Trimebutine suppresses Toll-like receptor 2/4/7/8/9 signaling pathways in macrophages.

Because of the critical roles of Toll-like receptors (TLRs) and receptor for advanced glycation end-products (RAGE) in the pathophysiology of various acute and chronic inflammatory diseases, continuous efforts have been made to discover novel therapeutic inhibitors of TLRs and RAGE to treat inflammatory disorders. A recent study by our group has demonstrated that trimebutine, a spasmolytic drug, suppresses the high mobility group box 1‒RAGE signaling that is associated with triggering proinflammatory signaling pathways in macrophages. Our present work showed that trimebutine suppresses interleukin-6 (IL-6) production in lipopolysaccharide (LPS, a stimulant of TLR4)-stimulated macrophages of RAGE-knockout mice. In addition, trimebutine suppresses the LPS-induced production of various proinflammatory cytokines and chemokines in mouse macrophage-like RAW264.7 cells. Importantly, trimebutine suppresses IL-6 production induced by TLR2-and TLR7/8/9 stimulants. Furthermore, trimebutine greatly reduces mortality in a mouse model of LPS-induced sepsis. Studies exploring the action mechanism of trimebutine revealed that it inhibits the LPS-induced activation of IL-1 receptor-associated kinase 1 (IRAK1), and the subsequent activations of extracellular signal-related kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and nuclear factor-κB (NF-κB). These findings suggest that trimebutine exerts anti-inflammatory effects on TLR signaling by downregulating IRAK1‒ERK1/2‒JNK pathway and NF-κB activity, thereby indicating the therapeutic potential of trimebutine in inflammatory diseases. Therefore, trimebutine can be a novel anti-inflammatory drug-repositioning candidate and may provide an important scaffold for designing more effective dual anti-inflammatory drugs that target TLR/RAGE signaling.

Trimebutine attenuates high mobility group box 1-receptor for advanced glycation end-products inflammatory signaling pathways.

We previously identified papaverine as an inhibitor of receptor for advanced glycation end-products (RAGE) and showed its suppressive effect on high mobility group box 1 (HMGB1)-mediated responses to inflammation. Here, we found trimebutine to be a 3D pharmacophore mimetics of papaverine. Trimebutine was revealed to have more potent suppressive effects on HMGB1-induced production of pro-inflammatory cytokines, such as interleukin-6 and tumor necrosis factor-α in macrophage-like RAW264.7 cells and mouse bone marrow primarily differentiated macrophages than did papaverine. However, the inhibitory effect of trimebutine on the interaction of HMGB1 and RAGE was weaker than that of papaverine. Importantly, mechanism-of-action analyses revealed that trimebutine strongly inhibited the activation of RAGE downstream inflammatory signaling pathways, especially the activation of extracellular signal-regulated kinase 1 and 2 (ERK1/2), which are mediator/effector kinases recruited to the intracellular domain of RAGE. Consequently, the activation of Jun amino terminal kinase, which is an important effector kinase for the up-regulation of pro-inflammatory cytokines, was inhibited. Taken together, these results suggest that trimebutine may exert its suppressive effect on the HMGB1-RAGE inflammatory signal pathways by strongly blocking the recruitment of ERK1/2 to the intracellular tail domain of RAGE in addition to its weak inhibition of the extracellular interaction of HMGB1 with RAGE. Thus, trimebutine may provide a unique scaffold for the development of novel dual inhibitors of RAGE for inflammatory diseases.

Suppression of substrate inhibition in phenanthrene-degrading Mycobacterium by co-cultivation with a non-degrading Burkholderia strain.

In natural environments contaminated by recalcitrant organic pollutants, efficient biodegradation of such pollutants has been suggested to occur through the cooperation of different bacterial species. A phenanthrene-degrading bacterial consortium, MixEPa4, from polluted soil was previously shown to include a phenanthrene-degrading strain, Mycobacterium sp. EPa45, and a non-polycyclic aromatic hydrocarbon (PAH)-degrading strain, Burkholderia sp. Bcrs1W. In this study, we show that addition of phenanthrene to rich liquid medium resulted in the transient growth arrest of EPa45 during its degradation of phenanthrene. RNA-sequencing analysis of the growth-arrested cells showed the phenanthrene-dependent induction of genes that were predicted to be involved in the catabolism of this compound, and many other cell systems, such as a ferric iron-uptake, were up-regulated, implying iron deficiency of the cells. This negative effect of phenanthrene became much more apparent when using phenanthrene-containing minimal agar medium; colony formation of EPa45 on such agar was significantly inhibited in the presence of phenanthrene and its intermediate degradation products. However, growth inhibition was suppressed by the co-residence of viable Bcrs1W cells. Various Gram-negative bacterial strains, including the three other strains from MixEPa4, also exhibited varying degrees of suppression of the growth inhibition effect on EPa45, strongly suggesting that this effect is not strain-specific. Growth inhibition of EPa45 was also observed by other PAHs, biphenyl and naphthalene, and these two compounds and phenanthrene also inhibited the growth of another mycobacterial strain, M. vanbaalenii PYR-1, that can use them as carbon sources. These phenomena of growth inhibition were also suppressed by Bcrs1W. Our findings suggest that, in natural environments, various non-PAH-degrading bacterial strains play potentially important roles in the facilitation of PAH degradation by the co-residing mycobacteria.

Papaverine identified as an inhibitor of high mobility group box 1/receptor for advanced glycation end-products interaction suppresses high mobility group box 1-mediated inflammatory responses.

The interaction of high mobility group box 1 (HMGB1), which is secreted from immune and dying cells during cellular infection and injury, and receptor for advanced glycation end-products (RAGE) appears to be critical for acute and chronic inflammatory disorders. Here we designed a unique cyclic ß-hairpin peptide (Pepb2), which mimics the predicted RAGE-binding domain of HMGB1. Pepb2 competitively inhibited HMGB1/RAGE interaction. We then identified papaverine as a Pepb2 mimetic by in silico 3D-structural similarity screening from the DrugBank library. Papaverine was found to directly inhibit HMGB1/RAGE interaction. It also suppressed the HMGB1-mediated production of pro-inflammatory cytokines, IL-6 and TNF-α, in mouse macrophage-like RAW264.7 cells and bone marrow-derived macrophages. In addition, papaverine attenuated mortality in cecal ligation puncture-induced sepsis model mice. Taken together, these findings indicate that papaverine could become a useful therapeutic against HMGB1/RAGE-mediated sepsis and other inflammatory diseases.

Establishment of plasmid vector and allelic exchange mutagenesis systems in a mycobacterial strain that is able to degrade polycyclic aromatic hydrocarbon.

Plasmid vector and allelic exchange mutagenesis systems were established for the genetic analysis of a phenanthrene-degrading mycobacterial strain, Mycobacterium sp. EPa45. Successful application of these systems revealed the necessity of the EPa45 phdI gene for the degradation of 1-hydroxy-2-naphthoate, which has been proposed to be an intermediate product in the degradation pathway of phenanthrene.

Phase 1/2 study of venetoclax, a BCL-2 inhibitor, in Japanese patients with relapsed or refractory chronic lymphocytic leukemia and small lymphocytic lymphoma.

Patients with relapsed/refractory (R/R) chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL) have limited treatment options. Venetoclax is a potent BCL-2 inhibitor that induces apoptosis in CLL cells. This open-label, phase 1/2 study (NCT02265731) evaluated the safety, pharmacokinetics, and efficacy of venetoclax in Japanese patients with R/R CLL/SLL. Patients enrolled in phase 1 received 400 mg/day venetoclax monotherapy. Patients enrolled in phase 2 received 400 mg/day venetoclax, plus rituximab. Venetoclax was administered with a weekly stepwise ramp-up in doses. In phase 2, efficacy was evaluated by objective response rate (ORR). Twelve patients were enrolled, six in each arm. The most common grade ≥ 3 adverse events were neutropenia (83%), lymphopenia (67%), leukopenia (33%), and thrombocytopenia (17%). Patients receiving venetoclax monotherapy achieved an ORR of 100%, including a complete remission (CR) rate of 17%. Patients receiving combination therapy had an ORR of 67% and a CR rate of 50%. The venetoclax pharmacokinetics profile in Japanese patients was similar to that of Western patients. Venetoclax 400 mg/day monotherapy or in combination with rituximab was well-tolerated and induced promising responses in Japanese patients with R/R CLL/SLL. Although patient numbers were small, the safety profile was largely consistent with other Western studies. Clinical trial registration: clinicaltrials.gov; NCT02265731.

Crystallization and hardening of poly(ethylene-co-vinyl acetate) mouthguards during routine use.

Mouthguards (MGs) made from poly(ethylene-co-vinyl acetate) (EVA) are widely used in contact sports to prevent injuries such as breaking teeth and lip lacerations and to reduce brain concussion. However, the changes in morphology and the molecular mobility of EVA, which can affect its physical properties during practical usage, have not been precisely examined. Therefore, we attempted to determine the main factors which lead to changes in MG performance after one season of practical use by high school rugby players. Solid-state nuclear magnetic resonance (NMR) and pulse NMR measurements showed the hardening of MGs, which was associated with an increased crystallinity of the EVA resulting from prolonged usage. Furthermore, our data indicated that the increase in the relative amount of the crystalline phase may be primarily attributed to temperature fluctuations and repeated changes in pressure, which could cause the hardening of EVA and eventually diminish the protective ability of MGs.

Complete genome sequence of Burkholderia caribensis Bcrs1W (NBRC110739), a strain co-residing with phenanthrene degrader Mycobacterium sp. EPa45.

Complete genome sequence of Burkholderia caribensis Bcrs1W, isolated from a phenanthrene-degrading mixed culture, was determined. The genomic information of Bcrs1W will be beneficial to elucidating the mechanisms of its positive effects on phenanthrene degradation by co-residing Mycobacterium sp. Epa45, and to exploiting their degradation potentials.

Complete Genome Sequence of a Phenanthrene Degrader, Burkholderia sp. HB-1 (NBRC 110738).

The phenanthrene-degrading Burkholderia sp. HB-1 was isolated from a phenanthrene-enrichment culture seeded with a pristine farm soil sample. We report the complete genome sequence of HB-1, which has been deposited to the stock culture (NBRC 110738) at Biological Resource Center, National Institute of Technology and Evaluation (NITE), Tokyo, Japan. The genome of strain HB-1 comprises two circular chromosomes of 4.1 Mb and 3.1 Mb. The finishing was facilitated by the computational tools GenoFinisher, AceFileViewer, and ShortReadManager.

A new aldehyde oxidase catalyzing the conversion of glycolaldehyde to glycolate from Burkholderia sp. AIU 129.

We found a new aldehyde oxidase (ALOD), which catalyzes the conversion of glycolaldehyde to glycolate, from Burkholderia sp. AIU 129. The enzyme further oxidized aliphatic aldehydes, an aromatic aldehyde, and glyoxal, but not glycolate or alcohols. The molecular mass of this enzyme was 130 kDa, and it was composed of three different subunits (αßγ structure), in which the α, ß, and γ subunits were 76 kDa, 36 kDa, and 14 kDa, respectively. The N-terminal amino acid sequences of each subunit showed high similarity to those of putative subunits of xanthine dehydrogenase. Metals (copper, iron and molybdenum) and chelating reagents (α,α'-dipyridyl and 8-hydroxyquinoline) inhibited the ALOD activity. The ALOD showed highest activity at pH 6.0 and 50°C. Twenty mM glycolaldehyde was completely converted to glycolate by incubation at 30°C for 3 h, suggesting that the ALOD found in this study would be useful for enzymatic production of glycolate.

Complete Genome Sequence of a Phenanthrene Degrader, Mycobacterium sp. Strain EPa45 (NBRC 110737), Isolated from a Phenanthrene-Degrading Consortium.

A phenanthrene degrader, Mycobacterium sp. EPa45, was isolated from a phenanthrene-degrading consortium. Here, we report the complete genome sequence of EPa45, which has a 6.2-Mb single circular chromosome. We propose a phenanthrene degradation pathway in EPa45 based on the complete genome sequence.