Methods and Protocols

21 pages, 3088 KB

Open AccessArticle

An Efficient TetR/TetO-Integrated Packaging System for Fowl Adenovirus 4 Vector Carrying Toxic Transgene

by Qian-Wen Ma, Zhi Li, Zhi-Chao Zhang, Xiao-Juan Guo, Xiao-Hui Zou, Tao Hung and Zhuo-Zhuang Lu

Methods Protoc. 2026, 9(3), 100; https://doi.org/10.3390/mps9030100 - 22 Jun 2026

Viewed by 478

Abstract

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 [...] Read more.

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. Full article

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

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10 pages, 219 KB

Open AccessStudy Protocol

Exploring Barriers and Facilitators to COVID-19 Vaccination Uptake Among Individuals with Mental Illness in the Australian Healthcare System: A Qualitative Study Protocol

by Soumitra Das, James Killian, Mahesh Jayaram, Naveen Thomas and Chi Jonasi

Methods Protoc. 2026, 9(3), 99; https://doi.org/10.3390/mps9030099 - 16 Jun 2026

Viewed by 524

Abstract

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 [...] Read more.

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). Full article

(This article belongs to the Section Public Health Research)

20 pages, 4960 KB

Open AccessArticle

In Vitro Capacitation in Boar Sperm: Evaluation of Selected Detection Techniques

by Barbora Klusackova, Zuzana Pilsova, Katerina Nemeckova, Aneta Pilsova and Pavla Postlerova

Methods Protoc. 2026, 9(3), 98; https://doi.org/10.3390/mps9030098 - 15 Jun 2026

Viewed by 590

Abstract

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 [...] Read more.

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. Full article

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

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22 pages, 6096 KB

Open AccessProtocol

Multiparametric Flow Cytometry Panel for Characterization of Mouse T Cell Differentiation and NK Cell Maturation Following Inflammatory Challenge

by Tim Bozic, Bostjan Markelc, Simona Kranjc Brezar, Ziva Pisljar, Tanja Jesenko and Maja Cemazar

Methods Protoc. 2026, 9(3), 97; https://doi.org/10.3390/mps9030097 - 12 Jun 2026

Viewed by 690

Abstract

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. [...] Read more.

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. Full article

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

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11 pages, 415 KB

Open AccessStudy Protocol

Protocol for the Implementation of a Targeted Maternal and Newborn Service Delivery Bundle in Sierra Leone

by Robert B. Clark, Joseph Odu, Annette Ofodum and Rondi Anderson

Methods Protoc. 2026, 9(3), 96; https://doi.org/10.3390/mps9030096 - 10 Jun 2026

Viewed by 816

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Section Public Health Research)

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17 pages, 5667 KB

Open AccessReview

Contact Lens-Associated Ocular Surface and Corneal Disorders

by Omar Abdelaziz, Seyyedehfatemeh Ghalibafan, Raul E. Ruiz-Lozano, Jeffrey C. Peterson, Ryan A. Gallo and Ali R. Djalilian

Methods Protoc. 2026, 9(3), 95; https://doi.org/10.3390/mps9030095 - 10 Jun 2026

Cited by 1 | Viewed by 828

Abstract

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, [...] Read more.

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. Full article

(This article belongs to the Section Public Health Research)

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19 pages, 6550 KB

Open AccessProtocol

Methodological Framework for a Multimodal Rat Model of Bleomycin-Induced Fibrosis and Autologous Tissue Grafting

by 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 and Zorin Petrisor Crainiceanu

Methods Protoc. 2026, 9(3), 94; https://doi.org/10.3390/mps9030094 - 10 Jun 2026

Viewed by 523

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Section Tissue Engineering and Organoids)

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12 pages, 1224 KB

Open AccessArticle

Design, Preparation and Characterization of Nationally Representative Synthetic Food Waste for Reproducible Waste Valorization Research

by Ryan Scott Anderson, Sybil Sharvelle and Susan K. De Long

Methods Protoc. 2026, 9(3), 93; https://doi.org/10.3390/mps9030093 - 10 Jun 2026

Cited by 1 | Viewed by 591

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Section Biochemical and Chemical Analysis & Synthesis)

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Graphical abstract

17 pages, 666 KB

Open AccessArticle

Cumulative Frameworks as a Pragmatic Alternative to Multivariable Modeling in Rare-Event Clinical Settings: A Coronary Care Unit Case Study

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

Methods Protoc. 2026, 9(3), 92; https://doi.org/10.3390/mps9030092 - 10 Jun 2026

Viewed by 392

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Section Public Health Research)

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13 pages, 1800 KB

Open AccessArticle

A Lightweight Workflow for Targeted Long-Read Transcriptomic Profiling Using Oxford Nanopore Sequencing

by Mariya Levkova

Methods Protoc. 2026, 9(3), 91; https://doi.org/10.3390/mps9030091 - 4 Jun 2026

Viewed by 536

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Section Synthetic and Systems Biology)

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13 pages, 214 KB

Open AccessTechnical Note

Content Generalizability: Generalizing Qualitative Research to the General Study Population

by Costas S. Constantinou

Methods Protoc. 2026, 9(3), 90; https://doi.org/10.3390/mps9030090 - 4 Jun 2026

Viewed by 802

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Section Public Health Research)

14 pages, 1665 KB

Open AccessArticle

Comparative Study of Bacteriophage Pharmacokinetics by Different Enteral Administration Routes

by Maria Anurova, Aleksey Kuzmin, Aleksey Vorobev, Nataliya Feldman, Elena Zinurova and Andrey Aleshkin

Methods Protoc. 2026, 9(3), 89; https://doi.org/10.3390/mps9030089 - 3 Jun 2026

Viewed by 678

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue Advanced Methods and Technologies in Drug Discovery)

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20 pages, 4485 KB

Open AccessArticle

A Multiresolution Breast Cancer CIBERSORTx Resource Validated for Accuracy, Interpretive Limits, and Biological and Clinical Coherence in Tumor Microenvironment Deconvolution

by Toru Hanamura, Akinori Takase, Masanori Oshi and Naoki Niikura

Methods Protoc. 2026, 9(3), 88; https://doi.org/10.3390/mps9030088 - 2 Jun 2026

Viewed by 714

Abstract

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 [...] Read more.

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. Full article

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

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37 pages, 3082 KB

Open AccessReview

An Overview of In Vitro Release Methods for Long-Acting Injectable Products Based on PLGA

by Maja Lusina Kregar, Iva Krtalić and Ivana Šagud

Methods Protoc. 2026, 9(3), 87; https://doi.org/10.3390/mps9030087 - 1 Jun 2026

Viewed by 826

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue Feature Papers in Methods and Protocols 2026)

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20 pages, 11021 KB

Open AccessArticle

Boron-Doped Carbon Dots for Organelle Labeling and Mitochondrial Bioimaging

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

Methods Protoc. 2026, 9(3), 86; https://doi.org/10.3390/mps9030086 - 1 Jun 2026

Viewed by 700

Abstract

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 [...] Read more.

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. Full article

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

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13 pages, 994 KB

Open AccessArticle

Evaluation of Deep Eutectic Solvents for Cryopreservation of the Fish Pathogen Saprolegnia parasitica

by Sara Delimar, Ela Šarić, Marina Cvjetko Bubalo and Ana Bielen

Methods Protoc. 2026, 9(3), 85; https://doi.org/10.3390/mps9030085 - 1 Jun 2026

Viewed by 675

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Section Public Health Research)

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16 pages, 12063 KB

Open AccessProtocol

A Simple, Rapid and Reliable Protocol for Extraction of High Quality Bacterial Genomic DNA Directly from Potato Tubers for Efficient PCR-Based Surveillance and Molecular Characterization of Ralstonia solanacearum

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

Methods Protoc. 2026, 9(3), 84; https://doi.org/10.3390/mps9030084 - 31 May 2026

Viewed by 862

Abstract

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 [...] Read more.

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. Full article

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

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55 pages, 8287 KB

Open AccessReview

Regulated Cell Death in Fungi, the Role of Metacaspases and Assay Techniques

by Derek Wilkinson

Methods Protoc. 2026, 9(3), 83; https://doi.org/10.3390/mps9030083 - 31 May 2026

Viewed by 669

Abstract

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 [...] Read more.

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. Full article

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

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18 pages, 4448 KB

Open AccessProtocol

Triple-Survival Stereotactic Brain Surgeries for the Intracranial Injections of Glioblastoma Stem-like Cells and Oncolytic Herpes Simplex Viruses

by Sourav Chakraborty, Connor Howard, Checo J. Rorie, Samuel D. Rabkin, Hiroaki Wakimoto and Dipongkor Saha

Methods Protoc. 2026, 9(3), 82; https://doi.org/10.3390/mps9030082 - 31 May 2026

Viewed by 962

Abstract

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, [...] Read more.

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. Full article

(This article belongs to the Special Issue Current Methodology Advances in Cell Therapy Applications—2nd Edition)

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22 pages, 6807 KB

Open AccessProtocol

Optimized Wound Healing Assay to Study Extracellular Vesicle-Driven Glioblastoma Cell Migration

by Concetta D’Antonio, Francesca Mantile, Gabriella Pocsfalvi and Giovanna L. Liguori

Methods Protoc. 2026, 9(3), 81; https://doi.org/10.3390/mps9030081 - 31 May 2026

Viewed by 938

Abstract

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 [...] Read more.

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. Full article

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

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13 pages, 5102 KB

Open AccessArticle

A Simple Approach to Sample Preparation for Accurate and Reproducible Gas Phase Breakthrough Analysis of Adsorbent Materials

by Daniel A. Corbin, Christopher J. Breshike, Michael R. Papantonakis and R. Andrew McGill

Methods Protoc. 2026, 9(3), 80; https://doi.org/10.3390/mps9030080 - 29 May 2026

Viewed by 661

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Section Biochemical and Chemical Analysis & Synthesis)

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11 pages, 941 KB

Open AccessBrief Report

Folate Receptor Alpha Autoantibodies in Early Pregnancy: First-Trimester Reference Intervals and Proposed Clinical Thresholds

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

Methods Protoc. 2026, 9(3), 79; https://doi.org/10.3390/mps9030079 - 25 May 2026

Viewed by 562

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Section Biochemical and Chemical Analysis & Synthesis)

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9 pages, 1034 KB

Open AccessStudy Protocol

The ADAPT-HEAT Study: A Multi-Method Approach to Develop Recommendations for Drug Safety During Hot Weather (The CALOR List)—Study Protocol

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

Methods Protoc. 2026, 9(3), 78; https://doi.org/10.3390/mps9030078 - 25 May 2026

Viewed by 789

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Section Public Health Research)

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11 pages, 1677 KB

Open AccessArticle

A Divergent TaqMan RT-qPCR Strategy for Isoform-Resolved Detection of HIV-1 Circular RNAs

by Christopher Mauer, Sean Paz and Massimo Caputi

Methods Protoc. 2026, 9(3), 77; https://doi.org/10.3390/mps9030077 - 13 May 2026

Viewed by 449

Abstract

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 [...] Read more.

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. Full article

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

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10 pages, 475 KB

Open AccessArticle

Task-Specific Reliability and Measurement Error of Frontal Plane Kinematics in Individuals with Patellofemoral Pain: A Preliminary Study

by Hiraku Nagahori, Isabella Keefer, Derrick Smith, Brendan Yawn, Jing Nong Liang and Kai-Yu Ho

Methods Protoc. 2026, 9(3), 76; https://doi.org/10.3390/mps9030076 - 13 May 2026

Viewed by 557

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue Methods, Protocols, and Analytical Approaches for the Assessment of Human Movement)

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12 pages, 857 KB

Open AccessArticle

Comparative Evaluation of Methods of DNA Extraction from Cryopreserved Bovine Semen for Molecular Diagnostic Applications

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

Methods Protoc. 2026, 9(3), 75; https://doi.org/10.3390/mps9030075 - 9 May 2026

Viewed by 754

Abstract

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 [...] Read more.

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. Full article

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

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15 pages, 1550 KB

Open AccessArticle

Evaluating the Efficacy of Monoclonal Antibodies Against a Bioactive Peptide Involved in Alzheimer’s Disease: A Methodological Approach

by Georgina Bonny, Kashif Mahfooz, Sara Garcia-Rates, Sibah Hasan and Susan Adele Greenfield

Methods Protoc. 2026, 9(3), 74; https://doi.org/10.3390/mps9030074 - 9 May 2026

Viewed by 837

Abstract

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 [...] Read more.

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. Full article

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

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19 pages, 29617 KB

Open AccessProtocol

I-TEP: A Simple and Affordable Method to Measure Permeability in Reconstructed Tissues Combined with DAMO–TSC-Based Urea Assay

by Yudaï Sahuc, Elissa Elia, Christophe Caneparo, Nathan Félix, Marilou Hardy, Stéphane Chabaud and Stéphane Bolduc

Methods Protoc. 2026, 9(3), 73; https://doi.org/10.3390/mps9030073 - 3 May 2026

Viewed by 904

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Section Tissue Engineering and Organoids)

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21 pages, 2511 KB

Open AccessArticle

Comparative Analysis of Streptozotocin, Streptozotocin–Nicotinamide and Alloxan-Based Diabetes Models in Female Wistar Rats

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

Methods Protoc. 2026, 9(3), 72; https://doi.org/10.3390/mps9030072 - 2 May 2026

Viewed by 1773

Abstract

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 [...] Read more.

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. Full article

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

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14 pages, 2245 KB

Open AccessArticle

Morphological Assessment of Stage HH38 of the Japanese Quail (Coturnix japonica) Heart by Micro-Sonogram

by Jaden Roe, Ashlyn Benavides, Michael B. Filla, Douglas C. Bittel, Whitney Shae, Geetha Haligheri, James E. O’Brien, Jr. and Nataliya Kibiryeva

Methods Protoc. 2026, 9(3), 71; https://doi.org/10.3390/mps9030071 - 2 May 2026

Viewed by 692

Abstract

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 [...] Read more.

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. Full article

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

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