Expression of trehalose synthase gene from Pseudomonas putida P06 in Pichia pastoris.

The trehalose synthase (TreS) gene from Pseudomonas putida P06 was successfully ligated with pPICZaA expression vector by the EcoRI and XbaI and was transformed into Pichia pastoris GS115 by electrotransformation. The trehalose synthase gene was fused to the genome of Pichia pastoris GS115 and was controlled by AOX1 promoter. The TreS protein was successfully expressed in intracellularly. SDS-PAGE results illustrated that a specificity protein band was observed at about 76 kDa. The cell lysates could convert 60% maltose into trehalose at 50 °C and pH 7.5 in 10% maltose substrate for 24 h. The Pichia pastorisas exogenous gene expression host is safer to produce endotoxin free TreS than E.coli.

Biomechanical characteristics of thoracic T10 bone tumor metastasis at different locations:three-dimensional finite element analysis / 中国组织工程研究

BACKGROUND:With the innovation of examination technique,the number of patients with spinal metastases in different stages is increasing year by year.Percutaneous vertebroplasty is an important treatment for spinal metastases;however,there is no report on the biomechanical effect in different stages and different activities after operation. OBJECTIVE:To simulate thoracic T10 bone stress and displacement of the different locations of the tumor metastasis based on the three-dimensional finite element model. METHODS:According to thoracic three-dimensional CT images of a 30-year-old healthy male,Mimics software was used to construct a three-dimensional geometric model of thoracic vertebrae(T9-T11),including ribs,ligaments and intervertebral discs.Three-dimensional models of T9-T11 vertebral bodies and different parts of the posterior thoracic vertebrae invaded by thoracic metastatic tumors were simulated,including the control group with intact vertebral structure,unilateral metastasis involving the vertebral body area(experimental group 1),unilateral metastasis involving the vertebral body and pedicle area(experimental group 2),unilateral metastasis involving the vertebral body,pedicle and transverse process area(experimental group 3),and bilateral metastasis involving the vertebral body,pedicle and transverse process area(experimental group 4).Abaqus software was used to create a three-dimensional finite element model.The von Mises stress distribution and the displacement of the model were analyzed under the loading condition,buckling condition,extension condition,and rotation condition. RESULTS AND CONCLUSION:(1)In the study of the maximum total displacement of loading points in different experimental groups under loading,flexion,extension,and rotation conditions,with the increase of metastatic tumor invasion site and invasion surface,the total displacement of loading points increased,and the overall stiffness decreased,especially the total displacement of loading points in experimental group 4 was the largest.(2)Under flexion condition,the maximum Von Mises stress value increased significantly after vertebral body and pedicle destruction,while the maximum Von Mises stress value was almost unchanged when the thoracocostal joint destruction was added.(3)On the basis of finite element analysis and simulation of bone tumor model,the elements in the bone cement region were set as a single set,and the bone cement region was set as the corresponding material properties to simulate bone cement filling.The results showed that the maximum total displacement under loading,flexion,extension,and rotation conditions was less than that of each experimental group.(4)The maximum stress values of the simulated percutaneous vertebroplasty patients in the loading,flexion,extension and rotation conditions were significantly lower than those of the femoral model.(5)It is concluded that the three-dimensional finite element model based on thoracic T9-T11 conducive to the biomechanics characteristics of thoracic vertebrae tumor metastasis,and on the basis of the thoracic vertebrae tumor metastasis model can accurately simulate load point after percutaneous vertebral body under different conditions of total displacement and the maximum Von Mises stress situation.

Regulative role of nitric oxide synthase in myocardial ischemia-reperfusion injury of ovariectomized rats / 中华老年心脑血管病杂志

Objective To investigate the role of nitric oxide synthase(NOS)in the regulation of myocardial ischemia-reperfusion(IR)injury in ovariectomized(OVX)rats.Methods A total of 132 female SD rats were subjected,and 48 of them were randomly divided into sham operation group,IR group,OVX group and combined group,with 12 in each group.In order to explore the role of endothelous NOS(eNOS)and inducible NOS(iNOS)in ovariectomization increasing myo-cardial IR injury,another 84 mice were divided into negative sham group,negative IR group,nega-tive combined group,eNOS+IR group,eNOS combined group,iNOS small interfering RNA(si-iNOS)+IR group and si-iNOS combined group,with 12 in each group.The mice of the corre-sponding groups were injected with adeno-associated virus(AAV)overexpressing eNOS or knoc-king down iNOS via tail vein before OVX modeling.Myocardial infarct size,serum levels of lac-tate dehydrogenase(LDH)and creatine phosphokinase isoenzyme(CK-MB),LVEF,LVFS,and expression levels of eNOS and iNOS in the myocardial tissues were measured.Results The com-bined group had significantly increased level of iNOS in myocardium,larger myocardial infarct size and elevated serum LDH and CK-MB levels,but decreased myocardial expression of eNOS and LVEF and LVFS values than the IR group(P＜0.05).When compared with the negative combined group,the myocardial infarct size and serum LDH and CK-MB levels were decreased[(23.51±3.22)％and(26.21±2.93)％vs(58.78±5.42)％,(176.31±15.48 and 169.52±17.12 vs 328.85±37.12 U/L,35.41±6.41 and 34.77±5.94 vs 88.73±9.14 U/L,P＜0.05],and the LVEF and LVFS values were increased[(41.31±3.12)％and(42.09±3.41)％vs(30.77± 2.15)％,(21.47±1.57)％and(21.32±1.42)％vs(15.92±1.33)％,P＜0.05]in the eNOS com-bined group and si-iNOS combined group.Conclusion The decrease of eNOS expression and in-crease of iNOS expression are related to the aggravation of myocardial IR injury in OVX rats.

Dynamic expression of insulin-like growth factor-binding proteins during chondrogenesis / 中华整形外科杂志

Objective@#To clarify the status of proliferation and differentiation of RCJ3.1C5.18 cells in chondrogenesis and explore insulin-like growth factor binding proteins (Igfbps) gene expression profile during the process. The results provided a research foundation for understanding the role of Igfbps in chondrogenesis.@*Methods@#Fetal rat derived mesenchymal chondrogenic cell line RCJ3.1C5.18 was used, which progresses spontaneously to differentiated growth plate chondrocytes. The proliferation and differentiation status of cells were determined by cell staining, real-time PCR and Muse™ Cell Analyzer at days 1, 4, 7 and 10 of culture respectively. Igfbps gene expression was assessed by real-time PCR.@*Results@#In the early period (1-4 days), cells proliferated rapidly and the expression of chondrocytes marker genes, such as typeⅡ collagen, SRY-box 9 and aggrecan increased gradually. Igfbp4 and Igfbp6 gene expression paralleled the expression of chondrocytes marker genes. On days 7-10 of culture, cells viability decreased and gradually developed hypertrophic-like and osteogenic-like characteristics, which was confirmed by specific staining and gene expression of alkaline phosphatase, matrix metallopeptidase 13.Igfbp1, Igfbp3 and Igfbp5 mRNA levels were up regulated gradually at this stage.@*Conclusions@#The status of proliferation and differentiation of cells are different in the process of chondrogenesis. Changes in Igfbp gene expression are associated with chondrocyte differentiation, suggesting that they have different role during chondrocyte proliferation and differentiation.

Overpression of miR-29b suppresses the proliferation and induces apoptosis of cholangiocarcinoma cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the expression of miR-29b in cholangiocarcinoma and explore its effects on cell proliferation and apoptosis of cholangiocarcinoma cells.</p><p><b>METHODS</b>Real-time PCR was used to detect the expression of miR-29b in cholangiocarcinoma cells line QBC939 and cholangiocarcinoma tissues. The lentiviral vector LV-hsa-miR-29b and blank vector were constructed to infect QBC939 cells. MTT assay and cell clone formation assay were performed to assess the changes in the cell proliferation and clone formation, respectively; flow cytometry was employed to evaluate the effect of miR-29b overexpression on cell cycle and apoptosis.</p><p><b>RESULTS</b>The expression of miR-29b was significantly down-regulated in QBC939 cells and cholangiocarcinoma tissues as compared with H-69 cells and normal tissues ( < 0.01). Compared with the blank vector, the lentiviral vector LV-hsa-miR-29b caused significantly increased expression of miR-29b in QBC939 cells ( < 0.01), which exhibited suppressed cell proliferation and clone formation ( < 0.01 or 0.05), cell cycle arrest at the S phase ( < 0.05), and significantly increased cell apoptosis ( < 0.01).</p><p><b>CONCLUSIONS</b>As a tumor-suppressing miRNA, miR-29b is down-regulated in cholangiocarcinoma, and its overexpression can suppress the proliferation and induce apoptosis of cholangiocarcinoma cells.</p>

Effect of ctpxa1 gene deletion in Candida tropicalis on long chain dicarboxylic acid accumulation / 生物工程学报

Candida tropicalis uses alkanes and fatty acids to produce long chain dicarboxylic acids. However, the yield can be affected by β-oxidation in peroxisomes. Pxa1p was a membrane protein of Saccharomyces cerevisiae peroxisomes. Pxa1p and Pxa2p form a dimer that is involved in transporting of long chain fatty acids into peroxisomes, but the similar transporting system of Candida tropicalis has not yet been reported. In this study, a ctpxa1 gene deletion strain named C. tropicalis 1798-pxa1 was constructed by homologous single exchange method using PCR fragment. The expression of ctpxa1 gene in C. tropicalis 1798, C. tropicalis 1798-pxa1 was detected by semi-quantitative RT-PCR, and the ratio of gray value was 2.03, implying that the expression of ctpxa1 in C. tropicalis 1798-pxa1 was weakened. After 144 h fermentation, the dodecanedioic acid production of C. tropicalis 1798-pxa1 was increased 94.3% than the former strain, the maximum yield was 10.3 g/L.

Preparation, characterization of hydrophilic and hydrophobic drug in combine loaded chitosan/cyclodextrin nanoparticles and in vitro release study.

The compound nanoparticles of chitosan (CS) and cyclodextrin (CD) loading with hydrophilic and hydrophobic drug simultaneously were prepared via the cross-linking method. Methotrexate (MTX) and calcium folinate (CaF) were selected as the model drugs. The prepared nanoparticles were characterized by FT-IR spectroscopy to confirm the cross-linking reaction between CS and cross-linking agent. X-ray diffraction (XRD) was performed to reveal the form of the drug after encapsulation. The average size of nanoparticles ranged from 308.4 ± 15.22 to 369.3 ± 30.01 nm. The nanoparticles formed were spherical in shape with high zeta potentials (higher than +30mV). In vitro release studies in phosphate buffer saline (pH 7.4) showed an initial burst effect and followed by a slow drug release. Cumulative release data were fitted to an empirical equation to compute diffusional exponent (n), which indicated the non-Fickian trend for drug release.

Expression of Arabidopsis thaliana thioesterase gene in Pichia pastoris / 生物工程学报

Thioesterase catalyzes the hydrolysis of acyl-ACP and saturated fatty acyl chain. It plays a key role in the accumulation of medium chain fatty acids in vivo. In this study, to construct an engineering strain to produce MCFAs, the Arabidopsis acyl-ACP thioesterase gene AtFatA was amplified by PCR from cDNA of arabidopsis and double digested by EcoR I/Xba I, then linked to the plasmid digested with same enzymes to get the recombinant plasmid pPICZaA-AtFatA. We transformed the gene into Pichia pastoris GS115 by electroporation and screened positive colonies by YPD medium with Zeocin. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results showed that the recombinant enzyme had a molecular of 45 kDa band which was consistent with the predicted molecular mass and we constructed the expression system of gene AtFatA in fungus for the first time. Under shake-flask conditions, Gas Chromatograph-Mass Spectrometer-computer results indicated that recombinant strain produced 51% more extracellular free MCFAs than the wild and its yield reached 28.7% of all extracellular fatty acids. This figure is 10% higher than the control group. The result provides a new way to produce MCFAs.

Determination of Au and Ag in Geological Samples by Loaded Polyurethane Foam-Inductively Coupled Plasma-Mass Spectrometry / 分析化学

The relatively high abundance of geochemical elements such as Nb, Zr, Y and other elements shows serious interferences in the determination of trace silver in geochemical samples by inductively coupled plasma-mass spectrometry ( ICP-MS) . Thus it will lead to large deviation in the determination of geochemical samples without separation and enrichment. The traditional emission spectrum or graphite furnace atomic absorption method is only single-element analysis to the silver and with bad sample representativeness. In this study, load diphenylthiourea ( DPTU) foam selective enrichment was used for the separation of Au and Ag from other interfering elements in geological samples, and thiourea liberation-ICP-MS method was developed for the simultaneous determination of Au and Ag. The samples were first decomposed by 1:1 aqua regia. After addition of 50 mL of water, the samples were adsorbed under oscillation for 30 min at 20℃. The detection limits of the Au and Ag were 0. 02 ng/g and 0. 007μg/g, respectively. The proposed method was successfully applied to the determination of Au and Ag in eight national standard materials.

Protective effects of Trimetazidine on ischemia-reperfusion injury of fatty liver in rats / 天津医药

Objective To investigate the protective effects of Trimetazidine(TMZ)on the ischemia reperfusion injury (IRI)of fatty liver in autotransplantation model. Methods Fatty liver model was established by feeding high fat diet. Male Wistar rats (n=30) were randomized into three groups;Sham group, TMZ group and Model group. Liver was autotransplanted in both TMZ group and Model group. Serum levels of ALT, SOD, MDA, Bcl-2 and activated Caspase-3 were assessed 6 hours after the operation. The pathological performances of liver were also determined. Results Compared with the Model group, serum levels of ALT,AST, MDA and SOD levels decreased significantly in the TMZ group(P<0.05). Serum level of Bcl-2 was higher while level of activated Caspase-3 was lower in TMZ group than those in Model group(P<0.05). Histo?logical assay and TUNEL staining showed reduced hepatocyte swelling and narrowed sinusoid as well as decreased hepatic apoptosis in TMZ group compared with Model group. Conclusion TMZ can reduce oxidative stress, promote the expression of Bcl-2 and inhibit the activation of Caspase-3, which all contribute to its protective effect on fatty liver with ischemia-re?perfusion injury.