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During the 21st century pandemic, it has become difficult for students and teachers to engage physically in the aftermath of the epidemic, the process of assessing and grading students has become cumbersome. Multiple Choice Questions (MCQs) have increasingly become a more popular method of assessing a person's knowledge, but the questions which are generated directly from the chapter provided are readily available on the internet. Thus evaluer, the proposed approach solves this problem by paraphrasing the text, and thus the questions, so as to make it difficult to look it up on the internet. Usage of OMR(Optical Mark Recognition) sheets or manual correction had been prominent in the pre-pandemics situation. The evaluer automates the process of generation of MCQs which are difficult to search on the internet, and speeds up the task of grading a candidate's OMR answer sheet.

The objective of the paper was to reinforce security and to make privacy a priority in mailing services. There has been an excellent effort over a few decades to enhance the security of emails. ProtonMail has made an enormous breakthrough in the security field by using encryption as a base to reinforce the user’s data privacy and digital wellbeing. The security constraints prevent ProtonMail itself from deciphering the messages. ProtonMail has implemented various algorithms like SSL, TLS, TOR, and Open PGP to upgrade privacy. ProtonMail helps the field of Information Technology as it provides a secure e-mail experience with zero data sharing, which other mailing platforms cannot ensure. An analytical comparison was conducted to demonstrate how ProtonMail eradicates the vulnerabilities that other mailing services allow. The results demonstrated that the algorithms effectively prevent data breaches and are protected with the keys provided. The scope for threat significantly decreases and reflects on how it is an ideal platform to adopt in the coming future to seize possibilities of misconduct.

The paper proposes a method to make roads a safer place for driving by taking advantage of Internet of Things (IoT) enabled vehicles. The proposed method is based on assessing the trust levels of each vehicle and the behavioral patterns of the driver. Proposed method also utilizes a mobile agent(MA) which would help detecting Misbehaving nodes in the IoT enabled Vehicular Adhoc NETwork (VANET). The proposed method works on the assumption that all the nodes in VANET comprises of the sensors which can give the required information about the user and sensor behavior. We also presume that the IoT backbone required to accomplish the architecture is laid down.

The IEEE 802.16e [Mobile Worldwide Interoperability for Microwave Access(MWiMAX)] standard supports the mobility of Subscriber Station (SS), thus making them Mobile Station (MS). This incurs one of the most challenging issues in IEEE 802.16e which is handover (HO). HO occurs whenever MS migrates from one cell to another. Reducing HO delay is significant to improve the Quality of Service (QoS). To reduce the delay, we propose a method where the amount of intersection between the coverage area of Serving Base Station (SBS) and the coverage area of Neighboring Base Station (NBS) is considered as one set of NBS (Set A). The NBS which are in the direction in which MS is moving forms another set of NBS (Set B). The NBS which exist in Set A as well as Set B will then be scanned by MS for finding a suitable TBS. The simulation is implemented in MATLAB and graphs are provided to show the improvement in terms of lesser packet drop and reduced handover delay than the standard approach as well as the adaptive approach.

While several therapeutic options exist, the need for more effective, safe, and convenient treatment for a variety of autoimmune diseases persists. Targeting the Janus tyrosine kinases (JAKs), which play essential roles in cell signaling responses and can contribute to aberrant immune function associated with disease, has emerged as a novel and attractive approach for the development of new autoimmune disease therapies. We screened our compound library against JAK3, a key signaling kinase in immune cells, and identified multiple scaffolds showing good inhibitory activity for this kinase. A particular scaffold of interest, the 1H-pyrrolo[2,3-b]pyridine series (7-azaindoles), was selected for further optimization in part on the basis of binding affinity (Ki) as well as on the basis of cellular potency. Optimization of this chemical series led to the identification of VX-509 (decernotinib), a novel, potent, and selective JAK3 inhibitor, which demonstrates good efficacy in vivo in the rat host versus graft model (HvG). On the basis of these findings, it appears that VX-509 offers potential for the treatment of a variety of autoimmune diseases.

VX-787 is a novel inhibitor of influenza virus replication that blocks the PB2 cap-snatching activity of the influenza viral polymerase complex. Viral genetics and X-ray crystallography studies provide support for the idea that VX-787 occupies the 7-methyl GTP (m 7 GTP) cap-binding site of PB2. VX-787 binds the cap-binding domain of the PB2 subunit with a K D (dissociation constant) of 24 nM as determined by isothermal titration calorimetry (ITC). The cell-based EC 50 (the concentration of compound that ensures 50% cell viability of an uninfected control) for VX-787 is 1.6 nM in a cytopathic effect (CPE) assay, with a similar EC 50 in a viral RNA replication assay. VX-787 is active against a diverse panel of influenza A virus strains, including H1N1pdm09 and H5N1 strains, as well as strains with reduced susceptibility to neuraminidase inhibitors (NAIs). VX-787 was highly efficacious in both prophylaxis and treatment models of mouse influenza and was superior to the neuraminidase inhibitor, oseltamivir, including in delayed-start-to-treat experiments, with 100% survival at up to 96 h postinfection and partial survival in groups where the initiation of therapy was delayed up to 120 h postinfection. At different doses, VX-787 showed a 1-log to >5-log reduction in viral load (relative to vehicle controls) in mouse lungs. Overall, these favorable findings validate the PB2 subunit of the viral polymerase as a drug target for influenza therapy and support the continued development of VX-787 as a novel antiviral agent for the treatment of influenza infection.

The precise role(s) and topological organization of different factors in the hepatitis C virus (HCV) RNA replication complex are not well understood. In order to elucidate the role of viral and host proteins in HCV replication, we have developed a novel in vitro replication system that utilizes a rolling-circle RNA template. Under close-to-physiological salt conditions, HCV NS5BΔ21, an RNA-dependent RNA polymerase, has poor affinity for the RNA template. Human replication protein A (RPA) and HCV NS5A recruit NS5BΔ21 to the template. Subsequently, NS3 is recruited to the replication complex by NS5BΔ21, resulting in RNA synthesis stimulation by helicase. Both RPA and NS5A (S25-C447) , but not NS5A (S25-K215) , enabled the NS5BΔ21-NS3 helicase complex to be stably associated with the template and synthesize RNA product in a highly processive manner in vitro . This new in vitro HCV replication system is a useful tool that may facilitate the study of other replication factors and aid in the discovery of novel inhibitors of HCV replication. IMPORTANCE The molecular mechanism of hepatitis C virus (HCV) replication is not fully understood, but viral and host proteins collaborate in this process. Using a rolling-circle RNA template, we have reconstituted an in vitro HCV replication system that allows us to interrogate the role of viral and host proteins in HCV replication and delineate the molecular interactions. We showed that HCV NS5A (S25-C447) and cellular replication protein A (RPA) functionally cooperate as a processivity factor to stimulate HCV replication by HCV NS5BΔ21 polymerase and NS3 helicase. This system paves the way to test other proteins and may be used as an assay for discovery of HCV inhibitors.

Pim-1 is an oncogene-encoded serine/threonine kinase primarily expressed in hematopoietic and germ cell lines. Pim-1 kinase was originally identified in Maloney murine leukemia virus-induced T-cell lymphomas and is associated with multiple cellular functions such as proliferation, survival, differentiation, apoptosis, and tumorigenesis (Wang, Z., Bhattacharya, N., Weaver, M., Petersen, K., Meyer, M., Gapter, L., and Magnuson, N. S. (2001) J. Vet. Sci. 2, 167-179). The crystal structures of Pim-1 complexed with staurosporine and adenosine were determined. Although a typical two-domain serine/threonine protein kinase fold is observed, the inter-domain hinge region is unusual in both sequence and conformation; a two-residue insertion causes the hinge to bulge away from the ATP-binding pocket, and a proline residue in the hinge removes a conserved main chain hydrogen bond donor. Without this hydrogen bond, van der Waals interactions with the hinge serve to position the ligand. The hinge region of Pim-1 resembles that of phosphatidylinositol 3-kinase more closely than it does other protein kinases. Although the phosphatidylinositol 3-kinase inhibitor LY294002 also inhibits Pim-1, the structure of the LY294002.Pim-1 complex reveals a new binding mode that may be general for Ser/Thr kinases.

Differential scanning calorimetry (DSC) and x-ray diffraction have been used to study the structural and thermal properties of totally synthetic D-erythro-N-palmitoyl-lactosyl-C(18)-sphingosine (C16:0-LacCer). Over the temperature range 0-90 degrees C, fully hydrated C16:0-LacCer shows complex thermal transitions characteristic of polymorphic behavior of exclusively bilayer phases. On heating at 5 degrees C/min, hydrated C16:0-LacCer undergoes a complex two-peak endothermic transition with maxima at 69 degrees C and 74 degrees C and a total enthalpy of 14.6 kcal/mol C16:0-LacCer. At a slower heating rate (1.5 degrees C/min), two endothermic transitions are observed at 66 degrees C and 78 degrees C. After cooling to 0 degrees C, the subsequent heating run shows three overlapping endothermic transitions at 66 degrees C, 69 degrees C, and 71.5 degrees C, followed by a chain-melting endothermic transition at 78 degrees C. Two thermal protocols were used to completely convert C16:0-LacCer to its stable, high melting temperature (78 degrees C) form. As revealed by x-ray diffraction, over the temperature range 20-78 degrees C this stable phase exhibits a bilayer structure, periodicity d approximately 65 A with an ordered chain packing mode. At the phase transition (78 degrees C) chain melting occurs, and C16:0-LacCer converts to a liquid crystalline bilayer (L(alpha)) phase of reduced periodicity d approximately 59 A. On cooling from the L(alpha) phase, C16:0-LacCer converts to metastable bilayer phases undergoing transitions at 66-72 degrees C. These studies allow comparisons to be made with the behavior of the corresponding C16:0-Cer (. J. Lipid Res. 36:1936-1944) and C16:0-GluCer and C16:0-GalCer (. J. Lipid Res. 40:839-849). Our systematic studies are aimed at understanding the role of oligosaccharide complexity in regulating glycosphingolipid structure and properties.

SAP, the product of the gene mutated in X-linked lymphoproliferative syndrome (XLP), consists of a single SH2 domain that has been shown to bind the cytoplasmic tail of the lymphocyte coreceptor SLAM. Here we describe structures that show that SAP binds phosphorylated and nonphosphorylated SLAM peptides in a similar mode, with the tyrosine or phosphotyrosine residue inserted into the phosphotyrosine-binding pocket. We find that specific interactions with residues N-terminal to the tyrosine, in addition to more characteristic C-terminal interactions, stabilize the complexes. A phosphopeptide library screen and analysis of mutations identified in XLP patients confirm that these extended interactions are required for SAP function. Further, we show that SAP and the similar protein EAT-2 recognize the sequence motif TIpYXX(V/I).

The structural and thermal properties of aqueous dispersions of the totally synthetic cerebrosides, d-erythro-N-palmitoyl galactosyl- and glucosyl-C18-sphingosine (C16:0-GalCer and C16:0-GluCer, respectively) have been studied using differential scanning calorimetry (DSC) and X-ray diffraction. Over the temperature range 0–100°C, both C16:0-GalCer and C16:0-GluCer show complex thermal transitions characteristic of polymorphic behavior of exclusively bilayer phases. On heating, hydrated C16:0-GalCer undergoes an exothermic bilayer–bilayer transition at 59°C to produce a stable bilayer crystal form. X-ray diffraction at 70°C reveals a bilayer structure with an ordered hydrocarbon chain-packing arrangement. This ordered bilayer phase undergoes an endothermic chain-melting transition at 85°C to the bilayer liquid crystalline state. Similar behavior is exhibited by hydrated C16:0-GluCer which undergoes the exothermic transition at 49°C and a chain-melting transition at 87°C. The exothermic transitions observed on heating hydrated C16:0-GalCer and C16:0-GluCer are irreversible and dependent upon previous chain melting, prior cooling rate, and time of incubation at low temperatures. Thus, the structure and properties of totally synthetic C16:0-GalCer and C16:0-GluCer with identical sphingosine (C18:1) and fatty acid (C16:0) chains are quite similar, suggesting that the precise isomeric structure of the linked sugar plays only a minor role in regulating the properties of hydrated cerebrosides. Further, these studies indicate that the complex thermal behavior and bilayer phase formation exhibited by these single-suga cerebrosides are intrinsic properties and not due to the heterogeneity of the sphingosine base found in natural and partially synthetic cerebrosides.—Saxena, K., R. I. Duclos, P. Zimmermann, R. R. Schmidt, and G. G. Shipley. Structure and properties of totally synthetic galactoand gluco-cerebrosides. J. Lipid Res. 1999. 40: 839–849.

Our knowledge of the large family of proteins that contain the WD repeat continues to accumulate. The WD-repeat proteins are found in all eukaryotes and are implicated in a wide variety of crucial functions. The solution of the three-dimensional structure of one WD-repeat protein and the assumption that the structure will be common to all members of this family has allowed subfamilies of WD-repeat proteins to be defined on the basis of probable surface similarity. Proteins that have very similar surfaces are likely to have common binding partners and similar functions.

The βγ subunits of the heterotrimeric GTP-binding proteins (G proteins) that couple heptahelical, plasma membrane-bound receptors to intracellular effector enzymes or ion channels directly regulate several types of effectors, including phospholipase Cβ and adenylyl cyclase. The β subunit is made up of two structurally different regions: an N-terminal α helix followed by a toroidal structure made up of 7 blades, each of which is a twisted β sheet composed of four anti-parallel β strands (Wall, M. A., Coleman, D. E., Lee, E., Iniguez-Lluhi, J. A., Posner, B. A., Gilman, A. G., and Sprang, S. R. (1995)Cell 83, 1047–1058; Lambright, D. G., Sondek, J., Bohm, A., Skiba, N. P., Hamm, H. E., and Sigler, P. B. (1996) Nature 379, 311–319). We have previously shown that sites for activation of PLCβ2, PLCβ3, and adenylyl cyclase II overlap on the “top” surface of the propeller, where Gα also binds (Li, Y., Sternweis, P. M., Charnecki, S., Smith, T. F., Gilman, A. G., Neer, E. J., and Kozasa, T. (1998)J. Biol. Chem. 273, 16265–16272). The present study was undertaken to identify the regions on the side of the torus that might be important for effector interactions. We made mutations in each of the outer β strands of the G protein β1 propeller, as well as mutations in the loops that connect the outer strands to the adjacent β strands. Our results suggest that activation of PLCβ2 involves residues in the outer strands of blades 2, 6, and 7 of the propeller. We tested three of the mutations that most severely affected PLCβ2 activity against two forms of adenylyl cyclase (ACI and ACII). Both inhibition of ACI and activation of ACII were unaffected by these mutations, suggesting that if ACI and ACII contact the outer strands, the sites of contact are different from those for PLCβ2. We propose that distinct sets of contacts along the sides of the propeller will define the specificity of the interaction of βγ with effectors.

After deacylation of bovine brain sulfatide under mild alkaline conditions and reacylation using palmitoyl chloride (, Chem. Phys. Lipids. 34:41-53), the anionic glycosphingolipid N-palmitoyl galactosulfatide (C16:0-GalSulf) has been synthesized. By differential scanning calorimetry (DSC), anhydrous C16:0-GalSulf exhibits an endothermic transition, T(M) = 93 degrees C (DeltaH = 5. 5 kcal/mol C16:0-GalSulf) on heating. With increasing hydration (50 mM sodium phosphate buffer, pH 7.0; 50 mM NaCl), T(M) decreases, reaching a limiting value of 49 degrees C (DeltaH = 8.2 kcal/mol C16:0-GalSulf) at 20 wt% buffer. X-ray diffraction data have been recorded over the hydration range 0-62% at temperatures below (20 degrees C) and above (60 degrees C) T(M). At 20 degrees C, sharp wide-angle reflections at approximately 1/4.4 A(-1), approximately 1/4.1 A(-1), and approximately 1/3.8 A(-1) indicate the presence of an ordered-chain gel phase, whereas at 60 degrees C a broad reflection at 1/4.5 A(-1) characteristic of a melted-chain phase is observed. Lamellar diffraction patterns consistent with the presence of bilayer phases are observed at both temperatures. At 60 degrees C, in the liquid-crystalline L(alpha) phase, the bilayer periodicity increases with hydration, in both water and 100 mM Na(+) buffer. Interestingly, in the gel phase at 20 degrees C, the bilayer periodicity (d = 64 A) is insensitive to hydration (over the range 30-60 wt%) with either water or buffer. The continuous swelling behavior exhibited by the L(alpha) bilayer phase of C16:0-GalSulf is typical of lipids bearing a net negative charge and confirms that the presence of 100 mM Na(+) is insufficient to shield the charge contributed by the sulfate group. In contrast, the lack of continuous swelling behavior of the bilayer gel phase of C16:0-GalSulf is unusual and resembles that of Na(+) soaps. Thus, presumably, alterations in the surface charge characteristics of the C16:0-GalSulf bilayer occur on hydrocarbon chain melting and lead to major changes in lipid hydration.

The low-density lipoprotein (LDL) receptor plays a key role in maintaining circulating and cellular cholesterol homeostasis. The LDL receptor is a transmembrane glycoprotein whose biochemical and genetic properties have been extensively studied notably by Brown, Goldstein and colleagues [Brown, M. S.,Goldstein, J. L., (1986) Science 232, 34-47]. However, few if any structural studies of the LDL receptor have been reported, and details of its secondary and tertiary structure are lacking. In an attempt to determine the low-resolution structure of the LDL receptor, we have purified the receptor from bovine adrenal cortices using modifications of the method of Schneider et al. [Schneider, W. J., Goldstein, J. L.,Brown, M. S. (1985) Methods in Enzymol.109, 405-417]. Using circular dichroism, the secondary structure of the detergent-solubilized bovine LDL receptor at 25 degrees C was shown to be 19% alpha-helix, 42% beta-sheet, and 39% random coil. Interestingly, the detergent-solubilized receptor appeared to be quite resistant to changes in secondary structure over the temperature range 10-90 degrees C, with only minor but reversible changes being observed. In contrast, a more pronounced unfolding of the detergent-solubilized receptor was observed in the presence of guanidinium hydrochloride. Using the complete sequence of the human LDL receptor, sequence analysis by the Chou-Fasman prediction algorithm showed quite good agreement with the experimentally determined secondary structure of the bovine LDL receptor at 25 degrees C. Finally, the purified, bovine LDL receptor was reconstituted into large unilamellar vesicles of egg yolk phosphatidylcholine using a procedure exploiting preformed vesicles and detergent dialysis. We showed previously using negative stain electron microscopy that reconstituted vesicles bind LDL. Now, using cryoelectron microscopy of frozen hydrated reconstituted vesicles evidence of an extended, stick-like morphology (length approximately 120 A) for the extracellular domain of the LDL receptor has been obtained. Successful purification of the receptor, its incorporation into single bilayer vesicles, and its direct visualization by cryoelectron microscopy pave the way for more detailed structural studies of the LDL receptor and the receptor-LDL complex.

WD repeat proteins are a family of proteins that contain a series of highly conserved internal repeat motifs, usually ending with WD (Trp-Asp). The G beta subunit of heterotrimeric guanine nucleotide binding protein is a member of this family, and its crystal structure has been recently solved at high resolution (Wall et al. (1995) Cell 83, 1047-1058; Sondek et al. (1996) Nature 379, 369-374). Based on the coordinates of G beta, we have constructed a model for the structure of Sec13, a 33 kDa WD repeat protein from Saccharomyces cerevesiae essential for vesicular traffic. The model has been tested using a combination of biophysical and biochemical methods. Sec13 was expressed in Escherichia coli as a hexa-His-tagged protein (H6Sec13) and purified to homogeneity. In contrast to some other WD repeat proteins that are unable to fold into monomeric structures when expressed in E. coli, H6Sec13 was soluble and monomeric in the absence of detergent. The far-UV circular dichroism (CD) spectra of H6Sec13 indicated less than 10% alpha-helix consistent with the model which predicts primarily beta-sheets. H6Sec13 shows a cooperative and irreversible thermal denaturation curve consistent with a tightly packed structure. The CD spectrum shows an unusual positive ellipticity at 229 nm that was attributed to interactions of surface tryptophans since the 229 nm maximum could be abolished by modification of 6.3 +/- 0.3 (n = 3) tryptophans (out of 15 total in the molecule) with N-bromosuccinimide. Our model predicts that three sets of tryptophans are clustered near the surface. As predicted by the model, purified H6Sec13 was completely resistant to trypsin digestion. The concordance of the model of Sec13 presented in this paper with the biochemical and biophysical studies suggests that this model can be useful as a guide to further experiments designed to elucidate the function of Sec13 in vesicular traffic.