Methods and Protocols

Objectives: The present study mainly aimed to identify whether the envelope and triangular flaps affected wound healing and patient quality of life differently. Secondarily, the study aimed to investigate whether some anatomical and operational variables may also affect healing. Study design: A prospective randomized study was conducted with 56 fully impacted lower third molars, randomly divided into two groups, one treated with the envelope flap and the other with the bayonet flap. Qualitative variables were transformed into quantitative ones and then analyzed using independent samples t-tests or analysis of variance. An analysis of bivariate correlations with Pearson’s coefficient was also used. The chi-square test was used to verify the association between each flap and the categorical variables considered. Results: No statistically significant associations were found between flap types and dehiscence, although the mean dehiscence diameter was consistently greater in the envelope flap group. The maximum diameter of the dehiscence at 14 days was found to be significantly and negatively related to the 14-day wound healing indices. Analyses relating to the quality of life did not show significant associations. Conclusions: Despite some significant healing differences between the two considered flaps exist, they do not have relevant effects on the patient’s post-operative quality of life. Full article

Cannabis sativa (C. sativa) is a psychoactive plant that has been used for millennia for medicinal, recreational, and industrial purposes. The main constituents of cannabis are the cannabinoids, with other constituents including terpenes and flavonoids that contribute to its bioactivity. Among the flavonoid class, there is a subclass, specific to cannabis, namely the cannflavins (A, B, and C), which are biologically active. This study is directed to the analysis of these constituents in various cannabis chemovars. In this study, an HPLC-PDA method was validated and applied to determine the content of cannflavins, namely, cannflavin A (CF-A), cannflavin B (CF-B), and cannflavin C (CF-C), in six different cannabis chemovars. The HPLC separation was achieved using a Luna^® C18 (150 × 4.6 mm × 3 μm) with isocratic elution using a mobile phase consisting of acetonitrile and water (65:35, v/v), both containing 0.1% formic acid at a flow rate of 1 mL/min, with the detector set at 342.4 nm. The method was validated according to the ICH guidelines and exhibited a linear relationship in the 5–500 ppm range with R² > 0.99. The method showed good recovery, ranging from 82% to 98%. The intra-day and inter-day relative standard deviations (% RSDs) were ≤5.29%. Consequently, the method was applied for the determination of all these cannflavins in the different cannabis chemovars. CF-A was the most abundant cannflavin in the examined samples (15.2–478.38 ppm). The method was shown to be simple, accurate, and selective. Full article

Pharmaceutical formulations, in addition to the medicinal substance(s), contain added excipients that make it possible to create a pharmaceutical product that exhibits required properties in terms of mechanical, physical, chemical, and microbiological stability. Additionally, these substances can act as release modifiers or improve bioavailability parameters. Literature data indicate that excipients, especially polymeric ones, can also affect the polymorphism of the active substance, resulting in drug bioavailability enhancement or reduction. This influence can be evaluated using thermal and spectroscopic methods. In the study, differential scanning calorimetry (DSC), vibrational spectroscopic studies (Fourier transform infrared spectroscopy, FTIR), Raman spectroscopy, and X-ray diffraction (XRD) assay of ibuprofen, naproxen, and naproxen sodium standards and pharmaceutical preparations containing these medicinal substances in their compositions were carried out. DSC results indicated that a sharp melting peak was observed on the DSC curves of the standards, confirming their crystalline form. DSC results obtained for pharmaceutical formulations also indicated that the enthalpy of melting is sometimes lower than calculated from the percentage of active ingredients in the formulations. In addition, the melting peak is often broadened and shifted toward lower temperatures, suggesting the influence of excipients on the polymorphism of drug substances. The FTIR and Raman spectra of pharmaceutical formulations contained all characteristics of the active substances. XRD analysis was also performed. Therefore, possible chemical interactions between the components of the preparations have been excluded. At the same time, FTIR and Raman spectroscopy results as well as XRD assay showed a reduction in the height of signals corresponding to the crystalline API form, confirming the possibility of reducing API crystallinity in pharmaceutical formulations. Full article

Yeasts of the Candida genus are part of the normal human microbiota but can cause infections (candidiasis) under certain conditions. While Candida albicans remains the most common etiological agent, the prevalence of non-albicans Candida species—such as C. glabrata, C. tropicalis, C. krusei, C. parapsilosis, C. kefyr, C. lusitaniae, and the emerging multidrug-resistant C. auris—has been increasing. Effective treatment of candidiasis requires rapid and accurate identification of the causative species, particularly due to species-specific antifungal agent resistance patterns. The aim of this study was to evaluate the usefulness of five chromogenic media for the differentiation of Candida species: BD CHROMagar Candida (Becton Dickinson), CHROM ID Candida (bioMérieux), CHROMAgar Candida Plus (CHROMAgar France, Biomaxima), CHROMAgar Candida Plus (GRASO Biotech), and Brilliance Candida Agar (OXOID). A total of 175 strains from the following species were tested: C. albicans, C. parapsilosis, C. dubliniensis, C. lusitaniae, C. tropicalis, C. glabrata, C. kefyr, C. krusei, and C. auris. Species identification was confirmed by MALDI-TOF mass spectrometry using the MALDI Biotyper system (Bruker). Colony morphology, especially color characteristics, was assessed on each medium. The morphological features of most Candida species were consistent with the manufacturer’s descriptions and allowed for presumptive species-level identification. However, some species showed reproducible but previously undescribed morphological traits, including variations in colony shade. Notably, C. auris could not be reliably identified using BD, bioMérieux, or OXOID media. In conclusion, while chromogenic media are a helpful preliminary diagnostic tool, subtle differences in colony coloration can complicate interpretation. Diagnostic caution is recommended, and confirmatory methods such as MALDI-TOF remain essential for reliable identification, especially for emerging or less common Candida species. Full article

Bone remodeling relies on the coordinated activity of osteoblasts (OBs) and osteoclasts (OCs). Disruptions in OB-OC balance can lead to diseases such as periodontitis, a chronic microbial-induced inflammatory disease. To investigate how inflammation affects OB-OC interactions, we standardized an in vitro 2D indirect co-culture system using primary human OB and OC precursors from peripheral blood mononuclear cells in a transwell setup, which allows paracrine signaling and separate analysis of each cell type. When exposed to bacterial lipopolysaccharides (Aa LPS and E. coli LPS) and proinflammatory cytokines (IL-6 and TNF-α), we observed that inflammatory stimuli significantly increased OC differentiation, particularly TNF-α, while E. coli LPS specifically suppressed OB activity as observed by the expression of key markers and cellular staining. These results demonstrate that microbial and host-derived inflammatory factors can differentially modulate bone cell behavior. This approach offers a physiologically relevant and ethically advantageous alternative to animal models to screen dual-targeted bone therapies to restore perturbed metabolism. Full article

Background: Urethral strictures impact millions, causing significant morbidity and millions in healthcare costs. Testing new interventions is limited by the lack of inexpensive urethral healing models. We developed an ex vivo model of early urethral wound healing using explanted rabbit urethral tissue. This was used to test the impact of six growth factors (GFs). Methods: The rabbit urethra was detubularized by cutting it between the corpora cavernosa, and then it was stitched flat using a custom 3D-printed platform. The tissue was carefully scratched to produce a visible wound, and the specimens were placed in media containing growth factors at 100 ng/mL and 10 ng/mL. Images were taken at 0, 24, 48, 72, and 96 h, and the wound area was measured by blinded reviewers to determine the rate of wound contraction. Results: Specimens with IGF at 100 ng/mL showed a statistically significant difference in wound contraction when compared to those with GF-free control medium, showing that IGF-1 supports early urethral epithelization and may improve healing. Conclusions: The developed protocol provides a simple explant platform that can be used to investigate methods of enhancing early phases of urethral healing or used to investigate other areas of urethral health, including drug delivery, infection, and mechanical properties. Full article

The initial molecular events mediating mammalian sperm binding to the zona pellucida (ZP) of the oocyte are highly complex and still not fully elucidated. Recent advances have identified multiple candidate sperm surface proteins, often functioning as part of high-molecular-weight complexes that mediate this critical fertilization event in a species-specific and coordinated manner. To address a significant gap in the literature, we provide an in-depth overview of the functional assays employed to investigate sperm–ZP interactions, emphasizing their underlying principles, potential applications, and key methodological strengths and limitations. The techniques discussed range from classical in vitro sperm–oocyte and hemizona binding assays, including antibody-blocking and competitive strategies, to cutting-edge in vivo genetic models, each contributing unique insights into the physiological relevance of the proposed ZP receptors. Robust experimental design, including the use of appropriate controls and validation strategies, is essential for accurately interpreting the role of candidate sperm receptors. This review provides a structured overview of current methodologies to support researchers in critically evaluating and applying functional assays in future studies. Full article

Data on the genetic and environmental factors underlying the co-occurrence of Cognitive Disengagement Syndrome (CDS), Attention Deficit Hyperactivity Disorder (ADHD), and Emotional Dysregulation (ED) are limited. This study aimed to explore the nature of the associations between CDS, ADHD with ED, and to assess the role of shared etiological factors in explaining their comorbidity. We analyzed a sample of 400 Italian twin pairs aged 8–18, from Northern Italy and enrolled in the Italian Twin Registry. Bivariate genetic analyses were conducted using parent-rated CBCL scores for CDS, ADHD, and ED. For both CDS–ED and ADHD–ED associations, the best-fitting models were Cholesky AE models (−2LL = −849.167 and −339.030, respectively; p > 0.05), suggesting that the covariation was mainly due to additive genetic factors (CDS–ED—A = 0.81, 95% CI [0.66–0.95]; ADHD–ED—A = 0.86, 95% CI [0.75–0.95]). More than half of the genes were shown to be shared among the phenotypes. Non-shared environmental contributions were smaller (CDS–ED—E = 0.19, 95% CI [0.05–0.34]; ADHD–ED—E = 0.14, 95% CI [0.05–0.25]), indicating interrelated but distinct constructs. Despite some limitations, particularly the exclusive use of the CBCL, findings highlight the importance of monitoring ED symptoms in individuals with CDS or ADHD, and vice versa. Full article

Background: Accurate assessment of drug cytotoxicity in vitro is essential for preclinical evaluation of anticancer agents. Methodological parameters such as cell density and solvent concentrations can significantly influence the reproducibility and reliability of cell-based assay results. Objective: This study aims to optimize cell seeding density and evaluate the cytotoxic effects of common solvents (DMSO and ethanol) on different cancer cell lines, complemented by in silico analysis to elucidate underlying mechanisms. Materials and Methods: Six cancer cell lines (HepG2, Huh7, HT29, SW480, MCF-7, and MDA-MB-231) were seeded at different densities to determine the optimal cell seeding number ideal for cell viability assay at 24, 48, and 72 h. The cytotoxicity of DMSO and ethanol was assessed in these cell lines using an MTT assay at multiple time points. In silico docking studies were conducted to investigate the interactions between solvents and key proteins involved in apoptosis, membrane function, and metabolism. Results: A cell density of 2000 cells per well yielded consistent linear viability across cell lines and time points. DMSO at 0.3125% showed minimal cytotoxicity across all cell lines (except MCF-7) and time points; the cytotoxic effect at higher concentrations is variable depending on cell type and exposure duration. Ethanol exhibited rapid and concentration-dependent cytotoxicity, reducing viability by more than 30% at as low as 0.3125% concentration after 24 h. Docking analyses revealed that DMSO binds specifically to apoptotic and membrane proteins, suggesting a role in inducing apoptosis. In contrast, ethanol primarily interacts with metabolic proteins, consistent with its effect on membrane disruption and rapid cell death. Conclusion: DMSO at 0.3125% is a good choice as a solvent since it has low toxicity in most tested cell lines; however, the safe concentration limit is dependent on cell type and exposure duration. Ethanol exhibited higher cytotoxicity, necessitating careful concentration management. The in silico analysis supports these findings, indicating that DMSO interacts with apoptosis-related proteins, whereas ethanol primarily affects metabolic processes. These results highlight the importance of precise cell density optimization and solvents for reliable cytotoxicity assessment in cell-based assays. Full article

This protocol paper outlines a robust and reproducible framework for a ¹H nuclear magnetic resonance (NMR) metabolomics analysis of rodent plasma, designed to facilitate preclinical biomarker discovery. The protocol details optimised steps for plasma collection in a preclinical rodent model, sample preparation, and NMR data acquisition using presaturation Carr–Purcell–Meiboom–Gill (PRESAT-CPMG) pulse sequences, ensuring high-quality spectral data and effective suppression of macromolecule signals. Comprehensive spectral processing and metabolite assignment are described, with guidance on multivariate and univariate statistical analyses to identify metabolic changes and potential biomarkers. The framework emphasises methodological rigour and reproducibility, enabling accurate quantification and interpretation of metabolites relevant to disease mechanisms or therapeutic interventions. By providing a standardised approach, this protocol supports longitudinal and translational studies, bridging findings from rodent models to clinical applications and advancing the reliability of metabolomics-based biomarker discovery in preclinical research. Full article

In vitro cartilage explant culture has been used to assess nutraceuticals on cartilage responses to inflammatory stimuli. However, applying extracts of nutraceuticals directly to cartilage explants does not account for effects of digestion and hepatic biotransformation, or selective exclusion of product metabolites from joint fluid by the synovial membrane. The current study produced a simulated biological extract of a common nutraceutical (glucosamine; G_sim) by exposing it to a simulated upper gastrointestinal tract digestion, hepatic biotransformation by liver microsomes, and purification to a molecular weight cut-off of 50 kDa. This extract was then used to condition cartilage explants cultured for 120 h in the presence or absence of an inflammatory stimulus (lipopolysaccharide). Media samples were analyzed for prostaglandin E₂ (PGE₂), glycosaminoglycan (GAG), and nitric oxide (NO). Tissue was digested and analyzed for GAG content and stained for viability. Conditioning of explants with G_sim significantly reduced media GAG in stimulated and unstimulated explants and reduced nitric oxide production in unstimulated explants. These data provide evidence for the value of glucosamine in protecting cartilage from deterioration following an inflammatory challenge, and the model improves applicability of these in vitro data to the in vivo setting. Full article

Serial block-face scanning electron microscopy (SBEM) is a powerful technique for three-dimensional ultrastructural analysis of biological samples, though its application to in vitro cultured human cells remains underutilized. In this study, we present an optimized SBEM sample preparation protocol using human dermal fibroblasts and induced pluripotent stem cells (iPSCs). The method includes key modifications to the original protocol, such as using only glutaraldehyde for fixation and substituting the toxic cacodylate buffer with a less hazardous phosphate buffer. These adaptations result in excellent preservation of cellular ultrastructure, with high contrast and clarity, as validated by transmission electron microscopy (TEM). The loss of natural cell morphology resulted from fixation during passage, when cells formed a precipitate, rather than from fixation directly within the culture medium. The protocol is time-efficient, safe, and broadly applicable to both stem cells and differentiated cells cultured under 2D conditions, providing a valuable tool for ultrastructural analysis in diverse biomedical research settings. Full article

The expression of recombinant proteins in heterologous hosts is a common strategy to obtain larger quantities of the “protein of interest” (POI) for scientific, therapeutic or commercial purposes. However, the experimental success of such an approach critically depends on the choice of an appropriate host system to obtain biologically active forms of the POI. The correct folding of the molecule, mediated by disulfide bond formation, is one of the most critical steps in that process. Here we describe the recombinant expression of hirudin, a leech-derived anticoagulant and thrombin inhibitor, in the yeast Komagataella phaffii (formerly known and mentioned throughout this publication as Pichia pastoris) and in two different strains of Escherichia coli, one of them being especially designed for improved disulfide bond formation through expression of a protein disulfide isomerase. Cultivation of the heterologous hosts and expression of hirudin were performed at different temperatures, ranging from 22 to 42 °C for the bacterial strains and from 20 to 30 °C for the yeast strain, respectively. The thrombin-inhibitory potencies of all hirudin preparations were determined using the thrombin time coagulation assay. To our surprise, the hirudin preparations of P. pastoris were considerably less potent as thrombin inhibitors than the respective preparations of both E. coli strains, indicating that a eukaryotic background is not per se a better choice for the expression of a biologically active eukaryotic protein. The hirudin preparations of both E. coli strains exhibited comparable high thrombin-inhibitory potencies when the strains were cultivated at their respective optimal temperatures, whereas lower or higher cultivation temperatures reduced the inhibitory potencies. Full article

In establishing the safety or tolerability profile of bioactive plant extracts, it is important to perform toxicity studies using appropriate, accessible, and sustainable methods. The Allium cepa model is well known and frequently used for accurate environmental risk assessments, as well as for evaluating the toxic potential of the bioactive compounds of plant extracts. The present review focuses on this in vivo cytogenetic model, highlighting its widespread utilization and advantages as a first assessment in monitoring the genotoxicity and cytotoxicity of herbal extracts, avoiding the use of animals for testing. This plant-based assay allows for the detection of the possible cytotoxic and genotoxic effects induced on onion meristematic cells. The outcomes of the Allium cepa assay are comparable to other tests on various organisms, making it a reliable screening test due to its simplicity in terms of implementation, as well as its high sensitivity and reproducibility. Full article

Mitochondria play a crucial role in adapting to fluctuating energy demands, particularly in various heart diseases. In addition to functional analyses such as the measurement of ROS or ATP, analysis of mitochondrial ultrastructure can be used to draw further conclusions about their functions and effects in tissue. In this protocol, we introduce a set of measurements to compare the ultrastructural and functional characteristics of human left ventricular mitochondria, using transmission electron microscopy (TEM). Measured parameters included mean size in µm², elongation, count, percental mitochondrial area in the measuring frame, and a conglomeration score. We also introduce a novel method of defining hydropic mitochondria as a comparable evaluation standard. With this cluster of measurement parameters, we aim to contribute a protocol for studying human mitochondrial morphology, distribution, and functionality. Full article

Erythrocyte (RBC) aging involves significant structural and nanomechanical alterations crucial to their function. This study aims to bridge the gap between analyses based on statistical morphometric parameters, e.g., membrane roughness, and those based on point-dependent nanomechanical properties, e.g., stiffness or Young’s modulus. Using Atomic Force Microscopy, we investigated morphology, membrane roughness, and nanomechanical properties on the very same RBCs under dehydrated (air) and hydrated (physiological buffer) conditions. The cells were studied at different stages of in vitro aging: one, seven, and 12 days. Our results quantitatively show that across dehydration, as well as along the aging pathway, RBCs become progressively more rigid while their membrane roughness decreases, a trend observed in both environments. Notably, the differences between the hydrated and dehydrated states were large in young cells but diminished when erythrocytes aged. Despite these parallel trends, high-resolution mapping on the nanoscale revealed that roughness and Young’s modulus do not correlate, indicating that these parameters are linked to different properties. In conclusion, this work provides a comprehensive protocol for a biophysical description of RBC aging and establishes that the simultaneous measurement of membrane roughness and nanomechanical properties offers a complementary approach, yielding a more complete characterization of cellular properties. Full article

Vitamin D is the only vitamin that is conditionally essential, as it is synthesized from precursors after UV light exposure, whilst also being obtained from the diet. It has numerous health benefits, with deficiency becoming a major concern globally, such that dietary supplementation has more recently achieved vital importance to maintain satisfactory levels. In recent years, measurements made from blood have, therefore, become critical to determine the status of vitamin D levels in individuals and the larger population. Tests for vitamin D have routinely relied on laboratory analysis with sophisticated equipment, often being slow and costly, whilst rapid immunoassays have suffered from poor specificity and sensitivity. Here, we have evaluated a new rapid immunoassay test on the market (Rapi-D & IgLoo) to quickly and accurately measure vitamin D levels in small capillary blood specimens and compared this to measurements made using the standard laboratory method of liquid chromatography and mass spectrometry. Our results show that vitamin D can be measured very quickly and over a broad range using the new method, as well as correlate relatively well with standard laboratory testing; however, it cannot be fully relied upon currently to accurately diagnose deficiency or sufficiency in individuals. Our statistical and comparative analyses find that the rapid immunoassay with digital quantification significantly overestimates vitamin D levels, leading to diminished diagnosis of vitamin D deficiency. The speed and simplicity of the rapid method will likely provide advantages in various healthcare settings; however, further calibration of this rapid method and testing parameters for improving quantification of vitamin D from capillary blood specimens is required before integration of it into clinical decision-making pathways. Full article

Air pollution is linked to childhood mortality and morbidity in low- and middle-income countries globally. There is growing evidence linking air pollution to asthma and other respiratory diseases in children. Studies have shown that children are likely to experience asthma due to their narrow airways and their heightened sensitivity to environmental irritants. This study aims to investigate the relationship between ambient air pollution and respiratory diseases in children under the age of 5. The study will be conducted in the informal township of Alexandra, north of Johannesburg, South Africa. A quantitative approach will be used in this cross-sectional analytical study. Data will be collected using different tools that include a questionnaire to determine the prevalence of asthma and respiratory disease and potential risk factors. While environmental air pollution will be measured using Radiello passive samplers and Gillian pumps. Data will be analyzed using the latest version of the STATANow/MP 19.5 software. Furthermore, health risk assessment will be conducted for lifetime non-carcinogenic and carcinogenic risk estimation following the USEPA framework. The study will identify environmental triggers that exacerbate asthma and other respiratory conditions in other similar community settings and will contribute to the body of knowledge in public health. Ethical approval was obtained from the Research Ethics Committee, Faculty of Health Sciences at the University of Johannesburg. Full article

Faecal microbiota transplantation (FMT) is an emerging therapy for gastrointestinal and neurological disorders, acting via the microbiota–gut–brain axis. Altering gut microbial composition may influence cognitive function, but this has not been tested in cognitively healthy adults. This randomised, double-blinded, placebo-controlled pilot trial investigates whether FMT is feasible and improves cognition in adults with irritable bowel syndrome (IBS). Participants receive a single dose of FMT or placebo via rectal retention enema. Cognitive performance is the primary outcome, assessed using the Cambridge Neuropsychological Test Automated Battery (CANTAB). Secondary outcomes include IBS symptom severity and mood. Tertiary outcomes include microbiome composition and plasma biomarkers related to inflammation, short-chain fatty acids, and tryptophan metabolism. Outcomes are assessed at baseline and at one, three, six, and twelve months following treatment. We hypothesise that FMT will lead to greater improvements in cognitive performance than placebo, with benefits extending beyond practice effects, emerging at one month and persisting in the long term. The findings will contribute to evaluating the safety and efficacy of FMT and enhance our understanding of gut–brain interactions. Full article