Current protocols最新文献

{"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":"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":2.2,"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":"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":2.2,"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":"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":2.2,"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":"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":2.2,"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":"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":2.2,"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":"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":2.2,"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":"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":2.2,"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}

{"title":"Streaming Long-Read Sequence Alignments for HLA Predictions Using HLAminer","authors":"René L. Warren,&nbsp;Inanc Birol","doi":"10.1002/cpz1.70124","DOIUrl":"10.1002/cpz1.70124","url":null,"abstract":"<p>Long-read sequencing platforms such as the Oxford Nanopore Technologies (ONT) and Pacific Biosciences (PacBio) platforms now offer sufficient read lengths, throughput, and accuracy at competitive costs to analyze polymorphic regions of the human genome, including the highly complex human leukocyte antigen (HLA) gene cluster—a cornerstone of human immunity. Here, we present a streamlined protocol for predicting HLA signatures from whole-genome shotgun (WGS) long-read sequencing data by directly streaming sequence alignments into HLAminer. This method is as simple as running minimap2, scales efficiently with the number of sequences, and works with any read aligner compatible with the SAM file format—eliminating the need to store bulky alignment files on disk. We provide a step-by-step guide for predicting HLA class I and class II alleles from third-generation long-read sequencing data and demonstrate the robustness of predictions even with older, less accurate WGS nanopore datasets and relatively low (10×) sequencing coverage. Code availability: HLAminer is available under the BC Cancer software license agreement (academic use) at https://github.com/bcgsc/HLAminer. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p>This article was corrected on 25 April 2025. See the end of the full text for details.</p><p><b>Basic Protocol</b>: HLA prediction from streamed ONT or PacBio long-read alignments</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 3","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70124","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143707570","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":"Isolation and Characterization of Human Tissue Resident Memory T cells","authors":"Isaac J. Jensen,&nbsp;Steven B. Wells,&nbsp;Julien Gras,&nbsp;Donna L. Farber","doi":"10.1002/cpz1.70120","DOIUrl":"10.1002/cpz1.70120","url":null,"abstract":"<p>The majority of immune cells in the human body exist within the tissues rather than in the circulation. Nevertheless, most of our knowledge of the human immune system is biased towards the characterization and understanding of circulating immune cell populations because the latter are readily sampled, whereas cells in tissues are difficult to obtain and/or are limited to single sites of disease. Tissue-resident cells differ from circulating cells due to tissue-specific niche adaptations that influence their phenotype and function. For instance, T cells in tissues, resident memory (T_RM cells), exhibit tissue-specific properties that allow optimal protection from infection due to an acquired ability to coordinate rapid and efficacious pathogen clearance. Thus, to fully understand T-cell responses in various pathological conditions one must focus on the properties of T_RM cells and how they have been shaped by their environment. Moreover, one must sample and analyze T cells from multiple tissues, optimally from the same individual, to determine how infectious, cancer, or autoimmune challenge is affecting homeostatic function. Our longstanding collaboration with the organ procurement organization, LiveOnNY, provides unique access to multiple lymphoid, mucosal, and peripheral tissues from organ donors where consent for research use has been obtained. These samples have enabled characterization of human tissue resident memory T cells and other immune cell types across a variety of tissues. Concomitant with this endeavor, we developed and refined a series of methodologies critical for extracting immune cells from tissue for the purpose of phenotypic and mechanistic interrogation. Here, we describe our optimized protocols for processing select human tissues and the requisite coordination and considerations for their maximal yield and tissue quality. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Isolation of immune cells from blood-rich sites [spleen (SPL), blood (BLD), bone marrow (BOM)]</p><p><b>Basic Protocol 2</b>: Isolation of immune cells from lymph nodes, tonsils, and thymus [iliac lymph nodes (ILN), lung lymph nodes (LLN), mesenteric lymph nodes (MLN), colonic lymph nodes (CLN), hepatic lymph nodes (HLN), tonsils (TON), thymus (THY)]</p><p><b>Basic Protocol 3</b>: Isolation of immune cells from the lungs [lung (LNG), bronchioalveolar lavage (BAL)]</p><p><b>Basic Protocol 4</b>: Isolation of immune cells from the intestines [jejunum epithelial layer (JEL), jejunum lamina propria (JLP), colon epithelial layer (CEL), colon lamina propria (CLP)]</p><p><b>Basic Protocol 5</b>: Isolation of immune cells from the liver (LVR)</p><p><b>Basic Protocol 6</b>: Immune cell staining for flow cytometry</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 3","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143707569","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":"Correction: Establishment and Culture of Human Intestinal Organoids Derived from Adult Stem Cells","authors":"Cayetano Pleguezuelos-Manzano,&nbsp;Jens Puschhof,&nbsp;Stieneke van den Brink,&nbsp;Veerle Geurts,&nbsp;Joep Beumer,&nbsp;Hans Clevers","doi":"10.1002/cpz1.70132","DOIUrl":"10.1002/cpz1.70132","url":null,"abstract":"<p><i>Current Protocols</i> is issuing corrections for the following protocol article.</p><p>Pleguezuelos-Manzano, C., Puschhof, J., van den Brink, S., Geurts, V., Beumer, J., &amp; Clevers, H. (2020). Establishment and culture of human intestinal organoids derived from adult stem cells. <i>Current Protocols in Immunology</i>, <i>130</i>, e106. doi: 10.1002/cpim.106</p><p>In the above-referenced article:</p><p>In step 3 of Basic Protocol 1, collagenase concentration has been changed from “5 mg/ml” to “50 µg/ml”.</p><p>The current version online now includes this correction and may be considered the authoritative version of record.</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 3","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70132","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143707480","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}