Hemizygous Le-Cre Transgenic Mice Have Severe Eye Abnormalities on Some Genetic Backgrounds in the Absence of LoxP Sites

Eye phenotypes were investigated in Le-Cre(Tg/-); Pax6(fl/+) mice, which were expected to show tissue-specific reduction of Pax6 in surface ectoderm derivatives. To provide a better comparison with our previous studies of Pax6(+/-) eye phenotypes, hemizygous Le-Cre(Tg/-) and heterozygous Pax6(fl/+)mice were crossed onto the CBA/Ca genetic background. After the Le-Cre transgene had been backcrossed to CBA/Ca for seven generations, significant eye abnormalities occurred in some hemizygous Le-Cre(Tg/-); Pax6(+/+) controls (without a floxed Pax6(fl) allele) as well as experimental Le-Cre(Tg/-); Pax6(fl/+) mice. However, no abnormalities were seen in Le-Cre(-/-); Pax6(fl/+) or Le-Cre(-/-); Pax6(+/+) controls (without the Le-Cre transgene). The severity and frequency of the eye abnormalities in Le-Cre(Tg/-); Pax6(+/+) control mice diminished after backcrossing Le-Cre(Tg/-) mice to the original FVB/N strain for two generations, showing that the effect was reversible. This genetic background effect suggests that the eye abnormalities are a consequence of an interaction between the Le-Cre transgene and alleles of unknown modifier genes present in certain genetic backgrounds. The abnormalities were also ameliorated by introducing additional Pax6 gene copies on a CBA/Ca background, suggesting involvement of Pax6 depletion in Le-Cre(Tg/-); Pax6(+/+) mice rather than direct action of Cre recombinase on cryptic pseudo-loxP sites. One possibility is that expression of Cre recombinase from the Pax6-Le regulatory sequences in the Le-Cre transgene depletes cofactors required for endogenous Pax6 gene expression. Our observation that eye abnormalities can occur in hemizygous Le-Cre(Tg/-); Pax6(+/+) mice, in the absence of a floxed allele, demonstrates the importance of including all the relevant genetic controls in Cre-loxP experiments

HBx protein modulates PI3K/Akt pathway to overcome genotoxic stress-induced destabilization of cyclin D1 and arrest of cell cycle

37-44Growth arrest represents an innate barrier to carcinogenesis. DNA damage and replicational stress are known to induce growth arrest and apoptotic death to avert genomic instability and consequently carcinogenesis. In this study, working on the genotoxic stress induced by hydroxyurea and methylmethanesulfone, we observed a growth arrest at G1/S-phase that was mediated by destabilization of cyclin D1. The growth arrest was independent of the stability of cdc25A and preceded transcriptional up-regulation of p21waf1. Cyclin D1 destabilization involved its phosphorylation by GSK-3β at threonine-286, since overexpression of the kinase-dead mutant of GSK-3β or cyclin D1T286A mutant conferred stability to cyclin D1. Further, overexpression of cyclin D1T286A also helped in bypassing G1/S phase growth arrest. We also observed a rapid inactivation of Akt/PKB kinase in the presence of hydroxyurea. Enforced expression of the constitutively active Akt or viral oncoprotein HBx (Hepatitis B virus X protein) was sufficient to overcome growth arrest, independent of ATR signaling and stabilized cyclin D1. Thus, the present work not only establishes cyclin D1 to be a novel mediator of genotoxic stress signaling, but also explains how a deregulated mitogenic signaling or a viral oncoprotein can help bypass growth arres

Thin layer chromatographic study of Indian bauxites

65-73Thin layer chromatography (TLC) has been used for the detection and separation of Al³⁺, Fe²⁺, Si⁴⁺ and Ti⁴⁺ in bauxite. Thirty bauxite samples of different geological origin have been studied to identify the optimized chromatographic systems capable of selective separations of the constituents. Binary and ternary separations of Al³⁺, Fe²⁺, Ti⁴⁺ and Si⁴⁺ in bauxite were realized with chromatographic systems comprising various combinations of silica gel G, silica gel H and microcrystalline cellulose coated plates and aqueous mobile phases consisting alkali salt solutions, formic acid and hydrochloric acid etc. The chromatographic system consisting of silica gel H and mixture of 10% aqueous solutions of sodium chloride and formic acid in 8:2 (v/v) ratio was found to be the ideal combination for the analogous separation of Al³⁺, Fe²⁺ and Ti⁴⁺ in bauxites. The efficacy of optimized chromatographic system has been investigated by monitoring reproducibility of RF values and spot compactness with respect to change in bauxite geology, sample concentration and change of mobile phase composition. Chromatograms of the cations were quantitatively evaluated by scanning densitometry in comparison with wet analysis method

Sierra: discovery of differential transcript usage from polyA-captured single-cell RNA-seq data

High-throughput single-cell RNA-seq (scRNA-seq) is a powerful tool for studying gene expression in single cells. Most current scRNA-seq bioinformatics tools focus on analysing overall expression levels, largely ignoring alternative mRNA isoform expression. We present a computational pipeline, Sierra, that readily detects differential transcript usage from data generated by commonly used polyA-captured scRNA-seq technology. We validate Sierra by comparing cardiac scRNA-seq cell types to bulk RNA-seq of matched populations, finding significant overlap in differential transcripts. Sierra detects differential transcript usage across human peripheral blood mononuclear cells and the Tabula Muris, and 3 'UTR shortening in cardiac fibroblasts. Sierra is available at https://github.com/VCCRI/Sierra

A Comparative Analysis Study of Partial Replacement of Rubber Tyre pieces and Broken Bricks to the Aggregate

Concrete is one of the most popular building material, the construction industry is always increase its use and applications that’s why it is necessary to find alternative material to reduce the cost of concrete materials. On the other hand, the natural coarse aggregate are extracted from quarries and the result this quarries will be soon depleted in few decades if the rate of their uses continuous at this speed. As a result there was a need of permanent solution of this problem. The disposal of tire elastic or broken blocks for the creation of cement has been thought as an elective transfer of such waste to secure the earth. In this research paper tyre rubber and broken bricks were used as a coarse aggregate with 30% replacement of conventional aggregate. The compressive strength of concrete is observed at 7, 14 and 28 days and comparative analysis between waste tyre rubber and waste broken brick were analyzed. After 28 days comparison which waste material gives high strength is to preferred as best replacement of waste material with the aggregate

Single-cell expression profiling reveals dynamic flux of cardiac stromal, vascular and immune cells in health and injury

Besides cardiomyocytes (CM), the heart contains numerous interstitial cell types which play key roles in heart repair, regeneration and disease, including fibroblast, vascular and immune cells. However, a comprehensive understanding of this interactive cell community is lacking. We performed single-cell RNA-sequencing of the total non-CM fraction and enriched (Pdgfra-GFP+) fibroblast lineage cells from murine hearts at days 3 and 7 post-sham or myocardial infarction (MI) surgery. Clustering of >30,000 single cells identified >30 populations representing nine cell lineages, including a previously undescribed fibroblast lineage trajectory present in both sham and MI hearts leading to a uniquely activated cell state defined in part by a strong anti-WNT transcriptome signature. We also uncovered novel myofibroblast subtypes expressing either pro- fibrotic or anti-fibrotic signatures. Our data highlight non-linear dynamics in myeloid and fibroblast lineages after cardiac injury, and provide an entry point for deeper analysis of cardiac homeostasis, inflammation, fibrosis, repair and regeneration