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Journal articleNoble E, Kumar S, Gorlitz F, et al., 2017,
In vivo label-free mapping of the effect of a photosystem II inhibiting herbicide in plants using chlorophyll fluorescence lifetime
, Plant Methods, Vol: 13, ISSN: 1746-4811BackgroundIn order to better understand and improve the mode of action of agrochemicals, it is useful to be able to visualize their uptake and distribution in vivo, non-invasively and, ideally, in the field. Here we explore the potential of plant autofluorescence (specifically chlorophyll fluorescence) to provide a readout of herbicide action across the scales utilising multiphoton-excited fluorescence lifetime imaging, wide-field single-photon excited fluorescence lifetime imaging and single point fluorescence lifetime measurements via a fibre-optic probe.ResultsOur studies indicate that changes in chlorophyll fluorescence lifetime can be utilised as an indirect readout of a photosystem II inhibiting herbicide activity in living plant leaves at three different scales: cellular (~μm), single point (~1 mm2) and macroscopic (~8 × 6 mm2 of a leaf). Multiphoton excited fluorescence lifetime imaging of Triticum aestivum leaves indicated that there is an increase in the spatially averaged chlorophyll fluorescence lifetime of leaves treated with Flagon EC—a photosystem II inhibiting herbicide. The untreated leaf exhibited an average lifetime of 560 ± 30 ps while the leaf imaged 2 h post treatment exhibited an increased lifetime of 2000 ± 440 ps in different fields of view. The results from in vivo wide-field single-photon excited fluorescence lifetime imaging excited at 440 nm indicated an increase in chlorophyll fluorescence lifetime from 521 ps in an untreated leaf to 1000 ps, just 3 min after treating the same leaf with Flagon EC, and to 2150 ps after 27 min. In vivo single point fluorescence lifetime measurements demonstrated a similar increase in chlorophyll fluorescence lifetime. Untreated leaf presented a fluorescence lifetime of 435 ps in the 440 nm excited chlorophyll channel, CH4 (620–710 nm). In the first 5 min after treatment, mean fluorescence lifetime is observed to have increased to 1 ns and then to 1.3 ns after 60 min. For
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Journal articleMurray JI, Flodén NJ, Bauer A, et al., 2017,
Kinetic Resolution of 2-Substituted Indolines by N -Sulfonylation using an Atropisomeric 4-DMAP- N -oxide Organocatalyst
, Angewandte Chemie, Vol: 129, Pages: 5854-5858, ISSN: 0044-8249The first catalytic kinetic resolution by N‐sulfonylation is described. 2‐Substituted indolines are resolved (s=2.6–19) using an atropisomeric 4‐dimethylaminopyridine‐N‐oxide (4‐DMAP‐N‐oxide) organocatalyst. Use of 2‐isopropyl‐4‐nitrophenylsulfonyl chloride is critical to the stereodiscrimination and enables facile deprotection of the sulfonamide products with thioglycolic acid. A qualitative model that accounts for the stereodiscrimination is proposed.
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Journal articleMagness 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: 2057-1739Using 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.
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Journal articleClulow JA, Storck EM, Lanyon-Hogg T, et al., 2017,
Competition-based, quantitative chemical proteomics in breast cancer cells identifies new target profiles for sulforaphane
, Chemical Communications, Vol: 53, Pages: 5182-5185, ISSN: 1364-548XSulforaphane is a small molecule isothiocyanate which exhibits anticancer potential, yet its biological targets remain poorly understood. Here we employ a competition-based chemical proteomics strategy to profile sulforaphane's targets and identify over 500 targets along with their relative affinities. These targets provide a new set of mediators for sulforaphane's bioactivity, and aid understanding of its complex mode of action.
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Conference paperAndrews N, Davis S, Hay C, et al., 2017,
Functional imaging of live Zebrafish using fluorescence lifetime optical projection tomography
, Conference on Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XV, Publisher: Society of Photo-Optical Instrumentation Engineers (SPIE), ISSN: 0277-786X -
Conference paperYuan Q, Gould I, Kidley N, 2017,
Density functional theory study on triplet intermolecular hydrogen transfer between cycloxydim and chlorothalonil
, 253rd National Meeting of the American-Chemical-Society (ACS) on Advanced Materials, Technologies, Systems, and Processes, Publisher: AMER CHEMICAL SOC, ISSN: 0065-7727 -
Conference paperKhanna T, Barter L, Gould I, 2017,
Development and application of the AMBER molecular mechanics force field to investigate herbicide interaction in plants
, 253rd National Meeting of the American-Chemical-Society (ACS) on Advanced Materials, Technologies, Systems, and Processes, Publisher: AMER CHEMICAL SOC, ISSN: 0065-7727 -
Journal articleBarlow NE, Smpokou E, Friddin MS, et al., 2017,
Engineering plant membranes using droplet interface bilayers
, Biomicrofluidics, Vol: 11, ISSN: 1932-1058Droplet interface bilayers (DIBs) have become widely recognised as a robust platform for constructing model membranes and are emerging as a key technology for the bottom-up assembly of synthetic cell-like and tissue-like structures. DIBs are formed when lipid-monolayer coated water droplets are brought together inside a well of oil, which is excluded from the interface as the DIB forms. The unique features of the system, compared to traditional approaches (e.g., supported lipid bilayers, black lipid membranes, and liposomes), is the ability to engineer multi-layered bilayer networks by connecting multiple droplets together in 3D, and the capability to impart bilayer asymmetry freely within these droplet architectures by supplying droplets with different lipids. Yet despite these achievements, one potential limitation of the technology is that DIBs formed from biologically relevant components have not been well studied. This could limit the reach of the platform to biological systems where bilayer composition and asymmetry are understood to play a key role. Herein, we address this issue by reporting the assembly of asymmetric DIBs designed to replicate the plasma membrane compositions of three different plant species; Arabidopsis thaliana, tobacco, and oats, by engineering vesicles with different amounts of plant phospholipids, sterols and cerebrosides for the first time. We show that vesicles made from our plant lipid formulations are stable and can be used to assemble asymmetric plant DIBs. We verify this using a bilayer permeation assay, from which we extract values for absolute effective bilayer permeation and bilayer stability. Our results confirm that stable DIBs can be assembled from our plant membrane mimics and could lead to new approaches for assembling model systems to study membrane translocation and to screen new agrochemicals in plants.
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Journal articleFrampton CS, Murray JI, Spivey AC, 2017,
Crystal structure of 1-methylimidazole 3-oxide monohydrate.
, Acta Crystallographica Section E: Crystallographic Communications, Vol: 73, Pages: 372-374, ISSN: 2056-98901-Methylimidazole 3-N-oxide (NMI-O) crystallizes as a monohydrate, C4H6N2O·H2O, in the monoclinic space group P21 with Z' = 2 (mol-ecules A and B). The imidazole rings display a planar geometry (r.m.s. deviations = 0.0008 and 0.0002 Å) and are linked in the crystal structure into infinite zigzag strands of ⋯NMI-O(A)⋯OH2⋯NMI-O(B)⋯OH2⋯ units by O-H⋯O hydrogen bonds. These chains propagate along the b-axis direction of the unit cell.
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Journal articleFrench PMW, Görlitz F, Kelly D, et al., 2017,
Open source high content analysis utilizing automated fluorescence lifetime imaging microscopy
, Jove-Journal of Visualized Experiments, Vol: 119, ISSN: 1940-087XWe present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using Förster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe herethe functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in μ Manager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.
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