Search Results (2,156)

While the Ankom RF system facilitates efficient high-throughput in vitro fermentation studies, its high cost and limited flexibility constrain its broader applicability. To address these limitations, we developed and validated a low-cost, modular gas monitoring system (FerME), assembled from commercially available components. To evaluate its performance and reproducibility relative to the Ankom RF system (Ankom Technology, Macedon, NY, USA), in vitro rumen fermentation experiments were conducted under strictly controlled and identical conditions. Whole rumen contents were collected approximately 2 h post-feeding from individual mid- or late-lactation dairy cows and immediately transported to the laboratory. Each fermenter received 50 mL of processed rumen fluid, 100 mL of anaerobically prepared artificial saliva buffer, and 1.2 g of the donor cow’s diet. Bottles were sealed with the respective system’s pressure sensors, flushed with CO₂, and incubated in a 50 L water bath maintained at 39 °C. FerME (New Taipei City, Taiwan) and Ankom RF fermenters were placed side-by-side to ensure uniform thermal conditions. To assess the effect of filter bag use, an additional trial employed Ankom F57 filter bags (Ankom Technology, Macedon, NY, USA; 25 μm pore size). Trial 1 revealed no significant differences in cumulative gas production, volatile fatty acids (VFAs), NH₃-N, or pH between systems (p > 0.05). However, the use of filter bags reduced gas output and increased propionate concentrations (p < 0.05). Trial 2, which employed filter bags in both systems, confirmed comparable results, with the FerME system demonstrating improved precision (CV: 4.8% vs. 13.2%). Gas composition (CH₄ + CO₂: 76–82%) and fermentation parameters remained consistent across systems (p > 0.05). Importantly, with 12 pressure sensors, the total cost of FerME was about half that of the Ankom RF system. Collectively, these findings demonstrate that FerME is a reliable, low-cost alternative for real-time rumen fermentation monitoring and could be suitable for studies in animal nutrition, methane mitigation, and related applications. Full article

The kinetics of insulin aggregation and fibril formation were studied in vitro using Scanning Electron Microscopy (SEM) and Fourier Transform Infrared (FTIR) spectroscopy. Our investigation centered on the protein’s morphological and structural changes to better understand the transient molecular configurations that occur during the lag phase. SEM images showed that, already at early incubation stages, a network of disordered pseudo-filaments, ranging in length between 200 and 500 nanometers, develops on the surface of large aggregates. At later stages, fibrils catalyzed by protein aggregates were observed. Principal Component Analysis (PCA) of the FTIR data identified signatures of intramolecular β-sheet secondary structures forming during the lag phase and at the onset of the exponential growth phase. These absorption bands are linked to secondary nucleation mechanisms due to their transient nature. This interpretation is further supported by a chemical equilibrium model, which yielded a reliable secondary nucleation rate constant, K₂, on the order of 10⁴ M⁻² s⁻¹. Full article

The aim of this work is to evaluate the effectiveness of antimicrobial photodynamic therapy (aPDT) against oral MDR (multi-drug-resistant) Candida spp. infections related to orthodontic treatment with palatal expanders through in vitro study. Methods: PDT protocol: Curcumin + H₂O₂ was used as a photosensitizer activated by a 460 nm diode LED lamp, with an 8 mm blunt tip for 2 min in each spot of interest. In vitro simulation: A palatal expander sterile device was inserted into a custom-designed orthodontic bioreactor, realized with 10 mL of Sabouraud dextrose broth plus 10% human saliva and infected with an MDR C. albicans clinical isolate CA95 strain to reproduce an oral palatal expander infection. After 48 h of incubation at 37 °C, the device was treated with the PDT protocol. Two samples before and 5 min after the PDT process were taken and used to contaminate a Petri dish with a Sabouraud field to evaluate Candida spp. CFUs (colony-forming units). Results: A nearly 99% reduction in C. albicans colonies in the palatal expander biofilm was found after PDT. Conclusion: The data showed the effectiveness of using aPDT to treat palatal infection; however, specific patient oral micro-environment reproduction (Ph values, salivary flow, mucosal adhesion of photosensitizer) must be further analyzed. Full article

Background/Objectives: Tamoxifen, a selective estrogen receptor modulator widely used as an adjunct in the treatment of breast cancer, has known effects on bone metabolism, although its impact on osseointegration and cellular responses during early bone healing remains unclear. Understanding these effects is essential given the increasing use of dental implants in cancer survivors. The study aimed to observe the influence of tamoxifen on human osteosarcoma (SAOS-2) cells lines, as well on the osseointegration of titanium implants in ovariectomized female rats. Methods: SAOS-2 cells were incubated with Dulbecco’s modified growth medium. Six titanium (Ti) disks were used at each time point. The samples were divided into groups with the presence (TAM, n = 36) or not (CTR, n = 36) of tamoxifen in a concentration of 2 μM. In vivo, 72 animals were divided in groups with bilateral ovariectomy or SHAM and tamoxifen administration or not (15 mg/kg). Cell viability, mineralization rate, and collagen synthesis were assessed, as well as bone/implant contact (BIC) and bone ingrowth (BIN). Results: Tamoxifen caused a decrease in SAOS-2 viability, although an increase in the mineralization rate was observed. In vivo, the TAM groups presented higher BIC and BIN when compared to their control, but a lower percentage of mature collagen cells. Conclusions: Based on our findings, in vitro, the therapy with TAM slightly reduced the viability of SAOS-2 cells while significantly increasing the mineralization rate. In vivo, the therapy positively influenced BIC and BIN during the osseointegration phase. Full article

Plant latex is a rich source of proteolytic enzymes with potential biomedical applications, particularly in hemostasis. Among them, thrombin-like enzymes (TLEs) have garnered interest in their ability to mimic thrombin by catalyzing the conversion of fibrinogen to fibrin, facilitating clot formation. While TLEs from snake venoms have been well-characterized and applied clinically, their plant-derived counterparts remain underexplored. This review critically examines the structural and functional characteristics of TLEs from plant latex, comparing them to animal-derived TLEs and evaluating their role in both procoagulant and fibrinolytic processes. Emphasis is placed on dual fibrinogenolytic and fibrinolytic activities exhibited by latex proteases, which often vary with concentration, incubation time, and protease type. In vitro coagulation assays and electrophoretic analyses are discussed as critical tools for characterizing their multifunctionality. By addressing the knowledge gaps and proposing future directions, this paper positions plant latex proteases as promising candidates for development in localized hemostatic and thrombolytic therapies. Full article

Objective: To investigate the neuroprotective effects of a Rho-associated kinase (ROCK) inhibitor on retinal ganglion cells (RGCs) in vitro and in vivo. Methods: For in vivo studies, a unilateral optic nerve crush mouse model was established. Then, 100 mM Y-27632 (a ROCK inhibitor) or saline was applied to the experimental eyes once a day for 14 days. The effects of the ROCK inhibitor were evaluated by counting the surviving RGCs in the enucleated flat retina tissues and measuring the inner retinal thickness using optical coherence tomography (OCT), the amplitude of the electroretinogram (ERG), and the change in intraocular pressure (IOP). For the in vitro study, RGCs were isolated from five-day-old mice using a modified immunopanning method with magnetic beads. The isolated RGCs were incubated for 72 h with various concentrations of Y-27632, after which TUNEL assays were performed to determine the number of surviving RGCs. Results: Y-27632 has neuroprotective effects, as it significantly increased the number of surviving RGCs by approximately 6.3%. OCT and ERG data also revealed that Y-27632 induced neuroprotective effects in vivo; furthermore, Y-27632 reduced IOP by approximately 18.3%. The in vitro study revealed the dose-dependent neuroprotective effects of Y-27632, with the highest dose of Y-27632 (1000 nM) increasing the RGC survival rate after 72 h of incubation compared with that of the control. Conclusions: The ROCK inhibitor Y-27632 may exert some neuroprotective effects on RGCs when it is used as an eye drop through an IOP-independent mechanism. Full article

Background: Obesity is associated with insulin resistance (IR) and characterized by impaired activation of the PI3K/AKT route and glucose uptake. Elevated plasma levels of palmitic acid (PA) diminish insulin signaling in vitro and in vivo. Origanum vulgare L. essential oil (OVEO) is rich in monoterpenes with protective effects against IR. Objective: The study aimed to assess total phenols content and antioxidant activity of OVEO and its cytotoxicity, as well as its effect on insulin signaling and glucose uptake in PA-treated adipocytes. Methods: The quantification of total phenolic content was determined using the Folin–Ciocalteu method, while the antioxidant capacity of OVEO was assessed by DPPH (2,2-diphenyl-1-picrylhydrazyl) and FRAP (ferric reducing antioxidant power) methods. The cytotoxicity of OVEO (0.1–10 µg/mL) was assessed using the MTS assay. SW872 adipocytes were incubated with 0.4 mM PA for 24 h, with or without a 2 h preincubation of OVEO, and then stimulated with insulin (100 nM, 10 min) or a vehicle. Phosphorylation of Tyr-IRS-1, Ser-AKT, and Thr-AS160 was analyzed by Western blot, and glucose uptake was measured using 2-NBDG. Results: OVEO contained phenols and exhibits antioxidant capacity. All the concentrations of OVEO assessed were not cytotoxic on SW872 adipocytes. PA decreased basal phospho-AS160 as well as insulin-stimulated phospho-IRS1, phospho-AKT, phospho-AS160 and glucose uptake, while OVEO co-treatment enhanced these markers. Conclusions: These findings suggest a beneficial effect of OVEO on the PA-impaired insulin pathway and glucose uptake, which might be explained by its phenolic content and antioxidant capacity, highlighting its potential as a complementary therapeutic agent for IR and related metabolic disorders. Full article

Background: snoRNAs have traditionally been known for their role as guides in post-transcriptional rRNA modifications. Previously, our research group identified several RNAs that may bind to PIP2 with LIPRNA-seq. Among them, snR191 stood out due to its potential specific interaction with this lipid, distinguishing itself from other snoRNAs. However, a detailed study is needed to define the molecular interactions between RNA and lipids, which remain unknown but may serve as a mechanism for transport or liquid–liquid phase separation. This study aimed to determine the interaction between a snoRNA called snR191 and PIP2. Method: A novel methodology for RNA-PIP2 interaction was carried out. Total RNA from Saccharomyces cerevisiae was incubated with PIP2-bound nitrocellulose membranes and RT-PCR reactions. We performed the prediction of snR191-PIP2 interaction by molecular docking and in silico mutations of snoR191. Results: From LIPRNA-seq analysis, we identified that PIP2-bound RNAs were significantly enriched in diverse biological processes, including transmembrane transport and redox functions. Our RNA-PIP2 interaction approach was successful. We demonstrated that snR191 specifically interacts with PIP2 in vitro. The elimination of DNA ensured that the interaction assay was RNA-specific, strengthening the robustness of the experiment. PIP2 was docked to snR191 in a stem–loop–stem motif. Six hydrogen bonds across four nucleotides mediated the PIP2-snR191 interaction. Finally, mutations in snR191 affected the structural folding. Conclusions: In this study, we demonstrate the effectiveness of a new methodology for determining RNA–lipid interactions, providing strong evidence for the specific interaction between snR191 and PIP2. Integrating biochemical and computational approaches has allowed us to understand the binding of these biomolecules. Therefore, this work significantly broadens our understanding of snR191-PIP2 interactions and opens new perspectives for further research. Full article

F₄-Neuroprostanes (F₄-NeuroPs), oxidative metabolites of docosahexaenoic acid, act as bioactive lipid mediators enhancing sperm motility and induce capacitation-like changes in vitro. Their biological action is proposed to involve sperm ion channels, in particular ryanodine receptors (RyRs), which regulate intracellular calcium homeostasis. We evaluated the effects of dantrolene, a RyR inhibitor, on motility and vitality of a selected spermatozoa at different concentrations (10, 30, 50, 100 μM). Then sperm motility, acrosome integrity, and RyR localization following co-incubation with dantrolene (D50 or D100 μM) and 4-/10-F_4t-NeuroPs (7 ng) were investigated. Acrosomal status was assessed using Pisum sativum agglutinin (PSA) staining and RyR localization by immunofluorescence. D50 was identified as the minimum effective dose to induce significant reductions in sperm motility. F₄-NeuroPs significantly increased rapid progressive motility versus controls. Co-incubation with F₄-NeuroPs + D50 reduced rapid motility and increased in situ and circular movement. The acrosome staining appeared altered or absent to different percentages, and RyR localization was also seen in the midpiece. These findings suggested that F₄-NeuroPs enhance sperm motility via RyR-mediated pathways, as confirmed by dantrolene inhibition. Accordingly, our results underscore the physiological relevance of RyRs in sperm function and suggest new insights into lipid-based mechanisms regulating sperm motility. Full article

Mabroom dates (Phoenix dactylifera L.) are recognized as one of the most important crops in Qatar. Fresh fruit dates are susceptible to mould and post-harvest spoilage, resulting in a significant financial loss. Octanoic fatty acid (OFA) has been shown to regulate the growth of mould-causing organisms such as fungi and bacteria. It is known to have antibacterial properties. The objective of the current study was to evaluate the in vitro effect of OFA on the post-harvest pathogens of Mabroom fruits. Fresh, apparently healthy, and fully ripe Mabroom dates were obtained from the National Agriculture and Food Corporation (NAFCO). The chosen fruits were packed in sterile, well-ventilated plastic boxes and transported to the lab under controlled conditions. The fruits were distributed into five groups (G1 to G5). The groups G1, G2, and G3 received 1%, 2%, and 3.5% OFA, respectively, while G4 was left untreated and G5 was washed only with tap water as a positive control treatment. Each group contained 200 g of fresh and healthy semi-soft dates. The samples were then dried and incubated in a humidity chamber at 25 °C ± 2 for seven days. The signs and symptoms of decay were monitored and recorded. The presence of pathogens was confirmed via phenotypic and microscopic-based methods. The results showed a significant difference (p ≤ 0.05) among the groups. OFA at 3.5% had the strongest inhibitory action against post-harvest pathogens, followed by OFA2%. However, there were no differences (p ≤ 0.05) between OFA1% and the control groups. Aspergillus spp., Penicillium spp., Rhizopus spp., and Botrytis spp. were most abundant in the control group, followed by OFA2% and OFA1%, respectively. In conclusion, octanoic fatty acid at 3.5% may improve the quality of date fruits through its high antimicrobial activity, reduce the effect of post-harvest decay, minimize the loss of date fruits during storage, and improve the sustainability of date fruits. Further experiments are necessary to confirm the effectiveness of OFA as a green solution for sustainable date fruit production. Full article

This study aimed to investigate the tyrosine kinase inhibitor Nintedanib and its potential role in reversing epithelial–mesenchymal transition (EMT) induced by transforming growth factor beta 2 (TGF-β2) in retinal pigment epithelial (RPE) cells, along with its therapeutic potential using a mouse model of subretinal fibrosis. We hypothesized that the blockade of angiogenesis promoting and fibrosis inducing signaling using the receptor tyrosine kinase inhibitor Nintedanib (OfevTM) can prevent or reverse EMT both in vitro and in our in vivo model of subretinal fibrosis. Primary human retinal pigment epithelial cells (phRPE) and adult retinal pigment epithelial cell line (ARPE-19) cells were treated with TGF-β210 ng/mL for two days followed by four days of Nintedanib (1 µM) incubation. Epithelial and mesenchymal phenotypes were assessed by morphological examination, quantitative real-time polymerase chain reaction(qPCR) (ZO-1, Acta2, FN, and Vim), and immunocytochemistry (ZO-1, vimentin, fibronectin, and αSMA). Metabolites were measured using luciferase-based assays. Extracellular acidification and oxygen consumption rates were measured using the Seahorse XF system. Metabolic-related genes (GLUT1, HK2, PFKFB3, CS, LDHA, LDHB) were evaluated by qPCR. A model of subretinal fibrosis using the two-stage laser-induced method in C57BL/6J mice assessed Nintedanib’s therapeutic potential. Fibro-vascular lesions were examined 10 days later via fluorescence angiography and immunohistochemistry. Both primary and ARPE-19 RPE stimulated with TGF-β2 upregulated expression of fibronectin, αSMA, and vimentin, and downregulation of ZO-1, consistent with morphological changes (i.e., elongation). Glucose consumption, lactate production, and glycolytic reserve were significantly increased in TGF-β2-treated cells, with upregulation of glycolysis-related genes (GLUT1, HK2, PFKFB3, CS). Nintedanib treatment reversed TGF-β2-induced EMT signatures, down-regulated glycolytic-related genes, and normalized glycolysis. Nintedanib intravitreal injection significantly reduced collagen-1+ fibrotic lesion size and Isolectin B4+ neovascularization and reduced vascular leakage in the two-stage laser-induced model of subretinal fibrosis. Nintedanib can induce Mesenchymal-to-Epithelial Transition (MET) in RPE cells and reduce subretinal fibrosis through metabolic reprogramming. Nintedanib can therefore potentially be repurposed to treat retinal fibrosis. Full article

Porcine endogenous retroviruses (PERVs) are integrated into the genome of all pigs. As they can be released as infectious virus particles capable of infecting human cells in vitro, they pose a potential risk for xenotransplantation involving pig cells or organs. To assess whether pigs produce infectious human-tropic viruses, infection assays with human cells are required. There are three main types of assays. First is the incubation of human target cells with gamma-irradiated pig cells. This method ensures that viral transmission is assessed in the absence of replicating pig cells. However, gamma irradiation may alter gene expression in pig cells, potentially affecting the results. Second is the co-culture in a double-chamber system in which pig and human cells are separated by a porous membrane, preventing direct cell-to-cell contact. While this method allows for the detection of infection by free virus particles, it does not account for infection via cell-to-cell transmission, which is a common mode of retroviral infection. And third is the co-culture of pig cells with human cells expressing a resistance gene. The resistance gene allows selective elimination of pig cells upon the addition of a selection medium. This assay enables both free virus and cell-to-cell transmission as well as complete removal of pig cells, which may not be fully achieved in the first type of assay. The third assay best simulates the conditions of in vivo xenotransplantation. However, in all cases the selection of donor and recipient cells is crucial to the experimental outcome. Results only indicate whether a specific pig cell type releases PERVs and whether a specific human cell type is susceptible to infection. A negative infection result does not necessarily reflect the in vivo situation, in which a transplanted organ consists of multiple pig cell types interacting with a diverse range of human cells within a living organism. Knowledge of these limitations is important for authorities regulating clinical applications for xenotransplantation. Full article

Background/Objectives: The emergence of cathinone-based psychostimulants necessitates ongoing research and analysis of the characteristics and properties of novel derivatives. The metabolic fate of five novel cathinone-derived substances (ASProp, MASProp, MASPent, PySProp, and PySPent) containing a selenophene moiety was investigated in vitro and in vivo. Methods: All compounds were incubated individually with pooled human liver S9 fraction. A monooxygenase activity screening investigating the metabolic contribution of eleven recombinant phase I isoenzymes was conducted. Rat urine after oral administration was prepared by urine precipitation. Liquid chromatography–high-resolution tandem mass spectrometry was used for the analysis of all samples. Reversed-phase liquid chromatography (RPLC) and zwitterionic hydrophilic interaction liquid chromatography (HILIC) were used to evaluate and compare the metabolites’ chromatographic resolution. Results: Phase I reactions of ASProp, MASProp, MASPent, PySProp, and PySPent included N-dealkylation, hydroxylation, reduction, and combinations thereof. The monooxygenase activity screening revealed the contribution of various isozymes. Phase II reactions detected in vivo included N-acetylation and glucuronidation. Both chromatographic columns complemented each other. Conclusions: All substances revealed metabolic reactions comparable to those observed for other synthetic cathinones. Contributions from isozymes to their metabolism minimized the risk of drug–drug interactions. The identified metabolites should be considered as targets in human biosamples, especially in urine screening procedures. RPLC and HILIC can both be recommended for this purpose. Full article

Vibrio parahaemolyticus is the leading cause of bacterial diarrhea in humans from shellfish consumption. In Thailand, blood clam is a popular shellfish, but homemade cooking often results in insufficient heating. Therefore, consumers may suffer from food poisoning due to Vibrio infection. This study aimed to determine the effect of chitooligosaccharide conjugated with epigallocatechin gallate (COS-EGCG) at different concentrations (200 and 400 ppm) combined with high-voltage atmospheric cold plasma (HVACP) on inhibiting V. parahaemolyticus in vitro and in challenged blood clam meat. Firstly, HVACP conditions were optimized for gas composition and treatment time (20 and 60 s); a 70% Ar and 30% O₂ gas mixture resulted in the highest ozone formation and a treatment time of 60 s was used for further study. COS-EGCG conjugate at 400 ppm with HVACP (ACP-CE400) completely killed V. parahaemolyticus after incubation at 37 °C for 6 h. Furthermore, an antibacterial ability of ACP-CE400 treatment against bacterial cells was advocated due to the increased cell membrane damage, permeability, and leakage of proteins and nucleic acids. Scanning electron microscopy (SEM) showed cell elongation and pore formation, while confocal microscopy revealed disrupted biofilm formation. Additionally, the shelf life of challenged blood clam meat treated with ACP-CE400 was extended to nine days. SEM analysis revealed damaged bacterial cells on the meat surface after ACP-CE400 treatment, indicating the antibacterial activity of the combined treatment. Thus, HVACP combined with COS-EGCG conjugate, especially at a highest concentration (400 ppm), effectively inhibited microbial growth and extended the shelf life of contaminated blood clam meat. Full article

Balantioides coli is the only ciliate currently described as an intestinal parasite of humans, although it can also infect other animals, particularly pigs. Its in vitro cultivation remains challenging, and no axenic culture system is currently available. Cultures are initiated by adding small amounts of feces containing cysts or trophozoites to the culture medium. Implantation success is lower when starting from cysts, and the mechanisms and early events of excystation remain poorly understood. In this study, we describe the sequence of events involved in excystation and identify factors potentially important for culture establishment. Cysts were obtained from orangutan feces and genetically confirmed as B. coli. Only viable cysts, determined by trypan blue or methylene blue exclusion, were used. After artificial digestion with pepsin and trypsin, cysts were incubated at 28 °C for up to 72 h in DMEM supplemented with L-glutamine, yeast extract, fetal bovine serum, and starch granules. Excystation began with a fissure in the cyst wall, allowing for bacterial entry. This appeared to stimulate the trophozoites, the increased motility of which progressively weakened and ruptured the wall, allowing for their emergence. Wall rupture and bacterial entry were critical for activation., whereas starch type had no apparent influence. Excystation occurred within the first hours; otherwise, cysts degenerated. Full article