Expression levels of Fv1: effects on retroviral restriction specificities

Background The mouse protein Fv1 is a factor that can confer resistance to retroviral infection. The two major Fv1 alleles from laboratory mice, Fv1 n and Fv1 b , restrict infection by different murine leukaemia viruses (MLVs). Fv1n restricts B-tropic MLV, but not N-tropic MLV or NB-tropic MLV. In cells expressing Fv1b at natural levels, only N-MLV is restricted, however restriction of NB-MLV and partial restriction of B-MLV were observed when recombinant Fv1b was expressed from an MLV promoter in Fv1 null Mus dunni tail fibroblast cells. To investigate the relationship between expression level and restriction specificity we have developed new retroviral delivery vectors which allow inducible expression of Fv1, and yet allow sufficient production of fluorescent reporter proteins for analysis in our FACS-based restriction assay. Results We demonstrated that at concentrations close to the endogenous expression level, Fv1b specifically restricts only N-MLV, but restriction of NB-MLV, and to a lesser extent B-MLV, could be gained by increasing the protein level of Fv1b. By contrast, we found that even when Fv1n is expressed at very high levels, no significant inhibition of N-MLV or NB-MLV could be observed. Study of Fv1 mutants using this assay led to the identification of determinants for N/B tropism at an expression level close to that of endogenous Fv1n and Fv1b. We also compared the recently described restriction activities of wild mice Fv1 proteins directed against non-MLV retroviruses when expressed at different levels. Fv1 from M. spretus restricted N-MLV, B-MLV and equine infectious anaemia virus equally even at low concentrations, while Fv1 from M. macedonicus showed even stronger restriction against equine infectious anaemia virus than to N-MLV. Restriction of feline foamy virus by Fv1 of M. caroli occurred at levels equivalent to MLV restriction. Conclusions Our data indicate that for some but not all Fv1 proteins, gain of restriction activities could be achieved by increasing the expression level of Fv1. However such a concentration dependent effect is not seen with most Fv1s and cannot explain the recently reported activities against non-MLVs. It will be interesting to examine whether overexpression of other capsid binding restriction factors such as TRIM5α or Mx2 result in novel restriction specificities

Improvement of rheological and functional properties of milk protein concentrate by hydrodynamic cavitation

Spray drying at higher solids concentrations improves drying efficiency, and reduces the overall energy cost of milk powder production. As the performance of the evaporator prior to spray drying is limited by viscosity, several methods can be employed to reduce feed viscosity such as thermal pre-treatment or ultrasound. The method employed in this study was hydrodynamic cavitation (HC) on milk protein concentrate (MPC80). Rheological properties of the protein milk were observed to improve, with a reduction in viscosity by 20% and 56% upon the application of a cavitation rotor speed of 25 Hz and 50 Hz, respectively, due to the breakdown in protein gel structure and hence a decrease in the elastic modulus of the proteins. While HC did not adversely affect solubility, with the powders having on average a solubility of 97.5% at a reconstitution temperature of 50 °C, both bulk and tapped density increased when the emulsion was subjected to HC, owing to a reduction in particle size. This study therefore suggests the potential of using HC for a more efficient drying of high solids milk, while maintaining and/or improving the physicochemical properties of powders

A Novel Unsupervised Method to Identify Genes Important in the Anti-viral Response: Application to Interferon/Ribavirin in Hepatitis C Patients

Background: Treating hepatitis C with interferon/ribavirin results in a varied response in terms of decrease in viral titer and ultimate outcome. Marked responders have a sharp decline in viral titer within a few days of treatment initiation, whereas in other patients there is no effect on the virus (poor responders). Previous studies have shown that combination therapy modifies expression of hundreds of genes in vitro and in vivo. However, identifying which, if any, of these genes have a role in viral clearance remains challenging. Aims: The goal of this paper is to link viral levels with gene expression and thereby identify genes that may be responsible for early decrease in viral titer. Methods: Microarrays were performed on RNA isolated from PBMC of patients undergoing interferon/ribavirin therapy. Samples were collected at pre-treatment (day 0), and 1, 2, 7, 14 and 28 days after initiating treatment. A novel method was applied to identify genes that are linked to a decrease in viral titer during interferon/ribavirin treatment. The method uses the relationship between inter-patient gene expression based proximities and inter-patient viral titer based proximities to define the association between microarray gene expression measurements of each gene and viral-titer measurements. Results: We detected 36 unique genes whose expressions provide a clustering of patients that resembles viral titer based clustering of patients. These genes include IRF7, MX1, OASL and OAS2, viperin and many ISG's of unknown function. Conclusion: The genes identified by this method appear to play a major role in the reduction of hepatitis C virus during the early phase of treatment. The method has broad utility and can be used to analyze response to any group of factors influencing biological outcome such as antiviral drugs or anti-cancer agents where microarray data are available. © 2007 Brodsky et al

Picophytoplankton biomass distribution in the global ocean

The smallest marine phytoplankton, collectively termed picophytoplankton, have been routinely enumerated by flow cytometry since the late 1980s during cruises throughout most of the world ocean. We compiled a database of 40 946 data points, with separate abundance entries for Prochlorococcus, Synechococcus and picoeukaryotes. We use average conversion factors for each of the three groups to convert the abundance data to carbon biomass. After gridding with 1? spacing, the database covers 2.4% of the ocean surface area, with the best data coverage in the North Atlantic, the South Pacific and North Indian basins, and at least some data in all other basins. The average picophytoplankton biomass is 12 ± 22 µg Cl-1 or 1.9 g Cm-2. We estimate a total global picophytoplankton biomass of 0.53–1.32 Pg C (17–39% Prochlorococcus, 12–15% Synechococcus and 49–69% picoeukaryotes), with an intermediate/best estimate of 0.74 Pg C. Future efforts in this area of research should focus on reporting calibrated cell size and collecting data in undersampled regions

Potentiation of Synaptic GluN2B NMDAR Currents by Fyn Kinase Is Gated through BDNF-Mediated Disinhibition in Spinal Pain Processing

In chronic pain states, the neurotrophin brain-derived neurotrophic factor (BDNF) transforms the output of lamina I spinal neurons by decreasing synaptic inhibition. Pain hypersensitivity also depends on N-methyl-D-aspartate receptors (NMDARs) and Src-family kinases, but the locus of NMDAR dysregulation remains unknown. Here, we show that NMDAR-mediated currents at lamina I synapses are potentiated in a peripheral nerve injury model of neuropathic pain. We find that BDNF mediates NMDAR potentiation through activation of TrkB and phosphorylation of the GluN2B subunit by the Src-family kinase Fyn. Surprisingly, we find that Cl−-dependent disinhibition is necessary and sufficient to prime potentiation of synaptic NMDARs by BDNF. Thus, we propose that spinal pain amplification is mediated by a feedforward mechanism whereby loss of inhibition gates the increase in synaptic excitation within individual lamina I neurons. Given that neither disinhibition alone nor BDNF-TrkB signaling is sufficient to potentiate NMDARs, we have discovered a form of molecular coincidence detection in lamina I neurons

Rhesus TRIM5α disrupts the HIV-1 capsid at the inter-hexamer interfaces

TRIM proteins play important roles in the innate immune defense against retroviral infection, including human immunodeficiency virus type-1 (HIV-1). Rhesus macaque TRIM5α (TRIM5αrh) targets the HIV-1 capsid and blocks infection at an early post-entry stage, prior to reverse transcription. Studies have shown that binding of TRIM5α to the assembled capsid is essential for restriction and requires the coiled-coil and B30.2/SPRY domains, but the molecular mechanism of restriction is not fully understood. In this study, we investigated, by cryoEM combined with mutagenesis and chemical cross-linking, the direct interactions between HIV-1 capsid protein (CA) assemblies and purified TRIM5αrh containing coiled-coil and SPRY domains (CC-SPRYrh). Concentration-dependent binding of CC-SPRYrh to CA assemblies was observed, while under equivalent conditions the human protein did not bind. Importantly, CC-SPRYrh, but not its human counterpart, disrupted CA tubes in a non-random fashion, releasing fragments of protofilaments consisting of CA hexamers without dissociation into monomers. Furthermore, such structural destruction was prevented by inter-hexamer crosslinking using P207C/T216C mutant CA with disulfide bonds at the CTD-CTD trimer interface of capsid assemblies, but not by intra-hexamer crosslinking via A14C/E45C at the NTD-NTD interface. The same disruption effect by TRIM5αrh on the inter-hexamer interfaces also occurred with purified intact HIV-1 cores. These results provide insights concerning how TRIM5α disrupts the virion core and demonstrate that structural damage of the viral capsid by TRIM5α is likely one of the important components of the mechanism of TRIM5α-mediated HIV-1 restriction. © 2011 Zhao et al

Quantifying single nucleotide variant detection sensitivity in exome sequencing

BACKGROUND: The targeted capture and sequencing of genomic regions has rapidly demonstrated its utility in genetic studies. Inherent in this technology is considerable heterogeneity of target coverage and this is expected to systematically impact our sensitivity to detect genuine polymorphisms. To fully interpret the polymorphisms identified in a genetic study it is often essential to both detect polymorphisms and to understand where and with what probability real polymorphisms may have been missed. RESULTS: Using down-sampling of 30 deeply sequenced exomes and a set of gold-standard single nucleotide variant (SNV) genotype calls for each sample, we developed an empirical model relating the read depth at a polymorphic site to the probability of calling the correct genotype at that site. We find that measured sensitivity in SNV detection is substantially worse than that predicted from the naive expectation of sampling from a binomial. This calibrated model allows us to produce single nucleotide resolution SNV sensitivity estimates which can be merged to give summary sensitivity measures for any arbitrary partition of the target sequences (nucleotide, exon, gene, pathway, exome). These metrics are directly comparable between platforms and can be combined between samples to give “power estimates” for an entire study. We estimate a local read depth of 13X is required to detect the alleles and genotype of a heterozygous SNV 95% of the time, but only 3X for a homozygous SNV. At a mean on-target read depth of 20X, commonly used for rare disease exome sequencing studies, we predict 5–15% of heterozygous and 1–4% of homozygous SNVs in the targeted regions will be missed. CONCLUSIONS: Non-reference alleles in the heterozygote state have a high chance of being missed when commonly applied read coverage thresholds are used despite the widely held assumption that there is good polymorphism detection at these coverage levels. Such alleles are likely to be of functional importance in population based studies of rare diseases, somatic mutations in cancer and explaining the “missing heritability” of quantitative traits

Synergistic melanoma cell death mediated by inhibition of both MCL1 and BCL2 in high-risk tumors driven by NF1/PTEN loss

Melanomas driven by loss of the NF1 tumor suppressor have a high risk of treatment failure and effective therapies have not been developed. Here we show that loss-of-function mutations of nf1 and pten result in aggressive melanomas in zebrafish, representing the first animal model of NF1-mutant melanomas harboring PTEN loss. MEK or PI3K inhibitors show little activity when given alone due to cross-talk between the pathways, and high toxicity when given together. The mTOR inhibitors, sirolimus, everolimus, and temsirolimus, were the most active single agents tested, potently induced tumor-suppressive autophagy, but not apoptosis. Because addition of the BCL2 inhibitor venetoclax resulted in compensatory upregulation of MCL1, we established a three-drug combination composed of sirolimus, venetoclax, and the MCL1 inhibitor S63845. This well-tolerated drug combination potently and synergistically induces apoptosis in both zebrafish and human NF1/PTEN-deficient melanoma cells, providing preclinical evidence justifying an early-stage clinical trial in patients with NF1/PTEN-deficient melanoma

Structure of hadron resonances with a nearby zero of the amplitude

We discuss the relation between the analytic structure of the scattering amplitude and the origin of an eigenstate represented by a pole of the amplitude.If the eigenstate is not dynamically generated by the interaction in the channel of interest, the residue of the pole vanishes in the zero coupling limit. Based on the topological nature of the phase of the scattering amplitude, we show that the pole must encounter with the Castillejo-Dalitz-Dyson (CDD) zero in this limit. It is concluded that the dynamical component of the eigenstate is small if a CDD zero exists near the eigenstate pole. We show that the line shape of the resonance is distorted from the Breit-Wigner form as an observable consequence of the nearby CDD zero. Finally, studying the positions of poles and CDD zeros of the KbarN-piSigma amplitude, we discuss the origin of the eigenstates in the Lambda(1405) region.Comment: 7 pages, 3 figures, v2: published versio