Methods Protoc., Volume 8, Issue 3 (June 2025) – 19 articles

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. Full article

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. Full article

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. Full article

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. Full article

Nile tilapia (Oreochromis niloticus) 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. Full article

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. Full article

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. Full article

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 Allium fistulosum, 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. Full article

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, p < 0.001), while ICW was correlated with the percentage of body fat in women (r = 0.579, p < 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. Full article

Klebsiella pneumoniae 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 Klebsiella pneumoniae 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 Klebsiella pneumoniae. Full article

Pleural carcinomatosis (PC) and malignant pleural effusion (MPE) affect up to 20% of patients with non-small cell lung cancer (NSCLC) and are usually synonymous with poor prognosis. Pressurized Intra-Thoracic Aerosol Chemotherapy (PITAC) is a novel and promising technique to control MPE in PC-NSCLC. This pilot study aimed to assess the feasibility, safety, and efficacy of PITAC in terms of palliative pleurodesis and evaluate the local antineoplastic control by analyzing patient-derived primary cell cultures. From January to December 2023, seven patients underwent PITAC with tailored doses of cisplatin and doxorubicin. There were four males and three females, with a median age of 65 (IQR:19) years. No operating room contamination by aerosolized chemotherapeutics was observed. No intraoperative complications occurred, and 30-day mortality was nil. One patient developed a postoperative prolonged air leak. The median chest tube stay was 2 (IQR:2) days, and the median hospital stay was 4 (IQR:2) days. No systemic toxicity nor hypersensitivity to chemotherapeutics were observed. All patients developed effective pleurodesis in 30 days. Cell cultures obtained from biopsy of PC-NSCLC sampled before PITAC formed confluent and monolayer sheets of attached tumor cells, while after 30 min from PITAC, cultures exhibited a significant reduction in the cancer cells’ growth. Effective pleurodesis was observed three and six months after surgery in all patients. PITAC is a safe and effective technique to control MPE recurrence and might revolutionize loco-regional therapy for PC-NSCLC. Further research should assess its oncological role. Full article

Efficient extraction of high-quality DNA from plants is a critical challenge in molecular research, especially in species such as Gossypium barbadense L., native to Peru, due to the presence of inhibitors such as polysaccharides and phenolic compounds. This study presents a modified CTAB-based protocol with silica columns that is designed to overcome these limitations without the need for liquid nitrogen or expensive reagents. Native cotton samples were collected in Lambayeque, Peru, and processed using a simplified procedure that optimizes the purity and concentration of the extracted DNA. Eight cultivars of G. barbadense L. with colored fibers (cream, fifo, light brown, dark brown, orange-brown, reddish, fine reddish, and white) were evaluated, yielding DNA with A260/A280 ratios between 2.14 and 2.19 and A260/A230 ratios between 1.8 and 3.14; these values are higher than those obtained with the classical CTAB method. DNA quality was validated by PCR amplification using ISSR and RAPD molecular markers, which yielded clear and well-defined banding patterns. Furthermore, the extracted DNA was suitable for advanced applications, such as Sanger sequencing, by which high-quality electropherograms were obtained. The results demonstrate that the proposed protocol is an efficient, economical, and adaptable alternative for laboratories with limited resources, allowing the extraction of high-quality DNA from Gossypium barbadense L. and other plant species. This simplified approach facilitates the development of genetic and biotechnological research, contributing to the knowledge and valorization of the genetic resources of Peruvian native cotton. Full article

Background: Oral diseases represent a constant burden for health care and socio-economic systems as they are correlated to other non-communicable diseases. The aim of the proposed intervention is to test the effect of daily tooth brushing and oral health education on the oral health status of kindergarten children. Methods: The protocol will be conducted based on a previous epidemiological survey and conducted over 24 months; it has been developed on different levels. Dental hygienists will receive specific training to deliver oral health promotion to children and nursery educators. Training will focus on tailoring key messages to the specific age at visit; this will be outlined in the care pathway and offer practical preparation for delivering interventions and a toothpaste/toothbrush scheme. It will also, involving involve offering free daily tooth brushing to every 4–6-year-old child attending nursery. Data will be collected in four kindergartens in the capital or metropolitan areas, two kindergartens each in two large cities, and one kindergarten each in four villages from different geographic areas. Procedures used to assess the outcomes of each activity will be tailored to specific outcomes. Daily tooth-brushing activities will be monitored using qualitative research. A cost analysis including the distribution of necessary materials and correct delivery of products that shows price trends and percentage differences over the time span as well as consumer price index evaluation for the given time span will be conducted. Clinical outcomes will be evaluated using the caries incidence rate; this will be calculated for each tooth as the unit of analysis and evaluated using a multi-step approach. Discussion: Downstream oral health prevention interventions, like clinical prevention and oral health promotion, aim to enhance children’s quality of life. The program’s goal is to progress towards upstream interventions for a more significant impact. Full article

Background: Lafora disease (LD) is an ultra-rare and fatal neurodegenerative disorder with limited therapeutic options. Current treatments primarily address symptoms, with modest efficacy in halting disease progression, thus highlighting the urgent need for novel therapeutic approaches. Gene therapy, antisense oligonucleotides, and recombinant enzymes have recently been, and still are, under investigation. Drug repurposing may offer a promising approach to identify new, possibly effective, therapies. Methods: This study aims to investigate the conditions for repurposing empagliflozin, an SGLT2 (sodium/glucose cotransporter-2) inhibitor, as a potential treatment for LD and to establish a clinical protocol. Clinical phase: This 12-month prospective observational study will assess the safety and clinical efficacy of empagliflozin in two patients with early to intermediate LD stage. The primary endpoints will include changes in the severity of epilepsy and cognitive function, while the secondary endpoints will assess motor function, global function, and autonomy. Multiple clinical and instrumental evaluations (including MRI and PET with ¹⁸F-fluorodeoxyglucose) will be performed before and during treatment. Safety monitoring will include regular clinical assessments and reports of adverse events. Preclinical phase: In silico studies (using both molecular docking calculations and reverse ligand-based screening) and in vitro cell-based assays will allow us to investigate the effects of empagliflozin (and other gliflozins) on some key targets likely implicated in LD pathogenesis, such as GLUT1, GLUT3, glycogen synthase (hGYS), and glycogen phosphorylase (GP), as suggested in the literature and digital platforms for in silico target fishing. Results: The expected outcome of this study is twofold, i.e., (i) assessing the safety and tolerability of empagliflozin in LD patients and (ii) gathering preliminary data on its potential efficacy in improving clinical and neurologic features. Additionally, the in silico and in vitro studies may provide new insights into the mechanisms through which empagliflozin may exert its therapeutic effects in LD. Conclusion: The findings of this study are expected to provide evidence in support of the repurposing of empagliflozin for the treatment of LD. Full article

The Eurasian lynx (Lynx lynx) is listed in CITES Appendix II and is protected under the Bern Convention and the EU Habitats Directive, yet it remains a frequent target of wildlife crime, highlighting the urgent need for reliable identification methods. This study focuses on determination and DNA quantification of the Lynx spp. using quantitative real-time PCR (qPCR). The Llynx Qplex quantification multiplex system effectively distinguishes Lynx spp. from other Feliformia species by targeting mitochondrial and nuclear markers. Additionally, we present the results of the developmental validation of the Llyn STRplex system for individual identification and databasing using six STR loci. This study followed ISFG recommendations for non-human DNA testing and developmental validation guidelines. Both systems demonstrate high sensitivity (5 pg genomic DNA for Llynx Qplex and 30 pg of mtDNA for Llyn STRplex) and high specificity to Lynx spp., confirmed by testing against 16 related Feliformia species. Robustness was evaluated, showing sensitivity to temperature variation, and both repeatability and reproducibility were successfully tested across replicates and conditions. Given that forensic casework often involves degraded and limited biological material, molecular tools must be both sensitive and specific to ensure accurate results. Developing precise and efficient tools is essential for supporting investigations of wildlife crime involving the Eurasian lynx, as well as efforts aimed at conserving the species. Full article

Replicating in vivo conditions is essential for understanding immune responses and measuring immune biomarkers in blood. Sampling immune biomarkers in plasma or serum often fails to detect disease-relevant signals, possibly because these markers are sequestered in immune cells or extracellular vesicles. Furthermore, traditional whole blood cultures using external media may not accurately mimic the physiological environment of blood cells. To address these limitations, we developed a strategy using whole blood cell lysates and supernatants to optimize biomarker detection. Additionally, we employed neat whole blood culture methods, preserving the natural cellular and biochemical environment to assess sensitivity to immune modulators, such as lipopolysaccharide (LPS). This cost-effective approach minimizes variability and contamination risks. By utilizing Luminex multiplex immunoassays, we profiled immune biomarkers with higher sensitivity and efficiency than traditional ELISAs. Blood samples from individuals with high alcohol consumption validated our method by assessing biomarker levels before and after LPS stimulation, providing insights into intracellular responses and inflammatory pathways. This method enhances our understanding of inflammatory processes in blood cells, demonstrating the advantages of cell lysates, supernatants, and advanced multiplex assays in immunological research. Full article

Early initiation of palliative care in patients with advanced chronic conditions significantly improves their quality of care; however, variability in disease trajectories complicates such interventions’ timing. The NECPAL CCOMS-ICO prognostic tool was developed as a straightforward instrument to help healthcare providers in all clinical settings promptly identify patients with advanced chronic conditions who require palliative care, thereby enhancing service planning and delivery. Its latest version, 4.0, 2021, for the first time, incorporates a patient survival estimation. Nevertheless, validation is necessary. This study aims to validate the NECPAL version 4.0 tool in an independent cohort. It is an observational, prospective study involving outpatients and hospitalized non-randomized patients at Hospital Universitario Infanta Leonor–Virgen de la Torre in Madrid, Spain, all of whom have at least one advanced chronic condition. The study is scheduled to last 6 years, including a recruitment period of 30 months starting 1 February 2024, followed by a 12-month follow-up period for each patient. This is the first prospective study designed to validate the NECPAL version 4.0 instrument. Implementing this tool would allow the identification of patients with advanced chronic conditions and unmet palliative care needs and determine the more appropriate care pathway at the proper moment. Full article

Myocardial infarction with non-obstructive coronary arteries (MINOCA) is a working diagnosis encompassing several pathophysiological mechanisms with specific treatments and different prognoses. Despite the absence of obstructive coronary artery disease, MINOCA has proven to be associated with a significant risk of mortality, angina burden, and socioeconomic costs. However, due to the heterogeneous nature of this clinical condition and the absence of randomized clinical trials, evidence supporting a standardized diagnostic algorithm and the clinical management of these patients is lacking. The PROMISE trial is the first randomized clinical trial evaluating the effectiveness of a precision medicine approach strategy in improving the outcomes and quality of life of patients with MINOCA, offering new insights into personalized treatment strategies. This review article discusses the promise of a precision medicine approach in patients with MINOCA, highlighting the potential innovations and challenges of a personalized medicine strategy in MINOCA. Full article

Antigen presentation on major histocompatibility complex (MHC) molecules is central to the initiation of immune responses, and a lot of our understanding about the antigen processing and presentation pathway has been gained through studies in mice. MHC molecules are the most genetically diverse genes; consequently, mouse strains differ substantially in their MHC make up and resulting antigen presentation. Swiss mice are commonly used in pharmacological research, yet our understanding of antigen presentation in this strain is surprisingly limited. Here, we have tested a range of anti-MHC antibodies and present a range of clones suitable to analyse MHC class I and class II molecules in Swiss mice who have the H2-q MHC haplotype. Moreover, we demonstrate using immunopeptidomics that clones 28-12-8, 34-1-2, MKD6, and N22 are also suited to isolate MHC class I and class II ligands in this mouse strain. Thus, this work also establishes a first experimental account of the H2-q-derived thymus and spleen immunopeptidome in Swiss mice which bears strong resemblance with ligands isolated from the H2-d MHC haplotype of Balb/C mice. The analysis of source proteins shows common but also organ- and function-specific antigen presentation in line with the involvement of the thymus in tolerance induction and the function of the spleen as a site of immune responses. Full article