Journal Description
Methods and Protocols
Methods and Protocols
is an international, peer-reviewed, open access journal aiming to establish and describe new experimental techniques in the fields of Life Sciences, Chemistry, and Biomedical Sciences, published bimonthly online by MDPI.
- Open Access— free for readers, with article processing charges (APC) paid by authors or their institutions.
- High Visibility: indexed within Scopus, ESCI (Web of Science), PubMed, PMC, CAPlus / SciFinder, and other databases.
- Journal Rank: CiteScore - Q2 (Biochemistry, Genetics and Molecular Biology (miscellaneous))
- Rapid Publication: manuscripts are peer-reviewed and a first decision is provided to authors approximately 30.6 days after submission; acceptance to publication is undertaken in 4.8 days (median values for papers published in this journal in the second half of 2024).
- Recognition of Reviewers: reviewers who provide timely, thorough peer-review reports receive vouchers entitling them to a discount on the APC of their next publication in any MDPI journal, in appreciation of the work done.
Impact Factor:
2.3 (2023);
5-Year Impact Factor:
2.2 (2023)
Latest Articles
Improved RSV Neutralization Assay Using Recombinant RSV Expressing Reporter Fluorescent Protein
Methods Protoc. 2025, 8(3), 60; https://doi.org/10.3390/mps8030060 - 4 Jun 2025
Abstract
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
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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
(This article belongs to the Section Molecular and Cellular Biology)
Open AccessProtocol
Determination of the Minimum Cell-to-Cell Adhesion Time Using Optical Tweezers in Leukemia and Lymphoma Research
by
Kamila Duś-Szachniewicz and Sławomir Drobczyński
Methods Protoc. 2025, 8(3), 59; https://doi.org/10.3390/mps8030059 - 4 Jun 2025
Abstract
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
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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
(This article belongs to the Section Molecular and Cellular Biology)
Open AccessProtocol
Synthesis of DOTA-Based 43Sc Radiopharmaceuticals Using Cyclotron-Produced 43Sc as Exemplified by [43Sc]Sc-PSMA-617 for PSMA PET Imaging
by
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 and Satish K. Chitneni
Methods Protoc. 2025, 8(3), 58; https://doi.org/10.3390/mps8030058 - 4 Jun 2025
Abstract
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 [
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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
(This article belongs to the Section Biochemical and Chemical Analysis & Synthesis)
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Open AccessProtocol
Optimization of Nile Tilapia Artificial Breeding Using Human Chorionic Gonadotropin (hCG) Hormone
by
Golam Rbbani, Prabhugouda Siriyappagouder, Riaz Murshed, Rajesh Joshi, Artem Nedoluzhko, Jorge Galindo-Villegas and Jorge M. O. Fernandes
Methods Protoc. 2025, 8(3), 57; https://doi.org/10.3390/mps8030057 - 2 Jun 2025
Abstract
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
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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
(This article belongs to the Section Biomedical Sciences and Physiology)
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Open AccessArticle
Towards Automated Testing of Kynurenine for Point-of-Care Metabolomics
by
Dipanjan Bhattacharyya, Marcia A. LeVatte and David S. Wishart
Methods Protoc. 2025, 8(3), 56; https://doi.org/10.3390/mps8030056 - 1 Jun 2025
Abstract
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
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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
(This article belongs to the Section Omics and High Throughput)
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Open AccessProtocol
A Minimally Invasive Transthoracic Injection Technique for Reproducible Intrapleural Delivery in Mice
by
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 and Evelien Smits
Methods Protoc. 2025, 8(3), 55; https://doi.org/10.3390/mps8030055 - 28 May 2025
Abstract
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
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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
(This article belongs to the Section Biomedical Sciences and Physiology)
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Open AccessProtocol
A Method of Well-Spread Pachytene Chromosome Preparations for Plant Species with Large Genomes Suitable for the Immunolocalization of Meiotic Proteins
by
Natalya Kudryavtseva, Aleksey Ermolaev and Ludmila Khrustaleva
Methods Protoc. 2025, 8(3), 54; https://doi.org/10.3390/mps8030054 - 19 May 2025
Abstract
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
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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
(This article belongs to the Section Molecular and Cellular Biology)
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Open AccessArticle
New Bioelectrical Impedance-Based Equations to Estimate Resting Metabolic Rate in Young Athletes
by
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 and Athanasios Chatzinikolaou
Methods Protoc. 2025, 8(3), 53; https://doi.org/10.3390/mps8030053 - 19 May 2025
Abstract
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
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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.
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(This article belongs to the Section Biomedical Sciences and Physiology)
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Open AccessProtocol
Development and Characterization of a Ten-Plex Assay to Measure Klebsiella pneumoniae Antigen-Specific IgG in Human Sera
by
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 and Omar Rossi
Methods Protoc. 2025, 8(3), 52; https://doi.org/10.3390/mps8030052 - 19 May 2025
Abstract
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
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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.
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(This article belongs to the Section Public Health Research)
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Open AccessArticle
Safety and Efficacy of Pressurized Intra-Thoracic Aerosol Chemotherapy in Non-Small Cell Lung Cancer Pleural Carcinomatosis: Preliminary Results of a Pilot Study
by
Maria Giovanna Mastromarino, Vittorio Aprile, Gianmarco Elia, Diana Bacchin, Alessandra Lenzini, Stylianos Korasidis, Marcello Carlo Ambrogi, Silvia Martina Ferrari, Poupak Fallahi and Marco Lucchi
Methods Protoc. 2025, 8(3), 51; https://doi.org/10.3390/mps8030051 - 14 May 2025
Abstract
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.
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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.
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(This article belongs to the Section Biomedical Sciences and Physiology)
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Open AccessProtocol
Rapid and Efficient DNA Extraction Protocol from Peruvian Native Cotton (Gossypium barbadense L.) Lambayeque, Peru
by
Luis Miguel Serquén Lopez, Herry Lloclla Gonzales, Wilmer Enrique Vidaurre Garcia, Ricardo Leonidas de Jesus Velez Chicoma and Mendoza Cornejo Greta
Methods Protoc. 2025, 8(3), 50; https://doi.org/10.3390/mps8030050 - 7 May 2025
Abstract
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
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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.
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(This article belongs to the Section Molecular and Cellular Biology)
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Open AccessStudy Protocol
SmilebrightRO—Study Protocol for a Randomized Clinical Trial to Evaluate Oral Health Interventions in Children
by
Ruxandra Sava-Rosianu, Guglielmo Campus, Vlad Tiberiu Alexa, Octavia Balean, Ruxandra Sfeatcu, Alice Murariu, Alexandrina Muntean, Daniela Esian, Constantin Daguci, Simona Olaru-Posiar, Vanessa Bolchis, Antonia Ilin, Ramona Dumitrescu, Berivan Laura Rebeca Buzatu, Mariana Postolache, Nicoleta Toderas, Roxana Oancea, Daniela Jumanca and Atena Galuscan
Methods Protoc. 2025, 8(3), 49; https://doi.org/10.3390/mps8030049 - 7 May 2025
Abstract
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
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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.
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(This article belongs to the Section Public Health Research)
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Open AccessStudy Protocol
Empagliflozin Repurposing for Lafora Disease: A Pilot Clinical Trial and Preclinical Investigation of Novel Therapeutic Targets
by
Giuseppe d’Orsi, Antonella Liantonio, Paola Imbrici, Nicola Gambacorta, Giorgia Dinoi, Cosimo Damiano Altomare, DEFEAT-LD Study Group and Massimo Carella
Methods Protoc. 2025, 8(3), 48; https://doi.org/10.3390/mps8030048 - 6 May 2025
Abstract
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
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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.
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(This article belongs to the Section Biomedical Sciences and Physiology)
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Open AccessArticle
Molecular Tools for Lynx spp. qPCR Identification and STR-Based Individual Identification of Eurasian Lynx (Lynx lynx) in Forensic Casework
by
Karolina Mahlerová, Johana Alaverdyan, Lenka Vaňková and Daniel Vaněk
Methods Protoc. 2025, 8(3), 47; https://doi.org/10.3390/mps8030047 - 2 May 2025
Abstract
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
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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.
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(This article belongs to the Section Molecular and Cellular Biology)
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Open AccessProtocol
Multiplex Immunoassay for Biomarker Profiling of Whole Blood Cell Lysates and Supernatants and Pathogen Response in Neat Whole Blood Cultures
by
Irina Balan, Alejandro G. Lopez and A. Leslie Morrow
Methods Protoc. 2025, 8(3), 46; https://doi.org/10.3390/mps8030046 - 1 May 2025
Abstract
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
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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
(This article belongs to the Section Public Health Research)
Open AccessStudy Protocol
External Validation of the NECPAL CCOMS-ICO Prognostic Tool for Early Palliative Care and Mortality Prediction in Patients with Advanced Chronic Conditions: A Prospective Observational Study Protocol
by
Ana Bustamante-Fermosel, Elena Díaz-Sánchez, Natalia Pavón-Muñoz, Laetitia Hennekinne, Fuensanta Gil-Gil, Helena Notario-Leo, Alicia Sánchez-Pizarro, Marta Bustamante-Vega, Juan Torres-Macho, Anabel Franco-Moreno and on behalf of the COMPASS Research Group
Methods Protoc. 2025, 8(3), 45; https://doi.org/10.3390/mps8030045 - 1 May 2025
Abstract
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
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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.
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(This article belongs to the Section Public Health Research)
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Open AccessReview
The PROMISE of Precision Medicine in Myocardial Infarction with Non-Obstructive Coronary Arteries
by
Giulia La Vecchia, Vincenzo Scarica, Ludovica Leo and Rocco A. Montone
Methods Protoc. 2025, 8(3), 44; https://doi.org/10.3390/mps8030044 - 27 Apr 2025
Abstract
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,
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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.
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(This article belongs to the Special Issue Feature Papers in Methods and Protocols 2025)
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Open AccessArticle
The Validation of Antibodies Suitable for Flow Cytometric Analysis and Immunopeptidomics of Peptide–MHC Complexes in the Outbred Swiss Albino Mouse Strain
by
Shanzou Chung, Isambard G. Knox-Johnson, Sarah E. Gazzard, Runqiu Song, Ngoc H. Le, Luise A. Cullen-McEwen, John F. Bertram, Anthony W. Purcell and Asolina Braun
Methods Protoc. 2025, 8(3), 43; https://doi.org/10.3390/mps8030043 - 24 Apr 2025
Abstract
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
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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.
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(This article belongs to the Section Molecular and Cellular Biology)
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Open AccessArticle
Quantification of Circulating Cell-Free DNA as a NETosis Marker in Trauma Patients with Type 2 Diabetes Mellitus
by
Filiz Sahin, Regina Breinbauer, Caren Linnemann, Melike Tombaz, Andreas K. Nussler and Sabrina Ehnert
Methods Protoc. 2025, 8(2), 42; https://doi.org/10.3390/mps8020042 - 16 Apr 2025
Abstract
Type 2 diabetes mellitus (T2DM) significantly impairs fracture healing, with neutrophils playing a crucial role in this process. In T2DM, these immune cells are over-activated, leading to the excessive release of neutrophil extracellular traps (NETs), increasing inflammation and hindering recovery. Thus, a need
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Type 2 diabetes mellitus (T2DM) significantly impairs fracture healing, with neutrophils playing a crucial role in this process. In T2DM, these immune cells are over-activated, leading to the excessive release of neutrophil extracellular traps (NETs), increasing inflammation and hindering recovery. Thus, a need for markers to assess patients in the risk group arises. This study demonstrates that circulating cell-free DNA (cfDNA) can be efficiently quantified from serum samples by a single-step qPCR and be used as a marker for NETosis. Our results revealed that trauma patients with T2DM have the highest cfDNA levels, followed by trauma patients, and the healthy group has the lowest. The method shows strong correlations between cfDNA and neutrophil-specific markers such as MPO, citH3, AZU1, and α-defensin, highlighting its potential as a rapid indicator of NETosis. This approach could allow the timely interference for high-risk patients, ultimately improving healing outcomes and reducing complications such as chronic inflammation, non-union fractures, and diabetic foot ulcers.
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(This article belongs to the Section Public Health Research)
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Open AccessArticle
Concurrent Validity of Three Photogrammetric Methods for Assessing Knee Alignment in Sagittal Plane
by
Bruna Nichele da Rosa, Paula Andryelly Gomes Giendruczak, Marina Ziegler Frantz, Matias Noll and Cláudia Tarragô Candotti
Methods Protoc. 2025, 8(2), 41; https://doi.org/10.3390/mps8020041 - 14 Apr 2025
Abstract
Background: Evidence supporting the validity of photogrammetry for assessing body segment alignment remains limited, with most studies focusing on spinal evaluation. Thus, there is a lack of robust research examining its use for other body segments such as the lower limbs. Objective
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Background: Evidence supporting the validity of photogrammetry for assessing body segment alignment remains limited, with most studies focusing on spinal evaluation. Thus, there is a lack of robust research examining its use for other body segments such as the lower limbs. Objective: This study aimed to evaluate the concurrent validity of three photogrammetric methods for measuring knee alignment in the sagittal plane with and without corrections for potential rotational deviations in the participant’s thigh and leg. Methods: A total of 21 adults underwent sequential evaluations involving panoramic radiography of the lower limbs and photogrammetry at a private radiology clinic. Photogrammetric analysis involved identifying the following anatomical landmarks: the greater trochanter of the femur (GTF), the lateral condyle of the femur (LCF), the head of the fibula (HF), and lateral malleolus (LM). Three photogrammetric methods were employed: (1) the condylar angle (CA) defined by the GTF, LCF, and LM points; (2) the fibula head angle (FHA) defined by the GTF, HF, and LM points; and (3) the four-point angle (4PA) incorporating the GTF, LCF, HF, and LM. Concurrent validity was assessed using correlation analysis, agreement with radiographic measurements, and the root mean square error (RMSE). Each photogrammetric method was tested using raw (CA, FHA, and 4PA) and corrected (CAcorr, FHAcorr, and 4PAcorr) values, accounting for thigh and/or leg rotational deviations. Results: Correcting for thigh and leg rotations significantly improved the validity metrics for all methods. The best performance was observed with the corrected condylar angle (CAcorr: r = 0.746; adjusted r2 = 0.533; RMSE = 2.9°) and the corrected four-point angle (4PAcorr: r = 0.733; adjusted r2 = 0.513; RMSE = 3.0°); however, the measurements presented proportional errors, possible due the method of assessment of rotations. Conclusions: The findings validate the evaluated photogrammetric methods for assessing sagittal knee alignment. Accounting for thigh and leg rotational deviations is critical for achieving accurate measurements, raising the need of accurate tools for measuring rotational changes in the lower limbs to avoid errors.
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(This article belongs to the Section Biomedical Sciences and Physiology)
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