MassCode Liquid Arrays as a Tool for Multiplexed High-Throughput Genetic Profiling

Multiplexed detection assays that analyze a modest number of nucleic acid targets over large sample sets are emerging as the preferred testing approach in such applications as routine pathogen typing, outbreak monitoring, and diagnostics. However, very few DNA testing platforms have proven to offer a solution for mid-plexed analysis that is high-throughput, sensitive, and with a low cost per test. In this work, an enhanced genotyping method based on MassCode technology was devised and integrated as part of a high-throughput mid-plexing analytical system that facilitates robust qualitative differential detection of DNA targets. Samples are first analyzed using MassCode PCR (MC-PCR) performed with an array of primer sets encoded with unique mass tags. Lambda exonuclease and an array of MassCode probes are then contacted with MC-PCR products for further interrogation and target sequences are specifically identified. Primer and probe hybridizations occur in homogeneous solution, a clear advantage over micro- or nanoparticle suspension arrays. The two cognate tags coupled to resultant MassCode hybrids are detected in an automated process using a benchtop single quadrupole mass spectrometer. The prospective value of using MassCode probe arrays for multiplexed bioanalysis was demonstrated after developing a 14plex proof of concept assay designed to subtype a select panel of Salmonella enterica serogroups and serovars. This MassCode system is very flexible and test panels can be customized to include more, less, or different markers

AKT methylation by SETDB1 promotes AKT kinase activity and oncogenic functions

Aberrant activation of Akt disturbs proliferation, survival and metabolic homeostasis of various human cancers. Thus, it is critical to understand upstream signaling pathways governing Akt activation. Here, we report that Akt undergoes SETDB1-mediated lysine-methylation to promote its activation, which is antagonized by the Jumonji-family demethylase, KDM4B. Notably, compared with wild-type mice, mice harboring non-methylated mutant Akt1 not only exhibited reduced body size, but also were less prone to carcinogen-induced skin tumors in part due to reduced Akt activation. Mechanistically, Phosphatidylinositol (3,4,5)-trisphosphate (PIP3) interaction with Akt facilitates its interaction with SETDB1 for subsequent Akt methylation, which in turn sustains Akt phosphorylation. Pathologically, genetic alterations including SETDB1 amplification aberrantly promote Akt methylation to facilitate its activation and oncogenic functions. Thus, Akt methylation is an important step synergizing with PI3K signaling to control Akt activation, suggesting that targeting the SETDB1 signaling could be a potential therapeutic strategy for combatting hyperactive Akt-driven cancers.Accepted Manuscrip

Nanosized Materials in Amperometric Sensors

The use of nanosized materials nowadays constitutes one of the most diffused approach to modify electrode surface when aiming at obtaining efficient amperometric sensors; quite spontaneously, this trend has also involved the field of environmental monitoring. The chapter aims at discussing the properties of nanosized materials, the most widespread strategies for their deposition on the electrode surface as well as the main advantages and limitations of their use in electroanalysis. Metal and carbon nanostructures, and the relevant composite materials, are particularly discussed