Sensitive, multiplex and direct quantification of RNA sequences using a modified RASL assay.

A sensitive and highly multiplex method to directly measure RNA sequence abundance without requiring reverse transcription would be of value for a number of biomedical applications, including high throughput small molecule screening, pathogen transcript detection and quantification of short/degraded RNAs. R NA A: nnealing, S: election and L: igation (RASL) assays, which are based on RNA template-dependent oligonucleotide probe ligation, have been developed to meet this need, but technical limitations have impeded their adoption. Whereas DNA ligase-based RASL assays suffer from extremely low and sequence-dependent ligation efficiencies that compromise assay robustness, Rnl2 can join a fully DNA donor probe to a 3'-diribonucleotide-terminated acceptor probe with high efficiency on an RNA template strand. Rnl2-based RASL exhibits sub-femtomolar transcript detection sensitivity, and permits the rational tuning of probe signals for optimal analysis by massively parallel DNA sequencing (RASL-seq). A streamlined Rnl2-based RASL-seq protocol was assessed in a small molecule screen using 77 probe sets designed to monitor complex human B cell phenotypes during antibody class switch recombination. Our data demonstrate the robustness, cost-efficiency and broad applicability of Rnl2-based RASL assays.

Corrosion behaviour of four handguns in aqueous environments: corrosion product characterization and effects on estimating the time since deposition.

When a firearm has been disposed of in a body of water and becomes corroded, its appearance is altered and determining a time-since-immersion may be of import to the investigation. Therefore, in this study, the corrosion and mass loss of four handgun slides over a period of 180days were examined. Solid-state characterization of the metals and their corrosion products via SEM/EDX and powder X-ray Diffraction (pXRD) was performed. The pXRDs were analyzed against the NIST Powder Diffraction Database to determine the crystalline phases. Filings from the SS416 standard, Llama and Ruger handgun slide predominantly consisted of iron alloys. After 180-days in solution, pXRD indicated that the adherent corrosion products consisted of 1) γ-FeOOH and 2) iron oxide (Fe3O4 or Fe2O3). Additionally, pXRD analysis indicated that the adherent corrosion products of the SS416 standard also consisted of CrO3. Metal filings from the Raven and Jennings handgun slides were a mixture of iron-nickel-zinc and EDX and pXRD analyses of the corrosion products, when submersed in deionized water, indicated that the products consisted of: 1) γ-FeOOH, 2) iron oxide (Fe3O4 or Fe2O3), and 3) ZnFe2O4 or ZnO; where the Jennings adherent rust contained ZnFe2O4 and the Raven adherent rust contained ZnO. Further, pXRD of the corrosion products from these alloys, when submersed in 25 PSU (Practical Salinity Unit) solution, indicated that the products consisted of: 1) ZnO, 2) Zn(OH)2, 3) α-Ni(OH)2, and 4) NaCl. The data thus indicated that both metal composition and the presence of chloride ions had significant impacts on rates and products of corrosion and suggest that the presence of Cl(-) changes not only the rate of corrosion, but also the corroding species itself. While mechanisms and rates of the chloride driven corrosion processes offer explanations as to the different oxides and hydroxides observed between immersion conditions, they do not offer an explanation for the differences observed between handguns. Therefore, utilizing a general approach where surface area coverage of corrosion products is the sole consideration is not sufficient to determine time-since-immersion. Attempts to determine a time-since-immersion would require a priori knowledge of the mechanism of corrosion for a given metal mixture within a specified environment. The results described herein give indications as to the possible corrosion mechanism driving the process in high and low Cl(-) environments and show the necessity of including the metal composition, rust composition and ion concentration in any models that attempt to elucidate the time-since-immersion of handguns for forensic applications.

Interstrain differences of in vitro metabolic stability and impact on early drug discovery.

With the extensive use of different strains of mice and rats in in vivo efficacy models, lack of relevant metabolic clearance data among strains has been a concern. Metabolic clearance is an important parameter impacting drug discovery, and it is often used as a compound selection filter. Metabolically stable compounds are often preferred, and will have a better chance to achieve the desired exposure in vivo. The present study examined strain differences in mouse and rat, using 96 compounds which spanned a wide range of intrinsic clearances. The in vitro clearances were determined using liver microsomes from commonly used strains of mouse (BALB/c, C57BL/6J, and CD-1) and of rat (Sprague-Dawley, Fischer, and Wistar Han). There were few discrepancies in the interpretation of the in vitro intrinsic clearance results within species for the 96 compounds tested in mouse and rat liver microsomes. This data gives us confidence that the phase I hepatic clearance can be determined using only one strain of mouse or rat liver microsomes.