Journal of biological methods最新文献

{"title":"Measurement of skeletal muscle glucose uptake in mice in response to acute treadmill running.","authors":"Lisbeth Liliendal Valbjørn Møller,&nbsp;Steffen Henning Raun,&nbsp;Andreas Mæchel Fritzen,&nbsp;Lykke Sylow","doi":"10.14440/jbm.2022.385","DOIUrl":"https://doi.org/10.14440/jbm.2022.385","url":null,"abstract":"<p><p>Skeletal muscle contractions stimulate glucose uptake into the working muscles during exercise. Because this signaling pathway is independent of insulin, exercise constitutes an important alternative pathway to increase glucose uptake, also in insulin-resistant muscle. Therefore, much effort is being put into understanding the molecular regulation of exercise-stimulated glucose uptake by skeletal muscle. To delineate the causal molecular mechanisms whereby muscle contraction or exercise regulate glucose uptake, the investigation of genetically manipulated rodents is necessary. Presented here is a modified and optimized protocol assessing exercise-induced muscle glucose uptake in mice in response to acute treadmill running. Using this high-throughput protocol, running capacity can accurately and reproducibly be determined in mice, and basal- and exercise-stimulated skeletal muscle glucose uptake and intracellular signaling can precisely and dose-dependently be measured in awake mice <i>in vivo</i> without the need for catheterization and with minimal loss of blood.</p>","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":" ","pages":"e162"},"PeriodicalIF":0.0,"publicationDate":"2022-08-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/89/dd/jbm-9-3-e162.PMC9668592.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40696726","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}

{"title":"An experimental method for evoking and characterizing dynamic color patterning of cuttlefish during prey capture.","authors":"Danbee Kim,&nbsp;Kendra C Buresch,&nbsp;Roger T Hanlon,&nbsp;Adam R Kampff","doi":"10.14440/jbm.2022.386","DOIUrl":"https://doi.org/10.14440/jbm.2022.386","url":null,"abstract":"<p><p>Cuttlefish are active carnivores that possess a wide repertoire of body patterns that can be changed within milliseconds for many types of camouflage and communication. The forms and functions of many body patterns are well known from ethological studies in the field and laboratory. Yet one aspect has not been reported in detail: the category of rapid, brief and high-contrast changes in body coloration (\"Tentacle Shot Patterns\" or TSPs) that always occur with the ejection of two ballistic tentacles to strike live moving prey (\"Tentacles Go Ballistic\" or TGB moment). We designed and tested a mechanical device that presented prey in a controlled manner, taking advantage of a key stimulus for feeding: motion of the prey. High-speed video recordings show a rapid transition into TSPs starting 114 ms before TGB (<i>N</i> = 114). TSPs are then suppressed as early as 470-500 ms after TGB (<i>P</i> < 0.05) in unsuccessful hunts, while persisting for at least 3 s after TGB in successful hunts. A granularity analysis revealed significant differences in the large-scale high-contrast body patterning present in TSPs compared to the camouflage body pattern deployed beforehand. TSPs best fit the category of secondary defense called deimatic displaying, meant to briefly startle predators and interrupt their attack sequence while cuttlefish are distracted by striking prey. We characterize TSPs as a pattern category for which the main distinguishing feature is a high-contrast signaling pattern with aspects of Acute Conflict Mottle or Acute Disruptive Pattern. The data and methodology presented here open opportunities for quantifying the rapid neural responses in this visual sensorimotor set of behaviors.</p>","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":"9 2","pages":"e161"},"PeriodicalIF":0.0,"publicationDate":"2022-06-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/89/83/jbm-9-2-e161.PMC9208852.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40224534","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}

{"title":"Methodology for measuring oxidative capacity of isolated peroxisomes in the Seahorse assay.","authors":"Brittany A Stork, Adam Dean, Brian York","doi":"10.14440/jbm.2022.374","DOIUrl":"10.14440/jbm.2022.374","url":null,"abstract":"<p><p>The regulation of cellular energetics is a complex process that requires the coordinated function of multiple organelles. Historically, studies focused on understanding cellular energy utilization and production have been overwhelmingly concentrated on the mitochondria. While mitochondria account for the majority of intracellular energy production, they alone are incapable of maintaining the variable energetic demands of the cell. The peroxisome has recently emerged as a secondary metabolic organelle that complements and improves mitochondrial performance. Although mitochondria and peroxisomes are structurally distinct organelles, they share key functional similarities that allows for the potential to repurpose readily available tools initially developed for mitochondrial assessment to interrogate peroxisomal metabolic function in a novel manner. To this end, we report here on procedures for the isolation, purification and real-time metabolic assessment of peroxisomal β-oxidation using the Agilent Seahorse<b>®</b> system. When used together, these protocols provide a straightforward, reproducible and highly quantifiable method for measuring the contributions of peroxisomes to cellular and organismal metabolism.</p>","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":"9 2","pages":"e160"},"PeriodicalIF":0.0,"publicationDate":"2022-06-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/d5/15/jbm-9-2-e160.PMC9208851.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40224533","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}

{"title":"Estimating somatic mutation rates by bottlenecked duplex sequencing in non-model organisms: Daphnia magna as a case study","authors":"E. Sobel, J. Coate, S. Schaack","doi":"10.1101/2022.05.31.494242","DOIUrl":"https://doi.org/10.1101/2022.05.31.494242","url":null,"abstract":"Somatic mutations are evolutionarily important as determinants of individual organismal fitness, as well as being a focus of clinical research on age-related disease, such as cancer. Identifying somatic mutations and quantifying mutation rates, however, is extremely challenging and genome-wide somatic mutation rates have only been reported for a few model organisms. Here, we describe the application of Duplex Sequencing on bottlenecked WGS libraries to quantify genome-wide somatic base substitution rates in Daphnia magna. Daphnia, historically an ecological model system, has more recently been the focus of mutation studies, in part because of its high germline mutation rates. Using our protocol and pipeline, we estimate a somatic mutation rate of 2.14 × 10−7 substitutions per site (in a genotype where the germline rate is 3.60 × 10−9 substitutions per site per generation). To obtain this estimate, we tested multiple dilution levels to maximize sequencing efficiency, and developed bioinformatic filters needed to minimize false positives when a high quality reference genome is not available. In addition to laying the groundwork for estimating genotypic variation in rates of somatic mutations within D. magna, we provide a framework for quantifying somatic mutations in other non-model systems, and also highlight recent innovations to single molecule sequencing that will help to further refine such estimates.","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"42787103","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}

{"title":"Site-specific nanobody-oligonucleotide conjugation for super-resolution imaging","authors":"L. Teodori, Marjan Omer, Anders Märcher, M. K. Skaanning, V. L. Andersen, J. Nielsen, E. Oldenburg, Yuchen Lin, K. Gothelf, J. Kjems","doi":"10.14440/jbm.2022.381","DOIUrl":"https://doi.org/10.14440/jbm.2022.381","url":null,"abstract":"Camelid single-domain antibody fragments, also called nanobodies, constitute a class of binders that are small in size (~15 kDa) and possess antigen-binding properties similar to their antibody counterparts. Facile production of recombinant nanobodies in several microorganisms has made this class of binders attractive within the field of molecular imaging. Particularly, their use in super-resolution microscopy has improved the spatial resolution of molecular targets due to a smaller linkage error. In single-molecule localization microscopy techniques, the effective spatial resolution can be further enhanced by site-specific fluorescent labeling of nanobodies owing to a more homogeneous protein-to-fluorophore stoichiometry, reduced background staining and a known distance between dye and epitope. Here, we present a protocol for site-specific bioconjugation of DNA oligonucleotides to three distinct nanobodies expressed with an N- or C-terminal unnatural amino acid, 4-azido-L-phenylalanine (pAzF). Using copper-free click chemistry, the nanobody-oligonucleotide conjugation reactions were efficient and yielded highly pure bioconjugates. Target binding was retained in the bioconjugates, as demonstrated by bio-layer interferometry binding assays and the super-resolution microscopy technique, DNA points accumulation for imaging in nanoscale topography (PAINT). This method for site-specific protein-oligonucleotide conjugation can be further extended for applications within drug delivery and molecular targeting where site-specificity and stoichiometric control are required.","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"48983183","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}

{"title":"SpheroidAnalyseR-an online platform for analyzing data from 3D spheroids or organoids grown in 96-well plates.","authors":"Rhiannon Barrow,&nbsp;Joseph N Wilkinson,&nbsp;Yichen He,&nbsp;Martin Callaghan,&nbsp;Anke Brüning-Richardson,&nbsp;Mark Dunning,&nbsp;Lucy F Stead","doi":"10.14440/jbm.2022.388","DOIUrl":"https://doi.org/10.14440/jbm.2022.388","url":null,"abstract":"<p><p>Spheroids and organoids are increasingly popular three-dimensional (3D) cell culture models. Spheroid models are more physiologically relevant to a tumor compared to two-dimensional (2D) cultures and organoids are a simplified version of an organ with similar composition. Spheroids are often only formed from a single cell type which does not represent the situation <i>in vivo</i>. However, despite this, both spheroids and organoids can be used in cell migration studies, disease modelling and drug discovery. A drawback of these models is, however, the lack of appropriate analytical tools for high throughput imaging and analysis over a time course. To address this, we have developed an R Shiny app called SpheroidAnalyseR: a simple, fast, effective open-source app that allows the analysis of spheroid or organoid size data generated in a 96-well format. SpheroidAnalyseR processes and analyzes datasets of image measurements that can be obtained <i>via</i> a bespoke software, described herein, that automates spheroid imaging and quantification using the Nikon A1R Confocal Laser Scanning Microscope. However, templates are provided to enable users to input spheroid image measurements obtained by user-preferred methods. SpheroidAnalyseR facilitates outlier identification and removal followed by graphical visualization of spheroid measurements across multiple predefined parameters such as time, cell-type and treatment(s). Spheroid imaging and analysis can, thus, be reduced from hours to minutes, removing the requirement for substantial manual data manipulation in a spreadsheet application. The combination of spheroid generation in 96-well ultra-low attachment microplates, imaging using our bespoke software, and analysis using SpheroidAnalyseR toolkit allows high throughput, longitudinal quantification of 3D spheroid growth whilst minimizing user input and significantly improving the efficiency and reproducibility of data analysis. Our bespoke imaging software is available from https://github.com/GliomaGenomics. SpheroidAnalyseR is available at https://spheroidanalyser.leeds.ac.uk, and the source code found at https://github.com/GliomaGenomics.</p>","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":"9 4","pages":"e163"},"PeriodicalIF":0.0,"publicationDate":"2022-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/1e/fa/jbm-9-4-e163.PMC10040300.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9213301","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}

{"title":"Multiplexed tape-stabilized cryohistology of mineralized large animal specimens.","authors":"Hannah M Zlotnick,&nbsp;Xi Jiang,&nbsp;Robert L Mauck,&nbsp;Nathaniel A Dyment","doi":"10.14440/jbm.2022.389","DOIUrl":"https://doi.org/10.14440/jbm.2022.389","url":null,"abstract":"<p><p>Tape-stabilized cryohistology is a powerful histological method to reinforce tissue samples during and after sectioning, enhancing the overall image quality. This technique has widely been applied to section mineralized small animal (<i>i.e.</i>, mice, rat, rabbit) specimens, but has only been sparsely implemented for large animal samples that have a greater tendency to tear due to their increased surface area. Here, we present an optimized protocol for tape-stabilized cryohistology of undecalcified minipig vertebral body, femoral head, and temporomandibular joint samples. This protocol further develops a pipeline for sequential staining and imaging of the tape-stabilized cryosections. Images from multiple rounds of staining (endogenous bone mineral labels, aligned collagen (polarized light), tartrate resistant phosphatase (TRAP), alkaline phosphatase (AP), and toluidine blue) are overlaid to provide insight into dynamic bone remodeling. Overall, the established multiplexed tape-stabilized cryohistology protocol provides step-by-step instructions and guidance to cryosection large, mineralized tissues, and maximize data output from a single histological section.</p>","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":"9 4","pages":"e166"},"PeriodicalIF":0.0,"publicationDate":"2022-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/7d/3c/jbm-9-4-e166.PMC10040302.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9219444","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}

{"title":"Special issue editorial: Methods to facilitate SARS-CoV-2 and COVID-19 research.","authors":"Keith T Gagnon,&nbsp;Vera Huang","doi":"10.14440/jbm.2021.387","DOIUrl":"https://doi.org/10.14440/jbm.2021.387","url":null,"abstract":"This special issue of Journal of Biological Methods presents methods related to SARS- CoV-2 research in responding to the current global COVID-19 pandemic.","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":"8 COVID 19 Spec Iss","pages":"e157"},"PeriodicalIF":0.0,"publicationDate":"2021-12-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/ab/c6/jbm-8-specissue-e157.PMC8753996.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39702466","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}

{"title":"Novel adaptation of a running suture technique in a mouse model of corneal transplantation.","authors":"Hayate Nakagawa,&nbsp;Tomas Blanco,&nbsp;Francesca Kahale,&nbsp;Rohan Bir Singh,&nbsp;Thomas H Dohlman,&nbsp;Reza Dana","doi":"10.14440/jbm.2021.373","DOIUrl":"https://doi.org/10.14440/jbm.2021.373","url":null,"abstract":"<p><p>Several murine models of corneal transplantation have been developed over the years to study the immunopathological processes that lead to the failure of grafted corneas. In all of them, the classic eight interrupted sutures technique is utilized for transplanting the donor cornea on the host bed. However, in clinical practice, a single continuous suture with a single knot is generally performed for corneal transplantation. Here, we describe the adaptation of the single continuous suture technique in a mouse model of corneal transplantation.</p>","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":"8 4","pages":"e156"},"PeriodicalIF":0.0,"publicationDate":"2021-10-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/d7/ed/jbm-8-4-e156.PMC8748801.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39909656","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}

{"title":"High throughput nanopore sequencing of SARS-CoV-2 viral genomes from patient samples.","authors":"Adrian A Pater,&nbsp;Michael S Bosmeny,&nbsp;Adam A White,&nbsp;Rourke J Sylvain,&nbsp;Seth B Eddington,&nbsp;Mansi Parasrampuria,&nbsp;Katy N Ovington,&nbsp;Paige E Metz,&nbsp;Abadat O Yinusa,&nbsp;Christopher L Barkau,&nbsp;Ramadevi Chilamkurthy,&nbsp;Scott W Benzinger,&nbsp;Madison M Hebert,&nbsp;Keith T Gagnon","doi":"10.14440/jbm.2021.360","DOIUrl":"https://doi.org/10.14440/jbm.2021.360","url":null,"abstract":"<p><p>In late 2019, a novel coronavirus began spreading in Wuhan, China, causing a potentially lethal respiratory viral infection. By early 2020, the novel coronavirus, called SARS-CoV-2, had spread globally, causing the COVID-19 pandemic. The infection and mutation rates of SARS-CoV-2 make it amenable to tracking introduction, spread and evolution by viral genome sequencing. Efforts to develop effective public health policies, therapeutics, or vaccines to treat or prevent COVID-19 are also expected to benefit from tracking mutations of the SARS-CoV-2 virus. Here we describe a set of comprehensive working protocols, from viral RNA extraction to analysis using established visualization tools, for high throughput sequencing of SARS-CoV-2 viral genomes using a MinION instrument. This set of protocols should serve as a reliable \"how-to\" reference for generating quality SARS-CoV-2 genome sequences with ARTIC primer sets and long-read nanopore sequencing technology. In addition, many of the preparation, quality control, and analysis steps will be generally applicable to other sequencing platforms.</p>","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":"8 COVID 19 Spec Iss","pages":"e155"},"PeriodicalIF":0.0,"publicationDate":"2021-09-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/fd/4f/jbm-8-specissue-e155.PMC8493558.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39503998","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}