Biomedical Sciences Research Complex (BSRC) Researchhttps://hdl.handle.net/10023/1762024-03-28T14:16:00Z2024-03-28T14:16:00ZHuman cytomegalovirus major immediate early 1 protein targets host chromosomes by docking to the acidic pocket on the nucleosome surfaceMücke, KatrinPaulus, ChristinaBernhardt, KatharinaGerrer, KatrinSchön, KathrinFink, AlinaSauer, Eva-MariaAsbach-Nitzsche, AlexandraHarwardt, ThomasKieninger, BärbelKremer, WernerKalbitzer, Hans RobertNevels, Michaelhttps://hdl.handle.net/10023/148712022-07-05T10:30:02Z2014-01-15T00:00:00ZThe 72-kDa immediate early 1 (IE1) protein encoded by human cytomegalovirus (hCMV) is a nuclearly localized promiscuous regulator of viral and cellular transcription. IE1 has long been known to associate with host mitotic chromatin, yet the mechanisms underlying this interaction have not been specified. In this study, we identify the cellular chromosome receptor for IE1. We demonstrate that the viral protein targets human nucleosomes by directly binding to core histones in a nucleic acid-independent manner. IE1 exhibits two separable histone-interacting regions with differential binding specificities for H2A-H2B and H3-H4. The H2A-H2B binding region was mapped to an evolutionarily conserved 10-amino-acid motif within the chromatin-tethering domain (CTD) of IE1. Results from experimental approaches combined with molecular modeling indicate that the IE1 CTD adopts a β-hairpin structure, docking with the acidic pocket formed by H2A-H2B on the nucleosome surface. IE1 binds to the acidic pocket in a way similar to that of the latency-associated nuclear antigen (LANA) of the Kaposi's sarcoma-associated herpesvirus. Consequently, the IE1 and LANA CTDs compete for binding to nucleosome cores and chromatin. Our work elucidates in detail how a key viral regulator is anchored to human chromosomes and identifies the nucleosomal acidic pocket as a joint target of proteins from distantly related viruses. Based on the striking similarities between the IE1 and LANA CTDs and the fact that nucleosome targeting by IE1 is dispensable for productive replication even in "clinical" strains of hCMV, we speculate that the two viral proteins may serve analogous functions during latency of their respective viruses.
2014-01-15T00:00:00ZMücke, KatrinPaulus, ChristinaBernhardt, KatharinaGerrer, KatrinSchön, KathrinFink, AlinaSauer, Eva-MariaAsbach-Nitzsche, AlexandraHarwardt, ThomasKieninger, BärbelKremer, WernerKalbitzer, Hans RobertNevels, MichaelThe 72-kDa immediate early 1 (IE1) protein encoded by human cytomegalovirus (hCMV) is a nuclearly localized promiscuous regulator of viral and cellular transcription. IE1 has long been known to associate with host mitotic chromatin, yet the mechanisms underlying this interaction have not been specified. In this study, we identify the cellular chromosome receptor for IE1. We demonstrate that the viral protein targets human nucleosomes by directly binding to core histones in a nucleic acid-independent manner. IE1 exhibits two separable histone-interacting regions with differential binding specificities for H2A-H2B and H3-H4. The H2A-H2B binding region was mapped to an evolutionarily conserved 10-amino-acid motif within the chromatin-tethering domain (CTD) of IE1. Results from experimental approaches combined with molecular modeling indicate that the IE1 CTD adopts a β-hairpin structure, docking with the acidic pocket formed by H2A-H2B on the nucleosome surface. IE1 binds to the acidic pocket in a way similar to that of the latency-associated nuclear antigen (LANA) of the Kaposi's sarcoma-associated herpesvirus. Consequently, the IE1 and LANA CTDs compete for binding to nucleosome cores and chromatin. Our work elucidates in detail how a key viral regulator is anchored to human chromosomes and identifies the nucleosomal acidic pocket as a joint target of proteins from distantly related viruses. Based on the striking similarities between the IE1 and LANA CTDs and the fact that nucleosome targeting by IE1 is dispensable for productive replication even in "clinical" strains of hCMV, we speculate that the two viral proteins may serve analogous functions during latency of their respective viruses.3-2: Invited paper: Color on Demand - Color-tunable OLEDs for lighting and displaysFröbel, MarkusSchwab, TobiasKliem, MonaLenk, SimoneLeo, KarlReineke, SebastianGather, Malte C.https://hdl.handle.net/10023/132742024-03-27T00:42:37Z2017-01-01T00:00:00ZA device concept for highly efficient OLEDs is introduced that allows to tune the emission color of the device over a broad range of the CIE color gamut. The approach exploits the different polarities of the positive and negative half-cycles of an alternating current driving signal to independently address two vertically stacked emission units with complementary color. Ultrathin metal electrodes fabricated by a wetting layer approach are used to achieve good electrical contact to each stack with minimal impact on optical performance.
2017-01-01T00:00:00ZFröbel, MarkusSchwab, TobiasKliem, MonaLenk, SimoneLeo, KarlReineke, SebastianGather, Malte C.A device concept for highly efficient OLEDs is introduced that allows to tune the emission color of the device over a broad range of the CIE color gamut. The approach exploits the different polarities of the positive and negative half-cycles of an alternating current driving signal to independently address two vertically stacked emission units with complementary color. Ultrathin metal electrodes fabricated by a wetting layer approach are used to achieve good electrical contact to each stack with minimal impact on optical performance.Carpe lucem: harnessing organic light sources for optogeneticsMorton, AndrewMurawski, CarolineGather, Malte Christianhttps://hdl.handle.net/10023/122362023-04-19T00:45:49Z2016-12-01T00:00:00ZWith the advent of optogenetics, numerous functions in cells have been rendered responsive to the experimental delivery of light. The most common implementation of this technique features neurons genetically modified to express light-sensitive ion channel proteins, which open specifically in response to pulses of blue light, triggering electrical impulses in neurons. Optogenetics now has matured to a point where in addition to answering fundamental questions about the function of the brain, scientists begin to consider clinical applications. Further progress in this field however will require new ways of delivering light. One of these involves the use of organic light-emitting diodes (OLEDs), a display technology increasingly common in modern-day smart phones, for the optical stimulation of cells.
2016-12-01T00:00:00ZMorton, AndrewMurawski, CarolineGather, Malte ChristianWith the advent of optogenetics, numerous functions in cells have been rendered responsive to the experimental delivery of light. The most common implementation of this technique features neurons genetically modified to express light-sensitive ion channel proteins, which open specifically in response to pulses of blue light, triggering electrical impulses in neurons. Optogenetics now has matured to a point where in addition to answering fundamental questions about the function of the brain, scientists begin to consider clinical applications. Further progress in this field however will require new ways of delivering light. One of these involves the use of organic light-emitting diodes (OLEDs), a display technology increasingly common in modern-day smart phones, for the optical stimulation of cells.Foundational and translational research opportunities to improve plant healthMichelmore, RichardCoaker, GittaBart, RebeccaBeattie, GwynBent, AndrewBruce, TobyCameron, DuncanDangl, JeffDinesh-Kumar, SavithrammaEdwards, RobEves-van den Akker, SebastianGassmann, WalterGreenberg, JeanHanley-Bowdoin, LindaHarrison, RichardHe, PingHarvey, JaggerHuffaker, AlisaHulbert, ScotInnes, RogerJones, JonathanKaloshian, IsghouhiKamoun, SophienKatagiri, FumiakiLeach, JanMa, WenboMcDowell, JohnMedford, JuneMeyers, BlakeNelson, RebeccaOliver, RichardQi, YipingSaunders, DianeShaw, MichaelSmart, ChristineSubudhi, PrasantaTorrance, LesleyTyler, BrettValent, BarbaraWalsh, Johnhttps://hdl.handle.net/10023/111422023-04-25T23:49:10Z2017-07-01T00:00:00ZThis whitepaper reports the deliberations of a workshop focused on biotic challenges to plant health held in Washington, D.C. in September 2016. Ensuring health of food plants is critical to maintaining the quality and productivity of crops and for sustenance of the rapidly growing human population. There is a close linkage between food security and societal stability; however, global food security is threatened by the vulnerability of our agricultural systems to numerous pests, pathogens, weeds, and environmental stresses. These threats are aggravated by climate change, the globalization of agriculture, and an over-reliance on non-sustainable inputs. New analytical and computational technologies are providing unprecedented resolution at a variety of molecular, cellular, organismal, and population scales for crop plants as well as pathogens, pests, beneficial microbes, and weeds. It is now possible to both characterize useful or deleterious variation as well as precisely manipulate it. Data-driven, informed decisions based on knowledge of the variation of biotic challenges and of natural and synthetic variation in crop plants will enable deployment of durable interventions throughout the world. These should be integral, dynamic components of agricultural strategies for sustainable agriculture.
2017-07-01T00:00:00ZMichelmore, RichardCoaker, GittaBart, RebeccaBeattie, GwynBent, AndrewBruce, TobyCameron, DuncanDangl, JeffDinesh-Kumar, SavithrammaEdwards, RobEves-van den Akker, SebastianGassmann, WalterGreenberg, JeanHanley-Bowdoin, LindaHarrison, RichardHe, PingHarvey, JaggerHuffaker, AlisaHulbert, ScotInnes, RogerJones, JonathanKaloshian, IsghouhiKamoun, SophienKatagiri, FumiakiLeach, JanMa, WenboMcDowell, JohnMedford, JuneMeyers, BlakeNelson, RebeccaOliver, RichardQi, YipingSaunders, DianeShaw, MichaelSmart, ChristineSubudhi, PrasantaTorrance, LesleyTyler, BrettValent, BarbaraWalsh, JohnThis whitepaper reports the deliberations of a workshop focused on biotic challenges to plant health held in Washington, D.C. in September 2016. Ensuring health of food plants is critical to maintaining the quality and productivity of crops and for sustenance of the rapidly growing human population. There is a close linkage between food security and societal stability; however, global food security is threatened by the vulnerability of our agricultural systems to numerous pests, pathogens, weeds, and environmental stresses. These threats are aggravated by climate change, the globalization of agriculture, and an over-reliance on non-sustainable inputs. New analytical and computational technologies are providing unprecedented resolution at a variety of molecular, cellular, organismal, and population scales for crop plants as well as pathogens, pests, beneficial microbes, and weeds. It is now possible to both characterize useful or deleterious variation as well as precisely manipulate it. Data-driven, informed decisions based on knowledge of the variation of biotic challenges and of natural and synthetic variation in crop plants will enable deployment of durable interventions throughout the world. These should be integral, dynamic components of agricultural strategies for sustainable agriculture.Orientation of OLED emitter molecules revealed by XRDMurawski, CarolineElschner, ChrisLenk, SimoneReineke, SebastianGather, Malte Christianhttps://hdl.handle.net/10023/98622023-04-19T00:41:20Z2016-11-14T00:00:00ZThin films of the phosphorescent emitters Ir(ppy)3 and Ir(ppy)2(acac) are investigated by GIXRD and GIWAXS. Both molecules form crystalline grains and exhibit a preferred orientation that is pertained even when doped into a host.
We thank Dr. Lutz Wilde at Fraunhofer IPMS, Center Nanoelectronic Technologies, Dresden for carrying out the GIXRD measurements. CM acknowledges funding by the Graduate Academy of the TU Dresden and by the European Commission through a Marie Skłodowska Curie individual fellowship (703387).
2016-11-14T00:00:00ZMurawski, CarolineElschner, ChrisLenk, SimoneReineke, SebastianGather, Malte ChristianThin films of the phosphorescent emitters Ir(ppy)3 and Ir(ppy)2(acac) are investigated by GIXRD and GIWAXS. Both molecules form crystalline grains and exhibit a preferred orientation that is pertained even when doped into a host.Organic light-emitting diodes for optogenetic stimulation of Drosophila larvaeMurawski, CarolineMorton, AndrewSamuel, Ifor David WilliamPulver, StefanGather, Malte Christianhttps://hdl.handle.net/10023/98612024-03-02T00:39:41Z2016-11-14T00:00:00ZOptogenetics is an emerging method in biology that enables controlling neurons with light. We use organic light-emitting diodes to stimulate neurons in Drosophila larvae and investigate subsequent behavioral changes at different light intensities.
We are grateful for financial support from the Scottish Funding Council (through SUPA), Human Frontier Science Program (RGY0074/2013), Wellcome Trust Institutional Strategic Support Fund St Andrews, the RS Macdonald Charitable Trust, and EPSRC via grant EP/J01771X/1. CM acknowledges funding by the European Commission through a Marie Skłodowska Curie individual fellowship (703387).
2016-11-14T00:00:00ZMurawski, CarolineMorton, AndrewSamuel, Ifor David WilliamPulver, StefanGather, Malte ChristianOptogenetics is an emerging method in biology that enables controlling neurons with light. We use organic light-emitting diodes to stimulate neurons in Drosophila larvae and investigate subsequent behavioral changes at different light intensities.A novel archaeal DNA repair factor that acts with the UvrABC system to repair mitomycin C-induced DNA damage in a PCNA-dependent mannerGiroux, XavierMacNeill, Stuarthttps://hdl.handle.net/10023/94242023-04-18T10:02:29Z2015-12-22T00:00:00ZThe sliding clamp PCNA plays a vital role in a number of DNA repair pathways in eukaryotes and archaea by acting as a stable platform onto which other essential protein factors assemble. Many of these proteins interact with PCNA via a short peptide sequence known as a PIP (PCNA interacting protein) motif. Here we describe the identification and functional analysis of a novel PCNA interacting protein NreA that is conserved in the archaea and which has a PIP motif at its C-terminus. Using the genetically tractable euryarchaeon Haloferax volcanii as a model system, we show that the NreA protein is not required for cell viability but that loss of NreA (or replacement of the wild-type protein with a truncated version lacking the C-terminal PIP motif) results in an increased sensitivity to the DNA damaging agent mitomycin C (MMC) that correlates with delayed repair of MMC-induced chromosomal DNA damage monitored by pulsed-field gel electrophoresis (PFGE). Genetic epistasis analysis in Hfx. volcanii suggests that NreA works together with the UvrABC proteins in repairing DNA damage resulting from exposure to MMC. The wide distribution of NreA family members implies an important role for the protein in DNA damage repair in all archaeal lineages.
This work was supported by the USAF Office of Scientific Research under award number FA9550-10-1-0421.
2015-12-22T00:00:00ZGiroux, XavierMacNeill, StuartThe sliding clamp PCNA plays a vital role in a number of DNA repair pathways in eukaryotes and archaea by acting as a stable platform onto which other essential protein factors assemble. Many of these proteins interact with PCNA via a short peptide sequence known as a PIP (PCNA interacting protein) motif. Here we describe the identification and functional analysis of a novel PCNA interacting protein NreA that is conserved in the archaea and which has a PIP motif at its C-terminus. Using the genetically tractable euryarchaeon Haloferax volcanii as a model system, we show that the NreA protein is not required for cell viability but that loss of NreA (or replacement of the wild-type protein with a truncated version lacking the C-terminal PIP motif) results in an increased sensitivity to the DNA damaging agent mitomycin C (MMC) that correlates with delayed repair of MMC-induced chromosomal DNA damage monitored by pulsed-field gel electrophoresis (PFGE). Genetic epistasis analysis in Hfx. volcanii suggests that NreA works together with the UvrABC proteins in repairing DNA damage resulting from exposure to MMC. The wide distribution of NreA family members implies an important role for the protein in DNA damage repair in all archaeal lineages.Lipidomic analysis of fats and oils – a lot more than just omega-3Smith, Terry Khttps://hdl.handle.net/10023/92432023-04-18T10:00:30Z2015-01-01T00:00:00ZEdible oils and fats are among the most abundant cooking ingredients in the world, and are an important part of a healthy balanced diet, especially if they are high in omega-6 and omega-3 polyunsaturated fatty acids. Rather than just the total fatty acid compositions, the analysis of individual lipid species within these oils and fats has become increasingly important. Within the past decade several mass spectrometric lipidomics methods have been adapted and applied to the analysis of edible oils and fats. These methodologies are vital for the analysis of a plethora of lipid species that will be important for numerous health and sustainability issues in the future.
TKS research is supported in part by the Wellcome Trust, SUSLA, BBSRC and the European Community’s Seventh Framework Programme under grant agreement No. 602773 (Project KINDReD)
2015-01-01T00:00:00ZSmith, Terry KEdible oils and fats are among the most abundant cooking ingredients in the world, and are an important part of a healthy balanced diet, especially if they are high in omega-6 and omega-3 polyunsaturated fatty acids. Rather than just the total fatty acid compositions, the analysis of individual lipid species within these oils and fats has become increasingly important. Within the past decade several mass spectrometric lipidomics methods have been adapted and applied to the analysis of edible oils and fats. These methodologies are vital for the analysis of a plethora of lipid species that will be important for numerous health and sustainability issues in the future.Taking a molecular motor for a spin : helicase mechanism studied by spin labelling and PELDORConstantinescu Aruxandei, DianaPetrovic-Stojanovska, BiljanaSchiemann, OlavNaismith, JimWhite, Malcolm Fhttps://hdl.handle.net/10023/79262023-04-18T10:04:50Z2015-12-10T00:00:00ZThe complex molecular motions central to the functions of helicases have long attracted attention. Protein crystallography has provided transformative insights into these dynamic conformational changes, however important questions about the true nature of helicase configurations during the catalytic cycle remain. Using pulsed EPR (PELDOR or DEER) to measure interdomain distances in solution, we have examined two representative helicases: PcrA from superfamily 1 and XPD from superfamily 2. The data show that PcrA is a dynamic structure with domain movements that correlate with particular functional states, confirming and extending the information gleaned from crystal structures and other techniques. XPD in contrast is shown to be a rigid protein with almost no conformational changes resulting from nucleotide or DNA binding, which is well described by static crystal structures. Our results highlight the complimentary nature of PELDOR to crystallography and the power of its precision in understanding the conformational changes relevant to helicase function.
Welcome Trust programme grant [WT091825MA to M.F.W., J.H.N.]; Wellcome Trust multi-user equipment grant [099149/Z/12/Z]. Royal Society Wolfseon Merit Award (to M.F.W., J.H.N.). Funding for open access charge: Wellcome Trust [WT091825MA].
2015-12-10T00:00:00ZConstantinescu Aruxandei, DianaPetrovic-Stojanovska, BiljanaSchiemann, OlavNaismith, JimWhite, Malcolm FThe complex molecular motions central to the functions of helicases have long attracted attention. Protein crystallography has provided transformative insights into these dynamic conformational changes, however important questions about the true nature of helicase configurations during the catalytic cycle remain. Using pulsed EPR (PELDOR or DEER) to measure interdomain distances in solution, we have examined two representative helicases: PcrA from superfamily 1 and XPD from superfamily 2. The data show that PcrA is a dynamic structure with domain movements that correlate with particular functional states, confirming and extending the information gleaned from crystal structures and other techniques. XPD in contrast is shown to be a rigid protein with almost no conformational changes resulting from nucleotide or DNA binding, which is well described by static crystal structures. Our results highlight the complimentary nature of PELDOR to crystallography and the power of its precision in understanding the conformational changes relevant to helicase function.Identification of a candidate Rad1 subunit for the kinetoplastid 9-1-1 (Rad9-Hus1-Rad1) complexMacNeill, Stuarthttps://hdl.handle.net/10023/67042023-04-18T09:56:36Z2014-12-19T00:00:00ZThe trimeric 9-1-1 (Rad9-Hus1-Rad1) complex plays an important role in the eukaryotic DNA damage response by recruiting DNA repair factors and checkpoint mediators to damaged sites. Extensively characterised in mammals and yeast, evidence is now emerging that 9-1-1 function is conserved beyond the relatively narrow evolutionary range of the Opisthokonts. Kinetoplastid Rad9 and Hus1 proteins have been identified and shown to be involved in the DNA damage response but Rad1 has remained elusive. In this study, PSI-BLAST iterative database searching, phylogenetic and structural modeling techniques are used to identify and characterise candidate Rad1 proteins in kinetoplastid organisms.
Date of Acceptance: 12/12/2014
2014-12-19T00:00:00ZMacNeill, StuartThe trimeric 9-1-1 (Rad9-Hus1-Rad1) complex plays an important role in the eukaryotic DNA damage response by recruiting DNA repair factors and checkpoint mediators to damaged sites. Extensively characterised in mammals and yeast, evidence is now emerging that 9-1-1 function is conserved beyond the relatively narrow evolutionary range of the Opisthokonts. Kinetoplastid Rad9 and Hus1 proteins have been identified and shown to be involved in the DNA damage response but Rad1 has remained elusive. In this study, PSI-BLAST iterative database searching, phylogenetic and structural modeling techniques are used to identify and characterise candidate Rad1 proteins in kinetoplastid organisms.Surface plasmon polariton modification in top-emitting organic light-emitting diodes for enhanced light outcouplingFuchs, C.Schwab, T.Wieczorek, M.Gather, M.C.Hofmann, S.Leo, K.Scholz, R.https://hdl.handle.net/10023/61582022-04-14T11:30:29Z2014-01-01T00:00:00ZWe report on the enhanced light outcoupling efficiency of monochrome top-emitting organic light-emitting diodes (OLEDs). These OLEDs incorporate a hole transport layer (HTL) material with a substantially lower refractive index (∼ 1:5) than the emitter material or the standard HTL material (∼ 1:8) of a reference device. This low-index HTL is situated between the opaque bottom metal contact (anode) and the active emission layer. Compared to an HTL with common refractive index, the dispersion relation of the surface plasmon polariton (SPP) mode from the opaque metal contact is shifted to smaller in-plane wavenumbers. This shift enhances the outcoupling efficiency as it reduces the total dissipated power of the emitter. Furthermore, the excitation of the coupled SPPs at the thin transparent metal top contact (cathode) is avoided by using an ultrathin top electrode. Hence, the coupling of the electroluminescence from the emitter molecules to all non-radiative evanescent modes, with respect to the emitter material, is reduced by at least a factor of two, additionally increasing the outcoupling efficiency. Furthermore, for sufficiently high refractive index contrast the shift of the SPP at the anode/organic interface can lead to in-plane wavenumbers smaller than the wavenumber within the organic emitter layer and outcoupling of all excited modes by high index light extraction structures, e.g. microlens, seems feasible. In accordance to optical simulations, the external quantum efficiency is enhanced by about 20% for monochrome green emitting OLEDs with low refractive index HTL compared to a reference sample.
2014-01-01T00:00:00ZFuchs, C.Schwab, T.Wieczorek, M.Gather, M.C.Hofmann, S.Leo, K.Scholz, R.We report on the enhanced light outcoupling efficiency of monochrome top-emitting organic light-emitting diodes (OLEDs). These OLEDs incorporate a hole transport layer (HTL) material with a substantially lower refractive index (∼ 1:5) than the emitter material or the standard HTL material (∼ 1:8) of a reference device. This low-index HTL is situated between the opaque bottom metal contact (anode) and the active emission layer. Compared to an HTL with common refractive index, the dispersion relation of the surface plasmon polariton (SPP) mode from the opaque metal contact is shifted to smaller in-plane wavenumbers. This shift enhances the outcoupling efficiency as it reduces the total dissipated power of the emitter. Furthermore, the excitation of the coupled SPPs at the thin transparent metal top contact (cathode) is avoided by using an ultrathin top electrode. Hence, the coupling of the electroluminescence from the emitter molecules to all non-radiative evanescent modes, with respect to the emitter material, is reduced by at least a factor of two, additionally increasing the outcoupling efficiency. Furthermore, for sufficiently high refractive index contrast the shift of the SPP at the anode/organic interface can lead to in-plane wavenumbers smaller than the wavenumber within the organic emitter layer and outcoupling of all excited modes by high index light extraction structures, e.g. microlens, seems feasible. In accordance to optical simulations, the external quantum efficiency is enhanced by about 20% for monochrome green emitting OLEDs with low refractive index HTL compared to a reference sample.Gammaherpesvirus infection modulates the temporal and spatial expression of SCGB1A1 (CCSP) and BPIFA1 (SPLUNC1) in the respiratory tractLeeming, Gail HKipar, AnjaHughes, David JBingle, LynneBennett, ElaineMoyo, Nathifa ATripp, Ralph ABigley, Alison LBingle, Colin DSample, Jeffery TStewart, James Phttps://hdl.handle.net/10023/60042024-03-26T00:41:48Z2015-06-01T00:00:00ZMurine γ-herpesvirus 68 (MHV-68) infection of Mus musculus-derived strains of mice is an established model of γ-herpesvirus infection. We have previously developed an alternative system using a natural host, the wood mouse (Apodemus sylvaticus), and shown that the MHV-68 M3 chemokine-binding protein contributes significantly to MHV-68 pathogenesis. Here we demonstrate in A. sylvaticus using high-density micro-arrays that M3 influences the expression of genes involved in the host response including Scgb1a1 and Bpifa1 that encode potential innate defense proteins secreted into the respiratory tract. Further analysis of MHV-68-infected animals showed that the levels of both protein and RNA for SCGB1A1 and BPIFA1 were decreased at day 7 post infection (p.i.) but increased at day 14 p.i. as compared with M3-deficient and mock-infected animals. The modulation of expression was most pronounced in bronchioles but was also present in the bronchi and trachea. Double staining using RNA in situ hybridization and immunohistology demonstrated that much of the BPIFA1 expression occurs in club cells along with SCGB1A1 and that BPIFA1 is stored within granules in these cells. The increase in SCGB1A1 and BPIFA1 expression at day 14 p.i. was associated with the differentiation of club cells into mucus-secreting cells. Our data highlight the role of club cells and the potential of SCGB1A1 and BPIFA1 as innate defense mediators during respiratory virus infection.
This work was supported by a Royal Society (London) University Research Fellowship (to JPS), by Biotechnology and Biological Sciences Research Council (UK) grants BB/K009664/1 (to JPS, AK, and GHL) and BB/K009737/1 (CDB and LB), US Public Health Service grant CA090208, the Penn State Hershey Cancer Institute, and a Biotechnology and Biological Sciences Research Council studentship (to GHL).
2015-06-01T00:00:00ZLeeming, Gail HKipar, AnjaHughes, David JBingle, LynneBennett, ElaineMoyo, Nathifa ATripp, Ralph ABigley, Alison LBingle, Colin DSample, Jeffery TStewart, James PMurine γ-herpesvirus 68 (MHV-68) infection of Mus musculus-derived strains of mice is an established model of γ-herpesvirus infection. We have previously developed an alternative system using a natural host, the wood mouse (Apodemus sylvaticus), and shown that the MHV-68 M3 chemokine-binding protein contributes significantly to MHV-68 pathogenesis. Here we demonstrate in A. sylvaticus using high-density micro-arrays that M3 influences the expression of genes involved in the host response including Scgb1a1 and Bpifa1 that encode potential innate defense proteins secreted into the respiratory tract. Further analysis of MHV-68-infected animals showed that the levels of both protein and RNA for SCGB1A1 and BPIFA1 were decreased at day 7 post infection (p.i.) but increased at day 14 p.i. as compared with M3-deficient and mock-infected animals. The modulation of expression was most pronounced in bronchioles but was also present in the bronchi and trachea. Double staining using RNA in situ hybridization and immunohistology demonstrated that much of the BPIFA1 expression occurs in club cells along with SCGB1A1 and that BPIFA1 is stored within granules in these cells. The increase in SCGB1A1 and BPIFA1 expression at day 14 p.i. was associated with the differentiation of club cells into mucus-secreting cells. Our data highlight the role of club cells and the potential of SCGB1A1 and BPIFA1 as innate defense mediators during respiratory virus infection.The protein coexpression problem in biotechnology and biomedicine : virus 2A and 2A-like sequences provide a solutionLuke, Garry AlecRyan, Martin Denishttps://hdl.handle.net/10023/55182024-03-28T00:41:20Z2013-10-01T00:00:00ZSynthetic biology enables us to create genes virtually at will. Ensuring that multiple genes are efficiently co-expressed within the same cell – to assemble multimeric complexes, to transfer biochemical pathways, to transfer ‘traits’, is more problematic. Viruses such as picornaviruses accomplish exactly this task: they generate multiple, different, proteins from a single open reading frame. The study of how foot-and-mouth disease virus (FMDV) controls it’s protein biogenesis lead to the discovery of a short oligopeptide sequence, ‘2A’, that is able to mediate a co-translational ‘cleavage’ between proteins. 2A and ‘2A-like’ sequences (from other viruses and cellular sequences) can be used to concatenate multiple gene sequences into a single gene, ensuring their co-expression within the same cell. These sequences are now being used in the treatment of cancer, in the production of pluripotent stem cells, to create transgenic plants and animals amongst a host of other biotechnological and biomedical applications.
The authors acknowledge the support of the UK Biotechnology and Biological Sciences Research Council (BBSRC), the Wellcome Trust and the UK Medical Research Council (MRC).
2013-10-01T00:00:00ZLuke, Garry AlecRyan, Martin DenisSynthetic biology enables us to create genes virtually at will. Ensuring that multiple genes are efficiently co-expressed within the same cell – to assemble multimeric complexes, to transfer biochemical pathways, to transfer ‘traits’, is more problematic. Viruses such as picornaviruses accomplish exactly this task: they generate multiple, different, proteins from a single open reading frame. The study of how foot-and-mouth disease virus (FMDV) controls it’s protein biogenesis lead to the discovery of a short oligopeptide sequence, ‘2A’, that is able to mediate a co-translational ‘cleavage’ between proteins. 2A and ‘2A-like’ sequences (from other viruses and cellular sequences) can be used to concatenate multiple gene sequences into a single gene, ensuring their co-expression within the same cell. These sequences are now being used in the treatment of cancer, in the production of pluripotent stem cells, to create transgenic plants and animals amongst a host of other biotechnological and biomedical applications.Optimisation of engineered Escherichia coli biofilms for enzymatic biosynthesis of L-halotryptophansPerni, S.Hackett, L.Goss, R.J.M.Simmons, M.J.Overton, T.W.https://hdl.handle.net/10023/52412022-04-12T14:30:25Z2013-11-04T00:00:00ZEngineered biofilms comprising a single recombinant species have demonstrated remarkable activity as novel biocatalysts for a range of applications. In this work, we focused on the biotransformation of 5-haloindole into 5-halotryptophan, a pharmaceutical intermediate, using Escherichia coli expressing a recombinant tryptophan synthase enzyme encoded by plasmid pSTB7. To optimise the reaction we compared two E. coli K-12 strains (MC4100 and MG1655) and their ompR234 mutants, which overproduce the adhesin curli (PHL644 and PHL628). The ompR234 mutation increased the quantity of biofilm in both MG1655 and MC4100 backgrounds. In all cases, no conversion of 5-haloindoles was observed using cells without the pSTB7 plasmid. Engineered biofilms of strains PHL628 pSTB7 and PHL644 pSTB7 generated more 5-halotryptophan than their corresponding planktonic cells. Flow cytometry revealed that the vast majority of cells were alive after 24 hour biotransformation reactions, both in planktonic and biofilm forms, suggesting that cell viability was not a major factor in the greater performance of biofilm reactions. Monitoring 5-haloindole depletion, 5-halotryptophan synthesis and the percentage conversion of the biotransformation reaction suggested that there were inherent differences between strains MG1655 and MC4100, and between planktonic and biofilm cells, in terms of tryptophan and indole metabolism and transport. The study has reinforced the need to thoroughly investigate bacterial physiology and make informed strain selections when developing biotransformation reactions.
This study was funded by a UK Biotechnology & Biological Sciences Research Council grant (BB/I006834/1) to MJS, RJMG and TWO and a quota PhD studentship to LH. The Accuri C6 instrument was awarded to TWO as a BD Accuri Creativity Award.
2013-11-04T00:00:00ZPerni, S.Hackett, L.Goss, R.J.M.Simmons, M.J.Overton, T.W.Engineered biofilms comprising a single recombinant species have demonstrated remarkable activity as novel biocatalysts for a range of applications. In this work, we focused on the biotransformation of 5-haloindole into 5-halotryptophan, a pharmaceutical intermediate, using Escherichia coli expressing a recombinant tryptophan synthase enzyme encoded by plasmid pSTB7. To optimise the reaction we compared two E. coli K-12 strains (MC4100 and MG1655) and their ompR234 mutants, which overproduce the adhesin curli (PHL644 and PHL628). The ompR234 mutation increased the quantity of biofilm in both MG1655 and MC4100 backgrounds. In all cases, no conversion of 5-haloindoles was observed using cells without the pSTB7 plasmid. Engineered biofilms of strains PHL628 pSTB7 and PHL644 pSTB7 generated more 5-halotryptophan than their corresponding planktonic cells. Flow cytometry revealed that the vast majority of cells were alive after 24 hour biotransformation reactions, both in planktonic and biofilm forms, suggesting that cell viability was not a major factor in the greater performance of biofilm reactions. Monitoring 5-haloindole depletion, 5-halotryptophan synthesis and the percentage conversion of the biotransformation reaction suggested that there were inherent differences between strains MG1655 and MC4100, and between planktonic and biofilm cells, in terms of tryptophan and indole metabolism and transport. The study has reinforced the need to thoroughly investigate bacterial physiology and make informed strain selections when developing biotransformation reactions.The conserved transmembrane proteoglycan Perdido/Kon-tiki is essential for myofibrillogenesis and sarcomeric structure in DrosophilaPérez-Moreno, J JBischoff, MMartín-Bermudo, M DEstrada, Bhttps://hdl.handle.net/10023/50402024-03-22T00:41:02Z2014-07-01T00:00:00ZMuscle differentiation requires the assembly of high-order structures called myofibrils, composed of sarcomeres. Even though the molecular organization of sarcomeres is well known, the mechanisms underlying myofibrillogenesis are poorly understood. It has been proposed that integrin-dependent adhesion nucleates myofibril at the periphery of the muscle cell to sustain sarcomere assembly. Here, we report a role for the gene perdido (perd, also known as kon-tiki, a transmembrane chondroitin proteoglycan) in myofibrillogenesis. Expression of perd RNAi in muscles, prior to adult myogenesis, can induce misorientation and detachment of Drosophila adult abdominal muscles. In comparison to controls, perd-depleted muscles contain fewer myofibrils, localized at the cell periphery. These myofibrils are detached from each other and display a defective sarcomeric structure. Our results demonstrate that the extracellular matrix receptor Perd has a specific role in the assembly of myofibrils and in sarcomeric organization. We suggest that Perd acts downstream or in parallel to integrins to enable the connection of nascent myofibrils to the Z-bands. Our work identifies the Drosophila adult abdominal muscles as a model to investigate in vivo the mechanisms behind myofibrillogenesis.
M.B. was funded by a Wellcome Trust Senior Investigator Award to P. Lawrence (WT096645MA).
2014-07-01T00:00:00ZPérez-Moreno, J JBischoff, MMartín-Bermudo, M DEstrada, BMuscle differentiation requires the assembly of high-order structures called myofibrils, composed of sarcomeres. Even though the molecular organization of sarcomeres is well known, the mechanisms underlying myofibrillogenesis are poorly understood. It has been proposed that integrin-dependent adhesion nucleates myofibril at the periphery of the muscle cell to sustain sarcomere assembly. Here, we report a role for the gene perdido (perd, also known as kon-tiki, a transmembrane chondroitin proteoglycan) in myofibrillogenesis. Expression of perd RNAi in muscles, prior to adult myogenesis, can induce misorientation and detachment of Drosophila adult abdominal muscles. In comparison to controls, perd-depleted muscles contain fewer myofibrils, localized at the cell periphery. These myofibrils are detached from each other and display a defective sarcomeric structure. Our results demonstrate that the extracellular matrix receptor Perd has a specific role in the assembly of myofibrils and in sarcomeric organization. We suggest that Perd acts downstream or in parallel to integrins to enable the connection of nascent myofibrils to the Z-bands. Our work identifies the Drosophila adult abdominal muscles as a model to investigate in vivo the mechanisms behind myofibrillogenesis.Numerical investigation of passive optical sorting of plasmon nanoparticlesPloschner, MartinMazilu, MichaelCizmar, TomasDholakia, Kishanhttps://hdl.handle.net/10023/47362023-04-18T09:44:02Z2011-07-06T00:00:00ZWe explore the passive optical sorting of plasmon nanoparticles and investigate the optimal wavelength and optimal beam shape of incident field. The condition for optimal wavelength is found by maximising the nanoparticle separation whilst minimising the temperature increase in the system. We then use the force optical eigenmode (FOEi) method to find the beam shape of incident electromagnetic field, maximising the force difference between plasmon nanoparticles. The maximum force difference is found with respect to the whole sorting region. The combination of wavelength and beam shape study is demonstrated for a specific case of gold nanoparticles of radius 40 nm and 50 nm respectively. The optimum wavelength for this particular situation is found to be above 700 nm. The optimum beam shape depends upon the size of sorting region and ranges from plane-wave illumination for infinite sorting region to a field maximising gradient force difference in a single point. (C) 2011 Optical Society of America
Funding: UK Engineering and Physical Sciences Research Council for funding, KD is a Royal Society-Wolfson Merit Award Holder.
2011-07-06T00:00:00ZPloschner, MartinMazilu, MichaelCizmar, TomasDholakia, KishanWe explore the passive optical sorting of plasmon nanoparticles and investigate the optimal wavelength and optimal beam shape of incident field. The condition for optimal wavelength is found by maximising the nanoparticle separation whilst minimising the temperature increase in the system. We then use the force optical eigenmode (FOEi) method to find the beam shape of incident electromagnetic field, maximising the force difference between plasmon nanoparticles. The maximum force difference is found with respect to the whole sorting region. The combination of wavelength and beam shape study is demonstrated for a specific case of gold nanoparticles of radius 40 nm and 50 nm respectively. The optimum wavelength for this particular situation is found to be above 700 nm. The optimum beam shape depends upon the size of sorting region and ranges from plane-wave illumination for infinite sorting region to a field maximising gradient force difference in a single point. (C) 2011 Optical Society of AmericaRapid regulation of protein activity in fission yeastBoe, Cathrine A.Garcia, IgnacioPai, Chen-ChunSharom, Jeffrey R.Skjolberg, Henriette C.Boye, ErikKearsey, StephenMacNeill, Stuart AndrewTyers, Michael D.Grallert, Beatahttps://hdl.handle.net/10023/46292023-04-18T09:40:07Z2008-05-05T00:00:00ZBackground: The fission yeast Schizosaccharomyces pombe is widely-used as a model organism for the study of a broad range of eukaryotic cellular processes such as cell cycle, genome stability and cell morphology. Despite the availability of extensive set of genetic, molecular biological, biochemical and cell biological tools for analysis of protein function in fissio n yeast, studies are often hampered by the lack of an effective method allowing for the rapid regulation of protein level or protein activity. Results: In order to be able to regulate protein function, we have made use of a previous finding that the hormone binding domain of steroid receptors can be used as a regulatory cassette to subject the activity of heterologous proteins to hormonal regulation. The approach is based on fusing the protein of interest to the hormone binding domain (HBD) of the estrogen receptor (ER). The HBD tag will attract the Hsp90 complex, which can render the fusion protein inactive. Upon addition of estradiol the protein is quickly released from the Hsp90 complex and thereby activated. We have tagged and characterised the induction of activity of four different HBD-tagged proteins. Here we show that the tag provided the means to effectively regulate the activity of two of these proteins. Conclusion: The estradiol-regulatable hormone binding do main provides a means to regulate the function of some, though not all, fission yeast proteins. This system may result in very quick and reversible activation of the protein of interest. Therefore it will be a powerful tool and it will open experimental approaches in fission yeast that have previously not been possible. Since fission yeast is a widely-used model organism, this will be valuable in many areas of research.
2008-05-05T00:00:00ZBoe, Cathrine A.Garcia, IgnacioPai, Chen-ChunSharom, Jeffrey R.Skjolberg, Henriette C.Boye, ErikKearsey, StephenMacNeill, Stuart AndrewTyers, Michael D.Grallert, BeataBackground: The fission yeast Schizosaccharomyces pombe is widely-used as a model organism for the study of a broad range of eukaryotic cellular processes such as cell cycle, genome stability and cell morphology. Despite the availability of extensive set of genetic, molecular biological, biochemical and cell biological tools for analysis of protein function in fissio n yeast, studies are often hampered by the lack of an effective method allowing for the rapid regulation of protein level or protein activity. Results: In order to be able to regulate protein function, we have made use of a previous finding that the hormone binding domain of steroid receptors can be used as a regulatory cassette to subject the activity of heterologous proteins to hormonal regulation. The approach is based on fusing the protein of interest to the hormone binding domain (HBD) of the estrogen receptor (ER). The HBD tag will attract the Hsp90 complex, which can render the fusion protein inactive. Upon addition of estradiol the protein is quickly released from the Hsp90 complex and thereby activated. We have tagged and characterised the induction of activity of four different HBD-tagged proteins. Here we show that the tag provided the means to effectively regulate the activity of two of these proteins. Conclusion: The estradiol-regulatable hormone binding do main provides a means to regulate the function of some, though not all, fission yeast proteins. This system may result in very quick and reversible activation of the protein of interest. Therefore it will be a powerful tool and it will open experimental approaches in fission yeast that have previously not been possible. Since fission yeast is a widely-used model organism, this will be valuable in many areas of research.MCM-GINS and MCM-MCM interactions in vivo visualised by bimolecular fluorescence complementation in fission yeastAkman, GoekhanMacNeill, Stuart Andrewhttps://hdl.handle.net/10023/46282023-04-18T09:40:07Z2009-02-19T00:00:00ZBackground: Each of the three individual components of the CMG complex (Cdc45, MCM and GINS) is essential for chromosomal DNA replication in eukaryotic cells, both for the initiation of replication at origins and also for normal replication fork progression. The MCM complex is a DNA helicase that most likely functions as the catalytic core of the replicative helicase, unwinding the parental duplex DNA ahead of the moving replication fork, whereas Cdc45 and the GINS complex are believed to act as accessory factors for MCM. Results:To investigate interactions between components of the CMG complex, we have used bimolecular fluorescence complementation (BiFC) in the fission yeast Schizosaccharomyces pombe for the first time, to analyse protein-protein interactions between GINS and MCM subunits expressed from their native chromosomal loci. We demonstrate interactions between GINS andMCM in the nuclei of exponentially-growing fission yeast cells and on chromatin in binucleate S-phase cells. In addition we present evidence of MCM-MCM interactions in diploid fission yeast cells. As with GINS-MCM interactions, MCM-MCM interactions also occur on chromatin in S-phase cells. Conclusion: Bimolecular fluorescence complementation can be used in fission yeast to visualise interactions between two of the three components of the CMG complex, offering the prospect that this technique could in the future be used to allow studies on replication protein dynamics in living S. pombe cells.
2009-02-19T00:00:00ZAkman, GoekhanMacNeill, Stuart AndrewBackground: Each of the three individual components of the CMG complex (Cdc45, MCM and GINS) is essential for chromosomal DNA replication in eukaryotic cells, both for the initiation of replication at origins and also for normal replication fork progression. The MCM complex is a DNA helicase that most likely functions as the catalytic core of the replicative helicase, unwinding the parental duplex DNA ahead of the moving replication fork, whereas Cdc45 and the GINS complex are believed to act as accessory factors for MCM. Results:To investigate interactions between components of the CMG complex, we have used bimolecular fluorescence complementation (BiFC) in the fission yeast Schizosaccharomyces pombe for the first time, to analyse protein-protein interactions between GINS and MCM subunits expressed from their native chromosomal loci. We demonstrate interactions between GINS andMCM in the nuclei of exponentially-growing fission yeast cells and on chromatin in binucleate S-phase cells. In addition we present evidence of MCM-MCM interactions in diploid fission yeast cells. As with GINS-MCM interactions, MCM-MCM interactions also occur on chromatin in S-phase cells. Conclusion: Bimolecular fluorescence complementation can be used in fission yeast to visualise interactions between two of the three components of the CMG complex, offering the prospect that this technique could in the future be used to allow studies on replication protein dynamics in living S. pombe cells.Functional mapping of the fission yeast DNA polymerase delta B-subunit Cdc1 by site-directed and random pentapeptide insertion mutagenesisGarcia, JSBaranovskiy, AGKnatko, EVGray, FCTahirov, THMacNeill, Stuart Andrewhttps://hdl.handle.net/10023/46272024-03-22T00:39:35Z2009-08-17T00:00:00ZBackground: DNA polymerase delta plays an essential role in chromosomal DNA replication in eukaryotic cells, being responsible for synthesising the bulk of the lagging strand. In fission yeast, Pol delta is a heterotetrameric enzyme comprising four evolutionarily well-conserved proteins: the catalytic subunit Pol3 and three smaller subunits Cdc1, Cdc27 and Cdm1. Pol3 binds directly to the B-subunit, Cdc1, which in turn binds the C-subunit, Cdc27. Human Pol d comprises the same four subunits, and the crystal structure was recently reported of a complex of human p50 and the N-terminal domain of p66, the human orthologues of Cdc1 and Cdc27, respectively. Results: To gain insights into the structure and function of Cdc1, random and directed mutagenesis techniques were used to create a collection of thirty alleles encoding mutant Cdc1 proteins. Each allele was tested for function in fission yeast and for binding of the altered protein to Pol3 and Cdc27 using the two-hybrid system. Additionally, the locations of the amino acid changes in each protein were mapped onto the three-dimensional structure of human p50. The results obtained from these studies identify amino acid residues and regions within the Cdc1 protein that are essential for interaction with Pol3 and Cdc27 and for in vivo function. Mutations specifically defective in Pol3-Cdc1 interactions allow the identification of a possible Pol3 binding surface on Cdc1. Conclusion: In the absence of a three-dimensional structure of the entire Pol d complex, the results of this study highlight regions in Cdc1 that are vital for protein function in vivo and provide valuable clues to possible protein-protein interaction surfaces on the Cdc1 protein that will be important targets for further study.
JSG was supported by a BBSRC Research Committee studentship, EVK by the Darwin Trust of Edinburgh, and FCG and SAM by a Wellcome Trust Senior Research Fellowship in Basic Biomedical Sciences. AGB and THT research was supported by UNMC Eppley Cancer Center Pilot Project LB595 to THT.
2009-08-17T00:00:00ZGarcia, JSBaranovskiy, AGKnatko, EVGray, FCTahirov, THMacNeill, Stuart AndrewBackground: DNA polymerase delta plays an essential role in chromosomal DNA replication in eukaryotic cells, being responsible for synthesising the bulk of the lagging strand. In fission yeast, Pol delta is a heterotetrameric enzyme comprising four evolutionarily well-conserved proteins: the catalytic subunit Pol3 and three smaller subunits Cdc1, Cdc27 and Cdm1. Pol3 binds directly to the B-subunit, Cdc1, which in turn binds the C-subunit, Cdc27. Human Pol d comprises the same four subunits, and the crystal structure was recently reported of a complex of human p50 and the N-terminal domain of p66, the human orthologues of Cdc1 and Cdc27, respectively. Results: To gain insights into the structure and function of Cdc1, random and directed mutagenesis techniques were used to create a collection of thirty alleles encoding mutant Cdc1 proteins. Each allele was tested for function in fission yeast and for binding of the altered protein to Pol3 and Cdc27 using the two-hybrid system. Additionally, the locations of the amino acid changes in each protein were mapped onto the three-dimensional structure of human p50. The results obtained from these studies identify amino acid residues and regions within the Cdc1 protein that are essential for interaction with Pol3 and Cdc27 and for in vivo function. Mutations specifically defective in Pol3-Cdc1 interactions allow the identification of a possible Pol3 binding surface on Cdc1. Conclusion: In the absence of a three-dimensional structure of the entire Pol d complex, the results of this study highlight regions in Cdc1 that are vital for protein function in vivo and provide valuable clues to possible protein-protein interaction surfaces on the Cdc1 protein that will be important targets for further study.Human tissue in systems medicineCaie, Peter DavidSchuur, KlaasOniscu, AncaMullen, PeterReynolds, Paul AndrewHarrison, David Jameshttps://hdl.handle.net/10023/43112024-02-29T00:41:43Z2013-12-01T00:00:00ZHistopathology, the examination of an architecturally artefactual, two dimensional, static image remains a potent tool allowing diagnosis and empirical expectation of prognosis. Considerable optimism exists that the advent of molecular genetic testing and other biomarker strategies will improve or even replace this ancient technology. A number of biomarkers add considerable value for prediction of whether a treatment will work. This short review argues that a systems medicine approach to pathology will not seek to replace traditional pathology, but rather augment it. Systems approaches need to incorporate quantitative morphological, protein, mRNA and DNA. A significant challenge for clinical implementation of systems pathology is how to optimise information available from tissue, which is frequently sub-optimal in quality and amount, and yet generate useful predictive models which work. The transition of histopathology to systems pathophysiology and the use of multiscale datasets ushers in a new era in diagnosis, prognosis and prediction based on analysis of human tissue.
2013-12-01T00:00:00ZCaie, Peter DavidSchuur, KlaasOniscu, AncaMullen, PeterReynolds, Paul AndrewHarrison, David JamesHistopathology, the examination of an architecturally artefactual, two dimensional, static image remains a potent tool allowing diagnosis and empirical expectation of prognosis. Considerable optimism exists that the advent of molecular genetic testing and other biomarker strategies will improve or even replace this ancient technology. A number of biomarkers add considerable value for prediction of whether a treatment will work. This short review argues that a systems medicine approach to pathology will not seek to replace traditional pathology, but rather augment it. Systems approaches need to incorporate quantitative morphological, protein, mRNA and DNA. A significant challenge for clinical implementation of systems pathology is how to optimise information available from tissue, which is frequently sub-optimal in quality and amount, and yet generate useful predictive models which work. The transition of histopathology to systems pathophysiology and the use of multiscale datasets ushers in a new era in diagnosis, prognosis and prediction based on analysis of human tissue.Crystal structure of a DNA containing the planar, phenoxazine-derived bi-functional spectroscopic probe ÇEdwards, Thomas E.Cekan, PavolReginsson, Gunnar WidtfeldtShelke, Sandip A.Ferre D'Amare, Adrian R.Schiemann, OlavSigurdsson, Snorrihttps://hdl.handle.net/10023/42702022-04-26T10:30:07Z2011-05-01T00:00:00ZElectron Paramagnetic Resonance (EPR) spectroscopy and fluorescence spectroscopy are complementary biophysical techniques used to examine the structure and dynamics of macromolecules. We have previously described the bi-functional spectroscopic probe Ç for the study of nucleic acid structure and dynamics using EPR and fluorescence spectroscopy. As with any newly designed spectroscopic probe, the utility, functionality, and the structural effects of the probe on the nucleic acid must be examined in detail. Initial EPR, fluorescence, and thermal denaturation studies indicated that the phenoxazine-derived spin-labeled deoxycytosine analog Ç forms a structurally non-perturbing base-pair with deoxyguanosine in DNA. Here we extend the analysis of the spectroscopic probe by presenting a detailed crystallographic study of this label based on small molecule crystal structures of the nucleoside base ç and its phenoxazine analog as well as a 1.7 Å resolution crystal structure of Ç within a decamer duplex A-form DNA. The DNA crystal structure confirms that the spin-labeled deoxycytosine analog forms a non-perturbing base-pair with deoxyguanosine. Interestingly, this structure and also the one of the phenoxazine base show the label in a planar conformation, whereas the structure of the free spin label base ç has a bend at the oxazine linkage. Density function theory (DFT) calculations reveal that both conformations are very close in energy and possess both the same frequency for bending at the oxazine linkage. These results are interpreted as a small degree of bending flexibility around the oxazine linkage, which may be a consequence of the antiaromaticity in this 16-pi electron ring system. Within DNA, the amplitude of the bending motion is likely to be restricted due to steric hindrance. This detailed structural analysis shows that the spin label base ç can be used with high confidence in EPR- or fluorescence-based structural and dynamics studies of oligonucleotides.
2011-05-01T00:00:00ZEdwards, Thomas E.Cekan, PavolReginsson, Gunnar WidtfeldtShelke, Sandip A.Ferre D'Amare, Adrian R.Schiemann, OlavSigurdsson, SnorriElectron Paramagnetic Resonance (EPR) spectroscopy and fluorescence spectroscopy are complementary biophysical techniques used to examine the structure and dynamics of macromolecules. We have previously described the bi-functional spectroscopic probe Ç for the study of nucleic acid structure and dynamics using EPR and fluorescence spectroscopy. As with any newly designed spectroscopic probe, the utility, functionality, and the structural effects of the probe on the nucleic acid must be examined in detail. Initial EPR, fluorescence, and thermal denaturation studies indicated that the phenoxazine-derived spin-labeled deoxycytosine analog Ç forms a structurally non-perturbing base-pair with deoxyguanosine in DNA. Here we extend the analysis of the spectroscopic probe by presenting a detailed crystallographic study of this label based on small molecule crystal structures of the nucleoside base ç and its phenoxazine analog as well as a 1.7 Å resolution crystal structure of Ç within a decamer duplex A-form DNA. The DNA crystal structure confirms that the spin-labeled deoxycytosine analog forms a non-perturbing base-pair with deoxyguanosine. Interestingly, this structure and also the one of the phenoxazine base show the label in a planar conformation, whereas the structure of the free spin label base ç has a bend at the oxazine linkage. Density function theory (DFT) calculations reveal that both conformations are very close in energy and possess both the same frequency for bending at the oxazine linkage. These results are interpreted as a small degree of bending flexibility around the oxazine linkage, which may be a consequence of the antiaromaticity in this 16-pi electron ring system. Within DNA, the amplitude of the bending motion is likely to be restricted due to steric hindrance. This detailed structural analysis shows that the spin label base ç can be used with high confidence in EPR- or fluorescence-based structural and dynamics studies of oligonucleotides.Femtosecond optoinjection of intact tobacco BY-2 cells using a reconfigurable photoporation platformMitchell, C.A.Kalies, S.Cizmár, T.Heisterkamp, A.Torrance, L.Roberts, A.G.Gunn-Moore, F.J.Dholakia, K.https://hdl.handle.net/10023/42342022-04-26T11:30:20Z2013-11-14T00:00:00ZA tightly-focused ultrashort pulsed laser beam incident upon a cell membrane has previously been shown to transiently increase cell membrane permeability while maintaining the viability of the cell, a technique known as photoporation. This permeability can be used to aid the passage of membrane-impermeable biologically-relevant substances such as dyes, proteins and nucleic acids into the cell. Ultrashort-pulsed lasers have proven to be indispensable for photoporating mammalian cells but they have rarely been applied to plant cells due to their larger sizes and rigid and thick cell walls, which significantly hinders the intracellular delivery of exogenous substances. Here we demonstrate and quantify femtosecond optical injection of membrane impermeable dyes into intact BY-2 tobacco plant cells growing in culture, investigating both optical and biological parameters. Specifically, we show that the long axial extent of a propagation invariant (“diffraction-free”) Bessel beam, which relaxes the requirements for tight focusing on the cell membrane, outperforms a standard Gaussian photoporation beam, achieving up to 70% optoinjection efficiency. Studies on the osmotic effects of culture media show that a hypertonic extracellular medium was found to be necessary to reduce turgor pressure and facilitate molecular entry into the cells.
2013-11-14T00:00:00ZMitchell, C.A.Kalies, S.Cizmár, T.Heisterkamp, A.Torrance, L.Roberts, A.G.Gunn-Moore, F.J.Dholakia, K.A tightly-focused ultrashort pulsed laser beam incident upon a cell membrane has previously been shown to transiently increase cell membrane permeability while maintaining the viability of the cell, a technique known as photoporation. This permeability can be used to aid the passage of membrane-impermeable biologically-relevant substances such as dyes, proteins and nucleic acids into the cell. Ultrashort-pulsed lasers have proven to be indispensable for photoporating mammalian cells but they have rarely been applied to plant cells due to their larger sizes and rigid and thick cell walls, which significantly hinders the intracellular delivery of exogenous substances. Here we demonstrate and quantify femtosecond optical injection of membrane impermeable dyes into intact BY-2 tobacco plant cells growing in culture, investigating both optical and biological parameters. Specifically, we show that the long axial extent of a propagation invariant (“diffraction-free”) Bessel beam, which relaxes the requirements for tight focusing on the cell membrane, outperforms a standard Gaussian photoporation beam, achieving up to 70% optoinjection efficiency. Studies on the osmotic effects of culture media show that a hypertonic extracellular medium was found to be necessary to reduce turgor pressure and facilitate molecular entry into the cells.Exploring the ultrashort pulse laser parameter space for membrane permeabilisation in mammalian cells.Rudhall, Andrew PeterAntkowiak, MaciejTsampoula, XanthiMazilu, MichaelMetzger, N KlausGunn-Moore, Frank JDholakia, Kishanhttps://hdl.handle.net/10023/39192023-04-18T09:46:20Z2012-01-01T00:00:00ZThe use of ultrashort femtosecond pulsed lasers to effect membrane permeabilisation and initiate both optoinjection and transfection of cells has recently seen immense interest. We investigate femtosecond laser-induced membrane permeabilisation in mammalian cells as a function of pulse duration, pulse energy and number of pulses, by quantifying the efficiency of optoinjection for these parameters. Depending on pulse duration and pulse energy we identify two distinct membrane permeabilisation regimes. In the first regime a nonlinear dependence of order 3.4-9.6 is exhibited below a threshold peak power of at least 6 kW. Above this threshold peak power, the nonlinear dependence is saturated resulting in linear behaviour. This indicates that the membrane permeabilisation mechanism requires efficient multiphoton absorption to produce free electrons but once this process saturates, linear absorption dominates. Our experimental findings support a previously proposed theoretical model and provide a step towards the optimisation of laser-mediated gene delivery into mammalian cells.
2012-01-01T00:00:00ZRudhall, Andrew PeterAntkowiak, MaciejTsampoula, XanthiMazilu, MichaelMetzger, N KlausGunn-Moore, Frank JDholakia, KishanThe use of ultrashort femtosecond pulsed lasers to effect membrane permeabilisation and initiate both optoinjection and transfection of cells has recently seen immense interest. We investigate femtosecond laser-induced membrane permeabilisation in mammalian cells as a function of pulse duration, pulse energy and number of pulses, by quantifying the efficiency of optoinjection for these parameters. Depending on pulse duration and pulse energy we identify two distinct membrane permeabilisation regimes. In the first regime a nonlinear dependence of order 3.4-9.6 is exhibited below a threshold peak power of at least 6 kW. Above this threshold peak power, the nonlinear dependence is saturated resulting in linear behaviour. This indicates that the membrane permeabilisation mechanism requires efficient multiphoton absorption to produce free electrons but once this process saturates, linear absorption dominates. Our experimental findings support a previously proposed theoretical model and provide a step towards the optimisation of laser-mediated gene delivery into mammalian cells.Characterization and downstream mannose phosphorylation of human recombinant α-L-iduronidase produced in Arabidopsis complex glycan-deficient (cgl) seedsHe, XuPierce, OwenHaselhorst, ThomasKolarich, DanielPacker, Nicolle H.Gloster, TraceyVocadlo, David J.Qian, YiBrooks, DougKermode, Allison R.https://hdl.handle.net/10023/39102024-02-27T00:41:32Z2013-12-01T00:00:00ZMucopolysaccharidosis (MPS) I is a lysosomal storage disease caused by a deficiency of α-L-iduronidase (IDUA) (EC 3.2.1.76); enzyme replacement therapy is the conventional treatment for this genetic disease. Arabidopsis cgl mutants are characterized by a deficiency of the activity of N-acetylglucosaminyl transferase I (EC 2.4.1.101), the first enzyme in the pathway of hybrid and complex N-glycan biosynthesis. To develop a seed-based platform for the production of recombinant IDUA for potential treatment of MPS I, cgl mutant seeds were generated to express human IDUA at high yields and to avoid maturation of the N-linked glycans on the recombinant human enzyme. Enzyme kinetic data showed that cgl-IDUA has similar enzymatic properties to the commercial recombinant IDUA derived from cultured Chinese hamster ovary (CHO) cells (AldurazymeTM). The N-glycan profile showed that cgl-derived IDUA contained predominantly high-mannose-type N-glycans (94.5%), and the residual complex/hybrid N-glycan-containing enzyme was efficiently removed by an additional affinity chromatography step. Furthermore, purified cgl-IDUA was amenable to sequential in vitro processing by soluble recombinant forms of the two enzymes that mediate the addition of the mannose-6-phosphate (M6P) tag in mammalian cells—UDP-GlcNAc:lysosomal enzyme N−acetylglucosamine (GlcNAc)−1−phosphotransferase—and GlcNAc−1−phosphodiester α−N−acetylglucosaminidase (the ‘uncovering enzyme’). Arabidopsis seeds provide an alternative system for producing recombinant lysosomal enzymes for enzyme replacement therapy; the purified enzymes can be subjected to downstream processing to create the M6P, a recognition marker essential for efficient receptor-mediated uptake into lysosomes of human cells.
This work was supported by a Wellcome Trust award to TMG.
2013-12-01T00:00:00ZHe, XuPierce, OwenHaselhorst, ThomasKolarich, DanielPacker, Nicolle H.Gloster, TraceyVocadlo, David J.Qian, YiBrooks, DougKermode, Allison R.Mucopolysaccharidosis (MPS) I is a lysosomal storage disease caused by a deficiency of α-L-iduronidase (IDUA) (EC 3.2.1.76); enzyme replacement therapy is the conventional treatment for this genetic disease. Arabidopsis cgl mutants are characterized by a deficiency of the activity of N-acetylglucosaminyl transferase I (EC 2.4.1.101), the first enzyme in the pathway of hybrid and complex N-glycan biosynthesis. To develop a seed-based platform for the production of recombinant IDUA for potential treatment of MPS I, cgl mutant seeds were generated to express human IDUA at high yields and to avoid maturation of the N-linked glycans on the recombinant human enzyme. Enzyme kinetic data showed that cgl-IDUA has similar enzymatic properties to the commercial recombinant IDUA derived from cultured Chinese hamster ovary (CHO) cells (AldurazymeTM). The N-glycan profile showed that cgl-derived IDUA contained predominantly high-mannose-type N-glycans (94.5%), and the residual complex/hybrid N-glycan-containing enzyme was efficiently removed by an additional affinity chromatography step. Furthermore, purified cgl-IDUA was amenable to sequential in vitro processing by soluble recombinant forms of the two enzymes that mediate the addition of the mannose-6-phosphate (M6P) tag in mammalian cells—UDP-GlcNAc:lysosomal enzyme N−acetylglucosamine (GlcNAc)−1−phosphotransferase—and GlcNAc−1−phosphodiester α−N−acetylglucosaminidase (the ‘uncovering enzyme’). Arabidopsis seeds provide an alternative system for producing recombinant lysosomal enzymes for enzyme replacement therapy; the purified enzymes can be subjected to downstream processing to create the M6P, a recognition marker essential for efficient receptor-mediated uptake into lysosomes of human cells.Fluorescence suppression using wavelength modulated Raman spectroscopy in fiber-probe-based tissue analysisBalagopal, BavishnaAshok, Praveen CheriyanMazilu, MichaelRiches, Andrew CliveHerrington, C SimonDholakia, Kishanhttps://hdl.handle.net/10023/33032024-02-17T00:40:25Z2012-07-09T00:00:00ZIn the field of biomedical optics, Raman spectroscopy is a powerful tool for probing the chemical composition of biological samples. In particular, fiber Raman probes play a crucial role for in vivo and ex vivo tissue analysis. However, the high-fluorescence background typically contributed by the auto fluorescence from both a tissue sample and the fiber-probe interferes strongly with the relatively weak Raman signal. Here we demonstrate the implementation of wavelength-modulated Raman spectroscopy (WMRS) to suppress the fluorescence background while analyzing tissues using fiber Raman probes. We have observed a significant signal-to-noise ratio enhancement in the Raman bands of bone tissue, which have a relatively high fluorescence background. Implementation of WMRS in fiber-probe-based bone tissue study yielded usable Raman spectra in a relatively short acquisition time (∼30 s), notably without any special sample preparation stage. Finally, we have validated its capability to suppress fluorescence on other tissue samples such as adipose tissue derived from four different species.
The work was funded by CR-UK/EPSRC/MRC/DoH (England) imaging programme.
2012-07-09T00:00:00ZBalagopal, BavishnaAshok, Praveen CheriyanMazilu, MichaelRiches, Andrew CliveHerrington, C SimonDholakia, KishanIn the field of biomedical optics, Raman spectroscopy is a powerful tool for probing the chemical composition of biological samples. In particular, fiber Raman probes play a crucial role for in vivo and ex vivo tissue analysis. However, the high-fluorescence background typically contributed by the auto fluorescence from both a tissue sample and the fiber-probe interferes strongly with the relatively weak Raman signal. Here we demonstrate the implementation of wavelength-modulated Raman spectroscopy (WMRS) to suppress the fluorescence background while analyzing tissues using fiber Raman probes. We have observed a significant signal-to-noise ratio enhancement in the Raman bands of bone tissue, which have a relatively high fluorescence background. Implementation of WMRS in fiber-probe-based bone tissue study yielded usable Raman spectra in a relatively short acquisition time (∼30 s), notably without any special sample preparation stage. Finally, we have validated its capability to suppress fluorescence on other tissue samples such as adipose tissue derived from four different species.The N-terminus of Bunyamwera orthobunyavirus NSs protein is essential for interferon antagonismVan Knippenberg, Ingeborg ChristineCarlton-Smith, CharlesElliott, Richard Michaelhttps://hdl.handle.net/10023/11482023-04-18T09:42:04Z2010-04-01T00:00:00ZBunyamwera virus NSs protein is involved in the inhibition of cellular transcription and the interferon (IFN) response, and it interacts with the Med8 component of Mediator. A spontaneous mutant of a recombinant NSs-deleted Bunyamwera virus (rBUNdelNSs2) was identified and characterized. This mutant virus, termed mBUNNSs22, expresses a 21 aa N-terminally truncated form of NSs. Like rBUNdelNSs2, mBUNNSs22 is attenuated in IFN-deficient cells, and to a greater extent in IFN-competent cells. Both rBUNdelNSs2 and mBUNNSs22 are potent IFN inducers and their growth can be rescued by depleting cellular IRF3. Strikingly, despite encoding an NSs protein that contains the Med8 interaction domain, mBUNNSs22 fails to block RNA polymerase II activity during infection. Overall, our data suggest that both the interaction of NSs with Med8 and a novel unidentified function of the NSs N-terminus, seem necessary for Bunyamwera virus to counteract host antiviral responses.
This work is supported by UK MRC and BBRC
2010-04-01T00:00:00ZVan Knippenberg, Ingeborg ChristineCarlton-Smith, CharlesElliott, Richard MichaelBunyamwera virus NSs protein is involved in the inhibition of cellular transcription and the interferon (IFN) response, and it interacts with the Med8 component of Mediator. A spontaneous mutant of a recombinant NSs-deleted Bunyamwera virus (rBUNdelNSs2) was identified and characterized. This mutant virus, termed mBUNNSs22, expresses a 21 aa N-terminally truncated form of NSs. Like rBUNdelNSs2, mBUNNSs22 is attenuated in IFN-deficient cells, and to a greater extent in IFN-competent cells. Both rBUNdelNSs2 and mBUNNSs22 are potent IFN inducers and their growth can be rescued by depleting cellular IRF3. Strikingly, despite encoding an NSs protein that contains the Med8 interaction domain, mBUNNSs22 fails to block RNA polymerase II activity during infection. Overall, our data suggest that both the interaction of NSs with Med8 and a novel unidentified function of the NSs N-terminus, seem necessary for Bunyamwera virus to counteract host antiviral responses.The archaeo-eukaryotic GINS proteins and the archaeal primase catalytic subunit PriS share a common domainSwiatek, AgnieszkaMacNeill, Stuart Andrewhttps://hdl.handle.net/10023/10472024-02-26T00:40:45Z2010-04-12T00:00:00ZPrimase and GINS are essential factors for chromosomal DNA replication in eukaryotic and archaeal cells. Here we describe a previously undetected relationship between the C-terminal domain of the catalytic subunit (PriS) of archaeal primase and the B-domains of the archaeo-eukaryotic GINS proteins in the form of a conserved structural domain comprising a three-stranded antiparallel beta-sheet adjacent to an alpha-helix and a two-stranded beta-sheet or hairpin. The presence of a shared domain in archaeal PriS and GINS proteins, the genes for which are often found adjacent on the chromosome, suggests simple mechanisms for the evolution of these proteins.
This work was funded by the Scottish Universities Life Sciences Alliance (SULSA).
2010-04-12T00:00:00ZSwiatek, AgnieszkaMacNeill, Stuart AndrewPrimase and GINS are essential factors for chromosomal DNA replication in eukaryotic and archaeal cells. Here we describe a previously undetected relationship between the C-terminal domain of the catalytic subunit (PriS) of archaeal primase and the B-domains of the archaeo-eukaryotic GINS proteins in the form of a conserved structural domain comprising a three-stranded antiparallel beta-sheet adjacent to an alpha-helix and a two-stranded beta-sheet or hairpin. The presence of a shared domain in archaeal PriS and GINS proteins, the genes for which are often found adjacent on the chromosome, suggests simple mechanisms for the evolution of these proteins.Identification of a novel class of mammalian phosphoinositol-specific phospholipase C enzymes.Stewart, AJMukherjee, JRoberts, SJLester, DFarquharson, Chttps://hdl.handle.net/10023/9562019-04-01T08:50:44Z2005-01-01T00:00:00ZPhosphoinositol (PhoIns)-specific phospholipase C enzymes (PLCs) are central to the inositol lipid signaling pathways and contribute to intracellular Ca2+ release and protein kinase C activation. Five distinct classes of PhoIns-specific PLCs are known to exist in mammals, which are activated by membrane receptor-mediated events. Here we have identified a sixth class of PhoIns-specific PLC with a novel domain structure, which we have termed PLC-eta. Two putative PLC-eta enzymes were identified in humans and in mice. Sequence analysis revealed that residues implicated in substrate binding and catalysis from other PhoIns-specific PLCs are conserved in the novel enzymes. PLC-eta enzymes are most closely related to the PLC-delta class and share a close evolutionary relationship with other PLC isozymes. EST analysis and RT-PCR data suggest that PLC-eta enzymes are expressed in several cell types and, by analogy with other mammalian PhoIns-specific PLCs, are likely to be involved in signal transduction pathways.
2005-01-01T00:00:00ZStewart, AJMukherjee, JRoberts, SJLester, DFarquharson, CPhosphoinositol (PhoIns)-specific phospholipase C enzymes (PLCs) are central to the inositol lipid signaling pathways and contribute to intracellular Ca2+ release and protein kinase C activation. Five distinct classes of PhoIns-specific PLCs are known to exist in mammals, which are activated by membrane receptor-mediated events. Here we have identified a sixth class of PhoIns-specific PLC with a novel domain structure, which we have termed PLC-eta. Two putative PLC-eta enzymes were identified in humans and in mice. Sequence analysis revealed that residues implicated in substrate binding and catalysis from other PhoIns-specific PLCs are conserved in the novel enzymes. PLC-eta enzymes are most closely related to the PLC-delta class and share a close evolutionary relationship with other PLC isozymes. EST analysis and RT-PCR data suggest that PLC-eta enzymes are expressed in several cell types and, by analogy with other mammalian PhoIns-specific PLCs, are likely to be involved in signal transduction pathways.