Search Results (1,747)

This review article focuses on the role of choline in ovarian follicular development, regulated by nutrient–epigenetic interactions. Choline, a key feed additive, participates in DNA methylation and steroid hormone synthesis via its methyl donor function. However, its role in follicular hierarchy and maturation is unclear. Research lacks an understanding of species-specific choline metabolism, follicular fluid methylation dynamics, and toxicity thresholds. This study combines animal nutrition, epigenetics, and reproductive endocrinology. Using in vitro follicle culture models, metabolomics analysis, and cytochrome P450 family 19 subfamily A member 1 (CYP19a1) methylation site screening, it reveals that choline regulates follicle hierarchy through the betaine-S-adenosylmethionine (SAM) pathway. Proper dietary choline reduces homocysteine (HCY) and boosts CYP19a1 demethylation, enhancing theca cell estradiol (E₂) production and accelerating follicle maturation. In contrast, inadequate or excessive choline causes mesoderm-specific transcript (MEST) gene methylation abnormalities or trimethylamine N-oxide (TMAO)-mediated β-oxidation inhibition, increasing follicle atresia. A phenomenon of steroidogenic factor 1 (SF-1) methylation has been observed in poultry, showing that choline affects offspring egg-laying persistence by altering the adrenal–ovarian axis DNA methylation imprint. Future research should establish a precise choline supply system based on the HCY/TMAO ratio in follicular fluid and the CYP19a1 methylation map to improve animal reproduction. Full article

Background: Peptide-loaded antigen-presenting cell (APC)-based vaccines have been under investigation as a therapeutic approach for treating cancer. However, in general they have demonstrated limited efficacy in clinical trials. Dendritic cells (DCs) have been the primary choice for APC-based vaccines given their ability to cross-present antigens. B cells have been less studied as APCs for vaccines. Here we compare the phenotype and anti-tumor activity of activated T cells that result from peptide-specific priming using either B cells or DCs. Methods: B cells and DCs were isolated from C57Bl/6 mice, and either treated or not treated with lipopolysaccharide (LPS) for maturation, and then either loaded or not loaded with SIINFEKL peptide to prime CD8+ T cells from OT-1 mice. Activated T cells were then analyzed for their phenotype and anti-tumor efficacy. Results: We report that both immature B cells and immature DCs were similarly capable of activating antigen-specific CD8+ T cells. However, LPS-matured DCs generated a stronger CD8+ T cell activation profile in vitro compared to LPS-matured B cells. Immature B cells, mature DCs and immature DCs all generated a similar anti-tumor response upon adoptive transfer of primed CD8+ T cells to tumor-bearing mice. Conclusions: Collectively, our data suggests that B cells and DCs are each capable of priming CD8+ T cells and generating anti-tumor responses. Given that B cells are relatively easier to culture and expand compared to DCs, our study suggests that, following further validation, B cells could be further investigated as APCs for peptide-based human cancer vaccines. Full article

To accelerate maturation of intestinal function and promote growth and development, the effect of maternal clofibrate on intestinal fatty acid (FA) metabolism was investigated in suckling piglets. Twenty-seven pregnant sows were fed either 0, 0.25, or 0.5% clofibrate, a peroxisome proliferator-activated receptor α (PPARα) agonist, during late gestation and early lactation. [1-¹⁴C]-Oleic acid metabolism was measured in vitro in intestinal mucosa of piglets with/without L-carnitine and/or malonate. Clofibrate increased oleic acid metabolism on d1, and the increase was higher from 0.5% than 0.25% of maternal clofibrate (p < 0.005). Flux to CO₂ increased with age, while flux to acid-soluble products (ASP) remained constant after d1. Flux to esterified products (ESP) increased on d7, but the increase was dampened by clofibrate (p < 0.0001). Carnitine increased flux to CO_2, and malonate decreased it (p < 0.0001), but neither affected ASP or ESP. Intestinal non-esterified FA and triglyceride levels decreased linearly, and carnitine palmitoyl-transferase (CPT) activity increased quadratically with age. Clofibrate increased acyl-CoA oxidase (ACOX) abundance but decreased the ratio of CPT1A and CPT1B on d1. Postnatal age increased FA binding protein 2 (FABP2) but decreased PPARα. In conclusion, maternal supplementation of clofibrate promotes intestinal energy generation from fat oxidation in postnatal piglets, but the stimulation is influenced by age, in which ACOX, FABP2, and CPT1 might play modulatory roles. Full article

Extraintestinal pathogenic Escherichia coli causes community-acquired and nosocomial pneumonia and poses a risk of infection, especially for patients with impaired lung function, such individuals with cystic fibrosis (CF). When chronic infection develops, eradication of the pathogen is difficult even with aggressive antibacterial therapy and targeted CF treatment. A new agent, Fluorthiazinone (CL-55), an inhibitor of bacterial virulence, was registered in Russia in 2024. The aim of our study was to characterize the genomes of E. coli ST648 isolated from long-term-infected CF patients, describe virulence factors, and investigate the effect of CL-55 on two CF isolates in vitro. Comparison of the genomes of hypermucoviscous isolates showed that, in the presence of a large number of core genes, the isolates have adaptive differences both in their chromosomes and the composition and genes of their plasmidomes. Both isolates formed mature biofilms on an abiotic surface and were able to survive and proliferate inside macrophages. CL-55 in in vitro experiments was effective in suppressing E. coli ST648 in both the aggregate and intracellular states, allowing us to propose the use of Fluorthiazinone as a combinative therapy to facilitate eradication of pathogenic microorganisms in the respiratory tract in patients with CF. Full article

Liver-expressed antimicrobial peptide 2 (LEAP2) is a cationic peptide that is integral to the innate immune system. However, its regulatory function in the immunity of fish against pathogens remains largely unexplored. This study identifies a LEAP2 homolog (AoLEAP2) in clownfish, Amphiprion ocellaris. Our phylogenetic analysis places AoLEAP2 within the fish LEAP2 cluster, closely related to Tachinotus anak LEAP2. AoLEAP2 mRNA is present across various tissues, with the highest expression in the spleen. Following Vibrio harveyi infection, the AoLEAP2 mRNA levels significantly increased in the liver, spleen, gills, and intestine. The chemically synthesized AoLEAP2 mature peptide exhibits broad-spectrum antibacterial activity without displaying toxicity to FHM cells in vitro. The intraperitoneal injection of AoLEAP2 significantly suppresses the mRNA expression of the pro-inflammatory factors IL1β and TNFα induced by V. harveyi infection, demonstrating its anti-inflammatory and immune-regulatory role. Full article

The in vitro production of embryos has significant potential to enhance animal productivity. However, further refining of this technology is required for its widespread adoption and cost-effectiveness. The objectives were to evaluate the effects of L-carnitine (LC) on the maturation, lipid content, and Hippo signaling of oocytes, and the cryotolerance of the resulting embryos. Abattoir-derived oocytes were in vitro matured using fetal bovine serum (FBS), bovine serum albumin (BSA), or FBS + 1.5 or 3.0 mM LC. The maturation rates did not differ among FBS (83%) and FBS with LC (1.5 or 3.0 mM; 82 and 80%, respectively). In contrast, the BSA group exhibited a significantly lower maturation rate of 71% compared to the other groups. The lipid content of matured oocytes (assessed using Nile red staining) was significantly reduced in the BSA group and in the FBS + LC groups, compared to the FBS group. The blastocyst-stage embryos were cryopreserved, and their cryotolerance was evaluated by assessing their ability to re-expand and hatch after thawing. The embryos from the FBS + LC groups showed a numerically higher re-expansion rate at 24 h (78.8%), compared to the BSA (74.0%) and FBS groups (57.7%). The expression of Hippo signaling pathway genes was not significantly affected by LC, indicating that LC enhanced cryotolerance and reduced lipid content without impacting oocyte maturation or the Hippo signaling pathway. Full article

RNA virus populations exist as quasispecies-complex, dynamic clouds of closely related but genetically diverse variants generated by high mutation rates during replication. Assessing quasispecies structure and diversity is crucial for understanding viral evolution, adaptation, and response to antiviral treatments. However, comparing single quasispecies observations from individual biosamples, especially at different infection or treatment time points, presents statistical challenges. Traditional inferential tests are inapplicable due to the lack of replicate observations, and resampling-based approaches such as the bootstrap and jackknife are limited by biases and non-independence, particularly for diversity indices sensitive to rare haplotypes. In this study, we address these limitations by applying the delta method to derive analytical variances for a set of quasispecies structure indicators specifically designed to assess the quasispecies maturation state. We demonstrate the utility of this approach using high-depth next-generation sequencing data from hepatitis C virus (HCV) quasispecies evolving in vitro under various conditions, including free evolution and exposure to antiviral or mutagenic treatments. Our results reveal that with highly fit HCV quasispecies, sofosbuvir inhibits quasispecies genetic diversity, while mutagenic treatments accelerate maturation, compared to untreated controls. We emphasize the interpretation of results through absolute differences, log-fold changes, and standardized effect sizes, moving beyond mere statistical significance. This framework enables robust, quantitative comparisons of quasispecies diversity from single observations, providing valuable insights into viral adaptation and treatment response. The R code and session info with required libraries and versions is provided in the supplementary material. Full article

Since the late 1970s, brown spot of pear (BSP), a fungal disease caused by Stemphylium vesicarium (Wallr.) Simmons, has been one of the most important pear fungal diseases in Italy. To protect orchards from BSP, frequent fungicide application is essential throughout the period spanning petal fall to the onset of fruit maturation. In Italy, boscalid was the first succinate dehydrogenase inhibitor (SDHIs) fungicide authorised against BSP; subsequently, penthiopyrad and fluxapyroxad were authorised against the disease. In 2016 and 2017, SDHI compounds were applied against BSP as solo products at the University of Bologna’s experimental farm, showing a reduction in efficacy. Stemphylium vesicarium strains were isolated from leaves and fruit, and sensitivity assays and molecular analyses were performed. In vitro tests confirmed resistance to SDHIs, and two specific single-nucleotide polymorphisms were discovered, SDHB P230L and SDHC H134R, both leading to amino acid substitutions in succinate dehydrogenase subunits and confirming the resistant phenotype. Full article

Background: Ovarian cancer is an immunologically cold tumor that is treated with surgery and a chemotherapy regimen of platinum agents with taxanes. Paradoxically, elevated levels of several immune markers are effective at predicting prognosis for patients with ovarian cancer, though it is not clear how chemotherapy might influence this. Chemotherapy elicits immunogenic cell death, yet tumor-controlling doses of chemotherapy are also immunotoxic. Objectives: To evaluate interactions of chemotherapy with the immune system, we studied the impact of chemotherapy in an aggressive mouse model of ovarian cancer developed within our lab. Methods: Using a single-cell transcriptomics sequencing approach, supported by flow cytometry, we evaluated the influence of a first-line therapy, cisplatin and docetaxel, and a second-line therapy, pegylated liposomal doxorubicin (PLD), on control of tumor growth and on tumor-associated immune populations of cells. Results: Both chemotherapy approaches were effective at controlling tumor growth and selectively depleted tumor cells from distinct transcriptional clusters. Both chemotherapies also resulted in relative increases in immune populations compared to untreated tumor-bearing mice, but immune populations from PLD-treated mice were more abundant and expressed a greater fraction of maturity-associated transcripts and increased proportions of tumor resident macrophage populations. PLD treatment selectively upregulated MHC class II on tumor cells, and this could be replicated in vitro across ovarian cancer cell lines and in patient tumor cells ex vivo. Conclusions: Altogether, the results support the notion that PLD has a greater capacity for immunopotentiation, which may be important to consider if immunotherapy approaches are adapted for ovarian tumors in the future. Full article

This study evaluated the effectiveness of individual clean-in-place (CIP) steps in removing biofilms from reverse osmosis (RO) membranes under dynamic flow conditions using the Centers for Disease Control (CDC) biofilm reactor. Biofilms were developed in the laboratory under continuous flow, using mixed-species bacterial isolates obtained from 10-month-old RO membrane biofilms from a commercial facility. Individual CIP chemicals, representative of those used in commercial protocols, were tested against 24 h-old biofilms. Additionally, a complete six-step sequential CIP process was conducted under dynamic conditions, consisting of treatments with alkali, surfactant, acid, enzyme, a secondary surfactant, and sanitizer. All experiments were performed in quadruplicate, and data were subjected to statistical analysis. Among individual treatments, the acid step was the most effective, significantly outperforming the other CIP cleaning steps by reducing bacterial counts from 5.62 to 4.10 log units, a 96.98% reduction. The full six-step CIP protocol reduced counts to 2.24 log units, indicating the persistence of resistant cells. The presence of viable cells post-treatment highlights the limited efficacy of the tested CIP chemicals in fully eradicating mature biofilms. Additionally, skipping any step in the membrane cleaning can significantly compromise the efficiency and performance during production. These findings suggest that biofilms grown in vitro under dynamic conditions using the CDC reactor exhibit a more robust assessment of the CIP treatments in accomplishing the biofilm control. This study highlights the need for optimized, scientifically validated CIP protocols targeting biofilms to improve cleaning efficacy and food safety. Full article

Advancements in bioinks and three-dimensional (3D) printing and bioprinting have significantly advanced cardiovascular tissue engineering by enabling the fabrication of biomimetic cardiac and vascular constructs. Traditional 3D printing has contributed to the development of acellular scaffolds, vascular grafts, and patient-specific cardiovascular models that support surgical planning and biomedical applications. In contrast, 3D bioprinting has emerged as a transformative biofabrication technology that allows for the spatially controlled deposition of living cells and biomaterials to construct functional tissues in vitro. Bioinks—derived from natural biomaterials such as collagen and decellularized matrix, synthetic polymers such as polyethylene glycol (PEG) and polycaprolactone (PCL), or hybrid combinations—have been engineered to replicate extracellular environments while offering tunable mechanical properties. These formulations ensure biocompatibility, appropriate mechanical strength, and high printing fidelity, thereby maintaining cell viability, structural integrity, and precise architectural resolution in the printed constructs. Advanced bioprinting modalities, including extrusion-based bioprinting (such as the FRESH technique), droplet/inkjet bioprinting, digital light processing (DLP), two-photon polymerization (TPP), and melt electrowriting (MEW), enable the fabrication of complex cardiovascular structures such as vascular patches, ventricle-like heart pumps, and perfusable vascular networks, demonstrating the feasibility of constructing functional cardiac tissues in vitro. This review highlights the respective strengths of these technologies—for example, extrusion’s ability to print high-cell-density bioinks and MEW’s ultrafine fiber resolution—as well as their limitations, including shear-induced cell stress in extrusion and limited throughput in TPP. The integration of optimized bioink formulations with appropriate printing and bioprinting platforms has significantly enhanced the replication of native cardiac and vascular architectures, thereby advancing the functional maturation of engineered cardiovascular constructs. Full article

Catalpa bungei C.A.Mey is an economically significant deciduous tree valued for timber production and landscaping applications. An efficient regeneration system is crucial for clonal propagation and serves as a foundation for future molecular breeding in C. bungei. This study established two in vitro regeneration pathways—indirect somatic embryogenesis and shoot organogenesis utilizing mature zygotic embryos as explants. Primary callus was induced from cotyledon, hypocotyl, and plumule explants. A high frequency (45.73%) of yellow-green compact callus was achieved on De-Klerk and Walton (DKW) medium supplemented with 2.0 mg/L 6-BA, 1.0 mg/L zeatin (ZT), and 0.1 mg/L NAA. Subsequent transfer to 1.5× Murashige and Skoog (MS) medium containing 1.5 mg/L 6-BA, 0.2 mg/L ZT, and 0.1 mg/L NAA yielded the highest embryogenic callus induction rate (16.67%). Embryogenic callus demonstrated bipotent potential, generating both adventitious shoots and somatic embryos under specific hormonal conditions. Histological analyses confirmed the typical developmental stages of somatic embryos, from globular to cotyledonary forms, validating the embryogenic origin of regenerated structures. Furthermore, hormone or osmotic additives such as abscisic acid (ABA), Phytagel, and polyethylene glycol 4000 (PEG4000) significantly enhanced somatic embryo induction, with Phytagel at 5.0 g/L achieving the highest rate (76.31%). For shoot organogenesis, the optimal hormonal combination of the 0.6 mg/L 6-BA, 0.4 mg/L KT, and 0.15 mg/L NAA achieved the highest bud induction rate (88.89%) and produced an average of 4.07 adventitious buds per explant. This study presents an efficient regeneration system for C. bungei, providing a practical platform for large-scale propagation and basis for biotechnological applications in woody plants. Full article

Chimeric antigen receptor (CAR) T-cell therapy directed to CD19 and B-cell maturation antigen has revolutionized treatment of B-cell leukemia and lymphoma, and multiple myeloma. However, identifying suitable targets for acute myeloid leukemia (AML) remains challenging due to concurrent expression of potential target antigens on normal hematopoietic stem cells or tissues. As the stress-induced B7H6 molecule is rarely found on normal tissues but expressed on many cancers including AML and melanoma, the NKp30-ligand B7H6 emerges as a promising target for NKp30-based CAR T therapy for these tumors. In this study, we report a comprehensive B7H6 expression analysis on primary AML and melanoma as well as on different tumor cell-lines examined by RT-qPCR and flow cytometry, and efficient anti-tumor reactivity of NKp30-CAR T cells to AML and melanoma. To overcome limitations of autologous CAR T-cell fitness-dependent efficacy and patient-tailored production, we generated CRISPR/Cas9-mediated TCR-knockout (TCR^KO) NKp30-CAR T cells as an off-the-shelf approach for CAR T therapy. Functional studies comparing NKp30-CD28 CAR or NKp30-CD137 CAR TCR⁺ and TCR^KO T lymphocytes revealed superior anti-tumoral immunity of NKp30-CD28 CAR TCR^KO T cells to AML and melanoma cell lines in vitro, and effective control of tumor burden in an NSG melanoma-xenograft mouse model. In conclusion, these findings highlight the therapeutic potential of NKp30 CAR TCR^KO T cells for adoptive T-cell therapy to B7H6-expressing cancers, including melanoma and AML. Full article

Mitochondrial-targeted antioxidant supplementation is promising for in vitro culture of mammalian embryos. Elamipretide (SS-31) is a synthetic tetrapeptide that binds to the inner mitochondrial membrane, contributing to the prevention of oxidative stress. In this study, the effects of SS-31 supplementation in maturation medium on the developmental competence of porcine oocytes and its protective function were evaluated. Porcine oocytes were matured in maturation medium with SS-31 at 0.001, 0.01, 0.1, 0.5, 1, 1.5, 2.5, and 5 µM, with 0 µM and DMSO-treated groups established as controls. In vitro fertilization and embryo culture were performed to analyze developmental potential. Oocytes cultured in medium with 1 µM SS-31 exhibited higher maturation and blastocyst formation rates than the control (0 µM) (78.3 ± 3.8% vs. 55.2 ± 4.1% and 7.6 ± 1.6% vs. 2.8 ± 1.8%, respectively). Oocytes treated with 1 µM SS-31 showed significantly lower reactive oxygen species, higher glutathione content, and improved mitochondrial membrane potential than those without treatment. DNA fragmentation in oocytes after in vitro maturation was significantly lower in the 1 µM SS-31 supplemented group than in the control. This study demonstrates that SS-31 exerts beneficial effects on the in vitro production of porcine embryos via the enhancement of the mitochondrial antioxidant capacity. Full article

Objectives: To prepare porous scaffolds combining hydrogel and hydroxycarbonateapatite, enriched with a promising therapeutic agent, gold nanoparticles, to improve bone regeneration. The fabrication procedure is conducted under mild conditions, without toxic or aggressive chemicals, at physiological pH, and low temperatures; Methods: Gold nanoparticles (15–20 nm), were obtained by the Turkevith method. The scaffolds were fabricated by the GELPOR3D method, which has demonstrated its ability to integrate thermal labile molecules, during the scaffold fabrication process. The role of these nanoparticles in promoting cell adhesion, proliferation, and mineralization processes in vitro has been studied using osteoprogenitor MC3T3-E1 cells; Results: The scaffold fabrication conditions, combined with the surface functionalization of the gold nanoparticles with poly(ethylene glycol), ensure their uniform distribution throughout the scaffold and facilitate their gradual release over 48 h in a physiological medium. A significant increase in the mean cell area and a significant decrease in the circularity index during the early stages of osteoblast differentiation are observed. These pieces of evidence suggest that adequate cell spreading could lead to enhanced proliferation and matrix deposition activity; Conclusions: Scaffolds containing these gold nanoparticles exhibited a marked improvement in adhesion, proliferation, and mineralization of preosteoblasts (MC3T3 cells) at the concentrations studied. The functionalization of the nanoparticles, along with the shaping procedure employed, is critical for their homogeneous dispersion throughout the scaffold and their progressive release. The findings confirm the crucial role of gold nanoparticles in the early stages of osteoblast differentiation, which is essential for the transition from premature osteoblasts to mature osteoblasts. Full article