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 improve the accurate ultrasonography of the portal vein abnormal echoes .Methods 30 cases of portal vein abnormal echo with conventional ultrasound examination were contrast-enhanced by ultrasound microbubble .6 cases were con-firmed by biopsy or surgical pathology ,24 cases were confirmed after clinical data and enhanced CT or MR .Results 27 cases of tumor thrombus in the arterial phase ,of which 21 cases were overall uniformity enhanced ,6 cases was part of the high enhance-ment ,partial filling defect ;In portal venous phase all was equal or lower enhancement ;in delayed phase all showed low enhance-ment .3 cases of thrombosis were contrast-enhanced process with 2 cases without enhancement ,1 case of portal vein segmental en-hancement .Conclusion Contrast-enhanced ultrasound microbubble can accurately identify tumor thrombus and thrombosis ,and provid the reference value to further clear of the disease detected in nature ,staging ,treatment and prognosis .

Objective To find out the cerebral blood flow velocity and its relationship with attention and execu?tion function in patients with major depressive episode. Methods A total of 70 patients with major depressive episode and 65 healthy controls were included. The 24 items Hamilton depression scale (HAMD-24) was adopted for patients' depressive symptoms assessment. The Cancellation Test (CT) and Wisconsin Card Sorting Test (WCST) were adopted for cognitive function assessment. And the blood flow velocity of cerebral arteries were detected by transcranial Doppler ul?trasonography (TCD). Results Compared with the controls, the patients showed significantly slower average blood flow ve?locity in basilar artery, left middle cerebral artery, right middle cerebral artery, left anterior cerebral artery and right ante?rior cerebral artery (P<0.05). Compared with those in the controls, the net score of each stage and the total net score of CT were significantly lower in the patients, while the total number of responses, response errors, perseverative errors and the necessary response number to complete the first category of WCST were significantly higher (P<0.01). The blood flow velocity in the cerebral basilar artery (r=0.25), left middle cerebral artery (r=0.46), right middle cerebral artery (r=0.25) and right posterior cerebral artery (r=0.26) showed positive correlations with total net score of CT (P<0.05), and the cere?bral artery average blood flow velocity showed negative correlations with the total number of responses, response errors and perseverative errors of WCST in the patients (P<0.05). Conclusions Compared with healthy controls, patients with major depressive episode suffer from lower average cerebral artery blood flow velocity, bad attention and executive func?tion. The change of average cerebral artery blood flow velocity may be responsible for impaired cognitive function in epi?sode major depression patients.

Objective To assess apoptosis mediated by the cell death receptor Fas in peripheral T lymphocytes of patients with abdominal aortic aneurysm. Methods The apoptotic pathway was triggered by anti-Fas monoclonal antibody in cultured and activated peripheral T-cells from 20 AAA patients. Control groups consisted of 15 patients with aortic atherosclerotic occlusive disease(AOD)and 25 healthy individuals. Cell survival and death rate were assessed. Results Cross-linkage of Fas receptor exerted a strong apoptotic response on T cells from AOD patients and healthy controls, while the effect on T cells was very limited from that of AAA patients. The evaluation of cell Survival rate showed a significantly higher percentage in AAA group(98.9%±10.3%)than in the AOD subjects(58.9%±15.2%)or the healthy group(59.4%±12.9%;P＜0.001＝.Apoptosis assessment by annexin V and propidium iodide staining and flow cytometry showed similar results. The defect in AAA group was not due to decreased fas expressed at normal levels. Moreover,it specifically involved the Fas system because cell death was induced in the normal way by methylprednisolone. Conclusions Fas-induced apoptosis in activated T cell from AAA patients is impaired. This may disturb the normal down-regulation of the immune response and thus provide a new insight into possible mechanisms and routes in the pathogenesis of AAA.

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.

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.

The aim of this paper is to compare the cytotoxicity and cellular uptake efficiency of three kinds of poly(b-benzyl-L-amino) block-poly(ethylene glycol) nanoparticles (PXA-PEG-NPs) using Calu-3 cells, and select one as a nasal drug delivery vector for curcumin (Cur). Poly(gamma-benzyl-L-glutamate) block-poly(ethylene glycol) nanoparticles (PBLG-PEG-NPs), poly(gamma-benzyl-L-lysine) block-poly(ethyleneglycol) nanoparticles (PZLL-PEG-NPs) and poly(gamma-benzyl-L-aspartate) block-poly(ethylene glycol) nanoparticles (PBLA-PEG-NPs) were prepared by emulsion-solvent evaporation method. MTT assays were used to evaluate the cytotoxicity of PXA-PEG-NPs against Calu-3 cells. The cellular uptake of nanoparticles was visualized by an inverted fluorescence microscope and quantified by a flow cytometer. The results indicated that even at high concentration of 2 mg x mL(-1) the three nanoparticles had no cytotoxicity on Calu-3 cells. Compared to the curcumin solution, the three curcumin-loaded PXA-PEG-NPs showed significantly higher cellular uptake efficiency on Calu-3 cells (at equal concentration of curcumin with 5 microg x mL(-1) Cur solution), PBLG-PEG-NPs group was the highest. The cellular uptake increased with incubation time, and has positive correlation with nanoparticle concentration. In brief, PXA-PEG-NPs are conducive to delivery Cur into cells, and PBLG-PEG-NPs might be provided as a good nasal drug delivery carrier.