Early diagnosis of Niemann–Pick diseases (NPDs) is important for better prognosis of such diseases. N-Palmitoyl-O-phosphocholine-serine (PPCS) is a new NPD biomarker possessing high sensitivity, and with its combination with sphingosylphosphocholine (SPC) it may be possible to distinguish NPD-C from NPD-A/B. In this study, a rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method (method 1) and a validated LC-MS/MS analysis (method 2) of PPCS and SPC were developed, and we have proposed a diagnostic screening strategy for NPDs using a combination of serum PPCS and SPC concentrations. Nexera and API 5000 were used as LC-MS/MS systems. C18 columns with lengths of 10 and 50 mm were used for method 1 and 2, respectively. ²H₃-Labeled PPCS and nor-SPC were used as internal standards. Selective reaction monitoring in positive-ion mode was used for MS/MS. Run times of 1.2 and 8 min were set for methods 1 and 2, respectively. In both methods 1 and 2, two analytes showed high linearity in the range of 1–4000 ng/mL. Method 2 provided high accuracy and precision in method validation. Serum concentrations of both analytes were significantly higher in NPD-C patients than those of healthy subjects in both methods. Serum PPCS correlated between methods 1 and 2; however, it was different in the case of SPC. The serum PPCS/SPC ratio was different in healthy subjects, NPD-C, and NPD-A/B. These results suggest that using a combination of the two LC-MS/MS analytical methods for PPCS and SPC is useful for diagnostic screening of NPDs.

Gene and nucleic medicines have recently gained attention as novel drugs with the advancement of molecular biology and genetics; however, they have low bioavailability and low target delivery due to their low stability and poor membrane permeability. Therefore, the development of an effective drug delivery system (DDS) is necessary for the practical use of gene and nucleic acid medicines; however, despite considerable research, both safety and efficiency remain poor. Furthermore, the healthcare needs are not met by traditional DDS. Therefore, we developed an effective multi-functional DDS, which is constructed using materials that are safe for human consumption. This DDS involves several ternary complexes as novel gene delivery carriers constructed by coating the cationic complex of the gene and nucleic acid medicines as well as the biodegradable cationic polymer with a biocompatible anionic polymer. Early implementation of the ternary complex in clinical studies is expected due to their efficacy and safety. Furthermore, these complexes may be prepared using large-scale manufacturing. In addition, personalized DDS may be prepared according to the patient’s disease stage, which is useful for advanced therapy.

The sodium salt of isosteviol (STVNa) is a beyerane diterpene synthesized through acid hydrolysis of stevioside. STVNa improves multiple types of tissue injuries. However, it is not known how isosteviol sodium affects high-fat and high cholesterol diet (HFD)-induced kidney. Therefore, in this study we examined the potential molecular mechanism underlying STVNa mediated protective effect against high fat/high cholesterol-induced kidney dysfunction in HFD-induced kidney injury. Sprague-Dawley (SD) rats were allocated into six groups: the normal group, HFD group and HFD treated with three doses of STVNa, fenofibrate treatment group. The results indicated that HFD induced kidney injury evident by a 60% increase in serum creatinine (CRE) leves. In addition, there was a significant accumulation of triglycerides (approx. 60%), fatty acids (approx. 50%) and total cholesterol (approx. 2.5 fold) in the kidneys. STVNa inhibited HFD-induced kidney injury evident by reducing the increased levels of serum CRE. Specifically, STVNa attenuated HFD-induced kidney injury by inhibiting inflammation, oxidative stress, and apoptosis. These findings indicate that STVNa has a therapeutic potential for HFD-induced kidney dysfunction. The mechanisms of this pharmacological effect are through the inhibition of inflammation, oxidative stress and apoptosis.

The immunostimulatory activity of unmethylated cytosine-phosphate-guanine oligodeoxynucleotide (CpG ODN) could be improved via delivery to immune cells expressing Toll-like receptor 9 (TLR9). Previously, we showed that the polypod-like structured nucleic acid (polypodna), a nanostructured DNA comprised of three or more ODNs, was an efficient system for the delivery of CpG ODNs to immune cells. Because some TLR9-positive immune cells express mannose receptors (MR), the uptake of polypodna by immune cells can be further increased by its modification with mannose. In this study, we selected the phosphodiester CpG ODN, ODN1668, which has a sequence identical to CpG1668, and a hexapodna, a polypodna with six pods, to design a hexapodna that harbored ODN1668 or the mannosylated CpG ODN (Man-ODN1668) synthesized via modification of the 5′-terminal of ODN1668 with a synthesized mannose motif. By mixing ODN1668 or Man-ODN1668 with the hexapodna, ODN1668/hexapodna and Man-ODN1668/hexapodna were successfully formed with high yields. However, Man-ODN1668/hexapodna was found to induce a greater tumor necrosis factor-α release from TLR9- and MR-positive mouse peritoneal macrophages and macrophage-like J774.1 cells than Man-ODN1668 or ODN1668/hexapodna. These results indicate that the combination of mannose modification and incorporation into nanostructured DNA is a useful approach for enhancing the immunostimulatory activity of CpG ODN.

The detailed epidemiology of invasive mycoses and superficial mycoses has not been clarified in Japan. In addition, treatment options have increased because of novel antifungals and/or guidelines for fungal infection. In the present study, we aimed to clarify the trends of antifungal use in Japan from 2006 to 2015 based on sales data to serve as an alternative indicator of fungal infection trends. We found that the total antifungal use decreased over time (r = −0.057, P_{for trend} < 0.0001). Oral and parenteral use significantly decreased by 44.1% (r = −0.056, P_{for trend} < 0.0001) and 27.1% (r = −0.0012, P_{for trend} = 0.00061), respectively. The trend of antifungal use for superficial mycoses significantly decreased by 49.8% (r = −0.061, P_{for trend} < 0.0001). However, the trend of antifungal use for invasive mycoses was significantly increased by 19.9% (r = 0.0032, P_{for trend} = 0.00045). In Japan, the increase in the number of immunocompromised patients might be associated with the increase in the frequency of antifungal use for invasive mycoses. This is the first study to clarify the trends of antifungal use in Japan. Further research is needed to establish a continuous surveillance system to compare fungal infections between Japan and the world.

A genetically modified (GM) soybean kernel detection system using combination of DNA preparation from individual soybean kernels and event-specific real-time PCR was developed to simultaneously identify GM soybean events authorized for food after safety assessments in Japan. Over 100 kernels in the non-identity-preserved soybean samples imported from the United States of America (two U.S.A. lots) and Brazil (one lot) were randomly selected and examined. In total, 98 and 96% of the two independent U.S.A. lots, and 100% of the Brazilian lot contained GM soybean kernels. Herbicide-tolerant events, MON89788 (trade name Genuity^® Roundup Ready 2 Yield™), GTS 40-3-2 (trade name Roundup Ready™ soybean) and A2704-12 (trade name Liberty Link^® soybean), were detected similarly in both U.S.A. lots. In the Brazilian lot, in addition to GTS 40-3-2, a stacked GM event, MON87701 × MON89788, having insect-resistance and herbicide-tolerance, was detected. There were no unauthorized GM soybeans comingled, and the ratio of GM soybean events detected was consistent with statistical reports on the cultivated GM soybean events in both countries.

This study aims to evaluate the association between passive smoking and high-grade squamous intraepithelial lesion (HSIL) at the sample of Chinese women. We conducted a case-control study to analyze the effect of passive smoking on the incidence that patients diagnosed with HSIL. The participants had undergone cervical cancer screening by cytology and human papillomavirus (HPV) co-testing within a year before the study. Multiple logistic regression was used to explore the effect and interactive effect of risk factors on HSIL. The odds ratio (OR) and 95% confidence interval (CI) were calculated. Passive smokers were 1.57 times (95% CI 1.05–2.35) higher than non-smokers to occur HSIL. The medium of the combined smoking index divided patients into low and high exposure, with the ORs of 1.64 (95%CI 1.02–2.64) and 1.71 (95%CI 1.06–2.77) relative to non-smokers, respectively. The combined smokers in the high exposure group experienced the most considerable risk of HSIL (OR = 4.67; 95%CI 1.17–18.70). The OR of HPV positive passive smoker relative to that of HPV negative non-smokers was 5.28 (95%CI 2.25–14.52;). Passive smokers who reported adolescent exposure history was 4.04 times (95%CI 1.44–11.37) more at risk of the disease than non-smokers. This study supported that passive smoking was a significant independent risk factor on the occurrence of HSIL and showed a positive correlated dose-response relationship. HPV infection interacting with passive smoking led to an even higher risk of the disease. Adolescent exposure to passive smoking persistent for more than 20 years would also increase the risk of HSIL.

Latifolin, a natural flavonoid found in Dalbergia odorifera T. Chen, has been reported to exhibit anti-inflammatory and anticarcinogenic activities in vitro. However, the anti-aging effects of latifolin are unknown. In this study, we selected a model in vitro system, hydrogen peroxide (H₂O₂)-induced senescence in human dermal fibroblasts (HDFs), to examine the protective effects of latifolin against senescence and the detailed molecular mechanisms involved. Latifolin reversed the senescence-like phenotypes of the oxidant-challenged model, including senescence-associated β-galactosidase (SA-β-gal) staining, cell proliferation, and the expression of senescence-related proteins, such as caveolin-1, ac-p53, p21^Cip1/WAF1, p16^Ink4α, pRb, and cyclinD1. We also found that latifolin induced the expression of silent information regulator 1 (SIRT1) in a concentration- and time-dependent manner, and the anti-senescence effect of latifolin was abrogated by SIRT1 inhibition. Latifolin also suppressed the activation of Akt and S6K1 and attenuated the increase in SA-β-gal activity after H₂O₂ exposure. Our results indicate that latifolin exerts protective effects against senescence in HDFs and that induction of SIRT1 and inhibition of the mammalian target of rapamycin (mTOR) pathway are key mediators of its anti-aging effects.

Patients with cholestatic liver diseases, such as primary biliary cirrhosis, usually suffer from pruritus. However, the pathogenesis of cholestatic pruritus is unclear, and there is no current effective treatment for it. In order to find a treatment for the condition, an appropriate mouse model should be developed. Therefore, here, we established a surgically-induced mouse model of cholestatic pruritus. The bile duct was ligated in order to block bile secretion from the anterior, right, and left lobes, with the exception of the caudate lobe. Serum levels of total bile acid increased after bile duct ligation (BDL). The spontaneous hind paw scratching was also increased in BDL mice. Spontaneous scratching was reduced in BDL mice by naloxone (µ-opioid receptor antagonist), U-50,488H (κ-opioid receptor agonist), and clonidine (α2-adrenoceptor agonist). Azelastine (H₁ receptor antagonist with membrane-stabilizing activity) slightly reduced scratching. However, terfenadine (H₁ receptor antagonist), methysergide (serotonin (5-HT)₂ receptor antagonist), ondansetron (5-HT₃ receptor antagonist), proteinase-activated receptor 2-neutralizing antibody, fluvoxamine (selective serotonin reuptake inhibitor), milnacipran (serotonin-noradrenalin reuptake inhibitor), and cyproheptadine (H₁ and 5-HT₂ receptor antagonist) did not affect scratching. These results suggested that partial obstruction of bile secretion in mice induced anti-histamine-resistant itching and that central opioid system is involved in cholestatic itching.

Large conductance Ca²⁺-activated K⁺ (BK_Ca) channels are ubiquitously expressed in plasma membrane of both excitable and non-excitable cells and possess significant physiological functions. A tetrameric complex of α subunit (BKα) forms a functional pore of BK_Ca channel. The properties of BK_Ca channel, such as voltage-dependence, Ca²⁺ sensitivity and pharmacological responses, are extensively modulated by co-expressing accessory β subunits (BKβ), which can associate with BKα in one to one manner. Although the functional significance of newly identified γ subunits (BKγ) has been revealed, the stoichiometry between BKα and BKγ1 remains unclear. In the present study, we utilized a single molecule fluorescence imaging with a total internal reflection fluorescence (TIRF) microscope to directly count the number of green fluorescent protein (GFP)-tagged BKγ1 (BKγ1-GFP) within a single BK_Ca channel complex in HEK293 cell expression system. BKγ1-GFP significantly enhanced the BK channel activity even when the intracellular Ca²⁺ concentration was kept lower, i.e., 10 nM, than the physiological resting level. BKγ1-GFP stably formed molecular complexes with BKα-mCherry in the plasma membrane. Counting of GFP bleaching steps revealed that a BK_Ca channel can contain up to four BKγ1 per channel at the maximum. These results suggest that BKγ1 forms a BK_Ca channel complex with BKα in a 1 : 1 stoichiometry in a human cell line.

Rotavirus (RV) induced diarrhea has been a major reason affecting children healthy under 5 years old especially in developing countries. Although specific vaccines have preventive effects, antiviral therapy is essential for the diarrhea patients. Ziyuglycoside II is a traditional Chinese herb which has been proven to possess anti-virus effects. This study aimed to investigate the roles of Ziyuglycoside II in rotavirus-induced diarrhea and the underlying molecular mechanism. We found that normal MA104 cells treated with RV became swollen and gather together. However, Ziyuglycoside II treatment inhibited cell growth in a dose- and time dependent manner and suppressed RV replication. Moreover, Ziyuglycoside II reversed RV-induced downregulation of anti-inflammatory cytokine interleukin (IL)-10 and upregulation of pro-inflammatory factors, such as interferon-γ (IFN-γ), IL-1β, IL-6, and tumor necrosis factor (TNF-α). Moreover, Ziyuglycoside II administration and ribavirin blocked toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NF-κB) signaling pathway both in mRNA and protein level, which was paralleled with immunohistochemical assay. Additionally, Ziyuglycoside II administration improved diarrhea symptoms and decreased diarrhea scores. Ziyuglycoside II and ribavirin inhibited the apoptosis of small intestine epithelial cells induced by RV. Taken together, RV treatment induced diarrhea. Ziyuglycoside II administration inhibited TLR4/NF-κB pathway and inflammatory response and improved RV-induced diarrhea. The inhibitory effects of Ziyuglycoside II on RV-induced diarrhea predicted Ziyuglycoside II may be a promising drug for diarrhea.

In this study, we investigated the physicochemical properties and composition of monosaccharidex from Polygonatum sibiricum. Simultaneously, we explored the in vivo and in vitro immunomodulatory activity and mechanism of Polygonatum sibiricum polysaccharide (PSP) activity by monitoring changes in immune organs, immune cells, and cytokines. The average molecular weight (M_w) of PSP was 9.514 × 10⁴ Da. The monosaccharide components of PSP were galactose, rhamnose, arabinose, mannose, and glucose at a molar ratio of 11.72 : 1.78 : 4.15 : 1.00 : 2.48. PSP increased thymus and spleen indices, enhance the proliferative responses of splenocytes, and increased the phagocytosis of mononuclear macrophages. Simultaneously, PSP could recover the body mass of immunosuppressed mice, and increased blood erythrocyte counts in the sera of cyclophosphamide (Cy)-treated and normal mice, whilst blood leukocytes and platelet counts of Cy-treated mice recovered. PSP elevated the CD4⁺/CD8⁺ ratio is a dose-dependent manner and increased the levels of interleukin-2 (IL-2) and tumor necrosis factor-α (TNF-α) in the sera of Cy-treated mice. PSP further enhanced the expression of IL-2 and TNF-α in spleen lymphocytes. Additionally, PSP treatment accelerated the recovery of natural killer cell activity in a dose-dependent manner. Taken together, PSP not only regulated the immune function of normal mice, but participated in the protection against immunosuppression in Cy-treated mice, highlighting its potential as an immunostimulant.

The purpose of the present study was to investigate the protective effects and the underlying mechanisms of Danshensu on liver injury induced by iron overload. The mouse model was induced by injection of iron dextran intraperitoneally for 14 d. Danshensu significantly ameliorated liver injury by decreasing iron accumulation in the liver, possibly by down-regulating the expression of iron uptake-related proteins: divalent metal ion transporters-1 (DMT-1), transferrin receptor (TfR), and L-type calcium channel α1C subunit. Furthermore, Danshensu alleviated oxidative stress injury through potentiating glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) activities; Immunohistochemistry results demonstrated that Danshensu reduced the expression of inflammatory cytokines: interleukin-6 (IL-6) and transforming growth factor-beta (TGF-β). Moreover, Danshensu prominently inhibited hepatocyte apoptosis through decreasing Bax and Caspase-3 and increasing Bcl-2 expression levels. The present results suggest that Danshensu possess significant hepatic-protection at least partly through inhibition of iron uptake, oxidative stress, inflammatory, and apoptosis. Therefore, we believe that Danshensu could be used as a promising therapeutic agent for preventing and treating iron overload diseases.

The interaction of human leukocyte antigen (HLA) with specific drugs is associated with delayed-type hypersensitivity reactions, which cause severe cutaneous toxicity. Such interactions induce structural alterations in HLA complexes via several different mechanisms such as the hapten theory, p-i concept, and altered peptide repertoire model, leading to the activation of cytotoxic T cells. To date, comprehensive detection of such structural alterations in preclinical studies has been difficult. Here, we evaluated structural alterations in HLA complexes focusing on the interaction between the HLA-B*57 : 01 allele and abacavir (an anti-human immunodeficiency virus drug), representing a model of abacavir hypersensitivity syndrome induced by changes in the peptide repertoire on the HLA molecule. We employed a phage display method using a commercially available antibody library to screen specific phage antibodies able to recognize HLA-B*57 : 01. The affinity of selected phage antibodies increased because of structural alterations in HLA-B*57 : 01 following exposure to abacavir, indicating that specific phage antibodies can identify drug-mediated structural changes in HLA complexes. We also identified an unreported structural change in HLA-B*57 : 01 using the phage display method, whereby abacavir increased the expression of peptide-deficient HLA-B*57 : 01 on the cell surface. These results suggest that phage display technology is a useful method for detecting structural changes in HLA complexes. This technology represents a potential novel strategy for predicting HLA-associated hypersensitivity reactions by drugs in pre-clinical studies.

Efficiency (speed and cost) and animal welfare are important factors in the development of new drugs. A novel method (the half-life method) was developed to predict the human plasma concentration–time profile of a monoclonal antibody (mAb) after intravenous (i.v.) administration using less data compared to the conventional approach; moreover, predicted results were comparable to conventional method. This new method use human geometric means of pharmacokinetics (PK) parameters and the non-human primates (NHP) half-life of each mAb. PK data on mAbs in humans and NHPs were collected from literature focusing on linear elimination, and the two-compartment model was used for analysis. The following features were revealed in humans: 1) the coefficient of variation in the distribution volume of the central compartment and at steady state of mAbs was small (22.6 and 23.8%, respectively) and 2) half-life at the elimination phase (t1/2_β) was the main contributor to plasma clearance. Moreover, distribution volume showed no significant correlation between humans and NHPs, and human t1/2_β showed a good correlation with allometrically scaled t1/2_β of NHP. Based on the features revealed in this study, we propose a new method for predicting the human plasma concentration–time profile of mAbs after i.v. dosing. When tested, this half-life method showed reasonable human prediction compared with a conventional empirical approach. The half-life method only requires t1/2_β to predict human PK, and is therefore able to improve animal welfare and potentially accelerate the drug development process.

Ethenzamide (ETZ), an antipyretic analgesic categorized as a non-steroidal anti-inflammatory drug (NSAID), is widely used as an OTC drug in combination with other NSAIDs. However, its site of action and mechanism underlying its analgesic action have not yet been fully elucidated. In this study, we performed in vitro pharmacological assays to identify the mechanism underlying the analgesic action of ETZ, and also conducted the rat formalin test to investigate its analgesic effect and site of action. Of the 85 receptors, ion channels, transporters and enzymes tested, we found that ETZ binds to the 5-hydroxytryptamine (5HT)_2B receptor in concentration-dependent manner with modest inhibitory effects on monoamine oxidase-A and transient potential vanilloid 1 channel. The 5HT_2B receptor antagonist activity of ETZ was also confirmed in a cellular functional assay. Furthermore, the drug exerted no inhibitory effects on cycrooxygenase-1 and -2. In the rat formalin test, oral administration of ETZ significantly reduced the nociceptive responses of the second phase and also the number of c-Fos-expressing cells in the spinal dorsal horn, in a dose-dependent manner. Moreover, intrathecal administration of ETZ significantly reduced the nociceptive responses. These results suggest that the analgesic effect of ETZ is exerted at least in the spinal cord, and the effect would be attributed to multiple mechanisms of action including 5HT_2B receptor blockade.

Pathological angiogenesis is a leading cause of blindness in several retinal diseases. The key driving factor inducing pathological angiogenesis is the pronounced hypoxia leading to a marked, increased production of vascular endothelial growth factor (VEGF). The aim of this study was to determine whether the abnormal vascular growth occurs in a manner dependent on the degree of the vascular defects. Vascular defects of two different degrees were created in the retina by subcutaneously treating neonatal rats with the VEGF receptor (VEGFR) tyrosine kinase inhibitor KRN633 on postnatal day (P) 4 and P5 (P4/5) or P7 and P8 (P7/8). The structure of the retinal vasculature changes was examined immunohistochemically. Prevention of vascular growth and regression of some preformed capillaries were observed on the next day, after completion of each treatment (i.e., P6 and P9). The vascular regrowth occurred as a result of eliminating the inhibitory effect on the VEGFR signaling pathway. KRN633 (P4/5)-treated rats exhibited a retinal vasculature with aggressive intravitreal neovascularization on P21. On the other hand, the appearance of tortuous arteries is a representative vascular pathological feature in retinas of KRN633 (P7/8)-treated groups. These results suggest that an interruption of the retinal vascular development at different time points induces different vascular pathological features in the retina. Pharmacological agents targeting the VEGF signaling pathway are useful for creating an abnormal retinal vasculature with various pathological features in order to evaluate the efficacy of anti-angiogenic compounds.

The objective of this study was to evaluate the influence of cancer cachexia on pain control in cancer patients receiving a transdermal fentanyl patch (FP) and to investigate whether dry skin was a factor related to cancer cachexia and uncontrolled pain. We retrospectively reviewed the medical records of 77 patients receiving FP treatment for the first time, who were classified into cancer cachexia and non-cancer-cachexia groups, according to European Palliative Care Research Collaborative criteria. On day 7 after FP administration, the mean FP dose and morphine equivalent dose (MED) in the cancer cachexia group were significantly higher than in the non-cancer-cachexia group. Additionally, in the cancer cachexia group, there was a significantly larger degree of variation in pain intensity over 7 d than in the non-cachexia group. In patients who were switched from FP to morphine injection, the mean pain intensity and MED on day 3 after morphine injection were significantly lower than those immediately before morphine injection. Subsequently, to investigate whether dry skin was involved in poor pain control in the cancer cachexia group, transepidermal water loss (TEWL) was compared between 15 additional patients classified into cancer cachexia and non-cancer cachexia groups; the mean TEWL in the cancer cachexia group was found to be significantly lower. Our data suggest that cancer cachexia may be a risk factor for poor pain control in patients receiving FP treatment, and that uncontrolled pain in FP treatment may be caused by the inhibition of fentanyl transdermal absorption due to dry skin.

Bone loss and bone-related disease are associated with the deregulation of osteoclast function, and therefore agents that affect osteoclastogenesis have attracted attention. The purpose of the present study was to discover modified kavalactone analogs as potential anti-osteoclastogenic agents. We assessed the effect of 26 analogs on osteoclast differentiation in vitro. The most potent compound, (E)-6-(2-fluorostyryl)-4-methoxy-2H-pyran-2-one (22), suppressed receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenic differentiation of RAW264 cells with IC₅₀ values of 4.3 µM. A partial structure–activity relationship study revealed the importance of fluorine and its position within the 5,6-dehydrokawain skeleton. The results of a pit formation assay suggested that compound 22 prevents osteoclastic bone resorption by inhibiting osteoclastogenesis. Moreover, compound 22 downregulated mRNA expression levels of RANKL-induced nuclear factor of activated T cells c1 (NFATc1) and osteoclastogenesis-related genes. These results suggest that (E)-6-(2-fluorostyryl)-4-methoxy-2H-pyran-2-one scaffold could lead to the identification of new anti-resorptive agents.