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1.
Nat Microbiol ; 4(8): 1378-1388, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31110366

RESUMO

Mycobacterium tuberculosis (Mtb)-derived components are usually recognized by pattern recognition receptors to initiate a cascade of innate immune responses. One striking characteristic of Mtb is their utilization of different type VII secretion systems to secrete numerous proteins across their hydrophobic and highly impermeable cell walls, but whether and how these Mtb-secreted proteins are sensed by host immune system remains largely unknown. Here, we report that MPT53 (Rv2878c), a secreted disulfide-bond-forming-like protein of Mtb, directly interacts with TGF-ß-activated kinase 1 (TAK1) and activates TAK1 in a TLR2- or MyD88-independent manner. MPT53 induces disulfide bond formation at C210 on TAK1 to facilitate its interaction with TRAFs and TAB1, thus activating TAK1 to induce the expression of pro-inflammatory cytokines. Furthermore, MPT53 and its disulfide oxidoreductase activity is required for Mtb to induce the host inflammatory responses via TAK1. Our findings provide an alternative pathway for host signalling proteins to sense Mtb infection and may favour the improvement of current vaccination strategies.

2.
BMC Biol ; 17(1): 7, 2019 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-30683096

RESUMO

BACKGROUND: The emergence of drug-resistant strains of Mycobacterium tuberculosis (Mtb), especially those that are multidrug resistant poses a serious threat to global tuberculosis control. However, the mechanism underlying the occurrence of drug resistance against more than one drug is poorly understood. Given that the Beijing/W strains are associated with outbreaks and multidrug resistance, they may harbor a genetic advantage and provide useful insight into the disease. One marker found in all Beijing/W Mtb strains is a deletion of RD105 region that results in a gene fusion, Rv0071/74, with a variable number (3-9 m) of VDP (V: Val, D: Asp; P: Pro) repeats (coded by gtggacccg repeat sequences) at the N-terminal. Here, we report that this variable number of VDP repeats in Rv0071/74 regulates the development of multidrug resistance. RESULTS: We collected and analyzed 1255 Beijing/W clinical strains. The results showed that the number of VDP repeats in Rv0071/74 was related to the development of multidrug resistance, and the deletion of Rv0071/74-9 m from Beijing/W clinical strain restored drug susceptibility. Rv0071/74-9 m also increased resistance to multiple drugs when transferred to different mycobacterial strains. Cell-free assays indicate that the domain carrying 4-9 VDP repeats (4-9 m) showed a variable binding affinity with peptidoglycan and Rv0071/74 cleaves peptidoglycan. Furthermore, Rv0071/74-9 m increased cell wall thickness and reduced the intracellular concentration of antibiotics. CONCLUSIONS: These findings not only identify Rv0071/74 with VDP repeats as a newly identified multidrug resistance gene but also provide a new model for the development of multiple drug resistance.


Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Mycobacterium tuberculosis/genética , Deleção de Sequência , Genótipo , Mycobacterium tuberculosis/efeitos dos fármacos
3.
Nat Commun ; 9(1): 4072, 2018 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-30287856

RESUMO

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb), and remains a leading public health problem. Previous studies have identified host genetic factors that contribute to Mtb infection outcomes. However, much of the heritability in TB remains unaccounted for and additional susceptibility loci most likely exist. We perform a multistage genome-wide association study on 2949 pulmonary TB patients and 5090 healthy controls (833 cases and 1220 controls were genome-wide genotyped) from Han Chinese population. We discover two risk loci: 14q24.3 (rs12437118, Pcombined = 1.72 × 10-11, OR = 1.277, ESRRB) and 20p13 (rs6114027, Pcombined = 2.37 × 10-11, OR = 1.339, TGM6). Moreover, we determine that the rs6114027 risk allele is related to decreased TGM6 transcripts in PBMCs from pulmonary TB patients and severer pulmonary TB disease. Furthermore, we find that tgm6-deficient mice are more susceptible to Mtb infection. Our results provide new insights into the genetic etiology of TB.

4.
Nature ; 563(7729): 131-136, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30356214

RESUMO

Accurate repair of DNA double-stranded breaks by homologous recombination preserves genome integrity and inhibits tumorigenesis. Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor that activates innate immunity by initiating the STING-IRF3-type I IFN signalling cascade1,2. Recognition of ruptured micronuclei by cGAS links genome instability to the innate immune response3,4, but the potential involvement of cGAS in DNA repair remains unknown. Here we demonstrate that cGAS inhibits homologous recombination in mouse and human models. DNA damage induces nuclear translocation of cGAS in a manner that is dependent on importin-α, and the phosphorylation of cGAS at tyrosine 215-mediated by B-lymphoid tyrosine kinase-facilitates the cytosolic retention of cGAS. In the nucleus, cGAS is recruited to double-stranded breaks and interacts with PARP1 via poly(ADP-ribose). The cGAS-PARP1 interaction impedes the formation of the PARP1-Timeless complex, and thereby suppresses homologous recombination. We show that knockdown of cGAS suppresses DNA damage and inhibits tumour growth both in vitro and in vivo. We conclude that nuclear cGAS suppresses homologous-recombination-mediated repair and promotes tumour growth, and that cGAS therefore represents a potential target for cancer prevention and therapy.

5.
Nat Commun ; 9(1): 4295, 2018 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-30327467

RESUMO

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) kills millions every year, and there is urgent need to develop novel anti-TB agents due to the fast-growing of drug-resistant TB. Although autophagy regulates the intracellular survival of Mtb, the role of calcium (Ca2+) signaling in modulating autophagy during Mtb infection remains largely unknown. Here, we show that microRNA miR-27a is abundantly expressed in active TB patients, Mtb-infected mice and macrophages. The target of miR-27a is the ER-located Ca2+ transporter CACNA2D3. Targeting of this transporter leads to the downregulation of Ca2+ signaling, thus inhibiting autophagosome formation and promoting the intracellular survival of Mtb. Mice lacking of miR-27a and mice treated with an antagomir to miR-27a are more resistant to Mtb infection. Our findings reveal a strategy for Mtb to increase intracellular survival by manipulating the Ca2+-associated autophagy, and may also support the development of host-directed anti-TB therapeutic approaches.

6.
Cell Death Discov ; 4: 17, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29531814

RESUMO

The epithelial-mesenchymal transition (EMT) is a multifunctional cell process involved in the pathogenesis of numerous conditions, including fibrosis and cancer. Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal disease characterized by fibroblast accumulation and collagen deposition in the lungs. The fibroblasts involved in this process partially originate from lung epithelial cells via the EMT. Evidence suggests that the EMT contributes to progression, invasion, and metastasis of various types of cancer. We screened a series of 80 compounds for the ability to interfere with the EMT and potentially be applied as a therapeutic for IPF and/or lung cancer. We identified 2-aminopurine (2-AP), a fluorescent analog of guanosine and adenosine, as a candidate in this screen. Herein, we demonstrate that 2-AP can restore E-cadherin expression and inhibit fibronectin and vimentin expression in TGF-ß1-treated A549 lung cancer cells. Moreover, 2-AP can inhibit TGF-ß1-induced metastasis of A549 cells. This compound significantly attenuated bleomycin (BLM)-induced pulmonary inflammation, the EMT, and fibrosis. In addition, 2-AP treatment significantly decreased mortality in a mouse model of pulmonary fibrosis. Collectively, we determined that 2-AP could inhibit metastasis in vitro by suppressing the TGF-ß1-induced EMT and could attenuate BLM-induced pulmonary fibrosis in vivo. Results of this study suggest that 2-AP may have utility as a treatment for lung cancer and pulmonary fibrosis.

7.
Emerg Microbes Infect ; 7(1): 34, 2018 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-29559631

RESUMO

Tuberculosis caused by Mycobacterium tuberculosis (Mtb) infection remains a large global public health problem. One striking characteristic of Mtb is its ability to adapt to hypoxia and trigger the ensuing transition to a dormant state for persistent infection, but how the hypoxia response of Mtb is regulated remains largely unknown. Here we performed a quantitative acetylome analysis to compare the acetylation profile of Mtb under aeration and hypoxia, and showed that 377 acetylation sites in 269 Mtb proteins were significantly changed under hypoxia. In particular, deacetylation of dormancy survival regulator (DosR) at K182 promoted the hypoxia response in Mtb and enhanced the transcription of DosR-targeted genes. Mechanistically, recombinant DosRK182R protein demonstrated enhanced DNA-binding activity in comparison with DosRK182Q protein. Moreover, Rv0998 was identified as an acetyltransferase that mediates the acetylation of DosR at K182. Deletion of Rv0998 also promoted the adaptation of Mtb to hypoxia and the transcription of DosR-targeted genes. Mice infected with an Mtb strain containing acetylation-defective DosRK182R had much lower bacterial counts and less severe histopathological impairments compared with those infected with the wild-type strain. Our findings suggest that hypoxia induces the deacetylation of DosR, which in turn increases its DNA-binding ability to promote the transcription of target genes, allowing Mtb to shift to dormancy under hypoxia.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Lisina/metabolismo , Mycobacterium tuberculosis/metabolismo , Oxigênio/metabolismo , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Tuberculose/microbiologia , Acetilação , Motivos de Aminoácidos , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Humanos , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/genética , Proteínas Quinases/genética
8.
J Infect Dis ; 218(2): 312-323, 2018 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-29228365

RESUMO

Tuberculosis, caused by Mycobacterium tuberculosis infection, remains a global threat to human health, but knowledge of the molecular mechanisms underlying the pathogenesis of tuberculosis is still limited. Although Notch4, a member of the Notch receptor family, is involved in the initiation of mammary tumors, its function in M. tuberculosis infection remains unclear. In this study, we found that Notch4-deficient mice were more resistant to M. tuberculosis infection, with a much lower bacterial burden and fewer pathological changes in the lungs. Notch4 inhibited M. tuberculosis-induced production of proinflammatory cytokines by interaction with TAK1 and inhibition of its activation. Furthermore, we found that Notch intracellular domain 4 prevented TRAF6 autoubiquitination and suppressed TRAF6-mediated TAK1 polyubiquitination. Finally, Notch inhibitors made mice more resistant to M. tuberculosis infection. These results suggest that Notch4 is a negative regulator of M. tuberculosis-induced inflammatory response, and treatment with a Notch inhibitor could serve as a new therapeutic strategy for tuberculosis.

9.
Chest ; 153(5): 1187-1200, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29224833

RESUMO

BACKGROUND: Exacerbated immunopathology is a frequent consequence of TB that is complicated by diabetes mellitus (DM); however, the underlying mechanisms are still poorly defined. METHODS: In the two groups of age- and sex-matched patients with TB and DM (DM-TB) and with TB and without DM, we microscopically evaluated the areas of caseous necrosis and graded the extent of perinecrotic fibrosis in lung biopsies from the sputum smear-negative (SN) patients. We scored acid-fast bacilli in sputum smear-positive (SP) patients and compiled CT scan data from both the SN and SP patients. We compared inflammatory biomarkers and routine hematologic and biochemical parameters. Binary logistic regression analyses were applied to define the indices associated with the extent of lung injury. RESULTS: Enlarged caseous necrotic areas with exacerbated fibrotic encapsulations were found in SN patients with DM-TB, consistent with the higher ratio of thick-walled cavities and more bacilli in the sputum from SP patients with DM-TB. Larger necrotic foci were detected in men compared with women within the SN TB groups. Significantly higher fibrinogen and lower high-density lipoprotein cholesterol (HDL-C) were observed in SN patients with DM-TB. Regression analyses revealed that diabetes, activation of the coagulation pathway (shown by increased platelet distribution width, decreased mean platelet volume, and shortened prothrombin time), and dyslipidemia (shown by decreased low-density lipoprotein cholesterol, HDL-C, and apolipoprotein A) are risk factors for severe lung lesions in both SN and SP patients with TB. CONCLUSIONS: Hemostasis and dyslipidemia are associated with granuloma necrosis and fibroplasia leading to exacerbated lung damage in TB, especially in patients with DM-TB.

10.
Clin Drug Investig ; 37(12): 1125-1136, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28900877

RESUMO

BACKGROUND: Drug transporters and drug-metabolizing enzymes have been linked to drug-induced hepatotoxicity. Solute carrier organic anion transporter family member 1B1 (SLCO1B1), cytochrome P450 2E1 (CYP2E1), and UDP glucuronosyltransferase 1A1 (UGT1A1) were selected as candidate genes to explore their association with susceptibility to anti-tuberculosis drug-induced hepatotoxicity (ATDH). METHODS: Thirty-four tag single nucleotide polymorphisms (tagSNPs) in SLCO1B1, CYP2E1, and UGT1A1 with 10-kb expansion up- and down-stream were genotyped in 461 patients with ATDH and 466 patients without ATDH in a prospective 1:1 matched case-control study. The frequencies and distributions of genotypes and haplotypes were compared between the groups using three genetic models (dominant, recessive, and additive) to identify associations with susceptibility to ATDH. RESULTS: Patients with the rs4149034 G/A, rs1564370 G/C, and rs2900478 T/A genotypes of SLCO1B1 had a significantly lower risk of ATDH, while those carrying the rs2417957 T/T and rs4149063 T/T genotypes had an increased risk. The rs4148323 A/A genotype of UGT1A1 was found to significantly reduce the risk of ATDH. Haplotype analysis showed the TGTG, TTTC, and GTTC haplotypes of SLCO1B1 were associated with an increased ATDH risk, whereas the GACC haplotype was related to a reduced risk. The ATG haplotype of UGT1A1 reduced the risk of ATDH. Moreover, treatment outcomes in tuberculosis patients were further affected by genetic variants of SLCO1B1. CONCLUSIONS: Genetic polymorphisms of SLCO1B1 and UGT1A1 were found to be associated with susceptibility to ATDH. Molecular identification of susceptibility genes provides a theoretical foundation for predicting the likelihood of ATDH and predicting treatment outcomes in tuberculosis patients.


Assuntos
Antituberculosos/efeitos adversos , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Citocromo P-450 CYP2E1/genética , Glucuronosiltransferase/genética , Adolescente , Adulto , Idoso , Grupo com Ancestrais do Continente Asiático/genética , Estudos de Casos e Controles , Doença Hepática Induzida por Substâncias e Drogas/genética , Feminino , Predisposição Genética para Doença , Genótipo , Haplótipos , Humanos , Transportador 1 de Ânion Orgânico Específico do Fígado/genética , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Estudos Prospectivos , Risco , Adulto Jovem
11.
PLoS One ; 12(1): e0169922, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28076390

RESUMO

Interleukin-37 (IL-37), a novel member of the IL-1 family, plays fundamental immunosuppressive roles by broadly reducing both innate inflammation and acquired immunity, but whether it is involved in the pathogenesis of tuberculosis (TB) has not been clearly elucidated. In this study, single nucleotide polymorphism (SNP) analysis demonstrated an association of the genetic variant rs3811047 of IL-37 with TB susceptibility. In line with previous report, a significant elevated IL-37 abundance in the sera and increased expression of IL-37 protein in the peripheral blood mononuclear cells (PBMC) were observed in TB patients in comparison to healthy controls. Moreover, release of IL-37 were detected in either macrophages infected with Mycobacterium tuberculosis (Mtb) or the lung of BCG-infected mice, concurrent with reduced production of proinflammatory cytokines including IL-6 and TNF-α. Furthermore, in contrast to wild-type mice, BCG-infected IL-37-Tg mice manifested with reduced mycobacterial burden and tissue damage in the lung, accompanied by higher frequency of Th1 cell and less frequencies of regulatory T cells and Th17 cells in the spleen. Taken together, our findings demonstrated that IL-37 conferred resistance to Mtb infection possibly involving suppressing detrimental inflammation and modulating T cell responses. These findings implicated that IL-37 may be employed as a new molecular target for the therapy and diagnosis of TB.


Assuntos
Inflamação/genética , Interleucina-1/fisiologia , Ativação Linfocitária/genética , Infecções por Mycobacterium/prevenção & controle , Células Th1/imunologia , Animais , Estudos de Casos e Controles , Células Cultivadas , Citoproteção/genética , Citoproteção/imunologia , Feminino , Frequência do Gene , Predisposição Genética para Doença , Humanos , Interleucina-1/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Infecções por Mycobacterium/genética , Infecções por Mycobacterium/imunologia , Infecções por Mycobacterium/veterinária , Mycobacterium bovis/imunologia , Mycobacterium tuberculosis/imunologia , Polimorfismo de Nucleotídeo Único , Células Th17/imunologia , Tuberculose/genética , Tuberculose/imunologia , Tuberculose/prevenção & controle
12.
Luminescence ; 32(5): 806-811, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27995761

RESUMO

In this paper, a novel and ultrasensitive electrochemiluminescent sensor employing a solvothermal-synthesized CdS nanorod-modified pencil graphite electrode (CdS/PGE) for the determination of chlorogenic acid (CA) is fabricated. In the first step, the PGE surface is modified using CdS nanorods. In the next step, the developed electrode is used to detect CA using a electrochemiluminescent (ECL) technique, in which potassium persulfate (K2 S2 O8 ) served as a co-reactant. The possible ECL mechanism is investigated, and the influences of pH and cyclic voltammetric scanning rate on the signal response are studied. The ECL intensity decreases quantitatively in relation to the concentration of the target molecule. Under optimized conditions, the linear correlation between the quenched ECL intensity and the logarithm of CA concentration is observed in the range from 2 × 10-9 to 8 × 10-7  mol L-1 with a limit of detection of 1 × 10-9  mol L-1 . This proposed method is applied to the analysis of CA in honeysuckle flower, giving recoveries of 99-107%. The experimental results demonstrate that this ECL sensor shows good stability and reproducibility.


Assuntos
Ácido Clorogênico/análise , Eletroquímica/métodos , Lonicera/química , Medições Luminescentes/métodos , Nanotubos/química , Tampões (Química) , Compostos de Cádmio/química , Calibragem , Eletroquímica/instrumentação , Eletrodos , Etilenodiaminas/química , Flores/química , Grafite/química , Concentração de Íons de Hidrogênio , Medições Luminescentes/instrumentação , Nitratos/química , Compostos de Potássio/química , Reprodutibilidade dos Testes , Sulfatos/química , Sulfetos/química , Difração de Raios X
13.
Cell Signal ; 28(9): 1145-51, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27259691

RESUMO

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, invades and replicates within susceptible hosts by disturbing host antimicrobial mechanisms. Although G protein-coupled receptors (GPCRs) are involved in most physiological and pathological activities of mammalian cells, the roles of GPCRs in Mtb invasion into host cell remain elusive. Here, we report that GPR160 expression is elevated at both mRNA and protein level in macrophages in response to BCG infection. Both the PiggyBac (PB) transposon-mediated mutation of gpr160 gene in mouse primary macrophages and siRNA-mediated knockdown of GPR160 in the human macrophage cell line THP-1 markedly reduced the entry of green fluorescent protein (GFP) expressing BCG (BCG-GFP), also operative in vivo. BCG infection-induced phosphorylation of ERK1/2 was significantly reduced in gpr160 mutated (gpr160(-/-)) macrophages relative to levels observed in wild type macrophages, while inhibition of ERK by specific inhibitor or knockdown ERK1/2 by specific siRNA markedly reduced entry of BCG. Finally, lower bacteria burdens and attenuated pathological impairments were observed in the lungs of BCG-infected gpr160(-/-) mice. Furthermore, gpr160(-/-) macrophages also exhibits reduced uptake of Escherichia coli and Francisella tularensis. Taken together, these findings suggest an important role of GPR160 in regulating the entry of BCG into macrophages by targeting the ERK signaling pathway. As GPCRs have proven to be successful drug targets in pharmaceutical industry, it's tempting to speculate that compounds targeting GPR160, a G protein-coupled receptor, could intervene in Mtb infection.


Assuntos
Macrófagos Peritoneais/enzimologia , Macrófagos Peritoneais/microbiologia , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Mycobacterium/fisiologia , Receptores Acoplados a Proteínas-G/metabolismo , Animais , Linhagem Celular , Ativação Enzimática , Feminino , Humanos , Camundongos , Mutação/genética , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Acoplados a Proteínas-G/deficiência
14.
Tuberculosis (Edinb) ; 95(4): 497-504, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25937126

RESUMO

Tuberculosis (TB) remains a major global health problem and host genetic factors play a critical role in susceptibility and resistance to TB. The aim of this study was to identify novel candidate genes associated with TB susceptibility. We performed a population-based case-control study to genotype 13 tag SNPs spanning Epstein-Barr virus-induced gene 3 (EBI3), colony stimulating factor 2 (CSF2), IL-4, interferon beta 1 (IFNB1), chemokine (C-X-C motif) ligand 14 (CXCL14) and myeloid differentiation primary response gene 88 (Myd88) genes in 435 pulmonary TB patients and 375 health donors from China. We observed that EBI3 gene rs4740 polymorphism was associated with susceptibility to pulmonary tuberculosis (PTB) and the allele G was associated with a protective effect against PTB. Furthermore, EBI3 deficiency led to reduced bacterial burden and histopathological impairment in the lung of mice infected with Mycobacterium bovis BCG. Meanwhile, higher abundance of EBI3 was observed in the granuloma of PTB patients and in the lung tissue of BCG-infected mice. Of note, the expression of EBI3 in macrophages was remarkably induced by mycobacteria infection at both mRNA and protein level. In conclusion, EBI3 gene rs4740 polymorphism is closely associated with susceptibility to PTB and the elevation and enrichment of EBI3 in the lung which at least partially derived from macrophages may contribute to the exacerbation of mycobacterial infection.


Assuntos
Interleucinas/genética , Pulmão/metabolismo , Mycobacterium tuberculosis/patogenicidade , Polimorfismo de Nucleotídeo Único , Receptores de Citocinas/genética , Tuberculose Pulmonar/genética , Animais , Carga Bacteriana , Estudos de Casos e Controles , Linhagem Celular , China , Modelos Animais de Doenças , Predisposição Genética para Doença , Interações Hospedeiro-Patógeno , Humanos , Interleucinas/metabolismo , Pulmão/microbiologia , Macrófagos/microbiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Antígenos de Histocompatibilidade Menor , Mycobacterium bovis/patogenicidade , Fenótipo , Fatores de Proteção , Receptores de Citocinas/deficiência , Fatores de Risco , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/metabolismo , Tuberculose Pulmonar/microbiologia , Tuberculose Pulmonar/prevenção & controle
15.
BMC Infect Dis ; 14: 200, 2014 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-24725975

RESUMO

BACKGROUND: Multidrug-resistant tuberculosis (MDR-TB) is a major public health problem. Early diagnosis of MDR-TB patients is essential for minimizing the risk of Mycobacterium tuberculosis (MTB) transmission. The conventional drug susceptibility testing (DST) methods for detection of drug-resistant M. tuberculosis are laborious and cannot provide the rapid detection for clinical practice. METHODS: The aim of this study was to develop a pyrosequencing approach for the simultaneous detection of resistance to rifampin (RIF), isoniazid (INH), ethambutol (EMB), streptomycin (SM), ofloxacin (OFL) and amikacin (AMK) in M. tuberculosis clinical isolates and sputum samples from re-treatment pulmonary tuberculosis (PTB) patients. We identified the optimum conditions for detection mutation of rpoB, katG, rpsl, embB, gyrA and rrs gene by pyrosequencing. Then this approach was applied to detect 205 clinical isolates and 24 sputum samples of M. tuberculosis from re-treatment PTB patients. RESULTS: The mutations of rpoB and gyrA gene were detected by pyrosequencig with the SQA mode, and the mutations of katG, rpsl, embB, gyrA and rrs gene were detected by pyrosequencing with SNP mode. Compared with the Bactec MGIT 960 mycobacterial detection system, the accuracy of pyrosequencing for the detection of RIF, INH, EMB, SM, AMK and OFL resistance in clinical isolates was 95.0%, 79.2%, 70.3%, 84.5%, 96.5% and 91.1%, respectively. In sputum samples the accuracy was 83.3%, 83.3%, 60.9%, 83.3%, 87.5% and 91.7%, respectively. CONCLUSIONS: The newly established pyrosequencing assay is a rapid and high-throughput method for the detection of resistance to RIF, INH, SM, EMB, OFL and AMK in M. tuberculosis. Pyrosequencing can be used as a practical molecular diagnostic tool for screening and predicting the resistance of re-treatment pulmonary tuberculosis patients.


Assuntos
Antituberculosos/farmacologia , Mycobacterium tuberculosis/genética , Escarro/microbiologia , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Tuberculose Pulmonar/microbiologia , DNA Bacteriano/genética , Genes Bacterianos/genética , Humanos , Testes de Sensibilidade Microbiana/métodos , Tipagem Molecular/métodos , Mutação , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/isolamento & purificação , Análise de Sequência de DNA/métodos , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/tratamento farmacológico
16.
ScientificWorldJournal ; 2014: 617087, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24696650

RESUMO

The trust levels of cloud services should be evaluated to ensure their reliability. The effectiveness of these evaluations has major effects on user satisfaction, which is increasingly important. However, it is difficult to provide objective evaluations in open and dynamic environments because of the possibilities of malicious evaluations, individual preferences, and intentional praise. In this study, we propose a novel unfair rating filtering method for a reputation revision system. This method uses prior knowledge as the basis of similarity when calculating the average rating, which facilitates the recognition and filtering of unfair ratings. In addition, the overall performance is increased by a market mechanism that allows users and service providers to adjust their choice of services and service configuration in a timely manner. The experimental results showed that this method filtered unfair ratings in an effective manner, which greatly improved the precision of the reputation revision system.


Assuntos
Armazenamento e Recuperação da Informação
17.
ScientificWorldJournal ; 2014: 531032, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24672326

RESUMO

Routing in wireless sensor networks (WSNs) is an extremely challenging issue due to the features of WSNs. Inspired by the large and single-celled amoeboid organism, slime mold Physarum polycephalum, we establish a novel selecting next hop model (SNH). Based on this model, we present a novel Physarum-based routing scheme (P-bRS) for WSNs to balance routing efficiency and energy equilibrium. In P-bRS, a sensor node can choose the proper next hop by using SNH which comprehensively considers the distance, energy residue, and location of the next hop. The simulation results show how P-bRS can achieve the effective trade-off between routing efficiency and energy equilibrium compared to two famous algorithms.


Assuntos
Physarum polycephalum/química , Tecnologia sem Fio
18.
Cell Signal ; 26(5): 942-50, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24462705

RESUMO

Cytokine induction in response to Mycobacterium tuberculosis (Mtb) infection is critical for pathogen control, by (i) mediating innate immune effector functions and (ii) instructing specific adaptive immunity. IL-10 is an important anti-inflammatory cytokine involved in pathogenesis of tuberculosis (TB). Here, we show that TLR3, a sensor of extracellular viral or host RNA with stable stem structures derived from infected or damaged cells, is essential for Mtb-induced IL-10 production. Upon Mycobacterium bovis Bacillus Calmette-Guérin (BCG) infection, TLR3(-/-) macrophages expressed lower IL-10 but higher IL-12p40 production, accompanied by reduced phosphorylation of AKT at Ser473. BCG-infected TLR3(-/-) mice exhibited reduced IL-10 but elevated IL-12 expression compared to controls. Moreover, higher numbers of splenic Th1 cells and reduced pulmonary bacterial burden and tissue damage were observed in BCG-infected TLR3(-/-) mice. Finally, BCG RNA induced IL-10 in macrophages via TLR3-mediated activation of PI3K/AKT. Our findings demonstrate a critical role of TLR3-mediated regulation in the pathogenesis of mycobacterial infection involving mycobacterial RNA, which induces IL-10 through the PI3K/AKT signaling pathway.


Assuntos
Interleucina-10/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Bacteriano/metabolismo , Receptor 3 Toll-Like/metabolismo , Animais , Subunidade p40 da Interleucina-12/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Mycobacterium bovis/genética , Mycobacterium bovis/patogenicidade , Fosforilação , Transdução de Sinais , Receptor 3 Toll-Like/deficiência , Receptor 3 Toll-Like/genética
19.
PLoS One ; 8(9): e73955, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24058507

RESUMO

BACKGROUND: Accurate and early diagnosis of tuberculosis (TB) is of major importance in the control of TB. One of the most important technical advances in diagnosis of tuberculosis is the development of nucleic acid amplification (NAA) tests. However, the choice of the target sequence remains controversial in NAA tests. Recently, interesting alternatives have been found in hypothetical protein coding sequences from mycobacterial genome. METHODOLOGY/PRINCIPAL FINDINGS: To obtain rational biomarker for TB diagnosis, the conservation of three hypothetical genes was firstly evaluated in 714 mycobacterial strains. The results showed that SCAR1 (Sequenced Characterized Amplified Region) based on Rv0264c coding gene showed the highest conservation (99.8%) and SCAR2 based on Rv1508c gene showed the secondary high conservation (99.7%) in M. tuberculosis (MTB) strains. SCAR3 based on Rv2135c gene (3.2%) and IS6110 (8%) showed relatively high deletion rate in MTB strains. Secondly, three SCAR markers were evaluated in 307 clinical sputum from patients in whom TB was suspected or patients with diseases other than TB. The amplification of IS6110 and 16SrRNA sequences together with both clinical and bacteriological identification was as a protocol to evaluate the efficacy of SCAR markers. The sensitivities and specificities, positive predictive value (PPV) and negative predictive value (NPV) of all NAA tests were higher than those of bacteriological detection. In four NAA tests, IS6110 and SCAR3 showed the highest PPV (100%) and low NPV (70% and 68.8%, respectively), and SCAR1 and SCAR2 showed the relatively high PPV and NPV (97% and 82.6%, 95.6% and 88.8%, respectively). CONCLUSIONS/SIGNIFICANCE: Our result indicated that SCAR1 and SCAR2 with a high degree of sequence conservation represent efficient and promising alternatives as NAA test targets in identification of MTB. Moreover, the targets developed from this study may provide more alternative targets for the development of a multisite system to effectively detect MTB in samples.


Assuntos
DNA Bacteriano/genética , Tipagem de Sequências Multilocus/métodos , Mycobacterium tuberculosis/genética , Fases de Leitura Aberta , Tuberculose/diagnóstico , Técnicas de Tipagem Bacteriana , Sequência de Bases , Sequência Conservada , Primers do DNA , DNA Bacteriano/química , Humanos , Dados de Sequência Molecular , Mycobacterium tuberculosis/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Escarro/microbiologia , Tuberculose/microbiologia
20.
J Clin Microbiol ; 51(5): 1374-80, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23390285

RESUMO

Pyrazinamide (PZA) is a first-line antituberculosis (anti-TB) drug capable of killing nonreplicating, persistent Mycobacterium tuberculosis. However, reliable testing of the susceptibility of M. tuberculosis to PZA is challenging. Using 432 clinical M. tuberculosis isolates, we compared the performances of five methods for the determination of M. tuberculosis susceptibility to PZA: the MGIT 960 system, the molecular drug susceptibility test (mDST), the pyrazinamidase (PZase) activity assay, the resazurin microtiter assay (REMA), and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction test. The sensitivities of the MGIT 960 system, the PZase activity assay, the mDST, the REMA, and the MTT assay were 98.8%, 88.8%, 90.5%, 98.8%, and 98.2%, respectively. The sensitivities of the PZase activity assay and the mDST were lower than those of the other three methods (P < 0.05). The specificities of the MGIT 960 system, the PZase activity assay, the mDST, the REMA and the MTT assays were 99.2%, 98.9%, 90.9%, 98.5%, and 100%, respectively. The specificity of the mDST was lower than those of the other four methods (P < 0.05). In conclusion, the MGIT 960 system, the MTT assay, and the REMA are superior to the PZase activity assay and the mDST in determining the susceptibility of M. tuberculosis to PZA. The MTT assay and the REMA might serve as alternative methods for clinical laboratories without access to the MGIT 960 system. For rapid testing in well-equipped laboratories, the mDST might be the best choice, particularly for small quantities of M. tuberculosis. The PZase activity assay has no obvious advantage in the assessment of M. tuberculosis susceptibility to PZA, as it is less accurate and requires larger quantities of bacteria.


Assuntos
Antituberculosos/farmacologia , Testes de Sensibilidade Microbiana/métodos , Mycobacterium tuberculosis/efeitos dos fármacos , Pirazinamida/farmacologia , Amidoidrolases/análise , Amidoidrolases/genética , Farmacorresistência Bacteriana , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Oxazinas/análise , Sais de Tetrazólio/análise , Tiazóis/análise , Xantenos/análise
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