Current protocols最新文献

{"title":"An Aryl Diazonium-Based Enrichment Method for Deep Coverage of Monomethyllysine Proteomics","authors":"Lufeng Yan,&nbsp;Manqian Zheng,&nbsp;Mingzhu Fan,&nbsp;Shan Feng,&nbsp;Mingxuan Wu","doi":"10.1002/cpz1.70170","DOIUrl":"10.1002/cpz1.70170","url":null,"abstract":"<p>Lysine methylation is an important post-translational modification (PTM) that regulates diverse cellular processes. Proteomic analysis is a robust method to study PTMs, but a lack of good enrichment tool limits current understanding of lysine methylation. In a previous study, we demonstrated that aryl diazonium containing 2,6-dimethoxy substitutions can conjugate monomethyllysine-modified (Kme1) peptides with high selectivity and that the reaction is reversible under acidic conditions, allowing the release of Kme1 peptides. Therefore, such a warhead has great potential for the enrichment of low-abundance Kme1 peptides from biological samples. Here, we report the preparation of aryl diazonium-functionalized resins as enrichment tools and their application for mass spectrometry-based proteomic studies of Kme1 peptides. In this procedure, aniline with a PEG linker as a precursor is synthesized and then coupled to a hydrophilic solid phase. After preparation of tryptic peptides from cell samples, the aniline groups on the resin are converted to aryl diazonium for Kme1 peptide capture. After sufficient treatment and washing, the covalent linkage is broken under acidic conditions to release the original Kme1 peptides from the resin. Finally, the enriched samples are processed by mass spectrometry scanning and data analysis to identify ∼10,000 Kme1 sites in cell or mouse tissue samples. Herein, we demonstrate an efficient Kme1 peptides enrichment strategy for deep coverage of the Kme1 proteome in biological samples. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Preparation of aryl aniline-functionalized Sepharose resin</p><p><b>Basic Protocol 2</b>: Preparation of tryptic peptides from biological samples</p><p><b>Basic Protocol 3</b>: Enrichment of Kme1 peptides from whole-cell lysate tryptic peptides</p><p><b>Basic Protocol 4</b>: Data acquisition and analysis for Kme1 proteomics</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 7","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-07-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144573923","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":"An Aqueous-Compatible On-Column Approach for the Conjugation of Nucleic Acids Using Amino Modifiers","authors":"Jagandeep S. Saraya,&nbsp;Derek K. O'Flaherty","doi":"10.1002/cpz1.70169","DOIUrl":"10.1002/cpz1.70169","url":null,"abstract":"<p>Nucleic acid conjugation has emerged as a powerful strategy for enhancing the chemical and biological versatility of synthetic oligonucleotides. Solid-support synthesis of oligonucleotides provides an avenue for nucleic acid conjugation, so long as the method is inherently compatible with the synthesis cycle. Many methods exist, post-synthetically (i.e., after strand extension via the DNA synthesizer), to add ligands to the strand while it is still bound to the solid support. These, however, tend to require stringent reaction conditions (i.e., anhydrous, degassed solvents, special apparatus), making them sometimes impractical for routine use. This article describes a streamlined aqueous-compatible on-column conjugation strategy for preparing nucleic acids containing site-specific chemical modifications. This method utilizes commercially available or easily synthesized monophosphate or carboxylate-containing ligands and solid-phase synthesized oligonucleotides containing amino-modified termini. Coupling is enabled by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and <i>N</i>-methylimidazole (<i>N</i>-MeIm) chemistry in buffered aqueous-organic mixtures. The resulting conjugates are processed using typical deprotection and solid-support cleavage protocols, purified by standard techniques such as strong anion exchange high-performance liquid chromatography (SAX-HPLC), and are 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 zidovudine 5′-<i>O</i>-monophosphate</p><p><b>Basic Protocol 2</b>: On-column phosphoramidate-mediated conjugation of 5′-<i>O</i>-phosphorylated AZT to 5′-amino DNA</p><p><b>Basic Protocol 3</b>: On-column amide-mediated conjugation of Fmoc-protected glycine to 5′-amino modified DNA</p><p><b>Basic Protocol 4</b>: On-column amide-mediated conjugation of potassium benzoate to 5′-amino modified RNA</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 7","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-07-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70169","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144550961","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":"PASTa: An Open-Source Analysis and Signal Processing Toolbox for Fiber Photometry Data","authors":"Rachel M. Donka,&nbsp;Maxine K. Loh,&nbsp;Vaibhav R. Konanur,&nbsp;Mitchell F. Roitman,&nbsp;Jamie D. Roitman","doi":"10.1002/cpz1.70161","DOIUrl":"10.1002/cpz1.70161","url":null,"abstract":"<p>Fiber photometry is a rapidly growing technique to record real-time neural signaling in awake, behaving subjects. However, the processing and analysis of photometry data streams can be complicated, and there is wide divergence in methods across the field. Although several open-source signal processing tools exist, platforms can be inflexible in accommodating experimental designs, lack consistency in signal peak detection, and difficult to use for novice users. In addition, more recent sensors produce fluorescence that may be detected on both signal and control streams, confounding signal processing. To remedy these challenges, we developed PASTa (Photometry Analysis and Signal Processing Toolbox), an open-source MATLAB-based toolbox and protocol for the processing and analysis of fiber photometry data. PASTa includes a full analysis pipeline from data preparation through signal processing and transient event detection. Default parameters were selected to provide users with a conservative starting place, with optional inputs to include other commonly used methods and techniques in the field. Additionally, the transient detection protocol adopts options for determining peak detection thresholds and pre-peak baselines to allow more reliable detection of events and characterization of transient kinetics. Although operating through MATLAB, the code is annotated to be readable, accessible for new users, and adaptable. Ultimately, the PASTa protocol introduces a customizable, user-friendly platform to process fiber photometry signals and detect and quantify transient events. Novel methods for the analysis of signal processing will continue to be integrated into the toolbox, seeking to provide fiber photometry users with access to a comprehensive analysis suite that can accommodate the wide diversity of sensors and experimental paradigms used in photometry. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Software installation</p><p><b>Basic Protocol 2</b>: Data preparation</p><p><b>Basic Protocol 3</b>: Signal processing</p><p><b>Basic Protocol 4</b>: Transient detection and analysis</p><p><b>Support Protocol</b>: Function default customization</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 7","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-07-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70161","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144536978","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":"An Introduction to Causal Inference Methods with Multi-omics Data","authors":"Minhao Yao,&nbsp;Zhonghua Liu","doi":"10.1002/cpz1.70168","DOIUrl":"10.1002/cpz1.70168","url":null,"abstract":"<p>Omics biomarkers play a pivotal role in personalized medicine by providing molecular-level insights into the etiology of diseases, guiding precise diagnostics, and facilitating targeted therapeutic interventions. Recent advancements in omics technologies have resulted in an increasing abundance of multimodal omics data, providing unprecedented opportunities for identifying novel omics biomarkers for human diseases. Mendelian randomization (MR) is a practically useful causal inference method that uses genetic variants as instrumental variables to infer causal relationships between omics biomarkers and complex traits/diseases by removing hidden confounding bias. In this article, we first present current challenges in performing MR analysis with omics data and then describe four MR methods for analyzing multi-omics data, including epigenomics, transcriptomics, proteomics, and metabolomics data, all executable within the R software environment. © 2025 Wiley Periodicals LLC.</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 6","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-06-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144473121","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":"Analysis of Autophagy Under Abiotic Stress in Arabidopsis Seedlings Expressing the GFP-ATG8 Autophagosome Marker","authors":"William Agbemafle,&nbsp;Vishadinie Jayasinghe,&nbsp;Diane C. Bassham","doi":"10.1002/cpz1.70166","DOIUrl":"10.1002/cpz1.70166","url":null,"abstract":"<p>Plant autophagy is a catabolic process where cellular components such as protein aggregates and dysfunctional organelles are degraded and recycled to maintain homeostasis and facilitate stress resilience. Autophagy relies on a double-membrane vesicle called the autophagosome, which delivers cellular cargo to the vacuole for degradation. The Arabidopsis GFP-ATG8 reporter line is a valuable tool widely used to visualize and quantify autophagosomes via microscopy and monitor autophagic degradation via immunoblotting. Consistent assessment of autophagic activity requires standardized protocols for sample preparation, imaging, and data analysis. Here, we present protocols for monitoring autophagy in Arabidopsis seedlings expressing GFP-ATG8, including treatments to induce or inhibit autophagic flux, as well as imaging and image analysis procedures. These methods enable reliable evaluation of autophagic activity and can be adapted for diverse experimental conditions and genotypes. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Growth of Arabidopsis seedlings</p><p><b>Basic Protocol 2</b>: Activation of autophagy in Arabidopsis seedlings by abiotic stresses</p><p><b>Basic Protocol 3</b>: Inhibition of vacuolar degradation by concanamycin A treatment</p><p><b>Basic Protocol 4</b>: Quantification of GFP-ATG8-labeled autophagosomes in Arabidopsis seedlings via microscopy</p><p><b>Basic Protocol 5</b>: Analysis of autophagic degradation of GFP-ATG8 via immunoblotting</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 6","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-06-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70166","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144472890","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":"Phosphoramidite Platform for the Automated Synthesis of Morpholino Antisense Oligonucleotides and Their Chimeras","authors":"Atanu Ghosh,&nbsp;Maria Mukherjee,&nbsp;Arpan Banerjee,&nbsp;Shreyasi Acharya,&nbsp;Surajit Sinha","doi":"10.1002/cpz1.70162","DOIUrl":"10.1002/cpz1.70162","url":null,"abstract":"<p>Phosphorodiamidate morpholino oligonucleotides (PMOs) are antisense oligonucleotides that modulate gene expression through a steric blocking mechanism. Despite their increasing use in antisense therapeutics, the synthesis of PMOs remains challenging, thus limiting their accessibility. As a result, a modular synthesis protocol for PMOs, compatible with DNA/RNA synthesizers, is highly anticipated to facilitate the development of next-generation PMO chimeras incorporating other oligonucleotide backbones with proven therapeutic efficacy. In this article, we demonstrate a streamlined 5′→3′ phosphoramidite approach for synthesizing PMOs using 3′-<i>N</i>-MMTr (monomethoxytrityl)-5′-<i>^tBu</i> (tert-butyl)-morpholino phosphoramidites and 3′-<i>N</i>-Tr(trityl)-5′-CE(cyanoethyl)-morpholino phosphoramidites, enabling the synthesis of thiophosphoramidate (TMO) and phosphoramidate (MO) morpholino oligonucleotides. The phosphoramidite building blocks were synthesized from 5′-OH morpholino monomers derived from commercially available ribonucleosides. The solid-phase oligonucleotide synthesis was carried out in acetonitrile solvent on controlled pore glass (CPG) support using an automated DNA/RNA synthesizer, resulting in excellent overall yields. The synthesis process also incorporated regular amide protecting groups for the exocyclic amines of cytidine and adenosine, as well as dimethylformamidine for guanosine. The solid-phase synthesis cycle consists of the following steps: (a) deblocking the 3′-N protecting group of the morpholino ring, (b) neutralization, (c) coupling and oxidation of the 5′-phosphoramidite (oxidative coupling), and (d) capping unreacted morpholino-NH. Afterward, cleavage from the solid support is achieved through treatment with aqueous ammonia. This method provides a convenient and efficient approach for synthesizing various biologically relevant antisense oligonucleotide (ASO) designs, including PMO–TMO and PMO–MO chimeras, with reproducible and high yields. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Synthesis of 3′-<i>N</i>-Tr-5′-CE-morpholino phosphoramidites</p><p><b>Basic Protocol 2</b>: Synthesis of 3′-N-MMTr-5′-<i>^tBu</i>-morpholino phosphoramidites</p><p><b>Basic Protocol 3</b>: Solid phase synthesis of PMO, TMO, PMO–TMO, and other chimeras in an automated DNA synthesizer</p><p><b>Basic Protocol 4</b>: HPLC purification of synthesized ONs</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 6","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-06-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144473116","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":"Semi-Automated Method for Analyzing Lymphatic Vessels From Immunofluorescence Images","authors":"Victor Rojas-Henríquez,&nbsp;Alexandra Olate-Briones,&nbsp;Celia Salazar,&nbsp;Noelia Escobedo,&nbsp;Andrés A. Herrada","doi":"10.1002/cpz1.70160","DOIUrl":"10.1002/cpz1.70160","url":null,"abstract":"<p>The lymphatic vasculature (LV) plays a crucial role in regulating the trafficking of immune cells into lymph nodes during homeostasis and under inflammatory conditions. LV dysfunction has been linked to various inflammatory and neuroinflammatory diseases. Thus, assessing the LV coverage area using confocal immunofluorescence microscopy is essential for accurately determining the extent of inflammation in the tissue. However, manual selection processes may require considerable time to finalize and are inherently more prone to errors, which can compromise the consistency of results among various users and lead to imprecise measurements. Here, we have developed an improved protocol to determine the area of LV covering the intestinal and meningeal tissues. By using this protocol, regions of interest can be identified more accurately and efficiently than through manual measurement, allowing for efficient analysis of changes in LV coverage during inflammation. Colon tissue and meninges obtained from mice under different experimental conditions were stained with LV-specific antibodies. Confocal microscopy was used to capture all images. Here, the manual selection protocol was compared with our semi-automatic selection protocol using the Fiji software Threshold tool. Using this tool, we successfully automated the selection of regions of interest, achieving greater accuracy in our values and minimizing both errors and the time spent by the operator. Additionally, this tool produces much more specific selections than those performed manually, and the results are more accurate. Finally, it is essential to note that this protocol can be adapted for use with other microscopy techniques, provided the image has sufficient contrast against the background and the scale size is known. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Immunofluorescence of Colon Tissue Section and Whole-Mount Meninges</p><p><b>Basic Protocol 2</b>: Semi-Automated Analysis of Lymphatic Vasculature Using the Fiji Software</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 6","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-06-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144473125","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":"Two-Photon Microscopy Functional Assays for Serial Imaging of Brain Microvessels and the Neurovascular Unit Through Disease Progression","authors":"Kareem El-Ghazawi,&nbsp;William A. Mills (III),&nbsp;Shayn M. Peirce,&nbsp;Ukpong B. Eyo","doi":"10.1002/cpz1.70167","DOIUrl":"10.1002/cpz1.70167","url":null,"abstract":"<p>Microvascular impairments are a major issue in many diseases of the brain. In fact, they are often considered the earliest pathological phenomena in neurodegenerative diseases like Alzheimer's Disease or the locus of pathology as in stroke. Still, little is known about the mechanistic and cellular level events that contribute to these impairments. Given the importance of the neurovascular unit (NVU) in maintaining functional brain tissue, alterations to NVU cell types are important to study in the context of disease progression. With the use of two-photon microscopy, microvessels and cells can be imaged and evaluated throughout disease progression. Herein we aim to provide a comprehensive protocol for setting up and using two-photon microscopy for serial imaging of neurovascular unit cell types (i.e., pericytes, astrocytes, and microglia). We also describe interpreting results from cell and vessel changes based on disease or functional tests through multiple timepoints. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Mouse set up for serial, live 2-photon imaging</p><p><b>Basic Protocol 2</b>: Field of view acquisitions over multiple timepoints</p><p><b>Basic Protocol 3</b>: Line scan acquisitions for acquiring red blood cell metrics</p><p><b>Basic Protocol 4</b>: Quantitative measurements of vessel and cell changes</p><p><b>Basic Protocol 5</b>: Documenting changes in astrocyte morphology following induction of transient focal photothrombotic stroke using Rose Bengal</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 6","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-06-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70167","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144473119","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":"An Alternative Procedure for the Storage and Preservation of Vermamoeba vermiformis and Other Amoebae in the Laboratory","authors":"Nidia Esther Colquehuanca Arias,&nbsp;Matheus Felipe dos Reis Rodrigues,&nbsp;Bruna Nascimento Neiva,&nbsp;Juliana Miranda Tatara,&nbsp;Gabriel Magno de Freitas Almeida,&nbsp;Jônatas Santos Abrahão","doi":"10.1002/cpz1.70164","DOIUrl":"10.1002/cpz1.70164","url":null,"abstract":"<p>Free-living amoebae (FLA), particularly <i>Vermamoeba vermiformis</i> and <i>Acanthamoeba</i> spp., have emerged as indispensable models for the study of giant viruses. These organisms provide a unique and efficient system for the isolation and replication of giant viruses, serving as known permissive hosts and thus playing a pivotal role in advancing research in this field. FLA can alternate between vegetative (trophozoite) and resistant (cyst) forms during their life cycle. Current methods for FLA storage and preservation face limitations, including prolonged excystment times and low rates of trophozoite viability. To address these challenges, these protocols provide instructions for the storage and preservation of <i>Vermamoeba vermiformis</i> at refrigeration temperatures, allowing feasible and rapid reactivation of cultures as an alternative to the more fastidious cyst-based storage methods. This method is also applicable to other FLA species, such as <i>Acanthamoeba castellanii</i> and <i>Acanthamoeba polyphaga</i> © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: <i>Vermamoeba vermiformis</i> propagation</p><p><b>Basic Protocol 2</b>: Preparation of <i>Vermamoeba vermiformis</i> stocks for cold room storage</p><p><b>Basic Protocol 3</b>: Reactivation of cold-stored <i>Vermamoeba vermiformis</i></p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 6","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144367518","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":"Mapping Motor Learning Stages: A Longitudinal fNIRS-Based Assessment of Cortical Activation","authors":"Xiaoli Li,&nbsp;Yongxin Zhu,&nbsp;Hongman Wei,&nbsp;Nan Zhang,&nbsp;LianHui Fu,&nbsp;Qi Qi","doi":"10.1002/cpz1.70147","DOIUrl":"10.1002/cpz1.70147","url":null,"abstract":"<p>Here we describe a protocol to measure changes in cortical activation over stages of motor learning. Participants are recruited and assigned to either simple or complex motor tasks, performed using their non-dominant hand over 10 days. Motor performance is measured using the Minnesota Manual Dexterity Test, while cortical activation is assessed using functional near-infrared spectroscopy (fNIRS). The primary goal is to assess how varying levels of task complexity affect motor learning and to characterize the corresponding neural activity. Generalized estimating equations (GEE) are used to establish marginal models for the statistical analysis of factors influencing motor learning. This protocol has potential use for therapeutic applications, particularly in neurological rehabilitation contexts, where the findings could help inform recovery protocols for patients with motor impairments, such as stroke survivors. By establishing a clear understanding of motor learning stages and associated brain activity, this research could guide the development of noninvasive brain stimulation techniques, such as transcranial magnetic stimulation (TMS) and transcranial direct current stimulation (tDCS), in rehabilitative treatments. Beyond its therapeutic applications, use of this protocol may contribute to the broader understanding of how task complexity influences motor learning in real-world settings. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Task design for motor learning and cortical activation</p><p><b>Basic Protocol 2</b>: fNIRS set up and data collection</p><p><b>Support Protocol</b>: Data analysis using GEE</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 6","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-06-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144315035","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}