Paramyxovirus Activation and Inhibition of Innate Immune Responses

Paramyxoviruses represent a remarkably diverse family of enveloped nonsegmented negative-strand RNA viruses, some of which are the most ubiquitous disease-causing viruses of humans and animals. This review focuses on paramyxovirus activation of innate immune pathways, the mechanisms by which these RNA viruses counteract these pathways, and the innate response to paramyxovirus infection of dendritic cells (DC). Paramyxoviruses are potent activators of extracellular complement pathways, a first line of defense that viruses must face during natural infections. We discuss mechanisms by which these viruses activate and combat complement to delay neutralization. Once cells are infected, virus replication drives type I interferon (IFN) synthesis that has the potential to induce a large number of antiviral genes. Here we describe four approaches by which paramyxoviruses limit IFN induction: by limiting synthesis of IFN-inducing aberrant viral RNAs, through targeted inhibition of RNA sensors, by providing viral decoy substrates for cellular kinase complexes, and through direct blocking of the IFN promoter. In addition, paramyxoviruses have evolved diverse mechanisms to disrupt IFN signaling pathways. We describe three general mechanisms, including targeted proteolysis of signaling factors, sequestering cellular factors, and upregulation of cellular inhibitors. DC are exceptional cells with the capacity to generate adaptive immunity through the coupling of innate immune signals and T cell activation. We discuss the importance of innate responses in DC following paramyxovirus infection and their consequences for the ability to mount and maintain antiviral T cells.     

Biophysical and Ultrastructural Characterization of Adeno-Associated Virus Capsid Uncoating and Genome Release

We describe biophysical and ultrastructural differences in genome release from adeno-associated virus (AAV) capsids packaging wild-type DNA, recombinant single-stranded DNA (ssDNA), or dimeric, self-complementary DNA (scDNA) genomes. Atomic force microscopy and electron microscopy (EM) revealed that AAV particles release packaged genomes and undergo marked changes in capsid morphology upon heating in physiological buffer (pH 7.2). When different AAV capsids packaging ss/scDNA varying in length from 72 to 123% of wild-type DNA (3.4 to 5.8 kb) were incrementally heated, the proportion of uncoated AAV capsids decreased with genome length as observed by EM. Genome release was further characterized by a fluorimetric assay, which demonstrated that acidic pH and high osmotic pressure suppress genome release from AAV particles. In addition, fluorimetric analysis corroborated an inverse correlation between packaged genome length and the temperature needed to induce uncoating. Surprisingly, scAAV vectors required significantly higher temperatures to uncoat than their ssDNA-packaging counterparts. However, externalization of VP1 N termini appears to be unaffected by packaged genome length or self-complementarity. Further analysis by tungsten-shadowing EM revealed striking differences in the morphologies of ssDNA and scDNA genomes upon release from intact capsids. Computational modeling and molecular dynamics simulations suggest that the unusual thermal stability of scAAV vectors might arise from partial base pairing and optimal organization of packaged scDNA. Our work further defines the biophysical mechanisms underlying adeno-associated virus uncoating and genome release.     

Integrating telemetry with a predictive model to assess habitat preferences and juvenile survival in an endangered freshwater turtle

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Two New Species of Caddisflies of the Genus Lepidostoma Rambur (Trichoptera: Lepidostomatidae) from the Great Basin

Two new species of Caddisflies from the Great Basin region in California and Nevada are described. Lepidostoma castalianum sp. nov., collected at a spring in the White Mts. of Mono Co., California, is closely related to L. verodum Ross. L. ojanum sp. nov., collected at three separate springs in the White Mts. of Inyo Co., California, and one spring in Mineral Co., Nevada, is a member of the unicolor species group, similar to L. frosti (Milne) and L. unicolor (Banks).     

Phylogeny of the cycads based on multiple single-copy nuclear genes: congruence of concatenated parsimony,likelihood and species tree inference methods

Background and aims

Despite a recent new classification, a stable phylogeny for the cycads has been elusive, particularly regarding resolution of Bowenia, Stangeria and Dioon. In this study, five single-copy nuclear genes (SCNGs) are applied to the phylogeny of the order Cycadales. The specific aim is to evaluate several gene tree–species tree reconciliation approaches for developing an accurate phylogeny of the order, to contrast them with concatenated parsimony analysis and to resolve the erstwhile problematic phylogenetic position of these three genera.

Methods

DNA sequences of five SCNGs were obtained for 20 cycad species representing all ten genera of Cycadales. These were analysed with parsimony, maximum likelihood (ML) and three Bayesian methods of gene tree–species tree reconciliation, using Cycas as the outgroup. A calibrated date estimation was developed with Bayesian methods, and biogeographic analysis was also conducted.

Key Results

Concatenated parsimony, ML and three species tree inference methods resolve exactly the same tree topology with high support at most nodes. Dioon and Bowenia are the first and second branches of Cycadales after Cycas, respectively, followed by an encephalartoid clade (Macrozamia–Lepidozamia–Encephalartos), which is sister to a zamioid clade, of which Ceratozamia is the first branch, and in which Stangeria is sister to Microcycas and Zamia.

Conclusions

A single, well-supported phylogenetic hypothesis of the generic relationships of the Cycadales is presented. However, massive extinction events inferred from the fossil record that eliminated broader ancestral distributions within Zamiaceae compromise accurate optimization of ancestral biogeographical areas for that hypothesis. While major lineages of Cycadales are ancient, crown ages of all modern genera are no older than 12 million years, supporting a recent hypothesis of mostly Miocene radiations. This phylogeny can contribute to an accurate infrafamilial classification of Zamiaceae.     

Morph-dependent resource acquisition and fitness in a polymorphic bird

Understanding genetic colour polymorphism has proved a major challenge, both in terms of the underlying genetic mechanisms and the evolutionarily forces maintaining such genetic variation. In this context, genetic differences in aggression or competitive-related traits may covary with the expression of alternative phenotypes, and affect the evolutionary stability and maintenance of colour polymorphisms. Genetic red and black head-colour morphs of the Gouldian finch (Erythrura gouldiae) co-occur in temporally and geographically stable frequencies in sympatric populations. Gouldian finches are obligate cavity-nesters with highly specific preferences for nest-site morphometry that directly affect reproductive success. Because intra- and interspecific competition for high quality nest-sites is prevalent, and fitness is directly related to nest-site quality, we investigated the relative access (and consequences for reproductive success) of alternative morphs to this critical limiting resource in the wild. Red males defended higher quality nest-sites, and overcame greater levels of nest-site competition against conspecifics and superior heterospecific competitors than black males. Red-headed males also produced more fledglings (especially with red-headed females) than black-headed males, independent of nest-site quality. Finally, the independent (positive) effect of nest-site quality on reproductive success was confirmed. Such competitive asymmetries are important to relative selection among coexisting morphs, and are likely to contribute to the maintenance of alternative sympatric colour-morphs in wild populations.     

Oxime-dipeptides as anticholinesterase,reactivator of phosphonylated-serine of AChE catalytic triad: probing the mechanistic insight by MM-GBSA,dynamics simulations and DFT analysis

Neuropathological cascades leading to reduced cholinergic transmission in Alzheimer’s disease led to development of AChE-inhibitors. Although lethal dose of some inhibitors cause interruption with AChE mediated mechanism but reversible AChE inhibitors can assist in protection from inhibition of AChE and hence in an aim to probe potential molecules as anticholinesterase and as reactivators, computationally structure-based approach has been exploited in this work for designing new 2-amino-3-pyridoixime-dipeptides conjugates. We have combined MD simulations with flexible ligand docking approach to determine binding specificity of 2-amino-3-pyridoixime dipeptides towards AChE (PDB 2WHP). PAS residues are found to be responsible for oxime-dipeptides binding along with π–π interactions with Trp86 and Tyr286, hydrogen bonding with side chains of Asp74 and Tyr341 (Gscore –10.801 and MM-GBSA free energy –34.89?kcal/mol). The docking results depicted complementary multivalent interactions along with good binding affinity as predicted from MM-GBSA analysis. The 2-amino-3-pyridoxime-(Arg-Asn) AChE systems subjected to MD simulations under explicit solvent systems with NPT and NVT ensemble. MD simulations uncovered dynamic behavior of 2-amino-3-pyridoxime-(Arg-Asn) and exposed its mobile nature and competence to form strong long range-order contacts towards active site residues to approach inhibited serine residue and facilitated via large contribution from hydrogen bonding and water bridges along with slow and large movements of adjacent important residues. In an effort to evaluate the complete potential surface profile, 2-amino-3-pyridoxime induced reactivation pathway of sarin–serine adduct has been investigated by the DFT approach at the vacuum MO6/6–311G (d, p) level along with the Poisson-Boltzmann solvation model and found to be of relatively low energy barrier. The pKa evaluation has revealed the major deprotonated 2-amino-3-pyridoixime species having pKa of 6.47 and hence making 2-amino-3-pyridoxime-(Arg-Asn) potential anticholinesterase and reactivator for AChE under the physiological pH.     

Reconstitution of KCNE1 into lipid bilayers: comparing the structural, dynamic, and activity differences in micelle and vesicle environments

KCNE1 (minK), found in the human heart and cochlea, is a transmembrane protein that modulates the voltage-gated potassium KCNQ1 channel. While KCNE1 has previously been the subject of extensive structural studies in lyso-phospholipid detergent micelles, key observations have yet to be confirmed and refined in lipid bilayers. In this study, a reliable method for reconstituting KCNE1 into lipid bilayer vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho(1'-rac-glycerol) (sodium salt) (POPG) was developed. Microinjection of the proteoliposomes into Xenopus oocytes expressing the human KCNQ1 (K(V)7.1) voltage-gated potassium channel led to nativelike modulation of the channel. Circular dichroism spectroscopy demonstrated that the percent helicity of KCNE1 is significantly higher for the protein reconstituted in lipid vesicles than for the previously described structure in 1.0% 1-myristoyl-2-hydroxy-sn-glycero-3-phospho(1'-rac-glycerol) (sodium salt) (LMPG) micelles. SDSL electron paramagnetic resonance spectroscopic techniques were used to probe the local structure and environment of Ser28, Phe54, Phe57, Leu59, and Ser64 of KCNE1 in both POPC/POPG vesicles and LMPG micelles. Spin-labeled KCNE1 cysteine mutants at Phe54, Phe57, Leu59, and Ser64 were found to be located inside POPC/POPG vesicles, whereas Ser28 was found to be located outside the membrane. Ser64 was shown to be water inaccessible in vesicles but found to be water accessible in LMPG micelle solutions. These results suggest that key components of the micelle-derived structure of KCNE1 extend to the structure of this protein in lipid bilayers but also demonstrate the need to refine this structure using data derived from the bilayer-reconstituted protein to more accurately define its native structure. This work establishes the basis for such future studies.     

Probing oxygen activation sites in two flavoprotein oxidases using chloride as an oxygen surrogate

A single basic residue above the si-face of the flavin ring is the site of oxygen activation in glucose oxidase (GOX) (His516) and monomeric sarcosine oxidase (MSOX) (Lys265). Crystal structures of both flavoenzymes exhibit a small pocket at the oxygen activation site that might provide a preorganized binding site for superoxide anion, an obligatory intermediate in the two-electron reduction of oxygen. Chloride binds at these polar oxygen activation sites, as judged by solution and structural studies. First, chloride forms spectrally detectable complexes with GOX and MSOX. The protonated form of His516 is required for tight binding of chloride to oxidized GOX and for rapid reaction of reduced GOX with oxygen. Formation of a binary MSOX·chloride complex requires Lys265 and is not observed with Lys265Met. Binding of chloride to MSOX does not affect the binding of a sarcosine analogue (MTA, methylthioactetate) above the re-face of the flavin ring. Definitive evidence is provided by crystal structures determined for a binary MSOX·chloride complex and a ternary MSOX·chloride·MTA complex. Chloride binds in the small pocket at a position otherwise occupied by a water molecule and forms hydrogen bonds to four ligands that are arranged in approximate tetrahedral geometry: Lys265:NZ, Arg49:NH1, and two water molecules, one of which is hydrogen bonded to FAD:N5. The results show that chloride (i) acts as an oxygen surrogate, (ii) is an effective probe of polar oxygen activation sites, and (iii) provides a valuable complementary tool to the xenon gas method that is used to map nonpolar oxygen-binding cavities.