Angiogenesis during endochondral ossification is crucial to skeletal development, bone regeneration, and fracture healing. Vascular buds at the chondro-osseous border represent key markers of angiogenic activity. Conventional quantification approaches, such as global vessel density measurements or manual counting of buds, are time-consuming and often lack reproducibility. Therefore, we developed SACOB (Semi-Automated Counting of Buds), a modular Python-based software pipeline designed for reproducible and anatomically guided quantification of vascular buds in histological sections of chondro-osseous tissues. The SACOB workflow integrates image import, user-defined path annotation along the cartilage-bone interface, extraction and optional smoothing of intensity profiles, and algorithmic peak detection to identify vascular buds. Validation was performed on Endomucin-stained femoral sections from C57BL/6 mice aged 4, 12, and 18 months (n = 18). Four sections per animal, derived from central and peripheral regions, were analyzed to assess both age-related and spatial differences in angiogenesis (n = 6 animals/group; 4 sections/animal (total sections per age group = 24), balanced by sex). The software reliably detected vascular buds and reproduced biological patterns of vascular decline with aging. Bud density was highest in 4-month-old mice (22.23 ± 3.06 mm[-1]), significantly reduced at 12 months (9.97 ± 1.54 mm[-1], p < 0.001), and further declined at 18 months (6.71 ± 2.11 mm[-1], p < 0.001). No significant differences were observed between central and peripheral femoral sections, indicating that angiogenesis activity is spatially consistent across the growth plate. Using SACOB workflow enables rapid, reproducible determination of vascular buds at the chondro-osseous border in tissue sections.

Stockert and Blázquez-Castro have skilfully changed the perspective on melanin, turning it from a histological pigment to a possible therapeutic substrate in melanoma. However, if melanin is considered a binary and/or spatially uniform property, then translation may be restricted. The amount of pigment, its molecular structure, redox status, and intracellular localization and interactions with living tumour and immune cells could account for the ability of melanin to shield from photodynamic damage, enhance photothermal damage, or represent a photodynamic damage-resistant state. We suggest a spatial melanin phenotype, based on pigment burden, physicochemical state, cellular compartment, microenvironmental context and optical response. A combination of validated histochemistry and image analysis (digital image) with selected spectroscopic or spatial method might be a resource adaptive workflow. The hypothesis of prospective studies is whether this phenotype predicts temperature distribution, and oxidative injury, immune activation and residual viable tumour more accurately than conventional pigmentation grading. This would adjust the place of histochemistry as a tool of phototherapy selection and dosimetry that can be used for action.

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.

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.

Ambystoma mexicanum has emerged as an important experimental model in regenerative biology due to its remarkable regenerative capacity and neotenic condition. Although histological descriptions of several organs have been previously reported, an integrative characterization of the tissues and organ systems of neotenic axolotls remains limited. To investigate the structural features and cellular organization of axolotl organs, we applied a systematic histological approach with comparative perspectives. Organ samples from neotenic axolotls were processed using conventional histological techniques and analyzed by light microscopy. Fundamental tissues and multiple organ systems, including digestive, cardiorespiratory, urinary, reproductive, tegumentary, nervous, endocrine, and lymphohematopoietic systems, were characterized. The analysis revealed several distinctive features, including cartilage characterized by predominantly isolated chondrocytes, trabeculated myocardial organization, epithelial transitions in the lung, the organization of exocrine pancreatic acini and endocrine islets, distinctive retinal organizational features, and extensive hematopoietic tissue associated with the liver capsule and pericardial serosa. In addition, this study provides detailed histomorphological descriptions of the pancreas and urinary bladder in neotenic axolotls. Overall, the findings demonstrate that while many tissues share organizational similarities with other amphibians and vertebrates, A. mexicanum exhibits particular structural adaptations potentially associated with neoteny and regenerative biology. This work expands the existing histological knowledge of the species and supports future studies in regeneration, developmental biology, comparative anatomy, pathology, and conservation.

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] [0]boron and [1] [57]gadolinium 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.

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.