Tissue under- and over-processing in the histology laboratory, while decreasing in frequency, is still a valid concern and typically results from user or instrument error. This study evaluated the effects of extended exposure of histological tissues to processing reagents on specimen quality, including embedding, microtomy, histochemical staining, and immunohistochemistry (IHC). All specimens were fixed in neutral buffered formalin for 3-5 weeks prior to processing except for a subset of organs which underwent prolonged fixation for 1 year. Although overprocessing is traditionally associated with tissue hardening, brittleness, and shrinkage, these effects are often noted when fixation timing is not clearly defined. To simulate processing delays, specimens were subjected to an additional 24 h in absolute ethanol, xylene, molten paraffin, or a heated embedding station. Extended exposure to these reagents did not result in observable morphologic changes. Embedding quality, microtomy performance, and histochemical staining were consistent across all experimental conditions. Additionally, IHC antigenicity was largely preserved under most conditions. However, reduced or absent immunoreactivity was observed in select markers in organs fixed for 1 year, consistent with prior reports of fixation-related antigen masking and deterioration of antigenic sites. Unexpectedly, extended exposure to ethanol, xylene, or paraffin did not independently compromise IHC performance in adequately fixed specimens.

Skeletal muscle regeneration depends on satellite cells, which proliferate, differentiate, and fuse to repair damaged myofibers. Satellite-cell dysfunction contributes to defective regeneration in Duchenne muscular dystrophy (DMD), but the contribution of circadian regulators to this process remains incompletely understood. Here, we investigated the role of the CLOCK-interacting protein CIPC in satellite-cell differentiation and muscle regeneration in mdx mice. Satellite cell-specific deletion of Cipc increased centrally nucleated fibers and embryonic myosin heavy chain-positive regenerating fibers in dystrophic and ischemia-reperfusion-injured tibialis anterior muscles, and enhanced MyHC-positive myotube formation in primary cultures. Cipc deletion was accompanied by increased MyoD and myogenin mRNA expression and a higher number of myogenin-positive cells. RNA sequencing of day-3 differentiated satellite cells identified Mapk14/p38α and Elk1 as candidate upstream regulators associated with increased myogenin expression. Immunoblotting in three independent experiments further showed increased p38 and ELK-1 phosphorylation in Cipc-deficient satellite cells, without an apparent genotype-dependent change in total p38 or total ELK-1 abundance. These findings indicate that Cipc deletion enhances regenerative myogenesis in mdx mice and is associated with increased p38/ELK-1 signaling and myogenin expression. Because pathway inhibition, ELK-1 promoter-occupancy, and myogenin loss-of-function experiments were not performed, the p38/ELK-1-myogenin axis should be regarded as a candidate mechanism rather than an established causal pathway.

Sertoli cells (SCs), the essential functional units of the testis, are responsible for supporting spermatogenesis. Heat stress impairs SCs by triggering oxidative injury, apoptosis, and ferroptosis, consequently suppressing spermatogenesis. Nevertheless, the dynamic alterations of SCs upon sustained heat stress remain poorly defined. Although HSP72 is recognized to exert a vital pro-survival function under heat stress, its detailed molecular mechanism remains largely elusive. The present study aimed to investigate the protective effect of HSP72 against heat stress-induced damage in SCs. Our results revealed that heat stress treatment for 1-6 h markedly suppressed cell viability, with a highly significant elevation in the apoptotic rate observed at the 6-h time point. Furthermore, either knockdown of Hsp72 or perturbation of the components of its functional complex markedly increased cellular susceptibility to heat stress, wherein a significant rise in apoptosis was detected as early as 1 h post heat exposure. We additionally evaluated the influence of heat stress on cell proliferation; prolonged heat stress for 6 h caused highly significant repression of cell proliferation, and Hsp72 knockdown resulted in pronounced proliferation inhibition regardless of heat stress exposure status. Further mechanistic assays demonstrated that HSP72 depletion dramatically upregulated P21 expression, leading to cell cycle arrest at the G2 phase. Moreover, intracellular galactosidase activity was significantly elevated following 1-6 h of heat stress, and HSP72 silencing further increased the percentage of galactosidase-positive cells. Collectively, these findings highlight the crucial protective role of HSP72 in counteracting heat stress-elicited cellular injury, laying a fundamental theoretical foundation for alleviating heat-induced cellular damage.

Iodine-based staining techniques are commonly used in histological imaging and micro-computed tomography (μ CT) because iodine binds to specific molecules. However, the basis for tissue-specific contrast has not yet been sufficiently explored. In this study, we analysed the human proteome at four levels: individual proteins, protein families, tissues with additional expression values for selected proteins, and organs as distinct combinations of tissues. At each level, we identified groups with high potential for iodine binding, particularly those rich in aromatic heterocyclic amino acids. We evaluated the occurrence of aromatic and non-aromatic heterocyclic and carbocyclic amino acids in 20,650 proteins, 1487 protein families, 57 tissues, and 16 organs using bioinformatic methods. This evaluation was based on curated human protein sequences and integrated expression information from the Human Protein Atlas and ProteomicsDB. At the protein level, structural proteins such as titin, nebulin, obscurin, mucin, filaggrin, and hornerin exhibited a high absolute number of aromatic heterocyclic amino acids, which could explain the high μ CT contrast in muscle, skin, and mucosal tissues. A smaller set of proteins displayed strong relative enrichment. At the family level, the average absolute values were highest in families containing very large proteins. However, relative values highlighted sequence-level enrichment in selected enzyme- and membrane-associated families. In contrast, tissue- and organ-level enrichment showed only small, non-significant differences depending on the expression dataset, and was highly similar across groups. Furthermore, enrichment was not robustly associated with iodine staining intensity in organs. Our results provide a foundation for iodine-based tissue imaging and could serve as a starting point for future research, such as cross-species applications and the structural and functional effects of iodination.

This study investigated the ability of quercetin (QU) alone or with sildenafil (Sild) or pentoxifylline (PTX) to modulate carbon tetrachloride (CCl4)-induced renal fibrosis in Wistar albino rats. Renal fibrosis was induced by intraperitoneal injection of CCl4 (1.5 ml/kg, three times weekly for 10 weeks). Rats received oral doses of QU (50 mg/kg once daily), PTX (50 mg/kg once daily), and Sild (10 mg/kg twice daily) separately or in combination from week 7 to week 10. CCl4 induced severe renal injury as indicated by urinary electrolytes disturbance, renal function impairment, increased oxidative stress, inflammation, up-regulation of endoplasmic reticulum (ER) stress markers (CHOP, GRP75), and apoptosis (Bax), as well as down-regulation of defense proteins (Bcl2, Nrf2, HO-1). QU, Sild, and PTX, alone or in combination, improved renal function and alleviated histological changes. The combination of QU with Sild or PTX resulted in greater renoprotection by regulating several key pathways, including reductions in ER stress, enhanced antioxidative defenses, and regulation of apoptosis. In conclusion, a combination of QU with Sild or PTX ameliorates renal fibrosis by inhibiting ER stress-induced cellular apoptosis through modulation of the GRP75/CHOP/Bax/Bcl2 and oxidative stress pathways.

The widespread presence of nanoplastics in the environment has raised concerns regarding their potential adverse effects on reproductive health, particularly during the early developmental stages. Among these contaminants, polystyrene nanoplastics (PSNPs) can cross biological barriers and accumulate in tissues; however, the reproductive consequences of lactational exposure remain insufficiently understood. This study investigated the effects of maternal exposure to PSNPs during lactation on testicular development, oxidative stress, spermatogenesis, and sperm quality in male rat offspring on postnatal days (PD) 30 and 60. Lactating Sprague Dawley rats were orally administered PSNPs at doses of 0.1, 1, or 10 mg/kg/day from postnatal days 1 to 21. Male offspring were evaluated on PD30 and PD60. Testicular morphometry, histopathological alterations, oxidative stress biomarkers, reproductive hormone levels, sperm characteristics, and immunohistochemical expression of p53 and BCL-2 were also assessed. Histopathological examination revealed degeneration of seminiferous tubules, reduction in germinal epithelium height, and impaired spermatogenic organization, particularly at higher exposure doses. Significant alterations in oxidative stress biomarkers were observed, characterized by an increased total oxidant status and oxidative stress index, accompanied by a reduced antioxidant capacity. Sperm analysis revealed reductions in sperm count, motility, and viability, along with increased morphological abnormalities in the exposed groups. Hormonal evaluation indicated disturbances in serum testosterone, luteinizing hormone, and follicle-stimulating hormone (FSH) levels. Immunohistochemical analyses further demonstrated increased p53 expression and reduced BCL-2 immunoreactivity, suggesting the activation of apoptosis-related pathways. Lactational exposure to PSNPs adversely affects testicular development and sperm quality in male offspring, potentially through oxidative stress and apoptosis-related mechanisms. These findings suggest that early life exposure to nanoplastics during critical developmental periods may contribute to reproductive dysfunction, highlighting the need for further investigation into the long-term biological effects of environmental nanoplastic exposure.

A one-cell embryo called a zygote develops into a blastocyst through several successive cell divisions and lineage specification, this process is called early embryo development. Both embryonic genome activation (EGA) and the first lineage specification during early embryonic development depend on tightly coordinated epigenomic organization. Regulation of the epigenome is primarily governed by DNA methylation mediated through DNA methyltransferase (Dnmt) enzymes. Dnmt1 is responsible for the maintenance of methylation during cellular division, while Dnmt3a/Dnmt3b enzymes play a role in the establishment of de novo methylation particularly during gametogenesis and early embryo development. Despite its lack of catalytic activity, Dnmt3l functions as a cofactor enhancing Dnmt3a/3b activity. Dnmt3b deficiency results in global hypomethylation and ultimately embryonic lethality. In this study, we aim to elucidate the effect of Dnmt3b and Dnmt3l silencing on early embryo development. For this purpose, our experimental groups were established using an in vitro mouse embryo development model: control, Dnmt3b small interfering RNA (siRNA), Dnmt3l siRNA, and a nontargeting siRNA group. Following gene silencing at the one-cell stage, embryonic developmental competence, the expression pattern of nonsilenced Dnmt enzymes, global DNA methylation levels, and transcriptome profiles were analyzed at the blastocyst stage. Dnmt3b/3l silencing resulted in decreased global DNA methylation and Dnmt1/3a expression, and reduced blastocyst rate. Differentially expressed genes included those involved in X-chromosome inactivation (Xist), transcriptional regulation (Rn7sk), translation (Eef1a1, Eef2), trophoblast development (Hsd3b1), compaction (Gja1), and oxidative phosphorylation (CYTB, COX1, mt-Rnr1). Our findings indicate that siRNA-mediated knockdown of Dnmt3b and Dnmt3l is associated with reduced blastocyst development, impaired embryo quality, and alterations in DNA methylation-related processes during early embryonic development.

Pulmonary fibrosis is a chronic progressive disease, caused by numerous factors, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Damage to the alveolar epithelial barrier (formed by junctions between alveolar epithelial cells I) has been described as a key mechanism of early fibrosis and therefore could be crucial for COVID-19-associated sequelae. The integrity of adherens junctions is regulated by p120 catenin and is crucial for barrier regulation in epithelial-mesenchymal-transition-driven diseases such as fibrogenesis and cancer. Abundance and expression of p120 catenin and intercellular contact regulating proteins (caveolin-1, P2X7R) were investigated retrospectively in COVID-19 lungs (acute, chronic) compared with healthy controls and interstitial lung disease of different etiologies (n = 6). p120 Catenin was further investigated in early injury models using profibrotic agents in murine lung and human alveolar epithelial cell culture. p120 Catenin abundance was higher in chronic COVID-19 compared with acute COVID-19, suggesting a role in fibrotic development. The interacting proteins caveolin-1 and P2X7R were simultaneously decreased in COVID-19, indicating a role in early pathophysiology. Early epithelial damage showed no changes in p120 catenin and its phospho-Y228 site in cell culture whereas p120 catenin was reduced in murine lung culture. Thus, alterations in and interactions between p120 catenin and caveolae associated proteins may represent a potential mechanism underlying COVID-19-induced pulmonary fibrosis.

Endothelial dysfunction is a central mechanism linking type 2 diabetes (T2D) to cardiovascular complications, and environmental endocrine-disrupting chemicals may further perturb vascular homeostasis. Bisphenol S (BPS), widely used as a bisphenol A substitute, is increasingly detected in human and environmental samples, but its impact on endothelial morphology in the context of pre-existing metabolic disease remains poorly defined. Here, we used high-content imaging and Cell Painting to determine whether exposure to 0.1 µM BPS for 96 h induces distinct morphology-based responses in primary human arterial endothelial cells from healthy and T2D donors. Multiplex staining of DNA, RNA-rich regions, RER, Golgi/membrane structures, and mitochondria was combined with automated segmentation, multidimensional feature extraction, sparse partial least-squares discriminant analysis, and inferential statistics. Metabolic status was the main determinant of the endothelial morphology-based profile, clearly separating healthy and T2D-derived cells. In addition, BPS induced a pronounced multidimensional phenotypic shift in healthy endothelial cells, involving nuclear, mitochondrial, RNA-associated, and membrane/perinuclear descriptors. In contrast, T2D-derived cells retained a dominant disease-associated profile, with a less evident whole-cell response to BPS. However, selected RER-associated descriptors remained BPS-responsive, indicating that BPS can still modulate stress-related subcellular organization under diabetic conditions. These findings show that BPS perturbs endothelial morphology under exposure-relevant low-dose conditions but that baseline metabolic status strongly shapes the magnitude and compartmental distribution of this response. The study identifies the diabetic endothelial phenotype as a key modifier of BPS-induced cellular remodeling and supports Cell Painting as a sensitive strategy to resolve context-dependent endothelial responses to environmental pollutants.

Obesity is a metabolic disorder characterized by the accumulation of excess body fat, which has been linked to skeletal muscle and motor behavior changes. Despite the high prevalence of obesity among children and adolescents in Mexico, it has mostly been studied using adult obese animal models. The objective of the present study was to analyze open-field locomotor activity and dendritic length and dendritic spine density in layer V pyramidal neurons of the primary motor cortex (M1) in adolescent (60-day-old) obese Zucker diabetic fatty (ZDF) rats of both sexes. Long-Evans (LE) rats were used as the control group. Female ZDF rats exhibited increased body weight, Lee index, and abdominal circumference. In contrast, male ZDF rats showed a shorter body length together with increased Lee index and abdominal circumference. However, ZDF rats of both sexes displayed elevated triglyceride and cholesterol levels. In addition, hyperglycemia was observed in male ZDF rats. Female ZDF rats showed decreased exploratory activity. The dendritic length of the basilar arbor of M1 neurons was significantly shorter in female and male ZDF rats, with ZDF males exhibiting a smaller basilar arbor than ZDF females. This last result was also observed in the apical arbor of these neurons. There was also a decrease in basilar and apical dendritic spine density in ZDF rats of both sexes. Our results indicate that female ZDF rats show alterations in exploratory behavior and dendritic arborization, whereas ZDF males are affected in terms of dendritic arborization in M1 neurons, which suggests sex differences in the effects of obesity in adolescent Zucker rats.

Lipopolysaccharide (LPS) is a component of the Gram-negative bacterial cell wall and is considered a potent inflammatory inducer. Empagliflozin (EMPA) is a sodium-glucose co-transporter 2 inhibitor used in treatment of type 2 diabetes mellitus. Interleukin-6/Janus kinase/signal transducer and activator of transcription 3 (IL-6/JAK/STAT3) signaling pathway is essential in driving inflammation, fibrosis, and epithelial-mesenchymal transition (EMT). Tracheal basal stem cells are responsible for epithelial regeneration after injury. This study aimed to investigate the potential protective effects of EMPA against LPS-induced tracheal injury, with emphasis on EMT and tracheal basal stem cells. Sixty adult male rats were allocated into four groups: control; EMPA, which received EMPA (10 mg/kg/day) via oral gavage for 28 days; LPS, which received single intratracheal dose of LPS (5 mg/kg) on day 1; and LPS + EMPA, which received single LPS dose and EMPA for 28 days. Tracheal specimens were processed for biochemical, histological, immunohistochemical and RT-quantitative real-time PCR analysis. Compared to the LPS group, the LPS + EMPA group showed marked improvement of histological, ultrastructural ,and biochemical alterations including maintenance of epithelial and cilia integrity, decreased lipid peroxidation, stimulated antioxidant enzyme activities, reduced serum inflammatory cytokines, increased percentage area of E-cadherin (an epithelial marker) immunostaining, decreased percentage area of fibrosis, vimentin (a mesenchymal marker), and pro-apoptotic Bax; and enhanced percentage area of cytokeratin 5/6 (CK5/6) (stem cell marker). Moreover, EMPA downregulated the IL-6/JAK/STAT3 pathway involved in EMT. In conclusion, EMPA alleviated LPS-induced tracheal injury through its antioxidant and anti-inflammatory properties, downregulation of IL-6/JAK/STAT3 signaling pathway, and preservation of the tracheal basal stem cells.

DNA methylation is considered a well-regulated mechanism involved in cellular differentiation, particularly in the differentiation of mesenchymal stem cells into chondrocytes. TET family proteins potentially regulate demethylation by the oxidative conversion of 5-methylcytosine (5-mC) into hydroxymethyl cytosine (5-hmC) and then into formylcytosine (5-fC) and carboxyl cytosine (5-caC) in a series of reactions. However, the precise role of TET proteins and the impact of 5-hmC dynamics during chondrocyte differentiation remain unclear. This study aimed to investigate the potential association of 5-hydroxymethylation with TET family proteins and human telomerase reverse transcriptase (hTERT) gene expression during the differentiation of human mesenchymal stem cells (hMSCs) into chondrocytes. Methodology involves hMSCs differentiation into chondrocytes over 16 days of culture, with successful differentiation confirmed by Alcian Blue staining and chondrogenic marker gene (ACAN and COL2A1). Gene and protein expressions of TET1, TET2, TET3, and hTERT were analyzed through real-time polymerase chain reaction (RT-PCR) and western blot. Global changes in DNA 5-hmC level quantified using dot blot and enzyme-linked immunosorbent assay (ELISA). Locus-specific changes within the hTERT promoter gene were analyzed through locus-specific PCR. We observed a substantial increase in the expression levels of TET family proteins TET1 and TET2, as well as high global 5-hmC levels during chondrocyte differentiation. However, human telomerase reverse transcriptase (hTERT) expression was reduced both at the messenger RNA (mRNA) and protein levels throughout the differentiation process. Furthermore, epigenetic analysis revealed that elevated 5-hmC enrichment at the hTERT promoter region was strongly associated with the downregulation of hTERT expression. Our findings suggest that 5-hmC may serve as a potential epigenetic biomarker and regulatory element in chondrocyte differentiation, offering new insights into skeletal development and cartilage-related disorders.

Anaplastic thyroid carcinoma (ATC) is among the most aggressive malignancies. Patients with ATC have poor prognoses and require novel therapies. Cleaved caspase-3 (CC3) has pivotal functions in apoptosis and is usually present in the nuclei and cytoplasm of apoptotic cells. We investigated the aberrant membranous localization of CC3 in thyroid carcinoma (TC) and its diagnostic and clinicobiological significance. We examined the expression of nuclear/cytoplasmic CC3 (n/cCC3) and membranous CC3 (mCC3) in 80 primary and one metastatic TC samples. Receiver operating characteristic curve analysis was used to evaluate their diagnostic efficacy for ATC. Multivariate logistic regression analysis was used to identify risk factors for large tumor size. The n/cCC3 and mCC3 indices were significantly greater in ATC than in other types of TC (P < 0.001). Moreover, the area under the curve and sensitivity of mCC3 were greater than those of n/cCC3 for identifying ATC (0.961 versus 0.948 and 95.0% versus 90.0%). A preabsorption test confirmed the specificity of CC3 immunostaining. However, Western blotting indicated that the anti-CC3 antibody crossreacted with an unknown protein with epitopes common to those of CC3. Significant correlations were detected between Ki-67 and n/cCC3 (ρ = 0.536, P < 0.001) and mCC3 (ρ = 0.680, P < 0.001). Multivariate analysis revealed that mCC3 was an independent risk factor for large tumor size. Double immunofluorescence staining demonstrated that mCC3-expressing ATC cells were Ki-67-negative but occasionally SOX2-positive. Our results suggest that mCC3 may have diagnostic value for ATC and that mCC3-expressing ATC cells may be involved in cell proliferation and stemness.

Merkel-cell carcinoma (MCC) is known for its high metastatic potential. Studies in several tumor types have proposed a role for up- or downregulation of nuclear A- and B-type lamins in facilitating the process of invasion and metastasis, but such studies are lacking for MCC. Therefore, lamin expression levels were examined in 38 tissue samples from 28 patients with MCC using immunohistochemistry. The series comprised primary tumors and different metastases and both Merkel cell polyomavirus (MCPyV) positive and MCPyV-negative cases. This study revealed that primary MCC tumors express variable, but mostly low levels of A-type lamins, while the B-type lamins are overall highly expressed. No obvious differences in lamin expression were observed between the MCPyV-positive and MCPyV-negative cases. A generally higher A-type lamin expression was detected in MCC metastases, although comparison of primary tumors versus metastases from the same patients showed either no change or up- or downregulation of A-type lamins. Normal Merkel cells were found to express both A- and B-type lamins. Conclusively, our data suggest a flexible A-type lamin expression in the process of MCC carcinogenesis and metastasis, which is an important finding regarding the aggressiveness of MCC and in the debate about the cellular origin of MCC.