Binding of Distinct Substrate Conformations Enables Hydroxylation of Remote Sites in Thaxtomin D by Cytochrome P450 TxtC

Cytochromes P450 (CYPs) catalyze various oxidative transformations in drug metabolism, xenobiotic degradation, and natural product biosynthesis. Here we report biochemical, structural, and theoretical studies of TxtC, an unusual bifunctional CYP involved in the biosynthesis of the EPA-approved herbicide thaxtomin A. TxtC was shown to hydroxylate two remote sites within the Phe residue of its diketopiperazine substrate thaxtomin D. The reactions follow a preferred order, with hydroxylation of the α-carbon preceding functionalization of the phenyl group. To illuminate the molecular basis for remote site functionalization, X-ray crystal structures of TxtC in complex with the substrate and monohydroxylated intermediate were determined. Electron density corresponding to a diatomic molecule (probably dioxygen) was sandwiched between the heme iron atom and Thr237 in the TxtC-intermediate structure, providing insight into the mechanism for conversion of the ferrous-dioxygen complex into the reactive ferryl intermediate. The substrate and monohydroxylated intermediate adopted similar conformations in the active site, with the π-face of the phenyl group positioned over the heme iron atom. Docking simulations reproduced this observation and identified a second, energetically similar but conformationally distinct binding mode in which the α-hydrogen of the Phe residue is positioned over the heme prosthetic group. Molecular dynamics simulations confirmed that the α-hydrogen is sufficiently close to the ferryl oxygen atom to be extracted by it and indicated that the two substrate conformations cannot readily interconvert in the active site. These results indicate that TxtC is able to hydroxylate two spatially remote sites by binding distinct conformations of the substrate and monohydroxylated intermediate.</p

A Double-Blind, Randomized, Placebo-Controlled Clinical Trial on Benfotiamine Treatment in Patients With Diabetic Nephropathy

OBJECTIVE - To investigate the effect of benfotiamine on urinary albumin excretion (UAE) and the tubular damage marker kidney injury molecule-1 (KIM-1) in patients with type 2 diabetes and nephropathy. RESEARCH DESIGN AND METHODS - Patients with type 2 diabetes and UAE equivalent to 15-300 mg/24 h, despite ACE inhibitors (ACE-Is) or angiotensin receptor blockers (ARBs), were randomly assigned to 12 weeks of benfotiamine (900 mg/day) (n = 39) or placebo (n = 43). RESULTS - Compared with placebo, benfotiamine treatment resulted in significant improvement of thiamine status (P <0.001). Benfotiamine treatment did not significantly decrease 24-h UAE or 24-h KIM-1 excretion. CONCLUSIONS - In patients with type 2 diabetes and nephropathy, high-dose benfotiamine treatment for 12 weeks in addition to ACE-Is or ARBs did not reduce UAE or KIM-1 excretion, despite improvement of thiamine status

Development and external validation of a model to predict complex treatment after RFA for Barrett's esophagus with early neoplasia

Background & Aims: Endoscopic eradication therapy for Barrett's esophagus (BE)-related neoplasia is safe and leads to complete eradication in the majority of patients. However, a subgroup will experience a more complex treatment course with a risk for failure or disease progression. Early identification of these patients may improve patient counseling and treatment outcomes. We aimed to develop a prognostic model for a complex treatment course. Methods: We collected data from a nationwide registry that captures outcomes for all patients undergoing endoscopic eradication therapy for early BE neoplasia. A complex treatment course was defined as neoplastic progression, treatment failure, or the need for endoscopic resection during the radiofrequency ablation treatment phase. We developed a prognostic model using logistic regression. We externally validated our model in an independent registry. Results: A total of 1386 patients were included, of whom 78 (6%) had a complex treatment course. Our model identified patients with a BE length of 9 cm or longer with a visible lesion containing high-grade dysplasia/cancer, and patients with less than 50% squamous conversion after radiofrequency ablation were identified as high risk for a complex treatment. This applied to 8% of the study population and included 93% of all treatment failures and 76% of all patients with advanced neoplastic progression. The model appeared robust in multiple sensitivity analyses and performed well in external validation (area under the curve, 0.84). Conclusions: We developed a prognostic model that identified patients with a BE length of 9 cm or longer and high-grade dysplasia/esophageal adenocarcinoma and those with poor squamous regeneration as high risk for a complex treatment course. The good performance in external validation suggests that it may be used in clinical management (Netherlands Trial Register: NL7039)

Endoscopic Resection Without Subsequent Ablation Therapy for Early Barrett’s Neoplasia: Endoscopic Findings and Long-Term Mortality

Introduction: After endoscopic resection (ER) of neoplasia in Barrett’s esophagus (BE), it is recommended to ablate the remaining BE to minimize the risk for metachronous disease. However, we report long-term outcomes for a nationwide cohort of all patients who did not undergo ablation of the remaining BE after ER for early BE neoplasia, due to clinical reasons or performance status. Methods: Endoscopic therapy for BE neoplasia in the Netherlands is centralized in 8 expert centers with specifically trained endoscopists and pathologists. Uniformity is ensured by a joint protocol and regular group meetings. We report all patients who underwent ER for a neoplastic lesion between 2008 and 2018, without further ablation therapy. Outcomes include progression during endoscopic FU and all-cause mortality. Results: Ninety-four patients were included with mean age 74 (± 10) years. ER was performed for low-grade dysplasia (LGD) (10%), high-grade dysplasia (HGD) (25%), or low-risk esophageal adenocarcinoma (EAC) (65%). No additional ablation was performed for several reasons; in 73 patients (78%), the main argument was expected limited life expectancy. Median C2M5 BE persisted after ER, and during median 21 months (IQR 11–51) with 4 endoscopies per patient, no patient progressed to advanced cancer. Seventeen patients (18%) developed HGD/EAC: all were curatively treated endoscopically. In total, 29/73 patients (40%) with expected limited life expectancy died due to unrelated causes during FU, none of EAC. Conclusion: In selected patients, ER monotherapy with endoscopic surveillance of the residual BE is a valid alternative to eradication therapy with ablation.</p

Pharmacokinetics of high-dose oral thiamine hydrochloride in healthy subjects

Background: High dose oral thiamine may have a role in treating diabetes, heart failure, and hypermetabolic states. The purpose of this study was to determine the pharmacokinetic profile of oral thiamine hydrochloride at 100 mg, 500 mg and 1500 mg doses in healthy subjects. Methods: This was a randomized, double-blind, single-dose, 4-way crossover study. Pharmacokinetic measures were calculated. Results: The $AUC_{0-10 hr}$ and $C_{max}$ values increased nonlinearly between 100 mg and 1500 mg. The slope of the $AUC_{0-10 hr}$ vs dose, as well as the $C_{max}$ vs dose, plots are steepest at the lowest thiamine doses. Conclusion: Our study demonstrates that high blood levels of thiamine can be achieved rapidly with oral thiamine hydrochloride. Thiamine is absorbed by both an active and nonsaturable passive process

A deep learning system for detection of early Barrett's neoplasia:a model development and validation study

BACKGROUND: Computer-aided detection (CADe) systems could assist endoscopists in detecting early neoplasia in Barrett's oesophagus, which could be difficult to detect in endoscopic images. The aim of this study was to develop, test, and benchmark a CADe system for early neoplasia in Barrett's oesophagus.METHODS: The CADe system was first pretrained with ImageNet followed by domain-specific pretraining with GastroNet. We trained the CADe system on a dataset of 14 046 images (2506 patients) of confirmed Barrett's oesophagus neoplasia and non-dysplastic Barrett's oesophagus from 15 centres. Neoplasia was delineated by 14 Barrett's oesophagus experts for all datasets. We tested the performance of the CADe system on two independent test sets. The all-comers test set comprised 327 (73 patients) non-dysplastic Barrett's oesophagus images, 82 (46 patients) neoplastic images, 180 (66 of the same patients) non-dysplastic Barrett's oesophagus videos, and 71 (45 of the same patients) neoplastic videos. The benchmarking test set comprised 100 (50 patients) neoplastic images, 300 (125 patients) non-dysplastic images, 47 (47 of the same patients) neoplastic videos, and 141 (82 of the same patients) non-dysplastic videos, and was enriched with subtle neoplasia cases. The benchmarking test set was evaluated by 112 endoscopists from six countries (first without CADe and, after 6 weeks, with CADe) and by 28 external international Barrett's oesophagus experts. The primary outcome was the sensitivity of Barrett's neoplasia detection by general endoscopists without CADe assistance versus with CADe assistance on the benchmarking test set. We compared sensitivity using a mixed-effects logistic regression model with conditional odds ratios (ORs; likelihood profile 95% CIs).FINDINGS: Sensitivity for neoplasia detection among endoscopists increased from 74% to 88% with CADe assistance (OR 2·04; 95% CI 1·73-2·42; p&lt;0·0001 for images and from 67% to 79% [2·35; 1·90-2·94; p&lt;0·0001] for video) without compromising specificity (from 89% to 90% [1·07; 0·96-1·19; p=0·20] for images and from 96% to 94% [0·94; 0·79-1·11; ] for video; p=0·46). In the all-comers test set, CADe detected neoplastic lesions in 95% (88-98) of images and 97% (90-99) of videos. In the benchmarking test set, the CADe system was superior to endoscopists in detecting neoplasia (90% vs 74% [OR 3·75; 95% CI 1·93-8·05; p=0·0002] for images and 91% vs 67% [11·68; 3·85-47·53; p&lt;0·0001] for video) and non-inferior to Barrett's oesophagus experts (90% vs 87% [OR 1·74; 95% CI 0·83-3·65] for images and 91% vs 86% [2·94; 0·99-11·40] for video).INTERPRETATION: CADe outperformed endoscopists in detecting Barrett's oesophagus neoplasia and, when used as an assistive tool, it improved their detection rate. CADe detected virtually all neoplasia in a test set of consecutive cases.FUNDING: Olympus.</p

Association of eGFR-Related Loci Identified by GWAS with Incident CKD and ESRD

Family studies suggest a genetic component to the etiology of chronic kidney disease (CKD) and end stage renal disease (ESRD). Previously, we identified 16 loci for eGFR in genome-wide association studies, but the associations of these single nucleotide polymorphisms (SNPs) for incident CKD or ESRD are unknown. We thus investigated the association of these loci with incident CKD in 26,308 individuals of European ancestry free of CKD at baseline drawn from eight population-based cohorts followed for a median of 7.2 years (including 2,122 incident CKD cases defined as eGFR <60ml/min/1.73m2 at follow-up) and with ESRD in four case-control studies in subjects of European ancestry (3,775 cases, 4,577 controls). SNPs at 11 of the 16 loci (UMOD, PRKAG2, ANXA9, DAB2, SHROOM3, DACH1, STC1, SLC34A1, ALMS1/NAT8, UBE2Q2, and GCKR) were associated with incident CKD; p-values ranged from p = 4.1e-9 in UMOD to p = 0.03 in GCKR. After adjusting for baseline eGFR, six of these loci remained significantly associated with incident CKD (UMOD, PRKAG2, ANXA9, DAB2, DACH1, and STC1). SNPs in UMOD (OR = 0.92, p = 0.04) and GCKR (OR = 0.93, p = 0.03) were nominally associated with ESRD. In summary, the majority of eGFR-related loci are either associated or show a strong trend towards association with incident CKD, but have modest associations with ESRD in individuals of European descent. Additional work is required to characterize the association of genetic determinants of CKD and ESRD at different stages of disease progression

Computer-aided Diagnosis Improves Characterization of Barrett's Neoplasia By General Endoscopists

BACKGROUND &amp; AIMS: Characterization of visible abnormalities in Barrett esophagus (BE) patients can be challenging, especially for unexperienced endoscopists. This results in suboptimal diagnostic accuracy and poor inter-observer agreement. Computer-aided diagnosis (CADx) systems may assist endoscopists. We aimed to develop, validate and benchmark a CADx system for BE neoplasia.METHODS: The CADx system received pretraining with ImageNet with consecutive domain-specific pretraining with GastroNet which includes 5 million endoscopic images. It was subsequently trained and internally validated using 1,758 narrow-band imaging (NBI) images of early BE neoplasia (352 patients) and 1,838 NBI images of non-dysplastic BE (173 patients) from 8 international centers. CADx was tested prospectively on corresponding image and video test sets with 30 cases (20 patients) of BE neoplasia and 60 cases (31 patients) of non-dysplastic BE. The test set was benchmarked by 44 general endoscopists in two phases (phase 1: no CADx assistance; phase 2: with CADx assistance). Ten international BE experts provided additional benchmark performance.RESULTS: Stand-alone sensitivity and specificity of the CADx system were 100% and 98% for images and 93% and 96% for videos, respectively. CADx outperformed general endoscopists without CADx assistance in terms of sensitivity (p=0.04). Sensitivity and specificity of general endoscopist increased from 84% to 96% and 90 to 98% with CAD assistance (p&lt;0.001), respectively. CADx assistance increased endoscopists' confidence in characterization (p&lt;0.001). CADx performance was similar to Barrett experts.CONCLUSION: CADx assistance significantly increased characterization performance of BE neoplasia by general endoscopists to the level of expert endoscopists. The use of this CADx system may thereby improve daily Barrett surveillance.</p

Transanal minimally invasive surgery (TAMIS) versus endoscopic submucosal dissection (ESD) for resection of non-pedunculated rectal lesions (TRIASSIC study):study protocol of a European multicenter randomised controlled trial

BACKGROUND: In the recent years two innovative approaches have become available for minimally invasive en bloc resections of large non-pedunculated rectal lesions (polyps and early cancers). One is Transanal Minimally Invasive Surgery (TAMIS), the other is Endoscopic Submucosal Dissection (ESD). Both techniques are standard of care, but a direct randomised comparison is lacking. The choice between either of these procedures is dependent on local expertise or availability rather than evidence-based. The European Society for Endoscopy has recommended that a comparison between ESD and local surgical resection is needed to guide decision making for the optimal approach for the removal of large rectal lesions in Western countries. The aim of this study is to directly compare both procedures in a randomised setting with regard to effectiveness, safety and perceived patient burden. METHODS: Multicenter randomised trial in 15 hospitals in the Netherlands. Patients with non-pedunculated lesions > 2 cm, where the bulk of the lesion is below 15 cm from the anal verge, will be randomised between either a TAMIS or an ESD procedure. Lesions judged to be deeply invasive by an expert panel will be excluded. The primary endpoint is the cumulative local recurrence rate at follow-up rectoscopy at 12 months. Secondary endpoints are: 1) Radical (R0-) resection rate; 2) Perceived burden and quality of life; 3) Cost effectiveness at 12 months; 4) Surgical referral rate at 12 months; 5) Complication rate; 6) Local recurrence rate at 6 months. For this non-inferiority trial, the total sample size of 198 is based on an expected local recurrence rate of 3% in the ESD group, 6% in the TAMIS group and considering a difference of less than 6% to be non-inferior. DISCUSSION: This is the first European randomised controlled trial comparing the effectiveness and safety of TAMIS and ESD for the en bloc resection of large non-pedunculated rectal lesions. This is important as the detection rate of these adenomas is expected to further increase with the introduction of colorectal screening programs throughout Europe. This study will therefore support an optimal use of healthcare resources in the future. TRIAL REGISTRATION: Netherlands Trial Register, NL7083 , 06 July 2018

A polymorphism in the gene encoding carnosinase (CNDP1) as a predictor of mortality and progression from nephropathy to end-stage renal disease in type 1 diabetes mellitus

Aims/hypothesis Homozygosity for a five leucine repeat (5L-5L) in the carnosinase gene (CNDP1) has been found to be cross-sectionally associated with a low frequency of diabetic nephropathy (DN), mainly in type 2 diabetes. We prospectively investigated in patients with type I diabetes whether: (1) 5L-5L is associated with mortality; (2) there is an interaction of 5L-5L with DN or sex for prediction of mortality; and (3) 5L-5L is associated with progression to end-stage renal disease (ESRD). Methods In this prospective study in white European patients with type 1 diabetes, individuals with DN were defined by persistent albuminuria >= 300 mg/24 h. Controls without nephropathy were defined by persistent (>15 years) normoalbuminuria Results The study involved 916 patients with DN and 1,170 controls. During follow-up for 8.8 years, 107 patients (14%) with 5L-5L died compared with 182 patients (13.8%) with other genotypes (p=0.99). There was no significant interaction of 5L-5L with DN for prediction of mortality (p=0.57), but a trend towards interaction with sex (p=0.08). In patients with DN, HR for ESRD in 5L-5L vs other genotypes was not constant over time, with increased risk for 5L-5L beyond 8 years of follow-up (p=0.03). Conclusions/interpretation CNDP1 polymorphism was not associated with mortality, and nor was there an interaction of this polymorphism with DN for prediction of mortality in patients with type 1 diabetes. CNDP1 polymorphism predicts progression to ESRD in patients with DN, but only late after baseline measurements