Cloning, over-expression, and characterization of a new carboxypeptidase A gene of Bacillus pumilus ML413 in Bacillus subtilis 168

Carboxypeptidase A (CPAs) are a well-studied group of zinc-containing exopeptidases that facilitate thebreakdown of proteins and peptides during metabolism. Carboxypeptidase A is typically produced in mammalian pancreatic, brain and other tissues. A new gene encoding carboxypeptidase A in the prokaryote Bacillus pumilus was amplified by polymerase chain reaction (PCR), ligated into the shuttle vector pMA5, and cloned in a GRAS bacteria-Bacillus subtilis 168 host. This gene sequence contained a 1621 bp open reading frame that encodes a protein of 540 amino acids. The optimum pH and temperature for enzyme activity were 7.5 and 50°C, respectively. The enzyme was quite stable at neutral pH and maintained about 65% activity following a 24 h incubation at 40°C. The Km of this CPA was 0.1 mM, much higher than in mammalian species. Glycerol, ammonium sulfate, and sodium citrate improved enzyme activity under optimal culture condition. The carboxypeptidase activity in recombinant B. subtilis 168 reached a maximum of 179 U ml-1 in a 5 L fermentator when cultured on improved medium. The over expression of  carboxypeptidase A in Bacillus subtilis has commercial applications.Key words: Bacillus pumilus, Bacillus subtilis 168, over-expression, orthogonal arrays, carboxypeptidase A,metallocarboxypeptidase

Over-expression of Mycobacterium neoaurum 3-ketosteroid-\u3941-dehydrogenase in Corynebacterium crenatum for efficient bioconversion of 4-androstene-3,17-dione to androst-1,4-diene-3,17-dione

Background: 3-Ketosteroid-\u3941-dehydrogenase (KSDD), a flavoprotein enzyme, catalyzes the bioconversion of 4-androstene-3,17-dione (AD) to androst-1,4-diene-3,17-dione (ADD). To date, there has been no report about characterization of KSDD from Mycobacterium neoaurum strains, which were usually employed to produce AD or ADD by fermentation. Results: In this work, Corynebacterium crenatum was chosen as a new host for heterologous expression of KSDD from M. neoaurum JC-12 after codon optimization of the KSDD gene. SDS-PAGE and western blotting results indicated that the recombinant C. crenatum harboring the optimized ksdd (ksddII) gene showed significantly improved ability to express KSDD. The expression level of KSDD was about 1.6-fold increased C. crenatum after codon optimization. After purification of the protein, we first characterized KSDD from M. neoaurum JC-12, and the results showed that the optimum temperature and pH for KSDD activity were 30\ub0C and pH 7.0, respectively. The Km and Vmax values of purified KSDD were 8.91 \u3bcM and 6.43 mM/min. In this work, C. crenatum as a novel whole-cell catalyst was also employed and validated for bioconversion of AD to ADD. The highest transformation rate of AD to ADD by recombinant C. crenatum was about 83.87% after 10 h reaction time, which was more efficient than M. neoaurum JC-12 (only 3.56% at 10 h). Conclusions: In this work, basing on the codon optimization, overexpression, purification and characterization of KSDD, we constructed a novel system, the recombinant C. crenatum SYPA 5-5 expressing KSDD, to accumulate ADD from AD efficiently. This work provided new insights into strengthening sterol catabolism by overexpressing the key enzyme KSDD, for efficient ADD production

Strongly coupled multicore fiber with FBGs for multipoint and multiparameter sensing

[EN] A compact optical fiber sensor by embedding fiber Bragg gratings (FBGs) in strongly coupled multicore fiber (SCMCF) is proposed for multipoint and multiparameter sensing. To build the device, two FBGs with different peak wavelengths were inscribed in a segment of SCMCF. Then one end of the SCMCF was fusion spliced to a single-mode fiber (SMF) and the other end of the SCMCF was cleaved. In the SMF-SCMCF structure, two supermodes are excited, as a result, the reflection spectrum exhibits a sinusoidal pattern with two sharp peaks. The wavelength position of the FBGs and the supermode coupler can be extracted simultaneously. Two distinct FBGs inscribed in different positions of the SCMCF were used to demonstrate quasi-distributed multipoint sensing in the proposed structure. To the best of our knowledge, this is the first demonstration of an optical fiber sensor that combines FBGs with SCMCF. The sensor here proposed has the advantage of compact size, low-cost, good mechanical strength and ease of interrogation.This work was supported in part by National Key Research and Development Program of China (2019YFB2203204), National Natural Science Foundation of China (61922069, 61775185), the "111" Plan (B18045), Sichuan Science and Technology Program (2020YJ0329), and the Spanish Ministry of Economy and Competitiveness under the project DIMENSION TEC2017 88029-R. J.V. acknowledges funding from the Fondo Europeo de Desarrollo Regional (FEDER) and the Ministerio de Economia y Competitividad (Spain) under project PGC2018-101997-B-I00. DocumentLiu, Z.; Zheng, D.; Madrigal-Madrigal, J.; Villatoro, J.; Antonio-Lopez, E.; Schuelzgen, A.; Amezcua-Correa, R.... (2020). Strongly coupled multicore fiber with FBGs for multipoint and multiparameter sensing. Optical Fiber Technology. 58:1-7. https://doi.org/10.1016/j.yofte.2020.102315S175

Design evolution and knowledge handling for product conceptualization in new product development

The highly competitive and rapidly changing global business environment requires companies to deliver new products with better performance, lower cost, higher quality and shorter time-to-market. To develop a successful product, conceptualization of the product plays an extremely important role and, hence, deserves much more attention. In essence, this work views product concept development (PCD) as a strategic task that has a major impact on subsequent production related activities. In PCD, design evolution and evaluation are of great importance to generate satisfactory design concepts. To facilitate the efficiency of PCD and, hence, shorten the time-to-market, this work aims at establishing new approaches and methodologies to address the decisive issues of design evolution and evaluation in product conceptualization. In so doing, it is found that current practices and literature have not adequately addressed the issue of design information reuse in conceptual design evolution. Such difficulties as indexing, retrieval and modification of prior design knowledge remain to be dealt with. On the other hand, a knowledge-based approach has been recognized as a promising and logical alternative to support design evaluation. However, it is well-known that knowledge acquisition is the most time-consuming phase, and, therefore, the bottleneck in constructing a knowledge-based system (KBS). There are two major omissions in existing work. First, the quantitative characteristics of information and interrelationships between various knowledge elements, which are of critical importance in constructing a KBS, have not been investigated. Second, an integrated and easy-to-use knowledge acquisition process with a well-organized knowledge representation scheme to facilitate rule generation needs to be explored. Accordingly, a feature-based design evolution approach via case-based reasoning (CBR) is proposed to facilitate the evolution process of concept generation. In this approach, a feature relationship decomposition (FRD) procedure is postulated. The FRD provides an indexing scheme of CBR for design reuse with more flexibility of case retrieval. The performance of analytical hierarchy process (AHP) is enhanced with FRD via reducing the lengthy calculations of pair-wise comparisons. To arm the proposed design evolution approach with the capability of innovation, a TRIZ-enhanced innovative adaptation procedure (IAP) is investigated. Additionally, a matrix representation and mapping approach (MRM) is proposed to tackle the difficulty of knowledge acquisition in constructing a KBS to support design evaluation. The MRM approach establishes a systematic procedure to model the rule generation process mathematically. It uses matrix representation and mapping techniques to represent the knowledge attributes and reveal their interrelationships. It addresses the quantitative characteristics of relationship strength between various knowledge elements using a relationship estimation procedure. Three case studies on wood golf club design, hard disk design, and diagnosing automotive systems are employed to verify the proposed approaches, i.e. FRD, IAP, MRM, respectively. The economic impact of this work is to enhance design efficiency in new product development so as to reduce design time and hence cost by effectively making use of historical design data. In this work, the proposed CBR-based design system has been tested on tangible product design problems. Given the proposed benefits, the possibility of extending the current work to a broader scope will merit further exploration.DOCTOR OF PHILOSOPHY (MAE

Gene based message passing for drug repurposing

Summary: The medicinal effect of a drug acts through a series of genes, and the pathological mechanism of a disease is also related to genes with certain biological functions. However, the complex information between drug or disease and a series of genes is neglected by traditional message passing methods. In this study, we proposed a new framework using two different strategies for gene-drug/disease and drug-disease networks, respectively. We employ long short-term memory (LSTM) network to extract the flow of message from series of genes (gene path) to drug/disease. Incorporating the resulting information of gene paths into drug-disease network, we utilize graph convolutional network (GCN) to predict drug-disease associations. Experimental results showed that our method GeneDR (gene-based drug repurposing) makes better use of the information in gene paths, and performs better in predicting drug-disease associations

Fairness and Smiling Mediate the Effects of Openness on Perceived Fairness: Beside Perceived Intention

Previous studies have shown that smiling, fairness, intention, and the results being openness to the proposer can influence the responses in ultimatum games, respectively. But it is not clear that how the four factors might interact with each other in twos or in threes or in fours. This study examined the way that how the four factors work in resource distribution games by testing the differences between average rejection rates in different treatments. Two hundred and twenty healthy volunteers participated in an intentional version of the ultimatum game (UG). The experiment used a 2 × 2 × 2 × 2 mixed design with “openness” as a between subjects factor and the other three as within subjects factors, and the participants were assigned as recipients. The results revealed that fairness or perceived good intention reduced the subject’s average rejection rates. There was a significant interaction between facial expressions and openness. With fair offers, the average rejection rate for informed was lower than that of uninformed; but when unfair, no difference between the corresponding average rejection rates was found. The interaction effect of smiling and openness was also significant, the average rejection rate for informed offers was lower when the proposer was smiling and no rejection rate difference between uninformed offers and informed offers when no smiling. No other interaction effect was found

Over-expression of Mycobacterium neoaurum 3-ketosteroid-Δ1-dehydrogenase in Corynebacterium crenatum for efficient bioconversion of 4-androstene-3,17-dione to androst-1,4-diene-3,17-dione

Background: 3-Ketosteroid-Δ1-dehydrogenase (KSDD), a flavoprotein enzyme, catalyzes the bioconversion of 4-androstene-3,17-dione (AD) to androst-1,4-diene-3,17-dione (ADD). To date, there has been no report about characterization of KSDD from Mycobacterium neoaurum strains, which were usually employed to produce AD or ADD by fermentation. Results: In this work, Corynebacterium crenatum was chosen as a new host for heterologous expression of KSDD from M. neoaurum JC-12 after codon optimization of the KSDD gene. SDS-PAGE and western blotting results indicated that the recombinant C. crenatum harboring the optimized ksdd (ksddII) gene showed significantly improved ability to express KSDD. The expression level of KSDD was about 1.6-fold increased C. crenatum after codon optimization. After purification of the protein, we first characterized KSDD from M. neoaurum JC-12, and the results showed that the optimum temperature and pH for KSDD activity were 30°C and pH 7.0, respectively. The Km and Vmax values of purified KSDD were 8.91 μM and 6.43 mM/min. In this work, C. crenatum as a novel whole-cell catalyst was also employed and validated for bioconversion of AD to ADD. The highest transformation rate of AD to ADD by recombinant C. crenatum was about 83.87% after 10 h reaction time, which was more efficient than M. neoaurum JC-12 (only 3.56% at 10 h). Conclusions: In this work, basing on the codon optimization, overexpression, purification and characterization of KSDD, we constructed a novel system, the recombinant C. crenatum SYPA 5-5 expressing KSDD, to accumulate ADD from AD efficiently. This work provided new insights into strengthening sterol catabolism by overexpressing the key enzyme KSDD, for efficient ADD production

Effects of Different Aging Methods on the Phenolic Compounds and Antioxidant Activity of Red Wine

In this study, oak barrels, glazed pottery altars, unglazed pottery altars, and stainless-steel tanks were selected as aging containers for red wine, and phenolic compounds and antioxidant activity were analyzed and compared. The color of red wine in unglazed pottery altars and glazed pottery altars changed to brick red and brownish yellow, respectively; the color of red wine in oak barrels did not change significantly; and color retention was best in stainless-steel tanks. The total content of anthocyanins and nonanthocyanin phenolic compounds was higher in the unglazed pottery altar group (227.68 mg/L and 288.88 mg/L, respectively) than in the oak barrel group (209.46 mg/L and 273.42 mg/L), the stainless-steel tank group (221.92 mg/L and 213.23 mg/L), or the glazed pottery altar group (74.71 mg/L and 204.43 mg/L). After aging, DPPH (1,1-diphenyl-2-picrylhydrazine free radical scavenging ability), I confirm. (ABTS+ free radical scavenging ability), and FRAP (a ferric ion-reducing antioxidant power reduction of Ion Ability) were decreased by 8.8%, 0.5%, and 17.1%, respectively, in the unglazed pottery altar group; by 15.2%, 1.7%, and 19.5%, respectively, in the oak barrel group; by 18.0%, 1.8%, and 20.0%, respectively, in the stainless-steel tank group; and by 18.7%, 4.2%, and 34.9%, respectively, in the glazed pottery altar group. In conclusion, antioxidative ability decreased less in the unglazed pottery altar group than in the other three groups, indicating that unglazed pottery altars retain antioxidant components in red wine well