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Search Results: 1 - 10 of 300337 matches for " Nicola J. Stonehouse "
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RNA Multimerisation in the DNA Packaging Motor of Bacteriophage φ29
Nicola J. Stonehouse
Computational and Mathematical Methods in Medicine , 2005, DOI: 10.1080/10273660500149802
Abstract: The use of bacteriophages as experimental tools allows the investigation of interactions between components at the molecular level that are often not possible in more complex virus systems. The bacteriophage φ29 acts as a molecular machine to package its own genomic DNA during viral assembly. Self-associating RNA molecules, called pRNA, have an essential role in the function of this machine. This paper reports the characterization of this self-association (which leads to multimerisation of wild-type and truncated variant pRNAs) by analytical ultracentrifugation (including determination of the partial specific volume of the pRNA), together with an investigation into the domains of the molecule important for multimerisation by the use of complementary DNA probes.
Conformational Changes in the Connector Protein Complex of the Bacteriophage φ29 DNA Packaging Motor
Arron C. Tolley,Nicola J. Stonehouse
Computational and Mathematical Methods in Medicine , 2008, DOI: 10.1080/17486700802168486
Abstract: DNA packaging in the bacteriophage φ29 involves a molecular motor. It is proposed that dsDNA is packaged through a channel in a connector located at the 5-fold vertex of a preformed prolate icosahedral capsid. The packaging motor also consists of virally-encoded RNA molecules (pRNA) coupled to ATPases. Data obtained from studies using surface plasmon resonance, fluorescence quenching and circular dichroism are presented to demonstrate the importance of the N-termini of the connector protein subunits in pRNA interaction and in conformational change. Based on our findings, we propose a model of DNA packaging based on connector conformational change.
An RNA Aptamer Provides a Novel Approach for the Induction of Apoptosis by Targeting the HPV16 E7 Oncoprotein
Clare Nicol, ?zlem Cesur, Sophie Forrest, Tamara A. Belyaeva, David H. J. Bunka, G. Eric Blair, Nicola J. Stonehouse
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0064781
Abstract: Background Human papillomavirus 16 (HPV16) is a high-risk DNA tumour virus, which is a major causative agent of cervical cancer. Cellular transformation is associated with deregulated expression of the E6 and E7 oncogenes. E7 has been shown to bind a number of cellular proteins, including the cell cycle control protein pRb. In this study, RNA aptamers (small, single-stranded oligonucleotides selected for high-affinity binding) to HPV16 E7 were employed as molecular tools to further investigate these protein-protein interactions. Methodology/Principal Findings This study is focused on one aptamer (termed A2). Transfection of this molecule into HPV16-transformed cells resulted in inhibition of cell proliferation (shown using real-time cell electronic sensing and MTT assays) due to the induction of apoptosis (as demonstrated by Annexin V/propidium iodide staining). GST-pull down and bead binding assays were used to demonstrate that the binding of A2 required N-terminal residues of E7 known to be involved in interaction with the cell cycle control protein, pRb. Using a similar approach, A2 was shown to disrupt the interaction between E7 and pRb in vitro. Furthermore, transfection of HPV16-transformed cells with A2 appeared to result in the loss of E7 and rise in pRb levels, as observed by immunoblotting. Conclusions/Significance This paper includes the first characterisation of the effects of an E7 RNA aptamer in a cell line derived from a cervical carcinoma. Transfection of cells with A2 was correlated with the loss of E7 and the induction of apoptosis. Aptamers specific for a number of cellular and viral proteins have been documented previously; one aptamer (Macugen) is approved for clinical use and several others are in clinical trials. In addition to its role as a molecular tool, A2 could have further applications in the future.
Capsid Protein VP4 of Human Rhinovirus Induces Membrane Permeability by the Formation of a Size-Selective Multimeric Pore
Anusha Panjwani,Mike Strauss,Sarah Gold,Hannah Wenham,Terry Jackson,James J. Chou,David J. Rowlands,Nicola J. Stonehouse,James M. Hogle,Tobias J. Tuthill
PLOS Pathogens , 2014, DOI: doi/10.1371/journal.ppat.1004294
Abstract: Non-enveloped viruses must deliver their viral genome across a cell membrane without the advantage of membrane fusion. The mechanisms used to achieve this remain poorly understood. Human rhinovirus, a frequent cause of the common cold, is a non-enveloped virus of the picornavirus family, which includes other significant pathogens such as poliovirus and foot-and-mouth disease virus. During picornavirus cell entry, the small myristoylated capsid protein VP4 is released from the virus, interacts with the cell membrane and is implicated in the delivery of the viral RNA genome into the cytoplasm to initiate replication. In this study, we have produced recombinant C-terminal histidine-tagged human rhinovirus VP4 and shown it can induce membrane permeability in liposome model membranes. Dextran size-exclusion studies, chemical crosslinking and electron microscopy demonstrated that VP4 forms a multimeric membrane pore, with a channel size consistent with transfer of the single-stranded RNA genome. The membrane permeability induced by recombinant VP4 was influenced by pH and was comparable to permeability induced by infectious virions. These findings present a molecular mechanism for the involvement of VP4 in cell entry and provide a model system which will facilitate exploration of VP4 as a novel antiviral target for the picornavirus family.
RNA Packing Specificity and Folding during Assembly of the Bacteriophage MS2
Ottar Rolfsson,Katerina Toropova,Victoria Morton,Simona Francese,Gabriella Basnak,Gary S. Thompson,Stephen W. Homans,Alison E. Ashcroft,Nicola J. Stonehouse,Neil A. Ranson,Peter G. Stockley
Computational and Mathematical Methods in Medicine , 2008, DOI: 10.1080/17486700802168445
Abstract: Using a combination of biochemistry, mass spectrometry, NMR spectroscopy and cryo-electron microscopy (cryo-EM), we have been able to show that quasi-equivalent conformer switching in the coat protein (CP) of an RNA bacteriophage (MS2) is controlled by a sequence-specific RNA–protein interaction. The RNA component of this complex is an RNA stem-loop encompassing just 19 nts from the phage genomic RNA, which is 3569 nts in length. This binding results in the conversion of a CP dimer from a symmetrical conformation to an asymmetric one. Only when both symmetrical and asymmetrical dimers are present in solution is assembly of the T = 3 phage capsid efficient. This implies that the conformers, we have characterized by NMR correspond to the two distinct quasi-equivalent conformers seen in the 3D structure of the virion. An icosahedrally-averaged single particle cryo-EM reconstruction of the wild-type phage (to ∼9 Å resolution) has revealed icosahedrally ordered density encompassing up to 90% of the single-stranded RNA genome. The RNA is seen with a novel arrangement of two concentric shells, with connections between them along the 5-fold symmetry axes. RNA in the outer shell interacts with each of the 90 CP dimers in the T = 3 capsid and although the density is icosahedrally averaged, there appears to be a different average contact at the different quasi-equivalent protein dimers: precisely the result that would be expected if protein conformer switching is RNA-mediated throughout the assembly pathway. This unprecedented RNA structure provides new constraints for models of viral assembly and we describe experiments aimed at probing these. Together, these results suggest that viral genomic RNA folding is an important factor in efficient assembly, and further suggest that RNAs that could sequester viral CPs but not fold appropriately could act as potent inhibitors of viral assembly.
A Complex Extracellular Sphingomyelinase of Pseudomonas aeruginosa Inhibits Angiogenesis by Selective Cytotoxicity to Endothelial Cells
Michael L. Vasil ,Martin J. Stonehouse,Adriana I. Vasil,Sandra J. Wadsworth,Howard Goldfine,Robert E. Bolcome III,Joanne Chan
PLOS Pathogens , 2009, DOI: 10.1371/journal.ppat.1000420
Abstract: The hemolytic phospholipase C (PlcHR) expressed by Pseudomonas aeruginosa is the original member of a Phosphoesterase Superfamily, which includes phosphorylcholine-specific phospholipases C (PC-PLC) produced by frank and opportunistic pathogens. PlcHR, but not all its family members, is also a potent sphingomyelinase (SMase). Data presented herein indicate that picomolar (pM) concentrations of PlcHR are selectively lethal to endothelial cells (EC). An RGD motif of PlcHR contributes to this selectivity. Peptides containing an RGD motif (i.e., GRGDS), but not control peptides (i.e., GDGRS), block the effects of PlcHR on calcium signaling and cytotoxicity to EC. Moreover, RGD variants of PlcHR (e.g., RGE, KGD) are significantly reduced in their binding and toxicity, but retain the enzymatic activity of the wild type PlcHR. PlcHR also inhibits several EC-dependent in vitro assays (i.e., EC migration, EC invasion, and EC tubule formation), which represent key processes involved in angiogenesis (i.e., formation of new blood vessels from existing vasculature). Finally, the impact of PlcHR in an in vivo model of angiogenesis in transgenic zebrafish, and ones treated with an antisense morpholino to knock down a key blood cell regulator, were evaluated because in vitro assays cannot fully represent the complex processes of angiogenesis. As little as 2 ng/embryo of PlcHR was lethal to ~50% of EGFP-labeled EC at 6 h after injection of embryos at 48 hpf (hours post-fertilization). An active site mutant of PlcHR (Thr178Ala) exhibited 120-fold reduced inhibitory activity in the EC invasion assay, and 20 ng/embryo elicited no detectable inhibitory activity in the zebrafish model. Taken together, these observations are pertinent to the distinctive vasculitis and poor wound healing associated with P. aeruginosa sepsis and suggest that the potent antiangiogenic properties of PlcHR are worthy of further investigation for the treatment of diseases where angiogenesis contributes pathological conditions (e.g., vascularization of tumors, diabetic retinopathy).
Do Individual Differences Moderate the Cognitive Benefits of Chewing Gum?  [PDF]
Richard Stephens, Nicola M. J. Edelstyn
Psychology (PSYCH) , 2011, DOI: 10.4236/psych.2011.28127
Abstract: Recent experiments investigating whether chewing gum enhances cognitive performance have shown mixed results and a recent replication failed to reproduce earlier findings. The present experiment aimed to investigate whether participant individual differences underlie the discrepant findings. Therefore, in addition to examining differences in Digit Span and Spatial Span performance across gum and control groups, chronotype, extraversion, habitual tiredness, current stress, current arousal and current thirst were assessed using questionnaires. Task difficulty was also manipulated. While there were no chewing gum effects under standard testing conditions, chewing gum enhanced Digit Span performance in the more difficult dual task condition. Furthermore, Spatial Span performance was improved by chewing gum in introverts but not extraverts and chewing gum was shown to eliminate the negative relationship between thirst and Digit Span performance. In explaining these data it is proposed that chewing gum may act both to reduce stress and to alleviate thirst.
Bulk Aluminum at High Pressure: A First-Principles Study
Michael J. Tambe,Nicola Bonini,Nicola Marzari
Physics , 2007, DOI: 10.1103/PhysRevB.77.172102
Abstract: The behavior of metals at high pressure is of great importance to the fields of shock physics, geophysics, astrophysics, and nuclear materials. In order to further understand the properties of metals at high pressures we studied the equation of state of aluminum using first-principles techniques up to 2500 GPa, pressures within reach of the planned L.L.N.L. National Ignition Facility. Our simulations use density-functional theory and density-functional perturbation theory in the generalized gradient approximation at 0K. We found core overlaps to become relevant beyond pressures of 1200 GPa. The equations of state for three phases (fcc, bcc, and hcp) were calculated predicting the fcc-hcp, fcc-bcc, and hcp-bcc transitions to occur at 215 GPa, 307 GPa, and 435 GPa respectively. From the phonon dispersions at increasing pressure, we predict a softening of the lowest transverse acoustic vibrational mode along the [110] direction, which corresponds to a Born instability of the fcc phase at 725 GPa.
Air or 100% oxygen for asphyxiated babies? Time to decide
Nicola J Robertson
Critical Care , 2004, DOI: 10.1186/cc3059
Abstract: Around 5–10% of all newborn infants born worldwide require some degree of resuscitation at birth, ranging from simple stimulation to assisted ventilation. Defining the optimum technique for neonatal resuscitation is an extremely important challenge and has the potential to improve neonatal outcome globally. In the past two decades, neonatal research has established that, if assisted ventilation is required, room air is as efficient as 100% oxygen for newborn resuscitation. The meta-analysis published in the Lancet [1] goes further and suggests that mortality is lower in newborn infants resuscitated in room air compared with those resuscitated in 100% oxygen. One death would be prevented for every 20 babies resuscitated with air versus 100% oxygen. Several hundred thousand deaths might be prevented by avoiding 100% oxygen during resuscitation. It is astonishing that a brief exposure of only a few minutes to 100% oxygen may be so toxic to newborn infants. A brief review of the experimental, basic science and clinical literature on this subject adds weight to this finding. It is imperative that newborn resuscitation guidelines are now revised.The process of childbirth is accompanied by increased oxidative stress, as birth itself is a hyperoxic challenge. The foetus transfers from an intra-uterine hypoxic environment with a pO2 of 20–25 mmHg to an extra-uterine environment with a pO2 of 100 mmHg.During the 20th century it became traditional to use 100% oxygen to resuscitate infants in hospitals because labour wards only had oxygen as a compressed gas [2,3]. It seemed logical and intuitive to reverse the anaerobic state as quickly as possible with 100% oxygen because 18–19 times more ATP is produced from glucose during aerobic metabolism than during anaerobic metabolism. Howere, following the discovery of the link between retinopathy of prematurity and the liberal use of supplemental oxygen therapy, controversy has surrounded the use of oxygen in neonatology [4,5].There
Dissecting the Fine Details of Assembly of a T?=?3 Phage Capsid
P. G. Stockley,A. E. Ashcroft,S. Francese,G. S. Thompson,N. A. Ranson,A. M. Smith,S. W. Homans,N. J. Stonehouse
Computational and Mathematical Methods in Medicine , 2005, DOI: 10.1080/10273660500149869
Abstract: The RNA bacteriophages represent ideal model systems in which to probe the detailed assembly pathway for the formation of a T = 3 quasi-equivalent capsid. For MS2, the assembly reaction can be probed in vitro using acid disassembled coat protein subunits and a short (19 nt) RNA stem-loop that acts as the translational operator of the replicase gene and leads to sequence-specific sequestration and packaging of the cognate phage RNA in vivo. Reassembly reactions can be initiated by mixing these components at neutral pH. The molecular basis of the sequence-specific RNA–protein interaction is now well understood. Recent NMR studies on the protein demonstrate extensive mobility in the loops of the polypeptide that alter their conformations to form the quasi-equivalent conformers of the final capsid. It seems reasonable to assume that RNA binding results in reduction of this flexibility. However, mass spectrometry suggests that these RNA–protein complexes may only provide one type of quasi-equivalent capsid building block competent to form five-fold axes but not the full shell. Work with longer RNAs suggests that the RNA may actively template the assembly pathway providing a partial explanation of how conformers are selected in the growing shell.
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