9 research outputs found
Stacks off tracks: a role for the golgin AtCASP in plant endoplasmic reticulum-Golgi apparatus tethering
No full textPublished onlineThis is the author accepted manuscript. The final version is available from Oxford University Press via the DOI in this record.The plant Golgi apparatus modifies and sorts incoming proteins from the endoplasmic reticulum (ER) and synthesizes cell wall matrix material. Plant cells possess numerous motile Golgi bodies, which are connected to the ER by yet to be identified tethering factors. Previous studies indicated a role for cis-Golgi plant golgins, which are long coiled-coil domain proteins anchored to Golgi membranes, in Golgi biogenesis. Here we show a tethering role for the golgin AtCASP at the ER-Golgi interface. Using live-cell imaging, Golgi body dynamics were compared in Arabidopsis thaliana leaf epidermal cells expressing fluorescently tagged AtCASP, a truncated AtCASP-ΔCC lacking the coiled-coil domains, and the Golgi marker STtmd. Golgi body speed and displacement were significantly reduced in AtCASP-ΔCC lines. Using a dual-colour optical trapping system and a TIRF-tweezer system, individual Golgi bodies were captured in planta. Golgi bodies in AtCASP-ΔCC lines were easier to trap and the ER-Golgi connection was more easily disrupted. Occasionally, the ER tubule followed a trapped Golgi body with a gap, indicating the presence of other tethering factors. Our work confirms that the intimate ER-Golgi association can be disrupted or weakened by expression of truncated AtCASP-ΔCC and suggests that this connection is most likely maintained by a golgin-mediated tethering complex.We thank Janet Evins for help with growing plants. The work was supported by a BBSRC grant (BB/J000302/1) and a Royal Society Travel grant to CH
Preprint: Near infrared-light treatment alters mitochondrial homeostasis to induce senescence in breast cancer cells
No full textThe application of near infrared (NIR)-light to living systems has been suggested as a potential method to enhance tissue repair, decrease inflammation, and possibly mitigate cancer therapy-associated side effects. In this study, we examined the effect of exposing three cell lines: breast cancer (MCF7), non-cancer breast cells (MCF10A), and lung fibroblasts (IMR-90), to 734 nm NIR-light for 20 minutes per day for six days, and measuring changes in cellular senescence. Positive senescent populations were induced using doxorubicin. Flow cytometry was used to assess relative levels of senescence together with mitochondria-related variables. Exposure to NIR-light significantly increased the level of senescence in MCF7 cells (13.5%; P<0.01), with no observable effects on MCF10A or IMR-90 cell lines. NIR-induced senescence was associated with significant changes in mitochondria homeostasis, including raised ROS level (36.0%; P<0.05) and mitochondrial membrane potential (14.9%; P<0.05), with no changes in mitochondrial Ca2+. These results suggest that NIR-light exposure can significantly arrest the proliferation of breast cancer cells via inducing senescence, while leaving non-cancerous cell lines unaffected
Re and<sup> 99m</sup>Tc complexes of BodP<sub>3</sub> – multi-modality imaging probes
No full textA fluorescent tridentate phosphine, BodP3 (2), forms rhenium complexes which effectively image cancer cells. Related technetium analogues are also readily prepared and have potential as dual SPECT/fluorescent biological probes
High yield vesicle packaged recombinant protein production from E. coli.
No full textWe describe an innovative system that exports diverse recombinant proteins in membrane bound vesicles from E. coli. These recombinant vesicles compartmentalise proteins within a micro-environment that allows production of otherwise challenging insoluble, toxic, or disulphide-bond containing proteins from bacteria. The release of the inducible vesicle packaged soluble protein supports isolation from the culture within an environment allowing long term storage of active protein. This technology results in high-yields of vesicle packaged soluble functional proteins for efficient downstream processing for a wide range of applications from discovery science to applied biotechnology and medicine
Directly interrogating single quantum dot labelled UvrA2 molecules on DNA tightropes using an optically trapped nanoprobe
No full textIn this study we describe a new methodology to physically probe individual complexes formed between proteins and DNA. By combining nanoscale, high speed physical force measurement with sensitive fluorescence imaging we investigate the complex formed between the prokaryotic DNA repair protein UvrA2 and DNA. This approach uses a triangular, optically-trapped "nanoprobe" with a nanometer scale tip protruding from one vertex. By scanning this tip along a single DNA strand suspended between surface-bound micron-scale beads, quantum-dot tagged UvrA2 molecules bound to these '"DNA tightropes" can be mechanically interrogated. Encounters with UvrA2 led to deflections of the whole nanoprobe structure, which were converted to resistive force. A force histogram from all 144 detected interactions generated a bimodal distribution centered on 2.6 and 8.1 pN, possibly reflecting the asymmetry of UvrA2's binding to DNA. These observations successfully demonstrate the use of a highly controllable purpose-designed and built synthetic nanoprobe combined with fluorescence imaging to study protein-DNA interactions at the single molecule level
Fluorescence and phosphorescence lifetime imaging reveals a significant cell nuclear viscosity and refractive index changes upon DNA damage
No full textCytoplasmic viscosity is a crucial parameter in determining rates of diffusion-limited reactions. Changes in viscosity are associated with several diseases, whilst nuclear viscosity determines gene integrity, regulation and expression. Yet how drugs including DNA-damaging agents affect viscosity is unknown. We demonstrate the use of a platinum complex, Pt[L]Cl, that localizes efficiently mostly in the nucleus as a probe for nuclear viscosity. The phosphorescence lifetime of Pt[L]Cl is sensitive to viscosity and provides an excellent tool to investigate the impact of DNA damage. We show using Fluorescence Lifetime Imaging (FLIM) that the lifetime of both green and red fluorescent proteins (FP) are also sensitive to changes in cellular viscosity and refractive index. However, Pt[L]Cl proved to be a more sensitive viscosity probe, by virtue of microsecond phosphorescence lifetime versus nanosecond fluorescence lifetime of FP, hence greater sensitivity to bimolecular reactions. DNA damage was inflicted by either a two-photon excitation, one-photon excitation microbeam and X-rays. DNA damage of live cells causes significant increase in the lifetime of either Pt[L]Cl (HeLa cells, 12.5–14.1 µs) or intracellularly expressed mCherry (HEK293 cells, 1.54–1.67 ns), but a decrease in fluorescence lifetime of GFP from 2.65 to 2.29 ns (in V15B cells). These values represent a viscosity change from 8.59 to 20.56 cP as well as significant changes in the refractive index (RI), according to independent calibration. Interestingly DNA damage localized to a submicron region following a laser microbeam induction showed a whole cell viscosity change, with those in the nucleus being greater than the cytoplasm. We also found evidence of a by-stander effect, whereby adjacent un-irradiated cells also showed nuclear viscosity change. Finally, an increase in viscosity following DNA damage was also observed in bacterial cells with an over-expressed mNeonGreen FP, evidenced by the change in its lifetime from 2.8 to 2.4 ns
Carbon Nanotubes: Interactions Between Amino Acid‐Tagged Naphthalenediimide and Single Walled Carbon Nanotubes for the Design and Construction of New Bioimaging Probes (Adv. Funct. Mater. 3/2012)
No full textInteractions Between Amino Acid-Tagged Naphthalenediimide and Single Walled Carbon Nanotubes for the Design and Construction of New Bioimaging Probes
No full textA new synthetic route to functionalized single walled carbon nanotubes (SWNTs) via supramolecular interactions using a specifically designed naphthalenediimide (NDI) nanoreceptor is demonstrated. The tendency of the NDI to spontaneously form composites with carbon nanomaterials leads to fluorescent amino acid tagged SWNTs, which are dispersible in widely accessible organic solvents (CHCl 3, DMSO) as well as in biocompatible cell medium (EMEM, Eagle's modified essential medium). The X-ray crystal structure of the first iodine-tagged and amino acid-functionalized NDI molecule, designed especially to facilitate the high resolution transmission electron microscopy (HR TEM) imaging whilst retaining its ability to self-assemble into a nanodimensional receptor in weakly polar solvents, is also described. A new hybrid material, NDI@SWNT, was prepared and characterized as dispersed in organic solvents and aqueous media and in the solid state by HR TEM, tapping mode atomic force microscopy (TM AFM), scanning electron microscopy (SEM), circular dichroism, Raman and fluorescence spectroscopies (steady-state single and two-photon techniques). Combined microscopy techniques, density functional theory (DFT) calculations using the Spanish Initiative for Electronic Simulations with Thousands of Atoms (SIESTA) program and spectroscopic measurements in solution indicate that amino acid-functionalized NDI interacts strongly with SWNTs and forms a donor-acceptor complex. Density functional theory (DFT) calculations predicted the geometry and the binding energies of an NDI molecule loaded onto a SWNT strand and the possibility of charge transfer interactions within the hybrid. The NDI@SWNT composite translocates into cells (e.g. FEK-4, HeLa, MCF-7) as an intact object and localizes in the cells' cytoplasm and partially in the nucleus. The NDI coating enhances the biocompatibility of SWNTs and mediates its intracellular localization as shown by confocal fluorescence imaging and fluorescence lifetime imaging (FLIM) techniques. The excited state fluorescence lifetime of the probes in cells versus solution phase indicates that the probes remain unaffected by the change in their chemical environment within the experimental timescale (2 h). © 2012 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim
Actualising economic development through privatisation legal reform: a general assessment of privatisation in Africa with a specific case study of Nigeria and sub focus on the Nigerian electricity sector
No full textPhDThis dissertation analysed the outcome of the adoption and implementation of
privatisation by Nigeria, of which a legal framework has been put in place by the
government to legalise the process of transferring the ownership and/or control of
public enterprises to private entrepreneurs with a view to facilitating economic
development in the country. Many other African countries have pursued similar reform
paths with similar objectives and the thesis undertakes a general analysis of the outcome
of adopting and implementing privatisation within the continent. Within Nigeria, the
proposed power sector privatisation is specifically analysed. The dissertation focuses on
the economic development outcome of privatisation, which encompasses key benefits
that have been attributed to privatisation including the beneficial impact of privatisation
on the public sector as well as the privatised enterprises, privatisation’s contribution to
overall private sector development, the benefit of privatisation to the citizens of the
country and finally privatisation’s usefulness as a conduit for beneficial foreign
investment inflow to the country. These benefits are viewed collectively, of which
achieving some of them at the expense of others may not augur well for broad based
economic development in Nigeria specifically or Africa in general.
Using the analytical framework created in the thesis, various issues that have adversely
affected the full realisation of these key economic development benefits and created a
gap between the policy objectives behind privatisation law and the reality of
implementation were analysed. The approach of International Financial Institutions
(IFIs) (specifically the World Bank and the International Monetary Fund) to
privatisation was also considered in the thesis owing to the fact that they have had some
influence in its adoption and implementation in Nigeria, and Africa more broadly.
Privatisation entails more than just legal reform, thus, the research is interdisciplinary in
nature and principally touches on legal issues, public policy issues and issues pertaining
to economic development, including social issues
