Gender differences in human immunodeficiency virus type 1-specific CD8 responses in the reproductive tract and colon following nasal peptide priming and modified vaccinia virus Ankara boosting

Induction of mucosal anti-human immunodeficiency virus type 1 (HIV-1) T-cell responses in males and females will be important for the development of a successful HIV-1 vaccine. An HIV-1 envelope peptide, DNA plasmid, and recombinant modified vaccinia virus Ankara (rMVA) expressing the H-2D(d)-restricted cytotoxic T lymphocyte P18 epitope were used as immunogens to test for their ability to prime and boost anti-HIV-1 T-cell responses at mucosal and systemic sites in BALB/c mice. We found of all prime-boost combinations tested, an HIV-1 Env peptide subunit mucosal prime followed by systemic (intradermal) boosting with rMVA yielded the maximal induction of gamma interferon (IFN-gamma) spot-forming cells in the female genital tract and colon. However, this mucosal prime-systemic rMVA boost regimen was minimally immunogenic for the induction of genital, colon, or lung anti-HIV-1 T-cell responses in male mice. We determined that a mucosal Env subunit immunization could optimally prime an rMVA boost in female but not male mice, as determined by the magnitude of antigen-specific IFN-gamma responses in the reproductive tracts, colon, and lung. Defective mucosal priming in male mice could not be overcome by multiple mucosal immunizations. However, rMVA priming followed by an rMVA boost was the optimal prime-boost strategy for male mice as determined by the magnitude of antigen-specific IFN-gamma responses in the reproductive tract and lung. Thus, prime-boost immunization strategies able to induce mucosal antigen-specific IFN-gamma responses were identified for male and female mice. Understanding the cellular and molecular basis of gender-determined immune responses will be important for optimizing induction of anti-HIV-1 mucosal immune responses in both males and females.     

The genome sequence of a reassortant bluetongue virus serotype 3 from India

All 10 genome segments (Seg-1 to 10-a total of 19,188 bp) were sequenced from a strain of bluetongue virus serotype 3 (BTV-3) from India (strain IND2003/08). Sequence comparisons showed that nine of the genome segments from this virus group with other eastern topotype strains. Genome Seg-2 and Seg-6 group with eastern BTV-3 strains from Japan. However, Seg-5 (the NS1 gene) from IND2003/08 belongs to a western lineage, demonstrating that IND2003/08 is a reassortant between eastern and western topotype bluetongue viruses. This confirms that western BTV strains have been imported and are circulating within the subcontinent.     

Identification and differentiation of the twenty six bluetongue virus serotypes by RT-PCR amplification of the serotype-specific genome segment 2

Bluetongue (BT) is an arthropod-borne viral disease, which primarily affects ruminants in tropical and temperate regions of the world. Twenty six bluetongue virus (BTV) serotypes have been recognised worldwide, including nine from Europe and fifteen in the United States. Identification of BTV serotype is important for vaccination programmes and for BTV epidemiology studies. Traditional typing methods (virus isolation and serum or virus neutralisation tests (SNT or VNT)) are slow (taking weeks, depend on availability of reference virus-strains or antisera) and can be inconclusive. Nucleotide sequence analyses and phylogenetic comparisons of genome segment 2 (Seg-2) encoding BTV outer-capsid protein VP2 (the primary determinant of virus serotype) were completed for reference strains of BTV-1 to 26, as well as multiple additional isolates from different geographic and temporal origins. The resulting Seg-2 database has been used to develop rapid (within 24 h) and reliable RT-PCR-based typing assays for each BTV type. Multiple primer-pairs (at least three designed for each serotype) were widely tested, providing an initial identification of serotype by amplification of a cDNA product of the expected size. Serotype was confirmed by sequencing of the cDNA amplicons and phylogenetic comparisons to previously characterised reference strains. The results from RT-PCR and sequencing were in perfect agreement with VNT for reference strains of all 26 BTV serotypes, as well as the field isolates tested. The serotype-specific primers showed no cross-amplification with reference strains of the remaining 25 serotypes, or multiple other isolates of the more closely related heterologous BTV types. The primers and RT-PCR assays developed in this study provide a rapid, sensitive and reliable method for the identification and differentiation of the twenty-six BTV serotypes, and will be updated periodically to maintain their relevance to current BTV distribution and epidemiology (http://www.reoviridae.org/dsRNA_virus_proteins/ReoID/rt-pcr-primers.htm).     

Effect of different biosynthetic precursors on the production of nargenicin A1 from metabolically engineered Nocardia sp. CS682

Nargenicin A1 is a 28-membered polyketide macrolide, with antibacterial activity against methicillin-resistant Staphylococcus aureus, produced by Nocardia sp. CS682. In this study, the production of nargenicin A1 was improved by enhancing the supply of different biosynthetic precursors. In Nocardia sp. CS682 (KCTC11297BP), this improvement was ~4.62-fold with the supplementation of 30 mM methyl oleate, 4.25-fold with supplementation of 15 mM sodium propionate, and 2.81-fold with supplementation of 15 mM sodium acetate. In Nocardia sp. metK18 and Nocardia sp. CS682 expressing S-adenosylmethionine synthetase (MetK), the production of nargenicin A1 was improved by ~5.57-fold by supplementation with 30 mM methyl oleate, 5.01-fold by supplementation with 15 mM sodium propionate, and 3.64-fold by supplementation with 15 mM sodium acetate. Furthermore, supplementing the culture broth of Nocardia sp. ACC18 and Nocardia sp. CS682 expressing the acetyl-CoA carboxylase complex (AccA2 and AccBE) with 30 mM methyl oleate, 15 mM sodium propionate, or 15 mM sodium acetate resulted in ~6.99-, 6.46-, and 5.58-fold increases, respectively, in nargenicin A1 production. Our overall results showed that among the supplements, methyl oleate was the most effective precursor supporting the highest titers of nargenicin A1 in Nocardia sp. CS682, Nocardia sp. metK18, and Nocardia sp. ACC18.     

Retake the center stage--new development of rat genetics

The rat is a powerful model for the study of human physiology and diseases,and is preferred by physiologists,neuroscientists and toxicologists.However,the lack of robust genetic modification tools has severely limited the generation of rat genetic models over the last two decades.In the last few years,several gene-targeting strategies have been developed in rats using N-ethyl-N-nitrosourea(ENU), transposons,zinc-finger nucleases(ZFNs),bacterial artificial chromosome(BAC) mediated transgenesis,and recently established rat embryonic stem(ES) cells.The development and improvement of these approaches to genetic manipulation have created a bright future for the use of genetic rat models in investigations of gene function and human diseases.Here,we summarize the strategies used for rat genetic manipulation in current research.We also discuss BAC transgenesis as a potential tool in rat transgenic models.     

Inhibition of an endogenous growth-related proteinase enhances the recovery of a negative growth regulator from cultured human cells.

The possibility that a growth-related proteinase may act by degrading a negative growth regulatory protein has been investigated. Proteinase inhibitors which inhibit the enzyme also enhance the accumulation of the growth regulator by human fibroblasts. The negative growth regulator shows a similar specificity of inhibition of cellular growth to inhibitors of the growth-related proteinase.     

Evolution and Phylogenetic Analysis of Full-Length VP3 Genes of Eastern Mediterranean Bluetongue Virus Isolates

Bluetongue virus (BTV) is the ‘type’ species of the genus Orbivirus within the family Reoviridae. The BTV genome is composed of ten linear segments of double-stranded RNA (dsRNA), each of which codes for one of ten distinct viral proteins. Previous phylogenetic comparisons have evaluated variations in genome segment 3 (Seg-3) nucleotide sequence as way to identify the geographical origin (different topotypes) of BTV isolates. The full-length nucleotide sequence of genome Seg-3 was determined for thirty BTV isolates recovered in the eastern Mediterranean region, the Balkans and other geographic areas (Spain, India, Malaysia and Africa). These data were compared, based on molecular variability, positive-selection-analysis and maximum-likelihood phylogenetic reconstructions (using appropriate substitution models) to 24 previously published sequences, revealing their evolutionary relationships. These analyses indicate that negative selection is a major force in the evolution of BTV, restricting nucleotide variability, reducing the evolutionary rate of Seg-3 and potentially of other regions of the BTV genome. Phylogenetic analysis of the BTV-4 strains isolated over a relatively long time interval (1979–2000), in a single geographic area (Greece), showed a low level of nucleotide diversity, indicating that the virus can circulate almost unchanged for many years. These analyses also show that the recent incursions into south-eastern Europe were caused by BTV strains belonging to two different major-lineages: representing an ‘eastern’ (BTV-9, -16 and -1) and a ‘western’ (BTV-4) group/topotype. Epidemiological and phylogenetic analyses indicate that these viruses originated from a geographic area to the east and southeast of Greece (including Cyprus and the Middle East), which appears to represent an important ecological niche for the virus that is likely to represent a continuing source of future BTV incursions into Europe.