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.
Endoplasmic reticulum (ER) stress is the accumulation of misfolded or defective proteins in the ER. ER stress is capable of inducing both anti-apoptotic and pro-apoptotic cellular response, and at the same time plays an important role in metabolism and progression of many types of tumors. Current understanding of the role of ER stress in changing functional parameters of normal and tumor cells is lacking. This study investigated how ER stress inducers bortezomib, dithiothreitol, and tunicamycin influence proliferation, cell cycle, and changes in ploidy of normal and tumor cells of epidermal origin HaCaT and A431 in vitro following incubation with the agent as well as after its removal from the culture medium. Bortezomib caused a cell cycle arrest in the G2 phase in HaCaT cells, as well as polyploidization in both cell lines. Dithiothreitol induced apoptosis in HaCaT cells. Tunicamycin caused a decrease in proliferative index, cell cycle arrest, as well as apoptosis and necrosis in the A431 cells. In conclusion, induction of ER stress by different mechanisms has different effects on normal and tumor cells and can lead to both polyploidization and, presumably, cell differentiation or senescence.
Immunophenotyping of tumor-infiltrating immune cells is increasingly important for understanding the tumor microenvironment (TME), particularly in the diagnosis and treatment of skin cancer. CD1a+ dendritic cells (DC) initiate T cell activation by presenting tumor antigens, while T cells directly target tumor cells. Analyzing their spatial distribution in different types of skin cancer can provide insights into immune response patterns. To characterize the immune cell composition within the TME, we performed immunofluorescence staining for CD1a and CD3 on formalin-fixed, paraffin-embedded (FFPE) samples from actinic keratoses (n = 18), squamous cell carcinoma (n = 23), basal cell carcinoma (n = 19), and melanoma (n = 22), with nevi (n = 16) and healthy skin (n = 9) as controls. Immune cells were quantified across four tumor compartments: intratumoral, tumor margin, intraepidermal, and intradermal. Both CD1a+ DC and CD3+ T cells were detected across all tumor entities, displaying distinct spatial distribution patterns. DC were enriched intratumorally and within the epidermis, whereas T cells predominantly accumulated at the tumor margin (main effect of region, p < 0.001). Melanoma exhibited significantly fewer DC at the tumor margin while maintaining strong T cell infiltration. Overall, the immune architecture of skin tumors is highly compartmentalized, characterized by region-specific DC and T cell distributions. These findings underscore the relevance of spatial immune profiling for understanding immune escape mechanisms and informing immunotherapeutic strategies.
Impaired wound healing in type 2 diabetes is largely attributed to dysregulated cellular responses, defective extracellular matrix (ECM) remodeling, insufficient angiogenesis, and a prolonged inflammatory microenvironment. Understanding how these processes can be modulated at the cellular and tissue levels remains essential for improving diabetic wound repair. In this study, we examined the effects of a decellularized dermal scaffold (DDS) combined with photobiomodulation therapy (PBM), applied as an adjunct biophysical stimulus, on angiogenic, inflammatory, and matrix remodeling responses in a type 2 diabetic rat wound model. Full-thickness excisional skin wounds were created and assigned to control, DDS-treated, PBM-treated, or combined DDS + PBM groups. Wound tissues were harvested on days 8 and 16 post-injury for macroscopic evaluation, biomechanical testing, histological and histochemical analyses, and cytokine quantification. Morphometric assessment revealed that wounds receiving the combined intervention exhibited significantly accelerated wound contraction compared with single-modality and untreated groups at both timepoints. Biomechanical analyses demonstrated improved tissue integrity in treated wounds, with the combined group showing the highest values of tensile strength, maximum force, energy absorption, and bending stiffness, indicative of enhanced structural organization. Histological evaluation showed that DDS combined with PBM markedly increased fibroblast density and neovascularization while reducing inflammatory cell infiltration. Histochemical staining further demonstrated more advanced and organized collagen deposition in the combined group, reflecting accelerated ECM maturation and remodeling. At the molecular level, treated wounds displayed elevated levels of pro-regenerative mediators, including transforming growth factor-β1 (TGF-β1) and vascular endothelial growth factor (VEGF), with maximal expression observed in the combined group. Conversely, the expression of proinflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) was significantly attenuated. Collectively, these findings indicate that integrating a decellularized dermal scaffold with adjunct photobiomodulation effectively modulates cellular behavior, angiogenic signaling, inflammatory responses, and ECM remodeling in diabetic wounds. This study provides mechanistic insight into scaffold-based microenvironmental regulation of impaired wound healing under diabetic conditions.
Mesenchymal stem cells (MSCs) are widely utilized in regenerative medicine owing to their differentiation potential and paracrine effects. Although numerous tissues have been identified as sources of MSCs, the search for novel, noninvasive sources continues. To date, the stem cell content of colostrum has not been investigated. To address this gap, this study aimed to comparatively evaluate the biological properties, proliferative dynamics, and functional potential of cell populations derived from colostrum and mature milk. Cells isolated from both sources (n = 3) were morphologically assessed under in vitro culture conditions and were induced to undergo multilineage differentiation. Phenotypic characterization was performed by flow cytometric analysis. Proliferation capacity was assessed by determining population doubling time (PDT) and performing colony formation assays, while cell viability was evaluated using the methyl thiazolyl tetrazolium (MTT) assay. Cells initially exhibited an epithelial-like morphology and adopted a fibroblast-like phenotype after passaging. Colostrum-derived and mature milk-derived cells displayed multilineage differentiation potential, and flow cytometric profiling confirmed the presence of cells positive for CD73, CD90, and CD105 in both sources. Colostrum-derived cells exhibited higher cell densities; however, population doubling times showed no statistically significant difference between the groups. MTT analysis demonstrated a progressive increase in metabolic activity in both groups, with colostrum-derived cells exhibiting significantly higher optical density values from day 2 onward. No statistically significant difference in colony-forming efficiency was observed between the groups. Consequently, these preliminary findings suggest that colostrum may harbor cell populations exhibiting MSC-like properties and could represent a potential area of interest for MSC research.
Pregnancy induces marked metabolic and physiological adaptations in the maternal liver to maintain systemic homeostasis and support fetal development. Nuclear factor erythroid 2-related factors 1 and 2 (Nrf1 and Nrf2) are Cap'n'Collar (CNC) transcription factors that regulate antioxidant response element (ARE)-dependent gene expression and play essential roles in redox homeostasis and cellular stress responses; however, their gestational stage-dependent intracellular distribution in the maternal liver remains insufficiently characterized. This study investigated the immunohistochemical expression and subcellular localization of Nrf1 and Nrf2 in the maternal rat liver during mid and late gestation. Liver samples were obtained from non-pregnant control rats and pregnant Wistar albino rats on gestational days (GD) 13, 18, and 20 (n = 6 per group). Immunohistochemistry was performed using anti-Nrf1 and anti-Nrf2 antibodies, and staining intensities in nuclear and cytoplasmic compartments were quantified using Fiji (ImageJ). Nuclear-to-cytoplasmic (N/C) ratios were also calculated. Nrf1 immunoreactivity showed gestational stage-dependent changes, with stronger nuclear and cytoplasmic staining at GD13 and reduced staining at GD18 and GD20, accompanied by lower N/C ratios during mid gestation. In contrast, Nrf2 nuclear immunoreactivity remained relatively stable throughout most of gestation, with a significant decrease observed only between GD18 and GD20, whereas cytoplasmic immunoreactivity increased at GD13 and GD18 and declined at GD20. Statistical analysis demonstrated significant gestational stage-related differences in nuclear immunoreactivity, cytoplasmic immunoreactivity, and the N/C ratio for Nrf1. For Nrf2, significant differences were also observed in nuclear immunoreactivity, cytoplasmic immunoreactivity, and the N/C ratio, although pairwise differences in nuclear immunoreactivity were limited to GD18 and GD20. These findings demonstrate distinct gestational stage-dependent patterns of Nrf1 and Nrf2 immunoreactivity in the maternal liver, suggesting differential involvement of these transcription factors in maternal hepatic adaptation during pregnancy.
This investigation sought to determine whether the formation and progressive buildup of advanced glycation end products (AGEs) within hyperglycemic environments impairs the biological functions of stem cells from human exfoliated deciduous teeth (SHEDs) through a mechanism mediated by the receptor for advanced glycation end products (RAGE), endoplasmic reticulum (ER) stress, and oxidative dysregulation. An in vitro hyperglycemic model was established by culturing SHEDs under high glucose concentrations, AGE exposure, or their combination. Multiple experimental approaches were employed to assess cellular behaviors: proliferative capacity was measured via cell counting kit-8 (CCK-8) assay, migratory ability was evaluated through scratch wound assays, the proportion of apoptotic cells was quantified through annexin V/propidium iodide (PI)-based flow cytometry, while senescence was assessed using senescence-associated β-galactosidase (SA-β-Gal) staining. Critical molecular mediators encompassing RAGE/nuclear factor-κB (NF)-κB signaling components, ER stress biomarkers, oxidative stress parameters, stemness regulators, and osteogenic differentiation markers were quantified through quantitative real-time polymerase chain reaction (qRT-PCR), immunoblotting, immunofluorescence microscopy, and enzyme-linked immunosorbent assay (ELISA) methodologies. Concurrent treatment with high glucose and AGEs substantially suppressed SHED proliferative and migratory capacity while simultaneously enhancing apoptotic cell death and cellular senescence. These functional impairments correlated with elevated RAGE expression, NF-κB pathway engagement, and intensified ER stress responses, as evidenced by augmented GRP78 and CHOP abundance. Oxidative stress parameters were similarly elevated, manifesting as increased reactive oxygen species (ROS) generation and malondialdehyde (MDA) accumulation alongside glutathione (GSH) depletion. The expression of the stemness-associated factors SOX2 and OCT4, together with osteogenic markers RUNX2 and OCN, exhibited marked downregulation. Notably, pharmacological RAGE blockade using FPS-ZM1 successfully reversed these pathological alterations, attenuating ER stress responses, oxidative dysregulation, and functional deterioration triggered by the combined treatment regimen. Our investigation demonstrates that AGEs exacerbate SHED dysfunction under high-glucose conditions by triggering RAGE-dependent ER stress and oxidative stress cascades. This pathological sequence ultimately compromises cellular self-renewal capacity and differentiation potential. Consequently, therapeutic targeting of the RAGE signaling axis represents a promising approach for maintaining dental stem cell functionality and promoting regenerative outcomes in pediatric populations with high-sugar dietary habits.
Phosphoinositides are low-abundance regulatory lipids that control a broad range of cellular processes, from membrane trafficking and cytoskeletal remodeling to transcriptional regulation and RNA processing. These lipids are distributed across distinct subcellular compartments, where they carry out compartment-specific regulatory functions. Dysregulation of phosphoinositide metabolism is associated with cancer, neurodegenerative diseases, and immune dysfunction. However, their roles remain difficult to investigate owing to technical limitations in lipid detection and manipulation. This review outlines current strategies for modulating, visualizing, and quantifying phosphoinositide pools, including genetic manipulation techniques such as RNA interference, clustered regularly interspaced short palindromic repeats (CRISPR)-based approaches, and optogenetics. It also evaluates visualization tools such as fluorescent biosensors and live-cell imaging techniques, including superresolution microscopy. In parallel, quantitative methods such as thin-layer chromatography and mass spectrometry for profiling phosphoinositide species, including isomer- and acyl-specific variants, are discussed. By comparing the strengths and limitations of these approaches and highlighting how they can be combined, this review provides a practical framework for dissecting phosphoinositide function in defined subcellular contexts.
Replication stress (RS) and oxidative stress (OS) are two main types of endogenous stress (ES) which, by inducing various forms of DNA damage lead to genome destabilization and disruption of cell division control mechanisms. Methotrexate (MTX) is a compound that inhibits dihydrofolate reductase (DHFR), thereby blocking DNA replication and exhibiting antiproliferative effects. The aim of the study was to investigate how 72-h exposure to 0.75 mM MTX on meristematic cells of Vicia faba roots affects the morphology of cell nuclei and mitotic chromosomes, population change of cells in interphase, DNA replication dynamics, cell viability, and hydrogen peroxide (H2O2) production. Furthermore, in order to assess epigenetic changes induced by MTX, associated with DNA damage and the replication process, histone H3 acetylation at lysine 56 (H3K56Ac) and histone H4 acetylation at lysine 5 (H4K5Ac) were examined. It was demonstrated that root meristematic cells treated with MTX exhibited abnormal chromosome structure, sustained DNA biosynthesis, and elevated intracellular H2O2 levels. Immunocytochemical studies revealed an increased number of fluorescent H3K56Ac foci. It was observed that in the case of H4K5Ac, MTX significantly reduced the frequency of cell populations characterized by euchromatin immunofluorescence and limited the occurrence of heterochromatin-type nuclei. Furthermore, in cells treated with MTX, a significant increase in the number of nuclei with marked nucleoli was observed in addition to the gap 1 (G1) phase. In summary, continuous 3-day exposure to low concentrations of MTX induced a cellular response to reactive oxygen species and DNA replication stress conditions.
Spinal cord injury (SCI) is a devastating neurological condition characterized by severe neuronal loss, inflammation, oxidative stress, and various forms of regulated cell death that collectively impair functional recovery. The present study aimed to develop a bioactive therapeutic platform based on an adipose-derived extracellular matrix (ECM) hydrogel incorporating cytokine-releasing PLGA microspheres to promote spinal cord repair. The hydrogel was fabricated from decellularized adipose tissue and combined with microspheres encapsulating interleukin-10 (IL-10), vascular endothelial growth factor (VEGF), and glial cell line-derived neurotrophic factor (GDNF) to achieve sustained cytokine delivery. Seventy-five male Sprague-Dawley rats were randomly allocated into five experimental groups, including control, SCI, hydrogel, microsphere, and Hydrogel + Mic groups. Tissue specimens were subsequently harvested from the lesion site for further analyses. In a rat model of SCI, treatment with the cytokine-releasing microsphere-loaded hydrogel significantly improved electrophysiological conduction and locomotor recovery compared with untreated SCI animals and groups receiving individual treatments. Molecular analyses demonstrated that the combined treatment markedly suppressed the expression of pro-inflammatory cytokines TNF-α and IL-1β. Additionally, apoptosis-related markers showed substantial modulation, characterized by decreased Caspase-3 and Bax expression and increased Bcl-2 levels. The therapy also improved the oxidative balance by increasing antioxidant markers including GSH, SOD, and CAT while reducing the lipid peroxidation marker MDA. Furthermore, ferroptosis-associated biomarkers were significantly regulated, with elevated levels of GSH, GPX4, and SLC7A11 and reduced ACSL4 expression. Histological analyses revealed significant preservation of spinal cord architecture, reduced cavity formation, enhanced neuronal survival, and decreased glial activation in animals treated with the composite hydrogel system. Collectively, these findings demonstrate that adipose-derived ECM hydrogel loaded with cytokine-releasing microspheres provides a multifunctional therapeutic strategy that attenuates inflammation, apoptosis, oxidative stress, and ferroptosis, ultimately promoting structural and functional recovery following spinal cord injury.
Renal tubular damage and interstitial fibrosis are highly linked to diabetic kidney disease (DKD) progression. Ferroptosis in renal tubular epithelial cells has emerged as one of the key mechanisms of DKD. Spermidine/spermine N1-acetyltransferase 1 (SAT1) knockdown has been found to alleviate repetitive low-dose cisplatin-induced kidney damage and fibrosis, and importantly, SAT1 silencing represses cellular sensitivity to ferroptosis. However, the effect of SAT1 on DKD-associated ferroptosis and its potential mechanism remain understood. In this study, we constructed a high-fat diet/streptozotocin-induced DKD mouse model and a high glucose (HG)-injured HK-2 cell model with the aim of verifying whether SAT1 silencing attenuates DKD tubular damage by regulating ferroptosis. We found that SAT1 was upregulated in DKD mouse kidneys and HG-treated HK-2 cells. Significant tubular damage, fibrosis, ferroptosis, and oxidative stress were observed in DKD mouse kidneys. In an in vitro loss-of-function assay, SAT1 silencing suppressed HG-induced HK-2 cytotoxicity, extracellular matrix (ECM) synthesis, and inflammation. Additionally, SAT1 silencing decreased HG-activated MDA and 4-HNE production, while restoring GSH levels. SAT1 silencing also abrogated HG-activated ferroptosis in HK-2 cells, as evidenced by a reduction in iron overload, inhibition of lipid peroxidation, and upregulation of ferroptosis-related protein (SLC7A11, GPX4, and TFR1) expression. Mechanistically, SAT1 silencing facilitated nuclear translocation and expression of NRF2. Impairment of NRF2 function abrogated the inhibitory effects of SAT1 silencing on HG-stimulated HK-2 cytotoxicity, ferroptosis, and ECM accumulation. Overall, the SAT1/NRF2 axis is a critical regulator of tubular damage in DKD, and suppression of SAT1 may be an underlying target for DKD treatment.
Lipofuscin is a subcellular pigmented granule that has long been considered a sign of wear-and-tear and aging. This granule cannot be degraded but may be diluted by cell division. In the heart, lipofuscin can be easily identified in cardiomyocytes where proliferative capacity is limited, and to a lesser extent in other cardiac cell types, by their characteristic morphological features. They appear as yellow-brown granules in histopathology sections reflecting their lipid origins, and as heterogenous membrane-bound bodies containing a mix of lipid and electron-lucent content in transmission electron microscopy. Lipofuscin was originally considered a mere byproduct of age-related cell senescence but studies over the past three decades have indicated that its role and mechanism of formation may be far more complex. In this review, we examine how lipofuscin is formed and its implications in aging and diseases of the heart. While the focus of this review is on lipofuscin in the two most predominate cell types of the heart, cardiomyocytes and fibroblasts, we also touch on the presence of lipofuscin in the vasculature in certain disease phenotypes that are directly relevant to cardiovascular health.
Polycystic ovarian syndrome (PCOS) is a prevalent metabolic and neuroendocrine disease affecting females of childbearing age. Irregular ovulation, elevated androgen levels, and the presence of multiple ovarian cysts characterize it. This study aimed to investigate the prolonged impact of RA on female fertility during three oestrus cycles, utilizing biochemical, histopathological, and immunohistochemical analyses. Twelve female mice were classified into two groups of 6 animals each: (1) the negative control group that received DMSO diluted with sunflower oil, and (2) the positive control group that received a 10 mg/kg dose of retinoic acid. For 15 days, intraperitoneal injections were given daily. The animals were physically euthanized by slaughter on day 16 after treatment. Histopathological and immunohistochemical expression of EGFR, as well as hormonal level investigations, including FSH and LH, were performed on day 16. A 10 mg/kg RA daily dose for 15 days significantly induces FSH and reduces LH. In addition, we revealed that exogenous excess RA leads to PCOS, which causes an increase in cystic follicles and a reduction in antral follicles and corpus luteum. Immunohistochemically, excessive RA suppresses the expression of EGFR, which is localized in granulosa cells. Our investigation concluded that inhibition of epidermal growth factor receptor (EGFR) signaling resulting from long-term high-dose treatment with retinoic acid (RA) affects cumulus granulosa cell proliferation and oocyte maturation. So, vitamin A may harm female fertility.
In humans, testicular peritubular cells (TPCs) form a small compartment surrounding the seminiferous tubules and, as shown previously, undergo age-related changes. How they may contribute to the age-associated decline of testicular functions is not well known. Likewise, the mechanisms of testicular aging in humans are not well examined. This is in part due to the lack of appropriate cellular models. We aimed to establish an aging model of TPCs, which is based on immortalized nonhuman primate (NHP) peritubular cells (iMKTPCs) from Callithrix jacchus. As shown previously by a comprehensive proteomic approach, they strongly resemble human TPCs but lack the human cell-intrinsic heterogeneity. Cellular senescence was robustly induced within 10 days upon exposure to 25 µg/mL bleomycin for 24 h. This resulted in increased β-galactosidase activity, enlarged cells, and elevated transcript levels of senescence-associated secretory phenotype (SASP) molecules (IL1b, TNFa, CCL2). The ability to contract upon a stimulus was reduced, as shown in live cell imaging studies. Reduced proliferation among others was indicated by a decreased abundance of proliferating cell nuclear antigen (PCNA), evident in a proteomic analysis, which also revealed massive changes (341 significantly increased and 372 decreased proteins). Bioinformatics analysis indicated a marked reduction in several cell-motility-associated proteins, whereas proteins linked to extracellular exosomes, the cytoskeleton, and lysosomal pathways were increased in abundance. To conclude, bleomycin treatment causes a rapid and robust induction of cellular senescence in a translational testicular cell model of peritubular cells. This model will enable future studies aiming to explore mechanisms and consequences of testicular aging.
In this review, we summarize the data on the cellular pigment lipofuscin that accumulates in liver tissue over time, due to aging and cellular stress. Despite the presence of these typical subcellular inclusions under various conditions, relatively little is known about their origins, roles, and effects on liver cell and tissue health. Pathologists use the presence of lipofuscin, in combination with other markers, to achieve differential diagnosis across various diseases. Routine histological stains reveal characteristic irregular shaped intracellular inclusions of lipofuscin that cannot be missed. Moreover, lipofuscin is autofluorescent and in transmission electron microscopy it appears in the cytoplasm as irregularly shaped structures containing fat and floccular material with varying electron density. Herein, we discuss the current state of knowledge concerning the origin and function of this pigment in the liver. Lipofuscin can distinctively be found in liver, although it has also been reported in cells in the heart, brain, and eye. Its biochemical composition is heterogeneous and varies depending on the tissue and the age of the organism. The liver parenchymal cells have efficient cellular waste disposal mechanisms, but they are still susceptible to aging. Lipofuscin accumulation in the liver may result from ongoing oxidative damage and impaired hepatic detoxification leading to cellular stress.
Endothelial cell senescence represents a critical mechanistic driver in the initiation and progression of cardiovascular diseases. Senescent endothelial cells exhibit characteristic features, including cell cycle arrest-mediated primarily through the p53/p21 and p16 pathways-morphological transformations such as increased cell volume, elevated caveolin-1 expression, and loss of LaminB1, as well as activation of the senescence-associated secretory phenotype (SASP). The SASP facilitates the secretion of numerous inflammatory cytokines and chemokines, thereby fostering a state of chronic inflammation and contributing to tissue dysfunction. Key molecular regulators of endothelial senescence include transcription factors such as NF-κB and p53, along with the p38 MAPK signaling pathway, which collectively modulate inflammatory responses, cell cycle progression, and stress adaptation. This review offers a comprehensive and integrative perspective on endothelial senescence as a central element in cardiovascular pathophysiology. Its novelty stems from a systematic synthesis of classical pathways, including p53/p21 and p16, with more recently implicated players such as mammalian target of rapamycin (mTOR) signaling and associated microRNAs (miRNAs), accompanied by a focused examination of the SASP as a core pathological mechanism in chronic inflammation and vascular impairment. Moving beyond singular pathways, this work constructs a multidimensional framework that integrates cell cycle arrest, morphological changes, SASP activation, and transcriptional regulation to delineate a cohesive pathological sequence through which endothelial senescence promotes cardiovascular disease.
Small tissue biopsies, including renal core biopsies, bone marrow trephines, gastrointestinal endoscopic samples, prostate needle cores, liver biopsies, and skin punch or shave specimens, are fundamental to contemporary diagnostic pathology. Their limited volume, focal sampling, and susceptibility to technical artifacts impose distinct interpretive challenges, often requiring semi-quantitative assessment within a restricted architectural context. Although artificial intelligence (AI) has rapidly expanded in digital pathology, most models have been developed using large surgical resection specimens, with comparatively limited attention to small biopsy material. This minireview examines current and emerging applications of AI in small biopsy diagnostics across renal, hepatic, gastrointestinal, hematopathological, dermatopathological, and urological pathology. Reported applications include glomerular segmentation and fibrosis quantification in renal biopsies; automated cellularity, blast detection, and fibrosis grading in bone marrow trephines; dysplasia and microorganism detection in gastrointestinal biopsies; quantitative steatosis and fibrosis assessment in liver samples; tumor detection and grading in prostate cores; and neoplastic pattern recognition in skin specimens. Despite encouraging performance in research settings, substantial barriers to routine clinical implementation remain, including limited dataset size, class imbalance, pre-analytical variability, inter-institutional heterogeneity, and insufficient external validation. We discuss methodological considerations relevant to diagnostic practice, including multi-institutional validation, stain normalization, multimodal integration with histochemistry and ancillary testing, explainability, and regulatory oversight. In the context of small biopsies, AI should be regarded as a quantitative adjunct to morphological interpretation rather than an autonomous diagnostic system. Careful integration within established histopathological workflows is essential to ensure reproducibility, safety, and clinical accountability.
In type 2 diabetes mellitus (T2DM), skeletal muscle is a major site of metabolic and microvascular dysfunction, yet most human data derive from large locomotor muscles, whereas postural and respiratory muscles remain less well characterised. We examined whether T2DM alters fibre morphology, intramyocellular lipid (IMCL) content, and three-dimensional (3D) capillary architecture across functionally distinct muscles. Postural (splenius capitis [SC]), respiratory (diaphragm [DIA]; external intercostal [EXT]), and locomotor (vastus lateralis [VL]) muscles from adult male individuals (T2DM versus control, n = 24/group) were sampled < 24 h post-mortem. Analysis included myosin heavy chain fibre typing, Sudan Black B intramyocellular lipid (IMCL) quantification, and 3D capillary morphometry (length, tortuosity, anisotropy, branching density). Groups were age-matched (T2DM 70.8 ± 7.4 versus 69.7 ± 11.8 years; p = 0.684), but body mass index (BMI) was higher in T2DM (31.9 ± 4.7 versus 24.8 ± 2.7 kg m-2; p < 0.0001). Fibre-type profiles were similar, except for elevated 2a/2x hybrids in T2DM VL (p = 0.014). Mean fibre diameters were preserved, though type 1 fibres were larger in T2DM SC (p = 0.0238). IMCL was higher in T2DM SC and EXT (p < 0.05), with non-significant differences in VL and DIA. Type 1 and 2a fibres had higher IMCL than glycolytic fibres, with no group-by-fibre-type interaction. BMI strongly predicted VL IMCL (p < 0.0001), while age was negatively associated with IMCL in respiratory muscles (p ≤ 0.05). Capillary length per fibre volume was selectively reduced in DIA (p = 0.0115); other indices were preserved, except for higher anisotropy in EXT (p = 0.0495). Overall, these functionally diverse muscles showed subtle muscle-specific remodelling in T2DM, with adiposity-linked IMCL accumulation and reduced DIA capillary supply, although findings should be interpreted in the context of the post-mortem study.
Hirschsprung's disease (HSCR) is a congenital disorder of the distal intestine characterized by aganglionosis of the enteric nervous system. While defective migration of neural crest-derived precursors is a well-established hallmark, how the intestinal microenvironment contributes to impaired neuronal support remains poorly understood. Here, we combined histochemical analyses of human HSCR colon with functional assays to investigate the role of muscularis macrophages (MMs) and a miR-93-5p-AHNAK pathway in regulating enteric neurons. Using immunohistochemistry and immunofluorescence on ganglionic and aganglionic segments, we mapped the density, spatial distribution, and phenotype of MMs, and quantified the expression of miR-93-5p and its predicted target AHNAK. In vitro, polarized M2-like macrophages were cocultured with enteric neuronal cell lines to assess neuronal migration, proliferation, and apoptosis. Gain-of-function and loss-of-function approaches for miR-93-5p, together with a dual-luciferase reporter assay, were used to validate AHNAK as a direct target. M2-like MMs (CD163+/CD206+) were abundant around myenteric ganglia in ganglionic colon but reduced in aganglionic segments, where AHNAK expression was increased. M2 macrophages enhanced neuronal migration and proliferation and protected against apoptosis, whereas disruption of the miR-93-5p-AHNAK axis impaired these neuro-supportive effects. Together, our data identify a muscularis macrophage-miR-93-5p-AHNAK axis that supports enteric neurons and demonstrates that loss of M2-like MMs and dysregulated miR-93-5p-AHNAK signaling compromise neuronal homeostasis in HSCR. These findings add a histochemical microenvironment perspective to HSCR pathogenesis and support a candidate neuroimmune pathway for restoring immune-neuronal balance, pending in vivo and clinical validation.
Periodontitis is a chronic inflammatory disease characterized by collagen degradation and alveolar bone loss. Although estrogen contributes to periodontal homeostasis, the role of the membrane-bound G protein-coupled estrogen receptor 30 (GPR30) in periodontitis remains unclear. This study investigated the temporal involvement of GPR30 in ligature-induced periodontitis, focusing on alveolar bone changes, collagen integrity, fibroblast activation, and epigenetic regulation. Twenty male Wistar rats were allocated to control and periodontitis groups and evaluated at days 7, 14, and 21 following placements of a stainless-steel ligature around the maxillary first molars. Alveolar bone loss was assessed radiographically, while inflammatory changes and collagen organization were examined using hematoxylin-eosin and Masson's trichrome staining. Immunohistochemistry was performed to evaluate GPR30, collagen I/III, α-smooth muscle actin (α-SMA), and histone modifications. Statistical analysis was conducted using one-way ANOVA with Tukey's post hoc test. Alveolar bone loss and collagen degradation were detectable on day 7 and peaked at day 14. GPR30 expression increased during the active inflammatory phase, accompanied by elevated α-SMA and histone H4 lysine 8 crotonylation (H4K8cr), and declined by day 21. Fibroblast transiently adopted a myofibroblast phenotype during inflammation and regressed during early tissue repair. Temporal changes in H4K8cr closely paralleled GPR30 expression. These findings indicate that GPR30 is dynamically associated with inflammatory and remodeling phases of periodontitis and may contribute to epigenetic regulation during periodontal tissue remodeling.