Search Results (24,000)

Vascularization remains a central challenge in thick tissue engineering. Building on our prior demonstration that carbonate buffer concentration governs multi-channel collagen gel (MCCG) architecture and perfusion culture performance, this study aimed to establish non-contact, orthogonal control of pore size and density in riboflavin-sensitized Type I collagen hydrogels via UV irradiation intensity and preparation temperature. UV intensity was modulated by varying the source-to-sample distance (25–52 mm); preparation temperature was set at 5, 25, or 40 °C; gelation kinetics were quantified using a vial-tilt assay. Pore area fraction ranged from 0.9% to 8.6% and Young’s modulus from 16 to 49 kPa depending on UV dose. Higher preparation temperatures accelerated gelation and produced smaller, more densely distributed pores, consistent with kinetically arrested phase separation. NIH/3T3 fibroblasts cultured on intermediate- and low-intensity UV scaffolds achieved >80% confluency by Day 7, with three-dimensional tissue-like organization and directionally aligned cellular bundles within large pores; cell metabolic activity, assessed by CCK-8 assay, remained consistently high throughout the culture period. These results demonstrate that UV irradiation intensity and preparation temperature are independently tunable, non-contact parameters for reproducible fabrication of collagen scaffolds with tunable vascular-like pore networks, complementing and extending the chemical (buffer concentration) design space of MCCG-based perfusion culture systems. Full article

Ferroptosis of mesenchymal stem cells (MSCs) is a critical bottleneck restricting the efficiency of ruminant biological breeding. Phloretin, a natural bioactive polyphenol, exhibits potential ferroptosis-inhibitory activity. However, the regulatory effects and underlying mechanisms of phloretin on ruminant MSCs remain poorly understood. This study aimed to investigate the effects of phloretin on ferroptosis and elucidate its underlying molecular mechanisms. Herein, we isolated and cultured adipose-derived mesenchymal stem cells (AD-MSCs) from adipose tissue of a 9-day-old Leizhou goat and established a ferroptosis model in these cells using RSL3. We detected cell viability, proliferation, migration, ferroptosis-related indexes and key protein expression. The results showed that phloretin (25 and 50 μM) dose-dependently inhibited ferroptosis in goat AD-MSCs, reducing intracellular ferrous ion (Fe²⁺), reactive oxygen species (ROS) and lipid peroxidation levels, restoring glutathione content, and ameliorating mitochondrial structural damage. Mechanistically, phloretin exerted its anti-ferroptosis effects through direct antioxidant activity, activation of the Nrf2/HO-1/GPX4 signaling pathway and Fe²⁺ chelation. Nrf2 and GPX4 were key targets in this process. These results provide preliminary in vitro evidence and a theoretical basis for the potential application of phloretin in future research related to meat goat production and ruminant breeding. Full article

Chondroitin sulfate (CS) chains are major components of the extra- and pericellular matrix in the central nervous system (CNS), and their sulfation patterns influence CNS development and function. Highly sulfated CS preparations, including CS-D- and CS-E-enriched forms, have been shown to facilitate neurite outgrowth in cultured mouse hippocampal neurons. Notably, neurons cultured on CS-D- or CS-E-enriched substrates exhibited the following distinct morphological characteristics: CS-D promoted the extension of multiple short neurites, whereas CS-E induced the formation of a single elongated neurite with a polarization-like morphology. These features are consistent with early stages of neuronal polarization. However, the specific roles of these highly sulfated CS forms in polarization-like morphology remain unclear. In this study, we demonstrate that polarization-like morphological transitions in hippocampal neurons can be modulated on mixed CS-D/CS-E substrates by varying their ratios. Compared with CS-D-enriched substrates, CS-E-enriched substrates more effectively promoted polarization-like neuronal morphology, accompanied by enhanced activation of Src-family kinases. Furthermore, forced activation of Fyn kinase induced morphological changes resembling polarization-like features in a neuroblastoma cell line, even in the absence of CS-D/CS-E mixed substrates. In conclusion, highly sulfated CS subtypes may function as extracellular cues that regulate neuronal morphology via Src-family signaling, with likely involvement of Fyn. Full article

Three new, previously undescribed tanzawaic acids, steckwaic acids H–J (1–3), and twenty-three known natural products (4–26) were isolated from the marine algicolous fungus Penicillium steckii SCSIO 41040. Structurally, compound 3 underwent a rare hydration reaction at the double bond of its carboxylic acid side chain. The chemical structures and stereochemistry were determined using comprehensive spectroscopic analyses, including NMR, electronic circular dichroism (ECD) calculations, and high-resolution electrospray ionization mass spectrometry (HRESIMS), and verified by literature comparison. The protective effect of tanzawaic acids on inflammatory damage to the intestinal epithelial barrier was assessed using an LPS-stimulated Caco-2/THP-1 co-culture model. Notably, immunofluorescence and Western blotting assays showed that compound 10 significantly enhanced the fluorescence signals and protein expression of ZO-1 and occludin, alleviated lipopolysaccharide (LPS)-induced intestinal barrier damage in Caco-2 cells, and contributed to the re-establishment of intestinal barrier homeostasis. Our findings demonstrate the critical role of tanzawaic acids in maintaining intestinal barrier integrity, identifying them as promising lead compounds for UC treatment. Full article

This study investigates the degradation characteristics, pathways, and mechanisms of ochratoxin A (OTA) by Weizmannia coagulans CGMCC 9951 (W. coagulans CGMCC 9951), as well as its detoxification effect on Cornus officinalis pulp through fermentation. The strain efficiently degraded 300 ng/mL of OTA within 72 h (98% degradation) under optimal conditions of 37 °C, pH 5.0, and 180 rpm. Active degradation substances were primarily localized in the cell-free supernatant (CF). The degradation activity was significantly inhibited by heat treatment, proteinase K, EDTA, Cu²⁺, and organic reagents, suggesting an enzymatic mechanism. UHPLC-MS and MS/MS analysis indicated that OTA appears to be degraded to a product consistent with ochratoxin α (OTα). Based on homology to known OTA-degrading carboxypeptidases, the gene encoding WGU28473.1 was selected, expressed in E. coli, and confirmed to possess OTA-degrading activity. Molecular docking suggested potential interactions between the enzyme and OTA. Under optimal conditions, co-fermentation with Cornus officinalis pulp contaminated with 300 ng/mL OTA for 96 h resulted in a 74% degradation of OTA. The fermentation process increased the pulp’s sugar content and ABTS⁺ free radical scavenging capacity, reduced acidity, and improved the safety of the pulp. These findings demonstrate that W. coagulans CGMCC 9951 efficiently degrades OTA and improves pulp quality, highlighting its potential as a starter culture for detoxifying OTA-contaminated food. Full article

High-quality genomic DNA extraction remains a major bottleneck for fungal genomics, particularly for worldwide aerobic and non-photosynthetic mushroom species that rely on their rigid cell walls, interference between metabolites, polysaccharides, etc., and complex genomes. This study systematically compares five DNA extraction protocols involving four distinct sample preparation procedures (fresh (A), filtered (B), frozen (C) and cryogenic mycelium (D)) across mycelial cultures of eight Tunisian fungal strains representing Ascomycota and Basidiomycota to identify the optimal combination for genomic DNA extraction from mycelium. The eight phylogenetically diverse fungal species were analyzed using short-read (MiSeq and NextSeq550) and/or long-read (MinION Mk1C) sequencing technologies, giving a depth coverage between 3.7× and 83×. The generation and quality of the assemblies were assessed within the Galaxy platform, which revealed a gap percentage of 0–0.509%. Taxonomic characterization and phylogenetic inference were performed with SANGER technology using the Internal Transcribed Spacer (ITS) and D1/D2 region of the 26S rRNA gene, assigning the species to our eight different strains: Clitopilus baronii (BS6), Porostereum spadiceum (BS200), Trametes versicolor (BS22-9), Schizophyllum commune (BS23-13), Gloeophyllum abietinum (BS23-14), Irpex laceratus (BS100), Trichoderma asperellum (GC9) and Trichoderma harzianum (S3). The optimized DNeasy Plant Pro Kit protocol with cryogenic biomass treatment presents a safe and cost-effective method for fungal genome sequencing and taxonomic resolution. This integrated comparative evaluation of extraction for sequencing identifies an optimal Qiagen-based extraction strategy combined with cryogenic treatment for eight diverse Tunisian fungal species, guiding method selection based on specific cell wall characteristics rather than proposing a universal protocol limited by unequal replication and strain numbers. Full article

Background/Objectives: Triple-negative breast cancer (TNBC) is frequently characterized by notably elevated Ki-67 expression, a hallmark of uncontrolled rapid cell-cycle progression. However, the underlying mechanisms remain unclear, leading to limited therapeutic options. Methods: In this study, hub gene was identified through integrated bioinformatic analysis of public datasets (TCGA-BRCA and METABRIC). Subsequent functional validation was performed both in vitro and in vivo using siRNA-mediated knockdown and small-molecule inhibitors. Phenotypic effects—including cell viability, cell cycle distribution, DNA synthesis, and clonogenic survival—were comprehensively assessed using MTT assays, flow cytometry, EdU, and colony formation assays. Protein-level changes were confirmed by Western blotting and immunohistochemistry (IHC). To dissect the transcriptional regulation of the key hub gene TTK, we first predicted potential upstream transcription factors using the JASPAR database; binding specificity was then validated through in silico motif analysis, luciferase reporter assays, and chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR). Results: The mitotic kinase TTK is significantly overexpressed in TNBC compared with non-TNBC breast cancers. Notably, TTK overexpression exhibited a strong positive correlation with elevated Ki-67 indices and reduced overall survival in TNBC patients. Functional validation demonstrated that pharmacological or genetic inhibition of TTK effectively induced G2/M cell-cycle arrest and potently suppressed TNBC proliferation in both in vitro cell cultures and in vivo xenograft models. Mechanistically, TTK overexpression stems from enhanced transcriptional initiation driven by the transcription factor NFYA binding to the CCAAT box in the TTK promoter—an interaction newly identified here. Concurrently, TTK blockade disrupted spindle assembly checkpoint (SAC) signaling via BUB1B/MAD1L1 downregulation, triggering mitotic arrest and catastrophe. Conclusions: Collectively, these findings establish TTK as a key cell-cycle regulator driving TNBC proliferation. More importantly, targeting mitotic control through TTK inhibition represents an efficient strategy to impede the aberrantly fast cell cycle progression in TNBC. Full article

Pulmonary bioengineering holds significant promise for the development of functional lungs suitable for transplantation in patients with terminal lung diseases; however, it encounters considerable challenges. The inherent structural complexity, diverse cellular composition, and the intricate process of re-endothelialization the pulmonary vasculature complicate efforts to reconstruct viable lungs for transplantation. This study aimed to establish an innovative re-endothelialization technique utilizing decellularized scaffolds, integrating canine yolk sac-derived endothelial precursor cells with mechanical respiratory stimuli within a bioreactor framework. Wistar rat lungs were subjected to a decellularization protocol employing SDS + Triton X-100 0.5% and subsequently assessed for cytocompatibility with murine fibroblasts (3T3) and yolk sac (YS) cells in fragments. Following this, the recellularization of the whole-lung scaffold was evaluated under constant mechanical respiratory stimulation with YS cells. Each stage of the process was rigorously analyzed using histological staining, DAPI, scanning electron microscopy (SEM), and genomic DNA quantification. The findings reveal that the implemented alternating decellularization protocol resulted in a structured scaffold conducive to the culture of various cell types in fragments. When subjected to the complete scaffold recellularization model, the results indicated that YS cells are advantageous for the re-endothelialization process. Moreover, when employed in conjunction with the bioreactor model incorporating respiratory stimulation, these cells demonstrated enhanced cellular diffusion capacity and facilitated more homogeneous recellularization of the entire organ. These results signify a notable advancement in the reconstruction of new tissues for pulmonary transplantation. Full article

Growth media for human cell culture were developed in the twentieth century, when the first immortal human cell lines were established. The nutrient compositions of these media arose not from a desire to reproduce the microenvironment of the cells in vivo, but rather to encourage continuous replicative growth. Armed with comprehensive datasets detailing the metabolomes of the various fluid compartments within which cells reside, cell culturists are now exploring the effects of media designed to reproduce the in vivo environment on cell biology. The early results of this research indicate the media composition has profound impacts on cell form and function. In parallel, taking care to maintain oxygen at the relatively low levels found in vivo also affects many cellular activities. The lessons learned from ‘physiological cell culture’ should be applied to the culture of human embryos in the in vitro fertilization (IVF) clinic, where a critical stage of growth and development might be best supported by recreating, to the greatest extent possible, the environment of the oviduct and uterus. In this review, we translate recent advancements in physiological cell culture to emerging approaches in human embryo culture. Full article

This study investigated the bactericidal effect and examined the associated cellular damage of low temperature plasma (LTP) combined with slightly acidic electrolyzed water (SAEW) against Listeria monocytogenes. Single-factor experiments were conducted to assess the bactericidal efficacy under individual treatment conditions, followed by the evaluation of three different combination sequences. An orthogonal experimental design was performed to optimize the key parameters, and the optimal treatment conditions were determined as LTP at 45 W with an electrode spacing of 1 mm for 2 min, combined with SAEW at an available chlorine concentration (ACC) of 30 mg/L. Under these conditions, confocal laser scanning microscopy (CLSM) with SYTO 9/PI staining confirmed that the combined treatment caused cell death, as indicated by loss of membrane integrity in treated cells. A resuscitation assay further ruled out the viable but non-culturable (VBNC) state, as no bacterial growth was detected after 48 h of enrichment. The leakage of intracellular proteins and nucleic acids was measured using the Coomassie Brilliant Blue method combined with a microplate reader, and changes in cellular morphology were observed by scanning electron microscopy (SEM). The results demonstrated that SAEW+LTP treatment exerted a distinct effect, significantly disrupting bacterial cell membrane integrity, inducing the leakage of intracellular contents, and causing obvious morphological damage to the bacterial cells. In conclusion, the combined treatment of LTP and SAEW significantly improved the bactericidal efficiency against L. monocytogenes, which may be due to the combined disruptive effects on membrane integrity and subsequent structural and functional damage to the cells. Future investigations are needed to unravel the precise mechanisms, establish the efficacy against a wider panel of strains, and explore the potential for practical application in food matrices. Full article

Background: Bone metastasis is a major determinant of morbidity and therapeutic failure in advanced prostate cancer (PCa); however, the transcriptional programs and tumor microenvironmental alterations driving metastatic progression remain incompletely understood. This study aimed to systematically characterize transcriptomic differences between non-metastatic and bone-metastatic PCa and to identify key microenvironmental signaling pathways involved in tumor survival and chemoresistance. Methods: Bulk RNA sequencing was performed on 49 non-metastatic and 28 bone-metastatic PCa specimens. Differential expression analysis was integrated with weighted gene co-expression network analysis (WGCNA), gene set enrichment analysis, and immune/stromal deconvolution. Key findings were validated using in vitro functional assays, including Transwell co-culture models, small interfering RNA (siRNA)-mediated gene silencing, cell viability, apoptosis, and docetaxel resistance analyses. Results: Transcriptomic profiling identified 574 differentially expressed genes. Bone-metastatic tumors were enriched in ribosome-related and translational pathways, whereas non-metastatic tumors displayed immune-associated signatures, including natural killer (NK) cell-mediated cytotoxicity and cytokine signaling. WGCNA revealed immune-related gene modules preferentially enriched in non-metastatic disease. Immune deconvolution demonstrated significantly higher infiltration of NK cells and endothelial cells in non-metastatic tumors. Chemokine-receptor analysis highlighted upregulation of the CXCL10-CXCR3 axis in non-metastatic PCa. In vitro, PCa cells expressed CXCR3, while endothelial cells markedly increased CXCL10 expression upon co-culture. Functional assays showed that endothelial-derived CXCL10 promoted PCa cell survival, suppressed apoptosis, and conferred resistance to docetaxel via CXCR3-dependent signaling; these effects were reversed by CXCL10 or CXCR3 knockdown. Conclusions: These findings uncover a context-dependent endothelial-immune chemokine network distinguishing non-metastatic from bone-metastatic PCa and identify the CXCL10-CXCR3 axis as a critical mediator of tumor survival and chemoresistance, suggesting a potential therapeutic vulnerability in advanced prostate cancer. Full article

Candida albicans (C. albicans) is an opportunistic fungal pathogen capable of causing a wide range of infections, including mucosal and systemic candidiasis. In the oral cavity, fungi represent a minor component of the microbiome but can significantly contribute to morbidity, particularly under conditions of dysbiosis or immunosuppression. Treatment remains challenging due to increasing multidrug resistance. This study investigates the in vitro antifungal potential of Viroelixir, a standardized polyphenol blend derived from green tea and pomegranate and enriched in catechins (including epigallocatechin gallate, EGCG), ellagitannins (notably punicalagin), ellagic acid, and flavonoids, with particular focus on its potential anti-virulence mechanisms. Methods: The effect of Viroelixir on C. albicans growth was assessed using MTT assay, optical density measurements, colony formation, carbohydrate quantification, and pH variation analysis. Biofilm formation, morphological transition, ROS production, necrosis, virulence gene expression, adhesion, and host immune responses were also evaluated. Results: Viroelixir significantly inhibited C. albicans growth and reduced colony formation compared with untreated controls. The formulation also inhibited biofilm formation and markedly reduced pseudohyphal development, reaching up to 94% reduction under specific treatment conditions. Flow cytometry analysis showed an increase in dead fungal cells, reaching approximately 88% following exposure to Viroelixir at the highest tested concentration. In addition, Viroelixir reduced the transcript levels of several virulence-associated genes, including SAP1–SAP9 and EAP1. In epithelial cell co-culture models, pre-treatment of C. albicans with Viroelixir reduced fungal adhesion and attenuated epithelial inflammatory responses, including IL-6, IL-8, and hBD-2 production, and was associated with reduced activation of the TLR4-NF-κB signaling pathway. Conclusions: These findings suggest that the antifungal and anti-virulence effects observed may be associated with the polyphenolic compounds present in the Viroelixir formulation, highlighting its potential as a promising in vitro antifungal candidate against C. albicans. Full article

Toxoplasma gondii is an intracellular protozoan transmitted via environmentally resistant oocysts present in food and water, as well as through the consumption of meat containing infective bradyzoites. This study evaluated the inactivation of T. gondii oocysts and bradyzoites (ME-49 strain) by Pulsed Electric Field technology (PEF). Treatment efficacy was determined by mouse bioassay combining brain qPCR and indirect immunofluorescence (IFA), with complementary qPCR in Hs27 cells. The infectious dose (ID₅₀) of T. gondii was estimated at 34.6 oocysts. PEF-treated oocysts (15 kV/cm; 50 kJ/kg; 225 µs) showed a significant reduction in infectivity compared with untreated controls; accordingly, the dose required to establish infection increased to 85.3 oocysts after PEF treatment. Brain qPCR and IFA were highly correlated, whereas heart tissue was less sensitive. Bradyzoites recovered from PEF-treated meat (3.3 kV/cm; 27 kJ/kg; 1600 µs) showed a 50% infectivity reduction compared with untreated samples. In vitro assays confirmed an in vivo reduction in infectivity, indicating that cell cultures can serve as an ethical and efficient tool for preliminary viability assessment. This is the first evidence of T. gondii inactivation by PEF, highlighting its potential as a non-thermal strategy. Further studies are needed to optimize treatment parameters. Full article

Peritoneal dialysis (PD) is limited by insufficient phosphate removal, leading to adverse cardiovascular outcomes in patients with chronic kidney disease. To advance the understanding of the molecular mechanisms of peritoneal phosphate transport, RNAseq data of phosphate transporters in four PD-relevant cell lines were analyzed. The expression and localization of the respective proteins were validated by immunostaining in these cells. The transcriptomics of omental arterioles from children on PD were analyzed. In vitro Transwell models of an immortalized mesothelial cell line (MeT-5A) and human umbilical vein endothelial cells (HUVECs) and respective co-cultures were established, enabling quantification of phosphate transport across mesothelial and endothelial monolayers. Sodium phosphonoformate tribasic hexahydrate (PFA) and Tenapanor were used to inhibit transcellular and paracellular transport pathways. Cell viability and integrity markers were measured over the experimental periods. SLC20A1 and SLC20A2 were expressed across all studied cell types, while SLC34A2 and SLC34A3 were mesothelial cell-specific. Omental arterioles of children on low-glucose-degradation-product (GDP) PD showed higher SLC20A1 expression vs. stage 5 chronic kidney disease (CKD5) and healthy controls. Permeability for phosphate was lower across MeT-5A compared with HUVEC monolayers and was not further reduced in co-culture. Inhibitors reduced both transcellular and paracellular transport to 75% in MeT-5A and 65% in co-cultures, while no effects were observed in HUVEC alone, suggesting the mesothelial cell layer as a significant barrier for phosphate transport. Our studies provide first analyses combining findings on molecular phosphate transporters in peritoneal cells and arterioles and introducing a Transwell model for quantitative studies of phosphate kinetics. Full article

Microorganisms represent the Earth’s most abundant biomass and a vast reservoir of genetic diversity. However, traditional agar plate methods fail to recover the vast majority of these species, leaving a “microbial dark matter” that holds immense potential for the discovery of novel antibiotics and bioactive compounds. While conventional techniques such as selective media and enrichment culture remain foundational, they are inherently limited by community biases and the inability to support low-abundance, oligotrophic species. To address these bottlenecks, a diverse array of innovative isolation strategies has emerged. This review systematically categorizes and evaluates these methodologies, ranging from in situ cultivation to high-resolution single-cell manipulation. We first examine membrane diffusion-based cultivation (e.g., iChip), which mimics natural microenvironments to resuscitate recalcitrant microbes. Subsequently, we explore high-throughput single-cell technologies, including microfluidics for physicochemical separation, optical tweezers for precise manipulation, and fluorescence-activated cell sorting (FACS). Special attention is given to Raman-activated cell sorting (RACS) as a label-free functional screening tool and reverse genomics for targeted capture. By synthesizing the strengths and limitations of these approaches, we propose integrated workflows designed to accelerate the mining of untapped microbial resources. Full article