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1.
J Cell Mol Med ; 24(2): 1541-1552, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31793207

RESUMO

Both PNPLA3 I148M and hepatic inflammation are associated with nonalcoholic fatty liver disease (NAFLD) progression. This study aimed to elucidate whether PNPLA3 I148M is involved in NF-kB-related inflammation regulation in NAFLD. HepG2 cells homozygous for the PNPLA3 I148M mutation were used. The human PNPLA3 promoter sequence was screened for NF-kB binding sites using the MATCH and PATCH tools. NF-kB-mediated transcriptional regulation of the PNPLA3 gene was assessed by luciferase reporter assay, EMSA and ChIP-qPCR. Wild-type (I148I) and mutant (M148M) PNPLA3 were overexpressed using stable lentivirus-mediated transfection. The pCMV vector and siRNA were transiently transfected into cells to direct NF-kB overexpression and PNPLA3 silencing, respectively. A putative NF-kB binding site in the human PNPLA3 promoter was shown to be necessary for basal and NF-kB-driven transcriptional activation of PNPLA3 and protein/DNA complex formation. Supershift analysis demonstrated a protein/DNA complex specifically containing the NF-kB p65 and p50 subunits. ChIP-qPCR confirmed the endogenous binding of NF-kB to the human PNPLA3 promoter in response to NF-kB overexpression and palmitic acid (PA) challenge. The silencing of PNPLA3 blocked the overexpression of NF-kB or PA-induced TNF-α up-regulation. Moreover, mutant PNPLA3 overexpression prevented NF-kB inhibitor-induced down-regulation of TNF-α expression in PA-treated HepG2 cells. Finally, the overexpression of mutant but not wild-type PNPLA3 increased TNF-α expression and activated the ER stress-mediated and NF-kB-independent inflammatory IRE-1α/JNK/c-Jun pathway. Human PNPLA3 was shown to be a target of NF-kB, and PNPLA3 I148M mediated the regulatory effect of NF-kB on inflammation in PA-treated HepG2 cells, most likely via the IRE-1α/JNK/c-Jun ER stress pathway.

2.
BMJ Open ; 9(8): e025378, 2019 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-31455696

RESUMO

OBJECTIVE: The limited existing asthma control questionnaires that are available for children 5 years of age or younger in China mostly assess only the impairment domain of asthma control. Here, the English version of the Test for Respiratory and Asthma Control in Kids (TRACK) was translated into Chinese and validated for its application in asthma control in preschool children. DESIGN: Prospective validation study. SETTING AND PARTICIPANTS: A total of 321 Chinese preschool children suffering from asthma completed the study from December 2017 to February 2018. METHOD: The TRACK translation into Chinese employed the translation and back translation technique. The caregivers of the preschool children with asthma symptoms completed TRACK during two clinical visits over 4-6 weeks. Moreover, the physicians completed a Global Initiative for Asthma (GINA)-based asthma control survey at both visits. The utility of TRACK for assessing the change in asthma control status and its reliability and discriminant validity were evaluated. RESULTS: The Chinese version of TRACK showed internal consistency reliability values of 0.63 and 0.71 at each visit, respectively (Cronbach's α). The test-retest reliability was 0.62 for individuals whose GINA-based assessment results were the same at both visits (n=206). The TRACK scores for the children in the various asthma control categories were significantly different (p<0.001). Children recommended for increased treatment by the physicians had lower TRACK scores than those recommended for no change in treatment or decreased treatment (p<0.001). CONCLUSION: The study verifies the validity and reliability of the Chinese version of TRACK. Changes in the TRACK scores effectively reflected the level of asthma control in preschool children and guided further treatment strategies. TRIAL REGISTRATION NUMBER: NCT02649803.

3.
Artigo em Inglês | MEDLINE | ID: mdl-30988144

RESUMO

Chlorhexidine gluconate (CHG) is a topical antiseptic widely used in health care settings. In Staphylococcus spp., the pump QacA effluxes CHG, while the closely related QacB cannot due to a single amino acid substitution. We characterized 1,050 cutaneous Staphylococcus isolates obtained from 173 pediatric oncology patients enrolled in a multicenter CHG bathing trial. CHG susceptibility testing revealed that 63 (6%) of these isolates had elevated CHG MICs (≥4 µg/ml). Screening of all 1,050 isolates for the qacA/B gene (the same qac gene with A or B allele) by restriction fragment length polymorphism (RFLP) yielded 56 isolates with a novel qacA/B RFLP pattern, qacA/B273 The CHG MIC was significantly higher for qacA/B273 -positive isolates (MIC50, 4 µg/ml; MIC range, 0.5 to 4 µg/ml) than for other qac groups: qacA-positive isolates (n = 559; MIC50, 1 µg/ml; MIC range, 0.5 to 4 µg/ml), qacB-positive isolates (n = 17; MIC50, 1 µg/ml; MIC range, 0.25 to 2 µg/ml), and qacA/B-negative isolates (n = 418, MIC50, 1 µg/ml; MIC range, 0.125 to 2 µg/ml) (P = 0.001). A high proportion of the qacA/B273 -positive isolates also displayed methicillin resistance (96.4%) compared to the other qac groups (24.9 to 61.7%) (P = 0.001). Whole-genome sequencing revealed that qacA/B273 -positive isolates encoded a variant of QacA with 2 amino acid substitutions. This new allele, named qacA4, was carried on the novel plasmid pAQZ1. The qacA4-carrying isolates belonged to the highly resistant Staphylococcus epidermidis sequence type 2 clone. By searching available sequence data sets, we identified 39 additional qacA4-carrying S. epidermidis strains from 5 countries. Curing an isolate of qacA4 resulted in a 4-fold decrease in the CHG MIC, confirming the role of qacA4 in the elevated CHG MIC. Our results highlight the importance of further studying qacA4 and its functional role in clinical staphylococci.

4.
mSphere ; 3(2)2018.
Artigo em Inglês | MEDLINE | ID: mdl-29564400

RESUMO

Clinical isolates of Pseudomonas aeruginosa from patients with cystic fibrosis (CF) are known to differ from those associated with non-CF hosts by colony morphology, drug susceptibility patterns, and genomic hypermutability. Pseudomonas aeruginosa isolates from CF patients have long been recognized for their overall reduced rate of antimicrobial susceptibility, but their intraclonal MIC heterogeneity has long been overlooked. Using two distinct cohorts of clinical strains (n = 224 from 56 CF patients, n = 130 from 68 non-CF patients) isolated in 2013, we demonstrated profound Etest MIC heterogeneity in CF P. aeruginosa isolates in comparison to non-CF P. aeruginosa isolates. On the basis of whole-genome sequencing of 19 CF P. aeruginosa isolates from 9 patients with heterogeneous MICs, the core genome phylogenetic tree confirmed the within-patient CF P. aeruginosa clonal lineage along with considerable coding sequence variability. No extrachromosomal DNA elements or previously characterized antibiotic resistance mutations could account for the wide divergence in antimicrobial MICs between P. aeruginosa coisolates, though many heterogeneous mutations in efflux and porin genes and their regulators were present. A unique OprD sequence was conserved among the majority of isolates of CF P. aeruginosa analyzed, suggesting a pseudomonal response to selective pressure that is common to the isolates. Genomic sequence data also suggested that CF pseudomonal hypermutability was not entirely due to mutations in mutL, mutS, and uvr. We conclude that the net effect of hundreds of adaptive mutations, both shared between clonally related isolate pairs and unshared, accounts for their highly heterogeneous MIC variances. We hypothesize that this heterogeneity is indicative of the pseudomonal syntrophic-like lifestyle under conditions of being "locked" inside a host focal airway environment for prolonged periods. IMPORTANCE Patients with cystic fibrosis endure "chronic focal infections" with a variety of microorganisms. One microorganism, Pseudomonas aeruginosa, adapts to the host and develops resistance to a wide range of antimicrobials. Interestingly, as the infection progresses, multiple isogenic strains of P. aeruginosa emerge and coexist within the airways of these patients. Despite a common parental origin, the multiple strains of P. aeruginosa develop vastly different susceptibility patterns to actively used antimicrobial agents-a phenomenon we define as "heterogeneous MICs." By sequencing pairs of P. aeruginosa isolates displaying heterogeneous MICs, we observed widespread isogenic gene lesions in drug transporters, DNA mismatch repair machinery, and many other structural or cellular functions. Coupled with the heterogeneous MICs, these genetic lesions demonstrated a symbiotic response to host selection and suggested evolution of a multicellular syntrophic bacterial lifestyle. Current laboratory standard interpretive criteria do not address the emergence of heterogeneous growth and susceptibilities in vitro with treatment implications.

5.
Minerva Pediatr ; 70(5): 438-443, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28922909

RESUMO

BACKGROUND: We explored the correlation between the TGFBR2 gene that is mediated by NF-κb signaling pathways and the pathogenesis of Kawasaki disease in children. METHODS: In this study, 43 children with Kawasaki disease from April 2014 to January 2016 at our hospital were selected as the observation group, and 42 healthy children were selected as the control group. The mRNA expression levels of NF-κb gene and TGFBR2 gene in different groups were detected using fluorescence quantitative PCR. The protein expression levels of the NF-κb and TGFBR2 were detected using enzyme-linked immunosorbent assay (ELISA) in different groups. The expression levels of NF-κb and TGFBR2 in the observation group and the control group were detected using immunohistochemistry. RESULTS: Compared to the control group, the mRNA expression levels of NF-κb and TGFBR2 were 12.3 times and 27.5 times as high as those in the control group respectively and there were significant differences between the two groups (P<0.05). ELISA results showed that there were significant differences between the protein expression levels of NF-κb and TGFBR2 in the control group (0.87±0.12, 1.25±0.18 µg/L) and those in the observation group (3.27±0.17, 8.16±0.22 µg/L) (P<0.05). Western-blotting results showed that the expression levels of the NF-κB and TGFBR2 in children with Kawasaki disease were significantly higher than those in healthy subjects (P<0.05). Immunohistochemistry results showed that the positive cell rate of TGFBR2 (89.7%) was significantly higher in children with Kawasaki disease than that in healthy children (4.5%); there was significant difference between the two groups (P<0.05). CONCLUSIONS: The TGFBR2 may be involved in the pathogenesis of Kawasaki disease in children through NF-κb signaling pathways.


Assuntos
Síndrome de Linfonodos Mucocutâneos/fisiopatologia , NF-kappa B/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II/genética , Transdução de Sinais/genética , Adolescente , Estudos de Casos e Controles , Criança , Pré-Escolar , Ensaio de Imunoadsorção Enzimática , Feminino , Regulação da Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Síndrome de Linfonodos Mucocutâneos/genética , NF-kappa B/genética , RNA Mensageiro/metabolismo
6.
Pediatr Pulmonol ; 52(8): 1020-1028, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28608624

RESUMO

AIM: Cystic fibrosis (CF) is an extremely rare disease in Asians. Here, we report four Chinese children with CF and review the literature about Chinese CF patients. METHODS: The cystic fibrosis transmembrane conductance regulator (CFTR) gene testing was performed on four suspected patients for CF screening. We also reviewed the literature about Chinese CF patients from 1970s. The clinical data of all these CF patients were summarized. RESULTS: We diagnosed four CF patients who had mutations in the CFTR gene. We identified six different mutations in the four patients. The c.1766+5G>T, c.595C>T, c.2909G>A, and c.4056G>C had been reported already. The two splicing mutations of c.579+1_579+2insACAT and c.1117-1G>C were novel mutations. There have been 46 Chinese CF patients reported in literature from 1974 up to present (2016.12). The clinical manifestations of CF involved several systems. The most common symptom was recurrent pulmonary infections. Thirty-three different mutations were identified; c.1766 + 5G>T was the most common mutation among Chinese CF patients. Only one of these mutations (R553X) was in the Caucasian CF screening panel. The spectrum of CFTR mutations in Chinese was highly different from that of Caucasian. CONCLUSIONS: There was a high risk of misdiagnosis or delayed diagnosis of CF even in suspected cases in China. It is necessary to educate Chinese clinicians about the signs, symptoms, and diagnosis of cystic fibrosis and promote the implementation of the sweat chloride test.


Assuntos
Grupo com Ancestrais do Continente Asiático/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/diagnóstico , Fibrose Cística/genética , Adolescente , Criança , Pré-Escolar , Grupo com Ancestrais do Continente Europeu/genética , Feminino , Humanos , Masculino , Mutação
7.
J Cell Physiol ; 230(9): 2224-32, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25655569

RESUMO

Patatin-like phospholipase domain containing 3 (PNPLA3) is a non-secreted protein primarily expressed in liver and adipose tissue. Recently, numerous genetic studies have shown that PNPLA3 is a major susceptibility gene for nonalcoholic fatty liver disease (NAFLD). However, the mechanism involved in transcriptional regulation of the PNPLA3 gene remains unknown. We performed a detailed analysis of the human PNPLA3 gene promoter and identified two novel cis-acting elements (SRE and NFY binding motifs) located at -97/-88 and -26/-22 bp, respectively. Overexpression of SREBP-1c in HepG2 cells significantly increased PNPLA3 promoter activity. Mutation of either of the putative SRE or NFY binding motifs blocked the transactivation effects of SREBP-1c on the promoter. Overexpression of SREBP-1c and NFY together increased PNPLA3 promoter activity twice as much as that of SREBP-1c or NFY expression alone. This result suggests that SREBP-1c and NFY synergistically transactivate the human PNPLA3 gene. The ability of SREBP-1c and NFY to bind these cis-elements was confirmed using gel shift analysis. Putative SRE and NFY motifs also mediated synergistic insulin-induced transactivation of the PNPLA3 promoter in HepG2 cells. Additionally, the ability of SREBP-1c to bind to the PNPLA3 promoter was increased by insulin in a dose-dependent manner. Moreover, the treatment of HepG2 cells with the PI3K inhibitor LY294002 led to reduced insulin promoter-activating ability accompanied by a decrease in PNPLA3 and SREBP-1c protein expression. These results demonstrate that SREBP-1c is a direct activator of the human PNPLA3 gene and insulin transactivates the PNPLA3 gene via the PI3K-SREBP-1c/NFY pathway in HepG2 cells.


Assuntos
Lipase/genética , Proteínas de Membrana/genética , Hepatopatia Gordurosa não Alcoólica/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Ativação Transcricional/genética , Fator de Ligação a CCAAT/biossíntese , Regulação da Expressão Gênica/genética , Células Hep G2 , Humanos , Insulina/metabolismo , Lipase/biossíntese , Fígado/metabolismo , Fígado/patologia , Proteínas de Membrana/biossíntese , Mutação , Hepatopatia Gordurosa não Alcoólica/patologia , Regiões Promotoras Genéticas , Ligação Proteica , Proteína de Ligação a Elemento Regulador de Esterol 1/biossíntese
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