Immunogenic Properties of Lactobacillus plantarum Producing Surface-Displayed Mycobacterium tuberculosis Antigens

Tuberculosis (TB) remains among the most deadly diseases in the world. The only available vaccine against tuberculosis is the bacille Calmette-Guerin (BCG) vaccine, which does not ensure full protection in adults. There is a global urgency for the development of an effective vaccine for preventing disease transmission, and it requires novel approaches. We are exploring the use of lactic acid bacteria (LAB) as a vector for antigen delivery to mucosal sites. Here, we demonstrate the successful expression and surface display of a Mycobacterium tuberculosis fusion antigen (comprising Ag85B and ESAT-6, referred to as AgE6) on Lactobacillus plantarum. The AgE6 fusion antigen was targeted to the bacterial surface using two different anchors, a lipoprotein anchor directing the protein to the cell membrane and a covalent cell wall anchor. AgE6-producing L. plantarum strains using each of the two anchors induced antigen-specific proliferative responses in lymphocytes purified from TB-positive donors. Similarly, both strains induced immune responses in mice after nasal or oral immunization. The impact of the anchoring strategies was reflected in dissimilarities in the immune responses generated by the two L. plantarum strains in vivo. The present study comprises an initial step toward the development of L. plantarum as a vector for M. tuberculosis antigen delivery. IMPORTANCE This work presents the development of Lactobacillus plantarum as a candidate mucosal vaccine against tuberculosis. Tuberculosis remains one of the top infectious diseases worldwide, and the only available vaccine, bacille Calmette-Guerin (BCG), fails to protect adults and adolescents. Direct antigen delivery to mucosal sites is a promising strategy in tuberculosis vaccine development, and lactic acid bacteria potentially provide easy, safe, and low-cost delivery vehicles for mucosal immunization. We have engineered L. plantarum strains to produce a Mycobacterium tuberculosis fusion antigen and to anchor this antigen to the bacterial cell wall or to the cell membrane. The recombinant strains elicited proliferative antigenspecific T-cell responses in white blood cells from tuberculosis-positive humans and induced specific immune responses after nasal and oral administrations in mice

Early detection of breast cancer based on gene-expression patterns in peripheral blood cells

INTRODUCTION: Existing methods to detect breast cancer in asymptomatic patients have limitations, and there is a need to develop more accurate and convenient methods. In this study, we investigated whether early detection of breast cancer is possible by analyzing gene-expression patterns in peripheral blood cells. METHODS: Using macroarrays and nearest-shrunken-centroid method, we analyzed the expression pattern of 1,368 genes in peripheral blood cells of 24 women with breast cancer and 32 women with no signs of this disease. The results were validated using a standard leave-one-out cross-validation approach. RESULTS: We identified a set of 37 genes that correctly predicted the diagnostic class in at least 82% of the samples. The majority of these genes had a decreased expression in samples from breast cancer patients, and predominantly encoded proteins implicated in ribosome production and translation control. In contrast, the expression of some defense-related genes was increased in samples from breast cancer patients. CONCLUSION: The results show that a blood-based gene-expression test can be developed to detect breast cancer early in asymptomatic patients. Additional studies with a large sample size, from women both with and without the disease, are warranted to confirm or refute this finding

Differential transcript isoform usage pre- and post-zygotic genome activation in zebrafish

BACKGROUND: Zebrafish embryos are transcriptionally silent until activation of the zygotic genome during the 10(th) cell cycle. Onset of transcription is followed by cellular and morphological changes involving cell speciation and gastrulation. Previous genome-wide surveys of transcriptional changes only assessed gene expression levels; however, recent studies have shown the necessity to map isoform-specific transcriptional changes. Here, we perform isoform discovery and quantification on transcriptome sequences from before and after zebrafish zygotic genome activation (ZGA). RESULTS: We identify novel isoforms and isoform switches during ZGA for genes related to cell adhesion, pluripotency and DNA methylation. Isoform switching events include alternative splicing and changes in transcriptional start sites and in 3’ untranslated regions. New isoforms are identified even for well-characterized genes such as pou5f1, sall4 and dnmt1. Genes involved in cell-cell interactions such as f11r and magi1 display isoform switches with alterations of coding sequences. We also detect over 1000 transcripts that acquire a longer 3’ terminal exon when transcribed by the zygote compared to their maternal transcript counterparts. ChIP-sequencing data mapped onto skipped exon events reveal a correlation between histone H3K36 trimethylation peaks and skipped exons, suggesting epigenetic marks being part of alternative splicing regulation. CONCLUSIONS: The novel isoforms and isoform switches reported here include regulators of transcriptional, cellular and morphological changes taking place around ZGA. Our data display an array of isoform-related functional changes and represent a valuable resource complementary to existing early embryo transcriptomes

Normalization of RNA-Sequencing Data from Samples with Varying mRNA Levels

<div><p>Methods for normalization of RNA-sequencing gene expression data commonly assume equal total expression between compared samples. In contrast, scenarios of global gene expression shifts are many and increasing. Here we compare the performance of three normalization methods when polyA⁺ RNA content fluctuates significantly during zebrafish early developmental stages. As a benchmark we have used reverse transcription-quantitative PCR. The results show that reads per kilobase per million (RPKM) and trimmed mean of M-values (TMM) normalization systematically leads to biased gene expression estimates. Biological scaling normalization (BSN), designed to handle differences in total expression, showed improved accuracy compared to the two other methods in estimating transcript level dynamics. The results have implications for past and future studies using RNA-sequencing on samples with different levels of total or polyA⁺ RNA.</p></div

Comparison of normalization methods.

<p>Log2-tranformed fold-changes comparing RT-qPCR and RNA-seq data normalized using RPM, TMM and BSN for transcripts increasing pre-ZGA (a), decreasing pre-ZGA (b), decreasing post-ZGA (c) and increasing post-ZGA (d).</p

Distribution of gene expression values.

<p>Box plot of distribution of transcript counts or values before (not normalized) and after normalization (BSN, RPM and TMM).</p

cDNA template and primer comparison.

<p>Comparison of RT-qPCR results based on polyA⁺ and total RNA and oligo(dT) and random primers for <i>stat3</i>. The increase pre-ZGA is only detected in the polyA⁺ RNA-based cDNA libraries. PolyA  =  polyA⁺ RNA, Total  =  total RNA, OdT  =  oligo(dT) primers, RP  =  random primers.</p

Relative polyA⁺ RNA amounts.

<p>Measurements of polyA⁺ RNA determined by a standard laboratory method (full line) and using trimmed mean of M-values (TMM) (stippled line) display an almost identical pattern during early embryogenesis with an early increase and subsequent decrease. The levels are relative to the 1-cell stage.</p

Restriction spectrum imaging of white matter and its relation to neurological disability in multiple sclerosis.

BACKGROUND:Restriction spectrum imaging (RSI) is a recently introduced magnetic resonance imaging diffusion technique. The utility of RSI in multiple sclerosis (MS) is unknown. OBJECTIVE:To investigate the association between RSI-derived parameters and neurological disability in MS. METHODS:Seventy-seven relapsing-remitting MS patients were scanned with RSI on a 3-T scanner. RSI-derived parameters: fast and slow apparent diffusion coefficient (sADC), fractional anisotropy, restricted fractional anisotropy, neurite density (ND), cellularity, extracellular water fraction, and free water fraction, were obtained in white matter lesions (WML) and normal appearing white matter (NAWM). Patients were divided into three groups according to their expanded disability status scale (EDSS): with minimal, low, and substantial disability (&lt;2.5, 2.5-3, and &gt;3, respectively). Group comparisons and correlation analyses were performed. RESULTS:All tested RSI-derived parameters differed between WML and NAWM ( p &lt; 0.001 for all pairwise comparisons). The sADC in WML showed largest difference across disability subgroups (analysis of variance (ANOVA): F = 5.1, η2 = 0.12, p = 0.008). ND in NAWM showed strongest correlation with disability (ϱ = -0.39, p &lt; 0.001). CONCLUSION:The strongest correlation with EDSS of ND obtained in NAWM indicates that processes outside lesions are important for disability in MS. Our study suggests that RSI-derived parameters may help understand the "clinico-radiological paradox" and improve disease monitoring in MS