Altered Gene Expression in Asymptomatic SHIV-infected Rhesus Macaques (Macacca Mulatta)

Virology Journal. 2006  |  Pubmed ID: 16956415

Simian-Human immunodeficiency virus is a chimeric virus which, in rhesus macaques (Macacca mulatta) closely imitates immunodeficiency virus infection in human (HIV). A relatively new way to study pathogenesis of viral infection is to study alterations in host gene expression induced by the virus. SHIV infection with certain strains does not result in clinical signs. We hypothesized that alterations in gene expression relating to the immune system would be present in SHIV-infected animals despite the lack of clinical signs. Splenic tissue from four adult male Indian-origin Rhesus monkeys serologically positive for non-pathogenic SHIV 89.6 was processed by cDNA microarray analysis. Results were compared with the corresponding outcome using splenic tissues from four unexposed adult male Rhesus monkeys. Subsequent gene analysis confirmed statistically significant variations between control and infected samples. Interestingly, SHIV-infected monkeys exhibited altered expression in genes related to apoptosis, signal transduction, T and B lymphocyte activation and importantly, to immune regulation. Although infected animals appeared asymptomatic, our study demonstrated that SHIV-infected monkeys cannot reliably be used in studies of other infectious agents as their baseline gene expression differs from that of normal Rhesus monkeys. The gene expression differences in SHIV-infected animals relative to uninfected animals offer additional clues to the pathogenesis of altered immune function in response to secondary infection.

A Contemporary Case Study Illustrating the Integration of Health Information Technologies into the Organisation and Clinical Practice of Radiation Oncology

The HIM Journal. 2006  |  Pubmed ID: 18216418

The development of software in radiation oncology departments has seen the increase in capability from the Record and Verify software focused on patient safety to a fully-fledged Oncology Information System (OIS). This paper reports on the medical aspects of the implementation of a modern Oncology Information System (IMPAC MultiAccess, also known as the Siemens LANTIS) in a New Zealand hospital oncology department. The department was successful in translating paper procedures into electronic procedures, and the report focuses on the changes in approach to organisation and data use that occurred. The difficulties that were faced, which included procedural re-design, management of change, removal of paper, implementation cost, integration with the HIS, quality assurance and datasets, are highlighted along with the local solutions developed to overcome these problems.

Structure and Dynamics of the Abeta(21-30) Peptide from the Interplay of NMR Experiments and Molecular Simulations

Journal of the American Chemical Society. May, 2008  |  Pubmed ID: 18412346

We combine molecular dynamics simulations and new high-field NMR experiments to describe the solution structure of the Abeta(21-30) peptide fragment that may be relevant for understanding structural mechanisms related to Alzheimer's disease. By using two different empirical force-field combinations, we provide predictions of the three-bond scalar coupling constants ((3)J(H(N)H(alpha))), chemical-shift values, (13)C relaxation parameters, and rotating-frame nuclear Overhauser effect spectroscopy (ROESY) crosspeaks that can then be compared directly to the same observables measured in the corresponding NMR experiment of Abeta(21-30). We find robust prediction of the (13)C relaxation parameters and medium-range ROESY crosspeaks by using new generation TIP4P-Ew water and Amber ff99SB protein force fields, in which the NMR validates that the simulation yields both a structurally and dynamically correct ensemble over the entire Abeta(21-30) peptide. Analysis of the simulated ensemble shows that all medium-range ROE restraints are not satisfied simultaneously and demonstrates the structural diversity of the Abeta(21-30) conformations more completely than when determined from the experimental medium-range ROE restraints alone. We find that the structural ensemble of the Abeta(21-30) peptide involves a majority population (approximately 60%) of unstructured conformers, lacking any secondary structure or persistent hydrogen-bonding networks. However, the remaining minority population contains a substantial percentage of conformers with a beta-turn centered at Val24 and Gly25, as well as evidence of the Asp23 to Lys28 salt bridge important to the fibril structure. This study sets the stage for robust theoretical work on Abeta(1-40) and Abeta(1-42), for which collection of detailed NMR data on the monomer will be more challenging because of aggregation and fibril formation on experimental timescales at physiological conditions. In addition, we believe that the interplay of modern molecular simulation and high-quality NMR experiments has reached a fruitful stage for characterizing structural ensembles of disordered peptides and proteins in general.

Suppression of RNA Interference Increases Alphavirus Replication and Virus-associated Mortality in Aedes Aegypti Mosquitoes

BMC Microbiology. 2009  |  Pubmed ID: 19265532

Arthropod-borne viruses (arboviruses) can persistently infect and cause limited damage to mosquito vectors. RNA interference (RNAi) is a mosquito antiviral response important in restricting RNA virus replication and has been shown to be active against some arboviruses. The goal of this study was to use a recombinant Sindbis virus (SINV; family Togaviridae; genus Alphavirus) that expresses B2 protein of Flock House virus (FHV; family Nodaviridae; genus Alphanodavirus), a protein that inhibits RNAi, to determine the effects of linking arbovirus infection with RNAi inhibition.

Virulence Variation Among Isolates of Western Equine Encephalitis Virus in an Outbred Mouse Model

The Journal of General Virology. Aug, 2009  |  Pubmed ID: 19403754

Little is known about viral determinants of virulence associated with western equine encephalitis virus (WEEV). Here, we have analysed six North American WEEV isolates in an outbred CD1 mouse model. Full genome sequence analyses showed < or =2.7 % divergence among the six WEEV isolates. However, the percentage mortality and mean time to death (MTD) varied significantly when mice received subcutaneous injections of 10(3) p.f.u. of each virus. Two WEEV strains, McMillan (McM) and Imperial 181 (IMP), were the most divergent of the six in genome sequence; McM caused 100 % mortality by 5 days post-infection, whereas IMP caused no mortality. McM had significantly higher titres in the brain than IMP. Similar differences in virulence were observed when McM and IMP were administered by aerosol, intranasal or intravenous routes. McM was 100 % lethal with an MTD of 1.9 days when 10(3) p.f.u. of each virus was administered by intracerebral inoculation; in contrast, IMP caused no mortality. The presence of IMP in the brains after infection by different routes and the lack of observed mortality confirmed that IMP is neuroinvasive but not neurovirulent. Based on morbidity, mortality, MTD, severity of brain lesions, virus distribution patterns, routes of infection and differences in infection of cultured cells, McM and IMP were identified as high- and low-virulence isolates, respectively.

Hemodynamic Response to Lower Body Negative Pressure in Children: a Pilot Study

Autonomic Neuroscience : Basic & Clinical. Jun, 2010  |  Pubmed ID: 20171939

Although head-up tilt and upright standing are common methods used to induce orthostatic stress, lower body negative pressure (LBNP) is another safe and easy technique that induces orthostatic stress independently of gravity. However, the use of LBNP in children has never been investigated. The purpose of this pilot study was to determine whether LBNP was capable of inducing hemodynamic adaptations in pre-pubertal boys. Ten healthy pre-pubertal boys (9+/-1 years) were recruited and randomly exposed to 3 levels of LBNP (15, 20 and 25 mm Hg). Heart rate, manual and beat-by-beat systolic (SBP), diastolic and mean arterial blood pressure were monitored continuously. Aortic diameter was measured at rest and peak aortic blood velocity was recorded continuously for at least 1 min during each condition. R-R interval (RRI), heart rate, stroke volume (SV), cardiac output (Q), total peripheral resistance (TPR), low frequency (LF) and high frequency (HF) baroreceptor sensitivity (BRS), as well as LF/HF RRI ratio were calculated. With increasing LBNP TPR increased while SBP decreased (P< or =0.05). As well, a trend towards a decrease in SV (P=0.054) and Q (P=0.06) was found. However, LF and HF BRS, and LF/HF RRI ratio did not significantly change from baseline to LBNP 15, 20 or 25 mm Hg. In conclusion, the results of this pilot study suggest that low levels of LBNP are capable of inducing hemodynamic adaptations in children that are to be expected when undergoing an orthostatic stress. As well, LBNP is a safe and effective method of inducing orthostatic stress in children.

Treatment with Cationic Liposome-DNA Complexes (CLDCs) Protects Mice from Lethal Western Equine Encephalitis Virus (WEEV) Challenge

Antiviral Research. Aug, 2010  |  Pubmed ID: 20452378

Having recently characterized a CD-1 outbred mouse model of pathogenesis for Western equine encephalitis virus, we examined the possible protective effects of cationic liposome-DNA complexes (CLDCs) against encephalitic arboviral infection. In this investigation, mice were pre-treated, co-treated, or post-treated with CLDC then challenged with a subcutaneous or aerosol dose of the highly virulent WEEV-McMillan strain, lethal in mice 4-5 days after inoculation. Pre-treatment with CLDCs provided a significant protective effect in mice, which was reflected in significantly increased survival rates. Further, in some instances a therapeutic effect of CLDC administration up to 12h after WEEV challenge was observed. Mice treated with CLDC had significantly increased serum IFN-gamma, TNF-alpha, and IL-12, suggesting a strong Th1-biased antiviral activation of the innate immune system. In virus-infected animals, large increases in production of IFN-gamma, TNF-alpha, MCP-1, IL-12, and IL-10 in the brain were observed by 72h after infection, consistent with neuroinvasion and viral replication in the CNS. These results indicate that strong non-specific activation of innate immunity with an immune therapeutic such as CLDC is capable of eliciting significant protective immunity against a rapidly lethal strain of WEEV and suggest a possible prophylactic option for exposed individuals.

Probing the Conformation of a Prion Protein Fibril with Hydrogen Exchange

The Journal of Biological Chemistry. Oct, 2010  |  Pubmed ID: 20679344

A fragment of the prion protein, PrP(89-143, P101L), bearing a mutation implicated in familial prion disease, forms fibrils that have been shown to induce prion disease when injected intracerebrally into transgenic mice expressing full-length PrP containing the P101L mutation. In this study, we utilize amide hydrogen exchange measurements to probe the organization of the peptide in its fibrillar form. We determined the extent of hydrogen exchange first by tandem proteolysis, liquid chromatography, and mass spectrometry (HXMS) and then by exchange-quenched NMR. Although single amide resolution is afforded by NMR measurements, HXMS is well suited to the study of natural prions because it does not require labeling with NMR active isotopes. Thus, natural prions obtained from infected animals can be compared with model systems such as PrP(89-143, P101L) studied here. In our study, we find two segments of sequence that display a high level of protection from exchange, residues 102-109 and 117-136. In addition, there is a region that displays exchange behavior consistent with the presence of a conformationally heterogeneous turn. We discuss our data with respect to several structural models proposed for infectious PrP aggregates and highlight HXMS as one of the few techniques well suited to studying natural prions.

Paramagnetic Relaxation Assisted Docking of a Small Indole Compound in the HIV-1 Gp41 Hydrophobic Pocket

ACS Chemical Biology. Mar, 2011  |  Pubmed ID: 21155611

The hydrophobic pocket contained within the gp41 coiled coil is an important target for small molecules designed to inhibit HIV-1 fusion. While various screening experiments have identified molecules purported to bind in this pocket, few have confirmed details of the interaction, instead relying on computational docking to predict the binding mode. This is made more challenging by the fact that residues lining the hydrophobic pocket are highly flexible, as is typical for a protein-protein interaction site, limiting the predictive power of computational tools. In this study, we report on an NMR method to define the binding mode of 1-5i, a compound in a series of newly developed indole inhibitors. We show that paramagnetic relaxation enhancement of ligand protons due to an MTSL group positioned close to the binding pocket could be applied quantitatively to distinguish between more than 30 different computational poses, selecting a single pose that agreed with the NMR data. In this pose, important hydrophobic and polar contacts occur with pocket lysine, tryptophan, and glutamine residues, including putative hydrogen bonds between the ligand carboxylate and the lysine ε-amino group. A study of the ligand orientation suggests directions for optimization.

A Sterile Alpha-motif Domain in NafY Targets Apo-NifDK for Iron-molybdenum Cofactor Delivery Via a Tethered Domain

The Journal of Biological Chemistry. Feb, 2011  |  Pubmed ID: 21156797

NafY participates in the final steps of nitrogenase maturation, having a dual role as iron-molybdenum cofactor (FeMo-co) carrier and as chaperone to the FeMo-co-deficient apo-NifDK (apo-dinitrogenase). NafY contains an N-terminal domain of unknown function (n-NafY) and a C-terminal domain (core-NafY) necessary for FeMo-co binding. We show here that n-NafY and core-NafY have very weak interactions in intact NafY. The NMR structure of n-NafY reveals that it belongs to the sterile α-motif (SAM) family of domains, which are frequently involved in protein-protein interactions. The presence of a SAM domain in NafY was unexpected and could not be inferred from its amino acid sequence. Although SAM domains are very commonly found in eukaryotic proteins, they have rarely been identified in prokaryotes. The n-NafY SAM domain binds apo-NifDK. As opposed to full-length NafY, n-NafY impaired FeMo-co insertion when present in molar excess relative to FeMo-co and apo-NifDK. The implications of these observations are discussed to offer a plausible mechanism of FeMo-co insertion. NafY domain structure, molecular tumbling, and interdomain motion, as well as NafY interaction with apo-NifDK are consistent with the function of NafY in FeMo-co delivery to apo-NifDK.

The Impact of Parkinson's Disease As a Comorbid Diagnosis

Age and Ageing. May, 2011  |  Pubmed ID: 21454348

Clinicians, departments and trusts are compared using Hospital Standardised Mortality Ratios. The benchmarking process makes corrections for patients' past medical histories, using a tool called the Charlson Index of Co-morbidity. Patients with any of 17 conditions receive an adjustment to their expected mortality and length of stay. Although the weighting of these conditions has been altered to take account of advances in medical practice, there have been no conditions added to the list since Charlson's original paper of 1986. The Charlson Index of Co-morbidity does not make a correction for Parkinson's disease. We describe evidence of the impact of Parkinson's disease as a co-morbid condition and recommend that, in future, benchmarking processes should take account of this important and common diagnosis.

Homogeneous and Heterogeneous Tertiary Structure Ensembles of Amyloid-β Peptides

Biochemistry. Sep, 2011  |  Pubmed ID: 21797254

The interplay of modern molecular simulation and high-quality nuclear magnetic resonance (NMR) experiments has reached a fruitful stage for quantitative characterization of structural ensembles of disordered peptides. Amyloid-β 1-42 (Aβ42), the primary peptide associated with Alzheimer's disease, and fragments such as Aβ21-30 are both classified as intrinsically disordered peptides (IDPs). We use a variety of NMR observables to validate de novo molecular dynamics simulations in explicit water to characterize the tertiary structure ensemble of Aβ42 and Aβ21-30 from the perspective of their classification as IDPs. Unlike the Aβ21-30 fragment that conforms to expectations of an IDP that is primarily extended, we find that Aβ42 samples conformations reflecting all possible secondary structure categories and spans the range of IDP classifications from collapsed structured states to highly extended conformations, making it an IDP with a far more heterogeneous tertiary ensemble.

Letter to the Editor: "Left Ventricular Mechanical Limitations to Stroke Volume in Healthy Humans During Incremental Exercise"

American Journal of Physiology. Heart and Circulatory Physiology. Jan, 2012  |  Pubmed ID: 22201176