Improve protective efficacy of a TB DNA-HSP65 vaccine by BCG priming

Vaccines are considered by many to be one of the most successful medical interventions against infectious diseases. But many significant obstacles remain, such as optimizing DNA vaccines for use in humans or large animals. The amount of doses, route and easiness of administration are also important points to consider in the design of new DNA vaccines. Heterologous prime-boost regimens probably represent the best hope for an improved DNA vaccine strategy. In this study, we have shown that heterologous prime-boost vaccination against tuberculosis (TB) using intranasal BCG priming/DNA-HSP65 boosting (BCGin/DNA) provided significantly greater protection than that afforded by a single subcutaneous or intranasal dose of BCG. In addition, BCGin/DNA immunization was also more efficient in controlling bacterial loads than were the other prime-boost schedules evaluated or three doses of DNA-HSP65 as a naked DNA. The single dose of DNA-HSP65 booster enhanced the immunogenicity of a single subcutaneous BCG vaccination, as evidenced by the significantly higher serum levels of anti-Hsp65 IgG2a Th1-induced antibodies, as well as by the significantly greater production of IFN-γ by antigen-specific spleen cells. The BCG prime/DNA-HSP65 booster was also associated with better preservation of lung parenchyma. The improvement of the protective effect of BCG vaccine mediated by a DNA-HSP65 booster suggests that our strategy may hold promise as a safe and effective vaccine against TB.     

Two levels of resting potential in cardiac purkinje fibers

In an appropriate ionic environment, the resting potential of canine cardiac purkinje fibers may have either of two value. By changing the external K concentration, [K](0), in small steps, it was shown that, in the low (1 mM) Cl, Na-containing solutions used in this study, the two levels of resting potential could be obtained only within a narrow range of [K](0) values; that range was usually found between 1 and 4 mM. Within the critical [K](0) range the resting potential could be shifted from either level to the other by the application of small current pulses. It was shown that under these conditions the steady-state current- voltage relationship was “N-shaped,” and that a region of both negative slope, and negative chord conductance lay between the two stable zero-current potentials. The negative chord conductance was largely due to inward sodium current, only part of which was sensitive to tetrodotoxin (TTX). Under appropriate conditions, the negative chord conductance could be abolished by several experimental interventions and the membrane potential thereby shifted from the lower to the higher resting level: those interventions which were effective by presumably diminishing the steady-state inward current included reducing the external sodium concentration, adding TTX, or adding lidocaine; those which presumably increased the steady-state outward current included small increases in [K](0), brief depolarizations to around -20 mV, or the addition of acetylcholine chloride.     

Rat toxicogenomic study reveals analytical consistency across microarray platforms

To validate and extend the findings of the MicroArray Quality Control (MAQC) project, a biologically relevant toxicogenomics data set was generated using 36 RNA samples from rats treated with three chemicals (aristolochic acid, riddelliine and comfrey) and each sample was hybridized to four microarray platforms. The MAQC project assessed concordance in intersite and cross-platform comparisons and the impact of gene selection methods on the reproducibility of profiling data in terms of differentially expressed genes using distinct reference RNA samples. The real-world toxicogenomic data set reported here showed high concordance in intersite and cross-platform comparisons. Further, gene lists generated by fold-change ranking were more reproducible than those obtained by t-test P value or Significance Analysis of Microarrays. Finally, gene lists generated by fold-change ranking with a nonstringent P-value cutoff showed increased consistency in Gene Ontology terms and pathways, and hence the biological impact of chemical exposure could be reliably deduced from all platforms analyzed.     

Performance evaluation of commercial short-oligonucleotide microarrays and the impact of noise in making cross-platform correlations

Background  

Despite the widespread use of microarrays, much ambiguity regarding data analysis, interpretation and correlation of the different technologies exists. There is a considerable amount of interest in correlating results obtained between different microarray platforms. To date, only a few cross-platform evaluations have been published and unfortunately, no guidelines have been established on the best methods of making such correlations. To address this issue we conducted a thorough evaluation of two commercial microarray platforms to determine an appropriate methodology for making cross-platform correlations.     

Real-time PCR quantitation of glucocorticoid receptor alpha isoform

Background  

The expression of glucocorticoid-receptor (GR) seems to be a key mechanism in the regulation of glucocorticoid (GC) sensitivity and is potentially involved in cases of GC resistance or hypersensitivity. The aim of this study is to describe a method for quantitation of GR alpha isoform (GRα) expression using real-time PCR (qrt-PCR) with analytical capabilities to monitor patients, offering standard-curve reproducibility as well as intra- and inter-assay precision.     

GidA Expression in Salmonella is Modulated Under Certain Environmental Conditions

Bacillus subtilis endospores have applications in different fields including their use as probiotics and antigen delivery vectors. Such specialized applications frequently require highly purified spore preparations. Nonetheless, quantitative data regarding both yields and purity of B. subtilis endospores after application of different growth conditions and purification methods are scarce or poorly reported. In the present study, we conducted several quantitative and qualitative analyses of growth conditions and purification procedures aiming generation of purified B. subtilis spores. Based on two growth media and different incubations conditions, sporulation frequencies up to 74.2 % and spore concentrations up to 7 × 10⁹ spores/ml were achieved. Application of a simplified spore isolation method, in which samples were incubated with lysozyme and a detergent, resulted in preparations with highly purified spores at the highest yields. The present study represents, therefore, an important contribution for those working with B. subtilis endospores for different biotechnological purposes.     

The <Emphasis Type="Italic">Tribolium spineless</Emphasis> ortholog specifies both larval and adult antennal identity

The morphology of insect antennae varies widely among species, but our understanding of antennal development comes almost solely from studies of one species—the fruit fly, Drosophila melanogaster. Moreover, this knowledge applies mostly to adult structures, since Drosophila lacks external larval appendages. In contrast to Drosophila, the red flour beetle, Tribolium castaneum, has both larval and adult antennae, which are very different from one another in morphology. Thus, Tribolium provides an ideal system to compare modes of antennal development both within and between species. Here, we report that the Tribolium ortholog of spineless (Tc-ss) is required in both the larval and adult antennae. Knockdown of Tc-ss by RNAi during either larval or imaginal development causes transformation of the distal portion of the antennae to legs. Thus, the function of ss is conserved between Drosophila and Tribolium with respect to adult antennal specification and also between Tribolium larval and adult antennal development. The similarity of the Tc-ss RNAi phenotype to that of a classically described Tribolium mutation, antennapedia (ap) (of no relationship to the Drosophila Hox gene of the same name), led us to characterize the original ap mutation and two newly identified ap alleles. Our mapping and phenotypic data suggest that Tc-ss is the best candidate for the ap locus. These results represent a first step in characterizing larval and adult antennal patterning in Tribolium, which should provide important insights into the evolution of insect antennal development.     

The hairpin ribozyme

The hairpin ribozyme is a member of a family of small RNA endonucleases, which includes hammer-head, human hepatitis delta virus, Neurospora VS, and the lead-dependent catalytic RNAs. All these catalytic RNAs reversibly cleave the phosphodiester bond of substrate RNA to generate 5'-hydroxyl and 2',3'-cyclic phosphate termini. Whereas the reaction products from family members are similar, large structural and mechanistic differences exist. Structurally the hairpin ribozyme has two principal domains that interact to facilitate catalysis. The hairpin ribozyme uses a catalytic mechanism that does not require metals for cleavage or ligation of substrate RNA. In this regard it is presently unique among RNA catalysts. Targeting rules for cleavage of substrate have been determined and required bases for catalysis have been identified. The hairpin ribozyme has been developed and used for gene therapy and was the first ribozyme to be approved for human clinical trials.