A trapped covalent intermediate as a key catalytic element in the hydrolysis of a GH3 ß-glucosidase: An X-ray crystallographic and biochemical study.

Glycoside hydrolases (GHs) are industrially important enzymes that hydrolyze glycosidic bonds in glycoconjugates. In this study, we found a GH3 ß-glucosidase (CcBgl3B) from Cellulosimicrobium cellulans sp. 21 was able to selectively hydrolyze the ß-1,6-glucosidic bond linked glucose of ginsenosides. X-ray crystallographic studies of the ligand complex ginsenoside-specific ß-glucosidase provided a novel finding that support the catalytic mechanism of GH3. The substrate was clearly identified within the catalytic center of wild-type CcBgl3B, revealing that the C1 atom of the glucose was covalently bound to the Oδ1 group of the conserved catalytic nucleophile Asp264 as an enzyme-glycosyl intermediate. The glycosylated Asp264 could be identified by mass spectrometry. Through site-directed mutagenesis studies with Asp264, it was found that the covalent intermediate state formed by Asp264 and the substrate was critical for catalysis. In addition, Glu525 variants (E525A, E525Q and E525D) showed no or marginal activity against pNPßGlc; thus, this residue could supply a proton for the reaction. Overall, our study provides an insight into the catalytic mechanism of the GH3 enzyme CcBgl3B.

[A single center study：An analysis of the safety and validity of delaying repeated biopsy for patients with atypical small acinar proliferation].

OBJECTIVE: To explore the association between atypical small acinar proliferation (ASAP) and subsequent diagnosis of intermediate and high risk prostate cancer (PCa), and analyze whether delaying repeat biopsy timing is safe and effective. METHODS: From June 2000 to June 2022, we retrospectively analyzed the clinical data of 276 patients accepting prostatic biopsy and diagnosed with ASAP in China-Japan Union Hospital of Jilin University. 54.7% (151/276) patients had a repeat biopsy. We used statistic methods to process the data. RESULTS: 25.2%（38/151）patients were diagnosed with PCa on repeat biopsy. Among them, 78.9%（30/38）patients had Gleason score (GS) 3+3 and 21.1% (8/38) had GS 3+4 disease. There were 4 and 6 patients got RP respectively in the two cohorts. Only 5.3% (8/151) of ASAP patients were diagnosed as intermediate risk PCa in repeated biopsy and specially, no high risk PCa was identified in our study. CONCLUSION: It was safe and valid to delay the repeat biopsy.

A TP53-based immune prognostic model for muscle-invasive bladder cancer.

BACKGROUND: Muscle-invasive bladder cancer (MIBC) patients are subject to unfavorable treatment options and a high recurrence rate. The status of TP53 mutations played an essential role in the progression and the prognosis of MIBC. The present study proposed to investigate the association between TP53 mutations and immunophenotype in MIBC. RESULTS: We established an immune prognostic model (IPM) ground on the immune-associated genes derived from variation analysis between wild-type TP53 and mutated TP53 TCGA-MIBC patients, and validated in another cohort from GEO database. Based on IPM, we divided MIBC patients into low and high risk subgroups. The high risk MIBC patients had higher proportions of macrophages M1, and lower proportions of T cells regulatory (Tregs) and activated dendritic cells than the low risk MIBC patients. Moreover, the expression of immune checkpoints genes (PD1, CTLA4, LAG3, HAVCR2 and TIGIT) was higher in the high risk patients than the low risk patients. In clinical application, IPM exhibited better survival prediction than conventional clinical characteristics. CONCLUSIONS: Our investigation presented practical prognostic significance for MIBC patients and displayed the overarching landscape of the immune response in the MIBC microenvironment. METHODS: Data were obtained from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). Differentially expressed genes (DEGs) analysis between the TP53 mutated and wild-type MIBC patients was conducted. The CIBERSORT algorithm was performed to evaluate the proportion of immune cell types. Gene expression profiles from the TCGA and GEO were used as training and testing cohorts to build and validate an immune-related prognostic model (IPM). Genes in the IPM model were first screened by univariate Cox analysis, then filtered by the least absolute shrinkage and selection operator (LASSO) Cox regression. A nomogram was finally established and evaluated by combining both the IPM and other clinical factors.

Compound K, a final intestinal metabolite of ginsenosides, enhances insulin secretion in MIN6 pancreatic ß-cells by upregulation of GLUT2.

Compound K (CK) is a final intestinal metabolite of protopanaxadiol-type ginsenosides from Panax ginseng and shows various bioactivities. Although it has also been found to have the property of anti-diabetes, the long-term effect of CK on insulin secretion in ß-cells is still unclear. In this study, CK was prepared from ginsenoside Rd by snailase hydrolysis and its effect on the insulin secretion activity in MIN6 pancreatic ß-cell lines in vitro was assessed. The expression of glucose transporter isoform-2 (GLUT2) and the cellular ATP content were also examined by western blot and HPLC analysis, respectively. The results showed that CK significantly enhanced insulin secretion, increased cellular ATP content, and upregulated the expression of GLUT2. These findings indicate that CK exerts prominent stimulatory effects on insulin secretion in the MIN6 cells partly via upregulating the expression of GLUT2.