Methods and Protocols

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

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

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

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

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

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

Epigenetic research has made notable progress in recent years, yet our ability to explore the human brain at a cellular level remains limited. One of the main obstacles has been the difficulty of isolating specific neuronal populations from postmortem tissue—particularly interneurons, which play a central role in many psychiatric disorders. In this study, we present a practical and reproducible protocol for isolating GAD-positive interneurons from human brain samples. We isolate permeabilized cell-like structures suitable for downstream epigenetic analysis. To ensure specificity, we validated the isolated cells by comparing them with interneurons derived from human iPSCs. This approach allows for high-quality DNA extraction suitable for downstream epigenetic analysis, including methylation-specific PCR. By targeting a well-defined neuronal subtype, our method provides a solid foundation for studying the molecular changes associated with disorders such as schizophrenia and autism. This protocol opens new doors for cell-specific investigations in brain tissue, a step forward in understanding how epigenetic mechanisms contribute to neuropsychiatric pathophysiology. Full article

Human respiratory syncytial virus (RSV) causes acute respiratory illness, attributing to deaths among young children and older adults worldwide. RSV neutralization assay is an important tool to measure RSV neutralization antibody that can prevent infection and severe complication of RSV. Conventional RSV neutralization assays have some limitations of speed and cost, especially for expensive kits, reagents or instruments required for detection. To solve this problem, this paper describes an improved simple and economical RSV neutralization assay protocol using recombinant RSV (rRSV) expressing reporter fluorescent protein to measure RSV growth as reporter activity with plate reader. The condition of 3 days culture demonstrated sufficient fluorescent activity even when small amounts of rRSV were used to inoculate Hep-2 cells. In addition, white 96-well cell culture plate showed better stable reporter activities than black plate. Furthermore, RSV neutralization assay protocol using rRSV-reporter fluorescent protein demonstrated similar signal detection capacity for RSV antibody titer detection compared to other protocols, such as rRSV-Luciferase and ELISA assay. The new RSV neutralization assay protocol can be applied to RSV antibody titration of numerous samples necessary for RSV surveillance or antiviral testing. Full article