Restriction and Sequence Alterations Affect DNA Uptake Sequence-Dependent Transformation in Neisseria meningitidis

Transformation is a complex process that involves several interactions from the binding and uptake of naked DNA to homologous recombination. Some actions affect transformation favourably whereas others act to limit it. Here, meticulous manipulation of a single type of transforming DNA allowed for quantifying the impact of three different mediators of meningococcal transformation: NlaIV restriction, homologous recombination and the DNA Uptake Sequence (DUS). In the wildtype, an inverse relationship between the transformation frequency and the number of NlaIV restriction sites in DNA was observed when the transforming DNA harboured a heterologous region for selection (ermC) but not when the transforming DNA was homologous with only a single nucleotide heterology. The influence of homologous sequence in transforming DNA was further studied using plasmids with a small interruption or larger deletions in the recombinogenic region and these alterations were found to impair transformation frequency. In contrast, a particularly potent positive driver of DNA uptake in Neisseria sp. are short DUS in the transforming DNA. However, the molecular mechanism(s) responsible for DUS specificity remains unknown. Increasing the number of DUS in the transforming DNA was here shown to exert a positive effect on transformation. Furthermore, an influence of variable placement of DUS relative to the homologous region in the donor DNA was documented for the first time. No effect of altering the orientation of DUS was observed. These observations suggest that DUS is important at an early stage in the recognition of DNA, but does not exclude the existence of more than one level of DUS specificity in the sequence of events that constitute transformation. New knowledge on the positive and negative drivers of transformation may in a larger perspective illuminate both the mechanisms and the evolutionary role(s) of one of the most conserved mechanisms in nature: homologous recombination

GATA3 induces mitochondrial biogenesis in primary human CD4+ T cells during DNA damage

GATA3 has been considered to be primarily associated with CD4+ Th2 cell function. Using CD4+ effector memory that re-express CD45RA (EMRA) T cells the authors show that in response to DNA damage GATA3 can regulate increase of mitochondrial mass and biogenesis involving AMPK

Biopolymers as potential carrier for effervescent reaction based drug delivery system in gastrointestinal condition

Biopolymers are naturally occurring materials formed in nature during the life cycles of organisms. Biopolymers include the polysaccharides, carbohydrates and protein such as cellulose, starch, wool, silk, gelatine and collagen. In recent years, biopolymer-based hydrogels become important area of research in pharmaceutical aspects because of their promising properties in drug delivery system. These properties include low toxicity, biodegradability, stability and renewable nature. Numerous studies have been carried out in order to develop carrier from biopolymers with better controlled release properties. This is important to ensure precisely desired concentration of drug or essential nutrient absorption into the blood or tissue could be achieved. Among other different approaches, floating system is one of the most convenient, economical, and effective drug delivery system. Floating delivery system could potentially achieve longer retention time of carrier with capsulated bioactive drug or functional nutrients in the gastrointestinal tract. The floating behaviour of carrier could be induced by effervescent reactions. Effervescent reaction occurs between acidic gastric content and pore forming agent such as carbonates or bicarbonates salts incorporated into the carrier. This chapter discusses some of the use of biopolymers in drug delivery systems for effervescent reaction in gastrointestinal tract

Quantitative assessment of the robustness of next-generation sequencing of antibody variable gene repertoires from immunized mice

Background Next-generation sequencing (NGS) of antibody variable regions has emerged as a powerful tool in systems immunology by providing quantitative molecular information on polyclonal humoral immune responses. Reproducible and robust information on antibody repertoires is valuable for basic and applied immunology studies: thus, it is essential to establish the reliability of antibody NGS data. Results We isolated RNA from antibody-secreting cells (ASCs) from either 1 mouse or a pool of 9 immunized mice in order to simulate both normal and high diversity populations. Next, we prepared three technical replicates of antibody libraries by RT-PCR from each diversity scenario, which were sequenced using the Illumina MiSeq platform resulting in >106 250 bp paired-end reads per replicate. We then assessed the robustness of antibody repertoire data based on clonal identification defined by amino acid sequence of either full-length VDJ region or the complementarity determining region 3 (CDR3). Leveraging modeling approaches adapted from mathematical ecology, we found that in either diversity scenario both CDR3 and VDJ detection nears completeness indicating deep coverage of ASC repertoires. Additionally, we defined reliability thresholds for accurate quantification and ranking of CDR3s and VDJs. Importantly, we show that both factors-(i) replicate sequencing and (ii) sequencing depth-are crucial for robust CDR3 and VDJ detection and ranking. Conclusions In summary, we established widely applicable experimental and computational guidelines for robust antibody NGS and analysis, which will help advance systems immunology studies related to the quantitative profiling of antibody responses following infection and vaccination.ISSN:1471-217