Plasma free amino acid profiles are associated with serum high molecular weight adiponectin levels in Japanese medical check-up population without type 2 diabetes mellitus.

To prevent the progression of type 2 diabetes mellitus (T2DM), early detection and intervention are important. Several studies have already shown that the serum adiponectin level could be useful for evaluating the future risk of T2DM. Recently, plasma free amino acid (PFAA) concentrations have also emerged as potential biomarkers that predict the future onset of T2DM. In this study, we aimed to further characterise PFAA profiles by elucidating the association with the serum high molecular weight (HMW) adiponectin level in this cross-sectional study. A total of 1000 Japanese subjects who underwent medical check-ups were enrolled, and their plasma concentrations of 21 amino acids and clinical parameters were measured. The subjects without T2DM were divided into quartiles (Q1-4) by serum HMW adiponectin level, and the association with between PFAA concentrations was analysed. Concentrations of glutamate, alanine, proline, tyrosine, histidine, methionine, lysine, branched-chain amino acids (BCAAs) and tryptophan varied significantly according to the adiponectin quartile. Furthermore, serum adiponectin levels showed significant inverse correlations with these amino acids. The change in the PFAA profile in the group with the lowest adiponectin concentrations (Q1) was similar to that of T2DM patients. Although both adiponectin levels and PFAA concentrations are known to be altered by the accumulation of visceral fat and insulin resistance, the levels of glutamate, BCAA, lysine and tryptophan remain significantly associated with adiponectin level after adjustment for age, body mass index and homeostasis model assessment of insulin resistance, showing the direct association between PFAA concentrations and the serum HMW adiponectin level. Registration number: University Hospital Medical Information Network Clinical Trials Registry (UMIN-CTR) UMIN000029920, registered on Nov 13th 2017 (prospectively registered).

Actinorhodin Biosynthesis Terminates with an Unprecedented Biaryl Coupling Reaction.

A plethora of dimeric natural products exist with diverse chemical structures and biological activities. A major strategy for dimerization is aryl coupling catalyzed by cytochrome P450 or laccase. Actinorhodin (ACT) from Streptomyces coelicolor A3(2) has a dimeric pyranonaphthoquinone structure connected by a C-C bond. In this study, we identified an NmrA-family dimerizing enzyme, ActVA-ORF4, and a cofactor-independent oxidase, ActVA-ORF3, both involved in the last step of ACT biosynthesis. ActVA-ORF4 is a unique NAD(P)H-dependent enzyme that catalyzes the intermolecular C-C bond formation using 8-hydroxydihydrokalafungin (DHK-OH) as the sole substrate. On the other hand, ActVA-ORF3 was found to be a quinone-forming enzyme that produces the coupling substrate, DHK-OH and the final product, ACT. Consequently, the functional assignment of all essential enzymes in the biosynthesis of ACT, one of the best-known model natural products, has been completed.

Comparative Analysis of Serum microRNA in Diagnosed Ocular Sarcoidosis versus Idiopathic Uveitis with Ocular Manifestations of Sarcoidosis.

"Idiopathic" is the most common category of uveitis, representing cases in which a specific diagnosis has not been established despite work-up. Sarcoidosis is a systemic granulomatous disorder affecting multiple organs including the lungs, skin, kidneys, and eyes. We used microRNA (miRNA) microarrays to investigate serum miRNA profiles of patients with ocular sarcoidosis as diagnosed by specific criteria (diagnosed ocular sarcoidosis), and patients with idiopathic uveitis characterized by ocular manifestations of sarcoidosis (suspected ocular sarcoidosis). Principal component analysis (PCA) and hierarchical clustering showed that serum miRNA profiles of diagnosed ocular sarcoidosis and suspected ocular sarcoidosis were both clearly distinguishable from healthy controls. Furthermore, comparative analysis of the miRNA profiles showed highly similar patterns between diagnosed ocular sarcoidosis and suspected ocular sarcoidosis. Pathway analysis revealed common pathways were involved in the two groups, including those of WNT signaling and TGF-beta signaling. Our study demonstrated a high overlap of differentially expressed serum miRNAs in patients with diagnosed ocular sarcoidosis and suspected ocular sarcoidosis, suggesting that these groups share a similar underlying pathology and may represent possible variants of the disease. Characterization of serum miRNA profiles may provide an opportunity for earlier diagnosis and treatment, and may inform more accurate clinical prognosis in patients with an ocular sarcoidosis phenotype.

Characterization of stereospecific enoyl reductase ActVI-ORF2 for pyran ring formation in the actinorhodin biosynthesis of Streptomyces coelicolor A3(2).

Actinorhodin (ACT) is a benzoisochromanequinone antibiotic produced by Streptomyces coelicolor A3(2), which has served as a favored model organism for comprehensive studies of antibiotic biosynthesis and its regulation. (S)-DNPA undergoes various modifications as an intermediate in the ACT biosynthetic pathway, including enoyl reduction to DDHK. It has been suggested that actVI-ORF2 encodes an enoyl reductase (ER). However, its function has not been characterized in vitro. In this study, biochemical analysis of recombinant ActVI-ORF2 revealed that (S)-DNPA is converted to DDHK in a stereospecific manner with NADPH acting as a cofactor. (R)-DNPA was also reduced to 3-epi-DDHK with the comparable efficacy as (S)-DNPA, suggesting that the stereospecificity of ActVI-ORF2 was not affected by the stereochemistry at the C-3 of DNPA. ActVI-ORF2 is a new example of a discrete ER, which is distantly related to known ERs according to phylogenetic analysis.

Machine learning using clinical data at baseline predicts the efficacy of vedolizumab at week 22 in patients with ulcerative colitis.

Predicting the response of patients with ulcerative colitis (UC) to a biologic such as vedolizumab (VDZ) before administration is an unmet need for optimizing individual patient treatment. We hypothesized that the machine-learning approach with daily clinical information can be a new, promising strategy for developing a drug-efficacy prediction tool. Random forest with grid search and cross-validation was employed in Cohort 1 to determine the contribution of clinical features at baseline (week 0) to steroid-free clinical remission (SFCR) with VDZ at week 22. Among 49 clinical features including sex, age, height, body weight, BMI, disease duration/phenotype, treatment history, clinical activity, endoscopic activity, and blood test items, the top eight features (partial Mayo score, MCH, BMI, BUN, concomitant use of AZA, lymphocyte fraction, height, and CRP) were selected for logistic regression to develop a prediction model for SFCR at week 22. In the validation using the external Cohort 2, the positive and negative predictive values of the prediction model were 54.5% and 92.3%, respectively. The prediction tool appeared useful for identifying patients with UC who would not achieve SFCR at week 22 during VDZ therapy. This study provides a proof-of-concept that machine learning using real-world data could permit personalized treatment for UC.

Endoscopic severe mucosal atrophy indicates the presence of gastric cancer after Helicobacter pylori eradication -analysis based on the Kyoto classification.

BACKGROUND: Gastric cancer after Helicobacter pylori (HP) eradication is a crucial clinical challenge today as HP eradication therapy is widely performed. Detecting gastric cancer after HP eradication tends to be difficult with normal white-light endoscopy. In the present study, we aimed to identify easily-evaluated endoscopic findings that indicate the presence of gastric cancer after HP eradication so that endoscopists can consider additional detailed examinations at the site. METHODS: We analyzed the endoscopic images of 43 patients who underwent endoscopic submucosal dissection for early gastric cancer after HP eradication and 119 patients with an HP eradication history who underwent esophagogastroduodenoscopy for a medical checkup. Endoscopic findings were evaluated according to the Kyoto classification of gastritis (atrophy, intestinal metaplasia, enlarged folds, nodularity, and diffuse redness) and map-like redness. RESULTS: Patients with gastric cancer had significantly higher total Kyoto risk scores; more atrophy, intestinal metaplasia, and diffuse redness; and a significantly higher prevalence of map-like redness compared with those without gastric cancer, in the univariate analyses. We used logistic regression analysis with forward selection based on the likelihood ratio to develop a model using atrophy and diffuse redness. Receiver operating characteristic analysis showed that a score of A2 in the Kyoto classification of gastritis (open-type atrophic pattern in the Kimura-Takemoto classification) was an endoscopic marker for the presence of post-HP-eradication gastric cancer. CONCLUSIONS: Endoscopic severe gastric mucosal atrophy is useful to screen patients for gastric cancer after HP eradication.

Unveiling Two Consecutive Hydroxylations: Mechanisms of Aromatic Hydroxylations Catalyzed by Flavin-Dependent Monooxygenases for the Biosynthesis of Actinorhodin and Related Antibiotics.

Flavin-dependent monooxygenases are ubiquitous in living systems and are classified into single- or two-component systems. Actinorhodin, produced by Streptomyces coelicolor, is a representative polycyclic polyketide that is hydroxylated through the action of the two-component ActVA-5/ActVB hydroxylase system. These homologous systems are widely distributed in bacteria, but their reaction mechanisms remain unclear. This in vitro investigation has provided chemical proof of two consecutive hydroxylations via hydroxynaphthalene intermediates involved in actinorhodin biosynthesis. The ActVA-5 oxygenase component catalyzed a stepwise dihydroxylation of the substrate, whereas the ActVB flavin reductase not only supplied a reduced cofactor, but also regulated the quinone-hydroquinone interconversion of an intermediate. Our study provides clues for understanding the general biosynthetic mechanisms of highly functionalized aromatic natural products with structural diversity.

Analysis of free radical production capacity in mouse faeces and its possible application in evaluating the intestinal environment: a pilot study.

Complex interplay between the intestinal environment and the host has attracted considerable attention and has been well studied with respect to the gut microbiome and metabolome. Oxygen free radicals such as superoxide and the hydroxyl radical (•OH) are generated during normal cellular metabolism. They are toxic to both eukaryotic and prokaryotic cells and might thus affect intestinal homeostasis. However, the effect of oxygen free radicals on the intestinal environment has not been widely studied. Herein, we applied electron spin resonance spectroscopy with spin trapping reagents to evaluate oxygen free radical production capacity in the intestinal lumen and the faeces of mice. •OH was generated in faeces and lumens of the small and large intestines. There were no remarkable differences in •OH levels between faeces and the large intestine, suggesting that faeces can be used as alternative samples to estimate the •OH production capacity in the colonic contents. We then compared free radical levels in faecal samples among five different mouse strains (ddY, ICR, C57BL/6, C3H/HeJ, and BALB/c) and found that strain ddY had considerably higher levels than the other four strains. In addition, strain ddY was more susceptible to dextran sulphate sodium-induced colitis. These differences were possibly related to the relative abundance of the gut bacterial group Candidatus Arthromitus, which is known to modulate the host immune response. From these results, we suggest that the production capacity of oxygen free radicals in mouse faeces is associated with intestinal homeostasis.

5-Aminosalicylic acid aggravates colitis mimicking exacerbation of ulcerative colitis.

Ulcerative colitis (UC) is one of the major clinical phenotypes of inflammatory bowel diseases. Although 5-aminosalicylic acid (5-ASA) is widely used for UC and its efficacy and safety have been demonstrated, a few patients paradoxically develop a severe exacerbation of colitis by 5-ASA administration. It is crucial to know clinical features including endoscopic findings in this condition for making a correct diagnosis and a prompt decision to withdraw the medication. Here, we report case series with UC exacerbated by 5-ASA. Medical records of 8 UC patients experiencing an exacerbation of colitis after induction of 5-ASA that was improved by the withdrawal of 5-ASA but also re-aggravated by dose increase or re-administration of 5-ASA were reviewed. The patients were newly diagnosed with UC, started 5-ASA and developed an exacerbation in approximately 2 to 3 weeks. They did not appear to have systemic allergic reactions. Seven of the 8 patients had a high fever. Three of 5 patients who undertook total colonoscopy showed right-side-dominant colitis. These findings suggest clinical characteristics in this condition. Further assessment of clinical and endoscopic features in more cases is necessary for establishing diagnostic criteria and understanding underlying mechanisms in those cases where 5-ASA aggravates the colitis.

Depressive Status in Patients With Chronic Thromboembolic Pulmonary Hypertension.

BACKGROUND: The present comparative study with healthy volunteers was conducted to investigate the depressive status and temperament in patients with chronic thromboembolic pulmonary hypertension (CTEPH).Methods and Results:The results of the temperament and personality scale test, and the Quick Inventory of Depressive Symptomatology-Self Report revealed that CTEPH patients have a significantly higher depressive status than healthy volunteers. CONCLUSIONS: It may be that CTEPH patients are more likely to have a depressive temperament in origin. It is expected that the relationship between the biological traits of CTEPH (e.g., genetics) and patients' depressive temperament will be elucidated in the future.

Computer-Aided Prediction of Long-Term Prognosis of Patients with Ulcerative Colitis after Cytoapheresis Therapy.

Cytoapheresis (CAP) therapy is widely used in ulcerative colitis (UC) patients with moderate to severe activity in Japan. The aim of this study is to predict the need of operation after CAP therapy of UC patients on an individual level using an artificial neural network system (ANN). Ninety UC patients with moderate to severe activity were treated with CAP. Data on the patients' demographics, medication, clinical activity index (CAI) and efficacy of CAP were collected. Clinical data were divided into training data group and validation data group and analyzed using ANN to predict individual outcomes. The sensitivity and specificity of predictive expression by ANN were 0.96 and 0.97, respectively. Events of admission, operation, and use of immunomodulator, and efficacy of CAP were significantly correlated to the outcome. Requirement of operation after CAP therapy was successfully predicted by using ANN. This newly established ANN strategy would be used as powerful support of physicians in the clinical practice.

Combination therapy with infliximab and thiopurine compared to infliximab monotherapy in maintaining remission of postoperative Crohn's disease.

BACKGROUND AND AIMS: Infliximab is an efficacious agent used for the induction and maintenance of remission in Crohn's disease (CD), and recent studies suggested that it may also prevent the recurrence of this disease after surgery. The present study was performed to assess the efficacy and safety of infliximab in the postoperative setting, and to identify whether combination treatment with thiopurines had any additional beneficial effect as compared to mono-therapy. METHODS: We performed a retrospective cohort study to compare the efficacy of infliximab mono-therapy and combination treatment with a thiopurine in preventing recurrence after surgery. RESULTS: Forty-one patients who received infliximab as maintenance treatment following surgery from May 2002 to April 2010 were identified. Twenty-four were naive to infliximab, and 17 who underwent surgery during infliximab treatment were continued on it following surgery. The median follow-up period was 27 months (range 12-66 months). All patients continued infliximab as maintenance treatment, but 10 required dose intensification due to clinical recurrence. Kaplan-Meier analysis demonstrated that the use of concomitant thiopurine was correlated with the continuation of infliximab treatment at an 8-week interval (log-rank test p = 0.018). The rate of adverse event was 9.8% with no patient experiencing severe adverse reactions. CONCLUSION: Infliximab appears to be safe and it prevented clinical recurrence after surgery. Concomitant thiopurine use predicted response toward continuation of therapy at an 8-week interval. Prospective controlled studies to assess the efficacy of combination treatment in the postoperative setting are warranted.

Extensive amplification of GI-VII-6, a multidrug resistance genomic island of Salmonella enterica serovar Typhimurium, increases resistance to extended-spectrum cephalosporins.

GI-VII-6 is a chromosomally integrated multidrug resistance genomic island harbored by a specific clone of Salmonella enterica serovar Typhimurium (S.Typhimurium). It contains a gene encoding CMY-2 ß-lactamase (bla CMY-2), and therefore contributes to extended-spectrum cephalosporin resistance. To elucidate the significance of GI-VII-6 on adaptive evolution, spontaneous mutants of S. Typhimurium strain L-3553 were selected on plates containing cefotaxime (CTX). The concentrations of CTX were higher than its minimum inhibition concentration to the parent strain. The mutants appeared on the plates containing 12.5 and 25 mg/L CTX at a frequency of 10(-6) and 10(-8), respectively. No colonies were observed at higher CTX concentrations. The copy number of bla CMY-2 increased up to 85 per genome in the mutants, while the parent strain contains one copy of that in the chromosome. This elevation was accompanied by increased amount of transcription. The bla CMY-2 copy number in the mutants drastically decreased in the absence of antimicrobial selection pressure. Southern hybridization analysis and short-read mapping indicated that the entire 125 kb GI-VII-6 or parts of it were tandemly amplified. GI-VII-6 amplification occurred at its original position, although it also transposed to other locations in the genome in some mutants, including an endogenous plasmid in some of the mutants, leading to the amplification of GI-VII-6 at different loci. Insertion sequences were observed at the junction of the amplified regions in the mutants, suggesting their significant roles in the transposition and amplification. Plasmid copy number in the selected mutants was 1.4 to 4.4 times higher than that of the parent strain. These data suggest that transposition and amplification of the bla CMY-2-containing region, along with the copy number variation of the plasmid, contributed to the extensive amplification of bla CMY-2 and increased resistance to CTX.

Risk factors for decreased bone mineral density in inflammatory bowel disease: A cross-sectional study.

BACKGROUND & AIM: Although inflammatory bowel disease (IBD) patients are at risk for metabolic bone disease, studies analyzing this correlation have identified various risk factors, including disease phenotype, age, sex and steroid therapy. Furthermore, few studies have assessed risk factors for bone loss in Japanese IBD patients. This study analyzed risk factors for metabolic bone disease in Japanese IBD patients. METHODS: This cross-sectional study assessed 388 patients with IBD aged 20-50 years, including 232 with ulcerative colitis (UC) and 156 with Crohn's disease (CD). Bone mineral density of the femoral neck, total femur and lumbar spine was quantified by dual-energy X-ray absorptiometry. The blood concentrations of bone metabolism markers were measured. History of smoking and bone fracture, and nutritional intake were assessed using questionnaires. RESULTS: Of the 388 patients with IBD, 78 (20.1%; UC, 17.2%; CD, 24.4%) had osteopenia and 17 (4.4%; UC, 3.4%; CD, 5.8%) had osteoporosis, as assessed by T-score. Bone mineral density of the lumbar vertebrae was lower in males than in females. Multivariate regression analysis showed that risk factors for bone loss in UC patients were male sex, low body mass index (BMI), high steroid dose and disease location. Risk factors for bone loss in CD patients were male sex and low BMI. CONCLUSION: Among Japanese patients with IBD, male sex and low BMI were associated with increased risk for metabolic bone disease. In addition, Steroid therapy shouldn't be indiscriminate in UC patients. These findings may help identify patients at particularly high risk of metabolic bone disease and may help implement appropriate therapies in a timely manner and improve long-term quality of life.

Early intervention with adalimumab may contribute to favorable clinical efficacy in patients with Crohn's disease.

BACKGROUND: We evaluated the clinical efficacy of adalimumab (ADA) for Crohn's disease (CD) and analyzed predictive factors for clinical remission and long-term prognosis. METHODS: We retrospectively reviewed the medical records of 45 patients treated with ADA for CD at Keio University Hospital between October 2010 and March 2014. Clinical remission was defined as a Harvey-Bradshaw index of ≤4. RESULTS: Twenty-eight of 45 patients (62.2%) achieved clinical remission at week 4. Among these 28 patients, 18 patients (64.3%) maintained clinical remission at week 26, and among these, 16 patients (88.9%) maintained clinical remission at week 52. Absence of a history of bowel resection and absence of prior anti-tumor necrosis factor (anti-TNF) therapy were significant predictive factors for clinical remission at week 4 upon multivariate logistic regression analyses. Younger age and a disease duration of ≤3 years correlated with clinical remission at week 26 upon univariate analyses. Patients without a history of bowel resection showed significantly better long-term prognosis than those with a history of bowel resection (p = 0.01). None of the patients contracted a serious infectious disease. CONCLUSIONS: Younger age, shorter duration of disease, being naive to anti-TNF antagonists, and absence of a history of bowel resection were associated with the efficacy of ADA in CD patients.

Risk and management of intra-abdominal abscess in Crohn's disease treated with infliximab.

BACKGROUND AND AIMS: Infliximab (IFX) is a monoclonal antibody used to treat patients with Crohn's disease (CD). Intra-abdominal abscess formation is a major complication of CD with negative effects on patient prognosis. We have analyzed risk factors for abscess formation in CD patients treated with IFX. METHODS: CD patients who received IFX between January 2000 and April 2011 at Keio University Hospital were analyzed retrospectively. Risk factors for abscess formation were assessed by univariate and multivariate logistic regression analyses. RESULTS: Intra-abdominal abscess was seen in 15 of 258 patients. Univariate analyses showed serum C-reactive protein (CRP) concentration at 14 weeks after initiation of IFX (p = 0.021), serum albumin concentration at week 0 (p = 0.022) and week 14 (p = 0.004), the presence of anal lesions (p = 0.036), progression of intestine deformation (p = 0.015) and early loss of response to IFX (p < 0.0001) to be risk factors. Multivariate analysis showed that CRP concentration at 14 weeks [odds ratio (OR) 1.361] and loss of IFX response within 6 months (OR 5.361) were independent risk factors. CONCLUSIONS: Abscess formation should be suspected in patients with symptoms of CD recurrence during IFX therapy. Uncontrolled CRP concentration and early loss of response to IFX are risk factors.

Biosynthesis of pyranonaphthoquinone polyketides reveals diverse strategies for enzymatic carbon-carbon bond formation.

Pyranonaphthoquinones synthesized by Streptomyces bacteria via type II polyketide pathways are aromatic compounds build around a common three-ring structure, which is composed of pyran, quinone and benzene rings. Over the years, actinorhodin in particular has served as a model compound for studying the biosynthesis of aromatic polyketides, while some of the other metabolites such as granaticin, medermycin, frenolicin and alnumycin A have enabled comparative studies that complement our understanding how these complex biological systems function and have evolved. In addition, despite the similarity of the aglycone units, pyranonaphthoquinones in effect display remarkable diversity in tailoring reactions, which include numerous enzymatic carbon-carbon bond forming reactions. This review focuses on the current status of molecular genetic, biochemical and structural investigations on this intriguing family of natural products.

Mucosal healing with oral tacrolimus is associated with favorable medium- and long-term prognosis in steroid-refractory/dependent ulcerative colitis patients.

BACKGROUND: Oral administration of tacrolimus is an effective remission induction therapy for steroid-refractory/dependent ulcerative colitis (UC). AIM: This study aimed to evaluate the short- as well as medium- and long-term effectiveness of tacrolimus therapy. METHODS: The medical records of 51 patients treated with tacrolimus for UC at our hospital between July 2009 and December 2011 were reviewed retrospectively. Clinical remission and improvement were defined as a Lichtiger score of 4 or less and as a Lichtiger score of ≤10 and a reduction in the score of ≥3 compared with the baseline score, respectively. Endoscopic findings were evaluated based on the endoscopic activity index and Mayo endoscopic score. RESULTS: The clinical effectiveness combining clinical remission and improvement was observed in 62.7% of the patients at 3 months. Thirty-six patients underwent colonoscopy at 3 months, and 12 (33.3%) and 10 patients (27.8%) showed Mayo endoscopic scores of 0 and 1, respectively. On Kaplan-Meier analysis, the overall percentage of event-free survivors, who did not require colectomy nor switching to other induction therapy such as infliximab, was 73.0% at 6 months, 49.9% at 1 year, and 37.8% at 2 years. Patients with a Mayo endoscopic score of 0-1 at 3 months showed significantly better medium- and long-term prognosis than those with a score of 2-3 (p<0.01). All adverse events, including infections in 2 patients, were reversible. CONCLUSIONS: Tacrolimus therapy was effective for inducing clinical and endoscopic remission of steroid-refractory/dependent UC. Endoscopic improvement was associated with favorable medium- and long-term prognosis.

Establishment of novel prediction system of intestinal absorption in humans using human intestinal tissues.

The objective of this study was to establish a novel prediction system of drug absorption in humans by utilizing human intestinal tissues. Based on the transport index (TI), a newly defined parameter, calculated by taking account of the change in drug concentrations because of precipitation on the apical side and the amounts accumulated in the tissue and transported to the basal side, the absorbability of drugs in rank order as well as the fraction of dose absorbed (Fa) in humans were estimated. Human intestinal tissues taken from ulcerative colitis or Crohn's disease patients were mounted in a mini-Ussing chamber and transport studies were performed to evaluate the permeation of drugs, including FD-4, a very low permeable marker, atenolol, a low permeable marker, and metoprolol, a high permeable marker. Although apparent permeability coefficients calculated by the conventional equation did not reflect human Fa values for FD-4, atenolol, and metoprolol, TI values were well correlated with Fa values, which are described by 100 · [1 - e (- f · (TI - α)) ]. Based on this equation, Fa values in humans for other test drugs were predicted successfully, indicating that our new system utilizing human intestinal tissues would be valuable for predicting oral drug absorption in humans.