Steady-State NTPase Activity of Dengue Virus NS3: Number of Catalytic Sites,Nucleotide Specificity and Activation by ssRNA

Dengue virus nonstructural protein 3 (NS3) unwinds double stranded RNA driven by the free energy derived from the hydrolysis of nucleoside triphosphates. This paper presents the first systematic and quantitative characterization of the steady-state NTPase activity of DENV NS3 and their interaction with ssRNA. Substrate curves for ATP, GTP, CTP and UTP were obtained, and the specificity order for these nucleotides - evaluated as the ratio (k_cat/K_M)- was GTPATPCTP UTP, which showed that NS3 have poor ability to discriminate between different NTPs. Competition experiments between the four substrates indicated that all of them are hydrolyzed in one and the same catalytic site of the enzyme. The effect of ssRNA on the ATPase activity of NS3 was studied using poly(A) and poly(C). Both RNA molecules produced a 10 fold increase in the turnover rate constant (k_cat) and a 100 fold decrease in the apparent affinity (K_M) for ATP. When the ratio [RNA bases]/[NS3] was between 0 and 20 the ATPase activity was inhibited by increasing both poly(A) and poly(C). Using the theory of binding of large ligands (NS3) to a one-dimensional homogeneous lattice of infinite length (RNA) we tested the hypothesis that inhibition is the result of crowding of NS3 molecules along the RNA lattices. Finally, we discuss why this hypothesis is consistent with the idea that the ATPase catalytic cycle is tightly coupled to the movement of NS3 helicase along the RNA.     

Symptoms in Response to Controlled Diesel Exhaust More Closely Reflect Exposure Perception Than True Exposure

Background

Diesel exhaust (DE) exposures are very common, yet exposure-related symptoms haven’t been rigorously examined.

Objective

Describe symptomatic responses to freshly generated and diluted DE and filtered air (FA) in a controlled human exposure setting; assess whether such responses are altered by perception of exposure.

Methods

43 subjects participated within three double-blind crossover experiments to order-randomized DE exposure levels (FA and DE calibrated at 100 and/or 200 micrograms/m³ particulate matter of diameter less than 2.5 microns), and completed questionnaires regarding symptoms and dose perception.

Results

For a given symptom cluster, the majority of those exposed to moderate concentrations of diesel exhaust do not report such symptoms. The most commonly reported symptom cluster was of the nose (29%). Blinding to exposure is generally effective. Perceived exposure, rather than true exposure, is the dominant modifier of symptom reporting.

Conclusion

Controlled human exposure to moderate-dose diesel exhaust is associated with a range of mild symptoms, though the majority of individuals will not experience any given symptom. Blinding to DE exposure is generally effective. Perceived DE exposure, rather than true DE exposure, is the dominant modifier of symptom reporting.     

LRP6 Enhances Glucose Metabolism by Promoting TCF7L2-Dependent Insulin Receptor Expression and IGF Receptor Stabilization in Humans

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Highlights? Nondiabetic LRP6 mutation carriers are hyperinsulinemic and insulin resistant ? IR expression is reduced in skeletal muscles of the LRP6 mutation carriers ? Wnt/LRP6 regulate the insulin receptor and IGFR expression ? The LRP6 mutation reduces TCF7L2-dependent IR expression and enhances mTOR activity     

Neuronal ER Stress Impedes Myeloid-Cell-Induced Vascular Regeneration through IRE1α Degradation of Netrin-1

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Highlights? Canonical ER stress pathways are activated in central neurons during hypoxia/ischemia ? The ER stress endoribonuclease IRE1α degrades the neurovascular guidance cue netrin-1 ? Neuronal-derived netrin-1 activates a reparative proangiogenic program in microglial cells ? Neuronal ER stress prevents reparative angiogenesis in the ischemic neural retina     

Studying the collective motions of the adenosine A2A receptor as a result of ligand binding using principal component analysis

Abstract

Adenosine receptors (ARs) belong to family A of GPCRs that are involved in many diseases, including cerebral and cardiac ischemic diseases, immune and inflammatory disorders, etc. Thus, they represent important therapeutic targets to treat these conditions. Computational techniques such as molecular dynamics (MD) simulations permit researchers to obtain structural information about these proteins, and principal component analysis (PCA) allows for the identification of collective motions. There are available structures for the active form (3QAK) and the inactive form (3EML) of A2AR which permit us to gain insight about their activation/inactivation mechanism. In this work, we have proposed an inverse strategy using MD simulations where the active form was coupled to the antagonist caffeine and the inactive form was coupled to adenosine agonist. Moreover, we have included four reported thermostabilizing mutations in the inactive form to study A2AR structural differences under different conditions. Some observations stand out from the PCA studies. For instance, the apo structures showed remarkable similarities, and the principal components (PCs) were rearranged in a ligand-dependent manner. Additionally, the active conformation was less stable compared to the inactive one. Some PCs inverted their direction in the presence of a ligand, and comparison of the PCs between 3EML and 3EML_ADN showed that adenosine induced major changes in the structure of A2AR. Rearrangement of PCs precedes and drives conformational changes that occur after ligand binding. Knowledge about these conformational changes provides important insights about the activity of A2AR.     

The peptide motif of the single dominantly expressed class I molecule of the chicken MHC can explain the response to a molecular defined vaccine of infectious bursal disease virus (IBDV)

In contrast to typical mammals, the chicken MHC (the BF-BL region of the B locus) has strong genetic associations with resistance and susceptibility to infectious pathogens as well as responses to vaccines. We have shown that the chicken MHC encodes a single dominantly expressed class I molecule whose peptide-binding motifs can determine resistance to viral pathogens, such as Rous sarcoma virus and Marek’s disease virus. In this report, we examine the response to a molecular defined vaccine, fp-IBD1, which consists of a fowlpox virus vector carrying the VP2 gene of infectious bursal disease virus (IBDV) fused with β-galactosidase. We vaccinated parental lines and two backcross families with fp-IBD1, challenged with the virulent IBDV strain F52/70, and measured damage to the bursa. We found that the MHC haplotype B15 from line 15I confers no protection, whereas B2 from line 6₁ and B12 from line C determine protection, although another locus from line 6₁ was also important. Using our peptide motifs, we found that many more peptides from VP2 were predicted to bind to the dominantly expressed class I molecule BF2*1201 than BF2*1501. Moreover, most of the peptides predicted to bind BF2*1201 did in fact bind, while none bound BF2*1501. Using peptide vaccination, we identified one B12 peptide that conferred protection to challenge, as assessed by bursal damage and viremia. Thus, we show the strong genetic association of the chicken MHC to a T cell vaccine can be explained by peptide presentation by the single dominantly expressed class I molecule.     

On Circuit Functionality in Boolean Networks

It has been proved, for several classes of continuous and discrete dynamical systems, that the presence of a positive (resp. negative) circuit in the interaction graph of a system is a necessary condition for the presence of multiple stable states (resp. a cyclic attractor). A positive (resp. negative) circuit is said to be functional when it “generates” several stable states (resp. a cyclic attractor). However, there are no definite mathematical frameworks translating the underlying meaning of “generates.” Focusing on Boolean networks, we recall and propose some definitions concerning the notion of functionality along with associated mathematical results.     

Anticarcinogenic effect of brucine in diethylnitrosamine initiated and phenobarbital-promoted hepatocarcinogenesis in rats

We evaluated the effects of brucine on N-nitrosodiethylamine (DENA)-induced hepatocarcinogenesis in rats. Initiation of hepatocarcinogenesis was done by intraperitoneal injection of diethylnitrosamine (DENA) followed by promotion with phenobarbital. The rats were exposed to dietary brucine for 4 weeks prior to initiation, and the treatment was continued for 22 consecutive weeks. Brucine decreased the incidence, total number, multiplicity, size and volume of preneoplastic hepatic nodules in a dose-dependent manner. Administration of DENA induced hepatocellular carcinoma (HCC), as evidenced by changes in histopathological architecture, increased activity of cytochrome P450, decreased activity of glutathione Stransferase (GST) as well as decreased antioxidant status, enhanced lipid peroxidation, increased liver marker enzymes. Western blot analysis showed decreased expression of cyclin D1 and Bcl-2 with activation of caspase-3 and increased expression of Bax. Immunohistochemical demonstrated the decreased expression of the PCNA and VEGF. These results indicate that brucine prevents lipid peroxidation and hepatic cell damage and also protects the antioxidant system in DENA-induced hepatocarcinogenesis.