Antibacterial Effects of Glycyrrhetinic Acid and Its Derivatives on Staphylococcus aureus

Staphylococcus aureus is a major pathogen in humans and causes serious problems due to antibiotic resistance. We investigated the antimicrobial effect of glycyrrhetinic acid (GRA) and its derivatives against 50 clinical S. aureus strains, including 18 methicillin-resistant strains. The minimum inhibitory concentrations (MICs) of GRA, dipotassium glycyrrhizate, disodium succinoyl glycyrrhetinate (GR-SU), stearyl glycyrrhetinate and glycyrrhetinyl stearate were evaluated against various S. aureus strains. Additionally, we investigated the bactericidal effects of GRA and GR-SU against two specific S. aureus strains. DNA microarray analysis was also performed to clarify the mechanism underlying the antibacterial activity of GR-SU. We detected the antimicrobial activities of five agents against S. aureus strains. GRA and GR-SU showed strong antibacterial activities compared to the other three agents tested. At a higher concentration (above 2x MIC), GRA and GR-SU showed bactericidal activity, whereas at a concentration of 1x MIC, they showed a bacteriostatic effect. Additionally, GRA and GR-SU exhibited a synergistic effect with gentamicin. The expression of a large number of genes (including transporters) and metabolic factors (carbohydrates and amino acids) was altered by the addition of GR-SU, suggesting that the inhibition of these metabolic processes may influence the degree of the requirement for carbohydrates or amino acids. In fact, the requirement for carbohydrates or amino acids was increased in the presence of either GRA or GR-SU. GRA and GR-SU exhibited strong antibacterial activity against several S. aureus strains, including MRSA. This activity may be partly due to the inhibition of several pathways involved in carbohydrate and amino acid metabolism.     

Distribution and microhabitats of freshwater mussels in waterbodies in the terrestrialized floodplains of a lowland river

Even when anthropogenically altered, river floodplains continue to contribute to biodiversity. This study examined the distribution of freshwater mussels in relation to environmental factors in waterbodies in the terrestrialized floodplain of a lowland river. Mussels were captured, and environmental measurements were taken in November of 2013 and 2014 in quadrats established in three floodplain waterbodies (FWBs), which were isolated from the main river channel. Among the three FWBs, mussel abundance was highest in a shallow FWB (depth range 18–45 cm) that had intermediate conditions of mud depth and fine sediment rate. Mussel abundance showed a hump-shaped relationship with water depth (the peak 45–50 cm) and mud depth (the peak 8–12 cm). Mussel abundance was also negatively related to the abundance of benthic litter. Litter abundance was positively related to branch abundance and the presence of tree cover, and negatively related to the distance to tree cover, indicating that benthic litter was derived from riparian trees. Our results indicate that relatively shallow (≤ 50 cm) FWBs with moderately accumulated mud, which are not scoured even during flooding, appear to be suitable habitats for mussels. Moreover, it is possible that riparian trees negatively impact mussel distribution in FWBs. Possible short-term measures for improving mussel habitat in FWBs may include the elimination of riparian trees and benthic litter.     

HTLV-1 bZIP Factor Impairs Anti-viral Immunity by Inducing Co-inhibitory Molecule,T Cell Immunoglobulin and ITIM Domain (TIGIT)

Human T-cell leukemia virus type 1 (HTLV-1) infects CD4⁺ T cells and induces proliferation of infected cells in vivo, which leads to the onset of adult T-cell leukemia (ATL) in some infected individuals. The HTLV-1 bZIP factor (HBZ) gene, which is encoded in the minus strand of HTLV-1, plays critical roles in pathogenesis. In this study, RNA-seq and ChIP-seq analyses using HBZ transduced T cells revealed that HBZ upregulates the expression and promoter acetylation levels of a co-inhibitory molecule, T cell immunoglobulin and ITIM domain (TIGIT), in addition to those of regulatory T cells related genes, Foxp3 and Ccr4. TIGIT was expressed on CD4⁺ T cells from HBZ-transgenic (HBZ-Tg) mice, and on ATL cells and HTLV-1 infected CD4⁺ T cells of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in vivo. Expression of Blimp1 and IL-10 was upregulated in TIGIT⁺CD4⁺ cells of HBZ-Tg mice compared with TIGIT^-CD4⁺ T cells, suggesting the correlation between TIGIT expression and IL-10 production. When CD4⁺ T cells from HBZ-Tg mice were stimulated with TIGIT’s ligand, CD155, their production of the inhibitory cytokine IL-10 was enhanced. Furthermore, dendritic cells from HBZ-Tg mice produced high levels of IL-10 after stimulation. These data suggest that HBZ alters immune system to suppressive state via TIGIT and IL-10. Importantly, TIGIT suppressed T-cell responses to another HTLV-1 virus protein, Tax, in vitro. Blocking of TIGIT and PD-1 slightly increased anti-Tax T-cell activity in some HAM/TSP patients. These results suggest that HBZ-induced TIGIT on HTLV-1 infected cells impairs T-cell responses to viral antigens. This study shows that HBZ-induced TIGIT plays a pivotal role in attenuating host immune responses and shaping a microenvironment favorable to HTLV-1.     

Mtu1-Mediated Thiouridine Formation of Mitochondrial tRNAs Is Required for Mitochondrial Translation and Is Involved in Reversible Infantile Liver Injury

Reversible infantile liver failure (RILF) is a unique heritable liver disease characterized by acute liver failure followed by spontaneous recovery at an early stage of life. Genetic mutations in MTU1 have been identified in RILF patients. MTU1 is a mitochondrial enzyme that catalyzes the 2-thiolation of 5-taurinomethyl-2-thiouridine (τm⁵s²U) found in the anticodon of a subset of mitochondrial tRNAs (mt-tRNAs). Although the genetic basis of RILF is clear, the molecular mechanism that drives the pathogenesis remains elusive. We here generated liver-specific knockout of Mtu1 (Mtu1^LKO) mice, which exhibited symptoms of liver injury characterized by hepatic inflammation and elevated levels of plasma lactate and AST. Mechanistically, Mtu1 deficiency resulted in a loss of 2-thiolation in mt-tRNAs, which led to a marked impairment of mitochondrial translation. Consequently, Mtu1^LKO mice exhibited severe disruption of mitochondrial membrane integrity and a broad decrease in respiratory complex activities in the hepatocytes. Interestingly, mitochondrial dysfunction induced signaling pathways related to mitochondrial proliferation and the suppression of oxidative stress. The present study demonstrates that Mtu1-dependent 2-thiolation of mt-tRNA is indispensable for mitochondrial translation and that Mtu1 deficiency is a primary cause of RILF. In addition, Mtu1 deficiency is associated with multiple cytoprotective pathways that might prevent catastrophic liver failure and assist in the recovery from liver injury.     

Study of biological communities subject to imperfect detection: bias and precision of community <Emphasis Type="Italic">N</Emphasis>-mixture abundance models in small-sample situations

Community N-mixture abundance models for replicated counts provide a powerful and novel framework for drawing inferences related to species abundance within communities subject to imperfect detection. To assess the performance of these models, and to compare them to related community occupancy models in situations with marginal information, we used simulation to examine the effects of mean abundance \((\bar{\lambda }\): 0.1, 0.5, 1, 5), detection probability \((\bar{p}\): 0.1, 0.2, 0.5), and number of sampling sites (n _site : 10, 20, 40) and visits (n _visit : 2, 3, 4) on the bias and precision of species-level parameters (mean abundance and covariate effect) and a community-level parameter (species richness). Bias and imprecision of estimates decreased when any of the four variables \((\bar{\lambda }\), \(\bar{p}\), n _site , n _visit ) increased. Detection probability \(\bar{p}\) was most important for the estimates of mean abundance, while \(\bar{\lambda }\) was most influential for covariate effect and species richness estimates. For all parameters, increasing n _site was more beneficial than increasing n _visit . Minimal conditions for obtaining adequate performance of community abundance models were n _site  ≥ 20, \(\bar{p}\) ≥ 0.2, and \(\bar{\lambda }\) ≥ 0.5. At lower abundance, the performance of community abundance and community occupancy models as species richness estimators were comparable. We then used additive partitioning analysis to reveal that raw species counts can overestimate β diversity both of species richness and the Shannon index, while community abundance models yielded better estimates. Community N-mixture abundance models thus have great potential for use with community ecology or conservation applications provided that replicated counts are available.     

Expression of a putative dioxygenase gene adjacent to an insertion mutation is involved in the short internodes of columnar apples (<Emphasis Type="Italic">Malus</Emphasis> × <Emphasis Type="Italic">domestica</Emphasis>)

Determining the molecular mechanism of fruit tree architecture is important for tree management and fruit production. An apple mutant ‘McIntosh Wijcik’, which was discovered as a bud mutation from ‘McIntosh’, exhibits a columnar growth phenotype that is controlled by a single dominant gene, Co. In this study, the mutation and the Co gene were analyzed. Fine mapping narrowed the Co region to a 101 kb region. Sequence analysis of the Co region and the original wild-type co region identified an insertion mutation of an 8202 bp long terminal repeat (LTR) retroposon in the Co region. Segregation analysis using a DNA marker based on the insertion polymorphism showed that the LTR retroposon was closely associated with the columnar growth phenotype. RNA-seq and RT-PCR analysis identified a promising Co candidate gene (91071-gene) within the Co region that is specifically expressed in ‘McIntosh Wijcik’ but not in ‘McIntosh’. The 91071-gene was located approximately 16 kb downstream of the insertion mutation and is predicted to encode a 2-oxoglutarate-dependent dioxygenase involved in an unknown reaction. Overexpression of the 91071-gene in transgenic tobaccos and apples resulted in phenotypes with short internodes, like columnar apples. These data suggested that the 8202 bp retroposon insertion in ‘McIntosh Wijcik’ is associated with the short internodes of the columnar growth phenotype via upregulated expression of the adjacent 91071-gene. Furthermore, the DNA marker based on the insertion polymorphism could be useful for the marker-assisted selection of columnar apples.     

Structure-activity relationship study of estrogen receptor down-regulators with a diphenylmethane skeleton

Selective estrogen receptor (ER) down-regulators (SERDs) are pure ER antagonists that also induce ER degradation upon binding to the receptor. Although SERDs have been developed for the treatment of ER-positive breast cancers for nearly a decade, their precise mechanism(s) of action and structure-activity relationship are still unclear. Generally, Western blotting is used to examine the effects of SERDs on ER protein levels, but the methodology is low-throughput and not quantitative. Here, we describe a quantitative, high-throughput, luciferase-based assay for the evaluation of SERDs activity. For this purpose, we established stable recombinant HEK-293 cell lines expressing ERα fused with emerald luciferase. We also designed and synthesized new diphenylmethane derivatives as candidate SERDs, and evaluated their SERDs activity using the developed system in order to examine their structure-activity relationship, taking EC₅₀ as a measure of potency, and E_max as a measure of efficacy.     

Significance of early morning insomnia for keeping the work continuity of shift workers

Sleep and Biological Rhythms -     

Crystal structure of unautoprocessed precursor of subtilisin from a hyperthermophilic archaeon: evidence for Ca2+-induced folding

The crystal structure of an active site mutant of pro-Tk-subtilisin (pro-S324A) from the hyperthermophilic archaeon Thermococcus kodakaraensis was determined at 2.3 A resolution. The overall structure of this protein is similar to those of bacterial subtilisin-propeptide complexes, except that the peptide bond linking the propeptide and mature domain contacts with the active site, and the mature domain contains six Ca2+ binding sites. The Ca-1 site is conserved in bacterial subtilisins but is formed prior to autoprocessing, unlike the corresponding sites of bacterial subtilisins. All other Ca2+-binding sites are unique in the pro-S324A structure and are located at the surface loops. Four of them apparently contribute to the stability of the central alphabetaalpha substructure of the mature domain. The CD spectra, 1-anilino-8-naphthalenesulfonic acid fluorescence spectra, and sensitivities to chymotryptic digestion of this protein indicate that the conformation of pro-S324A is changed from an unstable molten globule-like structure to a stable native one upon Ca2+ binding. Another active site mutant, pro-S324C, was shown to be autoprocessed to form a propeptide-mature domain complex in the presence of Ca2+. The CD spectra of this protein indicate that the structure of pro-S324C is changed upon Ca2+ binding like pro-S324A but is not seriously changed upon subsequent autoprocessing. These results suggest that the maturation process of Tk-subtilisin is different from that of bacterial subtilisins in terms of the requirement of Ca2+ for folding of the mature domain and completion of the folding process prior to autoprocessing.