Adipokines and the Right Ventricle: The MESA-RV Study.

ObjectiveObesity is associated with changes in both right (RV) and left (LV) ventricular morphology, but the biological basis of this finding is not well established. We examined whether adipokine levels were associated with RV morphology and function in a population-based multiethnic sample free of clinical cardiovascular disease.MethodsWe examined relationships of leptin, resistin, TNF-α, and adiponectin with RV morphology and function (from cardiac MRI) in participants (n = 1,267) free of clinical cardiovascular disease from the Multi-Ethnic Study of Atherosclerosis (MESA)-RV study. Multivariable regressions (linear, quantile [25th and 75th] and generalized additive models [GAM]) were used to examine the independent association of each adipokine with RV mass, RV end-diastolic volume (RVEDV), RV end-systolic volume (RVESV), RV stroke volume (RVSV) and RV ejection fraction (RVEF).ResultsHigher leptin levels were associated with significantly lower levels of RV mass, RVEDV, RVESV and stroke volume, but not RVEF, after adjustment for age, gender, race, height and weight. These associations were somewhat attenuated but still significant after adjustment for traditional risk factors and covariates, and were completely attenuated when correcting for the respective LV measures. There were no significant interactions of age, gender, or race/ethnicity on the relationship between the four adipokines and RV structure or function.ConclusionsLeptin levels are associated with favorable RV morphology in a multi-ethnic population free of cardiovascular disease, however these associations may be explained by a yet to be understood bi-ventricular process as this association was no longer present after adjustment for LV values. These findings complement the associations previously shown between adipokines and LV structure and function in both healthy and diseased patients. The mechanisms linking adipokines to healthy cardiovascular function require further investigation

Identification of an HIV-1 mutation in spacer peptide 1 that stabilizes the immature CA-SP1 lattice

Upon release of HIV-1 particles from the infected cell, the viral protease cleaves the Gag polyprotein at specific sites, triggering maturation. During this process, which is essential for infectivity, the capsid protein (CA) reassembles into a conical core. Maturation inhibitors (MIs) block HIV-1 maturation by interfering with protease-mediated CA-spacer peptide 1 (CA-SP1) processing, concomitantly stabilizing the immature CA-SP1 lattice; virions from MI-treated cells retain an immature-like CA-SP1 lattice, whereas mutational abolition of cleavage at the CA-SP1 site results in virions in which the CA-SP1 lattice converts to a mature-like form. We previously reported that propagation of HIV-1 in the presence of MI PF-46396 selected for assembly-defective, compound-dependent mutants with amino acid substitutions in the major homology region (MHR) of CA. Propagation of these mutants in the absence of PF-46396 resulted in the acquisition of second-site compensatory mutations. These included a Thr-to-Ile substitution at SP1 residue 8 (T8I), which results in impaired CA-SP1 processing. Thus, the T8I mutation phenocopies PF-46396 treatment in terms of its ability to rescue the replication defect imposed by the MHR mutations and to impede CASP1 processing. Here, we use cryo-electron tomography to show that, like MIs, the T8I mutation stabilizes the immature-like CA-SP1 lattice. These results have important implications for the mechanism of action of HIV-1 MIs; they also suggest that T8I may provide a valuable tool for structural definition of the CA-SP1 boundary region, which has thus far been refractory to highresolution analysis, apparently because of conformational flexibility in this region of Gag. IMPORTANCE HIV-1 maturation involves dissection of the Gag polyprotein by the viral protease and assembly of a conical capsid enclosing the viral ribonucleoprotein. Maturation inhibitors (MIs) prevent the final cleavage step at the site between the capsid protein (CA) and spacer peptide 1 (SP1), apparently by binding at this site and denying the protease access. Additionally, MIs stabilize the immature-like CA-SP1 lattice, preventing release of CA into the soluble pool. We previously found that T8I, a mutation in SP1, rescues a PF-46396-dependent CA mutant and blocks CA-SP1 cleavage. In this study, we imaged T8I virions by cryo-electron tomography and showed that T8I mutants, like MI-treated virions, contain an immature CA-SP1 lattice. These results lay the groundwork needed to understand the structure of the CA-SP1 interface region and further illuminate the mechanism of action of MIs

The Primary Enveloped Virion of Herpes Simplex Virus 1: Its Role in Nuclear Egress

Many viruses migrate between different cellular compartments for successive stages of assembly. The HSV-1 capsid assembles in the nucleus and then transfers into the cytoplasm. First, the capsid buds through the inner nuclear membrane, becoming coated with nuclear egress complex (NEC) protein. This yields a primary enveloped virion (PEV) whose envelope fuses with the outer nuclear membrane, releasing the capsid into the cytoplasm. We investigated the associated molecular mechanisms by isolating PEVs from US3-null-infected cells and imaging them by cryo-electron microscopy and tomography. (pUS3 is a viral protein kinase in whose absence PEVs accumulate in the perinuclear space.) Unlike mature extracellular virions, PEVs have very few lycoprotein spikes. PEVs are ~20% smaller than mature virions, and the little space available between the capsid and the NEC layer suggests that most tegument proteins are acquired later in the egress pathway. Previous studies have proposed that NEC is organized as hexamers in honeycomb arrays in PEVs, but we find arrays of heptameric rings in extracts from US3-nullinfected cells. In a PEV, NEC contacts the capsid predominantly via the pUL17/pUL25 complexes which are located close to the capsid vertices. Finally, the NEC layer dissociates from the capsid as it leaves the nucleus, possibly in response to pUS3- mediated phosphorylation. Overall, nuclear egress emerges as a process driven by a program of multiple weak interactions

Subassemblies and Asymmetry in Assembly of Herpes Simplex Virus Procapsid

The herpes simplex virus 1 (HSV-1) capsid is a massive particle (~200 MDa; 1,250-Å diameter) with T=16 icosahedral symmetry. It initially assembles as a procapsid with ~4,000 protein subunits of 11 different kinds. The procapsid undergoes major changes in structure and composition as it matures, a process driven by proteolysis and expulsion of the internal scaffolding protein. Assembly also relies on an external scaffolding protein, the triplex, an α2β heterotrimer that coordinates neighboring capsomers in the procapsid and becomes a stabilizing clamp in the mature capsid. To investigate the mechanisms that regulate its assembly, we developed a novel isolation procedure for the metastable procapsid and collected a large set of cryo-electron microscopy data. In addition to procapsids, these preparations contain maturation intermediates, which were distinguished by classifying the images and calculating a three-dimensional reconstruction for each class. Appraisal of the procapsid structure led to a new model for assembly; in it, the protomer (assembly unit) consists of one triplex, surrounded by three major capsid protein (MCP) subunits. The model exploits the triplexes’ departure from 3-fold symmetry to explain the highly skewed MCP hexamers, the triplex orientations at each 3-fold site, and the T=16 architecture. These observations also yielded new insights into maturation

Distribution and Redistribution of HIV-1 Nucleocapsid Protein in Immature, Mature, and Integrase-Inhibited Virions: a Role for Integrase in Maturation

During virion maturation, HIV-1 capsid protein assembles into a conical core containing the viral ribonucleoprotein (vRNP) complex, thought to be composed mainly of the viral RNA and nucleocapsid protein (NC). After infection, the viral RNA is reverse transcribed into double-stranded DNA, which is then incorporated into host chromosomes by integrase (IN) catalysis. Certain IN mutations (class II) and antiviral drugs (allosteric IN inhibitors [ALLINIs]) adversely affect maturation, resulting in virions that contain “eccentric condensates,” electron-dense aggregates located outside seemingly empty capsids. Here we demonstrate that in addition to this mislocalization of electron density, a class II IN mutation and ALLINIs each increase the fraction of virions with malformed capsids (from ∼12% to ∼53%). Eccentric condensates have a high NC content, as demonstrated by “tomo-bubblegram” imaging, a novel labeling technique that exploits the susceptibility of NC to radiation damage. Tomo-bubblegrams also localized NC inside wild-type cores and lining the spherical Gag shell in immature virions. We conclude that eccentric condensates represent nonpackaged vRNPs and that either genetic or pharmacological inhibition of IN can impair vRNP incorporation into mature cores. Supplying IN in trans as part of a Vpr-IN fusion protein partially restored the formation of conical cores with internal electron density and the infectivity of a class II IN deletion mutant virus. Moreover, the ability of ALLINIs to induce eccentric condensate formation required both IN and viral RNA. Based on these observations, we propose a role for IN in initiating core morphogenesis and vRNP incorporation into the mature core during HIV-1 maturation

Bootstrap-after-Bootstrap Model Averaging for Reducing Model Uncertainty in Model Selection for Air Pollution Mortality Studies

Ba c k g r o u n d: Concerns have been raised about findings of associations between particulate matter (PM) air pollution and mortality that have been based on a single “best ” model arising from a model selection procedure, because such a strategy may ignore model uncertainty inherently involved in searching through a set of candidate models to find the best model. Model averaging has been proposed as a method of allowing for model uncertainty in this context. Objectives: To propose an extension (double BOOT) to a previously described bootstrap modelaveraging procedure (BOOT) for use in time series studies of the association between PM and mortality. We compared double BOOT and BOOT with Bayesian model averaging (BMA) and a standard method of model selection [standard Akaike’s information criterion (AIC)]. Me t h o d: Actual time series data from the United States are used to conduct a simulation study to compare and contrast the performance of double BOOT, BOOT, BMA, and standard AIC. Re s u l t s: Double BOOT produced estimates of the effect of PM on mortality that have had smaller root mean squared error than did those produced by BOOT, BMA, and standard AIC. This performance boost resulted from estimates produced by double BOOT having smaller variance than those produced by BOOTand BMA. Co n c l u s i o n s: Double BOOT is a viable alternative to BOOT and BMA for producing estimates of the mortality effect of PM. Key w o r d s: air pollution, Bayesian, bootstrap, model averaging, mortality, particulate matter. Environ Health Perspect 118:131–136 (2010). doi:10.1289/ehp.0901007 available vi

Ultra-strong Adhesion of Graphene Membranes

As mechanical structures enter the nanoscale regime, the influence of van der Waals forces increases. Graphene is attractive for nanomechanical systems because its Young's modulus and strength are both intrinsically high, but the mechanical behavior of graphene is also strongly influenced by the van der Waals force. For example, this force clamps graphene samples to substrates, and also holds together the individual graphene sheets in multilayer samples. Here we use a pressurized blister test to directly measure the adhesion energy of graphene sheets with a silicon oxide substrate. We find an adhesion energy of 0.45 \pm 0.02 J/m2 for monolayer graphene and 0.31 \pm 0.03 J/m2 for samples containing 2-5 graphene sheets. These values are larger than the adhesion energies measured in typical micromechanical structures and are comparable to solid/liquid adhesion energies. We attribute this to the extreme flexibility of graphene, which allows it to conform to the topography of even the smoothest substrates, thus making its interaction with the substrate more liquid-like than solid-like.Comment: to appear in Nature Nanotechnolog

Risk algorithm using serial biomarker measurements doubles the number of screen-detected cancers compared with a single-threshold rule in the United Kingdom collaborative trial of ovarian cancer screening

PURPOSE: Cancer screening strategies have commonly adopted single-biomarker thresholds to identify abnormality. We investigated the impact of serial biomarker change interpreted through a risk algorithm on cancer detection rates. PATIENTS AND METHODS: In the United Kingdom Collaborative Trial of Ovarian Cancer Screening, 46,237 women, age 50 years or older underwent incidence screening by using the multimodal strategy (MMS) in which annual serum cancer antigen 125 (CA-125) was interpreted with the risk of ovarian cancer algorithm (ROCA). Women were triaged by the ROCA: normal risk, returned to annual screening; intermediate risk, repeat CA-125; and elevated risk, repeat CA-125 and transvaginal ultrasound. Women with persistently increased risk were clinically evaluated. All participants were followed through national cancer and/or death registries. Performance characteristics of a single-threshold rule and the ROCA were compared by using receiver operating characteristic curves. RESULTS: After 296,911 women-years of annual incidence screening, 640 women underwent surgery. Of those, 133 had primary invasive epithelial ovarian or tubal cancers (iEOCs). In all, 22 interval iEOCs occurred within 1 year of screening, of which one was detected by ROCA but was managed conservatively after clinical assessment. The sensitivity and specificity of MMS for detection of iEOCs were 85.8% (95% CI, 79.3% to 90.9%) and 99.8% (95% CI, 99.8% to 99.8%), respectively, with 4.8 surgeries per iEOC. ROCA alone detected 87.1% (135 of 155) of the iEOCs. Using fixed CA-125 cutoffs at the last annual screen of more than 35, more than 30, and more than 22 U/mL would have identified 41.3% (64 of 155), 48.4% (75 of 155), and 66.5% (103 of 155), respectively. The area under the curve for ROCA (0.915) was significantly (P = .0027) higher than that for a single-threshold rule (0.869). CONCLUSION: Screening by using ROCA doubled the number of screen-detected iEOCs compared with a fixed cutoff. In the context of cancer screening, reliance on predefined single-threshold rules may result in biomarkers of value being discarded

Apparently synonymous substitutions in FGFR2affect splicing and result in mild Crouzon syndrome

BACKGROUND: Mutations of fibroblast growth factor receptor 2 (FGFR2) account for a higher proportion of genetic cases of craniosynostosis than any other gene, and are associated with a wide spectrum of severity of clinical problems. Many of these mutations are highly recurrent and their associated features well documented. Crouzon syndrome is typically caused by heterozygous missense mutations in the third immunoglobulin domain of FGFR2. CASE PRESENTATION: Here we describe two families, each segregating a different, previously unreported FGFR2 mutation of the same nucleotide, c.1083A>G and c.1083A>T, both of which encode an apparently synonymous change at the Pro361 codon. We provide experimental evidence that these mutations affect normal FGFR2 splicing and document the clinical consequences, which include a mild Crouzon syndrome phenotype and reduced penetrance of craniosynostosis. CONCLUSIONS: These observations add to a growing list of FGFR2 mutations that affect splicing and provide important clinical information for genetic counselling of families affected by these specific mutations

Identification of sVSG117 as an immunodiagnostic antigen and evaluation of a dual-antigen lateral flow test for the diagnosis of human african trypanosomiasis

The diagnosis of human African trypanosomiasis (HAT) caused by Trypanosoma brucei gambiense relies mainly on the Card Agglutination Test for Trypanosomiasis (CATT). There is no immunodiagnostic for HAT caused by T. b. rhodesiense. Our principle aim was to develop a prototype lateral flow test that might be an improvement on CATT.Pools of infection and control sera were screened against four different soluble form variant surface glycoproteins (sVSGs) by ELISA and one, sVSG117, showed particularly strong immunoreactivity to pooled infection sera. Using individual sera, sVSG117 was shown to be able to discriminate between T. b. gambiense infection and control sera by both ELISA and lateral flow test. The sVSG117 antigen was subsequently used with a previously described recombinant diagnostic antigen, rISG65, to create a dual-antigen lateral flow test prototype. The latter was used blind in a virtual field trial of 431 randomized infection and control sera from the WHO HAT Specimen Biobank.In the virtual field trial, using two positive antigen bands as the criterion for infection, the sVSG117 and rISG65 dual-antigen lateral flow test prototype showed a sensitivity of 97.3% (95% CI: 93.3 to 99.2) and a specificity of 83.3% (95% CI: 76.4 to 88.9) for the detection of T. b. gambiense infections. The device was not as good for detecting T. b. rhodesiense infections using two positive antigen bands as the criterion for infection, with a sensitivity of 58.9% (95% CI: 44.9 to 71.9) and specificity of 97.3% (95% CI: 90.7 to 99.7). However, using one or both positive antigen band(s) as the criterion for T. b. rhodesiense infection improved the sensitivity to 83.9% (95% CI: 71.7 to 92.4) with a specificity of 85.3% (95% CI: 75.3 to 92.4). These results encourage further development of the dual-antigen device for clinical use