186 results found
Klug D, Osman S, Bendtsen C, et al., 2021, Evaluation of FOXO1 target engagement using a single-cell microfluidic platform, Analytical Chemistry, Vol: 93, Pages: 14659-14666, ISSN: 0003-2700
The cellular thermal shift assay (CETSA) has been used extensively since its introduction to study drug–target engagement within both live cells and cellular lysate. This has proven to be a useful tool in early stage drug discovery and is used to study a wide range of protein classes. We describe the application of a single-cell CETSA workflow within a microfluidic affinity capture (MAC) chip. This has enabled us to quantitatively determine the active FOXO1 single-molecule count and observe FOXO1 stabilization and destabilization in the presence of three small molecule inhibitors, including demonstrating the determination of EC50. The successful use of the MAC chip for single-cell CETSA paves the way for the study of precious clinical samples owing to the low number of cells needed by the chip. It also provides a useful tool for studying any underlying population heterogeneity that exists within a cellular system, a feature that is usually masked when conducting ensemble measurements.
Ho V, Willison K, Baker J, et al., 2021, Microfluidic single cell analysis of microRNA levels in small airway epithelial cells and fibroblasts from COPD patients, Publisher: EUROPEAN RESPIRATORY SOC JOURNALS LTD, ISSN: 0903-1936
Pardo O, Chrysostomou S, Roy R, et al., 2021, Repurposed floxacins targeting RSK4 prevent chemoresistance and metastasis in lung and bladder cancer, Science Translational Medicine, Vol: 13, ISSN: 1946-6234
Lung and bladder cancers are mostly incurable because of the early development of drug resistance and metastatic dissemination. Hence, improved therapies that tackle these two processes are urgently needed to improve clinical outcome. We have identified RSK4 as a promoter of drug resistance and metastasis in lung and bladder cancer cells. Silencing this kinase, through either RNA interference or CRISPR, sensitized tumor cells to chemotherapy and hindered metastasis in vitro and in vivo in a tail vein injection model. Drug screening revealed several floxacin antibiotics as potent RSK4 activation inhibitors, and trovafloxacin reproduced all effects of RSK4 silencing in vitro and in/ex vivo using lung cancer xenograft and genetically engineered mouse models and bladder tumor explants. Through x-ray structure determination and Markov transient and Deuterium exchange analyses, we identified the allosteric binding site and revealed how this compound blocks RSK4 kinase activation through binding to an allosteric site and mimicking a kinase autoinhibitory mechanism involving the RSK4’s hydrophobic motif. Last, we show that patients undergoing chemotherapy and adhering to prophylactic levofloxacin in the large placebo-controlled randomized phase 3 SIGNIFICANT trial had significantly increased (P = 0.048) long-term overall survival times. Hence, we suggest that RSK4 inhibition may represent an effective therapeutic strategy for treating lung and bladder cancer.
Driver T, Cooper B, Ayers R, et al., 2020, Two-dimensional partial covariance mass spectrometry of large molecules based on fragment correlations, Physical Review X, Vol: 10, Pages: 041004 – 1-041004 – 13, ISSN: 2160-3308
Covariance mapping [L. J. Frasinski, K. Codling, and P. A. Hatherly, Science 246, 1029 (1989)] is a well-established technique used for the study of mechanisms of laser-induced molecular ionization and decomposition. It measures statistical correlations between fluctuating signals of pairs of detected species (ions, fragments, electrons). A positive correlation identifies pairs of products originating from the same dissociation or ionization event. A major challenge for covariance-mapping spectroscopy is accessing decompositions of large polyatomic molecules, where true physical correlations are overwhelmed by spurious signals of no physical significance induced by fluctuations in experimental parameters. As a result, successful applications of covariance mapping have so far been restricted to low-mass systems, e.g., organic molecules of around 50 daltons (Da). Partial-covariance mapping was suggested to tackle the problem of spurious correlations by taking into account the independently measured fluctuations in the experimental conditions. However, its potential has never been realized for the decomposition of large molecules, because in these complex situations, determining and continuously monitoring multiple experimental parameters affecting all the measured signals simultaneously becomes unfeasible. We introduce, through deriving theoretically and confirming experimentally, a conceptually new type of partial-covariance mapping—self-correcting partial-covariance spectroscopy—based on a parameter extracted from the measured spectrum itself. We use the readily available total ion count as the self-correcting partial-covariance parameter, thus eliminating the challenge of determining experimental parameter fluctuations in covariance measurements of large complex systems. The introduced self-correcting partial covariance enables us to successfully resolve correlations of molecules as large as
Ying L, Tahirbegi B, Magness A, et al., 2020, A novel Aβ40 assembly at physiological concentration, Scientific Reports, Vol: 10, ISSN: 2045-2322
Aggregates of amyloid-β (Aβ) are characteristic of Alzheimer’s disease, but there is no consensus as to either the nature of the toxic molecular complex or the mechanism by which toxic aggregates are produced. We report on a novel feature of amyloid-lipid interactions where discontinuities in the lipid continuum can serve as catalytic centers for a previously unseen microscale aggregation phenomenon. We show that specific lipid membrane conditions rapidly produce long contours of lipid-bound peptide, even at sub-physiological concentrations of Aβ. Using single molecule fluorescence, time-lapse TIRF microscopy and AFM imaging we characterize this phenomenon and identify some exceptional properties of the aggregation pathway which make it a likely contributor to early oligomer and fibril formation, and thus a potential critical mechanism in the etiology of AD. We infer that these amyloidogenic events occur only at areas of high membrane curvature, which suggests a range of possible mechanisms by which accumulated physiological changes may lead to their inception. The speed of the formation is in hours to days, even at 1 nM peptide concentrations. Lipid features of this type may act like an assembly line for monomeric and small oligomeric subunits of Aβ to increase their aggregation states. We conclude that under lipid environmental conditions, where catalytic centers of the observed type are common, key pathological features of AD may arise on a very short timescale under physiological concentration.
Mickleburgh TG, Salehi-Reyhani A, Magness AJ, et al., 2020, A miniaturized microfluidic assay for single plant cell protein quantitation, Pages: 183-184
We present a method based on a miniaturized antibody capture chip to perform protein quantitation in plants with single cell single molecule resolution. We demonstrate the versatility of the method in capturing and analyzing membrane, cytosolic, and chloroplast stroma located proteins, specifically demonstrated by SNARE 12: YFP, 35S: GFP, and Ribulose-1, 5-bisphosphate carboxylase/oxygenase, respectively. Single cell protein expression quantification is achieved through a bi or tri-molecular antibody microarray. We have also reported single cell experimental evidence that a reduction in chamber volume enhances assay sensitivity.
Prischi F, Chrysostomou S, Roy R, et al., 2019, Targeting RSK4 prevents both chemoresistance and metastasis in lung and bladder cancer, FEBS Open Bio, Publisher: WILEY, Pages: 330-330, ISSN: 2211-5463
Chrysostomou S, Roy R, Prischi F, et al., 2019, Abstract 1775: Targeting RSK4 prevents both chemoresistance and metastasis in lung cancer, AACR Annual Meeting on Bioinformatics, Convergence Science, and Systems Biology, Publisher: American Association for Cancer Research, Pages: 1-2, ISSN: 0008-5472
Lung cancer is the commonest cause of cancer death worldwide with a five-year survival rate of less than five percent for metastatic tumors. Non-small cell lung cancer (NSCLC) accounts for 80% of lung cancer cases of which adenocarcinoma prevails. Patients almost invariably develop metastatic drug-resistant disease and this is responsible for our failure to provide curative therapy. Hence, a better understanding of the mechanisms underlying these biological processes is urgently required to improve clinical outcome.The 90-kDa ribosomal S6 kinases (RSKs) are downstream effectors of the RAS/MAPK cascade. RSKs are highly conserved serine/threonine protein kinases implicated in diverse cellular processes, including cell survival, proliferation, migration and invasion. Four isoforms exist in humans (RSK1-4) and are uniquely characterized by the presence of two non-identical N- and C-terminal kinase domains. RSK isoforms are 73-80% identical at protein level and this has been thought to suggest overlapping functions.However, through functional genomic kinome screens, we show that RSK4, contrary to RSK1, promotes both drug resistance and metastasis in lung cancer. This kinase is overexpressed in the majority (57%) of NSCLC biopsies and this correlates with poor overall survival in lung adenocarcinoma patients. Genetic silencing of RSK4 sensitizes lung cancer cells to chemotherapy and prevents their migration and invasiveness in vitro and in vivo. RSK4 downregulation decreases the anti-apoptotic proteins Bcl2 and cIAP1/2 which correlates with increased apoptotic signalling, whilst it also induces mesenchymal-epithelial transition (MET) through inhibition of NFκB activity. A small-molecule inhibitor screen identified several floxacins, including trovafloxacin, as potent allosteric inhibitors of RSK4 activation. Trovafloxacin reproduced all biological and molecular effects of RSK4 silencing in vitro and in vivo, and is predicted to bind a novel allosteric site revealed
Sowley H, Liu Z, Davies J, et al., 2019, Detection of drug binding to a target protein using EVV 2DIR spectroscopy, Journal of Physical Chemistry B, Vol: 123, Pages: 3598-3606, 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
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
Intrinsic doping of hematite through the inclusion of oxygen vacancies (VO) is being increasingly explored as a simple, low temperature route to preparing active water splitting α-Fe2O3−x photoelectrodes. Whilst it is widely accepted that the introduction of VO leads to improved conductivities, little else is verified regarding the actual mechanism of enhancement. Here we employ transient absorption (TA) spectroscopy to build a comprehensive kinetic model for water oxidation on α-Fe2O3−x. In contrast to previous suggestions, the primary effect of introducing VO is to block very slow (ms) surface hole – bulk electron recombination pathways. In light of our mechanistic research we are also able to identify and address a cause of the high photocurrent onset potential, a common issue with this class of electrodes. Atomic layer deposition (ALD) of Al2O3 is found to be particularly effective with α-Fe2O3−x, leading to the photocurrent onset potential shifting by ca. 200 mV. Significantly TA measurements on these ALD passivated electrodes also provide important insights into the role of passivating layers, that are relevant to the wider development of α-Fe2O3 photoelectrodes.
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
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, The Journal of Physical Chemistry C: Energy Conversion and Storage, Optical and Electronic Devices, Interfaces, Nanomaterials, and Hard Matter, Vol: 117, Pages: 25837-25844, ISSN: 1932-7447
There is an increasing level of interest in the use of black TiO2 prepared by thermal hydrogen treatments (H:TiO2) due to the potential to enhance both the photocatalytic and the light-harvesting properties of TiO2. Here, we examine oxygen-deficient H:TiO2 nanotube arrays that have previously achieved very high solar-to-hydrogen (STH) efficiencies due to incident photon-to-current efficiency (IPCE) values of >90% for photoelectrochemical water splitting at only 0.4 V vs RHE under UV illumination. Our transient absorption (TA) mechanistic study provides strong evidence that the improved electrical properties of oxygen-deficient TiO2 enables remarkably efficient spatial separation of electron–hole pairs on the submicrosecond time scale at moderate applied bias, and this coupled to effective suppression of microsecond to seconds charge carrier recombination is the primary factor behind the dramatically improved photoelectrochemical activity.
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
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