Development of biosensors highly responsive to N-acetylneuraminic acid in Bacillus subtilis / 生物工程学报

Bacillus subtilis is recognized as a generally-regarded-as-safe strain, and has been widely used in the biosynthesis of high value-added products, including N-acetylneuraminic acid (NeuAc) which is widely used as a nutraceutical and a pharmaceutical intermediate. Biosensors responding to target products are widely used in dynamic regulation and high-throughput screening in metabolic engineering to improve the efficiency of biosynthesis. However, B. subtilis lacks biosensors that can efficiently respond to NeuAc. This study first tested and optimized the transport capacity of NeuAc transporters, and obtained a series of strains with different transport capacities for testing NeuAc-responsive biosensors. Subsequently, the binding site sequence of Bbr_NanR responding to NeuAc was inserted into different sites of the constitutive promoter of B. subtilis, and active hybrid promoters were obtained. Next, by introducing and optimizing the expression of Bbr_NanR in B. subtilis with NeuAc transport capacity, we obtained an NeuAc-responsive biosensor with wide dynamic range and higher activation fold. Among them, P535-N2 can sensitively respond to changes in intracellular NeuAc concentration, with the largest dynamic range (180-20 245) AU/OD. P566-N2 shows a 122-fold of activation, which is 2 times of the reported NeuAc-responsive biosensor in B. subtilis. The NeuAc-responsive biosensor developed in this study can be used to screen enzyme mutants and B. subtilis strains with high NeuAc production efficiency, providing an efficient and sensitive analysis and regulation tool for biosynthesis of NeuAc in B. subtilis.

Functional analysis of functional membrane microdomains in the biosynthesis of menaquinone-7 / 生物工程学报

Functional membrane microdomains (FMMs) that are mainly composed of scaffold proteins and polyisoprenoids play important roles in diverse cellular physiological processes in bacteria. The aim of this study was to identify the correlation between MK-7 and FMMs and then regulate the MK-7 biosynthesis through FMMs. Firstly, the relationship between FMMs and MK-7 on the cell membrane was determined by fluorescent labeling. Secondly, we demonstrated that MK-7 is a key polyisoprenoid component of FMMs by analyzing the changes in the content of MK-7 on cell membrane and the changes in the membrane order before and after destroying the integrity of FMMs. Subsequently, the subcellular localization of some key enzymes in MK-7 synthesis was explored by visual analysis, and the intracellular free pathway enzymes Fni, IspA, HepT and YuxO were localized to FMMs through FloA to achieve the compartmentalization of MK-7 synthesis pathway. Finally, a high MK-7 production strain BS3AT was successfully obtained. The production of MK-7 reached 300.3 mg/L in shake flask and 464.2 mg/L in 3 L fermenter.

Construction and optimization of ergothioneine-producing Escherichia coli / 生物工程学报

Ergothioneine (ERG) is a natural antioxidant that has been widely used in the fields of food, medicine and cosmetics. Compared with traditional plant extraction and chemical synthesis approaches, microbial synthesis of ergothioneine has many advantages, such as the short production cycle and low cost, and thus has attracted intensive attention. In order to engineer an ergothioneine high-yielding Escherichia coli strain, the ergothioneine synthesis gene cluster egtABCDE from Mycobacterium smegmatis and egt1 from Schizosaccharomyces pombe were introduced into E. coli BL21(DE3) to generate a strain E1-A1 harboring the ergothioneine biosynthesis pathway. As a result, (95.58±3.2) mg/L ergothioneine was produced in flask cultures. To further increase ergothioneine yield, the relevant enzymes for biosynthesis of histidine, methionine, and cysteine, the three precursor amino acids of ergothioneine, were overexpressed. Individual overexpression of serAT410STOP and thrA resulted in an ergothioneine titer of (134.83±4.22) mg/L and (130.26±3.34) mg/L, respectively, while co-overexpression of serAT410STOP and thrA increased the production of ergothioneine to (144.97±5.40) mg/L. Eventually, by adopting a fed-batch fermentation strategy in 3 L fermenter, the optimized strain E1-A1-thrA-serA* produced 548.75 mg/L and 710.53 mg/L ergothioneine in glucose inorganic salt medium and rich medium, respectively.

Effect of key enzymes ubiquitination sites on the biosynthesis of naringenin / 生物工程学报

Flavonoids have a variety of biological activities and have important applications in food, medicine, cosmetics, and many other fields. Naringenin is a platform chemical for the biosynthesis of many important flavonoids. Ubiquitination plays a pivotal role in the post-translational modification of proteins and participates in the regulation of cellular activities. Ubiquitinated proteins can be degraded by the ubiquitin-protease system, which is important for maintaining the physiological activities of cells, and may also exert a significant impact on the expression of exogenous proteins. In this study, a real-time in-situ detection system for ubiquitination modification has been established in Saccharomyces cerevisiae by using a fluorescence bimolecular complementation approach. The ubiquitination level of protein was characterized by fluorescence intensity. By using the approach, the potential ubiquitination sites of proteins involved in the naringenin biosynthesis pathway have been obtained. The lysine residues of the relevant ubiquitination sites were mutated to arginine to reduce the ubiquitination level. The mutants of tyrosine ammonia-lyase (FjTAL) and chalcone synthase (SjCHS, SmCHS) showed decreased fluorescence, suggested that a decreased ubiquitination level. After fermentation verification, the S. cerevisiae expressing tyrosine ammonia-lyase FjTAL mutant FjTAL-K487R accumulated 74.2 mg/L p-coumaric acid at 72 h, which was 32.3% higher than that of the original FjTAL. The strains expressing chalcone synthase mutants showed no significant change in the titer of naringenin. The results showed that mutation of the potential ubiquitination sites of proteins involved in the naringenin biosynthesis pathway could increase the titer of p-coumaric acid and have positive effect on naringenin biosynthesis.

Strategies and tools for metabolic engineering in Bacillus subtilis / 生物工程学报

As a typical food safety industrial model strain, Bacillus subtilis has been widely used in the field of metabolic engineering due to its non-pathogenicity, strong ability of extracellular protein secretion and no obvious codon preference. In recent years, with the rapid development of molecular biology and genetic engineering technology, a variety of research strategies and tools have been used to construct B. subtilis chassis cells for efficient synthesis of biological products. This review introduces the research progress of B. subtilis from the aspects of promoter engineering, gene editing, genetic circuit, cofactor engineering and pathway enzyme assembly. Then, we also summarized the application of B. subtilis in the production of biological products. Finally, the future research directions of B. subtilis are prospected.

Current status and future perspectives of metabolic network models of industrial microorganisms / 生物工程学报

Genome-scale metabolic network model (GSMM) is an extremely important guiding tool in the targeted modification of industrial microbial strains, which helps researchers to quickly obtain industrial microbes with specific traits and has attracted increasing attention. Here we reviewe the development history of GSMM and summarized the construction method of GSMM. Furthermore, the development and application of GSMM in industrial microorganisms are elaborated by using four typical industrial microorganisms (Bacillus subtilis, Escherichia coli, Corynebacterium glutamicum, and Saccharomyces cerevisiae) as examples. In addition, prospects in the development trend of GSMM are proposed.

Interdisciplinary education of fermentation engineering graduates / 生物工程学报

Fermentation engineering is an industrial process that uses the transformation of microorganisms or other cells to produce a specific product in a specific bioreactor. Fermentation engineering has developed from an ancient food fermentation relying solely on experience accumulation to an important production mode of food, agriculture, medicine, chemical industry and other means of production and life. It has become a key technology to support the sustainable development of human beings, and is inseparable from the continuous progress of interdisciplinary technology. The interdisciplinary integration and the continuous upward movement of China's global industrial chain will inevitably put forward higher requirements for the cultivation of fermentation engineering composite talents in the new situation. In order to constantly improve the interdisciplinary fermentation engineering compound talent training system, in recent years, the research lab has been refining and improving the concept of talent training, and actively deepening the reform of talent training system. Systematic research and practice have been carried out around the aspects of training program, enrollment system, teacher background, subject setting, scientific research practice, evaluation system, etc., which has promoted the technological progress of fermentation engineering and related supporting industries, and contributed an important force to the transformation of China from a big fermentation country to a powerful fermentation country.

Expression optimization and molecular modification of heparin C5 epimerase / 生物工程学报

Heparin and heparan sulfate are a class of glycosaminoglycans for clinical anticoagulation. Heparosan N-sulfate-glucuronate 5-epimerase (C5, EC 5.1.3.17) is a critical modifying enzyme in the synthesis of heparin and heparan sulfate, and catalyzes the inversion of carboxyl group at position 5 on D-glucuronic acid (D-GlcA) of N-sulfoheparosan to form L-iduronic acid (L-IdoA). In this study, the heparin C5 epimerase gene Glce from zebrafish was expressed and molecularly modified in Escherichia coli. After comparing three expression vectors of pET-20b (+), pET-28a (+) and pCold Ⅲ, C5 activity reached the highest ((1 873.61±5.42) U/L) with the vector pCold Ⅲ. Then we fused the solution-promoting label SET2 at the N-terminal for increasing the soluble expression of C5. As a result, the soluble protein expression was increased by 50% compared with the control, and the enzyme activity reached (2 409±6.43) U/L. Based on this, site-directed mutations near the substrate binding pocket were performed through rational design, the optimal mutant (V153R) enzyme activity and specific enzyme activity were (5 804±5.63) U/L and (145.1±2.33) U/mg, respectively 2.41-fold and 2.28-fold of the original enzyme. Modification and expression optimization of heparin C5 epimerase has laid the foundation for heparin enzymatic catalytic biosynthesis.

Directed evolution of chitinase Chisb and biosynthesis of chitooligosaccharides / 生物工程学报

Chitinase has a wide industrial application prospect. For example, it can degrade shrimp shells, crab shells and other crustacean waste into high value-added chitooligosaccharides. However, the low catalytic efficiency of chitinase greatly limits the production of chitooligosaccharides. In previous study, the we expressed a chitinase Chisb with high catalytic efficiency and studied its enzymatic properties. In order to further improve the catalytic efficiency of Chisb, with R13NprB-C-SP-H as the parent, here error-prone PCR was used to construct random mutant library to conduct directed evolution of chitinase Chisb. Two mutants C43D and E336R were obtained with 96-well plate primary screening and shaker-screening, and their enzymatic properties were also studied. The optimum temperature of C43D and E336R was 55 °C, and the optimum pH of C43D was 5.0, while that of E336R was 9.0. The catalytic efficiency of C43D and E336R was 1.35 times and 1.57 times higher than that of control. The chitooligosaccharide concentration of E336R and C43D was 2.53 g/L and 2.06 g/L, improved by 2.84 times and 2.31 times compared with the control (0.89 g/L), respectively. In addition, the substrate conversion rate of mutants E336R and C43D was 84.3% and 68.7%, improved by 54.6% and 39% compared with the control (29.7%), respectively. In summary, the study indicates that random mutation introduced by error-prone PCR can effectively improve the catalytic efficiency of chitinase Chisb. The positive mutants with higher catalytic efficiency obtained in the above study and their enzymatic property analysis have important research significance and application value for the biosynthesis of chitooligosaccharides.

Research and prospect of hospital scientific research performance management / 中华医院管理杂志

Accelerating the management of scientific research performance for hospitals can not only mobilize the enthusiasm of scientific researchers, but also effectively optimize their scientific research work. The present academia is plagued in general by such setbacks as indeterminacy of definition and scope of scientific research performance, confusion of the evaluation index system of scientific research performance, and lack of unification of the evaluation principles of scientific research performance of the hospitals. Therefore, it is imperative to define the scope of hospital scientific research performance scientifically, eliminate performance indicator deviation efficiently, accurately analyze hospital scientific research performance management cases, and encourage the application of third-party evaluation methods, which are trendy in research.

Relationship between social anxiety and acceptance of disability in breast cancer patients with mastectomy / 中国实用护理杂志

Objective To explore the relationship between social anxiety and acceptance of disability in breast cancer patients with mastectomy. Methods Totally 325 patients with breast cancer were investigated with general information questionnaire, Interaction Anxiety Scale and Acceptance of Disability Scale.Results The total score of Interaction Anxiety Scale was(40.01±9.38)points.The total score of Acceptance of Disability Scale was (78.02 ± 11.61) points. One-way ANOVA showed that age, education level, marital status, economic level, whether the spouse care about the appearance or not, therapy types affected social anxiety significantly(t/F=-4.696-35.694,all P<0.01).Significantly negative correlation was found between social anxiety and acceptance of disability (r =-0.469--0.371, P<0.01). Multiple regression analysis showed that acceptance of disability,age,whether the spouse care about the appearance or not, therapy types were influencing factors of social anxiety. Conclusions Nurses and doctors should explore effective psychological intervention mode to rebuild the patient′s self-confidence and return to normal social interaction in order to improve the acceptance of disability.

Effect of the intervention based on Information-Motivation-Behavioral skills on the preoperative anxiety in patients with gynecological malignancies / 中国实用护理杂志

Objective To investigate the effect of interventions based on Information-Motivation-Behavioral skills on the preoperative anxiety in patients with gynecological malignancies. Methods A total of 64 patients with malignant tumor were divided into two groups by random digits table method with 32 cases each. The patients in the two groups received routine nursing. In addition, interventions based on Information-Motivation-Behavioral skills were provided in the intervention group. All patients were investigated by the following indexes such as the Self-Rating Anxiety Scale (SAS), sleep quality scale, blood pressure and heart rate before and after the intervention. Results After the intervention, the SAS scores in the intervention group was (49.47 ± 3.81) points, sleep quality score was (3.66 ± 0.97) points, and systolic blood pressure and heart rate were (128.56±5.93) mmHg (1 mmHg=0.133 kPa), (75.09 ± 3.78) beats/min, which were lower than those in the control group (57.38 ± 3.75) points, (5.50 ± 1.50) points, (134.97 ± 7.19) mmHg, (81.34 ± 4.88) beats/min, the differences were statistically significant (t=-8.350--3.887, P<0.05). Conclusions The intervention focused on Information-Motivation-Behavioral Skills can help patients to relieve anxiety, reduce stress response, improve quality of sleep.

The effect of written expression on perceived stress, anxiety and depression in patients with gynecologic malignant tumor during the stage of chemotherapy / 中国实用护理杂志

Objective To evaluate the effect of written expression on perceived stress,anxiety and depression in patients with gynecologic malignant tumor during chemotherapy. Methods From June 2016 to December 2016,90 chemotherapy patients with gynecologic malignant tumor were extracted from North China University of Science and Technology and divided into intervention group and control group by random digits table method,45 cases in each group.The control group received routine psychological intervention and while the intervention group received written expression lasting for 4 weeks in addition. Before and after the intervention,all patients were measured using the Chinese Version Perceived Stress Scale(CPSS), Self-Rating Anxiety Scale(SAS) and Self-Rating depression Scale(SDS). Results The intervention group showed a significant decrease in CPSS(32.12 ± 3.75) points, SAS(43.67 ± 5.23) points, SDS(46.18±4.37)points,compared with the control group(37.26±4.42),(50.88±3.15),(52.47±3.60)points, the difference was significant(t=-5.782,-7.667,-7.230,all P<0.05). Conclusions Written expression can effectively reduce perceived stress, anxiety and depression in patients with gynecologic malignant tumor undergoing chemotherapy.

Metabolic engineering of Saccharomyces cerevisiae for production of glucaric acid / 生物工程学报

Glucaric acid, a high value-added organic acid, is widely used in food, pharmaceutical and chemical industries. For microbial production of glucaric acid in Saccharomyces cerevisiae, we constructed a synthetic glucaric acid biosynthetic pathway by coexpressing the genes encoding myo-inositol oxygenase from mice and uronate dehydrogenase from Pseudomonas putida. Moreover, myo-inositol-1-phosphate synthase was identified as a rate-limiting enzyme in glucaric acid pathway and was upregulated, resulting in the production of glucaric acid of (107.51±10.87) mg/L, a 2.8-fold increase compared to the parent strain. Then, by repressing the activity of phosphofructokinase, the concentration of glucaric acid further increased to (230.22±10.75) mg/L. The strategy could be further used to construct cell factories for glucaric acid production.

Chemokine CXCL12 and its receptor CXCR4 expression and significance in hypopharynx cancer / 中国医师杂志

Objective To investigate the chemokine 12 (CXCL12) and chemokine receptor 4 (CXCR4) expressions in hypopharyngeal carcinoma and its place in the disease development,invasion and metastasis of significance.Methods Immunohistochemistry was used to detect the expressions of CXCL12 and CXCR4 in 35 cases of hypopharyngeal cancer tissues and in 28 cases of tumor-adjacent non-tumor tissues.Results The expressions of CXCL12 and CXCR4 in the hypopharynx carcinomas were significantly higher (P ＜ 0.05).Both expressed in hypopharyngeal carcinomas was significantly positively correlated (P ＜ 0.01).Both hypopharynx cancer in lymph node metastasis group were significantly higher than the expression of cervical lymph node metastasis group,the difference was significant (P ＜ 0.05).Conclusions CXCL12 and CXCR4 are involved in hypopharynx cancer development,invasion and metastasis,and there is a positive feedback regulation mechanism between two factors.Moreover,CXCL12 and CXCR4 have synergistic effect in development,invasion and metastasis of hypopharynx cancers.

Mutagenesis of Methylobacterium extorquens AM1 for increasing pyrroloquinoline quinone production by atmospheric and room temperature plasma / 生物工程学报

As a novel cofactor of oxidoreductase, pyrroloquinoline quinone (PQQ) has a great potential of application in medicine, food industries. In order to improve the efficiency of the PQQ production by Methylobacterium extorquens AM1, the strain was treated by atmospheric and room temperature plasma (ARTP). Positive mutants with changes in PQQ yield were obtained based on a high-throughput screening approach. After ARTP treatment, analysis data show that the positive mutation rate was 31.6%. Furthermore, we obtained an excellent positive mutant M. extorquens AM1 (E-F3) with the yield of 54.0 mg/L PQQ, which was approximately 3 times as much compared with that of the wild-type strain. The robust high-throughput screening method for mutagenesis by ARTP improves PQQ production. In addition, this method also provides a new strategy for further strain improvement.

MicroRNA-146 a promotes proliferation of rat vascular smooth muscle cells by up-regulating cyclin D1 expression / 中国免疫学杂志

Objective:To detect and verifica the gene profile difference of microRNA-146a (miR-146a) and its role in the pro-liferation of vascular smooth muscle cells (VSMCs) by gene chip technology. Methods: Artificially synthesized miR-146a mimics(50 nmol/L) ,miR-146 inhibitor ( 50 nmol/L ) , scramble ( 50 nmol/L ) and PBS were transfected into cultured primary rat VSMCs in vitro. After transfection,Real time PCR was used to measure the levels of miR-146a and the cell counting kit 8(CCK8) was employed to investigate the proliferation of VSMCs. The VSMCs interfered by miR-146a inhibitor or miR-146a control were examined by gene chips and the profile of gene were analyzed by bioinformatics technology to detect the different genes and signal transduction pathway. The changes in mRNAs and proteins were accessed separately by Real time PCR and Western blot. Results: Compared with sham and control VSMCs,miR-146a expression level was significantly decreased in treatment with miR-146a inhibitor(P0. 05),however,the mRNA and protein expression levels of cyclin D1 significantly increased in treatment with miR-146a mimics VSMCs group and decreased in miR-146a inhibitor VSMCs group ( compared with sham VSMCs group, both P<0. 05 ) . Conclusion: Our data indicated that miR-146a may promote the proliferation of rat VSMCs by up-regulating cyclin D1 expression.

Comparison of the hemostatic effect of total knee arthroplasty in intravenous and intra-articular injection of tranexamic acid / 重庆医学

Objective To find a more suitable approach for the application of tranexamic acid(TXA)on total knee arthroplas‐ty (TKA) .Methods Totally 60 patients who met the inclusion criteria from January 2014 to August 2015 in the First Affiliated Hospital of Shihezi University were selected and divided into two groups according to the different route of administration .Group A (n=30) was intravenously injected with 100 mL TXA ,and group B(n=30) was locally injected with 100 mL TXA .Three hours drainage tubes occlusion were carried out after operation in the two groups .The intraoperative and postoperative dominant blood loss ,hidden blood loss indexes and the amount of total blood loss were recorded ,and coagulation indexes and D‐2 polymer were reg‐ularly monitored ,the incidence of thrombosis and postoperative adverse events were also observed .Results The amount of total blood loss in group B[(895 .41 ± 239 .02)mL] was lower than that in group A[(1 020 .89 ± 210 .83)mL] ,and the difference was statistically significant (P0 .05) .No blood trans‐fusion ,symptomatic deep venous thrombosis and fatal pulmonary embolism occurred in the two groups .Conclusion The hemostatic effect of local application of TXA is better than that of intravenous injection in patients′initial TKA .

Application of polyetheretherketone cages and titanium cages combined with pedicle screw fixation operation in lumbar degenerative disease / 局解手术学杂志

Objective To explore the clinical efficacy of polyetheretherketone( PEEK) cages and titanium cages combined with pedicle screw fixation operation in treatment of lumbar degenerative disease,and provide reference for clinic. Methods The data of 50 patients with lumbar degenerative disease in our hospital from March 2011 to March 2013 were retrospectively analyzed. All patients were treated with pos-terior decompression,bone graft and transpedicular screw internal fixation,according to the different cages,they were divided into PEEK group and titanium group. The PEEK group with 25 patients used polyetheretherketone cages,and the titanium group with 25 patients used titanium cages. The JOA scores of patient before surgery,1 month,6 months and 1 year after surgery between two groups were recorded and compared. The ROM of fusion levels and adjacent segment of patients before and after operation between two groups were compared. Results There was no statistical significance of difference in JOA score,Oswestry score and fusion rate between two groups(P>0. 05). There were statistical significance of differences intervertebral disc height change between 2 groups(P<0. 05). The ROM of fusion levels and adjacent segment of patients in PEEK group was better than those in titanium group(P<0. 05). Conclusion The polyetheretherketone cages combined with pedicle screw fixation operation have a good effect in the treatment of patients with lumbar degenerative disease,especially in matter of lumbar spinal motion and intervertebral height lost.

Chemerin promotes proliferation of mouse vascular smooth muscle cells by up-regulating p-JNK / 中国病理生理杂志

AIM:To investigate the proliferation property of stable chemerin gene knockdown vascular smooth muscle cells ( VSMCs) and to explore its mechanism .METHODS:The normal VSMCs , chemerin gene interfering control VSMCs and stable chemerin gene knockdown VSMCs were divided into normal group , PDGF group, control group and knockdown group .The VSMCs in PDGF group were given platelet-derived growth factor-BB ( PDGF-BB) to initiate proli-feration.The cell counting and BrdU assay were employed to investigate the proliferation property of VSMCs .The mitogen-activated protein kinase ( MAPK) signal pathway was determined by Western blot .RESULTS:The cell number and BrdU A value in PDGF-BB-treated VSMCs significantly increased as compared with the normal VSMCs ( P<0.05 ) .Compared with the normal VSMCs , the chemerin knockdown VSMCs showed obviously decreased cell number and BrdU A value ( P<0.05).Simultaneously, no significant difference in the proliferation of VSMCs between the normal VSMCs and the control VSMCs was observed.No significant difference of the protein levels of p-ERK1/2, ERK1/2, p-p38 MAPK and p38 MAPK among 4 kinds of VSMCs was found .The protein level of p-JNK in PDGF-BB-treated VSMCs was up-regulated, while it was down-regulated in chemerin knockdown VSMCs compared with the normal VSMCs .CONCLUSION: Chemerin pro-motes the proliferation of mouse vascular smooth muscle cells by up-regulating p-JNK production .