Cytotoxic brain edema is a life-threatening condition arising from pathological water accumulation within brain parenchymal cells, primarily astrocytes, following metabolic or toxic insults. Aquaporin-4 (AQP4), the predominant water channel in the central nervous system, is highly enriched in the perivascular endfeet of astrocytes and is considered a key facilitator of bidirectional water flux. Despite extensive molecular and light-microscopic studies, the precise nanoscale distribution of AQP4 within endfoot membranes and its spatial reorganization during the development of cytotoxic edema remain insufficiently characterized. We employed a high-resolution post-embedding immunogold electron microscopy protocol on brain tissue from an established murine model of water-intoxication-induced cytotoxic edema. Adult C57BL/6 J mice were subjected to acute systemic hypo-osmolality by intraperitoneal water loading, and perfused at defined time points. Ultrathin sections of frontoparietal cortex embedded in hydrophilic resin were labeled with primary anti-AQP4 antibodies and secondary antibodies conjugated to ultra-small (1.4 nm) or 10 nm gold particles, followed by silver enhancement for size-based discrimination of two gold particle populations. Quantitative analyses included gold particle density mapping along astrocytic plasma membrane domains, nearest-neighbour distance measurements, polarization indices, and double-label colocalization studies with the inwardly rectifying potassium channel Kir4.1. In control brains, AQP4 immunoreactivity was strikingly polarized, with a 12.7-fold higher gold particle density on perivascular endfoot membranes (62.4 ± 4.8 particles/µm) compared to the parenchymal-facing membranes of the same cells (4.9 ± 1.2 particles/µm; p < 0.001). Double immunogold labeling revealed that 73.5% of AQP4-positive clusters in endfeet colocalized with Kir4.1 signals. Following induction of cytotoxic edema, the density of AQP4 gold particles on endfoot membranes decreased by 41% within 15 min and by 64% at 60 min (p < 0.001), accompanied by a reciprocal increase in labeling on non-endfoot membranes. The polarization index collapsed from 12.7 to 2.1 at 60 min. Morphometric analysis documented a 3.2-fold increase in endfoot cross-sectional area and a progressive loss of orthogonal array-like particle clusters. Across pooled groups, AQP4 polarization was inversely associated with endfoot cross-sectional area; however, within-group analyses indicated that this association was largely attributable to treatment-group differences. This study provides a time-resolved quantitative ultrastructural analysis of AQP4 labeling across astrocytic membrane domains during the evolution of cytotoxic edema. These findings demonstrate a marked time-dependent loss of perivascular AQP4 polarization during acute cytotoxic edema and provide a quantitative framework for future studies examining the mechanisms underlying changes in AQP4 membrane organization.
Hepatitis B virus (HBV) infection remains a major global health challenge, requiring accurate diagnostic tools and precise therapeutic drug monitoring (TDM) to guide antiviral treatment and prevent disease progression. Conventional laboratory‑based assays, while reliable, are often limited by long turnaround times, high operational costs, and the need for specialized infrastructure. Recent advances in nanomaterials have transformed the landscape of HBV diagnostics and TDM by enabling highly sensitive, rapid, and cost‑effective analytical platforms. Nanostructures such as gold nanoparticles, quantum dots, carbon nanotubes, graphene derivatives, and magnetic nanomaterials offer unique physicochemical properties including large surface area, tunable optical signatures, and enhanced biorecognition capabilities that significantly improve assay performance. These materials have been integrated into diverse sensing modalities, including electrochemical biosensors, fluorescence‑based assays, plasmonic platforms, and point‑of‑care devices, achieving ultralow detection limits for HBV DNA, HBsAg, and other clinically relevant biomarkers. In therapeutic monitoring, nanomaterial‑enabled sensors facilitate real‑time quantification of antiviral drugs such as tenofovir and entecavir, supporting personalized treatment strategies and improving adherence assessment. Despite promising advancements, challenges remain regarding clinical translation, standardization, biocompatibility, and large‑scale manufacturing. This narrative review summarizes current nanomaterial‑based approaches for HBV detection and TDM, highlights emerging technologies, and discusses future directions for integrating nanodiagnostics into routine clinical practice to enhance HBV management and global disease control.
Hydroxyurea (HU) is a growth-inhibiting agent used to treat conditions characterized by excessive cellular proliferation. As an antimetabolite, HU inhibits ribonucleotide reductase, reducing the deoxyribonucleotide pool and thereby slowing DNA synthesis. Pharmacological inhibition of cell division is closely associated with the development of a cellular senescence phenotype, which may contribute to therapeutic resistance and tumor-associated inflammation. This phenotype has extensive effects, potentially inducing bystander effects and fostering tumor immunosuppression, thereby increasing the risk of cancer recurrence or relapse. Furthermore, HU has been shown to induce cellular senescence in non-malignant cells in laboratory settings. This review examines the principal molecular mechanisms underlying HU's cytotoxic and genotoxic effects, as well as the salient features of cellular senescence induction. A comprehensive understanding of the pathways through which HU prompts cellular senescence in normal cells may enhance current chemotherapy strategies and mitigate adverse effects in patients with cancer and other pathologies.
BACKGROUND:Tuft cells are specialised chemosensory epithelial cells involved in mucosal immunity and epithelial homeostasis, while leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) regulates Wnt signalling, essential for post-mitotic neuronal maintenance. However, within the human colon the impact of ageing on tuft cell abundance in the mucosa and LGR5 distribution in the ganglionic area remains poorly characterised. The aims of this study were to quantify tuft cell populations in the adult human colonic epithelium and assess LGR5 presence within the ganglionic area. METHODS:Colonic tissue samples were obtained from younger adults (Ascending colon: AC, n = 6, aged 30-52 years; Descending colon: DC, n = 10, aged 42-60 years) and older adults (AC: n = 12, aged 70-89 years; DC: n = 5, aged 67-82 years). Immunolabelling was performed on formalin-fixed, paraffin-embedded sections using antibodies against cyclooxygenase-1 (COX-1) to identify tuft cells within epithelial crypts and LGR5 presence within the myenteric ganglia. RESULTS:Tuft cells exhibited a characteristic flask-shaped morphology with intense apical COX-1 staining. Their numbers per crypt and also per mucosal area were higher in younger adults compared with older adults (respectively, mean numbers per mm2 mucosal area: AC, 109.7 ± 8.9 vs. 44.9 ± 2.1; DC, 103.2 ± 5.1 vs. 58.6 ± 3.7; P < 0.05 each), with a more pronounced decline in the AC. LGR5-positive structures were consistently observed within myenteric ganglia located between the circular and longitudinal muscle layers in both colonic regions. Compared with younger adults LGR5-positive structures within the ganglia was markedly reduced in the ageing AC (younger: 0.63 ± 0.07 ×10-3, older: 0.26 ± 0.03 ×10-3 per ganglionic area; P < 0.05) but not clearly in the DC (younger: 0.83 ± 0.05 ×10-3, older: 0.58 ± 0.05 ×10-3 per ganglionic area; P > 0.05). CONCLUSION:In both regions of human colon, ageing reduced tuft cell abundance in the mucosal epithelium. Ageing also reduced LGR5-IR structures within the ganglionic area in the AC. These structural changes may contribute to age-dependent impairments of neuromodulatory functions of the epithelium and the mechanisms for maintaining myenteric ganglion homeostasis.
Tuberculosis (TB), caused by Mycobacterium tuberculosis (M.tb), remains a major global health threat. The tripartite motif (TRIM) family of E3 ligases plays crucial roles in various cellular processes, yet the involvement of TRIM32 in TB remains poorly understood. This study explores the function of TRIM32 in M.tb infection and its underlying mechanisms. We found that M.tb infection significantly reduced TRIM32 expression in mouse macrophages, suggesting a regulatory role in TB progression. TRIM32 inhibited NLRP3 inflammasome activation and pyroptosis of M.tb-infected macrophages. Furthermore, TRIM32 overexpression mitigated mitochondrial dysfunction, evidenced by decreased reactive oxygen species (ROS) generation, inhibited mitochondrial ROS (mtROS), and restored mitochondrial membrane potential. Importantly, TRIM32 directly interacted with TLR4 and promoted its degradation via ubiquitination at the K48 site, a critical process for proteasomal degradation. The inhibitory effects of TRIM32 on pyroptosis and mitochondrial dysfunction were partially reversed by TLR4 overexpression, confirming TLR4 as a key mediator of TRIM32's actions. Our findings uncover TRIM32 as a critical regulator of mitochondrial health and pyroptosis in M.tb-infected macrophages through TLR4-mediated ubiquitylation, Moreover, silencing of endogenous TRIM32 aggravated NLRP3 inflammasome activation, pyroptosis and mitochondrial injury, and AAV-mediated TRIM32 overexpression alleviated lung pathology, lowered bacterial burden and improved survival in M.tb-infected mice, with these benefits being partially abrogated by TLR4 co-overexpression. Our findings identify TRIM32 as a candidate regulatory node of macrophage pyroptosis in TB that merits further preclinical evaluation.
Cre-loxP systems provide powerful tools for cell-specific manipulation and lineage tracing, but their interpretation depends on the assumption that reporter activation faithfully reflects cell-autonomous recombinase activity. Recent evidence that Cre recombinase protein may transfer from oligodendroglial cells to neurons under pathological stress raises an important methodological question: does reporter positivity invariably establish lineage identity? We propose a four-gate lineage-certainty framework that distinguishes driver confirmation, recombination confirmation, independent cell-identity confirmation, and a stress-transfer challenge. This framework separates four biologically distinct events, recombinase transcription, recombinase protein localization, genomic recombination, and lineage identityand encourages orthogonal validation when unexpected reporter-positive populations emerge. A resource-adaptive implementation, ranging from conventional immunohistochemistry and transcript localization to cell-resolved recombination analysis and spatial or single-cell approaches, could improve applicability across laboratory settings. Reporter activation should therefore be regarded as evidence of recombination-dependent reporter activity rather than automatic proof of lineage derivation. This distinction may strengthen the reproducibility and interpretability of Cre-loxP histochemistry across experimental models of development, injury, degeneration, and regeneration.
Melanin is an ancestral biopigment with strong adaptive value and diverse functions across all phyla. In mammals, melanin appears in the skin, retinal-pigmented epithelium, central nervous system and in the malignant melanoma, one of the most aggressive and therapy-resistant tumors. Owing to the occurrence of melanin in melanomas, structural and physico-chemical properties can be considered to develop improved treatments. Several chemical structures have been suggested for melanin, but the model that best aligns with all known features, is the planar or spiral catechol/benzoquinone derivative of the porphycene ring. This model contains the 2,2'-bipyrrole motif as revealed by Ru(II)-induced luminescence, accounting for melanin's broadband light and ultrasound absorption, metal, dye and drug binding, antioxidant capacity, radical scavenging, electrical conductivity and multilayered graphite-like structure, observed by X-ray crystallography and electron microscopy. Due to this properties, endogenous melanin is an ideal target for treating melanotic melanomas by near infrared (NIR)-driven photothermal therapy (PTT). In addition to PTT with melanin itself and its complexes with red- or NIR-absorbing intercalating and redox dyes, further perspectives include PTT using mixed-valence and inclusion compounds. Recently, even more exotic approaches involve employing isotopes ¹ ⁰boron and ¹ ⁵⁷gadolinium for enhanced neutron-capture therapy, lanthanide-based up-conversion, ultrasonic-piezoelectric and tetrazine photo-explosive effects. Excited π-electrons after NIR or electrochemical production of reactive oxygen species and radicals following ultrasound absorption induce efficient thermal and/or oxidative responses, leading to tumor cell death. The ability of melanin to bind metal cations, dyes and drugs with high affinity has seminal implications for Cell Biology, Histochemistry, Pathology and Pharmacology of melanomas. Ultimately, this review explores melanin's structure as a therapeutic target, leveraging its striking biophysical, biochemical and biological properties to propose and implement new strategies for melanoma treatment.
OBJECTIVE:Histone deacetylases (HDACs) act as transcriptional repressors and play essential roles in mammalian development. However, data on the expression and subcellular localization of HDACs in the tongue and oral mucosa remain limited. This study aimed to investigate the spatiotemporal expression patterns of HDACs in the mouse tongue and oral mucosa during postnatal development and aging. METHODS:Tongue and oral mucosal tissues were collected from C57BL/6 J mice at postnatal day 1 (P1), postnatal day 21 (P21), 3 months, 10 months, and 18 months. Hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) were performed to assess tissue morphology and HDAC expression. RESULTS:HDAC1 and HDAC6 were not detected in either tissue. In contrast, HDAC2 showed distinct nuclear localization in epithelial cells. The remaining HDAC isoforms exhibited diverse spatiotemporal expression patterns with marked age-related changes. Specifically, HDAC2-5 and HDAC7-11 were expressed at higher levels in adult mice (3 and 10 months) than at P21 and 18 months. In addition, these HDACs were not detected in basal epithelial cells at P21 and 18 months. CONCLUSION:This study characterizes the spatiotemporal expression patterns of HDAC family members in the mouse tongue and oral mucosa during postnatal development and aging, providing a valuable reference for future studies on the functional roles of HDACs in oral tissue homeostasis and aging.
This study examined the dynamic progression of DNA damage following status epilepticus (SE) using three complementary in situ techniques-PANT, Klenow, and TUNEL-combined with Fluoro-Jade B (FJB) staining in the susceptible hippocampal neurons. SE was induced by intracerebroventricular injection of kainic acid, which triggered early oxidative DNA lesions characterized by single-strand breaks (ssDNA) detectable by PANT and Klenow as early as 6 h post-insult. A transient decline in ssDNA labeling at 16 h suggested the possible activation of DNA repair mechanisms during the latent phase preceding irreversible degeneration. From day 1 onward, DNA damage intensified, with PANT and Klenow signals resurging and temporally coinciding with increased neuronal death. TUNEL, which detects double-strand DNA (dsDNA) breaks, consistently exhibited lower signal intensity than PANT/Klenow but followed a similar temporal pattern. Quantitatively, PANT demonstrated higher sensitivity for early ssDNA lesions (6-16 h), while PANT and Klenow showed comparable sensitivity at later stages (1-7 d), indicating progression toward complex DNA fragmentation. The convergence of DNA damage signals with FJB staining from day 3 post-SE highlighted the transition from reversible DNA lesions to irreversible neurodegeneration. Notably, TUNEL's limited specificity-due to its detection of non-apoptotic 3'-OH ends-was evident in its lower yield compared to ssDNA-specific methods. These findings reveal SE-induced DNA damage as a dynamic continuum-from early ssDNA breaks to delayed dsDNA fragmentation-that drives delayed neuronal loss, providing morphological insights into the neurodegenerative mechanisms following epileptic insults.
The striped dolphin (Stenella coeruleoalba) is a small pelagic dolphin. The melon, present in most odontocetes, is a rounded structure located in the center of the forehead, between the blowhole and the tip of the head. The dolphin's melon, as well as the blubber, consists of a lipid component similar to that of adipose tissue, with large, rounded cells closely related to each other and a muscular component. The melon primarily plays a role in echolocation, but this organ can also be involved in immune function. Numerous studies have demonstrated the accumulation of contaminants, such as heavy metals, in the fatty tissues of cetaceans (melon and blubber), which alter the immune system and stimulate an inflammatory response. This study aims to describe the involvement of Stenella coeruleoalba melon in immune function. Histological samples of the melon of Stenella coeruleoalba, were used in this study. To describe the morphology of this organ, the sections of the tissue were stained with Giemsa, Masson, and Mallory staining techniques. Immunoperoxidase and immunofluorescence techniques using specific antibodies such as MHC II, Langerin/CD 207, TLR2, and CD14 were used to characterize melon immune cells, providing novel insights into their immunological features. The results showed scattered immune cells among melon adipocytes immunoreactive to the tested antibodies; macrophages are the principal immune cells infiltrating adipose tissue, and their function is to secrete proinflammatory mediators. Furthermore, the adipocytes themselves appear to be labeled with the antibodies used. This study demonstrates the involvement of the dolphin melon in immune function, helping us better understand the immune system of cetaceans, which is still little understood. Furthermore, it could also demonstrate that analyses of blubber collected from deceased individuals of highly protected species can provide valuable information on their health status and exposure to contaminants.
BACKGROUND:Pancreatic ductal adenocarcinoma (PDAC) resistance to PARP inhibitors (PARPi) remains poorly understood, the mechanism of resistance remain poorly understood. MATERIAL AND METHODS:We integrated multi-omics data from The Cancer Genome Atlas-Pancreatic Adenocarcinoma (TCGA-PAAD) datasets, Cancer Cell Line Encyclopedia (CCLE), and the Genomics of Drug Sensitivity in Cancer (GDSC) performed weighted gene co-expression network analysis (WGCNA) to identify hub genes linked to PARPi resistance. GSE86394 from the Gene Expression Omnibus (GEO) database was utilized to differential expressed genes (DEGs) and performed functional enrichment analyses. CCK8, flow cytometry, Transwell assay were conducted to detected cell viability, apoptosis, and migration. Xenograft models was conducted to investigate the effect of YARS2 on PARPi-resistant in vivo. Immunohistochemistry (IHC) was used to detect the expression of various proteins. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) was utilized to assess cells apoptosis. Gene and protein expression were detected by quantitative reverse transcription PCR (qRT-PCR) and western blotting (WB), respectively. RESULTS:YARS2 was identified as a PARPi resistance hub gene, whose high expression correlated with poor prognosis in PDAC. YARS2 was significantly upregulated in PARPi-resistant PDAC cells, and its knockdown reversed PARPi resistance in vitro and in vivo. Mechanistically, YARS2 knockdown suppressed the expression of the c-MYC, E2F1 and elevated levels of phosphorylated histone H2AX (γ-H2AX). However, the overexpression of c-MYC was reversed the inhibitory role of YARS2 knockdown in PARPi-resistant PDAC cells. CONCLUSION:Our findings reveal that YARS2 mediates PARPi resistance via the MYC/E2F1 pathway in PDAC, highlighting YARS2 may serve as a potential target for therapy to counteract PARPi resistance.
Casein kinase 1 alpha (CK1α), a conserved serine/threonine kinase, regulates diverse cellular processes including signal transduction, cell cycle progression, and DNA replication. To define its spatiotemporal dynamics during mammalian ovarian development, we systematically analyzed CK1α protein expression from embryonic day (E) 14.5 to postnatal day (P) 56 in the mouse ovary using Western blotting, immunofluorescence (IF) on tissue sections, and IF on isolated primary granulosa cells and oocytes. Reanalysis of published single-cell RNA sequencing data revealed progressive upregulation of Csnk1a1 transcripts from E9.5 through early postnatal stages. At the protein level, CK1α expression significantly increased from E14.5 to P56, with stabilization after P28. Immunofluorescence showed CK1α localized predominantly to oocytes within primordial follicles from E14.5 to P7. Starting at P7, CK1α expression expanded to include granulosa cells of developing primary and secondary follicles. Quantitative fluorescence analysis confirmed that CK1α signal in granulosa cells increased significantly from P7 onward, while the signal in the oocytes gradually decreased throughout development process. In vitro validation confirmed robust CK1α localization in both isolated granulosa cells and oocytes, co-localizing with FSHR and DDX4, respectively. This study provides the first comprehensive spatiotemporal atlas of CK1α expression in the wild-type developing mouse ovary, revealing stage-specific localization patterns that suggest distinct roles in oocyte maintenance and granulosa cell differentiation during folliculogenesis.
Several acrylate-based materials are commercially available as embedding resins for the histological analysis of non-decalcified hard tissues, including implants. However, commercial embedding resins are imported, expensive, and take a long time to buy. We conducted several tests to apply a mixture of methyl methacrylate and oleyl alcohol (oleyl Alc) as an embedding resin on a non-decalcified bone specimen. Further, we observed differences in the compressive strength of the block resin and the fused bone sample, changes in crystallinity, and a hydrophilic change in the block. Hematoxylin and eosin (H&E) and Masson's Trichrome (MT) staining were performed for the histological analysis of dog bones to compare staining characteristics. The block to which the resin with the optimal composition ratio was applied for embedding was confirmed experimentally to achieve optimal performance.
