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.

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. [

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.

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 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.

Objective To explore the circadian rhythm of neurally mediated syncope (NMS)in children. Methods There were 21 6 children with NMS included in the study,including 91 male and 1 25 female,aged from 4 to 1 7 years old with a mean age of (1 1 .34 ±2.65)years,who came from the Specialist Syncope Outpatient Department or Inpatient Department of the Second Xiangya Hospital of Central South University from December 201 3 to October 201 5. The patients were divided into vasovagal syncope (VVS)group and postural tachycardia syndrome (POTS)group ac-cording to head -up tilt test (HUTT)results,including 1 78 VVS patients and 38 POTS patients.Ninety -four patients with NMS were in the <1 2 years old group[(8.88 ±1 .88)years old]and 1 22 subjects with NMS were in the ≥1 2 years old group[(1 3.24 ±1 .1 8)years old].All patients or guardians were carefully asked about the number of synco-pal attacks and the periods in which episodes occurred in before HUTT [24 hours of a day were divided into 4 periods:morning (0600 AM-1 200 AM),afternoon (1 200 AM-1 800 PM),evening (1 800 PM-2400 PM), night (0000 AM-0600 AM)].Results (1 )General data:the total syncopal episodes of 21 6 children with NMS were 61 4 episodes,including 1 78 VVS patients with 471 syncopal episodes in total and 1 43 attacks of 38 children with POTS.There were 273 episodes of 94 patients in the <1 2 years old group and 341 episodes of 1 22 subjects in the ≥1 2 years old group.There were no significant differences in the diurnal variation of syncopal episodes between the VVS group and POTS group regarding age and gender (P >0.05).(2)The number of syncopal episodes in patients with VVS which occurred in the morning hours was strikingly higher than that of afternoon,evening or nighttime (P <0.05).But there was no significant difference in the frequency of episodes in different periods through the day in the POTS group (P >0.05).Patients with VVS had a higher proportion of episodes in the morning but a lower proportion in the evening when compared with the POTS group (P <0.05).(3)The male children with NMS tended to have a higher proportion of episodes in the morning than the female patients(χ2 =1 1 .001 ,P =0.01 2).(4)There seemed to be no difference in the frequency of syncopal episodes through the day between the <1 2 years old group and the ≥1 2 years old group(χ2 =1 .995,P =0.573).Conclusions The frequency of syncopal episodes in children with VVS displayed a clear circadian rhythm,with a peak in the morning,but the POTS patients did not show a circadian variation.The male children with NMS tended to have a higher proportion of episodes in the morning than the female patients.

Objective To investigate the role of vitamin D3 in anti-pancreatic cancer.Methods After treatment of different concentrations of vitamin D3 on PANC1 cells (25,50,75,100μmol/L),MTT assay was used to detect the growth inhibition rates of PANC1 cells,the early apoptotic rates of the cell were detected by flow cytometry,PTCH and Gli-1 mRNA expression were detected by RT-PCR method,and cells without treatment were used as control.Results The vitamin D3 inhibited the proliferation of PANC1 cells in a dose-dependent manner,the highest inhibition rate was at 48 hours.After 48 hours,the control group,25,50,75,100 μmol/L vitamin D3 groups' inhibition rates were 0,16.1％,18.8％,31.8％ and 39.4％,the differences among these groups were statistically significance (P ＜ 0.05).After 24 hours,the control group,25,50,75,100μmol/L vitamin D3 groups' early apoptotic rates were (5.89 ±0.57)％,(6.06 ±0.44)％,(16.21 ± 1.62)％,(16.94± 0.91)％ and (20.96 ± 0.98)％,early apoptotic rates were inhibited in a dose-dependent manner,and the differences was statistically significance (P ＜ 0.05).After 24 hours,the control group,25,50,75,100μmol/L vitamin D3 groups' PTCH mRNA expression were 0.117 ± 0.009,0.104 ± 0.011,0.069 ± 0.011,0.052 ± 0.009 and 0.056 ± 0.007,meanwhile the Gli-1 mRNA expressions were 0.323 ± 0.007,0.312 ± 0.015,0.299 ± 0.015,0.233 ± 0.007 and 0.175 ± 0.014,all in a declining trend with the increase of concentration,and the difference was statistically significant (P ＜ 0.05).After 75 μmol/L vitamin D3's intervention in 0,12,24,36 and 48 hours,the expression of PTCH mRNA were 0.142±0.008,0.127± 0.009,0.111± 0.010,0.115± 0.003 and 0.102± 0.007,meanwhile the expression of Gli-1 mRNA were 0.341 ± 0.011,0.317 ± 0.017,0.320 ± 0.018,0.226 ± 0.011 and 0.191 ±0.010,all in a declining trend with time,and the difference was statistically significance (p＜0.05).Conclusions Vitamin D3 can effectively inhibit the proliferation of PANC1 cells and promote its apoptosis,and these effects may be related to blocking of hedgehog signaling pathway.