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1.
J Pathol ; 248(2): 155-163, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30680725

RESUMO

The cellular mechanisms by which hepatitis C virus (HCV) replication might mediate cytopathic effects are controversial and not entirely clear. In this study, we found that blood-borne HCV (bbHCV) infection could lead to endoplasmic reticulum (ER)-stress and mitochondria-related/caspase-dependent apoptosis at the early stages of infection based on use of the highly efficient bbHCV cell culture model established previously. Sections of bbHCV-infected human fetal liver stem cells (hFLSCs) revealed convolution and nonlinear ER, cell vacuolization, swelling of mitochondria, and numerous double membrane vesicles (DMVs). The percentage of apoptotic hFLSCs infected by bbHCV reached 29.8% at 16 h postinfection, and the amount of cytochrome c increased remarkably in the cytosolic protein fraction. However, over time, apoptosis was inhibited due to the activation of NF-κB. The expression of NF-κB-p65, Bcl-xL, XIAP, and c-FLIPL in hFLSCs was increased significantly 24 h after in infection by bbHCV. The accelerated cell death cycles involving apoptosis, regeneration and repair by bbHCV infection might give rise to the development of cirrhosis, and ultimately to hepatocellular carcinogenesis. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Assuntos
Apoptose , Estresse do Retículo Endoplasmático , Células-Tronco Fetais/patologia , Hepacivirus/crescimento & desenvolvimento , Hepatite C Crônica/virologia , Fígado/patologia , Replicação Viral , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular , Células-Tronco Fetais/metabolismo , Células-Tronco Fetais/virologia , Hepatite C Crônica/metabolismo , Hepatite C Crônica/patologia , Humanos , Fígado/metabolismo , Fígado/virologia , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Hepáticas/patologia , Mitocôndrias Hepáticas/virologia , Estresse Oxidativo , Transdução de Sinais
2.
Hepatology ; 66(4): 1045-1057, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28407288

RESUMO

The development of pathogenic mechanisms, specific antiviral treatments and preventive vaccines for hepatitis C virus (HCV) infection has been limited due to lack of cell culture models that can naturally imitate the entire HCV life cycle. Here, we established an HCV cell culture model based on human fetal liver stem cells (hFLSCs) that supports the entire blood-borne hepatitis C virus (bbHCV) life cycle. More than 90% of cells remained infected by various genotypes. bbHCV was efficiently propagated, and progeny virus were infectious to hFLSCs. The virus could be passed efficiently between cells. The viral infectivity was partially blocked by specific antibodies or small interfering RNA against HCV entry factors, whereas HCV replication was inhibited by antiviral drugs. We observed viral particles of approximately 55 nm in diameter in both cell culture media and infected cells after bbHCV infection. CONCLUSION: Our data show that the entire bbHCV life cycle could be naturally imitated in hFLSCs. This model is expected to provide a powerful tool for exploring the process and the mechanism of bbHCV infection at the cellular level and for evaluating the treatment and preventive strategies of bbHCV infection. (Hepatology 2017;66:1045-1057).


Assuntos
Células-Tronco Fetais , Hepacivirus/fisiologia , Fígado/citologia , Modelos Biológicos , Replicação Viral , Humanos , Fígado/virologia , Cultura Primária de Células , Proteínas Virais/biossíntese , Liberação de Vírus
3.
Environ Sci Technol ; 48(12): 6947-56, 2014 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-24865258

RESUMO

Exposure to various infectious viruses in environmental drinking water can constitute a public health risk. However, it is difficult to detect viruses in water due to their low concentration. In this study, we have developed a novel filter cartridge system containing electropositive granule media (EGM). Viruses present in large volumes of environmental samples were adsorbed onto the EGM, and then recovered by elution and poly(ethylene glycol) (PEG) concentration. To evaluate the system's efficiency in viral recovery, poliovirus (PV-1), a surrogate for enteric viruses, was used to artificially contaminate river water samples which were then assayed by quantitative real-time PCR. To optimize the concentration procedure, the eluent type, water flow rate and properties (e.g., pH, bacterial, and viral loads), were evaluated. The highest virus recovery was obtained by pumping river water at a flow rate of 300 mL/min and then pushing 3 L of an eluent containing 3× broth [1.5% (w/v) NaCl, 3% (w/v) tryptone, 1.5% (w/v) beef powder] with 0.05 mol/L glycine through the filter. Using this procedure, the recovery efficiencies of PV-1 from 10 to 100 L of spiked river water were up to 99%. In addition, this method is virus load and pH dependent. Virus recovery was maximal at a load of between 10(3.5) and 10(5.5) TCID50 and a pH ranging from 5 to 7. The bacterial load in the water has no effect on virus recovery. Different types of viruses and surface water were tested to validate the system's applicability. Results revealed that the EGM filter cartridge was able to concentrate PV-1, human adenoviruses (HAdVs) and noroviruses (HuNoVs) with high efficiency from river, lake, and reservoir water. Furthermore, it showed more efficient recovery than glass wool and 1MDS filters. These data suggest that this system provides rapid and efficient virus recovery from a large volume of natural surface water and, as such, could be a useful tool in revealing the presence of viruses in surface water.


Assuntos
Filtração/instrumentação , Filtração/métodos , Vírus/isolamento & purificação , Microbiologia da Água , Adsorção , Óxido de Alumínio/química , Animais , Linhagem Celular , Precipitação Química , Eletrodos , Escherichia coli/crescimento & desenvolvimento , Humanos , Concentração de Íons de Hidrogênio , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Reologia , Rios/virologia , Vírus/genética , Qualidade da Água
4.
J Med Virol ; 84(3): 526-35, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22246842

RESUMO

Chemical disinfection is the most common method used to inactivate viruses from drinking water throughout the world. In this study, cell culture, ELISA, RT-PCR, and spot hybridization were employed to investigate the mechanism underlying chlorine dioxide (ClO(2) )-induced inactivation of Poliovirus type 1 (PV1), which was also confirmed by recombinant viral genome RNA infection models. The results suggested that ClO(2) inactivated PV1 primarily by disrupting the 5'-non-coding region (5'-NCR) of the PV1 genome. Further study revealed that ClO(2) degraded specifically the 40-80 nucleotides (nt) region in the 5'-NCR. Recombinant viral genome RNA infection models confirmed that PV1 RNA lacking this 40-80 nt region was not infectious. This study not only elucidated the mechanism of PV1 inactivation by ClO(2), but also defined the critical genetic target for the disinfectant to inactivate Poliovirus. This study also provides a strategy by which rapid, accurate, and molecular methods based on sensitive genetic targets may be established for evaluating the effects of disinfectants on viruses.


Assuntos
Regiões 5' não Traduzidas , Compostos Clorados/farmacologia , Desinfetantes/farmacologia , Genoma Viral , Óxidos/farmacologia , Poliovirus/efeitos dos fármacos , Poliovirus/genética , Inativação de Vírus/efeitos dos fármacos , Desinfecção , Células HeLa , Humanos , Poliovirus/imunologia
5.
Environ Sci Technol ; 46(24): 13448-54, 2012 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-23215020

RESUMO

Antibiotic resistance poses a significant challenge to human health and its rate continues to rise globally. While antibiotic-selectable synthetic plasmid vectors have proved invaluable tools of genetic engineering, this class of artificial recombinant DNA sequences with high expression of antibiotic resistance genes presents an unknown risk beyond the laboratory setting. Contamination of environmental microbes with synthetic plasmid vector-sourced antibiotic resistance genes may represent a yet unrecognized source of antibiotic resistance. In this study, PCR and real-time quantitative PCR were used to investigate the synthetic plasmid vector-originated ampicillin resistance gene, ß-lactam antibiotic (blá), in microbes from six Chinese rivers with significant human interactions. Various levels of blá were detected in all six rivers, with the highest levels in the Pearl and Haihe rivers. To validate the blá pollution, environmental plasmids in the river samples were captured by the E. coli transformants from the community plasmid metagenome. The resultant plasmid library of 205 ampicillin-resistant E. coli (transformants) showed a blá-positive rate of 27.3% by PCR. Sequencing results confirmed the synthetic plasmid vector sources. In addition, results of the Kirby-Bauer disc-diffusion test reinforced the ampicillin-resistant functions of the environmental plasmids. The resistance spectrum of transformants from the Pearl and Haihe rivers, in particular, had expanded to the third- and fourth-generation of cephalosporin drugs, while that of other transformants mainly involved first- and second-generation cephalosporins. This study not only reveals environmental contamination of synthetic plasmid vector-sourced blá drug resistance genes in Chinese rivers, but also suggests that synthetic plasmid vectors may represent a source of antibiotic resistance in humans.


Assuntos
Coleta de Dados , Genes Bacterianos/genética , Vetores Genéticos/genética , Plasmídeos/genética , Rios , Resistência beta-Lactâmica/genética , Antibacterianos/farmacologia , Sequência de Bases , China , DNA Recombinante/genética , Poluição Ambiental/análise , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Geografia , Humanos , Metagenoma/genética , Reação em Cadeia da Polimerase , Reprodutibilidade dos Testes , Análise de Sequência de DNA
6.
Front Microbiol ; 12: 604245, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33613479

RESUMO

Given its excellent performance against the pathogens, UV disinfection has been applied broadly in different fields. However, only limited studies have comprehensively investigated the response of bacteria surviving UV irradiation to the environmental antibiotic stress. Here, we investigated the antibiotic susceptibility of Pseudomonas aeruginosa suffering from the UV irradiation. Our results revealed that UV exposure may decrease the susceptibility to tetracycline, ciprofloxacin, and polymyxin B in the survival P. aeruginosa. Mechanistically, UV exposure causes oxidative stress in P. aeruginosa and consequently induces dysregulation of genes contributed to the related antibiotic resistance genes. These results revealed that the insufficient ultraviolet radiation dose may result in the decreased antibiotic susceptibility in the pathogens, thus posing potential threats to the environment and human health.

7.
ISME J ; 14(7): 1847-1856, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32327733

RESUMO

Chlorine disinfection to drinking water plays an important role in preventing and controlling waterborne disease outbreaks globally. Nevertheless, little is known about why it enriches the antibiotic resistance genes (ARGs) in bacteria after chlorination. Here, ARGs released from killed antibiotic-resistant bacteria (ARB), and culturable chlorine-injured bacteria produced in the chlorination process as the recipient, were investigated to determine their contribution to the horizontal transfer of ARGs during disinfection treatment. We discovered Escherichia coli, Salmonella aberdeen, Pseudomonas aeruginosa and Enterococcus faecalis showed diverse resistance to sodium hypochlorite, and transferable RP4 could be released from killed sensitive donor consistently. Meanwhile, the survival of chlorine-tolerant injured bacteria with enhanced cell membrane permeabilisation and a strong oxidative stress-response demonstrated that a physiologically competent cell could be transferred by RP4 with an improved transformation frequency of up to 550 times compared with the corresponding untreated bacteria. Furthermore, the water quality factors involving chemical oxygen demand (CODMn), ammonium nitrogen and metal ions (Ca2+ and K+) could significantly promote above transformation frequency of released RP4 into injured E. faecalis. Our findings demonstrated that the chlorination process promoted the horizontal transfer of plasmids by natural transformation, which resulted in the exchange of ARGs across bacterial genera and the emergence of new ARB, as well as the transfer of chlorine-injured opportunistic pathogen from non-ARB to ARB. Considering that the transfer elements were quite resistant to degradation through disinfection, this situation poses a potential risk to public health.


Assuntos
Cloro , Desinfecção , Antagonistas de Receptores de Angiotensina , Inibidores da Enzima Conversora de Angiotensina , Antibacterianos/farmacologia , Bactérias/genética , Cloro/farmacologia , Farmacorresistência Bacteriana , Resistência Microbiana a Medicamentos , Genes Bacterianos
8.
J Hazard Mater ; 365: 340-345, 2019 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-30448547

RESUMO

Antibiotic resistance genes (ARGs) have gained global attention due to their public health threat. Extracelluar ARGs (eARGs) can result in the dissemination of antibiotic resistance via free-living ARGs in natural environments, where they promote ARB transmission in drinking water distribution systems. However, eARG pollution in tap water has not been well researched. In this study, concentrations of eARGs and intracellular ARGs (iARGs) in tap water, sampled at Tianjin, China, were investigated for one year. Fourteen eARG types were found at the highest concentration of 1.3 × 105 gene copies (GC)/L. TetC was detected in 66.7% of samples, followed by sul1, sul2, and qnrA with the same detection frequency of 41.7%. Fifteen iARGs (including tetA, tetB, tetM, tetQ, tetX, sul1, sul2, sul3, ermB, blaTEM, and qnrA) were continuously detected in all collected tap water samples with sul1 and sul2 the most abundant. Additionally, both eARG and iARG concentrations in tap water presented a seasonal pattern with most abundant prevalence in summer. The concentration of observed intracellular sulfonamide resistance genes showed a significantly positive correlation with total nitrogen concentrations. This study suggested that eARG and iARG pollution of drinking water systems pose a potential risk to human public health.


Assuntos
Água Potável/microbiologia , Resistência Microbiana a Medicamentos/genética , Genes Bacterianos , Microbiologia da Água , China , Monitoramento Ambiental
9.
Water Res ; 156: 366-371, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30928530

RESUMO

Adaption to adverse environments plays an important role in bacterial survival and is receiving increasing globe attention now. Here, cultivable chlorine-injured Pseudomonas aeruginosa, produced on the chlorination process, was investigated about their resistance to antibiotics. Then, global transcriptional analyses, quantitative PCR (qPCR) validation and antioxidant enzymes measurement were performed to explore the underlying mechanisms. The results showed that chlorine injury enhanced antibiotic resistance in P. aeruginosa and cultivable chlorine-injured P. aeruginosa exposed to 4 mg/L sodium hypochlorite (half of the lethal dose) improved antibiotic resistance against ceftazidime, chloramphenicol and ampicillin by 1.4-5.6 fold. This increase in antibiotic resistance was not hereditable and over expression of the MexEF-OprN efflux pump resulting from oxidative stress contributed to it. These results demonstrate temporal physiological persistence to antibiotics in cultivable chlorine-injured pathogens, suggesting their survival from adverse environments with antibiotic exposure and thereby posing lasting hazards to human health.


Assuntos
Cloro , Pseudomonas aeruginosa , Antibacterianos , Cloranfenicol , Resistência Microbiana a Medicamentos , Humanos
10.
Biomed Environ Sci ; 32(5): 324-333, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31217049

RESUMO

OBJECTIVE: To investigate the mechanisms underlying ozone-induced inactivation of poliovirus type 1 (PV1). METHODS: We used cell culture, long-overlapping RT-PCR, and spot hybridization assays to verify and accurately locate the sites of action of ozone that cause PV1 inactivation. We also employed recombinant viral genome RNA infection models to confirm our observations. RESULTS: Our results indicated that ozone inactivated PV1 primarily by disrupting the 5'-non-coding region (5'-NCR) of the PV1 genome. Further study revealed that ozone specifically damaged the 80-124 nucleotide (nt) region in the 5'-NCR. Recombinant viral genome RNA infection models confirmed that PV1 lacking this region was non-infectious. CONCLUSION: In this study, we not only elucidated the mechanisms by which ozone induces PV1 inactivation but also determined that the 80-124 nt region in the 5'-NCR is targeted by ozone to achieve this inactivation.


Assuntos
Genoma Viral/efeitos dos fármacos , Oxidantes Fotoquímicos/farmacologia , Ozônio/farmacologia , Poliovirus/efeitos dos fármacos , Inativação de Vírus , Regiões 5' não Traduzidas , Animais , Chlorocebus aethiops , Células Vero
11.
BMC Cancer ; 8: 383, 2008 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-19099597

RESUMO

BACKGROUND: Epidemiological and in vitro studies suggest that antioxidants such as quercetin and vitamin E (VE) can prevent lung tumor caused by smoking; however, there is limited evidence from animal studies. METHODS: In the present study, Swiss mouse was used to examine the potential of quercetin and VE for prevention lung tumor induced by smoking. RESULTS: Our results suggest that the incidence of lung tumor and tumor multiplicity were 43.5% and 1.00 +/- 0.29 in smoking group; Quercetin has limited effects on lung tumor prevention in this in vivo model, as measured by assays for free radical scavenging, reduction of smoke-induced DNA damage and inhibition of apoptosis. On the other hand, vitamin E drastically decreased the incidence of lung tumor and tumor multiplicity which were 17.0% and 0.32 +/- 0.16, respectively (p < 0.05); and demonstrated prominent antioxidant effects, reduction of DNA damage and decreased cell apoptosis (p < 0.05). Combined treatment with quercetin and VE in this animal model did not demonstrate any effect greater than that due to vitamin E alone. In addition, gender differences in the occurrence of smoke induced-lung tumor and antioxidant intervention were also observed. CONCLUSION: We conclude that VE might prevent lung tumor induced by smoking in Swiss mice.


Assuntos
Antioxidantes/farmacologia , Neoplasias Pulmonares/prevenção & controle , Pulmão/efeitos dos fármacos , Quercetina/farmacologia , Fumar , Vitamina E/farmacologia , Análise de Variância , Animais , Apoptose/efeitos dos fármacos , Carcinógenos/toxicidade , Ensaio Cometa , Dano ao DNA/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Sequestradores de Radicais Livres/farmacologia , Radicais Livres/metabolismo , Pulmão/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Quercetina/administração & dosagem , Espécies Reativas de Oxigênio/sangue , Fatores Sexuais , Vitamina E/administração & dosagem
12.
Water Res ; 136: 131-136, 2018 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-29501757

RESUMO

The emergence and spread of antibiotic resistance has posed a major threat to both human health and environmental ecosystem. Although the disinfection has been proved to be efficient to control the occurrence of pathogens, little effort is dedicated to revealing potential impacts of disinfection on transmission of antibiotic resistance genes (ARGs), particularly for free-living ARGs in final disinfected effluent of urban wastewater treatment plants (UWWTP). Here, we investigated the effects of chlorine disinfection on the occurrence and concentration of both extracellular ARGs (eARGs) and intracellular ARGs (iARGs) in a full-scale UWWTP over a year. We reported that the concentrations of both eARGs and iARGs would be increased by the disinfection with chlorine dioxide (ClO2). Specifically, chlorination preferentially increased the abundances of eARGs against macrolide (ermB), tetracycline (tetA, tetB and tetC), sulfonamide (sul1, sul2 and sul3), ß-lactam (ampC), aminoglycosides (aph(2')-Id), rifampicin (katG) and vancomycin (vanA) up to 3.8 folds. Similarly, the abundances of iARGs were also increased up to 7.8 folds after chlorination. In terms of correlation analyses, the abundance of Escherichia coli before chlorination showed a strong positive correlation with the total eARG concentration, while lower temperature and higher ammonium concentration were assumed to be associated with the concentration of iARGs. This study suggests the chlorine disinfection could increase the abundances of both iARGs and eARGs, thereby posing risk of the dissemination of antibiotic resistance in environments.


Assuntos
Cloro/farmacologia , Desinfetantes/farmacologia , Farmacorresistência Bacteriana , Proteínas de Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Antibacterianos/farmacologia , Cloro/análise , Desinfetantes/análise , Desinfecção , Resistência Microbiana a Medicamentos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Halogenação , Tetraciclina/farmacologia , Águas Residuárias/microbiologia , Purificação da Água
13.
J Microbiol Methods ; 139: 143-149, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28577899

RESUMO

Bacteriophages are widely used to the treatment of drug-resistant bacteria and the improvement of food safety through bacterial lysis. However, the limited investigations on bacteriophage restrict their further application. In this study, a novel and highly efficient method was developed for isolating bacteriophage from water based on the electropositive silica gel particles (ESPs) method. To optimize the ESPs method, we evaluated the eluent type, flow rate, pH, temperature, and inoculation concentration of bacteriophage using bacteriophage f2. The quantitative detection reported that the recovery of the ESPs method reached over 90%. The qualitative detection demonstrated that the ESPs method effectively isolated 70% of extremely low-concentration bacteriophage (100 PFU/100L). Based on the host bacteria composed of 33 standard strains and 10 isolated strains, the bacteriophages in 18 water samples collected from the three sites in the Tianjin Haihe River Basin were isolated by the ESPs and traditional methods. Results showed that the ESPs method was significantly superior to the traditional method. The ESPs method isolated 32 strains of bacteriophage, whereas the traditional method isolated 15 strains. The sample isolation efficiency and bacteriophage isolation efficiency of the ESPs method were 3.28 and 2.13 times higher than those of the traditional method. The developed ESPs method was characterized by high isolation efficiency, efficient handling of large water sample size and low requirement on water quality.


Assuntos
Bacteriófagos/isolamento & purificação , Sílica Gel , Virologia/métodos , Microbiologia da Água , Adsorção , Bactérias/virologia , Técnicas Eletroquímicas/métodos , Filtração/métodos , Concentração de Íons de Hidrogênio , Rios/virologia , Temperatura , Qualidade da Água
14.
Environ Sci Process Impacts ; 19(5): 720-726, 2017 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-28406501

RESUMO

Underestimation of Escherichia coli in drinking water, an indicator microorganism of sanitary risk, may result in potential risks of waterborne diseases. However, the detection of disinfectant-injured or genetically modified (GM) E. coli has been largely overlooked so far. To evaluate the accuracy of culture-dependent enumeration with regard to disinfectant-injured and GM E. coli, chlorine- or ozone-injured wild-type (WT) and GM E. coli were prepared and characterized. Then, water samples contaminated with these E. coli strains were assayed by four widely used methods, including lactose tryptose broth-based multiple-tube fermentation (MTF), m-endo-based membrane filtration method (MFM), an enzyme substrate test (EST) known as Colilert, and Petrifilm-based testing slip method (TSM). It was found that MTF was the most effective method to detect disinfectant-injured WT E. coli (with 76.9% trials detecting all these bacteria), while this method could not effectively detect GM E. coli (with uninjured bacteria undetectable and a maximal detection rate of 21.5% for the injured). The EST was the only method which enabled considerable enumeration of uninjured GM E. coli, with a detection rate of over 93%. However, the detection rate declined to lower than 45.4% once the GM E. coli was injured by disinfectants. The MFM was invalid for both disinfectant-injured and GM E. coli. This is the first study to report the failure of these commonly used enumeration methods to simultaneously detect disinfectant-injured and GM E. coli. Thus, it highlights the urgent requirement for the development of a more accurate and versatile enumeration method which allows the detection of disinfectant-injured and GM E. coli on the assessment of microbial quality of drinking water.


Assuntos
Técnicas Bacteriológicas/métodos , Desinfetantes/toxicidade , Água Potável/microbiologia , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/genética , Microbiologia da Água/normas , Cloro/toxicidade , Água Potável/normas , Enterobacteriaceae/genética , Enterobacteriaceae/crescimento & desenvolvimento , Fermentação , Filtração , Ozônio/toxicidade , Sensibilidade e Especificidade , Qualidade da Água
15.
Water Res ; 92: 188-98, 2016 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-26854607

RESUMO

Extracellular antibiotic resistance genes (eARGs) that help in the transmission and spread of antibiotic-resistant bacteria are emerging environmental contaminants in water, and there is therefore a growing need to assess environmental levels and associated risks of eARGs. However, as they are present in low amounts, it is difficult to detect eARGs in water directly with PCR techniques. Here, we prepared a new type of nucleic acid adsorption particle (NAAP) with high capacity and developed an optimal adsorption-elution method to concentrate eARGs from large volumes of water. With this technique, we were able to achieve an eARG recovery rate of above 95% from 10 L of water samples. Moreover, combining this new method with quantitative real-time PCR (qPCR), the sensitivity of the eARG detection was 10(4) times that of single qPCR, with the detection limit lowered to 100 gene copies (GCs)/L. Our analyses showed that the eARG load, virus load and certain water characteristics such as pH, chemical oxygen demand (CODMn), and turbidity affected the eARGs recovery rate. However, high eARGs recovery rates always remained within the standard limits for natural surface water quality, while eARG levels in water were lower than the detection limits of single qPCR assays. The recovery rates were not affected by water temperature and heterotrophic plate counts (HPC). The eARGs whatever located in the plasmids or the short-length linear DNAs can be recovered from the water. Furthermore, the recovery rate was high even in the presence of high concentrations of plasmids in different natural water (Haihe river, well water, raw water for drinking water, Jinhe river, Tuanbo lake and the Yunqiao reservoir). By this technology, eARGs concentrations were found ranging from (2.70 ± 0.73) × 10(2) to (4.58 ± 0.47) × 10(4) GCs/L for the extracellular ampicillin resistance gene and (5.43 ± 0.41) × 10(2) to (2.14 ± 0.23) × 10(4) GCs/L for the extracellular gentamicin resistance gene in natural water for the first time, respectively. All these findings suggest that NAAPs have great potential for the monitoring of eARGs pollution in water.


Assuntos
Resistência Microbiana a Medicamentos/genética , Espaço Extracelular/química , Microbiologia da Água , Purificação da Água/métodos , Água/química , Adsorção , Análise da Demanda Biológica de Oxigênio , Precipitação Química , DNA/análise , Concentração de Íons de Hidrogênio , Ácidos Nucleicos/análise , Reprodutibilidade dos Testes , Reologia , Espectrometria por Raios X , Fatores de Tempo , Eliminação de Resíduos Líquidos , Poluentes Químicos da Água/análise
16.
Artigo em Zh | MEDLINE | ID: mdl-27255030

RESUMO

OBJECTIVE: To investigate the relationship between the changes of the copy numbers of mtDNA in peripheral blood mono-nucle- ar cell(PBMC) and the disordered of antioxidant capacity of hepatocellular carcinoma (HCC) patients. METHODS: The Ficoll Hypaque method was used to isolate the PBMC from blood specimens. The ND1 gene of the mitochondrial was amplified by real-time PCR; meantime ß-actin was served as a quantitative standard marker; the difference of mtDNA copy number in PBMC was compared between HCC and healthy control group. The level of reactive oxygen species (ROS) in PBMC was determined by flow cytometry. The change of total antioxidant capacity (T- AOC) of plasma was detected by the biochemistry examination. RESULTS: The copy numbers of ND1 gene in PBMC of HCC was 73% that of the healthy control group,which suggested a decrease of the copy numbers of mtDNA in HCC. The levels of ROS of PBMC in HCC was (417. 82 ± 110.62) and (301.82 ± 75.54) in control group, which showed that the levels of ROS of PBMC in HCC were significant higher than that in control group (P < 0.01).Plasma T-AOC in HCC was (1.30 ± 0.85), and (3.20 ± 1.62) in control. The T-AOC of plasma of HCC was significantly lower than in control group (P < 0.01). CONCLUSION: There was a certain relationship between the decrease of the copy numbers of mtDNA and the disordered antioxidant capacity in hepatocellular carcinoma, which may be associated with the development of hepatocellular carcinoma.


Assuntos
Carcinoma Hepatocelular/genética , Variações do Número de Cópias de DNA , DNA Mitocondrial/genética , Leucócitos Mononucleares/metabolismo , Neoplasias Hepáticas/genética , Espécies Reativas de Oxigênio/metabolismo , Actinas , Antioxidantes/metabolismo , Carcinoma Hepatocelular/sangue , Estudos de Casos e Controles , Humanos , Neoplasias Hepáticas/sangue
17.
Zhongguo Zhong Yao Za Zhi ; 30(9): 653-8, 2005 May.
Artigo em Zh | MEDLINE | ID: mdl-16075725

RESUMO

The function for cardiac vascular system of taurine is extensive, and the mechanism is complicated. Taurine protects the cells from the cell injury caused by ischemia etc. Through repressing apoptosis, prevents endothelial dysfunction caused by hyperglycemia, hypercholesterolemia, smoking and homocysteine; suppresses the proliferation and calcification in vascular smooth muscle cells, promotes metabolization and excretion of cholesterol in the animal models of hyperlipemia, and confers the resistance to an oxidant, hypochlorous acid, produced by neutrophil on cells, and taurine chrolamine to inhibit activation of NF-kappaB, which might be associated with anti-atherosclerotic effect. Taurine mainly acts inside the cell. However, taurine transport system becomes aberrant in pathological myocardial and vascular tissue. In addition, taurine improves cardiovascular function in fructose-induced hypertension and an iron-overload murine animal models.


Assuntos
Apoptose/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Materia Medica/farmacologia , Músculo Liso Vascular/citologia , Taurina/farmacologia , Animais , Antioxidantes/farmacologia , Proliferação de Células/efeitos dos fármacos , Humanos , Miócitos Cardíacos/patologia
18.
Rejuvenation Res ; 18(3): 211-24, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25556695

RESUMO

Pluripotent human hepatic stem cells have broad research and clinical applications, which are, however, restricted by both limited resources and technical difficulties with respect to isolation of stem cells from the adult or fetal liver. In this study, we developed a convenient and efficient method involving a two-step in situ collagenase perfusion, gravity sedimentation, and Percoll density gradient centrifugation to enrich and maintain highly proliferative human fetal liver stem cells (hFLSCs). Using this method, the isolated hFLSCs entered into the exponential growth phase within 10 days and maintained sufficient proliferative activity to permit subculture for at least 20 passages without differentiation. Immunocytochemistry, immunofluorescence, and flow cytometry results showed that these cells expressed stem cell markers, such as c-kit, CD44, epithelial cell adhesion molecule (EpCAM), oval cell marker-6 (OV-6), epithelial marker cytokeratin 18 (CK18), biliary ductal marker CK19, and alpha-fetoprotein (AFP). Gene expression analysis showed that these cells had stable mRNA expression of c-Kit, EpCAM, neural cell adhesion molecule (NCAM), CK19, CK18, AFP, and claudin 3 (CLDN-3) throughout each passage while maintaining low levels of ALB, but with complete absence of cytochrome P450 3A4 (C3A4), phosphoenolpyruvate carboxykinase (PEPCK), telomeric repeat binding factor (TRF), and connexin 26 (CX26) expression. When grown in appropriate medium, these isolated liver stem cells could differentiate into hepatocytes, cholangiocytes, osteoblasts, adipocytes, or endothelial cells. Thus, we have demonstrated a more economical and efficient method to isolate hFLSCs than magnetic-activated cell sorting (MACS). This novel approach may provide an excellent tool to isolate highly proliferative hFLSCs for tissue engineering and regenerative therapies.


Assuntos
Biomarcadores/metabolismo , Diferenciação Celular , Proliferação de Células , Células-Tronco Fetais/citologia , Feto/citologia , Hepatócitos/citologia , Fígado/citologia , Adulto , Moléculas de Adesão Celular/metabolismo , Técnicas de Cultura de Células , Células Cultivadas , Conexina 26 , Conexinas , Feminino , Células-Tronco Fetais/metabolismo , Feto/metabolismo , Citometria de Fluxo , Imunofluorescência , Perfilação da Expressão Gênica , Hepatócitos/metabolismo , Humanos , Técnicas Imunoenzimáticas , Imunofenotipagem , Fígado/metabolismo , Fenótipo , Gravidez , Segundo Trimestre da Gravidez , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
19.
World J Gastroenterol ; 20(24): 7955-63, 2014 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-24976732

RESUMO

AIM: To investigate the seroprevalence and evolutionary dynamics of hepatitis E virus (HEV) and assess the ancestor of HEVs in China's Shandong Province. METHODS: A total of 2028 serum, 60 fecal and 82 bile samples were collected from the general human population, patients and swine, respectively. This seroepidemiological study was conducted using an immunnosorbent assay and HEV RNA was detected by the reverse transcription-nested polymerase chain reaction (RT-nPCR) method. Complete genome sequences of the prevalent strains (CH-YT-HEV01, CH-YT-HEV02 and CH-YT-sHEV01) were determined, and the sequences were analyzed phylogenetically. In addition, the evolutionary dynamics of three HEV isolates were determined using the framework of coalescent analysis in the program package BEAST, and the time of the most recent common ancestors (TMRCAs) of China-indigenous genotype 4 HEV isolates was calculated. RESULTS: The overall viral burden in the general human population was 0.1%, and the positive rates of anti-HEV IgG and IgM in the serum specimens were 25.1% (509/2028) and 2.3% (51/2028), respectively. In addition, IgG positivity increased with age. The phylogenetic analysis based on the full-length nucleotide sequences showed that the strain CH-YT-HEV02 was directly related to CH-YT-sHEV01 with a 94% identity, suggesting that they were involved in cross-species transmission. The isolate CH-YT-HEV01 was close to HB-3 and CHN-SD-sHEV with a bootstrap value of 100%, sharing a 96.1%-96.4% identity with each other. Surprisingly, the HB-3 strain was a representative strain prevalent in swine in Hubei, and the isolate CHN-SD-sHEV was obtained from swine in Shandong in a previous report. TMRCA for the clade of CH-YT-HEV01 and HB-3 was 2003, which was consistent with the TMRCA for the clade of CHN-SD-sHEV and HB-3, and they were both earlier than the TMRCA for the clade of CH-YT-HEV01 and CHN-SD-sHEV (2004). CONCLUSION: The strains CH-YT-HEV01, CHN-SD-sHEV and HB-3 are involved in trans-regional transmission, and the ancestors of HEVs in Shandong come from Hubei Province.


Assuntos
Vírus da Hepatite E/genética , Hepatite E/epidemiologia , Hepatite E/virologia , Adolescente , Adulto , Idoso , Animais , Bile/virologia , Biomarcadores/sangue , Criança , Pré-Escolar , China/epidemiologia , Estudos Transversais , Evolução Molecular , Fezes/virologia , Feminino , Genótipo , Anticorpos Anti-Hepatite/sangue , Hepatite E/sangue , Hepatite E/diagnóstico , Hepatite E/transmissão , Vírus da Hepatite E/imunologia , Vírus da Hepatite E/patogenicidade , Humanos , Imunoglobulina G/sangue , Masculino , Pessoa de Meia-Idade , Epidemiologia Molecular , Fenótipo , Filogenia , Prevalência , RNA Viral/sangue , Estudos Soroepidemiológicos , Suínos , Carga Viral , Adulto Jovem , Zoonoses
20.
J Microbiol Biotechnol ; 22(2): 256-63, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22370359

RESUMO

A strain of bacterium producing antifungal antibiotic was isolated and identification of the strain was attempted. We could identify the bacterium as being a Bacillus sp., based on morphological observation, physiological characteristics, and 16S rDNA sequence analysis, thus leading us to designate the strain as Bacillus sp. AH-E-1. The strain showed potent antibiotic activity against phytopathogenic and human pathogenic fungi by inducing mycelial distortion and swelling and inhibiting spore germination. The antibiotic metabolite produced by the strain demonstrated excellent thermal and pH (2-11) stability, but was labile to autoclaving. From these results, we could find a broader antifungal activity of Bacillus genus. Isolation and characterization of the active agent produced by the strain are under progress.


Assuntos
Antifúngicos/metabolismo , Bacillus/isolamento & purificação , Bacillus/metabolismo , Fungos/efeitos dos fármacos , Bacillus/classificação , Bacillus/genética , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Fungos/isolamento & purificação , Humanos , Dados de Sequência Molecular , Micoses/microbiologia , Filogenia , Doenças das Plantas/microbiologia , Plantas , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
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