growing clinical significance. Acinetobacter baumannii has an exceptional ability to rapidly develop drug resistance and to adhere to abiotic surfaces, including medical equipment, significantly promoting bacterial spread and also limiting our ability to control A. baumannii infections. Consequently, A. baumannii is frequently responsible for ventilator-associated pneumonia in clinical settings. In order to develop an effective treatment strategy, understanding host-pathogen interactions during A. baumannii infection is crucial. Various A. baumannii virulence factors have been identified as targets of host innate pattern-recognition receptors, which leads to activation of downstream inflammasomes to develop inflammatory responses, and the recruitment of innate immune effectors against A. baumannii infection. To counteract host immune attack, A. baumannii regulates its expression of different virulence factors. This review summarizes the significance of mechanisms of host-bacteria interaction, as well as different bacteria and host defense mechanisms during A. baumannii infection.


Assuntos
Infecções por Acinetobacter/imunologia , Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/imunologia , Acinetobacter baumannii/patogenicidade , Interações Hospedeiro-Patógeno , Imunidade Inata , Fatores de Virulência/metabolismo , Humanos
11.
Physiol Rep ; 6(7): e13669, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29654633

RESUMO

Airway remodeling is an important process in response to repetitive inflammatory-mediated airway wall injuries. This is characterized by profound changes and reorganizations at the cellular and molecular levels of the lung tissue. It is of particular importance to understand the mechanisms involved in airway remodeling, as this is strongly associated with severe asthma leading to devastating airway dysfunction. In this study, we have investigated the transforming growth factor-ß (TGFß, a proinflammatory mediator)-activated fibroblast to myofibroblast transdifferentiation pathway, which plays a key role in asthma-related airway remodeling. We show that TGFß induces fibroblast to myofibroblast transdifferentiation by the expression of αSMA, a specific myofibroblast marker. Furthermore, Smad2/Smad3 gene and protein expression patterns are different between fibroblasts and myofibroblasts. Such a change in expression patterns reveals an important role of these proteins in the cellular phenotype as well as their regulation by TGFß during cellular transdifferentiation. Interestingly, our data show a myofibroblastic TGFß-mediated increase in glucocorticoid receptor (GR) expression and a preferential localization of GR in the nucleus, compared to in fibroblasts. Furthermore, the GRß (nonfunctional GR isoform) is increased relative to GRα (functional isoform) in myofibroblasts. These results are interesting as they support the idea of a GRß-mediated glucocorticoid resistance observed in the severe asthmatic population. All together, we provide evidence that key players are involved in the TGFß-mediated fibroblast to myofibroblast transdifferentiation pathway in a human lung fibroblast cell line. These players could be the targets of new treatments to limit airway remodeling and reverse glucocorticoid resistance in severe asthma.


Assuntos
Remodelação das Vias Aéreas/fisiologia , Transdiferenciação Celular/fisiologia , Fibroblastos/metabolismo , Miofibroblastos/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Linhagem Celular , Fibroblastos/citologia , Humanos , Pulmão/citologia , Pulmão/metabolismo , Miofibroblastos/citologia , Receptores de Glucocorticoides/metabolismo
12.
PLoS One ; 13(2): e0191360, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29415062

RESUMO

Type 1 diabetes (T1D) is an autoimmune disease in which insulin-producing beta cells in pancreatic islets are progressively destroyed. Clinical trials of immunotherapies in recently diagnosed T1D patients have only transiently and partially impacted the disease course, suggesting that other approaches are required. Our previous studies have demonstrated that heparan sulfate (HS), a glycosaminoglycan conventionally expressed in extracellular matrix, is present at high levels inside normal mouse beta cells. Intracellular HS was shown to be critical for beta cell survival and protection from oxidative damage. T1D development in Non-Obese Diabetic (NOD) mice correlated with loss of islet HS and was prevented by inhibiting HS degradation by the endoglycosidase, heparanase. In this study we investigated the distribution of HS and heparan sulfate proteoglycan (HSPG) core proteins in normal human islets, a role for HS in human beta cell viability and the clinical relevance of intra-islet HS and HSPG levels, compared to insulin, in human T1D. In normal human islets, HS (identified by 10E4 mAb) co-localized with insulin but not glucagon and correlated with the HSPG core proteins for collagen type XVIII (Col18) and syndecan-1 (Sdc1). Insulin-positive islets of T1D pancreases showed significant loss of HS, Col18 and Sdc1 and heparanase was strongly expressed by islet-infiltrating leukocytes. Human beta cells cultured with HS mimetics showed significantly improved survival and protection against hydrogen peroxide-induced death, suggesting that loss of HS could contribute to beta cell death in T1D. We conclude that HS depletion in beta cells, possibly due to heparanase produced by insulitis leukocytes, may function as an important mechanism in the pathogenesis of human T1D. Our findings raise the possibility that intervention therapy with dual activity HS replacers/heparanase inhibitors could help to protect the residual beta cell mass in patients recently diagnosed with T1D.


Assuntos
Biomarcadores/metabolismo , Diabetes Mellitus Tipo 1/patologia , Heparitina Sulfato/metabolismo , Ilhotas Pancreáticas/metabolismo , Adolescente , Adulto , Estudos de Casos e Controles , Células Cultivadas , Criança , Pré-Escolar , Diabetes Mellitus Tipo 1/metabolismo , Progressão da Doença , Feminino , Humanos , Lactente , Ilhotas Pancreáticas/citologia , Masculino , Sensibilidade e Especificidade , Adulto Jovem
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