Journal of pharmacological and toxicological methods最新文献

{"title":"Evaluation of the Effects of cypermethrin Toxicity on Liver Mitochondria of Male Albino Rats","authors":"Venkataramanaiah Poli ,&nbsp;Srinivasulu Reddy Motireddy","doi":"10.1016/j.vascn.2025.108388","DOIUrl":"10.1016/j.vascn.2025.108388","url":null,"abstract":"<div><div>The purpose of this study is to look into the toxic effects of giving male albino rats cypermethrin (CPM). In order to achieve this, three groups of rats were formed. Every group received the same amount of saline, including the control group, while the experimental groups received 4.5 and 5.5 mg/kg body weight of cypermethrin. After that, the rats are sacrificed, and tissue samples are pooled. In all the treatments, rats were administered orally for an experimental period of 30 days. Every week, the body weight of every individual rat was recorded. In order to achieve this goal, indicators of toxicity such as body and organic weight, ATP and ATP/ADP ratio, antioxidant enzymes and oxidative stress, complex activity (complex I–IV), plasma biochemical parameters, and levels of pro-inflammatory cytokines are utilized. Rats injected with cypermethrin showed a substantial (<em>p</em> &lt; 0.05) drop in their ultimate weight and organ weight when compared to control rats. The administration of cypermethrin resulted in a significant (<em>p</em> &lt; 0.05) drop in adenosine triphosphate (ATP) and the ATP/ADP ratio, indicating the involvement of activity of liver mitochondria. The administration of cypermethrin oxidative stress biomarkers resulted in a substantial increase (<em>p</em> &lt; 0.05) in malondialdehyde (MDA), glutathione (GSH), reactive oxygen species (ROS), and protein carbonylated content (PCC). Nevertheless, there was a significant (<em>p</em> &lt; 0.05) drop in the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione S-transferase (GST) in the mitochondria of rat liver. In the present study, the complex activity (complex I–IV) and mitochondrial membrane potential (MMP) of rat liver mitochondria decreased significantly (<em>p</em> &lt; 0.05). The administration of Cypermethrin resulted in a significant (p &lt; 0.05) increase in plasma levels of triglycerides (TAG), alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin (BIL), total cholesterol (TC), high-density lipoprotein (HDL-C), low-density lipoprotein (LDL-C), and alkaline phosphatase (ALP). Pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, and IL-10) were observed to have higher plasma levels after cypermethrin injection (<em>p</em> &lt; 0.05) when compared to the control. The current study's findings demonstrated that rat liver tissue suffered mitochondrial damage as a result of cypermethrin intoxication.</div></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"135 ","pages":"Article 108388"},"PeriodicalIF":1.8,"publicationDate":"2025-07-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144739316","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Legacy hERG study data in contemporary integrated risk assessments – mitigating the risk of protocol and positive control article differences","authors":"Richard W. Daniels ,&nbsp;James W. Kramer ,&nbsp;Matthew M. Abernathy ,&nbsp;Derek D. Best ,&nbsp;Jessica C. Brimecombe ,&nbsp;Derek J. Leishman","doi":"10.1016/j.vascn.2025.108387","DOIUrl":"10.1016/j.vascn.2025.108387","url":null,"abstract":"<div><div>Testing new molecules against the hERG potassium channel has been a routine component of secondary pharmacology assessments for more than a quarter of a century. In 2005, the ICH S7B guidance required a GLP hERG study prior to first administration of new chemical entities to man. In 2022, the ICH S7B Q&amp;As introduced some ‘best practice’ recommendations for the hERG assessment. A required component of a QTc integrated risk assessment is an evaluation of how well the hERG assessment met the ‘best practice’ recommendations. For studies conducted under the 2005 ICH S7B guidance, it is unclear how to assess the differences between study protocols. Many sponsors had been conducting the GLP hERG assay in a manner which was largely compliant with these recommendations but differed subtly in the recording temperature, the voltage-clamp protocol employed and/or in the positive control used in the assay (‘Legacy Protocol’). This led many sponsors to consider repeating their earlier hERG assessment using the ‘best practice’ (‘Revised Protocol’) recommendations. The current analyses sought to examine whether the differences in practice warranted retesting the compounds or could dispel a concern that prior practice had been less sensitive.</div><div>The three key positive control articles, dofetilide, moxifloxacin and ondansetron were compared under the ‘Legacy Protocol’ and best practice ‘Revised Protocol’. Furthermore, two prior positive control articles, terfenadine and cisapride, were tested under both protocols. Terfenadine data under the ‘Legacy Protocol’ were available from separate studies (<em>n</em> = 6) dating from 2003 to 2025. Terfenadine was also tested in four studies using the ‘Revised Protocol’ in 2023. In addition, the hERG inhibition data for 3627 single concentration positive control article tests with 60 nM terfenadine from studies conducted from 2002 to 2018 were collated.</div><div>There was no difference for the 3 positive control articles tested under the two different protocols for pooled hERG safety margin. Similarly, the pooled hERG IC₅₀ values for terfenadine under the two protocols were not different, although the variability in hERG potency was greater under the ‘Revised Protocol’. The inhibition of hERG current for 60 nM terfenadine in the 3627 historical examples was also consistent with predictions based on the hERG IC₅₀ and Hill slope data under the two protocols.</div><div>Based on the similarity between results collected under ‘Legacy’ and ‘Revised’ protocols we conclude that if the ‘Legacy Protocol’ has been used and the positive control examined was 60 nM terfenadine, there appears to be no need to repeat the hERG assessment.</div></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"135 ","pages":"Article 108387"},"PeriodicalIF":1.3,"publicationDate":"2025-07-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144669182","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A retrospective analysis of the concordance between cardiovascular effects in telemetry studies, toxicity studies, and early clinical trials","authors":"Andrea Greiter-Wilke ,&nbsp;Jan Attig ,&nbsp;Simon Bassett","doi":"10.1016/j.vascn.2025.108385","DOIUrl":"10.1016/j.vascn.2025.108385","url":null,"abstract":"<div><div>This work aimed to compare the ability of several ECG technologies used in toxicity studies to identify cardiovascular effects. A retrospective analysis of 41 drug development projects across the Roche portfolio from 2005 to 2023 was conducted to evaluate the concordance of snapshot ECG (SS-ECG) and jacket external telemetry (JET, with or without blood pressure assessment) to detect effects on heart rate, ECG, and blood pressure in toxicity studies, when compared to implanted telemetry as the gold standard. Overall, 132 non-clinical studies were investigated. For 24 projects, translation to phase 1 safety data under consideration of plasma exposures could be followed up. Company-internal strategic decisions on advancing certain molecules despite cardiovascular findings were subject to a risk-benefit assessment and not evaluated in this investigation.</div><div>SS-ECGs never detected changes in heart rate (HR) previously revealed in telemetry studies, whereas JET identified these in all telemetry-positive cases. JET (or M11 implants) detected 44 % and SS-ECGs identified 33 % of QTc increases in telemetry-positive molecules, indicating that stand-alone telemetry is still the most reliable in vivo study in identifying QTc-effects. No clear species differences between beagle dogs, Göttingen minipigs or cynomolgus monkeys were evident when comparing heart rate and QTc detection. HR increases were noted clinically in about 50 % of the non-clinical positive cases, whereas HR decreases were never detected in clinical trials. Non-clinical blood pressure decreases showed higher translatability to humans (at least 80 %) than blood pressure increases (29 %), with a high prevalence of dogs and minipigs showing a signal. Non-clinical QTc increases detected by JET and/or telemetry were confirmed in 3 clinical studies analyzed with concentration/QTc modeling, however in 7 clinical studies employing other methodologies to measure QT, the non-clinical QTc effects did not translate.</div></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"135 ","pages":"Article 108385"},"PeriodicalIF":1.3,"publicationDate":"2025-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144669181","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Spatial regulation of CD14-like epitope in hematopoietic and ocular tissues of cultured North Atlantic lumpfish (Cyclopterus lumpus) and spotted wolffish (Anarhichas minor) during bacterial infection and temperature variations","authors":"Rebecca R. Kwabiah ,&nbsp;Lauren E. Murphy ,&nbsp;Hélène Paradis ,&nbsp;Rand Al-Badoosh ,&nbsp;Trung Cao ,&nbsp;Vimbai I. Machimbirike ,&nbsp;Hajarooba Gnanagobal ,&nbsp;Ignacio Vasquez ,&nbsp;Aqsa Maqsood ,&nbsp;Kenneth Kao ,&nbsp;Javier Santander ,&nbsp;Robert L. Gendron","doi":"10.1016/j.vascn.2025.108383","DOIUrl":"10.1016/j.vascn.2025.108383","url":null,"abstract":"<div><div>The development of new tools for a better understanding of innate immune mediated responses to tissue damage in emerging model organisms could offer novel approaches to inform toxicology, disease mitigation and/or drug discovery. Innate immunity is one of the first lines of defence in response to tissue damage, with macrophages being key players in these processes. The expression of macrophage protein markers in North Atlantic marine teleost fish has not been extensively studied. Here we used an anti-mammalian-CD14 antibody to explore CD14 epitope expression in hematopoietic and ocular tissues of lumpfish and spotted wolffish. Western blotting indicated that proteins reactive with an anti-mammalian-CD14 antibody with similar molecular weights to mammalian CD14 isoforms exist in both lumpfish and spotted wolffish. Immunohistochemistry (IHC) analysis using this anti-mammalian-CD14 indicated CD14-like reactive epitope expression in head kidney and ocular choroid rete mirabile in both normal healthy lumpfish and in a <em>Vibrio anguillarum</em> marine bacterial pathogenesis paradigm. As infection proceeded in lumpfish, CD14-like expression increased in head kidney and ocular choroid rete mirabile vasculature as revealed by IHC. In addition, we also used a temperature challenge paradigm in spotted wolffish to assess CD14-like reactivity in ocular choroid rete mirabile. A six-week hypothermal condition led to higher levels of CD14-like expression in the wolffish choroid rete mirabile than control temperature or a six-week hyperthermal condition as revealed by IHC. The tissue spatial regulation of CD14 epitope in lumpfish and spotted wolffish we observed herein might serve as a tool in emerging novel model organism-based technologies toward a better understanding of the mechanisms involved in responses to tissue damage.</div></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"135 ","pages":"Article 108383"},"PeriodicalIF":1.3,"publicationDate":"2025-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144669183","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Improved translation of Nav1.5 channel inhibition to in vivo QRS interval prolongation via the hIPSC cardiomyocyte model","authors":"John P. Imredy ,&nbsp;Holly Clouse ,&nbsp;Mei Zhang ,&nbsp;Spencer Dech ,&nbsp;Qiuwei Xu ,&nbsp;Jeremy Ellis ,&nbsp;Shaun Gruver ,&nbsp;Julia C. Hotek ,&nbsp;Christopher P. Regan","doi":"10.1016/j.vascn.2025.108384","DOIUrl":"10.1016/j.vascn.2025.108384","url":null,"abstract":"<div><h3>Introduction</h3><div>Drug risk assessment to ventricular conduction typically involves measuring functional inhibition of the cardiac sodium channel (Na_v1.5) followed by nonclinical in vivo assessment of prolongation of the electrocardiographic QRS interval. The Na_v1.5 IC₅₀ concentration, however, underpredicts the threshold concentrations of in vivo QRS prolongation by 10–20-fold. We here develop and implement a novel human induced pluripotent stem cell derived cardiomyocyte (hIPSC-CM) field potential spike analysis paradigm that facilitates the use of this model for accurate forecasting of QRS prolongation concentration thresholds.</div></div><div><h3>Methods and results</h3><div>Using multi-electrode arrays we record the extracellular field potential spike of hIPSC-CM monolayers. Large variations in the field potential spike amplitudes across the array, however, confound translation of this parameter. To solve this shortcoming we derive a novel time parameter, T_A/Vmax, defined as the quotient of the amplitude (A) and the peak rate of change (V_max) of the of the cardiomyocyte field potential spike. T_A/Vmax normalizes effects on spike amplitude independent of Na_v1.5 inhibition, such as cell density, amplitude drift, and variable attachment of the monolayer to the field potential electrode. Small changes (&lt; 5 %) in T_A/Vmax become statistically significant and directly comparable to threshold QRS interval changes in early in vivo screening models. Characterization of a set of 12 compounds including Class I antiarrhythmics and internal test compounds demonstrates that the T_A/Vmax EC5% more consistently and accurately predicts both clinical and non-clinical QRS prolongation than the Na_v1.5 IC₅₀; accuracy of threshold concentration forecasting improved 16-fold and the correlation coefficient, R, increased from 0.76 to 0.88.</div></div><div><h3>Conclusion</h3><div>Calculation of T_A/Vmax enables use of the hIPSC-CM field potential spike to predict in vivo QRS prolongation. Use of this in vitro model in early screening or mechanistic evaluation of risk to ventricular conduction should facilitate a broader cardiac in vitro electrophysiologic assessment strategy for new molecular entities.</div></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"135 ","pages":"Article 108384"},"PeriodicalIF":1.3,"publicationDate":"2025-07-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144644485","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Metabolomics using high-resolution magic angle spinning nuclear magnetic resonance: Neurotoxicological applications","authors":"Rachel Neita ,&nbsp;Kenna L.R. Hynes ,&nbsp;Grace V. Mercer ,&nbsp;Céline Schneider ,&nbsp;Lindsay S. Cahill","doi":"10.1016/j.vascn.2025.108382","DOIUrl":"10.1016/j.vascn.2025.108382","url":null,"abstract":"<div><div>High-resolution magic angle spinning nuclear magnetic resonance (HRMAS NMR) is a cutting-edge technology for metabolomics studies that has found application in pharmacology and toxicology. Metabolomics provides insight into how biological systems function, providing a unique chemical fingerprint of the physiological state of an organism. Using the high signal sensitivity and spectral resolution of HRMAS NMR, metabolic screening of intact tissue samples can be performed for complex organs such as the brain. In this study, we present a detailed HRMAS NMR methodology including sample preparation and experimental conditions to ensure data repeatability and reproducibility. We present the results of a neurotoxicology study, involving exposure of adult mice to 50 ppm of a legacy perfluoroalkyl substance, perfluorooctanoic acid (PFOA), through their drinking water for one month (<em>n</em> = 8/group). The PFOA exposed group had a significant increase in the relative concentration of glutamate compared to controls (<em>p</em> &lt; 0.05), illustrating the potential for HRMAS NMR metabolomics to be used to determine the mode of action of neurotoxins and to provide an understanding of the molecular biochemical pathways impacted by toxicant exposure.</div></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"135 ","pages":"Article 108382"},"PeriodicalIF":1.3,"publicationDate":"2025-07-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144628359","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Thermal shift assays in drug discovery – Troubleshooting from biochemical to cellular applications","authors":"Danielle Hanke,&nbsp;Jennifer I. Brown,&nbsp;Brent D.G. Page","doi":"10.1016/j.vascn.2025.108380","DOIUrl":"10.1016/j.vascn.2025.108380","url":null,"abstract":"<div><div>Thermal shift assays (TSAs) have become valuable tools within drug discovery and development pipelines. These techniques detect direct physical interactions between small, drug-like molecules and their target proteins in biochemical and cellular settings. While there are several recent articles that outline TSA protocols and highlight their utility in drug discovery projects, this article aims to highlight common issues and solutions for challenges within cutting-edge TSAs. Herein, we have described three commonly used thermal stability assays, differential scanning fluorimetry (DSF), the protein thermal shift assay (PTSA) and the cellular thermal shift assay (CETSA), highlighting specific considerations for their use and implementation. We have also described frequently experienced challenges, including irregular melt curves, challenges with protein detection, and the impacts of buffer and test compounds on TSA performance. Overall, this troubleshooting guide serves to complement established methods and protocols to make TSAs more accessible and of greater use to the scientific community. We believe this will facilitate the use of thermal stability assays in biochemical (cell-free) and biological (cell-based) settings within drug discovery projects and support researchers seeking to develop the next-generation of therapeutic agents.</div></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"135 ","pages":"Article 108380"},"PeriodicalIF":1.3,"publicationDate":"2025-07-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144568279","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Anticoagulant activity of Eucalyptus essential oils: An in vitro approach and a bioinformatics-based pharmacokinetic-pharmacodynamic analysis","authors":"Jhonatan Felipe dos Santos ,&nbsp;Maria Eduarda Dias Possate ,&nbsp;Maysa Barbosa de Almeida ,&nbsp;Eveline Maria de Melo ,&nbsp;Ricardo Andrade Furtado ,&nbsp;Lays da Silva Moreira Santos ,&nbsp;Isabela Cristina Gomes Honório ,&nbsp;Silvio de Almeida-Junior","doi":"10.1016/j.vascn.2025.108381","DOIUrl":"10.1016/j.vascn.2025.108381","url":null,"abstract":"<div><div>Coagulation disorders pose significant health risks, requiring effective therapies. Natural products emerge as promising anticoagulant agents, standing out in the search for innovative and safe alternatives, integrating <em>in vitro studies</em>, pharmacological analyses and bioinformatics for therapeutic advances. In this context, a study was conducted on the anticoagulant and toxicological activity of three essential oils of <em>Eucalyptus spp.</em>: <em>Eucalyptus citriodora</em>, <em>Eucalyptus globulus</em> and <em>Eucalyptus staigeriana</em>, commercially acquired. The chemical composition was analyzed by gas chromatography coupled to mass spectrometry. The anticoagulant activity was evaluated by <em>in vitro</em> assays of prothrombin time (PT) and activated partial thromboplastin time (aPTT) at concentrations of 0.19 % to 100 %, performed in triplicate. The mechanism of action was investigated by <em>in silico</em> analysis of activated Factor X, and the toxicological potential was evaluated with bioinformatics tools. The major compound identified was citronellal (57.77 %) in <em>E. citriodora</em> essential oil, eucalyptol (77.03 %) in <em>E. globulus</em> oil, and <span>d</span>-limonene (20.89 %) and geranial (10.54 %) in <em>E. staigeriana</em> oil. <em>E. globulus</em> showed greater anticoagulant efficacy, with prolongation of 256 % in aPTT and 147 % in PT, while the other oils showed prolongation of 117 % in PT. The pharmacokinetic parameters showed similarity with commercial anticoagulants, and the ΔG values in the <em>in-silico</em> model for activated Factor X were comparable to those of warfarin. The results confirm the anticoagulant potential of the three essential oils, especially <em>E. globulus</em> oil, which is more indicated for the prospection of new drugs.</div></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"135 ","pages":"Article 108381"},"PeriodicalIF":1.3,"publicationDate":"2025-06-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144522950","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Standardizing high-throughput RNA extraction: Modifying commercial kits for improved purity, yield, and efficiency","authors":"Ruwini D. Rajapaksha,&nbsp;Catherine Brooks,&nbsp;Adriana Rascon,&nbsp;Adam Fadem,&nbsp;Ivy Nguyen,&nbsp;Philip J. Kuehl,&nbsp;John T. Farmer","doi":"10.1016/j.vascn.2025.108379","DOIUrl":"10.1016/j.vascn.2025.108379","url":null,"abstract":"<div><div>High-throughput extraction kits are widely use in clinical and preclinical settings to extract nucleic acids from biological samples due to their scalability, repeatability, and reduced labor. However, achieving high nucleic acid yield and purity while maintaining reliability and reproducibility remains challenging in molecular biology labs, partly due to lack of standardized guidelines for nucleic acid extractions and purification. Variations in extraction kits and extraction chemistries often lead to inconsistence results across experiments.</div><div>To address this variability, we modified manufacturers' extraction protocols by introducing additional chloroform and ethanol extraction steps. These modifications aimed to optimize RNA purity, yield, and extraction efficiency (EE) using Xeno Internal Positive Control (IPC) spiking as a benchmark. We evaluated the performance of three commercially available magnetic-bead-based RNA extraction kits across six naïve non-human primate (NHP) tissue types: brain, heart, kidney, liver, lung, and spleen, using the KingFisher™ Flex automated extraction platform.</div><div>The modified protocols demonstrated significant improvements in RNA purity, yield, and EE across the selected kits. These additional steps enhanced the suitability of extracted RNA for downstream applications, underscoring the importance of considering both kit performance and tissue characteristics in experimental design.</div><div>We further assessed the MagMAX™ <em>mir</em>Vana™ Total RNA Isolation Kit for its ability to remove interfering nucleic acids, such as plasmid DNA and single-stranded DNA (ssDNA), from adeno-associated viral (AAV) vector preparations. Specifically, we invesigated AAV serotype 8 (AAV8) due to its popularity in use for gene and cell therapies. The kit demonstrated high efficiency, removing ≥98 % of non-encapsidated genomes, plasmid DNA, and other impurities, yielding RNA suitable for downstream applications and removing contaminating DNA that would create a false-positive signal and result in over quantification.</div><div>These results emphasize the value of optimized, standardized protocols to improve reproducibility and reliability in RNA extraction workflows, with broad applications in molecular biology, gene therapy, and cell biology research.</div></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"135 ","pages":"Article 108379"},"PeriodicalIF":1.3,"publicationDate":"2025-06-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144517504","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"From cellular waste to biomarkers; insights into past, present, and future methods to detect immune cell-derived extracellular vesicles using flow cytometry","authors":"Jennifer L. Zagrodnik ,&nbsp;Craig S. Moore","doi":"10.1016/j.vascn.2025.108376","DOIUrl":"10.1016/j.vascn.2025.108376","url":null,"abstract":"<div><div>Extracellular vesicles (EVs) often possess both ubiquitous and unique tetraspanin molecules that can help elucidate their cell-of-origin. Furthermore, the presence and/or absence of specific tetraspanins can be used to phenotype and identify specific subpopulations of EVs. In the context of immune-related disorders (<em>i.e.</em> multiple sclerosis, rheumatoid arthritis, and various cancers), specific immune cell-derived EVs are now being investigated in the context of biomarker exploration, identifying novel disease mechanisms, and monitoring therapeutic responses in patients. Flow cytometry (FCM) is a technique that uses the light scattering properties of cells and/or subcellular particles (<em>e.g.</em> EVs), while combining fluorescent signals that can detect the presence, absence, or abundance of surface and/or intracellular molecules. To date, however, using FCM to accurately quantify EV populations has been challenging due to their relatively small size and weak light scattering and fluorescence properties compared to intact cells. Historically, the application of calibration beads of known sizes, refractory indices, a violet-side scatter, and standardized methodologies have made positive contributions towards the accurate detection and quantification of EVs while also permitting exploration into their biological properties. This review provides a summary and perspective of current FCM methodologies that are used to assess immune cell-derived EVs within biological fluids and cell supernatants. While acknowledging past and current limitations, as well as the recent successes, improvements, and efficiencies of assays used in EV-related research, the field will inevitably continue to advance through the implementation of standards and guidelines to enhance discovery and reproducibility.</div></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"135 ","pages":"Article 108376"},"PeriodicalIF":1.3,"publicationDate":"2025-06-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144522949","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}