Computational design of an imine reductase: mechanism-guided stereoselectivity reversion and interface stabilization.

Imine reductases (IREDs) are important biocatalysts in the asymmetric synthesis of chiral amines. However, a detailed understanding of the stereocontrol mechanism of IRED remains incomplete, making the design of IRED for producing the desired amine enantiomers challenging. In this study, we investigated the stereoselective catalytic mechanism and designed an (R)-stereoselective IRED from Paenibacillus mucilaginosus (PmIR) using pharmaceutically relevant 2-aryl-substituted pyrrolines as substrates. A putative mechanism for controlling stereoselectivity was proposed based on the crucial role of electrostatic interactions in controlling iminium cation orientation and employed to achieve complete inversion of stereoselectivity in PmIR using computational design. The variant PmIR-Re (Q138M/P140M/Y187E/Q190A/D250M/R251N) exhibited opposite (S)-stereoselectivity, with >96% enantiomeric excess (ee) towards tested 2-aryl-substituted pyrrolines. Computational tools were employed to identify stabilizing mutations at the interface between the two subunits. The variant PmIR-6P (P140A/Q190S/R251N/Q217E/A257R/T277M) showed a nearly 5-fold increase in activity and a 12 °C increase in melting temperature. The PmIR-6P successfully produced (R)-2-(2,5-difluorophenyl)-pyrrolidine, a key chiral pharmaceutical intermediate, at a concentration of 400 mM with an ee exceeding 99%. This study provides insight into the stereocontrol elements of IREDs and demonstrates the potential of computational design for tailored stereoselectivity and thermal stability.

Comprehensive Analysis Identified ASF1B as an Independent Prognostic Factor for HBV-Infected Hepatocellular Carcinoma.

Purpose: Hepatitis B (HBV)-infected hepatocellular carcinoma is one of the most common cancers, and it has high incidence and mortality rates worldwide. The incidence of hepatocellular carcinoma has been increasing in recent years, and existing treatment modalities do not significantly improve prognosis. Therefore, it is important to find a biomarker that can accurately predict prognosis. Methods: This study was analyzed using the The Cancer Genome Atlas (TCGA) database and validated by the International Cancer Genome Consortium (ICGC) database. The STRING database was used to construct a gene co-expression network and visualize its functional clustering using Cytoscape. A prognostic signature model was constructed to observe high and low risk with prognosis, and independent prognostic factors for HBV-infected hepatocellular carcinoma were identified by Cox regression analysis. The independent prognostic factors were then analyzed for expression and survival, and their pathway enrichment was analyzed using gene set enrichment analysis (GSEA). Results: 805 differentially expressed genes (DEGs) were obtained by differential analysis. Protein-protein interaction (PPI) showed that DEGs were mostly clustered in functional modules, such as cellular matrix response, cell differentiation, and tissue development. Prognostic characterization models showed that the high-risk group was associated with poor prognosis, while Cox regression analysis identified ASF1B as the only independent prognostic factor. As verified by expression and prognosis, ASF1B was highly expressed in HBV-infected hepatocellular carcinoma and led to a poor prognosis. GSEA showed that high ASF1B expression was involved in cell cycle-related signaling pathways. Conclusion: Bioinformatic analysis identified ASF1B as an independent prognostic factor in HBV-infected hepatocellular carcinoma, and its high expression led to a poor prognosis. Furthermore, it may promote hepatocellular carcinoma progression by affecting cell cycle-related signaling pathways.

Efficient synthesis of bepotastine and cloperastine intermediates using engineered alcohol dehydrogenase with a hydrophobic pocket.

(S)-4-Chlorophenylpyridylmethanol and (R)-4-chlorobenzhydrol are key pharmaceutical intermediates for the synthesis of bepotastine and cloperastine, respectively. However, the biocatalytic approach to prepare these bulky diaryl ketones remains challenging because of the low activity of naturally occurring alcohol dehydrogenases (ADH). In the present study, ADH seq5, which has an adequate binding pocket volume and accepts bulky diaryl ketones, was further engineered with a binding pocket of increased hydrophobicity. Based on molecular simulation and binding free energy analyses, a small mutation library was constructed, and mutant seq5-D150I with a threefold increase in kcat and a low Km was obtained successfully. The comparison of kinetic parameters, binding free energy, docking conformation, and critical catalytic distances calculated by molecular dynamic simulations revealed the source of increased activity. To develop a practical approach with seq5-D150I, reaction conditions including pH, temperature, buffer, and metal ions were optimised and applied to synthesise (S)-4-chlorophenylpyridylmethanol and (R)-4-chlorobenzhydrol with high enantiomeric excess. The space-time yields for (S)-4-chlorophenylpyridylmethanol and (R)-4-chlorobenzhydrol increased dramatically to as high as 263.4 g∙L-1 day-1 and 150 g∙L-1 day-1, respectively, which, to our knowledge, is the highest reported yield to date. These results show that the biocatalytic approach with seq5-D150I may be practical for future industrial applications.Key points An alcohol dehydrogenase was engineered based on binding free energy analysis. The mutant seq5-D150I obtained a threefold increase in kcat and a low Km. Two important pharmaceutical intermediates were obtained with high space-time yield.

Corrigendum: Carbapenem-Resistant E. cloacae in Southwest China: Molecular Analysis of Resistance and Risk Factors for Infections Caused by NDM-1-Producers.

[This corrects the article DOI: 10.3389/fmicb.2018.00658.].

Carbapenem-Resistant E. cloacae in Southwest China: Molecular Analysis of Resistance and Risk Factors for Infections Caused by NDM-1-Producers.

Carbapenem-resistant Enterobacteriaceae (CRE) has been considered a serious global threat, but carbapenem resistance remains relatively uncommon in E. cloacae, especially in China. The aim of this study was to characterize carbapenem-resistant E. cloacae (CR-ECL) isolates from 2012 to 2016 in Southwest China. Our study revealed that 20 (15.2%) of the 132 CR-ECL isolates obtained from patients were identified as NDM-1, with most isolates carrying the IncFIIA plasmids. Notably, we initially observed that the E. cloacae strain co-harbored NDM-1 and IMP-8 carbapenemases simultaneously. Analysis of the genetic environment of these two genes has revealed that the highly conserved regions (blaNDM-1-bleMBL-trpF-tat) are associated with the dissemination of NDM-1, while IS26, intI1, and tniC could be involved in the spread of IMP-8. Molecular epidemiology studies showed the nosocomial outbreak caused by NDM-1-producing E. cloacae ST88. Transferring from another hospital and previous carbapenem exposure were identified as independent risk factors for the acquisition of NDM-1-producing E. cloacae. These findings emphasize the need for intensive surveillance and precautions to monitor the further spread of NDM-1 in China.

Multidrug Resistance Mechanisms of Carbapenem Resistant Klebsiella pneumoniae Strains Isolated in Chongqing, China.

BACKGROUND: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is considered a serious global threat. However, little is known regarding the multidrug resistance (MDR) mechanisms of CRKP. This study investigated the phenotypes and MDR mechanisms of CRKP and identified their clonal characteristics. METHODS: PCR and sequencing were utilized to identify antibiotic resistance determinants. Integron gene cassette arrays were determined by restriction fragment length polymorphism (RFLP) analysis. Multi-locus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were used for epidemiological analysis. Plasmids were typed by using a PCR-based replicon typing and analyzed by conjugation and transformation assays. RESULTS: Seventy-eight strains were identified as resistant to at least one carbapenem; these CRKP strains had a high prevalence rate (38.5%, 30/78) of carbapenemase producers. Additionally, most isolates harbored MDR genes, including Extended spectrum ß-lactamases (ESBLs), AmpC, and quinolone and aminoglycoside resistance genes. Loss of porin genes was observed, and Class 1 integron was detected in 66.7% of the investigated isolates. PFGE and MLST results excluded the occurrence of clonal dissemination among these isolates. CONCLUSIONS: A high prevalence of NDM-1 genes encoding carbapenem resistance determinants was demonstrated among the K. pneumoniae isolates. Importantly, this is the first report of bla(NDM-1) carriage in a K. pneumoniae ST1383 clone in China and of a MDR CRKP isolate co-harboring bla(NDM-1), bla(KPC-2), bla(CTX-M), bla(SHV), acc(6')-Ib, rmtB, qnrB, and acc(6')-Ib-cr.

Evaluation of Six Phenotypic Methods for the Detection of Carbapenemases in Gram-Negative Bacteria With Characterized Resistance Mechanisms.

BACKGROUND: We compared the performance of the modified Hodge test (MHT), Triton Hodge test (THT), Carba NP test (CNPt), simplified Carba NP test (CNPt-direct), blue-Carba NP test (BCT), and carbapenem inactivation method (CIM) for rapid and accurate carbapenemase detection. METHODS: The methods were evaluated by using 256 gram-negative isolates, including 197 Enterobacteriaceae (79 Enterobacter spp., 74 Klebsiella spp., 33 Escherichia coli, 10 Citrobacter spp., and 1 Serratia marcescens), 51 Acinetobacter baumannii, and 8 Pseudomonas aeruginosa strains. The collection included 117 non-carbapenemase, 18 Klebsiella pneumoniae carbapenemases (KPC) producers, 46 New Delhi metallo-ß-lactamases (NDM) producers, 11 imipenemases (IMP) producers, and 51 oxacillinases (OXA) producers, and 13 strains harboring two different carbapenemase genes. RESULTS: The specificity of the THT (91.5%) was significantly lower than other methods, each of which had 100% specificity (P<0.003). This can be attributed to the false detection of Ampler class C ß-lactamases (AmpC) carriers. The CNPt-direct and CIM yielded the highest sensitivities (P<0.003), which were comparable (92.8% vs 93.5%, P>0.999). Because of improved detection of NDM carriers, THT showed significantly higher sensitivity than the MHT (84.9% vs 75.5%, P<0.001). However, poor performances in detecting OXA still influenced the sensitivities of the CNPt (66.2%) and BCT (82.0%), as well as the MHT and THT. CONCLUSIONS: CNPt-direct and CIM demonstrated the best performance for the efficient detection of carbapenemase among the six evaluated methods. Except the MHT and THT, the detection of carbapenemase-producing Enterobacteriaceae by all the other methods was acceptable, when the OXA-type carbapenemase was not prevalent.

Hyperglycemia during induction therapy is associated with poorer survival in children with acute lymphocytic leukemia.

OBJECTIVES: To investigate whether children with acute lymphocytic leukemia (ALL) who have development of hyperglycemia during induction may have worse relapse-free (RFS) and overall survival (OS) rates. STUDY DESIGN: A review of 167 children diagnosed with ALL between 1999 to 2002 at Texas Children's Hospital was performed. Blood glucose concentrations during induction therapy were reviewed; patients were assigned to 3 groups: euglycemia (blood glucose < 140 mg/dL), mild hyperglycemia (blood glucose between 140-200 mg/dL), and overt hyperglycemia (blood glucose > 200 mg/dL). RFS and OS among groups were compared by use of Kaplan-Meier and Cox-proportional hazard analyses, adjusting for potential confounding variables. RESULTS: The median follow-up in survivors was 6 years; there were 18 deaths and 36 relapses. Overt hyperglycemia was seen in 56 (34%) patients. Patients with overt hyperglycemia had poorer RFS (68% +/- [SE] 6.7 vs 85% +/- 3.6, P = .025) and OS (74% +/- 6.1 vs 96% +/- 1.9, P < .0001) at 5 years than their counterparts. Patients with overt hyperglycemia had 6.2 times (95% CI 1.6-24.7, P = .01) greater risk for death, independent of risk group and type of steroid. CONCLUSIONS: Overt hyperglycemia may be an independent predictor of survival in children with ALL.

Hyperglycemia during induction therapy is associated with increased infectious complications in childhood acute lymphocytic leukemia.

BACKGROUND: Children with acute lymphocytic leukemia (ALL) are at high risk for developing hyperglycemia. Hyperglycemic adult ALL patients have shorter remissions, more infections, and increased mortality. No corresponding data are available in children. We hypothesized that children with ALL who become hyperglycemic during induction chemotherapy have an increased risk for infection during their first year of treatment. PROCEDURE: We conducted a retrospective chart review of 135 patients diagnosed with ALL during 1999-2001 at Texas Children's Hospital. Infectious outcomes during the first year of therapy were compared in three groups patients based on blood glucose concentrations during induction therapy: euglycemic (<140 mg/dl), mild hyperglycemic (MH) (140-199 mg/dl) and overt hyperglycemic (OH) (blood glucose >200 mg/dl). RESULTS: Seventy-five (56%) patients met criteria for either MH (21%) or OH (35%). Hyperglycemia was more prevalent in older children (P < 0.001) and those at risk for being overweight (BMI% >85%) at diagnosis (P < 0.01). Patients with MH and OH were 2.5 times (95% CI 1.0-6.2) and 2.1 times (95% CI 1.0-4.6) more likely to have documented infections, respectively. Patients with OH were 4.2 times (95% CI 1.5-12) more likely to have bacteremia/fungemia, 3.8 times (95% CI 1.2-11.6) more likely to have cellulitis, and 4.0 times (95% CI 1.7-9.3) more likely to be admitted for fever and neutropenia than the euglycemia group. CONCLUSION: Hyperglycemia, especially when overt, may be a previously unrecognized risk factor of infectious complications in children with ALL during the first year of treatment.