Methods Protoc., Volume 9, Issue 4 (August 2026) – 17 articles

Osteocalcin (OC), a bone protein, is evaluated by its proteoforms, which are implicated in various illnesses. Rapid, clinically used immunoassays are well-established. Epitope dependence, fragment cross-reactivity, uneven identification across carboxylation states, and lack of standardization restrict their efficacy. This study aims to characterize various OC proteoforms in patients with type 2 diabetes mellitus (T2DM) and vitamin D insufficiency and to develop a mass spectrometric immunoassay (MSIA) for their detection. The first MRM-MSIA test to specifically detect OC proteoforms in plasma with vitamin D deficiency-induced alterations in T2DM patients was developed and validated. A precise and selective MRM-MSIA was developed to assess OC fragments in plasma from T2DM patients with normal (DN) or deficient (DD) vitamin D levels. The approach examined OC-1 (aa64–71), OC-2 (aa64–70), and OC-3 (aa72–93) proteoforms. It met international linearity, precision, and accuracy criteria with variability <15% and intra/inter-day accuracy of 88.8% to 110.1%. The DD group had significantly higher OC-1 levels than the DN group. The receiver operating characteristic (ROC) curve for OC-1 produced an AUC of 0.8263 (95% confidence interval [CI], p = 0.0004). The developed MRM-MSIA measures plasma OC proteoforms with good specificity and sensitivity, making it a promising clinical diagnostic tool. Full article

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 Ptf1a^CreER/+; Kras^LSL-G12D/+; Trp53^LSL-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. Full article

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. Full article

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. Full article

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. Full article

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. Full article

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. Full article

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. Full article

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. Full article

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. Full article

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. Full article

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. Full article

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. Full article

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. Full article

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. Full article

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. Full article

(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. Full article