Turbidity and RI Dependency of a Polymer Optical Fiber-Based Chromatic Sensor.

An in-line and real time chromatic sensor for liquids based on plastic optical fiber was developed. It uses an air gap, fiber to fiber, transmission principle. Its dependency to turbidity and refractive index is studied and characterized. This information will provide the necessary knowledge for future implementation of more complex auto-compensations routines. Due to the predictable behavior of the sensor to variations of turbidity and refractive index, it is shown that a posterior compensation could be applied for the discrimination of color. The real-time color sensor can be used in different turbid liquids and contain different refractive indices.

Signal Transduction Reaction Monitoring Deciphers Site-Specific PI3K-mTOR/MAPK Pathway Dynamics in Oncogene-Induced Senescence.

We report a straightforward strategy to comprehensively monitor signal transduction pathway dynamics in mammalian systems. Combining targeted quantitative proteomics with highly selective phosphopeptide enrichment, we monitor, with great sensitivity, phosphorylation dynamics of the PI3K-mTOR and MAPK signaling networks. Our approach consists of a single enrichment step followed by a single targeted proteomics experiment, circumventing the need for labeling and immune purification while enabling analysis of selected phosphorylation nodes throughout signaling pathways. The need for such a comprehensive pathway analysis is illustrated by highlighting previously uncharacterized phosphorylation changes in oncogene-induced senescence, associated with diverse biological phenotypes and pharmacological intervention of the PI3K-mTOR pathway.

Btk SH2-kinase interface is critical for allosteric kinase activation and its targeting inhibits B-cell neoplasms.

Bruton's tyrosine kinase (Btk) is critical for B-cell maturation and activation. Btk loss-of-function mutations cause human X-linked agammaglobulinemia (XLA). In contrast, Btk signaling sustains growth of several B-cell neoplasms which may be treated with tyrosine kinase inhibitors (TKIs). Here, we uncovered the structural mechanism by which certain XLA mutations in the SH2 domain strongly perturb Btk activation. Using a combination of molecular dynamics (MD) simulations and small-angle X-ray scattering (SAXS), we discovered an allosteric interface between the SH2 and kinase domain required for Btk activation and to which multiple XLA mutations map. As allosteric interactions provide unique targeting opportunities, we developed an engineered repebody protein binding to the SH2 domain and able to disrupt the SH2-kinase interaction. The repebody prevents activation of wild-type and TKI-resistant Btk, inhibiting Btk-dependent signaling and proliferation of malignant B-cells. Therefore, the SH2-kinase interface is critical for Btk activation and a targetable site for allosteric inhibition.

Molecular Characterization of Trypanosoma evansi Mevalonate Kinase (TeMVK).

The mevalonate pathway is an essential part of isoprenoid biosynthesis leading to production of a diverse class of >30,000 biomolecules including cholesterol, heme, and all steroid hormones. In trypanosomatids, the mevalonate pathway also generates dolichols, which play an essential role in construction of glycosylphosphatidylinositol (GPI) molecules that anchor variable surface proteins (VSGs) to the plasma membrane. Isoprenoid biosynthesis involves one of the most highly regulated enzymes in nature, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), which catalyzes the conversion of HMG-CoA to mevalonic acid. The enzyme mevalonate kinase (MVK) subsequently converts mevalonic acid to 5-phosphomevalonic acid. Trypanosoma evansi is a flagellate protozoan parasite that causes the disease "Surra" in domesticated large mammals, with great economic impact. T. evansi has only a trypomastigote bloodstream form and requires constant modification of the variant surface glycoprotein (VSG) coat for protection against the host immune system. We identified MVK of T. evansi (termed TeMVK) and performed a preliminary characterization at molecular, biochemical, and cellular levels. TeMVK from parasite extract displayed molecular weight ~36 kDa, colocalized with aldolase (a glycosomal marker enzyme) in glycosomes, and is structurally similar to Leishmania major MVK. Interestingly, the active form of TeMVK is the tetrameric oligomer form, in contrast to other MVKs in which the dimeric form is active. Despite lacking organized mitochondria, T. evansi synthesizes both HMGCR transcripts and protein. Both MVK and HMGCR are expressed in T. evansi during the course of infection in animals, and therefore are potential targets for therapeutic drug design.