Construction of stabilized (R)-selective amine transaminase from Aspergillus terreus by consensus mutagenesis.

Amine transaminases are a class of efficient and industrially-desired biocatalysts for the production of chiral amines. In this study, stabilized variants of the (R)-selective amine transaminase from Aspergillus terreus (AT-ATA) were constructed by consensus mutagenesis. Using Consensus Finder (http://cbs-kazlab.oit.umn.edu/), six positions with the most prevalent amino acid (over 60% threshold) among the homologous family members were identified. Subsequently, these six residues were individually mutated to match the consensus sequence (I77 L, Q97E, H210N, N245D, G292D, and I295 V) using site-directed mutagenesis. Compared to that of the wild-type, the thermostability of all six single variants was improved. The H210N variant displayed the largest shift in thermostability, with a 3.3-fold increase in half-life (t1/2) at 40 °C, and a 4.6 °C increase in T5010 among the single variants. In addition, the double mutant H210N/I77L displayed an even larger shift with 6.1-fold improvement of t1/2 at 40 °C, and a 6.6 °C increase in T5010. Furtherly, the H210N/I77L mutation was introduced into the previously engineered thermostable AT-ATA by the introduction of disulfide bonds, employing B-factor and folding free energy (ΔΔGfold) calculations. Our results showed that the combined variant H210N/I77L/M150C-M280C had the largest shift in thermostability, with a 16.6-fold improvement of t1/2 and a 11.8 °C higher T5010.

A meta-analysis of association between serum iron levels and lung cancer risk.

Many studies conducted on the relationship between serum iron levels and lung cancer risk had produced inconsistent results. We therefore conducted a meta-analysis to determine whether serum iron levels were lower in lung cancer patients compared to those in controls.A literature survey was conducted by searching the PubMed, WanFang, CNKI, and SinoMed databases for articles published as of Mar 1, 2018. Standard mean differences (SMD) with the corresponding 95% confidence intervals (CI) were executed by Stata 12.0 software. A total of 13 publications involving 1118 lung cancer patients and 832 controls were included in our study. The combined results showed that serum iron levels in lung cancer cases had no significantly lower when compared to those in controls [summary SMD = -0.125, 95%CI= -0.439, 0.189, Z = 0.78, p for Z test= 0.435], with high heterogeneity (I2= 89.9%, P< 0.001) found. In the stratified analysis by geographic locations, consistent results were found for serum iron levels between lung cancer patients and controls both in Asian populations [summary SMD = -0.113, 95%CI= -0.471, 0.245] and European populations [summary SMD = -0.215, 95%CI= -0.835, 0.404]. Publication bias was not found when evaluated by Begg's funnel plot and Egger's regression asymmetry test.In summary, the current study showed that serum iron levels had no significant association on lung cancer risk.

Association between BIM polymorphism and lung cancer outcomes: a meta-analysis.

Accumulating evidences have indicated that BIM expression largely decides the development of lung cancer and outcome of EGFR-mutant lung cancers after TKI treatments. BIM polymorphism is a 2,903-bp deletion in the second exon. To clarify the relationship between this BIM polymorphism and clinical outcomes of lung cancers, we conducted this meta-analysis and observed the survival and responses to TKIs. Sixteen cohort studies, covering 4393 WT and 916 BIM deletion patients were included. Overall, BIM deletion polymorphism was associated with significantly shorter progression-free survival (PFS) and slightly shorter overall survival (OS), compared to the WT group. Moreover, patients with BIM deletion polymorphism showed significantly inferior response to EGFR TKIs. In conclusion, our analysis confirmed that lung cancer patients harboring the BIM deletion have inferior survival and TKI responses. Examination of the novel biomarker BIM deletion in lung cancer patients, especially for the EGFR mutant cohort, could provide some prognostic utility.

Combined detection of CEA and CA125 for the diagnosis for lung cancer: A meta-analysis.

This study aimed to systematically evaluate the value of combined detection of serum CEA and CA125 concentrations for the diagnosis of lung cancer. Related studies regarding the diagnosis of lung cancer were searched in PubMed, Embase, CNKI, and Wanfang using a computer. The number of patients who were true-positive, false-positive, false-negative, and true-negative were extracted from each study. Meta-analysis was performed using the Meta-Disc l.4, RevMan 5.3. Seven studies involving 2,216 cases were finally included. Regarding the diagnosis of lung cancer, the sensitivity, specificity, and diagnostic odds ratio of combined CEA and CA125 detection were higher than those of CEA detection alone. The area under the curve (AUC) of combined detection was 0.90, whereas the independently detected AUC was 0.73. Combined CEA and CA125 detection has higher diagnostic efficiency for lung cancer than CEA detection alone. The significance of combined serum CEA and CA125 detection in lung cancer is confirmed.

Improving thermostability of (R)-selective amine transaminase from Aspergillus terreus through introduction of disulfide bonds.

To improve the thermostability of (R)-selective amine transaminase from Aspergillus terreus (AT-ATA), we used computer software Disulfide by Design and Modelling of Disulfide Bonds in Proteins to identify mutation sites where the disulfide bonds were most likely to form. We obtained three stabilized mutants (N25C-A28C, R131C-D134C, M150C-M280C) from seven candidates by site-directed mutagenesis. Compared to the wild type, the best two mutants N25C-A28C and M150C-M280C showed improved thermal stability with a 3.1- and 3.6-fold increase in half-life (t1/2 ) at 40 °C and a 4.6 and 5.1 °C increase in T5010 . In addition, the combination of mutant R131C-D134C and M150C-M280C displayed the largest shift in thermostability with a 4.6-fold increase in t1/2 at 40 °C and a 5.5 °C increase in T5010 . Molecular dynamics simulation indicated that mutations of N25C-A28C and M150C-M280C lowered the overall root mean square deviation for the overall residues at elevated temperature and consequently increased the protein rigidity. The stabilized mutation of R131C-D134C was in the region of high mobility and on the protein surface, and the disulfide bond constraints the flexibility of loop 121-136.

Engineering thermostable (R)-selective amine transaminase from Aspergillus terreus through in silico design employing B-factor and folding free energy calculations.

Amine transaminases have recently gained a lot of attention for the synthesis of chiral amines. Using (R)-selective amine transaminase from Aspergillus terreus (AT-ATA) as a transaminase model, in silico design was applied employing B-factor and folding free energy (ΔΔGfold) calculations. Mutation sites were selected by targeting flexible regions with the greatest B-factors, and were substituted with amino acids that were determined by folding free energy calculations (ΔΔGfold < 0) to be more rigid than the original ones. By site-directed mutagenesis, we obtained four stabilized mutants (T130M, T130F, E133F and D134L) with improved stability from 19 candidates. Compared to the wild type, the best single mutant (T130M) showed an increase in thermal stability with a nearly 2.2-fold improvement of half-life (t1/2) at 40 °C and a 3.5 °C higher T1/210 min. The optimum catalytic temperature of T130F was increased by 10 °C. In addition, the T130M/E133F double mutant displayed the largest shift in thermostability with 3.3-fold improvement of t1/2 at 40 °C and a 5.0 °C higher T1/210 min. Modeling analysis showed that new hydrophobic interactions and hydrogen bonds might contribute to the observed thermostability improvement.

Preparation and copper ions adsorption properties of thiosemicarbazide chitosan from squid pens.

Chitosan was prepared by alkaline N-deacetylation of ß-chitin from squid pens. Thiosemicarbazide group was introduced to chitosan via formaldehyde-derived linkages, and thiosemicarbazide chitosan (TSFCS) with different degrees of substitution (DS) was synthesized. The DS values of TSFCS calculated by elemental analysis were 0.19, 0.36 and 0.63. The structure of the TSFCS was confirmed by elemental analysis, FTIR, XRD, TGA and SEM. The adsorption capacity of Cu(II) ions by TSFCS showed good correlation with the DS and pH (pH range 2.2-5.8). The maximum Cu(II) ions adsorption capacity of all three TSFCS samples reached 134.0mgg-1 at pH 3.6, but chitosan showed no adsorption at this pH. The adsorption equilibrium process of Cu(II) ions onto TSFCS was better described by the Langmuir model than the Freundlich isotherm model. Cu(II) ions adsorbed by TSFCS could be released using 0.01M Na2EDTA and the adsorption capacity could retain above 80% after five adsorption-desorption cycles. TSFCS exhibited good potential for heavy metal removal because of its high adsorption capacity at the low pH.

Identification of a small molecule 1,4-bis-[4-(3-phenoxy-propoxy)-but-2-ynyl]-piperazine as a novel inhibitor of the transcription factor p53.

AIM: To identify novel small compound inhibitor of p53 protein. METHODS: Mouse embryonic fibroblasts (MEF) and mouse embryonic stem (ES) cells were tested. Cell proliferation rate was determined using a Cell Proliferation Kit. The mRNA and protein levels of p53-related genes were measured using real-time PCR and Western blotting, respectively. Global response in the p53 signaling network was analyzed using Illumina whole-genome expression BeadChips. RESULTS: Treatment of MEF cells with a small molecule 1,4-bis-[4-(3-phenoxy-propoxy)-but-2-ynyl]-piperazine (G5) at 10 µmol/L for 24 h markedly reduced the mRNA and protein levels of the p53 downstream genes MDM2 and p21. In G5-treated ES cells, a total of 372 differentially expressed genes were identified, and 18 among them were direct downstream genes of p53; 6 out of 9 p53-repressed genes were upregulated, and 5 out of 9 p53-activated genes were downregulated. In both MEF cells and ES cells, treatment of with G5 (10 µmol/L) up to 48 h neither affected the proliferation rate nor caused morphological alterations. CONCLUSION: G5 inhibits p53 activity and simultaneously preserves the normal growth and proliferation of cells, therefore is a new compound for studies of p53-mediated cell manipulation.

[Cloning, sequencing of G3PD gene from Brugia malayi and prediction of B cell epitopes in its amino acid sequence].

Specific primers were designed and synthesized based on the reported glyceraldehyde-3-phosphate dehydrogenase (BmG3PD) gene of Brugia malayi (GenBank Accession No. U18137). Total RNA was extracted from Brugia malayi and its BmG3PD gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR). The PCR product was purified and cloned into plasmid pGEM-T, then transformed into Escherichia coli DH5alpha. The recombinant plasmids were screened and identified by digestion with restriction enzyme and PCR amplification. The positive recombinant plasmid pGEM-T-BmG3PD was confirmed by sequencing and homology comparison. Five parameters and methods were used to predict B-cell epitopes in amino acid sequence of BmG3PD. The amplified DNA fragment (1,020 bp) had a high identity of 99% with the BmG3PD gene sequence of Brugia malayi. B-cell epitopes of BmG3PD were probably at or adjacent to 22-36, 242-255, 303-318 and 326-336 in its amino acid sequence.

[Cloning and eukaryotic expression of the gene encoding myosin from Brugia malayi].

Total RNA was extracted from periodic microfilariae of Brugia malayi and its myosin partial gene (Bm-M55) was amplified by RT-PCR. The PCR product was cloned and then subcloned into pcDNA3.1 (+)vector. The recombinant eukaryotic plasmids were screened and identified by digestion with restriction enzyme and PCR amplification, and was transfected into COS-7 cells subsequently. The expressed protein was identified by SDS-PAGE. Bm-M55 mRNA was highly expressed in transfected COS-7 cells. The deduced amino acid sequence showed to be identical with that of Bm-M55, and the recombinant protein was about Mr 55000.

[Construction of eukaryotic expression plasmids with paramyosin gene of periodic Brugia malayi].

Total RNA was extracted from periodic Brugia malayi Specific primers were designed on the basis of known sequences of paramyosin gene from B. malayi (BmPmy). The desired gene was amplified by PCR technique from cDNA. The PCR products were purified and cloned into plasmid pGEM-T by T-A cloning method, transformed into Escherichia coli (E. coli) strain DH5alpha. The recombinant plasmids were screened and identified by digestion with restriction enzyme and PCR amplification. The right gene fragments encoding BmPmy in positive clones for prokaryotic and eukaryotic expression plasmids were digested with restrictive endonuclease, and were subcloned into pcDNA3.1(+). The recombinant eukaryotic plasmid (pcDNA3.1-BmPmy) was then transfected into COS-7 cells. The transient expression of BmPmy was examined with RT-PCR. BmPmy mRNA was highly expressed in transfected COS-7 cells.

Modified physiological and operative score for the enumeration of mortality and morbidity risk assessment model in general surgery.

AIM: To establish a scoring system for predicting the incidence of postoperative complications and mortality in general surgery based on the physiological and operative severity score for the enumeration of mortality and morbidity (POSSUM), and to evaluate its efficacy. METHODS: Eighty-four patients with postoperative complications or death and 172 patients without postoperative complications, who underwent surgery in our department during the previous 2 years, were retrospectively analyzed by logistic regression. Fifteen indexes were investigated including age, cardiovascular function, respiratory function, blood test results, endocrine function, central nervous system function, hepatic function, renal function, nutritional status, extent of operative trauma, and course of anesthesia. Modified POSSUM (M-POSSUM) was developed using significant risk factors with its efficacy evaluated. RESULTS: The significant risk factors were found to be age, cardiovascular function, respiratory function, hepatic function, renal function, blood test results, endocrine function, nutritional status, duration of operation, intraoperative blood loss, and course of anesthesia. These factors were all included in the scoring system. There were significant differences in the scores between the patients with and without postoperative complications, between the patients died and survived with complications, and between the patients died and survived without complications. The receiver operating characteristic curves showed that the M-POSSUM could accurately predict postoperative complications and mortality. CONCLUSION: M-POSSUM correlates well with postoperative complications and mortality, and is more accurate than POSSUM.