Journal of biological methods最新文献

{"title":"Getting two birds with one stone: Combining immunohistochemistry and Azan staining in animal morphology.","authors":"Patrick Beckers,&nbsp;Claudia Müller,&nbsp;Christiane Wallnisch,&nbsp;Thomas Bartolomaeus","doi":"10.14440/jbm.2021.354","DOIUrl":"https://doi.org/10.14440/jbm.2021.354","url":null,"abstract":"<p><p>Classical histological stained sections have the disadvantage that fine structures, like individual neurites, or specific macromolecules, like neurotransmitters cannot be visualized. Due to its highly specific staining of only one target molecule within the cell, the visualization of delicate structures, which would be superimposed by other tissue layers in classical Azan staining, is possible with immunohistochemistry. However, using immunohistological methods not all tissues of a specimen can be visualized at once. In contrast, density specific stains like Azan allow for a whole staining of the tissues. We provide a step by step protocol of how to combine immunohistochemistry and Azan staining in the same serial paraffin sections. The combination of both methods allows for a highly detailed investigation of structures of interest. The spatial detection of the previous, to Azan staining, gained antibody-labeled signal allows for a much better understanding of animal organ systems. By using serial sections, it is possible to create an aligned image stack that is both Azan stained and also antibody-labeled. Thus enabling a correlative approach that bridges traditional histology with immunohistochemistry in animal morphology.</p>","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":"8 3","pages":"e153"},"PeriodicalIF":0.0,"publicationDate":"2021-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/5a/54/jbm-8-3-e153.PMC8487864.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39503549","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":"A method for the efficient iron-labeling of patient-derived xenograft cells and cellular imaging validation.","authors":"Natasha N Knier,&nbsp;Veronica P Dubois,&nbsp;Yuanxin Chen,&nbsp;John A Ronald,&nbsp;Paula J Foster","doi":"10.14440/jbm.2021.356","DOIUrl":"https://doi.org/10.14440/jbm.2021.356","url":null,"abstract":"<p><p>There is momentum towards implementing patient-derived xenograft models (PDX) in cancer research to reflect the histopathology, tumor behavior, and metastatic properties observed in the original tumor. To study PDX cells preclinically, we used both bioluminescence imaging (BLI) to evaluate cell viability and magnetic particle imaging (MPI), an emerging imaging technology to allow for detection and quantification of iron nanoparticles. The goal of this study was to develop the first successful iron labeling method of breast cancer cells derived from patient brain metsastases and validate this method with imaging during tumor development. The overall workflow of this labeling method is as follows: adherent and non-adherent luciferase expressing human breast cancer PDX cells (F2-7) are dissociated and concurrently labeled after incubation with micron-sized iron oxide particles (MPIO; 25 μg Fe/ml), with labeling validated by cellular imaging with MPI and BLI. In this study, NOD/SCID/ILIIrg^-/- (<i>n</i> = 5) mice Received injections of 1 × 10⁶ iron-labeled F2-7 cells into the fourth mammary fat pad (MFP). BLI was performed longitudinally to day 49 and MPI was performed up to day 28. <i>In vivo</i> BLI revealed that signal increased over time with tumor development. MPI revealed decreasing signal in the tumors over time. Here, we demonstrate the first application of MPI to monitor the growth of a PDX MFP tumor and the first successful labeling of PDX cells with iron oxide particles. Imaging of PDX cells provides a powerful system to better develop personalized therapies targeting breast cancer brain metastasis.</p>","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":"8 3","pages":"e154"},"PeriodicalIF":0.0,"publicationDate":"2021-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/64/dd/jbm-8-3-e154.PMC8487865.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39503550","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":"Tissue-specific DamID protocol using nanopore sequencing.","authors":"Georgina Gómez-Saldivar,&nbsp;Dominique A Glauser,&nbsp;Peter Meister","doi":"10.14440/jbm.2021.362","DOIUrl":"https://doi.org/10.14440/jbm.2021.362","url":null,"abstract":"<p><p>DNA adenine methylation identification (DamID) is a powerful method to determine DNA binding profiles of proteins at a genomic scale. The method leverages the fusion between a protein of interest and the Dam methyltransferase of <i>E. coli</i>, which methylates proximal DNA <i>in vivo</i>. Here, we present an optimized procedure, which was developed for tissue-specific analyses in <i>Caenorhabditis elegans</i> and successfully used to footprint genes actively transcribed by RNA polymerases and to map transcription factor binding in gene regulatory regions. The present protocol details <i>C</i>. <i>elegans</i>-specific steps involved in the preparation of transgenic lines and genomic DNA samples, as well as broadly applicable steps for the DamID procedure, including the isolation of methylated DNA fragments, the preparation of multiplexed libraries, Nanopore sequencing, and data analysis. Two distinctive features of the approach are (i) the use of an efficient recombination-based strategy to selectively analyze rare cell types and (ii) the use of Nanopore sequencing, which streamlines the process. The method allows researchers to go from genomic DNA samples to sequencing results in less than a week, while being sensitive enough to report reliable DNA footprints in cell types as rare as 2 cells per animal.</p>","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":"8 3","pages":"e152"},"PeriodicalIF":0.0,"publicationDate":"2021-08-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/97/58/jbm-8-3-e152.PMC8411031.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39411353","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":"Development of a reproducible porcine model of infected burn wounds","authors":"S. Said, S. Jatana, A. Ponti, Erin E. Johnson, Kimberly A. Such, Megan T. Zangara, M. Madajka, Francis Papay, C. McDonald","doi":"10.1101/2021.08.16.456568","DOIUrl":"https://doi.org/10.1101/2021.08.16.456568","url":null,"abstract":"Severe burns are traumatic and physically debilitating injuries with a high rate of mortality. Bacterial infections often complicate burn injuries, which presents unique challenges for wound management and improved patient outcomes. Currently, pigs are used as the gold standard of pre-clinical models to study infected skin wounds due to the similarity between porcine and human skin in terms of structure and immunological response. However, utilizing this large animal model for wound infection studies can be technically challenging and create issues with data reproducibility. We present a detailed protocol for a porcine model of infected burn wounds based on our experience in creating and evaluating partial thickness burn wounds infected with Staphylococcus aureus on six pigs. Wound healing kinetics and bacterial clearance were measured over a period of 27 days in this model. Enumerated are steps to achieve standardized wound creation, bacterial inoculation, and dressing techniques. Systematic evaluation of wound healing and bacterial colonization of the wound bed is also described. Finally, advice on animal housing considerations, efficient bacterial plating procedures, and overcoming common technical challenges is provided. This protocol aims to provide investigators with a step-by-step guide to execute a technically challenging porcine wound infection model in a reproducible manner. Accordingly, this would allow for the design and evaluation of more effective burn infection therapies leading to better strategies for patient care. Graphical Abstract","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":"9 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-08-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"49431685","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":"Comparison of CRISPR and adenovirus-mediated Myd88 knockdown in RAW 264.7 cells and responses to lipopolysaccharide stimulation.","authors":"Alexander L Kolb,&nbsp;Marinaliz Reynoso,&nbsp;Ronald W Matheny","doi":"10.14440/jbm.2021.359","DOIUrl":"https://doi.org/10.14440/jbm.2021.359","url":null,"abstract":"<p><p>Genomic manipulation offers the possibility for novel therapies in lieu of medical interventions in use today. The ability to genetically restore missing inflammatory genes will have a monumental impact on our current immunotherapy treatments. This study compared the efficacy of two different genetic manipulation techniques: clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) transfection to adenoviral transduction to determine which method would provide the most transient and stable knockdown of myeloid differentiation primary response 88 (MyD88). MyD88 is a major regulator of nuclear factor kappa light chain enhancer of activated B cells (NFκB) pathway in Raw 264.7 macrophages. Following genetic manipulation, cells were treated for 24 h with Lipopolysaccharide (LPS) to stimulate the inflammatory pathway. Confirmation of knockdown was determined by western immunoblotting and quantification of band density. Both CRISPR/Cas9 and adenoviral transduction produced similar knockdown efficiency (~64% and 60%, respectively) in MyD88 protein 48 h post adenoviral transduction. NFκB phosphorylation was increased in CRISPR/Cas9-mediated MyD88 knockdown and control cells, but not in adenovirus-mediated MyD88 knockdown cells, following LPS administration. CRISPR/Cas9-mediated MyD88 knockdown macrophages treated with LPS for 24 h showed a 65% reduction in tumor necrosis factor alpha (TNFα) secretion, and a 67% reduction in interleukin-10 (IL-10) secretion when compared to LPS-stimulated control cells (<i>P</i> ≤ 0.01 for both). LPS did not stimulate TNFα or IL-10 secretion in adenovirus-mediated control or MyD88 knockdown cells. These data demonstrate that Raw 264.7 macrophages maintain responsiveness to inflammatory stimuli following CRISPR/Cas9-mediated reductions in MyD88, but not following adenovirus-mediated MyD88 knockdown.</p>","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":"8 3","pages":"e151"},"PeriodicalIF":0.0,"publicationDate":"2021-07-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/a9/80/jbm-8-3-e151.PMC8411032.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39411352","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":"Assessment of galactose-1-phosphate uridyltransferase activity in cells and tissues.","authors":"Megan L Brophy,&nbsp;John E Murphy,&nbsp;Robert D Bell","doi":"10.14440/jbm.2021.355","DOIUrl":"https://doi.org/10.14440/jbm.2021.355","url":null,"abstract":"<p><p>Galactosemias are a family of autosomal recessive genetic disorders resulting from impaired enzymes of the Leloir pathway of galactose metabolism including galactokinase, galactose uridyltransferase, and UDP-galactose 4-epimerase that are critical for conversion of galactose into glucose-6-phosphate. To better understand pathophysiological mechanisms involved in galactosemia and develop novel therapies to address the unmet need in patients, it is important to develop reliable assays to measure the activity of the Leloir pathway enzymes. Here we describe in-depth methods for indirectly measuring galacose-1-phosphate uridyltransferase activity in cell culture and animal tissues.</p>","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":"8 2","pages":"e149"},"PeriodicalIF":0.0,"publicationDate":"2021-06-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/81/a5/jbm-8-2-e149.PMC8270791.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39183537","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 efficient method to generate kidney organoids at the air-liquid interface.","authors":"Ashwani Kumar Gupta, David Z Ivancic, Bilal A Naved, Jason A Wertheim, Leif Oxburgh","doi":"10.14440/jbm.2021.357","DOIUrl":"10.14440/jbm.2021.357","url":null,"abstract":"<p><p>The prevalence of kidney dysfunction continues to increase worldwide, driving the need to develop transplantable renal tissues. The kidney develops from four major renal progenitor populations: nephron epithelial, ureteric epithelial, interstitial and endothelial progenitors. Methods have been developed to generate kidney organoids but few or dispersed tubular clusters within the organoids hamper its use in regenerative applications. Here, we describe a detailed protocol of asynchronous mixing of kidney progenitors using organotypic culture conditions to generate kidney organoids tightly packed with tubular clusters and major renal structures including endothelial network and functional proximal tubules. This protocol provides guidance in the culture of human embryonic stem cells from a National Institute of Health-approved line and their directed differentiation into kidney organoids. Our 18-day protocol provides a rapid method to generate kidney organoids that facilitate the study of different nephrological events including <i>in vitro</i> tissue development, disease modeling and chemical screening. However, further studies are required to optimize the protocol to generate additional renal-specific cell types, interconnected nephron segments and physiologically functional renal tissues.</p>","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":"8 2","pages":"e150"},"PeriodicalIF":0.0,"publicationDate":"2021-06-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/93/f6/jbm-8-2-e150.PMC8270790.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39183538","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":"Virtual screening on the web for drug repurposing: a primer.","authors":"Yu Wai Chen,&nbsp;Chin-Pang Bennu Yiu,&nbsp;Kwok-Yin Wong","doi":"10.14440/jbm.2021.351","DOIUrl":"https://doi.org/10.14440/jbm.2021.351","url":null,"abstract":"<p><p>We describe a procedure of performing <i>in silico</i> (virtual) screening using a web-based service, the MTiOpenScreen, which is freely accessible to non-commercial users. We shall use the SARS-CoV-2 main protease as an example. Starting from a structure downloaded from the Protein Data Bank, we discuss how to prepare the coordinates file, taking into account the known biochemical background information of the target protein. The reader will find that this preparation step takes up most of the effort before the target is ready for screening. The steps for uploading the target structure and defining the search volume by critical residues, and the main parameters to use, are outlined. When this protocol is followed, the user will expect to obtain a ranked list of small approved drug compounds docked into the target structure. The results can be readily examined graphically on the web site or downloaded for studying in a local molecular graphics program such as PyMOL.</p>","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":"8 2 COVID 19 Spec Iss","pages":"e148"},"PeriodicalIF":0.0,"publicationDate":"2021-05-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/1e/8c/jbm-8-2-e148.PMC8175336.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39007717","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":"<i>In vitro</i> fermentation test bed for evaluation of engineered probiotics in polymicrobial communities.","authors":"Steven Arcidiacono,&nbsp;Amy M Ehrenworth Breedon,&nbsp;Michael S Goodson,&nbsp;Laurel A Doherty,&nbsp;Wanda Lyon,&nbsp;Grace Jimenez,&nbsp;Ida G Pantoja-Feliciano,&nbsp;Jason W Soares","doi":"10.14440/jbm.2021.347","DOIUrl":"https://doi.org/10.14440/jbm.2021.347","url":null,"abstract":"<p><p><i>In vitro</i> fermentation systems offer significant opportunity for deconvoluting complex metabolic dynamics within polymicrobial communities, particularly those associated with the human gut microbiome. <i>In vitro</i> gut models have broad experimental capacity allowing rapid evaluation of multiple parameters, generating knowledge to inform design of subsequent <i>in vivo</i> studies. Here, our method describes an <i>in vitro</i> fermentation test bed to provide a physiologically-relevant assessment of engineered probiotics circuit design functions. Typically, engineered probiotics are evaluated under pristine, mono- or co-culture conditions and transitioned directly into animal or human studies, commonly resulting in a loss of desired function when introduced to complex gut communities. Our method encompasses a systematic workflow entailing fermentation, molecular and functional characterization, and statistical analyses to validate an engineered probiotic's persistence, plasmid stability and reporter response. To demonstrate the workflow, simplified polymicrobial communities of human gut microbial commensals were utilized to investigate the probiotic <i>Escherichia coli</i> Nissle 1917 engineered to produce a fluorescent reporter protein. Commensals were assembled with increasing complexity to produce a mock community based on nutrient utilization. The method assesses engineered probiotic persistence in a competitive growth environment, reporter production and function, effect of engineering on organism growth and influence on commensal composition. The <i>in vitro</i> test bed represents a new element within the Design-Build-Test-Learn paradigm, providing physiologically-relevant feedback for circuit re-design and experimental validation for transition of engineered probiotics to higher fidelity animal or human studies.</p>","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":"8 2","pages":"e147"},"PeriodicalIF":0.0,"publicationDate":"2021-05-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/a0/0f/jbm-8-2-e147.PMC8175340.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39007718","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":"Fabricating spatially functionalized 3D-printed scaffolds for osteochondral tissue engineering.","authors":"Paula Camacho,&nbsp;Matthew Fainor,&nbsp;Kelly B Seims,&nbsp;John W Tolbert,&nbsp;Lesley W Chow","doi":"10.14440/jbm.2021.353","DOIUrl":"https://doi.org/10.14440/jbm.2021.353","url":null,"abstract":"<p><p>Three-dimensional (3D) printing of biodegradable polymers has rapidly become a popular approach to create scaffolds for tissue engineering. This technique enables fabrication of complex architectures and layer-by-layer spatial control of multiple components with high resolution. The resulting scaffolds can also present distinct chemical groups or bioactive cues on the surface to guide cell behavior. However, surface functionalization often includes one or more post-fabrication processing steps, which typically produce biomaterials with homogeneously distributed chemistries that fail to mimic the biochemical organization found in native tissues. As an alternative, our laboratory developed a novel method that combines solvent-cast 3D printing with peptide-polymer conjugates to spatially present multiple biochemical cues in a single scaffold without requiring post-fabrication modification. Here, we describe a detailed, stepwise protocol to fabricate peptide-functionalized scaffolds and characterize their physical architecture and biochemical spatial organization. We used these 3D-printed scaffolds to direct human mesenchymal stem cell differentiation and osteochondral tissue formation by controlling the spatial presentation of cartilage-promoting and bone-promoting peptides. This protocol also describes how to seed scaffolds and evaluate matrix deposition driven by peptide organization.</p>","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":"8 1","pages":"e146"},"PeriodicalIF":0.0,"publicationDate":"2021-03-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/c0/1a/jbm-8-1-e146.PMC8054918.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38900999","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}