Journal of Biological Methods <p>The <em>Journal of Biological Methods (JBM) </em>(ISSN 2326-9901) is a multidisciplinary and open-access journal committed to publishing peer-reviewed papers on cutting-edge and innovative biological techniques, methods and protocols.</p> <p>JBM has been included by the following indexing and archiving services: Google Scholar, CrossRef, OCLC, Portico and SHERPA/RoMEO, BIOSIS Previews and Biological Abstracts.</p> <p><sup><span style="background-color: #ffff00;">New</span></sup> JBM has now been indexed by PubMed Central (PMC) and all papers are also searchable in PubMed.</p> <div style="margin: 0 auto; text-align: center; overflow: hidden; border-radius: 0px; background: #367e32; border: 0px solid #000000; padding: 5px; max-width: calc(100% - 10px); width: 740px;"> <div style="display: inline-block; text-shadow: #decf1b 4px 4px 4px; position: relative; vertical-align: middle; padding: 9px; font-size: 30px; color: #ffffff; font-weight: bold;">COVID 19 Special Issue - Call for Papers</div> <div style="display: inline-block; position: relative; vertical-align: middle; padding: 17px; font-size: 16px; color: #ffffff; font-weight: normal;">JBM is now accepting manuscripts to be published in a COVID 19 Special Issue</div> </div> en-US <p>Authors who publish with JBM agree to the following terms:</p> <ol> <li>Authors retain copyright and grant JBM right of first publication with the work simultaneously licensed under a <a href="">Creative Commons Attribution License</a> that allows others to share the work with an acknowledgement of the work's authorship and initial publication in this journal.</li> <li>Authors are able to enter into separate, additional contractual arrangements for the non-exclusive distribution of the journal's published version of the work (e.g., post it to an institutional repository or publish it in a book), with an acknowledgement of its initial publication in this journal.</li> <li>Authors are permitted and encouraged to post their work online (e.g., in institutional repositories or on their website) prior to and during the submission process, as it can lead to productive exchanges, as well as earlier and greater citation of published work (See <a href="" target="_new">The Effect of Open Access</a>).</li> </ol> (JBM Editorial Office) (JBM Technical Support) Fri, 29 Jan 2021 00:00:00 -0800 OJS 60 Using a variant of the optomotor response as a visual defect detection assay in zebrafish <p>We describe a visual stimulus that can be used with both larval and adult zebrafish (Danio rerio). This protocol is a modification of a standard visual behavior analysis, the optomotor response (OMR). The OMR is often used to determine the spatial response or to detect directional visuomotor deficiencies. An OMR can be generated using a high contrast grated pattern, typically vertical bars. The spatial sensitivity is measured by detection and response to a change in grating bar width and is reported in cycles per degree (CPD). This test has been used extensively with zebrafish larvae and adults to identify visual- and/or motor-based mutations. Historically, when tested in adults, the grated pattern was presented from a vertical perspective, using a rotating cylinder around a holding tank, allowing the grating to be seen solely from the sides and front of the organism. In contrast, OMRs in zebrafish larvae are elicited using a stimulus projected below the fish. This difference in methodology means that two different experimental set-ups are required: one for adults and one for larvae. Our visual stimulus modifies the stimulation format so that a single OMR stimulus, suitable for use with both adults and larvae, is being presented underneath the fish. Analysis of visuomotor responses using this method does not require costly behavioral tracking software and, using a single behavioral paradigm, allows the observer to rapidly determine visual spatial response in both zebrafish larvae and adults.</p> Matthew K. LeFauve, Cassie J. Rowe, Mikayla Crowley-Perry, Jenna L. Wiegand, Arthur G. Shapiro, Victoria P. Connaughton Copyright (c) Mon, 01 Feb 2021 00:00:00 -0800 Fabricating spatially functionalized 3D-printed scaffolds for osteochondral tissue engineering <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> Paula Camacho, Matthew Fainor, Kelly B. Seims, John W. Tolbert, Lesley W. Chow Copyright (c) Fri, 26 Mar 2021 00:00:00 -0700 Visualization of subdiffusive sites in a live single cell <p>We measured anomalous diffusion in human prostate cancer cells which were transfected with the Alexa633 fluorescent RNA probe and co-transfected with enhanced green fluorescent protein-labeled argonaute2 protein by laser scanning microscopy. The image analysis arose from diffusion based on a “two-level system”. A trap was an interaction site where the diffusive motion was slowed down. Anomalous subdiffusive spreading occurred at cellular traps. The cellular traps were not immobile. We showed how the novel analysis method of imaging data resulted in new information about the number of traps in the crowded and heterogeneous environment of a single human prostate cancer cell. The imaging data were consistent with and explained by our modern ideas of anomalous diffusion of mixed origins in live cells. Our original research presented in this study is significant as we obtained a complex diffusion mechanism in live single cells.</p> Zeno Földes-Papp, Gerd Baumann, Long-Cheng Li Copyright (c) Sat, 30 Jan 2021 00:00:00 -0800 High-yield purification of exceptional-quality, single-molecule DNA substrates <p>Single-molecule studies involving DNA or RNA, require homogeneous preparations of nucleic acid substrates of exceptional quality. Over the past several years, a variety of methods have been published describing different purification methods but these are frustratingly inconsistent with variable yields even in the hands of experienced bench scientists. To address these issues, we present an optimized and straightforward, column-based approach that is reproducible and produces high yields of substrates or substrate components of exceptional quality. Central to the success of the method presented is the use of a non-porous anion exchange resin. In addition to the use of this resin, we encourage the optimization of each step in the construction of substrates. The fully optimized method produces high yields of a hairpin DNA substrate of exceptional quality. While this substrate is suitable for single-molecule, magnetic tweezer experiments, the described method is readily adaptable to the production of DNA substrates for the majority of single-molecule studies involving nucleic acids ranging in size from 70–15000 bp.</p> Yue Lu, Piero Bianco Copyright (c) Wed, 24 Feb 2021 00:00:00 -0800 Optimization of small-scale sample preparation for high-throughput OpenArray analysis OpenArray is one of the most high-throughput qPCR platforms available but its efficiency can be limited by sample preparation methods that are slow and costly. To optimize the sample workflow for high-throughput qPCR processing by OpenArray, small-scale sample preparation methods were compared for compatibility with this system to build confidence in a method that maintains quality and accuracy while using less starting material and saving time and money. This study is the first to show that the Cells-to-CT kit can be used to prepare samples within the dynamic range of OpenArray directly from cultured cells in a single well of a 96-well plate when used together with a cDNA preamplification PCR step. Use of Cells-to-CT produced results of similar quality and accuracy to that of a preparation method using purified RNA in less than half the sample preparation time. While Cells-to-CT samples also exhibited slightly increased variance, which affects the ability of OpenArray to distinguish small differences in gene expression, overall gene expression mean results correlated well between small-scale methods. This work demonstrates that Cells-to-CT with preamplification can be used to reliably prepare samples for OpenArray analysis while saving time, money, and starting material. Neeta A. Abraham, Anne C. Campbell, Warren D. Hirst, Catherine L. Nezich Copyright (c) Mon, 01 Feb 2021 00:00:00 -0800