Improving the limit of detection for Sanger sequencing: A comparison of methodologies for KRAS variant detection

Fluorescent dye terminator Sanger sequencing (FTSS), with detection by automated capillary electrophoresis (CE), has long been regarded as the gold standard for variant detection. However, software analysis and base-calling algorithms used to detect mutations were largely optimized for resequencing applications in which different alleles were expected as heterozygous mixtures of 50%. Increasingly, the requirements for variant detection are an analytic sensitivity for minor alleles of <20%, in particular, when assessing the mutational status of heterogeneous tumor samples. Here, we describe a simple modification to the FTSS workflow that improves the limit of detection of cell-line gDNA mixtures from 50%-20% to 5% for G>A transitions and from 50%-5% to 5% for G>C and G>T transversions. In addition, we use two different sample types to compare the limit of detection of sequence variants in codons 12 and 13 of the KRAS gene between Sanger sequencing and other methodologies including shifted termination assay (STA) detection, single-base extension (SBE), pyrosequencing (PS), high- resolution melt (HRM), and real-time PCR (qPCR).     

Development of small-molecule probes that selectively kill cells induced to express mutant RAS

Synthetic lethal screening is a chemical biology approach to identify small molecules that selectively kill oncogene-expressing cell lines with the goal of identifying pathways that provide specific targets against cancer cells. We performed a high-throughput screen of 303,282 compounds from the National Institutes of Health-Molecular Libraries Small Molecule Repository (NIH-MLSMR) against immortalized BJ fibroblasts expressing HRAS(G12V) followed by a counterscreen of lethal compounds in a series of isogenic cells lacking the HRAS(G12V) oncogene. This effort led to the identification of two novel molecular probes (PubChem CID 3689413, ML162 and CID 49766530, ML210) with nanomolar potencies and 4-23-fold selectivities, which can potentially be used for identifying oncogene-specific pathways and targets in cancer cells.     

Interaction of the Bacillus subtilis chaperone CsaA with the secretory protein YvaY

Bacillus subtilis CsaA was previously characterised as a molecular chaperone with export-related activities. In order to elucidate the functionality of CsaA further, interaction with its postulated substrate YvaY was investigated. Similar binding to carrier immobilised mature and preYvaY revealed that the interaction was not mediated via the signal peptide of preYvaY. Higher affinity to denatured peptides compared to native peptides indicated preferred binding to unfolded proteins. To characterise affinity of CsaA more detailed, binding to preYvaY derived peptides was analysed. CsaA showed affinity to multiple peptides in the scan, mainly correlated to a positive net charge. Affinity of export-specific Escherichia coli chaperone SecB to the carrier immobilised peptides indicated partially overlapping binding characteristics of SecB and CsaA.     

Influence of zinc on the calcium carbonate biomineralization of Halomonas halophila

Background

The salt tolerance of halophilic bacteria make them promising candidates for technical applications, like isolation of salt tolerant enzymes or remediation of contaminated saline soils and waters. Furthermore, some halophilic bacteria synthesize inorganic solids resulting in organic–inorganic hybrids. This process is known as biomineralization, which is induced and/or controlled by the organism. The adaption of the soft and eco-friendly reaction conditions of this formation process to technical syntheses of inorganic nano materials is desirable. In addition, environmental contaminations can be entrapped in biomineralization products which facilitate the subsequent removal from waste waters. The moderately halophilic bacteria Halomonas halophila mineralize calcium carbonate in the calcite polymorph. The biomineralization process was investigated in the presence of zinc ions as a toxic model contaminant. In particular, the time course of the mineralization process and the influence of zinc on the mineralized inorganic materials have been focused in this study.

Results

H. halophila can adapt to zinc contaminated medium, maintaining the ability for biomineralization of calcium carbonate. Adapted cultures show only a low influence of zinc on the growth rate. In the time course of cultivation, zinc ions accumulated on the bacterial surface while the medium depleted in the zinc contamination. Intracellular zinc concentrations were below the detection limit, suggesting that zinc was mainly bound extracellular. Zinc ions influence the biomineralization process. In the presence of zinc, the polymorphs monohydrocalcite and vaterite were mineralized, instead of calcite which is synthesized in zinc-free medium.

Conclusions

We have demonstrated that the bacterial mineralization process can be influenced by zinc ions resulting in the modification of the synthesized calcium carbonate polymorph. In addition, the shape of the mineralized inorganic material is chancing through the presence of zinc ions. Furthermore, the moderately halophilic bacterium H. halophila can be applied for the decontamination of zinc from aqueous solutions.     

Design and characterization of a protein superagonist of IL‐15 fused with IL‐15Rα and a high‐affinity T cell receptor

To avoid high systemic doses, strategies involving antigen‐specific delivery of cytokine via linked antibodies or antibody fragments have been used. Targeting cancer‐associated peptides presented by major histocompatibility complex (MHC) molecules (pepMHC) increases the number of potential target antigens and takes advantage of cross‐presentation on tumor stroma and in draining lymph nodes. Here, we use a soluble, high‐affinity single‐chain T cell receptor Vα‐Vβ (scTv), to deliver cytokines to intracellular tumor‐associated antigens presented as pepMHC. As typical wild‐type T cell receptors (TCRs) exhibit low affinity (K_d = 1–100 μM or more), we used an engineered TCR, m33, that binds its antigenic peptide SIYRYYGL (SIY) bound to the murine class I major histocompatability complex protein H2‐K^b (SIY/K^b) with nanomolar affinity (K_d = 30 nM). We generated constructs consisting of m33 scTv fused to murine interleukin 2 (IL‐2), interleukin 15 (IL‐15), or IL‐15/IL‐15Rα (IL‐15 linked to IL‐15Rα sushi domain, called “superfusion”). The fusions were purified with good yields and bound specifically to SIY/K^b with high affinity. Proper cytokine folding and binding were confirmed, and the fusions were capable of stimulating proliferation of cytokine‐dependent cells, both when added directly and when presented in trans, bound to cells with the target pepMHC. The m33 superfusion was particularly potent and stable and represents a promising design for targeted antitumor immunomodulation. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Long chain ceramides and very long chain ceramides have opposite effects on human breast and colon cancer cell growth

Ceramides are known to be key players in intracellular signaling and are involved in apoptosis, cell senescence, proliferation, cell growth and differentiation. They are synthesized by ceramide synthases (CerS). So far, six different mammalian CerS (CerS1-6) have been described. Recently, we demonstrated that human breast cancer tissue displays increased activity of CerS2, 4, and 6, together with enhanced generation of their products, ceramides C(16:0), C(24:0), and C(24:1). Moreover, these increases were significantly associated with tumor dignity. To clarify the impact of this observation, we manipulated cellular ceramide levels by overexpressing ceramide synthases 2, 4 or 6 in MCF-7 (breast cancer) and HCT-116 (colon cancer) cells, respectively. Overexpression of ceramide synthases 4 and 6 elevated generation of short chain ceramides C(16:0), C(18:0) and C(20:0), while overexpression of ceramide synthase 2 had no effect on ceramide production in vivo, presumably due to limited substrate availability, because external addition of very long chain acyl-CoAs resulted in a significant upregulation of very long chain ceramides. We also demonstrated that upregulation of CerS4 and 6 led to the inhibition of cell proliferation and induction of apoptosis, whereas upregulation of CerS2 increased cell proliferation. On the basis of our data, we propose that a disequilibrium between ceramides of various chain length is crucial for cancer progression, while normal cells require an equilibrium between very long and long chain ceramides for normal physiology.     

The amyloid precursor protein forms plasmalemmal clusters via its pathogenic amyloid-β domain

The amyloid precursor protein (APP) is a large, ubiquitous integral membrane protein with a small amyloid-β (Aβ) domain. In the human brain, endosomal processing of APP produces neurotoxic Aβ-peptides, which are involved in Alzheimer's disease. Here, we show that the Aβ sequence exerts a physiological function when still present in the unprocessed APP molecule. From the extracellular site, Aβ concentrates APP molecules into plasmalemmal membrane protein clusters. Moreover, Aβ stabilization of clusters is a prerequisite for their targeting to endocytic clathrin structures. Therefore, we conclude that the Aβ domain directly mediates a central step in APP trafficking, driving its own conversion into neurotoxic peptides.     

The evolution of patch selection in stochastic environments

A null model for habitat patch selection in spatially heterogeneous environments is the ideal free distribution (IFD), which assumes individuals have complete knowledge about the environment and can freely disperse. Under equilibrium conditions, the IFD predicts that local population growth rates are zero in all occupied patches, sink patches are unoccupied, and the fraction of the population selecting a patch is proportional to the patch's carrying capacity. Individuals, however, often experience stochastic fluctuations in environmental conditions and cannot respond to these fluctuations instantaneously. An evolutionary stability analysis for fixed patch-selection strategies reveals that environmental uncertainty disrupts the classical IFD predictions: individuals playing the evolutionarily stable strategy may occupy sink patches, local growth rates are negative and typically unequal in all patches, and individuals prefer higher-quality patches less than predicted by their carrying capacities. Spatial correlations in environmental fluctuations can enhance or marginalize these trends. The analysis predicts that continually increasing environmental variation first selects for range expansion, then selects for persisting coupled sink populations, and ultimately leads to regional extinction. In contrast, continually increasing habitat degradation first selects for range contraction and may select for persisting coupled sink populations before regional extinction. These results highlight the combined roles of spatial and temporal heterogeneity on the evolution of habitat selection.