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Current Protocols in Microbiology最新文献

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Infection and Propagation of Astrovirus VA1 in Cell Culture. 天体病毒 VA1 在细胞培养中的感染和繁殖。
Current Protocols in Microbiology Pub Date : 2019-02-01 Epub Date: 2018-11-16 DOI: 10.1002/cpmc.73
Andrew B Janowski, David Wang
{"title":"Infection and Propagation of Astrovirus VA1 in Cell Culture.","authors":"Andrew B Janowski, David Wang","doi":"10.1002/cpmc.73","DOIUrl":"10.1002/cpmc.73","url":null,"abstract":"<p><p>Astrovirus VA1/HMO-C (VA1) is the representative genotype of mamastrovirus 9, a species of the single-stranded, positive-sense RNA viral family, Astroviridae. Astroviruses have been traditionally considered pathogens of the gastrointestinal tract but they have been recently associated with neurological diseases in humans, cattle, mink, sheep, and pigs. VA1 is the astrovirus genotype most commonly identified from human cases of meningoencephalitis and has been recently propagated in cell culture. VA1 can now be used as a model system to study pathogenesis of the neurological diseases associated with astrovirus infection. In this article, we describe two fundamental assays to quantify replication and propagation of VA1, a quantitative reverse transcription-PCR (qRT-PCR) to measure viral RNA and a 50% tissue culture infectious dose (TCID<sub>50</sub> ) assay to measure infectious viral particles. © 2018 by John Wiley & Sons, Inc.</p>","PeriodicalId":39967,"journal":{"name":"Current Protocols in Microbiology","volume":"52 1","pages":"e73"},"PeriodicalIF":0.0,"publicationDate":"2019-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6340763/pdf/nihms-994544.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36675265","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: 0
Issue Information TOC 发布信息TOC
Current Protocols in Microbiology Pub Date : 2019-01-21 DOI: 10.1002/cpmc.67
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引用次数: 0
Cryptococcus neoformans Mating and Genetic Crosses 新型隐球菌的交配与遗传杂交
Current Protocols in Microbiology Pub Date : 2019-01-20 DOI: 10.1002/cpmc.75
Sheng Sun, Shelby J. Priest, Joseph Heitman
{"title":"Cryptococcus neoformans Mating and Genetic Crosses","authors":"Sheng Sun,&nbsp;Shelby J. Priest,&nbsp;Joseph Heitman","doi":"10.1002/cpmc.75","DOIUrl":"10.1002/cpmc.75","url":null,"abstract":"<p>The <i>Cryptococcus</i> pathogenic species complex is a group of opportunistic human fungal pathogens that cause cryptococcal meningoencephalitis, an infection associated with unacceptably high mortality rates. The public health relevance of these pathogens has galvanized extensive research over the past several decades and led to characterization of their sexual cycles. This research has allowed several <i>Cryptococcus</i> species to develop into model fungal organisms for both pathogenesis and basic science studies. Many of these studies require observation of the meiotic process and its associated mating structures as well as generation of meiotic progeny with novel phenotypes and genotypes. Herein, we describe how to set up genetic crosses between <i>Cryptococcus</i> strains and observe their mating phenotypes as well as how to recover progeny from these crosses for further analysis. © 2019 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":39967,"journal":{"name":"Current Protocols in Microbiology","volume":"53 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-01-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmc.75","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36869420","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: 22
Analyzing Diguanylate Cyclase Activity In Vivo using a Heterologous Escherichia coli Host 异源大肠杆菌宿主体内二胍酸环化酶活性分析
Current Protocols in Microbiology Pub Date : 2018-11-29 DOI: 10.1002/cpmc.74
Nicolas Fernandez, Christopher M. Waters
{"title":"Analyzing Diguanylate Cyclase Activity In Vivo using a Heterologous Escherichia coli Host","authors":"Nicolas Fernandez,&nbsp;Christopher M. Waters","doi":"10.1002/cpmc.74","DOIUrl":"10.1002/cpmc.74","url":null,"abstract":"<p>Bacterial biofilms are notorious for their deleterious effects on human health and industrial biofouling. Key processes in biofilm formation are regulated by the second messenger signal cyclic dimeric guanosine monophosphate (c-di-GMP); accumulation of c-di-GMP promotes biofilm formation, while lowering c-di-GMP promotes motility. Complex networks of modular enzymes are involved in regulating c-di-GMP homeostasis. Understanding how these enzymes function in bacterial cells can help enlighten how bacteria use environmental cues to modulate c-di-GMP and cell physiology. In this article, we describe a workflow that utilizes <i>Escherichia coli</i> as a heterologous host to allow the researcher to identify genes encoding potential c-di-GMP-metabolizing proteins, to express the gene of interest from an inducible plasmid, and to directly detect changes in intracellular c-di-GMP using ultra-performance liquid chromatography-tandem mass spectrometry. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":39967,"journal":{"name":"Current Protocols in Microbiology","volume":"52 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-11-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmc.74","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36717039","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 TOC 发布信息TOC
Current Protocols in Microbiology Pub Date : 2018-10-31 DOI: 10.1002/cpmc.66
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引用次数: 0
Isolation and Whole-Genome Sequencing of Environmental Campylobacter 环境弯曲杆菌的分离与全基因组测序
Current Protocols in Microbiology Pub Date : 2018-10-25 DOI: 10.1002/cpmc.64
Brittni R. Kelley, J. Christopher Ellis, Doug Hyatt, Dan Jacobson, Jeremiah Johnson
{"title":"Isolation and Whole-Genome Sequencing of Environmental Campylobacter","authors":"Brittni R. Kelley,&nbsp;J. Christopher Ellis,&nbsp;Doug Hyatt,&nbsp;Dan Jacobson,&nbsp;Jeremiah Johnson","doi":"10.1002/cpmc.64","DOIUrl":"10.1002/cpmc.64","url":null,"abstract":"<p>As a leading cause of bacterial-derived gastroenteritis worldwide, <i>Campylobacter</i> has a significant impact on human health. In the developed world, most campylobacteriosis cases are attributed to the consumption of undercooked, contaminated poultry; however, it has been shown that <i>Campylobacter</i> can be transmitted to humans through contaminated water and other types of food, including beef and milk. As such, high-resolution microbial source-tracking is essential for health department officials to determine the source(s) of <i>Campylobacter</i> outbreaks. For these reasons, this protocol provides the techniques needed for isolation of <i>Campylobacter</i> from agricultural and environmental sources, as well as human clinical specimens. Additionally, we describe a simple method for preparing high-quality genomic DNA that can be used for whole-genome sequencing and downstream bioinformatics analyses of <i>Campylobacter</i> genotypes. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":39967,"journal":{"name":"Current Protocols in Microbiology","volume":"51 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-10-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmc.64","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36669635","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: 5
Propagation and Purification of Ectromelia Virus 电虫病毒的繁殖与纯化
Current Protocols in Microbiology Pub Date : 2018-10-03 DOI: 10.1002/cpmc.65
Geeta Chaudhri, Georgina Kaladimou, Pratikshya Pandey, Gunasegaran Karupiah
{"title":"Propagation and Purification of Ectromelia Virus","authors":"Geeta Chaudhri,&nbsp;Georgina Kaladimou,&nbsp;Pratikshya Pandey,&nbsp;Gunasegaran Karupiah","doi":"10.1002/cpmc.65","DOIUrl":"10.1002/cpmc.65","url":null,"abstract":"<p>Ectromelia virus (ECTV) is an orthopoxvirus that causes mousepox in mice. Members of the genus orthopoxvirus are closely related and include variola (the causative agent of smallpox in humans), monkeypox, and vaccinia. Common features of variola virus and ECTV further include a restricted host range and similar disease progression in their respective hosts. Mousepox makes an excellent small animal model for smallpox to investigate pathogenesis, vaccine and antiviral agent testing, host-virus interactions, and immune and inflammatory responses. The availability of a wide variety of inbred, congenic, and gene-knockout mice allows detailed analyses of the host response. ECTV mutant viruses lacking one or more genes encoding immunomodulatory proteins are being used in numerous studies in conjunction with wild-type or gene-knockout mice to study the functions of these genes in host-virus interactions. The methods used for propagation of ECTV in cell culture, purification, and quantification of infectious particles through viral plaque assay are described. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":39967,"journal":{"name":"Current Protocols in Microbiology","volume":"51 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-10-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmc.65","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36594769","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: 6
Building (Viral) Phylogenetic Trees Using a Maximum Likelihood Approach 使用最大似然方法构建(病毒)系统发生树
Current Protocols in Microbiology Pub Date : 2018-09-28 DOI: 10.1002/cpmc.63
Kelly M. King, Koenraad Van Doorslaer
{"title":"Building (Viral) Phylogenetic Trees Using a Maximum Likelihood Approach","authors":"Kelly M. King,&nbsp;Koenraad Van Doorslaer","doi":"10.1002/cpmc.63","DOIUrl":"10.1002/cpmc.63","url":null,"abstract":"<p>Phylogenetic analyses allow for inferring a hypothesis about the evolutionary history of a set of homologous molecular sequences. This hypothesis can be used as the basis for further molecular and computational studies. In this unit, we offer one specific method to construct a Maximum Likelihood phylogenetic tree. We outline how to identify homologous sequences and construct a multiple sequence alignment. Following alignment, sequences are screened for potentially confounding factors such as recombination and genetic saturation. Finally, a Maximum Likelihood phylogenetic tree can be constructed implementing a rigorously tested model of evolution. The workflow outlined in this unit provides sufficient background for inferring a robust phylogenetic tree starting from a particular gene of interest. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":39967,"journal":{"name":"Current Protocols in Microbiology","volume":"51 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-09-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmc.63","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36533163","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: 7
Quantification of HIV DNA Using Droplet Digital PCR Techniques 用微滴数字PCR技术定量HIV DNA
Current Protocols in Microbiology Pub Date : 2018-09-25 DOI: 10.1002/cpmc.62
Elizabeth M. Anderson, Frank Maldarelli
{"title":"Quantification of HIV DNA Using Droplet Digital PCR Techniques","authors":"Elizabeth M. Anderson,&nbsp;Frank Maldarelli","doi":"10.1002/cpmc.62","DOIUrl":"10.1002/cpmc.62","url":null,"abstract":"<p>HIV persists, despite effective antiretroviral therapy, in long-lived cells, posing a major barrier toward a cure. A key step in the HIV replication cycle and a hallmark of the <i>Retroviridae</i> family is the integration of the viral DNA into the host genome. Once integrated, HIV expression is regulated by host machinery and the provirus persists until the cell dies. A reservoir of cells harboring replication-competent proviruses can survive for years, and mechanisms that maintain that reservoir are under investigation. The majority of integrated proviruses, however, are defective or have large deletions, and the composition of the proviral landscape during therapy remains unknown. Methods to quantify HIV proviruses are useful in investigating HIV persistence. Presented in this unit is a method for total HIV DNA quantification of various HIV genome targets that utilizes the next-generation PCR platform, digital PCR. The abundance of various HIV gene targets reflects the overall proviral composition. In this protocol, total genomic DNA is isolated from patient-derived cells and then used as a template for droplet digital PCR, in which the PCR reaction is partitioned into approximately 20,000 individual droplets, PCR amplified to an end point, and subjected to absolute quantification by counting the number of positive and negative droplets. Copy number is directly calculated using straightforward Poisson correction. Additionally, this methodological approach can be used to obtain absolute quantification of other DNA targets. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":39967,"journal":{"name":"Current Protocols in Microbiology","volume":"51 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmc.62","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36524374","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: 15
Human Papillomavirus Integration: Analysis by Molecular Combing and Fiber-FISH 人乳头瘤病毒整合:分子梳理和纤维- fish分析
Current Protocols in Microbiology Pub Date : 2018-08-20 DOI: 10.1002/cpmc.61
Catherine J. Redmond, Haiqing Fu, Mirit I. Aladjem, Alison A. McBride
{"title":"Human Papillomavirus Integration: Analysis by Molecular Combing and Fiber-FISH","authors":"Catherine J. Redmond,&nbsp;Haiqing Fu,&nbsp;Mirit I. Aladjem,&nbsp;Alison A. McBride","doi":"10.1002/cpmc.61","DOIUrl":"10.1002/cpmc.61","url":null,"abstract":"<p>Human papillomaviruses (HPVs) are frequently integrated in HPV-associated cancers. HPV genomes can be integrated in three patterns: A single integrated HPV genome (type I), multiple, tandemly integrated HPV genomes (type II), and multiple, tandemly integrated HPV genomes interspersed with host DNA (type III). Analysis of the organization of type II and type III integration sites is complicated by their repetitive nature, as sequences of individual repeats are difficult to distinguish from each other. This article presents a method for directly visualizing HPV integration sites using molecular combing combined with fluorescent <i>in situ</i> hybridization, also known as fiber-FISH. In this technique, genomic DNA is stretched across a glass coverslip and individual integrated HPV sequences are detected and directly visualized by <i>in situ</i> hybridization with a resolution of ∼1 kb. Fiber-FISH allows comprehensive characterization of the genomic organization of HPV integration sites containing type II and type III integration. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":39967,"journal":{"name":"Current Protocols in Microbiology","volume":"51 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmc.61","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36416347","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: 5
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