Innate Intracellular Antiviral Responses Restrict the Amplification of Defective Virus Genomes of Parainfluenza Virus 5.

During the replication of parainfluenza virus 5 (PIV5), copyback defective virus genomes (DVGs) are erroneously produced and are packaged into "infectious" virus particles. Copyback DVGs are the primary inducers of innate intracellular responses, including the interferon (IFN) response. While DVGs can interfere with the replication of nondefective (ND) virus genomes and activate the IFN-induction cascade before ND PIV5 can block the production of IFN, we demonstrate that the converse is also true, i.e., high levels of ND virus can block the ability of DVGs to activate the IFN-induction cascade. By following the replication and amplification of DVGs in A549 cells that are deficient in a variety of innate intracellular antiviral responses, we show that DVGs induce an uncharacterized IFN-independent innate response(s) that limits their replication. High-throughput sequencing was used to characterize the molecular structure of copyback DVGs. While there appears to be no sequence-specific break or rejoining points for the generation of copyback DVGs, our findings suggest there are region, size, and/or structural preferences selected for during for their amplification.IMPORTANCE Copyback defective virus genomes (DVGs) are powerful inducers of innate immune responses both in vitro and in vivo They impact the outcome of natural infections, may help drive virus-host coevolution, and promote virus persistence. Due to their potent interfering and immunostimulatory properties, DVGs may also be used therapeutically as antivirals and vaccine adjuvants. However, little is known of the host cell restrictions which limit their amplification. We show here that the generation of copyback DVGs readily occurs during parainfluenza virus 5 (PIV5) replication, but that their subsequent amplification is restricted by the induction of innate intracellular responses. Molecular characterization of PIV5 copyback DVGs suggests that while there are no genome sequence-specific breaks or rejoin points for the generation of copyback DVGs, genome region, size, and structural preferences are selected for during their evolution and amplification

Temperature-dependent changes in neuronal dynamics in a patient with an SCN1A mutation and hyperthermia induced seizures

Dravet syndrome is the prototype of SCN1A-mutation associated epilepsies. It is characterised by prolonged seizures, typically provoked by fever. We describe the evaluation of an SCN1A mutation in a child with early-onset temperature-sensitive seizures. The patient carries a heterozygous missense variant (c3818C > T; pAla1273Val) in the NaV1.1 brain sodium channel. We compared the functional effects of the variant vs. wild type NaV1.1 using patch clamp recordings from channels expressed in Chinese Hamster Ovary Cells at different temperatures (32, 37, and 40 °C). The variant channels produced a temperature-dependent destabilization of activation and fast inactivation. Implementing these empirical abnormalities in a computational model predicts a higher threshold for depolarization block in the variant, particularly at 40 °C, suggesting a failure to autoregulate at high-input states. These results reveal direct effects of abnormalities in NaV1.1 biophysical properties on neuronal dynamics. They illustrate the value of combining cellular measurements with computational models to integrate different observational scales (gene/channel to patient)

The geochemistry and petrogenesis of the Paleoproterozoic du Chef dyke swarm, Québec, Canada

This is the author accepted manuscript. The final version is available from Elsevier via the DOI in this record.The du Chef dyke swarm in southern Québec, Canada is composed of numerous northeast trending, greenschist-amphibolite facies, gabbronoritic dykes that crop out either side of the Grenville Front. The age of the du Chef swarm (2408 ± 3 Ga) has led previous authors to suggest a genetic link between the du Chef dykes and coeval swarms (including the Ringvassøy, Scourie, Widgemooltha and Sebanga) preserved on other Archean cratons. These now disparate dyke swarms are proposed to have formed in response to mantle plume-induced continental breakup during the early Proterozoic. This work represents the first geochemical study of the du Chef dykes and shows that the swarm evolved through fractional crystallisation of a tholeiitic parent magma that remained largely uncontaminated during its residence in, and ascent through, the crust. We also show that the primary magma for the du Chef swarm was derived through partial melting of an enriched region of the mantle, with a similar trace element composition to the modern-day HIMU reservoir and that the magma produced was significantly hotter than the ambient mantle at the time. We contend that the du Chef dykes are the product of early Proterozoic mantle plume magmatism and may help pinpoint an ancient hotspot centre that initiated continental break up along the margin of the Superior Craton at ∼2.4 Ga. Other dyke swarms proposed to be genetically linked with the du Chef dykes record a distinctly different petrogenetic history to that of the du Chef dykes, as evidenced by their more volcanic arc-like geochemical signature. These contrasting geochemical signatures in supposedly cogenetic continental tholeiitic rocks may be evidence of early Proterozoic mantle heterogeneity sampled by the rising du Chef mantle plume.This study forms part of a Ph.D. dissertation undertaken by T.J.R.C. at the University of Cardiff, United Kingdom. A. Okrugin's assistance in the field is acknowledged. J. Strongman, J. Fletcher and J. Pett are thanked for their permission of use of the petrographic equipment at Petrolab Ltd. L. Badham, A. Oldroyd, L. Woolley and P. Fisher are thanked for their help in preparation and analysis of samples. This is publication number 38 of the Large Igneous Provinces, Supercontinent Reconstruction, Resource Exploration Project (www.supercontinent.org)

Ketamine Restores Thalamic-Prefrontal Cortex Functional Connectivity in a Mouse Model of Neurodevelopmental Disorder-Associated 2p16.3 Deletion

2p16.3 deletions, involving heterozygous NEUREXIN1 (NRXN1) deletion, dramatically increase the risk of developing neurodevelopmental disorders, including autism and schizophrenia. We have little understanding of how NRXN1 heterozygosity increases the risk of developing these disorders, particularly in terms of the impact on brain and neurotransmitter system function and brain network connectivity. Thus, here we characterize cerebral metabolism and functional brain network connectivity in Nrxn1α heterozygous mice (Nrxn1α+/− mice), and assess the impact of ketamine and dextro-amphetamine on cerebral metabolism in these animals. We show that heterozygous Nrxn1α deletion alters cerebral metabolism in neural systems implicated in autism and schizophrenia including the thalamus, mesolimbic system, and select cortical regions. Nrxn1α heterozygosity also reduces the efficiency of functional brain networks, through lost thalamic “rich club” and prefrontal cortex (PFC) hub connectivity and through reduced thalamic-PFC and thalamic “rich club” regional interconnectivity. Subanesthetic ketamine administration normalizes the thalamic hypermetabolism and partially normalizes thalamic disconnectivity present in Nrxn1α+/− mice, while cerebral metabolic responses to dextro-amphetamine are unaltered. The data provide new insight into the systems-level impact of heterozygous Nrxn1α deletion and how this increases the risk of developing neurodevelopmental disorders. The data also suggest that the thalamic dysfunction induced by heterozygous Nrxn1α deletion may be NMDA receptor-dependent

Analysis of Paramyxovirus Transcription and Replication by High-Throughput Sequencing.

We have developed a high-throughput sequencing (HTS) workflow for investigating paramyxovirus transcription and replication. We show that sequencing of oligo(dT)-selected polyadenylated mRNAs, without considering the orientation of the RNAs from which they had been generated, cannot accurately be used to analyze the abundance of viral mRNAs because genomic RNA copurifies with the viral mRNAs. The best method is directional sequencing of infected cell RNA that has physically been depleted of ribosomal and mitochondrial RNA followed by bioinformatic steps to differentiate data originating from genomes from viral mRNAs and antigenomes. This approach has the advantage that the abundance of viral mRNA (and antigenomes) and genomes can be analyzed and quantified from the same data. We investigated the kinetics of viral transcription and replication during infection of A549 cells with parainfluenza virus type 2 (PIV2), PIV3, PIV5, or mumps virus and determined the abundances of individual viral mRNAs and readthrough mRNAs. We found that the mRNA abundance gradients differed significantly between all four viruses but that for each virus the pattern remained relatively stable throughout infection. We suggest that rapid degradation of non-poly(A) mRNAs may be primarily responsible for the shape of the mRNA abundance gradient in parainfluenza virus 3, whereas a combination of this factor and disengagement of RNA polymerase at intergenic sequences, particularly those at the NP:P and P:M gene boundaries, may be responsible in the other viruses.IMPORTANCE High-throughput sequencing (HTS) of virus-infected cells can be used to study in great detail the patterns of virus transcription and replication. For paramyxoviruses, and by analogy for all other negative-strand RNA viruses, we show that directional sequencing must be used to distinguish between genomic RNA and mRNA/antigenomic RNA because significant amounts of genomic RNA copurify with poly(A)-selected mRNA. We found that the best method is directional sequencing of total cell RNA, after the physical removal of rRNA (and mitochondrial RNA), because quantitative information on the abundance of both genomic RNA and mRNA/antigenomes can be simultaneously derived. Using this approach, we revealed new details of the kinetics of virus transcription and replication for parainfluenza virus (PIV) type 2, PIV3, PIV5, and mumps virus, as well as on the relative abundance of the individual viral mRNAs

Persistent Replication of a Chikungunya Virus Replicon in Human Cells is Associated with Presence of Stable Cytoplasmic Granules Containing Non-structural Protein 3

Chikungunya virus (CHIKV), a mosquito-borne human pathogen, causes a disabling disease characterized by severe joint pain that can persist for weeks, months or even years in patients. The non-structural protein 3 (nsP3) plays essential roles during acute infection, but little is known about the function of nsP3 during chronic disease. Here, we used sub-diffraction multi-color microscopy for spatial and temporal analysis of CHIKV nsP3 within human cells that persistently replicate replicon RNA. Round cytoplasmic granules of various sizes (i) contained nsP3 and stress granule assembly factors 1 and 2 (G3BP1/2); (ii) were next to double-stranded RNA foci and nsP1-positive structures; and (iii) were close to the nuclear membrane and the nuclear pore complex protein Nup98. Analysis of protein turnover and mobility by live-cell microscopy revealed that granules could persist for hours to days, accumulated newly synthesized protein, and moved through the cytoplasm at varying speeds. Granules also had a static internal architecture and were stable in cell lysates. Refractory cells that had cleared the non-cytotoxic replicon regained the ability to respond to arsenite-induced stress. In summary, nsP3 can form uniquely stable granular structures that persist long-term within the host cell. This continued presence of viral and cellular protein-complexes has implications for the study of the pathogenic consequences of lingering CHIKV infection and the development of strategies to mitigate the burden of chronic musculoskeletal disease brought about by a medically important arthropod-borne virus (arbovirus).ImportanceChikungunya virus (CHIKV) is a re-emerging alphavirus transmitted by mosquitos and causes transient sickness but also chronic disease affecting muscles and joints. No approved vaccines or antivirals are available. Thus, a better understanding of the viral life cycle and the role of viral proteins can aid in identifying new therapeutic targets. Advances in microscopy and development of non-cytotoxic replicons (Utt, Das, Varjak, Lulla, Lulla, Merits, J Virol 89:3145-62, 2015, doi:10.1128/JVI.03213-14) have allowed researchers to study viral proteins within controlled laboratory environments over extended durations. Here we established human cells that stably replicate replicon RNA and express tagged non-structural protein 3. The ability to track nsP3 within the host cell and during persistent replication can benefit fundamental research efforts to better understand long-term consequences of the persistence of viral protein complexes and thereby provide the foundation for new therapeutic targets to control CHIKV infection and treat chronic disease symptoms

Psychometrics of new scales of parenting practices to encourage or discourage Hispanic preschool children's physical activity

Conference Theme: Promoting Healthy Eating and activity worldwidePoster - Personal and environmental determinants of physical activity in children and adolescents: abstract P077PURPOSE: Develop and assess the psychometrics of a new instrument for parenting practices (PP) that encourage or discourage physical activity (PA) in Hispanic preschool children. METHOD: Cross--‐sectional study of 240 Hispanic parents who reported their demographics and frequency of using PP that encourage (structure and encouragement) or discourage (promoting inactivity, psychological control, safety …postprin

Association of parenting practices to encourage or discourage physical activity with Hispanic preschool children's objectively measured physical activity

Oral Session - Determinants of physical activity in children and adolescents: no. O.002Conference Theme: Promoting Healthy Eating and Activity WorldwidePURPOSE: Assess the association of parenting practices (PP) to encourage or discourage physical activity (PA) with Hispanic 3-5 year old children’s objectively measured PA METHOD: Cross-sectional study of Hispanic parent-child dyads (n= 84) who reported their demographics and frequency of using PP that encourage (structure/encouragement) or discourage (promote inactive transport, promote screen time, psychological control, and safety concerns) child PA using verified scales. Children wore Actigraph GT3X accelerometers recording 15 second epochs for 7 days. Allowing for re-wears …postprin

A new method for the estimate of H_0 from quadruply imaged gravitational lens systems

We present a new method to estimate the Hubble constant H_0 from the measured time delays in quadruply imaged gravitational lens systems. We show how it is possible to get an estimate of H_0 without the need to completely reconstruct the lensing potential thus avoiding any a priori hypotheses on the expression of the galaxy lens model. Our method only needs to assume that the lens potential may be expressed as r^{\alpha} F(\theta), whatever the shape function F(\theta) is, and it is thus able to fully explore the degeneracy in the mass models taking also into account the presence of an external shear. We test the method on simulated cases and show that it does work well in recovering the correct value of the slope \alpha of the radial profile and of the Hubble constant H_0. Then, we apply the same method to the real quadruple lenses PG1115+080 and B1422+231 obtaining H_0 = 58_{-15}^{+17} km/s/Mpc (68% CL).Comment: 12 pages, 5 figures, accepted for publication on Astronomy & Astrophysic

ON-bipolar cell gene expression during retinal degeneration: Implications for optogenetic visual restoration.

PURPOSE: Retinal bipolar cells survive even in the later stages of inherited retinal degenerations (IRDs) and so are attractive targets for optogenetic approaches to vision restoration. However, it is not known to what extent the remodelling that these cells undergo during degeneration affects their function. Specifically, it is unclear if they are free from metabolic stress, receptive to adeno-associated viral vectors, suitable for opsin-based optogenetic tools and able to propagate signals by releasing neurotransmitter. METHODS: Fluorescence activated cell sorting (FACS) was performed to isolate labelled bipolar cells from dissociated retinae of litter-mates with or without the IRD mutation Pde6brd1/rd1 selectively expressing an enhanced yellow fluorescent protein (EYFP) as a marker in ON-bipolar cells. Subsequent mRNA extraction allowed Illumina® microarray comparison of gene expression in bipolar cells from degenerate to those of wildtype retinae. Changes in four candidate genes were further investigated at the protein level using retinal immunohistochemistry over the course of degeneration. RESULTS: A total of sixty differentially expressed transcripts reached statistical significance: these did not include any genes directly associated with native primary bipolar cell signalling, nor changes consistent with metabolic stress. Four significantly altered genes (Srm2, Slf2, Anxa7 & Cntn1), implicated in synaptic remodelling, neurotransmitter release and viral vector entry had immunohistochemical staining colocalising with ON-bipolar cell markers and varying over the course of degeneration. CONCLUSION: Our findings suggest relatively few gene expression changes in the context of degeneration: that despite remodelling, bipolar cells are likely to remain viable targets for optogenetic vision restoration. In addition, several genes where changes were seen could provide a basis for investigations to enhance the efficacy of optogenetic therapies