Current Protocols in Microbiology最新文献_第3页

Human Bocavirus 1 Infection of Well-Differentiated Human Airway Epithelium. 人博卡病毒1型感染高分化人气道上皮。

Current Protocols in Microbiology Pub Date : 2020-09-01 DOI: 10.1002/cpmc.107

Ziying Yan, Xuefeng Deng, Jianming Qiu

{"title":"Human Bocavirus 1 Infection of Well-Differentiated Human Airway Epithelium.","authors":"Ziying Yan, Xuefeng Deng, Jianming Qiu","doi":"10.1002/cpmc.107","DOIUrl":"https://doi.org/10.1002/cpmc.107","url":null,"abstract":"Human bocavirus 1 (HBoV1) is a small DNA virus that belongs to the Bocaparvovirus genus of the Parvoviridae family. HBoV1 is a common respiratory pathogen that causes mild to life-threatening acute respiratory tract infections in children and immunocompromised individuals, infecting both the upper and lower respiratory tracts. HBoV1 infection causes death of airway epithelial cells, resulting in airway injury and inflammation. In vitro, HBoV1 only infects well-differentiated (polarized) human airway epithelium cultured at an air-liquid interface (HAE-ALI), but not any dividing human cells. A full-length HBoV1 genome of 5543 nucleotides has been cloned from DNA extracted from a human nasopharyngeal swab into a plasmid called HBoV1 infectious clone pIHBoV1. Transfection of pIHBoV1 replicates efficiently in human embryonic kidney 293 (HEK293) cells and produces virions that are highly infectious. This article describes protocols for production of HBoV1 in HEK293 cells, generation of HAE-ALI cultures, and infection with HBoV1 in HAE-ALI. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Human bocavirus 1 production in HEK293 cells Support Protocol 1: HEK293 cell culture and transfection Support Protocol 2: Quantification of human bocavirus 1 using real-time quantitative PCR Basic Protocol 2: Differentiation of human airway cells at an air-liquid interface Support Protocol 3: Expansion of human airway epithelial cell line CuFi-8 Support Protocol 4: Expansion of human airway basal cells Support Protocol 5: Coating of plastic dishes and permeable membranes of inserts Support Protocol 6: Transepithelial electrical resistance measurement Basic Protocol 3: Human bocavirus 1 infection in human airway epithelium cultured at an air-liquid interface Support Protocol 7: Isolation of infected human airway epithelium cells from inserts Basic Protocol 4: Transduction of airway basal cells with lentiviral vector.","PeriodicalId":39967,"journal":{"name":"Current Protocols in Microbiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmc.107","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38137599","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 12

The Propagation, Quantification, and Storage of Vesicular Stomatitis Virus. 水泡性口炎病毒的繁殖、定量和储存。

Current Protocols in Microbiology Pub Date : 2020-09-01 DOI: 10.1002/cpmc.110

Alaa A Abdelmageed, Maureen C Ferran

引用次数: 10

Improved Method for Transformation of Vibrio vulnificus by Electroporation. 电穿孔法转化创伤弧菌的改进方法。

Current Protocols in Microbiology Pub Date : 2020-09-01 DOI: 10.1002/cpmc.106

Jane M Jayakumar, Orr H Shapiro, Salvador Almagro-Moreno

{"title":"Improved Method for Transformation of Vibrio vulnificus by Electroporation.","authors":"Jane M Jayakumar, Orr H Shapiro, Salvador Almagro-Moreno","doi":"10.1002/cpmc.106","DOIUrl":"https://doi.org/10.1002/cpmc.106","url":null,"abstract":"Vibrio vulnificus, an emergent human pathogen, causes fulminant septicemia with a mortality rate of over 50%. Unlike for other pathogenic Vibrio species, the factors to conclusively indicate the virulence potential of V. vulnificus strains remain largely unknown. Understanding the pathogenesis of this bacterium at a molecular level is severely hindered by inefficiencies in transformation, for instance, due to the presence of a periplasmic nuclease, Vvn. Currently, successful transformation of V. vulnificus is nearly impossible due to lack of mobilizable plasmids for the bacterium, requiring (i) very high DNA concentrations, (ii) plasmid linearization, (iii) development of novel V. vulnificus-derived plasmids, or (iv) time-consuming conjugation-based methods. To overcome these limitations, we describe a rapid, efficient, and reproducible electroporation protocol to effectively transform widely available plasmids, with different copy numbers and antibiotic resistances, into phylogenetically distant strains of V. vulnificus. Cells are made competent in high concentrations of sucrose devoid of cations and recovered from electroporation using a high-salinity recovery medium. Compared to existing methods for transformation of V. vulnificus, significantly higher efficiencies are obtained using this improved protocol. Rapid and effective transformations can markedly improve molecular analyses of V. vulnificus leading to a greater understanding of its virulence potential. This is crucial to develop rapid detection methods which have the potential to prevent future outbreaks. The electroporation protocol described here may be particularly useful for optimizing transformation of other nuclease-producing bacteria. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Preparation of competent cells Basic Protocol 2: Transformation of cells by electroporation.","PeriodicalId":39967,"journal":{"name":"Current Protocols in Microbiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmc.106","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38110417","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 2

Issue Information 问题信息

Current Protocols in Microbiology Pub Date : 2020-09-01 DOI: 10.1002/cpmc.84

引用次数: 0

An In Vitro Microneutralization Assay for SARS-CoV-2 Serology and Drug Screening. 体外微量中和法检测SARS-CoV-2血清学及药物筛选

Current Protocols in Microbiology Pub Date : 2020-09-01 DOI: 10.1002/cpmc.108

Fatima Amanat, Kris M White, Lisa Miorin, Shirin Strohmeier, Meagan McMahon, Philip Meade, Wen-Chun Liu, Randy A Albrecht, Viviana Simon, Luis Martinez-Sobrido, Thomas Moran, Adolfo García-Sastre, Florian Krammer

引用次数: 0

Corynebacterium diphtheriae Virulence Analyses Using a Caenorhabditis elegans Model. 利用秀丽隐杆线虫模型分析白喉棒状杆菌的毒力。

Current Protocols in Microbiology Pub Date : 2020-09-01 DOI: 10.1002/cpmc.109

Yi-Wei Chen, Hung Ton-That

{"title":"Corynebacterium diphtheriae Virulence Analyses Using a Caenorhabditis elegans Model.","authors":"Yi-Wei Chen, Hung Ton-That","doi":"10.1002/cpmc.109","DOIUrl":"https://doi.org/10.1002/cpmc.109","url":null,"abstract":"Corynebacterium diphtheriae is the leading cause of pharyngeal diphtheria, a respiratory disease characterized by formation of a pseudomembrane at the site of infection. Although outbreaks of C. diphtheriae infections are rare nowadays, the emergence of multidrug-resistant C. diphtheriae strains is one of the most significant public health concerns worldwide. Although C. diphtheriae has been studied for more than a century and diphtheria toxin and pili have been identified as major virulence factors, little is known about factors involved in bacterial colonization and development of disease. Here, we describe the utilization of Caenorhabditis elegans as a cost-effective, versatile model of infection to evaluate C. diphtheriae virulence. We provide detailed protocols for nematode synchronization and for evaluation of nematode survival and formation of a deformed anal region induced by C. diphtheriae infection. These protocols will permit future high-throughput screenings of virulence factors in C. diphtheriae and advance our knowledge of C. diphtheriae pathogenesis. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Synchronization of nematodes Basic Protocol 2: Assay for nematode survival following C. diphtheriae infection Basic Protocol 3: Assays for bacterial colonization and formation of deformed anal region.","PeriodicalId":39967,"journal":{"name":"Current Protocols in Microbiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmc.109","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38146359","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 3

Genetic Manipulation of Corynebacterium diphtheriae and Other Corynebacterium Species 白喉棒状杆菌和其他棒状杆菌的遗传操作

Current Protocols in Microbiology Pub Date : 2020-08-31 DOI: 10.1002/cpmc.111

Chungyu Chang, Minh Tan Nguyen, Hung Ton-That

引用次数: 2

A Low-Cost Tebuconazole-Based Screening Test for Azole-Resistant Aspergillus fumigatus 以低成本替布康唑为基础的耐唑烟曲霉筛选试验

Current Protocols in Microbiology Pub Date : 2020-08-28 DOI: 10.1002/cpmc.112

Amelie P. Brackin, Jennifer M. G. Shelton, Alireza Abdolrasouli, Matthew C. Fisher, Thomas R. Sewell

引用次数: 4

Genetic Manipulation and Virulence Assessment of Fusobacterium nucleatum 核梭杆菌的遗传操作及毒力评价

Current Protocols in Microbiology Pub Date : 2020-06-15 DOI: 10.1002/cpmc.104

Emily A. Peluso, Matthew Scheible, Hung Ton-That, Chenggang Wu

{"title":"Genetic Manipulation and Virulence Assessment of Fusobacterium nucleatum","authors":"Emily A. Peluso, Matthew Scheible, Hung Ton-That, Chenggang Wu","doi":"10.1002/cpmc.104","DOIUrl":"10.1002/cpmc.104","url":null,"abstract":"Considered a commensal, the Gram-negative anaerobe Fusobacterium nucleatum is a key member of the oral microbiome due to its wide range of interactions with many oral microbes. While the periodontal pathogenic properties of this organism have widely been examined, its connotation with extra-oral infections, including preterm birth and colorectal cancer, has now become apparent. Nonetheless, little is known about the mechanisms of pathogenicity and the associated virulence factors of F. nucleatum, most likely due to limited genetic tools and facile methodology. Here, we describe molecular techniques for the genetic manipulation of F. nucleatum, including markerless, nonpolar gene deletion, complementation, and Tn5 transposon mutagenesis. Further, we provide methodology to assess virulence potential of F. nucleatum using a mouse model of preterm birth. © 2020 Wiley Periodicals LLC.Basic Protocol 1: Generation of a galK mutant strainBasic Protocol 2: Complementation of a mutant strainBasic Protocol 3: Tn5 transposon mutagenesis of F. nucleatumBasic Protocol 4: Mouse model of preterm birth","PeriodicalId":39967,"journal":{"name":"Current Protocols in Microbiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-06-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmc.104","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38044889","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 17

Giardia lamblia: Laboratory Maintenance, Lifecycle Induction, and Infection of Murine Models 兰氏贾第鞭毛虫:实验室维护、生命周期诱导和小鼠模型感染

Current Protocols in Microbiology Pub Date : 2020-06-09 DOI: 10.1002/cpmc.102

Marc Y. Fink, Danielle Shapiro, Steven M. Singer

引用次数: 10