Protective Effects of Inulin on Stress-Recurrent Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD) is a chronic inflammatory condition of the digestive tract and is closely associated with the homeostasis of the gut microbiota. Inulin, as a natural prebiotic, displays anti-inflammatory activity and maintains equilibrium of the intestinal microbiota. In this study, our research aimed to explore the potential of inulin in enhancing intestinal immunity and reducing inflammation in stress-recurrent IBD. In this study, a co-culture intestinal epithelium model and a stress-recurrent IBD mouse model was used to examine the protective effects of inulin. It was observed that inulin digesta significantly reduced pro-inflammatory cytokine expression (CXCL8/IL8 and TNFA) and increased MUC2 expression in intestinal epithelial cells. In vivo, our findings showed that Inulin intake significantly prevented IBD symptoms. This was substantiated by a decrease in serum inflammatory markers (IL-6, CALP) and a downregulation of inflammatory cytokine (Il6) in colon samples. Additionally, inulin intake led to an increase in short-chain fatty acids (SCFAs) in cecal contents and a reduction in the expression of endoplasmic reticulum (ER) stress markers (CHOP, BiP). Our results highlight that inulin can improve stress-recurrent IBD symptoms by modulating microbiota composition, reducing inflammation, and alleviating ER stress. These findings suggested the therapeutic potential of inulin as a dietary intervention for ameliorating stress-recurrent IBD.

Small-Molecule Inhibitor of 8-Oxoguanine DNA Glycosylase 1 Regulates Inflammatory Responses during Pseudomonas aeruginosa Infection.

The DNA repair enzyme 8-oxoguanine DNA glycosylase 1 (OGG1), which excises 8-oxo-7,8-dihydroguanine lesions induced in DNA by reactive oxygen species, has been linked to the pathogenesis of lung diseases associated with bacterial infections. A recently developed small molecule, SU0268, has demonstrated selective inhibition of OGG1 activity; however, its role in attenuating inflammatory responses has not been tested. In this study, we report that SU0268 has a favorable effect on bacterial infection both in mouse alveolar macrophages (MH-S cells) and in C57BL/6 wild-type mice by suppressing inflammatory responses, particularly promoting type I IFN responses. SU0268 inhibited proinflammatory responses during Pseudomonas aeruginosa (PA14) infection, which is mediated by the KRAS-ERK1-NF-κB signaling pathway. Furthermore, SU0268 induces the release of type I IFN by the mitochondrial DNA-cGAS-STING-IRF3-IFN-ß axis, which decreases bacterial loads and halts disease progression. Collectively, our results demonstrate that the small-molecule inhibitor of OGG1 (SU0268) can attenuate excessive inflammation and improve mouse survival rates during PA14 infection. This strong anti-inflammatory feature may render the inhibitor as an alternative treatment for controlling severe inflammatory responses to bacterial infection.

Within- and between-laboratory reproducibility and predictive capacity of amino acid derivative reactivity assay (ADRA) using a 0.5 mg/mL test chemical solution: Results of the study for reproducibility confirmation implemented in five participating laboratories.

The amino acid derivative reactivity assay (ADRA) is an in chemico alternative assay for skin sensitization listed in OECD test guideline 442C. ADRA evaluates the reactivity of sensitizers to proteins, which is key event 1 in the skin sensitization adverse outcome pathway. Although the current key event 1 evaluation method is a simple assay that evaluates nucleophile and test chemical reactivity, mixtures of unknown molecular weights cannot be evaluated because a constant molar ratio between the nucleophile and test chemical is necessary. In addition, because the nucleophile is quantified by HPLC, the frequency of co-eluting the test chemical and nucleophile increases when measuring multi-component mixtures. To solve these issues, test conditions have been developed using a 0.5 mg/mL test chemical solution and fluorescence-based detection. Since the practicality of these methods has not been substantiated, a validation test to confirm reproducibility was conducted in this study. The 10 proficiency substances listed in the ADRA guidelines were tested three times at five different laboratories. The results of both within- and between-laboratory reproducibility were 100%, and the results of ultraviolet- and fluorescence-based measurements were also consistent. In addition to the proficiency substances, a new positive control, squaric acid diethyl ester, was tested three times at the five laboratories. The results showed high reproducibility with N-(2-(1-naphthyl)acetyl)-l-cysteine depletion of 37%-52% and α-N-(2-(1-naphthyl)acetyl)-l-lysine depletion of 99%-100%. Thus, high reproducibility was confirmed in both evaluations of the 0.5 mg/mL test chemical and the fluorescence-based measurements, validating the practicability of these methods.

The within- and between-laboratories reproducibility and predictive capacity of Amino acid Derivative Reactivity Assay using 4 mM test chemical solution: Results of ring study implemented at five participating laboratories.

Amino acid derivative reactivity assay (ADRA) for skin sensitization was adopted as an alternative method in the 2019 OECD Guideline for the Testing of Chemicals (OECD TG 442C). The molar ratio of the nucleophilic reagent to the test chemicals in the reaction solution was set to 1:50. Imamura et al. reported that changing this molar ratio from 1:50 to 1:200 reduced in false negatives and improved prediction accuracy. Hence, a ring study using ADRA with 4 mM of a test chemical solution (ADRA, 4 mM) was conducted at five different laboratories to verify within- and between-laboratory reproducibilities (WLR and BLR, respectively). In this study, we investigated the WLR and BLR using 14 test chemicals grouped into three classes: (1) eight proficiency substances, (2) four test chemicals that showed false negatives in the ADRA with 1 mM test chemical solution (ADRA, 1 mM), but correctly positive in ADRA (4 mM), and (3) current positive control (phenylacetaldehyde) and a new additional positive control (squaric acid diethyl ester). The results showed 100% reproducibility and 100% accuracy for skin sensitization. Hence, it is clear that the ADRA (4 mM) is an excellent test method in contrast to the currently used ADRA (1 mM). We plan to resubmit the ADRA (4 mM) test method to the OECD Test Guideline Group in the near future so that OECD TG 442C could be revised for the convenience and benefit of many ADRA users.

Polygalae Radix shortens the circadian period through activation of the CaMKII pathway.

CONTEXT: The mammalian circadian clock system regulates physiological function. Crude drugs, containing Polygalae Radix, and Kampo, combining multiple crude drugs, have been used to treat various diseases, but few studies have focussed on the circadian clock. OBJECTIVE: We examine effective crude drugs, which cover at least one or two of Kampo, for the shortening effects on period length of clock gene expression rhythm, and reveal the mechanism of shortening effects. MATERIALS AND METHODS: We prepared 40 crude drugs. In the in vitro experiments, we used mouse embryonic fibroblasts from PERIOD2::LUCIFERASE knock-in mice (background; C57BL/6J mice) to evaluate the effect of crude drugs on the period length of core clock gene, Per2, expression rhythm by chronic treatment (six days) with distilled water or crude drugs (100 µg/mL). In the in vivo experiments, we evaluated the free-running period length of C57BL/6J mice fed AIN-93M or AIN-93M supplemented with 1% crude drug (6 weeks) that shortened the period length of the PERIOD2::LUCIFERASE expression rhythm in the in vitro experiments. RESULTS: We found that Polygalae Radix (ED50: 24.01 µg/mL) had the most shortened PERIOD2::LUCIFERASE rhythm period length in 40 crude drugs and that the CaMKII pathway was involved in this effect. Moreover, long-term feeding with AIN-93M+Polygalae Radix slightly shortened the free-running period of the mouse locomotor activity rhythm. DISCUSSION AND CONCLUSIONS: Our results indicate that Polygalae Radix may be regarded as a new therapy for circadian rhythm disorder and that the CaMKII pathway may be regarded as a target pathway for circadian rhythm disorders.

Changes in sleep phase and body weight of mobile health App users during COVID-19 mild lockdown in Japan.

OBJECTIVE AND METHOD: The stay-at-home order during the COVID-19 pandemic has restricted individuals' social behaviors, and therefore, effected their lifestyle including sleep, diet, and physical activity. Using the cross-sectional study design with a large sample size (N = 30,275) from the mobile health App users in Japan, we show age-dependent lifestyle changes during a nonpunitive "mild lockdown" (from April to May 2020). RESULTS: Sleep onset and offset were delayed on work-days but not on free-days with increased sleep duration and decreased social jetlag, and the changes were more evident in the younger population. Although average weight change was close to none because of the users' characteristic (95% of App users try to lose weight), we investigated an association between lifestyle change and body-weight change. Participants who reported advanced sleep phase during mild lockdown described a weight decrease. In contrast, the delayed sleep phase reported a weight gain. The results were significant after adjustment of confounding factors including physical activity and meal changes. CONCLUSIONS: Although there is cumulative evidence showing a relationship between late chronotype and obesity, it is still unclear about the potential benefit of the chronotype management to control body weight. Thus, to the best of our knowledge, this is the first study investigating the association between chronotype and weight changes by leveraging a large cohort.

Melatonin treatment of repetitive behavioral deficits in the Cntnap2 mouse model of autism spectrum disorder.

Nighttime light pollution is linked to metabolic and cognitive dysfunction. Many patients with autism spectrum disorders (ASD) show disturbances in their sleep/wake cycle, and may be particularly vulnerable to the impact of circadian disruptors. In this study, we examined the impact of exposure to dim light at night (DLaN, 5 lx) in a model of ASD: the contactin associated protein-like 2 knock out (Cntnap2 KO) mice. DLaN was sufficient to disrupt locomotor activity rhythms, exacerbate the excessive grooming and diminish the social preference in Cntnap2 mutant mice. On a molecular level, DLaN altered the phase and amplitude of PER2:LUC rhythms in a tissue-specific manner in vitro. Daily treatment with melatonin reduced the excessive grooming of the mutant mice to wild-type levels and improved activity rhythms. Our findings suggest that common circadian disruptors such as light at night should be considered in the management of ASD.

The circadian clock is disrupted in mice with adenine-induced tubulointerstitial nephropathy.

Chronic Kidney Disease (CKD) is increasing in incidence and has become a worldwide health problem. Sleep disorders are prevalent in patients with CKD raising the possibility that these patients have a disorganized circadian timing system. Here, we examined the effect of adenine-induced tubulointerstitial nephropathy on the circadian system in mice. Compared to controls, adenine-treated mice showed serum biochemistry evidence of CKD as well as increased kidney expression of inflammation and fibrosis markers. Mice with CKD exhibited fragmented sleep behavior and locomotor activity, with lower degrees of cage activity compared to mice without CKD. On a molecular level, mice with CKD exhibited low amplitude rhythms in their central circadian clock as measured by bioluminescence in slices of the suprachiasmatic nucleus of PERIOD 2::LUCIFERASE mice. Whole animal imaging indicated that adenine treated mice also exhibited dampened oscillations in intact kidney, liver, and submandibular gland. Consistently, dampened circadian oscillations were observed in several circadian clock genes and clock-controlled genes in the kidney of the mice with CKD. Finally, mice with a genetically disrupted circadian clock (Clock mutants) were treated with adenine and compared to wild type control mice. The treatment evoked worse kidney damage as indicated by higher deposition of gelatinases (matrix metalloproteinase-2 and 9) and adenine metabolites in the kidney. Adenine also caused non-dipping hypertension and lower heart rate. Thus, our data indicate that central and peripheral circadian clocks are disrupted in the adenine-treated mice, and suggest that the disruption of the circadian clock accelerates CKD progression.

Oxidation of a cysteine-derived nucleophilic reagent by dimethyl sulfoxide in the amino acid derivative reactivity assay.

The amino acid derivative reactivity assay (ADRA), which is an in chemico alternative to the use of animals in testing for skin sensitization potential, offers significant advantages over the direct peptide reactivity assay (DPRA) in that it utilizes nucleophilic reagents that are sensitive enough to be used with test chemical solutions prepared to concentrations of 1 mm, which is one-hundredth that of DPRA. ADRA testing of hydrophobic or other poorly soluble compounds requires that they be dissolved in a solvent consisting of dimethyl sulfoxide (DMSO) and acetonitrile. DMSO is known to promote dimerization by oxidizing thiols, which then form disulfide bonds. We investigated the extent to which DMSO oxidizes the cysteine-derived nucleophilic reagents used in both DPRA and ADRA and found that oxidation of both N-(2-(1-naphthyl)acetyl)-l-cysteine (NAC) and cysteine peptide increases as the concentration of DMSO increases, thereby lowering the concentration of the nucleophilic reagent. We also found that use of a solvent consisting of 5% DMSO in acetonitrile consistently lowered NAC concentrations by about 0.4 µm relative to the use of solvents containing no DMSO. We also tested nine sensitizers and four nonsensitizers having different sensitization potencies to compare NAC depletion with and without 5% DMSO and found that reactivity was about the same with either solvent. Based on the above, we conclude that the use of a solvent containing 5% DMSO has no effect on the accuracy of ADRA test results. We plan to review and propose revisions to OECD Test Guideline 442C based on the above investigation.

Cause of and countermeasures for oxidation of the cysteine-derived reagent used in the amino acid derivative reactivity assay.

The amino acid derivative reactivity assay (ADRA) is an in chemico alternative to animal testing for skin sensitization that solves certain problems found in the use of the direct peptide reactivity assay (DPRA). During a recent validation study conducted at multiple laboratories as part of the process to include ADRA in an existing OECD test guideline, one of the nucleophilic reagents used in ADRA-N-(2-(1-naphthyl)acetyl)-l-cysteine (NAC)-was found to be susceptible to oxidation in much the same manner that the cysteine peptide used in DPRA was. Owing to this, we undertook a study to clarify the cause of the promotion of NAC oxidation. In general, cysteine and other chemicals that have thiol groups are known to oxidize in the presence of even minute quantities of metal ions. When metal ions were added to the ADRA reaction solution, Cu2+ promoted NAC oxidation significantly. When 0.25 µm of EDTA was added in the presence of Cu2+ , NAC oxidation was suppressed. Based on this, we predicted that the addition of EDTA to the NAC stock solution would suppress NAC oxidation. Next, we tested 82 chemicals used in developing ADRA to determine whether EDTA affects ADRA's ability to predict sensitization. The results showed that the addition of EDTA has virtually no effect on the reactivity of NAC with a test chemical, yielding an accuracy of 87% for predictions of skin sensitization, which was roughly the same as ADRA.

The within- and between-laboratory reproducibility and predictive capacity of the in chemico amino acid derivative reactivity assay: Results of validation study implemented in four participating laboratories.

The amino acid derivative reactivity assay (ADRA) is an in chemico alternative method that focuses on protein binding as the molecular initiating event for skin sensitization. It is a simple and versatile method that has successfully solved some of the problems of the direct peptide reactivity assay (DPRA). The transferability and within- and between-laboratory reproducibility of ADRA were evaluated and confirmed as part of a validation study conducted at four participating laboratories. The transfer of ADRA technology from the lead laboratory to the four participating laboratories was completed successfully during a two-step training program, after which the skin sensitization potentials of 40 coded chemicals were predicted based on the results of ADRA testing. Within-laboratories reproducibility was 100% (10 of 10), 100% (10 of 10), 100% (7 of 7) and 90% (9 of 10), or an average of 97.3% (36 of 37); between-laboratory reproducibility as calculated on the results of three laboratories at the time was 91.9%. The overall predictive capacity comprised an accuracy of 86.9%, sensitivity of 81.5% and specificity of 98.1%. These results satisfied the targets set by the validation management team for demonstrating transferability, within- and between-laboratory reproducibility, and predictive capacity as well as gave a clear indication that ADRA is easily transferable and sufficiently robust to be used in place of DPRA.

Potent and Selective Inhibitors of 8-Oxoguanine DNA Glycosylase.

The activity of DNA repair enzyme 8-oxoguanine DNA glycosylase (OGG1), which excises oxidized base 8-oxoguanine (8-OG) from DNA, is closely linked to mutagenesis, genotoxicity, cancer, and inflammation. To test the roles of OGG1-mediated repair in these pathways, we have undertaken the development of noncovalent small-molecule inhibitors of the enzyme. Screening of a PubChem-annotated library using a recently developed fluorogenic 8-OG excision assay resulted in multiple validated hit structures, including selected lead hit tetrahydroquinoline 1 (IC50 = 1.7 µM). Optimization of the tetrahydroquinoline scaffold over five regions of the structure ultimately yielded amidobiphenyl compound 41 (SU0268; IC50 = 0.059 µM). SU0268 was confirmed by surface plasmon resonance studies to bind the enzyme both in the absence and in the presence of DNA. The compound SU0268 was shown to be selective for inhibiting OGG1 over multiple repair enzymes, including other base excision repair enzymes, and displayed no toxicity in two human cell lines at 10 µM. Finally, experiments confirm the ability of SU0268 to inhibit OGG1 in HeLa cells, resulting in an increase in accumulation of 8-OG in DNA. The results suggest the compound SU0268 as a potentially useful tool in studies of the role of OGG1 in multiple disease-related pathways.

Pathophysiology in the suprachiasmatic nucleus in mouse models of Huntington's disease.

Disturbances in sleep/wake cycle are a common complaint of individuals with Huntington's disease (HD) and are displayed by HD mouse models. The underlying mechanisms, including the possible role of the circadian timing system, are not well established. The BACHD mouse model of HD exhibits disrupted behavioral and physiological rhythms, including decreased electrical activity in the central circadian clock (suprachiasmatic nucleus, SCN). In this study, electrophysiological techniques were used to explore the ionic underpinning of the reduced spontaneous neural activity in male mice. We found that SCN neural activity rhythms were lost early in the disease progression and was accompanied by loss of the normal daily variation in resting membrane potential in the mutant SCN neurons. The low neural activity could be transiently reversed by direct current injection or application of exogenous N-methyl-d-aspartate (NMDA) thus demonstrating that the neurons have the capacity to discharge at WT levels. Exploring the potassium currents known to regulate the electrical activity of SCN neurons, our most striking finding was that these cells in the mutants exhibited an enhancement in the large-conductance calcium activated K+ (BK) currents. The expression of the pore forming subunit (Kcnma1) of the BK channel was higher in the mutant SCN. We found a similar decrease in daytime electrical activity and enhancement in the magnitude of the BK currents early in disease in another HD mouse model (Q175). These findings suggest that SCN neurons of both HD models exhibit early pathophysiology and that dysregulation of BK current may be responsible.

Enantioselective Formal C-H Conjugate Addition of Acetanilides to ß-Substituted Acrylates by Chiral Iridium Catalysts.

The Ir-catalyzed enantioselective reaction of substituted acetanilides with ß-substituted α,ß-unsaturated esters provided chiral 3,3-disubstituted propanoates in high yield with good-to-excellent enantiomeric excess (up to 99 % ee). This transformation, initiated by sp2 C-H bond activation, is the first example of enantioselective formal C-H conjugate addition to ß-substituted α,ß-unsaturated carbonyl compounds. The starting materials are commercially available and/or readily accessible.

Design, synthesis, and taste evaluation of a high-intensity umami-imparting oxazole-based compound.

Umami taste is imparted predominantly by monosodium glutamate (MSG) and 5'-ribonucleotides. Recently, several different classes of hydrophobic umami-imparting compounds, the structures of which are quite different from MSG, have been reported. To obtain a novel umami-imparting compound, N-cinnamoyl phenethylamine was chosen as the lead compound, and a rational structure-optimization study was conducted on the basis of the pharmacophore model of previously reported compounds. The extremely potent umami-imparting compound 2-[[[2-[(1E)-2-(1,3-benzodioxol-5-yl)ethenyl]-4-oxazolyle]methoxy]methyl]pyridine, which exhibits 27,000 times the umami taste of MSG, was found. Its terminal pyridine residue and linear structure are suggested to be responsible for its strong activity. The time taken to reach maximum taste intensity exhibited by it, as determined by the time-intensity method, is 22.0 s, whereas the maximum taste intensity of MSG occurs immediately. This distinct difference in the time-course taste profile may be due to the hydrophobicity and strong receptor affinity of the new compound.

Strategies for the Total Synthesis of Clavicipitic Acid.

Clavicipitic acid is an ergot alkaloid, which was isolated from Claviceps strain and Claviceps fusiformis. Its unique tricyclic azepinoindole skeleton has attracted synthetic chemists, and various strategies have been developed for its total synthesis. These strategies can be generally categorized into two types based on the synthetic intermediates, namely, 4-substituted gramine derivatives and 4-substituted tryptophan derivatives. This Minireview summarizes the reported total syntheses from the point of these two key intermediates.

Catalytic and Enantioselective Synthesis of Chiral Multisubstituted Tribenzothiepins by Intermolecular Cycloadditions.

The first catalytic and highly enantioselective synthesis of tribenzothiepin derivatives was achieved. Two types of intermolecular cycloadditions using either diphenyl-sulfide-tethered diynes or 2-phenyl sulfanylbenzene-tethered diynes with a monoalkyne successfully gave chiral multisubstituted tribenzothiepins in good to excellent ee values under mild conditions. The inversion energy of this saddle-shaped molecule was calculated by measurement of the racemization rate of chiral tribenzothiepins using the Eyring kinetic equation under heating conditions. The present protocol could also be used to prepare a chiral tribenzoselenepin.

Total Synthesis of cis-Clavicipitic Acid from Asparagine via Ir-Catalyzed C-H bond Activation as a Key Step.

4-Substituted tryptophan derivatives and the total synthesis of cis-clavicipitic acid were achieved in reactions in which Ir-catalyzed C-H bond activation was a key step. The starting material for these reactions is asparagine, which is a cheap natural amino acid. The reductive amination step from the 4-substituted tryptophan derivative gave cis-clavicipitic acid with perfect diastereoselectivity.

Circadian regulation of allergic reactions by the mast cell clock in mice.

BACKGROUND: It remains elusive how allergic symptoms exhibit prominent 24-hour variations. In mammals the circadian clocks present in nearly all cells, including mast cells, drive the daily rhythms of physiology. Recently, we have shown that the circadian clocks drive the daily rhythms in IgE/mast cell-mediated allergic reactions. However, the precise mechanisms, particularly the specific roles of the mast cell-intrinsic clockwork in temporal regulation, remain unclear. OBJECTIVE: We determined whether the mast cell clockwork contributes to the temporal regulation of IgE/mast cell-mediated allergic reaction. METHODS: The kinetics of a time of day-dependent variation in passive cutaneous anaphylactic reactions were compared between mast cell-deficient mice reconstituted with bone marrow-derived cultured mast cells generated from mice with a wild-type allele and a dominant negative type mutation of the key clock gene Clock. We also examined the temporal responses of wild-type and Clock-mutated bone marrow-derived cultured mast cells to IgE stimulation in vitro. Furthermore, factors influencing the mast cell clockwork were determined by using in vivo imaging. RESULTS: The Clock mutation in mast cells resulted in the absence of temporal variations in IgE-mediated degranulation in mast cells both in vivo and in vitro associated with the loss of temporal regulation of FcεRI expression and signaling. Additionally, adrenalectomy abolished the mast cell clockwork in vivo. CONCLUSION: The mast cell-intrinsic clockwork, entrained by humoral factors from the adrenal gland, primarily contributes to the temporal regulation of IgE/mast cell-mediated allergic reactions. Our results reveal a novel regulatory mechanism for IgE-mediated mast cell responses that might underlie the circadian pathophysiology in patients with allergic diseases.

Chrono-biology, chrono-pharmacology, and chrono-nutrition.

The circadian clock system in mammals drives many physiological processes including the daily rhythms of sleep-wake behavior, hormonal secretion, and metabolism. This system responds to daily environmental changes, such as the light-dark cycle, food intake, and drug administration. In this review, we focus on the central and peripheral circadian clock systems in response to drugs, food, and nutrition. We also discuss the adaptation and anticipation mechanisms of our body with regard to clock system regulation of various kinetic and dynamic pathways, including absorption, distribution, metabolism, and excretion of drugs and nutrients. "Chrono-pharmacology" and "chrono-nutrition" are likely to become important research fields in chrono-biological studies.