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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: 2.0 (2024); 5-Year Impact Factor: 2.2 (2024)

Imprint Information Journal Flyer Open Access ISSN: 2409-9279

Latest Articles

1 pages, 129 KiB

Open AccessExpression of Concern

Expression of Concern: König, B.; Kirchner, J.O. Methodological Considerations Regarding the Quantification of DNA Impurities in the COVID-19 mRNA Vaccine Comirnaty^®. Methods Protoc. 2024, 7, 41

by Methods and Protocols Editorial Office

Methods Protoc. 2025, 8(4), 68; https://doi.org/10.3390/mps8040068 - 25 Jun 2025

Following publication [...] Full article

(This article belongs to the Section Biomedical Sciences and Physiology)

9 pages, 1004 KiB

Open AccessTechnical Note

A Simplified Method for Extracting the Movement Trajectories of Small Aquatic Animals

by Xin Liu, Huanan Gao, Aimin Hao and Yasushi Iseri

Methods Protoc. 2025, 8(4), 67; https://doi.org/10.3390/mps8040067 - 20 Jun 2025

Understanding the motion behaviors of animals is crucial for unraveling the mechanisms underlying ethology across various domains, such as movement patterns, food detection, and defense strategies. In this study, we devised a simplified method enabling the movement of small animals to be tracked [...] Read more.

Understanding the motion behaviors of animals is crucial for unraveling the mechanisms underlying ethology across various domains, such as movement patterns, food detection, and defense strategies. In this study, we devised a simplified method enabling the movement of small animals to be tracked conveniently using high-resolution smartphone videos and freely available tracking software. Employing a laboratory video setup, we traced the swimming trajectory of the small copepod zooplankton Eodiaptomus japonicus, which has a body size of approximately 1 mm. From the tracked position data, we analyzed key motion parameters, including swimming distance, speed, and jump frequency. The results of our video analysis showed that adult female E. japonicus exhibited an average swimming speed of 9.8 mm s⁻¹, displaying a predominant cruising pattern with speeds of around 5.0 mm s⁻¹, punctuated by sporadic jumps, showcasing maximum instantaneous speeds reaching a remarkable 190.1 mm s⁻¹. Our successful tracking of the high-speed swimming copepod not only sheds light on its locomotion dynamics but also underscores the potential to refine this method to study the motion trajectories of diverse animal species. Full article

(This article belongs to the Section Biomedical Sciences and Physiology)

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21 pages, 3724 KiB

Open AccessProtocol

Expression and Site-Specific Biotinylation of Human Cytosolic 5′-Nucleotidase 1A in Escherichia coli

by Nataliya Slater, Anuradha Sooda, Frank L. Mastaglia, Sue Fletcher, Mark Watson, Merrilee Needham and Jerome D. Coudert

Methods Protoc. 2025, 8(3), 66; https://doi.org/10.3390/mps8030066 - 18 Jun 2025

Autoantibodies targeting cytosolic 5′-nucleotidase 1A (cN1A) are found in several autoimmune diseases, including inclusion body myositis (IBM), Sjögren’s syndrome, and systemic lupus erythematosus. While they have diagnostic relevance for IBM, little is known about the autoreactive B cells that produce these antibodies. To [...] Read more.

Autoantibodies targeting cytosolic 5′-nucleotidase 1A (cN1A) are found in several autoimmune diseases, including inclusion body myositis (IBM), Sjögren’s syndrome, and systemic lupus erythematosus. While they have diagnostic relevance for IBM, little is known about the autoreactive B cells that produce these antibodies. To address this, we developed a robust protocol for the expression and site-specific biotinylation of recombinant human cN1A in Escherichia coli. The resulting antigen is suitable for generating double-labelled fluorescent baits for the isolation and characterisation of cN1A-specific B cells by flow cytometry. Site-specific biotinylation was achieved using the AviTag and BirA ligase, preserving the protein’s structure and immunoreactivity. Western blot analysis confirmed that the biotinylated cN1A was recognised by both human and rabbit anti-cN1A antibodies. Compared to conventional chemical biotinylation, this strategy minimises structural alterations that may affect antigen recognition. This approach provides a reliable method for producing biotinylated antigens for use in immunological assays. While demonstrated here for cN1A, the protocol can be adapted for other autoantigens to support studies of antigen-specific B cells in autoimmune diseases. Full article

(This article belongs to the Section Molecular and Cellular Biology)

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20 pages, 3665 KiB

Open AccessFeature PaperArticle

Evaluating the Effectiveness of Various Small RNA Alignment Techniques in Transcriptomic Analysis by Examining Different Sources of Variability Through a Multi-Alignment Approach

by Xinwei Zhao and Eberhard Korsching

Methods Protoc. 2025, 8(3), 65; https://doi.org/10.3390/mps8030065 - 17 Jun 2025

DNA and RNA nucleotide sequences are ubiquitous in all biological cells, serving as both a comprehensive library of capabilities for the cells and as an impressive regulatory system to control cellular function. The multi-alignment framework (MAF) provided in this study offers a user-friendly [...] Read more.

DNA and RNA nucleotide sequences are ubiquitous in all biological cells, serving as both a comprehensive library of capabilities for the cells and as an impressive regulatory system to control cellular function. The multi-alignment framework (MAF) provided in this study offers a user-friendly platform for sequence alignment and quantification. It is adaptable to various research needs and can incorporate different tools and parameters for in-depth analysis, especially in low read rate scenarios. This framework can be used to compare results from different alignment programs and algorithms on the same dataset, allowing for a comprehensive analysis of subtle to significant differences. This concept is demonstrated in a small RNA case study. MAF is specifically designed for the Linux platform, commonly used in bioinformatics. Its script structure streamlines processing steps, saving time when repeating procedures with various datasets. While the focus is on microRNA analysis, the templates provided can be adapted for all transcriptomic and genomic analyses. The template structure allows for flexible integration of pre- and post-processing steps. MicroRNA analysis indicates that STAR and Bowtie2 alignment programs are more effective than BBMap. Combining STAR with the Salmon quantifier or, with some limitations, the Samtools quantification, appears to be the most reliable approach. This method is ideal for scientists who want to thoroughly analyze their alignment results to ensure quality. The detailed microRNA analysis demonstrates the quality of three alignment and two quantification methods, offering guidance on assessing result quality and reducing false positives. Full article

(This article belongs to the Section Omics and High Throughput)

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16 pages, 3616 KiB

Open AccessProtocol

An Efficient Electroporation Protocol Supporting In Vitro Studies of Oligodendrocyte Biology

by Yugo Ishino, Shoko Shimizu and Shingo Miyata

Methods Protoc. 2025, 8(3), 64; https://doi.org/10.3390/mps8030064 - 13 Jun 2025

Oligodendrocytes form myelin in the central nervous system, and their dysfunction can cause severe neurological symptoms, as large-scale analyses have highlighted numerous gene expression alterations in pathological conditions. Although in vivo functional gene analyses are preferable, they have several limitations, especially in large-scale [...] Read more.

Oligodendrocytes form myelin in the central nervous system, and their dysfunction can cause severe neurological symptoms, as large-scale analyses have highlighted numerous gene expression alterations in pathological conditions. Although in vivo functional gene analyses are preferable, they have several limitations, especially in large-scale studies. Therefore, standardized in vitro systems are needed to facilitate efficient and reliable functional analyses of genes identified in such studies. Here, we describe a practical and efficient method for oligodendrocyte precursor cell (OPC) isolation from mouse brains on postnatal day 6–8 and a gene delivery method for the isolated OPCs. By modifying the magnetic-activated cell sorting (MACS) procedure with reduced processing volumes, we simplified OPC isolation, allowing simultaneous handling of multiple samples and improving workflow efficiency. We also optimized electroporation parameters to achieve robust transfection efficiency with minimal cell death. Transfected OPCs are suitable for both monoculture-based differentiation assays and co-culture with dorsal root ganglion (DRG) explants, in which they reliably differentiate into mature oligodendrocytes and myelinate along the axons. This system enables stable and reproducible in vitro analysis of oligodendrocyte function, supports investigations into both intrinsic differentiation and neuron–glia interactions, and provides a powerful platform for oligodendrocyte research with efficient and timely gene manipulation. Full article

(This article belongs to the Section Molecular and Cellular Biology)

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8 pages, 2115 KiB

Open AccessCommunication

Cystic Lung Phantom to Validate Clinical CT Protocols

by Shefra Shah, Farah Hussaini, Dumitru Mazilu, Eric E. Bennett and Han Wen

Methods Protoc. 2025, 8(3), 63; https://doi.org/10.3390/mps8030063 - 13 Jun 2025

In computed tomography (CT)-based evaluation of the extent of cystic changes in the lungs of patients with cystic lung diseases, such as Lymphangioleiomyomatosis (LAM), there is a lack of a lung phantom containing air-filled cavities that mimic pulmonary cysts to calibrate the measurement [...] Read more.

In computed tomography (CT)-based evaluation of the extent of cystic changes in the lungs of patients with cystic lung diseases, such as Lymphangioleiomyomatosis (LAM), there is a lack of a lung phantom containing air-filled cavities that mimic pulmonary cysts to calibrate the measurement of cystic volumes from CT scans. We describe an easy-to-replicate cystic lung phantom consisting of basic lung structures of a trachea and two lung compartments. The lung compartments contain air cavities of varying sizes to mimic cystic lesions. The lung compartments are made of a foam material recommended by NIST to simulate the radiodensity of human lung parenchyma. In tests performed on a clinical scanner, various structures in the lung phantom were correctly recognized by two types of lung analysis software. The resulting cystic volume measurements revealed the relationship between the size of the cysts and the accuracy of the measurement. The significant finding was that the volumes of individual cysts were underestimated for small cysts. The error increased with decreasing cyst sizes. Such underestimation has not been mentioned previously and deserves the attention of clinicians using CT scans to assess the cyst burden in the lungs, particularly in patients presenting with numerous small pulmonary cysts. Full article

(This article belongs to the Section Public Health Research)

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15 pages, 5385 KiB

Open AccessStudy Protocol

Investigating the Impact of Pressure Relief Performance on the Occurrence of Pressure Injuries and Shoulder Pain in Wheelchair Users with Spinal Cord Injury (PRperf Study): Study Protocol for a Prospective Observational Study

by Yannik Schürch, Anneke Hertig-Godeschalk, Inge Eriks-Hoogland, Anke Scheel-Sailer, Martin W. G. Brinkhof and Ursina Arnet

Methods Protoc. 2025, 8(3), 62; https://doi.org/10.3390/mps8030062 - 6 Jun 2025

Background: Pressure injuries (PIs) and shoulder pain (SP) are frequent problems in individuals with spinal cord injury (SCI), affecting both quality of life and healthcare use. Although pressure relief (PR) is recommended to prevent PIs, it is often not performed regularly, and its [...] Read more.

Background: Pressure injuries (PIs) and shoulder pain (SP) are frequent problems in individuals with spinal cord injury (SCI), affecting both quality of life and healthcare use. Although pressure relief (PR) is recommended to prevent PIs, it is often not performed regularly, and its long-term benefits remain unclear. Furthermore, some PR methods may contribute to SP, resulting in conflicting clinical guidelines. This study aims to objectively measure PR performance and investigate its long-term relationship with PI and SP. Methods: This study is a longitudinal observational study involving 70 manual wheelchair users with complete SCI. Over one year, participants attend five study visits to assess confounding factors such as comorbidities and shoulder range of motion. PR performance (technique, frequency, duration) is continuously monitored for three weeks after each of the first four visits using textile measurement mats, while SP is assessed weekly with a questionnaire. Causal associations with PI and SP will be examined using directed acyclic graphs and multivariable regression modelling. Results: The study is ongoing. Long-term objective data on PR performance will provide insights into its relationship with PI and SP. Conclusions: Findings will inform clinical practice and contribute to improved evidence-based PR guidelines for individuals with SCI. Full article

(This article belongs to the Section Public Health Research)

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19 pages, 5119 KiB

Open AccessArticle

A Refined Approach to Isolate Interneurons for High-Validity Epigenetic Studies in Human Brain Tissue

by Ariel Cariaga-Martínez, Kilian Jesús Gutierrez, Ignacio Regidor, Marta Del Álamo, Jerónimo Saiz-Ruiz and Raúl Alelú-Paz

Methods Protoc. 2025, 8(3), 61; https://doi.org/10.3390/mps8030061 - 5 Jun 2025

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 [...] Read more.

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

(This article belongs to the Section Molecular and Cellular Biology)

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10 pages, 1099 KiB

Open AccessArticle

Improved RSV Neutralization Assay Using Recombinant RSV Expressing Reporter Fluorescent Protein

by Yutaro Yamagata, Michiko Toizumi, Jean-Francois Eleouet, Marie-Anne Rameix-Welti, Makoto Takeda and Lay-Myint Yoshida

Methods Protoc. 2025, 8(3), 60; https://doi.org/10.3390/mps8030060 - 4 Jun 2025

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 [...] Read more.

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)

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17 pages, 3579 KiB

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

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 [...] Read more.

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)

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26 pages, 2541 KiB

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

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 [ [...] Read more.

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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8 pages, 742 KiB

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

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 [...] Read more.

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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22 pages, 1849 KiB

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

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 [...] Read more.

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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13 pages, 10557 KiB

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

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 [...] Read more.

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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10 pages, 33536 KiB

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

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 [...] Read more.

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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13 pages, 769 KiB

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

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 [...] Read more.

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

(This article belongs to the Section Biomedical Sciences and Physiology)

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17 pages, 2445 KiB

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

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 [...] Read more.

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

(This article belongs to the Section Public Health Research)

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12 pages, 3428 KiB

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

Cited by 1

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. [...] Read more.

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

(This article belongs to the Section Biomedical Sciences and Physiology)

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13 pages, 1936 KiB

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

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 [...] Read more.

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

(This article belongs to the Section Molecular and Cellular Biology)

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17 pages, 1898 KiB

Open AccessStudy Protocol

Smilebright^RO—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

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 [...] Read more.

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

(This article belongs to the Section Public Health Research)

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