MiST: A new approach to variant detection in deep sequencing datasets

MiST is a novel approach to variant calling from deep sequencing data, using the inverted mapping approach developed for Geoseq. Reads that can map to a targeted exonic region are identified using exact matches to tiles from the region. The reads are then aligned to the targets to discover variants. MiST carefully handles paralogous reads that map ambiguously to the genome and clonal reads arising from PCR bias, which are the two major sources of errors in variant calling. The reduced computational complexity of mapping selected reads to targeted regions of the genome improves speed, specificity and sensitivity of variant detection. Compared with variant calls from the GATK platform, MiST showed better concordance with SNPs from dbSNP and genotypes determined by an exonic-SNP array. Variant calls made only by MiST confirm at a high rate (>90%) by Sanger sequencing. Thus, MiST is a valuable alternative tool to analyse variants in deep sequencing data.     

A female with five chambers

Netherlands Heart Journal -     

C6-Ceramide Nanoliposomes Target the Warburg Effect in Chronic Lymphocytic Leukemia

Ceramide is a sphingolipid metabolite that induces cancer cell death. When C6-ceramide is encapsulated in a nanoliposome bilayer formulation, cell death is selectively induced in tumor models. However, the mechanism underlying this selectivity is unknown. As most tumors exhibit a preferential switch to glycolysis, as described in the “Warburg effect”, we hypothesize that ceramide nanoliposomes selectively target this glycolytic pathway in cancer. We utilize chronic lymphocytic leukemia (CLL) as a cancer model, which has an increased dependency on glycolysis. In CLL cells, we demonstrate that C6-ceramide nanoliposomes, but not control nanoliposomes, induce caspase 3/7-independent necrotic cell death. Nanoliposomal ceramide inhibits both the RNA and protein expression of GAPDH, an enzyme in the glycolytic pathway, which is overexpressed in CLL. To confirm that ceramide targets GAPDH, we demonstrate that downregulation of GAPDH potentiates the decrease in ATP after ceramide treatment and exogenous pyruvate treatment as well as GAPDH overexpression partially rescues ceramide-induced necrosis. Finally, an in vivo murine model of CLL shows that nanoliposomal C6-ceramide treatment elicits tumor regression, concomitant with GAPDH downregulation. We conclude that selective inhibition of the glycolytic pathway in CLL cells with nanoliposomal C6-ceramide could potentially be an effective therapy for leukemia by targeting the Warburg effect.     

The choreography of multidrug export

Multidrug transporters have a crucial role in causing the drug resistance that can arise in infectious micro-organisms and tumours. These integral membrane proteins mediate the export of a broad range of unrelated compounds from cells, including antibiotics and anticancer agents, thus reducing the concentration of these compounds to subtoxic levels in target cells. In spite of intensive research, it is not clear exactly how multidrug transporters work. The present review focuses on recent advancements in the biochemistry and structural biology of bacterial and human multidrug ABC (ATP-binding cassette) transporters. These advancements point to a common mechanism in which polyspecific drug-binding surfaces in the membrane domains are alternately exposed to the inside and outside surface of the membrane in response to the ATP-driven dimerization of nucleotide-binding domains and their dissociation following ATP hydrolysis.     

Biosorption of Cadmium by Live and Dead <Emphasis Type="Italic">Spirulina</Emphasis>: IR Spectroscopic,Kinetics, and SEM Studies

Cadmium is an important environmental pollutant and a potent toxicant to bacteria, algae, and fungi. Mechanisms of Cd⁺² toxicity and resistance are variable, depending on the organism. The present work reports the use of live and dead Spirulina sp. for sorption of Cd⁺². This investigation shows that this biomass takes up substantial amount of Cd⁺² ions. IR spectroscopic study, kinetics models, Langmuir & Freundlich adsorption isotherms, scanning electron microscopic analysis of Spirulina sp., and the Spirulina sp. treated with different metal ions have been employed to understand the sorption mechanism. Infrared spectra of live Spirulina treated with Cd⁺² ions for different lengths of time have been taken to understand the time dependency of metal interaction.     

Anthocyanin expression in marker free transgenic wheat and triticale embryos

Wheat and triticale plants were transformed by bombardment of isolated scutella with a genetic construct consisting of the two anthocyanin biosynthesis regulatory genes, C1 and Bperu, each under the control of the Ltp1 embryo-specific promoter. Transgenic plants were obtained in the absence of selective pressure and selectable marker gene at a transformation frequency of 0.93% and 1.55% in triticale and wheat, respectively. Initial screening of T₀ lines was performed by polymerase chain reaction (PCR), and further confirmation of PCR positives was done using real-time PCR and by phenotypic observation. In this study, quantitative real-time PCR (qRT-PCR) was developed to determine the transgene copy number in transgenic wheat and triticale. A conserved wheat housekeeping gene, puroindoline-b, was used as an internal control to calculate the transgene copy number in wheat and the SYBR green detection method with a standard curve, constructed on the basis of serially diluted plasmid, was used to calculate the transgene copy in triticale. Estimated transgene copies varied from 3 to 8 in wheat and 4 to 7 in triticale lines. The presence of anthocyanin regulatory genes, promoter, and termination sequences was detected in six wheat lines and four triticale lines. However, anthocyanin-pigmented embryos were only observed visually in mature T₁ seeds of two transgenic wheat lines and a single triticale line. Multisite insertion and reorganization of transgenes was likely the explanation for the failure of expression for the anthocyanin genes in the remaining wheat and triticale transgenic lines.     

Crystal structures of c-Src reveal features of its autoinhibitory mechanism.

Src family kinases are maintained in an assembled, inactive conformation by intramolecular interactions of their SH2 and SH3 domains. Full catalytic activity requires release of these restraints as well as phosphorylation of Tyr-416 in the activation loop. In previous structures of inactive Src kinases, Tyr-416 and flanking residues are disordered. We report here four additional c-Src structures in which this segment adopts an ordered but inhibitory conformation. The ordered activation loop forms an alpha helix that stabilizes the inactive conformation of the kinase domain, blocks the peptide substrate-binding site, and prevents Tyr-416 phosphorylation. Disassembly of the regulatory domains, induced by SH2 or SH3 ligands, or by dephosphorylation of Tyr-527, could lead to exposure and phosphorylation of Tyr-416.     

Wound healing from a cellular stress response perspective

This meeting review highlights areas of mutual interest to investigators in the cellular stress response field and to those carrying out wound-healing research. Inflammation, perhaps the major unifying theme of this meeting, is an essential component of the adult wound response and understanding the control of inflammation is a common interest shared with researchers of the cellular stress response. The particular interest of the authors of this review is in chronic non-healing wounds that frequently occur in patients with major illnesses such as diabetes and diseases of the blood vessels. This orientation has undoubtedly influenced the selection of topics. It is fair to say that the authors were often surprised and certainly impressed with the overlapping interests and possibilities for collaboration among investigators of these two research areas.