Engineering and process management-based design of Comprehensive Biotechnology Experiment course / 生物工程学报

Based on the demand of enterprise talents and the characteristics of manufacturing process management in biotechnology, in order to make the students acquire the ability to solve complex engineering problems in the production process, we developed a "Comprehensive Biotechnology Experiment" course, where two-step enzymatic production of l-aspartate and l-alanine were the key processes. In this course, we drew lessons from the site management of the production enterprise, performed the experimental operation mode of four shifts and three operations. The content of this course includes principles, methods and experimental techniques of several core curricula and the site management mode of enterprises. As to the evaluation, the summary of the experimental staff's handover records and the content of teamwork were examined and scored. Through teaching practice and continuous improvement, we developed a complete experimental teaching process and assessment mechanism. Overall, the Comprehensive Biotechnology Experiment course achieved good teaching effect, which may serve as a reference to promote the development of experimental teaching of biotechnology.

Enzyme ancestral sequence reconstruction and directed evolution / 生物工程学报

The amino acid sequence of ancestral enzymes from extinct organisms can be deduced through in silico approach termed ancestral sequence reconstruction (ASR). ASR usually has six steps, which are the collection of nucleic acid/amino acid sequences of modern enzymes, multiple sequence alignment, phylogenetic tree construction, computational deduction of ancestral enzyme sequence, gene cloning, and characterization of enzyme properties. This method is widely used to study the adaptation and evolution mechanism of molecules to the changing environmental conditions on planetary time scale. As enzymes play key roles in biocatalysis, this method has become a powerful method for studying the relationship among the sequence, structure, and function of enzymes. Notably, most of the ancestral enzymes show better temperature stability and mutation stability, making them ideal protein scaffolds for further directed evolution. This article summarizes the computer algorithms, applications, and commonly used computer software of ASR, and discusses the potential application in directed evolution of enzymes.

Expressions of CD133 and p53 in colorectal carcinoma and the clinical relvance / 实用医学杂志

Objective To investigate the expressions of CD133 and p53 in colorectal cancer and their clin-ical significances. Methods The expressions of CD133 and p53 in 74 colorectal cancer patients were detected by the immunohistochemistry method. The relationships of CD133 and p53 with the clinicopathological parameters and prognosis were analyzed. Results The positive expression rates of CD133 and p53 in colorectal cancer tissues were 33.8%and 55.4%,respectively. The expression levels of CD133 and p53 were not related to age,sex,tumor location and histological type ,and but were significantly related to the histological differentiation ,TNM stage and distant metastasis(P<0.05,respectively). The Spearman correlation analysis showed that there was no correlation between the levels of CD133 and p53. Conclusions The high expressions of CD133 and p53 in colorectal cancer tissues were closely related to the histological differentiation and TNM stage. CD133 and p53 could be used as important biomarkers for the evaluation of malignant biological behavior,and the diagnosis and prognosis of colorectal cancer.

Clinic study of dose-dense cisplatin and 5-fluorouracil in patients with distant metastatic nasopharyngeal carcinoma / 国际肿瘤学杂志

Objective To investigate the efficacy and tolerability of dose-dense chemotherapy with cisplatin plus 5-fluorouracil( PF regimen)in distant metastatic nasopharyngeal carcinoma( NPC)patients. Methods From April 1,2008 to April 30,2014,168 patients were assigned to traditional group(n = 83) and dose-dense group(n = 85)using digital random table in 1 : 1 ratio. All patients received PF regimen,and once every 28 days in traditional group and once every 14 days in dose-dense group. The primary endpoint was progression-free survival(PFS)and the secondary endpoint was overall survival(OS),toxicity and response rate. Results The median PFS,median OS,1,2,3-year survival rate,complete response rate and objective response rate were significantly improved in dose-dense group which were 13. 3 months,20. 2 months,80. 2% , 36. 0% ,16. 1% ,16. 5% ,84. 7% ,and those in control group were 10. 0 months,16. 1 months,59. 6% , 10. 1% ,0,3. 6% ,54. 2% respectively(χ2 = 24. 47,P = 0. 000;χ2 = 16. 65,P = 0. 000;χ2 = 8. 41,P =0. 004;χ2 = 16. 96,P = 0. 000;χ2 = 14. 91,P = 0. 000;χ2 = 7. 63,P = 0. 006;χ2 = 18. 47,P = 0. 000). The rates of grade 3-4 adverse events in dose-dense and traditional group were 38. 8% and 6. 0%(χ2 = 25. 81, P = 0. 000). Conclusion Dose-dense chemotherapy of PF is more efficient and acceptable toxic than the tra-ditional one. It can be a new treatment option for patients with distant metastases of NPC.

Effect of co-expression of nicotinic acid phosphoribosyl transferase and pyruvate carboxylase on succinic acid production in Escherichia coli BA002 / 生物工程学报

Escherichia coli BA002, in which the ldhA and pflB genes are deleted, cannot utilize glucose anaerobically due to the inability to regenerate NAD+. To restore glucose utilization, overexpression of nicotinic acid phosphoribosyltransferase (NAPRTase) encoded by the pncB gene, a rate-limiting enzyme of NAD(H) synthesis pathway, resulted in a significant increase in cell mass and succinate production under anaerobic conditions. However, a high concentration of pyruvate was accumulated. Thus, co-expression of NAPRTase and the heterologous pyruvate carboxylase (PYC) of Lactococcus lactis subsp. cremoris NZ9000 in recombinant E. coli BA016 was investigated. Results in 3 L fermentor showed that OD600 is 4.64 and BA016 consumed 35.00 g/L glucose and produced 25.09 g/L succinate after 112 h under anaerobic conditions. Overexpression of pncB and pyc in BA016, the accumulation of pyruvic acid was further decreased, and the formation of succinic acid was further increased.

Succinic acid production with Escherichia coli AFP111 recovered from fermentation / 生物工程学报

During the anaerobic fermentation by Escherichia coli AFP111 for succinic acid production, the viable cell concentration and productivity were decreased with the raising of succinic acid concentration. In order to restore cellular succinic acid productivity and prolong fermentation time, we collected strains and refreshed medium for repetitive succinic acid production. However, productivity is lower than that in the anaerobic fermentation before reusing strains. To enhance the productivity, strains were aerobically cultivated for 3 h in pure water before anaerobic fermentation. The activities of key enzymes were enhanced for better performance in producing succinic acid at anaerobic stage. After three rounds of repetitive fermentations, succinic acid concentration and yield reached to 56.50 g/L and 90% respectively. The succinic acid productivity was 0.81 g/(L x h), which was 13% higher than the repetitive fermentations without aerobic activation of the strains.

Mutating Escherichia coli by atmospheric and room temperature plasmas for succinic acid production from xylose / 生物工程学报

Escherichia coli AFP111 is a spontaneous mutant with mutations in the glucose specific phosphotransferase system (ptsG) in NZN111 (delta pflAB deltaldhA). In AFP111, conversion of xylose to succinic acid generates 1.67 molecule of ATP per xylose. However, the strain needs 2.67 molecule ATP for xylose metabolism. Therefore, AFP111 cannot use xylose due to insufficient ATP under anaerobic condition. Through an atmospheric and room temperature plasma (ARTP) jet, we got a mutant strain named DC111 that could use xylose under anaerobic condition in M9 medium to produce succinic acid. After 72 h, DC111 consumed 10.52 g/L xylose to produce 6.46 g/L succinic acid, and the yield was 0.78 mol/mol. Furthermore, the reaction catalyzed by the ATP-generating PEP-carboxykinase (PCK) was enhanced. The specific activity of PCK was 19.33-fold higher in DC111 than that in AFP111, which made the strain have enough ATP to converse xylose to succinic acid.

Construct the recombinant plasmid of recombinant human basic fibroblast growth factor / 中国医师杂志

Objective To obtain the sequence of recombinant human basic fibroblast growth factor (rhbFGF) with endonuclease sites of BamHI and Pst Ⅰ by PCR based gene assembly and construct the recombining vector of rhbFGF gene by TA cloning technique.Methods The rhbFGF gene sequence which was designed for lactococcus with endonuclease sites of BamHI and Pst Ⅰ was divided into 22 oligonucleotides by DNASTAR 6.0 (bFGFl-bFGF22).The 22 oligonucleotides were spliced by PCR based gene assembly to get the rhbFGF eDNA with endonuclease sites of BamHI and Pst Ⅰ.The PCR product was inserted into the PMD18-T VECTOR.The recombining vector were converted to the competent E.coli TOP10.The clones generated from LAB were analyzed by miniprep isolation from LAB host.They were identified by the restriction enzyme cuuing and sequencing.Results The rhbFGFcDNA synthesized by PCR based gene assembly with endonuclease sites of BamHI and Pst Ⅰ was verified by 2％ agarose electrophoresis.The recombining vector of rhbFGF gene by TA cloning technique was identified by enzyme digestion and gene sequencing.Conclusions The TA cloning vector of recombining hbFGF with endonuclease sites of BamHI and Pst Ⅰ was constructed successfully.

Effect of overexpression of nicotinic acid mononucleotide adenylyltransferase on succinic acid production in Escherichia coli NZN111 / 生物工程学报

Escherichia coli NZN111 is a promising strain with ldhA and pflB genes inactivated for the production of succinic acid. However, with these mutations, NAD+ could not be regenerated from NADH, and an unbalanced NADH/NAD+ ratio eliminated cell growth and glucose utilization under anaerobic conditions. Nicotinic acid mononucleotide adenylyltransferase (NAMNAT), encoded by the nadD gene, catalyzes the reaction from nicotinic acid mononucleotide (NaMN) to nicotinic acid adenine dinucleotide (NaAD) during the synthetic pathway of NAD(H). Overexpression of the nadD gene could enhance the concentration of NAD(H) and maintain a suitable NADH/NAD+ ratio. In this study, we constructed a recombinant strain E. coli NZN111/pTrc99a-nadD, and overexpressed NAMNAT with 1.0 mmol/L of IPTG under anaerobic conditions in sealed bottles. Compared to E. coli NZN111, the concentrations of NAD+ and NADH in the recombinant strain increased by 3.21-fold and 1.67-fold, respectively. The total concentration of NAD(H) was increased by 2.63-fold, and the ratio of NADH/NAD+ decreased from 0.64 to 0.42. The recombinant strain restored the cell growth and glucose utilization under anaerobic conditions. After 72 h, the recombinant strain could consume 14.0 g/L of glucose to produce 6.23 g/L of succinic acid, and the concentration of succinic acid was 19-fold higher than in E. coli NZN111.

Isolation of high osmotic-tolerant mutants of Escherichia coli for succinic acid production by metabolic evolution / 生物工程学报

Succinic acid production was inhibited by high osmotic pressure caused by the accumulation of sodium ions in the process of two-stage fermentation by Escherichia coli using Na2CO3 as the pH regulator. To enhance the resistance of this strain to osmotic stress, the possibility to isolate high NaCl-tolerant mutant strain of Escherichia coli for succinic acid production by metabolic evolution was investigated. The metabolic evolution system was used as a mutant-generating system, allowing the cells to be continuously cultured at the maximum specific growth rate. The mutant strain can grow at maximum rate in the condition of high osmotic by gradually improving the concentration of NaCl in a continuous culture. Then the high osmotic-tolerant mutant strain of E. coli XB4 was selected with NaCl as the osmo-regulator. When using Na2CO3 as the pH regulator, E. coli XB4 was used in a 7.0 L fermenter during two-stage fermentation. After 60 h anaerobic fermentation, the mutant strain XB4 produced 69.5 g/L succinic acid with a productivity of 1.18 g/(L x h), which were increased by 18.6% and 20% compared with that of the parent strain.

Properties of sucrose phosphorylase from recombinant Escherichia coli and enzymatic synthesis of alpha-arbutin / 生物工程学报

Sucrose phosphorylase (EC 2.4.1.7, Sucrose phosphorylase, SPase) can be produced by recombinant strain Escherichia coli Rosetta(DE3)/Pet-SPase. Crude enzyme was obtained from the cells by the high pressure disruption and centrifugation. Sucrose phosphorylase was purified by Ni-NTA affinity column chromatography and desalted by ultrafiltration. The specific enzyme activity was 1.1-fold higher than that of the crude enzyme, and recovery rate was 82.7%. The purified recombinant SPase had a band of 59 kDa on SDS-PAGE. Thermostability of the enzyme was shown at temperatures up to 37 degrees C, and pH stability between pH 6.0 and 6.7. The optimum temperature and pH were 37 degrees C and 6.7, respectively. The K(m) of SPase for sucrose was 7.3 mmol/L, and Vmax was 0.2 micromol/(min x mg). Besides, alpha-arbutin was synthesized from sucrose and hydroquinone by transglucosylation with recombinant SPase. The optimal conditions for synthesis of alpha-arbutin were 200 U/mL of recombinant SPase, 20% of sucrose, and 1.6% hydroquinone at pH 6-6.5 and 25 degrees C for 21 h. Under these conditions, alpha-arbutin was obtained with a 78.3% molar yield with respect to hydroquinone, and the concentration of alpha-arbutin was about 31 g/L.

Clinical significance of antinucleosome antibody in patients with lupus nephritis / 中国医师进修杂志

Objective To analyze the correlation between serum antinucleosome antibody (AnuA) and renal pathological characteristic,disease activity as well as some laboratory tests in patients with lupus nephritis (LN).Methods Serum AnuA levels were detected by enzyme-linked immunosorbant assay in 40 patients with LN (observation group) and 40 healthy people (control group).Renal biopsy was examined in all LN patients.The relationships between serum AnuA level and systemic lupus erythematosus disease activity index (SLEDAI),renal pathohistology,laboratory parameters were analyzed.Results The serum AnuA level in observation group before treatment was significantly higher than that in control group [ ( 110.23 ± 80.48) kU/L vs. ( 10.45 ± 8.20) kU/L,P ＜ 0.05 ].Four cases of renal biopsies were class Ⅱ,8 cases were class m,23 cases were class Ⅳ,and 5 cases were class V.Serum AnuA level had significant difference between each class by Kruskal-Wallis rank sum test (P ＜ 0.05),and serum AnuA level of class Ⅳ was the highest (P ＜ 0.05).Serum AnuA level had positive correlation with SLEDAI,urine protein quantitation and anti-double strands DNA antibody (r =0.462,0.521,0.394,P ＜0.05),negative correlation with complement C3 and C4 levels (r =-0.403,-0.489,P ＜ 0.05 ).Serum AnuA level after treatment [ (32.45 ± 18.31) kU/L] was significantly decreased than that before treatment [(110.23 ± 80.48) kU/L](P＜0.05).Conclusions Serum AnuA level is not only a good index of LN activity,but also reflect renal involvement.That serial measurement of serum AnuA level may provide better clinical strategies for the therapy.

Breeding of ammonium-tolerant mutants of Actinobacillus succinogenes for succinic acid production and effect of ammonium / 生物工程学报

An ammonium-tolerant mutant of Actinobacillus succinogenes, YZ25, was obtained in the medium containing 61-242 mmol/L NH4+ after DES mutagenesis. Succinic acid produced by the mutant YZ25 reached 32.68 g/L when the medium contains 50 g/L glucose and 121 mmol/L ammonium, which was increased by 180.5% compared with that of the parent strain. The effects of different ammonium salts on the growth of the mutant and its metabolic response to high ammonium concentrations were investigated. The results showed that low ammonium concentration could improve the specific growth rates of the mutants, while high ammonium concentration inhibited cell growth. The ammonia-nitrogen half-inhibition constants (Ki) for different ammonium salts were as follows: 215 mmol/L for (NH4)2SO4, 265 mmol/L for NH4HCO3, 235 mmol/L for NH4Cl, and 210 mmol/L for NH4NO3. The process of ammonium inhibition on the mutant YZ25 was investigated in 3.0 L stirred fermenter. When NH4OH was used to buffer the pH, cell growth was not inhibited. However, production of succinic acid and consumption of glucose gradually decreased when cells entered the stationary phase, and the glucose could not be utilized completely at the end of fermentation. The possible ammonium inhibition mechanism was discussed based on the metabolic pathway of A. succinogenes.

Recycle of spent cells from anaerobic succinate fermentation / 生物工程学报

Spent cells recovered from anaerobic fermentation by Actinobacillus succinogenes were used as nitrogen source for succinic acid production. Three methods were investigated for cell wall-breaking. The results showed that enzymatic hydrolysis was more effective for higher succinic acid yield. When the enzymatic hydrolysate of spent cells was added to reach a total nitrogen concentration 1.11 g/L (equivalent to 10 g/L yeast extract), the succinic acid concentration was 42.0 g/L, but it increased slightly when enhancing the level of enzymatic hydrolysate. However, when 5 g/L yeast extract was supplemented with the enzymatic hydrolysate of spent cells, the succinic acid concentration reached 75.5 g/L after 36 hours and, the succinic acid productivity was 2.10 g/(L x h), which increased by 66.7% compared with the fermentation using 10 g/L yeast extract. Therefore, enzymatic hydrolysate of spent cells could replace 50% yeast extract in the original medium for succinic acid production.

The experimental study on effects of nuclear factor-κBp65 antisense oligonueleotide on liver fibrosis / 中华消化杂志

Objective To investigate the effect of the nuclear factor (NF)-κBp65 antisense oligonucleotide (ASODN) on NF-κB activity and expression of interleukin(IL)-6 in hepatic stellate cells (HSC). Methods The HSC were separated from rats and cultured. The toxicity of NF-κBp65 ASODN on HSC were detected by Trypan blue exclusion staining and the NF-κB activity was determined by EMSA. The expressions of IL-6 mRNA and protein were meaured by RT-PCR and ELISA, respectively. Results In vitro, no toxicity of ASODN on HSC was observed at the concentrations of 0. 001 to 1.0 μmol/L. NF-κB activity was increased after stimulating HSC with tumor necrosis factor (TNF)α, whereas it was weakened in a dose dependent manner when HSC were cultured with ASODN (concentration from 0. 001 to 1.0 μmol/L). At the same time, the expressions of IL-6 mRNA and protein induced by TNFα were decreased after transfected with ASODN at concentrations of 0.001- 1. 0 μmol/L in a dose dependent manner. Conclusion ASODN may specifically inhibit either the activiy of NF-κB or expression of IL-6, which provides the theoretical basis that ASODN may use to treat fibrosis of the liver.

AUTORADIOGRAPHIC OBSERVATION OF GOSSYPOL EFFECT ON REABSORPTION AND EXCRETION OF ~(42)KCL IN THE RENAL TUBULES / 解剖学报

The present study was designed to investigate the effect of gossypol on the reabsorption and excretion of ~(42)K in rats and guinea pigs by using autoradiographic technique, and selected the well known tubulo-toxic agent, gentamicin as a positive control for a comparative study to evaluate whether gossypol exerts nephrotoxic effect. Our results confirmed that gentamicin could induce significant decrease or inhibit ~(42)K reabsorption and cause structural damage of renal tubules. Gossypol could also affect the reabsorption function of proximal tubule, but did not appear to act as a tubulotoxic agent comparable with gentamicin to cause injury of the renal tubules.

STAGE-DEPENDENT EXPRESSION OF ANDROGEN RECEPTOR AND FSH RECEPTOR IN ADULT RAT TESTIS / 解剖学报

Objective To determine the cellular localization and expression pattern of AR and FSHR in adult rat testis must be helpful to understand the action site and mechanisms that T and FSH regulate spermatogenesis. Methods We applied in situ Hybridization to detect the expression of AR and FSHR on adult testis, in which Dig labeled cRNA probe was used to carry out the experiment on frozen sections; at the same time, following the technique of transillumination assisted microdissection we separated seminiferous epithelium into four stages(Ⅱ Ⅵ,Ⅶ Ⅷ,Ⅸ Ⅻ and ⅩⅢ Ⅰ), extracted total RNA and carried out dot hybridization, using ? 32 P labeled cDNA probe, in order to test qualitatively and quantitatively the location of AR and FSHR mRNA and their expression pattern in adult rat testis. Results Our results showed that the positive signal of AR mRNA was located in Sertoli cells and Leydig cells. The signal in Sertoli cells began to appear in Ⅱ Ⅵ stages, strongest in Ⅶ Ⅷ stages and weakest in Ⅸ Ⅰ stages ( P