Methods and Protocols最新文献

Background: Effective cetylpyridinium chloride (CPC)-based mouthwashes critically depend on maintaining maximum levels of bioavailable CPC to deliver optimum antimicrobial benefits. While this is traditionally assessed using cellulose-based methods, from economic and efficiency perspectives, there remains a need to identify other potential methods of assessing bioavailable CPC. Here, we explored whether quaternary ammonium compound (QAC) test strips are sensitive to CPC-based formulations, and if so, whether there might exist a possible correlation with glycolysis outcomes. Methods: Quantitative color parameters were obtained using spectrophotometric assessments of QAC test strips immersed in simple CPC solutions and eight commercial CPC-based mouthwashes available in the USA. Then, using our established glycolysis model, we assessed the glycolytic response of both the simple CPC solutions and commercial CPC-based mouthwashes, and compared these data sets. Results: Significant differences (p < 0.05) among the CPC simple solutions were found. Importantly, spectrophotometric assessments and glycolysis trials produced good correlations. Evaluations of the commercial mouthwashes further underlined this correlation, even though those that comprise zinc salts may impact QAC-based color. Conclusions: Based on these results, we believe the use of QAC test strips provides an attractive option to formulators and brands specializing in the development and/or testing of CPC-based oral care formulations.

Accurate quantification of iron is essential in biological, chemical, and nanomaterial research, yet commonly used ferrozine-based assays suffer from safety hazards, inconsistent reduction efficiency, and unstable absorbance readings. To address these issues, we systematically optimized the classical protocol and validated improvements that enhance both operational safety and analytical reproducibility. In this work, samples were digested using perchloric acid and hydrogen peroxide, reduced with hydroxylamine, and complexed with ferrozine, with all steps quantitatively evaluated to identify conditions that minimize variability. The optimized assay introduces three key refinements: combining the two traditional hydroxylamine additions into a single reduction step, extending the post-complexation incubation to 2 h to ensure complete formation of the Fe²⁺-ferrozine complex, and performing digestion exclusively in 5 mL screw-cap polypropylene tubes to eliminate tube-bursting events frequently observed with flip-cap formats. Kinetic analysis confirmed that absorbance at 562 nm reaches a stable plateau after 2 h, and the resulting standard curve exhibited excellent linearity (R² = 0.9999). These improvements significantly enhance precision, safety, and ease of implementation. The refined method is broadly applicable and enables reliable quantification of iron in tissues, cultured cells, aqueous solutions, and iron-containing nanomaterials.

Cerebrospinal fluid (CSF) is a key biological matrix that reflects the physiological and pathological states of the central nervous system (CNS). It supports brain function by regulating ionic balance, facilitating molecular transport, and clearing metabolic waste. In this article, we present a standardized protocol for CSF collection along with an integrative multiplatform metabolomic workflow that combines proton nuclear magnetic resonance spectroscopy (¹H-NMRS) and high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS). Integrating these complementary analytical modalities enhances metabolite coverage and improves analytical robustness, enabling a more comprehensive and reliable characterization of the CSF metabolome. This workflow supports the discovery of potential biomarkers and advances our understanding of neurochemical alterations within the CNS.

Metabolic syndrome is a disorder of energy metabolism characterized by persistently high prevalence and significant medical and economic burden on society. An effective animal model that closely replicates the key features of the syndrome in humans is essential for evaluating therapeutic strategies aimed at improving health outcomes. High-calorie diet-induced animal models of metabolic syndrome are preferred by many research groups for studying its pathogenesis, prevention and therapy. However, there are numerous variations in the types and proportions of carbohydrates and/or fats in the diets used. In 2015, our research team developed a diet-induced model of metabolic syndrome in young adult male Wistar rats that was based on adding 17% animal fat and 17% fructose to the standard rat chow and 10% fructose to the drinking water. This model reliably induced the morphometric and biochemical alterations that represent the core diagnostic features of the syndrome in humans. Since its initial introduction, we have utilized the high-fat high-fructose diet-induced model of metabolic syndrome/obesity in ten experimental studies. The current paper provides a protocol for applying the model, presents its repeatability and discusses the variability in the morphometric, biochemical, histopathological, immunohistochemical, and behavioral data of 10 experimental studies on Wistar rats.

Setting is a fundamental movement in volleyball. While there are several optimal interpreters of the role in professional play, there is a surprising lack of advanced measurement techniques for the evaluation of the movement from a biomechanical perspective. We proposed a comprehensive motion analysis protocol based on kinematics and motor coordination assessment (muscle synergies) for an in-depth analysis of the setting gesture. We also quantified the test-retest performance and discussed in detail the potential of the method. A single experienced player (age 27) tested and retested the protocol. The protocol was quite rapid to perform (about 30 min, including placement of kinematic and electromyography sensors on the patient's body); we found high test and re-test consistency in different sessions within this participant (ICC > 0.90). These preliminary results suggest that the protocol could support the use of the state-of-the-art methods for motion analysis and biomechanics in volleyball and sports in general.

Amniotic membrane (AM) has gained wide application in regenerative medicine due to its biocompatibility and extracellular matrix (ECM) composition. Effective decellularization is essential to minimize immunogenicity while preserving tissue architecture. This study standardized AM procurement and compared a simplified alkaline-based decellularization protocol with a conventional detergent-alkaline method, emphasizing practicality, histological integrity, and collagen preservation.

Methods: Human AM was aseptically obtained from placental tissue and processed using either method. Histological analysis with hematoxylin eosin and Masson's trichrome staining quantified nuclear content and collagen integrity.

Results: The alkaline method achieved the greatest nuclear clearance but retained epithelial outlines, indicating partial persistence of cellular structures. In contrast, the detergent method achieved complete morphological decellularization but showed slightly higher residual nuclear signal. Masson's trichrome staining revealed that the detergent-based method preserved collagen intensity most closely to native tissue (mean gray values: 128.3 ± 28.2 vs. 140.2 ± 23.4), while the alkaline group exhibited significantly reduced staining (177.8 ± 17.2; p < 0.001).

Conclusions: the simplified alkaline method provided efficient decellularization with reduced cost, time, and cytotoxic risk, making it a practical approach for AM processing. However, partial ECM alteration suggests that detergent-based methods remain preferable when optimal structural preservation is required.

The emergence and spread of coronavirus infections have created a necessity to develop serological methods for assessing population immunity. The enzyme-linked immunosorbent assay (ELISA) remains one of the most accessible and informative approaches for these purposes. The choice of recombinant proteins plays an important role in the sensitivity and specificity of the test system, and in this regard, the creation of a domestic ELISA based on the chimeric SM protein to the SARS-CoV-2 virus is relevant. In this work, a recombinant chimeric SM protein expressed in the E. coli system and purified using metal-affinity chromatography on Ni-NTA agarose was constructed and presented for the first time. An ELISA test system was developed and tested using panels of positive and negative sera, including samples obtained before the COVID-19 pandemic. The obtained sensitivity (90.48%) and specificity (93.65%) indicators with a ROC curve AUC = 0.9623 (OD450 = 0.213) indicate the diagnostic accuracy of the test system. The positive diagnostic ratio (LR+) = 14.25.0 indicates the reliability of a positive result. The domestically developed ELISA test system can be used for serological monitoring and assessment of the immune status of the population.

The creation of bioenergy based on the biomass wood pellet industry, which accounts for the majority of the global biomass supply, is one of the most common and important ways to utilize waste wood, wood dust, and other byproducts of wood manufacturing, known as forestry residues. Pellet production processes might greatly benefit from fast monitoring systems that may allow for at least a semi-quantitative measurement of crucial parameters such as lignin and cellulose. The determination of lignin and cellulose is complicated and time-consuming because it usually requires time-demanding and labor-intensive sample preparation. This, however, might be a crucial problem. In this context, the application of Raman spectroscopic techniques is considered a promising approach, as it enables rapid, reliable, and label-free analysis of wood pellets, providing information about the chemical composition of the biomass, specifically lignin and cellulose. The purpose of this article is to report on the application of Raman spectroscopy exemplified by the detection of the lignin/cellulose ratio. In our methodological approach, we integrated the area under the selected Raman bands to avoid a large scatter of data when only the intensities of the bands were used. Moreover, the acquired Raman spectra displayed very strong signals from both substances, which contributes to the feasibility of the analysis even with a portable instrument. This study is expected to be of assistance in situations when the monitoring of the chemical changes and the quick inspection of pellets are required in near real time, online, and in situ.

Ordinary linear regression is the most common approach for modeling relationships between continuous outcomes and explanatory variables in epidemiological research. However, this method relies on restrictive assumptions-normality, homoscedasticity, and linearity-that are often violated in real-world biomedical data. When these assumptions fail, mean-based estimates may obscure important heterogeneity across the outcome distribution. This study aims to illustrate the methodological and interpretive advantages of quantile regression over ordinary regression in the analysis of epidemiological data. Secondary data were derived from a cross-sectional study of 1415 healthy Greek adults aged 25-82 years. Body mass index (BMI) served as the outcome variable, while sex, age, physical activity, dieting status, and daily energy intake were considered predictors. Both ordinary and quantile regression models were applied to estimate associations between BMI and its determinants across the 25th, 50th, 75th, and 90th quantiles. Ordinary regression identified positive associations of BMI with age and energy intake and a negative association with physical activity. Quantile regression revealed that these relationships were not constant across the BMI distribution. The inverse association with physical activity intensified at higher quantiles, and the gender effect reversed direction at the upper tail, suggesting heterogeneity was not captured by mean-based models. Quantile regression provides a distribution-sensitive alternative to ordinary regression, offering insight into covariate effects across different points of the outcome distribution and serving as both a robust analytical tool and an educational framework for applied epidemiological research.

Live imaging has been instrumental in understanding cellular dynamics in Drosophila tissues, but technical limitations have prevented the long-term visualization of cell competition in adult brains. Here, we describe a simple ex vivo protocol that enables extended live imaging of adult Drosophila brains for up to 32 h. The method relies on non-supplemented Schneider's Drosophila medium and hydrophobic interactions to maintain brain stability during imaging, eliminating the need for complex culture conditions or embedding procedures. We validate this approach by studying cell competition in the optic lobes following traumatic brain injury, where cell competition is expected to occur with a peak at 48 h after damage. We demonstrate the value of this method by visualizing the expression of the fitness checkpoint Azot in a loser cell and its subsequent elimination. This protocol offers a versatile platform for studying cell competition and other cellular processes requiring extended observation of the adult Drosophila brain.