A small molecule iCDM-34 identified by in silico screening suppresses HBV DNA through activation of aryl hydrocarbon receptor.

IFN-alpha have been reported to suppress hepatitis B virus (HBV) cccDNA via APOBEC3 cytidine deaminase activity through interferon signaling. To develop a novel anti-HBV drug for a functional cure, we performed in silico screening of the binding compounds fitting the steric structure of the IFN-alpha-binding pocket in IFNAR2. We identified 37 compounds and named them in silico cccDNA modulator (iCDM)-1-37. We found that iCDM-34, a new small molecule with a pyrazole moiety, showed anti-HCV and anti-HBV activities. We measured the anti-HBV activity of iCDM-34 dependent on or independent of entecavir (ETV). iCDM-34 suppressed HBV DNA, pgRNA, HBsAg, and HBeAg, and also clearly exhibited additive inhibitory effects on the suppression of HBV DNA with ETV. We confirmed metabolic stability of iCDM-34 was stable in human liver microsomal fraction. Furthermore, anti-HBV activity in human hepatocyte-chimeric mice revealed that iCDM-34 was not effective as a single reagent, but when combined with ETV, it suppressed HBV DNA compared to ETV alone. Phosphoproteome and Western blotting analysis showed that iCDM-34 did not activate IFN-signaling. The transcriptome analysis of interferon-stimulated genes revealed no increase in expression, whereas downstream factors of aryl hydrocarbon receptor (AhR) showed increased levels of the expression. CDK1/2 and phospho-SAMHD1 levels decreased under iCDM-34 treatment. In addition, AhR knockdown inhibited anti-HCV activity of iCDM-34 in HCV replicon cells. These results suggest that iCDM-34 decreases the phosphorylation of SAMHD1 through CDK1/2, and suppresses HCV replicon RNA, HBV DNA, and pgRNA formation.

Establishment of a Rapid Detection System for ISG20-Dependent SARS-CoV-2 Subreplicon RNA Degradation Induced by Interferon-α.

Inhaled nebulized interferon (IFN)-α and IFN-ß have been shown to be effective in the management of coronavirus disease 2019 (COVID-19). We aimed to construct a virus-free rapid detection system for high-throughput screening of IFN-like compounds that induce viral RNA degradation and suppress the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We prepared a SARS-CoV-2 subreplicon RNA expression vector which contained the SARS-CoV-2 5'-UTR, the partial sequence of ORF1a, luciferase, nucleocapsid, ORF10, and 3'-UTR under the control of the cytomegalovirus promoter. The expression vector was transfected into Calu-3 cells and treated with IFN-α and the IFNAR2 agonist CDM-3008 (RO8191) for 3 days. SARS-CoV-2 subreplicon RNA degradation was subsequently evaluated based on luciferase levels. IFN-α and CDM-3008 suppressed SARS-CoV-2 subreplicon RNA in a dose-dependent manner, with IC50 values of 193 IU/mL and 2.54 µM, respectively. HeLa cells stably expressing SARS-CoV-2 subreplicon RNA were prepared and treated with the IFN-α and pan-JAK inhibitor Pyridone 6 or siRNA-targeting ISG20. IFN-α activity was canceled with Pyridone 6. The knockdown of ISG20 partially canceled IFN-α activity. Collectively, we constructed a virus-free rapid detection system to measure SARS-CoV-2 RNA suppression. Our data suggest that the SARS-CoV-2 subreplicon RNA was degraded by IFN-α-induced ISG20 exonuclease activity.

Design, synthesis, and target identification of new hypoxia-inducible factor 1 (HIF-1) inhibitors containing 1-alkyl-1H-pyrazole-3-carboxamide moiety.

Hypoxia-inducible factor 1 (HIF-1) is a promising drug target for cancer chemotherapy. In our screening program aimed at identifying new HIF-1 inhibitors by using a hypoxia-responsive luciferase reporter gene assay, KUSC-5001 containing the 1-alkyl-1H-pyrazole-3-carboxamide moiety was found as a potential hit molecule. During an extensive structure-activity relationship (SAR) study, we developed a more effective HIF-1 inhibitor KUSC-5037 (IC50 = 1.2 µM). Under hypoxic conditions, KUSC-5037 suppressed the HIF-1α (a regulatory subunit of HIF-1) mRNA, causing decreases in the gene expression of HIF-1 target genes such as carbonic anhydrase 9 (CA9) and vascular endothelial growth factor (VEGF) genes. Furthermore, by applying our fluorescent and bifunctional probes, ATP5B, a catalytic ß subunit of mitochondrial FoF1-ATP synthase, was identified as a target protein of KUSC-5037. These results indicate that the derivatives of KUSC-5037 containing the 1-alkyl-1H-pyrazole-3-carboxamide moiety are promising lead molecules for the inhibition of HIF-1 signaling via FoF1-ATP synthase suppression.

An interferon-like small chemical compound CDM-3008 suppresses hepatitis B virus through induction of interferon-stimulated genes.

Oral administration of nucleotide analogues and injection of interferon-α (IFNα) are used to achieve immediate suppression in replication of hepatitis B virus (HBV). Nucleotide analogs and IFNα inhibit viral polymerase activity and cause long-term eradication of the virus at least in part through removing covalently closed circular DNA (cccDNA) via induction of the APOBEC3 deaminases family of molecules, respectively. This study aimed to explore whether the orally administrable low molecular weight agent CDM-3008 (RO8191), which mimics IFNα through the binding to IFNα/ß receptor 2 (IFNAR2) and the activation of the JAK/STAT pathway, can suppress HBV replication and reduce cccDNA levels. In primary cultured human hepatocytes, HBV DNA levels were decreased after CDM-3008-treatment in a dose-dependent manner with a half-maximal inhibitory concentration (IC50) value of 0.1 µM, and this was accompanied by significant reductions in cellular cccDNA levels, both HBeAg and HBsAg levels in the cell culture medium. Using a microarray we comprehensively analyzed and compared changes in gene (mRNA) expression in CDM-3008- and IFNα-treated primary cultured human hepatocytes. As reported previously, CDM-3008 mimicked the induction of genes that participate in the interferon signaling pathway. OAS1 and ISG20 mRNA expression was similarly enhanced by both CDM-3008 and IFNα. Thus, CDM-3008 could suppress pgRNA expression to show anti-HBV activity. APOBEC3F and 3G mRNA expression was also induced by CDM-3008 and IFNα treatments, suggesting that cccDNA could be degraded through induced APOBEC3 family proteins. We identified the genes whose expression was specifically enhanced in CDM-3008-treated cells compared to IFNα-treated cells. The expression of SOCS1, SOCS2, SOCS3, and CISH, which inhibit STAT activation, was enhanced in CDM-3008-treated cells suggesting that a feedback inhibition of the JAK/STAT pathway was enhanced in CDM-3008-treated cells compared to IFNα-treated cells. In addition, CDM-3008 showed an additive effect with a clinically-used nucleoside entecavir on inhibition of HBV replication. In summary, CDM-3008 showed anti-HBV activity through activation of the JAK/STAT pathway, inducing the expression of interferon-stimulated genes (ISGs), with greater feedback inhibition than IFNα.

EVALUATION OF A RADON AIR MONITOR IN THE MEASUREMENT OF RADON CONCENTRATION IN WATER IN COMPARISON WITH A LIQUID SCINTILLATION COUNTER.

The World Health Organisation (WHO) recommends that the concentration of radon in water should be no more than 100 kBq m-3 (100 BqL-1) and the Codex Alimentarius Commission states that the limit of quantification (LOQ) of a method should be no more than one-fifth of this value. In this study, a degassing method with an RAD7 device was used to measure radon concentrations in water, compared to a liquid scintillation counter (LSC) method used as the reference, to investigate whether the numerical value of the LOQ of this method was more than 1/5 (20 kBq m-3) of 100 kBq m-3. The degassing method with leak prevention was shown to reach a target value of 20 kBq m-3 or less under a relative humidity of 6% or lower in the chamber of the RAD7 device. Accordingly, the RAD7 degassing method with leak prevention can be used to accurately measure radon concentrations in water within the guidance level set out by the WHO.

The mitochondrial ancestor of bonobos and the origin of their major haplogroups.

We report here where the most recent common ancestor (MRCA) of bonobos (Pan paniscus) ranged and how they dispersed throughout their current habitat. Mitochondrial DNA (mtDNA) molecular dating to analyze the time to MRCA (TMRCA) and the major mtDNA haplogroups of wild bonobos were performed using new estimations of divergence time of bonobos from other Pan species to investigate the dispersal routes of bonobos over the forest area of the Congo River's left bank. The TMRCA of bonobos was estimated to be 0.64 or 0.95 million years ago (Ma). Six major haplogroups had very old origins of 0.38 Ma or older. The reconstruction of the ancestral area revealed the mitochondrial ancestor of the bonobo populations ranged in the eastern area of the current bonobos' habitat. The haplogroups may have been formed from either the riparian forests along the Congo River or the center of the southern Congo Basin. Fragmentation of the forest refugia during the cooler periods may have greatly affected the formation of the genetic structure of bonobo populations.

Draft Genome Sequences of Amoeba-Resistant Aeromonas spp. Isolated from Aquatic Environments.

Amoeba-resistant Aeromonas veronii ARB3 and Aeromonas media ARB13 and ARB20, which may be important intracellular pathogens of eukaryotic hosts, were isolated from pond and river waters. The draft genome sequences indicate that the strains harbor multiple protein secretion systems and toxins that induce disruption of the actin cytoskeleton.

Genetic structure of wild bonobo populations: diversity of mitochondrial DNA and geographical distribution.

Bonobos (Pan paniscus) inhabit regions south of the Congo River including all areas between its southerly tributaries. To investigate the genetic diversity and evolutionary relationship among bonobo populations, we sequenced mitochondrial DNA from 376 fecal samples collected in seven study populations located within the eastern and western limits of the species' range. In 136 effective samples from different individuals (range: 7-37 per population), we distinguished 54 haplotypes in six clades (A1, A2, B1, B2, C, D), which included a newly identified clade (D). MtDNA haplotypes were regionally clustered; 83 percent of haplotypes were locality-specific. The distribution of haplotypes across populations and the genetic diversity within populations thus showed highly geographical patterns. Using population distance measures, seven populations were categorized in three clusters: the east, central, and west cohorts. Although further elucidation of historical changes in the geological setting is required, the geographical patterns of genetic diversity seem to be shaped by paleoenvironmental changes during the Pleistocene. The present day riverine barriers appeared to have a weak effect on gene flow among populations, except for the Lomami River, which separates the TL2 population from the others. The central cohort preserves a high genetic diversity, and two unique clades of haplotypes were found in the Wamba/Iyondji populations in the central cohort and in the TL2 population in the eastern cohort respectively. This knowledge may contribute to the planning of bonobo conservation.

Catalog of 178 variations in the Japanese population among eight human genes encoding G protein-coupled receptors (GPCRs).

We screened DNAs from 48 Japanese individuals for single-nucleotide polymorphisms (SNPs) in eight genes encoding G protein-coupled receptors (GPCRs) by directly sequencing the entire relevant genomic regions except for repetitive-sequence elements. This approach identified 147 SNPs and 31 insertion/deletion polymorphisms among the eight GPCR genes. On average, we identified one SNP in every 584 nucleotides. Of the 147 SNPs, 69 were identified in AGTR1, 12 in AGTR2, nine in AGTRL1, 20 in AVPR1A, nine in AVPR2, 16 in DRD1, six in ITGA2B, and six in PTGIR. Twenty-one SNPs were located in 5' flanking regions, 76 in introns, 32 in exons, and 18 in 3' flanking regions. These variants should contribute to investigations of possible correlations between genotypes and phenotypes as regards susceptibility to disease or responsiveness to drug therapy.

Catalog of 680 variations among eight cytochrome p450 ( CYP) genes, nine esterase genes, and two other genes in the Japanese population.

We screened DNAs from 48 Japanese individuals for single-nucleotide polymorphisms (SNPs) in eight cytochrome p450 ( CYP) genes, nine esterase genes, and two other genes by directly sequencing the relevant genomic regions in their entirety except for repetitive elements. This approach identified 607 SNPs and 73 insertion/deletion polymorphisms among the 19 genes examined. Of the 607 SNPs, 284 were identified in CYP genes, 302 in esterase genes, and 21 in the other two genes ( GGT1, and TGM1); overall, 37 SNPs were located in 5' flanking regions, 496 in introns, 55 in exons, and 19 in 3' flanking regions. These variants should contribute to studies designed to investigate possible correlations between genotypes and phenotypes of disease susceptibility or responsiveness to drug therapy.

Catalog of 238 variations among six human genes encoding solute carriers ( hSLCs) in the Japanese population.

We screened DNAs of 48 Japanese individuals for single-nucleotide polymorphisms (SNPs) in six genes encoding proteins of the solute carrier (SLC) family by direct sequencing of their entire genomic regions except for repetitive-sequence elements. This approach identified 213 SNPs and 25 insertion/deletion polymorphisms among the six genes. On average, we identified 1 SNP in every 509 nucleotides. Of the 213 SNPs, 14 were identified in the SLC10A1 gene, 51 in SLC15A1, 29 in SLC22A1, 27 in SLC22A2, 54 in SLC22A4, and 38 in SLC22A5. Eight were located in 5' flanking regions, 172 in introns, 25 in exons, and 8 in 3' flanking regions. These variants should contribute to investigations of possible correlations between genotypes and phenotypes as regards disease susceptibilities or responsiveness to drug therapy.

906 variations among 27 genes encoding cytochrome P450 (CYP) enzymes and aldehyde dehydrogenases (ALDHs) in the Japanese population.

We screened DNAs from 48 Japanese individuals for single-nucleotide polymorphisms (SNPs) in genes encoding 13 cytochrome P450 (CYP) enzymes and 14 aldehyde dehydrogenases (ALDHs) by directly sequencing their entire genomic regions except for repetitive elements. This approach identified 810 SNPs and 96 insertion/deletion polymorphisms among the 27 genes. Of the 810 SNPs, 229 were identified among the CYP genes and 581 in the ALDH genes; of the total, 48 SNPs were located in 5' flanking regions, 619 in introns, 91 in exons, and 52 in 3' flanking regions. These variants should contribute to studies designed to investigate possible correlations between genotypes and phenotypes of disease susceptibility or responsiveness to drug therapy.

Identification of 779 genetic variations in eight genes encoding members of the ATP-binding cassette, subfamily C (ABCC/MRP/CFTR.

We screened DNAs from 48 Japanese individuals for single-nucleotide polymorphisms (SNPs) in eight genes encoding the ATP-binding cassette, subfamily C (ABCC/ MRP/CFTR), by direct sequencing of their entire genomic regions, except repetitive sequence elements. This approach identified 688 SNPs and 91 insertion/deletion polymorphisms among the eight genes. Of the 688 SNPs, 81 were identified in the ABCC1 gene, 41 in ABCC2, 30 in ABCC3, 230 in ABCC4, 76 in ABCC5, 58 in CFTR, 102 in ABCC8. and 70 in ABCC9. Six SNPs were located in the 5' flanking regions, 617 in introns, 46 in exons, and 19 in the 3' flanking regions. These variants should contribute to studies that investigate possible correlations of genotypes with disease-susceptibility phenotypes and responsiveness or adverse effects to drugs.

Characterization of a VNTR polymorphism in the coding region of the CEL gene.

Human carboxyl ester lipase (CEL) secreted by the pancreas into the duodenum is a glycoprotein playing an essential role in the intestinal processing of cholesterol and lipid-soluble vitamins. The gene encoding CEL was known to contain a tandemly repeated sequence of the 11-amino-acid motif in the C-terminal region. We characterized its polymorphic features and found that there are five different alleles in Japanese populations and six in Caucasians. The allele containing 16 repeats is the most common in both populations. Although the distribution of the alleles seemed to be different in the two populations, the difference was not statistically significant. This polymorphism may influence the function of this enzyme and be a useful genetic marker to study diseases associated with cholesterol absorption.

Three hundred twenty-six genetic variations in genes encoding nine members of ATP-binding cassette, subfamily B (ABCB/MDR/TAP), in the Japanese population.

We screened DNAs from 48 Japanese individuals for single-nucleotide polymorphisms (SNPs) in nine genes encoding components of ATP-binding cassette subfamily B (ABCB/MDR/TAP) by directly sequencing the entire applicable genomic regions except for repetitive elements. This approach identified 297 SNPs and 29 insertion/deletion polymorphisms among the nine genes. Of the 297 SNPs, 50 were identified in the ABCB1 gene, 14 in TAP], 35 in TAP2, 48 in ABCB4, 13 in ABCB7, 21 in ABCB8, 21 in ABCB9, 13 in ABCB10, and 82 in ABCB11. Thirteen were located in 5' flanking regions, 237 in introns, 37 in exons, and 10 in 3' flanking regions. These variants may contribute to investigations of possible correlations between genotypes and disease-susceptibility phenotypes or responsiveness to drug therapy.