Recombinant Flag-tagged E1E2 glycoproteins from three hepatitis C virus genotypes are biologically functional and elicit cross-reactive neutralizing antibodies in mice

Hepatitis C virus (HCV) is a globally disseminated human pathogen for which no vaccine is currently available. HCV is highly diverse genetically and can be classified into 7 genotypes and multiple sub-types. Due to this antigenic variation, the induction of cross-reactive and at the same time neutralizing antibodies is a challenge in vaccine production. Here we report the analysis of immunogenicity of recombinant HCV envelope glycoproteins from genotypes 1a, 1b and 2a, with a Flag tag inserted in the hypervariable region 1 of E2. This modification did not affect protein expression or conformation or its capacity to bind the crucial virus entry factor, CD81. Importantly, in immunogenicity studies on mice, the purified E2-Flag mutants elicited high-titer, cross-reactive antibodies that were able to neutralize HCV infectious particles from two genotypes tested (1a and 2a). These findings indicate that E1E2-Flag envelope glycoproteins could be important immunogen candidates for vaccine aiming to induce broad HCV-neutralizing responses

Change in Nephrometry Scoring in Small Renal Masses (<4cm) on Active Surveillance: Preliminary Observations from Tayside Active Surveillance Cohort (TASC) Study

Rationale and Objectives: - Prediction of growth, in particular knowing the possibility of aggressive cancer in small renal masses on active surveillance, remains poorly understood. The study was designed to determine whether serial nephrometry score measurements could predict possibility of aggressive malignancy (grade of cancer) in patients with small renal masses opting for active surveillance initially. Materials and Methods: - One hundred sixteen patients between January 2000 and December 2016 undergoing partial nephrectomy were recruited. Out of these, 97 were analyzed using different nephrometry scoring systems. Measurement of nephrometry scores (Radius of tumors, Exo/Endophytic; Nearness of tumors to the collecting system or sinus; Anterior/posterior; Location in relation to polar lines, Preoperative Aspects and Dimensions Used for Anatomical, Centrality Index) was performed by two researchers. Among the patients opting for partial nephrectomy, 40 were on active surveillance for at least 12 months (mean 32; 12-60 months) before partial nephrectomy. Computed tomography scan images of these patients were retrieved and analyzed including comparison to histopathology. Results: - Nephrometry scores measured on serial computed tomography scan images showed a significant correlation between change in score and grade of cancer on multivariate analysis (P value .001). Addition of multivariate analysis to nomogram based on change in size alone did not improve predictive value of area under the curve significantly. Conclusions: - Change in nephrometry scoring measurements correlates with grade of cancer in small renal masses but falls short of significantly predicting presence of malignancy or grade of cancer on nomogram in patients opting for active surveillance for small renal masses. At present, this approach may be inadequate for decision-making

Magnetization Measurements on LHC Superconducting Strands

When using superconducting magnets in particle accelerators like the LHC, persistent currents in the superconductor often determine the field quality at injection, where the magnetic field is low. This paper describes magnetization measurements made on LHC cable strands at the Technical University of Vienna and the Institute of Physics of the Polish Academy of Sciences in collaboration with CERN. Measurements were performed at T=2K and T=4.2K on more than 50 strands of 7 different manufacturers with NbTi filament diameter between 5 and 7 micrometer. Two different measurement set-ups were used: vibrating sample magnetometer, with a sample length of about 8mm, and an integrating coil magnetometer, with sample length of about 1m. The two methods were compared by measuring the same sample. Low field evidence of proximity effect is discussed. Statistics like ratio of the width of the magnetization loop at 4.2K and 2K, and the initial slope dM/dB after cooldown are presented. Decrease of the magnetization with time, of the order of 2% per hour, was observed in some samples

Microlensing optical depth toward the Galactic Bulge using bright sources from OGLE-II

We present a measurement of the microlensing optical depth toward the Galactic Bulge based on 4 years of the OGLE-II survey using Red Clump Giant (RCG). Using 32 events we find tau=2.55_{-0.46}^{+0.57}* 10^{-6} at (l,b)=(1.16, -2.75). Taking into account the measured gradient along the Galactic latitude b, tau = [ (4.48+/- 2.37) + (0.78+/- 0.84)* b]* 10^{-6}, this value is consistent with previous measurements using RCG sources and recent theoretical predictions. We determine the microlensing parameters and select events using a model light curve with the flux blending. We find that ~38% of the OGLE-II events which appear to have RCG sources are actually due to much fainter stars blended with a bright companion. We show explicitly that model fits without blending result in similar tau estimates through partial cancellation of contributions from higher detection efficiency, underestimated time-scales and larger number of selected events. This approach, however, leads to biased time-scale distributions and event rates. Consequently, microlensing studies should carefully consider source confusion effects even for bright stars.Comment: 49 pages and 18 figures, ApJ in press, the value changed due to the systematic correctio

Planetary Detection Efficiency of the Magnification 3000 Microlensing Event OGLE-2004-BLG-343

OGLE-2004-BLG-343 was a microlensing event with peak magnification A_{max}=3000+/-1100, by far the highest-magnification event ever analyzed and hence potentially extremely sensitive to planets orbiting the lens star. Due to human error, intensive monitoring did not begin until 43 minutes after peak, at which point the magnification had fallen to A~1200, still by far the highest ever observed. As the light curve does not show significant deviations due to a planet, we place upper limits on the presence of such planets by extending the method of Yoo et al. (2004b), which combines light-curve analysis with priors from a Galactic model of the source and lens populations, to take account of finite-source effects. This is the first event so analyzed for which finite-source effects are important, and hence we develop two new techniques for evaluating these effects. Somewhat surprisingly, we find that OGLE-2004-BLG-343 is no more sensitive to planets than two previously analyzed events with A_{max}~100, despite the fact that it was observed at ~12 times higher magnification. However, we show that had the event been observed over its peak, it would have been sensitive to almost all Neptune-mass planets over a factor of 5 of projected separation and even would have had some sensitivity to Earth-mass planets. This shows that some microlensing events being detected in current experiments are sensitive to very low-mass planets. We also give suggestions on how extremely high-magnification events can be more promptly monitored in the future.Comment: 50 pages, 13 figures, published in The Astrophysical Journa

Varicellovirus UL 49.5 proteins differentially affect the function of the transporter associated with antigen processing, TAP

Cytotoxic T-lymphocytes play an important role in the protection against viral infections, which they detect through the recognition of virus-derived peptides, presented in the context of MHC class I molecules at the surface of the infected cell. The transporter associated with antigen processing (TAP) plays an essential role in MHC class I–restricted antigen presentation, as TAP imports peptides into the ER, where peptide loading of MHC class I molecules takes place. In this study, the UL49.5 proteins of the varicelloviruses bovine herpesvirus 1 (BHV-1), pseudorabies virus (PRV), and equine herpesvirus 1 and 4 (EHV-1 and EHV-4) are characterized as members of a novel class of viral immune evasion proteins. These UL49.5 proteins interfere with MHC class I antigen presentation by blocking the supply of antigenic peptides through inhibition of TAP. BHV-1, PRV, and EHV-1 recombinant viruses lacking UL49.5 no longer interfere with peptide transport. Combined with the observation that the individually expressed UL49.5 proteins block TAP as well, these data indicate that UL49.5 is the viral factor that is both necessary and sufficient to abolish TAP function during productive infection by these viruses. The mechanisms through which the UL49.5 proteins of BHV-1, PRV, EHV-1, and EHV-4 block TAP exhibit surprising diversity. BHV-1 UL49.5 targets TAP for proteasomal degradation, whereas EHV-1 and EHV-4 UL49.5 interfere with the binding of ATP to TAP. In contrast, TAP stability and ATP recruitment are not affected by PRV UL49.5, although it has the capacity to arrest the peptide transporter in a translocation-incompetent state, a property shared with the BHV-1 and EHV-1 UL49.5. Taken together, these results classify the UL49.5 gene products of BHV-1, PRV, EHV-1, and EHV-4 as members of a novel family of viral immune evasion proteins, inhibiting TAP through a variety of mechanisms

Comprehensive linker-scanning mutagenesis of the hepatitis C virus E1 and E2 envelope glycoproteins reveals new structure–function relationships

Despite extensive research, many details about the structure and functions of hepatitis C virus (HCV) glycoproteins E1 and E2 are not fully understood, and their crystal structure remains to be determined. We applied linker-scanning mutagenesis to generate a panel of 34 mutants, each containing an insertion of 5 aa at a random position within the E1E2 sequence. The mutated glycoproteins were analysed by using a range of assays to identify regions critical for maintaining protein conformation, E1E2 complex assembly, CD81 receptor binding, membrane fusion and infectivity. The results, while supporting previously published data, provide several interesting new findings. Firstly, insertion at amino acid 587 or 596 reduced E1E2 heterodimerization without affecting reactivity with some conformation-sensitive mAbs or with CD81, thus implicating these residues in glycoprotein assembly. Secondly, insertions within a conserved region of E2, between amino acid residues 611 and 631, severely disrupted protein conformation and abrogated binding of all conformation-sensitive antibodies, suggesting that the structural integrity of this region is critical for the correct folding of E2. Thirdly, an insertion at Leu-682 specifically affected membrane fusion, providing direct evidence that the membrane-proximal ‘stem’ of E2 is involved in the fusion mechanism. Overall, our results show that the HCV glycoproteins generally do not tolerate insertions and that there are a very limited number of sites that can be changed without dramatic loss of function. Nevertheless, we identified two E2 insertion mutants, at amino acid residues 408 and 577, that were infectious in the murine leukemia virus-based HCV pseudoparticle system