We constructed plasmid pMTac to overexpress 3-ketosteroid-Δ1-dehydrogenase (KSDD) in Mycobacterium neoaurum JC-12 for improving androst-1,4-diene-3,17-dione (ADD) production. To construct pMTac, pACE promoter on pMF41 was replaced by tac promoter, and then four recombinants were constructed, which were M. neoaurum JC-12/pMF41-gfp, M. neoaurum JC-12/pMTac-gfp, M. neoaurum JC-12/pMF41-ksdd and M. neoaurum JC-12/pMTac-ksdd. Fluorescence detection results show that much more green fluorescent protein (GFP) was expressed in M. neoaurum JC-12/pMTac-ksdd than M. neoaurum JC-12/pMF41-ksdd. The activity of KSDD was 2.41 U/mg in M. neoaurum JC-12/pMTac-ksdd, 6.53-fold as that of M. neoaurum JC-12 and 4.36-fold as that of M. neoaurum JC-12/pMF41-ksdd. In shake flask fermentation, ADD production of M. neoaurum JC-12/pMTac-ksdd was 5.94 g/L, increased about 22.2% compared to the original strain M. neoaurum JC-12 and 12.7% to M. neoaurum JC-12/pMF41-ksdd. AD (4-androstene-3,17-dione) production of JC-12/pMTac-ksdd was 0.17 g/L, decreased 81.5% compared to M. neoaurum JC-12 and 71.2% to M neoaurum JC-12/pMF41-ksdd. In the 5 L fermenter, 20 g/L phytosterols was used as substrate, ADD production of M. neoaurum JC-12/pMTac-ksdd was improved to 10.28 g/L. pMTac is favorable for expressing KSDD in M. neoaurum JC-12, and overexpression of KSDD has beneficial effect on ADD producing, and it is the highest level ever reported using fermentation method in M. neoaurum.
Androstadienes ; metabolism ; Fermentation ; Industrial Microbiology ; Mycobacterium ; Oxidoreductases ; genetics ; metabolism ; Phytosterols ; metabolism ; Plasmids

Objective To investigate cognitive function and behavior characteristics in the childhood onset schizo?phrenia patients with or without obsessive-compulsive symptoms. Methods One hundred forty-nine schizophrenia chil?dren were recruited and 72 healthy children served as children control group. According to the Schedule for Affective Dis?orders and Schizophrenia for School-Age Children-Present and Lifetime Version (K-SADS-PL), the patients were fur?ther divided into two groups, the children with obsessive-compulsive disorder group (70 cases) and the children without obsessive-compulsive disorder group (79 cases). All the children were retrospectively surveyed and the Achenbach’s Child Behavior Check-list (CBCL) was used to assess their social competence and behavioral characteristics at the age from 6-year-old to 10-year-old. The cognitive function was estimated with WISC, STROOP color and word test, trail making test, visal spatial memory test and maze test. Results Compare to the control group, the CBCL factor scores of behavior problems were higher (P<0.01), social ability factor scores were lower (P<0.05) and all factor scores in the cog?nitive function tests were lower (P<0.05) in the two patients groups. Compare to the patients without obsessive-compul?sive symptoms, the CBCL factor scores of the school situation (P<0.01), splitting force (P<0.01), and physical complaints (P<0.01) were significantly higher and the scores were significantly lower in the test of common sense (P<0.01), wood (P<0.01), STROOP (P<0.01) and BVMT-R (P<0.01) in those with obsessive-compulsive symptoms. Correlation analysis showed that social competence total scores were positively correlated with similarity in the patients with obsessive-com?pulsive symptoms (r=0.31, P<0.01);while behavior problems total scores were negatively correlated with wood (r=-0.31, P<0.01) and patchwork (r=-0.32, P<0.01) in the patients without obsessive-compulsive symptoms. Conclusion The schizophrenia children with obsessive-compulsive symptoms have more behavioral problems in pre-symptom period and their cognitive dysfunction are more severe following onset of the disease. Cognitive function is related to behavioral prob?lems and social competence in the schizophrenia children with or without obsessive-compulsive symptoms .

Objective To investigate the characteristics and relationship of cognitive function and resting state functional magnetic resonance imaging(fMRI) amplitude of low-frequency fluctuations (ALFF)in childhood and adolescence-first-onset obsessive-compulsive disorder(OCD).Methods Twenty-four childhood and adolescence-first-onset OCD patients (patient group) and thirty-three age/gender/education-matched healthy controls (healthy control group) were enrolled in this study.The participants' cognitive function was tested with trail making test (TMT),symbol coding,Hopkins verbal learning test-revised (HVLT-R),brief visuospatial memory test-revised (BVMT-R),verbal fluency test (VF),digital span test,maze test and Stroop test.Then a resting-state fMRI scan was conducted for each participant.Amplitude of low-frequency fluctuation approach was used to explore the differences of resting-state brain function between patients and controls,and analyze the significant brain regions by adopting t test.The cognitive functions of patients and healthy control group in the significant brain regions were compared by using Pearson correlation analysis.Results The patient group had lower scores than healthy control group in symbol coding,BVMT-R and digital span test,and the differences were statistically significant [(45.24 ± 10.32)scores vs.(56.85 ± 9.57) scores,(21.94 ± 6.99) scores vs.(30.58 ± 3.92) scores,(64.06 ± 20.00) scores vs.(78.03 ±18.08) scores;t =3.96,4.90,2.50;all P ＜ 0.05].Corrected by Alphasim,patient group showed significantly increased ALFF in the left and fight temporal lobe,left and fight posterior lobe of cerebellum,right middle temporal gyrus,but ALFF decreased in the area of left and right lenticula,left putamen compared with the healthy control group (all P ＜ 0.05).The patients group showed that TMT scores were positively correlated with the value of ALFF in the right temporal lobe (r =0.67),and color scores were positively correlated with the value of ALFF in the right middle temporal gyrus (r =0.53),but colour scores were negatively correlated with the value of ALFF in the right tcmporal lobe (r =-0.54);the healthy control group showed that BVMT-R and digital span test scores were positively correlated with the value of ALFF in the left temporal lobe (r =0.47,0.41),but digital span test scores were negatively correlated with the value of ALFF in the right temporal lobe (r =-0.49),and colour scores were negatively correlated with the value of ALFF in the left temporal lobe (r =-0.35),while word and color-word scores were negatively correlated with the value of ALFF in the right middle temporal gyrus (r =-0.39,-0.36);the correlation coefficient above had statistical significance (all P ＜ 0.05).Conclusions The childhood and adolescence-first-onset OCD patients had cognitive impairment,including speed of processing,visual learning and memory,working memory;and the patients showed abnormal spontaneous neural activity at some brain regions.The cognitive dysfunction may be associated with abnormal spontaneous neuronal activity in childhood and adolescence-first-onset OCD.

Objective To explore the classification and characteristics of common syndromes of primary liver cancer and provide reference for clinical treatment.Methods Collect the four diagnostic information of patients with primary liver cancer from two top three TCM hospitals in Henan Province,and build a database.Using Lantern 5.0 software,based on two-step hidden tree analysis,a hidden structure model was constructed,and common syndromes of primary liver cancer were extracted through comprehensive clustering.SPSS 23.0 software was used for factor analysis and systematic cluster analysis to infer the potential syndromes.Combined with the results of different methods and professional knowledge,the syndrome classification of primary liver cancer was determined.Results A total of 1353 patients with 105 symptoms of primary liver cancer were included.59 symptoms with an analysis frequency≥40 were included to construct a hidden structure model,24 hidden variables were obtained,and 5 common syndromes were obtained by comprehensive clustering,namely,qi deficiency syndrome,liver depression and qi stagnation syndrome,blood stasis syndrome,water dampness stagnation syndrome,liver and gallbladder damp heat syndrome.20 common factors were obtained by factor analysis for symptoms with frequency>3%,and 8 common syndromes were inferred by cluster analysis with common factors.7 common syndromes and characteristics were finally determined by combining different methods and expertise.Conclusion The common syndromes of primary liver cancer are qi deficiency syndrome,liver depression and qi stagnation syndrome,blood stasis syndrome,water dampness stagnation syndrome,yin deficiency syndrome,liver and gallbladder damp heat syndrome,spleen deficiency and dampness stagnation syndrome.The results objectively reflect the actual situation of patients with primary liver cancer,and can provide reference for the treatment of primary liver cancer based on syndrome differentiation.

Objective To investigate the relationship between brain white matter volume (WMV) and cognitive function (CF) in childhood and adolescence-onset schizophrenia (CAOS).Methods Forty childhood and adolescence-onset first-episode schizophrenia patients (case group) and thirty-nine healthy volunteers (control group) were selected.All subjects underwent a 3.0 T magnetic resonance imaging(MRI) scans for data acquisition.The Chinese version of MATRICS consensus cognitive battery(MCCB) was used to assess the cognitive function.The psychiatric symptoms of schizophrenia were assessed with the positive and negative syndrome scale (PANSS).And t test was applied to compare whether there were differences in WMV,CF between the two groups.Pearson correlation analysis was used to analyze whether the differences were related with cognitive function and mental symptoms.Results Compared with healthy controls,the case group exhibited WMV deficits in the region of left frontal lobe (LFL) (MNI:x =-27,y =12,z =21),right side limbic lobe (RSLL) (MNI:x =18,y =-9,z =39) and left cingulate gyrus (LCG) (MNI:x =-21,y =-18,z=45) (P＜0.05).The WMV of LFL was negatively related with the total score (r=-0.344,P＜0.05) and the WMV of LCG was negatively related with the positive symptoms score (r=-0.326,P＜0.05) and total score (r=-0.348,P＜0.05) of the PANSS.Besides,compared with the controls,the patients showed significant increase in the scores of Trail Making Test (t =3.56,P＜ 0.01),while significant decrease in the scores of the HVLT-R and BVMT-R(t=-5.67,-8.66,P＜0.05).The WMV of RSLL was positively correlated with verbal fluency test in the case group (r=0.40,P＜0.05).Conclusion CAOS patients exist multiple reduced WMV and cognitive impairment,suggesting the importance of the WMV abnormalities in the pathophysiological mechanism of cognitive impairment.

Objective To construct a CaME141G fusion protein-expressing plasmid,and to express,purify,and identify the activity of the recombinant protein. Methods The 141st site of the wild type CaM,E (GAG),was mutated to G (GGG),using site-specific mutagenesis technology. Escherichia coli BL-21 was transformed with the mutant plasmid. The GST-CaME141G fusion protein was mass-cultured and induced for expression. Subsequently,the GST-CaME141G fusion protein was purified using GS-4B beads. PreScission protease was applied to remove the GST,the Bradford method used to determine the concentration of purified protein,and SDS-PAGE used to detect its relative molecular weight and purity. The GST pull-down assay was used to study the protein's biological activity. Results The CaME141G protein was successfully purified at a high concentration and purity. The protein could interact with PreIQ protein fragments from the myocardial CaV1. 2 calcium channel C terminal,in a CaME141G concentration-dependent manner. Therefore,CaME141G has the ability to bind with the CaV1. 2 calcium channel. Conclusion This study successfully constructed a CaME141G fusion protein-expressing plasmid and purified the CaME141G protein. This lays a foundation for regulating the function of CaM mutations in the myocardial CaV1. 2 calcium channel,and for the study of its relationship with diseases of the cardiovascular system.

Objective:To investigate the relationships of rs4658971 polymorphism of disrupted-in-schizophrinia-1 ( DISC1) gene with schizophrenia in children and adolescents and with the brain structure of such patients. Methods:(1) A total of 577 children and adolescents with schizophrenia were recruited from the Inpatient Department of the Second Affiliated Hospital of Xinxiang Medical University from October 2010 to December 2018, and 516 healthy children were selected as the control group.The rs4658971 polymorphism of DISC1 gene was detected by restriction fragment length polymorphism (PCR-RFLP). The differences of DISC1 genotype and allele frequencies between schizophrenia patients and healthy children were compared by Chi-square test.(2)One hundred and eighteen patients with the first-episode schizophrenia whose illness duration was no more than 12 months and 101 healthy children and adolescents were detected by 3.0T magnetic resonance imaging (MRI). The craniocerebral structures of patients with different DISC1 genotypes were analyzed by covariance with age as the covariant. Results:(1) The rs4658971 genotype frequencies(TT 6.6% vs.5.2%, TC 37.4% vs.38.4%, CC 56.0% vs.56.4%, χ2=0.91, P=0.63) and allele frequencies(C 74.70% vs.75.58%, T 25.30% vs.24.42%, χ2=0.23, P=0.63) of DISC1 gene in children and adolescents with schizophrenia were not significantly different from those in the healthy children.(2) Compared with the control group, the transverse diameter of the third ventricle was significantly enlarged in children and adolescent with schizophrenia [(4.68±1.41) mm vs.(4.06±1.36) mm, F=8.534, P=0.004], while the distance between the anterior feet of the lateral ventricle, the width of the caudate nucleus head and the thickness of the hippocampus were significantly reduced [(30.69±2.51) mm vs.(32.02±2.41) mm, (8.17±1.01) mm vs.(9.22±1.14) mm, (8.93±0.97) mm vs.(9.93±1.14) mm], and the differences were statistically significant( F=15.435, 40.201, 44.650, all P<0.001). The covariance analysis (with age as the covariant) showed that there was significant difference in the width of the caudate nucleus head between patients with CC genotype and those with CT+ TT genotype[(8.73±0.85) mm vs.(7.39±0.61) mm, F=41.376, P<0.001]. Conclusions:There are multiple brain structural changes in children and adolescents with first-episode schizophrenia.T allele carriers of DISC1 gene rs4658971 locus may be related to the reduced width of the caudate nucleus head in patients.

OBJECTIVE:To explore an effective method to formulate management-related strategies for off-lable use of drugs by the evidence-based medicine. METHODS:The process of guideline formulation included seven procedures,i.g. establishment ofguideliesformulation workgroup;investigation and selection of the status quo on off-label drug use;identification of the clinical problems;retrieval and evaluation and comprehensing of evidence;applification of GRADE in evidence quality grading;formation of the recommendations consensus;peer review and result publication. And eventually guidelines were formed based on the steps. This study took off-label use of rheumatoid immunoprotective subjects as a case to explore. RESULTS & CONCLUSIONS:Based on the evidence evaluation system and above 7 steps,the methods and process of guideline formulation on off-label use of rheuma-toid immunoprotective subjects that integrated administration,law,clinical medicine,pharmacy subjects were made .The process of guideline formulation fully reflects multidisciplinary characteristics of the workgroup,the advanced nature of the process,the comprehensiveness of evidence ,the rigor of evidence quality grading,and the normalization of consensus. It provides reference in methodology for establishing a comprehensive evidence-based evaluation and management system of off-label use of drugs for all clinical specialist disease. Therefore,this scientific research results may promote the standardization and legalization of the off-label use of drugs management in China.

OBJECTIVE To evaluate the cost-effectiveness of sintilimab combined with chemotherapy than single-use chemotherapy in the first-line treatment of advanced， recurrent or metastatic esophageal squamous cell carcinoma （ESCC） from the perspective of health system of our country， and provide reference for rational use of drug in clinic. METHODS Based on ORIENT-15 study data， TreeAge Pro 2011 software was used to establish a three-state Markov model of non-progressive survival （PFS）， disease progression and death for cost-utility analysis. The model period was 3 weeks， the research time limit was 10 years， and the discount rate was 5%. The main outputs of the model were total cost， quality-adjusted life year （QALY） and incremental cost-effectiveness ratio （ICER）. The 1-3 times of China’s GDP per capita in 2021 was taken as the threshold of willing- ness to pay （WTP）. The uncertainty of the parameters was analyzed by single factor sensitivity analysis and probability sensitivity analysis， and the cost-effectiveness of the two schemes was discussed under three situations： different discount rates， comparison with other similar treatment schemes and charitable drug donation schemes. RESULTS The results of basic analysis showed that compared with chemotherapy plan alone， the ICER of sintilimab combined with chemotherapy was 64 208.75 yuan/QALY， which was less than WTP threshold. The results of single factor sensitivity analysis show that PFS state utility value， cycle cost of sintilimab and discount rate had relatively great influence on the results. Probability sensitivity analysis showed that when WTP≥120 000 yuan， the economic probability of sintilimab combined with chemotherapy plan was 100%. The results of situational analysis showed that sintilimab combined chemotherapy was more cost- effective than single-use chemotherapy. CONCLUSIONS Sintilimab combined with chemotherapy is more cost-effective than single-use chemotherapy in the first-line treatment of advanced， recurrent or metastatic ESCC.

By organizing and studying on the original works of LI Wenrong， this paper aimed to explore his theories and methods for treating liver diseases. LI Wenrong believes that the liver is easily prone to physiological disturbances， difficult to pacify， and can be compared to a “chariot” that moves horizontally and vertically， acting as a “general” for both civil and military affairs. Pathologically， liver diseases tend to spread to other organs， resulting in ever-changing pathological changes， often affecting emotional health. In terms of therapeutic methods， LI Wenrong proposes ten methods including draining fire to calm the liver， nourishing water to moisten wood， clearing metal to calm the liver， calming gallbladder and harmonizing liver， subduing yang with shell-drug， treating liver diseases by supplementing spleen， soothing the liver with sweetness， astringing the liver with sourness， relieving the liver with spiciness， and draining the liver with excess-fire. These methods have great guidance and reference value for contemporary clinical practice in traditional Chinese medicine.