Structural basis of negative regulation of CRISPR-Cas7-11 by TPR-CHAT.

CRISPR‒Cas7-11 is a Type III-E CRISPR-associated nuclease that functions as a potent RNA editing tool. Tetratrico-peptide repeat fused with Cas/HEF1-associated signal transducer (TPR-CHAT) acts as a regulatory protein that interacts with CRISPR RNA (crRNA)-bound Cas7-11 to form a CRISPR-guided caspase complex (Craspase). However, the precise modulation of Cas7-11's nuclease activity by TPR-CHAT to enhance its utility requires further study. Here, we report cryo-electron microscopy (cryo-EM) structures of Desulfonema ishimotonii (Di) Cas7-11-crRNA, complexed with or without the full length or the N-terminus of TPR-CHAT. These structures unveil the molecular features of the Craspase complex. Structural analysis, combined with in vitro nuclease assay and electrophoretic mobility shift assay, reveals that DiTPR-CHAT negatively regulates the activity of DiCas7-11 by preventing target RNA from binding through the N-terminal 65 amino acids of DiTPR-CHAT (DiTPR-CHATNTD). Our work demonstrates that DiTPR-CHATNTD can function as a small unit of DiCas7-11 regulator, potentially enabling safe applications to prevent overcutting and off-target effects of the CRISPR‒Cas7-11 system.

Association of iron status with all-cause and cause-specific mortality in individuals with diabetes.

AIMS: Current evidence regarding iron status and mortality risk among patients with diabetes is limited. This study aimed to evaluate association of iron indices with all-cause and cause-specific mortality risk among patients with diabetes. METHODS: The current study included 2080 (with ferritin data), 1974 (with transferrin saturation (Tsat) data), and 1106 (with soluble transferrin receptor (sTfR) data) adults with diabetes from NHANES 1999-2018. Death outcomes were obtained from National Death Index through December 31, 2019. Cox proportional hazards models were employed to calculate hazard ratios and 95% confidence intervals for mortality. RESULTS: Association with all-cause mortality was demonstrated to be J-shaped for serum ferritin (Pnonlinearity < 0.01), U-shaped for Tsat (Pnonlinearity < 0.01) and linear for sTfR (Plinearity < 0.01). Ferritin 300-500 ng/mL possessed lower all-cause mortality risk than ferritin ≤ 100 ng/mL, 100-300 ng/mL, and > 500 ng/mL. Tsat 25-32 % showed a protective effect on all-cause mortality risk compared with Tsat ≤ 20 %, 20-25 %, and > 32 %. Individuals with sTfR < 4 mg/L were associated with a lower risk of all-cause mortality than those with higher sTfR. CONCLUSIONS: Moderate levels of serum ferritin (300-500 ng/mL), Tsat (25 %-32 %) and a lower concentration of sTfR (< 4 mg/L) identified adults with diabetes with lower all-cause mortality risk, adding novel modifiers to diabetes management.

Gut microbiota regulates postprandial GLP-1 response via ileal bile acid-TGR5 signaling.

The gut microbiota interacts with intestinal epithelial cells through microbial metabolites to regulate the release of gut hormones. We investigated whether the gut microbiota affects the postprandial glucagon-like peptide-1 (GLP-1) response using antibiotic-treated mice and germ-free mice. Gut microbiome depletion completely abolished postprandial GLP-1 response in the circulation and ileum in a lipid tolerance test. Microbiome depletion did not influence the GLP-1 secretory function of primary ileal cells in response to stimulators in vitro, but dramatically changed the postprandial dynamics of endogenous bile acids, particularly ω-muricholic acid (ωMCA) and hyocholic acid (HCA). The bile acid receptor Takeda G protein-coupled receptor 5 (TGR5) but not farnesoid X receptor (FXR), participated in the regulation of postprandial GLP-1 response in the circulation and ileum, and ωMCA or HCA stimulated GLP-1 secretion via TGR5. Finally, fecal microbiota transplantation or ωMCA and HCA supplementation restored postprandial GLP-1 response. In conclusion, gut microbiota is indispensable for maintaining the postprandial GLP-1 response specifically in the ileum, and bile acid (ωMCA and HCA)-TGR5 signaling is involved in this process. This study helps to understand the essential interplay between the gut microbiota and host in regulating postprandial GLP-1 response and opens the foundation for new therapeutic targets.

Fecal short chain fatty acids modify therapeutic effects of sleeve gastrectomy.

Aims: We aimed to investigate changes of fecal short chain fatty acids (SCFAs) and their association with metabolic benefits after sleeve gastrectomy (SG). Specifically, whether pre-surgery SCFAs modify surgical therapeutic effects was determined. Methods: 62 participants with measurements of fecal SCFAs and metabolic indices before and 1, 3, 6 months after SG were included. Changes of fecal SCFAs and their association with post-surgery metabolic benefits were calculated. Then, participants were stratified by medians of pre-surgery fecal SCFAs and modification effects of pre-surgery fecal SCFAs on surgical therapeutic effects were investigated, through calculating interaction of group by surgery. Results: Fecal SCFAs were markedly changed by SG. Changes of propionate and acetate were positively correlated with serum triglycerides and total cholesterol, respectively. Notably, high pre-surgery fecal hexanoate group showed a better effect of SG treatment on lowering body weight (P=0.01), BMI (P=0.041) and serum triglycerides (P=0.031), and low pre-surgery fecal butyrate had a better effect of SG on lowering ALT (P=0.003) and AST (P=0.019). Conclusion: Fecal SCFAs were changed and correlated with lipid profiles improvement after SG. Pre-surgery fecal hexanoate and butyrate were potential modifiers impacting metabolic benefits of SG.

Structural basis of BAM-mediated outer membrane ß-barrel protein assembly.

The outer membrane structure is common in Gram-negative bacteria, mitochondria and chloroplasts, and contains outer membrane ß-barrel proteins (OMPs) that are essential interchange portals of materials1-3. All known OMPs share the antiparallel ß-strand topology4, implicating a common evolutionary origin and conserved folding mechanism. Models have been proposed for bacterial ß-barrel assembly machinery (BAM) to initiate OMP folding5,6; however, mechanisms by which BAM proceeds to complete OMP assembly remain unclear. Here we report intermediate structures of BAM assembling an OMP substrate, EspP, demonstrating sequential conformational dynamics of BAM during the late stages of OMP assembly, which is further supported by molecular dynamics simulations. Mutagenic in vitro and in vivo assembly assays reveal functional residues of BamA and EspP for barrel hybridization, closure and release. Our work provides novel insights into the common mechanism of OMP assembly.

Isocaloric-restricted Mediterranean Diet and Chinese Diets High or Low in Plants in Adults With Prediabetes.

CONTEXT: Calorie restriction plus dietary advice is suggested as a preventive strategy for individuals with obesity and prediabetes; however, optimal diet is still debatable. We aimed to compare the effects of Mediterranean diet (MD) and Chinese diets high or low in plants on body weight and glucose homeostasis among high-risk Chinese. SUBJECTS AND METHODS: In this parallel-arm randomized controlled trial, 253 Chinese adults aged 25 to 60 years with a body mass index ≥ 24.0 kg/m2 and fasting blood glucose ≥ 5.6 mmol/L were randomly assigned to 3 isocaloric-restricted diets: MD (n = 84), a traditional Jiangnan diet high in plants (TJD, n = 85), or a control diet low in plants (CD, n = 84). During the 6-month trial, a 5-weekday full-feeding regimen was followed, along with mobile app-based monitoring. Abdominal fat measurement (magnetic resonance imaging), oral glucose tolerance test (OGTT), and continuous glucose monitoring (CGM) were conducted at baseline and 3 and 6 months. RESULTS: With a 25% calorie restriction for 6 months, weight deduction was 5.72 kg (95% confidence interval, 5.03-6.40) for MD, 5.05 kg (4.38-5.73) for TJD, and 5.38 kg (4.70-6.06) for CD (Ptime < 0.0001). No between-group differences were found for fasting glucose, insulin, and the Matsuda index from OGTT. Notably, CD had significantly longer time below range (glucose < 3.9 mmol/L) than MD (0.81% [0.21-1.40], P = 0.024) and marginally longer time than TJD (0.56% [-0.03 to 1.15], P = 0.065), as measured by CGM. CONCLUSIONS: With the 6-month isocaloric-restricted feeding, TJD and MD achieved comparable weight deduction and improved glucose homeostasis, whereas CD showed a higher risk for hypoglycemia.

Structural basis for bacterial lipoprotein relocation by the transporter LolCDE.

Lipoproteins in the outer membrane of Gram-negative bacteria are involved in various vital physiological activities, including multidrug resistance. Synthesized in the cytoplasm and matured in the inner membrane, lipoproteins must be transported to the outer membrane through the Lol pathway mediated by the ATP-binding cassette transporter LolCDE in the inner membrane via an unknown mechanism. Here, we report cryo-EM structures of Escherichia coli LolCDE in apo, lipoprotein-bound, LolA-bound, ADP-bound and AMP-PNP-bound states at a resolution of 3.2-3.8 Å, covering the complete lipoprotein transport cycle. Mutagenesis and in vivo viability assays verify features of the structures and reveal functional residues and structural characteristics of LolCDE. The results provide insights into the mechanisms of sorting and transport of outer-membrane lipoproteins and may guide the development of novel therapies against multidrug-resistant Gram-negative bacteria.

Rectal NSAIDs in the prevention of post-endoscopic retrograde cholangiopancreatography pancreatitis in unselected patients: Systematic review and meta-analysis.

BACKGROUND AND AIM: Efficacy of rectal non-steroidal anti-inflammatory drugs (NSAIDs) for post-endoscopic retrograde cholangiopancreatography (ERCP) pancreatitis (PEP) prophylaxis in unselected patients remained controversial. The aim of the present study was to evaluate the efficacy of rectal NSAIDs in the prevention of PEP in unselected patients. METHODS: An electronic literature search in the Cochrane Library, Embase, and PubMed was carried out for randomized controlled trials comparing rectal indomethacin or diclofenac with placebo in the prevention of PEP in unselected patients. Cochrane risk of bias tool was used to evaluate methodological quality. The data were carried out in forest plots using fixed-effect methods, and random-effect methods were used when heterogeneity was significant. RESULTS: A total of nine trials were included in our final analysis. Rectal NSAIDs were effective to reduce the incidence of PEP in unselected patients (RR, 0.61; 95% CI, 0.46-0.79), especially for moderate-to-severe PEP (RR, 0.37; 95% CI, 0.17-0.79). Both indomethacin (RR, 0.67; 95% CI, 0.50-0.88) and diclofenac (RR, 0.29; 95% CI, 0.12-0.69) were significantly efficacious. Rectal NSAIDs given pre-ERCP showed significant efficacy (RR, 0.53; 95% CI, 0.39-0.71), whether when given within 30 min pre-ERCP (RR, 0.62; 95% CI, 0.42-0.92) or not (RR, 0.43; 95% CI, 0.28-0.67). CONCLUSION: Rectal NSAIDs are effective in the prevention of PEP in unselected patients.