Biology Methods and Protocols最新文献_第8页

A methodological primer of extracellular vesicles isolation and characterization via different techniques. 通过不同技术分离和表征细胞外囊泡的方法论入门。

IF 3.6

Biology Methods and Protocols Pub Date : 2024-02-13 eCollection Date: 2024-01-01 DOI: 10.1093/biomethods/bpae009

Farhang Aliakbari, Noah B Stocek, Maxximuss Cole-André, Janice Gomes, Giovanni Fanchini, Stephen H Pasternak, Gunna Christiansen, Dina Morshedi, Kathryn Volkening, Michael J Strong

{"title":"A methodological primer of extracellular vesicles isolation and characterization via different techniques.","authors":"Farhang Aliakbari, Noah B Stocek, Maxximuss Cole-André, Janice Gomes, Giovanni Fanchini, Stephen H Pasternak, Gunna Christiansen, Dina Morshedi, Kathryn Volkening, Michael J Strong","doi":"10.1093/biomethods/bpae009","DOIUrl":"10.1093/biomethods/bpae009","url":null,"abstract":"We present four different protocols of varying complexity for the isolation of cell culture-derived extracellular vesicles (EVs)/exosome-enriched fractions with the objective of providing researchers with easily conducted methods that can be adapted for many different uses in various laboratory settings and locations. These protocols are primarily based on polymer precipitation, filtration and/or ultracentrifugation, as well as size-exclusion chromatography (SEC) and include: (i) polyethylene glycol and sodium chloride supplementation of the conditioned medium followed by low-speed centrifugation; (ii) ultracentrifugation of conditioned medium; (iii) filtration of conditioned media through a 100-kDa exclusion filter; and (iv) isolation using a standard commercial kit. These techniques can be followed by further purification by ultracentrifugation, sucrose density gradient centrifugation, or SEC if needed and the equipment is available. HEK293 and SH-SY5Y cell cultures were used to generate conditioned medium containing exosomes. This medium was then depleted of cells and debris, filtered through a 0.2-µM filter, and supplemented with protease and RNAse inhibitors prior to exosomal isolation. The purified EVs can be used immediately or stably stored at 4°C (up to a week for imaging or using intact EVS downstream) or at -80°C for extended periods and then used for biochemical study. Our aim is not to compare these methodologies but to present them with descriptors so that researchers can choose the \"best method\" for their work under their individual conditions.","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"9 1","pages":"bpae009"},"PeriodicalIF":3.6,"publicationDate":"2024-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10902684/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139997669","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

Teaching at the intersection of science and society: an activity on healthcare disparities 科学与社会交汇处的教学：关于医疗保健差异的活动

IF 3.6

Biology Methods and Protocols Pub Date : 2024-01-05 DOI: 10.1093/biomethods/bpad041

Paula E Adams, Enya Granados, Abby E. Beatty, C. Ballen

{"title":"Teaching at the intersection of science and society: an activity on healthcare disparities","authors":"Paula E Adams, Enya Granados, Abby E. Beatty, C. Ballen","doi":"10.1093/biomethods/bpad041","DOIUrl":"https://doi.org/10.1093/biomethods/bpad041","url":null,"abstract":"\u0000 Understanding the relationship between science and society is an objective of science education and included as a core competency in the AAAS Vision and Change guidelines for biology education. However, traditional undergraduate biology instruction emphasizes scientific practice and generally avoids potentially controversial issues at the intersection of biology and society. By including these topics in biology coursework, instructors can challenge damaging ideologies and systemic inequalities that have influenced science, such as biological essentialism and health disparities. Specifically, an ideologically aware curriculum highlights how ideologies and paradigms shape our biological knowledge base and the application of that knowledge. Ideologically aware lessons emphasize the relationship between science and society with an aim to create more transparent, scientifically accurate, and inclusive postsecondary biology classrooms. Here we expand upon our ideologically aware curriculum with a new activity that challenges undergraduate biology students to consider the impacts of healthcare disparities. This lesson allows instructors to directly address systemic inequalities and allows students to connect biomedical sciences to real-world issues. Implementing an ideologically aware curriculum enables students to challenge prevailing worldviews and better address societal problems that lead to exclusion and oppression.","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"24 1","pages":""},"PeriodicalIF":3.6,"publicationDate":"2024-01-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139383809","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: 0

Long amplicon Nanopore sequencing of Botrytis cinerea and other fungal species present in infected grapevine leaf samples 对受感染葡萄叶片样本中的葡萄孢菌和其他真菌物种进行长扩增片段纳米孔测序

IF 3.6

Biology Methods and Protocols Pub Date : 2024-01-05 DOI: 10.1093/biomethods/bpad042

Vladimer Baramidze, Luca Sella, Tamar Japaridze, Nino Abashidze, Daviti Lamazoshvili, Nino Dzotsenidze, Giorgi Tomashvili

{"title":"Long amplicon Nanopore sequencing of Botrytis cinerea and other fungal species present in infected grapevine leaf samples","authors":"Vladimer Baramidze, Luca Sella, Tamar Japaridze, Nino Abashidze, Daviti Lamazoshvili, Nino Dzotsenidze, Giorgi Tomashvili","doi":"10.1093/biomethods/bpad042","DOIUrl":"https://doi.org/10.1093/biomethods/bpad042","url":null,"abstract":"\u0000 Botrytis cinerea is a well-known plant pathogen responsible for grey mould disease infecting more than 500 plant species. It is listed as the second most important plant pathogen scientifically and economically. Its impact is particularly severe in grapes since it affects both the yield of grape berries and the quality of wines. While various methods for detecting B. cinerea have been investigated, the application of Oxford Nanopore Technology (ONT) for complete ribosomal operon sequencing, which has proven effective in human and animal fungal research and diagnostics, has not yet been explored in grapevine (Vitis vinifera) disease research. In this study, we sequenced complete ribosomal operons (∼5.5 kb amplicons), which encompass the 18S, ITS1, 5.8S, ITS2, and 28S regions, from both pure cultures of B. cinerea and infected grapevine leaf samples. Minimap2, a sequence alignment tool integrated into the EPI2ME software, served as a taxonomy classifier, utilising the custom reference database FRODO. The results demonstrate that B. cinerea was detectable when this pathogen was not the dominant fungal species in leaf samples. Additionally, the method facilitates host DNA-free sequencing and might have a good potential to distinguish other pathogenic and non-pathogenic fungal species hosted within grapevine’s infected leaves, such as Alternaria alternata, Saccharomyces cerevisiae, Saccharomyces boulardii, Mucor racemosus, Ascochyta rabie, etc The sequences were uploaded to the NCBI database. Long Amplicon sequencing method has the capacity to be broadened to other susceptible crops and pathogens, as a valuable tool for early grey rot detection and mycobiome research. Future large-scale studies are needed to overcome challenges, such as comprehensive reference databases for complete fungal ribosomal operons for grape mycobiome studies.","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"80 3","pages":""},"PeriodicalIF":3.6,"publicationDate":"2024-01-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139383598","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: 0

Bringing science to the public in the light of evolution. 从进化论的角度向公众介绍科学。

IF 3.6

Biology Methods and Protocols Pub Date : 2023-12-18 eCollection Date: 2023-01-01 DOI: 10.1093/biomethods/bpad040

Marie-Claude Blatter, Monique Zahn-Zabal, Samuel Moix, Béatrice Pichon, Christophe Dessimoz, Natasha Glover

{"title":"Bringing science to the public in the light of evolution.","authors":"Marie-Claude Blatter, Monique Zahn-Zabal, Samuel Moix, Béatrice Pichon, Christophe Dessimoz, Natasha Glover","doi":"10.1093/biomethods/bpad040","DOIUrl":"10.1093/biomethods/bpad040","url":null,"abstract":"Evolution stands as a foundational pillar within modern biology, shaping our understanding of life. Studies related to evolution, for example constructing phylogenetic trees, are often carried out using DNA or protein sequences. These data, readily accessible from public databases, represent a treasure trove of resources that can be harnessed to create engaging activities with the public. At the heart of our project lies a collection of \"stories\" about evolution, each rooted in genuine scientific publications that furnish both biological context and supporting evidence. These narratives serve as the focal point of our LightOfEvolution.org website. Each story is accompanied by a dedicated \"Your Turn to Play\" section. Within this section, we furnish user-friendly activities and step-by-step guidelines, equipping visitors with the means to replicate analyses showcased in the highlighted publications. For example, the website OhMyGenes.org, relying on authentic scientific data, provides the capability to compute the proportion of shared genes across different species. Here, visitors can address the captivating question: \"How many genes do we share with a banana?\" To extend the educational reach, we have developed a series of modular activities, also related to the stories. These activities have been thoughtfully designed to be adaptable for face-to-face workshops held in classrooms or presented during public events. We aim to create stories and activities that resonate with participants, offering a tangible and enjoyable experience. By providing opportunities that reflect real-world scientific practices, we seek to offer participants valuable insights into the current workings of scientists \"in the light of evolution.\"","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"8 1","pages":"bpad040"},"PeriodicalIF":3.6,"publicationDate":"2023-12-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10752581/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139049460","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

tANCHOR fast and cost-effective cell-based immunization approach with focus on the receptor-binding domain of SARS-CoV-2. tANCHOR 以 SARS-CoV-2 的受体结合域为重点的快速、经济的细胞免疫方法。

IF 3.6

Biology Methods and Protocols Pub Date : 2023-12-12 eCollection Date: 2023-01-01 DOI: 10.1093/biomethods/bpad030

Hubert Bernauer, Anja Schlör, Josef Maier, Norbert Bannert, Katja Hanack, Daniel Ivanusic

引用次数: 0

Improved Shorea robusta genomic DNA extraction protocol with high PCR fidelity 具有高 PCR 保真度的改良娑罗双树基因组 DNA 提取方案

IF 3.6

Biology Methods and Protocols Pub Date : 2023-12-09 DOI: 10.1093/biomethods/bpad039

Garima Mishra, R. Meena, Rama Kant, S. Pandey, H. Ginwal, M. Bhandari

{"title":"Improved Shorea robusta genomic DNA extraction protocol with high PCR fidelity","authors":"Garima Mishra, R. Meena, Rama Kant, S. Pandey, H. Ginwal, M. Bhandari","doi":"10.1093/biomethods/bpad039","DOIUrl":"https://doi.org/10.1093/biomethods/bpad039","url":null,"abstract":"\u0000 Shorea robusta (Dipterocarpaceae), commonly known as Sal, is an economically and culturally-important timber species, known to contain a wide spectrum of polyphenols, polysaccharides, and other secondary metabolites in the tissues, which can interfere with the extraction of high-quality genomic DNA. In order to screen simple sequence repeat (SSR) markers and carry out other DNA-based analyses for this species in our laboratory, a high-throughput DNA extraction methodology was needed. Hence, we have optimised a simple, rapid, safe and reliable high-throughput protocol for DNA extraction suitable for both fresh and dry leaves. The standardized protocol delivered good DNA yield of approximately ∼1500 µg from one gram of leaf tissue, with purity indicated by 260 nm/280 nm absorbance ratio ranging from 1.70–1.91, which validated the suitability of extracted DNA and revealed reduced levels of contaminants. Additionally, the protocol that we developed was found to be suitable for polymerase chain reaction (PCR) amplification using microsatellite markers. Genome-wide characterization with SSR markers has been established in S. robusta, which further validates the protocol and its usefulness in DNA-based studies across the genus and/or family.","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"1 5","pages":""},"PeriodicalIF":3.6,"publicationDate":"2023-12-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138585923","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: 0

COVID's long shadow: How SARS-CoV-2 infection, COVID-19 severity, and vaccination status affect long-term cognitive performance and health. COVID 的长影：SARS-CoV-2 感染、COVID-19 严重程度和疫苗接种状况如何影响长期认知能力和健康。

IF 3.6

Biology Methods and Protocols Pub Date : 2023-12-07 eCollection Date: 2023-01-01 DOI: 10.1093/biomethods/bpad038

Jaroslav Flegr, Ashkan Latifi

{"title":"COVID's long shadow: How SARS-CoV-2 infection, COVID-19 severity, and vaccination status affect long-term cognitive performance and health.","authors":"Jaroslav Flegr, Ashkan Latifi","doi":"10.1093/biomethods/bpad038","DOIUrl":"10.1093/biomethods/bpad038","url":null,"abstract":"COVID-19 affects a variety of organs and systems of the body including the central nervous system. Recent research has shown that COVID-19 survivors often experience neurological and psychiatric complications that can last for months after infection. We conducted a large Internet study using online tests to analyze the effects of SARS-CoV-2 infection, COVID-19 severity, and vaccination on health, intelligence, memory, and information processing precision and speed in a cohort of 4445 subjects. We found that both SARS-CoV-2 infection and COVID-19 severity were associated with negative impacts on patients' health. Furthermore, we observed a negative association between COVID-19 severity and cognitive performance. Younger participants had a higher likelihood of SARS-CoV-2 contraction, while the elderly had a higher likelihood of severe COVID-19 and vaccination. The association between age and COVID-19 severity was primarily mediated by older participants' impaired long-term health. Vaccination was positively associated with intelligence and the precision of information processing. However, the positive association between vaccination and intelligence was likely mediated by achieved education, which was itself strongly associated with the likelihood of being vaccinated.","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"8 1","pages":"bpad038"},"PeriodicalIF":3.6,"publicationDate":"2023-12-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10733723/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138832011","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

Is recovery just the beginning? Persistent symptoms and health and performance deterioration in post-COVID-19, non-hospitalized university students-a cross-sectional study. 康复仅仅是开始吗？COVID-19后未住院大学生的持续症状与健康和成绩下降--一项横断面研究。

IF 2.5

Biology Methods and Protocols Pub Date : 2023-12-06 eCollection Date: 2023-01-01 DOI: 10.1093/biomethods/bpad037

Ashkan Latifi, Jaroslav Flegr

{"title":"Is recovery just the beginning? Persistent symptoms and health and performance deterioration in post-COVID-19, non-hospitalized university students-a cross-sectional study.","authors":"Ashkan Latifi, Jaroslav Flegr","doi":"10.1093/biomethods/bpad037","DOIUrl":"10.1093/biomethods/bpad037","url":null,"abstract":"Many individuals experience persistent symptoms such as deteriorated physical and mental health, increased fatigue, and reduced cognitive performance months after recovering from coronavirus disease 2019 (COVID-19). There is limited data on the long-term trajectory and prevalence of these symptoms, especially in milder cases. Our study aimed to assess the persistent effects of COVID-19 on physical and mental health, fatigue, and cognitive performance in a cohort of 214 students, averaging 21.8 years of age. Of these, 148 had contracted COVID-19 but were not hospitalized, with the time since infection ranging from 1 to 39 months. We utilized a comprehensive panel of cognitive tests to measure intelligence, memory, and psychomotor skills, and a detailed anamnestic questionnaire to evaluate physical and mental health. While contracting COVID-19 did not significantly impact overall health and performance, it was associated with increased reports of fatigue. However, the reported severity of the disease had a pronounced negative influence on physical health, mental well-being, fatigue, and reaction time. Trends of improvement in physical and mental health, as well as error rate, were observed within the first 2 years post-infection. However, fatigue and reaction time showed a trend of deterioration. Beyond the 2-year mark, physical health and error rate continued to improve, while mental health began to deteriorate. Fatigue and reaction time continued to decline. Overall, our findings suggest that some effects of contracting COVID-19 can persist or even deteriorate over time, even in younger individuals who had mild cases that did not require hospitalization.","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"8 1","pages":"bpad037"},"PeriodicalIF":2.5,"publicationDate":"2023-12-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10739555/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139032641","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

An accurate spectrophotometric method for chitosan quantification. 准确的壳聚糖分光光度法。

IF 3.6

Biology Methods and Protocols Pub Date : 2023-11-29 eCollection Date: 2023-01-01 DOI: 10.1093/biomethods/bpad036

Oscar González-Davis, Itandehui Betanzo, Rafael Vazquez-Duhalt

引用次数: 0

Exogenous spike-in mouse RNAs for accurate differential gene expression analysis in barley using RT-qPCR. 利用 RT-qPCR 对大麦中的外源性尖峰小鼠 RNA 进行精确的差异基因表达分析。

IF 3.6

Biology Methods and Protocols Pub Date : 2023-11-28 eCollection Date: 2023-01-01 DOI: 10.1093/biomethods/bpad034

Marcus A Vinje, David A Friedman

{"title":"Exogenous spike-in mouse RNAs for accurate differential gene expression analysis in barley using RT-qPCR.","authors":"Marcus A Vinje, David A Friedman","doi":"10.1093/biomethods/bpad034","DOIUrl":"10.1093/biomethods/bpad034","url":null,"abstract":"Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) followed by the 2-ΔΔCt method is the most common way to measure transcript levels for relative gene expression assays. The quality of an RT-qPCR assay is dependent upon the identification and validation of reference genes to normalize gene expression data. The so-called housekeeping genes are commonly used as internal reference genes because they are assumed to be ubiquitously expressed at stable levels. Commonly, researchers do not validate their reference genes but rely on historical reference genes or previously validated genes from an unrelated experiment. Using previously validated reference genes to assess gene expression changes occurring during malting resulted in extensive variability. Therefore, a new method was tested and validated to circumvent the use of internal reference genes. Total mouse RNA was chosen as the external reference RNA and a suite of primer sets to putatively stable mouse genes was created to identify stably expressed genes for use as an external reference gene. cDNA was created by co-amplifying total mouse RNA, as an RNA spike-in, and barley RNA. When using the external reference genes to normalize malting gene expression data, standard deviations were significantly reduced and significant differences in transcript abundance were observed, whereas when using the internal reference genes, standard deviations were larger with no significant differences seen. Furthermore, external reference genes were more accurate at assessing expression levels in malting and developing grains, whereas the internal reference genes overestimated abundance in developing grains and underestimated abundance in malting grains.","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"8 1","pages":"bpad034"},"PeriodicalIF":3.6,"publicationDate":"2023-11-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10728042/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138810077","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