Methods and Protocols最新文献

{"title":"A Refined Approach to Isolate Interneurons for High-Validity Epigenetic Studies in Human Brain Tissue.","authors":"Ariel Cariaga-Martínez, Kilian Jesús Gutierrez, Ignacio Regidor, Marta Del Álamo, Jerónimo Saiz-Ruiz, Raúl Alelú-Paz","doi":"10.3390/mps8030061","DOIUrl":"10.3390/mps8030061","url":null,"abstract":"<p><p>Epigenetic research has made notable progress in recent years, yet our ability to explore the human brain at a cellular level remains limited. One of the main obstacles has been the difficulty of isolating specific neuronal populations from postmortem tissue-particularly interneurons, which play a central role in many psychiatric disorders. In this study, we present a practical and reproducible protocol for isolating GAD-positive interneurons from human brain samples. We isolate permeabilized cell-like structures suitable for downstream epigenetic analysis. To ensure specificity, we validated the isolated cells by comparing them with interneurons derived from human iPSCs. This approach allows for high-quality DNA extraction suitable for downstream epigenetic analysis, including methylation-specific PCR. By targeting a well-defined neuronal subtype, our method provides a solid foundation for studying the molecular changes associated with disorders such as schizophrenia and autism. This protocol opens new doors for cell-specific investigations in brain tissue, a step forward in understanding how epigenetic mechanisms contribute to neuropsychiatric pathophysiology.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 3","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-06-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12196513/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144485103","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":"Synthesis of DOTA-Based ⁴³Sc Radiopharmaceuticals Using Cyclotron-Produced ⁴³Sc as Exemplified by [⁴³Sc]Sc-PSMA-617 for PSMA PET Imaging.","authors":"Jason P Meier, Mohammed Bhuiyan, Richard Freifelder, Hannah J Zhang, Lucas Gonzalez, Antonino Pusateri, Hsiu-Ming Tsai, Lara Leoni, Kaustab Ghosh, Erica Markiewicz, Christopher Henning, Yuhan Zhang, Ralph Weichselbaum, Jerry Nolen, David A Rotsch, Chien-Min Kao, Russell Z Szmulewitz, Chin-Tu Chen, Satish K Chitneni","doi":"10.3390/mps8030058","DOIUrl":"10.3390/mps8030058","url":null,"abstract":"<p><p>The implementation of theranostics in oncologic nuclear medicine has exhibited immense potential in improving patient outcomes in prostate cancer with the implementation of [⁶⁸Ga]Ga-PSMA-11 PET and [¹⁷⁷Lu]Lu-PSMA-617 into clinical practice. However, the correlation between radiopharmaceutical biodistributions seen with [⁶⁸Ga]Ga-PSMA-11 PET imaging and downstream [¹⁷⁷Lu]Lu-PSMA-617 therapy remains imperfect. This suggests that prostate cancer theranostics could potentially be further refined through the implementation of true theranostics, tandem pairs of diagnostic and therapeutic radiopharmaceuticals that utilize the same ligand and element, thus yielding identical pharmacokinetics. The radioscandiums are one such group of true theranostic radiopharmaceuticals. The radioscandiums consist of two β+ emitting scandium isotopes (⁴³Sc/⁴⁴Sc), as well as a β^- emitting therapeutic isotope (⁴⁷Sc), which can all conjugate with PSMA-targeting PSMA-617. This potential has led to extensive investigations into the production of the radioscandiums as well as pre-clinical assessments with several ligands; however, there is a lack of literature extensively describing the complete synthesis of scandium radiopharmaceuticals. which therefore limits the accessibility of radioscandium research in theranostics. As such, this work aims to present an easily translatable protocol for the synthesis of [⁴³Sc]Sc-PSMA-617 from a [⁴²Ca]CaCO₃ starting material, including target formation, nuclear production via ⁴²Ca(d,n)⁴³Sc reaction, chemical separation, radiolabeling, solvent reformulation, and target recycling.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 3","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-06-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12196382/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144485114","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":"Improved RSV Neutralization Assay Using Recombinant RSV Expressing Reporter Fluorescent Protein.","authors":"Yutaro Yamagata, Michiko Toizumi, Jean-Francois Eleouet, Marie-Anne Rameix-Welti, Makoto Takeda, Lay-Myint Yoshida","doi":"10.3390/mps8030060","DOIUrl":"10.3390/mps8030060","url":null,"abstract":"<p><p>Human respiratory syncytial virus (RSV) causes acute respiratory illness, attributing to deaths among young children and older adults worldwide. RSV neutralization assay is an important tool to measure RSV neutralization antibody that can prevent infection and severe complication of RSV. Conventional RSV neutralization assays have some limitations of speed and cost, especially for expensive kits, reagents or instruments required for detection. To solve this problem, this paper describes an improved simple and economical RSV neutralization assay protocol using recombinant RSV (rRSV) expressing reporter fluorescent protein to measure RSV growth as reporter activity with plate reader. The condition of 3 days culture demonstrated sufficient fluorescent activity even when small amounts of rRSV were used to inoculate Hep-2 cells. In addition, white 96-well cell culture plate showed better stable reporter activities than black plate. Furthermore, RSV neutralization assay protocol using rRSV-reporter fluorescent protein demonstrated similar signal detection capacity for RSV antibody titer detection compared to other protocols, such as rRSV-Luciferase and ELISA assay. The new RSV neutralization assay protocol can be applied to RSV antibody titration of numerous samples necessary for RSV surveillance or antiviral testing.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 3","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-06-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12196034/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144485111","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":"Determination of the Minimum Cell-to-Cell Adhesion Time Using Optical Tweezers in Leukemia and Lymphoma Research.","authors":"Kamila Duś-Szachniewicz, Sławomir Drobczyński","doi":"10.3390/mps8030059","DOIUrl":"10.3390/mps8030059","url":null,"abstract":"<p><p>Single-cell adhesion assays can be divided into studies on attachment and detachment events, and several methods that enable the characterization of both processes have been established in the past. Due to their low invasiveness, label-free principles, and contactless operation, optical methods are especially beneficial for this purpose. Historically, optical tweezers (OTs) have been used to explore single-cell detachment events, allowing for the precise determination of minute physical forces. However, it has been noted that OTs can also be used to study single-cell attachment dynamics, including the evaluation of minimum cell-to-cell contact times necessary to establish a stable adhesive bond. Here, we provide a step-by-step protocol to effectively evaluate minute changes in the adhesion of single leukemia-lymphoma cells using optical tweezers with low laser intensities. This serves as a valuable in vitro model to determine the effects of physical and chemical factors on the adhesive properties of leukemia-lymphoma (LL) cells.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 3","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-06-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12196108/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144485106","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":"Optimization of Nile Tilapia Artificial Breeding Using Human Chorionic Gonadotropin (hCG) Hormone.","authors":"Golam Rbbani, Prabhugouda Siriyappagouder, Riaz Murshed, Rajesh Joshi, Artem Nedoluzhko, Jorge Galindo-Villegas, Jorge M O Fernandes","doi":"10.3390/mps8030057","DOIUrl":"10.3390/mps8030057","url":null,"abstract":"<p><p>Nile tilapia (<i>Oreochromis niloticus</i>) is the most widely farmed tilapia species globally, making it one of the most important aquaculture species. To meet increasing demand, hatcheries occasionally use artificial breeding techniques such as hormonal induction to synchronize breeding. Despite the common use of human chorionic gonadotropin (hCG) in fish breeding, no detailed protocol has been established specifically for Nile tilapia. The objective of this study is to establish an effective hCG-induced artificial breeding protocol for gene editing and aquaculture production, optimizing fertilization, hatching, and survival rates. We employed a single intramuscular injection of 2 IU/g hCG to induce ovulation. The protocol achieved an average fertilization rate of 88.3% and a larval survival rate of 90.5%, demonstrating its potential for obtaining high-quality embryos for functional studies and enhancing reproductive performance on a commercial scale.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 3","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-06-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12195898/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144485113","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":"Towards Automated Testing of Kynurenine for Point-of-Care Metabolomics.","authors":"Dipanjan Bhattacharyya, Marcia A LeVatte, David S Wishart","doi":"10.3390/mps8030056","DOIUrl":"10.3390/mps8030056","url":null,"abstract":"<p><p>Our objective was to develop a simple, low-cost colorimetric assay to detect kynurenine (L-Kyn) in human biofluids, that would be compatible with a point-of-care (POC) system being developed in our lab. Elevated L-Kyn is associated with many pathological conditions. However, current detection methods are expensive, time-consuming, and unsuitable for resource-limited settings. Existing colorimetric L-Kyn assays lack specificity, require unusual reagents, or lack sensitivity, hindering their practical application. Here we report a two-step diazotization-based colorimetric assay that produces a red chromophore upon reaction with L-Kyn. To reduce background interference, we used dilution and anion exchange chromatography for urine samples and acid precipitation for serum samples. The assay detected 5-300 μM L-Kyn in urine (lower limit of detection (LLOD) 1.34 μM) and 5-125 μM L-Kyn in serum (LLOD 1.24 μM). Correlation studies achieved strong linearity (R² = 0.98 for spiked urine, 0.99 for spiked serum) and were highly correlated (>0.95) to liquid chromatography tandem mass spectrometry (LC-MS/MS) concentrations. Bland-Altman analysis confirmed agreement between L-Kyn assay and LC-MS/MS methods. To our knowledge, this is the first application of a diazotization reaction for L-Kyn quantification at physiologically relevant levels. The assay is now being ported to a low-cost, automated POC biosensor platform.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 3","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12196141/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144485115","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 Minimally Invasive Transthoracic Injection Technique for Reproducible Intrapleural Delivery in Mice.","authors":"Sophie Rovers, Pooyeh Farahmand, Dana Liu, Louize Brants, Christophe Hermans, Dieter Peeters, Danielle McKinven, Jennifer Doig, Filip Lardon, Jan van Meerbeeck, Elly Marcq, Daniel J Murphy, Evelien Smits","doi":"10.3390/mps8030055","DOIUrl":"10.3390/mps8030055","url":null,"abstract":"<p><p>The development of standardised, reproducible preclinical models is essential for advancing pleural mesothelioma (PM) research. Here, we present a simple and reliable minimally invasive transthoracic intrapleural injection technique that could improve the efficiency of orthotopic PM model generation. By incorporating a simple needle sleeve to control the injection depth, this method eliminates the need for surgery or general anaesthesia, reducing technical complexity and animal stress while ensuring precise delivery into the pleural cavity. We demonstrate the effectiveness of this approach by achieving a 100% tumour engraftment rate following the injection of AE17 tumour cells. Additionally, this technique has been successfully used for asbestos fibre injection in mesothelioma models, highlighting its versatility. By providing a more accessible, standardised alternative to existing methods, this protocol improves the reliability of PM models and facilitates broader adoption by researchers, including those with limited experience in invasive procedures.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 3","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-05-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12195961/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144485102","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 and Characterization of a Ten-Plex Assay to Measure <i>Klebsiella pneumoniae</i> Antigen-Specific IgG in Human Sera.","authors":"Luca Rovetini, Gianina Florentina Belciug, Luisa Massai, Francesca Nonne, Renzo Alfini, Heena Ranchod, Denasha L Reddy, Mariagrazia Molfetta, Davide Oldrini, Makrina Totsika, Miren Iturriza, Ziyaad Dangor, Carlo Giannelli, Shabir A Madhi, Francesca Micoli, Martina Carducci, Omar Rossi","doi":"10.3390/mps8030052","DOIUrl":"10.3390/mps8030052","url":null,"abstract":"<p><p><i>Klebsiella pneumoniae</i> is a leading cause of nosocomial infections, neonatal sepsis, and childhood mortality worldwide. A drastic rise in antibiotic-resistant isolates poses an urgent threat to humanity, and the World Health Organization (WHO) has classified this as a critical-priority antimicrobial-resistant (AMR) pathogen. Recent advancements in developing vaccines against <i>Klebsiella pneumoniae</i> have highlighted the lack of standardized assays to evaluate immunogenicity, complicating comparison among different vaccines under development and the establishment of a serological threshold of risk reduction (SToRR). Here, we describe the development of a ten-plex multiplex assay to measure IgG against capsular polysaccharides (K2, K25, K102, K149), O antigens (O1v1, O1v2, O2v1, O2v2 and O5), and a conserved protein (MrkA). A standard curve was established by pooling human sera from naturally exposed subjects and then calibrated in terms of Relative Luminex Units/mL. The assay was fully characterized in terms of specificity, precision, linearity, and repeatability. This immunoassay demonstrates performance suitable for future clinical trials, as well as to perform sero-epidemiological studies to gain insights into naturally occurring immunity, potentially contributing to the establishment of a serological threshold of risk reduction against <i>Klebsiella pneumoniae</i>.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 3","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-05-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12101422/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144128188","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":"New Bioelectrical Impedance-Based Equations to Estimate Resting Metabolic Rate in Young Athletes.","authors":"Theodoros Stampoulis, Alexandra Avloniti, Dimitrios Draganidis, Dimitrios Balampanos, Polyxeni Efthimia Chalastra, Anastasia Gkachtsou, Dimitrios Pantazis, Nikolaos-Orestis Retzepis, Maria Protopapa, Athanasios Poulios, Nikolaos Zaras, Maria Michalopoulou, Ioannis G Fatouros, Athanasios Chatzinikolaou","doi":"10.3390/mps8030053","DOIUrl":"10.3390/mps8030053","url":null,"abstract":"<p><p>Resting metabolic rate (RMR) significantly impacts total daily energy expenditure, particularly on training days, and varies among trained individuals. Studies estimating RMR in this population show notable discrepancies. This study aimed to develop and validate new bioelectrical impedance analysis-based (BIA) RMR equations for young athletes, using a calibration and a validation group of 219 and 51 participants, respectively. RMR was measured via indirect calorimetry, while body composition was assessed through DXA and BIA. Correlation and agreement were evaluated by using Pearson's correlation coefficients and Bland-Altman analysis. Multiple linear regression was applied for the estimation of RMR and a one-way ANOVA was used to compare the new BIA-based equations with other specific formulas. A significant correlation was noted between the BIA and DXA measurements. The final equation, applicable to both genders, was significantly correlated with intracellular water (ICW) and trunk fat, predicting 71.1% of RMR variance. When analyzed separately, body weight and protein displayed a moderate correlation with RMR in men (r = 0.616, <i>p</i> < 0.001), while ICW was correlated with the percentage of body fat in women (r = 0.579, <i>p</i> < 0.001). In the validation group, the values obtained through the three BIA-based equations were similar to the measured RMR, but differed significantly from those obtained through the four existing equations for trained individuals. In conclusion, the developed equations based on BIA-mediated body composition analysis provide a reliable method for estimating RMR in trained populations daily.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 3","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-05-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12101231/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144128197","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 Method of Well-Spread Pachytene Chromosome Preparations for Plant Species with Large Genomes Suitable for the Immunolocalization of Meiotic Proteins.","authors":"Natalya Kudryavtseva, Aleksey Ermolaev, Ludmila Khrustaleva","doi":"10.3390/mps8030054","DOIUrl":"10.3390/mps8030054","url":null,"abstract":"<p><p>Well-spread pachytene chromosomes are critical for studying the location of meiotic proteins along individual chromosomes. However, producing good spreads in species with large genomes is challenging due to the tangling of pachytene chromosomes. Existing protocols often fail to achieve proper separation of large chromosomes in spreads. Here, we describe in detail an improved protocol that ensures the effective separation of large pachytene chromosomes and demonstrates its suitability for protein immunodetection. To develop the protocol, pollen mother cells at the middle-late pachytene stage from <i>Allium fistulosum</i>, a species with a large genome and chromosomes, were used. The protocol involved three main steps: fixing anthers in Clark's solution (ethanol-acetic acid, 3:1), digestion in an enzyme mixture, and gentle squashing in 45% acetic acid. A clear ZYP1 signal on all separated chromosomes was observed. The high quality of well-spread pachytene chromosomes obtained with the modified protocol allowed for the easy extraction of individual chromosomes for more precise detection and analysis of the proteins of interest.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 3","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-05-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12101289/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144128186","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}