Chitosan enhanced gene delivery of cationic liposome via non-covalent conjugation

Two new types of stable ternary complexes were formed by mixing chitosan with DOTAP/pDNA lipoplex and DOTAP with chitosan/pDNA polyplex via non-covalent conjugation for the efficient delivery of plasmid DNA. They were characterized by atomic force microscopy, gel retarding, and dynamic light scattering. The DOTAP/CTS/pDNA complexes were in compacted spheroids and irregular lump of larger aggregates in structure, while the short rod- and toroid-like and donut shapes were found in CTS/DOTAP/pDNA complexes. The transfection efficiency of the lipopolyplexes showed higher GFP gene expression than DOTAP/pDNA and CTS/pDNA controls in Hep-2 and Hela cells, and luciferase gene expression 2–3-fold than DOTAP/pDNA control and 70–120-fold than CTS/pDNA control in Hep-2 cells. The intracellular trafficking was examined by confocal laser scanning microscopy. Rapid pDNA delivery to the nucleus enchanced by chitosan was achieved after 4 h transfection.     

Highly efficient genetic transduction of primary human synoviocytes with concentrated retroviral supernatant

We are developing retroviral-mediated gene transfer to human fibroblast-like synovial cells (FLS) as one approach to characterizing genetic pathways involved in synoviocyte pathophysiology. Prior work has suggested that FLS are relatively refractory to infection by Moloney murine leukemia virus based vectors. To determine if viral titer influenced the transduction efficiency of FLS, we optimized a rapid, efficient, and inexpensive centrifugation method to concentrate recombinant retroviral supernatant. The technique was evaluated by measurement of the expression of a viral enhanced green fluorescent protein transgene in transduced cells, and by analysis of viral RNA in retroviral supernatant. Concentration (100-fold) was achieved by centrifugation of viral supernatant for four hours, with 100% recovery of viral particles. The transduction of FLS increased from approximately 15% with unconcentrated supernatant, to nearly 50% using concentrated supernatant. This protocol will be useful for investigators with applications that require efficient, stable, high level transgene expression in primary FLS.     

Coupling between catalytic site and collective dynamics: a requirement for mechanochemical activity of enzymes

Growing evidence supports the view that enzymatic activity results from a subtle interplay between chemical kinetics and molecular motions. A systematic analysis is performed here to delineate the type and level of coupling between catalysis and conformational mechanics. The dynamics of a set of 98 enzymes representative of different EC classes are analyzed with the Gaussian network model (GNM) and compared with experimental data. In more than 70% of the examined enzymes, the global hinge centers predicted by the GNM are found to be colocalized with the catalytic sites experimentally identified. Low translational mobility (< 7%) is observed for the catalytic residues, consistent with the fine-tuned design of enzymes to achieve precise mechanochemical activities. Ligand binding sites, while closely neighboring catalytic sites, enjoy a moderate flexibility to accommodate the ligand binding. These findings could serve as additional criteria for assessing drug binding residues and could lessen the computational burden of substrate docking searches.     

Th17 cell induction and immune regulatory effects

The T help 1 (Th1) and Th2 cell classification have provided the framework for understanding CD4⁺ T cell biology and the interplay between innate and adaptive immunity for almost two decades. Recent studies have defined a previously unknown arm of the CD4⁺ T cell effector response, the Th17 lineage, which promises to change our understanding of immune regulation, immune pathogenesis and host defense. The factors that specify differentiation of IL‐17 producing effector T cells from naïve T cell precursors are being rapidly discovered and are providing insights into mechanisms by which signals from cells of the innate immune system guide alternative pathways of Th1, Th2, or Th17 development. In this review, we will focus on recent studies that have identified new subsets of Th cells, new insights regarding the induced generation and differentiation mechanisms of Th17 cells and immune regulatory effects. J. Cell. Physiol. 211: 273–278, 2007. © 2007 Wiley‐Liss, Inc.     

Functional analysis of the split Synechocystis DnaE intein in plant tissues by biolistic particle bombardment

The DnaE intein of Synechocystis sp. PCC6803 (Ssp DnaE intein) is the first split intein identified in nature. Its N-terminal fragment (Int-n) is attached to the end of the N-terminal half of the DnaE protein (DnaE-n) to form the precursor DnaE-n/Int-n, while the C-terminal fragment (Int-c) precedes the C-terminal half of the DnaE protein (DnaE-c) to form the precursor Int-c/DnaE-c. Int-n and Int-c fragments in the separate precursors catalyze, in concert, a protein trans-splicing process to splice the flanking DnaE-n and DnaE-c into a functional catalytic subunit of DNA polymerase III. They then release themselves from the precursors. Previously, the Ssp DnaE intein has been used to reconstitute a protein trans-splicing mechanism in stably transformed Arabidopsis thaliana, resulting in successful reassembly of an intact and functional GUS from two halves of a split GUS protein. In this report, transient expression using a biolistic particle bombardment approach is described for functional analysis of Ssp DnaE intein. Analyses confirmed that the Ssp DnaE intein could catalyze protein trans-splicing not only in model plants but also in monocot and dicot crops. It also demonstrated that when up to 45 amino acid residues were removed from the C-terminus of the Int-n fragment, the Int-n fragment was still able to function in the protein trans-splicing process.     

Next-generation sequencing to generate interactome datasets

Next-generation sequencing has not been applied to protein-protein interactome network mapping so far because the association between the members of each interacting pair would not be maintained in en masse sequencing. We describe a massively parallel interactome-mapping pipeline, Stitch-seq, that combines PCR stitching with next-generation sequencing and used it to generate a new human interactome dataset. Stitch-seq is applicable to various interaction assays and should help expand interactome network mapping.     

Characterization and development of EST-SSR markers to study the genetic diversity and populations analysis of Jerusalem artichoke (<Emphasis Type="Italic">Helianthus tuberosus</Emphasis> L.)

In recent years, Jerusalem artichoke has received widespread attention as a novel source of sugar, biofuel, and animal feed. Currently, only few gDNA-SSRs derived from sunflower were verified in the Jerusalem artichoke; therefore, it is particularly important to develop SSR primer markers that belonged to Jerusalem artichoke resources. Using EST data to develop EST-SSR markers is simple and effective. In order to understand the general characteristics of SSR markers in Jerusalem artichoke EST sequences and accelerate the use of SSR markers in Jerusalem artichoke research. This study used 40,370 sequenced unigene fragments and MISA software to identify SSR loci. The 48 pairs of EST-SSR primers assessed for the identification of 45 varieties of Jerusalem artichoke. Cluster, genetic diversity parameters and AMOVA analysis was conducted using the genetic similarity coefficient, revealing genetic differences between 48 genetic material. A total of 1204 SSR loci were identified with 13 different types of repeats, distributed among 1020 EST sequences, of which trinucleotide repeats were the most common, accounting for 38.21% of the total SSR loci. Among the 44 repeat motifs, AG/CT, AAG/CTT, and ATC/ATG motifs had the highest frequencies, accounting for 22.45, 14.71, and 7.84% of all motifs, respectively. From these sequences, 48 pairs of EST-SSR primers were designed, and 22 primer pairs for loci with high polymorphism were selected to analyze the genetic diversity of 45 Jerusalem artichoke germplasm sources. The results indicated that the variation range of the effective number of alleles for 22 primers ranged between 1.7502 and 4.5660. The Shannon’s information index ranged between 0.6200 and 1.6423. The variation range of PIC ranged between 0.3121 and 0.6662 with an average of 0.5184. Cluster analysis was conducted using the genetic similarity coefficient, revealing significant genetic differences between Asian and European genetic material. Cluster analysis revealed a relationship between the genotypes and geographic origins of the Jerusalem artichoke. The results of AMOVA as well as the genetic identity and genetic distance in the Jerusalem artichoke population showed that there presented certain genetic heterogeneity in Jerusalem artichoke genetic structure of 45 samples from seven different geographic populations. The Jerusalem artichoke EST-SSR marker system established in this study provides an effective molecular marker system for future research focused on Jerusalem artichoke genetic diversity and the breeding of new varieties.     

昆虫多次交配的策略和利益

简要回顾昆虫交配系统的研究历史,并简述Bateman的性选择原理,讨论雌雄昆虫在交配过程中的角色和性选择压力的相对关系。一般而言,由于雌雄两性在交配过程中的角色和对后代投入的不同,往往是雄虫试图与尽可能多的雌虫交配,即雄虫使自己被尽可能多的雌虫接受,雌虫选择最好的雄虫进行交配,即雌虫选择交配对象。雄虫因而发展出各种类型的交配策略,以获取与更多雌虫的交配机会;而雌虫也通过交配选择权,选择对自己更有利的雄虫进行交配,雌虫在交配过程中除了可以获得基因利益,还可以获取物质利益。文章分别就昆虫的交配策略和利益展开叙述和讨论。     

Specific primer design for the polymerase chain reaction

The design of primers has a major impact on the success of PCR in relation to the specificity and yield of the amplified product. Here, we introduce the applications of PCR as well as the definition and characteristics for PCR primer design. Recent primer design tools based on Primer3, along with several computational intelligence-based primer design methods which have been applied in primer design, are also reviewed. In addition, characteristics of population-based methods used in primer design are discussed in detail.