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Peptaibols are a class of short peptides, typically 7 to 20 amino acids long, characterized by noncanonical amino acid residues such as aminoisobutyric acid (Aib). Although the helix length is shorter than the membrane thickness, the 11-residue peptaibol trichorovin-XII (TV-XII) can form ion channels in membranes. Assuming that a higher proportion of isoleucine (Ile) relative to leucine (Leu) residues is crucial for maintaining the ion channel activity of TV-XII, peptide analogs of TV-XII with varying Ile content were designed, synthesized, and evaluated. The secondary structure of all derivatives under hydrophobic conditions was confirmed by CD measurement as an α-helix-like β-bend ribbon spiral structure. The most stable ion channel activity was found in compound 4a with maximum Ile. Furthermore, the C-terminal Ile analog showed greater ion channel activity compared to the Leu analog. This suggests that the choice between Leu and Ile can influence the expression of ion channel activity, which will be crucial for the de novo designed functional peptides., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Tubulin C-terminal tail (CTT) is a disordered segment extended from each tubulin monomer of αβ tubulin heterodimers, the building blocks of microtubules. The tubulin CTT contributes to the cellular function of microtubules such as intracellular transportation by regulating their interaction with other proteins and cell shape regulation by controlling microtubule polymerization dynamics. Although the mechanical integrity of microtubules is crucial for their functions, the role of tubulin CTT on microtubule mechanical properties has remained elusive. In this work, we investigate the role of tubulin CTTs in regulating the mechanical properties of microtubules by estimating the persistence lengths and investigating the buckling behavior of microtubules with and without CTT. We find that microtubules with intact CTTs exhibit twice the rigidity of microtubules lacking tubulin CTTs. Our study will widen the scope of altering microtubule mechanical properties for its application in nano bio-devices and lead to novel therapeutic approaches for neurodegenerative diseases with altered microtubule properties., Competing Interests: Declaration of competing interest The authors declare no conflict of interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The development of drugs targeting gene products associated with insulin resistance holds the potential to enhance our understanding of type 2 diabetes mellitus (T2DM). The virtual screening, based on a three-dimensional (3D) protein structure, is a potential technique to accelerate the development of molecular target drugs. Among the targets implicated in insulin resistance, the genetic characterization and protein function of Grb14 have been clarified without contradiction. The Grb14 gene displays significant variations in T2DM, and its gene product is known to inhibit the function of the insulin receptor (IR) by directly binding to the tyrosine kinase domain. In the present study, a virtual screening, based on a 3D structure of the IR tyrosine kinase domain (IRβ) in complex with part of Grb14, was conducted to find compounds that can disrupt the complex formation between Grb14 and IRβ. First, ten compounds were selected from 154,118 compounds via hierarchical in silico structure-based drug screening, composed of grid docking-based and genetic algorithm-based programs. The experimental validations suggested that the one compound can affect the blood glucose level. The molecular dynamics simulations and co-immunoprecipitation analysis showed that the compound did not completely suppress the protein-protein interaction between Grb14 and IR, though competitively bound to IR with the tyrosine kinase pseudosubstrate region in Grb14.

This case-control study evaluated the gene expression levels of interleukin (IL)-4, macrophage inflammatory protein type 1 alpha (MIP-1α), and metalloproteinase (MMP)-9, factors involved in the formation of giant cells in healthy peri-implant tissue and peri-implantitis. Thirty-five subjects (15 healthy and 20 with peri-implantitis), who met the inclusion and exclusion criteria, were included in this study. The peri-implant tissue biopsies were subjected to total RNA extraction, DNAse treatment, and cDNA synthesis. Subsequently, the reaction of real-time PCR was performed to evaluate the gene expression levels of IL-4, MIP-1α, and MMP-9 concerning the reference gene. IL-4 gene expression showed higher (18-fold) values in the Peri-Implantitis Group of Patients when compared with the Healthy (Control) Group (p<0.0001). Although MIP- 1α and MMP-9 gene expression levels were higher in diseased implants, they showed no significant differences (p=0.06 and p=0.2337), respectively. Within the limitations of this study, the results showed that in tissues affected by peri-implantitis, only levels of Il-4 were increased when compared with tissues in the control group.

The low-level antioxidant activity of pancreatic islets causes type 1 diabetes due to oxidative stress, which is also the cause of failure in the pancreatic islets' isolation and cell transplantation. In our previous study, pteryxin was found to be a natural product as a nuclear factor-erythroid-2-related factor (Nrf2) activator. This study focused on elucidation that the potentiality of pteryxin can activate the antioxidant enzymes, even under oxidative stress, by hydrogen peroxide (H 2 O 2 ). Pteryxin treated with mouse insulinoma MIN6 cells was enhanced the antioxidant gene expressions in the ARE (antioxidant response element) region for HO-1 (Heme Oxygenase-1), GCLC (Glutamate-cysteine ligase catalytic subunit), SOD1 (Super Oxide dismutase1), and Trxr1 (Thioredoxin reductase1), and those enzymes were also expressed during the nuclei transference of cytoplasmic Nrf2. In fact, the cells exposed to H 2 O 2 concentrations of a half-cell lethal in the presence of pteryxin were then induced main antioxidant enzymes, HO-1, GCLC, and Trxr1 in the ARE region. The increased glutathione (GSH) levels associated with the GCLC expression also suggested to be cytoprotective against oxidative stress by activating the redox-metabolizing enzymes involving their increased antioxidant activity in the cells. In addition, Akt is a modulator for Nrf2, which may be responsible for the Nrf2 activation. These results allowed us to consider whether pteryxin or its synthesized congeners, an Nrf2 activator, is a potential preservative agent against islet isolation during cell transplantation.

Identifying small compounds capable of inhibiting Mycobacterium tuberculosis polyketide synthase 13 (Pks13), in charge of final step of mycolic acid biosynthesis, could lead to the development of a novel antituberculosis drug. This study screened for lead compounds capable of targeting M. tuberculosis Pks13 from a chemical library comprising 154,118 compounds through multiple in silico docking simulations. The parallel compound screening (PCS), conducted via two genetic algorithm-based programs was applied in the screening strategy. Out of seven experimentally validated compounds, four compounds showed inhibitory effects on the growth of the model mycobacteria (Mycobacterium smegmatis). Subsequent docking simulation of analogs of the promising leads with the assistance of PCS resulted in the identification of three additional compounds with potent antimycobacterial effects (compounds A1, A2, and A5). Further, molecular dynamics simulation predicted stable interaction between M. tuberculosis Pks13 active site and compound A2, which showed potent antimycobacterial activity comparable to that of isoniazid. The present study demonstrated the efficacy of in silico structure-based drug screening through PCS in antituberculosis drug discovery., (© 2022. The Author(s), under exclusive licence to the Japan Antibiotics Research Association.)

The mechanism and physiological functions of heme oxygenase-2 (HO-2)-mediated carbon monoxide (CO) production, accompanied by heme metabolism, have been studied intensively in recent years. The enzymatic activity of constitutively expressed HO-2 must be strictly controlled in terms of the toxicity and chemical stability of CO. In this study, the molecular interaction between HO-2 and caveolin-1 and its effect on HO action were evaluated. An enzyme kinetics assay with residues 82-101 of caveolin-1, also called the caveolin scaffold domain, inhibited HO-2 activity in a competitive manner. Analytical ultracentrifugation and a hemin titration assay suggested that the inhibitory effect was generated by direct binding of caveolin-1 to aromatic residues, which were defined as components of the caveolin-binding motif in the HO-2 heme pocket. Herein, we developed a HO-2-based fluorescence bioprobe, namely EGFP-Δ19/D159H, which was capable of quantifying heme binding by HO-2 as the initial step in the CO production. The fluorescence of EGFP-Δ19/D159H decreased in accordance with 5-aminolevulinic acid-facilitated heme biosynthesis in COS-7 cells. In contrast, expression of the N-terminal cytosolic domain of caveolin-1 (residues 1-101) increased the probe fluorescence, suggesting that the cytosolic domain of caveolin-1 potently inhibits the binding of heme to the heme pocket of EGFP-Δ19/D159H. Taken together, our results suggest that caveolin-1 is a negative regulator of HO-2 enzymatic action. Moreover, our bioprobe EGFP-Δ19/D159H represents a powerful tool for use in future studies addressing HO-2-mediated CO production.

Background: The emergence of frequent hitters (FHs) remains a challenge in drug discovery. We have previously used in silico structure-based drug screening (SBDS) to identify antimycobacterial candidates. However, excluding FHs has not been integrated into the SBDS system., Methods: A dataset comprising 15,000 docking score (protein-compound affinity matrix) was constructed by multiple target screening (MTS): DOCK-GOLD two-step docking simulations with 154,118 compounds versus the 30 target proteins essential for mycobacterial survival. After extraction of 141 compounds from the protein-compound affinity matrix, compounds determined to be FHs or false positives were excluded. Antimycobacterial properties of the top nine compounds selected through SBDS were experimentally evaluated., Results: Nine compounds designated KS1-KS9 were selected for experimental evaluation. Among the selected compounds, KS3, identified as adenosylhomocysteinase inhibitor, showed a potent inhibitory effect on antimycobacterial growth (inhibitory concentration [IC] 50 = 1.2 M). However, the compound also showed potent cytotoxicity., Conclusion: The MTS method is applicable in SBDS for the identification of enzyme-specific inhibitors., Competing Interests: None

The initial defensive secretory compounds emitted from a live millipede have not yet been clarified. This study focused on elucidating the initial secretory compounds emitted from a live millipede. Pre-concentration of the defensive secretory volatile organic compounds (VOC) from the live Polidesmida millipedes, Chamberlinius hualienensis and Oxidus gracilis, was performed using a three-stage VOC concentration technique by an on-line GC/MS system. As a result, the monoterpenes derived from the plant metabolite; i.e., α-pinene, α-thujene, β-pinene, 3-carene, β-myrcene, β-phellandrene, γ-terpinene, o,m,p-cymenes, limonene and camphene were first detected as the initial secretory substances. It was elucidated that some plant monoterpenes have a repellent effect and antifungal and antibacterial actions which are used as defensive substances. In addition, this study also confirmed that these monoterpenes induced apoptotic cell death involved in the induction of the caspase 3/7 activity. The millipede feeds on fallen or withered leaves containing the monoterpenes. Thus, the millipede accumulates the plant defensive secretions in the exocrine defense glands of the body somites, which would be used as against predators.

Cancer patients are regarded as highly vulnerable to severe acute respiratory syndrome coronavirus (SARS-CoV)-2. However, little is known regarding how cancer treatments should be restarted for cancer patients after coronavirus disease (COVID)-19. We herein report a pancreatic cancer case in which chemotherapy was able to be reinstituted after COVID-19. The patient was a 67-year-old man diagnosed with pancreatic cancer. On day 7 after first chemotherapy, he was infected with COVID-19. A SARS-CoV-2 test was negative after one month of treatment, and we reinstituted chemotherapy. The patient has received three cycles of chemotherapy without recurrence of COVID-19. It may be feasible to reinstitute chemotherapy for cancer patients after a negative SARS-CoV-2 test.

Heme oxygenase (HO) catalyzes heme degradation using electrons supplied by NADPH-cytochrome P450 oxidoreductase (CPR). Electrons from NADPH flow first to FAD, then to FMN, and finally to the heme in the redox partner. Previous biophysical analyses suggest the presence of a dynamic equilibrium between the open and the closed forms of CPR. We previously demonstrated that the open-form stabilized CPR (ΔTGEE) is tightly bound to heme-HO-1, whereas the reduction in heme-HO-1 coupled with ΔTGEE is considerably slow because the distance between FAD and FMN in ΔTGEE is inappropriate for electron transfer from FAD to FMN. Here, we characterized the enzymatic activity and the reduction kinetics of HO-1 using the closed-form stabilized CPR (147CC514). Additionally, we analyzed the interaction between 147CC514 and heme-HO-1 by analytical ultracentrifugation. The results indicate that the interaction between 147CC514 and heme-HO-1 is considerably weak, and the enzymatic activity of 147CC514 is markedly weaker than that of CPR. Further, using cryo-electron microscopy, we confirmed that the crystal structure of ΔTGEE in complex with heme-HO-1 is similar to the relatively low-resolution structure of CPR complexed with heme-HO-1 in solution. We conclude that the "open-close" transition of CPR is indispensable for electron transfer from CPR to heme-HO-1.

InhA or enoyl-acyl carrier protein reductase of Mycobacterium tuberculosis (mtInhA), which controls mycobacterial cell wall construction, has been targeted in the development of antituberculosis drugs. Previously, our in silico structure-based drug screening study identified a novel class of compounds (designated KES4), which is capable of inhibiting the enzymatic activity of mtInhA, as well as mycobacterial growth. The compounds are composed of four ring structures (A-D), and the MD simulation predicted specific interactions with mtInhA of the D-ring and methylene group between the B-ring and C-ring; however, there is still room for improvement in the A-ring structure. In this study, a structure-activity relationship study of the A-ring was attempted with the assistance of in silico docking simulations. In brief, the virtual chemical library of A-ring-modified KES4 was constructed and subjected to in silico docking simulation against mtInhA using the GOLD program. Among the selected candidates, we achieved synthesis of seven compounds, and the bioactivities (effects on InhA activity and mycobacterial growth and cytotoxicity) of the synthesized molecules were evaluated. Among the compounds tested, two candidates (compounds 3d and 3f) exhibited superior properties as mtInhA-targeted anti-infectives for mycobacteria than the lead compound KES4.

Folic acid (FA) can be protected the neural tube defects (NTDs) causing nitric oxide (NO) induction, but the alleviation mechanism of the detailed FA function against NO has not yet been clarified. This study focused on elucidation of the interaction of FA and NO. FA suppressed nitrite accumulation as the NO indicator in lipopolysaccharide (LPS)-stimulated RAW264.7 cells, then the expression of the i NOS gene due to the LPS treatment was not inhibited by FA, suggesting that FA can modulate against NO or nitrogen radicals. NOR3 (4-Ethyl-2-hydroxyamino-5-nitro-3-hexenamide) as the NO donor was used for evaluation of the NO scavenging activity of FA. FA suppressed the nitrite accumulation in a dose-dependent manner. To confirm the reaction product of FA and NO (FA-NO), liquid chromatography-mass spectrometry (LC/MS) was used to measure a similar system containing NOR3 and FA, and then detected the mass numbers of the FA-NO as m/z 470.9 (M + H) + and m/z 469.1 (M - H) - . In addition, the adducts of the FA-NO derived from 14 NO and 15 NO gave individual mass numbers of the isotopic ratio of nitrogen for the following products: FA- 14 NO, m/z 471.14 (M + H) + ; m/z 469.17 (M - H) - and FA- 15 NO, m/z 472.16 (M + H) + ; m/z 470.12 (M - H) - . To clarify the detailed NO scavenging action of FA, an electron spin resonance (ESR) study for radical detecting of the system containing carboxy-PTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) as an NO detection reagent in the presence of NOR3 and FA was performed. The carboxy-PTI (2-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl) radical produced from the reaction with NO reduced in the presence of FA showing that FA can directly scavenge NO. These results indicated that NO scavenging activity of FA reduced the accumulation of nitrite in the LPS-stimulated RAW264.7 cells. The NO modulation due to FA would be responsible for the alleviation from the failure in neural tube formation causing a high level of NO production.

Background: Mycobacterium tuberculosis enoyl-acyl carrier protein reductase (mtInhA) is involved in the biosynthesis of mycolic acids, a major component of mycobacterial cell walls, and has been targeted in the development of anti-tuberculosis (TB) drugs. In our previous in silico structure-based drug screening study, we identified KES4, a novel class of mtInhA inhibitor. KES4 is composed of four ring structures (A-D-rings) and molecular dynamic simulation predicted that the D-ring is essential for the interaction with mtInhA., Methods: The structure-activity relationship study of the D-ring was attempted and aided by in silico docking simulations to improve the mtInhA inhibitory activity of KES4. A virtual chemical library of the D-ring-modified KES4 was then constructed and subjected to in silico docking simulation against mtInhA using the GOLD program. The candidate compound showing the highest GOLD score, referred to as KEN1, was synthesized, and its biological properties were compared with those of the lead compound KES4., Results: We achieved the synthesis of KEN1 and evaluated its effects on InhA activity, mycobacterial growth, and cytotoxicity. The antimycobacterial activity of KEN1 was comparable to that of the lead compound (KES4), although it exhibited superior activity in mtInhA inhibition. \., Conclusions: We obtained a KES4 derivative with high mtInhA inhibitory activity by in silico docking simulation with a chemical library consisting of a series of D-ring-modified KES4., Competing Interests: None

Objective This study aims to elucidate the association between the clinical characteristics of post-colonoscopy colorectal cancer (PCCRC) and quality indicators (QIs) of colonoscopy. Methods Patients with PCCRC who underwent total colonoscopy (TCS) and were histologically diagnosed with adenocarcinoma within six months to five years of the last examination were included in this study. PCCRC and normally detected cancer (NDC) identified within the same period were compared in terms of their clinicopathological characteristics. Furthermore, the QIs at PCCRC detection were compared to those at the last examination. Results Patients with PCCRC had a significantly higher rate of colon surgery history than those with NDC (PCCRC: 25/76, 32.9%; NDC: 31/1,437, 2.2%; p<0.001), but the invasion depth in these patients was significantly shallower (PCCRC: ≤Tis/≥T1, 37/39; NDC: ≤Tis/≥T1, 416/1,021; p<0.001). Among patients with PCCRC, the T1b group had significantly more non-polypoid growth (NPG)-type cases than PG-type CRC cases (p=0.018). The adenoma detection rate (ADR) of colonoscopists performing TCS was 30.2-52.8%. Furthermore, the ADR of colonoscopists at the time of PCCRC detection (36.7%±5.9%) was significantly higher than that of colonoscopists who performed the last examination (34.9%±4.4%; p=0.034). The withdrawal time for negative colonoscopy (WT-NC) at detection was significantly longer than that at the last examination (at detection: 494.3±253.8 s; at last examination: 579.5±243.6 s; p=0.010). Conclusion Given that these PCCRC cases were post-colon surgery cases, had a long WT-NC, and were detected by colonoscopists with a high ADR, most cases showed lesions that were missed during the previous colonoscopy. Caution should be practiced in order to avoid missing flat, NPG-type tumors.

This study focused on exploring the nuclear factor-erythroid-2-related factor (Nrf2) active compound to avoid oxidative stress related to various diseases, such as obesity and diabetes mellitus. The activity of the Nrf2-ARE (antioxidant response element) signaling was evaluated by a reporter assay involving over five hundred various edible medicinal herbs, and the highest Nrf2 activity was found in the ethanol extract of Peucedanum japonicum leaves. The active compound in the extract was isolated by high performance liquid chromatography (HPLC), and the chemical structure was identical to pteryxin based on 1 H, 13 C-NMR spectra and liquid chromatography/time-of-fright mass spectrometer (LC/TOF/MS). From the pteryxin, the transcription factor Nrf2 was accumulated in the nucleus and resulted in the expression of the antioxidant protein, heme oxygenase-1 (HO-1). In addition, the Nrf2 activity involving HO-1 expression due to coumarin derivatives was evaluated together with pteryxin. This suggested that the electrophilicity, due to the α,β-carbonyl and/or substituted acyl groups in the molecule, modulates the cysteine residue in Keap1 via the Michel reaction, at which point the Nrf2 is dissociated from the Keap1. These results suggest that pteryxin will be a useful agent for developing functional foods.

The physical interaction of the human growth factor receptor-bound protein 14 (hGrb14) and the insulin receptor (IR) represses insulin signaling. With respect to the recruiting mechanism of hGrb14 to IR respond to insulin stimulus, our previous reports have suggested that phosphorylation of Ser 358 , Ser 362 , and Ser 366 in hGrb14 by glycogen synthase kinase-3 repressed hGrb14-IR complex formation. In this study, we investigated phosphatase-mediated dephosphorylation of the hGrb14 phosphoserine residues. An in vitro phosphatase assay with hGrb14-derived synthetic phosphopeptides suggested that protein phosphatase 1 (PP1) is involved in the dephosphorylation of Ser 358 and Ser 362 . Furthermore, coimmunoprecipitation experiments suggested that insulin-induced hGrb14-IR complex formation was repressed by the substitution of Ser 358 or Ser 362 with glutamic acid. These findings suggested that phosphate groups on Ser 358 and Ser 362 in hGrb14 are dephosphorylated by PP1, and the dephosphorylation facilitates hGrb14-IR complex formation., (© 2019 European Peptide Society and John Wiley & Sons, Ltd.)

Extant eukaryote ecology is primarily sustained by oxygenic photosynthesis, in which chlorophylls play essential roles. The exceptional photosensitivity of chlorophylls allows them to harvest solar energy for photosynthesis, but on the other hand, they also generate cytotoxic reactive oxygen species. A risk of such phototoxicity of the chlorophyll must become particularly prominent upon dynamic cellular interactions that potentially disrupt the mechanisms that are designed to quench photoexcited chlorophylls in the phototrophic cells. Extensive examination of a wide variety of phagotrophic, parasitic, and phototrophic microeukaryotes demonstrates that a catabolic process that converts chlorophylls into nonphotosensitive 13 2 ,17 3 -cyclopheophorbide enols (CPEs) is phylogenetically ubiquitous among extant eukaryotes. The accumulation of CPEs is identified in phagotrophic algivores belonging to virtually all major eukaryotic assemblages with the exception of Archaeplastida, in which no algivorous species have been reported. In addition, accumulation of CPEs is revealed to be common among phototrophic microeukaryotes (i.e., microalgae) along with dismantling of their secondary chloroplasts. Thus, we infer that CPE-accumulating chlorophyll catabolism (CACC) primarily evolved among algivorous microeukaryotes to detoxify chlorophylls in an early stage of their evolution. Subsequently, it also underpinned photosynthetic endosymbiosis by securing close interactions with photosynthetic machinery containing abundant chlorophylls, which led to the acquisition of secondary chloroplasts. Our results strongly suggest that CACC, which allowed the consumption of oxygenic primary producers, ultimately permitted the successful radiation of the eukaryotes throughout and after the late Proterozoic global oxygenation.

Our current study demonstrated that the marine peroxy sesquiterpenoids isolated from the Okinawan soft coral Sinularia sp. have an antitumor activity in human colon cancer cell (HCT) 116 colon cancer cells with their induction of apoptosis due to H₂O₂ production derived from the compounds. This study clarified that peroxy sesquiterpenoids ( 1 and 2 ) inhibited anti-apoptosis proteins, such as B-cell lymphoma-extra large (Bcl-xL) and phosphoAkt (pAkt). In addition, the heme oxygenase-1 (HO-1), nuclear factor-erythroid-2-related factor (Nrf2), and phosphoNrf2 (pNrf2) proteins related to the cell survival regulation signal of Nrf2-ARE (antioxidant response element) were also suppressed in the presence of these compounds. While the cells treated with the compounds and trolox as an antioxidant expressed the inhibited proteins, such as HO-1, Nrf2, and Bcl-xL, it was suggested that the H₂O₂ involving free radical reactions derived from the molecule would be a trigger of apoptosis with the modulation of Nrf2-ARE signaling in the cells.

Synchronous spawning as mass reproduction is well known to occur in many hermatypic corals, which is one of the mysterious life birth events. However, its contributing mechanism has not yet been clarified. This study placed focus on elucidating a neurotransmitter as endocrine signals that contribute to the synchronous spawning. First, the determination method of the neurotransmitters in coral was established by LC/MS in the selective ion mode together with a solid phase extraction method. As a result, the similar contents of the neurotransmitters for dopamine (DA), adrenaline (AD) and noradrenaline (NR) were detected in both the hermatypic corals of Acropora intermedia and Acropora digitifera. More interestingly, these neurotransmitters increased through the reproductive event during the synchronous spawning of A. intermedia, particularly, remarkable changes in the NR and DA were observed. In addition, hydrogen peroxide is known as the spawning stimulant and the metabolic by-product of the neurotransmitters, which was exposed to A. digitifera, then the neurotransmitters increased as well as those of the synchronization of spawning. All of the results suggested that the neurotransmitters contribute to the synchronous spawning in the hermatypic corals., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Vanillin is valuable and popular flavor used in foods and cosmetics. Many bacteria species have the ability to decarboxylate substituted cinnamic acids in order to form vanillin. However, the phenolic biotransformation including vanillin production in a common fungus, the Aspergillus luchuensis, which is used in distilled beverages, has not yet been clarified. This study focused on elucidating the vanillin production due to phenolic biotransformation in A. luchuensis during fermentation. The phenolic metabolites were extracted by a solid phase column and they were determined using on LC/MS and LC/MS/MS in a selective ion mode. As a result, ferulic acid, vanillin and vanillic acid, were detected in the rice koji fermentationed by A. luchuensis and also fermentated with yeast. In addition, the accurate molecular formula of vanillin glucoside (C 14 H 17 O 8 , 313.0927, (M-H) - and its production ions was also determined by HRESI-mass spectrometry. Based on the results including the phenolic metabolites and related genes found in A. luchuensis genome, this study proposed the vanillin production mechanism due to the side chain cleavage of ferulic acid through Coenzyme A (CoA) and feruloyl-CoA hydratase/lyase, to form vanillin and acetyl-COA. In this study, another possible vanillin production pathway also was proposed due to the neutral hexose hydrolysis of vanillin glucoside. The subsequent dehydrogenation of vanillin produced vanillic acid. In addition, vanillin was detected in the distilled alcohol indicating its contribution to the aroma profile of beverages. It has been unknown that the vanillin in the distilled solution is derived from the vanillin produced during rice-koji and/or moromi mash fermentations.

The enzymes responsible for biotin biosynthesis in mycobacteria have been considered as potential drug targets owing to the important role in infection and cell survival that the biotin synthetic pathway plays in Mycobacterium tuberculosis. Among the enzymes that comprise mycobacterium biotin biosynthesis systems, 7,8-diaminopelargonic acid synthase (DAPAS) plays an essential role during the stationary phase in bacterial growth. In this study, compounds that inhibit mycobacterial DAPAS were screened in the virtual chemical library using an in silico structure-based drug screening (SBDS) technique, and the antimycobacterial activity of the selected compounds was validated experimentally. The DOCK-GOLD programs utilized by in silico SBDS facilitated the identification of a compound, referred to as KMD6, with potent inhibitory effects on the growth of model mycobacteria (M. smegmatis). The subsequent compound search, which was based on the structural features of KMD6, resulted in identification of three additional active compounds, designated as KMDs3, KMDs9 and KMDs10. The inhibitory effect of these compounds was comparable to that of isoniazid, which is a first-line antituberculosis drug. The high antimycobacterial activity of KMD6, KMDs9 and KMDs10 was maintained on the experiment with M. tuberculosis. Of the active compounds identified, KMDs9 would be a promising pharmacophore, owing to its long-term antimycobacterial effect and lack of cytotoxicity.

In this study, the function of fucoxanthinol (FxOH) as a bioavailable marine carotenoid together with the pre-metabolite, fucoxanthin (Fx), was examined through the Nrf2-ARE pathway. The antioxidant activity in the low concentration range of the compounds (1-4 μM) with a peroxyl radical scavenging capacity was proved by the ORAC (Oxygen Radical Absorbance Capacity) method and an ESR study. Similar concentrations of the compound also activated the Nrf2-ARE signaling with the Nrf2 translocation into the nuclear, then the expression of the antioxidant protein HO-1 increased. On the other hand, the high concentrations of both compounds (>10 μM) induced apoptosis with caspase 3/7 activation during suppression of the anti-apoptotic proteins, such as Bcl-XL and phosphorous Akt (pAkt). The Nrf2 expression was then activated in the nuclear, indicating that the Nrf2 plays a significant role in the cytoprotective effect against the toxicity of the compounds. These results indicated that the compounds have the dual functions of a cytoprotective effect and the apoptosis induction dependent on the treated concentrations through the Nrf2 activation. In addition, the results of all the assays involved in our previous studies suggested that the metabolite FxOH having a higher activity than the Fx, will be a bioavailable compound in biological systems., Competing Interests: All the authors declare that there is no conflict of interest for any of them.

Growth factor receptor-bound protein 14 (Grb14) is a negative regulator of insulin receptor (IR) and is involved in a negative feedback mechanism of insulin signaling. Grb14 associates with IR and inhibits its tyrosine kinase activity through the between pleckstrin homology and Src homology-2 (BPS) domain. We previously reported that the pharmacological inhibition and knockdown of glycogen synthase kinase-3 (GSK-3) facilitates the insulin-induced complex formation of human Grb14 (hGrb14) and IR, suggesting that GSK-3 suppresses hGrb14 recruitment to IR. This study further investigated a functional phosphorylation of the serine residues in hGrb14 BPS domain, identified as putative GSK-3 targets to verify an effect of GSK-3 on the hGrb14-IR complex formation. In vitro kinase assay using the motif-derived peptides showed that the serine residues located in N-terminal (Ser358, Ser362 and Ser366) and C-terminal (Ser419 and Ser423) regions of the BPS domain were phosphorylated by GSK-3. Co-immunoprecipitation and yeast two-hybrid (Y2H) experiments suggested that the negative charges genetically introduced on the Ser358, Ser362 and Ser366 suppressed the association of hGrb14 to IR. Surface plasmon resonance experiment gave Kd values of 8 nM for recombinant hGrb14 with respect to the interaction with IR β-subunit, and this affinity was lost after the replacements of the Ser358, Ser362 and Ser366 with glutamic acid residues. Y2H experiment with the BPS domain alone; however, did not show any difference owing to the same mutations. It is therefore evident that the N-terminus of the BPS domain plays an important role in the regulation of hGrb14-IR complex formation through phosphorylation, in addition to other domains., (© The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Background: Enzymes responsible for cell wall development in Mycobacterium tuberculosis are considered as potential targets of anti-tuberculosis (TB) agents. Mycobacterial cyclopropane mycolic acid synthase 1 (CmaA1) is essential for mycobacterial survival because of its critical role in synthesizing mycolic acids., Materials and Methods: We screened compounds that were capable of interacting with the mycobacterial CmaA1 active site using a virtual compound library with an in silico structure-based drug screening (SBDS). Following the selection of such compounds, their antimycobacterial activity was examined., Results: With the in silico SBDS, for which we also used DOCK-GOLD programs and screening methods that utilized the structural similarity between the selected active compounds, we identified two compounds with potent inhibitory effects on mycobacterial growth. The antimycobacterial effect of the compounds was comparable to that of isoniazid, which is used as a first-line anti-TB drug., Conclusion: The compounds identified through SBDS were expected to be a novel class of anti-TB pharmacophores.

In this study, the isolates of the peroxy sesquiterpenoids (1-3) from the Okinawan soft coral, Sinularia sp., indicated cytotoxicity in HCT116 colon cancer cells. The apoptotic cells with a nuclear condensation were detected in the presence of these compounds, then the caspase 3/7 activity was induced, indicating that the compounds have a potential antitumor activity by apoptosis-induction. The cells treated with these compounds were generated reactive oxygen species (ROS), indicating that the ROS is related to the induction of apoptosis. The ROS production reduced in the presence of catalase or trolox, indicating that hydrogen peroxide (H2O2) is generated through a certain free radical reaction derived from the compound. In fact, the accumulation of intracellular H2O2 was also confirmed in the presence of these compounds. Based on all the results, this study proposed the apoptosis-inducing mechanism due to the compounds that the H2O2 produced involving free radical reactions derived from cleavage of the end or hydro-peroxide in the molecule induced cell death., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Aim: Protein and energy malnutrition is a severe problem for patients with liver cirrhosis (LC) and fasting often induces starvation which is a vitally important outcome. Dietary restriction is essential for endoscopic injection sclerotherapy (EIS) in patients with risky esophageal varices, thereby creating the possible exacerbation of nutritional state and inducing liver dysfunction. Whether EIS induces nutritional deficiency in LC patients and the effects of branched-chain amino acid (BCAA)-enriched nutrient are prospectively investigated., Methods: A total of 61 LC patients were randomly divided into an EIS monotherapy group (non-BCAA group, n = 31) and an EIS combined with BCAA therapy group (n = 30). Platelet count, blood chemistry and somatometry values were prospectively measured at five time points., Results: The platelet counts before treatment were at the same level in both groups (P = 0.72). Three months after treatment, the counts decreased in the non-BCAA group; however, they increased in the BCAA group (P = 0.019). Body mass index, triceps skin fold thickness and arm muscle circumference significantly decreased in both groups. The BCAA and tyrosine ratio value increased only in the BCAA group (P < 0.01). The skeletal muscle volume measured by InBody720 significantly decreased in the non-BCAA group (P < 0.001)., Conclusion: EIS induced protein-energy malnutrition, however, skeletal muscle volume was maintained by taking BCAA. Administration of BCAA had some effect in maintaining the nutritional state, and may improve the platelet count. Taking a greater amount of nutrients and shorter dietary restriction period or hospitalization was desirable., (© 2016 The Japan Society of Hepatology.)

The acquisition of drug resistance mediated by the interaction of tumor cells with the extracellular matrix (ECM), commonly referred to as cell adhesion-mediated drug resistance (CAM-DR), has been observed not only in hematopoietic tumor cells but also in solid tumor cells. We have previously demonstrated that a 22-mer peptide derived from fibronectin, FNIII14, can inhibit cell adhesion through the inactivation of β1 integrin; when coadministered with cytarabine, FNIII14 completely eradicates acute myelogenous leukemia by suppressing CAM-DR. In this study, we show that our FNIII14 peptide also enhances chemotherapy efficacy in solid tumors. Coadministration of FNIII14 synergistically enhances the cytotoxicity of doxorubicin and aclarubicin in mammary tumor and melanoma cells, respectively. The solid tumor cell chemosensitization induced by FNIII14 is dependent upon the upregulation and activation of the pro-apoptotic protein, Bim. Furthermore, the metastasis of tumor cells derived from ventrally transplanted mammary tumor grafts is suppressed by the coadministration of FNIII14 and doxorubicin. These results suggest that the coadministration of our FNIII14 peptide with chemotherapy could achieve efficient solid tumor eradication by increasing chemosensitivity and decreasing metastasis. The major causes of tumor recurrence are the existence of chemotherapy-resistant primary tumor cells and the establishment of secondary metastatic lesions. As such, coadministering FNIII14 with anti-cancer drugs could provide a promising new approach to improve the prognosis of patients with solid tumors., Competing Interests: The authors have declared that no competing interests exist.

Three cadinane-type sesquiterpenoids, endoperoxide (1) and hydroperoxides (2, 3) together with three known sesquiterpenoids (4-6) were isolated from an Okinawan soft coral, Sinularia species. Structures of these isolates were elucidated by spectroscopic analyses (NMR, IR and MS) and molecular modeling. In addition, the isolates 1-3 as new compounds were examined for biological activities, resulting that they have antibacterial activity and weak cytotoxicity against HCT116 cells as well as anti-inflammatory effect on LPS/IFN-γ-stimulated RAW 264.7 macrophage cells.