Current protocols最新文献

{"title":"Optimized Purification of Human Amyloid-beta (Aβ) 40 and Aβ42 Using an E. coli Expression System","authors":"Nathan Lehman,&nbsp;Pasan Gaminda Kuruppu Achchige,&nbsp;Jun Zhang","doi":"10.1002/cpz1.70197","DOIUrl":"10.1002/cpz1.70197","url":null,"abstract":"<p>Amyloid-beta (Aβ) peptides, primarily Aβ40 and Aβ42, are central to the formation of amyloid plaques, a pathological hallmark of Alzheimer's disease (AD). These peptides, derived from the amyloid precursor protein (APP), are aggregation prone and neurotoxic. Experimental studies aimed at understanding Aβ aggregation and interaction require pure, monomeric peptides with the native sequences, including the absence of an N-terminal methionine. We present an optimized protocol for producing recombinant human Aβ40 and Aβ42 using a SUMO fusion system in <i>Escherichia coli</i>. Cleavage of the SUMO tag enables recovery of native-sequence peptides, producing physiologically relevant monomers with high yield and purity. This method eliminates the need for chemical synthesis and offers a reliable and cost-effective approach to producing recombinant Aβ suitable for aggregation studies, structural analyses, and interaction assays. The resulting peptides closely mimic endogenous Aβ, facilitating accurate models of Alzheimer's disease pathogenesis and supporting future therapeutics development. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol</b>: Expression and purification of Aβ40 and Aβ42 from <i>Escherichia coli</i></p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 8","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-08-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://currentprotocols.onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70197","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144832513","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":"Comprehensive Tools for Culturing Blastocystis: A Standardized Resource for Research and Diagnostics","authors":"Daisy Shaw,&nbsp;Constance Denoyelle,&nbsp;Kevin S. W. Tan,&nbsp;C. Graham Clark,&nbsp;Hisao Yoshikawa,&nbsp;Eric Viscogliosi,&nbsp;Eleni Gentekaki,&nbsp;Kateřina Jirků,&nbsp;Anastasios D. Tsaousis","doi":"10.1002/cpz1.70175","DOIUrl":"10.1002/cpz1.70175","url":null,"abstract":"&lt;p&gt;&lt;i&gt;Blastocystis&lt;/i&gt; spp. is a widely prevalent anaerobic protozoan of uncertain pathogenicity found in the gastrointestinal tracts of over 1 billion people worldwide. Despite its potential significance in health and disease, &lt;i&gt;Blastocystis&lt;/i&gt; spp. remains challenging to culture axenically due to its anaerobic nature and the diversity of its genetic subtypes. This manuscript presents a standardized toolkit for culturing &lt;i&gt;Blastocystis&lt;/i&gt; spp. in xenic and axenic conditions, detailing protocols for the preparation of appropriate liquid and solid media, cryopreservation, and inoculation. By providing a comprehensive set of tools and methodologies, this work aims to streamline research on &lt;i&gt;Blastocystis&lt;/i&gt; spp., enabling reproducibility, subtype characterization, and advancements in understanding its role in the gut microbiome and host health. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Basic Protocol 1&lt;/b&gt;: Establishment of xenic &lt;i&gt;Blastocystis&lt;/i&gt; culture from stool samples in liquid medium&lt;/p&gt;&lt;p&gt;&lt;b&gt;Basic Protocol 2&lt;/b&gt;: Subculturing from existing &lt;i&gt;Blastocystis&lt;/i&gt; liquid culture&lt;/p&gt;&lt;p&gt;&lt;b&gt;Support Protocol 1&lt;/b&gt;: Preparation of modified Jones’ medium for &lt;i&gt;Blastocystis&lt;/i&gt; culturing&lt;/p&gt;&lt;p&gt;&lt;b&gt;Support Protocol 2&lt;/b&gt;: Preparation of trypticase-yeast extract-serum-gastric mucin-9 (TSGYM-9) medium for &lt;i&gt;Blastocystis&lt;/i&gt; culturing&lt;/p&gt;&lt;p&gt;&lt;b&gt;Support Protocol 3&lt;/b&gt;: Preparation of liver extract-yeast extract-serum-gastric mucin (LYSGM) medium for &lt;i&gt;Blastocystis&lt;/i&gt; culturing&lt;/p&gt;&lt;p&gt;&lt;b&gt;Basic Protocol 3&lt;/b&gt;: Subculturing xenic &lt;i&gt;Blastocystis&lt;/i&gt; cultures in diphasic medium&lt;/p&gt;&lt;p&gt;&lt;b&gt;Support Protocol 4&lt;/b&gt;: Preparation of Robinson's medium for &lt;i&gt;Blastocystis&lt;/i&gt; culturing&lt;/p&gt;&lt;p&gt;&lt;b&gt;Support Protocol 5&lt;/b&gt;: Preparation of Ringer's agar slants for &lt;i&gt;Blastocystis&lt;/i&gt; culturing&lt;/p&gt;&lt;p&gt;&lt;b&gt;Basic Protocol 4&lt;/b&gt;: Axenization of &lt;i&gt;Blastocystis&lt;/i&gt; cultures&lt;/p&gt;&lt;p&gt;&lt;b&gt;Basic Protocol 5&lt;/b&gt;: Subculturing axenic &lt;i&gt;Blastocystis&lt;/i&gt; cultures in diphasic medium&lt;/p&gt;&lt;p&gt;&lt;b&gt;Support Protocol 6&lt;/b&gt;: Preparation of Boeck and Drbohlav's Locke-egg serum (LES) medium for &lt;i&gt;Blastocystis&lt;/i&gt; culturing&lt;/p&gt;&lt;p&gt;&lt;b&gt;Support Protocol 7&lt;/b&gt;: Preparation of Boeck and Drbohlav's diphasic modified medium (BDMM) for &lt;i&gt;Blastocystis&lt;/i&gt; culturing&lt;/p&gt;&lt;p&gt;&lt;b&gt;Basic Protocol 6&lt;/b&gt;: Establishment of axenic &lt;i&gt;Blastocystis&lt;/i&gt; cultures in Iscove's modified Dulbecco's medium (IMDM)&lt;/p&gt;&lt;p&gt;&lt;b&gt;Basic Protocol 7&lt;/b&gt;: Establishment of axenic &lt;i&gt;Blastocystis&lt;/i&gt; cultures in soft IMDM agar&lt;/p&gt;&lt;p&gt;&lt;b&gt;Basic Protocol 8&lt;/b&gt;: Establishment of axenic &lt;i&gt;Blastocystis&lt;/i&gt; cultures on solid IMDM agar&lt;/p&gt;&lt;p&gt;&lt;b&gt;Basic Protocol 9&lt;/b&gt;: Optimized method for establishing axenic &lt;i&gt;Blastocystis&lt;/i&gt; cultures on solid IMDM agar&lt;/p&gt;&lt;p&gt;&lt;b&gt;Basic Protocol 10&lt;/b&gt;: Establishment of axenic &lt;i&gt;Blastocystis&lt;/i&gt; cultures in semi-solid Locke's agar&lt;/p&gt;&lt;p&gt;&lt;b&gt;Basic Protocol 11&lt;/b&gt;: Cryopreservation of xenic &lt;i&gt;Blastocystis&lt;/i&gt; cultures&lt;/p&gt;&lt;p&gt;&lt;b&gt;Basic Protocol 12&lt;/b&gt;: Cryopreservation of axenic &lt;","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 8","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-08-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://currentprotocols.onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70175","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144833298","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":"Simple Passive Environmental DNA Samplers for Marine Biomonitoring in Resource-Limited Programs","authors":"Patrick K. Nichols,&nbsp;Cécile M. Vimond,&nbsp;Peter B. Marko","doi":"10.1002/cpz1.70186","DOIUrl":"10.1002/cpz1.70186","url":null,"abstract":"<p>The analysis of environmental DNA (eDNA) is a powerful tool for rapidly assessing biodiversity across aquatic ecosystems. Its implementation remains limited, however, by the logistical complexity of standard eDNA workflows, which often require specialized equipment and expertise. This protocol presents passive environmental DNA samplers (PEDS) as a simplified, low-cost alternative to conventional active water filtration methods. PEDS are designed for ease of use, enabling ambient eDNA capture without pumps or filtration systems, and allowing for rapid deployment and retrieval with short field exposures (∼15 min). We detail procedures for construction, deployment, and retrieval, alongside recommendations for minimizing contamination and optimizing DNA recovery. DNA is extracted from cotton membranes housed within the PEDS unit using a modified Qiagen DNeasy Blood &amp; Tissue protocol. The protocol was validated at the Papahānaumokuākea Marine National Monument, Hawaiʻi, where PEDS were used to detect <i>Chondria tumulosa</i>, a cryptogenic nuisance alga, as part of ongoing conservation efforts. The use of simplistic PEDS and cotton membranes provides a cost-effective and scalable method for researchers seeking to implement eDNA-based monitoring in marine and other aquatic environments. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Assembly and deployment of PEDS on stationary buoys</p><p><b>Alternate Protocol</b>: Assembly and deployment of PEDS on roving surveyors</p><p><b>Basic Protocol 2</b>: Processing and storage of sample membranes</p><p><b>Support Protocol 1</b>: Extraction of DNA from PEDS membranes</p><p><b>Support Protocol 2</b>: Amplification of extracted DNA by PCR</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 8","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-08-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144814645","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 and Application of BRD4-Targeting ByeTACs (Bypassing E-Ligase Targeting Chimeras)","authors":"Cody A. Loy,&nbsp;Timothy J. Harris Jr.,&nbsp;Darci J. Trader","doi":"10.1002/cpz1.70196","DOIUrl":"10.1002/cpz1.70196","url":null,"abstract":"<p>Targeted protein degradation (TPD) has revolutionized the way we think of drug discovery and has the potential for substantial therapeutic benefits. Traditional mechanisms rely on taking advantage of the cells endogenous protein degradation pathway known as the ubiquitin proteasome system (UPS). Traditional proteolysis targeting chimeras (PROTACs) rely on this mechanism by developing heterobifunctional molecules, which are compounds that contain two different ligands that bind two different proteins linked together with varying linker lengths. These compounds typically contain a ligand to a protein of interest that is to be degraded and a linker to the other ligand that binds to an E3 ligase. Once these compounds bind both proteins of interest with the proper confirmation, the E-ligase complex can facilitate the ubiquitination of the protein, leading to its recognition by the proteasome for degradation. This approach has been effective at developing degraders for a wide variety of proteins, yet there remain several challenges, such as limited ligands to E3 ligases, selectivity, and degrading proteins that cannot be ubiquitinated. To overcome these limitations, we developed a new targeted protein degradation approach that can bypass the need for E3 ligases and ubiquitination that we have named ByeTACs. This was accomplished by developing a bifunctional molecule that recruits proteins directly to the 26S proteasome, no longer requiring the E ligase cascade. The protocols presented here describe the synthesis and application of a ByeTAC targeting bromodomain-containing protein 4 (BRD4), that can be generalized to other POIs to assess their “ByeTACability.” © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Synthesis and characterization of a ByeTAC library targeting BRD4</p><p><b>Basic Protocol 2</b>: Assessing degradation of BRD4 ByeTACs in cells</p><p><b>Basic Protocol 3</b>: Validating BRD4 ByeTACs mechanism of action</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 8","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-08-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144811146","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":"Stroke Modeling and Assessment in Mice Using Internal Carotid Artery Occlusion","authors":"Sumana Chakravarty,&nbsp;Shashikant Patel,&nbsp;Mydhili Radhakrishnan,&nbsp;Roli Kushwaha,&nbsp;Priya Jhelum,&nbsp;Arvind Kumar","doi":"10.1002/cpz1.70193","DOIUrl":"10.1002/cpz1.70193","url":null,"abstract":"<p>The internal carotid artery occlusion (ICAO) model in mice replicates a clinically relevant subtype of stroke (mild to moderate ischemic stroke). The ICAO model represents a significant advancement in preclinical stroke research, providing a more accurate representation of human strokes caused by internal carotid artery occlusion. This model has facilitated novel insights into epigenetic modifications following stroke, specifically the dynamics of histone lysine methylation and demethylation, which are crucial in ischemia-induced brain damage and recovery. In contrast to the widely used middle cerebral artery occlusion (MCAO) model, which primarily induces extensive cortical damage, the ICAO model more precisely mimics the striatal and hippocampal injury seen in these stroke cases. Here, we describe the establishment and utilization of the ICAO model in adult CD1 mice, highlighting its reliability and reproducibility in inducing mild to moderate ischemic injury. Our method involves a temporary occlusion of the internal carotid artery for 90 min, followed by reperfusion, leading to localized neural damage. This article details the surgical procedure for inducing ICAO in mice, followed by methods for characterizing the resulting ischemia. These methods include laser doppler perfusion imaging and neurobehavioral assessments, such as neurological deficit scoring and motor function tests. Additionally, the model also emphasizes the importance of considering sex-specific differences in the response to ICAO. This model's ability to yield reproducible and localized neural damage makes it a valuable tool for studying stroke pathophysiology and evaluating potential therapeutic interventions. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Detailed surgical procedure for inducing internal carotid artery occlusion</p><p><b>Basic Protocol 2</b>: Laser doppler perfusion imaging</p><p><b>Basic protocol 3</b>: Neurobehavioral assessments</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 8","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-08-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144805440","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":"Measuring Plasmodesmata-mediated Intercellular Trafficking Using Microparticle Bombardment in Arabidopsis and Crops","authors":"Zhongpeng Li,&nbsp;Connor Thorpe,&nbsp;Shan Jiang,&nbsp;Kyaw Aung","doi":"10.1002/cpz1.70194","DOIUrl":"10.1002/cpz1.70194","url":null,"abstract":"<p>Plasmodesmata (PD) are highly specialized, nanoscopic pores that traverse the cell wall to connect the cytoplasm of adjacent plant cells, enabling direct cell-to-cell communication. PD provides the continuity of three key cellular components: the plasma membrane, the endoplasmic reticulum (ER), and the cytosol. The compressed ER within PD is known as the desmotubule. PD mediates the intercellular trafficking of ions, metabolites, hormones, proteins, and RNA molecules between adjacent cells. Although several methods have been developed to quantify PD-mediated molecular trafficking, it remains a technical challenge. Among these, PD-mediated movement of fluorescent proteins is one of the most commonly used approaches. Here we present a microparticle bombardment method using a biolistic particle delivery system to investigate the PD-mediated movement of fluorescent proteins. We equipped the delivery system with a flow guiding barrel to improve bombardment efficiency and consistency. We demonstrated the effects of gold particle aggregation and plant age on transformation efficiency and protein movement in <i>Arabidopsis</i>. We also showed the feasibility of the method in determining PD-mediated movement in tomato, pepper, and soybean. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol</b>: Microparticle bombardment assay for measuring plasmodesmata-mediated trafficking</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 8","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-08-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12330778/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144796484","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":"Whole Nerve Electrophysiology and Analyses of A-alpha/beta, A-delta, and C Fiber Action Potentials","authors":"Alyssa Flippen,&nbsp;Maggie Dubus,&nbsp;Micaela Dugan,&nbsp;Mark Rainier Macaligiw Catapusan,&nbsp;Namrata Sangwan,&nbsp;Michael Klukinov,&nbsp;Cholawat Pacharinsak,&nbsp;David Yeomans,&nbsp;Thomas Anderson","doi":"10.1002/cpz1.70190","DOIUrl":"10.1002/cpz1.70190","url":null,"abstract":"<p><i>Ex vivo</i> whole peripheral nerve electrophysiology provides a powerful tool for evaluating the effects of current and potential clinical treatments on nerve impulse transmission. It allows quantitative assessment of nerve conduction through compound action potential (AP) recordings, which can be correlated with sensory and motor changes. However, existing literature lacks a comprehensive methodology for isolating and analyzing APs from individual nerve fiber subtypes (e.g., A-alpha/beta, A-delta, and C fibers). This article details the optimal setup conditions and nerve stimulator parameters required to capture APs from specific nerve fibers reliably and provides a detailed and systematic approach for their analysis. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Setup conditions for obtaining individual nerve fiber action potentials</p><p><b>Basic Protocol 2</b>: Nerve handling and parameters for optimal nerve health</p><p><b>Basic Protocol 3</b>: Nerve stimulation to obtain nerve fiber action potentials</p><p><b>Support Protocol 1</b>: Parameters for A-alpha/beta fiber stimulation and recording</p><p><b>Support Protocol 2</b>: Parameters for A-delta and C fiber stimulation and recording</p><p><b>Basic Protocol 4</b>: Quantitative analysis of nerve fiber action potentials</p><p><b>Support Protocol 3</b>: A-alpha/beta fiber analysis</p><p><b>Support Protocol 4</b>: A-delta fiber analysis</p><p><b>Support Protocol 5</b>: C fiber analysis</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 8","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-08-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144796485","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":"Derivatization of Solid Supports Using a Carbamate-Linked Tether Containing a Disulfide Functional Group","authors":"Jagandeep S. Saraya,&nbsp;Derek K. O'Flaherty","doi":"10.1002/cpz1.70174","DOIUrl":"10.1002/cpz1.70174","url":null,"abstract":"<p>Chemical modifications of oligonucleotides are widely used to improve their functional properties. Amino modifiers provide versatile chemical handles for post-synthetic (bio)conjugation, nucleic acid immobilization on solid supports, and studies of nonenzymatic genome replication related to the origins of life. This protocol describes an economical disulfide-containing solid-support linker that enables on-column synthesis of nucleic acids bearing 3′-amino or 3′-phosphate modifications. The orthogonal nature of this linker allows for an on-column protecting group strategy, facilitating the synthesis of DNA and RNA containing 3′-amino-2′,3′-dideoxyribosides from commercially available unprotected mononucleosides. This protocol further extends the on-column construction of oligomers containing 3′-amino-2′,3′-dideoxyribosides to those containing a 2′-amino-2′-deoxynucleoside at the 3′-end, using an orthogonal protecting group strategy. An improved on-column deprotection procedure for DNA and RNA is also presented, eliminating the precipitation step typically utilized in conventional RNA workflows, resulting in an enhanced strand recovery for certain sequences. Finally, the disulfide-containing solid support is applied to the preparation of amino acid–oligonucleotide conjugates, wherein the identity of the final product was contingent upon the specific deprotection conditions employed. All oligonucleotide conjugates and derivatives were purified using standard methods [(e.g., strong anion exchange high-performance liquid chromatography (SAX-HPLC)] and characterized by mass spectrometry (MS). © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Synthesis of 4,4′-dimethoxytrityl (DMTr) disulfide succinimide and solid-support functionalization reaction</p><p><b>Basic Protocol 2</b>: Solid-phase synthesis of DNA containing a 3′-amino-2′,3′-dideoxyguanosine using 3′-Disulfide CPG</p><p><b>Alternate Protocol 1</b>: Synthesis of DNA containing terminal 2′-amino-2′-deoxyadenosine</p><p><b>Alternate Protocol 2</b>: Alternate RNA (terminated with 3′-amino modifiers or phosphate) workflow using <i><b>D</b></i>-TentaGel</p><p><b>Basic Protocol 3</b>: Amino acid–oligonucleotide conjugate synthesis using Lys and <i><b>D</b></i>-TentaGel</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 8","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-08-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12330986/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144801289","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":"Murine Models of Klebsiella pneumoniae Gastrointestinal Infection","authors":"Juan D. Valencia-Bacca,&nbsp;Md. Maidul Islam,&nbsp;Karen M. Haas,&nbsp;M. Ammar Zafar","doi":"10.1002/cpz1.70187","DOIUrl":"10.1002/cpz1.70187","url":null,"abstract":"<p><i>Klebsiella pneumoniae</i> (<i>Kpn</i>) is a Gram-negative opportunistic pathogen and a leading cause of hospital-acquired infections. The gastrointestinal (GI) tract serves as a critical site for <i>Kpn</i> colonization and a reservoir for host-to-host spread that primarily occurs through the fecal-oral route. The emergence of multidrug-resistant strains, coupled with virulence factors such as enhanced capsule production, presents significant challenges for clinical management and public health. Animal models—particularly murine models of GI colonization—are essential for advancing our understanding of <i>Kpn</i> pathogenesis, host-pathogen interactions, and for identifying therapeutic strategies. In this manuscript, we describe standardized protocols for studying <i>Kpn</i> GI colonization and systemic dissemination. These methods are designed to facilitate reproducibility and consistency across laboratories investigating <i>Kpn</i> colonization dynamics, dissemination routes, and immune evasion mechanisms. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p>By providing detailed and optimized experimental approaches, this work aims to support the development of uniform methodologies for <i>Kpn</i> GI colonization and host response studies</p><p><b>Basic Protocol 1</b>: Murine model of <i>Kpn</i> gastrointestinal colonization via oral feeding</p><p><b>Basic Protocol 2</b>: Murine model of <i>Kpn</i> gastrointestinal colonization via gavage</p><p><b>Basic Protocol 3</b>: Murine model of <i>Kpn</i> intraperitoneal challenge</p><p><b>Basic Protocol 4</b>: Murine model of systemic <i>Kpn</i> infection via intravenous injection</p><p><b>Basic Protocol 5</b>: <i>Kpn</i> transmission studies following gastrointestinal colonization with intact microbiota</p><p><b>Basic Protocol 6</b>: Transmission studies of <i>Kpn</i> gastrointestinal colonization in antibiotic-pretreated mice</p><p><b>Support Protocol 1</b>: Preparation of <i>Kpn</i> inoculum</p><p><b>Support Protocol 2</b>: Preparation of mixed <i>Kpn</i> inoculum for competitive infection</p><p><b>Support Protocol 3</b>: Assessment of <i>Kpn</i> bacterial burden</p><p><b>Support Protocol 4</b>: Preparation of cecal filtrate</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 8","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70187","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144773619","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":"Evaluating Neuropsychiatric Disease in Mouse Models of Systemic Lupus Erythematosus","authors":"Baruh Polis,&nbsp;Melodie Zaknoun,&nbsp;Doaa Tehawey,&nbsp;Maria Gulinello,&nbsp;Chaim Putterman","doi":"10.1002/cpz1.70185","DOIUrl":"10.1002/cpz1.70185","url":null,"abstract":"<p>Primary neuropsychiatric manifestations in systemic lupus erythematosus (NPSLE) affect 20% to 40% of patients, significantly impacting quality of life and patient prognosis. However, the complex pathophysiology underlying these manifestations remains incompletely understood, partly due to challenges in distinguishing direct disease effects from secondary complications. Animal models are essential for investigating these mechanisms, but their effective utilization requires standardized behavioral assessment protocols, an area where the field currently lacks sufficient consensus. This article presents seven validated behavioral paradigms optimized for NPSLE mouse models, covering four key behavioral domains: activity levels (open field test); anxiety-like behavior (open field test, dark–light box, elevated plus maze); depression-like behavior (tail suspension test, Porsolt swim test); and cognitive function (novel objects, Barnes maze). We provide detailed protocols for each test, including equipment specifications, procedural steps, parameter measurements, and troubleshooting guidance specifically tailored for NPSLE research. Special considerations for NPSLE mouse models, including potential confounding factors like light sensitivity, motor impairments, and stress sensitivity, are addressed throughout. Additionally, we offer strategic recommendations for test selection based on specific research objectives, emphasizing the importance of test sequencing, proper habituation, and consistent handling techniques. We additionally recommend correlating behavioral outcomes with immunological markers to establish relationships between immune dysfunction and neuropsychiatric manifestations. By standardizing behavioral assessment methodologies in NPSLE research, these protocols enable more reliable cross-laboratory comparisons, enhance reproducibility, and ultimately improve translational relevance. The rigorous behavioral phenotyping approaches detailed herein will facilitate more accurate modeling of neuropsychiatric manifestations in lupus, advance our understanding of underlying disease mechanisms, and accelerate the development of targeted therapeutic interventions for this challenging aspect of SLE. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Open field test: Assessment of locomotor activity and anxiety-like behavior</p><p><b>Basic Protocol 2</b>: Novel object assays: Assessment of learning and memory</p><p><b>Basic Protocol 3</b>: Dark–light box: Assessment of anxiety-like behavior</p><p><b>Basic Protocol 4</b>: Elevated plus maze: Assessment of anxiety-like behavior</p><p><b>Basic Protocol 5</b>: Barnes maze: Assessment of spatial learning and memory</p><p><b>Basic Protocol 6</b>: Tail suspension test: Assessment of depression-like behavior</p><p><b>Basic Protocol 7</b>: Porsolt swim test: Assessment of depression-like behavior</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 7","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-07-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70185","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144725563","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}