Current protocols最新文献

{"title":"The Effort-Based Forage Task: An Ethological Behavioral Test for Assessing Motivation and Apathy-Related Behavior in Mice","authors":"Eleanor W. Grayson,&nbsp;Foteini Xeni,&nbsp;Caterina Marangoni,&nbsp;Megan G. Jackson","doi":"10.1002/cpz1.70195","DOIUrl":"10.1002/cpz1.70195","url":null,"abstract":"<p>Apathy and other disorders of motivation represent a significant clinical problem but do not have an agreed treatment approach. The use of translational animal models could facilitate drug development and advance treatment approach. The effort-based forage task provides a readout of motivational state in mouse models based on their intrinsic drive to forage for nesting material. In this task the mouse is placed in an arena composed of an enclosed home area and a foraging area joined via a tube. Throughout the session the mouse can freely choose to traverse the tube to reach the forage area, obtain nesting material, and shuttle it back to the home area. The nesting material requires effort to obtain and is pulled through apertures in a custom designed nesting material box. The amount of nesting material foraged provides a readout of motivational state, where a deficit in foraging indicates a reduction in motivation. The task does not require physiological (food/water) restriction to motivate the animal to perform in the task, and it does not require training beyond initial habituation to the task environment. The task has been used in behavioral phenotyping of disease models and has been used to test the effects of a wide range of pharmacological manipulations on motivational state. The task environment can be altered to test additional behavioral components that contribute to motivational deficit including effort-based modulation of behavior and affective reactivity. Overall, the task provides a rapid, translationally relevant method for understanding changes in motivated behavior independent of physiological restriction at the preclinical level. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Support Protocol</b>: Animal husbandry and arena set up</p><p><b>Basic Protocol</b>: Habituation and acute pharmacological manipulation</p><p><b>Alternate Protocol 1</b>: The effort curve paradigm</p><p><b>Alternate Protocol 2</b>: Affective reactivity test</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 9","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://currentprotocols.onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70195","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144929633","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":"Optimized Enrichment of CD71+ Reticulocytes From Whole Blood for Single-Cell RNA Sequencing","authors":"Hanneda A. Fomukong,&nbsp;Idowu A. Aimola,&nbsp;Abubakar Sani,&nbsp;Mamman Mohammed,&nbsp;Halima Bello-Manga,&nbsp;Gloria Chechet,&nbsp;Joy U. Ameloko,&nbsp;Reuben S. Baba,&nbsp;Abduljabar Musa,&nbsp;Pecular Nwanyibunwa Okoro","doi":"10.1002/cpz1.70203","DOIUrl":"10.1002/cpz1.70203","url":null,"abstract":"<p>Reticulocytes are a group of immature red blood cells (RBCs) produced in the bone marrow and released into the bloodstream where they mature into RBCs within 1 to 2 days. They play a crucial role in assessing bone health and many hematological diseases. Isolating viable reticulocytes for use in single-cell transcriptomics has been a challenge due to their similarity to mature RBCs. Their low concentration and continuous maturation to RBCs with time also poses a great challenge. In this article, we optimized a method for enrichment of CD71⁺ reticulocytes from whole blood, with a high yield and viability for downstream studies. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Blood tissue preparation and reticulocyte enrichment</p><p><b>Basic Protocol 2</b>: Visualization and determination of cell viability</p><p><b>Alternate Protocol</b>: Counting cells using a hemocytometer</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 9","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144929634","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":"Preparation of Cuvette-Based Sorters for Sorting Submicron Microbial Cells and Viruses from Environmental and Biological Samples","authors":"Jamie C. Tijerina,&nbsp;Francisco Martinez-Hernandez,&nbsp;Vera Beilinson,&nbsp;Olivia Finney,&nbsp;Victoria J. Orphan,&nbsp;Rochelle A. Diamond","doi":"10.1002/cpz1.70176","DOIUrl":"10.1002/cpz1.70176","url":null,"abstract":"&lt;p&gt;This protocol set focuses on the preparation of the BD FACSAria II/III/Fusion, a cuvette-based cell sorting system commonly found in shared resource settings, to sort submicron samples, including but not limited to virus-like particles (VLPs) and bacteria. This is meant to serve as a proven workflow for staff in general shared resource laboratories (SRL) and individual labs. It is also useful for labs purchasing cuvette-based sorters with similar fluidic paths to the FACSAria Fusion from BD Biosciences, such as the BD FACSSymphony S6 and BD FACSDiscover S8, as well as for specialized SRLs that will need to move away from Influx and MoFlo platforms that are approaching end of life. VLPs and submicron-sized cells (e.g., ultramicrobacteria and archaea) are found at or near the limit of detection of most flow cytometers and cell sorters. With VLPs, the small quantity of DNA recovered requires amplification before downstream sequencing. Thorough cleaning of the fluidic system and careful sample preparation are necessary both to improve detection and to prevent genomic contamination from amplifiable free DNA or microorganisms. These protocols include instructions for the preparation of sheath fluid, decontamination of the cell sorter, and removal from the fluidic system of free DNA and endotoxin that could interfere with the high-throughput amplification and sequencing of the target DNA in downstream processes. To minimize noise when sorting submicron-sized samples, clean PBS filtered with a 0.1-µm-pore-size filter is prepared, minimizing microbubbles and particulates; use of commercially available sheath fluids is not recommended, as they contain preservatives and surfactants that can affect microbial viability and are not filtered at the optimal pore size for this experimentation. In addition, detailed steps are provided for cleaning the instrument, tanks, and related media to prepare the sort, along with guidance for setting up the software, voltages, and gating strategies for successful experiments. © 2025 Wiley Periodicals LLC.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Basic Protocol 1&lt;/b&gt;: Preparation of the BD FACSAria II/III/Fusion cell sorter fluidics and software to perform sorts for validation by culture or microscopy&lt;/p&gt;&lt;p&gt;&lt;b&gt;Basic Protocol 2&lt;/b&gt;: Preparation of the BD FACSAria II/III/Fusion cell sorter fluidics and software to perform sorts for high-throughput whole-genome amplification and genomic sequencing&lt;/p&gt;&lt;p&gt;&lt;b&gt;Support Protocol 1&lt;/b&gt;: Autoclaving the BD FACSAria II/III/Fusion stainless-steel sheath tank&lt;/p&gt;&lt;p&gt;&lt;b&gt;Support Protocol 2&lt;/b&gt;: Chemical decontamination and maintenance of the BD FACSAria II/III/Fusion stainless-steel sheath fluid tank&lt;/p&gt;&lt;p&gt;&lt;b&gt;Support Protocol 3&lt;/b&gt;: Preparation of 1 L of 1× PBS&lt;/p&gt;&lt;p&gt;&lt;b&gt;Support Protocol 4&lt;/b&gt;: Inspection of the BD FACSAria II/III/Fusion cell sorter for contamination&lt;/p&gt;&lt;p&gt;&lt;b&gt;Support Protocol 5&lt;/b&gt;: Manual aseptic sorting procedure using BD FACSAria II/III/Fusion cell sorter&lt;/p&gt;&lt;p&gt;&lt;b&gt;Support Protocol 6&lt;/b&gt;: Chemical decon","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 8","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-08-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144888318","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":"Novel In Vitro Culture of Microglia from Aged Mice: Implications for the Future of Aging Neurobiology Research","authors":"Katie L. Reagin,&nbsp;Rae-Ling Lee,&nbsp;Kristen E. Funk","doi":"10.1002/cpz1.70199","DOIUrl":"10.1002/cpz1.70199","url":null,"abstract":"<p>Aging is associated with elevated levels of inflammation across tissues, a status recognized as “inflammaging.” Within the brain, microglia are the resident phagocytic immune cells that are important in both homeostatic and disease states. Aged microglia are susceptible to processes of “inflammaging,” which can include higher expression of baseline levels of inflammatory signals, decline in functional activity, and contribution to neurodegenerative processes. Information about microglial function has been gained using <i>in vitro</i> cell culture methods; however, most studies described previously have used microglia cultured from neonatal mice. More recent studies have used microglia cultured from young adult mice, but those using microglia from aged mice are lacking. Considering the distinct changes that come with aging and the important role of microglia in age-related neurologic disorders, there is a need for reliable protocols for studying aged cells specifically. Here, we describe a method to culture primary microglia from aged mice. Collected brain tissue is digested using enzymatic and mechanical techniques and then cultured in specific medium that supports the continued survival and proliferation of adult and aged microglia. To confirm microglial identity, cultured cells were immunostained for microglia-specific markers and imaged by microscopy and flow cytometry. We also compared the activation status of adult and aged microglia that were cultured versus those that were assessed directly after collection. Microglial cultures can easily be manipulated via genetic modifications or pharmacologic intervention to test specific functions. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol</b>: Culturing primary microglia from adult and aged mice</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 8","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://currentprotocols.onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70199","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144869885","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":"Integrating Prism-based TIRF with Confocal Microscopy: An Economical Approach for smFRET Experiments","authors":"Pratibha Agarwala,&nbsp;Dibyendu K. Sasmal","doi":"10.1002/cpz1.70165","DOIUrl":"10.1002/cpz1.70165","url":null,"abstract":"<p>Total internal reflection fluorescence (TIRF) microscopy enables the observation of complex bioassemblies and macromolecular dynamics in high spatial-temporal resolution at the single-molecule level in real time. Through TIRF illumination, fluorophores near a sample substrate are selectively excited within an evanescent field, thereby overcoming the axial diffraction limit of light. Prism-based TIRF (p-TIRF) microscopes are relatively straightforward to construct and can be readily adapted to accommodate a wide range of experimental applications, including the examination of macromolecular complexes, the study of the behavior of vesicles and small organelles, and the investigation of protein-DNA complexes at the single-molecule level. These experiments can give unique insights into the mechanisms driving the molecular interactions that underline many fundamental activities within the cell by providing information on fluctuation distributions and unusual events. In this paper, we present a detailed and cost-effective protocol for constructing a p-TIRF setup using an existing confocal microscope, utilizing the same light source for both modalities. Additionally, we provide a step-by-step tutorial on building, assembling, and aligning the p-TIRF setup and preparing the sample for single-molecule fluorescence resonance energy transfer (smFRET) experiments. This article will be particularly helpful for laboratories equipped with a confocal microscope seeking to expand their experimental capabilities by integrating TIRF-based approaches. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol</b>: Microscopy setup for TIRF</p><p><b>Support Protocol 1</b>: Construction of prism holder and prism holder carrier arm</p><p><b>Support Protocol 2</b>: Preparation of sample chamber with sample</p><p><b>Support Protocol 3</b>: Slide preparation and KOH etching</p><p><b>Support Protocol 4</b>: Preparation of biotinylated DNA Holliday junctions immobilized on slides</p><p><b>Support Protocol 5</b>: Preparation of DNA Holliday junctions</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 8","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144869886","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":"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}