Search Results (471)

This study presents a rapid, eukaryotic impedimetric biosensor that applies the yeast Saccharomyces cerevisiae as a robust, cost-effective biorecognition element for monitoring the acute toxicity of two representative pharmaceuticals, naproxen and isoniazid, in aquatic systems. The biosensor utilizes a previously developed three-electrode system made from type 316 stainless steel. Yeast cells seeded onto these electrodes serve as the biosensing element. By monitoring changes in electrical impedance, the system quantifies the cellular stress induced by pharmaceutical exposure. Electrochemical Impedance Spectroscopy (EIS) revealed a concentration-dependent decrease in both resistance and capacitance, attributed to cell death and subsequent desorption from the working electrode surface. These findings were validated through optical density at 600 nm (OD₆₀₀) growth curve analysis and methylene blue viability staining, which confirmed metabolic inhibition and membrane damage. Results indicate a linear response for naproxen within the 2.5 mM to 20 mM range, with a LOD of 0.509 mM, and for isoniazid within the 10 mM to 100 mM range, with a LOD of 0.684 mM. Naproxen demonstrated a more pronounced cytotoxic effect, with cell viability dropping to 41.08% at 10 mM compared to 68.79% for isoniazid. While conventional analytical methods focus on chemical quantification, this proof-of-concept biosensor provides a rapid toxic/non-toxic signal, offering a biologically relevant tool for real-time monitoring of industrial waste streams and acute environmental contamination. Full article

Pollution of rivers and large water bodies, including reservoirs, by wastewater from various sources is one of the most critical issues in the Donetsk region, requiring continuous monitoring and assessment of surface water quality. The research aims to assess the state of the Kalmius River under anthropogenic pressure, as well as to find correlations between the species composition, photosynthetic activity of phytoplankton, and the degree of water pollution. This study presents the results of biomonitoring of the Kalmius River and its tributaries within Donetsk City, which are under intense anthropogenic pressure. Pollution of the river channel by phenol, anionic surfactants, Ferrum ions, chlorides, and sulfates was identified. Based on the combinatorial pollution index, the water in the Kalmius River and its tributaries can be classified as polluted. The pigment composition of water samples was analyzed, and the species composition of river phytoplankton was determined. Dominant species include Chlorella vulgaris Beij., Dictyosphaerium pulchellum H.C.Wood, Scenedesmus quadricauda Brébisson, and Oscillatoria agardhii M.A.Gomont. Photosynthetic activity of the river’s algal flora was assessed based on chlorophyll fluorescence induction curves of natural phytoplankton. A correlation was established between surface water pollution levels and changes in the photosynthetic apparatus of microalgae cells. A strong negative correlation was found between the content of nitrate nitrogen in the aquatic environment and the photosynthetic activity, pigment composition, and abundance of the main dominant forms of phytoplankton, particularly the microalgae of the genus Cyclotella. The data obtained shows that the Kalmius River’s pollution has a significant impact on phytoplankton biodiversity, leading to the growth of cyanobacteria species. Full article

Background: Hepatocellular carcinoma (HCC) shows a high rate of early recurrence after curative resection, indicating a critical contribution of tumor microenvironment-driven molecular mechanisms. Early recurrence of hepatocellular carcinoma is defined as recurrence within 6 months after curative resection, with a prevalence exceeding 30%. Hypoxia signaling and immune dysregulation have been implicated, yet their compartment-specific relevance remains unclear. Methods: This multicenter nested case–control study included 49 HCC patients to evaluate associations between hypoxia-inducible factor-1 alpha (HIF-1α), vascular endothelial growth factor (VEGF), tumor-infiltrating lymphocytes (TILs), CD4⁺ T cells, CD8⁺ T cells, regulatory T cells (Tregs), programmed cell death protein 1 (PD-1), and programmed death-ligand 1 (PD-L1) and early recurrence after resection. TIL density was assessed using hematoxylin and eosin staining, while immunohistochemistry was performed to quantify intratumoral and peritumoral expression of the studied markers. Receiver operating characteristic (ROC) curve analysis was used to evaluate the predictive performance. Recurrence-free survival (RFS) was analyzed using the Kaplan–Meier, and independent predictors were identified using multivariate Cox proportional hazards regression. Results: Early recurrence occurred in 11 of 49 patients (22.4%) of Child–Pugh A patients. Recurrent tumors were characterized by elevated VEGF expression despite absent HIF-1α, alongside significant depletion of intratumoral TILs (HR 5.02; 95% CI 1.09–23.26), CD4⁺ (HR 7.68; 95% CI 1.66–35.60) and CD8⁺ cells (HR 6.68; 95% CI 1.77–25.23) and reduced peritumoral CD8⁺ infiltration (HR 4.20; 95% CI 1.11–15.91). Multivariable analysis identified low intratumoral CD4⁺ (HR 7.98; 95% CI 1.63–39.07) and reduced peritumoral CD8⁺ expression (HR 4.98; 95% CI 1.14–21.70) as independent predictors, whereas HIF-1α, VEGF, Treg, PD-1, and PD-L1 were not significantly associated. Conclusions: Early HCC recurrence shows HIF-1α-independent angiogenesis alongside spatial immune depletion, supporting integrated immune profiling over single angiogenic markers. Full article

The response of tree growth to environmental (climatic) changes has largely been analyzed through ring width–climate relationships, yet such analyses often lack the dynamic process of radial growth in response to environmental changes. Therefore, this study focuses on Korean pine (Pinus koraiensis Siebold & Zucc.) and white birch (Betula platyphylla Sukaczev) at three elevations (750 m, 950 m, and 1150 m) in the broadleaved Korean pine forest on the northern slope of Changbai Mountains, China. We systematically monitored cambial activity and the dynamics of xylem formation stages to analyze the different adaptation strategies of the two species in terms of phenology, cellular characteristics, growth rates, and climatic responses during cambial and xylem formation stages. The results showed that the phenological stages of xylem formation in Korean pine were more sensitive to elevation, while the phenological changes in birch were smaller, indicating greater growth stability. The seasonal dynamics of the number of xylem cell layers in both species followed a unimodal or sigmoid curve, but high elevations significantly inhibited the number of mature cell layers. Gompertz model fitting revealed that the maximum growth rate of Korean pine decreased significantly with increasing elevation, whereas no significant change was observed in birch. With increasing elevation, temperature emerged as the primary factor influencing cambial phenology and growth duration in both species, while precipitation dominated changes in growth rates. Xylem growth in Korean pine was co-regulated by growth rate (R² = 0.62) and growth duration (R² = 0.35), with tracheid diameter closely related to the duration of expansion (R² = 0.36). The regulatory pattern of xylem growth in birch was similar to that in Korean pine but with weaker correlations. In summary, Korean pine, as a coniferous dominant species, is more sensitive to temperature changes induced by elevation and adapts to elevational variations by adjusting phenology and cell development. In contrast, birch, as a broadleaved pioneer species, exhibits a high buffering capacity in xylem formation in response to elevational changes, thereby maintaining growth stability. The divergent growth strategies of the two species reveal the potential response pathways of temperate forest tree species to environmental changes and provide important insights for predicting the dynamics of broadleaved Korean pine forests. Full article

This study evaluates how titanium and polymethyl methacrylate (PMMA) Boston Keratoprosthesis backplate substrates influence human corneal fibroblast proliferation, cytotoxicity, morphology, activation phenotype, and mechanotransductive signaling. Human corneal fibroblasts were cultured on titanium and PMMA, with tissue culture plastic or glass as controls. Proliferation was assessed over 7 days using metabolic assays, and cytotoxicity was measured by lactate dehydrogenase release. Cell morphology and surface coverage were examined by scanning electron microscopy, while immunofluorescence quantified fibroblast-specific protein 1 (FSP-1) and α-smooth muscle actin (α-SMA). Gene expression of α-SMA, collagen I, FSP-1, and focal adhesion kinase (FAK) was analyzed by quantitative PCR. Cells cultured on both substrates maintained stable viability with modest increases in estimated cell numbers and comparable proliferation curves, indicating preserved metabolic activity without growth suppression. Cytotoxicity remained low and similar between groups. SEM demonstrated broader and more continuous cell spreading on titanium, whereas cells on PMMA were more sparsely distributed. Immunofluorescence showed higher FSP-1 expression on titanium and increased α-SMA on PMMA. Gene expression analysis revealed higher FAK transcripts on PMMA, with no significant differences in α-SMA, FSP-1, or collagen I. These results confirm the cytocompatibility of both titanium and PMMA backplates with human corneal fibroblasts and support their use with the Boston Keratoprosthesis. Full article

Since MDCK cells are inherently tumorigenic, their safety in vaccine production has long been a concern; thus, establishing a screening method for low-tumorigenic cells is of great significance for influenza vaccine development. This study successfully obtained a low-tumorigenic MDCK cell line through monoclonal screening and systematically evaluated its potential as a cellular substrate for influenza vaccines using male nude mice (BALB/c nu/nu, 4–7 weeks old) for tumorigenicity assessment. Comprehensive analysis of the biological characteristics of the screened cells—including growth curves and transcriptomic features—showed that the cell line exhibits stable growth and consistent traits. Transcriptomic comparison was performed between two defined biological states: parental MDCK cells (SQ group) and the low-tumorigenic clone MDCK-20B9 (SH group). Transcriptomic analysis revealed good dispersion among samples and an overall consistent gene expression distribution. Differential expression analysis identified a total of 2198 differentially expressed genes, including 902 upregulated and 1296 downregulated genes. GO functional enrichment analysis indicated that these genes are mainly involved in biological processes such as acute-phase response, retinol metabolism, mitotic chromosome condensation, and cell migration; are enriched in cellular components such as kinetochores and the extracellular matrix; and are associated with molecular functions including calcium ion binding and the Wnt signaling pathway. KEGG pathway analysis further revealed that the differentially expressed genes are significantly enriched in key pathways such as cancer pathways, cell cycle, and cell adhesion molecules. The expression trends of five key differentially expressed genes were validated by RT-qPCR. In summary, this study successfully screened a stable and consistent low-tumorigenic MDCK cell line, providing a theoretical basis and practical foundation for its use as a cellular substrate in influenza vaccine development. Full article

Background/Objectives: Clear cell renal cell carcinoma (ccRCC) involves complex interactions between immune evasion and metabolic reprogramming. This study aimed to characterize ccRCC through integrated immunometabolic profiling, develop a prognostic signature, and investigate the functional role of the key driver gene UCN using in vitro and in vivo approaches. Methods: Integrated immunometabolic profiling was performed to identify molecular subtypes and establish a prognostic gene signature. Two distinct molecular subtypes were identified, and a 9-gene Immune Metabolic Index (IMI) was constructed. The functional role of the key driver gene UCN was investigated through in vitro functional assays and in vivo xenograft models in BALB/c mice, including combination with PD-1 blockade. Results: Two molecular subtypes with significant survival differences (p < 0.001) were identified. The established IMI demonstrated high prognostic accuracy, with Area Under the Curve (AUC) values of 0.813, 0.751, and 0.779 at 1-, 3-, and 5-year intervals, respectively. UCN was identified as the highest-risk gene in the signature. Functional assays showed that UCN silencing significantly inhibited cell proliferation and migration (p < 0.05). In BALB/c mouse xenograft models, UCN silencing remodeled the tumor microenvironment by increasing CD8+ T cell infiltration and reducing regulatory T cells (p < 0.01). Furthermore, UCN knockdown significantly suppressed tumor growth and synergized with PD-1 blockade to enhance antitumor efficacy (p < 0.001). Conclusions: The IMI is a robust tool for risk stratification in ccRCC. Targeting the UCN-driven immunometabolic axis represents a promising therapeutic strategy to overcome immune resistance in ccRCC. Full article

Xanthomonas species are Gram-negative bacterial pathogens responsible for diseases in over 400 plant hosts, including numerous economically important crops such as Brassica species. The limited efficacy and environmental concerns associated with chemical control strategies underscore the need for sustainable and targeted alternatives. In this study, we evaluated the suitability and biocontrol efficacy of phages Phi1 and Phi3 to combat Xanthomonas campestris pv. campestris (Xcc) in broccoli plants. Kill-curve assays demonstrated that both phages effectively suppressed Xcc growth across a range of multiplicities of infection. Transmission electron microscopy further confirmed their lytic activity, revealing pronounced structural damage to Xcc cells following phage treatment, accompanied by the subsequent release of phage progeny. To assess host specificity and biosafety, the phages were tested against 41 bacterial isolates that were isolated and taxonomically characterized from broccoli and cauliflower in this study. Neither Phi1 nor Phi3 exhibited lytic activity against any non-target isolate, indicating high host specificity and minimal risk to the native Brassica-associated microbiota. In planta assays demonstrated that the combined application of Phi1 and Phi3 reduced Xcc-induced symptom severity in broccoli plants by 80%. Collectively, these results demonstrate that phages Phi1 and Phi3 represent effective and biologically precise agents for the control of black rot disease in Brassica crops. Full article

As an emerging oilseed crop in China, peony seed oils account for 0.41% of the annual production of Chinese edible vegetable oils, and the oil-type peony seed is rich in alpha-linolenic acid (ALA). Moisture content and temperature are key factors in the storage of oilseeds. In this study, the adsorption and desorption isotherms of ten species of peony seeds and one species of cake were determined in the range of 20–30 °C and 10–90% equilibrium relative humidity (ERH). The adsorption and desorption isotherms of peony seeds and cake were type II (sigmoidal) or type III curves. Nine equilibrium moisture content (EMC) equations were used to fit the isotherms of peony samples, with the optimal equations being our developed 7-parameter polynomial (Poly), modified Halsey equation (MHAE), and modified Oswin equation (MOE). For Poly, the fitting parameter determination coefficient (R²) was 0.9816–0.9986, and the mean relative error (MRE) was 0.83–6.52%; for MHAE, R² was 0.7815–0.9973, and MRE was 4.18–17.84%. Poly contains the terms of temperature and ERH interaction; therefore, Poly could analyze the safe moisture content of peony seeds and cake during storage and transportation, and the three-parameter reversible MHAE could be used for calculating the sorption isosteric heats. The adsorption monolayer moisture content (M₀) in peony seeds and cake estimated by MGAB were 3.64 ± 0.42% and 4.28%, respectively, while their desorption M₀ values, respectively, were 6.21 ± 0.47% and 4.83%. At ERH ≤ 65%, for preventing the growth of storage pests and fungi, the absolutely safe storage moisture content (MC) predicted by Poly at 25 °C and 65% ERH was 12.48% wet basis (w.b.) for seeds and 11.92% for cake. The heat of sorption of peony seeds and cake approached that of pure water at about 11% and 15% w.b. MC estimated by the MHAE model, respectively. Microstructure analysis showed that the rich liposomes in peony seeds were attached to the inner surface of the cell wall and the outer surface of the protein storage vacuole, and the rich protein bodies and hydrophilic polysaccharides explained why the safe storage moisture for yellow peony seeds was higher than for Ziyan Feishuang seeds. This study provides the basic data for drying simulation, and the safe storage and transportation of peony seed and cake products. Full article

Diseases caused by pathogenic microorganisms in Bombyx mori have long been a major constraint on the sustainable development of sericulture. Current preventive strategies remain substantially constrained by issues of drug resistance and environmental compatibility. In recent years, the application of nanomaterials for pathogenic microorganism control has garnered escalating attention. Among these, chitosan–silver nanoparticles (CS-Ag NPs), as an emerging class of nanocomposites, integrate the biocompatibility and biodegradability of chitosan with the robust antimicrobial activity of silver nanoparticles, thereby exhibiting considerable potential for preventing pathogenic infections. Nevertheless, the efficacy of CS-Ag NPs against B. mori pathogens has not previously been documented. In this study, CS-Ag NPs were successfully synthesized via chemical reduction. Their antiviral activity was validated using quantitative PCR. The inhibitory efficacy of CS-Ag NPs against Bacillus bombysepticus and Serratia marcescens was evaluated through in vitro inhibition zone assays and bacterial growth curve analysis, with the minimum inhibitory (MIC) concentration for both pathogens determined. Notably, CS-Ag NPs exhibited no significant inhibitory effect on filamentous fungi, potentially due to the impaired ability of nanoparticles to penetrate fungal cell walls. Preliminary mechanistic investigations into the antimicrobial mechanism of CS-Ag NPs were conducted from the perspectives of oxidative stress. Our data showed that CS-Ag NPs could effectively alleviate ROS accumulation induced by the pathogen. In summary, our work systematically investigates the potential of CS-Ag NPs in controlling pathogens and enables the preliminary elucidation of their antibacterial mechanisms. These findings establish a theoretical foundation for the development of pharmaceuticals against pathogenic microorganisms and also offer novel insights into the ecofriendly management of diseases. Full article

The anticancer ruthenium complex indazolium trans-[tetrachlorobis(1H-indazole) ruthenate (III)—also known as KP1019—inhibits cancer cell proliferation in vitro, causes tumor regression in animal models, and showed no dose-limiting toxicity in a phase I clinical trial. Previous studies found that KP1019 damages DNA in both cancer cells and the budding yeast Saccharomyces cerevisiae. To identify other potential targets of KP1019 along with pathways that modulate the drug’s cellular effects, we screened the yeast gene deletion strain library by quantitative high-throughput cell array phenotyping (Q-HTCP). Fitness differences, as judged by growth curve analysis, identified genes for which loss of function (gene deletion) interacts with (enhances or suppresses) KP1019 effects. Drug-enhancing deletions were enriched for DNA repair functions, consistent with DNA damage being a primary target of KP1019 in yeast. pH homeostasis also modified the effects of KP1019. Drug-suppressing deletions prominently involved ribosomal proteins. A mechanistic link between ribosomal protein function and KP1019 toxicity was supported by dose-dependent accumulation of Rpl7a-GFP in the nucleolus, which is a hallmark of ribosomal biogenesis stress. Furthermore, KP1019 acted synergistically with the TOR pathway inhibitor everolimus to inhibit cell proliferation. The resulting model, wherein KP1019 perturbs ribosome assembly, can inform the design of future combination therapies. Full article

Background/Objectives: Many products that claim to have anti-aging effects have been reported, but their relative potency is not clear. In this study, the in vitro replicative lifespan extension (RLE) activity of various groups of physiologically active substances was compared by using the updated “overlay method”. Methods: Human dermal and periodontal ligament fibroblasts (HDFa, HPLF) were inoculated into the inner 60 wells of 96-well microplate, surround by sterile water to prevent the water evaporation. At Day 1 and Day 8, the cells were overlayed with wide ranges of concentrations (0.01–100 µM) of samples without medium change. Viable cell number was measured by the MTT method at Day 15 and then corrected for the variation in cell growth due to the location of inoculated cells. The RLE value was calculated as the maximum cell proliferation rate relative to the control. Results: Cell density of HDFa and HPLFs at subculture decreased with the passage number, and their growth was stopped at 56 or 85 population doubling levels (PDLs), respectively. Hydrocortisone showed the highest RLE values among six hormones, followed by three plant extracts, sodium ascorbate and quercetin. On the other hand, other antioxidants, chlorogenic acid, phenylpropanoids, vanilloids, and bacterial products showed little or no RLE effects. However, for HPLF cells, hydrocortisone did not show RLE effects while oxytocin showed slight stimulation. Conclusions: When differences in proliferation due to cell seeding position were corrected, the biphasic dose response curve of most of the compounds significantly reduced. The present study suggests the significant role of hormones for the regulation of the long-term aging process. To confirm systemic or clinical anti-aging effects, further in vitro and in vivo experiments are needed. Full article

Mitochondrial dysfunction profoundly alters cellular metabolism, yet its systems-level consequences remain incompletely characterized. Here, we present a comprehensive untargeted metabolomics analysis of respiratory-deficient (ρ⁰) and competent (ρ⁺) Saccharomyces cerevisiae prototrophic cells using ultra-high-performance liquid chromatography coupled to Orbitrap Fusion™ Tribrid™ high-resolution mass spectrometry. By integrating hydrophilic interaction and reversed-phase chromatography in both ionization modes, we detected ~7000 features per chromatographic condition, of which ~12% were structurally annotated through MSⁿ fragmentation and in silico spectral matching. Principal component analysis revealed distinct metabolic signatures between ρ⁰ and ρ⁺ cells, with ~73% of total variance explained by the first two components. Volcano plot and hierarchical clustering analyses identified a marked accumulation of phosphate-containing metabolites, sphingolipids, ceramides, and fatty acid residues in ρ⁰ cells, whereas amino acids, excluding arginine, cysteine, and aromatics, were enriched in ρ⁺ cells. Notably, branched-chain amino acid depletion in ρ⁰ cells correlated with impaired growth and mitochondrial stress. Pathway enrichment analysis, supported by transcriptomic integration, prompted us to further investigate reprogramming of polyamine biosynthesis and aromatic amino acid metabolism. Calibration curves constructed from certified standards validated the accuracy of the LC–MS platform and reinforced annotation confidence. Our findings demonstrate that advanced untargeted metabolomics, coupled with MS³ fragmentation and multi-omics integration, enables high-resolution mapping of metabolic reconfiguration under mitochondrial dysfunction, offering mechanistic insights into mitochondrial retrograde signaling and adaptation. Full article

Listeria monocytogenes (Lm) is a foodborne pathogen whose virulence depends on the coordinated action of multiple virulence factors. Although deletion of either LIPI-4 or inlB reduces the virulence of Listeria monocytogenes, it remains unknown whether these two factors are functionally or regulatory connected. Therefore, we constructed an inlB deletion mutant and its complemented strain in the Lm928 and ΔLIPI-4 backgrounds. We assessed bacterial growth, biofilm formation, motility, host cell interactions (adhesion, invasion, intracellular proliferation), plaque formation, mouse organ colonization. Growth curve analysis showed no significant differences among strains. qPCR revealed that LIPI-4 modulates inlB expression in a cell-type-specific manner: inlB was downregulated in ΔLIPI-4 under culture and HTR-8 infection, but upregulated during hCMEC/D3 infection—yet functional defects persisted in all cases. Biofilm assays showed that ΔLIPI-4 and the double mutant exhibited enhanced biofilm formation, with the double mutant exceeding ΔLIPI-4, demonstrating synergistic enhancement. Motility assays indicated that LIPI-4 dominates bacterial movement, with ΔLIPI-4 and the double mutant showing identical severe defects. Plaque formation analysis showed that LIPI-4 is essential for cell-to-cell spread, while inlB deletion unexpectedly enhanced plaque formation—an effect completely abolished in the absence of LIPI-4. Host cell assays across Caco-2, HTR-8, and hCMEC/D3 models revealed that LIPI-4 is the core determinant of adhesion, invasion, and intracellular proliferation, whereas inlB contributes in the context of LIPI-4 and its effects vary with the specific cellular process examined. In mice, LIPI-4 was essential for systemic colonization of the liver and spleen, with inlB acting as a co-factor, whereas inlB unexpectedly promoted higher bacterial burdens in the brain, suggesting that inlB modulates LIPI-4-mediated neuroinvasion. Overall, our results establish LIPI-4 as the central determinant of Lm virulence, with inlB acting as a context-dependent co-factor that modulates LIPI-4-mediated pathogenesis in a cell type- and tissue-specific manner. Full article

Stress caused by suboptimal temperatures (ST) represents a stress that limits growth in all grasses without inhibiting their activity and induces alterations in photosynthetic performance. We evaluated the responses of photosynthetic parameters and leaf elongation between two groups of grass genotypes with different levels of tolerance to ST, belonging to phylogenetically distant species. Responses to ST depended on the type of parameter and on the genotypic group. Leaf elongation traits showed discriminatory power, especially the area under the leaf elongation curve, which integrated the early and transient effects of stress over time. The photosynthetic parameter PI_ABS showed lower discriminatory power compared with the area under the leaf elongation curve. However, a deeper analysis of other photosynthetic parameters revealed an increase in energetic connectivity between Photosystem II centers in tolerant, but not in sensitive, genotypes. A subsequent analysis of leaf and cellular parameters of early leaf elongation dynamics indicated that ST reduced meristematic activity in all genotypes, but the tolerant genotype group maintained a greater accumulation of mature cells compared with the sensitive genotype group. Overall, the results suggested a response to ST in tolerant genotypes, but not in sensitive genotypes, related to the early dynamics of leaf and cellular growth parameters to partially compensate for the restrictive effect of ST on leaf elongation not recorded. In parallel, they also indicated a response of the tolerant genotypes to ST in terms of photosynthetic parameters, probably as a pathway to maintain cellular homeostasis, to prevent photooxidative damage in PSII under stress. However, the relationship between both responses does not appear to be strictly linear, but rather would be mediated by coordinated adjustments in the temporal dynamics of growth, suggesting a functional integration between photosynthetic performance and the cellular mechanisms that regulate leaf expansion under ST stress. Full article