A novel and highly efficient production system for recombinant adeno-associated virus vector

Recombinant adeno-associated virus(rAAV) has proven to be a promising gene delivery vector for human gene therapy. However, its application has been limited by difficulty in obtaining enough quantities of high-titer vector stocks. In this paper, a novel and highly efficient production system for rAAV is described. A recombinant herpes simplex virus type 1(rHSV-1) designated HSV1-rc/△UL2, which expressed adeno-associated virus type2(AAV-2) Rep and Cap proteins, was constructed previously. The data confirmed that its functions were to support rAAV replication and packaging, and the generated rAAV was infectious. Meanwhile, an rAAV proviral cell line designated BHK/SG2, which carried the green fluorescent protein(GFP) gene expression cassette, was established by transfecting BHK-21 cells with rAAV vector plasmid pSNAV-2-GFP. Infecting BHK/SG2 with HSV1-rc/△UL2 at an MOI of 0.1 resulted in the optimal yields of rAAV, reaching 250 transducing unit(TU) or 4.28×104 particles per cell. Therefore, compared with the conventional transfection method, the yield of rAAV using this "one proviral cell line, one helper virus" strategy was increased by two orders of magnitude. Large-scale production of rAAV can be easily achieved using this strategy and might meet the demands for clinical trials of rAAV-mediated gene therapy.     

中国大鲵研究进展

中国大鲵是我国特有濒危的两栖物种,是研究生物进化、生物多样性、性别决定分子机制等问题的好材料。近年来,人们对它的研究力度不断加大,著述颇多。本文对大鲵的生态保护、解剖发育、生理生化、遗传进化等方面的近期研究资料进行了整理和回顾,也简要探讨了今后大鲵研究的主要工作,以期为研究者们提供有价值的资料。     

Resin gathering in neotropical resin bugs (Insecta: Hemiptera: Reduviidae): Functional and comparative morphology

Apiomerini (Reduviidae: Harpactorinae) collect plant resins with their forelegs and use these sticky substances for prey capture or maternal care. These behaviors have not been described in detail and morphological structures involved in resin gathering, transfer, and storage remain virtually undocumented. We here describe these behaviors in Apiomerus flaviventris and document the involved structures. To place them in a comparative context, we describe and document leg and abdominal structures in 14 additional species of Apiomerini that represent all but one of the 12 recent genera in the tribe. Based on these morphological data in combination with the behavioral observations on A. flaviventris, we infer behavioral and functional hypotheses for the remaining genera within the tribe Apiomerini. Setal abdominal patches for resin storage are associated with maternal care so far only documented for species of Apiomerus. Based on the occurrence of these patches in several other genera, we propose that maternal care is widespread within the tribe. Ventral abdominal glands are widespread within female Apiomerini. We propose that their products may prevent hardening of stored resins thus providing long‐term supply for egg coating. Judging from the diverse setal types and arrangements on the front legs, we predict six different behavioral patterns of resin gathering within the tribe. J. Morphol., 2011. © 2010 Wiley‐Liss, Inc.     

PerR-Regulated Manganese Ion Uptake Contributes to Oxidative Stress Defense in an Oral Streptococcus

Metal homeostasis plays a critical role in antioxidative stress. Streptococcus oligofermentans, an oral commensal facultative anaerobe lacking catalase activity, produces and tolerates abundant H₂O₂, whereas Dpr (an Fe²⁺-chelating protein)-dependent H₂O₂ protection does not confer such high tolerance. Here, we report that inactivation of perR, a peroxide-responsive repressor that regulates zinc and iron homeostasis in Gram-positive bacteria, increased the survival of H₂O₂-pulsed S. oligofermentans 32-fold and elevated cellular manganese 4.5-fold. perR complementation recovered the wild-type phenotype. When grown in 0.1 to 0.25 mM MnCl₂, S. oligofermentans increased survival after H₂O₂ stress 2.5- to 23-fold, and even greater survival was found for the perR mutant, indicating that PerR is involved in Mn²⁺-mediated H₂O₂ resistance in S. oligofermentans. Mutation of mntA could not be obtained in brain heart infusion (BHI) broth (containing ∼0.4 μM Mn²⁺) unless it was supplemented with ≥2.5 μM MnCl₂ and caused 82 to 95% reduction of the cellular Mn²⁺ level, while mntABC overexpression increased cellular Mn²⁺ 2.1- to 4.5-fold. Thus, MntABC was identified as a high-affinity Mn²⁺ transporter in S. oligofermentans. mntA mutation reduced the survival of H₂O₂-pulsed S. oligofermentans 5.7-fold, while mntABC overexpression enhanced H₂O₂-challenged survival 12-fold, indicating that MntABC-mediated Mn²⁺ uptake is pivotal to antioxidative stress in S. oligofermentans. perR mutation or H₂O₂ pulsing upregulated mntABC, while H₂O₂-induced upregulation diminished in the perR mutant. This suggests that perR represses mntABC expression but H₂O₂ can release the suppression. In conclusion, this work demonstrates that PerR regulates manganese homeostasis in S. oligofermentans, which is critical to H₂O₂ stress defenses and may be distributed across all oral streptococci lacking catalase.     

Identifying differentially expressed genes in meta-analysis via Bayesian model-based clustering

A Bayesian model-based clustering approach is proposed for identifying differentially expressed genes in meta-analysis. A Bayesian hierarchical model is used as a scientific tool for combining information from different studies, and a mixture prior is used to separate differentially expressed genes from non-differentially expressed genes. Posterior estimation of the parameters and missing observations are done by using a simple Markov chain Monte Carlo method. From the estimated mixture model, useful measure of significance of a test such as the Bayesian false discovery rate (FDR), the local FDR (Efron et al., 2001), and the integration-driven discovery rate (IDR; Choi et al., 2003) can be easily computed. The model-based approach is also compared with commonly used permutation methods, and it is shown that the model-based approach is superior to the permutation methods when there are excessive under-expressed genes compared to over-expressed genes or vice versa. The proposed method is applied to four publicly available prostate cancer gene expression data sets and simulated data sets.     

Structure-based functional inference in structural genomics

The dramatically increasing number of new protein sequences arising from genomics 4 proteomics requires the need for methods to rapidly and reliably infer the molecular and cellular functions of these proteins. One such approach, structural genomics, aims to delineate the total repertoire of protein folds in nature, thereby providing three-dimensional folding patterns for all proteins and to infer molecular functions of the proteins based on the combined information of structures and sequences. The goal of obtaining protein structures on a genomic scale has motivated the development of high throughput technologies and protocols for macromolecular structure determination that have begun to produce structures at a greater rate than previously possible. These new structures have revealed many unexpected functional inferences and evolutionary relationships that were hidden at the sequence level. Here, we present samples of structures determined at Berkeley Structural Genomics Center and collaborators laboratories to illustrate how structural information provides and complements sequence information to deduce the functional inferences of proteins with unknown molecular functions.Two of the major premises of structural genomics are to discover a complete repertoire of protein folds in nature and to find molecular functions of the proteins whose functions are not predicted from sequence comparison alone. To achieve these objectives on a genomic scale, new methods, protocols, and technologies need to be developed by multi-institutional collaborations worldwide. As part of this effort, the Protein Structure Initiative has been launched in the United States (PSI; www.nigms.nih.gov/funding/psi.html). Although infrastructure building and technology development are still the main focus of structural genomics programs [1–6], a considerable number of protein structures have already been produced, some of them coming directly out of semi-automated structure determination pipelines [6–10]. The Berkeley Structural Genomics Center (BSGC) has focused on the proteins of Mycoplasma or their homologues from other organisms as its structural genomics targets because of the minimal genome size of the Mycoplasmas as well as their relevance to human and animal pathogenicity (http://www.strgen.org). Here we present several protein examples encompassing a spectrum of functional inferences obtainable from their three-dimensional structures in five situations, where the inferences are new and testable, and are not predictable from protein sequence information alone.