Project description:Hydrogen/deuterium exchange-mass spectrometry (HDX-MS) has emerged as a powerful tool to probe protein dynamics. As a bottom-up technique, HDX-MS provides information at peptide-level resolution, allowing structural localisation of dynamic changes. Consequently, HDX-MS data quality is largely determined by the number of peptides that are identified and monitored after deuteration. Integration of ion mobility (IM) into HDX-MS workflows has been shown to increase data quality by providing an orthogonal mode of peptide ion separation in the gas-phase. This is of critical importance for challenging targets such as integral membrane proteins (IMPs), which often suffer from low sequence coverage and/or redundancy in HDX-MS analyses. The increasing complexity of samples being investigated by HDX-MS, such as membrane mimetic reconstituted and in vivo IMPs, has generated need for instrumentation with greater resolving power. Recently, Giles et al. developed cyclic ion mobility (cIM), an IM device with racetrack geometry that enables scalable, multi-pass IM separations. Using 1-pass and multi-pass cIM routines, we use the recently commercialised SELECT SERIES™ Cyclic™ IM spectrometer for HDX-MS analyses of 4 detergent solubilised IMP samples and report its enhanced performance. Furthermore, we develop a novel processing strategy capable of better handling multi-pass cIM data. Interestingly, use of 1-pass and multi-pass cIM routines produced unique peptide populations, with their combined peptide output being 31 to 222% higher than previous generation SYNAPT G2-Si instrumentation. Thus, we propose a novel HDX-MS workflow with integrated cIM which has the potential to enable the analysis of more complex systems with greater accuracy and speed.
Project description:We treated primary keratinocytes and SCC cells with IM for 24h to identify differentially expressed genes after treatment compared to DMSO contorl 3x DMSO keratinocytes, 3x IM 100 nM IM keratinocytes, 3x 10 uM IM keratinocytes, 3x DMSO SCC cells, 3x 1 nM IM SCC cells, 3x 100 nM IM SCC cells, 3x 10 uM IM SCC cells
Project description:Cyclic Py-Im polyamides containing two GABA turn units exhibit enhanced DNA binding affinity, but extensive studies of their biological properties have been hindered due to synthetic inaccessibility. A facile modular approach toward cyclic polyamides has been developed via microwave-assisted solid-phase synthesis of hairpin amino acid oligomer intermediates followed by macrocyclization. A focused library of cyclic polyamides 1-7 targeted to the androgen response element (ARE) and the estrogen response element (ERE) were synthesized in 12-17% overall yield. The Fmoc protection strategy also allows for selective modifications on the GABA turn units that have been shown to improve cellular uptake properties. The DNA binding affinities of a library of cyclic polyamides were measured by DNA thermal denaturation assays and compared to the corresponding hairpin polyamides. Fluorescein-labeled cyclic polyamides have been synthesized and imaged via confocal microscopy in A549 and T47D cell lines. The IC(50) values of compounds 1-7 and 9-11 were determined, revealing remarkably varying levels of cytotoxicity.
Project description:An approximately 40% of chronic myeloid leukemia (CML) patients who discontinued imatinib (IM) therapy maintained undetectable minimal residual disease (UMRD) for more than one year (STOP-IM). We set out to examine plasma miRNAs expression in CML patients who could discontinue IM, to seek the possible distinguishable biomarker in STOP-IM CML patients. We compared CML patients who sustained UMRD for more than one year after discontinuation of imatinib (STOP-IM group) with healthy volunteers (controls).
Project description:An approximately 40% of chronic myeloid leukemia (CML) patients who discontinued imatinib (IM) therapy maintained undetectable minimal residual disease (UMRD) for more than one year (STOP-IM). We therefore set out to examine exosomal miRNAs expression in CML patients who could discontinue IM, to seek the possible distinguishable biomarker in STOP-IM CML patients. We compared CML patients who sustained UMRD for more than one year after discontinuation of imatinib (STOP-IM group) with healthy volunteers (controls).
Project description:We treated primary keratinocytes and SCC cells with IM for 24h to identify differentially expressed genes after treatment compared to DMSO contorl
Project description:RPMI8226 Cells were treated with various concentrations of CDDO-2P-Im for 6 hours and then RNA was extracted for gene expression changes