Phosphorylation of DNA-binding domains of CLOCK-BMAL1 complex for PER-dependent inhibition in circadian clock of mammalian cells.

In mammals, CLOCK and BMAL1 proteins form a heterodimer that binds to E-box sequences and activates transcription of target genes, including Period (Per). Translated PER proteins then bind to the CLOCK-BMAL1 complex to inhibit its transcriptional activity. However, the molecular mechanism and the impact of this PER-dependent inhibition on the circadian clock oscillation remain elusive. We previously identified Ser38 and Ser42 in a DNA-binding domain of CLOCK as phosphorylation sites at the PER-dependent inhibition phase. In this study, knockout rescue experiments showed that nonphosphorylatable (Ala) mutations at these sites shortened circadian period, whereas their constitutive-phospho-mimetic (Asp) mutations completely abolished the circadian rhythms. Similarly, we found that nonphosphorylatable (Ala) and constitutive-phospho-mimetic (Glu) mutations at Ser78 in a DNA-binding domain of BMAL1 also shortened the circadian period and abolished the rhythms, respectively. The mathematical modeling predicted that these constitutive-phospho-mimetic mutations weaken the DNA binding of the CLOCK-BMAL1 complex and that the nonphosphorylatable mutations inhibit the PER-dependent displacement (reduction of DNA-binding ability) of the CLOCK-BMAL1 complex from DNA. Biochemical experiments supported the importance of these phosphorylation sites for displacement of the complex in the PER2-dependent inhibition. Our results provide direct evidence that phosphorylation of CLOCK-Ser38/Ser42 and BMAL1-Ser78 plays a crucial role in the PER-dependent inhibition and the determination of the circadian period.

GM-CSF Promotes the Survival of Peripheral-Derived Myeloid Cells in the Central Nervous System for Pain-Induced Relapse of Neuroinflammation.

We recently discovered a (to our knowledge) new neuroimmune interaction named the gateway reflex, in which the activation of specific neural circuits establishes immune cell gateways at specific vessel sites in organs, leading to the development of tissue-specific autoimmune diseases, including a multiple sclerosis (MS) mouse model, experimental autoimmune encephalomyelitis (EAE). We have reported that peripheral-derived myeloid cells, which are CD11b+MHC class II+ and accumulate in the fifth lumbar (L5) cord during the onset of a transfer model of EAE (tEAE), play a role in the pain-mediated relapse via the pain-gateway reflex. In this study, we investigated how these cells survive during the remission phase to cause the relapse. We show that peripheral-derived myeloid cells accumulated in the L5 cord after tEAE induction and survive more than other immune cells. These myeloid cells, which highly expressed GM-CSFRα with common ß chain molecules, grew in number and expressed more Bcl-xL after GM-CSF treatment but decreased in number by blockade of the GM-CSF pathway, which suppressed pain-mediated relapse of neuroinflammation. Therefore, GM-CSF is a survival factor for these cells. Moreover, these cells were colocalized with blood endothelial cells (BECs) around the L5 cord, and BECs expressed a high level of GM-CSF. Thus, GM-CSF from BECs may have an important role in the pain-mediated tEAE relapse caused by peripheral-derived myeloid cells in the CNS. Finally, we found that blockade of the GM-CSF pathway after pain induction suppressed EAE development. Therefore, GM-CSF suppression is a possible therapeutic approach in inflammatory CNS diseases with relapse, such as MS.

Temperature-Sensitive Substrate and Product Binding Underlie Temperature-Compensated Phosphorylation in the Clock.

Temperature compensation is a striking feature of the circadian clock. Here we investigate biochemical mechanisms underlying temperature-compensated, CKIδ-dependent multi-site phosphorylation in mammals. We identify two mechanisms for temperature-insensitive phosphorylation at higher temperature: lower substrate affinity to CKIδ-ATP complex and higher product affinity to CKIδ-ADP complex. Inhibitor screening of ADP-dependent phosphatase activity of CKIδ identified aurintricarboxylic acid (ATA) as a temperature-sensitive kinase activator. Docking simulation of ATA and mutagenesis experiment revealed K224D/K224E mutations in CKIδ that impaired product binding and temperature-compensated primed phosphorylation. Importantly, K224D mutation shortens behavioral circadian rhythms and changes the temperature dependency of SCN's circadian period. Interestingly, temperature-compensated phosphorylation was evolutionary conserved in yeast. Molecular dynamics simulation and X-ray crystallography demonstrate that an evolutionally conserved CKI-specific domain around K224 can provide a structural basis for temperature-sensitive substrate and product binding. Surprisingly, this domain can confer temperature compensation on a temperature-sensitive TTBK1. These findings suggest the temperature-sensitive substrate- and product-binding mechanisms underlie temperature compensation.

Computer model of IL-6-dependent rheumatoid arthritis in F759 mice.

The interleukin-6 (IL-6) amplifier, which describes the simultaneous activation of signal transducer and activator of transcription 3 (STAT3) and NF-κb nuclear factor kappa B (NF-κB), in synovial fibroblasts causes the infiltration of immune cells into the joints of F759 mice. The result is a disease that resembles human rheumatoid arthritis. However, the kinetics and regulatory mechanisms of how augmented transcriptional activation by STAT3 and NF-κB leads to F759 arthritis is unknown. We here show that the STAT3-NF-κB complex is present in the cytoplasm and nucleus and accumulates around NF-κB binding sites of the IL-6 promoter region and established a computer model that shows IL-6 and IL-17 (interleukin 17) signaling promotes the formation of the STAT3-NF-κB complex followed by its binding on promoter regions of NF-κB target genes to accelerate inflammatory responses, including the production of IL-6, epiregulin, and C-C motif chemokine ligand 2 (CCL2), phenotypes consistent with in vitro experiments. The binding also promoted cell growth in the synovium and the recruitment of T helper 17 (Th17) cells and macrophages in the joints. Anti-IL-6 blocking antibody treatment inhibited inflammatory responses even at the late phase, but anti-IL-17 and anti-TNFα antibodies did not. However, anti-IL-17 antibody at the early phase showed inhibitory effects, suggesting that the IL-6 amplifier is dependent on IL-6 and IL-17 stimulation at the early phase, but only on IL-6 at the late phase. These findings demonstrate the molecular mechanism of F759 arthritis can be recapitulated in silico and identify a possible therapeutic strategy for IL-6 amplifier-dependent chronic inflammatory diseases.

Zoobiquity experiments show the importance of the local MMP9-plasminogen axis in inflammatory bowel diseases in both dogs and patients.

Using a zoobiquity concept, we directly connect animal phenotypes to a human disease mechanism: the reduction of local plasminogen levels caused by matrix metalloproteinase-9 (MMP9) activity is associated with the development of inflammation in the intestines of dogs and patients with inflammatory bowel disease. We first investigated inflammatory colorectal polyps (ICRPs), which are a canine gastrointestinal disease characterized by the presence of idiopathic chronic inflammation, in Miniature Dachshund (MD) and found 31 missense disease-associated SNPs by whole-exome sequencing. We sequenced them in 10 other dog breeds and found five, PLG, TCOF1, TG, COL9A2 and COL4A4, only in MD. We then investigated two rare and breed-specific missense SNPs (T/T SNPs), PLG: c.477G > T and c.478A>T, and found that ICRPs with the T/T SNP risk alleles showed less intact plasminogen and plasmin activity in the lesions compared to ICRPs without the risk alleles but no differences in serum. Moreover, we show that MMP9, which is an NF-κB target, caused the plasminogen reduction and that intestinal epithelial cells expressing plasminogen molecules were co-localized with epithelial cells expressing MMP9 in normal colons with the risk alleles. Importantly, MMP9 expression in patients with ulcerous colitis or Crohn's disease also co-localized with epithelial cells showing enhanced NF-κB activation and less plasminogen expression. Overall, our zoobiquity experiments showed that MMP9 induces the plasminogen reduction in the intestine, contributing to the development of local inflammation and suggesting the local MMP9-plasminogen axis is a therapeutic target in both dogs and patients. Therefore, zoobiquity-type experiments could bring new perspectives for biomarkers and therapeutic targets.

Rewired Cellular Metabolic Profiles in Response to Metformin under Different Oxygen and Nutrient Conditions.

Metformin is a metabolic disruptor, and its efficacy and effects on metabolic profiles under different oxygen and nutrient conditions remain unclear. Therefore, the present study examined the effects of metformin on cell growth, the metabolic activities and consumption of glucose, glutamine, and pyruvate, and the intracellular ratio of nicotinamide adenine dinucleotide (NAD+) and reduced nicotinamide adenine dinucleotide (NADH) under normoxic (21% O2) and hypoxic (1% O2) conditions. The efficacy of metformin with nutrient removal from culture media was also investigated. The results obtained show that the efficacy of metformin was closely associated with cell types and environmental factors. Acute exposure to metformin had no effect on lactate production from glucose, glutamine, or pyruvate, whereas long-term exposure to metformin increased the consumption of glucose and pyruvate and the production of lactate in the culture media of HeLa and HaCaT cells as well as the metabolic activity of glucose. The NAD+/NADH ratio decreased during growth with metformin regardless of its efficacy. Furthermore, the inhibitory effects of metformin were enhanced in all cell lines following the removal of glucose or pyruvate from culture media. Collectively, the present results reveal that metformin efficacy may be regulated by oxygen conditions and nutrient availability, and indicate the potential of the metabolic switch induced by metformin as combinational therapy.

Establishment of a novel experimental model for muscle-invasive bladder cancer using a dog bladder cancer organoid culture.

In human and dogs, bladder cancer (BC) is the most common neoplasm affecting the urinary tract. Dog BC resembles human muscle-invasive BC in histopathological characteristics and gene expression profiles, and could be an important research model for this disease. Cancer patient-derived organoid culture can recapitulate organ structures and maintains the gene expression profiles of original tumor tissues. In a previous study, we generated dog prostate cancer organoids using urine samples, however dog BC organoids had never been produced. Therefore we aimed to generate dog BC organoids using urine samples and check their histopathological characteristics, drug sensitivity, and gene expression profiles. Organoids from individual BC dogs were successfully generated, expressed urothelial cell markers (CK7, CK20, and UPK3A) and exhibited tumorigenesis in vivo. In a cell viability assay, the response to combined treatment with a range of anticancer drugs (cisplatin, vinblastine, gemcitabine or piroxicam) was markedly different in each BC organoid. In RNA-sequencing analysis, expression levels of basal cell markers (CK5 and DSG3) and several novel genes (MMP28, CTSE, CNN3, TFPI2, COL17A1, and AGPAT4) were upregulated in BC organoids compared with normal bladder tissues or two-dimensional (2D) BC cell lines. These established dog BC organoids might be a useful tool, not only to determine suitable chemotherapy for BC diseased dogs but also to identify novel biomarkers in human muscle-invasive BC. In the present study, for the 1st time, dog BC organoids were generated and several specifically upregulated organoid genes were identified. Our data suggest that dog BC organoids might become a new tool to provide fresh insights into both dog BC therapy and diagnostic biomarkers.

Mass spectrometry-based absolute quantification reveals rhythmic variation of mouse circadian clock proteins.

Absolute values of protein expression levels in cells are crucial information for understanding cellular biological systems. Precise quantification of proteins can be achieved by liquid chromatography (LC)-mass spectrometry (MS) analysis of enzymatic digests of proteins in the presence of isotope-labeled internal standards. Thus, development of a simple and easy way for the preparation of internal standards is advantageous for the analyses of multiple target proteins, which will allow systems-level studies. Here we describe a method, termed MS-based Quantification By isotope-labeled Cell-free products (MS-QBiC), which provides the simple and high-throughput preparation of internal standards by using a reconstituted cell-free protein synthesis system, and thereby facilitates both multiplexed and sensitive quantification of absolute amounts of target proteins. This method was applied to a systems-level dynamic analysis of mammalian circadian clock proteins, which consist of transcription factors and protein kinases that govern central and peripheral circadian clocks in mammals. Sixteen proteins from 20 selected circadian clock proteins were successfully quantified from mouse liver over a 24-h time series, and 14 proteins had circadian variations. Quantified values were applied to detect internal body time using a previously developed molecular timetable method. The analyses showed that single time-point data from wild-type mice can predict the endogenous state of the circadian clock, whereas data from clock mutant mice are not applicable because of the disappearance of circadian variation.

Establishment of TSH ß real-time monitoring system in mammalian photoperiodism.

Organisms have seasonal physiological changes in response to day length. Long-day stimulation induces thyroid-stimulating hormone beta subunit (TSHß) in the pars tuberalis (PT), which mediates photoperiodic reactions like day-length measurement and physiological adaptation. However, the mechanism of TSHß induction for day-length measurement is largely unknown. To screen candidate upstream molecules of TSHß, which convey light information to the PT, we generated Luciferase knock-in mice, which quantitatively report the dynamics of TSHß expression. We cultured brain slices containing the PT region from adult and neonatal mice and measured the bioluminescence activities from each slice over several days. A decrease in the bioluminescence activities was observed after melatonin treatment in adult and neonatal slices. These observations indicate that the experimental system possesses responsiveness of the TSHß expression to melatonin. Thus, we concluded that our experimental system monitors TSHß expression dynamics in response to external stimuli.

Exercise effect on pain is associated with negative and positive affective components: A large-scale internet-based cross-sectional study in Japan.

Pain is a global health problem that leads to sedentary behavior and tends to cause negative emotion. In contrast, exercise is widely recommended for a health promotion, while pain often worsens with physical activity. Although exercise therapy is often prescribed to people with pain, the mechanisms of exercise effect on pain remains unclear. In this study, we tried to identify a universal association factor between regular exercise and pain intensity utilizing a cross-sectional web-based survey involving 52,353 adult participants from a large national study conducted in Japan. Using principal component analysis, we uncovered a mediation model of exercise effect on pain through psychological components. Analyses were performed in half of the population with pain (n = 20,330) and validated in the other half (n = 20,330), and showed that high-frequency exercise had a significant association with reduction in pain intensity. We also found Negative Affect and Vigor, two psychological components, are fully associating the exercise effect on pain (indirect effect = - 0.032, p < 0.001; association proportion = 0.99) with a dose-dependent response corresponding to the frequency of exercise. These findings were successfully validated (indirect effect of high-frequency exercise = - 0.028, p < 0.001; association proportion = 0.85). Moreover, these findings were also identified in subpopulation analyses of people with low back, neck, knee pain, and the tendency of the exercise effect on pain was increased with older people. In conclusion, the effect of exercise on pain is associated with psychological components and these association effects increased in parallel with the frequency of exercise habit regardless pain location.

Germ aversion is a risk factor for chronic low back pain and shoulder pain under the COVID-19 pandemic: an internet-based panel study.

The COVID-19 pandemic has increased germ aversion, an aversive affective response to a high likelihood of pathogen transmission. While psychological factors are associated with chronic pain, the relationship between germ aversion and chronic pain remains unexplored. This study aimed to examine the relationship between germ aversion and new-onset and prognosis of chronic pain using longitudinal data collected during the COVID-19 pandemic. We conducted web-based surveys of full-time workers at baseline and after three months. Data were collected on demographic characteristics, psychological factors, and chronic pain. Germ aversion was assessed using a modified Perceived Vulnerability to Disease scale. We analyzed responses from 1265 panelists who completed the survey twice. The prevalence of chronic low back pain (CLBP) and chronic neck and shoulder pain (CNSP) was associated with sex, short sleep duration, psychological distress, loneliness, and germ aversion. Stratified analyses showed that germ aversion was a risk factor for CLBP at three months in both individuals with and without CLBP at baseline, and for CNSP at three months in those with CNSP at baseline, even after adjustment for confounders. In conclusion, this preliminary study suggests that high germ aversion is a risk factor for CLBP and CNSP in young and middle-aged workers.

Cortical parvalbumin neurons are responsible for homeostatic sleep rebound through CaMKII activation.

The homeostatic regulation of sleep is characterized by rebound sleep after prolonged wakefulness, but the molecular and cellular mechanisms underlying this regulation are still unknown. In this study, we show that Ca2+/calmodulin-dependent protein kinase II (CaMKII)-dependent activity control of parvalbumin (PV)-expressing cortical neurons is involved in homeostatic regulation of sleep in male mice. Prolonged wakefulness enhances cortical PV-neuron activity. Chemogenetic suppression or activation of cortical PV neurons inhibits or induces rebound sleep, implying that rebound sleep is dependent on increased activity of cortical PV neurons. Furthermore, we discovered that CaMKII kinase activity boosts the activity of cortical PV neurons, and that kinase activity is important for homeostatic sleep rebound. Here, we propose that CaMKII-dependent PV-neuron activity represents negative feedback inhibition of cortical neural excitability, which serves as the distributive cortical circuits for sleep homeostatic regulation.

GGT1 is a SNP eQTL gene involved in STAT3 activation and associated with the development of Post-ERCP pancreatitis.

Post-ERCP pancreatitis (PEP) is an acute pancreatitis caused by endoscopic-retrograde-cholangiopancreatography (ERCP). About 10% of patients develop PEP after ERCP. Here we show that gamma-glutamyltransferase 1 (GGT1)-SNP rs5751901 is an eQTL in pancreatic cells associated with PEP and a positive regulator of the IL-6 amplifier. More PEP patients had the GGT1 SNP rs5751901 risk allele (C) than that of non-PEP patients at Hokkaido University Hospital. Additionally, GGT1 expression and IL-6 amplifier activation were increased in PEP pancreas samples with the risk allele. A mechanistic analysis showed that IL-6-mediated STAT3 nuclear translocation and STAT3 phosphorylation were suppressed in GGT1-deficient cells. Furthermore, GGT1 directly associated with gp130, the signal-transducer of IL-6. Importantly, GGT1-deficiency suppressed inflammation development in a STAT3/NF-κB-dependent disease model. Thus, the risk allele of GGT1-SNP rs5751901 is involved in the pathogenesis of PEP via IL-6 amplifier activation. Therefore, the GGT1-STAT3 axis in pancreas may be a prognosis marker and therapeutic target for PEP.

Detection of anti-feline infectious peritonitis virus activity of a Chinese herb extract using geneLEAD VIII, a fully automated nucleic acid extraction/quantitative PCR testing system.

The geneLEAD VIII is a fully-automated nucleic acid extraction/quantitative PCR equipment developed by Precision System Science Co., Ltd., (PSS). To take advantage of its capability, we developed a quantitative assay system to measure growth of animal viruses. The system was used to assay one of the Chinese herbal extracts whose anti-malarial activities were previously reported and demonstrated its dose-dependent anti-viral activity against feline infectious peritonitis virus (FIPV), a feline coronavirus causing the fatal diseases in cats, and relatively low cell toxicity. The assay developed in this study is useful to screen antiviral drugs and the anti-FIPV activity of the herbal extract identified have a potential to lead to development of new drugs against FIPV and other coronaviruses, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Anti-cancer activity of Chaga mushroom (Inonotus obliquus) against dog bladder cancer organoids.

Despite its disadvantages, chemotherapy is still commonly used for the treatment of bladder cancer (BC). Developing natural supplements that can target cancer stem cells (CSCs) which cause drug resistance and distant metastasis is necessary. Chaga mushrooms are popular to have several health-promoting and anti-cancer potentials. Organoid culture can recapitulate tumor heterogeneity, epithelial environment, and genetic and molecular imprints of the original tissues. In the previous study, we generated dog bladder cancer organoids (DBCO) as a novel experimental model of muscle-invasive BCO. Therefore, the present study aimed to examine the anti-tumor potentials of Chaga mushroom extract (Chaga) against DBCO. Four strains of DBCO were used in the present study. Treatment with Chaga inhibited the cell viability of DBCO in a concentration-dependent way. Treatment of DBCO with Chaga has significantly arrested its cell cycle and induced apoptosis. Expression of bladder CSC markers, CD44, C-MYC, SOX2, and YAP1, declined in the Chaga-treated DBCO. Also, Chaga inhibited the phosphorylation of ERK in DBCO. Expression of downstream signals of ERK, C-MYC, and Cyclins (Cyclin-A2, Cyclin-D1, Cyclin-E1, and CDK4) was also inhibited by Chaga in DBCO. Interestingly, the combinational treatment of DBCO with Chaga and anti-cancer drugs, vinblastine, mitoxantrone, or carboplatin, showed a potentiating activity. In vivo, Chaga administration decreased tumor growth and weight of DBCO-derived xenograft in mice with the induction of necrotic lesions. In conclusion, Chaga diminished the cell viability of DBCO by inhibiting proliferation-related signals and stemness conditions as well as by arresting the cell cycle. Collectively, these data suggest the value of Chaga as a promising natural supplement that could potentiate the effect of adjuvant chemotherapy, lower its adverse effects, and thus, limit the recurrence and metastasis of BC.

Establishment of an experimental model of canine malignant mesothelioma organoid culture using a three-dimensional culture method.

Canine malignant mesothelioma (cMM) is a rare and drug-resistant malignant tumor. Due to few patients and experimental models, there have not been enough studies to demonstrate the pathogenesis of the disease and novel effective treatment for cMM. Since cMM resembles human MM (hMM) in histopathological characteristics, it is also considered a promising research model of hMM. Compared with conventional 2-dimensional (2D) culture methods, 3-dimensional (3D) organoid culture can recapitulate the properties of original tumor tissues. However, cMM organoids have never been developed. In the present study, we for the first time generated cMM organoids using the pleural effusion samples. Organoids from individual MM dogs were successfully generated. They exhibited the characteristics of MM and expressed mesothelial cell markers, such as WT-1 and mesothelin. The sensitivity to anti-cancer drugs was different in each strain of cMM organoids. RNA sequencing analysis showed cell adhesion molecule pathways were specifically upregulated in cMM organoids compared with their corresponding 2D cultured cells. Among these genes, the expression level of E-cadherin was drastically higher in the organoids than that in the 2D cells. In conclusion, our established cMM organoids might become a new experimental tool to provide new insights into canine and human MM therapy.

Derivation of a new model of lung adenocarcinoma using canine lung cancer organoids for translational research in pulmonary medicine.

Canine primary lung cancer (cPLC) is a rare malignant tumor in dogs, and exhibits poor prognosis. Effective therapeutic drugs against cPLC have not been established yet. Also, cPLC resembles human lung cancer in histopathological characteristics and gene expression profiles and thus could be an important research model for this disease. Three-dimensional organoid culture is known to recapitulate the tissue dynamics in vivo. We, therefore, tried to generate cPLC organoids (cPLCO) for analyzing the profiles of cPLC. After samples from cPLC and the corresponding normal lung tissue were collected, cPLCO were successfully generated, which recapitulated the tissue architecture of cPLC, expressed lung adenocarcinoma marker (TTF1), and exhibited tumorigenesis in vivo. The sensitivity of cPLCO to anti-cancer drugs was different among strains. RNA-sequencing analysis showed significantly upregulated 11 genes in cPLCO compared with canine normal lung organoids (cNLO). Moreover, cPLCO were enriched with the MEK-signaling pathway compared with cNLO. The MEK inhibitor, trametinib decreased the viability of several strains of cPLCO and inhibited the growth of cPLC xenografts. Collectively, our established cPLCO model might be a useful tool for identifying novel biomarkers for cPLC and a new research model for dog and human lung cancer.

High-precision rapid testing of omicron SARS-CoV-2 variants in clinical samples using AI-nanopore.

A digital platform that can rapidly and accurately diagnose pathogenic viral variants, including SARS-CoV-2, will minimize pandemics, public anxiety, and economic losses. We recently reported an artificial intelligence (AI)-nanopore platform that enables testing for Wuhan SARS-CoV-2 with high sensitivity and specificity within five minutes. However, which parts of the virus are recognized by the platform are unknown. Similarly, whether the platform can detect SARS-CoV-2 variants or the presence of the virus in clinical samples needs further study. Here, we demonstrated the platform can distinguish SARS-CoV-2 variants. Further, it identified mutated Wuhan SARS-CoV-2 expressing spike proteins of the delta and omicron variants, indicating it discriminates spike proteins. Finally, we used the platform to identify omicron variants with a sensitivity and specificity of 100% and 94%, respectively, in saliva specimens from COVID-19 patients. Thus, our results demonstrate the AI-nanopore platform is an effective diagnostic tool for SARS-CoV-2 variants.

Effect of the liquid form of traditional Chinese medicine, Hozen-S, on gastric motility in dogs.

Juzen-taiho-to, a traditional Chinese herbal medicine, is used for patients with anorexia and fatigue in human medicine. In our previous study, granulated Juzen-taiho-to improved vincristine-induced gastrointestinal adverse effects through increasing gastric motility in dogs. As the effect of Hozen-S, the sweet liquid form of Juzen-taiho-to, on dog gastric motility has not been investigated, we examined the effect of administration of Hozen-S on gastric motility. Furthermore, we assessed dog plasma ghrelin level to further elucidate the mechanism of the effect of Hozen-S on gastric contraction. Finally, we assessed the palatability of Hozen-S compared to granulated Juzen-taiho-to and its effect on body weight in dogs. Administration of Hozen-S significantly increased gastric motility, plasma ghrelin concentration, and body weight. A palatability evaluation revealed that the dogs preferred Hozen-S to granulated Juzen-taiho-to. In conclusion, Hozen-S administration to dogs promoted gastric motility by raising plasma ghrelin levels. Considering these functional and palatability data, Hozen-S may replace granulated type Juzen-taiho-to and become a prominent traditional Chinese veterinary medicament.

Anti-malarial activity in a Chinese herbal supplement containing Inonotus obliquus and Panax notoginseng.

Plasmodium falciparum, the most virulent human malaria parasite, causes serious diseases among the infected patients in the world and is particularly important in African regions. Although artemisinin combination therapy is recommended by the WHO for treatment of P. falciparum-malaria, the emergence of artemisinin-resistant parasites has become a serious issue which underscores the importance of sustained efforts to obtain novel chemotherapeutic agents against malaria. As a part of such efforts, thirty-nine herbal extracts from traditional Chinese medicine (TCM) were assayed for their anti-malarial activity using 3D7 strain of P. falciparum. Three herbal supplements appeared to possess higher specific anti-malarial activity than the others. One of them (D3) was separated by two sequential fractionations with reverse-phase (the first step) and normal-phase (the second step) liquid chromatography, in which some fractions resulted in higher specific activities than those of D3 or the previous fractions. Cell toxicity assay was performed with the fractions of the first fractionation and demonstrated no obvious cell toxicity. These results suggest that structure determination of the major compound for the anti-malarial activity in D3 may help the development of more potent chemicals in the future.