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> POL Scientific, LLC en-US Journal of Biological Methods 2326-9901 <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> Development of a reproducible porcine model of infected burn wounds <p>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 full thickness burn wounds infected with <em>Staphylococcus aureus</em> on six pigs. Wound healing kinetics and bacterial clearance were measured over a period of 27 d 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.</p> Sayf Said Samreen Jatana Andras Ponti Erin Johnson Kimberly Such Megan Zangara Maria Madajka Francis Papay Christine McDonald Copyright (c) 2022 Sayf Al-deen Said, Samreen Jatana, Andras K. Ponti, Erin E. Johnson, Kimberly A. Such, Megan T. Zangara, Maria Madajka, Francis Papay, Christine McDonald 2022-02-21 2022-02-21 9 1 e158 e158 10.14440/jbm.2022.379 Site-specific nanobody-oligonucleotide conjugation for super-resolution imaging <p>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-<em>L</em>-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.</p> Laura Teodori Marjan Omer Anders Märcher Mads K. Skaanning Veronica L. Andersen Jesper S. Nielsen Emil Oldenburg Yuchen Lin Kurt V. Gothelf Jørgen Kjems Copyright (c) 2022 Laura Teodori, Marjan Omer, Anders Märcher, Mads K. Skaanning, Veronica L. Andersen, Jesper S. Nielsen, Emil Oldenburg, Yuchen Lin, Kurt V. Gothelf , Jørgen Kjems 2022-03-01 2022-03-01 9 1 e159 e159 10.14440/jbm.2022.381