Histochemistry and Cell Biology最新文献

Among nuclear compartments, interchromatin granule clusters (IGCs) are widely regarded as biomolecular condensates implicated in the regulation of gene expression, leading to the production of distinct mRNA species. Nevertheless, their functional dynamics within the nuclear environment remain largely elusive. In this study, we employed multiple transmission electron microscopy approaches to investigate the spatial and structural relationships between IGCs and chromatin. Our observations in HeLa cells demonstrate that IGCs establish physical connections with chromatin fibers. Furthermore, we show that the periphery of IGCs is enriched in decondensed chromatin domains and transcriptional sites. Quantitative analyses reveal that, upon α-amanitin treatment, the number of decondensed chromatin sites near IGCs is significantly reduced compared with untreated cells. In untreated conditions, a positive correlation emerges between IGC size and the abundance of adjacent decondensed chromatin regions. Based on these findings, we propose a model of IGC organization, considering their contacts with chromatin.

This study investigated whether ovarian adipokines exhibit uniform or stage-specific expression patterns across different follicular stages under hyperandrogenic conditions using a letrozole-induced polycystic ovary syndrome (PCOS) mouse model. Adult female mice received oral letrozole treatment for 21 days to induce hyperandrogenism, and ovarian tissues were analyzed by immunohistochemistry and western blot to examine the localization and expression of adiponectin (ADPN), adipoR1, adipoR2, leptin (Ob), leptin receptor (ObR), apelin (APLN), apelin receptor (APJ), chemerin, CMKLR1, and visfatin. Intense immunostaining for Ob, ObR, APJ, APLN, adipoR2, and visfatin was observed in primary, secondary, and Graafian follicles, whereas ADPN, adipoR1, and CMKLR1 showed reduced reactivity. In follicular cysts, adipoR2, APLN, APJ, and Ob were markedly upregulated compared with the corpus luteum of control ovaries, whereas ADPN, adipoR1, chemerin, CMKLR1, and ObR were downregulated. These findings indicate that hyperandrogenism disrupts adipokine signaling in a follicle-dependent manner, with differential expression patterns contributing to altered follicular maturation and cyst formation. The enhanced activation of adiponectin, apelin, and leptin signaling observed in cystic follicles may indicate disrupted adipokine-mediated regulation of ovarian physiology in letrozole-induced PCOS. Given the established roles of these adipokines in folliculogenesis and steroidogenesis, their dysregulation may contribute to follicular arrest and impaired ovarian function. These alterations are likely reflective responses to an altered endocrine and metabolic environment rather than direct causal mechanisms. Nonetheless, they may participate in the pathophysiological processes underlying cyst formation in PCOS.

The vitamin D receptor (VDR) has been implicated in anti-inflammatory and antifibrotic effects, but its role in regulating TGF-β1/Smad signaling and fibroblast activation in pulmonary fibrosis remains unclear. This study investigates the regulatory effects of VDR on TGF-β1/Smad signaling and its impact on fibrogenic responses in lung fibroblasts. MRC-5 cells were treated with L-lactate sodium to generate a fibrotic model, and VDR and TGF-β1 expression were manipulated using plasmids and siRNA. Fibroblast activation, TGF-β1/Smad signaling, and ECM remodeling were assessed using qRT-PCR, western blot, and immunofluorescence, while cell proliferation, migration, invasion, oxidative stress, and inflammation were also evaluated. Lactate stimulation increased α-SMA and collagen I/III expression, confirming fibroblast activation. VDR overexpression reduced fibrotic markers, downregulated ECM-degrading enzymes (MMP2, MMP9), and upregulated TIMP-1, while inhibiting migration, invasion, and reducing ROS and inflammatory cytokines (IL-6, IL-1β). In contrast, VDR knockdown enhanced fibrotic marker expression and fibroblast activity. Phosphorylation of Smad2/3 decreased with VDR overexpression and increased with knockdown. TGF-β1 overexpression elevated fibrotic markers and Smad signaling, while TGF-β1 knockdown reduced these markers and alleviated the activated phenotype. Exogenous TGF-β1 treatment reversed the antifibrotic effects of VDR overexpression, linking VDR to TGF-β1/Smad signaling. VDR suppresses fibroblast activation and fibrotic responses in lung fibroblasts by downregulating the TGF-β1/Smad signaling pathway, highlighting its potential as a therapeutic target for pulmonary fibrosis.

Achilles tendon ruptures are recognized as one of the most widespread musculoskeletal injuries. Tendon injuries are a notable clinical challenge, primarily due to the restricted regenerative capacity of the tissue and the associated risks of fibrosis and incomplete functional recovery. Recent studies suggest that cell-free therapies, including stem cell-derived secretomes, may facilitate tendon regeneration. Additionally, mechanical stimulation through exercise can enhance tissue remodeling. This study aimed to investigate the combined effects of tendon-derived stem cell (TDSC) secretome and treadmill-based rehabilitation on Achilles tendon regeneration in a rat model. TDSCs were isolated from rat Achilles tendon and characterized using morphology, cytochemical staining, and flow cytometry. In vitro scratch assays were performed to assess cell migration and wound healing in a laboratory setting. The secretome was collected from fourth-passage TDSCs and incorporated into a collagen-based, injectable hydrogel. A total of 42 adult female Wistar rats were categorized into eight distinct experimental groups, including injury-only, treadmill-only, secretome-only, and treadmill + secretome groups. A surgical procedure was performed to induce a partial rupture of the Achilles tendon, followed by the injection of a secretome-loaded hydrogel at the site of injury. Subsequently, a structured treadmill training program was initiated post-surgery. Regenerative outcomes were evaluated using footprint analysis, histological staining (hematoxylin and eosin, periodic acid-Schiff, and Masson's trichrome), and biomechanical testing. In vitro scratch assays demonstrated that TDSCs treated with secretome exhibited enhanced migratory capabilities. Flow cytometry confirmed the identity of these mesenchymal stem cells (MSCs). In vivo studies showed that the combination therapy group (secretome-loaded hydrogel and treadmill training) achieved superior histological recovery. This group exhibited organized collagen bundles, aligned spindle-shaped tenocytes, minimal inflammation, and restored extracellular matrix integrity. PAS staining indicated reduced glycosaminoglycan degradation, while Masson's trichrome staining revealed partial collagen maturation. Additionally, footprint analysis showed improved functional performance, with the combination group achieving significantly higher Achilles functional index scores. Biomechanical testing confirmed enhanced tensile strength and elastic modulus, approaching values comparable to those of intact tendon healing. The synergistic application of TDSC-derived secretome along with treadmill training significantly improved tendon regeneration, matrix remodeling, and functional recovery. This cell-free, bioactive approach offers a promising therapeutic alternative for enhancing tendon recovery.

Peroxisomes are dynamic organelles vital for lipid metabolism and redox homeostasis. In Saccharomyces cerevisiae, the expression of peroxisomal proteins is tightly regulated in response to metabolic conditions. Here, we provide the first absolute quantification of the yeast peroxisomal proteome under peroxisome-inducing (oleate) and fermentative (glucose) conditions using a label-free mass spectrometry approach. We determined protein copy numbers for ~ 4500 proteins, including 99 peroxisomal and peroxisome-associated proteins. Our data reveal that the overall peroxisomal proteome is approximately threefold more abundant in oleate-grown cells, constituting 2.8% (2.01 × 10⁶ protein copies) of the total proteome compared to 0.8% (6.67 × 10⁵ protein copies) in glucose. Considering only peroxisomal core proteins, i.e., proteins exclusively or predominantly localized in peroxisomes, total copy numbers for peroxisomal proteins were even ninefold higher on oleate (0.9%, 6.29 × 10⁵ protein copies) compared to glucose (0.1%, 7.78 × 10⁴ protein copies), reflecting the necessity for peroxisomal functions such as fatty acid beta-oxidation. Enzymes of the beta-oxidation and glyoxylate cycle showed up to > 500-fold higher abundance in oleate. In contrast, core components of the peroxisomal protein import machinery (e.g., Pex5, Pex14) exhibited only moderate changes (~ 2- to 8-fold). In addition to metabolic enzymes and components of the peroxisomal protein import pathways, we provide copy number data for proteins involved in cellular stress response, peroxisome proliferation, division and organization, peroxisome-associated membrane contact sites, and metabolite transporter. Taken together, our dataset offers a quantitative framework of peroxisomal remodeling under different metabolic conditions and highlights the organelle's adaptive flexibility, providing a valuable resource for future studies on peroxisome biology.

Acyl-CoA synthetase long-chain family member 4 (ACSL4) is a lipid-metabolizing enzyme implicated in ferroptosis regulation and tumor aggressiveness. Although ACSL4 overexpression has been reported in various malignancies, its immunohistochemical profile in primary cutaneous melanoma has not been fully characterized. This study aimed to evaluate ACSL4 expression in melanoma compared with normal skin using quantitative digital image analysis. A total of 80 formalin-fixed paraffin-embedded samples were analyzed, including 50 primary cutaneous melanoma specimens and 30 control skin samples obtained from benign dermatologic excisions. Hematoxylin-eosin staining was used to assess histopathologic features, and ACSL4 immunostaining was performed using a standardized protocol. Quantitative evaluation was conducted with QuPath software by calculating the percentage of positive cells, mean intensity scores (0-3), and H-scores (0-300) in epidermal and dermal compartments. Group comparisons were performed using the independent t test, with p < 0.05 considered statistically significant. Control tissues exhibited minimal ACSL4 expression (epidermal H-score 12; dermal H-score 9), whereas melanoma specimens demonstrated markedly increased ACSL4 immunoreactivity. Dermal atypical melanocytic tumor cells showed the highest expression levels (mean intensity 2.10 ± 0.35; H-score 168; p < 0.001), while epidermal layers also exhibited moderately elevated staining (H-score 58; p < 0.001). Histopathologic evaluation revealed characteristic features of invasive melanoma, including atypical melanocytic nests, pagetoid spread, cytologic atypia, and architectural disorder. Overall, ACSL4 expression was significantly upregulated in primary cutaneous melanoma compared with normal skin, particularly within dermal atypical melanocytic tumor cells, suggesting that ACSL4 may contribute to melanoma biology through lipid metabolic pathways and may represent a potential biomarker of tumor aggressiveness, warranting further investigation into its diagnostic and prognostic relevance.

Peroxisomes are eukaryotic organelles primarily known for their conserved roles in fatty acid β-oxidation and hydrogen peroxide detoxification. These organelles are also involved in a diverse range of other metabolic and non-metabolic functions. We recently compared the transcriptome and proteome of Saccharomyces cerevisiae wild-type and peroxisome-deficient (pex3) cells. This study uncovered the major processes and metabolic pathways that are influenced by peroxisomes. Here we performed a mass spectrometry-based analysis of intracellular metabolites of the same two strains. This led to the identification of 160 compounds, of which seven exhibited significant differences between wild-type and pex3 cells (glycerol-3-phosphate, carnitine, pantothenate, acetyl-spermidine, propionyl-carnitine, and aminolevulinic acid). Notably, we observed elevated lysine levels in pex3 cells, consistent with previous findings, which confirms the reliability and accuracy of our analytical approach. In addition, changes in carnitine compounds were measured, aligning with the proposed occurrence of a carnitine shuttle across the peroxisomal membrane. By integration of the current metabolomic data with the previously obtained transcriptomic and proteomic data, we provide a broader view of the metabolic impact of peroxisome deficiency. We show that, in addition to the well-known function of yeast peroxisomes in lipid and fatty acid degradation, these organelles are also involved in lipid synthesis. Furthermore, our study revealed that peroxisome deficiency affects polyamine homeostasis.

Peroxidasin (PXDN) is a multi-domain heme peroxidase that catalyzes sulfilimine cross-links in type IV collagen, a reaction essential for basement membrane stability. In addition to its structural role, PXDN has been implicated in oxidative stress regulation, fibrosis, and tumorigenesis. Antibody-based detection is critical for defining PXDN expression and localization, yet few commercial reagents have been rigorously validated, limiting reproducibility. Here, we assessed the specificity and performance of a commercially available anti-PXDN antibody (Abbexa, abx101906) in immunohistochemistry and immunocytochemistry applications. Validation was performed in formalin-fixed, paraffin-embedded human kidney tissue and in a primary kidney fibroblast line under PXDN silencing and overexpression conditions. The antibody yielded reproducible labeling patterns and demonstrated specificity across cellular contexts. These findings establish abx101906 as a reliable tool for immunofluorescence-based detection of PXDN. This validation provides a foundation for future studies of PXDN biology in kidney development, disease, and potential therapeutic targeting.

The development of immunohistochemical and immunohistofluorescence assays is essential for investigating the tissue and cellular distribution of target proteins. In this study, we identify specific anti-ESR1 antibodies against rodent ESR1 proteins and evaluate their applicability for immunohistochemistry and dual immunohistofluorescence with the specific anti-ESR2 antibody PPZ0506. We assessed the specificity and cross-reactivity of six commercially available anti-ESR1 antibodies (Clones MC-20, C1355, E115, H4624, SP1, and F-10) against mouse and rat ESR1 proteins using immunoblotting and immunocytofluorescence assays. Among them, MC-20, C1355, E115, and H4624 exhibited specific immunoreactivity to mouse and rat ESR1 proteins. These four antibodies were subsequently applied to paraffin-embedded ovarian and uterine sections from mice and rats. Heat-induced antigen retrieval and an appropriate antibody dilution were required to obtain specific and adequate signals. MC-20 and E115 were suitable for immunohistochemical detection of ESR1 proteins, while C1355 was effective for uterine tissue staining. H4624 showed utility only in mouse tissues. Furthermore, rabbit-derived MC-20 and E115 antibodies were successfully employed in dual immunohistofluorescence assays with the mouse monoclonal PPZ0506 antibody, enabling simultaneous visualization of ESR1 and ESR2 proteins in paraffin-embedded ovarian sections. Notably, little cellular co-localization of ESR1 and ESR2 proteins was observed in mouse and rat ovarian sections. These findings provide a validated set of antibodies for ESR1 immunohistochemical detection and demonstrate their compatibility with ESR2 co-labeling, facilitating detailed analysis of estrogen receptor distribution in rodent tissues.