Inhibitory effect of anthraquinones isolated from the Noni (Morinda citrifolia) root on animal A-, B- and Y-families of DNA polymerases and human cancer cell proliferation

During the course of our studies to develop new uses for the Noni (Morinda citrifolia) root, 10 anthraquinones, rubiadin (1), rubiadin 1-methyl ether (2), lucidin (3), damnacanthol (4), 1,3-dihydroxy-2-methoxymethylanthraquinone (5), 3-hydroxy-1-methoxy-2-methoxymethylanthraquinone (6), nordamnacanthal (7), damnacanthal (8), sorandidiol (9) and morindone (10), were isolated. Compounds 5, 6, 7, 8 and 10 exhibited remarkable inhibition against the activities of animal pols, and compound 10 was the strongest inhibitor in the anthraquinones investigated. Among mammalian pols, compound 10 inhibited the pol activities of A- (pol γ), B- (pols α, δ and ε) and Y- (pols η, ι and κ) families, but did not influence the activities of X-family pols (pols β, λ and terminal deoxynucleotidyl transferase). The tendency of pol inhibition showed a positive correlation with the suppression of human colon cancer cell HCT116 growth. These results suggested that the Noni root containing anthraquinones may be used as an anticancer functional food.     

Nitrogen removal from wastewater using simultaneous nitrate reduction and anaerobic ammonium oxidation in single reactor

The effects of C/N ratio and total organic carbon (TOC) loading on nitrogen removal through simultaneous nitrate reduction and anaerobic ammonium oxidation in a single reactor were examined. Granular sludge taken from a methane fermentation reactor was placed in an upflow reactor and supplied with synthetic wastewater containing nitrate at a C/N ratio of 1 to grow heterotrophic denitrifying bacteria. When nitrogen removal ratio reached 30%, anammox sludge attached to nonwoven-carrier was added into the same reactor and then ammonia was added to the synthetic wastewater. Nitrogen removal ratio was markedly increased to 80-94%. In this system, nitrogen removal ratio was affected by C/N ratio and TOC loading, not by the amount of granular sludge. A stable isotopic analysis using 15N-labeled nitrate showed that N2 gas was formed by anammox reaction.     

Electrochemical detection and control of interactions between DNA and electroactive intercalator using a DNA-alginate complex film modified electrode

The surface of a glassy carbon electrode was modified with a DNA-alginate complex film in which water-soluble DNA was encapsulated into a calcium-alginate gel. The resulting modified electrode (DAFE) was used to detect ethidium bromide (EtBr), after its accumulation on the electrode. The intercalative behavior of EtBr for dsDNA in the film was investigated by measuring the electrode response based on the intercalation of EtBr separated from nonspecific interactions (electrostatic interaction). The accumulation of EtBr in the dsDAF was enhanced by applying a negative potential below -200 mV at the dsDAFE. When a positive potential above +700 mV was applied to the dsDAFE for a constant time with stirring in a Tris buffer solution, the amount accumulated decreased. These results indicate that it is possible to electrochemically control the accumulation and release of EtBr when a dsDAFE is used. In addition, the accumulation and detection of EtBr in spiked river water samples and daunomycin, an antitumor agent, is described.     

Polyphasic characterization of a PCP-to-phenol dechlorinating microbial community enriched from paddy soil

Dechlorination of PCP has been observed previously under anaerobic condition in paddy soil. However, there is poor information about the dechlorination pathway of PCP and the microbial community associated with the PCP dechlorination in paddy soil. In this study, an anaerobic microbial community dechlorinating PCP was enriched by serial transfers from a paddy soil using a medium containing PCP, lactate and the steam-sterilized paddy soil. The enriched microbial community dechlorinated PCP completely to phenol under the anaerobic condition by a dechlorinating pathway as follows; PCP-->2,3,4,5-tetrachlorophenol-->3,4,5-trichlorophenol-->3,5-dichlorophenol-->3-chlorophenol-->phenol. Intermediate products such as 3-chlorophenol were not accumulated, which were immediately dechlorinated to phenol. The enriched microbial community was characterized physiologically by testing the effects of electron donors and electron acceptors on the dechlorinating activity. The dechlorinating activity was promoted with lactate, pyruvate, and hydrogen as electron donors but not with acetate. Electron acceptors, nitrate and sulphate, inhibited the dechlorinating activity competitively but not iron (III). The microbial group associated with the anaerobic dechlorination was characterized by the effect of specific inhibitors on the PCP dechlorination. Effects of specific metabolic inhibitors and antibiotics indicated the involvement of Gram-positive spore-forming bacteria with the PCP dechlorinating activity, which was represented as bacteria of phylum Firmicutes. The structure of the microbial community was characterized by fluorescence in situ hybridization, quinone profiling, and PCR-DGGE (denaturing gel gradient electrophoresis). The combined results indicated the predominance of Clostridium species of phylum Firmicutes in the microbial community. Desulfitobacterium spp. known as anaerobic Gram-positive spore-forming bacteria dechlorinating PCP were not detected by PCR using a specific primer set. These indicated a probable presence of novel anaerobic Gram-positive spore-forming bacteria dechlorinating PCP in the microbial community.     

Production of anti-prion scFv-Fc fusion proteins by recombinant animal cells

We constructed a replication-defective retroviral vector plasmid for the expression of a single-chain antibody fragment (scFv), derived from a chicken anti-human prion protein monoclonal antibody, fused with the Fc region of human IgG1. CHO-K1 and NS-1 cells were transformed with the viral vector pseudotyped with vesicular stomatitis virus G protein (VSV-G), and scFv-Fc producer clones were established. Among the established clones, CHO-2A9 cells produced a large amount of the product with an antibody-like dimerized structure in serum-free culture that facilitated the purification of scFv-Fc. The scFv-Fc specifically recognized the epitope sequence of prion protein in solid-phase enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. The injection test into quails revealed that the scFv became more stable in vivo by fusion with the Fc region. The scFv-Fc will be a useful tool for the detection of mammalian prion proteins.     

Quantitative measurements of amino acids by terahertz time-domain transmission spectroscopy

The quantitative analysis of amino acids by terahertz (THz) time-domain absorption spectroscopy is demonstrated. The optical densities of the amino acids were found to be linearly proportional to the concentration. The molar absorption coefficients of L-glutamic acid (L-Glu), L-glutamic acid sodium salt (Na-L-Glu), L-glutamic acid hydrochloric salt (HCl-L-Glu), L-cysteine (L-Cys), and L-histidine (L-His) were calculated by averaging the THz spectra of the amino acids at several different concentrations in approximately the 0.2-1.0 mol L(-1) range. The concentrations of L-Glu, L-Cys, and L-His mixed samples were successfully calculated with errors of less than 11% and 20% when their concentrations were higher than 0.45 and 0.22 mol L(-1), respectively, by using the obtained molar absorption coefficient.     

Roles of Macrophages in Advanced Liver Fibrosis,Identified Using a Newly Established Mouse Model of Diet-Induced Non-Alcoholic Steatohepatitis

Macrophages play critical roles in the pathogenesis of non-alcoholic steatohepatitis (NASH). However, it is unclear which macrophage subsets are critically involved in the development of inflammation and fibrosis in NASH. In TSNO mice fed a high-fat/cholesterol/cholate-based diet, which exhibit advanced liver fibrosis that mimics human NASH, we found that Kupffer cells (KCs) were less abundant and recruited macrophages were more abundant, forming hepatic crown-like structures (hCLS) in the liver. The recruited macrophages comprised two subsets: CD11c⁺/Ly6C⁻ and CD11c⁻/Ly6C⁺ cells. CD11c⁺ cells were present in a mesh-like pattern around the lipid droplets, constituting the hCLS. In addition, CD11c⁺ cells colocalized with collagen fibers, suggesting that this subset of recruited macrophages might promote advanced liver fibrosis. In contrast, Ly6C⁺ cells were present in doughnut-like inflammatory lesions, with a lipid droplet in the center. Finally, RNA sequence analysis indicates that CD11c⁺/Ly6C⁻ cells promote liver fibrosis and hepatic stellate cell (HSC) activation, whereas CD11c⁻/Ly6C⁺ cells are a macrophage subset that play an anti-inflammatory role and promote tissue repair in NASH. Taken together, our data revealed changes in liver macrophage subsets during the development of NASH and shed light on the roles of the recruited macrophages in the pathogenesis of advanced fibrosis in NASH.     

A Nano-Emulsion Containing Ceramide-like Lipo-Amino Acid Cholesteryl Derivatives Improves Skin Symptoms in Patients with Atopic Dermatitis by Ameliorating the Water-Holding Function

Because ceramide-like lipo-amino acid cholesteryl derivatives can exert a bound water-holding function due to their lamellae-forming properties, in this study, we determined if topical application of those derivatives to atopic dry skin would elicit an ameliorative effect on skin symptoms, at least on its water-holding function. In this clinical study, daily treatment with a nano-emulsion containing 10% phytosteryl/octyldodecyl lauroyl glutamate (POLG) significantly (p < 0.0001) improved skin symptoms, including dryness/scaling, itchiness and stimulus sensations, in the non-lesional skin of patients with atopic dermatitis (AD) at 3 and at 6 weeks compared with week 0. Those significant improvements in skin symptoms were accompanied by a significantly enhanced water content (conductance) and a significant improvement of roughness (SESC) and smoothness (SESM) values measured using a Visioscan at 3 and 6 weeks. Those effects appeared concomitant with a significantly increased corneocyte size, a significantly down-regulated degree of thick abrasions, and a significant impairment of the corneocyte lipid envelope at 6 weeks. Thus, our clinical study suggests, for the first time, that topical application of the POLG nano-emulsion has the distinct potential to ameliorate atopic dry skin symptoms, particularly scaling and itchiness, in the skin of patients with AD. Those effects result from alleviation of the disrupted water-holding function probably due to the increased supply of lamellae structures into the stratum corneum despite the failure to improve barrier function.     

TRPC3-Nox2 Protein Complex Formation Increases the Risk of SARS-CoV-2 Spike Protein-Induced Cardiomyocyte Dysfunction through ACE2 Upregulation

Myocardial damage caused by the newly emerged coronavirus (SARS-CoV-2) infection is one of the key determinants of COVID-19 severity and mortality. SARS-CoV-2 entry to host cells is initiated by binding with its receptor, angiotensin-converting enzyme (ACE) 2, and the ACE2 abundance is thought to reflect the susceptibility to infection. Here, we report that ibudilast, which we previously identified as a potent inhibitor of protein complex between transient receptor potential canonical (TRPC) 3 and NADPH oxidase (Nox) 2, attenuates the SARS-CoV-2 spike glycoprotein pseudovirus-evoked contractile and metabolic dysfunctions of neonatal rat cardiomyocytes (NRCMs). Epidemiologically reported risk factors of severe COVID-19, including cigarette sidestream smoke (CSS) and anti-cancer drug treatment, commonly upregulate ACE2 expression level, and these were suppressed by inhibiting TRPC3-Nox2 complex formation. Exposure of NRCMs to SARS-CoV-2 pseudovirus, as well as CSS and doxorubicin (Dox), induces ATP release through pannexin-1 hemi-channels, and this ATP release potentiates pseudovirus entry to NRCMs and human iPS cell-derived cardiomyocytes (hiPS-CMs). As the pseudovirus entry followed by production of reactive oxygen species was attenuated by inhibiting TRPC3-Nox2 complex in hiPS-CMs, we suggest that TRPC3-Nox2 complex formation triggered by panexin1-mediated ATP release participates in exacerbation of myocardial damage by amplifying ACE2-dependent SARS-CoV-2 entry.     

Serine metabolism contributes to cell survival by regulating extracellular pH and providing an energy source in Saccharomyces cerevisiae

Changes in extracellular pH affect the homeostasis and survival of unicellular organisms. Supplementation of culture media with amino acids can extend the lifespan of budding yeast, Saccharomyces cerevisiae, by alleviating the decrease in pH. However, the optimal amino acids to use to achieve this end, and the underlying mechanisms involved, remain unclear. Here, we describe the specific role of serine metabolism in the regulation of pH in a medium. The addition of serine to synthetic minimal medium suppressed acidification, and at higher doses increased the pH. CHA1, which encodes a catabolic serine hydratase that degrades serine into ammonium and pyruvate, is essential for serine-mediated alleviation of acidification. Moreover, serine metabolism supports extra growth after glucose depletion. Therefore, medium supplementation with serine can play a prominent role in the batch culture of budding yeast, controlling extracellular pH through catabolism into ammonium and acting as an energy source after glucose exhaustion.