Structure and Dynamics of the Second CARD of Human RIG-I Provide Mechanistic Insights into Regulation of RIG-I Activation

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Highlights? The structure of a CARD of human RIG-I ? The mechanism and structural role of phosphorylation are revealed ? In trans interaction with the Helicase and regulatory domains is shown ? Lys172 lies in proximity to the CARD2:helicase-CTD interface     

A computational study of the protein-ligand interactions in CDK2 inhibitors: using quantum mechanics/molecular mechanics interaction energy as a predictor of the biological activity

We report a combined quantum mechanics/molecular mechanics (QM/MM) study to determine the protein-ligand interaction energy between CDK2 (cyclin-dependent kinase 2) and five inhibitors with the N(2)-substituted 6-cyclohexyl-methoxy-purine scaffold. The computational results in this work show that the QM/MM interaction energy is strongly correlated to the biological activity and can be used as a predictor, at least within a family of substrates. A detailed analysis of the protein-ligand structures obtained from molecular dynamics simulations shows specific interactions within the active site that, in some cases, have not been reported before to our knowledge. The computed interaction energy gauges the strength of protein-ligand interactions. Finally, energy decomposition and multiple regression analyses were performed to check the contribution of the electrostatic and van der Waals energies to the total interaction energy and to show the capabilities of the computational model to identify new potent inhibitors.     

Protection of Mice against Lethal Challenge with 2009 H1N1 Influenza A Virus by 1918-Like and Classical Swine H1N1 Based Vaccines

The recent 2009 pandemic H1N1 virus infection in humans has resulted in nearly 5,000 deaths worldwide. Early epidemiological findings indicated a low level of infection in the older population (>65 years) with the pandemic virus, and a greater susceptibility in people younger than 35 years of age, a phenomenon correlated with the presence of cross-reactive immunity in the older population. It is unclear what virus(es) might be responsible for this apparent cross-protection against the 2009 pandemic H1N1 virus. We describe a mouse lethal challenge model for the 2009 pandemic H1N1 strain, used together with a panel of inactivated H1N1 virus vaccines and hemagglutinin (HA) monoclonal antibodies to dissect the possible humoral antigenic determinants of pre-existing immunity against this virus in the human population. By hemagglutinination inhibition (HI) assays and vaccination/challenge studies, we demonstrate that the 2009 pandemic H1N1 virus is antigenically similar to human H1N1 viruses that circulated from 1918–1943 and to classical swine H1N1 viruses. Antibodies elicited against 1918-like or classical swine H1N1 vaccines completely protect C57B/6 mice from lethal challenge with the influenza A/Netherlands/602/2009 virus isolate. In contrast, contemporary H1N1 vaccines afforded only partial protection. Passive immunization with cross-reactive monoclonal antibodies (mAbs) raised against either 1918 or A/California/04/2009 HA proteins offered full protection from death. Analysis of mAb antibody escape mutants, generated by selection of 2009 H1N1 virus with these mAbs, indicate that antigenic site Sa is one of the conserved cross-protective epitopes. Our findings in mice agree with serological data showing high prevalence of 2009 H1N1 cross-reactive antibodies only in the older population, indicating that prior infection with 1918-like viruses or vaccination against the 1976 swine H1N1 virus in the USA are likely to provide protection against the 2009 pandemic H1N1 virus. This data provides a mechanistic basis for the protection seen in the older population, and emphasizes a rationale for including vaccination of the younger, naïve population. Our results also support the notion that pigs can act as an animal reservoir where influenza virus HAs become antigenically frozen for long periods of time, facilitating the generation of human pandemic viruses.     

HIV neuromuscular disease and mitochondrial function

The pathogenesis of neuromuscular syndromes in HIV-infected patients is multifactorial. Of recent concern is the mitochondrial-mediated neuromuscular pathology in HIV and its treatment. We present currently available evidence supporting the role of mitochondrial pathology in the peripheral nerve and muscle disorders due to HIV infection and antiretroviral (ARV) therapy. Three neuromuscular syndromes are discussed: distal symmetric polyneuropathy (DSP), myopathy, and HIV-associated neuromuscular weakness syndrome (HANWS). Myopathy has the most in-vivo data relating to HIV-related mitochondrial pathology, while DSP and HANWS have growing evidence of mitochondrial pathology, particularly in the context of ARV use. It is likely that these neuromuscular disorders result from a combination of mitochondrial and immunological mechanisms due to HIV and ARV therapy.     

BRN3-type POU Homeobox Genes Maintain the Identity of Mature Postmitotic Neurons in Nematodes and Mice

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Post-fire natural regeneration of a <Emphasis Type="Italic">Pinus pinaster</Emphasis> forest in NW Spain

The aim of this study was to analyse the regeneration of Pinus pinaster after wildfire and the possible inter and intraspecific competition during the first 3 years after fire. The study area is located in a P. pinaster stand in León province (NW Spain). Three study sites (S1, S2 and S3) were established in an area burned in 1998. In each site, three permanent plots (20 × 1 m) were marked. A total of 20 quadrats of 1 m ² were studied in each plot. The number and height of pine seedlings 1, 2 and 3 years after fire was recorded in each quadrat. The regeneration of understorey vegetation in the quadrats was analysed concurrently. The significance of linear correlations among the number and height of seedlings and understorey vegetation cover was tested by calculating Pearson correlation coefficients.Seed germination and seedling emergence took place massively during the first year after the fire and decreased through time. The height growth was constant over the 3 years at site S2, while a growth burst could be observed between years 2 and 3 at sites S1 and S2. Also, pines from site S2 reached shorter maximum heights in all years compared to pines from site S1 and S3. The understorey vegetation showed minimal regeneration during the first year but then increased greatly with time. Woody understorey cover and total vegetation cover were negatively correlated with pine seedling density in sites with a high number of seedlings (e.g. S1 and S3). When woody cover, total cover and pine seedling density were low (e.g. S2), there were no correlations. There was a positive correlation between vegetation cover and the maximum height of Pinus seedlings in all study sites.     

A Versatile Vector for In Vivo Monitoring of Type I Interferon Induction and Signaling

Development of reporter systems for in vivo examination of IFN-β induction or signaling of type I interferon (IFN-I) pathways is of great interest in order to characterize biological responses to different inducers such as viral infections. Several reporter mice have been developed to monitor the induction of both pathways in response to different agonists. However, alternative strategies that do not require transgenic mice breeding have to date not been reported. In addition, detection of these pathways in vivo in animal species other than mice has not yet been addressed. Herein we describe a simple method based on the use of an adeno-associated viral vector (AAV8-3xIRF-ISRE-Luc) containing an IFN-β induction and signaling-sensitive promoter sequence controlling the expression of the reporter gene luciferase. This vector is valid for monitoring IFN-I responses in vivo elicited by diverse stimuli in different organs. Intravenous administration of the vector in C57BL/6 mice and Syrian hamsters was able to detect activation of the IFN pathway in the liver upon systemic treatment with different pro-inflammatory agents and infection with Newcastle disease virus (NDV). In addition, intranasal instillation of AAV8-3xIRF-ISRE-Luc showed a rapid and transient IFN-I response in the respiratory tract of mice infected with the influenza A/PR8/34 virus lacking the NS1 protein. In comparison, this response was delayed and exacerbated in mice infected with influenza A/PR/8 wild type virus. In conclusion, the AAV8-3xIRF-ISRE-Luc vector offers the possibility of detecting IFN-I activation in response to different stimuli and in different animal models with no need for reporter transgenic animals.     

A quantum mechanic/molecular mechanic study of the wild-type and N155S mutant HIV-1 integrase complexed with diketo acid

Integrase (IN) is one of the three human immunodeficiency virus type 1 (HIV-1) enzymes essential for effective viral replication. Recently, mutation studies have been reported that have shown that a certain degree of viral resistance to diketo acids (DKAs) appears when some amino acid residues of the IN active site are mutated. Mutations represent a fascinating experimental challenge, and we invite theoretical simulations for the disclosure of still unexplored features of enzyme reactions. The aim of this work is to understand the molecular mechanisms of HIV-1 IN drug resistance, which will be useful for designing anti-HIV inhibitors with unique resistance profiles. In this study, we use molecular dynamics simulations, within the hybrid quantum mechanics/molecular mechanics (QM/MM) approach, to determine the protein-ligand interaction energy for wild-type and N155S mutant HIV-1 IN, both complexed with a DKA. This hybrid methodology has the advantage of the inclusion of quantum effects such as ligand polarization upon binding, which can be very important when highly polarizable groups are embedded in anisotropic environments, for example in metal-containing active sites. Furthermore, an energy terms decomposition analysis was performed to determine contributions of individual residues to the enzyme-inhibitor interactions. The results reveal that there is a strong interaction between the Lys-159, Lys-156, and Asn-155 residues and Mg²⁺ cation and the DKA inhibitor. Our calculations show that the binding energy is higher in wild-type than in the N155S mutant, in accordance with the experimental results. The role of the mutated residue has thus been checked as maintaining the structure of the ternary complex formed by the protein, the Mg²⁺ cation, and the inhibitor. These results might be useful to design compounds with more interesting anti-HIV-1 IN activity on the basis of its three-dimensional structure.