Despite advances in oral cancer treatment, therapeutic resistance remains a major challenge. Cancer-associated fibroblasts (CAFs) play a pivotal role in shaping the tumor microenvironment. Building on our previous findings on immune responses in gingival fibroblasts, we investigated the immune characteristics of CAFs. Myofibroblast CAF-like cells (myCAF-like) were established using a co-culture system and compared with gingival fibroblast-derived myofibroblasts. MyCAF-like cells exhibited markedly reduced expression of TLR3 and TLR4, whereas RIG-I, COX-2, IL-1β, IL-6, and IL-8 were significantly upregulated. Activation of p38 MAPK signaling contributed to the increased expression of COX-2 and pro-inflammatory cytokines, and TGF-β was involved in the phenotypic transformation of gingival fibroblasts into myCAF-like cells. Microarray analysis revealed upregulation of IL-1α, IL-1β, CXCL8, PTGS2, and CXCL5, alongside downregulation of OMD, COL15A1, and ASPN in myCAF-like cells. Collectively, these findings demonstrate that CAFs acquire a distinct inflammatory signature that differs from gingival fibroblasts and promotes tumor invasion and progression. Targeting CAF-associated inflammatory and signaling pathways may represent a promising strategy to overcome tumor infiltration and treatment resistance in oral cancer.
Ultrastructural analysis has declined in diagnostic pathology since the advent of immunohistochemistry for formalin-fixed paraffin-embedded tissues. To reevaluate the utility of ultrastructural analysis, we adopted the osmium maceration method for scanning electron microscopy (SEM) of formalin-fixed surgical specimens: renal oncocytoma, chromophobe renal cell carcinoma (ChRCC), and tumors categorized as other oncocytic tumors of the kidney (OOT). Light microscopy and immunohistochemistry have revealed that these tumors have overlapping features. SEM observation of the formalin-fixed tumors highlighted distinct morphological features: The oncocytomas contained large mitochondria with vesicular cristae; ChRCCs harbored small mitochondria with microvesicles, and OOTs showed abundant but small mitochondria with lamellar cristae. Quantitative morphometry confirmed that the number of mitochondria in oncocytomas and OOTs exceeded that of non-neoplastic lesions, whereas ChRCCs contained fewer mitochondria. In addition, mitochondria in ChRCCs and OOTs were smaller than those of non-neoplastic lesions, whereas mitochondria in oncocytomas were larger. Thus, SEM of formalin-fixed tissues, optimized by osmium maceration, can provide diagnostically valuable information beyond conventional histology and immunohistochemistry that may help to clarify the biology of challenging oncocytic and chromophobe renal tumors.
This study aimed to elucidate the relationship between the spatiotemporal behavior of macrophages during exercise and axonal regeneration following peripheral nerve injury. A sciatic nerve crush model was created using male C57BL/6J mice, which were divided into sedentary and exercise groups. The exercise group performed low-intensity treadmill running (10 m/min, 60 min/day, 5 days/week) starting 3 days post-injury. Immunohistochemical analyses of Neurofilament 200 kDa (NF200), Growth Associated Protein-43 (GAP-43), F4/80, and arginase-1 (Arg-1), a representative marker of M2 macrophages and a downstream effector of the JAK-STAT6 signaling pathway, were conducted in the proximal and distal regions of the injured nerves. Functional recovery was assessed using the sciatic functional index (SFI) and compound muscle action potential (CMAP). At 7 and 14 days post-injury, the exercised group exhibited a significant increase in axonal number and Arg-1-positive area, specifically in the distal region. SFI and CMAP analyses also demonstrated enhanced functional recovery in the exercise group. These findings suggest that low-intensity treadmill exercise may enhance axonal regeneration, potentially through transient activation and local accumulation of M2-polarized macrophages. Therefore, exercise-induced regulation of macrophage dynamics may represent a novel therapeutic strategy for peripheral nerve repair.
Myo-inositol (MI) reduces hepatic histone acetylation in the enhancer-promoter regions of Elovl6 (ELOVL fatty acid elongase 6) in high-fructose diet (HFD)-fed rats. We examined whether dietary MI supplementation altered the epigenetic modifications in the transcribed regions of Fasn (fatty acid synthase) and Elovl6 in fatty livers of HFD-fed rats. MI supplementation alleviated HFD-induced hepatic expression of Fasn and Elovl6 probably by decreasing the acetylation of histones H3 and H4, and binding of cyclin-dependent kinase 9 and RNA polymerase II in the transcribed regions of these genes. Therefore, dietary MI supplementation can alleviate fatty liver disease by altering epigenetic modifications.
The cGAS-STING pathway is an innate immune pathway that senses microbial and host-derived double-stranded DNA in the cytosol, and triggers type I interferon responses and proinflammatory cytokine production. Recently, activation of the cGAS-STING pathway in tumors is reported to enhance anti-tumor effects, while it is often inactivated in tumors. Therefore, to develop strategies to activate STING in tumors has become imperative in cancer therapy. Here we report that the cGAS-STING signaling is inactivated in DU145 prostate cancer cell line and find that the inactivation mechanism is distinct from the previously reported mechanisms such as DNA methylation. Treatment of DU145 cells with a lysosomal inhibitor Baf-A1 increased a STING protein level. Knockdown of VPS4, the essential factor in lysosomal microautophagy, suppresses STING degradation. Most importantly, knockdown of cGAS, the enzyme responsible for the production of the endogenous STING ligand cGAMP, fully restores a STING protein level and a response to a synthetic membrane-permeable STING agonist MSA-2. Thus, these results suggest that the constant cGAS activation causes the lysosomal degradation of STING and underlies the unresponsiveness of the STING signaling in DU145 cells. Our results may lead to new strategies to enhance STING-targeted cancer immunotherapy.
Chromosomes undergo dynamic structural reorganization during the cell cycle and become highly condensed during mitosis. This condensation process involves the assembly of chromatin fibers, in which chromosome-associated proteins (e.g., condensin) and post-translational modifications (e.g., histone deacetylation) play key roles. Although an increasing number of studies have advanced our understanding of chromatin fiber assembly into chromosomes, detailed morphological information on chromatids remains limited. In this report, we used atomic force microscopy to examine the nanoscale structure of chromosomes preserved in liquid and found that mitotic chromosomes are composed of a series of thin filamentous structures. Quantitative analysis of chromosomes from the chromokinesin KIF4A-depleted cells and under histone deacetylase inhibition revealed that the filament width and inter-filament spacing are regulated by KIF4A and histone deacetylation, likely reflecting their specific contributions at different hierarchical levels of chromosome assembly. These findings demonstrate that atomic force microscopy enables the detection of fine nanoscale structural alterations beyond the resolution of optical microscopy and provides new morphological insights into the organization of mitotic chromosomes.
Intracerebral hemorrhage (ICH) is a devastating form of stroke with limited treatment options. Although exercise is beneficial in ischemic stroke, its preconditioning effects in hemorrhagic stroke remain unclear. We examined whether voluntary wheel running prior to ICH modulates acute outcomes and gene expression. Male ICR mice underwent three weeks of voluntary running or sedentary housing before ICH induction. Neurological function was assessed 3 days post-ICH, and perihematomal tissue was analyzed by quantitative PCR. ICH increased expression of Iba1, GFAP, C3, MMP9, TIMP1, and Caspase3, while reducing BDNF expression. Exercise preconditioning did not improve acute behavioral deficits at the group level. However, running distance strongly predicted molecular and pathological responses. BDNF expression correlated positively with exercise volume, whereas C3 showed a negative correlation, with a similar trend for GFAP. Caspase3 and Bax expression were negatively correlated with running distance. Although hematoma volume did not differ significantly between groups, greater running distance was associated with smaller hematoma volume within the exercise group. These findings indicate that habitual voluntary exercise does not attenuate acute functional deficits after ICH but induces dose-dependent molecular adaptations related to neuroplasticity, glial inflammation, and cell death, potentially priming the brain for enhanced recovery in later phases.
B cell antigen receptor (BCR) signaling plays a critical role in regulating B cell fate, including activation, tolerance, and apoptosis. In immature B cells, strong BCR engagement induces apoptosis, which contributes to the elimination of autoreactive clones during negative selection. However, the molecular mechanisms linking BCR signaling to apoptotic pathways remain incompletely understood. In this study, we investigated the role of Ca2+/calmodulin-dependent protein kinase II (CaMKII) in BCR-induced apoptosis using the immature B cell line WEHI-231. Stimulation of the BCR with anti-IgM antibody induced a rapid increase in intracellular Ca2+ levels and promoted apoptosis in WEHI-231 cells. BCR engagement also induced phosphorylation of CaMKII, indicating activation of this kinase downstream of Ca2+ signaling. Pharmacological inhibition of CaMKII with KN-93, a CaMKII inhibitor, attenuated BCR-induced apoptosis, whereas overexpression of CaMKII enhanced cell death. We further found that BCR stimulation resulted in downregulation of the anti-apoptotic protein Bcl-xL, and inhibition of CaMKII prevented this reduction. Conversely, CaMKII overexpression further enhanced Bcl-xL downregulation following BCR stimulation. Importantly, restoration of Bcl-xL expression significantly rescued CaMKII-mediated apoptosis. These findings identify a Ca2+-CaMKII-Bcl-xL signaling axis that promotes BCR-induced apoptosis in immature B cells.
Disuse induces capillary regression in skeletal muscle, which is associated with excessive production of reactive oxygen species (ROS), impaired mitochondrial oxidative capacity, and a shift from slow- to fast-twitch muscle fibers. Green tea is rich in catechins with potent antioxidant properties. We hypothesized that compressed residual green tea extract, a byproduct of green tea manufacturing, may attenuate capillary regression during muscle disuse. Twenty female Wistar rats were randomly assigned to four groups: control (CON), compressed residual green tea supplementation (GTs), hindlimb unloading (HU), and hindlimb unloading with compressed residual green tea supplementation (HU+GTs). Hindlimb unloading was performed for 2 weeks. Compressed residual green tea extract (850 mg/kg/day) or distilled water was administered orally. HU induced significant reductions in soleus muscle mass, capillary-to-fiber (C/F) ratio, slow-twitch fiber proportion, and succinate dehydrogenase (SDH) activity, along with increased ROS production. Compressed residual green tea supplementation significantly attenuated capillary regression, preserved slow fiber composition, restored SDH activity, and suppressed ROS overproduction under unloaded conditions. These findings suggest that compressed residual green tea may serve as a practical nutritional strategy to maintain skeletal muscle capillarity during disuse by preserving oxidative fiber phenotype and mitochondrial function.
We previously showed that tenascin-XB (TNXB) contributes to tumor suppressor function. The present study aimed to assess the tumor-suppressive mechanism of TNXB by focusing on immune cell infiltration into the tumor microenvironment (TME). We revealed that B16-OVA melanoma cells (MO5)-bearing TNXB-deficient (Tnxb-/-) mice exhibited significant tumor progression and a poor survival rate. Allogeneic mixed lymphocyte reaction showed reduced numbers and increased activation of both CD4+ and CD8+ T cells from Tnxb-/- spleens. Moreover, T cell activation assay further proved that CD4+ and CD8+ T cells from Tnxb-/- mice were more activated than those from WT mice. RT-qPCR analysis showed that expression of T cell activation-related cytokines and chemokines was significantly decreased in tumor tissues from Tnxb-/- mice. Flow cytometry analysis revealed a reduced infiltration level of CD8+ T cells in both naïve spleens and tumor tissues in Tnxb-/- mice. Ultimately, total activation of CD8+ T cells was decreased in tumor tissues in Tnxb-/- mice. In conclusion, we found that although Tnxb-/- CD4+ and CD8+ T cells tend to be activated more than WT CD4+ and CD8+ T cells, CD8+ T cell infiltration and activation level were attenuated in tumor sites of Tnxb-/- mice.
Serial section scanning electron microscopy (SEM) is useful for revealing the three-dimensional (3D) architecture of organelles by acquiring backscattered electron images of ultrathin serial sections of resin-embedded tissues on solid substrates. However, comprehensive analyses of organelle function require a combination of ultrastructural and molecular localization data. In the present study, we developed a novel 3D immuno-electron microscopy (immuno-EM) approach that combines Tokuyasu cryosectioning with serial section SEM to elucidate the spatial distribution of organelle-associated proteins. Thick cryosections of tissues were immunolabeled with primary antibodies and FluoroNanogold-conjugated secondary antibodies, followed by gold enhancement, resin embedding, and serial sectioning and SEM. Serial tomographic images of organelles were aligned and segmented to generate 3D reconstructions. To demonstrate the effectiveness of the method, we visualized the localization of GM130, a representative cis-Golgi matrix protein, in a 3D model of the Golgi apparatus in rat pituitary gonadotropes. The 3D model revealed a spherical Golgi apparatus composed of five cisternae arranged in cis-trans order, with GM130 localized on the outer cisternae, consistent with previous findings. Our 3D immuno-EM technique enables the detailed 3D visualization of the Golgi apparatus and other organelles as well as analyses of the spatial distribution of target proteins in their 3D reconstructions.
Per- and poly fluoroalkyl substances (PFAS) pose significant global health risks. Although the use of classical PFAS such as perfluorooctane sulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) is regulated, the toxicological effects of alternative PFASs remain unknown. Cleft palate is a congenital condition influenced by both environmental and genetic factors. Although PFOS has been linked to cleft palate, the effects of other PFAS compounds remain unexplored. The aim of this study was to clarify the involvement of classical and alternative PFAS (PFHxA and PFHxS) in human embryonic palatal mesenchymal cell (HEPM) proliferation. Following PFAS treatment for 48 h, cell viability, apoptosis, and expression of cell cycle-related proteins were tested. In addition, miRNA levels and predicted target genes were measured, and a rescue experiment against PFHxS was conducted using an miR-374a-5p inhibitor. Among the four PFASs, PFHxS decreased the number of cells showing cyclin- and cyclin-dependent kinase reduction. In addition, PFHxS treatment upregulated miR-374a-5p and downregulated its downstream genes. Furthermore, miR- 374a-5p inhibitor alleviated the PFHxS-induced reduction in cell proliferation. These findings therefore indicate that miR-374a-5p plays a key role in the development of PFHxS-induced cleft palate and that alternative PFAS may have a highly toxic effect on HEPM cells.
Ghrelin-producing cells (ghrelin cells) in rat fundic glands were analyzed three-dimensionally by combining serial section scanning electron microscopy with immunogold labeling to elucidate their ultrastructural characteristics. This approach enabled unambiguous identification of ghrelin cells and the three-dimensional (3D) reconstruction of their organelles with special reference to primary cilium. Rat ghrelin cells were morphologically classified into two types: ciliated ghrelin cells possessing primary cilia and non-ciliated ghrelin cells. In ciliated cells, primary cilia protruded from basal bodies located near the Golgi apparatus and were largely or entirely enclosed within ciliary pockets. Ghrelin-positive secretory granules were electron-dense and spherical. Ciliated and non-ciliated cells contained both large (250-350 nm) and small (100-200 nm) ghrelin-positive granules. In ciliated cells, the granules were densely accumulated in a localized region of the cytoplasm, being opposite the Golgi apparatus across the nucleus; in non-ciliated cells they were densely distributed throughout the cytoplasm. The ultrastructure of the basement membrane and overall 3D configuration of the Golgi apparatus also differed between the two cell types. Our results provide novel insights into the morphological organization of ghrelin cells, whose 3D ultrastructural features have been unclear in conventional two-dimensional transmission electron microscopy using single ultrathin sections.
Radiological and nuclear accidents require reliable biomarkers for rapid detection of radiation exposure and dose estimation. While conventional biodosimetry has focused on nuclear DNA damage in lymphocytes, mitochondrial DNA (mtDNA) may provide an additional index because of its high copy number and limited repair capacity. Previous studies using Epstein-Barr virus-transformed lymphocytes and HeLa-FUCCI cells demonstrated radiation-induced changes in mtDNA copy number (mtDNAcn), suggesting compensatory replication as a characteristic response, but in vivo evidence has been limited. In this study, we analyzed peripheral blood from C57BL/6N male mice (8 weeks old) exposed to 0, 0.05, 0.2, 0.5, or 2 Gy of X-rays. MtDNAcn and intact copy ratio, defined as the proportion of undamaged copies, were quantified at 1 day and 1 week post-irradiation. We found that mtDNAcn was significantly increased only in the 2 Gy group at both 1 day and 1 week, whereas the intact copy ratio was significantly decreased in the 0.5 Gy and 2 Gy groups at 1 day but returned to baseline by 1 week. These findings indicate that peripheral blood-derived mtDNA indices are promising biomarkers for radiation biodosimetry, and that intact copy ratio may be particularly useful for detecting low-dose exposure.
The health benefits of apple polyphenols are well-documented. Phlorizin (Pz), an apple-derived polyphenol, is a known inhibitor of sodium-glucose cotransporters (SGLTs); however, the effects of other apple constituents on SGLTs remain unclear. In this study, we examined the inhibitory effects of an unripe apple polyphenol extract, ApplePhenon® (AP), and its constituents-including Pz, phloretin, procyanidins B1 and B2, chlorogenic acid, gallic acid, catechin, epicatechin, epigallocatechin gallate, and quercetin-on SGLTs using the Ussing chamber in mouse small intestinal mucosa. AP exhibited noncompetitive inhibition of glucose-induced short-circuit current (Isc), reflecting electrogenic Na+ transport via SGLTs (apparent Ki = 22.23 ± 3.06 μg·mL-1). A similar inhibitory effect was observed in the case of Pz (Ki = 1.88 ± 0.32 μg·mL-1), phloretin (Ki = 18.82 ± 3.52 μg·mL-1), and quercetin (Ki = 220.9 ± 3.52 μg·mL-1) but not in other components. Based on the total Pz content of 6.2%, 86.0% of the inhibitory effect can be attributed to Pz. Although Pz is known to inhibit SGLT1 competitively, AP and its constituents, including Pz, phloretin, and quercetin, showed noncompetitive inhibition in this study. These findings warrant further investigation into the mechanisms underlying SGLT inhibition by AP.
In this study, 300 immune-related genes were analyzed in tumor tissues from 6,130 Japanese patients. Human leukocyte antigen (HLA) class I subunit B2M exhibited the highest fold change in mutation frequency in tumors with high tumor mutational burden (TMB). Stratified gene expression analysis revealed that tumors with a high TMB (10-100 mutations/Mb) exhibited increased expression of interferon-gamma (IFN-gamma) and upregulation of downstream genes, including CXCL9, TAP, PD-L1, and LAG3. In tumors with TMB > 100, expression of these genes was modest and accompanied by a marked increase in immunoregulatory gene expression and a high frequency of mutations in genes involved in antigen presentation and the IFN-gamma signaling pathway, such as B2M, TAP2, TYK2, and JAK2. To assess whether hotspot mutations in cancer driver genes are subject to immune pressure, the frequency of gene mutations and HLA allele genotype combinations were evaluated, resulting in the identification of three hotspot mutations in EGFR, KRAS, and TP53 that may be recognized by the immune system. Furthermore, comprehensive HLA allele genotyping of 369 patients identified the HLA-C*15:02 allele, present in 6% of Japanese patients, as a potential prognostic marker.
Trace amine-associated receptor 1 (TAAR1) is highly expressed in rat parotid acinar cells; however, its role in exocrine Ca2+ signaling remains unclear. We herein demonstrated that the selective TAAR1 agonist RO5256390 induced rapid and transient increases in intracellular Ca2+ levels that were completely abolished by the TAAR1 antagonist EPPTB, confirming receptor specificity. Pharmacological dissection revealed dual contributions from extracellular Ca2+ influx via L-type, T-type, and receptor-operated channels and Ca2+ release from intracellular stores mediated by phospholipase C, inositol trisphosphate receptors, and ryanodine receptors. Inhibition of adenylyl cyclase or protein kinase A (PKA) nearly abolished the Ca2+ response, and displacement of PKA from AKAPs using the cell-permeable AKAP-displacing peptide st-Ht31 produced a comparable suppression, underscoring the requirement for microdomain-restricted PKA activity. Exchange protein directly activated by cAMP 2 (Epac 2) and downstream calmodulin-dependent protein kinase II were also indispensable, whereas Epac 1 was dispensable. The present study identified a TAAR1-centered, 3',5'-cyclic adenosine monophosphate microdomain network that co-ordinates Ca2+ entry and release, thereby providing novel targets for the modulation of salivary secretion.
The distribution of transient receptor potential melastatin 7 (TRPM7) was investigated in the trigeminal (TG), petrosal (PG), jugular (JG), and nodose ganglia (NG) in a rat model. Approximately half of the sensory neurons contained TRPM7-immunoreactivity in the TG, PG, JG, and NG. TRPM7-immunoreactive (-IR) sensory neurons had small- to medium-sized cell bodies. Triple immunofluorescence analysis revealed that TRPM7-IR sensory neurons showed mainly transient receptor potential vanilloid 1 (TRPV1) immunoreactivity, whereas less than 10% of TRPM7-IR neurons contained TRPV2-immunoreactivity. In the facial skin, some nerve fibers contained TRPM7-immunoreactivity and formed free nerve endings. Using a retrograde tracing method, 56% of cutaneous TG neurons contained TRPM7-immunoreactivity. Approximately half of TRPM7-IR cutaneous TG neurons contained TRPV1-immunoreactivity. Approximately two-thirds of PG, JG, and NG neurons innervating the external ear canal skin contained TRPM7-immunoreactivity. Most TRPM7-IR cutaneous PG, JG, and NG neurons contained TRPV1-immunoreactivity. However, TRPM7-IR neurons that innervate the tooth pulp and pharynx are relatively rare. This study suggests that the distribution of TRPM7 depends on the size of nerve cells and peripheral innervation. TRPM7 may be involved in pain sensation in the trigeminal, glossopharyngeal, and vagal ganglia sensory neurons.
Cluster of differentiation 36 (CD36) is a cell-surface receptor that exhibits multifunctional properties with diverse ligands. A short segment of CD36, consisting of amino acids 149-168 (CD36149-168), has been shown to constitute a site for recognizing its lipid ligands (e.g., distinct forms of oxidized phospholipids). Recently, lauric acid was found to bind to the CD36 segment. Of interest, the fatty acid and some of its derivatives such as laurylamine and 1-monolaurin are well known to have a cytotoxic activity. This study aimed to assess whether lipids with cytotoxicity, including lauric acid derivatives, could be CD36 ligands by conducting an assay in which a fluorescently-labeled peptide containing human CD36149-168 was utilized. The assay results indicated that laurylamine and 1-monolaurin could bind to CD36149-168. Conversely, no evidence was obtained for non-cytotoxic derivatives (e.g., lauryl mercaptan) interacting with the CD36 segment. Other cytotoxic fatty amines (e.g., myristylamine) and 1-monoglycerides (e.g., 1-monocaprin) were also shown to be recognized by CD36149-168. Identification of these lipids offers an additional category for CD36 ligands, namely, "cytotoxic lipids," which further supports the role of this receptor as a multipurpose player.
Myocardial ischemia/reperfusion (I/R) injury can lead to substantial cardiac damage and pose a significant threat to human health and survival. Micheliolide (MCL), a sesquiterpene lactone extracted from Michelia (Magnoliaceae), has been reported to exhibit anti-inflammatory, anti-fibrotic and anti-oxidant properties. However, the potential regulatory role of MCL in the progression of myocardial I/R injury remains unclear. In this study, we demonstrated that MCL improved cell viability in H9c2 cells subjected to oxygen-glucose deprivation/reoxygenation (OGD/R). Furthermore, MCL treatment significantly attenuated the inflammatory response induced by OGD/R in H9c2 cells. OGD/R stimulation markedly increased reactive oxygen species (ROS) production, which was effectively reduced by MCL administration. In addition, MCL inhibited the elevated apoptosis observed in OGD/R-treated H9c2 cells. Mechanistically, MCL suppressed the activation of the NF-κB signaling pathway following OGD/R exposure. In summary, this study provides the first evidence that MCL mitigates OGD/R-induced inflammation and oxidative stress in H9c2 cells, suggesting its potential as a therapeutic candidate for the treatment of myocardial I/R injury.
