A Novel Approach for Introducing Bio-Materials Into Cells

A novel approach was developed to introduce biological materials into cells for gene transfection and gene therapy applications. The method is based on the technique of electrospraying bio-materials into cells. A prototype apparatus was constructed for a feasibility study. The features of the gene transfector include: (1) A dual-capillary assembly to spray suspensions of biological materials. The outer capillary provided sheathing liquid that controlled the charge level on individual particles without altering the properties of suspensions. (2) An air–CO₂ gas mixture was used for suppressing possible corona discharge and kept the same gas composition as those in incubators. (3) The designed chamber enabled the spray to operate at reduced pressure for increasing sprayed particle velocity. In the feasibility study, both suspensions of plasmid and plasmid-coated gold particles were used. The plasmid used was the commercially available Enhanced Green Fluorescent Protein gene. COS-1 cells were used as the target and the liquid media was evacuated immediately prior to the spraying process. Electrospraying was conducted at ambient pressure and the duration was no more than 2 min. After the spray transfection, the media was immediately replaced and the cell samples were returned to the incubator for 36 h. Transgene expression was detected by cellular fluorescence. This technology promises to have great potential for gene transfection and therapy studies.     

Nogo-B诱导oxLDL降解与肝癌基因激活

本文基于Hill动力学与Michaelis-Menten方程,建立理论模型研究内质网定位的蛋白Nogo-B诱导调节氧化修饰低密度脂蛋白(oxidized low density lipoprotein(oxLDL))降解与肝癌基因激活.理论模型考虑oxLDL*(降解的oxLDL)-Nogo-B-Yes-associated protein (YAP)通路,研究发现,oxLDL的降解,促进了大量的Lysopho-sphatidic acid (LPA)产生,之后便会提高Hippo信号通路YAP活性,激活了癌基因的表达;经过约5小时Nogo-B表达上调,Nogo-B决定着Nogo-B与Autophagy-related 5 gene(ATG5)的复合体NA,NA调控oxLDL的降解,未降解的oxLDL会诱导Nogo-B表达上调,激活了oxLDL*-Nogo-B-YAP通路,理论结果符合实验结果,并揭示非酒精性脂肪肝病诱发的肝癌的致病机理,可以为设计阻断肝炎向肝癌转变的通路治疗方案提供理论依据.     

从具体例子看惯性流形概念的推广

惯性流形的概念要求所有轨道指数收敛于唯一吸引子[5],这对于很多物理问题,例如sine-Gordon方程是很难满足的[4],本文中给出的人工例子建议了惯性流形的推广形式,这个推广形式去掉了整体吸引子是唯一的预先要求,该推广概念使用于sine-Gordon方程。     

基于纳米ZnO/壳聚糖复合膜DNA电化学传感器用于HIV基因的免标记检测

以室温固相合成法制备纳米ZnO,通过壳聚糖(CHIT)的成膜效应将纳米ZnO固定在玻碳电极(GCE)表面,制得的ZnO/CHIT/GCE电极成为DNA固定和杂交的良好平台。DNA的固定和杂交通过电化学交流阻抗进行表征。以电化学交流阻抗免标记法检测目标DNA,固定于电极表面的DNA探针与目标DNA杂交后使电极表面的电子传递电阻增大,以此作为检测信号可以高灵敏度地测定目标DNA。电化学阻抗谱检测人类免疫缺陷病毒(HIV)基因片段的线性范围为2.0×10-11~2.0×10-6mol/L,检出限为2.0×10-12mol/L。     

Electrochemical biosensor based on nanogold-modified poly-eriochrome black T film for BCR/ABL fusion gene assay by using hairpin LNA probe

A novel electrochemical biosensor is described for detection of breakpoint cluster region gene and a cellular abl (BCR/ABL) fusion gene in chronic myelogenous leukemia (CML) by using thiolated-hairpin locked nucleic acids (LNA) as the capture probe. The hairpin LNA probe was immobilized on the nanogold (NG)/poly-eriochrome black T (EBT) film-modified glassy carbon electrode (GCE). The immobilized LNA probe could selectively hybridize with its target DNA on LNA/NG/EBT/GCE surface. The immobilization and hybridization of the LNA probe were characterized with cyclic voltammetry and electrochemical impedance spectroscopy. The hybridization of the immobilized LNA probe with the target DNA was detected by differential pulse voltammetry with the electroactive methylene blue as an indicator. The results indicated this new method has excellent specificity for single-base mismatch and complementary after hybridization, and a high sensitivity. This novel electrochemical biosensor has been used for assay of PCR real sample with satisfactory result.     

Improving Gene Delivery Efficiency of Bioreducible Poly(amidoamine)s via Grafting with Dendritic Poly(amidoamine)s

Dendritic poly(amidoamine)s (PAMAM)s were introduced into the side chains of disulfide‐containing poly(amidoamine)s via repetitive Michael addition and amidation. The bioreducible poly(amidoamine)s grafted with dendritic polyamidoamines showed high buffer capacity, low cytotoxicity and strong DNA binding ability at low N/P ratio. They were able to condense DNA into small sized polycation/DNA complexes, which degraded and released the incorporated DNA under reductive conditions. In comparison to the original disulfide‐containing poly(amidoamine) with aminoethyl side chain, the grafting of the bioreducible poly(amidoamine) with dendrimer greatly improved the transfection efficiencies of 293T and HeLa cells with foreign DNA at various N/P ratios. The structure–gene delivery property relations of dendrimer‐grafted polycations will provide valuable insight into the design of highly efficient and less toxic polycationic gene carriers.

     

Trace LC/MS/MS quantitation of 17β‐estradiol as a biomarker for selective estrogen receptor modulator activity in the rat brain

A sensitive LC/MS/MS method has been developed by derivatization of 17β‐estradiol (E2) with dansyl chloride to quantitate 17β‐E2 in female rat serum. The use of E2‐d₅ minimized interferences from endogenous 17β‐E2 in order to achieve a limit of quantitation (LOQ) of 2.5 pg/ml using 150 µl of female rat serum. The recovery of the dansyl derivative was 95% or greater in quality control samples. The intra and interday assay precision was better than 8.2 and 6.2%, respectively, with accuracies ranging from 97 to 101% in the quality control samples. The assay was used for the quantitation of serum E2 as a biomarker for the estrogen receptor (ER) antagonist activity of small molecule SERMs (selective estrogen receptor modulators) in the female rat brain. The study revealed that a statistically significant upregulation of serum 17β‐E2 occurred for rats dosed with SERMs that are known to penetrate the brain and disrupt the hypothalamic‐pituitary‐ovarian (HPO) axis. Variations in 17β‐E2 in ascending dose studies also correlated with the corresponding trends in CYP17a1 levels, an mRNA biomarker for ovarian hyperstimulation. This biomarker assay has provided a useful screen for medicinal chemistry optimization to produce SERMs that do not interfere with negative feedback of estrogens on the brain and for biological hypothesis testing. Copyright © 2009 John Wiley & Sons, Ltd.     

Novel deletion of the E3A ubiquitin protein ligase gene detected by multiplex ligation-dependent probe amplification in a patient with Angelman syndrome

Angelman syndrome (AS) is a severe neurobehavioural disorder caused by failure of expression of the maternal copy of the imprinted domain located on 15q11-q13. There are different mechanisms leading to AS: maternal microdeletion, uniparental disomy, defects in a putative imprinting centre, mutations of the E3 ubiquitin protein ligase (UBE3A) gene. However, some of suspected cases of AS are still scored negative to all the latter mutations. Recently, it has been shown that a proportion of negative cases bear large deletions overlapping one or more exons of the UBE3A gene. These deletions are difficult to detect by conventional gene-scanning methods due to the masking effect by the non-deleted allele. In this study, we have used for the first time multiplex ligation-dependent probe amplification (MLPA) and comparative multiplex dosage analysis (CMDA) to search for large deletions affecting the UBE3A gene. Using this approach, we identified a novel causative deletion involving exon 8 in an affected sibling. Based on our results, we propose the use of MLPA as a fast, accurate and inexpensive test to detect large deletions in the UBE3A gene in a small but significant percentage of AS patients.     

Effects of adenovirus-mediated bFGF, IL-1Ra and IGF-1 gene transfer on human osteoarthritic chondrocytes and osteoarthritis in rabbits

The study investigated the effects of adenovirus-mediated gene transfection of basic fibroblast growth factor (bFGF), bFGF combined with interleukin-1 receptor antagonist protein (IL-Ra) and/or insulin-like growth factor-1 (IGF-1) both in human osteoarthritis (OA) chondrocytes and rabbits OA model. Human OA chondrocytes were delivered by adenovirus-mediated bFGF, IL-Ra and IGF-1 vectors, respectively. Chondrocyte proliferation, glycosaminoglycan (GAG) content, expression of type II collagen, ADAMTS-5, MMP-13, MMP-3 and TIMP-1 were determined. Rabbit OA model was induced by anterior cruciate ligament transaction (ACLT) in knees. Adenoviral vectors encoding human bFGF, IL-Ra and IGF-1 were injected intraarticularly into the knee joints after ACLT. The effects of adenovirus- mediated gene transfection on rabbit OA were evaluated. In vitro, the transfected genes were expressed in cell supernatant of human OA chondrocytes. AdbFGF group significantly promoted chondrocyte proliferation, and increased GAG and type II collagen synthesis than in the OA group. As two or three genes were transfected in different combinations, there was significant enhancement on the GAG content, type II collagen synthesis, and TIMP-1 levels, while ADAMTS-5, MMP-13, and MMP-3 levels were reduced. In vivo, the transfected genes were expressed in synovial fluid of rabbits. Intraarticular delivery of bFGF enhanced the expression of type II collagen in cartilage and decreased cartilage Mankin score compared with the OA control group (P = 0.047; P < 0.01, respectively). Multiple-gene transfection in different combinations showed better results than bFGF transfection alone. This study suggests that bFGF gene transfection is effective in treating experimental OA. Multiple gene transfection has better biologic effects on OA.