Methods and Protocols

The increasing frequency, duration, and intensity of wildfires over the past decade have raised significant concerns about widespread exposure to wildfire smoke. Inhalation of wildfire smoke poses a substantial risk to human health, with epidemiological studies linking exposure to cardiovascular, respiratory, and neurological dysfunction. Wildfire smoke contains hundreds of chemical compounds across diverse classes, with concentrations varying by fuel type and combustion conditions. Phenolic compounds are prominent constituents of wood smoke, and catechol is especially abundant under smoldering conditions that produce dense smoke. In this study, ¹⁴C-labeled catechol was spiked into smoldering eucalyptus wood smoke extract (WSE) and administered to rats via intranasal instillation. Plasma was collected at 5 min and 2 h post-exposure. Samples were analyzed using parallel accelerator and molecular mass spectrometry (PAMMS). Major catechol-derived metabolites identified included benzene oxide, catechol-cysteine conjugate, and catechol-glutamine conjugate; the parent compound was not detected. These results indicate that inhaled catechol in wood smoke is quickly metabolized upon entry into circulation. PAMMS enabled both identification and relative quantification of circulating catechol metabolites, demonstrating feasibility for biomarker discovery and exposure assessment. Full article

A straightforward whole-mount approach has been developed that uses fluorescence imaging, mouse trachea, and a range of off-the-shelf reagents for rapidly evaluating substance toxicity within the ciliated respiratory epithelium. Using this protocol, the lumen of control trachea samples displays a typical cobblestone epithelial structure, a high density of ciliated cells, and minimal evidence of cell death, as visualized by phalloidin, acetylated tubulin, and fixable live/dead staining, respectively. In contrast, trachea subjected to treatments that induce injury show disrupted epithelial architecture and increased cell death, indicating substance toxicity. These results support the utility of this protocol for rapidly detecting and quantifying respiratory epithelial toxicity and differential cell-type susceptibility. Full article

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. Full article

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. Full article

Drug discovery remains a time-consuming and costly process, necessitating innovative computational approaches to accelerate early stage target identification and compound development. We introduce AgentMol, a modular multimodel AI system that integrates large language models, chemical language modeling, and deep learning–based affinity prediction to automate the discovery pipeline. AgentMol begins with disease-related queries processed through a Retrieval-Augmented Generation system using the Large Language Model to identify protein targets. Protein sequences are then used to condition a GPT-2–based chemical language model, which generates corresponding small-molecule candidates in SMILES format. Finally, a regression convolutional neural network (RCNN) predicts the drug-target interaction by estimating binding affinities (pKi). Models were trained and validated on 470,560 ligand–protein pairs from the BindingDB database. The chemical language model achieved high validity (1.00), uniqueness (0.96), and diversity (0.89), whereas the RCNN model demonstrated robust predictive performance with R² > 0.6 and Pearson’s R > 0.8. By leveraging LangGraph for orchestration, AgentMol delivers a scalable, interpretable pipeline, effectively enabling the end-to-end generation and evaluation of drug candidates conditioned on protein targets. This system represents a significant step toward practical AI-driven molecular discovery with accessible computational demands. Full article

We previously developed a bead-coupled ligase detection reaction (LDR) assay that enables simple and rapid detection of single-nucleotide variations (SNVs) using synthetic oligonucleotide templates. In the present study, this approach was extended to genomic DNA extracted from colorectal cancer cell lines to evaluate its applicability to clinically relevant samples. Targeting codon 12 of the KRAS gene, PCR-amplified products served as templates for bead-coupled LDR, and fluorescence excitation–emission matrix (EEM) analysis was employed for signal readout. The four fluorophores used in the assay exhibited distinct spectral properties, allowing their signals to be clearly resolved within the EEM profiles. This mapping provided characteristic fluorescence signatures that revealed the underlying genotypes, enabling not only the distinction between homozygous and heterozygous states but also the precise identification of allele compositions, as exemplified by G/A, T/T, G/G, and G/C in colorectal cancer cell lines. The single-tube workflow, integrating magnetic bead capture with fluorescence-based detection, demonstrated robustness, speed, and cost-effectiveness compared with conventional mutation detection methods. These findings confirm that the LDR–EEM platform can be successfully applied to genomic DNA analysis, underscoring its potential as an accessible and reliable tool for SNV detection in both research and diagnostic contexts. Full article

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. Full article

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. Full article

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. Full article

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. Full article

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. Full article

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. Full article

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. Full article

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. Full article

Background: Shoulder soft tissue disorders, such as rotator cuff tears and subacromial impingement, are among the most common causes of musculoskeletal disability. Both physical examination tests and imaging techniques are routinely used in clinical settings; however, their respective contributions to patient outcomes and their potential complementarity remain underexplored. Methods: A systematic review and meta-analysis were conducted following PRISMA 2020 guidelines. Controlled clinical studies comparing pre- and post-intervention outcomes in adults with suspected or confirmed shoulder soft tissue pathology were included. Two groups were analyzed: studies using musculoskeletal imaging (ultrasound or MRI) and studies applying orthopedic physical examination tests (e.g., Neer, Hawkins, and Jobe). Functional outcomes were converted into standardized mean differences (SMDs) and synthesized using a random-effects model. Heterogeneity was quantified using the I² statistic. Results: In total, 11 studies met the inclusion criteria (n = 6 imaging, n = 5 orthopedic tests). Imaging-based studies showed a pooled SMD of 4.85 (95% CI: 2.77–6.93), indicating substantial clinical improvement. Orthopedic test-based studies yielded a pooled SMD of 2.34 (95% CI: 1.27–3.41). Heterogeneity was high across both groups (I² > 90%). Conclusions: Imaging was associated with a larger overall clinical effect, while orthopedic tests provided functional insight valuable for screening and monitoring. These findings support the complementary use of both strategies to enhance diagnostic accuracy and treatment planning in shoulder care. Full article

Background: Developing clinically relevant experimental models of the human airway can significantly advance our understanding of the mechanisms underlying airway diseases and aid in translating potential therapies to clinical settings. The aim of this study is to establish an ex vivo human airway tissue culture model. Methods: Human donor airway tissues were obtained from clinical cases of lung transplantation. Our established method is based on the concept of scavenging metabolic activity and controlling bacterial growth and includes increased media volume, frequent media exchange, and antifungal additives to efficiently maintain the homeostatic culture environment. After a 3-day culture period, the airway was investigated, and its viability and function were compared with a standard cell culture method. Results: Control tissue exhibited significant acidosis after 3 days, suggesting high metabolic activity of airway tissue and bacterial contamination. The airway epithelial viability—after culturing in our established method for 3 days—was better than that of the controls. We only performed an acute but early investigation of the cultures as airway complications have been known to start early at the proximal bronchus after transplantation. H&E and alcian blue staining showed intact morphology of the epithelium of airway tissue and mucus layers after 3 days in our model, while controls showed remarkable damage to the epithelial layer. Newly synthesized glycoproteins were detected in the epithelial layer using metabolic labeling and the click chemistry technique, suggesting cellular protein synthesis of the airway tissue in our established ex vivo model. Conclusions: We successfully established a reproducible model of human ex vivo airway tissue culture (n = 3 independent biological samples) that may be useful for investigating airway complications and developing their therapies. Full article

In the nuclear industry, the decontamination of nuclear metallic structures is an essential process to reduce radiation exposure during maintenance or dismantling. The oxide layer, such as chromium (III) oxide (Cr₂O₃), formed on stainless steel and nickel-based alloys, contributes significantly to surface radioactivity by trapping radioactive contaminants. To address this, permanganic acid (HMnO₄) has proven to be a promising oxidizing agent for dissolving these oxide layers—particularly chromium oxide—on stainless steel and nickel-based alloys. In this study, HMnO₄ was synthesized via ion exchange using AmberLite IRN97 H resin and potassium permanganate (KMnO₄). The optimized process yielded a highly acidic solution (pH~1.6) with potassium concentrations below 0.1 ppm, indicating near-complete exchange efficiency. Dissolution kinetics were investigated at HMnO₄ concentrations ranging from 240 to 1920 ppm and temperatures from 30 °C to 80 °C. At a constant temperature, increasing HMnO₄ concentration significantly improved Cr dissolution, with up to 31% of total chromium solubilized after 33 h. Lower temperatures favored higher dissolution efficiency, likely due to improved thermal stability of HMnO₄. For durations shorter than 4 h, the influence of temperature was limited compared to the effect of acid concentration. To assess post-treatment options, HMnO₄ decomposition was studied using oxalic acid (H₂C₂O₄) at 80 °C. Results showed that a minimum H₂C₂O₄/HMnO₄ molar ratio above 2.75 was necessary to achieve effective reduction while preventing MnO₂ precipitation. However, even under strongly acidic conditions and with a large excess of reductant, Mn²⁺ yields remained below 55%, suggesting that thermal degradation of oxalic acid and possible formation of undetected manganese species limited the reduction process. Full article

Hemodynamics significantly impact the biology of endothelial cells (ECs) lining the blood vessels. ECs are exposed to various hemodynamic forces, particularly frictional shear stress from flowing blood. While physiological flows are critical for the normal functioning of ECs, abnormal flow dynamics, known as disturbed flows, may trigger endothelial dysfunction leading to atherosclerosis and other vascular conditions. Such flows can occur due to sudden geometrical variations and vascular abnormalities in the cardiovascular system. In the current study, a microfluidic system was used to investigate the impact of different flow conditions (i.e, normal vs. disturbed) on ECs in vitro. We particularly explored the relationship between specific flow patterns and cellular pathways linked to oxidative stress and inflammation related to atherosclerosis. Here, we utilized a 2D cell culture perfusion system featuring an immortalized human vascular endothelial cell line (EA.hy926) connected to a modified peristaltic pump system to generate either steady laminar flows, representing healthy conditions, or disturbed oscillatory flows, representing diseased conditions. EA.hy926 were exposed to an oscillatory flow shear stress of 0.5 dynes/cm² or a laminar flow shear stress of 2 dynes/cm² up to 24 h. Following flow exposure, cells were harvested from the perfusion chamber for quantitative PCR analysis of gene expression. Reactive oxygen species (ROS) generation under various shear stress conditions was also measured using DCFDA/H2DCFDA fluorescent assays. Under oscillatory shear stress flow conditions (0.5 dynes/cm²), EA.hy926 ECs showed a 3.5-fold increase in the transcription factor nuclear factor (NFκ-B) and a remarkable 28.6-fold increase in cyclooxygenase-2 (COX-2) mRNA expression, which are both proinflammatory markers, compared to static culture. Transforming growth factor-beta (TGFβ) mRNA expression was downregulated in oscillatory and laminar flow conditions compared to the static culture. Apoptosis marker transcription factor Jun (C-Jun) mRNA expression increased in both flow conditions. Apoptosis marker C/EBP homologous protein (CHOP) mRNA levels increased significantly in oscillatory flow, with no difference in laminar flow. Endothelial nitric oxide synthase (eNOS) mRNA expression was significantly decreased in cells exposed to oscillatory flow, whereas there was no change in laminar flow. Endothelin-1 (ET-1) mRNA expression levels dropped significantly by 0.5- and 0.8-fold in cells exposed to oscillatory and laminar flow, respectively. ECs subjected to oscillatory flow exhibited a significant increase in ROS at both 4 and 24 h compared to the control and laminar flow. Laminar flow-treated cells exhibited a ROS generation pattern similar to that of static culture, but at a significantly lower level. Overall, by exposing ECs to disturbed and normal flows with varying shear stresses, significant changes in gene expression related to inflammation, endothelial function, and oxidative stress were observed. In this study, we present a practical, optimized system as an in vitro model that can be employed to investigate flow-associated diseases, such as atherosclerosis and aortic aneurysm, thereby supporting the understanding of the underlying molecular mechanisms. Full article

This study discusses the challenges encountered in implementing a detailed protocol for upper abdominal imaging using magnetic resonance imaging (MRI), ranging from patient preparation and sequence selection to clinical applications. MRI is a valuable non-invasive imaging modality employed both in the early detection of diseases and as a complementary tool for the detailed characterization of various pathologies. Nevertheless, performing an abdominal MRI examination can be challenging; therefore, the understanding of sequences is particularly important, as changing the parameters can not only influence the quality of the images but also optimize scanning time improve patient experience during the examination. The methodology illustrates the purpose of each sequence and the critical role of appropriate patient preparation. Results highlighted the significance of these factors in the evaluation of hepatic lesions, showing that the proper choice of sequences and parameters is essential for distinguishing benign from malignant findings and for achieving an accurate diagnosis. It was also shown that MRI plays an important role as a complementary technique in investigation of upper abdominal pathologies in order to avoid overexposure to radiation. Full article