Methods and Protocols最新文献

{"title":"Analysis of Osteocalcin Hormone Proteoforms with Vitamin D Deficiency in Patients with Diabetes Mellitus Using Multiple-Reaction Monitoring-Mass Spectrometric Immunoassay Approach.","authors":"Refat M Nimer, Hicham Benabdelkamel, Afshan Masood, Maha Al Mogren, Salini Scaria Joy, Anas M Abdel Rahman, Assim A Alfadda","doi":"10.3390/mps9040117","DOIUrl":"10.3390/mps9040117","url":null,"abstract":"<p><p>Osteocalcin (OC), a bone protein, is evaluated by its proteoforms, which are implicated in various illnesses. Rapid, clinically used immunoassays are well-established. Epitope dependence, fragment cross-reactivity, uneven identification across carboxylation states, and lack of standardization restrict their efficacy. This study aims to characterize various OC proteoforms in patients with type 2 diabetes mellitus (T2DM) and vitamin D insufficiency and to develop a mass spectrometric immunoassay (MSIA) for their detection. The first MRM-MSIA test to specifically detect OC proteoforms in plasma with vitamin D deficiency-induced alterations in T2DM patients was developed and validated. A precise and selective MRM-MSIA was developed to assess OC fragments in plasma from T2DM patients with normal (DN) or deficient (DD) vitamin D levels. The approach examined OC-1 (aa64-71), OC-2 (aa64-70), and OC-3 (aa72-93) proteoforms. It met international linearity, precision, and accuracy criteria with variability <15% and intra/inter-day accuracy of 88.8% to 110.1%. The DD group had significantly higher OC-1 levels than the DN group. The receiver operating characteristic (ROC) curve for OC-1 produced an AUC of 0.8263 (95% confidence interval [CI], <i>p</i> = 0.0004). The developed MRM-MSIA measures plasma OC proteoforms with good specificity and sensitivity, making it a promising clinical diagnostic tool.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 4","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-08-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13515257/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148830206","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":"Single-Dose L-Lysine-Induced Pancreatitis Followed by Pancreatic Neoplastic Progression in Adult <i>Kras/Trp53</i>-Driven Mice.","authors":"Akihiro Kaneda, Masahiro Yoshida, Yoshiya Kawaguchi, Kenichiro Furuyama","doi":"10.3390/mps9040116","DOIUrl":"10.3390/mps9040116","url":null,"abstract":"<p><p>Pancreatic ductal adenocarcinoma (PDAC) remains a highly lethal malignancy, highlighting the need for reproducible experimental systems that model pancreatic injury and subsequent neoplastic progression. Adult-onset genetically engineered models in which oncogenic <i>Kras</i> and mutant <i>Trp53</i> are induced in the pancreas provide a relevant platform for studying PDAC pathogenesis, but efficient neoplastic progression often requires concomitant pancreatic injury. Cerulein-induced pancreatitis is widely used to provide an injury-associated inflammatory stimulus that promotes PDAC progression; however, repeated-injection regimens are labor-intensive and increase cumulative handling stress in animals. Here, we describe a dose-optimized, single-dose L-Lysine protocol designed to induce acute pancreatitis and characterize subsequent pancreatic neoplastic progression in adult-onset <i>Kras/Trp53</i>-driven mice. Tamoxifen-treated <i>Ptf1a</i>^CreER/+; <i>Kras</i>^LSL-G12D/+; <i>Trp53</i>^LSL-R172H/+; Rosa26-RFP mice received a single intraperitoneal injection of L-Lysine, followed by biochemical, histological, and immunohistochemical assessment of pancreatic injury and tumor development. A single 2.0 g/kg L-Lysine injection induced sublethal acute pancreatitis characterized by increased serum pancreatic enzymes, interstitial edema, inflammatory cell infiltration, acinar cell necrosis, and rapid mitochondrial alterations. During 3-6 months of follow-up, mice developed multifocal acinar-to-ductal metaplasia, PanIN lesions, invasive PDAC, and peritoneal dissemination. This protocol provides a simple, synchronized, and less labor-intensive model for studying pancreatic injury and subsequent neoplastic progression in adult-onset <i>Kras/Trp53</i>-driven mice.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 4","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-08-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13516760/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148830489","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":"Screening for Sleep-Disordered Breathing Risk in Pediatric Dental Care: A Protocol Combining Questionnaire-Based Stratification and Wearable Home Sleep Monitoring.","authors":"Parker Norman, Jaclyn Bain, Linda Sangalli, Mitchell Levine, Caroline M Sawicki","doi":"10.3390/mps9040115","DOIUrl":"10.3390/mps9040115","url":null,"abstract":"<p><p>Pediatric sleep-disordered breathing (SDB) is underdiagnosed despite its associations with adverse neurobehavioral, psychosocial, and cardiometabolic outcomes. Pediatric dental providers routinely evaluate craniofacial growth and maintain longitudinal contact with children, yet practical approaches for integrating SDB risk assessment into dental settings remain limited. This protocol describes a prospective, cross-sectional observational study that will enroll 60 school-aged children aged 8-13 years from a university-based pediatric dental clinic and classify them as low-risk or high-risk for SDB using the Pediatric Sleep Questionnaire (PSQ; threshold ≥ 0.33). The primary aim is to examine associations between PSQ-based risk classification and objective physiologic sleep parameters, including the Apnea-Hypopnea Index, Respiratory Disturbance Index, Sleep Apnea Indicator, and Sleep Quality Index, obtained from a U.S. Food and Drug Administration-cleared wearable home sleep monitor-SleepImage Ring (MyCardio LLC, Denver, CO, USA)-worn for a minimum of three consecutive nights. Secondary aims will evaluate associations between SDB risk classification and body mass index, Mallampati score, Brodsky tonsillar grade, and psychosocial functioning (anxiety, depression, perceived stress, and daytime sleepiness). Exploratory craniofacial analyses will be conducted among participants with clinically available lateral cephalometric radiographs. This protocol could position pediatric dental visits as an accessible touchpoint for early identification of children with unrecognized SDB and inform pathways for timely referral. Protocol Version: 1.4, dated 13 May 2026. Trial Registration: ClinicalTrials.gov NCT07581938.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 4","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13516765/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148830498","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":"Development of a Direct Cell-to-PCR Lysis Buffer Using Optimized Non-Ionic Detergents for RNA-Extraction-Free RT-qPCR.","authors":"Mahmoud Zhra, Rawan Awni Alarawi, Shaimaa Abdelrahman Mohamed, Abdel Naser Daoud, Hana Fakhoury, Ahmad Aljada","doi":"10.3390/mps9040114","DOIUrl":"10.3390/mps9040114","url":null,"abstract":"<p><p>Column-based RNA extraction is time-consuming, involves cumulative losses during transfer, wash, and elution steps, and has reduced recovery from sub-microliter inputs. These limitations restrict gene-expression analysis in small cell populations and low-titre pathogen detection in clinical specimens. We developed the Direct Cell-to-PCR Lysis Buffer, a defined non-ionic detergent formulation containing Tween 20 (0.3%), Triton X-100 (0.1%), and NP-40 (0.1%), for RNA-extraction-free one-step RT-qPCR. Lysates are added directly to reactions without pretreatment, heating, or column purification. Non-ionic detergents produced Ct values comparable to detergent-free controls, whereas SDS, Sarkosyl, and sodium deoxycholate abolished amplification. The method was benchmarked against the standard column-based PureLink RNA Mini Kit using SaOS-2 cells, MCF7 cells, peripheral blood mononuclear cells, and nasal and throat swabs. Direct lysates showed Ct offsets of 0.6 to 2.85 cycles compared with purified RNA, depending on sample type. Ubiquitin-C mRNA was detected down to a single-cell-equivalent input, and plasmid templates were detected to approximately 100 copies per reaction. The buffer was compatible with multiplex ScriptTaq COVID PCR, with unchanged RdRP, N, and RPP30 Ct values, and tolerated sodium azide up to 0.1%, supporting rapid direct RT-qPCR workflows.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 4","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-07-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13516879/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148830323","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":"Agarose-Based 3D Invasion Assay for Simultaneous Quantification of Tumor Cell Invasion and Extracellular Matrix Degradation.","authors":"Andreas R Thomsen, Pascaline Kouam-Daniel, Bettina Priesch-Grzeszkowiak, Anja Grillenberger, Sandra Kumbruch, Ali H Acikelli, Helmut Bühler, Christian Baues","doi":"10.3390/mps9040112","DOIUrl":"10.3390/mps9040112","url":null,"abstract":"<p><p>Tumor cell invasion is a critical step in local tumor progression, recurrence, and metastasis. Conventional two-dimensional migration assays and many existing three-dimensional invasion models often assess cell migration, invasion into the extracellular matrix and matrix degradation as separate endpoints, although these processes are tightly coupled in vivo. Therefore, robust and reproducible in vitro models are needed to investigate tumor cell invasion under defined extracellular matrix conditions. We developed an agarose-based three-dimensional invasion assay, termed the Freiburg 3D invasion assay, for the simultaneous analysis of tumor cell migration, invasion, and extracellular matrix degradation. The system consists of a 2.8% agarose matrix containing defined microcavities connected by a common loading channel. Tumor cells are seeded into these microcavities, where they form compact cell aggregates. The cavities are subsequently filled with collagen type I or extracellular matrix gel. After polymerization, the matrix-containing agarose strips are transferred into parking pockets, cultured for several days, and monitored by microscopy. Invasion distance, single-cell migration, and ECM-cleared area are quantified from serial microscopic images using image analysis software. The system distinguished weakly invasive MCF7 breast cancer cells from highly invasive MDA-MB-231 cells. In addition, treatment with a protease inhibitor and irradiation reduced tumor cell invasion and extracellular matrix remodeling, demonstrating the suitability of the assay for pharmacological and radiation-response studies. The Freiburg 3D invasion assay provides a practical and reproducible three-dimensional in vitro model for analyzing tumor cell invasion and protease-associated extracellular matrix degradation.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 4","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-07-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13515452/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148830086","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":"Single Radial Immunodiffusion (SRID) Assay for Quantitative Determination of Recombinant SOD Protein in a Brucellosis Vaccine Candidate: Method Development and Validation.","authors":"Gulnur Nakhanova, Olga Chervyakova, Kamshat Shorayeva, Aigerim Zhakypbek, Sabina Moldagulova, Aknur Ulankyzy, Alisher Omurtay, Yeraly Shayakhmetov, Temirlan Baiseit, Zharkinay Absatova, Aisha Issabek, Gaukhar Shynybekova, Sandugash Sadikaliyeva, Aziz Nakhanov, Kuanysh Jekebekov, Ainar Kossylganova, Karlygash Zhaparkulova, Assem Kalykova, Tolkyn Bekezhanova, Albina Atakanova, Zakir Yershebulov, Kuandyk Zhugunissov","doi":"10.3390/mps9040113","DOIUrl":"10.3390/mps9040113","url":null,"abstract":"<p><p>Recombinant superoxide dismutase (SOD) protein is a promising antigen candidate for brucellosis vaccines, and its quantitative determination is essential for vaccine quality control, standardization, and regulatory compliance. Although enzyme-linked immunosorbent assay (ELISA) is widely used for antigen quantification, its application to vaccine formulations may be limited due to chemical modifications of antigenic determinants occurring during detoxification and adjuvantation processes. Therefore, alternative analytical methods, such as single radial immunodiffusion (SRID), which enable direct antigen quantification independent of antigenic determinant modifications, represent valuable tools for vaccine analysis. The aim of this study was to develop and validate a single radial immunodiffusion (SRID) assay for the quantitative determination of recombinant SOD protein and to obtain hyperimmune sera in sheep. Sheep were immunized with recombinant SOD protein formulated with aluminum hydroxide or AddaS03 adjuvants. Antibody responses were evaluated using agar gel immunodiffusion (AGID) and enzyme-linked immunosorbent assay (ELISA). Immunization with aluminum hydroxide induced higher antibody titers than AddaS03. By day 42, ELISA titers reached 1:25,600 in the aluminum hydroxide group compared with 1:3200 in the AddaS03 group. The obtained hyperimmune sera were used for SRID assay development. The optimized SRID assay demonstrated high reproducibility and specificity, with no cross-reactivity against unrelated proteins. A linear relationship was observed between the square of the precipitation ring diameter (D²) and antigen concentration (R² = 0.956-0.989). The assay showed a limit of detection of 0.312 μg/mL, a linear range of 0.625-10 μg/mL, high precision (CV < 2%), and robustness under varying analytical conditions. The developed SRID assay represents a specific, reproducible, and robust tool for quantitative control of recombinant SOD protein in vaccine formulations.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 4","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-07-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13515976/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148830543","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":"A One-Step RT-PCR-Coupled Cysteamine-Functionalized Gold Nanoparticle Assay for Colorimetric Detection of Tobacco Mosaic Virus.","authors":"Thuy-Duong Thi Tran, Quy Thi Vu, Hoa Thi Hoang, Phan Thi Ngoc Hoa, Nguyen Pham Thi Thao, Truong T N Lien","doi":"10.3390/mps9040111","DOIUrl":"10.3390/mps9040111","url":null,"abstract":"<p><p>Viral diseases are one of the most destructive threats to global agriculture. Among plant viruses, tobacco mosaic virus (TMV) is a highly contagious pathogen infecting numerous economically vital crops. With no curable treatments, the only strategy to mitigate virus spread is early detection and plant removal. The gold standard for TMV detection is reverse transcriptase-polymerase chain reaction (RT-PCR), followed by agarose gel electrophoresis. To reduce TMV diagnosis time and eliminate the requirement for expensive equipment, this study developed and optimized a one-step RT-PCR-coupled cysteamine-functionalized gold nanoparticle assay for the colorimetric determination of the virus. By integrating cDNA synthesis and PCR amplification into a single tube and analyzing the results using cysteamine- functionalized gold nanoparticles (Au@Cys), the diagnosis turnaround time was significantly reduced. Furthermore, this AuNPs-based colorimetric assay enabled straightforward visual inspection with the naked eye, eliminating the need for costly optical devices. Additionally, our regression equation linking RT-PCR product color values, extracted as mean A value based on the CIELAB color space (green-red axis), and viral load allows for the accurate quantification of TMV infection levels in field samples. Our research lays the groundwork for the further development of more cost-effective, quantitative and rapid plant virus diagnosis methods.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 4","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-07-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13515312/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148830109","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":"Gas Chromatography Method for Quantitation of Residual Solvent Impurities in Nanoformulations.","authors":"Krishna Kattel, Rachael M Crist, Jeffrey D Clogston","doi":"10.3390/mps9040110","DOIUrl":"10.3390/mps9040110","url":null,"abstract":"<p><p>The development and validation of a sensitive, rapid, and specific gas chromatography method for the evaluation of 19 common Class 2 and Class 3 solvents frequently used in nanomedicine formulation is described. Method validation was performed using PerkinElmer's headspace gas chromatograph system with flame ionization detection and an Elite 624 Crossbond 6% cyanopropylphenyl-94% dimethylpolysiloxane or DB-Fatwax-Ultra Inert column with helium as the carrier gas. Validation characteristics such as linearity, spike recovery, method precision, specificity, sensitivity, limit of detection/quantitation, and analyte stability were evaluated. The validated methods showed excellent linearity, with a correlation coefficient > 0.99, and good precision, with intra-day precision < 7.4% for all tested analytes. The percent recoveries ranged 83-104% within the method's quantitation range. In comparison to previously reported methods, the current method has a much shorter equilibration time, higher sensitivity, better separation for many solvents, and a wide concentration detection range. The current method is also perfectly suitable to analyze short chain fatty acids such as formic acid, acetic acid, butyric acid, and valeric acid without requiring additional extraction or derivatization steps. Notably, the method was found to be suitable for analysis of formic acid-a common solvent in certain nanoformulations and one in which there is no prior gas chromatography method available which does not require this additional sample manipulation-down to approximately 75 ppm. Herein, the method is demonstrated using various nanoformulations, including the commercial Doxil formulation as well as several research nanoformulations, including polymeric, cross-linked polymeric, and dendrimer platforms.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 4","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-07-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13515910/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148830259","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":"A Simple Method to Evaluate the Length of Poly(A) Tails in mRNA.","authors":"Jonas Mumenthaler, Maximilian Feldmann, Shahab Mamaghani, Rocco Roberto Penna, Julia Frei, Natalia Jarzebska, Mark Mellett, Emmanuella Guenova, Thomas Kündig, Severin Lauchli, Steve Pascolo","doi":"10.3390/mps9040109","DOIUrl":"10.3390/mps9040109","url":null,"abstract":"<p><p>Synthetic mRNA produced by in vitro transcription is the active pharmaceutical ingredient of approved vaccines and of many drugs under development. It typically contains a 3' poly(A) tail required for optimal stabilization and translation of the mRNA. The average length of the poly(A) tail in the produced mRNA cannot be precisely predicted and consequently must be measured using complicated technologies such as reverse transcription and sequencing or cleavage of the poly(A) tail and analysis by capillary electrophoresis or mass spectrometry. We report an accelerated method to evaluate the average poly(A) tail length in synthetic mRNA, which benefits from the fact that thiazole orange is fluorescent only when in close proximity of nucleic acid. An oligo(dT)₁₂ oligonucleotide having thiazole orange at its extremities emits a fluorescence signal proportional to the amount of poly(A) sequence available. Using a titration curve made with known amounts of poly(A) RNA oligonucleotide, the thiazole orange oligo(dT)₁₂ oligonucleotide can instantly indicate the average length of the poly(A) tail in an mRNA sample. This fast and easy method can be used in any laboratory to determine the size of the poly(A) tail in in vitro-transcribed mRNA produced for research, pre-clinical studies and clinical trials.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 4","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-07-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13398250/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148579371","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":"Preliminary Evaluation of the Virtual Reality-Based Gait Sensory Interaction Test (GaitSIT) for Quantifying Sensory Reweighting During Walking Balance.","authors":"Priyo Ranjan Kundu Prosun, Shafique Chaudhry, Masudul H Imtiaz, Poorna Raavi, David DiSalvo, Kwadwo O Appiah-Kubi","doi":"10.3390/mps9040108","DOIUrl":"10.3390/mps9040108","url":null,"abstract":"&lt;p&gt;&lt;p&gt;&lt;b&gt;Background:&lt;/b&gt; Walking is a dynamic activity that relies on inputs from the multisensory system, i.e., somatosensory, vision, and vestibular. These inputs are processed and integrated in the central nervous system to produce motor impulses for efficient walking balance. The Sensory Organization Test (SOT) is established as the gold standard for assessing sensory contributions to standing balance. However, no comparable assessments have been developed for the clinical evaluation of balance during gait. This study evaluated the Gait Sensory Interaction Test (GaitSIT), a novel virtual reality (VR)-based assessment for characterizing sensory-condition-specific changes in walking balance. &lt;b&gt;Methods:&lt;/b&gt; The GaitSIT comprises a VR environment with a physical compliant foam walking surface that evaluates gait-balance by systematically manipulating and evaluating the sensory systems. Twenty-nine healthy young adults (mean age 24.9 ± 6.4 years) were instructed to complete 6 m walking trials under six standardized conditions (C): eyes open, eyes closed/dark scene, and rotating visual scenes on a firm surface, then repeated on a foam surface. Wearing an Oculus VR headset, participants were instructed to walk in a straight line at their preferred speed, as naturally as possible, in two test sessions on the same day, followed by a third test session 24 h later. Headset-derived sway measures, including position, velocity, and acceleration data, were recorded, and the continuous trajectory deviation angle (i.e., directional control) and sensory ratios were calculated. Linear mixed-effects models included trial-level walking speed as a covariate. Additionally, participants completed the modified Clinical Test of Sensory Interaction on Balance (mCTSIB) as a clinical standing-balance reference measure; its concurrent-validity findings will be reported separately. &lt;b&gt;Results:&lt;/b&gt; Significant condition effects were observed for position, velocity, acceleration, and CTDA after adjustment for trial-level walking speed (all p&lt;0.001), indicating that the six sensory conditions elicited distinct gait-balance responses. Significant differences relative to the baseline condition (C1) were observed across conditions C2-C6 for position, C3-C6 for velocity, and C2 and C5 for acceleration. Session effects were not significant for any primary kinematic outcome after speed adjustment. A significant condition × session interaction was observed for position (p&lt;0.001), whereas velocity, acceleration, and CTDA demonstrated no significant interactions. Walking speed was significantly associated with position, acceleration, and CTDA, but not velocity. Sensory-ratio analyses revealed larger visual and vestibular ratios relative to somatosensory ratios, with the visual and vestibular ratios generally decreasing across sessions. &lt;b&gt;Conclusions:&lt;/b&gt; GaitSIT successfully manipulated sensory conditions during overground walking and produced significant changes in gait-related swa","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 4","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-07-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13398260/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148579374","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}