Heterologous expression of nattokinase from B. subtilis natto using Pichia pastoris GS115 and assessment of its thrombolytic activity.

BACKGROUND: Nattokinase is a fibrinolytic enzyme that has huge market value as a nutritional supplement for health promotion. In order to increase nattokinase yields, fermentation conditions, strains, cultivation media, and feeding strategies have been optimized. Nattokinase has been expressed using several heterologous expression systems. Pichia pastoris heterologous expression system was the alternative. RESULTS: This report aimed to express high levels of nattokinase from B. subtilis natto (NK-Bs) using a Pichia pastoris heterologous expression system and assess its fibrinolytic activity in vivo. Multicopy expression strains bearing 1-7 copies of the aprN gene were constructed. The expression level of the target protein reached a maximum at five copies of the target gene. However, multicopy expression strains were not stable in shake-flask or high-density fermentation, causing significant differences in the yield of the target protein among batches. Therefore, P. pastoris bearing a single copy of aprN was used in shake-flask and high-density fermentation. Target protein yield was 320 mg/L in shake-flask fermentation and approximately 9.5 g/L in high-density fermentation. The recombinant nattokinase showed high thermo- and pH-stability. The present study also demonstrated that recombinant NK-Bs had obvious thrombolytic activity. CONCLUSIONS: This study suggests that the P. pastoris expression system is an ideal platform for the large-scale, low-cost preparation of nattokinase.

High-level extracellular production and immobilisation of methyl parathion hydrolase from Plesiomonas sp. M6 expressed in Pichia pastoris.

Methyl parathion hydrolase (MPH) hydrolyses methyl parathion efficiently and specifically. Herein, we produced MPH from Plesiomonas sp. M6 using a Pichia pastoris multi-copy expression system. The original signal peptide sequence of the target gene was removed, and a modified coding sequence was synthesised. Multi-copy expression plasmids containing MPH were constructed using pHBM905BDM, and used to generate recombinant strains containing 1, 2, 3 or 4 copies of the MPH gene. The results showed that a higher target gene copy number increased the production of recombinant MPH (MPH-R), as anticipated. The expression level of the recombinant strain containing four copies of the MPH gene was increased to 1.9 U/ml using 500 ml shake flasks, and the specific activity was 15.8 U/mg. High-density fermentation further increased the target protein yield to 18.4 U/ml. Several metal ions were tested as additives, and Ni2+, Co2+ and Mg2+ at a concentration of 1 mM enhanced MPH-R activity by 196%, 201% and 154%, respectively. Enzyme immobilisation was then applied to overcome the difficulties in recovery, recycling and long-term stability associated with the free enzyme. Immobilised MPH-R exhibited significantly enhanced thermal and long-term stability, as well as broad pH adaptability. In the presence of inhibitors and chelating agents such as sodium dodecyl sulphate (SDS), immobilised MPH-R displayed 2-fold higher activity than free MPH-R, demonstrating its potential for industrial application.

Structure-based engineering of PET hydrolase from Ideonella sakaiensis / 生物工程学报

Polyethylene terephthalate (PET) is a synthetic polymer consisting of ester bond-linked terephthalate and ethylene glycol. Tremendous amounts of PET have been produced and majority of them enters terrestrial and marine environment as wastes, posing serious threats to the global ecosystems. In 2016, a PET hydrolase from a PET-assimilating bacterium Ideonalla sakaiensis was reported and termed as IsPETase. This enzyme outperforms other PET-hydrolyzing enzymes in terms of its PET hydrolytic activity at ambient temperature, thus holds a great promise for PET biodegradation. In order to improve IsPETase activity, we conducted structure-based engineering to modify the putative substrate-binding tunnel. Among the several variants to the N233 residue of IsPETase, we discovered that the substitution of N233 with alanine increases its PET hydrolytic activity, which can be further enhanced when combined with a R280A mutation. We also determined the X-ray crystal structure of the IsPETase N233A variant, which shares nearly identical fold to the WT protein, except for an open end of subsite Ⅱ. We hypothesized that the smaller side chain of N233A variant might lead to an extended subsite Ⅱ for PET binding, which subsequently increases the enzymatic activity. Thus, this study provides new clues for further structure-based engineering of PETase.

Advances of CRISPR/dCas9 system in live cell imaging / 生物工程学报

The study of distinct genes, chromosomes and the spatio-temporal relationships between them is of great significance in genetics, developmental biology and biomedicine. CRISPR/Cas9 has become the most widely used gene editing tool due to its excellent targeting ability. Recently, researchers have developed a series of advanced live cell imaging techniques based on the nuclease-inactivated mutant of Cas9 (dCas9), providing rapid and convenient tools for high-resolution imaging of specific sites in the chromatin and genome. This review summarizes the advances of CRISPR/dCas9 system in live cell imaging from three aspects, including the strategies of cell delivery, optimization of the fluorescence signals, as well as orthogonal and multicolor imaging. Furthermore, we shed light on the development trends and prospects of this field.

Advances in molecular biology research on human parvovirus B19 / 生物工程学报

Human parvovirus B19 (B19 virus) is one of the two parvoviruses that cause human diseases. As an important pathogen to humans, it causes infectious erythema in children, acute aplastic anemia, fetal edema and death. In this review, we focus on the recent advances in the molecular virology of B19V, such as viral genotypes, viral receptor, genomic features and viral replication, viral transcription and post-transcription regulation, viral nonstructural and structural protein features and functions, viral diagnosis and antiviral agents, to provide reference for further study of B19 pathogenesis mechanisms, treatment and diagnostic strategies.

Relationship between dormitory environment and respiratory tract infection among college students / 中国学校卫生

Objective@#To analyze the relationship between dormitory environment and respiratory tract infection among college students.@*Methods@#A total of 890 dormitory rooms and 1 727 college students were investigated on symptoms including cough, hemoptysis and dyspnea or chest pain, as well as room sanitation(wet stain, mildew, damp, water loss and suspicious windows condensate), cleaning frequency and resident population. The data were analyzed by descriptive statistics, correlation analysis and Logistic regression analysis by SPSS.@*Results@#63.0% of the school dorms were found of dampness, mustiness and water loss, 67.3% of students had the subjective perception of odor. Except for the dryness of air, the rate of subjective perception of odor of the damp dorms was higher than that of dry dorms, and the differences were of statistical significance(P<0.01). Factors such as sex, age, dorm orientation, bathroom equipment, were partially related to symptoms of students’ self-perception and diseases confirmed by the doctors(P<0.05). High humidity were significantly related to symptoms including cough, expectoration, dyspnea, asthma and bronchiectasia(P<0.05), while subjective perception of odor associated with risk of respiratory infections and symptoms.@*Conclusion@#Multipe dormitory evvironmental problems may cause respiratory tract infection and symptoms of college students, dorm sanitation should be promoted among college students.

Expression and correlation of Toll-like receptor 2, LC3B and Beclin1 in glioma / 中国医师进修杂志

Objective To explore the relationship between Toll-like receptor 2(TLR2) and autophagy marker protein in glioma. Methods Glioma tumors of a total of 74 patients from June 2012 to December 2017 were surgically resected, including WHO gradeⅠtoⅡ32 cases, grade Ⅲ 20 cases, gradeⅣ22 cases. Immunohistochemistry and Western blot were used to detect the expression of TLR2, autophagy related protein LC3B, Beclin 1 and apoptosis related protein Bax and Bcl-2. The correlation between TLR2 and autophagy related protein LC3B and Beclin 1 were analyzed. Results In high grade glioma (HGG) tissue and low grade glioma (LGG) tissue, the TLR2 positive expression rates were 92.9％(39/42) and 75.0％(24/32), and there was significant difference (P<0.05). In HGG tissue, autophagy related protein LC3B, Beclin1 protein was strongly positive and the positive expression rates were 45.2％(19/42) and 52.4％(22/42). In LGG tissue, LC3B and Beclin1 protein positive expression rates were 18.8％(6/32) and 15.6％(5/32), and there were significant differences (P<0.05). Spearman correlation analysis showed that TLR2 protein was closely related to autophagy related protein LC3B (r=0.5638, P<0.05) and Beclin1 (r=0.6101, P<0.05). Conclusions TLR2 is highly expressed in HGG tissue, and its expression level may be related to autophagy, which has potential value as a targeted therapy.

Progress of exosomes in tumor immune escape / 肿瘤研究与临床

Exosomes are a type of nanoscale vesicles that are actively secreted by various type of cells, and are considered as a new way of cell communication. The exosomes can shuttle bioactive molecules including proteins, lipids, miRNAs and mRNAs from one cell to another, resulting in the exchange of genetic information between cells and the reprogramming of recipient cells. Many evidences show that tumor cells can secrete a large amount of exosomes and regulate tumor progression,metastasis,immune escape, resistance and many other aspects through a variety of ways. In the tumor microenvironment, exosomes transmit between tumor cells,immune cells,and stromal cells,contributing to the escape from immune surveillance.This review summarizes recent advances in exosomes in tumor immune escape.

High-level expression of two thermophilic ß-mannanases in Yarrowialipolytica.

Two thermophilic ß-mannanases (ManA and ManB)were successfully expressed in Yarrowialipolytica using vector pINA1296I. The sequences of manA from Aspergillus niger CBS 513.88 and manB from Bacillus subtilis BCC41051 were optimized based on codon-usage bias in Y.lipolytica and synthesized by overlapping polymerase chain reaction (PCR). We utilized the pINA1296I vector, which allows inserting and expression of multiple copies of an expression cassette, to engineer recombinant strains containing multiple copies of manA or manB. Following verification of target-gene expression by quantitative PCR, fermentation experiments indicated that recombinant protein levels and enzyme activity increased along with increasing manA/manB copy number.After production in a 10 l fermenter, we obtained maximum enzyme activity from strains YLA6 and YLB6 of3024 U/mL and 1024 U/mL, respectively. Additionally, purification and characterization results revealed that the optimum pH and temperature for manA activity were pH∼5 and ∼70 °C, and for manB activity were pH∼7 and 60 °C, respectively. These results indicated that the thermo stabilities of these two enzymes were higher than most other mannanases, making them potentially useful for industrial applications.

High-level expression of l-glutamate oxidase in Pichia pastoris using multi-copy expression strains and high cell density cultivation.

l-glutamate oxidase (GLOD), encoded by the gox gene, catalyses the transformation of l-glutamic acid into α-ketoglutaric acid (α-KG). In the present study, Pichia pastoris was used for heterologous production of GLOD following optimization of the gox coding sequence for expression in the yeast host. A series of constructs based on the pHBM905BDM plasmid were engineered and transformed into P. pastoris to increase the gox copy number. The results indicated that GLOD protein levels and enzyme activity increased with increasing gox copy number. Strain PGLOD4, which contained four copies of the target gene, was chosen for subsequent fermentation experiments, and a fermentation strategy involving two exponential feeding phases was developed. During the preinduction phase, glycerol was fed exponentially at µG = 0.15/h. When the cell density reached 300 g/l, methanol was fed exponentially at µM = 0.03/h to induce GLOD production. After 84 h of cultivation, the final cell density and total enzyme activity reached 420 g/L and 247.8 U/mL, respectively. The recombinant enzyme displayed an optimum temperature of 40 °C, which was higher than recombinant enzyme expressed in E. coli. This is important because increasing the temperature could accelerate enzymatic transformation of l-glutamic acid to α-KG. Experiments also demonstrated superior thermo-stability for the enzyme produced in yeast, which further enhances its potential for industrial applications.

Dosimetry and efficacy between RapidArc and intensity-modulated radiotherapy for locally advanced cervical carcinoma / 中华放射医学与防护杂志

Objective To analyze the difference of dosimetry and evaluate clinical efficacy and acute toxicity reaction between RapidArc and fixed gantry angle dynamic intensity modulated radiotherapy (IMRT) in advanced cervical carcinoma.Methods A total of 43 patients with locally advanced cervical cancer were studied,including 22 patients treated with RapidArc and 21 patients with IMRT.All plans were prescribed 50.4 Gy in 28 fractions.The conformity index and homogeneity index of the targets,the monitor units(MUs) and delivery time were compared.Incidence of acute intestinal and bladder side effects and rates of efficacy were calculated.Results The conformity index of RapidArc was better compared to IMRT.The V40 and V50 of bladder and V30,V40 and V50 of rectum planned by RapidArc was significantly lower than that by IMRT(t =-2.386,-2.397,P ＜0.05;t =-5.525,-2.883,-2.686,P ＜0.05).The mean dose of femoral head planned by RapidArc was also significantly lower (t =-2.395,P ＜ 0.05).For RapidArc,mean MU and treatment time were reduced by 53.15％,and 62.14％,respectively.There was no difference in the incidence of acute intestinal and bladder toxicity and rates of complete remission and efficacy between the two groups.Conclusions In dosimetric analysis,RapidArc showed advantage in protecting organs at risk and reducing treatment time in radical radiotherapy for locally advanced cervical carcinoma.

Expression of the thermostable carboxypeptidase Taq gene in Pichia pastoris GS115 / 生物工程学报

To express recombinant carboxypeptidase from Thermus aquaticus (Cpase Taq) in Pichia pastosis, the open reading frame coding thermostable Cpase Taq was optimized based on the preference of P. pastoris codon usage and synthesized in vitro. The novel gene was cloned into P. pastoris expression vector pHBM905A and the sequence coding 6xHis tag was fused with the ORF of Cpase Taq gene. The recombinant plasmid was named pHBM905A-Cpase Taq and transformed into P. pastoris GS 115. Transformants were induced with 1% methanol for 72 h until the enzyme yield reached 0.1 mg/ml. The enzyme was purified and its enzymatic properties were analyzed. The results showed that the specific enzyme activity reached maximum at 75 °C and pH 7.5, which was about 80 U/mg. It was the first report about the secretory expression of Cpase Taq in P. pastoris GS115. Because of its large-scale preparation, this enzyme may be applied in industrial hydrolysis of peptides into amino acids in the future.

Construction of directional T vector for gene cloning and expression / 生物工程学报

Traditional T vector cloning method requires onerous procedures for identifying recombinant, and directional cloning was impossible. In order to overcome these problems, we have devised a directional T vector pETG based on pET-23a(+). For gene cloning, 7 bp partial LacO sequence was introduced into DNA fragment to reconstitute a full length LacO with Bfu I digested T vector. After transformation, blue colonies were selected on LB plate supplemented with X-gal. Restriction enzyme digestion and PCR identification showed that all blue colonies contained the directionally inserted recombinants and the recombinant efficiency was nearly 100%. We have successfully cloned 103 genes from human liver cDNA; in the study complicated procedures for screening of recombinant were not required. Eight pETG clones were picked for protein expression, and all the clones successfully produced corresponding proteins. We demonstrated that the directional T vector was successfully constructed, and it was very suitable for gene cloning and expression.

Systematic review on methodology of randomized controlled trials of post-marketing Chinese patent drugs for treatment of type 2 diabetes / 中西医结合学报

Randomized controlled trial (RCT) is considered as the gold standard for the efficacy assessment of medicines. With the increasing number of Chinese patent drugs for treatment of type 2 diabetes, the methodology of post-marketing RCTs evaluating the efficacy and specific effect has become more important.

Parenteral organ injury and Immune status in children with rotavirus enteritis / 中国小儿急救医学

Objective To evaluate the parenteral complications and their risk factors and to study the immune status by detecting immunoglobulin and lymphocyte subsets in children with rotavirus enteritis. Methods Sixty-four children with rotavirus enteritis who were treated in Shengjing Hospital of China Medical University between January 2007 and December 2008(observation group) and 18 healthy chiIdren for health screening at pediatric health care center (normal control group) were reeruimd.Clinical manifestations were collected,stool specimens were detected for rotavirus using ELISA,lymphocyte subsets were detected using flow cytometer,and immunoglobulin.liver enzyme and myocardial enzyme were detected.Results Rotavirus enteritis may be complicated by parenteral complications(liver,myocardium,respiratory and nervous system).The activity of ALT,AST,LDH,CK and CK-mB were higher in observation group than that in normal control group(P<0.05).In observation group,liver injury rate in children younger than 12 months was higher than that in children older than 12 months,and the activity of CK and CK-mB were highcr in severe diarrhea cases[CK(324.5±995.5)U／dl,CK-mB(93.8 4±61.5)U／dl]than that in mild diarrhea cases [CK(252.8±130.4)U／dl,CK.mB(59.6±32.6)U／dl](P<0.05).The level of IgG was lower in observation group[(4.46±1.56)g／L]than that in control group[(5.80±1.67)g／L](P<0.05).Lymphocytes subsets study revealed that the activity of CD4+ in observation group[(29.0±4.18)％]was lower than that in control group[(38.6±3.97)％](P<0.05),the activity of CDl9+ [(38.8±3.94)％]was higher than that in control group[(23.1±7.70)％](P<0.05)and the CD4+／CD8+ ratio was reverse in observafion group.Conclusion Rotavirus enteritis may be complicated by parenteral injuries which get liver,myocardium,respiratory and nervous system involved.Children with rotavirus enteritis Call lead to low immune function.Determination of liver enzyme,myocardial enzyme,immunoglobulin and lymphocyte subsets has important clinical significance to monitoring the change of condition and guiding treatment.

Expression of phytase gene phyA in Yarrowia lipolytica po1h / 生物工程学报

Using the polymmerse chain reaction (PCR), we amplified the phytase gene phyA from Pichia pastoris GS115-phyA in Aspergillus niger NRRL3135 without the signal peptide sequence and intron sequence,. Then, it was cloned into pINA1297 vector to generate a recombinant vector of pINA1297-phyA. pINA1297-phyA was linearized and transformed into Yarrowia lipolytica po1h by the lithium acetate method. The positive transformants were obtained by YNB(casa) and PPB plates, after induced in YM medium at 28 degrees C for 6 day. The activity of the expressed phytase phyA reached 636.23 U/mL. The molecular weight of the enzyme was 130 kDa measured with SDS-PAGE analysis, whereas its molecular size reduced to 51 kDa after deglycosylation which is correspond with theoretical value. The enzymatic analysis of the recombinant phytase phyA revealed its optimal pH and temperature was 5.5 and 55 degrees C, which had high activity after incubated in pH ranged from 2.0 to 8.0 for 1 h. Moreover, its activity remained 86.08% after exposure to 90 degrees C for 10 min. It also was resistant to pepsin or trypsin treatment.

High expression of antimicrobial peptide Cecropin AD in Escherichia coli by fusion with EDDIE / 生物工程学报

In this study, we efficiently expressed the active antimicrobial peptide (CAD), which fused with the site-mutated coat protein (EDDIE) of the classical swine fever virus, in Escherichia coli. First, we obtained the e-cad fusion gene from the CAD gene and the EDDIE gene using overlapping PCR. Then to get the recombinant expression vector (pETED), the e-cad fusion gene was cloned into the pET30a vector by a site-directed homologous recombination technique. The EDDIE-CAD fusion protein expressed in E. coli as inclusion bodies, and its yield was more than 40% of total bacterial proteins. After renaturated in vitro and self-cleavage of the fusion protein, we obtained the antimicrobial peptide Cecropin AD. Antimicrobial experiments showed that the Cecropin AD efficiently inhibited the growth of G+ and G- bacteria, but it weakly inhibited the growth of Saccharomyces. This method provides an excellent way for high expression of antimicrobial peptides when fused with EDDIE.

Effect of surrounding-acupuncture on scalp on hemiplegia / 中国康复理论与实践

@#ObjectiveTo investigate the effect of surrounding-acupuncture on scalp on hemiplegia.Methods90 patients were divided into three groups. First group were treated with surrounding-acupuncture on scalp, second group with traditional scalp-acupuncture, the third group was control group. ADL and Mannal Function Test (MFT) were used to evaluate the function pre- and post treatment. ResultsThere were no significant differences among the three groups on ADL and MFT at beginning, that were improved significantly after treatment. However, the first group improved more significantly than that of the other two groups for ADL and low limb MFT. The first group showed significantly improvement for Hamilton Rating Scale for Depression pre- and post treatment.ConclusionThe surrounding-acupuncture on scalp is effective method for hemiplegia.

Target Monitoring of Nosocomial Infection in ICU of 12 Hospitals in Shandong Province / 中华医院感染学杂志

OBJECTIVE To investigate the incidence of nosocomial infection in ICU patients and its risk factors to take measures to prevent from infection. METHODS The nosocomial infection of ICU patients in 12 hospitals from Oct to Dec 2007 using the method of target monitoring was investigated. The nosocomial infection rate was regalated by the method of ASA. The invasive procedure and the associated infection rate were analyzed. RESULTS Among 2087 inpatients in ICU,236 suffered from nosocomial infection,The nosocomial infection incidence was 11.31%,and the nosocomial infection rate per day was 2.38% after regulated by the method of ASA. The patient incidence of nosocomial infection was 3.57%,and the nosocomial infection rate per day was 0.67%. CONCLUSIONS The patients in ICU are susceptible population of nosocomial infection,target monitoring in ICU is an effective surveillance method to reduce the prevalence of nosocomial infection.