Current protocols最新文献

{"title":"Reverse-Transcriptase Loop-Mediated Isothermal Amplification (RT-LAMP) for Viral Detection — A Case Study","authors":"Ashley Hassman,&nbsp;Renée Richie Casey,&nbsp;Colby Rouchka,&nbsp;Diego Sunino,&nbsp;Felix Veloz Espinal,&nbsp;Mona Youssef","doi":"10.1002/cpz1.70127","DOIUrl":"https://doi.org/10.1002/cpz1.70127","url":null,"abstract":"<p>An assay and protocol framework are provided for utilizing reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) at the point-of-care for diagnosing and monitoring a hypothetical zoonotic viral outbreak in a resource-limited area. This manuscript utilizes a previously published decision tree algorithm to determine an appropriate molecular diagnostic point-of-care test that can effectively address the outbreak presented in the hypothetical case study. © 2025 Wiley Periodicals LLC.</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 4","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-04-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143852717","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":"Flow Cytometric Bone Marrow Evaluation in Suspected Myelodysplastic Neoplasms","authors":"Veronika Ecker,&nbsp;Martha-Lena Müller,&nbsp;Wolfgang Kern","doi":"10.1002/cpz1.70137","DOIUrl":"https://doi.org/10.1002/cpz1.70137","url":null,"abstract":"<p>Myelodysplastic neoplasms (MDS) are acquired heterogeneous clonal hematopoietic stem cell neoplasms, clinically characterized by progressively ineffective hematopoiesis and an increased risk of acute myeloid leukemia. MDS are accompanied by an inflammatory microenvironment and genome instability. Signs of dysplasia can occur in the erythroid, myeloid, monocytic, and megakaryocytic cell lineages and result in anemia, neutropenia, and thrombocytopenia. Multi-parameter flow cytometry can be used to detect aberrant antigen expression patterns typical of MDS, which correlate with cytomorphologically identified dysplasias and provide important information for diagnosis and prognosis. Characteristic findings include an increase in myeloid progenitor cells; aberrant myeloid and erythroid maturation; aberrant marker expression on progenitor cells, granulocytes, and monocytes, which corresponds to lineage infidelity, under-/overexpression, or asynchronous expression; and an increase in monocytes and progenitor cells in chronic myelomonocytic leukemia. The latter represents an independent disease entity with a similar phenotype. In addition, flow cytometry can rule out other causes of cytopenia, such as lymphoma, acute leukemias, paroxysmal nocturnal hemoglobinuria, or systemic mastocytosis with associated hematologic neoplasm. To analyze those features, the European LeukemiaNet recommends a set of markers together with important technical aspects. At least three distinct aberrations in at least two lineages are associated with a high likelihood of MDS. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol</b>: Flow cytometric bone marrow evaluation in suspected myelodysplastic neoplasms</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 4","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-04-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143852718","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":"Isolation and Characterization of Extracellular Vesicles from Natural Killer Cells","authors":"Sara G. Dosil,&nbsp;Francisco Sánchez-Madrid,&nbsp;Lola Fernández-Messina","doi":"10.1002/cpz1.70125","DOIUrl":"https://doi.org/10.1002/cpz1.70125","url":null,"abstract":"<p>Natural killer (NK) cells are critical components of the immune system, recognized for their ability to identify and eliminate target cells undergoing various forms of stress. Beyond their direct cytotoxic activity, NK cells secrete extracellular vesicles (EVs), which serve as mediators of intercellular communication. These vesicles play an essential role in modulating immune responses and influencing tumoral and post-infection microenvironments. In this study, we present a detailed methodology for the isolation and expansion of human primary NK cells, the accumulation of their EVs, and the subsequent procedures for their isolation and characterization. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: NK cell isolation and expansion</p><p><b>Basic Protocol 2</b>: NK-EV purification using ultracentrifugation</p><p><b>Alternate Protocol</b>: NK-EV isolation using size exclusion chromatography</p><p><b>Basic Protocol 3</b>: Characterization of NK-EVs</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 4","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-04-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143822270","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":"Synthesis of Prodrug-Type Oligonucleotides Modified With a Galactosylated Self-Immolative Linker Cleavable by β-Galactosidase","authors":"Kento Miyaji,&nbsp;Yoshiaki Masaki,&nbsp;Kohji Seio","doi":"10.1002/cpz1.70128","DOIUrl":"https://doi.org/10.1002/cpz1.70128","url":null,"abstract":"<p>This protocol describes procedures for the preparation of a modified thymidine conjugated with galactose via a self-immolative linker at the <i>O</i>4-position, and the synthesis of β-galactosidase-responsive prodrug-type oligodeoxynucleotides (ONs) containing these modified thymidines. These prodrug-ONs are designed to be activated in response to β-galactosidase, enabling targeted activation in specific cells or tissues and potentially contributing to the reduction of adverse effects. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Preparation of <i>O</i>4-modified thymidine phosphoramidite <b>7</b></p><p><b>Basic Protocol 2</b>: Preparation of <i>O</i>4-modified thymidine phosphoramidite <b>15</b></p><p><b>Support Protocol 1</b>: Preparation of triazolyl thymidine derivative <b>1</b></p><p><b>Support Protocol 2</b>: Preparation of benzyl alcohol derivative <b>2</b></p><p><b>Basic Protocol 3</b>: Preparation of β-galactosidase-responsive <b>ODNs 1</b> to <b>5</b></p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 4","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-04-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143786886","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":"Cover Image, Volume 5, Issue 3","authors":"","doi":"10.1002/cpz1.70134","DOIUrl":"https://doi.org/10.1002/cpz1.70134","url":null,"abstract":"<p>The cover image is based on the article <i>Optimizing In Situ Proximity Ligation Assays for Mitochondria, ER, or MERC Markers in Skeletal Muscle Tissue and Cells</i> by Amber Crabtree et al., https://doi.org/10.1002/cpz1.70043.\u0000\u0000 <figure>\u0000 <div><picture>\u0000 <source></source></picture><p></p>\u0000 </div>\u0000 </figure></p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 3","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70134","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143770101","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 Culture and DNA Isolation Methods for Aspergillus fumigatus","authors":"Francisca C. Reyes Márquez,&nbsp;Ben Auxier,&nbsp;Bo Briggeman,&nbsp;Eveline Snelders","doi":"10.1002/cpz1.70112","DOIUrl":"https://doi.org/10.1002/cpz1.70112","url":null,"abstract":"<p><i>Aspergillus fumigatus</i> is an opportunistic human fungal pathogen that is widely spread in the environment. The need to screen a large number of environmental or clinical isolates for phenotypic and/or genotypic purposes can be challenging and time consuming when using the protocols that are currently available. We have developed a novel approach that allows one to inoculate cultures, grow individual <i>A. fumigatus</i> isolates, and subsequently isolate DNA of sufficient quality for PCR and/or whole genome sequencing, all in a 96-well format. Compared to currently available methods, our protocols reduce both cost and labor significantly and are compatible with any experimental setup that uses a 96-well format. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: 96-well-format mini slant culture and preparation of spore suspension for <i>Aspergillus fumigatus</i></p><p><b>Basic Protocol 2</b>: PCR-quality DNA isolation</p><p><b>Basic Protocol 3</b>: High-purity DNA isolation after mini mycelial mat culture</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 4","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-04-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70112","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143749832","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":"Preparation of Boranophosphate DNA via the Boranophosphotriester Method and Synthesis of P-modified DNAs from Boranophosphate DNA via an Acyl Phosphite Intermediate","authors":"Yuhei Takahashi,&nbsp;Kazuki Sato,&nbsp;Takeshi Wada","doi":"10.1002/cpz1.70117","DOIUrl":"https://doi.org/10.1002/cpz1.70117","url":null,"abstract":"<p>Boranophosphate DNAs, in which one of the nonbridging oxygen atoms in the phosphate moiety is replaced with a borano group, are promising antisense oligonucleotide candidates because of their high nuclease resistance and low cytotoxicity. In Basic Protocol 1, we describe the synthesis of a dithymidylate boranophosphodiester via a boranophosphotriester method. Using the boranophosphotriester method, the condensation of a deoxyribonucleoside 3’-<i>O</i>-boranophosphodiester, which contains a borano group with a 5’-hydroxy group of another deoxyribonucleoside, followed by the deprotection of a protecting group at the phosphorus center, is conducted to yield a boranophosphodiester. A boranophosphodiester is also a useful synthetic precursor, and several <i>P</i>-modified nucleic acid derivatives can be successfully synthesized via the conversion reaction of a boranophosphodiester. In Basic Protocol 2, we describe the conversion reaction of the boranophosphodiester through an acyl phosphite intermediate. During the conversion reaction, various <i>P</i>-modified nucleic acid derivatives, such as a phosphorothioate diester, phosphotriester, phosphoramidate, phosphorothioate triester, and phosphorothioamidate, are obtained in good yields (69% to 93%). © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Synthesis of dithymidylate boranophosphodiester</p><p><b>Support Protocol 1</b>: Preparation of 3-[(trimethylsilyl)oxy]propanenitrile</p><p><b>Support Protocol 2</b>: Preparation of PyNTP</p><p><b>Support Protocol 3</b>: Preparation of compound <b>7</b></p><p><b>Basic Protocol 2</b>: One-pot synthesis of <i>P</i>-modified nucleic acid derivatives</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 3","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143707434","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":"Pipeline for Antimicrobial Resistance Gene Quantification from Host Tissue","authors":"Levi M. Svaren,&nbsp;Wenli Li","doi":"10.1002/cpz1.70116","DOIUrl":"https://doi.org/10.1002/cpz1.70116","url":null,"abstract":"<p>Antibiotics are frequently used in food production animals to control disease and improve productivity, but this promotes the development of antimicrobial resistance (AMR) and subsequent broader spread of AMR bacteria throughout food chain, endangering the well-being and health of both animals and humans. In humans, the gut microbiome harbors a diverse range of AMR bacteria, known as the resistome. To effectively mitigate AMR in food animals requires first determining the expression and abundance of AMR-related genes in the gut resistome. Currently, such knowledge in regard to food animals is largely lacking. Gut tissue RNA sequencing (GTRS) can capture metabolically active transcripts from both the host and the microbes attached to the gut epithelium. Ideally, AMR genes can be quantified using GTRS data, making it possible to study the relationship between host and microbe. For the majority of these GTRS studies, only host transcriptome changes have been reported, while the microbial AMR remains largely unexamined, mainly due to the lack of easily implementable bioinformatics tools. Here we present a straightforward workflow to accomplish that using common command-line bioinformatics tools. With this pipeline, the host is considered noise, and host data are filtered out from the microbial reads. Transcript quantification of the AMR genes is then performed. The pipeline then continues through AMR transcript quantification, differential gene expression, and SNP analysis. Using open-source tools, we made this analytical pipeline easy to implement and able to generate results ready to be incorporated into publishable reports. Published 2025. This article is a U.S. Government work and is in the public domain in the USA.</p><p><b>Basic Protocol</b>: Running the gene quantification pipeline</p><p><b>Support Protocol 1</b>: Downloading FASTQ files from the NCBI database</p><p><b>Support Protocol 2</b>: Building a genome reference index of the host</p><p><b>Support Protocol 3</b>: Differential gene expression analysis</p><p><b>Support Protocol 4</b>: Single-nucleotide polymorphism (SNP) analysis</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 3","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143707507","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":"Synthesis of O-Nicotinoyl-Substituted Depot Forms of Biologically Active N6-Benzyladenosine Analogs","authors":"Anastasia A. Zenchenko,&nbsp;Irina V. Varizhuk,&nbsp;Mikhail S. Drenichev,&nbsp;Vladimir E. Oslovsky","doi":"10.1002/cpz1.70121","DOIUrl":"https://doi.org/10.1002/cpz1.70121","url":null,"abstract":"<p>This unit describes an effective method for the preparation of <i>N</i>⁶-benzyl-2′,3′,5′-tri-<i>O</i>-nicotinoyl adenosine and <i>N</i>⁶-(3-fluorobenzyl)-2′,3′,5′-tri-<i>O</i>-nicotinoyl adenosine. These compounds are depot forms of biologically active <i>N</i>⁶-benzyladenosine (BAR) and its fluorinated analog <i>N</i>⁶-(3-fluorobenzyl)adenosine (FBAR), which had previously shown pronounced antiviral activity against human enterovirus EV-A71. BAR and FBAR bearing biodegradable <i>O</i>-nicotinoyl ester moieties were obtained by a three-step synthesis starting from inosine. An attractive feature of this strategy is the possibility of obtaining biodegradable depot forms of various biologically active ribonucleoside derivatives, particularly <i>N</i>⁶-substituted adenosine derivatives. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol</b>: Preparation of <i>N</i>⁶-benzyl-2′,3′,5′-tri-<i>O</i>-nicotinoyl adenosine (4a).</p><p><b>Alternate Protocol</b>: Preparation of <i>N</i>⁶-(3-fluorobenzyl)-2′,3′,5′-tri-<i>O</i>-nicotinoyl adenosine (4b).</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 3","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143707435","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":"Developing Monosodium Urate Monohydrate Crystals–Induced Gout Model in Rodents and Rabbits","authors":"Yufang Wang,&nbsp;Ya Zhang,&nbsp;Zhengrong Yu,&nbsp;Yige Bai,&nbsp;Mengxing Zhu,&nbsp;Yan Lei,&nbsp;Baoli Dong,&nbsp;Qiyao Zhang,&nbsp;Qingyang Gu,&nbsp;Jian Xiang","doi":"10.1002/cpz1.70114","DOIUrl":"https://doi.org/10.1002/cpz1.70114","url":null,"abstract":"<p>Gout is a chronic disease caused by the deposition of monosodium urate monohydrate (MSU) crystals within the body, particularly in one or more joints, which can lead to sudden severe attacks of pain, swelling, redness, and tenderness, known as gout flares. Historically termed the “disease of kings,” gout is one of the oldest joint diseases and remains the most common form of inflammatory arthritis haunting humans in the 21^st century. It is associated with cardiovascular, metabolic, and renal comorbidities and can lead to reduced mobility and impaired physical function and contributing to work absenteeism. Given its increasing global incidence, novel therapies for gouty arthritis disease are urgently needed. Experimental gout models are indispensable tools for deciphering disease pathogenesis and evaluating the efficacy and side effect of newly developed therapeutics at preclinical stage. Herein, we described a series of highly reproducible acute gout flare and air pouch models in rodents and rabbits that can be used to address various scientific questions relevant to pathological changes and immune responses during and after a gout attack. Animal gout flare models, elicited by MSU crystals, mimic the main histopathological features of human gouty arthritis, including damage to cartilage and joint swelling. Meanwhile, air pouch models serve as a tool to evaluate robust inflammatory cytokine secretion and neutrophil infiltration. This article provides a detailed description of procedures and troubleshooting tips required to reproducibly induce gout flare and air pouch models in animals and critically evaluate the pathogenesis of the disease. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Preparation of monosodium urate crystalline</p><p><b>Basic Protocol 2</b>: Development of MSU-induced gout flare model in mice</p><p><b>Support Protocol 1</b>: Histological assessment of mouse ankle tissues</p><p><b>Basic Protocol 3</b>: Development of MSU-induced gout flare model in rats</p><p><b>Basic Protocol 4</b>: Development of MSU-induced gout flare model in rabbits</p><p><b>Basic Protocol 5</b>: Development and validation of reference articles in MSU-induced air pouch model in rats</p><p><b>Basic Protocol 6</b>: Development and validation of reference articles in MSU-induced air pouch model in mice</p><p><b>Support Protocol 2</b>: Flow cytometry of mouse neutrophils in air pouch lavage samples</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 3","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143707506","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}