Search Results (99)

Background: Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia and is strongly associated with atrial structural remodeling driven by activated cardiac fibroblasts. Autophagy has been implicated in AF-related atrial remodeling; however, the non-coding RNA mechanisms that govern autophagic activation in atrial fibroblasts under rapid electrical stress remain poorly understood. Methods: Human cardiac fibroblasts from adult atria (HCF-aa) were subjected to rapid electrical stimulation (RES) at 0.5 V/cm and 10 Hz. Expression levels of exosomal metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), cytoplasmic miR-204-5p, and microtubule-associated protein light chain 3B (LC3B) were measured using quantitative real-time PCR and Western blot analyses. Luciferase reporter assays were performed to confirm direct molecular interactions. The functional roles of MALAT1 siRNA, miR-204-5p mimics/antagomirs, rapamycin, and 3-methyladenine (3-MA) on LC3B expression and autophagic activation were assessed by Western blot and immunofluorescence confocal microscopy for LC3B puncta formation. Results: RES significantly induced exosomal MALAT1 expression in a voltage- and time-dependent manner, peaking at 2 h post-stimulation, while cytoplasmic MALAT1 levels remained unchanged. Cytoplasmic miR-204-5p exhibited an initial transient rise followed by a significant decline at 2 h, inversely correlating with peak MALAT1 levels. LC3B mRNA and protein expression subsequently increased, peaking at 6 and 16 h, respectively. Luciferase reporter assays confirmed that miR-204-5p directly binds both the MALAT1 transcript and the 3′-UTR of LC3B mRNA. MALAT1 knockdown augmented miR-204-5p levels and suppressed LC3B expression, while miR-204-5p overexpression attenuated RES-induced LC3B upregulation and LC3B puncta accumulation. Conversely, miR-204-5p inhibition further enhanced autophagic activation, as evidenced by increased LC3B puncta density. Conclusions: In HCF-aa subjected to RES, MALAT1 functions intracellularly as a competing endogenous RNA to putatively sequester miR-204-5p, thereby de-repressing LC3B expression and promoting autophagic activation. Concurrent exosomal secretion of MALAT1 may additionally serve as a paracrine signal to neighboring cells, though this requires future conditioned-media transfer experiments to confirm. Full article

Orthoebolavirus causes severe Ebola virus disease (EVD) and deadly outbreaks in humans. This infection occurs through macropinocytosis and trafficking to late endosomes or lysosomes that utilize the receptor Niemann–Pick C1 (NPC1) to enter the cell cytoplasm. We designed peptide inhibitors based on the NPC1 receptor to target the NPC1 binding site to block viral entry. The results indicated that the ligand-based peptide inhibitors showed potent inhibition activities in vitro studies against pseudotyped or replication-competent Orthoebolavirus. Therefore, we further evaluated one of them in a mouse model challenged with mice-adapted Ebola viruses, which showed some protection efficacy compared with the control group. This study suggests that ligand-based peptides are encouraging inhibitors in the development of inhibitors against Ebola virus infection. Full article

Lentiviral vectors (LVs) are indispensable tools in cell and gene therapy. Rising demand has created a global shortage of LVs, driving the development of novel packaging approaches. We report a novel vector packaging approach using autonomously replicating cytoplasmic RNAs (replicons) to express packaging proteins. Yellow fever virus (YFV) was used as a source of replicons encoding the HIV-1 Gag–Pol polyprotein together with reporter or selectable markers. YFV replicons were able to establish chronic infection in HEK293FT cells. Replicons expressing HIV-1 Gag–Pol containing the wild-type HIV-1 protease caused strong cytotoxicity, which prevented the selection of polyclonal cell pools harboring the replicon. In contrast, a replicon carrying the T26S mutation in the HIV-1 protease gene showed no measurable cytotoxic effects, enabling the generation of stable replicon-containing cell pools. The replicon cell pools were established using antibiotic selection and maintained Gag-Pol expression for at least ten passages under selection pressure. Using these first-generation replicon cell pools as packaging cells, LV production required only transient transfection of a transfer vector, a Tat/Rev plasmid, and an envelope plasmid. Yields reached ~10⁶ TU/mL prior to concentration and ~10⁹ TU from multilayer cell stacks, which fall within the range typically reported for conventional transient transfection systems under similar culture conditions. The resulting vectors efficiently transduced target cells, and no replication-competent lentivirus (RCL) was detected using a two-phase RCL assay with p24 ELISA detection. This demonstrator platform utilizing replicon cell pools represents a novel approach for LV packaging. Full article

The preservation of rooster semen quality during short-term liquid storage remains a challenge in poultry reproductive biotechnology because sperm cells rapidly lose functional competence under ambient conditions. This deterioration is largely associated with oxidative stress and lipid peroxidation of sperm membranes, which are particularly vulnerable in avian species due to their high polyunsaturated fatty acid content and limited cytoplasmic antioxidant defenses. Selenium is an essential trace element involved in cellular antioxidant protection through its incorporation into several selenoproteins that regulate redox balance and protect cellular structures from oxidative injury. The present study evaluated the effects of selenium methionine supplementation on rooster semen quality during liquid storage at 25 °C. Semen was diluted using a standard poultry semen extender composed of sodium glutamate, glucose, potassium acetate, magnesium acetate, and potassium citrate. Selenium methionine was incorporated into the semen extender at concentrations of 0.5%, 1%, and 2% (w/v) at the time of semen dilution prior to storage. Semen quality was assessed at 0, 4, 8, 12, and 24 h of storage. Functional parameters, including total sperm motility, sperm viability, and dead sperm percentage, together with kinematic variables (VSL, VCL, VAP, ALH, LIN, and STR), were analyzed using computer-assisted sperm analysis (CASA). Structural integrity was evaluated through acrosome and plasma membrane integrity tests, while sperm physiological status and apoptotic progression were assessed using Annexin V-FITC/propidium iodide flow cytometry. Significant effects of storage time, selenium methionine concentration, and their interaction were observed for multiple semen quality parameters (p < 0.05). Among the tested concentrations, supplementation with 0.5% selenium methionine consistently produced the most favorable results, maintaining higher sperm motility, viability, and membrane integrity while reducing dead sperm percentage and apoptotic progression during storage, with protective effects particularly evident at 8, 12, and 24 h compared with the control and higher concentrations. Polynomial contrast analysis indicated predominantly non-linear dose–response relationships, with quadratic and cubic components providing the best model fit (R² = 0.90–0.99; p < 0.0001), suggesting a hormetic antioxidant effect. Overall, these findings indicate that selenium methionine supplementation in semen extender improves the stability of rooster semen during short-term liquid storage at ambient temperature, with 0.5% showing the most consistent protective effects among the concentrations evaluated. Full article

High-efficiency Ovum Pick-Up (OPU) and in vitro embryo production (IVP) are critical for the genetic improvement of high-value Wagyu cattle. However, oxidative stress and mitochondrial dysfunction during oocyte maturation remain major bottlenecks limiting blastocyst yield. This study investigated the role of inhibin in Wagyu oocyte competence through two independent proof-of-concept approaches. In the in vivo active immunization model, thirty Wagyu donors were immunized with a recombinant inhibin protein (INHA group), resulting in a significant increase in the number of recovered cumulus–oocyte complexes (COCs) (461 vs. 279, p < 0.05) and the proportion of high-quality oocytes compared to controls. Oocytes from the INHA group exhibited improved cytoplasmic maturation and mitochondrial function, characterized by higher membrane potential (ΔΨm, JC-1 ratio: 1.55 ± 0.06 vs. 0.83 ± 0.08, p < 0.05), elevated ATP content (2.35 ± 0.07 vs. 1.63 ± 0.03 pmol/oocyte, p < 0.05), and increased NADPH levels. Furthermore, the INHA group showed significantly reduced reactive oxygen species (ROS) accumulation and an increased GSH/GSSG ratio (8.48 ± 0.18 vs. 6.25 ± 0.09, p < 0.05), indicating restored redox homeostasis. Independently, in the in vitro anti-inhibin antibody (AIA) supplementation model, AIA supplementation during oocyte maturation significantly improved the nuclear maturation rate (92.96% ± 1.04%), blastocyst formation rate (56.63% ± 2.36%), and total cell number compared to controls (p < 0.05). Notably, AIA-derived blastocysts achieved a significantly higher pregnancy rate (78.65% ± 1.57%) following transfer. Collectively, these findings demonstrate that targeting inhibin mitigates oxidative injury and stabilizes mitochondrial bioenergetics, providing two distinct, physiology-based strategies for optimizing Wagyu oocyte yield and embryo production. Full article

The cytoplasmic accumulation of TDP-43 aggregates remains a persistent pathological hallmark of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and limbic-predominant age-related TDP-43 encephalopathy (LATE). The cell’s natural clearance mechanisms, the Ubiquitin-Proteasome System (UPS) and the autophagy-lysosome pathway (ALP), are hypothesized to fail, at least in part, due to the sequestration of key components of these pathways by pathological TDP-43 species, thereby impairing autophagosome-lysosome fusion and lysosomal competence. Classical autophagic activators (e.g., rapamycin) can initiate upstream steps in the pathway but cannot address downstream flux bottlenecks, limiting their ability to restore effective TDP-43 clearance. This review revisits classical strategies and discusses newer approaches to modulate TDP-43 clearance, including transcription factor EB (TFEB) activators, proteolysis-targeting chimeras (PROTACs), and antisense oligonucleotides (ASOs). We propose that adopting multi-targeting strategies and developing better biomarkers are vital for clinical success. Full article

Reproductive success in dogs and cats remains limited despite advances in assisted reproductive technologies, highlighting the need for a clearer understanding of the ultrastructural processes governing oocyte maturation. This review critically examines current knowledge on the ultrastructural and cytoplasmic mechanisms underlying oocyte maturation in dogs, with particular emphasis on cytoskeletal dynamics, organelle redistribution, mitochondrial activity, and lipid metabolism. Comparative insights from other mammalian species, especially the cat, are integrated to highlight fundamental differences in the timing and regulation of oocyte competence. The evidence suggests that inadequate cytoplasmic maturation, not meiotic progression, is the principal bottleneck affecting fertilization and early embryonic development in dogs. We conclude that future improvements in canine fertility and reproductive technologies will require a shift from predominantly hormonal and nuclear-focused approaches toward strategies targeting oocyte cytoplasmic quality and ultrastructural maturation. Full article

Mast cells (MCs) are innate immune cells that are derived from CD34⁺ hematopoietic stem/progenitor cells (HSPCs) and mature in peripheral tissues such as skin and mucosa. Mature human MCs can be generated from peripheral blood, but this process requires substantial blood volumes as HSPC frequencies are typically very low. The aim of this study was to validate a new in-house-developed protocol for the generation of MCs from less than 20 mL of peripheral blood. To this end, we used a magnetic bead-based procedure to isolate ‘untouched’ HSPCs from 14 to 16 mL peripheral blood (PB). In total, 12 cultures were set up with blood from seven healthy donors, wherein HSPCs were first expanded for 4 weeks, followed by another 8 weeks of culture in MC maturation-inducing medium. Flowcytometric analysis, histochemical staining, and degranulation assays were used to assess their phenotypic and functional features. Our data show comparable expression of cytoplasmic granules and cell-surface expression of MRGPRX2, FcεR1α, and CD117 in 8/12 blood-derived MCs (PB-MCs) and buffy coat-derived HSPCs (BC-MCs). PB-MCs responded to classic stimulating agents like IgE/anti-IgE and C48/80. Hence, our novel MC generation protocol yields functionally competent MCs with no compromise in their maturation or activation potential despite 12 weeks of in vitro culture. Full article

Endometriosis is a chronic, estrogen-dependent inflammatory disorder that is increasingly recognized as a systemic condition with profound implications for female reproductive potential. In addition to pelvic distortion and impaired folliculogenesis, growing evidence indicates that intrinsic alterations in oocyte morphology, mitochondrial function, and developmental competence contribute to infertility. The disease is driven by a multifactorial interplay of somatic mutations, epigenetic remodeling, immune dysregulation, and aberrant steroid signaling, which together create a pro-inflammatory, oxidative, and fibrotic microenvironment. Elevated cytokines, reactive oxygen species, and disrupted granulosa-cell function within the follicular niche impair meiotic progression, cytoplasmic maturation, and mitochondrial integrity, potentially accelerating oocyte aging and diminishing reproductive longevity. Epigenetic and post-transcriptional disturbances—including altered DNA methylation, histone modifications, and RNA-splicing defects—further reinforce estrogen dominance, progesterone resistance, and impaired decidualization, with downstream consequences for ovarian–endometrial communication. Although morphological abnormalities have been documented in oocytes from women with endometriosis, clinical outcomes remain heterogeneous, highlighting the need for integrative models that connect molecular alterations to functional reproductive endpoints. A deeper understanding of these mechanisms is essential for identifying biomarkers of oocyte competence and modifiable strategies—ranging from nutritional optimization to reduction of environmental risk factors—in clinical care to safeguard the reproductive potential of women with endometriosis. Full article

In vitro oocyte maturation (IVM) is a pivotal process influencing the success of embryo production in laboratory and clinical settings. However, oxidative stress (OS) often compromises oocyte quality during IVM. Antioxidants such as melatonin and epigallocatechin-3-gallate (EGCG) are known to mitigate OS by neutralizing reactive oxygen species (ROS) and bolstering antioxidant defenses. Despite extensive studies on their individual effects, the synergistic impact of melatonin and EGCG remains underexplored. Utilizing a mouse model, this study evaluated their combined effect on oocyte maturation, focusing on nuclear and cytoplasmic development, intracellular ROS, glutathione (GSH) levels, and subsequent embryonic competence. The results demonstrated that melatonin and EGCG significantly enhanced the polar body extrusion rate (p < 0.05), with the combination group achieving the highest rate of 91.96%. Cumulus expansion was observed to improve across all treated groups, with the combination treatment showing the highest cumulus expansion index (CEI) of 3.06. Furthermore, the combination treatment significantly reduced ROS levels and increased GSH content, indicating enhanced antioxidant capacity (p < 0.01). Embryonic development outcomes, including cleavage and blastocyst rates, were markedly higher in the combination group at 75.23% and 53.97%, respectively, demonstrating superior developmental potential (p < 0.01). These findings suggest that the melatonin–EGCG combination offers a novel and effective strategy to combat oxidative damage during IVM, thereby improving oocyte quality and embryonic development potential in mice. Full article

Oocytes cultured in vitro are exposed to high oxygen tension and lack follicular antioxidants, leading to redox imbalance. Eicosapentaenoic acid (EPA), a marine long-chain n-3 polyunsaturated fatty acid, possesses strong antioxidant activity. Here, using pigs as a model, we examined the effects of EPA on oocyte in vitro maturation (IVM) and subsequent developmental competence. Cumulus–oocyte complexes were cultured with EPA, followed by assessment of nuclear and cytoplasmic maturation and embryonic development; transcriptomic and proteomic analyses were conducted to explore underlying mechanisms. Supplementation with 10 µM EPA significantly improved maturation and blastocyst rates by reducing spindle defects, facilitating a more uniform organization of cortical granules and mitochondria. EPA increased resolvin E1 accumulation and reduced cumulus-cell apoptosis through downregulation of TNF-α and BAX and upregulation of BCL2. In MII oocytes, EPA lowered apoptosis, DNA damage, and ROS levels while enhancing SOD2 and GPX4 expression. Mitochondrial quality and turnover were improved via upregulation of PPARGC1A, NDUFS2, PINK1, LC3, FIS1, MUL1, and OPA1, alongside strengthened ER–mitochondria contacts. These findings demonstrate that EPA alleviates oxidative stress, optimizes mitochondrial function, and enhances porcine oocyte maturation and developmental competence in a parthenogenetic model, highlighting its potential as a marine-derived functional additive for reproductive biotechnology. Future studies will be required to validate these effects under fertilization-based embryo production systems and to further refine dose–response relationships using expanded embryo-quality endpoints. Full article

The Xenopus oocyte has long served as a versatile and powerful model for dissecting the molecular underpinnings of reproductive and developmental processes. Its large size, manipulability, and well-characterized cell cycle states have enabled generations of researchers to illuminate key aspects of oocyte maturation, fertilization, and early embryogenesis. This review provides an integrated overview of the cellular and molecular events that define the Xenopus oocyte’s transition from meiotic arrest to embryonic activation—or alternatively, to programmed demise if fertilization fails. We begin by exploring the architectural and biochemical landscape of the oocyte, including polarity, cytoskeletal organization, and nuclear dynamics. The regulatory networks governing meiotic resumption are then examined, with a focus on MPF (Cdk1/Cyclin B), MAPK cascades, and translational control via CPEB-mediated cytoplasmic polyadenylation. Fertilization is highlighted as a calcium-dependent trigger for oocyte activation. During fertilization in vertebrates, sperm-delivered phospholipase C zeta (PLCζ) is a key activator of Ca²⁺ signaling in mammals. In contrast, amphibian species such as Xenopus lack a PLCZ1 ortholog and instead appear to rely on alternative protease-mediated signaling mechanisms, including the uroplakin III–Src tyrosine kinase pathway and matrix metalloproteinase (MMP)-2 activity, to achieve egg activation. The review also addresses the molecular fate of unfertilized eggs, comparing apoptotic and necrotic mechanisms and their relevance to reproductive health. Finally, we discuss recent innovations in Xenopus-based technologies such as mRNA microinjection, genome editing, and in vitro ovulation systems, which are opening new avenues in developmental biology and translational medicine. By integrating classic findings with emerging frontiers, this review underscores the continued value of the Xenopus model in elucidating the fundamental processes of life’s origin. We conclude with perspectives on unresolved questions and future directions in oocyte and early embryonic research. Full article

Nascent polypeptides selected for export are synthesized in the cytoplasm by ribosomes and inserted into or translocated across membranes to reach their correct location. Exported proteins delay their folding and remain soluble during their cytoplasmic transit to the membrane. In bacteria, most secretory proteins require additional support from cytosolic chaperones such as trigger factor (TF) and SecB to promote their translocation competence. Here, we investigate the effect of TF and SecB on the folding dynamics and in vitro translocation of secretory and cytoplasmic model proteins PpiA and PpiB, respectively. Global hydrogen—deuterium exchange mass spectrometry (HDX-MS) experiments reveal that SecB delays the folding of slow-folding PpiA proteins but has no effect on fast folders like PpiB. In vitro protein translocation results show that TF inhibits the Sec-dependent translocation of mature PpiA/B and derivative proteins, as well as some secretory preproteins carrying a signal peptide (SP), whereas SecB has no clear effect under the same conditions. However, SecB proves to be dominant over TF in protein translocation in vitro. Finally, for the secretory preprotein proPpiA, SecB prevents SP-induced aggregation. Our findings indicate that the combined properties of signal peptides and mature domains dictate chaperone specificity and translocation efficiency, with both TF and SecB acting in a substrate-specific manner. Full article

Metabolic immune evasion is a major factor limiting the long-term efficacy of intravesical Bacillus Calmette–Guérin (BCG) therapy in non-muscle-invasive bladder cancer (NMIBC). TIA1 is a stress granule RNA-binding protein with liquid–liquid phase separation (LLPS) capacity. Its role in tumor metabolism and immunotherapy response has been unclear. Here, we demonstrated that high TIA1 expression was independently associated with favorable survival across multiple cohorts. Full-length TIA1 formed cytoplasmic condensates, repressed LDHA/PKM2/HK2, reduced lactate, and lowered extracellular acidification. A condensate-defective ΔLCD (deletion of the low-complexity domain) mutant was inactive. TIA1 showed physical association with these glycolytic mRNAs in human cells, consistent with mRNA-linked control. Condensate-competent TIA1 promoted CD8⁺ T-cell proliferation, increased CD69 and Granzyme-B, and reduced PD-1 in co-culture. TIMER (Tumor Immune Estimation Resource) and spatial-omics supported co-localization with tumoral CD8A. BCG induced this metabolic–immune signature in cell lines, murine models, and patient explants, but the effects were abolished by TIA1 knock-down. Conversely, TIA1 over-expression alone limited tumor growth and recapitulated BCG-mediated glycolytic restraint and T-cell activation. Together, these results support an LLPS-linked, mRNA-associated regulation of tumor glycolysis. BCG-driven glycolytic suppression and CD8⁺ T cell activation track with the condensate-forming capacity of TIA1. TIA1 emerges as a prognostic biomarker and a potential therapeutic axis to improve intravesical immunotherapy in NMIBC. Full article

Background: Tissue-specific long non-coding RNAs (lncRNAs) represent potential biomarkers. The testis-enriched lncRNA41584, previously identified as downregulated in male-sterile silkworm mutants (JMS, GMS), is associated with male sterility, but its functional mechanism remained unknown. Subcellular localization, dual-luciferase reporter assays, MTT, and flow cytometry were employed to examine lncRNA41584–miR-3047-z–BmCDK20 interactions. In vivo functional validation included lncRNA41584 knockdown and miR-3047-z overexpression in Bombyx mori. lncRNA41584 localizes predominantly to the cytoplasm and acts as a competing endogenous RNA (ceRNA) by sponging miR-3047-z, thereby upregulating the cyclin-dependent kinase BmCDK20. Perturbation of this axis impaired BmN cell proliferation, causing G1 phase arrest, and led to spermatocyst malformation, reduced fertilization rates, and increased unfertilized eggs. The lncRNA41584–miR-3047-z–BmCDK20 ceRNA network is essential for testicular cell cycle progression and spermatogenesis in silkworms, offering mechanistic insights into lepidopteran male sterility and potential targets for pest fertility regulation. Full article