169 results found
Sowley H, Liu Z, Davies J, et al., Detection of drug binding to a target protein using EVV 2DIR spectroscopy, Journal of Physical Chemistry B, ISSN: 1520-5207
We demonstrate that Electron-Vibration-Vibration Two Dimensional Infrared Spectroscopy (EVV 2DIR) can be used to detect the binding of a drug to a target protein active site. The EVV 2DIR spectrum of the FGFR1 Kinase target protein is found to have ~200 detectable crosspeaks in the spectral region 1250 - 1750cm-1/2600 - 3400cm-1, with an additional 63 caused by the addition of a drug, SU5402. Of these 63 new peaks, it is shown that only 6 are due to protein-drug interactions, with the other 57 being due to vibrational coupling within the drug itself. Quantum mechanical calculations employing density functional theory are used to support assignment of the 6 binding-dependent peaks, with one being assigned to a known interaction between the drug and a backbone carbonyl group which forms part of the binding site. None of the 57 intramolecular coupling peaks associated with the drug molecule change substantially in either intensity or frequency when the drug binds to the target protein. This strongly suggests that the structure of the drug in the target binding site, is essentially identical to that when it is not bound.
Tahirbegi B, Magness AJ, Boillat A, et al., 2018, Probing synaptic amyloid-beta aggregation promoted by copper release, 62nd Annual Meeting of the Biophysical-Society, Publisher: Biophysical Society, Pages: 430A-430A, ISSN: 0006-3495
Whether or not the metal ions released during synaptic transmission induce amyloid-beta oligomer formation in the vicinity of synapses is a central question pertinent to the molecular mechanism of Alzheimer's disease. Recently, through a combination of experimental kinetics studies and coupled reaction-diffusion simulations, we predicted that Cu(II) rather than Zn(II) plays an important role in the very early stages (i.e., dimer formation) of Aβ aggregation in the synapse. Single molecule photobleaching analysis is a powerful tool to determine the stoichiometry of amyloid-beta oligomers which enables us to examine the time course of small amyloid-beta oligomer formation in solution, immobilised to a solid-phase substrate or artificial lipid membrane, and in live neurons in the presence of Cu(II). Preliminary results indicate that small amyloid-beta oligomers can be locked in their oligomeric state without dissociation on a poly-lysine coated surface and that Cu(II) increases the diversity and abundance of amyloid-beta oligomers.
Sim S, Sowley H, Kidley N, et al., 2017, Investigation of inhibitor-protein interactions in plants & mammalians from EVV 2DIR data, 254th National Meeting and Exposition of the American-Chemical-Society (ACS) on Chemistry's Impact on the Global Economy, Publisher: AMER CHEMICAL SOC, ISSN: 0065-7727
Lakatos E, Salehi-Reyhani S, Barclay M, et al., 2017, Protein degradation rate is the dominant mechanism accounting for the differences in protein abundance of basal p53 in a human breast and colorectal cancer cell line, PLOS One, Vol: 12, ISSN: 1932-6203
We determine p53 protein abundances and cell to cell variation in two human cancer cell lines with single cell resolution, and show that the fractional width of the distributions is the same in both cases despite a large difference in average protein copy number. We developed a computational framework to identify dominant mechanisms controlling the variation of protein abundance in a simple model of gene expression from the summary statistics of single cell steady state protein expression distributions. Our results, based on single cell data analysed in a Bayesian framework, lends strong support to a model in which variation in the basal p53 protein abundance may be best explained by variations in the rate of p53 protein degradation. This is supported by measurements of the relative average levels of mRNA which are very similar despite large variation in the level of protein.
Magness AJ, Squires J, Griffiths B, et al., 2017, Multiplexed single cell protein expression analysis in solid tumours using a miniaturised microfluidic assay, Convergent Science Physical Oncology, Vol: 3, ISSN: 2057-1739
Using patient-derived colorectal cancer xenografts, we demonstrate a practicable workflow for single cell proteomics in clinically relevant samples and thus a potential translational route for single cell proteomics into medical diagnostics. Using a microfluidic antibody capture [MAC] chip we measured the expression of the tumour suppressor protein p53 and of its post-translationally modified form phosphorylated at serine-15. Aberrant expression of these has commonly been found in colorectal cancers and has been widely investigated for prognostic significance. Our results show that the MAC technology is viable for quantitatively assessing protein expression and phosphorylation at the single cell level in microscopic amounts of clinically relevant tumour material. Thus, this could become a useful tool in therapeutic-associated single cell protein analysis. We also found dramatic variability of p53 and phosphorylated p53 quantities between individual cancer cells from the same sample, demonstrating the power of this single cell technology to study functional intratumour heterogeneity.
Cilibrizzi A, Terenghi M, Fedorova M, et al., 2017, Small-molecule optical probes for cell imaging of protein sulfenylation and their application to monitor cisplatin induced protein oxidation, Sensors and Actuators B: Chemical, Vol: 248, Pages: 437-446, ISSN: 0925-4005
Reactive oxygen species (ROS) are considered versatile second messengers mediating fundamental biological functions. A molecular pathway by which ROS determine functional diversity is the selective oxidation of cysteine residues to form sulfenic acid (SOH) products, known as sulfenylation or S-hydroxylation. This crucial post-translational modification is responsible for the alteration of protein stability, function and signalling. Despite considerable advances on the identification of sulfenic residues on individual proteins, improved methods are needed for direct visualization and accurate quantification of the extent of total protein sulfenylation. Herein we present the synthesis of two new cell-permeable fluorescent probes containing dimedone (a cyclic β-diketone with high specificity for sulfenic acids), and apply them to study oxidation processes in individual cells via microscopy. The low cytotoxicity, cell permeability and optical features of the probes allowed us to visualize and quantify the oxidation of cysteine residues in live cells during H2O2-mediated oxidative burst (i.e. exogenously administered H2O2). We present preliminary cellular imaging studies with these probes to analyse the oxidation process in cells treated with the anticancer drug cisplatin.
We study the influence of acoustic fields on the evaporative self-assembly of solute particles suspended inside sessile droplets of complex fluids. The self-assembly process often results in an undesirable ring-like heterogeneous residue, a phenomenon known as the coffee-ring effect. Here we show that this ring-like self-assembly can be controlled acoustically to form homogeneous disc-like or concentrated spot-like residues. The principle of our method lies in the formation of dynamic patterns of particles in acoustically excited droplets, which inhibits the evaporation-driven convective transport of particles towards the contact line. We elucidate the mechanisms of this pattern formation and also obtain conditions for the suppression of the coffee-ring effect. Our results provide a more general solution to suppress the coffee-ring effect without any physiochemical modification of the fluids, the particles or the surface, thus potentially useful in a broad range of industrial and analytical applications that require homogenous solute depositions.
Willison KR, Salehi-Reyhani A, Burgin E, et al., 2015, Absolute quantification of protein copy number in single cells using single molecule microarrays, EUROPEAN BIOPHYSICS JOURNAL WITH BIOPHYSICS LETTERS, Vol: 44, Pages: S179-S179, ISSN: 0175-7571
Salehi-Reyhani A, Gesellchen F, Mampallil D, et al., 2015, Chemical-Free Lysis and Fractionation of Cells by Use of Surface Acoustic Waves for Sensitive Protein Assays, ANALYTICAL CHEMISTRY, Vol: 87, Pages: 2161-2169, ISSN: 0003-2700
Forster M, Potter RJ, Ling Y, et al., 2015, Oxygen deficient alpha-Fe2O3 photoelectrodes: a balance between enhanced electrical properties and trap-mediated losses, CHEMICAL SCIENCE, Vol: 6, Pages: 4009-4016, ISSN: 2041-6520
Casey D, Wylie D, Gallo J, et al., 2015, A novel, all-optical tool for controllable and non-destructive poration of cells with single-micron resolution, Bio-Optics: Design and Application 2015, Publisher: Optical Society of America
We demonstrate controllable poration within ≈1 µm regions of individual cells, mediated by a near-IR laser interacting with thin-layer amorphous silicon substrates. This technique will allow new experiments in single-cell biology, particularly in neuroscience.
Salehi-Reyhani A, Burgin E, Ces O, et al., 2014, Addressable droplet microarrays for single cell protein analysis, ANALYST, Vol: 139, Pages: 5367-5374, ISSN: 0003-2654
Valim LR, Davies JA, Jensen KT, et al., 2014, Identification and Relative Quantification of Tyrosine Nitration in a Model Peptide Using Two-Dimensional Infrared Spectroscopy, Journal of Physical Chemistry B, Vol: 118, Pages: 12855-12864, ISSN: 1520-6106
Nitration of tyrosine in proteins and peptides is a post-translationalmodification that occurs under conditions of oxidative stress. It is implicated in a varietyof medical conditions, including neurodegenerative and cardiovascular diseases. However,monitoring tyrosine nitration and understanding its role in modifying biological functionremains a major challenge. In this work, we investigate the use of electron-vibration-vibration(EVV) two-dimensional infrared (2DIR) spectroscopy for the study of tyrosine nitration inmodel peptides. We demonstrate the ability of EVV 2DIR spectroscopy to differentiatebetween the neutral and deprotonated states of 3-nitrotyrosine, and we characterize theirspectral signatures using information obtained from quantum chemistry calculations andsimulated EVV 2DIR spectra. To test the sensitivity of the technique, we use mixed-peptidesamples containing various levels of tyrosine nitration, and we use mass spectrometry toindependently verify the level of nitration. We conclude that EVV 2DIR spectroscopy is ableto provide detailed spectroscopic information on peptide side-chain modifications and todetect nitration levels down to 1%. We further propose that lower nitration levels could be detected by introducing a resonantRaman probe step to increase the detection sensitivity of EVV 2DIR spectroscopy.
Schrems A, Phillips J, Casey D, et al., 2014, The grab-and-drop protocol: a novel strategy for membrane protein isolation and reconstitution from single cells (vol 139, pg 3296, 2014), ANALYST, Vol: 139, Pages: 4382-4382, ISSN: 0003-2654
Schrems A, Phillips J, Casey D, et al., 2014, Erratum: The grab-and-drop protocol: A novel strategy for membrane protein isolation and reconstitution from single cells (Analyst (2014) DOI: 10.1039/C4AN00059E), Analyst, Vol: 139, ISSN: 0003-2654
Burgin E, Salehi-Reyhani A, Barclay M, et al., 2014, Absolute quantification of protein copy number using a single-molecule-sensitive microarray, ANALYST, Vol: 139, Pages: 3235-3244, ISSN: 0003-2654
Schrems A, Phillips J, Casey D, et al., 2014, The grab-and-drop protocol: a novel strategy for membrane protein isolation and reconstitution from single cells, ANALYST, Vol: 139, Pages: 3296-3304, ISSN: 0003-2654
Salehi-Reyhani A, Sharma S, Burgin E, et al., 2014, Scaling advantages and constraints in miniaturized capture assays for single cell protein analysis (vol 13, pg 2066, 2013), LAB ON A CHIP, Vol: 14, Pages: 3430-3430, ISSN: 1473-0197
Pastor E, Pesci FM, Reynal A, et al., 2014, Interfacial charge separation in Cu2O/RuOx as a visible light driven CO2 reduction catalyst, Physical Chemistry Chemical Physics, ISSN: 1463-9076
We employ transient absorption spectroscopy to record the absorption spectrum of photogenerated charge carriers in Cu2O. We have found that CO2 reduction in Cu2O is limited by fast electron-hole recombination. The deposition of RuOx nanoparticles on Cu2O results in a twofold increased yield of long-lived electrons, indicating partially reduced electron-hole recombination losses. This observation correlates with an approximately sixfold increase in the yield of CO2 reduction to CO.
Pesci FM, Wang G, Klug DR, et al., 2013, Efficient Suppression of Electron Hole Recombination in Oxygen-Deficient Hydrogen-Treated TiO2 Nanowires for Photoelectrochemical Water Splitting, JOURNAL OF PHYSICAL CHEMISTRY C, Vol: 117, Pages: 25837-25844, ISSN: 1932-7447
Willison KR, Klug DR, 2013, Quantitative single cell and single molecule proteomics for clinical studies, CURRENT OPINION IN BIOTECHNOLOGY, Vol: 24, Pages: 745-751, ISSN: 0958-1669
Cowan AJ, Leng W, Barnes PRF, et al., 2013, Charge carrier separation in nanostructured TiO2 photoelectrodes for water splitting, PHYSICAL CHEMISTRY CHEMICAL PHYSICS, Vol: 15, Pages: 8772-8778, ISSN: 1463-9076
Salehi-Reyhani A, Sharma S, Burgin E, et al., 2013, Scaling Advantages and Constraints in Miniaturized Capture Assays for Single Cell Protein Analysis, Lab on A Chip, Vol: 13, Pages: 2066-2074, ISSN: 1473-0197
Measuring protein expression in single cells is the basis of single cell proteomics. The sensitivity and dynamic range of a single cell immunoassay should ideally be such that proteins that are expressed between 1 – 106 copies per cell can be detected and counted. We have investigated the effect of miniaturizing antibody microarrays by reducing capture spot sizes from 100 μm to 15 μm using dip pen nanolithography. We demonstrate that protocols developed for printing and passivating antibody capture spots using conventional pin based contact printing can be directly transferred to dip-pen lithography whilst retaining the capture activity per unit area. Using a simple kinetic model, we highlight how the limit of detection and dynamic range of a sandwich immunoassay, respectively, increase and decrease when spot size is reduced. However, we show that reducing spot size is more effective than increasing assay chamber volume when seeking to multiplex such a microfluidic immunoassay. Although, we make particular reference to single cell microfluidic immunoassays, the topics discussed here are applicable to capture assays in general.
Barroso M, Mesa CA, Pendlebury SR, et al., 2012, Dynamics of photogenerated holes in surface modified alpha-Fe2O3 photoanodes for solar water splitting, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 109, Pages: 15640-15645, ISSN: 0027-8424
Gierakowski L, Guo R, Kornau KM, et al., 2012, Study of protein-tyrosine nitration by two-dimensional infrared vibrational spectroscopy, FREE RADICAL BIOLOGY AND MEDICINE, Vol: 53, Pages: S214-S214, ISSN: 0891-5849
Salehi-Reyhani A, Barclay M, Ces O, et al., 2012, Towards Practical Single Cell Proteomics: A Microfluidic Antibody Capture Chip with TIRF Detection, FREE RADICAL BIOLOGY AND MEDICINE, Vol: 53, Pages: S128-S128, ISSN: 0891-5849
Wylie D, Casey D, Phillips J, et al., 2012, Novel nanotechnologies for multiple spatially and temporally resolved live single cell membrane sampling and analysis, FREE RADICAL BIOLOGY AND MEDICINE, Vol: 53, Pages: S127-S128, ISSN: 0891-5849
Goyder MS, Willison KR, Klug DR, et al., 2012, Affinity chromatography and capillary electrophoresis for analysis of the yeast ribosomal proteins, BMB REPORTS, Vol: 45, Pages: 233-238, ISSN: 1976-6696
Guo R, Mukamel S, Klug DR, 2012, Geometry determination of complexes in a molecular liquid mixture using electron-vibration-vibration two-dimensional infrared spectroscopy with a vibrational transition density cube method, PHYSICAL CHEMISTRY CHEMICAL PHYSICS, Vol: 14, Pages: 14023-14033, ISSN: 1463-9076
Pendlebury SR, Cowan AJ, Barroso M, et al., 2012, Correlating long-lived photogenerated hole populations with photocurrent densities in hematite water oxidation photoanod, Energy and Environmental Science
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