Methods and Protocols最新文献

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.