To express feruloyl esterase A from Aspergillus oryzae in Pichia pastoris expression system and study its hydrolysis function, explore the conditions and effects of purification for ferulic acid extracts by macroporos resin. Using the total RNA from A. oryzae CICC 40186 as the template, we amplified coding sequence AorfaeA encoding a mature feruloyl esterase A (AorFaeA) by RT-PCR technique. Then, the coding sequence AorfaeA was successfully expressed in Pichia pastoris GS115 mediated by an expression plasmid pPIC9K. The purified recombinant AorFaeA (reAorFaeA) showed one single band on SDS-PAGE with an apparent molecular weight of 39.0 kDa. The maximum activity of reAorFaeA to methyl ferulate, measured by high-performance liquid chromatography (HPLC), was 58.35 U/mg. Then, reAorFaeA was used to release ferulic acid from de-starched wheat bran in the presence of xylanase. The purification tests for ferulic acid from the enzymatic hydrolysate were carried out with preselected macroporous resins. The results showed that macroporous resin HPD-300 had much higher adsorption and desorption capacities. Ferulic acid could be quantitatively recovered by 50% of the eluent concentration at a flow speed of 1 mL/min. Under the purification condition, the recovery ratio of ferulic acid was 92%, and the content of ferulic acid was increased from 0.13% in the raw material to 10.55%. This work exploits the breakdown of ferulic acid by recombinant enzymeand provids a good strategy to its "green production".
Aspergillus oryzae ; enzymology ; Carboxylic Ester Hydrolases ; biosynthesis ; genetics ; Cloning, Molecular ; Coumaric Acids ; chemistry ; Electrophoresis, Polyacrylamide Gel ; Hydrolysis ; Molecular Weight ; Pichia ; genetics ; metabolism

ObjectiveTo evaluate the clinical efficacy and safety of astragalus injection on the immune function in patients with senile sepsis.Methods Sixty patients with old age sepsis in Critical Care Medicine Department of Guangdong Provincial Traditional Chinese Medicine Hospital were enrolled and randomly assigned into control and treatment groups according to the table of random numbers, 30 cases in each group. According to 2012 sepsis guidelines for treatment, including antibacterial drug, mechanical ventilation, visceral function support, etc., the therapy was given to the control group; besides the treatment in the control group, intravenous drip of 60 mL astragalus injection(10 mL per ampoule) in 250 mL 0.9% normal saline was additionally given in the treatment group, once a day for 7 days. Before and after treatment, the immunological indexes, acute physiology and chronic health evaluationⅡ(APACHEⅡ) score, sequential organ failure assessment(SOFA) score, duration of mechanical ventilation and time of stay in intensive care unit(ICU), 28-day mortality and adverse drug reactions were compared between the two groups.Results Before treatment, there were no statistically significant differences in CD3+, CD3+CD4+, CD3+CD8+ and T helper cells /T suppressor cells(Th/Ts)levels between the two groups(allP>0.05), while CD3-NK+ of the control group was significantly higher than that in the treatment group〔(10.47±6.22)% vs. (6.26±4.13)%,P<0.05〕. After treatment in treatment group, CD3+, CD3+CD4+ and CD3-NK+ were increased, CD3+CD8+,Th/Ts were decreased compared with those before treatment; in the control group after treatment, CD3+,CD3+CD8+ and CD3-NK+ were decreased and CD3+CD4+ and Th/Ts increased compared with those before treatment. In the comparisons between the treatment group and control group after treatment, the differences in CD3+, CD3+CD4+ and CD3+CD8+ had statistical significance〔CD3+:(30.30±17.17)% vs.(41.91±22.29)%, CD3+CD4+:(31.54±13.24)% vs.(40.08±15.28)%, CD3+CD8+:(14.25±8.10)% vs.(9.52±9.33)%,allP<0.05〕; while the differences in Th/Ts and CD3-NK+ had no statistical significance(bothP>0.05). After treatment in the treatment group, IgG was increased compared with that in the control group〔IgG(g/L): 13.07±5.43 vs. 10.10±3.96,P<
0.05〕. The differences in IgA, IgM, complement(C3,C4) and total serum complement activity(CH50) in the comparisons between the two groups had no statistical significance after treatment(allP>0.05). The differences in APACHEⅡ score(13.83±6.18 vs. 15.90±7.48), SOFA score(7.38±4.66 vs. 6.89±4.19), time of stay in ICU(day: 11.63±5.13 vs. 13.62±8.08), invasive ventilation time(hour: 155.44±119.68 vs. 224.08±174.15) and noninvasive ventilation time(hour: 55.55±42.24 vs. 98.57±43.17) had no statistical significance in comparisons between the treatment group and control group after treatment(allP>0.05). The difference in 28-day mortality had no statistical significance in comparison between the treatment group and control group〔16.7%(5/30) vs. 20.0%(6/30),P>0.05〕. In 60 cases, there were 2 patients with adverse drug reaction, one diarrhea and another little rashes, the rest of the patients did not appear any drug side effect.ConclusionAstragalus injection combined with conventional western medicine therapy possibly has certain effect on adjustment of disturbance of immunologic functions in old patients with sepsis, and its therapeutic safety is well.

15mm~2)of dural sac cross-sectional area to values smaller than 75 mm~2 was found during examination in axial loading,or if a suspected disc herniation,narrow lateral recess,narrow intervertebral foramen,or intraspinal synovial cyst changed to being obvious at the axial loading examination,they were regarded as additional information.Results After axial loading CT examination,AVI was found in 16 of 40 patients.A significant decrease of dural sac area was found in 13 patients.Intervertebral disc herniation was more severe in 7 patients,lateral recess or interverbral foramen narrowed in 4 patients,no intraspinal synovial cyst was found.After axial loading MRI examination,AVI was found in 19 of 60 patients.A significant decrease of dural sac area was found in 13 patients.Intervertebral disc herniation became severe in 10 patients,lateral recess or interverbral foramen narrowed in 8 patients,no intraspinal synovial cyst was found.AVI was found in 32 of 79(40.5%)patients with sciatica and 2 of 20(10.0%)patients with low back pain(?~2=7.45 P

Objective To understand the related factors of rabies epidemic and provide the basic data for rabies control and prevention in China by statistic and retrospective analysis of rabies surveillance data in 2010.Methods We used descriptive epidemiology method and statistic analysis to analyze the epidemiological characteristics of rabies in 2010 of China.Results 2048 rabies cases were rabies cases were reported in 817 counties (districts) in 2010,which dropped 7.46％ compares to 2009.The incidences in children and elder people were high; farmers are main occupation of the cases,the male to female ratio of the cases was 2.44:1.Children and older people are higher acquired rabies than other age population.640 cases reported through national rabies sentinel surveillance system,87.50％ cases were caused by exposed to dogs,bite was the main exposure reason.The situation of deposing wounds was poor,and the use of vaccine was still low in individual cases,but in the rabies clinic cases under surveillance,the vaccine usage can reach 98％,the usage of immunoglobulin (RIG) or anti-serum for category Ⅲ exposure in either group cases was not high.Conclusion The epidemic of the rabies in 2010 was eased,Out-patient post-exposure prophylaxis was in good station,but there are still lots of problem existed: post-exposure prophylaxis of individual case was not desirable yet.

Objective To investigate the surgical treatment strategies and prognostic factors of cT4bM0 primary rectal cancer.Methods The clinical data of 53 patients with cT4bM0 primary rectal cancer who were admitted to the First Hospital of Peking University from January 2000 to December 2010 were retrospectively analyzed.All the patients received en-bloc multivisceral resection and postoperative chemotherapy.The survival and prognostic factors were analyzed.The patients were followed up via out-patient examination,phone call or mail,and the follow-up was ended till December 2012.The survival curve was drawn using the Kaplan-Meier method,and the survival was analyzed using the Log-rank test.Uni-and multivariate analysis were done using chisquare test and COX's proportional hazard model.Results Of all the 53 patients,20 received posterior pelvic exenteration (PPE),20 received total pelvic exenteration (TPE),3 received low anterior resection (LAR) +local resection of ballder,2 received LAR + ovariectomy,2 received LAR + local resection of ureter,1 received LAR + local resection of posterior vaginal wall,1 received LAR + vesiculectomy and vesectomy,3 received abdominoperineal resection (APR) + local resection of posterior vaginal wall,1 received APR + sacrectomy.R0 resection was achieved in all the patients.No intraoperative death was observed,and the incidence of postoperative complication was 9.4％ (5/53).The results of postoperative pathological examination showed that 2 patients were with well-differentiated adenocarcinoma,41 with moderate-differentiated adenocarcinoma,and 10 with poorly differentiated adenocarcinoma.Twenty-four patients were with lymph node metastasis.Four patients were in TNM stage Ⅰ,25 in TNM stage Ⅱ and 24 in TNM stage Ⅲ.Fifty-three patients were followed up postoperatively,and the median time for follow-up was 33 months (range,4-116 months).The overall 5-year survival rates was 57.3％.The 5-year survival rate for patients with or without lymph node metastasis were 77.1％ and 30.4％,respectively,with significant difference between the 2 groups (x2 =7.374,P ＜ 0.05).The 5-year survival rates of patients with malignant infiltration and inflammatory adhesion mere 51.0％ and 68.5％,with no significant difference (x2=1.148,P ＞0.05).The 5-year survival rates of patients with malignant infiltration and inflammatory adhesion in stage Ⅱ were 74.6％ and 85.7％,with no significant difference between the 2 groups (x2=0.118,P ＞ 0.05).The 5-year survival rates of patients with malignant infiltration and inflammatory adhesion in stage Ⅲ were 28.8％ and 37.5％,with no significant difference between the 2 groups (x2 =0.959,P ＞ 0.05).The results of univariate analysis showed that lymph node metastasis and TNM stage were the risk factors influencing the prognosis of patients with cT4bM0 primary rectal cancer (x2=6.468,6.596,P ＜ 0.05).The results of multivariate analysis showed that lymph node metastasis was the independent risk factor (RR =3.797,P ＜ 0.05).Conclusions En-bloc multivisceral resection should be the first surgical treatment choice for patients with cT4bM0 primary rectal cancer,and lymph node metastasis is the independent risk factor.Under the same N stage,the prognosis of patients with malignant infiltration or inflammatory adhesion is similar if R0 resection is achieved.

ObjectiveTo explore the clinical diagnosis and surgical treatment of primary retroperitonealneurilemoma(schwannoma). MethodsClinicaldataof 47patientsof primary retroperitoneal schwannoma admitted and surgically treated from January 1995 to December 2009 were retrospectivelly reviewed.ResultsAs diagnosed by pathology there were 36 cases of Benign schwannoma,with a median age at onset of 41years, among those 11 patients were symptomatic, and 25 were asymptomatic. There were 11 malignant 11 cases, the median age was 38 years, among those 6 patients were symptomatic, and 5 were asymptomatic. The positive diagnostic rate of preoperative CT and MRI were 36. 2％ ( 17/47 ) and 58. 3％ ( 7/12 ) respectively. Immunohistochemically positive rates of S-100 were 100％ and 81.8％(9/11) in benign and malignant group respectively.All cases underwent surgical treatment. Surgical resection rates for benign and malignant groups were 100％ and 90. 9％(10/11)respectively. There was no perioperative death, Overall 5-year survival rates were 100％ and 45.5％ for benign and malignant tumors groups respectively. In benign group 2 cases recurred, in malignant group 4 cases recurred, and 3 had distant metastasis.ConclusionsPrimary retroperitoneal schwannomas are less common. It is difficult to make an accurate preoperative diagnosis. Surgery is the most effective therapy.Prognosis is good for benign and poor for malignant retroperitoneal neurilemomas.

U1 trastuctrural changes of myocardium and lungs from 6 cases died of cranioce-rebral penerating gunshot wound 2 hours after injury is reportcd.In all cases theelectron microscopy of the myocardial and lung tissue samples showed the similar ultrastructural morphological changes of the cells and interstitial tissues.The mostpr-ominent ultrastructural changes of myocardium were disorderly arrangement of the Zband.focal dissociation of the myofibrills,mitochondrial swelling with decreasing ofmatrix density and disruption of cristae,and interstial edema.The changes of theung tissue were increasing of width of alveolar septa with decreasing of the electron density.Aggregation of neutrophils in the capillaries of alveolar septa and some alveolar space was observed.The significance and the pathogenesis of the mainpathological changes were discussed.It is suggested that the pulmonary interstitialedema was neurogenic.The pulmonary edema may be manifested as interstitial edemaor intra-lveolar edema depending upon the time elapsed after the gunshot injury.

Objective To study the clinical application of the peak expiratory flow rate( PEFR) in children with asthma.Methods The PEFRs of 43 cases of asthma at the acute stage were measured to guide the clinical grading and therapy by the optimum individual PEFR of the patients at the remission stage, and the descending rate and warning value of individual PEFR were determined to investigate the relationship between the individual PEFR descending rate and the asthma attack conditions in the standard and nonstandard monitoring groups.Results At the asthma attack stage, the clinical symptoms became severe with PEFR declining; at the remission stage, the preventive application of drugs was based on the changes of the individual PEEF descending rate . The case number and frequency of asthma attack and the cases subjected to moderate or severe attacks in the standard group were 15 %,23.5 % and 25 % respectively; while those in the nonstandard group were 43.5 %, 75.5 % and 76.9 % respectively, which showed a significant difference( P

OBJECTIVE:To develop a method for rapid,accurate and simultaneous determination of dexzopiclone,zolpidam and zaleplon in human plasma. METHODS:After the plasma sample was processed by liquid-liquid extraction,UPLC-MS/MS was established to detect plasma sample with carbamazepine as internal standard. The separation was performed on a Waters ACQUITY UPLC HSS T3 column with mobile phase composed of 0.1% ammonium solution-acetonitrile (gradient elution) at flow rate of 0.2 ml/min. The column temperature was set at 40 ℃,and sample size was 5 μl. The detection was performed by multiple reaction monitoring (MRM) via electrospray ionization (ESI) source in positive mode. The mass transition ion-pairs were as follows:m/z 308.2→263.1(zolpidem),m/z 389.3→245.0(dexzopiclone),m/z 306.2→236.1(zaleplon),m/z 237.3→151.2(internal standard). RE-SULTS:The linear range of zolpidem,dexzopiclone and zaleplon were 0.02-20.00,0.50-20.00,0.02-20.00 ng/ml,respectively (r=0.990 1,0.996 8,0.991 7). LLOQ of them were 0.02,0.50,0.02 ng/ml,and detection limit were 0.01,0.20,0.01 ng/ml. RS-Ds of intra-day and inter-day were all lower than 15% ;extraction recovery were 88.9% -106.5% ;matrix effect were 94.8%-106.3%. CONCLUSIONS:The method is simple,rapid,sensitive and specific,and it can be used for simultaneous deter-mination of zaleplon,zolpidem and dexzopiclone in human plasma.

The important progress of the relationship between genomics and cardiovascular diseases has elucidated several abnormalities of genes leading to familial cardiomyopathy and ion channel diseases.Investigations of genomics show that the difference including resistance and susceptivity to diseases among human is determined by over 3 million base pairs of all 3,000 million.Single nucleotide polymorphism plays an essential role in elucidating hereditary cardiovascular diseases.