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Purpose: This study aimed to investigate the relationship between diclofenac sodium ophthalmic solution (DFNa) and corneal epithelial cell damage and to evaluate the preventive effect of rebamipide (RBM) on it. Methods: DFNa, DFNa/preservative-free (PF), or 0.5% chlorobutanol (CB) solution was instilled into the conjunctival sac of a normal rabbit eye, and corneal resistance measurement (using a corneal resistance device [CRD]) was performed 120 min after the end of instillation. Then, fluorescent staining (FL), corneal tissue staining (hematoxylin and eosin [H&E]), and immunostaining (zona occlusion-1) were performed (RBM-untreated group). However, RBM was instilled into the eyes of another group of normal rabbits, followed by each of the solutions; 120 min after the end of instillation, all evaluations were performed for this group (RBM treatment group). Results: Using the CRD method, in the RBM-untreated group, corneal resistance (CR; %) was found to be significantly reduced in DFNa (79.9 ± 19.4%), DFNa/PF (89.1 ± 17.3%), and 0.5% CB (83.8 ± 10.6%). In addition, DFNa and 0.5% CB solutions showed positive staining in the FL staining method. In the H&E staining method, some clear voids were observed in the outermost layer of the cornea using DFNa and 0.5% CB solutions. However, corneal epithelial damage was suppressed in the RBM treatment group. ZO-1 immunostaining in DFNa and 0.5% CB solutions revealed discontinuous localization of ZO-1 at the cell periphery. Conclusions: RBM eye drops were effective in preventing corneal epithelial damage caused by DFNa eye drops, and CB was considered to be the main causative agent of this damage.

Purpose: Sensory nerve terminals are highly distributed in the cornea, and regulate ocular surface sensation and homeostasis in response to various endogenous and exogenous stimuli. However, little is known about mediators regulating the physiological and pathophysiological activities of corneal sensory nerves. The aim of this study was to investigate the presence of cholinergic regulation in sensory nerves in the cornea., Methods: Localization of choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (vAChT) was evaluated using western blotting and immunohistochemical analysis. The synthesis and liberation of acetylcholine from the cornea were assessed using corneal segments pre-incubated with [ 3 H]choline. The responsiveness of corneal neurons and nerves to cholinergic drugs was explored using calcium imaging with primary cultures of trigeminal ganglion neurons and extracellular recording from corneal preparations in guinea pigs., Results: ChAT, but not vAChT, was highly distributed in the corneal epithelium. In corneal segments, [ 3 H] acetylcholine was synthesized from [ 3 H]choline, and was also released in response to electrical stimuli. In cultured corneal neurons, the population sensitive to a transient receptor potential melastatin 8 (TRPM8) agonist exhibited high probability of responding to nicotine in a calcium imaging experiment. The firing frequency of cold-sensitive corneal nerves was increased by the application of nicotine, but diminished by an α4 nicotinic acetylcholine receptor antagonist., Conclusions: The corneal epithelium can synthesize and release acetylcholine. Corneal acetylcholine can excite sensory nerves via nicotinic receptors containing the α4 subunit. Therefore, corneal acetylcholine may be one of the important regulators of corneal nerve activity arranging ocular surface condition and sensation., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Histological analysis is a morphological technique and an effective method for understanding the pathology of rheumatoid arthritis (RA). RA is an inflammatory disease characterized by increased synovial tissue and osteoclasts, angiogenesis, infiltration of inflammatory cells, and pannus formation. These pathologies can be observed in a collagen-induced arthritis model mouse using formaldehyde-fixated paraffin-embedded (FFPE) samples. For the preparation of FFPE samples, the conditions of the fixation and decalcification process significantly affect tissue staining results. Since the lesion sites include bone tissue, a decalcification process is necessary when preparing an FFPE sample. Therefore, selecting an optimal condition for the fixating and decalcifying solution is important. In this chapter, we describe the procedures of preparing paraffin samples, including fixation, decalcification, embedding, and sectioning from the RA model mouse, as well as different staining methods (hematoxylin and eosin, tartrate-resistant acid phosphatase)., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Photosensitivity disorder caused by sunlight, including ultraviolet (UV) rays, often occurs in connective tissue diseases such as lupus erythematosus. In addition, UVA (320-400 nM) and UVB (280-320 nM) trigger the progression of skin inflammation in the patients. Therefore, it is crucial to evaluate skin damage under UV exposure using experimental animals to clarify the relationship between connective tissue disease and photosensitivity disorder. In this chapter, our original protocol for evaluating UVA-dependent skin damage, which is known as photoaging via oxidative stress, is described., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Bone homeostasis depends on the balance between bone deposition and bone resorption, which are mediated by the activity of osteoblasts and osteoclasts, respectively. Blocking osteoclast activity can be a therapeutic strategy in rheumatoid arthritis (RA) to reduce subsequent bone erosion. Therefore, investigating the activity of osteoclasts is essential for understanding the pathology of RA. Bone-resorption pits, which are caused by activated osteoclasts, are significantly increased in RA. Scanning electron microscopic analysis of bone-resorption pits is an effective method for understanding the pathology of RA. This chapter describes the method for observing the surface microstructure of pit formation on bone slices., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Aim: Molecular hydrogen is not only expected to be used as an energy-generating resource, but also to have preventive effects on a variety of clinical manifestations related to oxidative stress through scavenging radicals or regulating gene expression. In the current study, we investigated the influence of intermittent environmental exposure to hydrogen gas at a safe concentration (1.3%) on photoaging using an ultraviolet A (UVA)-irradiated murine model., Methods: To mimic the expected human daily activity cycle, UVA exposure in the daytime and hydrogen exposure in the night-time, an original design, UVA-transmission, hydrogen-exposure system was established. Mice were bred under experimental conditions of UVA irradiation and normal air for 8 h (outdoor time 09.00-17.00 hours), and UVA non-irradiation and inhalation of hydrogen gas for 16 h (indoor time 17.00-09.00 hours), and the daily cycle was continued for up to 6 weeks. The progression of photoaging, including morphological changes, collagen degradation and UVA-related DNA damage, was evaluated., Results: Intermittent administration of hydrogen gas by our system prevented UVA-induced epidermal signs, such as hyperplasia, melanogenesis and appearance of senescence cells, and UVA-induced dermal signs, such as collagen degradation. In addition, we detected attenuation of DNA damage in the hydrogen exposure group as indirect evidence that intermittent exposure to hydrogen gas reduced oxidative stress., Conclusions: Our findings support the notion that long-term, intermittent environmental exposure to hydrogen gas in daily life has a beneficial effect on UVA-induced photoaging. Geriatr Gerontol Int 2023; 23: 304-312., (© 2023 Japan Geriatrics Society.)

Trehalose is the nonreducing disaccharide of glucose, evolutionarily conserved in invertebrates. The living skin equivalent (LSE) is an organotypic coculture containing keratinocytes cultivated on fibroblast-populated dermal substitutes. We demonstrated that human primary fibroblasts treated with highly concentrated trehalose promote significantly extensive spread of the epidermal layer of LSE without any deleterious effects. The RNA-seq analysis of trehalose-treated 2D and 3D fibroblasts at early time points revealed the involvement of the CDKN1A pathway, the knockdown of which significantly suppressed the upregulation of DPT, ANGPT2, VEGFA, EREG, and FGF2. The trehalose-treated fibroblasts were positive for senescence-associated β-galactosidase. Finally, transplantation of the dermal substitute with trehalose-treated fibroblasts accelerated wound closure and increased capillary formation significantly in the experimental mouse wounds in vivo, which was canceled by the CDKN1A knockdown. These data indicate that high-concentration trehalose can induce the senescence-like state in fibroblasts via CDKN1A/p21, which may be therapeutically useful for optimal wound repair., (© 2023. The Author(s).)

Recent clinical studies indicate that dry eye is closely associated with psychiatric disorders such as depression and anxiety. Here, we investigated whether two types of mouse dry eye models showed depressive-like behavior in forced swim and sucrose preference tests, and whether voluntary wheel-running helped ameliorate depressive states. To reproduce the dry eye models, the exorbital lacrimal glands (ELG) or exorbital and intraorbital lacrimal glands (ELG+ILG) were bilaterally excised from male C57BL/6J mice. Tear volume was persistently reduced in both models, but the ELG+ILG excision mice exhibited more severe corneal damage than the ELG excision mice. In the forced swim and sucrose preference tests, the gland excision mice showed longer immobility and shorter climbing times, and lower sucrose preference than sham-operated mice, respectively, which appeared earlier in the ELG+ILG excision mice. Wheel-running activities were significantly lower in the ELG+ILG excision mice, but not in the ELG excision mice. After short-period wheel-running, the longer immobility times and the shorter climbing times in the forced swim completely disappeared in both models. Our results suggest that dry eyes might directly cause a depressive disorder that depends on the severity and duration of the ocular surface damage, and that voluntary motor activity could help recovery from a depressive state induced by dry eye., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Nakano, Nakazawa, He, Uwada, Kiyoi, Ishibashi and Masuoka.)

The long-term effect of changes in maternal dietary composition during pregnancy on the offspring's metabolic homeostasis is uncertain. We aimed to investigate the long-term effects of maternal balanced low-fat interventions on metabolic homeostasis of the offspring using a mouse model of gestational obesity induced by a high-fat diet (HFD). Male newborns in the balanced low-fat intervention group had significantly lower serum insulin and higher serum adiponectin levels than those in the HFD group. Changes in maternal dietary composition improved glucose tolerance in pups at 3 and 12 weeks of age. We also performed transcriptomic analysis of the liver in neonatal and 3-week-old pups. Genes in the peroxisome proliferator-activated receptor signaling pathway were significantly down-regulated in neonates in the balanced low-fat intervention group compared with the HFD group. A maternal balanced low-fat diet fully compensated for the detrimental effects of a maternal HFD on glucose metabolism, insulin tolerance, circulating insulin, dyslipidemia, and body weight gain in male offspring by changing the gene expression profile. These data suggest that maternal balanced low-fat intervention is critical for improving the metabolic health of future generations., (Copyright © 2022. Published by Elsevier Inc.)

Aim: Diabetes confers a high risk of developing poor health in later life in women. Based on the Developmental Origins of Health and Disease theory, the present study was undertaken to investigate the efficacy of perinatal fat restriction in maternal high-fat-exposed female offspring to maintain glucose homeostasis in later life between adulthood and aging., Methods: Low-fat dietary intervention during either gestation or lactation was performed using a high-fat diet-induced maternal obesity mouse model (HFD mice). Physiological metabolic parameters, including body weight and serum levels of total cholesterol and triglycerides, were monitored. Glucose tolerance test and insulin sensitivity test were performed in 12- and 70-week-old offspring. Insulin-positive islet cells were also observed using immunohistochemical staining., Results: HFD significantly induced abnormal weight gain, hyperlipidemia and impairment of both glucose tolerance and insulin sensitivity in offspring. Standard diet intake after weaning improved weight gain, serum total cholesterol level and glucose tolerance, but not insulin sensitivity, in 70-week-old offspring. Only perinatal fat restriction during both gestation and lactation, followed by standard food intake for the rest of their life, provided adequate efficacy to restore insulin sensitivity in aging female progeny., Conclusions: Perinatal low-fat intervention may prevent deterioration of glucose metabolism. To improve the health status over a female's lifespan, appropriate nutritional intervention during the early developmental stage may reset the disease trajectory and prevent the onset and development of diabetes. Geriatr Gerontol Int 2022; 22: 441-448., (© 2022 Japan Geriatrics Society.)

Background: Prostate-specific membrane antigen (PSMA) is highly expressed in poorly differentiated, metastatic, and castration-resistant prostate cancers. Recently, 68Ga-PSMA positron emission tomography/computed tomography has been successfully developed as an effective diagnostic tool for prostate cancer. However, the pathophysiological functions of PSMA in prostate tumors remain unclear., Methods: We examined the protein expression of PSMA in tumor endothelial cells in human prostate tumors by immunohistochemistry. Prostate cancer tissues were resected by robotic surgery in 2019 at Ehime University from patients with prostate cancer. In vitro, we prepared conditioned medium (CM) derived from a PSMA-positive human prostate cancer cell line, LNCaP, cultured on collagen I gels. We then examined PSMA expression in human umbilical vascular endothelial cells (HUVECs) cultured with the CM. We assessed angiogenic activities by treatment of HUVECs with LNCaP-derived CM using a tube formation assay that mimics angiogenesis., Results: Immunohistochemistry of PSMA and CD31, a marker of endothelial cells, and PSMA-expressing tumor endothelial cells were observed in 4 of 33 prostate cancer patients (12.1%). We also found that the 10,000g pellet fraction of the LNCaP-derived CM containing PSMA-positive membranes, such as microvesicles transformed HUVECs "PSMA-negative" into "PSMA-positive." Furthermore, treatment of HUVECs with the 10,000g pellet fraction of the LNCaP-derived CM significantly promoted tube formation, mimicking angiogenesis in a PSMA-dependent manner., Conclusions: Our findings revealed the existence of PSMA-positive tumor endothelial cells in human prostate tumors, which enhances tumor angiogenesis in prostate cancer tissues., (© 2021 The Authors. The Prostate published by Wiley Periodicals LLC.)

Although Bruton's tyrosine kinase (BTK) has been recognized as a validated drug target for the treatment of B-cell malignances, the emergence of clinical resistance to the first-generation covalent BTK inhibitors is becoming a serious concern. As a part of our effort to develop noncovalent BTK inhibitors, a series of novel pyrrolopyrimidines was identified as noncovalent inhibitors of both the wild-type and C481S mutant BTKs. Subsequent lead optimization led to the identification of an orally available, potent, and selective BTK inhibitor 13f (AS-1763) as a next-generation noncovalent BTK inhibitor. With significant efficacies in vivo tumor xenograft models, AS-1763 has advanced to phase 1 clinical trials.

The epidermal growth factor receptor is the only available tyrosine kinase molecular target for treating oral cancer. To improve the prognosis of tongue squamous cell carcinoma (TSCC) patients, a novel molecular target for tyrosine kinases is thus needed. We examined the expression of interleukin-2-inducible T-cell kinase (ITK) using immunohistochemistry, and the biological function of ITK was investigated using biochemical, phosphoproteomic, and metabolomic analyses. We found that ITK is overexpressed in TSCC patients with poor outcomes. The proliferation of oral cancer cell lines expressing ITK via transfection exhibited significant increases in three-dimensional culture assays and murine inoculation models with athymic male nude mice as compared with mock control cells. Suppressing the kinase activity using chemical inhibitors significantly reduced the increase in cell growth induced by ITK expression. Phosphoproteomic analyses revealed that ITK expression triggered phosphorylation of a novel tyrosine residue in trifunctional purine biosynthetic protein adenosine-3, an enzyme in the purine biosynthesis pathway. A significant increase in de novo biosynthesis of purines was observed in cells expressing ITK, which was abolished by the ITK inhibitor. ITK thus represents a potentially useful target for treating TSCC through modulation of purine biosynthesis.

Epidermal growth factor receptor (EGFR) and human EGFR 2 (HER2) phosphorylation drives HER2-positive breast cancer cell proliferation. Enforced activation of phosphatases for those receptors could be a therapeutic option for HER2-positive breast cancers. Here, we report that degradation of an endosomal small GTPase, RhoB, by the ubiquitin ligase complex cullin-3 (CUL3)/KCTD10 is essential for both EGFR and HER2 phosphorylation in HER2-positive breast cancer cells. Using human protein arrays produced in a wheat cell-free protein synthesis system, RhoB-GTP, and protein tyrosine phosphatase receptor type H (PTPRH) were identified as interacting proteins of connector enhancer of kinase suppressor of Ras1 (CNKSR1). Mechanistically, constitutive degradation of RhoB, which is mediated by the CUL3/KCTD10 E3 complex, enabled CNKSR1 to interact with PTPRH at the plasma membrane resulting in inactivation of EGFR phosphatase activity. Depletion of CUL3 or KCTD10 led to the accumulation of RhoB-GTP at the plasma membrane followed by its interaction with CNKSR1, which released activated PTPRH from CNKSR1. This study suggests a mechanism of PTPRH activation through the exclusive binding of RhoB-GTP to CNKSR1., (© 2021 Nishiyama et al.)

Mast cell (MC) exocytosis is organized by prenylated protein, including Rab families. Among Rab proteins, Rab3a, Rab27a, and Rab11 are responsible for exocytosis arrangement. Rab3a and Rab27a are contributed to exocytosis by interacting with other exocytosis proteins. Zoledronate administration disrupted the Rab prenylation process that affected its interaction with other proteins, and finally, its function. The present study has investigated the effect of zoledronate on the histamine release (HR) from RBL-2H3 cells. The main focus is to answer the question of whether zoledronate affects Rab27a/Doc2a interaction. Histamine release on RBL-2H3 cells after zoledronate or clodronate administration was measured using HPLC-fluorometry. Dinitrophenylated bovine serum albumin (DNP-BSA) (20 ng/mL) or ionomycin (1 µM) are used as secretagogues. Calcium (Ca 2+ ) influx observation was performed using Fura-2A/M. In situ proximity ligation assay (PLA) is used to investigate Rab27a/Doc2a interaction after bisphosphonates (BPs) treatment. Histamine concentration measurement with HPLC-fluorometry showed that zoledronate (30, 100 µM) inhibited HR from antigen-activated RBL-2H3 cells. Zoledronate showed less inhibition in cells activated with ionomycin. Intracellular Ca 2+ concentration and Ca 2+ flux rate from the extracellular compartment was not changed by zoledronate administration. No changes in Rab27a/Doc2a interaction after zoledronate treatment. Histamine release inhibition by zoledronate in DNP-BSA-activated RBL-2H3 cells is not related to the disruption of Rab27a/Doc2a interaction and is not involve the change in Ca 2+ influx.

The incidence of colon cancer increased worldwide in 2019 and its treatment is urgent from a quality of life perspective. A relationship has been reported between elevated numbers of tumor-associated macrophages (TAMs) in the tumor microenvironment and a poor prognosis in cancer patients, and M2 TAMs have been shown to promote tumor growth by immunosuppression through the stimulation of programmed death-1 (PD-1, an immune check point receptor), interleukin (IL)-1β, and monocyte chemoattractant protein (MCP)-1. We herein examined the effects of three synthetic dihydroxystilbenes (2,3-, 3,4-, and 4,4'-dihydroxystilbenes) on colon carcinogenesis, colon tumor growth, and colon cytokines (IL-1β, IL-6, and tumor necrosis factor (TNF)-α), a chemokine (MCP-1), vascular endothelial growth factor (VEGF), and PD-1 levels in azoxymethane (AOM) plus dextran sulfate sodium (DSS)-treated C57BL/6J mice. The three dihydroxystilbenes inhibited colon carcinogenesis and tumor growth as well as increases in colon IL-1β, IL-6, MCP-1, and PD-1 levels in AOM/DDS-treated mice (in vivo). The three dihydroxystilbenes also suppressed COX-2 expression in colon tumors (in vivo). The results obtained also revealed that the three dihydroxystilbenes inhibited PD-1 elevations in M2-THP-1 macrophages (in vitro). Therefore, the inhibition of AOM/DSS-induced colon carcinogenesis and colon tumor growth by 2,3-, 3,4-, and 4,4'-dihydroxystilbenes appears to be due to the suppression of M2 TAM differentiation and activation and PD-1 expression (immunosuppression) via reductions in COX-2 expression levels in the colon tumor microenvironment., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Objective: Estrogen deficiency caused by bilateral ovariectomy (OVX) has been reported to lead to morphological changes in otoconia. Thus, we examined the morphological changes in the otoconial layer after OVX. We also investigated whether micro-computed tomography (µCT) is useful for the detection of morphological changes in the otoconial layer., Methods: The otic capsules of C57BL/6 J mice were removed and evaluated using histological techniques and µCT at 2, 4, and 8 weeks after OVX or sham surgery. The volume of the utricle otoconial layer was measured and compared between the OVX and sham groups. The µCT scan and histological study results were also compared., Results: The volume of the utricle otoconial layer was significantly increased 4 weeks after OVX compared to the sham group in both histological and µCT studies (p < 0.05). The volume of the otoconial layer measured using µCT was significantly correlated with the histological study results (p < 0.05)., Conclusion: The volume of the utricle otoconial layer increased after OVX. These morphological changes could be detected by µCT., Competing Interests: Declaration of Competing Interest None declared., (Copyright © 2020. Published by Elsevier B.V.)