Methods and Protocols最新文献

Investigation of molecular mechanisms that underlie the toxicity of reactive oxidants requires the usage of reductionist cellular models, where laboratory cultures are treated by known doses of the target compounds in strictly controlled conditions. In recent years, much attention has been focused on hypothiocyanous acid (HOSCN), a pseudohypohalous acid that is one of the main products of chordata heme peroxidases. Due to its instability, HOSCN cannot be purchased and stored, so it has to be enzymatically synthesized before each experiment. For the first time, we systematically classified the published protocols for HOSCN synthesis, compared them by product yield, and found that the highest achievable concentration was about 1.9 mM. This value is not convenient for large-scale experiments with high cell density. Therefore, we developed an improved protocol for HOSCN preparation by optimizing reagent ratios, incubation times, and temperature. The current paper describes all steps from scratch, namely lactoperoxidase purification via a combination of cation exchange, hydrophobic interaction, and size exclusion chromatography, HOSCN synthesis from SCN^- and H₂O₂, as well as HOSCN concentration measurement. The main advantage of the current protocol is that the product yield reaches 2.9 mM, which is 60% higher than published alternatives.

L-Carnitine (L-CAR) and acetyl-L-carnitine (Acetyl L-CAR) are the essential cofactor compounds in lipid metabolism and are used in the treatment of various diseases. The European Food Safety Authority (EFSA) has reported that Acetyl-L-CAR contributes to normal cognitive function and has a beneficial physiological effect. Therefore, the sensitive separation and determination of L-CAR and Acetyl-L-CAR in foodstuffs can provide critical information. A notable trend in modern food analysis is the increasing use of miniaturized analytical columns with a narrow inner diameter (ID). In this study, a new, green analytical method for food analysis was developed to analyze L-CAR and Acetyl-L-CAR in food samples by nano-LC/UV with a hydrophilic monolithic 100 µm ID capillary. This is the first time that the preparation and application of a hydrophilic monolithic nano-column for the analysis of L-CAR and Acetyl-L-CAR in food samples by nano LC/UV has been reported. The hydrophilic monolith was prepared using in situ co-polymerization of glyceryl methacrylate (GMM) and ethylene dimethacrylate (EDMA). Following preparation and characterization, the hydrophilic monolith was used to analyze L-CAR and Acetyl-L-CAR in food samples, including three infant powdered milk samples and five supplements using nano LC/UV. The developed method was validated in terms of precision, sensitivity, linearity, recovery, and repeatability. The LOD and LOQ values were found to be in the range of 0.04-0.09 µg/kg, respectively. In short, the proposed method proved to be suitable for the routine analysis of L-CAR and Acetyl-L-CAR in food samples.

Retinal detachment (RD) disrupts the eye's immune-privileged status, causing a local inflammatory response that contributes to adverse clinical outcomes, including proliferative vitreoretinopathy and suboptimal visual recovery. Comprehensive profiling of intraocular immune cells will offer mechanistic insights and support the development of personalized immunomodulatory strategies. Here, we describe a robust and standardized protocol for the collection and high-dimensional analysis of the intraocular immune infiltrate from patients undergoing RD surgery, using state-of-the-art spectral cytometry. Vitreous and retinal tissue samples were obtained during standard surgical procedures, without the need for additional invasive interventions. Our approach integrates two complementary protocols: one that enables selective isolation of immune cells by sorting for CD45⁺ populations, and a second one that applies a 39-color spectral cytometry panel to profile the general landscape of immune subpopulations. The panel can identify up to 62 distinct viable immune subsets per sample, along with their functional status, as it includes expression of 13 functional markers. Hence, we hereby detail sample preparation, staining, and acquisition workflow, as well as the gating strategy and essential steps necessary for reproducible immunophenotyping. Our protocol, which enables high-dimensional immune profiling from minimal biological material, provides a valuable platform for studying ocular inflammation in RD and other retinal diseases.

We have developed a method for in vivo quantitation of lung delivery of inhaled nebulized drugs by measuring a fluorescent-labeled analog in bronchioalveolar lavage fluid (BALF) collected immediately after inhalation dosing. The effectiveness of delivery of an aerosolized formulation of our proprietary water-miscible vitamin A product to the deep lung (target organ) was studied; the product is being developed for prevention of bronchopulmonary dysplasia (BPD) in preterm infants. The fluorescent retinol analog was incorporated by spiking into a standard formulation, remaining fully compatible with existing nebulizer administration procedures for animal exposure. The method provides quantitation of the delivered dose (DD) to the lung within a few minutes after dosing; fluorescence in BAL in a plate reader allows for simple rapid quantitation of the delivered drug, while avoiding the complexities of other labeling methods (e.g., heavy labels or radioactivity). Data from newborn rat and lamb models showed linear dose responses, validating the method. Approximately 5-10% of the inhaled drug was recovered in BALF in both models, consistent with reports in the literature. The ease of use of the method facilitated various aspects of our project, including the transition to more clinically relevant animal models and aerosol exposure systems. The formulation of this approach could be spiked into other formulations, allowing application of the method to other aerosol drug development programs.

The presence of antioxidants in food contributes to the preservation of its taste and technological qualities, preventing its spoilage for a longer time, which is important at all stages of production and storage. The major antioxidants are vitamins, proteins (primarily, enzymes), peptides, amino acids, fatty acid residues of lipids, etc. There is currently an explosive growth in the development of methods for assessing the content and effectiveness of particular antioxidants but not the total antioxidant activity (AOA) in raw milk and food systems. This article provides a critical overview of the most important AOA methods, their mechanisms and applicability, advantages, and limitations (primarily, for antioxidants of milk and dairy products). Among all the antioxidant indicators of milk, the simplest and sufficiently informative is the detection of the total amount of water-soluble antioxidant (TAWSA), which is confirmed by comparison of numerous publications and practical results of various methods (as summarized in this review). It is important to emphasize that the TAWSA of milk is an "integral characteristic" of the most valuable biosubstances (possessing AOA) together. Therefore, the TAWSA method is recommended for assessing AOA in raw milk as an "integrated indicator" in dairy husbandry.

For many therapeutic agents to be effective against intracellular targets, they must first be able to penetrate the cell membrane. Current methodologies for assessing internalization, such as confocal microscopy and conventional flow cytometry, are limited by low throughput or an inability to provide precise spatial information on signal localization. Here, we present a comprehensive, semi-automated analytical pipeline for investigating compound internalization based on imaging flow cytometry, which is designed to address these limitations. Our workflow details the procedure from sample preparation and data acquisition on an Amnis FlowSight cytometer to analysis using IDEAS 6.2 software with a custom-designed template. Key features of our approach include the automated discrimination of signal between the plasma membrane and cytoplasmic compartments, the calculation of an internalization coefficient, and the introduction of a novel parameter-signal distribution entropy-to quantify the uniformity of the compound distribution within cells. For the statistical analysis, we developed FluoSta v1.0, a software tool that automates descriptive statistics and analysis of variance (ANOVA with Tukey's post hoc test) and facilitates data visualization. The pipeline's utility was demonstrated in a series of model experiments, including a comparative assessment of the internalization efficiency of PS- versus PS/LNA-modified compounds in MT-4 cell cultures.

The fatty acid composition of some seed oils from plants of emerging interest was studied. The benefits towards human health were evaluated by taking into account current recommendations regarding dietary intake of essential, polyunsaturated, and monounsaturated fatty acids and by discussing the pathologies for which such fatty acids exert protective action. Species studied were Hemp (Cannabis sativa), Flax (Linum usitatissimum), Milk Thistle (Silybum marianum), Perilla (Perilla frutescens), Borage (Borago officinalis), and Black Cumin (Nigella sativa). Seeds were subjected to cold milling in order to maintain their original nutritional characteristics. Chemical analyses were performed via the dual-detector gas chromatography technique by means of Flame Ionization Detection (FID) and mass spectrometry (MS) and by applying a modified version of the AOAC Official Method 991.39, thanks to which it was possible to obtain the fatty acid composition expressed as mg per gram of oil: such information is not always available in the literature for the species studied here. Comparison with the fatty acid international guidelines about the recommended intakes in g/day was made. This allowed us to evaluate whether such oils are suitable to be used as fatty acid food supplements to rebalance the Western diet, which is shown to be inadequate by numerous studies. Results show that seed oils from Cannabis sativa, Linum usitatissimum, and Perilla frutescens are suitable to be used as food supplements while seed oils from Silybum marianum, Borago officinalis, and Nigella sativa are not. It is important to note that any possible benefits from other parts of the plant (leaves, stems, flowers, and roots) are not studied or questioned by the present research, which focuses solely on fatty acids in the oil extracted from the seeds.

Intravenous medications are frequently administered through shared catheter lines in neonatal intensive care units (NICUs) due to the limited venous access in preterm infants, raising concerns about drug incompatibilities that may cause serious complications. Hydrocortisone sodium (HDC), ampicillin (ABPC), and cefotaxime (CTX) are commonly used in NICUs and are often co-administered with unfractionated heparin (UFH), which is routinely infused to prevent catheter occlusion. This study evaluated the physicochemical compatibility of HDC, ABPC, and CTX when mixed with UFH. Each drug was combined with UFH at equal volumes, and the mixtures were assessed immediately and after 3 h of storage by visual inspection, pH measurement, UV absorbance, and HPLC-UV analysis. No precipitation, turbidity, or color changes were observed in any mixture, and UV absorbance showed no relevant deviations compared with controls. Slight pH variations were detected but remained within acceptable limits. In semi-quantitative HPLC analysis, relative peak area changes were all below 10%, indicating no major degradation of the drugs. These findings suggest that HDC, ABPC, and CTX maintain acceptable physicochemical compatibility when co-administered with UFH, supporting their safe concomitant use in NICU practice.

Blood flow restriction (BFR) and body cooling (BC) have been investigated separately during exercise, but little is known about their concurrent use. This study examined acute metabolic responses, respiratory physiology, and rate of perceived exertion (RPE) during interval training (IT) performed with combined BFR and BC (VASPER ON) compared with IT without BFR and BC (VASPER OFF). It was hypothesized that VASPER ON would elicit greater physiological demands. A total of 7 female and 19 male participants (20.2 ± 2.4 years) completed a 21-min IT exercise. In VASPER ON, the participants wore cuffs that simultaneously applied BFR and BC. Total oxygen consumption (TVO₂), total carbon dioxide production (TVCO₂), total breaths (BRTH), and total ventilation (TVE) were measured during exercise (EX) and for 10 min post-exercise (Post-EX). RPE was recorded during EX. During EX, TVE and ventilatory equivalents for both oxygen and carbon were significantly higher in VASPER ON. Post-EX, all variables remained significantly elevated in VASPER ON except for the ventilatory equivalent for carbon dioxide. Sprint interval RPE was significantly lower in VASPER OFF. These findings suggest that concurrent BFR and BC increase post-exercise metabolic and ventilatory demands without attenuating each other's effect.

The Transdermal Drug Delivery System (TDDS) offers several benefits, such as enhanced patient adherence, controlled release, reduced gastric irritation, and the bypassing of the first-pass metabolism. However, not all drugs can be delivered through this route in effective doses. Biodegradable microneedles (BMn) are designed to improve TDDS. This review outlines various types of BMn and their fabrication methods. BMn are produced in different forms, including hollow, solid, dissolve, and hydrogel-forming versions, which have garnered significant attention. These innovative BMn do not contain drugs themselves but instead absorb interstitial fluid to create continuous channels between the dermal microcirculation and a drug-containing patch. Several types of BMn have been tested and approved by regulatory bodies. The use of BMn technology is rapidly growing in point-of-care applications, attracting significant interest from both researchers and healthcare providers. BMn-based Point-of-care (POC) devices have high efficacy for finding various analytes of clinical interests and transdermal drug administration in a minimally invasive manner owing to BMn' micro-size sharp tips and ease of use. Porous BMn technology may have a very rising future in the case of a vaccine delivery system.