Biodiesel, a nontoxic,cleaning, renewable and biodegradable fuel, is expected as a substitute for conventional fossil diesel. There are three main approaches to produce biodiesel, alkali-catalysis processing, enzymatic-catalysis processing and supercritical processing. With the unique property of energy-saving and environment-friendly, enzymatic-catalysis appears a great potential for industrial application. The main bottleneck of this technology is high cost and low stability of the lipase, as well as the inactivation of lipase by methanol and so on. To settle the problem, several methods have been used including the fixed-bed bioreactor, enzyme immobilized processing, whole-cell biocatalyst, changing addition method of methanol, developing of novel acyl acceptor, enhancing methanol resistance of lipase. The main problems and the relative strategy research of the enzymatic-catalysis technology were sum up.

The inverted terminal repeat (ITR) is the only cis element of AAV genome essential for rAAV rescue, replication and packaging. It is prone to mutation or loss when it is latent in host cell or in plasmid. Plasmids with different ITR types were cloned to compare the influence of ITR types on the AAV packaging and infectivity. The vector plasmids were transformed the competent SURE cells to get different colonies. The ITR types of plasmids were screened by digestion with SmaⅠ. AAV vector plasmid pScGFPud has two ITRs at both ends of AAV genome and plasmid pScGFPu has only one ITR at upstream end of AAV genome. When the two plasmids were co-transfected 293 cells to prepare rAAVs, 1.08?1013 viral particles (AAV1-GFPud) were produced from 20-dishes of 293 cells cotransfected with plasmid pScGFPud, 4.28?1012 viral particles (AAV1-GFPu) were produced from 20-dishes of 293 cells cotransfected with plasmid pScGFPu. Virus AAV1-GFPud infected 293, HeLa and NCI H446 cells more efficiently than did virus AAV1-GFPu. This suggests that defect ITRs in AAV genome is deleterious to AAV packaging and AAV infectivity and vector with complete ITRs is favorable to the yield and activity of rAAV.

AIM: To provide experimental evidence for gene therapy of thrombophilia disease, we constructed the eukaryotic expression plasmid with human thrombomodulin (hTM) gene and observed the alteration of hTM expression on the surface of human umbilical vein endothelial cells (HUVECs) with and without the reconstructive plasmid. METHODS: The whole expressive fragment of hTM gene was amplified by PCR from human genome. Both hTM gene and pcDNA3.1(+)/neo empty vector was digested by HindⅢ and EcoRⅠ. Two digested fragments were ligated into pcDNA3.1/hTM with T_4DNA ligase. After identifying, the reconstructive plasmid transfected into HUVECs using lipofectin. The hTM antigen on the HUVECs was detected by immunohistochemistry. RESULTS: The hTM reconstructive plasmid was confirmed by double endonuclease redigesting and sequencing. About 10% HUVECs were transfected by pcDNA3.1/hTM plasmid with lipofectin and the high-level hTM was detected on the transfected cells. CONCLUSION: We constructed the pcDNA3.1/hTM plasmid successfully, and it could be expressed on the HUVECs. [

Objective To describe an intelligent three-dimensional technique for automatic visualization of the fetal cranial mid-sagittal view to allow for the differential diagnosis of fetal midline anomalies.Methods Two hundred and twenty pregnant women with singleton pregnancies were imaged to display the mid-sagittal view of fetal head using a new 3D program (Smart MSP) developed by our team.Results The mid-sagittal view of the fetal head was successfully visualized in 190 normal cases (95％) and 18 abnormal cases (90％) by Smart MSP program.The total successful rate was 94.5％ (208/220).Conclusions Smart MSP is a novel and feasible method for the automatic visualization of fetal cranial midsagittal plane and may become a potential tool for routinely screening the fetal midline anomalies.

Objective To assess the characteristic of left ventricular(LV) twist changed from infancy to the elder,in order to determine the normal value of LV twist in different age group.Methods After acquired basal and apical LV short-axis images in 293 healthy volunteers (aged 15 days to 72 years) by 2-dimensional echocardiography, speckle tracking echocardiography was used to analysis LV twist motion offline.The peak apical rotation (PAr), peak basal rotation (PBr), peak LV twist (Ptw), peak LV twist normalized by LV length ( PtwN), peak untwisting velocity ( PutwV), isovolumic untwisting% ( Iutw% ) in different group were compared.Results Reliable LV twist measurements were obtained in 274 volunteers.Different LV twist values were noted according to age.Ptw increased gradually with advancing age, when normalized by LV length, PtwN showed higher values before 3 years old and 25 to 45 years old.PutwV increased with aging from infancy to 25 years old, and leveled off from 25 to 45 years old, then decreased with advancing age.Iutw% was lowest before 3 years old,and increased gradually from 25 to 45 years old,then decreased with aging.Conclusions speckle tracking echocardiography can be used to noninvasively and exactly assess LV twist with high feasibility.The effect of aging on LV twist may reflect the process of maturational and adaptive modulation of LV torsional biomechanics from infancy to the elder.

Lipase gene from Penicillium expansum(lip07) was cloned and over-expressed in Pichia pastoris.a random mutant named ep8,which contained a single amino acid substitution,was obtained by using the lip07 as an error-prone PCR template in previous study.ep8 shows higher thermostability than that of lip07,To further improve the thermostability of the lipase,the Lys of wild-type(lip07) and mutant(ep8) in 202 were substituted by Ala using the Overlap extension PCR technique respectively.The mutant genes(lip07-K202A and ep8-K202A) were subcloned into pAO815,and then transformed into the Pichia pastoris GS115 for extracelluar expression,respectively.15% SDS-PAGE analysis indicated that the molecular mass of PEL-ep8-K202A and PEL-lip07-K202A are both about 28kDa,which is same with the wild-type lipase.The Tm of PEL-ep8-K202A is 41.66℃,2.63℃ higher than that of the wild-type(39.03℃) and 1.21℃ higher than the random mutant(PEL-ep8:40.45℃);the Tm of single mutant(PEL-lip07-K202A) is 37.08℃,2℃ lower than that of the wild-type lipase.

OBJECTIVE o facilitate the basic acquaintance with the bioactive lycodine-type alka-loids biosynthetic pathways, we conducted the transcriptome analysis of L. casuarinoides by illumina sequencing.METHODS The plant of L.casuarinoides was collected and was subjected to RNA isolation, cDNA library construction, and illumina sequencing before bioinformatics analysis. After sequencing, the clean reads were obtained for de novo assembly by using Trinity software,and then further processed with TGICL sequencing clustering software to generate unigenes,The unigenes are aligned by Blast X alignment to six public protein database. In addition, all unigenes are functionally annotated by GO, KEGG and characterized putative genes involved in lycopodium alkaloids biosynthesis. RESULTS In total, 124, 524 high-quality unigenes were obtained with an average sequence length of 601 bp. Among the L. casuarinoides transcripts, 61,304 showed significant similarity (E-value<1 e-5) to the known proteins in the public database.Among the total 124 524 unigenes,47,538 open reading frame (ORFs) were predicted. Based on the bioinformatics analysis, all possible enzymes involved in the Lycodine-type alkaloids biosynthetic pathway of L. casuarinoides were identified, including primary amine oxidase (PAO), and Malonly-CoA decarboxylase. In addition, a total of 64 putative cytochrome P450 (CYP450) and 827 transcription factors were selected as the candidates of Lycodine-type alka-loids modifiers. Furthermore, a total of 13 352 simple sequence repeats (SSRs) were identified from the 124, 524 unigenes, of which dinucleotide motifs AG/CT (50.1%), were the most abundant. CONCLU-SION This transcriptome analysis of L.casuarinoides,provides many valuable candidate genes involv-ing in the biosynthesis of novel secondary metabolites but also lays the foundation for genetic diversity analysis via SSRs markers in L.casuarinoides.

Objective To investigate the clinical value of velocity vector imaging (VVI) in the assessment of cardiac torsion in fetuses with gestational diabetes mellitus(GDM).Methods Digital dynamic four-chamber views of 98 fetuses with gestational diabetes mellitus and 135 normal fetuses were collected and analyzed using VVI.Left ventricular(LV) peak apical rotation of endocardium and epicardium at basal and apical level were measured,LV peak torsion was calculated.Results 140 of 233 cases were analyzed successfully.Direction of LV peak apical and basal rotation of endocardium and epicardium in fetus could be clockwise or counter clockwise,however apical and basal level was opposite.LV peak apical and basal rotation of endocardium and epicardium in fetus were stable throughout gestation ( P ＞ 0.05).LV peak rotation of endocardium was higher than that of epicardium in both apical and basal level.The peak rotation　in apical level of endocardium and epicardium was higher than that in basal level( P ＜0.05).LV peak　torsion of endocardium was significantly higher than that of epicardium( P ＜0.05).Compared with normal　group,LV peak rotation of endocardium and LV peak torsion in fetus with GDM were higher( P ＜0.05).　Conclusions VVI can measure the LV rotation and torsion parameters,and evaluate the cardiac function of　GDM fetus.

Objective To investigate the value of the bedside index for severity in acute pancreatitis (BISAP),Ranson's,APACHE Ⅱ and computed tomography severity index (CTSI) scoring system in evaluating the severity of acute pancreatitis.Methods The clinical data of 385 patients with acute pancreatitis who were admitted to the Zhongnan Hospital of Wuhan University from 2005 to 2011 were retrospectively analyzed.The values of 4 scoring systems including BISAP,Ranson's,APACHE Ⅱ and CTSI in predicting the incidences of severe acute pancreatitis,local complications and death were investigated by Chi-square test and receiver operating characteristic curv e.Odds ratio (OR) was calculated.The differences of areas under the curves (AUC) were analyzed using the Z test.Results The incidences of severe acute pancreatitis,local complications and mortality of patients with BISAP score ≥ 3 were 64.4％ (56/87),16.1％ (14/87) and 8.0％ (7/87),which were significantly higher than 13.4％ (40/298),6.4％ (19/298) and 0.3 ％ (1/298) of patients with BISAP score ≤ 2 (x2 =93.4,8.1,19.7,P ＜ 0.05).The incidences of severe acute pancreatitis,local complications and mortality of patients with Ranson's score≥3 were 52.7％ (48/91),22.0％ (20/91) and 7.7％ (7/91),which were significantly higher than 16.3％ (48/294),4.4％ (13/294) and 0.3％ (1/294) of patients with Ranson's score ≤2 (x2 =49.2,27.3,18.5,P ＜0.05).The incidences of severe acute pancreatitis,local complications and mortality of patients with APACHE Ⅱ score ≥ 8 were 46.6％ (27/58),20.7％ (12/58) and 8.6％ (5/58),which were significantly higher than 21.1％ (69/327),6.4％ (21/327) and 0.9％ (3/327) of patients with APACHE Ⅱ score≤7 (x2 =17.0,12.8,14.4,P ＜0.05).The incidences of severe acute pancreatitis,local complications and mortality of patients with CTSI score ≥4 were 51.4％ (19/37),51.4％ (19/37),16.2％ (6/37),which were significantly higher than 22.2％ (77/347),4.0％ (14/347),0.6％ (2/347) of patients with CTSI score≤3 (x2 =15.1,95.3,40.1,P ＜ 0.05).The sensitivity,specificity,positive and negative predictive values of BISAP were 58％,89％,64％,86％,respectively,and the AUC was 0.848,which were significantly higher than the other 3 systems (Z =2.02,4.22,4.78,P ＜ 0.05).The sensitivity,specificity,positive and negative predictive values of CTSI were 58％,95％,51％ and 96％,respectively,and the AUC was 0.926,which was significantly higher than the other 3 systems (Z =3.99,3.24,4.06,P ＜ 0.05).The sensitivity,specificity,positive and negative predictive values of BISAP were 88％,79％,8％ and 100％,respectively,and the AUC was 0.855,with no significant difference compared with the other 3 systems (Z =0.81,0.03,0.14,P ＞ 0.05).Conclusions The accurate rate of BISAP in predicting the severe acute pancreatitis is higher than Ranson's,APACHE Ⅱ and CTSI.The accurate rate of CTSI in predicting the incidence of local complications is higher than the other 3 systems.There is no significant difference of the 4 systems in predicting the mortality.The BISAP scoring system is helpful in early diagnosis of severe acute pancreatitis,and making the individualized treatment plan,thus improving the prognosis of patients.

Objective To investigate the situation of prevalence,treatment and control of hypertension in patients with chronic kidney disease(CKD)by CROSS-sectional study. Methods Nine hundred out-patients with CKD in our department from November 2006 to March 2007 were enrolled in the study,including 480 male and 420 female.Among 900 CKD cases,354 patients underwent maintenance dialysis,including 228 on hemodialysis and 126 on peritoneal dialysis.Results The prevalence of hypertension in CKD patients was 80.2%(nude 83.5%vs female 76.4%,P<0.01).The prevalence of hypertension in patients on dialysis was significantly higher than that in non-dialysis patients(90.1%vs 73.8%,P<0.01),but there was no significant difference between hemodialysis and peritoneal dialysis cases.Antihypertensive treatment rate was 92.4%in CKD patients with hypertension.and was significantly higher in patients on dialysis than that in non-dialysis patients(95.6%vs 89.8%.P<0.01).The control rate according to current recommendations for CKD patients (BP<130/80 mm Hg) was very low. Control of both SBP and DBP was only achieved in 20.4% of non- dialysis patients. The control rate of hypertension (BP< 125/75 mm Hg) in patients with proteinuria >1 g/24 h was 8.4%. The proportion of dialysis patients with BP<140/90 mm Hg was significantly lower than that of non-dialysis patients (45.2% vs 55.5%, P<0.01). The percentage of hemodialysis patients with BP < 140/90 mm Hg was significantly higher than that of peritoneal dialysis patients (49.8% vs 36.5%, P<0.05). The prevalence of hypertension was associated with the decrease of renal function and the increase of age. The prevalence of hypertension in diabetic nephropathy was higher than that in primary glomerular diseases. Patients received 1, 2, 3 and 4 or more kinds of antihypertensive drugs accounted for 37.2%, 37.5%, 19.3% and 5.9% respectively. The combination of calcium channel blocker (CCB) and renin-angiotensin-aldosterone system (RAAS) inhibitors was more frequently used in CKD patients. The CCB was the most frequently prescribed drug (74.1% ), followed by angiotensin Ⅱ receptor blockers (ARB) (48.4%), angiotensin-converting enzyme inhibitors (ACEI) (25.6%) and alpha, beta-blockers (24.7%). Conclusions The prevalence of hypertension in CKD patients is quite high, which is associated with the progression of renal function, increase of age, the type of underlying kidney disease, obesity and diabetes mellitus. The control of hypertension is unsatisfied in CKD patients, especially in dialysis patients and those with overt proteinuria.