Methods Protoc., Volume 8, Issue 4 (August 2025) – 15 articles

Every year, diarrhoea is responsible for >1 million deaths in children with ages from 0 to 5 years, with rotavirus as the leading cause. The regions most affected lack routine rotavirus diagnosis due to high cost, lack of necessary equipment and shortage of trained-personnel for Enzyme-Link-Immunosorbent-Assay (ELISA) and molecular methods. We report the development and evaluation of a cheap, nanoparticle-based immunoassay for routine machine-free rotavirus diagnosis. In this work, optimal conditions for oxidation of cotton swabs and aldehyde production for kit development was confirmed by Fourier-Transform Infrared Spectroscopy (FTIR). Lactoferrin (LF) needed to bind the virus to the cotton swab was immobilised on activated cotton swabs, followed by the capture of commercial rotavirus antigen on LF-immobilised swabs. This was dipped in coloured nanobeads covalently coupled to rotavirus-group-specific monoclonal antibody for visual rotavirus detection. Subsequently, rotavirus detection by nanoassay, commercial ELISA and quantitative reverse transcription PCR were compared using same set of 186 stool samples and subjected to statistical analyses. Optimal oxidisation condition was observed using 48 mg/mL NaIO₄ in 0.1 M sodium acetate buffer at 35 °C for 9 h. Rotavirus detection was confirmed visually by blue colour retention on swabs after several washings. Sensitivity, specificity, positive-predictive-value and negative-predictive-value of ELISA in rotavirus detection were 60%, 84%, 53% and 88%, respectively, while our immunoassay showed performance at 88%, 94%, 82% and 96%. This immunoassay will provide effective rotavirus public health interventions in low-and-middle-income countries with high morbidity/mortality. Full article

Bats, as key ecological players, interact with a diverse array of organisms and perform essential roles in ecosystems, including pollination, pest control, and seed dispersal. However, their populations face significant threats from habitat contamination, particularly from microplastics (MPs). This study introduces a novel, efficient, and cost-effective method for visualizing transparent microplastics using ultraviolet (UV) light. By employing handheld UV flashlights with a wavelength range of 312 to 400 nm, we enhance the detection of MPs that may otherwise go unnoticed due to color overlap with filtration membranes. All necessary precautions were taken during sampling and analysis to minimize the risk of contamination and ensure the reliability of the results. Our findings demonstrate that the application of UV light significantly improves the visualization and identification of MPs, particularly transparent fibers. This innovative approach contributes to our understanding of plastic contamination in bat habitats and underscores the importance of monitoring environmental pollutants to protect bat populations and maintain ecosystem health. Full article

Burn wounds present a significant challenge to both the medical and scientific communities, contributing to the global economic burden on healthcare systems. Due to the complexity and highly variability of burn injuries, along with intricate pathophysiological mechanisms, the development of appropriate and effective treatment strategies remains particularly demanding. The development of robust pre-clinical models that recapitulate specific molecular and cellular events underlying burn injury are essential to advance the understanding of associated biological mechanisms and facilitate the screening of innovative therapeutic interventions. While conventional in vivo models can replicate the key aspects of human burn wound pathology, they are often associated with ethical, logistical, and cost-related limitations. In this context, this study aims to explore the potential of the chicken chorioallantoic membrane (CAM) as an alternative model for burn wound research. Thus, we describe a reproducible and ethically favorable protocol for establishing standardized burn injuries on the CAM and provide a comprehensive evaluation of tissue responses through macroscopic, morphometric, and histological analyses. Our findings support the CAM as a viable pre-clinical platform for the study of burn wound healing and for the early-stage screening of candidate therapeutic agents. Full article

Fusarium spp. represent a critical threat to maize production and food safety due to their mycotoxin production. This study introduces a refined molecular identification protocol integrating four genomic regions—ITS1, IGS, TEF-1α, and β-TUB—for robust species differentiation of Fusarium spp. isolates from post-harvest maize in Bulgaria. The protocol enhances species resolution, especially for closely related taxa within the Fusarium fujikuroi species complex (FFSC). A newly optimized multiplex PCR strategy was developed using three primer sets, each designed to co-amplify a specific pair of toxigenic genes: fum6/fum8, tri5/tri6, and tri5/zea2. Although all five genes were analyzed, they were detected through separate two-target reactions, not in a single multiplex tube. Among 17 identified isolates, F. proliferatum (52.9%) dominated, followed by F. verticillioides, F. oxysporum, F. fujikuroi, and F. subglutinans. All isolates harbored at least one toxin biosynthesis gene, with 18% co-harboring genes for both fumonisins and zearalenone. This dual-protocol approach enhances diagnostic precision and supports targeted mycotoxin risk management strategies. Full article

Core stability is fundamental to posture, balance, and force transmission throughout the kinetic chain. Although traditionally associated with athletic performance, emerging research highlights its broader applicability to recreational fitness. This study investigates the effects of an eight-week core training program on muscle hypertrophy, static balance, and neuromuscular control in recreationally active, non-athletic adults. Participants will undertake a structured intervention comprising progressive triads targeting core stability, strength, and power. Assessment methods include surface electromyography (EMG), ultrasound imaging, three-dimensional force plates, Kinovea motion analysis, and the Satisfaction With Life Scale (SWLS) questionnaire. Expected outcomes include enhanced core muscle activation, improved static balance, and increased core-generated force during overhead medicine ball slam trials. Additionally, the intervention aims to facilitate hypertrophy of the transverse abdominis, internal oblique, and lumbar multifidus muscles, contributing to spinal resilience and motor control. This protocol bridges gaps in core training methodologies and advances their scalability for recreational populations. The proposed model offers a structured, evidence-informed framework for improving core activation, postural stability, muscle adaptation, movement efficiency, and perceived quality of life in recreationally active individuals. Full article

The study of neuronal electrical activity and spatial organization is essential for uncovering the mechanisms that regulate neuronal electrophysiology and function. Mathematical models have been utilized to analyze the structural properties of neuronal networks, predict connectivity patterns, and examine how morphological changes impact neural network function. In this study, we aimed to explore the role of the actin cytoskeleton in neuronal signaling via primary cilia and to elucidate the role of the actin network in conjunction with neuronal electrical activity in shaping spatial neuronal formation and organization, as demonstrated by relevant mathematical models. Our proposed model is based on the polygamma function, a mathematical application of ramification, and a geometrical definition of the actin cytoskeleton via complex numbers, ring polynomials, homogeneous polynomials, characteristic polynomials, gradients, the Dirac delta function, the vector Laplacian, the Goldman equation, and the Lie bracket of vector fields. We were able to reflect the effects of neuronal electrical activity, as modeled by the Van der Pol equation in combination with the actin cytoskeleton, on neuronal morphology in a 2D model. In the next step, we converted the 2D model into a 3D model of neuronal electrical activity, known as a core-shell model, in which our generated membrane potential is compatible with the neuronal membrane potential (in millivolts, mV). The generated neurons can grow and develop like an organoid brain based on the developed mathematical equations. Furthermore, we mathematically introduced the signal transduction of primary cilia in neurons. Additionally, we proposed a geometrical model of the neuronal branching pattern, which we described as ramification, that could serve as an alternative mathematical explanation for the branching pattern emanating from the neuronal soma. In conclusion, we highlighted the relationship between the actin cytoskeleton and the signaling processes of primary cilia. We also developed a 3D model that integrates the geometric organization unique to neurons, which contains soma and branches, such that the mathematical model represents the interaction between the actin cytoskeleton and neuronal electrical activity in generating action potentials. Next, we could generalize the model into a cluster of neurons, similar to an organoid brain model. This mathematical framework offers promising applications in artificial intelligence and advancements in neural networks. Full article

Pancreatic adenocarcinoma remains one of the deadliest cancers, with limited treatment options and high chemoresistance. Traditional 2D cell cultures fail to accurately replicate the tumor architecture. Our study introduces three-dimensional (3D) pancreatic adenocarcinoma spheroid models using magnetic aggregation of pancreatic cancer cells and immortalized fibroblasts in either liquid culture medium or embedded in hydrogels. The spheroids’ growth was characterized using optical imaging, while viability was assessed using ATP quantification and flow cytometry. Results demonstrated successful spheroid formation and growth. Further analysis suggested that on one hand, culture in liquid medium and ATP-based viability assessment are practical for initial experiments. On the other hand, hydrogel culture and flow cytometry, although being more resource- and labor-intensive, provided both a more reproducible and detailed viability analysis. Full article

Sophisticated voluntary movements are essential for everyday functioning, making the study of how the brain controls muscle activity a central challenge in neuroscience. Investigating corticomuscular control through non-invasive electrophysiological recordings is particularly complex due to the intricate nature of neuronal signals. To address this challenge, we present a novel experimental methodology designed to study corticomuscular control using electroencephalography (EEG) and electromyography (EMG). Our approach integrates a serious gaming biofeedback system with a specialized experimental protocol for simultaneous EEG-EMG data acquisition, optimized for corticomuscular studies. This work introduces, for the first time, a method for assessing brain–muscle functional connectivity during the execution of a demanding motor task. By identifying neuronal sources linked to muscular activity, this methodology has the potential to advance our understanding of motor control mechanisms. These insights could contribute to improving clinical practices and fostering the development of novel brain–computer interface technologies. Full article

Several of the integrin family of cell adhesion receptors have been popular targets for the development of anticancer agents, but with little clinical success to date. Cancer cells usually express multiple redundant integrins; one hypothesis for the lack of efficacy of current antagonists is their high selectivity for a single integrin. To address this, we developed a functional dual-β3-expressing cell model to investigate the effects of combined αIIbβ3/αvβ3 antagonism. We established that treating K562 chronic myeloid leukemia cells with 0.04 μM phorbol 12-myristate 13-acetate (PMA) for 40 h significantly upregulates functional αIIbβ3 and αvβ3 integrins. This optimized method provides a reliable platform for adhesion and detachment assays, enabling the characterization of dual integrin targeting strategies. Using this model, we demonstrate that combining αIIbβ3 and αvβ3 antagonists (GR144053 and cRGDfV) synergistically enhances inhibition of cell adhesion and promotes cell detachment compared to single-agent treatments. Our findings establish a reproducible approach for studying dual β3 integrin targeting, which can be used to investigate potential strategies for overcoming integrin redundancy in cancer therapeutics. Full article

Xanthomonas phaseoli pv. dieffenbachiae (Xpd), the causal agent of bacterial blight in Anthurium within the Araceae family, is listed as an EPPO A2 quarantine organism. Although the whole genome of Xpd has been sequenced, the molecular mechanisms underlying anthurium bacterial blight (ABB) remain unknown. Selecting an optimal reference gene is crucial for obtaining accurate and reliable gene expression profiles during the initial interactions between Xpd and Anthurium. The stability of four reference genes was evaluated by applying three statistical methods—BestKeeper, geNorm, and delta Ct (ΔCt)—using reverse-transcription quantitative PCR (RT-qPCR) data. The rpoD and gyrB genes exhibited the most consistent gene expression profiles, whereas atpD and thyA were less stable at four time points (0, 0.5, 1, and 2 h) during the interactions between Xpd and susceptible A. andreanum cultivar ‘Marian Seefurth.’ The suitability of these reference gene candidates was validated by normalizing the gene expression levels of four pathogenicity-related genes. The highly upregulated expression of gumD, which encodes xanthan biosynthesis glycosyltransferase, observed after 1 h of interaction, suggests it may be a key virulence determinant in the Xpd–Anthurium pathosystem. The stable reference genes identified here will facilitate more accurate and comprehensive gene expression studies in the Xpd–Anthurium pathosystem going forward. Full article

This research addresses critical challenges in clinical gait analysis by developing an automated video quality assessment framework to support Edinburgh Visual Gait Score (EVGS) scoring. The proposed methodology uses the MoveNet Lightning pose estimation model to extract body keypoints from video frames, enabling detection of multiple persons, tracking the person of interest, assessment of plane orientation, identification of overlapping individuals, detection of zoom artifacts, and evaluation of video resolution. These components are integrated into a unified quality classification system using a random forest classifier. The framework achieved high performance across key metrics, with 96% accuracy in detecting multiple persons, 95% in assessing overlaps, and 92% in identifying zoom events, culminating in an overall video quality categorization accuracy of 95%. This performance not only facilitates the automated selection of videos suitable for analysis but also provides specific video improvement suggestions when quality standards are not met. Consequently, the proposed system has the potential to streamline gait analysis workflows, reduce reliance on manual quality checks in clinical practice, and enable automated EVGS scoring by ensuring appropriate video quality as input to the gait scoring system. Full article

Background/Objectives: Cigarette smoking has been causally linked to 90% of all cases of lung cancer, contributing to its high mortality rate. Lung cancer screening centers offer low-dose computed tomography, the only recommended diagnostic screening tool for lung cancer detection. A previous Texas-based study found that centers with lung cancer screening programs failed to consistently provide evidence-based tobacco cessation and relapse prevention interventions recommended by clinical practice guidelines to their patients, who are primarily people who currently or previously smoked. This represents a missed opportunity to assist patients by providing evidence-based tobacco use care during a particularly relevant clinical encounter. Methods: To improve cigarette smoking cessation care delivery and relapse prevention in this setting, this protocol paper seeks to provide a framework for adapting Taking Texas Tobacco Free, a comprehensive, evidence-based tobacco-free workplace program, to lung cancer screening centers. The adapted program, Project SWITCH, will be developed through a formative evaluation process with center stakeholders to identify proactive adaptations to programming based on center-specific contexts. Project SWITCH is expected to be implemented in at least nine lung cancer screening centers in Texas and will be disseminated more broadly to centers statewide. Results: Quantitative and qualitative data will be collected from multiple stakeholders throughout the intervention using a convergent parallel mixed methods design to make additional program adaptations and comprehensively evaluate the achievement of the project’s implementation and dissemination goals. Conclusions: Results from this project’s implementation and dissemination phases are expected to reduce lung cancer morbidity and mortality in Texas by providing an evidence-based, sustainable framework for tobacco-free workplace programs in this specific setting that improves cancer prevention and control practices. Full article

Background: This study aimed to evaluate and compare the survival rates of immediate dental implants (type 1) in patients who received different types of prophylactic antibiotics. Methods: This retrospective analysis examined data from 3351 immediate implants placed in 2391 patients (mean age 59.56 ± 13.42 years, 75.9% white, 53.6% female, 7.8% smokers, 6.7% with diabetes) within the BigMouth network between 2011 and 2022. Patient demographics, medical history, and the type of prophylactic antibiotic administered (amoxicillin, amoxicillin and clavulanic acid (Augmentin), clindamycin, azithromycin, ciprofloxacin, doxycycline, metronidazole) were analyzed in relation to implant survival or failure. Statistical analyses included descriptive statistics, chi-square tests, t-tests, Kaplan–Meier survival analysis, and Cox regression. All statistical analyses were performed with a significance level at p < 0.05. Results: The overall implant failure rate was 3.2% at the patient level (77 out of 2391 patients) and 1.9% at the implant level (65 out of 3351 implants), with a mean follow-up of 77 months. No significant associations were found between patient-related characteristics or implant position and implant failure, such as age (p = 0.84), gender (p = 0.30), or tobacco use (p = 0.83). Amoxicillin was the most frequently prescribed antibiotic (86.4%). Kaplan–Meier survival analysis revealed significantly shorter survival times for implants in patients who received ciprofloxacin and clindamycin compared to amoxicillin (n = 2894 implants) (p < 0.001). Cox regression analysis indicated a significantly increased risk of implant failure with ciprofloxacin (n = 5 implants) (HR: 16.50, p = 0.006) and clindamycin (n = 290 implants) (HR: 3.70, p < 0.001) compared to amoxicillin. Conclusion: The choice of prophylactic antibiotic significantly impacted the survival of immediate dental implants. Ciprofloxacin and clindamycin were associated with higher failure rates compared to amoxicillin. These findings underscore the importance of antibiotic selection in immediate implant procedures and highlight the need for further research to establish evidence-based guidelines for antibiotic prophylaxis in this context. Full article

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