A Novel High-Throughput Method for Molecular Detection of Human Pathogenic Viruses Using a Nanofluidic Real-Time PCR System

Human enteric viruses are recognized as the main causes of food- and waterborne diseases worldwide. Sensitive and quantitative detection of human enteric viruses is typically achieved through quantitative RT-PCR (RT-qPCR). A nanofluidic real-time PCR system was used to develop novel high-throughput methods for qualitative molecular detection (RT-qPCR array) and quantification of human pathogenic viruses by digital RT-PCR (RT-dPCR). The performance of high-throughput PCR methods was investigated for detecting 19 human pathogenic viruses and two main process controls used in food virology. The conventional real-time PCR system was compared to the RT-dPCR and RT-qPCR array. Based on the number of genome copies calculated by spectrophotometry, sensitivity was found to be slightly better with RT-qPCR than with RT-dPCR for 14 viruses by a factor range of from 0.3 to 1.6 log₁₀. Conversely, sensitivity was better with RT-dPCR than with RT-qPCR for seven viruses by a factor range of from 0.10 to 1.40 log₁₀. Interestingly, the number of genome copies determined by RT-dPCR was always from 1 to 2 log₁₀ lower than the expected copy number calculated by RT-qPCR standard curve. The sensitivity of the RT-qPCR and RT-qPCR array assays was found to be similar for two viruses, and better with RT-qPCR than with RT-qPCR array for eighteen viruses by a factor range of from 0.7 to 3.0 log₁₀. Conversely, sensitivity was only 0.30 log₁₀ better with the RT-qPCR array than with conventional RT-qPCR assays for norovirus GIV detection. Finally, the RT-qPCR array and RT-dPCR assays were successfully used together to screen clinical samples and quantify pathogenic viruses. Additionally, this method made it possible to identify co-infection in clinical samples. In conclusion, given the rapidity and potential for large numbers of viral targets, this nanofluidic RT-qPCR assay should have a major impact on human pathogenic virus surveillance and outbreak investigations and is likely to be of benefit to public health.     

Spatio-Temporal Patterns of Demyelination and Remyelination in the Cuprizone Mouse Model

Cuprizone administration in mice provides a reproducible model of demyelination and spontaneous remyelination, and has been useful in understanding important aspects of human disease, including multiple sclerosis. In this study, we apply high spatial resolution quantitative MRI techniques to establish the spatio-temporal patterns of acute demyelination in C57BL/6 mice after 6 weeks of cuprizone administration, and subsequent remyelination after 6 weeks of post-cuprizone recovery. MRI measurements were complemented with Black Gold II stain for myelin and immunohistochemical stains for associated tissue changes. Gene expression was evaluated using the Allen Gene Expression Atlas. Twenty-five C57BL/6 male mice were split into control and cuprizone groups; MRI data were obtained at baseline, after 6 weeks of cuprizone, and 6 weeks post-cuprizone. High-resolution (100μm isotropic) whole-brain coverage magnetization transfer ratio (MTR) parametric maps demonstrated concurrent caudal-to-rostral and medial-to-lateral gradients of MTR decrease within corpus callosum (CC) that correlated well with demyelination assessed histologically. Our results show that demyelination was not limited to the midsagittal line of the corpus callosum, and also that opposing gradients of demyelination occur in the lateral and medial CC. T₂-weighted MRI gray/white matter contrast was strong at baseline, weak after 6 weeks of cuprizone treatment, and returned to a limited extent after recovery. MTR decreases during demyelination were observed throughout the brain, most clearly in callosal white matter. Myelin damage and repair appear to be influenced by proximity to oligodendrocyte progenitor cell populations and exhibit an inverse correlation with myelin basic protein gene expression. These findings suggest that susceptibility to injury and ability to repair vary across the brain, and whole-brain analysis is necessary to accurately characterize this model. Whole-brain parametric mapping across time is essential for gaining a real understanding of disease processes in-vivo. MTR increases in healthy mice throughout adolescence and adulthood were observed, illustrating the need for appropriate age-matched controls. Elucidating the unique and site-specific demyelination in the cuprizone model may offer new insights into in mechanisms of both damage and repair in human demyelinating diseases.     

Using fish guilds to assess community responses to temperature and flow regimes in unregulated and regulated Canadian rivers

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An essential role for Grk2 in Hedgehog signalling downstream of Smoothened

The G‐protein‐coupled receptor kinase 2 (adrbk2/GRK2) has been implicated in vertebrate Hedgehog (Hh) signalling based on the effects of its transient knock‐down in mammalian cells and zebrafish embryos. Here, we show that the response to Hh signalling is effectively abolished in the absence of Grk2 activity. Zebrafish embryos lacking all Grk2 activity are refractory to both Sonic hedgehog (Shh) and oncogenic Smoothened (Smo) activity, but remain responsive to inhibition of cAMP‐dependent protein kinase (PKA) activity. Mutation of the kinase domain abrogates the rescuing activity of grk2 mRNA, suggesting that Grk2 acts in a kinase‐dependent manner to regulate the response to Hh. Previous studies have suggested that Grk2 potentiates Smo activity by phosphorylating its C‐terminal tail (CTT). In the zebrafish embryo, however, phosphomimetic Smo does not display constitutive activity, whereas phospho‐null mutants retain activity, implying phosphorylation is neither sufficient nor necessary for Smo function. Since Grk2 rescuing activity requires the integrity of domains essential for its interaction with GPCRs, we speculate that Grk2 may regulate Hh pathway activity by downregulation of a GPCR.     

A role for septin 2 in Drp1‐mediated mitochondrial fission

Mitochondria are essential eukaryotic organelles often forming intricate networks. The overall network morphology is determined by mitochondrial fusion and fission. Among the multiple mechanisms that appear to regulate mitochondrial fission, the ER and actin have recently been shown to play an important role by mediating mitochondrial constriction and promoting the action of a key fission factor, the dynamin‐like protein Drp1. Here, we report that the cytoskeletal component septin 2 is involved in Drp1‐dependent mitochondrial fission in mammalian cells. Septin 2 localizes to a subset of mitochondrial constrictions and directly binds Drp1, as shown by immunoprecipitation of the endogenous proteins and by pulldown assays with recombinant proteins. Depletion of septin 2 reduces Drp1 recruitment to mitochondria and results in hyperfused mitochondria and delayed FCCP‐induced fission. Strikingly, septin depletion also affects mitochondrial morphology in Caenorhabditis elegans, strongly suggesting that the role of septins in mitochondrial dynamics is evolutionarily conserved.     

Deciphering structure-activity relationships in a series of Tat/TAR inhibitors

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Impact of deforestation on the abundance,diversity, and richness of Culex mosquitoes in a southwest Cameroon tropical rainforest

Deforestation is a major threat to biodiversity but little data exist on how deforestation in real‐time affects the overall mosquito species community despite its known role in the transmission of diseases. We compared the abundance and diversity of Culex mosquitoes before and after deforestation along a gradient of three different anthropogenic disturbance levels in a tropical rainforest in southwestern Cameroon. The collections were conducted in unlogged forest (January, 2016), selectively logged forest (January, 2017), and within a young palm plantation (October, 2017) using net traps, sweep nets, resting traps, and dipping for immature stages in water bodies. Mosquitoes were morphologically identified to subspecies, groups, and species. A total of 2,556 mosquitoes was collected of which 1,663 (65.06%) belong to the genus Culex, (n=427 (25.68%) in the unlogged forest; n=900 (54.12%) in the selectively logged forest; and n=336 (20.2%) in the young palm plantation) with a significant difference among the habitats. Diversity and richness of mosquitoes varied significantly among habitats with the highest values found in the selectively logged forest (H=2.4; DS=0.87; S=33) and the lowest value in the unlogged forest (H=1.37; DS=0.68; S=13). The results of this study showed that deforestation affects the abundance and diversity of Culex mosquitoes and favors the invasion of anthropophilic mosquitoes. Higher mosquito abundance and diversity in the selectively logged forest than in the pristine forest is notable and some explanations for these differences are discussed.     

Eimeria tenella ROP kinase EtROP1 induces G0/G1 cell cycle arrest and inhibits host cell apoptosis

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