Immunoassays enable the sensitive determination of various compounds and have been widely utilized in pharmaceutical and medical sciences. To develop practical assays, it is essential to obtain antibodies that capture the target analytes with high specificity and affinity. To date, we have generated high-performance antibodies and developed immunoassays for determining bioactive compounds, particularly focusing on haptens, such as steroids and synthetic drugs. In previous studies, we have produced specific anti-hapten antibodies by immunizing animals with reasonably prepared hapten–carrier conjugates. However, the resulting antibodies sometimes lacked sufficient affinity for a sensitive determination. Therefore, we challenged genetic engineering to produce artificially modified antibodies with improved affinity. Therein, native antibodies with insufficient affinities were converted into single-chain Fv fragments (scFvs), to which random point mutations were introduced to generate diverse scFv libraries. Mutated scFv species with increased affinities were selected and isolated with the aid of phage-display system combined with panning. Using this strategy, we obtained scFvs specific to several haptens, such as estradiol-17β (E₂) and cotinine, that show significantly improved affinity (K_a) than that of the parental scFv, enabling more sensitive enzyme-linked immunosorbent assays. However, the panning step often fails in straightforward selection and requires laborious trial-and-error work. Thus, we developed a “clonal array profiling (CAP)” system for more efficient isolation of the mutants with enhanced affinities, which successfully functioned generating multiple anti-cortisol scFvs with the K_a improved up to 63-fold and an anti-E₂ scFv with 372-fold larger K_a. In this study, we identified new strategies that allow for efficient site-directed mutagenesis to improve affinity. We expect that the engineered antibodies described here will open the door to next-generation immunoassays that will enable simpler and more reliable determination of bioactive compounds.

Epstein–Barr virus (EBV) is one of the most pervasive viruses worldwide, and EBV infection is inextricably linked to a multitude of lymphoid and epithelial neoplasms. EBV is responsible for the advancement of malignant disease by modifying the tumor microenvironment (TME), which is a sophisticated and evolving system that facilitates tumor growth, invasion, and metastasis. EBV infection has a profound impact on the cellular and noncellular components that constitute the TME. Our review presents a summary of the composition of the EBV-remodeled TME, with a particular focus on EBV-induced functional phenotypes in non-tumor cells. Furthermore, we discuss the potential for reversing EBV-driven TME remodeling as a therapeutic strategy for treating the malignancies associated with EBV infection.

Herbal medicines are widely used in clinical practice. Several herbal medicines are prescribed in clinical practice to improve mental symptoms. Yokukansan is an effective prescription for irritability and aggression, which are behavioral and psychological symptoms of dementia (BPSD) or autism spectrum disorder (ASD). However, because herbal medicines contain many components, their pharmacological effects have not been analyzed in detail. Risperidone and quetiapine are prescribed in severe cases; however, their side effects of oversedation are problematic. Tokishigyakukagoshuyushokyoto (TSGST) is a herbal medicine prescribed to improve blood circulation and relieve headaches, back pain, or chilblains associated with hemodynamic insufficiency. Interestingly, most of the individual components of TSGST are known to exert sedative or analgesic effects. In this study, we investigated whether TSGST ameliorates aggressive behavior induced by social isolation in mice. The mice were isolated for 5 or 6 weeks immediately after weaning and given TSGST via a water bottle during this period. Long-term administration of TSGST suppressed the onset of aggression induced by isolation rearing. This aggressive phenotype was significantly reversed by intraperitoneal (i.p.) administration of the 5-hydroxytryptamine 1A (5-HT_1A) receptor antagonist WAY-100635 in TSGST-isolated mice. We also showed that TSGST had similar effects as risperidone, a commonly used antipsychotic for irritability and aggression. These results suggest that TSGST may be effective for irritability or aggression in BPSD or ASD.

The rates of risperidone in precipitates from 5 risperidone oral solutions and tea beverages (green, black, and oolong teas) against risperidone used were measured by quantitative (q)NMR, indicating that as the amount of caffeine in the beverages increased, the rate decreased significantly. A solution of caffeine (0, 5, 10, and 30 μmol) in tartaric acid buffer (pH 3.0) was added to a solution of risperidone and (−)-epigallocatechin-3-O-gallate (EGCg) (each 3.0 μmol) in tartaric acid buffer (pH 3.0). Judging from the amounts of risperidone and EGCg in the precipitates, it was considered that risperidone formed a 1 : 1 complex with EGCg. As the amount of caffeine increased, the amounts of risperidone and EGCg in precipitates decreased significantly, suggesting that caffeine has a solubilizing effect on precipitates of the 1 : 1 complex of risperidone and EGCg. Furthermore, the amount of risperidone in precipitates formed when compounds with a xanthine derivative, such as diprophylline, theobromine, and theophylline, were added was less than when they were not added, indicating that such compounds with a xanthine derivative have the same solubilizing effect as caffeine on precipitate of the 1 : 1 complex of risperidone and EGCg.

Astragalus polysaccharide (APS) is a biologically active water-soluble polysaccharide extracted from stems or roots, which has been proven to have antiaging effects. The aim of this study was to investigate the effects of APS on cognitive function in d-galactose (d-gal)-induced aging rats and explore the potential underlying molecular mechanisms. The rats were induced to age by intraperitoneal injection with 400 mg/kg/d d-gal for 8 weeks. Aging of rats was assessed through the Morris water maze test, step-down test, open field test, and grip strength test. Pathological changes in the hippocampal CA3 and CA1 regions were determined by Hematoxylin and eosin and Nissl staining. The superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), malondialdehyde (MDA), and total antioxidant capacity (T-AOC) in the serum were measured. Telomere length, dual oxidase 1 (Duox1), dual oxidase 2 (Duox2), peroxiredoxin 1 (Prdx1), p21, p16, p53, telomerase reverse transcriptase (TERT), phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT), nicotinamide phosphoribosyl transferase (NAMPT), and sirtuin 1 (SIRT1) were detected via real-time PCR, Western blotting, and immunohistochemical staining. The results indicated that APS ameliorated the general status in d-gal-induced aging rats, mitigated neuronal degeneration in the CA3 and CA1 regions, reduced the oxidative stress levels, modulated senescence-related β-GAL and protein expression, and maintained telomere length. Furthermore, APS significantly reduced p53 expression and increased p-PI3K, p-AKT, NAMPT, SIRT1, and TERT expression. Therefore, d-gal-induced aging and cognitive impairment in rats can be prevented by APS, likely through regulation of the TERT/p53 signaling axis via the PI3K/Akt and NAMPT/SIRT1 signaling pathways.

This study aimed to clarify the optimal value for the unbound cefmetazole concentration to remain above the minimum inhibitory concentration (MIC) (fT ≥ MIC) for efficacy of de-escalation to cefmetazole in patients with bacteremic urinary tract infection by extended-spectrum β-lactamase-producing Escherichia coli. This double-center retrospective observational study was conducted at Tokyo Bay Urayasu Ichikawa Medical Center and Keio University Hospital from January 2012 to October 2022. Efficacy was determined via clinical evaluation (mortality rate, recurrence rate, vital changes) and bacteriological evaluation, and the optimal fT ≥ MIC was calculated via receiver operating characteristic curve analysis. As a result, the number of patients evaluated were 40 (35 and 5 in the treatment success and treatment failure groups, respectively). Univariate analysis showed that fT ≥ MIC, recurrence rate, and MIC for cefmetazole against bacteria were significantly different for the two groups (p < 0.05). Receiver operating characteristic curve analysis showed that the optimal fT ≥ MIC indicating efficacy was 57% (area under the curve: 0.94, 95% confidence interval: 0.86–1.00, p = 0.002). All patients with fT ≥ MIC ≥ 57% had successful treatment, whereas the frequency of treatment failure was high among those with fT ≥ MIC <57%. The optimal fT ≥ MIC for the clinical efficacy of de-escalation to cefmetazole in patients with bacteremic urinary tract infection by extended-spectrum β-lactamase-producing E. coli was fT ≥ MIC ≥ 57%. This finding would be useful for optimal dosing of cefmetazole.

The central histamine system is involved in several physiological behaviors and neurological disorders, including the sleep–wake cycle, anxiety-related behaviors (both high and low anxiety), and attention deficit hyperactivity disorder (ADHD). Histamine is synthesized from l-histidine by histidine decarboxylase (HDC) and primarily metabolized by histamine-N-methyltransferase (HNMT) in the central nervous system. We previously reported that mice with intermittent sleep deprivation may exhibit impulsive-like symptoms resembling ADHD and low-anxiety behavior. However, the specific role of histaminergic systems in these behaviors remains unclear. In this study, we evaluated HDC expression levels in the hypothalamus as well as the expression of histamine H1 to H4 receptors and HNMT in the hypothalamus and frontal cortex of sleep-deprived mice. Moreover, the effects of administering histidine, a histamine precursor, and inhibitors of each histamine receptor on sleep deprivation-induced low-anxiety and impulsive-like behaviors were examined using an elevated plus maze test. The expressions of HDC and histamine H1 and H3 receptors in the hypothalamus increased, while that of histamine H1 receptors in the frontal cortex of sleep-deprived mice decreased. The low-anxiety and impulsive-like behaviors in intermittent sleep-deprived mice significantly decreased and increased, respectively, following the administration of histamine H1 and H3 receptor blockers and histidine. Collectively, these findings suggest that the low-anxiety behavior and impulsive-like ADHD symptoms induced by intermittent sleep deprivation may result from the overstimulation of histamine H1 and H3 receptors by elevated histamine, together with increased hypothalamic HDC expression. Furthermore, they suggest that sufficient sleep may contribute to ameliorating ADHD symptoms.

Pulmonary arterial hypertension (PAH) is a progressive disorder that lacks a validated and effective therapy. Thus, further investigation of the pathogenesis of PAH will help explore novel treatments. The increase in T helper 17 (Th17) cell-mediated pro-inflammatory response and reduction of regulatory T (Treg) cell-mediated anti-inflammatory effect exacerbates PAH progression. Increasing evidence indicates that 5-hydroxytryptamine (5-HT) is closely related to Th17 and Treg polarization. Here, a decrease of 5-HT was found in hypoxia-induced CD4 + T cells. Hypoxia also resulted in a reduction in Treg cells and an increase in Th17 cells, but the addition of 5-HT rescued Th17/Treg balance, confirming that hypoxia destroyed Th17/Treg balance by inducing a 5-HT decrease. Furthermore, we found that 5-HT-restored Th17/Treg balance mitigated primary pulmonary artery smooth muscle cell (PASMC) proliferation, migration, and contraction, which are important factors in vascular remodeling in PAH. In summary, our findings demonstrate that hypoxia-induced 5-HT decline interferes with the balance of Th17/Treg, which affects the biofunction of PASMCs, thus accelerating PAH development. 5-HT-mediated Th17/Treg balance is expected to act as a novel immunotherapy for PAH treatment.

In rats, platelet-activating factor (PAF) has been reported to increase mechanical activity in various gastrointestinal smooth muscles (SMs) except for esophagus SM. The aim of this study was to examine whether PAF increases mechanical activity in rat esophagus longitudinal SM (LSM) and to compare PAF actions in esophagus LSM with those in other gastrointestinal LSMs. PAF (10⁻⁹–10⁻⁶ M) increased esophagus LSM mechanical activities in a concentration-dependent manner; PAF mainly elicited basal tension increases that were almost eliminated by a PAF receptor antagonist CV-6209 (10⁻⁵ M; against 10⁻⁶ M PAF). In the LSM of the gastric fundus, which is similar to esophagus LSM in that it is derived from the foregut during development, PAF (10⁻⁶ M) increased basal tension to a comparable, albeit significantly different, magnitude as in esophagus LSM. In contrast, in LSMs of the duodenum–jejunum, ileum, and ascending colon, which are derived from the midgut, and the descending colon, which is derived from the hindgut, the ability of PAF (10⁻⁶ M) to increase basal tension was less than that in esophagus and gastric fundus LSMs. Interestingly, in ascending colon LSMs, PAF (10⁻⁶ M) induced oscillatory contractions with a small increase in basal tension. PAF-induced contractions were positively correlated with the mRNA expression levels of the PAF-degrading enzymes Pafah2 (R = 0.82) and Pafah1b3 (R = 0.51). These results suggest that PAF strongly stimulates mechanical activities that are mainly accompanied by basal tension increases in rat LSMs of the gastrointestinal tracts that are derived from the foregut during embryogenesis.

Multidrug-resistant bacteria pose a major challenge in healthcare, and antimicrobial stewardship teams (ASTs) play a crucial role in optimizing antimicrobial use, particularly for last-resort drugs like meropenem (MEPM) and tazobactam/piperacillin (TAZ/PIPC). This study evaluated the impact of enhanced interventions, which included a hospital-wide policy restricting MEPM and TAZ/PIPC use to 5 d and mandating pre-treatment culture testing. A before-and-after study was conducted at a public hospital in Japan, comparing the pre- (June 2021–May 2022) and post-enhanced intervention (June 2022–May 2023) periods. The primary outcome was days of therapy (DOT) per 1000 patient days for MEPM and TAZ/PIPC. Secondary outcomes included antimicrobial use density (AUD), monthly number of patients receiving MEPM and TAZ/PIPC, 30-d mortality, and AST intervention proposals. Overall, 1896 patients received MEPM (pre: 591; post: 527) or TAZ/PIPC (pre: 411; post: 367). As a result, MEPM DOT decreased from 19.4 to 17.2 per 1000 patient days (p = 0.019), and AUD from 14.4 to 11.7 defined daily doses per 1000 patient days (p = 0.017). TAZ/PIPC DOT remained unchanged (p = 0.219), while AUD decreased from 8.7 to 7.6 (p = 0.046). Furthermore, the monthly number of patients receiving MEPM and TAZ/PIPC and their 30-d mortality showed no significant change. AST proposals increased from 209 to 359 for MEPM and from 116 to 238 for TAZ/PIPC (both p < 0.001). In conclusion, enhanced interventions effectively reduced MEPM use without increasing TAZ/PIPC use or worsening 30-d mortality, suggesting that structured guidelines may enhance antimicrobial stewardship in resource-limited settings.

Nephropathy II Decoction (NED) is a widely used Chinese medicinal formulation for managing chronic kidney disease (CKD). Despite its extensive application, the precise mechanisms underlying its therapeutic effects remain poorly understood. This study aims to elucidate the role of NED in attenuating renal fibrosis and to explore its impact on the gut-kidney axis. The principal constituents of NED were analyzed using ultra-performance LC-tandem mass spectrometry (UPLC-MS/MS). A bilateral renal ischemia-reperfusion injury (bIRI) model was employed to induce fibrosis. RT-qPCR was utilized to assess the expression of mRNA related to the toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88) and nuclear factor-κB (NF-κB) signaling pathway. Western blotting analysis was performed to identify changes in renal fibrosis markers, TLR4/MyD88/NF-κB pathway proteins, and the colon proteins ZO-1 and Occludin-1. Serum levels of uremic toxins were quantified using enzyme-linked immunosorbent assay (ELISA), and 16S ribosomal RNA (rRNA) gene sequencing was conducted to explore changes in the gut microbiome of the mice. Our study demonstrated that mice in the NED group exhibited reduced serum creatinine, blood urea nitrogen, and urinary protein levels, alongside improvements in kidney damage and a decrease in renal fibrosis markers. In the bIRI group, TLR4/MyD88/NF-κB protein and mRNA levels, as well as intestinal tight junction proteins and enterogenic uremic toxins, were significantly reduced. NED treatment reversed these changes and modified the gut microbiota. Furthermore, fecal microbial transplantation (FMT) alleviated kidney damage and fibrosis in bIRI mice. In summary, NED ameliorates kidney injury and fibrosis by modulating the gut microbiota and may further attenuate fibrosis through the inhibition of TLR4 expression, thereby influencing the gut-kidney axis.

Transmembrane protein 16F (TMEM16F), identified as the causative gene for Scott syndrome, which causes blood coagulation disorders, is known to function as not only a scramblase that bi-directionally transports phospholipids in the lipid bilayer but also a Ca²⁺-activated ion channel with low intracellular Ca²⁺ sensitivity. However, how the dual functions of TMEM16F are controlled remains poorly understood. In this study, we investigated the properties of amino acid residues in human TMEM16F involved in the linkage between phospholipid and ion transports and the regulation of their transports using flow cytometry and whole-cell patch-clamp recordings. We demonstrated that ion and phospholipid transports induced by elevation of intracellular Ca²⁺ concentration were tightly coupled in human embryonic kidney HEK293T cells overexpressing wild-type TMEM16F or its mutants. Mutations of amino acid residues in the hydrophilic subunit cavity of TMEM16F indicated that both substrates were transported through its subunit cavity. Importantly, the tail current analysis suggests that conformational changes of TMEM16F by the channel gating are required for its phospholipid transport. These results suggest that ion channel activities of human TMEM16F modulate its scramblase activities.

Detecting low-frequency genetic mutations is crucial for genetic testing, especially in cancer diagnostics. Wild-type blocking PCR identifies these genetic mutations using a blocking oligonucleotide that is fully complementary to wild-type DNA. The blocking oligonucleotide selectively binds to wild-type DNA, inhibiting its amplification by DNA polymerase and allowing preferential amplification of mutant DNA. Bridged nucleic acids (BNAs), with high binding affinities for cDNA, are often incorporated into the blocking oligonucleotide to enhance inhibition. However, the effects of BNA positioning within the blocking oligonucleotide on wild-type DNA amplification inhibition are poorly understood. To address this issue, we evaluated the effects of different BNA positions on amplification inhibition efficacy by comparing blocking oligonucleotides with varying numbers of BNAs at the 5′ end, 3′ end, and central region. Results indicated that BNAs at the 5′ end enhanced the inhibition efficacy, whereas BNAs at the 3′ end notably diminished the inhibition efficacy. Likewise, increasing the number of BNAs in the central region generally decreased the inhibition efficacy. This is one of the first studies to report the importance of BNA positioning in the amplification inhibition efficacy of blocking oligonucleotides.

Chemical modification of mRNA regulates its stability and translation efficiency. The most prevalent modification is the N⁶-methyladenosine (m⁶A) modification. YT521-B homology domain-containing proteins (YTHDCs) are “reader” proteins of m⁶A modification and contribute to various cell functions by regulating their target m⁶A-containing mRNAs. Although m⁶A modification dynamically changes throughout the cell cycle, the role of the m⁶A pathway in cell division remains unclear. In this study, we found that YTHDC1, one of the YTHDCs, is important for cell division. Mitotic progression is delayed in YTHDC1-knockdown cells, and the mitotic delay is mitigated by the re-expression of wild-type YTHDC1, indicating the YTHDC1 function in mitotic progression. Time-lapse imaging analysis showed that prolonged mitotic duration caused by YTHDC1 knockdown is due to the retardation of chromosome alignment and segregation. Treatment of AZ3146, an inhibitor of the spindle assembly checkpoint (SAC), mitigates the mitotic delay in YTHDC1-knockdown cells, suggesting that YTHDC1 knockdown results in SAC activation, leading to a slowdown of mitotic progression. Furthermore, increased TPX2 protein expression and the subsequent overabundance of Aurora A at the centrosomes is partly involved in YTHDC1 knockdown-induced mitotic delay. Since YTHDC1 knockdown does not affect the TPX2 mRNA level and the inhibition of protein synthesis by cycloheximide treatment impairs the YTHDC1-knockdown effect on the TPX2 protein level, YTHDC1 may post-transcriptionally regulate the TPX2 expression. Considering that TPX2 or Aurora A overexpression causes mitotic failure, these results suggest that YTHDC1 contributes to mitotic progression partly through the precise regulation of TPX2–Aurora A signaling.

Eight species of the genus Dendrobium were collected from the Solomon Islands. The morphological and compositional characteristics of their stems and leaves were studied and compared with two Japanese medicinal species of Dendrobium by using multivariate analysis of their components. As a result, we discovered that some types of Dendrobium plants from the Solomon Islands have a composition pattern comparable to that of the Japanese medicinal species, D. catenatum. Within the same species, distinct varieties with different compositions exist based on their locations in the Solomon Islands. Certain types also have different composition patterns depending on the stem and leaf sections. Our findings show that Dendrobium plants in the Solomon Islands have a distinct stem and leaf morphology and composition, while they also share similar compositional patterns with existing medicinal species. As a result, these plants are regarded as valuable medicinal resources with the potential to be used as novel crude drug ingredients.

Autism spectrum disorder (ASD), a neurodevelopmental disorder of unknown etiology with limited treatment options, has emerged as a significant public health concern. Studies have demonstrated that prenatal vitamin D deficiency is a risk factor for ASD development in offspring; however, the underlying mechanism remains unclear. In this project, vitamin D was administered orally to pregnant mice with/without the subsequent administration of polyriboinosinic polyribocytidylic acid (Poly(I:C)), which induced the maternal immune activation (MIA). Our results showed that vitamin D supplementation during pregnancy alleviated MIA-induced ASD-like behaviors in offspring. Moreover, vitamin D supplementation reduced the MIA-induced elevation of interleukin-6 (IL-6) and IL-17a levels in both the maternal ileum and fetal brains. It also suppressed signal transducer and activator of transcription 3 (Stat3) activation and the elevated expression of serum amyloid A1 and A2 (SAA1/2) in the ileum of MIA-affected pregnant mice. This study revealed that vitamin D may reduce the expression of IL-17a by inhibiting the IL-6/Stat3/SAA signaling pathway, thereby improving ASD-like behavior in offspring mice, and provide a new theoretical support for the prevention and treatment of ASD by scientific dietary interventions and nutritional supplement during pregnancy.

Long coronavirus disease (COVID) is characterized by symptoms persisting or reappearing at least 2 months post-recovery from acute coronavirus disease 2019 (COVID-19). Although Long COVID symptoms have been widely studied, data on drug prescriptions for patients with Long COVID in Japan remain limited. Therefore, this study aimed to analyze drug utilization patterns for Long COVID treatment using a nationwide database in Japan, with the goal of providing basic data to support the establishment of standard treatments in the future. The Medical Data Vision COVID-19 dataset was used to identify patients diagnosed with Long COVID between January 15, 2020 and December 31, 2022. Symptoms and prescribed medications were extracted, and descriptive statistics were used to analyze the relationship between symptoms and drug prescriptions. Among 652016 patients with COVID-19, 3769 (0.6%) developed Long COVID. Common symptoms included fatigue, bronchial asthma-like symptoms, and insomnia. Acetaminophen was the most prescribed drug in the first month of diagnosis. Other frequently prescribed drugs included dextromethorphan, l-carbocisteine, and polaprezinc. From 3 months post-diagnosis, prescriptions for Hochu-ekki-to (a traditional Japanese herbal medicine; Kampo medicine) and polaprezinc increased, especially among patients aged 30–50 years. Long COVID in Japan is characterized by a wide range of symptoms, leading to symptom-based drug prescriptions, particularly fatigue, respiratory issues, and taste disturbances. These findings offer insights into the pharmacological management of Long COVID in Japan, highlighting the need for further research on optimal treatments in the future.

This study aimed to evaluate published population pharmacokinetic models of unbound cefazolin to assess their predictive performance using an independent dataset. A systematic literature search was conducted on PubMed to identify studies evaluating the population pharmacokinetics of unbound cefazolin in patients. Subsequently, the selected models were used for external validation. Predictive bias was visually assessed by plotting the prediction errors (PEs) and relative PEs. Predictive precision was evaluated by calculating the mean absolute error (MAE), root mean square error (RMSE), and mean relative error (MRE). The predictive performance of the 4 unbound population pharmacokinetic models was evaluated using clinical data from 64 patients and 218 unbound concentration samples. The PEs for unbound cefazolin concentrations in the Komatsu model indicated a positive bias, while the RPEs demonstrated similar predictive distributions along the y = 0 line, regardless of the predicted values. In contrast, the other 3 models showed a negative bias for both PE and RPE at unbound cefazolin concentrations. The best MAE, RMSE, and MRE (%) values were 4.71, 9.02, and 30.2 in Komatsu et al.’s model, while the next best values were 11.5, 16.1, and 107.2 in Chung et al.’s model. Both models, which performed best regarding bias and accuracy, were also utilized in studies on unbound concentrations and the correlation between total concentrations and protein-binding sites. This study identified these models as the most suitable for predicting unbound cefazolin concentration profiles in surgical patients.

Basal cells, goblet cells, and ciliated cells compose the human airway epithelium and protect the respiratory tract from foreign substances through their involvement in mucociliary clearance (MCC). In particular, ciliary beat frequency (CBF) is an important indicator of MCC efficiency. Although several herbal medicines have been reported to increase CBF, it is still unclear which compounds in these medicines are responsible for this effect. In the present study, we performed experiments to identify compounds that directly increase CBF and elucidated their mechanisms of action. We screened ginsenosides and their derivatives owned by Kyoto Pharmaceutical University and found that ginsenoside Rd was the most effective in increasing CBF and intracellular cAMP concentration ([cAMP]_i). Furthermore, we identified the estrogen receptor β (ERβ) as a new target of ginsenoside Rd-mediated increases in [cAMP]_i and CBF. We also found that ginsenoside Rd increased [Ca²⁺]_i and potentiated the positive effect of ATP on CBF by acting as a positive allosteric modulator (PAM) for the P2X7 receptor in murine airway ciliated cells. In conclusion, the results suggest that ginsenoside Rd increases CBF and that its mechanism of action is as an agonist of ERβ and a PAM of the P2X7 receptor.

Ergothioneine (ERGO) has antioxidant and anti-inflammatory activities in UV-irradiated skin cells in vitro; however, there is no evidence about the effects of dietary ERGO on UV-induced skin damage or ERGO skin distribution in vivo. This study examined the protective effects of ERGO-rich edible mushrooms Pleurotus species against UVB-induced skin damage and the exposure to ERGO in the plasma and skin. Hos : HR-1 hairless mice were fed with or without freeze-dried cross-bred Pleurotus species (PS) or Pleurotus eringii (PE) and were exposed to UVB. Dietary intake of PS or PE significantly alleviated UVB-induced reductions in skin moisture content, increases in transepidermal water loss and oxidative stress markers, and epidermal thickening at plasma ERGO concentrations of 30–40 μM. Additionally, ingestion of PS significantly suppressed UVB-induced expression of pro-inflammatory cytokines. These results suggest that ingesting PS and PE may protect against UVB-induced skin disorders through antioxidant and anti-inflammatory activities at clinically relevant ERGO concentrations. Ingestion of PS and PE led to an increase in epidermal ERGO concentration to levels that were approx. 100 times higher than the ERGO concentration required for significant suppression of UVB-induced intracellular reactive oxygen species in immortalized human keratinocyte HaCaT cells. This suggests that the beneficial effects of PS and PE may be at least partly due to the antioxidant effects of ERGO in murine skin. Overall, ingestion of ERGO-rich Pleurotus species resulted in efficient distribution of ERGO to the skin and protective effects against UVB-induced skin damage, suggesting that these mushrooms may have beneficial effects in humans.

Opioid receptors and their endogenous ligands are novel targets for the treatment of depression. The nociception (NOP) receptor is structurally similar to the opioid receptor, but NOP is known to have a low affinity for the opioid receptor subtypes μ, δ, and κ. In previous studies, we synthesized peptides with a high affinity for opioid receptors and investigated their antidepressant-like effects in mice. However, we have not yet examined whether NOP-related analogs have antidepressant-like effects. Herein, we synthesized NOP analogs (peptide-1–peptide-8) by solid-phase peptide synthesis using the 9-fluorenylmethyloxycarbony (Fmoc) method with Acetyl-Arg-Tyr-Tyr-Arg-Ile-Arg-NH₂ (Ac-RYYRIR-NH₂) as the lead compound. We examined the affinities and antagonistic activities of the analogs for the NOP receptor using receptor-binding and mouse vas deferens assays, and their effects on the duration of immobile behavior in a tail suspension test. Peptide-6 showed a high affinity and antagonistic activity for the NOP receptor. The intracerebroventricular administration of peptide-6 in mice shortened the duration of immobile behavior, whereas the co-administration of NOP inhibited this effect. Moreover, intracerebroventricular administration of the selective NOP receptor antagonist J-113397 showed antidepressant-like effects in mice. These data suggest that peptide-6 exerts an antidepressant-like effect via inactivation of the central NOP receptor in mice and may represent a lead compound for the development of antidepressant drugs in the future.

AHCC^®, a standardized extract of cultured Lentinula edodes mycelia, suppresses the proliferation of both cancer cell line-derived and patient-derived xenografts transplanted into mice. However, the mechanism of action underlying the suppressive effect of AHCC on spontaneous carcinogenesis remains unclear. This study investigated the effects of long-term AHCC ingestion on spontaneous carcinogenesis and the health of C3H/HeJ mice. The mice were divided into three groups: A (2% AHCC ingestion continuously 2 d a week), B (2% AHCC ingestion daily), and C (water ingestion). The ingestion of AHCC or water was started when mice were 5 weeks old and were observed until 24 months of age. The occurrence of the first death was delayed in the AHCC-ingestion groups, and the survival rates were significantly higher in the AHCC-ingestion groups than in the control group. The proportion of “healthy mice” with no morphological abnormalities in their organs was also significantly higher in the AHCC-ingestion groups than in the control group. Furthermore, the incidence of cancer-bearing mice, particularly breast cancer and liver cancer, was significantly reduced in the AHCC-ingestion groups. The reduced rates of breast cancer were particularly higher among females of the AHCC-ingestion groups, whereas the reduced rates of liver cancer were higher among males of the AHCC-ingestion groups. These results suggest that continuous AHCC ingestion maintains health and prevents spontaneous carcinogenesis.

Voriconazole (VRCZ), an azole-based, deep-seated antifungal agent, is used as a 1st-line treatment for aspergillosis in Japan. VRCZ exhibits nonlinear pharmacokinetic (PK) behavior with relatively large inter-individual variability in plasma concentration. Additionally, genetic polymorphisms of CYP2C19 have been reported to influence the metabolic variability of VRCZ. The purpose of this study was to search for and identify clinically relevant potential factors influencing the PK and plasma concentration of VRCZ to better inform VRCZ dosing regimens. Thirty patients receiving VRCZ were enrolled. Total (C_t) and unbound (C_u) trough plasma concentrations of VRCZ were determined by the HPLC-UV method. Univariate and multivariate correlation analyses were used to evaluate the relationships between C_t or C_t/dose per body weight (C_t/D) and individual demographic and laboratory characteristics. Since the increasing trend of C-reactive protein (CRP) inversely correlated with the classification of CYP2C19 gene polymorphisms, it was suggested that the inflammation counteracted the trend of C_t according to CYP2C19 gene polymorphisms. Spearman’s rank-order correlation analysis showed significant correlations between C_t and dose per body weight, CRP, and α1-acid glycoprotein (α1-AGP). Multivariate linear regression analysis showed that age, dose per body weight, CRP, and α1-AGP were significant explanatory factors for C_t. In particular, elevated α1-AGP levels were found to have significant explanatory value for decreased C_t. Although the present study has critical limitations, such as the patient sample was small in size and being limited to a single medical institution, this finding may explain some of the inter-individual variability in plasma VRCZ concentration.

mRNA vaccines have emerged as promising platforms for the prevention of infectious diseases and cancer treatment. The antigenic protein has a signal peptide added to the N-terminus for extracellular secretion. However, it remains unclear whether the optimization of signal peptides has been sufficiently compared and examined for antigen protein secretion and immunogenicity. This study investigated the effects of various signal peptides on the extracellular secretion of a model protein, NanoLuc luciferase (Nluc), in different cell lines. We compared the secretion efficiency of Nluc fused to artificial (#38 and #34) and natural signal peptides (cystatin S, lactotransferrin, and tissue plasminogen activator) in human embryonic kidney 293, C2C12, and HepG2 cells. Luciferase assays and Western blot analysis revealed that the cystatin S signal peptide consistently induced the highest secretion of Nluc among all cell types tested. Notably, the cystatin S signal peptide outperformed previously reported tissue plasminogen activator signal peptides in terms of secretion efficiency. Furthermore, we observed no correlation between Nluc secretion and mRNA expression levels for each signal peptide, suggesting that enhanced secretion was not attributable to increased transcription. Our findings highlight the potential of the cystatin S signal peptide in enhancing the extracellular secretion of antigenic proteins in mRNA vaccines by improving the efficiency of protein translation.

Thiopurines, such as 6-mercaptopurine (6-MP) and azathioprine, are converted to the inactive metabolites 6-thioxanthin (6-TX) and 6-thiouric acid (6-TUA). Molybdenum-containing oxidoreductases, aldehyde oxidase (AOX) and xanthine oxidase (XO), are involved in the oxidation of 6-MP to 6-TX; XO inhibitors affect the therapeutic efficacy of thiopurines and the incidence of adverse effects, such as liver and blood disorders. However, the role of AOX in the pharmacokinetics of 6-MP remains unclear. To clarify the clinical importance of AOX-mediated drug–drug interactions, we evaluated whether drugs that inhibit AOX affect 6-MP metabolism. The metabolism of 6-MP to 6-TX was strongly inhibited by AOX inhibitors (amitriptyline, chlorpromazine, clomipramine, clozapine, hydralazine, quetiapine, and raloxifene) in a reaction mixture containing human liver cytosol. The inhibition of 6-TX production rate by each AOX inhibitor was 60–70% at high concentrations, although the XO inhibitor febuxostat showed an inhibition rate of 10–30%. Furthermore, the combination of febuxostat and each AOX inhibitor showed greater inhibition than when each compound was added alone. The AOX inhibitor did not alter 6-MP oxidation by recombinant XO. These results suggest that AOX inhibition may affect the pharmacokinetics of thiopurines. However, because of the lower activity of AOX in rats than that in humans, the contribution of AOX could not be assessed using in vivo experiments. Further studies are needed to evaluate the contribution of AOX to the therapeutic and adverse effects of thiopurines, both in clinical studies and in animal models of liver humanization.

Duchenne muscular dystrophy (DMD) is a hereditary disease that requires gene or nucleic acid therapy, which involves muscle-targeted delivery of therapeutic material. We previously developed liposomes targeting muscle tissue in DMD model mice (mdx) using an A2G80 peptide, which has an affinity for α-dystroglycan abundantly expressed on the muscle cell membrane. However, these liposomes did not carry gene or nucleic acids. In this study, we aimed to develop muscle-targeting lipid nanoparticles (LNPs) encapsulating luciferase mRNA and evaluate gene expression levels after systemic administration of these LNPs. We first evaluated the efficiency of mRNA delivery based on luciferase activity using polyethylene glycol (PEG)-dimyristoyl glycerol (DMG) and PEG-distearoyl glycerol (DSG) in mdx systemic administration. PEG-DSG-LNPs showed lower luciferase expression in the liver and spleen and higher expression in mdx muscle tissue than PEG-DMG-LNPs. The addition of the A2G80 peptide to LNPs using PEG-DSG (A2G80-DSG-LNPs) significantly increased their activity in mdx but not in normal mice. These results suggest that A2G80-DSG-LNPs allow for muscle-targeted mRNA delivery and are useful tools for DMD treatment.

Ninjinyoeito (NYT) is a Japanese herbal medicine that is prescribed for individuals with depleted mental and physical energy. In this study, we examined the mechanism underlying the effect of NYT on depleted mental and physical energy in mice following a simulated infection. Injection of lipopolysaccharide (LPS), a bacterial endotoxin, decreased the motivational behaviors and body weight of the mice. One of the decreased motivational behaviors, reduced self-grooming, was ameliorated by the continuous administration of NYT. But NYT did not ameliorate reductions in the other motivational behaviors of nest building and food consumption. Furthermore, a single administration of NYT also ameliorated the reduction in self-grooming. PNU282987, an agonist of the alpha7 nicotinic acetylcholine receptor (α7 nAChR), also ameliorated the reduction in self-grooming. Blocking α7 nAChR by methyllycaconitine, an α7 nAChR antagonist, canceled any improvements in self-grooming induced by NYT. Together, these results suggest that NYT ameliorates diminished motivation for self-care via the activation of α7 nAChR.

Decanoic acid/arginine supramolecular hydrogels (C10/Arg) were prepared and evaluated for use as skin-application formulations to improve the skin permeation of hydrophilic middle-to-high-molecular-weight drugs. Dextran (Mw; 6000 Da [Dex]) was used to evaluate the physicochemical properties of the hydrogels, and fluorescein isothiocyanate-Dex (Mw; 4000 Da [FD4], Mw; 40000 Da [FD40]) was used as a model drug to evaluate skin permeability. The addition of 1% Dex to C10/Arg resulted in a translucent supramolecular hydrogel with a lamellar liquid crystal structure, and the addition of a low-molecular-weight model drug to this hydrogel did not significantly affect its physicochemical properties. However, the addition of middle-molecular-weight Dex (6000 Da) changed the rheological properties of the gel, strengthening its internal structure and increasing its elasticity. In vitro skin permeation experiments of model drugs released from C10/Arg gels were performed using Yucatan micropig skin. C10/Arg increased the cumulative amount of FD4 permeation by 40 times after 24 h compared to the application of aqueous FD4 solution. No skin permeation from the aqueous FD40 solution was observed, but permeation was observed following treatment of the skin sample with C10/Arg. The enhanced skin permeation of model drugs by hydrogels was largely due to the contribution of C10 in the hydrogels.