Search Results (896)

Histone lysine demethylases are mainly known as transcriptional regulators for their demethylase activity, but catalysis-independent functions are increasingly recognized. This work focuses on lysine-specific demethylase 5B (KDM5B), targeting H3K4me3. The canonical functional isoform is PLU-1. A catalytically inactive N-terminally truncated isoform (NTT) was previously identified in human cell lines. NTT lacks the Jumonji N-terminal (JmjN) domain, responsible for demethylase activity, and part of the AT-Rich Interaction Domain (ARID), but retains the plant homeodomains (PHDs) involved in chromatin interaction. The study aimed to investigate NTT chromatin association, its potential regulatory role, and its phenotypic effects. NTT-FLAG was overexpressed in MCF7 cells, using PLU-1-FLAG and empty vector for comparison. Chromatin association and H3K4me3 distribution were analyzed by Cleavage Under Targets and Release Using Nuclease (CUT&RUN), while migration and invasion were assessed by wound-healing, live-imaging microscopy, and invasion assays. NTT-FLAG and PLU-1-FLAG displayed similar genomic localization, preferentially associating with active promoters enriched in H3K4me3. Both isoforms increased promoter-associated H3K4me3, suggesting that chromatin binding alone is insufficient to promote demethylation. Functionally, NTT overexpression was associated with increased directional migration and invasive capacity compared with PLU-1. Overall, these findings support a catalysis-independent regulatory role for NTT and suggest a model in which chromatin interaction is driven by PHD domains, while co-interactor availability may be required for proper PLU-1 demethylase activity. Full article

Cardiac pacemaker and valve cells operate within a heterogeneous interstitial environment, but the identities and observable behaviors of many constituent cells remain incompletely defined. We integrated complementary imaging observations from a single experimental platform into an exploratory analysis of cultured cells isolated from the adult mouse sinoatrial node (SAN) and atrioventricular valves of S100B-EGFP reporter mice. Cellular morphology, endogenous NAD(P)H fluorescence, tetramethylrhodamine methyl ester (TMRM) fluorescence, correlative light and electron microscopy (CLEM), time-lapse imaging, transmission electron microscopy (TEM), and 5-ethynyl-2′-deoxyuridine (EdU) labeling were used to describe selected reporter-positive and reporter-negative cells. S100B-promoter-associated EGFP+ cells displayed heterogeneous forms, including cells with small bodies and long processes. In representative fields, EGFP+ cells showed greater NAD(P)H autofluorescence intensity and lower TMRM signal than adjacent EGFP− cells. CLEM related selected fluorescence phenotypes to ultrastructure in the same cells and documented mitochondrial, vesicular, nuclear, and membrane-associated profiles. One live sequence captured movement of a TMRM-positive, mitochondrion-like structure through a thin intercellular bridge toward an EGFP− cell, followed by bridge disassembly. Additional images showed mitochondrial and vesicular profiles near cell surfaces or in extracellularly situated regions, elongated NAD(P)H-bright structures, reporter-positive cytoplasmic fragments, nuclear size heterogeneity, a narrow connection between nuclear profiles, and unequal EdU labeling among nuclear fragments. These findings are descriptive and hypothesis-generating. They do not establish definitive cell identity, phenotype prevalence, active or selective secretion, tunneling-nanotube identity, mitochondrial metabolic competence, regulated nuclear remodeling, recipient-cell uptake, or physiological consequence. The study provides an integrated morphological framework and a set of candidate events for future quantitative investigation of S100B-associated cardiac interstitial-cell biology. Full article

Sweetpotato yield depends strongly on storage root development, but live-cell analysis of cytoskeletal dynamics in Ipomoea is limited by the lack of suitable in vivo reporters. Here, we chose the diploid wild relative Ipomoea cordatotriloba (I. cordatotriloba) as a model and constructed a pMAS::DsRed-MBD recombinant vector for microtubule visualization. The pMAS::DsRed-MBD construct was first evaluated by transient co-expression with the microtubule marker StayGold-TUB6 in Nicotiana benthamiana, where the two signals showed overlapping filamentous patterns. Cut-dip-budding (CDB) transformation yielded one PCR-positive primary T₀ plant (L1). In L1, confocal imaging visualized filamentous microtubule-like networks and real-time filamentous dynamics in roots, stems and leaves. PCR screening of five T₁ progeny detected the transgene in four individuals, supporting germline transmission from the T₀ plant. No significant differences were detected between WT and L1 in the measured vegetative traits under the tested conditions. This study generates a candidate microtubule reporter plant within the genus Ipomoea. It offers a prospective imaging resource in vivo for future investigations into potential cytoskeletal contributions to sweetpotato storage root development. Full article

Drug resistance in TNBC is associated with metabolic plasticity, yet little is known about how mitochondrial and cellular states evolve after chemotherapeutic withdrawal. We used optical redox imaging (ORI) microscopy and complementary functional measurements to characterize post-Taxol recovery in two TNBC cell lines with different Taxol sensitivities. Whole-dish nuclear counting following 48 h Taxol treatment confirmed that HCC1806 cells responded at lower Taxol concentrations than MDA-MB-436 cells. Recovery was subsequently monitored following extracellular Taxol withdrawal using mitochondrial redox indices, mitochondrial reactive oxygen species (mito-ROS), mitochondrial membrane potential (MMP), multinucleation, and live- and dead-cell counts. HCC1806 cells showed substantial recovery of several mitochondrial parameters and resolution of multinucleation by 96 h; however, a late reductive shift in the optical redox ratio (ORR), reduced MMP, and attenuated live-cell population expansion indicated incomplete redox and functional recovery. In contrast, MDA-MB-436 cells exhibited persistent elevations in Fp and ORR through 144 h, residual multinucleation, and little sustained live-cell population expansion. These findings demonstrate that mitochondrial, multinucleation, and population-level recovery can proceed on different timescales and that post-treatment recovery differs markedly between TNBC models with different Taxol sensitivities. Longitudinal ORI provides a non-destructive approach for characterizing persistent post-treatment metabolic states and, together with functional measurements, may facilitate investigation of recovery-associated phenotypes relevant to therapeutic response. Full article

Hypoxia poses a significant threat to hippocampal neurons, but the temporal dynamics of mitophagy pathways in neuronal models remain incompletely characterized. This study aims to delineate time-dependent mitochondrial structural damage, fragmentation, and mitophagic responses in hippocampal neuronal cells under sustained hypoxia and to explore the key mitophagy receptors involved. Murine HT22 immortalized hippocampal neuronal cells were exposed to 1% O₂ for 6–72 h. Mitochondrial integrity was assessed using Western blot and transmission electron microscopy. Mitochondrial fragmentation was quantified via HSP60 intensity, mitochondrial length, and number of fragments per cell using immunofluorescence and MitoTracker live-cell labeling. Mitophagic activity was evaluated using LC3B puncta quantification, LC3B–Tom20 colocalization, and mitochondria–lysosome proximity via LysoTracker co-labeling. The transcriptional responses of five mitophagy-related genes (Bnip3, Bnip3l, Pink1, Fundc1, and Atg5) were screened using RT-qPCR, with protein validation by Western blot and immunofluorescence. Prolonged hypoxia progressively reduced Tim23 and Tom20, with ultrastructural damage and mitochondrial fragmentation seen. Mitophagy-associated phenotypes were evident, as shown by increased LC3B puncta, LC3B–Tom20 colocalization, and mitochondria–lysosome proximity. Bnip3 and Bnip3l showed the earliest and most pronounced transcriptional upregulation, with BNIP3 and BNIP3L protein levels elevated from 12 to 48 h. BNIP3 and BNIP3L intensities increased after 48 h, exhibiting granular and filamentous morphologies, respectively. This study provides a temporal framework for hypoxia-induced mitophagy in hippocampal neurons, identifying BNIP3/BNIP3L as early and prominent responders, which may inform therapeutic targeting in hypoxic brain injury. Full article

Foot-and-mouth disease (FMD) is a highly contagious viral disease that seriously threatens livestock health. Protective immunity induced by vaccination is primarily associated with the generation of neutralizing antibodies; however, conventional virus neutralization tests are time-consuming and require the handling of live virus, limiting their suitability for rapid and repeated evaluation in routine settings. In this study, we established a surrogate neutralization assay for the functional evaluation of neutralizing antibody activity against FMD virus (FMDV) serotype O. The assay measures changes in luciferase-based luminescent signals generated following exposure of LgBiT-expressing cells to HiBiT-tagged virus-like particle (VLP) preparations. FMDV serotype O-derived VLPs exhibited stable capsid protein expression, assembly characteristics, and morphological integrity similar to those of virus particles, as confirmed by sucrose gradient fractionation and electron microscopy. In the presence of neutralizing antibodies, a reduction in luminescent signals was observed, enabling functional discrimination of antibody activity. The surrogate assay results showed a strong correlation with those of the conventional virus neutralization test for FMDV serotype O (R² = 0.9068). This study demonstrates the feasibility of the surrogate neutralization assay for assessing the functional activity of neutralizing antibodies and its potential applicability to vaccine immunogenicity assessment and functional antibody analysis. Full article

Background: This study aims to develop an amyloid-beta-induced three-dimensional (3D) Alzheimer’s disease model using the Magnetic Levitation (MagLev) technique and rat pheochromocytoma (PC-12) (rat pheochromocytoma) cells. Methods: Initially, optimization studies were performed to determine the appropriate paramagnetic agent concentration and cell number required for the formation of 3D cellular structures. The formation of these structures was monitored by light microscopy, and cell viability was evaluated using a live/dead assay. Filamentous actin (F-actin) expression was assessed by immunostaining. Subsequently, amyloid beta 1–42 (Aβ1–42) aggregates were introduced into the 3D cellular structures to establish a 3D Alzheimer’s disease model. The 3D disease model was characterized by immunostaining of Choline Acetyltransferase (ChAT). Finally, the applicability of the developed model for therapeutic evaluation was demonstrated using curcumin as a representative neuroprotective compound. Results: The optimization studies successfully enabled the formation of 3D cellular structures using the magnetic levitation technique. Immunostaining analysis showed an increase in F-actin expression in 3D structures. Following the introduction of Aβ1–42 aggregates, a significant decline in cell viability was observed by day 21. ChAT immunostaining revealed a decrease in fluorescence intensity (F.I.) to 28% of the control level, corresponding to an approximately 72% reduction in ChAT expression. Curcumin treatment increased cell viability by 22%, 23%, and 13% on days 7, 15, and 21, respectively. Conclusion: Overall, the findings showed that the 3D Aβ 1–42 induced 3D Alzheimer’s disease model can provide a valuable experimental platform for investigating neurodegenerative diseases and evaluating potential therapeutic interventions. Full article

Escherichia coli is a common porcine diarrheal pathogen transmitted via the fecal–oral route through contaminated environments and feed. This study evaluated the antimicrobial potential of a bacteriocin-like substance produced by Loigolactobacillus coryniformis WYQ-2 isolated from pickled vegetables. Its physicochemical stability, molecular mass, and antimicrobial spectrum were characterized, and the mode of action against Escherichia coli was investigated through intracellular ATP quantification, live/dead staining, and scanning electron microscopy (SEM). The substance exhibited broad-spectrum antibacterial activity, with the strongest inhibition against Escherichia coli (the diameter of the inhibition zone reached 41.35 mm). It remained stable under prolonged UV irradiation (0–2.5 h), under heating at 100 °C, and within the pH range of 3.0–9.0. Molecular mass determination revealed a low-molecular-weight peptide (<2.7 kDa). Protease susceptibility tests showed activity was reduced by trypsin and papain, slightly sensitive to proteinase K, yet relatively stable toward other tested proteases. Mechanistically, ATP levels declined markedly within 0.5–2 h of treatment, while live/dead staining and SEM confirmed membrane disruption, deformation, and surface indentations in treated Escherichia coli cells. These findings demonstrate that the WYQ-2-derived bacteriocin-like substance possesses favorable physicochemical traits and exerts antibacterial action primarily by compromising membrane integrity, highlighting its promise as a natural alternative for controlling porcine diarrheal diseases. Full article

Integrin-mediated adhesion is essential for leukocyte trafficking and contributes to hematogenous tumor metastasis. We recently showed that metastatic B16 melanoma cells undergo VLA-4-dependent rolling and rapid arrest on endothelial cells under physiological shear flow without classical chemokine-induced integrin activation. However, the molecular basis of this chemokine-independent adhesion remained unresolved. Here, atomic force microscopy (AFM)-based single-molecule force spectroscopy was used to characterize VLA-4-mediated adhesion between living B16 melanoma cells and bEnd.3 endothelial cells at the level of individual receptor–ligand interactions. Rupture-force histograms revealed a dominant population centered at approximately 33 pN, consistent with single VLA-4/VCAM-1 bonds. VLA-4 blockade reduced adhesion frequency, whereas VCAM-1 blockade shifted the rupture-force distribution toward lower forces; both effects support molecular specificity. VLA-4-directed antibody and pharmacological inhibition further indicated that regulation primarily affects bond-formation probability rather than the strength of individual receptor–ligand interactions. Homotypic VLA-4-mediated adhesion between melanoma cells was also detected. These findings provide a functional link between single-molecule receptor interactions and the rolling-to-arrest transition of metastatic melanoma cells under physiological flow. Together with our recent flow-chamber studies, they support a model in which VLA-4/VCAM-1 bond formation contributes to vascular arrest without requiring classical integrin activation, providing molecular insight into an early step of metastatic dissemination. Full article

Oral supplementation of the Hafnia alvei (H. alvei) HA4597 probiotic strain has been shown to reduce appetite, body weight and fat content in overweight humans and obese mice. However, it remains unknown if H. alvei may also influence the gut digestive function. Thus, in the present study, the structural parameters of the intestine and the activity of digestive enzymes of the brush border enterocytes were analyzed in mice after they received a suspension of live H. alvei bacteria or their total protein extract for 16 days. Using electron microscopy, bacteria resembling H. alvei were found on the surface of microvilli of colonic enterocytes of mice treated with H. alvei suspension. In the group receiving H. alvei protein, there was a trend toward an increase in the number of mucus-producing goblet cells on the villi of the jejunum, and in both groups receiving H. alvei preparations, the number of intraepithelial lymphocytes on the villi of the ileum increased (in the group receiving H. alvei protein, there was a tendency towards an increase; in the group receiving H. alvei suspension, there was a significant increase). Treatment with both H. alvei preparations led to an increased activity of alkaline phosphatase in the chyme of the duodenum and jejunum and also in the jejunal mucosa, correlating negatively with visceral body fat content. The use of both H. alvei preparations had no significant effect on the activity of glucoamylase, maltase and aminopeptidase N in the intestinal mucosa. Thus, the 16-day administration of H. alvei bacteria or their protein extract increases the protective properties of the intestine, including the mucous barrier. Moreover, increased activity of alkaline phosphatase, a fat absorption-limiting enzyme, may contribute to the known anti-obesity effects of H. alvei. Taken together, H. alvei preparations display beneficial effects on gut health. Full article

Background: Pathogenic variants in SLC6A1, which encodes the γ-aminobutyric acid (GABA) transporter GAT-1, cause developmental and epileptic encephalopathies (DEEs) through reduced GABA uptake, impaired transporter trafficking, and functional haploinsufficiency. 4-phenylbutyrate (PBA) is a clinically available small molecule with chemical-chaperone and histone-deacetylase-inhibitor activities that can rescue misfolded GABAergic proteins, but variant-level rescue data are needed to guide precision treatment. Methods: We report a novel de novo missense mutation p.Ala305Val in GAT-1 encoding SLC6A1, in a patient with myoclonic-atonic epilepsy and a developmental and epileptic encephalopathy phenotype. Ala305Val was compared with the residue-matched comparator p.Ala305Thr (Ala305Thr). Variant effects were evaluated by (i) protein-structure prediction across nine stability-prediction algorithms using the cryo-EM-derived human GAT-1 template (PDB 7Y7W); (ii) 3H-GABA uptake assays in HEK293T cells and in human iPSC-derived astrocytes and cortical neurons; (iii) live-cell confocal microscopy of ER colocalization; (iv) pharmacologic rescue with PBA, TUDCA and salubrinal (v) and GAT-1 cDNA gene-augmentation, alone and in combination with PBA. Results: AI-based stability predictors uniformly indicated destabilization of GAT-1 p.Ala305Val and GAT-1 p.Ala305Thr. GAT-1 p.Ala305Val reduced 3H GABA uptake across HEK293Ts, astrocytes, and neurons. The mutant transporter accumulated within the endoplasmic reticulum (ER), with ER colocalization rising from approximately 30% in wildtype to ~80% in GAT-1 p.Ala305Val; PBA reduced ER retention to approximately ~40% and restored total GAT-1 fluorescence toward wildtype levels. Pharmacochaperones (PBA, TUDCA) restored GABA uptake for the mutant transporters. Wildtype GAT-1 gene augmentation improved GABA uptake in the heterozygous condition but combined PBA plus wildtype allele augmentation produced rescue greater than either intervention alone in the available dose-response ranges. Conclusions: GAT-1 p.Ala305Val is a trafficking-impaired, loss-of-function variant whose dysfunction is amenable to two convergent therapeutic axes: pharmacologic correction of folding and trafficking, and augmentation of functional transporter expression. These findings support a two-pronged precision-medicine framework for SLC6A1-related DEEs in which PBA increased the transporter function augmented by genetic approaches. Full article

Mitochondrial mechanisms are increasingly implicated in complex neurological conditions, including Autism Spectrum Disorder (ASD). Propionic acid (PPA) is widely used to study mitochondrial dysfunction in preclinical models of ASD. However, the molecular mechanisms that drive PPA-induced neurotoxicity are unresolved. Here, we examined mitochondrial remodeling under PPA-induced stress in neuroblastoma SH-SY5Y cells. PPA systemically altered the transcriptional regulation of mitochondrial dynamics and disrupted canonical proteins involved in mitochondrial fusion (L-OPA1, MFN2), fission (DRP1) and quality control (LC3-II). Confocal microscopy revealed an upregulation of both fission and fusion events and impairments to mitochondrial integrity, connectivity and turnover. Live-cell respirometry demonstrated consequent deficits in both oxidative and glycolytic energy production, while respiratory chain electron flow assays illustrated a shift in TCA cycle flux driven by a remodeling of mitochondrial substrate utilization. This work describes a molecular signature of metabolic stress in the SH-SY5Y system, providing novel insights into the mechanisms and manifestations of PPA-induced neurotoxicity. Full article

Background: Surgical removal of jaw cysts may result in bone defects that compromise local structural integrity and delay spontaneous regeneration. Photobiomodulation (PBM) has been proposed as a minimally invasive intraoperative adjunct capable of modulating osteogenic activity; however, translational evidence derived from freshly harvested human jawbone tissue remains limited. Objective: This study evaluated the effects of an intraoperative 980 nm PBM protocol on early osteogenic activity, osteoblast-like cell abundance, morphometric outcomes, and cell viability in primary human jawbone explants obtained during cystectomy. Materials and Methods: A comparative translational ex vivo study was conducted using cortical bone explants harvested from 40 patients undergoing surgical treatment of medium- to large-sized maxillary or mandibular cystic lesions. For each patient, one explant was exposed immediately after harvesting to pulsed 980 nm laser irradiation (20 J total energy; 40 J/cm² fluence), whereas the paired explant served as an untreated control. Explants were cultured using a primary outgrowth technique and subjected to osteogenic induction for 14 days. Osteogenic activity was evaluated by immunofluorescence staining for alkaline phosphatase (ALPL) and osteocalcin (OCN), followed by confocal microscopy and quantitative morphometric analysis. Cell viability was assessed using a live/dead fluorescence assay. Results: Viable cultures were successfully established from 34 patients (85% experimental yield). PBM-treated explants exhibited significantly greater osteoblast-like cell counts than paired controls (median: 2820 vs. 1525 cells; p < 0.001), together with greater cumulative osteoblastic area (median: 537,028 vs. 325,166 relative units; p < 0.001) and higher osteoblastic area occupancy (median: 19.00% vs. 11.75%; p < 0.001). Immunofluorescence analysis demonstrated qualitatively stronger ALPL and OCN expression in PBM-treated cultures. Cell viability did not differ significantly between paired PBM-treated and control cultures (Wilcoxon signed-rank test, p = 0.18). Conclusions: In this comparative ex vivo model, intraoperative 980 nm PBM significantly enhanced early osteogenic activity and increased osteoblast-like cellular abundance in human jawbone explants while maintaining cellular viability. These findings provide translational support for further investigation of PBM as a biostimulatory adjunct to enhance bone regeneration following cyst surgery. Full article

Background: Nucleic acid vaccines adjuvanted with lipid nanoparticles (LNPs) are a simple and effective means of protection against infectious diseases through the delivery of antigen(s) that elicit(s) specific immunity. DNA vaccines are not commercially available for poultry mainly due to low protective efficacy and the associated production cost. Methods: We introduced here a novel nanoadjuvant system (namely, QTAP) comprising DOTAP and Quil-A for efficient delivery of plasmid DNA (pDNA) constructs into cells and boosting immune responses of chickens. Variable QTAP-pDNA-LNPs formulas were investigated for their physicochemical characteristics, cellular transfection, and in vivo efficacy in a vaccine/challenge model using pDNA encoding both S and N proteins of avian Infectious Bronchitis Virus (IBV). Results: Under electron microscopy, all LNP formulas were spherical in shape and had an overall size range of 45.4–376.3 nm. The formula with the smallest size had the lowest pDNA encapsulation and the highest polydispersity index (PDI). On the contrary, formulas with larger sizes and lower PDI values encapsulated a higher concentration of plasmid payload. Interestingly, the high-payload vaccine formula was stable at both 25 °C and 4 °C for up to 6 weeks of storage, but its gene expression dropped beyond this point at both temperatures. Following immunization of chickens, all groups were challenged with a virulent IBV. Surprisingly, both formulas were able to reduce the IBV viral shedding, indicating their effectiveness even when low concentrations of pDNA were incorporated in LNPs. However, pre-challenge sera from immunized chicks did not elicit IBV-specific antibodies, compared to birds immunized with a commercial live-attenuated IBV vaccine. Conclusions: This study suggests that the QTAP nanoadjuvant system is a safe and effective adjuvant for DNA immunization in poultry that could also be further developed for commercialization. Full article

The metastrongyloid parasite Angiostrongylus vasorum is currently considered an emerging gastropod-borne lungworm species of canids and has gained growing scientific attention in recent years. Nonetheless, knowledge of gastropod innate immune reactions and how A. vasorum larvae are attacked by gastropod-derived haemocytes is still very sparse. The current study aims to investigate gastropod haemocyte innate immune reactions in response to A. vasorum L1 larvae and antigen (AvAg) in vitro. Gastropod haemocytes were isolated from the giant African snail (Lissachatina fulica) via cardiac puncture, confronted with AvAg or A. vasorum L1, and thereafter assessed for cell activation status, ROS production and extrusion of invertebrate extracellular phagocyte traps (InEPT). Haemocyte–parasite interactions were assessed via flow cytometry, live cell 3D-holotomographic microscopy as well as scanning electron microscopy (SEM). Overall AvAg stimulation activated haemocytes and induced a time-dependent increase in haemocyte ROS production. Moreover, SEM analyses illustrated the formation of ‘InEPT-like’ structures in response to A. vasorum L1. These novel findings on neglected invertebrate innate immune responses will help to better understand the complex mollusc–metastrongyloid parasite interactions, which contribute to the epizootiology of not only canine angiostrongylosis but also of closely related metastrongyloid nematodes of public and veterinary health concern. Full article