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><em>JBM</em> 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><em>JBM</em> has been indexed by PubMed Central (PMC) and all papers are also searchable in PubMed.</p> 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) Tue, 07 Feb 2023 06:21:35 +0000 OJS 60 An experimental workflow for identifying RNA m6A alterations in cellular senescence by methylated RNA immunoprecipitation sequencing <p>N<sup>6</sup>-methyladenosine (m<sup>6</sup>A), the most prevalent mRNA modification in eukaryotic cells, is known to play regulatory roles in a wide array of biological processes, including aging and cellular senescence. To investigate such roles, the m<sup>6</sup>A modification can be identified across the entire transcriptome by immunoprecipitation of methylated RNA with an anti-m<sup>6</sup>A antibody, followed by high-throughput sequencing (meRIP-seq or m<sup>6</sup>A-seq). Presented here is a protocol for employing meRIP-seq to profile the RNA m6A landscape in senescent human cells. We described, in detail, sample preparation, mRNA isolation, immunoprecipitation, library preparation, sequencing, bioinformatic analysis and validation. We also provided tips and considerations for the optimization and interpretation of the results. Our protocol serves as a methodological resource for investigating transcriptomic m<sup>6</sup>A alterations in cellular senescence as well as a valuable paradigm for the validation of genes of interest.</p> Yue Shi, Zeming Wu, Weiqi Zhang, Jing Qu, Weimin Ci, Guang-Hui Liu Copyright (c) 2023 Yue Shi, Zeming Wu, Weiqi Zhang, Jing Qu, Weimin Ci, Guang-Hui Liu Fri, 25 Aug 2023 00:00:00 +0000 Prevention, Diagnosis and Eradication of Mycoplasma Contamination in Cell Culture <p>Mycoplasmas, which are the smallest and simplest prokaryotes, lack a cell wall but possess the ability to undergo self-replication. Mycoplasma contamination is a common problem for laboratories engaging in cell culture. Due to their small size, Mycoplasmas can easily permeate filters designed to prevent bacterial and fungal contamination in cell culture. Although Mycoplasma contamination usually does not result in cell death, it can significantly affect cell proliferation, metabolism, and cause chromosomal aberrations. Therefore, it is crucial to detect and eliminate Mycoplasma contamination in cell culture. This step-by-step protocol presents a comprehensive approach to prevent Mycoplasma contamination in cell culture, as well as to detect and eradicate Mycoplasma to ensure accurate experimental and sequencing results.</p> Xuefeng Huang, Minghang Yu, Bingbing Wang, Yanlong Zhang, Junjing Xue, Yu Fu, Xi Wang Copyright (c) 2023 Xuefeng Huang, Minghang Yu, Bingbing Wang, Yanlong Zhang, Junjing Xue, Yu Fu, Xi Wang Wed, 01 Nov 2023 00:00:00 +0000 Proximity Protein Labeling In Dictyostelium With Engineered Ascorbic Acid Peroxidase 2 <p>To fully understand any cellular process, we not only need to identify the proteins implicated, but also how the protein network is structurally and spatially organized and changes over time. However, the dynamic nature of many protein interactions involved in cellular signaling pathways continues to be the bottleneck in mapping and studying protein networks. Fortunately, a recently developed proximity labeling method using engineered ascorbic acid peroxidase 2 (APEX2) in mammalian cells allows the identification of weak and/or transient protein interactions with spatial and temporal resolution. Here, we describe a protocol for successfully using the APEX2-proximity labeling method in <em>Dictyostelium</em>, using the cAMP receptor cAR1 as example. Coupled to the identification of the labeled proteins by mass spectrometry, this method expands <em>Dictyostelium’s</em> proteomics toolbox and should be widely useful for identifying interacting partners involved in a variety of biological processes in <em>Dictyostelium</em>.</p> Jamie A. Takashima, Helena A. Woroniecka, Pascale G. Charest Copyright (c) 2023 Jamie A. Takashima, Helena A. Woroniecka, Pascale G. Charest Thu, 16 Mar 2023 00:00:00 +0000 A strategy to recover a poor-quality ligase product <p>Over the last decades, PCR and molecular cloning have profoundly impacted various biological areas, from basic to pharmaceutical sciences. Presented in this study is a simple and step-by-step protocol that uses PCR to recover a poor-quality ligase product. In fact, a classic step that can be problematic in typical recombinant DNA manipulations can be the recovery of a product from a T4 DNA ligase reaction between two or more suitably prepared DNA fragments (sticky ends, blunt ends, TA cloning, etc.). This reaction can result in poor yields of the ligation product, due to various causes, mainly the preparation of the DNA fragments, and the poor yield can severely invalidate all subsequent steps. To overcome this problem, we designed a pair of PCR primers to amplify the entire ligase product into satisfactory amount. Of course, high-fidelity DNA polymerase must be used to obtain a faithful copy of the DNA of interest. The fragment thus amplified can then be inserted into a suitable vector and propagated by bacterial transformation.<br />We applied this procedure to modify a synthetic gene by adding a His-Tag to its 5’ end, and to insert this new construct into an expression cassette. This last step was achieved by employing a PCR cloning system. In our practical example, comprehensive PCR-based protocol with important tips were introduced. This methodological paper can serve as a roadmap for biologists who want to quickly/fully exploit the potential of the PCR-cloning to get desired constructs.</p> Sonia Del Prete, Marta Gogliettino, Gianna Palmieri, Ennio Cocca Copyright (c) 2023 Sonia Del Prete, Marta Gogliettino, Gianna Palmieri, Ennio Cocca Thu, 16 Nov 2023 00:00:00 +0000 An experimental workflow for enrichment of low abundant proteins from human serum for the discovery of serum biomarkers <p>Serum contains proteins that possess important information about diseases and their progression. Unfortunately, these proteins, which carry the information in the serum are in low abundance and are masked by other serum proteins that are in high abundance. Such masking prevents their identification and quantification. Therefore, removal of high abundance proteins is required to enrich, identify, and quantify the low abundance proteins. Immunodepletion methods are often used for this purpose, but there are limitations in their use because of off-target effects and high costs. Here we presented a robust, reproducible and cost-effective experimental workflow to remove immunoglobulins and albumin from serum with high efficiency. The workflow did not suffer from such limitations and enabled identification of 681 low abundance proteins that were otherwise undetectable in the serum. The identified low abundance proteins belonged to 21 different protein classes, namely the immunity-related proteins, modulators of protein-binding activity, and protein-modifying enzymes. They also played roles in various metabolic events, such as integrin signalling, inflammation-mediated signalling, and cadherin signalling. The presented workflow can be adapted to remove abundant proteins from other types of biological material and to provide considerable enrichment for low-abundance proteins.</p> Mehmet Sarihan, Merve Gülsen Bal Albayrak, Murat Kasap, Gurler Akpinar, Elifcan Kocyigit Copyright (c) 2023 Mehmet Sarihan, Merve Gülsen Bal Albayrak, Murat Kasap, Gurler Akpinar, Elifcan Kocyigit Tue, 07 Feb 2023 00:00:00 +0000 Optimizing factors for large-scale production of Arbuscular Mycorrhizal Fungi consortia using root organ cultures <p>Large-scale production of Arbuscular Mycorrhizal Fungi (AMF) consortia is a crucial stride in harnessing their potential for sustainable agriculture and plant growth enhancement. However, establishing optimal production conditions is challenging due to their obligate nature, variability, lack of standardized protocols, and limited understanding of their specific requirements. Previous attempts to standardize Root Organ Cultures (ROC) for AMF overlooked challenges related to viable inoculum production for field applications. This current investigation reported, for the first time, the optimization of various factors during large-scale production of AMF using ROC. By optimizing factors like gelling agents, media preparation, medium-to-inoculum ratios, incubation conditions, age, harvesting method and drying temperatures, we achieved significant yields of viable propagules. The standardized protocol outlined in this study will greatly influence commercial-scale AMF production. These standardized protocols are poised to contribute to larger-scale AMF production worldwide, with the potential to support sustainable agriculture and ecosystem management.</p> Maunata Ghorui, Shouvik Chowdhury, Keshab Das , Kiran Sunar, Balu Prakash Copyright (c) 2023 Maunata Ghorui, Shouvik Chowdhury, Keshab Das , Kiran Suanr, Balu Prakash Wed, 08 Nov 2023 00:00:00 +0000 Utilizing a human TLR selective ligand in a humanized immune system mouse model to investigate human TLR4 signaling <p>Mouse models with humanized immune systems are becoming increasingly prevalent in pharmaceutical research as a platform for preclinical testing with potential for greater translatability to clinical applications. However,the presence of both mouse and human cells that respond to TLR ligands poses a challenge for investigating therapeutic modalities targeting TLR signaling. AZ617 is a human TLR4 agonist,which has been shown <em>in vitro</em> to preferentially induce human cytokines via the TLR4 signaling pathway. We sought to examine the ability of AZ617 to preferentially induce human cytokines in CD34+ stem cell-engrafted NOG-EXL mice (huNOG-EXL), to determine its suitability as an <em>in vivo</em> human functional readout. AZ617 elicited a strong human TNFα and IL-6 response <em>in vivo</em> that demonstrated a 10- and 5-fold preference, respectively, over the mouse TNFα and IL-6. To assess efficacy of inhibiting a key protein in the TLR4 signaling pathway, PF-06650833, a small molecule inhibitor of IRAK4, was used as a tool molecule. PF-0660833 was found to effectively inhibit AZ617-induced human TNFα release <em>in vitro</em>. Likewise, PF-06650833 reduced AZ617-induced human TNFα in the huNOG-EXL mouse model, with a weaker effect on human IL-6. A longitudinal study tracking functionality of monocytes revealed that the ability of monocytes to respond to <em>ex vivo</em> stimuli was increased by 21 weeks after engraftment. Taken together, our data suggests that human selective TLR ligands could preferentially drive cytokine production from human cells in huNOG-EXL mice. This model will allow for investigation of pharmacological inhibition of human TLR signaling pathways in an <em>in vivo</em> model system.</p> Rachel Twomey, Sean Graham, Joseph S. Spina, Xiaoming Wu, Philip E. Dube, Courtney Ferrebee, William Housley Copyright (c) 2023 Rachel Twomey, Sean Graham, Joseph S. Spina, Xiaoming Wu, Philip E. Dube, Courtney Ferrebee, William Housley Mon, 20 Nov 2023 00:00:00 +0000 Benchmarking assembly free nanopore read mappers to classify complex millipede gut microbiota via Oxford Nanopore Sequencing Technology <p>Millipedes are key players in recycling leaf litter into soil in tropical ecosystems. To elucidate their gut microbiota, we collected millipedes from different municipalities of Puerto Rico. Here we aim to benchmark which method is best for metagenomic skimming of this highly complex millipede microbiome. We sequenced the gut DNA with Oxford Nanopore Technologies’ (ONT) MinION sequencer, then analyzed the data using <em>MEGAN-LR</em>, <em>Kraken2</em> protein mode, <em>Kraken2</em> nucleotide mode, <em>GraphMap</em>, and <em>Minimap2</em> to classify these long ONT reads. From our two samples, we obtained a total of 87,110 and 99,749 ONT reads, respectively. <em>Kraken2</em> nucleotide mode classified the most reads compared to all other methods at the phylum and class taxonomic level, classifying 75% of the reads in the two samples, the other methods failed to assign enough reads to either phylum or class to yield asymptotes in the taxa rarefaction curves indicating that they required more sequencing depth to fully classify this community. The community is hyper diverse with all methods classifying 20‒50 phyla in the two samples. There was significant overlap in the reads used and phyla classified between the five methods benchmarked. Our results suggest that <em>Kraken2</em> nucleotide mode is the most appropriate tool for the application of metagenomic skimming of this highly complex community.</p> Orlando J. Geli-Cruz, Carlos J. Santos-Flores, Matías J. Cafaro, Alexander J. Ropelewski, Alex Van Dam Copyright (c) 2023 Orlando J. Geli-Cruz, Carlos J. Santos-Flores, Matías J. Cafaro, Alexander J. Ropelewski, Alex Van Dam Fri, 04 Aug 2023 00:00:00 +0000