CTAB-PEG DNA Extraction from Fungi with High Contents of Polysaccharides

Polysaccharides influence concentration and purity of extracted DNA. Here we present rapid and efficient protocol for DNA extraction from samples rich in polysaccharides. The technique has been developed using cultures of Schizophyllum commune and involves a modification of known Cetyltrimethyl Ammonium Bromide (CTAB) protocol. To remove polysaccharides, Polyethylene Glycol (PEG) 8000 was added during DNA precipitation. Genomic DNA obtained with the CTAB-PEG method had high integrity, with average fragment size >30 kb, the concentration higher than 100 ng/μL, and the yield more than 30 μg/g. Presented technique is suitable for DNA extraction from fungi, bacteria, archaea or even mollusks with high polysaccharide content.     

The Downregulation of MiR-182 Is Associated with the Growth and Invasion of Osteosarcoma Cells through the Regulation of TIAM1 Expression

BackgroundOsteosarcoma is the most common primary bone malignancy in children and young adults. Increasing results suggest that discovery of microRNAs (miRNAs) might provide a novel therapeutical target for osteosarcoma.MethodsMiR-182 expression level in osteosarcoma cell lines and tissues were assayed by qRT-PCR. MiRNA mimics or inhibitor were transfected for up-regulation or down-regulation of miR-182 expression. Cell function was assayed by CCK8, migration assay and invasion assay. The target genes of miR-182 were predicated by bioinformatics algorithm (TargetScan Human).ResultsMiR-182 was down-regulated in osteosarcoma tissues and cell lines. Overexpression of miR-182 inhibited tumor growth, migration and invasion. Subsequent investigation revealed that TIAM1 was a direct and functional target of miR-182 in osteosarcoma cells. Overexpression of miR-182 impaired TIAM1-induced inhibition of proliferation and invasion in osteosarcoma cells.ConclusionsDown-expression of miR-182 in osteosarcoma promoted tumor growth, migration and invasion by targeting TIAM1. MiR-182 might act as a tumor suppressor gene whose down-regulation contributes to the progression and metastasis of osteosarcoma, providing a potential therapy target for osteosarcoma patients.     

Recent advances in synthesis and surface modification of lanthanide-doped upconversion nanoparticles for biomedical applications

Lanthanide (Ln)-doped upconversion nanoparticles (UCNPs) with appropriate surface modification can be used for a wide range of biomedical applications such as bio-detection, cancer therapy, bio-labeling, fluorescence imaging, magnetic resonance imaging and drug delivery. The upconversion phenomenon exhibited by Ln-doped UCNPs renders them tremendous advantages in biological applications over other types of fluorescent materials (e.g., organic dyes, fluorescent proteins, gold nanoparticles, quantum dots, and luminescent transition metal complexes) for: (i) enhanced tissue penetration depths achieved by near-infrared (NIR) excitation; (ii) improved stability against photobleaching, photoblinking and photochemical degradation; (iii) non-photodamaging to DNA/RNA due to lower excitation light energy; (iv) lower cytotoxicity; and (v) higher detection sensitivity. Ln-doped UCNPs are therefore attracting increasing attentions in recent years. In this review, we present recent advances in the synthesis of Ln-doped UCNPs and their surface modification, as well as their emerging applications in biomedicine. The future prospects of Ln-doped UCNPs for biomedical applications are also discussed.     

Enzymatic synthesis of glutathione using engineered Saccharomyces cerevisiae

Two single gene cassettes, each containing one of the individual gene (γ-glutamylcysteine synthetase gene GSH1 or glutathione synthetase gene GSH2), were constructed under the control of alcohol dehydrogenase (ADH1) promoter and their respective native terminators. The recombinant plasmids constructed with Kan ^r or Hyg ^r as the selective markers and were transformed into Saccharomyces cerevisiae separately and jointly. Three engineered strains, GSH1-enhanced strain S.TS013/GSH1, GSH2-enhanced strain S.TS013/GSH2 and GSH1+GSH2 double-enhanced strain S.TS013/GSH1+GSH2, were constructed. Glutathione production using the recombinant strains to improve was then determined. By the cell dosage proportions of two engineered strains (S.TS013/GSH1, S.TS013/GSH2) and a two-stage reaction, GSH productivity increased by 84 and 59 % over that of the host strain and the S.TS013/GSH1+GSH2 strain, respectively.     

登革3型病毒prM-E基因重组质粒DNA的构建和真核表达

构建登革 3型病毒 prM E基因的真核表达重组质粒 ,并进行体外表达 ,为登革DNA疫苗的研究奠定基础。用RT -PCR法获得 prM -E基因片段 ,然后将其克隆到真核表达载体中。用电穿孔法将重组质粒DNA转入BHK细胞 ,通过免疫荧光法检测外源基因在真核细胞中的表达。结果 ,通过酶切和序列测定证实了构建的重组质粒DNA含序列正确的 prM- E基因。用免疫荧光法检测到转染了重组质粒DNA的BHK细胞的胞浆中有登革 3型病毒特异蛋白的表达。说明含有登革 3型病毒prM -E基因的真核表达重组质粒可以在BHK细胞中表达 ,该结果为观察该重组质粒的免疫原性奠定了基础。     

ITE and TCDD Differentially Regulate the Vascular Remodeling of Rat Placenta via the Activation of AhR

Vascular remodeling in the placenta is essential for normal fetal development. The previous studies have demonstrated that in utero exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, an environmental toxicant) induces the intrauterine fetal death in many species via the activation of aryl hydrocarbon receptor (AhR). In the current study, we compared the effects of 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) and TCDD on the vascular remodeling of rat placentas. Pregnant rats on gestational day (GD) 15 were randomly assigned into 5 groups, and were exposed to a single dose of 1.6 and 8.0 mg/kg body weight (bw) ITE, 1.6 and 8.0 µg/kg bw TCDD, or an equivalent volume of the vehicle, respectively. The dams were sacrificed on GD20 and the placental tissues were gathered. The intrauterine fetal death was observed only in 8.0 µg/kg bw TCDD-exposed group and no significant difference was seen in either the placental weight or the fetal weight among all these groups. The immunohistochemical and histological analyses revealed that as compared with the vehicle-control, TCDD, but not ITE, suppressed the placental vascular remodeling, including reduced the ratio of the placental labyrinth zone to the basal zone thickness (at least 0.71 fold of control), inhibited the maternal sinusoids dilation and thickened the trophoblastic septa. However, no marked difference was observed in the density of fetal capillaries in the labyrinth zone among these groups, although significant differences were detected in the expression of angiogenic growth factors between ITE and TCDD-exposed groups, especially Angiopoietin-2 (Ang-2), Endoglin, Interferon-γ (IFN-γ) and placenta growth factor (PIGF). These results suggest ITE and TCDD differentially regulate the vascular remodeling of rat placentas, as well as the expression of angiogenic factors and their receptors, which in turn may alter the blood flow in the late gestation and partially resulted in intrauterine fetal death.     

水稻白叶枯病菌基因XOO2193的突变体构建及其毒力和胞外多糖分析

水稻白叶枯病菌是一种引起水稻白叶枯病的植物病原细菌,水稻白叶枯病是世界水稻生产中最严重的细菌性病害之一.本研究采用携带同源序列的自杀质粒pK18MobGⅡ整合的办法构建了水稻白叶枯病菌中国菌株13751编码6-磷酸葡糖酸内酯酶的基因XOO2193的非极性突变体GNM2193.对突变体的表型分析发现其毒力在杂交水稻品种特优63上显著减弱,突变体在非寄主植物蓖麻上不能引起过敏反应.此外,突变体胞外多糖的产量是野生型的43.4%.用一段含有XOO2193基因的DNA 片段对GNM2193进行功能互补,互补菌株在水稻上的毒力、引起过敏反应的能力和胞外多糖产量恢复到野生型水平.说明XOO2193基因与病菌的毒力和胞外多糖的产生有关.     

Formation of Adeno-Associated Virus Circular Genomes Is Differentially Regulated by Adenovirus E4 ORF6 and E2a Gene Expression

A central feature of the adeno-associated virus (AAV) latent life cycle is persistence in the form of both integrated and episomal genomes. However, the molecular processes associated with episomal long-term persistence of AAV genomes are only poorly understood. To investigate these mechanisms, we have utilized a recombinant AAV (rAAV) shuttle vector to identify circular AAV intermediates from transduced HeLa cells and primary fibroblasts. The unique structural features exhibited by these transduction intermediates included circularized monomer and dimer virus genomes in a head-to-tail array, with associated specific base pair alterations in the 5′ viral D sequence. In HeLa cells, the abundance and stability of AAV circular intermediates were augmented by adenovirus expressing the E2a gene product. In the absence of E2a, adenovirus expressing the E4 open reading frame 6 gene product decreased the abundance of AAV circular intermediates, favoring instead the linear replication form monomer (Rf_m) and dimer (Rf_d) structures. In summary, the formation of AAV circular intermediates appears to represent a new pathway for AAV genome conversion, which is consistent with the head-to-tail concatemerization associated with latent-phase persistence of rAAV. A better understanding of this pathway may increase the utility of rAAV vectors for gene therapy.     

Expression of modified xynA gene fragments from Bacillus subtilis BE-91

Expression of modified xynA gene fragments in Escherichia coli BL21 was studied, using the complete xynA gene from Bacillus subtilis BE-91 as the positive control. The technical workflow consisted of the following steps: (1) predicting protein structures relative to the xynA gene; (2) designing primers for modifiers; (3) amplifying the modifiers; (4) integrating the modifiers with the pET-28a(+) vector; (5) transferring the recombinant plasmids into E. coli BL21; (6) evaluating and analyzing the expression of modified cells. The results were: (1) the xynA gene from BE-91 with the untranslated region deleted on both ends was able to promote XynA activity by 28.9 %; (2) deletion of the 1- to 16-amino acid (AA) coding sequence in the open reading frame on the 5′-end, deletion of the 209- to 213-AA fragment on the 3′-end and deletion of the 20 AA on both ends could promote XynA activity by 27.2, 27.7 and 24.0 %,respectively; (3) deletion of the 1- to 29-AA fragment on the 5′-end and deletion of the 197- to 213-AA fragment on the 3′-end could reduce XynA activity dramatically by 95.6 and 74.8 %, respectively; (4) inactivation factors of XynA would be either the first β-fold and the hydrophilic structure domain or the last two α-screws and the seventeenth turn region. The results mean that any deletion in the catalytic domain would lead to a decline or inactivation in XynA activity while the deletion of any sequence outside the catalytic domain could effectively promote XynA activity, as such sequences are unnecessary for XynA function.     

Dynamic distribution of epidermal growth factor during mouse embryo peri-implantation

Embryo implantation depends on the synchronized development of the blastocyst and the endometrium. This process is highly controlled by the coordinated action of the steroid hormones: estrogen and progesterone. By autocrine, paracrine or juxtacrine routes, some growth factors or cytokines are involved in this steroidal regulation pathway. Here we report the effects of epidermal growth factor (EGF) on embryo implantation in the mouse, the expression and distribution patterns of EGF protein in the mouse blastocyst, ectoplacental cone (EPC) and peri-implantation uterus on days 1-8 of gestation.By RT-PCR and dot blot, we found that EGF and its receptor (EGFR) are co-expressed in the blastocyst and peri-implantational uteri of pregnant days 2-8 (D2-D8) mice. Injection of EGF antibody into a uterine horn on the third day of pregnancy (D3) significantly reduced the number of mouse embryos that implanted on D8, indicating EGF have a function in the mouse embryo implantation.Further investigation by using indirect immunofluorescence and confocal microscope was made to trace EGF and EGFR protein localization during the mouse embryo implantation. EGF and EGFR are co-localized in the blastocyst, and in the secondary trophoblastic giant cells (SGC) of the EPC. At the pre-implantation stage, the distribution of EGF protein in the mouse uterus changes from epithelium to stroma. On D1 of pregnancy, EGF is mainly distributed in uterine stroma and myometrium. On D2, it is present in the uterine epithelium. On D3, it changes again from the uterine epithelium to the stroma. By D4, EGF is predominantly in the stroma. This dynamic distribution correlates with the proliferation activity of uterine cells at each period. On D6-D8 of embryo implantation, EGF 3 protein accumulates at the uterine mesometrial pole, a region that contributes to the trophoblastic invasiveness and placentation.This temporal and spatial localization of EGF protein in the mouse uterus implicates the cytokine in the regulation of trophoblastic invasiveness and uterine receptiveness.