Methods and Protocols doi: 10.3390/mps9040116

Authors: Akihiro Kaneda Masahiro Yoshida Yoshiya Kawaguchi Kenichiro Furuyama

Pancreatic ductal adenocarcinoma (PDAC) remains a highly lethal malignancy, highlighting the need for reproducible experimental systems that model pancreatic injury and subsequent neoplastic progression. Adult-onset genetically engineered models in which oncogenic Kras and mutant Trp53 are induced in the pancreas provide a relevant platform for studying PDAC pathogenesis, but efficient neoplastic progression often requires concomitant pancreatic injury. Cerulein-induced pancreatitis is widely used to provide an injury-associated inflammatory stimulus that promotes PDAC progression; however, repeated-injection regimens are labor-intensive and increase cumulative handling stress in animals. Here, we describe a dose-optimized, single-dose L-Lysine protocol designed to induce acute pancreatitis and characterize subsequent pancreatic neoplastic progression in adult-onset Kras/Trp53-driven mice. Tamoxifen-treated Ptf1aCreER/+; KrasLSL-G12D/+; Trp53LSL-R172H/+; Rosa26-RFP mice received a single intraperitoneal injection of L-Lysine, followed by biochemical, histological, and immunohistochemical assessment of pancreatic injury and tumor development. A single 2.0 g/kg L-Lysine injection induced sublethal acute pancreatitis characterized by increased serum pancreatic enzymes, interstitial edema, inflammatory cell infiltration, acinar cell necrosis, and rapid mitochondrial alterations. During 3–6 months of follow-up, mice developed multifocal acinar-to-ductal metaplasia, PanIN lesions, invasive PDAC, and peritoneal dissemination. This protocol provides a simple, synchronized, and less labor-intensive model for studying pancreatic injury and subsequent neoplastic progression in adult-onset Kras/Trp53-driven mice.

Methods and Protocols doi: 10.3390/mps9040115

Authors: Parker Norman Jaclyn Bain Linda Sangalli Mitchell Levine Caroline M. Sawicki

Pediatric sleep-disordered breathing (SDB) is underdiagnosed despite its associations with adverse neurobehavioral, psychosocial, and cardiometabolic outcomes. Pediatric dental providers routinely evaluate craniofacial growth and maintain longitudinal contact with children, yet practical approaches for integrating SDB risk assessment into dental settings remain limited. This protocol describes a prospective, cross-sectional observational study that will enroll 60 school-aged children aged 8–13 years from a university-based pediatric dental clinic and classify them as low-risk or high-risk for SDB using the Pediatric Sleep Questionnaire (PSQ; threshold ≥ 0.33). The primary aim is to examine associations between PSQ-based risk classification and objective physiologic sleep parameters, including the Apnea–Hypopnea Index, Respiratory Disturbance Index, Sleep Apnea Indicator, and Sleep Quality Index, obtained from a U.S. Food and Drug Administration-cleared wearable home sleep monitor—SleepImage Ring (MyCardio LLC, Denver, CO, USA)—worn for a minimum of three consecutive nights. Secondary aims will evaluate associations between SDB risk classification and body mass index, Mallampati score, Brodsky tonsillar grade, and psychosocial functioning (anxiety, depression, perceived stress, and daytime sleepiness). Exploratory craniofacial analyses will be conducted among participants with clinically available lateral cephalometric radiographs. This protocol could position pediatric dental visits as an accessible touchpoint for early identification of children with unrecognized SDB and inform pathways for timely referral. Protocol Version: 1.4, dated 13 May 2026. Trial Registration: ClinicalTrials.gov NCT07581938.

Methods and Protocols doi: 10.3390/mps9040114

Authors: Mahmoud Zhra Rawan Awni Alarawi Shaimaa Abdelrahman Mohamed Abdel Naser Daoud Hana Fakhoury Ahmad Aljada

Column-based RNA extraction is time-consuming, involves cumulative losses during transfer, wash, and elution steps, and has reduced recovery from sub-microliter inputs. These limitations restrict gene-expression analysis in small cell populations and low-titre pathogen detection in clinical specimens. We developed the Direct Cell-to-PCR Lysis Buffer, a defined non-ionic detergent formulation containing Tween 20 (0.3%), Triton X-100 (0.1%), and NP-40 (0.1%), for RNA-extraction-free one-step RT-qPCR. Lysates are added directly to reactions without pretreatment, heating, or column purification. Non-ionic detergents produced Ct values comparable to detergent-free controls, whereas SDS, Sarkosyl, and sodium deoxycholate abolished amplification. The method was benchmarked against the standard column-based PureLink RNA Mini Kit using SaOS-2 cells, MCF7 cells, peripheral blood mononuclear cells, and nasal and throat swabs. Direct lysates showed Ct offsets of 0.6 to 2.85 cycles compared with purified RNA, depending on sample type. Ubiquitin-C mRNA was detected down to a single-cell-equivalent input, and plasmid templates were detected to approximately 100 copies per reaction. The buffer was compatible with multiplex ScriptTaq COVID PCR, with unchanged RdRP, N, and RPP30 Ct values, and tolerated sodium azide up to 0.1%, supporting rapid direct RT-qPCR workflows.

Methods and Protocols doi: 10.3390/mps9040113

Authors: Gulnur Nakhanova Olga Chervyakova Kamshat Shorayeva Aigerim Zhakypbek Sabina Moldagulova Aknur Ulankyzy Alisher Omurtay Yeraly Shayakhmetov Temirlan Baiseit Zharkinay Absatova Aisha Issabek Gaukhar Shynybekova Sandugash Sadikaliyeva Aziz Nakhanov Kuanysh Jekebekov Ainar Kossylganova Karlygash Zhaparkulova Assem Kalykova Tolkyn Bekezhanova Albina Atakanova Zakir Yershebulov Kuandyk Zhugunissov

Recombinant superoxide dismutase (SOD) protein is a promising antigen candidate for brucellosis vaccines, and its quantitative determination is essential for vaccine quality control, standardization, and regulatory compliance. Although enzyme-linked immunosorbent assay (ELISA) is widely used for antigen quantification, its application to vaccine formulations may be limited due to chemical modifications of antigenic determinants occurring during detoxification and adjuvantation processes. Therefore, alternative analytical methods, such as single radial immunodiffusion (SRID), which enable direct antigen quantification independent of antigenic determinant modifications, represent valuable tools for vaccine analysis. The aim of this study was to develop and validate a single radial immunodiffusion (SRID) assay for the quantitative determination of recombinant SOD protein and to obtain hyperimmune sera in sheep. Sheep were immunized with recombinant SOD protein formulated with aluminum hydroxide or AddaS03 adjuvants. Antibody responses were evaluated using agar gel immunodiffusion (AGID) and enzyme-linked immunosorbent assay (ELISA). Immunization with aluminum hydroxide induced higher antibody titers than AddaS03. By day 42, ELISA titers reached 1:25,600 in the aluminum hydroxide group compared with 1:3200 in the AddaS03 group. The obtained hyperimmune sera were used for SRID assay development. The optimized SRID assay demonstrated high reproducibility and specificity, with no cross-reactivity against unrelated proteins. A linear relationship was observed between the square of the precipitation ring diameter (D2) and antigen concentration (R2 = 0.956–0.989). The assay showed a limit of detection of 0.312 μg/mL, a linear range of 0.625–10 μg/mL, high precision (CV < 2%), and robustness under varying analytical conditions. The developed SRID assay represents a specific, reproducible, and robust tool for quantitative control of recombinant SOD protein in vaccine formulations.

Methods and Protocols doi: 10.3390/mps9040112

Authors: Andreas R. Thomsen Pascaline Kouam-Daniel Bettina Priesch-Grzeszkowiak Anja Grillenberger Sandra Kumbruch Ali H. Acikelli Helmut Bühler Christian Baues

Tumor cell invasion is a critical step in local tumor progression, recurrence, and metastasis. Conventional two-dimensional migration assays and many existing three-dimensional invasion models often assess cell migration, invasion into the extracellular matrix and matrix degradation as separate endpoints, although these processes are tightly coupled in vivo. Therefore, robust and reproducible in vitro models are needed to investigate tumor cell invasion under defined extracellular matrix conditions. We developed an agarose-based three-dimensional invasion assay, termed the Freiburg 3D invasion assay, for the simultaneous analysis of tumor cell migration, invasion, and extracellular matrix degradation. The system consists of a 2.8% agarose matrix containing defined microcavities connected by a common loading channel. Tumor cells are seeded into these microcavities, where they form compact cell aggregates. The cavities are subsequently filled with collagen type I or extracellular matrix gel. After polymerization, the matrix-containing agarose strips are transferred into parking pockets, cultured for several days, and monitored by microscopy. Invasion distance, single-cell migration, and ECM-cleared area are quantified from serial microscopic images using image analysis software. The system distinguished weakly invasive MCF7 breast cancer cells from highly invasive MDA-MB-231 cells. In addition, treatment with a protease inhibitor and irradiation reduced tumor cell invasion and extracellular matrix remodeling, demonstrating the suitability of the assay for pharmacological and radiation-response studies. The Freiburg 3D invasion assay provides a practical and reproducible three-dimensional in vitro model for analyzing tumor cell invasion and protease-associated extracellular matrix degradation.

Methods and Protocols doi: 10.3390/mps9040111

Authors: Thuy-Duong Thi Tran Quy Thi Vu Hoa Thi Hoang Phan Thi Ngoc Hoa Nguyen Pham Thi Thao Truong T. N. Lien

Viral diseases are one of the most destructive threats to global agriculture. Among plant viruses, tobacco mosaic virus (TMV) is a highly contagious pathogen infecting numerous economically vital crops. With no curable treatments, the only strategy to mitigate virus spread is early detection and plant removal. The gold standard for TMV detection is reverse transcriptase-polymerase chain reaction (RT-PCR), followed by agarose gel electrophoresis. To reduce TMV diagnosis time and eliminate the requirement for expensive equipment, this study developed and optimized a one-step RT-PCR-coupled cysteamine-functionalized gold nanoparticle assay for the colorimetric determination of the virus. By integrating cDNA synthesis and PCR amplification into a single tube and analyzing the results using cysteamine- functionalized gold nanoparticles (Au@Cys), the diagnosis turnaround time was significantly reduced. Furthermore, this AuNPs-based colorimetric assay enabled straightforward visual inspection with the naked eye, eliminating the need for costly optical devices. Additionally, our regression equation linking RT-PCR product color values, extracted as mean A value based on the CIELAB color space (green-red axis), and viral load allows for the accurate quantification of TMV infection levels in field samples. Our research lays the groundwork for the further development of more cost-effective, quantitative and rapid plant virus diagnosis methods.

Methods and Protocols doi: 10.3390/mps9040110

Authors: Krishna Kattel Rachael M. Crist Jeffrey D. Clogston

The development and validation of a sensitive, rapid, and specific gas chromatography method for the evaluation of 19 common Class 2 and Class 3 solvents frequently used in nanomedicine formulation is described. Method validation was performed using PerkinElmer’s headspace gas chromatograph system with flame ionization detection and an Elite 624 Crossbond 6% cyanopropylphenyl-94% dimethylpolysiloxane or DB-Fatwax-Ultra Inert column with helium as the carrier gas. Validation characteristics such as linearity, spike recovery, method precision, specificity, sensitivity, limit of detection/quantitation, and analyte stability were evaluated. The validated methods showed excellent linearity, with a correlation coefficient > 0.99, and good precision, with intra-day precision < 7.4% for all tested analytes. The percent recoveries ranged 83–104% within the method’s quantitation range. In comparison to previously reported methods, the current method has a much shorter equilibration time, higher sensitivity, better separation for many solvents, and a wide concentration detection range. The current method is also perfectly suitable to analyze short chain fatty acids such as formic acid, acetic acid, butyric acid, and valeric acid without requiring additional extraction or derivatization steps. Notably, the method was found to be suitable for analysis of formic acid—a common solvent in certain nanoformulations and one in which there is no prior gas chromatography method available which does not require this additional sample manipulation—down to approximately 75 ppm. Herein, the method is demonstrated using various nanoformulations, including the commercial Doxil formulation as well as several research nanoformulations, including polymeric, cross-linked polymeric, and dendrimer platforms.

Methods and Protocols doi: 10.3390/mps9040109

Authors: Jonas Mumenthaler Maximilian Feldmann Shahab Mamaghani Rocco Roberto Penna Julia Frei Natalia Jarzebska Mark Mellett Emmanuella Guenova Thomas Kündig Severin Lauchli Steve Pascolo

Synthetic mRNA produced by in vitro transcription is the active pharmaceutical ingredient of approved vaccines and of many drugs under development. It typically contains a 3′ poly(A) tail required for optimal stabilization and translation of the mRNA. The average length of the poly(A) tail in the produced mRNA cannot be precisely predicted and consequently must be measured using complicated technologies such as reverse transcription and sequencing or cleavage of the poly(A) tail and analysis by capillary electrophoresis or mass spectrometry. We report an accelerated method to evaluate the average poly(A) tail length in synthetic mRNA, which benefits from the fact that thiazole orange is fluorescent only when in close proximity of nucleic acid. An oligo(dT)12 oligonucleotide having thiazole orange at its extremities emits a fluorescence signal proportional to the amount of poly(A) sequence available. Using a titration curve made with known amounts of poly(A) RNA oligonucleotide, the thiazole orange oligo(dT)12 oligonucleotide can instantly indicate the average length of the poly(A) tail in an mRNA sample. This fast and easy method can be used in any laboratory to determine the size of the poly(A) tail in in vitro-transcribed mRNA produced for research, pre-clinical studies and clinical trials.

Methods and Protocols doi: 10.3390/mps9040108

Authors: Priyo Ranjan Kundu Prosun Shafique Chaudhry Masudul H. Imtiaz Poorna Raavi David DiSalvo Kwadwo O. Appiah-Kubi

Background: Walking is a dynamic activity that relies on inputs from the multisensory system, i.e., somatosensory, vision, and vestibular. These inputs are processed and integrated in the central nervous system to produce motor impulses for efficient walking balance. The Sensory Organization Test (SOT) is established as the gold standard for assessing sensory contributions to standing balance. However, no comparable assessments have been developed for the clinical evaluation of balance during gait. This study evaluated the Gait Sensory Interaction Test (GaitSIT), a novel virtual reality (VR)-based assessment for characterizing sensory-condition-specific changes in walking balance. Methods: The GaitSIT comprises a VR environment with a physical compliant foam walking surface that evaluates gait–balance by systematically manipulating and evaluating the sensory systems. Twenty-nine healthy young adults (mean age 24.9 ± 6.4 years) were instructed to complete 6 m walking trials under six standardized conditions (C): eyes open, eyes closed/dark scene, and rotating visual scenes on a firm surface, then repeated on a foam surface. Wearing an Oculus VR headset, participants were instructed to walk in a straight line at their preferred speed, as naturally as possible, in two test sessions on the same day, followed by a third test session 24 h later. Headset-derived sway measures, including position, velocity, and acceleration data, were recorded, and the continuous trajectory deviation angle (i.e., directional control) and sensory ratios were calculated. Linear mixed-effects models included trial-level walking speed as a covariate. Additionally, participants completed the modified Clinical Test of Sensory Interaction on Balance (mCTSIB) as a clinical standing-balance reference measure; its concurrent-validity findings will be reported separately. Results: Significant condition effects were observed for position, velocity, acceleration, and CTDA after adjustment for trial-level walking speed (all p<0.001), indicating that the six sensory conditions elicited distinct gait–balance responses. Significant differences relative to the baseline condition (C1) were observed across conditions C2–C6 for position, C3–C6 for velocity, and C2 and C5 for acceleration. Session effects were not significant for any primary kinematic outcome after speed adjustment. A significant condition × session interaction was observed for position (p<0.001), whereas velocity, acceleration, and CTDA demonstrated no significant interactions. Walking speed was significantly associated with position, acceleration, and CTDA, but not velocity. Sensory-ratio analyses revealed larger visual and vestibular ratios relative to somatosensory ratios, with the visual and vestibular ratios generally decreasing across sessions. Conclusions: GaitSIT successfully manipulated sensory conditions during overground walking and produced significant changes in gait-related sway, directional control, and sensory-ratio measures. These findings support the feasibility of GaitSIT as a portable, low-cost, and immersive assessment framework for characterizing sensory-condition-specific gait–balance responses after accounting for walking speed and providing indirect behavioral indices related to sensory reweighting.

Methods and Protocols doi: 10.3390/mps9040107

Authors: Agnieszka Staniszewska Agnieszka Piastowska-Ciesielska

Ensuring viral safety is a critical aspect of biopharmaceutical production, requiring sensitive and reliable methods for detecting adventitious agents. In this study, we systematically evaluated the performance of selected cell line–virus combinations to identify optimal models for in vitro viral detection assays. Three cell lines (Vero, MRC-5 and BHK-21 [C-13]) and representative model viruses (Reovirus type 3, Adenovirus type 5, Human parainfluenza virus type 3, and Herpes simplex virus) were analyzed in terms of cytopathic effect (CPE) kinetics, morphology, and detection sensitivity. All tested systems demonstrated high analytical sensitivity, with limits of quantification (LOQ) reaching 0.01 TCID50/mL for selected viruses. However, substantial differences were observed in infection dynamics and CPE morphology depending on the cell line–virus combination. BHK-21 [C-13] cells exhibited the most rapid and pronounced CPE for Reovirus type 3, enabling early and unambiguous detection. Vero cells provided robust and reproducible detection of Adenovirus type 5, characterized by well-defined cytopathic progression. MRC-5 cells showed controlled and consistent infection kinetics for both Human parainfluenza virus type 3 and Herpes simplex virus, allowing improved temporal resolution and interpretability. These findings demonstrate that assay performance depends not only on sensitivity but also on the kinetics and morphology of infection. Based on combined evaluation criteria, the following optimal cell line–virus pairs were identified: BHK-21 [C-13]/Reovirus type 3, Vero/Adenovirus type 5, and MRC-5/Human parainfluenza virus type 3 and Herpes simplex virus. The proposed approach supports rational selection of detection models and provides a preliminary descriptive framework for the development of routine visual screening assays in biopharmaceutical quality control.

Methods and Protocols doi: 10.3390/mps9040106

Authors: Danieli Soares de Oliveira Clainer Bravin Donadel

Natural coagulants have emerged as potential alternatives to synthetic chemicals in water treatment, especially for decentralized and low-resource applications. However, many previously reported Aloe vera-based coagulant preparation methods rely on drying, powder production, distilled water extraction, refrigeration, or other laboratory-dependent procedures that increase operational complexity and limit practical implementation. This study presents a simplified and rapid protocol for producing an Aloe vera-based natural coagulant using accessible materials and simplified preparation steps. The proposed methodology consists of extracting Aloe vera g13el, homogenizing 2 g of fresh gel with 50 mL of tap water using a household blender, and applying simple paper filtration to obtain the liquid coagulant. The protocol can be completed in less than 10 min without specialized laboratory infrastructure, energy-intensive processing, or laboratory-grade reagents. Coagulation performance was evaluated using synthetic turbid water with initial turbidity levels of 100, 200, and 300 NTU. Significant turbidity reduction was observed under all tested conditions, with several samples reaching residual turbidity values close to or equal to 0 NTU after 50–60 min of sedimentation. The results demonstrate the potential of the proposed protocol as a rapid, reproducible, and accessible approach for future investigation in point-of-use and decentralized water treatment applications.

Methods and Protocols doi: 10.3390/mps9040105

Authors: Colm P. Roster James C. Morris

The pathogenic free-living amoeba Naegleria fowleri is the cause of primary amebic meningoencephalitis (PAM), a central nervous system infection that is almost always lethal. One of the unusual features of the amoebae is the presence of ~4000 copies of a nucleolar-localized closed circular extrachromosomal ribosomal DNA element (CERE) that encodes the cell’s ribosomal RNA repertoire. It has historically been challenging to purify large quantities of CERE, limiting our understanding of the nucleic acid. Here, we describe a methodology for CERE purification that improves yield, reduces processing times, and maintains the integrity of the plasmid. This approach will enable the study of this unique DNA architecture, advancing our understanding of the pathobiology of the organism.

Methods and Protocols doi: 10.3390/mps9040104

Authors: Lyubov A. Rzhanova Ekaterina V. Kuzmenko Alena A. Permyakova Andrei A. Riabinin Evgenii S. Ruchko Maria B. Chernysheva Ekaterina A. Vorotelyak Elena I. Morgun

Background: This study focuses on developing a model of a non-healing wound that recapitulates the pathogenesis of the corresponding human pathology. Methods: A non-healing wound was modeled in mice with streptozotocin-induced diabetes. The following parameters were assessed: re-epithelialization, epidermal hypertrophy, wound contraction, relief index, angiogenesis, and granulation tissue maturation. These parameters were compared between diabetic mice and healthy controls. Results: The proposed model demonstrated a significant delay in regenerative processes compared to healthy animals. Conclusions: These findings support the relevance of this model to human pathology and indicate that it may be applicable for preclinical studies of drugs aimed at promoting wound regeneration.

Methods and Protocols doi: 10.3390/mps9040103

Authors: Ardian Rizal Mohammad Saifur Rohman Fatchiyah Fatchiyah Hidayat Sujuti Anna Fuji Rahimah Wella Karolina Victor Alvianoes Guterez Hose Mokhammad Afifudin

Background: Small animal models are essential for atrial fibrillation (AF) research. Researchers in AF use an electrocardiogram (ECG), echocardiography and invasive electrophysiology study (EPS) to assess atrial structural and electrical remodeling. In relatively smaller cardiac structures and rapid heart rates, the examination can be challenging without special tools designed for animal study. Moreover, conventional invasive EPSs often cause significant trauma, alter autonomic tone, and limit longitudinal evaluations. This study aimed to evaluate the feasibility of repurposing hospital-grade medical devices for the non-invasive, multi-modality assessment of atrial myopathy in a rat model of metabolic syndrome (MetS). Methods: A total of 12 male Sprague-Dawley rats underwent the multi-modality assessment. Structural remodeling was evaluated using hospital-grade echocardiography (8–12 MHz) to measure left atrial (LA) dimensions and volume. Surface ECG was used to determine P-wave duration. Electrical remodeling and AF inducibility were assessed using transesophageal pacing (TEP)-based EPS, evaluating the atrial effective refractory period (AERP), sinus node recovery time (SNRT), and response to rapid atrial burst pacing. Results: The protocols showed high procedural safety (survival rate 91.67%) and successfully characterized atrial myopathy. Surface ECG showed marked intra-atrial conduction delay with prolonged P-wave duration in the MetS group (30.17 ± 4.62 vs. 22.33 ± 1.86 ms, p < 0.05). Echocardiography revealed signs of structural remodeling in the MetS group, evidenced by marked prolonged Isovolumic Relaxation Time (IVRT: 35.602 ± 3.043 vs. 19.187 ± 3.631 ms; p < 0.001) and increased Left Atrial Area (0.223 ± 0.0556 vs. 0.134 ± 0.033; p = 0.007). Furthermore, TEP-based EPS quantified electrical remodeling. The MetS group had shorter AERP (73.33 ± 10.33 ms vs. 120.00 ± 34.06 ms; p = 0.010) and Corrected SNRT (100.67 ± 53.98 ms) versus controls (208.33 ± 76.97 ms; p = 0.018). The MetS group exhibited a higher absolute AF inducibility rate (50%, three out of six rats) compared to the SH group (33.3%, two out of six rats). Conclusions: The integration of surface ECG, echocardiography, and TEP-based EPS provides a safe, highly reproducible, and comprehensive method for evaluating both structural and electrical components of atrial myopathy in small animal models, allowing for robust longitudinal studies.

Methods and Protocols doi: 10.3390/mps9040102

Authors: Izabela Antepowicz Antonia Despotidi Emma Rowan Mbuyiselwa Shadrack Moloi Silke Aulhorn Harry Esmonde Konstantinos Grintzalis Eberhard Küster

Daphnids, commonly known as water fleas, are freshwater planktonic microcrustacean species used as model organisms in ecotoxicology, particularly in regulatory frameworks that adhere to OECD and ISO standards. Mortality is the most common endpoint in toxicity testing; however, more sensitive indicators are required to assess sublethal acute effects of pollutants. The use of feeding impairment as a toxicity phenotypic endpoint in daphnids is considered a cost-effective approach that aligns with the 3Rs principle (Replace, Reduce, Refine) and is more physiologically and environmentally relevant. Current feeding methods are inefficient due to the large test volumes and extended incubation periods required. In this paper, we present a miniaturised protocol to assess feeding behaviour following exposure to chemicals in daphnids. The method is based on the consumption of algae, which is measured with chlorophyll fluorescence. The optimised protocol is more robust and rapid, and results can be obtained in 30 min and in a 96-well plate. Responses in feeding rate were investigated using this miniaturised protocol following exposure to a range of prevalent pollutants, which include two metals and, as a more realistic sample, a leachate from smoked cigarette filters. All three pollutants were tested at sublethal concentrations. This method provides an efficient approach to assess the toxicity of chemicals and water quality.

Methods and Protocols doi: 10.3390/mps9040101

Authors: Taavi Rand Henri Tilga Ángel Abós Luis García-González Sergio Diloy Peña Rafael Burgueño-Mengibar Andre Koka

(1) Background: Previous school-based interventions have addressed adolescent health behaviors such as physical activity, screen time, and sleep, but have predominantly targeted these behaviors independently rather than simultaneously. The Erasmus+ project FiTeens developed an integrated intervention combining theoretical content, videos, infographics, and interactive tasks to promote multiple health behaviors concurrently. The objective of the current article is to present the protocol for a school-based intervention study designed to examine the effects of the FiTeens program on adolescents’ physical activity, screen time, and sleep behaviors. We hypothesize that students receiving the FiTeens intervention will demonstrate increased physical activity, reduced screen time, and improved sleep outcomes compared with students in the control group. (2) Methods: Teachers will be introduced to the FiTeens tools prior to delivering the intervention to students in grades 5–9. Students will participate in an eight-week intervention program combining structured lessons and behavior-change challenges. Primary outcomes include changes in physical activity, screen time, and sleep duration and quality. Secondary outcomes include psychological determinants such as motivation and behavioral intentions. Data will be collected at baseline and at 1-, 3-, and 6-month follow-ups and analyzed using repeated measures ANOVA. (3) Expected results: The study will evaluate whether the intervention may contribute to improvements in health-related behaviors among adolescents, including increased physical activity, reduced screen time, and improved sleep outcomes. (4) Conclusions: The intervention based on FiTeens tools could have the potential to promote healthier lifestyle behaviors among students by increasing physical activity during leisure time, supporting the effective limitation of screen time and enhancing bedtime routines to improve sleep quality.

Methods and Protocols doi: 10.3390/mps9030100

Authors: Qian-Wen Ma Zhi Li Zhi-Chao Zhang Xiao-Juan Guo Xiao-Hui Zou Tao Hung Zhuo-Zhuang Lu

Adenoviral vectors are widely used for gene therapy and vaccine development. To circumvent pre-existing immunity against commonly used human adenovirus type 5, vectors based on rare human serotype or animal adenoviruses have attracted increasing interest. Previously, we constructed vectors based on fowl adenovirus 4 (FAdV-4) and replaced the knob of FAdV-4 fiber2 with that of FAdV-1 fiber1 to generate FAdV4-CF1K vectors with enhanced transduction efficiency in human cells. In this study, we aimed to modify the packaging system to efficiently produce FAdV-4 vectors carrying transgenes toxic to viral replication. Chicken LMH cells failed to form colonies at low seeding densities. We collected used medium from LMH cell cultures and used it as a supplement to adapt LMH cells, generating the colony-competent subclone LMH-C3532. A lentiviral vector encoding a codon-optimized tetracycline repressor (tetR) was transduced into LMH-C3532 to establish a tetR-integrated cell line, LMH-tetR24. An adenoviral plasmid, pKFAV4-CF1K-CtG, was constructed in which a tetracycline operator (tetO)-bearing CMV promoter controlled GFP expression. The SwaI-flanked GFP in this plasmid was replaced with the HA gene from an H5N1 influenza virus to generate pKFAV4-CF1K-CtHA. Linearized adenoviral plasmids were transfected into LMH-tetR24 cells, and recombinant FAdV4-CF1K-CtG and FAdV4-CF1K-CtHA viruses were successfully rescued, amplified, and purified. When infected with FAdV4-CF1K-CtG at various multiplicities of infection (MOI), the progeny virus yield from LMH-tetR24 cells was 4–10 times higher than that from LMH-C3532 cells. For FAdV4-CF1K-CtHA, the yield difference between the two cell lines was even more pronounced, reaching 3–4 orders of magnitude. Overexpression of HA in LMH-C3532 cells negatively affected FAdV4-CF1K-CtHA replication, resulting in smaller and fewer plaques. In conclusion, by separately integrating tetR into packaging cells and TetO into the adenoviral plasmid, we established a system that can be routinely used to package FAdV-4 vectors. Notably, this system facilitates the propagation of FAdV-4 vectors carrying toxic transgenes.

Methods and Protocols doi: 10.3390/mps9030099

Authors: Soumitra Das James Killian Mahesh Jayaram Naveen Thomas Chi Jonasi

Individuals living with mental illness face disproportionately higher COVID-19 morbidity and mortality than the general population. Despite their prioritisation in Australia’s national vaccination rollout, vaccination rates among this population are significantly lower than those without mental illness. No previous study has employed a qualitative research paradigm to explore the barriers and facilitators to COVID-19 vaccination among people with mental illness in the Australian context. This qualitative study will employ Braun and Clarke’s reflexive thematic analysis framework. Participants will be recruited through Western Health Mental Health and Wellbeing Services in Victoria, Australia. Semi-structured individual interviews will be conducted with approximately 17–20 participants (aged 18–65) who have a DSM-5 diagnosed mental illness and any experience (vaccinated or unvaccinated) with COVID-19 vaccination. Interviews will be audio-recorded, transcribed, and then analysed and collated into themes using NVivo software. Code saturation will guide the final sample size. This study aims to produce a rich thematic map that captures the experience of individual, illness, and system-level barriers and facilitators to COVID-19 vaccination in this cohort. Findings are anticipated to inform targeted public health interventions to improve equitable vaccine uptake and contribute to closing the mortality gap for those with mental illness. This protocol has been approved by the Western Health Low Risk Ethics Panel (ERM ID: 113351).

Methods and Protocols doi: 10.3390/mps9030098

Authors: Barbora Klusackova Zuzana Pilsova Katerina Nemeckova Aneta Pilsova Pavla Postlerova

Sperm capacitation is essential for fertilization and involves coordinated changes in membrane organization, ion fluxes, and intracellular signaling. However, commonly used detection methods may reflect different biological events, which can be strongly influenced by experimental methodology. This study critically evaluated fluorescence-based approaches for assessing capacitation in boar spermatozoa, focusing on their specificity, interpretative limits, and methodological sensitivity. Ejaculated boar spermatozoa were incubated under in vitro capacitating conditions in TALP medium. Selected samples were subsequently treated with calcium ionophore to induce the acrosome reaction (AR). Phosphotyrosine (PTyr) immunofluorescence was assessed using five fixation and labeling protocols, acrosin redistribution was evaluated with the ACR.2 antibody, calcium ion redistribution was assessed using chlortetracycline (CTC) fluorescence, and acrosomal responsiveness was monitored by peanut agglutinin (PNA) lectin labeling. PTyr immunofluorescence was highly dependent on fixation protocol, indicating marked methodological sensitivity. Acrosin immunodetection revealed a clear capacitation-associated redistribution from weak or diffuse staining to a well-defined acrosomal pattern, whereas ionophore treatment caused a pronounced signal loss consistent with acrosomal exocytosis. PNA labeling confirmed that capacitation alone did not increase spontaneous acrosome loss, whereas ionophore treatment induced a robust AR. CTC staining showed a significant shift from whole-head pattern to acrosome in TALP-treated spermatozoa, indicating capacitation-associated Ca2+ redistribution. Together with CTC and Western blot data, these findings show that sperm capacitation status should be evaluated using multiple complementary markers rather than a single gold-standard assay.

Methods and Protocols doi: 10.3390/mps9030097

Authors: Tim Bozic Bostjan Markelc Simona Kranjc Brezar Ziva Pisljar Tanja Jesenko Maja Cemazar

Lymph nodes are central hubs of immune regulation and coordination, serving as primary sites for antigen presentation, lymphocyte activation, and the orchestration of adaptive immune responses. The composition and activation state of lymph node-resident immune cells critically shape both local and systemic immunity. Comprehensive immunophenotyping of these populations is therefore essential for understanding immune organization and functional heterogeneity. Here, we present an optimized protocol for the characterization of mouse lymph node-associated immune populations using 14-color multiparametric flow cytometry. The method combines lymph node isolation based on anatomical landmarks with mechanical dissociation and enzymatic digestion to generate high-quality single-cell suspensions suitable for downstream analysis. Furthermore, the described flow cytometry panel and gating strategy enable reliable identification and quantification of major lymphoid subsets, including helper CD4+ and cytotoxic CD8+ T cells with their differentiation states, as well as natural killer (NK) cells across distinct maturation stages. Although optimized for assessing lymphocyte maturation after lipopolysaccharide (LPS) challenge, the protocol serves as a reproducible platform for broad immunophenotyping of T and NK cell subsets in mouse lymphoid tissues under experimental conditions.

Methods and Protocols doi: 10.3390/mps9030096

Authors: Robert B. Clark Joseph Odu Annette Ofodum Rondi Anderson

Sierra Leone faces persistently high neonatal and maternal mortality rates, driven largely by delayed recognition and treatment of newborn respiratory distress and postpartum hemorrhage. In this protocol, we describe the planned implementation of a bundle of maternal and newborn clinical practices over a 36-month period across nine public health facilities in the Greater Freetown area and Bo District to address these critical gaps. The service delivery improvements include the World Health Organization (WHO) Essential Newborn Care Course (ENCC) Parts 1 and 2; Vayu bubble continuous positive airway pressure (bCPAP) and oxygen blenders for respiratory support; the WHO Postpartum Hemorrhage package; and obstetric risk stratification using point-of-care ultrasound (POCUS) and complementary diagnostics for maternal care improvement. We anticipate that this bundle of evidence-based clinical tools and training, reinforced by mentorship, structured checklists, and low-dose high-frequency (LDHF) practice, will significantly reduce perinatal and maternal mortality and morbidity. The bundle will be evaluated using a Hybrid Type 1 effectiveness-implementation design, utilizing routine health information system data, supplemented by project registers, skills assessments, and observations. By aligning with the Ministry of Health’s Child Survival Action Plan, the aim of this project protocol is to provide a sustainable and scalable model for reducing preventable maternal and newborn deaths in resource-constrained settings.

Methods and Protocols doi: 10.3390/mps9030095

Authors: Omar Abdelaziz Seyyedehfatemeh Ghalibafan Raul E. Ruiz-Lozano Jeffrey C. Peterson Ryan A. Gallo Ali R. Djalilian

Contact lens wear is widely used for vision correction by millions of individuals worldwide; however, it remains associated with a spectrum of ocular complications ranging from mild inflammatory conditions to vision-threatening infections. Common contact lens-related complications are predominantly noninfectious, including contact lens discomfort, dry eye syndromes, and papillary conjunctivitis. These conditions are typically mild and manageable with conservative measures. In contrast, corneal inflammatory conditions, such as contact lens-induced acute red eye and peripheral ulcers, represent an intermediate spectrum and may clinically overlap with early infection, creating diagnostic uncertainty. The most serious complication is microbial keratitis, a vision-threatening infection that remains challenging to recognize in its early stages due to its variable and often subtle presentation. Delayed identification may lead to rapid progression and significant visual morbidity. Patients with contact lens-related complaints often present to frontline settings, where early recognition is essential. Distinguishing benign from infectious conditions can be challenging; a risk-based approach with prompt triage and referral, along with proper lens hygiene and patient education, is key.

Methods and Protocols doi: 10.3390/mps9030094

Authors: Razvan George Bogdan Iulian-Alexandru Ciprian Blidisel Ionut Ciobota Anca Maria Campean Alina Helgiu Claudiu Helgiu Ioan Catalin Bodea Dan Ionel Orbulescu Rodica Elena Heredea Zorin Petrisor Crainiceanu

Reproducible experimental models of localized dermal–hypodermal fibrosis are essential for standardized investigation of regenerative interventions. Variability in bleomycin dosing, anatomical targeting, and assessment strategies limits cross-study comparability. This study describes a methodological framework for standardized induction of early dermal–hypodermal remodeling in a rat model followed by autologous subcutaneous tissue grafting and multimodal longitudinal evaluation. Female Wistar rats underwent subcutaneous bleomycin administration at 1 mg/kg/day for three consecutive days. Clinical documentation, high-frequency ultrasonography with fixed imaging parameters, and sequential biopsies from a predefined thoracic anatomical site were performed at baseline, intermediate reassessment, and final evaluation. Autologous subcutaneous tissue grafting was conducted at Day 17 after study initiation. The protocol enabled controlled induction of early structural remodeling and consistent longitudinal documentation of dermal–hypodermal thickness, echogenicity changes, and histological architecture within a standardized anatomical region. This protocol development study establishes a reproducible and spatially consistent experimental platform integrating imaging and histological assessment, facilitating future hypothesis-driven investigations of fibrotic remodeling and regenerative strategies.

Methods and Protocols doi: 10.3390/mps9030092

Authors: Daniela Mirela Vîrtosu Simina Crișan Oana Pătru Angela Dragomir Silvia Luca Ruxandra-Maria Băghină Mihai-Andrei Lazăr Alina-Ramona Cozlac Stela Iurciuc Constantin Tudor Luca

Background: Risk stratification models are widely used in clinical research; however, their development becomes methodologically challenging in settings characterized by low outcome incidence. In coronary care unit (CCU) populations, healthcare-associated infections (HAIs) occur relatively infrequently, limiting the feasibility of conventional multivariable predictive modeling. Methods: A retrospectively assembled CCU cohort comprising 870 patients with 16 HAI events (1.8%) was used as an illustrative example to examine methodological constraints associated with low events-per-variable (EPV) ratios. The implications of limited event frequency for multivariable logistic regression were evaluated, including risks of overfitting, coefficient instability, and reduced reproducibility. As an alternative strategy, a prespecified cumulative additive framework integrating baseline vulnerability and exposure-related variables was conceptually and analytically explored. Results: With four candidate predictors and 16 outcome events, the resulting EPV was approximately four, indicating a high risk of instability for conventional multivariable modeling. The cumulative framework allowed structured cumulative stratification without coefficient optimization. Infection occurrence increased progressively across cumulative framework levels, illustrating a graded pattern of increasing HAI occurrence with accumulating vulnerability and exposure-related burden. Conclusions: In clinical datasets with limited outcome events, modeling strategies should be aligned with the informational capacity of the data. Cumulative additive frameworks may represent a pragmatic structural alternative to coefficient-based modeling approaches in rare-event clinical settings. The present work provides a structured methodological framework for risk stratification under low-events-per-variable conditions rather than proposing a novel clinical scoring system.

Methods and Protocols doi: 10.3390/mps9030093

Authors: Ryan Scott Anderson Sybil Sharvelle Susan K. De Long

Food waste is a readily digestible and fermentable feedstock for waste to energy bioprocesses. Approximately one third of food is wasted, thus making improvements in food waste valorization is essential for a circular economy. Laboratory results must be reproducible and as representative of scaled performance as possible to facilitate knowledge sharing between research groups. Food waste used in laboratory studies is often collected in situ or overly simplistic synthetic mixtures are used. Food waste collected in situ from any one local source at a single time point (e.g., grab samples from a cafeteria or restaurant) are not reproducible or nationally representative; additionally, overly simple synthetic mixtures are reproducible, but lack the complexity of real food waste and are not nationally representative. Thus, an adequately complex, reproducible, and nationally representative food waste recipe is needed to standardize the feedstocks used in laboratory scale food waste digestion and fermentation studies. In this work, we developed a food waste recipe made from widely and commercially available ingredients which is based on national-scale food wastage data in the United States. The nationally representative food waste mixture was 45.4% carbohydrates, 32.5% lipids, and 13.4% proteins. The biomethane potential was 495 ± 44 mL CH4/g VS and the food waste mixture was suitable for use in low-pH bench-scale arrested anaerobic digesters. This design approach can be adapted for other regions and countries where food loss data are available.

Methods and Protocols doi: 10.3390/mps9030091

Authors: Mariya Levkova

Long-read sequencing technologies provide portable and flexible service, making them attractive for small-scale sequencing studies. However, many existing RNA-sequencing analysis frameworks are designed for transcriptome-wide analyses and require substantial computational resources. Here we present a lightweight and reproducible computational pipeline for targeted long-read transcriptomic profiling using Oxford Nanopore Technologies (ONT) cDNA sequencing data. The pipeline was evaluated using targeted long-read transcriptomic datasets generated from formalin-fixed paraffin-embedded (FFPE) colorectal carcinoma samples previously classified as microsatellite instability—high (MSI-high) by PCR-based testing. Libraries were sequenced on the Oxford Nanopore MinION platform using R10.4.1 flow cells. Application of the workflow enabled rapid quantification of mismatch repair gene expression and detection of immune-related transcripts including CD8A, PDCD1, and HAVCR2 across multiplexed barcode samples. The pipeline performs targeted alignment of long-read sequencing data to a custom transcript reference panel using minimap2, followed by gene-level read counting and normalization using reads-per-million (RPM). Optional modules enable immune marker profiling, detection of reads aligning to multiple genes, exploratory variant analysis, and visualization of expression patterns. By combining simplicity, reproducibility, and minimal computational overhead, the present pipeline provides an accessible framework for targeted transcriptomic analysis of long-read sequencing data. It may facilitate adoption of ONT-based transcriptomic profiling in settings with restricted computational resources.

Methods and Protocols doi: 10.3390/mps9030090

Authors: Costas S. Constantinou

This paper highlights an important topic in qualitative research: the generalizability of qualitative findings to the general study population. While the concept of generalizability in qualitative inquiry has been discussed, with types such as analytical generalizability and transferability being proposed, the direct generalization of qualitative results to the general population has received limited attention. This oversight stems largely from the prevailing belief among researchers that qualitative research cannot be generalized due to its reliance on small, non-randomized samples. This perception has led many scientists, particularly those outside the social sciences, to regard qualitative research primarily as exploratory or supplementary to quantitative findings. This paper is a technical note that presents a new concept and procedure. Based on concept analysis, content generalizability is proposed, which offers a new type of generalizability through which qualitative findings can be applied to the general population, thereby enabling them to inform policy and drive social change. Content generalizability could expand the uses of qualitative research within the scientific community by providing knowledge and insights into the experiences of the general study population. This paper also contributes to developing empirical research by testing and applying content generalizability.

Methods and Protocols doi: 10.3390/mps9030089

Authors: Maria Anurova Aleksey Kuzmin Aleksey Vorobev Nataliya Feldman Elena Zinurova Andrey Aleshkin

The effect of administration routes (oral and rectal) on the pharmacokinetics of Salmonella phage SE40 and Escherichia phage V18 was studied. To detect phages in biomaterial (blood, urine, and feces), the Spot-test, Gratia, and double-nested polymerase chain reaction methods were used. Systemic action of the studied phages with both routes of administration, beginning 15–30 min after administration, was demonstrated. The phages persisted for up to 24 h after both oral and rectal administration. However, the concentration in the blood was higher after oral administration, while concentrations in urine and feces were higher after rectal administration. The need to protect phages from the acidic contents of the stomach was confirmed.

Methods and Protocols doi: 10.3390/mps9030088

Authors: Toru Hanamura Akinori Takase Masanori Oshi Naoki Niikura

Accurate deconvolution of bulk transcriptomes is essential for characterizing the breast cancer tumor microenvironment (TME), yet existing reference matrices incompletely capture tumor-specific cellular diversity. Here, we developed breast cancer–specific multiresolution CIBERSORTx signature matrices from single-cell RNA sequencing data and systematically evaluated their analytical performance and interpretability. Major-, minor-, and subset-level matrices were constructed and assessed using pseudo-bulk mixtures and pure cell profiles, while biological and clinical coherence were evaluated in TCGA-BRCA and the I-SPY2 cohort. All matrices demonstrated high accuracy in reconstructing pseudo-bulk compositions, with performance declining at finer resolution. Spillover increased with granularity but was largely restricted within related lineages. Lineage-wise deconvolution modestly reduced spillover but consistently decreased accuracy, highlighting the importance of cross-lineage transcriptional contrast. In external datasets, most inferred cell populations showed biologically coherent associations with canonical markers and pathways, whereas some fine-resolution subsets exhibited non-canonical patterns, likely reflecting intra-lineage trade-offs or context-dependent transcriptional states. In the I-SPY2 cohort, plasmablasts and selected myeloid populations were positively associated with pathological complete response, whereas fibroblastic and perivascular-like populations showed negative associations. These findings establish a validated and interpretable resource for breast cancer TME deconvolution and clarify its performance characteristics and limitations.

Methods and Protocols doi: 10.3390/mps9030087

Authors: Maja Lusina Kregar Iva Krtalić Ivana Šagud

Long-acting injectables (LAIs) are widely used for chronic conditions such as schizophrenia, opioid use disorder, and HIV. Their prolonged efficacy improves adherence and reduces dosing frequency. Among these systems, poly(lactide-co-glycolide) (PLGA)-based formulations are commonly used to deliver drugs ranging from small molecules to peptides and proteins. In vitro release (IVR) tests play a critical role in evaluating drug product performance for both immediate- and prolonged-release dosage forms. However, there is a lack of standardized compendial IVR methods for the assessment of LAIs. This lack impedes the development of new drug products in this area and also complicates their regulatory approval process. Considering the complexity of drug release mechanisms and the diversity of various formulation design approaches, it is not possible to devise a universal IVR method that is applicable to all LAI products. The in vitro release test applied for quality control should be simple, robust, reproducible, and discriminatory. On the other hand, more complex biorelevant media and methods are often used during development to better reflect physiological conditions. This article provides a comprehensive review of compendial and non-compendial methods used for in vitro release testing of PLGA-based LAIs (microspheres and in situ forming implants), with the goal of aiding the development and standardization of future methodologies.

Methods and Protocols doi: 10.3390/mps9030086

Authors: Aasia Bibi Daniela De Benedictis Giuseppe Capitanio Alessandra Gabriele Alessandro Buccolieri Mariapompea Cutroneo Lorenzo Torrisi Daniela E. Manno Antonio Serra Domenico De Rasmo Anna Signorile

Background: Carbon dots (CDs) are promising fluorescent nanomaterials with great application potential in bioimaging and organelle-targeted diagnostics. This study compares nitrogen-doped (N-CDs) and boron–nitrogen co-doped CDs (BN-CDs) in normal NIH3T3 fibroblasts and KRAS-transformed cells. Methods: CDs were synthesized via a microwave-assisted method. Their fluorescence, cytocompatibility, and intracellular localization were evaluated using confocal microscopy, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, and organelle colocalization. Cellular metabolism was assessed by Seahorse analysis. Oxidative stress and cAMP levels were pharmacologically modulated. Results: BN-CDs exhibited stronger intracellular fluorescence than N-CDs, indicating enhanced uptake and imaging performance, with no cytotoxicity up to 100 µg/mL. They localized to multiple organelles, particularly mitochondria. However, fluorescence was significantly reduced in KRAS-transformed cells despite similar mitochondrial mass. BN-CDs did not affect mitochondrial respiration or glycolytic activity. Induced oxidative stress or elevated cAMP in normal cells reduced BN-CD fluorescence. Conclusions: Boron doping improves N-CD imaging properties without affecting cell viability or metabolism. Reduced fluorescence in KRAS cells is associated with altered intracellular conditions, suggesting that BN-CDs could be used to discriminate between normal and cancer cells.

Methods and Protocols doi: 10.3390/mps9030085

Authors: Sara Delimar Ela Šarić Marina Cvjetko Bubalo Ana Bielen

Saprolegnia parasitica (Oomycota) causes saprolegniosis and poses significant ecological and economic challenges in aquaculture. Experimental research on this pathogen is constrained by the lack of reliable long-term preservation methods, as routine maintenance by serial subculturing is labor-intensive and may result in genetic and phenotypic instability. Deep eutectic solvents (DESs), tunable low-melting mixtures, have recently gained attention as alternative cryoprotectants. However, their application has not been evaluated in oomycetes. Here, twelve glycerol-based two- and multicomponent DESs were assessed for cryopreservation of S. parasitica at −80 °C and compared with glycerol as a conventional cryoprotectant. Cryopreservation efficiency was assessed based on post-thaw survival and mycelial regeneration. Several two-component DESs, particularly glycerol-trehalose, supported 100% survival and high post-thaw mycelial regeneration, performing comparably to glycerol under the tested conditions. Shorter pre-incubation (30 min vs. 1 h and 3 h) and controlled-rate freezing (vs. direct freezing) significantly improved post-thaw growth. Although survival remained 100% under optimized conditions, extending storage from 7 to 32 days significantly reduced mycelial regeneration in the glycerol–trehalose treatment, indicating that survival alone, as done in existing literature, does not reflect physiological recovery. Overall, our results support the use of selected DESs as alternative cryoprotectants in oomycetes and contribute to the development of cryopreservation strategies for S. parasitica.

Methods and Protocols doi: 10.3390/mps9030083

Authors: Derek Wilkinson

Caspases orchestrate metazoan apoptosis, regulating processes such as embryogenesis, the death of old and infected cells and immune tolerance. Structural orthologs of caspases have been identified in bacteria, plants, protists and fungi and regulated cell death has been demonstrated in these organisms. This led some researchers to conclude that fungal metacaspases might perform a similar function to caspases. This review discusses regulated cell death, beginning with an account of RCD and the central role of caspases in mammalian RCD. It goes on to give examples of RCD in fungi, compares the structure and activity of caspase orthologs and outlines examples of metacaspase-dependent and metacaspase-independent cell death in fungi, focusing on S. cerevisiae. Finally, it addresses the question “are metacaspases caspases?”, identifies alternative cell death proteases and recommends future research objectives.

Methods and Protocols doi: 10.3390/mps9030084

Authors: Brian Mwangi Joshua M. Njiru Sarah A. Wandili Kennedy K. Gachoka Kenneth Mburu Geoffrey Muriira Henry Rotich Elvince Ager Evans N. Nyaboga

Potato (Solanum tuberosum L.) is an important staple and food security crop to many communities in the world. However, potato production and quality is greatly constrained by bacterial wilt, a disease caused by a soil-borne pathogen, Ralstonia solanacearum. Ralstonia solanacearum can be managed through clean seed systems and therefore laboratory testing is a pre-requisite for seed certification to confirm the absence of the pathogen in potato seeds before planting. Molecular diagnostics is the gold standard for detection of R. solanacearum in potato seeds. However, the extraction of genomic DNA from R. solanacearum for molecular diagnostics is complex, tedious, lengthy and/or costly procedure. A simple, rapid and reliable DNA extraction protocol is required for use in routine molecular diagnosis of R. solanacearum, a high-risk quarantine pathogen. In this study, we developed a simple and rapid protocol for extracting genomic DNA from symptomatic and asymptomatic potato tubers infected with R. solanacearum and verified its efficiency for the detection and molecular characterization of the pathogen. The protocol was developed from the evaluation of distilled water, Tris-EDTA (TE) and Tris buffer as a base solution for tissue maceration. The DNA quantity and integrity was determined using the NanoDrop 2000C spectrophotometer and agarose gel electrophoresis, respectively. Both hot and cold solutions produced intact high molecular weight genomic DNA of sufficient yield and purity for molecular-based applications. The detection and determination of phylotypes of R. solanacearum, based on conventional and multiplex polymerase chain reaction (PCR), amplified the expected 280 and 372 bp amplicons, respectively, confirming that the quantity and quality of the extracted pathogen genomic DNA was sufficient for molecular diagnostic applications. The sequencing of the amplified products of the endoglucanase gene produced good quality sequences, which confirmed the R. solanacearum isolates to be members of phylotype II sequevar 1. This protocol is a simple, fast and reliable tool for the extraction of sufficient genomic DNA with high quality, directly from R. solancearum-infected potato tubers for PCR and sequencing applications. Its simplicity and throughput make it valuable for use in routine diagnostics and can be adopted by certification programs to ensure distribution of clean potato seeds to farmers.

Methods and Protocols doi: 10.3390/mps9030082

Authors: Sourav Chakraborty Connor Howard Checo J. Rorie Samuel D. Rabkin Hiroaki Wakimoto Dipongkor Saha

Glioblastoma (GBM) is an aggressive primary brain tumor associated with poor prognosis and resistance to therapy, underscoring the need for reliable preclinical models to evaluate emerging treatments. The orthotopic implantation of GBM stem-like cells (GSCs), combined with the intratumoral delivery of therapeutic agents, represents a widely used approach for modeling GBM tumor growth and studying treatment response. In particular, oncolytic herpes simplex viruses (oHSVs) have emerged as a promising strategy to selectively target malignant cells while inducing antitumor immune responses with minimal systemic toxicity. However, performing repeated survival stereotactic neurosurgeries in the same animal poses significant technical challenges. Here, we describe a comprehensive and reproducible protocol for triple survival stereotactic neurosurgery in mice. This approach involves (i) the intracranial implantation of GSCs to establish orthotopic tumors, (ii) the intratumoral delivery of oHSV using the same stereotactic coordinates, and (iii) contralateral intracranial rechallenge with GSCs to evaluate therapeutic efficacy and resistance to tumor rechallenge as a measure of immune memory. Using this protocol, consistent tumor establishment was achieved, and mice tolerated repeated neurosurgical procedures with stable postoperative recovery. Successful intracranial rechallenge in the same animal demonstrates the technical feasibility of multiple survival surgeries while minimizing procedure-related variability and complications. This method enables longitudinal assessment of tumor progression, therapeutic response, and durable memory protection within a single subject. Furthermore, this protocol provides a versatile platform for evaluating oncolytic virotherapy and other localized treatment strategies for GBM.

Methods and Protocols doi: 10.3390/mps9030081

Authors: Concetta D’Antonio Francesca Mantile Gabriella Pocsfalvi Giovanna L. Liguori

Cell migration is a fundamental process in cancer progression, playing a central role in tumor invasion and metastasis. This highly coordinated behavior is regulated by dynamic interactions between cancer cells and extracellular environment. Among the different tumor types, glioblastoma (GB) represents a particularly aggressive form of cancer in which enhanced migratory capacity is a key determinant of diffuse brain infiltration, tumor recurrence, and poor prognosis. In this context, extracellular vesicles (EVs) have emerged as important mediators, regulating cell migration in several cancer types, including GB. EVs are lipid bilayer-enclosed nano- and micro-sized particles, containing various bioactive molecules that can target specific recipient cells, thereby modulating cellular properties, including the migratory behavior. Among the available methods for studying cell migration, the wound healing assay is the most widely used. Although simple, cost-effective and not requiring sophisticated equipment, its reliability and reproducibility can be affected by technical variability and the diversity of existing protocols. Here, we present an optimized protocol for executing and analyzing a cellular wound healing assay designed to assess EV-mediated migration in GB cells. The protocol incorporates the use of silicone culture inserts to enhance wound homogeneity and reproducibility, together with continuous Mitomycin C incubation to inhibit cell proliferation without inducing cytotoxicity, enabling specific assessment of cell migration. We outline a step-by-step description of the procedure, detailing all required materials and equipment and highlighting critical steps, checkpoints, and key parameters. This method provides a robust framework for reproducible wound healing assays to investigate EV effects on GB cell migration.

Methods and Protocols doi: 10.3390/mps9030080

Authors: Daniel A. Corbin Christopher J. Breshike Michael R. Papantonakis R. Andrew McGill

A simple approach to sample preparation for gas phase breakthrough analysis of adsorbent materials such as metal–organic frameworks is reported. To circumvent issues related to particle size, MOF powders are coated onto glass beads using only the adhesion forces between the glass surface and the particles themselves. These coatings are sufficiently stable for the coated beads to be packed into columns and used for breakthrough measurements of the pure solids. Samples prepared in this manner are compared to analogous samples coated using a binder to attach the MOF to the surface of the beads. In many cases, the approach reported here achieves higher uptake capacities and longer breakthrough times than when a binder is used, presumably because the binder partially clogs the porous structure of the MOF. In addition, an example is discussed that highlights the possibility of a reaction between the sorbent and the binder, highlighting the advantage of a simplified sample preparation method that does not require additional chemical additives.

Methods and Protocols doi: 10.3390/mps9030079

Authors: Claudio Giorlandino Marina Cupellaro Katia Margiotti Francesca Giorlandino Francesco Pignataro Maria Luisa Mastrandrea Raffaella Raffio Laura D’Emidio Alvaro Mesoraca Vincenzo Milite

Maternal folate receptor alpha autoantibodies (FRAA) have been associated with impaired placental folate transport, fetal cerebral folate deficiency (CFD) and neurodevelopmental risks including autism spectrum disorder (ASD); however, first-trimester-specific reference intervals remain undefined. This prospective single-center study of 534 healthy pregnant women at 10 + 0 to 15 + 6 weeks’ gestation (week 10 n = 26; week 11 n = 155; week 12 n = 203; week 13 n = 105; week 14 n = 39; week 15 n = 6) used a CE-IVDR FRAA ELISA, following CLSI EP28-A3c and IFCC C-RIDL protocols, to establish week-specific percentiles (P5, P50, P95, P99) via non-parametric estimation and log-smoothed regression with a 1-week rolling window. An internal-consistency ROC analysis was performed against the within-dataset ≥P99 designation and is therefore not interpretable as discrimination against an independent clinical outcome. Median FRAA declined from 29 ng/mL (week 10) to 25 ng/mL (week 14), with provisional clinically actionable thresholds of P95 ≈ 120 ng/mL and P99 ≈ 150 ng/mL. These data provide the first first-trimester normative percentile curves for maternal FRAA and may support prioritizing FRAA assessment before 15 weeks (onset of accelerated transplacental IgG transfer). Given the cross-sectional, single-center design, the small samples at weeks 14–15, and the absence of long-term neurodevelopmental outcome data, the proposed thresholds and any downstream clinical implications, including folinic acid intervention and ASD risk mitigation, should be considered hypothesis-generating and require external and longitudinal validation.

Methods and Protocols doi: 10.3390/mps9030078

Authors: Maxie Bunz Pascal Nohl-Deryk Heike van de Sand Katharina van Baal Svenja Arendt Alina Herrmann Ingo Meyer Adriana Poppe Olaf Krause Johannes Heck Beate Sigrid Müller

Certain medications may adversely affect health during hot days and heatwaves by altering chronic conditions, comorbidities, fluid balance, or impairing heat adaptation. This study aims to develop evidence-based cross-sectoral recommendations for the safe administration of heat-sensitive medications, compiled into a so-called ‘CALOR’ list (calor: Latin for ‘heat’). Development of the CALOR list will follow a four-pillar process. First, a scoping review of scientific literature and best practices will identify potentially inadequate medications during heat events (heat-PIMs) and adaptation measures, resulting in a first draft. Second, an expert panel will refine this draft through a Delphi process to reach consensus on clinically relevant recommendations. Third, German statutory health insurance (SHI) claims data will be analysed to determine heat-PIMs prevalence; data from Cologne residents will additionally be linked with climate data to investigate health outcomes during heat events. Fourth, thirty health professionals (i.e., medical doctors, nurses, pharmacists) will field-test the CALOR list in summer, providing qualitative feedback on feasibility, leading to further refinement of the CALOR list. To our knowledge this study protocol presents the first study attempting to collate a comprehensive and actionable list of recommendations for drug safety management during hot days and heatwaves.

Methods and Protocols doi: 10.3390/mps9030077

Authors: Christopher Mauer Sean Paz Massimo Caputi

The HIV-1 genome is initially transcribed as a single primary RNA that undergoes extensive splicing to produce over 40 linear and 15 circular RNA (circRNA) isoforms sharing common sequences. Conventional methods for circRNA detection, such as Northern blotting and hybridization-based assays, are inadequate for distinguishing specific circRNA isoforms when multiple circular and linear species originate from the same transcript. We previously identified 15 HIV-1 circRNAs generated by backsplicing and demonstrated that some enhance viral replication by sequestering cellular miRNAs. PCR-based approaches using divergent primers (RT-qPCR) offer greater specificity for detecting individual circular RNAs under these conditions. Building on this, we have developed a TaqMan qPCR assay capable of specifically detecting 14 HIV circRNA isoforms using backsplicing junction-directed divergent primers coupled to a hydrolysis probe for signal confirmation. Compared with matched SYBR Green assays, the TaqMan platform showed lower background in non-infected controls and reduced variance across donor-derived samples. This method provides a robust platform for selective and qualitative analysis of HIV-1 circRNAs.

Methods and Protocols doi: 10.3390/mps9030076

Authors: Hiraku Nagahori Isabella Keefer Derrick Smith Brendan Yawn Jing Nong Liang Kai-Yu Ho

This study evaluated the test–retest reliability, standard error of measurement (SEM), and minimal detectable change (MDC) of frontal plane projection angles (FPPAs) across five single-leg tasks in individuals with patellofemoral pain (PFP). Two-dimensional video data was collected from ten individuals with predominantly unilateral PFP. Participants performed single-leg squat, single-leg landing, single-leg hop, forward step-down, and lateral step-down across two testing sessions. FPPAs were measured at peak knee flexion for each task, including trunk lean angle, knee FPPA, hip FPPA, and dynamic valgus index. Test–retest reliability was assessed using intraclass correlation coefficients (ICCs). Our findings indicate that test–retest reliability and measurement error for trunk and lower limb FPPA varied across tasks in individuals with PFP. The lowest ICC was observed for hip FPPA, particularly during single-leg squat and lateral step-down tasks. Among the five tasks tested, the single-leg squat appeared to be the most demanding task, demonstrating the lowest ICCs, and highest SEM and MDC values across all four outcome measures (trunk lean angle, knee and hip FPPAs, and dynamic valgus index). The dynamic valgus index consistently showed larger SEM and MDC values than isolated hip or knee FPPAs, likely reflecting compounded measurement errors across segments. These findings provide preliminary insights, though confirmation in larger samples in persons with PFP is warranted.

Methods and Protocols doi: 10.3390/mps9030075

Authors: Carlos A. Ramos-Jonapá Lily X. Zelaya-Molina Luis Felipe Guzmán Edgar I. González-Jiménez David Urbán-Duarte Horacio Álvarez-Gallardo Francisco J. Padilla-Ramírez

Cryopreserved bovine semen represents an accessible source of genetic material due to its widespread use in assisted reproductive technologies and the conservation of genetically valuable animals. However, DNA extraction from spermatozoa within this type of sample remains challenging due to the high protein content and the complex structure of the ejaculate, which can affect DNA yield and quality. The aim of this study was to identify and validate an efficient method for obtaining high-quality DNA from spermatozoa present in cryopreserved bovine semen for molecular diagnostic applications. Five DNA extraction protocols were evaluated: TRIzol™, MagMax™ Nucleic Acid Purification Kit, Rapid DNA™ Fecal/Soil Microbe Kit, a conventional phenol–chloroform protocol, and a modified phenol–chloroform–isoamyl alcohol protocol. All extracted genetic material was assessed by spectrophotometry (concentration and purity), and DNA integrity was evaluated by agarose gel electrophoresis. Statistical analysis revealed significant differences in DNA concentration among extraction methods (Friedman test, χ2 = 22.0, df = 4, p = 0.0002). Post hoc comparisons indicated that the modified phenol–chloroform–isoamyl alcohol protocol yielded significantly higher DNA concentrations compared to selected methods. This protocol showed the highest DNA concentration (1006.2 ± 829.4 ng/μL) and favorable purity values, and enabled consistent amplification in both conventional PCR and qPCR assays targeting the β-actin gene and Tritrichomonas foetus, respectively. These findings suggest that the modified protocol represents a suitable and promising approach for extracting genomic DNA from spermatozoa in cryopreserved bovine semen, with potential applications in molecular diagnostics and reproductive biotechnology.

Methods and Protocols doi: 10.3390/mps9030074

Authors: Georgina Bonny Kashif Mahfooz Sara Garcia-Rates Sibah Hasan Susan Adele Greenfield

Antibody treatment for Alzheimer’s disease is an evolving therapeutic strategy that ensures high affinity and specificity to the target antigen; however, current approaches have proven only partially successful. A 14-mer peptide, T14, is twice as high in Alzheimer’s brains and has been identified as a primary driver in the neurodegenerative process. Previously, the polyclonal antibody Ab-19 was shown to be as effective as the T14 receptor blocker (NBP-14) in reducing the toxic calcium influx in PC12 cells. The aim of this study was to establish a thorough validation process in order to evaluate the efficacy of respective anti-T14 monoclonal antibodies in T14 detection and rescuing potential from T14-induced toxicity in PC12 cells. Subsequently, we assessed the binding affinity of the most promising antibody, THK-117, via quantitative indirect conjugated T14 ELISA assays. The level of efficacy shown proved to be comparable to the polyclonal antibody, yet with the additional advantage of robust manufacturing reproducibility and high binding specificity toward the T14 epitope. With a notably low EC50, THK-117 can be viewed as a promising candidate for humanization, offering a strong potential as a therapeutic monoclonal antibody for the treatment and prevention of Alzheimer’s disease.

Methods and Protocols doi: 10.3390/mps9030073

Authors: Yudaï Sahuc Elissa Elia Christophe Caneparo Nathan Félix Marilou Hardy Stéphane Chabaud Stéphane Bolduc

Trans-epithelial permeability is a critical functional parameter for reconstructed tissues, particularly in genitourinary tissue engineering, where urine leakage must be avoided. Although Franz diffusion cells are considered the gold standard for permeability measurements, their cost and limited accessibility restrict their widespread use. In parallel, the reliable quantification of urea in culture media remains challenging due to protein interference and assay cost. The Inexpensive Trans-Epithelial Permeability (I-TEP) test is a simple and a low-cost Franz-like permeability system which can be combined with an optimized diacetyl monoxime–thiosemicarbazide (DAMO–TSC) colorimetric urea assay. I-TEP system relies on readily available laboratory components to create physically separate donor and receiver compartments, with the tissue acting as the sole diffusion interface. The DAMO–TSC assay was optimized through systematic evaluation of deproteinization, incubation time, storage conditions, and serum interference. The I-TEP test showed a strong correlation with conventional Franz diffusion cells when testing similar tissue samples. Deproteinization was identified as a mandatory step for accurate urea quantification in serum-containing media. The combined approach was successfully applied on engineered genitourinary tissues, demonstrating sensitivity to tissue maturation and cellular composition. This protocol provides a proof of concept for an affordable, robust, and autonomous method for routine permeability assessment, bridging the gap between costly commercial systems and high-throughput experimental needs.

Methods and Protocols doi: 10.3390/mps9030072

Authors: Sabrina-Gabriela Mîndruț Cristina Pop Sorin-Marian Mârza Alexia-Teodora Hoța Flaviu-Alexandru Tăbăran Ibrahima Mamadou Sall Ana Uifălean Emilia-Laura Mogoșan Oliviu Voștinaru Cristina-Ionela Mogoșan

Experimental diabetes models induced by streptozotocin (STZ) and alloxan are widely used in preclinical research; however, direct standardized comparisons in female rodents remain limited. The present study evaluated multiple chemical induction protocols in female Wistar rats, including STZ (40 and 65 mg/kg), STZ at the same doses combined with nicotinamide (110 mg/kg), and alloxan (130 mg/kg). Glycemic progression, oral glucose tolerance test, body weight evolution, oxidative stress markers, and multi-organ histopathology were assessed over a 14-day period. High-dose STZ (65 mg/kg) and alloxan produced rapid, sustained hyperglycemia (p < 0.0001), significant body weight reduction, increased lipid peroxidation (elevated MDA), nitric oxide overproduction, thiol depletion, and pronounced pancreatic and renal structural damage. In contrast, STZ–nicotinamide protocols generated moderate but stable hyperglycemia with partial preservation of islet architecture, attenuated oxidative imbalance, and improved systemic tolerability. Oral glucose tolerance test confirmed impaired glucose handling in the STZ–nicotinamide group, consistent with a type 2 diabetes-like phenotype rather than complete insulin deficiency. These results demonstrate that induction strategy critically determines metabolic stability, oxidative stress burden, and tissue remodeling patterns, supporting model selection according to specific experimental objectives.

Methods and Protocols doi: 10.3390/mps9030071

Authors: Jaden Roe Ashlyn Benavides Michael B. Filla Douglas C. Bittel Whitney Shae Geetha Haligheri James E. O’Brien Nataliya Kibiryeva

A challenge of studying mammalian cardiac embryogenesis is the limited ability to perform experimental manipulations in animal models. The avian embryo is widely accepted as a model for mammalian heart developmental studies. In this study, we establish the methodology and protocols for studying the Japanese quail (Coturnix japonica) heart at embryonic day 10 (HH38) using the FUJIFILM VisualSonics Vevo 3100 ultrasound system equipped with a MX550D small animal cardiology transducer. These protocols were designed to measure right ventricular wall thickness, pulmonary artery diameter, and the outflow velocities of the right ventricular outflow tract (RVOT) and the pulmonary artery (PA), thereby establishing baseline parameters of the normally developing quail morphology. Quail embryos are an ideal model for cardiovascular research due to their short incubation period (16–17 days), experimental accessibility, and strong similarities to mammalian heart development. These developmental similarities include, but are not limited to, looping, chamber septation, and the development of a true four-chamber heart. High-resolution imaging modalities, including ultrasound and optical coherence tomography, enable noninvasive, real-time visualization of cardiac morphology and function throughout development. Echocardiography allows for quantitative and qualitative assessments of myocardial structure and cardiac hemodynamics. The similarity to the mammalian heart, combined with rapid embryogenesis, makes quail embryos a valuable model for investigating congenital heart defects, genetic modifications, and fundamental cardiac developmental processes. In this study, we describe reproducible incubation protocols and baseline echocardiographic parameters used to evaluate morphological and physiological changes in the developing embryonic quail heart on embryonic day 10.

Methods and Protocols doi: 10.3390/mps9030070

Authors: Tong Yang Luke Stasikelis Alexander J. Knights

Intra-articular soft connective tissues such as synovium and adipose tissue play a crucial role in governing joint homeostasis and disease progression in various forms of arthritis. In the knee, like many synovial joints, adipose tissue forms an integrated anatomic and functional unit with the joint-lining synovium, and the most prominent adipose depot is the infrapatellar fat pad (IFP). With growing evidence that lipid profiles in the synovium–IFP unit shift during progression of joint diseases like osteoarthritis (OA), there is strong impetus for consistent tissue collection approaches and reproducible subsequent lipid characterization. Here, we present a standardized dissection and low-input untargeted lipidomics workflow optimized for mouse knee synovium and IFP, to enable comprehensive lipid profiling. Synovium/IFP from multiple joints are pooled to increase input mass and guarantee robust lipid yield, followed by lipid extraction and high-resolution liquid chromatography-mass spectrometry (LC–MS) acquisition for global, untargeted lipidomic profiling. The analysis workflow encompasses robust feature detection, accurate lipid annotation, data transformation and normalization. These steps enhance comparability across samples, particularly those with low input amounts, while minimizing technical variance and batch effects. Using this approach, we detect a broad spectrum of lipid species spanning the major lipid categories. As expected for untargeted discovery, a subset of non-lipid species is also observed. This protocol provides a practical framework for robust, reproducible lipidomics in murine intra-articular soft tissues to support future disease-specific biomarker and drug target discovery in OA and other joint diseases.

Methods and Protocols doi: 10.3390/mps9030069

Authors: Achilleas Livieratos Junjing Lin Paraskevi Chasani Mina Gaga Fotios S. Fousekis Charalambos Gogos Karolina Akinosoglou Konstantinos H. Katsanos Margaret Gamalo

Large language models (LLMs) have rapidly advanced natural language processing and are increasingly used to analyze clinical narratives. Their ability to extract information, summarize records, and support clinical workflows makes them potential tools for enhancing documentation efficiency and the secondary application in the analysis of electronic health record (EHR) data. The aim of this work is to synthesize recent evidence on methodological approaches and applications of LLMs for clinical narrative processing, and to assess their performance, benefits, limitations, and implications for clinical practice. Across 2022–2026 studies, LLMs demonstrated strong performance in information extraction, summarization, triage prediction, section classification, and synthetic text generation, often surpassing traditional machine-learning models. Overall, LLMs improved the conversion of unstructured notes into actionable clinical insights, reduced documentation burden, and supported decision-making tasks. Key challenges included hallucinations, variable reproducibility, sensitivity to prompting, domain adaptation gaps, and limited transparency. Our findings indicate that LLMs show substantial promise for transforming clinical narrative processing, but safe adoption requires rigorous evaluation and continuous model auditing. This work provides a structured, non-systematic synthesis of representative studies and is intended as a high-level overview of emerging applications rather than a comprehensive systematic review.

Methods and Protocols doi: 10.3390/mps9030068

Authors: Dalchand Ahirwar Kun Xie Philip O’Herron

Neuronal activity and cerebral blood flow are tightly coupled to support the high metabolic demands of the brain. Disruption of neurovascular coupling is a defining feature of many neurodegenerative disorders such as Alzheimer’s disease, stroke, small vessel disease, Parkinson’s disease, and aging. Progress in understanding the mechanisms underlying neurovascular coupling requires experimental approaches that can simultaneously measure neuronal activity and vascular dynamics with high spatial and temporal resolution, while also enabling targeted perturbations of the system. Here, we present a methodological framework that combines chronic electrophysiological recordings with two-photon imaging of cerebral blood flow and optogenetic manipulation of the vasculature in vivo. Using a chronically implanted flexible electrode array, we obtain measurements of the single- and multi-unit spiking activity, as well as local field potentials. Concurrently, two-photon microscopy enables high-resolution measurements of vessel diameter and blood flow within individual vascular segments. In addition, optogenetic control of vascular smooth muscle cells allows for rapid and reversible manipulation of the vessel diameter through the same cranial window while simultaneously recording the neural and vascular activity. We provide detailed protocols for surgical implantation, data acquisition, and analysis, and discuss experimental considerations and limitations. This combined platform offers a powerful tool for mechanistic studies of neurovascular coupling and its dysfunction in disease models.

Methods and Protocols doi: 10.3390/mps9030067

Authors: Vivian K. O’Donnell Andrew Schoepke Heather Petrowski Leslie Blakemore Douglas P. Gladue Bonto Faburay Manuel V. Borca

African swine fever (ASF) is a highly contagious and often fatal viral disease of pigs that poses serious economic consequences to the swine industry due to its high mortality rate and rapid spread. Currently, the identification of infectious ASF virus (ASFV) is the confirmatory test when clinical samples are positive for ASFV by any other diagnostic methods. Detection of infectious ASFV requires the availability of primary swine macrophage cultures as a cell substrate. We demonstrate here that cryopreserved swine primary macrophages are a suitable cell substrate for the detection, isolation and propagation of ASFV, showing similar results as when fresh swine macrophages are utilized. The possibility of using cryopreserved macrophages for detecting infectious ASFV would improve the efficacy of diagnostics in ensuring the availability of macrophage cell cultures during an emergency response.

Methods and Protocols doi: 10.3390/mps9020066

Authors: Giovanni Visco Maria Pia Sammartino Angela Marchetti Mauro Castrucci Mauro Tomassetti

Portable devices are powered in direct current (DC) or by batteries (primary battery), accumulators (secondary battery), and now supercapacitors, which can also be used for energy storage. The European Portable Battery Association states that approximately 239,000 tons of batteries were placed on the market in the European Economic Area (EEA) plus Switzerland in 2022. Even if they were all disposed of correctly respecting the 3R paradigm (Reduce, Reuse and Recycle), non-rechargeable batteries create an environmental problem because they do not discharge completely with an obvious waste of energy. Secondary batteries and supercapacitors can be recharged because they use reversible chemical/physical processes while primary batteries cannot be recharged because they are based on irreversible redox reactions; nevertheless, it is possible to try to recover their residual charge if this is higher than a threshold beyond which the reactions can be reversible. The most used batteries are alkaline zinc/manganese dioxide and they are non-rechargeable; an inappropriate recharge attempt can lead to serious harm to the operator and the environment. This paper describes a simple Arduino-based circuit and the protocol to measure and graph the residual charge of an alkaline battery in order to establish if it can be recharged. The circuit, design, the Arduino Uno R3 sketch (i.e., microprocessor software) and the full protocol are here presented under the open source license (Copyright Creative Commons Public license, CC BY-NC-ND 4.0 EN) so that they could become a pilot system and then a commercial product. The residual charge of 158 batteries, obtained after discharging those that, by eye, appeared damaged, was measured. Results evidenced that 49% of batteries had a residual voltage, under low load, between 1.2 and 1.6 V, making them good candidates for a recharge attempt.

Methods and Protocols doi: 10.3390/mps9020065

Authors: Nicolas De Azevedo Anthony Lozano Ramon E. Parsons Tiphaine C. Martin

Background: Highly lipidic tissues (e.g., adipose tissue, brain, and liver) are challenging for transcript and protein extraction and for next-generation sequencing. Lipids can clog filters, columns, and pipettes; cause autofluorescence and quenching in imaging; and interfere with centrifugation-based separation. Aim: To identify the most suitable method for extracting total RNA for RT-qPCR and an alternative method for extracting total protein for quantification in mice fed a regular or high-fat diet. Methods: We compared three total RNA extraction methods and two total protein extraction methods. Results: The highest total RNA yield and purity were obtained with TRIzol and chloroform, with optimized steps added to the original protocol to address the challenges posed by highly lipid-rich tissues. For total protein extraction, an adipose tissue-specific kit from Invent Biotechnologies yielded higher protein levels than the classical RIPA-based method. Among lipid-rich tissues, we observed that adipose tissue was more challenging to process than the brain and the liver. Conclusions: Adipose tissue, particularly under a high-fat diet, is the most challenging lipid-rich tissue, followed by the brain and then the liver. We highlight protocols that improve total RNA and protein yields and purity, which may benefit other researchers working with these tissues.

Methods and Protocols doi: 10.3390/mps9020064

Authors: Marco Scotto Alessandro Galgani Marina Boido Nooria Mohammady Alessandro Vercelli Filippo S. Giorgi

Unbiased stereology represents the most accurate approach for estimating the total number of neurons of specific brain regions; however, its reliability critically depends on the use of rigorously defined and anatomically appropriate sampling parameters. The brain nucleus Locus Coeruleus (LC) plays a key role in several brain functions. LC impairment has been associated with a range of disorders affecting individuals across the lifespan, from infancy to adulthood. In animal models of these conditions, precise estimation of LC neuronal number is essential. The LC analysis poses specific methodological challenges due to its small size, indistinct anatomical boundaries, and age-dependent changes in neuronal density. In this study, we present a detailed and reproducible stereological workflow for the quantification of LC neurons in the mouse brain across the lifespan. Using C57BL/6J mice at postnatal, adult, and aged stages, we optimized all key components of the Optical Fractionator method, LC neurons were identified by immunoperoxidase staining for tyrosine hydroxylase (TH) and quantified using systematic-random sampling implemented in Stereo Investigator® software. We show that age-specific adjustment of stereological parameters is necessary to obtain reliable estimates, particularly at early postnatal stages characterized by high neuronal packing density. With the optimized protocols described here, TH+ LC neuron counts consistently met accepted precision criteria, as assessed by the Gundersen coefficient of error.

Methods and Protocols doi: 10.3390/mps9020063

Authors: Chloe Wright Gowri Chellappan Abigail Mydland Gowthami Jagruthi Penumaka Geetha Karengil Harshita Seth Anup Datta Subhash V. Kapre

Background/Objectives: Infection from Streptococcus pneumoniae can lead to serious complications, such as meningitis and pneumonia, in children under 2 years of age, older adults, and immunocompromised populations. Conjugate vaccines against the pathogen have been licensed for the prevention of invasive pneumococcal disease. Conjugate vaccine development is an involved process demanding extensive characterization of both the polysaccharide (PS) and protein (Pr) moieties in complex structures. One powerful tool in our analytical tool kit that can shed light on various analytical attributes of conjugate vaccines, such as molecular weight and composition and conjugation efficiency, is the size-exclusion chromatography-multi-angle light scattering detector (SEC-MALS) technique. Herein, we demonstrate the applicability of the SEC-MALS approach for pneumococcal conjugate vaccine product characterization. Methods: Capsular polysaccharides for serotypes (STs) 1, 3, 5, 10 A, 18 C, 24 F, and 33 F conjugated to rCRM197 carrier protein were chosen for this study. Results: The technique was very straightforward, with a high degree of accuracy (>90% based on standards) and repeatability (<2% RSD) for conjugate molar mass measurements. In addition, leveraging the capability of SEC-MALS for compositional analysis, we were able to get detailed information on the molecular assembly and conformation of the conjugates and further tweak the conjugation process to yield conjugates of a desired molar mass. Conclusions: Thus, this study highlights the usefulness of the SEC-MALS technique for in-depth conjugate vaccine biophysical characterization, which is critical for achieving optimal product attributes, driving manufacturing consistency and vaccine potency.

Methods and Protocols doi: 10.3390/mps9020062

Authors: Liam J. Houlton Jeremy A. Moody Theodoros M. Bampouras Joseph I. Esformes

This study aimed to evaluate the performance expression stability (PES) of sixteen alternative complex–contrast training (CCT) set strategies. Three separate cross-sectional studies (n = 14–17) evaluated the effects of different intra-contrast rest periods (ICRP; ≤300 s) and rest redistribution (RR) strategies (≤60 s) within CCT sets on the application of vertical jump propulsive force were examined using dual force platforms. To establish PES for propulsive force–time variables, repetitions one and two of the baseline set were analyzed using within-participant (coefficient of variation, CV; standard error of measurement; smallest worthwhile change; relative mean bias) and between-participant (intra-class correlation coefficient, ICC3,1; Pearson’s correlation coefficient, r) stability metrics. Results showed that all CCT set strategies facilitate stable performance expression between participants and facilitated the detection of practically meaningful changes for propulsive impulse, peak force, mean force, and propulsion time (ICC3,1 = 0.64–0.99, r = 0.80–0.99, CV = 1.12–9.98%), while rate of force development metrics demonstrated less consistent between- and within-participant stability (ICC3,1 = 0.55–0.97, r = 0.46–0.96, CV = 7.52–27.66%). The findings indicate that alternative CCT set strategies facilitate the stable expression of propulsive force–time performance in vertical jumps, although individualized prescriptions are essential for optimizing rate of force development outcomes. Performance expression stability insights provide a practical tool for balancing the effectiveness and potential for performance enhancement of vertical jump propulsion across alternative CCT set strategies. Practitioners may use these results to improve the prescription and monitoring of CCT-based strength and power mesocycles.

Methods and Protocols doi: 10.3390/mps9020061

Authors: Giuseppa D’Amico Alessandra Maria Vitale Martina Di Marco Alessandro Lo Giudice Francesca Chiara Cecala Francesco Cappello Celeste Caruso Bavisotto

Glioblastoma (GBM) is one of the most aggressive and therapy-resistant primary brain tumors, mainly due to its pronounced intratumoral heterogeneity and highly invasive phenotype. Patient-derived three-dimensional (3D) culture models, including tumor spheroids, represent valuable tools to preserve the cellular complexity, phenotypic plasticity, and microenvironmental features of GBM ex vivo. However, standardized and reproducible protocols for the generation and maintenance of GBM spheroids from surgical specimens are still limited. Here, we describe a detailed and robust protocol for the isolation, 3D cultures, and expansion of primary GBM cells obtained from patient biopsies, leading to the formation of stable and morphologically consistent spheroids. The protocol provides step-by-step instructions for tissue dissociation, cell seeding under low-adhesion conditions, optimization of culture density, and long-term spheroid maintenance. In addition, we include guidelines for the morpho-phenotypical characterization of the resulting 3D structures. This methodological workflow offers a reproducible platform for modeling GBM in vitro, enabling the study of tumor biology and supporting translational applications such as drug screening, biomarker validation, and patient-specific therapeutic testing in a 3D context.

Methods and Protocols doi: 10.3390/mps9020060

Authors: Miroslav Lisjak Marija Špoljarević Jelena Ravlić Zdenko Lončarić Lucija Galić

Phenolic compounds are plant-derived antioxidants crucial for human health and food preservation. Their bioactive potential including anti-inflammatory, antimicrobial, and anti-carcinogenic properties makes them a vital focus in nutritional, pharmaceutical, and agricultural research. This review critically evaluates the methodologies for their extraction, detection, and quantification to accurately assess antioxidant activity. Oxidative stress in biological systems and food matrices necessitates accurate analytical methodologies for assessing antioxidant behavior, which include both in vitro, in vivo and ex vivo approaches. Sample pretreatment and extraction techniques are critical for reliable analysis and vary depending on the matrix, compound polarity, and target phenolic subclass. We compare conventional extraction techniques (Soxhlet, maceration) with advanced methods like ultrasound-assisted, microwave-assisted, and supercritical fluid extraction. Detection methods reviewed include spectrophotometric assays (e.g., DPPH, FRAP, ORAC), electrochemical sensors, and chromatographic techniques (e.g., HPLC, HPLC−MS). While each method has distinct advantages, a lack of standardization remains the primary challenge, driven by variations in protocols and the vast chemical diversity of phenolics. This review underscores the critical need for integrated, standardized approaches to ensure the accurate and comparable evaluation of antioxidant activity in research and industry.

Methods and Protocols doi: 10.3390/mps9020059

Authors: Alessandra Helena da Silva Hellwig Gisele Menezes Ewald Grazielle Motta Rodrigues William Latosinski Matos Juliana Bergmann Viviane Horn de Melo Rodrigo Minuto Paiva Dariane Castro Pereira Eduardo Wandame Gomez

Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy. AML classification is currently based on cytogenetic and molecular alterations as well as immunophenotyping, although risk stratification still relies primarily on cytogenetic findings. However, approximately 45% of AML patients present with a normal karyotype, which makes accurate risk classification and treatment stratification more challenging. Therefore, the identification of molecular prognostic markers described in the literature has become essential in routine diagnostic laboratories, enabling the more precise categorization of patients into risk groups. In this study, we present a simple, rapid, step-by-step multiplex PCR protocol combined with capillary electrophoresis for the detection of two of the most prevalent molecular alterations in AML: nucleophosmin 1 (NPM1) mutations and Fms-like tyrosine kinase 3 internal tandem duplications (FLT3/ITD). This protocol provides a practical workflow that can assist diagnostic laboratories in implementing and optimizing multiplex mutation detection in routine practice.

Methods and Protocols doi: 10.3390/mps9020057

Authors: Dmitry Yakovlev Kanamat Efendiev Polina Alekseeva Vladimir Makarov Kirill Linkov Anna Lukianova Vladimir Oleinikov Victor Loschenov

This study presents a comparative analysis of the effect of methylene blue (MB) loading strategy on the physicochemical and colloidal properties of ethosomes prepared by the cold method. Two synthesis protocols differing in the phase of introduction of the cationic hydrophilic dye were investigated: a classical approach with MB loading into the aqueous phase and an alternative approach involving MB incorporation into the ethanolic lipid phase. It is shown that the loading strategy is a critical technological factor determining vesicle size, encapsulation efficiency, loading capacity, and electrokinetic properties of the systems. The alternative method results in the formation of smaller ethosomes (Rh ≈ 78 nm) compared to the classical protocol (Rh ≈ 96 nm), but is accompanied by a lower encapsulation efficiency (EE ≈ 36% versus 48%). The results indicate that a reduction in vesicle size does not necessarily lead to higher encapsulation of hydrophilic cationic MB and may be associated with a decrease in the total internal aqueous volume as well as an increased contribution of a weakly bound surface-associated dye fraction. Spectral analysis indicates the preservation of a predominantly monomeric form of MB within ethosomes, while differences in ζ-potential suggest distinct localization of the dye within the vesicular systems. Overall, the results highlight the importance of optimizing the loading protocol in the development of ethosomal drug delivery systems for photodynamic therapy and topical applications.

Methods and Protocols doi: 10.3390/mps9020058

Authors: Daniel Cozzolino

The concepts of food matrix and holistic analysis have been used in a wide range of scientific disciplines to describe the sum of the parts of a whole that provide a specific property or functionality to the sample. Traditional chemical and physical analysis needs to destroy the sample (e.g., dilution, extraction, drying) before analysis. The utilization of vibrational spectroscopy techniques, like near (NIR), mid infrared (MIR) and Raman spectroscopy, allows for the non-destructive analysis of food ingredients and products. The resulting output of this analysis is based on the information provided by the vibrational modes of atoms present in the different molecules, allowing the measurement of different chemical and physical characteristics of the food. The objective of this paper is to discuss the ability of vibrational spectroscopy methods to provide robust tools to analyze the food matrix holistically, moving away from the traditional analysis of individual compounds or chemical parameters. Studies discussed and presented in this review demonstrated the ability of vibrational spectroscopy (e.g., NIR, MIR and Raman spectroscopy, hyperspectral imaging) to assess the whole food matrix beyond the traditional notion of developing a calibration model.

Methods and Protocols doi: 10.3390/mps9020056

Authors: Maria Bernadette Cilona Serena Nannipieri Arianna Ferlito Giulia Orsi Monica Ronzoni Giorgia Mangili Emanuela Rabaiotti Fabio Ciceri Michele Reni Mona-Rita Yacoub

Background: Hypersensitivity reactions to antineoplastic agents are an increasing clinical challenge due to their rising incidence and potential severity. Early allergological assessment and tailored drug re-exposure strategies may allow continuation of essential therapies, although real-world data remain limited. Methods: We conducted a monocentric retrospective observational study including adult oncologic patients with suspected chemotherapy-induced hypersensitivity reactions. Clinical features, allergological work-up, and management strategies were analyzed. The primary outcome was the success rate of drug reintroduction using desensitization or enhanced premedication. Secondary outcomes included skin test positivity rates and the association between cumulative chemotherapy exposure and anaphylaxis. Results: Forty-two patients were included (95% female; median age 57.5 years). Re-exposure was required in 18 patients, and was successful in all patients undergoing desensitization and in 92% of those managed with enhanced premedication. Skin testing was positive in 71% of suspected platinum reactions, 30% of taxanes, and 40% of biologic agent reactions. Anaphylaxis occurred in 26.2% of patients, and a trend toward an association with cumulative chemotherapy exposure was observed; each additional cycle was associated with a 28% increase in the odds of anaphylaxis (adjusted OR 1.28; 95% CI, 1.00–1.63). Conclusions: Desensitization and enhanced premedication allow safe reintroduction of antineoplastic agents. Cumulative chemotherapy exposure is associated with an increased risk of anaphylaxis.

Methods and Protocols doi: 10.3390/mps9020055

Authors: Diana Guerrero-Jaramillo Ricardo de la Caridad Montero Oscar Campo

Background: Work at heights is a high-risk occupational activity, with falls being a leading cause of fatal accidents in construction and industrial maintenance. Conventional safety training often does not fully prepare workers for real-world hazards. Immersive virtual reality (IVR) has emerged as a promising training tool, providing controlled and realistic simulations of hazardous scenarios. This hypothesis-generating pilot study evaluates the feasibility and effectiveness of IVR in enhancing practical skills, safety perception, and physiological responses during work-at-height training. Methods: This controlled trial will recruit first-time trainees from the National Learning Service (SENA) of Colombia. Participants will be assigned to an intervention group, receiving IVR training before field-based practical sessions, or a control group, receiving standard theoretical instruction. Outcomes include practical skill acquisition, ergonomic risk, cognitive performance, and physiological responses, including heart rate variability measured with validated devices. Assessments will be performed using standardized tools, and data will be analyzed with repeated-measures ANOVA and regression models to compare groups. Conclusions: By integrating practical, cognitive, ergonomic, and physiological measures, this study will provide evidence on whether IVR improves the effectiveness of work-at-height training beyond conventional methods. Findings may inform future strategies to enhance occupational safety training in high-risk work environments.

Methods and Protocols doi: 10.3390/mps9020054

Authors: Prakash T. Parvatkar Nishikant Satam

The rapid expansion of click chemistry reflects its transformative influence on contemporary drug discovery. This review highlights major advances in the application of click reactions within the kinetic target-guided synthesis (KTGS) paradigm for identifying potent inhibitors across a broad range of biological targets. KTGS constitutes a methodological shift that leverages the inherent dynamics of biomolecular systems, enabling biological targets to direct the in situ assembly of high-affinity bidentate ligands from a diverse repertoire of reactive building blocks. The review systematically classifies the principal bond-forming reactions that underpin effective inhibitor generation via KTGS. Collectively, it provides a comprehensive and scholarly analysis of how click-chemistry-enabled KTGS is redefining drug discovery and expediting the development of next-generation therapeutics.

Methods and Protocols doi: 10.3390/mps9020053

Authors: Lenka Fialova Ivana Marova

Singleplex and multiplex real-time PCR-HRM (polymerase chain reaction with high resolution melting), both with specific and non-specific amplicon detection, are used for a wide range of applications, from clinical diagnostics to food authentication. However, their results can be influenced by the quality of the template DNA and composition of the reaction mixture. The methods used for the analysis of these results then influence the conclusions drawn. In this work we present an example from our laboratory practice, where the results of singleplex and multiplex real-time PCR differed, despite using the same reaction conditions, primers and analyzed plant material. We show the influence of a singleplex and multiplex PCR setup on the results, as well as the influence of template contamination on the melting behaviour of amplicons. We also discuss the usefulness of cluster analysis for the clarification of real-time PCR-HRM results which appear unclear when only melting and difference curves or similarity scores are used for the analysis of these results. We provide a discussion of problems which we encountered during an analysis of commercial teas and which should be considered by researchers new to PCR-based analysis of plant material, especially if the studied material is rich in various contaminants.

Methods and Protocols doi: 10.3390/mps9020052

Authors: Sinethemba H. Yakobi Uchechukwu U. Nwodo

Pseudomonas aeruginosa causes severe healthcare-associated infections, yet no vaccine has been licenced. To circumvent the antigenic variability of classical surface antigens, we evaluated LolB—an essential outer-membrane lipoprotein whose periplasmic orientation favours T-cell-dominant mechanisms with potential antibody access via outer-membrane vesicles (OMVs) or bacteriolysis. An integrative in silico pipeline combined multi-strain conservation (20 isolates), epitope discovery (B- and T-cell), safety filters, physicochemical profiling, de novo/refined 3D modelling, molecular dynamics (MD), and docking to TLR4/MD-2. LolB was highly conserved (95–100% identity) under strong purifying selection (dN/dS = 0.15). A conformational B-cell hotspot centred on Q72 mapped to a solvent-accessible flexible loop. Two class II epitopes—LAAQNSPLT and FLGSAAAVS—showed predicted high affinity (IC50 < 10 nM), non-toxicity, and broad coverage, with the pooled set achieving 98.6% global HLA coverage in silico. The final 119-aa construct (N-terminal hBD-3 adjuvant; GPGPG linkers) was compact and tractable (MW = 12.7 kDa; instability index < 40; near-neutral GRAVY) and scored higher for antigenicity than native LolB (VaxiJen 0.82 vs. 0.41). MD supported thermal stability up to 350 K, linker RMSF < 1.5 Å, and a stable 18.2 ± 2.8 Å interdomain spacing. Docking predicted a 1420 Å2 interface and ΔG = −10.2 kcal·mol−1 (Kd = 28 nM) with reproducible polar contacts, suggesting productive TLR4/MD-2 engagement. A conservative R42A/K variant is proposed to temper IFN-γ bias. This work therefore suggests an essentiality-anchored LolB-derived multi-epitope construct as a computational vaccine candidate against multidrug-resistant P. aaeruginosa and defines specific experimentally testable hypotheses for future in vitro/in vivo assessment. Essentiality-anchored epitope selection plus adjuvant-surface engineering yielded a structurally coherent, immunologically rational LolB-derived multi-epitope vaccine warranting experimental validation.

Methods and Protocols doi: 10.3390/mps9020051

Authors: Emma Klemola Joona Tapio Rasmus I. P. Valtonen Mikko P. Tulppo Janne Hukkanen Peppi Koivunen

Hypoxia activates hypoxia-inducible factors (HIFs), which regulate genes involved in erythropoiesis, angiogenesis, and metabolism. HIF stability is controlled by oxygen-dependent HIF prolyl 4-hydroxylases (HIF-P4Hs). Pharmacological HIF-P4H inhibitors are approved for the treatment of anaemia in chronic kidney disease (CKD). Beyond erythropoiesis, these drugs have been linked to improved lipid profiles in CKD, and preclinical studies suggest benefits for glucose tolerance and cardiovascular protection. However, cardiometabolic effects of HIF-P4H inhibitors have not been systematically examined in healthy or non-anaemic individuals. This investigator-initiated, double-blind, placebo-controlled, randomised crossover trial evaluates the systemic effects of roxadustat, an orally administered pan-HIF-P4H inhibitor. The study consists of two 10-day study arms separated by a minimum 4-week washout. Participants receive 70 mg of roxadustat or a placebo thrice a week. The primary hypothesis is that roxadustat lowers plasma total cholesterol. Secondary outcomes include changes in LDL cholesterol, triglycerides, insulin sensitivity, glucose tolerance, body composition, 24 h blood pressure, exercise capacity, autonomic cardiovascular regulation, and skeletal muscle microcirculation. Healthy volunteers (n = 24) aged 18–40 years will be enrolled. This study will provide insights into the potential of HIF-P4H inhibitors for obesity, dyslipidaemia, insulin resistance, and hypertension, and may inform future therapeutic strategies for metabolic syndrome, type 2 diabetes, and cardiovascular disease.

Methods and Protocols doi: 10.3390/mps9020050

Authors: Mathilde Rodriguez Claire Brunet Franz Bruckert Marianne Weidenhaupt

Prefilled syringes are valuable drug delivery systems, offering convenience and precision dosing. Among the critical factors influencing their performance is the stability of the silicone oil layer, which acts as a lubricant, guaranteeing the gliding properties of the plunger. The silicone oil, if it comes in contact with therapeutic formulations, can be subject to drug–container interactions, potentially leading to silicone oil release into the solution, thereby altering the gliding properties of the syringe and leading to unwanted particle formation, compromising drug efficacy and safety. Different measurement techniques, such as visual inspection, dynamic light scattering and spectroscopic analysis, are used to assess silicone oil layer stability in prefilled syringes. However, a quantitative, rapid and low-volume screening method to rapidly evaluate container compatibility for therapeutic formulations is not available. Here, we present a multi-well-based screening protocol allowing users to quantify, through fluorescence, the silicone oil released into a solution upon contact with liquid formulations. Fluorescently labeled uniform silicone oil layers of the desired thickness are deposited in glass-bottom wells and exposed to typical formulations, containing surfactants and monoclonal antibodies. The release of silicon oil as a function of contact time is quantified using fluorescence calibration. Beyond its use as a screening tool to evaluate drug–container compatibility, our protocol can contribute to the fundamental understanding of the factors and mechanisms influencing silicone oil layer stability and, furthermore, to the optimization of drug delivery systems.

Methods and Protocols doi: 10.3390/mps9020049

Authors: Ee Wern Tan Tien Yang Goh Shi Hui Law Kuan Onn Tan Bey Hing Goh

Three-dimensional (3D) spheroid cancer models provide enhanced physiological relevance relative to traditional monolayer cultures but often demonstrate restricted drug responsiveness due to their dense architecture, hypoxic gradients, and diminished therapeutic penetrance. This study overcomes these limitations by establishing a sensitized 3D spheroid cancer cell model that employs the adenovirus-mediated gene expressions of tumor-suppressor and pro-apoptotic genes consisting of MOAP-1, BAX, and RASSF1A. The optimization of adenoviral infectivity led to the discovery of an intermediate multiplicity of infection (MOI) that facilitated effective and uniform transduction while reducing cytotoxicity. Adenovirus-infected 3D spheroid cells demonstrated enhanced apoptotic activities, evidenced by increased cell death relative to untreated spheroids. When exposed to the anti-cancer compound such as piperonal and pyrazole, the sensitized spheroids exhibited significantly enhanced drug responsiveness and synergistic effects over a five-day treatment period, surpassing the effects of adenovirus or anti-cancer drug treatment alone. Notably, similar responses were noted between low- and high drug doses, suggesting an enhancement of therapeutic efficacy at lower concentrations. This sensitized 3D spheroid model constitutes a more predictive in vitro system for anti-cancer drug discovery, facilitating enhanced mechanistic evaluation and the identification of potent drug candidates with greater translational significance.

Methods and Protocols doi: 10.3390/mps9020048

Authors: Tomohiro Shinozaki Takuya Hasegawa MST Tahmina Akter Kazuyuki Kumagai Youichi Suzuki Taiichi Sakamoto

Antisense oligonucleotides (ASOs) are nucleic acid therapeutics that regulate gene expression through sequence-specific hybridization with target RNA. Under physiological conditions, many target RNAs adopt higher-order structures, which can strongly influence ASO accessibility and binding behavior. Although UV melting analysis is widely used to evaluate the thermal stability of ASO/RNA duplexes, this approach does not adequately account for the structural features of target RNAs. In this study, we investigated the utility of surface plasmon resonance (SPR) analysis as an in vitro method to evaluate ASO binding while considering RNA structural constraints. Multiple ASOs were designed to target PRF84, an 84-nucleotide RNA motif that induces −1 programmed ribosomal frameshifting in HIV-1 gag-pol expression. SPR analyses were performed to compare ASO interactions with complementary RNA fragments and with structurally folded PRF84. The results demonstrated that identical ASOs exhibited distinct binding behaviors depending on whether the target was a complementary RNA or PRF84, indicating that RNA structure significantly affects ASO binding. These findings suggest that SPR analysis enables the evaluation of ASO–RNA interactions taking structure into account, and may be a useful alternative approach to conventional UV melting analysis-based ASO screening.

Methods and Protocols doi: 10.3390/mps9020047

Authors: Licínio Manco Helena Correia Dias Lara Palmeira

DNA methylation (DNAm) age estimation is one of the hottest topics in forensic contexts. However, there is growing evidence that DNAm can be affected by several factors, including many clinical conditions. In this study, we analyzed the methylation levels within the FHL2 gene in Portuguese women using the droplet digital PCR (ddPCR) methodology to develop age prediction models (APMs). We hypothesized that obesity could affect the accuracy of APMs and would be associated with the advancement in epigenetic aging. We collected blood samples from 62 women (aged 21–58 years old) with overweight and obesity. DNA extracts were subjected to bisulfite conversion followed by ddPCR using dual-labeled probes targeting the methylated and unmethylated FHL2 CpG site cg06639320. The developed APM yielded a mean absolute deviation (MAD) of 4.72 years between predicted and chronological ages in the total sample. When applying the developed APM to women classified as overweight, the MAD was 3.64 years, while, for those with obesity class 1, it was 3.93 years, and, for those with obesity class 2, 6.29 years. The same pattern of accuracy was observed when we developed APMs specifically for the groups categorized by overweight and obesity, obtaining MAD values of 3.75 years (overweight), 3.69 years (obesity class 1) and 6.24 years (obesity class 2). Our study indicates that severe obesity may impact the accuracy of DNA methylation-based age estimators. We did not find evidence of an association between BMI and accelerated epigenetic aging. However, we found signals of epigenetic age acceleration in younger subjects and epigenetic age deceleration in the older participants.

Methods and Protocols doi: 10.3390/mps9020046

Authors: Subhadeep Das Sagar M. Utturkar Roshnee Bose Elizabeth J. Tran

Small cell lung cancer (SCLC) is an aggressive malignancy characterized by rapid progression, early metastasis, and high relapse rates due to acquired chemoresistance. The human DEAD-box RNA helicase DDX5 is overexpressed in SCLC and has recently gained attention as a viable therapeutic target. Supinoxin (RX-5902), a selective small-molecule inhibitor of DDX5, exhibits strong anti-tumor activity. Recent evidence suggests that its cytotoxic effects are mediated through the disruption of mitochondrial respiration. In this study, transcriptomic profiling via RNA sequencing (RNA-seq) revealed significant downregulation of genes involved in cellular respiration following Supinoxin treatment and DDX5 knockdown in chemoresistant H69AR cells. To functionally validate these findings, we employed the Seahorse XF Cell Mito Stress Test, which measures key parameters of mitochondrial bioenergetics through oxygen consumption rate (OCR) analysis. Supinoxin-treated cells exhibited marked mitochondrial dysfunction, supporting the hypothesis that DDX5 inhibition disrupts cellular energy metabolism. These findings illuminate a previously underappreciated role of DDX5 in mitochondrial regulation and offer mechanistic insights into Supinoxin’s cytotoxic effects, underscoring its potential as a targeted therapy in SCLC.

Methods and Protocols doi: 10.3390/mps9020045

Authors: Marc Follmer Korbinian Pürckhauer Klaus Neuhaus

Ribosome profiling, or Ribo-Seq, is a powerful tool for studying translation. It maps the positions of translating ribosomes on mRNAs, providing insights into actively expressed genes. Unlike mass spectrometry, Ribo-Seq is not affected by the same biases that limit mass spectrometry, such as protein size, concentration, trypsin digestibility, or hydrophobicity. Thus, the translatome has previously been used to discover unannotated genes, including small and overlapping ones that were missed by mass spectrometry or gene prediction models. However, a major limitation of classical ribosome profiling is its complexity, involving multiple steps such as sucrose density gradient centrifugation and gel electrophoresis. These make the method costly, time-consuming, and limit its throughput. Here, we compared the classical method using gradient centrifugation and size exclusion by gel electrophoresis with shortened versions to evaluate experimental performance and achieved reductions. Our results show that the sucrose density gradient centrifugation is essential for obtaining accurate Ribo-Seq data, whereas gel electrophoresis for size selection can be omitted (although this requires increased sequencing depth). Thus, future experiments can be conducted with reduced sample input and hands-on time while still achieving a reliable quantification of translation.

Methods and Protocols doi: 10.3390/mps9020044

Authors: Pál Szarvas Judit Csabai Anzhela Kolesnyk Judit Dobránszki

Acmella oleracea (L.) R.K. Jansen is an herbaceous plant cultivated globally as an annual ornamental species. While conventional propagation methods exist, the lack of a standardized in vitro protocol limits research and industrial applications that require genetically and morphologically uniform plant material. In this study, in vitro cultures of A. oleracea were established via seed germination. Well-developed in vitro shoots were dissected into individual nodal segments to serve as explants. Multiple media were evaluated for regeneration and growth, including full-, half, and quarter-strength Murashige and Skoog (MS) media, as well as full- and half-strength McCown Woody Plant media. Two carbohydrate sources, saccharose and glucose, were tested at concentrations of 1%, 2%, and 3% (w/v) in the multiplication medium. Subsequently, the effects of different cytokinins were assessed at concentrations of 4.4 µM and 13.2 µM. The findings demonstrated that 13.2 µM meta-Topolin with 3% saccharose, or 13.2 µM Benzyladenine with 2% glucose was most beneficial for shoot multiplication of A. oleracea. The multiplied shoots were rooted in vitro within 13 days, then potted and acclimatized within 8 days. This protocol facilitates future industrial applications of A. oleracea, particularly in the cosmetics sector, where the use of standardized biomass is essential.

Methods and Protocols doi: 10.3390/mps9020043

Authors: Thereza Piloya-Were Catherine Nyangabyaki Elizabeth Pappenfus Expeditus Ahimbisibwe Ezrah Trevor Rwakinanga Lin Zhang Silver Bahendeka Antoinette Moran

Metabolic control is poor in East Africa for youth with type1 diabetes (T1D). Self-monitoring of blood glucose (SMBG) by fingerstick 2–3 times daily is routine care. This randomized controlled trial (RCT) will test the hypothesis that providing continuous glucose monitoring (CGM) to Ugandan youth with T1D will improve glucose time-in-range (TIR glucose 3.9–10.0 mmol/L) and be cost effective in this setting. Ugandan youth with T1D (n = 180, age 4–26 years) will be divided into four 12-month cohorts (August 2022–August 2027). Half will receive unblinded Freestyle Libre 2 Flash CGM for 12 months. For six months, control subjects received sufficient test strips for SMBG three times daily while wearing blinded Freestyle Libre Pro CGM (for endpoint assessment), and then they switch to unblinded CGM for six months. Everyone receives monthly diabetes education. The primary endpoints are as follows: (1) the six-month change from baseline in glucose TIR, unblinded CGM versus SMBG; (2) a cost analysis of CGM versus SMBG. The TIR hypothesis will be tested by linear mixed effects models. Cost analysis assumptions include direct material and indirect costs like hospitalizations, missed school/work, and diabetes complications. The study will inform T1D management guidelines in a low resource setting using evidence-based recommendations.

Methods and Protocols doi: 10.3390/mps9020042

Authors: Elena Di Nisio Chiara Frigerio Valerio Licursi Sara Castelli Benedetta Caraba Rodolfo Negri Michela Clerici

Eukaryotic DNA is wrapped around octamers of four core histones, forming nucleosomes. Histone post-translational modifications (PTMs) influence chromatin structure and the recruitment of regulatory factors, thereby affecting gene expression and DNA repair, including the response to DNA double-strand breaks (DSBs). Here, we describe a robust chromatin immunoprecipitation protocol combined with micrococcal nuclease digestion and DNA sequencing (MNase-ChIP-seq) to map histone modifications and their genome-wide distribution after the induction of a single DSB by the HO endonuclease in Saccharomyces cerevisiae. We validate the method by detecting changes in histone H3 methylation following HO transcriptional activation and DSB induction. This protocol enables reliable analysis of histone PTMs across mutant strains or stress conditions, supporting studies of chromatin dynamics in yeast.

Methods and Protocols doi: 10.3390/mps9020041

Authors: Anna Dziubina Dominika Szkatuła Małgorzata Szafarz Agata Siwek Marek Kowalski Joanna Gdula-Argasińska

The present study was to evaluate the analgesic activity of two newly synthesized 1H-pyrrolo[3,4-c]pyridine-1,3(2H)-dione derivatives, designated DSZ-13 and DSZ-19. To achieve the desired result, the in vitro XTT cell proliferation assay, serotonin 5-HT1A receptor affinity and COX-1 and COX-2 enzyme inhibition potential of the compounds were conducted by real-time qPCR. Non-compartmental analysis was used to estimate the pharmacokinetic parameters of the compounds in serum and brain tissue. The analgesic activity was evaluated using various in vivo pain models, encompassing acute pain (hot plate test), tonic pain (formalin test), neurogenic pain (capsaicin test), carrageenan-induced acute inflammation, and neuropathic pain models. Both compounds showed moderate affinity for serotonin 5-HT1A receptors, a lack of cytotoxic activity, desirable pharmacokinetic parameters and slightly reduced mRNA expression for COX-1 and COX-2. Only the DSZ-19 revealed central/supraspinal analgesic activity and did not affect movement. Both compounds attenuated tonic and neurogenic pain, in the formalin and capsaicin tests, respectively. In addition, the involvement of the 5-HT1A receptors in the formalin test was confirmed. Both compounds also showed antiallodynic activity in the oxaliplatin- and streptozotocin-induced neuropathy models. Slightly weaker than indomethacin, DSZ-13 and DSZ-19 attenuated carrageenan-induced inflammation (edema) and hyperalgesia in rat models.

Methods and Protocols doi: 10.3390/mps9020040

Authors: Jamie Cooper Scott Tait Airey Eric Patino Theo Andriot Mousumi Ghosh Damien D. Pearse

Reliable characterization of circulating extracellular vesicles (EVs) in rodents may be significantly influenced by how blood is collected, yet systematic comparisons of commonly used sampling methods remain limited. Here, we directly evaluate the effects of cardiac puncture and perfusate blood collection on EV yield and surface-marker profiles in naïve mice, as well as in mice subjected to neurotrauma using a contusion spinal cord injury (SCI) model. Using matched isolation procedures and MACSPlex immunophenotyping, we analyzed newly generated cardiac puncture plasma alongside previously published perfusate-derived datasets, with both cohorts matched for age, sex, weight, injury severity, and post-injury timepoint. Cardiac puncture produced substantially higher particle concentrations due to access to undiluted blood, whereas perfusate samples exhibited modest increases in select markers, such as CD9, consistent with method-associated influences on platelet-derived vesicles. Despite these quantitative differences, both approaches yielded broadly similar EV phenotypes, and SCI-associated marker patterns remained stable across sampling methods. The consistency between cardiac puncture and perfusate datasets validates the robustness of our earlier perfusate-based findings and demonstrates that key biological signatures are preserved regardless of collection technique. These results provide practical guidance for optimizing murine EV studies and underscore the importance of methodological transparency and standardization in preclinical EV research.

Methods and Protocols doi: 10.3390/mps9020039

Authors: Mathilde Hoareau Grégorie Lebeau Luce Muzi Jérémy Fontaine Pascale Krejbich-Trotot Olivier Meilhac Christine Robert-Da Silva Wildriss Viranaicken

During ischemia, endothelial cell integrity is compromised, as a consequence, blood barrier homeostasis is disrupted. Therefore, the structural and functional preservation of endothelial cells is paramount when trying to improve outcomes after ischemic injury. Endoplasmic reticulum (ER) stress is increasingly recognized as a key player in ischemic injury through unfolded protein response (UPR) signalling, and its crosstalk with mitochondrial death pathways. This study provides a cost-effective and straightforward method to delve into the relationship between ER stress and ischemia in human microvascular endothelial cells-1 (HMEC-1). HMEC-1 was exposed to 8 h of oxygen–glucose deprivation (OGD) in glucose-free medium with rapidly induced hypoxia. Hypoxia, oxygen consumption, cell viability, apoptosis, and ER stress markers (BiP/GRP78, PERK, ATF6, IRE1/XBP1s, CHOP) were assessed by RT-qPCR and Western blot. Cell viability decreased by approximately 33% following OGD, while CHOP expression increased ~4-fold, indicating significant ER stress induction. The model enables quantification of metabolic stress (OCR), as well as evaluation of viability loss, membrane integrity, apoptotic commitment, and discrimination between ER stress resolution versus maladaptation. Overall, GasPak EZ Pouch Systems provide a reproducible and practical in vitro platform to study ischemic injury down to the mechanistic details of ER-mitochondria signalling. They give the opportunity to evaluate therapeutic approaches that target ER homeostasis to limit apoptosis and/or recovery of metabolic function after ischemia. This method could allow rapid screening of ER stress-modulating interventions aimed at preserving endothelial barrier function, in various ischemic contexts.

Methods and Protocols doi: 10.3390/mps9020038

Authors: Sivananthan Manoharan

The process of developing a drug is complex and involves many steps, from basic research (bench) to patient applications (bedside), which are conducted to ensure the drug is both safe and effective. In cancer research, the failure rate is high when translating basic findings to clinical trials. One of the main factors probably contributing to high failure rates is the basic quality of in vitro and in vivo study designs. Advanced basic cancer research techniques, including various types of 3D cell culture, the use of valuable organoids, organs, or tumors on chips, traditional or automated Western blots, omics research, advanced imaging techniques, usage of cutting-edge preclinical models and others, may produce inaccurate results for translational research if the basic study design is not carefully planned, especially when drugs or compounds are involved. In this manuscript, the author discussed (i) the importance of understanding and applying pharmacokinetic data in basic research, (ii) a proper comparison of the efficacy and safety of investigational drugs with the standard of care, (iii) the importance of following the actual route of drug administration as experienced by patients, the cruciality of human-to-animal dose conversion, and dose frequencies in animal models, (iv) significance of the age, gender, and strain of mice, along with adherence to the ARRIVE guidelines for ensuring transparency in conducting and reporting preclinical research, (v) benefits of having both subcutaneous and metastasis models in preclinical studies, (vi) the impact of comorbidities and related cancer drug studies in animal models and (vii) the importance of testing drug candidate/s in model mimicking acidic tumor microenvironment.

Methods and Protocols doi: 10.3390/mps9020037

Authors: Aglaia Zafeiroudi Ioannis Tsartsapakis Charilaos Kouthouris

Recreational dance offers significant psychological well-being potential. However, traditional instruction emphasizes technique while limiting attention to nervous system development and embodied meaning-making. Despite empirical support for polyvagal theory, motor learning science, somatic education, and phenomenology, their systematic integration into unified structures is not clearly established in recreational dance contexts. This protocol integrates nervous system regulation, motor learning, and creative expression within structured Imperial Society of Teachers of Dancing (ISTD) modern dance syllabus for recreational adults. It presents a 12-week integrated dance-mindfulness intervention addressing this gap through a three-phase structure grounded in neuroscience and embodied pedagogy. The intervention comprises eight standardized components delivered weekly. The randomized controlled trial evaluates intervention effects using the Satisfaction With Life Scale (SWLS), Depression Anxiety Stress Scales-21 (DASS-21), the Mindful Attention Awareness Scale (MAAS), the Subjective Happiness Scale (SHS), and the Leisure Involvement Scale (LIS). Qualitative assessment via semi-structured phenomenological interviews (Weeks 8 and 12) and weekly journaling captures somatic awareness, nervous system resilience, technical confidence, creative expression, relational and social belonging, and embodied meaning-making. Intervention participants are expected to show significantly greater improvements compared to controls. Results will establish evidence-based practice standards for recreational dance and demonstrate neuroscience integration’s efficacy for psychological wellbeing and embodied meaning-making.

Methods and Protocols doi: 10.3390/mps9020036

Authors: Weiqi Li Yongjie Wang Borui Qi Lin Yang Xin Ma Xinyu Dang Yayin Qi Shilei Zhang

This study aimed to establish an indirect ELISA for detecting the avian reovirus (ARV) epidemic strain xj-1.1 by using the purified recombinant protein pET-σC as the coating antigen. To optimize assay performance, key parameters were systematically evaluated, including antigen-coating concentration, serum dilution, blocking reagent and duration, serum incubation time, and the dilution and reaction time of the HRP-conjugated secondary antibody. The optimized conditions identified were a coating antigen dilution of 1:100, serum dilution of 1:1600, coating at 37 °C for 1 h followed by overnight incubation at 4 °C, and blocking with 5% skim milk for 2 h. The optimal serum incubation time was 1.5 h, with the secondary antibody diluted 1:1000 and incubated for 2 h, followed by a 20-min color development step. The cut-off value for distinguishing positive and negative samples was determined to be 0.121. Validation of the assay demonstrated favorable specificity, sensitivity, and repeatability, indicating that the developed indirect ELISA provides a reliable method for detecting ARV xj-1.1 infection.

Methods and Protocols doi: 10.3390/mps9020035

Authors: Borislav Arabadjiev Ivelina Vassileva Georgi Nikolaev Roumen Pankov

The differentiation of human embryonic stem cells (hESCs) into primordial germ cell-like cells (PGC-LCs) provides a robust in vitro model to study human germline specification. Here, we present a simple, reproducible, and cost-effective protocol for generating DEAD-box helicase 4 (DDX4)/VASA and Deleted in Azoospermia-Like (DAZL)-positive PGC-LCs from hESCs using a combination of bone morphogenetic protein 4 (BMP4) and conditioned medium (CM) derived from Stage-Specific Embryonic Antigen-4 (SSEA4)-positive human amniotic fluid stem cells (hAFSC-4). Importantly, unlike conventional protocols that rely on embryoid body formation, our method employs adherent cultures for germ cell differentiation. This approach enhances reproducibility by avoiding the spontaneous and stochastic variability inherent to embryoid body formation. This protocol provides a reproducible and physiologically relevant platform for studying human germ cell development in vitro.

Methods and Protocols doi: 10.3390/mps9020034

Authors: Bilgehan A. Geylani Stephen M. Parris Jhulia Gelain Guido Schnabel Ksenija Gasic

Pseudomonas syringae pv. syringae (Pss) is the causal agent of bacterial canker, a disease that can result in yield losses, aerial tissue damage, and tree mortality in stone fruits worldwide. Peach, one of the major stone fruit crops, experiences significant yield losses and tree mortality attributed to bacterial canker in the United States. As the second-largest peach-producing state, South Carolina faces direct and significant impacts due to Pss. Early evaluations of peach scion responses to Pss infection have relied primarily on circumstantial field observations in rootstock trials. Although laboratory evaluations in peach have been reported, these studies primarily focused on pathogen virulence testing or small accession sets and did not establish a standardized, scalable detached twig protocol for systematic germplasm phenotyping. The absence of a clearly described laboratory assay has limited reproducible and large-scale evaluation of bacterial canker tolerance in peach. To address this gap, a detached dormant twig assay, previously developed for cherry, was adapted and optimized for peach. Dormant shoots from nine peach accessions were cut into 10 cm segments, surface-sterilized, and inoculated with a Pss suspension prepared in 10 mM MgCl2 buffer or with the buffer alone. After six weeks of incubation, inner bark lesion size was evaluated visually and quantified using ImageJ. A newly developed visual rating scale was established and compared with quantitative lesion measurements. Spearman correlation analysis showed strong positive correlations between visual disease scores and ImageJ-based lesion measurements across two independent replicates (ρ = 0.80–1.00, p < 0.01), while shoot segment diameter showed weak-to-moderate negative correlations with disease severity. This adapted and consolidated dormant twig assay provides a practical, reproducible, and scalable method for phenotyping bacterial canker tolerance in peach and supports future germplasm screening and breeding efforts.

Methods and Protocols doi: 10.3390/mps9020033

Authors: Jackson Spieser Ali Balapour Jarek Meller Krushna C. Patra Behrouz Shamsaei

This review evaluates the emerging paradigm of multi-agent systems (MASs) for biomedical and clinical data analysis, focusing on their ability to overcome the reasoning and reliability limitations of standalone large language models (LLMs). We synthesize findings from recent architectural frameworks, specifically LangGraph, CrewAI, and the Model Context Protocol (MCP), to examine how specialized agent teams divide labor, utilize precision tools, and cross-verify outputs. We find that MAS architectures yield significant performance gains in various domains: recent implementations improved oncology decision-making accuracy from 30.3% to 87.2% and reached a peak of 93.2% accuracy on USMLE-style benchmarks through simulated clinical evolution. In clinical trial matching, multi-agent frameworks achieved 87.3% accuracy and enhanced clinician screening efficiency by 42.6% (p < 0.001). However, we also highlight critical operational challenges, including an unreliability tax of 15–50× higher token consumption compared to standalone models and the risk of cascading errors where initial hallucinations are amplified across the agent collective. We conclude that while MAS enables a shift toward collaborative intelligence in biomedicine, its clinical and research adoption requires the development of deterministic orchestration and rigorous cost-utility frameworks to ensure safety and expert-centered oversight.

Methods and Protocols doi: 10.3390/mps9020032

Authors: Stylianos Syropoulos Maria Moutzouri Eirini Grammatopoulou Irini Patsaki

Gastroesophageal reflux disease (GERD) is a common chronic condition mainly caused by the dysfunction of the antireflux mechanism at the gastroesophageal junction. This is composed of the lower esophageal sphincter and the crural diaphragm. Increasing evidence suggests that diaphragmatic dysfunction and reduced inspiratory muscle strength may contribute to the persistence of GERD symptoms. Although respiratory physiotherapy has shown beneficial effects, the role of a structured inspiratory muscle training (IMT) program has not been sufficiently examined. This study aims to investigate the effects of an inspiratory muscle training program on inspiratory muscle strength and secondary clinical outcomes in individuals with GERD. A total of thirty adults with a confirmed GERD diagnosis will be enrolled in a two-arm randomized controlled trial. These volunteers will be randomly assigned either to the experimental group, which will undergo a 3-month inspiratory muscle training (IMT) using tapered flow resistive loading at 40% of maximal inspiratory pressure (MIP), or to the control group, which will receive sham IMT with a consistent low resistance. Primary outcomes will include maximal inspiratory pressure (MIP) and maximal dynamic inspiratory pressure (S-index). Secondary outcomes will assess GERD symptoms, disease-related quality of life, and pulmonary function. Measurements will be performed at baseline, at three months of intervention, and at six months from recruitment (follow-up). IMT is expected to lead to significant improvements in inspiratory muscle strength, symptom burden, and quality of life compared with sham training. This trial will provide novel evidence regarding the role of inspiratory muscle training as a non-pharmacological intervention in the management of GERD. Trial registration: ClinicalTrials.gov Identifier: NCT07131397.

Methods and Protocols doi: 10.3390/mps9020031

Authors: Chantal Wunderlin Flavia Bürgisser Armin Gemperli Claudio Perret Mario Widmer

Robot-assisted therapy (RT) is increasingly implemented in rehabilitation, yet evidence on its effectiveness in improving upper extremity function after cervical spinal cord injury (cSCI) remains limited. Therefore, this randomised crossover study aims to investigate the effects of unilateral RT compared to conventional unilateral occupational therapy (OT) on upper extremity function in individuals with cSCI. 40 participants with traumatic or non-traumatic cSCI (16–81 days post-injury, neurological level of injury: C1–T1) will be randomised (1:1), stratified by their predicted recovery profile, to receive 6 weeks of RT (ArmeoSpring) and 6 weeks of OT in random order, each 3 × 30 min/week in addition to the clinical routine therapy. Assessments are conducted before (t0), between (t1) and after both intervention blocks (t2 and t3). The primary outcome is the Quantitative Grasping Subtest of the Graded Redefined Assessment of Strength, Sensibility, and Prehension (GRASSP-QtG); primary analysis uses a linear mixed model to estimate the treatment effect based on change scores. Recruitment is currently ongoing. This randomised crossover study allows the collection of a comprehensive dataset to generate knowledge about treatment effectiveness, enabling future individuals with cSCI to benefit from improved and individualised therapy schedules.

Methods and Protocols doi: 10.3390/mps9010030

Authors: Masao Yoshida Waku Hatta Tomohiro Nakamura Naoki Nakaya Satoki Shichijo Yasuyuki Tanaka Hiromitsu Kanzaki Kingo Hirasawa Ichiro Oda Takashi Hirose Motohiko Kato Kohei Takizawa Yosuke Toya Takuto Hikichi Hiroaki Sawai Naohiro Yoshida Osamu Dohi Atsushi Masamune Seiichiro Abe Tomonori Yano

Background: Underwater endoscopic mucosal resection (UEMR) is a relatively new treatment method for sporadic non-ampullary duodenal epithelial tumors (SNADETs), and its usefulness has been reported for SNADETs ≤ 20 mm. However, its effectiveness and safety compared with conventional endoscopic mucosal resection (CEMR) remain controversial. This study aims to assess the treatment outcomes and evaluate the beneficial effects and safety of UEMR for SNADETs ≤ 20 mm. Methods: This is an open-label, multicenter collaborative, non-inferiority randomized controlled trial with two parallel groups conducted across 40 institutions in Japan. The study subjects will be patients with SNADETs ≤ 20 mm. A total of 320 patients will be randomized to either the CEMR or UEMR group in a 1:1 allocation ratio. The primary endpoint is the 1-year recurrence-free survival rate, defined as the number of cases with no recurrence of SNADET or death from any cause within one year of EMR. The secondary endpoints include the en bloc resection rate, histological complete resection rate, adverse events, technical success rate, total procedure time, resection time, mucosal closure time, complete mucosal closure rate, complete mucosal closure rate with standard clips, 1-year duodenum preservation survival rate, and device cost. Discussion: This multicenter, open-label, randomized controlled trial (RCT) with 320 subjects aims to determine whether the 1-year recurrence-free survival rate of underwater endoscopic resection is not inferior to that of conventional endoscopic mucosal resection for SNADETs ≤ 20 mm. If the efficacy and safety of UEMR are proven in this RCT, it is expected to be recognized as the standard treatment for SNADETs ≤ 20 mm. Owing to the absence of submucosal injection, UEMR is a simpler and more cost-effective technique compared to CEMR and is anticipated to become the primary method of EMR.

Methods and Protocols doi: 10.3390/mps9010029

Authors: Wathuto Ogopotse Valentine Musabyimana Pamela M. Khasandi Dennis Kotti Maina Ngotho John M. Kagira George O. Oluoch

Caprylic acid (CA) fractionation of serum is a simple and cost-effective method of producing high-quality immunoglobulins. While standardised procedures exist for CA purification of IgG for various animals, no published protocol exists for baboon IgG. This study aimed to optimise an efficient protocol for purifying IgG from baboon serum using CA through a stepwise one-factor-at-a-time (OFAT) approach. The effects of serum pH, CA concentration, stirring time and intensity, dialysis buffer, and lyophilisation were evaluated based on the protein content, with SDS-PAGE profiles and albumin–globulin ratios distinguishing IgG from residual albumin. Serum at pH 5.0 with 7% CA (v/v) produced the highest yield, minimising albumin while maximising IgG content. Lower pH (4.0–4.5) and CA (5–6%) reduced protein content, while a higher pH (5.5–6.0) and CA (8–15%) increased protein, but with elevated albumin and contaminants. Stirring serum vigorously at 1200 rpm for 60 min provided effective precipitation of non-IgG proteins. Lower intensities and shorter times resulted in higher albumin and residual proteins, while excessive stirring caused protein denaturation. Dialysis buffer composition had little impact, while lyophilisation significantly enhanced IgG concentration. The optimal protocol involved serum at pH 5.0, 7% CA (v/v), vigorous stirring (1200 rpm) for 60 min, and dialysis against sodium phosphate buffer (pH 7.4) followed by lyophilisation. The resulting IgG enrichment and purity were comparable to commercial-grade products. This study thus established optimal conditions for the purification of baboon IgG with CA, which could be used to support research in this animal model of immunology.

Methods and Protocols doi: 10.3390/mps9010028

Authors: Kejin Hu

High-throughput sequencing generally results in a list of genes. Which functional groups of genes among the DEGs are meaningful underlying factors to the differential biological/biomedical conditions under investigation? The process to find answers to this question can be called biological functional class enrichment analysis (FunCEA). R is a robust platform for FunCEA due to its accessibility by general users and availability of well-developed R packages for enrichment analysis and visualization, as well as for knowledge databases. Bench scientists in biomedical sciences need accessible and easy-to-understand protocols for FunCEA. This R tutorial provides detailed R scripts or command lines for FunCEA, as well as for data processing and visualization of the enrichment results. It keeps bench scientists in mind and provides supportive and apprehensible descriptions of the R scripts for each task (enrichment analysis, enrichment data processing, and visualization). It describes detailed procedures for the two popular FunCEA methods, the so-called over-representation analysis (ORA) and functional class scoring (FCS). The introduced FunCEA here uses three basic knowledge databases: gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and reactome. R codes for various visualizations (dot plot, term-gene network plot, enrichment map plot, ridge plot, and GSEA plot) are presented and annotated. Since all analyses are conducted in R, no commercial software is needed, yet clusterProfiler can directly access the latest KEGG knowledge database.

Methods and Protocols doi: 10.3390/mps9010027

Authors: Karthika Muthuraj Iwona Scheliga Dunja M. Baston-Buest Jana Bender-Liebenthron Jan-Steffen Kruessel Alexandra P. Bielfeld

Three-dimensional (3D) cell culture models provide physiologically relevant systems that mimic the native endometrial environment better than 2D models and offer reliable platforms to study embryo implantation and maternal–embryo interactions. One widely used 3D culture model is the generation of spheroids. However, standardized and reproducible methods for generating uniform spheroids from trophoblast and endometrial stromal cells are limited. In this study, we established and validated a robust protocol for spheroid formation using human trophoblast (HTR8/SVneo, JEG3) and endometrial stromal (St-T1b, tHESC) cell lines. The protocol was further extended to generate spheroids from decidualized tHESC, representing a novel approach that closely reflects the receptive endometrial environment. Key parameters, including cell concentration and methyl cellulose supplementation, were optimized to produce compact and homogeneous spheroids. Spheroid formation was monitored at defined intervals (0, 8, 24, 32, and 48 h), and decidualized spheroids were assessed up to 72 h. Long-term cryopreservation over 11 months demonstrated high post-thaw viability across all spheroid types, as confirmed by Calcein-AM staining. This standardized workflow provides a reliable 3D model incorporating hormonally primed stromal cells and offers a practical platform to investigate the mechanisms underlying normal and trophoblast invasion in vitro.

Methods and Protocols doi: 10.3390/mps9010026

Authors: Mayu Enomoto-Kusano Kyoko Furuita Takashi S. Kodama Chojiro Kojima

Liquid–liquid phase separation (LLPS) underlies the formation of membraneless cellular compartments, yet experimental strategies that directly connect quantitative LLPS behavior with residue-level structural information remain limited. Here, we present an integrated protocol that combines quantitative LLPS assays with nuclear magnetic resonance (NMR) spectroscopy and structure-guided mutagenesis to regulate protein phase separation. Using the VAPB MSP domain as a representative example, this workflow links residue-specific structural features to macroscopic LLPS behavior and enables suppression or enhancement of phase separation through targeted amino acid substitutions. This protocol provides a generalizable framework for systematic, residue-level regulation of protein LLPS.

Methods and Protocols doi: 10.3390/mps9010025

Authors: Eva Vargas Inés de la Torre Francisco J. Esteban

We describe a comprehensive methodology for the application of game theory to omics data analysis, with a particular focus on coalitional games and Shapley values. This approach evaluates the cooperative distribution of genes within high-dimensional transcriptomics datasets, providing a complementary perspective to conventional statistical methods. We present the mathematical framework, implementation details, and references for applications that demonstrate its ability to improve the detection of biologically meaningful signals that may not be explicitly modeled by many conventional statistical methods. Our results highlight the potential of coalitional game theory as a powerful tool for enhancing reproducibility and interpretability in omics research, opening new perspectives in systems biology and precision medicine.

Methods and Protocols doi: 10.3390/mps9010024

Authors: Elías Hernández-Cruz Lorena Jacqueline Gómez-Godínez José Martín Ruvalcaba-Gómez Ramón Ignacio Arteaga-Garibay

Soil harbors the highest concentration of microorganisms in ecosystems, and their molecular characterization through high-throughput sequencing is essential for metagenomic studies. However, obtaining high-quality, high-concentration DNA is limited by physicochemical properties (pH, heavy metals, humic acids) and adsorption to clay minerals. Although standardized commercial protocols exist, they present variable limitations depending on soil type. This study developed and validated the National Center for Genetic Resources—Microorganism Collection (CNRG-CM) method, which incorporates innovative pre-washing steps using phosphate-buffered saline (PBS) and sodium phosphate to effectively remove inhibitory humic acids and metal ions, combined with cetyltrimethylammonium bromide (CTAB)/chloroform extraction to achieve high-molecular-weight metagenomic DNA isolation. The CNRG-CM method was applied to three diverse soil types with variable physicochemical properties, recovering DNA concentrations ranging from 1000 to 1300 ng/μL ith a yield of 30 to 48 µg/g−1, significantly exceeding those obtained with a standard commercial kit with maximum DNA concentrations of 360 ng/μL and a yield of 43 µg/g−1. The CNRG-CM protocol is established as an effective and adaptable alternative for metagenomic DNA extraction across diverse agricultural and ecological contexts. It enables subsequent metagenomic studies of soil microbial communities.

Methods and Protocols doi: 10.3390/mps9010023

Authors: Skyelar Reuter Peter Geevarghese Alex Casey Weisfuss Britt Burton-Freeman Indika Edirisinghe

Postprandial inflammation, characterized by elevated cytokines, is linked to metabolic diseases. Polyphenol-rich fruits like strawberries possess anti-inflammatory properties that may help reduce this response. Because clinical studies are often costly and time-consuming, this study aimed to develop an in vitro model using whole blood to examine the effect of bioactive components such as polyphenols on postprandial inflammation. Whole blood from healthy adults was exposed to lipopolysaccharides (LPS) and high glucose concentrations (250/500 mg/dL), as well as strawberry extract (100 ng/mL). Cytokines (interleukin-6 and -1 beta; IL-6, IL-1β, and Tumor Necrosis Factor-alpha; TNF-α) were quantified using the Luminex multiplex assay. High glucose levels caused non-significant increases in IL-6 (p > 0.05), while strawberry extracts significantly reduced LPS-induced cytokines (p < 0.05). These findings demonstrate the potential of using whole blood in vitro systems to model inflammation and to explore the anti-inflammatory effects of fruit components such as polyphenols from strawberries.

Methods and Protocols doi: 10.3390/mps9010022

Authors: Ekaterina Sergeevna Arshinova Nataliia Sergeevna Karpova Olga Leonidovna Terekhina Malik Nurbekov Maria Ivanovna Burtovskaya

Telomere length is a crucial marker of cellular aging and genomic stability, with significant implications for age-related diseases and cancers. This study introduces an improved quantitative PCR (qPCR) method for measuring absolute telomere length, addressing the need for accurate and high-throughput assessment in both clinical and research settings. Novel primers were designed for the single-copy gene interferon beta (IFNB1) to serve as an internal control, alongside a series of single-stranded oligonucleotide standards to establish a calibration curve. This approach allows for precise quantification of telomere length in kilobases per single copy gene copy number per chromosome. We validated this method using DNA samples from peripheral blood and buccal swabs from 17 healthy human volunteers, as well as umbilical cord blood from 9 healthy newborn babies, demonstrating its high linearity and reproducibility. Our findings indicate that this improved qPCR technique provides a rapid, cost-effective, and accurate means of measuring absolute telomere length, thereby facilitating large-scale studies and enhancing clinical diagnostics related to telomere biology.

Methods and Protocols doi: 10.3390/mps9010021

Authors: Pius N. Amartey Jocelyn S. Kim Yetunde I. Kayode Glenn E. Simmons

Three-dimensional (3D) culture systems that recapitulate the tumor microenvironment are essential for studying cancer cell behavior, drug response, and cell–matrix interactions. Here, we present a detailed protocol for generating 3D spheroid cultures from murine breast cancer cells using methacrylated gelatin (GelMA)-based bioink and a CELLINK BioX bioprinter. This method enables precise deposition of spheroid-laden GelMA droplets into low-attachment plates, facilitating high-throughput and reproducible 3D culture formation. The protocol includes steps for spheroid formation, GelMA preparation, bioprinting, and post-printing analysis using a customized CellProfiler pipeline. The analysis pipeline takes advantage of the functionality of CellProfiler and ImageJ software (version 2.14.0) packages to create a versatile and accessible analysis tool. This approach provides a robust and adaptable platform for in vitro cancer research, including studies of metastasis, drug resistance, cancer cell lipid metabolism, and TME-associated hypoxia.

Methods and Protocols doi: 10.3390/mps9010020

Authors: Dantong Huang Kaewbucha Manus Apiwathnasorn Chalisa Tianyi Liu Chuyun Yan Yumeng Gu

Rosin, a renewable natural resin derived from pine trees, is a promising biomass material for sustainable product development, though its distinct intrinsic odor limits broader use. This study implemented a comprehensive analytical strategy to mitigate the odor by incorporating essential oils (EOs)—eucalyptus (EUC) and peppermint (MINT)—and to conduct a multi-analytical characterization of the modified rosin jewelry. By integrating complementary analytical techniques, including LC-Q/TOF-MS for non-volatile components and GC-Q/TOF-MS for volatile organic compounds (VOCs), we achieved a systematic chemical profiling of the materials. The core composition of rosin, dominated by abietic acid (>48%), remained stable across all samples. The incorporation of EOs significantly altered the VOC profiles: The total VOC signal (summed peak area) in MINT-modified rosin was 2.57-fold that of the EUC-modified sample, with monoterpenoids comprising 87.62% of its VOC signature. Eucalyptol and limonene were tentatively identified as the major components in the EUC sample, whereas menthone, menthol, and limonene predominated in the MINT sample. Multivariate statistical analysis highlighted that variations in specific VOCs—particularly menthone, menthol, eucalyptol, and allo-ocimene—were closely associated with differences in the scent profiles of each modification. This work illustrates how a multi-technique analytical strategy can both guide and assess the functional modification of sustainable biomass materials. The findings offer a practical approach to improving rosin’s functional properties while providing a methodological framework for the integrated characterization of complex biomaterials, supporting the development of eco-friendly products aligned with green chemistry and sustainable design principles.

Methods and Protocols doi: 10.3390/mps9010019

Authors: Nina Handzewniak Abid Mahmood Canan Metin Shahnawaz Khan Tanvir Khan Henry Atkinson

Introduction: Management of trochanteric fractures in revision hip surgery has a high incidence of non-union and complications. Fixation devices are often bulky, prone to breakage, and necessitate reoperation. This study describes a novel soft tissue and bony abductor repair that reduces the forces on bony fragments without the need for prominent metalwork. Methods: This novel surgical technique involves fixation of the abductor mechanism with polyester and polyethylene sutures that are woven through the abductors and secured to the femoral shaft with a proprietary suture cerclage tape with cerclage wire supplementation in select cases. All patients undergoing fixation were retrospectively reviewed with a minimum follow-up period of 12 months. Outcomes relating to dislocation, reoperation, fracture union and the incidence of symptomatic Trendelenburg gait were recorded. Results: A total of 17 patients underwent this novel intervention. There were no dislocations or reoperations for prominent metalwork at the last follow-up. One patient had evidence of greater trochanter (GT) non-union, and three had GT displacement of over 3 mm. Eight (47.1%) patients were independently mobile and seven (41.2%) were mobile with only one walking aid. No patients required plate or bolt fixation. Conclusions: GT fractures and abductor deficiency are difficult to manage, with most reported methods utilising bulky metalwork to treat a soft tissue injury. We describe a novel combined soft tissue and bony fixation without the need for excessive metalwork. Our pilot study demonstrates satisfactory outcomes of this intervention that are technically reproducible and more appropriately addresses the deforming forces involved with a low complication profile.

Methods and Protocols doi: 10.3390/mps9010018

Authors: Nikoleta Eleftheriadou Efstathia D. Mantzari Natasa Kiorapostolou Christodoulos I. Sazeides Georgios Xanthopoulos Nikos Markos Gavriil Spyroglou Evdoxia Bintsi-Frantzi Alexandros Gouvas Panayiotis G. Dimitrakopoulos Mariangela N. Fotelli Kalliopi Radoglou Nikolaos M. Fyllas

This article describes a field- and laboratory-based framework that can be used to monitor the C balance in Mediterranean pine forest ecosystems under different management practices that determine their structure and function. By jointly monitoring stand structure, gas exchange, litter, and decomposition dynamics, this protocol enables the assessment of how management-driven changes regulate carbon uptake, turnover, and losses, thereby affecting carbon sequestration potential. As an example, we suggest the implementation of the protocol at ten (10) permanent monitoring plots across three study areas located in Greece. The first group of plots represents a post-fire chronosequence in pine stands with no management interventions. The second group includes pine stands that exhibit variation in overstory and understory density driven by differences in microclimate and management history. The third group consists of peri-urban pine stands subjected to thinning of varying intensity. The monitoring protocol is implemented across all plots and the collected data can be classified into three analytical domains: (a) demography, encompassing measurements of tree growth and mortality; (b) litter and decomposition dynamics, involving the quantification of litterfall and its seasonality and the estimation of its decomposition rates; and (c) gas exchange, focusing on measurements of leaf photosynthesis and respiration (including relevant leaf functional traits) and monitoring of soil respiration. These three data domains can be used to comparatively consider the effect of forest management on key ecosystem processes and to constrain local-scale vegetation dynamics models.

Methods and Protocols doi: 10.3390/mps9010017

Authors: Loredana Lopez Michele Antonio Savoia Loretta Daddiego Paolo Facella Elio Fantini Linda Bianco Simone Maci Francesco Panara

Taraxacum kok-saghyz (Tks) is a promising plant species for natural rubber (NR) production and represents a model for studying NR biosynthesis in the Asteraceae family. The generation of transgenic plants overexpressing a gene of interest is a well-established strategy to investigate gene function and potential interactions. Here, we present a comprehensive workflow—from the construction of an overexpression vector to the generation, identification, and propagation of stable transgenic Tks lines. In addition, we describe a rapid and reliable method for quantifying NR content in transformed plants, providing essential phenotypic characterisation in this species.