Search Results (3,939)

Hepatocellular carcinoma (HCC) is a highly aggressive malignancy with a poor prognosis. While sorafenib serves as the first-line therapy for advanced HCC, its efficacy is frequently hampered by side effects and the development of drug resistance, necessitating the development of novel agents to enhance HCC sensitivity to sorafenib. In this study, we demonstrate that the antihistamine astemizole significantly enhanced the antitumor efficacy of sorafenib in HCC cell lines. This combination treatment cooperatively inhibited HCC cells’ proliferation and induced cell cycle arrest at the G1 phase, as evidenced by decreased cyclin D1 and p-Rb levels and increased p27 expression. Furthermore, the combination of astemizole and sorafenib synergistically inhibited HCC cells’ migration, invasion, and adhesion. It also reduced F-actin polymerization and the expression of metastasis-regulating proteins, including p-Integrinβ1, FAK, and MMP1. Additionally, the combination treatment suppressed tube formation in HUVECs, accompanied by downregulation of HIF-1α and reduced VEGF secretion. Co-inhibition of Eag1 and the ERK/MAPK signaling pathway may underlie the enhanced anti-HCC effects of sorafenib by astemizole. Collectively, these findings indicate that astemizole significantly enhanced the antitumor activity of sorafenib by inhibiting proliferation, metastasis, and angiogenesis in HCC cells, suggesting its potential as a promising adjuvant to improve sorafenib-based therapy in HCC. Full article

Toxoplasma gondii, the causative agent of toxoplasmosis, a disease widely distributed, is an intracellular parasite that invades host cells of different tissues using specialized endocytic activity. Recent studies suggest that tunneling nanotubes (TNTs), thin cell-surface projections, may participate in the parasite–host cell interaction. Here we report results that suggest the involvement of host-cell TNTs in the adhesion of T. gondii tachyzoites to epithelial LLC-MK² cells. Microscopy analysis showed that incubating cells in a medium containing 0.45 M sucrose induces reversible assembly of TNTs without affecting cell viability. The presence of extended TNTs correlated with increased parasite adhesion and reduced parasite entry, thus suggesting a structural or signaling role in mediating adhesion. TNTs assembled following sucrose incubation contain both actin and tubulin components as determined by immunofluorescence microscopy. These results highlight a possible functional relevance of TNTs in T. gondii host cell interaction, especially in parasite adhesion, opening new perspectives for understanding T. gondii-host cell interaction. Full article

The deficiency of TCOF1 is closely associated with multiple cellular dysfunctions, but its function in mitochondrial homeostasis and cytoskeletal regulation remains unclear. First, our research revealed that TCOF1 deficiency significantly inhibits tumor cell migration, suggesting TCOF1 plays a crucial role in cellular motility. Further studies demonstrated that TCOF1 deficiency disrupts normal F-actin polymerization, compromises cytoskeletal structural integrity, and impairs the dynamic assembly of F-actin, thereby affecting cell morphology and motility functions. Additionally, TCOF1 deficiency leads to mitochondrial dysfunction characterized by aberrant energy metabolism. Mechanistically, TCOF1 deficiency decreased the protein levels of p53, subsequently affecting mitochondrial biogenesis and functional maintenance, suggesting TCOF1 may regulate mitochondrial homeostasis via a p53-dependent pathway. Collectively, our study reveals TCOF1’s role in regulating tumor cell migration by influencing F-actin assembly and the p53-mitochondrial axis, playing a critical role in maintaining cytoskeletal dynamics and energy metabolism. Full article

Aflatoxin B1 (AFB1) and patulin (PAT) are prevalent foodborne mycotoxins with hepatotoxic potential, but the hepatic effects of combined exposure remain largely unclear. This study investigated the hepatotoxic consequences of co-exposure to AFB1 and PAT using no-observed adverse effect levels (NOAELs) in C57BL/6 mice and low-cytotoxic concentrations in HepG2 cells selected by viability screening. Mice and cells were assigned to four groups: control, AFB1, PAT and AFB1 + PAT. Exposure to either toxin individually did not cause evident liver injury, whereas co-exposure significantly elevated alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, reduced liver index, and induced clear histopathological alterations. Co-exposure markedly aggravated oxidative stress, characterized by increased reactive oxygen species (ROS) and malondialdehyde (MDA) and decreased superoxide dismutase (SOD). In parallel, the levels of interleukin-6 (IL-6), interleukin-1 beta (IL-1β), and tumor necrosis factor-alpha (TNF-α) were elevated, together with the early fibrosis-related markers alpha-smooth muscle actin (α-SMA) and vimentin. The apoptotic response was characterized by increased Bcl-2-associated X protein (Bax) and reduced B-cell lymphoma-2 (Bcl-2), together with cysteine-dependent aspartate-specific protease-3 (caspase-3) activation. These findings indicate that co-exposure to AFB1 and PAT elicits hepatotoxicity through amplified oxidative stress, inflammation, and caspase-dependent apoptosis, supporting the need to further consider mycotoxin co-exposure in toxicological evaluation. Full article

Formalin-fixed paraffin-embedded (FFPE) tissues represent an invaluable resource for both basic and clinical research due to their stable preservation of tissue architecture and molecular integrity. Multiplex immunofluorescence (mIF) using tyramide signal amplification (TSA) enables the simultaneous detection of multiple antigens within a single FFPE section. Here, we describe a kit-independent and customizable TSA-based mIF protocol that utilizes commercially available horseradish peroxidase (HRP)-conjugated secondary antibodies and tyramide–fluorophore reagents. The method was applied using FFPE endometriosis tissue, targeting estrogen receptor alpha (ERα), progesterone receptor (PR), α-smooth muscle actin (αSMA), CD20 and CD31. Each staining round was followed by heat-induced epitope removal (HIER) of the bound antibodies while preserving covalently deposited signals. Fluorescence imaging was performed using a multi-channel slide scanner with carefully selected fluorophores to enable optical separation between detection channels. Under the conditions described, the protocol enabled clear visualization of maker-specific staining patterns with preserved tissue morphology. This study provides a practical and flexible TSA-based mIF protocol as a qualitative proof of concept, offering an accessible alternative to commercial kit-based approaches. Further studies will be required to establish quantitative performance and a broader applicability across tissue types. Full article

Background: Actinic keratoses (AKs) are considered early in situ forms of cutaneous squamous cell carcinoma (cSCC). However reliable histopathological or molecular markers for predicting the risk of progression are still lacking. The aim of this study was to investigate the relationship between immunohistochemical markers and basal proliferation patterns of AKs in order to identify histopathological associations that may be relevant for malignant transformation. Methods: A total of 97 AK samples from facial and scalp areas were retrospectively analyzed and classified according to their basal proliferation pattern (Pro I: non-proliferative and Pro III: proliferative). Immunohistochemical staining was performed for Ki-67, p53, p16 and podoplanin. In addition, histopathological parameters such as Röwert-Huber grade, inflammatory infiltrate, parakeratosis, elastosis and the presence of acantholysis were evaluated. Results: Pro III lesions were significantly more frequently associated with higher Röwert-Huber grades (AK III: 47.9% vs. 14.3%; p = 0.0004) and with acantholysis (p = 0.0004). No significant differences between the groups were found for Ki-67, p53 and p16. Podoplanin expression, however, was significantly higher in Pro III lesions (93.7% vs. 57.1%, p < 0.0001) and was predominantly localized basally. The combination of a PRO III pattern and podoplanin positivity identified a distinct histopathological subgroup associated with features linked to progression. Conclusions: Podoplanin expression, especially in combination with PRO III pattern and acantholysis, characterizes a histologically and biologically distinct AK subgroup. In contrast, Ki-67, p53 and p16 showed no additional discriminative value in this cohort. Podoplanin could therefore be a useful addition to existing classification systems and in the future support risk-adapted treatment decision. However, prospective longitudinal studies are required to determine its prognostic value. Full article

Betaine-homocysteine methyltransferase (BHMT) is an enzyme involved in one-carbon metabolism and plays a crucial role in maintaining liver health. In this study, we investigated the impact of liver-specific deletion of BHMT on liver dysfunction using a mouse model. We generated BHMT floxed mice and bred them with albumin Cre to generate liver-specific BHMT knockout (BHMT LKO) mice. Liver tissues harvested from six-month-old chow-fed BHMT floxed and LKO mice were characterized through histological, biochemical, and molecular analyses. BHMT LKO mice displayed a complete loss of hepatic expression of BHMT mRNA, protein and enzyme activity. Histopathological analysis revealed the development of hepatic steatosis in BHMT LKO mice compared to the floxed mice. These morphological changes were supported by biochemical analysis showing elevated levels of hepatic triglycerides in conjunction with a profound decrease in the methylation potential (i.e., reduced S-adenosylmethionine (SAM): S-adenosylhomocysteine (SAH) ratio), which was mainly driven by a six- to sevenfold increase in SAH levels. BHMT LKO mice also exhibited increased lipid peroxidation and lysosomal dysfunction compared to floxed mice. Early signs of inflammation were seen in the livers of BHMT LKO mice of both sexes, as evident from significant increase in CD68-positive cells and interleukin 1β levels. Additionally, there was a moderate increase in fibrosis, as evidenced by the upregulated expression of α-smooth muscle actin and collagen II levels and the histological assessment of picrosirius red-stained liver sections of BHMT LKO mice of both sexes compared to their respective counterparts. These findings demonstrate that hepatic BHMT deficiency promotes lipid accumulation, lysosomal/proteasomal dysfunction, and early inflammatory and fibrotic changes in the liver by reducing the methylation potential. Collectively, our results underscore BHMT as a critical regulator of liver homeostasis and a potential therapeutic target in liver-related disorders. Full article

Titanium and its alloys are widely used for orthopedic implants, but their intrinsic bioinertness may hinder osseointegration. In this study, titanium dioxide nanotube (TNT) arrays were fabricated on Ti-6Al-4V scaffolds via anodization, and their effects on the adhesion behavior of human bone marrow mesenchymal stem cells (hBMSCs) were investigated. Surface characterization showed that anodization successfully generated ordered TNT layers, increased surface roughness, enhanced protein adsorption, and induced an apparent superhydrophilic wetting response. Compared to the untreated scaffold and TNT50, the small-diameter TNT10 surface significantly promoted hBMSC adhesion and proliferation. Microscope imaging further revealed enhanced cell spreading, F-actin organization, and vinculin expression on TNT surfaces, with the most prominent focal adhesion-related staining observed in TNT10. Quantitative proteomic analysis showed that TNT10 was associated with coordinated remodeling of adhesion- and cytoskeleton-related molecular programs, including focal adhesion, cell–substrate junction, and regulation of the actin cytoskeleton. In contrast, TNT50, despite supporting obvious cytoskeletal remodeling, was more compatible with a dynamic, higher-turnover adhesion state. Overall, these findings suggest that small-diameter TNTs provide a more favorable interfacial microenvironment for stable early hBMSC adhesion on porous titanium scaffolds. Full article

Opioid analgesics are essential in the management of severe and chronic pain; however, their prolonged use is limited by the onset of analgesic tolerance and opioid-induced hyperalgesia (OIH). Recent studies increasingly implicate both synaptic functional and structural plasticity within nociceptive pathways as crucial mechanisms in OIH and tolerance. This review integrates current mechanistic understanding of how opioids alter synaptic transmission throughout the dorsal root ganglia (DRG), spinal dorsal horn, and supraspinal nociceptive networks. Peripherally, μ-opioid receptor (MOR) activation on TRPV1-positive nociceptors initiates presynaptic long-term potentiation (LTP), forming an early substrate for central sensitization. In the spinal dorsal horn, chronic opioid exposure drives NMDAR-dependent LTP, TRPC-mediated calcium influx, and actin cytoskeleton remodeling, leading to persistent increases in synaptic strength and excitatory connectivity. In supraspinal regions—including the ventral hippocampus, prefrontal cortex, and amygdala—opioids promote experience-dependent plasticity and predictive coding, which link environmental cues to reduced analgesic effectiveness. In addition to synaptic functional plasticity, opioid-induced synaptic structural plasticity within nociceptive pathways has been shown to underlie the long-term nature of opioid analgesic tolerance. Collectively, these data define a distributed network of opioid-responsive synapses whose pathological potentiation underpins the development of tolerance and hyperalgesia. Elucidating these mechanisms underlying OIH and tolerance paves the way for targeted therapeutic strategies that maintain analgesic efficacy while minimizing adverse synaptic remodeling and negative outcomes. Full article

Adenomyosis is a benign gynaecological disorder in which endometrial glands and stroma enter the uterine myometrium with varying degrees of spreading. To analyse the presence of developmentally displaced endometrial glands and stroma in the foetal myometrium, a retrospective cohort of 420 foetal uteri, including one monozygotic twin pair, was histopathologically evaluated. The gestational age ranged between 18 and 37 weeks; the clinical characteristics included various foetal malformations with a predominantly normal karyotype, except in one case with trisomy 18. Ectopic endometrial tissue enclosed in the myometrium was discovered in twelve individual foetuses from the cohort (12/420). The investigation of the histogenetic attributes of the misplaced endometrial tissue in both monozygotic twins’ (MZ) foetal uteri revealed isolated glands and thin channels containing cords of endometrial-type glands penetrating the myometrium. Through immunohistochemistry, low levels of oestrogen receptors (ERs) were detected, whereas a moderate level of progesterone receptor (PR) expression was observed in the ectopic glandular and stromal cell nuclei in all cases. Additionally, the surrounding periglandular component consistently expressed the vimentin and CD10 stromal cell markers, while the myometrial smooth muscle cells revealed the strong expression of both alpha-Smooth Muscle Actin (α-SMA) and desmin marker proteins. Full article

The lateral organization of the plasma membrane (PM) is vital for cellular signaling, yet the specific mechanisms by which the internal cortical actin meshwork templates the organization of the external lipid leaflet remain poorly understood. While established models like the ‘picket-fence’ emphasize physical barriers to diffusion, recent observations of fiber-like “ghost” structures in the distribution of glycosylphosphatidylinositol-anchored proteins (GPI-APs) suggest a more intricate mode of spatial coordination. In this study, we utilize imaging total internal reflection fluorescence correlation spectroscopy (ITIR-FCS) and variable-angle TIRF to resolve whether these filamentous patterns represent genuine membrane-proximal features or optical artifacts of cytosolic transport. Our results demonstrate that these fiber-like tracks are strictly confined to the immediate PM interface and disappear as the evanescent field probes deeper into the cytosol. While the spatial distribution of GPI-APs is templated by the underlying actin meshwork, quantitative diffusion mapping shows that the lateral dynamics of the probe remains largely uniform and is not significantly modulated by these filamentous patterns. By pharmacologically perturbing the actin scaffold and membrane cholesterol, we show that this transbilayer coupling is contingent upon a cholesterol-dependent cytoskeletal pinning mechanism. These findings demonstrate a decoupling of spatial organization and molecular dynamics, providing evidence for how the actin scaffold patterns nanoscale membrane organization without imposing long-range barriers to diffusion. Full article

Severe full-thickness corneal lacerations disrupt the tight cellular and extracellular matrix (ECM) organization required for corneal transparency. Following injury, an influx of transforming growth factor beta (TGFβ) into the corneal stroma signals the formation of haze-inducing myofibroblasts, resulting in excessive stromal remodeling and corneal haze. We hypothesized that MasR activation using NorLeu³Angiotensin (1-7) (NLE) engages the pro-resolving arm of the renin–angiotensin system (RAS) to minimize fibrotic corneal repair. In this study, 6 mm stellate-shaped, full-thickness corneal lacerations were induced in New Zealand Black (NZB) rabbits and treated with topical vehicle, or 0.1%, 0.3%, or 0.45% NLE. Corneal healing was evaluated using noninvasive corneal imaging, histology, and the gene expression of RAS- and fibrosis-related targets (MasR, AT1R, TGFβR1). Corneal imaging revealed significantly decreased corneal haze (p < 0.05) and increased keratocyte density with 0.1% NLE treatment (p < 0.05). Immunofluorescence showed significantly reduced α-smooth muscle actin (αSMA), indicating decreased myofibroblast formation (p < 0.05). Additionally, 0.1% NLE reduced stromal TGFβR1, suggesting that NLE mediates its activity by disrupting the TGFβ/TGFβR axis. MasR and AT1R gene expression were downregulated, which contributes to a reduction in fibrosis. Collectively, these findings suggest that the NLE activation of MasR modulates RAS and TGFβ/TGFβR signaling to reduce myofibroblast activity and fibrosis following severe corneal trauma. Full article

Background/Objectives: Hernia and pelvic organ prolapse often involve defects necessitating reinforcement with synthetic materials. Polypropylene meshes of varying weights are commonly used in abdominal wall repair; however, their impact on tissue response, potentially linked to adverse events during wound healing, remains incompletely understood. This study aimed to evaluate and compare the tissue response to lightweight polypropylene (LWPP) and heavyweight polypropylene (HWPP) grafts used for abdominal wall defect closure in Wistar rats. Methods: Abdominal wall defects were surgically created in male Wistar rats and repaired using either LWPP (43 g/m²) or HWPP (76 g/m²) mesh. Three weeks post-implantation, tissue response and wound closure were evaluated across different phases by assessing expression of CD68, CD56, GATA-3, CD138, vimentin, α-smooth muscle actin, and collagen deposition. Results: HWPP promoted a more pronounced foreign body response, as evidenced by increased CD68, CD56, and CD138 expression, while LWPP improved tissue compliance, enhanced fibroblast migration, and increased vimentin-associated cellular activity. Conclusions: Three weeks post-implantation, HWPP was found to induce a stronger inflammatory response, whereas LWPP implantation was associated with increased vimentin expression, indicating differences in the balance between inflammation and extracellular matrix remodelling during early wound healing. Full article

High ammonia nitrogen stress significantly compromises the survival of Litopenaeus vannamei under low-salinity conditions. However, existing studies predominantly focus on ammonia nitrogen responses under single stressors or normal seawater salinity. The molecular regulatory mechanisms, metabolic remodeling patterns, and key pathway interactions in shrimp subjected to high ammonia nitrogen stress under low-salinity environment remain unclear. In this study, we employed integrated transcriptomic and metabolomic analyses to unveil the underlying molecular responses and metabolic biomarkers in the gills of L. vannamei to ammonia stress under low-salinity conditions. First, L. vannamei underwent low-salinity acclimation from 30‰ to 5‰ salinity and was then reared for one week to acclimate to the experimental environment. Subsequently, shrimp were treated with 42.32 mg/L ammonia nitrogen for a consecutive 96 h period. Integrated transcriptomic and metabolomic analyses elucidated the stress response patterns in the gills of L. vannamei under low-salinity ammonia nitrogen exposure. Specifically, 352, 802, and 140 differentially expressed genes (DEGs) were identified at 12 h, 48 h, and 96 h post-exposure, respectively. GO and KEGG enrichment analyses revealed that the significant DEGs were primarily enriched in six major pathways: autophagy, immune-related pathway, ABC transporter, fatty acid degradation and metabolism, metabolic pathway, and PPAR signaling pathway. Metabolomic profiling identified numerous differentially accumulated metabolites (DAMs) in both positive and negative ion modes, with significantly altered DAMs mainly consisting of organic acids and their derivatives, phospholipids, and other related metabolites. Key DAMs included taurine, guanosine, 1-palmitoyl-sn-glycero-3-phosphocholine, pseudouridine, and betaine. Integrative multi-omics analysis revealed that L. vannamei mediates stress responses by modulating five core pathways under low-salinity/high-ammonia-nitrogen dual stress: fatty acid degradation and metabolism (e.g., acyl-CoA dehydrogenase short chain (Acads), acetyl-CoA acetyltransferase 2 (ACAT2)), autophagy (e.g., autophagy-related protein 101-like (atg101)), immune regulation pathway (e.g., V-type proton ATPase subunit H-like (VhaSFD), actin-5C-like (Act5C)), metabolic pathway (e.g., molybdopterin synthase catalytic subunit-like (Mocs2B), cytochrome P450 2U1-like (Cyp2b1)), and ABC transporter (e.g., ATP-binding cassette sub-family D member 3-like (ABCD3), ATP-binding cassette sub-family B member 10 (ABCB10)). Through characterization of these core pathways, this study reveals the fundamental mechanisms by which L. vannamei responds to high ammonia nitrogen stress following low-salinity acclimation, providing a theoretical foundation for estuarine shrimp farming. Full article

Although the parental recombinant protein rLj-RGD3 exhibits antitumor activity, it carries immunogenicity risks owing to its large molecular size (13.5 kDa). We generated a genetically truncated mutant, rLj-RGD4 (6.27 kDa, four RGD motifs), which inhibited B16 melanoma cell proliferation, migration, and invasion in vitro. However, the in vivo efficacy and mechanisms of action remain unclear. Here, B16 xenograft mice were treated with rLj-RGD4 (5, 10, and 20 μg/kg i.p. daily for 14 days). Tumor growth was measured, and histopathology/apoptosis was evaluated using hematoxylin and eosin (HE), Masson’s dye, Hoechst, and TUNEL staining. Apoptotic pathways (mitochondrial, death receptor, and MAPK) were analyzed via Western blotting, whereas endocytosis mechanisms were explored using inhibitors (filipin III, NaN₃, cytochalasin D), and EGFR (epidermal growth factor receptor) interactions via fluorescence co-localization and phosphoprotein assays. The results demonstrated dose-dependent tumor growth inhibition (21.60–89.26% volume reduction, 41.03–86.51% weight reduction), with histological evidence of tissue loosening, fibrosis, and apoptosis. rLj-RGD4 induced apoptosis by activating the mitochondrial (Bax/Bcl-2 upregulation), death receptor (caspase-8 activation), and MAPK (JNK/p38 phosphorylation) pathways. Internalization was blocked by NaN₃ and cytochalasin D, indicating actin-dependent macropinocytosis. Direct EGFR binding was confirmed, accompanied by reduced EGFR expression and the inhibition of FAK/AKT/Src signaling. In conclusion, rLj-RGD4 exerts potent in vivo antitumor activity via two mechanisms: induction of multi-pathway apoptosis and EGFR-targeted suppression of pro-survival signaling. RGD4 exerts its antitumor function in vivo by targeting and co-internalizing with EGFR. Full article