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.     

"Silent" sites in Drosophila genes are not neutral: evidence of selection among synonymous codons

The patterns of synonymous codon usage in 91 Drosophila melanogaster genes have been examined. Codon usage varies strikingly among genes. This variation is associated with differences in G+C content at silent sites, but (unlike the situation in mammalian genes) these differences are not correlated with variation in intron base composition and so are not easily explicable in terms of mutational biases. Instead, those genes with high G+C content at silent sites, resulting from a strong "preference" for a particular subset of the codons that are mostly C- ending, appear to be the more highly expressed genes. This suggests that G+C content is reduced in sequences where selective constraints are weaker, as indeed seen in a pseudogene. These and other data discussed are consistent with the effects of translational selection among synonymous codons, as seen in unicellular organisms. The existence of selective constraints on silent substitutions, which may vary in strength among genes, has implications for the use of silent molecular clocks.      

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.     

Intratumor heterogeneity of biomarker expression in breast carcinomas

Small biopsy samples are used increasingly to assess the biomarker expression for prognostic information and for monitoring therapeutic responses prior to and during neoadjuvant therapy. The issue of intratumor heterogeneity of expression of biomarkers, however, has raised questions about the validity of the assessment of biomarker expression based on limited tissue samples. We examined immunohistochemically the expression of HER-2neu (p185^erbB-2), epidermal growth factor receptor (EGFR), Bcl-2, p53, and proliferating cell nuclear antigen (PCNA) in 30 breast carcinomas using archived, paraffin embedded tissue and determined the extent of intratumor heterogeneity. Each section was divided into four randomly oriented discrete regions, each containing a portion of the infiltrating carcinoma. For each tumor, the entire lesion and four regions were analyzed for the expression of these markers. Scores of both membrane and cytoplasmic staining of HER-2neu and EGFR, scores of cytoplasmic staining of Bcl-2, and scores of nuclear staining of both p53 and PCNA were recorded. The intensity of staining and the proportion of immunostained cells were determined. A semiquantitative immunoscore was calculated by determining the sum of the products of the intensity and corresponding proportion of stained tumor cells. We analyzed both invasive (IDC) and in situ (DCIS) carcinomas. The Wilcoxon signed-rank test was used for paired comparisons between overall and regional immunoscores and between overall and regional percentages of stained cells. Spearman's correlation coefficients were used to assess the level of agreement of overall biomarker expression with each of the regions. Generalized linear models were used to assess overall and pair-wise differences in the absolute values of percent changes between overall and regional expression of biomarkers. For IDCs, there were no statistically significant differences in the expression of the biomarkers in terms of either the percentage of cells staining or the immunoscores when comparing the entire tumor with each region except for the lower EGFR expression of arbitrarily selected region 1 and lower p53 expression of region 1 compared to that of the entire tumor section. For DCIS, there were no statistically significant differences in the expression of the biomarkers between the entire tumor and each region except in PCNA of region 2 compared to that of entire tumor section. Positive correlation of immunoscores was observed between the entire tumor and each region as well as across all four regions for IDC. Similar observations were noted with DCIS except for HER-2neu and PCNA. No statistically significant differences were observed in the absolute values of percent changes of biomarker expression between overall and the four regions for both DCIS and IDC. Therefore, no significant intratumor heterogeneity in the expression of HER-2neu, Bcl-2, and PCNA was observed in IDC. Minor regional variations were observed for EGFR and p53 in IDC. Similarly, no significant regional variation in the expression of markers was observed in DCIS except for PCNA.     

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.     

Development of wheat–Aegilops speltoides recombinants and simple PCR-based markers for Sr32 and a new stem rust resistance gene on the 2S#1 chromosome

Key message

Wheat– Aegilops speltoides recombinants carrying stem rust resistance genes Sr32 and SrAes1t effective against Ug99 and PCR markers for marker-assisted selection.

Abstract

Wild relatives of wheat are important resources for new rust resistance genes but underutilized because the valuable resistances are often linked to negative traits that prevent deployment of these genes in commercial wheats. Here, we report ph1b-induced recombinants with reduced alien chromatin derived from E.R. Sears’ wheat–Aegilops speltoides 2D-2S#1 translocation line C82.2, which carries the widely effective stem rust resistance gene Sr32. Infection type assessments of the recombinants showed that the original translocation in fact carries two stem rust resistance genes, Sr32 on the short arm and a previously undescribed gene SrAes1t on the long arm of chromosome 2S#1. Recombinants with substantially shortened alien chromatin were produced for both genes, which confer resistance to stem rust races in the TTKSK (Ug99) lineage and representative races of all Australian stem rust lineages. Selected recombinants were back crossed into adapted Australian cultivars and PCR markers were developed to facilitate the incorporation of these genes into future wheat varieties. Our recombinants and those from several other labs now show that Sr32, Sr39, and SrAes7t on the short arm and Sr47 and SrAes1t on the long arm of 2S#1 form two linkage groups and at present no rust races are described that can distinguish these resistance specificities.     

Quantitative comparisons ofin situ microbial biodiversity by signature biomarker analysis

Microscopic examinations have convinced microbial ecologists that the culturable microbes recovered from environmental samples represent a tiny proportion of the extant microbiota. Methods for recovery and enzymatic amplification of nucleic acids from environmental samples have shown that a huge diversity existsin situ, far exceeding any expectations which were based on direct microscopy. It is now theoretically possible to extract, amplify and sequence all the nucleic acids from a community and thereby gain a comprehensive measure of the diversity as well as some insights into the phylogeny of the various elements within this community. Unfortunately, this analysis becomes economically prohibitive if applied to the multitude of niches in a single biome let alone to a diverse set of environments. It is also difficult to utilize PCR amplification on nucleic acids from some biomes because of coextracting enzymatic inhibitors. Signature biomarker analysis which potentially combines gene probe and lipid analysis on the same sample, can serve as a complement to massive environmental genome analysis in providing quantitative comparisons between microniches in the biome under study. This analysis can also give indications of the magnitude of differences in biodiversity in the blome as well as provide insight into the phenotypic activities of each community in a rapid and cost-effective manner. Applications of signature lipid biomarker analysis to define quantitatively the microbial viable biomass of portions of an Eastern USA deciduous forest, are presented.     

Familial Clustering of Abdominal Visceral Fat and Total Fat Mass: The Québec Family Study

The evidence for common familial factors underlying total fat mass (estimated from underwater weighing) and abdominal visceral fat (assessed from CT scan) was examined in families participating in phase 2 of the Québec Family Study (QFS) using a bivariate familial correlation model. Previous QFS investigations suggest that both genetic (major and polygenic) and familial environmental factors influence each phenotype, accounting for between 55% to 71% of the phenotypic variance in fat mass, and between 55% to 72% for abdominal visceral fat The current study suggests that the bivariate familial effect ranges from 29% to 50%. This pattern suggests that there may be common familial determinants for abdominal visceral fat and total fat mass, as well as additional familial factors which are specific to each. The relatively high spouse cross-trait correlations usually suggest that a large percent of the bivariate familial effect may be environmental in origin. However, if mating is not random, then the spouse resemblance may reflect either genetic or environmental causes, depending on the source [i.e., through similar genes or cohabitation (environmental) effects]. Finally, there are significant sex differences in the magnitude of the familial cross-trait correlations involving parents, but not offspring, suggesting complex generation (i.e., age) and sex effects. For example, genes may turn on or off as a function of age and sex, and/or there may be an accumulation over time of effects due to the environment which may vary by sex. Whether the common familial factors are genetic (major and/or polygenic), environmental, or some combination of both, and whether the familial expression depends on sex and/or age warrants further investigation using more complex models.