Hepatitis C virus NS4B carboxy terminal domain is a membrane binding domain

<p>Abstract</p> <p>Background</p> <p>Hepatitis C virus (HCV) induces membrane rearrangements during replication. All HCV proteins are associated to membranes, pointing out the importance of membranes for HCV. Non structural protein 4B (NS4B) has been reported to induce cellular membrane alterations like the membranous web. Four transmembrane segments in the middle of the protein anchor NS4B to membranes. An amphipatic helix at the amino-terminus attaches to membranes as well. The carboxy-terminal domain (CTD) of NS4B is highly conserved in Hepaciviruses, though its function remains unknown.</p> <p>Results</p> <p>A cytosolic localization is predicted for the NS4B-CTD. However, using membrane floatation assays and immunofluorescence, we now show targeting of the NS4B-CTD to membranes. Furthermore, a profile-profile search, with an HCV NS4B-CTD multiple sequence alignment, indicates sequence similarity to the membrane binding domain of prokaryotic D-lactate dehydrogenase (d-LDH). The crystal structure of E. coli d-LDH suggests that the region similar to NS4B-CTD is located in the membrane binding domain (MBD) of d-LDH, implying analogy in membrane association. Targeting of d-LDH to membranes occurs via electrostatic interactions of positive residues on the outside of the protein with negative head groups of lipids. To verify that anchorage of d-LDH MBD and NS4B-CTD is analogous, NS4B-CTD mutants were designed to disrupt these electrostatic interactions. Membrane association was confirmed by swopping the membrane contacting helix of d-LDH with the corresponding domain of the 4B-CTD. Furthermore, the functionality of these residues was tested in the HCV replicon system.</p> <p>Conclusion</p> <p>Together these data show that NS4B-CTD is associated to membranes, similar to the prokaryotic d-LDH MBD, and is important for replication.</p

Mutagenesis of the transmembrane domain of the SARS coronavirus spike glycoprotein: refinement of the requirements for SARS coronavirus cell entry

Abstract Background The spike protein (S) of SARS Coronavirus (SARS-CoV) mediates entry of the virus into target cells, including receptor binding and membrane fusion. Close to or in the viral membrane, the S protein contains three distinct motifs: a juxtamembrane aromatic part, a central highly hydrophobic stretch and a cysteine rich motif. Here, we investigate the role of aromatic and hydrophobic parts of S in the entry of SARS CoV and in cell-cell fusion. This was investigated using the previously described SARS pseudotyped particles system (SARSpp) and by fluorescence-based cell-cell fusion assays. Results Mutagenesis showed that the aromatic domain was crucial for SARSpp entry into cells, with a likely role in pore enlargement. Introduction of lysine residues in the hydrophobic stretch of S also resulted in a block of entry, suggesting the borders of the actual transmembrane domain. Surprisingly, replacement of a glycine residue, situated close to the aromatic domain, with a lysine residue was tolerated, whereas the introduction of a lysine adjacent to the glycine, was not. In a model, we propose that during fusion, the lateral flexibility of the transmembrane domain plays a critical role, as do the tryptophans and the cysteines. Conclusions The aromatic domain plays a crucial role in the entry of SARS CoV into target cells. The positioning of the aromatic domain and the hydrophobic domain relative to each other is another essential characteristic of this membrane fusion process.</p

Cholesteric color filter and method of manufacturing such

\u3cp\u3eThe invention pertains to a method of manufacturing a layer of a cholesterically ordered polymer material, in which the material is oriented in such a way that the axis of the molecular helix of the cholesterically ordered material extends transversely to the layer, wherein the method comprises the following steps: a) providing a layer comprising a cholesterically ordered material, which material comprises a quantity of a convertible compound, the conversion of said compound being inducible by radiation; b) irradiating the layer to convert at least a part of the convertible compound in the irradiated parts of the layer; c) optionally at least partially polymerizing and/or cross-linking the cholesterically ordered material to form a three-dimensional polymer; d) applying a diffusion agent onto the layer to transversely swell the layer as a function of the concentration variation of the diffusion agent; e) fixing the diffusion agent to the polymer to freeze in the formed structure.\u3c/p\u3

GxxxG Motif of Severe Acute Respiratory Syndrome Coronavirus Spike Glycoprotein Transmembrane Domain Is Not Involved in Trimerization and Is Not Important for Entry▿

Recently, a paper was published in which it was proposed that the GxxxG motif of the severe acute respiratory syndrome (SARS) coronavirus spike (S) protein transmembrane domain plays a vital role in oligomerization of the protein (E. Arbely, Z. Granot, I. Kass, J. Orly, and I. T. Arkin, Biochemistry 45:11349-11356, 2006). Here, we show that the GxxxG motif is not involved in SARS S oligomerization by trimerization analysis of S GxxxG mutant proteins. In addition, the capability of S to mediate entry of SARS S-pseudotyped particles overall was affected moderately in the mutant proteins, also arguing for a nonvital role for the GxxxG motif in SARS coronavirus entry

Formation of optical films by photo-polymerisation of liquid crystalline acrylates and application of these films in liquid crystal display technology

\u3cp\u3ePhoto-polymerisation of liquid crystalline di-acrylates is a very versatile method to produce homogeneously aligned polymer films. The orientation of the liquid crystals and hence the anisotropic optical properties of the films are frozen in by the photo-polymerisation process. Homogeneously aligned nematic liquid crystals form birefringent films that find application as retarders, while splayed nematic liquid crystalline mixtures form, angle dependent birefringent films that find application as wide viewing angle foils. In addition, cholesteric liquid crystals form reflecting films that can be used as colour filters or polarisers and smectic liquid crystals form highly ordered films that find application as polarisers. Moreover, modulating the light during the manufacturing process either in lateral or in-depth direction of the film can create complex structures not attainable with liquid crystalline polymers. All these films are mechanically, thermally and chemically stable and therefore very suitable to be processed in liquid crystal display manufacturing as additional films or as in-cell optical components.\u3c/p\u3

Predicting menopausal age with anti-Mullerian hormone: a cross-validation study of two existing models

Objective This study aimed to cross-validate two comparable Weibull models of prediction of age at natural menopause from two cohorts, the Scheffer, van Rooij, de Vet (SRV) cohort and the Tehran Lipid and Glucose Study (TLGS) cohort. It summarizes advantages and disadvantages of the models and underlines the need for achieving correct time dependency in dynamic variables like anti-Mullerian hormone. Methods Models were fitted in the original datasets and then applied to the cross-validation datasets. The discriminatory capacity of each model was assessed by calculating C-statistics for the models in their own data and in the cross-validation data. Calibration of the models on the cross-validation data was assessed by measuring the slope, intercept and Weibull shape parameter. Results The C-statistic for the SRV model on the SRV data was 0.7 (95% confidence interval (CI) 0.7-0.8) and on the TLGS data it was 0.8 (95% CI 0.8-0.9). For the TLGS model on the TLGS data, it was 0.9 (95% CI 0.8-0.9) and on the SRV data it was 0.7 (95% CI 0.6-0.8). After calibration of the SRV model on the TLGS data, the slope was 1, the intercept -0.3 and the shape parameter 1.1. The TLGS model on the SRV data had a slope of 0.3, an intercept of 12.7 and a shape parameter of 0.6. Conclusions Both models discriminate well between women that enter menopause early or late during follow-up. While the SRV model showed good agreement between the predicted risk of entering menopause and the observed proportion of women who entered menopause during follow-up (calibration) in the cross-validation dataset, the TLGS model showed poor calibration

High aspect ratio surface relief structures by photoembossing

Photoembossing is a convenient and economical process to form complex surface relief structures in polymer thin films. We have improved the aspect ratio of photoembossed microstructures by adding t-butyl hydroquinone (TBHQ) to the polymerization mixture. The mechanism that is proposed is based on the radical transfer principle, where TBHQ converts acrylate radicals into stable phenol radicals that at elevated temperatures act as latent initiators, thereby controlling the kinetics without changing the number of polymerization active sites. As a result, the aspect ratio can be improved with a factor of 5–7 in comparison with previously proposed similar processe