Ocular diseases that result in blindness impair the QOL of patients as well as cause significant socioeconomic losses. Glaucoma is the leading cause of blindness, followed by retinitis pigmentosa, diabetic retinopathy, and age-related macular degeneration (AMD). Although the pathogeneses of these ocular diseases differ, they are all retinal diseases that lead to blindness owing to progressive retinal damage. However, the mechanisms underlying the pathogenesis and progression of these diseases have not yet been fully elucidated. In addition, treatment methods for these diseases have not yet been fully established, and their pathophysiology should be elucidated to establish novel treatment methods and drugs. Rodent pathological models, particularly mice and rats, have been established to elucidate the pathogenesis of these diseases. However, anatomical differences between the eyes of humans and rodents suggest that differences in pathogenic mechanisms may exist. In addition, species differences in drug responsiveness have become an issue in various respects, making it increasingly difficult to directly extrapolate the results obtained in rodents to humans. Therefore, evaluations using non-human primates, which are physiologically similar to humans, are required. This review outlines the basic research of AMD and glaucoma models using mice and non-human primates and their therapeutic strategies, focusing on the research findings.

The mucosal layer in various mucosal tissues acts as a barrier that protects the epithelial membrane from foreign substances. However, in the process of mucosal absorption of drugs, the mucus layer, a smart biological sieve to particles and molecules, can be an obstacle to effective drug delivery. Recently, functional nanoparticles (NPs) have attracted considerable interest in the field of biopharmaceutical science owing to their delivery potential and effectiveness. Among various pharmaceutical technologies, mucopenetrating NPs (MPP) and mucoadhesive NPs (MAP) are viable dosage options for controlling pharmacokinetic behavior by modifying drug absorption from the mucosal site. MPP and MAP can rapidly deliver encapsulated drugs to the absorption site by passing through the mucus layer and/or retaining NPs near the absorption membrane, possibly resulting in better drug delivery than that of conventional approaches. Modifying the surface properties of NPs is critical for determining their potential diffusiveness within the mucus layer owing to various types of interactions between the mucosal components and the surface of NPs. Additionally, the physiological characteristics of the mucus layer (thickness, viscosity, and turnover time) differ depending on the mucosal site. Thus, a deeper understanding of the design of NPs and the functional properties of the administration site is essential for developing mucosal drug delivery systems (mDDS) to maximize the potential of target drugs. This review summarizes the basic information and functions of the mucosal layer, highlights the recent progress in designing functional NPs for mDDS, and discusses the advantages and disadvantages of mucosal administration at major mucosal sites.

Cannabinoid receptor type 1 (CB1R) plays a key role in neuronal homeostasis, synaptic plasticity, and neuroprotection. CB1R antagonists typically protect against CB1R agonists-induced neurotoxicity. However, we previously found that the CB1R antagonists rimonabant and its analog AM251 can also be neurotoxic: under serum-free conditions, these compounds induce apoptosis in human neuroblastoma SH-SY5Y cells through mitochondrial damage and endoplasmic reticulum (ER) stress. To elucidate the mechanisms of this neurotoxicity, we examined the effects of CB1R agonists. We co-treated SH-SY5Y cells with rimonabant or AM251 in combination with either the CB1R agonist arachidonyl 2-chloroethylamide (ACEA) or WIN 55212-2 mesylate (WIN). ACEA, but not WIN, protected cells from rimonabant- and AM251-induced apoptosis. While ACEA had only a limited effect on mitochondrial damage, it significantly reduced phosphorylation of the eukaryotic initiation factor 2 alpha (eIF2α), a key marker of ER stress. Given that ACEA also functions as an agonist of transient receptor potential vanilloid 1 (TRPV1), we investigated its role in ACEA-mediated neuroprotection. The TRPV1 antagonist capsazepine blocked ACEA’s protective effects, suggesting that ACEA acts through TRPV1 rather than CB1R. ACEA also prevented apoptosis induced by camptothecin, a well-established apoptosis inducer, through a similar capsazepine-sensitive mechanism, demonstrating its broader protective effects against apoptosis. These findings indicate that rimonabant and AM251 induce neurotoxicity independently of CB1R under serum-free conditions and that ER stress is likely to be a key target of CB1R-independent neuroprotection by ACEA. Our study highlights the complexity of CB1R ligand-associated neurotoxicity and neuroprotection.

In the hospital settings, performing clinical trials is an intricate process that is generally hampered by several institutional, technical, and record-keeping challenges. Hence, we evaluated the effectiveness and efficiency of our established integrated clinical trial management system (CTMS) in terms of analyzing core functionalities, assessing integration with existing systems, measuring time, and cost efficiency. Our CTMS (version 10.1, August 2024) is integrated into one of the largest teaching hospitals with the human research audit system, biobank management system, biological sample management system, enterprise resource system service, institutional review, and single sign-on system. The total number of trials, subjects enrolled, products developed, investigators, new drug and indication, devices, and new medical technology are found to be 913, 53969, 851, 159, 784, and 98, respectively, with a total budget of 3539846777 New Taiwan Dollar (approx. 106881045 US$). Our CTMS is efficient in updating data, with improved user interface experience, and controlled access according to the defined policy. Integrating CTMS with other components provides effective tracking and monitoring of the clinical study. Conclusively, our integrated CTMS is designed for comprehensive evaluation and supervision of clinical trials, supporting full-process data management and seamless integration with clinical systems of hospitals through a unified interface. The increasing number of trials, subjects enrolled, products developed, investigators, new drugs and indications, devices, and new medical technology indicates its robustness and efficacy.

Light stress is a risk factor leading to retinal diseases such as age-related macular degeneration. However, the mechanism underlying the stress response to light in the retina has yet to be elucidated. We have reported that exposure to blue light-emitting diode light induces excessive production of reactive oxygen species and activates the unfolded protein response, robustly increasing activating transcription factor 4 (ATF4) expression. These processes result in photoreceptor cell death. This study investigates the effects of Pentadecyl, a bioactive product obtained from Aurantiochytrium limacinum, on either chemical-induced or blue light-induced endoplasmic reticulum (ER) stress. Pentadecyl suppressed cell death induced by either thapsigargin or tunicamycin in a concentration-dependent manner. Pentadecyl also suppressed the expression of unfolded protein response target genes, including Atf4 and ER chaperones. Consistently, immunoblotting revealed that Pentadecyl suppressed the increased expression of ATF4 at the protein level. Pentadecyl also protected 661W cells from blue light-induced damage but did not protect against hydrogen peroxide (H₂O₂)-induced oxidative stress. These results indicated that Pentadecyl has a novel function that protects against ER stress induced by photodamage.

Oversulfated chondroitin sulfate (OSCS) is a chondroitin sulfate ester in which all hydroxyl groups have been converted to sulfuric acid esters. Here, we describe a rapid, novel synthesis method for OSCS. ¹H-NMR analysis revealed that all signals derived from the protons of N-acetyl-galactosamine and glucuronic acid in the synthesized OSCS were shifted downfield due to sulfonation compared with the chondroitin sulfate (CS) starting material. Comparison of the edited heteronuclear single quantum correlation spectrum of the prepared OSCS with that of the Japanese Pharmacopoeia OSCS reference standard showed excellent agreement between the main correlation peaks. The proposed novel synthesis method for OSCS is faster and simpler than the conventional method.

Werner helicase-interacting protein 1 (WRNIP1) is a member of the AAA⁺ ATPase family and is conserved from Escherichia coli to Homo sapiens. Although a role for WRNIP1 in the S phase of the cell cycle has previously been reported, the present study demonstrates a novel function for this protein in G₁ phase. Deletion of WRNIP1 in non-homologous end joining (NHEJ)-deficient chicken DT40 cell lines resulted in slow growth, accumulation in G₁ phase, an increased population of dead cells, and accumulation of double-strand breaks (DSBs), which were indirectly evaluated by γH2AX. The data suggest that WRNIP1 prevents the generation of DSBs, executes DSB repair, and/or regulates DSB repair independently of NHEJ, particularly in G₁ phase. The potential molecular mechanism by which WRNIP1 functions in G₁ phase is discussed.

Transplantation of adipose-derived mesenchymal stromal/stem cells (ASCs) has successfully alleviated the severity of psoriasis. Although several therapeutic mechanisms of mesenchymal stromal/stem cells (MSCs) for psoriasis have been elucidated using the imiquimod (IMQ)-induced psoriasis-like dermatitis model, the effects of MSC transplantation on pathways other than the interleukin (IL)-23/T helper 17 (Th17) axis, including the IL-36 pathway, remain unclear. In this study, we aimed to investigate the efficacy of ASC transplantation for the IMQ-induced psoriasis-like dermatitis in male C57BL/6J mice, and to elucidate its effects on the IL-36 pathway as well as the IL23/Th17 axis. ASCs (2.0 × 10⁶ cells) from mouse inguinal white adipose tissue were subcutaneously injected into the dorsal skin of mice. After the topical application of IMQ cream for 5 consecutive days, objective severity scores, cytokine gene expression levels, and neutrophil infiltration grade were determined to evaluate their efficacy. Anti-IL-23p19 antibody treatment was used for comparison. ASCs slightly ameliorated IMQ-induced epidermal thickening, although anti-IL-23p19 antibodies had no effect on any skin manifestations. Anti-IL-23p19 antibody and ASC suppressed the expressions of Il17a, Il17f, and Il22 mRNAs and neutrophil infiltration in IMQ-applied skin, but not the expression of Il1f6 and Il1f9. ASC also suppressed the expressions of Il23, Il6, Il1b, Tnfa, Lipocalin-2, and Cxcl5 mRNAs, which were not suppressed by anti-IL-23p19 antibody treatment. In conclusion, ASC transplantation suppressed activation of the IL-23/Th17 axis and neutrophil infiltration, and inhibited the activation of a broader range of inflammatory mediators except for IL-36 expression in IMQ-applied skin compared with anti-IL-23p19 antibody treatment.

In rodents, the whiskers and paws are essential for somatosensory perception, yet their preference for specific textures remains unclear. Here, we examined the preference for rough versus smooth surfaces in rats using a custom open-field paradigm with a water-based motivator to ensure consistent and unbiased movement. We found that rats strongly preferred rough textures with longer interactions and increased dwell time. Notably, this preference persisted in darkness, confirming the tactile, but not visual, influence. These findings highlight the primacy of tactile input in behavioral choices and provide key insights into optimizing rodent environments.

Glutathione S-transferases (GSTs) are essential phase II detoxification enzymes encoded by a diverse gene superfamily. Among them, the pi class (GSTP) includes 2 isozymes, GSTP1 and GSTP2, which share a high degree of sequence similarity. In mice, hepatic GSTP1/2 expression is higher in males than in females. To investigate the regulatory mechanisms underlying this sex difference, we examined orchiectomized mice treated with testosterone. Orchiectomy reduced hepatic GSTP1/2 expression and associated enzyme activity, both of which were restored following testosterone administration. To assess the sensitivity of GSTP1/2 to testosterone fluctuations, we compared mice experiencing a serum testosterone surge with those maintaining baseline levels. Mice with elevated testosterone exhibited increased hepatic GSTP1/2 protein expression and enzyme activity, demonstrating the high responsiveness of GSTP1/2 to testosterone. To our knowledge, this is the first study to demonstrate that testosterone surges regulate both the expression and enzymatic function of a specific protein. These findings underscore testosterone’s critical role in the male-dominant expression of GSTP1/2 and highlight its sensitivity to physiological fluctuations in testosterone levels. Further studies are warranted to elucidate the molecular mechanisms by which testosterone surges influence gene expression.

Mechanical hyperalgesia is commonly evaluated using von Frey filaments (vFFs) in the rodent hind paw plantar. However, it is difficult to select the plantar location to stimulate with the vFFs. In the present study, we investigated the effective location of the plantar surface for the evaluation of mechanical hyperalgesia using vFFs in mice treated with paclitaxel (PTX). PTX or vehicle was injected intraperitoneally once daily, 4 times every other day. On Day 14, mechanical hyperalgesia was evaluated using vFFs. The evaluation was performed by stimulation with vFFs on the skin directly under the 3rd and 4th paw pads (base of the index and middle fingers) from closest to the heel on the thumb side, in the central skin surrounded by the pads, and in the plantar skin one-third of the way down from the heel toward the toe. In comparison to vehicle-treated mice, mechanical hyperalgesia was significantly observed in the skin directly under the 3rd and 4th paw pads counted from closest to the heel, and in the plantar skin one-third of the way down from the heel toward the toe. However, this was not significant in the central skin surrounded by the pads. Interestingly, in vehicle-treated mice, the paw withdrawal threshold varied for each location evaluated. In contrast, in PTX-treated mice, the thresholds were similar across all evaluated locations. These results suggest that the selection of the location on the plantar surface to be stimulated is important for pain evaluation using vFFs.

The high cost and limited scalability of monoclonal antibody (mAb) production necessitate the development of alternative systems. Transgenic chickens offer a promising platform for recombinant mAb production; however, efficient purification methods remain underexplored. This study investigated the initial purification of recombinant mAbs from transgenic chicken egg whites. We utilized Protein A chromatography, followed by ammonium sulfate precipitation and cation-exchange chromatography, to improve the purification efficiency. Although Protein A chromatography was able to purify mAbs, there was substantial egg-white contamination. Ammonium sulfate precipitation and cation-exchange chromatography successfully removed 84.6 and 93.8% of egg-white proteins, respectively, enhancing mAb purification efficiency. Overall, this study proposes an efficient method for initial mAb purification from transgenic chicken egg whites, providing a basis to develop a scalable, cost-effective biopharmaceutical production approach.

Spontaneous walking behavior is modulated by various factors, but it remains almost unknown whether and how moisturizing paw soles of rats alters their gait. To this end, we allowed rats to spontaneously walk on a disk-shaped treadmill before and after their paw soles were coated with white petrolatum (vaseline) and monitored the rat’s walking. Using DeepLabCut- and DeepEthogram-based pipelines offline, we calculated the shoulder-elbow-wrist angle and defined the distance as the trajectory of the wrist. The distance was shorter in the stance phase but longer in the swing phase after vaseline was treated than before. The duration of the stance and swing phases was shorter in the vaseline-treated period. The angle at forepaw landing and takeoff was smaller and larger, respectively, in the vaseline period. These findings underscore the dominance of moisturizing sole skins over the well-organized dynamics of a spontaneous gait.

Wenweishu (WWS), a traditional gastritis formula, was studied to elucidate its mechanisms in preventing water immersion restraint stress-induced gastric ulcers (GU) in rats. The degree of gastric tissue damage was assessed in experimental rats based on the gastric mucosal ulcer index and pathological observation. The antioxidant properties of malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-px) were detected in gastric tissues, along with expression levels of inflammatory factors, such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6. The core components, primary targets, and putative mechanisms of WWS were predicted using network pharmacology and molecular docking and validated via immunohistochemistry and immunoblotting analyses. WWS reduced the ulcer index, increased SOD and GSH-px, and decreased IL-1β, IL-6, TNF-α, and MDA levels. A total of 126 WWS components were identified and associated with 4920 GU-associated targets via network pharmacological analysis. Data from Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed associations of the core targets with multiple pathways, in particular, phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling. Molecular docking analysis confirmed significant docking activity of the main bioactive components with the core targets tumor protein (TP53) and protein kinase Bα (AKT1). Immunohistochemistry and Western blot analyses showed that WWS markedly suppressed phosphorylation levels of Akt and PI3K proteins in gastric tissues. WWS exerts a protective effect on the gastric mucosa by inhibiting the PI3K/Akt pathway, attenuating inflammatory factors and oxidative damage. The collective findings provide a scientific basis for the application of WWS in the management of GU.

Denosumab is used to reduce skeletal-related events such as fractures in cancer patients with bone metastasis, but may cause severe hypocalcemia. We previously developed and updated a risk prediction model for ≥ grade 2 denosumab-induced hypocalcemia from a hospital-based administrative database and clinical datasets from two facilities. The final risk-scoring system using only calcium, albumin, and alkaline phosphatase levels provided high performance. Here, we aimed to externally validate the scoring system’s performance using an independent clinical dataset from Gunma Prefectural Cancer Center. Clinical data (May 2017–November 2023) were retrospectively collected and the discriminant performance of the previously developed model (sensitivity, specificity, positive predictive value, negative predictive value and receiver operating characteristic-area under the curve (ROC-AUC)) was evaluated. For 161 cases analyzed, the model demonstrated a sensitivity of 85.7%, specificity of 72.1%, positive predictive value of 12.2%, and negative predictive value of 99.1%. ROC-AUC was 0.813. All performance parameters were comparable to those in the previous study. The results strongly support the generalizability of the scoring system. This straightforward, easily interpretable, high-performance risk prediction system is expected to enhance the safety of denosumab treatment.

Loss-of-function mutations in Janus kinase 1/2 (JAK1/2) cause low tumor immunogenicity through defects in the induction of the transcription factor interferon regulatory factor 1 (IRF1), resulting in non-responsiveness to cancer immunotherapy reagents. Therefore, the discovery of reagents that increase IRF1 expression independent of JAK activity is clinically important for the success of cancer immunotherapy reagents. We herein demonstrated that proteasome inhibitors activated IRF1 downstream pathways in a JAK-independent manner in various cancer types. Proteasome inhibitors increased IRF1 expression by inhibiting the degradation of IRF1 in melanoma. Furthermore, proteasome inhibitors induced the expression of the IRF1 downstream molecules, programmed death-ligand 1 (PD-L1), PD-L2, and human leukocyte antigen class I molecules. The induction of IRF1 expression by proteasome inhibitors was also detected in cancer types other than melanoma. Moreover, we showed that the induction of IRF1 expression was independent of JAK activity by genetic or chemical inhibition of JAK. Therefore, proteasome inhibitors may serve as adjuvants that potentiate the efficacy of cancer immunotherapy reagents by enhancing cancer immunogenicity.

Butyrate exerts strong anti-colorectal cancer effects via epigenetic regulation. However, whether butyrate has a negative impact on colorectal cancer in vivo remains unclear. Therefore, this study aimed to investigate whether butyric acid can regulate the growth of colorectal cancer cells using spheroids as an in vivo model. A three-dimensional (3D) culture system was used to form spheroids from the human colorectal cancer cell line HT-29. Spheroids formed in the 3D culture system exhibited some malignant cancer phenotypes (increased cancer stem cell marker levels, anticancer drug resistance, and G0/G1 phase accumulation in the cell cycle) compared with cells in the two-dimensional cell culture system. Sodium butyrate (SB) treatment suppressed cancer stem cell marker levels in spheroids and induced differentiation by increasing the alkaline phosphatase activity. Additionally, SB-induced changes in genes related to cell cycle control indicated that SB reactivated the cell cycle in spheroids, regulating the transition from the G0 to G1 phase. Furthermore, SB treatment induced cell death via apoptosis, which might be due to G1 arrest in the cell cycle. In conclusion, SB induced the differentiation and subsequent cell death of HT-29-derived cancer cells in spheroids, highlighting its potential as a novel anticancer agent for colorectal cancer.

As previously reported, vialinin A, inhibits tumor necrosis factor α (TNF-α) production and extracellular release from RBL-2H3 cells by targeting USP5. In this study, we examined its inhibitory effect and mechanism of action on immunoglobulin E (IgE)-mediated early-phase allergic reactions in RBL-2H3 cells. Vialinin A inhibited β-hexosaminidase release from RBL-2H3 cells and tended to suppress cytosolic calcium ion concentration elevations. Kinase analysis and results from immunoblotting showed that vialinin A inhibited Syk activation and its autophosphorylation. Consequently, the phosphorylation of Syk downstream signalling proteins, such as linker for activation of T cells family and phospholipase Cγ1/2, essential in calcium ion mobilization and cell activation were slightly inhibited. These results suggest that vialinin A affects degranulation of RBL-2H3 cells by inhibiting Syk.

Inflammation is a key factor that contributes to cartilage degeneration in osteoarthritis (OA). p-Synephrine has anti-inflammatory effects. Nevertheless, the effects of p-synephrine on OA remain to be elucidated. The objective is to explore the effects of p-synephrine on OA. First, cell counting kit-8 (CCK-8) assay and flow cytometry were used to assess the effects of p-synephrine on chondrocyte viability and apoptosis. Then, Western blot and quantitative real time-PCR (qRT-PCR) were employed to determine the expressions of matrix metalloproteinase-1 (MMP-1), MMP-3, and MMP-13, as well as collagen II and aggrecan, in OA chondrocytes induced by interleukin-1β (IL-1β). Furthermore, we created an injectable gelatin methacrylamide (Gelma) hydrogel incorporating p-synephrine and conducted evaluations of its drug release profile and the degradation properties of hydrogels, aiming to optimize the intra-articular application of p-synephrine in the mouse OA model. Finally, cartilage degradation was analyzed using safranine O and fast green staining. In vitro, p-synephrine protected chondrocytes and effectively inhibited IL-1β-induced chondrocyte apoptosis. Moreover, p-synephrine inhibited the expressions of MMP-1, MMP-3, and MMP-13, and increased the expressions of collagen II and aggrecan. p-Synephrine might exert its biological effects by suppressing the mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) signaling pathways. Gelma hydrogels with different degrees of amination could control the release rate of p-synephrine due to differences in their pore structure and degradation rates. Among these, Gelma 90 achieved a more stable and sustained p-synephrine release. In vivo, p-synephrine loaded by injectable Gelma hydrogel thwarted cartilage deterioration. In summary, p-synephrine may exhibit chondroprotective effects by suppressing the MAPK and NF-κB signaling pathways, providing a new treatment for OA.

The purpose of this study was to explore the potential therapeutic effect of sodium glucuronate (SG) on osteoporosis (OP). To achieve this aim, the optimal concentration of SG for stimulating MC3T3-E1 osteoblast cells derived from the calvaria of neonatal mice was determined using cell counting kit-8 and alkaline phosphatase (ALP) activity assays. Osteogenic markers were analyzed by qRT-PCR and Western blotting. The histopathological morphology of the tibial tissues was performed using hematoxylin and eosin staining. The levels of bone turnover markers (BTMs) were assessed using enzyme-linked immunosorbent assay (ELISA). SG treatment was found to effectively promote osteoblastic differentiation and mineralization in MC3T3-E1 cells, evidenced by enhanced ALP activity, increased calcium deposition, and upregulated expression of key osteogenic markers including runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), and osteopontin (OPN). Moreover, in ovariectomized rats, a model of postmenopausal OP, SG treatment significantly promoted bone formation, regulated the levels of BTMs, and augmented bone mineral density. Consistently, SG upregulated the expression of osteogenic genes (RUNX2, OCN, and OPN) in bone tissue, further supporting its osteogenic potential. Collectively, these findings suggest that SG possesses the ability to stimulate bone formation and may hold promise as a potential agent for the management of OP.

Herein, we present the rational design and development of an innovative nanoplatform for synergistic breast cancer therapy through cascade enzymatic reactions, integrating starvation therapy (ST) and photodynamic therapy (PDT). The system is constructed based on biocompatible calcium phosphate (CaP) nanoparticles, which function as nanocarriers for the photosensitizer indocyanine green (ICG), a critical agent for PDT. To overcome the clinical limitations of hypoxia-induced ICG inactivation, we engineered a sequential enzymatic system by co-immobilizing glucose oxidase (GOX) and catalase (CAT) on the CaP surface, followed by encapsulation within a sodium alginate matrix to prevent premature drug degradation. Systematic characterization demonstrates that the as-prepared nanosystem (denoted as ICG/CaP@GOX-CAT@SA) not only maintains excellent biocompatibility but also achieves tumor-specific accumulation through the enhanced permeability and retention effect, subsequently undergoing lysosomal degradation in tumor cells. Mechanistic investigations reveal that the released GOX initiates ST by catalyzing glucose depletion in the tumor microenvironment, while the simultaneously produced hydrogen peroxide is effectively converted to oxygen (O₂) by CAT, thereby ameliorating tumor hypoxia and maintaining sufficient O₂ levels for ICG-mediated PDT. This cascade enzymatic nanoplatform, which orchestrates the sequential reactions of GOX and CAT to synergistically enhance ST and PDT, demonstrates significantly improved anti-tumor efficacy both in vitro and in vivo, offering a promising paradigm for combinatorial cancer therapy.

Circadian rhythms regulate essential physiological functions, including body temperature and hormone secretion, in a 24-h cycle. These rhythms are synchronized with environmental cues, primarily light, through the suprachiasmatic nucleus. Disruptions, such as jet lag, misalign internal rhythms with external time, leading to fatigue and insomnia. This study explores the potential of dietary sweetening agents as non-pharmacological interventions to facilitate circadian re-entrainment in a mouse jet lag model. Male C57BL/6 mice, maintained on a 12-h light/dark cycle, underwent a 6-h phase advance to simulate jet lag. Mice received drinking water with or without sweeteners (sucrose, sucralose, xylitol, maltitol), and locomotor activity was assessed using wheel-running behavior and intraperitoneally implanted nanotags measuring 3dimensional acceleration and body temperature. Sucrose and sucralose significantly accelerated re-entrainment, with phase-shifting rates of 0.93 and 1.28 h/d, respectively, compared to 0.76 h/d in controls. Both sweeteners also enhanced post-shift activity, whereas xylitol had a minor effect and maltitol showed no significant impact. Sweeteners did not affect rest duration during the jet lag period. These findings indicate that sweet taste can facilitate circadian adaptation, offering a potential dietary approach to mitigate jet lag symptoms. This study provides insights into how taste perception influences circadian regulation, with implications for managing circadian misalignment in frequent travelers and shift workers.

Renal proximal tubular injury was reported as a result of Red Yeast Rice supplements intake, with puberulic acid identified as one of the causative agents. Although the compound has shown nephrotoxicity in animal models, the cytotoxicity of this compound on human kidney cells has not been investigated. In this study, we evaluated the cytotoxic effects of puberulic acid using three-dimensional cultured primary human renal proximal tubular epithelial cells (3D-RPTECs). Upon exposure to puberulic acid, the spheroid surface area and mitochondrial fluorescence intensity were reduced. Moreover, a time-dependent decrease in intracellular ATP levels was observed with the EC₅₀ value at 7 d of 24.7 μM. Based on the carboxylic acid structure of puberulic acid, we investigated the effect of probenecid, a non-selective organic anion transporter (OAT) inhibitor, on puberulic acid cytotoxicity. The ATP reduction effect of puberulic acid was partially alleviated by probenecid. Furthermore, the uptake of OAT1 substrate furosemide by OAT1-expressing HEK293 cells was reduced by puberulic acid with an IC₅₀ value of 5.4 μM. These findings suggest that puberulic acid has a highly cytotoxic effect on the proximal tubules.

We examined the contribution of L-type voltage-dependent Ca²⁺ channels (VDCCs) and non-VDCCs to platelet-activating factor (PAF)-induced contractions of rat esophageal smooth muscle (ESM). We also attempted to obtain more detailed information about the non-VDCC molecules involved in the PAF effect. PAF (10⁻⁶ M)-induced contractions were abolished in Ca²⁺-free solution containing ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid and were attenuated by diltiazem (10⁻⁵ M), a VDCC inhibitor. PAF-induced contractions in the presence of diltiazem were inhibited by approx. 50% by LOE-908 (3 × 10⁻⁵ M), an inhibitor of receptor-operated Ca²⁺ channels (ROCCs), and were strongly inhibited by LOE-908 plus SKF-96365 (3 × 10⁻⁵ M), an inhibitor of both ROCCs and store-operated Ca²⁺ channels (SOCCs). The contribution of each Ca²⁺ channel was estimated to be approx. 25% for VDCCs, approx. 30% for ROCCs, and approx. 35% for SOCCs. Among the non-VDCC-related molecular candidates examined in rat ESM, Trpv4, Trpc6, and Trpc3 were abundant ROCC-related mRNAs, and Orai1 was the most abundant SOCC-related mRNAs. However, PAF-induced contractions in the presence of diltiazem were not significantly inhibited by combination treatment with putative inhibitors of transient receptor potential V4 (TRPV4) (GSK 2193874, 3 × 10⁻⁷ M), TRPC6 (SAR7334, 10⁻⁶ M), and TRPC3 (Pyr10, 3 × 10⁻⁵ M). In contrast, PAF-induced contractions in the presence of both diltiazem and LOE-908 were completely inhibited by Synta66 (10⁻⁵ M), an Orai1 inhibitor, which also inhibited PAF-induced contractions by approx. 30% in the absence of Ca²⁺ channel inhibitors. These findings indicate that PAF-induced rat ESM contractions depend on extracellular Ca²⁺ influx through VDCCs, ROCCs, and SOCCs, with Orai1 being the key SOCC molecule.

Small interfering RNAs (siRNAs) hold great therapeutic promise due to their ability to selectively silence disease-associated genes. Although chemically modified siRNAs have demonstrated clinical efficacy, their development remains hindered by challenges such as stability and delivery under physiological conditions. Furthermore, in vitro screening of chemically modified siRNAs using cultured cells is cost-effective but often fails to recapitulate in vivo complexity, limiting predictive accuracy. To address this, we have developed a transfection-free siRNA evaluation platform using Gryllus bimaculatus (Gb), an insect model with natural RNA uptake capacity. We first demonstrated that 21-nucleotide siRNAs induced RNA interference upon abdominal injection under conditions of free uptake. We then generated transgenic lines harboring an EGFP reporter fused to the therapeutic siRNA target sequence and integrated into the β-actin locus via clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9-mediated knock-in. Two transgenic strains (s1 and d1) were established and validated. We compared unmodified and chemically modified siRNAs designed using enhanced stabilization chemistry (ESC), a clinically validated modification pattern incorporating 2′-O-methyl, 2′-fluoro, and phosphorothioate modifications. While ESC-modified siRNAs showed reduced activity compared to unmodified natural siRNAs in conventional cell-based assays requiring transfection reagents, they exhibited consistent gene silencing in Gb, reflecting their enhanced biochemical stability under free uptake conditions at picomole-scale doses. These results establish Gb as a scalable, cost-effective, and biologically relevant platform for evaluating therapeutic siRNAs, particularly those incorporating chemical modifications.