215 results found
Ho V, Baker J, Willison K, et al., 2023, Single cell quantification of microRNA from small numbers of non-invasively sampled primary human cells, Communications Biology, Vol: 6, Pages: 1-11, ISSN: 2399-3642
Expression levels of microRNAs (miRNAs) in single cells are low and conventional miRNA detection methods require amplification that can be complex, time-consuming, costly and may bias results. Single cell microfluidic platforms have been developed; however, current approaches are unable to absolutely quantify single miRNA molecules expressed in single cells. Herein, we present an amplification-free sandwich hybridisation assay to detect single miRNA molecules in single cells using a microfluidic platform that optically traps and lyses individual cells. Absolute quantification of miR-21 and miR-34a molecules was achieved at a single cell level in human cell lines and validated using real-time qPCR. The sensitivity of the assay was demonstrated by quantifying single miRNA molecules in nasal epithelial cells and CD3+ T-cells, as well as nasal fluid collected non-invasively from healthy individuals. This platform requires ~50 cells or ~30 µL biofluid and can be extended for other miRNA targets therefore it could monitor miRNA levels in disease progression or clinical studies.
Wilkinson MD, Ferreira JL, Beeby M, et al., 2022, The malaria parasite chaperonin containing TCP-1 (CCT) complex: Data integration with other CCT proteomes, FRONTIERS IN MOLECULAR BIOSCIENCES, Vol: 9
Ho V, Baker JR, Willison KR, et al., 2022, An amplification-free, innovative, multiplex assay to quantify microRNAs in single cells from COPD patients, Publisher: EUROPEAN RESPIRATORY SOC JOURNALS LTD, ISSN: 0903-1936
Tahirbegi IB, Magness A, Piersimoni ME, et al., 2022, Towards high throughput oligomer detection and classification for early-stage aggregation of amyloidogenic protein, Frontiers in Chemistry, Vol: 10, ISSN: 2296-2646
Aggregation kinetics of proteins and peptides have been studied extensively due to their significance in many human diseases, including neurodegenerative disorders, and the roles they play in some key physiological processes. However, most of these studies have been performed as bulk measurements using Thioflavin T or other fluorescence turn-on reagents as indicators of fibrillization. Such techniques are highly successful in making inferences about the nucleation and growth mechanism of fibrils, yet cannot directly measure assembly reactions at low protein concentrations which is the case for amyloid-β (Aβ) peptide under physiological conditions. In particular, the evolution from monomer to low-order oligomer in early stages of aggregation cannot be detected. Single-molecule methods allow direct access to such fundamental information. We developed a high-throughput protocol for single-molecule photobleaching experiments using an automated fluorescence microscope. Stepwise photobleaching analysis of the time profiles of individual foci allowed us to determine stoichiometry of protein oligomers and probe protein aggregation kinetics. Furthermore, we investigated the potential application of supervised machine learning with support vector machines (SVMs) as well as multilayer perceptron (MLP) artificial neural networks to classify bleaching traces into stoichiometric categories based on an ensemble of measurable quantities derivable from individual traces. Both SVM and MLP models achieved a comparable accuracy of more than 80% against simulated traces up to 19-mer, although MLP offered considerable speed advantages, thus making it suitable for application to high-throughput experimental data. We used our high-throughput method to study the aggregation of Aβ40 in the presence of metal ions and the aggregation of α-synuclein in the presence of gold nanoparticles.
Ho V, Baker JR, Willison KR, et al., 2022, Novel Single Cell Analysis of microRNA Levels in Response to Oxidative Stress and in COPD Using Microfluidic Technology, International Conference of the American-Thoracic-Society, Publisher: AMER THORACIC SOC, ISSN: 1073-449X
Teng X, Sheveleva A, Tuna F, et al., 2021, Front Cover: Acetylation Rather than H50Q Mutation Impacts the Kinetics of Cu(II) Binding to α‐Synuclein (ChemPhysChem 23/2021), ChemPhysChem, Vol: 22, Pages: 2378-2378, ISSN: 1439-4235
Ying L, Teng X, Sheveleva A, et al., 2021, Acetylation rather than H50Q mutation impacts the kinetics of Cu(II) binding to α-synuclein, ChemPhysChem: a European journal of chemical physics and physical chemistry, Vol: 22, Pages: 2413-2419, ISSN: 1439-4235
The interaction between α-synuclein (αSyn) and Cu2+ has been suggested to be closely linked to brain copper homeostasis. Disruption of copper levels could induce misfolding and aggregation of αSyn, and thus contribute to the progression of Parkinson's disease (PD). Understanding the molecular mechanism of αSyn-Cu2+ interaction is important and controversies in Cu2+ coordination geometry with αSyn still exists. Herein, we find that the pathological H50Q mutation has no impact on the kinetics of Cu2+ binding to the high-affinity site of wild type αSyn (WT-αSyn), indicating the non-involvement of His50 in high-affinity Cu2+ binding to WT-αSyn. In contrast, the physiological N-terminally acetylated αSyn (NAc-αSyn) displays several orders of magnitude weaker Cu2+ binding affinity than WT-αSyn. Cu2+ coordination mode to NAc-αSyn has also been proposed based on EPR spectrum. In addition, we find that Cu2+ coordinated WT-αSyn is reduction-active in the presence of GSH, but essentially inactive towards ascorbate. Our work provides new insights into αSyn-Cu2+ interaction, which may help understand the multifaceted normal functions of αSyn as well as pathological consequences of αSyn aggregation.
Teng X, Sheveleva A, Tuna F, et al., 2021, Acetylation Rather than H50Q Mutation Impacts the Kinetics of Cu(II) Binding to alpha-Synuclein, CHEMPHYSCHEM, ISSN: 1439-4235
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
Teng X, 2021, Probing the Interactions of Intrinsically Disordered Protein with Metal Ions and Lipid Membranes by Fluorescence Spectroscopy, 65th Annual Meeting of The Biophysical Society
Wang X, Wilkinson MD, Lin X, et al., 2020, Correction: Single-molecule nanopore sensing of actin dynamics and drug binding, Chemical Science, Vol: 11, Pages: 8036-8038, ISSN: 2041-6520
Correction for ‘Single-molecule nanopore sensing of actin dynamics and drug binding’ by Xiaoyi Wang et al., Chem. Sci., 2020, 11, 970–979, DOI: 10.1039/C9SC05710B.
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.
Wang X, Wilkinson MD, Lin X, et al., 2020, Single-molecule nanopore sensing of actin dynamics and drug binding, Chemical Science, Vol: 11, Pages: 970-979, ISSN: 2041-6520
Actin is a key protein in the dynamic processes within the eukaryotic cell. To date, methods exploring the molecular state of actin are limited to insights gained from structural approaches, providing a snapshot of protein folding, or methods that require chemical modifications compromising actin monomer thermostability. Nanopore sensing permits label-free investigation of native proteins and is ideally suited to study proteins such as actin that require specialised buffers and cofactors. Using nanopores, we determined the state of actin at the macromolecular level (filamentous or globular) and in its monomeric form bound to inhibitors. We revealed urea-dependent and voltage-dependent transitional states and observed unfolding process within which sub-populations of transient actin oligomers are visible. We detected, in real-time, filament-growth, and drug-binding at the single-molecule level demonstrating the promise of nanopores sensing for in-depth understanding of protein folding landscapes and for drug discovery.
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.
Willison K, 2019, An intracellular calcium frequency code model extended to the Riemann zeta function, Publisher: https://arxiv.org/
We have used the Nernst chemical potential treatment to couple the time domains of sodium and calcium ion channel opening and closing rates to the spatial domain of the diffusing waves of the travelling calcium ions inside single cells. The model is plausibly evolvable with respect to the origins of the molecular components and the scaling of the system from simple cells to neurons. The mixed chemical potentials are calculated by summing the concentrations or particle numbers of the two constituent ions which are pure numbers and thus dimensionless. Chemical potentials are true thermodynamic free Gibbs/Fermi energies and the forces acting on chemical flows are calculated from the natural logarithms of the particle numbers or their concentrations. The mixed chemical potential is converted to the time domain of an action potential by assuming that the injection of calcium ions accelerates depolarization in direct proportion to the amplitude of the total charge contribution of the calcium pulse. We assert that the natural logarithm of the real component of the imaginary term of any Riemann zeta zero corresponds to an instantaneous calcium potential. In principle, in a physiologically plausible fashion, the first few thousand Riemann zeta-zeros can be encoded on this chemical scale manifested as regulated step-changes in the amplitudes of naturally occurring calcium current transients. We show that pairs of Zn channels can form Dirac fences which encode the logarithmic spacings and summed amplitudes of any pair of Riemann zeros. Remarkably the beat frequencies of the pairings of the early frequency terms overlap the naturally occurring frequency modes in vertebrate brains. The equation for the time domain in the biological model has a similar form to the Riemann zeta function on the half-plane and mimics analytical continuation on the complex plane.
Much of the functionality of multicellular systems arises from the spatial organization and dynamic behaviours within and between cells. Current single-cell genomic methods only provide a transcriptional ‘snapshot’ of individual cells. The real-time analysis and perturbation of living cells would generate a step change in single-cell analysis. Here we describe minimally invasive nanotweezers that can be spatially controlled to extract samples from living cells with single-molecule precision. They consist of two closely spaced electrodes with gaps as small as 10–20 nm, which can be used for the dielectrophoretic trapping of DNA and proteins. Aside from trapping single molecules, we also extract nucleic acids for gene expression analysis from living cells without affecting their viability. Finally, we report on the trapping and extraction of a single mitochondrion. This work bridges the gap between single-molecule/organelle manipulation and cell biology and can ultimately enable a better understanding of living cells.
Willison KR, 2018, The structure and evolution of eukaryotic chaperonin containing TCP-1 and its mechanism that folds actin into a protein spring, Biochemical Journal, Vol: 475, Pages: 3009-3034, ISSN: 1470-8728
Actin is folded to its native state in eukaryotic cytosol by the sequential allosteric mechanism of the chaperonin-containing TCP-1 (CCT). The CCT machine is a double-ring ATPase built from eight related subunits, CCT1–CCT8. Non-native actin interacts with specific subunits and is annealed slowly through sequential binding and hydrolysis of ATP around and across the ring system. CCT releases a folded but soft ATP-G-actin monomer which is trapped 80 kJ/mol uphill on the folding energy surface by its ATP-Mg2+/Ca2+ clasp. The energy landscape can be re-explored in the actin filament, F-actin, because ATP hydrolysis produces dehydrated and more compact ADP-actin monomers which, upon application of force and strain, are opened and closed like the elements of a spring. Actin-based myosin motor systems underpin a multitude of force generation processes in cells and muscles. We propose that the water surface of F-actin acts as a low-binding energy, directional waveguide which is recognized specifically by the myosin lever-arm domain before the system engages to form the tight-binding actomyosin complex. Such a water-mediated recognition process between actin and myosin would enable symmetry breaking through fast, low energy initial binding events. The origin of chaperonins and the subsequent emergence of the CCT–actin system in LECA (last eukaryotic common ancestor) point to the critical role of CCT in facilitating phagocytosis during early eukaryotic evolution and the transition from the bacterial world. The coupling of CCT-folding fluxes to the cell cycle, cell size control networks and cancer are discussed together with directions for further research.
Willison KR, 2018, The substrate specificity of eukaryotic cytosolic chaperonin CCT, Philosophical Transactions B: Biological Sciences, Vol: 373, ISSN: 0962-8436
The cytosolic chaperonin CCT (chaperonin containing TCP-1) is an ATP-dependent double-ring protein machine mediating the folding of members of the eukaryotic cytoskeletal protein families. The actins and tubulins are obligate substrates of CCT because they are completely dependent on CCT activity to reach their native states. Genetic and proteomic analysis of the CCT interactome in the yeast Saccharomyces cerevisiae revealed a CCT network of approximately 300 genes and proteins involved in many fundamental biological processes. We classified network members into sets such as substrates, CCT cofactors and CCT-mediated assembly processes. Many members of the 7-bladed propeller family of proteins are commonly found tightly bound to CCT isolated from human and plant cells and yeasts. The anaphase promoting complex (APC/C) cofactor propellers, Cdh1p and Cdc20p, are also obligate substrates since they both require CCT for folding and functional activation. In vitro translation analysis in prokaryotic and eukaryotic cell extracts of a set of yeast propellers demonstrates their highly differential interactions with CCT and GroEL (another chaperonin). Individual propeller proteins have idiosyncratic interaction modes with CCT because they emerged independently with neo-functions many times throughout eukaryotic evolution. We present a toy model in which cytoskeletal protein biogenesis and folding flux through CCT couples cell growth and size control to time dependent cell cycle mechanisms.
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.
Baumann H, Matthews H, Li M, et al., 2018, A high-throughput in vitro translation screen towards discovery of novel antimalarial protein translation inhibitors, Publisher: BioRxiv
Drugs that target protein synthesis are well-validated for use as antimicrobials, yet specific high throughput (HTP) methods to screen for those targeting malaria are lacking. Here, we have developed a cell free in vitro translation (IVT) assay for the human malaria parasite, Plasmodium falciparum, which reconstitutes the native parasite protein translation machinery. Combining clarified IVT lysate with a click beetle luciferase reporter gene fused to untranslated regions of Pf histidine-rich proteins (hrp)-2 and 3, the HTP IVT assay accurately reports protein translation in a 384-well plate format using a standard spectrofluorometer. We validate the assay as effective in detecting compounds targeting the ribosome, ribosome co-factors (elongation factor 2) and cytosolic tRNA synthetases as well as its ability to find translation inhibitors in a blind screen using a high-density assay format amenable for high throughput. This demonstrates an ability to reconstitute the breadth of the parasite eukaryotic protein translation machinery in vitro and use it as a powerful platform for antimalarial drug discovery.
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.
Baum J, Olshina M, Baumann H, et al., 2016, Plasmodium actin is incompletely folded by heterologous protein-folding machinery and likely requires the native Plasmodium chaperonin complex to enter a mature functional state, The FASEB Journal, Vol: 30, Pages: 405-416, ISSN: 0892-6638
Actin filament turnover underpins several processes in the life cycle of the malaria parasite, Plasmodium falciparum. Polymerization and depolymerization are especially important for gliding motility, a substrate-dependent form of cell movement that underpins the protozoan parasite’s ability to disseminate and invade host cells. To date, given difficulties in extraction of native actins directly from parasites, much of our biochemical understanding of malarial actin has instead relied on recombinant protein extracted and purified from heterologous protein expression systems. Here, using in vitro transcription-translation methodologies and quantitative protein-binding assays, we explored the folding state of heterologously expressed P. falciparum actin 1 (PfACTI) with the aim of assessing the reliability of current recombinant-protein-based data. We demonstrate that PfACTI, when expressed in non-native systems, is capable of binding to and release from bacterial, yeast, and mammalian chaperonin complexes but appears to be incompletely folded. Characterization of the native Plasmodium folding machinery in silico, the chaperonin containing t-complex protein-1 complex, highlights key divergences between the different chaperonin systems that likely underpins this incomplete folded state. These results highlight the importance of characterizing actin’s folded state and raise concerns about the interpretation of actin polymerization kinetics based solely on protein derived from heterologous expression systems.—Olshina, M. A., Baumann, H., Willison, K. R., Baum, J. Plasmodium actin is incompletely folded by heterologous protein-folding machinery and likely requires the native Plasmodium chaperonin complex to enter a mature functional state.
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
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.
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
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
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